Final Report: A Multidisciplinary Effort Linking the Impact of Sediment Contaminant Mixtures on Toxicological Response with Genetic Adaptation

EPA Grant Number: R826399E02
Title: A Multidisciplinary Effort Linking the Impact of Sediment Contaminant Mixtures on Toxicological Response with Genetic Adaptation
Investigators: Coull, Bruce C. , Chandler, G. Thomas , Decho, Alan , Quattro, Joseph M. , Shaw, Timothy J.
Institution: University of South Carolina at Columbia
EPA Project Officer: Chung, Serena
Project Period: March 1, 1998 through August 31, 2000
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (1997) RFA Text |  Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)

Objective:

The goal of this study was to evaluate the impact of contaminant mixtures (as heavy metals and organic compounds) on toxicity and genetic adaptation in infaunal estuarine organisms. The first step towards meeting this goal was to expand our faculty with the addition of Dr. John Ferry, an environmental chemist who specializes in the behavior of organic contaminants in natural systems. Dr. Ferry joined our faculty in the fall of 1998 and has begun collaborative projects with the co-principal investigators (PIs) in support of the U.S. Environmental Protection Agency (EPA) research objectives (see below). The second step was to begin to integrate current studies of toxic impact of metal mixtures (Chandler, Shaw, and Decho) and genetic adaptation to contaminant loads (Chandler, Coull, and Quattro) into a broader study of contaminant mixtures. The initial results of these efforts are reflected in the expansion of our ongoing studies to include: the phase association and transport of organic contaminants in natural waters (Shaw, Alegria, and Cowan) and the fate and impact of pharmaceuticals (Ferry) in natural waters. While the component projects are detailed below, it is important to note that this collaboration has resulted in the submittal and funding of several multi-investigator proposals to both EPA and the National Oceanic and Atmospheric Administration (NOAA). The early success of this project, and the rapid integration of Dr. Ferry into the project, would not have been possible were it not for the ongoing research in environmental toxicology, environmental chemistry, genetics, and benthic ecology here at the University of South Carolina (USC).

Summary/Accomplishments (Outputs/Outcomes):

Investigators: Chandler, Decho, and Shaw

Findings. In 1998, extensive acute sediment toxicity tests were completed to determine both the sediment and pore-water LC50s of copper, lead, nickel, and zinc to our model estuarine benthic copepod Amphiascus tenuiremis cf. Mielke. Laboratory cultured copepods were exposed to clean 98% silt:clay sediment spiked with metal chloride salt solutions to produce five treatments and a control. Trimmed Spearman-Karber analysis gave sediment 96-hour LC50 values of 281.9 μg Cu/g, 335.6 μg Ni/g, 2462 μg Pb/g, 671.3 μg Zn/g, and pore-water 96-hour LC50 values of 124.6 μg/L, 685.6 μg/L, and 375.8 μg/L for Cu, Ni, and Zn, respectively. Male survival after exposure to Cu, Pb, and Ni was significantly less than female survival (α = 0.05), suggesting a possible sex-linked mechanism for metal resistance in female copepods. The combined effect of these metals in mixture on A. tenuiremis survival was studied through an acute (96-hour) sediment toxicity test using sediment spiked with a mixture of Cd, Cu, Ni, Pb, and Zn. The mixture toxic unit that produced a median lethal response was 0.72 (95% CIE: 0.64–0.85). Thus a sediment mixture of Cd, Cu, Pb, Ni, and Zn was not simply additive but had a significant synergistic effect on A. tenuiremis survival. The additive index that produced 50% mortality in the metal mixture test was 0.39 (95% CIE: 0.18–0.56). This indicates that the mixture was 1.4 times more toxic than would be expected if the toxic effects of these metals were simply additive.

