Lead Bioaccessibility In The Gastrointestinal Tract: Evaluation Of Five In Vitro Digestion Models Against In Vivo Data
Tom R. Van de Wiele, LabMET, Ghent University, Ghent, Belgium
Agnes G. Oomen, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
Joanna Wragg, British Geological Survey, Nottingham, United Kingdom
Mark Cave, British Geological Survey, Nottingham, United Kingdom
Mans Minekus, TNO Nutrition, Zeist, The Netherlands
Alfons Hack, Ruhr-Universität Bochum, Bochum, Germany
Christa Cornelis, Vito, Mol, Belgium
Ben Klinck, British Geological Survey, Nottingham, United Kingdom
Cathy Rompelberg, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
Loeckie De Zwart, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
Joop Van Wijnen, GG&GD, Amsterdam, The Netherlands
Willy Verstraete , LabMET, Ghent University, Ghent, Belgium
Adriënne J. Sips, National Institute of Public Health and the Environment, Bilthoven, The Netherlands

Bioavailability of Contaminants in Soils and Sediments: Processes, Tools, and Applications
Richard G. Luthy, Stanford University
Richelle M. Allen-King, Washington State University
Sally L. Brown, University of Washington
David A. Dzombak, Carnegie Mellon University
Scott E. Fendorf, Stanford University
John P. Giesy, Michigan State University
Joseph B. Hughes, Rice University
Samuel N. Luoma, U.S. Geological Survey
Linda A. Malone, College of William & Mary
Charles A. Menzie, Menzie-Cura & Associates, Inc.
Stephen M. Roberts, University of Florida
Michael V. Ruby, Exponent
Terry W. Schultz, University of Tennessee
Barth F. Smets, University of Connecticut
Laura J. Ehlers, National Research Council  

Bioavailability of Nickel from Port Colborne Soils: A Comparison of in vivo and in vitro Methodologies
Christopher A. Ollson, Jacques Whitford Environment Limited
Wai Chi Kwan, Cecile E. Willert, Eric Veska, Jacques Whitford Environment Limited
Iris Koch, Kenneth J. Reimer, Royal Military College of Canada

Measurement of Arsenic Bioavailability from Soil
Michael V. Ruby, Exponent
Stephen M. Roberts, University of Florida
Ronald C. Wester, University of California, San Francisco
Rosalind A. Schoof, Integral Consulting, Inc.
Yvette W. Lowney, Exponent

Comparison of Five In Vitro Digestion Models to Study the Bioaccessibility of Soil Contaminants
Agnes G. Oomen, RIVM-SIR
Alfons Hack, Ruhr-Universität Bochum
Mans Minekus, and Evelijn Zeijdner, TNO Nutrition
Christa Cornelis and Greet Schoeters, Vito
Willy Verstraete and Tom Van de Wiele,Ghent University
Joanna Wragg, British Geological Survey
Joop H. Van Wijnen, GG&GD Amsterdam

Human In Vitro Intestinal Cell Lines and Bioavailability
Desmond. I. Bannon, ATTN: MCHB-TS-THE 

The Effect of Phosphate Treatment on the Bioavailability of Soil Lead in Humans
Nancy Lo Iacono, Columbia University
Steven Chillrud, Columbia University
Conrad Blum, Columbia University
Mark Maddaloni, Environmental Protection Agency
Joseph Graziano, Columbia University

Lead Bioaccessibility In The Gastrointestinal Tract: Evaluation Of Five In Vitro Digestion Models Against In Vivo Data

Tom R. Van de Wiele, LabMET, Ghent University, Ghent, Belgium
Agnes G. Oomen, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
Joanna Wragg, British Geological Survey, Nottingham, United Kingdom
Mark Cave, British Geological Survey, Nottingham, United Kingdom
Mans Minekus, TNO Nutrition, Zeist, The Netherlands
Alfons Hack, Ruhr-Universität Bochum, Bochum, Germany
Christa Cornelis, Vito, Mol, Belgium
Ben Klinck, British Geological Survey, Nottingham, United Kingdom
Cathy Rompelberg, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
Loeckie De Zwart, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
Joop Van Wijnen, GG&GD, Amsterdam, The Netherlands
Willy Verstraete , LabMET, Ghent University, Ghent, Belgium
Adriënne J. Sips, National Institute of Public Health and the Environment, Bilthoven, The Netherlands

Soil ingestion is a predominant exposure route of environmental contaminants to the human body. Contaminant bioaccessibility can be defined as that fraction that mobilizes from the soil matrix in the gastrointestinal tract and that is at maximum available for intestinal absorption. Hence, it is an important prerequisite to oral bioavailability. Mechanistic information on which processes and parameters influence contaminant bioaccessibility in the gut is scarce. This paper presents a multi-laboratory comparison study of in vitro models assessing bioaccessibility of soil-bound lead in the human gastrointestinal tract. The bioaccessibility results of this round-robin were compared and evaluated against oral bioavailability data that were obtained from a previous in vivo study in which the oral lead bioavailability in adults for that same contaminated soil was investigated. Every in vitro method simulated fasted and fed conditions in analogy with the former in vivo study.

