Relative Bioavailability of Arsenic in the
Flat Creek Soil Reference Material
United States OLEM 9200.2-159
Environmental
Protection Agency
A \ Relative Bioavailability of Arsenic in the Flat
Creek Soil Reference Material
December 2015
-------
TJ UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
OFFICE OF
SOLID WASTE AND
JAM 9 n Oft*!? EMERGENCY RESPONSE
JMIX £ U £U|Q OLEM -9200.0-89
MEMORANDUM
SUBJECT: Transmittal of the Five-Year Review Recommended Template
n ^-i -jj A
FROM: James E. Wool ford, Director yhi~~- ^ 4- C^/i-^C^-
Office of Superfund Remediairon and Technology Innovation
TO: Superfund National Policy Managers, Regions 1-10
PURPOSE
The purpose of this memorandum is to transmit the Five-Year Review Recommended Template. The
Five-Year Review Recommended Template amends Appendix E of the "Comprehensive Five-Year
Review Guidance," OSWER 9355.7-03B-P, June 2001.
The purpose of this guidance for the recommended template is to provide an approach for preparing
Five-Year Review (FYR) reports in a manner that is intended to promote national consistency, to reduce
non-essential information and to decrease repetitiveness in the report. With such an approach, the Office
of Superfund Remediation and Technology Innovation (OSRTI) intends for the template to reduce the
time and cost associated with both writing and reviewing FYR reports. Also, OSRTI expects the
template's application will increase the efficiency and consistency of FYR data entry into the Superfund
Enterprise Management System (SEMS).
As stated in EPA's Comprehensive Five-Year Review Guidance (OSWER Directive 9355.7-03B-P,
June 2001) on page 1-1, "[t]he purpose of a five-year review is to evaluate the implementation and
performance of a remedy in order to determine if the remedy is or will be protective of human health
and the environment." The 2001 guidance addresses the recommended process for conducting FYRs.
BACKGROUND
OSRTI began working on pilot projects with the Regional programs in 2011 to streamline the FYR
report. The Superfund Remedial Program Review Action Plan from November 2013 reemphasized the
need to "...provide a 'streamlined' FYR template for national use" to increase efficiency in site cleanup
approaches. The goal of the FYR report streamlining pilots was to explore changes that can be made to
the report to reduce non-essential information and repetitiveness, while remaining focused on the
information and conclusions necessary to evaluate whether the remedy is or will be protective of human
health and the environment. The results and conclusions of these pilots informed the development of the
Five-Year Review Recommended Template.
Internet Address (URL) • http://www.epa.gov
Recycled/Recyclable • Printed with Vegetable Oil Based Inks on 100% Postconsumer Process Chlorine Free Hecycled Paper
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Prepared by:
Stan W. Casteel, DVM, PhD, DABVT
Laura Naught, MS
Veterinary Medical Diagnostic Laboratory
College of Veterinary Medicine
University of Missouri, Columbia
Columbia, Missouri
and
Amber Bacom, MS
William Brattin, PhD
SRC, Inc.
Denver, Colorado
December 9, 2015
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ACRONYMS AND ABBREVIATIONS
ABA
AF0
ANOVA
As
As+3
As+5
DMA
D
DF
FCRM
g
GLP
ICP-MS
ICP-OES
Kb
kg
Kt
Ku
MBW
mL
MMA
MSB
N
NRC
ORD
OSRTI
PE
QC
RBA
ref
RfD
RPD
SD
SF
SSE
TM
UEF
U.S. EPA
USGS
°C
°F
Absolute bioavailability
Oral absorption fraction
Analysis of variance
Arsenic
Trivalent inorganic arsenic
Pentavalent inorganic arsenic
Dimethyl arsenic
Ingested dose
Degrees of freedom
Flat Creek Soil Reference Material
Gram
Good Laboratory Practices
Inductively coupled plasma-mass spectrometry
Inductively coupled plasma-optical emission spectrometry
Fraction of absorbed arsenic that is excreted in the bile
Kilogram
Fraction of absorbed arsenic that is retained in tissues
Fraction of absorbed arsenic that is excreted in urine
Mean body weight
Milliliter
Monomethyl arsenic
Mean squared error
Number of data points
National Research Council
Office of Research and Development
Office of Superfund Remediation and Technical Innovation
Performance evaluation
Quality control
Relative bioavailability
Reference material
Reference dose
Relative percent difference
Standard deviation
Slope factor
Sum of squared standard error
Test material
Urinary excretion fraction
United States Environmental Protection Agency
United States Geological Survey
Microgram
Degrees Celsius
Degrees Fahrenheit
OLEM 9200.2-159 December, 2015.doc
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TABLE OF CONTENTS
EXECUTIVE SUMMARY v
INTRODUCTION 1
1.1 Overview of Bioavailability 1
1.2 Using RBA Data to Refine Risk Calculations 2
1.3 Purpose of this Study 2
2.0 STUDY DESIGN 2
2.1 Test Materials 3
2.1.1 Sample Description 3
2.1.2 Sample Preparation and Analysis 3
2.2 Experimental Animals 3
2.3 Diet 4
2.4 Dosing 4
2.5 Collection and Preservation of Urine Samples 5
2.6 Arsenic Analysis 5
2.7 Quality Control 5
3.0 Data Analysis 7
3.1 Overview 7
3.2 Data Fitting 9
3.3 Calculation of RBA Estimates 12
4.0 RESULTS 12
4.1 Clinical Signs 12
4.2 Dosing Deviations 12
4.3 Background Arsenic Excretion 12
4.4 Urinary Arsenic Variance 13
4.5 Dose-Response Modeling 13
4.6 Calculated RBA Values 20
4.7 Uncertainty 20
5.0 REFERENCES 21
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LIST OF TABLES
Table 2-1. Study Design and Dosing Information 3
Table 4-1. NAXCEL Treatments 12
Table 4-2. Background Urinary Arsenic 13
Table 4-3. Urine Excretion Fraction (UEF) Estimates 15
Table 4-4. Estimated Arsenic Relative Bioavailability (RBA) for FCRM Soil 20
LIST OF FIGURES
Figure 3-1. Conceptual Model for Arsenic Toxicokinetics 8
Figure 3-2. Urinary Arsenic Variance Model 11
Figure 4-1. FCRM Data Compared to Urinary Arsenic Variance Model 14
Figure 4-2. FCRM Urinary Excretion of Arsenic: Days 6/7 16
Figure 4-3. FCRM Urinary Excretion of Arsenic: Days 9/10 17
Figure 4-4. FCRM Urinary Excretion of Arsenic: Days 12/13 18
Figure 4-5. FCRM Urinary Excretion of Arsenic: All Days 19
APPENDICES
Appendix A: Group Assignments A-l
Table A-l. Group Assignments for FCRM Arsenic Study A-2
Appendix B: Body Weights B-l
Table B-l. Body Weights B-2
Appendix C: Typical Feed Composition C-l
Table C-l: Procine Grower Produced by the University of Missouri Feed
Mill C-2
Appendix D: Urinary Arsenic Analytical Results and Urine Volumes for FCRM Study
Samples D-l
Table D-l. Urinary Arsenic Analytical Results and Urine Volumes for D-2
Appendix E: Analytical Results for Quality Control Samples E-l
Table E-l. Blind Duplicate Samples E-2
Table E-2. Laboratory Spikes E-2
Table E-3. Laboratory Quality Control Standards E-3
Table E-4. Arsenic Performance Evaluation Samples E-3
Table E-5. Blanks E-4
Figure E-l. Urinary Arsenic Blind Duplicates E-4
Figure E-2. Performance Evaluation Samples E-5
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EXECUTIVE SUMMARY
A study using juvenile swine as test animals was performed to measure the gastrointestinal
absorption of arsenic (As) from a sample of the Flat Creek Soil Reference Material (FCRM). In
conjunction with the United States Environmental Protection Agency (U.S. EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI), FCRM was developed by the
United States Geological Survey (USGS) from soil containing high concentrations of metals due
to mining activity near an abandoned lead mine in Montana. The measured arsenic
concentration of FCRM is 740 ± 57 mg/kg (mean ± standard deviation [SD]).
