Analytica Chimica Acta 504 (2004) 217226

Assessment of CaCl2 , NaNO3 and NH4 NO3 extraction procedures for the study of Cd, Cu, Pb and Zn extractability in contaminated soils
M. Pueyo, J.F. Lpez-Snchez , G. Rauret
Departament de Qumica Analtica, Universitat de Barcelona, Diagonal 647, Barcelona, E-08028 Spain Received 24 July 2003; received in revised form 16 October 2003; accepted 16 October 2003

Abstract The variety of extraction procedures used in environmental studies makes it very difcult to compare the results obtained; therefore, harmonisation and standardisation is required. The extraction of heavy metals from soil by un-buffered salt solutions is a method used to estimate soil contamination and trace metal availability to plants. The present study assesses three of these methods. All the three methods are standardised or is undergoing standardisation in Europe: 0.01 mol l1 CaCl2 (The Netherlands), 0.1 mol l1 NaNO3 (Switzerland) and 1 mol l1 NH4 NO3 (Germany). The soil-reference material BCR CRM 483, with indicative values for CaCl2 , NaNO3 , NH4 NO3 extractable metals, was analysed for quality control purposes. The three methods were also applied to 10 contaminated soils and the extracted metals (Cd, Cu, Pb, and Zn) were determined. The procedures were found to be precise (typically <10%) for all metals, taking into account the low metal concentrations extracted. The metal extraction efciency obtained with each procedure was slightly different, and the three methods provided equivalent information while predicting the relative trace-metal mobility (Cd > Zn > Cu > Pb) in the soils studied. From the experience obtained, the 0.01 mol l1 CaCl2 extraction procedure seems to be the most suitable method for performing a harmonisation process, since this procedure combines an appropriate extraction capacity for this type of studies with the lowest salt concentration in the extracts and, consequently, with a more simple matrix for metal determination. 2003 Elsevier B.V. All rights reserved.
Keywords: Soils; Trace metals; Single extraction procedures; Mobility; Availability

1. Introduction Heavy metals in soils exist in different chemical forms or types of binding. In environmental studies, the determination of these forms gives more information on trace metal mobility, as well as on their availability and toxicity, in comparison to the total element content. To carry out such determinations, many approaches based on desorption or adsorption phenomena have been designed, but extraction procedures are the most widely accepted and used method in soil science. These procedures, using a single extractant, provide a relative empirical method for evaluating the potential availability of soil pollutants for plant uptake. Moreover, they are also well-established procedures for the evaluation of soil chemical fertility and nutrient deciency [1]. A large number of single extraction procedures are reviewed in [24]. They not only use different extracting
Corresponding author. Tel.: +34-93-403-4873; fax: +34-93-402-1233. E-mail address: fermin.lopez@apolo.qui.ub.es (J.F. L pez-S nchez). o a 0003-2670/$ see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.10.047

agents, but also different experimental conditions. Chelating agents and strong acids, which extract either a large portion or almost the entire total metal from the solid phase, are postulated as suitable for the prediction of plant uptake only in specic scenarios [5]. This fact has led extraction methods to use less aggressive solutions, such as neutral salts [6]. These solutions are often called soft or mild extractants and are based on un-buffered salt solutions, which mainly dissolve the cation exchangeable fraction, although in some cases the complexing ability of the anion can play a certain role. Additionally, several studies have demonstrated a good correlation between metal concentration in these extraction media and metal concentration in plants [68]. Lebourg et al. [9] studied the chemical speciation of Cd, Cu, Pb, and Zn in different neutral salts extracts and observed similarities between the qualitative composition of these extracts and the soil solution, which is the main regulatory factor in the soil system because plant roots and soil organisms are exposed to it. All these studies justify the use of un-buffered salt solutions to estimate the soil trace metal availability and dene guide values for risk assessment.

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The main problem in the use of single extraction methods lies in the lack of uniformity in the different procedures used. In consequence, the results obtained are operationally dened depending on the experimental conditions used (type and concentration of extracting agent, soil mass:volume ratio, shaking time and speed of shaking). This fact makes data comparison difcult and prevent the standardisation of these methods. To date, there is no generally accepted method of estimating the bio-availability of heavy metals in soils. However, three methods using un-buffered salt solutions are standardised or undergoing standardisation in several European countries. A 0.01 mol l1 CaCl2 solution has been recommended in The Netherlands [10,11], while a 0.1 mol l1 NaNO3 solution [12] and 1 mol l1 NH4 NO3 solution [13] have become standard national protocols in Switzerland and Germany, respectively. There are no certied soil reference materials available for the extractable trace metal content using these soft extractants, that could help the method validation and quality control of the measurements. Nevertheless, several inter-laboratory exercises applying mild extraction procedures have been organised in recent years. The Standards Measurements and Testing Programme (formerly BCR) of the European Commission reported indicative values for CaCl2 , NaNO3 and NH4 NO3 extractable metals in two sewage sludge amended soils (BCR CRM 483 and BCR CRM 484) [14]. The Austrian Governmental Agricultural Research Institutes (ALVA) tested different methods (CaCl2 and NH4 NO3 ) to evaluate the analytical inter-laboratory precision of the data obtained by 12 laboratories [15]. In order to evaluate the accuracy and precision of the analytical procedures used in soil science, an International Soil-analytical Exchange Programme (ISE) has been established. The ISE programme at present comprises almost 300 laboratories in many countries and is one of the prociency testing schemes of the Wageningen Evaluating Programmes for Analytical Laboratories (WEPAL), organised by The Wageningen University (The Netherlands). This inter-laboratory comparison scheme allows the laboratories that use the procedures studied in the present work (CaCl2 , NaNO3 and NH4 NO3 ) to compare their methods and analytical results continuously [16] (http://www.wepal.nl). To harmonise such methodologies, it is necessary to have available sufcient information about the behaviour of the different extraction procedures when they are applied to different soil-types and for a number of heavy metals. From an adequate database on trace metal extraction using common extraction procedures, similarities and relationships among the methods can be established. After this, the choice of the most appropriate extractant should be based on both the environmental information obtained and the analytical characteristics of the method. Here, we report the assessment of 0.01 mol l1 CaCl2 , 0.1 mol l1 NaNO3, and 1 mol l1 NH4 NO3 extraction procedures for metal (Cd, Cu, Pb and Zn) analysis in contaminated soils. For this purpose, different aspects are consid-

