Grantee Research Project Results

2011 Progress Report: Transatlantic Initiative for Nanotechnology and the Environment

EPA Grant Number: R834574
Title: Transatlantic Initiative for Nanotechnology and the Environment
Investigators: Bertsch, Paul M. , Unrine, Jason M. , Wiesner, Mark R. , Lowry, Gregory V. , Tsyusko, Olga V. , Casman, Elizabeth , Liu, Jie , Kabengi, Nadine
Current Investigators: Bertsch, Paul M. , Dorey, Robert A , Rocks, Sophie A , McNear, David H. , Unrine, Jason M. , Wiesner, Mark R. , Lowry, Gregory V. , Tsyusko, Olga V. , Neal, Andy , Jefferson, Bruce , Svendsen, Claus , Spurgeon, David , Casman, Elizabeth , Zhang, Hao , Harris, J. , Liu, Jie , Ritz, Karl , Kabengi, Nadine , McGrath, Steve , Lofts, Steve
Institution: University of Kentucky , Carnegie Mellon University , Duke University
Current Institution: University of Kentucky , Carnegie Mellon University , Centre for Ecology and Hydrology , Cranfield University , Duke University , Lancaster University , Rothamsted Research
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2010 through September 30, 2014 (Extended to June 30, 2016)
Project Period Covered by this Report: August 1, 2010 through September 30,2011
Project Amount: $2,000,000
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:

We have developed a life cycle perspective-inspired conceptual model (CM) that suggests the importance of terrestrial ecosystems as a major repository of ZnO, TiO₂, and Ag manufactured nanomaterials (MNMs) introduced via the land application of MNM-containing biosolids. In this project we are investigating the transport, fate, behavior, bioavailability, and effects of MNMs in agroecosystems under environmentally realistic scenarios organized around three key hypotheses:

Hypothesis (H1): Surface chemistry is the primary factor influencing the fate and transport of MNMs in the terrestrial environment as well as the bioavailability and effects to biological receptors;
Hypothesis (H2): Once released to the environment, pristine MNM surfaces will be modified by interactions with organic and inorganic ligands (macromolecules) or via other biogeochemical transformations (aging effects forming a-MNMs); and
Hypothesis (H3): Ecoreceptors will respond to interactions with pristine metal and metal oxide MNMs, a-MNMs, and/or dissolved constituent metal ions and bulk oxides by specific ecological and toxicogenomic responses that will reflect their combined effects.

The overall objectives are to: 1) compare the transport, fate, behavior, bioavailability, and effects of MNMs, a- MNMs, and/or dissolved free metals/bulk oxides to organisms with key terrestrial ecosystem functions, as well as exposure pathways involving humans; 2) determine MNM, surface modified MNM and a-MNM interactions with important biological targets relevant to the BLM and pBRM models and relate these interactions to physicochemical properties; 3) validate models with information generated from experiments designed to address objective 1 for MNMs introduced through a pilot scale Waste Water Treatment Process (WWTP) to key terrestrial ecoreceptors, including effects of MNMs on the WWTP itself; 4) determine realistic MNM emission scenarios for Tier 1 MNMs in wastewater from the WWT pilot plant data and develop first generation Life-Cycle-Analysis-inspired Risk Assessment (LCA-RA) model components for terrestrial effects of Tier 1 MNMs and a-MNMS based on data generated in experiments designed to address objectives 1, 2, and 3; and 5) provide tools for in situ detection, monitoring, and characterization of pristine MNMs and a-MNMs in environmental media and biota.

Progress Summary:

The tier 1 manufactured nanomaterials (MNMs) to be used in the project have been synthesized/selected and thoroughly characterized by Jie Liu’s laboratory. The TiO₂ and ZnO MNMs were obtained from commercial sources (Degussa and NanoSun from Micronisers, respectively) while the Ag particles were synthesized at Duke University by Jie Liu’s laboratory. The final selection of the TiO₂and ZnO MNMs were based on the fact that these materials are being used in larger studies being conducted in the European Union and United States by TĪNĒ team members. The ZnO, Ag, and TiO₂ MNMs have been distributed to all (UK and US) of the TĪNĒ team members conducting laboratory exposure experiments, as well as to Cranfield University for their ultimate addition to the wastewater treatment facility. Recently, 4 nm citrate-coated CeO₂ MNMs for use in experiments involving tier 2 materials have been synthesized at the University of Kentucky by Jason Unrine’s group. Other Ag, ZnO, TiO₂,and Au NPs have been obtained/synthesized for use in a variety of laboratory experiments examining the role of size and surface charge on bioavailability and toxicity.

