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Arsenic bioaccessibility in contaminated soils: Coupling in vitro assays with sequential and HNO3 extraction
Accepted Manuscript Title: Arsenic bioaccessibility in contaminated soils: coupling in vitro assays with sequential and HNO3 extraction Author: Shi–Wei Li Jie Li Hong–Bo Li Ravi Naidu L.Q. Ma PII: DOI: Reference:
S0304-3894(15)00293-9 http://dx.doi.org/doi:10.1016/j.jhazmat.2015.04.011 HAZMAT 16729
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
3-1-2015 29-3-2015 5-4-2015
Please cite this article as: ShindashWei Li, Jie Li, HongndashBo Li, Ravi Naidu, L.Q.Ma, Arsenic bioaccessibility in contaminated soils: coupling in vitro assays with sequential and HNO3 extraction, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Arsenic bioaccessibility in contaminated soils: coupling in vitro assays with sequential and HNO3 extraction
Shi‒Wei Lia, Jie Lia, Hong‒Bo Lia, Ravi Naidub, and L.Q. Maa,c,*
a
State Key Laboratory of Pollution Control and Resource Reuse, School of the
Environment, Nanjing University, Jiangsu 210023, China b
Centre for Environmental Risk Assessment and Remediation, University of South
Australia, Mawson Lakes, SA 5095, Australia c
Soil and Water Science Department, University of Florida, Gainesville, FL 32611
*
Corresponding author, Tel./fax: +86 025 8969 0631, E–mail: [email protected]
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Abstract Arsenic bioaccessibility varies with in vitro methods and soils. Four assays including unified BARGE method (UBM), Solubility Bioaccessibility Research Consortium method (SBRC), in vitro gastrointestinal method (IVG), and physiologically based extraction test (PBET), were used to determine As bioaccessibility in 11 contaminated soils (224,172 mg kg–1). The objective was to understand how bioaccessible As by different methods was related to different As pools based on sequential extraction and 0.43 M HNO3 extraction. Arsenic bioaccessibility was 7.625, 2.349, 7.344, and 1.338% in gastric phase (GP), and 5.753, 0.4633, 2.342, and 0.8643% in intestinal phase (IP) for UBM, SBRC, IVG, and PBET, respectively, with HNO3-extractable As being 0.9060%. Based on sequential extraction, As was primarily associated with amorphous (AF3; 1779%) and crystallized Fe/Al oxides (CF4; 6.473%) while non-specifically sorbed (NS1), specifically sorbed (SS2), and residual fractions (RS5) were 010%, 3.420% and 3.225%. Significant correlation was found between As bioaccessibility by PBET and NS1+SS2 (R2 = 0.550.69), and UBM-GP and NS1+SS2+AF3 (R2 = 0.58), indicating PBET mostly targeted As in NS1+SS2 whereas UBM in NS1+SS2+AF3. HNO3-extractable As was correlated to bioaccessible As by four methods (R2=0.42–0.72) with SBRC-GP being the best. The fact that different methods targeted different As fractions in soils suggested the importance of validation by animal test. Our data suggested that HNO3 may have potential to determine bioaccessible As in soils.
Keywords: contaminated soil; arsenic; bioaccessibility; sequence extraction; HNO3 extraction
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1. Introduction Arsenic is toxic and carcinogenic [1]. Anthropogenic sources such as mining and smelting are major contributors to soil As contamination [2]. Soil ingestion is an important pathway for human exposure to As, especially for children [3]. However, risk assessment based on total As may overestimate the risk so it is important to determine bioavailable As in soils. Bioavailable As is defined as the fraction of As that reaches the systemic circulation in human gastrointestinal (GI) tract [4]. Common animal models including juvenile swine and mouse have been used to assess As bioavailability in contaminated soils [5, 6]. However, they are costly and time consuming. As a result, in vitro assays including SBRC (Solubility Bioaccessibility Research Consortium method), PBET (physiologically based extraction test), IVG (in vitro gastrointestinal method) and UBM (unified BARGE method) have been developed [7-10]. These tests mimic human GI environments and determine bioaccessible As, which is the fraction of As dissolved in GI fluids, representing the As potentially available for absorption into the systemic circulation [4]. All four methods include gastric phase (GP) and intestinal phase (IP). Several studies have shown that arsenic bioaccessibility varies with different assays. Juhasz et al. [11] reported that As bioaccessibility in 12 contaminated soils varied with SBRC, IVG, PBET, and DIN methods, with As bioaccessibility in SBRC-GP best predicting in vivo As relative bioavailability (RBA) based on a swine model. Oomen et al. compared five assays (SBET, DIN, RIVM, SHIME, and TIM) and found pH in gastric phase is important in controlling As bioaccessibility [12]. However, Smith et al. [13] modified the gastric pH of PBET from 2.5 to 1.5 similar to that of SBRC-GP and found As bioaccessibility in two soils increased by up 60%, however, it is still markedly lower than SBRC. These results indicate that besides pH, GI fluid composition is important in controlling As bioaccessibility in soils. In addition to the above common in vitro assays, 0.43 M HNO3 extraction has been used to extract the reactive fraction of heavy metals, which are sorbed onto amorphous Fe/Al oxides and can become bioavailable. In recent years, due to its simplicity, the 0.43 M HNO3 extraction has been suggested as a potential in vitro method for bioaccessible metals [1416]. 3
However, limited studies focus on relationship between toxic metals in reactive pool and bioaccessible pool. Such information helps to evaluate its feasibility as an alternative method to measure metal bioaccessibility in contaminated soils. Rodrigues et al. compared bioaccessible Cd, Cu, Pb and Zn in urban soils between SBRC-GP and 0.43 M HNO3, showing a close 1:1 relationship. This result implied that 0.43 M HNO3 is comparable SBRCGP assay [17]. It is known that As bioavailability in soils is often <100%, partially because As is present in mineral phases and as sorbed species [5, 18]. Therefore, information on As fractions in contaminated soils is important. An improved sequence extraction procedure has been used to determine As fractions in soils [13, 19]. The method separates soil As into five operationally-defined fractionations, including: non-specifically sorbed (NS1), specifically sorbed (SS2), amorphous Fe/Al bound (AF3), crystallized Fe/Al-bound (CF4) and residual (RS5) fractions. Arsenic in NS1 is easily exchangeable and belongs to outer-sphere complexes, while in SS2 represents surface-bound As species and belongs to inner-sphere surface complexes [19]. Arsenic in different fractions has been commonly used to explain extractable As of in vitro assays via correlation. Tang et al. [3] reported that As in the first two fractions (NS1+ SS2) correlated well with bioaccessible As using PBET in 5 spiked soils after 3-month of aging. Whitacre et al. [20] obtained similar conclusion using IVG in 19 spiked soils after 3-month of aging. However, Smith et al. [21] reported that bioaccessible As by SBRC-GP is correlated with As in the AF3 fraction in 12 contaminated soils. Those studies either focused on one in vitro method or used spiked soils. So, how As in different fractions impacted As bioaccessibility in contaminated soils based on different assays has not been investigated. In addition, little information is available to compare 0.43 M HNO3 extraction with different in vitro assays. In this study, four in vitro assays (UBM, SBRC, IVG, and PBET) and 0.43 M HNO3 extraction were employed to determine As bioaccessibility in 11 contaminated soils in addition to arsenic fractionation based on sequential extraction. The objectives were to (1) determine As fraction contributing to bioaccessible As in different assays, and (2) compare bioaccessible As extracted by 0.43 M HNO3 and different in vitro assays.
