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: anel@mednet.ucla.edu

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,

2 www.small-journal.com © 2012 Wiley-VCH Verlag G

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-

small 2012, DOI: 10.1002/smll.201201700

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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T. Xia et al.essay

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.

8

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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

www.small-journal.com © 2012 Wiley-VCH Verlag G

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

im small 2012, DOI: 10.1002/smll.201201700

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 ]

www.small-journal.com © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Wein

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