ered for the evaluation of these methods: precision, extraction pH, extraction efciency, metal mobility predictions, soil characteristics and major element (Ca, Fe, K, Mg, Mn and Na) concentration in the extracts. 2. Experimental 2.1. Instruments A pH-meter Crison Model Basic 20 was used to measure the pH of soils and extracts. X-ray uorescence (XRF) measurements were carried out using a Philips PW2400 X-ray spectrophotometer with Rh excitation tubes. A Thermo Finnigan NA 2100 elemental analyser was used for C-determination. This model is equipped with an autosampler AS200, a combustion oven containing the oxidation catalyst and a reduction oven containing copper, as well as Porapak GC column and a thermal conductivity detector (TCD). For FAAS determinations, a single-beam atomic absorption spectrometer (Perkin-Elmer model 1100B) with airacetylene ame and a deuterium lamp for continuous background correction was used. Measurements were performed at 324.8 nm for Cu and at 213.9 nm for Zn. A Perkin-Elmer Model OPTIMA 3200RL ICP-atomic emission spectrometer equipped with a Perkin-Elmer AS-90 Plus autosampler and consisting of a radio-frequency source (working at a power of 1150 W and a frequency of 40 MHz), a cross-ow nebulizer, and a SCD (segmented-array charge coupled device) detector was used for ICPAES measurements. The following emission lines were used for each element determined (nm): Ca: 315.887 and 317.933; Cu: 324.752 and 327.393; Fe: 259.939; K: 766.490; Mg: 279.077 and 285.213; Mn: 257.610 and 259.372; Na: 330.237; Zn: 206.200 and 213.857. A Perkin-Elmer Model ELAN 6000 ICPMS equipped with a Perkin-Elmer AS-91 autosampler and consisting of a radio-frequency source working at a power of 1000 W and a frequency of 40 MHz, a cross-ow nebulizer, and a quadrupol mass spectrometer was used for Cd, Cu, and Pb determinations. Different element isotopes (111 Cd, 112 Cd and 114 Cd; 63 Cu and 65 Cu; 208 Pb) were measured to detect and control possible isobaric or poly-atomic interferences. 2.2. Reagents and standards All the reagents used to prepare the extracting solutions were products of analytical-grade quality (Merck pro-analysis). Concentrated HNO3 used for the experiments was for trace metal analysis (J.T Baker Instra-Analysed). All solutions and dilutions were prepared using doubly de-ionised water (18.2 M cm1 ) obtained from an USF PureLaB Plus. Standard stock solutions of 1000 mg l1 of Cd, Cu, Pb, and Zn were prepared from metal wires of purity higher

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than 99.998% from Alfa Johnson Matthey following the recommendations of the National Institute of Standards and Technology [17]. Diluted standard working solutions were prepared from these daily. All laboratory glassware and plastic ware were rinsed three times with double deionised water after being soaked in a HNO3 (10%, v/v) bath overnight. Preparation of reagents and standards were performed in a clean laboratory Class 100, dotted with a workbench with vertical airow, according to the US Federal Standard 209E norm. 2.3. Procedures 2.3.1. Characterisation of soil samples Cation Exchange Capacity (CEC), pH (in water) and particle size distribution were determined following the procedures described in the Ofcial Methods of Analysis for soils of the Spanish Ministry of Agriculture, Fishery and Food [18]. Total C and organic carbon (Corg ) contents in soils were determined by elemental analysis using tin capsules and V2 O5 as additive. A pre-treatment of the samples with HCl 2 mol l1 was performed before the organic carbon analysis. The inorganic carbon content was calculated from the difference between the two values and is expressed as % CaCO3 [19]. Major elements in the soil were determined by X-ray uorescence spectrometry (XRF) using 56 geological international reference samples for calibration. Samples were diluted (1:20) with lithium tetraborate and melted in a radio-frequency inductive oven (Philips PERLX2 Micro processing System) to obtain 30 mm diameter pearls. 2.3.2. Extraction procedures Soil extracts were obtained following the extracting conditions described in the original procedures [1013]: after shaking 100 ml of 0.01 mol l1 CaCl2 solution and 10 g soil, 100 ml of 0.1 mol l1 NaNO3 solution and 40 g soil, and 50 ml of 1 mol l1 NH4 NO3 solution and 20 g soil in an end-over-end shaker for 2 h at 30, 120 and 5060 rpm, respectively, in a room at 20 2 C, the pH was measured in the settling suspension and the extracts were separated from the solid residue by centrifugation at 3000 g for 10 min. For NaNO3 and NH4 NO3 extracts, the supernatant was ltered through a membrane unit lter (0.45 m) (For CaCl2 extracts, ltration is not recommended in the procedure). Then, the extracts were acidied with HNO3 to prevent adsorption to the polyethylene of the storage vessel and to prevent growth of bacteria. The acidied supernatant was collected in polyethylene bottles and stored at 4 C until metal analysis. Three replicates were performed for each sample and each procedure and in independent working sessions. Blanks were measured in parallel for each batch of analysis using the extracting reagents described above. Major and trace elements were determined in the extracts following the determination

