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Predictive Toxicology
Implementation of a Multidisciplinary Approach to Solve Complex Nano EHS Problems by the UC Center for the Environmental Implications of Nanotechnology
Tian Xia , Davin Malasarn , Sijie Lin , Zhaoxia Ji , Haiyuan Zhang , Robert J. Miller , Arturo A. Keller , Roger M. Nisbet , Barbara H. Harthorn , Hilary A. Godwin , Hunter S. Lenihan , Rong Liu , Jorge Gardea-Torresdey , Yoram Cohen , Lutz Mädler , Patricia A. Holden , Jeffrey I. Zink , and Andre E. Nel *
1wileyonlinelibrary.com© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/smll.201201700
Prof. A. E. NelDepartment of MedicineDivision of NanoMedicineUCLA School of Medicine, 52-175 CHS10833 Le Conte Ave, Los Angeles, CA 90095-1680, USA E-mail: [email protected]
Prof. T. Xia, Dr. S. Lin, Dr. Z. Ji, Dr. H. ZhangDivision of NanoMedicineDepartment of MedicineUCLA, Los Angeles, California 90095, USA
Dr. D. MalasarnCalifornia NanoSystems InstituteUCLA, Los Angeles, California 90095, USA
Dr. R. J. Miller, Prof. A. A. Keller, Prof. H. S. Lenihan, Prof. P. A. HoldenBren School of Environmental Science and ManagementUCSB, Santa Barbara, California 93106, USA
Prof. R. M. NisbetDepartment of EcologyEvolution and Marine BiologyUCSB, Santa Barbara, California 93106, USA
Prof. B. H. HarthornDepartments of Feminist StudiesAnthropology & SociologyUCSB, Santa Barbara, California 93106, USA
UC CEIN was established with funding from the US National Science Foundation and the US Environmental Protection Agency in 2008 with the mission to study the impact of nanotechnology on the environment, including the identifi cation of hazard and exposure scenarios that take into consideration the unique physicochemical properties of engineered nanomaterials (ENMs). Since its inception, the Center has made great progress in assembling a multidisciplinary team to develop the scientifi c underpinnings, research, knowledge acquisition, education and outreach that is required for assessing the safe implementation of nanotechnology in the environment. In this essay, the development of the infrastructure, protocols, and decision-making tools that are required to effectively integrate complementary scientifi c disciplines allowing knowledge gathering in a complex study area that goes beyond the traditional safety and risk assessment protocols of the 20th century is outlined. UC CEIN’s streamlined approach, premised on predictive hazard and exposure assessment methods, high-throughput discovery platforms and environmental decision-making tools that consider a wide range of nano/bio interfaces in terrestrial and aquatic ecosystems, demonstrates the implementation of a 21st-century approach to the safe implementation of nanotechnology in the environment.
Prof. H. A. GodwinDepartment of Environmental Health SciencesUCLA, Los Angeles, California 90095, USA
Dr. R. Liu, Prof. Y. CohenDepartment of Chemical & Biomolecular EngineeringUCLA, Los Angeles, California 90095, USA
Prof. J. Gardea-TorresdeyDepartment of ChemistryUniversity of TexasEl Paso, Texas 79902, USA
Prof. L. MädlerIWT Foundation Institute of Materials ScienceDepartment of Production EngineeringUniversity of BremenBremen, Germany
Prof. J. I. ZinkDepartment of Chemistry & BiochemistryUCLA, Los Angeles, California 90095, USA
small 2012, DOI: 10.1002/smll.201201700
T. Xia et al.essay
1. Introduction
The mission of the University of California Center for Envi-
ronmental Implications of Nanotechnology (UC CEIN) is to
use a multidisciplinary approach towards research, knowl-
edge acquisition, education and outreach to ensure the safe
implementation of nanotechnology in the environment. [ 1 ]
This mission is being accomplished by generating the funda-
mental knowledge that is necessary to understand the role
of the physicochemical properties of engineered nanomate-
rials (ENMs) in determining their environmental fate, trans-
port, bio-accumulation, and hazard generation at the nano/
bio interface. [ 1,2 ] The Center makes use of well-characterized
ENM libraries to study exposure in parallel with the materials’
bioavailability and potential to engage toxicological path-
ways in organisms and environmental life forms ( Figure 1 ).
Where possible, this exploration involves high-throughput
screening (HTS) approaches to develop structure–activity
relationships (SARs) that can be used to predict the impact
of ENMs on organisms in freshwater, marine, and terrestrial
environments. [ 3 ] In silico data transformation and decision-
making tools are used for data processing to provide hazard
ranking, exposure modeling, and development of SARs for
ENMs (nano-SARs). [ 3d , 4 ] The center also has an important
education and outreach mission to train the next generation
of nano EHS experts and to discuss the importance of our
work with the general public, scholars, government agencies,
policy makers and industrial stakeholders. [ 1a ] Collectively,
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Figure 1 . Multidisciplinary research in UC CEIN. Well-characterized compscreening, using HTS where possible, to establish structure–activity relincreasing trophic series of environmental life forms. Select ENMs are alsoexposure in terrestrial and aquatic ecosystems. Data across the centertransformation and decision-making tools to provide hazard ranking, exby-design strategies.
these activities contribute to evidence-based nanotechnology
environmental health and safety (nano EHS) for society. In
this communication we will review the scientifi c progress of
UC CEIN since its inception in 2008, with the view of dem-
onstrating the integration of materials science, chemistry,
biology, toxicology, ecology, engineering, computer science,
law, public health, occupational medicine, and social sci-
ence into the multidisciplinary platform required to make
advances in this important and complex study area.
2. UC CEIN Organization Supports Multidisciplinary Research
The science in UC CEIN is carried out in four major
thrusts ( Figure 2 ). The fi rst thrust involves nanomate-
rial acquisition with a view to use HTS of ENM libraries
to understand structure–activity relationships at the nano/
bio interface. [ 3b , c , 4c , 5 ] This task is carried out by material sci-
entists and chemists who acquire and synthesize compo-
sitional and combinatorial ENM libraries that are used to
assess the ENM physicochemical properties that could con-
tribute to hazard generation in cells, bacteria, yeast, zebrafi sh
embryos, terrestrial and aquatic life forms. [ 3b , c , 6 ] Where pos-
sible, the hazard assessment is carried out by automated HTS
in the Molecular Shared Screening Resource (MSSR) in the
California NanoSystems Institute (CNSI). [ 1c , 3a–c , 4c , 5 ] The rich
data sets emerging from the HTS are deposited into our
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ositional and combinatorial ENM libraries are being used in toxicological ationships that help to prioritize testing of the same ENM libraries in an used for fate and transport studies and multimedia analysis to determine
are being collected in a data repository, which is used for in silico data posure modeling, development of quantitative SARs (QSARs), and safe-
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A Multidisciplinary Approach to Solve Complex Nano EHS Problems by UC CEIN
Figure 2 . Integrated UC CEIN research thrusts and themes. The science in UC CEIN is carried out in four major thrusts. The fi rst involves the acquisition of ENM libraries for HTS and in silico data transformation to establish structure–activity relationships at the nano/bio interface. The second major thrust looks at the impacts of materials selected from the libraries on a series of trophic life forms in terrestrial and aquatic ecosystems. The third examines environmental modeling through the use of environmental fate and transport lifecycle analyses. The fourth is engaged in education, risk perception and outreach activities that that translate knowledge generation to students, experts, the public and industry stakeholders.
data repository, enabling computer scientists and environ-
mental engineers to use nanoinformatics tools, such as sta-
tistical analyses and machine learning for data visualization
(e.g., heat maps and Self-Organizing Map), hazard ranking,
and building structure–activity relationships (SARs). [ 3d , 4 ] The
second major thrust looks at the impacts of selected materials
from the hazard ranking in the fi rst thrust on terrestrial and
aquatic ecosystems. [ 7 ] The terrestrial theme emphasizes the
ENM impact on microbes and plants, while the aquatic theme
looks at freshwater and marine planktonic and invertebrate
organisms. [ 5a , 7a , e–h , 8 ] Both environmental themes are focused
on ENM impacts on ecosystem services (e.g., nutrient cycling,
food webs, and biodiversity) and ecological processes (e.g.,
growth, primary production, and trophic transfer). [ 7c–g , 8g , 9 ]
The ecosystems studies also involve the development of
dynamic energy budget (DEB) models that quantify and inte-
grate the ecosystem impacts across scales and life stages. [ 7g , 10 ]
The third major thrust examines environmental modeling
through the lens of environmental fate and transport life-
cycle analyses. [ 7a , j − l, 11 ] In combination with multimedia mod-
eling tools developed by the nanoinformatics group, this
research is used for ENM environmental decision analysis
and modeling of the environmental exposure scenarios. [ 3d , 4,12 ]
The fourth thrust is engaged in societal implications, educa-
tion and outreach activities that generate new knowledge
about societal contexts for ENM risk and also translates our
research, knowledge acquisition and decision-making to stu-
dents, experts, the public and industry stakeholders. [ 13 ] For
© 2012 Wiley-VCH Verlag Gmsmall 2012, DOI: 10.1002/smll.201201700
more comprehensive information on the organization and
integration of UC CEIN, please refer to our website (http://
www.cein.ucla.edu/).