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2. Materials and methods 2.1. Arsenic-contaminated soils A total of 11 As-contaminated soils were collected from eight cities in China. The soils were collected from top 20 cm from three land uses, including farming, mining, and smelting. The soils represented different geological locations and contamination sources. Soils were air-dried at room temperature, sieved < 2 mm using a nylon sieve and homogenized before storage in polyethylene containers. Soil samples were further sieved to < 250 μm for bioaccessibility and sequential extraction analysis. This fraction easily adheres to children’s hands, which can be ingested through hand‒mouth pathway [22]. The soil properties were characterized using the following methods. Soil digestion was performed using concentrated HNO3 and 30% H2O2 following USEPA Method 3050B. Total Fe oxides (FeT) was extracted by dithionite–citrate–bicarbonate (DCB) [23]. Amorphous Fe oxides (FeA) was extracted using acid ammonium oxalate [24] . Organic matter (OM) content was determined as loss on ignition at 500°C in an oven. Soil pH was determined in 0.01 M CaCl2 extracts (1:5 soil:solution). A laser diffractometer (Mastersizer 2000, Malvern, UK) was used to obtain soil clay content. Inductively coupled plasma mass spectrometry (ICP– MS, NexIONTM300X, Perkin Elmer, USA) was used to measure As concentration in the solution. Iron and Mn in the extracts was quantified using flame atomic absorption spectrometry (PinAAcle 900T, PerkinElmer, USA). Standard Reference Material (SRM) D061‒540 was performed for QA/QC during digestion process. The recovery for Fe, Mn and As in the soil were 101±5.37, 100±2.04, and 97.8±3.02%. 2.2．Arsenic bioaccessibility assessment using in vitro assays Four in vitro assays (UBM, SBRC, IVG, and PBET) were used to assess As bioaccessibility in soils. They have been validated against animal models and commonly used to determine As bioaccessibility in soils [79, 25]. The compositions and analysis parameters in gastric phase (GP) and intestinal phase (IP) was listed in Table 1 [11, 25]. For gastric phase extraction, 1.0 g soil was weighted into wide-mouth high density polyethylene bottles and 37.5 (UBM), 100 (SBRC and PBET) or 150 mL (IVG) of GP fluid was added. Then, the soil suspensions were put into a thermo-bath at 37°C shaking horizontally at 150 rpm for 1 h. During gastric phase extraction, pH was maintained at 1.2, 6
1.5, 1.8, and 2.5 for UBM, SBRC, IVG, and PBET with concentrated HCl. At the end of extraction, suspension solutions were centrifuged at 4,000 rpm for 10 min and an aliquot of the supernatant (10% of GP extraction) was filtered, and transferred into polyethylene tubes for storage at 4oC before As analysis by ICP‒MS. The rest of gastric phase solutions were adjusted to pH of 6.3 (UBM) or 7.0 (SBRC) with NaOH, and 5.5 (IVG) or 7.0 (PBET) with Na2CO3. Then bile and pancreatin were added for doing intestinal phase extraction. After shaking in a thermo-bath at 37oC, 150 rpm for 4 h (UBM, SBRC, and PBET), or 1 h (IVG), samples were centrifuged, filtered, and transferred into polyethylene tubes for storage at 4oC before As analysis by ICP-MS. Arsenic bioaccessibility in soil was calculated by dividing extractable As by total As content in soils. 2.3．Arsenic extracted via 0.43 M HNO3 and sequential extraction 0.43 M HNO3 extraction was performed according to Rodrigues et al. [14], representing reactive metals sorbed on solid phases [15]. Briefly 5 g of air-dried soil was added to 50 mL of 0.43 M HNO3 in a Polypropylene bottle and shaken horizontally at 150 rpm for 2 h at room temperature. Part of the extraction fluid was filtered through 0.45 μm filter for As analysis by ICP–MS. SRM D061‒540 was included, which contained 87.5 ± 1.74% extractable As. Extraction blanks were employed, showing negligible As. An improved sequential extraction procedure was used to determine As fractions in soils [19]. Regents with increasing dissolution strength, including (0.05 M (NH4)2SO4, 0.05 M NH4H2PO4, 0.2 M NH4‒oxalate, 0.2 M NH4‒oxalate and 0.1 M ascorbic acid, and concentrated HNO3 and 30% H2O2) were employed to extract As associated with nonspecifically sorbed (NS1), specifically sorbed (SS2), amorphous Fe/Al oxides (AF3), crystallized Fe/Al oxides (CF4) and residual fraction (RS5), respectively. After each extraction, suspension samples were centrifuged at 4000 rpm for 10 min before filtered through 0.45 μm filters. The filtered samples were stored at 4°C before As analysis by ICP‒MS. SRM D061‒540 and blanks were used for quality control. The recovery by summing the five As fractions in D061‒540 was 99 ± 2% compared to total analysis of 101 ± 13%, which was comparable to 97 ± 16% by Smith et al. [21]. To help better understand the relationship among the different extraction methods used in this experiment, including 4 in vitro assays, 0.43 M HNO3 and sequential extraction, a flow char 7
is provided ( Fig. 1). 2.4．Statistical analysis All experiments were run with three replicates. Results were presented by means±standard deviation. ANOVA based on least-significant difference was applied to determine the significant differences in As bioaccessibility between different methods using SPSS 10.0 for windows. Origin 8.0 was used for Linear correlation analysis to correlate extractable As by four in vitro method with 1) different fractions by sequential extraction and 2) those extracted by 0.43 M HNO3. 3. Results and discussion 3.1. Soil characteristics The properties of 11 contaminated soils are summarized in Table 2. Arsenic concentrations varied from 22.2 to 4,172 mg kg‒1. The total Fe in some samples was >8% whiles they were 1.877.46% in other samples. Total Fe oxides (FeT), amorphous Fe oxides (FeA), and total Mn were highly variable at 0.83‒7.42%, 0.17‒8.99%, and 78.9‒8,992 mg kg1. Soil organic matter and pH were variable at 1.239.92% and 2.607.5. Soils primarily consisted of silt (65‒80%) except for soil S10 and S11 where sand (5357%) was the dominant fraction. Soils from mining area had the highest clay (13‒14%) while soils from farming and smelting areas had low clay (2.8‒10%) (Table 2). Soil with different properties made them good candidates to investigate the relationship between As bioaccessibility and As fractionation in soils. 3.2. Arsenic bioaccessibility based on four in vitro assays Four common in vitro assays (UBM, SBRC, IVG, and PBET) were used to assess As bioaccessibility in 11 contaminated soils. Arsenic bioaccessibility in soils depended on assays, ranging from 0.46 to 49% (Table 3). In this study, As bioaccessibility in the gastric phase of UBM, SBRC, IVG, and PBET assay were 7.6‒52% (averaging 29%), 2.3‒49% (25%), 7.3‒44% (20%) and 1.3‒38% (16%). The bioaccessibility trend was consistent with their gastric pH. This was consistent with results of Oomen et al. [12] and Juhasz et al. [26]. In intestinal phase, As bioaccessibility were 5.7‒53%, 0.46‒32%, 2.3‒42%, and 0.86‒43% for UBM, SBRC, IVG and PBET. In general, As bioaccessibility in gastric phase 8
was higher than that in intestinal phase of SBRC and IVG, which was also observed by Juhasz et al. [11]. It is known that Fe oxides are important in controlling As bioaccessibility in soils. Based on IVG, 18% of Fe oxide is dissolved in gastric phase and so was the associated As [27, 28]. The Fe solubilized in acidic gastric phase (pH 1.2–2.5) is probably precipitated as amorphous Fe oxides in intestinal phase at higher pH (5.5–7.0) [29]. The soluble As in gastric phase was probably adsorbed onto Fe oxides and/or soil solid in intestinal phase, thereby reducing bioaccessible As. This may explain why As bioaccessibility decreased from gastric to intestinal phase by SBRC and IVG. Different from SBRC and IVG , bioaccessible As using UBM and PBET increased from gastric to intestinal phase in some soils (Table 3). Similar increase is also observed by Juhasz et al. [11]. Higher desorption of As from solid phases at higher pH in intestinal phase than gastric phase may partially explain the increase in bioaccessible As [27, 28]. 3.3. Correlation of bioaccessible As with As in different fractions in soils In soils, As is distributed in different solid phases, which has different As bioaccessibility. To understand how different fraction contributes to bioaccessible As, soil As was separated into five fractions via sequential extraction [19]. Among the five fractions, As was primarily distributed in the amorphous Fe/Al oxide fraction (AF3; 17–79%, averaging 48%) and crystallized Fe/Al oxide fraction (CF4; 6.4–73%, 31%) (Fig. 2). Together, they accounted for 64–93% of soil As. Arsenic in the specifically sorbed fraction (SS2; 3.4–20%, 11%) and residual fraction (RS5; 3.2–25%, 9.4%) were similar. The least amount of As was in the non-specifically sorbed fraction (NS1) at 0.00–1.1% excluding soil 11 at 10%, which had the highest total As at 4,172 mg kg‒1 with sandy loam texture (Table 2). In general, the binding strength of As associated with different fractions increases from fraction one (NS1) to five (RS5) [30]. Smith et al. compared changes in As fractions before and after SBCR-GP extraction in 12 contaminated soils [21]. They reported that bioaccessible As was mainly from the first three fractions, including NS1 (<1‒11%), SS2 (<1‒29%) and AF3 (30‒93%) with last two fractions CF4 and RS5 being relatively unchanged. Arsenic in NS1 fraction includes easily exchangeable As and outer-sphere As complexes while As in SS2 fraction includes inner-sphere As complexes [19]. As in different fractions was compared with bioaccessible As based on four assays (Fig. 3. ). While average arsenic concentrations in 9
