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Electron paramagnetic resonance and thermo-gravimetric characterization of humic acids in nutrient-rich soils from the raposa-serra do sol Indian reserve, Roraima, Brazil/ Caracterizacion de acidos humicos de suelos eutroficos en la reserva indigena del flechal, Roraima, Brasil, por resonancia electronica paramagnetica y termogravimetria/Caracterizacao de acidos humicos de solos eutroficos da area indigena do flechal, Roraima, Brasil....


Little is known about the organic matter status in nutrientrich eultivated soil in Amazonia since most studies dealt with slash-and-burn nutrient-poor soil. In Roraima (north Amazonia) Maeuxi Indians have cultivated food crops (maize, manihoc and beans) on nutrient-rich Alfisols for many years in the Flexal indian community. The attributes of humic acids fraction from Chernosols under different land uses in the Raposa Serra do Sol Indian reserve were characterized. The humic substances were extracted according to procedures recommended by the International Humic Substances Society and characterized by means of elemental analysis, thermogravimetry, and electron paramagnetic resonance (EPR). The cultivated Chernosol, under long-term burning practices, showed increasing production of oligomers of humic acids, indicated by the largest concentration of semiquinone-type free radicals. High fertility uncultivated soils in fallows showed less concentration of semiquinone-type free radicals compared with cultivated soils and less fertile soils from the same area. Positive correlations were obtained between the values of semiquinone-type free radicals concentration and the thermogravimetry indexes for temperature >350[degrees]C. The most weathered soils and those under more intense land use showed an overall higher degree of humification.


Poco es conocido sobre el estado de la materia organica en los suelos cultivados que presentan alta fertilidad de la Amazonia, eonsiderando que la mayoria de los trabajos se han desarrollado en tierras de fertilidad baja en los sistemas de agricultura de tala y quema. En Roraima los indigenas Macuxi han desarrollado cultivos en Nitosoles, Chernosoles y Cambisoles de fertilidad alta durante muchos anos, en la comunidad flexal. Esta practica puede provocar alteraciones en las substancias humicas de los horizontes Chernozemicos en diferentes sistemas de uso del suelo en la Reserva Indigena Raposa Serra do Sol. Las sustancias humicas se extrajeron de acuerdo con los procedimientos recomendados por la Sociedad Internacional de Sustancias Humicas y fueron caracterizadas usando analisis elemental, termogravimetria y resonancia electronica paramagnetiea. El Chernosuelo cultivado, con quemas de largo historial, mostro incremento de la polimerizacion de acidos humicos, indicada por una mayor concentracion de radicales libre del tipo semiquinona. Los suelos no cultivados y de buena fertilidad mostraron menor concentracion de radicales libre del tipo semiquinona comparado con los suelos de mas haja fertilidad y los suelos cultivados. Correlaciones positivas fueron obtenidas entre la concentracion de radicales libres y los indices termogravimetricos en temperaturas >350[degrees]C. Los suelos mas intemperizados y con mayor intensidad de uso presentaron mayor grado de humificacion.


Pouco e conhecido sobre o comportamento da materia organica nos solos cultivados da Amazonia que apresentam alta fertilidade, considerando que na sua maioria, os trabalhos tem sido desenvolvidos em solos de baixa fertilidade em sistemas de agricultura de derruba e queima. Em Roraima os indios Macuxi desenvolvem seus cultivos (milho, mandioca, feijao) em Nitossolos, Chernossolos e Cambissolos de alta fertiIidade por muitos anos, na comunidade indigena do Flexal. Esta pratica pode provocar alteracoes nas fracoes humicas de horizontes chernozemicos em diferentes sistemas de uso do solo na area indigena Raposa Serra do Sol. As substancias humicas foram extraidas de acordo com procedimentos recomendados pela Sociedade Internacional de Substancias Humicas e caracterizadas usando analise elementar, termogravimetria e ressonancia paramagnetica de eletrons. O Chernossolo cultivado, com queimar de restos culturais, mostrou incremento da polimerizacao de acidos humicos, indicada pelos maiores valores na concentracao de radicals livres do tipo semiquinona. Os solos nao cultivados e de melhor fertilidade mostraram menor concentracao de radicals livres do tipo semiquinona comparado com solos de mais baixa fertilidade e solos cultivados. Foram obtidas correlacoes positivas entre os valores de concentracao de radicals livres e os indices thermogravimetricos em temperatura >350[degrees]C. Os solos mais intemperizados e com maior intensidade de uso mostraram maior grau de humificacao da fracao acido humico.

