Nanomaterials in the Environment: From Materials to High-Throughput Screening to Organisms

Courtney R. Thomas, Saji George, Allison M. Horst, Zhaoxia Ji, Robert J. Miller, Jose R. Peralta-Videa, Tian Xia, Suman Pokhrel, Lutz Madler, Jorge L. Gardea Torresdey, Patricia A. Holden, Arturo A. Keller, Hunter S. Leniahn,

Andre E. Nel, and Jeffrey I. Zink. UC Center for Environmental Implications of Nanotechnology

Libraries of materials are first characterized to establish theproperties of the materials. After HTS toxicity screening, thedata can be analyzed and displayed as heat and self-organizing maps, with fluorescence signals seen in yellowand red corresponding to increased toxicity. At the sametime, the mechanism of toxicity will be determined and linkedto the physicochemical properties of nanomaterials.Nanomaterials are then prioritized with regard to furthertoxicity screening. Model organisms in various trophic levelswill be used to examine the toxicity of nanomaterials. This -information will be used to build the structure-activityrelationships established using cell studies and confirmed invivo; new materials can be synthesized based on safedesign principles. These new materials are added to thecombinatorial libraries, and tested to verify the hypothesizedreduced toxicity.

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ACS Nano 5(1) p.13-20, 2011

The reduction in the band gap energy of TiO2 with increasing Fe loading had no effect in the crystallinity and the homogeneousdistribution of the nanoparticles. Photochemical studies showed that band gap was reciprocally tuned proportional to the Fe contentand the photo-oxidation increased with Fe loading during irradiation. The cytotoxic and ROS production in the macrophage cell lineshowed increased oxidant injury and cell death with a decreased band gap energy. These findings reflect the potential of Fe-TiO2 togenerate adverse effects in humans and the environment during high intensity light exposure. (Work submitted for publication).

Effects of near-visible light irradiation to the Fe doped TiO2 exposed cellsS. George ς,δ, S. Pokhrel¥, Z. Jiχ, B. Hendersonχ, Tian Xia ς, δ, L. Li ς, δ, J. I. Zinkχ, A. E. Nel ς, δ, Lutz Mädler¥,δ

ςDepartment of Medicine-Division of NanoMedicine, University of California, Los Angeles, California, USA, δCalifornia NanoSystems Institute at University of California, Los Angeles, California, USA, ¥Foundation Institute of Materials Science (IWT), Department of Production Engineering, University of Bremen,

Germany, χDepartment of Chemistry and Biochemistry, University of California, Los Angeles, California, USA

IRG 1, L. Mädler, S. Pokhrel, J. I. Zink, Z. Ji, B. Henderson

IRG 2, A. E. Nel, S. George, T. Xia, L. Li

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Effects of Zn-containing compounds on sea urchin development. Elise A. Fairbairn, Arturo A. Keller, Lutz Mädler, Dongxu Zhou, Suman Pokhrel, Gary N. Cherra

UC Center for Environmental Implications of Nanotechnology

Newly fertilized embryos were exposed to Zn-containing compounds (ZnO nanomaterial, or 10% Fe-doped ZnO NM) until Control embryos reached the pluteus stage (approximately 96 hours after fertilization), then fixed and assessed for normal/ abnormal development. Fe-doped ZnO NM was less soluble than pure ZnO NM in seawater. At concentrations near those used in the toxicity assays, Fe-doped ZnO NM reached ~80% dissolution, compared to nearly 100% dissolution with the pure ZnO NM. However, in contrast to the reduced toxicity observed in the in vitro cell culture system, we observed no significant difference between toxicity with Fe-doped ZnO NM and the pure ZnO NM in our sea urchin developmental bioassay.

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J. Hazardous Materials (2011). Accepted.

Biomagnification of CdSe QDs: Pseudomonas to TetrahymenaR. Werlin, J. H. Priester,R. E. Mielke, S. Krämer, S. Jackson, P. K. Stoimenov, G. D. Stucky, G. N. Cherr, E. Orias, P. A. Holden

UC Center for Environmental Implications of Nanotechnology

Biomagnification of pollutants is a pinnacle concern in ecotoxicology. Here, using basal organisms in most food webs, the potential for biomagnifcation of an engineered nanomaterial was shown anew. A common protozoan predator, Tetrahymena thermophila, when feeding exclusively on its bacterial prey that had internalized quantum dots (QDs), Pseudomonas aeruginosa, biomagnified cadmium in the form of intact QDs (bar graph, with dark bars for QD cadmium). Intact QDs distributed throughout the predators, but ultimately stunted digestion, leaving undigested early food vacuoles (eFV) packed with QD-laced bacteria (right, arrow and triangle). These protozoans, owing to swimming cessation, are particularly susceptible to predation which could increase trophic transfer.

