EPA 9200.2-86
                                                                    April 2012
                          Standard Operating Procedure for an
                     In Vitro Bioaccessibility Assay for Lead in Soil

          1.1 The purpose of this standard operating procedure (SOP) is to define the
    proper analytical procedure for the validated in vitro bioaccessibility assay for lead in
    soil (U.S. EPA, 2007b), to describe the typical working range and limits of the assay,
    quality assurance, and to indicate potential interferences. At this time, the method
    described herein has only been validated for lead in soil (U.S. EPA, 2007b).

          1.2 The SOP described herein is typically applicable for the characterization of
    lead bioaccessibility in soil. The assay may be varied or changed as required and
    dependent upon site conditions, equipment limitations, or limitations imposed by the
    procedure. Users are cautioned that deviations in the method from the assay described
    herein may impact the results (and the validity of the method). Users are strongly
    encouraged to document any deviations as well as any comparisons with other
    methods and associated Quality Assurance (QA) in any report.

          1.3 This document is intended to be used as reference for developing site-
    specific Quality Assurance Project Plans (QAPPs) and Sampling and Analysis Plans
    (SAPs), but not intended to  be used as a substitute for a site-specific QAPP or a
    detailed SAP or laboratory Standard Operating Procedure.  The information contained
    in this method is provided by EPA as guidance to be used by the analyst and the
    regulatory community in making judgments necessary to generate results that meet the
    data quality objectives for the intended application.

          1.4 Mention of trade names or commercial products does not constitute
    endorsement or recommended use by U.S. EPA.


      Reliable analysis of the  potential hazard to children from ingestion of lead in the
environment depends on accurate information on a number of key parameters, including (1) lead
concentration in environmental media (soil, dust, water, food, air, paint, etc.), (2) childhood
intake rates of each medium, and (3) the rate and extent of lead absorption from each medium
("bioavailability").  Knowledge of lead bioavailability is important because the amount of lead
that actually enters  the blood and body tissues from an ingested medium depends on the


physical-chemical properties of the lead and of the medium.  For example, lead in soil may exist,
at least in part, as poorly water-soluble minerals, and may also exist inside particles of inert
matrix such as rock or slag of variable size, shape, and association. These chemical and physical
properties may tend to influence (usually decrease) the absorption (bioavailability) of lead when
ingested. Thus, equal ingested doses of different forms of lead in different media may not be of
equal health concern.

       The term bioavailability (BA) has many different meanings across various disciplines of
toxicology and pharmacology. For the purposes of this SOP, the term bioavailability means:

       The fraction of an ingested dose that crosses the gastrointestinal epithelium and
       becomes available for distribution to internal target tissues and organs.

Bioavailability expressed as a fraction (or percentage) of a dose is commonly referred to as
absolute bioavailability.  The term relative bioavailability (RBA) refers to a comparison of
absolute bioavailabilities. Relative bioavailability generally is important in risk assessment
because we are often most interested in knowing the extent to which the absolute bioavailability
of a metal increases or decreases in context with the exposure matrix (e.g., food vs.  water vs.
soil), or with the physical or chemical form(s) of the metal to which humans are exposed.  Often,
it is more feasible to assess relative bioavailability than absolute bioavailability (an  example of
this for lead is demonstrated in U.S. EPA, 2007b).  Thus, for the purposes of this  guidance
document,  relative bioavailability means:

       The ratio of the bioavailability of a metal in one exposure context (i.e., physical
       chemical matrix or physical chemical form of the metal) to that in another
       exposure context.
A related term, pertaining to bioavailability assessment, is bioaccessibility. For the purposes of
this SOP, this refers to an in vitro measure of the physiological solubility of the metal that may
be available for absorption into the body. Since solublization is usually required for absorption
across membranes, poorly soluble forms of metals, with low bioaccessibility, may also have low
bioavailability. In certain circumstances, if solubility is the major determinant of absorption at
the portal of entry, bioaccessibility may be a predictor of bioavailability. Lead is an example of
this, as is discussed in U.S. EPA (2007a).

