Application of chemometric methods in the evaluation of chemical

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Geoderma xxx (2009) xxx–xxx
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Application of chemometric methods in the evaluation of chemical and spectroscopic
data on organic matter from Oxisols in sewage sludge applications
Larissa Macedo dos Santos a,b,1, Marcelo Luiz Simões a, Wanderley José de Melo d,
Ladislau Martin-Neto a, Edenir Rodrigues Pereira-Filho c,⁎
a
Embrapa Instrumentação Agropecuária, P.O. Box 741, 13560-970, São Carlos-SP, Brazil
Universidade de São Paulo, Instituto de Química de São Carlos, P.O. Box. 369, 13560-970, São Carlos-SP, Brazil
Universidade Federal de São Carlos, Departamento de Química, P.O. Box. 676, 13565-905, São Carlos-SP, Brazil
d
Universidade Estadual Paulista, Departamento de Tecnologia, Via de Acesso Prof. Paulo Donato Castellane, Km 5, 14884-900, Jaboticabal-SP, Brazil
b
c
a r t i c l e
i n f o
Article history:
Received 3 August 2009
Received in revised form 3 December 2009
Accepted 8 December 2009
Available online xxxx
Keywords:
Chemometric
PCA
Humification degree
Soil organic matter
Sewage sludge
a b s t r a c t
Chemometric methods can contribute to soil research by permitting the extraction of more information from
the data. The aim of this work was to use Principal Component Analysis to evaluate data obtained through
chemical and spectroscopic methods on the changes in the humification process of soil organic matter from
two tropical soils after sewage sludge application. In this case, humic acids extracted from Typic Eutrorthox
and Typic Haplorthox soils with and without sewage sludge application for 7 consecutive years were studied.
The results obtained for all of the samples and methods showed two clusters: samples extracted from the
two soil types. These expected results indicated the textural difference between the two soils was more
significant than the differences between treatments (control and sewage sludge application) or between
depths. In this case, an individual chemometric treatment was made for each type of soil. It was noted that
the characterization of the humic acids extracted from soils with and without sewage sludge application
after 7 consecutive years using several methods supplies important results about changes in the humification
degree of soil organic matter. These important result obtained by Principal Component Analysis justify
further research using these methods to characterize the changes in the humic acids extracted from sewage
sludge-amended soils.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Soil organic matter (SOM) is an essential component of highquality agricultural soils because it affects many soil processes
(Causarano et al., 2008). According to Stevenson (1994), SOM consists
of a mixture of compounds at several stages of decomposition
resulting from the biological degradation of plant and animal residues
and microbial activity, and it can be divided into humic and non-humic
substances.
Humic substances are complicated mixtures of biologically transformed organic debris (Hayes and Clapp, 2001). These substances are
the major components (around 2/3) of the mixture of materials that
comprise SOM and are by far the most abundant organic materials in
the environment.
⁎ Corresponding author. Universidade Federal de São Carlos, Rodovia Washington
Luiz, Km 235, P.O. Box 676, 13565-905, São Carlos-SP, Brazil. Tel.: + 55 16 33518092;
fax: + 55 16 33518350.
E-mail address: [email protected] (E.R. Pereira-Filho).
1
Current address: Universidade Federal de São Carlos, Departamento de Química, P.O.
Box. 676, 13565-905, São Carlos-SP, Brazil.
An important parameter related to the qualitative and quantitative
changes in SOM and humic substances is the degree of humification.
According to Zech et al. (1997), humification is the transformation of
macromorphologically identifiable matter into amorphous compounds,
involving changes that occur in vegetal residues or SOM during the
humification process. Humification has been related to the preferential
oxidation of plant polysaccharides, to the selective preservation of more
recalcitrant organic compounds such as lignins and phenolic structures,
and to the incorporation of organic compounds of microbial origin.
As described in the previous paragraph, SOM and humic substances
are important constituents of soils, and they are an important source of
organic matter in sewage sludge.
Sewage sludge can be used as a fertilizer because of its high N, P,
and organic matter contents, making both a quantitative and
qualitative contribution to the SOM and acting as an organic amendment (Hue, 1988). The impacts of sewage sludge application on SOM
and humic substances have largely been analyzed using chemical and
spectroscopic approaches (Adani and Tambone, 2005; González-Pérez
et al., 2006; Senesi et al., 2007; Brunetti et al., 2007).
Results of the chemical and spectroscopy studies have shown that, in
general, HA-like substances from organic amendments differ from
native soil humic substances due to their lower aromatic and carboxyl
0016-7061/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.geoderma.2009.12.006
Please cite this article as: Santos, L.M., et al., Application of chemometric methods in the evaluation of chemical and spectroscopic data on
organic matter from Oxisols in sewage sludge applications, Geoderma (2009), doi:10.1016/j.geoderma.2009.12.006
ARTICLE IN PRESS
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L.M. Santos et al. / Geoderma xxx (2009) xxx–xxx
group contents and higher aliphatic character and levels of N-containing
and polysaccharide-like structures. During the maturation and stabilization of any organic amendment, organic matter mineralization and
humification occur, and the HA-like substances components undergo
concomitant changes. In particular, the chemical and spectroscopic
characteristics tend to approach those typical of native soil humic
substances, which indicate the occurred partial decomposition of
aliphatic, polypeptidic and polysaccharide-like components and increase of the degrees of aromatic ring polycondensation and polymerization (Senesi and Brunetti, 1996; Chen, 2003).
The chemical and spectroscopic techniques can provide important
information about the composition, functionalities and structural,
chemical and spectroscopic characteristics of SOM and humic
substances, including parameters related to the degree of humification, such as the C/N and C/H atomic ratios as revealed by elemental
composition; the E4/E6 ratio as determined by the absorption of
ultraviolet and visible light (Chen et al., 1977; Stevenson, 1994); the
identification of functional groups as established by Fourier transform
infrared spectroscopy (FTIR) (Schnitzer and Khan, 1972; Silverstein
et al., 1991; Stevenson, 1994); the degrees of aromaticity and
aliphaticity as revealed by solid-state 13C nuclear magnetic resonance
(13C NMR) (Stevenson, 1994; Skjemstad et al., 1998; Baldock and
Skjemstad, 2000); and the semiquinone-type free radical concentration as inferred by electron paramagnetic resonance (EPR) (MartinNeto et al., 1994).
