Variability Associated with As in Vivoâin Vitro Correlations When

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Variability associated with As in vivo-in vitro correlations when using different bioaccessibility methodologies Albert L. Juhasz, Euan Smith, Clay Nelson, David James Thomas, and Karen D. Bradham Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es502751z • Publication Date (Web): 26 Aug 2014 Downloaded from http://pubs.acs.org on August 28, 2014

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Environmental Science & Technology

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Variability associated with As in vivo-in vitro correlations when using different

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bioaccessibility methodologies

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Albert L. Juhasza*, Euan Smitha, Clay Nelsonb, David J. Thomasb, Karen Bradhamb

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a

Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095, Australia.

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b

United States Environmental Protection Agency, Office of Research and Development, Research Triangle Park, NC 27711, USA.

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*Corresponding Author

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Tel:

+618 8302 5045

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Fax:

+618 8302 3057

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Email: [email protected]

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Keywords

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Arsenic, Bioaccessibility, Correlation, Relative Bioavailability, SBRC

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Abstract

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To evaluate the capabilities of in vitro assays to predict arsenic (As) relative bioavailability

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(RBA), we examined the relationship between As bioaccessibility, determined using a

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number of in vitro bioaccessibility (IVBA) methodologies (SBRC, IVG, PBET, DIN and

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UBM) and As RBA determined in a mouse assay for nine As-contaminated soils and 1 NIST

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reference material (2710a). Significant differences (P < 0.05) in As IVBA were observed

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within and between assays indicating that different IVBA methodologies may not produce

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congruent data as a result of variability in the extracting medium constituents and/or

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differences in the pH of gastric and intestinal phases. When results of in vivo determinations

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of As RBA were compared with As IVBA results, there was no significant difference in

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slopes of the relationships (P = 0.49-0.88) when SBRC, IVG, PBET, DIN and UBM gastric

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and intestinal phase data were used. A significantly (P < 0.05) smaller y-intercept was,

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however, determined for the in vivo-SBRC gastric phase correlation compared to SBRC,

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IVG, PBET and DIN intestinal phase, a factor that may influence prediction of As RBA,

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especially for soils with low As RBA. When in vivo-in vitro relationships were compared to

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previously derived correlations from the literature, some differences were observed. These

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differences may be attributed to factors affecting both in vivo and in vitro data including

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physiological differences in animal models (e.g. mouse versus swine) which may influence

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As absorption, differences in the approach used to estimate As RBA, and variability arising

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from subtle interoperator differences in performance of in vitro assays.

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Introduction

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Arsenic (As) is a group 1 human carcinogen1 and the most common contaminant on the

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Priority List of Hazardous Substances, which includes substances determined to be of

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greatest public health concern to persons at or near U.S. National Priority Listing sites.2

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Human exposure to As in soils has serious health impacts, including increased cancer risk

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associated with ingestion of As-contaminated soil.3-5 Reliable analysis of human health risks

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from exposure to As depends on estimating its bioavailability in contaminated media, defined

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as the fraction of As that is absorbed into the systemic circulation. Arsenic bioavailability

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varies depending on the contamination source and is influenced by site-specific soil physical

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and chemical characteristics, as well as internal biological factors. Difficulties inherent in

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measuring site specific soil As bioavailability in humans6 have resulted in the development of

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animal bioassays to estimate As relative bioavailability (RBA; relative to a water soluble

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form of As such as sodium arsenate).7-14 However, time, cost and ethical considerations often

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limit their use in risk assessment.7,15

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As an alternative to in vivo bioassays, in vitro methodologies have been developed that

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measure the extent of As solubilisation in an extraction medium that resembles gastric or

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intestinal fluid. The amount of contaminant extracted in vitro is assumed to be available for

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absorption across the intestinal membrane (i.e., bioaccessible) into the systemic circulation.16

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In vitro bioaccessibility methods can be used to refine human exposure estimates to soil

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contaminants, providing data more economically than costly animal-based studies.17 For

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regulatory risk assessment purposes, in vitro bioaccessibility methods must be predictive of

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in vivo RBA to justify their use as an appropriate surrogate.18 To date, limited efforts have

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been made to establish the relationship between in vitro As bioaccessibility and in vivo As

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RBA7,9,10,12,19,20 possibly because of the prohibitive cost of animal-based studies.