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(Werlin, et al. 2011, Nat. Nano. 6, 65-71)

NM TiO2 reduces marine phytoplankton population growth rates in high UV light levels because ROS is generated. ROS causes multiple forms of cytotoxicity.

We are investigating the effects of TiO2 on population growth rates of marine phytoplankton, and DEB modeling was used to quantify effect parameters, including no-effect concentrations for four common species of coastal phytoplankton.

In experiments exposing phytoplankton to TiO2 NPs with (red dots) and without (black dots). environmentally relevant UV light exposure, TiO2 was shown to be toxic only with UV exposure. The toxicity levels were at relatively high concentrations (>4ppm).

Population tests will proceed with the next generation of CEIN NP libraries.

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Robert J. Miller1, Hunter S. Lenihan2, Scott Pease2, Edward Hu21Marine Science Institute, UCSB 2Bren School of Environmental Science and Management, UCSB

Submitted to Environmental Science & Technology.

Shannon Hanna1, Robert J. Miller2, Hunter S. Lenihan1, Erik Muller3, Roger Nisbet31Bren School of Environmental Science and Management, UCSB 2Marine Science Institute, UCSB 3Department of Ecology, Evolution

and Marine Biology, UCSB

Marine mussels are used extensively in marine pollution monitoring programs (e.g., The CA Mussel Watch program) to test for the presence and bioavailability of contaminants in marine systems. Mussels are a sentinel species for another ecosystem service, biofiltration by suspension feeders. Mussels are also critical links between phytoplankton and benthic consumers in coastal reef food webs.

1000+ mussels were exposed to ZnO ENMs over a 3 month period. Early data analysis shows exposure to ZnO ENMs inhibit mussel growth with increasing concentrations, but may promote growth at low levels. Additionally, mussels show bioaccumulation of Zn in soft tissue when exposed to ZnO ENMs, and survival of mussels decrease at an exposure concentration of 2 mg L-1 ZnO ENMs.

These data will be used to build a Dynamic Energy Budget model for mussels

Performance of mussels exposed to ZnO

y = - 0.20x + 0.76R2 = 0.82

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Submitted to Chemosphere.

Photoinduced DisaggregationSamuel Bennett, Dongxu Zhou, Arturo Keller

University of California, Santa Barbara

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• Natural sunlight and other light sources induce disaggregation of some nanoparticles from the cluster core. This can results in enhanced environmental mobility. We have shown that this enables transdermal penetration of TiO2. We have shown that disaggregation is also observed for CeO2, ZnO and CNTs. Our modeling of the behavior shows that it can be explained theoretically, and that there is a clear explanation for this behavior, which had not previously been observed. The diagram shows how the incident photons increase the energy and allow for partial disaggregation. The graph shows data from our experimental work.

Results to be submitted for publication.

Nanoparticle AggregationDongxu Zhou, Milka Montes, B. Reginald Thio, Arturo Keller

University of California, Santa Barbara

pH 8pH 8, IS = 100 mM

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• We are exploring the IRG 1 library for TiO2 NPs with different sizes, shapes and crystal structures. We find that aggregation behavior is a strong function of shape, and that the critical coagulation concentrations differs based on shape. This data will inform the IRG 6 aggregation model.

• Our preliminary studies indicate that the new CEIN NPs (Ag, Pd and Pt) aggregate rapidly in seawater; Ag coated with citrate is stable in freshwater. We are conducting stability studies with alginate, which indicate that stable suspensions can be formed. This information will help IRG 2 & 3 to design their experiments.

Submitted to ACS Nano

Use of a high throughput screening approach coupled with in vivo zebrafish embryo screening to develop hazard ranking for engineered nanomaterials

Saji George1,2, Tian Xia1,2, Robert Rallo2,5, Yan Zhao6, Zhaoxia Ji2, Xiang Wang2, Haiyuan Zhang2, Bryan France3, David Schoenfeld2,6, Robert Damoiseaux3,2, Rong Liu2,5, Shuo Lin6, Kenneth A

Bradley4,2, Yoram Cohen2,5, André E Nel1,21Department of Medicine, Division of NanoMedicine; 2Center for Environmental Implications of Nanotechnology, 3Molecular Shared Screening Resources, 4Department of Microbiology, Immunology and Mol Genetics, 5Chemical and Biomolecular Engineering, 6Department of Molecular,

Cell, and Developmental Biology, University of California, Los Angeles, CA, USA.

