Characterization of humic acids from a Brazilian Oxisol under different tillage systems by EPR, 13C...

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Geoderma 118 (2004) 181–190

Characterization of humic acids from a Brazilian Oxisol under

different tillage systems by EPR, 13C NMR, FTIR and

fluorescence spectroscopy

Martha Gonzalez Pereza,b, Ladislau Martin-Netoa,*, Sergio C. Saabb,c,Etelvino H. Novotnya,b, Debora M.B.P. Miloria, Vanderlei S. Bagnatod,

Luiz A. Colnagoa, Wanderley J. Meloe, Heike Knickerf

aEmbrapa Instrumentac�ao Agropecuaria, Rua XV de Novembro, 1452, P.O. Box 741, CEP: 13560-970, Sao Carlos, SP, BrazilbUniversidade de Sao Paulo, Instituto de Quımica de Sao Carlos (IQSC/USP), P.O. Box 780, CEP: 13560-250, Sao Carlos, SP, Brazil

cUniversidade Estadual de Ponta Grossa (UEPG), Departamento de Fısica, Ponta Grossa, PR, BrazildUniversidade de Sao Paulo, Instituto de Fısica de Sao Carlos (IFSC/USP), P.O. Box 369, CEP: 13560-970, Sao Carlos, SP, Brazil

eUniversidade Estadual Paulista (UNESP), Faculdade de Ciencias Agrarias e Veterinarias, Rodovia Carlos Tonanni, km 05,

CEP: 14870-000, Jaboticabal, SP, BrazilfTechnische Universitat Munchen, Department of Soil Science, 85350 Freising-Weihenstephan, Germany

Received 23 April 2002; accepted 1 May 2003

Abstract

The importance of soil organic matter functions is well known, but structural information, chemical composition and

changes induced by anthropogenic factors such as tillage practices are still being researched. In the present paper were

characterized Brazilian humic acids (HAs) from an Oxisol under different treatments: conventional tillage/maize-bare fallow

(CT1); conventional tillage/maize rotation with soybean-bare fallow (CT2); no-till/maize-bare fallow (NT1); no-till/maize

rotation with soybean-bare fallow (NT2); no-till/maize-cajanus (NT3) and no cultivated soil under natural vegetation (NC).

Soil HA samples were analyzed by electron paramagnetic resonance (EPR), solid-state 13C nuclear magnetic resonance

(13C NMR), Fourier transform infra-red (FTIR) and UV-Vis fluorescence spectroscopies and elemental analysis (CHNS).

The FTIR spectra of the HAs were similar for all treatments. The level of semiquinone-type free radical determined from

the EPR spectra was lower for treatments no-till/maize-cajanus (NT3) and noncultivated soil (1.74� 1017 and 1.02� 1017

spins g� 1 HA, respectively), compared with 2.3� 1017 spins g� 1 HA for other soils under cultivation. The percentage of

aromatic carbons determined by 13C NMR also decreases for noncultivated soil to 24%, being around 30% for samples of

the other treatments. The solid-state 13C NMR and EPR spectroscopies showed small differences in chemical composition

of the HA from soils where incorporation of vegetal residues was higher, showing that organic matter (OM) formed in this

cases is less aromatic. The fluorescence intensities were in agreement with the percentage of aromatic carbons, determined

by NMR (r = 0.97 P< 0.01) and with semiquinone content, determined by EPR (r = 0.97 P< 0.01). No important effect due

0016-7061/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0016-7061(03)00192-7

* Corresponding author. Fax: +55-16-272-59-58.

E-mail addresses: [email protected] (M. Gonzalez Perez), [email protected] (L. Martin-Neto).

M. Gonzalez Perez et al. / Geoderma 118 (2004) 181–190182

to tillage system was observed in these areas after 5 years of cultivation. Probably, the studied Oxisol has a high clay

content that offers protection to the clay–Fe–OM complex against strong structural alterations.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Tropical soil; No-till; Organic matter; Humification degree

1. Introduction quality and possible reduction of CO emission are

In tropical soils, the high turnover rates of organic

matter (OM) and a significant loss of soil organic

carbon due to intense mineralization generally lead to

faster soil degradation under conventional tillage

practices than is observed for soils in temperate

climatic zones (Shang and Tiessen, 1997). Tiessen et

al. (1992) reported losses of 30% of organic carbon in

an Oxisol from northeastern Brazil after 6 years of

conventional tillage. According to Silva et al. (1994),

after 5 years of soybean monoculture, organic matter

losses were 76% of the initial OM content in a Red

Yellow Latosol with 15–30% clay content, 41% for a

Red Yellow Latosol with >30% clay content and 80%

for a sandy quartz soil.

