University of California Center for Environmental ......HTS-2 Development of high content screening of nanoparticle toxicity using zebrafish models – Nel/Lin ... TER-5 DEB modeling

University of California

Center for Environmental Implications of Nanotechnology (UC CEIN)

NSF: DBI-0830117

4th Year Progress Report

April 1, 2011 – March 31, 2012

UC Center for Environmental Implications of Nanotechnology Year 4 Progress Report 2012

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UC CEIN Themes and Cores Themes 1. Compositional and combinatorial ENM Libraries for property-activity analysis (Zink) ENM-1 Toxicity of ENM with Cationic Surfaces - Zink ENM-2 Processing and Characterization of Single Walled and Multi-Walled Carbon Nanotubes - Hersam ENM-3 Systematic Synthesis of Nanoparticles of controllable morphology, composition, and porosity to

perform biological structure function analysis in mammalian cells and bacteria - Brinker ENM-4 FSP generated pure and Fe doped ZnO or TiO2 NP libraries for testing paradigms of environmental and

cellular responses- Madler ENM-5 Relationships Between ENM Geometry and Biological Outcomes: Nanorods ENM-6 Relationships Between ENM Geometry and Biological Outcomes: Nanowires

2. Molecular, cellular and organism high-throughput screening for hazard assessment(Nel) HTS-1 Use of multi-parametric HTS to study predictive ENM toxicological paradigms in mammalian and fish

cell lines – Nel/Xia HTS-2 Development of high content screening of nanoparticle toxicity using zebrafish models – Nel/Lin HTS-3 HTS to Determine the Mechanistic Toxicology of Engineered Nanomaterials in Bacteria -

Godwin/Holden HTS-4 Property-activity analysis of silica nanoparticles, including the relationship of surface chemistry to

toxicological potential – Nel/Xia HTS-5 Assessment of the role of metal oxide energy structure on the biological effects and potential toxicity

of this class of ENMs - Nel/Xia HTS-6 Linking the physicochemical characteristics of a library of multi-wall carbon nanotubes (CNTs) to

toxicological outcomes in vitro and in vivo – Xia/Nel HTS-7 Linking the physicochemical properties of a library of single-walled carbon nanotubes (SWCNTs) to

toxicological outcomes in vitro and in vivo - Nel/Xia HTS-8 Study of the role of long AR mesoporous silica nanoparticles (MSNPs) on cellular uptake and bio-

availability – Nel/Meng HTS-9 Study of the biological effects of long aspect ratio CeO2 on inflammasome activation and the generation

of cytotoxicity- Xia/Nel HTS-10 Reporter Gene Cell-based Assays for High Throughput Screening to Determine Sub-Lethal Toxicity of

Nanomaterials –Bradley/Damoiseaux (Completed Aug 2011) HTS-11 Toxicological assessment of cationic nanoparticles with different surface charge densities in

differentiated in undifferentiated bronchial epithelial cells- Nel 3. Fate, transport, exposure and life cycle assessment(Keller) FT-1 Role of material properties and environmental conditions on nanoparticle aggregation & dissolution -

Keller FT-2 Attachment of nanoparticles to mineral surfaces under different aqueous solution chemistries - Keller FT-3 Quantitative determination of fate and transport of nanoparticles in porous media - Walker FT-4 Effect of wettability on the transport and fate of metal oxide nanoparticles - Somasundaran FT-5 Photoactivity of nanomaterials in natural waters – Keller FT-6 Physicochemical nano-bio interactions at different scales that influence fate & transport of nanoparticles

- Keller FT-7 Life-cycle assessment of carbon nanotubes (new seed project) – Keller, Suh and Cohen


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4. Terrestrial ecosystems impact and hazard assessment(Holden) TER-1 Nanotoxicology in terrestrial microcosms - Holden/Schimel TER-2 Trophic Transfer, Bioaccumulation and Biomagnification of Engineered Nanomaterials in Basal Levels

of Environmental Food Webs - Holden TER-3 Engineered nanoparticle biosorption, toxicity, and toxicity mechanisms in planktonic and biofilm

bacteria - Holden TER-4 Toxicity and uptake of nanoparticles by terrestrial plant species - Gardea-Torresdey TER-5 DEB modeling of toxic effects of CdSe quantum dots - Nisbet 5. Marine and freshwater ecosystems impact and toxicology (Lenihan) MFW-1 Marine organismal nanotoxicology: Studying Nanomaterials’ (NMs) Interactions at the Molecular,

Cellular, Organ, and Systemic Levels - Cherr MFW-2 Impacts of engineered nanomaterials on marine ecosystems - Lenihan MFW-3 Decoupling and recoupling plant-herbivore systems to determine the fate and impact of nanomaterials

in freshwater environments - McCauley MFW-4 Impacts of TiO2 nanoparticles on freshwater food webs - Cardinale MFW-5 Dynamic energy budget (DEB) modeling to support design of aquatic microcosm and mesocosm

experiments - Nisbet 6. Environmental decision analysis for nanoparticles(Cohen) EDA-1 Machine Learning Analysis and Modeling of High Throughput Screening Data for Nanoparticles -

Telesca/Cohen EDA-2 QSARs of NP Toxicity and Physicochemical Properties - Cohen EDA-3 Modeling of the Environmental Multimedia Distribution of Nanoparticles - Cohen EDA-4 Environmental Impact Analysis - Cohen 7. Societal implications, risk perception and outreach activities (Harthorn/Godwin) SOC-1 Environmental Risk Perception - Harthorn/Satterfield SOC-2 Environmental Sociology of ENMs - Harthorn SOC-3 Environmental Risk Management and Regulation in the International Nanomaterials Industry -

Harthorn/Holden SOC-4 Risk Assessment and Nanomaterial Regulation - Kandlikar SOC-5 Nanomaterial Hazard Ranking and Nano Regulatory Policy - Godwin/Malloy SOC-6 California Nano Partnership (CANP) - Industry/Gov't/Academia Linkages - Godwin/Nel 8. Education, career development, knowledge dissemination, and integrative efforts (Godwin) ED-1 Student/Postdoctoral Professional Development - Godwin ED-2 Course development, Workshops, and Learning Tools - Godwin ED-3 Protocols development, harmonized and integrative efforts - Godwin ED-4 Synergistic/Integrative Center Activities - Godwin ED-5 Informal Science Education - Goodwin Cores

a) Administration - Avery b) ENM acquisition, characterization, distribution- Zink/Ji c) Data repository and Nano Collaboratory - Cohen/Hassan d) Molecular Shared Screening Resource (MSSR) for HTS - Bradley


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Theme 1: Compositional and combinatorial ENM Libraries for property-activity analysis Year 4 Progress - April 1, 2011 - March 31, 2012 Faculty Investigators: Jeffrey I. ZinkMark

, UCLA, Professor of Chemistry and Biochemistry – Theme leader Hersam

C. Jeffrey , Northwestern University, Professor of Chemistry

BrinkerLutz

, University of New Mexico and Sandia National Laboratory Mädler

, University of Bremen (Germany)

Updated Goals of Theme 1: Theme 1 is the successor of IRG1 (“Nanomaterial Standard Reference and Combinatorial Libraries and Physical-Chemical Characterization”), which was focused on the synthesis or procurement of libraries of nanomaterials and the thorough characterization of these materials for use by the entire Center. The primary goal of Theme 1 is to characterize property-activity relationships between fundamental physical/chemical properties of nanoparticles and biological responses, and to develop predictive models based upon these relationships. The fundamental task of nanomaterial acquisition and characterization, which had been an IRG1 activity, has been moved into Core B. In a highly interactive feedback process, nanoparticles with designed physical and chemical properties are synthesized, characterized, and studied in Theme 2 using high content or high throughput methods to assess injury paradigms in cells and organisms. Highly interactive collaboration and feedback between members of Themes 1 and 2 facilitates analysis of the property-activity relationships in the cells and organism could, in turn, has led to additional design and synthesis of nanomaterials for further testing of altered properties until the predictions of a model are validated (or rejected). Successful models are shared with investigators in themes 3-5 for incorporation in studies of transport and fate in the environment (Theme 3) and of terrestrial and aqueous ecosystems (Themes 4-5). Organization and Integration of Theme 1 Projects: We have moved the fundamental preparation and characterization service functions into Core B in order to focus on property-activity relationships in Theme 1. Core B is an integral part of Theme 1 because all materials being synthesized for Theme 1 projects are also thoroughly characterized as part of the Core’s activities. Most of the projects in Theme 1 have evolved from the compositional nanomaterial libraries in the Core because of the shift in focus from more general effects of material composition to the exploration of specific properties of those materials once initial biological screening or hazard ranking have taken place. Example Theme 1 projects include acquisition of more metal oxides to study the role of band energies, use of calcination to change the surface properties of fumed silica, and the introduction of aspect ratio and shape variation to a library of mesoporous silica nanospheres. This dynamic feedback process has been sped up by the accelerated rate of discovery in the high throughput studies in Theme 2. Table 1 summarizes the property-activity analyses that have been incorporated into the projects which evolved from the original compositional libraries. The table shows properties of nanoparticles that have emerged from the prior years’ studies as predictors of toxicity. For example, the electronic structure of nanoparticles (in particular, conduction band energy) has emerged as a unifying feature that is predictive of oxidative stress. Many metal oxides were used to test hypotheses about electronic structure, including those from the previous libraries and new materials that were synthesized specifically for these studies (see descriptions in the following section.) Likewise, selected sizes and shapes of ceria, titania and silica nanoparticles were synthesized to test morphological properties. Based upon quantitative biological response assessment (Theme 2) as a function of the variation in nanomaterial compositions and their combinatorial properties (Theme 1), Theme 6 uses feature


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selection tools (e.g., heatmaps and self organizing maps) to provide hazard ranking and structure-activity analysis for further project and hypothesis development. Table 1. Relationships between nanoparticles’ properties, physical state or chemical composition, and biological and environmental toxicity. The interplay between the physical/chemical focus of Theme 1 and the biological/environmental toxicology results from Theme 2 leads to development of predictive theoretical models. Nanoparticle Property Physical/Chemical Focus Biological response and

environmental hazard potential Electronic Structure Valence band energy (Metal

oxides) Inter-band energy (Fe and Pt doped titania)

Predictive model of toxic oxidative stress (cytotoxicity) Wavelength-dependent photo-toxicity; altered dissolution chemistry as a safer design feature

Surface (chemical structure) Silanol group/siloxane rings (polymorphs of silica) Cationic coatings (silica)

Correlation of toxicity from fumed silica with surface silanol coverage Surface membrane and lysosomal toxicity

Shape and Size Nanowires (ceria) Nanorods (silica) Diameter of spheres (titania)

Inflammasome activation, inflammation Length-dependent enhancement of cellular uptake; Size-dependent toxicity

ENM-1 Toxicity of ENM with Cationic Surfaces - Zink Project List for Theme 1

ENM-2 Processing and Characterization of Single Walled and Multi-Walled Carbon Nanotubes - Hersam ENM-3 Systematic Synthesis of Nanoparticles of Controllable Morphology, Composition, and Porosity to

Perform Biological Structure Function Analysis in Mammalian Cells and Bacteria - Brinker ENM-4 FSP-generated Pure and Fe-doped ZnO or TiO2 NP Libraries for Testing Paradigms of

Environmental and Cellular Responses - Madler ENM-5 Relationships between ENM Geometry and Biological Outcomes: Nanorods - Zink ENM-6 Relationships between ENM Geometry and Biological Outcomes: Nanowires - Zink CORE-B ENM Acquisition, Characterization and Distribution Core- Zink/Ji Major Accomplishments of Theme 1 since February 2011: Significant progress was made in six of the property-activity assessment areas. Our results are summarized below in order of the Project number. Project ENM-1 Toxicity of ENM with Cationic Surfaces - Zink Cationic surface charges on nanoparticles have been shown to cause toxicity. In order to study systematically the effect of surface charge and charge density, we synthesized a library of cationic


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polymer-coated mesoporous silica nanoparticles (MSNs). Polyethyleneimine was chosen because its highly branched structure has multiple cationic amine functional groups that increase with the molecular weight of the polymer. The MSNs (~100 nm diameter, non-toxic in the absence of poymer) were made by sol-gel methods. The PEI coating of different polymer sizes (MW 0.6, 0.8, 1.2, 1.8, 10 and 25 kD) was attached electrostatically to the MSNs. In order to monitor the location of the particles and their cellular uptake by confocal imaging, the polymer-coated particles were derivatized by covalent attachment of fluorescein. All of the MSNs were characterized for size, size distribution, shape and charge using TEM, DLS and electrophoretic mobility. The proposed synthetic methods were successful and resulted in a new library of cation-coated nanoparticles. Particles coated with the 10 and 25 kD PEI polymers were shown in Project HTS-11 to undergo increased cellular association compared to those with smaller polymers. The toxicity of the coated particles was measured using a rapid throughput multiparametric assay that discovered that 10 and 25 kD polymers were more toxic in differentiated cells than particles coated with shorter length polymers. The high impact of this discovery is described in Theme 2. Project ENM-2 Processing and Characterization of Single Walled and Multi-Walled Carbon Nanotubes – Hersam Single and multi-walled carbon nanotubes, a carbonaceous large aspect ratio nanomaterial, were purified and characterized in ENM-2. The following materials have been incorporated into the ENM library and are being studied by the Center: raw and purified HiPco single-walled carbon nanotubes; pluronic-dispersed, purified HiPco single-walled carbon nanotubes where large aggregates and impurities have been removed via centrifugal processing; as-prepared and purified arc discharge single-walled carbon nanotubes; purified P2 arc discharge single-walled carbon nanotubes; pluronic-dispersed, purified P2 arc discharge single-walled carbon nanotubes where large aggregates and impurities have been removed via centrifugal processing; purified CoMoCAT SG65 single-walled carbon nanotubes; and pluronic-dispersed and purified CoMoCAT arc discharge single-walled carbon nanotubes where large aggregates and impurities have been removed via centrifugal processing. The careful separation and dispersion methods proved to be vital for projects HTS-3 and HTS-7 because cellular and organismal screening studies demonstrated that the state of tube dispersion is extremely important for bioavailability. CNTs dispersed by BSA and DPPC are taken up into macrophages and epithelial cells and induce pro-fibrogenic effects. The pluronic-dispersed CNTs are less efficiently taken up because of steric hindrance and the cells are protected against the induction of pro-fibrogenic responses. The careful purification and dispersion of CNTs is a necessity in order for screening and environmental studies to be meaningful.

Project ENM-3 Systematic Synthesis of Nanoparticles of controllable morphology, composition, and porosity to perform biological structure function analysis in mammalian cells and bacteria – Brinker We made a major advance in the understanding of the origin of toxicity of certain forms of silica in Project ENM-3 and the corresponding project HTS-4 in Theme 2. Commercially synthesized silica is in general amorphous and often in the form of nanostructured powders that are used extensively in applications like fillers, catalysts, and desiccants. Amorphous silica nanoparticles are prepared by two main routes, high temperature flame pyrolysis to form fumed silica (in tonnage quantities) or by molecular condensation of silanol in aqueous solution or under hydrothermal conditions to form so-called precipitated or colloidal silicas. Silica is comprised of rings that make up their framework; the most stable are comprised of 5 or 6 silicon atoms while the smallest (2 and 3 members) are the least stable. The surface also contains silanol (Si-OH) groups. Both the rings and the silanols are monitored spectroscopically in our study by using vibrational (infrared or Raman) spectroscopy. Upon heating, the surface silanol concentration decreases due to condensation reactions to form siloxane bonds (and can


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form strained rings). The vibrational signatures of the rings are monitored in the low wavenumber region of the spectra and those of the silanols in the high frequency region. Upon exposure to water, rehydration occurs and silanols are regenerated as evidenced by the vibrational intensity increase. In our study of five types of manmade silica (fumed, amorphous, mesoporous, silicalite and quartz), fumed silica is by far the most toxic. Its toxicity (measured in HTS-4) decreases when it is heated to 500 oC to decrease the surface silanol coverage, but increases when the fumed silica is rehydrated in boiling water. Raman spectroscopy was used to quantify the surface coverage and verify that the coverage decreased after heating and recovered after rehydration. Specifically, isolated vicinal silanols are correlated with silica toxicty due to ROS. Because of these breakthrough results, research is continuing in order to better understand the role of ring structure (and the corresponding silanol groups on the rings) on toxicity. Project ENM-4 FSP-generated pure and Fe-doped ZnO or TiO2 NP libraries for testing paradigms of environmental and cellular responses- Madler The fundamental hypothesis underlying the studies is that spontaneous electron transfer will occur when the energy of the conduction band of a nanoparticle overlaps with the energy range of the oxidation potentials of the redox-active molecules the cell. The resulting transfer of an electron from a cellular biomolecule to the nanoparticle leads to the generation of oxidative stress injury. A series of metal oxide nanoparticles with conduction band energies above, equal to and below the biomolecular redox energies were chosen for investigation. The metal oxide nanoparticles Bi2O3, CuO Co3O4, Sb2O3, WO3 and Fe3O4 were synthesized using flame-spray pyrolysis. All were thoroughly characterized (XRD, BET, LRTEM, HRTEM and SAED) and have a primary particle size of around 10 nm. Single parameter toxicity assays (MTS, LDH and ATP) as well as a multi-parameter HTS assay that assesses toxic oxidative stress (HTS-6) were used to evaluate their toxicity in BEAS-2B and RAW 264.7 cell lines. The results obtained from the single parameter assays demonstrated high toxicity potential for CuO and Co3O4 (particles with conduction band energies within the energy range of cellular redox potentials) while low or no toxicity was found for Sb2O3, WO3 and Fe3O4. Similar results were obtained in the multi-parametric HTS assay indicating that both epithelial cells and macrophages exhibit sublethal responses to CuO and Co3O4 at 4-5 hours and then progressing to lethal cellular responses at a later point. Sb2O3, WO3 and Fe3O4 did not exert toxicity. The “safe-by-design” concept being developed in the CEIN is based on transforming nanomaterials from toxic into benign by introducing dopants into the structure. A major success described in prior reports utilized iron ion doping into the ZnO lattice to stabilize the crystal lattice, slow the rate of dissolution, and reduce the toxicity caused by the dissolved zinc ions. We therefore continued this line of investigation by studying aluminum ion doping. Aluminum has a much higher charge/size ratio and would be expected to provide more stabilization than iron. A library of to 1-10% of Al doped ZnO NPs was synthesized using flame spray pyrolysis. The particle size derived from BET (dBET) and XRD (dXRD) was found to be in the range of 16-12 nm and 18-12 nm respectively. The distribution of Al atoms in the ZnO matrix was also investigated with combined EELS and EFTEM analysis. HRTEM images of single nanoparticles of pure and Al-doped ZnO revealed highly crystalline nature with lattice spacing values of 2.81-2.91 Å corroborating a negligible doping effect on the crystal parameters even at high atomic loadings. The toxicity screening of this library was performed in Theme HTS-6. Cell death decreased with increasing Al-content. Comparing the cytotoxic effect achieved by Fe-doped and Al-doped ZnO nanoparticles, the latter was found to be more protective in terms of cellular oxidative stress pathways.


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Project ENM-5 Relationships between ENM Geometry and Biological Outcomes: Nanorods – Zink Spherical nanoparticles have been studied extensively in the Center to date. However, nanoparticles that are made in large industrial quantities are rarely perfect spheres. There is evidence that the aspect ratio of nanomaterials (i.e. the ratio of length to diameter) may change both their bioavailabiity and toxicity. For that reason, it became necessary for us to construct libraries of ENMs that show AR variation but also have a constant chemical composition. In order for us to focus on the effects of aspect ratio without complications from chemical toxicity, we chose to use nontoxic amorphous silica. A library of mesoporous silica rod-shaped nanoparticles (MSN) that covers a range of different lengths was synthesized using templated sol-gel synthesis methods. The particles were designed to have a diameter of about 100 nm and lengths up to 500 nm. The length of the rod was controlled by the concentration of an amphiphilic co-templating agent; we discovered that the rod length increased as a function of its concentration. The size measurements based on TEM images confirmed a very narrow length distribution of the nanorods. The particles were thoroughly characterized by TEM, XRD, gas adsorption/desorption isotherms, dynamic light scattering and electrophoretic mobility. When this library of particles was examined in Theme HTS-8, it was discovered that the AR has a profound influence on the rates and abundance of MSN taken up by cells. MSN with an AR of 2.1-2.5 showed a 40-fold increase in uptake compared to spheres. This discovery impacts environmental safety considerations; problems may occur when nanoparticles that are inherently toxic or have become toxic by adsorption of hazardous chemicals undergo enhanced uptake by cells. Mechanisms by which the AR impacts cellular function are unknown but of considerable importance in understanding how to improve nanomaterial safety through the use of a physical design feature. Project ENM-6 Relationships between ENM Geometry and Biological Outcomes: Nanowires – Zink Commercial cerium oxide (CeO2) contains a mixture of nanoparticles with shapes ranging from spherical to those of wires. It is unknown if there is a critical length that can induce deleterious biological responses. To address this question, we synthesized a cerium oxide (CeO2) nanowire library with a wide range of large aspect ratios using hydrothermal methods. An advantage of using CeO2 is that spherically shaped nanoparticles are biologically inert, and thus studies of the length and aspect ratio are not complicated by intrinsic toxicity. By controlling the hydrothermal synthesis composition and conditions, including (i) cerium precursor type and concentration; (ii) concentration of phosphate; (iii) pH of the initial synthesis mixture; (iv) synthesis temperature; and (v) seeding and secondary treatment, we obtained for the first time a series of CeO2 nanowires with precisely controlled lengths and aspect ratios. The size measurement based on TEM images confirmed a very narrow length distribution of the nanowires. The particles were thoroughly characterized using TEM, XRD, gas adsorption/desorption. The successful creation of the precisely tuned CeO2nanowire combinatorial library for the first time allowed a pure length and aspect ratio effect on biological response to be studied in a systematic manner. Theme 2’s in vitro toxicity studies on human cells (Theme HTS-10) showed that the short CeO2 nanowires with aspect ratios varying from 1 to 16 were all non-toxic, whereas longer nanowires with an aspect ratio of 30-50 and >100, respectively, induce inflammasome activation and eventually led to significant cell death. These results demonstrate that nanowire length plays a key role in high aspect ratio nanomaterial toxicity. Impacts on the Overall Goals of the Center The major progress made in Theme 1 over the last year has clarified the importance of metal oxide nanoparticle conduction band energy, nanomaterial aspect ratio and shape, surface cationic charge, and the surface chemistry by which silica leads to a toxicological outcome. Thus, one major impact has been to provide property-activity analysis of highly abundant nanomaterials such as metal oxides and fumed silica. Another major impact was the demonstration of the importance of size and shape, specifically the


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aspect ratio of rods and wires, on undesirable biological outcomes even if the underlying chemical composition is non-toxic when the materials have spherical shapes. In a highly interactive feedback process, nanoparticles with designed physical and chemical properties were designed, synthesized and characterized in Theme 1, and then studied in cells, bacteria, and organisms in Theme 2. Evaluation of the cellular and organism responses led to further synthesis of designed materials and further testing until the predictions of a model were validated (or rejected). These results, in turn, informed researchers in Themes 3-5 about key properties of engineered nanomaterials that will be important in fate and transport and for assessment of their effects in terrestrial and aqueous ecosysems. Furthermore, numerical quantification of energy levels of the nanoparticles and biomolecules, chemical descriptions of nanoparticle surfaces, numerical identifications of shape, and elemental (chemical) compositions of the materials enabled detailed refinement of machine learning analyses and ranking of hazards in Theme 6. Our results also enable us to inform concerned scientists and the public that there is more to environmental safety than simply identifying the chemical composition of nanomaterials, and show that we identify, understand and control relevant properties. We can reassure those interested in nanosafety that knowledgeable design changes can improve safety. Major Planned Activities for the Next Period During the next three-month period, we will continue our research involving the relationships between the valence band energies of nanoparticles and toxicity. Additional components that will be investigated include solvation energies, conduction band energies, lattice energies, and other thermodynamic properties in the Born-Haber cycle. We will also continue studies on the relationships between silica surfaces and toxicity, especially more detailed studies of the vibrational spectroscopic determination of ring sizes, properties of silanols as a function of ring size, and crystallinity. Finally, we will continue our studies of aspect ratio’s relationship to biological response by investigating other parameters (such as constant aspect ratio but varying the length) and composition (carbon nanotubes, vanadia rods and wires).


