Proceedings of the ISEA Bioavailability Symposium
Durham, North Carolina
Use of In Vitro Bioaccessibility/Relative Bioavailability
Estimates for Metals in Regulatory Settings:
What Is Needed?
Prepared for:
U.S. Environmental Protection Agency
Technical Workgroup Bioavailability Committee
October 2007
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Table of Contents
Panel Members 2
Introduction 3
Proceedings 4
Panel Discussion Session 1 9
Panel Discussion Session II 18
Summary 20
Appendix A: Questions for the Panel (submitted in advance) 22
Appendix B: Abstracts 25
Appendix C: Presentations 26
Appendix D: Agenda
Panel Members
Nick Basta, Ohio State University
Karen Bradham, U.S. Environmental Protection Agency; (co-chair)
Mike Beringer, U.S. Environmental Protection Agency
Stan Casteel, University of Missouri
Mark Cave, British Geological Survey (co-chair)
Heather Jamieson, Queen's University
Brian Laird, University of Saskatchewan
Yvette Lowney, Integral Consulting
Agnes Oomen, Rijksinstituut Voor Volksgezondheid En Milieu
Pat Rasmussen, Health Canada; (co-chair)
Sohel Saikat, UK Environment Agency
Kirk Scheckel, U.S. Environmental Protection Agency
Rosalind Schoof, Integral Consulting, Inc.; (co-chair)
Dave Thomas, U.S. Environmental Protection Agency
Joanna Wragg, British Geological Survey
Aaron Yeow, U.S. Environmental Protection Agency
Notes recorded and draft prepared by Mark Follansbee, Sycracuse Research Corporation,
Environmental Science Center
Abbreviations
% Percent
ABA Absolute Bioavailability
As Arsenic
BARGE Bioaccessibility Research Group of Europe
BGS British Geological Survey
Cr Chromium
Cu Copper
DMA Dimethylarsenic
DQO Data Quality Objective
g gram
g/kg gram per kilogram
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GI Gastrointestinal
HERA Human and Ecological Risk Assessment
HF Hydrofluoric acid
HHRS Human Health Risk Assessment
ICCVAM Interagency Coordinating Committee for Validation of Alternative Methods
IAEA International Atomic Energy Agency
ISEA International Society of Exposure Analysis
IVG In vitro gastrointestinal
k thousand
jig microgram
mg/kg milligram per kilogram
MMA methylarsonic acid
NaAS Sodium arsenate
Ni Nickel
NIST National Institute of Standards and Technology
OSWER Office of Solid Waste and Emergency Response
Pb Lead
PBET Physiologically Based Extraction Technique
ppm parts per million
PRG Preliminary Remediation Goal
Q&A Question and Answer
RA Risk Assessment
RBA Relative Bioavailability
RIVM Rijksinstituut Voor Volksgezondheid En Milieu
SBRC Solubility/Bioavailability Research Consortium
SERDP Strategic Environmental Research and Development Program
SOP Standard Operating Procedure
TIM TNO Gastrointestinal Model
TRV Toxicity Reference Value
UEF Urinary Excretion Fraction
UK United Kingdom
US EPA United States Environmental Protection Agency
XANES X-Ray Absorption Near-Edge Spectroscopy
XRD X-Ray Diffraction
Zn Zinc
Introduction
Oral ingestion of soil and dust is a key pathway for human exposures to metal and metalloid
contaminants. It is widely recognized that the site-specific bioavailability of metals in soil and
dust may be reduced relative to the metal bioavailability in media such as water and food, and
adjustments for oral relative bioavailability are becoming more accepted. Both animal models
and in vitro bioaccessibility models have been used to estimate relative bioavailability of metals
in soil and dust. Although animal models are often considered the "gold standard", they may be
costly or otherwise prohibitive at certain sites and may not be sensitive enough to test
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environmentally relevant samples for all contaminants. Routine application of in vitro metal
bioaccessibility models in regulatory settings is being held up by different perceptions of what is
required of these models in terms of validation.
This symposium provided the opportunity for international experts to exchange their views on
methods for assessing relative bioavailability/bioaccessibility for application in risk assessments
at contaminated sites. The symposium speakers presented recent developments in animal
models, new in vitro models, the role of mineralogical analyses in assessing relative
bioavailability, and the application of physiologically based models as research tools. In
addition, two panel discussions addressed specific research questions and discussed future
research needs in this area. Recognizing the multi-disciplinary nature of exposure assessment,
this symposium included representation from many disciplines including risk assessment,
toxicology, environmental geochemistry, geology, soil, and analytical chemistry from the U.S.,
Europe, and Canada.
Proceedings
Introduction and Overview
K. Bradham; U.S. Environmental Protection Agency, Research Triangle Park, NC
M. Beringer; U.S. Environmental Protection Agency, Kansas City, KS.
A. Yeow; U.S. Environmental Protection Agency, OSRTI, Washington, DC
P. E. Rasmussen; Health Canada, Ottawa, ON, CANADA.
R. A. Schoof; Integral Consulting, Inc., Mercer Island, WA.
M. R. Cave; British Geological Survey, Nottingham, UNITED KINGDOM.
Karen Bradham provided definitions and background for the discussions. Oral
bioavailability of metals is important for Human Health Risk Assessment (HHRA) of metals and
decision-making. Absorption from the gastrointestinal tract (GI) depends on the metal,
chemical, and physical form, as well as biological factors. Karen acknowledged the many
definitions of bioavailability, but she asked attendees to consider the definition in the Guidance
for Evaluating the Oral Bioavailability of Metals in Soils for Use in Human Health Risk
Assessment (OSWER 9285.7-80): "the fraction of an ingested dose that crosses the
gastrointestinal epithelium and becomes available for distribution to internal target tissues and
organs" Absolute bioavailability (ABA) and relative bioavailability (RBA) are also important
definitions. ABA: ratio of the amount of metal absorbed compared to the amount ingested.
RBA: the ratio of the bioavailability of a metal in one exposure context (i.e., physical chemical
matrix or physical chemical form of the metal) to that in another exposure context.
Bioaccessibility is a related term, typically referring to a measure of the physiological solubility
of the metal at the portal of entry into the body. Bioavailability information can be used to
improve the accuracy of risk calculations and inform decisions at hazardous waste sites. Karen
provided an overview of the three forms of studies: in vivo, in vitro, and mineralogical/speciation
studies. Highlights from the 2006 ISEA symposium: general agreement that criteria are needed
to assess in vivo and in vitro methods; some countries are allowing limited site-specific
adjustments; and few are comfortable with allowing existing site-specific methods to be
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universally applied. Karen reviewed the panel discussion questions for the audience, which were
disseminated to the panelists prior to the symposium.
Q&A: No time for questions
Evaluating the Unavailability of Soil-Borne Contaminants at Waste Sites
M. Beringer; U.S. Environmental Protection Agency, Kansas City, KS.
A. Yeow; U.S. Environmental Protection Agency, OSRTI, Washington, DC.
Mike Beringer discussed EPA's new guidance on using bioavailability studies to gather
site-specific information at Superfund sites. EPA developed this guidance because there was a
need for a consistent basis for approaching sites and evaluating new bioavailability methods.
The bioavailability guidance is limited in scope to oral ingestion of metals at hazardous waste
sites for HHRA. The assessment of bioavailability is consistent with existing EPA guidance.
The new guidance provides a decision framework for collecting site-specific bioavailability
information to make quantitative adjustments. The guidance also recommends using a validated
methodology and provides recommended evaluation criteria for the development of new
methods. The recommended evaluation criteria are based on the Interagency Coordinating
Committee for Validation of Alternative Methods (ICCVAM) criteria for method validation and
regulatory acceptance. The ICCVAM criteria are widely accepted internationally. Mike noted
that the evaluation criteria are not all critical to the validation or acceptance of a methodology.
Mike highlighted the importance of establishing a correlation between an in vitro method and an
in vivo method. Mike provided an overview of the methods for evaluating lead bioavailability
and bioaccessibility. The juvenile swine model and the in vitro bioaccessibility method for lead
have been evaluated and accepted for site-specific HHRA by EPA using the ICCVAM criteria.
These methods are described in a companion document to the guidance, referred to as the Lead
Technical Support Document. Mike noted that the evaluation covered a broad range of metal
forms and a range of bioavailability values. The documentation also outlines some limitations of
the accepted methodology. Mike listed some future activities to include support for
implementation of the guidance and evaluation of arsenic methodologies.
Q&A
What was the in vitro method?
o Mike responded that the in vitro assay published by John Drexler was
evaluated (Drexler and Brattin, 2007).
For the in vivo bioavailability: in vitro bioaccessibility correlation, how did you
derive the confidence interval?
o Mike responded that this was a prediction interval, not a confidence interval.
Do you recommend using the prediction interval in the RA?
o Mike responded that the EPA recommendation suggests that one should use
the best-fit line.
What is the mechanism for using new methods that are similar to the existing
method? Do we need to show equivalence or go through the entire process?
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Mike responded that the method described in Drexler and Brattin (2007) is
recommended because it has satisfied the ICCVAM criteria and suggested that
we discuss the process for similar methods later in the symposium.
Method Development and the Application of Oral Unavailability Data in U.S. Risk
Assessments
R. A. Schoof; Integral Consulting, Inc., Mercer Island, WA.
Rosalind Schoof posed several (5) questions related to EPA's new guidance on bioavailability.
1. How is validation defined?
2. What are validation requirements?
3. What are regulatory acceptance requirements?
4. What is the process for method development and validation?
5. What methods are already considered validated based on history of use?
Rosalind expressed concerns about how strictly the validation process will be defined.
Validation requires an understanding of relevance and reliability. Rosalind noted that the
ICCVAM criteria were developed for evaluating the alternative toxicity models, not for
bioavailability. These may or may not necessarily be applicable to bioavailability methods.
Rosalind provided an overview of the process leading from research to acceptance and
implementation. Validation occurs when its performance characteristics, advantages, and
limitations have been adequately documented for a specific purpose. Rosalind identified three
types of tests: definitive, screening, and adjunct. Are bioavailability test methods adjunct?
Rosalind offered that some test methods might be considered validated based on history of use
(e.g., in vivo pharmacokinetic studies of bioavailability). Rosalind noted that validated studies
still require scientific interpretation and oversight. GI physiology differs among species, so
animal models are predictive, but not perfect. Rosalind expressed concerns related to her
perception that EPA is going to require validation for all metals. A number of other metals that
are of concern in soil, are not typically found at high enough concentration in soil to allow for
animal study and may therefore not be evaluated using the EPA process (cost of method
validation is too high). Rosalind ended with four questions:
1. How reliable are the in vivo methods?
2. Are the in vivo methods considered validated?
3. Should validation of in vitro protocols be required on a metal-specific basis?
4. Should results of methods that have not been validated be considered in risk
assessment? If not then they may not be developed using site money. If so how?
Q&A
Related to in vivo testing of other metals, I agree that concentrations are too low to
establish a correlation. Can you comment on this limitation?
o Rosalind agreed that this would be difficult for metals other than lead and
arsenic.
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The concept of saying that concentrations are too low is not necessarily true. We can
develop methods to assess low concentrations. The document EPA developed is not
universal to all metals. We need to examine relevant samples.
o Rosalind agreed and responded that the concern is high background in diet
and other sources to distinguish additional exposure from site soil to detect
site exposure.
Arsenic Bioaccessibility Testing Using Various Extraction Methods: Results and
Relation to Relative Oral Bioavailability as Measured in the Cynomolgus Monkey
Y. W. Lowney; Exponent, Boulder, CO.
S. Roberts; University of Florida, Gainesville, FL.
S. Saikat; UK Environment Agency, Oxfordshire, UNITED KINGDOM.
Yvette Lowney discussed the in vivo bioavailability method using the Cynomolgus
monkey model and in vitro methods using a variety of methods. The Cynomolgus monkey
model was used because the oral bioavailability of the monkeys tends to correlate well with
humans. Excretion of As into urine is relatively rapid—within 48 hours nearly all of the As has
been excreted. The Cynomolgus monkey studies used a low arsenic diet: Soil dose <1 g/kg and
As dose <1 mg/kg. Five animals were used to develop an RBA estimate. Urinary and fecal
recoveries were very good. Controls (high and low RBA) were used. Sample RBA ranged from
5% to 31%. No RBA was found to be higher than 32%. Soluble NaAs was used and found to
have an RBA of nearly 100%. The study (published by Roberts et al., 2007) used a variety of
soil types. In vitro model development began with the Solubility/Bioavailability Research
Consortium (SBRC) method, with some alterations and consideration of The National Institute
for Public Health and the Environment (RIVM) and Bioavailability Research Group of Europe
(BARGE) methods. The SBRC method had good correlation for some samples, but not all (even
when different pH levels were used). It could not be determined why all soils did not have good
correlation. Phosphate additions improved the correlation, but did not work for all soil samples.
Other alterations to the method were made, but even with hydroxylamine additions correlation
was not good enough. When the RIVM and unified BARGE methods were used, none of these
methods worked (correlated) well for all soil samples. There is a need to develop a method that
is predictive for all soils or to understand which soils will work with the method. Available
methods do not provide a 1:1 correlation, but may be predictive. Yvette noted that the soil
samples have been extensively studied: mineralogy, speciation, etc. Mathematic modeling may
not necessarily work; however, the factors that control arsenic bioavailability can be stated.
Research status: data suggests RBA <30% from in vivo bioavailability testing, need more work
for the in vitro model.
Q&A
What is going on with the soils that affect the correlation? We need to define the
parameters for when soil samples will and will not work with the method.
o Yvette responded that she agrees; however, soil sample source (e.g., mine
tailings or orchard samples) and mineralogy cannot explain correlation.
Were the monkeys fasted?
o Yvette responded that the monkeys were fasted overnight.
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Can you give background as to why correlation is the standard as opposed to
predicting or over predicting in vitrol
o Yvette responded that she agrees that an in vitro assay either predicts or over
predicts (slope of the line is flatter than it should be). The objective is to have
the in vitro assay predictive of bioavailability.
Have you considered reabsorption of the arsenic? Should you consider a different
method, perhaps to add some resin beads to absorb the arsenic?
o Yvette agrees that reabsorption and precipitation could be a problem. Yvette
noted that the Hawaii volcanic soil sample had very low recovery.
Assessing Bioavailability Using the Swine Model
S. Casteel; University of Missouri, Columbia, MO.
G. Pent; University of Missouri, Columbia, MO.
C. Weis; US EPA, Denver, CO.
W. Brattin; Syracuse Research Corp, Denver, CO.
Stan Casteel gave an overview of the swine model (in vivo bioavailability) used for Pb,
As, Cd, and Cr. Juvenile swine were used as a surrogate for children. Naive animals are used,
so background is not typically a concern. The model allows multiple tissue endpoints to assess
exposure and absorption. The model is reproducible. Intravenous and oral routes can be used.
Most oral exposures are fed to the pig in a dough ball (moistened, powdered feed). Twice daily
fasted dosing is typically used for consecutive 12-14 days (subchronic exposure). Three (3)
levels of soil sample and three levels of reference standard are used (along with negative
controls). For arsenic, urinary arsenic (48 hours) approximates the oral absorption fraction
(ABA). After 5 days of exposure, arsenic excretion is a linear function of dose (independent of
time). RBA is the ratio of urinary excretion fraction (UEF) of test material to standard. In
general 80 ppm is lowest level in soil that they will use—because of the amount of soil that
needs to be used to achieve a measurable urinary arsenic concentration. With increasing
exposure dose, variability increases (heteroscedascity). Research has found RBAs of 26% to
72% for the test soils.
Q&A
Is it relevant to do an in vivo study when soil arsenic levels are as high as 4000 ppm?
o Stan said that they probably do not, but that is not his decision.
Can you explain the variability in the control group?
o Stan responded that there is some As in the low As feed, but that it is likely
due to biological variability.
Assessing Soil Arsenic Bioavailability in the Laboratory Mouse
D. Thomas; US EPA, Research Triangle Park, NC.
M. Hughes; US EPA, Research Triangle Park, NC.
K. Herbin-Davis; US EPA, Research Triangle Park, NC.
P. Seales; US EPA, Research Triangle Park, NC.
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Dave Thomas discussed the results of the pilot study in mice. Dave noted that this is a method
that is still in development. Goals are to develop a mouse model and to determine whether the
mouse is a good model for As absorption in humans. Mice were chosen because they have been
well studied, can be manipulated experimentally and a large body of As information is available
for this animal model. Dietary exposure was used. Total arsenic absorption (body burden and
excretion) was used. Dave noted that pharmacodynamic issues are significant for As
(methylated forms are more toxic); in the GI tract methylation and demethylation reactions do
occur, as well as thiol forms of As. Similarly in the tissues (post absorption) methylation and
thiolation occur. The method permits the evaluation of arsenic forms (inorganic As,
methylarsonic acid (MMA), dimethylarsenic (DMA)). Mice are fed purified diet which is non-
detect for As (cellulose in the diet is replaced with soil). The diet preparation method needs
further development. Eight (8) to 9 days of exposure are used (steady state). Food consumption
is not impacted by addition of soil to the diet. One limitation is stress involved with use of the
metabolism cage (mice lose weight). Future work will repeat existing research, examine other
soils, and refine methods of arsenic speciation, examine the relationship between soil source and
patterns of arsenic metabolism, and examine dietary factors that may impact absorption (dietary
fat, iron, and copper levels).
Q&A
Are you also interested in comparing the mouse model with other in vivo models?
o Dave replied that yes, for those soils that have been evaluated by others.
Why do they lose weight?
o Dave responded that this is likely the stress of the open bottom metabolism
cage.
Is a loss of 10% of body mass likely to impact the kinetics of absorption?
o Dave responded that we do not know and we would have to design a study to
evaluate that issue.
How many mice in each study?
o Dave responded there are three replicates per cage.
Panel Discussion Session I
Question for Stan: at 80 ppm As, how much soil is delivered to the pig?
Stan responded that is approximately 5-6 grams of soil per dose. Food impacts
absorption so this is a concern.
Question for Stan: are there different quantities of soil at different dose levels?
Yes.
Question for Stan: do the different quantities impact absorption?
No. However, at higher doses we have an increase in variability, but not a change in
absorption.
What is the cost per sample for various methods?
Yvette: monkeys are approximately $90-100k for three soils; in vitro assay for 10 soils is
approximately $10k.
Stan: swine are approximately $60k for two soils.
Dave: mice are probably several thousand dollars per sample. We have not looked at this
yet.
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Is the purpose of in vivo methods to calibrate the in vitro tests or to use data for a site?
Stan: no. the in vitro assay plays a role, as well as soil sample characterization
(mineralogy).
Aaron: EPA wants something cost effective and quick. We are looking to use in vivo
models for correlation/calibration to have less reliance on animal models.
Yvette: although in vitro methods will allow us to generate data, there are sites where
animal studies are warranted.
Which animal model is best? Primate vs. swine vs. mouse vs. human? Which is best for
developing a correlation?
