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P \ UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
| | WASHINGTON, D.C. 20460
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MAY ~ 5 2017 SOUD WASTE AND
EMERGENCY RESPONSE
NOW THE
OFFICE OF LAND AND
EMERGENCY MANAGEMENT
MEMORANDUM
SUBJECT: Release of Standard Operating Procedure lor an In Vitro Bioaecessihility Assay lor Lead
and Arsenic in Soil and Validation Assessment of the In Vitro Arsenic Bioaecessihility
Assay for Predicting Relative Bioavailability of Arsenic in Soils and Soil-like Materials
at Superfund Sites
FROM: Schatzi Fitz-James. Acting Director '•
Assessment and Remediation Division " (J
Ofllce of Superfund Remediation and Technology Innovation (OSRTI)
TO: Superfund National Program Managers. Regions 1-10
The purpose of this memorandum is to transmit the Technical Review Workgroup (TRW) for Metals
and Asbestos technical documents entitled "Standard Operating Procedure for an In Vitro
Bioaecessihility Assa\ for Lead and Arsenic in Soil" and "Validation Assessment of In Vitro Arsenic
Bioaecessihility Assay for Predicting Relative Bioavailability of Arsenic in Soils and Soil-like Materials
at Superfund Sites." The Standard Operating Procedure prov ides an update to EPA Method 1340
(Standard Operating Procedure for an In Vitro Bioaecessihility Assay for Lead in Soil. April 2012. LPA
9200.2-86) by including an assessment of arsenic bioaecessihility. The Validation Assessment Report
presents the basis for the Agency's determination that the In Vitro Bioaecessihility Assa> (IVBA)
method has satisfied the validation and regulatory acceptance criteria for application of the method for
arsenic.
LPA Method 1340 was first published as an SW-846 Method by LPA Office of Resource Conservation
and Recovery in 2013 for the assessment of lead bioaecessihility as a method to calculate Relative
Bioavailability (RBA) and is now regularly used at Superfund sites. Since then, the TRW has worked to
incorporate the assessment of arsenic bioaecessihility into this same method. Arsenic and lead are
commonly found together at Superfund sites and accurately measuring their RBA has a significant
impact on the risk assessment and on the selection of soil cleanup levels. The addition of arsenic to this
method allows the arsenic RBA to be measured rapidly and inexpensively. The method does not require
the use or sacrifice of animals, and the reduced cost per sample allows risk assessors to obtain a more
representative number of soil samples per exposure unit. Additionally, the incorporation of arsenic into
the already existing method for lead means that laboratories already have experience performing the
assay.
Internet Address (URL) • http://www.epa gov
Recycled/Recyclable • Printed with Vegetable Oil Based inks on 100% Postconsumer. Process Chlorine Free Reeycledfaperl 96 / 51
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I hese two documents can be accessed on the I (S HP A Supcrfund Website:
1IU[ U -\v, qv,>; n vt'p.-riiMi.i -hi.til\.'iilibiljl\ .,ir- ."iv. Please contact
Matt Lambert at la'•*»'.•)1 i-i.i.i'ie". ciu.th-y or 703-603-71 74 il \ou have any questions or concerns.
Attachments:
1. "Standard ()perating Procedure for an In Vitro BioaeeessifaiJity Assay for Lead and Arsenic in
Soil"
2. "Validation Assessment ot In Vitro Arsenic Rioaccessibilm Assav for Predicting Relative
Bioavailability ol Arsenic in Soils and Soil-like Materials at Supertund Sites,"
ec:
James Wool ford. ()l l \1 OSR I 1
Barbara Hostage, Ol.f AI OPM
Reggie Cheatham, OIJ APOHM
Barnes Johnson, Ol.l M ORCR
l)a\ id I.loyd. C)IJ-M/OBI,R
Charlotte Bertrand. OI.1MIFRR()
Carolyn Hoskinson, ( M.HM/Ol 1ST
Cyndy Macke>. Oi (\\ OSRI-
Sally Dal/ell. OKCA/FFR)
Karen Meh'in and Jill I.owe. Region 3 - Lead Region
I R\V Co min it tee Members
NARPM ("o-Cliairs
OI1IIRRAI* Members
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I 4m \
OLEM 9355.4-29
April 20, 2017
PRO**
Validation Assessment of In Vitro Arsenic Bioaccessibility Assay for Predicting Relative
Bioavailability of Arsenic in Soils and Soil-like Materials at Superfund Sites
1. Introduction
This report summarizes the basis for the Agency's determination that the IVBA method for
arsenic has satisfied the validation and regulatory acceptance criteria for application of the
method in an appropriate regulatory context. Validation and regulatory acceptance criteria
developed by the U.S. Environmental Protection Agency (U.S. EPA, 2007a), as adapted from the
Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM,
1997), have been applied to an in vitro arsenic bioaccessibility (IVBA) assay described in detail
by Brattin et al. (2013). The arsenic IVBA method estimates site-specific relative bioavailability
(RBA) of arsenic in soils quickly and inexpensively relative to in vivo methods. The arsenic
IVBA assay is well suited for regulatory use in arsenic risk assessment for several reasons:
(1) the assay does not sacrifice animals; (2) the reduced cost and analysis time from use of the
IVBA assay in place of in vivo RBA assays will facilitate greater numbers of soil samples
analyzed at each site to improve representativeness; (3) regulatory acceptance of the arsenic
IVBA assay would lower bioavailability assessment costs by enabling simultaneous assessments
of RBA for both arsenic and lead using the existing Standard Operating Procedure (SOP) for the
IVBA extraction protocol, which has been previously validated for assessment of RBA of lead in
soil (U.S. EPA 2009, 2012a); and (4) some of the U.S. EPA Regional laboratories and
commercial laboratories have analytical and quality control experience with the SOP gained
from use of the identical assay for lead.
2. Validation Assessment of the In Vitro Arsenic Bioaccessibility Assay
This section discusses the validation criteria established in the Agency soil bioavailability
guidance (U.S. EPA, 2007a). Criteria for method validation and regulatory acceptance were
consolidated because many of the criteria overlap.
2.1. Scientific and regulatory rationale for the test method, including a clear statement of
its proposed use, should be available.
The scientific and regulatory rationale for the arsenic IVBA method is presented in the
following:
U.S. EPA. (2007a) Guidance for Evaluating the Bioavailability of Metals in Soils for Use
in Human Health Risk Assessment. OSWER 9285.7-80. May 2007. Available online at
https://semspub.epa.gov/work/ll/175333.pdf
U.S. EPA. (2012b) Recommendations for Default Value for Relative Bioavailability of
Arsenic in Soil. OSWER 9200.1-113. December 2012. Available online at
https://semspub.epa.gov/work/ll/175338.pdf
Regulatory and scientific rationale. The Guidance for Evaluating the Bioavailability of Metals
in Soils for Use in Human Health Risk Assessment (U.S. EPA, 2007a) articulates the regulatory
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rationale for determining the bioavailability of metals from soils when assessing human health
risks at hazardous waste sites:
Accounting for potential differences in oral bioavailability of metals in different exposure
media can be important to site risk assessment (U.S. EPA, 1989). This is true for all
chemicals, but is of special importance for ingested metals. This is because metals can
exist in a variety of chemical and physical forms, and not all forms of a given metal are
absorbed to the same extent. For example, a metal in contaminated soil may be absorbed
to a lesser extent than when ingested in drinking water or food. Thus, if the oral RfD or
CSF for a metal is based on studies using the metal administered in water or food, risks
from ingestion of the metal in soil might be overestimated. Even a relatively small
adjustment in oral bioavailability can have significant impacts on estimated risks and
cleanup goals. (U.S. EPA, 2007a)
The Recommendations for Default Value for Relative Bioavailability of Arsenic in Soil (U.S.
