)

Ilaria Corsi,*, Gary N. Cherr, Hunter S. Lenihan, Jerome Labille,^ Martin Hassellov, Laura Canesi,#
Francesco Dondero,r Giada Frenzilli,[ Danail Hristozov, Victor Puntes,] Camilla Della Torre,
Annalisa Pinsino,z Giovanni Libralato,, Antonio Marcomini, Enrico Sabbioni, and Valeria Matranga*,z

Department of Physical, Earth and Environmental Sciences, University of Siena, Siena 53100, Italy, Bodega Marine Laboratory and Department of Environmental
Toxicology and Nutrition, University of California, Davis, Bodega Bay, California 94923, United States, Bren School of the Environment, University of California, Santa
Barbara, Santa Barbara, California 93106, United States, ^Aix-Marseille Universit, CNRS, IRD, CEREGE UM34, Aix en Provence cedex 04, France, Department of
Chemistry and Molecular Biology, University of Gothenburg, Gothenburg S-405 30, Sweden, #Department of Earth, Environment and Life Sciences, University of
Genova, Genova 16142, Italy, rDepartment of Earth, Environment and Life Sciences, University of Piemonte Orientale, x Alessandria 15100, Italy, [Department of
Clinical and Experimental Medicine, University of Pisa, Pisa 56126, Italy, Department of Environmental Sciences, Informatics and Statistics, University C Foscari
Venice, Venice 30123, Italy, ]Catalan Institute of Nanoscience and Nanotechnology (ICN2), Barcelona 08193, Spain, ECSIN - European Center for Sustainable
Impact of Nanotechnology, Veneto Nanotech S.C.p.A., Rovigo 45100, Italy, and zCNR  Institute of Biomedicine and Molecular Immunology Alberto Monroy,
Palermo 90146, Italy

NANO FOCUS

Common Strategies and Technologies

for the Ecosafety Assessment and
Design of Nanomaterials Entering
the Marine Environment

ABSTRACT The widespread use of engineered nanomaterials (ENMs) in a variety of technologies and consumer products inevitably causes their release

into aquatic environments and nal deposition into the oceans. In addition, a growing number of ENM products are being developed specically for marine
applications, such as antifouling coatings and environmental remediation systems, thus increasing the need to address any potential risks for marine
organisms and ecosystems. To safeguard the marine environment, major scientic gaps related to assessing and designing ecosafe ENMs need to be lled.
In this Nano Focus, we examine key issues related to the state-of-the-art models and analytical tools being developed to understand ecological risks and to
design safeguards for marine organisms.

ngineered nanomaterials (ENMs) are

used extensively in a variety of emerging technologies and commercial
products, including biomedicine, pharmaceuticals and personal care, renewable energies, and electronic devices.1,2 As these
materials are used, disposed of, and degraded, they can release ENMs into the
environment. Fate and transport models
indicate that ENMs entering soil and waterways will eventually reach the marine environment as nanowaste that can cause
human injuries as well as ecological impact with signicant socioeconomic consequences.3,4 In addition to accidental releases and exposures, a suite of new marine
nanotechnologies, including antifouling
paints and pollution remediation systems,
are also being developed with great uncertainty about their ecosafety and sustainability for the marine environment. An increasing
number of short-term, well-controlled laboratory studies have tested ENM's toxicity on
CORSI ET AL.

marine organisms and showed a wide variety

of potential biological injuries.5,6 Whether
ENMs cause similar injuries in the dynamic
natural marine environment is uncertain because the fate, transport, and behavior of
many ENMs in seawater, and thus their biological risks, remain poorly understood.
The chemistry of ENMs plays a crucial role
as their bioavailability, bioaccumulation,
and toxicity are dicult to predict in seawater because the materials undergo complex interactions/transformations when exposed to elevated ionic concentrations. For
example, metallic ENMs undergo aggregation, sedimentation, corrosion, and reprecipitation in seawater, but the rates at which
these processes occur depend heavily on
the inherent dissolution rate of the dominant metal. Therefore, developing tools to
predict, to estimate, and to compare the
long-term eects and risks of ENMs presents
many challenges. Meeting these challenges
is a fundamental objective of many marine
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* Address correspondence to
ilaria.corsi@unisi.it,
valeria.matranga@ibim.cnr.it.

Published online September 29, 2014

10.1021/nn504684k
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In this Nano Focus, we

examine the state-ofthe-art approaches used
to predict and to
measure the exposure
and toxicity of ENMs in
marine ecosystems
Characterization and Behavior in Seawater: From Models to Analytical Tools.
Predicting ENM exposure will lead to
a better understanding of their fate
and behavior in the marine ecosystem. Their dispersion stability is a
key factor that determines their residence time in the water column,
and thus their occurrence in the
benthic or pelagic systems. Dispersion is influenced by a variety of
parameters, including the intrinsic
characteristics of the ENMs, the solution chemistry, and the interaction
with surrounding components.712
Ranging from the basic theoretical
CORSI ET AL.