Aaron: we have accepted the in vitro model for lead based on in vivo bioavailability. For
As, we have not yet accepted an in vivo bioavailability model. Until we compare the
animal models we cannot identify an animal model.
Mike added that juvenile swine were used for lead to mimic the child for lead. Monkeys
may be good models for adult exposure and for As because As is a carcinogen.
Rosalind agreed. Arsenic absorption may be more applicable to adults.
Question for the panel: How can we determine the better model swine or primates?
Stan replied that the dosing methods are not the same. We are currently evaluating a
single soil for comparison. The age issue is pertinent to the discussion. Another
consideration is using a naive animal vs. using an animal that was exposed over and
over to the same metal.
Yvette noted that we have not yet cross-dosed different species with the same test soil.
That work needs to be done. We need to do this work and discuss the benefits of the
various models.
General agreement that this would be helpful.
Kirk Scheckel suggested that this would be a huge investment and an opportunity for
EPA to attack the problem. European researchers and regulators agree that this
would be helpful.
Mark Cave: what is the best way forward to work together to achieve this goal?
Mike agreed that the available data should be pulled together to identify what needs to be
done to fill in the data gaps.
Suggestion to plan the next phase: get real soil, characterize the soil, and compare the
in vivo bioavailability and in vitro results back to the epidemiological data.
Responses noted that the epidemiological data are confounded by background
exposures to arsenic exposures in the diet. This complicates the biomarker
analysis. The UK Environment Agency is working on this issue and the BARGE
group is also considering this.
Question for the Panel: Can the rat data and swine data be used for arsenic? A lot of research has
been done with swine. Yvette asked for what chemicals is there a preponderance of data.
Stan responded that he has dosed between 30-50 arsenic soil samples.
Rosalind suggested that we should not leave other animal models behind until we have a
correlation between juvenile swine and the adult human.
It was also noted that Toxicity Reference Values (TRY) are from rodents—that should be
a consideration for model selection.
Marc Stifelman noted that recoverability and mass balance were good for non-human primates,
less so with swine (older Region 10 studies).
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Stan replied that the urinary excretion fraction is not affected by total uptake. His
laboratory does not collect mass balance data, because they no longer have these
difficulties.
Dave Thomas noted that recovery in the mouse model is 80-85% (urinary and fecal, not
considering tissue retention, which is expected to account for the other 15%).
Stan noted that this information is necessary for ABA, not for KB A.
Mark Maddaloni noted that discrepancies in mass balance might be a cause for concern
because it is these tissue levels that are having the ultimate toxic effect.
Rosalind agreed that pharmacokinetic considerations need to be evaluated in developing
an animal model.
Stan agreed and noted that differences in distribution are easily observed with different
routes of exposure.
Yvette agreed and stated we need to be careful in selecting a single animal model because
our data set is based on a small subset of metals.
Questions for Panel Discussion Session I (evaluation/site application and in vivo research):
1 - For regulatory applications such as site clean-up decisions, is an in vitro model that is
predictive (i.e., correlates with an in vivo model) adequate even if we don't know why it is
predictive, or is it important to know why a model is predictive?
From the audience, a suggestion to not be closed minded about model acceptance without
complete understanding.
Kirk noted that complete understanding of the model may be the ultimate goal to help
evaluate long-term stability of contaminants at sites.
Rosalind would prefer not to need to understand mechanism as long as the model is
predictive.
Stan agrees. We do not need to know the answer—the utility of the in vitro method
depends upon consistency in correlation for soil types of interest. How much
confidence we have in adding a new soil type depends upon how well we understand
the factors controlling the bioavailability.
2 - Given that many animal models require use of soil concentrations exceeding those of
public health concern, what efforts are necessary, if any, to show that bioavailability at these
concentrations reflect oral absorption at lower environmental concentrations? In other
words, how important is it to evaluate whether bioavailability is concentration dependent?
Pat asked that if 80 ppm is the lower limit for arsenic studies in swine, what do we do for
soils between 20 ppm (Canadian soil level) and 80 ppm? It was agreed that this is a
concern for monkeys, too.
Nick offered that when you are below 100 ppm perhaps you are stuck with in vitro
assays. Nick noted that the soil type that doesn't work may be the most informative.
Why doesn't it work? That is the important piece of information.
Kirk suggested that rather than arsenic, can we look at a tracer.
Mark Maddaloni responded that we have to identify a tracer that we know is very similar
to arsenic.
3 - Should a single animal model be specified, or are studies in a variety of different animal
models more likely to improve our understanding of relative bioavailability in humans.
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No. Agreement to retain various animal models for further evaluation.
4 - Is there a single animal model that will work for all chemicals considering the widely
varying toxicokinetic profiles?
Not at this time.
In-vitro Bioaccessibility of Soil-borne Contaminants: An Environment Agency Perspective
S. Saikat; UK Environment Agency, Oxfordshire, UNITED KINGDOM.
Sohel Saikat gave a brief overview of contamination in the United Kingdom. The UK
must use in vitro methods because of ethical constraints. Five in vitro methods that have been
used in the UK were evaluated using an inter-laboratory approach (metals tested: As, Pb, and
Ni). Labs that used the same methods and same SOP produced consistent results; however,
different methods did not produce comparable results. They also investigated the utility of in
vitro methods for in vivo bioavailability for As (the Cynomolgus monkey was used). In general,
the results showed that the five in vitro methods evaluated are not adequate for these arsenic soils
(i.e., poor correlation between in vivo and in vitro). Sohel also reported that of the three methods
tested, none was adequate for all metals (As, Pb, and Ni). Sohel noted that bioavailability and
bioaccessibility are considered the same for many UK researchers. Sohel suggested that more
work is needed to develop the in vitro assay including appropriate validation with an in vivo
bioavailability model. Sohel suggested furthering the knowledge of geochemical
characterization (method screening, geochemical matching, geochemical classification, and
biomarkers) to further the in vitro method. Sohel suggested focused discussion on data gaps,
harmonization of approaches among international regulators, and sharing of data among the
research community.
Q&A
- What is a reasonable correlation to move forward?
o We don't know. It was also noted that the correlation will change (as will
p-value) when more data become available.
Mark Maddaloni noted that the important area is when the bioavailability is low.
Sohel agreed.
Measurement of Metal Bioaccessibility in Urban Household Dust and Corresponding
Garden Soils
P. E. Rasmussen; Health Canada, Ottawa, ON, CANADA.
Patricia Rasmussen noted high variability of bioaccessible metals in house dust and the many
geochemical differences between outdoor soil and indoor dust. Patricia noted that sample
preparation (size fraction) and analytical procedures must be consistent for comparisons of
outdoor soil and indoor dust. She observed that tracking-in models do not work in the city of
Ottawa (indoor dust metal concentrations are not predicted accurately using outdoor soil metal
concentrations). Patricia cautioned that dust may differ from outdoor soil (e.g., some metals are
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found at higher concentrations and with higher bioaccessibilities; dust also has higher
concentrations of organic carbon). Patricia reported data for Zn, Ni, Cu, inorganic carbon,
organic carbon and a variety of other metals in dust and corresponding garden soil, and noted
that the higher organic content of indoor dust is associated with greater variability in Ni and Zn.
Patricia reported that for some metals (e.g. copper) bioaccessibility is affected by particle size,
but noted that speciation can override the influence of particle size (likely due to different
species of the metal in the various size fractions). She indicated that although smallest size
fractions yield important information, she finds it necessary to sieve house dust to larger
fractions (e.g. 80 microns or 150 microns) due to the small amount of dust that is typically
obtained when sampling inside homes. Patricia noted that in her pilot study, the High Volume
Small Surface Sampler (HVS3) vacuum sampler was used (from Rasmussen et al., 2008 [HERA,
in press]). She provided an overview of the sources of variability in bioaccessibility (variability
in both the numerator and the denominator of the % bioaccessibility equation). Variability in the
numerator (bioaccessible metal) may result from differences in mass to volume ratio, pH, other
constituents in the sample such as buffers, complexing agents, means of physical mixing and
means of separation (filtration vs. centrifugation). Variability in the denominator (total metal
concentration) may result from differences in recovery from using alternative methods to
determine total metal concentrations (EPA 3051 vs. HF total digestion methods). Patricia
suggested qualitative statements (low [<20%], medium [20-59%], and high [>60%]) could be
used to categorize the bioaccessibility of metals for outdoor soil and indoor dust, in light of the
many sources of large variability. Patricia suggested that indoor-outdoor ratios need to be
determined to understand indoor exposures. She offered that representative indoor dust data is
being collected across Canada (Canadian House Dust Study) to develop a national baseline
against which site-related measurements can be compared.
Q&A
- What is the German VDI method?
o Patricia reported that this is a whole house vacuum method.
Assessment of the Use of Dynamic Human Stomach Models for In-vitro Measurement of
the Bioaccessibility of Arsenics and Chromium in Soils - Can They Replace Animal
Testing?
M. R. Cave; British Geological Survey, Nottingham, UNITED KINGDOM.
Mark Cave reported on his research to develop dynamic in vitro human stomach models:
TEVI-1 and Model Gut. These models have been used for pharmacology research. The TEVI-l
system (TIM) includes a stomach phase and a small intestinal phase. TIM seeks to mimic human
physiological processes (e.g, temperature, pH changes, peristalsis, secretions, and absorption).
While TIM has been validated for glycemic response, there has been limited testing to date using
soil samples. Results for soil samples showed good correlation for a single As and Pb soil, but
not for Cd. TIM compared well with human subject data for fed/fasted ratio for Pb. The British
Geological Survey (BGS) is developing a standard reference material: 104 ppm As, and 79 ppm
Pb (BGS 102). Data suggest that the arsenic in this sample is of low bioaccessibility. A
chromium (Cr VI) soil sample (approx 3500-ppm total Cr; 1400 ppm Cr VI) was also evaluated.
Low bioaccessibility was reported for this sample using BARGE and TIM assays. The dynamic
in vitro models show promising results, however, these methods are not intended to replace batch
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methods (they may be used as reference methods, to characterize factors underlying differences
in absorption, and provide an alternative to animal models).
Q&A
How much soil is used for TIM and how much do they cost?
o 10 g of soil and a few thousand Euros.
- Does TIM include a mastication process?
o No, but pH changes over time.
Is redox state controlled?
o I am not sure, but I don't think so.
The Use of In Vitro Bioavailability Studies in Human Health Risk Assessment: Scientific
Research and Application by Policy Makers
A. G. Oomen; RIVM, Bilthoven, THE NETHERLANDS.
W. I. Hagens; RIVM, Bilthoven, THE NETHERLANDS.
J. P. A. Lijzen; RIVM, Bilthoven, THE NETHERLANDS.
E. B. P. Kessels; Actief Bodembeheer de Kempen, Eindhoven, THE NETHERLANDS.
A. J. A. M. Sips; RIVM, Bilthoven, THE NETHERLANDS
Agnes Oomen noted that for most risk assessments oral bioavailability is equal to the
bioavailability in the studies underlying the reference toxicity study (default RBA is 100%).
Agnes reported that this has changed recently for lead, where RBA for soil lead is 74% (based on
80 percentile value). The intervention value (similar to Preliminary Remediation Goals) in the
Netherlands is 530 ppm. She stated the default RBA for lead may be adjusted with reliable site-
specific data (including in vitro data). Agnes discussed an area of the Netherlands contaminated
by an historical Zn smelter. Contaminated slags and soils from the area were evaluated for
bioavailability of Pb and As. RBA information was not applied because for lead it didn't have a
major consequence for the site; whereas for As the soil did not pose a human health risk, but an
ecological risk.
Q&A
Might the soils be impacted by lead-based paint, since they are so high?
o Agnes responded no.
Assessing Contaminant Bioavailability in Soil when In Vitro Gastrointestinal Methods are
the Only Option
N. T. Basta, Ohio State University, Columbus, OH.
K. G. Scheckel, U. S. Environmental Protection Agency, Cincinnati, OH.
K. D. Bradham; U.S. Environmental Protection Agency, Research Triangle Park, NC.
Nick Basta discussed the utility of in vitro methods when they are the only option. Nick
reported that the majority of samples to date have been from highly contaminated soils (often
>2000 ppm Pb; >1500 ppm As). Nick noted that at highly contaminated soils adjustment of a
cleanup level is likely to be influenced by RBA. Development of methods that can be used for
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moderately contaminated soils (near 400 ppm) may have greater utility for more sites. Nick
noted that most in vivo studies require highly contaminated soils (limit of detection issues). In
vitro methods can be used at moderately contaminates sites, even down to near background
levels. He questioned whether we are confident of in vitro methods at low levels. Nick
suggested that he has greater confidence if it has been validated at high levels for the same
contaminant. If the method will be used for a different contaminant, he is hesitant. Also, the
source of the arsenic (or metal of interest) and factors that control the bioavailability of the
sample (e.g., weathering) need to be better understood to use the method for other samples and
concentration ranges. Nick believes that different extraction methods can be used, but first these
factors must be understood. Nick provided an overview of some historical data on arsenic and
collaboration with other researchers, including data on scorodite, iron oxide, and phosphate.
When only in vitro methods can be used, As speciation can be used to identify the form of
arsenic in the sample and to determine whether the method has been calibrated for those species.
Nick suggested that more work is needed in speciation and in vivo bioavailability to in vitro
bioaccessibility correlation and round robin studies. Nick added that collaboration maximizes
resources and saves time. Thus, there is a need to share characterized soils and share data.
Q&A
- Which of the speciation methods is essential?
o Nick responded that advanced spectroscopy will be discussed later. You need
to understand the soil type and weathering of the contaminant to select a
method.
The Bioaccessibility of Nickel in Contaminated Soils, Can It Be Explained Using Solid
Phase Distribution Data?
J. Wragg, British Geological Survey, Nottingham, UNITED KINGDOM.
M. Cave, British Geological Survey, Nottingham, UNITED KINGDOM.
C. Ollson, Jacques Whitford Ltd., Ottawa, ON, CANADA.
K. J. Reimer, Royal Military College of Canada, Kingston, ON, CANADA.
Joanna Wragg discussed Ni bioaccessibility and factors that are known to impact Ni
bioaccessibility. Joanna reported on data showing differences in geogenically-influenced soils
and anthropogenically influenced soils. Geogenic soils tend to have less organic carbon and
lower levels of Ni. Joanna compared Physiologically Based Extraction Technique (Ruby) and In
Vitro Gastrointestinal (Basta) assays for soil samples that were characterized by chemical
speciation. For anthropogenically influenced soils, the two methods compared well: no
statistically significant difference in Ni bioaccessibility. Speciation identified 9 distinct soil
components. PBET and IVG likely measured a mixture of several of these 9 contaminants. For
geogenic-influenced soils, Joanna did not see a significant difference between stomach and
intestinal phase of the PBET and IVG assays. She used cluster analysis to understand the
speciation data for the anthropogenic soils. In general, anthropogenic soils were more
bioaccessible. Joanna reported that aging of the geogenic sources impacted the bioaccessibility
of iron complexes. Joanna believes that geochemical data can support the use of bioaccessibility
data for risk assessment.
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Q&A
- What pH was used for anthropogenic and glycine extractions?
o Joanna responded glycine was used and pH of 1.8.
Importance of Metal Speciation in Understanding Bioavailability
Kirk G. Scheckel; U.S. Environmental Protection Agency, Cincinnati, OH.
Kirk Scheckel noted that speciation can support remediation and bioavailability. Kirk
differentiated between the issues: what do we want to know and what do we need to know. Both
chemical and physiology factors influence bioavailability. Kirk noted that research time at the
synchrotron facility is free and the only cost is travel associated with getting to the facility. The
synchrotron allows for characterization on the atomic scale. For example x-ray absorption near-
edge spectroscopy (XANES) can measure down to 10 ppm. Kirk discussed a case study using
phosphate amended soil speciation at Joplin, MO. Changes in speciation were observed with
different soil treatments. Kirk reported on data showing alteration in bioavailability as a function
of time after phosphate treatment where there was a reduction in bioavailability as the time since
treatment increased. He noted that requirements are an appropriate measure (methodology and
samples) as well as knowledge of the reason for the observed measurement (outliers).
Q&A
Concerning the figure with the in vivo comparison, was what was shown the KB A or
the ABA?
o In vitro was RBA others were ABA.
pH has ranged from 1.5 to 2.5. What should be used?
o 2.5 was best correlation between PBET and in vivo (closer to 1:1 slope).
Is 250-micron size fraction only correct size fraction?
o Kirk replied that we need to harmonize our size fraction to allow
comparability among assays.
Direct Identification of Metal Compounds in Contaminated Soil Mine Tailings and House
Dust Using Synchrotron-based Methods
Heather E. Jamieson, Queen's University, Kingston, ON, CANADA.
S. R. Walker, Queen's University, Kingston, ON, CANADA.
S. E. Fawcett, Queen's University, Kingston, ON, CANADA.
A. Lanzirotti, University of Chicago, Chicago, IL.
P.E. Rasmussen, Health Canada, Ottawa, ON, CANADA.
S. Beauchemin, Natural Resources Canada, Ottawa, ON, CANADA.
M. Parsons, Geological Survey of Canada, Halifax, NS, CANADA.
Heather Jamieson noted that bioavailability is a function of mineral, grain size, and
encapsulation. She suggests researchers spend several hours characterizing soils under the
microscope for every hour of synchrotron X-ray beam time. Heather discussed two cases for As.
The first case was from the Giant mine (an abandoned gold mine in Yellowknife near Slave Lake
in Canada), while the second case was a series of sites such as Goldenville and Montague
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(abandoned gold mines in Nova Scotia). The Nova Scotia sites are used recreationally today.
They are working on paired speciation and bioaccessibility assays for the samples. Heather
noted that many As-bearing secondary minerals are nanocrystalline (tens of nanometers), but
porosity, cementation, and disaggregation can impact grain size. Heather also discussed the
Ottawa house dust samples that Pat Rasmussen discussed earlier. Heather believes that
synchrotron methods combined with classical mineralogical studies can provide valuable
characterization information on the metal form in the sample.
Q&A
- No time for questions.
Bioaccessibility of Arsenic Adsorbed onto or Incorporated within Freshly Synthesized Iron
Oxide Minerals Using the Simulator of the Human Intestinal Microbial Ecosystem
(SHIME)
Brian D. Laird, University of Saskatchewan, Saskatoon, SK, CANADA.
T. Van De Wiele, University of Ghent, Ghent, BELGIUM.
D. Peak, University of Saskatchewan, Saskatoon, SK, CANADA.
W. Verstraete, University of Ghent, Ghent, BELGIUM.
S. D. Siciliano, University of Saskatchewan, Saskatoon, SK, CANADA.
Brian Laird suggested that understanding the inverse relationship between
bioaccessibility and concentration is a data gap. Does it result from a thermodynamic limitation
(liquid:solid ratio) or a kinetic limitation (residence time)? Complex mineralogy in mine tailings
makes developing a relationship based on mineralogy challenging. Another hypothesis
considered was the impact of colon microbes on As bioavailability. Does microbial activity in
the colon also impact other metals? Brian found that for scorodite, stomach phase
bioaccessibility was determined by the liquid:solid ratio. By contrast, in the small intestine
scorodite bioaccessibility was determined by residence time. Isotherm analysis requires that the
sample mineralogy is identical. Other research has shown that scorodite bioaccessibility was
reduced in the sterile colon as compared to the microbially active colon. The opposite was seen
with ferrihydrite-As(V). This effect was greater at higher concentrations of ferrihydrite-As(V).