procedures described in the next section. The moisture content of each sample was determined in three independent aliquots by drying 1 g sample in an oven (105 2 C) until constant weight was attained. From this, a correction to dry mass was obtained which was applied to all analytical concentrations reported. 2.3.3. Determination of major and trace elements in the extracts FAAS measurements were carried out by aspirating the extracts directly. ICPAES measurements were performed by diluting 1:5 NaNO3 and NH4 NO3 extracts (in 1% HNO3 ) to minimise matrix interferences (such as high concentrations of Ca, Na, Mg or K), and consequently the background signal [20]. For CaCl2 extracts, measurements were carried out without dilution. The calibration method used for FAAS and ICPAES measurements was an external calibration procedure using matrix matching with the extracting reagent. For the measurement of the major element, the calibration graph was prepared in 1% HNO3 . ICPMS measurements were carried out by diluting 1:5 CaCl2 extracts and 1:10 NaNO3 and NH4 NO3 extracts (in 1% HNO3 ) to minimise, interferences from the calcium and sodium present in the extracts [21]. The calibration method used in all cases was an external calibration procedure (in 1% HNO3 ) with Rh as internal standard. No matrix matching was necessary due to the sufciently dilution of the extracts. For all analytical techniques used, the instrument was washed with HNO3 (1%, v/v) between samples and standards during 30 s. Since a long series of samples were analysed, calibration of every ten samples was performed in order to control the sensitivity of the instruments. 2.4. Reference materials and soil samples A sewage sludge amended soil certied reference material (BCR 483) was used for quality control purposes, since the extractable heavy metal contents applying the three un-buffered salt solutions studied here are given as indicative values for this soil [14]. Ten agricultural soil samples were used for the evaluation of the three procedures. These soils were affected by a toxic spill formed by ne particles of pyrite together with acid wastewater, originated by an accident in a mine located in Aznalcllar (South of Spain) in 1998. All these samples were air-dried, weakly rolled to break the aggregates and passed through a 2 mm sieve. More details of sampling and sample preparation are described in a previous work [22].

3. Results and discussion 3.1. Heavy metal contents and characterisation of samples Highly contaminated soils were selected for this study since for non-contaminated ones the extracted metal

220 Table 1 Metal content in the soil samplesa Code

M. Pueyo et al. / Analytica Chimica Acta 504 (2004) 217226

Mean S.D. values (mg kg-1 ) Cd Zn 0.08 0.02 0.03 0.01 0.2 0.1 0.08 0.4 0.2 0.05 647 4 844 1 1746 16 776 48 1277 62 911 47 1184 26 1248 13 1883 60 965 50 150300 720 Cu 288 8 171 4 344 5 278 3 241 10 214 14 253 6 308 15 341 7 63 2 50140 190 Pb 516 29 1353 20 1495 35 403 14 754 28 96 4 1153 20 245 19 1610 71 68 2 50300 530

1RIB1 2PILh 2QUEh 1DOB1 2SOBh 1QUE2 2AUTh 1PIL2 1DOB2 2DEP MAC values (EU-range)b Intervention limits (The Netherlands)c
a b c

2.11 2.74 6.09 1.69 4.1 3.7 4.00 5.9 6.0 2.50 13 12

Aqua regia extractable contents from [22]. MAC is the maximum allowable concentrations from [24]. Data from [25].

