Diversity of Diversity of SizeSize, , ShapeShape, and, and Composition Composition in the Nanomaterial Libraryin the Nanomaterial LibraryZhaoxia Ji and Jeffrey I Zink

Establishing a nanomaterial library thatencompasses a broad range of chemicalcompositions sizes and shapes is a necessity for

Cube TiO2d=10 nm

Zhaoxia Ji and Jeffrey I. ZinkUC Center for Environmental Implications of Nanotechnology, University of California, Los Angeles, California, 90095, USA.

compositions, sizes, and shapes is a necessity formechanistic and high throughput nanotoxicitystudies. Currently, the nanomaterial library has beenexpanded extensively from the initial three majorcommercial metal oxides (TiO2, CeO2, and ZnO) to a

CdSed=30 nm Rod( 2, 2, )

variety of new compositions including metals (Au,Ag), quantum dots (CdS, CdSe), and carbonnanotubes (SWCNT, MWCNT). For eachnanomaterial, the particle size has been well 20 nm

Ad 130 Wicontrolled from a few nanometers to severalhundred nanometers. In addition, nanomaterialswith shapes ranging from spheres, cubes, rods, towires were also successfully synthesized.

Aud=130 nm Wire

NSF: EF‐0830117 

SizeSize ShapeShape CompositionComposition50 nm

Optimization of Nanoparticle Dispersion and Stability in Cell Culture MediaZhaoxia Ji Xiang Wang and Jeffrey I ZinkZhaoxia Ji, Xiang Wang, and Jeffrey I. Zink

UC Center for Environmental Implications of Nanotechnology, University of California, Los Angeles, California, 90095, USA.

It is well known that nanoparticles agglomerateextensively upon addition to cell culture media. If

30 min 24 hrthe agglomerates were used withoutmodification for nanotoxicity studies, the doseestimation would be innacurate and theinterpretation of the toxicity results would be

Large Agglomerates

MajorSedimentation

Medium Alone complicated. It is very important to developeffective methods for preparing well‐ dispersednanoparticle suspensions. Our results show thatwhen proteins and serum, (natural components

NP Stock Solution

Nice 

30 min 24 hr

Stable Medium w/ 

of physiological fluids) are used as dispersingagents, highly dispersed nanoparticlesuspensions can be obtained. The resultingdispersions remain stable even after 24 hours.

NSF: EF‐0830117 

Dispersion SuspensionDispersing Agent

Effects of Soluble Cadmium Salts vs. CdSe Quantum Dots on the Growth of Planktonic Pseudomonas aeruginosa

John H. Priester1, Peter K. Stoimenov2, Randall E. Mielke3, Samuel M. Webb4, Christopher Ehrhardt5, Jin Ping Zhang6, Galen D. Stucky2, Patricia A. Holden11Donald Bren School of Environmental Science & Management, 2Department of Chemistry and Biochemistry, 5Earth Sciences, 6Materials Research Laboratory, University of California, Santa Barbara, CA 93106, 3Center for Life Detection, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 911094 Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, Menlo Park, CA 94025

The effects of CdSe QDs vs. Cd(II) ions on P. aeruginosa growth were investigated.  Above a total cadmium concentration threshold, QDs impaired growth more than Cd(II) ions.  Scanning transmission electron microscopy (STEM) images showed cellular destruction with QDs (below, right) not observed with Cd(II) (below, middle).  Reactive oxygen species (ROS) concentrations were also higher with QDs, supporting a specific and larger effect of nanoparticles above the threshold. (Priester et. al, 2009, Environmental Science & Technology).

d dControl Cd acetate –treated CdSe QD –treated

NSF: EF‐0830117 

500 nm500 nm 500 nm

Toxicity of nanomaterials in the marine environmentHunter Lenihan, Robert Miller, Shannon Hanna, Arturo Keller

Our work on marine organisms shows that impacts of nanoparticles (NP) depend on the type of NP and its behavior in the environment.  Zinc oxide NPs are toxic to marine 

Bren School of Environmental Science and Management University of California Santa Barbara, CA USA

Model fits to experimental data h i d dphytoplankton because they dissolve and release zinc into 

seawater.  Titanium dioxide particles, in contrast, do not dissolve and had no effect on phytoplankton growth.Top figure on shows toxicity of zinc oxide to phytoplankton: diamond‐shapes are highest concentrations the NP, open circles the lowest.  Lines on graph represent fits of results predicted by 

showing decreased growth rates in a marine diatom due to exposure to zinc oxide NPs.