The effect of GI microbial activity may pose a challenge for validation of in vitro models with
in vivo results. Brian acknowledged that the toxicological implications of the microbial activity
are unknown.
Q&A
- Does physical mixing affect bioaccessibility?
o Brian: Yes. These are kinetic constraints. Contact with solution is important.
These models are operationally defined.
Have you captured the dynamics of the GI tract?
o Brian: No. We have not. The kinetics of the absorption (pulling arsenic out
of the solution) is important, but how do you establish what the rate of
absorption is?
Do we know whether arsenic is absorbed in the colon?
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o Brian replied yes. Some data are available concerning rate of arsenic
absorption in various stages of the GI tract. For example, Brian found only
one study of absorption throughout the GI tract (a study from 50 years ago in
Japanese).
Do the animal models we use have microbial flora in the upper GI?
o Brian responded: yes. Some species have greater and other species have
lesser gut microflora. Brian noted that cecum of the mouse is relatively large,
so the microbial alteration may be more important to understand in this
species.
Panel Discussion Session II
1 - Is it sufficient for in vitro models to have correlation with animal results or is it necessary to
make the models accurate physiologic mimics of human gut dissolution?
Mark Maddaloni stated that good correlation across multiple soil types is the objective.
Research to develop a physiological model is not necessary (assuming we have validation);
however, we would like to have detailed information on physiological mechanisms. Nick
Basta agrees.
2 - Is it acceptable to use the terms "bioaccessible", "soluble", "migrateable", and "extractable"
interchangeably in the numerator of the % bioaccessibility equation?
In general the terminology has been consistent for the presentations today. Some differences
in sample preparation (e.g., pH of digestion and sieving) have come out today. We
should work to make these consistent.
Agnes noted that separation method (dialysis vs. centrifugation) also has a large influence on
results. This, too, should be standardized.
Nick noted that the methods with more colloidal material may be influenced by separation
method to a greater degree than other simpler methods.
Brian noted that we need to consider the role of concentration on kinetics of dissolution.
Kirk noted that we are not trying to establish equilibrium in the in vitro tests.
Yvette noted that for other metals (like barium) we may be reaching the solubility limitation.
Marc Stifelman noted that for risk assessment purposes we can probably establish a
reasonable upper bound for liquid:solid ratio. Others agreed that would be a start.
3 - Should bioaccessibility values be used on their own without supporting information? If not,
what additional information should be included for each soil sample analyzed (e.g.,
geochemistry)?
Rosalind believes that supporting information is important for better understanding the site.
Patricia asked for clarification of the question "be used for what?"
Are we using the bioaccessibility information to make site decisions or for comparing
sources? Bioaccessibility is operationally defined.
Beverly Hale agreed. We should collect as much information as possible.
Rosalind followed up that for many of the studies we need to understand the limitations and
how we can augment the information.
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Kirk noted that electron microprobe studies tell you about chemical composition, it does not
provide speciation. The results can be misleading for As. XRD and microscopy can give
you some very good information also.
Mark noted that the bioaccessibility and extraction methods are best used in combination.
Nick added that weathering is an important factor. The speciation of soil samples is
important and these methods should be used.
What can be done where we have limitations on methods for low concentration and in vivo
bioavailability methods?
Marc Stifelman noted that he agrees with testing unknowns in as many species as available.
We can probably feed monkeys low levels.
Yvette agrees—we should study soils where the metal concentration is of environmental
relevance (e.g., 50-100 ppm for As). Moving from there to the risk range is not a scary
extrapolation. Mark Maddaloni agrees.
Marc Stifelman noted that with the exception of Florida, most Superfund soil cleanup goals
range from 20-245 ppm As.
Nick suggested that if we can characterize the species that is controlling As solubility, then
the effect of solubility is likely to be the same at 30 ppm as it is at 3000 ppm.
Kirk and others raised concerns with this related to pH and other factors (as demonstrated
with scorodite research).
Particle size distribution issue: Rosalind noted that for oral studies many have sieved to 250-|im
particle size (based on adherence of soils to skin and incidental soil ingestion pathway). Recent
research on dust is looking at smaller particle size fraction (<150|im). There may or may not be
enrichment in the finer fraction. What is the appropriate particle size range cutoff?
Kirk offered that for consistency we should stick with one size fraction.
Rosalind agrees that harmonization is an important issue.
Yvette added that when they examined soils from SERDP, the majority of lead mass was in
the <75 |im size fraction.
Patricia noted that house dust has a different particle size distribution. A key issue is to use
the same procedure for indoor and outdoor dust. Patricia noted that it is physically
challenging to get down below 50 jam. Mark Richardson's paper (Health Canada)
encouraged the further characterization of size fractions (look at different cuts).
Agnes added that in the Netherlands they agree that smaller particles tend to stick to the
hand, but if even a few large particles adhere to the hand those can dramatically impact
the results. RIVM uses the <2 mm size fraction as a result.
Rosalind noted that the particle size fraction to keep in mind is the size fraction commonly
used for site characterization, <250 jim.
Patricia noted that the cost of sieving is relatively cheap (a few dollars for full
characterization of a sample). A more complete characterization may result in greater
consistency across sites. Patricia and Yvette agreed.
Yvette noted that the 3050 method found >100% bioaccessibility for barium (this is a known
limitation of the 3050 method). Agreement that consistency in digestion method is
important. Heather agrees that results from total digestion may be misleading.
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Summary
The EPA guidance also recommends using a validated methodology and provides
recommended evaluation criteria for the development of new methods.
Establishing a correlation between an in vitro method and an in vivo method was
generally considered an important step. A 1:1 correlation is not necessary, only that the
model is predictive for soil types and contaminant concentrations of interest.
Agreement that mimicking physiology is not necessary, nor is complete characterization
and understanding of processes, for an in vitro method to be accepted. What is important
is that the in vitro method is correlated with an in vivo model and that it is predictive for
soil types and contaminant concentrations of interest.
While not strictly required, insight into the factors underlying the absorption processes
for a given metal and complete characterization of the metal in the media of interest will
inform the limits of a test method for a given metal species (e.g., concentration range).
This information will also determine confidence in using the method for other forms of
the metal or soil phases.
Any candidate method for evaluation must be presented with bounds of valid use (e.g.,
metal forms) and methodological constraints (e.g., concentration range, pH, and
liquid:solid ratio).
Agreement that the next logical step is to develop an arsenic assay (in vivo and in vitro).
Cost is a factor for an in vitro assay. EPA is seeking in vitro methods that are cost
effective and relatively quick. The in vivo model against which it is correlated is not
necessarily similarly constrained.
At this time no decision has been made concerning which animal model is preferred for
metals other than soil borne lead from mining, milling, and smelting sites (for which EPA
has an SOP).
For selection of an in vivo model for As, comparisons among the available animal models
using the same soils are required to make this determination.
A single animal model may not work for all metals because of differences in
pharmacokinetics.
A more complete characterization of concentration and bioaccessibility for various
particle size fractions may result in greater consistency across sites.
There is a need for harmonization for digestion methods and sieving across sites.
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• A combination of laboratory, microscopic, and synchrotron methods may provide useful
information for bioaccessibility, bioavailability, and risk assessment.
• Agreement was made to identify, characterize, and share standard reference materials for
in vivo and in vitro assays.
• Agreement to share information and data, as well as soil samples, to identify data gaps
and research needs.
• Agreement to collect soil samples from a wide array of relevant sites, preferring
weathered soils over spiked soils (because they may differ).
• Agreement to meet again before the 2008 ISEA meeting. This will foster collaboration
and further discussion.
Although this work was reviewed by EPA and approved for publication, it may not necessarily
reflect official Agency policy.
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General References:
Basta, N., Foster, J., Dayton, E., Rodriguez, R., Casteel, S., The effect of dosing vehicle on
arsenic bioaccessibility in smelter-contaminated soils. Journal of Environmental Science and
Health, Part A 2007, 42, (9), 1275-1281.
Casteel, S., Evans, T., Turk, J., Basta, N., Weis, C., Henningsen, G., Hoffman, E., Refining the
risk assessment of metal-contaminated soils. InternationalJournal of Hygiene and
Environmental Health 2001, 203, (5-6), 473-474.
Cave, M., Taylor, H., Wragg, J., Estimation of the bioaccessible arsenic fraction in soils using
near infrared spectroscopy. Journal of Environmental Science and Health, Part A 2007, 42, (9),
1293- 1301.
Drexler, J., Brattin, W., An in vitro procedure for estimation of lead relative bioavailability: with
validation. Human and Ecological Risk Assessment 2007, 13, (2), 383 - 401.
EPA. 2007. Guidance for Evaluating the Oral Bioavailability of Metals in Soils for Use in
Human Health Risk Assessment. OSWER Report 9285.7-80.
EPA. 2007. Estimation of Relative Bioavailability of Lead in Soil and Soil-like Materials Using
in Vivo and in Vitro Methods. OSWER 9285.7-77.
ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods).
1997. Validation and Regulatory Acceptance of Toxicological Test Methods: A Report of the Ad
Hoc Coordinating Committee on the Validation of Alternative Methods. NIH Publication
973981. National Institute of Environmental Health Sciences, Research Triangle Park, N.C.
Kelly, M., Brauning, S., Schoof, R., Ruby, M., Assessing Oral Bioavailability of Metals in
Soil, A 1 &A2, 2002 pp. 75 -88, Battelle Press Columbus, OH.
Laird, B., Van De Wiele, T., Corriveau, M., Jamieson, H., Siciliano, S., Evaluation of the
bioaccessibility of metals and metalloids in Eastern Canadian mine tailings using an in vitro
gastrointestinal model, the simulator of the human intestinal microbial ecology (SHIME).
Epidemiology 2006, 17, (6), S40-S40.
Oomen, A., Hack, A., Minekus, M., Zeijdner, E^Cornelis, C., Schoeters, G., Verstraete, W.,
Van de Wiele, T., Wragg, J., Rompelberg, C., Sips, A., Van Wijnen, J., Comparison of five in
vitro digestion models to study the bioaccessibility of soil contaminants. Environ. Sci. Technol.
2002, 36, (15), 3326-3334.
Rodriguez, R., Basta, N., Casteel, S., Pace, L., An in vitro gastrointestinal method to estimate
bioavailable arsenic in contaminated soils and solid media. Environ. Sci. Technol. 1999, 33, (4),
642 -649.
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Roberts S., Munson J., Lowney Y., Ruby M. Relative Oral Bioavailability of Arsenic from
Contaminated Soils Measured in the Cynomolgus Monkey. Toxicological Sciences 2007
95(l):281-288
Ruby, M., Davis, A., Schoof, R., Eberle, S., Sellstone, C., Estimation of lead and arsenic
bioavailability using a physiologically based extraction test. Environ. Sci. Technol. 1996,30,
(2), 422-430.
Saikat, S., Barnes, B., Westwood, D., A review of laboratory results for bioaccessibility values
of arsenic, lead and nickel in contaminated UK soils. Journal of Environmental Science and
Health, Part A 2007, 42, (9), 1213-1221.
Scheckel, K., Ryan, J., Allen, D., Lescano, N., Determining speciation of Pb in phosphate-
amended soils: Method limitations. Sci. Total Environ. 2005, 350, (1-3), 261-272.
Wragg, J., Cave, M., Nathanail, P., A Study of the relationship between arsenic bioaccessibility
and its solid-phase distribution in soils from Wellingborough, UK. Journal of Environmental
Science and Health Part A-Toxic/Hazardous Substances & Environmental Engineering 2007, 42,
(9), 1303-1315.
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Appendix A: Questions for the Panel (submitted in advance)
Questions for Panel Discussion Session I (evaluation/site application and in vivo research):
1 - For regulatory applications such as site clean up decisions, is an in vitro model that is
predictive (i.e., correlates with an in vivo model) adequate even if we don't know why it is
predictive, or is it important to know why a model is predictive?
2 - Given that many animal models require use of soil concentrations exceeding those of
public health concern, what efforts are necessary, if any, to show that bioavailability at these
concentrations reflect oral absorption at lower environmental concentrations? In other
words, how important is it to evaluate whether bioavailability is concentration dependent?
3 - Should a single animal model be specified, or are studies in a variety of different animal
models more likely to improve our understanding of relative bioavailability in humans.
4 - Is there a single animal model that will work for all chemicals considering the widely
varying toxicokinetic profiles?
5 - Is relative oral bioavailability of chemicals in soil so routine that it is "ready" for
guidance/regulation that will inherently limit science-based research?
Questions for Panel Discussion Session II (in vitro and speciation/mineralogy research):
1 - Is it sufficient for in vitro models to have correlation with animal results or is it necessary
to make the models accurate physiologic mimics of human gut dissolution?
2 - Is it acceptable to use the terms "bioaccessible", "soluble", "migrateable", and
"extractable" interchangeably in the numerator of the % bioaccessibility equation?
3 - Should bioaccessibility values be used on their own without supporting information? If
not, what additional information should be included for each soil sample analyzed (e.g.,
geochemistry)?
4 - How can we leverage resources to answer specific research questions to advance the
understanding of bioavailability/bioaccessibility?
- Develop Standard Reference Materials or Certified Reference Materials?
- Round robin testing?
5 - In lieu of comparing in vitro model results to an in vivo model, what criteria should be
considered when evaluating whether a particular in vitro method is appropriate for providing
screening level data versus data to derive quantitative site-specific bioavailability
adjustments?
6 - What other metals, or metal species, are of interest for developing additional in vivo and
in vitro bioavailability assays? What criteria are used by regulatory agencies for ranking or
prioritizing contaminants?
7 - Is it feasible and cost effective to develop a single standardized in vitro method for each
metal for simulating oral bioaccessibility? If so, what steps would be needed for that to
happen? What criteria would be used to determine the difference between the methods?
- What is available (and in process) and how available/quantities (related to above) how
prioritized types of materials needed (ex: soils, tailings, etc.)
- Discussions of desired specifications by type (uses will drive Data Quality Objectives)
- Funding (ex: to National Institute of Standards and Technology for Standard Reference
Materials) vs. volunteer analysis via round robins (to develop consensus values) and how
determine "reliable" methods? International Atomic Energy Agency could be a model.
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Appendix B: Abstracts
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Abstract 580
Introduction and Overview
K. Bradham; U.S. Environmental Protection Agency, RTF, NC
In human health risk assessments, soil and dust ingestion can be a major route of
exposure to many soil contaminants, including metals and metalloids. Site-specific soil
physical and chemical characteristics, as well as internal biological factors, determine the
oral bioavailability of soil contaminants. Within a single sample, this contamination may
be from multiple sources of metals and may exist as different forms and species. Both
animal models and in vitro bioaccessibility models have been used to estimate relative
bioavailability of metals in soil and dust. The bioavailability estimates for soil have a
direct impact on current human health risk assessment and risk management practices.
This introduction and overview to the symposium will include definitions and
specification information necessary for setting the stage for the presentations on recent
developments in animal models, new in vitro models, the role of mineralogical analyses
in assessing relative bioavailability, and the application of physiologically-based models
as research tools. Information will also be presented regarding the panel discussions and
specific research questions provided to the panelists and presenters for discussion during
this symposium.
Although this work was reviewed by EPA and approved for publication, it may not
necessarily reflect official Agency policy.
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Abstract 520
Evaluating the Unavailability of Soil-Borne Contaminants at Waste Sites
M. Beringer1, A. Yeow2; *U.S. Environmental Protection Agency, Kansas City, KS,
2OSRTI, Washington, DC
Site-specific bioavailability is an important consideration in determining potential threats
to human health that are posed by metals-contaminated soils at waste sites. It is important
to consider bioavailability because metals may be absorbed to a lesser or greater extent
following ingestion of contaminated soils as compared to the fraction absorbed in the
studies used to establish toxicity values, such as a reference dose or a cancer slope factor.
U.S. EPA's Office of Superfund Remediation and Technology Innovation (OSRTI) has
led an effort to develop guidance on evaluating and incorporating bioavailability
adjustments into human health risk assessments. The guidance outlines a decision
framework for deciding when to collect and incorporate site-specific bioavailability
information; recommends a process for documenting the data collection, analysis, and
site-specific implementation of a validated method; as well as provides recommended
method validation and regulatory acceptance criteria for evaluating alternative
methodologies. U.S. EPA has used these criteria to evaluate two separate methodologies
for predicting the relative bioavailability of lead in soil and soil-like materials. The
Agency has determined that both an in vivo swine bioavailability bioassay and an in vitro
bioaccessibility assay have sufficiently satisfied these criteria. Thus, they are considered
regulatory methodologies appropriate for determining the relative bioavailability of lead
for quantitative use in site-specific risk assessments. This presentation will summarize the
bioavailability guidance document and the basis for the Agency's decision regarding the
two methodologies for predicting lead relative bioavailability.
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Abstract 514
Method Development and the Application of Oral Unavailability Data in U.S. Risk
Assessments
R. A. Schoof; Integral Consulting Inc., Mercer Island, WA
USEPA guidance includes provisions for site-specific adjustments in exposure estimates
to account for differing relative bioavailability of chemicals in soil and in the exposure
media of toxicity studies. The relative oral bioavailability of lead and arsenic in soil has
been assessed in a series of studies that have included both animal models and in vitro
test systems. USEPA recommends a default assumption that children will absorb only 60
percent as much lead from soil compared with absorption from water or diet. For arsenic,
no such default assumption has been generally accepted. In vivo bioavailability studies
should be designed to account for variations in metabolism and excretion of chemicals.
The absorption and disposition of lead and arsenic differ substantially. Consequently,
bioavailability studies to assess these two chemicals have been designed to reflect these
differences. In vivo methods used to assess the bioavailability of soil-borne chemicals are
typically modified versions of methods widely used in biomedical research. These
methods have been modified to address constraints associated with use of doses relevant
to environmental concentrations, the need to reflect weathering behavior in soils over
time, and the need to generate data applicable to human health risk assessments.
Regulatory acceptance of bioavailability data in a site-specific risk assessment is
generally dependent on the use of a validated test method or a careful scientific review of
the test method employed. In vivo bioavailability data generated by traditional study
designs is usually considered to be acceptable, but in vitro studies face a greater burden in
obtaining acceptance. In the US a process developed by the Interagency Coordinating
Committee on the Validation of Alternative Methods for validating newly developed
alternative toxicity methods provides relevant guidance for assessing in vitro methods.
Efforts to obtain regulatory acceptance of in vitro studies of relative bioavailability will
be reviewed.