Considering the increase of degraded soil cultivat-

ed with conventional tillage in Brazil, the application

of no-till systems has grown exponentially during the

last 5 years (Saturnino and Landers, 1997). Nowa-

days, there is an estimate that more than 14 million ha

are under no-till management in Brazil, accounting

for approximately 25% of its total grain cultivated

area.

Studies conducted with a sandy clay loam Acrisol

from Southern Brazil showed under high-residue

addition and no-till an increase in C and N total

contents (Bayer et al., 2000). Further improvements

in structural stability (Silva and Mielniczuk, 1997a,b),

cation exchange capacity (Testa et al., 1992; Burle et

al., 1997) and microbial activity (Cattelan and Vidor,

1990a,b) were observed. In consequence, those

improvements led to an increase in the yields of maize

(Burle et al., 1997).

In addition to these agricultural benefits, the pos-

sibility of reduced CO2 emission due to C retention in

the soil under no-till and high crop residue addition

could help to mitigate the greenhouse effect (Bayer et

al., 2002). The effects of tillage methods on decom-

position rates of OM and their consequence to soil

2

also influenced by soil type, mainly texture and

mineralogy (Parfitt et al., 1997).

There are many publications reporting quantitative

losses of total organic carbon as consequences of

conventional tillage practices; nevertheless, there are

only a few qualitative studies for Brazilian soils

studying changes of the chemical structures of humic

substances (Martin-Neto et al., 1991; Nascimento et

al., 1992; Dick and Burba, 1999; Dick et al., 1999;

Guimaraes et al., 2001; Bayer et al., 2002).

The use of spectroscopic techniques such as

nuclear magnetic resonance (NMR), electron para-

magnetic resonance (EPR), Fourier transform infra-

red (FTIR) and fluorescence allows identification of

functional groups and molecular structures providing

a better understanding of decomposition pathways of

OM and qualitative alterations induced by manage-

ment practices (Schulten and Schnitzer, 1993; Schul-

ten, 1995; Preston, 1996; Kogel-Knabner, 1997).

This approach was intended to identify possible

structural changes caused by management in soil

humic acids (HAs) from Oxisols under different

tillage practices making use of spectroscopic techni-

ques.

2. Materials and methods

2.1. Humic acids

Humic acids were extracted from the surface

layer (0–5 cm) of an Oxisol, containing 53% clay,

24% silt, 23% sand and 3.6% organic matter

content. The soil material was derived from field

experiments during which different tillage systems

were applied for 5 years. The Oxisols are located in

an area with geographic coordinates 21j15V22US,48j15V18UW and 610 m altitude (city of Jabotica-

bal, State of Sao Paulo, Brazil) in a subtropical

M. Gonzalez Perez et al. / Geoderma 118 (2004) 181–190 183

region with a mean annual temperature of 22 jC,having humid summers and dry winters. The fol-

lowing treatments were selected: conventional till-

age/maize-bare fallow (CT1); conventional tillage/

maize rotation with soybean-bare fallow (CT2); no-

till/maize-bare fallow (NT1); no-till/maize rotation

with soybean-bare fallow (NT2); no-till/maize-caja-

nus (NT3) and no cultivated soil under natural

vegetation (NC). The bare fallow treatment con-

sisted of remaining the soil to rest with residues

of the main crop and newborn weeds. Additional

informations about experiment can be found in

Souza and Melo (2000).

Humic acids were extracted with 0.5 M NaOH

using a soil: solvent ratio of 1:15 (Stevenson, 1994).

After centrifugation, the HAs were separated from the

supernatant by precipitation with 6 M HCl added to

the extract until a pH of 2.0 was reached. The

precipitated HAs were separated by centrifugation

and purified by successive repetitions of the extraction

procedure. The obtained HAs were then dialyzed by

Spectrapor membrane (size exclusion limit, 6000–

8000 D) and finally freeze-dried.

Elemental composition (C, H, N and S) of HAwas

determined using a Fisons Instruments Elemental

Analyzer EA 1110.