prediction of the colloidal stability to

the most relevant and holistic approach accounting for the system
heterogeneity, a number of physicochemical mechanisms appear determinant in the fate of ENMs in the
seawater column and sediments. In
the seawater column, both hydrodynamic flow (river outlet, wave
oscillations) and random diffusion
(Brownian) favor the colloidal transport of particulate matter, competing with gravitational sedimentation
(see Figure 1). The particle size and
density thus determine the persistence in suspension. The size is that
of the individual isolated nanoparticles (NP) or their homogeneous and
heterogeneous aggregates (nanoobjects and their aggregates and
agglomerates). The aggregation dynamics consist of a two-step process
where the suspended particles first
collide, and then may attach to each
other.13 The collision frequency and
the attachment efficiency, respectively, drive these two steps, together determining the aggregation rate. The attachment efficiency
results from the balance between
repulsive and attractive interparticle
forces, as predicted by DLVO theory
and its numerous extensions.1417
These interactions have been widely
studied for synthetic systems with
basic and homogeneous compositions, revealing how surface charge,
which strongly depends on pH and
salinity, plays a determining role in
the electrostatic dispersion stability
of particles, especially in those that
have no steric protection against
aggregation.18,19 Critical salt concentrations have been determined
for the counterion nature and valence and solution pH by measuring
the induced kinetics of aggregation.12,2023 Bare NPs often display
low dispersion stability in natural
water, while functionalized NPs (e.g.,
using polyethylene glycol or a polyvinylpyrrolidone coating) may remain very stable at NaCl concentrations higher than that of seawater.
Nevertheless, the marine environment is rarely homogeneous and of
basic composition. Natural mineral
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(clay, carbonates, etc.) and organic

matter (algaes, exopolymers, etc.) reside suspended in the seawater column as geogenic and biogenic colloids,24 potentially interacting with
the ENMs. The affinity between the
NPs and these naturally occurring
colloids, a consequence of the NPs'
high surface energy, drives the socalled heteroaggregation phenomenon. This has been studied under
certain solution chemistries.10,2123,25
As an example, metal oxide and silver
ENMs have been shown to undergo
high aggregation and sedimentation rates in seawater of high ionic
strength (IS) and low in natural organic matter (NOM) content while
they remained more stable in freshwater (low IS, high NOM).26,27 Similarly, metal28 and oxide ENMs29 aggregating at high salt concentrations
are actually stabilized by interactions
with proteins. Moreover, the kinetics
of ENMs removal appeared also to
correlate with their concentrations.
In natural water, both homo- and
heteroaggregation may occur simultaneously, their respective kinetics resulting directly from the corresponding collision frequencies and
attachment eciencies.25 In the seawater column, the natural colloids
mainly consist of dissolved and particulate organic carbon, among
which large and amorphous organic
aggregates displaying low settling
velocities are distinct from denser
particles with higher settling velocities, such as diatoms.30,31 Their concentrations fall in the microgram per
liter (g/L) range. However, the release of ENMs in seawater is of terrestrial origin, implying their prior
transport through surface water
wherein suspended particulate matter is 3 orders of magnitude more
abundant (mg/L), while the predicted ENMs concentrations range
below microgram per liter (g/L).
The most widely used ENMs, based
on TiO2 and Ag NPs, are expected
at 102-10 and 105-1 mg/L, respectively.32 These concentrations are
likely to favor the heteroaggregation and colloidal transport of persistent ENMs to the sea. Estuaries
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nanoecosafety focused research

programs, as there is little doubt
that organisms living in marine
waters and sediments will be exposed to ENMs in euents from
factories and households or from
urban runo. In this Nano Focus,
we examine the state-of-the-art approaches used to predict and to
measure the exposure and toxicity
of ENMs in marine ecosystems. We
focus on the inuence of key environmental conditions, such as salinity, chronic low-level exposures
that probably will dominate natural
coastal seascapes, the eects of
EMNs in concert with other multiple
environmental stressors, and the
varying eects of ENMs as they age
under natural environmental regimes. Our goal is to provide a synopsis of available methods, models,
and insights necessary to highlight
the gaps that exist and need to be
lled for the design and production
of ecosafe ENMs to be used for marine ecosystem sustainability.

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Figure 1. Potential fate scenarios encountered by engineered nanomaterials (ENMs) when released into seawater. Dispersion
stabilization in the water column is favored by certain manufactured functionalization, natural organic matter (NOM) coating,
or colloidal transport by natural suspended particulate matter (SPM). Sedimentation is favored when large enough aggregates
are formed following salt-induced coagulation, NOM-induced bridging occulation, or heteroaggregation with SPM.

and coastal marine environments

are thus areas of high interest,
where stable colloids brought by
river encounter increasing salinity
vgradients that may cause their
large agglomeration and sedimentation. This situation typically forms
siltation areas, where ENMs and
other pollutants bound to those colloids may bury and accumulate
within sediments or persist in the
water column.31,3335 The NOM
composition also plays a role in this
balance. Seasonal variations inuence phytoplankton and microbial
activity that produces exudates
made of brillar and acidic polysaccharide compounds. These biopolymers can remain dispersed or
assemble together vas gel networks.3640 Their structure and
abundance determine their potential roles in the fate of the suspended particulate matter, and
thus of ENMs. Electrostatic or steric
stabilization is often observed when
the particle surface is saturated with
adsorbed molecules.41,42 On the
contrary, lower surface coverage
added to large molecular weight
may favor bridging occulation4345 and formation of large
aggregates such as marine snow.
Moreover, because of a given NP's
CORSI ET AL.