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Theme 2: Molecular, Cellular and Organism High Throughput Screening for Hazard Assessment Year 4 Progress - April 1, 2011 - March 31, 2012 Faculty Investigators: André Nel, UCLA – Professor, Medicine; Chief, Division of NanoMedicine- Theme leader Kenneth Bradley, UCLA – Associate Professor, Microbiology Hilary Godwin, UCLA – Professor, Environmental Health Sciences Patricia Holden, UC Santa Barbara – Professor, Environmental Microbiology Shuo Lin, UCLA – Professor, Molecular, Cellular, and Developmental Biology Tian Xia, UCLA- Assistant Adjunct Professor, Medicine, Division of NanoMedicine Huan Meng, UCLA- Assistant Research Scientist, Medicine, Division of NanoMedicine Updated Goals of Theme 2: Theme 2 is an outgrowth of the activities previously incorporated in IRG2 (“Nanomaterial interactions at the molecular, cellular, organ and systemic levels”) and IRG 5 (“High throughput screening for nanomaterial properties and nanotoxicity”). The overarching goal of Theme 2 is to develop high throughput screening (HTS) approaches that can be carried out in tissue culture cells, bacteria, and yeasts to develop predictive toxicology paradigms that relate potentially hazardous engineered nanomaterial (ENM) properties to adverse biological outcomes in organisms and animals. To do so, we have developed a number of robust scientific platforms relating molecular and cellular injury responses to ENM properties that could pose biological risk and potentially useful for high content toxicological screening, dosimetry calculation and safe-by design approaches. High content or high throughput screening is done in close collaboration with other CEIN themes to prioritize CEIN’s ability to address important nanomaterials by expedited hazard ranking. We are utilizing OECD’s priority list of primary nanomaterials such as metals, metal oxides, silica and carbon nanotube (CNT) nanomaterials for acquisition, synthesis and characterization by Theme 1. The availability of these compositional and combinatorial ENM libraries has allowed us to develop a number of biological endpoints upon which to base predictive toxicological screening and HTS development. The integrated efforts of Themes 1, 2 and 6 inform investigators in Themes 3-5 about the key materials and property-activity relationships that should be considered for fate and transport studies as well as for introduction into terrestrial, marine and freshwater ecosystems. The rich data sets emerging from the HTS studies have been instrumental in the development of quantitative structure-activity relationships, machine learning analyses and hierarchical ranking of ENM hazard by Theme 6. Theme 2 is leveraging the automated high throughput screening (HTS) infrastructure of the UCLA Molecular Screening Shared Resource (MSSR), which is an associated Core facility (see Core Report). The MSSR Core performs: automated screening with robotic equipment that allows assessment of luminescence-based reporter gene activity, epifluorescence microscopy that tracks multi-parameter sub-lethal and lethal cellular responses, multiplex quantification of pro-inflammatory and cellular injury response markers in the supernatant, as well as high throughput screening and imaging of zebrafish embryos and larvae. Organization and Integration of Theme 2 Projects: We have restructured this theme to incorporate the automated analysis of biological injury pathways and mechanisms into the MSSR Core, which is overseen by Prof. Bradley and Technical Director, Dr. Damoiseaux. They work in close collaboration with the project leaders and research teams to facilitate development of methods that can be used for exploration of the nanomaterial libraries in the specific projects outlined below. The progress report for the recently completed Project HTS-10, developed in the MSSR Core, illustrates how the assessment of transcriptional gene activation can be used for high throughput discovery of cellular signaling pathways that are triggered in response to ENM injury. Additional HTS tools are being developed by the MSSR for bacterial HTS (Godwin and Holden), and marine organisms (e.g., mussel hemocytes, Cherr).) Theme 2


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includes 11 active projects that utilize mammalian cells, bacteria, a fish gill cell line and zebrafish embryos/ larvae for high throughput discovery of biological injury responses in response to specific ENM compositions and properties relevant to the environment. The high volume data sets emerging from these studies are being used by Theme 6 for the development of in silico feature analysis and decision-making tools that inform hazard ranking, dosimetry calculations and prioritizing more costly experiments at higher trophic levels. We will indicate how the integrated research in Themes 1, 2 and 6 has led to the development of quantitative structure-activity relationships (SARs) as well as predicting possible safe-by design approaches for nanomaterials. Professors Nel and Xia oversee the HTS analyses in mammalian tissue culture cells and a rainbow trout gill epithelial cell line and are also working in collaboration with Dr. Shoa Lin from the UCLA Zebrafish Core facility to conduct high content screening analyses of morphological abnormalities and GFP-transgene activation in zebrafish embryos and larvae. Professors Godwin and Holden are working jointly to oversee bacterial HTS assay development. Project List for Theme 2: HTS-1 Use of the Multi-Parametric HTS to Study in the Phototoxicity of Nano-TiO2 and Surface

Reactivity of Ag-nanoplates in Mammalian and Fish Cell Lines – Nel/Xia HTS-2 Development of High Content Screening of Nanoparticle Toxicity Using Zebrafish Models –

Nel/Lin HTS-3 HTS to Determine the Mechanistic Toxicology of Engineered Nanomaterials in Bacteria -

Godwin/Holden HTS-4 Property-activity Analysis of Silica Nanoparticles, Including the Relationship of Surface Chemistry

to Toxicological Potential – Nel/Xia HTS-5 Assessment of the Role of Metal Oxide Energy Structure on the Biological Effects and Potential

Toxicity of this Class of ENMs – Nel/Xia HTS-6 Linking the Physicochemical Characteristics of a Library Multi-wall Carbon Nanotubes (CNTs) to

Toxicological Outcomes in Vitro and in Vivo – Xia/Nel HTS-7 Linking the Physicochemical Properties of a Library of Single-walled Carbon Nanotubes

(SWCNTs) to Toxicological Outcomes in Vitro and in Vivo – Nel/Xia HTS-8 Study of the Role of Long Aspect Ratio Mesoporous Silica Nanoparticles (MSNPs) on Cellular

Uptake and Bio-availability – Nel/Meng HTS-9 Study of the Biological Effects of Long Aspect Ratio CeO2 on Inflammasome Activation and the

Generation of Cytotoxicity – Xia/Nel HTS-10 Reporter Gene Cell-based Assays for High Throughput Screening to Determine Sub-lethal

Toxicity of Nanomaterials – Bradley/Damoiseaux HTS-11 Toxicological Assessment of Cationic Nanoparticles with Different Surface Charge Densities in

Differentiated and Undifferentiated Bronchial Epithelial Cells – Nel Major Accomplishments since February 2011: In previous study reports we have delineated the development of a high content multi-parametric assay to assess cytotoxicity, oxidative stress and organelle injury in response to a diverse number of ENMs that are capable of stimulating ROS generation, toxic metal ion release, and lysosomal and mitochondrial injury. Over the last year, we have demonstrated how this assay could be advanced to the level of HTS for the hazard ranking of large batches of nanomaterials, including metals, metal oxides and quantum dots (QD) as outlined in HTS-1 and HTS-5. Assisted by the feature analysis tools developed in Theme 6 we demonstrated how to develop hazard ranking of an integrated series of sub-lethal (superoxide and H2O2 generation, intracellular calcium flux, mitochondrial membrane perturbation) and lethal (cytotoxicity leading to increased surface membrane permeability) cellular responses (1). We have supplemented this study with conventional screening of ENM effects on zebrafish embryo hatching and morphological


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abnormalities to compare the cellular to the organismal response (1). While nano-ZnO, Pt and quantum dots (QD) were equally hazardous in embryos as in the mammalian cells, nano-Ag was a clear outlier that induced severe toxicity and morphological abnormalities in zebrafish embryos but showing little effect on mammalian cells (1). This result prompted an additional toxicological study in which we looked for species differences by comparing the toxicity of nano-Ag in a mammalian epithelial cell line with the toxicity in a rainbow trout gill epithelial cell line, RT-W1 (HTS-1 and 2). We observed that nano-Ag was indeed more toxic to fish cells than to mammalian cells, and we are currently exploring the basis for this difference, including the role of silver ions and particle surface reactivity. Our multi-parametric HTS assay also allowed us to dissect toxicity differences between different QD formulations: QDs stabilized in toluene were most toxic, while dissolvable QDs (core-only structure) showed intermediate effects and dissolution-resistant QDs (core-shell structure) the least toxicity (1). An important extension of the above studies was the development of self-organizing maps (SOM) by Theme 6, which allows the comparison of similarities between different ENM types as well as response differences between different cell types subjected to HTS (2). In one study (HTS-10) comparing metal and metal oxides, Theme 6 showed the emergence of two clusters, namely: (i) a cluster reflecting sub-lethal oxidative stress and pro-inflammatory responses to Al2O3, Au, Ag, SiO2 nanoparticles, and (ii) a second cluster reflecting lethal genotoxic responses to ZnO and Pt nanoparticles (2). In addition to quantifying similarity measures, the SOM approach also aided the development of predictive quantitative structure-activity relationships (QSARs). In collaboration with Theme 6, we developed a classification-based nano structure-activity relationship for a set of 9 metal oxide nanoparticles that lead to cell death and increased surface membrane permeability in transformed bronchial epithelial cells (9). The more detailed review of this study by Theme 6 describes beginning with a set of 14 fundamental particle physicochemical parameters, from among which four descriptors emerged that could predict cytotoxicity: namely, atomization energy, period of the metal, nanoparticle primary size and nanoparticle volume (9). The nano-QSAR prediction for the role of atomization energy was further enhanced by a parallel development in Theme 1 (Project ENM-1), namely the proposal that metal oxide bandgap energy may play an important role in promoting oxidative stress injury through ROS generation. The abstract and project description of HTS-5 describes the major conceptual advance that we made by projecting the bandgap energy of the materials versus the cellular biological redox potential. The biological redox potential is determined by several cellular redox couples that determine electron transfer to and from the nanoparticle or its remnants. The major hypothesis is that the overlap of valence band energy levels with the biological redox potential predicts which materials are capable of engaging in electron transfers between the nanoparticles and one or more intracellular redox couples. The progress outlined in HTS-5 indicates that for an initial group of 9 different metal oxides, cellular toxicity ranking by the multi-parametric HTS assay could correctly predict which materials are capable of generating oxidative stress and inflammation in intact animals. ZnO, which does not exhibit any bandgap overlap, is an outlier in this prediction and will be discussed below. Similar cellular predictions are now being tested for 24 metal oxide nanoparticles using multi-parameter HTS analysis and our preliminary results show remarkable predictive accuracy, with the exception of predictions for ZnO, TiO2, and magnetite. In Project HTS-2 we used zebrafish embryos and larvae to perform toxicological screening of ZnO, CuO, NiO and Co3O4. The zebrafish is an organism that is increasingly being used for toxicological screening of ENMs because it is a fish that also exhibits a relatively high degree of homology with mammalian biology (4). While the zebrafish embryo studies failed to demonstrate a relationship between the examined nanomaterial properties and oxidative stress, nanoparticle solubility and shedding of transition metals


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showed good correlation to hatching interference in the embryos (4). A key finding was that the more soluble materials (ZnO, CuO, NiO) disrupted embryo hatching due to shedding of metal ions that could interfere in the activity of the hatching enzyme, ZHE1 (4). This novel environmental injury paradigm has evolved from Theme 2’s ecotoxicity studies. Different from the embryos, zebrafish larvae showed that the same metal oxides could induce expression of hsp70-GFP (heat shock protein70 gene linked to a GFP reporter) in transgenic animals, indicating that cellular stress is a relevant screening platform for comparative toxicity studies in this organism. The use of transgene expression in these animals is being extended to look at the induction of oxidative stress responses, including the activation of the antioxidant response element (4). A major advance was the development and implementation of high content brightfield and fluorescence-based imaging platforms for retroactive analysis of hatching interference, morphological abnormalities and transgene expression (4). By establishing an automated pick-and-plate robotic system that augments the number of embryos that can be assessed in one experiment, it is now possible to perform HTS in zebrafish at a level commensurate with cellular HTS. We are currently screening 24 metal nanoparticles. To facilitate zebrafish HTS, Theme 6 has developed a high throughput image recognition model that will be implemented in future studies to accelerate phenotype recognition. We have previously demonstrated that the toxicity of nano-ZnO is directly related to its dissolution characteristics. The high solubility of ZnO could possibly explain why the bandgap hypothesis does not work for this nanomaterial. In the previous progress report, we have shown how the change in the ZnO matrix by Fe-doping leads to a slower rate of dissolution and decreased cellular toxicity. In the current study period, HTS-1 has extended this observation by comparing the impact of doping in cells with both the hatching outcome in zebrafish embryos and the generation of oxidative stress and inflammation in the rodent lung (3). While synthesis and chemical characterization details can be found in Dr. Lutz Mädler’s progress report in ENM-4 (Theme 1), project HTS-1 describes how this safe-by-design feature could indeed be shown to exert lesser toxic effects in cells, zebrafish embryos and rodents (5). Although TiO2 band gap energy overlaps with the cellular biological redox potential, TiO2 nanoparticles

did not generate toxicity in the cellular HTS assay. However, it is important to consider that HTS is carried out under dark conditions and does not take into consideration the photoactivation requirements for electron-hole generation by this material. While to date it has been difficult to study photoactivation as a biological toxicity mechanism due to the inherent toxicity of UV radiation, Project MFW-1 has demonstrated that this is an important consideration because bright sunlight conditions can induce TiO2 toxicity in phytoplankton. Thus, to more rationally dissect TiO2 photoactivation from a biological perspective in Theme 2, Prof. Lutz Mädler and Dr. Suman Pokhrel have synthesized Fe-doped TiO2 nanoparticles that can be used to excite electron hole generation and ROS production under near-visible light conditions (see ENM-4). The availability of this library of materials has enabled Project HTS-1 to use the multi-parameter HTS assay to demonstrate that incremental iron doping leads to progressively more ROS generation under near-visible light conditions (6). This leads to increased cytotoxicity in cells endocytosing the particles due to an oxidative stress response that can be reversed by the antioxidant N-acetyl cysteine (5). This new library is being utilized by numerous investigators to evaluate TiO2 nanoparticle toxicity in bacteria and marine life forms. Considerable progress has been made in studying the toxicity of silica nanomaterials that were synthesized and characterized in Theme 1. Fumed silica was chosen because of its widespread use and high volume production as a desiccant in consumer products and the associated possibility of this nanomaterial reaching the environment. Introduction of fumed silica has allowed us to perform a comparative study of the intrinsic toxicity of different types of silica nanomaterials (HTS-4), explore the


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effect of nanoparticle shape in determining bioavailability and cellular uptake (HTS-8), and achieve better understanding of cationic toxicity by silica nanoparticles that are functionalized by polyethylenimine (PEI) (HTS-11). The progress report for HTS-4 describes important preliminary progress in the use of single-parameter and multi-parameter screening assays to compare quartz, amorphous (Stober), fumed, mesoporous and silicalite nanoparticles. An interesting finding was that fumed silica is the most toxic, and that surface display of silanols that are closely spaced in relation to the underlying siloxane ring structure may be the dynamic material principle that determines toxicity. Ongoing work is delineating the role of surface reconstruction of strained siloxane rings as an explanation of how the hydration status of this high temperature Si material may impact toxicity through reactive silanol display. Not only did we demonstrate that mesoporous silica nanoparticles (MSNP) have no intrinsic toxicity, but we were able to make use of this inert silica material to develop rod-shaped particles that can be compared to spheres for their bio uptake behavior (7). Project HTS-8 demonstrates that rod-shaped MSNP are preferentially taken up by macropinocytosis, which reflects the ability of these long-aspect ratio materials (LARM) to stimulate a cellular signaling pathway (the small GTPase protein, Rac1) that communicates with the cellular cytoskeleton. This leads to actin polymerization, which forms the basis for the formation of surface membrane filopodia that preferentially take up rod-shaped particles by macropinocytosis. Macropinocytosis plays an important role in particle and food uptake by environmental prokaryotes as well as eukaryotes. Finally, the use of inert MSNP has allowed us to use this material to attach a series of cationic polyethylenimine (PEI)-polymers to study the mechanism of cationic toxicity in epithelial cell lines (8) as well as yeasts. This work has also allowed us to delineate the importance of cell surface expression of anionic membrane heparin sulfate proteoglycans in cationic particle attachment and catalysis of biological injury (8). In addition to the interesting findings with rod-shaped MSNP, we have begun to work with another class of LARM over the last year, namely single- (SWCNT) and multiwall carbon nanotubes (MWCNTs). While there are indications that these materials are definitely harmful in the rodent lung, we are still uncertain about their environmental impact. Following the development of successful dispersion protocols for SWCNTs and MWCNTs in the previous report period, we were able to perform cellular and organismal screening studies in the current progress period. Utilizing materials provided by Theme 1 (ENM-2), we were able to demonstrate in Projects HTS-3 and HTS-7 that the state of tube dispersion is a critical factor in the bioavailability and the toxicological potential of the tubes. HTS-3 has just completed a study demonstrating that MWCNTs that are better dispersed by BSA plus the phospholipid, DPPC, are more prone to be taken up into epithelial cells and macrophages, where they are better capable of inducing pro-fibrogenic cellular effects (e.g., IL-1 PDGF and TGF-1) than poorly dispersed tubes. In a recently accepted paper (12), we indicate that the ex vivo cellular responses closely agree with the generation of similar biochemical responses as well as induction of fibrosis in the intact animal tissues. Another important development has been the demonstration that dispersion of both SWCNTs and MWCNTs by the tri-block copolymer Pluronic F408 leads to steric hindrance that interferes in cellular uptake and protects against the induction of pro-fibrogenic responses. In order to perform a study of more direct environmental relevance, project HTS-7 introduced SWCNT to zebrafish embryos but could not elucidate any toxicological effects that could be related to fibrogenesis. However, the supernatants obtained from one type of SWCNT that is capable of shedding abundant Ni could be seen to interfere in zebrafish embryo hatching. In Project HTS-5, Dr. Godwin has been working with the MSSR on transitioning assays from the Holden laboratory into high-throughput mode and performing the preliminary experiments required to determine which materials and conditions should be used in the HTS experiments. To date, they have identified optimal suspension conditions (using DLS) and screened toxicity in E. coli (using a parent/wild-


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type strain) of a set of (i) 24 metal oxide nanomaterials, (ii) five Ag nanomaterials, (iii) nine TiO2

nanomaterials and (iv) four polystyrene nanomaterials. Eight out of 24 metal oxide nanomaterials exhibited toxicity for the E. coli parent strain; all Ag nanomaterials showed toxic effects although at different levels primarily depending on their primary size; the library of TiO2 nanomaterials, including Fe-doped TiO2, exhibited toxicity only after illumination under UV light; and polystyrene materials were not toxic except for smallest sized (60 nm primary size) cationic (amino charged) material (PS-NH2). Theme 6 is currently analyzing data from these studies to determine whether we can also develop predictive QSARs for the cellular screening studies. Taken together, we have made considerable progress in the study of metals, metal oxides, silica and CNTs using high throughput screening approaches in cells and in embryos. Results of the completed studies can be used for structure-activity analysis and for exploring the key ENM properties that should be considered for toxicological predictions, hazard ranking and safer-by design approaches that lessen environmental impact. The rich data sets emerging from multiple assays in different cell types and zebrafish now allow us to: (i) correlate/contrast behavior of different cells, including cells from different species; (ii) perform quantitative comparison of oxidative responses in cells with in vivo outcomes; (iii) prioritize in vivo toxicological profiling; (iv) perform property-activity analysis that reflects the relationship of specific NP properties (e.g., primary size, metal periodicity, bandgap energy, surface cationic density, surface hydroxyl density) to response parameters included in multi-parametric assays. This progress means that decision makers now have available a range of HTS based toxicity models and decision tools for hazard ranking/environmental impact analysis. Theme 2 efforts have enabled transfer of information to Theme 1 to redesign nanoparticles in such a way as to either reduce toxicity or further explore pertinent NP properties, to explore toxicological profiling, clustering and QSARs with Theme 6, to study bacterial toxicity in Theme 4, and to expand our ENM data repository. Our data collection serves as a basis for developing web-based tools that support activities such as HTS data processing and hit identification. Impacts on the Overall Goals of the Center: The development and implementation of high content and high throughput screening platforms has allowed the UC CEIN to demonstrate a new approach to speeding up the safety assessment of ENMs. While it is impossible for the Center to perform the task of investigating all be available types of primary ENMs, our collective research efforts involving ENM libraries, HTS assays and in silico decision-making tools demonstrate the feasibility of using a predictive toxicological paradigm for hazard and risk assessment of a large numbers of ENMs. As an example of what can be accomplished based upon these outcomes, we were able to perform hazard ranking of 24 metal oxide nanoparticles that can now be prioritized for further research using more costly and labor-intensive approaches. This approach has attracted national and international attention, as reflected by the high citation index of our published papers and invitations to speak at premier national and international workshops and conventions. Examples that are included in individual project progress reports in Theme 2 include participation in the Bilateral Presidential Commission for nanotechnology cooperation between the U.S. and Russia, giving a plenary award lecture at the ChinaNano 2011 Convention, and participating in a joint US/EU nano safety workshop in Washington DC. In addition, we have been invited to write reviews for high visibility journals and to participate in the series of international workshops that culminated in the NSF report, “Nanotechnology Research Directions for Societal Needs in 2020” (10-13). CEIN not only oversaw the writing of the nano EHS Chapter 4 for this report (now published by Springer), but was also extremely pleased to observe the widespread international support for high throughput screening, nano informatics, modeling and safer-by-design strategies for ENMs.


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Major Planned Activities for the Next Period: Over the next three months, we will continue to develop the theme of bandgap energies in the toxicity of metal oxide nanoparticles. Because our current predictions are not yet 100% accurate, we also need to properly develop this hypothesis by considering the dissolution characteristics and matrix energies of the material and require additional sets of ENM descriptors from the computational analysis in Theme 6. We would also like to conclude the studies regarding the role of strained siloxane rings in the surface reconstruction and toxicity of fumed silica, with publication of the results. We will continue to work on developing a HTS platform on which to model CNT toxicity by assessing pro-inflammatory responses. By the end of year 5, we aim to complete the major milestones originally envisaged for IRG5. Most of our goals have been accomplished through the development of multiple screening approaches in cells, bacteria and the zebrafish.