The use of several techniques to characterize the same sample can
quickly generate a large amount of data. Chemometric methods can be
used to properly handle and analyze these data because they allow the
extraction of more information from data obtained through chemical
and spectroscopic techniques (Sena et al., 2000). One of the most
important chemometric methods of data exploration is PCA (Principal
Component Analysis) (Malinowski, 1991), which is based on the
correlations among variables. PCA is an exploratory methodology that
seeks to find similarities or differences among the samples in a dataset
(Panero and da Silva, 2008). PCA is used to visualize an n-dimensional
starting dataset in a smaller number of dimensions called principal
components (PCs), which represent variable combinations describing
the maximal variance of the dataset (Kemsley, 1996). Thus, chemometric methods are used in this work through PCA application,
providing useful data interpretation for a better understanding of the
changes in SOM quality due to sewage sludge application.
The aim of this study was to use chemometric methods and PCA to
evaluate data obtained through chemical and spectroscopic methods
concerning the changes in the humification process of soil organic
matter after sewage sludge applications. Two tropical soils treated
with sewage sludge for seven consecutive years were studied.
at 70% (2.5 Mg ha− 1), and the sewage sludge was incorporated into
the soil by gradation. In the first year of experimentation, the
treatments were: control (without sewage sludge or mineral fertilization) and three rates of sewage sludge (2.5, 5.0 and 10.0 Mg ha− 1
on a dry basis), with five replicates for each treatment. The rate
5.0 Mg ha− 1 was selected to supply the N required by Zea mays,
supposing that (1/3) of this element present in the sewage sludge
would be available to the plants. From the second year on, the control
plots were fertilized with mineral fertilizers according to soil chemical
analysis and the recommendations of Raij et al. (1997). At the
beginning of the fourth year, the rate 2.5 Mg ha− 1 was replaced with
20.0 Mg ha− 1. Consequently, in the seventh year of experimentation,
the accumulated rates of sewage sludge in the treatments were 0, 35.0,
70.0, and 87.5 Mg ha−1 on a dry basis. Sewage sludge was applied
annually to the soil surface and was incorporated to a 0–10 cm depth
by gradation. The furrows were then opened and the mineral fertilizers
and seeds were applied into of them. The crops tested were Z. mays in
the first six years and C. juncea in the seventh. Cultural wastes and
seeds were managed by herbicides.
2. Materials and methods
2.5. UV-visible absorption spectroscopy
2.1. Soil samples
UV-visible absorption spectra were obtained using a Shimadzu
UV-1601 PC spectrometer (Shimadzu, Kyoto, JA). The experiments
were conducted according to the method proposed by Stevenson
(1994). The E4/E6 ratio was calculated by determining the ratio of the
signal heights at 465 nm and 665 nm (Chen et al., 1977).
Soil samples were collected from two tropical soils cultivated with
Crotalaria juncea: a clay soil (61 g kg− 1 clay, 17 g kg− 1 silt; 22 g kg− 1
sandy at a depth of 0 to 60 cm) classified as a Typic Eutrorthox, and a
sandy soil (36 g kg− 1 clay; 4 g kg− 1 silt; 60 g kg− 1 sandy at a depth of
0 to 60 cm) classified as a Typic Haplorthox soil. Samples were taken at
depths of 0–10, 10–20, 20–40, and 40–60 cm in a seven-year field
experiment that began in the agricultural season of 1997/98 to
evaluate sewage sludge application. The experimental area was
located in Jaboticabal, São Paulo State, Brazil (21°15′22″S, 48°15′18″
W and 610 m altitude). The climate is Cwa (subtropical and
mesothermic, with a hot and humid summer, cold and dry winter,
an average annual temperature of 22 °C and average annual rainfall of
about 1400 mm) according to the Köppen climatic classification
(Rolim et al., 2007). Before installation of the field experiment, the
area was ploughed and lime was applied to elevate the basis saturation
2.2. Sewage sludge
Sewage sludge was obtained from the Barueri sewage treatment
plant located in the metropolitan region of São Paulo City, São Paulo
State, Brazil and operated by the Basic Sanitation Company of the
State of São Paulo (SABESP).
2.3. Chemical fractionation
The humic acid (HA) and HA-like substances were extracted from
the soil and sewage sludge samples, respectively, according to the
IHSS methodology (Swift, 1996). Briefly, the method included
extraction with 0.1 mol L− 1 NaOH using a sample: solvent ratio of
1:10. After centrifugation, the HA was separated from the supernatant
by precipitation by adding 6 mol L− 1 HCl to the extract until a pH of
2.0 was reached. The precipitated HA was separated through
centrifugation, purified through dialysis using a Spectrapor membrane (size exclusion limit, 6000–8000 D), and finally freeze-dried.
2.4. Elemental composition (C, N, H and S)
The contents of C, N, H and S in the HA were determined using
elemental analysis with a CE-Instruments EA 1110 (Carlo Erba,
Rodano, Milano, IT). The C/N and C/H atomic ratios were calculated
by determining the ratio between the C and N, and C and H contents,
respectively.
2.6. UV-visible fluorescence spectroscopy
Fluorescence spectra were recorded using a Perkin Elmer LS-50B
luminescence spectrophotometer (Perkin Elmer, New Jersey, USA). The
HA samples were brought to a concentration of 20 mg L− 1 at pH 8 by
diluting them in a solution of 0.05 mol L− 1 NaHCO3 (Milori et al., 2002).
