Comparison of three sequential extraction procedures for arsenic fractionation in highly polluted sites

Chemosphere 178 (2017) 402e410

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Comparison of three sequential extraction procedures for arsenic fractionation in highly polluted sites Xiang Wan a, Haochen Dong a, Liu Feng a, *, Zhijia Lin b, Qiuchen Luo a a b

Department of Environmental Sciences and Engineering, Beijing University of Chemical Technology, Beijing, 100029, PR China Hunan Institute of Geological Survey, Changsha, Hunan, 410116, PR China

h i g h l i g h t s
With satisfied total recovery, Shiowatana SEP showed higher extraction efficiency in potentially mobile arsenic fractions.
Shiowatana SEP was preferred and efficient for the most mobile arsenic extraction.
Bioavailability evaluation on different arsenic fractions provides an insight to SEPs comparison.
A case study applied by Shiowatana SEP shows potential risk in relevant areas.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 January 2017 Received in revised form 13 March 2017 Accepted 19 March 2017

Three sequential extraction procedures (SEPs) including Tessier, Rauret, and Shiowatana SEPs, were compared for arsenic fractionation using highly polluted soils. In the definition context of exchangeable, reducible, oxidizable and residual fractions, with similar arsenic recovery and reproducibility, Tessier and Rauret SEPs were comparable to each other, whereas Shiowatana SEP showed higher extraction efficiency in all the first three arsenic fractions, although it might overestimate the reducible arsenic. Pot experiment indicated three SEPs all could provide an estimation of the most bioavailable arsenic fraction, and the application of Shiowatana SEP should be preferred. Accordingly, a case study with Shiowatana SEP for a site near a realgar mine area is conducted. The results show that although arsenic in this area presents predominantly in the stable fractions, the sum of most bioavailable fractions was accounted around 11% of total arsenic, and moreover, about another 10% of the total arsenic, the fourth fraction in Shiowatana SEP is likely to be transferred into bioavailable species under suitable conditions, such as strong acid impact, revealing a real major risk source being formed. The study indicated that Shiowatana should be more suitable for arsenic fractionation to provide valuable information in the framework of risk assessment. © 2017 Elsevier Ltd. All rights reserved.

Handling Editor: Caroline Gaus Keywords: Arsenic Fractionation Sequential extraction procedure Risk assessment Soil

1. Introduction Soil arsenic pollution has become a world problem. Many arsenic polluted sites have been found around the world due to human activities (Hibiki and Arimura, 2004; Ahmad and Goni, 2010), and millions of people were exposed to varied degrees of soil pollution (Karim, 2000; Rodríguezlado et al., 2013). Because of their high-toxicity and potential carcinogenic effect, arsenic polluted sites have been increasingly concerned, and many researches on risk assessment for these sites have been reported.

* Corresponding author. E-mail address: [email protected] (L. Feng). http://dx.doi.org/10.1016/j.chemosphere.2017.03.078 0045-6535/© 2017 Elsevier Ltd. All rights reserved.

Among them, total-concentration-based methodology has been widely applied for heavy metal polluted sites in early stage (Bech et al., 1997; Chen et al., 1997; Doyle and Otte, 1997). Nevertheless, with the progress in the researches on environmental behaviors and ecological effects of heavy metals, it has been gradually recognized that environmental effects of heavy metals, such as toxicity, migration and geo-chemical cycle, should depend on their chemical forms rather than their total concentration (Georgiadis et al., 2006; Ruiz-Chancho et al., 2007). Due to disregarding the differences in environmental effects and bioavailability among various chemical fractions of heavy metals, the total concentration based method often overestimated the potential risk, and has gradually given way to chemical-form-oriented methods and procedures in latter risk assessment for heavy metal polluted sites

X. Wan et al. / Chemosphere 178 (2017) 402e410


n et al., 2012; Lou et al., 2015). (Hartley et al., 2010; Guille Many sequential extraction procedures (SEP) can be used to define and extract chemical fractions of heavy metals in soil. These methods classify heavy metals in soil into different operationally defined fractions with increasing metal binding strength. Among them, Tessier SEP is developed for the partitioning of heavy metals into the water soluble and exchangeable fraction, the fraction bound to carbonates, the fraction bound to Fe/Mn oxides, the fraction bound to organic matter and the residual fraction (Tessier et al., 1979), it has been widely used in fractionation partition of mostly heavy metals like Cd, Pb, Cu, Co, Ni, Zn, Fe and Mn in soil/
rezcid et al., 1999; Filgueiras et al., 2002; Frankowski sediment (Pe et al., 2010), but it is seldom used for arsenic fractionation, and a few researchers found Tessier SEP was not entirely suitable for the determination of arsenic fractions due to incomplete oxidation of some bound arsenic fractions like arsenopyrite by the oxidation agents H2O2 used (Mihaljevic et al., 2003). BCR (shorter form of the Community Bureau of Reference of the European Commission) SEP was first developed in 1993 (Ure et al., 1993) and improved by Rauret et al. (1999). In accordance with this modified BCR SEP, elements of interest are divided in 4 operationally defined steps as the acid extractable fraction, the reducible fraction, the oxidizable fraction and the residual fraction. BCR SEP is another mostly commonly used method after Tessier SEP in present €kel€ (Albores et al., 2000; Zhang et al., 2010; Ma a et al., 2011; Chakraborty et al., 2014; KerollieMustafa et al., 2015). However, similar to Tessier SEP, it has occasionally been used to evaluate arsenic fractionation (Sz
akov
a et al., 1999; Fernandez et al., 2004; Baig et al., 2009; Otones et al., 2011), and a few researches also indicated that it was not entirely applicable for fractionation of arsenic in soil for similar reasons (Larios et al., 2012). In contrast with the cationic nature of trace metals, arsenic is predominantly present in soils and sediments as oxyanions. Hence, traditional SEPs have not been recommended for arsenic and procedures especially designed for its study have been advised (Gruebel et al., 1988), and some SEPs (Mclaren et al., 1998; Shiowatana et al., 2001; Wenzel et al., 2001; Cappuyns et al., 2002) specially developed for arsenic fractionation have been proposed and applied in the past years. Among them, the SEP proposed by Mclaren et al. (1998) and improved by Shiowatana et al. (2001) is one of the best methods to date. Within this procedure, arsenic in soil/sediment is divided into the water-soluble fraction, the surface-adsorbed fraction, the Fe/Al associated fraction, the acid extractable fraction and the residual fraction. Up to now, its practical application reported in the literatures is very few (Hartley et al., 2009; Beesley et al., 2010), its efficiency for arsenic fractionation remains to be tested through more real case studies. In summary, both the well-recognized and experienced SEPs and the developing arsenic-specific SEPs have limitations in arsenic fractionation, and it is indispensable to compare different SEPs, selecting more suitable SEP for arsenic fractionation in arsenic contaminated sites. To be sure, some studies have already concerned comparisons among different SEPs, but perspectives they have focused on were the recovery rate and reproducibility, the redistribution/readsorption during extraction, the selectivity of reagents toward the targeted solid materials and so on (Mihaljevi c et al., 2003; Larios et al., 2012). Bioavailability of heavy metals is widely known to be a critical factor to either their transfer from soil to plant or their environmental risks (Bryan and Langston, 1992; Khan et al., 2008), however the evaluation on the bioavailability of different fractions defined by each SEP has hardly or rarely been involved in the comparison studies (Vandenhove et al., 2014), which should be addressed in future. To improve our understanding of the risks associated with

