Grantee Research Project Results
2011 Progress Report: Risk Assessment for Manufactured Nanoparticles Used in Consumer Products (RAMNUC)
EPA Grant Number: R834693Title: Risk Assessment for Manufactured Nanoparticles Used in Consumer Products (RAMNUC)
Investigators: Zhang, Junfeng , Georgopoulos, Panos G. , Di Giulio, Richard T. , Lioy, Paul J. , Isukapalli, Sastry S. , Kipen, Howard , Chung, Kian Fan , Garfunkel, Eric , Lee, Ki-Bum , Mainelis, Gediminas , Porter, Alexandra , Ryan, Mary P. , Schwander, Stephan K. , Tetley, Teresa D
Current Investigators: Zhang, Junfeng , Tetley, Teresa D , Chung, Kian Fan , Georgopoulos, Panos G. , Lioy, Paul J. , Schwander, Stephan K. , Ryan, Mary P. , Isukapalli, Sastry S. , Di Giulio, Richard T. , Porter, Alexandra , Garfunkel, Eric , Mainelis, Gediminas , Kipen, Howard , Lee, Ki-Bum
Institution: University of Medicine and Dentistry of New Jersey , Duke University , Imperial College
EPA Project Officer: Aja, Hayley
Project Period: April 1, 2011 through June 30, 2014 (Extended to June 30, 2016)
Project Period Covered by this Report: April 1, 2011 through March 31,2012
Project Amount: $1,999,995
RFA: Environmental Behavior, Bioavailability and Effects of Manufactured Nanomaterials - Joint US – UK Research Program (2009) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
Our US-UK center, ‘Risk Assessment for Manufactured Nanoparticles Used in Consumer Products (RAMNUC)’, provides a systematic, multidisciplinary approach for predicting potential human and environmental risks associated with the use of selected consumer products that incorporate zinc oxide, silver, and cerium dioxide nanoparticles. The overall hypothesis of the RAMNUC project is that “manufactured nanoparticles (MNPs) at the point of exposure for both humans and aquatic animals will substantially differ in both physicochemical and toxicological properties from MNPs at the source (synthesized in the laboratory or acquired commercially)”.
Our project includes both experimental and computational tools to analyze the selected MNPs in consumer products using both in vitro and in vivo experiments to assess intra- and extra-cellular bioavailability and toxicity. We have been characterizing MNPs for their physical (e.g., size, shape, state of agglomeration/aggregation) and chemical properties (e.g., composition, functionalization, surface chemistry). Thus, our in vitro and in vivo studies are producing mechanism-based results relating toxic effects to the physicochemical properties of the MNPs. Another aspect of our project includes an exposure simulation study that will produce realistic estimates of MNP exposures to consumers.
Data generated from these experiments are being integrated into mechanism-based computational modules of our two existing source-to-exposure-dose-to-effects modeling systems, allowing for rational extrapolation and generalization in MNP risk assessment. Built upon an inter-institutional structure, the RAMNUC consortium serves as a model for systematically addressing complex problems associated with MNP risk assessment. The results will contribute to the limited knowledge about health risks associated with the use of nano-technology based consumer products, particularly consumer sprays containing ZnO and Ag nanoparticles as well as a nano-ceria diesel fuel additive.
Progress Summary:
This center project has eight specific aims. Below is a summary of the progress made in Project Year 1 by aim.
Aim 1 (Risk Assessment Framework):
This aim relates to the development of a generalized conceptual risk assessment framework for risks from MNPs and adaptation of these modules using data from Aims 2 through 7. In year 1, this framework has been developed by adapting existing frameworks and incorporating MNP life cycle analysis approaches for the following stages of MNPs’ life cycle: (a) sources, (b) environmental fate and transport, (c) concentrations in environmental media, (d) human exposure, (e) dose at target organs and bioavailability. We are expanding upon this framework by defining a computational structure for studying toxicodynamic responses following exposures to MNPs. This is being actively developed in collaboration with the RAMNUC team and utilizes other data and mechanistic information from ongoing related research efforts.
Computational Implementation and Application of the risk assessment framework for MNPs:
• Modules for estimating realistic environmental and occupational exposures to MNPs are being developed: (a) modules for environmental levels of silver nanoparticles and carbon nanotubes have been developed by evaluating and applying this framework in complementary research efforts, and (b) modules for environmental levels of cerium dioxide (CeO2) and zinc oxide (ZnO) are being developed as part of Aim 1.
