Grantee Research Project Results
Final Report: Ecotoxicology of Underivatized Fullerenes (C60) in Fish
EPA Grant Number: R833333Title: Ecotoxicology of Underivatized Fullerenes (C60) in Fish
Investigators: Henry, Theodore B. , Sayler, Gary S. , Menn, Fu-Min , Compton, Robert N.
Institution: University of Tennessee
EPA Project Officer: Aja, Hayley
Project Period: May 15, 2007 through May 14, 2010
Project Amount: $396,807
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: Safer Chemicals , Nanotechnology
Objective:
Objectives: Investigate characteristics of aqueous C60 aggregates and the impact of dissolved organic material on behavior of nanoparticles. Evaluate bioavailability and toxicity of C60 (both aqueous C60 aggregates and dietary C60) in fish by evaluating changes in gene expression, histopathology, and bioaccumulation of C60 in tissues. The species we will consider are zebrafish Danio rerio and channel catfish Ictalurus punctatus.
Hypotheses: 1) Bioavailability of aqueous C60 aggregates is impacted by nanoparticle characteristics and presence of dissolved organic material; 2) Exposure of fish to C60 can be detected by changes in expression of biomarker genes; and 3) Toxic effects of C60 in fish are evident only after long-term chronic exposure.
Summary/Accomplishments (Outputs/Outcomes):
The organic solvent tetrahydrofuran (THF) has been used as a vehicle to facilitate formation of nC60 aggregates in water, and previously researchers published results that did not include appropriate THF vehicle controls and erroneously implicated C60 as the cause of toxicity. Unfortunately, results of THF-induced toxicity were widely cited as C60-induced toxicity within the scientific literature and the public media, and these reports contributed to a mis-impression of the toxicity of nC60. Our work used changes in global gene expression in larval zebrafish Danio rerio to demonstrate that gene expression profiles for fish exposed to THF vehicle controls were highly similar to profiles of THF-nC60 exposed fish, and that gene expression profiles in fish exposed to water stirred nC60 did not have any significant indications of toxicity. We attributed THF-induced toxicity to decomposition products of THF generated during the nC60 preparation process (e.g., γ-butyrolactone and tetrahydro-2-furanol), and subsequent investigations by independent researchers have confirmed these observations. Our findings have contributed to a re-direction in the approach towards investigating toxicity of nanoparticles and a re-evaluation of the early literature on toxicity of nC60. This is especially important in terms of correctly characterizing the toxicity of NPs and enabling the responsible development of nanotechnology.
Results of global gene expression studies in zebrafish exposed to nC60 indicated the NP was of low toxicity and led us to re-direct our scientific investigation to more relevant aspects including the impact of nC60 on the transport, environmental fate, and bioavailability of other toxicants (termed here “co-contaminants”). Interactions between NPs and co-contaminants are environmentally relevant and there is evidence in the literature that indicates NPs will interact with other substances in surface waters through adsorption de-sorption reactions. The interaction of NPs and co-contaminants is emerging as a potentially important effect of the release of NPs into the environment; however, investigating the interactions between NPs and co-contaminants is challenging because most traditional analytical techniques disturb the NP-co-contaminant complex such that results may be difficult to interpret. We have developed an approach to investigate the association of specific co-contaminants with NPs in water based on changes in the bioavailable fraction of the co-contaminant. This technique takes advantage of the highly sensitive response of changes in gene expression in fish that occur as a consequence of exposure to some bioavailable-biologically active substances. We also have demonstrated that assessment of changes in the bioavailable fraction of a co-contaminant can be combined with other measures of NP physicochemistry to provide a more complete understanding of NPs in the aqueous phase.
We found the association between 17α-ethinylestradiol (EE2), a biologically active synthetic oestrogen, and nC60 reduced bioavailability of EE2 in fish, and that nC60 aggregates became in general larger, had reduced particle charge (zeta potential), and had a greater tendency to sediment out of the water column. The association with EE2 was related to the computed surface area of nC60 initially; however, changes in the bioavailability of EE2 that occurred over time (28 d) when associated with nC60 suggested that EE2 was being adsorbed within the nC60 matrix. Subsequent experiments in which nC60 aggregates containing EE2 were fed to fish demonstrated that the EE2 did not become bioavailable and indicated that the association between EE2 and nC60 was sufficiently robust to withstand the fish digestive system. We have recently completed experiments with nC60 and Hg2+ (as co-contaminant) that indicate Hg2+ associates with nC60, the association changes aggregate characteristics, and bioavailability of Hg2+ (by measuring changes in expression of metallothionein genes) is altered by association with nC60. Interestingly, greater relative amounts of nC60 increased the amount of bioavailable Hg2+ in fish perhaps because nC60-Hg2+ complexes sedimented to the bottom of the water column and increased fish exposure to Hg2+ (in this experiment larval fish resided on the bottom of the container). Clearly EE2 and Hg2+ have considerably different physicochemical properties and consequently the nature of their association with nC60 differs. By selecting different NPs and co-contaminants with various physicochemical properties we can obtain a better understanding of how NPs behave in the aqueous phase and establish some principles that can be applied more broadly to the numerous other substances (co-contaminants) and NPs that currently exist or may be produced in the future.
