Grantee Research Project Results
2007 Progress Report: Assessment of the Environmental Impacts of Nanotechnology on Organisms and Ecosystems
EPA Grant Number: R832635Title: Assessment of the Environmental Impacts of Nanotechnology on Organisms and Ecosystems
Investigators: Bonzongo, Jean-Claude J. , Kopelevich, Dmity , Bitton, Gabriel
Institution: University of Florida
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2005 through September 30, 2008
Project Period Covered by this Report: October 1, 2006 through September 30, 2007
Project Amount: $375,000
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: A Joint Research Solicitation - EPA, NSF, NIOSH (2005) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
The overall goal of this project is to develop an understanding of the potentially complex interplay between manufactured nanomaterials (MN) and the health of organisms and ecosystems. The research hypothesis is that chemical elements used in the production of MN could lead to environmental dysfunctions due to: (1) the potential toxicity of these elements and their derivatives; and (2) the nanometer-size that make MN prone to bio-uptake/bioaccumulation and (3) the large surface area which might lead MN to act as carriers/delivers of pollutants adsorbed onto them. To address this hypothesis, toxicity studies using microbiotests as screening tool and experimental investigations on environmental implications and transport of toxic MN are being conducted. These experimental studies are complemented by molecular dynamic (MD) simulations to investigate events on very small time- and length-scales. MD simulations are also used to assess the contribution of different types of intermolecular interactions and chemical reactions to the permeation of MN into cell interior and the potential of MN to damage cell membranes and lead to cell death.
Progress Summary:
Experimental work conducted during this second year included the completion of MN toxicity screening using model microorganisms. In addition, studies focusing on toxicity of MN in natural waters and sediments as well as laboratory investigations on transport of selected toxic MN in porous media (natural sandy and clayey soils) were started and are still ongoing. In parallel, the modeling component has focused on the study of mechanisms of permeation of MN into cell interior and the assessment of the possibility of this specific MN to cause lethal damages to cell membranes. The main findings for this second year of the project are summarized below.
2.1. Experimental studies: Toxicity and environmental impacts of MN
Figure 1: Toxicity of common surfactants used for dispersing SWCNTs on: (A) C. dubia, and (B) P. subcapitata. Bars with >10ppm and >1000ppm showed no toxicity up to these tested concentrations. (nd = not determined)
During year-1 of this project, the toxicity component of the study focused primarily on screening a wide variety of MN for their potential toxicity as described in the 2006 annual report. Following these tests five different MN (i.e. C60, single-walled carbon nanotubes (SWNTs), nanoparticles of copper and silver, and CdSe quantum dots) were retained for further studies on (i) toxicity mechanisms and (ii) transport of these MN in porous media. In parallel, we assessed the fate of pollutants adsorbed onto MN in natural systems using mercury as example pollutant. Laboratory studies have also focused on the effect of natural water matrices on the toxicity of MN, and effects of MN on sedimentary microorganisms that drive the cycling of organic carbon. This research is ongoing and has so far resulted in two papers which are currently in press in “Environmental Toxicology and Chemistry” and a book chapter to be published in summer 2008.
2.1.1. Mechanisms of toxicity—we initiated laboratory studies investigating the mechanism(s) of MN interaction with the freshwater algae Pseudokirchneriella subcapitata also known as Selanastrum capricornutum. This unicellular micro-algae was selected because of its high sensitivity to toxic MN as determined in year-1 of this study. So far, only SWNT suspensions have been used in these algal growth experiments. To eliminate the negative effect on algal growth associated with the solvent, a surfactant selection study was first conducted on two aquatic model species (Fig. 1), and based on obtained results, Gum Arabic (GA), a non-ionic surfactant was selected for the preparation of SWNT suspensions used in later experiments. A comparative study of the effects of GA alone (Fig. 2a) and SWNT suspension in GA (Fig. 2b) on the growth of P. subcapitata showed a non linear growth response in the presence of SWNT, in that at low concentrations (up to 0.1 ppm), SWNTs lead surprisingly to growth stimulation, which is then followed by a progressive growth inhibition trend in culture media containing SWNTs at levels greater than 0.1 ppm (Fig. 2b). Similar studies using C60, nanoparticles of and Ag or CdSe quantum dots showed only growth inhibition with increasing MN concentrations in the culture medium. Currently, we are conducting studies to gain insight in the mechanisms of both biostimulation and toxicity of SWNT on P. subcapitata. The hypotheses driving our studies on potential mechanisms of algae-SWNT interactions follow two tracks: (i) the SWNT manufacturing process with emphasis on level of impurities, and (ii) the degree of SWNT aggregation when in contact with living cells.
