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- EPA Region 8 Studies on RBA
- Geochemical Speciation
- Region 8 Site-specific Relative Bioavailability Studies
- Region 8 Recommendations for Quantifying Bioavailability of Lead and Arsenic
Most oral toxicity values are based on the empirical relationship between the occurrence of toxic effects and the amount of chemical ingested. That is, the amount of chemical that is actually absorbed into the body (bioavailability) is not explicitly considered. Thus, if it is expected that the absorption of a chemical from an on-site medium is significantly different from the medium used in the study supporting the reference dose (RfD) or slope factor (SF), then it is appropriate to adjust the RfD or SF to account for this difference in absorption. This adjustment increases the accuracy of the subsequent risk calculations while still being protective of public health.
The ratio of the absorption fraction for a chemical in a site medium compared to the medium used in the key toxicity studies is referred to as the Relative Bioavailability (RBA):
RBA = (fraction absorbed from site medium) / (fraction absorbed in toxicity studies)
If reliable estimates of RBA are available for chemicals of potential concern in site media, these can be used to adjust the default RfD and SF values as follows:
RfDadjusted = RfDdefault / RBA
SFadjusted = SFdefault × RBA
The concept of RBA is potentially important for all types of contaminants of concern, but is especially important in the case of metals and other inorganics that commonly occur at mining-related sites. This is because metals in soil and mine waste may occur in a wide variety of chemical and physical forms, not all of which are readily absorbed when ingested. A good general discussion of bioavailability of metals in soil is presented in the document Assessing the Bioavailability of Metals in Soil for Use in Human Health Risk Assessment (PDF) (122 pp, 441 K, About PDF) Exit (National Environmental Policy Institute, Summer 2000). Other general references include the following:
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Assessing Relative Bioavailability in Soil at Superfund Sites
EPA website providing information on how EPA is incorporating relative bioavailability information for human exposures to contaminated soil via the oral pathway at Superfund sites.
Guidance for Evaluating the Oral Bioavailability of Metals in Soils for Use in Human Health Risk Assessment (OSWER 9285.7-80, May 2007)
Provides guidance to Regional risk assessors for evaluating the bioavailability of metals in soils for use in human health risk assessments.
EPA Region 8 Studies on RBA
EPA Region 8 has been engaged in a multi-year study to investigate the RBA of metals (mainly lead and arsenic), both in experimental animals and using in vitro methods, as described below.
In Vivo RBA Studies on Lead
The basic approach for measuring lead absorption in vivo is to administer an oral dose of lead to test animals and measure the increase in lead level in one or more body compartments (blood, soft tissue, bone). In order to calculate the RBA value of a test material, the increase in lead in a body compartment is measured both for that test material and a reference material (lead acetate). Equal absorbed doses of lead are expected to produce approximately equal increases in concentration in tissues regardless of the source or nature of the ingested lead, so the RBA of a test material is calculated as the ratio of doses (test material and reference material) that produce equal increases in lead concentration in the body compartment. This approach has been applied to a number of different soils and soil-like materials from mining-related sites in Region 8 and elsewhere, and the results have been summarized in Estimation of Relative Bioavailability of Lead in Soil and Soil-Like Materials Using In Vivo and In Vitro Methods (OSWER 9285.7-77, May 2007).
In Vivo RBA Studies on Arsenic
The approach for measuring RBA of arsenic is similar to that for lead, except that the best marker of arsenic absorption is the amount of arsenic excreted in the urine rather than the concentration of arsenic in blood or tissues. This is because most arsenic that is absorbed into the body is rapidly (within 1-2 days) excreted in urine. In brief, RBA is calculated in two steps:
- First, the amount of arsenic excreted in urine (μg/d) is plotted as a function of the amount of arsenic administered (μg/d), and the best fit linear regression line through the data is defined as the Urinary Excretion Fraction (UEF).
- Second, RBA is calculated as the ratio of the UEF for test material to that for reference material (sodium arsenate).