                                                   Pbext • Vext • 100
                       In vitro bioaccessiblity =  —	—	
                                                   * ®soil  ' ^^•'•


Pbext = in vitro  extractable Pb in the in vitro extract (mg/L)
Vext = extraction solution volume (L)
Pbsoti = Pb concentration in the soil sample being assayed (mg/kg)
Soilmass = mass of soil sample being assayed (kg)

The extraction solution volume in this SOP is 0.1 L. For additional definitions for
bioavailability-related terms (e.g., Relative Bioavailability) refer to U.S. EPA (2007a). The in
vitro bioaccessibility assay described in this SOP provides a rapid and relatively inexpensive
alternative to in vivo assays for predicting KB A of lead in soils and soil-like materials. The
method is based on the concept that lead solubilization in gastrointestinal fluid is likely to be an
important determinant of lead bioavailability in vivo.  The method measures the extent of lead
solubilization in an extraction solvent that resembles gastric fluid. The fraction of lead which
solubilizes in an in vitro  system is referred to as in vitro bioaccessibility (IVBA), which may
then be used as an indicator of in vivo KB A. Measurements of IVBA using this assay have been
shown to be a reliable predictor of in vivo KB A of lead in a wide range of soil types and lead
phases from a variety of different sites (U.S. EPA, 2007b).

For the purposes of this document, the term batch refers to a group of analytical and
control/QC samples that are extracted simultaneously.


       At present, it appears  that the predictive relationship between IVBA and RBA is widely
applicable, having been found to hold true for a wide range of different soil types and lead
phases from a variety of different sites. However, the majority of the samples tested have been
collected from mining and milling sites, and it is plausible that some forms of lead that do not
occur at these types  of sites might not follow the observed correlation.  Thus, whenever a sample
containing an unusual and/or untested lead phase is evaluated by the IVBA protocol, this sample
should be identified as a potential source of uncertainty. In the future, as additional samples with
a variety of new and different lead forms are tested by both in vivo and in vitro methods,  the
limits on applicability of the method will be more clearly defined. In addition, excess phosphate
in the sample medium may result in interference (i.e., the assay is not suited to phosphate-
amended soils). Interferences and potential problems are discussed under Procedures (Section

When working with potentially hazardous materials, follow U.S. EPA, OSHA, or corporate
health and safety procedures.

       The equipment that may be used for this procedure is the 1) extraction device shown in
Figure 1 OR 2) an end-over-end rotator placed inside of an incubator.

       1)  The device shown in Figure 1 is an electric motor (the same motor as is used in the
          Toxicity Characteristic Leaching Procedure or TCLP) that drives a flywheel, which in
          turn drives a Plexiglass block situated inside a temperature-controlled water bath.
          The Plexiglass block contains twelve 5-centimeter holes with stainless steel screw
          clamps, each of which is designed to hold a capped 125-mL wide-mouth high density
          polyethylene (HOPE) bottle.   The water bath should be filled such that the extraction
          bottles are completely immersed. Temperature in the water bath should be
          maintained at 37±2 °C using  an immersion circulator heater, and the water bath

          temperature should be monitored and recorded. The electric motor must be capable of
          30±2 rpm.

       2) An end-over-end rotator, capable of 30±2 rpm, should be designed to hold at least
          twelve capped 125-mL wide-mouth HDPE bottles (e.g., Glas-Col® from Terre Haute
          coupled with an Innova 4230 refrigerated incubator shaker from New Brunswick
          Scientific, or equivalent). The rotating device should be placed inside of an incubator
          capable of maintaining 37±2 °C; and the temperature inside of the incubator should
          be monitored and recorded.

       The 125-mL HDPE bottles should have air-tight screw-cap seals, and care should be
taken to ensure that the bottles do not leak during the extraction procedure. All equipment
should be properly cleaned, acid washed, and rinsed with deionized (DI) water prior to use.

An automated temperature compensation (ATC) pH electrode shall be used for measuring the pH
of the extraction fluid prior and post experiment. Additional equipment for this method includes
typical laboratory supplies and reagents, as described in Section 7.0.

All reagents shall be free of lead and the final extraction fluid shall be tested to confirm that lead
concentrations are <1A (

       All test soils should be prepared by drying (<40°C) and sieving to <250 um. The <250
um size fraction was used because this particle size is representative of that which adheres to
children's hands (U.S. EPA, 2000). Stainless steel sieves are recommended. Samples should
be thoroughly mixed prior to use to ensure homogenization.  Mixing and aliquoting of samples
using a riffle splitter is recommended.  Clean HDPE storage bottles are recommended. All
samples should be archived after analysis and retained for further analysis for a period of six
(6) months. No preservatives or special storage conditions are required.

For the purposes of this document, the term batch refers to a group of analytical and
control/QC samples that are extracted simultaneously.