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A number of in vitro assays may be used for assessment of As bioaccessibility;19,21-24

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however, variable results have been observed among methodologies.25-27 This was

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exemplified by the study of Oomen et al.26 that compared As bioaccessibility in 3

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contaminated materials (Flanders, Oker soils and NIST reference material 2710) using five in

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vitro methods (SBET, DIN, RIVM, SHIME, TIM). Among these methods, arsenic

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bioaccessibility ranged from 6-95%, 1-19%, and 10-59% for the three soils, respectively.

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Between-method variability may arise from differences in the composition of gastric and

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intestinal phase solutions, pH and in vitro operational parameters (e.g. solid-to-solution ratio,

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extraction time).25,28,29 In addition, variability in estimates of As bioaccessibility obtained

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using the identical methodologies may result from inter-laboratory differences in method

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execution as exemplified by large reproducibility relative standard deviations (up to 44%) in

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bioaccessibility round robin studies.24,27 Despite differences among different laboratories in

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estimates of As bioaccessibility, validation and standardization of methods is needed to

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establish the predictive relationship for As RBA. This is prerequisite to achieving confidence

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in the use of in vitro assays for the refinement of human health risk assessments.18

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Recent studies7,12,25 have described development of a cost effective, reproducible, in vitro

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assay for determination of site-specific As RBA. These studies have reported a strong

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correlation between As RBA determined in mouse and swine assays and As bioaccessibility

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[SBRC-G (Solubility and Bioavailability Research Consortium Gastric phase)] and illustrate

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the potential of the in vitro assay as a surrogate measure of As RBA. However, other in vitro

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methods have the potential to be used as surrogate assays. Elucidating their capacity to

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predict As RBA is critical to assure both scientific and regulatory acceptance. As a

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consequence, we have investigated the relationship between As RBA and As bioaccessibility

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determined using gastric and intestinal phases of SBRC, DIN, IVG, PBET, and UBM assays

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(Solubility and Bioavailability Research Consortium, Deutsches Institut fur Normung, In

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Vitro Gastrointestinal, Physiologically Based Extraction Technique, and the Unified BARGE

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Method, respectively). It was hypothesised that the As RBA predictive capabilities of these

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in vitro assays would differ as a consequence of the variability in As bioaccessibility

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determinations.

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Materials and Methods

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Arsenic-contaminated soils

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In a previous study, Bradham et al.7 determined As RBA in 9 contaminated soils (from

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residential and smelter slag sites) and 2 reference materials (NIST 2710 and NIST 2710a)

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with As concentrations ranging from 173 to 6899 mg kg-1. Using an in vivo mouse model,

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As RBA in the < 250 µm soil particle size fraction ranged from 11.2 ± 0.3% to 51.6 ± 2.4%

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with comparable values determined using an in vitro extraction methodology (SBRC-G). In

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this study, soils from Bradham et al.7 were used to assess the relationship between As RBA

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and bioaccessibility determined using several commonly used in vitro assays. Nine As-

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contaminated soils and 1 reference material (NIST 2710a) were used; NIST 2710 was not

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included in the study due to the lack of sufficient material. Table 1 details physico-chemical

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properties of soils including the concentration of key elements, soil pH, As speciation and As

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RBA.

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Assessment of As bioaccessibility

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A number of methodologies, varying in operation parameters, have been used to assess As

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bioaccessibility in contaminated soils. In this study, we compared the estimates of soil As

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bioaccessibility determined by commonly used assays. In vitro assays evaluated were the

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Solubility Bioaccessibility Research Consortium (SBRC) assay,22 In Vitro Gastrointestinal

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extraction method (IVG),19 the Physiologically Based Extraction Test (PBET),21 the German

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standard bioaccessibility methodology (DIN)23 and the Unified Bioaccessibility Research

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Group of Europe (BARGE) method (UBM)24 which utilised the < 250 µm soil particle size

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fraction.18 Table S1 provides details of constituents and operational parameters for each

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assay. Additional assay information can be sourced from the relevant references. For the

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assessment of As bioaccessibility, gastric (G) and intestinal phase (I) extractions were

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performed in triplicate with extracts (filtered using 0.45 µm mixed cellulose filters) analysed

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by ICP-AES or ICP-MS. Differences between As bioaccessibility data for each soil was

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determined using the Tukey-Kramer multiple comparison test (at the P < 0.05 significance

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level).

