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 difficult 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 efficiency 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 deficiency [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. Lopez-Sanchez).

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 specific 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 define
guide values for risk assessment.

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

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
defined 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 difficult 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 certified 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 proficiency 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 sufficient 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 efficiency, 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 fluorescence (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 flame 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-flow 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-flow 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

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

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 airflow, 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 Official 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
fluorescence 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 filtered through a membrane unit filter (0.45 m) (For CaCl2
extracts, filtration is not recommended in the procedure).
Then, the extracts were acidified with HNO3 to prevent adsorption to the polyethylene of the storage vessel and to prevent growth of bacteria. The acidified 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

219

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 sufficiently 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 certified 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 fine 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

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

Table 1
Metal content in the soil samplesa
Code

Mean S.D. values (mg kg-1 )

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

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

Cd

a
b
c

0.08
0.02
0.03
0.01
0.2
0.1
0.08
0.4
0.2
0.05

Zn

Cu

Pb

647 4
844 1
1746 16
776 48
1277 62
911 47
1184 26
1248 13
1883 60
965 50
150300
720

288 8
171 4
344 5
278 3
241 10
214 14
253 6
308 15
341 7
63 2
50140
190

516 29
1353 20
1495 35
403 14
754 28
96 4
1153 20
245 19
1610 71
68 2
50300
530

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 defined 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

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) significantly 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-

Table 2
Characteristics of the soil samplesa
Corg b
(%)

CEC c
(meq/
100 g)

Sand
(%)

Silt
(%)

3.55 n.d.d
6.50 0.6

3.26
0.80

19.4
63.2

44.1
20.9

43.2 12.6
45.1 34.0

2QUEh 6.61 0.8

1DOB1 6.80 n.d.d

0.96
1.14

48.3
9.2

47.3
57.2

37.0 15.8
30.3 13.5

2SOBh
1QUE2

6.92 1.4
7.20 4.5

0.98
1.11

30.9
10.3

43.5
60.4

36.6 19.9
26.3 13.3

2AUTh

7.23 7.5

0.86

37.6

19.5

52.2 28.3

1PIL2

7.32 7.1

1.27

10.2

61.5

25.4 13.1

1.05
1.66

8.5
59.0

50.4
1.2

39.0 10.6
40.4 58.7

Code

pH

1RIB1
2PILh

CaCO3
(%)

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.

Clay
(%)

Texture CaO
(%)
(%)
Loam
Clay
loam
Loam
Sandy
loam
Loam
Sandy
loam
Silty
clay
loam
Sandy
loam
Loam
Clay

Fe2 O3
(%)

MgO
(%)

MnO
(%)

Al2 O3
(%)

SiO2
(%)

TiO2
(%)

K2 O
(%)

Na2 O
(%)

P 2 O5
(%)

1.83 8.76
2.91 11.04

1.47
1.24

0.11
0.10

12.99
12.41

57.1
54.7

1.13
0.78

1.68
1.64

1.79
1.11

0.11
0.16

4.14
2.45

9.26
8.60

1.18
1.48

0.08
0.10

8.33
11.51

61.5
60.9

0.70
1.17

1.34
1.56

1.20
1.75

0.12
0.13

4.17
3.50

9.16
4.09

1.69
1.08

0.12
0.10

13.19
9.82

58.3
71.0

0.93
0.78

1.72
1.42

2.96
1.38

0.14
0.11

6.53 11.05

1.32

0.07

8.94

53.2

0.67

1.42

0.81

0.15

5.74

4.60

1.31

0.08

9.81

64.5

0.65

1.52

1.36

0.11

7.08
10.05

6.50
5.66

2.08
4.17

0.12
0.13

9.01
14.21

59.8
40.8

0.57
0.75

1.71
2.88

1.08
1.41

0.08
0.28

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

221

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.

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. Assessment of the mild extraction procedures

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.