403

arsenic in soil, this study would compare three SEPs (Tessier, Rauret and Shiowatana) for some arsenic-polluted soil samples and evaluate the bioavailability of each arsenic fraction defined by the three SEPs. As such, it is aimed to find the most appropriate SEP for arsenic-polluted sites and to provide valuable information in the framework of risk assessment. 2. Materials and methods 2.1. Soil source and its physicochemical properties Shimen realgar (As4S4) mine area is one of the five main pollution sources in China. Some studies indicated that total arsenic concentration in soil there reached up to 5240 mg kg1, revealing a great potential risks for human health (Tang et al., 2016). It could be selected as an ideal case of arsenic polluted sites for this study. Total 34 topsoil samples (about 0e30 cm in depth, numbered from S1 to S34) were collected by grid sampling method with a grid size of 50 m  50 m within about 1 km2 (Fig. 1). All soil samples were air-dried in natural conditions, grinded, and sieved with 2 mm screen for later use. For all samples, we measured some physical and chemical properties including: pH, organic matter (OM), oxidation-reduction potential (ORP), and the contents of Fe, Mn, S, Ca, Al. For pH and OM measurements, we followed the techniques of a previous study (Sungur et al., 2014). ORP was determined using a depolarization method by an automatic ORP analyser (FJA-5) (Dong et al., 2017). The contents of Fe, Mn, S, Ca, Al was measured by XRF (Jansen et al., 1998; Johnson et al., 1999). Preliminary test indicated that the soil in this region was weakly acidic except only four high pH values was neutral soils (pH ranging from 5.26 to 7.46), with moderate soil fertility (the content of organic matter in soil ranging from 0.35% to 3.59%) and relative high oxidation resistance (soil redox potential ranging from 412 mV to 723 mV). The contents of Fe, Mn, S, Ca and Al in soil range from 1.2% to 15.6%, 0.01% to 0.39%, 0.35% to 6.08%, 0.98% to 4.86% and 1.07% to 6.54% respectively (see supplementary document). 2.2. Extraction of arsenic in soil samples 2.2.1. Extraction of total arsenic Total arsenic in soil was measured by hydride generationdatomic fluorescence way (Liu, 2005). Proceed as follows: each soil sample was mixed evenly; a certain quantity of sample was taken by quarterly dividing method and ground to 0.149 mm or less, then was digested by chloroazotic acid (aqua regia) for 2 h at water bath of 96  C. After complete cooling, the digestion was collected through centrifugation and filtration for later arsenic analysis. 2.2.2. Extraction of different arsenic fractions by three SEPs Nine of the 34 soil samples were selected to conduct comparative study on the three SEPs, based on the distribution of total arsenic concentrations in each soil sample. The arsenic fractions defined by each SEP and their extraction procedures are listed in Table 1. All materials which were in contact with the soil samples were soaked overnight in 4 M HNO3 before use. Like the procedure for extraction of total arsenic, a certain quantity of samples (2 g for each SEP) were taken, air dried at room temperature and sieved (0.149 mm) prior to extraction. For each SEP, after each step of the procedure, extracts were centrifugated at 4000 rpm for 30 min, and filtered through a 0.1 mm cellulose filter with a vacuum. The soil residue of soil was suspended in 20 mL deionized water, shaken end-over-end for 15 min and centrifugated at 4000 rpm for 5 min before filtration with the

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X. Wan et al. / Chemosphere 178 (2017) 402e410

Fig. 1. Map of the sample points.

