Center for Environmental Implications of Nanotechnology
Project Abstracts
January 2012
Theme 1 – Compositional and combinatorial ENM Libraries for property-activity analysis (Zink)
Project ID Assignment: ENM-1: Toxicity of ENM with Cationic Surfaces
Jeffrey I. Zink
To test the hypothesis that cellular responses to nanoparticles are related to the charge of the particles
(cationic or anionic), we will synthesize libraries of polymer-coated mesoporous silica nanoparticles. The
cationic polymer polyethyleneimine will be chosen for initial studies because it is known to exert
differential cytotoxicity depending on its molecular weight and cationic density, with higher molecular
weight (25 kD) being the most toxic. Particles suitable for confocal imaging will derivatized with
fluorescein, rhodamine, or near-infrared emitting dyes. The particles will be characterized for size, size
distribution, shape and charge using TEM, DLS and electrophoretic mobility.
Preliminary results show that particles coated with the 10 and 25 kD Pei polymers have increased
cellular association compared to those with smaller polymers. The MSNs coated with 10 and 25 kD
polymers were also more toxic than particles coated with shorter length polymers. This library will be
studied in collaboration with Theme 2 to search for a selective difference in the response of
undifferentiated and differentiated cells to a cationic nanomaterial. Other cationic polymers that we
have synthesized will be attached to metal oxide nanoparticles for further study with theme 2. These
polymers include Diethylaminoethyl- Dextran, poly-(N-ethyl-4-vinylpyridine-Ilium bromide), poly((2Dimethylamino)ethyl methacrylate), polyamidoamine dendrimer, and poly(ethylene glycol)-co-poly(Llysine).
A new type of nanoparticle containing a cationic –R-NH2+-R interior and an anionic SiO exterior will be
synthesized and characterized. The general assumption that cationic particles are toxic is based on the
total charge as measured electrophoretic mobility (zeta potential). Nothing is known about the effects
of surface charge vs. total charge. The total charge on this type of particle is cationic (based on zeta
potential measurements) even though the silica component on the outside surface is anionic. High
throughput studies will differentiate between the effects of total particle charge versus surface charge
on toxicity.
Project ID Assignment: ENM-2: Processing and Characterization of Single-Walled and Multi-Walled
Carbon Nanotubes
Mark C. Hersam, Northwestern University
Carbon nanotubes exhibit unique physical, chemical and electrical properties that make them an
attractive material for use in industry, medical diagnostics, and drug delivery. However, enthusiasm for
their use has been tempered by relevant concerns regarding their toxicity. The high ratio of length to
diameter (aspect ratio) of single and multi-walled carbon nanotubes has led some investigators to
compare these particles with asbestos fibers, which are also characterized by a large aspect ratio but
with a much larger mean diameter. The failure of resident macrophages to clear and eliminate these
needle-like structures has been suggested to activate pro-inflammatory pathways in these cells that
induce lung fibrosis and increase the susceptibility to pulmonary malignancies.
Van der Waals interactions between individual nanotubes in air or in aqueous solutions cause them to
form large aggregates, which can be more than one hundred microns in diameter, and it is the
administration of these aggregates that has been associated with lung toxicity in rodents. While
accidental industrial exposure to these large aggregates is certainly relevant, effective dispersal of
carbon nanotubes at the nanoscale is required for them to exhibit many of their desirable physical
properties. In preliminary work, we found single-walled carbon nanotubes that were highly dispersed
using the biocompatible block copolymer Pluronic F108NF yielded exceeding low levels of pulmonary
toxicity.
While these previous studies suggested methods for the safe handling and processing of carbon
nanotubes, the precise biological mechanisms for their reduced toxicity when well dispersed with
Pluronic block copolymers remains an open question. This project will systematically explore these
mechanisms by performing a thorough exploration of the interaction between carbon nanotubes and
biological systems as a function of several materials parameters including the level of dispersion,
dispersant identity/concentration, and carbon nanotube structure (e.g., diameter, length, single-walled
versus multi-walled, etc.). Specific carbon nanotube types include: (1) Raw HiPco single-walled carbon
nanotubes; (2) Purified HiPco single-walled carbon nanotubes; (3) Pluronic-dispersed, purified HiPco
single-walled carbon nanotubes where large aggregates and impurities have been removed via
centrifugal processing; (4) As-prepared arc discharge single-walled carbon nanotubes; (5) Purified P2 arc
discharge single-walled carbon nanotubes; (6) Pluronic-dispersed, purified P2 arc discharge single-walled
carbon nanotubes where large aggregates and impurities have been removed via centrifugal processing;
(7) Purified CoMoCAT SG65 single-walled carbon nanotubes; (8) Pluronic-dispersed, purified CoMoCAT
arc discharge single-walled carbon nanotubes where large aggregates and impurities have been
removed via centrifugal processing.
To supplement the toxicity studies that are being performed in the laboratory of Andre Nel, a variety of
materials characterization methods will be employed including optical absorbance spectroscopy,
photoluminescence spectroscopy, Raman spectroscopy, atomic force microscopy, and electron
microscopy. A parallel and related effort on the toxicity of graphene and graphene oxide is also
occurring in collaboration with Gokhan Mutlu and Scott Budinger of the Northwestern University
Medical School. The ultimate goal of this project is to determine the role of carbon nanomaterial
structure and dispersant chemistry in toxicity and environmental testing.
Project ID Assignment: ENM-3. Systematic synthesis and characterization of silicon dioxide
nanoparticles for establishing processing-structure-toxicity relationships
Xingmao Jiang, Haiyuan Zhang, Darren R. Dunphy, Jeffrey I Zink, Andre Nel, and C. Jeffrey Brinker
Silicon dioxide silica is the most abundant mineral on earth - predominantly in the form of alphacrystalline quartz. Manmade silicas however tend to be amorphous and often nanostructured powders
used extensively in applications like fillers to control rheological and mechanical behaviors, catalysts,
and desiccants. Amorphous silica nanoparticles are prepared by two main routes, high temperature
flame pyrolysis to form so-called fumed silica or by molecular condensation of silanol groups (S-OH +
HO-Si  S-O-Si) in aqueous solution or under hydrothermal conditions at low temperature to form
so-called precipitated, colloidal or mesoporous silicas. Fumed silica is produced in tonnage quantities
and is the second most abundant synthetic nanoparticle on earth. Based on the abundance and
potential exposure to amorphous silica NPs, establishing their processing-structure-toxicity relationships
is important for understanding their environmental implications - in particular with respect to the longstanding problem of silicosis and whether it is dependent on crystallinity. Establishing structure-activity
relationships for amorphous silica is problematic, however. Whereas crystalline silica is by definition
well-defined structurally, amorphous silicas lack long range order; their structures are therefore lesswell defined and, due to a flat structural energy landscape, dependent on kinetic factors. Literature
studies often refer to non-crystalline silicas as merely amorphous silica without regard to their thermal
and chemical processing conditions and environmental exposure, which alter their surface chemistry
and thus their colloidal, biomolecular, and toxicological behaviors. To unify the understanding of the
structure of nanostructured amorphous silicas and establish a predictive structure-toxicology paradigm
for this important class of nanomaterials, the major aims of this project are to: 1) Develop hydrothermal,
aerosol, and solution techniques to synthesize well-defined, monodisperse libraries of crystalline and
amorphous silica NPs and 2) Employ combined spectroscopic (e.g. near and mid-IR, Raman, EPR) and
physical (e.g. TGA, BET, TEM, SANS) analyses to characterize NP physicochemical properties as a function
of environmental conditions such as temperature and water exposure. Over the past year, we have
conducted an extensive study comparing the physicochemical properties of a model spherical colloidal
silica NP, Stober silica (prepared in-house), with commercial fumed silica - each with a primary particle
diameter of ~15-nm. For each particle class, we determined the surface area normalized OH content,
extent of hydrogen bonding, and hydroxy radical concentrations along with the concentration of
strained and unstrained siloxane rings as a function of heat treatment temperature and re-exposure to
water. Based on HTS assays and cell culture studies of viability, ATP level, and membrane permeability of
epithelial and macrophage cells conducted by IRG2, we have discovered an important temperature and
hydration dependent pattern of toxicity in fumed silica, whereas solution prepared, colloidal (and
mesoporous) silicas are found to be essentially non-toxic under any condition. Specifically we find for
fumed silica a positive correlation of toxicity with silanol concentration (implicated in membrane
disruption and protein unfolding) and hydroxy radicals, which are the most toxic ROS. We believe this
toxicity is related to the intrinsic state of aggregation of fumed silica causing it to get trapped in the cell
membrane along with intermediate range order of the fumed silica framework, viz. the concentration of
strained rings established at high temperature, which could be a source of ROS. Importantly the toxicity
pattern we observe in cell culture and HTS, has been replicated in an inhalation toxicology model. It may
help to establish a needed predictive toxicological paradigm for silicosis and related disease.
Project ID Assignment: ENM-4: FSP generated pure and Fe doped ZnO or TiO2 NP libraries for testing
paradigms of environmental and cellular responses.
Lutz Mädler, Suman Pokhrel
Project 4a: A comparative study of the FSP produced Fe-doped and Al-doped ZnO nanoparticles in
mammalian and fish cell lines.
The impact of nanoparticles (NPs) in the terrestrial and aquatic environment is an important question in
ecotoxicology. NPs can reach the aquatic environment via wastewater, during manufacturing processes,
consumer use and improper disposal. With the production of 100,000 tons of ZnO NPs per year, metal
oxides (especially Al-doped ZnO) is likely to have a wide range of commercial applications ranging from
electronics (TCO) to chemical catalysts and cosmetics. Hence, there exists a probability of these NPs
entering into the environment and consequently an immediate need for cellular cytotoxicity screening
arises. We began our investigation by engineering ZnO NPs (doping 1-10% of Al) and determining their
particle sizes. The particle size derived from BET (dBET) and XRD (dXRD) was found to be in the range of 1612 nm and 18-12 nm respectively. HRTEM images of single NPs of pure and Al-doped ZnO revealed
highly crystalline structures with constant lattice spacing of 2.81-2.91 Å corresponding to 57% intensity
peak (1 0 0) observed in XRD patterns corroborating a negligible doping effect on the crystal system
even at high atomic loadings. The distribution of Al atoms in the ZnO matrix was also investigated with
combined EELS and EFTEM analysis in STEM mode. The elemental maps of the parent and doped NPs
show homogeneous distribution of Al in ZnO. The band gap energy of the Al doped ZnO (3.5 eV) and Fe-
doped ZnO (3.2 eV) was derived using UV-visible spectrophotometer in the reflection mode. The data
([F(R)h]) was plotted against h and the linear part of the curve was extrapolated to zero
reflectance. The investigation of the structural and electronic properties, formation energy of Fe doped–
ZnO NPs was evaluated (Prof. Heine, Jacob University, Bremen, Germany) using First-principles
calculations. Substitution of Zn by Fe, anion/cation vacancies and interstitial oxygen defects was studied
to calculate the stability of the dopant in ZnO crystal. High-resolution inner-shell electron energy loss
spectroscopy measurements and X-ray absorption near-edge structure calculations of Fe and O atoms
show that Fe-doped ZnO NPs are thermodynamically more stable than the isolated FeO or ZnO phases.
The Fe dopants distribute homogeneously in ZnO NPs and do not significantly alter the host ZnO lattice
parameters. Simulations of the absorption spectra demonstrate that Fe2+ dominates in the Fe -doped
ZnO NPs.
We have recently demonstrated that ZnO induces 3-tier oxidative stress in human macrophage cell lines
due to Zn2+ release in the cellular medium. On the basis of this observation we performed similar toxicity
screening process with RAW 264.7 and BEAS-2B cells lines using 0.3-100μg/mL dose levels exposed to
the cells for 1-24 h. Under dark conditions, cell death was found to be decreasing with increasing Alcontent. The decrease in the cell death is directly associated with ROS production (hydroxyl radical
generation) and the integrated toxic oxidative stress pathway including intracellular calcium flux,
mitochondrial depolarization, and plasma membrane leakage. These responses were chosen based on
the compatibility of the fluorescent dyes demonstrating cytotoxicity in a dose- and time-dependent
fashion. Comparing the purposeful reduction of ZnO cytotoxicity achieved by Fe-doped and Al-doped
ZnO NPs, the latter have more enhanced protective effect in terms of cellular oxidative stress pathways.
In the presence of light, the hydroxyl radical generation by 1% Al doped ZnO was about 5 times less than
the OH· generation by 1% Fe-doped ZnO. The increase in Al-content to 10%, The OH·-radical generation
was almost equal to the control. The same experiment was repeated in RAW 264.7 cell line and the cell
apoptosis induced by 10% Fe doped ZnO was almost 6 times more than those induced by 10% Al-ZnO.
To verify these observations in a fish cell line (RT gills W1), we exposed 100 μg/mL Al doped ZnO NP
solution in presence of both light and dark conditions and measured mitochondrial superoxide
generation with MitoSox red. Data showed that under dark condition, very little or no toxicity was
induced. However, in the presence of light, the superoxide generation was found to decrease
systematically with each loading of Al into ZnO. Contradictory to this observation, Fe doped ZnO was
associated with increased superoxide generation with each Fe loading. Consistent to these results, cell
death was decreasing with incremental Al-loading while with incremental Fe content; there was an
inverse in cytotoxicity.
In conclusion, we have developed two Fe- and Al-doped ZnO libraries. We observed that Al based ZnO
NPs are protective in dark as well as in light conditions. However Fe doped ZnO appears to be relatively
toxic in presence of light, even though it has protective effect in the dark. This finding is in line with what
we observe for band gap tuned Fe doped TiO2 NPs in presence of light. The results are of importance for
the use of cosmetics, household and material products that make use of ZnO NPs.
Project 4b: FSP generated TiO2-based engineered NPs for photooxidation induced cytotoxicity
evaluation
To predict the potential impact of engineered NPs, cellular toxicity evaluation must include a variety of
organisms and understanding of the responsible mechanisms. After demonstrating the reduced cellular
toxicity of ZnO by re-engineering the NPs (doping Fe in ZnO; safe-by design strategy), we studied the
role of TiO2 photoactivation by using non-doped and Fe doped particles. When light of sufficient
wavelength is applied to the TiO2 or engineered TiO2 particles, the electrons are released to the
conduction band or trap levels of the dopant, creating holes in the valence band. The generated charge
(electon or hole e-/h+) can then interact with H2O and molecular oxygen, respectively, to generate HO•
radical and superoxide radicals. To test the cellular injury due to the highly reactive species generated
during light exposure, Fe-doped TiO2 nanoparticle library using the FSP technique was synthesized and
characterized in our laboratory. The physicochemical characterization was performed using advanced
techniques such as XRD, HRTEM, SAED, EFTEM, and EELS. HRTEM images of single nanoparticle of pure
and Fe- doped TiO2 revealed highly crystalline structures with lattice spacings of 3.73 Å for undoped and
3.43 Å for 10% doped TiO2. The distribution of Fe atoms in the TiO2 matrix was investigated with
combined EELS and EDAX analyses. The elemental maps of the parent and doped NPs show a
homogeneous distribution of Fe in the TiO2 matrix. By doping Fe in TiO2, we were able to reduce the
band gap energy significantly (from 3.2 to 2.8 eV) maintaining a homogeneous crystalline structure. In
the close collaboration with the group of Prof. Nel (Theme 2), we were able to demonstrate and
evaluate visible light effect through these band gap tuned Fe doped TiO2 NPs in the cells. Studies with a
macrophage cell line for the evaluation of the cellular injury using multi-parametric assay showed
incremental cytotoxicity with increased Fe loading demonstrating the importance of band gap energy in
the phototoxic response to TiO2 NPs.
These results have lead to application of the materials in several laboratories in CEIN. First, we have
tested the NPs for antimicrobial activity of TiO2 in the presence of UV light (not visible light as described
above) with the group of Prof. Godwin (Theme 5). In this study we demonstrated that Fe loading in TiO2
resulted in decreased ROS production and bacterial toxicity during light irradiation. The effect was
observed with the increase in Fe doping, which is explained by the redox potential generated from
chemical reactions taking place during the electron transformation. The study further confirmed that
the toxicity induction was through hydroxyl radical generation on the particle surface during TiO2 photoactivation. In a second collaborative effort with the group of Prof. Holden (Theme 4), we have observed
that engineered TiO2 NPs (by Fe doping) minimizes adverse effects on bacterial growth at higher Fe
concentrations which is in line with the findings in Prof. Godwin’s laboratory. In a third collaborative
work with the group of Prof. Nel (Theme 2), we have introduced a new library of Pt doped TiO2 NPs with
the hypothesis that noble metal may have a strong influence in the surface adsorption during light
irradiation process. The results show that the maximum hydroxyl radical generation was observed for
1% Pt doped TiO2 and the radical generation ceased with the increase in Pt during light irradiation (λ =
280-350 nm). In the absence of light, the metabolic activity (measured by MTS assay) decreased slightly
when RAW 264.7 cells were exposed to Pt doped TiO2. The cell death (measured by PI uptake) increased
when RAW 264.7 cells were exposed to 0.1 and 1 % Pt doped TiO2. Higher Pt doping had no significant
effect on cell viability.
To summarize, our findings promote the understanding of light-induced cytotoxicity of pure and
engineered TiO2 NPs in the microphage cell line as well as in the bacterial cells during light irradiation
using different wavelengths. These research findings are very important for the industry especially to
those used in consumer products that come into contact with human and the environment.
Project 4c: Primary particle size dependent transport properties and toxicity screening of TiO2
aggregates obtained from flame spray pyrolysis
Most NPs have inherent aggregation properties due to their high mobility which strongly depends on
the primary particle size and adhesion forces compared to the other forces. The aggregation of the NPs
usually reduces the mobility of the particles in the environment. It is therefore important to
demonstrate the mechanism involved in the transport of the nanomaterials in different media.
Moreover, it is also known that environmental parameters including water chemistry (pH, ionic strength,
and oxidation state), as well as the presence of natural organic matter and bacteria may control the
transport and removal of nanomaterials. To test this hypothesis, TiO2 NPs of different average primary
particle sizes (6, 13 and 23 nm) were developed in our laboratory using versatile the FSP technique. The
particle size was evaluated using advanced characterization techniques such as BET, X-ray diffraction
and TEM. XRD and selected area diffraction patterns (SAED) suggest a high crystalline nature of these
particles. The lattice spacing of 3.73 Å was evaluated from high resolution transmission electron
microscopic (HRTEM) images of TiO2 NPs. In collaboration with the group of Prof. Walker (Theme 3), the
experiments on electrophoretic mobilities (EPM), aggregate size, and potentiometric titration were
performed to understand the surface and transport properties of NPs. EPM of all the TiO2 particles
decreased with the increase in pH in the presence of 1 and 10 mM KCl solution. Data showed that the
particles have similar electrophoretic mobility (-1.6х10-8) at pH 7 and similar aggregate size (300 nm) at
pH 10. The aggregate size was found to increase with the increase in the ionic strength of KCl which is
ascribed to the attractive electrostatic forces between the individual NPs in the media. A packed bed
column has been used to demonstrate the transport and removal mechanisms of NPs through the
complex, three-dimensional porous media. The kinetics of deposition was evaluated by systematically
varying the concentration of the KCl solution. At pH 10, in presence of 1 mM KCl, the deposition rate of
23 nm sized TiO2 NPs aggregate was found to be 20 times more than those of 6 and 13 nm sized TiO2
NPs aggregate, while at 10 mM KCl, the deposition rate of 13 nm sized TiO 2 aggregate is twice that of
the 6 and 13 nm particle aggregates. In Conclusion, the combination of the transport properties of these
FSP generated TiO2 NPs will help to understand the fundamental mechanisms involved in the fate and
transport of NPs in the environment.
A follow-up study on the toxicity evaluation for these differently sized TiO2 NPs in environmentally
relevant aqueous medium using an established dispersion protocol was performed by the group of Prof.
Holden (Theme 4). The experiments were carried out with recently developed high-throughput assays to
test sublethal effects of NPs in bacteria (membrane potential, membrane integrity, ROS, superoxide, and
dehydrogenase assays). Data showed a distinct size effect, with the smallest particle exerting greater
dose-dependent toxicity, resulting in decreased cell yield and decreased growth rates, compared to
those observed for larger primary particle sized NPs. The quantification of the TiO2 adsorption by mass
on a per-cell basis have been performed to establish a relationship between primary particle size, TiO2
adsorption, effects on bacterial growth, and a possible mechanism of toxicity.
In summary two independent TiO2 NP studies (aggregation followed by deposition and the toxicity
induced by 6, 13 and 23 nm) demonstrate the complexity of nanomaterial hazard. Due to absolute
dependence of NPs stability and bioavailability on bacterial cell and water chemistry, high-throughput
combinatorial screening to assess potential impacts of different NPs exposed to the humans and the
environment is very important.
Project ID Assignment: ENM-5: Relationships Between ENM Geometry on Biological Outcomes :
Nanorods
Jeffrey I. Zink
A simple geometrical property of nanoparticles, the aspect ratio (AR, the ratio of length to diameter),
could be a factor in determining biological outcome, but not much is understood about how this
material property generates biological effects. How the AR impacts cellular function is unknown but of
considerable importance in understanding how to improve nanomaterial safety through the use of a
physical design feature. Nanoparticles made in large industrial quantities are rarely perfect spheres.
Shape effects have been reported for gold and monomethacrylate nanorods with conflicting results
regarding amount of uptake as a function of AR. In order to understand the impact of AR on cellular
function, it is necessary to construct a series of ENMs of a constant chemical composition that show AR
variation.
A library of mesoporous silica (MSN) rod-shaped nanoparticles (MSN) that covers a range of different
lengths will be synthesized using templated sol-gel synthesis methods. The particles will have a
diameter of about 100 nm and lengths up to 1000 nm. The length of the rod will be controlled by the
concentration of an amphiphilic co-templating agent. The size measurements will be based on TEM
images. The particles will be thoroughly characterized by TEM, XRD, gas adsorption/desorption
isotherms, dynamic light scattering and electrophoretic mobility.
To further refine the studies, nanorods with the same length but different diameters will be synthesized.
New chemical compositions (manganese and vanadium oxides) will also be investigated. Results
showing different cellular uptakes or unexpected sublethal responses will be important because they
would point to a potential environmental safety problem when toxic nanoparticles or particles that have
adsorbed hazardous chemicals have enhanced uptake by cells.
Project ID Assignment: ENM-6: Relationships Between ENM Geometry on Biological Outcomes :
Nanowires
Jeffrey I. Zink
Interest in high aspect ratio nanomaterials evolved from the history of fiber toxicology. High aspect ratio
materials such as asbestos, carbon nanotubes and TiO2 nanowires lead to frustrated phagocytosis and
inflammatory responses in cells. However most research only explored a very limited number of lengths
and aspect ratios, and it is therefore not clear what is the critical length and aspect ratio that can induce
such an effect. To address this question, we will synthesize a cerium oxide (CeO2) nanorod library with a
wide range of aspect ratios. The advantage of using CeO2 is that it is biologically inert, and thus studies
of the length and aspect ratio will not be complicated by intrinsic toxicity. By controlling the
hydrothermal synthesis composition and conditions, CeO2 nanorods and nanowires with precisely
controlled lengths and aspect ratios will be made. Preliminary results show that nanorods obtained with
the CeCl3 concentration varying by a factor of ten while keeping other synthesis composition and
conditions constant range in length from 110 to 30 nm (with a constant diameter). The aspect ratios will
be further tuned by adjusting the other synthesis parameters. The successful creation of
nanorod/nanowire combinatorial libraries will allow a pure length and aspect ratio effect on biological
response to be studied in a systematic manner. We will also synthesize other nanowire libraries of
materials such as vanadia and manganese oxide to attempt to further demonstrate that nanowire
length plays an important role in high aspect ratio nanomaterial toxicity.
Theme 2 - Molecular, cellular and organism high-throughput screening for hazard assessment (Nel)
Project ID assignment: HTS-1: Use of the Multi-Parametric HTS to Study in the Phototoxicity of NanoTiO2 and Surface Reactivity of Ag-nano plates in Mammalian and Fish Cell Lines
Saji George, Tian Xia, André E Nel
The overall goal of this project is to develop and utilize in vitro high throughput screening (HTS)
platforms for hazard ranking of small to large series of engineered nanomaterials (ENMs), with a view to
develop predictive toxicological paradigms in which the in vitro analysis helps to prioritize in vivo
studies. Further, in collaboration with the nanochemistry expertise in Theme 1 and the analytical
decision-making tools in Theme 6 we address the key ENM properties that may lead to harmful effects
at the nano/bio interface with a view to developing property-activity relationships. Previously we have
developed a successful HTS assay that contemporaneously assesses sub-lethal cellular responses
(intracellular ROS generation, mitochondrial depolarization, and increased intracellular calcium flux) in
combination with cytotoxicity, reflected by the enhanced permeability of the plasma membrane in dying
cells. This series of interconnected cellular responses, originating from the study of the induction of toxic
oxidative stress by ENMs, has subsequently also been used successfully to study the sub-lethal and
lethal effects of ENMs that do not primarily involve the generation of oxidative stress. To date we have
successfully implemented this assay for hazard ranking of metals metal oxides, Q-dots, cationic
nanomaterials and different silica chemistries that display hazardous physicochemical properties such as
particle dissolution with toxic metal ion shedding, contamination with toxic organic chemicals, display of
membrane disruptive surface cationic groups or surface reconstruction with the display of reactive
silanol groups. The predictive value of the cellular responses has been demonstrated during the
execution of animal studies, including the relationship of oxidative stress to the acute inflammatory
effects, zebrafish embryo toxicity and phytoplankton toxicity. Since the last activity report, we have
implemented the multi-parametric HTS as assay to explore the toxicity of nano-TiO2 and Ag
nanoparticles from the perspectives of phototoxicity and the effects of nanoparticle surface defects in
hazard generation.
TiO2 is a photoactive ENM in which UV-induced electron-hole (e-/h+) pair generation and free radical
production has been proposed as a possible pathway that can lead to biological injury. However, the UV
requirement for shifting the TiO2 band-gap energy to formation of e-/h+ pairs is an impediment in
biological experimentation because of the high toxicity of UV inmost biological settings. Therefore, a
series of TiO2 nanoparticles was synthesized in Dr. Lutz Mädler’s laboratory to allow us to study the TiO2
photoactivation in near-visible light. This library was developed by TiO2 doping with incremental levels
(0-10 atomic wt%) of Fe (Theme 1, ENM 4). Doping led to a reduction in band-gap energy and was found
to increase the photo-oxidation capability of this material by near-visible light. This allowed us to study
the generation of reactive oxygen species (ROS) and cellular toxicity in macrophages through the use of
the multi-parametric HTS assay. Pre-treatment of macrophages with N-acetyl cysteine abolished the
photo-activation toxicity, confirming the role of oxidative stress in cell death. Not only did this study
demonstrates the importance of band gap energy in determining the phototoxic response to TiO2, but is
of considerable importance in the performance of eco-toxicity studies at UCSB. For instance, Dr. Hunter
Lenihan's group have demonstrated in Theme 5 (report MFW-1) the bright sunlight exposure leads to
TiO2-induced toxicity in phytoplankton.