In 1998, we also conducted two complete runs of our meiofaunal microcosm experiments where we used the above acute toxicity data to derive a metal mixture exposure regime of 0, 1, and 2 total metal toxic units. Intact sediment microcosms containing hundreds of benthic copepods, foraminifera, nematodes, polychates, ostracods, and others were exposed for 14 days (75% of their life-cycles) to Cd, Cu, Pb, Ni, and Zn sediment mixtures. Sediment phase partitioning and metal mixture toxicity were studied in meiofaunal microcosms from salt marsh sediments. Microcosms consisted of undisturbed cores (φ = 12 cm) from a pristine salt marsh at North Inlet, SC, USA. A pre-defined population of our model harpacticoid copepod (A. tenuiremis) was also added to microcosms. Metal exposure was accomplished by adding a layer of spiked sediment containing an equitoxic metal mixture to achieve toxic units of 0.5, 1, and 10. The equitoxic metal mixture was such that each metal’s concentration was 1/5 the 96-hour Amphiascus LC50 in single-metal toxicity tests. Microcosms were maintained under flow-through conditions in environmental chambers (20°C, 16:8 L:D photoperiod). Water quality parameters were measured every other day, and the microcosms were fed twice a week. Metal concentrations (pore water and sediment) and metal binding phases, such as organic carbon (dissolved and total) and iron- and manganese-oxides, were measured in addition to simultaneously extracted metal (SEM)/acid volatile sulfide (AVS). Native meiobenthos were analyzed for impact on community composition, and A. tenuiremis was analyzed for effects on age-structure, clutch-size, reproductive output, and net population growth. Results emphasize that in the sediment oxic horizon where AVS concentrations were low (0.l μM/g), other binding phases (Fe, Mn, dissolved organic carbon [DOC]) were likely controlling metal bioavailability to meiofauna. For example, cadmium spikes in sterile sediments up to two times the 96-hour Amphiascus LC50 produced little to no toxicity in whole-sediment microcosms. Significant mixture mortality was not observed even at toxic units of 1, but significant reproductive depressions were observed at toxic unit exposures of 0.5,1, and 10 in dose response fashion. We conducted a triplicate run of this experiment in March 1998.

We have upgraded our inductively coupled plasma mass spectrometry (ICP-MS) facility with a newly developed Merchantek laser ablation system (LAS). In July 1998, we ordered a custom-built LAS for our Finnegan Element high-resolution ICP-MS. It is now on-site and operational. In terms of our research capability, this new sampling system will allow, for the first time, direct measurements of total metal body burdens on target species of individual benthic copepods and foraminifera. This work has begun with evaluation of trace metal/Ca ratio determinations in benthic foraminifera. This will provide us with much better statistical rigor with respect to assessing toxic impact of metals on single animals, and also on multi-species meiobenthic communities. Furthermore, the development of these techniques will allow us to simultaneously determine for multiple metals the concurrent relationships among body burdens, sediment bio-geochemistry, bioavailability, and toxic response in estuarine sediment systems.

Investigators: Coull and Quattro

Findings. The effects of contaminants in the genetic population structure of the ubiquitous harpaetieoid copepod Microarthridion littorale (Poppe, 1881) were examined. Central to the successful completion of this project was the development of a DNA isolation procedure that provided a template of sufficient quality and quantity to consistently amplify DNA from single specimens (Schizas, et al., 1997).

Relationships among populations (southeast Atlantic and northern Gulf coast, USA) of M. littorale were estimated using sequence data from two loci: cyt b and ITS-1 (Schizas, et al., 1999). Phylogenetic trees based on both genes were generally concordant and suggested that M. littorale populations are structured over large geographic scales (hundreds of km). Three well-supported groups were found in both gene trees comprising clades of alleles sampled from South Carolina, Florida, and Louisiana.

Analysis of the resulting cyt b diversity within and between populations of the M. littorale from 10 estuarine creeks, mainly in South Carolina, revealed the presence of three distinct mitochondrial lineages. The distribution of the three M. littorale mitochondrial groups had no geographic pattern but were correlated with degree of contamination of each creek (Schizas et al., 2002)

The three mitochondrial lineages were subjected to high concentrations of a mixture of two pesticides, chlorpyrifos and DDT, to compare survival rates (Schizas, et al., 2001; Kovatch, et al., 2000). The most frequently encountered mitochondrial group in the field survived better in the presence of pesticides relative to the others. The other two types were significantly reduced in the presence of pesticides compared to the robust type. The experimental data corroborate the distribution patterns of the three mitochondrial groups sampled from clean and contaminated tidal creeks on the South Carolina coast and suggest that contamination plays an important role in structuring the genetic composition of M. littorale.

Future Activities. We are currently estimating the population structure of M. littorale with an independently evolving nuclear DNA marker, an intron from tubulin. We will continue testing the fitness of the three haplotypes in laboratory settings by analyzing the genetic composition of animals exposed to toxicant in laboratory tests.