The relative oral bioavailability of arsenic in FCRM was assessed by comparing the absorption
of arsenic from FCRM ("test material") to that of a reference material, sodium arsenate. Groups
of swine (five per dose group) were given oral doses of the reference material or the test material
twice a day for 14 days at three target dose levels (40, 80, and 120 mg As/kg body weight/day).
A group of three untreated swine served as a control for the arsenic test groups.
The amount of arsenic absorbed by each animal was evaluated by measuring the amount of
arsenic excreted in the urine (collected over 48-hour periods beginning on days 6, 9, and 12).
The urinary excretion fraction (UEF) is the ratio of the amount excreted per 48 hours divided by
the dose given per 48 hours. UEFs were calculated for the test material and sodium arsenate
using simultaneous weighted linear regression. The relative bioavailability (RB A) of arsenic in
the test material compared to sodium arsenate was calculated as follows:
RBA =
UEF(test soil}
UEF (sodium arsenate)
Estimated arsenic RBA values (mean and 90% confidence interval) are as follows:
Estimated RBA for FCRM
Measurement
Interval
Days 6/7
Days 9/10
Days 12/13
All Days
Estimated Arsenic RBA
(90% Confidence
Interval)
0.16(0.14-0.19)
0.17(0.14-0.20)
0.17(0.15-0.19)
0.17 (0.15-0.19)
The best fit point estimate for the arsenic RBA for FCRM soil is 17%.
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INTRODUCTION
1.1 Overview of Unavailability
Reliable analysis of the potential hazard to humans from ingestion of a chemical depends upon
accurate information on a number of key parameters, including the concentration of the chemical
in the exposure medium of interest (e.g., soil, dust, water, food, air, paint), intake rates of each
exposure medium, and the rate and extent of absorption ("bioavailability") of the chemical by the
body from each ingested medium. The amount of a chemical that actually enters the body from
an ingested medium depends on the physical-chemical properties of the chemical and of the
exposure medium. For example, some metals in soil may exist, at least in part, as poorly water-
soluble minerals, and may also exist inside particles of inert matrices such as rock or slag of
variable sizes, shapes, and compositions. These chemical and physical properties may influence
(usually decrease) the absorption (bioavailability) of the metals when ingested. Thus, equal
ingested doses of different forms of a chemical in different media may not be of equal health
concern.
Bioavailability of a chemical in a particular medium may be expressed either in absolute terms
(absolute bioavailability) or in relative terms (relative bioavailability).
Absolute bioavailability (ABA) is the ratio of the amount of the chemical absorbed to the amount
ingested:
Absorbed Dose
ABA=- - — -
Ingested Dose
This ratio is also referred to as the oral absorption fraction (AF0).
Relative bioavailability (RBA) is the ratio of the AF0 of the chemical present in some test
material ("fesf") to the AF0 of the chemical in an appropriate reference material ("re/") such as
sodium arsenate (e.g., either the chemical dissolved in water or a solid form that is expected to
fully dissolve in the stomach):
RBA(test vs ref} =
For example, if 100 micrograms (ug) of a chemical dissolved in drinking water were ingested
and a total of 50 ug were absorbed into the body, the AF0 would be 50/100, or 0.50 (50%).
Likewise, if 100 ug of the same chemical contained in soil were ingested and 30 ug were
absorbed into the body, the AF0 for this chemical in soil would be 30/100, or 0.30 (30%). If the
chemical dissolved in water was used as the frame of reference for describing the relative
bioavailability of the same chemical in soil, the RBA would be 0.30/0.50, or 0.60 (60%).
For additional discussion about the concept and application of bioavailability, see Gibaldi and
Perrier (1982), Goodman et al. (1990), and/or Klaassen et al. (1996).
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1.2 Using RBA Data to Refine Risk Calculations
When reliable data are available on the RBA of a chemical in an exposure medium (e.g., soil),
the information can be used to refine the accuracy of exposure and risk calculations at that site.
RBA data can be used to adjust default oral toxicity values (reference dose [RfD] and slope
factor [SF]) to account for differences in absorption between the chemical ingested as a soluble
form of arsenic (As) and the chemical ingested in the exposure media, assuming that the toxicity
factors are also based on a readily soluble form of the chemical. For noncancer effects, the
default reference dose (RfD default) can be adjusted (RfDadjmted) as follows:
default
For potential carcinogenic effects, the default slope factor (SFdefault) can be adjusted (SFadjmted) as
follows:
^adjusted = SFdefault ' ^^
Alternatively, it is also acceptable to adjust the dose (e.g., mg/kg body weight/day) rather than
the toxicity factors as follows:
D°SeadjuSted = D°Sedefault ' RBA
This dose adjustment is mathematically equivalent to adjusting the toxicity factors as described
above.
1.3 Purpose of this Study
The objective of this study was to use juvenile swine as a test system in order to determine the
RBA of arsenic in Flat Creek Soil Reference Material (FCRM) compared to a soluble form of
arsenic (sodium arsenate).
2.0 STUDY DESIGN
The test and reference materials were administered to groups of five juvenile swine at three
different dose levels for 14 days (doses were administered in two increments each day). The
study included a non-treated group of three animals to serve as a control for determining
background arsenic levels. Study details are presented in Table 2-1. All doses were
administered orally with the dosing material mixed into a small portion of feed, which was hand
fed to the animals (see Section 2.4). The study was performed as nearly as possible within
guidelines of Good Laboratory Practices (GLP: 40 CFR 792).
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Table 2-1. Study Design and Dosing Information
Group
4
5
6
7
8
9
10
Group Name
Test material
Test material
Test material
Sodium arsenate
Sodium arsenate
Sodium arsenate
Control
Dose Material
Administered
FCRM
FCRM
FCRM
Sodium arsenate
Sodium arsenate
Sodium arsenate
Negative control
Number of
Swine in
Group
5
5
5
5
5
5
o
J
Arsenic Dose3
Target
(jig/kg Body
Weight-Day)
40
80
120
40
80
120
0
Actual15
(jig/kg Body
Weight-Day)
42
85
125
42
83
125
0
bDoses were administered in two equal portions given at 9:00 AM and 3:00 PM each day. Doses were held constant based on the
expected mean weight during the exposure interval (14 days).
Calculated as the administered daily dose divided by the measured or extrapolated daily body weight, averaged over days 0-14 for
each animal and each group.
2.1 Test Materials
2.1.1 Sample Description
The test soil used in this investigation was a sample of FCRM. The FCRM was developed by
the United States Geological Survey (USGS), in conjunction with the United States
Environmental Protection Agency (U.S. EPA) Office of Superfund Remediation and Technical
Innovation (OSRTI), from soil containing high concentrations of metals due to mining activity
near an abandoned lead mine in Montana.
