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Biology and Fertility of SoilsCooperating Journal of InternationalSociety of Soil Science ISSN 0178-2762 Biol Fertil SoilsDOI 10.1007/s00374-014-0940-9

Selective extraction of humic acids from ananthropogenic Amazonian dark earth andfrom a chemically oxidized charcoal

Joyce R. Araujo, Braulio S. Archanjo,Katia R. de Souza, Witold Kwapinski,Newton P. S. Falcão, EtelvinoH. Novotny, et al.

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SPECIAL ISSUE

Selective extraction of humic acids from an anthropogenicAmazonian dark earth and from a chemically oxidized charcoal

Joyce R. Araujo & Braulio S. Archanjo & Katia R. de Souza & Witold Kwapinski &Newton P. S. Falcão & Etelvino H. Novotny & Carlos A. Achete

Received: 10 February 2014 /Revised: 30 May 2014 /Accepted: 19 June 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Spectroscopic techniques including X-ray photo-electron spectroscopy (XPS) can identify particular chemicalgroups of humic acids (HA) from “Terra Preta de Índios”(TPI) or Amazonian dark earth, the highly fertile anthropo-genic soil found in the Amazonian region. The high fertilityand resilience of these soils cannot be explained by theirchemically inert pyrogenic C content alone, but the naturalaging of this C generates reactive carboxyl functional groupsattached directly to the recalcitrant polycondensed aromaticbackbone. Through spectroscopic techniques used in thiswork, the HA fraction (the alkaline-soluble organic matterthat precipitates at low pH) of the TPI soil was compared withhumic and fulvic acids, obtained by oxidizing activated char-coal with sodium hypochlorite. The yields recovery of HA-like substances was 12 and 28 wt% by using 10 and20 cmol L−1 of oxidizing agent, respectively. X-ray photo-electron spectroscopy, energy dispersive X-ray, and solid-state13C nuclear magnetic resonance (13C NMR) spectroscopies

were used to evaluate the elements and structures present in allsamples. XPS C 1 s spectra of HA extracted from TPI soil andfrom prepared HA showed aromatic structures (C=C andπ–π* shake-up satellite peak) bounded to carboxyl groups(COOH). The morphology and polycondensation level ofaromatic C were evaluated by scanning electron microscopy(SEM). The similarities of the spectra indicated that the usedmethod was efficient to obtain an organic amendment similarto TPI soil organic matter.

Keywords Oxidized charcoal . Amazonian dark earth . Soilorganic matter . Humic acids . X-ray photoelectronspectroscopy . Pyrogenic C

Introduction

Soil organic matter (SOM) was found to play a fundamentalrole, and significant attention is given to the most stable SOMtypes, such as humic substances, and to carbonaceous mate-rials, as black C (pyrogenic C), pyrolyzed biomass, and, morerecently, biochar (Brady and Weil 1999; Jorio et al. 2012;Lehmann 2007; Lehmann et al. 2008; Marris 2006). Thisinterest primarily arises from the discovery of Terra Preta deÍndios (TPI) soils, also known as Amazonian dark earths,anthropogenic dark earths, Indian black earths, orarcheological dark earths. These soils, found in the AmazonBasin, were modified and formed by the agricultural andhousehold activities of the indigenous people during the pre-Columbian era (Jorio et al. 2012). In addition to having higherfertility than the adjacent non-anthropogenic soils, these soilsexhibit a peculiar resilience to intense degradative use(Novotny et al. 2009). These properties can be explained bythe organic matter of these soils—pyrogenic C (Cpy) withperipheral aromatic units that partially oxidize to yield acidic(carboxyl) substituents (Glaser et al. 2001; Kramer et al. 2004;

J. R. Araujo (*) :B. S. Archanjo :K. R. de Souza : C. A. AcheteInstituto Nacional de Metrologia, Qualidade e Tecnologia, Av. NossaSra. das Graças, 50, Duque de Caxias, RJ 25250-020, Brazile-mail: jraraujo@inmetro.gov.br

W. KwapinskiCarbolea Research Group, Department of Chemical andEnvironmental Science, University of Limerick, Limerick, Ireland

N. P. S. FalcãoDepartamento de Ciências Agronômicas, Instituto Nacional dePesquisas da Amazônia, Manaus, AM 69011-970, Brazil

E. H. NovotnyEmbrapa Solos, Jardim Botânico, Rio de Janeiro, RJ 22460-000,Brazil

C. A. AcheteDepartamento de Engenharia Metalúrgica e de Materiais,Universidade Federal do Rio de Janeiro, Rio de Janeiro,RJ 21941-972, Brazil

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Masiello 2004; Novotny et al. 2009)—and by the relativelyhigh total acidity, which contributes to soil fertility and sus-tainability (Glaser et al. 2001).

