EPA 9200. 1 -86
November 2008
Standard Operating Procedure for an
In Vitro Bio accessibility Assay for Lead in Soil
1.0 Scope and Application
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, and to indicate potential interferences.
At this time, the method described herein has only been validated for lead in soil (U.S.
EPA, 2007b).
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 the
comparison and associated Quality Assurance (QA) in any report.
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.
Mention of trade names or commercial products does not constitute endorsement or
recommended use by U.S. EPA.
2.0 Method Summary
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 body 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 bioavailability of lead in a particular medium may be expressed either in absolute
terms (absolute bioavailability) or in relative terms (relative bioavailability).
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• Absolute Bioavailability (ABA) is the ratio of the amount of lead absorbed compared
to the amount ingested:
ABA = (Absorbed Dose) / (Ingested Dose)
This ratio is also referred to as the oral absorption fraction (AFo).
• Relative Bioavailability (RBA) is the ratio of the absolute bioavailability of lead
present in some test material compared to the absolute bioavailability of lead in some
appropriate reference material:
RBA = ABA(test) / ABA(reference)
For example, if 100 jig of lead contained in soil were ingested and 30 jig entered the
body, the ABA for soil would be:
30 (Absorbed Dose) 7100 (Ingested Dose), or 0.30 (30%).
Likewise, if 100 micrograms (jig) of lead dissolved in drinking water were ingested and a
total of 50 jig entered the body, the ABA would be:
50 (Absorbed Dose) 7100 (Ingested Dose), or 0.50 (50%).
If the lead dissolved in water was used as the frame of reference for describing the
relative amount of lead absorbed from soil, the RBA would be:
0.30 (test) 7 0.50 (reference), or 0.60 (60%).
Usually the form of lead used as reference material is a soluble compound such as lead
acetate that is expected to completely dissolve when ingested.
The in vitro bioaccessibility assay described in this SOP provides a rapid and relatively
inexpensive alternative to in vivo assays for predicting RBA 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 RBA. Measurements of IVBA using this assay
have been shown to be a reliable predictor of in vivo RBA of lead in a wide range of soil types
and lead phases from a variety of different sites (U.S. EPA, 2007b).
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3.0 Sample Preparation, Preservation, Containers, Handling, and Storage
All test soils should be prepared by drying (<40°C) and sieving to <250 jim. The
<250 |im 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 plastic bags or 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.
4.0 Interferences and Potential Problems
At present, it appears that the 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 this type
of site 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 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 7).
5.0 Apparatus
The main piece of equipment used for this procedure is the extraction device shown in
Figure 1. An electric motor (the same motor as is used in the Toxicity Characteristic Leaching
Procedure, or TCLP) drives a flywheel, which in turn drives a Plexiglass block situated inside a
temperature-controlled water bath. The Plexiglass block contains ten 5-centimeter holes with
stainless steel screw clamps, each of which is designed to hold a 125-mL wide-mouth high
density polyethylene (HDPE) 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. 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 water prior to use.
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Circulating
Heater Plexiglass Tank
(Set at 37° C) /
Magnetic Flywheel
125 ml Nalgene wide mouth bottles
& motor
RPM)
Figure 1. In Vitro Bioaccessibility Extraction Apparatus.
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6.0 Reagents
All reagents should be free of lead and the final fluid should be tested to confirm that lead
concentrations are <% (
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7.5 Agitation
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
and Drexler, 1995; Drexler, 1997). Although no systematic comparison of agitation methods
was performed, an end-over-end method of agitation is recommended.
7.6 Solid/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 as noted by Sorenson etal. (1971) when lower ratios (1/5 and 1/25) were
used. Tests using Standard Reference Materials (SRM 2710a) 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.
7.7 Summary of Final Leaching Protocol
The extraction procedure is begun by placing 1.00±0.05 g of sieved test material
(<250 [im) and 100±0.5 mL of the buffered extraction fluid (0.4 M glycine, pH 1.5) into a 125-
mL wide-mouth HDPE bottle. 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 can be used to eliminate static electricity prior to adding the test substrate. 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.
Each bottle should be placed into the modified TCLP extractor (water temperature
37±2°C). Samples are extracted by rotating the samples end-over-end at 30±2 rpm for 1 hour.