We have previously shown that Ag nanospheres exert little or no toxicity in mammalian cells during the
use of the multi-parametric assay but are highly toxic in zebrafish embryos. This toxicity was associated
with interference in embryo hatching as well as the generation of morphological abnormalities in the
embryos and larvae. Similar effects in aquatic lifeforms have previously been ascribed to the dissolution
of nano-Ag, leading to the release of toxic Ag ions. In view of this apparent discrepancy between
mammalian cells and the zebrafish model, we were interested in comparing mammalian cells with a fish
cell line. For this purpose, we established a culture of rainbow trout gill epithelial cells (RT-W1), which
were exposed to a commercially acquired library of Ag nanoparticles that included nano-spheres (10
nm, 20 nm and 40 nm), nano-plates and nano-wires. While the nano-spheres induced a dose-dependent
increase in oxidative stress and cell death in RT- W1, the Ag-plates were comparatively more toxic. The
nano-wires had little toxic effect. Moreover, the toxicological response of the the fish cell line was
accentuated compared to the effect on mammalian cells. Likewise, Ag-plates were also more toxic in the
zebrafish embryos. Compared to the predominant mechanism of toxicity Ag-nanospheres, which
involves shedding of Ag ions, the toxicity of the Ag-plates was due to high surface reactivity and the
ability to damage the surface membrane of RT-W1 and red blood cells. In order to explain this finding,
high resolution transmission electron microscopy was used to examine the Ag-plates. This analysis
revealed a high level of crystal defects (stacking fault and point defects), suggesting that the Ag-plate
toxicity is catalyzed by the surface defects. Surface passivation by cysteine coating ameliorated the
toxicity of Ag-plates in vitro and in vivo. All considered, these studies demonstrate the role of crystal
defects in Ag-plate toxicity in addition to the well established effect of Ag-ion shedding by the
nanospheres. The excellent correlation between the in vitro and in vivo toxicity data illustrates the
usefulness of the fish cell line for predicting toxicity at the organism level.
Project ID Assignment: HTS-2: Development of high content screening of nanoparticle toxicity using
zebrafish models
Sijie Lin, Tian Xia and André E Nel
The goal of this project is to establish a high content screening platform for hazard ranking of
engineered nanomaterials (ENMs) using the zebrafish model. We have recently demonstrated in a
conventional toxicology study in zebrafish embryos that for a series of commercially acquired metal and
metal nanoparticles (Ag, Au, Pt, Al2O3, Fe3O4, SiO2, CdSe/ZnS and ZnO) there is an excellent correlation
between hatching interference in the embryos and cellular toxicity as determined by our multiparametric HTS assay (George et al. ACS Nano, 2011). However, the conventional screening procedures
in the embryos are labor-intensive and time-consuming, including performance of zebrafish husbandry,
embryo selection, as well as real-time reading and scoring of the toxicological endpoints. These
endpoints include real-time scoring of parameters such as hatching interference, mortality,
abnormalities etc., which could be made more efficient through the use of automated imaging
techniques for capture of morphology and developmental abnormalities. In order to develop high
content screening (HCS) in the zebrafish, we focused on three major steps to improve the efficacy of
greater the screening process, namely (i) the use of a robotic system for automated embryo selection
and placement, (ii) development of high content imaging platforms and (iii) computer-assisted image
and data analysis. We developed and integrated a robotic system that allows preparation of 5-10 times
the number of multi-well plates (at 50% of maximum speed) compared to what can be achieved through
handpicking of the embryos. This system automates embryo picking and placement by integrating a 6axis robotic arm (Denso Robotics), a vision recognition system (Cognex) and a syringe pump for liquid
handling (Hamilton). The progress to date includes acquisition of the major components, hardware
installation (mounting brackets and safety enclosure), testing each component’s performance, and
writing software programs that automate and integrate all the components. We have also developed
bright-field and fluorescence-based imaging capture to allow retroactive assessment of morphological
features and GFP expression in transgenic zebrafish larvae and embryos.
In order to demonstrate the potential utility of these imaging platforms, we used a series of transition
metal oxide nanoparticles (CuO, ZnO, NiO and Co3O4) that have acquired by the Nanomaterials Core or
prepared by Drs. Suman Pokhrel and Lutz Madler (Theme 1). Making use of the bright-field image
collection feature, we observed potent and dose-dependent hatching interference in the exposed
embryos for all the materials, except Co3O4 that was relatively inert. hypothesized that hatching
interference by CuO and NiO particles was due to ion shedding and interference in activity of the
hatching enzyme, ZHE1, as previously demonstrated for ZnO. Co-introduction of a metal ion chelator,
diethylene triamine pentaacetic acid (DTPA), reversed the hatching interference of Cu, Zn and Ni. While
neither the embryos nor the larvae demonstrated morphological abnormalities, fluorescence-based high
content imaging demonstrated that CuO, ZnO and NiO could induce the expression of the heat shock
protein 70: enhanced green fluorescence protein (hsp70:eGFP) in transgenic zebrafish larvae. Induction
of this response by CuO required a higher nanoparticle dose than for hatching interference. This
response was DTPA sensitive. Based on this progress, we have now begun to explore an expanded series
of 24 metal oxides (Al2O3, CeO2, CoO, Co3O4, Cr2O3, CuO, Fe2O3, Fe3O4, Gd2O3, HfO2, La2O3, In2O3, Mn2O3,
NiO, Ni2O3, Sb2O3, SiO2, SnO2, TiO2, WO3, Y2O3, Yb2O3, ZnO and ZrO2), as outlined in Projects ENM-1 and
HTS-6. The results of the zebrafish embryo screening will be compared to the cellular HTS of these
particles with a view to establish in vitro/in vivo correlation. This will include screening of the rainbow
trout gill epithelial cell line (RT-W1), as outlined in Project HTS-1. We will also generate the recombinant
ZHE1 to develop and enzymatic assay that can be used for abiotic screening of the various metal oxide
nanoparticles. Finally, in collaboration with Dr. Rong Liu and Dr. Yoram Cohen (Theme 6, EDA-1), we
have developed a phenotype recognition system for zebrafish embryos. This system provides visual
interrogation and automated scoring of the bright-field images into three outcome categories (hatched,
non-hatched/alive and non-hatched/dead) with an accuracy prediction of 97.40 ± 0.95%. This will allow
us to perform hazard ranking of the 24 metal oxides as well as other ENM libraries. The metal ion
shedding Taken together, these data demonstrate the utility of high content zebrafish screening to
perform hazard ranking of ENMs.
Project ID Assignment: HTS-3: High Throughput Screening to Determine the Mechanistic Toxicology of
Engineered Nanomaterials in Bacteria
Hilary Godwin, Patricia Holden, Angela Ivask, Chitrada Kaweeteerawat
This project aims to demonstrate that high-throughput screening (HTS) of nanoparticles (NPs) can be
performed in bacteria as models for environmentally relevant organisms and that these experiments can
be used to predict the hazards that different NPs pose to environmental systems. Because bacteria are
critical sentinel species and form a biological foundation for ecosystems, demonstration of effective use
of HTS for nanotoxicology in bacterial systems is a high priority for the UC CEIN. In this project, we are
capitalizing on the ground work that has been laid in bacterial nano high-throughput screening in UC
CEIN in performing the following experiments: (i) screening of a broad range of ENMs using a HT growth
assay with E. coli cells, (ii) screening of a broad range of ENMs using HT sub-lethal assays for
toxicological endpoints in E. coli and (iii) systematically studying which gene pathways are involved in
toxicological responses to highly toxic NPs using a genome-wide collection of E. coli gene deletion
strains.
To date, we have identified optimal suspension conditions (using DLS) and screened the toxicity of a sets
of:
1) nine different TiO2 nanomaterials including a series of Fe-doped TiO2, (ii) 24 metal oxide
nanomaterials, (iii) five different Ag nanomaterials, and (iv) four polystyrene nanomaterials. These
ENM libraries were selected to complement the studies in mammalian cells as well as in vivo studies.
Our major findings to date are that:
2) Under the conditions studied, the library of TiO2 nanomaterials, including Fe-doped TiO2, exhibited
toxicity only after UV illumination conditions; the antibacterial effects of this library of ENMs
decreased with increasing Fe doping as a result of decreased hydroxyl radicals production. This work
is complementary to phototoxicity studies performed on these materials using mammalian cells
under the Project ENM-4.
3) Eight out of 24 metal oxide nanomaterials exhibited toxicity in the E. coli parent strain; these results
are complementary to those obtained using mammalian cell lines and will be used to develop nanoSAR models for metal oxide nanomaterials (in collaboration with H. Zhang, Nel´s group).
4) All Ag nanomaterials showed toxic effects that differed according to the materials' primary size and
surface coating. To elucidate whether mechanistic differences underlie the observed differences in
toxicity, these Ag materials will be analyzed in an HT assay using 4000 E. coli mutant strains (see
below).
5) The set of 4 polystyrene materials were not toxic except for smallest sized (60 nm primary size)
cationic (amino charged) material (PS-NH2). This highly toxic PS-NH2 nanomaterial has also proven
highly toxic in the study of mammalian cells, which constitutes the basis of why this library was
chosen to develop and validate the HT assay with bacterial gene deletion mutants (see below).
We are currently in the process of developing HTS platforms to complement the bacterial growth
inhibition assays with sub-lethal assays. These assays are being developed premised on existing
protocols from Holden ´s group, which include assays for membrane integrity & permeability, ROS
generation, and electron transport. In addition, to systematically study which gene pathways are
involved in the toxicity of certain nanomaterials, we have developed a genome-wide HTS assay
exploiting 4000 E. coli mutant single gene mutant strains. In the last year, we have optimized the test
procedure for HTS with bacterial single gene deletion mutants using a toxic standard reference
nanomaterial, PS-NH2 and used this methodology to demonstrate that the two primary mechanisms of
toxicity for this material are generation of ROS and destabilization of the outer membrane. We are
currently applying this methodology to investigate the mechanisms of toxicity of Ag nanomaterials in E.
coli and to determine whether the differential levels of toxicity observed for Ag nanomaterials with
different sizes, shapes, and surface coatings reflect different mechanisms of toxicity. Based on the
results obtained from our genome-wide HTS toxicity screens, and earlier data from the literature, we are
currently developing a database of E. coli single gene mutants that are more sensitive or resistant
towards ENMs. This set of strains should serve as an important tool for defining the relationship
between physicochemical properties and mechanisms of toxicity in nanomaterials.
The impact of this work from a theme-wide perspective is that we are poised to compare bacterial HTS
results to those obtained in HTS studies in mammalian cell lines and zebrafish. What is already clear is
that not all particles that are toxic to organisms in one kingdom, class or phylum are necessarily toxic to
all others, which reinforces the importance of the holistic approach taken by the UC CEIN in studying
nanotoxicology in a broad range of platforms and organisms. At the same time, we have been able to
demonstrate that the “positive control NM” (60 nm PS-NH2 NM) which is used across the UC CEIN is not
only toxic across all organisms studied to date, but that at least one of the mechanisms of toxicity for
this NM (ROS generation) is conserved across all of the organisms studied to date – including ones from
different kingdoms. Going forward, our hope is that the sub-lethal assays in bacteria which we are
transitioning to high-throughput will provide a rapid way to prioritize testing for not only bacteria, but
other environmentally-relevant organisms.
Project ID Assignment: HTS-4: Property-activity analysis of silica nanoparticles, including the
relationship of surface chemistry to toxicological potential
Haiyuan Zhang, Tian Xia, Andre Nel
Silica nanoparticles can exist in crystalline as well as amorphous form. Although crystalline silica in the
form of quartz is capable of inducing silicosis, lung cancer and autoimmune diseases, there is a
considerable debate about the relative toxicity of amorphous, fumed, mesoporous and other crystalline
silica polymorphs, particularly as it pertains to nanoparticles. Due to the widespread use of silica
nanoparticles in consumer products (e.g., as desiccants), it is important to determine what the basis for
silica toxicity is and to explain material hazard in terms of the physicochemical properties of the
different silica types. Project ENM-3 describes the acquisition and physicochemical characterization of a
library of silica nanoparticles that includes amorphous colloidal (Stober) silica, fumed silica, mesoporous
silica, silicalite and Min-U Sil (quartz) for conducting in vitro and in vivo property-activity analysis. Quartz
toxicity in the human lung has been hypothesized to be dependent on particle properties that include
the surface display of silanols groups and surface defects capable of generating adverse biological
effects such as oxidative stress and cell membrane damage. Moreover, the presence of strained and
unstrained siloxane rings in the particle structure could determine the surface display of silanols and
hydroxyl groups that contribute to the biological activity of the material. Project ENM-3 describes the
physicochemical characterization of the silica nanoparticles in the library, including the assessment of
their crystalline phases, primary sizes, hydrodynamic sizes, display of various silanols groups and the
presence of strained and unstrained siloxane rings. These materials were dispersed in tissue culture
media and used for conducting MTS, LDH and ATP assays in BEAS-2B and RAW 264.7 cell lines. The
classic single parameter assays showed increased cytotoxicity for fumed silica and quartz compared to
amorphous colloidal silica, mesoporous nanoparticles and silicalite that showed little or no toxicity. This
hazard ranking was confirmed by in the multi-parameter HTS assay showing that fumed silica and quartz
could induce decreased plasma membrane integrity, increased intracellular calcium flux and increased
oxygen radical generation. We demonstrated that the inclusion of N-acetylcysteine (NAC, a thiol
antioxidant) could reduce the ROS production and cytotoxicity in response to Min-U-Sil but not change
fumed silica toxicity, indicating that the latter material type could be operating by a different
mechanism than Min-U-Sil. Confocal microscopy revealed most FITC-labeled fumed silica were bound to
the cell membrane rather than being taken up into cells in the same way as Stober silica. Since previous
work has suggested that high temperature calcination and the state of hydration affects the surface
properties of silica, we were interested in determining whether the synthesis of fumed silica under high
temperature flame spray conditions would change the toxic potential of this material. Fumed silica
nanoparticles calcined at 600 and 800oC showed a progressive decline of cytotoxic potential compared
to non-calcined particles. The reduced toxicity was accompanied by decreased density of vicinal silanols
groups as shown by the IR analysis (see Project ENM-3). Not only did the decrease in these closely
spaced silanols groups lead to decreased hydroxyl radical generation, but also decreased red cell
membrane disruption by fumed silica. Moreover, these changes were accompanied by an increase in the
number of strained three-membered siloxane rings as demonstrated by the Raman spectroscopy (see
Project ENM-3). In contrast, rehydration of calcined particles increased their cytotoxic potential in
parallel with increased surface vicinal silanol display, increased hydroxyl radical generation and
increased lytic potential of red cell membranes. Project ENM-3 further demonstrates that water reflux
decreased the number of three-membered siloxane rings in the calcined particles, suggesting that the
formation of the vicinal silanols is dependent on reconstruction of strained rings. In summary, the
toxicity of fumed silica nanoparticles, which are produced in large quantities for industrial use, is related
to the surface display of vicinal silanol groups through reconstruction of strained siloxane rings. We now
need to determine whether fumed silica pose environmental hazard by studying bacteria,
environmental organisms and rodents. We are currently characterizing other silica chemical constructs
to probe their structure-activity relationships. To date, we have not seen any toxicity related to
mesoporous silica nanoparticles that are being used in CEIN as stealth delivery particles (e.g., for
delivery of metal and metal oxides intracellular) or for studying the effect of long aspect ratio
nanomaterials on the cellular response (Project HTS-8).
Project ID Assignment: HTS-5: Assessment of the role of metal oxide energy structure on the biological
effects and potential toxicity of this class of ENMs
Haiyuan Zhang, Tian Xia, Andre Nel
Because metal oxide (MeO) nanoparticles are widely used in cosmetics, sunscreens, catalysts, water
treatment equipment, textiles and solar batteries, there is a good likelihood that either the primary or
derivatized materials could come into contact with humans and environment. Although some metal
oxide nanoparticles such as ZnO and CuO, have been shown to exhibit high potential toxicity in the
environment, most metal oxides have not been systematically explored for their hazard potential in a
nano EHS setting. Because of the potential large number of nanomaterials in this category, it would be
useful to develop a toxicological paradigm that is premised on property-activity relationships that will
allow metal oxide hazard ranking for execution of in vivo studies. Recently, a theoretical and conceptual
framework was suggested in which the toxicity of metal oxide nanoparticles in bacteria could be
correlated to their energy structures, including conduction band (Ec) and valence band energies (Ev)
(Burrello et al, Nanotoxicology, 2010, 5, 228-235). Moreover, it has been suggested that the overlap of
the Ec and Ev with biological redox potential could constitute a basis for electron transfer that leads to
oxygen radical generation and oxidative stress as the basis for the toxicological profiling. This is in
agreement with CEIN's efforts to develop nano-QSARs, including the finding that MOx toxicity analysis by
the multi-parametric HTS assay suggest that atomization energy is a key nanoparticle descriptor for
predicting the toxicity of these materials (Theme 6, EDA-2). Based on our hypothesis that bandgap and
solvation energies may constitute the basis for the nano-QSAR classification of MOx’s (EDA-2 and ENM1), we obtained a series of 24 different MOx nanoparticles and determined the Ec and Ev values by
measurement of UV-Vis absorbance in relation to a commodity that can be defined as "cellular
biological redox potential", which ranges from -4.12 to -4.84 eV. This resulted in the prediction that 6
types of materials (Co3O4, Cr2O3, Mn2O3, CoO, Ni2O3 and TiO2) should theoretically pose toxicity in a
mammalian cytotoxicity screen, while the rest (18 alternative MOx materials) should be devoid of
toxicity. A limitation of the study was that we had to acquire most of the particles from commercial
sources (other than a small number Dr Mädler could produce), and therefore had to work with a spread
of particle sizes (20-100 nm) that are not ideal. In spite of this shortcoming, we developed an initial data
set to correlate bandgap energy to cytotoxicity, which is quite helpful. This will now allow us to
synthesize some of these particles in-house for further modeling studies. Following particle
characterization and optimal dispersion in cellular tissue culture media by the methods developed in
Theme 1, the 24 MOx’s were subjected to cytotoxicity screening. This commenced through the use of
single parameter MTS, LDH and ATP assays in epithelial and macrophages cell lines. Additional
screening was employed to compare MOx bandgap to the multi-parameter assessment of intracellular
calcium influx, hydrogen peroxide and superperoxide generation, dissipation of mitochondrial
membrane potential and cell death (assessed as increased the podium iodide uptake), using multiple
doses and time points (1-6 h and 24 h) as described in Project HTS-1. The single parameter assays
showed a clear hierarchical ranking in which CuO, Co3O4, Cr2O3, Mn2O3, CoO, Ni2O3 and ZnO induced
more toxicity over the 24 h observation period, while the rest (i.e. 17 MOx’s) showed little or no toxicity.
There was a similar ranking of these materials in the multi-parametric HTS assay. Our ICP-MS analysis
showed CuO and ZnO had high metal dissolution rates (CuO: ~15%; ZnO: ~35%) while Co 3O4, Cr2O3,
Mn2O3, CoO and Ni2O3 had very low metal dissolution rates (<1%), demonstrating the toxicity of CuO and
ZnO was due to high metal dissolution rate rather than bandgap. Taken together, both single and
multiple-parameter assays demonstrated excellent correlation with the bandgap prediction for toxicity,
with the exception of TiO2 that did not generate toxicity in our screen. Our biophysical assay further
indicated that Co3O4, Mn2O3, and Ni2O3 could significantly oxidize reduced cytochrome c into its oxidized
form and Mn2O3 could significantly oxidize NADPH into NADP. These constitute two of the redox couples
that determine the biological redox potential. We are now in the process of doing in silico data analysis
with the nano-QSAR model developed in Theme 6 (R. Liu, et al, Small, 2011, 7, 1118-1126) and we will
supplement the data set by including different particle sizes for a few selected high and low toxic
materials. We have also just begun to explore the extrapolation of the in vitro to in vivo analysis in zebra
fish embryos and in rodents. Our preliminary in vivo toxicological assessment of acute inflammatory
responses in lung tissue have confirmed that the more cytotoxic CuO, Co3O4, Cr2O3, Mn2O3, CoO, Ni2O3
nanoparticles could generate more injury compared to Fe2O3, Fe3O4, WO3, NiO, CeO2, HfO2, In2O3, Y2O3
and ZrO2 nanoparticles. In summary, we have developed a predictive toxicological model from which
MeO bandgap energy emerges as an important characteristic that determines material toxicity.
However, we want to address why TiO2 does not adhere to the predictions of this paradigm. For TiO2, it
is important to consider that our assays are performed under "dark conditions" and that the
toxicological predictions may change during photoactivation, as outlined in Project HTS-1. The
provisional toxicological effects of some of the above MOx's in the zebra fish embryo are delineated in
Project HTS-2. The results demonstrated that dissolution chemistry and interference in the zebra fish
hatching enzyme is the appropriate toxicological paradigm for hazard assessment in that environmental
setting.
Project ID Assignment: HTS-6. Linking the physicochemical characteristics of a library of multiwall
carbon nanotubes (MWCNTs) to toxicological outcomes in vitro and in vivo
Xiang Wang, Tian Xia and André E Nel
The goal of this project is to link the physicochemical properties of MWCNTs to toxicological effects in
cells, using cellular perturbation of the NALP3 inflammasome to predict pro-inflammatory and fibrogenic
effects in vivo. Multiple CNT characteristics play a role in their toxicological effects, including tube
length, state of dispersion, presence of metal contaminants and hydrophobicity, leading to
agglomeration and formation of tube stacks that can damage the phagolysosome. We have recently
developed a dispersal method for as-prepared (AP), purified (PD) and carboxylated (COOH) MWCNTs
that allows us to quantitatively assess their interaction with epithelial cells and macrophages, which are
two of the key cell types involved in pulmonary fibrosis (Wang et al; ACS Nano. 2010, 4, (12), 72417252). We demonstrated that tube dispersal by bovine serum albumin (BSA) and
dipalmitoylphosphatidylcholine (DPPC) increases the production of TGF-β1 in BEAS-2B cells and IL-1β in
THP-1 cells. We hypothesized that the state of dispersion may determine cellular and tissue
bioavailability and demonstrated that the well-dispersed AP- and PD-MWCNTs are readily taken up by
BEAS-2B cells with the ability to induce more prominent TGF-β1 production than non-dispersed tubes.
Cellular uptake was identified by TEM and Raman microscopy. BEAS-2B cells also produced increased
levels of PDGF-AA (another pro-fibrogenic factor) in response to better dispersed tubes. COOHMWCNTs were poorly taken up in bronchial epithelial cells and induced little or no TGF-β1 and PDGF-AA
production. In contrast to the effects on epithelial cells, all tube types were taken up by pulmonary
macrophages cells, irrespective of their state of dispersal, but there was a clear functional difference
insofar as AP and PD-MWCNTs inducing more IL-1β production in a myeloid cell line than COOHMWCNTs. Since these data suggest that the state of tube dispersal determines bioavailability and proinflammatory cellular responses, we also asked whether similar biochemical responses can be detected
the lungs of intact mice. 21 days following the aspiration of AP, PD and COOH-MWCNTs in their welldispersed or non-dispersed states, TGF-β1, IL-1β and PDGF-AA levels were assessed in the
bronchoalveolar lavage (BAL) fluid. The data demonstrated that dispersed tubes induced increased TGFβ1 and PDGF-AA levels in BAL fluid and that these biochemical responses is accompanied by increased
lung collagen production in comparison to non-dispersed tubes. Moreover, Trichrome-stained lung
sections showed that well-dispersed AP- and PD-MWCNT induced more prominent peribronchiolar and
interstitial fibrosis than those of COOH-MWCNT. Instead, carboxylated tubes induced less fibrosis,
which is in keeping with the trend towards decreased growth factor production. While IL-1β levels were
not increased at 21 days, the BAL fluid did demonstrate increased levels of this cytokine at an earlier
time point (24 hr) following administration of dispersed AP- and PD-MWCNT. Thus, the in vivo
biomarker responses and collagen deposition accurately reflect pro-fibrogenic cellular responses in
vitro. These results indicate that the state of MWCNT dispersal affects pro-fibrogenic cellular responses
that correlate with the extent of pulmonary fibrosis. Taken together, our results demonstrate good
agreement between pro-fibrogenic responses in vitro and bio-markers in vivo. A key question is
whether similar effects are induced in simpler organisms living in the environment. A comparative study
in the zebrafish has not shown any discernable tissue injury in the embryos and larvae. We are
collaborating with Dr. Cherr in Theme 5 to determine whether CNTs induce inflammasome activation in
oyster hemocytes. These phagocytic cells play a role in protection of the oyster against infectious agents
and it is possible that triggering of hemocytes inflammasomes could alter innate immune defense.
Project ID Assignment: HTS-7. Linking the physicochemical properties of a library of single-walled
carbon nanotubes (SWCNTs) to toxicological outcomes in vitro and in vivo
Xiang Wang, Tian Xia and André E Nel
The goal of this project is to link the physicochemical properties of SWCNTs to toxicological effects at
cellular level that also reflect their pathogenic potential in vivo. A number of SWCNT characteristics
could contribute to nano EHS effects, including metal contaminants, hydrophobicity, state of dispersion,
surface functionalities etc. The method of synthesis (CoMoCAT®, HipCO and Arc discharge) could also
contribute to the display of these properties and the toxicological potential of the different types of
tubes. The main hypothesis is that the metal impurity, hydrophobicity and dispersibility determine the
toxicological outcomes in vitro and in vivo. We obtained a SWCNT library that is composed of raw and
purified tubes synthesized by three different methods: CoMoCAT®, HipCO and Arc discharge from Prof.
Mark Hersam (Project ENM-2). These materials exhibit different levels of metal impurity,
hydrophobicity as well as dispersal states to allow us to conduct cellular toxicity studies for discovery of
injury responses that also reflect the in vivo hazard potential of the tubes. Basic tube characterization to
determine their length, diameter, purity, hydrophobicity, zeta potential and hydrodynamic size in water
and cell culture media was performed by using TEM, TGA, ICP-MS, ZetaPALS and DLS. Dispersal states
(suspendability index) of SWCNTs in physiological buffers using BSA plus DPPC (Wang et al; ACS Nano.
2010, 4, (12), 7241-7252) or Pluronic F108 (by Matt Duch from Dr. Mark Hersam’s lab) were performed
using UV/Vis spectroscopy. Aim 1 addresses the cellular viability and pro-inflammatory effects of the
different formulations in the monocytoid cell line, THP-1, which is prototype cell line for studying the
mechanistic affect of fibers and nanoparticles on NALP3 inflammasome activation and ILTriggering of this pathway plays an important role in the chronic inflammatory effects of CNTs in the
lung and could also play an important role in affecting hemocyte function in oysters and other
phagocytic cell types that play a role in innate and primitive immunity in several environmental life
forms. Raw, unpurified SWCNTs induced the highest levels of IL-1β production, which went down
precipitously after isopicnic centrifugation of the tubes and returned to basal levels when the tubes
were coated with the tri-block copolymer, Pluronic F108. Similar effects on cytotoxicity were seen when
the single parameter MTS and LDH assays were used to study the different tube types in THP-1 cells. In
Aim 2, we used the high content screening platform developed for zebrafish embryos in Project HTS-2 to
study the possible environmental impact of SWCNTs. We demonstrated that the raw SWCNT produced
by Arc discharge (and which includes as much as 32 % Ni by weight) could inhibit zebrafish embryo
hatching due to shedding of nickel ions as demonstrated by ICP-MS. In contrast, purified tubes
synthesized by the same process (and only containing 6 % Ni by weight) did not induce any adverse
effect in the embryos. We are in the process of assessing inflammasome activation in oysters hemocytes
in collaboration with Prof. Gary Cherr and Theme 5. In summary, our results showed that the metal
impurity content and the state of dispersal in buffered media play a key role in the toxicological effects
of different SWCNT types in cells and zebrafish embryos. Our future work will address the contribution
of inflammasome activation and IL-
and DNA leverage project, lung injury in rodents. We also looking at the passivation of the tubes surface
by Pleuronic F108 as a potential safe design feature for CNTs from a biological perspective.