Incorporation of charred biomass maybe more efficientthan incorporating plant residues for organic C storage in soil(Jorio et al. 2012; Lehmann 2007; Lehmann et al. 2008;Marris 2006; Schmidt et al. 2011) because Cpy is more stableand increases the mean SOM residence time. Furthermore,Cpy can increase plant yield (Lehmann 2007; Novotny et al.2009; Schmidt et al. 2011), which further can increase Cpy

production. However, the turnover rate and extended resi-dence time of Cpy in the soil remain under debate(Brodowski et al. 2006; Oades 1988; Schimel et al. 1994;Schmidt et al. 2011; Six et al. 2002; Torn et al. 1997).Researchers agree that the partial oxidation of the Cpy aromat-ic rings (Brodowski et al. 2006; Oades 1988; Schimel et al.1994; Schmidt and Noack 2000; Schmidt et al. 2011; Six et al.2002) and Cpy’s interaction with soil minerals (Brodowskiet al. 2006; Kogel-Knabner et al. 2008; Oades 1988;Schimel et al. 1994; Schmidt et al. 2011; Six et al. 2002;Torn et al. 1997) are fundamental to Cpy stability.

Detailed investigations have yielded an efficient model forthe peculiar organic matter in TPI (Novotny et al. 2007, 2009;Linhares et al. 2012). Humic acid (HA) from TPI can besatisfactorily modeled as a binary mixture composed of ordi-nary HA and polycondensed aromatic structures with carbox-yl groups attached directly to the aromatic C backbone(Novotny et al. 2009). At the typical pH of TPI soil (4–6),carboxylic acids are deprotonated generating carboxylate an-ions which contribute to soil cation exchange capacity (CEC);these peculiar HA structures are not only extremely recalci-trant, which is important for C sequestration, but also reactive,with a high CEC, which is important to soil fertility. On theother hand, the HA of the adjacent soils contain mainly labilecompounds such as carbohydrates, amino acids, and ligninresidues (Novotny et al. 2009), which explains the low resi-dence time of organic matter under cultivation. Based on thisproposed model, an oxidized material was obtained whichshare several chemical properties and resilience with thepolycondensed aromatic structures, containing carboxylicfunctionality, present in TPI; however, after an ecotoxicolog-ical test using Daphnia similes, this product was classified asmoderately toxic, likely due to the production of aryl chlorideduring oxidation as inferred from the infrared spectroscopicanalysis (Linhares et al. 2012).

Recently, advanced spectroscopic techniques have beenused to study soil properties and determine the chemicalstructures of SOM properties that are responsible for therecalcitrance and reactivity of highly fertile soils such as TPI(Jorio et al. 2012; Lehmann 2007; Lehmann et al. 2008;Marris 2006; Schmidt et al. 2011). For example, chemicalcharacterization using X-ray photoelectron spectroscopy(XPS) provided the C, O, and Cl core-electron binding

energies of the laboratory-prepared fractions to allow a de-tailed study of the humic acid-like materials (HALM) andfulvic acid-like materials (FALM).

To our knowledge, this is the first report where a compar-ison of HA extracted from TPI soils with laboratory-preparedHALM from charcoal oxidation was performed. The use ofmany coupled spectroscopic techniques is a powerful tool todetermine the chemical structure of target materials, due to thedifferent sensitivity of each technique; the type of sample to beanalyzed for each technique is also important, for example,bulk vs. surface. Arenella et al. (2014) reported the effect ofsoil-borne humic substances (HS) on the identification ofmodel proteins with different properties, such as myoglobin(Mb), α-glucosidase (αG), and β-glucosidase (βG), by usingelectrophoretic and ESI- and MALDI-mass spectrometry(MS) methodologies. Results showed that the contact betweenproteins and HS did not alter protein electrophoretic mobilitybut led to protein modifications that affected protein identifi-cation by MS.