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Theme 3: Fate, transport, exposure and life cycle assessment Year 4 Progress - April 1, 2011 - March 31, 2012 Faculty Investigators: Arturo Keller, UCSB – Professor, Bren School of Environmental Science & Mgmt - Theme leader Ponisseril Somasundaran, Columbia University – Professor, Earth & Environmental Engineering Sharon Walker, UC Riverside – Associate Professor, Chemical Engineering Sangwon Suh – Assistant Professor, Bren School of Environmental Science & Mgmt Goals of Theme 3: The overarching goals of Theme 3 is to generate the experimental data to predict the concentration of nanoparticles (NPs) at which organisms will be exposed to in different environmental compartments, and the NP physicochemical processes that may have environmental implications, such as NP reactivity and its effect on environmental conditions. In coordination with other Themes, we have focused our studies on the metal, metal oxide, silica and carbon nanotube (CNT) nanomaterials that have been acquired, synthesized and characterized by Theme 1. To achieve our primary goals, we have focused our efforts during the first three years on experimental studies to correlate the key parameters that control NP fate and transport processes with NP properties modulated by natural conditions. These include parameters such as aggregation, dissolution, attachment to mineral and biological surfaces, persistence, reactivity, bioaccumulation and bioprocessing, particularly under environmentally relevant conditions. As we move forward into year four, we will initiate life cycle assessment of a limited number of NPs used for different commercial applications. Our first life cycle assessments will focus on CNTs. During the next two years, we also plan to develop methods for measuring environmental concentrations of NPs, which will allow us to use data previously collected by our Theme (3) and from other literature to evaluate the predictions of the Environmental Multimedia Distribution model developed by Theme 6. Organization and Integration of Theme 3 Projects: The first five projects in Theme 3 seek to characterize the abiotic interactions between NPs and the environment. During the first two years, these projects focused on the three metal oxide NPs that were also being studied by Theme 1 (TiO2, ZnO and CeO2). More recently, several projects have begun to generate results on metallic NPs (Ag, Pd, and Pt), metal oxides (CuO) and CNTs. These NPs are either currently being studied or are under consideration for ecotoxicity studies conducted by Themes 2, 4 and 5. Project FT-1 studies the factors that control NP aggregation and dissolution, which are two of the key processes in NP fate and transport. FT-1 studies seek to relate specific NP and surface modifier (e.g., coating or cap) characteristics such as surface charge and surface energy, which are strongly influenced by water chemistry (i.e. electrolytes, natural organic matter and other organic molecules), to rates of aggregation and dissolution in natural waters. Information about relationships between NP characteristics and aggregation and dissolution rates is key for determining the time frame during which NPs remain in particulate form in the water column. Project FT-2 focuses on the nano- and microscale processes that result in NP attachment to mineral surfaces, such as clays, silts and sands under the natural range of water chemistries relevant to the ecotoxicological studies being carried out by Themes 4 and 5. For example, if NPs attach strongly to mineral surfaces, they could become less bioavailable for many organisms, reducing the likelihood of exposure. However, if the attachment forces are weak, the NPs may be released and become bioavailable at a later stage. Project FT-3 evaluates NP transport mechanisms in groundwater under conditions relevant to Theme 4. Proven techniques are being applied to identify the parameters needed to predict NP filtration and straining in porous media. Better understanding of relevant parameters enables us to predict how far the NPs will travel in groundwater and whether they will be bioavailable to plants, microorganisms and other ecological receptors. Project


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FT-4 is developing new methods of NP characterization, such as NP wettability and surface energy, which can serve to generate robust correlations between NP properties and fate and transport processes. Project FT-5 studies the reactivity (in particular, photoactivity) of NPs in different media. Several NPs are photoactive, generating highly reactive free radicals and other oxidants that can react with natural organic matter (NOM), cell membranes, and other important biomolecules such as proteins, peptides, enzymes. NP photoactivity is a function of environmental conditions as well as NPs properties. Project FT-6 began in July 2010 in response to questions regarding NP uptake, metabolization and excretion. This project focuses on physicochemical aspects and the relationships between NP characteristics and the nature of these nano-bio interactions. FT-6 studies are done in close coordination with Theme 5, particularly the marine experiments which have suitable experimental platforms for evaluating NP bio-uptake (e.g. filter feeders). Project F-7 is a new seed project that was launched in October 2011 and focuses on determining the life cycle impacts of CNTs, considering their incorporation into data transmission cables and aerospace panels. Project List for Theme 3: FT-1 Role of material properties and environmental conditions on nanoparticle aggregation &

dissolution - Keller FT-2 Attachment of nanoparticles to mineral surfaces under different aqueous solution chemistries -

Keller FT-3 Quantitative determination of fate and transport of nanoparticles in porous media - Walker FT-4 Effect of wettability on the transport and fate of metal oxide nanoparticles - Somasundaran FT-5 Photoactivity of nanomaterials in natural waters – Keller FT-6 Physicochemical nano-bio interactions at different scales that influence fate & transport of

nanoparticles - Keller FT-7 Life-cycle assessment of carbon nanotubes (new seed project) – Keller, Suh and Cohen Major Accomplishments since February 2011: The results in this period build upon the robust base of findings generated over the past three years. Early studies to determine how a select number of metal oxide NPs (TiO2, ZnO and CeO2) behave when released into different environmental media such as seawater, freshwater, stormwater and other surface waters demonstrated that the water constituents play a very important role in overall fate and transport. Many plants and microorganisms exude significant amounts of organic molecules that can coat NP surfaces, presumably stabilizing them. We found that natural organic matter (NOM) in various forms plays a key role in stabilizing metal oxide NPs, and that only in high ionic strength conditions, such as those found in estuarine and marine environments and many groundwaters, will the suspensions become highly unstable, resulting in rapid deposition of nanoparticles. Recent studies in project FT-1 have shown that NOM, in the form of humic acid or alginate, can stabilize other metal oxide NPs such as CuO, metallic NPs such as Pd and Pt, as well as carbonaceous NPs like CNTs (Ref. 5, 8, 11). Studies in Project FT-2 showed that NOM also reduces the attachment of metal oxide NPs to mineral surfaces, even if these NPs do eventually aggregate and deposit on the sediments (Ref. 5, 11). Recent work by Project FT-3 confirmed the role of NOM in facilitating transport of metal oxide NPs (specifically TiO2) in groundwater, except under high ionic strength conditions (i.e. high hardness), where the TiO2 aggregates are more easily filtered and strained by the porous medium (Ref. 3). Because of their increased stability and mobility in the presence of NOM, which is ubiquitous in its various forms in environmental waters, these particles will be more bioavailable. These results are important to consider in the design of exposure studies in Themes 4 and 5, since even very low concentrations (< 1 mg/L) of NOM can result in stabilization and reduced attachment. NOM also reduces the photoactivity of metal oxide NPs, in part because it competes effectively for the photons and in part because it blocks the NP surface. However, additional studies are


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underway in Project FT-5 that seek to identify circumstances under which a metal oxide NP such as TiO2

can result in accelerated photooxidation of NOM, releasing nutrients and mineralizing them. This is important because the release of nutrients may result in faster growth of aquatic plants. Project FT-3 has begun a series of studies to evaluate the combined role of NOM and bacteria on TiO2 NP fate and transport in groundwater. E. coli HU1 fluorescently tagged with mCherry plasmid was used as a model organism and Suwanee River Humic Acid as the organic matter. Results showed a significant dependence on solution chemistry and ion valence (Ref. 9). A range of solution conditions (pH 5 and 7, and 10 mM KCl and CaCl2) were tested. Both SRHA and E. coli significantly reduced TiO2 NP 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. Because bacteria and NOM are present in all natural porous media (i.e. soils and sediments), these results are very significant because they indicate that these metal oxide NPs have the potential to travel further in groundwater and may result in higher exposure than predicted by simpler studies that do not consider these conditions. In collaboration with researchers in Theme 5, we have measured dissolution rates for ZnO, CuO and Ag NPs (Ref. 4, 11). Theme 2 has shown that these particles are toxic, in part due to the shedding of ions at concentrations that locally are too high for cells to tolerate. However, the rate of dissolution varies significantly for the various NPs, based on their surface energy. Project FT-4 has developed a novel instrument for measuring the surface energy of individual NPs, which can be correlated with their tendency to dissolve or aggregate (Ref. 2, 6). In addition, Project FT-1 has shown that the dissolution time of ZnO NPs is on the order of tens of hours, which is highly relevant for acute toxicity studies because it means that the organisms are most likely exposed to Zn2+ rather than the ZnO NPs. Preliminary studies by Project FT-1 with CuO and Ag NPs indicate that their dissolution is much slower, on the order of weeks. Dissolution and the bioavailability of the released ions is also a function of solution chemistry; under certain conditions, ions form complexes with and are sequestered by other water constituents as they are being released from the NP. Acidic conditions result in faster dissolution rates for ZnO, CuO and Ag NPs. The combinatorial nanoparticle libraries developed by Theme 1 provide us with information about property variations between nanoparticles of the same the same material composition, and this information has allowed us to test a number of hypotheses regarding NP aggregation, transport and photoactivity. Early work in Project FT-1 had shown that morphology played an important role in the aggregation of ZnO NPs. While spherical ZnO NPs behaved very close to theoretical predictions regarding the conditions that result in stability or high aggregation rates, plate-like ZnO NPs essentially aggregated under all conditions as a result of the availability of multiple contact points. Sixteen different TiO2 nanoparticles that vary in size, shape (spheres, wires/rods, nanodots), and crystalline structure (anatase, rutile and combinations) are currently under study. Project FT-1 has shown that TiO2 nanowires aggregate much more readily than TiO2 nanospheres and nanodots under all conditions (pub. in progress). This is likely due to the difference in numbers of contact points, which can be higher for nanowires than for other morphologies. Among TiO2 rods, increasing size leads to higher aggregation, even at lower ionic strength. Utilizing porous media transport studies, Project FT-3 found that different sized (6, 13 and 23 nm) TiO2 NPs were filtered out at different rates under the same conditions, even though their aggregate sizes and charges are very similar. This suggests that there are differences in the morphology of the fractal aggregates. Project FT-5 has evaluated OH• radical production in the presence and absence of natural organic matter (NOM) for TiO2 wires, nanodots and spheres. Preliminary results indicate that, for an equivalent dose, OH• radical production is greatest for the spheres and wires, and


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that nanodots have limited photoactivity. Since we have shown that photoactivity of TiO2 P25 spheres affects phytoplankton (Ref. 10), levels of photoactivity can be important measures to inform risk assessment. In summary, NP characteristics affect aggregate size, transport and photoactivity. These studies demonstrate the value of the NP libraries, and will help to guide the design of expansions of the Theme 1 libraries for the purpose of testing specific hypothesis. These results also highlight the need to consider additional NP characteristics in the fate and transport modeling under development in Theme 6. To control particle size, many NPs are coated with organic molecules during synthesis. During the past year, Projects FT-1 and FT-2 studied Ag NPs with two different coatings (citrate-Ag and PVP-Ag) that were characterized by Theme 1. Because these same materials are also being introduced for mesocosm studies in Theme 5, it is important to determine ahead of time how these coated NPs are expected to behave. Citrate is a relatively small molecule, while PVP is a large polymer. Although the Ag primary particle size is 40 nm, the hydrodynamic diameter of the PVP-Ag particles is approximately 60-80 nm and slightly dependent on pH and ionic strength, with smaller diameters at higher pH and lower ionic strength (Ref. 11). In contrast, the hydrodynamic diameter of the PVP-Ag particles is approximately 80-120 nm and much more dependent on pH, with a sharp decrease in aggregate size as pH increased from 4 to 10. In freshwater and seawater we observed additional aggregation, with mean hydrodynamic diameters of 150 and 250 nm, respectively. The very high ionic strength in seawater destabilizes the Ag NPs to some extent by neutralizing the charge of the organic coating. Thus, the particles are relatively stable in freshwater, but exhibit faster rates of sedimentation in seawater. NP attachment to silica surfaces, such as those found in sand, was very low, in part due to the coating and in part due to the presence of NOM. The results indicate that the organic coatings on the Ag particles and the presence of NOM in natural waters increase particle mobility by reducing aggregation and attachment to mineral surfaces. In seawater, deposition increases but the particles do not attach strongly to the mineral surfaces and may remain suspended. Thus, if coated Ag NPs are used in commercial products that shed Ag NPs, particles that enter freshwater or seawater systems are expected to be readily bioavailable. We are working with Theme 5 to incorporate these findings into their analysis of toxicity studies. Considering the large number of suspended particles in natural waters, there is a high likelihood that engineered NPs will interact with these naturally suspended particles. Project FT-1 has begun the study of NP-clay interactions, beginning with TiO2 NPs. Preliminary results indicate that the interaction between P25 TiO2 NPs and montmorillonite clay is strongly influenced by pH. At pH 4, the clay/P25 mixture was less stable than either P25 or clay alone. The enhanced aggregation between P25 and montmorillonite clay is due to electrostatic attraction between the positively charged P25 and the permanent negative charge on the basal planes of montmorillonite clays. We hypothesize that montmorillonite’s role in the stability of these NPs will vary with pH and ionic strength due to the complex charge behavior of montmorillonite’s face and edge sites. Project FT-4 evaluated the interactions between P25 TiO2 and kaolinite clay, and found that sedimentation of the NPs depends on the total surface energy loading density of the system (pub. in progress). In practical terms, kaolinite appears to reduce the rate of aggregation and sedimentation of the TiO2 NPs, making them more bioavailable to organisms in the water column. Thus, the presence of different clays can have very different effects on the overall mobility and bioavailability of these NPs. Additional studies are planned to elucidate the clay properties that control these interactions. The new library of carbon nanotubes that was made available by Theme 1 during the past year includes CNTs that vary in purity (with metal impurities up to 30% of total weight) and method of synthesis. Stability of the various CNTs in rainwater, groundwater, stormwater, deionized (DI) water, and DI with


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alginate, NOM, HA and bovine serum albumin was measured by Project FT-5 over 2 months (Ref. 15). With the exception of DI and alginate systems, where the CNTs did not readily disperse, all five CNTs were initially stable for up to a few weeks under the rest of the conditions. No significant differences in stability were found between the different CNTs. Waters rich in NOM (e.g., groundwater and surface water) generated much more stable suspensions for all CNTs, with > 90% of the CNTs suspended after a few weeks. Waters with high ionic strengths and low NOM concentrations (seawater) generated less stable suspensions and higher settling. Using electroparamagnetic spectroscopy, Project FT-5 demonstrated that CNTs produce superoxide in natural waters, but that there was no production of singlet oxygen. These results have led to collaboration with Theme 2 to evaluate the phototoxicity of CNTs in vivo using a fish cell line and zebrafish (Ref. 15). In preliminary experiments, the CNTs were found to be toxic in both light and dark conditions, with cell death slightly increased under light conditions. An assay for intracellular superoxide generation confirmed the increased generation of superoxide in the presence of light and CNTs. While dispersing the CNTs under different conditions, researchers in Project FT-5 discovered a new method for converting CNTs into rings known as nanotori (pub. in progress). Nanotori retain many of the unique physicochemical properties of CNTs, but also acquire new properties based on the ring formation. The stability in natural water, photoactivity and phototoxicity of the nanotori were evaluated and compared to the original CNTs. While nanotori form stable dispersions in deionized water, such stable dispersion was not observed for the original CNTs. Nanotori are also stable in all natural waters tested, with more than 99% of the initial mass remaining in suspension for months. The nanotori produced superoxide anion both in natural waters and in vivo. In collaboration with Theme 2, rainbow trout cells were exposed to 25 µg mL-1 nanotori and light from 350 to 1100 nm, which resulted in decreased viability in 30% of the cells after 24 hours. Since the nanotori induce levels of oxidative stress that are comparable to the stress related to similar concentrations of TiO2, a potent photocatalyst, it is likely that the rings are quite photoactive. The nanotori were not toxic under dark conditions, even though the original CNTs were toxic under all conditions. These nanotori have been made available to researchers in Themes 2 and 5 for toxicity screening. In general, fate and transport focuses on abiotic processes, such as aggregation, advective and diffusive/dispersive transport, deposition, attachment, and dissolution. However, biotic processes such as uptake, transformation/metabolization, bioaccumulation in different tissues, and excretion are important processes that influence nanoparticle fate and transport. Some key questions that focus on the role of these biotic processes are: (i) what are the biomolecules with which the NPs interact at the point of uptake, and how do they modify the physicochemical nature of the NP surface?; (ii) how are the NPs and their transformation products partitioned to different tissues within an organism?; and (iii) in what form are the NPs or their transformation products excreted? To address these questions, Project FT-6 has teamed up with researchers in Theme 5 to study the uptake, bioprocessing and excretion of CeO2 and ZnO NPs by mussels. To more closely examine uptake processes, CeO2, ZnO and Ag NPs were exposed to biomolecules present in mussel adhesive tissues, such as alanine, cysteine, glucosamine, lysine, and L-dihydroxyphenyalanine (L-DOPA) to determine their interactions. We observed significant interactions between 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. Ag nanoparticles formed similar complexes when exposed to cysteine due to charge-transfer interactions with the cysteine sulfide group. Interactions between other biomolecules studied and these NPs were rather limited, indicating that there are specific affinities between certain NPs and certain biomolecules. These findings are significant because they signal potentially specific uptake pathways for NPs at the small scale. On the other hand, bioprocessing of the two NPs at a larger scale was quite different. ZnO was


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partitioned to mussel tissue, pseudofeces, and the water column; in contrast, CeO2 was filtered almost completely from the water and was mostly excreted as NPs into pseudofeces, with much lower accumulation in the soft tissue (Ref. 13). The nature of bioprocessing at the organismal level plays an important role in the fate and transport of these NPs, because particles that accumulate in tissues become less bioavailable than those excreted in pseudofeces, which are likely to be distributed by organisms that feed on these materials. We are extending this work to determine whether or not these more bioavailable NPs accumulate in marine phytoplankton that is then taken up by the mussels. Results of these studies will allow researchers in Theme 5 to better quantify the actual doses that indicator species are exposed to in their studies. In summary, we have made important advances in quantifying key fate and transport processes of metal, metal oxide and CNT nanoparticles. In particular, we have identified and characterized aggregation and dissolution rates, photoactivity, attachment to mineral and biological surfaces, and bioprocessing. We have demonstrated the importance of considering NOM and NP coatings in determining the mobility, bioavailability and photoactivity of NPs; in fact, evidence is becoming stronger that molecules adsorbed on NP surfaces determine the interactions of the particles to a larger degree than do the NP surface characteristics themselves. We have found that shape, size and crystal structure are also important to consider for fate and transport predictions, and that bio-uptake and bioprocessing can be important determinants of the bioavailability of the NPs and their transformation products. This information will improve predictions regarding both the likelihood of nanoparticle exposure and the concentrations at which organisms may be exposed. Impacts on the Overall Goals of the Center: Theme 3 is providing the necessary data and results to: (1) inform experimental design (e.g. benthic vs. pelagic organisms, time frame for dissolution, stabilization techniques, photoactivity, bioavailability, and transport through mesocosm soils) and protocols (e.g. dispersion of NPs, monitoring of NPs) of Themes 4 and 5; (2) inform design and parameterization of the Multimedia Environmental Modeling tool under development by Theme 6, by providing information on the primary fate and transport processes, conditions and parameter values for the model; (3) guide in the selection of NPs to be acquired or synthesized by Theme 1 to test specific fate and transport hypotheses and (4) provide information for the conceptual models used by Theme 7 in designing their perception surveys. On a broader scale, this information was disseminated during this review period via presentations at international conferences (several of them invited talks) and published manuscripts. The Theme leader is also a participant in the Industrial Consortium on Environmental Monitoring of Nanomaterials (ICEMN), and Theme members presented results during this review period to the California Dept. of Toxic Substances as part of the ICEMN, to guide regulators in framing questions about dealing with NPs in the environment. Major Planned Activities for the Next Period: The studies focused on 16 different TiO2 NPs will be mostly completed in the next period. Because the work completed to date has shed significant light on the role of NP characteristics, we will perform additional studies using a wider range of types of TiO2 NPs from the Theme 1 library to validate emerging trends with regards to aggregation, mobility and photoactivity. Transport experiments with CNTs will be conducted to determine the influence of environmental conditions and method of dispersion on fate and transport of these materials, and we will also be completing studies on the photoactivity of CNTs and nanotori. Because our preliminary results point to very different NP behaviors in the presence of different types of clay, additional studies on the interactions between NPs and clays will be conducted. We have planned to collaborate with Theme 4 to determine the mobility and bioavailability of metal oxide NPs in planned mesocosm studies that will begin during the next period. In response to the observation that mussels are capable of direct


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uptake and bioprocessing of metal oxide NPs from the water column, we are working with Theme 5 on development of an indirect bioprocessing model in which nanoparticles are first adsorbed on phytoplankton surfaces, and these contaminated algae are then taken up by mussels. We anticipate that development of such a model will help us to determine how important these transfer mechanisms are and how they influence mussel bioprocessing of NPs.