EPA, 2012b) document articulates the regulatory rationale for site-specific assessment of arsenic
bioavailability in soils:
The current default assumption for assessing risk from arsenic in soil is that the
bioavailability of arsenic in soil is the same as the bioavailability of arsenic in water
(relative bioavailability [RBA] soil/water = 100%). However, recent bioavailability
studies conducted in animal models show that bioavailability of arsenic in soil is
typically less than that of highly water soluble forms of arsenic (e.g., sodium arsenate
dissolved in water). This suggests that bioavailability of arsenic in soil will typically be
lessthan that of arsenic dissolved in drinking water (i.e., RBA<100%). At sites where
this applies, the default assumption ofRBA=100% will result in an over estimation of
risk. (U.S. EPA, 2012b)
In general, the Agency (U.S. EPA, 2007a) recommends that efforts be made to collect
data that support site-specific estimates, rather than relying on the default value
recommended in this memorandum which may not accurately represent arsenic RBA at
any specific site. Use of the national default in place of site specific estimates may
underestimate or overestimate risk. Where development of site-specific RBA estimates is
not feasible (e.g., screening-level assessments), the default value of 60% can be used,
recognizing that the default value is an estimate that is not likely to be exceeded at most
sites and is preferable to the assumption of an RBA equal to 100%. (U.S. EPA, 2012b)
2.2. Relationship of the test method endpoint(s) to the endpoint of interest must be
described.
The endpoint of interest for risk assessment is a prediction of the oral RBA of arsenic in soil
(ratio of oral bioavailability of arsenic in soil to that of water-soluble arsenic) based on a
measurement of IVBA of arsenic in soil (solubility of arsenic in soil at gastric pH). The test soil
sample is assayed for IVBA, and the corresponding RBA is predicted from a regression model
relating IVBA and RBA. This same approach has been validated by EPA for predicting RBA of
lead in soil from IVBA (U.S. EPA, 2009).
The IVBA assay for predicting RBA of arsenic in soil is the same extraction procedure validated
for predicting the RBA of lead in soil (U.S. EPA, 2009, 2012a). In brief, the IVBA assay
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consists of incubating a 1 g soil sample with end-over-end mixing in 100 mL of 0.4 M glycine
buffer (pH 1.5) for 1 hour at 37°C (body temperature).
The regression model for predicting RBA of arsenic in soil from IVBA is based on a meta-
analysis of concordant data from studies in mice and swine (Bradham et al., 2011, 2013; Brattin
et al., 2013; Juhasz et al., 2009, 2014a). Data were combined into a validation dataset consisting
of paired IVBA and RBA measurements made on 83 soils collected from different sites and
mineral types, including mining, smelting, and pesticide or herbicide application (see Section 2.3
for mineral types). Paired measurements of IVBA and RBA for each of the 83 soil samples were
included in a weighted linear regression model (Equation 1) in which IVBA and RBA were
based on their respective variances (1/variance). The estimated slope is 0.79 ± 0.01 (SE) and
intercept is 3.0 ± 0.1 (SE). The equation of the model is:
RBA(%) = 0.79TVBA(%) + 3.0 Eq. (1)
This model explains approximately 87% of the variance in RBA (weight-adjusted R2 = 0.87).
The 95% prediction limit for a single RBA measurement was ±19% RBA. A detailed description
of the derivation of the regression model is provided in Diamond et al. (2016). This regression
model could be updated periodically by incorporating more data sets as they become available.
2.3. A detailed protocol for the test method must be available and should include a
description of the materials needed, a description of what is measured and how it is
measured, acceptable test performance criteria (e.g., positive and negative control
responses), a description of how data will be analyzed, a list of the materials for which
the test results are applicable, 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.
Standard Operating Procedure. The arsenic IVBA assay extraction protocol is the same as
SOP 92000.2-86 for the IVBA assay for lead in soil (U.S. EPA, 2012a, 2017). EPA has
developed an SOP specifically for arsenic that includes the SOP 09000.2-86 extraction protocol
along with the corresponding analytical procedures for measuring arsenic in the soil and soil-like
materials and extracts. The IVBA method is included under the validated methods tab on the
SW-846 website as Method 1340 for lead, which will be updated to include arsenic.
Aside from the standard laboratory glassware, reagents, supplies, and equipment, the materials
needed for the IVBA assay include 0.4 M glycine (free base, reagent-grade glycine in deionized
water, adjusted to a pH of 1.50 ± 0.05 at 37°C using trace metal-grade concentrated hydrochloric
acid), and either a water bath or an incubated air chamber with sample rotator is necessary for the
extraction of the samples at 37°C. In addition, reference standards NIST 2710a SRM or Flat
Creek SRM need to be purchased for use as the control soils in the QA/QC samples. These
materials and equipment do not require a large investment from laboratories interested in
performing the IVBA assay.
The IVBA assay is meant to measure the fraction of the amount of ingested arsenic that would be
solubilized at the low pH of the stomach. The samples are sieved at 150 |im to mimic the
fraction of soil that is likely to stick to human hands and thereby be ingested (U.S. EPA, 2016).
The samples are then extracted in a 0.4 M glycine solution, pH 1.5 at 37°C for 1 hour with
rotation to mimic gastric conditions. Following the extraction by IVBA assay, the concentration
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of arsenic in the extraction solution is measured by ICP-MS or ICP-AES. The total
concentration of arsenic in the sample is measured by SW-846 Method 3051 A.
As part of the quality control/quality assurance for the IVBA assay, the method requires that a
set of quality control samples be run in a batch of samples. Quality control samples are reagent
blank (extraction fluid that is not run through the extraction procedure), method blank (extraction
fluid that has been run through the extraction procedure), laboratory control sample (LCS;
extraction fluid spiked with arsenic that is run through the extraction procedure), matrix spike
(spiked matrix, e.g., soil, that is run through the extraction procedure), duplicate sample, and
control soil. Control limits and frequency for each quality control sample for arsenic are shown
in Table 1.
Table 1. Recommended Control Limits for Quality Control Samples for Arsenic
Quality Control Samples
Frequency
Control Limits for Arsenic
Reagent blank
once per batch
(minimum 1 in 20 samples)
<25 |ig/L arsenic
Method blank
once per batch
(minimum 1 in 20 samples)
<50 |ig/L arsenic
LCS (10 mg/L)
once per batch
(minimum 1 in 20 samples)
85-115% recovery
Matrix spike (10 mg/L)
once per batch
(minimum 1 in 10 samples)
75-125% recovery
Duplicate sample
once per batch
(minimum 1 in 10 samples)
±20% RPD
NIST 2710aa
once per batch
(minimum 1 in 20 samples)
32.9-49.1%
RPD = Relative percent difference
aAppendix A
The % IVBA for a sample is determined from the analytical results by Equation 2.
IVBA(%) = [(Asext X Vext)/(ASsoil x Soilmass) x 100 Eq. (2)
where:
ASext
= mass concentration of arsenic in the IVBA extract (mg/L)
Vext
= IVBA extract solution volume (L)
ASsoil
= total arsenic concentration (as determined by SW-846 Method 3051A or equivalent)
(mg/kg)
Soilmass
= mass of soil extracted by IVBA (kg)
Equation 1 is applied to the % IVBA results to determine the % RBA (see section 2.2).