The method for measuring the RBA of arsenic in soil and other soil-like media using young swine is described in Brattin and Casteel (2013). This approach has been applied to a number of different soils and soil-like materials from mining-related sites in Region 8 and elsewhere, and the results have been summarized in Relative Bioavailability of Arsenic in Soils at 11 Hazardous Waste Sites Using an In Vivo Juvenile Swine Method (PDF) (56 pp, 837 K) (OSWER 9200.0-76, June 2010).
In Vitro Methods for Lead and Arsenic
Because measurement of lead and arsenic RBA in animals is relatively slow and costly, a number of scientists have been working to develop alternative in vitro procedures that may provide a faster and less costly alternative for estimating the RBA of metals and metalloids in soil or soil-like samples. These methods are based on the concept that the rate and/or extent of metal ion solubilization in the gastrointestinal fluid are likely to be important determinants of the in vivo bioavailability of the metal in soil, and most in vitro tests are aimed at measuring the rate or extent of ion solubilization from soil into an extraction solvent that resembles gastric fluid. To help avoid confusion in nomenclature, the fraction of lead or arsenic which solubilizes in an in vitro system is referred to as in vitro bioaccessibility (IVBA), while the fraction that is absorbed in vivo is referred to as bioavailability.
EPA Region 8 has been working cooperatively with Dr. John Drexler at the University of Colorado in Boulder for a number of years to develop an in vitro method that can be used to obtain RBA data for lead, arsenic, and potentially other metals in soils. As a result of this collaboration, standard in vitro methods for lead and arsenic have been developed that are relatively fast, simple, and reproducible. The details of these test methods are available in the following standard operating procedures:
- Lead: Standard operating procedure for an in vitro bioaccessibility assay for lead in soil (PDF) (16 pp, 291 K) (EPA 9200.2-86, April 2012)
- Arsenic: Standard Operating Procedure, In Vitro Bioacessibility (IVBA) Procedure for Arsenic (Revision 0, September 25, 2012)
Both test procedures utilize a similar process. In brief, samples of test material (1 gram) are placed into a plastic bottle, and to this is added 100 mL of an extraction fluid (0.4 M glycine at pH 1.5). Each bottle is placed into a water bath maintained at 37°C, and samples are extracted by rotating the bottles end-over-end for 1 hour. After 1 hour, the bottles are removed, dried, and placed upright on the bench top to allow the soil to settle to the bottom. After a few minutes, a 15-mL sample of supernatant fluid is removed directly from the extraction bottle into a disposable 20-cc syringe, and then filtered through a 0.45-μm cellulose acetate disk filter (25-mm diameter) to remove any suspended particulate matter. This filtered sample of extraction fluid is then analyzed to quantify the fraction of lead or arsenic in the sample which had dissolved.
Use of these IVBA procedures is considered to be a useful strategy for obtaining estimates of lead and arsenic RBA at a site (see Assessing Relative Bioavailability in Soil at Superfund Sites).
IVBA Results for Lead
Lead IVBA data for 19 test materials from several different Superfund sites, including slag, tailing, and soil samples from various mining and smelting sites, are presented in Estimation of Relative Bioavailability of Lead in Soil and Soil-Like Materials Using In Vivo and In Vitro Methods (OSWER 9285.7-77, May 2007). The IVBA results are strongly correlated with the in vivo RBA values (R2=0.924). Comparison of results within and between laboratories indicates that the procedure is highly reproducible (Drexler and Brattin 2007).
