Fluorescence characterization of metal ion–humic acid interactions in soils amended with composted municipal solid wastes

ORIGINAL PAPER

Fluorescence characterization of metal ion–humic acidinteractions in soils amended with composted municipalsolid wastes

César Plaza & Gennaro Brunetti & Nicola Senesi &Alfredo Polo

Received: 3 July 2006 /Revised: 5 September 2006 /Accepted: 7 September 2006 / Published online: 17 October 2006# Springer-Verlag 2006

Abstract Fluorescence spectroscopy has been used toprobe the structural properties and Cu(II), Zn(II), Cd(II),and Pb(II)-binding behavior of humic acid (HA)-likefractions isolated from a municipal solid waste compost(MSWC) and HAs from unamended and MSWC-amendedsoils. The main feature of the fluorescence spectra, in theform of emission-excitation matrix (EEM) plots, was abroad peak with the maximum centered at an excitation/emission wavelength pair that was much shorter (340/437 nm) for MSWC-HA than for unamended and MSWC-amended soil HAs (455/513 and 455/512 nm, respectively).Fluorescence intensity for MSWC-amended soil HA wasless than that for unamended soil HA. These results wereindicative of more aromatic ring polycondensation andhumification of soil HAs, and of partial incorporation ofsimple and low-humified components of MSWC-HA intonative soil HA, as a result of MSWC amendment. Titrationsof HAs with Cu(II), Zn(II), Cd(II), and Pb(II) ions at pH 6and ionic strength 0.1 mol L−1 resulted in a markeddecrease of the fluorescence intensities of untreated HAs.By successfully fitting a single-site fluorescence-quenchingmodel to titration data, the metal ion complexing capacitiesof each HA and the stability constants of metal ion-HA

complexes were obtained. The binding capacities andstability constants of MSWC-HA were smaller than thoseof the unamended soil HA. Application of MSWC to soilslightly reduced the metal-ion-binding capacities andaffinities of soil HAs.

Keywords Composted municipal solid wastes . Soilamendment . Humic acids . Metal-complexing capacitiesand stability constants . Fluorescence spectroscopy

Introduction

The increasing accumulation in soil of potentially toxictrace metals, including Cu, Zn, Pb, and Cd, because ofindustrial, mining, and agricultural practices, is a severehazard to animal and human health [1–4]. Consequently,viable means of treatment of metal-contaminated soils havebecome a major focus of recent research. Because metalscannot be degraded, they must be either immobilized orremoved [5, 6]. Immobilization and removal are, however,complex processes that require understanding of metalbehavior in soil. The complexity arises mostly from thedependence of metal behavior on a variety of reactions thatmay occur in soil, including complexation with organic andinorganic ligands, ion exchange, adsorption and desorptionprocesses, precipitation and dissolution of solids, and acid-base equilibria [7, 8]. Among these, binding reactions tosoil organic matter and, especially, to its humified fractions,for example humic acids (HAs), are known to play a keyrole [9–11].

Land application of municipal solid waste compost(MSWC) is an important alternative to other municipalwaste disposal options, for example incineration and land-filling, which cause increasing environmental and econom-

Anal Bioanal Chem (2006) 386:2133–2140DOI 10.1007/s00216-006-0844-0

C. Plaza (*) :A. PoloCentro de Ciencias Medioambientales,Consejo Superior de Investigaciones Científicas,Serrano 115 dpdo.,28006 Madrid, Spaine-mail: [email protected]

G. Brunetti :N. SenesiDipartimento di Biologia e Chimica Agroforestale ed Ambientale,University of Bari,Via Amendola 165/A,70126 Bari, Italy

ical problems [12]. Soil application of MSWC increasessoil organic matter content, reduces the need for inorganicfertilizers, promotes higher yields of agricultural crops,facilitates reforestation, and helps suppress plant diseases[13]. MSWC may also cost-effectively remediate soilscontaminated by toxic metals by increasing their cation-exchange capacity, because of the increased organic mattercontent of the soil, thus preventing metal ion migration tosurface and subsurface water bodies and/or plant uptake[14, 15]. Depending on the compost feedstock, however,MSWC may contain relatively high levels of potentiallytoxic metals that pose serious threats to the soil environ-ment [16]. In both circumstances the HA fraction inMSWC-amended soil is of much interest, because it mayhave a powerful effect on metal transport, retention, andbioavailability.