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During the determination of As concentration in in vitro extracts, duplicate analysis, spiked

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sample recoveries and check samples (50 µg As l-1) were included. The average standard

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deviation between duplicate samples (n = 24) was 2.5% (0.3-11.7%), the average recovery

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from spiked samples (n = 24) was 101.5% (92.6-111.8%) whereas check sample recoveries (n

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= 24) ranged from 94.3-111.5% (102.6% average recovery).

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In vitro As bioaccessibility was calculated and expressed on a percentage basis according to

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equation 1.

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% =

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Equation 1

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Where:

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In vitro As = As extracted during the in vitro assay

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Total As = Amount of As in the contaminated soil used for bioaccessibility determination

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Prediction of arsenic relative bioavailability by in vitro assays

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For As-contaminated soils used in this study, As RBA was previously determined using an in

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vivo mouse model.7 Mice were exposed to As-contaminated soil incorporated into mouse

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chow for 9 days with urinary As excretion monitored for 10 days. A detailed description of

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the in vivo methodology can be found in Bradham et al.7. For each soil, urinary excretion

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factor (As excreted versus As consumed) was determined and compared to the sodium

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arsenate urinary excretion factor for the calculation of As RBA. In vivo data were then

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correlated to estimates of As bioaccessibility using the SBRC gastric phase (R2 = 0.92). In

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this study, a similar approach was used to determine the correlation between in vivo and in

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vitro data. The relationship between As RBA (data from Bradham et al.7) and As

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bioaccessibility (determined using SBRC, IVG, PBET, DIN and UBM assays) was

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determined using simple linear regression and included 95% confidence bands of the best fit

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line.

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Results and Discussion

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Assessment of As bioaccessibility in contaminated soils – reproducibility of SBRC-G

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In a previous study, Bradham et al.7determined As bioaccessibility in these soils using

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SBRC-G with values ranging from 6.8 ± 0.8% (soil 6) to 66.5 ± 1.2% (soil 2). As detailed by 7



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USEPA18 ‘Guidance for Evaluating Oral Bioavailability of Metals in Soils for Use in Human

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Health Risk Assessment’ a number of criteria must be met to establish the performance of a

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test method. Two criteria include the assessment of within-laboratory repeatability and

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between-laboratory reproducibility of the test. As detailed in Wragg et al.24 and Juhasz et

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al.,30 within-laboratory repeatability should be < 10% relative standard deviation (RSD) and

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between-laboratory reproducibility should be < 20% RSD. Although these experiments were

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not designed as an inter-laboratory trial, some information can be gleaned regarding the

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repeatability and reproducibility of SBRC-G.

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The within-laboratory repeatability of SBRC-G was illustrated by Bradham et al.7 with As

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bioaccessibility standard deviations (SD) for all soils being < 3% with RSD < 5% with the

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exception of samples 4 (14.8%) and 6 (11.8%). In this study, the within-laboratory

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repeatability of SBRC-G was similar to that of Bradham et al.7 with SD ≤ 2% for all soils

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analysed and RSD ranging from 0.5 to 8.7%. Similarly, the within-test variability for IVG,

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PBET, DIN and UBM assays was low; RSDs for gastric phase As bioaccessibility ranged

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from 0.5-9.5%, 0.1-3.6%, 0.1-13.6% and 0.4-10.7%, respectively, and 0.2-7.2%, 0.1-3.1%,

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0.4-8.0% and 0.7-15.3%, respectively, for intestinal phase analyses. Of the 100 assays

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undertaken in triplicate, 6 had RSDs exceeding 10%, with 2 of these resulting from gastric

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phase extraction (DIN and UBM) of sample 4. The higher RSDs for this sample may have

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arisen from higher variance in pH drift between sample replicates as a result of periodic HCl

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addition to maintain the required gastric phase pH (as a consequence of the buffering capacity

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of the soil).

