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Characterization of Heavy Metal Fractions

in Agricultural Soils by Sequential Extraction
Procedure: The Relationship Between Soil
Properties and Heavy Metal Fractions

Ali Sungur, Mustafa Soylak, Erkan Yilmaz, Selehattin Yilmaz & Hasan Ozcan

To cite this article: Ali Sungur, Mustafa Soylak, Erkan Yilmaz, Selehattin Yilmaz & Hasan
Ozcan (2015) Characterization of Heavy Metal Fractions in Agricultural Soils by Sequential
Extraction Procedure: The Relationship Between Soil Properties and Heavy Metal
Fractions, Soil and Sediment Contamination: An International Journal, 24:1, 1-15, DOI:
10.1080/15320383.2014.907238

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 Soil and Sediment Contamination, 24:1–15, 2015
Copyright © Taylor & Francis Group, LLC
ISSN: 1532-0383 print / 1549-7887 online
DOI: 10.1080/15320383.2014.907238

Characterization of Heavy Metal Fractions

in Agricultural Soils by Sequential Extraction
Procedure: The Relationship Between Soil
Properties and Heavy Metal Fractions
Downloaded by [Canakkale Onsekiz Mart Universitesi] at 10:05 20 November 2015

ALI SUNGUR,1 MUSTAFA SOYLAK,2 ERKAN YILMAZ,2

SELEHATTIN YILMAZ,3 AND HASAN OZCAN1
1
Çanakkale Onsekiz Mart University, Faculty of Agriculture, Department of Soil
Science and Plant Nutrition, Çanakkale, Turkey
2
Erciyes University, Faculty of Sciences, Department of Chemistry, Kayseri,
Turkey
3
Çanakkale Onsekiz Mart University, Faculty of Art and Science, Department of
Chemistry, Çanakkale, Turkey

The present research was conducted to determine heavy metals in agricultural soils from
Çanakkale, Turkey, using a sequential extraction procedure (acid soluble, reducible,
oxidizable, and residual) as proposed by the Community Bureau of Reference (BCR) of
the European Commission. Soil samples were taken from 12 different cultivated sites and
analyzed for Cd, Co, Cr, Cu, Mn, Ni, Pb, and Zn concentrations. The results revealed an
order of Mn > Cd > Pb > Co > Ni > Cu > Zn > Cr for the heavy metals based on the sum
of the first three fractions (acid soluble + reducible + oxidizable). The relationships
between soil properties and each metal fraction were identified through Pearsons’s
correlation analysis. Hierarchical cluster analysis was performed to determine the
behaviors and similarities of metals in each fraction. While Mn, Pb, and Zn exhibited
subjective behaviors in the acid-soluble fraction, Cd, Co, Cu, Cr, and Ni exhibited
similar behaviors with each other.

Keywords Agricultural soil, heavy metal, BCR, sequential extraction

Introduction
Heavy metal pollution in the environment (Duran et al., 2009; 2012; Karczewska, 1996;
Soylak et al., 2008; 2013) is a worldwide problem for agricultural lands (Soylak and
Turkoglu, 1999; Chlopecka et al., 1996). Soil is a dynamic system, resulting from the
weathering of the parent rocks, and is subjected to a number of pollutants from both natural
and anthropogenic sources (Antibachi et al., 2012). In general, soils are exposed to metal
pollutions the most since they are the largest receiving body among the other components
of the ecosystem (Sparks, 2005). Heavy metal pollution in agricultural soils mostly results
from sewage treatment sludge and excessive fertilizer, as well as mine, traffic, and similar

Address correspondence to Mustafa Soylak, Erciyes University, Faculty of Sciences, Department

of Chemistry 38039, Kayseri, Turkey. E-mail: soylak@erciyes.edu.tr

1
 2 A. Sungur et al.