We will be sequencing/restriction mapping the Cyt b gene and tubulin intron DNA for laboratory selection experiments. Results from these tests will be used to compare with those collected for natural populations in “contaminated” and “clean” sediments. Concurrent with this work, we will begin searching for other candidate nuclear genes to use in this research.

Shaw, Alegria, and Cowan

Findings. Particle Dynamics and Contaminant Partitioning: The dynamics of dissolved/particulate exchange have been studied in great detail in the upper estuary region for a number of compounds. This region is dominated by the physical and chemical processes induced by mixing of fresh riverine and coastal ocean water. This process initiates the trapping of a number of contaminants in the sedimentary reservoir of estuaries, and also effects a transition from toxic effects associated with dissolved phase uptake to solid phase effects. This study investigated pesticide partitioning in the estuary mixing zone of Winyah Bay, SC. Partition coefficients between dissolved, colloidal (Kc) and particulate phases (Kp) were measured for a group of pesticides with a range in polarities (as octanol-water partition coefficients, Kows), by spiking pesticides into natural river water. The partitioning of the pesticides in the spiked waters was measured after simulated estuarine mixing by artificially increasing salinity. An aggregate phase formed in the 0–3 ppt salinity range which sequestered the majority of the more hydrophobic pesticides used in the study. The colloidal bound pollutants comprised from as low as 16 to 75% of total pollutant concentrations in river surface waters depending on the compound.

Data presented here suggest flocculation of riverine colloids may release less hydrophobic pollutants (Kow ≤ 4) to the solution phase while scavenging pollutants with more hydrophobic character (Kow ≥ 4) from the solution phase. This suggests that sorption competition based on hydrophobic character is important to transport and fate studies of estuarine organic pollutants. The results have implications for bioavailability based on work by Decho (on metal uptake) suggesting that binding (higher partition coefficients) of contaminants to particles, retard or prevent uptake.

Future Activities. Ultimately, the partitioning of contaminants will play a significant role in transport, uptake, and persistence. Initial results of partitioning studies indicate that colloidal carrier phases play a major role in distribution coefficients. The impact of partitioning on mobility (and particulate/dissolved phase exchange) and bioavailability/trophic transfer need to be examined in greater detail. This is the focus of the Ph.D. research of Laura Darwin, a chemistry student who is working jointly with Drs. Shaw and Ferry.

Ferry

The proposal included funding for faculty development, specifically a Chemistry Department hire who specializes in Environmental Chemistry. This support enabled the appointment of Dr. John Ferry on August 15, 1998 at the Assistant Professor level.

Educational Commitment. A new class was offered in Fall semester 1998, Redox Processes in the Aqueous Environment. This was a graduate-level course directed toward instructing students in fundamental chemistry of free-radical processes encountered in natural and engineered environments (specifically the oxy-radical family), as well as an introduction to the chemistry of commonly used drinking water disinfectants such as ozone and hypochlorite. The course enrollment consisted of 11 students from the Departments of Chemistry and Biochemistry, Environmental Health Science, and Chemical Engineering, and it received very positive evaluations.

Ongoing Research. The Ferry group is contributing to investigations of the toxicity of contaminated sediments by engaging in exploratory research of the constituents of municipal wastewater, a significant source of hydrophilic contaminants. Currently, research is focused on determining the chemical fate of the model endocrine disruptor (and human pharmaceutical) 17β-estradiol as it passes through the chlorination/dechlorination procedure that is the terminus of traditional wastewater treatment in the United States, and also determining the role of the primary clarifier in scrubbing hydrophobic contaminants from wastewater. Research on the environmental fate of 17β-estradiol on photoactive mineral surfaces is in its initial stages. We are testing two hypotheses: (a) pharmaceutically active compounds that have been detected in wastewater treatment plant effluent may be chlorinated to yield halogenated analogues, still pharmaceutically active; and (b) the “grease” layer formed during the primary clarification of sanitary wastewater provides an environmental matrix where hydrophobic contaminants will be pre-concentrated, allowing their ready detection in wastewater without lengthy concentration steps in the laboratory.

Data Summary. Aqueous sodium hypochlorite (pH 7, phosphate buffer) has been demonstrated to react swiftly with 17-β estradiol under standard conditions. In these experiments, at an approximate 1:0.75 ratio of 17-β estradiol to hypochlorite, a by-product has been detected with a molecular ion corresponding to that proposed for monochloro-17-β estradiol, with the chlorine position undetermined. Efforts are underway to synthesize mg-scale quantities of monochloro-17-β estradiol, to verify its structure by nuclear magnetic resonance (NMR) spectroscopy, as well as to provide a sufficient quantity to Professor Tom Chandler for a toxicity evaluation. Stopped-flow techniques are being applied to determine the rate constant of the reaction between hypochlorite and 17-β estradiol.