2.2. Spectroscopic characterization of humic acids

2.2.1. FTIR

Spectra were recorded on an FTIR Perkin Elmer,

Paragon 1000 PC spectrophotometer. The KBr pellets

were obtained by pressing under 10000 kg cm� 2

during 2 min a mixture of 1 mg HA and 100 mg KBr,

spectroscopy grade (Stevenson, 1994). Spectra were

acquired at 4 cm� 1 resolution, and 64 scans were

averaged to reduce noise.

2.2.2. EPR

The measurements at room temperature were per-

formed in an E-109 Century Varian EPR spectrometer.

The spectrometer was operated in X-band (9 GHz).

The set of measurements for detection of semiquinone-

type free radicals was carried out under the following

experimental conditions: H0 = 0.34 T, m = 9.519 GHz,

DH = 10 mT, modulation amplitude 0.5 G peak to

peak, P= 0.2 mW. An additional set was used to

identify paramagnetic metals complexed with HA.

Those samples were measured using liquid N2 at

temperature of � 160 jC, H0 = 0.25 T, m = 9.14 GHz,

DH = 0.5 T, modulation amplitude 4 G peak to peak,

P= 10 mW, using an E-109 Century Varian EPR

spectrometer, equipped with a Varian E-257 tempera-

ture control accessory. The sample temperature was

measured with a Fluke 2100A digital thermometer

using a copper-constant thermocouple.

The absolute concentration for free radical signals

was measured using a ruby crystal as secondary

standard (Martin-Neto et al., 1991) calibrated with

Varian strong pitch, and the relative area of EPR

signals was calculated by the integration of the second

derivative of the absorption signal.

2.2.3. 13C CP/MAS NMR

Solid-state 13C NMR spectra were obtained at the13C resonance frequency of 100.58 MHz on a Varian

(Unity 400) spectrometer, equipped with a solid Doty

probe. The samples were confined in a zirconium

oxide rotor with an external diameter of 5 mm. The

variable amplitude cross polarization magic angle

spinning technique was applied with a contact time

of 1 ms, a spinning speed of 8 kHz and a pulse delay

of 1 s (Peersen et al., 1993; Knicker, 2000; Knicker

and Skjemstad, 2000).

In order to distinguish between protonated and

non-protonated carbons, the technique of dipolar

dephasing (DD) was applied (Preston, 1996). During

this experiment, a delay time of 70 As is inserted

between the contact time and the acquisition time

during which the decoupler is turned off. Due to

coupling between 13C and neighboring protons, the

signals of 13C species with strong dipolar interactions

will vanish after this delay time, while those of 13C

species with weak dipolar interactions will still be

visible in the spectrum.

2.2.4. Fluorescence

Fluorescence experiments were made with humic

acid aqueous solutions (200 mg l� 1) in a Perkin

Elmer LS 50B luminescence spectrometer using the

following conditions: excitation wavelength at 436

nm; spectral region at 450–650 nm. The humification

degree of HAs was estimated through total area under

emission spectrum centered at 520 nm, as it was

described by Bayer et al. (2002) and Milori et al.

(2002).

Fig. 1. FTIR spectra of HA of maize-bare fallow treatment,

comparing tillage systems: conventional, no-till and noncultivated.


3. Results

3.1. Elemental analysis

Table 1 lists the elemental composition (on an ash-

free basis), the C/N ratio and the atomic H/C and O/C

ratios of the humic acids. The C content of the HAs

ranges from 50% to 54%. The amount of N and H

varies between 4% and 5% and between 3% and 6%,

respectively. The determined elemental composition is

similar to those reported by Schnitzer and Khan

(1978) and Stevenson (1994) for HAs from tropical

soils. These results are also similar to those reported

by Nascimento et al. (1992) and Dick et al. (1999) for

other Brazilian soils.