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Round robin studies have detailed the variability associated with the assessment of

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contaminant bioaccessibility. In an extensive study, Koch et al.27 reported the within

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laboratory repeatability and between laboratory reproducibility of 17 extraction

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methodologies, 14 laboratories and the assessment of bioaccessibility (for 24 inorganic

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contaminants) in the standard reference material NIST 2710. For As bioaccessibility,

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repeatability RSDs were 3.0 and 5.1% for gastric and intestinal phase extractions illustrating

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the within laboratory performance of these assays. In an inter-laboratory trial using the

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unified Bioaccessibility Research Group of Europe (BARGE) method, Wragg et al.24

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reported repeatability and reproducibility for 7 participating laboratories and 33 samples (11

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analysed for As bioaccessibility). Within laboratory repeatability values (RSDs) for As

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bioaccessibility and NIST standards (2710 and 2711) ranged from 0.6-13.3%: however,

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RSDs of 26.7-35.7% were reported for a third reference material (BGS102) presumably due

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to the low concentration of bioaccessible As.24 When within laboratory repeatability was

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determined for the 11 As-contaminated soils, median RSDs for gastric and intestinal phase

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extractions were 5.7% and 6.9% respectively.

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In contrast to low RSDs estimates for repeatability, Koch et al.27 reported that reproducibility

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RSDs were up to 10-fold higher than within-laboratory RSDs with values of 38 and 24%

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determined for gastric and intestinal phase extractions respectively. Koch et al.27 concluded

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that the large variation in between-laboratory measurement of As (and other elements)

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bioaccessibility arose from the use of different in vitro methodologies that varied

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considerably in gastric phase pH (1.5-2.5), solid-to-solution ratio (1:20-1:2000) and

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extractant constituents. Similarly, Wragg et al.24 reported high between-laboratory RSDs for

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NIST 2710 (11.0-22.0%), BGS102 (28.2-44.5%) and the 11 As-contaminated soils (median

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values of 29.5 and 25.9% for gastric and intestinal phase extractions respectively). Wragg et

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al.24 suggested that the discrepancy between within- and between-laboratory RSDs arose

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from small differences in the way the UBM was applied among laboratories and the large

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effect this had on As bioaccessibility determination. Although a true reflection of between

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laboratory-reproducibility cannot be gleaned from this study with only 2 participating

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laboratories (IUPAC31 recommends a minimum of five laboratories), the relationship

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between As bioaccessibility derived by Bradham et al.7 and this study using SBRC-G can be

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seen as plotted in Figure 1. Linear regression demonstrated a strong relationship between

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SBRC-G As bioaccessibility measurements (slope = 1.12; y-intercept = 0.61; R2 = 0.98)

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suggesting the reproducibility of this assay. In a recent study, Brattin et al.10 determined the

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mean between-laboratory RSD of SBRC-G, using 4 participating laboratories and 12 As-

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contaminated soils, to be 8.8%. However, to verify the between-laboratory reproducibility

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of this in vitro assay, a structured round robin study should be devised that meets criteria

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detailed in ISO 5725-2.32

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Assessment of As bioaccessibility in contaminated soils – variation among methodologies

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Over the past 20 years, numerous in vitro methodologies have been used to assess As

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bioaccessibility.19,21-24 These assays have been proposed as surrogate methods to predict As

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RBA for refining human health exposure for the incidental soil ingestion pathway. However,

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as detailed in Table S1, these assays vary considerably with regard to constituents that

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comprise the extracting medium and differ in gastric and intestinal phase pH and extraction

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times. Although As bioaccessibility results may differ, depending on phase (gastric or

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intestinal) and the nature of the assay used, few comparative studies have been undertaken

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that detail variability between As bioaccessibility data.

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Figures 2 and 3 compare As bioaccessibility values for the 10 soils using gastric (G) and

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intestinal (I) phases of SBRC, IVG, PBET, DIN and UBM assays. For each soil, bars sharing

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the same letter indicate that bioaccessibility values are not significantly different (P > 0.05).