• Modules for consumer exposures and exposures in occupational settings have been developed by adapting existing consumer exposure models. Application case studies so far in Year 1 focused on silver nanoparticles.
• Modules for toxicokinetics and toxicological effects and occupational exposures to MNPs are being developed within the generalized framework. These are being evaluated with in vitro and in vivo experimental data on silver nanoparticles.
Aim 2 (Nanoparticle Synthesis and Characterization):
This aim focused on the preparation of well defined Ag and ZnO MNPs for initial cell exposure and toxicity assays. Appropriate protocols have been developed and characterization of the particles by size (and dispersion), phase and chemical purity has been achieved.
Ag Nanoparticles
Silver nanoparticles with sizes of ~10 nm diameter and ~50 nm diameter have been synthesized using chemical bath reduction with either sodium borohydride (10 nm) or sodium citrate (50 nm) as the reducing agent. The solution of NPs exhibits a yellow color characteristic of the surface plasmon resonance for Ag nanoparticles. The particles have been characterized using transmission electron microscopy (TEM) and Ultra-violet – visible light (UV-vis) spectroscopy. The particles are pseudo spherical, relatively monodispersed and non-aggregated (or easily redispersed by sonication). The particles have been purified and removed from solution using high speed centrifugation. Determination of Ag nanoparticle concentrations is ongoing using titration techniques based on the UV-vis data (and Beer Lambert Law).
ZnO Nanowires
High aspect ratio ZnO nanowires were fabricated by template assisted electro-deposition into high quality polycarbonate membranes (track etched, 6 x 108pores/cm2). One side of the membrane was coated with a layer of gold by sputter deposition to serve as a working electrode. The counter electrode used was a platinum mesh, and an AgCl electrode was used as the reference electrode. Electro-deposition was conducted using 0.1 M Zn(NO3)2 at a constant potential of -0.8 V vs. AgCl at 70 °C for 30 minutes. Wires were removed from the template by washing in dichloromethane; purification and separation of the wires via repeated solvent washing and centrifugation. The as deposited wires had a uniform diameter of 150 nm and are 2.5 μm long. Care had to be taken during centrifuging as there was a tendency for wires to break up and result in a wider distribution of wire lengths than desired. The dimensions of the wires (n= 565) were established using TEM and image analysis.
Characterization of Diesel Exhaust Particles
In Year 1, our major focus was centered on the characterization of diesel exhaust particles (DEPs) with and without CeO2 additives using a commercial product called Envirox (EnviroxTM, Energenics Ltd., UK). DEPs were generated from an internal combustion engine under Aim 4 of our project. It is unknown whether the addition of CeO2MNPs into diesel fuel will alter physicochemical (and toxicological) properties of diesel exhaust particles. Therefore, we investigated the effects of CeO2 additives on the physicochemical properties (size, shape, agglomeration, chemical composition, surface chemistry, surface charge) of DEPs. It was found that the addition of Envirox significantly affected the various physicochemical properties of the resulting DEPs, including size, morphology and the surface charge. The DEPs obtained after the addition of Envirox were significantly smaller in size as evidenced by the dynamic light scattering analysis and scanning electron microscopy (SEM), which could be due to the more effective combustion of the diesel fuel in presence of CeO2. In addition, it was noticed that the DEPs with Envirox had had a more negative surface charge as compared to DEPs without Envirox. This can be attributed to the increase in the amount of oxides as a result of the better combustion.
Aim 3 (Exposure Simulation Studies):
This aim was designed to investigate exposure to nanoparticles from commercially obtained consumer sprays that incorporate ZnO and Ag MNPs. Since many of the selected sprays are targeted for disinfection and cleaning, the spray simulation experiments are being conducted in a “simulated kitchen” chamber to observe effects under realistic conditions. This chamber has been fabricated and characterized in Year 1. The chamber is equipped with a small countertop, a sink, small furniture, and small appliances. This will provide data on personal exposures and a range of particle sizes and concentrations that a person could be exposed to as well as the total emission factor useful for experiments in Aims 2, 5, 6 and 7. We will also characterize the same properties of airborne particles when they are aerosolized using commercial nebulizers. The equipment being used for these experiments has been serviced and calibrated and set up for testing at the end of year 1. The following experiments are currently being initiated for characterization:
• Use of sprays using their original packaging (sprayers) in a small chamber to measure the size distribution of produced particles
• Release of some sprays (those where reservoir can be detached from the spraying mechanism) using commercially available atomizers to analyze the effect of spraying mechanism on the released particle size.
