A micro-sized model for the in vivo study of nanoparticle toxicity: What has Caenorhabditis elegans taught us?

Abstract

Recent years have seen a rapid increase in studies of nanoparticle toxicity. These are intended both to reduce the chances of unexpected toxicity to humans or ecosystems, and to inform a predictive framework that would improve the ability to design nanoparticles that are less likely to cause toxicity. Nanotoxicology research has been carried out using a wide range of model systems, including microbes, cells in culture, invertebrates, vertebrates, plants and complex assemblages of species in microcosms and mesocosms. These systems offer different strengths and have also resulted in somewhat different conclusions regarding nanoparticle bioavailability and toxicity. We review the advantages offered by the model organism Caenorhabditis elegans, summarise what has been learned about uptake, distribution and effects of nanoparticles in this organism and compare and contrast these results with those obtained in other organisms, such as daphnids, earthworms, fish and mammalian models.

Full text

A micro-sized model for the in vivo study of nanoparticle
toxicity: what has Caenorhabditis elegans taught us?
Jinhee Choi,
A
,
E
Olga V. Tsyusko,
B
,
C
,
E
Jason M. Unrine,
B
,
C
Nivedita Chatterjee,
A
Jeong-Min Ahn,
A
Xinyu Yang,
C
,
D
B. Lila Thornton,
C
,
D
Ian T. Ryde,
C
,
D
Daniel Starnes
B
,
C
and Joel N. Meyer
C
,
D
,
E
A
School of Environmental Engineering and Graduate School of Energy and Environmental
System Engineering, University of Seoul, Seoul 130-743, South Korea.
B
Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, USA.
C
The Center for Environmental Implications of Nanotechnology, Duke University, Durham,
NC 27708, USA.
D
Nicholas School of the Environment and Center for the Environmental Implications
of Nanotechnology, Duke University, Durham, NC 27708-0328, USA.
E
Corresponding authors. Email: jinhchoi@uos.ac.kr; olga.tsyusko@uky.edu; jnm4@duke.edu
Environmental context. The ability of the soil nematode Caenorhabditis elegans to withstand a wide range
of environmental conditions makes it an idea model for studying the bioavailability and effects of engi-
neered nanomaterials. We critically review what has been learned about the environmental fate of engineered
nanoparticles, their effects and their mechanisms of toxicity using this model organism. Future systematic
manipulation of nanoparticle properties and environmental variables should elucidate how their interaction
influences toxicity and increase the predictive power of nanomaterial toxicity studies.
Abstract. Recent years have seen a rapid increase in studies of nanoparticle toxicity. These are intended both to reduce
the chances of unexpected toxicity to humans or ecosystems, and to inform a predictive framework that would improve the
ability to design nanoparticles that are less likely to cause toxicity. Nanotoxicology research has been carried out using a
wide range of model systems, including microbes, cells in culture, invertebrates, vertebrates, plants and complex
assemblages of species in microcosms and mesocosms. These systems offer different strengths and have also resulted in
somewhat different conclusions regarding nanoparticle bioavailability and toxicity. We review the advantages offered by
the model organism Caenorhabditis elegans, summarise what has been learned about uptake, distribution and effects of
nanoparticles in this organism and compare and contrast these results with those obtained in other organisms, such as
daphnids, earthworms, fish and mammalian models.
Additional keywords: bioavailability, gene expression, mechanism of toxicity, uptake.
Received 17 October 2013, accepted 16 April 2014, published online 20 June 2014
The challenge of nanotoxicology
Nanotoxicological studies are of particular importance because
of the possibility that manufactured nanosized particles may
have unique biological effects, just as they have unique physical
and chemical properties. Nanosized particles are produced in
mass quantities anthropogenically and naturally. The focus in
this review is on the first category, which is often referred to as
‘manufactured’ or ‘engineered’ nanoparticles. For simplicity,
we will henceforth use the term ‘NPs’ to refer to all categories of
manufactured NPs, including carbon-based as well as metal-
based NPs. Past introductions of products with novel properties
(e.g. the persistent organic pollutants addressed by the Stock-
holm Convention) have taught us toxicological lessons the hard
way. Our current challenge is to gain critical insights about NP
toxicity ahead of time.
Toxicological studies of NPs are complicated by their uni-
que properties.
[1]
Chemical and toxicological paradigms are
frequently not applicable. For example, oil–water partition
coefficient (K
ow
) values inform our understanding of environ-
mental fate and transport as well as organismal uptake and
distribution of organic molecules. However, there are experi-
mental challenges in measuring K
ow
for NPs, such as the
distribution of NPs into the interface between octanol and water
due to high surface activity. K
ow
values for NPs have not been
extensively linked with environmental fate or bioavailability.
[2]
Other considerations (e.g. acid dissociation constants, pK
a
)
may have some application but must be interpreted somewhat
differently in the context of particles that may have a very large
number of potentially unevenly distributed (among and between
NPs) sites of protonation. Furthermore, we must incorporate
additional consideration of physicochemical properties that are
not often considered in the toxicology of discrete chemical
species, such as particle size, shape, crystallinity, complex
surface chemistry, aggregation state and inherent heterogeneity,
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RESEARCH FRONT

Dr Choi received her B.Sc. (1991) and Master’s in Environmental Planning (1993) from Seoul National University and moved
to France for study in graduate school. She earned a Ph.D. in Environmental Toxicology from University of Paris XI
(Paris-Sud) in 1998 and then carried out her postdoctoral research at the College of Medicine of Seoul National University
from 1999 to 2001. She serves as a professor of the School of Environmental Engineering at the University of Seoul from 2002.
Her laboratory studies the mechanism of eco- and human toxicity of various environmental contaminants, including
nanomaterials, using systems toxicology approaches.
Olga Tsyusko is Assistant Research Professor at the Department of Plant and Soil Sciences at the University of Kentucky. She
received her B.Sc. in Biology from Uzhgorod National University in Ukraine and her Ph.D. in Toxicology at the University of
Georgia in the United States. Her postdoctoral training was completed at the Savannah River Ecology Laboratory where she
later worked as Molecular Biologist. The focus of her research is on environmental toxicogenomics, examining effects and
toxicity mechanisms of engineered nanomaterials in soil invertebrates and plants. She is a member of the Center for
Environmental Implications of NanoTechnology.
Jason M. Unrine is Assistant Professor in the Department of Plant and Soil Sciences at the University of Kentucky. Prior to this
he served as a research scientist at the University of Georgia Savannah River Ecology Laboratory where he also undertook his
doctoral and postdoctoral training in toxicology and environmental analytical chemistry. He earned his B.Sc. in Biology from
Antioch College. His research focuses on understanding the fate, transport, bioavailability and adverse ecological effects of
trace-elements and metal-based manufactured nanomaterials. He is a member of the steering committee of the Center for
Environmental Implications of NanoTechnology (CEINT).
Dr Chatterjee received her B.Sc. (2001) and M.Sc. (2003) from University of Calcutta and moved to China to peruse her Ph.D.
with the fellowship of India Government and Chinese scholarship council. She received her Ph.D. in Environmental Science
(Environmental Toxicology) from China University of Geosciences, Wuhan, in 2009. Currently, she is a postdoctoral research
fellow in Dr Choi’s lab at the University of Seoul. She is engaged in the study of mechanisms of comparative (human and
C. elegans) toxicity of environmental contaminants, specifically nanomaterials.
Ms J.-M. Ahn received her B.Sc. (2010) from University of Incheon and her M.Sc. (2013) from University of Seoul. For her
M.Sc. she studied toxicity mechanisms of various nanomaterials in C. elegans. Since 2013, she has worked at the Risk
Assessment Division in the Korean National Institute of Environmental Research.
Xinyu Yang received her Bachelors degree in Environmental Engineering from Shanghai Jiaotong University in July 2007, and
then got her Master’s degree in Zoology with Jim Oris from Miami University in July 2009. She received her Ph.D. in
Environmental Toxicology from Duke University in 2014. Most of her Ph.D. work was focussed on the mechanistic toxicology
of silver nanoparticles both in laboratory and environmental settings. She has published nine peer-reviewed journal articles in
the field of environmental studies. With strong passion to apply her expertise in industrial settings, she currently joined Nalco-
Ecolab as a regulatory specialist in Naperville, IL.
Lila Thornton graduated from Duke University in 2013 with Bachelor degrees in Biology and Environmental Science. She is
currently an independent contractor for the US Environmental Protection Agency as part of the Chemical Safety for
Sustainability National Research Program. Ms Thornton plans on pursuing a higher degree in the field of toxicology.
Ian Ryde received his Bachelor of Science in Biology from Bowling Green State University in Ohio in 2002 and then moved to
the Raleigh–Durham area and started work in Dr Ted Slotkin’s lab at Duke University in 2005. After 5 years in the Slotkin Lab,
Ian moved on to Dr Joel Meyer’s laboratory at Duke, where he has been for over 4 years now, working as a Laboratory Analyst
II. He started working with the nematode C. elegans and on projects involving mitochondrial DNA damage and its effects on
things such as mtDNA copy number, mRNA expression and neurodegeneration.
Daniel Starnes is a Ph.D. candidate in Integrated Plant and Soil Sciences within the Department of Plant and Soil Sciences at
the University of Kentucky. He received his B.Sc. in Agriculture (2006) and M.Sc. in Biology (2009) from Western Kentucky
University, where his research focussed on Environmental Phytoremediation and Phyto-Nanotechnology. His current
research focuses on the environmental implications of manufactured nanoparticles on terrestrial ecosystems, specifically
soil invertebrates.
J. Choi et al.
B