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Theme 4: Terrestrial ecosystems impact and hazard assessment Year 4 Progress - April 1, 2011 - March 31, 2012 Faculty Investigators: Patricia Holden, UC Santa Barbara – Professor, Environmental Microbiology – Theme Leader Jorge Gardea-Torresdey, University of Texas, El Paso – Professor, Environmental Chemistry Roger Nisbet, UC Santa Barbara – Professor, Theoretical Ecology Joshua Schimel, UC Santa Barbara – Professor, Ecosystem Ecology Updated Goals of Theme: Theme 4 includes former IRG2 microbial and IRG3 plant and soil projects. The overarching goal of Theme 4 is to discover and understand the effects of engineered nanomaterials (ENMs) on terrestrial environments, with an aim to predict and avoid such effects. The societal drivers for studying effects of ENMs in terrestrial environments are: maintenance of ecosystem services (e.g. nutrient cycling, pollutant biodegradation, and plant fertility), agricultural crop production and food quality, and water quality (i.e. groundwater, used as a drinking water source). ENMs are expected to enter terrestrial environments in the U.S. mainly via land application of wastewater treatment plant (WWTP) biosolids; ENMs may also deposit to lands from the atmosphere (e.g. automobile exhaust carrying nano-CeO2 from catalytic converters) and from direct water-to-land processes (e.g. spills near manufacturing sites, painted facades, or personal care products released into recreational water bodies). Theme 4 researches the feedbacks between ENMs and terrestrial environments—and thus seeks to describe not only effects on organisms, but also bioprocessing of ENMs in soils and plants. In this regard Theme 4 results can inform prediction of ENM fates, which is the focus of Theme 3. Otherwise, Theme 4 research is comparatively source-blind, and seeks to understand what effects ENMs can have in terrestrial systems once ENMs have been transported into and have accumulated in soils. Terrestrial environments are comprised of soil minerals, organic matter, water, dissolved nutrients, microbiota, macrofauna, and plants. Theme 4 research appropriately addresses all relevant biological (macromolecular to whole organism) and ecological (individual, to population, to community, to ecosystem) scales, and is attentive to the physical, biological, and chemical processes influencing ENM impacts. To date, across published nanotoxicology, there have been relatively few studies focusing on terrestrial environments. Inaugural studies suggested that ENMs, if applied at realistic concentrations to soils, would not be sufficiently bioavailable to affect microbes or plants. However, work from Theme 4 is revealing that: (i) a broad range of ENMs are toxic to bacteria in the dark (as in soils); (ii) ENMs associate with, and damage, bacterial membranes; (iii) bacterial membrane damage inhibits bacterial growth with extents that can be predicted by mathematical models incorporating ROS amounts; (iv) ENMs attached to, or within, bacteria are trophically-transferred to protozoan predators which also bioprocess ENMs; (v) bacterial consortia in soils are altered by ENMs including losses of functionally-narrow taxa in N and C cycling; (vi) soil water holding characteristics are altered by ENMs with implications to drought tolerance of ENM-contaminated soils; (vii) plants bioaccumulate and process specific ENMs with variations by plant and ENM type; (viii) ENMs can genetically alter hydroponic plants; and (ix) ENMs negatively impact important crop plants and soil microbial processes relevant to soil fertility. Overall, the goals of Theme 4 are consistent with the Center goals of delivering understanding that is mechanistic and transferable. Theme 4 goals and findings are highly responsive to prior critiques that holistic, as well as reductionist, inquiries should be made to improve the relevance of our research. Organization and Integration of Theme 4 Projects: We have restructured to logically cluster projects from across the prior IRGs 2 and 3, and this restructuring was facilitated by the fact that research within terrestrially-themed projects was already highly synergistic. TER-1, previously within IRG3, is jointly led by Holden and Schimel, and recently includes Nisbet, Gardea-Torresdey, and Walker as the project has


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expanded from studying unplanted soils to both studying planted systems and to modeling plant populations. TER-1 and TER-4 (Gardea-Torresdey) are highly complementary, with the former originally emphasizing soil microbial interactions with ENMs in the absence of plants, and the latter emphasizing hydroponic (no soil) plant interactions. Now, both TER-1 and TER-4 include planted microcosm subprojects in addition to prior and ongoing microbial and plant research. Sensitive bacterial taxa identified in TER-1 are then used as test subjects for TER-3 (Holden), which concerns mechanistic effects of ENMs on bacteria. Effects screening approaches are recruited and tested in TER-3 and then applied to a wide variety of ENMs using HTS by Godwin in Theme 2. Band gap-modified metal oxides synthesized by Mädler in Theme 1 are among the ENMs studied in TER-3, and use of these particles allows us to test hypotheses regarding electron transfer between ENMs and energetic bacterial membranes. The growth-inhibitory actions of ENMs on bacteria are mechanistically modeled by Nisbet (TER-5), with TER-5 modeling reinforcing the oxidative stress paradigm forwarded by Nel in Theme 2. Model developments in TER-5 have fueled additional research questions and results in TER-2, and results of studies conducted by this project provide feedback to improve the model. Because TER-3 also examines bacterial bioaccumulation and processing of ENMs, it is logical to have TER-2 (Holden) within Theme 4. Additionally, trophic transfer between bacteria and protozoa (TER-3, Holden) indicates how ENMs can affect terrestrial food webs, which in turn informs TER-1 and TER-4. Project List for Theme 4: TER-1 Nanotoxicology in terrestrial microcosms - Holden/Schimel TER-2 Trophic transfer, bioaccumulation and biomagnification of engineered nanomaterials in basal

levels of environmental food webs - Holden TER-3 Engineered nanoparticle biosorption, toxicity, and toxicity mechanisms in planktonic and biofilm

bacteria - Holden TER-4 Toxicity and uptake of nanoparticles by terrestrial plant species - Gardea-Torresdey TER-5 DEB modeling of toxic effects of CdSe quantum dots - Nisbet Major Accomplishments since February 2011: In prior reporting periods we established the existence of a dose-response relationship for bacteria growing in the presence of ENMs in the dark, and that the relationship magnitude varied with bacterial strain, ENM, and media (Vukanti et al., in preparation)., Because of bacterial membrane exposure to ENMs in planktonic culture, and also because of our prior work showing uptake or at least high affinity (sorption) of ENMs with bacteria (Horst et al., 2010. AEM), we had hypothesized that bacterial membrane damage by oxygen free radicals (ROS) partly explains population growth reduction, but that there were also sublethal ENM effects on membranes, including cellular oxidation and membrane depolarization. In support of these complementary hypotheses, we had recruited 5 assays (ROS, superoxide, membrane potential, dehydrogenase activity, and membrane integrity), and optimized their use as a system (Horst et al., in preparation). We also demonstrated their simultaneous use within Theme 2 using HTS approaches at the UCLA MSSR Facility, with the aim that the assays are applied using environmentally-relevant bacteria, since HTS is generally effective using laboratory strains (5). Using low-throughput (96-well automated plate reader) approaches during this period, Project TER-3 discovered that the MeOs (TiO2, CeO2, and ZnO) provided by the Nanoparticle Acquisition and Characterization Core inhibit E. coli population growth by ROS-mediated membrane damage. We also observed membrane potential loss and increased electron transport chain activity—the latter of which was surprising, but is an indication of cellular stress. During this period we also completed the development of a dispersion protocol for nano-TiO2 in bacterial growth media (11), which we expect to be useful for environmentally-relevant HTS using oligotrophic media. Also during this period, Project TER-3 expanded the application of the assay suite to inform our prior observation that P. aeruginosa was more growth-inhibited by Ag exposure in minimal media than was E. coli; Ag NPs


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at 10 mg L-1 were slightly toxic to E. coli during growth experiments, but did not cause measurable membrane damage. P. aeruginosa showed increased toxicity to Ag as measured from the membrane assays and growth curves. In addition to compromised membranes, there was an increase in total ROS at all Ag concentrations. Whether toxicity was caused by intact particles or dissolved Ag2+ ions is yet to be determined, as are levels of Ag accumulation in, or on, cells. Toward the end of this period, growth and sublethal assay experiments with E. coli were performed in Project TER-3 to quantify effects of and mechanisms of toxicity for Fe-doped vs. undoped TiO2

in light vs. dark conditions. It is premature to draw conclusions from this early data, but dose-response relationships were observed for both types of nanoparticles (doped and undoped), and variations were observed in light vs. dark conditions. Because of the size differences between the doped and undoped particles, additional experiments are required to differentiate band-gap effects from surface area effects. During this period, Project TER-5 also refined our DEB model of Cd(II) toxicity to bacteria, where ROS—defined as a damage inducing compound—is newly incorporated as a variable. The model is unique in its formalization of oxidative damage to growing bacteria (12), and is currently being expanded to CdSe quantum dot-treated cultures. Given the consistent experimental results herein across a suite of bacteria and nanomaterials, we are optimistic that a DEB model with emphasis on ROS as a damage inducer will be applicable to many bacterial-nanomaterial combinations. Overall, the outcomes we are finding with our combined growth curve, mechanism assay, and modeling approach are revealing both when and how ENMs are detrimental for bacteria. We have used a spectrum of ENMs in our work, owing to the availability of Cd(II) and CdSe QD data with which to launch DEB modeling at the beginning of the UC CEIN; using the primary datasets for UC CEIN SRMs, analogous modeling efforts can occur.

We aim to make our mechanistic bacterial research relevant to understanding effects of ENMs on soil processes. Our work with unplanted soil microcosms, which were previously exposed to ZnO or TiO2 and for which community composition and diversity impacts were shown (3), was expanded in Project TER-1 this period to reveal which taxa were impaired. Using a sensitive pyrosequencing approach, functionally-narrow taxa were discovered to be particularly sensitive to these MeOs. Specifically, we discovered that free-living N2

-fixing taxa, methane-oxidizing taxa, and other taxa known for their important roles in organic matter decomposition, were relatively less abundant as a consequence of MeO nanoparticle exposure (10). This research has two important implications. First, as a matter of making our bacterial research increasingly relevant to environmental concerns, we can now recruit from within these sensitive taxa bacteria to perform population-based mechanistic research in Theme 4 or bacteria that can be used for HTS in Theme 2. This would allow demonstration of how ENMs are affecting sensitive taxa, and how sensitive these taxa are to a broader range of ENMs. Ultimately, these taxa—or others that are discovered through similar Theme 4 approaches—might be designated as ‘sentinel’ taxa that can be used as bioindicators of ENM environmental impacts. The second implication is ecological: specifically, soil N and C cycling processes may truly be adversely affected by these ENMs.

To further explore the potential for soil processes to be impacted by ENMs such that ecosystem or agricultural consequences result, Project TER-1 newly designed and completed a highly interdisciplinary and collaborative planted soil microcosm study during this period. In this study, soybeans were planted in organic farm soil amended with 3 concentrations of CeO2 or ZnO; unplanted controls were also established. We recruited additional intellectual capacity to perform the work, including adding a plant physiologist, a soil exoenzyme and fauna expert, and ultimately a soybean geneticist. Plants were grown to maturity so that the impacts on food (bean) quantity and quality could be evaluated. The results are just emerging but thus far striking: negative, dose-dependent effects are observed for many of the indicators including plant size, N2 fixation rates by symbiotic nodules, leaf chlorophyll abundance, pest (aphid) abundance, and soil microbial biomass. This project will yield a detailed and comprehensive


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examination of how two very high-production MeO ENMs could impact an agriculturally-significant plant. The expectation that either ZnO or CeO2 ENMs might impact and be bioaccumulated by soybeans was informed by work in prior periods, during which hydroponic soybeans were shown to be genetically altered by CeO2 and to accumulate Zn that dissolved from nanoparticles. During this period, Project TER-4 greatly expanded our work with hydroponic plants, working with other crop plants (e.g. corn and cucumber) and also native plants (i.e. mesquite) (1, 4, 14). We also published a high profile review of plant impacts of ENMs (6). Results from the absorption, distribution and possible biotransformation of Zn in mesquite treated with ZnO NPs showed that ZnO NPs increased catalase activity (CAT) in roots, stems, and leaves, while ascorbate peroxidase (APX) increased only in stems and leaves (photosynthetic parts of the plants) (1). Micro x-ray fluorescence (µXRF) analysis confirmed the presence of Zn in the vascular system of roots and leaves of ZnO NP treated plants. In addition, x-ray absorption near edge structure (XANES) spectra demonstrated that ZnO NPs were not present in mesquite tissues. Bright and diffuse spots of ionized Zinc(II) were observed in roots and leaves of ZnO NP-treated mesquite plants (4). Also during this period, corn plants were treated with FITC-stained nanoceria that by confocal microscopy analysis were shown to penetrate corn roots via the apoplast (14). Also FTIR analyses were newly performed on rice, wheat, and barley seedlings treated with nanoceria. Rice varieties varying in amylose content and wheat and barley were treated with nanoceria (62.5 -500 ppm), with preliminary results showing that lipids and lignin are modified in the low and medium amylose content rice, while only lipids are affected by the nanoceria in high amylose content rice. The IR spectra of barley and wheat show a differential response in the lipid band while no major changes are observed in the carbohydrates and protein bands. Finally during this period, roots and leaves of cucumber plants treated with 500 mg TiO2 NPs L-1 were analyzed with light microsat beamline ID21 at the European Synchrotron Research Facility (ESRF, Grenoble France). The µ-XRF images showed the presence of Ti within the root vascular region of the cucumber plant and within the leaf as well. µ-XANES spectra showed the pre-edge feature at 4985 eV and features at 4992 and 5008 eV, which resemble the spectrum from TiO2 NPs. By comparing the µ-XANES spectra obtained from the roots and leaves of the cucumber plant tissues with the spectra of the model compounds, we concluded that TiO2 NPs were not biotransformed within the root of cucumber plants and might be in the same chemical form as the TiO2

NPs. For the 4000 mg TiO2 NPs L-1 treated plants, light microscope histological analyses were performed in 30 measurements/replica of the root, stem, cotyledon and real leaf. The area average showed that, in general, cells of TiO2 treated plants were three times larger than cells of control plants, suggesting that cells swell in response to the presence of TiO2

. Taken together, our work during this period has significantly advanced the body of knowledge regarding plant uptake, bioprocessing, and effects of several MeO ENMs.

In the prior period, we reported our discovery that CdSe QDs were biomagnified when trophically transferred from bacteria to their protozoan predators (8). In this period, Project TER-2 expanded this work to include other configurations of bacteria with their ENM cargo: “natural” Se(0) ENMs formed inside bacterial cells, and TiO2 ENMs sorbed to the exterior of bacteria. We also studied how this cargo affects predator growth. We observed that all cargo diminishes protozoan growth relative to controls, and that—as we discovered with CdSe QDs—digestion impairment is a likely explanation. High resolution electron microscopy and energy dispersive spectroscopy (EDS) are important tools for describing the compartmentalization of biomagnified ENMs, and our techniques for using such approaches are advancing, including acquiring multiple types (stained and unstained) of images for comparative analysis and quantitative use of EDS data. Such methods are highly transferable to other


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nanotoxicology questions, and the overall finding that bacteria can initiate biomagnification has much broader implications to overall ecotoxicology, i.e. beyond “nano”. Impacts on the Overall Goals of the Center: The transference of sublethal assays for bacterial HTS to Theme 2 benefits the UC CEIN in accomplishing its goal of establishing HTS with environmentally-relevant microorganisms. Our collaborative work with Theme 2 to continue the implementation of bacterial sublethal assays at UCLA is ongoing. The discovery of sensitive taxa from within soil microcosms makes plausible the introduction of “sentinel species” into bacterial HTS research in Theme 2. We intend to work under “low throughput” conditions to help facilitate transitioning mechanistic research with these new taxa to the HTS platforms. While not described in detail above, our methods-development with bacterial-ENM exposures (i.e. environmentally-relevant media recruitment and dispersion protocols) are aimed at assisting Theme 2 bacterial HTS. To the degree that we are discovering the products of ENM bioprocessing by plants and microbes, our work can inform Theme 3 fate and transport modeling and experiments. Ultimately, our research—to the degree that it can enable Theme 2 bacterial HTS—can assist environmental hazard ranking by Theme 6. In the original proposal, the UC CEIN was committed to performing planted soil microcosms. We have successfully advanced this goal with our soybean plant microcosms and similar. Such work has high relevance for understanding the societal implications of ENM effects on the environment, as in Theme 7 (2). Lastly, Themes 4 and 5 share a strong interest in trophic transfer. As such, microscopy and other analytical methods are shared, and experimental approaches are cross-fertilizing. Major Planned Activities for the next Period: In the coming year, we will complete our interpretation of how SRM MeO ENMs affect the bacteria that we have studied. We will expand our studies to perform at least one growth and mechanistic study of the bacterial taxa found to be sensitive within our soil experiments. In this way, we will complete the originally-planned feedback between mechanistic and ecological research for terrestrial systems. We will complete the interpretation of our planted microcosm experiment with soybeans, and will follow up with additional experiments as needed to definitively interpret the major datasets. We will finish our work with nano-Ag effects on bacteria, including quantifying dissolution of Ag nanoparticles in our culture conditions so that we can ascribe effects to nanoparticles versus ions. We will complete our study of Fe-doped TiO2 nanoparticles, determining how modification of band gap influences bacterial growth and toxicity associated with electron transfer between particles and cells. We will conclude our triology of microbial trophic transfer studies, furthering our interpretation of how ENMs ultimately affect protozoans through digestive impairment. We will complete our studies of MeO nanoparticles within a range of native and agricultural plants, explaining the translocation and effects of ENMs to hydroponic and soil-cultivated plants. We will complete our DEB model of CdSe QD effects on growing bacterial populations. Through a seed grant, we will expand our ENM coverage to include MWCNTs and will assess the association of MWCNTs with bacteria, biodegradation of 14C-labeled MWCNTs, and trophic transfer of 14C-MWCNTs from bacteria to protozoan predators. Through this final new project, we aim to acquire 14C-MWCNTs for use within the UC CEIN for a range of nanotoxicological projects. We also plan to use sensitive accelerator mass spectrometry approaches—held by original UC CEIN collaborators at LLNL—to quantify MWCNT transfer and compartmentalization. Taken together, these final projects will fulfill our original objectives of describing and understanding nano-MeO impacts on bacteria, demonstrating approaches for performing that research, discovering if such impacts manifest in higher organisms (predators and plants) and in complex media (soil), and interpreting the findings towards predicting and avoiding impacts of ENMs in terrestrial environments.


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Theme 5: Marine and freshwater ecosystems impact and toxicology Year 4 Progress - April 1, 2011 - March 31, 2012 Faculty Investigators: Hunter Lenihan, UC Santa Barbara – Professor, Applied Marine Ecology- Theme leader Gary Cherr, UC Davis – Professor, Ecotoxicology Ed McCauley, UC Santa Barbara – Professor, Ecology Roger Nisbet, UC Santa Barbara – Professor, Ecology Robert Miller, UC Santa Barbara – Assistant Research Scientist, Marine Ecology Konrad Kulacki, UC Santa Barbara – Postdoctoral Researcher, Freshwater Ecology Updated Goals of Theme 5: The overarching goal of Theme 5 is to address questions about the impacts of engineered nanomaterials (ENMs) on individuals, populations, and communities of model organisms that are responsible for generating freshwater and marine ecosystem services, including primary production, the maintenance of biodiversity and water quality, support of food webs, nutrient cycling, and the provision of food and other economic resources for humans. Marine and other aquatic ecosystems are not presently exposed to large volumes of ENMs, yet many ENMs are toxic and therefore are of environmental concern. Our primary goals are to (1) assess potential environmental injury to model organisms before ENMs become a significant environmental problem, and (2) to help develop safe design for ENMs in aquatic ecosystems to facilitate the use of ENMs for the benefit of society. Our research is designed to help develop the CEIN ecotoxicological paradigm, in which key tests are made at various levels of biological organization in order to develop a general theoretical understanding of ENM environmental impacts. The ecotoxicological paradigm we envision is one that can be used to foresee, predict, and mitigate potential impacts caused by a wide spectrum of ENMs. We conduct our research at levels of biological organization ranging from subcellular to ecosystem, beginning with studies of ENM impacts on the development of early life stages (e.g., embryos), with a focus on model marine organisms. Work here is led by developmental biologist/ecotoxicologist Prof. Gary Cherr (UC Davis), who collaborates closely with Dr. Nel and his team studying freshwater embryos (e.g., zebrafish) in Theme 2. Prof. Cherr also leads in vivo and in vitro our research with organelles, cells, and tissues of adult marine animals, providing insight into toxicological mechanisms that influence whole organisms and populations. In addition to our connection with the high throughput screening (HTS) work in Theme 2, our research also has strong intellectual overlap with Themes 3 (fate and transport in aquatic systems) and 4 (HTS work with terrestrial bacteria and ENM trophic transfer). We study freshwater and marine phytoplankton and cyanobacteria, which are single celled organisms that form the basis of aquatic food webs and thus generate most of the world’s primary production. Our experiments with phytoplankton include high-content screening (HCS) of these single celled plants that is designed to identify multiple cytotoxic endpoints. Results from HCS experiments are used to understand the mechanisms of injury that lead to changes in phytoplankton population growth rates (and thus primary production). We also study suspension-feeding marine mussels that bioprocess and bioaccumulate nanomaterial contaminants, copepods that graze upon phytoplankton, marine and freshwater benthic invertebrates and plants that form important links in aquatic foods webs, and the California spiny lobster, a keystone predator in coastal marine food webs and an economically valuable seafood species.


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Our research is designed to generate information on toxicological effects that can be used in general ecological models that provide a synthetic understanding of ENM-environment interactions. We have chosen to pursue an ecological modeling approach because we cannot test all ENMs for toxicity; rather, we aim to understand general patterns of environmental injury from a circumscribed number of experiments, and then to use the resulting data in models designed to predict potential diverse outcomes in nature. Our primary modeling framework is Dynamic Energy Budget (DEB) modeling, which quantifies the effects of ENMs on the major energy transfer pathways within individual organisms. As such, many of our experiments are designed specifically to generate data describing the effects of ENMs on key physiological processes that regulate feeding, maintenance, growth, and fecundity. Data from those experiments are then utilized in DEB models to understand and predict whether or not ENM-related injuries propagate to influence population demographic rates. Results of DEB modeling can then be used in population dynamic models to explore potential impacts on population abundance, and also in community models that examine species interactions and nutrient cycling. We often employ an iterative approach in which results from experiments are used to parameterize models, which in turn generate simulated predictions that are subsequently tested in additional experiments. Organization and Integration of Theme 5 Projects: Theme 5 is composed of five projects (MFW 1-5) that address research questions in nano-ecotoxicology from the scale of molecules to ecosystems in both freshwater and marine systems. The five projects are: MFW-1 Marine organismal nanotoxicology: Studying Interactions of Nanomaterials (NMs) at the

Molecular, Cellular, Organ, and Systemic Levels – Cherr MFW-2 Impacts of engineered nanomaterials on marine ecosystems – Lenihan/Miller MFW-3 Decoupling and recoupling plant-herbivore systems to determine the fate and impact of

nanomaterials in freshwater environments – McCauley MFW-4 Impacts of TiO2 nanoparticles on freshwater food webs – Cardinale MFW-5 Dynamic energy budget (DEB) modeling to support design of aquatic microcosm and mesocosm

experiments – Nisbet The focus in Theme 5 has been on identifying paradigms of toxicity for specific metal oxide and CNT ENMs. Development of these paradigms is based on own cytotoxic high content screening (HCS) work, the high throughput screening (HTS) research conducted within Themes 2 and 3, and results reported in the literature including oxidative stress, mutagenesis, fibrogenesis, and DNA damage. We have used these paradigms to generate predictions and hypotheses about the responses of individuals, populations, and communities to ENM exposure. Because our focus is on specific key ecological processes, and not all cytotoxic/organellar mechanisms have been identified for ENMs, we also utilize a strategy that involves testing for the impacts of ENMs at the level of the whole individual (e.g., respiration and growth rate) or population (e.g., phytoplankton population growth rates). When an impact is detected using this strategy, we work “backwards” with HCS or HTS to identify the specific mechanisms of toxicity. This population-level screening approach is employed, in part, because one of our goals is to help develop less environmentally harmful forms of ENMs (e.g., via capping and doping) through collaboration with researchers in Theme 1 based on their expertise in compositional and combinatorial ENM libraries. ENMs have very different physicochemical behaviors in different environmental media that influence their bioavailability and toxicity in freshwater and marine environments. Therefore, Theme 5 researchers work closely with Arturo Keller and other researchers in Theme 3 to test questions that improve our understanding of ENM fate and transport in aquatic environments.