size ratio, the resulting heteroaggregates may sometimes consist of biopolymers decorated with adsorbed
NPs, as pearls on a necklace, or in
other cases, of biogel incorporating
the NP in their network.46,47 The
unique term heteroaggregation accounts for a very wide panel of
colloidal dynamics, ranging from
occulation to colloidal stabilization.
Very contrasting nal states may be
reached, depending on the respective size and concentration of the
natural colloids with regard to the
ENM.45,4850 It is the nal size and
density of the formed heteroaggregates that determine the persistence of ENMs in the seawater
column. Those remaining in the colloidal size range, typically below
1 m in size,24 promote further
transport of ENMs and their exposure to pelagic organisms, while the
formation and sedimentation of larger ocs instead concerns the
benthic ecosystem.
The State of the Art on Marine ENMs
Ecotoxicology. Many harmful effects
of ENMs have been reported for marine organisms, but factors including
physicochemical properties of ENMs,
seawater parameters, interactions
with both physical and chemical
factors (i.e., ultraviolet and marine
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pollutants), and organisms' physiology and ecology make ecotoxicological assessment difficult.4
Selected invertebrate organisms
have been recognized as potential
biological targets of ENMs exposure,
including biolm, phytoplankton,
bivalves, and bottom grazers (reviewed in ref 6 for inorganic NPs).
Little data are available on higher
trophic levels such as sh and
marine mammals.5154 Biological
responses and end points of toxicity
have been investigated as (1) mechanisms of uptake and of translocation inside the body, (2) organelles/
compartments/cells/tissues as targets of toxicity and/or retention, and
(3) cellular pathways/mechanisms of
toxicity.6 With few exceptions, there
is a lack of epidemiological studies
focusing on reproduction and development (e.g., early life stages, embryos, and larvae).5558
Increasing evidence supports the
hypothesis that the immune system
of marine bivalves represents a signicant target of ENMs. The blue
mussel Mytilus has been the species
most utilized so far for marine ecotoxicological studies on the eects
and mechanisms of action of ENMs
on innate immunity.59,60 In vitro
studies showed that dierent NPs
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Many harmful eects of

ENMs have been
reported for marine
organisms, but factors
including
physicochemical
properties of ENMs,
seawater parameters,
interactions with both
physical and chemical
factors, and organisms'
physiology and ecology
make ecotoxicological
assessment dicult.

are rapidly taken up by mussel

hemocytes, aecting a large number of functional parameters, from
lysosomal function to phagocytic
activity and oxyradical production,
and also inducing pro-apoptotic
processes; the eects of NPs were
mediated by stress-activated mitogen-activated protein kinase signaling, as in mammalian phagocytes.6163 In vivo exposure to
dierent NPs and, in particular,
to nano-TiO2 chosen as a model
NP type64,65 enabled formulation
of a hypothesis on the possible
pathways leading to nanoinduced
immunomodulation60 (see Figure 2).
Due to the physiological mechanisms involved in the feeding
process, nano-TiO2 agglomerates/
aggregates formed in seawater
are taken up by the gills and partly
directed to the digestive gland,
where intracellular uptake of NPs
induces lysosomal perturbations
and changes in the expression of
antioxidant and immune-related
genes. Nanoparticles can then potentially be translocated from the
digestive system to the hemolymph
and to circulating hemocytes, where
nano-TiO2 induced changes both
in functional parameters (lysosomal
CORSI ET AL.

Figure 2. Possible routes of engineered nanomaterial (ENM) uptake and disposal in

the model marine bivalve Mytilus leading to immunomodulation.

integrity, phagocytosis, reactive

oxygen species, ROS, and NO production, induction of preapoptotic
processes) and in transcription of
antimicrobial peptides. Interestingly, the in vivo eects of nanoTiO2 on mussel immune parameters
were observed at concentrations
(110 g/L) much lower than those
usually utilized in ecotoxicity tests
on aquatic species, and closer to
predicted environmental concentrations.65 Recently, the rapidly
expanding application of DNA microarrays and next generation sequencing (NGS) technologies oer
new and broader research perspectives, from the whole transcriptome
coverage to the Mytilus genome
sequencing, leading to the identication of an increasing number of
immune-related genes that could
be targeted by dierent ENMs.66,67
Likely candidates are the members
of the Toll receptor family recently
identied in mussels;68 among these,
transcription of the TRL-i isoform has
been shown to be down-regulated in
mussel hemocytes by in vivo exposure to nano-TiO2.69
Among invertebrates, the sea
urchin is a successful marine model,
globally distributed in almost all
depths, latitudes, temperatures,
and environments in the sea. The
key to its successful survival is its
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potent immune system, which provides protection, robustness, and

molecular plasticity.70 Thus, sea
urchins represent an excellent model to uncover molecular and regulatory mechanisms promoting roles
of the immune system on survival.
Sea urchin immune cells have been
demonstrated to activate their immune response machinery in response to dierent kinds of physical
and chemical stressors, such as temperature shocks; pH decreases; exposure to UVB radiation and heavy
metals; and exposure to tin (SnO2),
cerium (CeO2), iron (Fe3O4), and TiO2
NPs.7176 The utility of the heat
shock protein HSCP70/HSC70 as a
general stress response marker to
be used for monitoring both acute
and chronic stresses has been demonstrated, with the only exception
being NP exposure.76 In contrast, it is
expected that specic pathways and
biomarkers are selectively elicited in
response to NPs, as already demonstrated in human immune cells.77,78
The availability of the sea urchin
genome, which has been shown to
be closely phylogenetically related
to the human genome,79 oers the
possibility to analyze its complex
and sophisticated immune system
and to compare the biological effects observed in sea urchins and
human immune cells. Analysis of
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Figure 3. Sea urchin model to study immunity and development for the safe use of nanomaterials.

