Article pubs.acs.org/est
Independent Data Validation of an in Vitro Method for the Prediction of the Relative Bioavailability of Arsenic in Contaminated Soils Karen D. Bradham,*,† Clay Nelson,† Albert L. Juhasz,‡ Euan Smith,‡ Kirk Scheckel,§ Daniel R. Obenour,∥ Bradley W. Miller,§ and David J. Thomas† †
Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711, United States ‡ Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095, Australia § Office of Research and Development, United States Environmental Protection Agency, Cincinnati, Ohio 45224, United States ∥ Department of Civil, Construction, and Environmental Engineering, North Carolina State University, Raleigh, North Carolina 27695, United States S Supporting Information *
ABSTRACT: In vitro bioaccessibility (IVBA) assays estimate arsenic (As) relative bioavailability (RBA) in contaminated soils to improve accuracy in human exposure assessments. Previous studies correlating soil As IVBA with RBA have been limited by the use of few soil types and sources of As, and the predictive value of As IVBA has not been validated using an independent set of Ascontaminated soils. In this study, a robust linear model was developed to predict As RBA in mice using IVBA, and the predictive capability of the model was independently validated using a unique set of As-contaminated soils. Forty Ascontaminated soils varying in soil type and contaminant source were included in this study, with 31 soils used for initial model development and nine soils used for independent model validation. The initial model reliably predicted As RBA values in the independent data set, with a mean As RBA prediction error of 5.4%. Following validation, 40 soils were used for final model development, resulting in a linear model with the equation RBA = 0.65 × IVBA + 7.8 and an R2 of 0.81. The in vivo−in vitro correlation and independent data validation presented provide critical verification necessary for regulatory acceptance in human health risk assessment.
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based pharmacokinetic models that can be scaled for humans.7 For these assays, the bioavailability of soil As is expressed relative to the bioavailability of a completely water-soluble form of As (i.e., sodium arsenate). Currently, the USEPA requires the use of in vivo models for assessing the RBA of Ascontaminated soils.15 However, time and cost considerations often limit their use in risk assessment and result in the use of default values for As RBA.7 As an alternative to in vivo bioassays, in vitro bioaccessibility (IVBA) assays have been developed to measure the extent of As solubilization in simulated gastrointestinal fluids.7,12,16−21 IVBAs are attractive alternatives to in vivo assays because they are cost-effective and reduce reliance on animal studies. A prime assumption underlying these IVBAs is that the fraction of As solubilized in vitro is similar to the fraction of As that can cross the gastrointestinal barrier.22 If an IVBA method is an appropriate surrogate, then it must be shown to reliably predict in vivo RBA.5 While some studies have examined the relationship between As RBA and IVBA,7,12,16,19,20,23 validation
INTRODUCTION Arsenic (As) is the most frequently occurring contaminant on the Priority List of Hazardous Substances, which lists substances of greatest public health concern to people living at or near U.S. National Priority Listing sites.1 Human exposure to As via ingestion of As-contaminated soils can have serious health impacts, including increased cancer risk.2−4 Accurate assessment of human health risks from exposure to Ascontaminated soils depends on estimating its bioavailability, defined as the fraction of ingested As absorbed across the gastrointestinal barrier and available for systemic distribution and metabolism. Arsenic bioavailability varies among soils and is influenced by site-specific soil physical and chemical characteristics and internal biological factors. U.S. Environmental Protection Agency (USEPA) guidance describes the need for development of soil As bioavailability methods and data to improve the accuracy of human exposure and risk calculations of As-contaminated sites.5 Difficulties inherent in measuring site-specific soil arsenic bioavailability in humans6 have prompted development of in vivo animal bioassays to determine As relative bioavailability (RBA) in soil.7−14 Although mice and humans differ in metabolism and disposition of arsenicals, similarities are sufficient to permit use of mouse data to create physiologically © 2015 American Chemical Society
Received: Revised: Accepted: Published: 6312
February 19, 2015 April 21, 2015 April 22, 2015 May 12, 2015 DOI: 10.1021/acs.est.5b00905 Environ. Sci. Technol. 2015, 49, 6312−6318
Article