anthropogenic sources (Tuzen et al., 2009; Soylak et al., 2001; Unsal et al., 2013). Total
heavy metal concentrations in soils are also directly related to the soil parent material.
However, the mobility of human-originated metals is higher than that of pedogenic and
lithospheric sources (Kaasalainen and Yli-Halla, 2003; Singani and Ahmadi, 2012). Heavy
metal pollution is considered a chemical pollution. It is resistant against environmental
conditions, easily gets into the food chain, and accumulates in living organisms (O’Connell
et al., 2008; Nannoni et al., 2011; Sprynskyy et al., 2011; Liu et al., 2013).
The methods developed and modified to extract heavy metals from soils are gener-
ally evaluated in two groups: as single-stage extraction methods and sequential extraction
methods (Rauret, 1998; Zimmerman and Weindorf, 2010). The use of total element con-
centration does not represent an optimum approach in environmental impact assessment
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(Antibachi et al., 2012). Therefore, sequential extraction methods are commonly employed
currently for heavy metal concentrations. These procedures allow estimation of trace metal
partitioning among the various forms in which they may appear in the soil (Al-Hwaiti et al.,
2014).
The definition of heavy metal bonds through sequential extractions provides support in
understanding geochemical processes and bioavailability. Such methods also allow source-
based interpretation of heavy metals and provide information about the potential mobility
of heavy metals (Cook and Parker, 2006; Zhang et al., 2010; Dacera and Babel, 2013).
The standard prepared by The European Community Bureau of Reference (BCR) is used
as a standard sequential extraction method to reliably compare the results obtained through
different sequential extraction methods (Usero et al., 1998; Rauret et al., 1999; Soylak
et al., 2004). In the BCR method, currently known as SM&T (the Standards Measurements
and Testing Programme), heavy metal fractions are evaluated as exchangeable and bond to
carbonates, reducible (bound to Fe-Mn oxides), and oxidizable (bound to organic matter
and sulphides). The last step, the non-extractable residual portion, contains the metals
bound to minerals that are soluble in strong acid solutions. The mobility and immobility of
heavy metals, along with their availability, largely depends on the types of binding forms.
In general, mobility and availability decrease in the order of acid-soluble forms > reducible
forms > oxidizable forms > residual forms (Zimmerman and Weiindorf, 2010).
In the present study, single correlation analysis was performed to determine the rela-
tionships between soil physicochemical characteristics and heavy metal fractions extracted
by the BCR sequential extraction method. Also, hierarchical cluster analysis (HCA) was
performed to evaluate the differences and similarities of heavy metals in each fraction.
Therefore, the basic objectives of the current study are: (1) to gather information about
mobility and availability of metals; (2) to determine the relationships between soil physic-
ochemical characteristics and different metal forms; and (3) to evaluate the differences and
similarities of the metals in each fraction.

Materials and Methods

Apparatus
An AnalitikJena novAA-350 flame atomic spectrometer was used for heavy metal analy-
ses. Entire elemental analyses were performed in air-acetylene flame. A Heidalph Model
UNIMAX 2010 shaker, a Nüve Model NF 800 centrifuge, and an inoLab Model WTW pH
meter were used throughout the analyses.
 Sequential Extraction Procedure 3

Reagents
All chemicals used in the experiments were of analytical purity (Merck, Germany). Double-
deionized water was used during the experiments (TKA, GenPure, 18.2 MΩ/cm resistivity).
Plastic and glassware were kept in 1:1 HNO3 overnight, then washed with 1% HNO3 at
room temperature. Stock solutions of analyte elements (SCP SCIENCE, 1000 μg/ml, AA
Standard) were used after suitable dilutions. Standard Reference Material BCR 701 (Lake
Sediment) was used to validate the procedure.

Sampling and Samples Preparation

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Soil samples were taken from 12 different rural sections of Çanakkale, Turkey. All of the
samples were taken from surface layers (0-20 cm) of agricultural lands. Precautions were
taken to prevent the use of metal materials during the sampling. Plastic shovels and plastic
containers were used and samples were securely transferred to laboratories. Samples were
air-dried in plastic trays (20–25◦ C). Then, aggregates were crushed into small particles
with a wooden hammer and sieved through a 2 mm sieve. Those samples were used for soil
texture, pH, and electrical conductivity (EC) analyses. The remaining samples were crushed
into smaller pieces in a mortar, homogenized, and sieved through a 0.5 mm sieve. Then
those samples were used for organic matter, calcium carbonate, and elemental analyses.

Analysis of Some Soil Properties

Soil reaction (pH) was determined in a 1:2.5 (soil:water ratio, w/v) suspension with a pH
meter (Grewelling and Peech, 1960). EC was measured in 1:2.5 (soil:water ratio, w/v)
suspension using an EC-meter (Richards, 1954). Soil organic matter (OM) content was
determined by oxidation of organic matter with dichromate in acid medium (Nelson and
Sommers, 1982). A calcimeter was used to determine calcium carbonate (CaCO3 ) content
of soil samples (Nelson, 1982). Soil particle sizes were determined via the hydrometer
method (Bouyoucos, 1951).