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Abstract 461
Arsenic Bioaccessibility Testing Using Various Extraction Methods: Results and
Relation to Relative Oral Unavailability as Measured in the Cynomolgus Monkey
Y. W. Lowney1, S. Roberts2, S. Saikat3; Exponent, Boulder, CO, 2University of Florida,
Gainesville, FL, 3UK Environment Agency, Wallingford, UNITED KINGDOM
Recent research has established that the absorption of arsenic from soils following
ingestion exposures is lower than absorption of soluble arsenic from water. Because
regulatory toxicity values for arsenic are based on studies of human exposures to arsenic
in water, understanding the relative oral bioavailability (RBA) of arsenic from soils is
important for accurate assessment of exposure to arsenic associated with soil ingestion.
Because of the site-specific nature of the controls on arsenic bioavailability, site-specific
information is important in assessing potential risk associated with arsenic in soils. To
conduct an animal study of bioavailability for every site affected by arsenic would be
time- and cost-prohibitive, and may counter policies regarding the use of animals in
research. Therefore, recent efforts have focused on developing practical and economical
bench-top (in vitro) procedures to measure the fraction of contaminants in soils that,
following ingestion, would be available for absorption into systemic circulation.
This presentation will discuss recent in vivo testing of arsenic bioavailability in the
cynomolgus monkey, and the results of in vitro extraction tests that have been designed
to predict the in vivo bioavailability results. The in vitro methods include an extraction
protocol that has been validated as predictive of the RBA of lead from soil, other
extraction methods that have been reported in the literature, methodologies developed by
the Dutch RIVM, and the RIVM method as modified by BARGE. This presentation
provides the results of extraction testing of splits of several soils that were evaluated in
vivo, and discusses the differences in results between the extraction methods, and the
correlation between the results from extraction testing and RBA as measured in
cynomolgus monkeys. Results indicate that additional work remains to identify an
individual in vitro method that is able to predict the in vivo bioavailability satisfactorily
for all soils.
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Abstract 519
Assessing Unavailability Using the Swine Model
S. Casteel1, G. Pent1, C. Weis2, W. Brattin3; University of Missouri, Columbia, MO,
n o
Environmental Protection Agency, Region VIII, Denver, CO, Syracuse Research Corp,
Denver, CO
Bioavailability of site-specific environmental contaminants is critical to exposure
assessment. Determining the bioavailability of contaminants in a diverse range of soils,
allows scientifically derived data to dictate site-specific remedies to reduce the risk for
sensitive human populations. Based on a series of dosing trials in a juvenile swine model,
site-specific estimates of relative bioavailability of metals and organic compounds, is
highly variable and is matrix and chemical species dependent. Results for lead- arsenic-
and cadmium-contaminated soils support the view that soil metals are not always as well
absorbed as soluble forms; therefore use of default assumptions for assessing human
health risk may overestimate the hazard.
Since the selection of appropriate animal models enhances the science and reduces
uncertainty in human risk assessment it is critical to use the best available model with
reasonable constraints. Numerous rodent model studies are in the literature. Studies in
non-human primate (NHP) models have been fewer due to higher purchase and per diem
costs, housing availability, zoonotic concerns, and animal rights attention.
Criteria useful in selecting the appropriate animal model include behavior, age, size, ease
of bleeding, anatomical considerations and gastrointestinal physiology. Research
management factors such as historical database, costs, model availability, and animal
rights group interest in the model were also involved in selection.
The swine model has the versatility to assess the bioavailability of a wide variety of
materials, including metals, organic compounds, and biodistribution of gold-, palladium-
and silver-nanoparticles. When assessing site-specific contaminants, pigs are dosed 2
hours before each feeding twice daily for 14-to-15 consecutive days, at constant dosing
times. Multiple doses are provided as a more likely real-world reflection of exposure.
This approach has been successfully applied at numerous sites for estimation of relative
bioavailability for lead, arsenic, cadmium, vanadium, and chromium.
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Abstract 521
Assessing Soil Arsenic Unavailability in the Laboratory Mouse
D. Thomas, M. Hughes, K. Herbin-Davis, P. Seales; U.S. Environmental Protection
Agency, RTF, NC
Variation among soils in the bioavailability of arsenic can be a critical determinant of the
risk posed by exposure to these soils. Although in vitro techniques can provide vital data
on aspects of bioavailability of metals and metalloids from soils, these results must be
validated in an animal model. A useful animal model provides a measure of
bioavailability and allows comparison of bioavailability for different soil matrices. Inbred
strains of laboratory mice are potentially good models for development of a
bioavailability assay. Laboratory mice are well characterized physiologically and can be
manipulated experimentally (e.g., altered dietary components, altered genotype). There is
also a large body of data on the absorption, metabolism, disposition, and excretion of
inorganic and methylated arsenicals in the mouse which is germane to evaluating the
differences and similarities between mouse and human. Initial studies are comparing
arsenic bioavailabilities in soils with known arsenic contents with the bioavailability of
sodium arsenate. Here, soils (e.g. NIST SRM 2710) or sodium arsenate are added to a
standard powdered mouse chow (AIN-93G purified rodent diet) at the one percent
(weight/weight) level. Adult female C57BL/6 mice have had free access to this amended
chow and tap water for nine days. Urine and feces are collected on a daily basis and food
intake is monitored throughout this period. At the end of the nine-day exposure period,
mice are euthanized and tissues collected. Data on food consumption and arsenic contents
of excreta and selected tissues are used to calculate the bioavailability of arsenic in each
soil matrix. Development and refinement of this animal model should provide a
convenient and rapid means to assess the absolute and relative bioavailability of arsenic
in soils. These data may be of great value in risk assessment. (This abstract does not
reflect US EPA policy.)
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Abstract 518
In-vitro Bioaccessibility of Soil-borne Contaminants: An Environment Agency
Perspective
S. Saikat; Environment Agency, Oxfordshire, UNITED KINGDOM
In the UK, interest in the use ofin-vitro bioaccessibility data in risk assessment, has been
stemmed mainly from the problem associated with elevated arsenic in mineralised and
mining effected areas. There is an expectation that in-vitro bioaccessibility data can be a
'quick fit solution' to dealing with land contamination where contaminant level exceeds
corresponding generic assessment criteria (e.g. Soil Guideline Values).
Studies undertaken by the Environment Agency, however, indicate that a number of in-
vitro methods (e.g. physiologically based, semi-physiologically based and simple
chemical leaching) are currently available to measure in-vitro bioaccessibility but no
information to indicate that they are being validated with in-vivo data for UK soils. The
study also indicated that reproducibility of different in-vitro methods, operating
procedures and reporting of results could contribute to a large variation in in-vitro
bioaccessibility data. Laboratories use same in-vitro method irrespective of chemicals,
concentrations, mineralogy and soil types. Moreover, no reference material containing in-
vivo data is available to measure the accuracy ofin-vitro methods.
In order to appreciate and make the best of research progress achieved, a review of
outstanding issues is required to consolidate efforts and develop appropriate partnerships.
Scientists, risk assessors and regulators need to balance their expectations of the in-vitro
approach in terms of its capabilities and weaknesses in order to make it more useful in
risk assessment.
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Abstract 524
Measurement of Metal Bioaccessibility in Urban Household Dust and
Corresponding Garden Soils
P. E. Rasmussen; Health Canada, Ottawa, ON, CANADA
Large uncertainties are associated with the measurement of gastric bioaccessibility of
metals in household dust, caused in part by the heterogeneous nature of settled dust
samples, and in part by variations in analytical parameters. A modified version of
European Standard EN 71-3 Toy Safety Protocol was used as a rapid screening method
for estimating gastric bioaccessibility of metals in urban geochemical surveys of
household dust and corresponding garden soil samples in Ottawa, Canada. In this study,
gastric bioaccessibility is defined as the concentration of metal leached from the test
sample into 0.07 M HC1 (2 h at 37°C; pH 1.5), expressed as a percentage of the total
metal concentration. To improve measurements of the total metal concentration (the
denominator in the bioaccessibility equation) several modifications were made to the US-
EPA 3051 microwave digestion protocol. Increasing the microwave digestion time to 30
min ramp followed by 30 min hold (compared to 5.5 min total digestion time specified by
EPA3051) increased total metal recoveries by 15-20%. Increasing the acid volume to
sample mass ratio to 1000 (compared to ratios of 20 to 100 specified by EPA3051)
increased total metal recoveries by 30-60%. Similarly, for the simulated gastric extraction
(the numerator), increasing the acid volume to sample mass ratio to 2000 (compared to
ratios of 50 to 500 specified by the Toy Safety protocol) typically increased the extraction
efficiency by 20 to 50%. Analytical reproducibility is improved using smaller sieve
fractions (<60 micron is best); however, settled dust samples collected in this study were
typically very small (1-2 g) necessitating the use of a larger size fraction (<150 micron).
In light of the inherent variability associated with settled dust measurements, estimates of
gastric bioaccessibility are grouped into simple categories: low (19% and less), medium
(20 to 59%) and high (over 60%).
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Abstract 391
Assessment of the Use of Dynamic Human Stomach Models for In-vitro
Measurement of the Bioaccessibility of Arsenics and Chromium in Soils - Can They
Replace Animal Testing?
Mark Cave1, Helen Taylor1, Joanna Wragg1, Andrew Broadway21 British Geological
Survey, Key worth, Nottingham; University of Edinburgh, School of GeoSciences,
Edinburgh, UK
The development of methods for estimating the oral bioavailability of soil contaminants
may reduce costs of site remediation and soil cleaning, while still maintaining the
required protection level. Currently, simple batch in vitro extraction methods, which
broadly mimic the physico-chemical conditions in the human gastro intestinal tract, have
been developed as screening methods for bioaccessibility measurement. Regulatory
authorities, however, require that the in-vitro methods should produce data that is
demonstrated to be comparable to the in-vivo situation. It has been shown, however, that
the GI tract of young pigs is similar to humans and that they can be used to validate the
results of in-vitro tests. Animal studies are, however, time consuming, costly, have
ethical considerations and there are concerns regarding their relevance to the human. The
food and drug industry has worked to produce dynamic in-vitro systems specifically
designed to mimic the human gastrointestinal system (Wickham, 2007, Minekus, 1995 ).
Such systems may be as relevant as animal models for soil bioavailability studies and
have a part to play in either estimating bioaccessibility or in validating the simpler batch
tests. This paper will discuss the results obtained for the bioaccessibility of arsenic and
chromium from soils with both in-vivo bioavailability and batch in-vitro bioaccessibility
data.
Wickham, M. (2007): The Model Gut, 2007 (27April),
http://www.ifr.ac.uk/science/platform/MG/default.html.
Minekus, M, Marteau, P, Havenaar, R and Huisintveld, JHJ. (1995): "A
Multicompartmental Dynamic Computer-Controlled Model Simulating the Stomach and
Small-Intestine", Atla-Alternatives to Laboratory Animals, 23(2), 197-209.
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Abstract 517
The Use of In vitro Unavailability Studies in Human Health Risk Assessment:
Scientific Research and Application by Policy Makers
A. G. Oomen1, W. I. Hagens1, J. P. A. Lijzen1, E. B. P. Kessels2, A. J. A. M. Sips1;
National Institute for Public Health and the Environment, Bilthoven, THE
NETHERLANDS, 2Actief Bodembeheer de Kempen, Eindhoven, THE
NETHERLANDS
Today, a relative bioavailability factor of "1" is used for human health risk assessment of
contaminated soils. This implicates the assumption that there is no difference in the
bioavailability of a contaminant from soil compared to the bioavailability from the matrix
used in the studies underlying the Intervention Value for remediation, which is typically a
food or water matrix. However, there is ample evidence demonstrating that the
bioavailability of a contaminant from soil can be considerably lower than from food or
water. Integrating oral bioavailability of contaminants from soil in human health risk
assessment will increase the realistic outcome of risk assessment through soil ingestion.
The research in this presentation focuses on the contaminant lead, since lead is frequently
encountered at human lexicologically high concentrations in soil in the Netherlands.
Furthermore, soil ingestion is an important pathway of exposure for lead, especially for
children, leading to potential adverse effects. Therefore, the need for a realistic but still
protective risk assessment for human health is high.
The RIVM has developed a simple experimental tool, an in vitro digestion model, to
supply information on the bioavailability of a contaminant in the human body after
ingestion of contaminated soil. This model has been used to estimate the bioaccessibility
of lead in specific soils. With CSOIL, site specific risk assessment of human health can
be modeled to answer specific policy issues.
In this presentation, the experimental setup and outcome of such a project is given.
Furthermore, the implementation of the results and the scientific advice towards policy
makers is addressed. Actions taken by policy makers following the recommendations are
discussed.
Taken together, this presentation gives an overview on the involvement of the RIVM in
vitro digestion model in site specific risk assessment in the Netherlands.
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Abstract 516
Assessing Contaminant Unavailability in Soil when In Vitro Gastrointestinal
Methods are the Only Option
N. T. Basta1, K. G. Scheckel2, K. D. Bradham3; ^hio State University, Columbus, OH,
0 1
U. S. Environmental Protection Agency, Cincinnati, OH, U.S. Environmental
Protection Agency, Research Triangle Park, NC
Incidental soil ingestion is an important exposure pathway for assessing public health
risks associated with contaminated soils. The bioavailability of Pb, As, and possibly other
contaminants in soils can be determined by conducting dosing trials using acceptable
surrogate animal models. To overcome the difficulty and expense associated with in vivo
trials, in vitro gastrointestinal (IVG) methods, that simulate human gastrointestinal
conditions, have been developed. Bioaccessible Pb and As determined by several IVG
methods has been shown to be correlated with in vivo bioavailability data.
Soils must have a very high contaminant concentration, often > 500 or 1000 mg/kg, to
accurately measure bioavailability from animal dosing trials. Most contaminated soils are
not highly contaminated. These moderately contaminated soils require risk assessment
but are below the "detection limits" of animal models. IVG methods will be the only
methods that can be used for exposure assessment of moderately contaminated soils.
Soil chemistry, mineralogy, and other geomedia properties are likely to have more
influence on contaminant bioavailability in moderately contaminated soils than highly
contaminated soils. Can we rely on IVG methods to assess contaminant bioavailability in
moderately contaminated soil without method validation based on in vivo bioavailability
data? Soil and contaminant chemistry requirements necessary for accurate application of
IVG methods to access contaminant (bio)availability will be presented.
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Abstract 406
The Bioaccessibility of Nickel in Contaminated Soils, Can It Be Explained Using
Solid Phase Distribution Data?
J. Wragg1, M. Cave1, C. Ollson2, K. J. Reimer3; British Geological Survey, Nottingham,
UNITED KINGDOM, 2Jacques Whitford Ltd., Ottawa, ON, CANADA, 3Royal Military
College of Canada, Kingston, ON, CANADA
In recent years there has been increased use of bioaccessibility testing to determine the
fraction of potentially harmful elements (PHEs) available for uptake in the human
gastrointestinal tract, and which therefore may pose a risk to human health. The data
produced by such tests may be incorporated into human health risk assessments to
determine the risk posed to the critical receptor, by a given land use, for the ingestion
pathway.
In tandem, research has focussed on identifying the physico-chemical sources of
bioaccessible PHEs (and in some cases non-bioaccessible PHEs) in soils. Identification of
the physico-chemical hosts of PHEs can be achieved by the use sequential extractions.
Application of these techniques in conjunction with bioaccessibility methods can aid the
understanding of soil-contaminant relationships and how contaminant bioaccessibility
and mobility may impact on human health risk assessment and future land use. To date,
most interest has centred on arsenic but more recently focus has shifted to nickel (Ni).
The physico-chemical sources of Ni in soils collected from Sudbury, Ontario will be
described, after indentification by the use of the CISED (Chemometric Identification of
Substrates and Element Distribution) extraction technique with confirmatory X-ray
diffraction information. The information surrounding the solid phase distribution of Ni in
the soils will then be used to provide an understanding of measured bioaccessibility data.
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Abstract 407
Importance of Metal Speciation in Understanding Bioavailability
K. G. Scheckel; U.S. Environmental Protection Agency, Cincinnati, OH
The speciation or chemical form of metals governs their fate, toxicity, mobility, and
bioavailability in contaminated soils, sediments and water. To assess these chemical
properties and to accurately gauge their impact on human health and the environment we
need to characterize metals at the atomic level. One can employ an array of techniques to
address speciation including XRD, DRS, TEM, TGA, and XPS. In addition to these tools,
researchers have used synchrotron radiation methods to elucidate metal speciation.
The complexity of metal contaminated sites has and continues to be simplified to a
measure of the total metal content. While total metal content is a critical measure in
assessing risk of a contaminated site, total metal content alone does not provide
predictive insights on the bioavailability, mobility, and fate of the metal contaminants.
Our ability to determine metal speciation in soils enhances efforts to understand the
mobility, bioavailability, and fate of contaminant metals in environmental systems, to
assess health risks posed by them, and to develop methods to remediate metal
contaminated sites. To attain in situ atomic level information on the speciation of metals
we utilize high-energy synchrotron X-rays to probe chemical structure. At the Advanced
Photon Source (APS) of Argonne National Laboratory (Argonne, IL), we incorporate X-
ray absorption (XAS), X-ray fluorescence (XRF), and micro-tomography spectroscopies
to analyze environmental samples to determine the true, in situ speciation of metal
contaminants. These innovative research tools are expanding our ability to directly
identify the role of metal speciation on many dynamic processes that influence risk.
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Abstract 587
Direct Identification of Metal Compounds in Contaminated Soil, Mine Tailings and
House Dust Using Synchrotron-based Methods
H. E. Jamieson1, S. R. Walker1, S. E. Fawcett1, A. Lanzirotti2, P. Rasmussen3, S.
Beauchemin4, M. Parsons5; Queen's University, Kingston, ON, CANADA, 2University
of Chicago, Chicago, IL, 3Health Canada, Ottawa, ON, CANADA, 4Natural Resources
Canada, Ottawa, ON, CANADA, 5Geological Survey of Canada, Halifax, NS, CANADA
Contaminated soils can be expected to contain multiple hosts of the metal or metalloid of
concern, especially in the case of mine-impacted soils or tailings, where the
concentrations are orders of magnitude above soil quality guidelines. For example, we
have determined that a single sample of arsenic-rich gold mine tailings contains, in
addition to the primary arsenopyrite (FeAsS), five secondary oxidation products namely
scorodite (FeAsO4 2H2O) , amorphous Fe arsenate, kankite (FeAsO4 3.5H2O), yukonite
(Ca-Fe arsenate), and arsenic bound to iron oxyhydroxides. At sites where ore roasting
was used, tailings and soils contain AsIII-bearing roaster-generated iron oxides, as well
as AsV-bearing iron oxyhydroxides generated by sulfide weathering. We have also
determined that antimony is present in multiple mineral forms and oxidation states in
mine waste impacted sediments. The detailed and direct identification of these As- and
Sb-bearing phases was achieved using a combination of synchrotron-based techniques
microanalytical including microXRF (X-ray fluorescence), microXANES (X-ray near
edge spectroscopy) and microXRD (X-ray diffraction) on target grains in polished thin
sections with a <10 micron spatial resolution.