the first two fractions (NS1+ SS2, 12%) was lower than the average bioaccessible As (13‒29%), the sum of the first three fractions (NS1+SS2+AF3, 60%) was higher. The data suggested that bioaccessible As may primarily came from NS1 and SS2 fractions and part of AF3 fraction in the 11 As-contaminated soils. To explore how each As fraction contributed the bioaccessible As, we correlated As in different fractions with bioaccessible As by four assays (Table 4). It is known that RS5 fraction was the least bioavailable so it was not included. Correlation coefficient between As fractions and As bioaccessibility of UBM-GP increased from R2= 0.30 to 0.45 and to 0.58 when As in NS1, NS1+SS2, and NS1+SS2+AF3 fraction was employed, illustrating the first three fractions contributed 58% of the variability of bioaccessible As by UBM-GP. However when CF4 was added, the correlation reduced to R2= 0.17. The data illustrated that UBM-GP dissolved most of amorphous Fe/Al oxides (AF3) at pH of 1.2. For UBM-IP, only As in NS1+SS2 was correlated to bioaccessible As at R2=0.50 (Table 4). Thus, the first two fractions of As significantly contributed bioaccessible As by UBM. However, PBET behaved differently from UBM. Arsenic in NS1 and NS1+SS2 was correlated with bioaccessible As by PBET at R2=0.420.52 and 0.550.69. The data indicated that non-specifically (NS1) and specifically sorbed (SS2) As contributed the most to bioaccessible As by PBET. Similar to PBET, As in NS1 was correlated with bioaccessible As by SBRC at R2=0.410.43 (Table 4). However, bioaccessible As by IVG had poor correlation with As in different fractions with R2 < 0.32. The PBET results were consistent with Tang et al. [3] and Liang et al. [31] who reported that As in NS1+SS2 fraction was significantly correlated to bioaccessible As by PBET in 5 As-spiked soils. However, current IVG and SBRC results were different from literature. Whitacre et al. [20] reported that As in NS1+SS2 fraction correlated to bioaccessible As by IVG in 19 As-spiked soils while Smith et al. [21] reported no correlation between As in NS1+SS2 fraction and As bioaccessibility by SBRC-GP. The differences in contamination sources between the two studies were partially responsible. In Whitacre et al., [20], the 19 As-spiked soils was aged for three months while the contaminated soils in this study were collected from field, which were contaminated by pesticide application, and mining and smelting process. Insoluble minerals such as arsenopyrite, realgar, and pyrite 10
have been observed in mining soils [18] while As is mainly presented as sorbed species in spiked soils [31]. Differences in As speciation in spiked and contaminated soils were responsible for different relationship observed between As fraction and As bioaccessibility among different studies. The results highlight the importance of testing soils with different contamination sources. In addition, Smith et al. [13] attributed the presence of organic acids (citrate and malate) in PBET-GP to enhance As solubility. The presence of phosphate in UBM also played a role in As desorption [19]. Thus, the significant correlation between As in NS1+SS2 fractions with bioaccessible As was likely due to both organic acids and phosphate in desorbing As from specifically and non-specifically sorbed fractions in soils. However, they are absent in SBRC and IVG. Combining this study with those of Tang et al. [3] and Liang et al. [31], we concluded that As in NS1+ SS2 fractions mainly contributed to bioaccessible As by PBET and UBM in As-contaminated soils. 3.4. Correlation of bioaccessible As with 0.43 M HNO3 extractable As The 0.43 M HNO3 (HNO3) extractable As was 0.90‒60.1% (averaging 27.2%) in Ascontaminated soils, which was higher than bioaccessible As by gastric phase of SBRC, IVG, and PBET but comparable to UBM-GP. In general, HNO3 extractable As was correlated with bioaccessible As in gastric phase, with R2 being 0.42‒0.85, and that in intestinal phase with 0.37‒0.59 for the four methods ( Fig. 4). SBRC-GP showed the best correlation with R2 = 0.85. This was consistent with Rodrigues et al. [17] who showed good correlation between the two methods in 45 soils from grassland areas and vegetable gardens. They attribute the good correlation to their similarity in the extraction pH (SBRC-GP at pH=1.5 and 0.43 M HNO3 < 1). However the data from UBM-GP and PBET-GP were inconsistent with this hypothesis. Both showed good correlation with R2 = 0.70–0.72, yet the gastric pH of UBM is 1.2 while that of PBET is 2.5. Apparently, other factors have contributed to different bioaccessible As by different assays, including the different chemicals used in the gastric phase of different methods. For example, Li et al. [32] observed that, after removing lactic acid, acetic acid, and sodium citrate from PBET, the extractable Fe significantly decreased so was extractable As. This is because As is strongly associated with Fe mineral in soils [33]. By adding glycine, pepsin, or mucin to 11
diluted HCl solution at pH 1.5, Li et al. [34] found the extractable As of contaminated soil increased from 7.0% to 1222%, illustrating those chemicals contributed to bioaccessible As of in the assays. Since glycine is used in SBRC, lactic acid, acetic acid, sodium citrate, and pepsin in PBET, and pepsin and mucin in UBM, their differences in composition were responsible for different relationship between bioaccessible As in different assays and 0.43 M HNO3 extractable As. Among the four in vitro assays, gastric phase of SBRC extractable As was best correlated with in vivo data [11, 26]. Here, the strong correlation (R2 = 0.85) between 0.43 M HNO3 and SBRC-GP extractable As suggested that 0.43 M HNO3 extraction may have potential to determine bioaccessible As in soils.
4. Conclusion Bioaccessible As varied with different in vitro assays. Arsenic fractionation based on sequential extraction helped explain the sources of bioaccessible As in different assays. Bioaccessible As by UBM-GP mainly from first three fractions (NS1+SS2+AF3, 58%) while those by UBM-IP, PBET-GP, and PBET-IP mainly from first two fractions (NS1+SS2, 50– 69%). However, bioaccessible As by IVG and SBRC could not be explained by As in different fractions. In addition, bioaccessible As in gastric phase of UBM, SBRC, and PBET was well correlated to 0.43 M HNO3 extractable As, with R2 of 0.70–0.85, but not for IVG. Among the four in vitro assays, As in SBRC-GP was best correlated to 0.43 M HNO3 extractable As. Our data suggested that 0.43 M HNO3 has potential to determine bioaccessible As in soils. Based on its good correlation with As from the first two-three fractions and good correlation with in vivo data (r2=0.67; data not shown), UBM can be used to determine As bioaccessibility in contaminated soils.
Acknowledgements This work was supported in part by Jiangsu Provincial Innovation Program and Jiangsu Provincial Double Innovation Program.
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tailings and soil in gold mine districts of Nova Scotia, Environ. Sci. Technol. 44 (2010) 2667-2674. [19] W.W. Wenzel, N. Kirchbaumer, T. Prohaska, G. Stingeder, E. Lombi, D.C. Adriano, Arsenic fractionation in soils using an improved sequential extraction procedure, Anal. Chim. Acta 436 (2001) 309-323. [20] S.D. Whitacre, N.T. Basta, E.A. Dayton, Bioaccessible and non-bioaccessible fractions of soil arsenic, J. Environ. Sci. Health, Part A 48 (2013) 620-628. [21] E. Smith, R. Naidu, J. Weber, A.L. Juhasz, The impact of sequestration on the bioaccessibility of arsenic in long-term contaminated soils, Chemosphere 71 (2008) 773-780. [22] U.S. E.P.A. (Environmental Protection Agency), Standard operationg procedure for an in vitro bioaccessibility assay for lead in soil, 2008. [23] O. Mehra, M. Jackson, Iron oxide removal from soils and clays by a dithionite-citrate system buffered with sodium bicarbonate, in: Proc. 7th nat. Conf. Clays. (1960) 317327. [24] J. McKeague, J.H. Day, Dithionite-and oxalate-extractable Fe and Al as aids in differentiating various classes of soils, Can. J. Soil Sci. 46 (1966) 13-22. [25] S. Denys, J. Caboche, K. Tack, G. Rychen, J. Wragg, M. Cave, C. Jondreville, C. Feidt, In vivo validation of the unified BARGE method to assess the bioaccessibility of arsenic, antimony, cadmium, and lead in soils, Environ. Sci. Technol. 46 (2012) 62526260. [26] A.L. Juhasz, J. Weber, E. Smith, Influence of saliva, gastric and intestinal phases on the prediction of As relative bioavailability using the Unified Bioaccessibility Research Group of Europe Method (UBM), J. Hazard. Mater. 197 (2011) 161-168. [27] D.G. Beak, N.T. Basta, K.G. Scheckel, S.J. Traina, Bioaccessibility of arsenic (V) to Ferrihydrite using a simulated gastronintestinal system, Environ. Sci. Technol. 40 (2006) 1364-1370. [28] D.G. Beak, N.T. Basta, K.G. Scheckel, S.J. Traina, Bioaccessibility of arsenic bound to corundum using a simulated gastrointestinal system, Environ. Chem. 3 (2006) 208-214. [29] K.L. Mercer, J.E. Tobiason, Removal of arsenic from high ionic strength solutions: 15
effects of ionic strength, pH, and preformed versus in situ formed HFO, Environ. Sci. Technol. 42 (2008) 3797-3802. [30] E.J. Kim, J.C. Yoo, K. Baek, Arsenic speciation and bioaccessibility in arseniccontaminated soils: Sequential extraction and mineralogical investigation, Environ. Pollut. 186 (2014) 29-35. [31] S. Liang, D.X. Guan, J.H. Ren, M. Zhang, J. Luo, L.Q. Ma, Effect of aging on arsenic and lead fractionation and availability in soils: coupling sequential extractions with diffusive gradients in thin–films technique, J. Hazard. Mater. 273 (2014) 272-279. [32] H.B. Li, X.-Y. Cui, K. Li, J. Li, A.L. Juhasz, L.Q. Ma, Assessment of In Vitro Lead Bioaccessibility in House Dust and Its Relationship to In Vivo Lead Relative Bioavailability, Environ. Sci. Technol. 48 (2014) 8548-8555. [33] S. Goldberg, Competitive adsorption of arsenate and arsenite on oxides and clay minerals, Soil Sci. Soc. Am. J., 66 (2002) 413-421. [34] J. Li, H.B. Li, X.Y. Cui, K. Li, N.T. Basta, L.Q. Ma, Arsenic bioaccessibility in contaminated soils from China: assessment using four in vitro methods and correlation to in vivo bioavailability using a mouse model, Unpublished.