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Although similar As bioaccessibility values were obtained for some soils (i.e. soils 1, 2 and

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3), significantly different values were obtained within and between assays indicating that

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different in vitro methodologies may produce discrepant results. The SBRC-G assay yielded

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the most conservative (i.e. highest) measure of As bioaccessibility. For 5 of the 10 soils

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evaluated, the SBRC-G yielded As bioaccessibility values that were significantly higher (P
0.05)

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in sample 3; PBET-I As bioaccessibility was 13% greater than PBET-G values. The increase

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in As bioaccessibility in sample 3 corresponded with an increase in dissolved Fe

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concentrations in PBET-I by 1.8-fold (Figure S1). An increase in As bioaccessibility in

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PBET-I compared to PBET-G has previously been reported by Cave et al.,32 Palumbo-Roe

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and Klinck33 and Juhasz et al.25 Presumably, elevated As bioaccessibility in PBET-I

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extractions resulted from enhanced Fe solubilisation facilitated by organic acids (malate,

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citrate, lactic acid and acetic acid) in the extracting fluid. As the pH increases from the

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gastric phase (2.5) to the intestinal phase (7.0), these organic acids will contain more

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deprotonated functional groups which have a greater capacity to bind to Fe.34

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Relationship between As bioaccessibility and As relative bioavailability

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As described by Oomen et al.,26 Van de Wiele et al.34 and Juhasz et al.25 and as detailed

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above, the use of different in vitro methodologies may produce different As bioaccessibility

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results (see Figures 2 and 3). However, the suitability of bioaccessibility assays as a

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surrogate measure of As RBA is dependent on the strength of the correlation between As

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bioaccessibility and As RBA. Using these soils, Bradham et al.7 determined that SBRC-G

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results strongly predicted As RBA determined using an in vivo mouse model; As

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bioaccessibility was strongly correlated to As RBA (R2 = 0.92). In order to compare in vivo-

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in vitro correlations derived in this study, the relationship between As RBA and As

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bioaccessibility (SBRC-G) from Bradham et al.7 was recalculated using the 10 As-

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contaminated soils (of 11) used for As bioaccessibility assessment. Removal of sample #10

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(NIST 2710) did not alter the in vivo-in vitro correlation significantly (P > 0.05); the slope

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and goodness of fit of the As RBA-bioaccessibility relationship was 0.63 and 0.88

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respectively (Table 2).

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As detailed in Tables 2 and S2 and Figure S3, the in vivo-SBRC-G correlation determined in

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this study (R2 = 0.90) was similar to that of Bradham et al.7 There was no significant

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difference in the slope (P = 0.92) and y-intercepts (P = 0.43) of these relationships illustrating

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the robustness and reproducibility of SBRC-G for predicting As RBA (Figure S3A).

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Although limited in vivo-in vitro correlations have been described for As-contaminated soil

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using the mouse assay and SBRC-G, other relationships have been detailed using a swine in

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vivo model. Juhasz et al.12,25 determined the relationship between As RBA, using an in vivo

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swine model and area under the As blood time curve (AUC) following a single gavaged As

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dose and SBRC-G in 12 As-contaminated soils from Australia. Arsenic bioaccessibility

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determined using SBRC-G was a strong predictor of As RBA (slope = 0.99, y-intercept =

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1.69, R2 = 0.75). Similarly, Brattin et al.10 demonstrated a strong correlation between As

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RBA, determined using swine and steady state urinary excretion (SSUE) as the biomarker of

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As exposure and As bioaccessibility determined using SBRC-G for 20 As-contaminated soils

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from the U.S (slope = 0.62, y-intercept = 19.68, R2 = 0.72). When in vivo-in vitro

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correlations from this study and Brattin et al.10 were compared, the slope of the relationships

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were similar (P = 0.64) although the y-intercepts varied significantly (P < 0.001) (Figure

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S3B). In contrast, the slope of the in vivo-in vitro correlations were significantly different (P

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< 0.01) when compared to data from Juhasz et al. (Figure S3C).12,25

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There are notable differences in the physiological parameters associated with the in vivo