Applicable test materials. Application of the IVBA method SOP is expected to yield predictions
of RBA for individual soil samples that fall within the prediction interval of the assay
(±19 RBA%). The prediction interval was based on results from various sources, including
mining, smelting, or pesticide applications. Although arsenic mineralogy has not been
evaluated for all soils in the data set, the following arsenic mineral phases were identified:
sorbed Asv and As111, arsenic trioxide, arsenopyrite, lollingite, realgar, scorodite, and a variety
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of arsenic-metal oxides (Bradham et al., 2011, 2013, 2015; Brattin et al., 2013; Juhasz et al.,
2007). It is possible that some soils may fall outside of the established prediction interval as a
result of an unusual arsenic mineralogy or soil composition not represented in the validation
dataset. Therefore, whenever a sample is suspected of containing an unusual and/or untested
source material or arsenic mineralogy, this should be identified as a potential data gap and source
of uncertainty in the resulting prediction of RBA. As additional samples with a variety of new
and different arsenic forms are tested by both in vivo and in vitro methods, the range of
applicability of the method should be refined and expanded.
Assay limitations. The following uncertainties may apply to applications of the IVBA assay.
i. Sample arsenic concentration limits: The arsenic concentrations of soils tested in the
development of the regression model relating IVBA and RBA and its associated
prediction interval for the IVBA assay ranged from 40 to 13,000 ppm. This validation
range should be sufficient for most applications of the methodology. Although there is
no basis for predicting what errors would necessarily be introduced into the predictions of
RBA if sample concentrations outside this range were used in the IVBA assay, use of
such samples without validating comparisons with results of an in vivo assay will
introduce additional uncertainty into estimates of RBA. However, applications of the
IVBA assay to such high arsenic concentrations (e.g., >7,000 ppm) are unlikely to change
risk management decisions; thus, this limitation is not a serious constraint for the utility
of the method to support cleanup decisions. If additional data suggests modification of
the limits, then the Agency will issue additional guidance. In addition, the minimum soil
concentration in the sample is determined by that which is measurable in the assay using
the SOP.
ii. Particle size: Soil samples in the validation dataset were sieved for particles less than
250 [j,m. Particle size can be expected to affect dissolution of arsenic embedded in soil
particles (Kama et al., 2017). Therefore, additional uncertainty will be associated with
RBA estimates from IVBA assays of soil samples having particle sizes excluded from the
validation dataset (i.e., >250 (j,m) U.S. EPA recommends a sieving size of <150 [j,m to
represent the particle fraction having the highest likelihood of incidental ingestion (Ruby
and Lowney, 2012; U.S. EPA, 2016). Arsenic IVBA in soils sieved to <250 |im were not
different from IVBA measured in soils sieved to <150 |im (Kama et al., 2017).
iii. Uncertainty in predicted RBA value: The IVBA assay for arsenic measures IVBA for
a test soil and converts this to an estimate of RBA using a regression equation estimated
from a meta-analysis of 83 samples. The predicted RBA is the most likely (highest
probability) estimate corresponding to the IVBA, but the actual RBA (if measured in
vivo) might be either higher or lower than the predicted value. The 95% prediction limit
for the arsenic IVBA-RBA regression model is relatively narrow in the context of its
application to risk assessment, ±19 RBA%. This means that there will be a 95%
probability that individual RBA measurements will be ±19 of the RBA% predicted
from IVBA. In general, the most likely estimate of RBA is the most appropriate value
for use in risk assessments because there is an equal probability of the true RBA being
above or below the predicted value; however, other values from within the RBA
prediction interval could also be evaluated as part of an uncertainty analysis.
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iv. Predicting RBA in humans: The IVBA assay was developed to predict arsenic RBA in
humans, although there are no data in humans to provide a direct validation of RBA
predictions in humans. Therefore, the arsenic IVBA assay was evaluated with estimates
of RBA made from studies conducted in two different juvenile swine bioassays and a
mouse bioassay. The use of animals for establishing arsenic RBA values to be used in
regulatory contexts has several precedents: (1) a national default soil arsenic RBA, to
be used when site-specific estimates are not available (it is always better to collect and
analyze site-specific data than to rely on a default value), was derived based on a large
sample of soil RBA measurements made in mice, monkeys, and swine (U.S. EPA,
2012a,c); (2) an IVBA assay was validated for predicting lead RBA based on soil RBA
measurements made in a swine assay (U.S. EPA, 2009); and (3) animal bioassays (e.g.,
mice, monkeys, swine) remain valid for establishing site-specific soil arsenic and lead
RBA, but are not recommended because it is better to run IVBA analyses on many
samples (e.g., a statistical sample) than to rely on a smaller number of samples analyzed
in animal bioassays (U.S. EPA, 2007b, 2010). Significantly greater costs and time to
complete will limit the number of animal bioassays.
Although there is no quantitative support for discerning which animal bioassay provides a
more accurate prediction of arsenic RBA in humans, RBA estimates obtained from the
mouse and swine assays are in close agreement (Bradham et al., 2013; Juhasz et al.,
2014b).
2.4. The extent of within-test variability and the reproducibility of the test within and
among laboratories must have been demonstrated. The degree to which sample
variability affects this test reproducibility should be addressed.
Within-test variability . Precision of the IVBA protocol was assessed with analyses of soils
included in the validation dataset, which included contributions from three laboratories. Each
laboratory achieved consistent and relatively low coefficients of variation (CV=standard
deviation/mean): 2.1, 4.0, and <5% (Brattin et al., 2013; Diamond et al., 2016).
Inter-laboratory reproducibility. An inter-laboratory comparison of the IVBA was conducted
with four participating laboratories: ACZ Laboratories Inc.; EPA Region 7 laboratory; EPA
Region 8 laboratory; and University of Colorado at Boulder (Brattin et al., 2013). Each
laboratory applied the IVBA method to analyses (in triplicate) of 12 test soils. Average within-
laboratory variability (coefficient of variation, CV) ranged from 1.3 to 11.0%. The inter-
laboratory coefficient ranged from 2.2 to 15% (mean: 5.4%).
Effects of sample variability: The prediction interval for the IVBA assay was derived based on
analysis of 83 soil samples from a variety of site types: mining, smelting, or pesticide application.
The IVBA range for the soil samples was 0-80% (mean: 27.2 ± 20 SD). The within-laboratory
coefficient of variation for IVBA was <0.05 (Diamond et al., 2016).
2.5. The test method performance must have been demonstrated using reference materials
or test materials representative of the types of substances to which the test method
will be applied, and should include both known positive and known negative agents.
Performance with reference materials. Precision of the IVBA protocol was assessed with
replicate arsenic analyses of standard reference materials (SRMs; National Institute of Standards
and Technology [NIST] SRM 271 OA) conducted by the EPA Office of Research and
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Development National Exposure Research Laboratory [ORD NERL]) over several years
(Appendix B). The mean relative percent difference ranged from -10.2 to 9.6% (mean: -0.14 ±
5.3% SD).
Performance with representative materials. The prediction interval for the IVBA assay was
derived based on analysis of samples having a variety of arsenic mineral phases from a variety of
different types of sites: mining, smelting, and pesticide application.
2.6. Sufficient data should be provided to permit a comparison of the performance of a
proposed substitute test with that of the test it is designed to replace.