We are developing high-throughput screening and in silicodata transformation tools to speed up in vitro hazard ranking of nanomaterials. As a proof of principle, studies were conducted using seven different nanoparticles and eight incremental concentrations and durations of exposure against two cell lines and assayed for four interlinked cytotoxicity events. Using state-of-the-art statistical methods we analyzed, ranked and organized nanomaterials according to cytotoxic potential. Quantum dot (CdSe/ZnS) and ZnO showed the most prominent lethality, Pt, Ag, SiO2, Al2O3 and Au triggered sublethal effects without cytotoxicity. We then compared these results to in vivo response outcomes in zebrafish embryos. Among the results, Ag toxicity in zebrafish differed from in vitro results, which is congruent with this material’s designation as extremely dangerous in the environment.

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High throughput screening

Data analysis

In vivo screening using zebrafish embryos

ACS Nano 2011, 5 (3), 1805-1817

Haiyuan Zhang 1*, Tian Xia 2*, Huan Meng 2, Min Xue 3, Saji George 1, 2, Zhaoxia Ji 1, Xiang Wang 1, Rong Liu 4, Meiying Wang 2, Bryan France 6, Robert Rallo 4, Robert Damoiseaux 1, 5, Yoram Cohen 4,

Kenneth A. Bradley 1, 6, Jeffrey I. Zink 3, Andre E. Nel 1, 2, *

1 California NanoSystems Institute, 2 Division of NanoMedicine, Department of Medicine, 3 Department of Chemistry & Biochemistry, 4 Department of Chemical & Biomolecular Engineering,5 Molecular Shared Screening Resources, and 6 Department of Microbiology, Immunology & Molecular

Genetics, University of California, Los Angeles, California, United States

Differential Expression of Syndecan-1 mediates Cationic Nanoparticle Toxicity in Undifferentiated versus Differentiated Normal Human Bronchial Epithelial Cells

Primary cells can provide a more appropriateconnection to in vivo toxicity. We compared theresponse of undifferentiated and differentiatedprimary human bronchial epithelial cells (NHBE) tocationic mesoporous silica nanoparticles (MSNP) thatare coated with polyethyleneimine (PEI) A multi-parametric assay was used to screen for sub-lethaland lethal response outcomes. MSNP coated withhigh molecular weight (10 and 25 kD) polymers weremore toxic in differentiated cells than particles coatedwith shorter length polymers. Differentiated cellsshowed more cellular association with MSNP coatedwith high molecular weight due to more abundantexpression of a proteoglycan, syndecan-1. Thesedata demonstrate the importance of studying cellulardifferentiation as an important variable in theresponse of primary cells to toxic ENM properties.

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Undiff Diff

MSNP-PEI MSNP Colocalization of syndecan-1 and MSNP-PEI in differentiated cells

Different cellular association of MSNP-PEI

PEI layer

Differential toxicityACS Nano, 2011, DOI: 10.1021/nn200328m

HTS Data Analysis and Modeling WorkflowYoram Cohen1, Robert Rallo2, Kenneth Bradley1, Andre Nel1, Rong Liu1, Bryan France1,

Robert Damoiseaux1, Saji George1, Haven Liu1

University of California Los Angeles1, Universitat Rovira I Virgili2

Efforts in this project focus on knowledge extraction from high throughput screening data of nanoparticles toxicity, development ofpredictive nano-quantitative-structure-activity relations (nano-SARs), feature selection for nano-SAR development andidentification of pathway linkages. Based on newly developed feature selection approach, a classification based nano-SAR wasdeveloped for cytotoxicity of metal and metal oxide nanoparticles. This work revealed that atomization energy of the metal oxide,period of the nanoparticle metal, nanoparticle volume fraction (in solution), and the primary nanoparticle size were fundamentaldescriptors that enabled correlation of cytotoxicity at high level of accuracy without false negatives. The classification based nano-SAR enables one to identify decision boundaries which are crucial for use in hazard ranking of nanoparticles.

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Rallo et. al., ES&T (2011), 45, 1695-1702.

Modeling Transport and Fate of Engineered Nanomaterials (ENMs)Yoram Cohen1, Robert Rallo2, Haven Liu1, Sirikarn Surawanvijit1, Soomin Kim3, Arturo Keller3, Roger Nisbet3

1University of California, Los Angeles, 2Universitat Rovira I Virgili, 3Univerity of California, Santa Barbara

A multimedia modeling scheme is being developed to predict the tranposrt of nanoparticles. The first generation framework was evaluated for the partitioning of TiO2 nanoparticles between air, water and soil, using basic intermeidatransport processes such as wet/dry composition and sedimentation. Quantification of the fate and transport of nanoparticles requires information regarding their size distribution. A predictive computational “constant number” Monte Carlo model ws developed to model NP aggregation and to determine the stable particle size distribution under various environmental conditions. The model performance was successfully tested against experimental DLS data. Implementation of the fate and transport modeling efforts into user-friendly web-based software is an ongoing effort of the Center.