IVBA Results for Arsenic
Region 8 has performed a detailed analysis of arsenic IVBA data for 48 test materials from mining, smelting, herbicide, pesticide, wood-treating, and chemical plant sites across the United States in order to determine the best method for estimating a RBA value from an IVBA measurement. The results are presented in Validation of an In Vitro Bioaccessibility Test Method for Estimation of Bioavailability of Arsenic from Soil and Sediment (ESTCP Project ER-200916, Final Report, December 2012). These data are also summarized in Brattin et al. (2013). IVBA results for arsenic obtained using an extraction fluid of pH 1.5 correlate well with in vivo RBA measurements for arsenic in swine (R2=0.723), whereas in vivo RBA in monkeys tended to correlate better with IVBA results for arsenic obtained using an extraction fluid with phosphate added and a pH of 7 (R2=0.755) (Brattin et al. 2013). The arsenic IVBA method yields highly reproducible results, with intra-laboratory results typically within 0.7 percent and inter-laboratory results within 1.7 percent (Brattin et al. 2013).
A number of other researchers have also investigated methods for estimating arsenic RBA from IVBA values, including Bradham et al. 2011, 2013;, Denys et al. 2012, Juhasz et al., 2007a, b, 2009, 2011, 2014; Makris et al. 2008, Roberts et al., 2007, Rodriquez et al. 1999, Ruby et al., 1996, and Wragg et al. 2011. Recently, EPA’s Technical Review Workgroup (TRW) has reviewed these studies, along with the studies performed by Region 8, in an effort to derive a mathematical model that incorporates the finding from all relevant data. The results of this effort are currently undergoing review and comment, and a link to the report will be published here when the report is finalized.
The RBA value for lead or arsenic in any test soil is believed to depend on (at least) three important attributes of the metal-bearing grains in the soil:
Mineral Phase. Mineral phase refers to the chemical form in which the metal occurs in the soil. For example, lead might occur in grains of galena (PbS) or cerrusite (PbCO3). In general, the less soluble a mineral phase is, the more likely it is to be associated with a low RBA value.
Matrix Association. Some metal-bearing grains in soil are entirely free (“liberated”) in the soil, while others may exist in association with other mineral phases or matrices that may limit the contact of the grain with gastrointestinal fluid. For example, most slags contain the majority of their metal content in small grains or inclusions located within the glassy (vitreous) phase of the slag. When fully encased in the glassy phase, these grains are essentially isolated from gastrointestinal fluid, and are not believed to be available for dissolution or absorption. However, if the slag is crushed and the grains are released, then the metal in the grains become available for contact with gastrointestinal fluid and the RBA value would be expected to increase.
Particle Size. Metals occur in soils as particles that may have a wide range of sizes. Because the process of dissolving a metal from a particle into gastrointestinal fluid occurs only at the surface of the particle, it is thought that large particles are likely to be associated with a lower RBA value than smaller particles of the same mineral, since the ratio of surface area to volume (mass) tends to decrease as particle size increases.
Based on these general concepts of the factors that influence RBA of metals in soil, valuable, information on potential sources of contamination at a site and the potential for differences in RBA at different locations within a site can often be gained by observing the mineral forms, particle size distribution, and matrix associations for metal-bearing grains using.
One approach for geochemical speciation of a sample is electron microprobe analysis (EMPA). A detailed description of the EMPA method is provided at the following site:
Another technique that can be used to obtain information on the geochemical attributes of a sample is synchrotron-based x-ray absorption near edge structure (XANES). XANES measurements determine the arsenic oxidation state (i.e., whether the arsenic is coordinated by oxygen or sulfur). Using this technique, the elemental composition of a single grain in the soil can be examined, and the crystalline structure can be identified and compared to known minerals.
Scientists in Region 8 have investigated the possibility of combining speciation data collected by EMPA with IVBA data to improve the ability to predict reliable RBA values for arsenic in soil. While the approach appears to have merit, inter-laboratory testing of the EMPA method indicated that quantitative characterization of the arsenic phases in a sample is difficult, and the time and cost to obtain speciation data may be prohibitive (Brattin et al. 2013). Therefore, use of speciation data as an input for quantitative RBA models is not recommended at this time.
Region 8 Site-specific Relative Bioavailability Studies
As noted above, Region 8 has measured the in vivo RBA of lead and/or arsenic using swine at a number of sites and has used the results to help improve the accuracy of risk assessments for these two contaminants in soil. Links to several site-specific reports are provided below. Links for additional in vivo RBA studies can be found on the EPA Superfund Bioavailability Guidance website.
