Manufactured Nanomaterials

L
Fate of Manufactured Nanomaterials in the

Australian Environment
G.E. Batley and M.J. McLaughlin
CSIRO Niche Manufacturing Flagship Report
March 2010
Prepared for the Department of the Environment, Water, Heritage and

the Arts
Enquiries should be addressed to:
Graeme Batley
Centre for Environmental Contaminants Research
CSIRO Land and Water
Private Mailbag 7, Bangor NSW 2234
Phone 02 9710 6830
Fax 02 9719 6837
Email Graeme.batley@csiro.au
© Commonwealth of Australia 2008

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ii Fate of Manufactured Nanomaterials in the Australian Environment

EXECUTIVE SUMMARY
With growing production and use of manufactured nanoparticles in a large range of consumer
products, regulatory agencies worldwide are addressing the risk that these substances may
pose to both the environment and human health. An assessment with respect to ecosystem
health requires an ecological risk assessment that must take into account current knowledge
about nanomaterial uses, environmental concentrations, fate, and effects, to determine both
predicted environmental concentrations (PECs) and predicted no-effect concentrations
(PNECs).
This report reviews the available literature on the fate of manufactured nanomaterials in the
aquatic and terrestrial environment. Seven classes of nanomaterials were considered: (i) metal
oxides; (ii) carbon products (n-C60 fullerenes, carbon nanotubes); (iii) metals; (iv) quantum
dots and semiconductors; (v) nanoclays, (vi) dendrimers, and (vii) nanoemulsions.
The key processes that govern nanoparticle behaviour in the aquatic environment are
aggregation and dissolution, driven by size and surface properties of the materials. These
processes can be mediated by interactions with dissolved organic matter and other natural
colloids. Biological degradation processes and abiotic degradation via hydrolysis and
photolysis do not appear to be significant in waters, although oxidation/reduction reactions can
be significant for some metals.
Similar processes are operative in terrestrial systems, but mobility is much reduced compared
to aquatic environments. Interactions of nanoparticles with soil minerals and organic matter
have not been evaluated, but are likely to be a function of particle size, shape and surface
properties (specific surface area and surface charge). Small hydrophilic nanoparticles (<20 nm)
with net negative surface charges are likely to be mobile, while large hydrophilic positively-
charged particles will be sorbed by soil. Strongly hydrophobic nanoparticles are likely to be
strongly retained by soil organic matter.
Many parallels can be seen in the behaviour of natural colloids. In considering the behaviour
of manufactured nanomaterials, it is important that studies be carried out in natural waters as
the often orders of magnitude higher concentration of natural colloids can have a significant
impact. Aggregation results in growth of nanoparticles, often to sizes in excess of the
nanoparticle size definition of <100 nm, ultimately leading to sedimentation. This growth can
be prevented by the presence of surfactants and other surface coatings, or through the presence
of natural humic materials. Fibrillar colloids enhance precipitation. Any toxicity studies will
need to separately address particular nanomaterial formulations.
There is evidence to suggest that the impact of manufactured nanoparticles on aquatic

organisms differs compared to their macroparticle equivalents. In some instances such
assessments can be confounded due to nanoparticle solubility, with zinc oxide and cadmium-
containing quantum dots being cases in point.
Mechanisms of nanomaterial toxicity include cellular damage due to oxidative stress, physical
damage to the cell surface, dissolution at the cell surface, and impacts via bioaccumulation.
The latter involves interaction with the cell surface for unicellular organisms and uptake across
Fate of Manufactured Nanomaterials in the Australian Environment iii

the gill and other external surface epithelia for higher organisms. Bioaccumulation via the food
chain is also possible. The extensive literature on bacterial toxicity was considered
inappropriate for defining no effects concentrations of nanomaterials in waters, however, the
effects, both positive and negative, of some nanomaterials on bacteria present in sewage
treatment plants and on soil microorganisms have been discussed.
There are limited data on toxicity of nanoparticles to algae, invertebrates and fish. In the case
of n-C60 fullerenes, toxicity was highly dependent on the method of preparation, with the
particles dispersed by evaporation of tetrahydrofuran (THF) extracts being more toxic than
those dispersed by sonication, due supposedly to secondary effects of THF. Insufficient data
were available to derive high reliability environmental guidelines, but for freshwaters, low
reliability guidelines were derived for n-C60 and TiO2 nanoparticles. The calculated PNEC
values are only marginally above the concentrations estimated to be released to the
environment in calculations based on nanomaterial usage in the UK.
There is much less information on the behaviour and toxicity of nanoparticles in terrestrial
systems, due to difficulties in assessing dose against a background of natural nanoparticles in
the soil matrix. Heterogeneity and incorporation of nanoparticles into soil is also an issue for
ecotoxicological testing. There are a few reports of adverse effects of some nanoparticles to
terrestrial species cultured in vitro, but to date there is no strong evidence that nanomaterials
have significant adverse effects on terrestrial species in soil exposures. Further studies are
needed with a wide range of terrestrial species, and a wide range of nanoparticulate materials
in a range of soil environments, to determine if the preliminary data are sound.
There are numerous examples to demonstrate that nanomaterials can be bioaccumulated by

organisms. The extent to which this uptake exerts toxicity is less certain.
Current international activities in relation to nanoparticle risk assessment are discussed. In

summary, most countries see the need for more data gathering and research to improve the risk
assessment of these materials. This review indicates that the same is possibly true for
Australia, but the way ahead is reasonably clear. The basic recommendations for future
research are:
1. There is a need for measurements in natural water, sediment and soil samples of the
stability, and short- and long-term fate of the various likely formulations that might
reach these compartments of the environment. As well, techniques are needed to
distinguish natural from manufactured nanoparticles. These measurements should
focus on particle concentration, size and surface characteristics (area and charge).
2. Toxicity testing needs to be undertaken on nanoparticle formulations assessed in (1)

above. The tests should involve at least five species from four trophic levels as
required to derive PNECs using species sensitivity distributions. It is critical that
appropriate verification of particle and solute dose be undertaken in all ecotoxicity
testing, necessitating significant effort in (1) above.
3. As a precursor to toxicity testing, it will be necessary to develop standard (and valid)

methodologies for the hazard ranking of nanomaterial toxicity. These will need to
iv Fate of Manufactured Nanomaterials in the Australian Environment

ensure the stability of the nanoparticle suspensions over the duration of the
standardised toxicity tests.
4. Comparisons of toxicity testing in natural vs. synthetic soil and water samples
demonstrating the effects of natural colloids.
Understanding the fate of nanoparticles in the Australian environment will assist risk assessments by
guiding the toxicity testing of nanomaterial formulations under real environmental conditions, yielding
realistic PNECs. This should be coupled with the development of appropriate measurement
techniques that can quantify both concentrations and particle sizes with appropriate quality
assurance and quality control. As well as size and composition, it is evident that surface
properties of nanoparticles will be fundamental in determining fate and toxicity in the
environment and these properties will need to be considered in any hazard ranking.
A check list has been provided to incorporate fate considerations in assessing both
environmental exposure and effects of manufactured nanomaterials.
Fate of Manufactured Nanomaterials in the Australian Environment v

Contents
EXECUTIVE SUMMARY..................................................................iii
1. INTRODUCTION.....................................................................1
2. CLASSES OF NANOMATERIALS................................................2
3. NANOPARTICLE USAGE IN AUSTRALIA.....................................7
4. CHARACTERISTICS OF ENVIRONMENTAL NANOPARTICLES........9

4.1 Manufactured Nanomaterials...................................................................................9
4.2 Natural Nanoparticles.............................................................................................. 9
5. ENVIRONMENTAL SOURCES OF MANUFACTURED

NANOPARTICLES..................................................................12
6. FATE OF NANOMATERIALS IN AQUATIC SYSTEMS....................14
6.1 Key Pathways........................................................................................................ 14
6.2 Behaviour of Manufactured Nanoparticles.............................................................15
6.2.1 Aggregation..........................................................................................................15
6.2.2 Nanoparticle Solubility.........................................................................................17
6.2.3 Role of Nanomaterial Formulations and Impurities.............................................19
6.2.4 Fate in Natural Water Systems............................................................................20
6.2.5 Nanoparticles as Vectors for Contaminant Transport..........................................21
6.3 Fate of Manufactured Nanomaterials in Terrestrial Systems.................................22
6.3.1 Key Pathways.......................................................................................................22
6.3.2 Behaviour of Natural Colloids in Soils.................................................................23
6.3.3 Behaviour of Manufactured Nanoparticles in Soils..............................................24
7. ECOLOGICAL RISK ASSESSMENT OF MANUFACTURED

NANOPARTICLES..................................................................25
7.1 Polymeric Nanoparticles as a Separate Class.......................................................26
8. EXPOSURE ASSESSMENT......................................................26
8.1 What to Measure................................................................................................... 26
8.2 Methods for Measurement of Nanoparticles..........................................................27
8.2.1 Relevance of OECD Test Guidelines...................................................................28
8.3 Modelling Exposure............................................................................................... 29
9. ECOTOXICOLOGY OF NANOPARTICLES...................................33
9.1 Ecotoxicity and Nanoparticle Dose Metrics............................................................33
9.2 Toxicity to Aquatic Biota......................................................................................... 35
9.2.1 Mechanisms of Biological Uptake and Toxicity....................................................35
9.2.2 Ecotoxicity to Individual Species.........................................................................36
9.2.3 Developing Appropriate Guidelines for Nanomaterials in Waters.......................43
9.2.4 Bioaccumulation...................................................................................................44
9.2.5 Ecological Impacts...............................................................................................45
vi Fate of Manufactured Nanomaterials in the Australian Environment

9.3 Sediment Toxicity................................................................................................... 46
9.4 Toxicity to Terrestrial Biota.....................................................................................46
9.4.1 Ecotoxicity to Individual Species.........................................................................46
9.4.2 Development of Guidelines for Nanomaterials in Soils.......................................49
10. INTERNATIONAL PROGRESS ON NANOMATERIAL RISK

ASSESSMENT.......................................................................49
10.1 International Approaches.......................................................................................49
10.1.1 USA...................................................................................................................... 49
10.1.2 United Kingdom....................................................................................................51
10.1.3 Other International Activities................................................................................52
10.2 Australian Activities................................................................................................53
11. DEVELOPMENT OF TECHNICAL GUIDELINES FOR NANOMATERIAL

ASSESSMENT.......................................................................55
11.1 Exposure Assessment Incorporating Nanomaterial Fate.......................................55
11.2 Effects Assessment Incorporating Nanomaterial Fate...........................................57
11.3 Possible Approaches to Environmental Hazard Ranking of Nanomaterials..........58
12. RESEARCH NEEDS................................................................59

13. ACKNOWLEDGEMENTS.........................................................59
14. REFERENCES.......................................................................60
15. GLOSSARY............................................................................................ 73
Fate of Manufactured Nanomaterials in the Environment vii

List of Figures
Figure 1. Structures of (a) fullerene and (b) single-walled and (c) multi-walled carbon nanotubes
Figure 2. Schematic representation of mechanisms whereby surfactants help disperse SWCNTs. Top –
SWCNT encapsulated in a cylindrical surfactant micelle, middle – hemi-micellular adsorption of
surfactants on SWCNTs, and bottom – random adsorption of surfactants on SWCNT (from Ke and Qiao,
2007)
Figure 3. Typical structure of a dendrimer (first-generation polyphenylene dendrimer reported by Müllen

and coworkers in Chem.-Eur. J., 2002, 3858-3864).
Figure 4. Major types of aggregates formed in the three-colloidal component system: fulvic compounds
(or aggregated refractory organic material), small points; inorganic colloids, circles; rigid biopolymers,
lines. Both fulvics and polysaccharides can also form gels, which are represented here as gray areas into
which inorganic colloids can be embedded. (From Buffle et al., 1998)
Figure 5. Potential sources of manufactured nanoparticles to the environment
Figure 6. Pathways for manufactured metal oxide nanoparticles in natural waters
Figure 7. Electron micrographs illustrating aggregation of zinc oxide nanoparticles from dispersion of a
ZnO nanopowder (nominally 30 nm) in a freshwater algal medium, pH 7.5
Figure 8. Illustration of the solubility of amorphous silica as a function of radius of curvature (adapted
from Bjorn et al., 2006)
Figure 9. Key processes in soil relating to transformation and potential risk from manufactured
nanoparticulate particles
Figure 10. Framework for deriving mass flow data for silver flows from biocidal plastics and textiles
(from Blaser et al., 2008).
List of Tables
Table 1. Classes of manufactured nanomaterials
Table 2. Usage of nanomaterials in the commercial sector in Australia
Table 3. Aggregation data for manufactured nanomaterials in water (adapted from Boxall et al., 2007)
Table 4. Predicted environmental concentrations of manufactured nanoparticles in UK soil and waters

(from Boxall et al., 2007)
Table 5. Comparison of UK exposure data for manufactured nanoparticles with toxicity data (from Boxall
et al., 2007)
Table 6. Predicted environmental concentrations (PEC) of nano-Ag, nano-TiO 2 and CNTs in air, water
and soil. (RE: realistic scenario; HE: high emission scenario) (from Mueller and Nowack, 2008)
Table 7. Hazard quotients (PEC/PNEC) for nano-Ag, nano-TiO 2 and CNT in water (RE: realistic scenario;
HE: high emission scenario) (from Mueller and Nowack, 2008)
Table 8. Approach to toxicity testing of nanomaterials in waters
viii Fate of Manufactured Nanomaterials in the Australian Environment

Table 9. Summary of toxicity testing results for manufactured nanomaterials (expanded from Apte et al.,
2008)
Table 10. Data for estimation of guideline concentrations for n-C60 in freshwater
Table 11. Published evidence of nanoparticle uptake by aquatic organisms (from Apte et al., 2008)
Table 12. Toxic effects of nanomaterials on soil organisms (from Klaine et al., 2008)
Fate of Manufactured Nanomaterials in the Australian Environment ix

1. INTRODUCTION
The last decade has seen an amazing growth in nanoscale science and technology, to the
extent that nanomaterials are now a component of a wide range of manufactured
products, from sunscreens to sensors. Given that the production volumes of some of
these materials is already exceeding thousands of tonnes, there is growing public and
regulatory concern for the potential adverse effects that release of these materials to the
environment may have on both human and ecosystem health. Nanoparticles are already
present in our environment in large quantities, derived both from natural sources
(volcanic dusts or natural bushfire products in air, colloids in aquatic systems and soils),
and as a consequence of anthropogenic activities (e.g. smoking, motor vehicle exhausts,
industrial stack emissions). Nanoparticles in the size range 3–7 nm have been shown to
account for more than 36–44% of the total number of particles in some urban air
samples, with the total size of particles ranging from <10 to 10,000 nm(Shi et al., 2001).
The adverse effects on human health of such nanoparticles in the atmospheric
environment (usually referred to as fine and ultrafine particles) have been well studied
and there are clear concerns for the finer particles that can reach the deeper recesses of
the lungs. For terrestrial and aquatic environments, there has been extensive research on
natural colloids (Buffle and Leppard, 1995a), however, there have been few studies of
anthropogenic particles.
Manufactured nanomaterials can be defined as those that are deliberately produced rather
than materials that are by-products of other activities not targeted at nanomaterial
production. Nanomaterials are commonly based on nanoparticles, for which the
accepted definition is particles that have at least one dimension less than 100 nm, but the
term is also used to refer to materials such as surfaces with nanometre-sized features that
are not particulate in nature, or substances with nanometre size voids. Small size gives
materials properties that differ from those of bulk or macroscopic materials. In
particular, optical, electrical and magnetic properties can differ in ways that are subject to
the laws of quantum rather than classical physics. Nanoparticles have a large surface to
volume (and mass) ratio, and potentially greater reactivity and mobility. Surface areas
can be as high as 1000 m2/g, far higher than conventional catalysts for example. They
have the tendency to agglomerate into larger microparticles, losing their distinctive nano
properties, although manufacturers are devising coatings that can stabilise nanoparticles.
Smaller size carries with it the potential to be more bioavailable, able to penetrate
biological membranes or to enter cells by endocytosis (engulfing by the cell wall).
This report reviews the current state of knowledge with respect to nanoparticle fate and
effects in the environment, with a particular focus on aquatic and terrestrial systems, to
provide a foundation for the risk assessment of manufactured nanoparticles in Australia.
2. CLASSES OF NANOMATERIALS
Manufactured nanomaterials currently fall into one of at least seven different classes, as
shown in Table 1. The first class comprising metal oxides are common in their bulk, non-
Fate of Manufactured Nanomaterials in the Australian Environment 1

nanoparticulate forms, and they are now being produced in nanosized forms that
capitalise on their enhanced properties. A case in point is zinc oxide that has been used
for many years as an opaque sunscreen because of its UV-absorbing properties,
scattering light in the range 200–700 nm.
Table 1. Classes of manufactured nanomaterials
Class Component Use

Metal oxides Zinc oxide Cosmetic sunscreens and UV coatings; paints, plastics
and packaging
Titanium dioxide Cosmetics
Cerium dioxide Automobile catalyst
Mixed oxides Cosmetics
Carbon Fullerenes Plastics, catalysts, battery and fuel cell electrodes,
products super-capacitors, water purification systems, cosmetics,
orthopedic implants, conductive coatings, adhesives and
Single-walled and
composites, sensors, and components in electronics,
multi-walled
aircraft, aerospace and automotive industries
carbon nanotubes
Amorphous Inks, photocopier toner, automobile tyres
carbon
Metals Silver Bactericide in wound dressings, socks and other
textiles, air filters, toothpaste, baby products, vacuum
cleaners, and washing machines
Iron Remediation of groundwaters, sediments, soils
Gold Electronics in flexible conducting inks or films, and as
catalysts
Bimetallic Remediation of organics in waters; usually supported
nanoparticles Fe- nanoparticles
Pd, Fe-Ni, Fe-Ag
Quantum dots CdTe, CdSe/ZnS, Medical applications, photovoltaics, security inks, and
and CdSe, PbSe and photonics and telecommunications
semiconductors InP
Nanoclays Hectorites, Cosmetics, toothpaste, antacids, paint additives,
layered double catalyst supports, flame retardants, drug delivery agents
hydroxides
Dendrimers Coloured glasses, chemical sensors, and modified
electrodes; in medicine as DNA transfecting agents,
therapeutic agents for prion diseases, formation of
hydrogels, drug delivery, DNA chips, and ex vivo
amplification of human blood cells
Emulsions Acrylic latex and Paints and surface coatings; sunscreens and similar
2 Fate of Manufactured Nanomaterials in the Australian Environment

other formulations cream formulations; in medicine for drug delivery;
pesticide formulations
In a nanoparticulate form, ZnO is transparent to the eye, but retains much of its ability to
absorb UV radiation, albeit over a narrower spectrum. In Australia in 2005, of the 1200
sunscreens authorised by the Therapeutic Goods Administration (TGA), 228 contained
zinc oxide, 363 contained titanium dioxide and 73 contained both (TGA, 2006).
Nano-zinc oxide coatings on clear glass beer bottles prevent UV-degradation of the
contents, while making them appealingly visible to the consumer. Other metal oxides in
common use include titanium and cerium dioxides, while mixed-metal compounds such
as indium-tin oxide (ITO) are currently used in polishing agents for semiconductor
wafers, sunscreen formulas and scratch-resistant coatings for glass (Arabe, 2003).
Carbon-based nanoparticles comprise the second class (Figure 1). This includes
fullerenes, carbon nanotubes (CNTs) and amorphous carbon nanoparticles. The first
fullerene was discovered in 1985, a sixty carbon atom hollow sphere known as the
buckyball was produced by evaporating graphite (Kroto and Walton, 2007). It was
recently revealed that naturally-produced fullerenes have been around for over a billion
years, found in parts per million concentrations in ancient rock formations and believed
to be carried to earth by comets or asteroids (Becker et al., 1996).
a. b.
c.
Figure 1. Structures of (a) fullerene and (b) single-walled and (c) multi-walled carbon nanotubes
Carbon nanotubes, first produced in 1991, are cylindrical fullerene derivatives that can be
synthesised under controlled conditions to a particular diameter and size, either from
graphite using an arc discharge or laser ablation, or from a carbon-containing gas using
chemical vapour deposition. The multiwalled-products (MWCNTs) are concentric
cylinders up to 10 nm in length and 5–40 nm in diameter. It was later shown that it was
possible to produce single-walled CNTs in the presence of a cobalt-nickel catalyst.
Single-walled CNTs (SWCNTs) have a strength-to-weight ratio that is 460 times that of
steel (Lekas, 2005).