Fluorescence spectra were then recorded in emission and synchronous
scan mode. The emission spectra were measured with excitation at 240
and 465 nm (Zsolnay et al., 1999; Milori et al., 2002). The synchronous
scan was measured with Δλ = 55 nm, which was adapted from Kalbitz's
procedure (Kalbitz et al., 1999). The degree of humification of the HA
Please cite this article as: Santos, L.M., et al., Application of chemometric methods in the evaluation of chemical and spectroscopic data on
organic matter from Oxisols in sewage sludge applications, Geoderma (2009), doi:10.1016/j.geoderma.2009.12.006
ARTICLE IN PRESS
L.M. Santos et al. / Geoderma xxx (2009) xxx–xxx
samples was calculated according to three different procedures: Zsolnay
et al., 1999; Milori et al., 2002; and Kalbitz et al., 1999. Zsolnay et al.
(1999) calculated the degree of humification by determining the ratio
between the area of the last quarter (A4: 560–640 nm) and the area
of the first quarter (A1: 320–400 nm) of the emission spectrum, called
A4/A1. Milori et al. (2002) determined the degree of humification as the
area of the fluorescence spectrum obtained by excitation of the sample
at blue wavelengths, referred to as A465. Kalbitz et al. (1999) calculated
the degree of humification as the ratio between the fluorescence intensity at 460 and 399 nm (I460/I399) in the synchronous scan excitation
mode spectra.
2.7. Electron paramagnetic resonance (EPR) spectroscopy
The EPR spectra were acquired using a Bruker EMX spectrometer
operating at the X-band (9 GHz) at room temperature. The relative area
of semiquinone-type free radicals was obtained using the approximation I × ΔH2 (Poole, 1967), where I is the EPR derivative signal intensity
and ΔH is the peak-to-peak line width. The content of semiquinone-type
free radicals was obtained using a sample with a known free radicals
content acquired from Bruker. A secondary standard according to
Singer's method was also used to detect possible alterations in the
Q-value of the resonant cavity (Singer, 1959; Martin-Neto et al.,
1991). The experimental conditions were: modulation frequency of
100 kHz; microwave power (0.2 mW) in order to avoid semiquinonetype free radical signal saturation, adequate modulation amplitude
(0.1 mT), and a time constant (2.56 ms) of 1/4 of the conversion time to
guarantee no signal deformation by increasing the line width. The
analyses were carried out in duplicate to obtain an intermediate
deviation with at least sixteen sweeps per determination. The results
obtained were corrected for the sample C content (spins (g C)− 1).
2.8. Solid-state
13
C nuclear magnetic resonance (NMR) spectroscopy
The 13C NMR spectra were obtained with a Varian model Unity Inova
spectrometer (Varian, Palo Alto, California, USA) operating at a 13C
resonance frequency of 100 MHz. The cross-polarization (CP) and magicangle spinning (MAS) techniques were applied at 6.4 kHz. The 13C
chemical shift scale was referenced to hexamethyl benzene (HMB),
which has a well-defined resonance at 17.2 ppm. The experimental
parameters were an acquisition time of 0.0128 ms and a contact time of
1.0 ms. All of the integral regions were corrected for the areas of spinning
side bands when they appeared in the spectra. For quantification, the
spectra were divided into different chemical shift regions according to
Stevenson (1994). The chemical shift regions were: 0–45 ppm, alkyl
C; 45–60 ppm, methoxyl and N-alkyl; 60–110 ppm, O-alkyl C; 110–
140 ppm, aromatic C; 140–160 ppm, phenolic C; 160–185 ppm, carboxyl
C; and 185–220 ppm, carbonyl C, all of which were integrated to
determine the relative contribution of the respective C groups to the total
organic carbon C (relative intensity). The degree of aromaticity was
obtained by integrating the spectral regions using the integration routine
of the spectrometer. The following indexes were calculated relating the
respective areas (Stevenson, 1994):
Degree of aromaticity =
Aromatic peak area ð110−160 ppmÞ
× 100
Total peak area ð0−160 ppmÞ
Degree of aliphaticity =
Aliphacity peak area ð0−110 ppmÞ
× 100
Total peak area ð0−160 ppmÞ
2.9. Fourier transform infrared (FTIR) spectroscopy
The FTIR spectra were acquired using a Perkin Elmer model
Spectrum 1000 spectrometer (Perkin Elmer, New Jersey, USA). The
3
experiments were performed using compressed pellets that were
prepared using 1 mg of sample and 100 mg of KBr according to the
method proposed by Stevenson (1994).
2.10. Statistical analyses
The degrees of humification obtained through the elemental
composition (C, N, H and S), UV-visible absorption spectroscopy,
FTIR spectroscopy, 13C NMR spectroscopy, EPR spectroscopy, and UVvisible fluorescence spectroscopy for the HA extracted from the Typic
Eutrorthox and Typic Haplorthox soils were evaluated using PCA. The
PCA analyses were performed using Pirouette software version 4.0
(Infometrix, Seattle, Washington, USA).
3. Results and analyses
Table 1 describes the origin of the HA samples examined in this
study and Table 2 lists the variables obtained by chemical and spectroscopic methods applied.
Chemical methods were used to characterize the progress of
humification, including measurements of the C/H and C/N ratios,
which indicate the aromaticity and the level of organic material
decomposition (Stevenson, 1994; Rosa et al., 2005). According to the
literature (Stevenson, 1994), high values of C/N and C/H atomic ratios
are associated with high degrees of humification due to decreased
acid, carbohydrate, and amino acid/protein content.
Spectroscopic methods were used to characterize the progress of
humification, including measurement of the E4/E6 ratio. The decrease
in the E4/E6 ratio is directly related to the increase in molecular weight
and condensation of aromatic carbons, and aromaticity are inversely
related to the amount of aliphatic groups (Stevenson, 1994). Although
these relationships are widely used for characterization of humification degree, they present some controversies in the literature. Chen
et al. (1977) reported that the E4/E6 ratio is mainly controlled by the
size of humic substances, however, studies of Baes and Bloom (1990)
shown that humic substances do not exhibit the properties of light
scattering and, therefore, the ratio E4/E6 is not controlled by molecular
size. According to Stevenson (1994) and Baes and Bloom (1990) the
ratio E4/E6 is negatively correlated with the degree of condensation or
conjugation of the aromatic rings in humic substances. Thus, this ratio
has shown conflicting results and limitations, suggesting careful with
their use, however it was available in this study due to the fact that
they are often used in literature (Senesi et al., 2007; Saab and MartinNeto, 2007; Brunetti et al., 2007).
