Progress 07/01/04 to 06/30/09
Outputs
OUTPUTS: The major activities this period continued with characterization of nanoparticles of copper and mercury and their interactions with aquatic natural organic matter or NOM. In particular, the effects of NOM on particle dissolution and aggregation are being examined in more detail with respect to time dependence and aggregate stability. Overall, major activities of the full project period progressed from the study of whole water samples obtained from 7 sites in the Ogeechee River (Georgia) and characterization of the NOM in Ogeechee R. samples and its interactions with copper and mercury, including toxicity to Ceriodaphnia dubia (water flea) to laboratory based studies of commercially available aquatic NOM and its interactions with copper and mercury in two forms (dissolved ions and nanoparticles). The major types of experiments performed were 48-hr acute toxicity tests with C. dubia in Ogeechee R. waters amended with varying concentrations of copper and mercury ions, separation of Ogeechee R. waters containing NOM into small (< 3K) and large (> 3K) size fractions, extensive characterization of the fluorescence properties of Ogeechee R. NOM in order to relate changes to complexation by copper and mercury, kinetics experiments with purchased NOM (isolated from the Suwanee River) to better understand the time-based character of NOM complexation of copper and mercury, the preparation of nanometer-sized particles of copper oxide (CuO) and mercury sulfide (HgS), and studies of the solubility of these particles in the presence of NOM. A wide range of types of instruments and methods were employed in these activities and a number of Clemson University graduate students were involved. The students include Christina Pac McNaughton, Robert Hereford, Xin Xu, Alan Jones, and Shanna Estes; in addition, collaborations with Dr. Alan Johnson (Clemson University) were particularly beneficial for the Ogeechee R. work. Activities associated with this project contributed significantly to the Ph.D. dissertations of Christina Pac McNaughton (2007) and Xin Xu (2009). Dissemination of project activities and findings has been done primarily through presentations at local to international level meetings. Representative presentations are summarized below in the publications section (those for which abstracts are available). Most of the presentations have been made at meetings of the Society of Environmental Toxicology and Chemistry (SETAC) which are typically attended by scientists, engineers, and policy-makers from academic, industry and government groups. PARTICIPANTS: Major student participants: Christina Pac McNaughton, Ph.D. student, Environmental Toxicology, Clemson University Alan J. Jones, M.S. student, Environmental Toxicology, Clemson University Xin Xu, Ph.D. student, Environmental Engineering and Science, Clemson University Faculty: (PI) Elizabeth R. Carraway, Assoc. Professor, Dept. of Environmental Engineering and Earth Sciences, Graduate Program in Environmental Toxicology, Clemson University (Collaborator) Alan R. Johnson, Assoc. Professor, Dept. of Forestry and Natural Resources, Graduate Program in Environmental Toxicology, Clemson University TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major findings of this project are important for metal (copper and mercury) toxicity to aquatic freshwater species. In particular, the role of NOM in complexing or sequestering the metals so as to reduced bioavailability of the metals and hence reduce toxic effects. Relatively early results with Ogeechee R. samples indicated that while equilibrium complexation of copper by NOM may satisfactorily predict reductions in copper toxicity for C. dubia, the same could not be said for mercury. Further studies showed that the molecular weight or size of NOM did not play a significant role (at least not for mercury and for the size fractions investigated in this project), but that differences in kinetic or temporal behavior of NOM complexation of the metals might be a key factor in explaining the different behavior of mercury compared to copper for NOM and related ligands. In later stages of the project, nanoparticulate forms of copper and mercury have been investigated. Here we find that the effects of NOM will increased the total load of solubilized metal in freshwater systems if such particles should be released. Further, interactions between mercury and NOM may result in the formation of methylmercury which can bioaccumulate in aquatic species to harmful levels. Yearly reports have included more details on these findings. Future studies will further focus on nanoparticulates, NOM, and toxic mercury species.
Publications
- Hereford, R.S., Pac, C.R., Jones, A.J., Johnson, A.R., and Carraway, E.R. 2004. The attenuation of copper toxicity to Ceriodaphnia dubia by Ogeechee River dissolved organic matter. SETAC World Congress, Portland, OR.
- Pac, C.R., Hereford, R.S., Johnson, A.R., and Carraway, E.R. 2004. Assessing the Role of Mercury-Natural Organic Matter (NOM) Complexation on Toxicity to Ceriodaphnia dubia. SETAC World Congress, Portland, OR.