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 quantification limits are reported in Table 3. The results
are expressed as the mean value and the standard deviation of three independent replicates. The precision, defined
as (standard deviation/mean) 100, is also given in parentheses. The detection and quantification limits of each element in each extract were determined from the results of
analyte-concentration in 20 independent analytical blanks.
According to the IUPAC definition [26,27], detection limits and quantification 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 quantification 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 quantified 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 quantification
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

3.2.3. Extraction efficiency

The metal extraction efficiency 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 efficiency 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 efficiencies 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 findings 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 efficiencies 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

222

Cd (g kg1 )

Code

Zn (mg kg1 )

Cu (g kg1 )

Pb (g kg1 )

NaNO3

CaCl2

NH4 NO3

NaNO3

CaCl2

NH4 NO3

NaNO3

CaCl2

NH4 NO3

NaNO3

CaCl2

Technique

ICPMS

ICPMS

ICPMS

FAAS

ICPAES

FAAS

ICPAES

ICPMS

FAAS

ICPMS

ICPMS

ICPMS

LOQb
1RIB1
2PILh
2QUEh
1DOB1
2SOBh
1QUE2
2AUTh
1PIL2
1DOB2
2DEP

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

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)

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)

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)

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)

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)

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)

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)

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)

36
3053 61 (2.0)
121 13 (11)
BDLd

38
5974 700 (12)
133 20 (15)
48 6 (13)
BQL
44 7 (16)
BDL
153 10 (6.5)
BDL
BDL
BDL

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

a
b
c
d

34 4 (12)
6.8 0.3 (4.4)
8.7 0.2 (2.3)
19 3 (16)

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

BDL
BQL
BDL
BQL
BDL
BQL
BDL

NH4 NO3

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

Table 3
Extractable metal concentrations in the soils studied after applying the three mild extraction proceduresa

Code

1RIB1
2PILh
2QUEh
1DOB1
2SOBh
1QUE2
2AUTh
1PIL2
1DOB2
2DEP

Ca

Na

CaCl2

NaNO3

NH4 NO3

607
900
896
576
677
370
405
417
468
444

853
997
938
828
948
295
989
389
617
696

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)

(0.1)
(0.5)
(0.6)
(0.8)
(1.4)
(0.3)
(0.4)
(0.4)
(0.4)
(0.4)

(5.8)
(0.5)
(1.5)
(1.1)
(0.7)
(0.1)
(0.4)
(1.4)
(0.3)
(1.8)

CaCl2
BQLb
BQL
BQL
BQL
BQL
BQL
1.64 (1.9)
BQL
BQL
507 (0.3)

Mg

Mn

Fe

NaNO3

NH4 NO3

CaCl2

NaNO3

NH4 NO3

CaCl2

NaNO3

NH4 NO3

CaCl2

NaNO3

NH4 NO3

CaCl2

NaNO3

NH4 NO3

2339
2346
2314
2307
2309
2340
2308
2303
2335
3613

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)

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)

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)

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)

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

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)

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)

5.01 (1.0)
BQL
BQL
BQL
BQL
BQL
BQL
BQL
BQL
BQL

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

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)

9.27
4.94
1.79
5.91
6.55
5.81
13.3
6.99
11.1
24.2

28.3
16.2
10.6
17.7
19.8
17.4
22.1
21.0
33.2
61.0

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)

(1.0)
(1.4)
(0.2)
(0.1)
(0.2)
(0.1)
(0.3)
(0.2)
(0.1)
(0.6)

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

(2.6)
(1.0)
(2.1)
(1.3)
(3.5)
(0.3)
(0.5)
(0.3)
(0.2)
(0.5)

(6.9)
(0.6)
(1.5)
(1.3)
(0.8)
(0.3)
(0.8)
(0.3)
(0.2)
(1.2)

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

Table 4
Major element concentration (mg L1 ) in CaCl2 , NaNO3 and NH4 NO3 soil extractsa

223

224

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

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

Cu

Pb

Zn

CaCl2

Obtained
Indicative valuec

0.48 0.01 (2.1)

0.45 0.05 (11)

1.73 0.06 (3.5)

1.2 0.4 (33)

BDLd
<0.06

8.1 0.4 (4.9)

8.3 0.7 (8.4)

NaNO3

Obtained valueb
Indicative valuec

0.104 0.008 (7.7)

0.08 0.03 (38)

1.22 0.07 (5.7)

0.89 0.22 (25)

BDLd
<0.03

3.3 0.1 (3.0)

2.7 0.8 (30)

NH4 NO3

Obtained valueb
Indicative valuec

0.25 0.01 (4.0)

0.26 0.05 (19)

1.69 0.07 (4.1)

1.2 0.3 (25)

0.032 0.004 (12)

0.020 0.013 (65)

5.4 0.3 (5.6)

6.5 0.9 (14)

a
b
c
d

valueb

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 five replicates.
Below detection limit by ICPMS.

extraction efficiency 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].

