Grantee Research Project Results
Final Report: Characterization of the Potential Toxicity of Metal Nanoparticles in Marine Ecosystems Using Oysters
EPA Grant Number: R833337Title: 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 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:
While nanotechnology development has exploded, it is only recently that important issues regarding their potential toxicity and non-target impacts have been raised and there are numerous potential environmental risks of engineered nanoparticles that are not yet well-characterized or understood. Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release from their use in electronic, biological, and other applications. Therefore, this research plan was designed to address a number of important issues regarding nanoparticle toxicity in marine organisms, e.g. adverse effects on important fundamental cellular responses related to lysosomal integrity, effects on antioxidants and oxidative damage, relative sensitivity of different life history stages, and cellular and tissue accumulation patterns. A bivalve mollusk (oysters, Crassostrea virginica) was used as a valuable indicator species that can be used to assess the potential toxicity and impacts on ecological receptors of nanostructured materials. As a filter-feeding organism, they spend their lives processing particles, so they are especially relevant for considering the effects and environmental impacts of nanoparticles. Confocal and high resolution microscopy were used in some studies to characterize the cellular localization of the particles, and also how their structure could be affected by common environmental variables (e.g. salinity). These studies involved whole animal oyster studies as well as studies with primary cell preparations and isolated tissues, and embryos. While the primary focus of these studies was on aquatic organisms and environmental issues, these studies also provided valuable information regarding fundamental cellular responses relevant to humans as well as wildlife.
Summary/Accomplishments (Outputs/Outcomes):
These studies were initiated using fullerenes to establish the feasibility of the techniques, and because fullerenes are inherently fluorescent, intracellular localization approaches could be evaluated. Most of the work involved metal nanoparticles (primarily Ag and also Ti nanoparticles) of different shapes and coatings, and also involved evaluation of phototoxicity. All materials in this study were produced and characterized at the Center for Nanotechnology and Molecular Materials at Wake Forest University, and Wake Forest University Medical Center.
This body of work provides important new insights regarding the potential toxicity of a variety of types of nanoparticles (NP) on adult and embryonic oysters, a valuable filter feeding indicator organism. Lysosomal destabilization and lipid peroxidation assays proved to be valuable indicators of toxicity and also provided insights regarding bioreactivity and the underlying mechanisms of toxicity. Most of the work involved assessment of AgNPs. We chose to place a lot of emphasis on AgNPs because they are one of the most extensively used NPs in 2 commercial products. Overall, we did find that they are more toxic than fullerenes, and much more toxic than TiO2 NPs. As part of the AgNP studies, we frequently conducted studies with dissolved Ag (especially AgNO3 at comparable Ag concentrations) to compare the responses between dissolved Ag and Ag nanoparticles. These types of comparative studies enabled a consideration of whether the observed toxicity was likely to be due to simply the dissolution of the particles and the release of toxic Ag, or if indeed there was a true nanoparticle effect. In most cases, the responses to dissolved Ag were quite different from the AgNP treatments, reinforcing the finding that toxicity was not merely a reflection of dissolution, but was indeed distinct nanoparticle effects. In many cases, toxicity of the AgNPs was actually greater and / or observed at lower Ag concentrations than the dissolved Ag. In some cases there were completely opposite antioxidant responses (e.g. elevated in some cases while reduced in others) between dissolved Ag and AgNP responses. There were also quite remarkable tissue-specific differences. While gill tissues were often very sensitive to dissolved Ag, the most prominent toxicity or bioreactivity responses to AgNPs were observed in the hepatopancreas tissues. Prior to actually conducting this research, greater impacts on gill tissues might have been expected, as gill tissues would be the primary particle capturing mechanism and would be the tissues that first encounter the particles. As a result of these studies, we now believe that while the particles are likely captured by the gills, they are transferred with minimal cellular processing (like their phytoplankton food), probably largely entrained in the mucus productions, moved into the food groove which transports foods and nutrient particulates into the digestive tract, and are readily transported into the hepatopancreas (probably by endocytosis). This is the typical food processing pathway as digestion of food is largely intracellular in invertebrates. Our studies with fullerenes (and earlier studies with quantum dots) indicate accumulation and concentration of nanoparticles in lysosomes, further supporting this model. The hepatopancreas tissues (aka digestive gland tissues) are rich in lysosomes involved in digestion of food particles as well as autophagic processes, and lysosomes were found to be a primary target organelle for toxicity. In general, embryo toxicity and sensitivity to AgNPs were similar to that of the adults, and also tended to exhibit a distinct threshold type of response rather than gradual dose-dependent response, indicating that when some critical level of metal nanoparticles does affect embryos, the response is devastating; this was in stark contrast to the fullerene embryo studies which demonstrated a very strong linear dose-response curve, and more graded toxicity responses.