In aqueous systems, carbon nanoparticles aggregate, due to their inherent
hydrophobicity. This limits their use in aqueous and biomedical applications. Much
research has been done to surface modify these nanoparticles to stabilize aqueous
suspensions. Covalent modification, such as the attachment of polyethylene glycol to
SWCNTs (Holzinger et al., 2004), and non-covalent modifications such as the self-
assembly of SWCNTs and the phospholipids, lysophosphatidylcholine (Qiao and Ke,
2006), result in very stable carbon nanoparticle suspensions. These modifications have
implications for their use in certain applications as well as repercussions for their fate and
behaviour in the environment.
Figure 2. Schematic representation of mechanisms whereby surfactants help disperse SWCNTs. Top –
SWCNT encapsulated in a cylindrical surfactant micelle, middle – hemi-micellular adsorption of
surfactants on SWCNTs, and bottom – random adsorption of surfactants on SWCNTs (from Ke and
Qiao, 2007)
Annual worldwide production of SWCNT is estimated to exceed 1000 tonnes by 2011

(Lekas, 2005). Fullerenes and carbon tubes are produced in large quantities in factories
with capacities as high as 1,500 tonne/y (Frontier Carbon Corporation, www.f-
carbon.com; Fullerene International Corporation, www.fullereneinternational.com).
Carbon nanotubes and their derivatives are both used and proposed to be used in
plastics, catalysts, battery and fuel cell electrodes, super-capacitors, water purification
systems, orthopedic implants, cosmetics, conductive coatings, adhesives and composites,
sensors, and components in electronics, aircraft, aerospace and automotive industries.
Increased production results in an increased potential for release to the environment,
either deliberately in discharges or accidentally in spillages, and a greater possibility of
adverse environmental effects. Increased manufacturing volumes also increase the
absolute quantities discharged to the environment as a result of use in products, and
significantly, the quantities that must be disposed of.
The third class comprises nanoparticulate zerovalent metals such as silver, gold and iron.
Nanoparticulate zerovalent iron has been used for some time for the remediation of
waters, particularly groundwaters, as well as sediments and soils (Tratnyek and Johnson,
2006). It has been used to remove nitrates via reduction, and has most recently found
use in detoxifying organochlorine pesticides and polychlorinated biphenyls (Zhang et al.
2003). Mobile iron nanoparticles are effective in treatment of dissolved non-aqueous
phase liquids (DNAPL) (Tratnyek and Johnson, 2006).

Bimetallic nanoparticles such as Fe-Pd, Fe-Ni, Fe-Ag, Pd-Ru, etc., have found extensive
use as heterogeneous catalysts (Meyer et al., 2004; Raja et al., 1999).
There is effectively a (voluntary) moratorium on zerovalent iron being used in the UK,
due to unknown potential effects of release of free nanoparticles into the environment
(Royal Society/Royal Academy of Engineering, 2004).
Nanoparticulate silver is one of the most widely used nanomaterials in consumer

products, as indicated in the inventory developed by the Woodrow Wilson International
Centre for Scholars Project on Emerging Nanotechnologies (PEN, 2007a). Applications
are largely based on its bactericidal activity, and include wound dressings, socks and
other textiles, air filters, toothpaste, baby products, vacuum cleaners, and washing
machines. In some cases, the active ingredient is metallic nanoparticulate silver, in
others, ionic silver (Ag+) is electrochemically generated. Ionic silver is not really a
nanoparticle, but is highly particle reactive, so in natural waters is readily adsorbed by
both macroparticles and by colloidal particles such as iron oxyhydroxides or natural
organic matter, and ranges in size from <1 kDa to >0.45 µm (Kramer et al., 2002).
Silver is one element that has useful properties both as a solid and in the dissolved form.
Its antimicrobial activity is most often attributed to the dissolved cation, while it has
entirely different properties as a non-ionic nanoparticle. In both cases, however, the
instability of the monovalent cation and the non-ionic nanoparticle result in extremely
short half-lives of the desired form. This has resulted in research to stabilise silver
nanoparticles to make them useful in biological and other aqueous applications (Doty et
al., 2005). This has created ambiguity in how investigators describe test systems and
manufacturers describe products. For example, it is common for manufacturers to
describe colloidal silver as ‘nanosilver’, rather than metallic silver powder that is
commercially available as nanoparticles.
Colloidal elemental gold has been used for many years, especially in medical applications
as a vector in tumour therapy. Its size varies from 20-160 nm and the spectral properties
change with the classical colour variation from ruby red through purple to pale blue as
size increases (Turkevich et al., 1954). Newer applications of nanoparticulate gold
include its use in electronics in flexible conducting inks or films, and as catalysts (Haruta
et al., 1989).
Fluorescent semiconductor nanocrystals, also known as quantum dots (QDs) form a

fourth class of nanomaterials. Typical materials include CdTe, CdSe/ZnS, CdSe, PbSe
and InP with size ranges from 10 to 50 nm. They usually consist of a semiconductor
core surrounded by a shell, e.g. silica (Sass, 2007). Newer formulations are coming onto
the market that do not have Cd, Pb or Se in the structure and are composed of just
gallium and zinc. Electrons are excited to higher energy levels in the core and the shell,
then fall into the empty spaces left behind. The dot then forms an "exciton" and emits a
particle of light. Changing the size of a QD-based LED makes it emit a different
wavelength of light – producing red, orange, yellow, or green light. The devices are
useful in that they only need about 3 to 4 volts to operate and can run for over 300 hours
without losing any brightness. Their surface is usually functionalised by coatings to
ensure solubility in water.

Synthetic clays represent a large class of nanomaterials with over 9000 tonnes of
nanoclays being produced in 2007 (Electronics.ca, 2007). Both manufactured and
natural clays are starting materials in nanocomposites for use in polymer
nanocomposites, in packaging, paints and cosmetics. They typically range in size from
10 nm to 100 nm. Both negatively charged and positively charged platelets can be
obtained. The former include montmorillinite and hectorite clays, while synthetic layered
double hydroxides (LDHs) of magnesium and aluminium have exchangeable interlayer
anions (Choy et al., 2000; Xu et al., 2006a.b). The anion exchange properties of these
materials allow binding to negatively charged biomolecules between the hydroxide layers,
with hybridisation effectively neutralising the charge and facilitating penetration into
cells, hence their potential as drug delivery agents.
Dendrimers are monodisperse multifunctional polymers that have repeatedly branched

components that form a fifth class of nanomaterials. They are typically spheroid or
globular nanostructures designed to carry molecules encapsulated in their interior void
spaces or attached to their surface (Figure 3). They range in size from around 5 nm for
the simple molecule shown in Figure 3 to five times that and more in larger polymers.
Their synthesis uses repeating procedures to build up their branches from molecular to
the nanoscale (e.g. see Frechet and Tomalia, 2002; Dendritic Technologies Inc,
www.dnanotech.com). These macromolecules can be used for many useful applications
in different fields from biology, material sciences, surface modification, to
enantioselective catalysis.
Figure 3. Typical structure of a dendrimer (first-generation

polyphenylene dendrimer reported by Müllen and
coworkers in Chem.-Eur. J., 2002, 3858-3864)
Among the most outstanding applications of

dendrimers are the formation of nanotubes,
micro and macrocapsules, nanolatex, coloured
glasses, chemical sensors, and modified electrodes. Some of the uses of dendrimers in
biology include DNA transfecting agents, therapeutic agents for prion diseases,
formation of hydrogels, drug delivery, DNA chips, and ex vivo amplification of human
blood cells.

Nanoparticulate emulsions or nanoemulsions are a potential additional class of
nanoparticles, more recently referred to as soft nanoparticles. Emulsions are by
definition dispersions of one immiscible liquid in another and while we are accustomed to
thinking of particles as solid phases, the term really refers to ‘small amounts’ so could
include emulsions. A nanoemulsion has been defined as a type of emulsion in which the
sizes of the particles in the dispersed phase are less than 1000 nm. This includes particles
much larger than the accepted nanoparticle size of < 100 nm, and typically 20–200 nm,
and on that basis, nanoemulsions are excluded from this review. Nanoemulsions include
latex and other formulations used in paints and surface coatings. They are also used in
sunscreens and similar cream formulations, and in medicine for drug delivery. It is worth
noting that the term microemulsion is also used to describe oil/water emulsions in the
nanoemulsion size range and below. The distinction is a fine one, with microemulsions
being thermodynamically stable while nanoemulsions are kinetically stable (Lawrence and
Waresnoicharoen, 2006).
From the list of manufactured nanoparticles and their reported uses, apart from the use
of iron and related bimetallic nanoparticles for water and soil remediation, it appears that
there are few confirmed uses of nanoparticles as agricultural or veterinary chemicals. So
saying, there is potential for use in veterinary medicine for drug delivery uses and other
applications common to human medical uses. One reference highlighted the use of
nanoemulsions for crop applications (www.nanowerk.com/spotlight/spotid=5305.php).
A distinction has been made by some authors between nanosized particles and nanosized
molecules. The latter include fullerenes and dendrimers. If a molecule contains
segments or has an internal insoluble core, it is considered to be a particle. Where size is
determined by milling, the product will be a nanoparticle. The functional significance of
these separate definitions is not immediately obvious. There will be differences in fate
and toxicity just as there are between different types of non-molecular nanoparticles.
3. NANOPARTICLE USAGE IN AUSTRALIA

A voluntary call for information on nanoparticle usage in Australia was recently issued by
the National Industrial Notification and Assessment Scheme (NICNAS). In the absence
of publicly available information, the call was targeted at manufacturers and importers of
nanomaterials or products containing nanomaterials for industrial (including domestic
and cosmetic) use during 2005 and 2006. It was designed as a first step in understanding
the potential for exposure. In addition to issuing an open call in the Chemical Gazette of
February 2006, companies known to be involved with nanomaterials were directly
contacted by NICNAS. The results of the survey were published on the NICNAS
website (www.nicnas.com.au) in January 2007 and are summarised in Table 2.
Table 2. Usage of nanomaterials in the commercial sector in Australia
Chemical Name Application Total usage (tonne/y)

Acrylic latex Surface coatings 10,000-50,000
Aluminium oxide Printing 0.05-0.1

Aluminosilicates Water treatment 10-50
Carbon black pigment Surface coatings 10-50
Cerium oxide Catalysts 1-5
Iron oxide Surface coatings 1-5
Cosmetics <0.01
Pearl powder Cosmetics 0.01-0.05
Phthalocyanine Surface coatings 10-50
Polyurethane resin Surface coatings <0.01
Silica dimethylsilylate Cosmetics <0.01
Silicon dioxide Surface coatings 10-50
Water treatment 0.05-0.1
Sodium silicates Water treatment 0.1-0.5
Surface-treated silicon dioxide Printing 1-5
Surface-treated aluminium oxide Printing 0.1-0.5
Surface-treated titanium oxide Printing 0.5-1
Titanium dioxide Water treatment 5-10
Domestic products 1-5
Cosmetics 1-5
Zinc oxide Surface coatings 5-10
Cosmetics 1-5
The interesting finding from this survey, in addition to the expected high usage of acrylic
latex in nanoemulsions, was the fact that CNTs, fullerenes and silver were not imported
or manufactured (as chemicals or in products) at that time, given the high production
volumes projected for 2008-2009 internationally. A second survey is currently being
undertaken.
Despite the fact that many of the literature reports on CNTs may be on proposed uses,
the Woodrow Wilson Project on Emerging Nanotechnologies’ on-line inventory of
nanotechnology-based consumer products (PEN, 2007a) lists 45 carbon-based products
as of February 22, 2008. This is the second most common material after silver (143
references), and followed by zinc oxide (28), titanium dioxide (28), silica (27) and gold
(15). Similarly there is no reference to silver nanoparticle usage in Australia, when we
know it is a component of many consumer products, or to nanoclays. Further local
research is needed to confirm actual usage of CNTs, silver and nanoclays.

4. CHARACTERISTICS OF ENVIRONMENTAL
NANOPARTICLES
Nanoparticles are characterised by a number of key physical parameters, including size,
shape, surface area, molecular weight (in the case of polymeric particles), and by their
chemical composition. Measurement of these properties is not a trivial exercise as will
be discussed later. The challenge is to determine these properties when the
nanomaterials reach the particular environmental compartments, (atmospheric, aquatic,
terrestrial). Where the measurement technique requires the nanoparticles to be
separated, e.g. electron microscopy, the possibility exists that this will perturb the natural
properties from their form in the environment.
4.1 Manufactured Nanomaterials

The properties of manufactured nanomaterials, as produced, will vary greatly once
released to the environment as interactions occur with other chemicals, or as
transformations such as aggregation and dissolution take place. Such processes can
dramatically affect subsequent biological interactions. Because of this, the concept of
intrinsic toxicity of manufactured nanoparticles is not a useful one, and needs to be
linked with measurements in field or simulated field media. There is a parallel here in the
study of metal speciation in aquatic and terrestrial systems, where the guideline
framework uses a conservative, total dissolved metals as a first cut before a detailed
measurement of a bioavailable fraction. Here the conservative assumption might be to
first base assessments of biological impact on the smallest and potentially more
bioavailable particles in the absence of later more appropriate measurements of actual
size.
The situation becomes more complex because the formulations of manufactured

nanomaterials can often include other additives that can alter the physical behaviour of
nanoparticles in some media, as will be discussed later. Such a concept is not unfamiliar,
for example, in the regulation of pesticide formulations.
4.2 Natural Nanoparticles

It is important to recognise that in both soil, water, and indeed air, compartments there
are a range of natural nanoparticles. In air, there are dusts as well as aerosols comprising
fine particles associated with volatiles emissions from trees and other plants, or with
‘natural’ events such as bushfires, typically less than 1 µm, but formed by agglomeration
of much smaller particles. Natural clays can be a significant nanoparticulate component
of some soils, as can iron and manganese oxides and other high molecular weight mineral
phases as well as dissolved organic matter in soil pore waters. In natural waters, as well
as in soil pore waters, colloidal particles comprise clays, iron and manganese oxides and
organic matter. We can learn a lot about the expected behaviour of manufactured
nanoparticles from what is already known about natural nanoparticles
Colloids and macromolecules in natural waters comprise fulvic and humic acids, fibrillar
colloids (exopolymers) that are exudates from algae and other microorganisms (these are

largely polysaccharides and some proteins), and hydrous iron, manganese and aluminium
oxides (Wilkinson and Lead, 2007). Natural colloids fall in the size range from 1–1000
nm, depending on their degree of aggregation (Buffle and Leppard, 1995a; Lead and
Wilkinson, 2007). Fulvics, humics, and proteins are typically smaller than tens of nm
while polysaccharides and metal oxides are larger, although iron oxide colloids cover the
full size range. Typically these are not present as discrete particles or compounds, but
are heterogeneous mixtures of organic and inorganic species (Lead and Wilkinson,
2007). Microbial activity in natural waters is a continuous source of macromolecular
material (e.g. polysaccharides) (Buffle and Leppard, 1995b)
Over the wide range of colloid particle sizes, the largest particles have the greatest
percentage mass, but the smaller particles have the greatest number and percentage of
total surface area. Buffle and Leppard (1995a) showed that irrespective of the aquatic
system of interest, the size distribution based on particle number (N) follows Pareto's
Law (i.e. dN/ddp = A dp-b , where A and b are constants with a b value close to 3, and dp, is
particle diameter). The inverse linear relationship between log (particle number/particle
diameter) and log (diameter) means that there are orders of magnitude more smaller
particles than large ones in a water system.
The aggregation of colloids is dependent upon particle size, density, surface charge and
chemical properties (Buffle and Leppard, 1995a; Handy et al., 2008). Aggregation
occurs as a result of particle-particle collisions, involving natural Brownian motion,
different shear velocities in flowing systems and different settling behaviour of different
sized particles.
It has been shown both practically and theoretically, that for a mixture of colloids in
which each size fraction has the same volume, the smallest colloids (<100 nm) disappear
first by aggregation, and the largest by sedimentation, leaving a distribution of sizes over
the range 100 nm-1 µm. (Buffle and Leppard, 1995b).
The interactions between colloids will be governed by their charge and the nature of their
bonding (covalent vs electrostatic). The surface charge of clays at the pH of natural
waters is typically negative over a range of natural pH values. So too is the charge on
most natural organic matter due to ionisable functional groups (e.g. hydroxyl and
carboxylic acid). Iron and aluminium oxyhydroxides have a positive charge below the
pH values at which the surface charge is zero (pH 8-9), however, binding with natural
organic matter typically results in aluminium and iron colloids having a net negative
charge in natural waters (Kretzschemar and Schafer, 2005).
It is not easy to measure surface charge, but it is implied by measurements of the zeta
potential (the potential between the colloid particle surface and solution). As a measure
of the stability of colloidal particles, the zeta potential range between +30 mV and -30
mV is characterised by instability with aqueous dispersions being stable on either side of
that range.
Particles with near neutral charges aggregate rapidly. In natural systems, such
interactions of organic macromolecules and colloidal particles lead to the formation of
loose aggregates or flocs whose structure will be dependent on the relative

concentrations of each in the mix and of the density, shape of the particles and the
flexibility of the macromolecules. Their stability depends on their relative charges and
the nature of the bonding. The aggregates may be stabilised at small sizes that will not
sediment. Larger aggregates form more slowly. It is difficult to predict the behaviour of
such mixtures in terms of rates of reaction and stability, however, it appears that in low
ionic strength solutions, appreciable stability is generally achieved in the size range 100
nm-1 µm as discussed above (Buffle and Leppard, 1995b). Neutrally-buoyant sub-
micron particles can migrate with currents over large distances in fresh waters. The rate
of settling will be controlled by both hydrodynamics and particle size. Deeper, well-
mixed waters have reduced settling and larger colloidal aggregates. Once the aggregates
become sufficiently large (>1 µm) they exceed the buoyant mass of colloids and the
newly formed macroparticles will gradually sediment.
Aggregation or particle coagulation can be faster in higher ionic strength water