Table 1 Extraction procedure for three methods. Desired fraction Procedure 1: Tessier et al. (1979) FT1: water soluble/exchangeable FT2: carbonates FT3: oxides (Fe/Mn) FT4: organic matter

FT5: residual

Procedure 2: Shiowatana et al. (2001) FS1: water-soluble FS2: surface-adsorbed FS3: Fe/Al associated FS4: acid-extractable FS5: residual Procedure 3: Rauret et al. (1999) FR1: exchangeable FR2: reducible FR3: oxidisable

FR4: residual

Extractive reagents

Reaction time and temperature

Reagent/sample ratio (mL g1)

1.0 M MgCl2 (pH ¼ 7) 1.0 M NaOAc in 25% HOAc (pH ¼ 5) 0.04 M NH2OH,HCl in 25% HOAc 1) 0.02 M HNO3/30% H2O2 (pH ¼ 2) 2) 30% H2O2 3) 3.2 M NH4OAc in 20% HNO3 1) HClO4: HF in 1:5 2) HClO4: HF in 1:10 3) HClO4

1 h, 25  C 25  C, 5 h 96  C, 6 h 1) 85  C, 5 h 2) 85  C, 3 h 3) 25  C, 0.5 h 180  C, near dryness

8:1 8:1 20:1 1) 3:1/5:1 2) 3:1 3) 5:1 1) 12:1 2) 11:1 3) 1:1

Deionized water (pH ¼ 6) 0.5 M NaHCO3 (pH ¼ 9) 0.1 M NaOH (pH ¼ 13) 1 M HCl 14 M HNO3

25  C, 16 h 25  C, 16 h 25  C, 16 h 25  C, 16 h 200  C, 15 min

30:1 30:1 30:1 30:1 10:1

0.11 M CH3COOH (pH ¼ 2e3) 0.5 M NH2OH,HCl (pH ¼ 1.5) 1) 30% H2O2 (pH ¼ 2e3) 2) 30% H2O2 (pH ¼ 2e3) 3) 1 M NH4OAc Aqua regia/hydrofluoric acid

23 ± 2  C, 16 h 23 ± 2  C, 16 h 1) 25  C, 1 h 2) 85  C, 2 h 3) 23± 2  C, 16 h 180 ± 5  C, 20 min

20:1 20:1 1) 5:1 2) 5:1 3) 25:1 20:1/2:1

same filter. The filter was rinsed with deionized water and kept for the following step of extraction procedure, and both of the filtrates were added to the extract for measurement. 2.3. Pot experiment Pot experiment was designed to evaluate the bioavailability of each arsenic fraction defined by the three SEPs. Amaranth (Amaranthus tricolor) that has been widely used in previous studies on the

soil-to-plant transfer of arsenic (Yao et al., 2008; Mellem et al., 2012) was selected as an indicator for plant uptake of arsenic. For each soil sample used in the comparative study, 1.5 kg of dry weight soil was moistened to 50% the saturation point with deionized water and incubated for 3 weeks. At the end of the incubation period, about 500 g of moist soil (50% saturation) was transferred to a f22.5 cm  15 cm pot. Three replicates were prepared per soil sample. 0.15 g amaranth seeds (about 100 capsules) were sown on top and covered with 60 g of the respective moist

X. Wan et al. / Chemosphere 178 (2017) 402e410

soil. 27 pots (3 replicates, 9 soil samples) were randomly placed in a plant growth incubator. Day and night temperatures were on average 27  C and 22  C respectively, in a 12 h/12 h cycle, with a light intensity of 12 000 Lux. Soil moisture was controlled by weighing pots and adjusting the weight with deionized water every second day. After 50 days, amaranth was harvested. Roots were handpicked after soaking the soil with deionized water for 1 h. They were then rinsed 3 times with deionized water to remove adhering soil particles. Plant samples were freeze dried under vacuum at 60  C for 2 days and dry weights were recorded. Before arsenic analysis, all plant samples were digested with concentrated HNO3 and H2O2 in the presence of 400 W microwave for 1 h at 120  C (Rodushkin et al., 1999). 2.4. Arsenic analysis Arsenic in both soil and plant digestion/extracts was determined by the atomic fluorescence spectrophotometry with a doublechannel atomic fluorescence spectrometer (Beijing Haiguang Instruments, AFS3100). Specific operating parameters of the spectrometer read as follows: alkali 1% KBH4 solution is added with a flow rate of 4.80 mL min1, sample solution is fed using 10% HCl (mass percentage) carrier liquid with a flow rate of 2.50 mL min1, and high-purity argon is used as a carrier gas with a flow rate of 400 mL min1. 2.5. Data analysis Statistical analysis, inclusive of the Wilcoxon test (at 95 confidence) for paired samples when comparing the arsenic concentrations in corresponding fractions defined by each SEP, shall be conducted on experimental data with SPSS20. Surfer 8 software is used for drawing of the contour map of total arsenic concentration.

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in the environment. For most soils, 1.21% to 5.73% of the total extracted arsenic is associated with this fraction. The second fraction (FT2) contains arsenic complexes formed with carbonates. Large variations were observed among soils selected for the extraction by arsenic fraction FT2, ranging from 0.170% to 6.63%. The third target fraction (FT3) defined by this SEP is often reducible, which mainly includes the fraction bound to Fe/Mn oxides. The relative arsenic concentration extracted by FT3 ranges from 1.31% to 21.8%, second only to that extracted by the residual fraction. The fourth fraction (FT4) comprises mainly of the metals associated with OM, in which the metals bound to sulfides may also be extracted. For this SEP, the total arsenic extracted by FT4 ranges from 0.630% to 11.6%. The residual arsenic is determined in the fifth fraction (FT5). Due to the historically high arsenic pollution, very high relative concentrations of residual arsenic were observed for all the studied soil samples, ranging from 66.0% to 96.3%, which is in with the results of Müller (Müller et al., 2007). Metal recovery, defined as the ratio of the sum of all metal fractions associated with a SEP to the pseudo-total metal concentration in the soil (Table 2, arsenic recovery [%]), is often used to judge whether the SEP is suitable for the fractionation of the target metal. With Tessier's method, arsenic recovery ranges from 72.2% to 123%, while the undesired low arsenic recovery was observed for three soil samples (S3, S6 and S12) containing an extremely high concentration of arsenic. Furthermore, the coefficient of variation (CV) is also used to measure the reproducibility of a SEP (Table 4, CV [%]). CVs for the fractions defined by this SEP are generally low. Higher CVs are also found, although they mostly do not exceed 10.0%. As seen in Table 2, apart from the heterogeneity of polluted soil samples, these higher CVs could mainly be attributed to the low concentration of arsenic in the extracted fractions (i.e. FT1, FT2).