3. Establishment of the Basic Tools, Protocols, and Multidisciplinary Platforms to Assess the Environmental Impact of Nanotechnology
The materials science and chemistry group is responsible for
the acquisition and characterization of nanomaterials that
are produced in large volume and have been earmarked for
study by the Organization of Economic Cooperation and
Development (OECD), an international economic organi-
zation that works to stimulate economic progress and world
trade in more than 30 countries. [ 14 ] To date we have acquired
more than 30 material compositions from commercial sources
as well as in-house synthesis using sol-gel, hydrothermal, and
fl ame spray pyrolysis techniques. [ 3b , c , 4c , 6c , g , j ] These include
major categories of metal, pure metal oxide, doped metal
oxide and silica nanoparticles, as well as single- and multi-
wall carbon nanotubes (CNTs).
To standardize our procedures for the experimental use
of these materials, participants across the Center initially
focused on three metal oxides (TiO 2 , CeO 2 , and ZnO) to
develop protocols for the characterization, handling and
dispersion of these nanoparticles before conducting cel-
lular, bacterial, organismal, and ecological studies. [ 1c , 6j ] Key
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Figure 3 . The establishment of compositional and combinatorial libraries. The Center acquires, synthesizes, and characterizes silica, metal, metal oxide, and carbon libraries to understand the role of material compositions as well as accentuation of selected material properties in hazard generation at the nano/bio interface. The combinatorial libraries involve systematic variations in particle size, shape, aspect ratio, surface charge, functionalized surface groups, crystallinity, surface reconstruction, band gap energy, dissolution chemistry, photoactivation, and hydrophobicity to establish structure–activity relationships at the nano/bio interface.
to the implementation of these materials
was the requirement to develop appro-
priate dispersion protocols to allow the
safety assessment at the nanoscale level
in a range of biological and environmental
exposure media. [ 8c , 11h ] Systematic investi-
gations were carried out to assess particle
dispersion in six different biological media
as well as in a series of freshwater and sea-
water exposure conditions. [ 11h ] Because of
the complexity of studying colloidal par-
ticle suspensions one material at a time, we
implemented a high-throughput dynamic
light scattering (HT-DLS) approach
to expedite the assessment of particle
agglomeration and dispersal. All HT-DLS
analyses are performed in 384-well micro-
titer plates. Since each measurement only
takes 3–5 s, usually ten runs are collected
for each well and samples are loaded in
triplicate. The built-in kinetics feature also
allows us to evaluate the stability of nano-
particle suspensions under different condi-
tions in various media. [ 8c , 11g , h , 15 ]
For the purpose of cellular experi-
ments, we demonstrated that fetal bovine
serum (FBS) was the most effective dis-
persion agent, principally due to the synergistic effect of
various protein components, which attaches to most particle
surfaces to provide electrosteric hindrance. [ 15 ] We note that,
while serum albumin and FBS provide a rapid and conven-
ient way for stabilizing particle suspensions for the pur-
pose of high content and high throughput screening, this
approach does not deal with the dynamics and complexity
of multiple possible protein coronas, which depending on
the biological context could infl uence the uptake and fate
of nanoparticles. [ 2b , 16 ] However, the compromise was neces-
sary to allow ENM screening to proceed as a necessary step
towards wide-scale implementation of multiple materials in
the center. [ 3b , c , 5a , 6g ] The lessons learned from HTS to improve
nanoparticle suspension stability in cell culture media also
had an impact on the assessment and selection of environ-
mentally relevant dispersion agents, e.g., natural organic
matter (NOM), alginic acid, humic acid, fulvic acid and
tannic acid, for the performance of environmental exposure
studies, including for zebrafi sh experiments. [ 3c , 5b , 8c , 11h , 15 , 17 ]
It is also worth mentioning that serum albumin as an abundant
amphiphilic carrier protein in extracellular fl uid, including
in lung lining fl uid, has been used effectively to provide dis-
persion and suspension standardization of CNTs, thereby
allowing comparative studies of SWCNT and MWCNT effects
in the lung. [ 6f–h ] Although it is impossible to fi nd a universal
dispersing agent, some criteria that apply when selecting a dis-
persing agent are that the agent be environmentally or bio-
logically relevant, biocompatible with organisms of interest,
and able to adsorb to the nanoparticle surface via electrostatic
or (electro)steric binding. [ 15 ] The successful implementation
of the fi rst oxide nanoparticle library and the series of inter-
disciplinary activities following from there are described later.
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In addition to the introduction of different nanoparticle
compositions, the materials science group was responsible for
producing combinatorial ENM libraries in which deliberate
property accentuations are used to understand the contri-
bution of systematic variation in particle size, shape, aspect
ratio, surface charge, surface functionalization, crystallinity,
surface reconstruction, band gap energy, dissolution chem-
istry, photoactivation and hydrophobicity in ENM interac-
tions at the nano/bio interface ( Figure 3 ). [ 1b , c , 3a , 6n ] The use of
a large number of compositional and combinatorial libraries
has allowed the Center to establish several SARs that will be
discussed later.
The availability of compositional and combinato-
rial libraries enabled the HTS group to initiate a series of
mechanism-based toxicological assays carried out by robotic
handling of the nanoparticles as well as cells and bacteria in
the MSSR core facility ( Figure 4 ). [ 1b , c , 3a , 5a , 6n ] The implemen-
tation of HTS was facilitated by the discovery and explo-
ration of toxicological injury mechanisms at the cellular
and biomolecular level that could also be used for under-
standing the pathophysiology of disease and injury to intact
organisms. [ 3b , c , 4c , 5c ] This allowed us to establish a number
of predictive paradigms in which the data from the in vitro
screening assays are used to prioritize the intact animal
studies. [ 1b , 6n ] We therefore developed a mechanisms-based
rather than a descriptive approach for our toxicological
assessment. [ 1b , 3b , 6n ] The major advance in the Center was the
delineation of a number of the robust injury mechanisms that
could be implemented for ENM screening as will be discussed
later. One major technical advance was the implementation
of a multi-parameter HTS assay that quantitatively assesses
the generation of toxic oxidative stress by epifl uorescence
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A Multidisciplinary Approach to Solve Complex Nano EHS Problems by UC CEIN
Figure 4 . High-throughput toxicological screening using cells and zebrafi sh embryos. In vitro HTS assays are developed based on the mechanisms of ENM toxicity. This includes use of a multi-parameter HTS assay that has been implemented to quantitatively assess the generation of cellular toxic oxidative stress by epifl uorescence microscopy. Other techniques used for HTS include UV–vis spectroscopy (cell growth and red blood cell lysis), bioluminescence (reporter gene assays), and multiplex assays for cytokine and chemokine quantifi cation. Implementation of HTS in organisms also making use of robotic equipment to pick and plate zebrafi sh embryos, which is followed by automated bright fi eld and fl uorescence microscopy. Bright fi eld imaging is used to study interference in embryo hatching and development abnormalities, while fl uorescence microscopy is used for studying the induction of reporter gene responses in transgenic animals, e.g., expression of heat shock proteins.
microscopy, which uses a cocktail of fl uorescent dyes to detect
total cell number, nuclear size, reactive oxygen species (ROS)
production, perturbation of the mitochondrial membrane
potential, intracellular calcium fl ux, and cell death. [ 3b , c , 4c , 5c ]
A later section will delineate how the use of this screening
could be used to develop a predictive toxicological paradigm
for oxidative stress and infl ammation. In addition to HTS in
mammalian cell lines, we also implemented HTS on bacterial
cells using a library of E. coli gene deletion strains. [ 5a ] Using
a comparative assay, in which the growth of over 4000 gene
deletion strains in the presence and absence of nanomaterial
was compared to that of the parent strain, we were able to
determine that disruption of the outer membrane is a primary
mechanism of nanoparticle-induced damage for positively-
charged nanomaterials in bacteria. [ 5a ] We also introduced
zebrafi sh embryos as a model for organism-based HTS, which
can be performed by robotic equipment and automated
bright fi eld and fl uorescence microscopy that assesses inter-
ference in embryo hatching, developmental abnormalities
and induction of transgenic stress responses. [ 5b , 18 ] We discuss
the use of this platform for HTS for toxicological ranking of
metal oxides below.