the amazingly large repertoire of

innate pathogen recognition proteins, such as the 20-fold expansion
and diversication of the Toll-like
receptors relative to human genes,
would probably identify specic target genes for dierent NPs or ENMs.
Complementary to the use of adults
for studies on immune modulation,
embryos have emerged as valid
tools for studies on developmental
and molecular perturbations induced by environmentally relevant
classical and emerging contaminants, including metal NPs.8089 As
an example, the in vivo exposure to
nano-Ag causes dose-dependent
CORSI ET AL.

developmental defects as well as

alterations in swimming patterns in
sea urchin embryos.56 However, the
causeeect relationship remains
to be demonstrated. In conclusion,
the value of the sea urchin model is
twofold: rst, it is proposed as a
proxy to humans for the analysis of
the eects of ENMs on the immune
system, and second, it can be used
as an alternative model for ecotoxicological studies (see Figure 3).
Engineered nanomaterials could
give rise to genotoxic eects in exposed marine organisms; the loss of
DNA integrity, if not properly repaired, may lead to mutations, birth
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defects, and long-term eects, such

as cancer in vertebrates.90,91 The
mechanisms of ENMs genotoxicity
are still not well understood92 and it
is often unclear if an eect on DNA is
nanospecic. DNA strand breaks are
one of the major types of oxidative
damage to DNA via oxidative stress,
which is generally assessed by the
Comet assay,92,93 a technique widely
used to evaluate the genotoxic effects of contaminants in bivalves'
hemocytes, which are a potential
target for genotoxicity.59,64,94,95 Oxidative stress after long-term exposure to nano-Ag was evidenced in
mussel gills and digestive glands.96
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present in the environment.4,104

Among actors on the rst line of
defense, P-glycoprotein (P-gp) is
the most well-characterized membrane transport protein; it is also
involved in the eux of xenobiotics
generally known as multidrug resistance (MDR, previously known as
MXR), i.e., phase 0 and III of biotransformation.105,106 Several ENMbased approaches have recently
been proposed to reverse/overcome the eux-mediated resistance controlled by P-gp in human
cell lines to favor cytotoxic drugs
accumulation in tumor cells. Of
course, such complex medical ENMs
are transformed inside the body
before expulsion.107109 Little attention has been paid to marine organisms where MDR is actively involved
in xenobiotic biotransformation and
a similar interaction may occur.
One example is the reported increase in the eux of P-gp substrate
Rhodamine B in the gills of the
marine mussel Mytilus galloprovincialis caused by nano-TiO2.103 In
parallel, an increase of TCDD bioconcentration in the whole body was
observed in co-exposure conditions.
Possible involvement of nano-TiO2
on eux functionality that, in turn,
aects the disposition/cellular retention of other toxic compounds
can be hypothesized.103 Concerning
heavy metal biotransformation, metallothioneins (MTs) are ubiquitous,
low-molecular-weight soluble proteins with high heavy-metal-ionbinding capacity. Their role in essential trace elements (Zn2 and Cu)
homeostasis as well as detoxication
from noxious ions such as Hg2 and
Cd2 has been widely demonstrated
in eukaryotic cells, including those of
marine organisms.110 The increase of
MT in response to a variety of oxidebased and metallic NPs has been
reported in cells and/or tissues of
several marine organisms. Nano-Ag
increased MT gene expression in
embryo and adult hepatopancreas
cells of Crassostrea virginica55 in the
nanomolar range. Conversely, no increase in MT-like proteins was reported in tissues of the ragworm
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Hediste diversicolor exposed to

nano-Ag in sediments, whereas soluble Ag caused MT induction.111 Gold
NPs (0.1 mg/L) were eective in
tissues of the endobenthic clam Scrobicularia plana.112 Among nano oxides, despite the number of studies
on the eects of ZnO and nano-TiO2,
only reports on nano-TiO2 are available, demonstrating no eects on MT
transcription in the tissues of in
M. galloprovincialis.69,101 However,
several studies have focused on the
eects of nano-CuO, for which an
increase of MT proteins has been
documented in at least two molluscan species, M. galloprovincialis and
S. plana.113116 It is worth noting that
in the latter studies, environmental
Cu dissolution was very low or virtually absent, therefore MT induction
had three possible explanations: (1)
intracellular Cu dissolution to ionic
forms; (2) oxyl-radical formation; or,
most likely, (3) a combination of
both, since metal and oxidative stress
show overlapping features, cellular
outcomes, and biochemical responses.
It is widely accepted that MT genes
are transcriptionally activated by different heavy metal ions penetrating
into cells through a mechanism involving a zinc-sensitive transcription
factor (MTF-1) and zinc displacement from a physiological pool of
MTs by more electrophilic cations. In
addition, oxyl radicals and, in general, oxidative stress are promoters
of MT induction.117 Authors did not
report MT modulation along with
eects to the antioxidant enzymatic
system and/or the occurrence of
oxidative insults in the aforementioned works (except in ref 111,
where no NP dissolution was observed). Assaying both MT heavy
metal content and oxidation state
and its concentration in marine organisms' tissue thus represents a
powerful tool to provide mechanistic information on the mode of action of metallic and metal-oxide NPs.
The Need To Develop Models. A major need in marine ecotoxicology for
ENMs is the development of quantitative approaches for integrating
more realistic ENM exposure scenarios
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A loss of DNA integrity was found in