The use of mathematical tool (multivariate curve resolu-tion—MCR) on 13C nuclear magnetic resonance (13C NMR)spectra of HA from TPI and HA from control adjacent soilsfacilitated the identification of a HA fraction peculiar fromTPI, i.e., polycondensed aromatic backbone heavily carbox-ylated (Novotny et al. 2009). It is important to prove theexistence of this fraction in TPI soils. Here, we have used amodification, proposed by Hayes and Graham (2000), of thetraditional method of humic substance extraction suggested byInternational Humic Substances Society (IHSS) to obtain,preferentially, the peculiar HA from TPI substances estimatedby MCR (Novotny et al. 2009). In addition, we have com-pared the properties of this fraction with those of analogoussubstances, obtained from charcoal oxidation, using XPSanalysis. This modification only selectively extracts the recal-citrant and reactive structures (polycondensed aromatic struc-tures functionalized with carboxylic acids) characterizing TPI.The novelty of this study is to replicate the HA of TPI throughoxidation of charcoal sample and compare the final productwith HA extracted from TPI soil in NaOH solutions of twodifferent pHs.

Materials and methods

HALM and FALM fractions obtained from charcoal

Activated charcoal (P.A., Vetec Química Fina Ltda., SãoPaulo, Brazil) was chemically oxidized using sodium hypo-chlorite (NaOCl) at two concentrations (10 and 20 cmol L−1).Activated charcoal was obtained from carbonaceous sourcesas peat, wood, coir, lignite, coal, and petroleum pitch aftertheir incubation at 600–900 °C, under inert atmosphere withargon or nitrogen. Five-gram-activated charcoal samples were

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placed in glass beakers with 200 mL of NaOCl of differentconcentrations. Then, 2.4 g of sodium hydroxide (NaOH)were added. The mixture was heated and stirred on a magneticstirrer-heater (60±2 °C) for 3 h and then filtered (filter paperwhite ribbon, grade 389). The supernatant was acidified to pH~2 to precipitate the HALM, while the FALM fractionremained in the solution. In order to compare fractions isolat-ed by exactly the same procedures, the precipitate wasredissolved in 0.1 mol L−1 KOH under a N2 atmosphere,and KCl was added until the K+ concentration reached0.3 mol L−1. The flocculated colloidal particles were centri-fuged at 40,000g (Relative Centrifugal Force—RCF) for20 min. The supernatant was acidified to pH~2 with concen-trated HCl, and the precipitated HALM was separated fromthe supernatant by centrifugation and dialysis. The unpurifiedFALM was freeze-dried (Swift 1996) and then purified usingXAD-7 and Amberlite IR-120 resins. The purified HALMand FALM were then freeze-dried (Table 1; Linhares et al.2012).

Extracting humic acids (HA) from TPI

The 0- to 20-cm depth soil samples (Anthroposols—FAOWorld Reference Base for Soil Resources 2007) were firstH+-exchanged by treating them with 0.1 M HCl, followed bywashing with distilled water until chloride free. The soils werethen repeatedly and exhaustively extracted (until the opticaldensity of the extract at 400 nm was below 0.1) with aqueoussolutions, with the soil/solution suspension pH sequentiallyadjusted to 7 and then to 10.6 using 0.1 mol L−1 NaOH(Simpson et al. 2007). The obtained suspensions were shakenovernight under a N2 atmosphere. This procedure has beendescribed in detail by Hayes and Graham (2000). The super-natant was separated from the residue by centrifugation (5,000g for 10 min) and filtration, and HCl was added to the filtrateto reach pH~2 and precipitate the HA, which was separatedfrom the supernatant by centrifugation (5,000g for 10 min).The residue was redissolved in 0.1 mol L−1 KOH under a N2

atmosphere, and KCl was added until the K+ concentrationreached 0.3 mol L−1. The flocculated colloidal particles wereseparated by centrifugation at 40,000g for 20 min. The super-natant was acidified to pH~2 using concentrated HCl, and theprecipitated HA was separated from the supernatant by cen-trifugation. Afterwards, the HA was dialyzed to remove anysoluble salts and then it was freeze-dried.

