University of Colorado

Relative Bioavailability Leaching
Procedure: RBALP

Standard Operating Procedure

1.0 Purpose

An increasingly important property of contaminated media found at environmental sites is the bioavailabilty of individual contaminants. Bioavailability is the fraction of a contaminant that is absorbed by an organism via a specific exposure route. Many animal studies have been conducted to experimentally determine oral bioavailability of individual metals, particularly lead and arsenic. During the period 1989-97, a juvenile swine model developed by USEPA Region VIII was used to measure the relative bioavailability of lead and arsenic in approximately 20 substrates (Weis and LaVelle 1991; Weis et al. 1994). The bioavailability determined was relative (RBA) to that of a soluble salt (i.e. lead acetate trihydrate or sodium arsenate). The tested media had a wide range of mineralogy, and produced a range of lead and arsenic RBA values. In addition to the swine studies, other animal models (e.g. rats and monkeys) have been used for measuring the RBA of lead and arsenic from soils. However, to-date the swine model is still considered the most appropriate for measuring child exposure.

Several researchers have developed in vitro tests to measure the fraction of a chemical solubilized from a soil sample under simulated gastrointestinal conditions. The in vitro tests consist of an aqueous fluid, into which the contaminant is introduced. The solution than solubilizes the media under simulated gastric conditions. Once this procedure is complete, the solution is analyzed for lead and/or arsenic. The mass of the lead and/or arsenic found in the filtered extract is compared to the mass introduced into the test. The fraction liberated into the aqueous phase is defined as the bioaccessable fraction of lead or arsenic in that media (IVBA). To date, for lead-bearing materials tested in the USEPA swine studies, this in vitro assay has correlated well (R2 = 0.83, p= .0001) with relative bioavailability. Arsenic has yet to be fully validated but shows a promising correlation with in vivo results.

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It has been postulated that a simplified in vitro method could be used to estimate bioavailability of lead and arsenic. The method described in this SOP represents a simplified in vitro method, which has been formally validated by USEPA (2004) for lead.

2.0 Scope

This procedure has been validated based on contaminated media from animal studies, to determine the correlation between in vitro and in vivo (IVIVC). Only samples from which mineralogy has been fully characterized by EMPA techniques and for which bioavailability results from acceptable animal models are available have been used for this study. A total of 19 substrates have been tested in validating the relative bioavailability leaching procedure (RBALP) for lead.

3.0 Relevant Literature

Background on the development of in vitro test systems for estimating lead and arsenic bioaccessability can be found in; Ruby et al. (1993, 1996); Medlin (1972); Medlin and Drexler, 1997; Drexler, 1998; and Drexler and Brattin, 2007.

Background information for the USEPA swine studies may be found in (Weis and LaVelle, 1991; Weis et al. 1994; and Casteel et al., 1997) and in the USEPA Region VIII Center in Denver, Colorado.

4.0 Sample Preparation

All media are prepared for the in vitro assay by first drying (<40 C) all samples and then sieving to < 250 m. The <250 m size fraction was used because this is the particle size is representative of that which adheres to children’s hands. Samples were thoroughly mixed prior to use to ensure homogenization. Samples are archived after the study completion and retained for further analysis for a period of six months unless otherwise requested. Prior to obtaining a subsample for testing in this procedure, each sample must be homogenized in its sample container by end-over-end mixing.

5.0 Apparatus and Materials

5.1 Equipment

The main piece of equipment required for this procedure is the extraction device illustrated in Figure 1. The device can be purchased from the Department of Geological Sciences, University of Colorado. For further information contact Dr. John W. Drexler, at (303) 492-5251 or drexlerj@colorado.edu. The device holds ten 125 ml, wide-mouth high-density polyethylene (HDPE) bottles. These are rotated within a Plexiglas tank by a TCLP extractor motor with a modified flywheel. The water bath must be filled such that the extraction bottles remained immersed. Temperature in the water bath is maintained at 37 +/- 2 C using an immersion circulatory heater (Fisher Scientific Model 730).


The 125-ml HDPE bottles must have an airtight screw-cap seal (Fisher Scientific #02-893-5C), and care must be taken to ensure that the bottles do not leak during the extraction procedure.

5.2 Standards and Reagents

The leaching procedure for this method uses an aqueous extraction fluid at a pH value of 1.5. The pH 1.5 fluid is prepared as follows:

Prepare 2 L of aqueous extraction fluid using ASTM Type II deionized (DI) water. The buffer is made up in the following manner. To 1.9 L of DI water, add 60.06 g glycine (free base, reagent grade), and bring the solution volume to 2 L (0.4M glycine). Place the mixture in the water bath at 37 C until the extraction fluid reaches 37 C. Standardize the pH meter ( one should use both a 2.0 and a 4.0 pH buffer for standardization) using temperature compensation at 37 C or buffers maintained at 37 C in the water bath. Add trace metal grade, concentrated hydrochloric acid (12.1N) until the solution pH reaches a value of 1.50 +/_ 0.05 (approximately 60 mL).

All reagents must be free of lead and arsenic, and the final fluid must be tested to confirm that lead and arsenic concentrations are less than one-fourth the project required detection limits (PRDLs) of 10 and 20 g/L, respectively (e.g., less than 2 g/L lead and 5g/L arsenic in the final fluid.