Project ID Assignment: HTS-8. Study of the role of long aspect ratio mesoporous silica nanoparticles
(MSNPs) on cellular uptake and bioavailability
Huan Meng, Sui Yang, Zongxi Li, Tian Xia, Jeffery I. Zink, and Andre E. Nel
Besides spherical nanoparticles that have been intensively investigated, nonspherical ENMs with a high
aspect ratios (AR’s) are of great interest since this physicochemical feature has been shown to have a big
impact on bioavailability. The goal of this project is to study how AR impacts cellular function, including
the effect on the rate, abundance and the mechanism of cellular uptake. The hypothesis is that the
nanoparticle AR determines the abundance and mechanism of the uptake in cells and environmental life
forms. In order to test this hypothesis, it is necessary to construct an ENM library in which the same
base material is used to construct a series of different ARs. Theme 1 provided us with an in-housesynthesized mesoporous silica nanoparticle (MSNP) library that included nano spheres and nanorods
that express AR’s that very from 1 to 4.5. Extensive material characterization by TEM, BET, ZetaPALS and
DLS was carried out to assess size, length, diameter, pore size, surface area, zeta potential and
hydrodynamic size in water and cell culture media. Aim 1 of this project was to use the MSNP library to
study the impact of AR variation on cellular uptake in epithelial cell lines. By using particles that were
tagged with fluorescent dyes, we could visualize and quantify the intracellular uptake by confocal
microscopy and flow cytometry. We also used electron microscopy (EM) for ultrastructural resolution of
the cellular processing of the rods and spheres. Our results demonstrated that rod-shaped particles are
taken up in greater abundance and that the cells prefer rods with an intermediary AR of 2.1-2.5.
Interestingly, the rod-shaped particles were taken up by a process of macropinocytosis as demonstrated
by electron microscopy. In Aim 2, we performed mechanistic studies to understand why there is a
preferential uptake of the intermediary length rods, and demonstrated that this is an active process that
is sensitive to amiloride, cytochalasin D, azide, and low temperature conditions. Intermediary length
rods induced the maximal number of filopodia and activation of small GTP-binding proteins (e.g., Rac1,
CDC42) that stimulate actin polymerization and filopodia formation. Collectively all of these elements
play an active role in adjusting the cellular response to the rod length. This suggests that AR may
determine bioavailability and in future work will explore how long AR may affect ENM uptake in
bacteria, yeasts and zebrafish embryos. We will also explore the impact of AR in other particle types,
such as metal and metal oxide (e.g. Ag nanoparticle, CeO2 nanoparticle). Our preliminary data indicate
that CeO2 nanowires are capable of generating cellular toxicity through inflammasome activation that is
not seen by spherical shapes of the same composition. The work on long aspect ratio materials will
further supplemented the studies on CNTs described in other Theme 2 projects.
Project ID Assignment: HTS-9. Study of the biological effects of long aspect ratio CeO2 on
inflammasome activation and the generation of cytotoxicity
Xiang Wang, Zhaoxia Ji, Tian Xia and André E Nel
The goal of this project is to link the aspect ratio (AR) materials (LARM) other than CNTs to toxicological
effects at cellular level. We have previously demonstrated that the mesoporous silica nanoparticle
(MSNP) with different AR induced differential cellular uptake (Meng et al., ACS Nano, 2011). The
hypothesis for this project is that the AR of ENMs could play a key role in determining the cellular
uptake, inflammasome activation and cytotoxicity and will use a CeO2 library to perform a study in the
human acute monocytic leukemia cell line, THP-1, which has proven useful for studying inflammasome
activation to LARMs. Project ENM-6 (Theme 1) describes the in-house synthesis of a CeO2 nanoparticle
library that includes materials with ARs that vary from 1 to > 200. The relevance of considering LARM is
that some commercial sources of CeO2 nanoparticles may include LARM as an inadvertent component
that may distort the assumed toxicological analysis of nanospheres. This may explain some of the
discrepancies related to the study of CeO2 toxicity, in which some investigators report toxicological
effects in the environment while to date we have not found any toxicity with the use of CeO 2
nanospheres. The library materials were thoroughly characterized by the Nanoparticle Core to
determine rod length, diameter, purity, zeta potential and hydrodynamic size in water and cell culture
media. Aim 1 was to study the cytotoxicity of the CeO2 LARM library in THP-1 for their ability to induce
cytotoxicity as determined by MTS and LDH assays. This demonstrated that CeO2 nanoparticles with an
AR <50 did not induce cytotoxicity but that nanoparticles with AR >50 and length >300 nm induced
incremental cell death in THP-1 cells. In order to explore the mechanism by which these LARM induce
cytotoxicity, Aim 2 studied cellular uptake in THP-1 cells, using the SEM and TEM techniques. This
demonstrated while low AR particles (AR <50) are taken up into endosomes/lysosomes, leaving the
endosomal membrane intact, particles with an AR > 50 could damage endosomes/lysosomes
membranes as determined by TEM. Moreover, SEM demonstrated that the LARM protruded through
the surface membrane, which is reminiscent of frustrated phagocytosis. Because frustrated
phagocytosis has been associated with inflammasome activation in macrophages, Aim 3 looks at IL-1β
production as a product of the Nalp 3 inflammasome that processes pre- IL-1β. The results showed that
while CeO2 nanoparticles with an AR >50 induced IL-1β production, materials with an AR <50 showed
little effect. In summary, these results demonstrate that AR of CeO2 nanoparticles is critical in terms of
the ability to induce cytotoxicity and inflammasome activation in THP-1 cells. In our future work, we will
perform in vivo studies to determine whether to propensity towards inflammasome activation of
particles with an AR >50 also translate into toxicological effects in the zebrafish embryo and oyster
hemocytes.
Project ID Assignment: HTS-10. Reporter Gene Cell-based Assays for High Throughput Screening to
Determine Sub-Lethal Toxicity of Nanomaterials
Ken Bradley and Bryan France
The goal of this project has been to implement mammalian reporter cell lines for rapid assessment of
sub-lethal nanotoxicity and pathway delineation. We have developed a high throughput screening (HTS)
assay to assess reporter gene activation of genes response elements control downstream of cellular
signaling pathways. A luciferase pathway reporter system (Qiagen/SA Biosciences; CLA-004LA) was used
to transduced RAW 264.7 and BEAS-2B cells to develop a multi-parametric assay similar to the oxidative
stress and cytotoxicity assay described in Project HTS-1. The reporter system contains a luciferase
reporter gene expressed upon the induction of one of ten independent cellular stress pathways. Each
pathway is controlled by a unique transcription factor that drives luciferase expression in the cognate
reporter cell line. In total, 20 distinct reporter cell lines (two cell types, ten reporters per cell type) were
generated. This system was validated using eight metal and metal oxide ENMs also used in Project HTS-1
(Theme 2). The the cells were exposed to NPs for multiple times (3-24 hours) across ten two-fold
dilutions ranging from 200 µg/mL down to 0.39 µg/mL.
Zinc oxide and platinum were observed as the most toxic ENMs assayed in the reporter gene assay,
consistent with the study in Project HTS-1. Although both ZnO and Pt NPs induced stress responses, the
specific pathways involved were found to depend on cell type. The macrophage reporters show a ZnOand Pt-mediated, time- and dose-dependent induction in pathways correlating to DNA damage and cell
cycle control (p53, MYC, and E2F). These results correlate with published results from Sharma et al
(2009) as well as Jung et al (2007). ZnO also appeared to down-
and AP1 pathways in macrophages. In lung epitheal cells, ZnO down-regulated a similar set of
pathways, including NFkB, SMAD, SRE, CRE, HIF1a, and AP1, indicative of sublethal toxicity and
dysfunction in cAMP and inflammatory regulation.
Using these data sets, Rong Li (Theme 6) performed association modeling to correlate reporter gene
activation with the data sets generated in the oxidative stress and cytotoxicity HTS. In macropahges,
increased superoxide production (MitoSox fluorescence) was the best correlated with E2F, SMAD, Myc,
and p53 pathways at high ZnO and Pt doses (50-200 µg/mL), implicating a mitochondrial driven death
mechanism. For lung epithelial cells, association modeling placed SMAD, SRE, CRE, HIF1a, and propidium
iodide membrane integrity dye (Theme 2) in a high level of association at high dosage exposure (25-200
µg/mL) of ZnO and Pt NPs, suggesting a necrotic death mechanism. Notably, this analysis reveals that
the most informative toxicity or sub-lethal assays depend on cell type. Therefore, future assays to
ascertain toxicity of new NPs and/or cell types should probe a broad range of toxicity mechanisms to
avoid missing sub-lethal toxicity/stress events.
Project ID assignment: HTS-11: Toxicological assessment of cationic nanoparticles with different
surface charge densities in differentiated and undifferentiated bronchial epithelial cells
Haiyuan Zhang, Tian Xia, Andre Nel
One of the key physicochemical characteristics that determine ENM interactions with biological surfaces
is the surface charge of the materials. Previous research has demonstrated that the cationic surface
charge could generate cytotoxicity, including to environmental life forms such as bacteria and yeasts as
well as the human lung. The goal of this project is to fully understand the biological effects of charged
nanoparticles with different charge densities, including the possibility that the elucidation of a cationic
threshold could be used to design safer materials. In order to test this hypothesis, Theme 1 synthesized
a library of used mesoporous silica nanoparticles (MSNPs) in which there negatively charged surfaces
were used for the electrostatic attachment of a series of polyethylenimine (PEI) polymers that vary in
length from 0.6-25 kD. These materials were extensively characterized to obtain the zeta potential,
primary size and hydrodynamic size by the performance of TEM and DLS in the Nanoparticle Core. For
comparison to the effects of the cationic and phosphonate functionalized MSNP, we also generated a
particle with non-charged surface through PEG attachment.
In Aim 1, the cytotoxicity and cellular uptake of anionic, neutral and cationic MSNPs were compared in a
variety of different cell types. While MSNP-PEI 25 kD exhibited high cytotoxicity ,MSNP-Phos and MSNPPEG showed little or non-toxicity. The increased toxicity of the PEI coated particles was associated with
high cellular uptake as determined by flow cytometry and confocal microscopy. This demonstrated that
the relative increase in fluorescent particle uptake was 2 orders of magnitude higher with MSNP-PEI
than MSNP-Phos or MSNP-PEG. This finding was corroborated by confocal studies, which showed a
significant increase in the number of MSNP-PEI 25 kD in all cell types looked at. Decrease of the in PEI
polymer length below 10 KD had a definitive effect on reducing cytotoxicity, which was absent at
polymer lengths of 5 and 1.2 kD. The contribution of polymer length to cytotoxicity was demonstrated in
the multi-parametric HTS assay, which confirmed that clear difference in their rates of sub-lethal and
lethal toxicity when polymer length decreases below 10 KD. Interestingly, the particles coated with
polymers < 10 kD maintained a high level of cellular uptake and this finding became the basis for the
development of MSNP carrier that are capable of nucleic acid delivery to cancer cells. This demonstrates
the dynamic use of discovery at the nano/bio interface, allowing toxicological screening to prestage the
design of safer nanomaterials.
In Aim 2, the mechanistic effects of the cationic MSNP library were further explored in differentiated
and undifferentiated normal human bronchial epithelial cells (NHBE). The necessity to perform this
comparison became obvious in preliminary studies demonstrating that the state of NHBE differentiation
determine their susceptibility to cationic particles. Using the multi-parametric assay, cationic MSNPs
with higher charge density (MSNP-PEI 10 kD or 25 kD) showed increased intracellular calcium influx,
increased cell membrane leakage, increased superoxide generation and decreased mitochondrial
membrane potential compared to MSNPs coated with polymer lengths of 0.6-1.8 kD. Moreover,
differentiated cells showed more toxicity than undifferentiated cells, which could be explained by the
fact that cellular differentiation leads to increased expression of anionic proteoglycans on the cell
surface, including the heparin sulfate glycoprotein, syndecan-1. Syndecan-1 contains abundant heparin
sulfate groups that are capable of binding to the cationic MSNPs. Pretreatment with heparinase lowered
particle association as well as cytotoxicity in differentiated cells. In addition, cationic MSNPs lowered the
membrane potential of differentiated NHBE cells and affected the structural integrity of the RBC
membrane, suggesting the surface-bound cationic particles can lead to surface membrane damage as
the most immediate injury effect.
In summary, cationic nanoparticles with high charge density is associated with higher cellular uptake and
cytotoxicity than anionic or neutral nanoparticles. For cationic particles we could define a definitive
threshold above which positive charge contributes to toxicity. Detailed analysis of this threshold
demonstrated that both the number of cationic groups as well as their placement on the particle surface
determines their membrane damaging potential. Thus, longer length polymers exhibit an asymmetric
distribution of cationic charge that is capable of making contact with the surface membrane, compared
to shorter length polymers where these cationic groups are screened by culture medium proteins that
interact with the particle surface. Differentiated NHBE is more susceptible to cationic injury due to the
expression of anionic heparin sulfate proteoglycans.
Theme 3 - Fate, transport, exposure and life cycle assessment (Keller)
Project ID Assignment: FT-1: Role of Material Properties and Environmental Conditions on
Nanoparticle Aggregation and Dissolution
Dongxu Zhou and Arturo Keller
The overall goal of this project is to understand the mechanisms and environmental factors that control
aggregation and dissolution of nanoparticles (NPs). NP aggregation is important because it determines
their mobility in the environment, which is key for determining their bioavailability. The aggregation
state of the NPs will affect how long they may keep suspended in water column, and at what size they
will interact with aquatic organisms. For several NPs, their dissolution and shedding of ions can be an
important factor determining their toxicity and the ultimate fate of the NP. Specifically, the aims of the
project are to: (1) determine the effect of solution chemistry on NP aggregation rates; (2) correlate NP
properties (e.g., composition, size, morphology, crystal structure, and capping agents) and their
tendency to aggregate or remain stable; (3) study the interactions between NPs and suspended
particles; and (4) determine the rate of dissolution of metal and metal oxide NPs in natural waters. We
work with metal (Ag, Pd and Pt) and metal oxide (TiO2, CeO2, ZnO, CuO and Fe2O3) NPs provided by
Theme 1.
In previous periods we reported on (1) how pH, ionic strength and NP concentration control metal oxide
(TiO2, CeO2, ZnO) NP aggregation; (2) the destabilization of TiO2 NP suspensions by divalent cations such
as Ca2+, typical of hard groundwater; (3) the significant increase in mobility and bioavailability of metal
oxide NPs in the presence of natural organic matter (NOM) even at low NOM concentrations; (4) the
differences in aggregation for ZnO NPs with different morphologies, with spheres aggregating much
more slowly than plates; (5) the dissolution of ZnO NPs within 12-24 hours in different Theme 5 waters,
but the rate of ZnO dissolution can be reduced if the NP is doped with Fe. We also reported on the
combined effect of solution chemistry on aggregation and sedimentation of MeO NPs in nine different
natural waters, including seawater and freshwater used in Theme 5 experiments. These findings were
shared with other research groups in the CEIN, and they served to refine our current aims. Clearly, NOM
and ionic strength dominate metal oxide NP aggregation and need to be taken into consideration for all
fate and transport studies with other NPs. Our findings stress the dominant role of multivalent ions and
organic macromolecules in controlling stability and bioavailability of metal oxide NPs in aqueous
environments. Since NP morphology affects stability and aggregation, it needs to be studied
systematically. Dissolution of the NPs is a strong function of NP composition and surface area.
In the current period, we determined the stability of Pt, Pd, Ag, and CuO NPs in three natural waters
(seawater, freshwater, and groundwater). All of the four NPs aggregated rapidly in seawater, while CuO
and Pd NPs were not stable in freshwater. However, the addition of 10 mg/L alginate generated stable
NPs suspensions for these four NPs in the three natural waters. This confirms our finding with the initial
metal oxides that NOM, in this case in the form of alginate, can serve to stabilize many NPs, including
CuO and the metallic NPs. We also determined the dissolution rate of CuO and Ag NP in seawater and
freshwater. No dissolution of CuO NP was detected in seawater or pH 8-buffered deionized water in the
15 day experimental period. However, CuO NP dissolved slowly in pH 6-buffered deionized water,
indicating that more acidic environments will lead to faster dissolution rates. Ag NP dissolution was not
observed in freshwater after 30 days, while more than 6% Ag dissolved in seawater with no plateau
observed in the time course of 30 days. The results suggest that Ag NP dissolution is sensitive to the
presence of ions. More extended dissolution experiments are currently being carried out. Thus, in
contrast to our ZnO results where dissolution was within hours, it appears that the available Cu2+ and
Ag+ ions are released slowly, within days to weeks, from the corresponding NPs under most natural
conditions.
To investigate how material properties affect aggregation, TiO2 samples with different morphologies
(rods, dots, spheres, and wires), crystal structures (anatase, rutile, and amorphous), and sizes were
obtained from Theme 1. Results to date show that TiO2 nanowires aggregate at a much faster rate than
TiO2 nanospheres and nanodots. This is likely due to the difference in diffusion coefficients between
wires, spheres and dots. Among TiO2 rods, increasing size leads to higher aggregation even at lower ionic
strength. Thus, for particles with the same composition we expect very different mobility and
bioavailability, which is important to consider in Theme 4 and 5 studies.
In this period we began studies on the aggregation between engineered nanoparticles (we use P25 TiO2
and citrate coated Ag as proxies) and montmorillonite clay particles (heteroaggregation).
Heteroaggregation between NPs and clay is important because of the prevalence of suspended clays in
natural water columns. Our results show that montmorillonite’s role in the stability of engineered NPs
varies with pH and ionic strength due to the complex charge behavior of montmorillonite’s face and
edge sites. At pH 4, montmorillonite is capable of destabilizing P25 TiO2 at low/intermediate salt
concentrations due to the electrostatic attraction between the positive charges of P25 and the negative
charges on montmorillonite’s basal plane. In contrast, montmorillonite can only facilitate fast
coagulation of Ag-citrate at intermediate salt concentrations, where the dominant basal plane double
layer of montmorillonite is compressed enough that the positive edge sites can appear and attract
negatively charged Ag-citrate. At pH 8, no enhanced coagulation was observed for either
P25/montmorillonite or Ag-citrate/montmorillonite mixture since all the surfaces are negatively
charged. This information will be useful for Theme 5 studies where organisms are exposed to suspended
sediments in the water column.
Overall, the outcome of this project is contributing to (1) identify critical environmental and material
parameters affecting NP aggregation, which will help to predict the behavior of these NPs when
released in the environment; (2) provide detailed information to ecotoxicologists in Themes 4 and 5
regarding (a) particle size and aggregation/disaggregation process they should expect when conducting
toxicology studies; (b) rate of NP dissolution and availability of dissolved metal ions; and (c)
improvements to the dispersion protocols of these metal and metal oxide NPs. In addition, the
experimental aggregation data will be important input and validation components for the
environmental modeling endeavor of Theme 6.
Project ID Assignment: FT-2: Attachment of nanoparticles to mineral surfaces under different aqueous
solution chemistries
Reginald Thio, Adeyemi Adeleye and Arturo Keller
Attachment of a nanoparticle (NP) to a mineral surface removes it temporarily or permanently from the
water column, depending on the strength of the interaction forces. Attachment is a major factor in the
overall mobility of NPs in the environment, which controls their bioavailability. These interaction forces
are a function of water chemistry. The overall aim of this project is to quantify the attachment of
nanoparticles (NPs) to natural materials such as sands and clays. It is hypothesized that the interaction
forces are a function of NP physicochemical characteristics and aqueous solution chemistry, including
the presence of natural organic matter (NOM). Thus, quantitative measurement of these interactions
can be used to model the removal of the NPs from the water column by mineral surfaces.
Atomic Force Microscopy (AFM) and Quartz Cell Microbalance with Dissipation (QCM-D) were used to
measure the interaction forces of different NPs and mineral surfaces. In a previous study (Thio et al,
2010) we reported the interaction forces between a single NP and a clay surface, using AFM. The
attachment was strongest at high ionic strength (e.g. seawater). There was no attachment in the
presence of NOM. This highlighted the role of NOM in making the NPs more mobile and bioavailable.
Earlier this year we published a study (Thio et al., 2011) in which QCM-D was used to determine the
attachment kinetics of TiO2 NPs to silica (sand) surfaces over a broad range of solution (pH and ionic
strength) conditions typical of the mesocosms waters used by Theme 5. As observed in the single NP
experiment using AFM, the TiO2 NPs attached more strongly to silica at high ionic strength and low pH.
When NOM was introduced, there was no attachment of the TiO2 NPs to the silica surfaces, indicating
that they would be very mobile and bioavailable under these conditions. Thus, NOM not only stabilizes
the NPs in terms of their size, but also inhibits attachment. In freshwater conditions, the TiO 2 NPs will
also remain stable in the water column and not attach to surfaces. With additional studies underway,
we can now generalize these findings to uncoated metal oxide NPs, such as ZnO and CeO 2 provided by
Theme 1.
The incorporation of new NPs into the CEIN library by Theme 1 allowed us to study the effect of coatings
on NP attachment. In this reporting period we measured the interactions between Ag NPs with different
coatings, to understand the important role that coatings play in these processes. We hypothesized that
attachment would be a function of the coating. Results indicate that bulkier PVP coating is much more
effective in reducing aggregation and attachment of Ag NPs on silica than shorter citrate coatings.
However, in the presence of NOM the mobility of Ag-PVP and Ag-citrate NPs in natural waters is
enhanced, even at the high ionic strengths observed in seawater. We also developed a Thin Layer
Chromatography (TLC) method to measure the attachment efficiency of the Ag NPs to silica surfaces
(such as those found in sand). We showed that the results produced by detailed QCM-D work could be
corroborated using the much faster TLC setup. These results are being used in the design of Themes 4
and 5 mesocosm studies, to understand the attachment of NPs to sediments, soil particles and other
mineral surfaces within these systems.
NPs can be dispersed and stabilized in natural waters by different natural biomolecules, including NOM,
humic acid, alginate and exudates from plants and other organisms. We hypothesize that the method of
dispersion will affect the interaction forces that lead to attachment. Since CNTs are very hydrophobic,
researchers have developed several methods for dispersing them. Thus, we recently began to study the
influence of different dispersion methods on the attachment of CNTs to mineral surfaces. We intend to
evaluate them under conditions relevant to the Terrestrial and Marine and freshwater ecosystems
impact and toxicology themes.
We also carried out experiments to study the persistence of nanoscale zero-valent iron (nZVI) and their
transport in soil column. This study is relevant because very little is known about the potential impact of
nZVI which is now being widely considered for groundwater remediation. Using different types of
commercially available nanofer (a type of nZVI) suspended in water at different ionic strengths in both
aerobic and anaerobic conditions, we monitored pH, redox, temperature, size, zeta potential,
conductivity and concentrations of various iron species in the supernatant, suspended sediment and
sediment over 28 days. As nZVI oxidized in water, the electrons released brought about reducing
conditions. As such, redox potential was found to reduce over time while pH increased slightly. Some of
the nZVI particles remained in suspension while the bulk of it settled out as sediment. Particle size
increased rapidly to a micron scale and then the particle size in suspension decreased slowly, within
days to weeks, as the reactivity decreased and the particles settled. The iron deposits may partially
reduce the porosity and hydraulic conductivity of the soil into which they are injected for remediation.
Transport studies indicated that under low calcium concentrations, a high proportion of the nZVI would
transport in the first few meters. Increasing calcium concentrations would decrease the fraction of nZVI
that transport due to the rapidly increasing particle size.
In summary, we have found that the attachment of metal oxide NPs to different surfaces is more
strongly a function of environmental conditions, such as the concentration of NOM and ionic strength,
than the physicochemical characteristics of the metal oxide NPs. High NOM concentrations lead to high
mobility and bioavailability of these NPs. Low NOM and high ionic strength, as is typical of marine
environments, can lead to attachment of the metal oxide NPs to sediments. The coating on the NPs
leads to differences in attachment. However, the presence of NOM reduces the importance of the
coating, since it can coat the surface of the NPs, reducing or inhibiting attachment.
Project ID Assignment: FT-3: Quantitative determination of fate and transport of nanoparticles in
porous media
Sharon Walker, Indranil Chowdhury, Ryan J. Honda, Jacob Lanphere, Alicia A. Taylor, Jose Valle, Elizabeth
Horstman
The overall goal is to quantify the mechanisms involved in the transport and removal of nanoparticles
(NPs) in porous media, such as groundwater and sediments. Nanoparticles released to the atmosphere
will deposit on soils and eventually enter the subsurface. In addition, there are direct and indirect
pathways for NP releases to enter terrestrial systems, of example the application of residual sludges
from sewage treatment to agricultural fields, or the injection of nanoparticles into the subsurface for
groundwater remediation. These releases present important questions for researchers in Theme 4, who
need to predict the concentration of NPs present in soils and groundwater. The experimental data
collected from these studies will be utilized in modeling by Theme 6 to predict NP transport in this
environmental compartment.
We utilize macroscopic and microscopic experimental systems to measure the key parameters needed
to predict NP fundamental mechanisms of nanomaterial transport in the porous media. Fluorescent
microscopy is utilized in observing the deposition of nanomaterials on the surface within two types of
flow cells, the Radial Stagnation Point Flow (RSPF) and Parallel Plate Flow Cell (PPFC). From these
microscopic images, the attachment is quantified under a range of hydrodynamic conditions and
systematically varied solution chemistry simulating the natural aquatic environment. Similarly,
groundwater transport is evaluated in a packed bed column system, from which the kinetics of
deposition is evaluated.
Our hypothesis is that environmental parameters including solution chemistry (pH, ionic strength, and
ion valence), as well as the presence of natural organic matter (NOM) and bacteria will control the
transport and removal of nanomaterials. Specifically, this project involves extensive characterization
and transport studies using Theme 1 nanomaterials (TiO2, CeO2, ZnO, carbon nanotubes), E. coli and
Suwannee River Humic Acid (SRHA). E. coli HU1 fluorescently tagged with mCherry plasmid was used as
model organism and Suwanee River Humic Acid was used as the organic matter. Titanium dioxide
nanoparticles (TNPs) were labeled with FITC such that both particles and cells could be simultaneously
visualized with a fluorescent microscope. We use several characterization techniques, such as
electrokinetic characterization, particle size, and potentiometric titration, to understand the surface
properties of nanomaterials both in the presence and absence of various aquatic species, NOM, and
bacteria. Stability of nanomaterials in aqueous suspension has been determined by a combination of
aggregation and sedimentation tests. Transport studies are being conducted in both the column and the
parallel plate system. A range of solution conditions (pH 5 and 7, and 10 mM KCl and CaCl2) were tested.
Results showed a significant dependence on solution chemistry and ion valence. Both SRHA and E. coli
significantly reduced nanoparticle deposition, with SRHA having a greater stabilizing influence than
bacteria. Transport in the presence of both SRHA and E. coli resulted in much less deposition than either
alone, indicating a combination of factors involved in deposition. Over the aquatic conditions
considered, TNP deposition generally 15 follows: without NOM or bacteria > with bacteria only > with
NOM only > combined bacteria and 16 NOM. This trend should allow better prediction of the fate of
TNPs in complex aquatic systems. The significance of these results for Theme 4 is that subtle changes in
pH and ion valence can alter the interactions among TNP, NOM and bacteria. These interactions leadto
changes in nanoparticle aggregate and nanoparticle-bacterial aggregate development, which can
significantly affect the transport of TNPs. A manuscript on this study is under review.