Figure 2: Growth of P. subcapitata in the absence (a) and presence (b) of SWNTs in a modified PAAP culture medium, and growth stimulation and inhibition along a SWNT concentration gradient. Horizontal solid lines represent the average algalgrowth rate (n=3) in control media while doted lines represent ±1 standard deviation
2.1.2. Effect of natural water matrices on the toxicity of MN—suspensions of the selected MN were prepared using 3 natural water samples collected along the Suwannee River in Florida. The three water samples differed significantly in dissolved organic matter content and ionic strength (samples collected along a dissolved organic matter and salinity gradients from headwaters at the Florida/Georgia border to the River delta in the Gulf of Mexico). Laboratory experiments are being conducted by exposing model test aquatic organisms to MN suspended mechanically in these natural waters. Our preliminary results suggest that the use of drastic methods such as sonication, organic solvents, and ultrasound does affect the toxicity response of tested organisms leading to results that might not be representative of what could occur in natural aquatic systems. Detailed results are expected at the end of year-3 of the project.
2.1.3. Effects of MN on sedimentary microorganisms that drive the cycling of carbon—these experiments were started in year-1 of the project and are still ongoing. In addition to assessing the toxicity of selected MN on the ability of sedimentary microorganisms to degrade organic substrates, we are currently sorting out the types of microbial populations affected by specific MN using genomics.
2.1.4. Fate and transport of pollutants and MN in sedimentary environments—this component of the project has focused on two different research topics. First, the fate of pollutants adsorbed on MN following their use in pollutant removal technologies. Here we used mercury as example pollutant after its removal from flue gases using SiO2-TiO2 nanoparticles. The results show that depending on sediment pH, SiO2-TiO2 adsorbed Hg can become bioavailable and a source of methyl-Hg if released to soil/sediment environment. These results ring the bell on the currently overlooked issue of nanowastes and their environmental implications. Results obtained from this study have been the subject of a paper that is in press in Environmental Toxicology and Chemistry. Second, SWNTs suspensions prepared in a non-ionic (i.e. GA) and an ionic (i.e. SDS) surfactants are currently being used to investigate the transport of SWNTs in (i) sandy and (ii) clayey soils. Our preliminary results show that sandy soils have a very poor retention capacity when SWNTs are suspended in surfactants as compared to simple aqueous solutions. In contrast, earlier trends in clayey soils seem to indicate that SWNTs have no to very little mobility, regardless of the composition of the solvent. These studies are still ongoing and detailed results will be available at the end of year-3 of the project.
2.2. Molecular dynamic (MD) simulations
Task 1: In the current computational study, the cellular membrane is modeled as a lipid bilayer and the presence of membrane proteins is neglected. Therefore, the main focus of this work is on the direct diffusion of MN through the cellular membranes and the protein-mediated transport of nanoparticles into cells is neglected. Recent experiments by Rothen-Rutishauser et al. (2006) have shown that NP can permeate some cells by a currently unknown mechanism different from phagocytosis and endocytosis. It is expected that the direct diffusion mechanism investigated in this work is dominant in these systems and therefore the current study is expected to be relevant to a significant number of nanoparticle-cell membrane systems. Lipid membranes are modeled as DPPC lipid bilayers using a coarse-grained molecular model proposed by Marrink et al. (2004).