- Johnson, A.R., Hereford, R.S., and Carraway, E.R. 2005. Longitudinal changes in copper complexation and bioavailability along the Ogeechee River." US-IALE Annual Meeting, Syracuse, NY.
- Pac, C. R., Jones, A. J., and Carraway, E. R. 2005. Metal Toxicity to Ceriodaphnia dubia as a Probe of the Kinetic Lability of Metal Complexes. SETAC North America 26th Annual Meeting, Baltimore, MD.
- Jones, A. J., Pac, C. R., and Carraway, E. R. 2005. Alkalinity Effects on Metal Speciation and Toxicity to Ceriodaphnia dubia in Waters of Varying Hardness. SETAC North America 26th Annual Meeting, Baltimore, MD.
- Jones, A.J., and Carraway, E.R. 2006. Reproductive Toxicity of Cadmium, Copper, Mercury and Zinc to Ceriodaphnia dubia. SETAC North America 27th Annual Meeting, Montreal, Quebec, Canada.
- Estes, S.L. and Carraway, E.R. 2007. Evaporative light scattering detection coupled with high performance size-exclusion chromatography for the determination of molecular weight distributions of aquatic humic substances. Carolinas and Southeastern Regional Chapters of SETAC meeting, Athens, GA.
- Xu, X. and Carraway, E.R. 2007. Effect of Synthesis Methods on Metal Oxide Nanoparticles Properties. SETAC North America 28th Annual Meeting, Milwaukee, WI.
- McNaughton, C. P. 2007. The Influence of Mercury-Dissolved Organic (DOM) Complexation on Toxicity in Natural Waters. Ph.D. Dissertation, Environmental Toxicology, Clemson University.
- Xu, X. and Carraway, E.R. 2008. Are Ultrafiltration Methods Ready for NOM Separation. SETAC North America 29th Annual Meeting, Tampa, FL.
- Xu, X. and Carraway, E.R. 2008. Stability of Metal-Based Nanoparticles in Aqueous Media. SETAC North America 29th Annual Meeting, Tampa, FL. Xu, X. and Carraway, E.R. 2009. Behavior of Metal-Based Nanoparticles in Aqueous Media. Intl. Conf. Environmental Implications and Applications of Nanotechnology, Amherst, MA.
- Xu, X. 2009. Metal Oxide and Mercuric Sulfide NPs Synthesis and Characterization. Ph.D. Dissertation, Environmental Engineering and Earth Sciences, Clemson University.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The major activities this period were directed towards the dissolution and complexation of mercury and copper by NOM. The source of mercury used was nanometer sized mercury sulfide (nHgS) particles. Similar particles and clusters (e.g., zinc sulfide) have been found to exist in surface waters in environments such as wetlands and oxygenated river waters downstream of wetlands. The HgS particles were synthesized in the laboratory since they are not commercially available. Commercially available bulk (micron-sized) HgS was also used as a comparison. Solution phase sol-gel methods were adapted to prepare nHgS from mercury chloride and sodium sulfide. Copper oxide (nCuO) particles in the nanometer-sized range were synthesized and obtained from a commercial source (Sigma-Aldrich). Particles were characterized using 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). Particle solubility tests were performed under a range of solution conditions (pH, ionic strength, 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: Nothing significant to report during this reporting period.
