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Methods for Extracting Heavy Metals in


Soils from the Southwestern Amazon,
Brazil
ARTICLE in WATER AIR AND SOIL POLLUTION FEBRUARY 2013
Impact Factor: 1.69 DOI: 10.1007/s11270-012-1430-z

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Luis Alleoni

University of So Paulo

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Water Air Soil Pollut (2013) 224:1430


DOI 10.1007/s11270-012-1430-z

Methods for Extracting Heavy Metals in Soils


from the Southwestern Amazon, Brazil
Sabrina Novaes dos Santos &
Lus Reynaldo Ferracci Alleoni

Received: 20 August 2012 / Accepted: 18 December 2012 / Published online: 19 January 2013
# Springer Science+Business Media Dordrecht 2013

Abstract Heavy metals occur naturally in soil, at


concentrations that depend on the parent material from
which the soil was formed, the processes of formation,
and the composition and the proportion of the components of its solid phase. Quantifying these concentrations is important for environmental studies of soil
contamination and pollution, and choosing the methods for doing so is a key step in establishing heavy
metal contents in soil samples. We evaluated two
digestion methods (aqua regia and EPA 3051, both
microwave oven-assisted) for assessing pseudo-total
concentrations of Cd, Co, Cr, Cu, Ni, Pb and Zn in
the surface layer (020 cm) of soil samples from the
Brazilian agricultural frontier in the southwestern
Amazon. Nineteen composite samples of the most
representative soil classes for the states of Mato
Grosso and Rondnia were collected under native
vegetation undisturbed by human intervention.
Canonical discriminant analysis and principal component analysis were used for multivariate exploration of
the data. Aqua regia extracted higher amounts of Co,
S. N. dos Santos (*)
Graduate Student in Soil Science and Plant Nutrition,
University of Sao Paulo (ESALQ/USP),
Av. Pdua Dias, 11, C.P. 9,
Piracicaba, So Paulo 13418-900, Brazil
e-mail: sabrinanovaes@usp.br
L. R. F. Alleoni
Department of Soil Science, ESALQ/USP,
Av. Pdua Dias, 11, C.P. 9,
Piracicaba, So Paulo 13418-900, Brazil

Ni, Pb, and Zn than EPA 3051, while levels of Cr and


Cu did not differ between methods. In general, aqua
regia recovered more of the metals when compared to
reference soil samples.
Keywords Aqua regia . EPA 3051 . Background
concentrations . Soil contamination . Acid extractors

1 Introduction
Heavy metals occur naturally in soils, at concentrations that depend on the soil parent material, on the
processes by which the soil formed, and on the composition and proportion of the components of its solid
phase. Quantifying these concentrations is important
for studies of environmental pollution, and a key step
towards establishing soil quality reference values.
Environmental agencies require reference indicators in order to monitor environmental impacts and
determine pollution levels, as part of their mandate to
enforce environmental legislation. These indicators
are obtained by comparing concentrations of toxic
elements in soils with those observed in natural
(nonpolluted) soils, or with reference values.
To date, a number of studies have been carried out
to quantify natural concentrations and reference background values of heavy metals in Brazilian soils.
However, no studies have described natural concentrations of heavy metals in the soils of Mato Grosso
and Rondnia, Brazilian states in the southwestern

1430, Page 2 of 16

Amazon, which represent one of the largest agricultural frontiers on Earth.


Deciding which method to prepare and extract concentrations of elements in soil samples is a crucial step
in the process of describing the environmental conditions of a given area. There are several different methods of acidic digestion, ranging from aqua regia (AR)
(3:1 HCl/HNO3, v/v), with varying quantities of acids
and various digestion times and temperatures, in an
open system, and sometimes even hydrofluoric acid is
used in a closed system. This latter digestion is considered total, due to the destruction of the silicate
matrices (Chen and Ma 1998; Caires 2009).
A large number of studies (e.g., Akker and Delft
1991; Chen and Ma 1998; Tam and Yao 1999; Chen
and Ma 2001; Campos et al. 2003; Tighe et al. 2004;
Chander et al. 2008; Caires 2009; Nemati et al. 2010)
have documented marked differences in the amounts
of metals extracted by these different methods. This
means that government agencies must establish norms
regarding methods to extract naturally occurring concentrations in soils, in order to permit rigorous comparisons with pre-established values.
In Brazil, the analytic methods adopted in legislation
for quantifying total concentrations of inorganic substances (apart from mercury) in soil samples are two methods
of the United States Environmental Protection Agency
(USEPA), known as 3050B (HNO3 + H2O2) and 3051
(HNO3) (USEPA, 2007a, b, c), which are widely used to
digest samples of soils, sediments, and wastes.
In the 3051 method, the oxidation of organic matter
is performed by nitric acid without the solubilization
of the silicate fraction (pseudo-total). Conversely, in
the 3052 method the use of nitric and hydrofluoric
acids promotes a full dissolution of the sample.
However, hydrofluoric acid requires very careful handling, because it can cause severe burns in contact
with skin (Costa et al. 2008). It can also damage
analytical instruments (e.g., it attacks silicate materials, especially glass) and promote undesirable formation of insoluble fluoride precipitation with Al, Ca, Fe,
and Mg, and coprecipitation with Rb, Sr, Y, Cs, Ba,
Pb, Th, and U (Yokoyama et al. 1999; Vieira et al.
2005). The use of the 3052 method is hardly justified
in highly weathered tropical soils because mineral
reserves are not impressive and their silicate fraction
contains low amounts of heavy metals.
Microwave ovens have been used in soil chemistry
laboratories since the 1980s (Jassie and Kingston