Recommended quality assurance for the extraction procedure are as follows:

•      Reagent Blank — unprocessed (not run through the extraction procedure) extraction fluid
analyzed at a frequency of 1 in 20 samples (minimum of 1 per batch).

•      Bottle Blank — extraction fluid only (no test soil) run through the complete procedure at
a frequency of 1 in 20 samples (minimum of 1 per batch).

•      Blank Spike — extraction fluid spiked at 10 mg/L lead, and run through the complete
procedure at a frequency of 1 in 20 samples (minimum of 1 per batch).

•      Matrix Spikes — subsample of each material used for duplicate analyses used as matrix
spike. The matrix spike should be prepared at 10 mg/L lead and run through the extraction
procedure at a frequency of 1 in 10 samples (minimum of 1 per batch).

       Duplicate Sample — a duplicate sample extraction performed on 1 in 10 samples
(minimum of 1 per batch). The duplicate is treated exactly like a sample and its purpose is to
determine laboratory precision.

•      Control Soil — National Institute of Standards and Testing (NIST) Standard Reference
Material (SRM) 2710 or 2710a or 2711 or 271 la (Montana Soil) used as a control soil. The SRM
shall be analyzed at a frequency of 1 in 20 samples (minimum 1 per batch).

Recommended control limits for these quality control samples:
Reagent blank
Bottle blank
once per batch
(minimum 1 in 20
once per batch
(minimum 1 in 20
Control Limits
<25 ug/L lead
<50 ug/L lead
Corrective Action
Make new extraction fluid
and rerun all analyses
Make new extraction fluid
and rerun all analyses


Blank spike (10 mg/L)

Matrix spike (10 mg/L)

Duplicate sample

Control soil (NIST 27 10
or2710aor2711 or
271 la)

once per batch
(minimum 1 in 20

once per batch
(minimum 1 in 10
once per batch
(minimum 1 in 10

once per batch
(minimum 1 in 20

85-1 15% recovery

75-125% recovery

±20% RPD

NIST 27 lOa mean
67.5% (acceptable
range 60.7-74.2%)
NIST 271 la mean
85.7% (acceptable
range 75. 2-96.2%)
(for NIST 27 10 and
2711 values see section
Ensure dilutions and spike
concentrations are correct. If
no error is found, re-extract
the samples or flag the data.
Ensure dilutions and spike
concentrations are correct. If
no error is found, re-extract
the samples or flag the data.
Re-extract the samples or
flag the data.

Re-extract the samples or
flag the data.

RPD = Relative percent difference


An automated temperature compensation (ATC) pH electrode shall be used for measuring the pH
of the extraction fluid prior and post experiment.  Each instrument/electrode system must be
calibrated at a minimum of two points that bracket the expected pH (1.5) of the samples and are
approximately two pH units or more apart. Repeat adjustments on successive portions of the two
buffer solutions until readings are within 0.05 pH units of the buffer solution value as indicated
in SW-846 method 9045D for Soil and Waste pH
(http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/9045d.pdf). The pH meter should be
calibrated and checked with another standard solution within the calibration range (e.g., pH = 2)
according to the manufacturer's instructions. Thermometers capable of measuring 37 °C ± 2 are
needed. After calibration,  the meter is ready to analyze samples.

       11.1 The extraction fluid for this procedure is 0.4 M glycine (free base, reagent grade
glycine in deionized water), adjusted to a pH of 1.50±0.05 at 37±2°C using trace metal grade
concentrated hydrochloric acid (HC1). See Section 7.0 for extraction fluid preparation details.

       11.2 Pre-heat the TCLP extractor water bath OR incubator (See Section 6.0) to 37°C.
Record the temperature at the beginning and end of each extraction batch (an example of an
extraction data recording form is provided in Attachment A).

       11.3 Soil samples should be thoroughly mixed immediately prior to removing aliquots for
extraction to ensure homogenization (i.e., rotate sample bottles using X, Y, Z motion).

       11.4 The extraction procedure is begun by placing 1.00±0.05 g of sieved test material
(<250 um) into a 125mL wide-mouth HDPE bottle. Record weight of soil to nearest 0.0001 g.
Care should be taken to ensure that static electricity does not cause soil particles to adhere to the
lip or outside threads of the bottle; if necessary, an antistatic brush should be used to eliminate
static electricity prior to adding the test substrate.