• Simulated use of sprays in a room; determination of resulting personal exposures
Aim 4 (Diesel Exhaust Particle Collection):
The purpose of this aim was to collect and characterize diesel exhaust particles from an internal combustion engine combusting diesel fuels with and without Envirox to allow comparisons of resultant exhaust particle properties.In order to investigate the effect of CeO2 additives on the physicochemical properties of the resulting DEPs, we collected diesel exhaust particles with and without Envirox at various Envirox concentrations. We used a diesel-powered electrical generator (Yanmar, Model YDG 5500EE, 5.5 Kw, 406 cc, one-cylinder, two cycle) to combust low sulfur diesel (Raceway) with different concentrations of CeO2additive (EnviroxTM, Energenics Ltd., UK). The diesel exhaust was analyzed at four different concentrations of CeO2 additive: 0X (0 ppm), 0.1X (~0.5 ppm), 1X (~5 ppm or 5 mg/L) and 10X (~50 ppm). For each CeO2 concentration, we measured the diesel exhaust for (1) particle size distributions by particle number and mass, (2) total particle number concentration, (3) surface area of the particles deposited in tracheobronchial and alveolar regions of the lung, (4) CO concentrations, (5) CO2, (6)NO/NO2/NOx, (6)black carbon, (7) gas-phase PAHs, (8) particle phase PAHs, (9) particle-phase elemental carbon (EC) and organic carbon (OC), (10) particle-phase elements, and (11) gas-phase aldehydes. For each CeO2concentration, we performed 3-4 measurement sessions with each one having 45-60 min of stable mass concentration. For each measured parameter, averages were calculated for each session and the final results are averages between the measurement sessions.
The data show that the particle size distributions shifted to the lower end of the size range as the CeO2 concentration increased, indicating that CeO2 additive decreases the size of particles emitted from the engine. The mode particle size (particle size at the peak concentration) decreased from 150 nm at 0X of CeO2 to less than 100 nm at 10X of CeO2. Since the same mass concentration was maintained throughout the measurements, the overall particle number increased with increasing CeO2concentration. EC content of particles (per mg particles) decreased, while OC content increased, as CeO2 concentration increased. We also confirmed the manufacturer’s statement found that the use of CeO2lowered fuel consumption. Tentative conclusion is that the addition of CeO2 in diesel fuel leads to changes in particle size and composition.
Aim 5 (in vitro Studies):
The in vitro studies have been conducted in parallel in two laboratories (the Tetley Lab in London and the Schwander Lab in New Jersey).
Tetley Lab
In year 1, each of the ENMs tested in vitro used the unique human pulmonary alveolar epithelial type 1-like cell line (AT1). Ag nanoparticles with surface area 2.34 m2/g, with BET 244 nm, SEM 80-100 nm and zeta potential -24.29 were used. Cell viability: The highest dose of silver, 100μg/ml, caused approximately 30% cell death. The lower concentrations had no effect. Mediator release: There was a significant increase in IL-8 release following 4 hours exposure to Ag, which was not so marked at 24 hours. Ag stimulated IL-6 release, most notably at 4 hours following 10 and 100 μg/ml. At 24 and 48 hours, this dropped back towards normal, possibly reflecting some cell death. Nevertheless, even the lower doses stimulated IL-6 was dose- and time-related. MCP release is inhibited by high doses (50 and 100 μg/ml). Preliminary studies with polydispersed ZnO NPs showed that this was very cytotoxic at a low dose (> 80% cell death at 10 μg/ml) with a sharp dose-response between 5 and 10 μg/ml. Upcoming tests will involve comparing the findings with the effects of the engineered silver nanoparticles in different nanosize and surface modification.