Impacts
The nHgS particles were oblong or oval in shape with the long diameter of dimensions 20-15 nm (by SEM and TEM). DLS results showed the average hydrodynamic diameter to be 296 +/- 22 nm and that the distribution was approximately Gaussian. The nCuO particles were shown by SEM and TEM to be spherical in shape with diameters of 20-40 nm (laboratory). Commercial particles were reported to have diameters under 50 nm. DLS results indicate hydrodynamic diameters of 192 +/- 55 nm (commercial) and 462 +/- 330 nm (laboratory). The result for the laboratory-synthesized particles is skewed by a small number of significantly larger particles. Solubility studies for each particle were accomplished by mixing particles with the appropriate aqueous solution for a period of seven days; preliminary experiments indicated this time period was sufficient for equilibrium to be reached. Experiments were performed at different pH values (4.0, 5.2, 6.6, 9.4), ionic strengths (0.01, 0.1, and 1.0 M using sodium nitrate), and NOM concentrations (5, 20, and 50 mgC/L using Suwanee River reverse osmosis isolate (1R101N obtained from International Humic Substances Society). Solubility with varying pH exhibited characteristics generally consistent with the behavior of bulk particles. The lowest solubilities were often observed at moderate pH values, as expected. In particular, the solubility of HgS is low at acidic and moderate pH values, but increases significantly at the highest pH value tested. Experiments at additional pH values are needed to confirm this trend. Ionic strength effects were not significant. Results obtained for nCuO solubility with added NOM showed the most significant increases between 5 and 20 mgC/L, with smaller changes upon increasing NOM to 50mgC/L. Laboratory-synthesized particles were slightly more soluble than commercial ones. For nHgS, relatively small increases in solubility were observed except at the highest NOM levels. Future experiments will attempt to determine the relative contributions of true dissolution vs. incorporation of particles in the NOM matrix to the observed increases in solubility. These two possible routes are expected to have significantly different effects on the toxicity of the metals. This may aid in sorting out toxic effects due to dissolved metals vs. metal-containing nanometer-sized particles.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The major activities directed towards achievement of the project objectives were the completion and analysis of experiments investigating the kinetic or time-dependent interactions between mercury or copper and natural organic matter (NOM), as well as a few simpler ligands which contain some chemistry similar to NOM. The student who performed most of these experiments, Christina Pac McNaughton, graduated with a Ph.D. in Environmental Toxicology in December 2007. Publications based on this dissertation and accompanying work are in preparation. The kinetic lability of various metal complexes was quantified using competitive ligand-exchange reactions. The kinetics of complexation of copper and mercury were assessed using the simple organic ligands EDTA, NTA, and cysteine. These experiments were followed with studies of the kinetics exchange of copper and mercury with NOM. The competing ligand used was calcein. This ligand is very convenient for such studies because its
concentration can be monitored through fluorescence; the unbound or free calcein fluoresces but the metal-bound or complexed calcein does not. Thus, free and bound concentrations of calcein could be directly measured at low calcein concentrations. Indirectly, bound copper or mercury concentrations were determined since total ligand concentrations were much higher than total metal concentrations (resulting in negligible unbound copper or mercury). Results were analyzed for different metal concentrations (either copper or mercury), calcein concentrations, and ligand (EDTA, NTA, cysteine, or NOM) concentrations. Models derived or created using limiting assumptions were used to interpret the experimental results.
PARTICIPANTS: Christina Pac McNaughton, a graduate student in Environmental Toxicology at Clemson University, performed the laboratory experiments and much of the data analysis.
Impacts
Overall results of the experiments and analyses described in outputs show that, although copper and mercury have reasonably similar reaction rate constants with both the simple ligands and NOM, they differ dramatically in their dependence on concentration of the ligands tested. For copper, reaction rate constants are essentially unaffected by the varying concentrations of the ligands. In contrast, mercury shows a strong dependence on the ligand concentration for EDTA and NTA, but not for cysteine. The models used to analyze the data indicate that two fundamentally different mechanisms of ligand exchange can explain these differences. For copper, in which negligible dependence is observed, the ligand exchange process does not involve free or unbound copper. These mechanisms are also referred to as adjunctive or associative since the intermediate is postulated to be or can be modeled as a complex of the metal and both the incoming and outgoing ligands. For mercury and
EDTA or NTA, a disjunctive or dissociative mechanism is consistent with the observation that rate constants depend inversely on the excess concentration of the ligand; that is, the time dependence of calcein displacement is very much dependent on the concentration of the ligand. The disjunctive mechanism is consistent with dissociation of the metal complex before the metal is recomplexed with another ligand. Since the most common complexing groups in NOM are similar to EDTA and NTA, this kinetic behavior could be key in understanding the differences in toxicity between copper and mercury in the presence of NOM as indicated by our previous experiments (copper toxicity is significantly reduced whereas mercury toxicity is not). Because our experiments used multiple model ligands as well as NOM, we expect the conclusions to hold their significance as further studies continue. The long-term impact of these results may include changes in the types of data needed for incorporation into