16
Table 1 Composition and in vitro parameters for UBM, SBRC, IVG, and PBET bioaccessibility assays Method Extraction Composition (g L-1) Solid/solution pH Extraction phase ratio time (h) UBM
SBRC
IVG
PBET
Gastric
0.824 g KCl, 0.266 g NaH2PO4, 2.752 g NaCl, 0.4 g CaCl2, 0.306 g NH4Cl, 8.3 ml HCl (37%), 0.085 g urea, 0.65 g glucose, 0.02 g glucuronic acid, 0.33 g glucosamine hydrochloride, 3.0 g mucin, 1.0 g bovine serum albumin, 1.0 g pepsin
1:37.5
1.2
1
Intestinal
0.94 g KCl, 12.3 g NaCl, 11.4 g NaHCO3, 0.08 g KH2PO4, 0.05 g MgCl2, 0.36 ml HCl (37%), 0.35 g urea, 0.42 g CaCl2, 2.8 g bovine serum albumin 3.0 g pancreatin, 0.5 g lipase, 6.0 g bile
1:97.5
6.3
4
Gastric
30.03 g glycine
1:100
1.5
1
Intestinal
1.75 g bile, 0.5 g pancreatin
1:100
7.0
4
Gastric
10 g pepsin, 8.77 g NaCl
1:150
1.8
1
Intestinal
3.5 g bile, 0.35 g pancreatin
1:150
5.5
1
Gastric
1.25 g pepsin, 0.5 g sodium malate, 0.5 g sodium citrate, 420 µL lactic acid, 500µL acetic acid
1:100
2.5
1
Intestinal
1.75 g bile, 0.5 g pancreatin
1:100
7.0
4
17
1 2
Table 2 Physical and chemical properties of 11 soils from different locations and contamination sources ------------------Farming------------- ------------------Mining--------------- --------------------------------Smelting--------------------------------S1
S2
S3
S4
S6
S7
S8
S9
S10
S11
Total As (mg kg‒1)
36.0±1.00 41.1±7.32 171±1.00
Total Fe (g)
27.8±0.21 42.1±0.29 28.8±0.21 76.4±0.43 103±0.06
122±1.17
29.5±0.04 21.1±0.10 84.4±0.41 196±5.19
18.7±0.06
FeDCB (g)a
11.3±0.55 25.3±3.36 16.7±0.39 29.8±1.78 29.4±3.38 74.2±1.40
8.29±0.37 13.9±0.70 38.1±1.47 50.9±0.65
10.2±0.50
FeAO (g)b
1.69±0.01 6.41±0.00 13.1±0.06 9.75±0.28 4.88±0.04 37.1±0.14
1.57±0.05 5.93±0.02 43.4±1.81 89.9±2.27
2.20±0.14
Total Mn (mg kg‒1)
589±1.3
694±6.1
458±42
Organic matter (%)
4.27±0.15 3.50±0.05 4.45±0.15 7.02±0.01 9.04±0.23 5.27±0.27
3.08±0.09 5.51±0.20 7.87±0.09 9.92±0.23
1.34±0.02
pH
7.50
7.50
7.40
Sand (%)
13.2±1.15 19.0±2.69 27.6±1.81 18.9±1.23 20.3±1.43 13.4±2.73
10.0±0.73 19.7±0.52 28.0±0.32 52.6±3.78
56.5±3.64
Silt (%)
78.4±1.12 72.1±3.98 65.2±1.35 67.4±1.02 66.4±1.38 73.6±2.50
79.8±0.66 70.7±0.34 65.3±0.37 44.5±3.51
39.7±3.15
Clay (%)
8.37±0.03 8.91±1.29 7.15±0.46 13.8±0.20 13.4±0.05 13.1±0.24
10.2±0.11 9.66±0.18 6.75±0.04 2.83±0.28
3.80±0.49
78.9±0.69 239±2.4
3.50
6.60
75.2±1.14 743±22.0
495±2.3
4.10
Texture 3 4 5
S5
a
8,992±97
7.30
Silt loam
1470±23.9 22.2±1.16 86.8±2.00 861±34.0
109±2.5
2.60
216±1.3
5.50
1,251±16
6.10
2,556±61.5
2,751±28
6.70
4,172±62.5
Sandy loam Sandy loam
Values represent dithionite–citrate–bicarbonate extractable Fe Values represent acid ammonium oxalate extractable Fe
b
18
6 7 8
Table 3 Bioaccessible arsenic concentrations in 11 As-contaminated soils based on gastric (GP) and intestinal phase (IP) of UBM, SBRC, PBET, and IVG in vitro assays and 0.43 M HNO3 extraction (%). Soil UBM‒GP UBM‒IP SBRC‒GP SBRC‒IP IVG‒GP IVG‒IP PBET‒GP PBET‒IP 0.43 M HNO3 S1
43.9±2.87
52.9±0.28
37.2±4.27
17.7±1.48
33.8±1.59
30.9±0.38
33.6±1.38
31.2±3.43
28.8±0.71
S2
7.59±0.14
13.9±1.45
17.1±0.37
15.0±0.44
15.7±1.33
14.3±0.38
1.33±0.10
3.02±0.11
16.4±2.62
S3
52.4±1.98
44.4±5.21
40.3±0.69
26.6±0.91
17.5±3.92
20.0±3.92
29.6±1.60
28.5±1.40
47.1±1.42
S4
13.6±0.58
15.2±1.04
11.1±0.59
9.80±0.57
12.6±2.53
11.6±0.36
2.65±0.14
7.37±0.26
5.83±0.11
S5
10.7±0.10
13.0±0.33
2.33±0.06
1.47±0.08
8.04±0.45
5.46±0.14
2.80±0.01
4.03±0.14
0.90±0.05
S6
17.0±0.58
14.4±1.73
12.8±0.32
0.46±0.01
7.26±0.52
3.66±0.07
10.6±0.51
5.58±0.06
18.0±0.08
S7
30.0±2.07
51.3±1.92
28.3±0.39
24.6±0.98
44.1±1.74
42.3±1.52
24.2±0.39
21.8±1.23
39.9±3.45
S8
36.3±2.09
38.2±0.51
23.3±0.35
16.3±0.06
21.7±0.40
14.7±2.75
15.1±2.77
21.6±1.33
20.7±0.45
S9
33.1±5.29
18.2±1.01
18.1±0.60
2.26±0.11
11.8±0.99
9.28±0.15
12.5±0.81
12.3±0.37
30.5±0.70
S10
27.4±2.86
5.74±0.21
30.5±0.88
0.56±0.02
11.0±0.25
2.32±0.06
6.70±1.52
0.86±2.59
30.8±0.33
S11
51.9±1.07
41.1±1.27
49.2±2.13
32.6±0.78
39.1±0.30
37.3±0.80
37.7±1.08
42.8±0.86
60.1±4.34
Minimum
7.59
5.74
2.33
0.46
7.26
2.32
1.33
0.86
0.90
Maximum
52.4
52.9
49.2
32.6
44.1
42.3
37.7
42.8
60.1
Average
29.4
28.0
24.6
13.4
20.2
17.4
16.1
16.3
27.2
19
9 10 11
12 13 14 15 16
Table 4 Linear correlation between different As fractions and bioaccessible As based on the gastric (GP) and intestinal phases (IP) of four in vitro assays (UBM, SBRC, IVG, and PBET) in 11 contaminated soils --------NS1+SS2+AF3+CF4-------------------NS1a------------- -----------NS1+SS2---------- ---------NS1+SS2+AF3-------Extractable As Equation R2 Equation R2 Equation R2 Equation R2 UBM-GP
2.92x+25.8
0.30
1.82x+6.91
0.45
0.50x-0.61
0.58
1.08x-68.7
0.17
UBM-IP
2.11x+25.4
0.13
2.09x+2.20
0.50
0.19x+16.9
0.07
0.94x-57.5
0.11
SBRC-GP
3.08x+20.7
0.43
1.38x+7.49
0.33
0.34x+4.23
0.34
1.03x-68.4
0.20
SBRC-IP
2.45x+10.3
0.41
0.94x+1.74
0.23
0.06x+9.96
0.01
0.58x-39.1
0.10
IVG-GP
2.42x+17.2
0.32
1.11x+6.52
0.26
0.35x+18.2
<0.01
0.37x-14.0
0.03
IVG-IP
2.60x+14.2
0.32
1.23x+2.23
0.28
0.005x+17.1
<0.01
0.38x-17.4
0.03
PBET-GP
2.82x+12.6
0.42
1.86x-6.91
0.69
0.26x+0.40
0.23
0.59x-38.0
0.08
PBET-IP
3.31x+12.2
0.52
1.73x-5.18
0.55
0.23x+2.59
0.16
0.53x-32.0
0.06
a
NS1 = non-specifically sorbed, SS2 = specifically sorbed, AF3 = amorphous poorly-crystalline Fe/Al oxides, and CF4 = well-crystallized Fe/Al oxides. R2 > 0.50 are in bold text.
20
As Contaminated Soils
Physico-chemical properties chracterization
As Sequential Extraction
UBM, SBRC, IVG, PBET
0.43 M HNO3 Extraction
As Fraction Distribution
As Bioaccessibility
As Reactive Pool
One Way ANOVA
17 18 19 20 21
Explain
Varied with various methods
Relationship
Fig. 1. Flow chart for all extractions methods used in the experiment, including sequential extraction, 4 in vitro assays (UBM, SBRC, IVG and PBET) and 0.43 M HNO3 extraction.
21
100
RS5 CF4 AF3 SS2 NS1
90
Extractable As (%)
80 70 60 50 40 30 20 10 0 S1 22 23 24 25 26 27 28
S2
S3
S4
S5
S6 S7 Soil ID
S8
S9 S10 S11
Fig. 2. Arsenic fractionations in 11 contaminated soils. Error bars represent the standard deviation of triplicate. NS1 = non-specifically sorbed; SS2 = specifically sorbed; AF3 = amorphous and poorly-crystalline Fe/Al oxides; CF4, well-crystallized Fe/Al oxides; and RS5 = residual.