Analysis of the environmental impact of ENMs required
the implementation of in silico tools and information sources
that provide data on the toxicity of ENMs and levels of
human and ecological exposures (e.g., concentration ranges
and exposure periods). [ 1b ] The fi eld of nanoinformatics has
emerged over the last few years and aims to develop and
implement effective mechanisms for collecting, validating,
storing, sharing, analyzing, modeling, and applying information
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinhesmall 2012, DOI: 10.1002/smll.201201700
pertinent to nanotechnology and its envi-
ronmental and societal impacts. [ 1b ] A key
challenge in nanoinformatics lies in estab-
lishing interoperability of data reposi-
tories containing heterogeneous datasets.
The Center has established a computa-
tional cluster and a computerized data
repository to accommodate structured and
unstructured datasets with a specialized
search engine called the NanoCrawler.
The NanoCrawler and data repository are
integrated with the CEIN ENM library
(NanoCatalog) of now over one hundred
different types or compositional varia-
tions of nanomaterials. This integration is
accomplished via metadata and fi le con-
tent, which enable effi cient searching and
dynamic reporting of data, protocols and
other relevant experimental information.
The CEIN data repository includes ENM
toxicity data for various cell lines and
simple organisms, physicochemical proper-
ties of nanomaterials, experimental proto-
cols, project reports and published articles.
The data repository resides on a central
server, is web-accessible and can be linked
with models designed for cloud-based
computing. For example, the CEIN high
throughput data analysis tool (HDAT) is
accessible via the web (http://nanoinfo.cein.ucla.edu/public/
hdat/default.aspx) and interfaces with the CEIN data reposi-
tory to process HTS plate data (to identify and remove out-
liers and systematic plate-to-plate variability and to defi ne
statistically meaningful toxicity metrics across plates). This
tool can also be used for “hit” identifi cation and cluster
analysis to identify similarity/dissimilarity in toxic behavior/
pattern among ENMs in the repository. The analyzed data is
then accessible for the development of toxicity-based QSARs
for nanomaterials. [ 3d , 4b ] The CEIN computational cluster also
provides various in-house models such as the web-accessible
model for assessing the environmental multimedia environ-
mental distribution of nanomaterials (MendNano) and the
system for environmental hazard ranking nanomaterials
(EHR-Nano).
To estimate actual exposure concentrations in the envi-
ronment and in the CEIN’s exposure studies, the fate and
transport group is using several approaches, including life
cycle assessment (LCA), aggregation, dissolution and fi ltra-
tion studies, soil infi ltration and transport in groundwater,
as well as quantitative multimedia modeling of the dynamic
mass distribution of ENMs in different environmental com-
partments (e.g., water, sediments, and biological tissues)
( Figure 5 ). [ 7g , h , 11h , m–o , 17,19 ] LCA provides estimates of the
emissions of ENMs at different stages (e.g., synthesis, incor-
poration into different products, product use, and disposal
at the end of life) and into different environments (e.g.,
the atmosphere, water, surface soils, groundwater, or cov-
ered landfi lls). [ 20 ] Given the novelty of many applications
of ENMs, information on the incorporation and potential
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T. Xia et al.essay
Figure 5 . Fate and transport processes for ENMs in an aquatic environment. We study abiotic and biotic fate and transport processes for ENMs in freshwater and marine environments, in which the colloidal behavior determines, for example, bioprocessing by phytoplankton, interactions with water column fi lter feeders such as mussels, or benthic feeders such as amphipods. Modeling of these exposures is then used to plan ecosystem studies in pelagic and benthic organisms.
release of ENMs from different products is either not pub-
licly available or does not exist. A probabilistic framework
for estimating emissions based on available scientifi c and
market information is being constructed using CNTs as a
fi rst case study. [ 21 ] Once the ENMs enter the environment,
their distribution is governed by different fate and transport
processes. [ 11h ] Studies on aggregation, dissolution and overall
mobility of ten different metal and metal oxide ENMs in dif-
ferent natural waters (e.g., storm water, river, groundwater,
seawater, and wastewater), in combination with studies
in synthetic waters in a range of pH values, ionic strengths
and concentrations of natural organic matter, provide the
information needed to parameterize a numerical model that
estimates the concentration of ENMs as a function of time
in different environmental compartments. [ 11h ] Collaboration
with the scientists studying the biological response to ENMs
has provided information on the bioaccumulation and bio-
processing of ENMs as they are taken up, metabolized and
excreted by various organisms. [ 11i ] The group also developed
protocols for dispersing ENMs in natural waters, with natural
organic matter or alginate as the key natural dispersants for
studies with stabilized ENM suspensions. [ 3c , 11h , 17 ]
Hazard ranking of materials based on their proper-
ties and HTS results allows UC CEIN to study ecosystem
impact by using ecological function-oriented experiments
rather than acute mortality assays. [ 1b ] The Center evaluates
hazards of ENMs to biota in freshwater, marine, or terres-
trial environments, with a focus on impacts to ecological
functions such as primary production and, by extension,
the ecosystem services these functions support, including
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food production, nutrient cycling, and climate regulation.
Ecosystem services can be defi ned as the conditions and
processes of natural ecosystems that sustain human life. Eco-
systems providing these services are organized hierarchi-
cally, with levels of organization spanning from the organism
(species) to populations to communities made up of several
to hundreds of species. Our research is similarly organized,
focusing fi rst on ENM effects on organisms and populations
and the consequences of those effects on rates of ecosystem
functions and processes such as respiration, primary produc-
tion and grazing or predation rate. [ 7c , d , h , 8d , 11i , k ] Our research
is prioritized around determining the potential for hazards
and the nature of the impacts, including the underlying
mechanisms. In the terrestrial research, we focus on plants,
microorganisms, and plant-microbe symbioses that affect
agriculture. [ 7c , d , h , j–l , 8a , b , d–g , 9 , 11g ] Hydroponic plants (e.g., toma-
toes, cucumber, or native plants) are studied to determine
fundamental plant and population responses when ENMs
are fully bioavailable, i.e., not bound to soil clays or organic
matter. [ 7c , d , h , j–l , 8a , b , d–g ] Similarly, microbes are studied as popu-
lations to understand ENM impact mechanisms and effects
on population growth, which control functions such as
nutrient cycling. [ 5a , 7d , 9 , 11g ] Since microbial taxa can be func-
tionally narrow, e.g., nitrogen fi xation or methane oxidation,
delineating impacts in such taxa is only realistically per-
formed within community exposures, such as whole soils. [ 7c , d ]
We assess planted soil systems to determine if ENMs are bio-
available to plants and microbes in the complex soil environ-
ment, how effects vary from population studies that are more
suitable for screening purposes, and whether plant-microbe
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A Multidisciplinary Approach to Solve Complex Nano EHS Problems by UC CEIN
Figure 6 . Demonstration of the impact of the development of harmonized protocols and assays following the introduction of the fi rst MO x library. The fi rst nanoparticle library, which was composed of well-characterized TiO 2 , CeO 2 , and ZnO nanoparticles, were screened for their toxicity profi les in vitro and the major mechanisms of toxicity were identifi ed. The fi ndings were used to guide studies in zebrafi sh embryos and microcosms, which resulted in environmental modeling and decision making. The development for harmonized protocols to handle this small set of materials across different disciplines in the Center allowed to progress from single to multi-parameter HTS assays, increased ability to make predictions, test more materials, establish zebrafi sh HTS, elucidated an increased number of toxicity mechanisms (dissolution chemistry, photoactivation, long AR toxicity), and identifying a material property (dissolution) for safer design (doping).