The bioaccessibility data were significantly different for each in vitro method and ranged for the fasted models from 2% to 33% and for the fed models from 6% to 29%. The in vivo data from literature were 26.2% for the fasted conditions, compared to 2.5% for the fed conditions. Crucial parameters that determined lead bioaccessibility during fasted conditions were the liquid to soil ratio, pH of gastric and duodenal juice, and the separation step for estimating bioaccessible lead (centrifugation speed, filtration, dialysis). For the fed conditions, the nutrition type and the formation of lead complexes appeared to be important as well as the separation step. Lead bioaccessibility from a dynamic in vitro model fitted oral bioavailailability data the best, both in fasted and fed conditions. This model used hollow fibre membranes with a cut-off factor of 3000 Da to identify lead bioaccessibility. Results from the other in vitro methods provide important mechanistic information on factors determining lead bioaccessibility. The combination of in vitro data from several methods and in vivo data on the same matrix from literature give a better insight in the processes that determine the bioavailability of ingested contaminants.

Bioavailability of Contaminants in Soils and Sediments: Processes, Tools, and Applications

Richard G. Luthy, Stanford University, Dept. of Civil & Env. Engineering, Terman Engineering Center, Room M21, Stanford, California 94305-4020, Tel: 650-725-9170, Fax: 650-725-3164 
Richelle M. Allen-King, Washington State University, Department of Geology, 440 SE Dilke Street, Pullman, WA 99163, Tel: 509-335-1180, Fax: 509-335-7816
Sally L. Brown, University of Washington, Ecosystem Science Division, College of Forest Resources, 156B Bloedel Hall, Box 352100, Seattle, WA 98195, Tel: 206-616-1299, Fax: 206-685-3091
David A. Dzombak, Carnegie Mellon University, Dept. of Civil & Env. Engineering, Pittsburgh, PA  15213-3890, Tel: 412-268-2946, Fax: 412-268-7813 
Scott E. Fendorf, Stanford University, Dept. of Geol. & Env. Science, Stanford, CA  94305-2115,Tel: 650-723-5238, Fax: 650-725-2199 
John P. Giesy, Michigan State University, Department of Zoology, 22 Natural Science, East Lansing, MI 48824, Tel: 517-353-2000
Joseph B. Hughes, Dept. of Env. Science and Engineering, Rice University MS-317, 6100 Main, Houston, TX  77005-1892, Tel: 713-348-5903, Fax: 713-348-5203
Samuel N. Luoma, U.S. Geological Survey, MS 465, 345 Middlefield Road, Menlo Park, CA  94025, Tel: 650-329-4481, Fax: 650-329-4545
Linda A. Malone, College of William & Mary, School of Law, P.O. Box 8795, Williamsburg, VA,23187-8795, Tel: 757-221-3844, Fax: 757-221-2939
Charles A. Menzie, Menzie-Cura & Associates, Inc., One Courthouse Lane, Suite #2, Chelmsford, MA  01824, Tel: 978-322-2856, Fax: 978-970-2791
Stephen M. Roberts, University of Florida, Center for Env. & Human Toxicology, Box 110885, Bldg. 471, Mowry Road, Gainesville, FL 32611, Tel: 352-392-4700 x5500, Fax: 352-392-4707 
Michael V. Ruby, Exponent, 4940 Pearl East Circle, Suite 300, Boulder, CO  80301, Tel: 303-444-7270 Fax: 303-444-7528 
Terry W. Schultz, University of Tennessee, Department of Comparative Medicine, College of Veterinary Medicine, 2407 River Drive, Knoxville, TN  37996-4500, Tel: 865-974-5826, Fax: 865-973-8222
Barth F. Smets, University of Connecticut, Dept. of Civil and Env. Engineering, 261 Glenbrook Dr., U-2037, Storrs, CT  06269, Tel: 860-486-2270
Laura J. Ehlers, National Research Council, Water Science and Technology Board, 500 5th Street, NW, Washington, DC  20418, Tel: 202-334-3422, Fax: 202-334-1961