Synchrotron-based techniques have also been applied household dust and shown that for
a sample from a background urban environment Cu and Zn are associated with distinct
matrices. Copper is dominantly hosted in an organic phase while Zn is associated with
inorganic minerals.
Each solid host of a metal or metalloid may exhibit different response to bioaccessibility
tests, as these phases are known to vary in solubility. The multiplicity of mineral hosts
has significant implications for the design of sampling programs that aim to obtain
representative ingestable material. Where applicable, synchrotron-based microanalysis
provides a tool to unambiguously characterize contaminants in complex samples.
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Abstract 525
Bioaccessibility of Arsenic Adsorbed onto or Incorporated within Freshly
Synthesized Iron Oxide Minerals Using the Simulator of the Human Intestinal
Microbial Ecosystem (SHIME)
B. D. Laird1, T. Van De Wiele2, D. Peak1, W. Verstraete2, S. D. Siciliano1; University of
Saskatchewan, Saskatoon, SK, CANADA, 2University of Ghent, Ghent, BELGIUM
The bioaccessibility of arsenic adsorbed to amorphous ferrihydrite or incorporated within
amorphous scorodite was measured in the stomach, small intestine, and colon stages of
the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), an in vitro
gastrointestinal model that incorporates the microbial community found in the human
colon. Arsenic concentrations adsorbed to ferrihydrite ranged between 500 and 9500 ppm
while the arsenic concentrations of amorphous scorodite mixed with freeze-dried iron
oxide ranged between 4500 and 450,000 ppm. The SHIME digests of these arsenic-
bearing minerals were used to construct arsenic dissolution isotherms for the stomach,
small intestine, and colon SHIME. Subsequently, the Kd of arsenic in gastrointestinal
fluids and the mechanism of concentration-dependent constraints on arsenic
bioaccessibility was evaluated. Additionally, the colon digest was repeated with sterilized
colon SHIME suspension to investigate the role gastrointestinal microorganisms on the
bioaccessibility of arsenic adsorbed onto or incorporated into iron oxide minerals. These
experiments investigated the mechanisms by which concentration and gastrointestinal
microbes affect arsenic bioaccessibility.
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Appendix C: Presentations
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Introduction and Overview for Symposium "Use of In
Vitro Bioaccessibihty/Relative Bioavailabihty Estimates in
Regulatory Settings: What is Needed?
Karen Bradham, Mike Beringer,
Aaron Yeow (EPA)
Pat Rasmussen (Health Canada)
Rosalind Schoof (Integral Consulting, Inc.)
Mark Cave (British Geological Survey)
Symposium Roadmap
• Welcome
• Setting the stage
• Definitions of bioavailability
• ISEA 2006
• ISEA 2007 overview and logistics
- Panel discussion questions
• Closing remarks
AEPA
>EFA
Oral bioavailability of metals
• Site-specific human health risk
assessments
• Risk assessments used to
determine whether a contaminated
site poses a current or future threat
to human health that warrants
remedial action
• Oral ingestion of soil and dust -
"risk driver" for human exposure to
metal contaminants
Exposed to contaminated soil - oral ingestion
• Toxicity of an ingested chemical depends,
on the degree to which it is absorbed from
the gastrointestinal tract into the body
• Metals can exist in a variety of chemical
and physical forms
• Not all forms of a given metal are
absorbed to the same extent
• Physical, chemical, biological
• Matrix: metal from a contaminated soil
absorbed vs. ingestion from dietary
exposure
iii »i,v,i!,t> Simpson et al.
The Gut: Inside out.
Physiology and biology
SB*
Definitions of bioavailability and related terms
• Many different meanings across various disciplines
• Guidance for Evaluating the Oral Bioavailability of Metals in Soils
for Use in Human Health Risk Assessment (OSWER 9285.7-80)
• Bioavailability: "The fraction of an ingested dose that crosses the
gastrointestinal epithelium and becomes available for distribution
to internal target tissues and organs"
http://www.epa.gov/superfund/bioavailability/guidance.htm
Definitions of bioavailability and related terms continued
• Bioavailability of metals in a particular matrix may be expressed as
absolute bioavailability or relative bioavailability
• "Bioavailability expressed as a fraction (or percentage) of a dose is
commonly referred to as absolute bioavailability (ABA)"
• ABA - "ratio of the amount of metal absorbed compared to the amount
ingested"
Absolute
Bioavailability
http:/MMW.epa.gov/superfund/bioavailability/guidance.htm
-------
Definitions of bioavailability terms continued
• Relative bioavailability (RBA): "The ratio of the bioavailability of a metal in one
exposure context (i.e., physical chemical matrix or physical chemical form of
the metal) to that in another exposure context'
• RBA - ratio of the absolute bioavailability of metal present in a test material
compared to the absolute bioavailability of metal in some appropriate
reference material
• RBA is usually the most important for risk assessment
• extent to which the absolute bioavailability of a metal increases or
decreases in context with the exposure matrix (e.g., water vs. soil)
• "A related term, pertaining to bioavailability assessment, is bioaccessibility.
This usually refers to a measure of the physiological solubility of the metal at
the portal of entry into the body (IVBA)
http://mvw.epa.gov/superfund/bioavailability/guidance.htm
Use and application - oral bioavailability
* If the oral dose used for risk assessment is based
on studies using metal administered in food or
water, then the risk from ingesting metal
contaminated soil might be over or
underestimated
• Risk assessments that adjust for metal
bioavailability may reduce the burden of
unnecessary and costly remedial action
• Small adjustment in oral bioavailability-
significant impacts on estimated risks and
cleanup goals
• Bioavailability data used to improve the
accuracy of exposure and risk calculations at a
site
AEPA
>EFA
Methods for Assessing Bioavailability in Soil
- In vivo methodologies
• Quantification of metal present in various tissues and excrement
• Used to develop quantitative bioavailability adjustments
• In vitro methodologies
• Physiologically-based extraction tests
• Measures bioaccessibility (e.g. solubility)
• Used for screening purposes and reducing uncertainty
- Mineralogical/speciation studies
• Importance of solid phase distribution, speciation and particle size
• Provides supporting bioavailability/bioaccessibility information
2006 Symposium
"Childhood exposures to bioavailable and bioaccessible metals in soil and
household dust in residential environments"
Joint ISEE/ISEA Conference on Environmental Epidemiology and
Exposure, September 2-6, 2006, in Paris
Organized by Health Canada (Pat Rasmussen) and British Geological
Survey (Joanna Wragg and Mark Cave)
Abstracts published in Epidemiology 17 (6) pp S39-S42 (2006)
SERA
Some observations from the 2006 symposium
• Participants found the International Society of Exposure Assessment
(ISEA) to be perfect "home" for the cross-disciplinary nature of
bioaccessibility/bioavailability research
• Many countries were represented, and in some areas similar views were
expressed:
- Everyone agreed that a set of criteria is needed, to assess both in vivo
and in vitro methods
- In addition to Canada, some countries (UK, USA, and EU) were
allowing some limited site-specific adjustments
-At that time, few would be comfortable allowing existing site-specific
methods to be universally applied
- Everyone expressed support for the annual ISEA BA session in 2007
2007 ISEA Symposium "Use of In Vitro Bioaccessibility/Relative
Bioavailability Estimates in Regulatory Settings: What is Needed?"
• Morning session 9:00 am - 12:30 pm:
- Site evaluation and application
- In vivo research
- Panel discussion session I
• Afternoon session 1:30 pm - 6:00 pm:
- In vitro research
- Mineralogy and speciation
- Panel discussion session II
- Closing remarks
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&EFA
Panel discussion and post ISEA
• Two panel discussion sessions
• Questions provided to all presenters/panelists prior to symposium
• EPA plans to develop symposium proceedings to capture current state of
the science and research needs
- Proceedings will be posted on EPA website
http://epa.gov/superfund/bioavailability/links.htm
- Presentations included in symposium proceedings
• Plan to publish a series of papers from symposium as a special issue
- Some recommendations have been made for several journals
-Symposium organizers will contact presenters following ISEA
conference
vvEPA
Questions for Panel Discussion Session I:
Evaluation/site application and in vivo research
• For regulatory applications such as site clean up decisions, is an in vitro model
that is predictive (i.e., correlates with an in vivo model) adequate even if we
don't know why it is predictive, or is it important to know why a model is
predictive?
• Given that many animal models require use of soil concentrations exceeding
those of public health concern, what efforts are necessary, if any, to show that
bioavai I ability at these concentrations reflect oral absorption at lower
environmental concentrations? In other words, how important is it to evaluate
whether bioavai I ability is concentration dependent?
• Should a single animal model be specified, or are studies in a variety of
different animal models more likely to improve our understanding of relative
bioavai I ability in humans?
• Is there a single animal model that will work for all chemicals considering the
widely varying toxicokinetic profiles?
• Is relative oral bioavai I ability of chemicals in soil so routine that it is "ready" for
guidance/regulation?
AEPA
Questions for Panel Discussion Session I
and speciation/mineralogy research
AB*
Questions for Panel Discussion Session II: In vitro
and speciation/mineralogy research continued
• Is it sufficient for in vitro models to have correlation with animal results or is it
necessary to make the models accurate physiologic mimics of human gut
dissolution?
• Is it acceptable to use the terms "bioaccessible", "soluble", "migratable", and
"extractable" interchangeably in the numerator of the % bioaccessibility
equation?
• Should bioaccessibility values be used on their own without supporting
information? If not, what additional information should be included for each soil
sample analyzed (e.g., geochemistry)?
• How can we leverage resources to answer specific research questions to
advance the understanding of bioavailability/bioaccessibility?
- Develop Standard Reference Materials or Certified Reference Materials?
- Round robin testing?
• In lieu of comparing in vitro model results to an in vivo model, what criteria
should be considered when evaluating whether a particular in vitro method is
appropriate for providing screening level data versus data to derive quantitative
site-specific bioavai I ability adjustments?
• What other metals, or metal species, are of interest for developing additional in
vivo and in vitro bioavai I ability assays? What criteria are used by regulatory
agencies for ranking or prioritizing contaminants?
• Is it feasible and cost effective to develop a single standardized in vitro method
for each metal for simulating oral bioaccessibility? If so, what steps would be
needed for that to happen? What criteria would be used to determine the
difference between the methods?
For additional information, visit the following
websites:
http://epa.gov/superfund/bioavailability/links.htm
http://www.epa.gov/superfund/bioavailability/guidance.htm
http://www.epa.gov/heasd/mdab/mdab.htm
www.bgs.ac.uk/barge
To obtain Proceedings from Health Canada Sponsored Workshops
on Bioaccessibility/Bioavailability in Contaminated Site Assessment
(2005, 2006, and 2007) go to: http://www.cntc.ca
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11
Evaluating the Bioavailability
of Soil-Borne Contaminants
at Waste Sites
ISEA Symposium
Durham, N.C.
October 15,2007
Mike Beringer & Aaron Yeow
U.S. Environmental Protection Agency
Need for Additional Bioavailability Guidance
• Default assumption likely overestimates health risks
• Bioavailability is equal in soil, diet and water
• Relative bioa vail ability or RBA is 1.0
• Lead is the exception where default RBA is 0.60
• Existing guidance supports bioavailability adjustments
• Does not address when data collection should be pursued
• Does not address how to evaluate site-specific bioavailability
• Limited use of site-specific bioavailability information
• Absence of rapid and inexpensive tools
• Lack of criteria for evaluating alternative test methods
Guidance for Evaluating the Oral Bioavailability
of Metals in Soils for Use in
Human Health Risk Assessment
^^^•^••^^^H
• Limited in scope
• Outlines a decision framework - series of questions
• Is a validated method available?
• Does the added value exceed the costs?
• Addresses site-specific documentation
• Basis for relying on the selected method
• Data translation
• Sample collection
• Recommends criteria for evaluating alternative methods
I I
Validation of Bioavailability Test Methods
• Relying on ICCVAM criteria (Interagency Coordinating
Committee for Validation of Alternative Methods)
• http://iccvam.niehs.nih.gov/
• Method validation criteria
• Demonstrate method is reliable and relevant for its proposed use
• Regulatory acceptance criteria
• Method fulfills a specific regulatory need
• Regulatory methodologies
• Must satisfy both sets of criteria
• Appropriate for making quantitative site-specific adjustments
N
II
Method Validation Criteria
(ICCVAM, 1997)
• Scientific and Regulatory Rationale
• Relationship Between Test Method Endpoint and Biological Effect
• Detailed Protocol and Known Limitations
• Within-Test Variability and Reproducibility Among Labs
• Test Method Performance with Representative Agents
• Comparison to Existing Test Method
• Data in Accordance with Good Laboratory Practices (GLP)
• Validity Assessment Data Available for Review
• Independent Scientific Review
Regulatory Acceptance Criteria
(ICCVAM, 1997)
• Independent Scientific Peer Review
• Detailed Protocol with SOPs
• Adequately Predicts Bioavailability and Demonstrates a Linkage
• Representative Chemicals Tested
• Generates Data Useful for Risk Assessment Purposes
• Documentation of Strengths and Limitations
• Robust and Transferable
• Time and Cost Effective
• Can Be Harmonized
• Suitable for International Use
• Reduction of Animal Use
-------
II
Estimation of Relative Bioavailability of Lead
in Soil and Soil-Like Materials Using
In Vivo and In Vitro Methods (Lead TSD)
• Describes in vivo and in vitro methodologies
• Juvenile swine model
• Simplified bioaccessibility method
• Characterizes 19 soil and soil-like test materials
• Mineral phase
• Particle size distribution
• Matrix association
• Clear differences in RBA between materials
• Data not sufficient for predictions based on mineral content alone
• Evaluates the correlation between both methods
Lead TSD Transmittal Memo
• Evaluated both methods using ICCVAM criteria
• Broad range of relative bioavailability
• Variety of mineralogical forms
• Pairwise comparison shows a good fit (r2=0.92)
• Both methods considered regulatory methodologies
• Weight-of-evidence determination
• Method validation and regulatory acceptance criteria achieved
• Appropriate for use in site-specific risk assessment
• Outlines limitations and considerations for use
• Quality assurance
• Sample lead concentration limits
• Particle size and soil mineralogy
• Extrapolation to adults
• Valid for soil samples from mining and milling sites
11
Lead - Correlation Between In Vivo RBA
and In Vitro Bioaccessibility (IVBA)
I I
Future Activities
• Formation of Bioavailability Committee
• Information archive
• Provide technical support to the USEPA Regions
• Develop additional guidance
• Review new bioavai I ability methods
• Evaluation of other metals
• Formal consideration of arsenic bioavailability data
• Possible derivation of default values for other metals
-------
Method Development and the
Application of Oral Bioavailability
Data in Risk Assessments
Rosalind A. Schoof
Integral Consulting, Inc.
Mercer Island, WA
rschoof@integral-corp.com
New EPA bioavailability guidance
calls for use of validated methods
• How is validation defined?
• What are validation requirements?
• What are regulatory acceptance requirements?
« What is the process for method development
and validation?
• What methods are already considered validated
based on history of use?
ISEA 2007
Validation - Definitions
Scientific process designed to characterize
operational characteristics, advantages, and
limitations of a test method
The process by which the reliability and
relevance of a test method are evaluated for
the purpose of supporting a specific use
Validation requirements
Relevance - Linked to mechanism of
toxic effect and proposed uses
Reliability - Objective measure of
method's intra- and interlaboratory
reproducibility
Who oversees validation efforts?
• Interagency Coordinating Committee for
Validation of Alternative Methods (ICCVAM)
• http://iccvam.niehs.nih.gov
• Organization for Economic Cooperation and
Development (OECD)
• American Society for Testing and Materials
(ASTM)
ICCVAM validation criteria
http://iccvam.niehs.nih.gov/docs/guidelines/validate.pdf
•Scientific and regulatory
rationale
• Endpoint relation to
effect of interest
• Detailed protocol
•Within test variability
and reproducibility among
labs
• Performance demo with
reference chemicals
• Data comparison with
standard test
• Limitations described
•GLP data
• Data available for review
-------
ICCVAM regulatory acceptance criteria
http://iccvam.niehs.nih.gov/docs/guidelines/validate.pdf
• Peer review
• Protocol with SOPs
•Measures endpoint of
interest, and linkage with
existing test
• Representative chemicals
tested
• Useful for risk
assessment
• Strengths and limitations
identified
• Robust and transferable
•Time and cost effective
• Can be harmonized
• International acceptance
possible
•Minimizes animal use
What is the process and when is a
method validated and implemented?
• Process: Research > development >
pre-validation > validation > review >
agency consideration > implementation
• Validated when its performance
characteristics, advantages, and
limitations have been adequately
documented for a specific purpose.
Types and uses of test methods
• Definitive tests - Used to measure toxic
effects
• Screening methods - Support
preliminary hazard decisions
• Adjunct tests - Used to increase the
information base and/or aid in the
interpretation of results from definitive
methods
Evolution of test methods
• Development of study design
• Refinement of test protocol
• Assurance of transferability
• Determination of performance
characteristics
Currently accepted methods
• Considered validated based on history
of use
• Applies to many approaches to
measuring oral bioavailability of
chemicals
Currently accepted in vivo methods
of measuring bioavailability
• Blood concentration over time
(area under the curve, or AUC
• Absorbed fraction in urine
and/or tissues
• Comparison of tissue concentrations
• Unabsorbed fraction in feces
-------
Variations in gastric anatomy
Validation for toxicity tests vs.
bioavailability methods
• Are validation criteria for development of
alternative toxicological methods appropriate
for methods of testing relative bioavailability
of chemicals in soil?
Validation/acceptance issues for oral
bioavailability studies of metals in soil
• Endpoint of interest is relative
bioavailability for oral exposures
• For some metals there is no standard
method available for comparison
• SBRC in vitro method meets validation
and regulatory acceptance criteria
• Should animal studies continue to be
used?
Does the in vitro method meet
validation criteria?
> Rationale and
relationship of endpoint
to effect of interest are
documented
• Detailed protocol
available
• Reproducibility
established in
interlaboratory study
• Performance
demonstrated for
representative chemicals
• Comparison of
performance with
existing test established
• Limitations described
> Data quality documented
• Data reviewed both in
peer-reviewed
publications and in
independent peer review
process
Bioavailability method development
questions for metals
• How reliable are oral in vivo study methods?
• Should validation of in vitro protocols be
required on a metal-specific basis?
• Should results of unvalidated methods be
considered in risk assessments?
If yes, how?