KEYWORDS / Amazon / Humic Substances / Slash and Burn Agriculture / Soil /

Received: 06/22/2010. Modified: 04/21/2011. Accepted: 05/02/2011.


Soil management practices can affect the natural ecosystem equilibrium, modifying the organic compounds both in quality and amount, resulting in different chemical and physical dynamics from the original conditions, with positive or negative alterations (Stevenson, 1994). Changes in the natural ecosystems depend on anthropogenic actions such as burning and cultivation, and can affect the soil nutrient cycling and soil fertility (Cerri et al., 1996), even in the indigenous areas. These changes, in turn, will also affect the composition of soil organic matter, notably the humic substances (Melo, 2002; Melo et al., 2010). The changes can further lead to significant modifications in pedoclimate, enhancing C[O.sub.2] emissions (Fearnside, 1997), depending on the magnitude of the impacts.

Humic substances (HS) are natural organic compounds that influence the physical, chemical and biological soil properties (Stevenson, 1994). They represent important carbon and nutrients stocks for plants (Mangrich, 2001) and are characterized by various structures and reactions. These HS are separated based on their solubility under different pH values, and can be classified as humin, humic and fulvic acids (Swift, 1985; Camargo et al., 1999). Quantitative and qualitative studies of HS are made through fractionation and purification, by physical and chemical processes, followed by spectroscopic characterization (Stevenson, 1994). Humic acids (HA) are organic eompounds with prevalence of aromatic structures of high complexity that present various different forms in soils and waters (Jonas and Kozler, 1995).

The methods to evaluate the degree of humification of the soil are still up to debate, because there is not a clear definition of the HS structure (Piccolo, 2001). However, HS are usually studied through indirect methods that indicate the occurrence of structural changes during the humification process. Several techniques have been developed to characterize the degree of humification of soil organic matter, such as measurement of the E4/E6 ratio (the ratio of optical absorbance at 465nm and 665nm in aqueous extract; Chen, et al., 1977; Stevenson, 1994), aromatic C content by CP-MAS 13C nuclear magnetic resonance (Preston, 1996; Ykeya et al., 2004), condensed or substituted aromatic rings with a large nelectronic system by UV and visible fluorescente (Milori et al., 2002), and C:H, C:O e C:N ratios (Rosa et al., 2005), amongst others.

Thermogravimetry is based on the identification of the degree of complex formation of humic substances as a function of temperature changes up to combustion (Benites, 2002). This method has been extensively used in the characterization of organic material because of its simplicity. It precludes any special sample pre-treatment, providing information on the thermal behavior and structural properties of HS. Studies regarding the effect of temperature on thermal stability of HS are of particular interest, since thermal pre-treatment has a strong impact on HS water-content, structure, stability, microbial degradability, and solubility (Kolokassidou et al., 2007).

Electron paramagnetic resonance (EPR), a technique of molecular spectroscopy, estimates the degree of humification of organic matter as a function of the concentration of stable semiquinone-type free radicals (Martin-Neto, et al., 1998; Bayer et al., 2002). Complex aromatic structures are believed to stabilize semiquinone free radicals in humic substances (Riffaldi and Sehnitzer, 1972; Senesi, 1990; Stevenson, 1994) in coexistence with carbon-centered "aromatic" radicals (Paul et al., 2006), although contributions from methoxybenzene and nitrogen-associated radicals cannot be excluded (Senesi, 1990).

The present study aimed to characterize the soil humic acid fraction properties of nutrient-rich soils under different land use systems by aborigine inhabitants of Roraima, Amazonia, by means of combined studies of thermogravimetry, EPR and elemental analysis.

Materials and Methods

Study area and soil sampling

The studied area is located at 'Maloea do Flechal,' one of the main indian communities of the Raposa Serra do Sol indigenous reserve, Northeastern Roraima, Brazil, at 4[degrees]35'10"N and 60[degrees]11'25"W (Figure 1).


Five surface soil horizons were selected, all belonging to soils developed from nutrient-rich mafic rocks (Diabase, Pedra Preta Formation) long cultivated by the local indian community, having shifting agriculture, pastureland and fallows. The climate is characterized by well defined wet and dry seasons, a five month dry season and mean rainfall of 1300mm per year (Pinheiro, 1990).

Soils were collected along a toposequence across the main soils developed from mafic rocks. We observed pedological variations according to topography, land use and management applied, and eventually areas with pasture, slash-and-burn agriculture for more than 20 years by local indians, and a 20 year old fallow, were selected. The five soils were classified, according to the Brazilian system of soil classification (soil taxonomy orders in brackets), as: Eutrophic Red Nitosol (Alfisol); Ortic Ebanic Chernosol (Mollisol); Eutrophic Haplic Cambisol (Mollisol), in lowland; Vertic Orthic Ebanic Chernosol (Mollisol); and Eutrophic Haplic Cambisol (Mollisol), in slope relief.