The “grease” layer from the Columbia municipal wastewater plant has been sampled, and preliminary efforts have positively identified the volatile pollutants tetrabutyltin and 1,4- dichlorobenzene, in samples taken up in hexane and diluted by approximately a factor of 10. These compounds have been identified by the comparison of their gas chromatography (GC) retention times and mass spectra with those of standards, and are present at roughly the level of 50 and 10 mg/kg of dry grease, respectively. Efforts are underway to remove fatty acid esters out of the samples so that we may extend our observations to more concentrated samples. These results are especially notable in terms of their potential human health impact, since this grease layer is frequently sold for agricultural purposes to companies that produce animal feed.

Four Students Mentored on EPA-Related Research:

William H. Henley, an undergraduate, presented the clarifier papers and won an NSF predoctoral fellowship.

Quincy L. Ford, a minority masters student, presented the derivatization work, and he is finishing his thesis this semester.

Spencer Walse and Li Kong are working on photoprocessing of endocrine disrupters. Both of them are entering their second year; they have no papers published yet but probably will have some soon.

Future Activities:

This project has formed the seed for a number of new interdisciplinary projects with participants in the College of Math and Science and the School of Public Health as well as the Medical University of South Carolina (MUSC). (EPA endocrine project, Foram project, etc.) In addition, this project has provided a venue for Ph.D. students to develop cross-discipline projects in environmental chemistry and environmental toxicology. For example, Chris Hintz, a marine science graduate student, has begun to examine the utility of using foraminiferal shell calcite produced via reproduction in metals-contaminated sediments as a biomonitor of porewater metals bioavailabilities. Chris is funded in part by a new National Science Foundation (NSF) project between Dr. Joan Bernhard, Dr. Tom Chandler (School of Public Health), and Dr. Tim Shaw (School of Science and Mathematics).

(Ms. Tamar Hagopian was supported for 2 years on this project; she successfully completed her M.Sc.P.H. in Environmental Health Sciences in December 1998).

References:

Schizas NV, Street GT, Coull BC, Chandler GT, Quattro JM. An efficient DNA extraction method for small metazoans. Molecular Marine Biology and Biotechnology 1997;6(4):381-383.