2.1.2 Sample Preparation and Analysis
The USGS reported the arsenic soil concentration of FCRM sample as 740 ± 57 mg/kg soil
(mean ± standard deviation [SD]), determined using inductively coupled plasma-optical emission
spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (TCP-MS).
2.2 Experimental Animals
Juvenile swine were selected for use because they are considered to be a good physiological
model for gastrointestinal absorption in children (Weis and LaVelle, 1991; Casteel et al., 1996).
The animals were intact males of the Pig Improvement Corporation genetically defined Line 26,
and were purchased from Chinn Farms, Clarence, Missouri.
The number of animals purchased for the study was several more than required by the protocol.
These animals were purchased at an age of about 5-6 weeks (weaning occurs at age 3 weeks)
and housed in individual stainless steel cages. The animals were then held under quarantine for
1 week to observe their health before beginning exposure to dosing materials. Each animal was
examined by a certified veterinary clinician (swine specialist) and any animals that appeared to
be in poor health during this quarantine period were excluded from the study. To minimize
weight variations among animals and groups, extra animals that were most different in body
weight (either heavier or lighter) 5 days prior to exposure (day 5) were also excluded from the
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study. The remaining animals were assigned to dose groups at random (group assignments are
presented in Appendix A).
When exposure began (day 0), the animals were about 6-7 weeks old. The animals were
weighed at the beginning of the study and every 3 days during the course of the study. In each
study, the rate of weight gain was comparable in all dosing groups. Body weight data are
presented in Appendix B.
All animals were examined daily by an attending veterinarian while on study and were subjected
to detailed examination at necropsy by a certified veterinary pathologist in order to assess overall
animal health.
2.3 Diet
Animals were weaned onto standard swine chow (purchased from MFA Inc., Columbia,
Missouri) by the supplier. The feed was nutritionally complete and met all requirements of the
National Institutes of Health (NRC, 1988). The ingredients and nutritional profile of the feed are
presented in Appendix C. The measured arsenic concentration in a randomly selected feed
sample was 0.11 ug/g feed.
Beginning 5 days before the first day of dosing, each animal was given a daily amount of feed
equal to 4.0% of the mean body weight of all animals on study. Feed was reduced to 3.7% body
weight starting on day 8 of the study. Feed amounts were adjusted every 3 days, when animals
were weighed. Feed was administered in two equal portions, at 11:00 AM and 5:00 PM daily.
Drinking water was provided ad libitum via self-activated watering nozzles within each cage.
The arsenic concentration measured in six water samples from randomly selected drinking water
nozzles averaged 1.1 ug/L.
2.4 Dosing
Animals were exposed to dosing materials (sodium arsenate or test material) for 14 days, with
the dose for each day being administered in two equal portions beginning at 9:00 AM and
3:00 PM (2 hours before feeding). Swine were dosed 2 hours before feeding to ensure that they
were in a semi-fasted state. To facilitate dose administration, dosing materials were placed in a
small depression in a ball of dough consisting of moistened feed (typically about 5 g), and the
dough was pinched shut. This was then placed in the feeder at dosing time.
Target arsenic doses (expressed as jig of arsenic per kg of body weight per day) for animals in
each group were determined in the study design (see Table 2-1). The daily mass of arsenic
administered (either as sodium arsenate or as test material) to animals in each group was
calculated by multiplying the target dose (|ig/kg-day) for that group by the anticipated average
weight of the animals (kg) over the course of the study:
Mass (\igl day) = Dose (\iglkg-day) • Average Body Weight (kg)
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The average body weight expected during the course of the study was estimated by measuring
the average body weight of all animals 1 day before the study began, and then assuming an
average weight gain of 0.5 kg/day during the study. After completion of the study, the true mean
body weight was calculated using the actual body weights (measured every 3 days during the
study), and the resulting true mean body weight was used to calculate the actual dose achieved.
Any missed or late doses were recorded, and the actual doses were adjusted accordingly. Actual
doses (jig arsenic/day) for each group are shown in Table 2-1.
2.5 Collection and Preservation of Urine Samples
Samples of urine were collected from each animal for 48-hour periods on days 6-7 (U-l), 9-10
(U-2), and 12-13 (U-3) of the study. Collection began at 9:00 AM and ended 48 hours later.
The urine was collected in a plastic bucket placed beneath each cage, which was emptied into a
plastic storage bottle. Aluminum screens were placed under the cages to minimize
contamination with feces or spilled food. Due to the length of the collection period, collection
containers were emptied periodically (typically twice daily) into separate plastic bottles to ensure
that there was no loss of sample due to overflow.
At the end of each collection period, the total urine volume for each animal was measured (see
Appendix D) and three 60-mL portions were removed and acidified with 0.6 mL concentrated
nitric acid. All samples were refrigerated. Two of the aliquots were archived and one aliquot
was sent for arsenic analysis. Refrigeration was maintained until arsenic analysis.
2.6 Arsenic Analysis
Urine samples were assigned random chain-of-custody tag numbers and submitted to the
analytical laboratory for analysis in a blind fashion. The samples were analyzed for arsenic by
L.E.T., Inc. (Columbia, Missouri). In brief, 25-mL samples of urine were digested by refluxing
and then heated to dryness in the presence of magnesium nitrate and concentrated nitric acid.
Following magnesium nitrate digestion, samples were transferred to a muffle furnace and ashed
at 500°C. The digested and ashed residue was dissolved in hydrochloric acid and analyzed by
the hydride generation technique using a Perkin Elmer 3100 atomic absorption spectrometer.
This method has established that each of the different forms of arsenic that may occur in urine,
including trivalent inorganic arsenic (As+3), pentavalent inorganic arsenic (As+5), monomethyl
arsenic (MMA), and dimethyl arsenic (DMA), are all recovered with high efficiency.
Analytical results for the urine samples are presented in Appendix D.
2.7 Quality Control
A number of quality control (QC) steps were taken during this project to evaluate the accuracy of
the analytical procedures. The results for QC samples are presented in Appendix E and are
summarized below.
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Blind Duplicates (Sample Preparation Replicates)
A random selection of about 8% of all urine samples generated during the study were prepared
for laboratory analysis in duplicate and submitted to the laboratory in a blind fashion. Results
are shown in Appendix E (see Table E-l and Figure E-l).
Six of nine urine duplicate samples had relative percent differences (RPD) values that were <5%.
Values for the remaining three duplicates were 20, 29, and 180% (see Appendix E).
Spike Recovery
During analysis, water samples were spiked with known amounts of arsenic (sodium arsenate),
and the recovery of the added arsenic was measured. Results (see Table E-2) show that mean
arsenic concentrations recovered from spiked samples were within 10% of expected
concentrations.
Laboratory Duplicates
No duplicate urine samples were analyzed.
Laboratory Control Standards
Internal laboratory control standards were tested periodically during sample analysis. Recovery
of arsenic from these standards was generally good and within the acceptable range (see
Table E-3).
Performance Evaluation Samples
A number of Performance Evaluation (PE) samples (urine samples of known arsenic
concentration) were submitted to the laboratory in a blind fashion. The PE samples included
varying concentrations (20, 100, or 400 |ig/L) each of four different types of arsenic (As+3, As+5,
MMA, and DMA). The results for the PE samples are shown in Appendix E (see Table E-4 and
Figure E-2). All sample results were close to the expected values, indicating that there was good
recovery of the arsenic in all cases.
Blanks
Laboratory blank samples were run along with each batch of samples at a rate of about 10%.