BCR Sequential Extraction Procedure

A sequential extraction scheme was applied to samples to determine the binding forms of
heavy metals in sediment samples. The modified BCR sequential extraction method has
been explained in detail in other studies (Usero et al., 1998; Rauret et al., 1999; Soylak
et al., 2004; Fuentes et al., 2008; Ololade, 2009; Saracoglu et al., 2009). Cd, Co, Cr, Cu,
Fe, Mn, Ni, Pb and Zn contents of different fractions in soil samples were determined using
a modified BCR sequential extraction method.
Pearson’s correlation analysis was performed to determine the relationships between
heavy metal fractions and soil characteristics, while Hierarchical Cluster Analysis (HCA)
was performed to evaluate the differences and similarities in heavy metal behaviors of each
fraction. A single linkage method was used for HCA on the normalized data set via the
correlation distances used as a measure of distance. Statistical analyses were performed
using Minitab-16 software.

Analytical Accuracy and Precision

The reagent blanks were negligible and no detectable contamination was found when
aliquots of the sequential extraction reagents were processed and analyzed with the samples
 4 A. Sungur et al.

Table 1
Detection limits for metals in sequential extraction reagents (n = 3; μg/g)

Hydroxylammonium Ammonium Aqua

Metals Acetic acid chloride acetate regia
Cd 0.004 0.004 0.04 0.09
Co 0.06 0.12 0.06 0.12
Cr 0.03 0.11 0.18 0.18
Cu 0.03 0.05 0.1 0.3
Mn 0.05 0.06 0.03 0.1
Ni 0.004 0.02 0.1 0.32
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Pb 0.06 0.36 0.19 0.14

Zn 0.3 0.47 0.18 0.23

(Davidson et al., 1998). Detection limits (three times the standard deviation of the blank,
divided by the slope of the calibration graph) are given in Table 1.
BCR 701 (Lake Sediment) validates the BCR sequential extraction procedure. Valida-
tion results are presented in Table 2. The recovery rates for heavy metals in the standard
reference material for acid soluble fraction were between 89.6-96%. The value was be-
tween 94–104.7% for the reducible fraction, and between 90.5–103.3% for the oxidizable
fraction. Recovery rates were found to be satisfactory for entire fractions.
Recovery rates were calculated for the results obtained from BCR extractions of each
element as follows:
Recovery% = [(Fraction 1 + Fraction 2 + Fraction 3 + Residual) / Pseudo total] ×
100
The recovery rate was calculated as 99.25% for Cd, 92.72% for Co, 95.29% for Cr,
96.37% for Cu, 98.57% for Mn, 94.42% for Ni, 94.67% for Pb, and 92.86% for Zn. All of
these rates were found to be satisfactory.

Results and Discussion

Descriptive Statistics of Soil Properties

Descriptive statistics for some physicochemical parameters and heavy metal contents of
the analyzed soils are summarized in Table 3. The soil pH value changed from slightly
acidic to mild alkali and was neutral with an average of 7.49. Electrical conductivity values
of the samples varied between 86.8-145.8 μS/cm and an agricultural salinity problem
was not observed. Soil organic matter content was at a medium level and varied between
1.24–4.12% with an average value of 2.31%; CaCO3 contents varied between 0.2-20.25%
with an average value of 4.75% and were classified as moderately calcareous. Clay content
was between 13.70–43.69%, silt content was between 13.05-30.61%, and sand content was
between 35.01-71.12%. Such results indicated varying soil textures among the sampling
sites.
The mean value of heavy metal contents measured in the soils followed a descending
order of Mn > Ni > Cr > Zn > Cu > Pb > Co > Cd. In the present study, heavy metal
contents, except for Cd and Ni, were within an acceptable value for agricultural lands
(Table 3).
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Table 2
Recovery rates (R,%) for various extractable metals (mean, μg/g dry wt ± StDev∗ ) in Certified Reference Material (BCR 701), n = 3