The A4/A1, A465 and I460/I399 ratios (Zsolnay et al., 1999; Kalbitz
et al., 1999; Milori et al., 2002), indicate the presence of fluorophores
Table 1
Origin of the humic acid samples studied.
Samples
Soils
Treatments
Depths
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Typic Eutrorthox
Control*
Typic Eutrorthox
Sewage sludge application
Typic Haplorthox
Control*
Typic Haplorthox
Sewage sludge application
0–10 cm
10–20 cm
20–40 cm
40–60 cm
0–10 cm
10–20 cm
20–40 cm
40–60 cm
0–10 cm
10–20 cm
20–40 cm
40–60 cm
0–10 cm
10–20 cm
20–40 cm
40–60 cm
*Control: without sewage sludge.
Please cite this article as: Santos, L.M., et al., Application of chemometric methods in the evaluation of chemical and spectroscopic data on
organic matter from Oxisols in sewage sludge applications, Geoderma (2009), doi:10.1016/j.geoderma.2009.12.006
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Table 2
The variables obtained by chemical and spectroscopic methods.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Variables
Chemical and spectroscopic methods
Content of C
Content of N
Content of H
C/N ratio
C/H ratio
E4/E6 ratio
A4/A1
A465
I460/I399
Semiquinone-type free
radicals
Line width
0–45
45–60
60–110
110–140
140–160
160–185
185–230
Degree of aromaticity
Degree of aliphaticity
400–4000 cm− 1
Elemental analysis
UV-visible light absorption spectroscopy
UV-visible light fluorescence spectroscopy
Electron paramagnetic resonance spectroscopy
Solid-state 13C nuclear magnetic resonance
spectroscopy
Fourier transform infrared spectroscopy
in the HA. According to Milori et al. (2002) these fluorophores are
mainly related to the presence of highly conjugated systems, possibly
in aromatic structures (polycondensed systems), and to the substitution of these structures with oxygen- and/or N-containing functional
groups. Kalbitz et al. (1999), Milori et al. (2002) and Zsolnay et al.
(1999) have shown the potential of UV-visible fluorescence spectroscopy to evaluate the humification degree of organic materials in
solutions of humic substances. Their theoretical basis lies in the
assumption that a shift in the maximum fluorescence intensity from
shorter to longer wavelengths can be attributed to an increase of
aromatic group condensation or an increase in conjugation in these
molecules (Kalbitz et al., 1999); therefore, organic substances that are
more transformed or humified have a higher fluorescence intensity
signal at a lower wavelength and it becomes possible to associate this
signal alteration with chemical alterations in organic compounds and
to follow reactions in soil and water environments. Also, these more
humified materials generally have higher chemical stability, increasing the residence time of organic matter in the environment and
consequently improving the soil structure and fertility (mainly those
aspects associated with the cation exchange capacity) (Schnitzer and
Khan, 1972).
EPR spectroscopy has also been used to characterize the progress of
humification, including measurement of the content of semiquinonetype free radicals and line width (Singer, 1959; Schnitzer and Lévesque,
1979; Martin-Neto et al., 1991; Senesi and Brunetti, 1996; Jerzykiewicz
et al., 1999; Watanabe et al., 2005), which has demonstrated the
molecular properties of SOM related to the humification degree. Several
transformation processes of terrestrial and aquatic organic matter in the
environment are connected with reactions of organic free radicals.
Complex aromatic structures are believed to stabilize semiquinone-type
free radicals in humic substances (Stevenson, 1994) in coexistence with
carbon-centered “aromatic” radicals (Paul et al., 2006), although
contributions from methoxybenzene and N-associated radicals cannot
be excluded. The progress of humification has also been characterized
by FTIR spectroscopy and 13C NMR spectroscopy, which reveal the
chemical structure of HA (Preston, 1996; Kögel-Knabner, 1997;
Pajaczkowska et al., 2003). These methods can provide information
concerning the presence of aromatic and aliphatic groups of HA
(Stevenson, 1994; Jouraiphy et al., 2005).
FTIR absorption bands representing the progress of humidification
were identified using data published by Stevenson (1994). The most
important features of HA are a broad band at 3400 cm− 1 associated with
the OH stretch, a peak at 2933 cm− 1 due to aliphatic C–H stretching, a
shoulder at 1716 cm− 1 attributed to C O stretching of COOH and ketones,
a strong peak at 1650 cm− 1 associated with structural vibrations of
aromatic C C bonds and antisymmetrical stretching of COO− groups, a
band at 1230 cm− 1 attributed to C–O stretching and OH bending of COOH
groups, and an absorption signal at 1030–1035 cm− 1 due to carbohydrates and silicates. This signal can be attributed to deformation of the
carboxylic group C–O by polysaccharides, and the absorption at 500 cm− 1
is normally attributed to the presence of mineral impurities from humic
substances.
The results obtained for HA extracted from Typic Eutrorthox and
Typic Haplorthox soils with and without sewage sludge application
through elemental analysis, UV-visible light absorption spectroscopy,
UV-visible light fluorescence spectroscopy, and EPR spectroscopy are
presented in Table 3.
Table 4 presents the relative 13C intensity distributions in the soil
HA as determined by integration of the various chemical shift areas of
the 13C NMR spectra, the degree of aromaticity, and the degree of
aliphaticity.
Table 3
Concentrations of C, N, and H, C/N and C/H ratios, the indexes A4/A1, A465, and I460/I399, concentration and line width of semiquinone-type free radicals of the humic acids extracted
from Typic Eutrorthox and Typic Haplorthox soils without (control) (1 to 4 and 9 to 12) and with sewage sludge application (5 to 8 and 13 to 16) by elemental analysis, UV-visible
light absorption spectroscopy, UV-visible light fluorescence spectroscopy and electron paramagnetic resonance spectroscopy.