Wastewater Treatment Facility (UK partners)

Following the US/UK partner project meeting at Rothamstead, UK, in January, the wastewater treatment flow sheet was amended to include a primary sedimentation tank with the contaminant (MNM or metal salts/oxide) to be added prior to primary sedimentation. The settlement process was designed and equipment ordered the day after the meeting leading to delivery of the three additional process units at the end of March. All other equipment was in place; however, early commissioning trials showed that the yield of primary sludge was too low for all of the MNM or metal salts/oxide to be dosed into primary sludge. Larger primary tanks were ordered and are due for delivery at the end of August. All appropriate health and safety requirements will have to be met along with a final post-commissioning risk assessment. When this is done, acclimatization of the plant can commence. It is anticipated that because of these requirements acclimatization will begin toward the end of September, leading to an expected start date for sludge production of December 1^st, which constitutes a 6-month delay compared to the original timeline.

Fate and Transport of MNMs

Aging experiments of Ag and ZnO MNMs under conditions expected in the WWTP are under way. Initial results by the Ph.D. student working with Bertsch and Unrine suggest that ZnO nanoparticles readily react with phosphate at concentrations that bracket those expected in wastewater. The phosphate modifies the particles so that they are composed of both ZnO and Zn phosphate. Phosphate lowers the free Zn ion concentrations in solutions containing either ZnO NPs or ZnCl₂. Phosphate also alters the surface chemistry of the particles, conferring a large negative charge. The nature of the phases (ZnO coated with phosphate versus reprecipitation as Zn phosphate) currently is being explored via TEM, micro-XRD, and X-ray absorption spectroscopy. Additional work on the reactivity of ZnO with sulfide also is under way in collaboration with Lowry’s lab (Brian Reinsch, Ph.D. student) at Carnegie Mellon. Significant progress also has been made to 1) determine the speciation of Ag⁰ nanoparticles (AgNPs) and the impact of sulfidation on availability of Ag⁺ ion, and 2) to develop laboratory accelerated aging studies that can provide sulfidized materials with the speciation observed in biosolids by the Lowry lab and through collaborative efforts with Nadine Kabengi at the University of Kentucky. The speciation of Ag NPs added directly to biosolids taken from a municipal WWTP in Kentucky has been examined, as has the speciation of Ag added as Ag⁺ ion. In all instances, the Ag NPs aged in biosolids for 2 weeks are nearly fully sulfidized and present as Ag₂S. Preliminary evidence suggests that the silver sulphide is associated with solid material within the biosolid and that sulfidation reduces the availability of silver ion. Methods have been developed to sulfidize Ag NPs at varying Ag/S ratios and for different times up to 48 hours that appear to provide Ag NPs having varying degrees of sulfidation. These will be used for testing various hypotheses regarding the effect of the degree of sulfidation on toxicity of various end members.

In tandem with these speciation experiments, the mobility and availability of AgNPs added directly to biosolids and soil amended with biosolids have been examined. Significant progress has been made to 1) determine the potential for surface runoff of AgNPs to aquatic systems as assessed by Ag species in the water dispersible clay fraction (WDC), 2) assess the final concentration and speciation of Ag remaining in treated water from spiked biosolids through settling experiments, and 3) compare the mobility and availability of Ag⁺ ion and laboratory sulfidized Ag NPs. The data suggest that the Ag recovered in the WDC fraction obtained from soils amended with Ag NPs at 1:1 ratio exceeded 10% of the initial mass of Ag added, representing an enrichment factor of up to 5, and that significant differences are observed between different forms of Ag (AgNP vs Ag⁺ ion). Additionally, settling experiments designed to mimic processes at a WWTP demonstrate that less than 0.5% of the total Ag remains dissolved/suspended in the aqueous phase obtained from soil spiked with biosolids.