Acid-soluble fraction Reducible fraction Oxidizable fraction

Metals Certified Determined R (%) Certified Determined R (%) Certified Determined R (%)
Cd 7.34 ± 0.35 6.84 ± 0.62 93.19 3.77 ± 0.28 3.60 ± 0.30 95.49 0.27 ± 0.06 0.25 ± 0.08 92.59
Cr 2.26 ± 0.16 2.10 ± 0.22 92.92 45.70 ± 2.00 47.89 ± 3.95 104.79 143.0 ± 7.0 136.8 ± 7.0 95.71
Cu 49.3 ± 1.70 46.83 ± 3.14 94.99 124.0 ± 3.00 119.1 ± 7.14 96.10 55.20 ± 4.00 57.01 ± 5.16 103.28
Ni 15.4 ± 0.90 14.65 ± 1.25 95.13 26.60 ± 1.30 27.25 ± 2.05 102.44 15.30 ± 0.90 14.27 ± 1.28 93.27
Pb 3.18 ± 0.21 2.85 ± 0.33 89.62 126.0 ± 3.00 118.5 ± 6.54 94.08 9.30 ± 2.00 8.42 ± 0.72 90.54
Zn 205.00 ± 6.00 196.8 ± 8.76 96.01 114.0 ± 5.00 109.3 ± 5.47 95.90 45.70 ± 4.00 46.23 ± 4.88 101.16
∗
: Standard deviation.

5
 6 A. Sungur et al.

Table 3
Descriptive statistics for some physicochemical properties and pseudo-total concentrations
of heavy metal in soils, n = 12

Soil properties Mean ± StDev ∗ In agricultural soils∗∗

pH 7.49 ± 0.69 −
EC (μS/cm) 145.80 ± 42.30 −
CaCO3 (%) 4.75 ± 6.93 −
OM (%) 2.31 ± 0.85 −
Clay (%) 33.34 ± 9.97 −
Silt (%) 19.89 ± 5.15 −
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Sand (%) 46.77 ± 9.88 −

Cd (μg/g) 1.48 ± 0.72 0.2–1
Co (μg/g) 9.08 ± 2.96 10
Cr (μg/g) 43.83 ± 11.16 70–100
Cu (μg/g) 22.50 ± 4.98 20–30
Mn (μg/g) 629.80 ± 83.90 1000
Ni (μg/g) 61.11 ± 17.97 50
Pb (μg/g) 19.66 ± 1.93 10–30
Zn (μg/g) 41.24 ± 7.52 50
∗
: Standard deviation.
∗∗
: Common value in agricultural soil (Alloway, 1990).

Application of Sequential Extraction Scheme

The behavior of heavy metals through the BCR sequential extraction scheme is provided
in Table 4. Figure 1 presents the average amounts of heavy metal fractions obtained by the
BCR sequential extraction method. The fact that entire heavy metals existed in different
amounts in different fractions is clearly seen in Table 4 and Figure 1. The order of metals
in each fraction was observed as follows:
Acid soluble (F1 ); Cd > Co > Ni > Cu > Mn > Zn > Cr > Pb,
Reducible (F2 ); Mn > Co > Pb > Cd > Zn > Cu > Ni > Cr,
Oxidizable (F3 ); Pb > Cd > Ni > Cr > Co > Cu > Mn > Zn,
Residual (F4 ); Cr > Zn > Cu > Ni > Co > Pb > Cd > Mn.
The first two fractions (acid-soluble and reducible) constitute a more available fraction
(Alvarez et al., 2002). Considering the extractable amounts of heavy metals of the present
study, the order of more available (acid soluble + reducible) fractions was as follows: Mn
> Co > Cd > Pb > Ni > Cu > Zn > Cr.
With regard to the extractable amounts of heavy metals, the concentrations of the first
three fractions (acid soluble + reducible + oxidizable) were considered as the mobile
fractions (Rauret, 1998). Average amounts of heavy metal fractions through the BCR
sequential extraction method are presented in Figure 1. The extractability order of the
heavy metals based on the sum of the first three fractions (acid soluble + reducible +
oxidizable) for soil samples were as follows: Mn (68.9%) > Cd (65.9%) > Pb (63.9%) >
Co (58.3%) > Ni (37.6%) > Cu (31.3%) > Zn (25.9%) > Cr (18.2%).
It is observed that the most mobile elements in the soil samples are Mn, Cd, Pb, and
Co, whereas the least mobile elements are Ni, Cu, Zn, and Cr, as can be viewed in Figure 1
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Table 4
Results of BCR sequential extraction for heavy metals in soil samples (μg/g dry wt mean ± StDev∗ ) n = 12