amounts, while using mild extractants, are usually very low or below detection limits of most analytical techniques. Trace metal content in the studied soil samples (see Table 1) was determined in a previous work [22] from aqua regia extraction following the procedure recommended by ISO [23]. Data derived from national regulations and European directives are also shown in Table 1: the maximum allowable concentrations (MAC) range considered for the European Union and dened as permissible levels of trace elements in agricultural soils [24] and the intervention limits established by the Dutch legislation [25], which determines whether a remedial strategy is necessary in a contaminated site. All the soil samples used in this study had Cd, Cu, Pb, and Zn contents higher than their respective MAC value range and
Table 2 Characteristics of the soil samplesa Code pH CaCO3 (%) Corg b (%) 3.26 0.80 0.96 1.14 0.98 1.11 0.86 CEC c (meq/ 100 g) 19.4 63.2 48.3 9.2 30.9 10.3 37.6 Sand (%) 44.1 20.9 47.3 57.2 43.5 60.4 19.5 Silt (%) Clay (%) Texture CaO (%) (%) Loam Clay loam Loam Sandy loam Loam Sandy loam Silty clay loam Sandy loam Loam Clay

even above intervention limits for at least one of them (especially for Cu and Zn), indicating serious contamination in these soils. The characterisation of the soil samples in terms of major elements and other main parameters such as pH, carbonate and organic carbon contents, cation exchange capacity (CEC) and texture are summarised in Table 2. Results are the mean values of duplicate analysis. As shown, the soil samples presented different textures and CEC (8.563.2 meq/100 g). There was one sample (1RIB1) with a pH value (3.55) signicantly different from others (6.507.93). The organic carbon content in all the samples was normal, ranging from 0.8 to 3.3%. The major element contents (expressed as percentage of oxides) provided in-

Fe2 O3 (%)

MgO (%) 1.47 1.24 1.18 1.48 1.69 1.08 1.32

MnO (%) 0.11 0.10 0.08 0.10 0.12 0.10 0.07

Al2 O3 (%) 12.99 12.41 8.33 11.51 13.19 9.82 8.94

SiO2 (%) 57.1 54.7 61.5 60.9 58.3 71.0 53.2

TiO2 (%) 1.13 0.78 0.70 1.17 0.93 0.78 0.67

K2 O (%) 1.68 1.64 1.34 1.56 1.72 1.42 1.42

Na2 O (%) 1.79 1.11 1.20 1.75 2.96 1.38 0.81

P 2 O5 (%) 0.11 0.16 0.12 0.13 0.14 0.11 0.15

1RIB1 2PILh

3.55 n.d.d 6.50 0.6

43.2 12.6 45.1 34.0 37.0 15.8 30.3 13.5 36.6 19.9 26.3 13.3 52.2 28.3

1.83 8.76 2.91 11.04 4.14 2.45 4.17 3.50 9.26 8.60 9.16 4.09

2QUEh 6.61 0.8 1DOB1 6.80 n.d.d 2SOBh 1QUE2 2AUTh 6.92 1.4 7.20 4.5 7.23 7.5

6.53 11.05

1PIL2

7.32 7.1

1.27 1.05 1.66

10.2 8.5 59.0

61.5 50.4 1.2

25.4 13.1 39.0 10.6 40.4 58.7

5.74 7.08 10.05

4.60 6.50 5.66

1.31 2.08 4.17

0.08 0.12 0.13

9.81 9.01 14.21

64.5 59.8 40.8

0.65 0.57 0.75

1.52 1.71 2.88

1.36 1.08 1.41

0.11 0.08 0.28

1DOB2 7.46 8.5 2DEP 7.93 14.1

a b c d

Mean values of duplicate analysis. Organic carbon. Cation exchange capacity. Not detected.

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formation on the chemical composition of the soils. Iron contents were high in most samples, since the values commonly found for Fe in sedimentary rocks are 0.66.7% as Fe2 O3 [24], indicating the contribution of the pyritic sludge particles to the soil contamination. 3.2. Assessment of the mild extraction procedures The results obtained for Cd, Zn, Cu and Pb after the application of the three extraction procedures to the soil samples together with the technique used for determination and the quantication limits are reported in Table 3. The results are expressed as the mean value and the standard deviation of three independent replicates. The precision, dened as (standard deviation/mean) 100, is also given in parentheses. The detection and quantication limits of each element in each extract were determined from the results of analyte-concentration in 20 independent analytical blanks. According to the IUPAC denition [26,27], detection limits and quantication limits were calculated as three and 10 times the standard deviation of the blank concentration, respectively. As can be observed in Table 3, extractable Cd, Zn and Cu amounts were detected in the soil extracts, whereas for Pb the concentration extracted was below the quantication limit for most of the soil samples when CaCl2 and NaNO3 were applied. The major element (Ca, Fe, K, Mg, Mn and Na) concentration was quantied by ICPAES in the CaCl2 , NaNO3 and NH4 NO3 soil extracts and it is shown in Table 4. The results are expressed as the mean value of the three replicates (mg l1 ) with precision in parentheses. The quantication limits for Na, Mn and Fe are also reported. 3.2.1. Quality control of the analytical data The quality of the analytical data obtained for the three mild extraction procedures was evaluated by analysing the sewage sludge amended soil BCR CRM 483 which was extracted simultaneously with the soil samples. The obtained results and the indicative values for the extractable metal concentration for this soil are shown in Table 5. The quality of the data was considered good for Cd, Pb and Zn and for the three extractants, since the mean and the standard deviation of the obtained results overlapped with the indicative values reported for the CRM. Only slight deviations were obtained for Cu and the three extraction methods. The deviations observed can be attributed to the instability of the Cu-extractable contents as has been previously reported for other reference materials [28,29]. As expected, the precision obtained for the reference material was good with values for all the elements lower than 7% (except for extractable Pb with NH4 NO3 ), when compared with the uncertainty of the indicative values. The precision obtained with the application of the three extraction methods to the soil samples analysed was satisfactory with values in most cases lower than 10% (see Table 3). However, in some cases a poorer precision was obtained (up to 17%). This