Blanks never yielded a measurable level of arsenic (all results were <1 |ig/L). Results are shown
in Table E-5.
Summary of QC Results
Based on the results of all of the QC samples and the steps described above, it is concluded that
the analytical results are of sufficient quality for derivation of reliable estimates of arsenic
absorption from the test materials.
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3.0 DATA ANALYSIS
3.1 Overview
Figure 3-1 shows a conceptual model for the toxicokinetic fate of ingested arsenic. Key points
of this model are as follows:
• In most animals (including humans), absorbed arsenic is excreted mainly in the urine
over the course of several days. Thus, the urinary excretion fraction (UEF), defined as
the amount excreted in the urine divided by the amount given, is usually a reasonable
approximation of the AF0 or ABA. However, this ratio will underestimate total
absorption, because some absorbed arsenic is excreted in the feces via the bile, and some
absorbed arsenic enters tissue compartments (e.g., skin, hair) from which it is cleared
very slowly or not at all. Thus, the UEF should not be equated with the absolute
absorption fraction.
• The RBA of two orally administered materials (i.e., a test material and reference
material) can be calculated from the ratio of the UEF of the two materials. This
calculation is independent of the extent of tissue binding and of biliary excretion:
mA(tes, vs ref) =
AF0(ref) D-AF,(ref)-K, UEF(ref)
where:
D = ingested dose (ug)
Ku = fraction of absorbed arsenic that is excreted in the urine
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Figure 3-1. Conceptual Model for Arsenic Toxicokinetics
INGESTED DOSE (D)
Absorbed
» Blood
AF0
> Tissue (T)
11 » T T
tine (U)
Ki
1 "->• BilefBI
Hepatobilliary
circulation
1-AF0
Non-Absorbed
» Fo
^eo /•F^
where:
AF0 = oral absorption fraction
Kt = fraction of absorbed arsenic that is retained in tissues
Ku = fraction of absorbed arsenic that is excreted in urine
Kb = fraction of absorbed arsenic that is excreted in the bile
BASIC EQUATIONS:
Amount in Urine
UEF
Uoral-D -AFo'Ku
oral
UEForai= = AFo ' Ku
Doral
RBA
UEFx,oral_ AFo (x) -Ku_AFo
— , N
y^rai AF0(y}-Ku
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Based on the conceptual model above, the basic method used to estimate the RBA of arsenic in a
particular test material compared to arsenic in a reference material (sodium arsenate) is as
follows:
1. Plot the amount of arsenic excreted in the urine (ug per 48 hours) as a function of the
administered amount of arsenic (ug per 48 hours) for both the reference material and the
test material.
2. Find the best fit linear regression line through each data set. The slope of each line (ug
per 48 hours excreted per ug per 48 hours ingested) is the best estimate of the UEF for
each material.
3. Calculate the RBA for each test material as the ratio of the UEF for the test material
compared to UEF for the reference material:
nn,/ ~ UEF (test}
RBA(test vs ref) = ^ '
UEF(ref)
3.2 Data Fitting
A detailed description of the data-fitting methods and rationale and the methods used to quantify
uncertainty in the arsenic RBA estimates for a test material are summarized below. All data
fitting was performed in Microsoft Excel® using matrix functions.
Simultaneous Regression
The techniques used to derive linear regression fits to the dose-response data are based on the
methods recommended by Finney (1978). As noted by Finney (1978), when the data to be
analyzed consist of two dose-response curves (the reference material and the test material), it is
obvious that both curves must have the same intercept, since there is no difference between the
curves when the dose is zero. This requirement is achieved by combining the two dose-response
equations into one and solving for the parameters simultaneously, as follows:
Separate Models
Combined Model
where p(i) indicates the expected mean response of animals exposed at dose x(i), and the
subscripts r and t refer to reference and test material, respectively. The coefficients of this
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combined model are derived using multivariate regression, with the understanding that the
combined data set is restricted to cases in which one (or both) of xr and xt is zero (Finney, 1978).
Weighted Regression
Regression analysis based on ordinary least squares assumes that the variance of the responses is
independent of the dose and/or the response (Draper and Smith, 1998). It has previously been
shown that this assumption is generally not satisfied in swine-based RBA studies, where there is
a tendency toward increasing variance in response as a function of increasing dose
(heteroscedasticity) (U.S. EPA, 2007). One method for dealing with heteroscedasticity is
through the use of weighted least squares regression (Draper and Smith, 1998). In this approach,
each observation in a group of animals is assigned a weight that is inversely proportional to the
variance of the response in that group:
cr.
where:
Wi = weight assigned to all data points in dose group /'
-------
Figure 3-2. Urinary Arsenic Variance Model
^Historical Data- Controls
oHistorical Data- Sodium Arsenal
^Historical Data- Test Materials
ln(GroupMean Response)
Goodness of Fit
The goodness-of-fit of each dose-response model was assessed using the F test statistic and the
adjusted coefficient of multiple determinations (Adj R2) as described by Draper and Smith
(1998). A fit is considered acceptable if the p-value is <0.05.
Data Assessment
Arsenic data were assessed in two parts. First, the urine volumes and arsenic concentrations
were reviewed. A large volume of urine is typically indicative that a swine spilled its drinking
water into the urine collection trays. In these instances, the arsenic concentration in the diluted
urine will become very small and will be difficult to measure with accuracy. Furthermore,
because the response of the swine to arsenic dose is calculated from the product of urine
concentration and volume, the result becomes highly uncertain when the concentration is
multiplied by a volume that is not representative of the total urine volume. For this reason, in
cases where total urine volume per 24-hour period was >5 liters (more than twice the average
urine output of swine) and the measured urine concentration of arsenic was at or below the
quantitation limit (<2 |ig/L), the samples were judged to be unreliable and were excluded from
the quantitative analysis. No samples met these criteria for exclusion.
The full dataset was modeled and analyzed for individual measured responses that appeared
atypical compared to the responses from other animals in the same dose group. Responses that
OLEM 9200.2-159 December, 2015.doc
11
-------
yielded standardized weighted residuals >3.5 or <-3.5 were considered to be potential outliers
(Canavos, 1984).
3.3 Calculation of RBA Estimates
The arsenic RBA values were calculated as the ratio of the slope term for the test material data
set (bt) and the reference material data set (br):
The uncertainty range about the RBA ratio was calculated using Fieller's Theorem as described
byFinney(1978).
4.0 RESULTS
4.1 Clinical Signs
The doses of arsenic administered in this study are below a level that is expected to cause
toxicological responses in swine. No clinical signs of arsenic-induced toxicity were noted in any
of the animals used in the studies. However, one swine died prior to initiating dosing. This pig
showed no signs of illness and was replaced before dosing began. Five swine received 1 cc
Naxcel once per day for several days during the study (Table 4-1) to treat a systemic bacterial
infection (swine were found with fever >104°F).
Table 4-1. NAXCEL Treatments
Swine Number
927
908
944
946
934
Days of Treatment
-4 --2
-4 --2
-3--1
1-3
2-4
4.2 Dosing Deviations
One pig (Swine #946) missed the initial dose on day 0. This was noted during the study, but the
calculated dose amounts for days 6/7, 9/10, and 12/13 were not affected by this deviation.