(seawater) compared to freshwaters, where colloids can be naturally stabilised by organic
macromolecules (Gustafsson and Gschwend, 1997). In estuarine waters, increasing ionic
strength increases screening of the particle charge, resulting in increased aggregation and
coagulation of colloidal particles (Buffle and Leppard, 1995a). For example, the
aggregation and precipitation of colloidal iron at salinities above 15 ‰ (by comparison,
seawater has a salinity of 35 ‰) was greater than 75% complete within 30 minutes, with
particles larger than 1.2 µm being formed (Liang and Morgan, 1990).
A schematic diagram of aggregate formation involving natural colloids is shown in Figure

4 (from Buffle et al., 1998). This does not consider any living components such as
bacteria and viruses which would add a further layer of complexity.
Natural colloids are frequently in high concentrations in soil pore waters and in natural
water systems, as high as mg/L, so interactions of these particles with manufactured
nanoparticles will be an important fate pathway to consider, and one that is overlooked
in laboratory investigations in synthetic media. The basic behaviour of natural colloids
and macromolecules in soils has been known for decades (Cameron, 1915), and is
governed by the same processes as those in natural waters. High ionic strength (salt
content) in soils will promote flocculation of particles, as will soil pore waters dominated
by calcium and low in sodium (Rengasamy and Olsson, 1991). Many Australian soils are
sodic (sodium rich) (Naidu and Rengasamy, 1993), conditions which promote dispersion
of natural soil colloids when low ionic strength (i.e. low salt) solution wets the soil (i.e.
rainfall or good quality irrigation water) leading to adverse soil conditions for agriculture
e.g. crusting, clogging of soil pores reducing water flow, reduced aeration (due to poor
drainage), etc. These conditions are likely to act similarly on manufactured nanoparticles,
although this needs confirmation.

Figure 4. Major types of aggregates formed in the three-colloidal component system: fulvic compounds
(or aggregated refractory organic material), small points; inorganic colloids, circles; rigid biopolymers,
lines. Both fulvics and polysaccharides can also form gels, which are represented here as gray areas into
which inorganic colloids can be embedded. (From Buffle et al., 1998)
5. ENVIRONMENTAL SOURCES OF MANUFACTURED

NANOPARTICLES
Unlike many anthropogenically derived nanoparticles, it is reasonable to assume that
there will controls on the release of manufactured nanoparticles that minimise their
release to the environment. The obvious sources that require management are release to
the atmosphere and release via aqueous discharges. These and other potential input
sources are illustrated schematically in Figure 5.
Atmospheric nanoparticles are both a potential risk to the environment and an

occupational health and safety concern for workers engaged in nanomaterial
manufacture. Sources include motor vehicle exhausts and stack emissions from a range
of sources. Eliminating exposure to workplace respirable nanoparticles will be a priority
and is easily addressed through both the use of filters and appropriate protective clothing
and respiratory protection. Motor vehicle and stack emissions are more problematic.
Dealing with fine particle emissions has been an issue for the power industry for many
years, and it is fair to say, has not been adequately eliminated. Nanoparticle filtration
requires nanofilters, which are available, however, appropriate monitoring will be
necessary to ensure their effectiveness.

Figure 5. Potential sources of manufactured nanoparticles to the environment
Atmospheric deposition
Soil application
Effluent discharge
Surface runoff
Road runoff
Groundwater discharge
Accidental spillage
The potential for nanoparticles to end up in aqueous discharges is currently unknown.

Depending on the manufacturing process, there are likely to be both solid and liquid
wastes that may contain nanoparticles. These days, discharges of aqueous wastes are
licensed, although there is unlikely to yet be advice on nanoparticle concentrations.
Similarly uncontrolled disposal of solid chemical wastes is generally not permitted, but
guidance on nanomaterials is most likely absent, so this remains a potential source.
Water treatment plants may have the capability to treat and remove nanomaterials where
discharges are to the sewage system, but as yet there is no information on the ability of
water treatment plants to deal with nanoparticulate contaminants. In particular, anionic
and uncharged nanomaterials could pass through into sewage effluents and not be
retained in sewage biosolids. Several recent studies have indicated a potential for
nanomaterials to interact with bacteria in sewage treatment plants. Choi et al. (2008)
showed that silver nanoparticles were toxic to nitrifying bacteria and that this could
imply detrimental effects on the microorganisms in wastewater treatment. Titanium
dioxide nanoparticles in the presence of ultraviolet light were shown to be toxic to E.
coli, inhibiting the fouling of water treatment membranes (Kwak et al., 2001). Ghafari et
al. (2008) found that SWCNTs caused the protozoan Tetrahymena thermophilia, present
in sewage treatment plants to release excess exudates, which contribute to floc
formation, so they could be used to improve the efficiency of ciliates in wastewater
treatment although effective measures to control and monitor SWCNT release would be
necessary. By contrast, Nyberg et al. (2008) recently indicated little toxicity of fullerenes
in sewage treatment sludge to methanogenic bacteria.
There are instances where nanomaterials are added to aquatic or terrestrial systems for
remediation purposes, e.g. zerovalent iron addition to soils or sediments, and their fate
and impacts will be separately discussed.

The remaining sources are accidental release and release as a consequence of product
usage. The accidental release amounts to spillage of containers, drums, etc., where solid
nanomaterials can end up on land or in water systems. The issue of product usage
requires consideration for each nanomaterial category and for formulations within each
category and this will be discussed in more detail below.
The US Department of Energy has recently published an approach to nanomaterial

environmental safety and health that discusses in some detail the requirements for
nanomaterial transportation and for the management of nanomaterial-bearing waste
streams and nanomaterial spills that minimise the likelihood of releases of nanomaterials
to the environment (USDOE, 2007).
In addressing the risks posed by manufactured nanomaterials, a relevant question is

which nanoparticles have the highest potential for release. Intuitively, these are likely to
be those being produced in the greatest amounts, however, if these productions are from
a large number of widely-dispersed small scale activities, perhaps the risk is less than
from larger facilities with high volume throughputs. Silver fits into the small but
dispersed source category. In particular, the in situ generation of silver nanoparticles in
washing machines will be a highly dispersed source, that may end up in wastewater
treatment plants, and from there may reach the environment although may well be
recovered in the flocculation stages of such plants.
The particular formulation of the nanomaterials is also important for assessing potential
for diffuse releases into the environment. Where nanoparticulates are incorporated into
stable solid-phases, e.g. ZnO nanoparticles in coatings on glass for UV protection, then
the potential for release of the dispersed nanoparticles is low. Where the nanoparticle is
used in a dispersed form (e.g. zerovalent iron for groundwater remediation), then the
potential for movement and effects is much higher.
6. FATE OF NANOMATERIALS IN AQUATIC SYSTEMS
6.1 Key Pathways

The major physicochemical pathways that govern the fate of nanomaterials in the aquatic
environment are summarised in Figure 6. These comprise aggregation and subsequent
sedimentation, dissolution, adsorption to particulates and other solid surfaces, binding to
natural dissolved organic matter, and stabilisation via surfactants. Other processes
include biological degradation (aerobic and anaerobic), and abiotic degradation
(including hydrolysis and photolysis). Oxidation and reduction may also be of concern in
some environments for specific materials. Concentration in the surface microlayer of
water bodies is a possibility, but unlikely to be a major pathway. The ultimate fate is
likely to involve accumulation and burial in bottom sediments.
In general, the fate of manufactured nanomaterials in aquatic systems has not been that
well studied, however, what information is available, coupled with the extensive
literature on natural colloids in aquatic systems can provide a useful basis for prediction

of nanomaterial fate. The interactions of nanomaterials with natural colloids will play a
critical role in their fate.
Surfactant-stabilised
nanoparticles
Binding to suspended
particles/biota
Binding to NOM Aggregation Binding to NOM
and other colloids and other colloids
Dissolution
Biological degradation,
photolysis, hydrolysis
Mn+
Mn+
Mn+
Sedimentation
Figure 6. Pathways for manufactured metal oxide nanoparticles in natural waters
6.2 Behaviour of Manufactured Nanoparticles

Of the pathways identified in Figure 6, the two most important contributors to the
environmental impacts of manufactured nanomaterials in waters are aggregation and
dissolution.
6.2.1 Aggregation
As shown for natural nanoparticulate colloids (in Section 4.2), the behaviour of
nanoparticles in aqueous systems mimics colloid behaviour. There is a natural propensity
for nanoparticles to grow in size in aqueous solution. Particles that according to
manufacturers’ specifications are nanosized, when suspended in water at neutral pH, are
frequently aggregated (e.g. Figure 7), and the size of these aggregates is frequently
greater than 100 nm, the upper boundary of the nanoparticle size range.
In the case of nanoparticles with a surface charge, screening of the surface charge by
electrolyte ions, e.g. in seawater, overcomes the electrostatic forces and allows
aggregation, as for natural colloids. Steric stabilisation of nanoparticles against
aggregation can occur through surface modification by surfactants or bulky polymeric
additives.

Interactions of nanomaterials with natural colloids (organic macromolecules, inorganic
colloids or heterogeneous aggregates) will also occur in the same manner as discussed in
Section 4.2 (Saleh et al. 2008).
The rates at which manufactured nanoparticles aggregate is particularly important, since

the slower the aggregation the greater the potential for interaction with biota.
Unfortunately this has been poorly studied, although there are data available for natural
colloidal nanomaterials.
The terms aggregate and agglomerate have distinct meanings in particle science, but are
frequently confused. As discussed by Nichols et al. (2002), agglomerates are generally
considered to be an assemblage of particles that are rigidly bound by fusion sintering or
growth, while aggregates are loosely bound particles that are readily dispersed. The
word clump is also used but they proposed replacement of ‘clumps’ with ‘agglomerates’
that may be hard (not readily dispersed) or soft (readily dispersed).
A summary of the available data on particle aggregation is presented in Table 3.
Brant et al. (2005) reported that n-C60 fullerenes (i.e. nanoscale suspended aggregates
known as fullerene water suspensions), showed a strong tendency to aggregate in weak
electrolyte solutions greater than 0.001 M ionic strength. Below these concentrations,
aggregates were stable for over 15 weeks (Lyon et al., 2006). The same effect of ionic
strength on natural colloid aggregation was noted earlier. The n-C60 aggregates
eventually settle out of suspension, sorb to particles or become otherwise immobilised on
surfaces.
Table 3. Aggregation data for manufactured nanomaterials in water (adapted from Boxall et al., 2007)
Nanomaterial Water type Aggregate size Comments References

range, nm
Fullerenes Freshwater 25-500 (mean 75) This is with a THF- Lyon et al., 2006
culture based preparation.
medium Smaller mean size
using sonication
TiO2 Freshwater 177-810 (mean From an initial size Adams et al., 2006
330) of 66 nm
SiO2 Freshwater 135-510 (mean From an initial size Adams et al., 2006
205) of 14 nm
ZnO Freshwater 420-640 (mean From an initial size Adams et al., 2006
480) of 67 nm
Zerovalent Freshwater 1000 Rapidly aggregate Mondal et al.,
iron 2004; Schrick et
al., 2004

1 µm
100 nm
Figure 7. Electron micrographs illustrating aggregation of zinc oxide nanoparticles from dispersion of a
ZnO nanopowder (nominally 30 nm) in a freshwater algal medium, pH 7.5
Zerovalent iron nanoparticles in water grow rapidly to micron sizes or more, and quickly
lose reactivity, rapidly settling out of solution (Phenrat et al., 2004).
In general, the effect of basic water chemistry (pH, redox potential, hardness, salinity) on
nanoparticle stability has been poorly studied. Lead et al. (2007) showed, for example,
that aggregation of gold (and iron oxide) nanoparticles was minimised at low pH. While
such studies assist in understanding aggregation behaviour, they are of little value in
predicting the behaviour in natural water systems where pH variation is limited.
There have been attempts to develop predictive models for aggregation behaviour
(Mackey et al., 2006), but these are as yet untested, and given the complexity of natural
waters, their applicability may be problematic.
A number of papers have documented oxidation/reduction reactions of fullerenes, and

the potential for oxidation (hydroxylation) mediated by fungal enzymes has been
suggested (Wiesner et al., 2006). No such biotransformations have, however, yet been
observed.
6.2.2 Nanoparticle Solubility
With respect to solubility, the Gibbs-Thompson effect predicts that nanoparticles with a
smaller radius of curvature are energetically unfavourable and subject to preferential
dissolution, and have a higher equilibrium solubility than macroparticles (Figure 8)
(Borm et al., 2006). This solubility can exceed saturation conditions in some instances,
leading to growth and precipitation of particles in a phenomenon known as Ostwald
ripening, where with time, the rapid initial dilution and supersaturation solubility is
reduced by the growth of larger particles with lower solubility. The overall process is one
of destabilisation of nanoparticles in solution.
These phenomena raise questions about the overall stability of nanoparticles in aquatic
environments and highlight the need for measurements of both particle size and solubility
to reliably assess the fate of nanomaterials.

Most metal-based nanoparticles are hydrophilic and have a finite but often low solubility.
In many cases, this is not measured, but since the soluble ionic metal fraction is the most
toxic to aquatic biota, it is desirable that the extent of this solubility be determined. As a
case in point, Franklin et al. (2007), investigating the biological impacts of zinc oxide
nanoparticles, found that despite a common belief that zinc oxide was ‘insoluble’,
nanoparticulate ZnO rapidly dissolved to the extent of 6 mg/L of dissolved (dialyzable)
zinc within 6 h and 16 mg/L in 72 h in a buffered pH 7.5 algal medium. This was a
concentration well in excess of the 5 mg Zn/L that would be toxic to most aquatic biota.
By contrast, in similar experiments with nanoparticulate cerium oxide, a very low
solubility (ng/L) was observed, and so the effects of nanoparticle versus macroparticle
toxicity could be readily investigated (Franklin, unpublished results). Greater toxicity to
algae was observed for nanoparticulate CeO2 compared to its macroparticulate
equivalent.
Figure 8. Illustration of the solubility of amorphous silica as a function of radius of curvature (adapted
from Bjorn et al., 2006)
The toxicity of a range of metal nanoparticles to a range of aquatic organisms was

investigated by Griffitt et al. (2008). Toxicity was observed for silver and copper with
48-h LC50s to Daphnia pulex being 40 and 60 µg/L respectively. Here the role of
dissolution was demonstrated to be minor, however, solubility played a major role in the
toxicity of nickel nanoparticles.
Semiconductor quantum dots based on cadmium selenide have been shown to release
ionic cadmium as a result of selenide oxidation (Derfus et al., 2004). Solutions of 250

mg/L yielded as high as 80 mg Cd/L. The concentration of cadmium directly correlated
with cytotoxic effects to primary hepatocytes isolated from rats and grown in vitro.
CdSe/ZnS nanocrystals released a factor of 10 less cadmium, however only polyethylene-
silane coatings were effective in preventing release (Kirchner et al., 2005). In the case of
environmental release, such concentrations of cadmium would readily exceed water
quality guidelines (ANZECC/ARMCANZ, 2000) with severe consequences for
ecosystem health.
Carbon-based nanoparticles are typically lipophilic and are virtually insoluble in natural
waters. The solubility of fullerene has been calculated as 10-18 mol/L (Abraham et al.,
2000). The lipophilicity will vary with substitution on the basic fullerene or nanotube
formulations, and derivatives have been prepared with appreciable water solubility.
Sayes et al. (2004) showed that cytotoxicity to human liver carcinoma cells was inversely
related to the solubility of fullerene derivatives, largely as a consequence of the reduced
ability to generate oxygen free radicals that are the cause of cytotoxic effects via lipid
peroxidation.
It is important to recognise that the term ‘solubility’ has been loosely used by some
authors, especially in relation to carbon-based nanomaterials, often meaning forming
stabilised suspensions as distinct from truly dissolving as was the case with metal oxides
for example.
6.2.3 Role of Nanomaterial Formulations and Impurities
In many instances, the formulations of nanomaterials include additives (e.g. surfactants),

which are added to modify the surface properties, and to minimise aggregation. These
formulations may also result in different solubility characteristics. Carbon nanotubes are
extremely hydrophobic and subject to high van der Vaal’s forces along the length axis,
with a tendency to aggregate. To disperse CNTs in aqueous solution, a range of
chemical additives have been used including surfactants (sodium dodecylsulfate, sodium
dodecylbenzene sulfonate, Triton X-100) and polymers, acting either sterically or
electrostatically (Brant et al., 2005). The effect of these dispersant additives is usually to
sterically stabilise the nanomaterials, by physically hindering their aggregation (Handy et
al., 2008; Saleh et al., 2008). Terashima and Nagao (2007) showed that the surfactant
Triton X-100 and natural humic substances enhance the solubility of C60 nanoparticles by
8-540 times, while also decreasing the rate of aggregation (Chen and Elimelech, 2007).
In many cases the effect of additives on solubility and aggregation in commercial

nanomaterial formulations is unknown. In the case of zinc oxide formulations, for
example, a relevant concern might be whether the equilibrium water solubility of
nanoparticulate zinc oxide in sunscreens is different to that seen for the raw nanoparticles
by Franklin et al. (2007). Such questions have important implications for risk assessment
in aquatic systems.
Nanomaterials often contain impurities, for example, carbon nanotubes have been
reported to contain metal catalyst impurities (Haddon et al., 2004). Plata et al. (2008)
showed that metal and carbonaceous impurities could account for up to 70% of the
weight of the SWCNT formulations, with nickel up to 22%, yttrium 6%, cobalt 2-10%,