Dynamic Energy Budget model: the empirical data closely match the model predictions.  

Bottom‐dwelling marine amphipods, which live in soft‐sediments that can trap and accumulate NPs, died when exposed to low doses of zinc oxide NPs dissolved in seawater (an unnatural 

96hr water exposure) 80

100

10 day sediment exposure

90

100

(exposure regime; see figure below on left). However, when exposed to the zinc oxide in sediments (a natural exposure regime; see figure below on right), amphipods tolerated very high concentrations of NPs. This work suggests toxicity of metal oxide NPs in marine environments may be alleviated by binding to bottom sediments However NPs bound in sediments may

Sur

viva

l (%

)

0

20

40

60

100% mortality % S

urvi

val

40

50

60

70

80

NSF: EF‐0830117 

to bottom sediments. However, NPs bound in sediments may create problems in the future due to complex process such as metal diagenesis.  ZnO (mg L-1)

0 1 2 3 4 5

0

[ZnO] (mg L-1)

0 20 40 60 80 10040

Negative effect of TiO2 and ZnO nanoparticles on soil microbial communitiesYuan Ge1,2, Joshua Schimel3 and Patricia Holden1,2

1Institute for Computational Earth System Science, 2Bren School of Environmental Science & Management,3Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara

B th TiO d Z O h ti6

8

soil)

15 days incubation010D TiO2 ZnO

P=0.013Both TiO2 and ZnO show negativeeffects on extractable soil DNATiO2: linear relationshipZnO: nonlinear relationship

y = -0.936x + 4.215R² = 0.996

y = -3.512x + 4.136R² = 0.4510

2

4

DN

A (µ

g g-1

Toxicity: ZnO>TiO2Higher slope for ZnO dose‐response curveSignificantly lower DNA for ZnO at the sameexposure concentration (0.5 mg g‐1 soil)

0 0.5 1 1.5 2 2.5MeO (mg g-1 soil)

8

10so

il)60 days incubation

010D TiO2 ZnOP=0.000

Time dependency:Stronger dose‐response after 60, vs. 15, daysGreater change over time with TiO2

y = -2.299x + 6.389R² = 0.922

y = -5.968x + 4.897R² = 0.4650

2

4

6

DN

A (µ

g g-1

s

NSF: EF‐0830117 

00 0.5 1 1.5 2 2.5MeO (mg g-1 soil)

Bioaccumulation of nano‐TiO2 in freshwater herbivoresKonrad J Kulacki Bradley J CardinaleKonrad J. Kulacki, Bradley J. Cardinale

Department of Ecology, Evolution, and Marine Biology, University of California Santa Barbara

Monocultures of 20 of the most common species of freshwater phytoplankton in North America were cultured in media containing increasing concentrations of TiO

2.0

in media containing increasing concentrations of TiO2nanoparticles.  Each algal monoculture was then fed to Daphnia pulex (one of the most widespread freshwater herbivores) for 24h, after which, Daphnia were placed in spring water, allowed to clear their guts, and then removed 

1.5

aphn

ia p

u lex

and analyzed for TiO2 in tissues. Daphnia accumulated nano‐TiO2 in proportion to ambient concentrations, and this was true for nearly every species of algae fed to this consumer (each algal species is represented by a different line in graph at right). We are currently determining 0.5

1.0

g Ti

O2

per D

a

line in graph at right).  We are currently determining whether accumulation was due to trophic transfer or direct consumption.  Either way, our results show that nano‐TiO2has the potential to accumulate at higher trophic levels in a food web. 0 50 100 150 200 250 300 350