models of metal toxicity in natural waters; specifically, kinetic information may be required for better prediction of toxicity of certain metals, including mercury.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
This progress report is an update on studies related to the effects of two potentially toxic metals, copper and mercury, and how complexation by natural organic matter (NOM) may act to decrease metal bioavailability. It is well documented that complexation between certain metals and ligands decreases toxic effects. Data analysis based on equilibrium concentrations of ligand-bound and free or aqueous metals has generally been successful, however, previous work in this lab has showed that increasing the NOM concentration does not always decrease apparent Hg toxicity. Acquisition of data needed to evaluate possible explanations has been focused primarily on two types of experiments. First, toxicity tests using the water flea Ceriodaphnia dubia and various metal-ligand combinations have been performed to assess relationships between metal toxicity and ligand characteristics (type and concentration). Metal speciation modeling using equilibrium-based programs (i.e., Visual
MINTEQ) was performed in support of toxicity tests. Second, metal complex kinetic lability experiments have been performed. These experiments are designed to investigate the rapidity of metal exchange between ligand-bound and unbound forms. Many people mistakenly associate a high equilibrium formation or stability constant for a metal complex with kinetic stability. In fact, a metal complex with a large stability constant may exhibit rapid or slow exchange. For example, the thermodynamic stabilities of cyanide complexes of nickel, manganese, and chromium (Ni(CN)4-2, Mn(CN)6-2, and Cr(CN)6-2) are all high (formation constants of approximately 1030 or greater), but measures of cyanide exchange reveal time periods of 30 seconds (for the Ni complex) to 1 hour (Mn) to 24 days (Cr). While all are thermodynamically stable only one is kinetically inert. Together these data will indicate relationships between metal complex kinetic lability and toxicity. Comparison using literature data is
difficult because the published data on kinetic lability is sparse and solutions conditions can vary significantly (e.g., very low pH). The work currently underway is determining kinetic exchange of metal complexes of copper and mercury with three ligands of known structure - EDTA, NTA, and cysteine - and NOM. These three ligands were chosen as they represent ligands that coordinate to the metal through oxygen, nitrogen, and sulfur atoms. NOM contains similar functional groups and can potentially coordinate metals through all of these routes. As this data collection and analysis advances, we believe a basis for interpretation and prediction of metal toxicity through both kinetic and thermodynamic stability mechanisms will emerge and contribute to our understanding of metal toxicity in instances where an equilibrium treatment yields incorrect or contradictory results.
Impacts
The relationship between toxicity and metal speciation is widely noted yet not well understood. As this research progresses, it is apparent that both equilibrium effects (how a chemical reaction is balanced between reactants and products) and kinetic effects (how fast a chemical reaction proceeds in both forward and reverse directions) are important in understanding the toxicity of metals to living organisms. Both the type of metal (such as copper vs. mercury) and the materials, including the living organisms, that may potentially react or form "complexes" with those metals are important to explaining the variability of metal toxicity under different conditions. Equilibrium effects are treated using a general equilibrium approach in models such as the Biotic Ligand Model (BLM); models such as these are instrumental in developing and applying water quality regulations. This study is providing data needed to take a complementary view based on the kinetic behavior of
metal complexes and the consequences it has on metal toxicity. Ultimately, the kinetic approach may be incorporated into models such as the BLM to become an additional tool for predicting effects of metal toxicity under varying conditions, however, much data acquisition will be needed to support such an effort as the scientific literature is relatively sparse in this area.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
It is well documented that complexation of metals to certain types of ligands decreases the bioavailability of the metal, thus reducing its toxicity. Analyses using equilibrium descriptions of metal complexation have generally been successful in relating toxicity to metal speciation, but important questions remain. The current work is a continuation of previous research on mercury (Hg) and copper (Cu) complexation by natural organic matter (NOM) which showed that increases in NOM concentrations are not always accompanied by decreases in Hg toxicity. It was hypothesized that this may be indicative of the kinetic lability of the Hg-NOM complex. Due to the lack of comprehensive data on the kinetic lability of metal complexes, additional toxicity studies were completed under selected conditions in order to gain a better understanding of importance of speciation dynamics in aquatic environments. This hypothesis was tested using a series of complexation experiments where
Visual MINTEQ was used to determine the equilibrium speciation of copper, mercury, and aluminum in the presence of simple, well-studied ligands. Toxicity tests using Ceriodaphnia dubia and various metal-ligand combinations were then performed to determine if equilibrium speciation modeling and toxicity were well correlated. When possible, results were also compared to predictions made by the Biotic Ligand Model (BLM). Following these studies, Hg complexation to NOM was assessed in a similar manner. Aquatic NOM was separated into selected size fractions. Each fraction was then complexed with Hg and the toxicity of the Hg-NOM mixture to Ceriodaphnia dubia measured. Preliminary results indicate toxicity tests can be an indicator of the kinetic lability of metal complexes when performed under conditions of low free metal concentrations. Interactions and predictions become more complex when metals are bound to NOM due to the large number of functional group types and numbers present.
