Progress 10/01/06 to 09/30/11
Outputs
OUTPUTS: Tests continued of nonlethal endpoints or biomarkers in Daphnia magna upon exposure to nanosized particles of titanium dioxide (TiO2), copper oxide (CuO), and zinc oxide (ZnO). Iron oxide nanoparticles, which showed no signs of lethality even at high concentrations, were not included in this study. For the sublethal toxicity, a suite of biomarkers including TBARS (thiobarbituric acid reactive substances), GST (glutathione S-transferase), glutathione (as total glutathione, tGSH, and in oxidized, GSSG, and reduced, GSH, forms) and MT (metallothionein) were monitored. This selection reflects the possibility of oxidative damage to the organisms and potential response of the organism to produce MT for metal sequestration. For TBARs and GST, the method described by Barata et al. (2005) with some modifications was used. For the measurement of GSH, a glutathione fluorescent detection kit, catalog number K006-F1 from Arbor Assays was used. The 5-6 day old organisms in 6-9 replicates for each suspension concentration including the control were exposed to sublethal concentrations of the metal oxide suspensions for 72 h. At the end of the exposure period the organisms for all replicates for each concentration were pooled together and the selected biomarkers such as TBARS, GST, GSH and metallothionein were determined. These tests continued this project's investigations of metal oxide nanoparticle behavior in aquatic ecosystems and potential toxicity to a representative fresh water organism. PARTICIPANTS: Phenny Mwaanga, Ph.D. student, Environmental Toxicology TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
TBARS, GST and GSH results showed that there were insignificant effects for organisms exposed to TiO2 nanoparticles. Significant effects were observed for CuO and ZnO nanoparticles. The results indicated that at the concentrations of the nanoparticle suspensions the organisms had been exposed, there was possible damage to the lipids and also that there was inhibition of GST enzymes to the organisms. The results further showed that the reduced form of the substrate glutathione (GSH) was being converted into the oxidized form (GSSG), an indication of oxidative stress. In the presence of the dissolved NOM, as expected, the effects were relatively less severe. Taken together, the combined results for this project have indicated both solubilization of metal ions and persistence of nanoparticles aggregates or agglomerates when metal oxide particles (specifically, oxides of titanium, iron, copper and zinc) are placed in aqueous environments. Aquatic organisms are therefore potentially exposed to both forms. The biomarker results indicate organism responses to oxidative stress even at nonlethal doses. At this point, it is not possible to distinguish if the effects are due to dissolved or nanoparticulate forms. Further work will attempt to clarify these issues.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Toxicity tests using the freshwater crustacean Daphnia magna were conducted using nanoparticles (NPs) of copper oxide (nCuO), zinc oxide (nZnO), titanium oxide (nTiO2), and iron(III) oxide (nFe2O3) with and without added natural organic matter (NOM). Following understanding of NP solubility and aggregation behavior, toxicity tests can be better interpreted by potentially separating effects of soluble metals (e.g., toxic effects of dissolved copper or zinc) from NPs and NP aggregates. Three metal oxide NPs, nCuO, nZnO, and nFe2O3, were purchased from Sigma-Aldrich; nTiO2 was from Degussa. Supplier-reported particle sizes are <50 nm for nCuO, nTiO2, and nFe2O3, and <100 nm for ZnO. The NOM is a material isolated through reverse osmosis from Suwanee R. (GA, FL) and was purchased from the International Humic Substances Society (IHSS). Stock solutions of NPs were prepared in deionized water (DI) and in synthetic moderately hard water (MHW) and were sonicated for 60 minutes to promote disaggregation. The pH for stock suspensions were around 6.7 and 7.9 for DDI and MHW, respectively. Following storage (refrigerated) and prior to preparation of test solutions, the stock solutions were sonicated for 10 minutes. Solutions for nCuO and nZnO tests were prepared in MHW at concentrations of 0.0, 1.0, 2 .0, 5.0 and 10.0 mg/L. The same series was also prepared in MHW with NOM at 1.0 and 5.0 mg/L. The solutions with NOM were allowed to stand for 24 hrs so as permit