       11.5 Measure 100±0.5 mL of the 37±2°C buffered extraction fluid (0.4 M glycine, pH
1.5), using a graduated cylinder or automated dispenser, and transfer extraction fluid to the 125-
mL wide-mouth HDPE bottle.

       11.6 The bottle should be tightly sealed and then shaken or inverted to ensure that there is
no leakage and that no soil is caked on the bottom of the bottle.

       11.7 Fill the extractor (TCLP extractor OR rotating extractor inside of a pre-heated
incubator, see Section 6.0  for details) with 125-mL bottles containing test materials or Quality
Control samples (see Section 7.0). Record start time of rotation.

       11.8 Samples are extracted by rotating the samples at 30±2 rpm for 1 hour.

       11.9 After 1 hour, the bottles should be removed from the rotator, dried, and placed
upright on the bench top to allow the soil to settle to the bottom.

       11.10 A 15-mL sample of supernatant fluid is removed directly from the extraction bottle
into a disposable 20-cc syringe. After withdrawal of the sample into the syringe, a Luer-Lok
attachment fitted with a 0.45-um cellulose acetate disk filter (25 mm diameter) is attached, and
the 15 mL aliquot of fluid is  filtered through the attachment to remove any particulate matter into
a pre-acid washed 15-mL polypropylene centrifuge tube or other appropriate sample vial for

       11.11 Record the time that the extract is filtered (i.e., extraction is stopped). If the total
time elapsed for the extraction and filtration process exceeds 90 minutes, the test must be
repeated (i.e. Steps  11.1-11.11).

       11.12 Measure and record the pH of fluid remaining in the extraction bottle.  If the fluid
pH is not within ±0.5 pH units of the starting pH, the test must be discarded and the sample

       In some cases (mainly slag soils), the test material can increase the pH of the extraction
       buffer, and this could influence the results of the bioaccessibility measurement. To guard
       against this, the pH of the fluid should be measured at the end of the extraction step (just
       after a sample was withdrawn for filtration and analysis). If the pH is not within 0.5 pH
       units of the starting pH (1.5), the sample should be re-analyzed. If the second test also
       resulted in an increase in pH of >0.5 units, it is reasonable to conclude that the test
       material is buffering the solution. In these cases, the test should be repeated using manual
       pH adjustment during the extraction process, stopping the extraction at 5, 10, 15, and 30
       minutes and manually adjusting the pH down to pH 1.5 at each interval by drop-wise
       addition of HC1.

       11.13 Store filtered sample(s) in a refrigerator at 4±2°C until they are analyzed. This
filtered sample of extraction fluid is then analyzed for lead. The samples should be analyzed for
lead by ICP-AES or ICP-MS (U.S. EPA Method 6010C or Method 6020A). The method
detection limit (MDL) in extraction fluid should be approximately 20 ug/L for Method 6010 and
0.1-0.3 ug/L for Method 6020 (U.S. EPA2012a,b).

       11.14. A check list of minimum data recording requirements is provided at the end of
Appendix A.

       11.15. Once received by the laboratory, all samples and extracts should be checked-in,
verified, and maintained under standard chain-of-custody (e.g., U.S. EPA, 2012c).

A split of each solid material (<250 jum) that has been subjected to this extraction procedure
should be analyzed for total lead concentration using analytical procedures taken from the U.S.
EPA SW-846 (U.S. EPA 2012d) or a non-destructive method such as Instrumental Neutron
Activation Analysis.  If SW-846 methods are used, the solid material should be acid digested
according to SW-846 Method 3050B (December 1996  revision) or 3051A (microwave-assisted
digestion, February 2007 revision), and the digestate analyzed for lead concentration determined
by ICP-AES analysis (method 60IOC, February 2007 revision) or ICP-MS (method 6020A,
February 2007 revision).

       12.1  In vitro bioaccessibility (IVBA) is calculated and expressed on a percentage basis
using the following equation:
                                                 Pbext • Vext • 100
                       In vitro bioaccessiblity = —	——	
                                                  ""soil ' Sou-mass


Pbext = in vitro extractable Pb in the in vitro extract (mg/L)
Vext = extraction solution volume (L)
Pbsoii= Pb concentration in the soil sample being assayed (mg/kg)
Soilmass = mass of soil sample being assayed (kg)