In each type of different land use, soil pits were dug for morphological description, sampling and classification. Fifteen composite surface samples were collected at 0-10cm depth at each profile, from which we obtained a single sample at each site, used to carry out detailed studies of humic substances.

Soil classification

Soils were classified according to the Brazilian Soil Classification System (Embrapa, 2006). Five soil classes were identified, varying mainly in function of relief, as indicated above and shown in Table I.

In the plain and lowest arca, Orthic Ebanic Chernosol occurs, with deeper mollic A horizon richer in organic carbon, being cultivated for >20 years and with annual burning of crop remnants. In the mid-slope (8% slope) Eutrophic Red Nitosol occurs, being under pasture and with a greater weathering degree. In the upslopes, Eutrophic Tb Haplic Cambisols (Mollisol) and vertic Orthic Ebanic Chernosols (Mollisol) occur on steeper slopes under pasture; there, a reference Eutrophic Tb Haplic Cambisol was covered by a 20 year old fallow, formed by secondary forest. All soils studied were eutrophic, with pH >5.7, high base saturation, and had a comparable mollic A horizon (Chernozemic epipedon).

Extraction and purification of HA

The humic acids (HA) were extracted and purified following the recommendations of the International Humic Substances Society (Swift, 1996). HA were extracted with NaOH 0.1molx[l.sup.-1] under [N.sub.2] atmosphere. After shaking for 24h, the material was centrifuged ar 10000g for 30min. The solution was collected and the pH was immediately adjusted to 2.0 with HCl 6mol x [l.sup.-1]. After 18h the fulvic acid fraction was siphoned and discarded. The remaining material was centrifuged at 5000g for 10min and the supernatant was discarded. The precipitate was redissolved in 200ml of NaOH 0.1mol x [l.sup.-1] under [N.sub.2] atmosphere and centrifuged at 10000g for 30min. The solution was collected, and pH was immediately adjusted to 2 with HCl 6mol x [l.sup.-1] .The acidified solution was centrifuged at 5000g for 10min. Precipitated HA was treated twice with 0.5% HF+HCl solution for 24h and again centrifuged at 5000g for 10min. The purified samples were washed with 200ml of HCI 0.01molx[l.sup.-1], centrifuged at 5000g for 10min and transferred to 100ml cellophane bags, dialyzed and lyophilized.

Elemental analysis

The elemental composition of the HA was determined in two replicates using a Perkin Elmer 2400 CHN analyzer. The C, H and N values were corrected for dry ash free weight, using the amount of moisture and ashes obtained by the thermogravimetric analysis. Oxygen was determined from the difference in the corrected data. The H:C, C:N and O:C atomic ratios were then calculated, using the following equations: NS

C:H= (%C/12) / (%H/l)

C:N= (%C/12) / (%N/14)

O:C= (%O/16) / (%C/12)


The thermal decomposition of HA was studied by means of a TGA-50 SHIMADZU thermogravimetric analyzer using 3.3 [+ or -] 0.1mg of sample mass. The initial weight was stabilized at 30[degrees]C and the heating curve was obtained from 5[degrees]C x [min-.sup.-1]to 105[degrees]C, with a holding time of 10min, followed by heating at 5[degrees]C x [min.sup.-1] up to 650[degrees]C. The thermal decomposition values were acquired by a microcomputer using the TA-50 WSI standardized according to Benites (2002).

Electron paramagnetie resonance (EPR)

The EPR spectra were obtained in a Bruker EMX spectrometer operating in the X-band (~9GHz) at room temperature. For quantitative analysis, quartz tubes were filled with freeze-dried HA samples, registering their respective masses for posterior data normalization. To obtain the areas of the signals, the Ix[([DELTA][H.sub.PP]).sup.2] approximation was used (Poole, 1967), where I is the derivative signal intensity and [DELTA][H.sub.PP] is the peak-to-peak line width. To determine the relative concentration of semiquinone-type free radicals (in spin/g of C) the signal area of the HA samples was compared with a standard ('strong pitch') of known spin concentration supplied by the manufacturer. A secondary standard, according to Singer's method (Singer, 1959; Martin-Neto et al., 1998) was also used to detect possible alterations in the Q-values of the EPR cavity. The experimental parameters were 0.2mW microwave power, 100kHz modulation frequency and 1 Gauss modulation amplitude.