We started investigating the transport of three distinctly sized nano-TiO2 through porous media. The
particles were provided by Theme 1 as a collaboration with Dr. Lutz Mädler from University of Bremen,
Germany. The objective of this study is to determine the influence of primary particle size on transport
of nano-TiO2 through porous media. Three distinctly sized TiO2 nanoparticles (6 nm, 13 nm, 23 nm) were
synthesized, maintaining similar crystal structure. Transport studies were conducted in column packed
with homogeneous quartz sand. Extensive analyses have been conducted for these nanoparticles
including sedimentation, electrokinetic characterization and dynamic light scattering (DLS) over the
range of pH 4-10 and ionic strength 0.1-100 mM KCl. Preliminary results showed that aggregation and
electrokinetic properties of the nanoparticles are function of solution chemistry, particularly pH, and not
necessarily sensitive to primary particle size. Subsequently, the column experiments were run at pH 7
and 10, at which similar aggregate sizes and electrokinetic properties exist. Transport results revealed
that deposition rates of three distinctly sized TiO2 nanoparticles are significantly different, even though
the aggregate size and electrokinetic properties of these particles are very similar. Aggregate
morphology of distinctly sized TiO2 nanoparticles were also determined by small angle light scattering in
collaboration with Dr. Steven E. Mylon at Lafayette College. Both characterization and transport results
to date indicated that heterogeneity in nanoparticle aggregate - due to its composition of primary
nanoparticles - is playing a significant role in the transport.
This summer, we also started investigating the transport properties of 8 different single walled carbon
nanotubes (SWNTs) received from Theme 1. The goal of this project is to understand the influence of
synthesis methods and purifications on transport of SWNTs. Extensive characterization has been
conducted for SWNTs over the pH range of 4-10. Both monovalent (KCl) and divalent (CaCl2) ions were
used in characterization and transport experiments. Transport experiments have been conducted in
packed-bed column to understand the deposition and transport process of SWNTs through porous
media. SRHA was also added as NOM. Work to date has shown significant effect of purification
(primarily metal content) on transport of SWNTs. SWNTs synthesized by different methods resulted
distinctive breakthrough curves, indicating the role of synthesis methods in transport of SWNTs.
Addition of natural organic matter increased the transport of SWNTs. A manuscript in this project is in
progress.
Previously, we completed a study on titanium dioxide (Evonik Degussa Corporation, NJ), cerium dioxide
(Meliorum Technologies, NY) and zinc oxide (Meliorum Technologies, NY) nanoparticleshandling
approach and a manuscript has been published in Colloid and Surfaces A (2010, volume 368(1-3): 91-95).
This study showed the effects of handling approaches on the resulting metal oxide nanoparticles
suspensions, and proposed a guideline for an optimum handling approach for preparing reproducible
dispersion of nanoparticles in aqueous solution. This information was used in the development of a
CEIN-wide protocol for dispersion. Also, we completed a study on role of solution chemistry, TiO 2
nanoparticle concentration and hydrodynamic effects on transport of titanium dioxide nanoparticles
through porous media (Journal of Colloid and Interface Science, 2011, volume 362 (2) 548-555). The
significance of these results for Theme 4 is that subtle changes in pH ( 5 to 7) and IS (1 to 10 mM) can
lead to larger aggregate size, reducing the mobility of nano-TiO2 in porous media. Additionally, work
with the parallel plate flow system has been conducted with TiO2 nanoparticles. This study
demonstrated that the deposition of these model nanoparticles on glass surfaces was controlled by a
combination of DLVO and non-DLVO-type forces, shear rate, aggregation state, and gravitational force
acting on TiO2 nanoparticles. The significance of this study for Theme 4 is that increased nanoparticle
aggregate size can lead to dominance of gravitational force over diffusive force, which can significantly
affect attachment of nanoparticles to surfaces and its subsequent capacity to be transported in the
subsurface.
Working with Theme 6, an experimental plan has been developed through which data will be
systematically generated on particle charge, aggregate size, and solution chemistry. The new PhD
student, Jake Lanphere, will conduct the experiments under the advisement of Dr. Walker and Dr.
Cohen.
Project ID Assignment: FT-4: Effect of Wettability on the Transport and Fate of Metal Oxide
Nanoparticles
P. Somasundaran, X. Fang, S. Murthy Khandrika, I. Chernyshova, Partha Patra
To find out how aggregation properties of metal oxide nanoparticles (NPs) affect their fate and transport
in the environment and living organisms, we addressed the fate of ~30-nm TiO2, ZnO2 and CeO2 in the
N. Europea bacterial culture (as a model system demonstrating the route from formulation to
organelle/membrane surface). Results suggest that bacterial cell membranes were more densely
populated with the TiO2 NPs as compared to ZnO and CeO2. Furthermore, the population of TiO2 was
more prominent on the membranes compared to that in the interior of the cell, i.e., cytoplasm. CeO2
nanoparticles, unlike TiO2 and ZnO2, were observed in the form of aggregates at the bacterial and ‘cellwall’-media interface. TiO2 NPs are less aggregated due to higher surface energy compared to CeO2 and
thus gets entrapped in cell wall whereas CeO2 aggregated much before to their contact with the
bacterial cell wall and probably does not penetrate the cell wall. Thus, even though the particles were of
comparable sizes their fates were different, which can be attributed to their different trends to
aggregate.
Considering that the interaction of NPs with proteins and lipids is also of importance for their fate and
transport in biotic media, we studied the interactions of NPs with bovine serum albumin (BSA). We
found that in general for unbuffered solutions of 2.5mg/l BSA, pH of the ZnO TiO2 and CeO2 range from
8.5-9.0, 6.9-7.5 and 6.0-6.75. The pH values of ZnO and TiO2 suspensions decrease with time, while in
CeO2 particles an increase is observed. pH of ZnO suspension is less sensitive to particle concentration as
compared to TiO2 and CeO2 suspensions. ZnO and TiO2 particle suspensions also show similar behavior in
conductivity by showing incremental trend with time. CeO2 particle suspensions, however, shows a
decremental trend with time in conductivity. Addition of more particles enhances the conductivity in all
types of suspensions. Both the TiO2and CeO2 particles have incremental zeta potentials with time and
particle concentrations. The zeta potential for ZnO particles remains on the similar levels with time and
particle concentrations. Affinity of the NPs to adsorb on BSA varies in the order of ZnO> CeO2> TiO2.
Higher affinity of ZnO towards BSA correlates with its high surface energy. Similarly in case of CeO2 and
TiO2 their lower adsorption towards BSA could be reasoned with their lower surface energies. These
results show our hypothesis holds good for interaction of NPs with proteins. Thus particles with lower
surface energies tend to stay in bulk solution and pose the possibility of getting transported to longer
distances. In another study, mutual effects of interactions of NPs with common soil components are also
carried out.
We have also studied the interaction of ZnO, CeO2 and TiO2 with natural minerals, kaolinite and talc
under various minerals and particle concentrations. pH, conductivity, turbidity/sedimentation of the
suspensions and the charge/size of the particles in suspension were measured as function of time. A
parameter called total surface energy loading density (SELD) was defined to assess the interaction of the
particles with the minerals. It was observed that sedimentation rates vary inversely with SELD. Presence
of kaolinite and talc slow down the sedimentation rate allowing particles to stay in suspension for longer
times. Since particles remained in suspension for longer times, it is higher probable for them to interact
with aquatic organisms and other environmental components. Zeta potentials of ZnO 30nm
suspensions with and without kaolinite were investigated. Zeta potential vs. ZnO amount in suspension
was also studied. Suspension pH increased with increasing amounts of ZnO from 8.1 to 9.3 and zeta
potential dropped from 12 mV to about zero mV. Suspension pHs of kaolinite (0.01g) with different
amounts of ZnO (0.01, 0.02 and 0.05g) ranged from 8.4 to 9.4 and corresponding zeta potentials became
less negative and ranged from -23 mV to about zero mV. This could be due to particle dissolution,
dissolved species and physicochemical properties of particles and surrounding medium. Isoelectric
point (IEP) of ZnO is at pH: 9.5 from literature and zeta potential of ZnO with and without kaolinite is
approaching zero mV at about pH: 9.5. This indicates that system behavior is dominated by ZnO and its
species perhaps by coating kaolinite particles.
We specified the adsorption forms of laurate on hematite. It was found that protonation of the outersphere complexes does not influence the conformational order of the surfactant tails. One monolayer,
which is filled through the growth of domains and is reached at the micellization/precipitation edge of
laurate, makes the particles superhydrophobic. These results contradict previous models of the fatty
acid adsorption and suggest new interpretation of literature data. The molecular-level understanding of
the adsorption of fatty acids on hematite presents the basis for the current study of the effects of NP
size and morphology on their aggregation in the presence of fatty acids.
Employing a combination of in situ FTIR and ex situ X-ray photoelectron spectroscopy (XPS) and using
the Mn(II) oxygenation on hematite (α-Fe2O3) and anatase (TiO2) NPs as a model catalytic reaction, we
discovered that the catalytic and sorption performance of the semiconducting NPs in the dark can be
manipulated by depositing them on different supports or mixing them with other NPs. We introduce the
electrochemical concept of the catalytic redox activity to explain the findings and to predict the effects
of (co)aggregation and deposition on the catalytic and corrosion properties of ferric (hydr)oxides. These
results provide a new framework for modeling the fate and transport of semiconducting metal oxide
NPs in the environment and living organisms. Using the same experimental approach, we found that
oxidative catalytic performance of hematite NPs degrades with decreasing their size. This unusual trend
was rationalized within the electrochemical paradigm.
Project ID Assignment: FT-5: Photoactivity of nanomaterials in natural waters
Samuel Bennett and Arturo Keller
The main objective of this project is to determine the photoactivity of nanoparticles (NPs) in natural
media. Photoactivity is relevant because it generates reactive oxygen species (ROS) which can lead to
toxicological outcomes and alteration to abiotic processes such as nutrient cycling. The interaction of
natural water constituents with NPs may influence photoactivity, for example natural organic matter
(NOM) may act as a photosensitizer when adsorbed to metal oxide NPs and various ions have been
shown to scavenge photogenerated holes in the conduction band of TiO2. Many NPs are known to be
photoactive, yet it’s unclear how natural water constituents and ambient conditions will influence
photoactivity. Others NPs, such as CeO2, may quench photoactivity. In addition to investigating the
photoactivity of NPs with different band gaps in natural waters, the influence of material morphology,
size and stability on photoactivity is under investigation.
Previously we reported that the photoactivity of TiO2, CeO2 and Fe2O3 NPs was very different in
freshwater and seawater, with a decrease of almost 50% in seawater. In contrast ZnO photoactivity as
measured by an OH• probe was higher in seawater than freshwater by around 40%. The likely cause of
the reduced photoactivity for most metal oxides is under investigation but is partially due to rapid
aggregation driven by high ionic strength in seawater and the ability of the carbonate system to
scavenge radicals. Experiments are under way to investigate the influence natural water constituents
have on NP stability and photoactivity. Preliminary results show that TiO2 wires, rods, irregular spheres
(P25) and nanodots (10 nm dot-shaped) are stable when dispersed in NOM or humic acid (HA) with >
95% of the initial 100 mg L-1 suspended after a few weeks. Subsequent photoactivity trials with the
NOM and HA dispersed TiO2 show that P25 and wire photoactivity are increased by as much as 30% in
the presence of NOM and that nanodot photoactivity is quenched in the presence of NOM or HA.
Although the photoactivity of TiO2 decreases in seawater, recent studies in collaboration with Theme 5
indicate that under natural lighting conditions (i.e. sunlight) TiO2 photoactivity in seawater is orders of
magnitude higher than natural conditions without TiO2, even at a 1-7 mg/L TiO2. The steady state [OH•]
generated in the presence of TiO2is sufficient to significantly reduce the growth rates of four sentinel
phytoplankton species [Miller, Bennett, Pease, Keller and Lenihan 2011, Submitted].
TiO2 NPs with different particle characteristics were received from Theme 1. In specific, we are
evaluating different morphologies (rods, wires, irregular P25 and nanodots) and within each morphology
the effect of size (4 – 30 nm) and crystalline structure (rutile, anatase and rutile:anatase), for a total of
16 different TiO2 NPs. Thus far we have evaluated OH radical production for TiO2 wires, irregular and
nanodots in the presence and absence of natural organic matter (NOM). Preliminary experiments show
that, for an equivalent dose in the presence and absence of NOM, OH radical production is greatest for
the irregular and wires, and that nanodots have limited photoactivity. The initial photoactivity trend in
DI is as follows: irregular anatase:rutile> hybrid wires> anatase nanodots> rutile wires. The presence of
NOM effectively quenches the photoactivity of the nanodots, yet increases the photoactivity of the
irregular P25 and wire morphologies.
While conducting studies with different NPs, we determined that the energy imparted by photons to
metal oxide NPs leads to the disaggregation of some primary particles from the aggregates [Bennett,
Zhou, and Keller 2011, Submitted]. This photoinduced disaggregation is important because the released
NPs have much greater mobility and are more likely to penetrate deep into the skin of different
organisms. Studies were conducted to establish the conditions that lead to photodisaggregation and the
depth of penetration of the primary NPs into pig skin. After exposure to sunlight the average TiO2
aggregate size is reduced from 280 nm to 220 nm, shedding smaller clusters and primary particles.
While full spectrum light results in greater disaggregation, bands of light in the UV, Vis and NIR can also
induce disaggregation. Using electron microscopy we have found that TiO2 NPs can penetrate the
epidermis and pass into the dermal layers of pig skin. This is an important new phenomenon that has
not previously been reported in the literature. The NP aggregates appear to shed some primary
nanoparticles that may be able to more easily pass through biotissues, resulting in higher exposure to
the NPs than in the aggregate form.
The stability and photoactivity of five CNTs provided by Theme 1 was studied. The CNTs vary in purity
(with metal impurities up to 30% of total weight). Their stability was measured over 2 months in
rainwater, groundwater, stormwater, deionized (DI) water, and DI with alginate, NOM, HA and bovine
serum albumin. With the exception of DI and alginate systems where the CNTs did not readily disperse,
all five CNTs were initially stable for a few weeks in all other systems. No significant differences were
found in stability between the different CNTs. Natural waters rich in NOM generated much more stable
suspensions, with > 50% of 2 different CNTs suspended after 4 months in water with 10 mg L-1 NOM.
Groundwater and stormwater, also rich in NOM, generated stable dispersions for all 5 CNTs. Waters
high in ionic strength and low NOM (seawater) generated less stable suspensions, with higher settling.
CNTs are synthesized with metal catalysts and the metallic residues are likely to leach from the CNT
matrix under natural conditions. Because metals are toxic to aquatic biota, we investigated the ability of
CNTs to leach metals in different natural waters. In all 6 natural waters, (e.g. seawater, surface water
and groundwater) the metal catalysts readily leach from the CNTs; with an initial dose of 10 mg L -1 CNT,
the metal concentrations in solution were often in the low [mg L-1] after a few hours. The metal
leaching rates were much greater for the arc-discharge produced CNTs than for the HiPCO produced
CNTs.
To test the photoactivity of CNTs we then measured the ability of CNTs to photocatalytically produce
superoxide anion and singlet oxygen via energy transfer in various natural waters. Our work with
electroparamagnetic spectroscopy has shown that CNT-mediated photoactivity is not due to energy-
transfer reactions, but may progress photocatalytically. CNTs were found to effectively produce
superoxide in natural waters; the production of singlet oxygen was near background rates. Working with
Theme 2, we are investigating the photoactivity and phototoxicity of CNTs to planktonic algae. In
preliminary experiments, the CNTs were found to be toxic both in the light and dark conditions, with cell
death slightly increased under light conditions. Control experiments with various metal concentrations
have shown that the toxicity of the CNTs, is not solely related to metal dissolution and suggests CNT
mediated ROS generation may have a significant role in toxicity.
While dispersing the CNTs under different conditions, we discovered a new method that converts CNTs
into rings known as nanotori. Nanotori retain many of the unique physicochemical properties of CNTs,
but also have new properties based on their conformation. The stability in natural water, photoactivity
and phototoxicity of the nanotori were evaluated and compared to the original CNTs. Remarkably, the
nanotori are able to form stable dispersions in deionized water, a phenomenon not observed for the
CNTs. The nanotori are also stable in all natural waters tested, with more than 99% of the initial mass
remaining in suspension after months. Similar to the CNTs, the nanotori are able to produce superoxide
anion in natural water as well as in vivo. Working with Theme 2, we showed that rainbow trout cells
exposed to 25 ug mL-1 nanotori and light from 350 to 1100 nm resulted in 30% cells mortality after 24
hours. As indicated by the MitoSox assay the nanotori induce oxidative stress at levels similar to TiO 2, a
potent photocatalyst, which suggests that the rings may be highly photoactive. The nanotori were not
toxic under dark conditions, even though the original CNTs were toxic under all conditions.
Project ID Assignment: FT-6: Physicochemical nano-bio interactions at different scales that influence
fate & transport of nanoparticles
Jon Conway, Milka Montes and Arturo Keller
A fundamental aspect in the evaluation of the risk of exposure to nanoparticles (NPs) for different
organisms in terrestrial and aquatic environments is an understanding of the nano-bio interface
interactions at different scales. The goal of this project is to quantify the physicochemical interactions
between and biological systems that lead to bioaccumulation and biotransformation of the NPs. The
first aim of this project is to establish a quantitative relationship for the attachment of nanoparticles to
biomolecules that are representative of the biological surfaces, such as peptides. The second aim
addresses a larger scale, namely the bioprocessing of NPs by different organisms. In particular we aim to
quantitatively determine the uptake, bioaccumulation, biotransformation and excretion of different NPs
by indicator organisms in the CEIN Terrestrial (Theme 4) and Marine and freshwater ecosystems impact
and toxicology (Theme 5) studies. A third aim, begun with the incorporation of Jon Conway (a new PhD
student) to the project, seeks to determine the bioavailability and transport of NPs in the mesocosm
studies conducted by the CEIN Themes 4 and 5.
In the past nine months we studied the interactions between Ag, CeO2, and TiO2 NPs (from Theme 1)
and L-dihydroxyphenyalanine (L-DOPA) in aqueous environments. This biomolecule is one of several that
are representative of mussel adhesive tissues. The adsorption of L-DOPA to the NPs was determined
using high-performance liquid chromatography (HPLC). We observed significant interactions between
TiO2 and L-DOPA and CeO2 and L-DOPA that led to the formation of nanohybrid complexes between the
nanoparticle surface and the catechol groups of L-DOPA through charge-transfer. The amount of
adsorbed L-DOPA was also shown to increase predictably as the exposure time was increased. Ag
nanoparticles also showed interactions with L-DOPA, although these were not as predictable. The
formation of charge-transfer complexes between Ag, CeO2, and TiO2 nanoparticles and L-DOPA may be
quite significant for understanding the differences in uptake of these NPs in aquatic environments. Since
these biomolecules are present in many important biological surfaces, they may play a very important
role in the nature of these nano-bio interactions.
In a continuation of the collaboration with the Marine and freshwater ecosystems impact and
toxicology theme we are extending our work in which we studied the uptake and bioprocessing of ZnO
and CeO2 by mussels to determine whether these NPs are accumulated in marine phytoplankton that is
then taken up by the mussels. This is intended to be a longer term study to simulate chronic
environmental exposure where we can look at the accumulation behavior of mussels over time. With
this information, researchers in Theme 5 can better understand the actual doses that their indicator
species are exposed to in their studies. The information will also be useful for Modeling of the
Environmental Multimedia Distribution of Nanoparticles (Theme 6) as they construct their model of
the distribution of NPs.
We have begun working with the Terrestrial ecosystems impact and toxicology (Theme 4), to determine
the bioavailability of ZnO and CeO2 in the planted mesocosm studies. We are preparing soil samples
identical to the ones used in the planted mesocosm study to look at the transport and interactions of
ZnO, CeO2, and other NPs. We will evaluate the fraction of NPs that remain strongly sorbed on the soil
relative to the fraction that are mobile, as well as later studies looking at bioaccumulation by plants. This
will give additional information regarding the behavior of nanoparticles in the soil, which is useful when
considering transport in the terrestrial environment.
This addresses questions regarding the actual exposure of the organisms to the NPs, as well as the fate
and transport of nanoparticles in the aquatic and terrestrial environments. This quantitative information
will serve to inform the modeling efforts at the CEIN.
In summary, this project is providing information on the nano-bio interface at various scales. We are (1)
determining the biomolecules that are most likely responsible for the initial interaction with the NPs; (2)
the distribution of NPs and their components within and outside the organisms used in the CEIN studies;
and (3) the movement and behavior of NPs through the terrestrial environment and consequences of
exposure.
Project ID Assignment: FT-7: Life Cycle Impacts Assessment of Carbon Nanotubes
Sheetal Gavankar, Arturo Keller and Sangwon Suh
The overall goal of this project is to perform a screening Life Cycle Impacts Assessment (LCIA) of carbon
nanotubes (CNT) in two applications. Existing studies indicate that CNT manufacturing may be more
energy intensive compared to conventional materials on a mass basis. However, the possibility that
certain application may require less amount of CNTs or other resources (e.g. energy) compared to the
“conventional” counterpart, or that it may save resources from other parts of the product system
upstream or downstream, is unexplored. Moreover, existing studies also do not evaluate CNT release
(emission / fate & transport) during any of the life cycle stages. Hence our environmental evaluation of
a downstream application of CNTs as well as incorporation of CNTs’ fate and transport evaluation at
various life cycle stages holds special significance.
As a theoretical preparation for this project, a review paper was submitted to the International Journal
of Life Cycle Assessment. The purpose of this review was to evaluate how the nano-specific
environmental assessments are being conducted within the existing framework of life cycle inventory
and impact assessment, and whether these frameworks are valid and/or whether they can be modified
for nano-evaluations. We evaluated the major characteristics and mechanisms under which nanomaterials affect the environment, and whether these characteristics and mechanisms can be adequately
addressed with current Life Cycle Inventories and Impact Assessment practices. We also discuss whether
the current data and knowledge accumulated around fate, transport and toxicity of nano-materials can
be used to perform an interim evaluation while more data are being generated.
Realistic information on manufacturing and other logistics from the entire supply-chain is vital for a life
cycle assessment to be meaningful. This may or may not be a problem for a mature product, but poses a
significant challenge for a cutting edge technology for which the data are closely guarded. Hence our
first tasks were identification of CNT applications for LCIA based on their market potential and data
availability for the same.
A prominent CNT manufacturing company from Oklahoma, SouthWest NanoTechnologies (SWeNT)
responded to our proposal, and showed keen interest in partnering with us on this project. Accordingly,
a Non-Disclosure Agreement and other formalities were completed in October, and preliminary
meetings for initial information exchange were held soon after. Data collection sheet for CNT
manufacturing was sent to SWeNT in November.
As we await specific details from SWeNT on the manufacturing of CNT, CNT ink and thin film formation,
parallel efforts are underway to collect publicly available data on some of the sub-processes as well as
on agreed upon end application (iPhone/iPad) in order to evaluate the complete life cycle of CNTs.
Overall the project has followed the timeline thus far, and will stay on course assuming sufficient data
collection over next months.
Theme 4 - Terrestrial ecosystems impact and hazard assessment (Holden)
Project ID Assignment: TER-1: Nanotoxicology in terrestrial microcosms
Yuan Ge, John Priester, Allison Horst, Josh Schimel, Roger Nisbet, Jorge Gardea Torresdey, Patricia A.
Holden
In soils, ENMs could negatively impact microbial and plant populations, microbial communities, and
ecosystem processes in nutrient cycling. Impacts could occur through either proximate or distal
processes. However, little is known regarding the fates and effects of ENMs in soil, including agricultural
systems. This project aims to quantify the impacts of ENMs to model terrestrial ecological systems as a
function of ENM type and concentration. The hypotheses are: 1) effects on ecosystem-level processes
will be observable through the effects of bioavailable ENMs (specifically metal oxide nanoparticles) on
sensitive or “key” microbial taxa, 2) ENMs can affect soil physical properties that influence water and
nutrient availability, thereby indirectly impacting soil microbes, plants, and their interactions, and 3) the
effects of ENMs in planted, e.g. agricultural, systems, will be through effects on plant-microbe
interactions that exceed the effects on either alone. Our hypotheses are being tested in three related
domains, involving terrestrial environments of differing complexity: grassland soil microcosms to
evaluate the effects of ENMs on 1) microbial community composition and 2) soil – water relations
(which may in turn influence community composition), and agricultural microcosms to evaluate 3) plant
uptake and damage, plus plant-microbe interactions and interactive effects with soil microbial
communities.
We previously reported the time-course effects of nano-TiO2 and -ZnO nanoparticles at varying
concentrations (0, 0.5, 1.0 and 2.0 mg g-1 soil for TiO2, 0.05, 0.1 and 0.5 mg g-1 soil for ZnO) on a
California grassland soil (Sedgwick Reserve, Santa Barbara County) in unplanted microcosms. The now-
published (Ge et al., ES&T, 2011) results include that nano-TiO2 and -ZnO reduced microbial biomass (as
measured by total extractable DNA and substrate induced respiration or SIR) and altered soil bacterial
community diversity and composition (as measured by PCR-TRFLP profiles, genotypic richness and
Shannon index). We initiated a pyrosequencing approach in the Fall of 2010 to explore the taxa-specific
response of soil bacteria to nano-TiO2 and -ZnO. The voluminous data have been analyzed, which
reveal functionally-important, sensitive taxa; a manuscript has been submitted. The insights into ENMsensitive taxa--of significance to N and C cycling--will be extremely valuable to the theme “Molecular,
cellular and organism high-throughput screening for hazard assessment (Theme 2)” which can recruit
the environmentally-relevant, sensitive bacterial taxa for HTS. This project will next assess treatment
effects to ammonia oxidation, where amoA gene copies will be quantified by qPCR to examine the
abundance of the bacterial functional group associated with ammonium oxidization, TRFLP analysis and
associated multivariate statistics will be used to characterize the effects of nano-TiO2 and -ZnO on
ammonia oxidizer community structure, and net and gross N mineralization and nitrification analyses
will indicate ecosystem-level consequences. The linkage between nitrifier community and ecosystem
process (N cycle) will allow for testing the hypothesis that effects on ecosystem-level processes will be
observable through the effects of nanoparticles on sensitive or “key” microbial taxa.
The observed effects of SRMs on soil microbial community composition and function, as described
above, may be due to proximate or distal effects of ENMs, i.e. on organisms in direct contact with ENMs
or of ENMs affecting soil properties that, in turn, alter organism function. Because of their high specific
surface area, ENMs, even in relatively low amounts, may alter soil water relations or nutrient
availability, which could impose distal effects on biota. For example, increased water retention—due to
the presence of high specific surface area ENMs in soil-- could increase water content under very dry
conditions, thus promoting comparatively greater nutrient and water availability as soils dry.
Alternatively, comparatively lowered water activity due to higher surface interactions between water
and ENMs could impose water stress to soil organisms. However, little is known regarding the
fundamental effects of ENMs on altered soil water relations. Working from theoretical and empirical
frameworks in soil physics and hydrology, we designed and conducted an experiment to equilibrate
nano-TiO2–amended soils across a range of water potentials, both higher and lower than the native
water potential of the as-excavated (grassland, as above) study soil. The change in water content of the
soils was monitored over time, and equilibration was reached. Following equilibration, the soils were
evaluated for final soil water content, nutrient concentrations, and microbial activity; DNA was
extracted to characterize bacterial community shifts. Data are under analysis and manuscripts are in
preparation, but synthesis thus far indicates very modest effects of ENMs on soil water holding
characteristics, but measureable effects on microbial communities according to soil dryness and ENM
addition. This work will contribute to our understanding of possible “distal” effects that ENMs can have
in soil, i.e. of changing soil water holding characteristics relevant to plant growth and microbial
processes.