TiO2 exposure concentration (mg/L)

0.0µg

NSF: EF‐0830117 

p ( g )

Stability and Aggregation of Metal Oxide Nanoparticles in Natural WatersArturo A. Keller, Dongxu Zhou and Hongtao Wang

U i f C lif i S B bUniv. of California, Santa Barbara

There is a pressing need for information on the mobility of nanoparticles in the complex aqueous matrices found in realistic environmental conditions. To address these questions, we dispersed three metal oxide nanoparticles (TiO2, ZnO and CeO2) in samples taken from eight different aqueous media associated with seawater, lagoon, river, and groundwater (Fig. 1). The electrophoretic mobility of the particles in a given aqueous media was dominated by the presence of natural organic matter (NOM)d h d d d f d b d h l f l d hand ionic strength, and independent of pH. NOM adsorbed onto these nanoparticles significantly reduces their aggregation, 

stabilizing them under many conditions. The transition from reaction to diffusion limited aggregation occurs at an electrophoretic mobility from around ‐2 to ‐0.8 m s‐1 V‐1 cm (Fig. 2). These results are key for designing and interpreting nanoparticle ecotoxicity studies in various environmental conditions.

0.8

1.0

Fig. 10.8

1.0

ency

(-)

ZnO

CeO2

TiO2

Fig. 2

0.2

0.4

0.6

C/C

o

Mesocosm freshwaterStormwaterMesocosm effluentTreated EffluentLagoonGroundwaterS t Cl Ri

0.2

0.4

0.6

Atta

chm

ent e

ffici

NSF: EF‐0830117 0.0

0 100 200 300 400

Time (min)

Santa Clara RiverSeawater

0.0-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0

EPM (m s-1 V-1 cm)

Correlation between total nanoparticle surface energy and cell responsePonniseril Somasundaran and Xiaohua Fang 

C l bi U i iColumbia University

We explored the toxic effects of metal oxide nanoparticles on N. Europaea cultures. In general, cell size scales inversely with the total surface energy of the nanoparticles. When the total surface energy increases, cell size decreases (Fig. 1), except

Fig. 2

surface energy increases, cell size decreases (Fig. 1), except for the CeO2 particles. It is clear that the cells were subjected to severe stress by ZnO and TiO2 nanoparticles (Fig. 2). The cells became smaller upon contacting ZnO.  Also the membrane and cell walls became distorted and fragmented under the stress of these nanoparticles.

Fig. 1

NSF: EF‐0830117 

Highthroughput Screening for Rapid Cytotoxicity Assessment of NanomaterialsSaji George ¶, Tian Xia ¶, Suman Pokhrel ‡, Robert Damoiseaux †, Ken Bradley , Lutz Madler ‡, Andre Nel¶

¶ Dept. of Medicine ‐ Div. of NanoMedicine, University of California, Los Angeles, CA, USA  ‡IWT Foundation Institute of Materials Science, Department of Production Engineering, University of Bremen, Germany, †Molecular Shared Screening Resources, University of California, Los Angeles, CA, USA  Dept of Microbiology, Immunology & 

Mol Genetics, University of California, Los Angeles, CA, USA

In order to bridge the gap between the faster growing list of engineerednanomaterials and their safety assessment, we devised a high throughputcytotoxicity screening that used automated liquid handling techniques andautomated epifluorescence microscopy to assay for multiple cytotoxic events innanoparticle treated cells. The microscopic images of nanoparticle treated cellsft t i i ith fl i di t d (U l ft l) l dafter staining with fluorescence indicator dyes (Upper‐left panel), was analyzedto measure and score the percentage of cells affected for the sublethal andlethal cytotoxic events (heatmap‐upper right panel). Dissolution of ZnO andrelease of Zn2+ ions was found to mediate toxicity of ZnO NPs and we achievedreduction in cytotoxicity of ZnO by doping ZnO with iron that changed thereduction in cytotoxicity of ZnO by doping ZnO with iron that changed thematerial matrix to slow Zn2+ release (lower panel). This work demonstrated theutility of a high throughput, integrated biological oxidative stress responsepathway to perform hazard ranking of nanoparticles, in addition to showinghow this assay can be used to improve nanosafety by decreasing ZnO

NSF: EF‐0830117 

how this assay can be used to improve nanosafety by decreasing ZnOdissolution through iron doping.