Fluorescence spectroscopy is well known to be a powerfultechnique useful for chemical characterization of HAs [17–20]and metal ion-HA interactions. Further, comprehensivefluorescence titration analysis has also been successfully usedto determine metal-ion-binding capacities of HAs the andstability constants of metal ion-HA complexes [21–32].Although HAs isolated from MSWC and MSWC-amendedsoils have been studied by fluorescence spectroscopy [33–37],no quantitative fluorescence investigation has been publishedon metal-ion binding by these HAs.

The objectives of this work were thus to use fluores-cence titration analysis to determine:

1. the complexing capacities of MSWC-HA for Cu(II), Zn(II), Cd(II), and Pb(II) ions and the stability constantsof MSWC-HA complexes with these ions, comparedwith those for unamended soil HA; and

2. the effect of application of MSWC on the metal-ion-binding behavior of soil HAs.

Materials and methods

Municipal solid waste compost and soils

The MSWC sample used in this work was collected in acomposting facility operating in Becker (MN, USA) froma pile composed of MSW mixed with cardboard, paper,and food yard wastes as bulking agents, which wassubjected to composting over a period of 4 months. Soilamended with 40 t ha−1 yr−1 of MSWC for three years(MSWC40) and the corresponding unamended control soil(MSWC0) were sampled from the top layer (Ap horizon, 0–15 cm depth) of a loamy sand, Udorthentic Haploboroll, inthe Sand Plain Research Farm of the University ofMinnesota in Becker (MN, USA). Soil samples werecollected approximately nine months after MSWC applica-

tion, which is generally believed to be enough time foradequate incorporation of organic amendment into nativesoil organic matter.

Isolation of humic acids

Humic acids were isolated from the MSWC sample andfrom the unamended and MSWC-amended soils byconventional procedures [38]. Briefly, a solution of0.1 mol L−1 Na4P2O7 and 0.1 mol L−1 NaOH was addedto air-dried, 2-mm sieved MSCW or soil sample using aextractant-to-sample ratio of 10:1. The mixture was shakenmechanically under a nitrogen gas atmosphere for 24 h atroom temperature (RT, 20±2 °C). The supernatant solutionwas then separated from the residue by centrifugation at9,600 g for 30 min. The extraction procedure was repeatedthree times on the residue, which was then discarded.The combined alkaline supernatants were acidified with6 mol L−1 HCl to pH ∼1, left to stand for 24 h underrefrigeration, and then centrifuged at 30,400 g for 20 min.The HA precipitates were purified by repeating the stepsbelow three times:

1. dissolution of the HA in a minimum volume of alkalineextractant;

2. centrifugation as above;3. removal of the residue;4. acidification of the recovered alkaline supernatant with

6 mol L−1 HCl to pH ∼1;5. leaving the suspension to stand for 24 h at RT; and6. final centrifugation as above.

The centrifuged HAs were recovered with distilledwater and then dialyzed against distilled water using amembrane with a molecular weight cutoff of 6,000–8,000 Da, until the dialysis water gave a negative Cl− iontest with AgNO3. Finally, the dialyzed HAs were freeze-dried and stored at RT in plastic vials placed in a desiccatorover P2O5.

Fluorescence titrations

A stock solution of each HA was prepared by dissolving50 mg freeze-dried HA in 100 mL 1 mol L−1 KOH. Themixture was stirred for 30 min under N2 gas and then100 mL 1 mol L−1 HNO3 was added. Subsequently thesolution was diluted to 950 mL with deionized distilledwater, the pH was adjusted to 6 by addition of 0.1 mol L−1

KOH, and the volume was finally diluted to 1 L withdeionized distilled water. This stock solution of HA(100 mL) was titrated in 150-mL thermostatic vessels with0.01 mol L−1 Cu(NO3)2, Zn(NO3)2, Cd(NO3)2 and Pb(NO3)2 by use of an automatic syringe. To maintainconstant pH, 0.1 mol L−1 KOH was dispensed using

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another automatic syringe. Samples were maintained at aconstant stirring speed, at 25 °C, and under N2 atmospherethroughout the titrations. After each addition of titrant thesolution was circulated via a peristaltic pump through aquartz flow-through cell for fluorescence spectral recordingand back to the titration vessel for 15 min.