marine mussel cells after in vitro exposure to nano-iron95 and in blue
mussel after in vitro exposure to
nano-Ag2S and CdS quantum dots.97
Concerning marine species, the effects of nano-TiO2 have been investigated with respect to invertebrates,98101 and two articles have
reported the susceptibility to micrometric rutile and nanometric anatase
TiO2 genotoxic potential of the top
predator bottle-nose dolphin leukocytes51 and broblasts.54 Interactive
genotoxic eects of C60 fullerenes
and uoranthene were studied in
marine mussels,102 highlighting that
both uoranthene and C60 on their
own caused concentration-dependent
increases in DNA strand breaks,
while combined exposure to C60
and uoranthene additively enhanced the levels of DNA strand
breaks, likely related to oxidative
defense impairment. Recent evidence
also showed that nano-TiO2 and
2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) can exert synergistic or antagonistic eects depending on the
experimental condition, cell/tissue,
and type of measured response, as
evaluated by a battery of biomarkers
including DNA primary damage
(Comet assay), genome instability
(RAPD assay), and chromosomal damage (Micronucleus test).103
Biotransformation/detoxication
may play a signicant role in ENM
toxicity, including genotoxicity,
by aecting ENMs localization/
disposition inside the cell. The accumulation and consequent toxicity of
a compound is strongly inuenced
by the organism's detoxifying/clearance capabilities. Biotransformation
of ENMs might alter their life-cycle
metabolism, but also might interfere
with that of other compounds, as
occurs in coexposure scenarios in
the natural environment. Therefore,
it is crucial to investigate the interactions of ENMs with the so-called
cell defensome (i.e., the cellular
machinery activated in response to
chemical stressors) in marine organisms that are naturally exposed to
mixtures of compounds/toxicants

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Figure 4. Process using estuarine fate and transport modeling to predict realistic exposure scenarios to design focused fate
and transport (F&T), high-content screening assays (HCS), and microcosm experiments with individuals and populations of
estuarine fauna. Results of the experiments generate predictions for impacts under realistic estuarine environmental scenarios
in mesocosms. Results from F&T, HCS, microcosm, and mesocosms are then synthesized in multivariate machine-learning
statistical analyses, the results of which update the estuarine F&T model.

and predicting ecosystem impacts,

thus enhancing the ability to design
ecosafe ENMs. This can be accomplished through modeling exposure
scenarios specific to estuaries and
marine environments, testing those
predictions with environmental fate
and transport (F&T) experiments, and
testing toxicity with organisms that
could be exposed in both pelagic
and benthic marine habitats (see
Figure 4). This integration requires
specifically: (1) generating predictive
F&T models for specific ENMs and
environments, such as multimedia environmental distribution (MendNano)
models for coastal or estuarine ecosystems;similar models have been
produced for terrestrial environments118 and allow flows of ENMs
within and between environmental
and biological media; (2) testing the
predictions of the MendNano models
through a combination of F&T experiments, high-content-screening (HCS)
assays, microcosms, and computerregulated mesocosm experiments at
the level of individuals, populations,
and/or communities; (3) analyzing the
results of the multiple experiments for
correlations using machine learning
CORSI ET AL.

analysis and statistical modeling

framework;119 and (4) using the correlation output to parametrize and update the MendNano F&T model. A full
array of ENMs of different shapes and
sizes, such as Cu, CuO, coreshell, and
safe design materials as they are developed, can be studied when they
are exposed to water, sediment, and
biological matrices, and as they are
transformed by physical (e.g., temperature, salinity, and dissolved and
particulate organic material) and biological processes associated with estuarine environments and food webs
composed of bacteria, plants, invertebrates, and fish. Both the benthic and
pelagic communities, from sedimentdwelling invertebrates to fish and
important estuarine fauna, represent
potential targets of ENMs. The microbial community is crucial in determining ENMs' F&T and effects; impacts to
phytoplankton, for example, which
are the primary producers in estuaries,
probably have manifold ecological
effects. The objectives of this approach are structured to assess the
environmental implications of ENMs
through integration models that generate theory, experiments that test
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those theories, and subsequent improvement of the models to generate

better theory. Marine ecotoxicology
efforts use this approach by linking
ENMs' chemical properties, exposure,
and biochemical responses of injury
with the ecological and physical processes that ultimately regulate marine
ecosystem-level impacts and ecosystem services (see Figure 4). The general observations produced from this
approach so far are the following:
(1) Engineered nanomaterials
can be toxic to bacteria;5 in response
to external aggression, algae and
bacteria can release extracellular
polymeric substances (EPS)120 that
will potentially bind to metal ENMs,
perhaps reducing their bioavailability and toxicity.121,122 The role of
occulation of EPS (marine snow)
in the fate of ENMs remains vastly
unappreciated.
(2) Oysters are estuarine lter
feeders that provide many important ecosystem services.123 As lter
feeders, they process large volumes
of water and are exposed to estuarine ENMs through ventilation for
respiration, thus acting as potential
ENM bioaccumulators from the
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A major need in marine

ecotoxicology for
engineered
nanomaterials (ENMs)
is the development of
quantitative approaches
for integrating more
realistic ENM exposure
scenarios and
predicting ecosystem
impacts, thus
enhancing the ability to
design ecosafe ENMs.
Risk Assessment, Including Nanotechnology Applications in Marine Waters. The
increasing exposure of aquatic
organisms to ENMs may lead to extended ecological risks. Considerable research efforts in Europe
have focused on evaluating the risks
from ENMs in the aquatic environment (see Table 1). In the United
States, the Nanotechnology Environmental and Health Implications
Working Group of the National
Nanotechnology Initiative coordinates the research that aims to develop protocols, standards, instruments, models, and validated data
for ecological risk assessment (RA) of
ENMs.127
Despite the signicant research
eorts, a key issue in both the European Union and the United States
that most research has not suciently addressed is that pristine
ENMs undergo aging and transformation reactions during incorporation into products, and weathering
and aging when released into the
environment.121 Thus, at each step
of the supply chain (lifecycle), there
is a potential risk of exposure to
ENMs with dierent physicochemical properties, which would aect
their toxicity. Some of the few
results for textiles, paints, and
nanocomposites32 suggest that the
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released particles undergo signicant aging128 and exhibit dierent