Water Air Soil Pollut (2013) 224:1430

1988) and provide fast, secure, and efficient digestion


(Chen and Ma 2001).
Microwave heating is employed in the 3050B,
3051, and 3052 methods. Its advantages include a
shorter sample digestion, a more complete dissolution
of samples, and a smaller loss of volatile elements, in
addition to a lower risk of contamination than other
methods (Abreu et al. 2006).
AR is also very efficient at extracting pseudo-total
contents of heavy metals in soils. It is the standard
method for certifying soil samples in Britain and
France (Prez et al. 1997). Nitric acid oxidizes hydrochloric acid, giving rise to various oxidation products
such as molecular chlorine and nitrosyl chloride (3HCl +
HNO3 = 2H2O + Cl2 + NOCl) (Chen and Ma 2001).
This property, along with the presence of chloride
(complexing), makes AR a highly efficient extractor for
dissolving heavy metals (Costa et al. 2008).
In this study we assessed the AR and EPA 3051
methods of pseudo-total digestion for extracting Cd,
Co, Cr, Cu, Ni, Pb, and Zn. Concentrations were
subsequently correlated with physical and chemical
properties of soils from the states of Mato Grosso
and Rondnia, in Brazils southwestern Amazon
region.

2 Materials and Methods


The territories of Mato Grosso and Rondnia states
were first divided into 11 ecoregions or biogeoclimatic
macrozones intended to reflect large-scale similarities
and differences in soil, climatic, and management
conditions. This classification was performed with an
ArcView 9.0 Geographical Information System by
superimposing maps of soil conditions (scale of 1:5
million), native vegetation, geology, climate, and topography, with the goal of identifying homogeneous
areas that would allow our measurements of soil attributes at the study sites to be extrapolated across the
entire region (Maia et al. 2009). These homogeneous
zones illustrate biogeoclimatic conditions free of anthropogenic disturbances such as deforestation, agriculture, or ranching (Table 1).
Soils were sampled in June and July 2007. Two
townships in each of the regions were selected at
random, for a total of 19 townships. These yielded
19 soil samples, with three replicates each, collected under native vegetation free of anthropogenic

Water Air Soil Pollut (2013) 224:1430

Page 3 of 16, 1430

Table 1 Characteristics of macroregions in the states of Mato Grosso and Rondnia, southwestern Amazon region, Brazil
(Mello, 2007)
Region

Topography

Soil

Climate and average


annual rainfall (mm)

Vegetation

Alto Xingu

Flat with large plateaus

Oxisols

Amia (1,7502,250)

Seasonal semi-deciduous
forest to open Amazon
forest

Paran Basin

Flat with large plateaus

Oxisols Quartzipsamments Am,b Cwac (1,2501,750) Cerrado sensu stricto

Parecis Plateau

Flat with large plateaus

Quartzipsamments Oxisols Ami (1,5002,250)

Cerrado sensu stricto and


seasonal semi-deciduous
forest

Entisols Aquent
Entisols

Ami (1,2502,000)

Open cerrado (dominated


by grasses) and cerrado
sensu stricto

Am (1,5001,750)

Cerrado sensu stricto

Araguaia Depression Flat areas and rolling


hills; frequently
waterlogged
Cuiab Depression

Flat areas and rolling hills Inceptisols Entisols

Guapor Depression Mostly flat

Oxisols, Ultisols
Entisols

Ami (1,7502,250), Ami, Open Amazon forest


Am (1,5001,750)
(north) and seasonal
semi-deciduous forest
to Cerrado (south)

Northeastern
Mato Grosso

Irregular with rolling hills Ultisols

Amid (2,0002,500)

Cerrado to seasonal
semi-deciduous forest

Northern
Mato Grosso

Irregular with rolling hills Ultisols, Oxisols


Inceptisols

Awi, Ami (2,0002,750)

Open Amazon forest to


seasonal semi-deciduous
forest

Northern Rondnia

Predominately flat

Oxisols

Awi (2,0002,500)

Open Amazon forest

Central Rondnia

Varies between flat


areas and rolling hills
Mostly flat; frequently
waterlogged

Ultisols, Oxisols

Awi, Ami (1,7502,250)

Open Amazon forest

Entisols, Alfisols

Am (1,5001,750)

Open Cerrado and seasonal


semi-deciduous forest

Pantanal
a

Ami = very hot tropical monsoon climate

Am = tropical monsoon climate

Cwa = wet subtropical climate

Awi = tropical savanna climate

disturbance and considered to be representative of


soils in Mato Grosso and Rondnia. Within each
township ranches with areas of native vegetation
in which to collect soils were selected (Fig. 1).
Compound samples were formed from samples
taken at five collection sites within a 100100 m
plot at each ranch: one site at the center of the
plot and the other four on the square corners.
Samples were collected at a depth of 020 cm.
After being collected, samples were air-dried, homogenized, and sieved through 2-mm mesh. Most
of the soils collected belonged to the following
orders: Oxisols (74 %), Inceptsols (16 %), and
Entisols (10 %). More details regarding the study
are found in the work of Santos and Alleoni
(2012).