The IVBA assay is a cost-effective and time-saving alternative to in vivo RBA assays that can
improve data quality by increasing the number of samples analyzed while reducing costs and turn-
around time. For the dataset used to derive the regression model, the model accounted for
approximately 87% of the observed variance in RBA. The 95% prediction interval for the model
is ±19 RBA%, based on 83 soil samples from a variety of site types that are expected to be
typical applications of the assay for site risk assessment (mining, smelting, and or pesticide
application). The standard errors for the RBA estimates for this sample of 83 soils ranged from
0.2 to 20% (median 2%), and the ratios of the SE to the mean RBA (SE/mean) ranged from 0.02
to 0.48 (median 0.09).
2.7. Data supporting the validity of a test method should be obtained and reported in
accordance with Good Laboratory Practices (GLPs).
Data supporting validity of the IVBA assay are reported in detail in a published report (Diamond
et al., 2016). Data used in the analysis is provided in Appendix C.
2.8. Data supporting the assessment of the validity of the test method must be available for
review.
Data supporting the assessment of the validity of the IVBA assay are available online at
http://www.tandfonline.eom/doi/full/10.1080/15287394.2015.l 134038.
2.9. The methodology and results should have been subjected to independent scientific
review.
The arsenic IVBA methodology was reviewed by EPA scientists and evaluated in several peer-
reviewed publications (Bradham et al., 2011, 2013, 2015; Brattin et al., 2013; Juhasz et al., 2009,
2014a,b). The report describing derivation of the prediction regression model was reviewed by
the EPA Office of Superfund Remediation and Technology Innovation (OSRTI) Technical
Review Workgroup Bioavailability Committee, EPA ORD peer-review for release of
publication, and editorial peer-review for publication (Diamond et al., 2016).
2.10. The method should be time and cost effective.
Costs of assessment of a soil sample using the IVBA assay are expected to range from
approximately 10-fold to 100-fold less than the costs of a bioassay. Time requirements for the
IVBA assay are expected to range from approximately 10-fold to 50-fold less than that required
to conduct an in vivo bioassay (i.e., days compared to several weeks). Additional cost and time
efficiencies are expected for applications at sites where arsenic and lead are chemicals of interest
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because the same IVB A extraction protocol can be used to predict arsenic and lead RBA. These
efficiencies can be used to analyze a greater number of samples.
2.11. The method should be one that can be harmonized with similar testing requirements
of other agencies and international groups.
Other international efforts (e.g., Australia, Canada, European Union, United Kingdom) are
pursuing the development of methods for in vitro assessment of RBA of arsenic and of other
metals and inorganic contaminants in soil. The IVBA assay is directly applicable to these
national and international programs. It satisfies the Bioaccessibility Research Canada (BARC)
acceptance criteria for use in risk assessment (BARC, 2016; Koch and Reimer, 2012) and the
IVBA assay has been used widely to characterize soil arsenic bioaccessibility; recent examples
of international use include reports from Africa, Australia, Canada, China, and Great Britain
(Dodd et al., 2013; Ettler et al., 2012; Juhasz et al., 2015; Koch and Reimer 2012; Kribek et al.,
2014; Li et al., 2015a,b; Meunier et al., 2010; Morales et al., 2015; Silvetti et al., 2014; Wang et
al., 2012; Yang et al., 2015). The meta-analysis that forms the basis for the predictive regression
model for RBA included contributors from the United States and Australia (Diamond et al.,
2016). Various EPA and non-government laboratories provided data to support the validation.
2.12. The method should be suitable for international acceptance.
The IVBA assay is suitable for international acceptance (see section 2.11 for further discussion).
2.13. The method must provide adequate consideration for the reduction, refinement, and
replacement of animal use.
The IVBA assay replaces bioassays and will decrease the use of animals for assessing RBA of
arsenic in soil.
3. Summary
The IVBA assay for arsenic has been evaluated against validation criteria established by EPA
(U.S. EPA, 2007a) for validation of test methods to be used in a regulatory context. All
validation criteria have been satisfied. SOPs have been established and tested for intra-
laboratory precision and inter-laboratory reproducibility. The quantitative relationship between
the IVBA assay output and output from in vivo animal bioassays, which the IVBA assay is meant
to replace, has been reliably established. The description in the method SOP is expected to yield
predictions of RBA that fall within acceptable prediction limits for applications in arsenic site
risk assessment. The prediction interval is based on assays of samples collected from a variety of
arsenic mineral phases from a variety of different sites and, as a result, the method is expected to
be widely applicable to soil typically encountered at arsenic waste sites. Based on this
assessment, EPA concludes that the IVBA method is valid for predicting RBA of arsenic in soils
in support of site-specific risk assessments. The following regression model is recommended for
applications to risk assessment (Equation 1):
RBA(%)=IVBA(%)• 0.79+3.0(%) Eq. (1)
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The Agency strongly encourages use of this methodology when implemented in context with the
decision framework described in its soil bioavailability guidance (U.S. EPA, 2007a).
4. References
BARC (Bioaccessibility Research Canada). (2016) Checklist for minimum criteria for in vitro
bioaccessibility tests (June, 2014). Available online at
http://bioavailabilitvresearch.ca/downloads.html. Accessed January 26, 2016.
Bradham, KD; Scheckel, KG; Nelson, CM; Seales, PE; Lee, GE; Hughes, MF; Miller, BW;
Yeow, A; Gilmore, T; Serda, SM; Harper, S; Thomas, DJ. (2011) Relative bioavailability and
bioaccessibility and speciation of arsenic in contaminated soils. Environ Health Perspect
119:1629-1634.
Bradham, KD; Diamond, GL; Scheckel, KG; Hughes, MF; Casteel, SW; Miller, BW; Klotzbach,
JM; Thayer, WC; Thomas, DJ. (2013) Mouse assay for determination of arsenic bioavailability
in contaminated soils. J Toxicol Environ Health A 76:815-826.
Bradham, KD; Nelson, C; Juhasz, AL; Smith, E; Scheckel, K; Obenour, DR; Miller, BW;
Thomas, DJ. (2015) Independent data validation of an in vitro method for the prediction of the
relative bioavailability of arsenic in contaminated soils. Environ Sci Technol 49:6313-6318.
Brattin, W; Drexler, J; Lowney, Y; Griffin, S; Diamond, G; Woodbury, L. (2013) An in vitro
method for estimation of arsenic relative bioavailability in soil. J Toxicol Environ Health, Part
A: Current Issues 76(7):458-478.
Diamond, GD; Bradham, KD; Brattin, WJ; Burgess, M; JW; Griffin, S; Hawkins, CA; Juhasz,
AL; Klotzbach, JM; Nelson C; Lowney, YW; Scheckel, KG; Thomas, DJ. (2016) Predicting
oral bioavailability of arsenic in soil from in vitro bioaccessibility. J Toxicol Environ Health,
Part A: Current Issues. 79:165-173.
Dodd, M; Rasmussen, PE; Chenier, M. (2013) Comparison of Two In Vitro Extraction Protocols
for Assessing Metals' Bioaccessibility Using Dust and Soil Reference Materials. Hum Ecol Risk
Assess 19(4): 1014-1027.
Ettler, V; Kribek, B; Majer, V; Knesl, I; Mihaljevic, M. (2012) Differences in the
bioaccessibility of metals/metalloids in soils from mining and smelting areas (Copperbelt,
Zambia). J Geochem Explor 113:68-75.
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
97-3981. National Institute of Environmental Health Sciences, Research Triangle Park, N.C.
Available online at http://ntp.niehs.nih.gov/iccvam/docs/about docs/validate.pdf. Accessed
January 26, 2016.