22
100 90 80
Percent (%)
70 60 50 40 30 20 10
29
N S1 N S N S1 1+S S +S S2 2 +A F3 U BM -G P U BM SB -IP R CG SB P RC -I IV P G -G P IV G PB -IP ET -G PB P ET 0. 43 M IP H NO 3
0
30 31 32 33 34 35 36
Fig. 3. Extractable As in different fractions (NS1, SS2 and AF3), gastric (GP) and intestinal phase (IP) of UBM, SBRC, IVG, and PBET, and 0.43 M HNO3 extraction. Boxes represent 25th to 75th percentile, solid lines and squares in boxes are the median and mean values, crossing symbols represent 1th to 99th percentiles, error bars represent the minimum and maximum values. NS1 = non-specifically sorbed; SS2 = specifically sorbed; AF3 = poorlycrystalline Fe/Al oxides.
23
60
y=0.61x+11.52, R2=0.37 UBM-IP (%)
UBM-GP (%)
80 y=0.77x+8.39, R2=0.72 70 60 50 40 30 20 10 0
y=0.74x+4.47, R2=0.85
y=0.47x+0.65, R2=0.51 SBRC-IP (%)
SBRC-GP (%)
40 30 20 10 0 60
y=0.48x+7.16, R2=0.42
y=0.51x+3.42, R2=0.43
50 40
IVG-IP (%)
IVG-GP (%)
As bioaccessibility (%)
50
30 20 10 0 60
y=0.63x-0.989, R2=0.70
y=0.61x-0.249, R2=0.59
40 30 20 10 0
37 38 39 40 41 42
PBET-IP (%)
PBET-GP (%)
50
0
10 20 30 40 50 60 70
0
10 20 30 40 50 60 70
0.43 M HNO3 extractable As (%) Fig. 4. Linear relationship between 0.43 M HNO3 extractable As and bioaccessible As in gastric (GP) and intestinal phase (IP) of UBM, SBRC, IVG, and PBET assays. Dash lines represent 95% confidence interval.
24
43
25
S0304-3894(15)00293-9 http://dx.doi.org/doi:10.1016/j.jhazmat.2015.04.011 HAZMAT 16729
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
3-1-2015 29-3-2015 5-4-2015
Please cite this article as: ShindashWei Li, Jie Li, HongndashBo Li, Ravi Naidu, L.Q.Ma, Arsenic bioaccessibility in contaminated soils: coupling in vitro assays with sequential and HNO3 extraction, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Arsenic bioaccessibility in contaminated soils: coupling in vitro assays with sequential and HNO3 extraction
Shi‒Wei Lia, Jie Lia, Hong‒Bo Lia, Ravi Naidub, and L.Q. Maa,c,*
a
State Key Laboratory of Pollution Control and Resource Reuse, School of the
Environment, Nanjing University, Jiangsu 210023, China b
Centre for Environmental Risk Assessment and Remediation, University of South
Australia, Mawson Lakes, SA 5095, Australia c
Soil and Water Science Department, University of Florida, Gainesville, FL 32611
*
Corresponding author, Tel./fax: +86 025 8969 0631, E–mail: [email protected]
1
Abstract Arsenic bioaccessibility varies with in vitro methods and soils. Four assays including unified BARGE method (UBM), Solubility Bioaccessibility Research Consortium method (SBRC), in vitro gastrointestinal method (IVG), and physiologically based extraction test (PBET), were used to determine As bioaccessibility in 11 contaminated soils (224,172 mg kg–1). The objective was to understand how bioaccessible As by different methods was related to different As pools based on sequential extraction and 0.43 M HNO3 extraction. Arsenic bioaccessibility was 7.625, 2.349, 7.344, and 1.338% in gastric phase (GP), and 5.753, 0.4633, 2.342, and 0.8643% in intestinal phase (IP) for UBM, SBRC, IVG, and PBET, respectively, with HNO3-extractable As being 0.9060%. Based on sequential extraction, As was primarily associated with amorphous (AF3; 1779%) and crystallized Fe/Al oxides (CF4; 6.473%) while non-specifically sorbed (NS1), specifically sorbed (SS2), and residual fractions (RS5) were 010%, 3.420% and 3.225%. Significant correlation was found between As bioaccessibility by PBET and NS1+SS2 (R2 = 0.550.69), and UBM-GP and NS1+SS2+AF3 (R2 = 0.58), indicating PBET mostly targeted As in NS1+SS2 whereas UBM in NS1+SS2+AF3. HNO3-extractable As was correlated to bioaccessible As by four methods (R2=0.42–0.72) with SBRC-GP being the best. The fact that different methods targeted different As fractions in soils suggested the importance of validation by animal test. Our data suggested that HNO3 may have potential to determine bioaccessible As in soils.
Keywords: contaminated soil; arsenic; bioaccessibility; sequence extraction; HNO3 extraction
2
1. Introduction Arsenic is toxic and carcinogenic [1]. Anthropogenic sources such as mining and smelting are major contributors to soil As contamination [2]. Soil ingestion is an important pathway for human exposure to As, especially for children [3]. However, risk assessment based on total As may overestimate the risk so it is important to determine bioavailable As in soils. Bioavailable As is defined as the fraction of As that reaches the systemic circulation in human gastrointestinal (GI) tract [4]. Common animal models including juvenile swine and mouse have been used to assess As bioavailability in contaminated soils [5, 6]. However, they are costly and time consuming. As a result, in vitro assays including SBRC (Solubility Bioaccessibility Research Consortium method), PBET (physiologically based extraction test), IVG (in vitro gastrointestinal method) and UBM (unified BARGE method) have been developed [7-10]. These tests mimic human GI environments and determine bioaccessible As, which is the fraction of As dissolved in GI fluids, representing the As potentially available for absorption into the systemic circulation [4]. All four methods include gastric phase (GP) and intestinal phase (IP). Several studies have shown that arsenic bioaccessibility varies with different assays. Juhasz et al. [11] reported that As bioaccessibility in 12 contaminated soils varied with SBRC, IVG, PBET, and DIN methods, with As bioaccessibility in SBRC-GP best predicting in vivo As relative bioavailability (RBA) based on a swine model. Oomen et al. compared five assays (SBET, DIN, RIVM, SHIME, and TIM) and found pH in gastric phase is important in controlling As bioaccessibility [12]. However, Smith et al. [13] modified the gastric pH of PBET from 2.5 to 1.5 similar to that of SBRC-GP and found As bioaccessibility in two soils increased by up 60%, however, it is still markedly lower than SBRC. These results indicate that besides pH, GI fluid composition is important in controlling As bioaccessibility in soils. In addition to the above common in vitro assays, 0.43 M HNO3 extraction has been used to extract the reactive fraction of heavy metals, which are sorbed onto amorphous Fe/Al oxides and can become bioavailable. In recent years, due to its simplicity, the 0.43 M HNO3 extraction has been suggested as a potential in vitro method for bioaccessible metals [1416]. 3
However, limited studies focus on relationship between toxic metals in reactive pool and bioaccessible pool. Such information helps to evaluate its feasibility as an alternative method to measure metal bioaccessibility in contaminated soils. Rodrigues et al. compared bioaccessible Cd, Cu, Pb and Zn in urban soils between SBRC-GP and 0.43 M HNO3, showing a close 1:1 relationship. This result implied that 0.43 M HNO3 is comparable SBRCGP assay [17]. It is known that As bioavailability in soils is often <100%, partially because As is present in mineral phases and as sorbed species [5, 18]. Therefore, information on As fractions in contaminated soils is important. An improved sequence extraction procedure has been used to determine As fractions in soils [13, 19]. The method separates soil As into five operationally-defined fractionations, including: non-specifically sorbed (NS1), specifically sorbed (SS2), amorphous Fe/Al bound (AF3), crystallized Fe/Al-bound (CF4) and residual (RS5) fractions. Arsenic in NS1 is easily exchangeable and belongs to outer-sphere complexes, while in SS2 represents surface-bound As species and belongs to inner-sphere surface complexes [19]. Arsenic in different fractions has been commonly used to explain extractable As of in vitro assays via correlation. Tang et al. [3] reported that As in the first two fractions (NS1+ SS2) correlated well with bioaccessible As using PBET in 5 spiked soils after 3-month of aging. Whitacre et al. [20] obtained similar conclusion using IVG in 19 spiked soils after 3-month of aging. However, Smith et al. [21] reported that bioaccessible As by SBRC-GP is correlated with As in the AF3 fraction in 12 contaminated soils. Those studies either focused on one in vitro method or used spiked soils. So, how As in different fractions impacted As bioaccessibility in contaminated soils based on different assays has not been investigated. In addition, little information is available to compare 0.43 M HNO3 extraction with different in vitro assays. In this study, four in vitro assays (UBM, SBRC, IVG, and PBET) and 0.43 M HNO3 extraction were employed to determine As bioaccessibility in 11 contaminated soils in addition to arsenic fractionation based on sequential extraction. The objectives were to (1) determine As fraction contributing to bioaccessible As in different assays, and (2) compare bioaccessible As extracted by 0.43 M HNO3 and different in vitro assays.