Site-specific Arsenic Studies
- Relative Bioavailability of Arsenic in Soils at 11 Hazardous Waste Sites Using an In Vivo Juvenile Swine Method (PDF) (56 pp, 837 K)
Site-specific Lead Studies
- Estimation of Relative Bioavailability of Lead in Soil and Soil-Like Materials Using In Vivo and In Vitro Methods (OSWER 9285.7-77, May 2007).
Region 8 Recommendations for Quantifying the Bioavailability of Lead and Arsenic in Soil for Use in Human Health Risk Assessments
Based on the information presented above, this section summarizes the general strategies that Region 8 recommends for obtaining information to make RBA adjustments for lead and arsenic in soil at Region 8 Superfund sites.
Strategy for Lead
The default value used in the IEUBK model for the relative bioavailability of lead in soil is 60 percent (EPA 1994). That is, it is assumed that lead in soil is absorbed about 60 percent as well as soluble lead that is ingested in water. When risk calculations based on the default RBA for lead are close to (either above or below) a level of health concern, then acquisition of site-specific data may be needed to help increase the accuracy of the assessment. As always, measurement of site-specific RBA using a reliable in vivo study is considered to be the most reliable, but this option is usually only feasible for large sites. If in vivo studies will not be performed, the best option for obtaining this information is through the IVBA assay for lead (EPA 9200.2-86, April 2012 (PDF) (16 pp, 291 K)). This IVBA technique has been validated by EPA (OSWER 9200.3-51, June 2009 (PDF) (14 pp, 114 K)), it is relatively fast and inexpensive, and data may be obtained for a number of site samples. The site-specific lead RBA can then be estimated from the IVBA measurements using a lead-specific in vivo-in vitro correlation (ICIVC) model (OSWER 9285.7-77, May 2007 (PDF) (386 pp, 4.8 MB)).
Strategy for Arsenic
In the past, Region 8 utilized a default relative bioavailability factor of 80 percent for arsenic in soil from mining and smelting sources. More recently, the EPA TRW Bioavailability Committee compiled all available estimates of soil arsenic RBA and determined that values exceeding 60 percent are relatively uncommon. On this basis, the committee has recommended a revised default relative bioavailability factor of 60 percent for arsenic in soil (OSWER 9200.1-113, December 2012 (PDF) (4 pp, 37 K)).
In cases where risk predications based on this default RBA value are near a level of concern (either higher or lower), collection of site-specific RBA information may be appropriate to increase the accuracy of the assessment. As above, results obtained using a reliable in vivo study are preferred, but may not be feasible in all cases. If in vivo studies will not be performed, the best option for obtaining this information is through the IVBA assay for arsenic. The site-specific arsenic RBA can then be estimated from the IVBA measurements using an arsenic-specific ICIVC model (Brattin et al. 2013).
As always, Region 8 highly recommends that anyone interested in adjusting the bioavailability factor of lead and arsenic in soil work closely with the Region 8 toxicologists during the development of the risk assessment.
The following links exit the site Exit
Bradham, K.D., Scheckel, K.G., Nelson, C.M., Seales, P.E., Lee, G.E., Hughes, M.F., Miller, B.W., Yeow, A., Gilmore, T., Harper, S., and Thomas, D.J. 2011. Relative bioavailability and bioaccessibility and speciation of arsenic in contaminated soils. Environ Health Perspect 119(11):1629-1634.
Bradham, K.D., Diamond, G.L., Scheckel, K.G., Hughes, M.F., Casteel, S.W., Miller, B.W., Klotzbach, J.M., Thayer, W.C., Thomas, D.J. (2013) Mouse assay for determination of arsenic bioavailability in contaminated soils. J. Toxicol. Environ. Health A 76:815-826.