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Theme 5 is structured to integrate the HCS analysis of biological injury pathways overseen by Gary Cherr and conducted at the UC Davis Bodega Marine laboratory with the micro- and mesocosm studies involving individual whole organisms, populations, and communities from both freshwater (Cardinale and McCauley) and marine (Lenihan) environments. Freshwater ecologist Brad Cardinale moved out of the program and on to the University of Michigan during this reporting period, but CEIN gained a very prominent and collaborative freshwater ecologist in Ed McCauley. Cherr, Lenihan, Keller, and McCauley are working in close collaboration to complete in vivo HSC experiments with phytoplankton and in vitro HTS experiments with mussel cells (especially hemocytes), and the results of these studies will be used to generate specific predictions that will be tested in population-level microcosm studies and community-level mesocosm studies. Both the work conducted with phytoplankton and zooplankton by McCauley and Lenihan and the mussel work by Lenihan’s group are informed by and used to erect DEB models developed by Nisbet and his team. Theme 5 is composed of five active projects that examine the influence of a suite of metal oxides and CNTs on freshwater and marine organisms. Data generated through our many experiments, like those generated by Theme 2, will be used by Theme 6 for the development of in silico feature analysis and decision-making tools that inform hazard ranking, dosimetry calculations and prioritizing more costly experiments at higher trophic levels. Major Accomplishments since February 2011: Work conducted on marine organisms by MFW-1 during this period identified a suite of mechanisms by which CNTs cause injury to the cells of marine mussels. Cherr’s group found that in vitro exposure of mussel hemocytes to CNTs causes 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 CNT used. Specifically, HiPCO SWCNTs interfered with phagocytosis at concentrations as low as 0.4 mg L-1

seawater, whereas there was no effect of exposure to SG65 or P2 CNTs on phagocytosis, even at above 10 mg L-1. HiPCO and SG65 SWCNTs caused increased cellular production of ROS at concentrations as low as 1 and 6.5 mg L-1, respectively. This effect was apparent after just 3 hours when mussel tissues were exposed to SG65 and at 20 hours exposure time with HiPCO CNTs. Finally, P2 SWCNTs had no effect on mussel hemocytes, although greater concentrations (probably not environmentally relevant) still need to be tested.

MFW-1, in collaboration with MFW-2, used HCS assays that were developed in a CEIN seed funded project (Cherr and Lenihan) to examine multiple cytotoxicological endpoints of exposure to nano-ZnO, CuO, and Ag in two species of marine phytoplankton, Dunaliella tertiolecta and Isochrysis galbana. Results from this study are important because they clearly indicate that effects of ENMs in one biological system/habitat may not be similar to responses observed in other systems (2). This concept is essential to understanding the global toxicological impacts of engineered ENMs. In most cases, the two species of phytoplankton responded differently to a particular ENM, though all materials tested were found to have a negative effect at some concentration. ZnO nanoparticles were found to significantly impact mitochondrial membrane potential in Dunaliella at as low as 1 mg L-1, but did not cause damage to membranes. Conversely, there was no effect of ZnO on Isochrysis mitochondria, but membrane damage was observed at 10 mg L-1. CuO nanoparticles significantly impacted mitochondria and increased production of ROS in both phytoplankton species at all concentrations tested (1-10 mg L-1). Nano-Ag significantly impacted Dunaliella mitochondria, but neither induced cytotoxicity directly nor caused production of ROS. In Isochrysis, nano-Ag was found to be directly cytotoxic at concentrations above 5 mg L-1, and significantly increased ROS production at lower sublethal doses. Differences in responses between the two species may be related to differences in cell wall structure and/or physiology. Future work is being designed to identify the specific morphological/physiological mechanisms that generate the variable responses.


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Studies in Theme 2 by Nel, Lin, and Xia found that low concentrations of CuO nanomaterials inhibit hatching in zebrafish embryos. When zebrafish embryos are exposed to 0.5-200 mg L-1 CuO NM, there is a significant and dramatic decrease in hatching success with no corresponding increase in either mortality or gross developmental abnormalities. Furthermore, when zebrafish embryos were co-exposed to CuO NM and a metal chelator, there was no specific effect on hatching. Unlike results with zebrafish embryos, work in MFW-1 with sea urchin embryos found no adverse effect of nano-CuO on hatching success that was independent of developmental abnormalities (1). However, CuO NM was very toxic to developing sea urchin embryos, with toxicity apparent at low µg L-1 (ppb) levels. Toxicity is likely attributable to soluble Cu2+. Co-exposures with CuO NM and a metal chelator were not conducted because no specific effects of CuO NM on hatching were observed, and because the general effects of Cu on embryo development have already been documented. The influence of nano-Ag on sea urchin embryo development was also studied. Ag aggregated rapidly in seawater, and toxicity was not apparent until concentrations exceeded 1-5 mg L-1, levels that would rarely be found in natural marine waters. Therefore, nano-Ag is probably not an environmental hazard to embryo development in marine ecosystems. Several important research breakthroughs were made during this reporting period in MFW-2 studies with marine phytoplankton populations. In collaboration with Cherr and MFW-1, MFW-2 found that despite variation in the specific mechanisms of toxicity of ZnO, CuO, and Ag in the two model phytoplankton species, these nano-metal oxides caused significant reduction in population growth rates. ZnO is highly toxic to marine phytoplankton, reducing growth rates at concentrations ranging from 230-1000 µg L-1 for all species of phytoplankton tested. However, Ag and CuO were much less toxic than ZnO, reducing growth rates only at relatively high concentrations (2 mg L-1), a pattern likely attributable to slower dissolution rates of these materials. Previous MFW-2 work in collaboration with Nisbet’s group established no-effect concentrations for ZnO NPs and was used to infer that dissolution of Zn ions was the likely mechanism of ZnO NP toxicity. Because phytoplankton take up ionic zinc as it dissolves, they potentially influence both the dissolution rate and the toxicity levels of ZnO. We therefore predicted that the no-effect concentration of Zn on phytoplankton would be positively correlated with initial population density due to uptake of Zn. We have now tested this prediction using ionic Zn, and found that Zn toxicity is influenced by starting biomass. When exposed to increasing Zn concentrations, growth rates of Isochrysis galbana cultures that were started at 10,000 cells ml-1 declined greater than ten times faster than cultures started at 20,000 cells ml-1. We are now designing experiments that use chelators to maintain constant Zn concentration, regardless of uptake, to determine true (and currently unknown) no-effect concentrations of Zn. If these studies support our initial finding that starting biomass influences Zn toxicity, the results will ultimately redefine metal toxicity benchmarks for marine ecosystems and cause fundamental revision of common ecotoxicological methods. With CEIN seed funding, Lenihan and Miller (MFW-2) developed a very sensitive and powerful method for detecting the cytotoxicity of nano-metal oxides in marine phytoplankton based on Pulse Amplitude Modulated (PAM) fluorometry. The method has allowed them to test the hypothesis that population growth rates of phytoplankton, and therefore primary production, are impacted by metal oxides specifically because these materials disrupt the function of photosynthetic pathways. Work completed with CuO indicates that this ENM reduces photosynthetic efficiency in Dunaliella tertiolecta and Isochrysis galbana at concentrations ≥1.0 mg L-1, thus indicating damage to the photosynthetic pathway as a mechanism of injury. We are currently investigating how the phytoplankton compensated for lost


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photosynthetic yield/efficiency to maintain growth rates at 1.0 mg L-1. Work to complete tests with less-soluble CeO2 and Ag are underway. PAM fluorometry portends to be an excellent predictor of ENM impacts on primary production, thus signifying an important research breakthrough. Work with marine mussels in MFW-2, in collaboration with Theme 3, found that mussels took up and processed 53% by weight of the nano-ZnO that was introduced into microcosm experiments (2). Of the 53% of Zn (nano-ZnO and Zn++ ions) taken up by mussels, 38% was rejected pre-ingestion and excreted in pseudofeces, which contain natural organic material (NOM) upon which the Zn was bound through adsorption. The pseudofeces are expelled to the surrounding benthic habitat, and therefore constitute an important pathway for nano-Zn to enter marine soft-sediments and the benthic detrital food web. The research groups also found a robust positive linear relationship between nano-ZnO concentrations (O.1-2 mg L-1) in the seawater/phytoplankton feed mixture and the Zn++ accumulation in mussel somatic and reproductive tissues. Thus, mussels appear to be important in metal oxide bioprocessing. They also bioaccumulate metal oxides, and potentially expose mussel predators to ENMs through trophic transfer. Work with mussels also found a significant negative effect of nano-ZnO on mussel growth at concentrations as low as 1 mg L-1

(3).

The most important research breakthrough of MFW-2 (Lenihan and Miller) is the demonstration that nano-TiO2 depresses population growth of marine phytoplankton only when the organisms are exposed to environmentally relevant levels of UVR (4). Results of microcosm population-level experiments showed with high probability that ROS generation is the mechanism of toxicity. This link was solidified through measurement of ROS production in seawater during the experiment using an electron paramagnetic resonance spectroscopy spin-trapping technique, which was performed in collaboration with the Keller lab (Theme 3). This analysis demonstrated that ROS production in seawater increased with increasing TiO2 concentration. Lenihan and Miller, together with Keller and Bennett from Theme 3, showed convincingly that nano-TiO2 is highly toxic when experiments are run under levels of UVR that correspond with those in shallow ocean depths. For the first time, these results provide evidence that environmentally relevant levels of UVR can interact with nanomaterials to negatively affect living organisms, thus indicating that toxicologists must consider photoactivity when evaluating the potential impact of nanomaterials, despite the experimental complications that might result. Work by the two teams of freshwater ecotoxicity groups during this period focused on (i) developing new mesocosm experimental systems to test the influence of nano-Ag on coupled bacteria-phytoplankton-zooplankton (i.e., Daphnid) population and community dynamics (MFW-3, McCauley), and (ii) completing mesocosm studies on the community-wide impacts of nano-TiO2 in freshwater benthic stream communities (MFW-4, Cardinale). McCauley’s group worked with Nisbet to design DEB models that generated specific predictions about the population growth responses of a tri-trophic (Daphnia-algae-bacteria) system under varying concentrations of nano-Ag. The bacterial assemblage present in our algal cultures provided an excellent model system to test the impacts of nano-Ag on freshwater bacterial dynamics. Through DEB model predictions, we were able to conclude that a single chemostat system would not provide sufficient algal density to support a sustainable Daphnia population. Therefore, the group developed a continuous flow-through, semi-chemostat experimental setup to investigate the behavior and effect of nano-Ag (citrate-coated vs. non-coated) on single faunal-floral types and different combinations of organisms. Cardinale’s MFW-4 team focused on completing and reporting a large scale freshwater mesocosm experiment designed to investigate the effects of nano-TiO2 on diverse stream communities, as well as the effects of community diversity on the fate of nano-TiO2. For this project, we grew three common


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algae both as monocultures and together as polycultures in biofilms of stream mesocosms exposed to 0, 0.1 or 1.0 mg n-TiO2 L-1. Nano-TiO2 did not alter the growth trajectory of any biofilm over 10+ generations, in large part because light levels in the experiments were not high enough to generate phototoxicity (5). These results imply that shaded stream communities will not experience the negative impacts of TiO2 on primary production that were observed by MFW-2 in marine phytoplankton, which live in sunlit surface waters. However, Ti accrual in biofilms not only differed among species, but increased as a function of species diversity, which regulated benthic biomass. This work has important implications for understanding the fate and effects of n-TiO2 in surface freshwaters, and the mechanisms that underlie their behavior. Work in MFW-5 by Nisbet and his team focused on developing DEB models that support projects in Theme 5 involving aquatic microcosms and mesocosms. The major accomplishments of this group were to help interpret the effects of ZnO, CuO, and Ag on marine phytoplankton population growth rates, mainly by providing robust estimates of the impacts of the ENMs on concentrations that were not tested directly in experiments (4). Research in this group also included the development of a DEB model for marine mussels that generalized the ecological effects of nano-ZnO. Specifically, results of the individual-based microcosm experiments on mussels performed by MFW-2, which tested the sublethal effects nano-ZnO on mussel respiration, feeding, growth, and survival (3), where used to parameterize a DEB model. Once parameterized, the model predicted that exposure to relatively small concentrations of ZnO ENMs for only 4 days can significantly reduce reproductive output of mussel populations due to reduced feeding rates. Results from that model will be reported in a paper to be submitted next reporting period. Finally, Nisbet’s work supported the development of the novel freshwater bacteria-algae-Daphnia experiments being conducted by McCauley’s group. Other results from MFW-5’s work in terrestrial ecosystems can be found in the report from Theme 4. Impacts on the Overall Goals of the Center: The development and implementation of high content screening approaches with phytoplankton and mussel tissues has allowed UC CEIN to further develop our integrated ecotoxicological paradigm and ultimately accelerate the safety assessment of ENMs. Theme 5’s close collaboration on the research regarding ENM fate and transport that is being conducted in Theme 3 is rapidly expanding the boundaries of ENM environmental science and safety. Of major importance is our finding that metal oxide ENMS and CNTs can have radically different behaviors, and therefore radically different fates, bioavailability, and biological impacts in different ecosystems. We cannot examine all the environmental impacts of ENMs, but our comprehensive selection of experiments that examine key ecological process and test hypotheses with carefully chosen model organisms that provide ecosystem services has helped to generate a robust body of general ecotoxicological information that we are disseminating in the top scientific journals and conferences. Our Theme has relied heavily on interactions with Themes 1, 2, and 3 to develop specific hypotheses concerning ENM impacts on aquatic organisms, thus highlighting the fact that advancing our understanding of the environmental science and safety of ENMs is best accomplished working within the NSF Science Center framework. In summary, the results we have generated during this period are helping to transform the scientific community’s understanding impacts of ENMs on freshwater and marine ecosystems. Major Planned Activities for the Next Period: Over the next three months we will continue integrating the HCS assay, PAM, and phytoplankton population growth experiments to test the impacts of select metal oxides and CNTs. We will begin experiments to examine the uptake, bioprocessing, and toxicity of CNTs in marine mussels. Work with metal oxides and phytoplankton will focus on testing ENMs that have been modified to reduce their toxicity (e.g., through capping and coating) and improve


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environmental safety. We will complete our DEB models of the responses of mussels to nano-ZnO. Amphipod bioassays that examine the demographic effects of CuO, Ag, and CNTs will be completed. The freshwater mesocosm work by McCauley will come fully online. During this reporting period, we began trophic-transfer experiments between marine pelagic phytoplankton and grazing copepods with Gretchen Bielmyer of Valdosta State University, a former student of prominent EMN ecotoxicologist Prof. Klaine. During the next period, we will complete those experiments with the phytoplankton-copepod mesocosm system, which have been designed to test the impacts of photo-activated TiO2 and Ag. In this reporting period, we also studied the influence of metal oxide ENMs on benthic primary production in cyanobacteria and community interactions between cyanobacteria and bacterioplankton, and we will publish result of that work in the next reporting period. Finally, we will begin the marine food-web mesocosm experiments we have designed to explore the fate and transport and bioaccumulation of nano-Ag in a model benthic ecosystem consisting of mussels, amphipods, phytoplankton, and California spiny lobster. We welcome new PhD student Tyronne Martin, who will be helping to lead our work with PAM fluorometry.


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Theme 6: Environmental Decision Analysis for Nanoparticles Year 4 Progress - April 1, 2011 - March 31, 2012 Faculty Investigators: Yoram Cohen, UCLA – Professor, Chemical and Biomolecular Engineering – Theme Leader Donatello Telesca, UCLA – Assistant Professor, Department of Biostatistics Robert Rallo, URV – Associate Professor, Departament d’Enginyeria Informatica i Matematiques Sharon Walker, UCR – Associate Professor, Chemical and Environmental Engineering Kenneth Bradley, UCLA – Associate Professor, Environmental Health Sciences Andre Nel, UCLA – Professor, Medicine; Chief, Division of NanoMedicine Arturo Keller, UC Santa Barbara – Professor, School of Environmental Science and Management Updated Goals of Theme 6: Theme 6 is an outgrowth of the activities previously reported in IRG6 (“Modeling of the Environmental Multimedia NM Distribution and Toxicity”) motivated by generation of significant new CEIN high throughput screening (HTS) data and characterization of engineered nanomaterials (ENMs). Theme 6 is rooted in the need to establish a rational approach for identifying and ranking nanomaterials that could be of environmental concern. The approach is based on the premise that the environmental impact of ENMs is determined both by levels of exposure to ENMs and by the toxicities of these materials. Accordingly, this Theme focuses on the development of a computational platform for environmental hazard ranking of nanomaterials (EHR-Nano). EHR requires information on ENM toxicity and the potential concentrations of the nanomaterial in various environmental media. Environmental ENM concentrations are estimated through compartmental multimedia modeling (MM) of environmental distribution, and toxicity information is derived from CEIN toxicity screening analysis and quantitative-structure-activity relations (QSARs). The EHR-Nano approach incorporates both quantitative and qualitative CEIN information regarding the physicochemical properties of ENMs (Theme 1), their environmental release, fate and transport (Theme 4), toxicity (Themes 2, 4 and 5), as well as aspects of risk perception (Theme 7). In order to accomplish the above, Theme 6 investigators have been developing the building blocks of the EHR-Nano approach; specifically, the rich library of characterized metal and metal oxide nanoparticles (Theme 1 and Core A) and HTS data (Theme 2) have served as a basis for developing the needed robust statistical analysis and machine learning methods and tools for the analysis of large CEIN data sets on ENM toxicities. These methods and tools include knowledge extraction from both numerical data (1) and HTS images of zebrafish embryo (5). For example, methods and tools developed by Theme 6 have enabled the development of predictive quantitative-structure-activity relationships for nanomaterials (nano-SARs) toxicity (6, 13, 20), identification of relationships among cell signaling pathway activities (induced by exposure ENMs) based upon differing assays and multiple cell lines (12), and correlation of information from in vitro HTS data with in vivo response and automated phenotype recognition of whole organism HTS images (11). In order to achieve appropriate ranking of potential environmental impact, it is also necessary to assess the expected levels of ENM concentrations in the various environmental media (16). Thus, aided both by the experimental studies of Theme 3 that focus on characterization ENM aggregation/dissolution behaviors, surface interactions under environmental conditions, and life cycle analysis, and by the elucidation of ENM characteristics by Theme 1, Theme 6 has been leading the development of fate and transport models suitable for the assessment of the environmental multimedia distribution of nanomaterials. Integration of these fate and transport models as web-based tools with the CEIN data has been made possible by a Nanoinformatics effort that is now supported by the CEIN “Data repository and Nano Collaboratory” Core C function. Organization and Integration of Theme 6 Projects: Theme 6 has been restructured to focus its research activities on the development of methodology and tools for environmental impact assessment.


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Accordingly, Profs. Cohen and Telesca are co-leading projects for which the development of software is now integral: EDA-1 (“Machine Learning Analysis and Modeling of High Throughput Screening Data for Nanoparticles”), and EDA-2 (“QSARs of NP Toxicity and Physicochemical Properties”). The high volume HTS data sets emerging from Theme 2 (e.g., based on nanotoxicity studies involving mammalian cells, bacteria, and zebrafish embryos) are now being used to support development of feature analysis and QSARs, association rule mining of toxicity-related signaling pathway activities and correlation of pathway activation and cytotoxicity measures, dosimetry calculations, optimization of HTS experiments, hazard ranking, and development of environmental impact analysis tools. The software development component of project EDA-3 (“Modeling of the Environmental Multimedia Distribution of Nanoparticles”), previously considered a separate project, is now an integral component of this project. Project EDA-3, aided by the significant ENM characterization data generated in Themes 1 and 3, has already led to the development of significant computational predictive capability of ENM agglomeration. Projects EDA-3 and EDA-4 are guided by a workgroup formed by Themes 3, 6 and 7. We note that Projects EDA-1 through EDA-3 feed into project EDA-4, which focuses on developing the approach and tools necessary to incorporate both quantitative and qualitative information into a scoring methodology that is appropriate for environmental impact assessment of ENMs (10). Finally, in order to enable the development of the UC CEIN ENM impact analysis approach and software, seamless access and retrieval of ENM related data (e.g., ENM characterization, toxicity, environmental transport data, environmental releases) is required; this has been addressed through close coordination with the CEIN “Data repository and Nano Collaboratory” Core function (established previously by IRG6), which is now a significant contributor to the national Nanoinformatics effort and to a collaboration established with the USEPA ToxCast program. Project List for Theme 6: EDA-1 Machine Learning Analysis and Modeling of High Throughput Screening Data for Nanoparticles –

Telesca/Cohen/Rallo EDA-2 QSARs of NP Toxicity and Physicochemical Properties – Telesca/Cohen/Rallo EDA-3 Modeling of the Environmental Multimedia Distribution of Nanoparticles – Cohen/Keller/Walker EDA-4 Environmental Impact Analysis – Cohen Major Accomplishments since February 2011: In previous reports, we described the development of a number of approaches for knowledge extraction from High Throughput Screening (HTS) data (1) which included: (i) a novel feature selection method based on linear least-squares forward search (LFS-FS), which was validated based on standard machine learning databases; (ii) a systematic approach to identification of biological endpoints based on statistical analyses that include adaptation of the strictly-standardized mean difference (SSMD) for HTS involving a small number of replicates with flexible control to minimize the occurrence of false negatives (13, 20); and (iii) self-organizing maps (SOMs) as an alternative to conventional hierarchical clustered heatmap representations of HTS data and for similarity analysis of toxicity-associated cell signaling pathways (2,3). The above work demonstrated that quantitative information can be derived not only to support QSAR development, but also to identify and quantify relationships between cell responses (pathway activation and cytotoxicity measures) and association rules (12) among toxicity-related signaling pathway activities. In this period, we have made a number of significant advances that include: (i) improved feature selection approach to allow handling of non-linearly correlated features (i.e., model descriptors) (9), (ii) development of a flexible web-based tool for HTS data analysis and hit identification (14), (iii) application of the SOM analysis approach to the Theme 2 ENM metal and metal-oxides toxicity data set for the RAW cell line and exploration of the clustering of cell-signaling pathways for this cell line (2, 3), (iv) development of a classification-based QSAR for toxicity of metal oxide ENMs that enables establishment of decision boundaries suitable for