Solid-state 13C nuclear magnetic resonance (NMR)spectroscopy

Solid-state 13C NMR spectra were obtained using a VarianINOVA (11.74 T) spectrometer with 13C and 1H frequenciesof 125.7 and 500.0 MHz, respectively. A variable-amplitudecross-polarization pulse sequence was employed. The

experiments were performed using magic-angle spinning(MAS) of 15 kHz, cross-polarization time of 1 ms, acquisitiontime of 15 ms, recycle delay of 500 ms; and high-power two-pulse phase-modulation (TPPM) proton decoupling of70 kHz.

X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS, OmicronNanotechnology) was used to study the chemical compositionand chemical state of the prepared fractions (HALM, FALM,and HA-TPI fractions) after the acid-base fractionation. TheXPS analyses were performed under ultra-high vacuum(10−10 mbar) using an Al, Kα=1,486.7 eV X-ray sourcepowered by an emission current of 16 mA and a voltage of12.5 kV. High-resolution spectra were obtained for C and Clelements using analyzer pass energy of a 30- and 0.05-eVstep. The binding energies referenced the C 1 s level, whichwas set at 284.6 eV.

Semi-quantitative chemical analyses were obtained byXPS survey spectra considering the peak areas of elementsevaluated by the CasaXPS software and the sensitivity factorof each atom. The typical uncertainty in quantitative XPS isabout 10 % due mainly to uncertainties in the experimentaloptical data (Powell and Jablonski 2010). Gaussian/Lorentzian (70/30) line shapes were used to fit the C 1 s peakcurves. The Shirley background and a least-squares routinewere used for peak fitting. During the start of the C 1 s fittingprocedure, the full width at half-maximum (FWHM) valueswere limited to 1 eV for all peaks. The sp2 peak of the C 1 senvelope centered at 284.3 eV had a FWHMof 1.0±0.2 eV. Inaddition, near the sp2 component at 284.3 eV, we found atleast three broad components overlapping the C 1 s features.

Table 1 Description of samples analyzed by XPS and EDS. The humic/fulvic fractions were obtained using different concentrations of the oxi-dizing agent (NaOCl), whereas the TPI-humic fractions were exhaustive-ly extracted using NaOH solutions at two different pHs (in parentheses)

Sample NaOCl(cmol L−1)

Maximum pHof extraction

FALM-1a 20 –

FALM-2b 10 –

HALM-1a 20 –

HALM-2b 10 –

HA-TPI (7)c – 7

HA-TPI (10)c – 10

FALM fulvic acid-like material, HALM humic acid-like material, HA-TPIhumic acids extracted from TPIa 20 cmol L−1 NaOClb 10 cmol L−1 NaOClc Humic acids extracted at pH 7dHumic acids extracted at pH 10.6

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The bands appearing in the higher energy region tended to bemuch broader (FWHM≈1.7 eV) than that in the sp2 compo-nent. In particular, the FWHM of the components near the tailof the C 1 s envelopes was approximately 2 eV. These featuresagree with what is already reported (Bagri et al. 2010; Matteviet al. 2009; Stankovich et al. 2007; Yang et al. 2009).

High-resolution Cl peaks were not fitted due to the lowerresolution and lower intensity of their spectra. Some mole-cules containing O, such as O2, H2O, and CO2, may beadsorbed on the surface of porous samples, and thus O 1 speak was not analyzed.

Scanning electron microscopy (SEM)

The SEM images were collected using an FEI Nova Nanolab600 dual-beam microscope with a 5-keV accelerating energyand a 98-pA current. All samples were coated with a thin goldlayer (100-nm thick) using an Electron Microscopy Sciencessputter coater. The energy-dispersive X-ray spectroscopy(EDS) measurements were performed using an FEI Quanta200 with a tungsten filament operating in the environmentalmode (pressure≈1 Torr).

Results and discussion

Solid-state 13C nuclear magnetic resonance spectroscopy(NMR)

The NMR spectra for several Amazon soil HA weremodeled by MCR as a binary mixture with one componentexhibiting peaks typical of alkyl groups (such as n-alkyl),methoxy groups, carbohydrates, aromatics, O-aromatics,aliphatic carboxylic acids, and amides (Novotny et al.