9
-------
Juhasz, AL; Smith, E; Weber, J; Rees, M; Rofe, A; Kuchel, T; Sansom, L; Naidu, R. (2007) In
vitro assessment of arsenic bioaccessibility in contaminated (anthropogenic and geogenic) soils.
Chemosphere 69:69-78.
Juhasz, AL; Weber, J; Smith, E; Naidu, R; Rees, M; Rofe, A; Kuchel, T; Sansom, L. (2009)
Assessment of four commonly employed in vitro arsenic bioaccessibility assays for predicting in
vivo relative arsenic bioavailability in contaminated soils. Environ Sci Technol 43:9487-9494.
Juhasz, AL; Herde, P; Herde, C; Boland, J; Smith, E. (2014a) Validation of the predictive
capabilities of the Sbrc-G in vitro assay for estimating arsenic relative bioavailability in
contaminated soils. Environ Sci Technol 48:12962-12969.
Juhasz, AL; Smith, E; Nelson, C; Thomas, DJ; Bradham, K. (2014b) Variability associated with
As in vivo-in vitro correlations when using different bioaccessibility methodologies. Environ Sci
Technol 48:11646-11653.
Juhasz, A.L., Herde, P., Herde, C., Boland, J., Smith, E. (2015) Predicting Arsenic Relative
Bioavailability Using Multiple In Vitro Assays: Validation of in Vivo-in Vitro Correlations.
Environ Sci Technol 49(18): 11167-11175.
Kama, R.R., Noerpel, M., Betts, A.R., Scheckel, K.G. (2017) Lead and arsenic bioaccessibility
and speciation as a function of soil particle size. J. Environ. Qual. DOI: 10.2134/jeq2016.10.0387
Koch, I; Reimer, KJ. (2012) Bioaccessibility extractions for contaminant risk assessment. In:
Comprehensive Sampling and Sample Preparation, Volume 3; Pawliszyn, J; Le, XC; Li, X; et
al.; Eds. Elsevier, Academic Press: Oxford, UK, pp 487-507.
Kribek, B; Majer, V; Pasava, J; Kamona, F; Mapani, B; Keder, J; Ettler, V. (2014)
Contamination of soils with dust fallout from the tailings dam at the Rosh Pinah area, Namibia:
Regional assessment, dust dispersion modeling and environmental consequences. J Geochem
Explor PartC 144:391-408.
Li, HB; Li, J; Zhu, YG; Juhasz, AL; Ma, LQ. (2015a) Comparison of arsenic bioaccessibility in
house dust and contaminated soils based on four in vitro assays. Sci Total Environ 532:803-811.
Li, J; Li, K; Cui, XY; Basta, NT; Li, LP; Li, HB; Ma, LQ. (2015b) In vitro bioaccessibility and
in vivo relative bioavailability in 12 contaminated soils: Method comparison and method
development. Sci Total Environ 532:812-820.
Meunier, L; Wragg, J; Koch, I; Reimer, KJ. (2010) Method variables affecting the
bioaccessibility of arsenic in soil. J Environ Sci Health A Tox Hazard Subst Environ Eng
45(5):517-526.
Morales, NA; Martinez, D; Garcia-Meza, JV; Labastida, I; Armienta, MA; Razo, I; Lara, RH.
(2015) Total and bioaccessible arsenic and lead in soils impacted by mining exploitation of Fe-
oxide-rich ore deposit at Cerro de Mercado, Durango, Mexico. Environ Earth Sci 73(7):3249-
3261.
10
-------
Ruby, MV; Lowney YW. (2012) Selective soil particle adherence to hands: Implications for
understanding oral exposure to soil contaminants. Environ Sci Technol 46:12759-12771.
Silvetti, M; Castaldi, P; Holm, PE; Deiana, S; Lombi, E. (2014) Leachability, bioaccessibility
and plant availability of trace elements in contaminated soils treated with industrial by-products
and subjected to oxidative/reductive conditions. Geoderma 214-215:204-212.
U.S. EPA (U.S. Environmental Protection Agency). (1989) Risk Assessment Guidance for
Superfund. Volume I. Human Health Evaluation Manual (Part A). EPA/540/1-89/002 (as cited
in U.S. EPA 2007a).
U.S. EPA (U.S. Environmental Protection Agency). (2007a) Guidance for Evaluating the
Bioavailability of Metals in Soils for Use in Human Health Risk Assessment. OSWER 9285.7-
80. May 2007. Available online at http://semspub.epa.gov/src/document/HQ/175333. Accessed
January 26, 2016.
U.S. EPA (U.S. Environmental Protection Agency). (2007b) Estimation of Relative
Bioavailability of Lead in Soil and Soil-like Materials Using In Vivo and In Vitro Methods.
OSWER 9285.7-77. May 2007. Available online at
http://semspub.epa.gov/src/document/HQ/175416. Accessed January 26, 2016.
U.S. EPA (U.S. Environmental Protection Agency). (2009) Validation Assessment of In Vitro
Lead Bioaccessibility Assay for Predicting Relative Bioavailability of Arsenic in Soils and Soil-
like Materials at Superfund Sites. U.S. Environmental Protection Agency, Office of Solid Waste
and Emergency Response: Washington, DC. OSWER 9200.3-51. Available online at
http://semspub.epa.gov/src/document/HQ/175417. Accessed January 26, 2016.
U.S. EPA (U.S. Environmental Protection Agency). (2010) Relative Bioavailability of Arsenic in
Soils at 11 Hazardous Waste Sites using In Vivo Juvenile Swine. OSWER 9200.0-76. June 2010.
Available online at http://semspub.epa.gov/src/document/HQ/175341 and
http:// semspub .epa. gov/ src/document/HQ/175340 (Appendix A). Accessed January 26, 2016.
U.S. EPA (U.S. Environmental Protection Agency). (2012a) Standard Operating Procedure for
an In Vitro Bioaccessibility Assay for Lead in Soil. EPA 9200.2-86. April 2012. Available
online at http://semspub.epa.gov/src/document/HQ/174533. Accessed January 26, 2016.
U.S. EPA (U.S. Environmental Protection Agency). (2012b) Recommendations for Default
Value for Relative Bioavailability of Arsenic in Soil. U.S. Environmental Protection Agency,
Office of Solid Waste and Emergency Response: Washington, DC. OSWER 9200.1-113.
Available online at http:// semspub .epa. gov/work/11/175338. pdf. Accessed January 26, 2016.
U.S. EPA (U.S. Environmental Protection Agency). (2012c) Compilation and Review of Data on
Relative Bioavailability of Arsenic in Soil. U.S. Environmental Protection Agency, Office of
Solid Waste and Emergency Response: Washington, DC. OSWER 9200.1-113. Available online
at http://nepis.epa.gov/Exe/ZyPDF.cgi/P 100FKWO,PDF?Dockev=P 100FKWO.pdf. Accessed
January 26, 2016.
11
-------
U.S. EPA (U.S. Environmental Protection Agency). (2016) Recommendations for Sieving Soil
and Dust Samples at Lead Sites for Assessment of Incidental Ingestion. U.S. Environmental
Protection Agency, Office of Solid Waste and Emergency Response: Washington, DC. OLEM
Directive 9200.1-128. Available online at https://semspub.epa.gov/work/HQ/100000133.pdf.
Accessed October 5, 2016.