       12.2 In order for an in vitro bioaccessibility test system to be useful in predicting the
in vivo RBA of a test material, it is necessary to empirically establish that a strong correlation
exists between the in vivo and the in vitro results across many different samples. Because there
is measurement error not only in RBA but also in IVBA, linear fitting was  also performed
taking the error in both RBA and IVBA into account. There was nearly no  difference in fit, so
the results of the weighted linear regression were selected for simplicity (U.S. EPA, 2007b).
This decision may be revisited as more data become available. Based on this decision, the
currently preferred model is:

       RBA = 0.878'IVBA - 0.028

where RBA and IVBA are expressed as fractions (not percent).  It is important to recognize that
use of this equation to calculate RBA from a given IVBA measurement will yield the "typical"
RBA value expected for a test material with that IVBA, and the true RBA may be somewhat
different (either higher or lower).

       13.1  NIST SRM (NIST SRM 2710,271 Oa, 2711, or 2711 a) should be used as a control
soil. The SRM will be analyzed at a frequency of 1 in 20 samples (minimum 1 per batch). These
SRMs are available from the National Institute of Standards and Technology, Standard
Reference Materials Program (http://www.nist.gov/srm/).  Information on the recent round study
used to develop the following new lead IVBA means (calculation for percent IVBA located in
section 12.1) for 2710a and 271 la is provided in Appendix A.

       13.2 NIST SRM 2710: Analysis of The NIST SRM 2710 standard should yield an IVBA
result of 75.5% (see Figure 3.3 of U.S. EPA, 2007b).

NIST SRM 2710a: Analysis of The NIST SRM 2710a standard should yield a mean IVBA result
of 67.5% (acceptable  IVBA range 60.7-74.2%).

       13.3 NIST SRM 2711: The NIST SRM 2711 standard should yield an IVBA result
of 84.4% (see Figure  3.3 of U.S. EPA, 2007b).

NIST 271 la: The NIST SRM 271 la standard should yield a mean IVBA result of 85.7%
(acceptable IVBA range 75.2-96.2%).

       14.1  Pollution prevention encompasses any technique that reduces or eliminates the
quantity or toxicity of waste at the point of generation. Numerous opportunities for pollution
prevention exist in laboratory operations. The EPA has established a preferred hierarchy of
environmental management techniques that places pollution prevention as the management
option of first choice. Whenever feasible, laboratory personnel should use pollution prevention
techniques to address their waste generation. When wastes cannot be feasibly reduced at the
source, the Agency recommends recycling as the next best option.

       14.2  For information about pollution prevention that may be applicable to laboratories
and research institutions consult Less is Better: Laboratory Chemical Management for Waste
Reduction available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street, NW, Washington, DC 20036,

The Environmental Protection Agency requires that laboratory waste management
practices are consistent with all applicable rules and regulations. The Agency urges laboratories
to protect the air, water, and land by minimizing and controlling all releases from hoods and

bench operations, complying with the letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions. For further information on
waste management, consult The Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society's Department of Government Relations and
Science Policy,  1155 16th Street, NW, Washington, DC 20036, (202) 872-4477.
    1.  Casteel, S.W., R.P. Cowart, C.P. Weis, G.M. Henningsen, E.Hoffman and J.W.
       Drexler. 1997. Bioavailability of lead in soil from the Smuggler Mountain site of
       Aspen Colorado. Fund. Appl. Toxicol. 36: 177-187.

    2.  Dankwerts, P.V. 1951. Significance of liquid-film coefficients in gas
       absorption. Ind. Eng. Chem. 43:1460.

    3.  Drexler J.W. and WJ. Brattin. 2007. An In Vitro Procedure for Estimation of Lead
       Relative Bioavailability: With Validation. Human and Ecological Risk Assessment.

    4.  Medlin, E. A. 1997. An In Vitro method for estimating the relative bioavailability of
       lead in humans. Masters thesis. Department of Geological Sciences, University of
       Colorado, Boulder.

    5.  Nernst, W., and E. Brunner. 1904. Theorie der reaktionsgeschwindigkeit in
       heterogenen systemen. Z. Phys. Chem. 47:52.

    6.  Ruby, M.W., A. Davis, I.E. Link, R.  Schoof, R.L. Chaney, G.B. Freeman, and P.
       Bergstrom. 1993. Development of an in vitro screening test to evaluate the in vivo
       bioaccessibility of ingested mine-waste lead. Environ. Sci. Technol. 27(13): 2870-2877.

