Soil origin, processing, and physicochemical characterization.
For full description of soil origin, processing, and physicochemical characterization, see Supplemental Material (http://dx.doi.org/10.1289/ehp.1003352
). Soils used in this study were collected from sites affected by mining and smelter activities. Physicochemical properties were determined in duplicate samples of each soil.
Arsenic speciation in soils was examined using the Materials Research Collaborative Access Team’s beamline 10-ID (Sector 10, Advanced Photon Source, Argonne National Laboratory, Argonne, IL). A principal component analysis coupled with linear combination fitting was used to identify the major As species in the samples. Linear combination fits were performed using X-ray absorption spectroscopy k2 space spectra from reference standards to As phases in the soil samples.
Arsenic concentrations in all soil and biological samples were determined by Instrumental Neutron Activation Analysis (INAA) at the Department of Nuclear Engineering, North Carolina State University (Raleigh, NC; mean As mass detection limit, 0.035 μg). All bioavailability and bioaccessibility calculations were based on INAA values.
Mouse bioavailability assay.
The Institutional Animal Care and Use Committee of the U.S. EPA National Health and Environmental Effects Research Laboratory approved the protocol for mouse use, which assured humane treatment and alleviation of suffering. Female C57BL/6 mice 4–6 weeks of age (Charles River Laboratory, Raleigh, NC) were acclimated in groups of three in a 12/12-hr light/dark photocycle at 20–22°C. Mice had free access to rodent diet (TestDiet, Richmond, IN) and tap water that contained < 11 μg/L As (Kenyon et al. 2008
). Composition of AIN-93G purified rodent diet (Reeves et al. 1993
) obtained from Dyets (Bethlehem, PA) is given in Supplemental Material, (http://dx.doi.org/10.1289/ehp.1003352
). Soil-amended diets were prepared by thorough mixing of test soil with powdered AIN-93G purified rodent diet to a 1% (wt/wt) soil:diet ratio. Arsenate (AsV
)-amended diet prepared by addition of sodium arsenate heptahydrate (Sigma, St. Louis, MO) to powdered AIN-93G purified rodent diet was used to determine the bioavailability of a freely soluble As salt. Diets were stored at 4°C until used.
Description, elemental composition, and As speciation in test soils.a
At the start of an assay, three mice housed together during acclimation were transferred as a group to a metabolic cage that separated urine and feces (Nalgene, Rochester, NY). Twelve mice in four metabolic cages constituted an experimental run. Metabolic cages were maintained for 10 days under environmental conditions given above with unlimited access to test diet and drinking water. For sample collection and data analysis, the unit of observation was the cage and the standard assay for a soil had a sample size of four (except soil 9, which had a sample size of three). To examine assay variability and reproducibility, bioavailability of As in soils 4 and 10 were assayed two and three times, respectively, over a 2-year period.
Daily food consumption for each cage was calculated as the difference between the weight of the food hopper immediately after each morning’s filling and before replenishment the next morning. Cumulative food consumption for each cage was the sum of daily food consumption. Urine and feces were collected each morning from each metabolic cage. Combined body weights of the three mice in each metabolic cage were determined immediately before initial transfer into the metabolic cage and at termination. Mice were euthanized by carbon dioxide (CO2) anesthesia on day 10.
Daily urine or feces collections for each cage were stored at –20°C until processed to produce a single cumulative urine sample and single cumulative feces sample. After thorough mixing, multiple aliquots of the cumulative urine sample for each cage were taken for determination of As concentration by INAA. Cumulative urinary excretion of As was calculated as the product of As concentration in the cumulative urine sample and the volume of the cumulative urine sample. Cumulative feces samples were homogenized with a freezer/mill (model 6850; Spex CertiPrep, Metuchen, NJ). Multiple aliquots of cumulative feces sample were taken for determination of As concentration by INAA. Cumulative fecal excretion of As was calculated as the product of As concentration in the cumulative feces sample and the mass of the cumulative feces sample.
Absolute bioavailability (ABA) of As from ingestion of a soil- or AsV-amended diet was calculated as the ratio of cumulative excretion of As in urine and cumulative dietary intake of As (NRC 2003; U.S. EPA 2007c). ABA is commonly calculated and expressed on a percentage basis:
%ABA = (cumulative As excreted in urine ÷ cumulative As consumed) × 100, 
with As measured in micrograms. Relative bioavailability (RBA) was calculated as the ratio of the ABA for As in a specific soil-amended diet to the ABA for As in a diet containing sodium arsenate (NRC 2003; U.S. EPA 2007c). RBA is commonly expressed on a percentage basis:
%RBA = (ABA of As in a specific diet ÷ ABA of As in sodium arsenate) × 100. 
For a full description of bioaccessibility assays, see Supplemental Material (http://dx.doi.org/10.1289/ehp.1003352
). Bioaccessible As was determined using an in vitro
method developed by the Solubility/Bioavailability Research Consortium (SBRC) assay (Kelly et al. 2002
). In vitro
assays were performed in triplicate for each soil and included addition of 1 g test soil to 100 mL gastric fluid consisting of 0.4 M glycine at pH 1.5 in a 125-mL high-density polyethylene bottle and rotating end over end in a water bath at 37°C for 1 hr. All soils tested in the bioaccessibility protocol were identical to those administered to mice in the in vivo
studies and used in the mineralogy studies described above. All in vitro
extraction solutions were refrigerated at 4°C for preservation and subsequent analysis by Inductively Coupled Plasma–Optical Emission Spectroscopy (ICP-OES) (U.S. EPA 2007e).
In vitro bioaccessibility (IVBA) was calculated and expressed on a percentage basis:
%IVBA = (in vitro extractable mg As/kg soil ÷ total contaminant mg As/kg soil) × 100. 
Statistical analysis. Simple linear regression was used to evaluate the relationship between in vivo As RBA data and IVBA data and to examine the effect of selected soil physicochemical properties on As RBA and bioaccessibility. All analyses were performed using R software (version 2.9.1; R Development Core Team, Vienna, Austria), and figures were created using GraphPad Prism (version 5.0; GraphPad, San Diego, CA).