can be attributed to the low metal concentrations extracted in some samples or to sample heterogeneity. Thus, the good precision obtained for the BCR CRM 483 soil can be attributed to the exhaustive homogenisation process suffered by this sample in comparison to the other soils studied. 3.2.2. Extraction pH The extraction procedures compared here use un-buffered salt solutions, with a pH of 5.16 0.10 for NaNO3 , 5.45 0.11 for CaCl2 , and 4.640.08 for NH4 NO3 . These extracting solutions have low effect on the soil pH and, therefore, the pH during the extraction process is mainly determined by the soil and not by the extractant. This effect was observed in this work, since the pH measured in the extracts was generally close to the natural soil pH (Table 2), with variations lower than 0.5 pH units for most cases. While comparing the pH value among extractants, the pH of CaCl2 and NaNO3 extracts was the same for most of the samples. However, the pH of the NH4 NO3 extracts was generally lower than the pH of the NaNO3 and CaCl2 extracts, possibly due to the slightly acidic character of the NH4 + ion. 3.2.3. Extraction efciency The metal extraction efciency obtained with the three procedures studied can be compared by observing the results presented in Table 3. The extractability of Cd obtained with the NH4 NO3 procedure was in general higher than that obtained with the CaCl2 and NaNO3 procedures. However, for some soils (1DOB1 and 2SOBh) the Cd concentration extracted with CaCl2 was equivalent to that obtained with NH4 NO3 and even higher, as is the case with the most acidic soil (1RIB1). The lowest extraction efciency in all the samples was obtained with the NaNO3 procedure. A similar pattern was observed for Zn extractability (NH4 NO3 > CaCl2 > NaNO3 ), and higher extraction efciencies with CaCl2 than with NH4 NO3 were obtained for some samples (1RIB1, 2PILh and 2QUEh). The high Cd and Zn extractability obtained with the NH4 NO3 procedure in comparison with the other studied reagents can be attributed to the possible complexation of these elements by NH3 [9] and to the higher salt concentration of the NH4 NO3 solution. The other sequence of extractability obtained for Cd and Zn and for some soils (CaCl2 NH4 NO3 > NaNO3 ) is consistent with previous ndings in [7,30]. The fact that calcium chloride can extract higher amounts of both Cd and Zn than the other extractants, which use higher salt concentrations is probably due to the combined effect of complexation by chloride [9,31] and by the fact that Cd and Zn are normally adsorbed in cation-exchange positions and, therefore, the divalent cation Ca2+ can remove them more easily than can monovalent cations, such as Na+ and NH4 + [6,32]. In the case of Cu, similar extraction efciencies were obtained when CaCl2 and NaNO3 were used. On the other hand, the extractability with NH4 NO3 was higher than with the other extractants, probably due to the formation of strong amino complexes of Cu [6,9]. Therefore, the sequence of

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Table 3 Extractable metal concentrations in the soils studied after applying the three mild extraction proceduresa
Code Cd ( g kg1 ) NaNO3 Technique LOQb 1RIB1 2PILh 2QUEh 1DOB1 2SOBh 1QUE2 2AUTh 1PIL2 1DOB2 2DEP a b c d ICPMS 5.6 685 7 (1.0) 139 4 (2.9) 482 17 (3.5) 12.1 0.4 (3.3) 25 1 (4.0) BQLc 34 4 (12) 6.8 0.3 (4.4) 8.7 0.2 (2.3) 19 3 (16) CaCl2 ICPMS 6.6 932 54 (5.8) 154 9 (5.8) 711 64 (9.0) 27 1 (3.7) 98 2 (2.0) 29 1 (3.4) 105 8 (7.6) 46 1 (2.2) 29.5 0.4 (1.4) 31 5 (16) NH4 NO3 ICPMS 4.7 833 4 (0.5) 349 6 (1.7) 862 32 (3.7) 25 1 (4.0) 92 3 (3.3) 48 4 (8.3) 156 2 (1.3) 86 6 (7.0) 81 4 (4.9) 59 1 (1.7) Zn (mg kg1 ) NaNO3 FAAS 0.167 164 2 (1.2) 15.8 0.5 (3.2) 90.6 0.7 (0.8) 1.60 0.02 (1.3) 1.92 0.02 (1.0) BQL 2.22 0.01 (0.5) 0.18 0.03 (17) 0.31 0.03 (9.7) 0.20 0.03 (15) CaCl2 ICPAES 0.153 217 1 (0.5) 42 1 (2.4) 187 5 (2.7) 2.5 0.1 (4.0) 4.0 0.1 (2.5) 0.31 0.05 (16) 4.8 0.5 (10) 0.29 0.05 (17) 0.47 0.05 (10) 0.22 0.01 (4.5) NH4 NO3 FAAS 0.207 208 3 (1.4) 31.5 0.9 (2.9) 127 2 (1.6) 2.80 0.05 (1.8) 5.0 0.1 (2.0) 1.5 0.2 (13) 7.8 0.2 (2.6) 1.85 0.01 (0.5) 2.03 0.06 (3.0) 1.16 0.07 (6.0) Cu ( g kg1 ) NaNO3 ICPAES 71 5715 308 (5.4) 259 4 (1.5) 1071 107 (10) 129 11 (8.5) 164 3 (1.8) 202 6 (3.0) 142 4 (2.8) 255 4 (1.6) 155 16 (10) 145 13 (8.9) CaCl2 ICPMS 54 5589 73 (1.3) 168 14 (8.3) 503 67 (13) 132 9 (6.8) 197 20 (10) 109 10 (9.2) 144 9 (6.3) 144 10 (6.9) 101 10 (9.9) 170 21 (12) NH4 NO3 FAAS 0.156 11364 259 (2.3) 634 18 (2.8) 5131 58 (1.1) 416 20 (4.8) 483 16 (3.3) 848 11 (1.3) 1205 36 (3.0) 1680 36 (2.1) 1074 2 (0.2) 304 31 (10) Pb ( g kg1 ) NaNO3 ICPMS 36 3053 61 (2.0) 121 13 (11) BDLd BDL BQL BDL BQL BDL BQL BDL CaCl2 ICPMS 38 5974 700 (12) 133 20 (15) 48 6 (13) BQL 44 7 (16) BDL 153 10 (6.5) BDL BDL BDL NH4 NO3 ICPMS 19 28040 479 (1.7) 936 55 (5.9) 195 27 (14) 56 3 (5.4) 419 48 (11) BQL 210 24 (11) 59 6 (10) 322 50 (15) BDL