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models (i.e. swine versus mouse) in addition to differences in As RBA methodologies (single

321

versus multiple doses; AUC versus SSUE) which may contribute to differences in As RBA

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values and as a consequence influence in vivo-in vitro correlations. Arsenic RBA,

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determined using SSUE (as utilised by Bradham et al.7 and Brattin et al.10), measured As

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concentrations in the urine following administration of As-contaminated soil or sodium

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arsenate in feed or a dough ball for up to 14 days. In contrast, the method utilised by Juhasz

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et al.12,25 determined As RBA by monitoring blood As concentration following a single

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gavaged dose of As-contaminated soil or sodium arsenate in fasted swine. Utilising the

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single gavaged dose, AUC approach, feed is removed from the in vivo methodology that may

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influence As solubility (i.e. gastric phase pH) and/or As absorption (i.e. competition with

330

phosphorus for absorption). For example, the presence of inorganic phosphate in diet may

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alter arsenate absorption due to competition between arsenate and phosphate for sodium-

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coupled phosphate transporters in the gastrointestinal barrier.35,36 As a consequence, the

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single gavaged dose, AUC approach may result in higher As RBA values (i.e. worst case

334

scenario) which would influence (increase) the slope of the in vivo-in vitro correlation.

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However, as detailed by USEPA37, an advantage of steady state models is that they more

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closely mimic the status of receptors which receive continuous daily exposure to

337

contaminated soil and dust. In addition, when steady state has been reached, urinary As

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excretion will be constant and as a consequence, urinary excretion factors can be estimated

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by averaging As concentrations from multiple samples over time. Although As RBA

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comparisons have been made between mouse and swine models using the SSUE approach,8 a

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comparison of As RBA derived using different exposure endpoints (i.e. SSUE versus AUC)

342

is lacking.

343 344

In vivo-in vitro correlations were also determined when As bioaccessibility was measured

345

using other in vitro assays. Although As bioaccessibility varied between extraction phases

346

and in vitro assays (Figures 2 and 3), there was no significant difference in the slopes of in

347

vivo-in vitro correlations (P = 0.49-0.88) when SBRC, IVG, PBET, DIN and UBM gastric

348

and intestinal phases were utilised to derive the relationship (Tables 2 and S2, Figures S4

349

and S5). However, a significantly (P < 0.05) smaller y-intercept was determined for the in

350

vivo-in vitro correlation using SBRC-G compared to SBRC-I, IVG-I, PBET-I and DIN-I

351

(Figures S4 and S5). This is important to note as the use of in vivo-in vitro correlations with

352

large y-intercepts may over-predict As absorption particularly in soils with low As RBA.

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Limited studies have compared the correlation between As RBA and As bioaccessibility

354

using difference in vitro methodologies. Recent research by Juhasz et al.25,28 determined that

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SBRC, IVG, PBET, DIN and UBM assays (including gastric and intestinal phases) could all

356

predict As RBA with varying degrees of confidence (R2 = 0.52-0.75). However, for some in

357

vivo-in vitro relationships, there were significant differences in the slope of the correlation

358

(e.g. SBRC-G versus SBRC-I, PBET-I, DIN-G and IVG-G versus PBET-I, DIN-G) and y-

359

intercepts (both SRBC-G and UBM-G had y-intercepts < 2%).25,28

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When in vivo-in vitro correlations from this study were compared to those of Juhasz et al.25

362

significant differences (P < 0.05) were observed in the slope of the relationships when SBRC,

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PBET and DIN gastric and intestinal phase values were utilised. However, in vivo-in vitro

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relationships were similar (i.e. no significant difference in the slope [P = 0.06-0.21] and y-

365

intercepts [P = 0.10-0.81]) when gastric and intestinal phases of IVG and UBM assays were

366

utilised (Figure S6). In contrast, comparison of in vivo-in vitro (IVG and UBM) relationships

367

derived in this study to those of Basta et al.9 and Denys et al.20 respectively yielded

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significantly different (P < 0.05) slopes of the linear regression models. These differences

369

may be attributed to a number of factors affecting both in vivo and in vitro data. Variability

370

in in vivo estimates may stem from physiological differences in animal models (e.g. intestinal

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morphology, As distribution patterns) which may influence As absorption and therefore As

372

RBA values.22,38 In addition, the approach for measuring As RBA may influence in vivo

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outcomes in terms of whether single versus multiple As doses are administered and whether

374

absorption is determined using AUC or SSUE. To date, it is unknown to what extent the

375

aforementioned parameters may influence As RBA measurement. Similarly, variability

376

associated with in vitro analysis may stem from subtle differences in the way in vitro assays

377

are conducted which influences bioaccessibility outcomes.24 Significant differences (P
0.05).