-------
Exponent
_ pone
Relative Oral Unavailability of
Arsenic From Soils:
in vivo and beyond
Yvette Lowney
Presentation to ISEA
October 2007
Presentation Topics
in vivo research
- cynomolgus monkeys
in vitro extraction testing
SBRC method
Other chemical extractions
RIVM/Unified Barge
Geochemistry and soil characterization
Future efforts
Research Funding
SBRC
SERDP
Industry sponsors
UK Environment
Urinary Arsenic Excretion:
Timeline Following Exposure
Integrated Assessment of Beneficial Uses and Development of Candidate Cleanup Levels
1
-------
Exponent
Study Design
cynomolgus monkey
Low arsenic diet prior to dosing
Dosed with slurry of soil in water
Soil dose = 1 g/kg bw
Arsenic dose = 1 mg/kg bw
Collection of urine and feces
n=5
RBA = (% of soil dose in urine) - (background)
(% of NaAs dose in urine) - (background)
Corrections made on animal-specific basis
Results: RBA of arsenic in cynomolgus monkeys
Soil Sample A[meg™ °«« R[JAi Total Recovery
Results from in vivo Research
Using cynomolgus Monkey
RBA measured in 14 soil samples from 12
sites
smelter soils
slag
mine tailings.
pesticide facilities,
agricultural soils
cattle dip vat site
wood treatment site
RBA for arsenic in environmental soils
ranged from 5% to 31%
Indicate that site- or soil-specific factors
control the absorption of arsenic from soil
Roberts et a/., 2007 ES&T
Development of in vitro Methods
for Estimating Arsenic RBA, as
Measured in the Cynomolgus
Monkey
Goals for in vitro method
-Simple
Repeatable and reproducible
Captures rate-limiting step controlling RBA
Predictive of animal data
Development of in vitro method
SBRC was used as the starting point
Phosphate extractions
Hydroxylamine HCI
RIVM
Unified BARGE methods
Integrated Assessment of Beneficial Uses and Development of Candidate Cleanup Levels
2
-------
Exponent
in vitro Extraction Method
SBRC, phosphate, hydroxlamine
buffered "gastric" solution
<250 um particle size
1 g soil: 100 mL fluid
End-over-end rotation at 37°C
1 hr
Filter (0.45 um)
ICP-MS for As
SBRC Method at pH 1.5 and 2.5
ARSENIC BIOACCESSIBILITY (°
Integrated Assessment of Beneficial Uses and Development of Candidate Cleanup Levels
3
-------
Exponent
Integrated Assessment of Beneficial Uses and Development of Candidate Cleanup Levels
4
-------
Exponent
Status of iw r/'tro Method
Indications that in vitro methods may
be predictive of in vivo results for RBA
of arsenic
Method validated for lead correlates
well with arsenic for most soils
Need method that is predictive for all
soils across a diversity of soil types
RIVM and UBM correlate with each
other, poorer relation with in vivo data
Avaijable approaches don't provide 1:1
relation between in vitro and in vivo,
but could find good correlation
Soil Characterization Data
Conventional parameters
Arsenic concentration
TOO
- Metals
-SoilpH
Arsenic source
Particle size distribution
Arsenic mineralogy (18 phases)
Extractable iron oxide
Integrated Assessment of Beneficial Uses and Development of Candidate Cleanup Levels
5
-------
Exponent
Soil Arsenic Mineralogy Data
Research Status
' 117 VIVO
- Robust database suggests RBA <30%
More data?
• in vitro
- Method validated for lead correlates well with arsenic
for most soils
Need method that is predictive for all soil types
Available approach doesn't provide 1:1 relation
between in vitro and in vivo, but could find good
correlation
Progress is potentially rapid
1 Predictive models
Currently no model that is robust across all soil types
Theoretically possible
Likely to be 'informed' by in vitro method
development
Integrated Assessment of Beneficial Uses and Development of Candidate Cleanup Levels
6
-------
Exponent
Integrated Assessment of Beneficial Uses and Development of Candidate Cleanup Levels
7
-------
UilJjiy
VirDiiiiJiiy—iiDDiDD j/jii-iJ:; !A=, rjd, rjr,
Pb, '••]], o/:J-i/jJfj fjo;;j{jou/jd:; (dJo/J/j:;^
DD'fj iiud Au-, Pd- iiud AcHjiiijo^iiriidE:
JuviuiJE: DWiui DurrogiiiE: for 'jhUd/i/j,
jJil'j'VE: JLJViJjijE: {jicJD UDid i/J -ill 5PA il/jd
jJrjJ ={jojj=orsd ^UjdiiD,
OrnJ i/iJO^u/E: for 'J2-'J4 eoijDEJcU'fJys;
diiy= si! "J do^E: Jeveii;,
Statistical Power of the Study > 90%.
Doses selected to reflect low-dose
human exposure (25-160 ug/kg BW).
Multiple responses to assess RBA—
blood, urine, liver, kidney, and bone.
Absorption of Soil Lead
by
Immature Swine
PbMn(M)O
average RBAs (25% - 75%) are associated with PbO,
PbFe(M)O, PbPO4, and Pb-Slags
lower RBAs (<25%) are associated with PbS, PbSO4,
Pb(M)O, PbFe(M)SO4, and metallic Pb
-------
Conceptual Model
General Subchronic As Study Design
-------
coal fly ash
agricultural/orchard pesticides
wood preservation
Absorbed As primarily excreted in urine
Urinary Excretion Fraction fUFr) is an
approximation of trie oral AF or ABA,
USr does not account for As excreted in bile or
-\s distributed to tissue compartments.
"•'BA of 2 orally dosed materials ftest an
of UEF(As.test) / UEF(A fx. Keep in mind this is
really a ratio of slopes of As excreted as a
function of As dosed.
'kriamiJs 21 mJ
7-10 Qmups o/4-j
[jigs dosed for-12-14
corjsecuiive diiyj
Abaor-bsd As
=s'iirrjai=d by J-.s
e/.creied irj urirje (24 or
43 br;—US:^—urirjyry
excretion Traction
Urinary As excretion—a
linear function of dose
and independent of
time after day 5
-------
Rssulis Coal Combusion By-
Prod LldS
Using sodium
arsenate as si relative
frame of rsfsrerics,
trie arsenic R3A
sstirnatsa 5ire
for Test Material 1
and 50% for Test
Material 2.
1.jnoT.-.T«!Mawii:
Eidpoo:
D«i«1
;^?:.
Dri i: U
AC 5m
InnuttK R3A i'?;*> "cdii»i;« £Ur-*l>
7t.: .^as:: .
.TTCd
0*4^«-0.!J)
. ;i •.!:-; •'.
M cr-:.':.
;~:(ca-;"*i
T«tywR»;:
7?:-".
35IJ3-«-eJf]
r: .+; •. ••
0«CH5-C-!3J
}»[94C-cn}
Jiiiiwsi Tow;j3hj|j rJsri 3Jsi33
Usincj sodium
arssnais as a relsiii /3
frame of rsfsrsnoe,
She arssriio P.3A
sstirnaiss sirs
for Test Material 1
and 26% for Test
Material 2.
-------
Absorption
Gastrointestinal microbial metabolism
(methylation, demethylation, thiolation)
Postabsorptive metabolism
(methylation, thiolation)
-------
Water bottle Food hopper
Feces collector
Urine collector
Soils
Diet
110700 AIM 93G Pljl
-------
S 2
8
\\
\\
\ \
NaAsV 170/2 170/5 170/4
-------
Sift*
I «MA»
SDMAs
M*At(V) 170H 170/5 170/4
-------
Environment
taencj
In-vitro Bioaccessibihty of Soil-
borne Contaminants: An
Environment Aaencv Perspective
Environment
Agency
Outline
£01
Risk assessments and land contamination
Review of currently available in-vitro methods
Conclusion to date
Forward Look: what is required?
Risk assessment approach
Preliminary Screening
2RA (e.g. SG\
DQRA (site specific bio-availability
Environment
Agency
Definitions
Absolute bioava liability: Is
fraction of an administered
• Needs in-vivo studies
Relative bioavailability: Cor
different forms of a chemic;
containing the chemical
• Needs in-vivo studies
measure of the uptake or the
ose absorbed by the body.
ve bioavailabilities of
- different exposure medic
Oral bioaccessibility: Is a measure of dissolution, or the
fraction of contaminant released into solution from the sc
during digestion
• Attempt to measure in in-vitro model
nmeni
Agency
hy in-vitro?
**'*
Technical expertise
Reproducibility
Ethical reservation
Absorption is likely to be
dependent on solubility
-------
Environment Aaencvs work Droaramme
N Environment
FAgency
Questions
cm
1) What are the different in-vitro methods in the UK?
2) Can they produce comparable results?
3) Can they adequately predict bioavailability?
4) Can one method be suitable for different chemicals and
different soils?
5) Do we have sufficient awareness of these issues and
their importance?
Q 1: Methods available
Q 2: Inter-lab data comparability
Study undertaken with UK labs producing bioaccess
data - available at
ileven labs participated (two from overseas)
Three prepared samples from UK supplied in triplicate (
one human tested lead contaminated sample from
Maddaloni etal. 1998)
Labs were asked to analyse for As, Pb and Ni using the
normal protocol.
'CONTEST soil proficiency-testing scheme' followed
in-vitro data evaluated as 'consistent' or 'inconsistent'
-------
/5?X Environment
V^iV'Agency
Data comparability (summary)
(excludes overseas and one UK l
Test soils Arsenic
R: 13-88 R: 1462-8219 R: 1-5
Med: 18 Med: 1911 Med: 2
R: 121-7011 R: 2920-84979 R: 5-25
Med: 194 Med: 10480 Med: 9
R:5-9 R: 348-542 R: 1.35-2
Med: 5 Med: 477 Med: 2
Environment
Agency
em
Can UK labs produce comparable results?
No, largely due to variability in the types
of in-vitro methods used
(But labs using the same method and same
operating procedure produced comparable results)
Q 3: Predicting bioavailability
Study undertaken with Exponent USA (report in
prep.)
• Aim: Evaluate selected in-vitro methods for their
ability to predict bioavailability
• Used 13 in-vivo (Cynomolgus monkey) tested US
soils with arsenic bioavailability data obtained from
a previous study (Roberts etal. 2007)
• In-vitro data produced was studied against in-vivo
bioavailability data
Q 3: Predicting bioavailability for arsenic
7 (0.37 excl. outlier)
Intestine phase
5 (0.32 excl. outlier)
PBET (Ruby <* ol. 1996)" 0.18
SBRC/SBET (Drexler 1998)" 0.27
Environment
Agency
Can in-vitro data adequately predict
bioavailability?
Not adequately for the soils tested. For
UK soils it is unknown as none have
gone through in-vivo studies
(3& Environment
*9jf Agency
Q 4: Applicability of one method to
various chemicals
In-vitro methods
(Drexler 1998)
SBRC/SBET
(Drexler 1998)
R2 (range 0-1)
0.17a (0.37 <
-------
Environment
Agency
\ Environment
Xi Agency
Q 5: Do we have sufficient awareness
in the UK?
Can one method be suitable for different
chemicals and different soils?
Questionable based on evidence
currently available
Perception that bioaccessibility and bioavailability are
the same thing
There are reports of extrapolation of bioaccessibility
data from literature or different sites
Inconsistency in the practice (e.g. lab procedure) and
use of data in risk assessment
S Environment
Conclusions to date
Ability to predict bioavailability by m-vitro methods used in the
UK is uncertain for UK soils
Considerable inter-laboratory variability of in-vitro data
Laboratories use same method irrespective of
• chemical
• chemical form
• matrix
Contaminants of concern differ from country to country
In-vitro bioaccessibility testing is an ongoing research area
Forward look: what is required?
More needs to be done to develop in-vitro methods
including validation with appropriate in vivo data
What can be done to increase confidence in in-vitro
data?
• Multiple lines of evidence to compliment in-vitro
bioaccessibility methods/data
nmeni
Agency
required?
Focussed discussion to identify and define what are
the essential data gaps
International harmonisation through a framework of
partnership (repeat and working in part would delay
achieving the common shared objectives)
More linkage between research scientist and
regulatory scientist (and with Policy makers)
-------
HC Science Forum Oct 31 2006
Pat E. Rasmussen
Outline of Presentation
Consumer Safety
Branch
Measurement of Metal
Bioaccessibility in Urban
Household Oust and
Corresponding Garden Soil
Differences between house dust and soil
High variability of bioaccessible metals in dust
Solution - simple categories of bioaccessibility:
low, medium and high
What is needed? significance for risk assessment
CanadS
The "tracking-in" models to
estimate indoor exposures
Coarse fraction of soil
leaves, twigs
stones
agglomerates
natural and/or
manufactured
Coarse Fraction of House Dust
textiles
"I am Canadian"
beer cap
plastics
metals
organics
Sample preparation technique
used for soils ..
-------
HC Science Forum Oct 31 2006
....are applied to house dust
To compare indoor dust with
outdoor soil, analytical approaches
must be consistent
Same size fraction
To calculate indoor/outdoor
metal ratios (dust/soil)
Same analytical approach
Aggressive digestion for
"total metal" determination,
to ensure equally efficient
recoveries in different
media
Weak extraction to
estimate "bioaccessible
metal" fraction
Metals in Dust - References
Detailed Ottawa studies: the unique
geochemical profile of house dust
After drying and sieving to fine
fraction, settled dust may look
the same as soil.
But...
• Key metals have higher total
concentrations.
• Bioaccessibility of key metals
higher in dust.
• Organic content higher
Dust is very heterogeneous.
• Particle size
• Speciation / mineralogy
Ottawa: an example of "urban background"
dust
410
soil dust
10 18 16
soil dust
2.2 2.7
dust
28 soil
5.1 dust
1.1
soil
0.18
Zn (ppm) Ni (ppm) Cu (ppm) Org C (%) Inorg C (%)
High variability of bioaccessible
metal in dust compared to soil
-------
HC Science Forum Oct 31 2006
Influence of particle size
Bioaccessibility as a function of mineralogy
Zinc species
Zn hydroxyl carbonate
Zn/Fe-oxides
ZnS
<36ftm 80-150 fim
% of total zinc in house dust
52 65
22 16
26 19
Influence of Speciation (XAS)
Influence of Speciation (XAS)
Summary of histograms
Sources of variability
- Bioaccessible metals in house dust-
Particle size is an important control on metal
concentration.
Metal concentration commonly increases as particle
size decreases.
Metal bioaccessibility commonly increases as
particle size decreases.
However, particle size is not the only control on
concentration & bioaccessibility: the opposite
trends may occur depending on metal Speciation.
Analytical reproducibility is improved using smaller
size fractions.
-------
HC Science Forum Oct 31 2006
Selection of Sampling Method -
Depends on Purpose of Study
Adaptation of Toy Safety Protocol
for House Dust
European Standard EN 71-3: for the migration of
certain elements from toys
• Used in Product Safety lab at HC
• Children as target population
• Extraction uses only dilute HCI (pH 1.5) to
simulate stomach acid
• No added ingredients (complexing agents)
• Omits mouthing/mastication - assumes toy is
small enough to be swallowed
• Omits passage through intestine
Modified Toy Safety Protocol Yields Similar
Results to Other Gastric Simulations
100
80
60
40
20
0
Copper in NIST 2583 Indoor Dust
error bar = 1 sd; n = 6 replicates
RSD=18 %
at ratio of
5000
1000 2000 3000 4000 SOW 6000
-------
HC Science Forum Oct 31 2006
Modifications to US-EPA 3051
microwave digestion method for total metals
in dust and soil samples
Increase microwave digestion time to 30
minutes ramp time & 30 min hold time
(EPA3051 specifies 5.5 min total)
- recovery improved by 15-20%
Increase acid volume to sample mass
ratio to at least 1000 (EPA 3051 specifies
ratios from 20 to 100)
- recovery improved by 30-60%
Sample mass to acid volume ratio
HIST 1648
Urban PM
Ni Cu Zn
• 10 mg • 30 mg • 100 mg
-------
HC Science Forum Oct 31 2006
LOW
19% or less
LOW
19% or less
HIGH
60% and higher
HIGH
60% and higher
Zn
Indoor: Outdoor Ratio
Arguments for measuring metal
bioaccessibility in indoor dust
for risk assessments
Bioaccessible extraction 13 7 23
-------
HC Science Forum Oct 31 2006
What is needed?
Relevance to Risk Assessment
Thanks to our great lab team
Acknowledgments & Funding
Michelle Nugent, Christine Levesque, Marc Chenier,
Jianjun Niu, Monique Lanouette
http://www.cntc.ca
7
-------
The use of in vitro bioavailability
in human health risk assessment
Scientific research and application by policy makers
Human risk assessment of
contaminated soils in the Netherlands
• Intervention Value for substance in soil derived from MPR
• MPR: Maximum Permissible Risk (mg/kg/day)
- Exposure level above which there is a potential health risk
- Based on toxicological and epidemiological studies
• For most substances it is implicitly assumed that:
- Oral bioavailability in all exposure matrices = oral
bioavailability in the studies underlying the MPR
- Relative bioavailability =1
Human risk assessment of lead-
contaminated soils in the Netherlands
• Most substances: relative bioavailability = 1
•Lead
- Recently changed
- Intervention value for lead in soil 530 mg/kg
for scenario "living with garden" in the
Netherlands
- Relative oral bioavailability = 0.74 for soil
ingestion
• P80 for relative bioavailability of lead in tested
soils (soils low in organic matter)
• P80 for difference between fasted and average
state in gastrointestinal tract
Status application relative oral
bioavailability in risk assessment
• In the assessment of the risk of contaminated soils a
default value for the relative bioavailability of a
contaminant from soil is applied (0.74)
• The default value can be changed if reliable site-specific
information is available
• Recommendation by RIVM to government to accept the
use of in vitro determined bioaccessibility for estimation
of relative bioavailability factor (2006)
• Government will probably seek advise from other
institutes (Health Council, Technical Soil Committee)
Dutch Kempen area
• Zinc smelter for over a century
• Area contaminated with cadmium, lead, arsenic, etc
Experimental set-up
• Intention: relative bioavailability as input for
an area specific policy
• What is bioavailability of lead and arsenic in
soils and slags?
• 20 zinc slags, 16 soils
• In vitro determination of bioaccessibility
• Estimation of relative oral bioavailability
-------
In vitro determination of bioaccessibility
astric duodenal juic
Estimation relative oral bioavailability
5 min 2 h 2 h
pH ±6.5 pH ±1.2 pH±5.5
chyme Pellet
(destruction)
small intestine F Fraction of F absorbed by small intestine
portal vein
F = Fraction reach
Internal exposure F = Fb xFaxFh
Fh = Fraction of Fapassing liver without being metabolised
nic circulation = bioavailable fractio
Estimation relative oral bioavailability of
lead
Described in Oomen et al.
„ , • r Fsoll FbsoilxFasoilxFhsoi
Relative F= sml - ' ' '
Relative F=
0.5
Research on oral bioavailability lead
and arsenic from zinc slags
Relative bioavailability lead and arsenic from soils and
slags from the Dutch Kempen area
Soils
Slags
Lead
Arsenic
0.83 ±0.11 0.15 ±0.13
(n=13) (n=12)
0.35 ± 0.15 0.02 ± 0.01
(n=l7) (n=11)
Application in risk assessment
• Intention: relative bioavailability as input for an area specific
policy
• In this case not applied
• Lead
- Relative bioavailability of lead from Kempen soils (0.83) and slags
(0.35) was high
- Minor consequences for area specific policy
- Soil concentrations in the Dutch Kempen may lead to ecological
risks, but in almost all cases not to human health risks
Team
•RIVM
- Marco Blokland
- Esther Brandon
- Menno Duits
- Werner Hagens
- Johannes Lijzen
- Agnes Oomen
- Adrienne Sips
- Frank Swartjes
•ABdK
- Eric Kessels
V- ^
-------
Assessment of the use of dynamic human stomach models
for in-vitro measurement of the bioaccessibility of Arsenic
and Chromium in soils - Can they replace animal testing?