Characterization of MNMs in environmental samples

Considerable progress has been made toward development of techniques for in situ characterization of Ag nanoparticles in soils and biosolids using field flow fractionation with multi-detection by Unrine’s lab. Field flow fractionation techniques with low ng sensitivity have been developed and have been applied to the characterization of Ag distribution in the colloidal fraction of pore water from non-amended soils as well as soils amended with biosolids. Some aggregation takes place in In non-amended soils, but individualized primary particles can be observed both for PVP and citrate-coated Ag nanoparticles. More primary particles are observed in pore water for citrate than PVP-coated particles. When biosolids are added to soil spiked with Ag NPs or Ag NPs are spiked into biosolids and then added to soil, the Ag distribution in the colloidal phase changes and Ag becomes associated with a 10-20 nm Al rich phase. Ag particles synthesized for addition to the WWTP were shown to be 100% Ag metal using micro EXAFS. The particles were shown to be taken up by both earthworms and C.elegans. Concentrations in C.elegans were not high enough in initial experiments to determine speciation, but in earthworms, we have found intact Ag metal in tissues. The highest concentrations are in the skin, intestine and possibly in the chloragenous tissue (needs to be confirmed). Methods and techniques have been developed for sample preparation for Ag imaging by micro X-ray absorption spectroscopic methods, and it has been demonstrated that Ag can be detected and XANES spectra collected on earthworm and nematode tissues.

Bioavailability and toxicity to key ecoreceptors

Plants and plant derived vesicles: Jonathan Judy, a Ph.D. student with Paul Bertsch, has examined the uptake of different sized (10, 30 and 50 nm) Au MNMs having different capping agents (citrate and tannic acid) by Nicotiana tabacum L. cv Xanthi (tobacco) and Triticum aestivum (wheat) in hydroponic exposures. It has been found that tobacco plants bioaccumulate NPs of all sizes and with both capping agents, with no significant differences evident between size or capping agent. There was no bioaccumulation evident for any of the Au NP treatments in wheat. This suggests that monocots and dicots may differentially bioaccumulate NPs, a hypothesis currently being tested on a wider range of plants, including Medicago truncatula. In collaboration with Dr. George Wagner at the Unviersity of Kentucky, Nicotiana tabacum tonoplast membrane vesicles and artificial lipid vesicles have been used to investigate Au NP induced membrane perturbation. Artificial membrane vesicles composed of different proportions of azolectin and ergosterol were used to simulate plant and fungal membranes, respectively. All Au NP treatments induced membrane perturbation whereas no effects were observed for the Au chloride solution nor the supernatant from the treatment suspensions. The data demonstrate a strong concentration and size dependence in membrane purturbation. The NPs induced more membrane perturbation in ergosterol derived membranes and this is currently being examined in greater detail.

C. elegans: Daniel Starnes, a Ph.D. student working with Olga Tsyusko, has performed a range of toxicity tests on C. elegans with (using E.coli OP50) and without feeding to PVP coated Ag-NPs and AgNO₃. The endpoints used for toxicity were mortality measured over 24 hours and change in growth over a 3 day period, measured every 8 hours, with LC₁₀,LC₅₀, and LD₁₀ for growth. Microarray experiments using LC₁₀ (5.9 mg L^-1) of 4 nm citrate-capped Au-NPs were performed to examine transcriptomic responses of C. elegans to Au-NPs (particle specific effects). Expressions of six of the selected responsive genes were confirmed independently with qRT-PCR. Experiments on inhibition of gene functions via Au-NP exposure employing RNAi were performed as well as toxicity testing of the nematodes with knockdown genes. Microarray experiments using LC₁₀ for Ag-NP with two different coatings, citrate and PVP, and AgNO₃have been conducted to test for particle specific versus ions specific effects. Independent qRT-PCR analyses have been conducted to confirm levels gene expression for several key genes.

The LC₅₀ calculated from the established concentration -response relationships for PVP capped Ag-NPs and AgNO₃ are at least 10 times higher in experiments with vs. without food. To examine particle specific effects, global genome expression were determined for nematodes exposed to 4-nm citrate-coated Au-NPs at the LC₁₀. The data reveal significant differential expression of 1,041 genes. Seven common biological pathways associated with 38 of these genes were identified. Activation of 26 pqn/abu genes from noncanonical specific to C. elegans unfolded protein response pathway and up-regulation of 4 molecular chaperones were observed. Inhibition of abu-11 with RNAi revealed increased mortality in Au-NP exposed nematodes suggesting possible involvement of abu-11 (a gene associated with protein chaperones) in protective mechanisms against Au-NPs. Exposure to Au-NPs also caused activation of genes involved in apoptosis and necrosis.