3. Results and discussion 3.1. Total arsenic concentration and its spatial distribution in the case area The total arsenic concentration in all the soil samples from the case area ranges from 22 mg kg1 to 2462 mg kg1, with a median concentration of 773 mg kg1. Arsenic concentration in the soil at all the sampling sites exceeds the soil background level of 11 mg kg1 in China (Chen et al., 1991), with the highest reaching 220-fold, revealing great potential risks to environment and human health. The concentration contour map of total arsenic is as shown in Fig. 2. Three relatively concentrated parcels (A, B and C; Fig. 2) were found with abnormal levels of arsenic, two of which (i.e. A and B) showed extremely high arsenic concentration that can be attributed to the mining history. Parcel A was located close to the entrance of the mine pit, while B was near the tailings dumping site. Parcel C, with a slightly lower arsenic concentration, probably due to long-term surface runoff and rain erosion, was located about 700 m from the mine pit, below the tailings dumping site. 3.2. Fractionation of arsenic in selected soil samples by the three SEPs 3.2.1. Method of Tessier The results of the three SEPs are shown in Table 2. The first fraction (FT1) usually contains water soluble species, such as free arsenate, and weakly adsorbed species. Therefore, it represents the most mobile and potentially the most bioavailable arsenic species

3.2.2. Method of Rauret Arsenic in the first fraction (FR1) with this SEP is supposed to be water- and weak-acid-soluble, as well as bound to carbonates, which can be a measure for the arsenic most-readily released into the environment. The relative arsenic concentration associated with FR1 ranges from 1.34% to 8.83%. In the second fraction step (FR2), the reducible fraction, which is mostly represented by the fraction bound to Fe/Mn oxides, is determined. For most soil samples, a relatively significant part of the total arsenic is associated with FR2, ranging from 1.71% to 13.1%. The third extraction phase (FR3) represents the fractions bound to OM and sulfides. The relative content of arsenic extracted by FR3 varies greatly, from 0.520% to 9.76%. Similarly, a very high relative concentration of residual arsenic, extracted by FR4, was observed for all soil samples, ranging from 70.2% to 97.5%. Arsenic recovery for this SEP ranges from 73.4% to 102%, with the undesirable low recovery also having been found to be associated with the same three soil samples involved in the SEP of Tessier. Similarly, CVs of different extracted fractions following the SEP of Rauret are also generally low and acceptable. 3.2.3. Method of Shiowatana Within this SEP, arsenic in the first fraction (FS1) is water soluble and the most bioavailable in soil. Like the other two SEPs, a small portion of the total extracted arsenic is associated with this fraction, ranging from 0.931% to 4.48%. The surface adsorbed arsenic in the second fraction (FS2) is easily

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X. Wan et al. / Chemosphere 178 (2017) 402e410

Fig. 2. Spatial distribution of arsenic in the case study area.

released into the environment, which can be regarded as potentially less bioavailable. For most soil samples, a relatively small proportion of the total arsenic is also extracted by FS2, ranging from 0.422% to 7.88%. The third extraction phase (FS3) represents the arsenic species associated predominantly with Fe and Al materials (oxides and hydroxides). A very significant portion of the total arsenic is associated with FS3, ranging from 21.0% to 56.1%, second only to the total arsenic extracted by FS5. The fourth fraction step (FS4) is designed to extract the fractions bound to OM and sulfides, as well as the Ca-associated fraction. Relatively small proportion of arsenic is found to be extracted by FS4 overall, ranging from 2.06% to 18.2%. Compared with the other two SEPs, the relative concentration of residual arsenic extracted by the fifth fraction (FS5) is generally low, ranging from 28.4% to 73.7%. However, it is the largest portion of total arsenic extracted by this SEP. Arsenic recovery for this SEP, ranging between 83.3% and 114%, is satisfactory in general. Furthermore, CVs for all fractions defined by this SEP are generally low, ranging between 0.600% and 14.3%, mostly being less than 5.00%. This implies that this SEP is slightly better than the other two SEPs with regard to arsenic fraction for high arsenic pollution sites, in terms of arsenic recovery and producibility. 3.3. Bioavailability evaluation on different fractions by three SEPs The soil-to-plant transfer factor (TF), which is defined by the ratio of the metal concentration in plants to the metal concentration in soil, is often used to parameterize metal uptake by plants. For all except three soil samples (S3, S6 and S12), where the planted amaranth was found to grow abnormally with no germination or

death after 15 days, the TFs for amaranth were determined and are presented in Fig. 3. TFs varied between 0.108 ± 0.004 and 0.353 ± 0.006, comparable with that reported by some related studies on arsenic uptake by amaranth (Choudhury et al.; Yao et al., 2008). Correlation analysis between the metal concentration in the extracted fractions by single or sequential extraction procedures and the metal concentration up-taken by plants has been widely used to evaluate the bioavailability of metal in soil. A good correlation often implies that the corresponding metal fractions are bioavailable (Vandenhove et al., 2014; Novotn
a et al., 2015). As shown in Table 3, at 99% confidence, the arsenic concentration in amaranth is significantly correlated to the arsenic concentration extracted by the first fraction (FT1, FS1 and FR1) of each SEP, with the highest correlation coefficient of R2 ¼ 0.98 being for the SEP of Tessier, followed by the arsenic concentration extracted by the first two fractions (FS1þFS2) of Shiowatana SEP, with a correlation coefficient of R2 ¼ 0.93. In addition, at 95% confidence, a significant correlation was also found between the arsenic concentration in amaranth and the arsenic concentration extracted by the second fraction (FS2) of Shiowatana SEP, with a correlation coefficient of R2 ¼ 0.88, as well as the arsenic concentration extracted by the first two fractions (FT1þFT2) of Tessier SEP, with a correlation coefficient of R2 ¼ 0.85. Based on these results, it can be concluded that the first arsenic fraction defined by each SEP and the second arsenic fraction defined by the SEP of Shiowatana are potentially bioavailable, the second fraction (FS2) of Shiowatana SEP is surface-adsorbed, is easily mobile and absorbed by plants for some studies (Shiowatana et al., 2001; Hartley et al., 2009). While the arsenic concentration extracted by the first two fractions of Shiowatana SEP is possibly a good approximation for plant uptake, this should be confirmed by further tests using more soil samples and more plants.