Results and Discussion

Electronic paramagnetic resonance

The quantification of semiquinone-type free radicals, as spin/g of C (Table II), provided information about the qualitative evaluation of the HA of the studied soils. The obtained values of spin concentration varied from 0.36 to 12.10x[10.sup.16] spin/g of C, indicating substantial differences among soil types and land management systems. Larger spin concentration in Chernosol and Nitosol, both under cultivation, corroborate the effect of the agricultural practices and soil fertility on the HA quality. These values are lower than those obtained by Rosa et al. (2005) in low-fertility Amazon soils of the Rio Negro basin, demonstrating the influence of the soil chemical attributes in the quality of humic substances, in Amazonia. No EPR data on nutrient-rich soils in the Amazon was found in the literature to allow comparison.

The increasing concentration of semiquinone-type free radicals in the Chernosol under cultivation is consistent with the frequent burning performed just before planting. Long-term cultivation, therefore, systematically increases the HA aromaticity, with greater concentration of semiquinone-type free radicals. This is probably the result of oxidation of the [H.sub.2] from OH in phenol groups, according to Riffaldi and Schnitzer (1972), following burning of plant residues. This, in turn, increases the aromaticity of HA, with a decrease of carboxilic groups from aliphatic structures (Almendros et al., 1992). It is postulated that the formation of such "chernozemic" (mollic) A horizon on nutrient-rich soils is the direct result of burning and cultivation, similarly to the process described for Indian Black Earth from Amazonia (Neves et al., 2003; Cunha et al., 2010).


Aromatic structures are more resistant to mineralization processes and can indicate the presence of "black carbon" in areas with high frequency of fires and high oxidation rates (Haumaier and Zech, 1995). This information allows inferences about the organic matter quality in different environmental conditions and its implications to soil genesis. The uncultivated soils (soils 3, 4 and 5), with higher fertility, showed less spin/g of C, resulting in greater nutrient cycling.

The thermal resistance of HA of surface horizons (Table III) shows different events of decomposition between materials, with well defined phases. The mass losses at temperatures below 350[degrees]C correspond to the decomposition of functional groups of aliphatic structure, while at temperatures >350[degrees]C thermal decomposition of functional aromatic groups occurrs (Mangrich, 2001 and Benites, 2002).

The HA resistance to thermal decomposition indicates composition variations which may occur both in aliphatic and aromatic nuclei. Soil samples 3 and 4, with greater similarity of HA composition, were both nutrient-rich Chernosols with higher CEC and more stable organic matter, with humic substances with a lower degree of polymerization.

The presence of organic structures of high aromaticity was observed in the correlation between the thermogravimetry data and EPR, where a significant positive correlation between the concentration of the semiquinone-type free radicals (spin/g of C) and the thermal decomposition values at >350[degrees]C were detected. A positive correlation between spin/g of C and the percentage of thermal decomposition of HA >350[degrees]C was also obtained (Figure 2), indicating a relation between the resistance to decomposition and complexity of the organic structures in these nutrientrich soils.

Elemental composition

The analysis of the relative contents of C, H, N and O in the elementary composition of HA revealed its chemical composition and quantity. The atomic ratio has been used to indicate the aromaticity and the mineralization degree of soil organic matter. According to Stevenson (1994), larger values of C:H, C:O and C:N atomic ratios are associated with a higher humification degree and content of carbohydrate, amino acids and protein. Carbon values were lower than the mean values reported by Steelink (1985), and only sample 2 was within the range, with greater carbon and nitrogen contents, compared to the other samples. It indicates an increasing humification degree, as postulated by Canellas et al. (1999).


The results indicate that there was a tendency of greater carbon cycling in these Eutrophic soils, retarding humification. This also suggests that in the Chernosols under long-term cultivation and burning, formation of HA with a high polymerization degree was favored (Santos et al., 2001), increasing the stable carbon pool and leading to the formation of mollic (chernozemic) surface horizon by an anthropic effect. Figure 3 reveals a significant correlation between the EPR signal and the C:N ratio. However, there was no correlation between C:H and C:O values and semiquinone-type free radicals concentration. These data are consistent with Rosa et al. (2005), who showed a lack of correlation between semiquinone-type free radicals concentration and such ratios; these authors pointed out the need for complimentary studies with high temperature combustion of humic substances to induce the formation of alkaline carbonates (NaC[O.sub.3]) that are stable to temperatures up to 950[degrees]C (Rosa et al., 2005).