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 influence 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),

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

Table 6
Extractable metal percentage (%) after applying the three mild extraction proceduresa
Code

1RIB1
2PILh
2QUEh
1DOB1
2SOBh
1QUE2
2AUTh
1PIL2
1DOB2
2DEP
a

0.1 mol l1 NaNO3

0.01 mol l1 CaCl2

1 mol l1 NH4 NO3

Cd

Zn

Cu

Pb

Cd

Zn

Cu

Pb

Cd

Zn

Cu

Pb

32.5
5.1
7.9
0.70
0.62
0.85
0.11
0.14
0.76

25.4
1.9
5.2
0.20
0.15
0.19
0.015
0.017
0.020

2.0
0.15
0.31
0.050
0.070
0.090
0.060
0.083
0.050
0.23

0.59
0.010

44.1
5.6
11.7
1.6
2.4
0.79
2.6
0.77
0.49
1.2

33.6
5.0
10.7
0.32
0.31
0.030
0.41
0.024
0.025
0.020

1.9
0.10
0.15
0.050
0.080
0.051
0.057
0.047
0.030
0.27

1.2
0.010
0.003

0.006

0.013

39.5
12.8
14.2
1.5
2.3
1.3
3.9
1.5
1.3
2.3

32.1
3.7
7.2
0.36
0.39
0.17
0.65
0.15
0.11
0.12

3.9
0.37
1.5
0.15
0.20
0.40
0.48
0.55
0.30
0.48

5.4
0.069
0.013
0.014
0.056

0.018
0.024
0.020
-

Results are expressed as mean values.

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

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 coefficients (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 coefficients
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
coefficients 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 coefficients 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 quantified in the NaNO3 extracts,
since the concentration of this element in these extracts was
determined by the extracting solution itself. The Ca concentration quantified 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.

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

225

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 efficiency
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 simplifies 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 first 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 financial support of this
study, and the Serveis Cientfico-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.

References
[1] H.A. van der Sloot, L. Heasman, Ph. Quevauviller, Harmonisation of
leaching/extraction tests, Chap. 3, Elsevier, Amsterdam, The Netherlands, 1997, p. 41.
[2] W.F. Pickering, Ore Geol. Rev. 1 (1986) 83.
[3] A. Lebourg, T. Sterckeman, H. Ciesielski, N. Proix, Agronomie 16
(1996) 201.
[4] V.H. Kennedy, A.L. Sanchez, D.H. Oughton, A.P. Rowland, Analyst
122 (1997) 89R.
[5] A. Ure, Ph. Quevauviller, H. Muntau, B. Griepink, Int. J. Environ.
Anal. Chem. 51 (1993) 135.