Brattin, W., and Casteel, S. 2013. Measurement of arsenic relative bioavailability in swine. J Toxicol Environ Health, Part A 76(7):449-457.
Brattin, W., Drexler, J., Lowney, Y., Griffin, S., Diamond, G., Woodbury, L. 2013. An in vitro method for estimation of arsenic relative bioavailability in soil. J Toxicol Environ Health, Part A 76(7):458-478.
Denys, S., Caboche, J., Tack, K., Rychen, G., Wragg, J., Cave, M., Jondreville, C., Feidt, C. 2012. In vivo validation of the unified BARGE method to assess thebioaccessibility of arsenic, antimony, cadmium, and lead in soils. Environ. Sci. Technol. 46: 6252–6260.
Drexler, J. and Brattin, W. 2007. An in vitro procedure for estimation of lead relative bioavailability: with validation. Hum Ecol Risk Assessment 13(2):383-401.
EPA. 1994. Guidance Manual for the Integrated Exposure Uptake Biokinetic Model for Lead in Children. U.S. Environmental Protection Agency, Office of Emergency and Remedial Response. Publication Number 9285.7-15-1. EPA/540/R-93/081.
Griffin, S. and Lowney, Y. 2012. Final Report: Validation of an In Vitro Bioaccessibility Test Method for Estimation of Bioavailability of Arsenic from Soil and Sediment. Environmental Security Technology Certification Program (ESTCP) Project ER-200916. December 2012.
Juhasz, A. L., Smith, E., Weber, J., Rees, M., Rofe, A., Kuchel, T., Sansom, L., and Naidu, R.2007a.In vitro assessment of arsenic bioaccessibility in contaminated (anthropogenic and geogenic) soils. Chemosphere 69: 69–78.
Juhasz, A. L., Smith, E., Weber, J., Rees, M., Rofe, A., Kuchel, T., Sansom, L., and Naidu, R.2007b. Comparison of in vivo and in vitro methodologies for the assessment of arsenic bioavailability in contaminated soils. Chemosphere 69: 961–966.
Juhasz, A. L., Weber, J., Smith, E., Naidu, R., Rees, M., Rofe, A., Kuchel, T., and Sansom,L. 2009. Assessment of four commonly employed in vitro arsenic bioaccessibility assays for predicting in vivo relative arsenic bioavailability in contaminated soils. Environ. Sci. Technol. 43: 9487–9494.
Juhasz, A.L., Weber, J., Smith, E. 2011. Predicting arsenic relative bioavailability in contaminated soils using meta analysis and relative bioavailability – Bioaccessibilityregression models. Environ. Sci. Technol. 45L 10676-10683.
Makris, K.C., Quazi, S., Nagar, R., Sarkar, D., Datta, R., and Sylvia, V.L. 2008. In vitro model improves the prediction of soil arsenic bioavailability: Worst-case scenario. Environ Sci Technol 42(16):6278-6284.
Roberts, S.M., Munson, J.W., Lowney, Y.W. and Ruby, M.V. 2007. Relative Oral Bioavailability of Arsenic from Contaminated Soils Measured in the Cynomolgus Monkey. ToxSci 95(1):281-288.
Rodriguez, R. R., Basta, N. T., Casteel, S. W., and Pace, L. W. 1999. An in vitro gastrointestinalmethod to estimate bioavailable arsenic in contaminated soils and solid media. Environ. Sci. Technol. 33: 642–649.
Ruby, M. W., Davis, A., Schoof, R., Eberle, S., and Sellstone, C. M. 1996. Estimation oflead and arsenic bioavailability using a physiologically based extraction test. Environ. Sci. Technol. 30: 422–430.
Wragg, J., Cave, M., Basta, N., Brandon, E., Casteel, S., Denys, S., Gron, C., Oomen, A.,Reimer, K., Tack, K., and Van der Wiele, T. 2011. An inter-laboratory trial of the unified BARGE bioaccesssability method for arsenic, cadmium, and lead in soil. Sci. Total Environ. 409: 4016–4030.