iron 0.5% and molybdenum 0.7%, together with traces of copper, lead and chromium.
Amorphous carbon could be as high as 45%, while polycyclic aromatic hydrocarbons
(particularly naphthalene) were up to 60 µg/g in arc-produced nanotubes and even
higher in those prepared by chemical vapour deposition. These impurities can affect the
surface charge, reactivity, transport and ecotoxicology of the SWCNTs.
The presence of impurities was found to be responsible for oxidative stress damage to rat
epithelial cells (Pulskamp et al., 2007). Similarly, the presence of tetrahydrofuran (THF)
residues was shown to be responsible for observed toxicity of n-C60 fullerenes to large
mouth bass (Brant et al., 2005). While these impurities are not expected to significantly
affect nanoparticle fate, they raise interesting questions with respect to the effects on the
environment. It could be argued that THF-containing n-C60 and metal-free SWCNTs are
both unnatural forms, and therefore are not environmentally relevant, but if these are
present in the manufactured products then their behaviour is a valid concern.
6.2.4 Fate in Natural Water Systems
While there are data from laboratory studies on the behaviour of selected nanomaterials
in water, the behaviour is likely to differ in natural waters, where there is a possibility of
interaction with natural colloids including dissolved (and particulate) organic matter
(NOM). The importance of colloids cannot be underestimated. In freshwaters for
example, colloidal organic matter concentrations lie in the range 1-10 mg/L compared to
the concentrations that have been predicted for manufactured nanoparticles of 1-100
µg/L, which is at least several orders of magnitude lower (Boxall et al., 2007).
A recent study by Hyung et al. (2007) showed that the addition of standard Suwannee
River humic acid greatly enhanced the dispersion of multi-walled carbon nanotubes in
Milli-Q water, and that the same effects were also seen in suspensions in Suwannee River
water samples. The dispersion was greater than that observed with sodium
dodecylsulfate. The exact mechanism of the enhanced dispersion is likely to again
involve both steric and electrostatic components, as was seen for natural colloids.
Similar stabilisation of iron oxide nanoparticles by humic acids has also been
demonstrated (Tipping and Higgins, 1982; Baalousha et al., 2008).
By contrast, it has been suggested that natural fibrillar colloids are likely to increase
aggregation because of different binding characteristics, compared to the charge
stabilisation mechanism of humic substances (Buffle et al., 1998).
The findings to date suggest that in natural water systems, nanoparticles may have a
greater stability than in synthetic (NOM-free) waters, particularly in estuarine and marine
waters of higher ionic strength. In waters with a high suspended sediment load,
however, association of nanoparticles is likely to provide an effective removal mechanism
that could enhance transport to and accumulation in bottom sediments.
Given these many uncertainties, site-specific fate studies are recommended that use
actual nanomaterial formulations in a variety of natural waters (fresh and estuarine).
Where nanoparticles are released with wastewaters, it has been suggested that the
presence of household or industrial detergents would result in the disaggregation of

nanoparticles (Fernandes et al., 2006). In a related study of cerium oxide nanoparticles
in a model wastewater treatment system, Limbach et al. (2008) found that a small but
significant fraction (6%) avoided aggregation and was released in the effluent (at 2-5
mg/L concentrations) largely as a result of stabilisation in the presence of protein
breakdown products and surfactants in the wastewater changing the zeta potential.
These examples highlight the impact that surface modifications from wastewater
components can have on nanoparticle fate.
Sinks and issues of non-steady state thermodynamics influence the fate of nanoparticles.
Adsorption of molecules or ions on nanoparticles can catalyse or promote dissolution,
e.g. via chelating agents. This is a dynamic process.
A recent paper by Benn and Westerhoff (2008) revealed some interesting findings on the
fate of nanoparticle silver released into water from commercially available sock fabrics.
Repeated washings released most of the silver, with 70-90% in an ionic form, and the
remainder as large nanoparticles (100-200 nm). In a simulated water treatment process
they showed that all of the silver was removable to the sludge, raising concerns about the
impacts of application of sludge to land.
The behaviour of emulsions in natural waters has been poorly studied. In a report on
acrylic latex, NICNAS (2000) noted that ‘the fate of the aqueous residues released to the
sewer system is less predictable as the notified polymer may remain in the aqueous phase
as an emulsion at low concentrations’. In addition, ‘all solid residues will remain
associated with the soil and sediment due to the high molecular weight and the stability
of the cured paint matrix’.
6.2.5 Nanoparticles as Vectors for Contaminant Transport
While so far we have considered manufactured nanoparticles as potential sources of

toxic effects in the environment, as noted earlier with respect to colloids, nanoparticles
are excellent binding sites for other soluble contaminants and therefore have the potential
to act as vectors for the delivery of these contaminants. Again, this will be a function of
surface properties of the particular nanomaterial formulations.
A recent publication by Hu et al. (2008) showed that aqueous suspensions of fullerene

were able to effectively sorb polycyclic aromatic hydrocarbons (PAHs), a process that
was further enhanced by the addition of humic acids. This predictable behaviour
indicates that nanomaterials can affect the fate of hydrophobic organic contaminants in
natural waters.
In soils, groundwaters, rivers and lakes, natural colloids have been shown to play an
important role in trace metal retention and transport (Kretzschmer and Schafer, 2005).
Similar binding capacities exist for manufactured nanoparticles. Secondary toxicity
effects from these adsorbed contaminants will need to be considered in any toxicity
studies of nanoparticles.

6.3 Fate of Manufactured Nanomaterials in Terrestrial
Systems
There is currently very little information available with which to assess the environmental
risk of manufactured nanoparticles to terrestrial ecosystems. The key physico-chemical
properties of nanoparticles described above are also likely to play a major role in the fate,
transformation, and environmental effects in soils. Soils differ from fresh and marine
waters in that the solid phase provides a large and reactive “sink” for nanoparticles, so
that the applied dose may overestimate the actual dose to soil biota.
One of the key hurdles in examining nanoparticles in terrestrial systems is the detection
of the manufactured nanoparticles in the presence of natural nanoparticles, which are
ubiquitous in soil.
6.3.1 Key Pathways
A number of key processes are likely to affect the fate and bioavailability of nanoparticles
in the soil environment (Figure 9).
Nanoparticles have high surface reactivity and, depending on surface charge and
coatings, their adhesion to reactive soil surfaces may be strong –“partition coefficients”
for nanoparticulate contaminants in soil have yet to be published. Data from transport
studies of soil colloids however indicate that surface coatings on the nanoparticles are
important determinants of mobility and may enhance transport (Kretzschmar et al., 1995;
Seaman and Bertsch, 2000; Saleh et al. 2008), and this has also been found for
nanoparticles used in groundwater remediation (Hydutsky et al,. 2007). As yet, there are
few data on transport of nanoparticles through soils, and hence characterisation of
nanoparticle mobility and associated potential bioavailability remains to be elucidated.
Recent studies examined the transport of eight nanoparticles (fullerol (C60-OHm),
SWCNTs, silica (57 nm), alumoxane, silica (135 nm), n-C60, anatase and ferroxane)
through spherical glass beads and found the attachment efficiencies to fall in the order as
listed (Lecoanet et al., 2004). Another recent sand column study demonstrated the
importance of surface coatings in the transport of zerovalent iron nanoparticles (Saleh et
al., 2008). Similar studies in soils are now required.
1. Dissolution
2. Sorption/aggregation
3. Plant bioaccumulation
4. Invertebrate accumulation and toxicity
5. Microbial toxicity
6. Direct particle uptake/toxicity
7. Particle migration
MNPs
6
2
3
4
1 Dissolved
pool
7 5
Figure 9. Key processes in soil relating to transformation and potential risk from manufactured
nanoparticulate particles
6.3.2 Behaviour of Natural Colloids in Soils
Naturally-present colloids and macromolecules in soils are similar to those found in

freshwater systems, and nanoparticulate and microparticulate clays, organic mater, iron
oxides and other minerals play an important role in biogeochemical processes. Soil
colloids have been studied for decades in relation to their influence on soil development
(pedogenesis), and their effect on soil structural behaviour (dispersion and crusting)
(Cameron, 1915). Dispersion of soil is a key process affecting the quality of surface
waters in Australia, and studies have examined the factors responsible for colloid
generation and transport in soil systems (Noack et al., 2000; Siepmann et al., 2004;
Kaplan et al., 1996; Seaman et al., 1997).
A large body of literature exists on the aggregation/dispersion behaviour of soil colloids

in relation to soil physical and chemical properties (for a review see Rengasamy and
Olsson, 1991). Aggregation of colloids in soil is a function of surface charge, ionic
strength, particle size and chemical composition of the soil pore water and exchangeable
ions held on the surface of colloids. Systems dominated by sodium and with low ionic
strengths are likely to have dispersion of colloids, while those dominated by calcium and
high ionic strengths will tend to aggregate. Recent evidence confirms that manufactured
nanoparticles behave similarly to natural colloids (Saleh et al. 2008; Wang et al. 2008).
High water flow through soils will tend to mobilise colloids, while slow water flow will
tend to allow interaction and binding of colloids with soil minerals and organic matter.
6.3.3 Behaviour of Manufactured Nanoparticles in Soils
Of the pathways identified in Figure 9, the most important properties that will control
nanoparticles fate in soils are likely to be dissolution, aggregation and partitioning
between solution and solid phases.
Nanoparticle Solubility
Dissolution of nanoparticles in aqueous media has already been covered in Section 6.2.2
above. A key difference in soils is the large surface area and exchange capacity for
cations and anions that can promote dissolution of compounds through acting as a sink
for dissolution products, and providing protons to enhance dissolution of compounds

with a pH-dependent solubility. To date, no studies have examined the rate or extent of
dissolution of nanoparticulate materials in soils in relation to their bulk counterparts.
Aggregation
There are virtually no studies which have examined this topic for manufactured
nanoparticles in soils, but inferences from the behaviour of natural colloids can be made
(Section 4.2 above). Aggregation behaviour of nanoparticles in aquatic systems has been
covered in Section 4.2, and the same processes would be active in soils, except we can
speculate that the aggregation of nanoparticles in soil may be greater due to the higher
ionic strength of soil pore waters compared to most surface water systems (streams and
dams). Aggregation in soils also leads to particle entrapment in pores through which the
dispersed nanoparticles could have passed, thus restricting mobility (Wang et al. 2008).
Partitioning
There are virtually no studies which have examined this topic. We can speculate that the
high surface area and charge of many hydrophilic manufactured nanoparticles will cause
a strong binding to the predominantly negatively charged surfaces of soil minerals and
organic matter (Li et al. 2008), depending on the nature of the charge. Net positively
charged particles will be retained strongly, while those with net negative charge will be
highly mobile in most soils (Saleh et al., 2008).
Where nanoparticles are hydrophobic, retention to organic matter surfaces in soil may
inhibit mobility and availability to organisms.
The characteristics of the surface “functional” coatings used in nanoparticle manufacture

may be very important in explaining (and predicting) fate in soil, as it is these surfaces
that will interact with minerals and organic matter surfaces in soil.
Given that contaminant partitioning (Kd, Koc or Kow) is a key property used in risk
assessments for a wide range of inorganic and organic contaminants in terrestrial
systems, this characteristic is a key property requiring evaluation.
7. ECOLOGICAL RISK ASSESSMENT OF

MANUFACTURED NANOPARTICLES
Regulators worldwide are seeking to undertake ecological risk assessments of
manufactured nanoparticles to determine the significance of any impacts associated with
their manufacture and use. The ecological risk assessment framework for contaminants
in the environment, developed by the USEPA and adopted in Australia, has the following
components:
 Problem formulation
 Exposure assessment – Chemical assessment taking into account contaminant

fate (predicted environmental concentrations – PECs)

 Effects assessment – Measurement of toxicity, bioaccumulation, effects on
ecology (predicted no effect concentrations – PNECs)
 Risk characterisation (PEC/PNEC)
As discussed by Owen and Handy (2007), the issue of problem formulation is a critical
one. The initial anxiety that nanomaterials might represent the current equivalent of
genetically modified foods in terms of its environmental danger (Dowling, 2005) appears
to have now passed. These concerns were heightened by the findings that fullerenes
were capable of crossing the blood-brain barrier in fish (Oberdortser, 2003), which has
since been shown to be an experimental artefact (Brant et al., 2005). Nevertheless there
are a number of basic concerns that need addressing, starting with the basic issue of
whether nanosized materials pose a greater hazard to biota than the equivalent
macrosized materials. While there is good evidence for altered behaviour with smaller
size, only a handful of studies have demonstrated that this translates into greater toxicity.
The risk assessment needs to show connectivity between the source, the pathway, and
the receptor. In most instances in water and soil ecosystems, the evidence of this
connectivity has been indirect or absent.
For industrial chemicals, a manual providing guidance on ecological risk assessment was
recently released by the Department of the Environment and Water Resources (now
Department of the Environment, Water, Heritage and the Arts, DEWHA) (DEW, 2007).
This manual specifically discussed data requirements, data evaluation, environmental
exposure assessment, environmental effects assessment, assessment of persistent,
bioaccumulative and toxic substances, and risk characterisation and management.
Data requirements include melting point, specific gravity, vapour pressure, water
solubility, hydrolysis as a function of pH, octanol/water partition coefficient, adsorption
behaviour in soils, acid dissociation constant, and environmental fate data, especially on
biodegradation and bioaccumulation. For effects assessment, toxicity tests must be
undertaken using a fish acute test, Daphnia immobilisation and reproduction tests, an
algal growth inhibition test, and measures of biodegradability and bioaccumulation. As
the following pages will show, the majority of these requirements could not currently be
met for manufactured nanoparticles. This means that the determination of both PECs
and PNECs will not be possible as a prerequisite to assessing the potential environmental
hazard of manufactured nanoparticles in soil, water and sediment compartments. The
current state of knowledge in these areas is reviewed in the following pages of this
report.
7.1 Polymeric Nanoparticles as a Separate Class

Some authors have drawn the distinction between polymers such as dendrimers,
fullerenes and carbon nanotubes whose size is determined by their molecular weight and
other particles where size is a function of their degree of aggregation. There is potential
for the size of both nanoparticle types to affect their interactions with aquatic biota, but
to date few studies have investigated this. We see no reason to consider polymers as
warranting separate regulatory consideration from other nanoparticle types.

It is clear that assessment of hazard of either type on the basis of intrinsic chemical
properties is inappropriate and there must be some consideration of size, be it molecular
weight or other measures of size. The premise that nanoparticulate size fractions are
more toxic than larger size fractions needs to be tested for all nanoparticle classes with
respect to the natural environment into which they are released.
8. EXPOSURE ASSESSMENT
8.1 What to Measure

An exposure assessment seeks to determine the concentrations and bioavailable forms of
a contaminant in the environment that, with a consideration of fate and exposure
duration, can be linked to effects on target organisms. It will be important therefore that
measurements reflect the concentrations and physical and chemical properties of the
nanoparticles in the field that are truly representative of exposure. In assessing industrial
chemicals, the DEW manual (DEW, 2007) lists fate, partitioning behaviour, and
persistence as important parameters. These need to be combined with concentration data
in estimating likely exposure.
For nanomaterials, since it has been demonstrated that size is a critical parameter, any
measurement of concentration must be accompanied by data on the distribution of
particle sizes in the test water taking into account any aggregation that might occur
within the life cycles of the test organisms.
The requirement and the current status of methods for nanoparticle analysis and
characterisation have been well summarised in recent reviews by Hassellov et al. (2008)
and Tiede et al. (2008). Particle size measured as a diameter was not adequate when
particles were other than spherical, and other measures including aspect ratio (ratio of
their longer dimension to their shorter dimension) were also of value. They believed that
in addition to particle size distributions, measures of surface area were also important,
but not always reported. Nanoparticle net surface charge was also seen as an important
measure of the extent to which their dispersion is stabilised by electrostatic repulsive
forces.
An interesting issue is the extent to which nanoparticle size distributions reach steady
state, and whether this state is maintained throughout the duration of an experiment, e.g.
for chronic toxicity testing. Frederici et al. (2007) noted a change in distribution when
studying the effects of nanoparticulate titanium dioxide on rainbow trout. Hassellov et
al. (2008) recommended that monitoring be undertaken over the duration of any studies
to detect this, or any changes due to other reaction and/or degradation pathways.
8.2 Methods for Measurement of Nanoparticles

The measurement of nanoparticles in environmental media poses particular challenges.
Measurements are required of concentrations and size, and possibly also of surface area

and charge. Where possible, measurements should be of the state of the nanoparticles in
the particular medium (soil, sediment, water), rather than an assumption based on
dilution of a starting material. Even the measurement of concentration poses issues, as
we are concerned with the bioavailable concentration of nanoparticles. In many cases,
what is measured is a surrogate for this, e.g. total zinc concentration rather than
nanoparticulate zinc oxide. In the case of n-C60, UV absorbance was used to measure
concentrations (Oberdorster et al., 2004), while for carbon nanotubes, light scattering
techniques were used to correlate with concentration (Smith et al., 2007).
The bioavailable fraction can however include a dissolved, soluble fraction rather than a
nanoparticulate fraction, so ideally some measurement that discriminates this fraction is
required. Standard 0.45-µm membrane filtration will not retain most nanoparticles, so a
separation technique is required. Ultracentrifugation, size-based chromatographic
separations, ultrafiltration and dialysis are all appropriate, although the last two are
probably the preferred methods of separation. In soils, there is the additional
complication that any nanoparticulate material that dissolves will interact with the soil
solid phase, and some assessment of this pool may also be required to assess
bioavailability in addition to characterisation of the material in soil pore water.
For measuring particle size distributions, electron microscopy (EM) and dynamic light
scattering (DLS) are the most commonly used techniques. Both have advantages and
disadvantages (Bootz et al., 2004). EM gives the most direct information on the size
distribution and shapes of particles, however, there is concern about artefacts introduced
by the sample preparation step. With DLS, the presence of small amounts of large
aggregates can affect the distribution of a main component of a smaller size, with results
being misleading where the samples have a broad size distribution. More detailed
information on specific surface area, surface charge and zeta potential can be obtained by
a variety of techniques, but these are research techniques that are not likely to contribute
to routine risk assessment of nanomaterials in the near term.
For studies of nanoparticles in situ, field flow fractionation (FFF) has been advocated
(Hasselov et al., 2008; Tieded et al., 2008), in particular a variation called flow field flow
fractionation (FlFFF). Basically the technique uses two right-angled flow streams to
partition particles on the basis of their diameter (Giddings, 2003). For metal-containing
nanoparticles, the metal concentrations in the separated fractions can be analysed by
inductively coupled plasma mass spectrometry (ICPMS). Stolpe et al. (2005) have
described the application of high resolution ICPMS coupled to FlFFF to study metals in
(natural) nanoparticulate colloids. The FFF technique has been well established, but is
not that easily mastered, and is in use in only a handful of laboratories worldwide. The
universal interest in nanomaterials might lead to a wider acceptance.
Single particle ICPMS analysis has recently been applied to the detection of gold colloids
in water (Degueldre et al., 2006). The use of new generation ICPMS approaches for
analysing individual nanoparticles show considerable promise (Stolpe et al., 2005), but it
may be some time before they can be applied to routine environmental monitoring of
manufactured nanoparticles.

