Grantee Research Project Results
2005 Progress Report: Chemical and Biological Behavior of Carbon Nanotubes in Estuarine Sedimentary Systems
EPA Grant Number: R831716Title: Chemical and Biological Behavior of Carbon Nanotubes in Estuarine Sedimentary Systems
Investigators: Ferguson, P. Lee , Scrivens, W. A. , Chandler, G. Thomas
Institution: University of South Carolina at Columbia
EPA Project Officer: Hahn, Intaek
Project Period: August 15, 2004 through August 14, 2007 (Extended to August 14, 2008)
Project Period Covered by this Report: August 15, 2004 through August 14, 2005
Project Amount: $334,750
RFA: Exploratory Research to Anticipate Future Environmental Issues: Impacts of Manufactured Nanomaterials on Human Health and the Environment (2003) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals , Human Health
Objective:
The research supported by this Science To Achieve Results (STAR) grant is directed at elucidating the fate and effects of single-walled carbon nanotubes (SWNTs) in estuarine environments. Because of their unique physicochemical properties and potential for large-scale commercialization, concerns have emerged over potential adverse effects of nanomaterials such as SWNTs in the aquatic environment. These concerns include direct toxicity to aquatic organisms as well as potential effects on distribution of hydrophobic organic contaminants (HOC) through adsorptive sequestration. We hypothesize that these materials will be discharged to the aquatic environment through waste streams during manufacturing and in disposal of nanotube-containing consumer products, and that because of their hydrophobic nature, they will associate with sediments, where they may be accumulated and cause effects in benthic organisms. Specific objectives of the project are to:
- Determine factors controlling the fate of SWNTs and their synthetic byproducts in estuarine seawater, sediment, and sediment-ingesting organisms.
- Examine the impact of SWNTs and byproducts on the disposition of model organic contaminants in estuarine sediments.
- Determine whether the presence of SWNTs and byproducts in estuarine sediments affects the bioavailability of model organic contaminants to estuarine invertebrates.
- Assess the toxicity of SWNTs and byproducts to suspension- and deposit-feeding estuarine invertebrate models in seawater suspension alone and/or in combination with estuarine sediments.
Progress Summary:
In our initial work on this project, we have addressed specific objectives one and four, using well-characterized SWNTs generated through the arc-discharge route and subsequently purified by acid treatment and electrophoresis. Work on objective #1 has focused on determining the factors that control the aggregation behavior of SWNT under simulated estuarine conditions. SWNT aggregation over time was examined in buffered (pH = 8) solutions containing various concentrations and types of electrolytes. Aggregation of SWNTs was assayed by dynamic light scattering using a 90 Plus/Zeta phase analysis light scattering (PALS) particle size analyzer (Brookhaven Instruments, Inc., Holtsville, NY) in batch experiments designed to examine the stability of SWNT suspensions under simulated estuarine conditions (change in salinity, change in dissolved organic matter (DOM) concentration) and at various mixing times and SWNT concentration loadings. For these experiments, SWNT concentration was kept constant at 10 mg L-1, and ionic strength was adjusted over the range of 0–686 mM using NaCl (Figure 1A), synthetic sea-salt (Instant Ocean, Figure 1B), or NaCl/CaCl2 (ratio of Na+:Ca2+ held constant at 7.7:1, Figure 1C) and particle size measurements were made at 1-minute intervals over 10 minutes. Results indicate that ionic strength strongly influenced SWNT aggregation behavior, and that colloidal SWNT suspensions were destabilized in solutions containing monovalent electrolytes at concentrations above 100 mM.
Figure 1. SWNT Aggregation in Buffered NaCl Solution (A), Synthetic Sea Salt (B), and Buffered NaCl/CaCl2 Solution (C), as Measured by Dynamic Light Scattering Methods
(A) Purified SWNTs in Buffered NaCl | (B) Purified SWNTs in Instant Ocean | (C) Purified SWNTs in Buffered NaCl/CaCl2 |
Synthetic seawater solutions with ionic strengths as low as < 20 mM stongly destabilized SWNT colloids, and this effect seems to be due to the presence of divalent cations as the SWNT aggregation behavior in Instant Ocean solution was reproduced quantitatively by a simple mixture of NaCl/CaCl2. This enhanced aggregation in divalent electrolyte solutions was most likely due to salt-bridging of SWNTs.
The effect of DOM on SWNT aggregation in solution was tested in experiments utilizing Suwanee River DOM (0–50 mg L-1) that had been filtered (0.45 μm) to remove particulate matter. SWNTs (10 mg L-1) were added to solutions containing increasing DOM concentrations and total electrolyte ionic strengths of 157 mM adjusted by addition of either NaCl (Figure 2A) or synthetic sea salt (Instant Ocean, Figure 2B). Particle size measurements were made over at 10-minute period by dynamic light scattering. In monovalent electrolyte solutions, DOM reduced SWNT aggregation in a concentration-dependent manner, such that at the highest DOM concentration (50 mg L-1) aggregation of SWNTs was inhibited and a colloidal suspension was maintained (particle sizes remained below 200 nm over the 10-minute incubation time). However, in synthetic seawater at the same ionic strength, the presence of DOM had no influence on the aggregation behavior of SWNT—large (> 1 μm) particles formed after 10 minutes at all DOM concentrations tested. This is most likely due to extensive cross-linking of the DOM material with the negatively charged sites of the SWNT by divalent cations in the synthetic seawater solution. In monovalent cation solutions (e.g., NaCl), aggregation may have been limited by coating of nanotubes with DOM and reduction of hydrophobic-interaction among adjacent nanotube clusters.