Samples
C
H
N
C/N
C/H
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
48
49
47
47
46
44
45
47
44
45
40
37
43
43
41
41
4
4
5
5
5
5
5
5
3
4
4
4
4
5
4
4
E4/E6
A4/A1
A465
I460/I399
(Arbitrary units)
4
5
5
5
4
4
4
5
4
4
4
3
4
4
4
3
14.0
11.4
11.0
11.0
13.4
12.8
13.1
11.0
12.8
13.1
11.7
14.4
12.5
12.5
12.0
15.9
1.0
1.0
0.8
0.8
0.8
0.7
0.8
0.8
1.2
0.9
0.8
0.8
0.9
0.7
0.9
0.9
5.3
5.7
6.6
9.0
5.4
5.7
5.8
6.9
7.1
6.5
8.9
10
6.4
6.6
8.5
9.5
20
21
25
31
16
17
20
23
34
33
43
42
17
19
31
42
28
29
44
73
27
30
36
54
62
56
89
89
37
36
73
86
1.4
1.3
1.5
1.6
1.2
1.2
1.4
1.5
1.6
1.6
1.7
1.7
1.3
1.3
1.6
1.7
Concentration of
semiquinone-type free radicals
Line width of
semiquinone-type free radicals
Spins g C− 1
Gauss
4.4
7.8
6.3
8.2
3.3
3.6
4.2
7.0
2.3
2.4
1.1
1.6
1.4
1.0
1.2
1.6
4.7
4.2
4.6
4.6
5.1
5.2
5.0
4.7
4.9
4.8
5.1
5.1
5.0
4.9
5.1
3.7
Please cite this article as: Santos, L.M., et al., Application of chemometric methods in the evaluation of chemical and spectroscopic data on
organic matter from Oxisols in sewage sludge applications, Geoderma (2009), doi:10.1016/j.geoderma.2009.12.006
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5
Table 4
Distribution of 13C in humic acids extracted from Typic Eutrorthox and Typic Haplorthox soils without (control) (1 to 4 and 9 to 12) and with sewage sludge application (5 to 8 and 13
to 16), and the degree of aromaticity and aliphaticity determined by solid-state 13C nuclear magnetic resonance spectroscopy.
Samples
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Percentage distribution of
13
C within indicated ppm regions (%)
Degree (%)
0–45
45–60
60–110
110–140
140–160
160–185
185–230
Aromaticity
Aliphaticity
27
24
21
26
25
26
25
22
25
30
31
23
33
35
29
25
11
12
12
13
12
11
12
12
12
12
11
11
12
12
11
10
21
26
29
32
27
28
27
30
27
26
27
31
23
24
27
28
14
18
16
15
16
15
16
16
15
14
13
16
13
12
14
16
5
6
5
4
6
6
6
5
7
6
6
6
6
5
6
6
11
11
12
12
11
10
11
12
10
10
10
10
10
9
10
11
11
4
4
3
3
4
4
4
4
3
3
2
3
2
3
4
25
27
26
23
26
25
26
25
25
23
21
26
21
20
23
26
75
73
74
77
74
75
74
75
76
77
79
74
79
80
77
74
The values of the degree of humification obtained by the techniques
previous described were evaluated using PCA. The use of chemometric
methods can help to extract more information from these data. The
data have been autoscaled to give the same importance to all variables.
Figs. 1 and 2 show the scores and loadings plots, respectively,
obtained through PCA of the chemical and spectroscopic data on the
humification degree of the 16 samples of the HA extracted from Typic
Eutrorthox and Typic Haplorthox soils with and without sewage
sludge application. Principal Component 1 (PC1) and PC2 described
33% and 24% of the total variance, respectively. The scores plot shows
that PC1 presented a separation between the HA extracted from the
Typic Eutrorthox (samples 1 to 8) and Typic Haplorthox (samples 9 to
16) soils (Fig. 1). The loadings plot has information about variables
and is possible to detect some tendencies. The HA extracted from
Typic Eutrorthox (1 to 8 samples) were characterized by high A4/A1,
A465, E4/E6, 0–45 and aliphatic. On the other hand the HA extracted
from Typic Haplorthox were connected to high SFR, C, N, 160–185.
Fig. 3 presents the scores plot of the humification degree obtained
using the chemical and spectroscopic methods for the samples of HA
extracted from Typic Eutrorthox soil in order to observe if the depth
shows a difference in humification degree. PC1 and PC2 described 48%
and 24% of the total variance, respectively. The scores plot shows that
Fig. 1. Scores plot of the 16 samples of humic acids extracted from Typic Eutrorthox and
Typic Haplorthox soils with and without sewage sludge application.
PC1 presented a separation between the depths (the samples 1 and 5,
for example, are located in the same depth profile) and that PC2
presented a separation between the treatments (Fig. 3). A PCA
evaluation with Typic Haplorthox (9 to 16 samples) soils was also
calculated and the results will be discussed in the next section. The
other variables were also evaluated separately and the discussion of
the results is presented in the next section.
4. Discussion and conclusion
The scores plots obtained by PCA of the chemical and spectroscopic
characterization of the 16 samples of HA extracted from the Typic
Eutrorthox and Typic Haplorthox soils showed that PC1 describes
around 33% of the total variance and is responsible for the separation
between the samples (Fig. 1). In this Figure, an expected clustering is
observed between samples 1 to 8 and samples 9 to 16 from the Typic
Eutrorthox and Typic Haplorthox soils, respectively. This behavior
indicates that the textural difference that exists between the Typic
Eutrorthox (clay, 61%; silt, 17%; sand 22% at a depth of 0 to 60 cm) and
Typic Haplorthox soils (clay, 36%; silt, 4%; sand 60% at a depth of 0 to
60 cm) is more significant than the differences between treatments
Fig. 2. Loadings plot of the 20 variables used to determinate the humification degree of
humic acids extracted from Typic Eutrorthox and Typic Haplorthox soils with and
without sewage sludge application.