Table 2 Arsenic concentration in the extracted fractions according to the three SEPs. Soil samples

Total As concentration Method of Tessier FT1: water-slouble/ exchangable FT2: carbonates

FT3: Fe/Mn oxides

FT4: organic matter

FT5: residual

[mg kg

S(FT1~FT5) Arsenic recovery Method of Shiowatana FS1: water-slouble As [mg As [%] CV [%] FS2: surfaceAs [mg adsorbed As [%] CV [%] FS3: Fe/Al As [mg associated As [%] CV [%] FS4: acid extractable As [mg As [%] CV [%] FS5: residual As [mg As [%] CV [%] S(FS1~FS5) As [mg Arsenic recovery [%] Method of Rauret FR1: exchangable As [mg As [%] CV [%] FR2: reducible As [mg As [%] CV [%] FR3: oxidisable As [mg As [%] CV [%] FR4: residual As [mg As [%] CV [%] S(FR1~FR4) As [mg Arsenic recovery [%]

]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

kg1]

S1

S18

S9

S16

S15

S11

S3

S12

S6

45.51 ± 2.15

68.93 ± 2.18

98.68 ± 3.91

128.47 ± 4.15

230.82 ± 11.22

398.81 ± 11.73

1437.49 ± 42.95

1509.41 ± 79.49

2462.7 ± 119.44

0.78 ± 0.07 1.64 ± 0.19 9.0 0.08 ± 0.01 0.17 ± 0.02 12.5 0.62 ± 0.04 1.31 ± 0.08 6.5 0.30 ± 0.03 0.63 ± 0.06 10.0 45.69 ± 4.01 96.25 ± 8.58 8.8 47.47 ± 4.09 104.31 ± 4.73

1.40 ± 0.17 1.81 ± 0.09 12.1 0.19 ± 0.01 0.25 ± 0.01 3.1 1.87 ± 0.19 2.42 ± 0.25 10.2 2.33 ± 0.23 3.02 ± 0.30 9.8 71.35 ± 3.73 92.5 ± 4.89 5.2 77.15 ± 4.05 111.93 ± 3.53

1.03 ± 0.14 1.21 ± 0.16 13.6 1.83 ± 0.07 2.06 ± 0.08 8.4 6.29 ± 0.42 7.42 ± 0.49 6.6 6.16 ± 0.22 7.27 ± 0.25 3.5 69.52 ± 2.26 81.95 ± 2.67 3.3 84.83 ± 1.46 85.96 ± 3.48

1.78 ± 0.08 1.73 ± 0.08 4.5 0.20 ± 0.17 0.19 ± 0.02 8.5 9.97 ± 0.28 9.69 ± 0.31 2.8 3.99 ± 0.13 3.88 ± 0.13 3.3 86.90 ± 2.79 84.51 ± 2.85 3.2 102.84 ± 2.72 80.04 ± 2.52

4.43 ± 0.50 1.56 ± 0.02 11.3 0.82 ± 0.05 0.29 ± 0.02 6.5 15.54 ± 1.29 5.45 ± 0.46 8.3 8.05 ± 0.30 2.83 ± 0.11 3.7 256.05 ± 10.79 89.88 ± 3.84 4.2 284.89 ± 11.49 123.42 ± 6.03

4.94 ± 0.24 1.40 ± 0.07 4.9 3.89 ± 0.29 1.10 ± 0.08 7.5 35.33 ± 3.22 10.00 ± 0.91 9.1 8.76 ± 0.30 2.48 ± 0.08 3.4 300.55 ± 12.26 85.03 ± 3.47 4.1 353.47 ± 14.14 88.63 ± 2.62

37.82 ± 2.76 3.41 ± 0.30 7.3 62.44 ± 3.52 5.63 ± 0.38 5.6 75.42 ± 2.34 6.80 ± 0.36 3.1 128.59 ± 8.16 11.59 ± 0.90 6.3 804.86 ± 21.20 72.57 ± 2.33 2.6 1109.13 ± 10.11 77.16 ± 1.87

14.50 ± 1.68 1.33 ± 0.19 11.6 48.8 ± 4.68 4.48 ± 0.53 9.6 237.74 ± 8.12 21.82 ± 1.21 3.4 69.66 ± 2.13 6.39 ± 0.29 3.1 718.79 ± 21.08 65.97 ± 3.12 2.9 1089.49 ± 30.54 72.18 ± 3.05

108.50 ± 5.28 5.73 ± 0.29 4.9 125.60 ± 6.72 6.63 ± 0.37 5.4 214.30 ± 11.08 11.32 ± 0.62 5.2 86.90 ± 6.62 4.59 ± 0.37 7.6 1358.50 ± 50.42 71.73 ± 2.81 3.7 1893.80 ± 74.86 76.90 ± 3.44

1.17 ± 0.13 2.56 ± 0.27 11.1 2.25 ± 0.13 4.93 ± 0.23 5.8 10.05 ± 0.90 22.03 ± 1.97 9.0 2.54 ± 0.08 5.56 ± 0.17 3.2 29.63 ± 0.72 64.9 ± 1.58 2.4 45.64 ± 0.29 100.28 ± 4.15

0.89 ± 0.05 1.38 ± 0.07 5.6 1.15 ± 0.10 1.78 ± 0.16 8.7 13.58 ± 0.75 21.04 ± 1.16 5.5 1.33 ± 0.05 2.06 ± 0.07 3.5 47.58 ± 2.48 73.73 ± 3.91 5.2 64.53 ± 1.79 93.62 ± 2.95