environmental behavior and eects
compared to the pristine ENMs.45
Aged and pristine Cu ENMs showed
dierent fate and toxicity in an
aquatic compartment under the
same environmental conditions.129
In addition, aged nano-TiO2 from
sunscreen caused lower mortality
in Daphnia magna compared to
bare nano-TiO2.130 These studies
have conrmed that aged ENMs
can exhibit dierences in solubility,
aggregation, and reactivity that can
aect their aquatic mobility and
toxicity. Moreover, it is hypothesized
that many ENMs are (designed to
be) persistent, which might lead to
long-term exposure of aquatic and
sediment species. However, longerterm ecological eects have not yet
been comprehensively researched,
especially for ENMs used in real
products. Experiments with single
species have provided evidence of
changes being transferred across
generations, causing epigenetic, mutational, or reproductive eects.131
Therefore, testing of longer-term
genotoxicity on multiple species
should be developed. Quantitative
ecological risk analyses of ENMs
would typically involve deterministic modeling of exposuredoseresponse relationships. However, this
would be aected by severe uncertainty and data variability. Therefore, it is recommended that the
ecological risk modeling of ENMs is
addressed in a probabilistic manner
using stochastic approaches such as
the Monte Carlo and the Latin hypercube simulations.132 In this case,
distributions of hazard estimates
would be derived instead of single
values, which could be plotted
against distributions of exposure estimates in order to identify central
tendencies of expected risk and associated high-end probability of
exposure. With this approach, predicted no eect concentrations
(PNEC) can be estimated based on
species sensitivity distribution (SSD)
methodologies,32 which are well suited to deal with high uncertainty
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pelagic habitat. Benthic infaunal invertebrates, especially polychaete

worms, crustaceans, echinoderms,
and deposit-feeding clams, are probably exposed to ENMs deposited
onto sediments, detritus, and biolms.116,120 Together, these organisms, which are important in estuarine food webs, may store ENMs or
their dissolution products in soft or
hard tissue, and may also, through
excretion, enhance ENM bioavailability. This may or may not represent an important pathway for
human exposure, as well as for the
trophic transfer of ENMs or their dissolution products. Fish are top predators in coastal ecosystems and can
be exposed to metal ENMs directly
through water column ingestion or
gill tissue exposure. However, many
sh species are also predators on
numerous sediment-dwelling invertebrates that will have consumed ENMs,
so this trophic transfer may represent
an important route of exposure.
(3) Dynamic energy budget
(DEB) theory124 focuses on the individual organism, with dierential
equations describing the rates at
which an organism assimilates and
utilizes energy and materials from
food for maintenance, growth, reproduction, and development. Engineered nanomaterials may disrupt
energy utilization, as do many other
anthropogenic contaminants. Dynamic energy budget models use
theory to provide estimates of dosespecic responses of ENM concentrations on individual growth, survival,
and reproductive capacity.125,126
(4) The outputs of DEB models
can be used in population dynamic
model simulations to predict the
potential impact of ENM injuries on
populations. For small organisms
such as phytoplankton, for which
population dynamics can be measured in microcosms or mesocoms,
the population dynamic models can
be used as predictions and then
tested empirically with follow-up experiments. For larger organisms, for
which population level manipulations
are impractical, the models can provide theoretical impacts.

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Engineered Nanomaterials (ENMs) in the Aquatic Environmenta

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TABLE 1. Overview of European Union Research Projects Providing Data and Methods for Ecological Risk Assessment of

Reproduced unchanged with permission from NanoSafety Cluster; European NanoSafety Cluster Compendium: Compendium of Projects in the European NanoSafety Cluster, 2014
Edition; Lynch, I., Ed.; Karolinska Institutet on behalf of WG8 of the NanoSafety Cluster, access at http://www.nanosafetycluster.eu/uploads/files/pdf/2014_NSC_Compendium.
pdf, under the Creatives Commons Attribution license 3.0 (http://creativecommons.org/licenses/by/3.0/). Copyright 2014 EU NanoSafety Cluster.

and variability. Such SSD modeling

procedures produce the biological
responses of each species of a target
environmental system and combine
them into a cumulative SSD for the
aquatic compartment. Probabilistic
estimation of ecological risks can then
be conducted by linking this SSD
to the probability of critical predicted environmental concentrations
(PEC). Such modeling approaches for
ecological risk estimation and the resulting risk mitigation measures can
provide a strong basis for guidance in
industrial and regulatory contexts.
ENMs for Environmental Applications:
Smart Nanoparticles for Sea Water Remediation. Most of the remediation
CORSI ET AL.