4.3 Background Arsenic Excretion
Measured values for urinary arsenic excretion for control animals from days 6 to 13 are shown in
Table 4-2. Urinary arsenic concentration (mean ± SD) was 84 ± 130 |ig/L (42 ± 37 |ig/L after
excluding the outlier for swine 916, days 9 and 10). The values shown are generally within the
range of typical endogenous background urinary arsenic levels reported from other studies (see
OLEM 9200.2-159 December, 2015.doc
12
-------
Figure 3-2), although at the higher end of the detected range. This supports the view that the
animals were not exposed to any significant exogenous sources of arsenic throughout the study.
Table 4-2. Background Urinary Arsenic
Swine
Number
911
940
916
911
940
916
911
940
916
Urine Collection
Period
(Days)
6/7
6/7
6/7
9/10
9/10
9/10
12/13
12/13
12/13
Arsenic Dose
(ug per
Collection
Period)
0
0
0
0
0
0
0
0
0
Arsenic
Concentration
in Urine
(HS/L)
32
34
37
19
21
419
27
33
132
Urine
Volume
(mL)
3,520
3,400
2,520
4,085
3,340
3,300
4,600
3,940
1,320
Total Arsenic
Excreted
(ug/48 Hours)
112
114
92
76
71
1,383
124
130
174
4.4 Urinary Arsenic Variance
As discussed in Section 3.2, the urinary arsenic dose-response data are analyzed using weighted
least squares regression and the weights are assigned using an "external" variance model. To
ensure that the variance model was valid, the variance values from each of the dose groups were
superimposed on the historic data set (see Figure 4-1). As shown, aside from the control pig that
was identified as an outlier, the variance of the urinary arsenic data from this study is consistent
with the data used to generate the variance model.
4.5 Dose-Response Modeling
Urinary data for collection days 9 and 10 for control pig 916 were identified as outliers (see
Section 3.2) and were excluded from analysis. The remaining data set was analyzed (Figures 4-2
through 4-5).
All of the dose-response curves were approximately linear, with the slope of the best-fit straight
line being equal to the best estimate of the UEF. The resulting slopes (UEF estimates) for the
final fittings of the test material and corresponding reference material are shown in Table 4-3.
OLEM 9200.2-159 December, 2015.doc
13
-------
Figure 4-1. FCRM Data Compared to Urinary Arsenic Variance Model
16 -
o>
u
c
ro
n.
z;
o
C5
11 -
6 -
1 -
-4
Model-Controls
o Model- Sodium Arsenate
L. Model -Test Mate rials
* FCRM-Controls
• FCRM - Sodium Arsenate
*FCRM-Test Materials
A A
A**
A A
00
456
ln(Group Mean Response)
OLEM 9200.2-159 December, 2015.doc
14
-------
Table 4-3. Urine Excretion Fraction (UEF) Estimates
Urine Collection Period (Days)
Days 6/7
Days 9/10
Days 12/13
All days
Outliers
Excluded
0
1
0
0
Slopes (UEF Estimates)
br
0.77
0.70
0.74
0.74
bt
0.13
0.04
0.13
0.12
br = slope for reference material (sodium arsenate) dose-response; bt = slope for test material 1 (FCRM) dose-response
OLEM 9200.2-159 December, 2015.doc
15
-------
Figure 4-2. FCRM Urinary Excretion of Arsenic: Days 6/7
Reference Material (Sodium Arsenate)
4500
Dose-Response Curve
500
1000
1500 2000 2500
Arsenic Dose (ug / 48 hours)
3000
3500
4000
4500
4000 -
3500
3000 -
2500 -
2000 -
1500 -
1000 -
500 -
Test Material (FCRM)
Dose-Response Curve
500
1000
1500 2000 2500
Arsenic Dose (ug / 48 hours)
3000
3500 4000
1 4-
&
1 2
£ o<
a
T3
3 -2
1
-4 -
f
Residual Plot
O Control
» Sodium Arsenate
1 * t
* «
•
*
5 10 15
SQRT(W) " Dose
Summary of Fitting3
Parameter
a
br
bti
Covariance (br,bt)
Degrees of freedom
Estimate
100.5
0.77
0.13
0.1197
31
Standard Error
14.8
0.03
0.01
-
-
ANOVA
Source
Fit
Error
Total
SSE
662.31
31.91
694.23
DF
2
30
32
MSE
331.16
1.06
21.69
ay = a + br*Xr + bt*xt
where r = Reference Material, t = Test Material
ANOVA = analysis of variance;
DF = degrees of freedom;
MSE = mean squared error;
SSE = sum of squared
standard error
Statistic
F
P
Adjusted R2
Estimate
311.291
0.001
0.9510
TS 4
in
iY
-a 2 •
1 <
5 ° <
E
°> -4
(
I
'
)
OContro
ATest Material 1
5 10
Residual Plot
A
A
A A
1 t"
A
15 20 25 30 35 4
SQRT(W) * Dose
0
RBA and Uncertainty
RBA
Lower boundb
Upper boundb
Standard errorb
Test Material
0.16
0.14
0.19
0.015
b90% confidence interval calculated using Fieller's theorem
OLEM 9200.2-159 December, 2015.doc
16
-------
Figure 4-3. FCRM Urinary Excretion of Arsenic: Days 9/10
Reference Material (Sodium Arsenate)
Dose-Response Curve
1000 1500 2000 2500
Arsenic Dose (ug / 48 hours)
4500
4000 •
3500
3000
2500
2000
1500 -
1000
500
0
Test Material (FCRM)
0
Dose-Response Curve
1500 2000 2500
Arsenic Dose (ug / 48 hours)
m
S 2
I -
-4 •
(
Residual Plot
»
* >
t
) 5 10
SQRT(W) " Dose
o Control
»Sodium Arsenate
1
5
Summary of Fitting3
Parameter
a
br
bti
Covariance (br,bt)
Degrees of freedom
Estimate
74.6
0.70
0.12
0.1090
30
Standard Error
16.3
0.04
0.01
-
-
ANOVA
Source
Fit
Error
Total
SSE
699.09
45.51
744.60
DF
2
29
31
MSE
349.55
1.57
24.02
ay = a + br*xr + bt*xt
where r = Reference Material, t = Test Material
ANOVA = analysis of variance;
DF = degrees of freedom;
MSE = mean squared error;
SSE = sum of squared
standard error
Statistic
F
P
Adjusted R2
Estimate
222.734
0.001
0.937
4
OT
Q^ 9
T3
0)
I -2 -
^
-4 -
c
»
o Contra I
A Test Material 1
5 10
Residual Plot
A
±__ * |
t
A
15 20 25 30 35 40 45 5
SQRT(W) " Dose
0
RBA and Uncertainty
RBA
Lower boundb
Upper boundb
Standard errorb
Test Material
0.17
0.14
0.20
0.018
b90% confidence interval calculated using Fieller's theorem
OLEM 9200.2-159 December, 2015.doc
17
-------
Figure 4-4. FCRM Urinary Excretion of Arsenic: Days 12/13
Reference Material (Sodium Arsenate)
Dose-Response Curve
500
1000
1500 2000 2500 3000
Arsenic Dose (ug / 48 hours)
3500 4000
4500
4500 -
4000
3500 -
3000 -
2500 -
2000 -
Test Material (FCRM)
Dose-Response Curve
OContro I
A Test Material 1
500
1000
1500 2000 2500 3000
Arsenic Dose (ug / 48 hours)
3500 4000 4500
6 -
S 2 -
£ (
5J
5 o
n
(f)
-4 -
)
i
)
OContro
Residual Plot
•
•
T *
*
5101
SQRT(W) " Dose
5
Summary of Fitting3
Parameter
a
br
bt
Covariance (br,bt)
Degrees of freedom
Estimate
143.3
0.74
0.13
0.1459
31
Standard Error
14.4
0.02
0.01
-
-
ANOVA
ay = a + br*Xr + bt*xt
where r = Reference Material, t = Test Material
ANOVA = analysis of variance;
DF = degrees of freedom;
MSE = mean squared error;
SSE = sum of squared
standard error
Source
Fit
Error
Total
SSE
633.96
19.03
625.99
DF
2
30
32
MSE
316.98
0.63
20.41
Statistic
F
P
Adjusted R2
Estimate
499.679
0.001
0.9689
6 -
~D
<7J
S. 2 -
1 '
1 °«
"p ?