Results are expressed as mean concentration 1 S.D., with precision (%) in parentheses (three replicates). Limit of quantication. Below quantication limit. Below detection limit.

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Table 4 Major element concentration (mg L1 ) in CaCl2 , NaNO3 and NH4 NO3 soil extractsa
Code Ca CaCl2 1RIB1 2PILh 2QUEh 1DOB1 2SOBh 1QUE2 2AUTh 1PIL2 1DOB2 2DEP 607 900 896 576 677 370 405 417 468 444 (0.1) (0.5) (0.6) (0.8) (1.4) (0.3) (0.4) (0.4) (0.4) (0.4) NaNO3 853 997 938 828 948 295 989 389 617 696 (5.8) (0.5) (1.5) (1.1) (0.7) (0.1) (0.4) (1.4) (0.3) (1.8) NH4 NO3 1361 (0.8) 2721 (1.8) 2373 (1.7) 1441 (0.8) 1895 (1.1) 925 (0.3) 2550 (1.2) 1041 (0.4) 1169 (0.4) 1389 (0.5) Na CaCl2 BQLb BQL BQL BQL BQL BQL 1.64 (1.9) BQL BQL 507 (0.3) NaNO3 2339 2346 2314 2307 2309 2340 2308 2303 2335 3613 (1.0) (1.4) (0.2) (0.1) (0.2) (0.1) (0.3) (0.2) (0.1) (0.6) NH4 NO3 25.2 (0.3) BQL 7.76 (2.3) BQL BQL 4.89 (5.3) BQL 4.28 (5.8) 6.20 (2.5) 1905 (0.2) Mg CaCl2 30.1 (0.1) 72.8 (0.6) 69.9 (0.1) 34.4 (0.3) 32.6 (0.4) 7.49 (0.2) 44.7 (1.3) 10.9 (0.9) 16.1 (0.3) 112 (0.5) NaNO3 99.5 (4.8) 189(0.4) 218 (1.0) 103 (0.3) 102 (0.7) 22.5(0.2) 107 (0.2) 32.8 (0.9) 48.2 (0.3) 348 (0.6) NH4 NO3 126 (0.8) 318 (2.0) 277 (1.9) 150 (0.9) 153 (1.4) 57.4 (0.2) 177 (0.2) 72.8 (0.2) 75.9 (0.1) 619 (0.3) Mn CaCl2 17.8 (0.7) 19.3 (1.4) 8.43 (0.2) 0.904 (1.6) 5.17 (1.1) BQL 3.65 (8.3) BQL 0.197 (1.7) BQL NaNO3 77.2 (2.6) 53.8 (0.8) 27.0 (3.9) 4.42 (2.2) 9.4 (0.6) 0.121 (1.4) 3.64 (3.0) 0.146 (1.8) 0.449 (0.8) 0.04 (1.5) NH4 NO3 98.0 (1.2) 97.2 (2.2) 40.3 (1.1) 7.49 (1.9) 18.3 (0.6) 0.788 (1.8) 9.36 (2.2) 1.06 (2.6) 1.71 (1.0) 0.131 (5.1) Fe CaCl2 5.01 (1.0) BQL BQL BQL BQL BQL BQL BQL BQL BQL NaNO3 11.0 (9.4) 0.123 (11) 0.124 (1.6) 0.105 (13) 0.103 (11) BQL 0.114 (2.0) BQL BQL BQL NH4 NO3 17.1 (5.2) 2.39 (15) 3.19 (14) 2.47 (13) 1.81 (9.8) 0.937 (4.1) 1.14 (8.9) 1.99 (9.1) 2.37 (7.9) 0.354 (15) K CaCl2 9.27 4.94 1.79 5.91 6.55 5.81 13.3 6.99 11.1 24.2 (2.6) (1.0) (2.1) (1.3) (3.5) (0.3) (0.5) (0.3) (0.2) (0.5) NaNO3 28.3 16.2 10.6 17.7 19.8 17.4 22.1 21.0 33.2 61.0 (6.9) (0.6) (1.5) (1.3) (0.8) (0.3) (0.8) (0.3) (0.2) (1.2) NH4 NO3 52.0 (1.0) 59.2 (2.6) 29.7 (1.3) 43.4 (0.7) 57.3 (1.3) 57.9 (0.6) 83.7 (0.2) 63.1 (0.9) 67.4 (0.3) 266 (0.2)