Global genome expression was also employed to examine effects of Ag-NPs and Ag ions. Some of the commonality in gene expression profiles between Ag ions and each of the Ag-NPs, as well as the measured dissolution of Ag-NPs over the time frame of the exposures, indicate that some observed responses are likely related to the release of free Ag ions. However, the integrated free Ag ion exposure over time at the LC₁₀ was less for the particles than for Ag ions, suggesting some particle specific effects. In addition, the expression of numerous genes that were not altered by Ag ions were up- or down-regulated by exposure to Ag-NPs. Out of 981 genes differentially expressed in response to citrate-coated Ag-NPs, only 35 were common with the genes responding to PVP-coated Ag-NPs, suggesting not only a particle specific, but particle coating specific-responses.

Rhizobacteria: Three important soil rhizobacteria, Sinorhizobium meliloti, Bacillus subtilis, and Pseudomonas fluorescens are being used in the development of a high throughput live-dead cell count assay, employing the nucleic acid binding SYTO 9 and Propidium Iodide fluorophores. These same organisms will be used to study MNM binding to cells in the development phase of the particle biotic receptor model.

Risk Analysis and forecasting

Elizabeth Casman is conducting a literature review for tier 1 MNMs. Mark Wiesner’s group has devloped a TOrC tool for ranking and research prioritization of MNMs in biosolids (adapted from model developed by RTI International). A literature review was conducted to develop a Bayesian Network to predict attachment efficiency of MNMs, a source inventory was compiled and partitioning data was collected for project-relevant MNMs. An EPA biosolids model for modelling MNMs in WWTP is being adapted. Thus far, this work has resulted in estimates of environmental concentrations of MNMs (soil and biota, surface water, ground water), identifying affected populations and associated exposure potential, an exposure assessment framework for emerging contaminants, and identifying regulatory impacts based on exposure potential.

Future Activities:

The major objectives for the upcoming period include comprehensive studies on primary transformation pathways and products as well as environmental fate of ZnO and Ag MNMs under aging conditions expected in soil, wastewater treatment and biosolids; continued bioavailability/toxicity testing of pristine and aged Ag, ZnO MNMs to C. elegans and key rhizobacteria; and examination of MNM properties (size, charge, relative hydrophobicity) on plant uptake and interaction with membrane vesicles. Additionally, information will continue to be collected for use in identifying affected populations and associated exposure potential, an exposure assessment framework for emerging contaminants, and identifying regulatory impacts based on exposure potential.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Publications Views
Other project views:	All 69 publications	39 publications in selected types	All 39 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Judy JD, Unrine JM, Bertsch PM. Evidence for biomagnification of gold nanoparticles within a terrestrial food chain. Environmental Science & Technology 2011;45(2):776-781.	R834574 (2011) R834574 (Final) R833335 (2009) R833335 (Final)	Abstract from PubMed Full-text: ACS-Full Text HTML Exit Abstract: ACS-Abstract Exit Other: ACS-Full Text PDF Exit
Journal Article	Neal AL, Kabengi N, Grider A, Bertsch PM. Can the soil bacterium Cupriavidus necator sense ZnO nanomaterials and aqueous Zn2+ differentially? Nanotoxicology 2012;6(4):371-380.	R834574 (2011) R834574 (2012) R834574 (Final)	Abstract from PubMed Full-text: ResearchGate-Full Text HTML and PDF Exit Abstract: Taylor and Francis-Abstract Exit
Journal Article	von der Kammer F, Ferguson PL, Holden PA, Masion A, Rogers KR, Klaine SJ, Koelmans AA, Horne N, Unrine JM. Analysis of engineered nanomaterials in complex matrices (environment and biota): general considerations and conceptual case studies. Environmental Toxicology and Chemistry 2012;31(1):32-49.	R834574 (2011) R834574 (2012) R834574 (Final) R833859 (Final) R834857 (2011) R834857 (2012)	Abstract from PubMed Full-text: Wiley-Full Text PDF Exit Abstract: Wiley-Abstract Exit

Supplemental Keywords:

environmental nanotechnology, nanotoxicology, environmental chemistry, ecological and human health risks of manufactured nanomaterials, chemical speciation, biosensors, environmental chemistry, biogeochemistry

Relevant Websites:

http://www.research.uky.edu/odyssey/features/nanotech.html Exit

http://www.pratt.duke.edu/duke_ceint_tine Exit

http://www.rothamsted.ac.uk/ProjectDetails.php?ID=5094 Exit

http://www.cranfield.ac.uk/sas/cas/projects/page49553.html Exit

Progress and Final Reports:

Original Abstract

The 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.