-4 -
Residual Plot
O Control
A Test Material 1
A
' A
...^ A...
1 » J
) 5 10 15 20 25 30 35 40 45 50
SQRT(W) " Dose
RBA and Uncertainty
RBA
Lower boundb
Upper boundb
Standard errorb
Test Material
0.17
0.15
0.19
0.012
b90% confidence interval calculated using Fieller's theorem
OLEM 9200.2-159 December, 2015.doc
18
-------
Figure 4-5. FCRM Urinary Excretion of Arsenic: All Days
Reference Material (Sodium Arsenate)
Dose-Response Curve
4500
1000
2000 3000 4000
Arsenic Dose (ug / 48 hours)
Summary of Fitting3
Parameter
a
br
bt
Covariance (br,bt)
Degrees of freedom
Estimate
98.4
0.74
0.12
0.1208
96
Standard Error
9.5
0.02
0.01
-
-
5000
— 4
T>
•«>
i "
a, .
S l
°<
N |
1 -2 -
-4
(
Residual Plot
* fe *»
j | ^ £
! *| **
*
) 5 10
SQRT(W) * Dose
ocontrol
• Sodium Arse nate
15
ANOVA
ay = a + br*Xr + bt*xt
where r = Reference Material, t = Test Material
ANOVA = analysis of variance;
DF = degrees of freedom;
MSE = mean squared error;
SSE = sum of squared
standard error
Source
Fit
Error
Total
SSE
2022.20
118.20
2140.40
DF
2
95
97
MSE
1011.10
1.24
22.07
Statistic
F
P
Adjusted R2
Estimate
812.643
O.001
0.9436
4000 -
3500 -
3000 -
2500 -
2000 -
1500 -
1000 -
500 -
0
0
Test Material (FCRM)
Dose-Response Curve
2000 3000
Arsenic Dose (ug / 48 hours)
4 -
n
~o
in
0? 2 C
i 1
| °c
N ^
I "2 "
i
™ .4 -
(
Residual Plot
1 A AA A
A A
1 S A i .1 A
1 | A" ~"p " a
3 8
A
) 5 10 15 20 25 30 35
SQRT(W) * Dose
oControl
ATest Material 1
A
A
A
40 45 5
0
RBA and Uncertainty
RBA
Lower boundb
Upper boundb
Standard errorb
Test Material
0.17
0.15
0.19
0.009
b90% confidence interval calculated using Fieller's theorem
OLEM 9200.2-159 December, 2015.doc
19
-------
4.6 Calculated RBA Values
Estimated RBA values (mean and 90% confidence interval) are shown in Table 4-4. As shown,
the best fit point estimate RBA of arsenic in FCRM is 17%.
Table 4-4. Estimated Arsenic Relative Unavailability (RBA)
for FCRM
Urine Collection Period
(days)
Days 6/7
Days 9/10
Days 12/13
All Days
Estimated RBA
(90% Confidence Interval)
0.16(0.14-0.19)
0.17(0.14-0.20)
0.17(0.15-0.19)
0.17 (0.15-0.19)
4.7 Uncertainty
The bioavailability estimates above are subject to uncertainty that arises from several different
sources. One source of uncertainty is the inherent biological variability between different
animals in a dose group, which in turn causes variability in the amount of arsenic absorbed by
the exposed animals. The between-animal variability results in statistical uncertainty in the best-
fit dose-response curves and, hence, uncertainty in the calculated values of RBA. Such statistical
uncertainty is accounted for by the statistical models used above and is characterized by the
uncertainty range around the RBA estimates.
However, there is also uncertainty in the extrapolation of RBA values measured in juvenile
swine to young children or adults, and this uncertainty is not included in the statistical
confidence bounds above. Even though the immature swine is believed to be a useful and
meaningful animal model for gastrointestinal absorption in humans, it is possible that there are
differences in physiological parameters that may influence RBA; therefore, RBA values in swine
may not be identical to values in children. In addition, RBA may depend on the amount and type
of food in the stomach, since the presence of food can influence stomach pH, holding time, and
possibly other factors that may influence solubilization of arsenic. RBA values measured in this
study are based on animals that have little or no food in their stomach at the time of exposure
and, hence, are likely to yield high-end values of RBA. Thus, these RBA values may be
somewhat conservative for humans who ingest the site soils along with food. The magnitude of
this bias is not known.
OLEM 9200.2-159 December, 2015.doc
20
-------
5.0 REFERENCES
Canavos, C.G. 1984. Applied Probability and Statistical Methods. Little, Brown and Co., Boston.
Casteel, S.W., Cowart, R.P., Weis, C.P., Henningsen, G.M., Hoffman, E., Brattin, W.J., Starost,
M.F., Payne, J.T., Stockham, S.L., Becker, S.V., and Turk, J.R. 1996. A swine model for
determining the bioavailability of lead from contaminated media. In: Advances in Swine in
Biomedical Research. Volume 2, Tumbleson, M.E. and Schook, L.B. (editors). Plenum Press,
New York. pp. 637-646.
Draper, N.R. and H. Smith. 1998. Applied Regression Analysis. 3rd Edition. John Wiley & Sons,
New York, NY.
Finney, DJ. 1978. Statistical Method in Biological Assay. 3rd Edition. Charles Griffin and Co.,
London.
Gibaldi, M. and Perrier, D. 1982. Pharmacokinetics. 2nd edition. Marcel Dekker, Inc, New York,
NY, pp 294-297.
Goodman, A.G., Rail, T.W., Nies, A.S., and Taylor, P. 1990. The Pharmacological Basis of
Therapeutics. 8th edition. Pergamon Press, Inc. Elmsford, NY, pp. 5-21.
Klaassen, C.D., Amdur, M.O., and Doull, J. 1996. Cassarett and Doull's Toxicology: The Basic
Science of Poisons. McGraw-Hill, Inc. New York, NY, pp. 190.
NRC. 1988. Nutrient Requirements of Swine. A Report of the Committee on Animal Nutrition.
National Research Council. National Academy Press, Washington, DC.
U.S. EPA. 2007. Estimation of Relative Bioavailability of Lead in Soil and Soil-Like Materials
by In Vivo and In Vitro Methods. U.S. Environmental Protection Agency, Office of Solid Waste
and Emergency Response, Washington DC. OSWER 9285.7-77.
Weis, C.P. and LaVelle, J.M. 1991. Characteristics to consider when choosing an animal model
for the study of lead bioavailability. In: The proceedings of the international symposium on the
bioavailability and dietary uptake of lead. Science and Technology Letters 3:113-119.