A significant research objective for this project was to investigate long-term toxicity and bioaccumulation of nC60 in fish exposed through the diet. Accumulation of NPs by filter feeding organisms and subsequent consumption of organisms that have accumulated NPs by fish is environmentally relevant and a potential manner in which trophic transfer of NPs may occur. We investigated the chronic toxicity of nC60 added to formulated fish pellets (500 mg C60/ kg food) in juvenile rainbow trout Oncorhynchus mykiss and included a separate experimental treatment of single-walled carbon nanotubes (SWCNTs, 500 mg/ kg food) to enable comparison of toxicity based on differences in NP shape. The exposure occurred for six weeks during which time fish were sampled for analysis of numerous toxicological endpoints including growth, haematology, tissue ion concentrations, histopathology, osmoregulation, and biochemistry; and, after exposure, fish were allowed three weeks of depuration (fed control food without NPs). We found at week 4, but not on weeks 2 and 6, significant elevation in brain TBARS (an indication of lipid peroxidation) was observed in fish exposed to SWCNTs (16.2 ± 1.38 nmol mg-1 protein) compared to the control (9.11 ± 0.81 nmol mg-1 protein) and fish exposed to C60 (8.28 ± 0.56 nmol mg-1 protein). No other significant treatment-related differences were observed and these results indicated that dietary exposure to SWCNTs and C60 in rainbow trout did not result in overt toxicity. Similar to other investigators, we did not observe accumulation of C60 in tissues, which indicates little or no absorption of C60 across intestinal epithelia and reduces concern for this NP to biomagnify within food chains.
An important objective for the advancement of environmental nanoscience is the integration of international research initiatives and this objective was facilitated directly during this project by the move (Nov 2007) of Dr. Henry (project PI) to the University of Plymouth, Plymouth, United Kingdom. Dr. Henry accepted a position as a Research Council of the United Kingdom (RCUK) Academic Fellow working primarily in nanotoxicology, and through this position numerous genuinely collaborative research projects were established. Dr. Henry maintained his role as PI on this project and directly supervised project personnel through weekly electronic communications and quarterly on site visits to The University of Tennessee to engage with ongoing research. These visits facilitated the exchange of characterized nanomaterials and samples for testing and direct international involvement in environmental nanoscience projects occurring in the US and the UK. Although this current EPA project has ended, the established research collaborations will continue to provide an international perspective within environmental nanoscience.
Conclusions:
Perhaps the most significant finding of this project was to provide the first evidence that vehicle solvents could explain toxicity previously attributed to nC60 aggregates.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 25 publications | 6 publications in selected types | All 6 journal articles |
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Henry TB, Menn FM, Fleming JT, Wilgus J, Compton RN, Sayler GS. Attributing effects of aqueous C60 nano-aggregates to tetrahydrofuran decomposition products in larval zebrafish by assessment of gene expression. Environmental Health Perspectives 2007;115(7):1059-65. |
R833333 (Final) |
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Handy RD, Henry TB, Scown TM, Johnston BD, Tyler CR. Manufactured nanoparticles: their uptake and effects on fish—a mechanistic analysis. Ecotoxicology2008;17(5):396-409. |
R833333 (Final) |
Exit Exit |
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Park JW, Henry TB, Menn FM, Compton RN, Sayler G. No bioavailability of 17α-ethinylestradiol when associated with nC60 aggregates during dietary exposure in adult male zebrafish (Danio rerio). Chemosphere. 2010;81(10):1227-32. |
R833333 (Final) |
Exit Exit |
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FraseR TW, Reinardy HC, Shaw BJ, Henry TB, Handy RD. Dietary toxicity of single-walled carbon nanotubes and fullerenes (C60) in rainbow trout (Oncorhynchus mykiss). Nanotoxicology2011;5(1):98-108 |
R833333 (Final) |
Exit |
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Park JW, Henry TB, Ard S, Menn FM, Compton RN, Sayler GS. The association between nC60 and 17α-ethinylestradiol (EE2) decreases EE2 bioavailability in zebrafish and alters nanoaggregate characteristics. Nanotoxicology2011;5(3):406-16. |
R833333 (Final) |
Exit Exit |
Supplemental Keywords:
C60 fullerenes, nanoparticles, EE2, co-contaminant, nanoparticle associations, bioavailability, dietary exposure, aqueous exposure, nanotoxicology, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Risk Assessments, Biochemistry, Physical Processes, Risk Assessment, biological pathways, bioavailability, exposure, genetic analysis, nanotechnology, carbon fullerene, human exposure, nanomaterials, fish model, toxicologic assessment, histopathology, nanoparticle toxicity, carcinogenic, human health riskProgress 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.