Nanomaterials have
a number of
physicochemical
properties that make
them particularly
attractive for water
purication and
environmental
remediation.
technologies available today are
based on the adsorption, ion
VOL. 8

exchange, amalgamation, chemical

degradation or precipitation, and
bioremediation. While effective in
many cases, these methods are often costly and time-consuming, particularly pump-and-treat methods
that require expensive investments
and procedures. In this context, the
high adsorption capacity of ENMs
for certain pollutants has been demonstrated in many cases. There are
a variety of examples in the literature that exploit the use of NPs for
the removal of heavy metals from
aqueous solutions, such as using
-FeO(OH) nanocrystals to remove
As(V),133 SiO2 NPs for removal of
Cr(III),134 as well as Al2O3 NPs135
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may be used in a stepwise strategy

(see Figure 6), e.g., (i) cleaning very
polluted sites toward less polluted
soil, after which biota may be able to
remediate pollutants further; (ii)
cleaning less polluted sites with existing microbiota by developing synergies between the NPs and the
involved biota to increase the potential for natural attenuation; and
nally, (iii) cleaning low but hazardous concentrations of pollutants
(microcontaminants). Very polluted
areas, where all signs of life have
disappeared, and therefore precluding the option of bioremediation,
are a big challenge for remediation
and a wonderful opportunity to test
the reactivity, catalytic behavior,
and large surface area per unit
mass of NPs. In such cases, NPs are
needed so they can induce/catalyze
the initial chemical breakdown or
adsorb and then slowly release the
contaminants in order to achieve a
level of contamination where microbial processes can take place, enhancing the sequential synergy of
nanotechnology and bioremediation. Ideally, the ENM would degrade into harmless substances
once its job is nalized. In addition,
while cleaning very polluted water
into less polluted water is feasible,
cleaning low pollution levels in
water to safe water (especially in
the case of microcontaminants) is
also a challenge that could be overcome by nanoremediation. However, the intrinsic nature of the inorganic NPs may also cause damage
to the environment if dispersed
uncontrollably.
Marine pollution by petroleum
products (i.e., oil spills) often produces disastrous eects with negative impacts, aecting both the
environment and socioeconomic
development. In this context, the
use of ENMs for in situ remediation
represents a promising and cuttingedge solution, by ensuring a quick
and ecient removal of pollutants.2
However, the use of such materials
should not pose any additional risk
to the marine ecosystem in which
they are released.2,141
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Developing safe and eective remediation technologies for petroleum

products based on the design and
synthesis of new ecofriendly ENMs
might be a challenging solution for
promoting in situ nanoremediation
for the marine environment. Such
innovative ecofriendly ENMs should
meet the highest standards of
environmental safety and eectiveness, supporting economic development in terms of industrial competitiveness and innovation. Their
application will contribute to the
management, in more ecient and
sustainable ways, of several systematic sources of petroleum pollution in various productive sectors,
such as maritime transport, rening,
mining, and liqueed natural gas
terminals in marine waters. Finally,
the use of ecofriendly ENMs, which
provide rapid and eective removal
of petroleum products without
compromising the ecosystem, is a
priority for the safeguard of the
oceans.

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and supported Au NPs on alumina136

and as colloids,137 for removal of
Hg(II). Although the ability of inorganic NPs to trap pollutants is best
known for cations of elements such
as the previous and Ni(II) and Cd(II),
they can also be functionalized to
trap organic molecules specifically
and/or to act as photocatalysts to
promote the complete degradation
of toxic organic matter. A comprehensive overview of the different
manufactured nanomaterials along
with the pollutants they could potentially remediate has been reviewed elsewhere.2,138 The toxic
species once absorbed onto the
NPs can be removed by applying
mild (and affordable) gravitational
(centrifugation) or magnetic (in the
case of magnetic NPs) field gradients. Other strategy when dealing
with organic compounds is to promote their reduction (e.g., to transform perchlorates to Cl) with
sacrificial electrodes, as iron NPs that
will reduce halogenated compounds
during their oxidation. Moreover,
nanoremediation can present complementary properties to bioremediation, not just by decreasing pollutants, but also by dispersing the
pollutants, and by synergetic interactions with biota. The majority of
these works have been performed
in the lab with synthetic water
due to complexity and restrictions
to disperse NPs in the natural
environment.
All this research interest is because nanomaterials have a number
of physicochemical properties that
make them particularly attractive for
water purication and environmental remediation. Their capacity to be
dispersed in water allows them to
travel farther than larger, macrosized particles, thus achieving wider
distribution and permitting the whole
volume to be quickly scanned with a
relatively small amount of material.2,139,140 These unique properties
can be employed to degrade and to
scavenge pollutants (see Table 2).
Nanoremediation may address
dierent levels of pollution with specic targeted technologies, which

FUTURE PERSPECTIVES AND

FORESEEN CHALLENGES
As mentioned, the deliberate or
accidental release of ENMs into the
oceans will impact marine organisms, especially if solutions for their
safe manufacturing are not properly
considered. To this purpose, two
main organizations, the University
of California Center for Environmental Implications of Nanotechnology
(UC CEIN) and the Marine Focus
Group in the framework of Hazard
Working Group of the NanoSafety
Cluster of the European Commission, focused their mission on studying the impacts of ENMs on the
marine environment. The UC CEIN
was established in 2008 with funding from the U.S. National Science
Foundation and the U.S. Environmental Protection Agency, with the
aim of studying the impact of nanotechnology on the environment, including the identication of hazard
and exposure scenarios that take
into consideration the novel physicochemical properties of ENMs. The
UC CEIN has made great progress in
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mechanism of action

CATALYST
oxidation and reduction
e SOURCES
Known Active Ion PROVIDERS
ABSORBERS
HYBRIDES I
Heterodimer
HYBRIDES II
Coreshell
Tautomeric species (oxygen/electron sponges)