Figure 3: Free energy profiles for model nanoparticles inside a lipid membrane; values of σ correspond to diameters of spherical nanoparticles. The z coordinate in this plot represents the direction perpendicular to the membrane surface; the bilayer width is approximately 4-6 nm. Therefore, the regions with |z| > 4 nm correspond to bulk water.
In Year 1 of the project, transport of hydrophobic nanoparticles of spherical and nearly spherical shapes through the cellular membrane was investigated. The parameters of the model nanoparticles were chosen to mimic fullerene properties. In Year 2, these studies were extended to other non-spherical nanoparticles such as rod-shaped hydrophobic NPs mimicking carbon nanotubes. A summary of the free energy profiles of these nanoparticles inside the lipid bilayer are shown in Fig. 3.
The obtained profiles show several important features that are expected to play an important role in MN toxicity: (1) There is very small (~ 1 kBT) energy barrier to enter the membrane, even for relatively large NP; (2) There is a significant barrier for a NP to leave the membrane. This barrier increases as the NP size increases. Therefore, a NP can easily enter the membrane but will spend a significant amount of time inside the bilayer before leaving it. During its long residence within the membrane, the NP may significantly disrupt the integrity of the membrane either through chemical reactions of through inducing a local instability of a lipid bilayer by physical effects such as a lipid phase transition in the NP neighborhood.
(3) The general features of the free energy profiles obtained for the non-spherical and spherical particles are the same. The general features of the free energy profiles obtained for the non-spherical and spherical particles are the same. There is a very small energy barrier to enter the membrane and a very large barrier to leave the membrane. However, there is a significant qualitative difference in the free energy profile inside the membrane: for the spherical particles, the minima of the free energy (and hence the preferred location of the particle) are located away from the membrane center whereas for all considered non-spherical NP the minimum of the free energy is directly at the membrane center. This implies that the NPs will be localized in different parts of the membrane depending on their shape and therefore will act differently on different groups of the lipid molecules.
Task 2: Here we investigate the effects of MN-membrane interaction on the membrane stability. In Year 1, a preliminary study of effects of lipid peroxidation caused by NPs was performed. This investigation was motivated by experimental observations of Sayes et al. (2004, 2005) that fullerenes lead to lipid peroxidation that in turn leads to leaky membranes that cause cell death. In order to perform an initial assessment the effects of lipid peroxidation on the membrane stability, the following simple model for the peroxidation reaction was implemented: one of the hydrophobic groups of the lipid tails is replaced by a hydrophilic (oxide) group. A fraction of lipids within the membrane are selected at random to undergo this reaction. MD simulations of such a model of a peroxidized membrane show the membrane is quickly destroyed when the concentration of the reacted lipids is extremely high (≥50%) or undergoes significant structural changes if the concentration of the reacted lipids is lower. In the latter case, the peroxidized lipids tend to fold inside the membrane so that their tails point to the exterior of the membrane rather than to the membrane interior. It is observed that this folding takes place very quickly (within 100 ns) after the reaction has taken place and is very likely to lead to the membrane instability. In fact, although the membrane remains intact on the scale of MD simulations, it is observed visually that it becomes softer since the amplitude of its fluctuations increase with increased concentration of reacted lipids. A more detailed quantification of this result by computing elastic properties of a peroxidized membrane is currently in progress. It is expected that this analysis will yield a limit for fraction of reacted peroxidized lipids (and hence safe exposure to NP) that does not lead to membrane instability.
Figure 4: Pressure profiles in NP-free lipid membrane and in lipid membranes containing NP of different shapes.
In Year 2, an investigation of possible physical damage of NPs embedded into cellular membranes was undertaken. Possibility of changes in (i) bending modulus of the membrane and (ii) pressure profile inside the membrane due to presence of NP was considered. Bending modulus is an important measure of elastic properties of a membrane. A change in this modulus may indicate, for example, “softening” of a membrane leading to its instability. Investigations of a membrane with embedded spherical, nearly spherical, and rod-shaped particles indicate that the membrane bending modulus is insensitive to the presence of these particles.