variably stable coatings, impurities and, in some cases, dissolu-
tion.
[3]
Some of these are well studied in other fields (e.g. colloid
science), but are not familiar to most toxicologists. Finally,
nanotoxicological studies are complicated by some of the
same factors that remain challenging in the fields of human
health toxicology and ecotoxicology, including environmental
variables (temperature, sunlight, presence or absence of
other organisms, medium constitution including pH, salts,
natural organic matter, sediments, etc.) and the potential for
co-exposure to other stressors. Toxicologists must consider
effects not just of pristine NPs, but also of environmentally
modified NPs.
Nonetheless, some key toxicological concepts can still be
employed, and may in fact be of more rather than less impor-
tance in the context of NPs. In particular, we are increasingly
convinced that a fuller appreciation of the importance of the
ADME (absorption, distribution, metabolism, excretion) para-
digm for organismal toxicity
[4]
will be critical to a realistic
evaluation of NP toxicity. We argue that because of their size,
compared to chemicals, even the smallest NPs face very
significant barriers to uptake in most living test systems, with
the barriers being least significant for cells in culture because
they are protected only by a cell membrane. All organisms have
significant barriers to the environment: cell walls in the case of
microbes, and cuticles, exoskeletons, epidermal layers, scales
and so forth in the case of metazoans. However, even when NPs
cannot pass through most of the organismal barriers, they can
still bioassociate with the membranes and may cause toxicity
due to such contact. Although cell membranes may have pores
large enough to permit passage of smaller NPs, this is not
generally true of the portions of free-living organisms that are
in contact with the environment, with important exceptions such
as gills, lungs, sensory organs, mucous membranes and gastro-
intestinal cells. Similarly, endocytosis results in ready uptake of
NPs by cells in culture, but not across most epidermal barriers.
Nonetheless, some studies have demonstrated penetration by
NPs of some epidermal barriers, for instance through hair
follicles and sweat glands.
[5]
The integrity of the skin barrier
will also influence the uptake of NPs. The fact that life has
evolved with constant exposure to naturally occurring NPs
[6]
further suggests that many organisms may have developed
mechanisms to avoid or to adapt to the uptake of nanosized
particles, although of course these putative defences may fail,
depending on the exposure, and on the fact that the elemental
content of the core and coatings of manufactured NPs differ
from naturally occurring NPs. As a result, extrapolating data
from cells in culture to an in vivo context is even more
challenging than it is in traditional (chemical) toxicology, and
careful analysis of uptake is even more important for NPs than
for chemicals that may cross many biological barriers.
Extrapolation across biological levels and between models is
also problematic in nanotoxicology. In vitro toxicity experi-
ments are often conducted using only a few cell types. This
approach does not take into consideration variability in
sensitivity among different cell types and would also be unpre-
dictive of emergent organismal responses (e.g. reproduction,
behaviour). Results from in vitro studies are also not really
applicable for ecotoxicological studies, where endpoints that are
relevant to the population level responses (e.g. reproduction)
should be selected. Thus, although in vitro (cell culture) experi-
ments offer some strengths, it is critical to complement such
work with nanotoxicological studies performed using whole
organisms. Use of organisms with short generation times facil-
itates the ability to screen the effects of combinations of
interactions between physicochemical properties inherent to
the NPs and external environmental factors that are extrinsic
to NP properties. For mechanistic studies, it is helpful to use well
characterised organisms such as Caenorhabditis elegans, for
which plentiful genomic information and functional genomic
tools (mutant and transgenic strains and RNA interference
(RNAi)) are available. Caenorhabditis elegans can also serve
as a model for medium–high throughput screening (MTS-HTS)
of NP toxicity (e.g. for mortality, growth and reproduction
endpoints), as successfully shown previously with other tox-
icants.
[7,8]
In order to keep up with the rapid pace of innovation
in nanotechnology, regulatory toxicity testing regimes will
likely need at some point to also rely on MTS-HTS
approaches.
[9]
In the context of these challenges, we propose
that the nematode C. elegans is particularly suited to the study of
nanotoxicology.
Advantages of C. elegans in nanotoxicological studies
Caenorhabditis elegans is a free-living, transparent nematode
,1 mm in length with a life cycle of ,3 days and an average life
span of 2–3 weeks.
[10]
It was the first multicellular organism to
have its genome completely sequenced,
[11]
and thus became one
of the most important model organisms in various biological
fields. Important biological phenomena such as apoptosis and
RNAi
[12,13]
were first discovered in C.elegans. Its natural hab-
itat and population biology, however, are less understood.
Recent work has shown that C. elegans is often found in
decaying plant material in nature
[14]
rather than being princi-
pally a soil nematode as frequently stated in earlier literature.
Caenorhabditis elegans develops through four larval stages
(i.e. L1–L4) before reaching the adult stage. ‘Dauer’ larvae are a
stress resistant larval stage that develops in place of the 3rd
larval stage under conditions of crowding, food depletion and
high temperature.
[15]
After several decades of rapid growth and success as a model
organism in the fields of genetics and developmental biology,
the use of C. elegans in toxicology has increased greatly in
recent years.
[16–22]
In Table 1 we describe advantages and
disadvantages of C. elegans in nanotoxicity studies, with com-
parisons to other important model organisms. Several attributes
that make it particularly useful for toxicology are the short
reproductive life cycle, large number of offspring and ease
of maintenance, all of which make feasible systematic
Dr Meyer received his B.Sc. from Juniata College in 1992, and then moved to Guatemala where he worked in a number of fields
including appropriate technology and high school teaching. He earned a Ph.D. in Environmental Toxicology from Duke
University in 2003, carried out postdoctoral research with Dr Bennett Van Houten at NIEHS from 2003 to 2006, and joined the
Nicholas School of the Environment at Duke University in 2007. His laboratory studies the effects of stressors on health, in
particular studying the mechanisms by which environmental agents cause DNA damage and mitochondrial toxicity and the
genetic differences that may alter sensitivity.
Nanoparticle toxicity to C. elegans
C

investigations that may yield information permitting prediction
of NP toxicity. Caenorhabidits elegans can be cultured either on
solid or in liquid media, using either highly controlled media or
more natural, complex media, such as soils
[23,24]
or sedi-
ments.
[25]
Although C. elegans is not principally found in soil,
it can nonetheless be conveniently cultured in soil and exposed
to environmental stressors such as NPs. Full life cycle effects
including developmental and reproductive toxicity can be stud-
ied in a short period of time, and developmental nanotoxicity
may be particularly well suited for analysis in C. elegans
because of the normally invariant, and fully mapped, pattern
of cell division that occurs in all somatic tissues. The ability to
extrapolate results from C. elegans to ecotoxicology is improved
by the ability to manipulate environmental variables including
medium chemistry, temperature, pH, oxygen tension, etc.
Caenorhabditis elegans is able to tolerate a wide range of
environmental conditions, permitting analysis of the effects of
environmental variables including temperature and chemical
composition and pH of the medium.
[26,27]
Caenorhabditis ele-
gans also offers the ability to relate mechanistic insights to
human health, because of the high degree of molecular conser-
vation and outstanding molecular, genetic and genomic tools.
[12]
We highlight two particular strengths in the context of
nanotoxicology. First, C. elegans permits the study of organis-
mal uptake of NPs, and their distribution in whole organisms,
because of its small size and transparency (Fig. 1). This allows
efficient yet careful analysis of digestive tract absorption and
subsequent distribution (there is currently no evidence for cross-
cuticle uptake). For instance, as shown in Fig. 1, the distribution
of Au in nematodes exposed to Au NPs can be observed using
X-ray fluorescence microscopy; further, the composition of Au
was confirmed as elemental Au by X-ray absorption near-edge
spectroscopy (mXANES) indicating that the Au NPs were being
taken up by the nematodes as intact particles.
[28]
Second,
Table 1. Advantages and disadvantages of C. elegans for nanotoxicity studies in comparison with other animal model organisms
MTS-HTS, medium–high throughput screening; NP, nanoparticle
Organisms Advantages Disadvantages
C. elegans (i) Permits medium- to high-throughput toxicity experiments
(MTS-HTS) and long-term multi-generational studies because
of short generation time, ease of culturing, small size and large
brood sizes
(i) poor system for toxicity detection in some organ systems,
e.g. pulmonary
(ii) Allows performance of aquatic and soil nanotoxicity
experiments because of its ability to survive in both media; can
live in media with low ionic strength, which is required when
testing some NPs
(ii) less ecological relevance
(iii) can provide guidance for ecotoxicity and human health
studies with NPs because of high degree of molecular
conservation
(ii) small size limits individual NP uptake and elimination
studies
(iv) offers most of the genetic power of single-celled systems in
the context of the biological complexity of a multiple well
developed organ systems
(v) provides high mechanistic power because of well established
functional genomic tools (transgenic and mutant strains, RNAi)
(vi) RNAi is achieved easily through feeding
Daphnia spp. (i) suitable for rapid toxicity screening using mortality,
reproduction and also for multigenerational experiments
because of short life cycle and high number of offspring
(i) no functional genomic tools are available, limiting testing
of mechanistic hypotheses
(ii) toxicogenomic approaches available because of recently
sequenced Daphnia pulex genome
(iii) high ecological relevance thus suitable for bioaccumulation
and transfer of NPs in food chain studies
Earthworms (i) highly relevant for soil exposure (i) functional genomic tools are not available
(ii) larger size permits easier detection and analysis
of internal NP distribution
(ii) mortality and reproduction toxicity experiments takes
more than 10 times longer than in C. elegans, thus, not suitable
for HTS
(iii) better model for NP uptake and elimination assays
Zebrafish and
Japanese
medaka
(i) excellent model for developmental toxicity assays (i) not as easy and cheap to maintain as C. elegans
(ii) ecotoxicological relevance of chorion, the barrier
of embryos, for NP uptake studies.
(ii) nanotoxicity experiments are mainly performed on embryos
because of larger adult sizes
(iii) may need to remove chorion because of its impermeability
to NPs
(iv) limited functional genomic tools
Mammalian
models
(i) rich literature and database of biological information (i) do not reflect current trend of reducing animal use in toxicity
studies
(ii) high throughput screening power for in vitro models (ii) limitations when extrapolating results between in vitro and
in vivo models
(iii) most realistic in vivo models for estimating risk
and effects of NP exposure to humans
(iii) very high cost associated with maintenance and use of
in vivo mammalian models
(iv) limited sample sizes
(v) limited functional genomic tools
J. Choi et al.
D