Similarly the use of novel techniques such as liquid chromatography coupled to nuclear
magnetic resonance spectrometry may hold promise for the analysis of carbon-based
nanoparticles.
The analysis of manufactured nanoparticles in natural systems can be complicated by the

background of natural colloids. Where nanoparticle shape is distinctive, e.g. CNTs, this
may not be such an issue, but for others, techniques such as DLS and FFF will not be
able to discriminate between nanoparticles and natural colloids unless linked to some
nanoparticle element-specific analyses such as ICPMS. The solution is to use single
particle confirmatory analyses, such as EM, with energy-dispersive x-ray fluorescence
(EDX).
To date there have been few measurements of manufactured nanoparticles in natural

waters or soils because of the extreme difficulty in detecting environmental
concentrations. Details of techniques applied to environmental nanoparticles in aquatic
systems have been discussed by Wiggington et al. (2007).
The area of nanoparticle measurement is one that is being pursued internationally by a

number of agencies. In particular, there is a need to develop standard methods of
analysis, including methods for sample preparation that can be used to characterise
nanoparticles. As part of this exercise, the development of standard reference materials
that can be used for method quality assurance and quality control will be essential. In
Australia, the National Measurement Institute (NMI) has an active program in this area.
8.2.1 Relevance of OECD Test Guidelines
The assessment of the environmental fate of chemicals and polymers currently relies on a
few critical tests recommended by the Organisation for Economic Cooperation and
Development (OECD) (OECD, 2007), including those for water solubility,
adsorption/desorption, water/oil partition coefficient, hydrolysis, surface tension and fat
solubility. The applicability of each of these tests to nanomaterials is generally
inappropriate, and the methods will need to be considerably altered to adequately cater
for nanomaterials.
The test for water solubility (No. 105) (OECD, 2007) uses either a microcolumn
separation or a flask dissolution. Since the tests were not designed for use with colloidal
or nanosized particles, the separation of these from the ‘soluble’ fraction will be critical.
The test method indicates that the presence of colloids in the microcolumn effluent
invalidates the test. In studies of zinc oxide solubility, Franklin et al. (2008) used dialysis
to separate soluble zinc. Such procedures will be required as a finish to the OECD test.
The same applies to Test No. 120, for the solution/extraction behaviour of polymers in
water.
The tests looking at adsorption/desorption onto soils need to be relevant to the likely
environmental concentrations. Test No. 106 uses a soluble chemical fraction, however,
for a nanomaterial suspension, this would not be appropriate. Test No. 121 determines
the adsorbed fraction by HPLC. Determining whether the nanoparticles are retained by
filtration rather than adsorption will be problematic.

The octanol/water partitioning tests (Nos 107 and 123) are designed to measure the
equilibrium partitioning of a ‘dissolved’ substance between the two solvents, as distinct
from ‘solubility in octanol’ which is what will be obtained using nanoparticles. Even if it
were meaningful, the physical application of this test is likely to be seriously impaired by
clumping of nanoparticles at the solvent interface.
The hydrolysis test (No. 111) looks at hydrolysis in the range pH 4-9. With
nanomaterials, the result would test both dissolution and hydrolysis as a function of pH.
Surface tension (No. 115) is inappropriate for an insoluble chemical in nanoparticulate

form, however, the assessment of fat solubility (No 116) is a potentially useful measure
in relation to biological uptake.
OECD has an active interest in nanomaterials, and has a working group considering
appropriate test methods (see Section 10.1.3), including those for toxicity testing.
8.3 Modelling Exposure

Existing models of exposure for soluble contaminants have little applicability to
nanoparticles. As already discussed, there have been preliminary approaches to
predictive modelling of the suspension stability and kinetics of aggregation of
nanoparticles, however their applicability to real systems is, as yet, untested (Mackay et
al., 2006).
In an attempt to model likely concentrations of manufactured nanoparticles that might be

found in the environment, Boxall et al. (2007) used a series of simple algorithms to
predict the likely concentrations that might be found in soils and waters. For waters,
they considered five routes of entry:
(i) the direct entry of manufactured nanoparticles into water bodies from
bioremediation;
(ii) inputs from spray drift following use of agrochemicals;
(iii) runoff from contaminated soils;
(iv) aerial deposition; and
(v) emissions from wastewater treatment plants.
For soils, routes comprised:
(i) the application of remediation technologies;
(ii) the application of plant protection products;
(iii) the excretion of nanomedicines used in veterinary products;
(iv) aerial deposition; and

(v) the application of sewage sludge as a fertiliser.
They focussed mainly on cosmetics, personal care products and paint, and the
nanoparticle concentrations that they contained (based on limited European data). Three
hypothetical scenarios were modelled, where 10, 50 and 100% of a product type
contained the manufactured nanoparticle. Predicted concentrations for the 10% scenario
are shown in Table 4. Despite all of the uncertainties, the concentrations can be
compared to the toxic concentrations where these are known, to see whether these are in
the same range or not.
Table 5 shows the comparison of exposure data with known toxicity data, indicating that
the predicted environmental concentrations are orders of magnitude below those known
to have environmental effects on aquatic biota (as will be elaborated on later). This
scenario naturally does not take into account all possible sources, or accidental releases.
The results nevertheless give regulatory agencies some reassurance, especially since the
assumptions in estimations are conservative.
The challenge for modellers in the derivation of appropriate PECs is to be able to obtain
reliable estimates of the mass flow of nanomaterials to different compartments of the
environment. A good example of a life cycle assessment approach to this is shown in
Figure 10 (from Blaser et al., 2008). This example has been used for silver derived from
nanoparticulate biocidal plastics and textiles, but the approach has generic application.
In deducing mass flows, estimates of total product usage and estimated (or measured)
release rates must be obtained. These data are then related to the time of exposure.
Knowledge of the behaviour of silver in the aquatic environment (colloidal forms,
attachment to particles, etc.) is used in coupled river fate models to predict
sediment/water partitioning during treatment and in the aquatic environment.
Table 4. Predicted environmental concentrations of manufactured nanoparticles in UK soil and

waters (from Boxall et al., 2007)
Particle type Application Water, µg/L Soil, µg/kg

Aluminium oxide Paint 0.002 0.01
Cerium dioxide Scratch resistant coatings, catalysts <0.0001 0.01
Fullerenes Anti-inflammatory cream, eyeliner, face 0.31 44.7
powder, foundation, lipstick, mascara,
moisturizing cream, perfume
Gold Face cream 0.14 20.4
Organosilica Scratch resistant coatings 0.0005 0.07
Silver Biocidal coatings, shampoo, soap, 0.01 1.45
toothpaste

Titanium dioxide Paint, sunscreen 24.5 1030
Hydroxyapatite Toothpaste 10.1 422
Latex Laundry detergents 103 4310
Zinc oxide Paint, scratch resistant coatings, 76 3190
sunscreens
Table 5. Comparison of UK exposure data for manufactured nanoparticles with toxicity data (from Boxall
et al., 2007)
Predicted in Toxicity data, µg/L Other endpoints

water, µg/L
Invertebrate Fish Algae
EC50 LC50 EC50
n-C60 0.31 >35,000 >>5000 - Effects on invertebrate growth at
260 µg/L; bacterial growth
affected at 40µg/L; bacterial
phospholipids affected at 10 µg/L
TiO2 24.5 >100,000 >100,000 16,000 Effects on invertebrate growth at
2000 µg/L; bacterial growth
affected at 100,000 µg/L
SiO2 0.0007 - - No effect on bacterial growth at
500,000 µg/L
ZnO 76 - - No effect on bacterial growth at
100,000 µg/L
Figure 10. Framework for deriving mass flow data for silver flows from nano-functionalised biocidal
plastics and textiles (from Blaser et al., 2008). Arrows represent silver flows; dashed lines indicate
different environmental spheres. TWT=thermal waste treatment; STP=sewage treatment plant.
The model predictions can be verified by comparison with measured data from different
aquatic environments.
Mueller and Nowack (2008) have followed a similar approach in the determination of the
expected exposure concentrations in air, soil and water for nanoparticulate silver and
titanium dioxide and for CNTs. Literature production data are used to determine the

weighted concentrations of nanomaterials from each product type. Release of
contaminants and their transfer between the various compartments in the model are then
determined using literature-derivations or best estimates of transfer coefficients. In much
the same way as that used by Boxall et al. (2007), PECs were derived for particular
environmental compartments, however, the number of product categories was extended
beyond the personal care and cosmetic products, to include all possible uses, e.g. for
silver, the categories were textiles, cosmetics, metal products, sprays and cleaning
agents, plastics, and paints. The findings are shown in Table 6.
The results were then compared with available toxicity data. No data were available for
soil toxicity. The EC50 value (concentration causing a 50% effect) used for silver
toxicity was 20-40 mg/L, but this was from bacterial toxicity testing (E. coli and
Bacillus subtilis), and so are not necessarily applicable. The authors noted that, for ionic
silver, literature LC50 values were 0.7 µg/L for algae and 2 µg/L for
Daphnia. They indicated that there was a lack of reliable toxicity
data for TiO2. Their hazard quotients (PEC/PNEC) indicate a potential
concern for TiO2, compared to the conclusions of Boxall et al. (2007)
discussed above, but this may be a function of the application of
large assessment factors (1/1000) to the limited toxicity data.
Table 6. Predicted environmental concentrations (PEC) of nano-Ag, nano-TiO 2 and CNTs in air,
water and soil (RE: realistic scenario; HE: high emission scenario) (from Mueller and Nowack,
2008)
nano-Ag nano-TiO2 CNT

Compartment Unit RE HE RE HE RE HE
Water µg/L 0.03 0.08 0.7 16 0.0005 0.0008
Water affected µg/L 8 21 180 3933 na na
by wastewater
Soil µg/kg 0.02 0.1 0.4 4.8 0.01 0.02
Table 7. Hazard quotients (PEC/PNEC) for nano-Ag, nano-TiO 2 and CNT in water (RE: realistic scenario;
HE: high emission scenario) (from Mueller and Nowack, 2008)
nano-Ag nano-TiO2 CNT

Compartment RE HE RE HE RE HE
Water 0.0008 0.002 >0.7 >16 0.005 0.008
Water affected 0.2 0.5 >180 > 3900 naa na

by wastewater
a
Not available

9. ECOTOXICOLOGY OF NANOPARTICLES
9.1 Ecotoxicity and Nanoparticle Dose Metrics

In examining the ecotoxicity of nanoparticles to biological organisms, a critical question
is the determination of what influences the dose response. In traditional ecotoxicology
with soluble species, concentration is the dose measure, and specifically, the bioavailable
concentration, which may be some sub-set of the total concentration. When dealing with
nanoparticles, the concentration or mass metric may not apply, and alternative
considerations may involve particle number, surface area (shape), particle composition,
or surface reactivity.
In defining the applicable dose metric, some understanding of the mechanism of toxicity
of nanoparticles is required. Thus toxicity could be exerted by soluble species
dissociating from nanoparticles at a cell surface and crossing the cell membrane, or by
disruption of cell function by blockage of surface sites. If the nanoparticle is a
heterogeneous source of oxygen free radicals that are responsible for lipid peroxidation,
then it is likely that the dose will be dependent on the number of active sites on the
nanoparticles that are capable of free radical generation.
In studies of human toxicology of nanoparticles, there has been some debate about the
appropriate dose metric. Oberdorster et al. (2005) showed that surface area accounted
for differences in lung inflammatory effects of nanoparticulate TiO2 to rats and mice far
better than any mass considerations. Duffin et al. (2002) reached similar conclusions for
quartz nanoparticles, although noting the importance of surface reactivity. Wittmaack
(2007) disputed this interpretation, suggesting that particle number provided a better fit
for differently prepared carbon nanoparticles, although the interpretation was
complicated by the possibility that aggregated particles might disaggregate on contact
with the lung. The relevance of these studies with atmospheric nanoparticles to toxicity
in aquatic or soil systems is, however, questionable.
Particle morphology may also be an important metric (Buzea et al., 2007). Particles can
be classified as having either high or low aspect ratios. The former include nanowires,
nanotubes and the like, while spherical, oval and cubic type particles have a low aspect
ratio. In pulmonary toxicology, particles with a high aspect ratio have been shown to be
more toxic (Inoue et al., 2006). The importance of aspect ratio in aquatic or terrestrial
toxicity is unknown.
No such definitive studies appear to have been undertaken for ecotoxicological

receptors, although there is clear evidence of greater toxicity of nanoparticulate versus
lesser surface area or macro forms, e.g. the demonstrated toxicity to water fleas
(Daphnia magna) of 30 nm TiO2 particles compared to no observed toxicity for 100-500
nm aggregates of the same material (Lovern and Klaper, 2006).
To date, all toxicity data has been reported in terms of concentrations, but since
bioavailability will be dependent upon the physical properties, it will be necessary to
qualify all concentration data. Size is the next most critical parameter, since indications

are that when the nanoparticulate range is exceeded, properties approach those of the
parent macroparticles. Included in the size estimation should be a verification of the
fraction that is not in true solution, so that any observed effects are related only to the
particulate forms. It should be noted that the key environmental issue for any risk
assessment is to derive a no-effects concentration in the site-specific medium. Toxicity
determined in synthetic media might greatly over- (or under-) estimate toxicity because
of modification of bioavailability in the presence of colloids or other constituents.
The parallel in aquatic ecotoxicology to the consideration of site specific modifications to

water quality guidelines is a useful one (e.g. ANZECC/ARMCANZ, 2000). The toxicity
in synthetic media can be used to derive a conservative guideline trigger value (for a
given nanoparticle size) that might be modified by site specific chemistry. At this stage of
our knowledge, other physical and chemical measurements are probably superfluous,
given the already existing uncertainties in the measurements that are being made.
A major practical issue with toxicity testing of nanomaterials is the dispersion of

nanoparticles in the test solutions. In aquatic toxicity testing, the contaminant is usually
in true solution and homogeneously distributed throughout the sample. Any attempts to
use artificial dispersants or sonication are likely to affect the degree of aggregation from
its natural state and so stirring of the sample is the only acceptable means of maintaining
the nanoparticles in any way close to a dispersed state, acknowledging that prolonged
stirring may also break up nanoparticles. Intermittent stirring might be an option.
A framework for nanoparticle toxicity assessment in waters based on the above

discussion is given in Table 8. A similar approach could be devised for testing in soils.
Table 8. Approach to toxicity testing of nanomaterials in waters
Derivation of Nanomaterial Guideline Trigger Value

1. Suspend nanomaterial in synthetic water (at an appropriate concentration with an
appropriate mixing time to achieve equilibrium solubility)
2. Determine ‘soluble’ fraction (e.g. using dialysis)
3. Determine insoluble fraction
4. Determine particle size distribution on the sample from 1.
5. Undertake toxicity tests using different species on the sample from 1 and on the
‘soluble’ fraction. Determine the contribution of ‘soluble’ species to the total
nanomaterial toxicity.
6. Derive a guideline trigger value for the measured size distribution.
Derivation of a Site-specific Trigger Value
1. Repeat the above approach using the appropriate site water.

Toxicity Testing of a Nanomaterial Sample for Comparison with Guideline Trigger
Values
1. Repeat the above approach using either a synthetic or site water sample as
appropriate.
2. Compare result with trigger value, noting compatibility of particle size distribution.
Crane and Handy (2007) in a recent review of methods for characterising the
ecotoxicological hazard of nanomaterials suggested that rapid tests that identified
specific modes of toxicity, e.g. genotoxicity, immunotoxicity or oxidative stress assays
might be a useful addition to the standard suite of toxicity tests that uses algae,
invertebrates and fish. Because of the uncertainties in acute to chronic ratios in tests on
nanomaterials, it was recommended that where possible, chronic tests were preferable.
9.2 Toxicity to Aquatic Biota
9.2.1 Mechanisms of Biological Uptake and Toxicity
Studies in vitro at the cellular level point to oxidative stress as a key mechanism of
toxicity for many nanoparticles. Oxidative stress has been linked in a number of cases to
the ability of many nanoparticles to generate reactive oxygen species (ROS: oxygen ions,
peroxides and free radicals) (Oberdorster et al., 2005, Nel et al., 2006).
Physical damage to cell membranes is also possible as a consequence of the abrasive

nature of some nanoparticles leading to toxicity (Stoimenov et al., 2002). Adhesion of
nanoparticles to the cell surface and dissociation of soluble toxic species can also provide
a route of uptake (Klaine et al., 2008; Apte et al., 2008).
Franklin et al. (2007) were unable to demonstrate algal cellular uptake of zinc from
nanoparticulate ZnO because of the unexpectedly high solubility of ZnO. They
subsequently demonstrated enhanced toxicity of CeO2 nanoparticles compared to bulk
CeO2 (Franklin et al., unpublished results), suggesting enhanced uptake.
For aquatic biota, nanoparticle uptake and potential toxicity will be dependent on the
type of organism, its trophic level and whether it is uni- or multicellular. With unicellular
organisms, the issue of whether nanoparticles can cross cell membranes directly or via
endocytosis is still a major question. For eukaryotic organisms, most internalisation of
nanoparticles will occur via endocytosis (Moore, 2006; Nowack and Bucheli 2007), i.e.
with the cell membrane enclosing the nanoparticles leading to their deposition in the
cytoplasm and association with intracellular organelles, without directly passing through
the cell membrane.
For higher organisms, uptake across the gill and other external surface epithelia is also
possible and interactions with aquatic plants may include adsorption onto the root

surface, incorporation into the cell wall, or diffusion into the intercellular space (Nowack
and Bucheli, 2007).
A further pathway for contaminant uptake is via the food chain. Direct ingestion is a
possibility for many organisms. Water fleas (Dapnia magna) rapidly ingested lipid-
coated nanotubes via normal feeding behaviour, metabolizing the lipid coating as a food
source (Roberts et al., 2007). The toxic impact in many instances will depend on the
ability of the particles to promote cellular damage, e.g. by oxygen radical formation. For
example, SWCNTs observed in the gut lumen of fish exposed to sub-lethal
concentrations for 10-days, demonstrated an increase in oxidative stress markers and
ionoregulatory disturbance (Smith et al., 2007). More recently, direct evidence for a
dietary pathway of nanoparticle uptake has been demonstrated for uptake of quantum
dots in water fleas (Ceriodaphnia dubia) via a previously exposed algal food source
(Bouldin et al., 2008).
9.2.2 Ecotoxicity to Individual Species
Toxicity test data on manufactured nanomaterials from existing literature are summarized
in Table 9.
Bacterial toxicity
Many of the toxicity assessments of nanomaterials have focussed on bacteria, largely

undertaken using traditional growth media under optimum conditions. These data have
been well summarized elsewhere (Apte et al., 2008; Handy et al., 2008; Klaine et al.,
2008). There is no doubt that many nanomaterials show bactericidal properties,
especially silver (Morones et al., 2005; Sondi and Salopek-Sondi, 2004), where that
property is the reason for its extensive usage. Similar antimicrobial activity is shown by
titanium dioxide (Wolfrum et al., 2002). More recent studies have demonstrated strong
antimicrobial activity of SWCNTs (Kang et al., 2007).
While these studies have been useful in investigating mechanisms of toxicity and relative
toxicities of different formulations (e.g. Lyon et al. 2005; Fang et al., 2007; Yamamoto et
al., 2001; Reddy et al., 2007), they will not be discussed in detail here: (i) data from
tests in growth media are not relevant to natural ecosystems; and (ii) bacterial data are
not used in species sensitivity distributions to determine safe concentrations of
nanomaterials in waters (DEW, 2007).
As already discussed in Section 5, the potential for impact on sewage bacteria is a

separate question to protecting organisms in natural waters.
Algal toxicity
Limited data are available for algal toxicity. The response to TiO2 is not particularly
sensitive (Hund-Rinke and Simon, 2006; Warheit et al., 2007) and that to ZnO is a
response to soluble zinc (Franklin et al., 2007).