The third component of this project involves planted microcosms to evaluate plant-microbe
interactions, plant uptake and damage, soil microbial communities, and interactions between microbes
and plants in effecting ENM influences. This project builds upon the considerable progress made in TER4 in which Jorge Gardea-Torresdey’s research has shown ENM uptake and damage in hydroponicallygrown and soil-cultivated plants, with magnitudes varying with plant species and ENM type. During this
period, we designed and conducted a highly collaborative project whereby soybean plants were grown
in nano-CeO2 and –ZnO amended organic farm soil. Plants were grown to maturity (60 days, for dwarf
plants), and analyses were performed to assess food crop consequences, metal compartmentalization
and transformation, and plant damage, microbial community alterations, and N-fixing symbioses
impacts. Most datasets are now complete (e.g. ICP for total metals in all plant tissues and soils, all plant
growth data, plant ultrastructure, N2 fixation potential, plant genetic damage, leaf ROS and chlorophyll,
plant photosystem II, and plant macro/micronutrients); some measurements are in progress (e.g.
microbial community analysis, and plant oxidative damage indicators). Key findings include that Nfixation is interfered with by CeO2 in a dose-dependent fashion. The result is particularly interesting,
given that N2-fixing symbiotic units (nodules, on soybean root) were similar in number and mass across
all treatments, but their potential for N2 fixation (as measured by the acetylene reduction assay)
appeared significantly affected by CeO2 at high concentrations. The implications of this finding are that
ENMs could affect self-fertilization by soybean, thereby requiring increased synthetic N administration
which, practically, would have profound impacts economically and environmentally. We also observed
copious translocation of Zn into above-ground biomass, suggesting that the food supply is vulnerable.
Combined with the unplanted soil microcosm work described above, the results of this experiment will
provide much needed insight into the effects of ENMs on terrestrial agricultural ecosystems, with an
emphasis on food supply and crops that promote soil fertility.
Project ID Assignment: TER-2: Trophic Transfer, Bioaccumulation and Biomagnification of Engineered
Nanomaterials in Basal Levels of Environmental Food Webs
John Priester, Rebecca Werlin, Randy Mielke, Patricia Holden
A fundamental concern in nanotoxicology is the possible propagation of intact nanoparticles and their
associated ecological effects through food chains. Bioaccumulation of nanoparticles involves the
concentration of nanoparticles from aqueous media into organisms. Trophic transfer of nanoparticles
involves the exchange of bioaccumulated materials from one organism in a food web to another via
predation. When the exchange during trophic transfer results in a concentration increase,
biomagnification has occurred. Bioaccumulation, trophic transfer and biomagnification each enhance
the toxicological threats of nanoparticles. The fundamental conditions of these processes should be
understood, so that nanoparticles can be designed to avoid these outcomes. Previously, we studied the
trophic transfer of CdSe quantum dots from bacteria into their protozoan predators and published
(Werlin et al., 2011, Nat. Nanotech.) that biomagnification occurred, providing the first evidence for this
pinnacle concern in ecological outcomes of nanomaterials in the environment. Besides being an
inaugural report of biomagnification in nanoecotoxicology, the work was important because of the
organisms involved: they are ubiquitous and at the base of all food webs. Further, during the research
it became apparent how little is known regarding trophic transfer and the potential for biomagnification
that is initiated with bacterial prey. We therefore extended the work to other nanoparticle and bacterial
configurations. During this period, we built upon our initial work with two discrete studies: 1) trophic
transfer of bacterially-synthesized Se(0) nanoparticles, and 2) protozoan uptake and bioprocessing of
nano-TiO2 sorbed to its bacterial prey. Together, the three scenarios form a complete trilogy of
nanoparticle presentations into basal food webs: bacterially-formed NPs, bacterially-biosorbed NPs, and
bacterially-internalized NPs. Leveraging our prior discovery that Pseudomonas aeruginosa PG201
intracellularly reduces selenite to elemental selenium and thereby forms uniform intracellular Se(0)
nanoparticles, we fed endpoint bacteria to Tetrahymena. Compared to controls, we observed
protozoan growth inhibition; we also observed Se(0) accumulation in protozoan and apparent
bioprocessing. Final conclusions await full analysis of microscopy data. With nano-TiO2, we observed
that protozoa do not avoid bacteria that are circumscribed with biosorbed NPs. Rather, protozoa
voraciously consume TiO2-decorated bacteria and accumulate nano-TiO2 into food vacuoles. The
accumulation of nano-TiO2 in protozoan food vacuoles is strikingly visible in electron micrographs and
exceeds accumulations from a treatment where TiO2, but not bacterial cells, were presented in rich
media. Bacterial delivery of TiO2 in Tetrahymena appeared to increase toxicity, possibly through
alterations in prey digestion, and thus creating nutrient acquisition differences as compared to the
treatment with dissolved nutrients amended with nano-TiO2. The implication is that biomagnification
can initiate in basal food webs without bacterial intracellularization of NPs. Overall, this project has the
potential to have great impact in the following ways: 1) further understand the conditions under which
biomagnification occurs, 2) quantify biomagnification from microbial prey into predators, 3) elaborate
on the currently-sparse knowledge base regarding biomagnification of contaminants as initiated in
bacteria.
Project ID Assignment: TER-3: Engineered nanoparticle biosorption, toxicity, and toxicity mechanisms
in planktonic and biofilm bacteria
Allison M. Horst, John H. Priester, Raja Vukanti, Patricia A. Holden
The overall goal of this project is to understand engineered nanoparticle (ENP) interactions with
bacterial populations, including growth inhibition, cell damage, and ENP transformations. This research
applies to: a) predicting how ENPs could alter environmental nutrient cycling and biodegradation, b)
informing transport and fate models that predict bioavailability and toxicity to other receptors, c)
informing ENP safe design, and d) informing endpoints and approaches to high throughput screening
(HTS) with environmentally-relevant organisms. The research emphasizes catalysis-relevant growth
rather than killing, using environmentally-relevant chemistries and organisms. Six current subprojects
regard: 1) experimental conditions, 2) sublethal assays, 3) planktonic growth inhibition and responsible
mechanisms, 4) biofilm inhibition and modes, 5) ENP biosorption and consequences, and 6) functionallyrelevant wastewater treatment bacteria.
Building upon our prior experimental condition optimization research, we completed a step-wise ENP
dispersion protocol for use with environmentally-relevant growth media. Our idea is systematic and
novel, showing that knowledge of ENP characteristics (here, point of zero charge or PZC, in select
media), strategic use of an appropriate stabilizing agent, and ultrasonication, can achieve excellent and
stable ENP dispersions for bacterial exposure studies. We also advanced a system of cellular membraneassociated sublethal assays for use in HTS with environmental bacteria, and transferred the approach to
our colleagues in Theme 2 for ongoing use. Our methodological advances, overall, provide a platform
for exposure (i.e. via media and dispersion) and assessment (i.e. via HTS using the assay system) using a
spectrum of bacteria and ENPs.
Our re-analysis of growth curve data continues to support a dose-response relationship between three
metal oxide (TiO2, CeO2, and ZnO) ENPs and bacterial growth rate, with gram-positive bacteria being
more susceptible, and susceptibility enhanced in oligotrophic media. ENP association with the outer
membrane (biosorption), in conjunction with ROS formation and sublethal assay-based evidence of
membrane damage plus loss of membrane potential, support oxidative stress as the basis for toxicity.
This is closely related to the data generated in mammalian systems by Theme 2. Similar conclusions
were reached for Ag nanoparticles exposed to two gram-negative bacterial types, although the greater
sensitivity of Pseudomonas relative to Escherichia remains unexplained. Evidence for the involvement of
band gap and nanoparticle doping, as a physicochemical and modular ENP characteristic, contributing to
oxidative stress in bacteria rests in recent work with Fe-doped vs. undoped TiO2. Here, there is early
evidence for a protective effect of Fe-dopant under dark conditions, but ongoing experiments will
differentiate between band gap and surface area as explanatory variables. We are planning to quantify
the amount of biosorption, to assess if surface loading can be explained as the ‘dose’ causing
membrane-associated effects. Taken together, the “oxidative stress paradigm” (Nel et al., Science,
2006) appears applicable to bacteria, and our selection of screening assays is validated by the results
thus far. This work will supplement and extend the work reported in project HTS-6 in Theme 2.
While dispersant and assays methods for planktonic bacteria are highly applicable to HTS endeavors,
most bacteria are encased in exopolymers and attached to surfaces as biofilms. Our sole work with
biofilms shows that exopolymers have little protective effect on biofilm bacteria, but ongoing work will
test this finding with other ENPs and varied (e.g. dry) experimental conditions.
Lastly, the environmental relevance is highest for the subproject assessing a functionally-important
group of wastewater treatment bacteria: polyhydroxybutyrate (PHB) producers. Here, we leveraged
additional resources to discover negative effects of Ag ENPs to PHB-production, a process that enables
“luxury” phosphorous uptake by select bacterial populations in “activated sludge,” the prevalent aerobic
biomass in wastewater treatment. We draw from our monoculture assay results to infer that oxidative
stress is at work, with the societally-relevant implication that nutrient removal during wastewater
treatment can be diminished by Ag nanoparticles that are increasingly present in our waste streams.
Overall, this research is improving our understanding of relationships between ENP association with
bacterial membranes with effects on membrane-oriented processes, and the resulting effects on
population growth in two environmentally-important bacterial growth modes: suspended cells and as
surface-attached biofilms. Linking ENP adsorption to toxicity mechanisms, then to population effects,
will help in predicting the behavior and impacts of ENPs when released into the environment. Further,
visualization and quantification of the association of ENPs with planktonic cells or biofilms will provide
insight into the potential biosorption of ENPs by bacteria or bacterial EPS. The methods and approaches
developed in the course of this research are also providing transferable roadmaps for routine and largescale testing of ENPs in bacterial systems (e.g. as per Theme 2).
Project ID Assignment: TER-4: Toxicity and uptake of nanoparticles by terrestrial plant species
Jorge Gardea-Torresdey
Studies on the interaction of nanoparticles (NPs) with terrestrial plants are very limited. We began our
studies with germination/root-elongation toxicity tests using ZnO and CeO2 NPs at concentrations
varying from 0-4000 mg L-1. Representative edible terrestrial plants such as Lycopersicon esculentum
(tomato), Cucumis sativus (cucumber), Glycine max (soybean), Medicago sativa (alfalfa), Zea mays (corn)
and the desert species Prosopis sp. (mesquite), Salsola tragus (tumbleweed), and Parkinsonia sp.
(Mexican Palo Verde) were used at this experimental stage. Results showed that at 4000 mg L-1 both
CeO2 and ZnO NPs reduced seed germination of all tested plants. At germination, ZnO NPs reduced root
elongation in all studied plants while CeO2 NPs increased root elongation in cucumber, alfalfa and
soybean. Although no visible signs of toxicity have been observed in the NP treated plants, biochemical
assays have demonstrated that stress enzymes such as catalase and ascorbate peroxidase are altered.
The x-ray absorption spectroscopy (XAS) studies have demonstrated for the first time the presence of
CeO2 NPs but not ZnO NPs within plants. This implies that CeO2 NPs could be stored without
biotransformation within the plants and may be passed on to the next plant generation. Our studies
have also shown that ZnO and CeO2 NPs are potentially genotoxic. Random amplified polymorphic DNA
(RAPD) profiles of soybean root DNA, extracted from plants separately treated with ZnO and CeO 2 NPs,
have shown a new DNA-band in plants treated with ZnO at 4000 mg L-1 and three and four new bands in
plants treated with CeO2 NPs at 4000 and 2000 mg L-1, respectively.
The micro x-ray fluorescence spectra obtained from the roots and leaves of TiO2 NP treated cucumber
plants confirmed the presence of TiO2 in the vascular system of roots and leaves. Light microscope
histological analyses have shown that cells of TiO2 treated plants were three fold larger compared to
cells of control plants, which suggests that cells swelling as a response to the presence of TiO 2.Confocal
microscopy was used to look into the NP uptake mechanisms. Confocal images of corn roots treated
with Fluorescein isothiocyanate (FITC)-stained ZnO and CeO2 NPs have shown that these NPs (single or
aggregates) enter into the root organ via the apoplastic pathway. Confocal images of corn root
longitudinal segments have shown the presence of NPs within the transport system of the plant, which
suggest that the NPs are potentially able to reach the aerial plant parts, including the reproductive
organs. Untransformed NPs associated to edible or reproductive organs can enter into the food chain,
with unpredictable consequences. Also, nothing is known about the impact of plant-biotransformation
products of NPs in consumer organisms.
Our future studies will focus on determining (1) the site and mechanism(s) of biotransformation of ZnO
NPs by mesquite and other plant species, (2) continue studying the mechanisms of CeO2 uptake and
storage by plants, (3) study the fate and transport of TiO2 in Cucurbitaceae, and (4) continue performing
biochemical assays to determine the stress enzyme activity on NP treated plants. Our proposed studies
are expected to provide sufficient data on the biotransformation of ZnO NPs in cultivated and wild
plants, as well as the uptake and storage of CeO2 NPs, and the genotoxicity of ZnO, CeO2 and TiO2 NPs.
Project ID Assignment: TER-5: Dynamic energy budget modeling of toxic effects of CdSe quantum dots
Roger M Nisbet , Tin Klanjscek, John Priester, Patricia Holden
The extraordinary pace of nanotechnology development has thus far exceeded society’s capacity to
predict, and thus mitigate against, unwanted effects on the environment. Similarly, the myriad of
biological receptors in the environment includes a nearly infinite number of organisms, life stages, and
biochemical pathways. Yet interactions of nanomaterials with biological receptors are inherently
anchored in biochemical bases that should be amenable to hypothesis formulation and testing using
mathematical modeling of fundamental interactions between nanomaterials and biological systems.
Quantitative structure activity relationships (QSARs) have great value in relating suborganismal
processes to organismal performance, but QSARs have limited transferability and utility for addressing
concerns about nanomaterials’ ecological effects – especially those involving populations and
ecosystems.
This research responds to the need for transferable models of nanomaterial effects on ecological
processes through the use of dynamic energy budget (DEB) modeling. DEB models use a system of
differential equations to represent energy acquisition and transduction processes in individual
organisms. Energy and elemental matter are the universal currencies of organismal growth,
reproduction, and induction of stress responses. DEB modeling characterizes the flow and
transformations of energy and key elements within organisms, making it possible to build models that
can relate individual function, population growth, and ultimately community composition and function.
We anticipate that DEB models will be powerful tools in predictive nanotoxicology, and aim to develop
and demonstrate their utility in this project. An immediate research product is new theory that uses
DEB models to characterize the response of individual organisms to exposure to nanomaterials and to
relate these responses to population and ecosystem level phenomena that are being studied in
experimental mesocosms.
We formulated DEB models appropriate for modeling bacterial responses to chemical stressors, and are
currently modifying the models as needed for testing against experiments that compared effects of CdSe
quantum dots versus soluble cadmium salts. 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
experimentally-derived data. This is the first DEB model to invoke ROS as a mathematically-represented
damage inducing compound that impacts cell physiology and population dynamics. We also evaluated
the predictive power of the model , demonstrating that it can predict to good accuracy bacterial growth
for treatments of up to 150mg(Cd)/l using only parameters estimated from cadmium treatments of
20mg(Cd)/l and lower. The model provides the foundation for future representations of data from other
studies involving different nanomaterials and organisms, and for predicting responses in environments
for which data are unavailable.
We extended our modeling to the CdSe quantum dot-bacterial interactions where published
experimental data by Holden and collaborators demonstrated that toxic effects of the nanoparticles
transcended those of cadmium ions above a concentration threshold. We collaborated with the Holden
group to design and conduct experiments to obtain the information necessary to calibrate data from
optical density (OD) measurements and to characterize dissolution of QDs. These experiments are
complete and the new data allowed generation of hypotheses to relate data to models and thereby help
identify the mechanisms that distinguish the response to QDs versus dissolved Cd.
We expect our research to not only impact the development of modeling approaches in nanotoxicology
but to also contribute to broader understanding the potential energetic basis for nanoparticle-specific
effects on organisms. Specifically, we are now developing a new, DEB-based representation of ROS
dynamics in cells that allows tracking of ROS generation, transformation, and accumulation of the
associated cellular damage.
This research also maps closely onto our work in projects MWF-3 and MWF-5, where DEB modeling is
being developed for describing and predicting toxicological effects of nanomaterials in marine and
freshwater ecosystems.
Theme 5 - Marine and freshwater ecosystems impact and toxicology (Lenihan)
Project ID Assignment: MFW-1: Marine organismal nanotoxicology: Studying Nanomaterial
Interactions at the Molecular, Cellular, Organ, and Systemic Levels
Gary N. Cherr, Elise Fairbairn, Bryan Cole, Carol Vines
Engineered NMs are likely to exhibit differing toxicological effects on individual organisms, populations,
and ecosystems in different marine habitats. In this project we are utilizing ecologically relevant model
marine organisms that include algae and invertebrates, as well as both embryonic and adult life stages.
The differences in fate and transport of diverse NMs, as well as differences in their interactions with
structurally and physiologically diverse organisms must be accounted for when predicting NM impacts
to marine populations and ecosystems. The individual components within this project include
determining: 1) the physiological effects of NMs on adult marine organisms’ cells and tissues, 2) the
effects of NMs on developing marine embryos, and 3) effects of NMs on both the cellular and
population-level endpoints in marine phytoplanktion (in collaboration with Theme 5, Project 24). We
are utilizing a broad range of organisms that include single cell marine phytoplankton (diatoms and
green algae), developing embryos from marine invertebrates (sea urchin, abalone, mussel), and adult
marine bivalve molluscs in order to address the hypothesized diverse responses we expect across phyla,
as well as in different habitats (water column and benthic environments). The mechanistic effects of NM
exposure being investigated include endpoints and high content screening (HCS) approaches have been
directly adapted from high throughput screening (HTS) results from in vitro studies with mammalian
cells in culture by Theme 2. These include: generation of reactive oxygen species (ROS), mitochondrial
function, membrane integrity, apoptosis, phagocytosis, inflammation, etc. These basic parameters of
cytotoxicity and sublethal responses are being linked directly to other projects within Theme 5 at the
larger-scale organismal/population levels. Our overall goal is to understand differences and similarities
in the responses of very different organisms and life stages from distinct habitats to diverse NMs. These
aims are described below.
Research on this project is being conducted in close collaboration with CEIN researchers in Themes 1, 2,
and 3. Relating to Aim 1 (physiological effects of NMs on cells and tissues), collaborative studies with
Theme 3 on the behavior of metal oxide NMs in seawater have been published (Keller et al., 2010; Env.
Sci. Tech.) and new approaches for stabilizing CNTs in seawater and physiologically relevant media are
being developed by Theme 1 in collaboration with the current project. In addition, the trophic transfer
of NMs through a simplified system has been published in a collaborative study with Theme 4 (Werlin et
al., 2011; Nat. Nano.); our data contribution was on oxidative damage as a result of the consumption of
NM-exposed bacteria. Our current focus is on marine mussel (bivalve mollusc) blood cells or hemocytes,
both in vitro and in vivo, for the assessment of sublethal cellular endpoints of toxicity and function.
Since hemocytes are the immune cells of marine invertebrates, their functional capabilities are critical
for organism and population level health. In vitro exposure to ZnO NMs inhibits hemocyte phagocytosis
of fluorescent yeast particles. In vivo exposure of mussels to ZnO NMs (collaboration with Theme 5,
Project 24) did not reduce hemocyte phagocytosis and may relate to adaptive mechanisms for
sequestration of free Zn++ at the organismal level. For hemocytes, ZnO NMs alter plasma membrane
membrane integrity and induce reactive oxygen species, and these responses are related to the
decrease in phagocytosis. The impacts of selected metal oxide NMs (TiO2, CeO2, and ZnO ) are being
prepared for publication.
Recently we have studied other NMs in order to compare responses of hemocytes to different NMs.
The effects of SWCNTs on hemocyte function as well as toxicity have been investigated. These SWCNTs
were introduced as nanotori due to the solvent dispersion methodology. We are now initiating
exposures of cells to SWCNTs using a dispersion method with natural organic matter. In vitro exposure
of mussel hemocytes to SWCNTs caused increased production of reactive oxygen species (ROS) and in
some cases can impair the hemocytes’ ability to phagocytose foreign particles. These effects occur at
different concentrations, depending on the SWCNTs used:
 HiPCO SWCNTs interfered with phagocytosis at concentrations as low as 0.4 ppm whereas there
was no effect of exposure to SG65 or P2 CNTs even above 10 ppm;
 HiPCO and SG65 SWCNTs caused increased cellular production of ROS at concentrations as low
as 1 and 6.5 ppm, respectively. This effect was apparent after just 3 hours when exposed to
SG65 and at 20 hours exposure time with HiPCO CNTs.;
 The P2 SWCNTs had no effect on mussel hemocytes, although greater concentrations (probably
not environmentally relevant) still need to be tested.
While distinct differences in subcellular and celluar responses of mussel hemocytes, to different SWCNT
exposures was apparent, the basis for these differences is not clear at present. These differences may
be related to SWCNT size and form, purity, or the catalyst used. The results of these initial studies with
SWCNT nanotori need to be repeated with CNTs dispersed with natural organic matter rather than
solvents.
Based on new findings from Theme 2 regarding inflammasome activation in mammalian cells in culture
by CNT exposure, we have adapted the inflammasome assay (Magic Red detection of cathepsin enzyme
activity) to mussel hemocytes using scanning laser confocal microscopy. We have modified the assay for
a cooler, higher osmotic medium using a positive control and will now assess the effects of CNTs on
inflammasome activation in these invertebrate blood cells.
In Aim 2, we are investigating the responses of developing embryos to NMs as these early life stages are
typically more sensitive than the adults, and impacts on development direcly relate to population-level
changes. Since all externally developing embryos exhibit distinct phases of development (early cleavage
stages with little gene transcription and later differentiation/gene activation stages), toxicological
impacts of NMs on development can be very significant to normal embryo development and thus
recruitment of new individuals to the population. To date we have linked the physical behavior of metal
oxide NMs in seawater with embryo toxicity (Fairbairn et al., 2011, J. Haz. Mat.). Several key findings
were that ZnO NMs were toxic to developing sea urchin embryos at the low ppb range and the Fe-doped
ZnO NMs were not significantly less toxic than non-doped particles; this differs from what has been
observed in mammalian cells and underlines the importance of comparative toxicological studies.
Related to this study we have found that ZnO NMs induce apoptosis in developing embryos at specific
stages of development, and that this may be related to observed abnormal development in later stage
embryos. These experiments are being repeated and will be prepared for publication. Another metal
oxide NM, CuO, has also been investigated as recently CuO NMs have been found to specifically inhibit
hatching in zebrafish embryos (Theme 2, Project 8). When zebrafish embryos were exposed to 0.5-200
ppm CuO NM, there is a significant and dramatic decrease in hatching success with no corresponding
increase in either mortality or gross developmental abnormalities. We expanded on these results by
investigating the effects of CuO NM on marine invertebrate embryos, including sea urchins, abalone,
and mussels. The hatching enzyme in sea urchins shares homology with the zebrafish hatching enzyme
so we hypothesized that we would observe a similar inhibition of hatching in sea urchins. While little is
known regarding the hatching enzymes of mussels and abalone, the embryos of all three species are
known to be very sensitive to Cu2+, with EC50s in the low ppb range. Embryos were exposed to CuO NM
from the early cleavage stage until hatching, or from 9 hours post fertilization until hatching, and were
then assessed for mortality, developmental abnormalities, and hatch success. There was no specific
effect of the CuO NM on hatching, even though developmental abnormalities were observed at the low
ppb range. Unlike results with tropical freshwater zebrafish embryos where inhibition of hatching was
independent of developmental abnormalities, we did not observe an effect on hatching success in
temperate marine embryos but did observe developmental abnormalities at very low NM levels.. Once
again, these results highlight the importance of assessing NM effects on different biological systems
under different environmental conditions.
In Aim 3, we are focusing on the toxicological responses of marine phytoplankton to different NMs.
Phytoplankton are important organisms as they form the base of marine food webs and may also be a
route of exposure for higher trophic organisms. In fact phytoplankton are the primary source of food for
larval marine invertebrates such as sea urchins. We have been establishing for the first time high
content assays with phytoplankton for assessing impacts of NM exposure. These endpoints include
oxidative stress, membrane integrity, and mitochondrial function. Results from these fluorescencebased assays with phytoplankton (four species) are being linked to ecosystem-level responses including
phytoplankton growth and photosynthesis (collaboration with Theme 5, Project 24). In two species of
marine phytoplankton, Dunaliella tertiolecta and Isochrysis galbana, we have performed high-content
screening for multiple cytotoxicological endpoints of exposure to metal oxide nanomaterials (ZnO, AgO,
CuO). In most cases, each species responded differently to a particular nanomaterial, though all
materials tested were found to have an effect at some concentration. This work is being combined with
information on the effects of the same nanomaterials on the growth rates and primary production being
conducted by the Theme 5, Project 24, and is being prepared for publication. To date we have found:
 ZnO nanoparticles were found to significantly impact mitochondrial membrane potential in
Dunaliella at concentrations as low as 1 ppm, but did not cause damage to membranes over a
48 hr exposure period. Conversely, there was no effect of ZnO on Isochrysis mitochondria, but
membrane damage was observed at 10 ppm.
 CuO nanoparticles significantly impacted mitochondria and increased production of ROS in both
study species at all concentrations tested (1-10 ppm). Further work will examine the effects at
lower concentrations.
 Ag nanoparticles significantly impacted Dunaliella mitochondria, but were not directly cytotoxic,
nor did they cause production of ROS. In Isochrysis Ag NMs were found to be directly cytotoxic
at concentrations above 5 ppm, and significantly increased ROS production and impacted
mitochondrial function at lower, sublethal doses.
Differences in responses of phytoplankton may relate to physiological differences between species.
These include a formidable silica frustule protecting diatoms but the presence of a typical cell wall in
green algae. The approaches we have developed for phytoplankton (as well as for mussel hemocytes)
will be applied to organisms that are part of larger mesocosm experiments in theme 5, Project 24. We
are also expanding our CNT investigations using marine embryos and phytoplankton, and SWCNTs
dispersed in natural organic matter.
Our results in marine organisms highlight the importance of toxicity assessments from both in vitro and
in vivo models, and from organisms from a variety of ecosystems and developmental life stages. For
example, CuO NMs had very different results with freshwater zebrafish embryos compared to marine
invertebrate embryos. Furthermore, our results with ZnO NM indicated that Fe-doped ZnO NMs were
not less toxic to developing sea urchin embryos compared to pure ZnO NMs, in contrast to the results
observed in cell culture. However, the basic inflammasome response appears conserved between
mammalian and invertebrate systems and we hypothesize that both could be initiated by CNTs. It is
important that stakeholders understand that effects of NMs in one biological system/environmental
medium may not be similar to responses observed in other organisims from different environments.
This concept is essential to understanding/predicting the global toxicological impacts of engineered
NMs.