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2. Materials and methods 2.1. Arsenic-contaminated soils A total of 11 As-contaminated soils were collected from eight cities in China. The soils were collected from top 20 cm from three land uses, including farming, mining, and smelting. The soils represented different geological locations and contamination sources. Soils were air-dried at room temperature, sieved < 2 mm using a nylon sieve and homogenized before storage in polyethylene containers. Soil samples were further sieved to < 250 μm for bioaccessibility and sequential extraction analysis. This fraction easily adheres to children’s hands, which can be ingested through hand‒mouth pathway [22]. The soil properties were characterized using the following methods. Soil digestion was performed using concentrated HNO3 and 30% H2O2 following USEPA Method 3050B. Total Fe oxides (FeT) was extracted by dithionite–citrate–bicarbonate (DCB) [23]. Amorphous Fe oxides (FeA) was extracted using acid ammonium oxalate [24] . Organic matter (OM) content was determined as loss on ignition at 500°C in an oven. Soil pH was determined in 0.01 M CaCl2 extracts (1:5 soil:solution). A laser diffractometer (Mastersizer 2000, Malvern, UK) was used to obtain soil clay content. Inductively coupled plasma mass spectrometry (ICP– MS, NexIONTM300X, Perkin Elmer, USA) was used to measure As concentration in the solution. Iron and Mn in the extracts was quantified using flame atomic absorption spectrometry (PinAAcle 900T, PerkinElmer, USA). Standard Reference Material (SRM) D061‒540 was performed for QA/QC during digestion process. The recovery for Fe, Mn and As in the soil were 101±5.37, 100±2.04, and 97.8±3.02%. 2.2．Arsenic bioaccessibility assessment using in vitro assays Four in vitro assays (UBM, SBRC, IVG, and PBET) were used to assess As bioaccessibility in soils. They have been validated against animal models and commonly used to determine As bioaccessibility in soils [79, 25]. The compositions and analysis parameters in gastric phase (GP) and intestinal phase (IP) was listed in Table 1 [11, 25]. For gastric phase extraction, 1.0 g soil was weighted into wide-mouth high density polyethylene bottles and 37.5 (UBM), 100 (SBRC and PBET) or 150 mL (IVG) of GP fluid was added. Then, the soil suspensions were put into a thermo-bath at 37°C shaking horizontally at 150 rpm for 1 h. During gastric phase extraction, pH was maintained at 1.2, 6
1.5, 1.8, and 2.5 for UBM, SBRC, IVG, and PBET with concentrated HCl. At the end of extraction, suspension solutions were centrifuged at 4,000 rpm for 10 min and an aliquot of the supernatant (10% of GP extraction) was filtered, and transferred into polyethylene tubes for storage at 4oC before As analysis by ICP‒MS. The rest of gastric phase solutions were adjusted to pH of 6.3 (UBM) or 7.0 (SBRC) with NaOH, and 5.5 (IVG) or 7.0 (PBET) with Na2CO3. Then bile and pancreatin were added for doing intestinal phase extraction. After shaking in a thermo-bath at 37oC, 150 rpm for 4 h (UBM, SBRC, and PBET), or 1 h (IVG), samples were centrifuged, filtered, and transferred into polyethylene tubes for storage at 4oC before As analysis by ICP-MS. Arsenic bioaccessibility in soil was calculated by dividing extractable As by total As content in soils. 2.3．Arsenic extracted via 0.43 M HNO3 and sequential extraction 0.43 M HNO3 extraction was performed according to Rodrigues et al. [14], representing reactive metals sorbed on solid phases [15]. Briefly 5 g of air-dried soil was added to 50 mL of 0.43 M HNO3 in a Polypropylene bottle and shaken horizontally at 150 rpm for 2 h at room temperature. Part of the extraction fluid was filtered through 0.45 μm filter for As analysis by ICP–MS. SRM D061‒540 was included, which contained 87.5 ± 1.74% extractable As. Extraction blanks were employed, showing negligible As. An improved sequential extraction procedure was used to determine As fractions in soils [19]. Regents with increasing dissolution strength, including (0.05 M (NH4)2SO4, 0.05 M NH4H2PO4, 0.2 M NH4‒oxalate, 0.2 M NH4‒oxalate and 0.1 M ascorbic acid, and concentrated HNO3 and 30% H2O2) were employed to extract As associated with nonspecifically sorbed (NS1), specifically sorbed (SS2), amorphous Fe/Al oxides (AF3), crystallized Fe/Al oxides (CF4) and residual fraction (RS5), respectively. After each extraction, suspension samples were centrifuged at 4000 rpm for 10 min before filtered through 0.45 μm filters. The filtered samples were stored at 4°C before As analysis by ICP‒MS. SRM D061‒540 and blanks were used for quality control. The recovery by summing the five As fractions in D061‒540 was 99 ± 2% compared to total analysis of 101 ± 13%, which was comparable to 97 ± 16% by Smith et al. [21]. To help better understand the relationship among the different extraction methods used in this experiment, including 4 in vitro assays, 0.43 M HNO3 and sequential extraction, a flow char 7
is provided ( Fig. 1). 2.4．Statistical analysis All experiments were run with three replicates. Results were presented by means±standard deviation. ANOVA based on least-significant difference was applied to determine the significant differences in As bioaccessibility between different methods using SPSS 10.0 for windows. Origin 8.0 was used for Linear correlation analysis to correlate extractable As by four in vitro method with 1) different fractions by sequential extraction and 2) those extracted by 0.43 M HNO3. 3. Results and discussion 3.1. Soil characteristics The properties of 11 contaminated soils are summarized in Table 2. Arsenic concentrations varied from 22.2 to 4,172 mg kg‒1. The total Fe in some samples was >8% whiles they were 1.877.46% in other samples. Total Fe oxides (FeT), amorphous Fe oxides (FeA), and total Mn were highly variable at 0.83‒7.42%, 0.17‒8.99%, and 78.9‒8,992 mg kg1. Soil organic matter and pH were variable at 1.239.92% and 2.607.5. Soils primarily consisted of silt (65‒80%) except for soil S10 and S11 where sand (5357%) was the dominant fraction. Soils from mining area had the highest clay (13‒14%) while soils from farming and smelting areas had low clay (2.8‒10%) (Table 2). Soil with different properties made them good candidates to investigate the relationship between As bioaccessibility and As fractionation in soils. 3.2. Arsenic bioaccessibility based on four in vitro assays Four common in vitro assays (UBM, SBRC, IVG, and PBET) were used to assess As bioaccessibility in 11 contaminated soils. Arsenic bioaccessibility in soils depended on assays, ranging from 0.46 to 49% (Table 3). In this study, As bioaccessibility in the gastric phase of UBM, SBRC, IVG, and PBET assay were 7.6‒52% (averaging 29%), 2.3‒49% (25%), 7.3‒44% (20%) and 1.3‒38% (16%). The bioaccessibility trend was consistent with their gastric pH. This was consistent with results of Oomen et al. [12] and Juhasz et al. [26]. In intestinal phase, As bioaccessibility were 5.7‒53%, 0.46‒32%, 2.3‒42%, and 0.86‒43% for UBM, SBRC, IVG and PBET. In general, As bioaccessibility in gastric phase 8
