Grantee Research Project Results

2007 Progress Report: Characterization of the Potential Toxicity of Metal Nanoparticles in Marine Ecosystems Using Oysters

EPA Grant Number: R833337
Title: Characterization of the Potential Toxicity of Metal Nanoparticles in Marine Ecosystems Using Oysters
Investigators: Ringwood, Amy Huffman , Carroll, David Loren
Institution: University of North Carolina at Charlotte , Wake Forest University
EPA Project Officer: Packard, Benjamin H
Project Period: April 5, 2007 through April 4, 2010
Project Period Covered by this Report: April 5, 2007 through April 4, 2008
Project Amount: $399,843
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: a Joint Research Solicitation-EPA, NSF, NIOSH, NIEHS (2006) RFA Text |  Recipients Lists
Research Category: Nanotechnology , Safer Chemicals

Objective:

This research plan is designed to address a number of important issues regarding metal nanoparticle toxicity in marine organisms, e.g. morphological changes of metal nanoparticles in seawater, adverse effects on fundamental cellular responses related to lysosomal integrity, mitochondrial function, effects on antioxidants and oxidative damage, relative sensitivity of different life history stages, and cellular and tissue accumulation patterns.

Progress Summary:

The account for this program was established June 2007. Much of the effort during the first year of the award has been establishment of working protocols for the cooperative studies between the two laboratories (e.g. UNCC and WFU). The WFU team has extensive experience with producing nanoparticles and analyzing them in pure preparations, and some experience working with human breast cancer cells, but no experience working with more complex environmental samples or oyster cells. Therefore, some preliminary studies were conducted in conjunction with their in-progress work with fullerenes to work out the protocols for these studies, because of their experience with analyzing these materials in biological samples. This also provided good opportunities for training the new graduate student working on this project in the various exposure and toxicity assays for the oyster studies. After the initial training phase, experiments with Ag nanoparticles were initiated. Wake Forest investigators produced a stock concentration of Ag nanoparticles, characterized as “seeds” which are roughly spherical in shape. These studies have involved 48 hour exposures with both embryo and adult oysters to a range of concentrations. For the embryo and adult studies, the lowest concentration at which significant adverse effects were observed on embryo development and lysosomal destabilization of oyster hepatopancreas cells was similar. No significant effects on glutathione levels were observed. Samples of adult tissues (e.g. gill and hepatopancreas) were also analyzed by atomic absorption spectroscopy to characterize the concentration of Ag nanoparticles accumulated in the tissues. Analysis of frozen tissues for the other various toxicity assays are in progress.

Work Status and Work Progress

The account for this program was established June 2007. Because of the timing, the graduate student working on the project started in August 2007. Much of the effort during the first year of the award has been establishment of working protocols for the cooperative studies between the two laboratories (e.g. UNCC and WFU). The WFU team has extensive experience with producing nanoparticles and analyzing them in pure preparations, and some experience working with human breast cancer cells, but no experience working with more complex environmental samples or oyster cells. Therefore, some preliminary studies were conducted in conjunction with their in-progress work with fullerenes to work out the protocols for these studies, because of their experience with analyzing these materials in biological samples. This also provided good opportunities for training the new graduate student working on this project in the various exposure and toxicity assays for the oyster studies. After the initial training phase, experiments with Ag nanoparticles were initiated. In general, we are progressing with the research as planned.

Preliminary Data Results

Wake Forest investigators produced a stock concentration of Ag nanoparticles (Ag-NP), characterized as “seeds” which are roughly spherical in shape. These studies have involved 48 hour exposures with both embryo and adult oysters to a range of concentrations. The analysis of the various samples generated are in progress, and some of the preliminary results are provided in the paragraphs that follow.