dissolution and complexation to proceed towards equilibrium, and then sonicated for 5 minutes before toxicity tests. For nFe2O3, the test concentrations were 0.0, 50.0, 100.0, 150.0 and 250.0 mg/L. For TiO2 the test concentrations were 0.0, 10.0, 50.0, 100.0 and 150.0 mg/L. In all these test suspensions/solutions the dilution water was MHW. The Daphnia magna organisms were cultured in laboratories at Clemson Institute of Environmental Toxicology, in the artificially moderately hard water as specified by EPA (2007). The culture medium was renewed three times a week and daphnids were fed on algae suspensions and YCT (Yeast Cereal Leaves Tetramin) food mixture. The daphnia toxicity tests were conducted according to the standard toxicity tests as described by EPA (2007). The static nonrenewal tests were conducted using 5 test concentrations (inclusive of the control). The tests were conducted in 30 mL plastic containers, each containing 25 mL of test solution. Three replicates per concentration and one control were used and each contained 5 individual organisms (neonates ≤ 24 hrs). The tests were performed under 16h light: 8h dark at temperature of 25 (+/-1 C). The dead organisms were checked after 48 hrs under the dissecting microscope. The LC50 values and their confidence intervals (CI) were calculated using Toxstat software. All concentrations reported are nominal concentrations. PARTICIPANTS: Phenny Mwaanga, Ph.D. student, Environmental Toxicology TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Insignificant mortalities were found for nFe2O3 and nTiO2. The same was true for nCuO and nZnO in the presence of 5.0 mg/L NOM. In these cases, LC50 values could not be calculated. Significant mortality was observed for nCuO and nZnO in solutions without NOM and at 1.0 mg/L NOM. The LC50 results for 48 hour mortality (mg/L) for D. magna obtained (and 95% CI) are: nZnO (MHW), 3.11 (2.04, 4.75); nZnO (MHW + 1 mg/L NOM), 3.54 (2.28, 5.51); nCuO (MHW), 2.71 (1.83, 4.02); nCuO (MHW + 1 mg/L NOM), 7.07 (3.64, 13.8). The larger effect of NOM on copper is likely due to more effective complexation of Cu2+ ions by NOM. Additional studies to determine influences of solubility and aggregation are planned. Further studies may include longer term effects on daphnia reproduction and rates of uptake and elimination of NPs.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The major activities conducted this period further advanced the understanding of nanoparticle (NP) behavior in aqueous media. Work during this period has been continued by Phenny Mwaanga and have focused on commercially available NPs of zinc oxide and copper oxide (both from Sigma-Aldrich). More detailed time dependence of particle solubility and particle size distribution (obtained with dynamic laser light scattering, DLS) has been obtained. Additional experiments are under development to assess the importance and strength of agglomeration of individual nanoparticles or small clusters of nanoparticles into larger aggregates and the effects of aggregation on toxicity. Work continues with solutions of varying conditions (pH, ionic strength, media for toxicity tests, and with natural organic matter (NOM)). The dynamic light scattering (DLS) technique and scanning electron microscope (SEM) were used to characterize aggregation. The Zeta PALS was used to measure surface charge. Visual minteq soft ware was used to characterize the speciation. The dissolved ions were measured by the inductively coupled plasma- mass spectrometer (ICP-MS) or atomic absorption spectrophotometer (AAS). PARTICIPANTS: PI: Elizabeth R. Carraway, Assoc. Professor, Dept. Environmental Engineering and Earth Sciences, Graduate Program in Environmental Toxicology; Students: Xin Xu, Ph.D. student, Environmental Engineering and Earth Sciences; Phenny Mwaanga, Ph.D. student, Environmental Toxicology (began 2009) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results indicated that there was high variation in solubility and aggregation among different particles. The results also showed that solubility and aggregation were influenced by pH, the dissolved NOM and ionic strength. There was clear evidence that aggregation of particles was affected by the presence of the dissolved ions. It was further observed that though pH had a strong effect on the release of ions, the presence of free metal ions in solution was affected by the dissolved NOM probably through partitioning and sorption processes. The solubility of particles was shown to be affected by ionic strength. While some nanoparticles such as ZnO showed high solubility at moderate pH (6.62), others such as CuO showed very low solubility (below detection). Significantly, although extensive aggregation was observed, most aggregates remained suspended in solution except at ionic strength of 1.0M where most aggregates were observed to have settled out the solution.