The contents of available phosphorus and potassium were extracted with Mehlich-I (Mehlich 1953),
and calcium and magnesium were extracted with
1 M KCl (Anderson and Ingram 1992). Phosphorus
content was determined by colorimetry, Ca and Mg
were quantified in an atomic absorption spectrophotometer (AAS), and K in a flame photometer.
Exchangeable aluminum was removed with 1 moll1
KCl (Anderson and Ingram 1992), and determined by
titration with 0.025 moll1 NaOH. Total acidity (H +
Al) was extracted with a 1 M calcium acetate (pH7
buffered) solution and determined by titration with
0.025 M ammonium hydroxide.
pH was determined potentiometrically for suspensions of air-dried fine earth in 1 M KCl, H2O, and
0.01 M CaCl2 1:2.5 (Anderson and Ingram 1992). The

1430, Page 4 of 16

Water Air Soil Pollut (2013) 224:1430

Fig. 1 Map of southwestern Amazonian Brazil, showing townships selected for soil sampling (RO Rondnia, MT Mato Grosso)

difference between pH1 M KCl and H2O was used to


verify the sign of the net charge (Mekaru and Uehara
1972). Based on the contents of exchangeable cations,
the sum of exchangeable bases, the cation exchange
capacity (CEC) at pH7.0, and base and Al saturations
were then calculated. Granulometric analysis was performed by the densimeter method (Gee and Or 2002)
and organic carbon was determined with a LECO CN2000 dry combustion elemental analyzer.
The oxides (expressed as SiO2, Al2O3, Fe2O3,
TiO2, and MnO) were extracted using 9 M H2SO4.
Contents of Fe, Mn and Al were determined using
atomic absorption spectrophotometry (AAS), Ti by
colorimetry (Vettori 1969), and Si by gravimetry.
The Ki weathering index was calculated by the molar
relation method where Ki = (%SiO2/60)/(%Al2O3/
102). Sodium citratebicarbonatedithionite (NaCBD) solution was used to extract the free iron oxides
(Mehra and Jackson 1960) and the concentration of
the poorly crystalline oxides of Fe, Al, and Mn were

determined by the method described by Loeppert and


Inskeep (1996).
Pseudo-total concentrations of metals were
obtained via digestion by (1) the AR method
(Mcgrath and Cunliffe 1985) and (2) with concentrated acid in a microwave oven, under controlled temperature and pressure, following the EPA 3051 method
(USEPA, 1996).
AR: 0.5 g of samples sieved through 100 mesh was
transferred to Teflon tubes. Each tube received 9 ml of
concentrated HCl (32 %) and 3 ml of concentrated
HNO3 (65 %) (3:1) of analytical-level purity, and the
mixture was left undisturbed for 12 h of pre-digestion.
The tubes were subsequently placed in a closed system
in a microwave oven (Mars Xpress, CEM Corporation)
for 20 min at 180 C subsequently passing the temperature ramp. After cooling, samples were transferred to
certified 25-ml flasks (NBR ISO/IEC), the flask volume
was completed with ultrapure water, and the extracts
were filtered. All analyses were carried out with three

Water Air Soil Pollut (2013) 224:1430

replicates. Analysis quality control was performed with


certified soil samples (NIST SRM 2709 San Joaquin
soil and DO65-540 Metals in Soil Environmental
Resource Associates).
Calibration curves to determine metal concentrations were prepared based on 1,000 mg l 1
standards (TITRISOL, Merck), using ultrapure
water for dilution, cleaning, and decontamination
of the glassware, which was kept in 10 % nitric
acid solution for 24 h and rinsed with distilled
water and ultrapure water. Concentrations of Co,
Cr, Cu, Ni, Pb, and Zn were determined via atomic absorption spectrophotometry (FAAS) and Cd
concentrations in a graphite oven.
The AR method is normally carried out with digestion in an open system. However, methods in which a
closed system is heated with a microwave oven are
well established and widely employed (Nieuwenhuize
et al. 1991; Chen and Ma 1998, 2001; Nemati et al.
2010; Sakan et al. 2011). The advantages of the closed
system compared to other methods are: (1) a
shorter digestion time for samples, (2) a lower risk
of pollution, (3) a more complete dissolution of
samples, and (4) lower losses of volatile elements
(Sakan et al. 2011).
EPA 3051: 0.5 g of samples sieved through 100
mesh was transferred to Teflon tubes, to which 10 ml
of concentrated HNO3 (65 %) of analytical purity were
added. Samples were placed in a closed system microwave oven (Mars Xpress, CEM Corporation) for
4 min and 30 s at 175 C, subsequently passing
the temperature ramp. After cooling, samples were
transferred to certified 25-ml flasks (NBR ISO/
IEC), with flask volume completed with ultrapure
water, and the extracts were filtered with slow
filter paper. All analyses were carried out in three
replicates, and sample blanks were performed simultaneously. Analysis quality control was performed with certified soils (NIST SRM 2709 San
Joaquin soil and DO65-540 Metals in Soil
Environmental Resource Associates). The NIST
recommends comparing methods that do not use
hydrofluoric acid (3050, 3051, and their updated
versions) with recovery based on leachable concentrations (NIST, 2002), since certified concentrations
are determined based on methods to determine total
concentrations. Calibration curves and the determination of metal concentrations were carried out as
described in the previous paragraph.

Page 5 of 16, 1430

Results were examined via analysis of variance


(ANOVA) (F < 0.01) and means compared by
Students t-test (p<0.01) and Pearsons correlation
analysis (p < 0.01). Multivariate procedures were
based on principal components analysis (PCA),
using version 4.0 of the CANOCO software program [28], and canonical discriminant analysis
(CDA). CDA allows one to identify differences
between groups and/or treatments and quantify
relationships between all the attributes under study
and between groups and/or treatments (CruzCastillo et al. 1994; Baretta et al. 2005). The
multivariate analysis met the objective of identifying the best digestion method for pseudo-total
concentrations of heavy metals. All statistical procedures were performed using version 8.2 of the
SAS software program (SAS, 2002).