226

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

[6] S.K. Gupta, C.F. Aten, Int. J. Environ. Anal. Chem. 51 (1993) 25.
[7] I. Novozamsky, T.M. Lexmond, V.J.G. Houba, Int. J. Environ. Anal.
Chem. 51 (1993) 47.
[8] C.F. Aten, S.K. Gupta, Sci. Total Environ. 178 (1996) 45.
[9] A. Lebourg, T. Sterckeman, H. Ciesielski, N. Proix, J. Environ. Qual.
27 (1998) 584.
[10] V.J.G. Houba, I. Novozamsky, T.M. Lexmond, J.J. Van der Lee,
Commun. Soil. Sci. Plant Anal. 21 (1990) 2281.
[11] V.J.G. Houba, T.M. Lexmond, I. Novozamsky, J.J. Van der Lee, Sci.
Total Environ. 178 (1996) 21.
[12] V.S. Bo, Verordnung ber Schadstoffgehalt im Boden, Swiss Ordinance on Pollutants in Soils Nr.814.12, Publ. Eidg. Drucksachen und
Materialzentrale [EDMZ], 3000 Bern, Switzerland, 1986, p. 1.
[13] DIN, Deutsches Institut fr Normung, Bodenbeschaffenheit. Extraktion von Spurenelemente mit Ammonium-nitratlsung. Vornorm
DINV 19730, DIN Boden-Chemische Bodenuntersuchungs-verfahren, Berlin, Germany, 1995.
[14] Ph. Quevauviller, G. Rauret, A. Ure, J. Bacon, H. Muntau, Report
EUR 17127 EN, European Commission, 1997.
[15] M. Sager, Accred. Qual. Assur. 4 (1999) 299.
[16] V.J.G. Houba, J.J. Van der Lee, I. Novozamsky, Analusis 19 (1991)
i45.
[17] J.R. Moody, R.R. Greenberg, K.W. Pratt, T.C. Rains, Anal. Chem.
60 (21) (1988) 1203A.
[18] Ministerio de Agricultura, Pesca y Alimentacin (M.A.P.A), Plantas,
productos orgnicos fertilizantes, suelos, aguas, productos fitosanitarios, fertilizantes inorgnicos, Mtodos Oficiales de Anlisis, Vol.
3, Servicio de publicaciones, Madrid, Spain, 1994, p. 205.
[19] A. Abadia, C. Cadahia, M. Casado, M. Chaves, M. Lachica, M.
Lasala, B. Sanchez, L. Prat, M. Romero, An. Edaf. Agrob. 32 (1973)
1153.

[20] M. Thompson, J.N. Walsh, Handbook of Inductively Coupled Plasma

Spectrometry, 2nd edition, Chapman and Hall, New York, 1989.
[21] M.A. Vaughan, G. Horlick, Appl. Spectrosc. 40 (1986) 434.
[22] M. Vidal, J.F. Lpez-Snchez, J. Sastre, G. Jimnez, T. Dagnac, R.
Rubio, G. Rauret, Sci. Total Environ. 242 (1999) 131.
[23] International Organisation for Standardisation, Soil quality, Extraction of Trace Elements Soluble in Aqua Regia, ISO 11466, (1995).
[24] A. Kabata-Pendias, H. Pendias, Trace Elements in Soils and Plants,
3rd edition, CRC Press, Boca Raton, FL, USA, 2001.
[25] D.C. Adriano, A. Chlopecka, D.I. Kaplan, H. Clijsters, J. Vangronsveld, Soil contamination and remediation: philosophy, science
and technology, in: R. Prost, (Ed.), Contaminated Soils. Les Colloques no 85, INRA editions, Paris, France, 1997, p. 465.
[26] L.A. Currie, Anal. Chim. Acta 391 (1999) 127.
[27] M. Thompson, S.L.R. Ellison, R. Wood, Pure Appl. Chem. 74 (2002)
835.
[28] Ph. Quevauviller, G. Rauret, J.F. Lpez-Snchez, R. Rubio, A. Ure,
H. Muntau, Sci. Total Environ. 205 (1997) 223.
[29] J.F. Lpez-Snchez, A. Sahuquillo, H.D. Fiedler, R. Rubio, G. Rauret,
H. Muntau, Ph. Quevauviller, The Analyst 123 (1998) 1675.
[30] J. Szkov, P. Tlustos, J. Balk, D. Pavlkov, M. Balkov, Chem.
Listy 95 (2001) 179.
[31] V. Hornburg, G. Welp, G.W. Brmmer, Z. Pflanzenernhr. Bodenk.
158 (1995) 137.
[32] H.A. van der Sloot, R.N.J. Comans, O. Hjelmar, Sci. Total Environ.
178 (1996) 111.
[33] A. Lebourg, T. Sterckeman, H. Ciesielski, N. Proix, A. Gomez,
Environ. Technol. 19 (1998) 243.
[34] K. E. Jarvis, A. L. Gray, Handbook of Inductively Coupled Plasma
Mass Spectrometry, 1st edition, Chapman and Hall, New York,
1992.