This proposal is submitted in response to RFA ES-99-001, entitled Superfund Hazardous Substances Basic Research Program. The contamination of soils and drinking water with As and Pb are associated with major public health, remedial, and environmental policy problems. As is found in soil or water at the majority of Superfund sites, while Pb is a soil-borne contaminant of concern at approximately 300. This proposal seeks to obtain new knowledge, and train multi-disciplinary pre- and post-doctoral students, concerning the bioavailability of soil Pb in humans, and the bioavailability, health effects and geochemistry of As. The proposed work involves studies of bioavailability and/or geochemistry at four Superfund sites in the U.S., two contaminated with Pb and two with As. It also encompasses epidemiologic and geochemistry studies of As in drinking water in Bangladesh which focus on carcinogenic, reproductive, and childhood effects of As exposure. We also devote resources to the development of practical remediation strategies for As in wastewater and drinking water.
The proposal includes four biomedical research projects: 1) Bioavailability of Soil Pb and As in Humans; 2) Genotoxic Mechanisms of As in Mammalian Cells; 3) A Cohort Study of Arsenicosis in Bangladesh; 4) Environmental As, Pregnancy, and Children’s Health. The biomedical research is directly related to that which occurs in three non-biomedical projects: 5) As Mobilization in Bangladesh Groundwater; 6) Redistribution of As at Sites in NJ and Maine; and 7) Assessment and Remediation for As Enrichments in Groundwater. The research projects are supported by three Research Support Core Labs: 8) Trace Metals; 9) Geochemistry; and 10) Hydrology. An Administrative Core includes an Information Dissemination Program and a Government Liasison & Outreach Program. Finally, a Training Core coordinates multi-disciplinary education and interaction among pre- and post-doctoral trainees support by this proposal as well as other training grant.
Project 1 – Bioavailability of Soil Pb and As in Humans
PI - Conrad Blum
The contamination of soils with lead (Pb) and arsenic (As) has created potential health hazards at numerous sites throughout the country, including hundreds now designated as Superfund sites. The bioavailability (i.e., fraction absorbed) of these metals from ingested soil in humans is not known. Models aimed at estimating human exposure to Pb and As from soil currently use assumptions based on bioavailability data from animal or in vitro models. Using the technique of stable Pb isotope dilution, we have developed a model for estimating soil Pb bioavailability in humans. This model examines changes in the ratio of 206Pb to 207Pb in blood, following the ingestion of trace quantities of Pb-contaminated soils.
Thus, we propose to examine the human bioavailability of soils from a mining site, a smelter site, and an urban site. Through collaboration with EPA, we have already obtained soils from a mining site and a smelter site which are isotopically ideal for study. In addition, we propose to determine whether soil amending agents currently being tested in the field by EPA actually reduce soil Pb bioavailability in humans. This phase of our work is important because the same pre- and post-amended soils, from a former smelter site in Joplin, Missouri, are currently being evaluated in swine, rat and in vitro models, possibly allowing validation of the less expensive in vitro model. Finally, we will explore the possibility that the ingestion of these soils, some of which contain trace quantities of As, may allow us to estimate the urinary excretion fraction (UEF) for soil As. Collectively, these studies promise to improve the precision of risk assessments at site contaminated with Pb and As.
Project 2 – Genotoxic Mechanisms of As in Mammalian Cells
PI - Tom Hei
Although arsenic is a well established human carcinogen and induces cancers of the skin, liver, bladder, and lung, the underlying mechanism(s) is unknown. Using the human-hamster hybrid (AL) cells that are sensitive in detecting multilocus deletions, we show recently that arsenic is indeed mutagenic to endogenous genes in mammalian cells. The goal of this application is to elucidate the mutagenic pathways involved in arsenite-treated cells. The first objective of this proposal is to determine if reactive oxygen species, particularly hydroxyl radicals, generated by arsenite result in oxidative DNA damage and mutagenesis in AL cells. The induction of the oxidative DNA damage product, 8-OHdG, and the formation of hydroxyl radicals will be determined from arsenite-treated cultures using immunoperoxidase staining and the salicylate assay, respectively. The localization of the ROS formed by arsenite will be determined using confocal microscopy with the oxidative sensitive dye CM-H2DCFDA. To show that oxyradicals induced by arsenite actually mediate the mutagenic events, the incidence and types of S1 mutants induced by equitoxic doses of either arsenite or hydrogen peroxide will be determined. The second objective is to determine the role of mitochondria in mediating the genotoxic response of arsenite. Mitochondria in AL cells will be functionally inactivated using rhodamine 6G. Cultures will be treated with graded doses of arsenite and rescued by fusion with cytoplasts to determine the mutagenic response. The AL cells contain only one copy of human chromosome 11 and mutations on marker genes located on this chromosome can be readily scored using an antibody complement lysis technique. Since the AL cell also contain the HPRT gene located on the hamster X chromosome, mutations induced by arsenic on an essential (-X) versus a non-essential chromosome (human chromosome 11) will provide useful information on the types and sizes of the induced molecular alterations.