2009). These chemical groups indicate the presence ofplant material at different stages of humification (fattyacids, proteinaceous material, lignin, and cellulose). Incontrast, the other MCR component is characterized bypolycondensed aromatic structures and aromatic carboxyl-ic groups (Fig. 1), which can be modeled as a pyrogenicand partially oxidized C (polycondensed rings functional-ized with carboxylic groups). The NMR spectra of the HAextracted from TPI at pH 7 and 10.6 are similar to thoseestimated by the MCR model (Fig. 1); thus, the mathemat-ical methods estimate a real component of the TPI soil andthe proposed extraction method selectively extracts thispeculiar organic matter from the TPI. Similar results wereobtained by Mao et al. (2012) using different methods.These authors analyzed the whole organic matter of TPIsamples collected from the sub-surface layers (0.4- to 0.7-m deep), to prevent contamination with fresh material,after demineralization with HF.

X-ray photoelectron spectroscopy (XPS)

XPS counts electrons ejected from a sample surface whenirradiated by X-rays. The underlying assumption when quan-tifying XPS spectra is that the number of electrons recorded isproportional to the number of atoms in a given state. As aresult, the best way to compare XPS intensities is by the so-called percentage atomic concentrations, which is percentageof intensity of the target atom corrected by its relative sensi-tivity factor (RSF; Powell and Jablonski 2010).

Unlike charcoal, which only contains C and O (Fig. 2), TPIcontains a variety of elements (Fig. 3). Like other productivesoils, TPI contains Ca, P, N, Al, Mg, Si, and Na, and low levelsof K, Ti, Cr, Mn, Fe, Cu, and Zn (Jorio et al. 2012). The HAextracted from the TPI samples primarily contained C and O

Fig. 1 13C NMR spectra of humic acids extracted from TPI soils atpH 7—HA-TPI (7)—and at pH 10.6—HA-TPI (10), and the estimatedspectra by multivariate curve resolution analysis of 22 humic acidssamples (TPI and non-anthropogenic control soils)

Fig. 2 XPS spectra of charcoal samples containing C and O

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(Fig. 3), with O/C atomic ratios of 0.27 (pH 10.6) and 0.29(pH 7).

Survey XPS spectra of the FALM and HALM preparedthrough the oxidation of activated charcoal with twoNaOCl solutions (20 and 10 cmol L−1) are presented inFig. 4. The FALM exhibited O/C ratios of 0.37 and 0.33

for FALM-1 and FALM-2, respectively, whereas theHALM yielded lower O/C ratios, 0.28 and 0.29 forHALM-1 and HALM-2, respectively (Table 2). The O/Cratios of the HALM were similar to those of the TPI-HAsamples, indicating similar oxidation level in these sam-ples. The O/C ratio of the charcoal samples was 0.07

Fig. 3 XPS spectra of humicacids extracted from TPI soils atpH 7—HA-TPI (7)—and atpH 10.6—HA-TPI (10), a XPSspectrum of HA-TPI (7), b C 1 shigh-resolution XPS spectrum ofHA-TPI (7), c XPS spectrum ofHA-TPI (10), and d C 1 s high-resolution XPS spectrum of HA-TPI (10)

Fig. 4 XPS spectra of thefractions extracted from activatedcharcoal oxidized with NaOCl: aFALM-1 fulvic acid-like material(10 cmol L−1 NaOCl), b FALM-2fulvic acid-like material(20 cmol L−1 NaOCl), c HALM-1humic acid-like material(10 cmol L−1 NaOCl), and dHALM-2 humic acid-like material(20 cmol L−1 NaOCl)

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(Fig. 2), which indicates that functional groups containingoxygen were introduced into the charcoal sample afterpartial oxidation with sodium hypochlorite. The differ-ence between the HALM and FALM can be explainedby their differing solubilities: FALM are more soluble dueto their additional oxygenated functional groups (higherhydrophilicity) and lower molecular size than HALM.