Figure 2. SWNT Aggregation in Buffered NaCl Solution (A) and Synthetic Sea Salt (B) in the Presence of Increasing Concentrations of Suwanee River DOM
We have focused our initial effects assessment work (addressing specific objective #4) on determining the full life-cycle effects of SWNTs on a model estuarine meiobenthic crustacean copepod (Amphiascus tenuiremis). To this end, a 28-day lifecycle microplate bioassay (American Society for Testing and Materials [ASTM] method E-2317-04) was utilized to identify chronic toxicological responses by A. tenuiremis to SWNT exposure in seawater. Our experimental design was comprehensive in that we included “as prepared” (AP) SWNT materials as model toxicants, plus purified fractions of this raw synthetic material including SWNTs, and fluorescent low molecular-weight nanocarbon synthetic byproducts. For this work, we took advantage of previously developed electrophoretic methods for size-separation of raw AP-SWNT preparations. A synchronous cohort of naupliar larvae were assayed by culturing individual larvae to adulthood in individual 96-well microplate wells amended with SWNTs in seawater. Copepods were exposed to “as prepared” (AP) SWNTs, electrophoretically purified SWNTs, or a fluorescent fraction of nanocarbon synthetic byproducts. Copepods ingesting purified SWNTs showed no significant effects on mortality, development, and reproduction across exposures (p < 0.05) (Figures 3 & 4). In contrast, exposure to the more complex AP-SWNT mixture significantly increased life-cycle mortality rates, reduced fertilization rates, and reduced molting success in the highest exposure (10 mg L-1) (p < 0.05). Exposure to small fluorescent nanocarbon byproducts caused significantly increased life-cycle mortality rates at 10 mg·L-1 (p < 0.05). The fluorescent nanocarbon fraction also caused significant reduction in life-cycle molting success for all exposures (p < 0.05). These results suggest size-dependent toxicity of SWNT-based nanomaterials, with the smallest synthetic byproduct fractions causing increased mortality and delayed copepod development over the concentration ranges tested.
Figure 3. Full Life-Cycle Mortality Rates for Each Population Within Each of the Three Separate Chronic Bioassays. AP-SWNTs populations are shown by solid black bars (left), pure SWNTs populations are shown by hatched bars (middle), and fluorescent nanocarbon byproduct populations are shown by unfilled bars (right). Error bars represent one standard deviation of the mean.
* represents those exposures showing significantly elevated mortality relative to controls (p < 0.05).
Figure 4. Mean Percentage of Individuals From Each Population That Successfully Developed From the Nauplius Stage to the Adult Stage. AP-SWNTs populations are shown by solid black bars (left), pure SWNTs populations are shown by hatched bars (middle), and fluorescent nanocarbon byproduct populations are shown by unfilled bars (right). Error bars represent one standard deviation of the mean.
* represents those exposures showing significantly depressed developmental success relative to controls (p < 0.05).
Light and confocal microscopy allowed direct observation of SWNT aggregates, naupliar copepods, copepodite and adult A. tenuiremis, and fecal materials during and following exposure to purified SWNTs. As shown in Figure 5, SWNT clusters were prominent and easily distinguished visually throughout the gut of exposed individuals (Figure 5C, D), as well as within individual fecal pellets (Figure 5E, F). This indicates that A. tenuiremis ingested SWNTs as aggregated clusters along with microalgae. Furthermore, relative to the observed size and consistency of SWNT aggregates in microplate exposures (Figure 5A) A. tenuiremis appears to condense SWNT clusters during the ingestion process, creating smaller, more tightly packaged SWNT clusters that pass back to the seawater environment in their feces (Figure 5B, C, D, E, F).
Figure 5. Panels A) and B) Represent Inverted Light Microscope Images of Unperturbed Pure SWNT Aggregates and Pure SWNT Aggregates Following Feeding by a Nauplius Copepod. Red arrows in image B) show a strand of SWNTs and compacted SWNT spherical bundles following feeding by nauplii. Images C) and D) show light-level and false-color confocal microscopic images of an adult copepod with multiple ingested pure SWNT aggregates traveling through its gut. Images E) and F) show light-level and false-color confocal microscopic images of adult copepod fecal pellets with compacted pure SWNT bundles.
Future Activities:
Future work on this project will address specific objectives two and three of the proposed work (as described above). Specifically, we will perform experiments to quantify the sorption of model hydrophobic organic contaminants (HOCs) to SWNT from aqueous solutions, with the goal of assessing the potential for these carbonaceous materials to act as strong sorbents (and potential transport mediators) for HOCs in the environment. These experiments will be complimented by experiments to determine the effect of SWNT on HOC bioaccumulation from contaminated sediment by estuarine invertebrate organisms. Here, we will test the hypothesis that SWNT will sequester HOCs and thereby reduce the bioaccumulation of these contaminants in sediment-ingesting organisms. Finally, we will utilize newly synthesized 14C-SWNT materials to examine the phase-partitioning and biological uptake of carbon nanotubes in the context of estuarine sedimentary systems.
Journal Articles:
No journal articles submitted with this report: View all 3 publications for this projectSupplemental Keywords:
RFA, Scientific Discipline, INTERNATIONAL COOPERATION, TREATMENT/CONTROL, Sustainable Industry/Business, Sustainable Environment, Environmental Chemistry, Technology, Technology for Sustainable Environment, Ecological Risk Assessment, Chemicals Management, Environmental Engineering, fate and transport, clean technologies, environmental hazard assessment, nanotechnology, effect of seawater and sediment on carbon nanotubes, alternative materials, environmental exposure, engineering, environmentally applicable nanoparticles, nanomaterials, chemical behavior, single walled carbon nanotubes, nanoparticles, bioacummulationProgress 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.