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environmental impact analysis (6, 13, 20), (v) development of an automated phenotype recognition system for HTS images of zebrafish embryos (11), and (vi) identification of association rules for toxicity-related cell signaling pathways for RAW and Beas-2B cell lines exposed to metal and metal oxide nanoparticles (12). Given the need to better understand and predict the agglomeration state of nanoparticles, we have also developed the first successful theoretical model for describing the agglomeration of nanoparticles over a wide range of environmental conditions (8). Results from the above models support the development of a parameterized model for use in the environmental hazard ranking methodology being developed in this Theme (10, 19). Additionally, the basic modeling framework for both the multimedia fate and transport model (Project EDA-3) and the EHR-Nano tool (Project EDA-4) have been formulated, and software development is now underway. Development of the various HTS knowledge extraction approaches was based upon HTS data for metal and metal oxide ENMs for RAW and BEAS-2B mammalian cell lines generated by Theme 2 and particle characterization data. 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 (in Projects EDA-1 and EDA-2) and utilized both for the development of QSARs for ENMs (6) and for SOM cluster analysis of HTS data (3). Self-organizing map (SOM) analysis of metal and metal-oxide HTS assays (Project EDA-1), which focused on the induction of toxicity-associated cell signaling pathways using a set of RAW 264.7 luciferase reporter cell lines, revealed two major clusters (3). The first cluster corresponded to (i) sub-lethal pro-inflammatory responses to Al2O3, Au, Ag, SiO2 nanoparticles that are 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). The analysis revealed that oxidative stress, inflammation, and perturbation of cellular signal transduction pathways were three of the key mechanistic injury paradigms that are currently being pursued in Theme 2. In addition to identifying clusters and quantifying similarity measures, Project EDA-2, in collaboration with Themes 1 and 2, developed a classification-based cytotoxicity nano-SAR based on a set of nine metal oxide ENMs (6). The end points for this toxicity classification nano-SAR were cell death and increased surface membrane permeability in transformed bronchial epithelial cells exposed over a wide ENM dosage (6). In this study, a rigorous method for the identification of the most suitable set of descriptors was employed, resulting in a model requiring only four simple descriptors (atomization energy of the metal oxide, period of the nanoparticle metal, nanoparticle primary size, and nanoparticle volume fraction in solution) that demonstrated 100% classification accuracy in both internal and external validation. This classification Nano-SAR enables identification of decision boundaries that are crucial for use in hazard ranking of nanoparticles, which allows for direct incorporation into the environmental impact analysis (EIA) of Project EAD-4. A collaborative effort with Themes 1 and 2 (Project HTS-6) is also ongoing to develop Nano-SARs for cytotoxicity of a new set of 24 metal-oxide nanoparticles (assays: ATP, LDH, and MTS; BEAS-2B and RAW cell lines; nanoparticle concentration range of 0.39 - 200 ppm). Based on whether the slope of the dose-response curve 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). The nanoparticle descriptor set included agglomerate diameter, electrophilicity, chemical hardness, atomization and band gap energies, as well as previously developed nanoparticle descriptors. Application of our HTS data analysis and feature extraction methods to this dataset enabled the development of a predictive toxicity classification-based QSARs for metal-oxide ENMs that achieved 92% classification accuracy (20). The potential utility of the band gap energy structure as a criterion for predicting the toxicity of metal oxides in cellular HTS assays and the additional correlation of those predictions to responses at the organismal


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level is further described in projects of Themes 1 and 2 (HTS-6). It is noted that Nano-SARs developed in this project will be incorporated into decision analysis tools being developed in Project EDA-4 (“Environmental Impact Analysis”). A more recent QSAR for native and surface-modified iron-oxide and quantum dot ENMs with various chemical modifications was developed (3) based on multiple cell lines (human aorta endothelial cells, coronary artery vascular smooth muscle cells, HepG2 cells, and the murine RAW 264.7 leukemic monocyte/macrophage cell line) and an optimal descriptor set (particle size, relaxivities (R1 and R2) and zeta potential). This QSAR predicts outcomes with approximately 85% classification accuracy. Because acceptable levels of false negative and false positive toxicity predictions may different under regulatory decision making scenarios, false negative and false positive thresholds for the above data set were systematically explored to enable the definition of decision boundaries (i.e., with respect to nanoparticle toxicity classification) based on the developed Nano-SAR. Knowledge extraction via network analysis (Project EDA-1) was employed to explore the relationships among 14 toxicity-related signaling pathway activities for the murine macrophage (RAW) and transformed bronchial epithelial (BEAS-2B) cell lines (12) exposed to six different metal/metal oxide nanoparticles (Ag, Au, Pt, Al2O3, SiO2, ZnO). Analysis of the large experimental HTS dataset (55,296 plate data readings) generated by Theme 2 and Core D via association rule mining resulted in 6 association rules involving the E2F, p53, Myc, SMAD, and Mito pathways for the Raw cell line and 12 rules involving the SMAD, SRF, CRE, PI, and HIF1A pathways for the Beas-2B cell line. These association rules revealed that ENM triggering of one or more pathways can induce triggering of other associated pathways, consistent with the biological cross-talk between cellular signal transduction and transcriptional regulation pathways. The identified rules suggest that BEAS-2B cells respond to the tested nanoparticles through downregulation of the MAPK/ERK pathway, leading to repression of downstream genes and induction of necrosis. Also, for the RAW cell line, the association rules suggest that the observed increase in E2F and p53 reporter activities support an environment of oxidative stress created by Pt and ZnO. Interestingly, c-Myc overexpression increases intracellular ROS levels, and the increased MitoSox signal indicates that mitochondria have higher levels of superoxide in response to Pt and ZnO. While the respective roles of host proteins and nanoparticles themselves to the oxidative stress observed here are currently unknown, the association rule {c-Myc, E2F - -> p53} presented above both indicates that oxidative stress is important and provides insight into cellular pathways that may be responsible for ultimate cytotoxicity. The above nanoinformatics analysis also implies that identification of association rules for strongly associated activity pathways can assist in confirming consistency of multi-assay HTS results and can aid in reducing the number of assays needed to establish reliable and consistent toxicity measures for QSAR development. In addition to knowledge extraction from HTS assays for specific cell lines, we have been working collaboratively with Theme 2 (project HTS-2) to accelerate knowledge generation from in vivo toxicity screening of ENMs using zebrafish embryos. Accordingly, in order to accelerate the analysis of the high volume of zebrafish images, an automated phenotype recognition system was developed (Project EDA-1) using support vector machines (SVM) with a set of suitable image descriptors (11). 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 a predictive accuracy as high as 97.4%. The advantage of the present phenotype recognition approach is that it allows retroactive analysis of large number of images obtained during high throughput zebra fish embryo screening, and therefore enables hazard ranking of large batches of ENMs in Project HTS-2 (Theme 2).


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Results of the nano-SAR work (6) have suggested increased cytotoxicity (for metal oxide ENMs BEAS-2B cell line) with increased primary ENM size for the range of primary particle sizes studied (8-20 nm). It is interesting to note that detailed theoretical calculations of nanoparticle agglomeration (8; Project EDA-3) demonstrated decreased average agglomerate size and a larger tail of smaller aggregates with increasing primary nanoparticle size. This behavior suggests that exposure to a larger concentration of smaller aggregates may occur with increased primary size. Expanded computer-based simulations of nanoparticle agglomeration along with experimental measurements of the particle size distribution by Theme 3 are now in progress to map out the above behavior. These computations are based on a novel Constant-Number Monte-Carlo/DLVO nanoparticle agglomeration model (8) that accounts for nanoparticle sedimentation. Our model is the first to quantitatively predict the agglomeration state of nanoparticles, and we validated our predictions through comparison with agglomeration data reported in the literature for TiO2, CeO2, and C60. This validation showed good agreement between model-based predictions and empirical data (average error of approximately 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). Current work (Project EDA-3) focused on expanding the DLS data to cover a wider range of nanoparticles primary size (~15-500 nm) in order to further validate the theoretical approach is proceeding in collaboration with Theme 3. Based on additional validation data and expanded model simulations, a simple parameterized model of nanoparticle agglomerate size will be developed and incorporated into both the environmental impact analysis methods developed in EDA-4 and the multimedia fate and transport model (EDA-3). Theme 6 has been developing the major building blocks of a computational web-based system for environmental hazard ranking of nanomaterials (EHR-Nano; Project EDA-4). EHR-Nano follows a decision-based process for environmental impact assessment that involves a scoring methodology for hazard ranking (10, 19). In such an environmental impact assessment, the analyst goes through the process of ranking ENMs according to their physicochemical properties (Theme 1), fate and transport analysis (Themes 3 and 6), toxicity and ecological impact information (Themes 1-3 and 5), and user risk perception (Theme 7). Information on ENM toxicity is critical for EHR; however, EHR must first consider the agglomeration state and potential environmental concentrations of ENMs (1, 10, 19; Project EDA-3) since the environmental transport of ENMs (e.g., dry and wet deposition from air, sedimentation in water, surface attachment) will be affected by their agglomeration state. Therefore, a computational model of the multimedia environmental distribution of nanomaterials (Mend-Nano) that considers the aggregation state of nanoparticles (8, 21, 22) and their surface interactions (Themes 1 and 3) was developed by Theme 6 (14, 17; Project EDA-3). Initial multimedia fate and transport simulations (Project EDA-3) have shown that when ENM emissions are primarily to the atmosphere, their environmental mass accumulation is greatest in the terrestrial environment. The deposition of ENMs onto soil and water surfaces is due to dry and wet deposition, the latter being an episodic removal mechanism. In contrast, when ENMs are discharged directly to the aquatic environment, their mass distribution remains primarily in the water and sediment compartments. In summary, considerable progress has been made in the development of methods and tools for knowledge extraction from HTS data and in the applications to metal and metal oxide ENMs. The research has resulted in development of quantitative structure-activity relations (QSARs), identification of association rules among cell signaling pathways, and correlation of properties with cytotoxicity of single cell lines exposed to ENMs. Theme 6 research has enabled identification of the key parameters determining ENM toxicity, agglomeration, and environmental transport and has also served as the basis for devising a rational approach to environmental impact assessment that will support strategies for the safe design and use of ENMs.


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Impacts on the Overall Goals of the Center: In order to assess the potential environmental impact of ENMs, information is needed about nanomaterial toxicities and potential distributions in the environment. It is not feasible to study all nanoparticles of concern in all of the relevant environmental media and thereby comprehensively determine toxicity profiles. Moreover, there is a need to assess the potential impact of nanomaterials prior to the manufacturing, use and disposal of these materials. Therefore, it is necessary to develop in silico models that incorporate prediction methods of ENM toxicity and environmental fate and transport into environmental impact analysis tools. Accordingly, advances by Theme 6 have provided HTS data analysis tools (e.g., feature extraction, clustering, and hit identification) that have enabled, in close collaboration with Themes 1 and 2, the development of quantitative structure-activity-relations for nanoparticle toxicity and have demonstrated potential for streamlining HTS assays and accelerating toxicity data analysis and interpretation. 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. Our web-based tools for HTS data analysis are now available to accelerate knowledge extraction, and the CEIN has recently established collaboration with the USEPA ToxCast program to jointly explore HTS data generated by the EPA and by the CEIN using the Them 6 HTS Data Analysis Tool (HDAT). Our efforts have attracted national and international attention and acclaim as is evident by invitations to the recent European Union COST Exploratory Workshop on Quantitative Nanostructure Toxicity Relationships (QNTR), invited presentations at Nanoinformatics 2010 and 2011 (as well as co-organization of these two workshops), and an invitation present the CEIN HTS data analysis and Nanoinformatics developments at the upcoming 3rd annual meeting of the American Society for Nanomedicine (ASNM). The ENM aggregation modeling effort has provided a mechanistic understanding the agglomeration of nanoparticles distributed in NP suspensions and a basis for the interpretation of ENM characterization data that has been generated in Themes 1 and 3 or published in relevant literature. Our work is the first to provide a definitive quantitative prediction of nanoparticle agglomerations and is a promising approach that will serve other researchers interested in assessing the impact of nanoparticle aggregation on both toxicity behavior and environmental transport. The ENM aggregation modeling work is also a foundation for the multimedia fate and transport modeling work, which is guided by a collaborative workgroup formed by Themes 3 and 6. A central goal of Theme 6 is to establish a rational approach for identifying and ranking nanomaterials that could be of environmental concern. Accordingly, the development of a decision process for environmental impact analysis (DeP-EIA) will serve a central mission of the CEIN. The resulting DeP-EIA tools will be useful for both regulatory agencies and the nanotechnology industry, and will aid in designing rational strategies for addressing environmental concerns associated with nanotechnology. This tool will ultimately be made publically available and thus serve the education and knowledge dissemination (Theme 8) goals of the CEIN. It is noted that in developing the DeP-EIA, Theme 6 members have benefited from their active participation and growing contribution to the “community-owned” Nanoinformatics Roadmap; such efforts have focused on developing a roadmap and standardized approaches to collection, storage, sharing, analyzing, modeling and applying information relevant to ENMs in order to foster scientific discovery and safe use of nanomaterials. Major Planned Activities for the Next Period: During the next period Theme 6 will focus its activities on the ultimate development of a multimedia fate and transport model, nano-SARs of an expanded applicability domain, and a decision process for environmental impact analysis. Specifically, we will continue with knowledge extraction via association rule identification in collaboration with Themes 1


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and 2. We intend to further develop the approach to defining suitable toxicity end-points based on aggregation of HTS data from a variety of assays in order to increase the reliability of the nano-SARs used for hazard ranking. Furthermore, work to develop nano-SARs based on the newly generated toxicity data for 24 metal oxide nanoparticles will continue in collaboration with researchers in Theme 2. This modeling effort will include identification of the most suitable descriptor set from an expanded pool of nanoparticle descriptors that will include energy descriptors and other structural properties. In addition, we expect to commence a collaborative effort with USEPA ToxCast researchers to analyze new EPA-generated HTS data, with the goal of expanding the applicability domain of present toxicity nano-SARs. Parallel to the work on nano-SAR development and continued HTS analysis, we will continue compilation, evaluation and incorporation of nanoparticle intermedia transport parameters and equations into the multimedia fate and transport model as the software architecture is being refined. We plan to develop a prototype of the model graphical user interface and web-based implementation for remote software utilization. Information regarding potential emissions/discharges of various nanoparticles such as the compiled data and estimation methods being generated by Theme 3 will be incorporated into the model as such information becomes available. Our goal is to have completed a functional prototype of the multimedia fate and transport model during the next reporting period. As part of the fate and transport modeling work, we also plan to develop a parameterized nanoparticle agglomeration model (based on a combination of simulations with our numerical model and additional experimental data from Themes 3 and 1) that will enable rapid estimation of the average agglomerate size as required in the multimedia fate and transport model being developed in Project EDA-3. Finally, work on the DeP-EIA prototype will continue with the aim of integrating this tool within the CEIN Data Management System. The approach is based upon development of a question hierarchy (organized by increasing level of complexity) to guide the analyst toward qualitative risk scoring. The approach will be improved incrementally to incorporate our machine learning (Project EDA-1), nano-SARs (Project EDA-2), and fate & transport modeling (Project EDA-3) tools and algorithms as well as information from the literature and data from CEIN researchers regarding nanoparticle fate and transport (Themes 4 and 6), toxicity (Themes 2, 3 and 5) and life-cycle analysis (Theme 4). When completed, the DeP-EIA will allow users both to evaluate various environmental scenarios involving ENM releases and exposures and to assess expert opinion on the impact analysis process.


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Theme 7: Societal implications, risk perception and outreach activities Year 4 Progress - April 1, 2011 - March 31, 2012 Faculty Investigators: Hilary Godwin, UCLA - Professor, Environmental Health Sciences, Theme Co-Leader Barbara Herr Harthorn, UC Santa Barbara – Associate Professor, Feminist Studies, Anthropology & Sociology – Theme Co-Leader Patricia Holden, UC Santa Barbara – Professor, Environmental Microbiology Milind Kandlikar, University of British Columbia – Associate Professor, Institute for Global Issues & Institute for Resources, Environment & Sustainability Arturo Keller, UC Santa Barbara—Professor, Biogeochemistry Timothy Malloy, UCLA - Professor, Law and Environmental Health Andre Nel, UCLA - Professor, Medicine and Environmental Health; Chief, Division of Nanomedicine Terre Satterfield, University of British Columbia –Professor, Institute for Resources, Environment & Sustainability Updated Goals of Theme 7: The overarching goals of Theme 7 are (i) to generate knowledge about the ethical, legal, and societal implications of nanotechnology, particularly in the area of perceived risks of nanotechnology to the environment, (ii) to disseminate both that knowledge and other insights gained from the science conducted across the UC CEIN to critical stakeholders, including the US public, the ENM industry, and policy makers/regulators, and (iii) to aid in the feedback process from stakeholder engagement to UC CEIN. The 2011 NNI Environmental, Health, and Safety Research Strategy (NNI, Oct 20, 2011) asserts that ELSI (ethical, legal, and societal implications) considerations “are deeply embedded in the NNI’s commitment to responsible development of nanotechnology” (2011: 10). “The NNI seeks to generate ELSI knowledge and insights through (1) research in the areas of public perception and understanding expected benefits, anticipated risks, and safety that can help society assess potential impacts of nanotechnology and possible responses; (2) scientific meetings and workshops at the local, state, national and international levels; and (3) public engagement activities to identify stakeholder perspectives on nanoEHS and ELSI issues.” The activities in Theme 7 are organized around the principle of ensuring that the science performed and discoveries made within the UC CEIN reflect the priorities of society at large and that critical partnerships are created and leveraged to ensure that the science and discoveries produced in the Center benefit society. Specifically, activities within Theme 7 are designed to foster a greater understanding of the factors that contribute to the differences in perception that stakeholders have regarding the risks of nanotechnology to the environment, of how risk perceptions impact industrial practices and beliefs, and of the importance of equitable distribution of benefit and harm in the development and application of nanotechnologies. Whereas the activities in Theme 8 focus on the development of new educational materials and research protocols that are based on science and discoveries within the Center, the outreach activities in Theme 7 focus on the dissemination of science from the Center to broad stakeholder groups through strategic partnerships. As a result, we have not only surveyed critical stakeholder groups – including the general public, industry, nanoscientists, environmental health and safety professionals, and regulators – but we have also established critical partnerships to engage these stakeholder groups on topics related to the science and innovation being pursued in our Center. Organization and Integration of Theme 7 Projects: In reorganizing the center from Interdisciplinary Research Groups (IRGs) into Themes, we have intentionally linked our societal implications research (Soc-1 to Soc-4) with outreach activities (Soc-5 to Soc-6) that are informed by this work. Theme 7 activities are organized into a set of research and outreach activities consistent with the societal goals


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for responsible development of nanotechnologies. On the research side, Theme 7 aims to produce new knowledge about key factors likely to drive critical stakeholder groups’ perceptions of risks to the environment posed by specific ENMs and their enabled products. Our goal is to use this knowledge to inform and refine UC CEIN research, as well as our Center’s public and policy outreach strategies. Groups/sectors we study for our research include representative samples of the US public, the ENM industry, and a range of experts (nanoscale scientists and engineers, nanotoxicologists, regulators, and nano environmental remediation experts). Factors currently being analyzed as drivers of public perception include: views about environmental media of air, water, and soil; environmental values and worldviews; different framings of ENM and application risks and benefits; and perceived thresholds of under-regulation or over-regulation by government or industry of emerging environmental risks. Theme 7 also analyzes how industry’s perceptions of risk and regulation impact their environmental stewardship and workplace safety practices and their receptivity to the regulation of engineered nanomaterials. For example, in spite of viewing ENMs as risky materials, our work shows that industry is inconsistent in using risk avoidant practices and has a strong ‘go it alone’ attitude about regulation that signals potential problems for workers and the environment. Critically, the results of this research are used directly to inform the UC CEIN’s outreach activities to both the general public as well as those to specific stakeholder groups, including U.S. regulatory agencies, EH&S professionals, and nanoscience researchers. Project List for Theme 7: SOC-1 Environmental Risk Perception – Satterfield/Harthorn SOC-2 Sociology of ENM Environmental Risk and Perception – Harthorn [Freudenburg] SOC-3 Environmental Risk Management and Regulation in the International Nanomaterials Industry –

Harthorn/Holden/Satterfield SOC-4 Risk Assessment and Nanomaterial Regulation – Kandlikar/Satterfield SOC-5 Nanomaterial Hazard Ranking and Nano Regulatory Policy – Godwin/Malloy SOC-6 California Nano Partnership (CANP) - Industry/Gov't/Academia Linkages –

Godwin/Nel/Avery/Malloy Major Accomplishments since February 2011: Theme 7 has made significant advances in both societal implications research and outreach in the reporting period, and particularly in using the insights gained from risk perception research both from the UC CEIN and from our partner, the Center for Nanotechnology and Society at UCSB, to inform UC CEIN outreach activities. For instance, a critical insight from risk perception research is that the general public is more likely to be accepting of the possible risks associated with nanotechnology if they are engaged in substantive discourse about both the potential advantages and the potential risks of nanotechnology. This insight challenges the naïve assumption of most scientists and engineers that it is better not to talk about risks at all when discussing new technologies with the general public or when engaging with the media. As a result of this insight, the UC CEIN has specifically designed and held a series of “public conversations” about the benefits and risks of nanotechnology, which have been held in collaboration with the Santa Monica Public Library (SOC-7). These events are very popular and well attended. Below, we provide concrete examples of how critical accomplishments in the research projects in Theme 7 (SOC-1 to SOC-4) have informed the outreach activities and accomplishments in Theme 7 (SOC-5 to SOC-6) in the last year. A critical accomplishment for this Theme is that a novel survey instrument was developed and used to probe the factors that influence the risk that members of the U.S. public assign to ENMs in different environmental media (air, water, and soil) and how they feel about the prospect of introducing ENMs into those media (SOC-1). This study was developed in collaboration with researchers in Theme 3 and


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informed by the results of research conducted by that group. Initial analysis of the data generated by this survey has revealed that individuals’ perceptions of risk are shaped by their gender (women are more risk averse), their views of the different environmental media (how resilient, tangible and available to sensory information they are), and their set of attitudes about whether ‘dose makes the poison’ in the ENM case, about the reliability of scientific information, and about the safety of chemicals. The survey also allowed us to probe how context influences people’s perception of risk. For instance, if respondents are told that the materials are more toxic, then they become more concerned about the potential risk. These results show that ENM risk perceptions, rather than being firmly fixed, are subject to change depending on how information about the materials, their environmental location, and their potential uses is framed, along with level of stated risk. This insight has important implications not only for how results from our Center should be presented, particularly to the general public, but also for how the field of nanotoxicology should be framed in general. As a result, when researchers within the UC CEIN report their results, they are encouraged to refer to the toxicity of the nanomaterials relative to the toxicity of the bulk material (or other known toxic materials), as opposed to simply in relation to other nanomaterials. This is a critical lesson that needs to be more broadly distributed to the nanotoxicology community: it is important to classify nanomaterials as “highly toxic” only if they meet generally accepted criteria for highly toxic compounds, and not simply because they are more toxic than other nanomaterials, because how we frame our results can have critical impacts on how nanomaterials are perceived by the public in general and hence on the sustainability of this technology. We have also been careful to apply this concept to how we frame our recommendations for policy makers and regulators (SOC-5). While SOC-1 is focused on understanding individual risk perception processes in forming judgments about the riskiness of ENMs to different environmental compartments, SOC-2 focuses on how social and institutional practices shape and amplify public perceptions of risk. Over the last year, we have specifically sought to address the question of whether ensuring that the benefits and harms of nanotechnology are equitably distributed across society is critical to the sustainability of this technology. To address this issue, we have investigated whether EPA nano remediation sites are equitably located (i.e., as likely to be located in wealthy communities as in poor or vulnerable communities), and whether equitable locational distribution influences how acceptable they are to the general public. The first phase of research is complete and indicates that, thus far, equitable distribution of hazards is a predictor of likely public acceptance of specific nanoenabled applications. We anticipate that this insight will critically impact our discussions with policy makers and regulators going forward (SOC-5), and have already seen evidence that this insight has influenced how individuals at the national level think about the concept of “sustainable development” as it pertains to nanotechnology. The third project in this theme (SOC-3) has focused on studying how workplace safety practices and environmental stewardship in ENM businesses (internationally) correlate with industry characteristics (e.g., company size, ENMs handled) and the attitudes regarding risk and regulation of leaders within that company. This study has revealed a critical contradiction between what many individuals in industry believe and their actual practices: despite a high level of perceived risk or uncertainty about the risks of nanotechnology, only a minority of the individuals surveyed actually used heightened safety practices when working with nanomaterials, and the majority of those surveyed felt that industry was capable of self-regulating in the area of Nano EH&S in spite of only limited knowledge of recommended practices. The study provides strong evidence of the lack of effectiveness of published guidance documents in effecting safety in the nanomaterials workplace. Based on this insight, we spearheaded the effort to establish the California Nanosafety Consortium of Higher Education (CNCHE), which has developed a novel “Nanotoolkit” (SOC-6) which helps scientists and engineers working with nanomaterials to quickly