In addition, the XPS survey spectra of the laboratory-prepared fractions indicate that the HALM fractions containhigh quantities of Cl and K. After being oxidized with NaOCl,the functionalized charcoal samples were treated with KCl in aKOH solution to remove colloidal particles before acidifyingto pH~2. Thus, the HALM was contaminated with Cl and Kfrom the KCl salt, which was not removed by dialysis.Organic chlorine likely remained in the FALM, while the

chloride salts remained in the HALM fraction. This assump-tion was later confirmed by high-resolution XPS analysis.

Seven components are required to fit the C 1 s peak of theFALM and HALM fractions (Table 3). These seven compo-nents in the C 1 s spectrum (Fig. 5) contain the followingfunctional groups: sp2-hybridized C (C=C, 284.0 eV), sp3-hybridized C (C–C/C–H, 284.8 eV), hydroxyl/phenols (C–OH/C–Cl, 285.5 eV), epoxy/ether (C–O–C, 286.2 eV), car-bonyl (C=O, 287.5 eV), carboxyl (HO-C=O, 289.3 eV), anda shake-up satellite peak (π→π*, 291.3 eV) characteristic ofaromatic C structures (Filik et al. 2003; Han et al. 2013; Zhuet al. 2011). A binding energy of 284.3 eV essentially corre-sponds to non-functionalized sp2 C (Papirer et al. 1995). Filiket al. (2003) reported a binding energy of 284 eV in a graphitesample and a full width at half-maximum (FWHM) value of0.98. The HALM fractions contain more C–C and C=C bondsthan the FALM fractions; these C groups make up 58–61% ofthe FALM and 73-74 % of the HALM (Table 3). HALM-2exhibited the highest (33 %) and FALM-2 the lowest C=Ccontent (28 %). The polycondensed aromatic HALM fractionhas a more recalcitrant structure than the FALM fraction,which confirms the NMR data of Linhares et al. (2012), whoshowed that the HALM has more polycondensed aromaticstructure than the FALM.

The peak at binding energy of 285.5 eV is essentiallyattributed to sp2 C atoms linked to a chlorine atom (chlorine-substituted sp2 C atoms at the periphery of the polyaromaticstructures) (Pérez-Cadenas et al. 2003). Hydroxyl and phenolgroups can also contribute to this binding energy (~285.8 eV;Larciprete et al. 2012). The FALM fractions exhibited higherquantities of this component (~13 %), which confirms boththe inferred attribution and the results of the IR vibrationbands, and it indicates that the FALM contains more arylchlorides than HALM, and explains its toxicity (Linhares

Table 2 Atomic percentages calculated from the XPS survey spectra(details are provided in “Materials and methods”) and O/C and Cl/Catomic ratios for the FALM and HALM fractions, respectively

Element FALM-1a

(at.%)FALM-2b

(at.%)HALM-1c

(at.%)HALM-2d

(at.%)

C 61 63 56 54

O 30 28 21 21

N 1 1 1 1

K 5 5 10 11

Cl 1 2 11 12

Na 2 1 1 < 1

O/C ratio 0.37 0.33 0.28 0.29

Cl/C ratio 0.01 0.01 0.07 0.08

a fulvic acid-like material (10 cmol L−1 NaOCl)b fulvic acid-like material (20 cmol L−1 NaOCl)c humic acid-like material (10 cmol L−1 NaOCl)d humic acid-like material (20 cmol L−1 NaOCl)

Table 3 Binding energies (eV) of the C 1 s components and the percentage of each species (in parentheses) of the fulvic (FALM) and humic acid-likematerials (HALM) and the humic acids (HA) extracted from TPI at pH 7 or 10.6

Assignment FALM-1a FALM-2b HALM-1c HALM-2d HA-TPI (7)e HA-TPI (10)f

C=C 284.0 (35) 284.1 (28) 284.0 (31) 284.0 (33) 284.1 (36) 284.0 (30)

C–C/C–H 284.8 (23) 284.8 (33) 284.8 (42) 284.9 (41) 284.9 (32) 284.8 (36)

C–OH/C–Cl 285.5 (13) 285.5 (13) 285.6 (9) 285.7 (12) 285.7 (5) 285.5 (6)

C–O–C 286.3 (12) 286.2 (8) 286.3 (6) 286.5 (5) 286.3 (13) 286.1 (14)

C=O 287.4 (4) 287.5 (9) 287.5 (8) 287.5 (4) 287.6 (7) 287.3 (6)