Bioavailability refers to the processes that cause contaminants in soil or sediment to become sequestered in such a way as to reduce overall exposure of nearby organisms, including humans.  After two years of deliberation, a committee of the National Research Council recently weighed in on the use of bioavailability in soil and sediment management.  The resulting report notes that the potential for the consideration of bioavailability to influence decision-making is greatest where certain chemical, environmental, and regulatory factors align.  The first condition is that the contaminant whose bioavailability is under investigation must be and remain the risk driver at a site.  Second, consideration of bioavailability could make a significant difference if the default assumptions made during risk assessment that affect the final cleanup goal are inappropriate.  Third, experience has shown that considerations of contaminant bioavailability make sense where a significant change to remedial goals is likely, for example because substantial quantities of contaminated soil or sediment are involved.  Fourth, bioavailability arguments should only be used to alter cleanup goals when site conditions are unlikely to change substantially over time.  Finally, bioavailability concepts have not received widespread regulatory and public acceptance.

The report begins by defining “bioavailability processes” and examining historical use of the term and concept.  A chapter is devoted to demystifying the current use of bioavailability in risk assessment and hazardous waste cleanup regulations.  The report discusses acceptable tools and models for bioavailability assessment, and it ranks tools according to seven criteria.  Finally, the intimate link between bioavailability and bioremediation is explored.  The report concludes with suggestions for moving bioavailability forward in the regulatory arena for both soil and sediment cleanup.

Bioavailability of Nickel from Port Colborne Soils: A Comparison of in vivo and in vitro Methodologies

Christopher A. Ollson, Jacques Whitford Environment Limited, Suite 200 – 2781 Lancaster Road
Ottawa, Ontario, K1B 1A7, Tel: 613-738-0708, Email: collson@jacqueswhitford.com
Wai Chi Kwan, Cecile E. Willert, Eric Veska, Jacques Whitford Environment Limited, 1200 Denison Street, Markham, Ontario L3R 8G6, Tel: 416-495-8614 
Iris Koch, Kenneth J. Reimer, Environmental Sciences Group, Royal Military College of Canada, PO Box 17 000 STN Forces, Kingston, Ontario, Tel: 613-541-6000 ext 6161

Elevated levels of nickel (Ni) in soil span a 29 km² area of Port Colborne, Ontario, as a result of historical stack emissions from a nickel refinery. A confounding factor for the human health risk assessment is that the RfD for Ni is based on oral administration of soluble nickel sulphate (NiSO₄) to rats. However, the bioavailability of Ni (primarily nickel oxide) in soils is likely less than soluble NiSO₄.

In vivo determination of the relative oral bioavailability (ROB) of Ni from three soil types was determined through oral administration to rats. The Ni bearing soils and NiSO₄^.6H₂O were orally administered to male rats as follows: Group 1 – vehicle blank, Group 2: NiSO₄^.6H₂O 39.7 mgNi/Kg bw, Group 3: Welland Clay 28.5 mgNi/Kg bw, Group 4: Organic Soil 53.1 mgNi/Kg bw, Group 5: Fill Soil 58.1 mgNi/Kg bw. The ROB fraction of Ni from soils was 3.9% for Welland Clay, 3.2% Organic Soil and 2.1% Fill Soil.

In vitro bioaccessibility of Ni from the three soil types was carried out using a two stage simulated gastric fluid extraction(GFE): Stage 1 acidic gastric conditions, then pH raised to neutral (intestinal) in Stage 2. Two GFE experimental regimes were conducted; first with glycine added as a buffer and the second without glycine. No statistical difference (p>0.05) in the % bioaccessible fraction of Ni from the soils was seen between the two regimes in Stage 1. However, there was a significant decrease (p<0.05) in the bioaccessible fraction of Ni in the regime conducted without glycine in Stage 2 (intestinal). It was determined that the in vivo ROB of Ni from the soil types is actually between the in vitro bioaccessibility GFE regimes of Stage 2, with and without glycine.

The use of the ROB of Ni from soils has served to set a scientifically defensible, health based soil remediation criterion. Work continues on validating an in vitro extraction for Ni.

