Available online at www.sciencedirect.

com

Chemosphere 70 (2008) 14591467 www.elsevier.com/locate/chemosphere

Evaluation of various chemical extraction methods to estimate plant-available arsenic in mine soils
H.M. Anawar *, A. Garcia-Sanchez, I. Santa Regina
Department of Environmental Geochemistry, IRNASA-CSIC, Cordel de Merinas 40-52, Aptdo 257, Salamanca 37008, Spain Received 29 September 2006; received in revised form 24 August 2007; accepted 26 August 2007 Available online 22 October 2007

Abstract Elevated levels of bioavailable As in mining soils, agricultural areas and human habitats may cause potential toxicity to human health, plants and microbe. Therefore, it is essential to determine proper soil chemical extraction method in order to estimate plant-available As in mining soils and protect agricultural and environmental ecosystems by evaluation of environmental risk and implementation of remediation measures. In this study, six single soil chemical extraction processes and four-step sequential chemical extraction protocol were used to determine the relative distribution of As in dierent chemical forms of soils and their correlations with total As in plants grown in mining areas and greenhouse experiments. The strongest relationship between As determined by single soil chemical extraction and As in plant biomass was found for sodium acetate and mixed acid extractant. The mean percent of total As extracted was: ammonium oxalate (41%) > hydroxylamine hydrochloride (32%) > mixed acid (16%) > phosphate (6%) > sodium acetate (1.2%) > water (0.13%). This trend suggests that most of the As in these soils is inside the soil mineral matrix and can only be released when iron oxides and other minerals are dissolved by the stronger chemical extractant. Single soil chemical extraction methods using sodium acetate and mixed acids, that extract As fractions complexed to soil particles or on the surface of mineral matrix of hydrous oxides of Fe, Mn and Al (exchangeable + sorbed forms) can be employed to estimate and predict the bioavailable As fraction for plant uptake in mining aected soils. In sequential chemical extraction methods, ammonium nitrate and hydroxylamine hydrochloride may be used to provide closer estimates of plant-available As in mining soils. 2007 Elsevier Ltd. All rights reserved.
Keywords: Plant-available As; Chemical extractants; Sodium acetate; Mixed acid

1. Introduction Although the background concentrations of As typically range from 0.1 to 40 mg kg1 in soil (Bowen, 1979), some anthropogenic activities including mining and smelting of metal ores, coal y ash and use of agricultural fertilizers and pesticides (Adriano, 2001) led to assimilation of high As levels (10 00020 000 mg kg1) in some agricultural areas and human habitats causing high levels of risk to human health, plants, and microorganisms. The weathering of pyrite and sphalerite minerals in the mine tailings, spoils

Corresponding author. Tel.: +34 923 219 606; fax: +34 923 219 609. E-mail address: anawar4@hotmail.com (H.M. Anawar).

and sludge produces poorly crystalline Fe oxides and this weathering process has strong impact on soil quality and phytotoxicity in soils (Hita and Torrent, 2005). Arsenic can play a very biologically complex role. It is not an essential element for many biological processes, but concentrations above a certain level have adverse eects, thereby converting it to a toxin (Emsley, 1991). Recently, it is demonstrated that the total concentration of As in the soils does not necessarily represent its biological availability or potential toxicity measures (Newman and Jagoe, 1994; Anawar et al., 2006), which are much more important for assessing possible environmental impacts. Most risk from As is associated with the forms of As that are biologically available for absorption, or bioavailable to plants. Bioavailability is a function of

0045-6535/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.08.058

1460

H.M. Anawar et al. / Chemosphere 70 (2008) 14591467

the abundance, chemical form (i.e., oxidation state), and the nature of its binding to soil grains. For example, the element may be part of a very stable mineral, such as quartz, and thus is not available for biological processes (Newman and Jagoe, 1994). It is essential to determine this form of As in soils for evaluating the potential health risk and proper waste disposal strategies, evaluation and management of environmental risk and safety. Chemical fractionation methods, based on sequential extraction procedures, have been used to determine the amount of contaminant in specic chemical pools (Pierzynski, 1998). Most chemical fractionation methods have been used to measure heavy metals in ion-exchangeable, surcially adsorbed, precipitated, organic chelated, and occluded chemical pools in baseline soils (Tessier et al., 1979); in sewage sludgeamended soils (Sposito et al., 1982); and in contaminated soils (Hickey and Kittrick, 1984; Gibson and Farmer, 1986; Ma and Rao, 1997). Phosphate solutions have been widely used as an extraction medium for soil As (Manning and Martens, 1997; Loeppert et al., 2002). Extraction of As with phosphate is through ligand exchange that involves the desorption of As by phosphate and provides a mild extraction medium. Dierent soil chemical methods, including single chemical extraction and sequential extraction methods are available to extract various pools of soil As (Kabata-Pendias, 2004), but their ability to quantify the amount of plant-available As from mining soil is still unknown. The direct relationships of dierent chemical forms of As in soils extracted by single chemical extraction with plant As are seldom reported to determine the best chemical extractant and form of As in soils that indicate the soil availability, solubility and bioavailability of As for plant uptake. Therefore, it is necessary to develop an analytical technique to evaluate the As species in the more soluble and mobilizable fractions that is more bioavailable. Phytotoxicity and bioavailability of As to plants in mining aected soils have been seldom investigated. When the As level in soils increases, the chances of a phytotoxic response by plant also increases. Therefore, a method for estimating the amount of bioavailable As present in the soil and correlating this value to total As in plant is desirable. Vandecaveye et al. (1936) found no correlation between phytotoxicity and the solubility of soil As in distilled water, 0.1 N NH4Ac, 0.1 N NH4NO3, 0.1 N KNO3, or hot concentrated HNO3. The ability of soil chemical extraction methods to measure As phytoavailability has been reviewed by Adriano (2001). To the best of our knowledge, very few studies are reported to determine the suitable chemical extracting method, which characterizes most precisely the bioavailability of As in mine soils for plant uptake by exhibiting correlations between dierent chemical forms of As in mining soils and total contents in plants collected at the same sampling locations. In this study, we determined the relative distribution of As in dierent chemical forms and their correlations with plant As content by using soil-plant relationships. There-