Please cite this article as: Santos, L.M., et al., Application of chemometric methods in the evaluation of chemical and spectroscopic data on
organic matter from Oxisols in sewage sludge applications, Geoderma (2009), doi:10.1016/j.geoderma.2009.12.006
ARTICLE IN PRESS
6
L.M. Santos et al. / Geoderma xxx (2009) xxx–xxx
Table 5
Results obtained by PCA.
PCA Plots
Samples Variables Remarks
used in
PCA
1
1 to 16
1–20
Loadings 1 to 16
1–20
2
Scores
1–20
3
Loadings 1 to 8
Scores
9 to 16
Loadings 9 to 16
1–20
1–20
1–20
4
Scores
1 to 8
7
5
Scores
9 to 16
7
6
Scores
1 to 8
8
7
Scores
9 to 16
8
8
Scores
1 to 8
9
9
Scores
9 to 16
9
10
Scores
1 to 8
12–20
11
Scores
9 to 16
12–20
Scores
1 to 8
PC1 (33%) and PC2 (24%). A separation
between the soils was observed.
A cluster between the following variables
was observed: E4/E6, A4/A1, A465, and I460/I399.
PC1 (48%): responsible for the separation
between soil depths. PC2 (24%): responsible
for the separation between treatments.
No clustering between variables was observed.
PC1 (55%) and PC2 (23%).
No clustering between the variables
was observed.
PC1 (97%): responsible for the separation
between soil depths. PC2 (3%).
PC1 (99%): responsible for the separation
between soil depths. PC2 (1%): responsible
for the separation between treatments.
PC1 (71%). PC2 (20%): responsible for the
separation between treatments.
PC1 (70%) and PC2 (12%): responsible for
the separation between treatments.
PC1 (82%): responsible for the separation
between treatments. PC2 (7%).
PC1 (95%): responsible for the separation
between treatments. PC2 (3%).
PC1 (87%): responsible for the separation
between treatments. PC2 (7%).
PC1 (86%): responsible for the separation
between soil depths. PC2 (11%): responsible
for the separation between treatments.
(with and without sewage sludge application) and depths (0–10, 10–
20, 20–40 and 40–60 cm). The textural differences interfere significantly with the humic properties, including with the humification
degree of each type of soil. According to Saggar et al. (1996) and
Baldock and Skjemstad (2000), the stabilization of SOM is directly
related to the soil texture and mineralogy. Mahieu et al. (1999)
mentioned a phenomenon called “clay protection”, remarking that the
clay content and cation exchange capacity have a strong influence on
the rate of organic carbon decomposition and/or the proportion of
decomposition products that are stabilized in soil. Thus, new PCA's
were conducted separately for each soil.
Fig. 3. Scores plot of the humic acids extracted from Typic Euthorthox soil with and
without sewage sludge application.
The loadings plot obtained did not show a separation between the
variables obtained using the various chemical and spectroscopic
methods (Fig. 2). These results underscore the importance of using
several chemical and spectroscopic methods in the study of SOM and
justify further research using these methods.
The scores plot obtained by PCA of the data obtained from the
chemical and spectroscopic methods for the HA extracted from the
Typic Eutrorthox soil (1 to 8 samples) showed that PC1 (48%) was
responsible for the separation between the depths, and that PC2 (24%)
was responsible for the separation between the treatments (Fig. 3).
These results demonstrate that the characterization of the HA
extracted from soils under sewage sludge application by chemical
and spectroscopic methods supplies important results about SOM
dynamics and again justifies further research using these methods
(González-Pérez et al., 2006; Senesi et al., 2007). In the loadings plot,
no clustering was observed between the variables (Table 5).
The other results obtained by PCA for the samples and for the
separate variables and methods are presented in Table 5.
The scores plot obtained by PCA of the data obtained from the
chemical and spectroscopic methods for the HA extracted from the
Typic Haplorthox (9 to 16 samples) soil showed that PC1 and PC2
describe 55% and 23% of the total variance, respectively. PC1 and PC2
created a separation between the soil depths (Table 5). This result is
probably due to the textural differences that exist between the
shallow and deep soil. The results obtained by physical fractionation
from the Typic Haplorthox soil indicated a difference in clay content
between the 0–20 cm and 20–60 cm depths of around 7%. The textural
differences interfere significantly with the detection of the humic
properties, such as the humification degree of the soil. According to
Saggar et al. (1996) and Baldock and Skjemstad (2000) the
stabilization of SOM is directly related to the soil texture and
mineralogy. The loadings plot did not demonstrate clustering
between the variables (Table 5).
The scores plot obtained by PCA of the UV-visible fluorescence
spectroscopy data (Milori et al., 2002) of the HA extracted from the
Typic Eutrorthox soil showed that PC1 and PC2 described 71% and 20%
of the total variance, respectively. Principal Component 2 was
responsible for the separation between the treatments (control and
sewage sludge application; Table 3). Similar results were observed for
the scores plot obtained through PCA using data acquired with the
Milori method for the HA extracted from Typic Haplorthox (Table 5).
This result is from the spectroscopic properties of the sewage sludge
applied to the soils. According to González-Pérez et al. (2006), Senesi
et al. (2007) and Senesi et al. (1991), this can be attributed to the
presence of relatively small molecular components with a low level of
aromatic polycondensation and SOM with a low humification degree
originating from the application of sewage sludge to the soils. Similar
results have been obtained through PCAs of the UV-visible fluorescence (Zsolnay et al., 1999; Kalbitz et al., 1999) and 13C NMR
spectroscopy data (Table 5). These data show the importance of these
methods in evaluating the changes in the humification process of HA
extracted from two tropical soils after sewage sludge application.
From a qualitative point of view, were observed that in soils with
sewage sludge additions the SOM and humic substances generally
have a lower degree of humification than untreated soils indicated
that the residues of the sewage sludge were chemically different from
the native soil organic matter. In addition their incorporation in the
soil altered the overall characteristics of the indigenous organic
matter.