Mark Cave1, Helen Taylor1, Joanna Wragg1, Andrew Broadway2
1 British Geological Survey, Keyworth. Nottingham, NG12 SGG UK
2 University of Edinburgh, School of GeoSciences, Crew Building, West Mains Road, Edinburgh, EH9 3JN,
Scotland, UK.
Kingsley Dunham Centre
Keyworth
Nottingham NG12 SGG
Tel 0115 936 3100
Investigating human digestion
Human clinical studies - EXPENSIVE and
time consuming (not to mention gross
intervention effects and ethics)
Animal studies - may provide an alternative •
ethical considerations and concerns on
relevance to the human
In vitro models - a requirement for accurate
in-vitro screening tools - currently
oversimplified
Dynamic in Vitro models
• TIM-1 and Tiny TIM -TNO Quality of Life, Zeist, The
Netherlands
• Model Gut - Institute of Food Research, Norwich, UK
TIM-1 system
stomach and small intestine
body temperature
gastrointestinal pH curves
seccetion ol gastric add and salivary
and gas t FK enzymes
secretion of We. pancreatic juice
absorption of digested products and
mi*
continuous proces* control and data
modular set-up, htgh flexibility
_^k mf± it. fK
TIM Validation
Batch in-vitro
TIM
Minipig in vivo
-------
Maddaloni soil
FASTED
FED
BGS reference Material
.-
Total As104±3mgkg-1
Total Pb 79 ±3 mg kg-1
Chromium In Glasgow:
• Between 1830 and 1968 the Rutherglen area
of Glasgow was home to the world's largest
producer of chromium chemicals.
• Chromite ore processing residue (COPR) and
waste from the chemical works was used as
landfill in south-east Glasgow during the 19th
and 20th Centuries.
•An estimated 2.5 million tons dry weight of
COPR waste was sent to landfill around
Southern Glasgow and South Lanarkshire.
•In Glasgow, 15 contaminated sites have been
identified. The average Cr(VI) concentration
found was 700 mg/kg, with the highest being
15,600mg/kg.
sibilityin COPRContar
4000
3500
3000
2500
? 2000
1500
1000
500
Institute for Food Research (UK)
• First model to combine emerging
knowledge of the physical/
mechanical aspects of digestion
with the biochemistry in a single
predictive system
• It is the only simulation available
that can handle real food and
pharmaceutical preparations
• Also the only model that allows
access at any stage of 'digestion'
permitting sample collection and
analysis at anytime point
-------
Echo-Planar
t" ;l Magnetic
'<^*-'' Resonance Imaging
(EPI)
Meal in fundus
* «•; t." *l 0 I
Meal in an
§(• s.i s^ ci K ?
Meal in antrum
Dilution Maps and Gastric Mixing
36 min 48 min 72 min
Marciani etal. Journal of Nutrition,130. 2000
Analysed samples are composed of EQUAL vi
Water Blank and Soil Run w ilh Water
Volume of Acid and Enzyme will deper
British Geological Survey
C Samples
10.00 15.00 20.00 25.00 30.00 35.00 40.00
Time (min)
Soil 1 Digest in Water
IFR Model Gut
Conclusions
Dynamic in-vivo models show promising results.
As yet we do not have a definitive study for soils.
At present they probably would not replace batch in-
vitro methods but they could be used as a 'reference
methods'.
Commercial units are likely to be available in the near
future.
Could provide a more cost effective, ethical, and
scientifically valid alternative to animal testing.
-------
.ML.
Soil 1,11300 mg kg'1 As (Calciner soil)
.ML.
Soil 8,1180 mg kg'1 As (Iron slag soil)
3500
3000
f
„ 2500
; 2000
!
1
J 1500
1000
CISED extractiot
| 300
CISED extractio
S- Ca Ca- S- As Fe- As Fe Fe- S Fe- As
Pb-Ca Ca Ca Si-Fe Ca-S Si-Pb Fe-Ca Fe-Ca
IFR Model Gut
'olume against Tim
Soil Run with Mlk
^e of acid and enzyme will depend
on how the pH responds.
British Geological Survey
6 Samples
iDigea in Full Fat Milk
20.00 30.00 40.00 50.00 60.00 70.00
Time (min)
i 35
1 30
I 25
I 20
LU
15
y = 0.7337x +3.8731
R2 = 0.7753
5 10 15 20 25 30
Relative Bioav ail ability %
TIM dynamic
model
.1 t hi III I I' < Ul
-------
Tiny - TIM
Small intestine compartment
Hollow fibre MW cut-off filte
Simulated Peristalsis in the TIM model
Bioaccessibility of Chem icals
Not New
Jacob Helm (1797-1802) Investigations of the human digestive
system and the physiologic manifestation of digestion using
gastric and enterocutaneous fistula [Kisch B (1954) Jacob Helm's
Observations and Experiments on Human Digestion: A One
Hundred and Fiftieth Anniversary, J Hist Med Allied Sci, IX, 311-
328]
Carson and Woelfel (1913) The solubility of white lead in human
gastric juice and its bearing on the hygiene of the lead. Am J Public
Health. 3, 755-769.
Reiman C.K & MinotA.S. (1920) Absorption and Elimination of
manganese ingested as oxides and silicates. Journal of Biological
Chemistry, 45,133-143
-------
-------
Assessing Contaminant (Bio)availability in Soil
when In vitro Gastrointestinal Methods
are the Only Option
Nick Basta
Professor of Soil and Environmental Chemistry
School of Environment and Natural Resources
Ohio State University
Dr. Kirk Scheckel
National Risk Management Research Laboratory
U.S. EPA, Cincinnati, OH
Dr. Karen Bradham
National Exposure Research Laboratory
U.S. EPA, Research Triangle Park, NC
U.S. EPA
Guidance for Evaluating the Oral Bioavailability of
Metals in Soils for Use in Human Health Risk Assessment
OSWER 9285.7-80, May 2007
Recommended Criteria for Validation of Test Methods
adapted from ICCVAM
"Data generated adequately measure or predict the toxic endpoint of
interest and demonstrate a linkage between either the new test
and effects in the target species."
In vitro gastrointestinal (IVG) method must
be correlated with an acceptable in vivo model
IVG must be predictive
Correlation of IVG method with an in vivo model
ii
U) ra
§ m
at.
IVG Method Correlation Studies
most on highly contaminated soils
RBA As = 0.942 IVG + 7.11 r = 0.91*
IVG Gastric As
% Bioaccessible As
Basta et al. 2003.
Grant R825410 Final Report.
submitted to U.S. EPA ORD
Most correlation studies conducted
on highly contaminated wastes
often > 2,000 mg/kg contaminant of
concern
Estimating RBA of Pb in Soil and Soil-like materials
(OSWER 9285.7-77, May 2007)
Most of 19 solid waste materials from smelter origin
Pb content: 1,590 to 14,200 mg/kg, median 7,225 mg/kg
Estimating RBA of Arsenic in Contaminated Soils and Solid Media
(Rodriguez et al., 1999)
As content: 233 to 17,500 mg/kg, median 1,460 mg/kg
Contaminant Concentration in Soil / Solid Waste
when will bioavailability adjustments be made?
Assessing Bioavailability of
Moderately Contaminated Soil
Highly Contaminated
Risk= [Soil]
(EF)(ED)(IR)(BIO)
(BW) (AT)
example: 100 mg/kg As target
The greatest utility of IVG or in vivo methods may be
to assess risk for soils with mod. level contamination
Pb paint, pesticides, coal ash, CCA, cattle dips, etc.
Moderately
Contaminated
reasonable adjustment
Background
High level: 7,000 mg/kg total As
bioavailability < 2% to be < target
unreasonable adjustment
Moderate level: 300 mg/kg As
bioavailability < 33% to be < target
reasonable adjustment
Moderately contaminated
urban and/or old industrial sites
Page 1
-------
Bioavailable (in vivo) vs. Bioaccessible (in vitro)
Method Detection Limits and Contaminant Levels
most in vivo dosing studies require highly contaminated soil
> 500 to 5,000 mg/kg contaminant
Moderately contaminated soil levels
could be < 1000 mg/kg Pb; < 100 mg/kg As
Below in vivo detection limits
Below in vivo working range but
easily measured by IVG methods
A Strong Advantage of IVG methods
the ability to estimate (bio)availability
at moderate contaminant levels
Highly Contaminated
Moderately
Contaminated
only in vitro
Background
Are we confident to use IVG methods to Estimate
Contaminant (Bio)availability in Soil
for Moderately Contaminated Soils?
More confident to use IVG methods validated for highly
contaminated soil if the contaminant source is the same
IVG method validated for scorodite (As contaminant)
in highly contaminated smelter soil;
OK to use IVG method on soil with moderate levels of scorodite
Confident to use IVG method on soil with moderate levels of CCA
contamination? other sources of As contamination (pesticides?)
What do we need to establish confidence in using
the IVG method for CCA / other arsenic contaminated soils?
1. Evidence the IVG method is correlated with in vivo
for the CCA (other) arsenic sources
study conducted with highly contaminated CCA soil(s)
2. Contaminant (i.e., arsenic) speciation in soil
identify the source term for arsenic (CCA or other?)
How do we do it?
Contaminant Source and Speciation
contaminant source
mineral source: mineral speciation method
i.e., galena in smelter-contaminated soil
Soil-sequestered contaminant Fe OH
i.e., As sequestered by reactive soil Fe oxides o
Soil chemical speciation
methods should be used
when soil is the sink
Sir
o
O As OH
OH
soil sequestered
arsenic
Contaminant Speciation in Soil / Solid Waste
using Advanced Spectroscopic Investigation
more from Kirk Scheckel and H. Jamieson at 4 p.m.
Contaminant Speciation in Soil using Extraction
Extract Different Contaminant Pools
70
EXAFS for As
in soil
eV
Advanced Photon Source
Argonne National Laboratory
« 50
- 40
| 30
•520
5? 10
0
Soil A
Soil B
water
NaOAc
0.1 M PCM
NH2OH(HCI|
NH40X
easily dissolved
weakly adsorbed
strongly adsorbed
part of Fe oxide fraction
Based on "selective" extraction of soil contaminants
must be very carefully applied and interpreted
must have knowledge of extractant and soil / solid waste system
Page 2
-------
Contaminant Speciation is Essential
Knowledge of chemical form of the soil / solid waste
contaminant controlling bioaccessibility
contaminant species in old orchard soil same as
contaminant species in smelter soil (in vivo correlation study)?
Yes: then we are more confident to use the IVG (in vitro) method for
the orchard soil
^^^LL^/ Pesticides
in old orchards % "
A Case Study
OSU In Vitro Gastrointestinal Method
Simulated Gl extraction at 37°C
Gastric bioaccessibility and
Intestinal bioaccessibility
Development of Chemical Methods to Assess
the Availability of Arsenic in Contaminated
Media, R825410
U.S. EPA, Office of Research and Development
National Center for Environmental Research
N.T. Basta, R.R. Rodriguez, and S.W. Casteel
$431,677; Nov 1996 to October 2000.
Development and Evaluation of IVG Methods
Correlation Studies with Immature Swine
In vitro bioaccessible vs. in vivo bioavailable
Approach to Development of an IVG Method
Occam's Razor
15 As-Contaminated Soils
Swine Dosing Trial
Measure Urinary As
Bioavailable
Arsenic
Stan Casteel
Univ. of Missouri
In Vitro
Gastrointestinal Methods
Compare Bioaccessible
Arsenic
Nick Basta, Robin Rodriguez
Oklahoma State Univ.
single batch dynamic
extraction in vitro in vitro
Simple Complex
RBA Method complexity
"Pluralitas non est ponenda sine neccesitate" - William of Occam (1280-1349)
Plurality should not be assumed without necessity
If you have two equally likely solutions to a problem - pick the simplest
"Everything should be made as simple as possible, but not simpler"
-A. Einstein
Bioaccessible vs. Bioavailable
OSU IVG correlated with immature swine
OSU IVG correlation with in vivo
eo As with dosing vehicle
SS RBA AS = o 942 IVG + 7 11 r=09r* Rodriguez et al. 1999.
«f so ES&T 33:642-649
.£ 40 As without dosing vehicle
2 Basta et al., 2007. J. Environ.
5 so Health Sci. Part A 42:1275-1181.
g Special Publication (BARGE):
io 20 Bioaccessibility of Soil Contaminants
» C. Gr0n and J. Wragg (eds.)
| Pb with/out dosing vehicle
K "„ .„ ,„ ,„ „ Schroder etal., 2004
J. Environ, dual., 33:513-521.
Cd with/out dosing vehicle
Schroder et al., 2003.
ES&T37:1365-1370.
Research on OSU IVG USEPA Project
still continuing after 10 yr
% Bioaccessible As
Basta et al. 2003.
Grant R825410 Final Report.
submitted to U.S. EPA ORD
the soil isn't contaminated
1997
2007
PageS
-------
OSU IVG USEPA Project Productivity
Publications (Basta): 39
10 refereed publications
27 proceedings /abstracts (11 at international conferences)
2 (book chapter, technical report)
5 Conference Symposia (3 international)
4 Graduate Student Ph.D. dissertation and M.S. Theses
Collaborative research
Soil samples, reports, data (including bioavailability) sent to
13 research groups
many joint publications / proceedings / symposium
U.S. EPA ORD (NERL, NRMRL)
Round robin validation studies
Bioavailability Research Group of Europe (BARGE)
Arsenic Solid Phase Speciation of OSU IVG Soils
Four solid phase species identified by EXAFS
Scorodite (FeAsO4- H2O) 43 to 76% As; mean 61 % As
SorbedAs 7.3 to 28% As; mean 17% As
"Elemental" As 6.3 to 43%; mean 16%
Lollingite Oto8%; mean 4.6%
Bioavailable Arsenic and Solid Phase Speciation (EXAFS)
Arsenic identified as Scorodite inversely related
to Relative Bioavailable Arsenic
Koch et al. (2005): Soils containing scorodite had lowest bioaccessible As
of 6 soils from a military base in gastric solution
Bioavailable As and Soil Reactive Fe Oxide
Soil Chemical Extraction Methods
Intercept= 46.1
Slope= -4.91
/• = -0.91**
% As in Feox
Bioavailable As
inversely related
to As in Soil Feox pool
Arsenic Fractionation
Phosphate extractable arsenic
Phosphate will increase
bioavailable arsenic
and bioaccessible arsenic
Basta et al. 2007. JEHS Part A,
42:1275-1281
% Arsenic Extracted by Phosphate
Assessing Contaminant Bioaccessibility in Soil
when In vitro Gastrointestinal Methods
are the Only Option
We could extrapolate the OSU IVG methods for highly contaminated
smelter waste soils to soils/solid waste where scorodite or As
sequestered by Fe oxide was the source term to moderately
contaminated soils
As-sorbed to Fe oxide: A likely source term for many As-
contaminated soils and solid wastes
Arsenic Speciation by chemical extraction / EXAFS could be
performed to verify that the form of As is sorbed to Fe-oxides
Page 4
-------
U.S. EPA
Guidance for Evaluating the Oral Bioavailability of
Metals in Soils for Use in Human Health Risk Assessment
OSWER 9285.7-80, May 2007
"A detailed protocol for the test method and a description of
the known limitations of the test including a description of the
classes of materials that the test can and cannot accurately assess."
> Specify the contaminant chemical speciation and
> whether the IVG method has been correlated with in vivo for the
contaminant species in the test material
may be down the track?
Future Research Needs
Collaborative Efforts are Essential
Maximize Use of Resources ($$), Time
Research is expensive
Especially Bioavailability Data
> Share soils, bioavailability data
> Cooperative round robin studies
> etc
Thank you for your attention
More information? Please contact:
Nick Basta
School of Environment
and Natural Resources
basta.4@osu.edu
www.snr.osu.edu
Page 5
-------
-------
The bioaccessibility of soil Nickel, can it
be explained using solid phase
distribution data?
Joanna Wragg
Kingsley Dunham Centre
Keyworth
Nottingham NG12 5GG
Tel 0115 936 3100
Health Effects
ACUTE
Death
• 90 min exposure to c.400
mg rrr3 metallic Ni
• Ingestion (by a child) of
570 mg kg-1 bwNiSO4
Headache, nausea, vomiting
etc
• Inhalation of Ni carbonyl
Gl distress, muscular pain,
exacerbation of dermatitis
• After ingestion of Ni
compounds
CHRONIC
Respiratory effects
(bronchitis, emphysema,
asthma etc)
• Repeated inhalation
toxicity
Effects on organ weight and
nervous system,
reproductive toxicity etc
(animal studies)
• Repeated oral toxicity
Skin sensitivity from Ni and
its compounds
• Dermal studies
/hypersensitivity tests
Nickel in Soil
Geogenic and Anthropogenic
Sources "
• Mined predominantly as Ni-Fe
sulphide
• Dominates in ultramafic and
igneous rocks
Many industrial uses
• Ni-Steel alloys,
electroplating, jewellery,
catalyst etc...
Soil chemistry is based on the most
stable, divalent ion
Ni (II) is stable over a range of Eh and
PH conditions
Solubility increases with decreasing
l'n
Associations with numerous soil
components/minerals
Mn °xides
Sulphides
Carbonates
Organic matter
Etc
Soils Under Investigation
Organic
c
Anthropogenically Influenced
s |e Nj % H
• •
Soi| 1 c1 c 6 25 c 28%
Soil 2 c. 1.4 c. 6.4 c. 6.5%
SPeciati°"
Soil 1
Soil 2
c. 90% c. 10% Fe
Oxidic Ni* oxide & trace Ni
c. 99.6% c. 0.4% Fe
Oxidic Ni* oxide & trace Ni
*Oxidic Ni includes all fornis of Ni oxide/hydroxide,
Ni-Fe oxide/hydroxide and complex multi-metal
oxide/hydroxides
©NERC All rights rescued
Geogenically Influenced
Sample
1
2
3
4
5
6
Ni
mg kg-1
19.4
65.9
45.9
78.9
PH
7.2
6.5
7.2
6.0
7.2
5.8
Organic
C
3.52
1.69
4.70
3.02
2.15
2.62
Bioaccessibility Methods
In vitro gastric and small intestinal simulations
• <250um size fraction
• 37°C
• PBET modified from Ruby et al.,
intestinal pH
• Agitation via shaking, not Ar bubbling
IVG
• Fluids and residence times as per
Basta et al., 2007
Differences in Bioaccessibility Method
Parameters for anthropogenic Ni
Parameter
Soil:Solution Ratio
Stomach Fluid
Composition
Intestine pH
Intestine Fluid
Composition
PBET
1:100
1.25 g M pepsin
7.0
pH adjusted with
Na2C03
IVG
1:150
10 g M pepsin, 0.15M
NaCI, no citrate, malate
or acetic acid
5.5
pH adjusted with
NaHCO3
4 hours
1 hour
Intestine Residence
Time
Are the results likely to differ significantly?