fore, the objectives of this study are to (1) determine the bioavailability of As in mining soils, (2) measure the phytoavailable fraction of As in mining soils in comparision with plant response to As or As accumulation in plant and develop a chemical extraction method that can be used to determine the phytoavailable As in mining soils. 2. Materials and methods 2.1. Soil and plant sampling, preparation and characteristics Mining soils and plants were collected from several tungsten, antimony, zinc and gold producing smeltering and mining aected sites from dierent provinces of Spain. Control soil and plants were collected from Mun ovela experimental station of Salamanca (IRNASA) where the soil As content is <15 mg kg1. Agricultural soil and plants were collected from Valladolid potato elds. Mine tailings were deposited between 20 and 50 yr ago. The mineral paragenesis mainly consisted of arsenopyrite, pyrite, scheelite, wolframite, and minor chalcopyrite (Anawar et al., 2006) and the mining wastes consisted of ne-grained ore minerals including arsenopyrite, and ore weathering products (Fe-oxyhydroxides and scorodite) containing high As contents that polluted the top soils of the surrounding environment. Specimens of the plant species mainly growing in the vicinity of the mining area were sampled with their corresponding soils. The sampling for both plants and soil was repeated several times at each sampling point. For plant samples, the aerial parts of the plant (stems, branches and leaves) were collected; and the roots were not included in the samples. Plant samples were cleaned with freshwater, rinsed with deionized water and air-dried at room temperature for several days. The air-dried plant samples were powdered homogenously and prepared for analysis. The soil samples were dried at 50 C, mixed and homogeneized and sieved through a 2-mm screen. The <2-mm fraction was used to determine the main soil properties: pH was determined potentiometrically in a soil paste saturated with water; organic matter was determined by dichromate oxidation using the Tiurin method (Jackson, 1960); cation exchange capacity (CEC) was determined according to the ammonium acetate method by extracting with a 1.0 M NH4OAc solution (pH 7.0) (Tan, 1996); and particle size distribution (sand, silt, and clay) was analysed by the pipette method (Gee and Bauder, 1986). 2.2. Arsenic analysis in soil and plant samples Arsenic in soil samples collected from mining soils and control area was determined by both Atomic Absorption Spectrophotometer (AAS) and X-ray uorescence spectroscopy method (when the XRF arsenic detection limit permits As analysis in soil samples). The detailed method of As measurement using AAS method is written in Anawar et al. (2006). Analytical accuracy was checked with

H.M. Anawar et al. / Chemosphere 70 (2008) 14591467

1461

BCR (Bureau Community References) material CRM-320 (river sediment) and US Geological Survey reference G-2 (granite). The precision of the method was assessed by performing the analysis 10 times for a single sample. The relative standard deviation (RSD) was between 5% and 10%. Plant samples were digested using a CEM (MDS-2000) microwave oven at pressure of 9 kPa (10 min) and at 12 kPa (15 min). Arsenic concentrations were measured in duplicate by using VARIAN spectra AA-220 and hydride generator VGA-76 AAS with a commercial stock standard (Panreac Quimica SA) and the calibration curve t was of R2 > 0.98 in all cases. 2.3. Chemical extraction methods

step, the suspensions were shaken, heated for a specic time. The suspensions were centrifuged, ltered, and acidied like above and As was measured using the AAS method. 2.4. Statistical methods Statistical analysis was performed using ANOVA. All statistical analyses were performed using program SPSS 10.1.0 for Windows. Data are presented as geometric mean. Linear regression was used to determine agreement between soil chemical extraction and plant accumulation of As. 3. Results and discussion