After 1 hour, the bottles should be removed, dried, and placed upright on the bench top to allow
the soil to settle to the bottom. 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-|im 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. This filtered sample of extraction fluid is then analyzed for lead, as
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described below. If the total time elapsed for the extraction process exceeds 90 minutes, the test
must be repeated.
As noted above, 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.
7.8 Analysis of Extraction Fluid for Lead
The filtered samples of extraction fluid should be stored in a refrigerator at 4°C until they
are analyzed (within 1 week of extraction). Once received by the laboratory, all media should be
maintained under standard chain-of-custody. The samples should be analyzed for lead by ICP-
AES or ICP-MS (U.S. EPA Method 6010 or 6020, U.S. EPA, 1986). The method detection limit
(MDL) in extraction fluid should be approximately 20 |ig/L for Method 6010 and 0.1-0.3 |ig/L
for Method 6020.
8.0 Calculations
In order for an in vitro bioaccessibility test system to be useful in predicting the in vivo
KB A of a test material, it is necessary to establish empirically 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
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).
9.0 Quality Control/Quality Assurance
Recommended quality assurance for the extraction procedure are as follows:
• Reagent Blank — extraction fluid analyzed once 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).
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• 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 a 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 — duplicate sample extractions performed on
1 in 10 samples (minimum of 1 per batch).
• Control Soil — National Institute of Standards and Testing (NIST) Standard
Reference Material (SRM) 2710 or 2711 (Montana Soil) used as a control soil.
The SRM should be analyzed at a frequency of 1 in 20 samples (minimum 1 per
batch).
Recommended control limits for these quality control samples:
Analysis
Reagent blank
Bottle blank
Blank spike (10 mg/L)
Matrix spike (10 mg/L)
Duplicate sample
Control soil (NIST 27 10
or 2711)
Frequency
once per batch
5%*
5%*
10%*
10%*
5%*
Control Limits
<25 ng/L lead
<50 |ig/L lead
85-1 15% recovery
75-125% recovery
±20% RPD
±10%RPD
RPD = Relative percent difference
*Minimum of once per batch
10.0 Data Validation
NIST SRM 2710 or 2711 should be used as a control soil. To evaluate the precision of
the in vitro bioaccessibility extraction protocol, replicate analyses of standard reference materials
(NIST SRM 2710 or 2711) should be used. The SRM will be analyzed at a frequency of 1 in 20
samples (minimum 1 per batch).
The NIST SRM 2710 standard should yield a result of 75.5% for in vitro RBA (see
Figure 3.3 of EPA, 2007b).
The NIST SRM 2711 standard should yield a result of 84.4% for in vitro RBA (see
Figure 3.3 of EPA, 2007b).
11.0 Health and Safety
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When working with potentially hazardous materials, follow U.S. EPA, OSHA, or
corporate health and safety procedures.
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12.0 References
Casteel, S.W., R.P. Cowart, C.P. Weis, G.M. Henningsen, E.Hoffinan and J.W. Drexler. 1997.
Bioavailability of lead in soil from the Smuggler Mountain site of Aspen Colorado. Fund. Appl. Toxicol.
36: 177-187.
Dankwerts, P.V. 1951. Significance of liquid-film coefficients in gas absorption. Ind. Eng. Chem.
43:1460.
Drexler, J.W. 1998. An in vitro method that works! A simple, rapid and accurate method for
determination of lead bioavailability. EPA Workshop, Durham, NC.
Drexler JW and Brattin WJ. 2007. An In Vitro Procedure for Estimation of Lead Relative
Bioavailability: With Validation. Human and Ecological Risk Assessment. 13: 383-401.
Medlin, E., and Drexler, J.W. 1995. Development of an in vitro technique for the determination of
bioavailability from metal-bearing solids., International Conference on the Biogeochemistry of Trace
Elements, Paris, France.
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.
Nernst, W., and E. Brunner. 1904. Theorie der reaktionsgeschwindigkeit in heterogenen systemen. Z.
Phys. Chem. 47:52.
Ruby, M.W., A. Davis, T.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.
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.
U.S. EPA. 2000. Short Sheet: TRW Recommendations for Sampling and Analysis of Soil at Lead (Pb)
Sites. OSWER 9285.7-38.
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.
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.
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. 3:113-119.
Weis, C.P., R.H., Poppenga, B.J. 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.
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