Journal Articles on this Report : 14 Displayed | Download in RIS Format

Other project views: All 34 publications 14 publications in selected types All 14 journal articles
Type Citation Project Document Sources
Journal Article Alegria HA, Shaw TJ. Rain deposition of pesticides in coastal waters of the South Atlantic Bight. Environmental Science & Technology 1999;33(6):850-856. R826399E02 (Final)
  • Full-text: ACS Full Text
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  • Abstract: ACS Abstract
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  • Other: ACS PDF
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  • Journal Article Alegria HA, d'Autel JP, Shaw TJ. Offshore transport of pesticides in the South Atlantic Bight: preliminary estimates of export budgets. Marine Pollution Bulletin 2000;40(12):1178-1185. R826399E02 (Final)
  • Full-text: Science Direct Full Text
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  • Abstract: Science Direct Abstract
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  • Other: Science Direct PDF
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  • Journal Article Alegria HA, Bidleman TF, Shaw TJ. Organochlorine pesticides in ambient air of Belize, Central America. Environmental Science & Technology 2000;34(10):1953-1958. R826399E02 (Final)
  • Full-text: ACS Full Text
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  • Abstract: ACS Abstract
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  • Other: ACS PDF
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  • Journal Article Chandler GT, Green AS. Developmental stage-specific life-cycle bioassay for assessment of sediment-associated toxicant effects on benthic copepod production. Environmental Toxicology and Chemistry 2001;20(1):171-178. R826399E02 (Final)
    R825279 (Final)
    R827397 (2000)
    R827397 (2002)
    R827397 (Final)
  • Abstract from PubMed
  • Abstract: Wiley-Abstract
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  • Journal Article Ford QL, Burns JM, Ferry JL. Aqueous in situ derivatization of carboxylic acids by an ionic carbodiimide and 2,2,2-trifluoroethylamine for electron-capture detection. Journal of Chromatography A 2007;1145(1-2):241-245. R826399E02 (Final)
    R831042 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-PDF
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  • Abstract: ScienceDirect-Abstract
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  • Journal Article Hagopian-Schlekat T, Chandler GT, Shaw TJ. Acute toxicity of five sediment-associated metals, individually and in a mixture, to the estuarine meiobenthic harpacticoid copepod Amphiascus tenuiremis. Marine Environmental Research 2001;51(3):247-264. R826399E02 (Final)
    R825279 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
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  • Abstract: ScienceDirect-Abstract
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  • Other: ScienceDirect-Full Text PDF
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  • Journal Article Kovatch CE, Chandler GT, Coull BC. Utility of a full life-cycle copepod bioassay approach for assessment of sediment-associated contaminant mixtures. Marine Pollution Bulletin 1999;38(8):692-701. R826399E02 (Final)
    R825279 (Final)
  • Full-text: ScienceDirect-Full Text HTML
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  • Abstract: ScienceDirect-Abstract
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  • Other: ScienceDirect-Full Text PDF
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  • Journal Article Kovatch CE, Schizas NV, Chandler GT, Coull BC, Quattro JM. Tolerance and genetic relatedness of three meiobenthic copepod populations exposed to sediment-associated contaminant mixtures: role of environmental history. Environmental Toxicology and Chemistry 2000;19(4):912-919. R826399E02 (Final)
    R825439 (1999)
    R825439 (Final)
  • Abstract: SETAC Abstract
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  • Other: SETAC PDF
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  • Journal Article Schizas NV, Street GT, Coull BC, Chandler GT, Quattro JM. Molecular population structure of the marine benthic copepod Microarthridion littorale along the southeastern and Gulf coasts of the USA. Marine Biology 1999;135(3):399-405. R826399E02 (Final)
    R825439 (1999)
    R825439 (Final)
  • Abstract: SpringerLink Abstract
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  • Journal Article Schizas NV, Chandler GT, Coull BC, Klosterhaus SL, Quattro JM. Differential survival of three mitochondrial lineages of a marine benthic copepod exposed to a pesticide mixture. Environmental Science & Technology 2001;35(3):535-538. R826399E02 (Final)
    R825279 (Final)
    R825439 (1999)
    R825439 (Final)
    R827397 (2002)
    R827397 (Final)
  • Abstract from PubMed
  • Full-text: ResearchGate - Abstract & Full Text HMTL
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  • Abstract: ES&T-Abstract
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  • Other: ResearchGate - Full Text PDF
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  • Journal Article Schizas N, Coull B, Chandler G, Quattro J. Sympatry of distinct mitochondrial DNA lineages in a copepod inhabiting estuarine creeks in the southeastern USA. Marine Biology 2002;140(3):585-594. R826399E02 (Final)
    R825439 (1999)
    R825439 (Final)
    R827397 (2002)
    R827397 (Final)
  • Abstract: SpringerLink Abstract
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  • Journal Article Schlekat CE, Decho AW, Chandler GT. Sorption of cadmium to bacterial extracellular polymeric sediment coatings under estuarine conditions. Environmental Toxicology and Chemistry 1998;17(9):1867-1874. R826399E02 (Final)
  • Abstract: Wiley
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  • Journal Article Schlekat CE, Decho AW, Chandler GT. Dietary assimilation of cadmium associated with bacterial exopolymer sediment coatings by the estuarine amphipod Leptocheirus plumulosus: effects of Cd concentration and salinity. Marine Ecology Progress Series 1999;183:205-216. R826399E02 (Final)
    R825279 (Final)
  • Full-text: MEPS - Full Text PDF
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  • Abstract: USGS - Abstract
  • Other: USGS - Full Text PDF
  • Journal Article Schlekat CE, Decho AW, Chandler GT. Bioavailability of particle-associated silver, cadmium, and zinc to the estuarine amphipod Leptocheirus plumulosus through dietary ingestion. Limnology and Oceanography 2000;45(1):11-21. R826399E02 (Final)
    R825279 (Final)
  • Full-text: ASLO-Full Text PDF
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  • Abstract: ASLO-Abstract
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  • Other: ResearchGate - Abstract & Full Text HTML
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  • Supplemental Keywords:

    estuary, sediment, water, exposure, bioavailability, vulnerability, animal, aquatic, habitat, conservation, metals, SC, colloids, conservation, pesticides,

    Progress and Final Reports:

    Original Abstract
  • 1998
  • 1999