OLEM 9200.2-159 December, 2015.doc 21
-------
Appendix A: Group Assignments
OLEM 9200.2-159 December, 2015.doc A-l
-------
Table A-l. Group Assignments for FCRM Arsenic Study
Swine Number
914
948
929
952
905
906
949
942
907
946
904
917
934
939
924
903
927
945
909
935
908
910
902
912
922
944
919
928
943
951
911
940
916
Group
4
5
6
7
8
9
10
Treatment
FCRM
FCRM
FCRM
Sodium arsenate
Sodium arsenate
Sodium arsenate
Control
Target Arsenic Dose
(ug/kg-day)
40
80
120
40
80
120
0
OLEM 9200.2-159 December, 2015.doc
A-2
-------
Appendix B: Body Weights
OLEM 9200.2-159 December, 2015.doc B-l
-------
Table B-l. Body Weights
Group Info
4
TM1 40 (As)
5
TM1 80 (As)
6
TM1 120 (As)
7
NaAs 40
8
NaAs 80
9
NaAs 120
10
Control 0
Animal
Ear Tag
914
948
929
952
905
906
949
942
907
946
904
917
934
939
924
903
927
945
909
935
908
910
902
912
922
944
919
928
943
951
911
940
916
Weight (kg)
Day -5
4/4/12
11.2
13.2
12.6
12.8
12.1
12.1
12.3
12
12.2
10.5
12.5
13.9
12.1
11.2
12.2
12.2
10.2
13.1
12.5
10.1
12.3
12.7
11
13.4
13.1
12.5
13.4
10.7
11.9
10.9
11.9
10.5
12.1
Group
MBW
12.38
11.82
12.38
11.62
12.50
11.88
11.50
Day-1
4/8/12
12.1
14
13.1
13.3
12.7
13.1
12.8
12.6
14.2
10
13.3
14.1
12.7
11.8
13
13.1
11.3
13.7
13.2
11.1
13.2
13.1
12.2
14.5
13.9
12.8
14.4
12.1
12.5
11.7
12.7
11.2
12.6
Group
MBW
13.04
12.54
12.98
12.48
13.38
12.70
12.17
Day 2
4/11/12
13.2
14.9
14
14.4
13.7
14
13.9
13.8
14.8
9.6
14.7
15.2
12.6
12.7
13.9
13
11.8
14.7
14
11.8
14
14.2
13
14.8
14.7
13.6
14.9
12.8
13.4
12.4
13.6
12.8
13.5
Group
MBW
14.04
13.22
13.82
13.06
14.14
13.42
13.30
DayS
4/14/12
14
15.8
15.2
15.3
14.6
15
15
14.5
16
10.2
15.3
15.8
13.3
13.4
14.9
13.5
12.5
15.3
14.8
12.8
14.8
15.1
14
15.6
15.5
14.1
15.3
13.6
13.3
13.5
14
12
14.5
Group
MBW
14.98
14.14
14.54
13.78
15.00
13.96
13.50
DayS
4/17/12
15
16.5
15.7
16.5
15.8
15.7
16
15.3
17
11.7
16.2
16.8
14.9
14.5
15.7
14.2
13.6
16.8
16.3
14
15.9
15.9
15.3
16.6
16.5
15
16.6
14.6
14.8
15.8
15.1
13.1
15.4
Group
MBW
15.90
15.14
15.62
14.98
16.04
15.36
14.53
Day 11
4/20/1 2
16
17.8
17
17.4
16.7
17.1
16.9
16.3
17.5
12.8
17
18
15.4
15.4
16.6
14.8
17.3
17.2
14.5
14.8
16.3
16.8
16.2
17.4
17.2
16.2
18
15.5
16.1
15.5
15.8
14.3
16.6
Group
MBW
16.98
16.12
16.48
15.72
16.78
16.26
15.57
Day 14
4/23/12
17
19
18
18.4
17.9
18
18.1
18.3
17.8
14.3
18.3
19.2
16
16.6
18
16.1
15.5
18.5
18.3
16
17.5
18
17.1
18.2
18.4
17.7
18.7
16.4
18
18.2
16.6
15
17.4
Group
MBW
18.06
17.30
17.62
16.88
17.84
17.80
16.33
Group MBW = Mean body weight of each group.
OLEM 9200.2-159 December, 2015.doc
B-2
-------
Appendix C: Typical Feed Composition
OLEM 9200.2-159 December, 2015.doc C-l
-------
Table C-l. Procine Grower Produced by the University of Missouri Feed Mill
Corn
Bean Mill
Fat
Dicalcium phosphate
Limestone
Salt
Vitamins
Minerals
Zenepro
Biotin
1528 Ibs
350 Ibs
50 Ibs
34 Ibs
18 Ibs
6 Ibs
4 Ibs
3 Ibs
2 Ibs
2 Ibs
OLEM 9200.2-159 December, 2015.doc
C-2
-------
Appendix D: Urinary Arsenic Analytical Results and
Urine Volumes for FCRM Study Samples
OLEM 9200.2-159 December, 2015.doc D-l
-------
Table D-l. Urinary Arsenic Analytical Results and Urine Volumes for
FCRM Study Samples
Group
4
5
6
Material
TM
TM
TM
Collection
Period (days)
6/7
9/10
12/13
6/7
9/10
12/13
6/7
9/10
12/13
Sample ID
USGS-573
USGS-618
USGS-627
USGS-594
USGS-608
USGS-646
USGS-667
USGS-642
USGS-669
USGS-666
USGS-719
USGS-732
USGS-721
USGS-729
USGS-695
USGS-605
USGS-592
USGS-596
USGS-619
USGS-607
USGS-660
USGS-658
USGS-653
USGS-638
USGS-652
USGS-722
USGS-710
USGS-733
USGS-694
USGS-736
USGS-600
USGS-599
USGS-621
USGS-611
USGS-583
USGS-639
USGS-649
USGS-659
USGS-631
USGS-681
USGS-728
USGS-693
Swine
Number
914
948
929
952
905
914
948
929
952
905
914
948
929
952
905
906
949
942
907
946
906
949
942
907
946
906
949
942
907
946
904
917
934
939
924
904
917
934
939
924
904
917
Urinary Arsenic
Concentration
(HS/L)
7.38
65.7
57.5
146
153
7.21
60.2
51.1
125
171
15.6
88.7
52.3
189
123
219
224
221
36.6
113
226
171
54.2
88.8
689
108
248
1100
91.4
343
217
80.8
91.6
94.8
102
75.1
136
82.7
78.8
96.8
100
117
Urine
Volume
(mL)
33120
4240
4220
1420
1720
29040
3940
5220
1580
1600
19040
3660
6480
1980
2820
1580
1880
2320
10500
1860
1840
2000
7160
4115
840
4220
1780
600
5560
1380
3920
6440
6380
6115
6500
7000
4280
6380
3500
6660
8500
4700
OLEM 9200.2-159 December, 2015.doc
D-2
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Table D-l. Urinary Arsenic Analytical Results and Urine Volumes for
FCRM Study Samples
Group
7
8
9
Material
Sodium
arsenate
Sodium
arsenate
Sodium
arsenate
Collection
Period (days)
6/7
9/10
12/13
6/7
9/10
12/13
6/7
9/10
12/13
Sample ID
USGS-715
USGS-731
USGS-708
USGS-576
USGS-580
USGS-597
USGS-595
USGS-586
USGS-650
USGS-663
USGS-628
USGS-680
USGS-641
USGS-702
USGS-690
USGS-724
USGS-720
USGS-716
USGS-624
USGS-612
USGS-623
USGS-622
USGS-591
USGS-647
USGS-634
USGS-635
USGS-630
USGS-668
USGS-697
USGS-712
USGS-704
USGS-711
USGS-707
USGS-606
USGS-581
USGS-572
USGS-616
USGS-582
USGS-636
USGS-656
USGS-655
USGS-675
USGS-665
USGS-700
USGS-709
USGS-730
Swine
Number
934
939
924
903
927
945
909