Project 3 – A Cohort Study of Arsenicosis in Bangladesh
PI - Habib Ahsan
Evidence of the health effects of inorganic arsenic (InAs) comes from ecological or retrospective studies. The overall goal of this proposal is to assemble a large cohort of adults to prospectively examine the short-term and intermediate-term health effects of such exposure, with an initial focus on skin lesions, skin cancers, biomarkers of As toxicity and mortality. The creation of this cohort will also facilitate Project #4 which will study pregnancy outcomes and children’s health in the same families. Ultimately this cohort will serve as the base for future studies of long-term health effects of InAs exposure. We propose to recruit and follow 10,000 married men and women from Sonargaon, Bangladesh who have been exposed to a wide range of InAs (from <10 µg/L to > 1000 µg/L) in drinking water. Interview data, blood and urine samples will be collected by trained personnel at the baseline. Follow-up interviews with the subjects will be conducted in year 3 and in year 5. Incidences of skin lesions and skin cancers will be ascertained through physical examination by the study physicians followed by histopathologic confirmation by the study pathologist. The proposed study will be the first to prospectively examine the full dose-response relationship between arsenic exposure and skin lesions and skin cancers using individual measurement data. The study will also examine the biomarkers, urinary arsenic species and TGFα, in relation to arsenic exposure (cross-sectionally in the baseline cohort) and in relation to the risk of skin lesions and skin cancers (prospectively using a case-cohort design nested within the total cohort).
Project 4 – Environmental As, Pregnancy and Children’s Health
PI - Joseph Graziano
Remarkably little is known about the health consequences of chronic inorganic arsenic (InAs) exposure in pregnant women and children, typically perceived to be the most vulnerable to environmental exposures. InAs is a known teratogen in animals, particularly in species that are inefficient at methylating it to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA).
We have identified a region of Bangladesh (Sonargaon) where drinking water contains a uniquely wide range concentrations of InAs. We propose to conduct three studies which will test hypotheses concerning the health effects and metabolism of InAs in the pregnant women and children. In year -01, through a retrospective study of past pregnancy outcomes in approximately 2000 women, we will examine the relations between InAs exposure, assessed through measurements of drinking water and urinary As, and late spontaneous abortion and stillbirth. In years -02 and -03, we will recruit approximately 100 women at 12-20 weeks of gestation to test the hypothesis that As methylation can be facilitated through the administration of multivitamins, or multivitamins + additional folate. This randomized trial will examine urinary excretion of InAs, MMA and DMA before and after the provision of such supplements. Third, among the same pregnant women, we will describe at delivery the relationships between maternal and umbilical cord InAs, MMA and DMA; we will also examine the relation between plasma homocyst(e)ine (a marker of folate and B12 status) and InAs, MMA and DMA. Finally, in years -04 and -05, we will conduct cross-sectional studies of children aged 6- and 10-years, respectively, to examine association between InAs exposure and selected biomarkers or health outcomes.
Project 5 – Arsenic Mobilization in Bangladesh Groundwater
PIs - Yan Zheng and Martin Stute
The origin of As enrichments in groundwater of many parts of the world is poorly understood. The problem is particularly acute in Bangladesh where 120 million people draw their drinking water from about 4 million wells that tap into aquifers of the Ganges-Brahmaputra Delta. It is estimated that approximately two thirds of these wells supply water containing >50μg/L As, the current EPA limit. The situation, distressing as it is, provides a special opportunity to study dose-response relationships in humans. One difficulty of such a study is the extreme spatial variability of As concentrations in groundwater of Bangladesh. It also is not known to what extent there are temporal variations in groundwater As. The first objective of this project is therefore to collect detailed time series of groundwater As concentrations in the region where biomedical Project 3 and 4 will be conducted over a period of 4 years. The second objective is to use this information to test several hypotheses that have been proposed to explain the extremely wide range of As concentrations in groundwater in Bangladesh. An improved understanding of the still largely unknown geochemical and hydrological factors that regulate As levels in groundwater will be directly transferable to the many parts of the US where groundwater is enriched in As due to either natural processes or anthropogenic inputs (Projects 5 and 7).
Our working hypothesis is that As is mobilized and re-concentrated in different sediment layers through a series of oxidation and reduction reactions that do not proceed at the same rate for As and for Fe. Under certain conditions, such a sequence could lead to extremely high As levels in groundwater. This hypothesis, and others that have been proposed, will be tested with multi-disciplinary study of the hydrology, geophysics, and geochemistry of the region where the biomedical studies will be conducted. The overall groundwater flow regime will first be determined from a broad survey of geophysical properties and hydrologic tracers such as 3H, 3He, and 14C. On the basis of this information, six new multi-level wells will be installed an ∼15 existing drinking water wells spanning a range of redox conditions will be monitored intensively. In addition to As concentration and speciation, the suite of parameters to be measured in the wells will include hydraulic head, temperature, conductivity, pH, Eh, dissolved O2, NO3, Mn, Fe, SO4, 18O and D (2H).