3 Results and Discussion


Most soils were acidic, with a pH ranging from 3.5 to
7.4 and a mean of 4.7 (Table 2). Soil samples had a
clay content varying from 25 to 651 gkg1 (mean
307 gkg1), a high sand content ranging from 271 to
952 gkg1, and generally low silt content (<100 g
kg1). Concentrations of organic C were generally
low, with a mean of 13.3 gkg1. Mean CECt was
55.4 mmolc kg1, while effective CEC (CECe), which
reflects the soil capacity for retaining cations near its
natural pH level, averaged 19 mmolc kg1.
3.1 Metal Recovery in Certified Soils
In general, recovery rates of metals were satisfactory
both in the two certified soils and in the soil samples
from Mato Grosso and Rondnia (Table 3). Recovery
values varied from 40 % to 104 % for the 2709a
material and from 91 % to 107 % for the DO65
material (Table 3). For the DO65 material the elements
that were most effectively recovered were Ni, Pb, and
Zn (rate=107 %), while Co showed a slightly lower
recovery rate (91 %).
The recovered percentages of the NIST 2709a material, when compared to the recovered percentage of
the leachate, showed the following decreasing order of
recovery: Cr > Cd, Pb > Ni > Co, Zn > Cu. The ranges
of recovery were within those recommended for the
elements under study (USEPA, 1996).

1430, Page 6 of 16
Table 2 Selected physical and
chemical attributes of soil samples from the states of Rondonia
and Mato Grosso, Brazil (n=19)

Water Air Soil Pollut (2013) 224:1430

Variable

Mean

Minimum

Maximum

Standard Deviation

pH (H2O)

4.6

3.5

7.4

0.9

pH (0.01 M CaCl2)

4.3

3.2

7.2

0.9

pH (1 M KCl)

4.2

3.2

6.9

0.9

mmolc kg

CECe

19.0

2.3

96.4

17.9

CECt

55.4

17.8

158.0

29.0

%
V

24

0.2

99

26.9

46

0.1

98

34.6

g kg1
Org. C
CECe effective cation exchange
capacity, CECt totalcation exchange capacity, V base saturation, m saturation for aluminum

13.3

Sand

189

Silt

38

10

92

21

Clay

307

25

651

174

Certified soil

Cd

2709ac

0.3

84.7

DO65

Cr
Cu

Used to extract metals with the


aqua regia extractor

2709a

9.3

DO65

174.4

2709a

52.0

DO65

140.4

Certified valuea
(mg kg1)

Recovery
(determined)b (%)

Leach
recovery (%)

0.370.002

104

110

91.0

93

12.800.2

73

81

190.0

91.8

130.09.0

40

41

144.0

97

24.4

33.90.5

72

81

DO65

251.9

237.0

106

2709a

59.5

852

70

77

DO65

214.0

200.0

107

Pb

2709a

8.1

17.30.1

47

53

DO65

111.2

104.0

107

Zn

2709a

71.0

1034

69

77

DO65

314.4

341.0

107

Ni

Used to extract metals with extractor EPA 3051

Recovery level
(mg kg1)

2709a

Percentage of metals recovered


for DO65 and percentage of metals recovered in relation to the
leachate 2709a

and Mato Grosso were also generally lower than those


reported in the literature for other Brazilian states and
for other countries (MINEROPAR 2005; Salonen and
Korkka-Niemi 2007; Paye et al. 2010; Caires 2009;
Bini et al. 2011; Shah et al. 2012).
The low natural levels obtained by the two methods
may be related to the physical and chemical soil attributes and to the parent material of soils in the region.
Oxisols and Ultisols are highly weathered soils, and
their clay fraction is dominated by kaolinite, gibbsite,

Element

Co

Value of metal content for both


certified soils

7.1

953

The two methods of pseudo-total digestion yielded


different levels of naturally occurring heavy metals
in soils of Mato Grosso and Rondnia (Table 4). In
general, pseudo-total concentrations of metals
obtained via digestion by both methods were well
below the maximum levels considered acceptable for
soils worldwide (Alloway 1990; Reimann et al. 2000).
Mean concentrations observed in soils of Rondnia

35.1

271

3.2 Digestion Methods

Table 3 Average contents of


heavy metals recovered for certified reference materials (Biondi
2010)

4.4

654

Water Air Soil Pollut (2013) 224:1430

Page 7 of 16, 1430

Table 4 Heavy metal concentrations extracted by aqua regia and EPA 3051 in the surface layer (020 cm) of soils from Mato Grosso
and Rondnia, Brazil (n=19)
Aqua regia
Cd
Co
mg kg1