3.2. FTIR

The FTIR spectra of the HA of treatment maize-

bare fallow comparing tillage systems are shown in

Fig. 1. These spectra are representative of all samples,

so other spectra are not shown. The identification of

absorption bands was done based on data published

by Barancikova et al. (1997), Schnitzer and Khan

(1972, 1978), Silverstein et al. (1991) and Stevenson

(1994). The most important features are broad band at

3400 cm� 1 associated to OH stretch of OH groups,

peak at 2933 cm� 1 due to aliphatic CUH stretching,

shoulder at 1716 cm� 1 attributed to CjO stretching

of COOH and ketones, strong peak at 1650 cm� 1

associated to structural vibrations of aromatic CjC

and antisymmetrical stretching of COO� groups and

1230 cm� 1 CUO stretching and OH bending of

COOH groups. The FTIR spectra were similar for

Table 1

Elemental analysis of HA samples (given as percentages of ash-free

substance basis) from different soils under conventional tillage

(CT), no-till (NT) and noncultivated (NC) areas

Sample Ash

(%)

N

(%)

C

(%)

H

(%)

O

(%)

S

(%)

C/N O/Ca H/Ca

CT1 2.6 4.7 53.9 5.8 35.7 0.5 11.51 0.49 1.29

CT2 1.4 4.6 52.8 5.7 36.4 0.5 11.42 0.52 1.30

NT1 1.6 4.6 52.7 5.6 36.6 0.5 11.55 0.52 1.28

NT2 1.3 4.6 53.0 5.7 36.3 0.4 11.47 0.51 1.29

NT3 1.8 4.5 52.3 5.5 37.2 0.5 11.60 0.53 1.26

NC 6.4 5.3 50.5 3.6 38.3 2.3 9.52 0.57 0.85

a Atomic ratio.

samples obtained from the no-till and the conventional

tillage system. In the FTIR spectrum of noncultivated

soil (NC) (Fig. 1), small peaks that are characteristic

for kaolinite were observed at 3697, 3662 and 3621

cm� 1. The sharp peak at the 1035 cm� 1 is attributed

to SiUO vibrations of clay impurities (Stevenson,

1994; Olk et al., 1999) and indicates the presence of

clay material that was not completely removed during

the purification process (6.35% ash content).

3.3. EPR

Fig. 2a shows a complete EPR spectrum of the

humic acid NT2 that was measured at � 160jC with

the magnetic field centered at 0.25 T. Fig. 2b shows

the spectrum obtained under the same conditions, but

scanning the magnetic field over 0.2–0.4 T to allow a

better resonance line resolution. Fig. 2c represents an

EPR semiquinone-type free radical signal of NT2

sample. The spectra of Fig. 2 obtained for NT2

sample are representative of all HA samples.

In Table 2, the determined g-values and hyperfine

constants (A) for each resonance line are listed. The

EPR spectra show the following lines with different g-

values (Martin-Neto et al., 1991; Mc Bride, 1978;

Senesi and Schnitzer, 1977; Senesi et al., 1987;

Senesi, 1990; Olk et al., 1999): g = 4.3 is assigned

to Fe+ 3 with rhombic symmetry, g-value of 2.003 is

due to semiquinone-type free radical while gz = 2.26

and g? = 2.06 with Azc 17 mT are associated with

Cu+ 2 complexed with oxygen ligands. VO+ 2 com-

Table 2

EPR parameters from HA spectra: semiquinone-type free radical

concentration, g-value and hyperfine constant (A) of identified

metals

Sample Spins* AzCu+ 2

(G)

gzCu+ 2

g?Cu+ 2

g

Fe+ 3

A?VO+ 2

(G)

g?VO+ 2


plexed with oxygen ligands results in g?= 1.98 and

A?c 7.1–7.3 mT. The three principal EPR parame-

ters: concentration of free radicals, width of resonance

line and spectroscopic g-value, were determined from

semiquinone-type free radical spectra. The concentra-

tions of free radicals of each sample are also shown in

CT1 2.21 165 2.28 2.07 4.27 73 1.99

CT2 2.46 170 2.26 2.06 4.27 72 1.98

NT1 2.27 – – 2.06 4.30 72 1.98

NT2 2.56 167 2.26 2.06 4.26 73 1.98

NT3 1.74 – – 2.06 4.30 73 1.97

NC 1.02 – – – 4.25 – –

(– ) Not determined.

(spins*) Spin concentration expressed as spins g� 1 HA (1017).

Table 2. The width of the resonance line is around

0.467 mT and the spectroscopic g-value is around

2.0032 for all HA samples.

The level of semiquinone-type free radicals deter-

mined from the EPR spectra was around 2.3� 1017

spins g� 1 for HA from treatments CT1, CT2, NT1

and NT2. The treatments NT3 and noncultivated soil

had 1.74 and 1.02� 1017 spins g� 1 HA, respectively.