Project 6 – Redistribution of Arsenic and Other Contaminants at Sites in New Jersey and Maine
PI - H. James Simpson and Martin Stute
Arsenic behavior in the subsurface reflects redox conditions, interactions with particle surfaces, specific minerals phases present in the aquifer and groundwater recharge dynamics. We propose field studies at two Superfund sites with As contamination, both of which will be under active groundwater treatment through the period of our investigation. The Vineland Chemical Company (ViChem) site in southern NJ, experienced dispersal of at least hundreds of tons of waste AS over five decades into surface soils, aquifer formations, groundwater, surface waters, plus stream, lake, and estuary sediments far downstream from the original arsenical herbicide and fungicide production facilities. Our strategy will be to examine recharge dynamics of this altered flow regime, primarily through analyzing samples from monitoring wells for tracers already present in the environment and to relate behavior of As and other redox sensitive species to those changes. As part of our investigation, we would analyze groundwater for 3H/3He, total [As], As speciation, dissolved Fe, Mn, NO3, SO4, O2, PO43-, Si(OH)4, pH, Eh and major element chemistry. Samples would be collected periodically, to examine critical controls on As solubility and dynamics of subsurface As transport. We will also collect and analyze a series of sediment cores in Union Lake in order to reconstruct the history of As migration downstream, and to estimate the proportion of As sediment inventory mobilized upward towards the sediment surface. A similar set of groundwater measurements, directed at elucidating controls on As behavior, would be conducted at the Winthrop (ME) Landfill site. Sources of As to this strongly reducing plume are unresolved, but mobilization from surrounding natural soils appears to be a significant possibility, suggesting that similar As plumes could occur at many other landfills. Differing from the Vineland site, the hydrogeology at Winthrop is very complex due to large spatial variability of hydraulic conductivity. We propose to better constrain the groundwater flow regime by employing geophysical survey techniques (ground penetrating radar and resistivity) in addition to 3H/3He. We will study As mobilization processes by following the geochemical evolution along a flow path underneath the landfill and by tracing the compositions of a plume of re-injected water labeled with SF6. Our studies will contribute to a better understanding of the processes affecting As transport under different geochemical conditions ranging from oxidizing to highly reducing. They could potentially also contribute to an increase in efficiency of As remediation at a number of Superfund sites currently under investigation.
Project 7 – Remediation of Groundwater for As and Effluents for Pb
PIs- Lex van Geen and Ponisseril Somasundaran
Groundwater naturally or anthropogenically enriched in As poses serious health hazzards in many parts of the world, including the US. This project builds on the growing realization that the particularly high affinity of As for Fe-oxyhydroxides is a key factor regulating dissolved As concentrations in groundwater. The particular affinity of As for Fe-oxyhydroxides has been exploited for remediation by FeCl3 addition and co-precipitation. Because the US As drinking water standard may be lowered to the WHO-recommended value of 10 mg/L in the near future, a better fundamental understanding of different treatment options and the influence of groundwater composition on the efficacy of treatment is needed.
Theme(I) As mobilization into reducing groundwater: Field material from US Superfund sites at Winthrop, ME and Vineland, NJ (Project 6) and Bangladesh (Project 5) will be used to quantify the exchange of As between the dissolved and the particulate phase under oxic and reducing conditions. In a first set of experiments, the potential integrated release of As by reductive dissolution will be determined with hydroxylamine extraction from archived aquifer material covering a range of redox conditions. The spatial patterns of As and Fe released by reductive dissolution will be compared to the oxygen distribution in the aquifers. In a second set of experiments, fresh cores of aquifer material from selected sites will be incubated to quantify the rate of As and Fe exchange (adsorption or desorption). In a third set of experiments, state-of-the-art surface analytical techniques will be used to characterize arsenic binding at model Fe oxide surfaces under a range of conditions representative of the natural environment.
Theme (II) As removal from reducing groundwater: Coprecipitation treatment with ferric chloride is an effective technique commonly used for the removal of arsenic from water. A different approach for removing As from groundwater that is promising but has not yet been fully evaluated relies on the adsorptive properties of Fe filings. This method was found to be very effective at the Winthrop, ME, Superfund site and will be tested at another site in Vineland, NJ. The mechanisms of As removal by Fe filings will also be probed using surface spectroscopy. Another objective under this theme is to develop practical ways of reducing the scale of As remediation and hereby meet the needs of users of domestic wells in the US and Bangladesh. We will focus on Fe-oxyhydroxide-based treatment using, whenever possible, the natural Fe content of groundwater. The As-removal potential of existing Iron Removal Units in Bangladesh will be explored in collaboration with local government researchers. The potential of filings as an alternate source of Fe for coprecipitation to treat water from domestic wells in the US and Bangladesh will also be examined.