HO–C=O 288.7 (13) 288.5 (9) 288.5 (4) 288.3 (5) 288.9 (7) 288.7 (8)

The numbers in parentheses are the atomic percentages obtained from the relative contributions of each peak.a fulvic acid-like material (10 cmol L−1 NaOCl)b fulvic acid-like material (20 cmol L−1 NaOCl)c humic acid-like material (10 cmol L−1 NaOCl)d humic acid-like material (20 cmol L−1 NaOCl)e humic acids extracted from TPI in pH 7f humic acids extracted from TPI in pH 10

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et al. 2012). Because the binding energy of sp2 C–Cl(285.5 eV) overlaps with that of oxygen-containing C atoms,the final evaluated percentage corresponds to the overall con-tribution of these two chemical sites. The Cl 2p peak must beexamined to determine the percentage of Cl-aryl bonds in thehumic/fulvic fractions.

A binding energy of ~286.3 eV corresponds to both thesp3 C bound to Cl and, to a lesser extent, the sp2 and sp3 Clinked to a single O atom (epoxide or ether; Papirer et al.1995). The XPS peaks at 287.5 and 288.4 eV correspond tocarbonyl and carboxyl groups, respectively. The greatestdifference observed in the C 1 s peaks for the FALM andHALM fractions occurs in the carboxyl peak (COOH;Fig. 5a–d). The percentage of carboxyls attached to aro-matic C in the FALM-1 and 2 are 13 and 9 %, respectively,and the HALM 4–5 % (Table 3, Fig. 5). The high carbox-ylic content and lower molecular size explain the highsolubility of the FALM samples across all pH values andlow condensation rate in the aromatic ring, which agreeswith the previous NMR results (Linhares et al. 2012).

Chlorine atoms easily bind to C surfaces, which in-creases their Lewis acidity but decreases their Brönstedacidity due to resonance from the aromatic rings (Pérez-Cadenas et al. 2003). A binding energy of ~200.5 eV isindicative of chlorine atoms covalently bonded to sp2 C asreported for organo chlorine compounds (Papirer et al.1995). The high-resolution Cl 2p peak (Fig. 6) arises fromtwo major components: inorganic chlorine (binding energy<199 eV), from a chloride salt with Na, K, Ca, or Fe, andorganic chlorine (binding energy >200 eV), from chlorine

bonded to aromatic sp2 C atoms or due to Cl–C=O bonds(Fiedler and Herzschuc 1993; Wang et al. 2012). TheFALM contained more organic chloride, as aryl chloride(Fig. 6), than the HALM, which mainly contained chloridesalts, as sodium hypochlorite (NaOCl). Activated charcoaloxidized with various sodium hypochlorite concentrationsformed aryl chloride. Also, non-reacted sodium hypochlo-rite remained in FALM and HALM fractions. Thisbyproduct was partially removed during purification; how-ever, ecotoxicological testing of the FALM indicated thatthe chemically functionalized charcoal was moderatelytoxic, and infrared analysis indicated the presence of arylchloride compounds that were likely responsible for this

Fig. 5 High-resolution XPSspectra of the C 1 s for a FALM-1fulvic acid-like material(10 cmol L−1 NaOCl), b FALM-2fulvic acid-like material(20 cmol L−1 NaOCl), c HALM-1humic acid-like material(10 cmol L−1 NaOCl), and dHALM-2 humic acid-like material(20 cmol L−1 NaOCl). The C 1 speak fitting procedure wasperformed with the followingconsiderations: C=C (sp2), C–C(sp3), C–OH (phenol), C–O–C(ether/epoxide), C=O (carbonyl),HO–C=O (carboxyl), and π–π*shake-up (satellite peak of the Csp2 component)

Fig. 6 Comparison of the high-resolution XPS spectra in the Cl 2pregion for fulvic (FALM) and humic acid-like materials (HALM) bothobtained using NaOCl concentrations of 20 and 10 cmol L−1

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toxicity (Linhares et al. 2012). The presence of aryl chlo-ride was confirmed by XPS analysis.