3.4. 13C CP/MAS NMR

In Fig. 3a, the 13C CP/MAS NMR spectra of the

HAs from the treatment maize rotation with soybean-

bare fallow are shown. All spectra were similar for

HAs of different treatments and detailed discussions

of the interpretation of NMR chemical shift can be

found elsewhere (Preston, 1996; Stevenson, 1994;

Celi et al., 1997; Skjemstad et al., 1998; Knicker,

2000; Baldock and Skjemstad, 2000). The relative 13C

intensity distributions in these soil HAs as determined

by integration of the various chemical shift areas of

the 13C CP/MAS NMR spectra are given in Table 3.

For all spectra, the relative intensity of the signals

associated to aliphatic C (0–110 ppm) are higher than

Fig. 2. EPR spectra of HA (sample NT2). (a) Experimental

conditions: m= 9.14 GHz, microwave power = 10 mW, temperature

= � 160 jC, modulation amplitude = 0.4 mT, measured centering

magnetic field in 0.25 T and DH= 0.5 T; (b) measured in the same

conditions scanning magnetic field over 0.2–0.4 T; and (c) typical

EPR HA semiquinone-type free radical spectrum. Experimental

conditions: H0 = 0.34 T, DH = 10 mT, m= 9.519 GHz, microwave

power = 0.2 mW, room temperature, modulation amplitude = 0.05

mT.

Fig. 3. (a) CP/MAS 13C NMR spectra of humic acids comparing

tillage systems: conventional, no-till and noncultivated for treatment

maize rotation with soybean-bare fallow, (b) CP/MAS 13C NMR

dipolar-dephased spectra of HA for the same treatment described

in (a).


that determined for the region between 110 and 160

ppm (aromatic C and olefinic C), indicating a higher

aliphatic character of the samples studied here, if

compared to humic acids described in the literature

Table 3

Distribution of 13C in soil humic acids determined by CP/MAS 13C NMR

Percentage distribution of 13C within indicated ppm regions (%)

Sample 0–45 45–65 65–90 90–110 110–143

CT1 21 16 14 7 19

CT2 22 16 14 7 17

NT1 21 16 14 7 19

NT2 21 16 14 7 18

NT3 21 17 14 7 18

NC 26 16 15 7 14

a Aliphaticity (%)=[Aliphatic C peak area (0–110 ppm)]100/[Total peb Aromaticity (%)=[Aromatic C peak area (110–160 ppm)]100/[Total

(Malcom, 1990; Stevenson, 1994; Kogel-Knabner,

1997; Martin et al., 1998). Methoxyl C signals are

expected to appear at 56 ppm and overlap with

intensity derived from N-alkyl that have their chemical

shift region between 46 and 67 ppm (Knicker, 2000).

These signals are intense in all spectra probably due to

the incorporation of lignin and lignin-like components

to the soil (Malcom, 1990; Golchin et al., 1994).

Fig. 3b shows some of the dipolar-dephasing (DD)

NMR spectra of HAs. This experiment allows distin-

guishing between carbons that have strong (protonat-

ed C) or weak (non-protonated C and mobile C)

dipolar interactions with hydrogens. The latter can

be identified in spectra obtained with a dephasing

delay of >70 As. In the DD spectra in Fig. 3b, there

are two peaks at 20 and 32–33 ppm that are most

tentatively assignable to mobile terminal CH3 and

CH2 and quaternary carbon, respectively (Preston,

1996; Baldock et al., 1990; Knicker, 2000; Kogel-

Knabner, 2000).

Compared to the chemical shift region between 46

and 67 ppm of the 13C CP/MAS NMR spectra, there

is a decrease of the signal at 56 ppm at a dephasing

delay of 70 As. This indicates that the intensity of this

region is attributable not only to mobile methoxyl-C,

possibly resulting from lignin residues, but also from

N-alkyl C of protein residues (Preston, 1996; Baldock

et al., 1990; Knicker, 2000).

The small peak at 105 ppm that is observed in all

DD NMR spectra (with exception of sample from

noncultivated soil) together with the peak at 145 ppm,

which is partially resolved from the overlapping lignin

signal, give a strong evidence for the presence of

condensed tannins in the soil samples (Preston, 1996).