was higher than that in intestinal phase of SBRC and IVG, which was also observed by Juhasz et al. [11]. It is known that Fe oxides are important in controlling As bioaccessibility in soils. Based on IVG, 18% of Fe oxide is dissolved in gastric phase and so was the associated As [27, 28]. The Fe solubilized in acidic gastric phase (pH 1.2–2.5) is probably precipitated as amorphous Fe oxides in intestinal phase at higher pH (5.5–7.0) [29]. The soluble As in gastric phase was probably adsorbed onto Fe oxides and/or soil solid in intestinal phase, thereby reducing bioaccessible As. This may explain why As bioaccessibility decreased from gastric to intestinal phase by SBRC and IVG. Different from SBRC and IVG , bioaccessible As using UBM and PBET increased from gastric to intestinal phase in some soils (Table 3). Similar increase is also observed by Juhasz et al. [11]. Higher desorption of As from solid phases at higher pH in intestinal phase than gastric phase may partially explain the increase in bioaccessible As [27, 28]. 3.3. Correlation of bioaccessible As with As in different fractions in soils In soils, As is distributed in different solid phases, which has different As bioaccessibility. To understand how different fraction contributes to bioaccessible As, soil As was separated into five fractions via sequential extraction [19]. Among the five fractions, As was primarily distributed in the amorphous Fe/Al oxide fraction (AF3; 17–79%, averaging 48%) and crystallized Fe/Al oxide fraction (CF4; 6.4–73%, 31%) (Fig. 2). Together, they accounted for 64–93% of soil As. Arsenic in the specifically sorbed fraction (SS2; 3.4–20%, 11%) and residual fraction (RS5; 3.2–25%, 9.4%) were similar. The least amount of As was in the non-specifically sorbed fraction (NS1) at 0.00–1.1% excluding soil 11 at 10%, which had the highest total As at 4,172 mg kg‒1 with sandy loam texture (Table 2). In general, the binding strength of As associated with different fractions increases from fraction one (NS1) to five (RS5) [30]. Smith et al. compared changes in As fractions before and after SBCR-GP extraction in 12 contaminated soils [21]. They reported that bioaccessible As was mainly from the first three fractions, including NS1 (<1‒11%), SS2 (<1‒29%) and AF3 (30‒93%) with last two fractions CF4 and RS5 being relatively unchanged. Arsenic in NS1 fraction includes easily exchangeable As and outer-sphere As complexes while As in SS2 fraction includes inner-sphere As complexes [19]. As in different fractions was compared with bioaccessible As based on four assays (Fig. 3. ). While average arsenic concentrations in 9
the first two fractions (NS1+ SS2, 12%) was lower than the average bioaccessible As (13‒29%), the sum of the first three fractions (NS1+SS2+AF3, 60%) was higher. The data suggested that bioaccessible As may primarily came from NS1 and SS2 fractions and part of AF3 fraction in the 11 As-contaminated soils. To explore how each As fraction contributed the bioaccessible As, we correlated As in different fractions with bioaccessible As by four assays (Table 4). It is known that RS5 fraction was the least bioavailable so it was not included. Correlation coefficient between As fractions and As bioaccessibility of UBM-GP increased from R2= 0.30 to 0.45 and to 0.58 when As in NS1, NS1+SS2, and NS1+SS2+AF3 fraction was employed, illustrating the first three fractions contributed 58% of the variability of bioaccessible As by UBM-GP. However when CF4 was added, the correlation reduced to R2= 0.17. The data illustrated that UBM-GP dissolved most of amorphous Fe/Al oxides (AF3) at pH of 1.2. For UBM-IP, only As in NS1+SS2 was correlated to bioaccessible As at R2=0.50 (Table 4). Thus, the first two fractions of As significantly contributed bioaccessible As by UBM. However, PBET behaved differently from UBM. Arsenic in NS1 and NS1+SS2 was correlated with bioaccessible As by PBET at R2=0.420.52 and 0.550.69. The data indicated that non-specifically (NS1) and specifically sorbed (SS2) As contributed the most to bioaccessible As by PBET. Similar to PBET, As in NS1 was correlated with bioaccessible As by SBRC at R2=0.410.43 (Table 4). However, bioaccessible As by IVG had poor correlation with As in different fractions with R2 < 0.32. The PBET results were consistent with Tang et al. [3] and Liang et al. [31] who reported that As in NS1+SS2 fraction was significantly correlated to bioaccessible As by PBET in 5 As-spiked soils. However, current IVG and SBRC results were different from literature. Whitacre et al. [20] reported that As in NS1+SS2 fraction correlated to bioaccessible As by IVG in 19 As-spiked soils while Smith et al. [21] reported no correlation between As in NS1+SS2 fraction and As bioaccessibility by SBRC-GP. The differences in contamination sources between the two studies were partially responsible. In Whitacre et al., [20], the 19 As-spiked soils was aged for three months while the contaminated soils in this study were collected from field, which were contaminated by pesticide application, and mining and smelting process. Insoluble minerals such as arsenopyrite, realgar, and pyrite 10
have been observed in mining soils [18] while As is mainly presented as sorbed species in spiked soils [31]. Differences in As speciation in spiked and contaminated soils were responsible for different relationship observed between As fraction and As bioaccessibility among different studies. The results highlight the importance of testing soils with different contamination sources. In addition, Smith et al. [13] attributed the presence of organic acids (citrate and malate) in PBET-GP to enhance As solubility. The presence of phosphate in UBM also played a role in As desorption [19]. Thus, the significant correlation between As in NS1+SS2 fractions with bioaccessible As was likely due to both organic acids and phosphate in desorbing As from specifically and non-specifically sorbed fractions in soils. However, they are absent in SBRC and IVG. Combining this study with those of Tang et al. [3] and Liang et al. [31], we concluded that As in NS1+ SS2 fractions mainly contributed to bioaccessible As by PBET and UBM in As-contaminated soils. 3.4. Correlation of bioaccessible As with 0.43 M HNO3 extractable As The 0.43 M HNO3 (HNO3) extractable As was 0.90‒60.1% (averaging 27.2%) in Ascontaminated soils, which was higher than bioaccessible As by gastric phase of SBRC, IVG, and PBET but comparable to UBM-GP. In general, HNO3 extractable As was correlated with bioaccessible As in gastric phase, with R2 being 0.42‒0.85, and that in intestinal phase with 0.37‒0.59 for the four methods ( Fig. 4). SBRC-GP showed the best correlation with R2 = 0.85. This was consistent with Rodrigues et al. [17] who showed good correlation between the two methods in 45 soils from grassland areas and vegetable gardens. They attribute the good correlation to their similarity in the extraction pH (SBRC-GP at pH=1.5 and 0.43 M HNO3 < 1). However the data from UBM-GP and PBET-GP were inconsistent with this hypothesis. Both showed good correlation with R2 = 0.70–0.72, yet the gastric pH of UBM is 1.2 while that of PBET is 2.5. Apparently, other factors have contributed to different bioaccessible As by different assays, including the different chemicals used in the gastric phase of different methods. For example, Li et al. [32] observed that, after removing lactic acid, acetic acid, and sodium citrate from PBET, the extractable Fe significantly decreased so was extractable As. This is because As is strongly associated with Fe mineral in soils [33]. By adding glycine, pepsin, or mucin to 11
diluted HCl solution at pH 1.5, Li et al. [34] found the extractable As of contaminated soil increased from 7.0% to 1222%, illustrating those chemicals contributed to bioaccessible As of in the assays. Since glycine is used in SBRC, lactic acid, acetic acid, sodium citrate, and pepsin in PBET, and pepsin and mucin in UBM, their differences in composition were responsible for different relationship between bioaccessible As in different assays and 0.43 M HNO3 extractable As. Among the four in vitro assays, gastric phase of SBRC extractable As was best correlated with in vivo data [11, 26]. Here, the strong correlation (R2 = 0.85) between 0.43 M HNO3 and SBRC-GP extractable As suggested that 0.43 M HNO3 extraction may have potential to determine bioaccessible As in soils.
4. Conclusion Bioaccessible As varied with different in vitro assays. Arsenic fractionation based on sequential extraction helped explain the sources of bioaccessible As in different assays. Bioaccessible As by UBM-GP mainly from first three fractions (NS1+SS2+AF3, 58%) while those by UBM-IP, PBET-GP, and PBET-IP mainly from first two fractions (NS1+SS2, 50– 69%). However, bioaccessible As by IVG and SBRC could not be explained by As in different fractions. In addition, bioaccessible As in gastric phase of UBM, SBRC, and PBET was well correlated to 0.43 M HNO3 extractable As, with R2 of 0.70–0.85, but not for IVG. Among the four in vitro assays, As in SBRC-GP was best correlated to 0.43 M HNO3 extractable As. Our data suggested that 0.43 M HNO3 has potential to determine bioaccessible As in soils. Based on its good correlation with As from the first two-three fractions and good correlation with in vivo data (r2=0.67; data not shown), UBM can be used to determine As bioaccessibility in contaminated soils.
Acknowledgements This work was supported in part by Jiangsu Provincial Innovation Program and Jiangsu Provincial Double Innovation Program.