Figure 3. Comparison of As bioaccessibility values for soils 7-11 using gastric (G) and intestinal (I) phases of SBRC, IVG, PBET, DIN and UBM assays. For each soil, bars sharing the same letter indicate that data do not differ significantly (P > 0.05).

27



Page 28 of 31

As Bioaccessibility (%; this study)

Figure 1.

100

80

60

40

20

0 0

20

40

60

80

As Bioaccessibility (%; Bradham et al.7)

28


e

10

c

0 In Vitro Methodology

Sample #5

b b

6

4

In Vitro Methodology

ACS Paragon Plus Environment b

d

c d,e

b,d

g

UBM-I

UBM-G

DIN-I

DIN-G

0 PBET-I

20

PBET-G

a,c

UBM-I

15

UBM-G

DIN-I

10

DIN-G

5

PBET-I

b

IVG-I

IVG-G

40

c

UBM-I

b,d 8

b c

UBM-G

d,e

60

c

DIN-I

a c

DIN-G

30

a

PBET-I

Sample #3

PBET-G

0 SBRC-I

Sample #1

PBET-G

20

IVG-I

40

b

IVG-I

20

IVG-G

80 80

IVG-G

b SBRC-G

0

SBRC-I

a

SBRC-G

20

As Bioaccessibility (%)

40


UBM-I

60

SBRC-I

d,e

a


a

UBM-I

UBM-G

DIN-I

100

SBRC-G

b a

UBM-I

c

UBM-G

a,c

a,b

UBM-G

c

DIN-I

DIN-G

PBET-I

b a

DIN-I

c DIN-G

a,c

DIN-G

c

b

PBET-I

b

PBET-G

b

PBET-I

b

PBET-G

IVG-I

IVG-G

SBRC-I

b

PBET-G

c IVG-I

b

IVG-I

60

IVG-G

SBRC-G


a

IVG-G

20 SBRC-I

a

SBRC-I

SBRC-G

As Bioaccessibility (%) 80

SBRC-G


Page 29 of 31 Environmental Science & Technology

Figure 2.

100

Sample #2

d a

a Sample #4

a f

c c e b,d

0


10

Sample #6

a c,g

f

e

2

0


29

10

f e e

c b

0 30

c

20


ACS Paragon Plus Environment c b,c c

b

10

0

d

UBM-I

e

UBM-I

40

UBM-G

DIN-I

DIN-G

a

UBM-G

d,e

DIN-I

50

d

DIN-G

Sample #9 PBET-I

40

PBET-I

a a,c

PBET-G

Sample #7

PBET-G

In Vitro Methodology IVG-I

b

IVG-I

b

IVG-G

0

a,c

IVG-G

f

SBRC-I

i 60

SBRC-I

h

SBRC-G

25

SBRC-G

15


20


c

UBM-I

UBM-G

g

UBM-I

UBM-G

e DIN-I

DIN-G

e

DIN-I

b

DIN-G

d PBET-I

d

PBET-I

c PBET-G

10

PBET-G

5

IVG-I

IVG-G

SBRC-I

SBRC-G


c

IVG-I

5

IVG-G

SBRC-I

15

SBRC-G


Environmental Science & Technology Page 30 of 31

Figure 3.

80

Sample #8

a c

d e

20

0

b


a Sample #11

d,e e


30

Page 31 of 31


TOC ART

As Relative Bioavailability (%)

100

80

60

40

20

0 0

20

40

60

80

100

As Bioaccessibility (SBRC-G; %)

31


Variability Associated with As in Vivoâin Vitro Correlations When

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