interactions (symbioses only assessable
at this scale) are especially susceptible to
ENM exposure. Aquatic studies include
species of freshwater and marine phyto-
plankton as primary producers and
daphnia and copepods as primary con-
sumers linking photosynthesis to higher
trophic levels. [ 7e , f , 11i ] This framework
allows evaluation of a spectrum of pos-
sible effects, especially direct effects on
the organisms and population growth
rate and, consequently, on rates of photo-
synthesis or grazing, indirect effects on
grazers via trophic transfer of contami-
nants and impacts to higher trophic levels
due to decreased resource supplies. [ 7e , f , 11i ]
Further integrative approaches are
required for understanding the effects of
nanomaterials on populations and eco-
sytems. Given the diversity of potentially
impacted organisms, habitats and life
stages, and also the high cost and long
duration of most ecological experiments,
it is essential to approach this ecological
challenge through a quantitative, con-
ceptual framework that takes maximum
advantage of the deep understanding
of organismal and sub-organismal proc-
esses emerging from the studies described
above. The approach taken at UC CEIN recognizes that
many potential impacts are mediated through energy trans-
duction processes, and we use dynamic energy budget (DEB)
theory to make connections across levels of biological
organization. [ 8g , 10a ] DEB theory focuses on the individual
organism and uses systems of kinetic equations to describe
the rates at which organisms assimilate and utilize energy
and elemental matter for maintenance, growth, reproduc-
tion, development and toxicokinetics. Established modeling
methodology in ecology allows straightforward connections
to population dynamics through the use of “structured” or
“individual-based” modeling techniques. [ 7g , 10b ] DEB-based
models thus allow us to predict/project effects of nanomate-
rials in the environment on population growth rates and to
model the impact of multiple stressors acting simultaneously.
The connection to the mechanistic studies described above is
made by recognizing that some parameters in DEB models
may be directly related to sub-organism processes. We illus-
trate these later through case studies on the effects of CdSe
quantum dots on bacterial population growth, and ZnO on
growth of marine mussels.
Accompanying the research activities of the center are
educational and outreach activities, including mentoring and
professional development programs; course development,
workshops, and learning tools; a protocols working group, which
develops and disseminates standard protocols for studying the
environmental implications of nanotechnology used across
the Center; public outreach; and other synergistic/integrative
center activities. [ 1a , 13e ] The latter includes societal implications
research that has demonstrated the importance of surveying
© 2012 Wiley-VCH Verlag Gsmall 2012, DOI: 10.1002/smll.201201700
critical stakeholders about their perceptions and beliefs about
risks to the environment, conducting research to understand
the factors that contribute to those perceptions and beliefs, and
acting upon the insights generated from those studies. [ 1a , 13e ] As
a result, we now have an empirical base for understanding how
to engage the public in a way that builds trust for both academic
researchers and for nanotechnology, as well as the priorities of
critical stakeholders when it comes to both the regulation and
deployment of nanotechnology. [ 1a , 13e ] The impacts of both the
educational activities and the societal implications research are
discussed in the impact section below.
4. Case Studies of the Impact of the Multidisciplinary Nano EHS in UC CEIN
4.1. Demonstration of the Impact of Multidisciplinary Research and Harmonized Procedures following the Introduction of the First Oxide Nanoparticle Library
The fi rst nanoparticle library, which was composed of TiO 2 ,
CeO 2 , and ZnO nanoparticles, was synthesized in-house and
rigorously characterized following development of proto-
cols that allow ENM physicochemical characterization under
a variety of biological and environmental use conditions
( Figure 6 ). [ 6j ] One example was the development of single
parameter cellular, bacterial and yeast toxicity assays such as
cytotoxicity, metabolic activity, growth, oxidant injury and ini-
tiation of pro-infl ammatory responses. [ 6j ] While these assays
demonstrated a relatively high level of cellular toxicity for
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T. Xia et al.
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Figure 7 . ENM-related injury mechanisms that have been implemented to explore the relationship between ENM physicochemical properties and adverse biological events at the cellular and biomolecular level. A: Oxygen radical generation and oxidative stress in relation to material properties that determine redox activity, including electronic properties; B: Photoactivation, leading to electron hole pair generation and ROS, C: Dissolution and release of toxic metal ions; D: Cationic injury leading to surface membrane and organellar damage; E: Cell membrane lysis by high surface reactive materials such as Ag plates or surface silanols in fumed silica; F: Infl ammasome activation by long AR materials; G: Pro-fi brogenic responses by long AR material such as CNTs and CeO 2 nanowires; H: Zebrafi sh embryo hatching interference by metal ions that interfere in hatching gland metalloprotease activity (ZHE1).
ZnO but not TiO 2 and CeO 2 nanospheres
under tissue culture conditions (room light
or dark incubator), we did observe the
emergence of toxicity under environmental
conditions in which phytoplankton was
exposed to bright sunlight (which leads to
photoactivation of TiO 2 ), or where a mate-
rial shape change (e.g., CeO 2 nanowires)
could induce lysosomal damage that was
not seen with spherical nanoparticles. [ 6c , 7e ]
The nano-ZnO toxicity could be ascribed to
extra- and intracellular release of toxic Zn 2 + ,
which has the capacity to generate reactive
oxygen species (ROS), perturb mitochon-
drial function, and initiate IL-8, IL-6, and
TNF- α production. [ 3b , 6j , 18 , 22 ] Severe oxi-
dant injury culminates in cytotoxicity, oth-
erwise known as toxic oxidative stress. [ 2a ]
Many of these cellular injury responses are
duplicated in the human and animal lung,
where the acute pro-infl ammatory effects
of nano-ZnO result from oxygen radical
generation. [ 18 ] Based on the involvement
of oxidative stress as a key mechanism of
metal oxide (MO x ) toxicity, we developed a
multi-parameter HTS assay to track lethal
and sub-lethal oxidative stress responses
to ROS-generating or ROS-inducing ENMs. [ 3b ] The introduc-
tion of the HTS assay made it possible to screen an increasing
number of MOx, ultimately leading to the evaluation of 24
oxide nanoparticles in one experiment. [ 3c , 4c ] The high data
volume allowed us to develop a SAR based on cellular ROS
production, which will be discussed later. The lessons learned
from cellular and bacterial studies also allowed introduction of
the MO x library to terrestrial and aquatic ecosystems, including
the ability to perform comparative studies in bacteria, plants,
oysters, phytoplankton, and other organisms, and to relate the
nanoparticle effects to ROS production and particle dissolution
with shedding of toxic ions. [ 5b , e , f , 18 ] This includes a demonstra-
tion of hatching interference in zebrafi sh embryos by nano-
ZnO followed by development of a zebrafi sh HTS platform as
described below. This work identifi ed the role of the metallo-
protease, ZHE1, a target for Zn 2 + as well as other metal ions
(see below) in hatching interference of zebrafi sh embryos. [ 5b , 18 ]
The demonstration of the importance of dissolution chemistry
in ZnO toxicity was responsible for the construction of the fi rst
combinatorial library, in which Fe doping was used to change
the rate of ZnO dissolution, leading to the fi rst demonstration
of a safer design procedure in CEIN. [ 3b , 18,23 ] Taken together,
the integration of the Center’s activities around a limited set
of well-characterized ENMs and protocol development was
instrumental in the subsequent integration of progressively
more multidisciplinary science.