Environmental Science & Technology
atomic emission spectroscopy in accordance with USEPA SWMethod 6010C. A subset of test soils (soils 1−5 and 8−27) were also characterized for As speciation using the Materials Research Collaborative Access Team’s (MRCAT) beamline 10-ID, Sector 10, at the Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL. Additional information about As speciation determination is provided in the Supporting Information. Assessment of As Relative Bioavailability. Arsenic RBA was determined using an in vivo mouse model.7,8 All assays were performed in 4- to 6-week-old female C57BL/6 mice (Charles River Laboratories, Raleigh, NC). Additional details about the mouse assay methodology are provided in the Supporting Information. Data from each mouse assay were used to calculate the urinary excretion fraction (UEF) of As from ingestion of an amended diet as the ratio of cumulative excretion of As in urine (micrograms) to cumulative dietary intake of arsenic (micrograms) as shown in eq 1:
of this relationship using an independent set of soils is the next critical step for regulatory acceptance. Multiple in vivo animal models and in vitro methods have been proposed to assess As RBA and IVBA, respectively, in contaminated soils.7,12,16,19,20,23 A recent study described a mouse assay as a cost-effective and reproducible alternative to other animal assays.7,8 Until recently, precision of As RBA estimates determined from repeated assays of the same soils had not been reported for any animal model. Low betweenassay variation in urinary excretion fraction (UEF) and RBA estimates in the mouse assay results in a highly reproducible, inexpensive in vivo model.8 A strong relationship was noted between As RBA estimated from the mouse assay and As IVBA determined using a simplified gastric phase method7 hereafter termed the solubility/bioavailability research consortium (SBRC) method.24 A study evaluating the correlation between the mouse model and five commonly employed in vitro methods, which varied in operational parameters from simplified gastric methods24 to complex physiological methods aimed at replicating human digestive systems,25 reported that the strongest correlation was found between the results obtained with the mouse model and the SBRC method.26 A similar study comparing results from a juvenile swine model and the SBRC method also found a strong correlation.20 The objective of this study was to build upon a previously published linear regression model7 to predict As RBA in mice using an IVBA and to develop a more robust model across multiple soil types, As contaminant sources, and As concentrations. A second objective was to validate the predictive capability of this model using an independent set of As-contaminated soils. Although earlier studies have evaluated correlations between As RBA and IVBA, these studies have lacked model validation using an independent set of soils and been limited with respect to variety of soil types and contaminant sources used to construct the model. Validation of model performance using data independent from those used to construct the model is imperative for IVBA data to be used routinely for incorporation into human health risk assessments.27 This is particularly important because the predictive capability of the model may be overestimated when evaluated solely with samples used to construct the model.28
UEF% = 100
Cum Urinary As Cum As Dose
(1)
Arsenic RBA was calculated as the ratio of the UEF for As in a specific soil-amended diet to the UEF for As in a diet containing sodium arsenate heptahydrate (see eq 2): RBA% =
UEF Soil UEF Arsenate
(2)
Each UEF in eq 2 is derived from multiple estimates of UEF for groups of three mice housed together in a single metabolic cage (the unit of measure in the assay is data from a single cage). Assessment of As Bioaccessibility. Arsenic IVBA was determined using the SBRC in vitro method (USEPA Method 9200.86-2).24 See the Supporting Information for additional details of IVBA methodology. Arsenic IVBA was calculated and expressed on a percentage basis according to eq 3.
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As bioaccessibility (%) =
MATERIALS AND METHODS Test Soils and Standard Reference Materials. This study used 37 As-contaminated soils in which As was introduced by mining and smelting, pesticide or defoliant use in agricultural or orchard sites, railway corridors, or cattle tick dip sites or occurred as a natural soil constituent. Standard reference materials (SRMs), SRM 2710 and SRM 2710a (National Institute of Standards and Technology), and a USEPA reference material were also evaluated. No soils spiked or amended with As were included in this study. All test soils were collected from the top 1−2 in. of soil, dried (