Three-dimensional fluorescence spectra as excitation-emission matrix (EEM) plots were recorded using aPerkin-Elmer (Norwalk, CT, USA) LS 55 luminescencespectrometer equipped with WinLab 4.00.02 software(Perkin-Elmer, 2001) for data processing. Emission andexcitation slits were set at a 5-nm band width, and a scanspeed of 500 nm min−1 was selected for the emissionmonochromator. The wavelength emission range was from350 to 550 nm and the excitation wavelength was increasedsequentially from 300 to 500 nm by 5-nm steps. Excitation-emission matrix plots were generated from fluorescencespectral data by use of Surfer 8.01 software (GoldenSoftware, 2002, Golden, CO, USA).

Determination of complexing capacities and stabilityconstants

The complexing capacities of HAs for Cu(II), Zn(II), Cd(II), and Pb(II) ions and the stability constants of Cu(II), Zn(II), Cd(II), and Pb(II) complexes with HAs were deter-mined by use of the single-site fluorescence-quenchingmodel of Ryan and Weber [21]. This model is based on theassumption that metal-ion binding occurs at identical andindependent binding sites or ligands, and only 1:1 metal-ligand complexes are formed. Although the assumption of1:1 stoichiometry is not accurate for metal-ion binding toHAs [10, 11], and may result in small complexing capacityvalues compared with the corresponding acidic functionalgroup content, this assumption simplifies the theoreticalanalysis, and is used and justified in most studies on metal-ion binding to HS [21–32].

The complexation of a metal ion (M) by an organicligand (L) (i.e., the formation of an ML complex) at

constant pH and ionic strength may be described by theconditional stability constant

KM¼ ML½ �M½ � L½ � ð1Þ

where [M] denotes the molar concentration of the freemetal, [L] is the molar concentration of all forms of ligandwhich are not bound to M, and [ML] is the molarconcentration of the ML complex.

The mass balance equations for the metal ion and theligand are:

CM ¼ M½ � þ ML½ � ð2Þ

CL ¼ L½ � þ ML½ � ð3Þwhere CM and CL are the stoichiometric concentrations ofthe metal ion and the ligand, respectively. The complexa-tion capacity (CCM), i.e. the amount of active binding sitesper unit mass of HA, is related to CL by

CCM ¼ CL

HAð Þtotalð4Þ

where (HA)total is the total concentration of HA.Equations (1) and (3) may be combined to obtain the

fraction of ligand bound (ν) expressed in terms of thestability constant and free metal ion concentration

n ¼ ML½ �CL

¼ KM M½ �1þ KM M½ � ð5Þ

When combined with Eq. (2), Eq. (5) assumes the form:

n ¼ KM CM � nCLð Þ1þ KM CM � nCLð Þ ð6Þ

Equation (6) can then be solved for ν, giving:

ν ¼ 1

2KMCL

� �1þ KMCLþKMCM�

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1þ KMCLþKMCMð Þ2�4K 2

M CLCM

q� �ð7Þ

According to Ryan and Weber [21], the measuredfluorescence intensity (I) varies linearly with the fractionof the total ligand bound, yielding the relationship:

n ¼ I0 � Ið ÞI0 � IMLð Þ ð8Þ

where I0 is the fluorescence intensity at the beginning ofthe titration (i.e. the fluorescence intensity of the metal-free HA), and IML is the limiting value below which thefluorescence intensity does not decrease because ofaddition of metal ion (i.e. the fluorescence intensity ofthe metal-saturated complex).

Anal Bioanal Chem (2006) 386:2133–2140 2135

Combination of Eq. (7) and Eq. (8), and rearrangement,yields:

I ¼ I0 þ IML � I0ð Þ 1

2KMCL

� �1þ KMCL þ KMCM �

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1þ KMCL þ KMCMð Þ2 � 4K 2

M CLCM

q� �ð9Þ

By introducing fluorescence intensity values determinedexperimentally at the different total metal ion concentra-tions used, Eq. (9) can be solved by nonlinear regressionanalysis for KM, CL, and IML. The optimum set of fittingdata for each HA sample was thus obtained by iterativelyvarying the adjustable values until the sum of the squares ofthe differences between observed and fitted values of I wasminimized. Full, unconstrained optimization was achievedby use of the quasi-Newton algorithm. The computerprogram Statistica 4.0 for Windows (Statsoft, 1993, Tulsa,OK, USA) was used for calculations.