This is a very complex issue that needs further research to be
completely solved. The necessity of new research is also due to the fact
that the degree of humification concept was established with the
consideration that humic substances are formed by macromolecules
with very high chemical stability (Schnitzer and Khan, 1972;
Stevenson, 1994). Now, however, the concept of macromolecule to
humic substances has been seriously questioned (Piccolo, 2002;
Please cite this article as: Santos, L.M., et al., Application of chemometric methods in the evaluation of chemical and spectroscopic data on
organic matter from Oxisols in sewage sludge applications, Geoderma (2009), doi:10.1016/j.geoderma.2009.12.006
ARTICLE IN PRESS
L.M. Santos et al. / Geoderma xxx (2009) xxx–xxx
Simpson, 2002; Simpson et al., 2002) and consequently our
understanding about their dynamic and reactivity in different
environments must be reconsidered. In the present stage of
knowledge, it is considered beneficial to soils including incorporation
of newly formed humic substances into the indigenous SOM, in spite
of the decrease in the degree of humification of HAs in areas under
sewage sludge treatment.
The results obtained through statistical analysis (PCA) confirmed
the significant differences that exist between SOM from the Typic
Eutrorthox (clayey soil) and the Typic Haplorthox (sandy soil) soils.
These results show that the characterization of HA extracted from
soils with and without sewage sludge application for 7 consecutive
years using several methods provides important information about
changes in the humification degree of SOM. These findings justify
further research using these methods to characterize the HA extracted
from sewage sludge-treated soils.
Acknowledgments
The authors thank the Brazilian agencies FAPESP (project: 99/
09133-4), CEPOF (project: 98/14270-8), and CNPq (project: 476350/
2004-2) for financial support.
References
Adani, F., Tambone, F., 2005. Long-term effect of sewage sludge application on soil
humic acids. Chemosphere 60, 1214–1221.
Baes, A.V., Bloom, P.R., 1990. Fulvic acid ultraviolet-visible spectra: influence of solvent
and pH. Soil Sci. Soc. Am. J. 54, 1248–1254.
Baldock, J.A., Skjemstad, J.O., 2000. Role of the soil matrix and minerals in protecting
natural organic materials against biological attack. Org. Geochem. 31, 697–710.
Brunetti, G., Plaza, C., Clapp, C.E., Senesi, N., 2007. Compositional and functional features
of humic acids from organic amendments and amended soils in Minnesota, USA.
Soil Biol. Biochem. 39, 1355–1365.
Causarano, H.J., Franzluebbers, A.J., Shaw, J.N., Reeves, D.W., Raper, R.L., Wood, C.W.,
2008. Soil organic carbon fractions and aggregation in the southern Piedmont and
Coastal Plain. Soil Sci. Soc. Am. J. 72, 221–230.
Chen, Y., 2003. Nuclear magnetic resonance, infra-red and pyrolysis: application of
spectroscopic methodologies to maturity determination of composts. Comp. Sci.
Util. 11, 152–168.
Chen, Y., Senesi, N., Schnitzer, M., 1977. Information provided on humic substances by
E4/E6 ratios. Soil Sci. Soc. Am. J. 41, 352–358.
González-Pérez, M., Martin-Neto, L., Colnago, L.A., Milori, D.M.B.P., Camargo, O.A., Berton,
R., Bettiol, W., 2006. Characterization of humic acids extracted from sewage sludgeamended oxisols by electron paramagnetic resonance. Soil Till. Res. 91, 95–100.
Hayes, M.H.B., Clapp, C.E., 2001. Humic substances: considerations of compositions,
aspects of structure, and environmental influences. Soil Sci. 166, 723–737.
Hue, N.V., 1988. Residual effects of sewage sludge application on plant and soil-profile
chemical composition. Commun. Soil Sci. Plan. 19, 1633–1643.
Jerzykiewicz, M., Drozd, J., Jezierski, A., 1999. Organic radicals and paramagnetic metal
complexes in municipal solid waste composts. An EPR and chemical study.
Chemosphere 92, 253–268.
Jouraiphy, A., Amir, S., Winterton, P., El Gharous, M., Revel, J.-C., Hafidi, M., 2005.
Structural study of the fulvic fraction during composting of activated sludge-plant
matter: elemental analysis, FTIR and 13C NMR. Bioresour. Technol. 99, 1066–1072.
Kalbitz, K., Geyer, W., Geyer, S., 1999. Spectroscopic properties of dissolved humic
substances — a reflection of land use history in a fen area. Biogeochemistry 47, 219–238.
Kemsley, E.K., 1996. Discriminant analysis of high-dimensional data: a comparison of
principal components analysis and partial least squares data reduction methods.
Chemometr. Intell. Lab. 33, 47–61.
Kögel-Knabner, I., 1997. 13C and 15 N spectroscopy as a tool in soil organic matter
studies. Geoderma 80, 243–270.
Mahieu, N., Powlson, D.S., Randall, E.W., 1999. Statistical analysis of published carbon13 CPMAS NMR spectra of soil organic matter. Soil Sci. Soc. Am. J. 63, 307–319.
Malinowski, E.R., 1991. Factor Analysis in Chemistry. John Wiley & Sons, New York.
Martin-Neto, L., Andriulo, A.E., Traguetta, D., 1994. Effects of cultivation on ESR spectra
of organic matter from soil size fractions of a mollisol. Soil Sci. 157, 365–372.
Martin-Neto, L., Nascimento, O.R., Talamoni, J., Poppi, N.R., 1991. EPR of micronutrientshumic substances complexes extracted from a Brazilian soil. Soil Sci. 151, 369–376.
7
Milori, D.M.B.P., Martin-Neto, L., Bayer, C., Mielniczuk, J., Bagnato, V.S., 2002.
Humification degree of soil humic acids determined by fluorescence spectroscopy.
Soil Sci. 167, 739–749.
Pajaczkowska, J., Sulkowska, A., Sulkowski, W.W., Jedrzejczyk, M., 2003. Spectroscopic
study of the humification process during sewage sludge treatment. J. Mol. Struct.