C. elegans offers much of the genetic power of single-celled
systems such as yeast or microbes in the context of the biological
complexity of a metazoan with multiple well developed organ
systems. The availability of two RNAi libraries and two mutant
consortia that respectively cover ,90 and .30 % (and growing,
in each case) of the genome permits a very powerful approach to
mechanistic toxicity research.
[29]
For example, as described in
more detail below, there is controversy regarding the role of
oxidative stress in the toxicity of many NPs. Traditional
approaches to testing for a role of oxidative stress, although
readily employed in C. elegans, have significant shortcomings.
Most oxidative stress-response genes lack specificity because
they can be up-regulated by stressors other than oxidative stress;
conversely, oxidative stress can up-regulate other global stress-
responsive genes (e.g. p53 target genes).
[30]
Markers of oxida-
tive damage are more reliable, but it can be challenging to
determine whether the toxicity is the result of direct or indirect
oxidative stress (i.e. whether the toxicity is caused by oxidative
stress, or whether dysfunction results in oxidative damage).
Pharmacological rescue experiments using chemical agents (to
illustrate such a ‘rescue’ experiment: if co-exposure to an
antioxidant such as vitamin E protects against toxicity, this
supports the hypothesis that the mechanism of toxicity is
oxidative stress) frequently lack specificity because the agents
used typically have many effects, and the compounds used in
these experiments can affect the properties of the NPs. Genetic
approaches utilising RNAi simply through feeding,
[31]
trans-
genic (such as reporter green fluorescent protein, GFP) and
mutant strains are a powerful complement to these traditional
approaches (for protocols and description see http://www.
workbook.org, accessed February 2014). For instance, if toxicity
is exacerbated in vivo in the context of knockdown or knockout
(using RNAi or a mutant strain) of a gene involved in a particular
defensive pathway (e.g. an antioxidant protein), this strongly
suggests that the exposure is causing toxicity by the associated
stressor (e.g. oxidative stress). This approach has been termed
‘functional toxicogenomics’
[32]
and has been successfully used
to study and identify toxicity mechanisms of metals and complex
environmental mixtures.
[33–36]
It has also been applied to nano-
toxicity studies.
[37–39]
In those studies, NP-induced genes and
pathways were selected based on toxicogenomics, and their
physiological importance was investigated by observing organ-
ism level endpoints such as survival, growth or reproduction in
wild-type nematodes compared to nematodes lacking specific
protein functions due to mutations or RNAi knockdown.
Finally, we note that C. elegans studies, like research with
other species, will always require complementary investigations
in other systems; no single model organism is sufficient
(Table 1). Physiological differences between C. elegans and
other organisms are important; for example, C. elegans lacks
lungs, and may therefore be a poor model for high aspect ratio
nanomaterials (i.e. NPs with a very high height-to-width ratio)
such as carbon nanotubes that might exhibit asbestos-like
pulmonary toxicity.
[40]
Earthworms (e.g. Eisenia fetida) may
be more suitable for some of the NP uptake and elimination
studies because of their larger size, which allows work with
0.35
0.30
0.25
0.20
0.15
0.10
0.05
00 0.2 0.4
2000 4000 6000 8000 10 000
X distance (mm)
All marked groups
Normalised x µ(E) Distance Y (mm)
0.6 0.8 1.0
11 900
0
0.5 HAuCl4
Gold foil
Nematode
1
11 920
E (eV)
11 940
(a)
(b)
Fig. 1. Synchrotron X-ray fluorescence microprobe (mXRF) map of (a) Au at Au
La
in C. elegans exposed for 6 h
to 20 mg L
1
of 4-nm citrate-coated Au in 50 % K-Medium. (b) Speciation for a pixel from the area of high Au
abundance was determined with X-ray absorption near-edge spectroscopy (mXANES) as metallic Au
0
(adopted
from Unrine et al.
[28]
)(E, energy).
Nanoparticle toxicity to C. elegans
E

individual worms.
[41,42]
However, toxicity and reproduction
studies can be performed much faster with C. elegans. Also,
because of limited genomic information for organisms such as
E. fetida,C. elegans remains preferable for mechanistic NP
studies (i.e. those toxicology studies that attempt to identify not
just a toxic effect, but the mechanism by which such toxicity
occurs). Finally, despite a generally high conservation of sig-
nalling pathways, molecular and biochemical differences may
limit some extrapolations. For example, C. elegans has phyto-
chelatins that complement the function of its metallothionein
proteins, lack the CYP1 family cytochrome P450 enzymes and
possess an aryl hydrocarbon receptor homologue that lacks
binding affinity for typical xenobiotic ligands of mammalian
aryl hydrocarbon receptors.
[43–45]
In summary, C. elegans offers
a bridge between very high-throughput systems (e.g. cell cul-
ture) that are hampered by low physiological and environmental
relevance, and more physiologically complex organisms that
offer more relevance to human and wildlife health, but less
mechanistic power and lower throughput.
Here we review the state of the evidence based on nanotoxi-
city studies in C. elegans focussing on the following aspects:
– Factors influencing nanotoxicity in C. elegans
– Potential mechanism of NP uptake
– Potential mechanisms of NP toxicity in C. elegans
– Comparision of C. elegans with other model organisms in
nanotoxicity studies
What are the factors that influence nanotoxicity?
We reviewed currently available published nano(eco)toxico-
logical studies involving C. elegans (Table 2). Based on this
information, it is possible to tentatively identify NPs that are
highly toxic, harmful, non-toxic and even therapeutic in this
organism. Among the NPs listed in Table 2, platinum NPs, for
example, are tentatively defined as potentially therapeutic based
on evidence of their antioxidant properties.
[46]
Silver NPs, in
contrast, would rank as the most toxic NPs to C. elegans,
because mortality, inhibition of growth and reproduction have
been observed at much lower concentrations compared to the
other NPs so far tested. This is also true for Ag NPs in other
tested model organisms, including bacteria, algae, crustaceans,
ciliates, fish and yeast.
[47]
Current literature suggests that the
physicochemical attributes of NPs as well as various exposure
conditions are critical parameters in determining the degree of
nanotoxicity in C. elegans.
Physicochemical properties of NPs
Coatings
Coatings can significantly alter NP effects, frequently miti-
gating toxicity. For instance, we found that uncoated Ag NPs
caused higher mortality (,10-fold) in C. elegans than poly-
vinylpyrrolidone (PVP)-coated Ag NPs.
[48]
Citrate-, PVP- and
gum arabic (GA)-coated Ag NPs of very similar size ranges had
dramatically different growth-inhibition effects, with the GA
Ag NPs being nine times more toxic (based on growth inhibi-
tion) than PVP Ag NPs, whereas PVP Ag NPs were three times
more toxic than citrate Ag NPs, apparently due in part to
differences in dissolution.
[49]
In another comparative study on
stability of citrate-, PVP- and polyethylene glycol (PEG)-coated
Ag NPs in OECD standard media, PVP Ag NPs were the most
stable in terms of concentration, shape, aggregation and
dissolution
[50]
. In some cases, however, the toxicity of NPs
with different coatings cannot be explained by dissolution
alone, as we observed in C. elegans based on differences
in transcriptomic responses between citrate and PVP-coated
Ag NPs.
[51]
Size
Although there is evidence from many studies that particle
size and surface area can be important determinants of the
toxicity of NPs,
[52–54]
many in vitro studies have failed to show
any clear relationship between cytotoxicity and NP size.
[55–57]
In C. elegans, however, there is some evidence for size-
dependent toxicity. When nematodes were exposed to the same
concentration of different sizes of CeO
2
NPs (15 and 45 nm) and
TiO
2
NPs (7 and 20 nm), the smaller NPs were more toxic based
on survival, growth and reproduction in both cases.
[58]
It was
also found that when comparing the toxicity of PVP Ag NPs
with different sizes (i.e. 8 and 40 nm), smaller particles caused a
higher level of accumulation of 8-OHdG, an oxidised DNA
base, than did larger particles.
[48]
Thus, size seems to be an
important variable in toxicity of NPs to C. elegans, and a smaller
size typically results in greater uptake and thus toxicity. How-
ever, this is not universal for all Ag NPs in C. elegans,
[49]
and
there is evidence that size-dependent differences in toxicity of
NPs in general are typically observed only when the primary
particle size is smaller than 10–20 nm.
[59]
Release of metals
Many NPs can release metals by dissolution before, during
and after their uptake in tissues (see Fig. 2). Different metal ions
have varied and well studied mechanisms of toxicity.
[60]
Although a full discussion of those mechanisms is beyond the
scope of this review, some progress has been made in under-
standing the extent to which dissolution as such drives the
toxicity of specific nanomaterials in C. elegans. Qu et al.
[61]
found that release of toxic metals from quantum dots (QDs) was
important in QD toxicity (reproduction) in C. elegans, and the
use of metal-chelating deficient C. elegans strains as well as
pharmacological chelators demonstrated that Ag NPs were toxic
in part by Ag dissolution.
[49,62,63]
These approaches, however,
do not clarify whether dissolution occurred internally or exter-
nally, and if the dissolution is internal, where it occurs. Further
research progress on mechanisms of NP uptake will help inform
our understanding of target tissues; it will also be critical to
understand subcellular distribution. Ag NPs, for example, are
likely to dissolve much better in the acidic environment of
lysosomes than in most typical exposure medium conditions.
[64]
Other physicochemical factors
Other NP properties that may be important for toxicity, such
as shape and charge, influence uptake, toxicity or both in other
organisms and in in vitro studies.
[65,66]
We are aware of one
study describing how coatings with different surface charge
(positively, negatively and neutral) of CeO
2
NPs affected their
bioavailability and mortality in C. elegans.
[67]
In that case,
positively charged CeO
2
NPs showed the highest toxicity and
bioavailability. This result indicates that future similar studies
examining the interactions between the NP charge and toxicity
are warranted.
Exposure conditions
Exposure medium
One of the advantages of using C. elegans in toxicity testing
is that both solid and liquid media can be easily used, which is
particularly useful in the ecotoxicological context. The effect of
J. Choi et al.
F