1.05 ± 0.14 0.93 ± 0.13 13.3 2.27 ± 0.25 2.01 ± 0.13 11.0 25.21 ± 1.17 22.29 ± 1.04 4.7 9.46 ± 0.27 8.36 ± 0.23 2.8 75.13 ± 1.19 66.42 ± 1.03 1.6 113.12 ± 2.52 114.63 ± 4.28

1.82 ± 0.06 1.37 ± 0.04 3.3 0.56 ± 0.08 0.42 ± 0.06 14.3 47.96 ± 2.43 36.18 ± 1.85 5.1 11.20 ± 0.48 8.45 ± 0.36 4.3 71.00 ± 1.62 53.63 ± 1.23 2.3 132.54 ± 1.77 103.17 ± 3.32

6.80 ± 0.53 2.97 ± 0.06 7.8 8.50 ± 0.53 3.72 ± 0.15 6.2 48.50 ± 2.76 21.20 ± 1.24 5.7 8.02 ± 0.14 3.51 ± 0.06 1.8 156.95 ± 3.86 68.61 ± 1.74 2.5 228.77 ± 6.91 99.11 ± 4.76

6.65 ± 0.39 1.76 ± 0.10 5.9 10.61 ± 0.55 2.81 ± 0.15 5.2 95.56 ± 4.12 25.3 ± 1.09 4.3 28.98 ± 0.72 7.67 ± 0.19 2.5 235.86 ± 2.04 62.45 ± 0.54 0.9 377.66 ± 4.64 94.71 ± 2.81

41.99 ± 2.37 3.51 ± 0.20 5.6 88.60 ± 2.92 7.4 ± 0.24 3.3 259.46 ± 7.96 21.67 ± 0.67 3.1 218.01 ± 9.29 18.21 ± 0.78 4.3 589.34 ± 35.69 49.22 ± 2.98 6.1 1197.40 ± 28.70 83.3 ± 2.47

30.13 ± 1.87 2.18 ± 0.14 6.2 47.33 ± 4.37 3.43 ± 0.32 9.2 774.78 ± 32.42 56.12 ± 2.35 4.2 136.88 ± 2.41 9.92 ± 0.17 1.8 391.40 ± 11.27 28.35 ± 0.82 2.9 1380.52 ± 31.34 91.46 ± 4.74

124.95 ± 3.00 4.48 ± 0.11 2.4 219.45 ± 1.33 7.88 ± 0.05 0.6 1226.00 ± 24.97 44.0 ± 0.89 2.0 194.75 ± 7.50 6.99 ± 0.27 3.9 1021.25 ± 30.84 36.65 ± 1.11 3.0 2786.40 ± 27.05 113.14 ± 5.35

0.89 ± 0.11 2.06 ± 0.25 12.4 0.74 ± 0.10 1.71 ± 0.22 13.5 0.22 ± 0.04 0.52 ± 0.08 18.2 41.38 ± 2.29 97.53 ± 5.39 5.5 43.23 ± 2.21 94.99 ± 4.37

1.27 ± 0.15 1.88 ± 0.04 11.8 2.35 ± 0.18 3.48 ± 0.27 7.7 1.27 ± 0.09 1.88 ± 0.14 7.2 62.58 ± 4.03 92.75 ± 6.06 6.4 66.47 ± 4.33 96.43 ± 3.08

1.93 ± 0.04 2.26 ± 0.05 2.1 8.47 ± 0.43 9.90 ± 0.51 5.1 7.63 ± 0.39 8.92 ± 0.46 5.1 67.52 ± 2.56 78.92 ± 2.99 3.8 85.55 ± 3.39 86.69 ± 3.52

1.39 ± 0.32 1.34 ± 0.41 23.0 10.47 ± 0.90 10.09 ± 0.86 8.6 3.52 ± 0.13 3.39 ± 0.13 3.8 88.34 ± 2.99 85.17 ± 2.88 3.4 103.72 ± 2.7 80.73 ± 2.62

6.79 ± 0.5 2.86 ± 0.02 7.4 21.68 ± 1.52 9.14 ± 0.66 7.0 7.47 ± 0.27 3.15 ± 0.12 3.6 201.30 ± 8.02 84.85 ± 3.47 4.0 237.24 ± 6.34 102.78 ± 4.97

6.23 ± 0.39 1.80 ± 0.11 6.3 36.90 ± 1.07 10.69 ± 0.31 2.9 5.73 ± 0.28 1.66 ± 0.08 4.8 296.45 ± 11.96 85.85 ± 3.46 4.0 345.31 ± 13.1 86.59 ± 2.57

82.54 ± 3.92 7.43 ± 0.41 4.7 96.35 ± 4.61 8.68 ± 0.49 4.8 108.36 ± 4.81 9.76 ± 0.59 4.4 823.25 ± 13.05 74.13 ± 2.59 1.6 1110.50 ± 10.73 77.25 ± 1.88

53.20 ± 2.57 4.71 ± 0.28 4.8 226.30 ± 8.62 20.02 ± 0.95 3.8 57.44 ± 4.58 5.08 ± 0.27 7.8 793.70 ± 15.53 70.20 ± 1.91 2.0 1130.64 ± 30.83 74.91 ± 3.20

159.60 ± 3.40 8.83 ± 0.19 2.1 236.90 ± 9.25 13.11 ± 0.51 3.9 73.68 ± 3.15 4.08 ± 0.17 4.3 1336.89 ± 61.03 73.98 ± 3.38 4.6 1807.07 ± 68.57 73.38 ± 3.47 407

Arsenic concentration data [mg kg1] represent the mean ± SD for 3 replicates. In addition is the arsenic concentration in the different fractions expressed as a percentage of the total arsenic concentration, calculated as the sum of all the fractions. As indication for the reproducibility of the different fractions, the coefficient of variation (CV) is calculated based on the absolute arsenic concentration values.