The change in pressure distribution within the membrane may affect functionality of proteins imbedded into the cellular membrane, in analogy with with action of general anesthetics (Cantor, 1997). However unlike the anesthetics, nanoparticles embedded into the membranes may have an undesirable effect. Our investigations demonstrate that addition of nanoparticles leads to a consistent change in the pressure profile, as shown in Fig. 4. It is interesting to note that the shift is relatively independent of the shape of the nanoparticles. Possible implications of this pressure change will need to be investigated using a more detailed membrane model which would explicitly take membrane-bound proteins into account. Simulations with this more complicated model are outside the scope of the current project but are expected to be performed in an anticipated future project.Future Activities:
- With regard to the experimental component, future activities include the completion of laboratory studies on toxicity mechanisms, and most importantly, the fate and transport of selected MN in sedimentary environments.
- With regard to the modeling component, an important issue remains to be addressed for permeation of rod-shaped NP (such as carbon nanotubes) into cellular membranes. It was discovered that the time-scale of rotation of a nanorod is comparable to the time-scale of its permeation into the membrane. This raises a question of possible membrane damage due to rotation of the rod as it enters the membrane. Other aspects of the study to be considered are:
- Effects of electrostatic charges of NPs on their transport through and interactions with cellular membranes.
- NP interaction with a membrane composed of a mixture of different lipids. It is possible that NP have preferred interactions with certain types of lipids (depending on their headgroup structures and tail lengths) that may lead to transport and stability properties different from those observed in the current studies of a homogeneous DPPC lipid bilayer.
- Investigation of critical levels of lipid peroxidation that lead to membrane instability.
References:
Cantor, 1997. The lateral pressure profile in membranes: A physical mechanism of general anesthesia, Biochemistry, 36, 2339-2344.
Marrink, S. J., de Vries, A. H., and Mark A. E., 2004.Coarse grained model for semi-quantitative lipid simulations, J. Phys. Chem. B, 108, 750-760.
Sayes, C. M., Fortner, J. D., Guo, W., Lyon, D., Boyd, A. M. Ausman, K. D., Tao, Y. J., Sitharaman, B., Wilson, L. J., Hughes, J. B., West, J. L., and Colvin, V. L. 2004. The differential cytotoxicity of water-soluble fullerenes. Nano Lett. 4, 1881-1887.
Sayes, C. M., Gobin, A. M., Ausman, K. D., Mendez, J., West, J. L., and Colvin, V. L. 2005, Nano-C60 cytotoxicity is due to lipid peroxidation. Biomaterials, 26, 7587-7595.
Rothen-Rutishauser, B. M., Schurch, S., Haenni, B., Kapp, N., and Gehr, P. 2006. Interaction of fine particles with red blood cells visualized with advances microscopic techniques. Evniron. Sci. Technol. 40, 4353-4359.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 14 publications | 5 publications in selected types | All 4 journal articles |
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Gao J, Bonzongo J-CJ, Bitton G, Li Y, Wu C-Y. Nanowastes and the environment: using mercury as an example pollutant to assess the environmental fate of chemicals adsorbed onto manufactured nanomaterials. Environmental Toxicology and Chemistry 2008;27(4):808-810. |
R832635 (2007) R832635 (Final) |
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Griffitt RJ, Luo J, Gao J, Bonzongo J-C, Barber DS. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environmental Toxicology and Chemistry 2008;27(9):1972-1978. |
R832635 (2007) R832635 (Final) |
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Tasseff RA, Kopelevich DI. Molecular modeling of nanoparticle transport across lipid bilayers. University of Florida. Journal of Undergraduate Research 2006;7(4). |
R832635 (2006) R832635 (2007) R832635 (Final) |
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Supplemental Keywords:
Health, Scientific Discipline, Health Risk Assessment, Risk Assessments, Biochemistry, biological pathways, nanochemistry, bioavailability, nanotechnology, manufactured nanomaterials, nanomaterials, toxicologic assessment, biogeochemistry, cellular response to nanoparticles, nanoparticle toxicity, bioaccumulation, biochemical researchProgress 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.