Invertebrate toxicity
The freshwater crustacean Daphnia magna has been the most used invertebrate species
for nanomaterial toxicity testing. Daphnia were quite sensitive to n-C60 prepared by
tetrahydrofuran (THF) extraction (Zhu et al., 2006; Lovern and Klaper, 2006). It is
important to note that for these fullerenes, two preparation methods were followed, one
using sonication of fullerenes in water for 30 minutes to disperse the nanoparticles and
the second using the evaporation of THF from a THF extract added to water. The latter
were consistently more toxic to all organisms tested, and the question remains as to
whether the additional toxicity was due to THF, although these tests used THF only
controls. It has been suggested that sonication could enhance toxicity (Zhu et al., 2006).
Fish
Normally fish would be expected to show less sensitivity to dissolved contaminants than
algae or daphnids. This was not necessarily the case with nanomaterials, and may be
indicative of a different mechanism of toxicity, e.g. gill clogging, that would not occur
with dissolved contaminants.
Toxicity of nanoparticulate silver to zebrafish embryos has been demonstrated by Lee et

al., (2007). In this study, the only one to date of nanoparticulate silver toxicity to aquatic
biota, the endpoints were deformities and abnormalities in the embryos. No EC50 values
were quoted, but from the graphs were estimated to be in the range 10-20 ng/L. This is
far lower than the bacterial toxicity values used by Mueller and Nowack (2008)
discussed earlier.
The toxicity of soft nanoparticles has been poorly studied. The NICNAS (2000) report
on acrylic latex indicates that no toxicity data are available. They are generally believed
to have low toxicity.

Table 9. Summary of toxicity testing results for manufactured nanomaterials (from Apte et al., 2008)
Nanomaterial Size Fraction, Test Medium Test species Endpoint Reference

nm
n-C60 water- Nominally 10-200 Standard USEPA medium Water flea Daphnia magna 48-h LC50 >35 mg/L Zhu et al., 2006
solubilised
n-C60 THF- Nominally 10-200 Moderately hard freshwater Water flea Daphnia magna 48-h LC50 0.8 mg/L Zhu et al., 2006
extract USEPA protocol
n-C60 water- Average diameter Moderately hard freshwater Water flea Daphnia magna 48-h LC50 7.9 mg/L Lovern and Klaper
USEPA protocol (2006)
solubilised 30
n-C60 THF 10-20 Moderately hard freshwater Water flea Daphnia magna 48-h LC50 0.46 mg/L; NOEC Lovern and Klaper
extract USEPA protocol 180 µg/L (2006)
n-C60 water- Nominal 10-200 Synthetic hard water Water flea Daphnia magna 40% mortality at 2.5 mg/L over Oberdorster et al., 2006
solubilised, 21 days. No acute toxicity up
to 35 mg/L
n-C60 THF Nominally 10-200 Standard USEPA medium Fathead minnow Pimephales promelas 0.5 mg/L 100% mortality in 6- Zhu et al., 2006
18 h
extract
n-C60 water- Nominally 10-200 Standard USEPA medium Fathead minnow Pimephales promelas 0.5 mg/L no effects after 48 h Zhu et al., 2006
solubilised,
n-C60 THF Nominally 30-100 Synthetic hard water Juvenile large- Mycropterus salmoides 0.8 mg/L 100% mortality in 6- Oberdorster, 2004
mouth bass 18 h
extract
n-C60 water- Nominally 10-200 Synthetic hard water Freshwater Hyalella azteca No toxicity below 7 mg/L Oberdorster et al., 2006
crustacea
solubilised,

n-C60 water- Nominally 10-200 Synthetic hard water Japanese medaka Oryzias latipes No acute toxicity at 0.5 mg/L Oberdorster et al., 2006
solubilised, for 96h
n-C60 THF 100 nm Synthetic hard water Zebrafish embryos Danio rerio 1.5 mg/L was toxic Zhu et al., 2007
extract aggregates
SWCNT purified ? Seawater Meiobenthic Amphiascus tenuiremis No effects at 10 mg/L. Templeton et al., 2006
copepods Evidence of ingestion and
aggregation
SWCNT as ? Seawater Meiobenthic Amphiascus tenuiremis No effect at 1.6 mg/L; effects at Templeton et al., 2006
prepared copepods 10 mg/L.
SWCNT ? Freshwater with up to 0.15 mg/L Rainbow trout Oncorhynchus mykiss Effects on ventilation rate, gill Smith et al., 2007
SDS pathologies and gill mucus
secretion at 0.5 mg/L
SWCNT ? Freshwater and seawater Zebrafish embryos Danio rerio Hatching delay at 150 mg/L Cheng et al., 2007
TiO2 Nominal 25 Moderately hard water Algae Desmodesmus Chlorophyll fluorescence Hund-Rinke and
(small); 100 OECD 201 protocol subspicatus 72-h EC50 44 mg/L small; no Simon, 2006
(large) dose response large
TiO2 Average 140 Moderately hard water Algae Pseudokirchneriella Chlorophyll fluorescence Warheit et al., 2007
OECD 201 protocol subcapitata 72h EC50 16-21 mg/L
TiO2 Nominal 25 Moderately hard water Water flea Daphnia magna No concentration-effect curve Hund-Rinke and
(small); 100 OECD 202 protocol observed up to 3 m/L Simon, 2006
(large)
TiO2 THF 30 THF; 100-500 Moderately hard freshwater Water flea Daphnia magna 48-h LC50 THF 5.5 mg/L; Lovern and Klaper,
dispersed; sonicated USEPA protocol Sonicated >500 mg/L 2006
sonicated
TiO2 THF 30 THF; 100-500 Moderately hard freshwater Water flea Daphnia magna No significant behavioural Lovern et al., 2007
dispersed; sonicated USEPA protocol changes LOEC 2.0 mg/L
sonicated
TiO2 Average 140 Moderately hard water Water flea Daphnia magna 48-h EC50 >100 mg/L Warheit et al., 2007
OECD 202 protocol
TiO2 24 De-chlorinated tap water Rainbow trout Oncorhynchus mykiss No mortality during 14-day Federici et al., 2007
exposure up to 1.0 mg/L.
Sub-lethal effects including gill
damage, observed.
TiO2 140 Moderately hard water Rainbow trout Oncorhynchus mykiss 96-h EC50 >100 mg/L Warheit et al., 2007
OECD 201 protocol

TiO2 TEM : 50 -400 De-chlorinated tap water Carp Cyprinus carpio No mortality during 25 day Sun et al., 2007
exposure to 10 mg/L.
TiO2 Nominal 19 De-chlorinated tap water Carp Cyprinus carpio No mortality with 25 day Zhang et al., 2007
exposure to 10 mg/L TiO2.
Increased Cd accumulation
ZnO Average 178-361 USEPA, pH 7.5 Algae Pseudokirchneriella 72-h EC50 68µg/L due to Franklin et al., 2007
dissolved Zn
subcapitata
ZnO Mixed Spring water + food pellets Water flea Daphnia magna 8-day EC50 0.2-0.5 mg/L, Adams et al., 2006
possibly dissolved Zn
SiO2 Mixed Spring water + food pellets Water flea Daphnia magna 8-day EC50 <10 mg/L Adams et al., 2006
Cu Nominally 80 De-chlorinated tap water Zebrafish Danio rerio 48-h LC50 1.5 mg/L Griffit et al., 2007
Fe Average 70 USEPA protocol Water flea Daphnia magna 48-h LC50 55 mg/L Oberdorster et al., 2006
Ag Average 12 Dilute NaCl Zebrafish Danio rerio Embryo abnormalities EC50 Lee et al., 2007
10-20 ng/L
Quantum dots; Estimated 10-25 Moderately hard water Water flea Ceriodaphnia dubia 96-h LC50 >110 µg/L Bouldin et al., 2008
Cd/Se or Cd/Te
core with ZnS
shell
Quantum dots; Estimated 10-25 Moderately hard water Algae Pseudokirchneriella 96-h LC50 37.1 µg/L of Bouldin et al., 2008
subcapitata
Cd/Se or Cd/Te quantum dots, estimated as 9.6
core with ZnS µg/L Cd and 2.4 µg/L Se
shell

9.2.3 Developing Appropriate Guidelines for Nanomaterials in
Waters
The available toxicity data are insufficient to develop reliable guidelines for most
nanomaterials in waters, however, it is instructive to attempt to derive low reliability
guidelines for the nanomaterials for which we have the most data. For the data from
Table 6 for n-C60 and TiO2, chronic NOEC values were obtained using a factor of 10 on
acute LC50 values or on EC50 values from acute endpoints (Table 10). Following
ANZECC/ARMCANZ (2000) guidelines, data would be required for an alga, an
invertebrate and a fish and the lowest NOEC would be then divided by a factor of 100.
In this case of n-C60, algal data are missing, however, if this is ignored, a value of 7.9
µg/L would be derived for the water-solubilised n-C60. A value for THF-extract n-C60 is
more problematic and clearly <0.5 µg/L. The OECD approach would use a factor of
1000 on the lowest NOEC. For TiO2 the lowest result is for a THF-extracted sample.
Ignoring that, the PNEC for TiO2 dispersed by sonication would be 40 µg/L.
Table 10. Data for estimation of guideline concentrations for n-C60 in freshwater
Nanomaterial Formulation Species Endpoint, mg/L Estimated

chronic NOEC,
mg/L
n-C60 Water Daphnia magna 48-h LC50 7.9 0.79

solubilised by
sonication
n-C60 Water Pimephales No effects after >0.05
solubilised by promelas 48 h at 0.5
sonication
n-C60 THF extract Daphnia magna 48-h LC50 0.8, 0.08, 0.05
0.46 (acute
NOEC 180 µg/L)
n-C60 THF extract Pimephales 100 % mortality <0.05
promelas in 6-18 h 0.5
n-C60 THF extract Mycropterus 100% mortality <0.08
salmoides in 6-18 h 0.8
n-C60 THF extract Danio rerio <1.5 <0.15
TiO2 No THF Desmodesmus 72-h EC50 44 8.1
subspicatus
TiO2 No THF Pseudokirchneriella 72-h EC50 16- 4.0
subcapitata 21
TiO2 THF extract Daphnia magna 48-h LC50 THF 0.55
5.5
TiO2 No THF Daphnia magna >500 >50

With this extra conservatism, the PNEC value for both n-C60 and TiO2 are seen to be now
getting closer to the PEC values estimated in the UK (Table 5), with all of their
limitations. This highlights, if nothing else, the need for additional toxicity data.
9.2.4 Bioaccumulation
The published evidence to date for the bioaccumulation of manufactured nanomaterials

by aquatic organisms is limited and is summarised in Table 11. There is TEM evidence of
the presence, in the cytoplasm of bacterial cells (e.g. Escherichia coli, Bacillus subtillus,
Staphylococcus aureus), of MgO (Makhulf et al., 2005), SWCNTs (Kang et al., 2007),
ZnO (Brayner et al., 2006), quantum dots (Kloepfer et al., 2005) and silver (Xu et al.,
2004; Morenes et al., 2005). Many of these studies indicated cellular damage, however,
intracellular uptake was only indicated for MgO (<11 nm) and silver nanoparticles (<80
nm), and for quantum dots (<5 nm).
As noted earlier, an important finding was the food chain transfer of quantum dots via
exposed algae to water fleas (Bouldin et al., 2008). The quantum dots have a CdSe core
and a ZnS shell. The coatings appeared to provide protection from toxicity to cadmium
(or selenium), but transfer of core metals from intact nanocrystals occurred at levels well
above toxic thresholds to the water fleas.
Table 11. Published evidence of nanoparticle uptake by aquatic organisms (expanded from Apte et al.,
2008)
Nanoparticle Organism Target Evidence Reference

Organ
Bacteria
MgO Escherichia coli Membrane TEM images confirm damage Stoimenov,
Bacillus and leakage of cell contents. 2002
subtillus
SWCNT Escherichia coli Membrane Increased membrane Kang et al.,
permeability in cells in direct 2007
contact with SWCNT.
Physical damage to the
membrane and leakage of cell
contents is proposed.
MgO Escherichia coli Whole cell TEM shows ultrastructural Makhluf et al.,
Staphylococcus changes on exposure to 8±1 2005
aureus and 11±1 nm particles.
Elevated intracellular Mg
confirmed.
ZnO Escherichia coli Whole cell TEM reveal electron dense Brayner et al.,
areas in the cytoplasm. No 2006
elemental analysis.
Quantum dots Escherichia coli Whole cell TEM, fluorescence Kloepfer et al.,
Bacillus subtilis spectroscopy show adenine- 2005
conjugated QDs < 5 nm are

internalised. Intracellular Cd
and Se confirmed.
Ag Pseudomonas Whole cell Particles up to 80 nm Xu et al., 2004
aeruginosa transporting in and out of
cells. TEM images confirm
electron dense areas in the
cytoplasm.
Ag Escherichia coli Whole cell TEM images showing electron Morones et al.,
dense intracellular areas. 2005
EDS elemental mapping
confirms Ag distribution
throughout the cell. 1 -10 nm
particles interact preferentially
with the cell.
SWCNT lipid- Daphnia magna Gut Rapid (45-min) ingestion and Roberts et al.,
coated presence of lipid-coated 2007
SWCNT in the gut track
observed in time-course
micrographs.
Quantum dots Ceriodaphnia Gut Evidence for food chain Bouldin et al.,
dubia transfer of core metals from 2008
quantum dot-dosed algae
Fish
Cu Danio rerio Gill Histopathological analysis Griffitt et al.,
revealed damage to gill 2007
lamellae by proliferation of
epithelial cells and oedema of
gill filaments. Unclear if
effects mediated by particle
uptake.
TiO2 Oncorhynchus Gill Histopathological changes to Federici et al.,
mykiss the gill and gut but fish did 2007
Gut
not accumulate TiO2 in the
internal organs.
SWCNT Oncorhynchus Gill Histopathological changes to Smith et al.,
mykiss the gill and gut and liver. 2007
Gut
Aggregated SWCNTs
observed in the gut lumen.
9.2.5 Ecological Impacts
There have been no published studies on the broader ecological impacts of manufactured
nanoparticles.