X. Wan et al. / Chemosphere 178 (2017) 402e410

As [mg As [%] CV [%] As [mg As [%] CV [%] As [mg As [%] CV [%] As [mg As [%] CV [%] As [mg As [%] CV [%] As [mg [%]

1

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X. Wan et al. / Chemosphere 178 (2017) 402e410

Fig. 3. The transfer factors (TF) for arsenic from soil to Amaranthus.

Table 3 Linear regression equation and Pearson correlation coefficient of R2 between different arsenic fractions in soil and the arsenic concentrations in plant (n ¼ 6). SEP Shiowatana

Rauret Tessier

Fractions FS1 FS2 FS1þFS2 FR1 FT1 FT2 FT1þFT2

Linear regression equation a

PT PT PT PT PT PT PT

¼ 1.36 FS1þ10.95 ¼ 6.68 FS2þ10.40 ¼ 4.25 (FS1þFS2)þ10.77 ¼ 11.24 FR1þ7.06 ¼ 17.45 FT1-0.06 ¼ 11.07 FT2þ28.79 ¼ 9.14 (FT1þFT2)þ9.15

R2 (Pearson)

P

0.97 0.88 0.93 0.94 0.98 0.51 0.85

0.001 0.021 0.008 0.006 0.001 0.300 0.030

a Total arsenic concentration uptake by Amaranthus, expressed by the mean for 3 replicates.

3.4. Comparison of the three SEPs for equivalent fractions For comparison among the three SEPs, based on the fractions defined by each SEP and the bioavailability evaluation, all the arsenic extracted by the SEPs were classified into four categories as exchangeable fraction (i.e. FR1, FT1þFT2 and FS1þFS2), reducible fraction (i.e. FR2, FT3 and FS3), oxidizable fraction (i.e. FR3, FT4 and FS4) and residual fraction (i.e. FR4, FT5 and FS5), with the arsenic fraction within each category being equivalent to each other. The absolute arsenic concentration extracted by different fractions of each SEP can be found in Table 2. The three SEPs were compared for equivalent fractions across all soil samples using the Wilcoxon test for paired samples. The arsenic concentration associated with the exchangeable fractions extracted by Tessier SEP (FT1þFT2) for most samples was much higher than that extracted by the corresponding fraction of Rauret SEP (FR1). However, both were significantly lower than that extracted by the corresponding fractions of Shiowatana SEP (FS1þFS2). The acetic acid/sodium acetate buffer used by Tessier SEP and the acetic acid used by Rauret SEP can promote the dissolution of clays and most carbonate materials in soil for metal release, while the acetate can prevent re-adsorption or precipitation of the released metal ions. However, as many researchers have proved, this is mainly suitable for cations, rather than anions (Tessier et al., 1979; Pickering, 1986; Filgueiras et al., 2002; Gleyzes et al., 2002). However, for arsenic present in the soil/sediment environment in the form of anions, the solubilization of these anionic species is highly favored in alkaline conditions, as their adsorption on to most mineral surfaces is minimal at alkaline pH values (Dzombak and Morel, 1989; Gleyzes et al., 2002). This could be the main reason for the significant increase in the concentration of arsenic extracted by FS1þFS2, in which 0.5 M sodium bicarbonate solution was used

for the extraction of the surface-adsorbed fraction at pH ¼ 9.0. The bioavailability evaluation has demonstrated that the individual fractions being compared in this category are potentially the most bioavailable species that are of great significance for risk assessment. Thus, in terms of the extraction efficiency of bioavailable species, Shiowatana SEP would be better than the other two, becoming an essential tool for the risk assessment of arsenic polluted sites. Similarly, the arsenic concentration associated with the reducible fraction extracted by Rauret SEP (FR2) for most samples is slightly higher than that extracted by the corresponding fraction of Tessier SEP (FT3), with both being significantly (about 3e7 times) lower than that extracted by the corresponding fraction of Shiowatana SEP (FS3). Several studies have established that oxides/hydroxides of Al, Mn and Fe, in particular, are the main arsenic scavengers in environmental solid samples (Dzombak and Morel, 1989; Sullivan and Aller, 1996). Due to the relatively high concentration of Fe in the selected soil samples, high percentages of arsenic can be expected in the reducible fraction. The relatively low percentages of reducible arsenic extracted by Tessier SEP (FT3) and Rauret SEP (FR2) can be attributed to the insufficient dissolution of the aforesaid oxides/hydroxides (amorphous Fe and Mn oxides/ hydroxides) by hydroxylamine solution under the conditions used. It is known that the dissolution of these hydroxides and oxides increases with increasing reagent concentration and reaction time, or reduced pH (Gleyzes et al., 2002). In addition, iron extraction can be insufficient with this reagent for materials with a high Fe content (Coetzee, 1993; Gleyzes et al., 2001) or containing the mixture of amorphous and crystalline iron oxides (Gruebel et al., 1988; Gleyzes et al., 2001; Lock et al., 2016). Although this reagent has been included in some arsenic specific sequential procedures (Kim et al., 2003; Matera et al., 2003), harsher conditions were used in such cases, with the extraction of the reducible fraction involving two consecutive steps of increasing strength (Hall et al., 1996; Kim et al., 2003). Hence, the association of arsenic with hydroxides and oxides may be underestimated in the SEPs of Tessier and Rauret due to the inefficiency of hydroxylamine under the conditions used. NaOH has been included in many arsenic specific SEPs to dissolve Fe/Al oxides/hydroxides for arsenic release, which has proven to be an efficient reagent by many studies (Onken and Adriano, 1997; Mclaren et al., 1998; Gleyzes et al., 2002; Beesley et al., 2010). However, it can also partially dissolve organic matter to release the arsenic bound to organic matter under certain conditions (Herreweghe et al., 2003). Therefore, Shiowatana SEP is likely to overestimate the arsenic concentration associated with Fe/Al oxides/hydroxides due to the partial extraction of the oxidizable fraction. Both, Tessier and Rauret SEPs, use H2O2 to extract the oxidizable arsenic fraction. Some studies (Tessier et al., 1979; Filgueiras et al., 2002; Mihaljevic et al., 2003) have corroborated that not only can this reagent destroy organic matter, but also partially dissolve sulfides at the same time. Thus, the oxidizable fractions extracted by both SEPs will most likely be a combination of arsenic fractions bound to organic matter and sulfides. Due to the longer reaction time (8 h vs. 3 h) and higher temperature at the first step (85  C vs. 25  C), the oxidizable arsenic fraction extracted by Tessier SEP (FT4) is slightly higher than that extracted by Rauret SEP (FR3). Indeed, these samples contain substantial amounts of sulfides instead of organic matter, which means that the oxidizable fraction in the soil is mainly the arsenic bound to sulfides rather than to organic matter. This is why the oxidizable fraction extracted by Shiowatana SEP is significantly higher than that extracted by the other two SEPs, especially for samples with extremely high arsenic content, containing large amounts of sulfur, because 1 M HCl solution is used by the Shiowatana SEP to extract the corresponding fraction