Measurement of Arsenic Bioavailability from Soil

Michael V. Ruby, Exponent, 4940 Pearl East Circle, Suite 300, Boulder, CO  80301, Tel: 303-444-7270, Fax: 303-444-7528 
Stephen M. Roberts, Center for Environmental & Human Toxicology, University of Florida, Bldg. 471, Mowry Road, Gainsville, FL  32611, Tel: 352-392-4700 x 5505, Fax:  352-392-4707 
Ronald C. Wester, University of California, San Francisco, Dermatology Department, 90 Medical Center Way, Surge Bldg., Room 110, San Francisco, CA  94143-0989, Tel: 415-665-2236, Fax: 415-753-5304
Rosalind A. Schoof, Integral Consulting, Inc., 1500 112th Street, Suite 101, Bellevue, WA  98004, Tel: 425-451-3823
Yvette W. Lowney, Exponent, 4940 Pearl East Circle, Suite 300, Boulder, CO  80301, Tel: 303-444-7270, Fax: 303-444-7528

This presentation will discuss a study conducted to assess the oral and dermal absorption of arsenic from soil, for the purpose of human health risk assessment.  Both the oral and dermal assessments relied on non-human primates as a surrogate for humans, and evaluated the absorption of arsenic from the same test soils.  Measurement of urinary arsenic excretion over a 5- to 7-day period, post-dosing, was relied on for determination of the absorbed fraction of arsenic from soil.  The oral bioavailability study was conducted in the model of Roberts et al. (2002)[1], with the following modifications:  the test species was changed from Cebus to Cynomolgus monkeys, which allowed anesthesia to be eliminated from the study design, and the analytical method was changed to ICP/MS to allow for lower detection limits for arsenic in monkey urine.  The dermal absorption study utilized the model of Wester et al. (1993)[2], with the exceptions that environmentally contaminated (i.e., weathered) soils were studied, and that certain exposure parameters (e.g., exposure time, soil particle size, and loading rate) were changed to be more representative of human exposures to contaminated soils.  The test soils (surficial, 0- to 2-in.) were collected from orchard, mining, and smelting sites, and contain arsenic in the range of 300–700 mg/kg.  Soil parameters, including pH, total organic carbon, particle size, Fe/Mn oxide content, and arsenic mineralogy, were determined for each soil.  The relation between oral bioavailability and dermal absorption of arsenic will be presented for the individual test soils, and the effect of soil characteristics on controlling the magnitude of these endpoints will be discussed.

Comparison of Five In Vitro Digestion Models to Study the Bioaccessibility of Soil Contaminants

Agnes G. Oomen, RIVM-SIR, P.O.Box 1, NL-3720 BA Bilthoven, The Netherlands, Tel. +31 30 2742159, Fax: +31 30 2744451 
Alfons Hack, Ruhr-Universität Bochum, Universitätstrasse 150, D-44801 Bochum, Germany Tel. +49 23 43225487
Mans Minekus, and Evelijn Zeijdner, TNO Nutrition, Utrechtseweg 48, NL-3704 HE Zeist, The Netherlands, Tel. +31 30 6944616
Christa Cornelis and Greet Schoeters, Vito, Boeretang 200, B-2400 Mol, Belgium, Tel. +32 14335200
Willy Verstraete and Tom Van de Wiele, LabMET, Ghent University, Coupure L653, B-9000, Ghent, Belgium, Tel. +32 92646033
Joanna Wragg, British Geological Survey, Kingsley Dunham Centre, Keyworth, NG12 5GG Nottingham, United Kingdom, Tel. +44 (0)115 936 3100
Joop H. Van Wijnen, GG&GD Amsterdam, P.O.Box 20244, NL-1000 HE Amsterdam, The Netherlands Tel. +31 20 5555352

Soil ingestion can be a major exposure route for humans to many immobile soil contaminants. Exposure to soil contaminants can be overestimated if oral bioavailability is not taken into account. Several in vitro digestion models simulating the human gastrointestinal tract have been developed to assess mobilization of contaminants from soil during digestion, i.e., bioaccessibility. Bioaccessibility is a crucial step of the oral bioavailability for soil contaminants. To what extent in vitro determination of bioaccessibility is method dependent has, until now, not been studied. This study presents a multi-laboratory comparison and evaluation of five in vitro digestion models. Their experimental design and the results of a round robin evaluation of three soils, each contaminated with arsenic, cadmium, and lead, are presented and discussed. A wide range of bioaccessibility values were found for the three soils: for As 6-95%, 1-19%, and 10-59%; for Cd 7-92%, 5-92%, and 6-99%; and for Pb 4-91%, 1-56%, and 3-90%. Bioaccessibility in many cases was less than 50%, indicating that a reduction of bioavailability can have implications for health risk assessment. Although the experimental designs of the different digestion systems are distinct, the main differences in test results of bioaccessibility can be explained on the basis of the applied gastric pH. High values are typically observed for a simple gastric method, which measures bioaccessibility in the gastric compartment at low pHs of 1.5. Other methods that also apply a low gastric pH, and include intestinal conditions, produce lower bioaccessibility values. The lowest bioaccessibility values are observed for a gastrointestinal method which employs a high gastric pH of 4.0.