For the adult oyster studies, there was a dose-dependent increase in lysosomal destabilization rates in hepatopancreas cells of exposed oysters (Figure 1). While there was no difference between the controls and oysters exposed to the lowest Ag-NP concentration, the higher exposure concentrations were significantly different. The lowest concentration at which statistically significant adverse effects on lysosomal destabilization were observed was 0.015 μM Ag-NP. However, based on our extensive experience with this assay, the lysosomal destabilization rates observed for this concentration may not be biologically significant; we generally regard lysosomal destabilization rates that are > 35% as biologically significant. Therefore, the adverse effects observed at 0.15 μM were considered both statistically and biologically significant. There was a highly significant correlation between exposure concentration and lysosomal destabilization. (Figure 2). The antioxidant assays and analysis of frozen tissues for the other various toxicity assays are in progress. One interesting finding at this point is that glutathione levels were not significantly different between treatments in either gill or hepatopancreas tissues. The glutathione (GSH) levels in gill tissues are consistently lower than that of hepatopancreas tissues; gill levels ranged from approximately 600-700 nmol/g, while the range for hepatopancreas tissues was approximately 1000 – 1200 nmol/g. As the most abundant antioxidant present in cells, depletion of GSH levels can be a consequence of excess radical production and can also exacerbate oxidative damage, but these exposure regimes did not impact GSH levels. Samples of adult tissues (e.g. gill and hepatopancreas) were also analyzed by atomic absorption spectroscopy to characterize the concentration of Ag nanoparticles accumulated in the tissues.

For the embryo studies, we had expected that the embryos might be more sensitive so we used a lower range of exposure concentrations. At 0.015 μM, the % normal embryos dropped to 78% of controls. While levels of <80% of controls are sometimes used to identify biologically significant effects, these levels were not statistically significant. However, the results indicated that the lowest concentration at which statistically (as well as biologically) significant adverse effects on embryo development were observed was 0.15 μM Ag-NP (Figure 3). At this concentration, there was a dramatic drop in the % normal development, to approximately only 11% of control levels. Moreover, the nature of the toxic responses with embryos was much more of a threshold type of response, as none of the lower concentrations were statistically significant from the controls. Therefore both statistically and biologically relevant toxicity were observed at similar concentrations in both adults and embryos. The results of all analyses associated with these Ag nanoparticle studies will provide valuable information regarding the relative toxicity of these particles to different life history stages of oysters, along with the accumulation of the particles in tissues. Therefore, these studies will provide valuable insights regarding the potential risks of Ag nanoparticle exposures to estuarine bivalves and marine ecosystems.

Students and Post-doctoral Fellows:

The following students and post-doctoral fellows have been involved in this work:
Melissa McCarthy, Biology MS Student, UNC-Charlotte; Fall 2007 - Current
Tina Changela, Biology, Undergraduate Student, UNC-Charlotte; Fall 2007 – Current
Nicole Levi, Physics PhD Student, Wake Forest University; June – August 2007;
Post-doctoral Fellow, Wake Forest University School of Medicine, August 2007 - Present

Future Activities:

During Year 2, we will be involved in: 1) the analyses of the samples generated during Year 1; 2) continuation of Ag exposure studies with different forms; 3) Cu nanoparticle exposure and analyses of samples; 4) submission of manuscripts for publication.

During Year 2, the primary activities that are planned are the completion of the analyses of the samples generated during Year 1 (including electron microscopy analyses of seawater preparations to characterize the particle characteristics), continuation of Ag exposure studies with different forms, and 3) Cu nanoparticle exposures and analyses of samples. Similar to Year 1, we will be conducting toxicity studies with both adult and embryonic oysters, and the tissue concentrations of the primary metal constituent of the nanoparticle preparations will be analyzed by atomic absorption spectroscopy. We also expect to submit at least 2 publications over the next year.

Journal Articles:

No journal articles submitted with this report: View all 13 publications for this project

Supplemental Keywords:

aquatic, nanotechnology, susceptibility, mid-Atlantic,, Health, Scientific Discipline, Health Risk Assessment, Risk Assessments, Biochemistry, bioavailability, nanomaterials, carcinogenic, genetic analysis, human exposure, biological pathways, nanoparticle toxicity, nanotechnology, histopathology, human health risk, toxicologic assessment, carbon fullerene

Relevant Websites:

http://www.bioweb.uncc.edu/Faculty/Ringwood/index.htm Exit

Progress and Final Reports:

Original Abstract

2008 Progress Report

Final Report