Arsenic concentrations in dierent fractionations of soils were extracted by six single chemical extraction methods. All samples were extracted in triplicate and included appropriate reagent blanks and spikes. One g of soil was placed into a 50-ml polycarbonate centrifuge tube and mixed with 2075 ml of extracting solutions. The six following fractions were extracted: water-soluble (1); amorphous FeMn oxides (2); weakly adsorbed to surface of soil particles and water-soluble (3); strongly (specically) adsorbed, water-soluble and weakly adsorbed (4); amorphous FeMn oxides, surcial adsorbed and some As in the mineral matrix (5); amorphous and crystalline iron oxides (6). According to Rodriguez et al. (2003), Price and Pichler (2005) and Norra et al. (2005), the above six fractions were respectively leached by deionized water (1); 0.05 N HCl and 0.025 N H2SO4 (2); sodium acetate 1.0 M in acetic acid (3); 0.1 M Na2HPO4 and 0.1 M NaH2PO4 (4); 0.04 M NH2OH and HCl in 25% acetic acid (5); acid-ammonium-oxalate 0.2 M (6). The tube was sealed using a neoprene stopper and placed in a horizontal position on a reciprocal laboratory shaker. The tubes were shaken for dierent time and then centrifuged at 5000 rpm for 10 min. The supernatant was collected followed by ltration using 0.45 m membrane lter, and the ltrate was acidied to pH < 2 with concentrated HCl for preservation before As analysis. Arsenic measurement was performed using the above AAS method. A four-step sequential chemical extraction scheme was carried out to determine As concentrations in surface bound-water soluble, exchangeable and easily soluble metallo-organic complexes (S1), amorphous FeMn oxides (S2), organic fraction (S3) and residual phase (S4). This sequential chemical extraction scheme is a combination and modications of Tessier et al. (1979), Price and Pichler (2005) and Norra et al. (2005). In this procedure, one g of soil was placed into a 50-ml polycarbonate centrifuge tube and an amount of each extracting reagent was added sequentially. The four fractions were respectively leached by 1.0 M NH4NO3 (S1), 0.04 M NH2OH/HCl in 25% acetic acid (S2), 0.02 M HNO3 in 30% H2O2 (5:2, v/v) and 3.2 M ammonium acetate in 20% HNO3 adjusted to pH 2.0 (S3) and aqua regia, HCl + HNO3 (3:1) (S4). In each

3.1. Soil properties and minerals in soils The pH, organic carbon and matter, nitrogen, phosphorus, CEC (3.934.6 meq 100 g1) and soil texture have been measured for a few selected mining soils collected from Terrubias, Barruecopardo, Aznalcollar, Cabaco, and La Parrilla mines and a few samples from control area of Mun ovela, and Valladolid (VA Portillo) agricultural soils that are presented in the Table 1. Mining soils collected from dierent mining aected areas were of acidic characteristics (pH = 2.916.60), had low to medium concentrations of organic carbon (0.035.68) and organic matter (0.069.80), high concentrations of ne sand (2.582%) and low contents of silt (5.334.7%) and clay (7.865.5%) (Table 1). Soils collected from control area of Mun ovela were slightly acidic (pH = 5.055.79), had low to medium concentrations of organic carbon (0.891.59) and organic matter (1.532.75), high concentrations of sand (49.752.8) and low contents of silt (4.28.2) and clay (19.334.9) (Table 1). Agricultural soils were of alkaline characteristics (Table 1). The constituent minerals in soil and spoil samples measured by the X-ray diractometry (XRD) study (a Geiger Flex Rad-X, Rigaku Denki KK operating at 35 kV and 20 mA with a Cu target) were illite, kaolinite, muscovite, plagioclase, quartz and scorodite. The XRD study of Terrubias-1 mine soil that had the highest content of As (26 500 mg kg1) clearly exhibited scorodite mineral, had the lowest pH value of 2.91, higher organic carbon and organic matter, high P and medium contents of CEC. The XRD study showed the presence of very weak peaks of goethite, ferrihydrite and jarosite that are the most common minerals resulting from the oxidation of pyrite in mine soils and waste dump samples. 3.2. Total arsenic in soils and water solubility Total As contents in mining polluted soils were high (Table 2) that varied from 130 to 26 500 mg kg1 in Terrubias mine soils, 58 to 1540 mg kg1 in Barruecopardo, Aznalcollar, Cabaco, and La Parrilla mining aected soils,

1462

H.M. Anawar et al. / Chemosphere 70 (2008) 14591467

Table 1 Physico-chemical characteristics of Mun ovela control area and mining soils Sample name Mun ovela-1 Mun ovela-2 Mun ovela-3 Mun ovela-4 Mun ovela-5 Terrubias-1 Terrubias-2 Terrubias-3 Terrubias-4 Terrubias-5 Barruecopardo Terrubias Aznalcollar Cabaco K72-2 Cabaco K72-5 Cabaco Calvnes Cabaco K5 LaParrilla LS-10b La Parrilla ES-2 VA Portillo-1 VA Portillo-2 Soil type Total As (mg kg1) 9.1 9.7 10.1 12.2 13.0 26 500 760 252 198 130 1327 1215 107 1540 1007 128 58 620 1280 42 45 pH 5.05 5.73 5.66 5.68 5.79 2.91 5.87 6.26 6.60 6.48 4.6 3.8 3.4 4.8 5.2 4.6 5.3 3.6 3.5 7.7 7.6 OC (%) 0.91 1.12 1.59 0.89 0.9 4.94 2.83 0.62 1.91 0.59 0.15 0.30 0.46 5.68 1.62 2.26 1.04 0.03 0.15 0.74 0.77 OM (%) 1.56 1.94 2.75 1.53 1.55 8.51 4.88 1.06 3.30 1.01 0.26 0.52 0.80 9.80 2.80 3.90 1.80 0.06 0.25 1.28 1.30 N (%) 0.07 0.09 0.11 0.08 0.08 0.38 0.23 0.07 0.22 0.07 P (mg kg1) 28.0 18.6 21.4 14.5 13.2 28.5 7.3 12.0 4.7 19.5 CEC meq100 g1 5.6 8.1 9.6 14.5 12.0 15.3 28.5 11.1 34.6 3.9 5.2 8.1 8.1 21.5 13.2 6.2 4.3 4.7 8.6 10.8 12.0 Fine sand (%) 49.7 52.8 52.8 50.6 52.8 47.7 17.1 45.2 2.5 81.5 79.0 82.0 76.5 58.0 52.0 47.6 69.9 71.0 47.4 Coarse sand (%) 24.2 20.4 17.2 11.0 16.7 2.5 5.7 20.8 0.7 4.2 Silt (%) 8.2 6.1 5.3 5.5 4.2 21.0 26.5 14.3 30.7 5.3 12.9 7.4 12.7 12.7 14.8 22.8 17.1 20.7 34.7 Clay (%) 19.3 22.6 25.4 34.9 26.5 18.9 47.5 18.2 65.5 8.3 7.8 9.8 8.8 17.5 28.2 17.0 12.3 8.2 18.1