U.S. EPA (U.S. Environmental Protection Agency). (2017) Standard Operating Procedure for an
In Vitro Bioaccessibility Assay for Lead and Arsenic in Soil. OLEM 9200.2-164. February
2017. Available online at: not currently posted
Wang, C; Zhao, Y; Pei, Y. (2012) Investigation on reusing water treatment residuals to remedy
soil contaminated with multiple metals in Baiyin, China. J Hazard Mat 237-238:240-246.
Yang, K; Im, J; Jeong, S; Nam, K. (2015) Determination of human health risk incorporating
experimentally derived site-specific bioaccessibility of arsenic at an old abandoned smelter site.
Environ Res 137:78-84.
12
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APPENDIX A
Provisional Reference Values for Arsenic IVBA of NIST 2710A Standard Reference
Material
Consensus values for In Vitro Bioaccessibility (IVBA) of arsenic in soil reference materials (RM) are
needed to support the Standard Operating Procedures (SOP) for determination of arsenic IVBA in soil.
EPA intends to conduct multi-laboratory evaluations of arsenic IVBA for NIST 271 OA and USGS Flat
Creek RMs. and has conducted similar evaluations of lead IVBA for these RMs. Until the arsenic IVBA
evaluations are completed, EPA recommends using the provisional reference values for NIST 2710A in
Table A-l. Although, the provisional reference values are based on data from only two laboratories, the
estimated prediction interval (±20%) is in the range observed for lead IVBA reference values (Table A-2).
The data on which the arsenic IVBA reference values are based are provided in Tables A-3 (summary)
and A-4 (individual replicates).
A-l
-------
Table A-l. Recommended Provisional Reference Value for Arsenic IVBA% of NIST 2710A
Laboratory
Reference
Material
Laboratory
Analysis
Total Soil
Arsenic Method
Units
Number of
Replicates
Lower 99%
Prediction
Limit
Mean
Upper 99%
Prediction
Limit
PI as Percent
of Mean
All Labs3
NIST2710A
Arsenic IVBA
NIST Certificate13
0/
/O
131
32.9
41.0
49.1
± 19.8
aData provided by Karen Bradham (EPA PRD NERL) and John Drexler (University of Colorado)
bNIST certificate median soil arsenic concentration: 1400 mg/kg
Table A-2. Reference Values for Lead IVBA% of Standard Reference Materials
Laboratory
Reference
Material
Laboratory
Analysis
Total Soil Lead
Method
Units
Number of
Replicates
Lower 99%
Prediction
Limit
Mean
Upper 99%
Prediction
Limit
PI as Percent
of Mean
QATS Round Robin
NIST2710A
Lead IVBA
NIST Certificate
0/
/O
35
60.7
67.5
74.2
±10
QATS Round Robin
NIST2711A
Lead IVBA
NIST Certificate
0/
/o
35
75.2
85.7
96.2
±12.3
QATS Round Robin
Flat Creek
Lead IVBA
EPA 3051A
0/
/o
30, 35a
56.0
71.0
86.0
±21.1
aBased on n=35 estimates of total Pb (mg/kg) and 30 estimates of IVBA Pb (mg/kg)
A-2
-------
Table A-3. Values for Arsenic IVBA% of NIST 2710A Based Data from Individual Laboratories and Combined Data
Laboratory3
Reference
Material
Laboratory
Analysis
Total Soil
Arsenic Method
Units
Number of
Replicates
Lower 99%
Prediction
Limit
Mean
Upper 99%
Prediction
Limit
PI as Percent
of Mean
EPA NERL
NIST2710A
Arsenic IVBA
NIST Certificate
0/
/o
117
33.1
40.8
48.4
± 18.8
U Colorado
NIST2710A
Arsenic IVBA
NIST Certificate
0/
/o
14
30.7
43.0
55.2
±28.5
All Labs
NIST2710A
Arsenic IVBA
NIST Certificate
0/
/o
131
32.9
41.0
49.1
± 19.8
aData provided by Karen Bradham (EPA PRD NERL) and John Drexler (University of Colorado)
A-3
-------
Table A-4. NIST 2710A Arsenic IVBA Replicate Data Used in Calculation of Provisional Reference
Values
Replicate
Laboratory3
Soil Mass
(g)
Extracted As
(mg/L)
Total Soil Asb
(mg/kg)
As IVBA
(%)
1
EPA NERL
1.00
5.59
1400
39.9
2
EPA NERL
1.00
5.56
1400
39.6
3
EPA NERL
1.00
5.33
1400
38.0
4
EPA NERL
1.00
5.14
1400
36.7
5
EPA NERL
1.00
6.40
1400
45.6
6
EPA NERL
1.00
6.40
1400
45.6
7
EPA NERL
1.00
5.98
1400
42.7
8
EPA NERL
1.00
6.15
1400
43.9
9
EPA NERL
1.00
5.46
1400
38.9
10
EPA NERL
1.00
5.82
1400
41.4
11
EPA NERL
1.00
6.39
1400
45.5
12
EPA NERL
1.00
5.25
1400
37.5
13
EPA NERL
1.00
5.26
1400
37.6
14
EPA NERL
1.00
5.19
1400
37.1
15
EPA NERL
1.00
5.54
1400
39.5
16
EPA NERL
1.00
5.43
1400
38.8
17
EPA NERL
1.00
5.52
1400
39.3
18
EPA NERL
1.00
5.20
1400
37.0
19
EPA NERL
1.00
5.08
1400
36.3
20
EPA NERL
1.00
5.19
1400
37.0
21
EPA NERL
1.00
5.24
1400
37.4
22
EPA NERL
1.00
6.01
1400
42.9
23
EPA NERL
1.00
5.57
1400
39.7
24
EPA NERL
1.00
5.58
1400
39.6
25
EPA NERL
1.00
5.66
1400
40.4
26
EPA NERL
1.00
5.25
1400
37.4
27
EPA NERL
1.00
5.25
1400
37.5
28
EPA NERL
1.00
5.51
1400
39.4
29
EPA NERL
1.00
4.89
1400
35.0
30
EPA NERL
1.00
5.61
1400
40.0
31
EPA NERL
1.00
5.36
1400
38.2
32