X. Wan et al. / Chemosphere 178 (2017) 402e410

(FS4). Although the HCl solution cannot destroy organic matter, it is much more efficient than H2O2 in dissolving sulfides completely. The residual fraction extracted by Tessier SEP (FT5) is comparable to that extracted by Rauret SEP, with both being significantly higher than that extracted by Shiowatana SEP (FS5), especially for samples with extremely high arsenic. This is mainly because a significant part of the arsenic bound to sulfides remains in the residual fraction, given the inability of H2O2 to completely leach the arsenic associated with these sulfides (Filgueiras et al., 2002). 3.5. Fractionation of arsenic in the soil in the case area by Shiowatana SEP and its implications to risk assessment As shown above, due to its relatively high arsenic recovery and reproducibility, as well as its efficient extraction of bioavailable arsenic, Shiowatana SEP was selected for the fractionation of arsenic in the study area to provide more information for risk assessment. For all 34 topsoil samples, the relative concentration of arsenic in various fractions is shown in Fig. 4. Residual arsenic presents predominantly in most soil samples, followed by the Fe/Al associated arsenic. Exceptions are found for a few heavily polluted samples (i.e. soil sample S6, S12, S22 and S26), in which the Fe/Al associated fraction takes priority instead of the residual fraction. The reason could be that these samples contain a high iron content, leading to a relatively high concentration of arsenic fraction associated with iron oxides/hydroxides. The relative concentration of these two fractions in most soil samples exceeds 80.0%, with the highest percentage being 96.6%. The relative concentration of HCl extractable fraction in all soil samples takes the second position, ranging from 1.88% to 19.7%, with most being below 10.0%. The higher relative concentration of this fraction is often found to be associated with high sulfur and iron content in the samples. This may be ascribed to the soil samples being collected from the Realgar mine. The surface adsorbed and the water-soluble fractions account for a very low portion of the total arsenic in all soil samples. The relative concentration of these two fractions ranges between 0.730% and 12.4%, with the highest concentration of arsenic extracted by these two steps reaching up to 130 mg kg1. Given the reaction conditions for Shiowatana SEP and the bioavailability evaluation on different fractions, both, residual and Fe/Al associated arsenic under reducing conditions amorphous Fe (oxyhydr)oxides are dissolved releasing As associated with them will be stable enough in the natural environment, without causing any harm to human health and environmental safety. The surfaceadsorbed and water-soluble arsenic, in particular, are the most labile fractions, proven to be easily transferred into bioavailable

409

species. However, as the HCl extractable arsenic is not stable enough, it can transfer into bioavailable species under extreme adverse environmental conditions, such as the impact of strong acidic wastewater and precipitation. Hence, although the arsenic in the soil of this area presents predominantly in stable fractions (ranging from 89.1% to 97.1%), with a small amount of bioavailable fractions (11.0% and below), the highest absolute concentration of bioavailable arsenic is up to 130 mg kg1, which is 13 times higher than the recommended lowest-observed-effects concentration (10 mg kg1) for arsenic by EPA (Us Epa, 1996). Meanwhile, with the HClextractable fraction, about 10.0% of total arsenic in the soil is likely to be transferred into bioavailable species, increasing the absolute concentration of bioavailable species. This implies that the arsenic accumulated in soil has become a major source of concern in this area, with a high potential risk to environment and human health. 4. Conclusion Tessier, Rauret and Shiowatana SEPs were compared for arsenic fractionation in soil samples highly polluted with arsenic, from Realgar mine area. As other studies have indicated, both Tessier and Rauret SEPs, which are commonly used for cation metals, are not as appropriate for arsenic fractionation as the arsenic-specific Shiowatana SEP, because the NH2OH,HCl and H2O2 used in these two SEPs are unable to completely leach the target arsenic fractions, such as Fe/Al associated arsenic and the arsenic bound to sulfides in soil, especially in soils containing high iron and sulfide content. The bioavailability evaluation on different fractions indicates that all three SEPs can provide an estimation of potentially the most bioavailable arsenic fraction. Linear regression and correlation analyses have shown that the arsenic concentration in amaranth is significantly correlated to the arsenic concentration extracted by bioavailable fractions (FT1þFT2, FS1þFS2 and FR1) of each SEP, with the concentration of FS1þFS2 apparently being higher than FT1þFT2 or FR1. Overall, the application of Shiowatana SEP is preferred and more suitable for assessing the bioavailability of arsenic in soil, because it can characterize the partitioning of the most labile arsenic fractions in soil better and extract them more effectively than the other two SEPs. Acknowledgement The authors gratefully acknowledge the financial support for this work by the Ministry of Land and Resources of P. R. China (Grant No. 201411089). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2017.03.078. References

Fig. 4. The distribution of arsenic fractions by shiowatana SEP.

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