Schwander Lab
Following initial studies of the effects of Ag, CeO2and carbon black (CB) on the expression of cytokines and chemokine genes, this year has been used to lay major emphasis on assessing the biological effects of combustion exhaust particles obtained from a diesel engine from Aim 4. We received preparations of exhaust particles collected from diesel fuel combustion with and without addition of Envirox (DEPenv and DEP, respectively). DEP and DEPenv preparations were provided at predetermined concentrations in two different resuspension media: RPMI1640 (culture media) and H20 (HPLC grade). THP-1 (monocytic human leukemia cell line) cells were stimulated with DEP and DEPenv at identical concentrations and the expression of cytokine (IL-1ß, IL-6, IL-12p40, TNFα) and chemokine (CXCL10) genes and proteins were assessed by qRT-PCR and ELISPOT assays, respectively. As part of our commitment to assessing MNP effects on antimicrobial immune effector functions, we also infected THP-1 cells with avirulent M.tb strain (H37Ra) and assessed how manufactured nanoparticles (MNPs) may alter M.tb-induced gene and protein expressions. M.tb infections were done at a multiplicity of infection (MOI) 10, i.e. exposing each THP-1 cell to 10 M.tb bacteria. Results showed that DEP and DEPenv re-suspension material in RPMI and water differed significantly in its capability to induce cyto/chemokine gene and protein expression in THP-1 cells. All responses were dose dependent (we compared 0, 10 and 50 μg/mL doses of DEP and DEPenv), with greater concentrations inducing greater gene and protein expressions. The pattern of gene expression in THP-1 cells exposed to DEP and DEPenv in RPMI was: DEP >> DEPenv. The pattern of gene expression in THP-1 cells exposed to DEP and DEPenv in H2O was: DEP << DEPenv. For the DEP and DEPenv in H2O we found the same pattern for protein expression (IL-1ß and TNFα). MNP effects also appeared to be similar in THP-1 cells with or without M.tb infection. THP-1 cells exposed to MNP and M.tb simultaneously, however, showed gene expression levels that were significantly greater than in THP-1 cells exposed to MNP only.
Aim 6 (in vivo Studies):
This aim’s focus is on animal in vivo studies of respiratory effects. Year 1 was primarily used to obtain in vitro toxicity data first to guide the exact nanoparticle types to be used in the in vivo studies. Several meetings have been held with Rachel Smith and Bob Maynard’s group to finalize the exposure systems to be used, inhalation doses and the measurements to be made, so that the plans for these experiments will proceed at year 2 as designed. There has been substantial progress in the discussing and finalizing of protocols and obtaining data for exposure of rats to NP aerosols. The instruments for measuring lung physiology are in place and the assays of mediators in lung/lavage fluid have been set up.
Aims 7 and 8 have not started according to the defined time line.
Future Activities:
We are on schedule of project implementation as planned and do not foresee major changes in project activities for the next report period, July 1, 2012 to June 30, 2013. Our planned activities are described below by specific aims.
Aim 1:
The activity for this aim has been completed in Year 1.
Aim 2
For year 2, we will continue to characterize our consortium’s nanoparticles for various physicochemical properties including the following techniques TEM, SEM, Raman, XANES, XRD, Zeta-potential, UV-Vis and others. The information gathered from our experiments would be used in order to investigate and probe the in vitro and in vivo toxicity of such nanomaterial-based commercial products. Tests for the next year include (i) Cell exposures to compare ZnO, Ag and CeO2nanoparticles with similar diameter to test the effect of redox activity on the cellular reactivity of the particles and their effects on cell viability. (ii) Transmission electron microscopy to determine uptake of NPs by cells (following i). (iii) Determination of the dissolution behavior of the various particles in simulated body fluids containing proteins (ICP, UV-vis, DLS, XAS).
Aim 3
Future tasks will be the collection of the released MNPs for their subsequent analysis, such as using SEM and similar techniques. In separate tests, the airborne particles from selected sprays will be collected for ensuing in vitro investigation of the particle toxicity (Aim 5). In year 2, the following specific experiments which have been recently initiated will continue: use of sprays using their original packaging (sprayers) in a small chamber to measure the size distribution of produced particles, release of some sprays (those where reservoir can be detached from the spraying mechanism) using commercially available atomizers to analyze the effect of spraying mechanism on the released particle size and simulated use of sprays in a room determining personal exposure.