Project ID Assignment: MFW-2. Impacts of engineered nanomaterials on marine ecosystems
Hunter S. Lenihan, Robert J. Miller
Discharge of engineered nanomaterials (ENMs) into the environment is expected to increase with their
expanding use in manufacturing, and much of this material may enter marine coastal and estuarine
ecosystems. Our research is designed to address questions concerning the impacts of ENMs on key
ecological processes that help to control the abundance and distribution of coastal marine organisms,
especially those that provide important ecosystem services, including nutrient cycling, maintenance of
water quality, and the provision of seafood. As such, we have carefully selected model (“sentinel”)
organisms, including pelagic phytoplankton (primary producers), suspension-feeding mussels and
copepods (gazers), benthic amphipods (deposit feeders), and CA spiny lobsters (predators) that are
involved in representative ecological processes that directly or indirectly generate ecosystem services.
Focus on ecological processes also allows us to generalize about the results of our experiments to other
model systems, where the same process occur, thereby providing a general ecological framework for
understanding the environmental impacts of ENMs. Within this framework, we also examine key
ecotoxicological processes, namely the trophic transfer of ENMs, bioprocessing and biotransformation,
and bioaccumulation and biomagnification. We work with microcosms and two-species mesocosms, the
result from which we are scaling up to multi-species mesocosm experiments designed to examine the
fate and transport, and biological impacts of ENMs on a coastal marine food web.
The most important aspect of our work in terms of developing the CEIN toxicological paradigm is
Dynamic Energy Budget (DEB) modeling conducted in collaboration with Dr. Roger Nisbet and his team
in MFW-5. DEB models allow us to integrate data from our microcosm and mesocosm assays - for
example tests of the impacts of metal oxide ENMs on the respiration, feeding, and growth rates in
marine mussels - in structured mathematical equations that describe four basic energy-transfer
processes impacted by pollutants - food uptake, reserve storage, maintenance, and reproduction. Using
this framework, the results of individual-based assays are used to predict the influence of ENMs on
population-level outcomes – such as the lifetime reproductive output of mussels. In turn, by integrating
population-level impacts with information from HCS, HTS, and Fate and Transport studies, we can
generate predictions about ENM impacts (or benefits) at community and ecosystem levels. As such, DEB
models provide a cornerstone for understanding and predicting the general implications of ENMs in the
environment based on the results of a relatively circumscribed set of experiments and models.
The major habitats of the oceans can be grouped as benthic and pelagic, and in MFW-2 we include
organisms from both. Primary producers, which feed most of the organisms in marine food webs, are
represented by very common coastal phytoplankton comprising three major taxa (Diatoms,
Chlorophytes, Prymnesiophytes). Pelagic copepods and benthic bivalve mussels represent suspension
feeding herbivores (or omnivores) that graze on phytoplankton, thus providing a common form of
trophic transfer - primary producer to primary consumer- that can be used to test for ENM trophic
transfer and its ecological impacts in two-species mesocosm experiments. Deposit feeders, which
consume phytoplankton and benthic algae, mainly in bottom sediments, are represented by amphipod
crustaceans and infaunal sea urchins, which are used extensively in marine ecotoxicity assays in
governmental agency pollution monitoring and assessment. Predators that eat primary consumers, such
as the CA spiny lobster, will be used in our multi-species food web mesocosm study. Examination of
trophic interactions in model ecosystems are made possible through close collaboration with other CEIN
research teams, who benefit by receiving critical information about ENM fate and transport, as well as
the capacity to test whether injuries detected in vivo and in vitro experiments influence higher levels of
biological organization, for example whole organisms, populations, and communities.
The overarching goals of MFW-2 are to (1) use the results of HTS and high-content screening (HCS)
assays for cytotoxicological impacts as predictors for the impacts in our whole organism and populationlevel assays; (2) focus on ENMs as they influence the distribution and abundance of model organisms
through effects on individual demographic performance and when possible population dynamics; and
(3) measure levels and impacts of ENM uptake, bioprocessing, biotransformation, trophic transfer,
bioaccumulation, and biomagnification involving our sentinel organisms.
We have completed and published results of population-level tests on marine phytoplankton for five
CEIN core metal oxide ENMs, zinc oxide, titania, ceria, Ag, and CuO. These results, based on population
growth rates, indicate that a prevalent metal oxide ENM, zinc oxide (ZnO), can be toxic to the most
common coastal marine phytoplankton species at relatively very low concentrations (NEC = 0.2-1.0
ppm); that CuO and Ag are almost as toxic as ZnO (>10 ppm); that TiO2 ENMs were relatively much less
toxic (>500 ppm); and that ceria is non-toxic. However, we also found that when phytoplankton were
exposed to TiO2 under relatively high levels of UVR, levels equivalent to those near the sea surface,
nano-titania was relatively very toxic to phytoplankton (<3 ppm). We measured increased ROS
generation using the same experimental design, uncovering for the first time a real-life demonstration
of the cytotoxic potential of TiO2 through photoactivation. This result underscores the importance of
developing marine-specific HCS models. As such, we initiated a collaborative SEED grant program with
Dr. Gary Cherr to develop HCS multiple endpoint cytotoxicity assays for phytoplankton; these we are
running in tandem with our phytoplankton population growth experiments. The HCS cytoxicological
assays, in addition to a novel Pulse Amplitude Modulated (PAM) fluorometry assay that we developed to
measure impacts of ENMs on photosynthesis, are designed to identify the specific mechanisms of
toxicity that cause a change in phytoplankton population growth rates, and thus primary production. So
far we have used this integrated approach to examine the effects of nano- ZnO, CeO2, CuO, and Ag.
Results of HCS and PAM fluorometry assays indicate ZnO, CuO, and Ag cause significant subcellular
injury to mitochondria, photosynthectic mechanisms, and our through the generate of ROS, at
concentrations 1-2 magnitudes less than those that reduced population growth rates. We found that the
population abundance of phytoplankton exposed to metal oxides ENMs, specifically nano-ZnO, also is an
important factor controlling toxicity, as high numbers (i.e., concentrations) of phytoplankton reduce
toxicity by taking up, processing, and essentially sequestering Zn ions. Our work with phytoplankton and
PAM fluorometry has recently been expanded to include experiments to test the impacts of nano-ZnO,
CeO2, NiO, Fe3O4, MnO, and CoO on benthic bacterioplankton.
Phytoplankton form the base of marine food webs, and are a particularly important food resource for
marine suspension feeders. We have developed the marine mussel Mytilus galloprovincialis as a sentinel
suspension feeding organism to test the influence of ENMs on a because this marine organism provides
a suite of ecosystem services, including habitat provision, nutrient cycling, and seafood. Mussels feed
on suspended cells by filtering large quantities of water and thus are vulnerable to exposure to
suspended ENMs. We have conducted experiments to measure uptake, bioaccumulation, and the
influence of the ENMs on multiple physiological endpoints and individual demographic performance in
mussels in laboratory microcosms. However, the mode by which we expose mussels to ENMs is through
contaminated phytoplankton, so in essence these experiments are two-species mesocosms. So far we
have concentrated on hypothesis concerning ZnO, but have also initiated studies with SW CNTS, as well
as CuO and Ag. Mussels were exposed to concentrations of ZnO NPs up to 2 mg L-1 (2 ppm) for 12 weeks,
sampled through time, and were found to take up ZnO from grazing on phytoplankton. ZnO decreased
growth rates of large and small mussels at concentrations as low as 0.5 mg L -1. Survival of large mussels
decreased only in the highest exposure treatment, but small mussels were more vulnerable, with
survival declining at the lowest concentration tested, 0.1 mg L-1. Small mussels also took up zinc much
more rapidly than large mussels. Relatively low concentrations of ZnO also substantially increased
respiration rates and reduced feeding rates in mussels. This work shows that ZnO may severely impact
mussel populations by exerting a disproportionately negative effect on small individuals. Mussels may
have a refuge in size from mortality due to zinc exposure. This work also implies that using large
mussels as indicators of metal pollution, as is presently done through the Mussel Watch program, may
underestimate exposure.
Researchers in MFW-5, specifically Drs. Nisbet and Muller, constructed Dynamic Energy Budget (DEB)
models to quantify impacts of ZnO ENMs on the energy requirements of mussels. We are nearing
completion of the DEB model for ZnO, and so far the largest predicted impact of nano-Zno appears to a
50% decrease in lifetime reproductive output at nano-ZnO concentrations between 0.2-0.3 mg L-1. These
models will allow us to make predictions about the impacts of different ZnO (and eventually other metal
oxide) concentrations under different environmental conditions including temperature, food availability,
and the presence of other toxicants. We have initiated test of the influence of SW CNTS on mussel
respiration, feeding, and tissues, and will once again use DEB models to integrate and generalize our
results. Preliminary results indicate that CNT aggregates are rejected prior to ingestion by the mussel
and accumulate in mussel pseudofeces, where they may become available for uptake by other marine
organisms, especially benthic detritivores (e.g., amphipodsadn urchins). We also found that raw CNTs
significantly reduce the suspension-feeding rate of mussels.
Fate and transport studies have indicated that ENMs are prone to aggregation in many natural media,
particularly seawater due to its high ionic strength. Aggregation ultimately leads to deposition, and the
seafloor will thus be a major sink for discharged ENMs. Estuaries, where rapidly changing water
chemistry favors deposition of natural colloids, will likely also favor deposition of ENMs. For this reason
we have used a sediment-dwelling estuarine amphipod Leptocheirus plumulosus as a model organism to
represent benthic infauna, and so far have completed experiments examining the toxicity of ZnO and
SWCNTs. Survival of amphipods decreased with increasing nano-ZnO concentration in water, but a
similar relationship was not found in sediment. The LC50 of ZnO ENMs in water was 1.4 mg L-1, which is
comparable with that of ionic Zn found in a previous study. Amphipods exposed to moderately high
concentrations of ZnO (100 mg kg-1) in sediment showed no decrease in survival relative to controls,
while very high concentrations (1000 and 2000 mg kg-1) caused 78% and 100% mortality respectively,
indicating that zinc bioavailability is significantly decreased when sequestered in sediment. Amphipods
exposed to moderately high concentrations of ZnO ENMs in sediment, did, however, accumulate
sublethal amounts of Zn, suggesting possible exposure pathways to higher taxa. Our results show that
ZnO ENM are toxic to L. plumulosus at concentrations similar to that of ionic Zn, and that sediment can
mitigate negative biological effects considerably. Partitioning of Zn between sediment and pore water
depends on geochemical composition of the sediment, as well as environmental conditions (e.g., pH, Eh,
O2), and we are working with the Keller lab to determine key factors influencing bioavailability of ZnO in
the sedimentary environment. Results from assays with SW CNTS indicated that even extremely high
concentrations of this material have no deleterious effect on amphipod demographic performance. Next
we will use our amphipod bioassays to test the biological effects of Ag and CuO ENMs in the marine
sedimentary environment.
The trophic transfer of ENMs has been shown by Theme 4 to be an especially important mechanism of
ENM fate and transport as well as injury. We conducted laboratory meoscosm experiments with marine
phytoplankton and copepods (phytoplankton grazers) to test the hypothesis that the demographic
performance and population dynamics of this important marine grazer is affected by phytoplankton
contaminated with ZnO, Ag, and photoactivated TiO2 metal oxide nanomaterials through trophic
transfer. Nano-ZnO and titania were taken up from contaminated phytoplankton and reduced copepod
growth and survival. Results from Ag are not yet available. We also tested the hypothesis that Ag ENMs
influence zooplankton grazing rates on phyto- and bacterioplankton in estuarine mesocosm
experiments. Mortality due to grazing and Ag differentially affected community components: grazing
significantly reduced bacterioplankton growth, while Ag significantly reduced phytoplankton growth and
photosynthetic activity. Thus, Ag ENMs pushed the community towards microplankton (i.e.,
bacterioplankton) dominance.
Work with individual sentinel organisms must be brought together to the examine behavior (fate and
transport) and effects (injury) of ENMs in the environment under realistic conditions. Our multi-species
coastal ecosystem mesocosm studies will explore the fate and transport, uptake, bioprocessing, and
trophic transfer of ENMs (e.g., CuO and CNTs) in a simplified coastal marine food web consisting of the
major model organisms described above: phytoplankton, mussels inhabiting rocky reef habitat, and
deposit-feeding amphipods inhabiting soft sediments. An additional trophic level (top predator) will also
be added in the form of spiny lobsters, Panulirus interruptus, which consume mussels and are an
important fishery species. Choice of ENMs for large mesocosm experiments is important, and multiple
factors must be considered, most importantly scientific relevance, but also including availability, cost,
and detectability. Collaborative work with Arturo Keller’s group has determined that ZnO dissolves
relatively quickly in seawater, and therefore may not be the best general representative for
nanoparticles. TiO2 does not dissolve, but is difficult to digest for ICP-MS, and its toxicity appears to
depend on UVR exposure. CuO dissolves slowly, and CNTs accumulate in sediments and the tissues of
grazers, so are promising candidates for these studies. Focus on the mesocosms as our primary research
effort begins in Winter 2012.
Project ID Assignment: MFW-3: Decoupling and Recoupling Plant-Herbivore Systems to Determine the
Fate and Impact of Nanomaterials in Freshwater Environments
Ed McCauley, Roger Nisbet, Louise Stevenson, Helen Dickson
Ecological systems are dynamically coupled populations, composed of individuals of different species
acquiring resources, expending energy, and cycling materials. Novel chemical nanomaterials (NM)
introduced to environments could significantly alter any or all of these fundamental processes (Baun et
al., 2008 Ecotoxicology; Hartmann et al., 2010 Toxicology; Petersen et al., 2009 Environ. Sci. Technol.)
leading to changes in major features of population dynamics, including persistence (van der Ploeg et al.,
2011 Environ. Pollut.). A major challenge is to predict the effects of these novel nanomaterials in
environments of interacting populations, or more broadly how the dynamics of coupled populations
change as we move from one “environment” to another. There is evidence of trophic transfer (Bouldin
et al., 2008 Environ. Toxicol. Chem.; Ferry et al., 2009 Nat. Nanotechn.; Holbrook et al., 2008 Nat.
Nanotechnol.; Zhu et al., 2010 Chemosphere) and even bioaccumulation of nanomaterials in bacterial
(Werlin et al., 2011 Nat. Nanotechnol.) and terrestrial systems (Judy et al., 2011 Enviro. Sci.Technol.).
The challenge is made harder because the fate and transport of these NM’s are largely unknown in
many environments, and chemical and biological characteristics of natural systems can alter the
behavior and thus toxicity of NMs, rendering classical exposure concepts potentially ineffective (Baun et
al., 2008 Ecotoxicology; Christian et al., 2008 Ecotoxicology; Klaine et al., 2008 Environ. Sci. Technol.).
There are (at least) two missing components to address this challenge: 1) an experimental system that
we can use to investigate the dynamical implications of exposure to NM and to understand how changes
in major system properties emerge in the presence of NM as they move through the environment, and
2) a quantitative framework that can be used both to identify causal mechanisms producing these
responses and to generalize these results to different ecological systems and environments.
We have developed these components for freshwater plankton systems, where we have a tight linkage
between models at key levels of biological organization and real dynamical experimental systems
(McCauley et al. 2008 Nature, Nelson et al. 2006 Nature, McCauley et al. 1999 Nature). Our models
successfully predict the presence/absence of complex dynamical behavior as we change major
environmental variables, such as temperature or the availability of major nutrient resources (McCauley
et al. in prep, LaMontagne et al. in prep). Mechanisms controlling the equilibria and stability of these
systems have been independently tested, using novel experiments that measure requisite individual
responses in dynamically interacting populations. Our theories accurately capture these dynamics in
systems without NMs ranging from milliliters to kiloliters, thereby enabling us to study impacts of NMs
over a wide range of spatial scales.
With this tight coupling between models and experimental systems, we hope to provide insight into the
vexing problem of assessing both direct and indirect effects of NMs. The direct effects on simple
individual and population parameters are hard to assess, given problems in establishing exposure levels
and evaluating toxicity or mechanisms of action (Klaine et al., 2008 Environ. Sci. Technol.). However,
concurrently evaluating indirect effects manifested through subsequent feedback of processes that
could compensate or magnify direct effects seems like fantasy. We think we can accomplish this difficult
assessment through the use of novel dynamic experiments, where we will systematically “decouple”
then “recouple” resource-consumer interactions in the absence and presence of NMs in parallel
replicate environments. At the same time, performance of individuals will be assessed to assist in the
interpretation of the causal mechanisms being altered by exposure to NM’s (c.f .Nisbet et al. 2010
Phil.Trans.Royal Soc., Nisbet et al. 2004 Ecology) and to explore HTS approaches. Finally, the
information on the performance of individuals will be used to derive model predictions for the effects of
NM on the dynamics of our predator-prey system, and these predictions will be evaluated against the
results from the decoupled-coupled semi-chemostat experiments.
Our initial experiments use batch culture conditions followed by a continuous flow system, but the
approach will be expanded in years 2 and 3 to include larger-scale systems with richer microbial
processes. We have completed testing the effect citrate-coated silver nanoparticles on a bacterial-algal
system in batch culture and in semi-chemostat conditions. There are tremendous opportunities for
collaboration with ongoing research in Fate, transport and life cycle analysis and Terrestrial ecosystems
impact and hazard assessment that we have begun to take advantage of and look forward to continued
collaboration in the future. McCauley has built and equipped an aquatic lab at UCSB designed to
complete experiments ranging from microcosms through to mesocosms. He has invested in state-of-the
art equipment for the automated analysis of freshwater systems that will provide the rich data-stream
needed to monitor dynamics at all levels of biological organization. Other labs in the UC CEIN have also
utilized this equipment for analysis of other nanoparticle experiments. The comparison of results from
comparable freshwater and marine microcosms and mesocosms in our research group will provide
insight into how major environmental factors modify fate and impact in these resource-consumer
systems. Other experiments focused on the effects of silver nanoparticles on marine algae are currently
being conducted in the Lenihan lab in the CEIN. To our knowledge, our experiments will serve as the first
data on the effects of citrate-coated silver nanoparticles on algae or Daphnia, and one of few
experiments investigating the effects of nanoparticles on a dynamic system. Further, the setup and
design of the sequence of experiments described in the previous paragraph is novel in the field.
Project ID Assignment: MFW-4: The ecological impacts of TiO2 nanoparticles on freshwater food-webs
Bradley Cardinale, Konrad Kulacki, Raven Bier
In order to make broad predictions about the potential effects of these materials across environments
and taxa, toxicity testing must incorporate not only a variety of organisms and endpoints, but also an
understanding of the mechanisms that underlie nanoparticle toxicity. We began our investigations by
performing a laboratory experiment in which we examined how titanium dioxide nanoparticles
impacted production of biomass across a broad range of freshwater algae. We exposed 18 of the most
common species of North American freshwater pelagic algae (phytoplankton) to five increasing
concentrations of n-TiO2 (ranging from controls to 300 mg n-TiO2 L-1). We then examined effects of nTiO2 on the growth rates and biomass production of each algal species over a period of 25 days.
Increasing concentrations of n-TiO2 had minimal effects on algal growth rates; however the same
concentrations had significant positive effects on the maximum biomass achieved in cultures, causing a
2-4% increase in peak chlorophyll-a per each 10 mg L-1 increase in n-TiO2. This result was very consistent
across taxa, being evident in 16 of the 18 species. At the end of our experiment, we added the primary
consumer, Daphnia magna, to each algal culture and allowed them to feed on the algae-nanoparticle
solution. We found significant accumulation of Ti in D. magna across all algal taxa, with accumulation
being proportional to initial n-TiO2 concentration. Our results suggest that titanium dioxide
nanoparticles may increase the biomass of many types of freshwater phytoplankton, and that they have
potential to enter aquatic food-webs through primary consumers.
These results have given rise to several follow-up studies in our laboratory. First, with the help of high
school summer scholar Courtney Kwan, we performed an experiment to determine how n-TiO2 affects
the growth and metabolism of three species of algae used in the previous experiment. We found that
the effects of n-TiO2 on rates of algal respiration (R) and gross primary production (GPP) varied by
species, but that overall GPP was impacted either less positively or more negatively than R in the
presence of n-TiO2, which led to decreased growth rates in each species when exposed to n-TiO2. A
second experiment, led by postdoctoral researcher Konrad Kulacki, further addressed the mechanisms
underlying the effects of n-TiO2 on algae. This experiment investigated the potential for n-TiO2 to 1)
affect the competitive interactions between algae and bacteria; 2) interact with ultraviolet light to break
down natural organic matter and release nutrients (in collaboration with Sam Bennett from IRG4); 3)
alter algal physiology in response to shading; and 4) create additional habitat by way of settled
nanoparticle aggregates.
Finally, our lab has completed a large scale freshwater mesocosm experiment to investigate the effects
of n-TiO2 on diverse stream communities, as well as the effects of community diversity on the fate of nTiO2. We grew three common algae as monocultures, and together as polycultures, in biofilms of
stream mesocosms exposed to 0, 0.1 or 1.0 mg n-TiO2 L-1. n-TiO2 did not alter the growth trajectory of
any biofilm over 10+ generations. However, Ti accrual in biofilms not only differed among species, but
increased as a function of species diversity, which regulated benthic biomass. This work was done in
collaboration with members of the Holden and Nisbet labs (IRG 2).
Together, our results have important implications for understanding the fate and effects of n-TiO2 in
surface waters, and the mechanisms that underlie their behavior. Work on this project was completed
in Summer 2011, with manuscript preparation continuing into early 2012. No additional work is
planned.
Project ID Assignment: MFW-5. Dynamic energy budget (DEB) modeling to support design of aquatic
microcosm and mesocosm experiments
Roger Nisbet, Edward McCauley, Shannon Hanna, Tin Klanjscek, Hunter Lenihan, Erik Muller, Louise
Stevenson
Engineered nanomaterials entering the natural environment may have ecological effects by influencing
the fluxes of energy and material that regulate the abundance, distribution, and dynamics of organisms.
It is evident from previous experience in ecotoxicology that empirical data alone cannot provide the
understanding to guide policy or management action in response to this emerging technology. A small
proportion of chemical compounds currently in use have been subjected to rigorous toxicity tests, and
even then it is not possible to predict ecological consequences from such tests. With ENMs, there are
further dimensions to consider (size, shape, coating, impurities and more), which carry with them
unknown effects. Thus theory is essential. This abstract describes how theory is used to support other
projects involving aquatic microcosms and mesocosms. Related research, including much fundamental
research on dynamic energy budget models is described under project TER-5.
Dynamic Energy Budget (DEB) theory offers an interactive, conceptual framework for describing the
interplay of physico-chemical processes with the physiological dynamics of living organisms. This has
long been recognized in ecotoxicology; indeed some of the earliest work on DEB theory was motivated
by the recognition of the limited ecological insight that follows from traditional methods for the analysis
of toxicity data. Conclusions drawn from such analyses typically depend on the choice of endpoint,
exposure duration and/or laboratory conditions, such as feeding regime. These limitations are overcome
with DEB theory, as the results of a DEB based analysis are independent of endpoint and exposure
duration. Furthermore, DEB theory offers the advantage of generalization, with the simplest DEB model
providing a characterization of growth, development and reproduction over a complete life cycle of an
animal of arbitrary size through a “standard” model with 12 parameters. Variants of this “standard”
model describe the physiological performance of plants and bacteria. The model also predicts both
inter- and intra-specific variation of the many fluxes in any environment, notably respiration.
DEB theory offers the potential to model mechanistically the effects of ENMs on single organisms and
populations and to relate effects at these different levels of biological organization. Equally important,
DEB models allow representation of the combined impacts of ENMs with other environmental stressors.
They thus represent a practical approach to generate predictions about impacts on populations (growth
rates and dynamics), communities (biodiversity, trophic transfer, bioaccumulation, and
biomagnification), and ecosystems (fluxes of energy and elemental matter). Early in the planning phases
for UC CEIN, it was decided that DEB theory would be used to help integrate information from many of
the interdisciplinary studies. The use of DEB theory to analyze and interpret CEIN data is only possible
because DEB theory has influenced the design of a number of ongoing and planned studies.
CEIN investigators on aquatic systems use DEB theory and models in two ways:
 Use of limited data (e.g. from toxicity tests) to characterize effects of ENMs on DEB model
parameters. This yields metrics that characterize effects in a manner that is independent of
experimental conditions, and that allows inter-species and cross-environment comparisons. The
approach is similar to one known as DEBTOX, developed in the Amsterdam laboratory of
Professor S.A.L.M. Kooijman and described in an OECD (2006) guidance document. In CEIN, this
approach was applied to interpret data on the effects of metal oxide ENMs on marine
phytoplankton (project MFW-2; Miller et al. Environmental Science and Technology 2010), is
currently in use for further studies on freshwater phytoplankton (project #25), and will be
applied to emerging data on marine and freshwater invertebrates (projects #24 and #25).
 Individual  Population projection. Here, DEB theory allows the use of large body of
physiological data from individual organisms to predict/project effects on population growth
rates and on population dynamics. This approach allows projection of population level
consequences from short-term experiments (few months duration) on the response of marine
mussels to ZnO ENMs (project MFW-2, work in progress). It is also being used in the design and
analysis of experiments on interacting phytoplankton and zooplankton populations (project
#25). To our knowledge, these experiments will be the first studies of the effects of ENMs on
population interactions that run for multiple generations of both interacting species.
Interpretation of these data will require a mix of DEB and other modeling approaches as
previously demonstrated in similar experiments without nanomaterials (McCauley, Nelson,
Nisbet, Nature: 2008).
Theme 6 - Environmental Decision Analysis for nanoparticles (Cohen)
Project ID Assignment: EDA-1: Machine Learning Analysis and Modeling of High Throughput Screening
Data for Nanoparticles
Yoram Cohen, Donatello Telesca, Robert Rallo, Kenneth Bradley, Andre Nel, Rong Liu, Cecile Low-Kam,
Taimur Hassan
Analyses of high throughput screening (HTS) data of engineered nanomaterials (eNMs) toxicity and
development of predictive models relating the properties of eNMs to observed biological endpoints, for
both single cell lines and microorganisms across multiple toxicity assays, require clear and unambiguous
identification, quantification and classification of biological responses. Accordingly, the overall goals of
the project is to develop a robust statistical analysis and machine learning framework and tools for the
analysis of HTS data of for eNMs with the following specific aims: (a) Development of advanced methods
for hit identification and cluster analysis; (b) Identification of relationships among cell signaling pathway
activities induced by exposure eNMs considering differing assays and multiple cell lines; (c) Inference
and assessment of data reliability in an automated fashion; (d) Design of quantitative prediction scheme
aimed at assessing how the modularity of information from in-vitro HTS data correlates with in-vivo
response; and (e) Development of automated (high throughput) image recognition for phenotyping of
Zebrafish-based in-vivo toxicity HTS data.
Knowledge extraction from high throughput screening (HTS) of nanoparticle toxicity focused on
identification of toxicity outcomes (i.e., “hit identification”), pathway linkages and correlation of cell
signaling pathways and cytotoxicity. CEIN HTS data for metal and metal-oxide NPs for RAW and BEAS-2B
mammalian cell lines served as the basis for developing the various HTS knowledge extraction
approaches. Hit identification with strict control of false negatives or positives (following outlier removal
and inter/inter-plate normalization) and a series of corresponding analyses methods were implemented
and utilized for both the development of structure-activity relationships for nanomaterials (i.e., nanoSARs) and cluster analysis of HTS data. In earlier work we introduced, for the first time, the use of selforganizing map (SOM) analysis for exploring nanoparticle HTS toxicity data. The approach was applied to
metal and metal-oxide HTS assays focusing on the induction of toxicity-associated cell signaling
pathways using a set 122 of RAW 264.7 luciferase reporter cell lines. SOM cluster analysis revealed two
major clusters corresponding to (i) sub-lethal pro-inflammatory responses to Al2O3, Au, Ag, SiO2
nanoparticles possibly related to ROS generation, and (ii) lethal genotoxic responses due to exposure to
ZnO and Pt nanoparticles (at a concentration range of 25 µg/mL-100 µg/mL at 12 h exposure). We note
that oxidative stress, inflammation, and perturbation of cellular signal transduction pathways were
three of the key mechanistic injury paradigms being pursued by the CEIN (see Theme 2).