Control soil

Mining soils

Agr. soils

Note: OC Organic carbon; OM Organic matter; CEC Cation exchange capacity; Agr. Agricultural.

Table 2 Leachable As concentrations of soils by single chemical extraction process (mg kg1) Sample name Mun ovela-1 Mun ovela-2 Mun ovela-3 Mun ovela-4 Mun ovela-5 Terrubias-1 Terrubias-2 Terrubias-3 Terrubias-4 Terrubias-5 Barruecopardo Terrubias Aznalcollar Cabaco K72-2 Cabaco K72-5 Cabaco Calvnes Cabaco K5 LaParrilla LS-10b La Parrilla ES-2 VA Portillo-1 VA Portillo-2 Soil type Total As 9.1 9.7 10.1 12.2 13.0 26 500 760 252 198 130 1327 1215 107 1540 1007 128 58 620 1280 42 45 Water 0.17 0.06 0.04 0.02 0.03 3.35 0.58 0.21 0.39 0.92 0.44 0.27 0.10 0.38 0.35 0.17 0.16 0.27 0.46 0.53 0.55 Mixed acid 1.1 0.8 0.9 0.7 0.8 974 57 44.3 8.9 22.3 452 222 35 152 82.8 3.0 2.8 292 280 1.9 2.3 CHCOONa 0.33 0.29 0.34 0.27 0.30 154 7.29 3.85 1.15 2.93 21.38 17.36 1.52 8.96 1.48 0.20 0.17 21.22 16.98 0.45 0.47 Phosphate 0.26 0.26 0.27 0.34 0.32 109 286 14.8 12.64 1.36 30.92 26.76 8.80 31.64 31.7 1.15 0.42 18.3 50.18 0.31 0.36 NH2OH HCl 0.05 0.02 0.01 0.03 0.06 194 172 96.58 15.84 29.05 875 523 82.69 276 183 15.4 37.9 247 275 2.30 2.46 Oxalate 4.5 4.8 5.0 6.0 6.4 13 370 422 117 88.3 25.8 515 915 54.2 440 199 19.5 11.0 438 466 2.5 2.9

Control soil

Mining soil

Agricultural soil

Note: Mixed acid = 0.05 N HCl and 0.025 N H2SO4; CHCOONa = 1.0 M sodium acetate in acetic acid Phosphate = 0.1 M Na2HPO4 and 0.1 M NaH2PO4; NH2OH.HCl = hydroxylamine hydrochloride; Oxalate = 0.2 M acid-ammonium-oxalate.

9.113.0 mg kg1 in the control area and 4245 mg kg1 in Valladolid agricultural soils (VA Portillo) (Table 2). These concentrations in mining aected soils substantially exceeded the guideline limit of 50 mg kg1 for agricultural soil (MAFF, 1993), the world-wide As levels in soils (0.1 55 mg kg1, Boyle and Jonasson, 1973; 0.140 mg kg1,

Bowen, 1979) and the background As concentration in control area of this study. Compared to the total As contents, relatively lower water solubility of As in the mining soils (0.010.7% of the total As) than in the control area and agricultural soils (use of As-based pesticides, herbicides and fertilizers)