EPA 3051
Cr

Cu

Ni

Pb

Zn

Cd

Co

Cr

Cu

Ni

Pb

Zn

<0.05a

15.8

47.9

18.2

8.6

15.3

22.4

<0.05

20.3

39.4

16.5

1.3

8.1

Standard error

0.0

1.7

6.8

2.0

1.2

1.2

5.4

0.0

1.2

4.9

1.3

0.3

0.7

3.7

Median

0.0

14.1

39.7

15.4

7.8

15.1

13.2

0.0

18.3

31.2

14.3

0.4

7.5

0.6

Standard deviation

0.0

7.4

29.7

8.7

5.5

5.4

19.7

0.0

5.4

21.5

5.8

1.6

3.1

5.3

Variance

0.0

55.3

885.4

75.6

31.0

29.9

565.3

0.0

29.6

464.5

34.2

2.6

9.8

268.2

Minimum

<0.05

7.2

20.4

6.5

0.2

5.2

1.2

<0.05

16.6

19.2

9.7

0.0

2.7

0.0

Maximum

<0.05

38.9

142.1

32.9

24.3

25.8

100.9

<0.05

39.0

98.8

28.4

5.6

15.7

69.6

47

64

35

105

26

54

35

LC (%)b

Detection limit in g kg1

Confidence level

46

62

goethite, and iron and aluminum oxides (Fontes and


Weed 1991). They are also typically acidic (pH in H2O
varying from 4.3 to 6.2) and have very low concentrations of heavy metals. Parent material is one of the
principal determinants in the distribution of heavy
metals in soils. Even under severe weathering conditions (pedogenesis) such as those present in the wet
tropics, parent material may play an important role in
determining a large part of the heavy metal content of
soils (Baize and Sterckeman 2001).
Cd concentrations were below the detection level
for both methods. Co concentrations varied from 7.2
to 38.9 mgkg1 for digestion with AR and from 16.6
to 39.0 mgkg1 for EPA 3051 digestion. Mean Co
concentrations were higher for the EPA 3051 extraction than for AR. Mean Cr concentrations were higher
for the AR digestion (47.9 mgkg1) than for EPA 3051
(39.4 mgkg1). However, there was significant variation within methods, with AR showing results from
20.4 to 142.1 mgkg1 and EPA 3051 from 19.2 to
98.8 mgkg1. Mean Cu concentrations were similar
for the two methods: 18.3 and 16.6 mgkg1 for AR
and EPA 3051, respectively.
AR extracted much higher quantities of Ni, Pb, and
Zn than EPA 3051. Mean values varied from 0.2, 5.2,
and 1.2 mgkg1 to 24.3, 25.9, and 100.9 mgkg1 for
the AR method, and from 0, 2.7, and 0 mgkg1 to 5.6,
15.8, and 69.6 mgkg1 for the EPA 3051 method,
respectively. The methods did not differ (p<0.01) in
recovery of Co, Cr, and Cu. However, higher contents

126

6.8

38

238

Ni, Pb and Zn were extracted when AR was employed


(p<0.01) (Fig. 2).
AR extracted greater amounts of Cr, Cu, Zn, and Ni
than EPA 3051, while EPA 3051 extracted more Co,
and Cd was below the detection limit for both methods. Florian et al. (1998) compared the effectiveness
of the EPA 3051 method and the AR method with
microwave heating at determining heavy metals in
soil, sediment, and sludge. The authors concluded that
AR is the more appropriate method for digesting samples to determine concentrations of trace metals that
are potentially dangerous to the environment, and
100
a
80

Concentration (mg kg -1)

Mean

Aqua Regia
EPA 3051

a
60

a
40
a
b
20

a
a

a
a

Pb

Zn

b
0
Co

Cr

Cu

Ni

Fig. 2 Concentrations (mean standard deviation) of heavy


metals extracted by aqua regia and EPA 3051 methods from
surface layer (020 cm) soil samples. Means indicated by the
same letter for a given element do not differ (p <0.01) by
Student's t-test

1430, Page 8 of 16

especially so for elements that are involved in the


silicate matrix, such as Cr, Ni, and Pb. The variation
in Ni, Pb and Zn contents in this study probably
occurred because the amount and the nature of the
aluminosilicate matrix also varied.
AR is a mixture of the acids HNO3 and HCl.
They react to form nitrosyl chloride (NOCl) and
molecular chlorine (Cl2), which are highly reactive, have a high oxidizing power, and are capable
of dissolving even noble metals, but do not totally
dissolve silicates. In the European Union, AR is
the most commonly used solution for extracting
metals from polluted soils (Gleyzes et al. 2002;
Grotti et al. 2002; Quevauviller 2002), and is the
standard method for certifying soil samples in
Great Britain and France (Sakan et al. 2011).
The EPA 3051 method uses nitric acid (HNO3),
an oxidizing agent commonly used to extract metals, which are converted into soluble nitrates.
Hydrogen peroxide is used as an auxiliary agent
in the oxidation of samples with a high content of
organic carbon, which reacts to produce H 2O.
Oliveira et al. (2008) evaluated the effectiveness
of three methods of extracting total metal concentrations (AR, EPA 3051, and EPA 3052), and
concluded that AR was the most appropriate.
Although it does not extract total concentrations
of heavy metals, AR does provide a reasonable
estimate of the maximum amount that can potentially become available to plants or be leached
into groundwater (Diaz-Barrientos et al. 1991).
In our study, AR was also the most effective method for extracting naturally occurring heavy metals.
This may be due to the association of nitric acid and
hydrochloric acid in a closed system, creating a dissolving mixture that is extremely efficient for heavy
metals (Costa et al. 2008) and increases the extraction
power of AR. Extraction procedures with strong acids
such as AR with HNO3 and/or HCl typically seek to
reflect a pollutants potential availability and mobility,
factors which are related to its transfer from soils to
plants (Rauret 1998). While it did not extract higher
concentrations of heavy metals from Mato Grosso and
Rondnia soils, the EPA 3051 method is widely used
in the United States and by many environmental agencies worldwide, and its use is mandated under
Brazilian law by CONAMA.
The CDA model explained the variation between
the methods, with Canonical Discriminant Function

Water Air Soil Pollut (2013) 224:1430

(CDF1) (AR) responsible for 100 % of the difference


(Fig. 3). One important insight provided by the CDA
is the value of the parallel discrimination rate (PDR),
which is the product of the standardized canonical
coefficients (SCC) and the correlation (r). The parameter r provides univariate information for each metal
(i.e., its individual contribution), independent of the
method used. The PDR is more efficient when the
researcher wants to discriminate between areas
(Cruz-Castillo et al. 1994; Baretta et al. 2005) or
methods, as in this study. Positive values of the
PDR coefficient indicate a separation effect between the methods, while negative values indicate
similarities between methods for a given attribute
(Baretta et al. 2005).
Within the CDF1 the methods varied depending on
the heavy metals studied (Table 3). According to the
PDR values within the CDF1, Co, Ni, Pb, and Zn were
the elements that contributed the most to distinguishing between the digestion methods (0.21, 0.27, 0.37,
and 0.17, respectively), and showed excellent potential as indicators (Fig. 3 and Table 5). Pb
showed the highest PDR and was the element that
contributed the most to distinguishing between the
methods. In the CDF2, Ni had the highest PDR
value (0.26) of all the heavy metals analyzed. Cu
was less sensitive, had a lower PDR value, and
contributed less (0.04 and 0.06, respectively) in
the two functions (CDF1 and CDF2) to distinguishing between the areas studied (Table 5).