EDS

The qualitative elemental composition obtained from the EDSanalysis of the FALM and HALM is shown in Fig. 7a. BothFALM and HALM fractions contained Cl, K, and Na from thereagents (NaOCl, KCl, and KOH) used during the synthesisand purification. The apparent presence of Al was due to X-rays interacting with Al parts of the microscope and thesample holder. These results agreed with the XPS measure-ments, where the chlorine- and potassium-binding energies ofHALM fractions were mainly assigned to these elements insalt form, while the chlorine binding energy in the FALM

fractions identified organic chlorides, as potentially toxic arylchloride. These results highlight the need for either purificationafter synthesis or a change in the used oxidant. The EDS resultsfor the HA extracted from TPI (Fig. 7b) revealed an importantfeature of the TPI samples: the region between 0.75 and 10 keVshowed elements characteristic of TPI soils, such as Si, P, S, K,Ca, Ti, and Fe (Jorio et al. 2012), and small quantities of Na andCl residues remaining after extraction.

Scanning electron microscopy (SEM)

Figure 8 provides the SEM images of the laboratory-preparedfractions (FALM, HALM) and TPI extracts (pH 7 and 10.6).The soluble fulvic acid fraction (Fig. 8a) forms distinguishablesmall particles. This characteristic promotes a high metal

Fig. 7 a EDS spectra of fulvic acid-like material obtained using NaOClconcentrations of 10 and 20 cmol L−1 (FALM-1 and FALM-2, respec-tively) and humic acid-like material obtained with NaOCl concentrations

of 10 and 20 cmol L−1 (HALM-1 and HALM-2, respectively). b EDSspectra of humic acids extracted from TPI at pH 7 and 10.6 (HA-TPI (7)and HA-TPI (10), respectively)

Fig. 8 SEM images of thefollowing: a fulvic acid-likematerial (FALM-1); b humicacid-like material (HALM-1),both fractions were obtainedusing NaOCl concentrations of10 cmol L−1; c humic acidsextracted from TPI at pH 7 (HA-TPI (7)); d humic acids extractedfrom TPI at pH 10.6 (HA-TPI(10))

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adsorption (Xu et al. 2006). The metals in this fraction (Fe, K,and Ca) can form bridges with carboxyl groups, interact bychemisorption with oxidized C groups and form coordinationcompounds (Pandey et al. 2000) with other functional groupsof humic substances, such as hydroxyl, phenol, and methoxyfunctional groups.

Compacted microaggregates were found both in the HALMand HA-TPI samples; however, the aggregate size of theHALM (Fig. 8b) was greater than those of the HA-TPI frac-tions (Fig. 8c, d). Changlung et al. (2007) reported that theaggregation phenomenon is important to the transport of heavymetal ions in natural environments. Zhang et al. (2009) report-ed that humic substances in dilute solutions form thin thread-and net-like structures that grow into larger rings and sheetswith increasing humic and cation concentrations. The HAfraction is insoluble at low pH (pH~2) due to aggregation atthis pH value. This aggregation can be observed in SEM imageof HALM-1 fraction (Fig. 8b), confirming this hypothesis.

Conclusion

Aqueous NaOH solutions with pH values of 7 and 10.6 wereused to selectively extract the peculiar humic fraction of the TPIsample. Substances similar to these peculiar HAwere success-fully prepared by oxidizing activated charcoal. The XPS andSEM images showed a polycondensed structure in the TPI-HAsimilar to that of the laboratory-prepared fractions. The use ofspectroscopic techniques to compare chemically produced ma-terials, and humic fractions from fertile soils, may be used tomeasure the chemical composition of these new producedmaterials in order to recreate TPI soils. This expertise may beimportant to indicate the need for a purification process orecotoxicological tests. Fulvic and humic fractions were isolatedthrough the precipitation of the HALM, which is insoluble atacidic pH. The XPS analyses indicated that the FALM fractionscontained more carboxyl/carbonyl groups than the humic frac-tions, which contained more aromatic/aliphatic carbons. Arylchloride was formed in the FALM, and thus, a purificationprocess or change in the used oxidant is necessary beforeincorporating this synthetic fraction into the soil.

Acknowledgments We thankAustimM. Pimenta and Carlos Senna forthe EDS measurements. Katia R. de Souza thanks Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq) for the research fel-lowship (384692/2012-5) supporting her research.

Conflict of interest The authors declare no competing financialinterest.

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