Knowledge extraction via the application of complex network theory methods was also employed to
identify relationships between cell responses (pathway activation and cytotoxicity measures) as well as
physicochemical properties of nanoparticles. For example, the analysis based on the above cell lines
revealed a hierarchical activity-activity pattern where sub-lethal effects such as ROS generation and the
intracellular Ca2+ flux are strongly related to lethal effects (e.g. cell membrane damage and cell death)
and that eNM primary size, aggregation and its dissolution tendency were critical parameters affecting
the observed toxicity behavior. More recently, the above approach was extended to explore the
relationships among fourteen toxicity-related signaling pathway activities for the murine macrophage
(RAW) and transformed bronchial epithelial (BEAS-2B) cell lines exposed to six different metal/metal
oxide nanoparticles (dose of 0.39-200 µg/mL). Association rule mining for a large HTS data set (55,296
plate data readings) revealed 6 non-redundant association rules involving E2F, p53, Myc, SMAD, Mito
pathways identified for Raw cell line and 12 rules among SMAD, SRF, CRE, PI, HIF1A pathways for Beas2B cell line. The resulting non-redundant association rules revealed pathways that are co-regulated by
the nanoparticles, depicting that triggering of one or more pathways implies that some other pathways
may also be triggered. The above findings are compatible with the biological cross-talk between cellular
signal transduction and transcriptional regulation pathways. The present work suggests that identified
association rules for strongly associated activity pathways can assist in confirming multi-assay
consistency. In addition, such rules can also guide endpoint selections for Nano-Structure Activity
Relationships (Nano-SAR) development. For example, based on the above analysis the identified Beas-2B
association Rule {PI ==> SRF, HIF1A, CRE} implies that earlier CEIN work on nano-SAR development, in
which Propidium Iodide (PI) uptake for the above cell line was the model endpoint, would also be
predictive for the HIF1A and CRE pathways activities. As another example, the identified rules provide
insight into mechanisms of toxic injury by Pt and ZnO in macrophages. Specifically, E2F and p53 respond
to oxidative stress (PMID 17638884) (PNAS August 20, 1996 vol. 93 no. 17 9166-9171), and both
contribute to cell cycle and apoptotic cell death programs, the latter proceeding through the
mitochondrium. In a preliminary experimental validation of this causation, Core D tested a chemical
inhibitor of p53, Pifithrin α (PFTα), for inhibition of ZnO-mediated mitochondrial dysfunction (MitoSox).
PFTα showed dose-dependent repression of MitoSox signal, supporting a role for p53-dependent gene
expression altering mitochondrial function. This finding further indicates that the superoxide measured
by MitoSox is of mitochondrial, rather than nanoparticle origin.
In addition to knowledge extraction from HTS assays for specific cell lines, generation of high throughput
data for in-vivo toxicity screening of eNMs using zebrafish embryo requires automatic phenotype
recognition system. Accordingly, a phenotype recognition model was developed using support vector
machines (SVM) with a set of suitable image descriptors. Model development was based on images of
zebrafish embryos exposed to metal and metal oxide eNMs that were classified as “dead”, “hatched” or
“unhatched”. The optimal subset of three vectorial image descriptors was identified from a pool of
standard MPEG-7 visual descriptors together with constructed texture and color descriptors. The best
performing model achieved an average predictive accuracy as high as 97.40 (±0.95)%. The above
approach has been incorporated into a software package for automated image parsing as part of the
automated pipe line of zebrafish-based in-vivo HTS model. The advantage of this approach is that it
allows retroactive analysis of large number of images obtained during high throughput zebra fish
embryo screening, which enables hazard ranking of large batches of eNMs as depicted in Project HTS-2
(Theme 2).
Finally, the HTS analysis methods developed at the CEIN were implemented as a unique web-based HTS
data analysis tool (HDAT) with a friendly graphical user interface (GUI). HDAT was initially developed
primarily for hit identification and mapping with later addition of a self-organizing map (SOM) analysis
module for rapid identification of nanoparticles that induce similar cell responses. The hit-identification
feature enables the user to detect significant cell responses/activities induced by nanoparticle with the
capability to control false-negative level and to use user-defined cut-off values. The SOM also provides
neat and clutter-free similarity visualization and can facilitates identification of common mechanisms of
action for specific types of nanoparticles. The HDAT tool now includes the following sub-modules: data
normalization (such as Signal to Background, Signal to Noise Ratio, Percentage of Inhibition, Z-score,
Robust Z-score, B-score, Median-Polish), Summarization (Strictly Standardized Mean Difference (SSMD),
Z-factor etc.), and heatmap as well as SOM visualizations. There is widespread interest in these tools by
the US Nanoinformatics community and the USEPA has also sent representative to arrange for webbased accessibility to these CEIN data analysis tools.
Project ID Assignment: EDA-2:
Quantitative-Structure-Activity Relationships (QSARs) of
Nanomaterials Toxicity and Physicochemical Properties
Yoram Cohen, Donatello Telesca, Robert Rallo, Kenneth Bradley, Andre Nel, Rong Liu, Cecile Low-Kam,
Taimur Hassan
In-silico methods for correlating toxicity end points associated with exposure of cell lines and whole
organisms to nanoparticles are essential for environmental hazard assessment. Accordingly, the primary
goal of the project is to develop quantitative-structure-activity relationships (QSARs) for nanoparticles
toxicity (i.e., Nano-SARs) based on HTS data initially focusing on metal and metal-oxide nanoparticles.
Toxic outcomes can be quantified based on numerical or visual data (e.g., images depicting a response)
and approaches developed in Project EDA-1 (“Machine Learning Analysis and Modeling of High
Throughput Screening Data for Nanoparticles”) form the core of the mathematical and computer
algorithms utilized for Nano-SAR development. The development of predictive Nano-SARs require
identification and quantification of the relevant features (i.e., parameters) that correlate the identified
toxicity measures with nanoparticles properties and environmental conditions (e.g., concentration) via
appropriate feature selection methods. Based on our previously developed unsupervised feature
selection method (KLS-FS), which can handle both linear and non-linear feature selections, a
classification based cytotoxicity nano-SAR was developed based on a set of nine metal oxide
nanoparticles to which transformed bronchial epithelial cells (BEAS-2B) were exposed over a range of
concentrations of 0.375-200 μg/ml and exposure times up to 24 h. The nano-SAR was developed using
cytotoxicity data for BEAS-2B cells with the best performing model, utilizing only four descriptors
(atomization energy of the metal oxide, period of the nanoparticle metal, nanoparticle primary size, in
addition to nanoparticle volume fraction in solution), having a 100% classification accuracy in both
internal and external validation. This classification Nano-SAR enables one to identify decision boundaries
which are crucial for use in hazard ranking of nanoparticles. The above Nano-SAR as well as the KLS-FS
feature selection algorithm were implemented within the Weka data mining/machine learning package
and are available for use by others. More recently, the previously developed KLS-FS feature selection
method was also utilized to develop a Nano-SAR based on a literature HTS data set of 109 nanoparticles
(for nanoparticles uptake by PaCa2 cells) consisting of the same core (iron oxides/NH2 core based) but
with different organic chemical surface modifications. Application of the unsupervised KLS-FS feature
selection and a subsequent supervised genetic feature selection algorithm for an initial pool of 150 2D
molecular descriptors resulted in the selection of only ten features. The resulting Nano-SAR performed
with a Mean Absolute error of ~6%. The above KLS-FS method is now also being employed for arriving at
a Nano-SAR based on a new CEIN HTS data set being developed for 24 metal oxide nanoparticles (by the
CEIN “Molecular, cellular and organism high-throughput screening for hazard assessment” group
(Theme 2). In this collaborative effort nano-(Q)SAR development for the above nanoparticles (based on
three toxicity assays, the BEAS-2B and RAW cell lines, and nanoparticles concentration 0.39 - 200 ppm),
the slope of the dose response curve for the nanoparticles (i.e., the rate of response increase with
concentration) at the EC50 was introduced as a metric for labeling “safe” nanoparticles. Based on
whether the slope is significantly larger than 0, NPs were divided into a toxic group (7 NPs: ZnO, CuO,
Mn2O3, CoO, Ni2O3, Co3O4, Cr2O3) and a Nontoxic group (17 NPs). Subsequently, the modeling task was
formulated as a classification problem given that for the following data set: (a) There is greater
confidence/reliability in predicting whether a NP is toxic or not than to predict the toxicity level; and (b)
There are only marginal differences in the responses induced by “nontoxic” NPs (17 NPs). Twelve
nanoparticle descriptors were evaluated and these were classified as: (a) Basic nanoparticles
Descriptors: Average NP size in water (dw, nm), Atomization Energy (Ea, eV), Conduction Band Energy
(Ec, eV), Valence Band Energy (Ev, eV); and (b) Derived Descriptors: Chemical Hardness (η), Chemical
Potential (μ), Electrophilicity (ω)2, four formation enthalpies ΔHs in Born-Haber Cycle, and ΔHIE1.
Accordingly, an initial nano-SAR classification model (toxic versus non-toxic) was evaluated using the
above collection of NP descriptors was developed, along with applicability domain analysis,
demonstrating ~92% predictive accuracy (measured via a five-fold cross-validation). The above
development was based on an extensive effort that included the use of self-organizing map analysis for
selecting the training and validation data sets. Work is continuing in collaboration with Theme 2 to
further improve the nano-SAR accuracy. In the developed nan-SAR, the potential utility of the bandgap
energy structure as a criterion for predicting the toxicity of metal oxides in cellular HTS assays in
addition to other pertinent descriptors. Additional correlation of those predictions to the intact animal
level is further described in Projects ENM-1 (Theme 1) and HTS-6 (Theme 2). Finally, it is noted that
Nano-SARs developed in this project will be incorporated decision analysis tools being developed in
Project EDA-4 (“Environmental Impact Analysis”).
Project ID Assignment: EDA-3: Modeling Framework for Multimedia Analysis of the Environmental
Distribution of Nanoparticles
Yoram Cohen, Robert Rallo, Haven Liu, Sirikarn Surawanvijit , Rong Liu, Arturo Keller
Other CEIN Collaborators: Roger Nisbet
In order to assess the potential environmental impact associated with nanoparticles, knowledge about
their potential distribution in the environment is required. Nanoparticles may enter various
environmental compartments (e.g., air, soil, water, sediment) via accidental or chronic discharges. These
nanoparticles may disperse through the various media and transfer across environmental phase
boundaries due to complex intermedia transport mechanisms. It is infeasible to monitor all
nanoparticles of concern in the various environmental media. Moreover, there is a need to assess the
potential impact of nanomaterials prior to their manufacturing, use and disposal. Therefore, it is
necessary to develop predictive models to analyze the fate and transport of nanoparticles from a
multimedia perspective. Accordingly, the overall goal of the project is to develop a model of the
multimedia environmental distribution of nanoparticles (Mend-Nano). The model is based on the use of
uniform compartments (with one or more compartments per environmental medium), each described
by a detailed unsteady-state differential mass balance equation and a detailed quantification of
transport processes among environmental compartments. Along with the CEIN “Fate, transport,
exposure and life cycle analysis” group (Theme 3) intermedia transport pathways and associated
predictive mechanistic and empirical model equations for nanoparticle intermedia transport are being
compiled and evaluated for incorporation into a multimedia modeling scheme. Mend-Nano is designed
via an object oriented programming as a web-based tool that allows remote execution via any web
browser since the numerical model itself resides and runs on the CEIN server. This approach allows
remote users to run the model without having to install the software on their own computer and will
enable us to make updates to the model without having to distribute updates to users. Mend-Nano has
an easy to use graphical user interface (GUI) and web-based implementation for remote software
utilization. The software consists of parts: User interface (UI) with expert system (ES), multimedia model
solver, parameter database, and result visualization tool. The model architecture allow for integration of
the model with built-in and user-defined libraries of meteorological, geographical, nanoparticles and
emission data. A library of region-specific environmental parameters has been developed to encompass
the range of potential scenarios to allow rapid analysis of the dynamic distribution (and persistence) of
nanoparticles in the environment. It is also expected that information (compiled data and estimation
methods) being developed by the CEIN “Fate, transport, exposure and life cycle analysis” group (Theme
3) regarding potential emissions/discharges of various nanoparticles will be incorporated into the
model. Beta testing of the Model is planned to begin in January 2012.
A first generation model for the environmental partitioning of TiO2 nanoparticles indicated that
atmospheric dry deposition is a key intermedia transport process for exchange of TiO2 between the
atmosphere and the terrestrial environment. Preliminary analysis suggested that atmospheric dry and
wet deposition processes are expected to govern the transport of particles to the soil and aquatic
environment with the prevailing aerosol size distribution (e.g., specific to urban, rural or industrial
locations) dominating the transport processes. In contrast to the availability of data on the size
distribution of atmospheric aerosols, data are scarce regarding the agglomeration state (and size
distribution) of nanoparticles under environmental aquatic conditions (e.g., ionic strength, pH,
temperature). Therefore, in the present model formulation information regarding nanoparticles
agglomerate size is obtained from either available experimental data or theoretical predictions based
nanoparticles agglomeration model recently developed in Project EDA-3 (Theme 6). Specifically, a
parameterized correlation for nanoparticles agglomeration (considering basic nanoparticle properties
and aquatic medium conditions) is presently being developed based on simulation results using our
recently developed Constant-Number Monte-Carlo/DLVO nanoparticles agglomeration model. The
accuracy of the approach was demonstrated via comparison of predictions with literature reported data
for TiO2, CeO2, and C60, showing good agreement (average error of ~11%) over a wide range of
environmental conditions (e.g., ionic strength and pH ranges of 0.03–156 mM and 3–10.4, respectively).
The agglomeration model was shown to be capable of quantitatively predicting the peak in the average
agglomerate size with pH (i.e., at the isoelectric point). Current work is proceeding in expanding the DLS
data in collaboration with the CEIN “Compositional and combinatorial ENM Libraries for propertyactivity analysis” group (Theme 1) and Project FT-4 (Theme 3) to cover a wider range of nanoparticles
primary size (~15-500 nm) in order to further validate the theoretical approach. This effort will enable
the generation of a simple to use parameterized model of nanoparticles agglomerate size (i.e., a
predictive correlation based on basic solution and nanoparticle properties). The above fundamental
models will be incorporated into environmental impact analysis methods developed in EDA-4 as well as
the multimedia fate and transport model developed under Project EDA-3. Moreover, through a
collaboration with Projects FT-1 and FT-4 (Theme 3) the range of pertinent environmental conditions
and their associated nanoparticle agglomeration and surface interactions are being mapped to develop
the necessary database and tools for conducting the fate and transport as well as environmental impact
analyses under projects Project EDA-3 and EDA-4, respectively.
Project ID Assignment: EDA-4: Environmental Impact Analysis
Yoram Cohen, Robert Rallo, Rong Liu, Haven Liu, Taimur Hassan
Given the rising concern regarding the potential environmental impact of nanomaterials, there is a need
to establish a rational approach to identify and rank nanomaterials that could be of environmental
concern. Accordingly, the project focuses on the development of a decision-based process for
environmental hazard ranking of nanomaterials (EHR-Nano). The premise for the EHR-Nano is that the
environmental impact of engineered nanomaterials (eNMs) is governed by exposure to eNMs and their
toxicity. A given eNM would be of environmental concern if it is hazardous and there is exposure to
ecological receptors at concentration levels that may induce an adverse impact associated with this
hazard. Exposure monitoring for all of the large number of already existing commercial eNMs and for
the anticipated growth in the number and types of eNMs would be impractical on both technical and
economical grounds. In this regard, estimates of the range of expected eNMs concentrations in the
environment can be obtained via suitable fate and transport models (e.g., Project EDA-3: “Modeling of
the Environmental Multimedia Distribution of Nanoparticles”; and projects of Theme 3: “Fate, transport
and life cycle analysis”) and its likely hazard can be accessed via appropriate eNMs toxicity data (Themes
2, 4 and 5) and/or predictive quantitative-structure-activity relations (Project EDA-2, Theme 6). Using
the above information, the EHR-Nano tool will enable assessment of whether or not a given eNM should
be of environmental concern.
Specific objectives in this project are to: (a) Establish a range of questions and decision areas that are
pertinent for environmental impact analysis w.r.t nanomaterials; (b) Review and compile parameters
that are likely to affect decisions w.r.t nanoparticles (e.g., toxicity level, expected level of exposure,
concentrations in various media, emissions, etc.); (c) Develop the structure of the EHR-Nano approach
(e.g., decision/network) based on parameters identified in (a); (d) Develop a web-based NP
environmental impact analysis tool that can be interfaced with quantitative and qualitative structureproperty/activity relations, fate and transport model(s) and nanoparticle databases of physicochemical
properties and toxicity. It is noted that although this project was originally scheduled to commence in
Year 4 of the CEIN, the urgent need for a systematic approach of EHR-Nano compelled us to initiate this
project at about mid-year 3 of the CEIN. To date, a working framework for the EHR-Nano was designed
in collaboration with members of the CEIN “Fate, transport, exposure and life cycle analysis” theme.
Consequently, a web-based EHR-Nano prototype was developed (as a server integrated within the CEIN
collaboratory) which contains simple questions to guide the analyst toward qualitative risk scoring. The
approach will be improved incrementally to incorporate our machine learning (Project EDA-1), nanoSARs (Project EDA-2) and fate & transport modeling (Project EDA-3) results as well as literature
information and data from CEIN researchers regarding nanoparticles transport and fate (Themes 4 and
6), toxicity (Themes 2, 3 and 5) and life-cycle analysis (Theme 4). Based on the above approach a first
generation web-based tool for environmental hazard assessment (EHR) of nanomaterials (EHR-Nano)
was developed. This tool is being expanded and ultimately will be linked with CIEN nanoparticles
properties and toxicity data. When completed the EHR-Nano will be allow users to evaluate various
environmental scenarios of involving eNMs releases and exposures as well as assess expert opinion on
the impact analysis process. Thus, this tool will be useful for regulatory agencies as well as the
nanotechnology industry in designing rationale strategies for addressing environmental concerns
associated with nanotechnology. The EHR-Nano analysis tool will ultimately be made publically available
and it will be updated with the growth of available data regarding nanoparticles properties (Theme 1),
fate and transport and life cycle (Theme 3), and toxicity (Themes 2, 4 and 5). Finally, it is noted that in
developing the EHR-Nano, CEIN members of Theme 6 are benefitting from their active participation in
the “community-owned” Nanoinformatics Roadmap efforts to develop a roadmap and standardize
approaches to collection, storage, sharing, analyzing, modeling and applying information relevant to
eNMs to foster scientific discovery and safe use of nanomaterials.
Theme 7 - Societal Implications, risk perception and outreach activities (Harthorn/Godwin)
Project ID Assignment: Soc-1: Environmental Risk Perception
Barbara Herr Harthorn – UC Santa Barbara, Terre Satterfield – University of British Columbia, Mary
Collins, UCSB; Gwen D’Arcangelis, UCSB; Shannon Hanna, UCSB; Anton Pitts, UBC
Statement of goals. Project Soc-1 aims to assess public environmental risk perceptions in the US about
potential effects of engineered nanomaterials (ENMs) in different environmental media (i.e., air, water,
and soil). To accomplish this, the study develops a new measure of attitudes toward air, water and soil
media and then assesses how those views affect perceived environmental impacts of ENMs. As a way of
anticipating public responses to particular risk information about ENMs, the project systematically
studies how malleable (subject to change) or fixed public risk perceptions are depending on specific
ENM risk messages and applications, and individual demographic characteristics, environmental values
and worldviews, and levels of trust in science and regulatory processes.
Relatedness to CEIN. To ensure highest level of applicability to CEIN research, Soc-1 has solicited input
from researchers in all other Themes about: the selection of ENMs to include in the survey; attributes of
environmental media; scientific validity of scenarios and vignettes constructed for the survey to model
possible applications and environmental interactions of ENMs; and have sought opportunities to present
and discuss results and implications for CEIN. Project will repeat process in Stage 2 survey.
Summary of highlights/key progress. Project has developed and put in the field a novel environmental
survey tool based on mental models interviews that provide insight into the concepts and language used
by lay persons to understand different environmental media and the ways these concepts interact with
their attitudes about new engineered materials. Data from the survey are analyzed to examine the
interactions among perceived characteristics of the different media, characteristics of ENMs, and a
range of environmental values and world views, across a range of specific applications.
Stage 1 of the survey, conducted as a web survey in 2010 (n=750), provides validation of a set of new
project-generated psychometric (cognitive, affective and attitude) scales. Results show that public
acceptance of ENMs in the environment is affected both by the description of the nanotechnology in
question (with environmental risk signals in particular driving the level of public acceptability) and by
respondents’ perceptions of the basic characteristics of environmental media (with those respondents
seeing environmental media as more resilient (p<2.2x10-16) and those rating media as available to
sensory evaluation (p=0.01) consistently rating examples of nanotechnology as more acceptable).
Stage 2 of the survey will extend to a broader sample of participants and focus on a wider range of
ENMs, environmental, and technological risk objects in order to more clearly delineate likely factors in
public view on ENMs in the environment. This work builds on prior and concurrent deliberative and
survey research conducted by CNS-UCSB on a related set of nanotech risk perception issues. Will
incorporate attitudes relevant to nano remediation and nano products.
Implications for CEIN. Information on drivers of public perceptions will enable more effective public
outreach and engagement. Study provides CEIN with tailored understanding of public perceptions of
risks/benefits of ENMs that are likely to drive responses to risk information that CEIN will need to
provide as its research program matures. More specifically, our finding on high salience and impact of
risk signal (high to low risk) rather than specific ENM characteristics or application context should be
taken into account in reporting CEIN ENM risk information.
Project ID Assignment: Soc-2. Sociological Aspects of ENM Environmental Risk and Perception
Barbara Herr Harthorn, Mary Collins – UC Santa Barbara
Statement of goals. Project Soc-2 aims to conduct sociological analysis of risk management challenges
for nanotech research and governance organizations. Using a widely cited theoretical concept of
organizational failure (or recreancy) developed by environmental sociologist Freudenburg, project aims
to apply recreancy assessment to the emergent nanotech organizational field to anticipate (and hence
avert) problems experienced in past technological hazard management cases. In specific nanotech case
analyses, project aims to provide early warnings about recreancy by conducting focused comparative
analysis of risk message framing by the media of nanotechnologies and comparative other technologies.
And, in a key downstream case, aims to use spatial analysis (GIS) to study potential environmental
equity issues in US nano remediation efforts.
Relatedness to CEIN. Project Soc-2 connects to the CEIN as a whole (as a social institution, subject to
recreancy); in its study of downstream nanoremediation of environmental toxins in soil and water, it
links to work in Themes 3, 4, and 5; the implications of its work connect to CEIN societal implications and
Outreach program aims; and this project links closely with Soc-1 which will include a focus on
nanoremediation applications in its Phase 2.
Summary of highlights/key progress. Project develops a theoretical framework for studying nano
institutional recreancy and responsibility, now complete. Sources of likely institutional failure in
managing the risks of nanotechnologies include lack of attention to public concerns, lack of transparency
about risks, collusion between government and industry that compromises risk assessment reliability.
Project provides case analyses of potential recreancy/institutional failure and risk amplification—and
finds that nanotech risk messaging in the media has been more balanced in provision of risk and benefit
information about nanotechnologies than in predecessor nuclear technologies, and hence less likely to
produce amplification and mistrust; and that nanoremediation projects have also apparently balanced
risk and benefit demographically in an equitable way, in contrast to many past environmental
remediation efforts in the US.
Implications for CEIN. Project aims to assist CEIN in avoiding recreancy problems and conflict in
delivering on its mission. Baseline studies on nano risk messaging and nanoremediation indicate CEIN as
a partner in government risk assessment (and eventual risk management) enters a relatively level
playing field, without significant trust disrupting or risk amplifying processes. New case analysis on
nanoremediation intends to aid CEIN in considering locational and distributional issues about risks and
benefits of ENMs as factors in risk perception and institutional responsibility regarding safe
development, while adding to the Center’s profile of downstream, application-based research. This case
provides preliminary evidence of equitability in risk/benefit distribution that CEIN could call on in
providing examples of responsible development in response to public concerns.
Project ID Assignment: Soc-3. Environmental Risk Management and Regulation in the International
Nanomaterials Industry
Barbara Herr Harthorn, Patricia Holden, Terre Satterfield, Cassandra Engeman, Lynn Baumgartner– UC
Santa Barbara
Statement of goals. The main aim of Soc-3 is to assess current nanomaterials industry environmental
and workplace risk mitigation practices and ENM risk perceptions from a diverse international set of
nanomaterials companies engaged in the production and/or handling of ENMs. Project developed and
deployed a detailed survey of 78 companies in 14 countries in 2009-2010. Analyses of these survey
responses aim to: 1) assess current environmental health and safety (EH&S) practices and their
relationship with company leaders’ views on risk and regulation, 2) characterize the nanomaterial
workforce, including a detailed understanding of specific EH&S practices across the product life cycle, as
well as the proportion of the ENM company workforce handling nanomaterials and types of
nanomaterials handled, and 3) provide US-specific and cross-national and/or cross-regional comparison
of company-reported EH&S practices and needs for guidance.
Relatedness to CEIN. Soc-3 is fully partnered with Theme 5, linked to CEIN Outreach, and demonstrates
CEIN commitment to responsible development of ENMs across the global value chain.
Summary of highlights/key progress. The survey found relatively high levels of uncertainty and
perceived risk among industry leaders across all 6 types of ENMs, but unaccompanied by expected
evidence of risk avoidant practices or preferences for regulatory oversight. A majority of companies
(61%, n = 45) claimed “lack of information” as a significant impediment to implementing nano-specific
safety practices but were not found to consistently incorporate widely available guidance. While
industry reluctance toward regulation might be expected, their own reported unsafe practices and
recognition of possible risks suggest a more top-down approach from regulators is needed to protect
workers and the environment.
Companies participating in this study ranged in size from three employees to 250,000. Overall,
companies were small, with a majority (65%, n = 51) reporting fewer than 50 employees. Companies
participating in our survey employed a total of 967,309 employees worldwide; only 3,519 employees, or
0.4% of this workforce, actually handled nanomaterials. Companies reported handling 15 types of
nanomaterials. At least one-third of companies reported handling nano-silver (37%, n = 29), titanium
dioxide (36%, n = 28), or silica (35%, n = 27). These materials are diverse, and their novel properties and
their potential toxicity are dependent on their size, surface area, form, and other characteristics. When
broken down by these characteristics, companies are handling a wide variety of nanomaterials, posing a
significant challenge for EH&S development. Combined with the challenge of identifying workers
exposed to nanomaterials, our study shows that government agencies face a significant challenge in
identifying the nanomaterial workforce for any “hard” approach to regulation.
Implications for CEIN. The aim of Soc-3 is to provide CEIN with new knowledge about the mid-/
downstream ENM industry, a key stakeholder in ENM and nanotechnology R&D, whose EH&S practices
are likely to have signficant impact on the environment and on society. The study provides depth
understanding of industry practices and perceptions likely to produce risk or protection to environment;
and that predict industry responsiveness to CEIN and government regulatory actions.Overall, findings
suggest insufficiency in “soft” approaches to regulation while also suggesting challenges to
implementing regulation for this complex industry. By understanding industry practices and responses
to risk, we contribute to knowledge about how best to control environmental risks.