-------
.ML.
Solid Phase Distribution of Nickel
CISED Test
hemometric dentification of ubstratesand lement istributions
•Separate aliquots of aqua regia of increasing
concentration " " Centrifugation
•Passed through the sample under centrifugal
force
•Determination by ICP-AES
•Chemometric data processing 045u
•Identification of physico-chemical hosts and membrane
the metal distributions within the sample L'"""" "* ' leachate
under test
Anthropogenic Contamination
Differences
Between Method
Not significant
(Stomach or
Intestine, for both •*
soils) E :
Within method |
Significant g
differences between |
stomach and s
Intestine data
Between Soils
Significant
difference between
stomach or intestine <
data «
Although there are methodological differences
SoihSolution ratio
Fluid Chemistry etc...
There is no appreciable difference in the
measured Ni bioaccessibility
£JL*. Britu. . 4 •&& BHii*h
<£f> GeotajfcJi Sumy ^PVIkT 'VI^T ^tfl 1 V*7 Ge«lo(fc»l Sum*
CISED Components - Soil 2
™ 4™ ™° Component
am lm 133°
JOCD icoo Organic
Carbonate (1)
Ca/AI mixed
^ !™ ™ ™ " assemblage
| im ™ ™ AI/Fe oxyhydroxide
1 ™ ™ o Fe oxide (1)
™o 5 10 15 ™o 5 10 15 ™o 5 10 15 Ex changeable
™ ™ Fe oxide (2)
Z ™ Fe oxide (3)
» o " Carbonate (2)
i'" »•
Soil 2 - Ni distribution
Component Composition
S
Ca
Ca-AI-Ni
AI-Fe-Ni
Fe-Ca
Ca-Mg-Fe
Fe-P
Fe-AI
Ca-Mg
fek. 4p~
Ni mg kg'1
10.7
208
1070
865
0
45.6
220
553
0
-------
3000
2500
2000
Soil 2
.ML.
' 1000
500
Natural Contamination
Not
significantly
different
P>0.05
Cluster
1
2
3
4
5
5
7
8
9
10
11
12
Name
High Carbonate
Carbonate
High Carton ate
(2)
Carbonate (2)
Organic
Fertilizer
Exchangeable
Fe/AI
Phosphate
Fe/AI
oxyhydroxide
Fe/AI silicate
Mn oxide
Fe oxide
Ni
1.67
0-2.8
0.6-0.9
0
0
0.03-0.1
0-0.1
3.72
0.5-1.3
0.04-1 .4
0.06-2.6
0.01-1.9
Briifeh
Gesbfka! Sum,
Bioaccessible
Ni associated
with
components
extracted over
the 1st 7-8
extraction
phases
RELATED
TO
Clusters
1-8 and 11
2 3 4 5 6 7 8 9 10 11 12 13 14
Extract Number
Summary
Higher total and bioaccessibility data observed for the
anthropogenic soils
No appreciable differences between the PBET and the IVG
data
Soil components acting as sources of Ni bioaccessibility were
similar for both contaminant types
• Non Fe dominated components
• Organic, Fertilizer/Exchangeable/Carbonates/Mn oxides
• Naturally contaminated soils
• Additional contribution from Fe oxyhydroxide
components
• Thought to be an effect of the different ageing of the Ni
within the soil matrix
-------
Summary (2)
Extraction Efficiencies of the CISED
• Similar for the anthropogenically contaminated
soils, c. 32%
• > extraction efficiency for the soil with a higher
total Ni content
• 5-15% for the geogenically influenced soils
• Higher extraction rates are not observed in
soils that have the highest total Ni
concentrations or absolute Ni bioaccessibilities
Final Thoughts .....
Can solid phase distribution help explain
measured bioaccessibility data?
• YES, Inclusion of geochemical information and
testing provides a wealth of additional data
• Help explain the bioaccessibility data
• Support the Risk Assessment process
• Previously adopted method for As and Pb studies
in the UK
• Being applied in to Cr issues in the UK and
Canada
Summary (3)
Anthropogenic soil
bioaccessibility data
• Significant decrease in
Ni concentration in the
intestine phase
• Not observed for
geogenic Ni soils
• Function of Ni solubility
with pH???
t « Id I) 12
Acknowledgements
Mark Cave, Chemical & Biological Hazards
Programme, British Geological Survey, UK
Ken Reimer, Kim House, Ashley Campbell, Megan
Lord-Hoyle, Environmental Sciences Group, Royal
Military College, Canada
Bruce Conard, BRConard Consulting, Canada
Chris Ollson, Jacques Whitford Ltd, Canada
-------
Importance of Metal Speciation
in Understanding Bioavailability
..to protect human health & the natural environment.
SPECIATION
Kirk G. Scheckel
US EPA, Cincinnati, OH
Remediation
Indicative of metal fate and transport
Helps decide remediation strategy, i.e.
dig-and-haul, in-situ amendment,
monitored natural attenuation
Evaluate effectiveness of remediation
Understand the variability of biologically
available metal uptake
Ability to manipulate system to reduce
bioavailability
Develop comprehensive predictive
models based on speciation
Systematic Characterization of Exposure-Dose-
Response Continuum and the Evolution of
Protective to Predictive Dose-Response Estimates
Chemical Exposure
Protective
Exposure
Toxicological Response
Qualitative
Solution Specie
Predictive
Site of
biological
response
What
do we
need
and
what
do we
want
to
know?
EFFECT OF SOURCE OF Pb ON BIOAVAILABILITV TO RATS
Q- 300
§200
m SOIL: 3,900 ppm Pb
SOIL: 810 ppm Pb
°0 10 20 30
DOSE (mg Pb/Kg BW)
EFFECTOF SOURCEOFPh ON BIOAVAILABILITY TO HUMAN
Stsse
SOURCE
BLOOD Pb (Jlg/dL)
SOIL Pb (lOOOmg/kg)
SMELTER/URBAN 1.1 - 7.6
MINING 0 - 4.8
Components of Bioavailability
Levels of Detail in Research
PHYSIOLOGICAL
• Pharmacokinetics - Nutritional needs (Ca, P,
Fe, etc)
CHEMICAL
• Form of metal
• Geochemical matrix
• Particle size
Atomic Molecular Microscopic Macroscopic Field
XRF
XPS
XAS
Requires
synchrotron
radiation.
•XRD
•TGA
• FTIR
• DRS
• Enhanced
Visual
Analysis:
1. SEM
2. TEM
3. AFM
• Field Plots
• Equilibrium
Studies
• Kinetic
Studies
• Extractions
• Visual/
Intuitive
Insight
• Field Plots
-------
Advanced Photon Source
(Argonne National Laboratory, Argonne, IL)
Principal Synchrotron Techniques
Used in Environmental Science
• X-ray Fluorescence (XRF): chemical
composition (quantification, mapping)
• X-ray Absorption Fine Structure
(XAFS) Spectroscopy: chemical
:>tx-cii!."io;i * ex; dun - vi state, coordination,
nearest neighbors)
• Surface Scattering and Diffraction:
surface structure, sorption processes
• Microtomography: 3D imaging of
internal microstructure (porosity, fluid
flow, composition)
X-ray Absorption Spectroscopy
X-ray Absorption Spectroscopy: Measure
energy-dependence of the x-ray absorption
coefficient fi(E) [either Iog(l0/I) or (If/lo)]
of a core-level of a selected element
Element Specific: Ele
XANES = X-ray Absorption Near-Edge Sp
EXAFS = Extended X-ray Absorption Fine-Stnlc
Pb Immobilization & Bioavailability
• Smelter contaminated site in Joplin, MO
• Various P amendments
• In-vitro and in-vivo bioavailability
• Synchrotron speciation
X-ray Absorption Spectroscopy &
PCA/LCF
ion in a Smelter Contaminated Soil: Results of in-situ Remediation with Phospho
Lead (Pb) Speciation (%)
Effect of Time and 1% P Treatment on Soil
Lead Bioavailability Joplin Swine Ryan et ai 2004
Control Soil (all),y = 6.44 + 198(1 -e"'0021 x), R2 = .95
250 1 % P Treated Soil (3mo), y = 3.42 + 180(1 - a'-0022*), R2 = .95
1% P Treated Soil (18 mo), y=10.02 + 150(1 -e"°°19*,, R2 = .92
\ 1 % P Treated Soil (32 mo), y = 6.09 + S2(1 - e"0""1*,, R2 = .94
5J1 200 PbOAc(all),y=6.49 + 200(1 - e''0023x), R2 = .S4
10% Biosolids 63
< 100
200 400 600 800
Lead Dose (jig Pb (pg BW day)'1)
-------
Soil Lead Bioavailability
Joplin 18 mo Sample
Conclusion: Joplin Field Experiment
1 Bioavailability of soil lead is not a simple function of
total soil lead.
Rat Swine In vitro Human
Control
Treated
7.2 21.6 21pH2.5 13.1
39pH2.0
51 pH1.5
• Soil lead bioavailability can be measured by
• Swine
• Rat
• Human
• I n vitro
• Soil lead bioavailability can be changed by addition of
materials to soil.
• The addition of materials to the soil altered the
geochemistry of soil lead.
What's next?
Do we have enough information from expensive in-
vivo animal bioavailability studies tojustify
acceptance of affordable in-vitro extraction
bioaccessibility?
What role does spectroscopic speciation play in
support of in-vivo and in-vitro research?
Requirements for using bioavailability in
risk management decisions
1) An appropriate measure (methodology and
samples)
2) Knowledge of the reason for the observed
measurement
3) Knowledge of the long-term stability of the
measurement
Lessons learned
Total metal content is not a good indicator of exposure or risk
Soil chemistry important in determination of
bioavailability/phytoavai lability
Form is important
• Particle size is important
Adsorption is important
• Fe/Mn are important adsorptive surfaces
Organic matter is important adsorptive surface
Cannot always assume an increase concentration in the foodchain
equates to increase transfer through the foodchain (plant uptake)
Predicting the potential transfer of soil metals requires a holistic
evaluation of soil, plant, animal, and human processes which may
increase or reduce the transfer (bioavailability)
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Direct Identification of Metal Compounds
in
Contaminated Soil, Mine Tailings, and
House Dust
Using Synchrotron-based Methods
H. E. Jamieson, S.R. Walker, S.E. Fawcett, Queen's University
A. Lanzirotti, University of Chicago
P. Rasmussen, Health Canada
S. Beauchemin, Natural Resources Canada
M.B. Parsons, Geological Survey of Canada
Mineral Form
FeAsS
Grain Size
Modified from Ruby et al, 1999
AS20,
Fe-As oxides
Encapsulation or Rimming of Grains
qtz As
decreasing
BIOAVAI LABILITY
Analytical Techniques
Analytical Techniques
Synchrotron-based
• Micro X-ray Fluorescence
(jjXRF): Element mapping
• Micro X-ray Diffraction (jiXRD):
Identify microcrystalline
phases
• Micro X-ray Absorption Near
Edge Structure (jiXANES):
oxidation state of As & other
elements
• Macro X-ray Absorption
(XANES): local molecular
environment
Classical
Electron Microprobe (EMPA):
quantitative chemical analysis
Petrography: visual
characterization
Synchrotron-based
• Micro X-ray Fluorescence
(jjXRF): Element mapping
• Micro X-ray Diffraction (jiXRD):
Identify microcrystalline
phases lOmfofOfl
• Micro X-ray Absorption Near
Edge Structure (jiXANES):
oxidation state of As & other
elements 10 micron
• Macro X-ray Absorption
(XANES): local molecular
environment 10mm
Classical
• Electron Microprobe (EMPA):
quantitative chemical analysis
10 mteron
• Petrography: visual
characterization
1-2 micron
Results in
grain-scale
characterization
Giant Mine: Large abandoned site near Yellowknife, NWT
Arsenopyrite-bearing
ore was roasted to
liberate Au
Total arsenic in tailings, soils, se'jy nts 10>
,
Roaster-generated Fe oxides:
nanocrystalline maghemite yFe2O3and hematite aFe2O3
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Nanocrystalline
Maghemite or
Maghemite
& Hematite
Mixture of As+3
and As+6
Walker etal (2005) Canadian Mineralogist 43, 1205-1224
Abandoned gold mines (more than 65
sites) in Nova Scotia
Montague
Arsenic concentrations
Total As content in <150 um fraction of tailings, soil, and mill residue samples (ppm)
Site
Caribou
Goldenville
Montague
N Brookfield
Max
313,000
210,000
62,100
9,170
Min
15,200
7,200
318 (soil)
195 (soil)
Median
72,600
38,900
10,600
1,590
At North Brookfield
sulfide ore was roasted
to liberate gold
Roaster Chlorination
Ml" Stacks Plant
Fe-arsenate rimmed Fe-oxyhydroxide
Three different Fe-arsenates
Almost amorphous
- two broad weak rings
Mixture of lepidocrocite and goethite
(polymorphs of FeOOH)
As2O5 = 27.9%
SO4=2.4%
-All concentrations by EPMA
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Rimmed Grain - Scorodite
Yellowish grain - Kankite
£_
Kankite
FeAsO4-3.5H2O
Yukonite
Yukonite
-Ca3Fe7(As04)e(OH)9.18H20
M^^^E'r^^MHM
Transmitted and reflected light Calks (molar) = 0.3
Tailings from gold-ore roasting
North Brookfield
Abundant roaster derived As-
bearing Fe-oxide (hematite
aFe2O3 or maghemite yFe2O3)
in some samples
68% Asi+
32% As!+
Arsenic-bearing minerals identified in IMS gold mine tailings (to date)
Information on Grain Size from jaXRD
Kankite
FeAs04-3.5H20
As-Ca bearing Fe
oxyhydroxide = Yukonite
As-bearing Fe
oxyhydroxide
Amorphous Fe arsenate
As-bearing Hematite
Many As-bearing secondary minerals are nanocrystalline, but
porosity, cementation and disaggregation affect grain size
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Metal Speciation of House Dust: |aXRF mapping
Zn macro XANES spectra of known species
Example of fitting results forZn
In contrast, Cu in
house dust is
largely bound
to organic species
General Conclusions
A combination of synchrotron and classical
mineralogical analysis can provide direct
identification of metal-bearing grains
Information on grain size and intergrowth
textures may also be provided
Most samples contain at least three mineral
forms of a particular metal
Many particles are aggregates or mixtures of
more than one mineral
Acknowledgements
Funding for this project provided by
MITHE (Metals in the Human Environment) Research
Network
NSERC
Indian and Northern Affairs Canada
Geological Survey of Canada
Health Canada
Correspondence: jamieson@geol.queensu.ca
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Bioaccessibility of arsenic
adsorbed onto or incorporated
within freshly synthesized iron
oxide minerals using the
SHIME
B.D. Laird1-2, K. Dekker1-2, T.R. Van De Wiele3, D.
Peak2, W. Verstraete3, S.D. Siciliano2
^interdisciplinary Graduate Program of Toxicology
2University of Saskatchewan, Saskatoon, Canada
3University of Ghent, Belgium
Bioaccessibility and Risk Assesment
D Total contaminant
concentration in a mineral
may overestimate exposure
D Bioaccessibility measured
using In vitro GI models as a
surrogate for bioava liability
D Not yet widely incorporated
into risk assessment
• How do we go about
validating in vitro models?
Bioaccessibility vs Concentration:
Inverse Relationships
Two Possible Mechanisms: A Cause for concern?
Thermodynamic
limitation of As
dissolution
D Bioaccessibility a
function of LS Ratio
2. Kinetic limitation of
arsenic dissolution
D Bioaccessibility a
function of residence
time
Bioaccessibility vs Concentration
D How does mineralogy impact the
relationship between bioaccessibility and
concentration?
• Amorphous Scorodite
D Arsenic incorporated into iron oxide mineral
D Readily soluble at low pH
• As(V) - Ferrihydrite
D Arsenic adsorbed onto surface
D Ferrihydrite sparingly soluble at low pH
Isotherms in Soil Chemistry
GI Microbes and As Toxicokinetics
Shape of isotherm is a function of the chemical
processes controlling dissolution
D Gastrointestinal microbes increase arsenic
bioaccessibility of mine tailings in colon stage of
SHIME
D Gastrointestinal microbial community may contain
both arsenate reducing bacteria and iron reducing
bacteria
D Small intestine is the primary site of absorption
• Duodenum & Jejunum: Lower microbial densities
• Ileum: Higher microbial densities
D Arsenic is potentially absorbed in the proximal
colon
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Hypotheses
1. Arsenic dissolution in the SHIME is at
equilibrium
D HA: Kinetics limit dissolution of arsenic in the
SHIME
2. Gastrointestinal microbes impact the
bioaccessibility of arsenic absorbed to
ferrihydrite and incorporated within scorodite
Scorodite Bioaccessibility in the
Stomach
Scorodite - Stomach
000 §
Arsenic Concentrationmjneral ™g kg"1
Scorodite Bioaccessibility in the
Small Intestine
Scorodite Isotherm: Stomach
Scorodite Small Intestine
2 Oe+5 .O
E
§
Scorodite Stomach
Arsenic Concentrationmineral mg
Concentrationso|utjon ng L"1
Scorodite Isotherm: Small Intestine
Conclusions: Arsenic Bioaccessibility
versus Concentration
Scorodite Small Intestine
I 26«
|
§ 1645
O
0.0 5.06+4 1.0645 1.5645 2.0645 2.5645
Arsenic Concentrationso|utjon fj.g L"1
When an inverse relationship between
arsenic concentration and
bioaccessibility is observed:
• As bioaccessibility from scorodite in the
stomach likely determined by LS Ratio
• As bioaccessibility from scorodite in the
small intestine may be determined by
residence time
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Conclusions: Use of Isotherms in
Bioaccessibility Research
GI Microbe Impact on Arsenic
Bioaccessibility
Use of isotherms may not be applicable for use
with real-world samples
• Mineralogy MUST be constant between samples
for interpretation of isotherm results
D Should test impact of LS ratio and residence
time when inverse relationship observed
• Current recommendations focus on LS ratio
• May result in underestimation of bioaccessibility
and exposure in risk assessments
§ 100
o
GI Microbe Impact on Arsenic
Bioaccessibility
Conclusions: Impact of GI Microbes
on arsenic bioaccessibility
3.50
3.00
Ferrihydrite-As(V)
Active Colon
Sterile Colon
GI microbes capable of increasing or decreasing
arsenic bioaccessibility from arsenic-bearing
minerals
• GI microbial activity associated with iron reduction
• Effect of GI microbes likely mineralogy-dependent
Unknown toxicological implications of this
microbial impact on bioaccessibility
• Mechanisms potentially also active in ileum
• May pose challenging for the validation of in vitro
models using in vivo animal dosing experiments
Acknowledgements
D International Society of Exposure Analysis
D NSERC MITHE-RN network
D NSERC
D University of Saskatchewan Department of Soil
Science
D University of Saskatchewan Department of
Toxicology
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