Fig. 3 Relationship between the first and second discriminant


canonical function (DCF1 and DCF2) on the mean (centroid) of
the standardized canonical coefficients (SCC) for Zn, Ni, Pb, Cr,
Co, and Cu in soils of Mato Grosso and Rondnia, Brazil

Water Air Soil Pollut (2013) 224:1430

Page 9 of 16, 1430

Table 5 Values of the canonical correlation coefficient (r),


standardized canonical coefficient (SCC), and coefficient of
the parallel discrimination rate (PDR) within the first discriminant canonical function (DCF1 aqua regia) and the second
discriminant canonical function (DCF2 EPA 3051) for the
elements Zn, Ni, Pb, Cr, Co, and Cu
Element

DCF 1
r

DCF 2
SCC

PDR

SCC

PDR

Co

1.5

0.1

0.2

1.5

0.1

0.1

Cr

0.1

0.1

0.0

0.2

0.1

0.1

Cu

0.9

0.1

0.0

0.8

0.1

0.1

Ni

0.7

0.3

0.2

0.9

0.2

0.2

Pb

1.0

0.3

0.3

0.4

0.2

0.1

Zn

1.0

0.1

0.1

1.8

0.2

0.5

3.3 Correlations
Correlations between the heavy metal concentrations
in the 19 soil samples and the 34 physical and chemical attributes of those soils are presented in Table 6.
Metal concentrations extracted with AR, which had a
higher extraction power of the pseudo-total concentrations of heavy metals, were used in the correlations.
With the exception of Cr, there was generally a positive correlation between the concentrations of Co, Cu,
Ni, Pb, and Zn (Table 6). Biondi (2010) reported
similar results in a study of soils from Pernambuco,
Brazil, where Cu concentrations were positively correlated with concentrations of Zn (r=0.78; r=0.81),
Co (r=0.81; r=0.76), and Ni (r=0.73; r=0.71) in the
surface and subsurface horizons, respectively (p<
0.001). Co levels also showed positive correlations
with levels of Ni (r=0.72; r=0.77) and Mn (r=0.79;
r=0.92), revealing the geochemical affinity of these
metals.
The content of exchangeable Al only showed a significant correlation with Cr concentrations (0.38*).
Acidity (pH) only showed a significant positive correlation with Co and Zn (0.47*) concentrations and a
significant negative correlation with Cr (0.39*).
These coefficients were relatively low, probably due to
the extraction methodology, since the total concentrations were extracted in a strongly acidic medium, and
significant correlations with pH are more frequent when
considering available concentrations in the most common range of soil pH in agricultural soils (between 5.0
and 6.5). CECe was positively correlated with C content

(0.40*), with base saturation V% (0.82*), and with


aluminum saturation m% (0.65*).
Clay content was positively correlated with contents of all metals, with the highest correlation coefficients for Cu and Pb (> 0.7*). Lee et al. (1997)
studied soils in Oklahoma, USA collected near
the parent material, underlying sedimentary rocks
and concluded that the most influential attribute in the
distribution of heavy metals in the soil profile was the
sum of silt and clay contents. In our study, that sum
was also positively correlated with metal concentrations, independent of extraction method, and showed a
correlation coefficient varying from 0.31 to 0.73.
In general, Fe and Mn oxides were the variables
that showed the strongest correlations with heavy metals. Various authors have noted the importance of iron
oxides as determinants of heavy metal mobility in
soils under a tropical climate (Fontes et al. 2000;
Fontes and Weed 1991; Alloway 1990; Covelo et al.
2007). Oxides have varying capacities for adsorbing
metallic cations. For a given type of oxide, the capacity for adsorbing cations varies with the degree of
crystallization of the oxide, and can vary as the weathering process causes changes in the form of the crystal,
in the surface area, and in the chemical properties of
the surface of the oxides (Yu 1997). In general, poorly
crystallized substances have a large specific surface
area and a high capacity for adsorbing metals. By
contrast, the activity of well-crystallized substances
is comparatively lower (Yu 1997).
Mn oxides and hydroxides are rare in soils and, for
this reason, less studied than Fe oxides. Nonetheless,
they are efficient sorbents of heavy metals because of
their small size and large specific surface area
(McKenzie 1979). Like Fe oxides, Mn oxides are very
stable (i.e., they have low levels of solubility; Stumm
and Morgan 1996), their surfaces are highly reactive,
and they precipitate as tiny poorly crystallized or
amorphous crystals (Fortin et al. 1993; Tessier et al.
1996).
CO concentrations were not correlated with the
concentrations of any metal in this study (p<0.01).
This may be because the concentrations of the other
variables did not vary at the same magnitude as carbon
concentrations. For this reason, CO did not contribute
to explaining variation in metal concentrations and
was excluded from the multivariate analyses.
The PCA was carried out on the data matrix
consisting of 34 variables (physical and chemical

0.624*

ns

0.469*

ns

ns

0.284*

MnO (SA)