4.2. Use of Toxicological Injury Pathways to Develop HTS and Predictive Toxicology
The mechanistic toxicology and HTS group have identifi ed
a number of ENM-related injury mechanisms that can be
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used to explore the relationship between material properties
and adverse biological events at cellular and bio molecular
level. [ 1b , 6n ] We have chosen cellular mechanisms that are
engaged in disease pathogenesis or in vivo toxicological
responses ( Figure 7 ). The reason for focusing on toxicological
injury pathways rather than primary use of discovery tools
such as genomics and proteomics is the considerable data
reduction and pathway analysis that is required before these
analytical tools can be applied to HTS. [ 1b , 6n ] However, we do
envisage that these discovery platforms will become increas-
ingly important as new material properties emerge that could
elicit toxicological effects independent of currently known
injury pathways. Figure 7 depicts a few of the salient injury
pathways that we have implemented for in vitro screening
regarded as potentially useful for predictive toxicological
approaches. This includes the generation of ROS and oxida-
tive stress as the best described injury mechanism linked to
nanoparticles, both engineered as well as those present in
polluted air. [ 4c , 6i ] Examples of the redox-active ENMs include:
(i) MO x capable of cellular ROS production through catalysis
of electron transfers from biological redox couples (such as
CoO, Co 3 O 4 , Cr 2 O 3 , Ni 2 O 3 , Mn 2 O 3 ) to the material conduction
band (see below); [ 4c ] (ii) materials capable of photo activation
under UV exposure conditions, leading to the generation of
electron hole pairs (e.g., TiO 2 ); [ 6b , 7e ] (iii) material dissolution
or shedding of toxic ions that induce cellular ROS produc-
tion (e.g., ZnO, CuO); [ 4c , 6j ] (iv) material surface defects that
catalyze ROS production (e.g., silica made under high tem-
perature conditions). Based on the oxidative stress paradigm,
we developed a multi-parametric in vitro HTS assay that
can detect lethal and sublethal oxidative stress responses in
cells, including ROS generation, intracellular calcium fl ux,
mbH & Co. KGaA, Weinheim small 2012, DOI: 10.1002/smll.201201700
A Multidisciplinary Approach to Solve Complex Nano EHS Problems by UC CEIN
mitochondrial membrane depolarization, and membrane
damage. [ 3b , c , 4c , 5c , 6b , i , j ] Using this platform, we can contempo-
raneously assess oxidant injury responses at multiple time
points and doses to generate high content data for hazard
ranking and SAR analyses. [ 3b ] Since the critical steps in the
assay (e.g., cell seeding, liquid handling, imaging, image anal-
ysis, etc) are automated, we were able to complete the multi-
parametric assay in a single day compared to the 2–3 weeks
that are required to complete a full set of individual oxidative
stress assays at each tier of oxidative stress. [ 3b , 4c , 5c ] In addi-
tion to oxidative stress, we have also developed other mecha-
nistic assays including lysosomal damage by cationic particles
(e.g., cationic polystyrene and polyethylenimine coated silica
nanoparticles). [ 5c , 6k , l ] The so-called “proton sponge effect”
triggers a series of cellular responses that result in mitochon-
drial injury and cell death. [ 5c , 6k , l ] Another example is material
surface reconstruction or surface defects (e.g., silanol groups
on SiO 2 and surface defects on Ag-plates), which can disrupt
the integrity of the cell membrane, leading to hemolysis or
cell death. [ 6a ] Because cellular mediated ROS production
also occurs downstream of ENM mechanisms that does
not primarily involve oxygen radical generation, the multi-
parametric assay is also useful for performing HTS on some
of these nanomaterials. [ 5c , 6a ] Finally, high aspect ratio nano-
materials, (e.g., Ag, CeO 2 nanorods or nanowires and carbon
nanotubes) can induce lysosomal damage, infl ammasome
activation, and IL-1 β production in macrophages, which play
a role in lung infl ammation and fi brosis. [ 6c , f , h ] Currently we
are developing HTS assays to screen those long aspect ratio
(AR) materials.
4.3. Use of Compositional and Combinatorial ENM Libraries to Assist in the Development of Predictive Toxicological Paradigms
To establish predictive toxicological paradigms, it is necessary
to link the physicochemical properties of ENMs to biological
outcomes. [ 1b , 6n ] To do so in a systematic manner, it was nec-
essary to establish compositional as well as combinatorial
ENM libraries. [ 1b , 6n ] We began making toxicological com-
parisons for a number of well-characterized primary material
compositions, which mostly involved nanospheres of approxi-
mately the same size. [ 3c , 4c , 6j ] However, several properties
emerged other than chemical composition that could impact
toxicological outcomes as described above. To more clearly
relate specifi c physicochemical properties to biological out-
comes, we introduced combinatorial libraries in which mate-
rials with the same chemical composition were synthesized
to accentuate specifi c properties such as size, shape, aspect
ratio, crystal structure, surface functionalities, solubility, etc
(Figure 3 ). This has allowed us to relate characteristics such
as nanoparticle size to a biological outcome, including cel-
lular uptake and biodistribution. [ 6d ] Length and AR are addi-
tional important factors that determine ENM behavior at
the cell membrane and intracellularly. [ 6c , 6e ] For instance, we
have demonstrated in mammalian cells that, for a series of
silica nanorods, there is a preferred AR for cellular uptake
premised on rod length. [ 6e ] Rod length determined assembly
© 2012 Wiley-VCH Verlag Gmsmall 2012, DOI: 10.1002/smll.201201700
of the cortical cytoskeleton through mediating the activation
of small GTP-binding proteins that control fi lopodia forma-
tion and macropinocytosis. [ 6e ] We also constructed a CeO 2
nanorod and nanowire library to demonstrate that a sys-
tematic increase of rod/wire length could change relatively
inert nanospheres to a toxic material, in which an AR above
a critical threshold can induce lysosomal damage, IL-1 β pro-
duction, and cytotoxicity. [ 6c ] CNTs represent another long
AR material in which the state of tube purity, hydrophobicity,
aggregation or dispersion could be shown to determine the
ability of a number of CNT libraries to generate lysosomal
damage and IL-1 β production; these biological responses
ultimately determine the CNTs’ propensity to induce chronic
infl ammation and fi brosis in the lung. [ 6h ] Surface charge is
an important property that, in addition to affecting particle-
particle or particle-cell interactions, can determine biological
hazard at the surface membrane or the lysosome. [ 5c , 6k , l ] By
constructing a combinatorial library of silica nanoparticles
with different surface silanol and siloxane concentrations, a
quantitative correlation between silica surface properties and
their toxicological potential was established. A defi ning fea-
ture appears to be the temperature conditions under which
the silica nanomaterials are made and their state of hydra-
tion. Thus, both fumed silica and a crystalline material like
α -quartz contain highly energetic 3- or 4-membered sioxane
rings that can reconstruct under hydration conditions to dis-
play closely spaced, H-bonded silanol groups at the particle
surface. [ 24 ] These silanols participate in surface membrane
damage as well as generating hydroxyl radicals, which lead to
cellular toxicity and excitation of pro-infl ammatory effects. In
contrast, amorphous silica nanoparticles made under lower
temperature synthesis conditions (e.g., Stöber silica, and
mesoporous silica) do not display the same toxicological fea-
tures. When dealing with semiconducting materials (e.g., low
solubility oxide nanoparticles) it is possible to use band gap
energy as well as the conduction band energy levels to delin-
eate predictive toxicological effects. [ 4c , 6b ] This is discussed in
case study #5. In contrast, for soluble metal oxides like ZnO
and CuO, their adverse biological effects can be attributed
to ion shedding, which generates oxidative stress as well as
inhibits the zebrafi sh metalloprotease (ZHE1), responsible
for embryo hatching. [ 4c , 5b , 6j , 18 ] By doping ZnO with Fe and
Al, the ZnO dissolution rate could be effectively reduced,
leading to decreased cellular toxicity. [ 3b , 18 ] Feedbacks pro-
vided by the development of SARs have also allowed us to
demonstrate the introduction of possible safer by design fea-
tures for CNTs and Ag-nanoplates. [ 6a , f ]
4.4. The Development of (Q)SARs and Hazard Ranking Tools Allows the Development of Predictive Toxicological Paradigms
Assessment of the potential environmental impact of ENMs
requires identifi cation and acceptance criteria or measures of
environmental/human health risks or other suitable impact
metrics. [ 1b ] Such analysis requires information regarding the
modes and release rates of ENMs to the environment, ENM
physicochemical properties, likely concentration ranges
in various environmental media, exposure pathways, and
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Figure 8 . Schematic illustration of experimental and analytical components for assessment of the environmental impact of nanomaterials. In order to answer the question “Is this engineered nanomaterial environmentally safe?”, information is needed regarding ENM hazard and exposure. Experimental studies and development of in-silico methods for both hazard assessment and exposure analysis require physicochemical characterization of ENMs. ENM hazard can be ascertained via experimental toxicity data (in vitro or in vivo) using both high-throughput (HT) and low-throughput (LT) methods, or predicted by an in silico approach using structure–activity relationships. Dose-response or hazard thresholds can then established and used, along with estimated environmental exposure concentrations (via fate and transport modeling or fi eld monitoring), to quantify potential environmental risks. Based on the assessed risk, decision analysis can be performed for the ENM of concern in order to arrive at protective measures at various levels in the ENM life cycle, including design, manufacturing, use approval, and exposure control.