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identify the appropriate risk category for the work they are performing and provides clear, concise guidance on what engineering controls, work practices, and personal protective equipment should be used for different risk categories. We interpret the results of this work as a strong case for regulation, since the uncertainty and medium to high risk perception with which industry leaders view nanomaterials demonstrate lack of corresponding orientation to safety and the risk protective behaviors normally associated with high perceived risk. As a result, within the UC CEIN we have prioritized working hand in hand with regulators (SOC-5) to ensure that regulators have the most up-to-date information available about hazards of nanomaterials and to provide them with science-based guidelines for prioritization of nanomaterials for regulation. The fourth project in this theme (SOC-4) has focused on the area of risk assessment and nanomaterial regulation. A core deliverable in the last year has been the deployment and analysis of an expert web survey, the goal of which was to elucidate how views of ENM risks varied between nanoscientists, nano EHS researchers and regulators. Regulators were found to be most risk averse and nanoscientists as most risk tolerant, with EHS researchers’ views lying between them. All groups viewed regulatory agencies as ‘unprepared’ to manage risk, but regulators most strongly considered regulatory agencies as unprepared. Based on this concern, we have partnered directly with the California Department of Toxic Substances Control to provide them with feedback and tools based on this agency’s needs and requests to help them to prioritize nanomaterials for regulation within the State of California (SOC-6). Another critical deliverable from SOC-4 in the last year has been the development of a much needed life cycle analysis of nanomaterial regulation in the US that identifies significant specific regulatory gaps and/or impediments to full regulation across the life cycle of nanomaterials. We anticipate that this analysis will profoundly impact our discussions with regulatory agencies in the upcoming year, as a repeated request from them has been the development of more grounded life cycle analysis. Taken together, Theme 7 has made substantial progress in generating new societal findings on environmental risk issues and systematically incorporating them into UC CEIN outreach programs and activities that contribute directly to the achievement of NNI goals for responsible development of new technologies. Impacts on the Overall Goals of the Center: The mission of UC CEIN is to ensure that nanotechnology is introduced in a “responsible and environmentally-compatible manner” (CEIN website). Theme 7 contributes to that mission through multi-stakeholder research on ENM environmental risk perception and regulatory challenges to “responsible development” that can be incorporated to enhance UC CEIN research, decision-making, education and outreach activities, and for input into regulatory policy. In addition to a growing publication profile, Theme 7 has already established an extensive record of public, scientific, social science, governmental, and industry presentations that significantly extends the reach of the UC CEIN and demonstrates its attention to the concerns and views of these different stakeholders (a key aim of the NNI). The partnership in Theme 7 of societal implications researchers with outreach program personnel indicates the close connection between the two in the CEIN. In the research arena, research from Theme 7 suggests a number of approaches for UC CEIN, including the following. UC CEIN should: (1) be attentive to how our behaviors influence and fulfill our obligations to the public’s trust in institutions of higher education; (2) be careful not to compromise our perceived trustworthiness for unbiased environmental risk assessment and communication through direct (funded) partnerships with industry; (3) provide balanced risk-benefit rather than benefit-only environmental risk communication to avoid future amplification; (4) anticipate and adjust for diverging perceptions of ENM risk and need for regulation across the experts involved in the UC CEIN enterprise


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when forming UC CEIN policy and risk assessment and communication; (5) anticipate US nano environmental regulators’ comparative lower confidence in the sufficiency of regulatory mechanisms for ENMs; (6) participate in social/decision science based risk ranking exercises that will aid in the adaptation of risk regulation mechanisms to ENMs; (7) anticipate industry risk attenuation effects and desires for autonomy from regulation in working toward industry participation in ENM regulatory decisions; (8) pursue intervention with industry EH&S knowledge gap in conjunction with regulatory advances; (9) consider downstream industry ENM utilization patterns in determining specific ENM characterization work; (10) work with regulatory agencies to devise methods for outreach to small startup firms with likely higher environmental impacts; (11) devise plans to address cultural values with regard to different environmental media in reporting effects of specific ENMs in those media; (12) target science educational outreach and risk communication to different audiences depending on level of concerns about intangibility of ENMs and relative resilience of the environmental media. Critically, work in Theme 7 has also contributed to the overarching goals of the UC CEIN to serve specific external stakeholder groups, including U.S. regulatory agencies, EH&S professionals, and nanoscience researchers. Our outreach activities over the last year in nano hazard ranking and nanoregulatory policy (SOC-5) 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. These outreach activities have also 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. Our work supporting and guiding linkages between industry, government and academic researchers in nanotechnology (SOC-6) has resulted the development of an online course on Nanoecotoxicology (which leveraged science and results from all of the Themes in the Center) and of online safety training modules that we are developing in partnership with UCLA’s Office of 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. Major Planned Activities for the Next Year: Theme 7 environmental risk perception researchers (SOC-1 to SOC-4) will continue to work with team members of UC CEIN outreach projects to fine tune research activities of the greatest value to the UC CEIN and to enhance incorporation of research results and implications into center-wide research and activities (SOC-5 to SOC-7). In the upcoming year, researchers in SOC-1 will complete analysis and dissemination of Stage 1 public environmental risk perception survey findings about the interrelationships of perceived environmental resilience, attitudes about chemical risks, and attitudes about consumer product safety. The will also put into the field a planned Stage 2 survey, which will extend understanding of the plasticity of ENM environmental risk perception and demographic effects on the key environmental factors of resilience and lay toxicology. Better understanding of these factors will in turn provide CEIN with better predictive data on likely public response to specific types of risk information and risk management practices. Institutional/ organizational issues for the environmentally responsible development of nanotechnologies will be pursued by SOC-2’s completion of a study of equitable distribution of nanoremediation projects by the EPA, examining the sociodemographic composition of local communities surrounding such sites to determine if there are patterns related to the demographic distribution of technological risks and/or benefits in an emergent application of nanotechnology. The nano remediation project will be refined through better modeling of environmental exposures, different ENMs, attention to different media of soil and water, and the addition of more sites, if possible. The high


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benefit/high uncertainty effects already identified regarding nano remediation views by experts and publics will be modeled in the SOC-1 Stage 2 survey for reference to this project. This project will enhance CEIN’s performance by suggesting means of avoiding institutional failures among institutions the public depend on for responsible development and risk management, and will demonstrate the importance of attention to locational and distributional justice aspects of ENM environmental risks. Researchers working on the SOC-3 ENM Industry Survey project will complete all planned publications in Winter 2012, concluding the main work on this project. We are researching the viability of extending data collection to Chinese industry through CNS-UCSB researchers; if feasible, we will recruit Chinese business participants for a comparative sample and will extend the project for the period necessary to collect, analyze, and write up such data. As one outcome of SOC-3, project leader Harthorn will continue work with Nel et al. on using SOC-3 findings to assist with UC CEIN outreach activities to industry and government. In closely related risk governance research activity, the primary goals of researchers in SOC-4 for the next year are to complete publication of their expert survey results that clearly indicate important risk management and risk assessment implications of differences among risk and regulation views of experts. This project will also conduct the CEIN expert decision workshop at the beginning of the next year (Winter 2012) on regulatory decision processes, and develop an expert web survey to extend these results, per discussion with EPA Program Office Nora Savage at the June 2011 site visit. This workshop will draw on advanced expertise of Decision Research specialist Robin Gregory in running the workshop and analyzing results (Gregory’s effort will be contributed to this UC CEIN project by CNS-UCSB). UC CEIN researchers are invited participants in the workshop, so this is expected to integrate discussion about risk assessment tools across Themes in the UC CEIN. The individuals working on SOC-5 are currently preparing a manuscript that describes our scheme for prioritizing nanomaterials for regulatory action based upon their hazard and exposure potential to allow for broader impact of these important concepts. This publication will significantly further our goal of providing 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.

The individuals working on SOC-6 are currently preparing a manuscript describing our review of existing guidance documents on safe handling and disposal of nanomaterials, the exposure literature on engineered nanomaterials (ENMs), and the synthesis of these materials into the approach to safe handling of nanomaterials described in the Nanotoolkit. In addition, we plan to conduct pilot testing of the alpha version of Nanotoolkit with researchers at UCLA and use feedback to develop a beta version of the toolkit, which will be disseminated to the broader academic community. This work has already 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 Office of 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.


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Education, Career Development, Knowledge Dissemination, And Integrative Efforts 4th Year Progress (April 1, 2011 - March 31, 2012) Faculty participants: Hilary Godwin, UCLA, Professor of Environmental Health Sciences - Theme Leader Timothy Malloy, UCLA, Professor of Law Andre Nel, UCLA, Professor of Medicine; Chief, Division of Nanomedicine Updated Goals of Theme: In reorganizing the center from Interdisciplinary Research Groups (IRGs) into Themes, we integrated the outreach activities that are informed by the societal implications work in the Center into a new Theme 7 called “Societal Implications, Risk Perception And Outreach Activities”. The remaining activities that were previously part of the Education and Outreach core focus on integrative elements within the Center as well as educational and professional development activities for emerging scientists. These elements, which leverage science that is being conducted across the UC CEIN, are represented in the “Education, Career Development, Knowledge Dissemination, And Integrative Efforts” Theme discussed below. Organization and Integration of Theme 8 Projects: ED-1 Student/Postdoctoral Mentoring and Professional Development - Godwin/Nameth/Rebich Hespanha ED-2 Course development, Workshops, and Learning Tools - Godwin/Nameth ED-3 Protocols Working Group – UC CEIN Protocols Project - Godwin/Sokolow ED-4 Synergistic/Integrative Center activities - Godwin/Nameth/Rebich Hespanha ED-5 Informal Science Education - Goodwin/Nameth Major Accomplishments since February 2011: ED-1: Student/Postdoctoral Mentoring and Professional Development A Student/Postdoc Retreat and Leadership Workshop was held in conjunction with the Center

Retreat at Lake Arrowhead in March 2011. Nameth worked with students/postdocs to design a workshop that suited their needs: Networking across thematic groups and Professional Development/Career-building Skills. 22 students/postdocs attended. Each thematic group was represented.

Avery & Nameth held workshops in person (at UCSB and at UCLA) and via Skype to assist students/postdocs in preparing posters and presentations for ICEIN 2011. As a result, 7 talks and 16 posters by students/postdoc were presented successfully at ICEIN 2011.

Three UC CEIN students/postdocs were invited to give posters for the CNSI external advisory committee meeting at UCLA in July 2011, and we received informal feedback that they were some of the best-prepared and most professional presenters at the session.

A Student/Postdoc Seminar Series was initiated and five presentations were given: Shannon Hanna (UCSB) at UCLA, April 2011; Saji George (UCLA) at UCSB, July 2011; Yuan Ge (UCSB) at UCSB, August 2011; Christian Beaudrie (UBC) at UCLA, September 2011; and Courtney Thomas (UCLA) at UCSB, October 2011. Indranil Chowdhury (UCR) is scheduled to give a seminar at UCSB in March 2012.

Research-focused UC CEIN seminars were held at UCSB each month. Each monthly seminar features a short presentation by a student or postdoc representing one Theme, and an informal discussion led by a representative of another Theme. These seminars serve both professional development and research goals; students/postdocs are able to practice communication of their work, and they receive timely feedback on their research as it progresses. Because these seminars are well


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attended and broadcast via Blackboard Collaborate, they also serve as a venue for exchange of ideas between the diverse research Themes within the Center.

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.

Beginning in November 2011, Rebich Hespanha offered one-on-one Professional Development Consultations (in person or via Skype) for students and postdocs affiliated with the Center. As part of the consultations, researchers completed a professional skills self-assessment and created an Individual Development Plan (IDP) for the coming year that focuses on development in targeted areas. Five students and 1 postdoc from 3 affiliate campuses completed consultations during Fall 2011, and others are expected to participate in 2012. Follow-up with researchers who have participated in Professional Development Consultations is ongoing.

ED-2: Course development, Workshops, and Learning Tools We launched an online Nanoecotoxicology lecture series that includes 13 lectures by 10 Center

members (faculty, postdocs) on various topics informed by research in the UC CEIN. The online Nanoecotoxicology course was piloted by faculty and students at a partner institution in

Mexico (CINVESTAV) in Winter 2011. These partners have provided us with detailed feedback on the course that will be used to improve the course prior to broader dissemination.

We ran a 5-day Nanoecotoxicology Bootcamp in August 2011. This 5-day bootcamp was an outgrowth of the lecture series and of partnerships with US and Mexican universities and research institutes. The bootcamp was highly successful, and we anticipate that similar workshops will be targeted in the future towards scientists in developing countries who wish to build capacity in this field.

Godwin gave a presentation about the online course and bootcamp to a group of researchers in South Africa and members the US Armed Forces in September 2011.

The quiz feature for the Nanoecotoxicology online course was updated, allowing participants to review their quiz before submitting it, and to receive their quiz grade immediately.

A flier advertising the Nanoecotoxicology online course was developed to advertise the course for broader dissemination to the national and international scientific communities and was distributed at the NSF Nanoscale Science and Engineering Grantees meeting in Arlington, VA in December 2011.

The Nanoecotoxicology online course is being used by one of the Bootcamp participants, Professor Marin Robinson, as a teaching tool for a course at Northern Arizona University in Winter 2012 (enrollment: 17 students).

The Nanoecotoxicology online course is being offered as an independent study course “Nanomaterials in the Environment” at UCSB under the direction of Arturo Keller. Eight students are enrolled in this course for Winter 2012.

Nameth and Truong, in partnership with CEIN students/postdocs and faculty, have initiated a project to develop 5 interactive, tabletop activities for public outreach events. Three of the activities were piloted at the “Explore Your Universe” event in November 2011.

ED-3: Protocols Working Group – UC CEIN Protocols Project. • The protocols working group (PWG) has met monthly since October 2009. • 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 three publically-available protocols, a link to our protocol-development partner (the International Alliance for Nanoharmonization) and references to more than a dozen papers describing protocols developed in the UC CEIN. Several additional

http://www.cein.ucla.edu/research/UC_CEIN_research_Protocols.html�


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protocols are currently being edited and will be placed on the public website once they are approved.

• The Protocols Working Group has completed the development of the unified suspension protocol and is now working on a publication describing this approach, with Ivy Ji taking the lead on writing the manuscript.

• The Protocols Working Group has completed the initial phase of 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. Saji George, Sijie Lin, and Angela Ivask are taking the lead on writing the manuscript.

ED-4: Synergistic/Integrative Center activities • CEIN co-hosted the annual ICEIN meeting with CEINT and contributed 6 invited talks by faculty and

staff, seven talks by graduate students and postdocs, and sixteen posters to ICEIN 2011. • We held an annual retreat at Lake Arrowhead in March 2011 that involved 19 students/postdocs, 20

faculty, and 4 staff. The first part of the retreat was a SPAC Leadership Workshop, a 2-day event that was planned by Avery, Godwin, and Nameth in consultation with the SPAC. The second part of the retreat involved all 43 Center members.

• We used Elluminate/Blackboard Collaborate to webcast and record monthly Protocols Working Group (PWG) and IRG 5/HTS meetings, seminar speakers, visiting scholars, and other meetings. Webcasts of seminars are archived on the Center Data Management system.

• We partnered with UCLA’s CNSI Seminar Series to co-sponsor talks by Piotr Grodzinski (NCI) and Mark Hersam (Northwestern) during the performance period. These talks are critical in that they not only help to inform research across the UC CEIN, but also because they help us to engage and inform the larger nanoscience and engineering community at UCLA about important issues and related to the environmental implications of nanotechnology.

• We hosted Dr. Amy Wang from the TOXCAST program at the US EPA, at UCLA for a seminar on July 6, 2011, helping to solidify a key collaboration between the center and the TOXCAST program.

• Center member Hilary Godwin gave the first seminar of the academic year in the UCLA CNSI (jointly sponsored by UC CEIN) on safe handling and disposal of nanomaterials on September 2011. Dr. Godwin’s presentation summarized work performed in Project 27 with the California Nanosafety Consortium of Higher Education (CNCHE) to develop straightforward guidance for researchers on how to safely handle and dispose of ENMs in academic settings. Dr. Godwin gave a similar presentation at the NSF NSE Grantees Meeting in Arlington, VA in December 2011. These presentations served to disseminate these materials to the broader academic community, which we anticipate 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.

• Center Director Andre Nel gave the first seminar of the academic year at UCSB on the use of high throughput discovery in nano hazard ranking and the prioritization of nano safety assessment. Dr. Nel’s presentation drew an audience from across the UCSB campus and was part of a daylong event at UCSB focused on supporting a sense of community within the Center. Other events that day included a center-wide meeting, lab tours, and a beach barbecue. Approximately 35 Center members from the UCLA and UCSB campuses participated in this event

• We hosted Dr. Frank von der Kammer (University of Vienna), Dr. Vicki Grassian (University of Iowa), and Dr. Andrew Whitehead (Louisiana State University/UC Davis) as part of the monthly UC CEIN seminar series at UCSB. These visits and seminars helped to facilitate and strengthen collaborations and exchange of ideas between these researchers and Center members.


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• We held an Executive Retreat in Camarillo in November 2011 to start the planning process for our annual review and resubmission of the Center Grant to NSF and EPA.

Impacts on the Overall Goals of the Center: ED-1: Student/Postdoctoral Mentoring and Professional Development 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. The Center’s yearly Leadership Workshop and all other mentoring and professional development activities, workshops, and consultations for students and postdocs are participant-centered. 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. In addition, 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. ED-1: Course development, Workshops, and Learning Tools The goal of this project is to develop and disseminate educational outputs related to nanoscience and the environment. 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. In particular, the Center’s web-based lecture series 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. ED-3: Protocols Working Group – UC CEIN Protocols Project. 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. ED-4: Synergistic/Integrative Center activities 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. To promote cross-fertilization across UC-CEIN, Theme 8/Project 42 also facilitates the annual Executive Committee retreat and the annual Center-wide retreat. ED-5: Informal Science Education As a result of CEIN’s collaborative efforts regarding informal science education, the UC CEIN has made significant contributions to raising public awareness about nanotechnology, particularly in the greater Los Angeles area and in Santa Barbara. Our outreach programs leverage science developed in the UC CEIN and are informed by the important risk perception work performed in the UC CEIN.


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Major Planned Activities for the next Period: In the coming year, the Center will continue to offer a broad range of activities and workshops, both in-person and via Skype aimed at furthering the professional development of our students and postdocs. In addition, Hilary and Katy will work closely with Dr. Gardea-Torresdey (UTEP) to develop a formal plan for the minority-recruiting program and an implementation schedule. We will also reach out to other groups on the UCLA campus who are involved in minority recruiting to see if we can leverage their activities and experience to the benefit of the students and postdocs in our center. Based on the feedback that we received on our online Nanoecotoxiology course, we will be making modifications to the course in the upcoming project period. 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 online course. 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 UCLA Education/Outreach Coordinator is negotiating with 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. The primary goals of the of the Protocols Working Group (PWG) for the upcoming project period are to submit manuscripts describing the two major projects to date conducted in the PWG and to work with researchers within the UC CEIN to ensure that the methods that they are developing within the Center are being transitioned into standard protocols that are made publically accessible on our website. Specifically, the PWG has completed the development of the unified suspension protocol and has completed the initial phase of our brainstorming and discussions about a streamlined approach to transitioning nanotox and nanoecotox assays to high throughput, and the members of the PWG are now preparing manuscripts describing these approaches. Publishing the work of the PWG and making the protocols developed in the center publically available on the Center website are critical to ensuring that the advances made in the center are broadly disseminated to the scientific community and positively inform how nanoecotoxicology studies are performed both within and outside the Center. In the coming year, due to the refunding process, ICEIN will not be held. However, the UC CEIN will continue to provide speakers for the CEIN/CNSI (California NanoSystems Institute) seminar series at UCLA and to engage in informal science education partnerships with NISENet, the California Science Center, the Santa Monica Public Library, the SciArt program at UCLA, and local schools. 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. Through these activities, we will continue to serve as source of information about nanotechnology for the general public and to sponsor activities that engage the public in open discussions about both the benefits and the risks of nanotechnology.


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UC CEIN CORE Support Functions Year 4 Progress - April 1, 2011 - March 31, 2012 CORE A Participants: Andre Nel, UCLA - Director, UC CEIN; Professor, Medicine; Chief, Division of Nanomedicine David Avery, UCLA - Chief Administrative Officer, UC CEIN CORE B Participants: Jeffrey Zink, UCLA - Professor, Chemistry and Biochemistry Zhaoxia Ivy Ji, UCLA - Senior Research Scientist, UC CEIN (CORE B) CORE C Participants: Yoram Cohen, UCLA - Professor, Chemical and Biomolecular Engineering Taimur Hassan, UCLA - Data Manager/Programmer, UC CEIN (CORE C) CORE D Participants: Kenneth Bradley, UCLA - Associate Professor, Microbiology Robert Damoiseaux, UCLA - Scientific Director, MSSR (CORE D) Introduction: The UC CEIN has identified four core function areas that form the basis for the Center's research infrastructure and provide key support functions. The core activities of the Center play the valuable role of facilitating interaction across the Center, ensuring that the Center's infrastructure exists to support the development and execution of the variety of research projects being conducted by each of the 7 major research themes of the Center. Each core is led by a research staff who has the technical skills to interact across Center projects. Each of the cores is housed within the California NanoSystems Institute (CNSI) facility at UCLA. Ideas for future development of core activities arise through ongoing discussion with theme leaders based on the direction and findings of the Center's overall research agenda. CORE A: UC CEIN Center Administration Goal: UC CEIN Center Administration - The UC CEIN organizational strategy is to maintain a strong 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. Our organizational structure allows for selection, prioritization, distribution, and management of resources within a multi-institutional structure. Integration and Interactions Across the Center: The Administrative Core was established with the founding of the Center in 2008 and has operated under the continuous management of the Center CAO (Avery). Core A interacts with all projects across the Center and is responsible for coordinating the reporting of Center activities. Core A also provides coordination for all Centerwide activities (workshops, seminars, conferences, site visits, retreats) and the CAO provides joint supervision of the CEIN Education Coordinator (Theme 8), Senior Research Scientist (Core B), and Data Manager (Core C).