H.M. Anawar et al. / Chemosphere 70 (2008) 14591467

1463

(1.261.87% of the total As) (Table 2) is presumed due to the specic soil characteristics, chemistry and mineral composition of the soil and low weathering of ore minerals in the deposition of mine spoils (Anawar et al., 2006). Phytotoxicity of As depends on the relative distribution of dierent chemical forms, solubility and bioavailability of As, which is a function of soil properties, such as pH, CEC, texture (clay mineralogy), amorphous FeAl oxides, organic matter, sulfur content, phosphorus concentration, and soil redox conditions (Alva et al., 2000; Adriano, 2001). The transfer of As within the soilplant chain is a part of the biogeochemical cycling. 3.3. Relationship between soil As extracted by single chemical extractions and plant As The geometric mean percent of As extracted from mining soil by soil chemical extractants, in parentheses, followed the trend: ammonium oxalate (41%) > hydroxylamine hydrochloride (32%) > mixed acid (16%) > phosphate (6%) > sodium acetate (1.2%) > water (0.13%) (Table 2). These results suggest that most of the As in these mining soils is inside the soil mineral matrix and can only be released when iron oxides and other minerals containing As are dissolved by the chemical extractant (i.e., ammonium oxalate and hydroxylamine hydrochloride). Some extractants approach 78% of total As, such as the hydroxylamine hydrochloride or ammonium oxalate extractant for some of the study samples. Much lesser amounts of surcially adsorbed As were extracted by phosphate and acetate than reagents that dissolved mineral forms occluding As (ammonium oxalate and hydroxylamine hydrochloride) (Table 2). Chemical extraction results presented here demonstrate that the amount of As in chemical pools was: inside mineral matrix > surcially adsorbed > watersoluble. With the increase of total As concentrations in mine soils and control area, the concentration of the readily soluble As forms (exchangeable + sorbed forms) extracted by water and sodium acetate single extractants increased in the low pH soils, that is, from 0.02 to 3.35 mg kg1 (0.011.87% of total As) for water-soluble As and from 0.17 to 154.40 mg kg1 (0.13.6% of total As) for sodium acetate soluble As (Table 2). For various samples (all the mining and control samples) the phosphate solution extracted a greater or less portion of As than the sodium acetate solution; results ranged between 0.42 and 286 mg kg1 of As for mine soils and between 0.26 and 0.34 mg kg1 of As for Mun ovela control area. The phosphate-extracted fraction represents some of the strongly (specically) adsorbed fraction of As as well as the watersoluble and weakly adsorbed fractions (non-adsorbed fraction). All of three solutions (water, sodium acetate, and phosphate) extracted adsorbed As to the surface of soil particles. Mixed acids, which is stronger extractant and extracted more As than water, sodium acetate and phosphate, extract

surcially adsorbed As and some of the As adsorbed to the surface of Fe, Mn and Al oxides. Hydroxylamine hydrochloride, a much more aggressive extractant that dissolves amorphous Fe and Mn oxide (Chao and Zhou, 1983) and extracted more As than water, sodium acetate, phosphate and mixed acids, extracts surcially adsorbed As and some of the As in the mineral matrix; results ranged between 15.4 and 875 mg kg1 (0.7077.3%) for mine soils and 0.010.06 mg kg1 for Mun ovela control area (Table 2). Ammonium oxalate, which is more aggressive than hydroxylamine hydrochloride (Shuman, 1982), extracts As associated with both the amorphous and crystalline iron oxides. Ammonium oxalate extracted between 11 and 13 370 mg kg1 (15.275.3%) of As (Table 2) in mine soils. Single chemical extraction studies, XRD, dierential thermal analysis and IR analysis have shown that in mine soils, the major portion of the total As was absorbed on the surface of hydrous oxides of Fe (AsFeO or Fe oxide), lepidocrocite (c-FeOOH), Mn and Al bound extracted by hydroxylamine hydrochloride and residual forms (Hickey and Kittrick, 1984; Roussel et al., 2000; Basta et al., 2001) or as acid-soluble forms extracted by mixed acid and ammonium oxalate. Drexler (2000) reported that As associated with mineralogical forms categorized as Fe oxide forms have low (120% RBA-relative bioavailability) bioavailability. This nding is consistent with the low As bioavailability of the mining aected soils of <20% RBA in this study (Table 2). A homogenate of four soil samples from each location was used to determine dierent forms of As by single chemical extractions. The mean value of As content in plants for each sampling point was used to represent co-relationship with dierent forms of As in soils by linear regression. 3.3.1. Mun ovela control area From ve sampling points of Mun ovela control area, twenty plant samples (four from each location) and soil samples were collected. The collected plant species were Agrostis castellana, Daucus carota, Helichrysum stoechas, Eryngium campestre, Dactylis glomerata, Andryala ragusina, Holcus lanatus and Carlina corymbosa. The water, sodium acetate, mixed acid, phosphate, hydroxylamine hydrochloride and ammonium oxalate extractable As exhibited no relationship with As in plant biomass. 3.3.2. Five dierent mining area A lot of plant samples and soil samples were collected from nine sampling points of Terrubias, Barruecopardo, Aznalcollar, Cabaco, and La Parrilla mining aected areas. The plant species were A. castellana, H. stoechas, Rubus ulmifolius, E. campestre, Scolymus hyspanicus, Senecio jacobaea, D. carota, D. glomerata, Centaurea paniculata, Daphne gnidium, Mentha pulegium, H. lanatus, Juncus, Cirsium arvense, Mentha rotundifolia, A. ragusina and C. corymbosa. For three chemical extracting methods that showed signicant linear regression coecients (Fig. 1), R2 values were 0.54 for mixed acid (signicant at the 0.05

1464
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 60
R2 = 0.54

H.M. Anawar et al. / Chemosphere 70 (2008) 14591467

200

As ext. by phosphate (% )

As ext. by water (%)

150 100 50 0
0 10

y = -0.0053x + 0.2333 R2 = 0.04

y = 0.3243x + 14.873 R2 = 0.0097

10

20

30

40

50

20

30

40

50

As in plants (mg/kg)

As ext. by mixed acid (%)

40 30 20 10 0 0 10 20 30 40 50

As ext. by oxalate (%)

50

y = 0.776x + 12.849

80 70 60 50 40 30 20 10 0
0

y = 0.9061x + 29.569 R2 = 0.32

10

20

30

40

50

As in plants (mg/kg) As by hydroxylamine(%)

100 80 60 40 20 0
0 10 20 30 40 50 y = 0.2019x + 35.215 R2 = 0.013

Sodium acetate (%)