9.3 Sediment Toxicity
Given that sediments are the ultimate receptor of nanoparticles in aquatic systems,
benthic organisms are likely to be as big a concern as those in the overlying water. The
nanoparticles are likely to be highly aggregated in the sediments, so any unique toxic
properties associated with nano size are likely to be absent. Very few studies have
looked at nanomaterials in sediments. For example, Kennedy et al. (2008) showed that
the survival of several amphipods was affected by MWCNTs in whole sediment
bioassays, but at unrealistic concentrations exceeding 100 g/kg. More studies are
required to fully assess nanoparticle properties (aggregation, surface area), bioavailability
and toxicity in the more complex sediment environment.
9.4 Toxicity to Terrestrial Biota

9.4.1 Ecotoxicity to Individual Species
There are very few data by which to assess the potential environmental risk of
nanoparticles to the terrestrial environment and this is seen as a key knowledge gap by
regulators (US EPA , 2007). As yet, there are few reports in the peer-reviewed scientific
literature of the assessment of ecotoxicity of nanoparticles to soil biota, in soils. Several
reports have examined ecotoxicity to soil organisms, but the media used have been
simple aqueous media (Brayner et al., 2006; Yang and Watts, 2005; Zheng et al., 2005;
Lin and Xing, 2007) and persistence of the nanoparticles in the test media was not
assessed. These are summarized in Table 12.
Table 12. Toxic effects of nanomaterials on soil organisms (from Klaine et al., 2008).
Nanomaterial Toxic Effects References
Carbon-containing
A) Fullerenes
C60 granular and None. Endpoints tested were respiration (basal and Tong et al., 2007
substrate-induced), microbial biomass C, enzyme
C60 water suspension (n-
activities. Small shift in bacterial and protozoan
C60)
gene patterns by PCR-DGGE.
n-C60 No effect on respiration (basal), microbial biomass Johansen et al.,
C (measured by substrate-induced respiration) and 2008
protozoan abundance. Reduction in numbers of
bacteria. Small shift in bacterial and protozoan gene
patterns by PCR-DGGE.
B) Carbon nanotubes
Multi-walled No effect on seed germination and root growth of Lin and Xing,
corn, cucumber, lettuce, radish, and rape. Reduced 2007
root growth of ryegrass.
Metals

Aluminium No effect on seed germination of corn, cucumber, Lin and Xing,
lettuce, radish, rape, and ryegrass. Rhizotoxic to 2007
corn, lettuce, and ryegrass but stimulated radish and
rape root growth.
Zinc Reduced seed germination of ryegrass and reduced Lin and Xing
root growth of corn, cucumber, lettuce, radish, rape, 2007
and ryegrass
Metal oxides
Al2O3 Phytotoxic (germination and seedling growth) but Yang and Watts,
see text. 2005
No effect on seed germination of corn, cucumber, Lin and Xing,
lettuce, radish, rape, and ryegrass. No effect on root 2007
growth of cucumber, lettuce, radish, rape, and
ryegrass. Reduced root growth of corn.
TiO2 Stimulatory to spinach seed germination and Zheng et al., 2005
seedling growth at low dose, phytotoxic at high
doses
ZnO Reduced seed germination of corn and reduced root Lin and Xing,
growth of corn, cucumber, lettuce, radish, rape, and 2007
ryegrass
Yang and Watts (2005) reported the toxicity of alumina nanoparticles (13 nm, coated
with and without phenanthrene) to root growth of five plant species (cabbage, carrot,
corn, cucumber, and soybean) exposed to aqueous suspensions of the nanoparticles, but
only at high concentrations (2,000 mg/L). Loading of the alumina nanoparticles with
phenanthrene reduced the toxicity of the nanoparticles. The nanoparticles were not
physically characterised prior to dosing, doses were not analytically confirmed, and in a
letter to the Editor of Toxicology Letters, Murashov (2006) pointed out the experimental
protocol of Yang and Watts (2005) did not distinguish toxicity caused by application of
the aluminium in a nanoparticle form, and toxicity of solution aluminium derived from
the nanoparticle. Indeed aluminium is a major component of soil minerals, known to be
phytotoxic in acidic soils for almost a century (Magistad 1925) so the phytotoxicity
observed by Yang and Watts (2005) is not surprising, and clearly indicates the need to
accurately determine if the nanoparticulate form of a contaminant is toxic, or if the
soluble contaminant derived from the nanoparticle is toxic. Franklin et al. (2007)
reached similar conclusions for the toxicity of ZnO nanoparticles to aquatic biota.
Zheng et al. (2005) examined the effects of nano- and bulk-TiO2 on spinach seed
germination and early plant growth in simple Perlite media containing a complete
nutrient solution. Nano-TiO2 significantly increased seed germination and plant growth
at low concentrations, but decreased these parameters at high concentrations. Bulk-TiO2
had little effect. The manufactured nanoparticles in this study were not physically
characterised and no details of size or surface reactivity of the materials were provided.
Recently, Lin and Zing (2007) examined the toxicity of several nanoparticles (MWCNTs,
Al, Al2O3, Zn and ZnO) to germination and early root growth of six plant species in

simple aqueous media at pH 6.5–7.5. The nanoparticles were not physically characterised
prior to exposure and doses were not confirmed. The zinc-based nanoparticles had the
greatest effect on plant germination and root growth, with EC50 concentrations similar
for both zinc- and ZnO-nanoparticles of 20–50 mg/L depending on plant species. The
authors attempted to quantify the solution zinc dose in their experiments by
centrifugation (3000 G for 60 min) and filtration (0.7 μm). They reported that the
centrifugation procedure did not fully separate the nanoparticles from the solution phase
(assessed using TM-AFM), but they did not provide microscopic information on the
solutions after filtration. Surprisingly, a 2000 mg/L suspension of ZnO after
centrifugation and filtration returned a solution zinc concentration of only 0.3-3.6 mg/L,
significantly less than the concentration of Zn2+ in equilibrium with bulk ZnO at pH 6.5–
7.5, ~10–900 mg/L (Lindsay, 1979). Copper nanoparticles were also recently found to
be potentially phytotoxic (Lee et al., 2008).
To date, there are only two reports in the literature of the terrestrial effects of
nanoparticles performed in soil, both on fullerenes (Tong et al., 2007; Johansen et al.,
2008). Tong et al. (2007) examined the toxicity of n-C60 in aqueous suspension and in
granular form to soil microorganisms using soil respiration, microbial biomass,
phospholipid fatty acid analysis, and enzyme activities as endpoints. The authors also
examined the DNA profile of the microbial community. All tests were performed in the
laboratory at optimal moisture conditions. In contrast to the observed microbial toxicity
of n-C60 in vitro (Fortner et al., 2005), Tong et al. found no effect of n-C60 to any
endpoint in the soil medium used (silty clay loam, 4% organic matter, pH 6.9). They
suggested that this was due to the strong binding of n-C60 to soil organic matter,
although no evidence was provided that organic matter was the solid phase in soil
reducing the effective dose. A similar set of experiments was performed by Johansen et
al. (2008), who examined the effect of n-C60 added to a neutral soil (pH 6.7) with low
organic C content (1.5%) on soil respiration, biomass C, bacterial and protozoan
abundance and the PCR-DGGE profiling of bacterial and protozoan DNA. No effects of
exposure of n-C60 were found on soil respiration, biomass C, and protozoan abundance,
but reductions in bacterial abundance were observed through colony counts. The n-C60
also caused only a small shift in bacterial and protozoan DNA, indicating a small change
in community structure, similar to the results of Tong et al. (2007). Similar results from
the same group were recently published for anaerobic bacteria typical of wastewater
sludge treatment systems (Nyberg et al. 2008).
There have been few reports of bioaccumulation or trophic transfer of nanomaterials to

soil invertebrates or mammals. A recent study of bioaccumulation of SWCNTs by
earthworms indicated a very low bioaccumulation factor compared to pyrene (~100-fold
lower) (Petersen et al., 2008), and a study of TiO2 accumulation by isopods (Porcellio
scaber) also indicated a low bioaccumulation potential for these nanomaterials (Jemec et
al., 2008).
These data highlight the need for more information on the interaction of nanoparticles
with soil components, and more quantitative assessments of aggregation/dispersion,
adsorption/desorption, precipitation/dissolution, decomposition and mobility of
manufactured nanoparticles in the soil environment. This information will aid the

interpretation of terrestrial ecotoxicity test data, and will inform the correct protocols for
the assessment of the ecotoxicity of nanoparticles in soils.
9.4.2 Development of Guidelines for Nanomaterials in Soils
The available toxicity data are insufficient to develop reliable guidelines for most
nanomaterials in soils. Effects have been inconsistent and studies of high quality have, to
date, not demonstrated significant adverse effects when soil was the medium used for
testing. It is therefore premature to suggest any regulatory limit for any nanomaterial in
soils.
10. INTERNATIONAL PROGRESS ON NANOPARTICLE

RISK ASSESSMENT
10.1 International Approaches

With nanotechnology industries growing exponentially worldwide, the assessment of the
risks they pose to the environment is still being pursued by government agencies.
Although it is recognised that available toxicity data on macro-sized chemicals will not
necessarily apply at the nanoscale, the current approach is still largely one of information
gathering through funding of additional research and development that will provide a
more sound basis than currently exists for managing the environmental impacts of
manufactured nanomaterials.
The field is evolving extremely rapidly, and it is important to regularly check the
literature. CSIRO are part of an international Nanoparticles Advisory Group in the
Society of Environmental Toxicology and Chemistry that shares on a monthly basis the
latest research and regulatory developments, while the Nanosafety Theme in CSIRO’s
Niche Manufacturing Flagship has close links with Dr Andrew Maynard of the Woodrow
Wilson International Centre for Scholars (see below). Such linkages are vital to both
contributing to and accessing the latest information. CSIRO also has links into the
OECD Working Party on the Safety of Manufactured Nanomaterials, as discussed later.
10.1.1 USA
In the US, a National Nanotechnology Initiative (NNI, 2001) was launched by the
National Science and Technology Council in 2001. Funding was provided to support
nanoscience and technology research via a range of major agencies (e.g. NSF, NIH,
DOE, NASA, NIST, EPA, etc.,) in a number of different theme areas. Environmental
issues were only of marginal concern. The National Science Foundation (NSF) later
established six facilities as part of Nanoscale Science and Engineering Centers. The
Center for Biological and Environmental Nanotechnology at Rice University was the
facility focussing on environmental issues (CBEN, 2005).

The Woodrow Wilson International Centre for Scholars and the Pew Charitable Trusts,
based in Washington DC, established a Project on Emerging Nanotechnologies in 2005.
This project has had a leading input to the nanotechnology debate in the US and beyond
(PEN, 2007b). Its publications (e.g. Maynard, 2006; Greenwood, 2007) have been a
vehicle for some useful basic information. Its inventory on nanoparticle usage (PEN,
2007a) is particularly valuable.
The US Environmental Protection Agency (US EPA) has been coming to grips with how
to apply the Toxic Substances Control Act to nanotechnology (Greenwood, 2007). A
report prepared by the Woodrow Wilson Institute for Scholars investigated the dilemmas
facing manufacturers and the USEPA in trying to deal with nanomaterials under that Act,
using as an example, carbon nanotubes (WWIS, 2003). There were many uncertainties
as to whether management in this way would be effective. Nevertheless, the USEPA
recently successfully fined a technology company over $200,000 for selling unregistered
nanopesticides (PEN, 2007b). The fine was made under the Federal Insecticide,
Fungicide and Rodenticide Act (FIFRA).
A nano risk framework was prepared in 2007 in a partnership between the Environmental
Defense Fund and DuPont (Environmental Defense-DuPont, 2007). The framework
identified a basic set of environmental fate data including nanomaterial aggregation and
disaggregation in the exposure media and screens for persistence and biodegradability.
For exposure assessments, they recommended acute toxicity and bioaccumulation
testing, but identified a need for ecosystem level studies of effects on populations.
Chronic tests would be required if a nanoparticle was potentially persistent and
bioaccumulative. Depending on the fate, sediment testing might also be triggered.
The USEPA published a definitive Nanotechnology White Paper in 2007, following a

three-year review, to inform EPA management of the science needs associated with
nanotechnology. It included recommendations for addressing science issues and research
needs (USEPA, 2007). More recently they produced a Draft Nanomaterial Research
Strategy to guide the nanotechnology research program within the EPA’s Office of
Research and Development (USEPA, 2008). They identified four key research themes
and seven key scientific questions which highlight the limitations of our current
knowledge:
1. Sources, Fate, Transport and Exposure
a. Which nanomaterials have a high potential for release from a life-cycle

perspective?
b. What technologies exist, can be modified, or must be developed to detect

and quantify engineered nanomaterials in environmental media and
biological samples?
c. What are the major processes/properties that govern the environmental

fate of engineered nanomaterials, and how are these related to physical
and chemical properties of these materials?

d. What are the exposures that will result from the releases of engineered
nanomaterials?
2. Human Health and Ecological Research to Inform Risk Assessment and Test
Methods
a. What are the effects of engineered nanomaterials and their applications on

human and ecological receptors, and how can these effects be best
quantified and predicted?
3. Risk Assessment Methods and Case Studies
a. Do Agency risk assessment approaches need to be amended to

incorporate special characteristics of engineered nanomaterials?
4. Preventing and Mitigating Risks
a. What technologies or practices can be applied to minimize risks of

engineered nanomaterials through their life cycle, and how can
naonotechnology’s beneficial uses be maximised to protect the
environment?
10.1.2 United Kingdom
The Royal Society and Royal Academy of Engineering released a report in 2004 on
nanoscience and nanotechnologies that addressed the current state of environmental
assessment of nanomaterials. It proposed that nanoparticulate forms of chemicals should
be treated as new chemicals for regulatory purposes, and identified the need for new
research to determine routes of exposure and toxicity. The UK government has released
several reports investigating the potential risks posed by manufactured nanoparticles
(DEFRA, 2005, 2007). The reports place the UK research program overseen by a cross-
government Nanotechnology Research Coordination Group in an international context.
They are collaborating with the OECD and the International Standards Organisation
(ISO) to share data and experiences to maximise the speed with which potential risks can
be identified and managed.
Specific task forces are addressing: (i) Metrology, characterisation, standardisation and
reference materials, (ii) Exposures: sources, pathways and technologies, (iii) Human
health and hazard assessment, (iv) Environmental hazard and risk assessment, and (v)
Social and economic dimensions of nanotechnologies.
A regulatory gaps analysis undertaken by Frater et al. (2006) for the UK Department of
Trade and Industry identified a number of gaps in the application of environmental
regulations to nanomaterials. A lack of knowledge of toxicity data was a critical issue.

10.1.3 Other International Activities
The OECD’s Environment Directorate has been active in sponsoring a number of

meetings dealing with the safety of manufactured nanomaterials. Details of these are
available on their website (http://www.oecd.org/ehs). Reports on developments in
China, Japan, Italy and Germany were included at the 2005 workshop in Washington.
The OECD established a Working Party on the Safety of Manufactured Nanomaterials
(WPMN) in 2006.
Eight steering groups (SG) have been established within the WPMN to run the following
projects:
 SG1: Development of an OECD database on EHS research
 SG2: EHS Research Strategies on Manufactured Nanomaterials
 SG3: Safety Testing of a Representative Set of Manufactured Nanomaterials
 SG4: Manufactured Nanomaterials and Test Guidelines
 SG5: Co-operation on Voluntary Schemes and Regulatory Programmes
 SG6: Co-operation on Risk Assessments
 SG7: The Role of Alternative Methods in Nanotoxicology
 SG8: Exposure Measurement and Exposure Mitigation.
At the OECD WPMN Workshop in Tokyo in April 2007, attended by Drs Maxine
McCall and Simon Apte of CSIRO and NICNAS staff, a sponsorship program was
initiated whereby member countries volunteered to undertake work on specific
nanomaterials of national interest in collaboration with each other. Australia agreed to
undertake the study of zinc oxide, cerium dioxide and silver.
Nanoparticles are fully covered by REACH, the new European Community regulation on
chemicals and their safe use requirements. One of the first activities of the member State
Committee under the European Chemicals Agency (ECHA) was to institute a
Nanoparticles Working Group. Nominations for this Working Group were sent to ECHA
by member states and observers from industry and other countries including the US. .
The European Chemical Industries Council (CEFIC) is currently reviewing strength and
weaknesses of the REACH risk assessment framework for nanoparticles, building on the
EU SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks)
report which covers the nanoparticles risk assessment topic (SCENIHR, 2005).
A summary of activities in Canada as of 2005, (Bergeron and Archambault, 2005)

indicates a similar scarcity of information, and identified needs for research and data
collection and the need to benefit from European and US experiences.

10.2 Australian Activities
Australia has been following a path similar to other major international players in its
approach to nanomaterial risk assessment. NICNAS, the national regulator of industrial
chemicals, issued a voluntary call for information to importers and manufacturers of
nanomaterials in 2006, as discussed earlier. Industry was asked to provide information
on uses and quantities of nanomaterials imported or manufactured for industrial
purposes, including use in cosmetics and personal care products. The information was
designed to assist in understanding which nanomaterials are available on the market or
close to commercialisation, and help focus our efforts to ensure the adequacy of the
regulatory scheme to assess nanomaterials.
Nanomaterials used exclusively as therapeutic goods, pesticides or food additives do not

fall within the scope of NICNAS, and were consequently outside the voluntary call for
information. The results of its findings were published on its website
http://www.nicnas.gov.au. Information from a new call is currently being compiled.
Chemicals that are not listed on the Australian Inventory of Chemical Substances
(AICS), which is based on the chemical formula and CAS number of chemicals (with no
size definition), are generally regarded as "new" and must be notified to NICNAS and
assessed for human health and environmental risks prior to their introduction and use.
Nanoscale forms of chemicals already listed on AICS (i.e. having an identical chemical
formula and CAS number) are currently considered to be "existing" chemicals. These
nanoscale existing chemicals can be selected for assessment if they potentially present a
changed risk of adverse health and/or environment effects. To date, NICNAS has not
assessed any nanomaterials with novel properties.
NICNAS is currently examining the suitability of its regulatory framework and processes
to protect human health and the environment in association with the OECD WPMN, and
by engagement with Australian government agencies under the National Nanotechnology
Strategy. At the same time, NICNAS has convened a Nanotechnology Advisory Group
which has three members each from the community and industry, and two members from
academia and one from NICNAS, and which NICNAS chairs.
A national Nanotechnology Roundtable was hosted by the National Health and Medical
Research Council (NHMRC) in December 2006. The Roundtable was attended by
representatives from Australian academic institutions, health and environment
government departments, regulatory bodies and industry and a representative from the
New Zealand Health Research Council. The Chief Executive Officer of the NHMRC is
using the outcomes of the Roundtable to inform future directions for the NHMRC.
In June 2006, the National Nanotechnology Strategy Taskforce produced a report for the
government on "Options for a National Nanotechnology Strategy" (NNST, 2006). Its
major findings with respect to the environment were:
 nanomaterials do exhibit novel properties that will have health safety and
environmental implications, but the significance is unclear at the moment, and
cannot be easily predicted due to a gap in knowledge;

 measurement and assessment of nanoparticles is a key priority for further
research;
 there is a need for continued international cooperation in the field;
 the Australian regulatory system needs to be flexible to address the challenges

posed by nanoparticles, and that coordinated processes at the regulatory level
need to be put in place; and
 while no serious risk is evident now, potential real risks in the use of
nanotechnologies in Australia must be identified so that appropriate risk
management strategies can be employed for their safe use.
Following on from the Taskforce’s report, a HSE Working Group comprising federal
agencies with responsibility for policy and implementation of Australia's regulatory
frameworks was established to consider HSE issues in more detail. This group
commissioned a review of the capacity of Australia's regulatory frameworks to manage
any potential impacts of nanotechnology, which was produced in 2007 (Ludlow et al.,
2007).
Also in 2006, the TGA conducted a review of the scientific literature in relation to the
use of nanoparticulate zinc oxide and titanium dioxide in sunscreens, concluding that
they did not represent a major health threat. At that time, Food Standards Australia New
Zealand (FSANZ) had not received any applications to consider the regulation of any
nanomaterials under the Australia New Zealand Food Standards Code.
The APVMA have also recently published in the Gazette, a voluntary call for information
on nanomaterials in agricultural or veterinary chemicals, or agricultural and veterinary
chemical products. APVMA has published a position paper on nanotechnology
(APVMA, 2008).
The Department of Infrastructure, Transport, Regional and Local Government (transport

of hazardous materials) and the Department of the Environment, Water, Heritage and the
Arts (DEWHA) in conjunction with the above bodies are currently assessing
international research in this area.
11. DEVELOPMENT OF TECHNICAL GUIDELINES FOR

NANOMATERIAL ASSESSMENT
An Environmental Risk Assessment Guidance Manual for industrial chemicals was issued
by the then Department of Environment and Water Resources (now DEWHA) in 2007
(DEW, 2007). While this did not consider nanomaterials, the draft framework outlined
an approach that was consistent with NChEM, the discussion paper on a national
framework for chemicals management in Australia prepared by the Environment
Protection and Heritage Council (EPHC, 2006).