Au, Pt, Ag, Pd, Fe3O4, CoO, CeO2

Fe, Cu, Co, Ni, Fe3O4, CeO2
Fe3O4, Ag, Pt, CeO2, ZnO
Fe3O4, Fe2O3, TiO2, SiO2 (porous, partially methylated), functionalized Layered Double Hydroxides (LDH)
AuCeO2, Pd/CuCeO2, AuTiO2, AuFe3O4, CeO2Fe3O4
Au-SiO2, Fe3O4SiO2, FeFe3O4
CeO2, Fe3O4, FeMnOx (perovsquites), MnOx

NANO FOCUS

TABLE 2. Remediation Mechanisms of Actions of Different Engineered Nanomaterials

Figure 5. Integration of engineered nanomaterials (EMN) chemistry, exposure modeling, high-throughput screening (HTS)
and high-content screening (HCS) assays, and injury responses across dierent levels of biological organization. Dynamic
energy budget (DEB) models enable the linkage of individual responses to population and community levels.

assembling a multidisciplinary team

to develop the scientic underpinnings, research, knowledge, education, and outreach that is required
for assessing the safety of nanotechnology in the environment. The approach includes high-throughput/
high content screening (HTS and HCS)
studies to develop structureactivity
relationships (QSARs) that can be
used to predict the impact of ENMs
on organisms in freshwater, marine,
and terrestrial environments.142,143 A
major goal of UC CEIN is to integrate
research goals and themes to better
estimate realistic nanomaterial exposure scenarios (F&T), predict ecosystem impacts, and design safer
materials. UC CEIN's strategies for
accomplishing this goal include
CORSI ET AL.

iterative learning between environmental modeling, chemistry, toxicology, ecosystem impacts, and
sociological research eorts. Marine
studies have included species of
phytoplankton as primary producers, and copepods and mussels as
primary consumers linking photosynthesis as well as ENM transfer to
higher trophic levels. The eect of
ENMs on embryo development is
also a focus of the Center. The UC
CEIN has been renewed (20132018)
and new directions are planned to
address the integrated marine nanotoxicology eort. A central role is to
identify which ENMs have important
environmental impacts versus those
that are safe. UC CEIN's marine ecotoxicology eort uses this approach
VOL. 8

by linking ENM chemical properties,

exposure, and biochemical responses
of injury with the ecological and physical processes that ultimately regulate ecosystem-level impacts and
ecosystem services.
Within the NanoSafety Cluster
of the European Commission, the
Marine Ecotox Focus Group in the
framework of the Hazard Working
Group (http://www.nanosafetycluster.eu/working-groups/2-hazard-wg/
marine-ecotox.html) was established
in 2011. Its mission is based on the
following assumptions: (1) the marine environment is likely to be a sink
of ENMs as it is for most manmade
pollutants, and it is therefore possible
that certain ENMs may bioaccumulate and biomagnify along the
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NANO FOCUS

Figure 6. Combining nano- and bioremediation.

marine trophic chain, thus potentially aecting marine biological resources (wild and farmed); (2) ENMs
may be transferred to humans
through diet by consumption of contaminated seafood products; (3)
ENMs may lead to a deterioration
of marine environmental quality
(coastal areas including natural and
recreational interests) with social and
economic repercussions; (4) some
ENMs can, however, be used to reduce marine pollution, through selected applications, such as nanoremediation by binding and removing specic contaminants. The progress in the development of
nanotechnologies and nanoenabled
products is vast and continuously
progressing. The aim of the Focus
group is to oer a platform for linking
nanotechnologists with ecotoxicologists, environmental scientists, analytical chemists, biochemists, molecular
biologists, industries, and end users
(public) to provide the proper scenario suitable for an overall risk assessment of ENMs in the marine environment. By doing this, the focus
group will provide appropriate support for decision and policy makers,
including an understanding of the
risks that may occur for ENMs that
fall outside the denition, guidance,
further development of measurement techniques, and help dealing
with changes during the life cycle
CORSI ET AL.

in agreement with recent work.144

Again, at the EU level, the QualityNano pan-European infrastructure for
quality in nanomaterials safety testing
is providing support for a full assessment of potential impacts of past and
next generation of ENMs at all stages
of their lifecycle including interactions
with environmental matrices and
biota (http://www.qualitynano.eu/).
At the national level, the Italian
Marine Nano Ecotoxicology working
group was formally announced in
November 2012 and further enlarged at the International level
during the rst Marine NanoEcoToxicolgy Workshop (MANET) held in
Palermo in 2012 and fully dedicated
to addressing the ecotoxicology of
ENMs in the marine environment.
Currently, it groups more than 50
scientists from 23 countries including France, Germany, Sweden, the
United Kingdom, and the United
States, involving both universities
and research centers. Its mission is
to encourage applied and basic research as a support for a safe and
sustainable use of ENMs, fostering
interdisciplinary approaches for research and training in the eld of
nanoecotoxicology and promoting
informed discussion on ethical and
relevant social issues as well. In this
context, the Italian Marine Nano
Ecotoxicology working group acts
as a reference group for the
VOL. 8

evaluation of the health and environmental impacts of ENMs with regard

to scientic research, industrial innovation, and regulatory issues. It has the
potential to act as a growing network
integrating multidisciplinary skills and
acquire the necessary expertise to ll
the existing prioritized gaps.
Conflict of Interest: The authors declare
no competing nancial interest.

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