Zebrafish model for toxicity screening of nanomaterials David Schoenfeld, Saji George, Tian Xia, Yan Zhao, Lin Shuo, Andre NelDavid Schoenfeld, Saji George, Tian Xia, Yan Zhao, Lin Shuo, Andre Nel

UCLARapid development of nanotechnologyincreases the possibility of exposure tonanomaterials, however, their potentialt i it t h i l l k Wtoxicity to humans is largely unknown. Weuse zebrafish as a model organism becausethe National Institutes of Health recognizesit as a representative model for exploringhuman disease. We tested the toxicity of 8ymetal/metal oxide nanoparticles to zebrafishembryos. Silver, quantum dot, and ZnOnanoparticles showed substantial toxicity interms of morphological defects, hatchingrate and survival rate Overall the toxicityrate, and survival rate. Overall, the toxicityprofile of nanomaterials in zebrafish agreeswith that in mammalian cells and zebrafishis a good model organism for studying thetoxicity of nanomaterials.

NSF: EF‐0830117 

Data‐Driven Models of Engineered Nanomaterials (eNMs)CEIN Computational Infrastructurep• Collaboration Infrastructure• Designed CEIN Data Repository• Prototype eNM Database System

Data preprocessing• HTS data Normalization• Data Quality analysis• Hit detection (Active vs. non‐Active)• Feature Extraction and Selection

Developed and Implemented : (a) new Feature Selection methods suitable for HTS analysis , and  (b) tools for HTS data normalization and hit detection.

Data Mining andKnowledge Extraction

• Hierarchical Clustering: heat‐maps• Topology preserving clustering:Self‐Organizing Maps (SOM) Au

Al2O3

Data‐driven Models• Data‐driven Modeling• Screening Classifiers• nano‐QSAR development

ZnO Ag Pt SiO2

Developed a correlation analysis of Toxicity pathways and Toxicity effects (shown above for  RAW cells). The analysis reveals specific correlation patterns for ZnO, Ag, and Pt NPs.and Pt NPs.

Example: QSAR  to predict cell damage (induced by metal and metal oxide NP after 24h exposure in BEAS‐2B cells).  Input parameters:  NP concentration, IEP, ZPwater, ZPBEGM.

NSF: EF‐0830117 

Transport and Fate of Engineered Nanomaterials (eNMs)Microlayer

Atmospheric NP

• Environmental distribution of eNMs

0.0001

0.001

0.01

0.1

1

raction of TiO

2

NP input

Resuspension

Sedimentation

AdvectionAggregation

DisaggregationWater Body

Environmental distribution of eNMs (a multimedia mass balance analysis)

Environmental concentrations & intermedia fluxes

Intermedia transport (e g

Estimated distribution of TiO2 in LA County. Largest fraction in

0.0000001

0.000001

0.00001

Air Water Soil Sediment

Mass F

• eNMs transport is governed by their aggregation d i i i h h d d

Sediment

A simple aquatic multimedia system showing major intermedia transport processes

Intermedia transport (e.g., sedimentation, dry/wet deposition, aerosolization)

fraction in air, >0.99

state and interaction with other suspended matter & dissolved species. Computational model incorporating DLVO theory with 

Monte Carlo approach to simulate NP aggregation. S di i i i l d d bl i i h

(rmean ,PSD) F(C, , I )

Sedimentation is included to enable comparison with DLS measurements.  

Develop parametric models of NP aggregation for use in intermedia transport assessment, in support of toxicity studies and for data driven toxicity models &toxicity studies and for data‐driven toxicity models & decision tools.

Evolution of  TiO2 NP aggregate size (20 nm primary size) for a suspension of 20 ppm in water (pH=10, I=6x10‐4 M).