    7.  Ruby, M.W., A. Davis, R. Schoof, S.  Eberle. and C.M. Sellstone. 1996. Estimation of
       lead and arsenic bioavailability using a physiologically based extraction test. Environ.
       Sci. Technol. 30(2): 422-430.

    8.  U.S. EPA. 2000. Short Sheet: TRW Recommendations for Sampling and Analysis of
       Soil at Lead (Pb) Sites. OSWER 9285.7-38.

    9.  U.S. EPA. 2007a. Guidance for Evaluating the Oral Bioavailability of Metals in Soils for
       Use in Human Health Risk Assessment. OSWER 9285.7-80. Available on-line at:

    10. U.S. EPA. 2007b. Estimation of Relative Bioavailability of Lead in Soil and Soil-like
       Materials Using in Vivo and in Vitro Methods. OSWER 9285.7-77.

    11. U.S. EPA. 2012a. Method 6010C. available on-line at:

    12. U.S. EPA. 2012b. Method 620A. Available on-line

    13. U.S. EPA 2012c. Chain of Custody Procedures for Samples and Data. Available on-line
       at: http://www.epa.gov/apti/coc/

    14. U.S. EPA. 2012d. Test Methods for Evaluating Solid Waste, Physical/Chemical Methods,
       SW-846.  Available on-line at:

    15. Weis, C.P., and J.M. LaVelle. 1991. Characteristics to consider when choosing an
       animal model for the study of lead bioavailability. In: Proceedings of the International
       Symposium on the Bioavailability and Dietary Uptake of Lead. Sci. Technol. Let.

    16. Weis, C.P., R.H., Poppenga, BJ. Thacker, and G.M. Henningsen. 1994. Design of
       pharmacokinetic and bioavailability studies of lead in an immature swine model.  In: Lead
       in paint, soil, and dust: health risks, exposure studies, control measures, measurement
       methods,  and quality assurance, ASTM STP 1226, M.E. Beard and S.A. Iske (Eds.).
       American Society for Testing and Materials, Philadelphia, PA, 19103-1187.
Appendix A. Additional information on methods development for EPA Method 9200.2-86:

The dissolution of lead from a test material into the extraction fluid depends on a number of
variables including extraction fluid composition, temperature, time, agitation, solid/fluid ratio,
and pH. Any alterations in these parameters should be evaluated to determine the optimum
values for maximizing sensitivity, stability, and the correlation between in vitro and in vivo
values. Additional discussion of these procedures is available in U.S. EPA (2007b) and Drexler
and Brattin (2007).

Most previous in vitro test systems have employed a more complex fluid intended to simulate
gastric fluid. For example, Medlin (1997) used a fluid that contained pepsin and a mixture of
citric, malic, lactic, acetic, and hydrochloric acids. When the bioaccessibility of a series of test
substances were compared using 0.4 M glycine buffer (pH 1.5) with and without the inclusion of

these enzymes and metabolic acids, no significant difference was observed (p=0.196). This
indicates that the simplified buffer employed in the procedure is appropriate, even though it lacks
some constituents known to be present in gastric fluid.
Water vs. air extraction comparison

A statistical comparison (t-test) was made between the SRM data derived from IVBA extractions
that were performed by laboratories employing air (incubator type) as the temperature
controlling (37± 2°C) medium, versus water (aquarium type). The comparison showed that, for
this set of results, there was no statistical difference between the two (2) techniques of
controlling the temperature of sample bottles during the extraction.

Additional  testing to confirm these results was conducted by EPA's NERL and included four in
vitro scenarios using SRM NIST 2710a (n = 27 for each scenario):
           1.   Water bath + preheated gastric solution
           2.  Water bath  + room temperature gastric solution
           3.  Air incubator + preheated gastric solution
           4.  Air incubator + room temperature gastric  solution
Results of the t-tests indicate that there was no statistically significant difference in observed
mean Pb IVBA values for NIST 2710a SRM between scenarios 1 and 2; 1 and 3; and 2 and 3.
The mean Pb IVBA value from scenario 4 (air temperature controlled, gastric solution not-
preheated) was slightly lower. Therefore, the mean Pb IVBA value for scenario 4 was
statistically different from the other three scenarios.

Extraction fluid

The extraction fluid for this procedure is 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 (HC1)


A temperature of 37°C is used because this is approximately the temperature of gastric fluid in

Extraction time

The time that ingested material is present in the stomach (i.e., stomach-emptying time) is about 1
hour for a child, particularly when a fasted state is assumed (see U.S. EPA 2007a,  Appendix A).
Thus, an extraction time of  1 hour should be used. It was found that allowing the bottles  to stand
at room temperature for up to 4 hours after rotation at 37°C caused no significant variation
(<10%) in lead concentration.