a Results expressed as mean concentration in extracts with precision (%) in parentheses (three replicates). b Below quantication limit. Limit of quantication by ICPAES: Na (0.984 mg l1 ); Mn (0.0017 mg l1 ); Fe (0.0065 mg l1 ).

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Table 5 Indicative and obtained CaCl2 , NaNO3 and NH4 NO3 extractable concentrations and precision for Cd, Cu, Pb, and Zn in BCR 483 certied reference material (mgkg-1 )a Cd CaCl2 NaNO3 NH4 NO3
a b c d

Cu 1.73 0.06 (3.5) 1.2 0.4 (33) 1.22 0.07 (5.7) 0.89 0.22 (25) 1.69 0.07 (4.1) 1.2 0.3 (25)

Pb BDLd <0.06 BDLd <0.03 0.032 0.004 (12) 0.020 0.013 (65)

Zn 8.1 0.4 (4.9) 8.3 0.7 (8.4) 3.3 0.1 (3.0) 2.7 0.8 (30) 5.4 0.3 (5.6) 6.5 0.9 (14)

Obtained valueb Indicative valuec Obtained valueb Indicative valuec Obtained valueb Indicative valuec

0.48 0.01 (2.1) 0.45 0.05 (11) 0.104 0.008 (7.7) 0.08 0.03 (38) 0.25 0.01 (4.0) 0.26 0.05 (19)

Mean concentration 1 S.D., with precision (%) in parentheses. Data obtained based on three replicates. Data from [14] for different laboratories reporting results based on ve replicates. Below detection limit by ICPMS.

extraction efciency obtained for this element (NH4 NO3 > CaCl2 NaNO3 ) revealed that the Cu extractability with the CaCl2 and NaNO3 solutions was fully comparable and clearly different from that obtained with the NH4 NO3 solution. The concentration of Pb extracted with the NH4 NO3 procedure was always higher than with the other solutions, the sequence of extractability in all samples being: NH4 NO3 >CaCl2 >NaNO3 . The highest lead extractability with the three procedures was obtained for the most acidic soil sample (1RIB1), the extractable amounts with NH4 NO3 being 49 times higher than those obtained with NaNO3 and CaCl2 . The low extractability observed for this element while using these extraction procedures has also been reported in the literature [6,31]. Lead concentrations in contaminated soil extracts are controlled by precipitation processes (such as carbonates, hydroxides, sulphates and phosphates), limiting the use of un-buffered salt solutions for the estimation of lead availability in soils [33]. Thus, the effects of lead contamination in soils are rarely evaluated using these extractants [3]. 3.2.4. Metal mobility predictions In order to establish a relative sequence of heavy metal mobility, the amounts of Cd, Zn, Cu, and Pb extracted with

each procedure are expressed in Table 6 as the extractable percentage (%) with respect to the trace element content in samples (from Table 1). Regarding the extractability of metals with these extractants, it can be observed from Table 6 that the three procedures provided equivalent information on the relative trace metal mobility sequence. Cadmium and Zn were predicted as the most mobile elements with the three procedures, Cd being more extractable than Zn in all cases. Copper and Pb presented extraction percentages below 1% (except for the acidic soil), indicating the low mobility of these elements, especially in the case of Pb. For some samples (1QUE2, 1PIL2, 1DOB2 and 2DEP), the three procedures predicted a slightly higher mobility for Cu than for Zn, this fact being more evident when the NH4 NO3 solution is used probably due to the formation of strong amino complexes of Cu, as has been previously mentioned. 3.2.5. Metal extractability and soil characteristics Given the complete characterisation of the soils presented in Table 2, the objective of Section 3.2.5 was to evaluate whether and how the soil matrix characteristics inuence the metal extractabilities with the three extracting solutions. The highest metal extractabilities with the three procedures were obtained for the soil with the lowest pH (1RIB1),

Table 6 Extractable metal percentage (%) after applying the three mild extraction proceduresa Code 0.1 mol l1 NaNO3 Cd 1RIB1 2PILh 2QUEh 1DOB1 2SOBh 1QUE2 2AUTh 1PIL2 1DOB2 2DEP
a

0.01 mol l1 CaCl2 Cu 2.0 0.15 0.31 0.050 0.070 0.090 0.060 0.083 0.050 0.23 Pb 0.59 0.010 Cd 44.1 5.6 11.7 1.6 2.4 0.79 2.6 0.77 0.49 1.2 Zn 33.6 5.0 10.7 0.32 0.31 0.030 0.41 0.024 0.025 0.020 Cu 1.9 0.10 0.15 0.050 0.080 0.051 0.057 0.047 0.030 0.27 Pb 1.2 0.010 0.003 0.006 0.013