Fractions Sum

Acid-soluble (F1 ) Reducible (F2 ) Oxidizable (F3 ) Residual (F4 )  (F1 +F2 +F3 +F4 )

Mean ± Mean ± Mean ± Mean ± Mean ±

Metals StDev %Ratio StDev %Ratio StDev %Ratio StDev %Ratio StDev %Ratio
Cd 0.30 ± 0.32 20.59 0.36 ± 0.21 24.53 0.30 ± 0.27 20.80 0.50 ± 0.14 34.08 1.46 ± 0.70 100
Co 0.76 ± 0.66 9.11 3.05 ± 1.06 36.43 1.07 ± 0.85 12.78 3.49 ± 1.71 41.68 8.36 ± 2.60 100
Cr 0.49 ± 0.42 1.18 0.93 ± 0.47 2.22 6.18 ± 2.24 14.77 34.25 ± 9.73 81.84 41.85 ± 11.30 100
Cu 1.13 ± 0.53 5.22 3.16 ± 3.00 14.59 2.48 ± 0.65 11.46 14.90 ± 3.22 68.73 21.68 ± 4.66 100
Mn 28.94 ± 11.82 4.68 352.12 ± 57.00 57.00 44.61 ± 7.22 7.22 192.06 ± 58.49 31.09 617.72 ± 69.93 100
Ni 4.56 ± 1.61 7.91 7.30 ± 2.00 12.67 9.84 ± 3.58 17.06 35.96 ± 11.84 62.36 57.66 ± 17.02 100
Pb 0.20 ± 0.19 1.05 5.26 ± 2.78 28.19 6.47 ± 2.02 34.71 6.73 ± 1.45 36.05 18.65 ± 2.44 100
Zn 1.36 ± 0.81 3.53 6.10 ± 3.79 15.90 2.48 ± 1.10 6.47 28.43 ± 6.53 74.10 38.36 ± 7.51 100
∗
: Standard deviation.

7
 8 A. Sungur et al.
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Figure 1. Relative abundance of metals in each fraction.

and Table 4. Heavy metals in uncontaminated soils and sediments are mainly immobile
in their bonded to silicate and primary minerals forms. Therefore, heavy metals in con-
taminated samples are more mobile and bonded to other phases (Rauret, 1998). Immobile
fractions of Ni, Cu, Zn, and Cr were higher than their mobile fractions. Therefore, they
were considered as lithosphere-originated. On the other hand, Ni was found to be above
the allowable limits for agricultural lands (Table 3). However, the immobile fraction of
Ni was higher than the mobile fraction and therefore Ni in soil samples was considered
to be parent-material-originated (Figure 1 and Table 4). Considering the extractable rates
of heavy metals, we determined that mobile fractions of Mn, Cd, Pb, and Co were higher
than immobile fractions, and human-induced effects (especially agricultural activities)
were considered the main reasons for this. Higher mobility rates and consequently more
available fractions of Mn, Cd, Pb, and Co in F1 and F2 of the present study indicate envi-
ronmental pollution and possible toxicity of these heavy metals, mainly from agricultural
practices.

Relationship between Soil Properties and Different Fractions of Heavy Metals

Pearson correlation analysis was performed in the present study to determine the relation-
ships between soil physicochemical characteristics (pH, organic matter, CaCO3 , and clay
contents) and heavy metal contents of each fraction. The results are presented in Table 5.
Binding forms of heavy metals in different fractions exhibit significant differences with
regard to their bioavailability and chemical activities (Qian et al., 1996; Han et al., 2000;
Adamo et al., 2006). Considering the current results, it was observed that soil pH, CaCO3 ,
and clay contents played dominant roles in different binding forms and types of heavy
metals (Table 5).
The pH is a significant parameter affecting the extraction of metals from soil samples
(Ettler et al., 2007). While there were not any correlations between soil pH and fractional
distribution of Cd and Cu, negative and positive correlations were observed, respectively,
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Table 5
Pearson’s correlation analysis for soil properties and BCR sequential extracted heavy metal concentrations n = 12