651–653, 141–149.
Panero, F.S., da Silva, H.E.B., 2008. Application of exploratory data analysis for the
characterization of tubular wells of the North of Brazil. Microchem. J. 88, 194–200.
Paul, A., Stosser, R., Zehl, A., Zwirnmann, E., Vogt, R.D., Steinberg, C.E.W., 2006. Nature
and abundance of organic radicals in natural organic matter: effect of pH and
irradiation. Environ. Sci. Technol. 40, 5897–5903.
Piccolo, A., 2002. The supramolecular structure of humic substances: a novel
understanding of humus chemistry and implications in soil science. Adv. Agron.
75, 57–134.
Poole, C.P., 1967. Electron Spin Resonance: A Comprehensive Treatise on Experimental
Techniques. Wiley-Interscience, New York.
Preston, C.M., 1996. Applications of NMR to soil organic matter analysis: history and
prospects. Soil Sci. 161, 144–166.
Raij, B.Van., Cantarella, H., Quaggio, J.A., Furlani, A.M.C., 1997. Recomendações de
Adubação e Calagem para o Estado de São Paulo. Instituto Agronômico, Campinas.
285 pp. (Boletim Técnico 100).
Rolim, G.S., Camargo, M.B.P., Lania, D.G., Moraes, J.F.L., 2007. Climatic classification of
Köppen and Thornthwaite systems and their applicability in the determination of
agroclimatic zoning for the state of São Paulo, Brazil. Bragantia 66, 711–720.
Rosa, A.H., Simões, M.L., Oliveira, L.C., Rocha, J.C., Martin-Neto, L., Milori, D.M.B.P., 2005.
Multimethod study of the degree of humification of humic substances extracted
from different tropical soil profiles in Brazil's Amazonian region. Geoderma 127,
1–10.
Saab, S.C., Martin-Neto, L., 2007. Anéis aromáticos condensados e relação E4/E6: estudo
de ácidos húmicos de gleissolos por RMN de 13C no estado sólido utilizando a
técnica CP/MAS desacoplamento defasado. Quim. Nova 30, 260–263.
Saggar, S., Parshotam, A., Sparling, G.P., Feltham, C.W., Hart, P.B.S., 1996. 14C-labelled
rygrass turnover and residence times in soils varying in clay content and
mineralogy. Soil Biol. Biochem. 28, 1677–1686.
Schnitzer, M., Khan, S.U., 1972. Humic Substances in the Environment. Marcel Dekker,
New York.
Schnitzer, M., Lévesque, M., 1979. Electron spin resonance as a guide to the degree of
humification of peats. Soil Sci. 127, 140–145.
Sena, M.M., Poppi, R., Frighetto, R.T.S., Valarini, P.J., 2000. Avaliação do Uso de Métodos
Quimiométricos em Análise de Solos. Quim. Nova 23, 547–556.
Senesi, N., Plaza, C., Brunetti, G., Polo, A., 2007. A comparative survey of recent results on
humic-like fractions in organic amendments and effects on native soil humic
substances. Soil Biol. Biochem. 39, 1244–1262.
Senesi, N., Brunetti, G., 1996. Chemical and physico-chemical parameters for quality
evaluation of humic substances produced during composting. In: De Bertoldi, M.,
Sequi, P., Lemmes, B., Papi, T. (Eds.), he Science of Composting. Chapman & Hall,
London, UK, pp. 195–212.
Senesi, N., Miano, T.M., Provenzano, M.R., Brunetti, G., 1991. Characterization,
differentiation, and classification of humic substances by spectroscopy. Soil Sci.
152, 259–271.
Silverstein, R.M., Bassler, G.C., Morrill, T.C., 1991. Spectrometric Identification of Organic
Compounds. Wiley, New York.
Simpson, A.J., 2002. Determining the molecular weight, aggregation, structures and
interactions of natural organic matter using diffusion ordered spectroscopy. Magn.
Reson. Chem. 40, S72–S80.
Simpson, A.J., Kingery, W.L., Swaw, D.R., Spraul, M., Humpfer, E., Dvortsak, P., 2002.
Molecular structures and associations of humic substances in the terrestrial
environment. Naturwissenschaften 89, 84–88.
Singer, L.S., 1959. Synthetic ruby as a secondary standard for the measurement of
intensities in Electron Paramagnetic Resonance. J. Appl. Phys. 30, 1463–1464.
Skjemstad, J.O., Janik, L.J., Taylor, J.A., 1998. Non-living soil organic matter: what do we
know about it? Aust. J. Exp. Agric. 38, 67–680.
Stevenson, F.J., 1994. Humus Chemistry: Genesis, Composition, Reactions. John Wiley,
New York.
Swift, R.S., 1996. Organic matter characterization. In: Sparks, O.L. (Ed.), Methods of Soil
Analysis Part 3: Chemical Methods. Madison, Soil Sci. Soc. Am, pp. 1011–1020.
Watanabe, A., McPhail, D.B., Maie, N., Kawasaki, S., Anderson, H.A., Cheshire, M.V., 2005.
Electron spin resonance characteristics of humic acids from a wide range of soil
types. Org. Geochem. 36, 981–990.
Zech, W., Senesi, N., Guggenberger, G., Kaiser, K., Lehmann, J., Miano, T.M., Miltner, A.,
Schroth, G., 1997. Factors controlling humification and mineralization of soil
organic matter in the tropics. Geoderma 79, 117–161.
Zsolnay, A., Baigar, E., Jimenez, B., Steinweg, B., Saccomandi, F., 1999. Differentiating
with fluorescence spectroscopy the sources of dissolved organic matter in soils
subjected to drying. Chemosphere 38, 45–50.
Please cite this article as: Santos, L.M., et al., Application of chemometric methods in the evaluation of chemical and spectroscopic data on
organic matter from Oxisols in sewage sludge applications, Geoderma (2009), doi:10.1016/j.geoderma.2009.12.006