Human gastric pH values tend to range from about 1 to 4 during fasting (see U.S. EPA 2007b,
Appendix A). Excess phosphate in the sample may result in interference with the IVBA assay
and IVBA results for phosphate-treated soils have not been shown to correlate with extraction
results from the juvenile swine in vivo assays.


If the test material is allowed to accumulate at the bottom of the extraction apparatus, the
effective surface area of contact between the extraction fluid and the test material may be
reduced,  and this may influence the extent of lead solubilization. Depending on which theory of
dissolution is relevant (Nernst and Brunner, 1904, or Dankwerts, 1951), agitation will greatly
affect either the diffusion layer thickness or the rate of production of fresh surface. Previous
workers have noted problems associated with both stirring and argon bubbling methods (Medlin,
1987). Although no systematic comparison of agitation methods was performed, an end-over-end
method of agitation is recommended.

Soil/Fluid Ratio and Mass of Test Material

A solid-to-fluid ratio of 1/100 (mass per unit volume) should be used to reduce the effects of
metal dissolution when lower ratios (1/5 and 1/25) were used. Tests using Standard Reference
Materials (SRM 2710) showed no significant variation (within ±1% of control means) in the
fraction of lead extracted with soil masses as low as 0.2 gram (g) per 100 mL. However, use of
low masses of test material could introduce variability due to small scale heterogeneity in the
sample and/or to weighing errors. Therefore, the final method employs 1.0 g of test material in
100 mL of extraction fluid.

In special cases, the mass of test material may need to be <1.0 g to avoid the potential for
saturation of the extraction solution. Tests performed using lead acetate, lead oxide, and lead
carbonate indicate that if the bulk concentration of a test material containing these relatively
soluble forms of lead exceed approximately 50,000 ppm, the extraction fluid becomes saturated
at 37°C and, upon cooling to room temperature and below,  lead chloride crystals will precipitate.
To prevent this from occurring, the concentration of lead in the test material should not exceed
50,000 ppm, or the mass of the test material should be reduced to 0.50±0.01 g.

NIST 2710a and 271 la consensus values

The previous lots of these materials, which have the same SRM number without an "a" suffix,
became unavailable for purchase from NIST in late 2008.  Therefore, it was necessary to develop
new lead IVBA means and acceptance ranges for the recently released replacement SRMs NIST
SRMs 2710a and 271 la. A Round Robin study was conducted in late 2010 using seven (7)
participating laboratories. Each laboratory analyzed each of the SRMs in five (5) replicate
analyses, along with the EPA IVBA SOP-required Quality  Control (QC) samples.  Statistical
analysis of the Round Robin  sample results provided a mean and acceptable ranges (based on 99-
percentile prediction interval) for the each of the two (2) NIST SRMs that are consistent with
previous  studies. The extracted lead prediction interval was converted to the IVBA prediction
interval by  dividing by the strong leach digestion value presented in the respective SRM
certificates of analysis. The lead values for the EPA Method 3050 strong leach digestion of the
SRMs 2710a and 271 la, are 5100 mg/Kg and 1300 mg/Kg, respectively. No outlying sample

results were indentified within each laboratory (n=5), or collectively for the n=35 data set for the
individual SRMs, based on statistical analysis. The associated Quality Control (QC) sample
results provided by the laboratories for the reagent blank, bottle blank, spiked blank, matrix
spike, and Control Soil were all within the acceptance criteria presented in the EPA IVBA SOP

Figure 1. Example of In vitro Bioaccessibility Extraction Apparatus.
               (Set at 37° C)
             125 ml     wide mouth bottles
s'!| L
  j KJI
earbox and motor
 30±2 rpm
                                       _>• """%.-' "":.;."';";

Checklist of minimum reporting requirements for EPA 9200.2-86

   •  Each batch must include the following:

      Bottle blank

      Blank spike

      Control soil (NIST SRM 2710 or 2710a or 2711 or 271 la)

      Reagent blank


   •  The sample mass of control soil and soil samples

   •  TCP concentrations of QCs and sample extracts

   •  Minimum detection limit for ICP

   •  QCs run as part of ICP analysis

   •  "Total" Pb concentration of soil samples used to calculate % IVBA