Table 2. Published nanotoxicity studies in C. elegans
Nanomaterials (size) C. elegans strains
and life stage of
the exposure
Exposure media Endpoints References
CeO
2
(8.5 nm) N2 Nematode growth agar medium (NGM) and CeO
2
mixed
with broth
Life span, lipofuscin accumulation, oxidative stress (reactive
oxygen species (ROS) fluorescence)
[113]
L1 larva
CeO
2
(nanostructured and
amorphous materials, TX)
N2, mtl-2,pcs-1,sod-3 Moderately hard reconstituted water (MHRW) Growth
[159]
CeO
2
(4-nm core size) with positively
charged (diethylaminoethyl dextran,
DEAE), negatively charged
(carboxymethyl dextran, CM) and
neutral (dextran, DEX) coatings
N2 MHRW alone and with humic acid Mortality, bioavailability
[67]
L1 and L3
Nano-ZnO (60 25 nm) N2 K-medium Lethality, lipid peroxidation
[83]
Phototoxicity
ZnO (1.5 nm) N2 and mtl-2::gfp K-medium (acetate buffered or unbuffered) Lethality, Reproduction, locomotion (behaviour), mtl-2::gfp
expression
[63]
ZnO (20 nm), Al
2
O
3
(60 nm),
TiO
2
(50 nm)
N2 Ultrapure water Lethality, growth, reproduction
[79]
L1
Al
2
O
3
nanoparticle (NP) (60 nm) N2 NGM Intestinal auto-fluorescence, ROS production; stress response
measured by heat shock protein expression
[125]
phsp-16.2::gfp
L1, L4, young adult)
Al
2
O
3
NP (60 nm) N2,sod-3,sod-2,
Phsp-16.2::gfp and
Psod-3-sod-3
NGM (exposure solutions were added to NGM plates) Locomotion behaviour with head thrash and body bend; imaging
of glutamatergic, serotoninergic and dopaminergic systems;
stress response (heat-shock) and oxidative stress response
(ROS formation, superoxide dismutase (SOD) activity)
[124]
Al
2
O
3
NP (60 nm) N2 and phsp-16.2::gfp Ultrapure water Lethality, lipofuscin accumulation, stress response measured by
heat shock protein expression
[160]
L1, L4 and young adult
Dimercaptosuccinic acid (DMSA)-
coated Fe
2
O
3
NP (9 nm)
N2, sod-2 and sod-3 K-medium Lethality, development, reproduction, locomotion behaviour,
pharyngeal pumping, defecation, intestinal autofluorescence
and ROS production
[161]
L1 and L4
CeO
2
NP (15 and 45 nm), TiO
2
NP
(7 and 20 nm)
N2 (Young adult) K-medium Lethality, growth, reproduction, stress response gene expression
[58]
Fluorescent nanodiamond (FND)
(120 nm)
N2, daf-16::gfp,gcs-1::gfp NGM (feeding) Brood size, life span, ROS production; stress response assay
by gfp expression,
[114]
L4 Microinjection of FND into gonads of the worm NP uptake
N2, daf-16::gfp,gcs-1::gfp
Uncoated Ag NP (,100 nm) N2, sod-3,daf-12,mtl-2 K-medium Uptake, global changes in gene expression determined with
microarrays, lethality, growth, reproduction
[38]
Young adult
Uncoated Ag NP (,100 nm) N2, pmk-1 Young adult K-medium ROS formation,
[109]
Gene and protein expression
Oxidative stress signaling
Uncoated Ag NP (,100 nm) N2, pmk-1,hif-1,egl-9,
vhl-1,fmo-2 cyp35a2
MHRW Survival, ROS formation,
[37]
Young adult Gene and protein expression
Ag NP (10 nm) N2 Mixed in a ratio of 2 : 3 : 5 of milk to C. elegans habitation
reagent (CeHR) to treatment solution in water
(only water for control)
NP uptake, localisation, larval growth, morphology
and oxidative DNA damage (8-OH guanine level)
[162]
L1, L2 larvae
(Continued )
Nanoparticle toxicity to C. elegans
G

Table 2. (Continued)
Nanomaterials (size) C. elegans strains
and life stage of
the exposure
Exposure media Endpoints References
Uncoated Ag NP (3 and 8 nm)
from Amepox
N2 Simulated soil pore H
2
O with and without fulvic acid,
M9 buffer
Reproduction
[26]
Adult
Ag NP (citrate (7 11, 21 17 nm)
and polyvinylpyrrolidone (PVP)
(75 21 nm))
N2, nth-1,sod-2,mtl-2,
xpa-1,mev-1
K-medium Uptake, growth inhibition (toxicity), mechanism of toxicity
by using different mutant models
[62]
All developmental stages
started from L1
Ag NP (1 and 28nm), coated
with PVP
pha-1 K-medium containing cholesterol Survival with and without presence of Escherichia coli as
food source
[81]
Ag NP citrate (50.6 nm) from
ABC Nanotech
N2 NGM with NP suspensions Mortality, reproduction and biological surface interaction with
Ag NPs by scanning electron microscopy
[68]
Platinum NP (2.4 0.7 nm) N2 S-medium Life span, lipofuscin accumulation, ROS, internalisation
[46]
L4
Platinum NP (2.4 0.7 nm) N2 and mev-1 S-medium Lethality, life span, lipofuscin, ROS
[155]
L4
Silica NP fluorescent labelled)
(50 nm)
N2 NP containing NGM plates Translocation, life span, lipofuscin accumulation (4th and 10th
day), reproduction (progeny production, vulva development
defect)
[152]
Polystyrene NPs PS-YG, PS-YO,
carboxy
L4 and young adult
Fluorescent polystyrene
nanoparticles (100 nm)
N2 Microinjected into the syncytial gonads of gravid hermaphrodite,
recovery buffer (15min) and then in M9 buffer (1hr)
Particle-tracking intracellular microrheology
[163]
NaYF4:Yb,Tm NCs with strong
NIR UC emission
(28, 42 and 86 nm)
N2, elt-2::gfp,lag2::gfp Colloid solution was dropped onto NGM agar plates Life span
[115]
(used for bioimaging) L4 and young adult Egg production, viability and growth rate assays,
gfp expression assay
CdSe, ZnS core shell quantum dots
(QDs) and CdTe QDs (10 nm)
N2 and mtl-2:: gfp
L1- arrested and adult
worms
NGM plates with or without QDs Life span, brood size, larval developmental assay; imaging
[61]
(used in bioimaging) M9 buffer treatment was used as negative control
Graphite nano-platelets (GNPs)
(mean lateral size of the flakes
is 350 nm and the thickness
measured through scanning
electron microscopy is ,50 nm,
with several graphene layers
varying between 3 and 60
N2 GNPs suspended in distilled water, Pseudomonas aeruginosa
was used as food source
Longevity and reproductive capability
[164]
Mercaptosuccinic acid (MSA)-
capped QDs (QDs-MSA)
N2 K-medium and H
2
O Growth (larvae to adults) and reproduction egg-laying defects
and response to anticonvulsant ethosuximide and to
neurotransmitter serotonin
[165]
L3 and L4
Quantum dots, core (CdSe)
(3.4-nm core diameter) and
core–shell (CdSe, ZnS)
(4.1-nm core diameter);
hydrodynamic diameter of both
QDs are ,17 nm
N2 QDs in borate buer added to NGM agar plate with food (E. coli) Lifespan and fertility, body length and locomotion
[166]
L4
J. Choi et al.
H