Independent measurements of metal lability using a competitive ligand method monitored by fluorescence have up to this point yielded ambiguous results in that while test cases agree reasonably well with literature results, the results for the kinetic lability of Hg and Cu complexes are surprisingly similar.
Impacts
The relationship between toxicity and metal speciation is widely noted yet not well understood. As this research progresses, it is apparent that equilibrium and kinetic effects involving the metals investigated and all ligands present, including those associated with the test organisms, are important. While these effects are included generally in models such as the Biotic Ligand Model (BLM), specific and thorough experimental investigations are needed to serve as test cases and to provide calibration for various parameters that are frequently given assumed or typical values.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
In the initial phases of this project, we have focused on testing the effects of complexation of copper (Cu) and mercury (Hg) by aquatic humic substances or natural organic matter (NOM) on the toxicity of the metals to Ceriodaphnia dubia, a water flea specie. We used whole water samples obtained at 7 sites from the Ogeechee River, a river in Georgia naturally high in NOM. The primary objectives were to examine the effectiveness of NOM binding of Cu and Hg and the extent of reduction of the bioavailability of the metals. Some molecular weight (MW) fractionation of the NOM was performed and the variability in metal binding constants of the NOM from different sites was investigated using fluorescence quenching. Whole water samples collected from the 7 sites spanning the nearly the full length of the river were filtered using 0.45 um Millipore filters. Parameters such as DO, conductivity, pH, temperature, alkalinity, and water hardness were recorded. In addition, metal
and anionic constituents were measured. NOM was characterized using TOC and absorbance measurements. Toxicity experiments were 48-hr acute toxicity tests performed using < 24-hr old C. dubia neonates. Site water was allowed to equilibrate for 24-hr with added metals (Cu or Hg) prior to testing and LC50 values were calculated using a Trimmed-Spearman Karber method. Fluorescence excitation-emission matrix scans (EEMs) were obtained for waters from all 7 sites with varying Cu and Hg concentrations. The Stern-Volmer model was used to determine conditional binding constants of the metals to the aquatic DOM. For water from selected sites, centrifuge dialysis was used to obtain MW fractions of < 3K and > 3K. Fluorescence quenching results indicated strong association of the metals Cu and Hg with NOM. While the NOM concentration increased from upstream to downstream, the variability from site to site in the strength of metal binding was insignificant. The fluorescence quenching results as
well as literature values for the complexation of Cu and Hg with aquatic NOM indicated that over the range of metal concentrations tested in our toxicity experiments, varying amounts of Cu should be complexed by NOM, resulting in variable amounts of free or bioavailable Cu. In contrast, Hg is expected to be almost completely complexed by NOM. C. dubia toxicity tests showed dramatic differences in the behavior of Cu vs. Hg. Observed Cu toxicity decreased with increasing NOM, consistent with the hypothesis and previously published observations that Cu bioavailability is reduced via NOM complexation. Observed Hg toxicity was decreased only by approximately a factor of 3 and the LC50 values obtained for all sites were statistically identical. Since free Hg concentrations should have been reduced to very low levels (10-9 M), much larger changes in apparent toxicity were expected. Therefore, the concept that toxicity should be directly proportional to the free, uncomplexed metal
concentration is not supported by the behavior of Hg. Kinetic factors and ligand competition (NOM vs. C. dubia) may be controlling factors. Toxicity tests using MW fractions are ongoing.
Impacts
Humic substances (NOM) are important components of natural aquatic systems, playing roles in carbon and nitrogen cycling, nutrient availability, and transport. This work focuses on the interactions between humic substances and metals and how those interactions affect metal toxicity. In addition, humics are altered over time as sunlight tends to break them into smaller molecular weight units. Aquatic humic substances are components of a complex natural system that serves to buffer or moderate metal concentrations, and we must consider both changes in humics characteristics and effects on metal concentrations in order to understand its functions. Hg and Cu contamination are areas of concern in the southeastern United States, and Hg in particular is of national and global concern. Cu is important because, of all metals, its interaction with NOM is the most studied and characterized; thus it presents many opportunities for comparison to prior studies. Hg, in contrast, is
a focus because it is a much more acute problem that is much less understood. Improvements in our scientific understanding of toxic metals in aquatic environments will aid in predictions of environmental effects and in improvements to treatment strategies such as constructed wetlands. In particular, the current results will be interpreted from the perspectives of metal behavior and toxicity models such as the free ion activity model (FIAM) and biotic ligand model (BLM), and in turn, the results will be used to evaluate the applicability of those models to Cu and Hg.
Publications
- No publications reported this period
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