NSF: EF‐0830117 

Current Reported Practices and Perceived Risks Related to Health, Safety and Environmental Stewardship in Nanomaterials Industry1p y

Researchers seek to understand how private firms are adapting practices for safe development of ENMs in the context of absent regulation and indeterminate standards. Since September 2009, researchers have collected 60 surveys from nanomaterials firms internationally (14% response rate). Preliminary findings include:

Waste Management

1

1.2

panies

Reported Impediments to Implementing Nano‐specific EHS Programs

g

23% Report listing nanomaterials in waste manifests36% Report having a nano-specific waste program42% Report using separate containers for nanomaterials66% Report disposing nanomaterials as hazardous waste

0.4

0.6

0.8

Percen

t of C

omp

The smaller firms (1-19 employees) are less likely to:• Consider budget constraints or report “lack of

knowledge” as an impediment to implementing a nanospecific EHS program.

0

0.2

Lack of information

Lack of guidance/regulation

Budget constraints

Internal enforcement

p p gYounger firms (<10 years) are more likely than older firms

to:• Implement a nano-specific EHS program.• Disclose that their products contain nanomaterials.

NSF: EF‐0830117 NSF SES 0531184

1Engeman, C. (Soc, UCSB),  Baumgartner, L., Carr, B., Fish, A., Meyerhofer, J., Holden, P. (Bren School, UCSB), Harthorn, B. (Fem Studies, UCSB).  2010.  In Progress.  

g

The Impact of Testing Costs on the Regulation of NanoparticlesThe Impact of Testing Costs on the Regulation of NanoparticlesCosts of testing the toxicity of nanoparticles are important for determining how nanoparticles might be regulated. Here we analyze whether testing costs might reasonably be borne by industrycosts might reasonably be borne by industry.

Based on publicly available information we estimate that there are 265 distinct nanoparticle types for sale in the US.  Testing costs vary from $70,000 (Level 1 – physical characterization) to $4.48 million (Level IV – in‐vivo animal models)  depending on level of testing.  Four scenarios assumed different proportions (“distribution”) of nanomaterials that are tested at different levels. In the optimistic scenario only 10% of nanoparticles will need the full range of tests, while in the precautionary approach all nanoparticles need testing at all levels. Costs of testing range f $249 illi (O ti i ti ) t $ 1 18 billi (P ti ) At t l l f R&D di t i l t i it thi t l t i tfrom $249 million (Optimistic) to $ 1.18 billion (Precautionary) At current levels of R&D spending on nanomaterial toxicity this translates into between 11 and 43 years for testing currently existing nanoparticles.

Testing level Level I Level II Level III Level IVTotal

Testing cost per substance $0.07 $0.83 $2.15 $4.48

Distribution 0 60 0 15 0 15 0 10 1 00

New approaches that increase the efficiency of testing are needed, 

J Y C

Optimistic

Distribution 0.60 0.15 0.15 0.10 1.00

Number of materials 159 40 40 27 265

Costs of testing (a) $11.4 $33.0 $85.6 $118.8 $249

Neutral

Distribution 0.25 0.25 0.25 0.25 1.00

Number of materials 66 66 66 66 265

especially as the numbers of nanoparticle types increase.

Jae‐Young, C., Ramachandran, G. Kandlikar, M.  2009 "The Impact of Toxicity Testing Costs on Nanomaterial Regulation", Environmental Science

Neutral Number of materials 66 66 66 66 265

Costs of testing $4.7 $55.0 $142.7 $296.9 $500

Risk Averse

Distribution 0.10 0.20 0.20 0.50 1.00

Number of materials 27 53 53 133 265

Costs of testing $1.9 $44.0 $114.1 $593.9 $754 Environmental Science and Technology 43(9):3030‐3034.