assessment of toxic outcomes resulting from environmental
exposures to ENMs ( Figure 8 ). Given the rapid growth of
nanotechnology and thus additions of many different types
of ENMs, the CEIN has developed in silico approaches to
perform quantitative toxicity predictions as well as models
for ENM environmental fate and transport. [ 3c , d , 4 ] The use
of such models for environmental impact assessment (EIA)
considers cause and effect relationships involving multiple
interdependent ranking criteria. Accordingly, a signifi cant
part of the Center’s effort is devoted to developing nano-
structure–activity relationships (nano-SARs), using the large
center ENM toxicity data repository for a heterogeneous
library of nanoparticles and biological receptors. [ 3d ] Predic-
tions of either the probability of specifi c ENMs as being toxic
or of specifi c dose response metrics are based on knowledge
(which may be acquired through feature/descriptor selection)
of suitable nanoparticle descriptors (i.e., physicochemical and
structural properties) and environmental conditions (e.g.,
ENM concentrations and solution properties). For example,
nano-SARs that delineate the toxicity of metals and metal
oxide ENMs in various cell lines have enabled reliable pre-
dictions of toxicity based on information regarding ENM size
(e.g., primary and aggregate sizes), fundamental nanopar-
ticle properties (e.g., zeta potential and magnetic properties)
as well as energetic parameters (e.g., energy of atomization,
band-gap energy and energy of hydration). [ 4c ] To account
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for data uncertainties and the potential
impact on regulatory decisions that may
rely on nano-SARs, the UC CEIN nano-
SARs enable predictions under different
acceptance levels of false predictions of
toxic outcomes (i.e., false positive) or false
predictions of lack of toxic outcome (i.e.,
false negative). The developed nano-SARs
along with estimates of environmental
exposure concentrations can then be used
as an impact analysis process, combining
estimated exposures and toxicity metrics
to establish ENM hazard ranking. [ 3d , 4c ]
4.5. Elucidation of a Predictive Toxicological Paradigm Linking Metal Oxide Conduction Band Energy to Biological Redox Potential and Oxidant Injury
MO x nanoparticles represent an industri-
ally important category of nanomaterials
that is produced in high volume and fre-
quently used for their semiconducting
properties. [ 3d , 4c ] Although some metal
oxide nanoparticles, such as ZnO, have a
high toxicological potential, the majority
of metal oxides have not been systemati-
cally explored for hazard potential. [ 3b , c , 6j ]
It would be useful to develop a toxico-
logical paradigm that is premised on prop-
erty-activity relationships that will allow
us to predict the hazard of metal oxide
nanoparticles. Recently, it was suggested that the toxicity of
metal oxide nanoparticles could be correlated to their energy
structure property, such as conduction band energy (Ec) and
valence band energy (Ev). [ 4c ] The overlap of the Ec and Ev
with biological redox potential, which is determined by a
series of redox couples and ranges from − 4.12 eV to − 4.84 eV,
could promote electron transfer ( Figure 9 ). Based on this
theory, we acquired a metal oxide nanoparticle library con-
taining 24 types of metal oxides and experimentally deter-
mined their Ec and Ev, revealing that the Ec of CoO, Co 3 O 4 ,
Cr 2 O 3 , Ni 2 O 3 , Mn 2 O 3 and TiO 2 nanoparticles overlap with
the biological redox potential and therefore possibly capable
of generating oxidative stress in cellular systems. [ 4c ] In both
in vitro and in vivo toxicity assessment, these materials as
well as ZnO and CuO showed high toxicological potential
while the rest of the nanoparticles showed little or no toxicity.
Toxicity assessment demonstrated excellent correlation with
the original toxicity prediction, with the exception of TiO 2 ,
which was predicted to be toxic but did not generate cyto-
toxicity. This may be due to the fact that the Ec of TiO 2 is
too close to the boundary of the redox potential range, and
the boundary is not solid, leading to the Ec of TiO 2 being out
of the range. [ 4c ] Moreover, CuO and ZnO were not predicted
as toxic but showed toxicity, possibly due to their high metal
shedding in culture medium (based on ICP-MS analysis) and
metal ion release rather than electronic property involved in
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A Multidisciplinary Approach to Solve Complex Nano EHS Problems by UC CEIN
Figure 9 . Use of a band gap paradigm for predicting metal oxide toxicity in vitro and in vivo as a result of oxidative stress generation. A nanoparticle library including 24 types of metal oxides was established by in-house synthesis and commercially available sources. [ 4c ] The conduction band energy (Ec) and valence band energy (Ev) of each nanoparticle was experimentally determined and expressed in relation to the cellular redox potential range ( − 4.12 eV to − 4.84 eV). The biological redox potential is determined by a number of cellular redox couples such as cytochrome C (Fe 2 + /Fe 3 + ), NADP + /NADPH, etc. The overlap of Ec with the biological redox potential establishes permissible energy levels that may allow electron transfer from the redox couples to the metal oxide nanoparticles. Some of these electrons are transferred to molecular dioxygen, leading to the formation of ROS and oxidative stress. The induction of oxidative stress by metal oxide nanoparticles is a multi-tier event in which the generation of antioxidant defense (Tier 1) precedes the activation of pro-infl ammatory (Tier 2) and cytotoxic (Tier 3) responses at higher levels of oxidative stress. Based on this hierarchical oxidative stress paradigm, an in vitro multi-parametric high throughput screening (HTS) assay was developed in the center, and implemented for performing toxicological ranking of 24 metal oxide nanoparticles in a time and dose-dependent fashion. [ 4c ] This ranking was expressed in the form of a heatmap, which was used for prioritizing and performance of pulmonary exposures in mice, in which the propensity to generate oxidative stress in vitro accurately predicted the development of acute pulmonary infl ammation in mice.
their toxicity mechanism. [ 4c ] All of the above demonstrates
that it is indeed possible to establish a predictive toxicological
paradigm on the basis of conduction band energy to predict
the in vitro and in vivo toxicity of metal oxide nanoparticles.
4.6. Increased Environmental Mobility of Metal and MO x Nanoparticles Stabilized by NOM
Studies on the environmental factors that control aggregation
of metal (Ag, Pd, Pt, Fe), metal oxide (TiO 2 , CeO 2 , ZnO, CuO)
and metalloid (SiO 2 ) nanoparticles indicate that, for uncoated
spherical particles, aggregation is a strong function of particle
surface charge, as measured by their electrophoretic mobility
(EPM). [ 11h , 17 ] Above the EPM threshold, the nanoparticles
are stable and can remain suspended for days to weeks. [ 11h ]
However, NOM is ubiquitous in the environment and binds
strongly to all of these NPs. Once NOM binds to the surface
of the ENMs, the EPM for the composite is dominated by
adsorbed NOM, which is highly negative, resulting in stabi-
lization of the nanoparticles except under very high ionic
strength conditions such as in hard (high Ca 2 + and Mg 2 + )
groundwater or seawater. This indicates that for freshwater
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheismall 2012, DOI: 10.1002/smll.201201700
studies the ENMs will remain suspended
in the water column throughout the dura-
tion of the exposure, while for marine
studies the particles will settle out within
tens to hundreds of minutes, and thus the
exposure will be more signifi cant for ben-
thic organisms. [ 7b ] In addition, nano-ZnO
dissolves rapidly (within 12–24 h). [ 7f ] ZnO
NPs dissolve much more rapidly than
comparable bulk ZnO particles due to the
high surface area available for dissolution.
As shown in many CEIN toxicity studies,
the release of metal ions (e.g., Zn 2 + ) in
concentrated amounts results in toxic
outcomes. [ 7b , f , 8d , 18 ] In comparison, Ag, CuO
and Fe dissolve much more slowly, on the
order of weeks to months. [ 19b ] Their slow
dissolution combined with high mobility if
stabilized by NOM will result in a longer
term source of dissolved ions that can
have longer range impacts.