1 mol l1 NH4 NO3 Cd 39.5 12.8 14.2 1.5 2.3 1.3 3.9 1.5 1.3 2.3 Zn 32.1 3.7 7.2 0.36 0.39 0.17 0.65 0.15 0.11 0.12 Cu 3.9 0.37 1.5 0.15 0.20 0.40 0.48 0.55 0.30 0.48 Pb 5.4 0.069 0.013 0.014 0.056 0.018 0.024 0.020 -

Zn 25.4 1.9 5.2 0.20 0.15 0.19 0.015 0.017 0.020

32.5 5.1 7.9 0.70 0.62 0.85 0.11 0.14 0.76

Results are expressed as mean values.

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indicating that for this sample the main factor that determines metal mobility is the pH. For the other soils studied with a pH ranging from 6.50 to 7.93, a correlation analysis was applied in order to explore the relationships among the extractable metal percentages with the three procedures and different soil characteristics, such as soil pH, CEC, organic carbon content, % clay, % Fe2 O3 and % MnO. The corresponding correlation coefcients (r) were obtained. Since these extraction procedures were designed to dissolve the exchangeable fraction, the CEC was one of the soil characteristic that correlated better with the extractable metal percentages, obtaining positive correlation coefcients in all cases (r > 0.5 in most cases). As expected, a negative correlation was observed among the extractable percentages and the soil pH (r > 0.5 in most cases). Correlation coefcients higher than 0.5 were also obtained among the percentage of Fe2 O3 and the extractable Cd with the three procedures. A poor correlation was observed with the other characteristics explored, since correlation coefcients were in general lower than 0.5. 3.2.6. Major element concentration in the extracts As can be observed in Table 4, the diluted CaCl2 extracting medium (0.01 mol l1 ) does not extract very high amounts of major elements as compared to the concentrated extracting media such as NaNO3 (0.1 mol l1 ) and NH4 NO3 (1 mol l1 ). For all the samples analysed, the concentration of Mg, Mn, Fe and K in the extracts followed the same sequence: NH4 NO3 > NaNO3 > CaCl2 . Special attention should be given to the concentration of Ca and Na in the extracts, since they were present in either the extracting agents or the soil matrix itself. The highest sodium concentration was quantied in the NaNO3 extracts, since the concentration of this element in these extracts was determined by the extracting solution itself. The Ca concentration quantied in the CaCl2 extracts was similar to the concentration of the reagent (400 mg l1 ) for some soils, or higher (up to 900 mg l1 ) indicating that amounts of Ca can be extracted from the soil matrix. The higher calcium concentration obtained in the NaNO3 (up to 997 mg l-1 ) and the NH4 NO3 extracts (up to 2700 mg l1 ) indicated the high extraction capacity of these concentrated solutions for the extraction of Ca from the soil matrix. Therefore, the 0.01 mol l1 CaCl2 extracting reagent yields a matrix of the soil extracts that is more simple and consequently, presents lower interferences for the measuring step. Moreover, while using ICP techniques, it is strongly recommended to work with solutions containing the lowest salt amounts [20,34] so that the CaCl2 solution would be the most appropriate in this case.

evaluated by comparing the obtained extractable Cd, Cu, Pb and Zn concentrations with the indicative values reported for the BCR CRM 483 soil. Concerning the extraction capacity of the three methods, the use of the 1 mol l1 NH4 NO3 solution led in general to a greater metal extractability. However, for some soils the Cd and Zn extractability obtained was higher with the 0.01 mol l1 CaCl2 solution than with the 1 mol l1 NH4 NO3 solution. The lowest extractability for all elements was obtained with the 0.1 mol l1 NaNO3 solution, although in the case of Cu a similar extraction efciency was obtained with both CaCl2 and NaNO3 procedures. Despite the different experimental conditions of the procedures compared here, the application of the three extractants led not only to assess the same order of metal mobility (Cd > Zn > Cu > Pb) but also to comparable amounts of extracted metals in the most part of cases. Therefore, from an environmental point of view the three procedures can be used in a similar way because equivalent information would be obtained. As a general conclusion, the 0.01 mol l1 CaCl2 extraction procedure seems to be a suitable method for the determination of Cd, Cu, Pb, and Zn mobility in soils, since this procedure presents an appropriate extraction capacity for this type of studies and also uses the lowest salt concentration. This fact simplies the matrix of the extracts and facilitates the metal determination with analytical techniques, such as ICPAES and ICPMS. From the results, it is advisable to adopt a single procedure for the determination of trace metal mobility and/or availability to plants. This would be the rst step towards the harmonisation of such procedures in order to obtain comparable data among laboratories.

Acknowledgements The authors would like to thank CICYT (Project PB98-1259 project) for the nancial support of this study, and the Serveis Cientco-Tcnics (Universitat de Barcelona) for their help in Elemental Analysis, and XRF and ICP measurements. M. Pueyo is grateful to the Ministerio de Ciencia y Tecnologa for a Ph.D Grant.

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4. Conclusions The studied procedures presented good precision with most values lower than 10%. The quality of the data was

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