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The major activities conducted this period further advanced the understanding of nanoparticle (NP) behavior in aqueous media. Synthesis methods were standardized following investigations in the previous period. In summary, literature methods were modified to optimize NP purity and uniformity of shape. Zinc oxide NPs (nZnO) are prepared by base hydrolysis of zinc acetate in ethanol with controlled mixing and addition rates and temperatures. Copper oxide NPs (nCuO) are synthesized through base hydrolysis of copper nitrate, again under controlled conditions, followed by 24 hours calcination at 300 C. Titanium dioxide NPs (nTiO2) are formed by hydrolysis of titanium isopropoxide with slow addition and rapid mixing, followed by drying. Iron oxide (hematite) NPs (nFe2O3) were produced by slow addition of aqueous iron chloride to boiling water followed by dialysis at pH 3.5. Commercially available NPs of each type were obtained from Sigma-Aldrich (nZnO, nCuO, nFe2O3) and Degussa (nTiO2). Techniques including UV-visible absorbance, surface area determination, imaging by transmission and scanning electron microscopy (TEM and SEM), x-ray diffraction (XRD), and dynamic laser light scattering (DLS) have been used to characterize the characteristics of laboratory-synthesized and commercial NPs. During this period, more DLS results were obtained in order to compare hydrodynamic diameter distributions. NP solubility experiments were begun and were performed under a range of solution conditions (pH, ionic strength, media for toxicity tests, and with natural organic matter (NOM)). Activities and results were communicated to the broader scientific audience through presentation of two posters at the November 2008 meeting of the Society of Environmental Toxicology and Chemistry (SETAC); this meeting is typically attended by scientists, engineers, and policy-makers from academic, industry and government groups. One poster was "Are ultrafiltration methods ready for NOM separation" and the other was "Stability of Metal-Based Nanoparticles in Aqueous Media;" both were authored by Xin Xu and Elizabeth Carraway. Abstracts are available at www.setac.org/tampa/. PARTICIPANTS: PI: Elizabeth R. Carraway, Associate Professor, Clemson University, Dept. of Environmental Engineering and Earth Sciences. TARGET AUDIENCES: Presentations at scientific meetings for the purpose of dissemination of results and discussion with other environmental researchers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The results of the experiments described above (outputs) for DLS measurements show that, on average, laboratory synthesized NPs tend to have more particles of larger diameters. A notable exception is the hematite particles, for which the laboratory synthesized NPs are both smaller and exhibit a narrower size distribution. However, closer inspection of the DLS results (generally 3 scans of sizes from approximately 3 to 3000 nm, over a time period of about 15 minutes) indicate laboratory-samples frequently exhibit a bimodal distribution. At the lower end, the distribution of diameters is similar to the commercial NPs, but the presence of a number of larger particles skews the average hydrodynamic diameter to higher values. Often, these larger particles were not present in the second and third scans as they settled out. Therefore, in practice, toxicity tests conducted on laboratory-synthesized NP suspensions can be made more comparable to those using commercial NPs after a selective filtration or by decanting the upper solution after allowing larger particles to settle. In all cases, techniques such as DLS should be used to characterize NPs before and after toxicity tests. NP solubility tests were performed this period. These results provide necessary background for toxicity tests in that particle solubility may contribute as much or more to observed toxicity as NPs themselves. This has been shown in the literature for nZnO using