11.1 Exposure Assessment Incorporating
Nanomaterial Fate
The development of appropriate technical guidelines that cover the impacts of
manufactured nanomaterials in waters, sediments and soils first requires an evaluation of
the fate of nanomaterials in these environments. Only then can exposure parameters be
appropriately assessed for comparison against any available environmental quality
guidelines. It is evident from the information presented in this report, that the fate of
nanomaterials in the environment, and their toxicity to biota, is likely to be a function of
size, shape, surface properties and bulk composition. Inferring fate and toxicity from
bulk composition alone (e.g. zinc oxide, CNTs, nanodots) is likely to be inappropriate
due to the wide range of surface functional coatings applied to many nanomaterials.
Surface properties will therefore play a key role in interactions with all environmental
matrices (soils, sediments and waters).
It is possible to lay out the following key questions that incorporate basic considerations
of nanomaterial fate in the form of a required check list:
A. Nanomaterial Classification
(i) What is the class of the nanomaterial (e.g. metal oxides; carbon products (n-
C60 fullerenes, CNTs; metals; quantum dots and semiconductors; nanoclays;
dendrimers, and nanoemulsions)?
(ii) What is its core chemical component (e.g. zinc oxide, silver, SWCNT etc)?
(iii) Is the basic formulation modified by additives?
(iv) What is the nominal particle size of the solid phase component?
B. Fate in Waters
The following considerations are required if the nanomaterials are to enter aquatic
systems, noting that the behaviour may differ for different product formulations:
(i) What is the particle size of dispersed nanomaterials in a natural receiving

water system (i.e. extent of aggregation) as determined by appropriate
techniques? The key factor here is the existence of dispersed or aggregated
particles that are in the nano range (<100 nm) and likely to have properties
differing from equivalent bulk macroparticles.
(ii) Does this particle size change with time, and if so over what timescale (hours,
days, weeks)?
(iii) What fraction of the nanomaterial dispersion is soluble (as determined by

dialysis or ultrafiltration) and/or dissociated thereby having potentially
different biological availability to the insoluble fraction?
(iv) Does the soluble fraction change with time, and if so over what timescale?

(v) What is the estimated mass concentration of the insoluble aggregated
dispersion in the nano size range? If the concentration is low, are interactions
with natural colloids at higher concentrations likely to modify the form of the
nanomaterial through adsorption.
C. Fate in Sediments
If nanomaterials accumulate in sediments, it would be assumed that this is the

consequence of settling from the water column due to a loss of buoyancy. To do so
requires considerable aggregation of particles or association with other water-borne
particulates notionally to exceed 1 µm in size, although this is density dependent. Once
accumulated in the sediments, the key fate questions relate to dissolution and
transformation. The key questions might be:
(i) Is there any dissolution of nanomaterials in the sediment pore waters under
the redox, pH and microbial conditions existing in the sediments?
(ii) Does this soluble fraction change with time?
(iii) What is the particle size of the nanomaterials in the sediment, i.e. are there
any nano-sized manufactured particles that might pose a different threat to
natural nanoparticles, or natural or manufactured macroparticles.
D. Fate in Soils
As already noted, nanomaterial interactions in soils are poorly characterised. In terms of

key questions, similar issues to those discussed above will need to be considered:
(i) Is the nanomaterial soluble in soil pore waters and so able to exert effects in
that form? How is this solubility affected by soil pH, salinity, sodicity, redox
conditions and time?
(ii) What is the particle size of nanomaterials in soil after any natural aggregation
processes? How easily are nanomaterials sorbed and retained by soil minerals
and organic matter?
(iii) Are nanomaterials more mobile through soils than natural nanoparticles? Can
they be vectors for enhanced transport of contaminant solutes to
groundwaters, e.g. pesticides, metals, dioxins, etc?
11.2 Effects Assessment Incorporating Nanomaterial

Fate
The information obtained from the above check list provides the necessary input to the
second component of any hazard assessment, the effects assessment. It will dictate the
evaluation of potential toxicity based on the existing toxicity database and a knowledge
of how changes to basic nanomaterials in the environment affect their bioavailability.

In terms of defining guideline concentrations for nanomaterials in waters, sediments and
soils, data are required from an appropriate set of toxicity tests (DEW, 2007) that can be
used to derive an appropriate PNEC. For aquatic biota, data are needed from at least
five species from four taxonomic groups if a statistical extrapolation method is to be
applied (ANZECC/ARMCANZ, 2000), although this size dataset must be viewed as a
minimum. Alternatively a low reliability value can be estimated by the application of an
assessment factor to the lowest chronic no observed effects concentration (NOEC). For
water sample toxicity data, as shown in Table 10, this will currently be the only
alternative.
The limitations of these toxicity datasets have been discussed earlier, the principal
concern being that the nanomaterials used are appropriately characterised in terms of key
parameters, especially particle size (degree of aggregation), and any specific formulations
that may modify behaviour (e.g. surfactant additions or surface coatings). In addition,
there is a need for site-specific, or at best water-, sediment- or soil-specific guidelines
that take into account environmental chemistry and its effect on nanoparticle behaviour
(especially the effect of ionic strength on aggregation).
Based on findings to date, the following hypotheses are suggested with respect to the
bioavailability and potential toxicity of nanomaterials in the environment:
(i) The fate and bioavailability of a particular nanomaterial might be expected to

change in the presence of additives that affect surface properties;
(ii) Small dispersed nanoparticles (<20 nm) are likely to be more bioavailable and
potentially toxic than large aggregates (>100 nm). Aggregates can
nevertheless exert toxicity compared with bulk material especially in instances
related to ROS generation where the aggregate surface area may be only
marginally lower than that of its composite nanoparticles.
(iii) Interaction with other particles and aggregation will be dependent on both the
surface charge and the surface area of nanoparticles. Low surface area and
net negatively charged particles are less prone to aggregation and are
potentially more mobile (but perhaps less bioavailable) since most membranes
have negative trans-membrane potentials;
(iv) Interaction of nanoparticles with other naturally present colloids or organic

macromolecules will also affect reactivity. Such interactions will be favoured
by high nanoparticle surface areas and excess concentrations of natural
colloids, and would be expected to result in larger particles with reduced
bioavailability;
(v) Aggregation is faster in environments of high ionic strength, i.e. high

hardness or saline waters, saline soils, or saline sediments;
(vi) With greater aggregation, particle toxicity approaches that of the equivalent
bulk macroparticles;

(vii) For metallic nanomaterials, water-soluble and potentially dissociated
nanoparticles are likely to be more toxic than insoluble and undissociated
particles of the same material, although there are mechanisms by which
insoluble materials can exert toxicity; and
(viii) The kinetics of both aggregation and dissolution will influence the above
toxicity.
Current indications are that it will only be possible to provide low reliability PNECs for a
limited set of nanomaterials, however, an awareness of the factors affecting fate will
guide the design of field-relevant toxicity testing.
11.3 Possible Approaches to Environmental Hazard

Ranking of Nanomaterials
As has been discussed, there are currently too few data available to set appropriate limits
or environmental guideline threshold numbers that accommodate all aspects of the
various formulations, and their fate in the environment, although an approach has been
outlined that might allow these to be achieved. It might then be appropriate to develop
a basic matrix approach to assess and rank the potential environmental mobility and
hazard from nanomaterials, where bulk size, solubility and surface properties are
considered. As yet, there are insufficient data to develop such an approach.
The prediction of behaviour based on the physical or chemical properties of

nanomaterials is only possible in a very limited way. For example, the use of quantitative
structure activity relationships (QSARs) may be useful for a limited number of
comparable nanomaterial types whose structures differ in the nature of chemical
substitution on a base molecular structure (e.g. fullerenes or CNTs). Nothing has yet
been published in this area, although the Joint Research Centre (JRC) of the European
Commission in late 2006 funded the Computational Toxicology Group, within the
European Chemicals Bureau in Ispra, Italy, to review the applicability of (Q)SARs to
nanoparticles (http://ecb/jrc.it/QSAR/). While no publications have yet appeared, the
JRC website indicates that their activities are focused on the development and
harmonisation of methods for toxicity testing of nanomaterials, the in vitro test of a
representative set of manufactured nanomaterials on critical cell lines and encompass
related studies on nanometrology and reference materials as well as the development of
databases and studies on the applicability of 'in silico' methods adapting the traditional
QSAR paradigm.
A JRC report (Dearden and Worth, 2007) outlines the concept of QSPRs (quantitative
structure property relationships) as a cost-effective computational alternative to
measurement of fate and toxicity. This is being explored in relation to nanomaterials. It
is important that these models go as far as predicting actual fate rather than stopping
only with the raw material and not its in-field characteristics.

12. RESEARCH NEEDS
It is clear that there is a need for more research to improve nanoparticle risk assessment.
This has already been discussed in a number of publications and discussed in this report.
Our recommendations are as follows:
1. There is a need for measurements in natural water, sediment and soil samples of
the stability, and short- and long-term fate of the various likely formulations that
might reach these compartments of the environment, and the development of
techniques to distinguish natural from manufactured nanoparticles. These
measurements should focus on particle concentration, size and surface
characteristics (area and charge).
2. Toxicity testing needs to be undertaken on nanoparticle formulations assessed in

(1) above. The tests should involve at least five species from four trophic levels
as required to derive PNECs using species sensitivity distributions. It is critical
that appropriate verification of particle and solute dose be undertaken in all
ecotoxicity testing, necessitating significant effort in (1) above.
3. As a precursor to toxicity testing, it will be necessary to develop standard (and

valid) methodologies for the hazard ranking of nanomaterial toxicity. These will
need to ensure the stability of the nanoparticle suspensions over the duration of
the standardised toxicity tests.
4. Comparisons of toxicity testing in natural vs. synthetic water and soil samples
demonstrating the effects of natural colloids.
13. ACKNOWLEDGEMENTS
The authors acknowledge Drs Natasha Franklin, Nicola Rogers and Simon Apte for
useful discussions and information provided for this report. We are grateful to Dr Glen
Walker (DEWHA) for his careful and comprehensive refereeing. The project was
commissioned by DEWHA with funding received from the Department of Innovation,
Industry, Science and Research under the National Nanotechnology Strategy. A 50% in-
kind contribution was provided by CSIRO’s Niche Manufacturing Flagship.
.
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15. GLOSSARY
Aerobic: In the presence of oxygen
AFM: Atomic force microscopy
Agglomerate: An assemblage of particles that are rigidly bound by sintering or growth
Aggregate: An assemblage of particles that is loosely bound and are readily dispersed
AICS: Australian Inventory of Chemical Substances
Anaerobic: In the absence of oxygen
APVMA: Australian Pesticides and Veterinary Medicines Authority
Bioavailability: Available for uptake by biological organisms
n-C60: Definition of n-C60 here
CAS: Chemical Abstracts service

CNT: Carbon nanotubes
Colloid: A particle, which may be a molecular aggregate, with a diameter of 1 nm-1 µm
Cytoplasm: All of the substance of a cell outside of the nucleus
Dendrimer: A synthetic, three-dimensional molecule with branching parts, formed using a
nanoscale, multistep fabrication process. Each step results in a new “generation” that has twice
the complexity of the previous generation
DLS: Dynamic light scattering
EDX: Energy dispersive x-ray fluorescence
EM: Electron microscopy
Endocytosis: A process of cellular ingestion by which the plasma membrane folds inward to
bring substances into the cell.
Eukaryote: A single-celled or multicellular organism whose cells contain a distinct membrane-
bound nucleus.
FFF: Field flow fractionation
Fibril: A threadlike fibre or filament
FlFFF: Flow field flow fractionation
Genotoxicity: Toxicity altering the structure or function of genetic material in an organism
Hazard quotient: The ratio of PEC to PNEC
Hydrolysis: Decomposition by reaction with water
Hydrophilic: Dissolving in or having a high affinity for water
Hydrophobic: Repelling or not easily dissolving in water

ICPMS: Inductive coupled plasma mass spectrometry
Immunotoxicity: Toxicity affecting the functioning of the immune system
ISO: International Standards Organisation
Lipophilic: Capable of combining with or dissolving in lipids
Manufactured nanoparticles: Particles with at least one dimension smaller than 100 nm that
have been created due to deliberate human activity.
Microemulsion: An emulsion (dispersion of one immiscible liquid in another) where the particles
in the dispersed phase are less than 1000 nm, which is thermodynamically stable
MWCNT: Multi-walled carbon nanotubes
Nano: A prefix meaning one billionth (1/1,000,000,000).
Nanoclay: Naturally occurring plate-like clays with nanoparticle sizes
Nanoemulsion: An emulsion (dispersion of one immiscible liquid in another) where the particles
in the dispersed phase are less than 1000 nm. Nanoemulsions are kinetically but not
thermodynamically stable.
Nanomaterials: Materials having structured components that have one dimension lower than 100
nm. Nanoparticle threshold size can also be defined as the size leading to different physico-
chemical behaviours and properties than bulk material. They can be subdivided into
nanoparticles, nanofilms and nanocomposites.
Nanoparticle: Individual pieces of matter with one dimension lower than 100 nm.
Nanotechnology: Areas of technology where dimensions and tolerances in the range of 0.1 nm to
100 nm play a critical role.
Nanotube: A one-dimensional fullerene (a convex cage of atoms with only hexagonal and/or
pentagonal faces) with a cylindrical shape.
Nanowires: One-dimensional structures, with unique electrical and optical properties, that are
used as building blocks in nanoscale devices.
NICNAS: National Industrial Chemicals Notification and Assessment Scheme
NNI: National Nanotechnology Initiative in the US
OECD: Organisation for Economic Cooperation and Development
Organelle: A differentiated structure within a cell, such as a mitochondrion, vacuole, or
chloroplast, that performs a specific function
PEC: Predicted environmental concentration
Photolysis: Chemical decomposition induced by light
PNEC: Predicted no effects concentration
Quantum dot: A nano-scale crystalline structure that can transform the colour of light. The
quantum dot is considered to have greater flexibility than other fluorescent materials, which
makes it suited to use in building nano-scale computing applications where light is used to
process information. They are made from a variety of different compounds, such as cadmium
selenide.

ROS: Reactive oxygen species
Sonication: Treatment by high frequency sound waves
SWCNT: Single walled carbon nanotubes
TEM: Transmission electron microscopy
THF: Tetrahydrofuran
USEPA: United States Environmental Protection Agency
Zeta potential: The electrostatic potential between particles and a liquid


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Fate of Manufactured Nanomaterials in the

Prepared for the Department of the Environment, Water, Heritage and

© Commonwealth of Australia 2008

ii Fate of Manufactured Nanomaterials in the Australian Environment

There is evidence to suggest that the impact of manufactured nanoparticles on aquatic

Fate of Manufactured Nanomaterials in the Australian Environment iii

There are numerous examples to demonstrate that nanomaterials can be bioaccumulated by

Current international activities in relation to nanoparticle risk assessment are discussed. In

2. Toxicity testing needs to be undertaken on nanoparticle formulations assessed in (1)

3. As a precursor to toxicity testing, it will be necessary to develop standard (and valid)

iv Fate of Manufactured Nanomaterials in the Australian Environment

Fate of Manufactured Nanomaterials in the Australian Environment v

4. CHARACTERISTICS OF ENVIRONMENTAL NANOPARTICLES........9

5. ENVIRONMENTAL SOURCES OF MANUFACTURED

7. ECOLOGICAL RISK ASSESSMENT OF MANUFACTURED

vi Fate of Manufactured Nanomaterials in the Australian Environment

10. INTERNATIONAL PROGRESS ON NANOMATERIAL RISK

11. DEVELOPMENT OF TECHNICAL GUIDELINES FOR NANOMATERIAL

12. RESEARCH NEEDS................................................................59

Fate of Manufactured Nanomaterials in the Environment vii

Figure 3. Typical structure of a dendrimer (first-generation polyphenylene dendrimer reported by Müllen

Figure 5. Potential sources of manufactured nanoparticles to the environment

Figure 6. Pathways for manufactured metal oxide nanoparticles in natural waters

Table 1. Classes of manufactured nanomaterials

Table 2. Usage of nanomaterials in the commercial sector in Australia

Table 4. Predicted environmental concentrations of manufactured nanoparticles in UK soil and waters

Table 8. Approach to toxicity testing of nanomaterials in waters

viii Fate of Manufactured Nanomaterials in the Australian Environment

Fate of Manufactured Nanomaterials in the Australian Environment ix

Fate of Manufactured Nanomaterials in the Australian Environment 1

Table 1. Classes of manufactured nanomaterials

Class Component Use

2 Fate of Manufactured Nanomaterials in the Australian Environment

Fate of Manufactured Nanomaterials in the Australian Environment 3

Annual worldwide production of SWCNT is estimated to exceed 1000 tonnes by 2011

4 Fate of Manufactured Nanomaterials in the Australian Environment

Nanoparticulate silver is one of the most widely used nanomaterials in consumer

Fluorescent semiconductor nanocrystals, also known as quantum dots (QDs) form a

Fate of Manufactured Nanomaterials in the Australian Environment 5

Dendrimers are monodisperse multifunctional polymers that have repeatedly branched

Figure 3. Typical structure of a dendrimer (first-generation

Among the most outstanding applications of

6 Fate of Manufactured Nanomaterials in the Australian Environment

3. NANOPARTICLE USAGE IN AUSTRALIA

Table 2. Usage of nanomaterials in the commercial sector in Australia

Chemical Name Application Total usage (tonne/y)

Fate of Manufactured Nanomaterials in the Australian Environment 7

8 Fate of Manufactured Nanomaterials in the Australian Environment

4.1 Manufactured Nanomaterials

The situation becomes more complex because the formulations of manufactured

4.2 Natural Nanoparticles

Fate of Manufactured Nanomaterials in the Australian Environment 9

10 Fate of Manufactured Nanomaterials in the Australian Environment

Aggregation or particle coagulation can be faster in higher ionic strength water

A schematic diagram of aggregate formation involving natural colloids is shown in Figure

Fate of Manufactured Nanomaterials in the Australian Environment 11

5. ENVIRONMENTAL SOURCES OF MANUFACTURED

Atmospheric nanoparticles are both a potential risk to the environment and an

12 Fate of Manufactured Nanomaterials in the Australian Environment

The potential for nanoparticles to end up in aqueous discharges is currently unknown.

Fate of Manufactured Nanomaterials in the Australian Environment 13

The US Department of Energy has recently published an approach to nanomaterial

In addressing the risks posed by manufactured nanomaterials, a relevant question is