EPA NERL
1.01
5.94
1400
42.1
33
EPA NERL
1.00
5.86
1400
41.8
34
EPA NERL
1.00
5.84
1400
41.6
35
EPA NERL
1.00
4.83
1400
34.4
36
EPA NERL
1.00
5.12
1400
36.5
37
EPA NERL
1.00
5.29
1400
37.7
38
EPA NERL
1.00
5.88
1400
41.9
A-4
-------
Table A-4. NIST 2710A Arsenic IVBA Replicate Data Used in Calculation of Provisional Reference
Values
Replicate
Laboratory3
Soil Mass
(g)
Extracted As
(mg/L)
Total Soil Asb
(mg/kg)
As IVBA
(%)
39
EPA NERL
1.00
5.69
1400
40.6
40
EPA NERL
1.00
5.88
1400
41.8
41
EPA NERL
1.00
5.70
1400
40.6
42
EPA NERL
1.00
5.44
1400
38.8
43
EPA NERL
1.00
5.35
1400
38.2
44
EPA NERL
1.00
5.38
1400
38.3
45
EPA NERL
1.00
5.37
1400
38.3
46
EPA NERL
1.00
5.42
1400
38.7
47
EPA NERL
1.00
5.30
1400
37.9
48
EPA NERL
1.00
5.10
1400
36.3
49
EPA NERL
1.00
6.00
1400
42.7
50
EPA NERL
1.00
5.21
1400
37.1
51
EPA NERL
1.00
5.19
1400
37.0
52
EPA NERL
1.00
6.29
1400
44.8
53
EPA NERL
1.00
5.92
1400
42.1
54
EPA NERL
1.00
5.64
1400
40.1
55
EPA NERL
1.00
5.60
1400
39.9
56
EPA NERL
1.00
5.73
1400
40.8
57
EPA NERL
1.00
5.90
1400
42.0
58
EPA NERL
1.00
5.59
1400
39.9
59
EPA NERL
1.00
5.55
1400
39.5
60
EPA NERL
1.00
5.73
1400
40.7
61
EPA NERL
1.00
5.95
1400
42.4
62
EPA NERL
1.00
5.83
1400
41.6
63
EPA NERL
1.00
5.63
1400
40.2
64
EPA NERL
1.00
5.64
1400
40.2
65
EPA NERL
1.00
6.18
1400
44.1
66
EPA NERL
1.00
5.70
1400
40.6
67
EPA NERL
1.00
5.39
1400
38.3
68
EPA NERL
1.00
5.85
1400
41.6
69
EPA NERL
1.00
6.14
1400
43.7
70
EPA NERL
1.00
6.05
1400
43.1
71
EPA NERL
1.00
6.53
1400
46.6
72
EPA NERL
1.00
6.13
1400
43.7
73
EPA NERL
1.00
6.35
1400
45.3
74
EPA NERL
1.00
6.21
1400
44.2
75
EPA NERL
1.00
5.24
1400
37.3
76
EPA NERL
1.00
5.60
1400
40.0
A-5
-------
Table A-4. NIST 2710A Arsenic IVBA Replicate Data Used in Calculation of Provisional Reference
Values
Replicate
Laboratory3
Soil Mass
(g)
Extracted As
(mg/L)
Total Soil Asb
(mg/kg)
As IVBA
(%)
77
EPA NERL
1.00
6.05
1400
43.1
78
EPA NERL
1.00
5.99
1400
42.6
79
EPA NERL
1.00
5.45
1400
38.9
80
EPA NERL
1.00
5.73
1400
40.8
81
EPA NERL
1.00
5.79
1400
41.2
82
EPA NERL
1.00
5.55
1400
39.5
83
EPA NERL
1.01
6.09
1400
43.1
84
EPA NERL
1.00
5.68
1400
40.4
85
EPA NERL
1.00
5.28
1400
37.6
86
EPA NERL
1.00
5.26
1400
37.5
87
EPA NERL
1.00
5.50
1400
39.2
88
EPA NERL
1.01
5.67
1400
40.2
89
EPA NERL
1.00
5.36
1400
38.2
90
EPA NERL
1.01
5.70
1400
40.5
91
EPA NERL
1.00
5.68
1400
40.4
92
EPA NERL
1.01
5.48
1400
38.8
93
EPA NERL
1.01
5.35
1400
37.9
94
EPA NERL
1.00
5.62
1400
40.0
95
EPA NERL
1.00
5.63
1400
40.1
96
EPA NERL
1.01
5.94
1400
42.0
97
EPA NERL
1.00
6.57
1400
46.9
98
EPA NERL
1.00
5.77
1400
41.2
99
EPA NERL
1.00
6.14
1400
43.8
100
EPA NERL
1.00
6.50
1400
46.5
101
EPA NERL
1.01
6.36
1400
44.9
102
EPA NERL
1.01
6.14
1400
43.5
103
EPA NERL
1.01
6.62
1400
46.7
104
EPA NERL
1.01
6.21
1400
44.0
105
EPA NERL
1.01
6.70
1400
47.5
106
EPA NERL
1.00
6.45
1400
46.1
107
EPA NERL
1.00
5.73
1400
40.8
108
EPA NERL
1.01
5.87
1400
41.7
109
EPA NERL
1.01
5.98
1400
42.5
110
EPA NERL
1.00
6.04
1400
43.0
111
EPA NERL
1.00
5.42
1400
38.6
112
EPA NERL
1.00
5.49
1400
39.1
113
EPA NERL
1.01
6.15
1400
43.6
114
EPA NERL
1.01
6.63
1400
46.9
A-6
-------
Table A-4. NIST 2710A Arsenic IVBA Replicate Data Used in Calculation of Provisional Reference
Values
Replicate
Laboratory3
Soil Mass
(g)
Extracted As
(mg/L)
Total Soil Asb
(mg/kg)
As IVBA
(%)
115
EPA NERL
1.01
5.93
1400
42.0
116
EPA NERL
1.01
6.14
1400
43.5
117
EPA NERL
1.00
6.44
1400
45.9
118
U. Colorado
1.00
5.10
1400
36.3
119
U. Colorado
1.02
5.22
1400
36.7
120
U. Colorado
1.01
5.69
1400
40.3
121
U. Colorado
1.01
6.55
1400
46.5
122
U. Colorado
1.00
6.69
1400
47.7
123
U. Colorado
1.00
6.34
1400
45.1
124
U. Colorado
1.00
6.75
1400
48.2
125
U. Colorado
1.00
6.45
1400
46.1
126
U. Colorado
1.00
6.34
1400
45.2
127
U. Colorado
1.01
6.46
1400
45.8
128
U. Colorado
1.02
5.79
1400
40.4
129
U. Colorado
1.01
5.69
1400
40.3
130
U. Colorado
1.00
5.68
1400
40.4
131
U. Colorado
1.01
6.02
1400
42.4
aData provided by Karen Bradham *(EPA ORD NERL) and John Drexler, University of Colorado
bNIST certificate median soil arsenic concentration
A-7
-------
APPENDIX B
Replicate IVBA results for NIST2710A (March 2010 - January 2015)
EPA Office of Research and Development National Exposure Research Laboratory
Replicate
IVBA (%)
RPD
1
42.4
3.9
2
40.0
-1.9
3
38.5
-5.7
4
37.2
-9.2
5
40.9
0.3
6
37.6
-8.1
7
39.5
-3.2
8
43.7
6.9
9
42.5
4.1
10
42.8
4.8
11
40.9
0.3
12
39.6
-2.9
13
38.8
-5.0
14
40.9
0.3
15
41.6
2.0
16
39.0
-4.4
17
42.5
4.1
18
36.8
-10.2
19
43.4
6.2
20
43.3
6.0
21
42.5
4.1
22
42.8
4.8
23
40.9
0.3
24
39.9
-2.2
25
39.6
-2.9
26
44.9
9.6
27
38.4
-6.0
Mean
40.8.