Aim 4
We are currently and will continue to calculate both fuel mass based and energy output based emission factors for CO2, CO, NOx PM with various sizes, EC, OC, TC, aldehydes, and PAHs. For the work under this aim we have outlined a manuscript currently under preparation with a tentative title “Changes in Diesel Engine Emissions of Particulate and Gaseous Pollutants by a Nano-ceria-based Fuel Additive”. For this paper, we tentatively aim to submit at a high-impact environmental journal, Environmental Science and Technology. In year 2, particles will continue to be collected in several mg/ml and will be used as stock solutions’ for experiments in Aims 2, 5, 6, and 7. Toxicological consequences of these changes are being examined through our ongoing in vitro and in vivo experiments.
Aim 5
In year 2, the effects of CeO2 and diesel exhaust particles provided from Aim 4 will be compared. We will also test synthesized ZnO nanoparticles and nanowires provided by Aim 2 and compare with the striking cytotoxic effect of this polydisperse ZnO. Finally, once all the MNPs available have been screened using the AT1 cell model, we will assess oxidative stress and cell signaling pathways on primary human alveolar epithelial cells and alveolar macrophages. Future experiments will include assessing endotoxin concentrations and inadvertent contamination of MNP material with bacteria and fungi to continue making efforts to unravel the opposing biological effects observed with DEP and DEPenv in the two resuspension formats with RPMI and H20. We will continue assessing the effects of particles on M.tb-induced gene and protein expression eventually broadening the breadth of our gene expression studies to TLR pathway related genes and a greater number of inflammation related cyto-, and chemokines supernatants of cell cultures. We will also assess the toxicity of the MNPs in THP-1 and primary blood cells using several toxicity assays and examine whether MNPs affect growth control of M.tb in peripheral blood mononuclear cells (PBMCs).
Aim 6
In year 2, initial experiments will be performed by direct intratracheal instillation of test MNPs synthesized in Aim 2, those collected in Aim 3 (from sprays) and in Aim 4 (DEPs). The studies regarding MNPs by inhalation will be carried out towards the latter part of year 2. Aerosolization techniques will include the use of electrospray, nebulizer and spark generator.
Aim 7 (Ecotoxicity Studies):
Our aquatic toxicity experiments will begin in Year 3 and will be led by R. Di Guilio at Duke University. The goal of this aim is to determine the effects of the selected MNPs as released into the environment on aquatic organisms by evaluating MNPs collected in media under simulated conditions of MNP disposal in wastewater. The experiments under this aim will allow the completion of the “life-cycle” analyses for the three MNPs being studied in the RAMNUC project.
Aim 8 (Refinement of Computational Modules):
This aim involves refinement of the risk assessment framework by parameterization of computational modules developed as part of Aim 1 with data collected from Aims 3 – 7 and will begin to be actively pursued starting in Year 2. We are currently customizing the modules developed as part of Aim 1 so that they can address specific experimental scenarios from Aims 3 through 6. Specific efforts include customization of these modules to: (a) estimate cerium oxide levels corresponding to controlled experimental settings in Aims 3 and 4, and (b) describe the in vitro toxicodynamic responses corresponding to experimental settings of Aim 5 and in vivo toxicodynamic responses corresponding to experimental settings of Aim 6.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 47 publications | 28 publications in selected types | All 28 journal articles |
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Nazarenko Y, Zhen H, Han T, Lioy PJ, Mainelis G. Potential for inhalation exposure to engineered nanoparticles from nanotechnology-based cosmetic powders. Environmental Health Perspectives 2012;120(6):885-892. |
R834693 (2011) R834693 (2012) R834693 (2013) R834693 (2014) R834693 (2015) R834693 (Final) |
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Subramaniam P, Lee SJ, Shah S, Patel S, Starovoytov V, Lee K-B. Generation of a library of non-toxic quantum dots for cellular imaging and siRNA delivery. Advanced Materials 2012;24(29):4014-4019. |
R834693 (2011) R834693 (2012) R834693 (2013) R834693 (2014) R834693 (2015) R834693 (Final) |
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Supplemental Keywords:
Consumer products, manufactured nanoparticles, diesel exhaust particles, inhalation exposureProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- Final Report
- 2015 Progress Report
- 2014 Progress Report
- 2013 Progress Report
- 2012 Progress Report
- Original Abstract
28 journal articles for this project