NP input

Microlayer

Atmospheric NP

Resuspension

Sedimentation

AdvectionAggregation

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DisaggregationWater Body

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TiO2 pH8 ExperimentTiO2 pH8 SimulationCeO2 pH8 ExperimentCeO2 pH8 Simulation

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Std.Dev<0.5 nm

104 particles

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NSF: DBI-0830117 NSF: SES-0531184NSF: SES-0938099

Key findingsneed for regulation, education•Only 46% of participants reported having a nano-specific EHS program. •More (61%) cited “lack of information” as an impediment to implementing nano-specific EHS practices than any other obstacle. •Participants reported high levels of uncertainty about ENM risks•Despite reported lack of information and uncertainty about ENM risk, a majority of participants believes ‘industry knows best’

Engeman, Baumgartner, Carr, Fish, Meyerhofer, Holden, & Harthorn 2011. In Progress.

0%10%20%30%40%50%

Carbon nanotubes

Other carbonaceous

materials

Dry powders Quantum dots Metal Oxides Heavy metals

Almost no risk Slight risk Moderate riskHigh risk Don't know

22% 32% 29%

40%

27%22%

0% 25% 50% 75% 100%

1. It is reasonable to assume that industries working with nanomaterials will adapt or alter their safe-handling

practices when new hazards are discovered.2. Businesses are better informed about their own work-

place safety needs than are government agencies.3. Industries working with nanomaterials can be trusted

to regulate the safe-handling of these materials.4. Voluntary reporting approaches for risk manage-ment are effective for protecting human health and

the environment.5. Employees are ultimately responsible for their own

safety at work.

Strongly agree

AgreeDon’t know

Disagree

Strongly disagree

International Survey of EH&S in Nanomaterials Industry

Environmental Sociology Case StudiesMary Collins1, William Freudenburg1, Barbara Herr Harthorn1, Terre Satterfield2

1University of California, Santa Barbara, 2University of British Columbia

Nanoremediation: Will equity concerns arise?Collins, Harthorn (UCSB), Satterfield (UBC)Nanoremediation currently in use in 50 US sites--in situ mitigation rather than removal • Benefit or risk, dep. on safety and effectiveness• Is there balance in subpopulation distribution of nanoremediation siting? Spatial analysis• Preliminary results indicate likely balance, but…

Temporal Aspects of Public-Private PartnershipsCollins, Freudenburg (UCSB) •Relationships that are Unproblematic initially may become problematic in thecontext of risk management •Implication: Use caution in forming industry partnerships that could lead toa perception of compromised risk management Nanotechnology and RecreancyFreudenburg, Collins (UCSB) •Theoretical contribution addresses potential risks to safe nanotech, reminding CEIN to instill public trust through its own safety practices, and transparent and timely disclosure of risks, in accessible language(Expected Publication: 2011 Social Life of Nanotechnology eds: Harthorn & Mohr)

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Nanoecotoxicology Lecture SeriesHilary Godwin

UC CEIN Education/Outreach Director

Each lecture in this 13 lecture series includes learning objectives, required and recommended readings, and quizzes, which can all be accessed through a password-protected website. This lecture series is available to Center members and to external partners, like CINVESTAV and INSP in Mexico, interested in building research programs in this area.

As a result of this lecture series, UC-CEIN will host a “Nanoecotoxicology Bootcamp” at UCLA in August 2011 to provide capacity building for researchers in Mexico and to promote collaborations between women scientists in the United States and women scientists in Mexico.

Postdoctoral Fellow Saji George explains High Throughput Screening (HTS) to visitors at the UC-CEIN’s HTS facility. Through the Nanoecotoxicology online lecture series, researchers unable to visit Los Angeles can listen to Dr. George’s lecture.

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Public OutreachHilary Godwin

UC CEIN Education/Outreach Director

Partnerships with university-based and community-based organizations have resulted in public outreach events that focus on communicating key scientific concepts to the community. UC-CEIN Volunteer Educators lead interactive tabletop activities and engage the public in dialogue about nanoscale science and engineering.

Event Location Attendees CEIN Volunteers

NanoDays 2010, Los Angeles California ScienCenter 550 14

NanoDays 2010, Santa Barbara SB Museum of Natural History 500 4

Nanotechnology: Small is Big! Santa Monica Public Library 50 5

Small things from a big planet SB Public Library 50 3

SciArt summer camp UCLA 50 10

Explore your universe! UCLA 500 20

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