Ki

Kr

Fe2O3(OXA)

0.502*

Fe2O3
ns

0.399*

0.619*

0.721*

0.710*

0.333*

0.341*

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

0.542*

0.401* ns

ns

0.405*

0.426*

ns

ns

ns

0.670*

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

0.705*

0.893*

ns

0.880*

ns

ns

0.805*

0.854*

1.00

0.874*

0.978*

ns

0.995*

ns

0.533*

0.829*

1.00

Ca

ns

0.347*

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

0.581*

ns

ns

ns

ns

ns

ns

ns

ns

ns

0.452* 0.609* 0.704*

0.519*

ns

ns

ns

0.364* ns

ns

ns

ns

0.380* 0.400*

ns

ns

ns

ns

0.524* 0.697* 0.697* 0.316* 0.452* 0.731* ns

0.520*

0.444*

0.513*

ns

0.760*

0.402*

ns

0.07
0.15

ns

0.339*

0.484*

0.716*

0.589*

0.536*

ns

0.19

0.649*

ns

0.692*

ns

0.428*

ns

ns

ns

ns

ns

0.725* ns

0.924*

0.768*

0.09

0.824*

ns

0.490* ns

0.432*

ns

0.464*

1.00

ns

ns

ns

ns

ns

1.00

pH

0.628* ns

0.672*

0.834*

0.648*

0.520*

Sand

0.636*

0.333*

0.42

0.383*

ns

0.636*

ns

ns

ns

0.490*

ns

0.357*

0.23

ns

0.354* ns

ns

0.507*

0.377*

0.475*

ns

ns

1.00
ns

0.488*

0.695*

ns

ns

0.500*

ns

ns

0.398* 0.518*

ns

ns

ns

0.377*

0.387*

ns

0.478*

0.525*

ns

ns

pH

Silt

0.681*

0.524*

0.388*

0.322*

ns

0.337* ns

ns

ns

ns

ns

ns

1.00

Cu

0.511*

0,.563*

ns

0.339*

ns

ns

ns

0.524*

0.310*

0.748*

0.450*

0.557*

ns

0.668*

1.00

Co

0.390* 0.478*

ns

ns

1.00

Cr

0.320* 0.450*

ns

0.308*

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

0.628*

0.705*

ns

1.00

Pb

Clay

0.488*

ns

ns

Al2O3(OXA)

(CBD)

ns

Mn2O3(OXA) 0.790*

0.343*

ns

ns

ns

0.322*

0.425*

0.387*

ns

0.282*

ns

TiO2 (SA)

ns

CECt

ns

Fe2O3 (SA)

ns

SB

ns

0.573*

ns

H + Al

ns

ns

ns

Al

ns

0.524*

ns

Mg

ns

SiO2 (SA)

0.550*

Ca

ns

Al2O3 (SA)

0.499*

ns

ns

0.444*

ns

ns

ns

pH

ns

CO

0.476*

pH

0.780*

0.366* ns

0.718*

Cu

ns

ns

0.857*

Co

0.540*

ns

Cr

ns

1.00

0.485*

0.609*

Pb

CECe

0.559*

Ni

Ni

1.00

Zn

Zn

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

0.828*

0.815*

0.846*

ns

0.875*

ns

0.528*

1.00

Mg

ns

ns

ns

ns

ns

ns

ns

ns

0.600*

0.364*

0.327*
ns

0.463*

ns

ns

ns

0.358*

ns

ns

ns

ns

ns

0.329*

ns

ns

ns

ns

ns

ns

ns

ns

0.717*

0.494*

ns

0.368*
0.632*

ns

ns

1.00
ns

0.584*

H + Al

0.535*

1.00

Al

ns

0.474*

1.00

CECt

ns

ns

ns

ns

ns

0.554*

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

0.421*

ns

0.540*

ns

ns

ns

ns

ns

ns

ns

ns

ns

0.456*

0.735* ns

0.880*

0.982*

ns

1.00

SB

ns

ns

ns

ns

ns

0.537*

ns

ns

ns

ns

ns

ns

ns

ns

0.407*

0.650*

0.829*

1.00

CECe

Table 6 Pearson correlation matrix between natural levels of heavy metals (aqua regia method) and soil attributes, with significant values (p<0.01) marked with an asterisk (*)

1430, Page 10 of 16
Water Air Soil Pollut (2013) 224:1430

ns

ns

ns

ns

ns

ns

0.338* ns

ns

ns

ns

ns

ns

0.340* ns

Fe2O3 (SA)

TiO2( (SA)

MnO (SA)

Ki

Kr

Fe2O3(OXA)

1.00

ns

ns

ns

ns

Fe2O3(CBD)

Clay

Silt

Sand

ns

ns

ns

ns
0.491*

0.737*

0.662*

ns

ns

0.525*

0.747*

0.875*

ns

ns

0.462*

ns

0.530*

0.507*

ns

ns

0.714*

ns

0.485*

ns

ns

1.00

TiO2
(SA)

0.467* ns

0.552*

0.579*

1.00

Fe2O3
(SA)

0.480* ns

ns

0.507*

0.61*

1.00

Al2O3
(SA)

0.370*

0.349*

ns

ns

0.475*

ns

ns

ns

1.00

MnO
(SA)