4 3 2 1 0 0

y = 0.0563x + 0.7444 R2 = 0.69

10

20

30

40

50

As in plants (mg/kg)

As in plants (mg/kg)

Fig. 1. Relationship between dierent chemical phases of As in soils and As in plant biomass (mean values of many plants at each point).

probability level), 0.69 for sodium acetate (signicant at the 0.05 probability level), and 0.32 for ammonium oxalate (signicant at the 0.01 probability level). The water, phosphate, and hydroxylamine hydrochloride extractable As exhibited no relationship with As in plant biomass. The best correlationship was found between sodium acetate extractable As in soils and plant As followed by better relationship found between mixed acid soluble As and plant As. Single chemical extraction studies have shown that sodium acetate, which measures the weakly adsorbed pool of As as well as the water-soluble pool, and extracted on average 1.178.6 times more As than water (Table 2) from mine soils, presented the best co-relationship with As in plant biomass (Fig. 1). The mixed acid extractable As in soils exhibited also better relationship with As in plant biomass. The As content in plant biomass was positively correlated with the readily soluble form of As, thus suggesting that the phytotoxicity or bioavailability of As was most sensitive to an increase in the readily soluble form of As in the soil (exchangeable and sorbed). The critical concentration of readily soluble As varied from 1.7 to 2.5 mg kg1 in soils with pH value of 5.76.5. Plant roots excreted

organic acids increase the dissolved organic carbon concentrations and control the rhizosphere soil pH enhancing total Fe and As solubility due to complexation reactions (Fitz et al., 2003). 3.3.3. Terrubias mining area Twenty-ve plant samples and soil samples were collected from ve sampling points of Terrubias mining aected area. The plant species were A. castellana, R. ulmifolius, S. hyspanicus, Erynginum campestre, S. jacobaea, D. carota, C. paniculata, D. gnidium, M. pulegium, H. lanatus, Juncus, C. arvense, M. rotundifolia. The correlation coecients between dierent fractions of soil As determined by single extraction vs. As in plant biomass were R2 = 0.74 for water, R2 = 0.69 for mixed acid, R2 = 0.68 for sodium acetate, R2 = 0.022 for phosphate, R2 = 0.21 for hydroxylamine hydrochloride and R2 = 0.67 for ammonium oxalate, respectively. The linear regression coecients, R2 values for relationship between dierent fractions of soil As and As in plant roots were 0.78 for water (signicant at the 0.05 probability level), 0.83 for mixed acid, 0.83 for sodium acetate, 0.0002 for phosphate, 0.82 for ammonium oxalate and 0.40 for hydroxylamine hydrochloride extract-

H.M. Anawar et al. / Chemosphere 70 (2008) 14591467

1465

able As. Out of six chemical extractants, water, sodium acetate, mixed acid and ammonium oxalate extractable As exhibited stronger co-relationship with plant As contents. 3.4. Relationship between soil As extracted by sequential extraction and plant As With the increase of total As concentrations in mining soils, the concentration of the ammonium nitrate extractable readily soluble As forms (exchangeable + sorbed forms) increased in all of the mining soils. In the greenhouse experiment, various chemical phases of As in the rhizosphere mining soils extracted by sequential process were correlated with total As contents in alfalfa (Medicago sativa L.) plant biomass. The linear regression coecients, R2 values were 0.89 for ammonium nitrate (signicant at the 0.05 probability level), 0.013 for hydroxylamine hydrochloride, and 0.087 for organic phase of As (Fig. 2). The correlation coecients between dierent fractions of soil As determined by sequential extraction vs. As in plant root biomass of alfalfa were 0.76, 0.0045 and 0.028 for ammonium nitrate, hydroxylamine hydrochloride extractable and organic phase of As, respectively in the greenhouse
As by NH 4NO3 (mg kg-1)
5

mining soils (Fig. 3). The ammonium nitrate extractable As in soils, which measures the weakly adsorbed pool of As as well as the water-soluble pool, exhibited strong relationship with As in alfalfa plants grown in the mining soils of greenhouse experiments. The correlation coecients between dierent fractions of soil As determined by sequential extraction vs. As in plant biomass were 0.71, 0.69 and 0.71 for ammonium nitrate, hydroxylamine hydrochloride extractable and organic phase of As, respectively in Terrubias mining aected soils. Kabata-Pendias (2004) reported that there is a still ongoing debate for the choice of the best chemical soil extractant that correlates the metal levels in plants to those of soils. Ernst and Nelissen (in press) reported that obviously none of the soil extracts delivers a good correlation to all metallic elements in plants (Soon and Abboud, 1993). Siebe (1995) and Qian et al. (1996) measured Cd, Pb, Cu, Zn, Ni and Co in aqua regia, 1 M NH4NO3 and water extractable fractions of soil samples and correlated with total contents in winter wheat (Triticum aestivum L.) and alfalfa (M. sativa L.) grown in the eld and a greenhouse study. The results showed that plant uptake of Cd and Pb correlated best with aqua regia and 0.1 M HCl gave

As by NH 4NO3 (mg kg )

4 3 2 1 0 0 10 20 30 40 50

-1

5 4 3 2 1 0 0 60 50 40 30 20 10 0 0 100 100 200 300 400 500

y = 0.0701x + 1.1845 R = 0.89

2

y = 0.0051x + 2.0288 R = 0.76

2

60

As by HA (mg kg )

50 40 30 20 10 0 0 10 20 30 40 50 y = -0,1212x + 39,109 R = 0,0132

2

-1 As by HA (mg kg )

-1

y = 0.0055x + 34.689 R = 0.005

200 300 400 500
2

As in organic phase (mg kg )

-1

14 12 10 8 6 4 2 0 0 10 20 30
-1

As in organic phase (mg kg-1)

y = -0.074x + 10.869 R = 0.087 40 50

2

14 12 10 8 6 4 2 0 0 100

y = -0.0033x + 9.5523 R = 0.028

200 300 400 500
2

As in plant (mg kg )
Fig. 2. Relationship between As in soils leached by chemical extractants and As in plant biomass (mean values of many plants at each point). HA-hydroxylamine.