Cleanliness of all materials used to prepare and/or store the extraction fluid and buffer is essential. All glassware and equipment used to prepare standards and reagents must be properly cleaned, acid washed, and finally, triple-rinsed with deionized water prior to use.

6.0 Leaching Procedure

Add 1.00 +/- 0.5 g of test substrate (<250 m) to the bottle, ensuring that static electricity does not cause soil particles to adhere to the lip or outside threads of the bottle. If necessary, use an anti-static brush to eliminate static electricity prior to adding the media. Record the mass of substrate. When ready to begin the test-- measure 100 +/- 0.5 mL of the extraction fluid, using a graduated cylinder or auto pipette and transfer to the 125 mL wide-mouth HPDE bottles. Hand-tighten each bottle top and shake/invert to ensure that no leakage occurs, and that no media is caked on the bottom of the bottle.

Place the bottle into the modified TCLP extractor, making sure each bottle is secure and the lid(s) are tightly fastened. Fill the extractor with 125 mL bottles containing test materials or QA samples.

The temperature of the water bath must be 37 +/- 2 C.

Turn on the extractor and rotate end-over-end at 30 +/- 2 rpm for 1 hour. Record the start time of rotation.

When extraction (rotation) is complete, immediately stop the extractor rotation and remove the bottles. Wipe them dry and place upright on the bench top.

Draw extract directly from the reaction vessel into a disposable 20 cc syringe with a Luer-Lok attachment. Attach a 0.45 m cellulose acetate disk filter (25 mm diameter) to the syringe, and filter the extract into a clean 15 mL polypropylene centrifuge tube (labeled with sample ID) or other appropriate sample vial for analysis.

Record the time that the extract is filtered (i.e. extraction is stopped). If the total time elapsed is greater than 1 hour 30 minutes, the test must be repeated.

Measure the pH of the remaining fluid 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 reanalyzed as follows:
If the pH has changed more than 0.5 units, the test will be re-run in an identical fashion. If the second test also results in a decrease in pH of greater than 0.5 s.u. this will be recorded, and the extract filtered for analysis. If the pH has increased by 0.5 s.u. or more, the test must be repeated, but the extractor must be stopped at specific intervals and the pH manually adjusted down to pH of 1.5 with drop-wise addition of HCl (adjustments at 5, 10, 15, and 30 minutes into the extraction, and upon final removal from the water bath { 60 min}). Samples with rising pH values might better be run following the method of Medlin, 1997.

Store filtered samples in a refrigerator at 4 C until they are analyzed. Analysis for lead and arsenic concentrations must occur within 1 week of extraction for each sample.

Extracts are to be analyzed for lead and arsenic, as specified in EPA methods 6010B, 6020, or 7061A.

6.1 Quality Control/Quality Assurance

Quality Assurance for the extraction procedure will consist of the following quality control samples.

Bottle Blank-extraction fluid only run through the complete procedure at a frequency of 1 in 20 samples.

Blank Spike- extraction fluid will be spiked at concentrations of 2.5 mg/L lead and arsenic and run through the complete procedure at a frequency of 1 in 10 samples.

Matrix Spike-a subsample of each material used will be spiked at concentrations of 2.5 mg/L lead and arsenic and run through the extraction procedure (frequency of 1 in 10 samples).

Duplicate sample-duplicate sample extractions to be performed on 1 in 10 samples.

National Institute of Standards and Testing (NIST) Standard Reference Material (SRM) 2710 or 2711 will be used as a control soil. The SRM will be analyzed at a frequency of 1 in 20 samples.

Control limits for lead are listed below.


Analysis Frequency Control Limits
Bottle blank 5% - 1:20 < 25 g/L lead
Blank spike * 5% - 1:20 85-115% recovery
Matrix spike * 10% - 1:10 75-125% recovery
Duplicate sample 10% - 1:10 +/- 20% RPD**
Control soil *** 5% - 1:20 +/- 10% RPD


* Spikes contained 2.5 mg/L lead and arsenic.
** RPD= relative percent difference.
*** The National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2710 or 2711.

7.0 Chain-of-Custody Procedures

All media once received by the Laboratory must be maintained under standard chain-of-custody.

8.0 Data Handling and Verification

All sample weights, fluid concentrations, and calculations must be recorded on data sheets. Finally all key data will be entered into the attached EXCEL spreadsheet for final delivery and calculation of IVBA.

9.0 References

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.

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, J.W., and Brattin, W., 2007. An In Vitro Procedure for Estimation of Lead Relative Bioavailability: With Validation. Human and Ecological Risk Assessment. 13(2), pp. 383-401.

Medlin, E., and Drexler, J.W., 1995. Development of an in vitro technique for the determination of bioavalability 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.

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 bioaccessability 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 bioavailbilty using a physiologically based extraction test. Environ. Sci. Technol. 30(2): 422-430.

USEPA, 2004. Estimation of relative Bioavailability of lead in Soil and Soil-Like Materials Using In Vivo and In Vitro Methods. OSWER-9285.7-77, June 2004, Washington, DC.

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 parmacokinetic 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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