TiO
2
NPs (5 1nm (4nm),
10 1 nm (10 nm), 60 9nm
(60 nm)
N2 K-medium Lethality, growth and locomotion behaviour, (ROS) production
and N-acetylcysteine (NAC) treatment rescue, intestinal auto-
fluorescence
[167]
Young adult
TiO2 NP (,25 and ,100 nm) N2 Ultrapure water Mortality (24 h without food)
[78]
ZnO NP(,25 and ,100 nm L4
TiO
2
NPs, ZnO NPs and SiO
2
NPs
with the same nanosize (30 nm)
N2 K-medium Lethality, growth, reproduction and locomotion behaviour,
(ROS) production and NAC treatment rescue
[168]
L1
TiO
2
NPs (10 nm) N2 and mtl-1,mtl-2,sod-1,
sod-2,sod-3,sod-4,sod-5,
mev-1,aak-2,xpa-1,pcm-1,
hsp-16.48,hsp-16.2,gst-4,
gst-8,gst-24,gst-5,gst-42
and isp-1
K-medium Lethality, growth, reproduction and locomotion behaviour,
Intestinal auto-fluorescence, ROS production and gene
expression
[158]
Young adult
TiO
2
NPs (10 2 nm) N2 and Punc-47::gfp,lin-15 K-medium Uptake, growth, locomotion behaviour, pharyngeal pumping and
defecation, intestinal auto-fluorescence, ROS production,
analysis of triglyceride content, TEM imaging for translocation
[169]
Young adult and L1 larvae
TiO
2
NPs (4 and 10 nm) N2, Psod-2-sod-2,Psod-3-
sod-3 and Pdpy-30-sod-2
K-medium in the presence of food lethality, growth, reproduction, locomotion behaviour, intestinal
autofluorescence and ROS production, gene expression (clk-1,
clk-2,ctl-1,ctl-2,ctl-3,gas-1,isp-1,mev-1,sod-1,sod-2,sod-3,
sod-4,sod-5).
[170]
L1 and young adults
Au NPs N2 and chc-1,dyn-1,rme-2
and pqn-5
K-medium Mortality, global changes in gene expression determined from
microarrays; mortality of mutants for endocytosis and UPR
pathways
[39]
L3 and Adult
Hydroxylated fullerene, [fullerol,
C
60
(OH)
19–24
] (4.7 and 40.1 nm)
ced-3, ced-4 and elt-2::gfp NGM plates with or without fullerene, mixed with OP50
cell pellet
Lifespan, reproduction, body length, defective digestion system
and apoptosis (SYTO 12 staining and ced-3 and ced-4
expression)
[133]
L4 and adults
Pristine single-walled carbon
nanotubes (SWCNTs) (length
0.5–2.0 mm) and amide-modified
SWCNTs (a-SWCNTs) (length
0.7–1.0 mm)
N2 and daf-16:gfp L1 larvae NGM Survival, growth, reproduction, endocytosis, oxygen consump-
tion rate, DAF-16 nuclear translocation assay, genome-wide
gene expression analysis (microarray and quantitative reverse-
transcriptase real-time PCR, qRT–PCR)
[171]
Multi-walled carbon nanotubes
(MWCNTs) and PEGylated
modification in reducing
MWCNTs
N2 K-medium Translocation in targeted organs regulating the toxicity
[172]
Ag NPs Citrate-coated (CTL
7
),
PVP-coated (PVP
8
and PVP
38
)
and gum-arabic (GA)-coated
(GA
5
,GA
22
)
N2, VC433 (sod-3 deletion),
TM1748 (pcs-1 deletion,)
and TK22 (mev-1, mutation
uncertain,)
MHRW Mechanisms of toxicity by rescue with trolox and
N-acetylcysteine, analysis of metal-sensitive and oxidative
stress-sensitive mutants, growth assay
[49]
Pt NPs (2.5 1 nm) N2 (L4 and young adult for
ROS detection)
Electrolysed-reduced water (ERW) medium Life span assay and ROS detection
[157]
Pt NPs N2 and nuo-1 (LB25) (L4) S-medium Lethality, life span, lipofusion ROS measurement, NAD
þ
and NADH assays
[156]
Au NPs surface modification for
microscopic analysis (20 nm)
N2 Polyelectrolyte, NP coated worms were inoculated (100mL)
into NGM plates containing E. Coli
Viability and reproduction
[173]
Au NPs (10 nm) from Sigma Aldrich N2 E. coli was added to LB Broth containing NPs and exposed
E. coli were fed to C. elegans
Multigenerational effects on survival and reproduction over
four generations.
[174]
Nanoparticle toxicity to C. elegans
I

medium composition on the bioavailability of unstable NPs
should be taken into account or tested before designing experi-
ments. For instance, a recent study
[68]
used a suspension of Ag
NPs embedded into solid nematode growth agar medium
(NGM). The mortality of C. elegans in NGM could have been
under-estimated in comparison with liquid medium because
binding of Ag NPs to the agar probably limited their availability
to C. elegans; in addition the particles could have undergone
agar-mediated surface modification. The NP toxicity to
C. elegans can also differ depending on the composition of
liquid testing media. For instance, we and others have observed
dramatically less toxicity after exposure of C. elegans to Ag NPs
and Ag ions (AgNO
3
) in K-medium (a standard medium for
liquid C. elegans culture
[69]
)v. moderately hard reconstituted
water (MHRW, for composition see Cressman and Williams
[70]
(Table 2
[49]
). This is likely a result of the high Cl

concentration
in the K-medium facilitating precipitation of highly insoluble
AgCl, which would reduce or eliminate the toxicity from Ag
ions. The Cl

concentration is relatively low in MHRW (54 mM)
compared to K-medium (32 mM KCl and 51 mM NaCl).
The effect of exposure medium may be less important in the
cases of insoluble NPs. However, even in the case of poorly
soluble NPs, a high ionic strength may cause NP aggregation,
particularly in the presence of polyvalent cations. For instance,
Au NPs are also very insoluble (1.4 10
5
% dissolution after
24-h exposure in 50 % K-medium
[39]
), but full strength
K-medium caused aggregation of Au NPs, necessitating the
use of 50 % strength K-medium for toxicity studies.
[39]
Several toxicity studies have been conducted in C. elegans
exposed to soil or sediment contaminated with metals or organic
contaminants.
[71–73]
However, perhaps because of the complex-
ity of the soil matrix, to our knowledge, there are no published
nanotoxicity studies that have been conducted in soil/sediment
exposure conditions. Studies evaluating the toxicity of NPs to
C. elegans in different natural soils and also focussing on
sensitive endpoints will be required to better understand the
toxicity of NPs in soil media.
Developmental life stage and presence of food
Nematode developmental stage is a major factor influencing
toxicity. In studies published so far that included earlier and later
developmental stages, the first larva stage (L1) was the most
vulnerable to NP exposure; early life stages have generally been
observed to be more sensitive for many contaminants and many
species.
[74–77]
For example, TiO
2
NPs with sizes between 25 and
100 nm in pure water were non-toxic for L4s
[78]
but toxic to
L1s,
[79]
reducing survival, growth and reproduction.
Esherichia coli is typically used as a source of food in
C. elegans toxicity experiments and the presence or lack of
food during exposure significantly affects toxicity, possibly
because of altered bioavailability and the effect that feeding
can have on the physiological state of the nematodes. For
instance, 24-h exposure to Ag NPs and Ag ions resulted in
50 % lethal concentration (LC
50
) values at least 10-fold lower
compared to the experiments performed without feeding.
[80]
However, the reverse results were found in another Ag NP study
where higher C. elegans mortality was observed in fed v. unfed
nematodes exposed for 24–72 h.
[81]
The opposite results in these
two studies may be attributable to the fact that exposures were
initiated at different C. elegans developmental stages (older L3
and young L2 nematodes). Limitation in food resources at C.
elegans early developmental stages (L1 and L2) can result in
developmental arrest of the nematodes at the dauer (resistant)
larval stage, where nematodes do not feed and are characterised
by extended lifespan, lower metabolism, increased fat storage
and high levels of antioxidant enzymes.
[82]
Unfolded protein
response
Activation of various
stress response signaling
pathways
(i.e. PMK-1 P38 MAPK)
Endocytosis
Dissolution Mz⫹
Dissolution
Various response to
NP exposure
Caspase 9
Caspase 3
DNA damage
Stress response
transcription factors
(i.e. HIF-1, SKN-1, etc)
ROS
Protein binding
Mz⫹
Mz⫹
Ca2⫹
Apoptosis
Apoptosis
Fig. 2. Potential mechanisms for nanoparticle (NP) uptake and toxicity in C. elegans (M
þ
are dissolved metallic
ions released from nanoparticles; ROS, reactive oxygen species).
J. Choi et al.
J