Project ID Assignment: Soc-4. Risk Assessment and Nanomaterial Regulation
Milind Kandlikar, Terre Satterfield, Christian Beaudrie – University of British Columbia
Statement of goals. Project Soc-4 is investigating approaches that enable the analysis and management
of potential risks from engineered nanomaterials (ENMs) under high uncertainty. The goals of this
research are threefold: i) to investigate the adequacy of US federal regulations for assessing and
managing risks along the ENM life-cycle; ii) to understand nano-experts’ views on risk and regulation
with the aim of informing regulatory policy iii) to devise methods for: (a) eliciting expert judgments of
environmental health and safety (EHS) concerns for nanomaterials and (b) using these judgments in
decision-making and risk management.
Relatedness to CEIN. Soc-4 is closely linked to Theme 6, and discussions w/ Theme 6 researchers have
been part of the planning process for the expert workshop. Results also link closely to nanotoxicologists
and engineered nanomaterials experts in the CEIN and potential differences in their views that may
affect risk assessment and risk management processes.
Summary of highlights/key progress. To address our first goal, we conducted a rigorous review of US
federal EHS regulations and nano-regulation reports, and found significant gaps in regulatory coverage
along the nanomaterial life cycle. Consequently, many ENMs may go without a formal risk evaluation,
and ENM risks may not be managed under federal EHS regulations. These findings point to the need for
significant reforms to key environmental regulations, and for an increase in funding for EHS, policy, and
societal implications research to enable regulatory agencies to better understand and manage risks at
each life-cycle stage.
To address our second goal we conducted a survey of three classes of nanotechnology experts
(nanoscientists/engineers, nano EHS researchers, nano specialists in regulatory agencies) aimed at
assessing risk perceptions for various NM applications, at identifying factors that influence or bias
judgment, and at understanding experts’ attitudes towards regulation. This research found significant
differences in opinions on ENM risk and regulation between the three expert groups, and identified key
variables that drive judgments. Between 34% and 50% of the variance was partially described in a
multivariate regression by variables including: gender, social and political preferences, views on the
novelty of nanomaterial risk, and preferences for precaution and voluntary or market-based approaches
in regulation. Characteristic group attitudes – which are likely related to disciplinary practices and focus,
worldviews and risk framing – appear to drive differences in judgments. These findings suggest that
selection of experts from each disciplinary group will result in a greater range of opinions than with
experts selected from any single group. This expert survey builds on the work conducted within Theme 7
with other stakeholder groups (public, industry surveys).
To address our third goal we are utilizing decision-analytic techniques with nanotechnology EHS experts
from academic and regulatory institutions to elicit drivers of EHS concerns and areas of uncertainty over
the ENM life cycle. If successful this approach might lead to the development of an expert model to
support screening level risk assessment for regulators and industry. Lessons learned from this approach
will inform the development of improved elicitation techniques to aid with life-cycle assessment and risk
assessment tools and research, such as is carried out under Themes 4 and 6 of CEIN.
Implications for CEIN. Together these three research areas contribute to the CEIN’s goals of
understanding the environmental implications of ENMs, and developing approaches and support tools
for risk assessment and decision-making under high uncertainty.
Project ID Assignment: SOC-5. Nanomaterial Hazard Ranking & Nano Regulatory Policy
Hilary Godwin (UCLA), Timothy Malloy (UCLA), Kristin Yamada (UCLA)
The overarching goal of this project is to provide critical recommendations about nanomaterials to
policy-makers and regulators that are based our most up-to-date understanding of the hazards of
nanomaterials and the potential for human and environmental exposure to these materials. This work is
informed not only by science conducted within the UC CEIN, but also that of the broader scientific
community. To date, our work has focused on two areas: (1) the development of a comprehensive
review of the scientific literature regarding hazards associated with engineered nanomaterials and (2)
the development of a scheme for prioritizing nanomaterials for regulatory action based upon their
hazard and exposure potential. To date, these efforts have had two major positive impacts: they have
helped the California Department of Toxic Substances Control to dramatically improve their requests for
information from manufacturers of nanomaterials within the State of California and they have provided
critical insights into the gaps that exist in our current understanding of the risks associated with
engineered nanomaterials that need to be addressed if we wish to have effective, science-based
regulatory policies and regulations for engineered nanomaterials.
Project ID Assignment: SOC-6. California Nano Partnership (CANP) (industry links)
Hilary Godwin (UCLA), Andre Nel (UCLA), David Avery (UCLA), Timothy Malloy (UCLA), Catherine
Nameth (UCLA)
The CEIN has played an important leadership role in the State of California and nationally in creating a
partnership between industry, labor, government, non-profit organizations, and academia to address
concerns related to occupational exposure to nanomaterials and release of nanomaterials in the
environment. A critical measure of our success in this area is that we were asked to host the 2010
annual symposium on nano occupational health and safety sponsored by the California Department of
Toxic Substances Control (DTSC): “Nano VI: Progress in Protection”. In addition, we have played the lead
role in organizing the California Nanosafety Consortium of Higher Education, a working group of
environmental health and safety professionals from academic institutions across the state of California,
university faculty and students, and state and federal agencies involved in nano health and safety. The
California Nanosafety Consortium of Higher Education has performed a comprehensive review of both
existing guidance documents on safe handling and disposal of nanomertials and the exposure literature
on engineered nanomaterials (ENMs). A critical deliverable from this group is the development of a
streamlined Nanotoolkit that focuses on easy steps that researchers can take to lower their own risk of
exposure to nanomaterials and to decrease the possibility of inadvertently releasing materials that may
be hazardous into the environment. This work has informed the development of both our own online
course on Nanoecotoxicology and of online safety training modules that we are developing in
partnership with UCLA’s Environmental Health and Safety. We anticipate that these materials will have
a dramatic impact on how researchers in academic institutions handle and dispose of nanomaterials and
hence decrease the risk of unintended hazardous exposures to ENMs in the academic workplace.
Project ID Assignment: SOC-7: Public Outreach
Hilary Godwin (UCLA), Catherine Nameth (UCLA)
The goal of our public outreach projects provide formal and informal opportunities for dialogue
between the Center and its stakeholders, to expand the knowledge base on research, societal
implications, and risk perception related to the environmental implications of nanotechnology. The
Center engages in public outreach by hosting academic conferences, seminars, and symposia, and by
participating in public events.
The Center co-hosts a joint annual international meeting with CEINT, the International Conference on
the Environmental Implications of Nanotechnology (ICEIN). ICEIN provides a public forum for scientists,
researchers, government, and industry to discuss research, societal implications, and risk perceptions of
the environmental implications of nanotechnology. In 2011, CEIN contributed six invited talks by faculty
and staff, seven invited talks by graduate students and postdoctoral fellows, and sixteen posters.
Additionally, in partnership with other research centers, government agencies, and industry, the Center
contributes to a number of academic conferences and seminars each year.
By partnering with the Nanoscale Informal Science and Engineering Network (NISENet) on educational
materials and with the California Science Center, the Santa Monica Public Library, and local schools on
event locations, the Center reaches a broad range (age, socioeconomic status, educational levels, etc.)
of stakeholders through its informal science education events. In 2011, over 1,000 children and adults
learned about nanoscale science and engineering at CEIN-affiliated events. As a result of CEIN’s
collaborative efforts regarding informal science education, the Center has contributed to raising public
awareness about nanotechnology.
The Center maintains a system for web-based knowledge dissemination to the public through its
website and its Facebook page.
In the coming year, the UC CEIN will continue to provide speakers for the CEIN/CNSI (California
NanoSystems Institute) seminar series at UCLA and to maintain its web page and other web-based
applications. Informal science education partnerships with NISENet, the California Science Center, the
Santa Monica Public Library, the SciArt program at UCLA, and local schools will continue. In addition,
CEIN’s NanoDays partnership with the California Science Center, which is focused on the K-12 audience,
will expand to include a 30-minute session for adults. Furthermore, the Center will produce its own
interactive educational materials, and the Education/Outreach Coordinator is negotiating with the
California Science Center in Los Angeles and the Lied Discovery Children’s Museum in Las Vegas, Nevada
(a NISENet partner) to pilot-test the materials and to gather evaluation data.
Theme 8 - Education, career development, knowledge dissemination, and integrative efforts (Godwin)
Project ID Assignment: ED-1. Student/Postdoctoral Mentoring and Professional Development
Hilary Godwin (UCLA), Catherine Nameth (UCLA)
The primary goal of the UC CEIN’s student/postdoctoral fellow mentoring and professional development
program is to improve participants’ professional skills by offering mentoring activities and targeted
professional development workshops.
Our first Leadership Workshop was held in conjunction with ICEIN 2009 at Howard University. Thirtyone graduate students and postdocs, from each research area of CEIN and from our partner, CEINT,
participated in this day-long workshop focusing on the development and validation of standard
protocols. Our second Leadership Workshop preceded ICEIN 2010 at UCLA. Thirty students and
postdoctoral fellows, representing research areas in both CEIN and CEINT, participated. These students
and postdocs learned about high-throughput screening (HTS), toxicity screening, dynamic light
scattering (DLS), and data analysis. Our 2011 Leadership Workshop focused on the job search process,
and included tips on the application process, the interview process, and a Q&A panel of Center faculty.
The Center’s yearly Leadership Workshop, as well as all of its other activities and workshops for students
and postdocs, are participant-centered. Ideas for activities and workshops come from participants via
direct contact with the Education/Outreach Coordinator, the evaluation forms that accompany each
event, and from the Student/Postdoc Advisory Committee’s (SPAC) biannual conference call. After the
annual Leadership Workshop and after each biannual SPAC conference call, the Education/Outreach
Coordinator sends each participant a copy of the group’s notes, feedback, and suggestions for future
events. The Coordinator asks students/postdocs for edits before she publishes the final version for all
Center members.
Participant feedback and suggestions are integral to implementing relevant, timely, participant-focused
events. Student/Postdoc feedback from 2009 resulted in the agenda for the 2010 Leadership
Workshop, the offering of Presentation Skills Workshops (in person and online) before Center-related
conferences, not just before ICEIN, and in students seeking writing assistance from the
Education/Outreach Coordinator.
Feedback from 2010 resulted in the agenda and the location for the 2011 Leadership Workshop at Lake
Arrowhead, ongoing presentations skills and writing skills assistance (in-person and online), and a new
workshop in September 2011, “Finding a Postdoctoral Fellowship,” which was attended by nine
graduate students from three campuses. In addition, a face-to-face workshop, “Writing Science: How to
write papers that get funded and proposals that get funded,” was offered at UCLA in December 2011 by
Center member Josh Schimmel.
To help the Center’s students and postdoctoral fellows develop effective, professional communication
skills for presenting their research, the Center offers a variety of participant-centered activities and
workshops throughout the year. In the coming year, the Center will continue to offer these activities
and workshops, both in-person and via Skype. Additionally, after receiving support for presentation or
writing skills, participants will receive a rubric that evaluates their skills and indicates areas for
improvement. Education/Outreach will continue to communicate with students and postdocs in order
to offer them relevant mentoring and professional development support.
Project ID Assignment: ED-2. Course development, Workshops, and Learning Tools
Hilary Godwin (UCLA), Catherine Nameth (UCLA)
The goal of this project is to develop and disseminate educational outputs related to nanoscience and
the environment. Educational outputs include lectures, workshops, informal science activities, and
online learning modules related to Center research. These educational outputs contribute to
stakeholder understanding of concepts related to nanoscale science and engineering, fill gaps in the
stakeholder knowledge base, and provide a springboard from which the Center can build future
collaborations and partnerships.
The Nanoecotoxicology online course consists of 13 lectures by ten UC-CEIN faculty and postdoctoral
fellows. These materials directly stem from science generated in the center and are aimed at broad
dissemination of core concepts and advances developed within the center to students and faculty at
other institutions, in addition to providing a cohesive set of training materials for our own graduate
students and postdoctoral fellows. Learning objectives, required and recommended readings, and
quizzes accompany each lecture, and all materials can be accessed through the Center’s passwordprotected website. These lectures cover nanomaterial manufacturing, physicochemical properties of
nanomaterials, potential sources of pollution, the fate and transport of nanomaterials in the
environment, the impact of nanomaterials on cells, organisms, populations, and the stability of
ecosystems, and tools used to assess and reduce biological harm. The Center’s web-based lecture series
also provides a critical mechanism for reaching out to new potential partners and for building capacity in
this important emerging area for institutions and countries that are trying to establish themselves in this
area. The first group of researchers who pilot tested the lecture series are from partner universities and
institutions (CINVESTAV, INSP, UNAM) in Mexico. As a result of the pilot test, ten scientists from these
institutions came to the Center in August, 2011, for a week-long Nanoecotoxicology Bootcamp. The
online lecture series has now been made available and broadly disseminated to the international
nanoscience and engineering communities.
The Center is developing a series of online learning modules, in collaboration with UCLA’s Environmental
Health and Safety (EH&S) group, on the safe design and handling of nanomaterials in the laboratory.
These modules will be implemented at UCLA and will be disseminated to partner institutions and other
entities in the future.
The Center is developing informal science activities related to nanoscale science and engineering. In one
of these hands-on, tabletop activities, participants (the general public) learn how the size of an object
affects the force of static electricity on that object. In another activity, participants learn about an
important property at the nanoscale, hydrophobicity. In the third activity, participants investigate how
nanotechnology can help solve environmental problems by using nanotechnology (hydrophobic sand) to
clean up an oil spill.
The Education/Outreach Coordinator has worked closely with the
Education/Outreach Director, an undergraduate intern, and other Center members, in designing and
modifying these activities.
In the coming months, two new lectures—one on nanomaterial characterization and one on the safe
handling of nanomaterials in the laboratory—will be added to the Nanoecotoxicology series. By
October, the lecture series’ quiz feature will be updated, allowing participants to review their quiz
before submitting it, and to receive their quiz grade immediately.
Additionally, following the
educational model (lecture series, bootcamp) set for the Center’s Mexican partners, a partnership with
universities and institutions in South Africa is being planned.
In the coming months, the Center will finalize the three informal science activities, and the
Education/Outreach Coordinator is negotiating with the the Nanoscale Informal Science and Engineering
Network (NISENet), the California Science Center in Los Angeles and the Lied Discovery Children’s
Museum in Las Vegas, Nevada (a NISENet partner) to pilot-test the materials with the public, and to
gather evaluation data.
Project ID Assignment: ED-3. Protocols Working Group – UC CEIN Protocols Project
Hilary Godwin (UCLA), Sharona Sokolow (UCLA)
The primary goal of the CEIN protocols project and the protocols working group (PWG) is to develop
standard protocols for studying the environmental implications of nanotechnology used across the
Center and to disseminate these protocols to external stakeholders. This standardization is essential not
only to our efforts to integrate data from different sources and laboratories within the UC CEIN but also
to international efforts to produce a set of robust assays for studying the fate and transport of
nanomaterials in the environment and their biological and ecological impacts. The PWG consists of
faculty & student representatives from all of the IRGs in the UC CEIN and meets monthly to develop and
discuss standard protocols for the center. The Protocols Working Group has completed the
development of the unified suspension protocol and is now working on a publication describing this
approach. In addition, we have completed the initial phase of our brainstorming and discussions about
a streamlined approach to transitioning nanotox and nanoecotox assays to high throughput and has
begun preparation of a manuscript describing this approach. In addition, a new Protocols section has
been added to the UC CEIN public website:
http://www.cein.ucla.edu/research/UC_CEIN_research_Protocols.html
The website currently includes links to download publically-available protocols, a link to our protocoldevelopment partner (the International Alliance for Nanoharmonization) and references to more than a
dozen papers describing protocols developed in the UC CEIN.
Project ID Assignment: ED-4. Synergistic/Integrative Center Activities
Hilary Godwin (UCLA), Catherine Nameth (UCLA)
To promote collaboration, cross-fertilization, and interdisciplinary partnerships across the UC-CEIN and
with other research partners, Education/Outreach is a mechanism for support for designing and
facilitating face-to-face and web-based meetings.
The Center co-hosts a joint annual international meeting with CEINT, the International Conference on
the Environmental Implications of Nanotechnology (ICEIN). ICEIN provides a public forum for scientists,
researchers, government, and industry to discuss research, societal implications, and risk perceptions of
the environmental implications of nanotechnology. In 2011, CEIN contributed six invited talks by faculty
and staff, seven invited talks by graduate students and postdoctoral fellows, and sixteen posters.
Education/Outreach coordinates iterative meetings for core activities, thematic groups, and
committees, by providing administrative support (teleconference and room scheduling) and by
providing access, and real-time support, to Blackboard Collaborate (formerly Elluminate), a web-based
meeting space. For example, in Year Three, Education/Outreach ensured that monthly meetings for the
Protocols Working Group and the High Throughput Working Group were broadcast on Elluminate and
archived on the Center Data Management (CDM) system. Additionally, the Center’s internal seminars,
such as Arturo Keller’s June 14 seminar from UCSB, were broadcast live and are archived on the CDM.
To promote cross-fertilization across UC-CEIN, Education/Outreach facilitates the annual Executive
Committee retreat and the annual Center-wide retreat.
The Center co-sponsors speakers with UCLA’s CNSI (California NanoSystems Institute) for the CNSI’s
Seminar Series, thus ensuring that Center research is disseminated to a broader scientific audience. In
Year Three, the UC-CEIN was a cosponsor for five speakers, including Paul Schulte (NIOSH), Piotr
Grodzinski (NCI), and Mark Hersam (Northwestern).
In the coming year, due to the refunding process, ICEIN will not be held. However, Education/Outreach
will continue to provide administrative and technical support for core activities, thematic groups, and
for the Center’s committees, and Education/Outreach will plan and coordinate the annual Executive
Committee retreat and the annual Center-wide retreat in 2012. Additionally, the Center will continue to
partner with the CNSI on its seminar series, and it has scheduled Hilary Godwin to speak in September
2011 and Jeffery Steevens (US Army Corps of Engineers) to present in February 2012.
CORE Activities
Project ID Assignment: CORE-A: CEIN Center Administration
David Avery, CAO
The UC CEIN strategy is to maintain a strong organizational infrastructure that supports and integrates
our research, technology development, educational and diversity efforts, internal and external
stakeholders, as well as facilitating seamless communication among all these communities. To this end
our organizational structure allows for selection, prioritization, distribution, and management of
resources within a multi-institutional center structure.
An administrative staff has been compiled at UCLA to support streamlined operations of the Center.
David Avery serves as the Chief Administrative Officer of the CEIN. The CAO assists the Director by
overseeing the general administration, cooperation, communication, planning, financial
implementation, goals setting, and development of Center activities. The CAO is supported by the
following dedicated staff:
 Financial/Budget Coordinator (Vi Huynh)– responsible for financial management and reporting
systems across partner institutions
 Administrative Assistant (Nancy Neymark) – provides general support for all Center activities
including meeting coordination
 Education/Outreach Coordinator (Catherine Nameth) – under joint supervision of the CAO and
Education/Outreach Director, organizes the training, mentoring, communication, diversity, and
evaluation components of the program.
To assist in the administrative and education coordination of the UC Santa Barbara activities, a half time
support staff position has been allocated to UCSB (Stacy Rebich Hespanha).
The administration for the UC CEIN plays a key supporting role in ensuring that the Center meets and
exceeds the reporting requirements of our funding agencies and the University. The highest level of
attention is paid to detail in the execution of Center activities, including the compilation and submission
of complex reporting structures. Support is provided as needed to ensure the Center leadership, theme
leaders, External Science Advisory Committee, Education and Outreach, and Center sponsored events
are all carried out with the highest level of professionalism.
Project ID Assignment: CORE B: ENM Acquisition, Characterization, Distribution
Jeffrey I. Zink and Zhaoxia Ji
The main goal of our Core is to establish a nanomaterial library that can serve as the basis for
mechanistic and high-throughput studies designed to probe environmental fate and transport of these
materials as well as their cellular, organism, and ecosystem toxicity. Currently, more than 100 different
nanomaterials, varying from metals, metal oxides, to carbon nanotubes, have been introduced into the
library. Characterization of these nanomaterials are being actively conducted as they are synthesized by
our Core members or purchased from commercial sources. During the reporting period, several major
nanomaterials libraries were established to study the effects of key physical chemical properties of
nanomaterials on biological responses as described below.
The first library, a compositional library of 24 metal oxide nanoparticles, was established to investigate
the correlation of electronic properties of nanomaterials and their toxicity outcomes. It has been
suggested that when the energies of the conduction bands of metal oxides are close to that of the redox
potential of components of biological media, electron transfer to nanoparticles becomes possible and
thus induces oxidative stress and toxicity to cells. Preliminary toxicity results obtained in Project HTS-6
show a moderate but not perfect agreement with the band gap energy suggestion. More thorough
calculations based on the Born-Haber cycle that include the effects of other important parameters such
as sublimation energy, lattice energy, and salvation energy, are in progress in order to obtain more
quantitative understanding.
A silica library containing amorphous Stöber silica, amorphous fumed silica, mesoporous silica,
crystalline silicalite and crystalline α-quartz was assembled to study crystallinity and surface structure
effects of silicas. Preliminary studies conducted in HTS-4 show that fumed silica is the most toxic of all of
the silicas in the library. The toxicity can be reduced by heating and re-imposed by hydration. The
changes are most likely related to the number of surface silanol groups and strained three-member Si
rings. These surface structural properties were confirmed by vibrational (FTIR and Raman) spectroscopy
(see details in ENM-3). Current efforts are focused on characterizing the surface structures of other
forms of silica.
The SWCNT library was recently created to study the effects of hydrophobicity, metal impurity, and
dispersion state. All nanotubes were extensively characterized using various techniques such as TEM,
AFM, ICP-MS, UV-vis, Raman, ZetaPALS, and DLS. The materials were distributed to various research
groups (HTS-7, FT-5, FT-6, FT-7, MFW-1, MFW-2) for toxicity, fate and transport studies. The in vitro
toxicity studies conducted in Project HTS-7 revealed that both as-prepared and purified tubes induced
significant IL-1 cytokine release in THP-1 cells, whereas the well-dispersed tubes did not. These results
suggest the key role of dispersion state playing in SWCNT toxicity. The in vivo zebrafish study also
showed that metal ion leaching from these nanotubes could induce hatching inhibition. Fate and
transport and photocatalytic activity of these SWCNTs are also being actively conducted in various
research groups (FT-5, MFW-1, MFW-2).
Since metal ion dissolution is observed in many metal and metal oxide nanoparticles, it is important to
differentiate between the effect of direct contact of a particle’s surface with a cell and the dissolution of
toxic ions from the particle. For that, a set of “Trojan horse” nanoparticles, which are composed of a
metal or metal oxide core and a mesoporous silica shell, were synthesized. By encapsulating highly
dissolvable nanoparticles such as Ag, CuO, and ZnO in mesoporous silica, the toxicity due to the
dissolved metal ions can be studied without the effects of contact of the nanoparticles with the cell
membrane or zebra fish chorion. Currently, toxicity studies in mammalian cell lines (BEAS-2B and RAW
264.7) and zebrafish embryos are being conducted in Projects HTS-1 and HTS-2, respectively.
In studying the toxic effects of nanomaterials, it is important to understand how they interact with cells.
However the small size of nanomaterials often makes tracking these particles difficult. By labeling ENMs
with fluorescent tags, we can track their location in cells and organisms to help determine the
interactions that contribute to any lethal and sub-lethal effects. The first set of nanoparticles being
successfully labeled are CuO, silver with different sizes, and silica nanorods with various aspect ratios.
When the labeled CuO nanoparticles were introduced to zebrafish embryos four hours after fertilization,
it was clear that (1) the particles were readily detected by fluorescence, and (2) CuO nanoparticles do
not penetrate the chorion of the zebrafish embryo (HTS-2). These particles will be examined in other
cells/organisms to determine the nano-CuO uptake and its interaction with cells/organisms.
Project ID Assignment: CORE C. Data Repository and Nano Collaboratory
Yoram Cohen, Rong Liu, Haven Liu, Robert Rallo, Taimur Hassan
The development and maintenance of a multidisciplinary collaboration infrastructure and data
management system are of vital importance for the CEIN. The CEIN data management (CDM) team of
the CEIN provides core support for data management, storage, web-based collaborative infrastructure
and computational needs of the CEIN. The CDM system is based on the Microsoft SharePoint Server.
The CDM system now hosts all CEIN research group sites, as well as a Data Repository site for the
sharing of files/data among internal and external groups. The CDM system now allows individual users
and groups (CEIN and external) to build their own sub-sites with document management features and
appropriate security measures. It also provides hosting of course material with access to non-CEIN users.
A protocol for document (data and metadata) submission/uploading has been implemented with
respect to the CEIN Data Repository and an advanced search capability is now in place. In addition, a
capability was developed and implemented for web-based online submission and archiving of CEIN
research progress reports.
The CEIN computational/data management infrastructure including servers, workstations and backup
equipment is maintained by Theme 6 personnel. Support is provided in the form of hardware/system
software, as well as development and maintenance of web-based data repository/management
systems. This year the Rocks Cluster version 5.4 operating system and software of the CEIN cluster
(NAGIOS and GANGLIA) were installed and configured for automated and web-based monitoring and
supervision of all CEIN critical computational infrastructure. The system is configured as an expandable
computational cluster which uses the Oracle Grid Engine for job scheduling and with web-based
applications for high volume data management and storage of HTS data files.
A major recent addition to the CDM system is an online web based system dubbed ‘NanoCrawler’
developed to improve searching/organizing/mining of research data/information uploaded to the CEIN
Data Repository and to enable the creation of summary reports linked to the CEIN nanoparticles library.
The NanoCrawler retrieves information from the Data Repository and custom sorts and displays selected
information and enables direct downloading of nanoparticle data files. Information regarding the CDM
system features and usage has been made available via video instructions, help files, online help and a
periodic Newsletter. In addition to the above, a capability for hosting web-based tools was developed
and the CDM now hosts the CEIN high throughput Screening data analysis tool (HDAT) developed in
Project EAD-1 and also being adapted to host software for environmental impact analysis being
developed in Project EDA-4.
In summary, the Data Repository and Nano Collaboratory core is now an important function that
facilitates the integration of CEIN research themes. Moreover, it is a significant contributor to the
national Nanoinformatics effort while its capabilities have enabled collaborations with external groups
such as the US EPA ToxCast program.
PROJECT ID Assignment: CORE D. Molecular Share Screening Resource (MSSR) for HTS.
Kenneth Bradley, Robert Damoiseaux.
Abstract. Dr. Kenneth Bradley is the Director of the Molecular Shared Screening Resource (MSSR),
which will assume a core function role within the Center. Dr. Bradley and the MSSR Scientific Director,
Dr. Robert Damoiseaux, will provide scientific and technical consultation, and assistance in the planning
and execution of high throughput experiments proposed by CEIN researchers. Numerous high
throughput and multiplex assays implementing high content readouts have already been developed.
These assays will continue to be supported as new nanomaterials become available. Further, MSSR staff
will assist in translation of existing low throughput assays and de novo establishment of novel assays.
The expertise and technical capabilities available through the MSSR make this facility uniquely suited to
handle a wide variety of high throughput assays, including those aimed at examining interactions
between nanomaterials and bacteria, yeast, animal cells (including insect, marine invertebrate,
mammalian, etc…) and whole-animals such as zebrafish embryos. The MSSR operates as a user facility
that fully integrates and trains users rather than acting soley as a service core. Thus, CEIN students and
postdoctoral fellows receive specialized and inimitable training on high throughput screening using
industrial scale equipment, thereby educating the next generation of researchers highly trained in
cutting edge lab automation technologies.