4.7. Elucidation of the Potential Impact of ENMs on Food Production as a Result of Affecting Important Plant Species and Microbes
We learned early on that MOx ENMs asso-
ciate with membranes of planktonic bacte-
rial populations, leading to a change in the
nanoparticles agglomeration behavior. [ 11g ]
Through soil-only microcosms, we discov-
ered that such associations probably occur
in soil and then lead to hazard generation,
since MOx nanoparticles (ZnO and TiO 2 )
alter soil bacterial community structure and were particularly
inhibitory to taxa crucial to N 2 fi xation, methane oxidation,
and complex C decomposition. [ 7c , d ] Prior research of other
ENMs in soils did not reveal signifi cant impacts. However,
depending on ENM type, our terrestrial studies clearly signal
that ENMs can be bioavailable in soil, and thus soil commu-
nity-level and ecosystem-level impacts should be understood
and mitigated ( Figure 10 ). Since soil bacteria promote soil
fertility, such fi ndings fuel the larger concerns for ENM risks
to agriculture and the food supply. [ 7i ] In assessing the poten-
tial and mechanisms of ENM effects on plants, we researched
responses of hydroponic soybean plants [ 8d ] and native desert
plants [ 8a , b ] to ZnO ENMs, discovering that plant uptake
occurred at levels causing stress; [ 8a , d ] analogously, CeO 2
translocated into hydroponic soybean root tissue and caused
genetic damage. [ 8a , d ] Such studies of plant populations fully
reveal the potential effects and well-inform assessments of
risks to commodity hydroponics exposed to ENMs via water
supplies or through aerial routes (e.g., deposition to leaves).
Taken together, our separate investigations of soil microbes
and plants show the potential for ENM effects at the popula-
tion scales and have greatly advanced the understanding of
effects potentials, and our investigations of complex (soil and
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Figure 10 . UC CEIN research model to show the impact of ENMs on an agricultural, are representative of a terrestrial ecosystem studies. Three ecological scales are portrayed, namely populations (upper left), communities (middle), and an ecosystem (right), to understand the potential effects and mechanisms of ENMs to plant (top left) and bacterial (bottom left) populations, to microbial communities in soil (middle), and to the entire ecosystem including plant-microbe root symbioses involved in N 2 fi xation, including the mature food crop. The model allows for rapidly screening populations for prioritizing campaigns and conditions at higher ecological scales. The highest scale, i.e., the ecosystem, contains fi eld elements (i.e., fruit and nodules) that are most relevant to societal concerns, and whose impacts can be explored in reverse, i.e., at population or community scales.
planted soil) systems have the potential to show that such
effects can manifest at ecosystem levels with ecological and
2
societal consequences.
Figure 11 . Impact of TiO 2 photoactivation on marine phytoplankton under environmental UV exposure conditions. Suspended TiO 2 nanoparticles (red dots) in sea water can easily adhere to phytoplankton cell membranes. Under UV exposure conditions, TiO 2 will generate ROS, which could induce protein and DNA oxidation as well as cell wall/membrane damage. The toxic effects of TiO 2 nanoparticles on marine phytoplankton under UV could scale up from the cellular level to affect population growth rate and ultimately the oceanic carbon cycle.
4.8. Impact of TiO 2 Photoactivation on Phytoplankton Toxicity under Environmental UV Exposure Conditions
Marine phytoplankton requires sunlight
to fulfi ll their role as the most important
photoautotrophs on Earth. [ 7e , 7f ] The
photoactivation paradigm described above
identifi ed TiO 2 as a potential mechanism
for oxidation and reduction reactions at
nanoparticle surfaces, including in cells
and organisms. [ 6b ] The well-known role
of irradiation in stimulating electron hole
pair formation in TiO 2 suggested that
phytoplankton in the marine photic zone
could be vulnerable to the redox injury
under UV exposure conditions ( Figure 11 ).
Traditional experimental protocols for
chronic ecotoxicological assays on phy-
toplankton, and other organisms, were
unsuited for evaluating the phototoxic
potential of TiO 2 in seawater due to the
relatively low levels of UV radiation. We
used an illumination system designed for
realistic evaluation of weathering of mate-
rials, combined with UVR-blocking acrylic
as a control, to test whether levels of UVR
seen in the upper photic zone of the oceans
could affect the toxic potential of TiO 2 . [ 7e ]
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After population-level experiments dem-
onstrated that UVR could indeed induce
TiO 2 toxicity of phytoplankton, we worked
with environmental chemists to identify
increased ROS production during UVR
and use of TiO 2 concentrations employed
in our biological experiments. Production
of OH • at low [TiO 2 ] in seawater with
simulated sunlight, measured using a
coumarin probe, was approximately 10–20
times higher than natural OH • generation
in untreated coastal marine waters. [ 7e ] We
confi rmed the presence of OH • by moni-
toring the formation of the dimethyl-1-
pyrroline N -oxide (DMPO)-OH adduct
using an in situ electroparamagnetic
resonance (EPR) spin trap. The relevant
EPR spectra were evident after only
20 min of illumination and, coupled with
the absorbance and fl uorescence data,
demonstrate the ability of TiO 2 to pro-
duce OH • in seawater. The experimen-
tally derived steady state [OH • ] was up to
2.5 × 10 − 15 M, nearly three orders of magni-
tude higher than that in natural untreated
seawater. [ 7e ] Since oxidative stress is an important force
shaping natural selection and physiology of marine organisms,
heim small 2012, DOI: 10.1002/smll.201201700
A Multidisciplinary Approach to Solve Complex Nano EHS Problems by UC CEIN
Figure 12 . DEB models on bio-effects of ENMs. DEB models describe energy fl ows and transformations within a single organism, and also between the organism and its environment (including ENMs). These computations allow prediction of biological effects on populations, communities and ecosystems (left panel). For example, DEB models were used to investigate how ROS production impacts CdSe quantum dot and bacterial interactions. Based on the experimental data on bioaccumulation and toxic effect of Cd(II) and CdSe to bacteria, a new predictive model was developed where the effects of intact quantum dots were distinguished from the effects of dissolved cadmium (right panel).
particularly plankton, these results imply that widespread
contamination of surface waters with TiO 2 could signifi cantly
add to the multiple anthropogenic stressors experienced by
coastal marine ecosystems. [ 7e ]
4.9. Use of DEB Modeling for Studying the Toxicity of Quantum Dots in a Terrestrial Ecosystem and ZnO in a Marine Ecosystem
We already discussed that DEB theory offers an integra-
tive approach for relating information on organism and
sub-organism processes to population dynamics. [ 10c ] In a
“proof-of-concept” study, we applied our approach to inter-
pret experiments using Pseudomonas bacteria, in which the
effects on population growth of CdSe quantum dots (QDs)
were compared with those observed in parallel experiments
using soluble cadmium salts. [ 8g ] We successfully developed a
comprehensive DEB modeling framework of cadmium effects
on bacterial population growth, and, with a limited number of
discrete and biologically-relevant parameters, demonstrated
the excellent ability of DEB modeling to represent experi-
mentally-derived exposure data ( Figure 12 ). [ 10a ] This is the fi rst
DEB model to invoke ROS as a mathematically-represented,
damage-inducing compound that impacts cell physiology
and population dynamics. We are now working on general-
izing our fi ndings beyond this specifi c experimental system
to contribute to the broader understanding of the potential
energetic basis for effects of more environmentally relevant
ENMs. Specifi cally, we are now developing a new DEB-based
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheismall 2012, DOI: 10.1002/smll.201201700
representation of ROS dynamics in cells
that allows tracking of ROS generation,
transformation, and accumulation of the
associated cellular damage. DEB models
open the way to derive new metrics char-
acterizing population level effects of expo-
sure to ENMs, using data of physiological
responses in individual organisms. We
have estimated the effects of ZnO NPs on
marine mussel ( Mytilus galloprovincialis )
population growth rates using data on:
(i) shell length; (ii) weights of shell, gonad
and somatic tissue; (iii) Zn body burden;
(iv) food ingestion (not shown); and
(v) oxygen consumption (not shown). We
projected effects on lifetime reproduction
using methodology in Muller et al. [ 7g , 10b ]
and demonstrated that EC50 for lifetime
reproduction is much lower than that for
individual physiological rate processes. The
DEB models give “added value” to rela-
tively inexpensive, short-term physiological
measurements on individual organisms
by using the data from such experiments
lasting weeks or months to predict a popu-
lation property that would be expressed
over years or even decades.