a freshwater microalga (Franklin et al., 2007). Solubility studies for each of the four types of NPs with varying pH exhibit characteristics generally consistent with the pH solubility of bulk or non-nano-sized particles. That is, for each particle the trend of solubility with pH matches that of observations for the bulk material; often, lowest solubilities are exhibited at moderate pH. Additional experiments are needed to confirm this correspondence. Ionic strength effects were not significant. Solubility of NPs in a complex mixture of salts (mimicking media used in toxicity tests), showed results consistent with pH dependence. Results obtained for NP solubility with added NOM showed the most dramatic changes. Solubility increased by factors up to 10 times upon addition of NOM at 5 and 20 mgC/L. In general, laboratory-synthesized NPs showed higher increases at higher NOM levels, which may reflect differences in preparation methods. At this time, it is not clear if apparent increases in NP solubility are due to true dissolution or to complexation of NPs into the NOM matrix. NP solubility measurements were performed after seven days of mixing, which was determined in preliminary experiments. Future experiments will further evaluate the effect of NOM and the potential of NP incorporation into NOM. In addition, the morphology of NPs at time periods shorter than seven days will be investigated as particle dissolution and regrowth may be significant over this time period. The changes in NP behavior in the presence of NOM are significant for the prediction and understanding of NP fate and toxicity in waters containing NOM and related chemicals (e.g., peptides, sugars) released by organisms.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
The major activities conducted this period contribute to the objective of understanding nanoparticle behavior in aqueous media; the following types of experiments were performed and the results analyzed. First, nanoparticle preparation methods were further investigated for changes that affect particle characteristics such as agglomeration and size distribution. Second, the stability of aqueous suspensions of particles was measured. Mentoring of graduate students was performed throughout the project report period. The general preparation approach is the commonly used sol-gel or liquid method and this was used for nanoparticles of zinc, iron, copper, and titanium oxides. However, an alternate method based simply on heating solid state precursors was also used for zinc oxide. Changes in the preparation method initial temperature, stirring conditions, high temperature heating (calcination conditions: temperature and time), precursors or reactants used, ionic strength, and
reaction medium were investigated. The resulting nanoparticle purity was assayed by determination of chemical composition using acid digestion and AA or ICP-AES measurements. X-ray diffraction (XRD) measurements provided information on particle crystal structure and confirmation of particle purity results. UV-visible absorption was used as a real-time measure of particle formation, size, and extent of agglomerate formation or disruption (e.g., by sonication). Particle and agglomerate size distributions were measured using laser light scattering. Electron microscopy (SEM and TEM) was used to obtain images of particles and agglomerates. In particle preparation experiments, stabilizers such as starches, synthetic polymers, or surfactants were strictly avoided since these additives could affect the results of future toxicity studies. Instead, aqueous suspensions of nanoparticles were treated in an ultrasonic bath for up to 1 hour. UV-visible absorption was used to follow the stability of
suspensions subjected to ultrasonication. Activities and results were communicated to the broader scientific audience through presentation of a poster at the November 2007 meeting of the Society of Environmental Toxicology and Chemistry (SETAC); this meeting is typically attended by scientists, engineers, and policy-makers from academic, industry and government groups. The poster was "Effect of Synthesis Methods on Metal Oxide Nanoparticles Properties" by Xin Xu and Elizabeth Carraway at the SETAC North America 28th Annual Meeting (Urban Environmental Issues: Impacts on Ecological Systems), Milwaukee, Wisconsin, USA. 11 - 15 November 2007; abstracts are available at www.setac.org/htdocs/what_meet_setac.html.