Fractions

F1 F2 F3 F4
Elements Soil Characteristics (Acid-soluble) (Reducible) (Oxidizable) (Residual)
Cd pH ns∗ ns ns ns
∗∗
CaCO3 (%) p < 0.01 (r: 0.949) p < 0.01 (r: 0.841)∗∗∗ ns ns
OM (%) ns ns p < 0.05 (r: 0.589) ns
Clay (%) ns ns ns ns
Co pH ns ns p < 0.05 (r: 0.684) p < 0.05 (r: 0.659)
CaCO3 (%) p < 0.01 (r: 0.892) p < 0.01 (r: − 0.690) p < 0.01 (r: 0.714) p < 0.01 (r: 0.866)
OM (%) ns ns ns ns
Clay (%) ns ns ns p < 0.01 (r: 0.718)
Cr pH ns ns ns p < 0.01 (r: 0.829)
CaCO3 (%) p < 0.01 (r: 0.939) ns ns ns
OM (%) ns ns ns ns
Clay (%) ns ns ns p < 0.01 (r: 0.864)
Cu pH ns ns ns ns
CaCO3 (%) p < 0.01 (r: 0.914) ns ns ns
OM (%) ns ns ns ns
Clay (%) ns ns ns ns
Mn pH p < 0.01 (r: − 0.751) ns p < 0.05 (r: 0.673) p < 0.01 (r: 0.797)
CaCO3 (%) ns p < 0.01 (r: − 0.839) ns p < 0.01 (r: 0.714)
OM (%) ns ns ns ns
Clay (%) p < 0.01 (r: − 0.775) ns p < 0.05 (r: 0.668) p < 0.01 (r: 0.855)
(Continued on next page)

9
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10
Table 5
Pearson’s correlation analysis for soil properties and BCR sequential extracted heavy metal concentrations n = 12(Continued)

Fractions

F1 F2 F3 F4
Elements Soil Characteristics (Acid-soluble) (Reducible) (Oxidizable) (Residual)
Ni pH p < 0.05 (r: 0.604) ns p < 0.01 (r: 0.789) p < 0.01 (r: 0.861)
CaCO3 (%) p < 0.01 (r: 0.814) ns ns ns
OM (%) ns p < 0.05 (r: − 0.599) ns ns
Clay (%) p < 0.05 (r: 0.697) ns p < 0.01 (r: 0.832) p < 0.01 (r: 0.915)
Pb pH ns p < 0.01 (r: − 0.778) p < 0.05 (r: 0.660) ns
CaCO3 (%) ns p < 0.01 (r: − 0.830) ns p < 0.01 (r: 0.870)
OM (%) ns ns ns ns
Clay (%) ns p < 0.01 (r: − 0.777) p < 0.01 (r: 0.742) ns
Zn pH ns ns ns p < 0.01 (r: 0.767)
CaCO3 (%) ns p < 0.05 (r: − 0.599) ns ns
OM (%) ns ns ns ns
Clay (%) ns ns ns p < 0.01 (r: 0.871)
∗
ns: not significant.
∗∗
p: significant level (p < 0.01 and p < 0.05 = significant at 99 and 95% confidence level, respectively).
∗∗∗
r: correlation coefficient.
 Sequential Extraction Procedure 11

with Mn and Ni contents of the F1 fraction. On the other hand, a negative relationship
was observed between soil pH value and only Pb content of the F2 fraction and pH had
positive impacts on the binding of the Co, Mn, Ni, Pb, and Zn in the F3 fraction. Similar
positive correlations were also observed between pH and bindings of Co, Cr, Mn, Ni, and
Zn in the F4 fraction. Positive correlations of pH, especially in the less mobile F3 and
immobile F4 fractions, indicated that increasing pH allowed higher bindings of less mobile
forms.
Some metals (like Mn) can easily exchange with Ca in calcareous soils, and calcium
carbonate has a strong impact on metal mobility, creating a strong binding of the metal
(Adriano, 1986). In the present study, a significant positive relationship was observed
between CaCO3 contents of soils and binding of Cd, Co, Cr, Cu, and Ni in the F1 fraction.
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On the other hand, while the relationship of CaCO3 content only with Co of the F3 fraction
was positive, significant correlations of CaCO3 contents were also observed with Co, Mn,
and Pb concentrations of the F4 fraction (Table 5). Positive correlations of CaCO3 with the
metals of the F1 fraction indicate that the metals of the most mobile fraction and included in
the environment (especially from phosphorus/nitrogenous fertilizers and pesticides) were
bonded to carbonated environments.
With regard to clay contents of soil samples and heavy metal concentrations of the
Fı fraction, a negative correlation was observed between clay content and Mn. A positive
correlation was observed between clay content and Ni concentration. On the other hand, a
negative correlation was observed only with Pb of the F2 fraction and a positive correlation
was observed with Mn, Ni, and Pb of the F3 fraction. Positive correlations were also
observed with Co, Cr, Mn, Ni, and Zn concentrations of the F4 fraction (Table 5). Heavy
metals of soil matrices are held over the negatively charged surfaces of colloidal organic
matter and clay particles with an electrostatic attraction (Adriano, 1986; Alloway, 1990).
Therefore, positive correlations between clay contents and metals, especially of the F3 and
F4 fractions, indicate that metals were held over negative surfaces of clay particles and
between clay layers and clay adsorption was a dominant process over metal binding into
the soil matrix.