Silver NPs synthesized with two different coatings, citrate-coated particles and PVP (polyvinylpyrrolidone)-coated particles, were also evaluated. While lysosomal damage was observed with both types of coatings, increased lipid peroxidation was rarely observed with the PVP-AgNPs, suggesting that they may be slightly less toxic. Citrate-coated particles also tended to cause reduced levels of the important antioxidants (glutathione and catalase), which would tend to exacerbate oxidative damage or susceptibility to other stressors, whereas PVP-coated particles did not.
Studes were conducted with three different shapes of citrate-coated AgNPs - Spheres, Prisms, and Plates with whole adult oysters, isolated hepatopancreas and gill tissues, and oyster embryos. The Prisms tended to cause the most severe adverse effects, especially in the hepatopancreas tissues and embryos, and significant toxicity was also frequently observed with the Spheres and Plates. Overall these studies indicated that shape can affect toxicity. Studies were also conducted with an intracellular probe for detecting reactive oxygen species (ROS) production. In combination, tissue damage associated with lysosomal effects, lipid peroxidation, and the increased production of ROS in response to AgNP exposures suggest that oxidative stress is a major mechanism of toxicity. Two shapes of PVP-coated AgNPs were also evaluated – Spheres and Prisms, and again the Prism shape tended to be more toxic.
One of the purposes of conducting studies with isolated tissues (e.g. in vitro exposures) was to consider the potential utility of these types of studies for screening various nanoparticle preparations. In vitro approaches have some value in that they involve small volumes and could be particularly amenable for more rapid evaluations. However, while toxicity was sometimes observed in vitro exposures, these types of exposure approaches were not as sensitive as whole oyster or embryo studies.
Adult oysters and embryos were also exposed to Titanium dioxide (TiO2) nanoparticles. Because Ti02-NPs have been reported to have enhanced phototoxicity in the presence of UV light or sunlight, these studies were conducted under dark, indoor conditions as well as outdoor in natural sunlight. The light levels used for these studies were low levels, typical of those that would occur in natural estuarine environments. In some cases adult oysters or embryos were exposed directly to the experimental light conditions outside, but some studies were also conducted with “charged”water, e.g. water with TiO2-NPs placed in the natural sunlight for 4 hours, and then used for exposures in the laboratory without direct sunlight. Generally high levels of TiO2 Nanoparticles were required to cause toxicity, at high ppb or ppm levels – much higher than the AgNPs. Some photo-enhanced toxicity was observed with regard to lipid peroxidation in adult oysters and in oyster embryos during the exposures to natural sunlight. However, unlike the AgNPs or fullerenes, no adverse effects on lyososmal destabilization were observed. No adverse effects were observed with the Charged particle exposures. It is possible that the TiO2 nanoparticles do not readily traverse cell membranes (which might explain the lack of effect on lysosomal destabilization) or embryonic membranes, but cause toxicity by generation of free-radicals externally that cause lipid peroxidation and general membrane damage during exposure to sunlight.
Conclusions:
These studies have served to validate a suite of valuable biomarker responses in a filterfeeding organism that can be used to evaluate the toxicity and bioreactivity of nanoparticles, metal as well as carbon-based fullerenes. Information on the effects on fundamental cellular responses and target organelles are just emerging. In these studies, some of the things that we evaluated were lysosomal and antioxidant responses, which are fundamental to all living organisms – humans and vertebrates as well as invertebrates. Therefore while these studies provide information regarding potential impacts on estuarine habitats, they also provide information on fundamental cellular processes that help to inform responses of other living systems. We have demonstrated that the responses are differentially sensitive to nanoparticles as compared to the dissolved constituents. The biomarker assays and embryonic assays are well established, fairly low cost protocols that can readily be used to screen a variety of nanoparticle preparations. Since oysters, like other filter feeding organisms, process large amounts of water, phytoplankton, and particulates, they are a valuable model for evaluating bioavailability and effects, and for assessing potential environmental impacts. As important ecosystem engineers, adverse effects on oysters can readily translate into wider-spread impacts on estuarine habitats as well as higher trophic levels (including fish and birds as well as microbes).
Journal Articles on this Report : 3 Displayed | Download in RIS Format
| Other project views: | All 13 publications | 3 publications in selected types | All 3 journal articles |
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Johnson BD, Gilbert SL, Khan B, Carroll DL, Ringwood AH. Cellular responses of eastern oysters, Crassostrea virginica, to titanium dioxide nanoparticles. Marine Environmental Research 2015;111:135-143. |
R833337 (Final) |
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McCarthy MP, Carroll DL, Ringwood AH. Tissue specific responses of oysters, Crassostrea virginica, to silver nanoparticles. Aquatic Toxicology 2013;138-139:123-128. |
R833337 (Final) |
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Ringwood AH, Levi-Polyachenko N, Carroll DL. Fullerene exposures with oysters: embryonic, adult, and cellular responses. Environmental Science & Technology 2009;43(18):7136-7141. |
R833337 (2008) R833337 (Final) |
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Supplemental Keywords:
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 fullereneProgress 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.