Environmental conditions and interactions
Solar irradiation and phototoxicity
Phototoxicity can be an important factor affecting the toxi-
city of NPs. In the environment, NPs may co-occur with the
ultraviolet wavelengths associated with phototoxicity. Recently
several C. elegans toxicity studies with metallic oxide NPs have
considered the effects of phototoxicity. The toxicity (based on
mortality) of ZnO NPs increased significantly with natural
sunlight due to photocatalytic reactive oxygen species (ROS)
generation by ZnO.
[83]
The phototoxicity of ZnO NPs was
greater in the study of Ma et al.
[83]
than for bulk ZnO, even
though the ZnO NP aggregates were similar in size to the bulk
ZnO aggregates (respective average aggregate size of 2.8 and
2.4 mM), demonstrating that aggregation did not quench the
photoreactivity of the particles and that primary particle size (10
v. 55 nm for ZnO NPs and bulk ZnO) rather than aggregate size
dictated toxicity.
[83]
Weathering
Once NPs are released into the environment, they will
undergo modifications due to aging processes involving inter-
actions with various organic and inorganic ligands, oxidation–
reduction reactions and dissolution–precipitation reactions.
A key environmental transformation for Ag NPs, especially in
environmental compartments such as sediments and sewage
sludge that contain reduced sulfur (sulfide), is the strong binding
of Ag to sulfur (sulfidation), which might reduce toxicity of
these sulfidised Ag NPs due to the very low solubility of
Ag
2
S.
[84]
We carried out experiments on the toxicity of Ag
NPs with varying degrees of sulfidation including fully sulfi-
dised particles, and found that sulfidation decreased mortality,
and caused much less inhibition in growth of C. elegans when
compared with those of the pristine Ag NPs.
[85]
Other particles
are also likely to be altered by weathering, and for certain
nanoparticles, weathering can increase their toxicity due to
continued or accelerated release of metal ions, as has been
shown for bacteria exposed to quantum dots as a result of their
weathering under acidic or alkaline conditions.
[86]
Thus, under-
standing these processes will be critical to assessing the risk of
NPs in the environment.
Organic matter
When NPs are released into the environment they will
inevitably interact with natural organic matter (NOM). The
presence of NOM in exposure solutions can significantly
increase or decrease the toxicity of the NPs by several mechan-
isms including coating NP surfaces to reduce or augment
interaction with biological receptors, and altering aggregation,
charge or dissolution properties.
[80]
For example, fulvic acid
significantly decreased the acute toxicity of both of Ag NPs and
Ag ions to C. elegans, in some cases reducing mortality from
100 to 0 %.
[80]
When humic acid (HA) was added to the CeO
2
NPs exposure medium, Collin et al.
[67]
observed a significant
decrease in mortality of C. elegans. The authors also demon-
strated that the decreased bioavailability of CeO
2
NPs depended
on the ratio of CeO
2
NPs to humic acid.
Potential mechanisms of entry for NPs
Uptake of NPs by ingestion and subsequent translocation into
intestinal cells in particular, but also reproductive cells, has been
observed in C. elegans with a variety of NPs (reviewed in Zhao
et al
[20]
). There are several possible mechanisms, identified
from in vitro studies, for how NPs can enter cells including
direct diffusion of NPs across the plasma membrane without
lipid bilayer disruption
[87]
; creation of pores in the mem-
brane
[88]
; endocytosis
[39,65,89–93]
and by G-protein-coupled
receptors.
[94]
Surface chemistry, size, shape and charge of NPs
significantly influence their uptake. For instance, in an in vitro
study with mouse dendritic cells, Au NPs coated with alternating
anionic and hydrophobic groups were able to diffuse through the
cell membrane without disrupting the lipid bilayer, whereas
similar Au NPs that differ from the first ones only by random
distribution of the same groups in the coating were endocytosed
and trapped in endosomes.
[87]
Size dependence during NP
uptake was observed by Chithrani and colleagues
[65,89]
:
depending on the energetics of a single nanoparticle (such as
bending and adhesion energy), its wrapping by a cell membrane
and uptake through endocytosis occurs optimally when NPs are
20–50 nm, whereas smaller particles would reach the optimal
size for endocytosis only by clustering.
[89]
Xenobiotic uptake and distribution in C. elegans are not
well studied. Nonetheless, and although the above-described
mechanisms for NP entry into the cells were inferred from
in vitro studies, we have some evidence that these mechanisms
can also apply for NP uptake into C. elegans tissues and cells
(as shown in Fig. 2). For instance, a study of toxicogenomic
responses in C. elegans exposed to Au NPs identified clathryn-
mediated endocytosis as one of the pathways activated by Au
NPs.
[39]
In that study, two of the endocytosis mutants (chc-1 and
rme-2) were more resistant than wild type nematodes to Au NPs,
thus providing evidence for the functional importance of this
pathway to Au NP uptake and toxicity. In the same study,
electron-dense particles with Au elemental composition were
found only in the animal’s gut lumen and microvilli, where
endocytosis is plausible, and not near the cuticle surface,
suggesting that the Au NPs are likely to be absorbed from the
intestine.
[39]
However, the dermal route of exposure should not
be entirely excluded for NPs. For instance, organically modified
silica NPs were incorporated into cuticle and caused demelani-
sation in Drosophila.
[95]
In C. elegans, the cuticle has an evenly
distributed net negative charge at neutral pH,
[96]
which can
attract positively charged NPs. In addition, Ag NPs can cause
damage to the cuticle of C. elegans,
[68]
although this did not
result in detectably increased uptake.
How do NPs cause toxicity to C. elegans?
The toxicity of NPs may be mediated by multiple mechanisms or
modes of action, depending on the physicochemical properties
of the NPs as well as exposure conditions.
[97,98]
Many mecha-
nistic studies conducted on NPs, mainly using in vitro systems,
have reported that oxidative stress is associated with NP expo-
sure.
[20]
However, the evidence for oxidative stress as a mech-
anism of toxicity in C. elegans exposed to various NPs is
contradictory. Furthermore, it is unclear whether the lack of
information on other mechanisms of toxicity is because of
negative results that have been obtained, or from the fact that
many researchers investigating toxicity of many NPs with
C. elegans and other organisms have limited their mechanistic
investigations to oxidative stress-related endpoints. Among
other possible mechanisms of NP toxicity described in pub-
lished studies are endoplasmic reticulum (ER) stress and protein
toxicity resulting in an unfolded protein response (UPR), which
was observed in C. elegans after Au NP exposure.
[56]
Below we
review studies that provide evidence for oxidative stress and
other mechanisms of NP toxicity (Fig. 2). Overall, although
Nanoparticle toxicity to C. elegans
K

some mechanisms shown in Fig. 2 are specific to C. elegans,
many represent pathways of NP–cell interactions that have been
determined for other organisms, including daphnid, zebrafish,
medaka, earthworms and mammalian cells.
[55,99–103]
Oxidative stress
In theory, NPs can induce oxidative stress, characterised by a
pathologically high production of oxidants,
[104]
either directly
by their intrinsic ability to generate ROS or indirectly by their
interactions with biological systems. ROS can be generated by
NPs as a result of the presence of transition metal impurities, the
ability to release toxic metal ions, the presence of electronically
active surface or photoactivation.
[9,105,106]
Direct generation of
ROS by NPs may also result from exposure to an acidic envi-
ronment, such as the intestine or lysosomes, either from the
surface of the NPs or from leached ions.
[107,108]
Although there are multiple studies with C. elegans provid-
ing support for oxidative stress as the mechanism of NP toxi-
city
[38,49,83,109]
(Table S1, Supplementary material), not all
C. elegans studies support this conclusion. Whether the effects
will be pro-oxidant or antioxidant may also depend on the
concentrations used. For instance, CeO
2
NPs have been reported
to have pro-oxidant effects at higher concentrations,
[110–112]
whereas at lower concentrations CeO
2
NPs have exhibited ROS
scavenging and superoxide dismutase (SOD) mimetic activi-
ty.
[113]
Antioxidant activity has been also demonstrated for Pt
NP compounds.
[46]
Other studies detected no ROS generation
after C. elegans exposure to ZnO, Al
2
O
3
, or TiO
2
NPs,
[79]
perhaps because the exposures were performed in the dark.
Nanodiamond particles did not significantly affect the ROS
level in either germline or somatic cells of C. elegans, nor did
they cause detectable changes in either brood size or longevity
of the nematodes.
[114,115]
Meyer et al.
[62]
reported that toxicity
caused by several types of Ag NPs could not be attributed to
oxidative stress in C. elegans because oxidative stress-sensitive
mutants were not more sensitive to the toxicity of the tested NPs,
and Yang et al.
[49]
found that oxidative stress was one mecha-
nism of toxicity, but was less important than dissolution result-
ing in metal ion toxicity, for a variety of Ag NPs.
Biological interactions of NPs could also contribute to ROS
production. It is possible, therefore, that NPs devoid of intrinsic
ROS generation capacity can also give rise to ROS generation
by interaction with sub-organelles and biological systems. NPs
can directly interact with organelles such as the mitochondria by
destabilising the outer membrane, altering the mitochondrial
membrane potential and disrupting the electron transport chain
and oxidative phosphorylation,
[116]
which may increase pro-
duction of ROS
[117,118]
(Fig. 2). NPs can also cause activation
of nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase in cells of the immune system, such as macro-
phages and neutrophils, resulting in production of superoxide
anions.
[116,119,120]
The interaction of NPs with cell surface
receptors might lead to receptor activation and triggering of
intracellular signalling cascades, such as MAPK, finally result-
ing in altered expression of stress response genes that may affect
ROS production or quenching (Fig. 2). Accumulation of high
intracellular calcium levels due to NP exposure might also act as
an alternative mechanism for the induction of oxidative
stress.
[120,121]
Finally, oxidative stress can be caused indirectly
even by NPs that do not inherently produce ROS, as a result of
depletion of antioxidants (e.g. reduced glutathione) that are
consumed, bound or oxidised after exposure to dissolved metal
ions from NPs.
[122]
Thus, NPs may directly or indirectly induce a range of
responses associated with increasing levels of oxidative stress.
These responses have been characterised as induction of anti-
oxidant defenses at lower levels, followed by inflammatory
responses at intermediate levels and cytotoxic responses at high
levels.
[117,122]
It should also be noted that very low levels of
oxidative stress often serve important physiological functions,
such that altered production or abnormal quenching can alter
normal signalling pathways.
[123]
Among the most studied NPs shown to induce oxidative
stress in C. elegans are Ag NPs. A wide range of endpoints, from
growth inhibition or mortality to stress response gene expression
assays, have been used to examine oxidative stress by different
groups (Tables 2, S1). Roh et al.
[38]
reported greater reproduc-
tive failure and increased expression of various stress-response
genes due to Ag NP (,100 nm) exposure in a sod-3 mutant
strain (lacking a mitochondrial superoxide dismutase), com-
pared to wild type nematodes (N2). A recent study showed that
the antioxidant and metal chelator N-acetylcysteine (NAC)
completely rescued the growth inhibition of C. elegans caused
by Ag NPs with various coatings.
[49]
Two oxidant sensitive
C. elegans mutant strains (mev-1 and sod-3) demonstrated
increased sensitivity to several (but not all) of the tested Ag
NPs with different coatings but not to Ag ions, supporting the
role of oxidative stress in the toxicity of those Ag NPs.
[49]
Lim et al.
[109]
found not only increased ROS formation, but also
significant increase in the expression of p38 MAPK PMK-1 at
both gene and protein levels after Ag NP exposure in wild type
(N2) C. elegans. MAPK, which may be linked to oxidative
stress or a response to UPR (discussed below) was one of the
pathways induced after C. elegans exposure to Au NPs.
[39]
The
hypoxia signalling pathway (hif-1) was also activated by Ag NPs
in C. elegans.
[109]
Recently, Eom et al.
[37]
reported increased
sensitivity to Ag NPs in strains with loss-of-function mutations
in genes in the hif-1 pathway, which was completely rescued by
NAC treatment.
Exposure to ZnO (40–100 nm) and Al
2
O
3
(60 nm) NPs
caused photocatalytic ROS generation, intestinal lipofuscin
accumulation, decreased SOD activity levels and decreased
expression of sod-2 and sod-3 in N2 nematodes
[83,124,125]
(Table 2). Yu et al.
[125]
further confirmed that the accumulation
of intestinal autofluorescence is largely due to ROS production
in the intestines of Al
2
O
3
NP-exposed nematodes. Li et al.
[124]
found that antioxidant treatment after chronic exposure to Al
2
O
3
NPs suppressed oxidative stress. Zhang et al.
[113]
hypothesised
that ROS accumulation and oxidative damage might be the
cause of cyto- and genotoxicity of CeO
2
NPs in wild type
C. elegans.
In summary, it would appear that oxidative stress is a
common but not universal mechanism of toxicity of NPs in
C. elegans.
Endoplasmic reticulum stress
Endoplasmic reticulum (ER) stress is another possible mecha-
nism of NP toxicity. The ER is responsible for the biosynthesis
and folding of multiple proteins, and also stores calcium. Under
ER stress, protein denaturation, accumulation of misfolded
proteins and changes in Ca
2þ
homeostasis take place
[126]
(Fig. 2).
Protein denaturation has previously been suggested as one of
the possible effects of Au NPs.
[127]
As a result of ER stress, or to
respond to protein damage by helping with correct folding or
degradation of damaged proteins, the adaptive pathway described
as the ‘unfolded protein response’ (UPR) is activated.
[128]
In
J. Choi et al.
L