A possible source for this tannin may be maize

and aromaticity and aliphaticity degrees

Degree (%)

143–160 160–188 188–230 Aliphatica Aromaticb

7 11 6 70 30

8 11 6 70 30

7 10 6 70 30

7 11 6 70 30

7 10 6 71 29

6 11 5 76 24

ak area (0–160 ppm)].

peak area (0–160 ppm)].


residues that became incorporated into the soil (Wer-

shaw et al., 1996).

The 13C CP/MAS NMR spectra revealed similar

percentages of aromatic carbons (f 30%) for practi-

cally all HA samples in spite of the tillage systems

that were derived (Table 3). However, a decrease of

Fig. 4. Comparison between spectroscopic analysis of HA: (a) EPR

(semiquinone level); (b) 13C NMR (aromaticity degree) and (c)

fluorescence (area of emission spectra with maximum of intensity

around 520 nm, excitation wavelength at 436 nm).

this percentage of aromatic carbons to 24% was

observed for HA from noncultivated area together

with an increase of alkyl C.

3.4.1. Fluorescence

All fluorescence emission spectra were similar and

showed a single broad band extending from 480 to

600 nm (data not shown). The fluorescence areas (Fig.

4) showed a good correlation with the percentage of

aromatic carbons determined by 13C NMR (r = 0.97

P < 0.05) and with the amount of semiquinone-type

free radicals determined by EPR (r = 0.97 P < 0.05).

4. Discussion

The FTIR spectroscopy can be used for the iden-

tification of functional groups and the determination

of their relative distribution; nevertheless, this tech-

nique did not provide any detailed information about

chemical or structural changes in HAs derived from

different tillage systems. This fact was already re-

ported by Schnitzer and Khan (1978) and is in

accordance with the findings of Olk et al. (1999).

They reported that fertilizer treatment and site location

had little impact on the FTIR spectra, although differ-

ences between HA fractions were evident. Haberhauer

et al. (2000) applied nonlinear statistical models to

FTIR spectra for obtaining information about decom-

position stages of soil organic matter. In their study,

the FTIR spectra of a Tropodult soil that was con-

verted from forest to pasture, 11 years ago, also

showed no significant differences.

The percentage of aromatic carbons obtained by

NMR showed a good correlation with the level of

semiquinone-type free radicals measured by means of

EPR (r = 0.92 P < 0.05). HAs of noncultivated soil

(NC) (highest soil carbon content f 60 g kg� 1)

presents the lowest percentage of aromatic carbons

and the lowest level of semiquinone-type free radicals,

indicating that the OM formed in these cases is more

aliphatic than aromatic. Among cultivated soils, both

parameters were the lowest for HA of no-till/maize-

cajanus (NT3) (soil carbon content f 23.20 g kg� 1)

treatment. Both treatments, during which the highest

quantity of crop residues was returned to the soil,

exhibit the highest carbon content. Thus, the effect

of constant accumulation of plant residue was more


important in this soil than the effect of tillage systems

among various factors that influence the humification

process.

Mahieu et al. (1999) also reported that HAs from

cultivated soils have higher concentration of aromatic

C determined by 13CP MAS NMR than HAs from

noncultivated soils.

Bayer et al. (2000) also used EPR to study the

effect of no-till cropping system on the chemical

composition of HA and organo-mineral aggregates

from a sandy clay loam Acrisol in southern Brazil.

The lowest level of semiquinone-type free radicals

was reported for no-till treatment where incorporation

of crop residue and organic C was higher.

Fluorescence results confirmed that this spectrosco-

py can be used for studying structural changes caused

by management. As Senesi et al. (1991) pointed out,

fluorescence properties of HA can provide criteria for

their differentiation and classification. Bayer et al.

(2002) and Milori et al. (2002) also reported that the

emission fluorescence intensity of HA with excitation

in the region of 430 nm can be directly related to

condensation, and they concluded that fluorescence

intensity may be a good parameter for revealing the

humification degree. As can be seen in Fig. 4c, HAs

from soil samples of noncultivated and NT3 treatments

(that had higher incorporation of plant residues)

showed a decrease in the emission fluorescence area,

indicating a lower concentration of condense aromatic

carbon (Milori et al., 2002; Bayer et al., 2002), in

agreement also with our 13C NMR and EPR data.