As in roots (mg kg-1)

Fig. 3. Relationship between As in soils leached by chemical extractants and As in plant roots (mean values of many plants at each point). HA-hydroxylamine.

1466

H.M. Anawar et al. / Chemosphere 70 (2008) 14591467 Alva, A.K., Huang, B., Paramasivam, S., 2000. Soil pH aects copper fractionation and phytotoxicity. Soil Sci. Soc. Am. J. 64, 955962. Anawar, H.M., Garcia-Sanchez, A., Murciego, A., Buyolo, T., 2006. Exposure and bioavailability of arsenic in contaminated soils from the La Parrilla mine, Spain. Environ. Geol. 50, 170179. Basta, N.T., Rodriguez, R.R., Casteel, S.W., 2001. Development of Chemical Methods to Assess Bioavailability of Arsenic in Contaminated Media. National Center for Environmental Research Quality Assurance Grant Final Report, USEPA Natl. Center for Environ. Res., Washington, DC. <http://cfpub.epa.gov/ncer_abstracts/index. cfm/fuseaction/display.abstractDetail/abstract/676/report/F> (veried 31 January 2003). Bowen, H.J.M., 1979. Environmental Chemistry of the Elements. Academic Press, New York. Boyle, R.W., Jonasson, I.R., 1973. The geochemistry of As and its use as an indicator element in geochemical prospecting. J. Geochem. Explor. 2, 251256. Chao, T.T., Zhou, L., 1983. Extraction techniques for selective dissolution of amorphous iron oxides from soils and sediments. Soil Sci. Soc. Am. J. 47, 225232. Drexler, J.W., 2000. Bioavailability/bioaccessability of metals. In: The ITRC Fall 2000 Conference on New Environ. Technol. and Market Opportunities, San Antonio, TX. 1620 October 2000, Interstate Technol. and Regulatory Council. http://www.itrcweb.org/user/bioofmetalsdrexlerslides.pdf (veried 31 January 2003). Emsley, J. (Ed.), 1991. The Elements. Oxford Chemistry Guides. Clarendon Press, Oxford, p. 250. Ernst, W.H.O., Nelissen, H.J.M., in press. Bleeding sap and leaves of silver birch (Betula pendula) as bioindicator of metal contaminated soils. Int. J. Environ. Pollut. 42. Fitz, W.J., Wenzel, W.W., Zhang, H., Nurmi, J., Stipek, K., Fischerova, Z., Schweiger, P., Kollensperger, G., Ma, L.Q., Stingeder, G., 2003. Rhizosphere characteristics of the arsenic hyperaccumulator Pteris vittata Land monitoring of phytoremoval eciency.. Environ. Sci. Technol. 37, 50085014. Gee, G.W., Bauder, J.W., 1986. Particle-size analysis. In: Clute (Ed.), Methods of Soil Analysis, . In: Soil Sci. Soc.. Amer., Inc. Publ., Madison, WI. Gibson, M.J., Farmer, J.G., 1986. Multi-step sequential chemical extraction of heavy metals from urban soils. Environ. Pollut. Ser. B 11, 117 135. Hickey, M.G., Kittrick, J.A., 1984. Chemical partitioning of cadmium, copper, nickel, and zinc in soils and sediments containing high levels of heavy metals. J. Environ. Qual. 13, 372376. Hita, R., Torrent, J., 2005. Weathering of pyrite and sphalerite in soils contaminated with pyritic sludge. Soil Sci. Soc. Am. J. 69, 13141319. Jackson, M.L., 1960. Soil Chemical Analysis. Prentice-Hall, Inc., Englewood Clis, NJ. Kabata-Pendias, A., 2004. Soil-plant transfer of trace elements An environmental issue. Geoderma 123, 143149. Loeppert, R.H., Jain, A., El-Haleem, M.A.A., Biswas, B.K., 2002. Quantity and speciation of arsenic in soils by chemical extraction. In: Cai, Y., Braids, O.C. (Eds.), Biogeochemistry of Environmentally Important Trace Elements. American Chemical Society, p. 436. Ma, L.Q., Rao, G.N., 1997. Chemical fractionation of cadmium, copper, nickel, and zinc in contaminated soils. J. Environ. Qual. 26, 259264. MAFF (Ministry of Agriculture, Fisheries and Food), 1993. Code of Good Agricultural Practice for the Protection of Soil. MAFF publications, London UK, pp. 55. Manning, B.A., Martens, D.A., 1997. Speciation of arsenic(III) and arsenic(V) in sediment extracts by high-performance liquid chromatography-hydride generation atomic absorption spectrophotometry. Environ. Sci. Technol. 31, 171177. Newman, M.C., Jagoe, C.H., 1994. Ligands and the bioavailability of metals in aquatic environments. In: Hamelink, J.L., Landrum, P.F., Bergman, H.L., Benson, W.H. (Eds.), . In: Bioavailability: Physical, Chemical and Biological Interactions, Proceedings of the Thirteenth Pellston Workshop. Lewis Publishers, Pellston, MI, pp. 3961.