-0.14
SD
2.2
5.32
Min
36.8
-10.25
Maximum
44.9
9.63
B-l
-------
APPENDIX C
Data Used for Meta-analysis of IVBA Assay for Predicting Oral RBA of Arsenic
ID
As Source
Soil As
(PPm)
IVBA
(%)
IVBA SD
(%)
RBA
(%)
RBA SE
(%)
RBA Assay
1
Mining/smelting
676
13.0
0.7
38.1
1.6
Swine UEF
2
Mining/smelting
313
32.5
1.6
52.4
2.0
Swine UEF
3
Pesticide (orchard)
290
21.0
1.1
31.0
4.0
Swine UEF
4
Pesticide (orchard)
388
18.6
0.9
40.8
1.8
Swine UEF
5
Pesticide (orchard)
382
19.4
0.4
48.7
4.7
Swine UEF
6
Pesticide (orchard)
364
30.6
1.5
52.8
2.3
Swine UEF
7
Mining/smelting
234
8.8
0.3
17.8
3.2
Swine UEF
8
Mining/smelting
367
6.0
0.3
23.6
2.4
Swine UEF
9
Mining/smelting
181
50.4
2.5
50.7
5.9
Swine UEF
10
Mining
200
78.0
3.9
60.2
2.7
Swine UEF
11
Mining
3957
11.0
0.6
18.6
0.9
Swine UEF
12
Mining/smelting
590
55.1
2.8
44.1
2.3
Swine UEF
13
Mining/smelting
1400
42.2
0.6
41.8
1.4
Swine UEF
14
Mining/smelting
312
41.8
2.1
40.3
3.6
Swine UEF
15
Mining/smelting
983
33.2
1.7
42.2
3.8
Swine UEF
16
Mining/smelting
390
40.3
0.7
36.7
3.3
Swine UEF
17
Mining/smelting
813
22.0
1.1
23.8
2.4
Swine UEF
18
Mining/smelting
368
18.7
0.9
21.2
2.1
Swine UEF
19
Mining/smelting
516
18.6
0.9
23.5
2.6
Swine UEF
20
Herbicide (railway corridor)
267
57.3
2.2
72.2
19.9
Swine AUC
21
Herbicide (railway corridor)
42
42.7
0.8
41.6
6.6
Swine AUC
22
Herbicide (railway corridor)
1114
17.2
0.4
20.0
9.5
Swine AUC
23
Herbicide (railway corridor)
257
10.5
0.1
10.1
2.5
Swine AUC
24
Herbicide (railway corridor)
751
22.2
0.0
22.5
2.2
Swine AUC
25
Herbicide (railway corridor)
91
80.0
0.3
80.5
6.9
Swine AUC
26
Pesticide (dip site)
713
17.8
0.1
29.3
8.7
Swine AUC
27
Pesticide (dip site)
228
55.4
0.6
43.8
5.6
Swine AUC
28
Mining
807
40.0
0.1
41.7
4.4
Swine AUC
29
Mining
577
3.8
0.0
7.0
2.9
Swine AUC
30
Gossan
190
19.0
0.2
16.4
5.2
Swine AUC
31
Gossan
88
14.0
0.2
12.1
4.9
Swine AUC
32
Pesticide
275
5.7
0.2
10.8
0.7
Swine AUC
33
Pesticide
210
7.7
0.4
12.9
1.2
Swine AUC
34
Pesticide
81
41.7
1.1
6.8
1.2
Swine AUC
35
Pesticide
358
6.5
0.1
10.1
3.5
Swine AUC
36
Pesticide
200
13.1
0.3
10.9
3.9
Swine AUC
37
Pesticide
215
7.2
0.2
18.2
3.8
Swine AUC
C-l
-------
Data Used for Meta-analysis of IVBA Assay for Predicting Oral RBA of Arsenic
ID
As Source
Soil As
(PPm)
IVBA
(%)
IVBA SD
(%)
RBA
(%)
RBA SE
(%)
RBA Assay
38
Pesticide
981
9.7
0.2
16.4
3.6
Swine AUC
39
Pesticide
1221
15.1
0.6
15.7
1.9
Swine AUC
40
Mining
949
52.9
0.1
45.8
2.6
Swine AUC
41
Mining
1126
36.9
1.1
30.7
4.1
Swine AUC
42
Mining
1695
38.1
1.3
27.5
0.7
Swine AUC
43
Mining
1306
78.4
0.4
70.5
6.8
Swine AUC
44
Mining
2270
43.5
3.4
36.2
1.5
Swine AUC
45
Mining
244
18.1
0.40
15.5
1.3
Mouse UEF
46
Mining
173
6.8
0.80
14.1
1.2
Mouse UEF
47
Mining
6899
17.5
0.60
14.7
1.0
Mouse UEF
48
Mining
280
53.6
0.20
39.9
1.7
Mouse UEF
49
Mining
4495
8.8
0.10
14.5
1.6
Mouse UEF
50
Mining
448
22.8
0.6
17.2
0.5
Mouse UEF
51
Mining
195
25.7
3.4
18.8
2.7
Mouse UEF
52
Mining/smelting
837
18.2
2.70
11.2
0.3
Mouse UEF
53
Mining/smelting
182
32.9
0.20
26.7
1.8
Mouse UEF
54
Mining/smelting
990
73.1
0.60
48.7
2.4
Mouse UEF
55
Mining/smelting
829
74.3
1.30
49.7
2.1
Mouse UEF
56
Mining/smelting
379
53.2
0.50
51.6
2.1
Mouse UEF
57
Pesticide (orchard)
322
18.8
0.30
26.3
1.4
Mouse UEF
58
Pesticide (orchard)
462
16.1
0.40
35.2
2.0
Mouse UEF
59
Pesticide (orchard)
401
18.0
0.20
20.9
2.2
Mouse UEF
60
Pesticide (orchard)
422
27.9
0.80
35.0
1.8
Mouse UEF
61
Pesticide (orchard)
340
35.4
1.90
33.2
2.4
Mouse UEF
62
Pesticide (orchard)
396
48.1
0.80
46.4
1.4
Mouse UEF
63
Pesticide (dip site)
965
9.0
0.40
21.7
1.5
Mouse UEF
64
Pesticide (dip site)
313
36.4
1.30
29.1
1.7
Mouse UEF
65
Herbicide (railway corridor)
246
47.0
2.10
45.1
2.7
Mouse UEF
66
Herbicide (railway corridor)
108
27.0
0.80
23.8
1.9
Mouse UEF
67
Herbicide (railway corridor)
184
11.9
0.20
23.0
1.8
Mouse UEF
68
Herbicide (railway corridor)
981
54.3
2.50
36.3
1.3
Mouse UEF
69
Mining
573
3.5
0.30
6.4
0.3
Mouse UEF
70
Mining
583
21.2
0.20
14.2
0.3
Mouse UEF
71
Gossan
239
12.3
0.70
20.4
1.9
Mouse UEF
72
Mining
197
21.9
0.20
29.0
2.7
Mouse UEF
73
Mining
884
16.9
0.40
23.2
3.3
Mouse UEF
74
Mining
293
12.3
0.30
17.9
0.7
Mouse UEF
75
Mining
223
17.3
0.10
19.8
1.9
Mouse UEF
76
Mining
494
15.5
0.10
18.0
1.8
Mouse UEF
C-2
-------
Data Used for Meta-analysis of IVBA Assay for Predicting Oral RBA of Arsenic
ID
As Source
Soil As
(PPm)
IVBA
(%)
IVBA SD
(%)
RBA
(%)
RBA SE
(%)
RBA Assay
77
Mining
738
13.4
3.50
11.2
0.9
Mouse UEF
78
Mining
777
0.0
0.00
4.3
0.7
Mouse UEF
79
Mining
943
0.1
0.00
3.0
0.2
Mouse UEF
80
Mining
898
0.1
0.00
1.9
0.2
Mouse UEF
81
Mining
668
0.0
0.00
3.6
0.3
Mouse UEF
82
Mining/smelting (SRM)
601
54.0
4.10
42.9
1.2
Mouse UEF
83
Mining/smelting (SRM)
1513
41.8
1.70
42.1
1.1
Mouse UEF
84
Mining/smelting (SRM)
879
14.5
0.20
14.6
0.8
Mouse UEF
As, arsenic; AUC, area under the curve; ID, sample identification number; IVBA, in vitro
bioaccessibility; RBA, relative bioavailability; SD, standard deviation; SE, standard error; SRM,
standard reference material; UEF, urinary excretion fraction
C-3
------- |