Human In Vitro Intestinal Cell Lines and Bioavailability

Desmond. I. Bannon, Ph.D., ATTN: MCHB-TS-THE, 5158 Blackhawk Road, Aberdeen Proving Ground, MD 21210-5403, Tel: 410-436-3387, Fax: 410-436-8258

Although lead transport has been localized to the duodenum, the molecular mechanism(s) of transport is still largely unknown and has only been described in terms of its general energy requirements (active or passive). This lack of knowledge exists in spite of the cloning of several intestinal metal transporters over the last 10 years, including those for iron and copper.  Since it is presumed that lead uses mechanisms of transport that already exit for essential metals newly discovered metal transporters may provide the key to a mechanistic model of lead transport, and thereby inform models of risk assessment.  For mammals, a membrane barrier mediates mammalian transport, which in humans consists of a thin layer of absorbing enterocytes in the intestine.  Transport from the intestinal lumen to the portal blood takes place across these enterocytes, which are differentiated into functionally distinct apical and basolateral membranes.  In vitro human intestinal cell lines have been widely used to examine transcellular and paracellular transport of metals besides lead.  When grown in culture, these cells form intestinal like epithelia with tight-junctions and differentiate to form apical absorbing and basolateral transporting membranes typical of enterocytes.  In this presentation, the potential for human intestinal cell lines to elucidate mechanisms of lead and other metal transport will be reviewed in the light of recent discoveries of intestinal metal transporters.  The potential of these models to inform both in vivo and in vitro bioavailability methods is assessed.

The Effect of Phosphate Treatment on the Bioavailability of Soil Lead in Humans

Nancy LoIacono, M.P.H., Mailman School of Public Health, Columbia University, 60 Haven Ave., New York, NY   10032, Tel: 212-305-1623, Fax: 212-305-3857
Steven Chillrud, Ph.D., Lamont Doherty Earth Observatory, Columbia University, 7 Marine Biology, Palisades, NY   10964, Tel: 845-365-8893, Fax:845-365-8155
Conrad Blum, M..D., Columbia University, College of Physicians & Surgeons, 16 East 60^th Street, New York, NY   10022, Tel: 212-326-8421, Fax: 212-326-8580
Mark Maddaloni, Dr.P.H., U.S. Environmental Protection Agency, 290 Broadway, New York, NY 10007 Tel: 212-637-3590, Fax: 212-637-5045
Joseph Graziano, Ph.D., Mailman School of Public Health, Columbia University, 60 Haven Ave., New York, NY   10032, Tel: 212-305-1678, Fax: 212-305-3857

Lead (Pb) is a major soil contaminant at hundreds of Superfund sites.  Soil can be ingested by small children through hand to mouth activity, and some of the ingested Pb can subsequently pass into the bloodstream.  The bioavailability (i.e., fraction absorbed) of Pb from ingested soil in humans has, until recently, been unknown.  Models aimed at estimating human exposure to Pb from soil currently use assumptions based on bioavailability data from animal or in vitro models. 

Technologies or soil treatments that would reduce Pb bioavailability could potentially influence the clean-up level for Pb in soil.  Phosphate, as found in typical agricultural phosphate fertilizers, can form insoluble complexes with Pb.  For this reason, EPA scientists have been interested in evaluating the effect of phosphate treatment, i.e., the application of standard phosphate fertilizer, on Pb bioavailability.  Through collaboration with the EPA, we obtained and evaluated soil from a former smelter site in Joplin, Missouri.  Employing a human model we developed, using a method known as stable isotope dilution, we have been able to determine the bioavailability of Pb in an untreated soil sample, and a sample from an adjacent plot of land that had been treated with 1% phosphate 18 months earlier.  We assessed changes in the ratio of ²⁰⁶Pb to ²⁰⁷Pb in blood, following the ingestion of trace quantities of the Pb-contaminated soils. 

Preliminary findings are extremely interesting and potentially important.  Among the five subjects who received the non-treated soil, the mean Pb bioavailability was 36%, with a range of 15-54%.  In contrast, among those who received the phosphate-treated soil, mean Pb bioavailability was 15%, with a range of 8-26%.  This represents a 57% reduction in bioavailability.  Comparison of the study results with those evaluating the bioavailability of these soil in animals, and in an in vitro system, are underway.

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