response to 4-nm Au NPs, we found that C. elegans induced both
canonical and non-canonical UPR pathways.
[39]
The canonical
UPR comprised of up-regulation of molecular chaperones (heat
shock proteins) including hsp-4, a marker of ER stress. The
non-canonical (specific to C. elegans) UPR response included
up-regulation of 25 genes from the abu/pqn families.
[39]
A strain
with a mutation in one of these genes (pqn-5) showed much
higher mortality than N2 after exposure to Au NPs, supporting a
protective role for this gene.
[39]
The abu/pqn genes, controlled by
the apoptotic receptor ced-1, have also been shown to activate
after exposure of C. elegans to pathogens and are involved in
regulation of innate immunity.
[129]
Another result of ER stress is
destabilisation of Ca
2þ
homeostasis, which has also been
observed in C. elegans after exposure to Au NPs.
[39]
DNA damage and apoptosis
Despite a growing number of mechanistic nanotoxicology
studies in C. elegans, there are few reports on possible genotoxic
effects of NPs. Recently we found that Ag NPs caused both
oxidative DNA damage and strand breaks to C. elegans, and
induced the hus-1 DNA damage checkpoint pathway and ulti-
mately apoptosis. DNA damage intensity was higher in a pmk-1
(the nematode p38 MAPK homologue, involved in apoptosis)
mutant, and necrosis instead of apoptosis was observed in
pmk-1 mutants.
[130]
We also found oxidative DNA damage in
C. elegans exposed to AgNO
3
and uncoated and PVP-coated
Ag NPs.
[48]
DNA damage, oxidative stress and ER stress can all eventu-
ally result in either apoptosis, necrosis or both (Fig. 2). In
C. elegans, there are a fixed number of somatic cells in adults,
and apoptotic events occur in two waves: during development
(131 out of 1090 cells die) and in the germline of adults.
[131,132]
It is commonly accepted that toxicants can increase the rate of
germ cell apoptosis (a basal level also occurs physiologically)
but not the developmentally programmed apoptotic cell
deaths.
[132]
However, to our knowledge, this question has not
been investigated extensively, and the possibility of somatic cell
apoptotic death should not be completely excluded. An increase
in apoptotic germ cell corpses was observed in C. elegans
exposed to fullerol NPs through food for 3 days.
[133]
In the
same study C. elegans strains carrying mutations in genes
functioning in the regulation of apoptosis, ced-3 and ced-4,
showed higher resistance to fullerol and a significant decrease in
apoptotic body formations when compared to the wild type
nematodes. In addition, apoptosis was one of the pathways
identified in our transcriptomic analysis of genes induced in
response to Au NP-exposed nematodes.
[39]
In the same study the
authors also observed significant up-regulation of three genes
(ced-1,rab-7 and dyn-1) implicated in phagocytosis, suggesting
that the processes associated with removal of necrotic cells
might be also activated in response to Au NP exposure.
Other mechanisms
To our knowledge, other possible nanotoxicity mechanisms
(e.g. receptor activation or antagonism, altered signalling,
lysosomal destabilisation, mitochondrial toxicity, protein
damage; Fig. 2) have not been well investigated in C. elegans.
In addition, although molecular mechanisms and organism-
level apical endpoints (e.g. growth, mortality, reproduction and
behaviour) have been fairly well studied, cellular and tissue-
level studies have been less common and are an important area
for future investigation.
Comparison with other models
There are numerous studies published on NP toxicity using
models other than C. elegans. It is beyond the scope of this
review to address all of these. Rather, for comparative purposes,
we focus on several model organisms that have been used fre-
quently in nano(eco)toxicity studies, emphasising the similarity
and differences with C. elegans in their responses to NP expo-
sures (Table 1).
Daphnia species
Numerous nanotoxicity studies have been performed with spe-
cies of Daphnia, fresh water free-swimming crustaceans that
serve as indicator species for various environmental stressors,
including exposure to NPs. Daphnia species, for example, have
shown high sensitivity to Ag NPs with mortality LC
50
of
40 mgL
1
for D. magna
[134]
and half maximal effective con-
centration (EC
50
) for reproduction of 121 mgL
1
for D. magna
and even lower (9 mgL
1
) for D. pulex.
[135]
Similar to C. elegans
studies with Ag NPs, particle- and ion-specific mechanisms of
toxicity were identified in D. magna in response to exposure to
PVP Ag NPs. These mechanisms were associated with abnor-
malities in mitochondrial activity,
[136]
and the authors suggested
that the mechanisms of toxicity of the Ag ions and particles
can be complementary, possibly resulting in enhanced toxicity.
Although particle-specific effects were identified after exposure
to Ag NPs in both C. elegans and Daphnia, the toxicity observed
after ZnO NP exposure seems to be explained by ions in both
model organisms (e.g. Ma et al.,
[63]
Adam et al.
[137]
and Zhu
et al.
[138]
).
Like C. elegans,Daphnia species are also characterised by a
short life cycle and high number of offspring, allowing rapid
toxicity screening for mortality and reproduction. Multigenera-
tional experiments have also been conducted for Daphnia
with Ag NPs
[135]
and carbon nanomaterials.
[139]
Decrease in
endpoints that may affect population levels (growth and repro-
duction) of three Daphnia species after exposure to Ag NPs for
five consecutive generations were observed at Ag concentra-
tions of 2.5–10 mgL
1
Ag.
Daphnia pulex’s genome has also been recently (in 2011)
sequenced, so that a whole genome toxicogenomic approach is
now available for studying the toxicity of NPs. Partial-genome
toxicogenomic approaches with custom microarrays have been
used to study the effects of Ag NPs on D. magna,
[140]
and
revealed distinct patterns of altered gene expression for the NPs
and ions. However, although the D. pulex genome has been
sequenced, the genes are not functionally annotated yet to the
same extent as for C. elegans, and functional genomic tools are
not available yet for Daphnia. Thus, C. elegans is a better model
species for testing mechanistic hypotheses for nanotoxicity. In
contrast, it is important to note Daphnia’s ecological advantage
over C. elegans.Daphnia pulex is a key species in freshwater
ecosystems and its ecology is well studied
[141]
and thus, it is
more suitable for bioaccumulation and transfer of NPs in food
chain studies. For example, a study by Zhu et al.
[142]
demon-
strated a transfer of TiO
2
NPs from D. magna to zebrafish
through dietary exposure.
Earthworms
Important ecotoxicological data have been derived using earth-
worms (Eisenia fetida). Uptake and elimination,
[41,42,143,144]
mechanistic toxicity
[103]
and avoidance
[145]
studies have been
performed with E. fetida exposed to metal and metallic NPs in
Nanoparticle toxicity to C. elegans
M