935
903
927
945
909
935
903
927
945
909
935
908
910
902
912
922
908
910
902
912
922
908
910
902
912
922
944
919
928
943
951
944
919
928
943
951
944
919
928
Urinary Arsenic
Concentration
(HB/L)
110
117
60.3
208
338
133
650
512
238
375
96.4
305
694
277
436
112
527
413
274
1150
1770
628
261
405
799
1930
696
240
371
972
834
623
234
782
427
985
697
1470
432
475
853
361
1690
419
372
1320
Urine
Volume
(mL)
5920
4800
9860
3140
3140
7700
1940
2125
3220
2820
10000
2660
1560
3340
2560
8420
1980
2600
6860
2110
1360
3200
7320
2000
3120
1160
3140
7580
5840
2600
3140
3760
9600
3640
6260
2300
4320
2110
6560
6540
3180
8660
1940
7080
8000
2300
OLEM 9200.2-159 December, 2015.doc
D-3
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Table D-l. Urinary Arsenic Analytical Results and Urine Volumes for
FCRM Study Samples
Group
10
Material
Control
Collection
Period (days)
6/7
9/10
12/13
Sample ID
USGS-738
USGS-734
USGS-604
USGS-617
USGS-609
USGS-651
USGS-676
USGS-657
USGS-713
USGS-698
USGS-723
Swine
Number
943
951
911
940
916
911
940
916
911
940
916
Urinary Arsenic
Concentration
(HB/L)
1180
2040
31.7
33.6
36.7
18.5
21.3
419
27
33
132
Urine
Volume
(mL)
3000
1860
3520
3400
2520
4085
3340
3300
4600
3940
1320
OLEM 9200.2-159 December, 2015.doc
D-4
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Appendix E: Analytical Results for Quality Control
Samples
OLEM 9200.2-159 December, 2015.doc E-l
-------
Table E-l. Blind Duplicate Samples
Blind Duplicate
Sample ID
USGS-574
USGS-584
USGS-789
USGS-790
USGS-791
USGS-645
USGS-699
USGS-684
USGS-792
Sample
Type
Urine
Urine
Urine
Urine
Urine
Urine
Urine
Urine
Urine
Swine Number
942
940
934
944
911
949
912
922
929
Collection
Days
6/7
6/7
6/7
9/10
9/10
9/10
12/13
12/13
12/13
Original Sample
Concentration
(HB/L)
221
33.6
91.6
432
18.5
171
623
234
52.3
Duplicate Sample
Concentration
(HS/L)
165
33.2
91.3
23.3
15.2
169
648
231
54.4
RPD
29%
1.2%
0.3%
180%
20%
1.2%
3.9%
1.3%
3.9%
Table E-2. Laboratory Spikes
Spike Sample ID
P206030-MS1
P206030-MS2
P206030-MS3
P206031-MS1
P206029-MS1
P206029-MS2
P206029-MS3
P206029-MS4
P206029-MS5
Sample Type
Water
Water
Water
Water
Water
Water
Water
Water
Water
Original Sample
Concentration
(HB/L)
15.6
371
24
1.24
7.38
274
42.7
694
447
Added Spike
Concentration
(HB/L)
300
300
300
30
300
300
300
300
300
Measured Sample
Concentration
(HB/L)
309
688
349
37.4
295
580
351
1040
779
Recovery (%)a
98%
106%
108%
121%
96%
102%
103%
117%
111%
aValues reported by laboratory.
OLEM 9200.2-159 December, 2015.doc
E-2
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Table E-3. Laboratory Quality Control Standards
Sample ID
P206029-BS1
P206030-BS1
P206031-BS1
Associated
Sample Type
Water
Water
Water
Measured
Concentration
(HS/L)
58.7
59.7
61.4
Detection
Limit
(HS/L)
1
1
1
Analysis Date
06/16/2012
06/16/2012
06/17/2012
True Concentration
60
60
60
Recovery (%)
98%
99%
102%
Table E-4. Arsenic Performance Evaluation Samples
Sample ID
USGS-643
USGS-687
USGS-593
USGS-620
USGS-662
USGS-735
USGS-737
USGS-625
USGS-678
USGS-626
USGS-691
USGS-706
USGS-577
USGS-654
PEID
as3.100
as3.20
As3.400
as5.100
as5.20
as5.400
Ctrl
Ctrl
dmalOO
dma20
dma400
mmalOO
mma20
mma400
PE Standard
Sodium arsenite
Sodium arsenite
Sodium arsenite
Sodium arsenate
Sodium arsenate
Sodium arsenate
Control urine
Control urine
Disodium methylarsenate
Disodium methylarsenate
Disodium methylarsenate
Dimethyl arsenic acid
Dimethyl arsenic acid
Dimethyl arsenic acid
PE Concentration
(HS/L)
100
20
400
100
20
400
0
0
100
20
400
100
20
400
Sample
Concentration (jig/L)
151
60.6
498
144
57.1
493
24
34.9
139
44.1
455
149
42.7
447
Adjusted
Concentration (jig/L)
109.3
18.9
456.3
102.3
15.4
451.3
-17.7
-6.8
97.3
2.4
413.3
107.3
0.98
405.3
RPD
9%
6%
13%
2%
26%
12%
-200%
-200%
3%
158%
3%
7%
181%
1%
PE = performance evaluation. Sample concentration adjusted by subtracting mean of background arsenic (-41.7 ug/L) from sample concentration (excluding outlier for
swine 916, days 9 and 10); RPD = relative percent difference
OLEM 9200.2-159 December, 2015.doc
E-3
-------
Table E-5. Blanks
Sample ID
P206029-BLK1
P206030-BLK1
Associated Sample Type
Water
Water
Measured Concentration
<1
<1
Detection Limit
1
1
Units
Hg/L
Hg/L
Figure E-l. Urinary Arsenic Blind Duplicates
700 n
600 -
j 500 -
E
c
^ 400 -
U)
"ra
< 300 -
o
=5. 200 -
Q
100 -
0 -
{
>-x
,,-"\
,..-"" Line of Equality
S*
^
.,,*"'
,-•*'* *
+••'•'
S
) 100 200 300 400 500 600 700
Primary Analysis (ng/mL)
OLEM 9200.2-159 December, 2015.doc
E-4
-------
Figure E-2. Performance Evaluation Samples
600 -i
500 -
o> 400 -
e
o 300 -
O)
o
n
°? 200 -
o>
100 -
ro
5
04
(
Sodium Arsenite (As+5)
Line of Equality
/'
t*
/-»"
100 200 300 400 500 600
Expected (ng/mL)
MUA
600
2- 500 -
f
S" 400 -
S
o>
•g 300 -
CD
m
1
1> 200 -
3
M
(0
(1)
5 100 -
0 •<
Line of Equality
^/
tf
S
f'
,'
/'
/'
/£
}£.'
0 100 200 300 400 500 600
Expected (ng/mL)
600 -,
§ 400-
|
I 300 -
T3
0)
i 200-
o
100 -
04
(
DMA
Line of Equality
^ /
/+
s
s
f*
100 200 300 400 500 600
Expected (ng/mL)
OLEM 9200.2-159 December, 2015.doc
E-5
------- |