Hierarchical Cluster Analysis for Heavy Metals in Each Fraction

Hierarchical cluster analysis (HCA) was performed and dendrograms were created to
present the differences and similarities of heavy metals in each fraction. The dendrograms
are presented in Figure 2.
While Mn, Pb, and Zn of the Fı fraction exhibited subjective behaviors, Cd, Co, Cu,
Cr, and Ni exhibited similar behaviors. Such a case may be related to the CaCO3 content
of the soil samples. In Table 5, correlations were observed between clustering metals and
CaCO3 , but a correlation was not observed between CaCO3 and dissimilar metals of Mn,
Pb, and Zn. This reveals that Cd, Co, Cu, Cr, and Ni might be accumulated in calcareous
environments and those metals included into the environment through agricultural practices
exhibited highly similar behaviors in fractions bonded to carbonates.
With regard to HCA results of the F2 fraction, Cd exhibited relatively different behav-
iors from the other metals. Other metals formed two sub-groups, in which Co, Mn, Pb, Zn,
and Cu with similar behaviors were placed into the first group, and dissimilar Ni and Cr
were placed into the second group (Figure 2). As can be inferred from Table 4 and Figure 1,
while Ni and Cr had the least fractional distribution in the reducible fraction, metals of the
other group had relatively higher reducible fractions. Thus, these clusters were considered
as the reflection of metal contents in the reducible fraction. The groups formed in the
 12 A. Sungur et al.
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Figure 2. Dendrograms of hierarchical cluster analysis for metals of each fraction.

present study were mainly due to metal affinity against reduction. Therefore, metals of the
soil matrix, except for Ni and Cr, reduced faster and consequently their solubility increased
under saturated conditions.
Two sub-groups formed by the metals of the F3 fraction are clearly seen in Figure 2.
While Cd, Cu, and Zn were placed into the first group, highly similar Co, Cr, Ni, Pb, and
Mn were placed into the second group. Such similarities and differences were basically
because of metal affinities against organic matter.
With regard to HCA results of the residual fraction, it was observed (as in the F4
(residual) fraction) that Cd exhibited relatively different behaviors from the other metals.
This may be related to human-induced Cd presence in soils rather than in the soil parent
material. Although the other metals exhibited a full similarity, Cr, Cu, Zn, and Ni had
higher similarity rates than Co, Mn, and Pb. In this case, this is related to the soil parent
material and Cr, Cu, Zn, and Ni come mainly from the parent material, together with
human-induced impacts. All of these findings reflect the mobility and possible source of
the metals (Figure 1).
 Sequential Extraction Procedure 13

Conclusion
Although it is beneficial to use total metal concentrations to characterize environmental
pollution, sequential extractions of heavy metals provide detailed information about the
basic reactions controlling metal behaviors. Heavy metal fractions of soils may provide more
information about possible sources of heavy metals and available forms of these metals for
plants. Therefore, in the present study, statistical analyses of the relationships between soil
physical characteristics and heavy metal behaviors through sequential extractions provided
better and reliable data about these metals. The high mobility of Mn, Cd, Pb, and Co and
their greater availability in acid-soluble and reducible fractions revealed that these metals
marked the environmental pollution over agricultural lands and they had great potential to
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get into the food chain through agricultural practices.

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