soils. One advantage of this species is that its larger size permits
easier detection and analysis of internal NP distribution, and
these studies demonstrated that earthworms can take up intact or
oxidised NPs and distribute them within tissues. In contrast to
C. elegans studies, different coatings (PVP and oleic acid) and
sizes of Ag NPs did not result in differences in E. fetida toxic-
ity.
[41,42]
It is important to note that these exposures were carried
out in soil, and the particles were subject to transformations
within the soil. Earthworm exposures to Ag NPs, similarly to
C. elegans, have adversely affected reproduction but only at
high concentrations, such as 700–800 mg kg
1
. Interestingly,
earthworms avoided Ag NPs 48 h after the exposure at con-
centrations 100-fold lower than the concentrations where
reproductive effects were observed.
[145]
Avoidance behaviour
in response to NPs was also observed in C. elegans. For instance,
Li et al.
[124]
(Table 2) observed decreased locomotive behaviour
in C. elegans exposed to Al
2
O
3
NPs at concentrations more than
10-fold lower than for bulk Al
2
O
3
, further supporting the
hypothesis that behavioural response may serve as one of
the most sensitive endpoints when studying NP toxicity.
Our study examining mechanisms of Ag NP toxicity in
E. fetida
[103]
suggests that oxidative stress occurs with some
delay (3 days) after exposure to Ag NPs as indicated by the
increased level of protein carbonyls. In the same study simi-
larity in expression levels of nine stress-response genes after
exposure to both ions and particles suggested that Ag NP
toxicity to earthworms is driven by ions. However, given that
less than 15 % of Ag was oxidised in the soils that the
earthworms were exposed to,
[41]
the dissolution of Ag NPs is
likely occurring during their uptake, internally or both.
[103]
Down-regulation and decreased activity of catalase on day
three suggest that increased levels of H
2
O
2
could have pro-
moted dissolution of Ag NPs internally within the short
exposure period.
[146,147]
To our knowledge, Ag speciation for
soil exposures with C. elegans has not been investigated, so the
earthworm studies provide important complementary insight
into Ag NP behaviour in soil. In C. elegans some of the
toxicity can also be explained partially by dissolution that,
given the nature of the aquatic exposures, probably occurs both
internally and externally. However, mortality, dissolution and
transcriptomic studies in response to Ag NPs in C. elegans
suggest that the observed effects are due to both dissolution
and particle-specific effects.
[51]
Zebrafish and Japanese medaka
Other ecotoxicological models that have been used in NP tox-
icity research include zebrafish (Danio rerio) and medaka
(Oryzias latipes). Studies on the toxicity of nC
60
in D. rerio
revealed adverse effects on embryo hatching, survival and
development
[148]
and oxidative stress was proposed as a key
mechanism of toxicity of nC
60
in D. rerio.
[100]
Studies with
metal oxide NPs (TiO
2
, ZnO and Al
2
O
3
)inD. rerio showed
toxicity (decrease in survival and malformations) only for ZnO
NPs,
[149]
with similar toxicity resulting from exposure to ZnO
and bulk material suggesting that the toxicity was likely due to
dissolution. Similarly, in a C. elegans ZnO NP study there were
no differences observed in toxicity between ZnO NPs and
ZnCl
2
.
[63]
Exposure of zebrafish fry to Ag and Cu NPs revealed
LC
50
values of less than 10 mgL
1
.
[134]
However, in contrast to
results in C. elegans, the toxicities of Ag and Cu NPs were
greater than those of their corresponding metals. Similarly,
greater toxicity for Ag NPs than AgNO
3
was observed in
medaka, at least at higher concentrations.
[99]
In the same study,
gene expression patterns suggesting an activation of stress
response pathways were documented for six stress related bio-
markers after exposure to Ag ions and Ag NPs, as also observed
for C. elegans exposed to AgNO
3
and Ag NPs.
[94]
Gene
expression patterns in medaka suggested that the Ag NP toxicity
was associated with oxidative stress, DNA damage and repair
mechanisms and apoptosis.
[99]
Kashiwada et al.
[101]
observed
changes in genes related to oxidative stress, growth regulation,
embryogenesis and morphogenesis after exposing medaka to
nano-colloidal Ag. These mechanisms and processes induced by
Ag NPs in D. rerio and O. latipes show similarity to the toxicity
mechanisms described above for C. elegans exposed to Ag and
Au NPs, despite the significant differences in the organisms’
physiology.
In fish, embryos are surrounded by a chorion (an acellular
envelope), which can allow or delay passage of NPs. This barrier
is of ecotoxicological relevance because it is important for many
fish and other species, and it is therefore important to study it
using the organisms that actually have such a barrier. Fortunately,
the chorion can be easily removed to test whether it prevents NPs
from passing to the embryo, as has been shown with single-
walled carbon nanotubes (SWNTs) in D. rerio.
[150]
Lee et al.
[151]
examined transport of Ag NPs into the embryos of D. rerio
and demonstrated that Ag NPs of 5–46 nm can pass through the
chorion pore channels (0.5–0.7 mm in diameter) with some of the
particles being trapped inside these channels. By comparison,
QDs have been observed in the reproductive organs of
C. elegans but not in the eggs,
[61]
whereas silica NPs
[152]
and
Ag NPs
[62]
have been observed in C. elegans embryos. Move-
ment into the gonads themselves may occur by passive diffu-
sion, but the cuticle of C. elegans eggs is dense and appears to
lack pores, suggesting that NPs probably reach embryos through
a mechanism other than passive diffusion. It may be that NPs can
be loaded into eggs during maternal loading of biomolecules
such as vitellogenin; this may be facilitated in C. elegans by
the fact that egg constituents are produced in intestinal cells
(to which NPs have good access), and are then actively trans-
ported directly to the gonad.
Mammalian models
C. elegans can be used for both ecotoxicity and human health
studies with NPs. Although a full review of the mammalian
nanotoxicity literature is beyond the scope of this review, sev-
eral studies indicate that molecular mechanisms in C. elegans
have been generally similar to those obtained in mammalian
systems. For example, a correlation was observed in the tran-
scriptomic response in human cultured cells (HepG2 or Jurkat
T-cells) and C. elegans.
[93,153]
Exposure to SiO
2
NPs resulted in
significant changes in expression of oxidative stress related
genes (i.e. catalase, Cu–Zn SOD), and DNA damage repair
genes (i.e. Rad-51) in HepG2 cell and C. elegans. Interestingly,
however, significant changes in expression of these genes was
not observed in a mouse model,
[130]
and the reason for this
discrepancy will be important to explore. ER stress, which is
involved in Au NP-exposed C. elegans as described earlier,
[39]
was also identified as a primary response to Au NP-treated
human leukaemia cells through proteomic and transcriptomic
approaches.
[102]
A role for ER stress was also supported in an
in vitro study with liver human cells exposed to Ag NPs.
[154]
Although there are some similarities in responses to NPs
between C. elegans and mammalian systems, it is important to
note that the bulk of the research in mammalian systems so far
has been in cell culture rather than whole-organism studies,
J. Choi et al.
N

and direct comparisons between C. elegans and mammalian cell
cultures studies can only be made with caution.
Conclusion: ‘what has C. elegans taught us so
far about NP toxicity?’
Caenorhabdits elegans studies have yielded a wealth of insight
into the relative toxicity of various NPs, their mechanisms of
toxicity and the importance of physiological barriers in modu-
lating their effects. So far, the general rank order of toxicity of
different NPs, as well as the molecular-level mechanisms of
toxicity, appear to extrapolate fairly well between C. elegans
and other systems. However, the concentrations of NPs required
to cause toxicity in C. elegans are higher than those reported in
cell culture and are more comparable to those observed in other
whole organism studies, highlighting the protective roles of
physiological barriers when using in vivo models. Thus,
C. elegans is poised to continue to serve a key role in bridging
in vitro cell culture studies to whole-organism studies in more
complex organisms.
We have also learned that interaction of NPs with environ-
mental variables such as natural organic matter and aging can
have dramatic effects on NP toxicity. In addition, the exposure
conditions (composition of media, presence of food etc.) can
also result in differences in toxicity, and enough studies have
been conducted to work out some of the difficulties associated
with performing controlled and well characterised exposures.
However, because many experiments have been conducted
using different conditions affecting toxicity (for instance, dif-
ferent exposure media) and also using different NPs or the same
NPs with different surface chemistry, systematic comparison of
the results is often difficult and therefore, our ability to draw
clear conclusions or make generalisations about NP toxicity to
C. elegans based on these experiments is limited. In this review
we discussed the factors that may explain the differences in NP
toxicity to C. elegans and provided recommendations for future
nanotoxicity experiments emphasising a need for more studies
that systematically vary NP properties in the exposure media to
examine how these interactions affect NP toxicity.
Availability of RNAi, transgenic and mutant C. elegans
strains along with toxicogenomic approaches allowed us to
identify some of the mechanisms of NP toxicity to C. elegans,
which are not limited to oxidative stress. Overall, although
progress has been made in terms of understanding the role of
NP characteristics in modulating toxicity in limited subsets of
NPs, we are still far from true predictive capability. Important
areas of future research are investigation of additional potential
mechanisms of toxicity, further systematic probing of the effect
of NP characteristics on toxicity to develop predictive models,
extension of molecular-level mechanistic toxicity to an under-
standing of cellular, tissue and organism effects, and elucidation
of multigenerational effects.
Acknowledgements
The authors appreciate the assistance of Krithika Umakanth, Alexander
Simon and Elena A. Turner. This work was supported by the National Sci-
ence Foundation (NSF) and the Environmental Protection Agency (EPA)
under NSF Cooperative Agreement EF-0830093, Center for the Environ-
mental Implications of NanoTechnology (CEINT) and through the EPA
Science to Achieve Results Program (RD 834574 and 834857). This work
was also supported by a grant from Mid-career Researcher Program through
the National Research Foundation of Korea (NRF) funded by the Ministry of
Science, ICT and Future Planning (2013R1A2A2A03010980) and by the
Korea Ministry of Environment (‘Environmental Health R&D program,’
2012001370009). Portions of this work were performed at Beamline X26A,
National Synchrotron Light Source (NSLS), Brookhaven National Labora-
tory. X26A is supported by the Department of Energy (DOE) – Geosciences
(DE-FG02–92ER14244 to The University of Chicago – CARS). Use of the
NSLS was supported by DOE under Contract DE-AC02–98CH10886. Any
opinions, findings, conclusions or recommendations expressed in this
material are those of the author(s) and do not necessarily reflect the views of
the NSF or the EPA. This work has not been subjected to EPA review and no
official endorsement should be inferred. J. Choi and O. V. Tsyusko con-
tributed equally to this paper.
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