In no-till system, plowing is not applied for the

incorporation of crop residues into the soil, favoring

the continuous accumulation of plant residues at the

surface. Therefore, for soil microorganisms, ‘‘fresh’’

crop residues are always available at soil surface,

sometimes in quantities higher than their capacity to

metabolize them. This leads to a great input of

metabolizable organic compounds into the soil organ-

ic matter (Bayer et al., 2002). Thus, the OM formed

will be rich in aliphatic compounds with lower semi-

quinone-type free radical concentration and lower

percentage of aromatic carbons. Mahieu et al. (2002)

reported similar results: with increasing crop residues

addition, the original soil OM appears to become

diluted by additions of incompletely decomposed crop

residues resulting in an OM with lower concentration

of semiquinone-type free radicals.

In our study, we observed that aliphatic portions of

HA are preserved and increase from the incorporation

of plant residues, as evidenced in the high aliphaticity

percentage and the low level of semiquinone-type free

radicals of no-till/maize-cajanus and noncultivated

soil treatments. It seems to be that microbial activity

resulted in the synthesis of dominantly alkyl, O-alkyl

and carboxyl carbon as Baldock et al. (1990) pointed

out.

Many studies characterizing the quality and quanti-

ty of soil organic matter demonstrated that a prolonged

time is needed to reverse the effect of decreasing OM

contents caused by conventional tillage practices

(Bayer et al., 2000). The results of our study unveiled

only small changes in the chemical composition of soil

HAs as a consequence of different tillage practices

(conventional and no-till), after 5 years after the

beginning of the field experiment.

For a Brazilian Oxisol after 17 years of a tillage

experiment, Bayer et al. (2000) reported decomposition

rates of k = 0.014 y�1 and 0.012 y� 1 for conventional

tillage and no-till management practices, respectively.

This small change in decomposition rate was attributed

to a high physical stability of organic matter in this soil.

In line with these studies, for an Oxisol under tropical

pastures, Guggenberger et al. (1995) observed that the

content of OM associated with clay particles had no

significant change compared to the alterations in OM

associated to sand and silt particles.

Besides, Bayer et al. (2001) showed the negative

correlation among the decay rates of soil organic matter

and the concentrations of iron oxides and kaolinite

demonstrating the physical stability of soil organic

matter caused by interaction with variable change

minerals. Also in this work, from saturation curves of

the semiquinone-type free radical signal detected by

electron spin spectroscopy, it was suggested strong

interaction of soil organic matter with minerals.

Mahieu et al. (1999) mentioned the phenomenon

termed ‘‘clay protection’’, remarking that the clay

content and cation exchange capacity have a strong

influence on the rate of organic C decomposition and/

or the proportion of decomposition products that are

stabilized in soil.

Our results are probably also associated with soil

type. For an Oxisol with high clay content (f 53%),

where the physical protection of soil organic matter

hinders strong structural alterations, even convention-


al tillage practices cannot destroy the clay–Fe–OM

complex. Soil matrix and minerals protect natural

organic materials in such a way that even after UV

photooxidation, they were still present in soil OM

fraction. Encapsulation into the hydrophobic paraffin-

ic network of some biopolymers and incorporation of

OM within microaggregates could also be acting as

mechanisms of OM protection (Baldock and Skjem-

stad, 2000; Knicker and Skjemstad, 2000).

5. Conclusions

HAs from treatments noncultivated and no-till/

maize-cajanus were less aromatics, as shown by the

strong correlation among obtained 13C NMR, EPR and

fluorescence data. The effect of constant accumulation

of plant residue was more important than the effect of

tillage systems in this kind of Oxisol containing

f 53% clay. After 5 years of experiments, important

structural changes in humic acids were not observed

comparing no-till and conventional tillage systems.

Acknowledgements

The authors are grateful to Dr. Otaciro Rangel

Nascimento for providing the EPR facilities. The

FAPESP (Projects: 97/05356-3, 98/15614-2 and 1998/

14270-8) and PADCT/CNPq (project: 62 0324/98-8),

Brazilian Agencies, are also acknowledged for

financial support. Besides, authors are thankful to

both reviewers, one of them is Dr. Caroline Preston,

by their very helpful suggestions given.

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Characterization of humic acids from a Brazilian Oxisol under different tillage systems by EPR, 13C...

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