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Table 1 Composition and in vitro parameters for UBM, SBRC, IVG, and PBET bioaccessibility assays Method Extraction Composition (g L-1) Solid/solution pH Extraction phase ratio time (h) UBM
SBRC
IVG
PBET
Gastric
0.824 g KCl, 0.266 g NaH2PO4, 2.752 g NaCl, 0.4 g CaCl2, 0.306 g NH4Cl, 8.3 ml HCl (37%), 0.085 g urea, 0.65 g glucose, 0.02 g glucuronic acid, 0.33 g glucosamine hydrochloride, 3.0 g mucin, 1.0 g bovine serum albumin, 1.0 g pepsin
1:37.5
1.2
1
Intestinal
0.94 g KCl, 12.3 g NaCl, 11.4 g NaHCO3, 0.08 g KH2PO4, 0.05 g MgCl2, 0.36 ml HCl (37%), 0.35 g urea, 0.42 g CaCl2, 2.8 g bovine serum albumin 3.0 g pancreatin, 0.5 g lipase, 6.0 g bile
1:97.5
6.3
4
Gastric
30.03 g glycine
1:100
1.5
1
Intestinal
1.75 g bile, 0.5 g pancreatin
1:100
7.0
4
Gastric
10 g pepsin, 8.77 g NaCl
1:150
1.8
1
Intestinal
3.5 g bile, 0.35 g pancreatin
1:150
5.5
1
Gastric
1.25 g pepsin, 0.5 g sodium malate, 0.5 g sodium citrate, 420 µL lactic acid, 500µL acetic acid
1:100
2.5
1
Intestinal
1.75 g bile, 0.5 g pancreatin
1:100
7.0
4
17
1 2
Table 2 Physical and chemical properties of 11 soils from different locations and contamination sources ------------------Farming------------- ------------------Mining--------------- --------------------------------Smelting--------------------------------S1
S2
S3
S4
S6
S7
S8
S9
S10
S11
Total As (mg kg‒1)
36.0±1.00 41.1±7.32 171±1.00
Total Fe (g)
27.8±0.21 42.1±0.29 28.8±0.21 76.4±0.43 103±0.06
122±1.17
29.5±0.04 21.1±0.10 84.4±0.41 196±5.19
18.7±0.06
FeDCB (g)a
11.3±0.55 25.3±3.36 16.7±0.39 29.8±1.78 29.4±3.38 74.2±1.40
8.29±0.37 13.9±0.70 38.1±1.47 50.9±0.65
10.2±0.50
FeAO (g)b
1.69±0.01 6.41±0.00 13.1±0.06 9.75±0.28 4.88±0.04 37.1±0.14
1.57±0.05 5.93±0.02 43.4±1.81 89.9±2.27
2.20±0.14
Total Mn (mg kg‒1)
589±1.3
694±6.1
458±42
Organic matter (%)
4.27±0.15 3.50±0.05 4.45±0.15 7.02±0.01 9.04±0.23 5.27±0.27
3.08±0.09 5.51±0.20 7.87±0.09 9.92±0.23
1.34±0.02
pH
7.50
7.50
7.40
Sand (%)
13.2±1.15 19.0±2.69 27.6±1.81 18.9±1.23 20.3±1.43 13.4±2.73
10.0±0.73 19.7±0.52 28.0±0.32 52.6±3.78
56.5±3.64
Silt (%)
78.4±1.12 72.1±3.98 65.2±1.35 67.4±1.02 66.4±1.38 73.6±2.50
79.8±0.66 70.7±0.34 65.3±0.37 44.5±3.51
39.7±3.15
Clay (%)
8.37±0.03 8.91±1.29 7.15±0.46 13.8±0.20 13.4±0.05 13.1±0.24
10.2±0.11 9.66±0.18 6.75±0.04 2.83±0.28
3.80±0.49
78.9±0.69 239±2.4
3.50
6.60
75.2±1.14 743±22.0
495±2.3
4.10
Texture 3 4 5
S5
a
8,992±97
7.30
Silt loam
1470±23.9 22.2±1.16 86.8±2.00 861±34.0
109±2.5
2.60
216±1.3
5.50
1,251±16
6.10
2,556±61.5
2,751±28
6.70
4,172±62.5
Sandy loam Sandy loam
Values represent dithionite–citrate–bicarbonate extractable Fe Values represent acid ammonium oxalate extractable Fe
b
18
6 7 8
Table 3 Bioaccessible arsenic concentrations in 11 As-contaminated soils based on gastric (GP) and intestinal phase (IP) of UBM, SBRC, PBET, and IVG in vitro assays and 0.43 M HNO3 extraction (%). Soil UBM‒GP UBM‒IP SBRC‒GP SBRC‒IP IVG‒GP IVG‒IP PBET‒GP PBET‒IP 0.43 M HNO3 S1
43.9±2.87
52.9±0.28
37.2±4.27
17.7±1.48
33.8±1.59
30.9±0.38
33.6±1.38
31.2±3.43
28.8±0.71
S2
7.59±0.14
13.9±1.45
17.1±0.37
15.0±0.44
15.7±1.33
14.3±0.38
1.33±0.10
3.02±0.11
16.4±2.62
S3
52.4±1.98
44.4±5.21
40.3±0.69
26.6±0.91
17.5±3.92
20.0±3.92
29.6±1.60
28.5±1.40
47.1±1.42
S4
13.6±0.58
15.2±1.04
11.1±0.59
9.80±0.57
12.6±2.53
11.6±0.36
2.65±0.14
7.37±0.26
5.83±0.11
S5
10.7±0.10
13.0±0.33
2.33±0.06
1.47±0.08
8.04±0.45
5.46±0.14
2.80±0.01
4.03±0.14
0.90±0.05
S6
17.0±0.58
14.4±1.73
12.8±0.32
0.46±0.01
7.26±0.52
3.66±0.07
10.6±0.51
5.58±0.06
18.0±0.08
S7
30.0±2.07
51.3±1.92
28.3±0.39
24.6±0.98
44.1±1.74
42.3±1.52
24.2±0.39
21.8±1.23
39.9±3.45
S8
36.3±2.09
38.2±0.51
23.3±0.35
16.3±0.06
21.7±0.40
14.7±2.75
15.1±2.77
21.6±1.33
20.7±0.45
S9
33.1±5.29
18.2±1.01
18.1±0.60
2.26±0.11
11.8±0.99
9.28±0.15
12.5±0.81
12.3±0.37
30.5±0.70
S10
27.4±2.86
5.74±0.21
30.5±0.88
0.56±0.02
11.0±0.25
2.32±0.06
6.70±1.52
0.86±2.59
30.8±0.33
S11
51.9±1.07
41.1±1.27
49.2±2.13
32.6±0.78
39.1±0.30
37.3±0.80
37.7±1.08
42.8±0.86
60.1±4.34
Minimum
7.59
5.74
2.33
0.46
7.26
2.32
1.33
0.86
0.90
Maximum
52.4
52.9
49.2
32.6
44.1
42.3
37.7
42.8
60.1
Average
29.4
28.0
24.6
13.4
20.2
17.4
16.1
16.3
27.2
19
9 10 11
12 13 14 15 16
Table 4 Linear correlation between different As fractions and bioaccessible As based on the gastric (GP) and intestinal phases (IP) of four in vitro assays (UBM, SBRC, IVG, and PBET) in 11 contaminated soils --------NS1+SS2+AF3+CF4-------------------NS1a------------- -----------NS1+SS2---------- ---------NS1+SS2+AF3-------Extractable As Equation R2 Equation R2 Equation R2 Equation R2 UBM-GP
2.92x+25.8
0.30
1.82x+6.91
0.45
0.50x-0.61
0.58
1.08x-68.7
0.17
UBM-IP
2.11x+25.4
0.13
2.09x+2.20
0.50
0.19x+16.9
0.07
0.94x-57.5
0.11
SBRC-GP
3.08x+20.7
0.43
1.38x+7.49
0.33
0.34x+4.23
0.34
1.03x-68.4
0.20
SBRC-IP
2.45x+10.3
0.41
0.94x+1.74
0.23
0.06x+9.96
0.01
0.58x-39.1
0.10
IVG-GP
2.42x+17.2
0.32
1.11x+6.52
0.26
0.35x+18.2
<0.01
0.37x-14.0
0.03
IVG-IP
2.60x+14.2
0.32
1.23x+2.23
0.28
0.005x+17.1
<0.01
0.38x-17.4
0.03
PBET-GP
2.82x+12.6
0.42
1.86x-6.91
0.69
0.26x+0.40
0.23
0.59x-38.0
0.08
PBET-IP
3.31x+12.2
0.52
1.73x-5.18
0.55
0.23x+2.59
0.16
0.53x-32.0
0.06
a
NS1 = non-specifically sorbed, SS2 = specifically sorbed, AF3 = amorphous poorly-crystalline Fe/Al oxides, and CF4 = well-crystallized Fe/Al oxides. R2 > 0.50 are in bold text.
20
As Contaminated Soils
Physico-chemical properties chracterization
As Sequential Extraction
UBM, SBRC, IVG, PBET
0.43 M HNO3 Extraction
As Fraction Distribution
As Bioaccessibility
As Reactive Pool
One Way ANOVA
17 18 19 20 21
Explain
Varied with various methods
Relationship
Fig. 1. Flow chart for all extractions methods used in the experiment, including sequential extraction, 4 in vitro assays (UBM, SBRC, IVG and PBET) and 0.43 M HNO3 extraction.
21
100
RS5 CF4 AF3 SS2 NS1
90
Extractable As (%)
80 70 60 50 40 30 20 10 0 S1 22 23 24 25 26 27 28
S2
S3
S4
S5
S6 S7 Soil ID
S8
S9 S10 S11
Fig. 2. Arsenic fractionations in 11 contaminated soils. Error bars represent the standard deviation of triplicate. NS1 = non-specifically sorbed; SS2 = specifically sorbed; AF3 = amorphous and poorly-crystalline Fe/Al oxides; CF4, well-crystallized Fe/Al oxides; and RS5 = residual.
22
100 90 80
Percent (%)
70 60 50 40 30 20 10
29
N S1 N S N S1 1+S S +S S2 2 +A F3 U BM -G P U BM SB -IP R CG SB P RC -I IV P G -G P IV G PB -IP ET -G PB P ET 0. 43 M IP H NO 3
0
30 31 32 33 34 35 36
Fig. 3. Extractable As in different fractions (NS1, SS2 and AF3), gastric (GP) and intestinal phase (IP) of UBM, SBRC, IVG, and PBET, and 0.43 M HNO3 extraction. Boxes represent 25th to 75th percentile, solid lines and squares in boxes are the median and mean values, crossing symbols represent 1th to 99th percentiles, error bars represent the minimum and maximum values. NS1 = non-specifically sorbed; SS2 = specifically sorbed; AF3 = poorlycrystalline Fe/Al oxides.
23
60
y=0.61x+11.52, R2=0.37 UBM-IP (%)
UBM-GP (%)
80 y=0.77x+8.39, R2=0.72 70 60 50 40 30 20 10 0
y=0.74x+4.47, R2=0.85
y=0.47x+0.65, R2=0.51 SBRC-IP (%)
SBRC-GP (%)
40 30 20 10 0 60
y=0.48x+7.16, R2=0.42
y=0.51x+3.42, R2=0.43
50 40
IVG-IP (%)
IVG-GP (%)
As bioaccessibility (%)
50
30 20 10 0 60
y=0.63x-0.989, R2=0.70
y=0.61x-0.249, R2=0.59
40 30 20 10 0
37 38 39 40 41 42
PBET-IP (%)
PBET-GP (%)
50
0
10 20 30 40 50 60 70
0
10 20 30 40 50 60 70
0.43 M HNO3 extractable As (%) Fig. 4. Linear relationship between 0.43 M HNO3 extractable As and bioaccessible As in gastric (GP) and intestinal phase (IP) of UBM, SBRC, IVG, and PBET assays. Dash lines represent 95% confidence interval.
24
43
25