Costs of testing $1.9 $44.0 $114.1 $593.9 $754

Precautionary

Distribution 0.00 0.00 0.00 1.00 1.00

Number of materials 0 0 0 265 265

Costs of testing $0.0 $0.0 $0.0 $1,187.7 $1188 NSF: EF‐0830117 NSF SES 0531184

While existing regulations are widely considered to provide adequate authority to

While several US federal regulations are expected to apply to emerging nanomaterials, questions remain as to whether current regulatory frameworks are sufficient for managing risks that may

Emerging Nanotechnologies and Life Cycle RegulationEmerging Nanotechnologies and Life Cycle Regulation

considered to provide adequate authority to regulate nanomaterials, novel properties, low production volumes, sparse data, and a lack of standards and protocols severely challenge the applicability of regulations.  Furthermore, a shortage of resources and i d h i i i

remain as to whether current regulatory frameworks are sufficient for managing risks that may emerge.  This work investigates the federal health, safety, and environmental regulations that apply over the life cycle of a typical nanomaterial to determine whether novel properties and high uncertainty over risks significantly challenge the current regulatory system.

inadequate authority to require testing or recalls severely limit regulators’ effectiveness in managing risk. Many nano‐products as a result will go largely unregulated along their life cycle, while others may fall through gaps in regulation as they move from one stage of their life to the next.  Overall, improvements in authority to require testing of a wider range of products, a systems approach to regulation that better engages stakeholders in risk management, and improvements in regulatory oversight at 

Christian E.H. Beaudrie (2010), “Emerging Nanotechnologies and Life Cycle Regulation: An 

the ‘use’ stage are recommended. 

investigation of federal regulatory oversight from nanomaterial production to end‐of‐life”. Center for Contemporary History & Policy, Chemical Heritage Foundation, Studies in Sustainability White Paper Series.

Figure 1.  Federal health, safety, and environmental regulations that apply along the life cycle of a typical nanomaterial. Dashed boxes denote the life cycle stages at which each regulation’s primary regulatory mechanisms are in effect.

NSF: EF‐0830117; NSF SES 0531184

SPAC Leadership WorkshopHilar God inHilary Godwin

CEIN Education/Outreach Director

The Student/Postdoc Advisory Committee sponsors activities to ensure effective mentoring and professional development for the Center’s students and postdocs.  On September 8, 2009, twenty CEIN‐affiliated and ten CEINT‐affiliated students and postdocs participated in an interactive workshop that included small group discussions, problem‐solving activities, and presentations.  The three topics covered were: Getting p ese tat o s. e t ee top cs co e ed e e: Gett gthe Mentoring you Need; Introduction to Principles of Quality Control and Quality Assurance, and Guidelines for Development and Validation of Standard Protocols.  

F db k iti d it i l d d “A M t ’Feedback was positive, and it included, “As a Master’s student, it’s rare to touch on these sorts of topics in my field. But all of these topics are very pertinent to my research and career interests related to the safe handling & disposal of nanomaterials.”

NSF: EF‐0830117 

NanoDays 2010: Partnerships & OutreachHilary GodwinHilary Godwin

CEIN Education/Outreach DirectorNanoDays is a nationwide weeklong educational festival  which aims to introduce nanoscale science & engineering to the public.  NanoDays is an initiative from the Nanoscale Informal Science Education Network (NISE Net).Education Network (NISE Net).  

Saturday, March 27, Santa Barbara: The CEIN at UCSB partnered with the Center for Nanotechnology in Society (CNS) and the National Nanotechnology Infrastructure Network (NNIN) at UCSB for an eight‐hour NanoDays event at the Santa Barbara Museum of Natural History. Three CEIN‐affiliated graduate students and staff participated in this event which reached 500 people.  

Saturday, April 3, Los Angeles:  The CEIN at UCLA partnered with the California Science Center for a four‐hour NanoDays event at the museum Fourteen CEIN Volunteer Educators (from the CEIN CNSImuseum.  Fourteen CEIN Volunteer Educators (from the CEIN, CNSI at UCLA, California Teach at UCLA) lead interactive activities and interacted with the public.  Included in this volunteer group were three faculty members and two postdocs who answered any and all nanoscience‐related questions.  This successful event reached 550 people. 

NSF: EF‐0830117 

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