0.800* 0.732* 0.745* 0.517* ns

0.732*

0.782*

0.660*

ns

ns

0.480*

ns

ns

0.383*

0.336*

0.671*

0.562*

1.00

SiO2
(SA)

ns

ns

ns

ns

ns

ns

ns

ns

1.00

Ki

ns

ns
0.519*

0.356*

ns

ns

ns

ns

1.00

Mn2O

0.314* 0.522*

(OXA)

ns

0.448*

ns

1.00

Fe2O3

0.594*

ns

ns

ns

ns

1.00

Kr

SA sulfuric acid attack, OXA ammonium oxalate, CBD sodium citratebicarbonatedithionite, ns not significant

ns

ns

ns

ns

ns

ns

Al2O3(OXA)

ns

0.464* ns

ns

ns

ns

Mn2O3(OXA) 0.644*

ns

ns

ns

ns

Al2O3 (SA)

ns

ns

ns

ns

SiO2 (SA)

0.841* 1.00

CO

CO

1.00

Table 6 (continued)
3(OXA)

(OXA)

0.539*

0.339*

0.545*

ns

1.00

Al2O3
(CBD)

0.694*

0.525*

0.692*

1.00

Fe2O3

1.00

Silt

Sand

0.996* 0.754* 1.00

0.700*

1.00

Clay

Water Air Soil Pollut (2013) 224:1430


Page 11 of 16, 1430

1430, Page 12 of 16
Fig. 4 Principal components analysis of variables
related to physical and
chemical properties of soils
from Mato Grosso and
Rondnia, Brazil, for
extractions performed with
aqua regia (a) and EPA 3051
(b)

Water Air Soil Pollut (2013) 224:1430

(a)

(b)

attributes and the concentrations of heavy metals)


and 19 soil samples, with two principal components. The primary variables (Co, Cr, Cu, Ni, Pb,

sand, P, Ca, Al saturation, total, free, and amorphous Fe2O3, amorphous Al2O3 and Mn2O3 and
total TiO2 for the EPA 3051 method, plus Ki and

Water Air Soil Pollut (2013) 224:1430

silt for the AR method) were tested for possible


multicolinearity based on the inflation values between data and were selected by the Forward
Selection option in CANOCO software, in order
to restrict the analysis to the primary correlated
explanatory and response variables. The heavy
metal variables were used as response variables,
while the physical and chemical soil attribute variables were considered explanatory variables.
Principal component 1 (PC1) explained 21 % of
the variation in the data, while principal component 2 (PC2) explained 68 %, for a total variation
in the analyzed attributes of 75 % for the AR
method (Fig. 4a). For the EPA 3051 method, total
variation of the attributes was 77 %, with PC1
explaining 17 % and PC2 explaining 74 % of
the variation (Fig. 4b).
In the PCA, the soil attributes are represented
by arrows which indicate the direction of the maximum gradient and whose length is proportional to
the correlation of the attribute with the axes and
its importance in explaining the variance projected
in each axis (Theodoro et al. 2003). Cr content
had an especially strong relationship with PC1,
showing a positive eigenvector together with the
contents of Ni and Fe, Al, and Ti oxides, followed
by the contents of Cu, Co, and Pb, Mn oxides,
and Ca and P, which exhibited negative eigenvectors. Contents of sand also had a clear influence
on PC1, with largest absolute value of the negative
eigenvector. The influence of the sand fraction was
the opposite of that of the clay variable, given that
these variables are inversely related, and this could
have potentially resulted in an ill-conditioned matrix. For this reason, the Forward Selection command excluded clay from the results matrix to
reduce the redundancy between those variables.
In the PC2, Al saturation (m%) had a positive
eigenvector for the EPA 3051 method. Patterns
observed in the AR method was very similar to
those observed in the EPA 3051 method, with a
change in the correlation axis. Cr content showed
a strong correlation with PC2, showing a positive
eigenvector together with Ni and Cu contents and
Fe, Al, and Ti oxides, followed by Co and Pb
contents and Mn and Ca oxides, which with largest absolute value of the negative eigenvector.
There was an inverse relationship between the Cr
variable and the Pb, Co, and Mn oxide variables

Page 13 of 16, 1430

(extracted via ammonium oxalate) for both methods,


with the highest Cr contents corresponding to the
lowest percentages of Mn oxide in soils. There was
no relationship between the Cr and Ki variables for the
AR method.
In general, oxides had a strong influence on
variation in the components, thereby highlighting
the association between various heavy metals and
Fe, Mn, and Ti oxides. This result also reflects
the geochemical affinities among elements, which
is in agreement with the geochemical classification proposed by Goldschmidt (1958), which
groups chemical elements based on their phases.
Amorphous Fe oxides interacted strongly with Cr,
total Fe oxides with Ni, and free Fe oxides with
Cu, for both methods.

4 Conclusions

Choosing a digestion method for soil samples for


heavy metals extraction is an important step in
environmental studies and soil pollution. AR and
EPA 3051 are established methods of extracting
the pseudo-total metal contents in soil. The two
methods performed differently in the recovery of
Co, Ni, Pb and Zn metals from soils, with AR
extracting larger quantities of these elements.
Concentrations of extracted Cr and Cu did not
differ between the methods.
According to a CDA, CDF1 (AR) accounted for
all of the variation in the data presented. The
elements Co, Ni, Pb, and Zn were the most effective at distinguishing between the digestion methods used and showed good promise as indicators.
In general, Fe and Mn oxide levels were the
variables most strongly correlated with heavy
metal concentrations.
Organic carbon content did not account for
any variation in metal concentrations, and
was not significantly correlated with any of
the variables.

Acknowledgments To Brazils National Council for Scientific


and Technological Development (CNPq) for granting a masters
degree scholarship to the first author and a Research Productivity Grant to the second author, and for financial support of the
project. To Prof. Carlos Clemente Cerri, of the Center for
Nuclear Energy in Agriculture of the University of So Paulo,
who kindly provided soil samples.

1430, Page 14 of 16

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