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Major Accomplishments since February 2011: CORE A: Core A continues to provide financial, administrative, management, and organizational oversight to Center activities. In addition to the usual grants management and financial management requirements, the Center administration played a key role in the following activities this year:

• Co-organized and co-hosted the 3rd International Conference on the Environmental Implications of Nanotechnology, May 2011, Duke University

• Planned and executed CEIN Centerwide retreat and Student/Postdoctoral development workshop at Lake Arrowhead, March 2011

• Planned and executed the June 2011 CEIN NSF/EPA Site visit • Coordinated the centerwide progress reporting process • Developed a planning process for the 2012 Site Visit/Center renewal cycle • Supported the organization of Center's Education and Public Outreach events

CORE B: ENM Acquisition, Characterization, and Distribution The former IRG1 (Nanomaterial Standard Reference and Combinatorial Libraries and Physical-Chemical Characterization”) in the CEIN was the precursor of both the current Core B and Theme 1. IRG1 was focused on the synthesis and procurement of libraries of nanomaterials and the thorough characterization of the materials for use by the entire center. We have moved the fundamental preparation, characterization, dispersion and distribution service functions into Core B in order to focus on property-activity relationships in Theme 1. Core B is an integral part and foundation of the Center. All of the nanomaterials for the Center are thoroughly characterized as part of the Core’s activities. Most of the projects in Theme 1 have evolved from the compositional nanomaterial libraries in the former IRG1 because of the shift in the focus from more general effects of material composition to the exploration of specific properties of those materials once initial biological screening or hazard ranking have taken place. Examples include the acquisition of more metal oxides to study the role of band energies, the use of calcination to change the surface properties of fumed silica, and the introduction of aspect ratio and shape variation to the libraries of mesoporous silica and ceria. This dynamic process has been sped up by the accelerated rate of discovery in the high-throughput studies in Theme 2. Goal: Core B has four main goals: 1) to assemble nanomaterial standard reference and combinatorial libraries, 2) to characterize the physiochemical properties of the nanomaterials, 3) to develop efficient methods of dispersing the particles in relevant media, and 4) to provide easy accessibility to and distribution of the particles and data associated with them.

1. The standard reference and combinatorial nanomaterial libraries are the sources of materials 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, including metals, metal oxides, and carbon nanotubes, which have been introduced into the library. The libraries' contents are provided in Appendix 2.

2. The major “service” function of Core B involves characterization of the nanomaterials as they are synthesized or acquired. Its goals are to thoroughly characterize nanoparticles of commercial importance and to make them available in usable forms and quantities for in vitro and in vivo studies. “Conventional” particles of commercial importance (including both nanoparticles mass-produced in multi-kilogram quantities such as titania, ceria, silica, zinc oxide, and scientifically-important high value particles including gold, silver copper, cobalt)


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have been characterized. A list of the methods that are used and the information resulting from the measurements are given in Appendix 1.

3. Development of methods of dispersing nanoparticles in biologically relevant media is another major service function. A systematic study of TiO2 nanoparticle dispersion by the former IRG-1 provided an important insight: light scattering measurements taken during the study revealed that the high ionic strengths of cell culture media inhibited dispersion of TiO2 nanoparticles with respect to levels of dispersion in distilled water. If TiO2 suspensions in cell culture media were directly used for nanotoxicity studies, such suspensions would lead to incorrect dose metric estimations and complicate interpretation of toxicity results. It was found that when BSA, diphosphotidyl choline, or fetal bovine serum (FBS) was added, the TiO2 agglomerate size was greatly reduced. Based on these results, similar approaches were used to disperse other nanoparticles and nanotubes in the library. Core B continues identify the best methods for dispersing a wide variety of nanoparticle types. Examples of dispersed nanoparticle solutions available within the Center are shown in Appendices 3 and 4.

4. Core B is also responsible for the distribution and tracking of materials across Center projects. Inter- and intra-campus distribution of both particles and associated characterization information have been very reliable and efficient.

Integration and Interactions Across the Center: Core B is closely tied to the activities of Theme 1 and operates under the direction of Theme 1 leader Jeffrey I. Zink. Core B maintains the Center’s nanomaterials library and coordinates the synthesis or acquisition and subsequent distribution of engineered nanomaterials (ENMs) across research projects and themes. Efficient distribution of ENMs is facilitated by close interaction with both the toxicity groups (to understand the major findings of current ongoing studies) and the material synthesis projects (to redesign materials as needed to modify material properties). To ensure that material characterization is conducted under relevant exposure conditions, Core B is closely affiliated with the Center’s cellular and environmental study investigators to determine the relevant range of characterization procedures and media required for each material. Characterization parameters that are key to ongoing studies include size and distribution analysis in relevant media, agglomeration kinetics, sedimentation studies, and surface charge analysis (see Core B - Table 1). Major Accomplishments since February 2011:

• Developed a new compositional library containing 24 metal oxide nanoparticles that project valence and conduction band energies in terms of biological redox potential in mammalian and bacterial systems. Eight of the materials (ZnO, CuO, Co3O4, Fe3O4, TiO2, WO3, Sb2O3, SiO2) were synthesized by Dr. Madler’s and Dr. Brinker's groups; 16 materials (Al2O3, CeO2, Cr2O3, CoO, Fe2O3, Gd2O3, HfO2, La2O3, In2O3, Mn2O3, NiO, Ni2O3, SnO2, Y2O3, Yb2O3, ZrO2

• Established a SiO

) were acquired from commercial sources.

2

• Created a single-walled carbon nanotube (SWCNT) library to study the effects of hydrophobicity, metal impurity, and dispersion state. Materials were characterized and distributed to research groups (Projects HTS-7, FT-4, FT-5, MFW-1, MFW-2) for toxicity, fate, and transport studies.

combinatorial library to study crystallinity and surface structure effects. Silica from Dr. Brinker's Group (Project ENM-3), Dr. Zink's Group (ENM-1), and commercial sources were characterized and studied in toxicity studies in Project HTS-4. Studies indicate that fumed silica is the most toxic and toxicity can be correlated to the surface display of silanol groups and strained three-silicon rings.

• Developed protocols for efficient dispersibility of metal and metal oxide nanoparticles and carbon nanotubes.


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• Developed a tracking system to monitor usage of library materials across Center projects. Impacts on the Overall Goals of the Center: The major impacts on the overall goals of the center directly follow from the Core Bs’ major accomplishments. Efficient distribution of the nanomaterials necessary for the high throughput screening studies being carried out by several research groups has been made possible through the assembly of a nanoparticle library containing over 100 different types of particles (condensed version in Appendix 2). All Center researchers now have immediate access to information about the physical and chemical properties of available nanomaterials due to the characterization of all nanoparticles in the current library (methods in Appendix 1, library in Appendix 2). Identification of efficient and reliable dispersion methods was a major step forward in helping the researchers prepare samples in stable suspended forms for biological studies. Samples prepared and characterized by Core B are currently being used by all research groups in the Center.


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Core B - Table 1. PHYSICAL/CHEMICAL CHARACTERIZATION OF COMMERCIALLY IMPORTANT NANOPARTICLES Overview of important chemical and physical properties Upon arrival, commercial nanoparticles are thoroughly characterized and prepared for the in vitro and in vivo studies conducted by Theme 2. Characterization involves identification of the intrinsic properties that are defined by the chemical composition, structure and morphology (shape) of the particles themselves. Intrinsic properties of nanoparticles include:

1. Chemical composition (metal or metal oxide, mixed metal or metal oxide) 2. Structure (crystal (highly ordered), glass (amorphous), or porous glass structure) 3. Size and shape (sphere, rod, wire, tetrahedron, cube, plate) 4. Charge (isoelectric point) 5. Solubility (equilibrium) and solubilization rate

These properties are determined by using the physical methods discussed below. This analytical component of the research program is vital to its success; biological responses to the nanoparticles are expected to be influenced strongly by such factors as crystallinity, size and shape, surface area, degree of aggregation, charge and solubility. Manufacturers’ statements about primary particle size and chemical composition may be insufficient for the information required to perform in vitro and in vivo biological experiments. Analytical Methods for Physical and Chemical Characterization of Nanoparticles The most important physical methods that are used in the Center are identified and briefly defined below.

Transmission Electron Microscopy (TEM) & Scanning Electron Microscopy (SEM): The operation principles of electron microscopy are based on the interaction between an electron beam and a solid surface. In TEM, transmitted or forward-scattered electrons are used to obtain images; in SEM, images are obtained through analysis of back scattered or secondary electrons. With enough representative images, TEM and SEM can be used to obtain primary size, morphology, topography, state of agglomeration, and even some crystallographic information about nanoparticles.

X-ray Diffraction (XRD): When a coherent X-ray beam is directed at a sample, interaction of the X-rays with the sample creates diffracted beams that can be related to interplanar spacings in the crystalline sample. Following Bragg’s law (nλ=2dsinθ; where n is an integer, λ is the wavelength of the X-rays, d is the interplanar spacing, and θ is the diffraction angle), XRD can be used to identify crystalline phase and structure and to determine crystallinity. Primary size of nanoparticles can also be derived from XRD patterns using the Sherrer equation (S=λ/ωcosθ; where S is the particle size and w is full-width-at-half-maximum of the diffraction peak).

Dynamic Light Scattering (DLS): In DLS, a monochromatic light beam directed at a particle suspension is scattered. Due to the random Brownian motion of the particles, the intensity of the scattered light fluctuates with time. A translational diffusion coefficient, Dt, can be determined based on the scattered light intensity using an autocorrelation function. Hydrodynamic diameter (dH) of nanoparticles can then be estimated from the Stokes-Einstein equation (dH=kT/3πηDt; where k is the Boltzmann constant, T is the temperature, and η is the viscosity). Size distribution and state of agglomeration can also be derived from the DLS measurement.


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Zeta Potential & Electrophoretic Mobility (EPM): Surface charge can be determined indirectly by measuring the zeta potential (ζ) or electrophoretic mobility (EPM) of the particles. EPM is defined as the velocity of a particle per electric field unit and is obtained by applying an electric field to the particle suspension and measuring average particle velocity. Zeta potential can be calculated based on average particle velocity using the Smoluchowski or Huckel equations. The magnitude of zeta potential provides an indication of the potential stability of the nanoparticle suspension. Nanoparticles with zeta potential more positive than 30 mV or more negative than -30 mV are generally considered stable. By adjusting pH, an isoelectric point (pHiep) can also be determined. Nanoparticles are positively charged below pHiep and negatively charged above pHiep.

Gas Absorption/Desorption, Brunauer-Emmett-Teller (BET) Surface Area Analysis: The BET method is based upon the adsorption of gas (typically nitrogen) on a surface. The quantity of gas adsorbed at a given pressure is used to determine the specific surface area. Assuming that the particles have a solid and uniform spherical shape with a smooth surface, an average particle size can also be estimated.

Thermo-Gravimetric Analysis (TGA): TGA is typically used to determine a material’s thermal stability and its fraction of volatile components by monitoring mass change of the material as a function of temperature and time. Based on the weight loss or gain profile, kinetic process such as dehydration, oxidation and decomposition can be identified and the corresponding moisture content and organic content can be determined. Cross Polarization/Magic Angle Spinning Solid State NMR (CP/MAS SSNMR) This technique provides molecular information about solid samples similar to that obtained from conventional solution phase NMR. The attachment of linkers is observed by using 29Si, and information about the attached surface functional groups and molecular machines is obtained from 13C and 1H NMR. UV/Visible Absorption Spectroscopy Absorption spectroscopy provides information about molecules in pores or attached to the surface of particles. It is especially useful for monitoring attachment of molecules with chromophores that have large molar absorptivities in the ultraviolet or visible regions of the electronic spectrum. The technique can also be used for nanoparticle suspension stability evaluation. In this case, the absorbance at a characteristic wavelength, which can be linearly correlated to the nanoparticle concentration, is monitored as a function of time. Fourier Transform Infrared Spectroscopy (FTIR) Infrared spectroscopy provides information about molecules in pores or attached to the surface of particles. It is especially useful for monitoring attachment of molecules that have vibrational absorption bands in regions that are not obscured by absorptions of the particle itself. X-ray Photoelectron Spectroscopy (XPS) In XPS, the sample is illuminated with soft x-rays in an ultrahigh vacuum, which leads to the production of photoelectrons from the sample surface. The characteristic binding energy of each element can be calculated through energy analysis of the generated electrons. The binding energy of a particular electron is also affected by its surrounding environment, and therefore can also provide oxidation state information. Peak area for each element allows for quantitative analysis. In the case of analysis for nanoparticles, defect information can also be obtained from the spectra.


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Inductively coupled plasma mass spectroscopy (ICP-MS) ICP-MS employs a plasma (ICP) as the ionization source and a mass spectrometer (MS) analyzer to detect the ions produced. The instrument can perform multi-elemental analysis with high sample throughput and excellent sensitivity. It can determine analyte concentration down to part per trillion level (ppt). ICP-MS is therefore one of the most powerful techniques for elemental analysis and purity quantification in nanomaterials. Flame Photometry Flame photometry is used for determination of certain metal ions such as sodium, potassium, and calcium. It relies on the principle that an alkali metal salt drawn into a non-luminous flame will ionize, absorb energy from the flame, and then emit light of a characteristic wavelength as the excited atoms decay to the unexcited ground state. The intensity of emission is proportional to the concentration of the element in the solution. Flame photometry is therefore suitable for both qualitative and quantitative elemental determination. Manufacturers of commercial nanomaterial samples provide statements of varying levels of detail and accuracy about the composition. Core B carries out a rigorous set of replicate studies for each material and provides uniform information about each type of nanoparticle to Projects 1 and 2. In addition, core B provides CEIN projects with dry powder samples, particles suspended in requested media, and suggestions for preparations based on experiments using a variety of tissue culture in biological media together with dispersal agents that will be carried out in the core. High throughput dynamic light scattering (DLS) is used for these experiments. Specialized physical and chemical characterization of nanoparticlesSome of the metal nanoparticles may require additional characterization because of the likelihood of oxidation and dissolution; native oxide coatings are expected. To test that the metal interior is intact, ionization methods such as x-ray photoelectron spectroscopy (XPS) or Auger electron spectroscopy are carried out. Ion milling removes the outer oxide layers to expose the metal, and characteristic peaks in the spectra identify the oxidation state. Scattering from the crystalline metal based on X-ray diffraction (XRD) and the Sherrer method allows calculation of the diameter of the metal (inside the oxide layer). Dissolution is studied by suspending the nanoparticles in relevant media for a fixed period of time, separating the particles by centrifugation, and measuring the concentration of dissolved ions in the supernatant by ICP-MS or ICP-OES. These measurements are used not only to calculate the dissolution rates, but also to characterize the metal released from stealth mesoporous silica nanoparticles with metal cores.


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Core B - Table 2. The Nanoparticle Library in the CEIN

Composition Size (nm) Shape Phase/Structure

TiO2

~25 Spheres 80% Anatase & 20% Rutile

6, 10, 15, 40, 60, 100, 260 Spheres Anatase

5, 12, 60, 140 Spheres Rutile

4×15, 8×45, 12×60, 10×100, 30×100, 80×300 Rods Rutile

5×1000 Wires Rutile

5, 10, 15, 20, 30, 60, 130, 160, 220, 240, 460, 600 Spheres Amorphous

15×70 Rods Amorphous

CeO2 5, 7, 10-12, 15-30, 20-70 Cubes Crystalline

8×33, 7×39, 7×51, 7×75, 7×94, 12×500, 12×1000-2000 Rods Crystalline

ZnO ~20 Spheres Crystalline

Fe-ZnO 20, 15, 14, 12, 8, 8, 8 nm with 0, 1, 2, 4, 6, 8, 10 atomic weight% Fe Spheres Crystalline

SiO2

5, 8, 30, 50, 80, 130 (Commercial) Spheres Amorphous

6, 30, 40, 60, 70, 135, 200, 500, 600, 2000 (In-House) Spheres Amorphous

81×137, 94×209, 72×201, 65×308, 69×446 Rods Mesoporous

Ag 8, 21, 40 (Monodispersed); 57 (Polydispersed) Spheres Crystalline

Pd 13 Spheres Crystalline

Pt 5-10 Spheres Crystalline

SWNT

~3×1500 (Caboxylated) Nanotubes Crystalline

0.7-0.9×450-2000 (Purified) Nanotubes Crystalline

1.5×1000-5000 (As-prepared, Purified) Nanotubes Crystalline

0.8-1.2x100-1000 (As-prepared, Purified) Nanotubes Crystalline

MWNT

2-6 nm inner diameter, 5-20 nm outer diameter, 1-10 µm length (As-prepared) Nanotubes Crystalline

2-6 nm inner diameter, 5-20 nm outer diameter, 1-10 µm length (PEI-coated) Nanotubes Crystalline

68±20 nm outer diameter, up to 300-400 µm length (Aligned) Nanotubes Crystalline


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Core B - Table 3. SiO2 Nanoparticle Distribution in UC CEIN (2010-11).

Sample Name Size (nm)

Quantity (g) Requestor IRG # Shipping

Date Commercial 5 nm colloidal SO2 5 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010 Commercial 30 nm colloidal SO2 30 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010 Commercial 50 nm colloidal SO2 50 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010 Commercial 80 nm colloidal SiO2 80 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010

Commercial 130 nm colloidal SO2 130 3.6 Robert Miller 3 3/23/2010

0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010

AML silica #2 6 0.3 Robert Miller 3 3/23/2010

0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010 AML Aug 6 2010-2 silica 8 8 0.01 Sijie Lin 5 9/9/2010

AML silica colloid July 7 #2 29 0.45 Robert Miller 3 3/23/2010 0.03 Angela Ivask 5 3/24/2010 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010

AML silica #4 32 0.01 Sijie Lin 5 9/9/2010

AML Mar 31B Silica 65 10 Reginald Thio 4 5/4/2010 5 Reginald Thio 4 6/11/2010

AML Aug 6 2010-5 silica 65 10.8 Reginald Thio 4 8/20/2010

AML silica colloid July 2 #1 72 0.49 Robert Miller 3 3/23/2010 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010 0.01 Sijie Lin 5 9/9/2010

AML Mar 31A Silica 135 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010

AML April 1C Silica 200 9 Reginald Thio 4 5/4/2010

0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010 0.01 Sijie Lin 5 9/9/2010

AML Aug 6 2010-6 silica 200 10 Reginald Thio 4 8/20/2010

AML Mar 30B Silica 530 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010 0.01 Sijie Lin 5 9/9/2010

AML Mar 30A Silica 640 10 Reginald Thio 4 5/4/2010

AML Mar 31C Silica 2000 0.02 Haiyuan Zhang & Saji George 2 & 5 5/12/2010

0.01 Sijie Lin 5 9/9/2010


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Core B - Table 4. Silver Nanoparticle Distribution in UC CEIN.

Sample Name Size (nm)

Quantity (g) Requestor IRG # Shipping

Date

Silver NP Powder (Dr. Heok) ~20 0.5 Gary Cherr & Arturo Keller 3 & 4 3/19/2009 1 Gary Cherr & Arturo Keller 3 & 4 3/19/2009

0.5 Angela Ivask 5 3/26/2011

Silver NP Powder (Sigma Aldrich) 57 0.05 Dongxu Zhou 4 11/12/2010 0.25 Angela Ivask 5 11/23/2010

20 nm BioPure Ag Nanoparticles 21

0.03 Reginald Thio 4 10/5/2010 0.001 Saji George 2 11/1/2010

0.0015 Angela Ivask 5 11/23/2010 0.006 Angela Ivask 5 12/8/2010

40 nm BioPure Ag Nanoparticles 40

0.03 Reginald Thio 4 10/5/2010

0.001 Saji George 2 11/1/2010

0.0015 Angela Ivask 5 11/23/2010

0.006 Angela Ivask 5 12/8/2010


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CORE C: Data Repository and Nano Collaboratory Goal: 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 provides core support for data management, data storage, IT support, web-based collaborative infrastructure and computational needs of the CEIN. The CDM group also plays a key role in the national NanoInformatics effort. Our computational capabilities have enabled collaborations with external groups, including the EPA's ToxCast program. Integration and Interactions Across the Center: The Data Repository and Collaborative Infrastructure Core (Core C) is an outgrowth of the research in Theme 6. The technological infrastructure (servers, workstations, and backup equipment) was developed to keep pace with the data generated by Center projects and to meet the computational needs of the Center's data analysis and modeling work. Core C has implemented a center-wide file and data repository, hosts the Center's public website, and hosts software that allows for searching/organizing/mining of research data uploaded to the system. The data manager (Hassan) works with individuals from each Center project to facilitate the uploading of data into the data repository. Major Accomplishments since February 2011: CORE C:

• Continued to maintain and upgrade the hardware and software of the CEIN collaborative infrastructure.

• Developed the "Nanocrawler" online tool, which improves searching and mining of the research data and information that has been uploaded to the data repository. Currently, data for 126 nanoparticles (including physicochemical data and experimental condition data) can be indexed using the Nanocrawler.

• Created a separate sub-site on the collaborative infrastructure to host the Nanoecotoxicology online web course. Users who have been granted password access to a secure site can watch lectures, participate in online quizzes, and review course reading materials.

CORE D: Molecular Shared Screening Resource (MSSR) Goal: The MSSR provides scientific and technical consultation in the planning and execution of the high throughput experiments conducted by CEIN researchers. Numerous high throughput and multiplex assays implementing high content readouts have been developed. MSSR staff assists in the translation of existing low throughput assays and de novo establishment of novel assays. The expertise and technical capabilities available through the MSSR (housed in the UCLA California NanoSystems Institute) make this facility uniquely suited to handle a wide variety of assays, including those aimed at exploring the interactions between nanomaterials and bacteria, yeast, animal cells, and whole animals (zebrafish). Integration and Interactions Across the Center: The MSSR is currently engaged in a number of ongoing CEIN research projects. The MSSR provides support and input on the translation of assays from the HTS lab into the robotic format of the MSSR. The current range of CEIN assays that involve ongoing MSSR support are:


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Assay Name Cell Type NM cellular toxicity during photoactivation RAW 264.7 TiO2 Uptake of Ag plates RT-W1 Cells Ag Multiparametric Assay RAW 264.7 Silica Multiparametric Assay BEAS-2B Silica Multiparametric Assay BEAS-2B Metal Oxide Multiparametric Assay RAW 264.7 Metal Oxide

Multiparametric Assay MCG/7 Breast Cancer Cell MSNP

Brightforce Image Analysis Wildtype (AB) Embryos Metal Oxide HC Flourescence Imaging Transgencic Zebrafish Metal Oxide

Bacterial Deletion Strain E-Coli Ag-citrate (10 nm at 2mg/L)

Bacterial Deletion Strain E-Coli Ag-citrate (20nm at 4mg/L) Bacterial Deletion Strain E-Coli Ag-BPEI NP (1mg/L) Bacterial Deletion Strain E-Coli Ag-PVP NP (4mg/L)

Major Accomplishments since February 2011: CORE D:

• The MSSR continues to provide support in the development and translation of cellular and organism studies to high throughput. Currently the MSSR is actively involved in a dozen ongoing HTS studies, primarily in Theme 2.

• Through the seed funding mechanism of the Center, the MSSR worked with Patricia Holden's group at UCSB to translate bacterial studies to a high throughput format and conducted validation studies to ensure the accuracy of the translation. The assays were successfully translated to HTS, and this work will continue as HTS-3 under the direction of Hilary Godwin at UCLA.

Documents

University of California Center for Environmental ......HTS-2 Development of high content screening of nanoparticle toxicity using zebrafish models – Nel/Lin ... TER-5 DEB modeling