the best estimate of plant-available Ni and Co, while DTPA (diethylene-triamine-penta-acetic acid) was the most suitable for Cu and Pb. Heavy metals of Cd, Pb, Cu, Zn, Ni and Co exist as positively charged and are mostly associated with organic matter in soils. However, As is dominantly present as negatively charged anions, arsenate and arsenite in soils. Therefore, soil chemical extraction, solubility and bioavailability of As in soils for plant uptake should be dierent from heavy metals. The present study attempted to establish which extracting solution characterizes most precisely the availability of As to plants. Further statistical analysis showed that the percent As extracted by single chemical methods for the mining soils followed the trend: ammonium oxalate, hydroxylamine hydrochloride, phosphate > mixed acid, acetate > plantavailable > water. The percent As extracted by sequential extraction methods for the greenhouse rhizosphere soils followed the trend: hydroxylamine hydrochloride > organic phase > plant-available > ammonium nitrate. The fraction of As in mining soils that is bioavailable is comprised of As fractions between the surcially complexed (desorbable) As/readily soluble As forms (exchangeable + sorbed forms)/As adsorbed to the surface of soil particles and minerals of hydrous oxides of Fe, Mn and Al. 4. Conclusions One chemical extractant is unlikely to measure the fraction of bioavailable As in all contaminated soils under all conditions. However, a chemical extractant that is more closely related to bioavailable As than total As for most of the major groups of environmental media is desirable. The results of this study performed by single chemical extraction methods have shown that sodium acetate and mixed acid extractable As in mining soils exhibited good relationship with As in plant aerial biomass while sodium acetate is the best extractant. Sequential chemical extraction methods have shown that ammonium nitrate may eventually be designed to provide closer estimates of bioavailable As in mining soils and subsequently better extractant is hydroxylamine hydrochloride. Acknowledgements The rst author would like to thank the Ministry of Education and Science, Spain for supporting his study at IRNASA-CSIC, Salamanca, Spain. The suggestions and critical technical comments of the anonymous reviewers were greatly appreciated. References
Adriano, D.C., 2001. Trace Elements in Terrestrial Environments Biogeochemistry Bioavailability and Risk of Metals, second ed. Springer-Verlag, New York.

H.M. Anawar et al. / Chemosphere 70 (2008) 14591467 Norra, S., Berner, Z.A., Agarwala, P., Wagner, F., Chandrasekharam, D., Stuben, D., 2005. Impact of irrigation with As rich groundwaters on soil and crops: a geochemical case study in West Bengal Delta Plain, India. Appl. Geochem. 20, 18901906. Pierzynski, G.M., 1998. Past, present, and future approaches for testing metals for environmental concerns and regulatory approaches. Commun. Soil Sci. Plant Anal. 29, 15231536. Price, R.E., Pichler, T., 2005. Distribution, speciation and bioavailability of arsenic in a shallow-water submarine hydrothermal system, Tutum Bay, Ambitle Island, PNG. Chem. Geol. 224, 122135. Qian, J., Shan, X.-Q., Wang, Z.-J., Tu, Q., 1996. Distribution and plant availability of heavy metals in dierent particle-size fractions of soil. Sci. Total Environ. 187, 131141. Rodriguez, R.R., Basta, N.T., Casteel, S.W., Armstrong, F.P., Ward, D.C., 2003. Chemical extraction methods to assess bioavailable arsenic in soil and solid media. J. Environ. Qual. 32, 876884. Roussel, C., Bril, H., Fernandez, A., 2000. Arsenic speciation: involvement in evaluation of environmental impact caused by mine wastes. J. Environ. Qual. 29, 182188.

1467

Shuman, L.M., 1982. Separating soil iron- and manganese-oxide fractions for microelement analysis. Soil Sci. Soc. Am. J. 46, 10991102. Siebe, C., 1995. Heavy metal availability to plants in soils irrigated with wastewater from Mexico city. Water Sci. Technol. 32 (12), 2934, 6. Soon, Y.K., Abboud, S., 1993. Cadmium, chromium, lead, and nickel. In: Carter, M.R. (Ed.), Soil Sampling and Methods of Analysis. Lewis Publishers, Boca Raton, pp. 102108. Sposito, G., Lund, L.J., Chang, A.C., 1982. Trace metal chemistry in aridzone eld soils amended with sewage sludge: I Fractionation of Ni, Cu, Zn, Cd, and Pb in solid phases.. Soil Sci. Soc. Am. J. 46, 260264. Tan, K.H., 1996. Soil Sampling. In: Preparation and Analysis. Marcel Dekker, NY. Tessier, A., Campbell, P.G.C., Bisson, M., 1979. Sequential extraction procedure for the speciation of particulate heavy metals. Anal. Chem. 51, 844851. Vandecaveye, S.C., Horner, G.M., Keaton, C.M., 1936. Unproductiveness of certain orchard soils as related to lead arsenate spray accumulations. Soil Sci. 42, 203215.