Impacts
The results and analysis of the experiments described in outputs overall showed that suspensions of the iron, copper, titanium, and zinc oxide nanoparticles are not stable for more than a few days under conditions of neutral pH and low ionic strength. Additional conditions will be investigated in continuing experiments. It is important to note that toxic effects may still be possible if aquatic organisms present in the overlying water or in sediments ingest the settled particles. Specific findings include the following. The average primary particle diameters were about 20-35 nm for iron oxide, 20-40 nm for copper oxide, 10-20 nm for titanium dioxide, and 10-20nm for zinc oxide. However, aggregates or agglomerates of the primary particles were one hundred nanometers or more in diameter. The primary particles were spherical or rod-like in shape. Agglomeration effects were apparent and often extensive in some synthesis approaches. In particular the media or solvent
composition used in the zinc oxide sol-gel synthesis had significant influence on the extent of agglomeration. The agglomerates formed by zinc, copper, and titanium oxides appeared to be random combinations of primary particles, but iron oxides formed web-like structures. These results are consistent with the behavior of larger particles of the same oxides. Agglomerates of particles can be partially broken up by ultrasonication. The average diameters of agglomerates persisting after 30 min sonication measured by by laser light scattering were not significantly lower than the agglomerates found just after synthesis, but the distribution of sizes showed an increase in particles and/or agglomerates with diameters in the nanometer size range. Following ultrasonication, aqueous suspensions of nanoparticles were stable for approximately 1 day. There was no significant indication of a growth in primary particle size, indicating at these time scales agglomeration of nanoparticles and settling
of the agglomerates are the most important processes.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The overall goals of this project are to synthesize four types of metal oxide nanoparticles and to test the toxicity of those particles to a common aquatic organism, Ceriodaphnia dubia (a water flea). In this period, efforts have centered on the synthesis of the metal oxide nanoparticles and methods of reducing agglomeration of the particles before beginning toxicity tests. Toxicity tests using discrete nanoparticles or larger agglomerates of nanoparticles can lead to contradictory results. Future commercial products are likely to contain discrete nanoparticles, but agglomerates are possible. If released to the environment, particle agglomerates are likely to form if conditions are favorable. Thus, understanding the toxicity of both discrete and aggregated nanoparticles is necessary for understanding the environmental fate of these nanomaterials. Four types of metal oxide nanoparticles were prepared by sol-gel methods: iron oxide, copper oxide, titanium dioxide, and
zinc oxide. Physical and chemical characterization of the nanoparticles has been carried out using electron microscopy (SEM and TEM), X-ray diffraction (XRD), and UV-vis absorption and fluorescence spectroscopy. Laser light scattering was used to measure the particle size distribution. Agglomeration effects were apparent and often extensive with most synthesis approaches. Modifications to different synthesis methods were compared for effectiveness in reducing coagulation. The general approach to the sol-gel synthesis of each particle was as follows. Nano-α-Fe2O3 was produced by dropwise addition of FeCl3 to boiling water, followed by dialysis against HClO4 or HCl to stabilize the hematite. Nano-CuO was synthesized by base-hydrolysis of CuCl2 and calcined at different temperatures. Nano-TiO2 was prepared by acid and base treatments of TiCl4. One organic solvent (ethanol) was tested as the reaction medium. Nano-ZnO was synthesized in DDI or ethanol or 2-propanol; in water, the
control of the temperature and time is important. Following synthesis, particle characterizations were carried out by SEM, TEM, XRD, laser light scattering, UV-visible absorption, and fluorescence spectroscopy. Conclusions: (1) There are several controlling factors in sol-gel synthesis methods (including pH, reactants concentrations, temperature, aging time, storage conditions). (2) The as-prepared nanoparticles were nanometer-sized, but often in agglomerates of a few hundred nanometers in size. (3) Spectroscopic properties confirm primary particles of quantum size. (4) The degree of agglomeration can be reduced by a few minutes exposure to ultrasonic energy. ZnO particles prepared in alcohols exhibit less coagulation than those prepared in water.
Impacts
Four types of nanomaterials (particles of sizes in the nanometer range) containing iron, copper, titanium, and zinc oxides will be synthesized. Their behavior in natural waters and toxicity towards a common aquatic organism, Ceriodaphnia dubia (a water flea) will be tested. Knowledge of the environmental fate and toxic effects of nanomaterials is essential to rational and appropriate advancement of this important technology. Toxicity tests using separate, individual nanoparticles as opposed to larger agglomerates of nanoparticles can lead to ambiguous or contradictory results. Future commercial products are likely to contain discrete nanoparticles, but agglomerates might also be included in certain product designs. If released to the environment, individual particle agglomerates are likely to form in aquatic environments. Thus, understanding the toxicity of both individual and aggregated nanoparticles is necessary for understanding the environmental fate of these
nanomaterials.
Publications
- No publications reported this period
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