4.10. Development of a Zebrafi sh HTS Paradigm to Delineate the Importance of Metal Oxide Nanoparticles in Interfering with ZHE1 Activity as a Predictive Environmental Model
The use of zebrafi sh as a fresh water model organism allowed
us to establish an important environmental toxicological
paradigm for semiconductor and metal oxide nanomate-
rials ( Figure 13 ). As demonstrated in our previous studies,
the dissolution chemistry of transition MOx nanoparti-
cles plays a major role in hatching interference in zebrafi sh
embryos. [ 5b , 6j , 18 ] The mechanism of hatching interference was
postulated to be due to the inhibition of the hatching enzyme,
a zinc metalloprotease, by metal ions released from the
nanoparticles. [ 5b , 6j , 18 ] To prove this hypothesis and being able
to use hatching interference as a mechanistic screening par-
adigm for hazard ranking of other nanomaterials, we made
use of the purifi ed recombinant zebrafi sh hatching enzyme 1
(rec. ZHE1) and a fl uorogenic substrate to develop an abiotic
assay that quantifi es the enzymatic activity. [ 25 ] The abiotic
assay, performed in multiwell plates, allowed us to evaluate
and rank the impact of 24 metal oxide nanoparticles on the
hatching enzyme activity. Four MOx nanoparticles (ZnO,
CuO, Cr 2 O 3 , and NiO) were found to signifi cantly inhibit
the enzyme activity of rec. ZHE1. Through the use of our in
vivo HTS platform that utilizes a robotic system for embryo
pick-and-plate and high content imaging devices for image
acquisition, we also demonstrated that the same materials
exert profound hatching interference in intact embryos. [ 25 ]
The correlation between the abiotic assay and in vivo HTS
has therefore established a predictive toxicological paradigm
13m www.small-journal.com
T. Xia et al.
14
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Figure 13 . Zebrafi sh HTS platform delineates the mechanism of hatching interference exertedby metal shedding semiconductor and metal oxide nanoparticles. The impact of metal oxidenanoparticle dissolution was investigated by a combination of an abiotic assay as well as azebrafi sh HTS screening. The abiotic assay makes use of the purifi ed recombinant zebrafi shhatching enzyme 1 (rec. ZHE1) and a fl uorogenic peptide substrate to assess the enzymaticactivity in response to metal oxide nanoparticles exposure. The abiotic assay conducted inmultiwell plates allows us to evaluate and rank the impact of 24 metal oxide nanoparticles, inwhich four metal oxide nanoparticles (ZnO, CuO, Cr 2 O 3 , and NiO) were found to signifi cantlyinhibit the enzyme activity of rec. ZHE1. To validate the abiotic assay results, we screenedthese nanoparticle effects on hatching using our established in vivo zebrafi sh HTS platformthat utilizes a robotic system for automated embryo pick-and-plate and high content imagingdevices for automatic image acquisition. The in vivo HTS assay results on zebrafi sh embryohatching interference confi rmed that of the abiotic assay. More importantly, the correlationbetween the inhibition of enzyme activity and hatching interference in intact embryos allowedus to establish a predictive toxicological paradigm that may have wider implications for a broadrange of environmental species that express the evolutionary conserved metalloprotease.
based on the dissolution chemistry of nanomaterials and
interference in metalloprotease activity. More importantly,
this paradigm may have wider implications for a broad range
of environmental organisms that share an evolutionary con-
served hatching enzyme (manuscript in preparation). As
a proof-of-concept, we demonstrated that ZnO, CuO and
Cr 2 O 3 also exerted profound hatching interference in Japa-
nese medaka embryos that uses a related metalloprotease
homolog.
4.11. Risk Perception and Guidance for Safe Handling of ENMs in Research and Occupational Environments
Two critical impacts of UC CEIN’s education and outreach
activities have been in the arenas of risk perception and
guidance for scientists and policy makers regarding safe
handling of nanomaterials and prioritization of materials
for regulation. [ 1a ] A signifi cant portion of UC CEIN societal
implications work has focused on risk perception, because
numerous studies have demonstrated the importance of risk
perception as a factor in risk tolerant or avoidant judgment,
decisions, and behavior. [ 13e ] Such effects are not limited to the
www.small-journal.com © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Wein
public, but have also been shown to affect
scientists and other experts. UC CEIN
asked the empirical question: What are the
attitudes about ENM risk and regulation of
expert stakeholders in the nano-enterprise,
nanoscale scientists and engineers in the
university and industry settings that may
affect the risk assessment process? CEIN
societal implications researchers have sur-
veyed industry representatives about their
knowledge of and company adherence to
nano-specifi c EHS recommended practices
and their perceptions of ENM risk. [ 26 ] The
fi ndings from this study, a collaborative
effort between social science and ecotoxi-
cology researchers in UC CEIN, have indi-
cated that a majority of industry leaders
views ENMs either as having moderate
to high or uncertain risk. [ 26 ] The impedi-
ment most frequently reported by com-
panies to their implementing a nano EHS
program was “lack of information”. [ 26 ]
Based in part on the insights gained from
the industry survey that practitioners han-
dling nanomaterials felt that they needed
more concrete guidance on how to work
safely with nanomaterials, UC CEIN pro-
ceeded in partnership with the California
Department of Toxic Substances Control
(DTSC) and NIOSH to produce a “Nano-
toolkit” that helps researchers to rapidly
determine the risks associated with pro-
posed activities involving ENMs, including
how to safely mitigate those risks through
engineering controls, workplace practices,
and appropriate use of personal protective
equipment (http://www.cein.ucla.edu/resources_safety.html).
This toolkit has been broadly implemented in academic insti-
tutions in the state of California, universities across the US
and internationally. Through these activities, UC CEIN has
provided critical guidance to scientists, environmental health
and safety professionals, and policy makers on how to work
safely with ENMs. Likewise, UC CEIN has provided con-
crete guidance to the California DTSC on how to improve
the quality of information obtained from their mandatory
call-ins for information on ENMs produced and used in Cali-
fornia, including how to prioritize the ENMs for regulation
using information available in the scientifi c literature. [ 13c ]
This represents one of the fi rst successful attempts in the US
to gather information about commercial use of ENMs.
5. Conclusion and Future Outlook
Since its inception, UC CEIN has made great progress in
demonstrating how to assemble a multidisciplinary team
to develop the research, knowledge acquisition, education
and outreach that is required for the safe implementation
of nanotechnology in the environment. Instrumental to the
heim small 2012, DOI: 10.1002/smll.201201700
A Multidisciplinary Approach to Solve Complex Nano EHS Problems by UC CEIN
success is the integration of the multidisciplinary concepts
that are required to understand and make an impact in this
complex study area. This has allowed the Center to develop
and establish novel research tools, protocols and scientifi c
breakthroughs to develop a predictive approach to nano
EHS in which knowledge generation in disparate areas of
science are blended into environmental decision-making
that, in turn, leads to scientifi c advances in each contributing
fi eld. These include establishment of compositional and com-
binatorial nanomaterial libraries, mechanism-based toxico-
logical injury pathways, in vitro and in vivo high throughput
screening systems using bacteria, cells, and zebrafi sh embryos,
and in silico data transformation and decision-making tools
for data processing, hazard ranking, exposure modeling, and
development of QSARs. Use of this expertise has enabled
rapid progress toward understanding the impact of nano-
materials on important species and services in terrestrial and
aquatic ecosystems. Moreover, DEB modeling was used to
quantify and integrate the ecosystem impacts across scales
and life stages. The Center’s research programs allowed us
to train graduate students and postdoctoral fellows in the
fi eld of nano EHS, while the use of our outreach programs
has allowed knowledge dissemination to the general public,
scholars, government agencies, policy makers, and industrial
stakeholders.
In spite of the progress, we realize that since nano-
technology is still a relatively young area of science in which
rapid development and dissemination of large numbers of
new materials will require a nimble and vigilant response
to continue developing the scientifi c underpinnings for the
safe implementation of this technology. In addition to the
challenge posed by the introduction of new materials and
nano-enabled products, we require a great deal of informa-
tion about the new materials that are being introduced in the
marketplace, their physicochemical properties, utility, life-
cycle analysis and data about real-life exposures in the envi-
ronment. UC CEIN’s streamlined approach, high throughput
discovery platforms and modeling efforts will continue to
prepare us for future nano EHS challenges, including how to
utilize this knowledge for the development of a sustainable
technology.
Acknowledgements
This work was supported by the National Science Foundation and the Environmental Protection Agency under Cooperative Agreement Number DBI-0830117. Any opinions, fi ndings, conclusions or rec-ommendations expressed herein are those of the author(s) and do not necessarily refl ect the views of the National Science Foundation or the Environmental Protection Agency. This work has not been subjected to an EPA peer and policy review.
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Received: July 17, 2012Published online:
H & Co. KGaA, Weinheim small 2012, DOI: 10.1002/smll.201201700