The formation of a mollic (chernozemic) A horizon on eutrophic soils from this part of Northern Amazonia has a strong anthropic influence.

Chernosols under cultivation, with burning of crop remnants, showed increasing polimerization of the humic acid fraction, accompanied by consistent high values of semiquinone-type free radicals, obtained by EPR.

The soils of higher fertility, in fallows, with less anthropogenic pressure and not cultivated at present, showed a smaller concentration of semiquinone-type free radicals.

Positive correlations were obtained between the values of semiquinone-type free radicals concentration and the thermogravimetry indexes in temperatures above 350[degrees]C.

The most weathered soils, and with greater land use intensity, showed a higher degree of humification, observed by EPR and confirmed by thermogravirnetry.


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Valdinar Ferreira Melo. Agronomist. D.Sc. in Soil and Plant Nutrition, Universidade Federal de Vicosa (UFV), Brazil. Professor, Universidade Federal de Roraima (UFRR), Brazil. Address: Department of Soil and Agricul tural Engineering, UFRR. 69310-250, Boa Vista, Roraima, Brazil. e-mail: valdinar@

Carlos Ernesto G. R. Schaefer. Agronomist. Ph.D. in Soil Science, Reading University, UK. Professor, UFV, Brazil.

Sandra Catia Pereira Uchoa. Agronomist. D.Sc. in Soil and Plant Nutrition, UFV, Brazil. Professor, UFRR, Brazil.

Marcelo Luiz Simoes. Physicist. D.Sc. Researcher, Embrapa, Sao Carlos, SP. Brazil.

Ladislau Martin-Neto. Physicist. D.Se. Researcher, Embrapa, Sao Carlos, SP. Brazil.

Jose Frutuoso do Vale Junior. Agronomist. D.Sc. in Soil and Plant Nutrition, UFV, Brazil. Professor, UFRR, Brazil.

                                  CEC    [Ca.sup.2+]
Sample   Soil (a)   [H.sub.2]O    cmol x [kg.sup.-1]

  1      NVe           5.7       12.21      2.63
  2      MEo           6.1       14.84      6.87
  3      CXbe          6.8       14.39      8.69
  4      MEov          7.0       11.88      9.31
  5      CXbe          6.5       10.63      6.75

         [Mg.sup.2+]   [K.sup.+]
                                          P            C
Sample      cmol x [kg.sup.-1]     mg x [kg.sup.-1]    %

  1         2.48         0.10            0.96         2.5
  2         3.16         0.15            2.50         2.4
  3         2.79         0.05            1.33         1.5
  4         0.04         0.05            1.16         2.1
  5         0.37         0.04            1.22         1.9


Sample   Sand   Silt   Clay

  1       19     28     52
  2       49     23     28
  3       34     34     32
  4       42     29     29
  5       37     28     35

(a) NVe: Eutrophic Red Nitosol, MEo: Orthic Ebanic Chernosol,
CXbe: Eutrophic Haplic Tb  Cambisol, MEov: Vertic Orthic Ebanic
Chernosol, CEC: cation exchange capacity.


                    C      H      N       O
Samples   Soils                %                 C:N    C:H    C:O

   1      NVe     29.96   2.85   1.92   65.27   18.21   0.88   0.61
   2      MEo     52.28   3.90   2.82   41.00   21.63   1.12   1.74
   3      CXbe    29.99   3.49   2.98   63.54   11.74   0.72   0.63
   4      MEov    33.32   5.30   4.37   57.01    8.89   0.52   0.78
   5      CXbe    44.33   4.35   3.49   47.83   14.82   0.85   1.99

           Mass        Spin/g
          of C          of C
Samples    (mg)    (x [10.sup.16])

   1       7.891         3.73
   2      15.041        12.10
   3       7.286         0.98
   4       4.118         0.36
   5       9.136         1.12


Samples   Soil   Humidity   Ash


   1      NVe      4.30     0.50
   2      MEo      6.95     0.35
   3      CXbe     4.30     3.40
   4      Meov     2.80     4.40
   5      CXbe     5.10     3.00

Samples                   PPI %                       QM

          105-350[degrees]C   350-650[degrees]C   [degrees]C

   1            24.43               75.07            627
   2            33.24               66.41            647
   3            12.35               84.34            623
   4            50.27               49.72            590
   5            40.37               59.63            597

Median values. PPI: loss of weight by burning, QM: maximal
temperature  for total combustion.
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Author:Melo, Valdinar Ferreira; Schaefer, Carlos Ernesto G.R.; Uchoa, Sandra Catia Pereira; Simoes, Marcelo
Date:Jun 1, 2011
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