Results and discussion

Chemical and structural characteristics of humic acids

The compositional, structural, and functional chemicalproperties of the HAs examined in this work have beendescribed elsewhere [37, 39]. Briefly, MSWC-HA haslarger H, N and S content, aliphatic character and molecularheterogeneity, and smaller O and organic free radicalcontent and degree of aromatic polycondensation andhumification than MSWC0-HA [37]. As a result ofamendment the MSWC40-HA contains more N-containingand polysaccharide-like components and aliphatic natureand less organic free radical content than MSWC0-HA[37]. The total acidity and carboxyl and phenolic OH groupcontent of MSWC-HA (4.25, 2.59, and 1.66 mmol g−1,respectively) are also smaller than those for unamended soilHA (6.60, 4.08, and 2.52 mmol g−1, respectively) [39]. Theacidic functional group content of MSWC40-HA areintermediate (6.06, 3.84, and 2.22 mmol g−1, respectively)between the corresponding values for MSWC-HA andMSWC0-HA, but closer to those for the latter [39].

Fluorescence spectra of humic acids

Figure 1 shows the fluorescence EEM spectra of HAsisolated from MSWC, and from unamended and MSWC-amended soils. The fluorescence EEM spectrum of MSWC-HA is characterized by a unique main fluorophore centeredat an excitation/emission wavelength pair maximum(EEWPmax) of 340/437 nm whose relative intensity is 204arbitrary units (a.u.). The fluorescence EEM spectrum of

MSWC0-HA has a prominent peak of greater intensity (276a.u.) at long excitation/emission wavelengths (455/513 nm)accompanied by a broad shoulder that extends to shorterexcitation wavelengths, and a less intense peak (243 a.u.) atEEWPmax=325/508 nm. Similar to that of MSWC0-HA,the spectrum of MSWC40-HA features two fluorescencemaxima at EEWPmax=455/512 nm and 325/507 nm, but ofsmaller intensity (208 a.u. and 191 a.u., respectively).

The lower fluorescence intensity of MSWC-HA than ofsoil HAs may be attributed to the greater presence ofpolysaccharide-like structures and lower content of reducedquinone-like fluorophores, organic free radicals, and elec-tron-donating substituents, for example hydroxyl andmethoxy groups [17, 19, 40]. The prevalence of fluores-cence peaks at short wavelength, for example that measuredfor MSWC-HA, can also be associated with the presence ofsimple structural components of wide molecular heteroge-neity and low molecular weight, low aromatic polyconden-sation, low level of conjugated chromophores, and lowhumification [17]. The prevalence of fluorescence peaks atlong wavelength may be ascribed to the presence of extended,linearly-condensed aromatic ring networks and other unsatu-rated bond systems capable of much conjugation in largemolecular weight units with a high degree of humification[17]. This seems to be true for MSWC0-HA, especially, andMSWC40-HA, to a lesser extent. The lower fluorescenceintensity of the main peak (i.e. most intense fluorophore) ofMSWC40-HA compared with that of MSWC0-HA suggestspartial incorporation of simple and low-humified componentsof MSWC-HA into native soil HA.

Changes in the fluorescence spectra of humic acidsas a result of metal-ion binding

Titration of HAs with Cu(II), Zn(II), Cd(II), or Pb(II) ionscauses significant modification of fluorescence EEMspectra which depends both on the metal ion and on theorigin of HA sample (Fig. 1). In particular, the fluorescence

bFig. 1 Fluorescence excitation-emission matrix spectra of humicacids (HAs) isolated from municipal solid waste compost (MSWC),from soil amended with MSWC at 40 t ha−1 yr−1 (MSWC40), andfrom the corresponding unamended control soil (MSWC0), in theabsence and presence of Cu(II), Zn(II), Cd(II), and Pb(II) ions at atotal concentration of 40 μmol L−1. EEWPmax denoted the excitation/emission wavelength pairs at maximum fluorescence intensity

2136 Anal Bioanal Chem (2006) 386:2133–2140

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MSWC-HA EEWPmax: 340/437

MSWC-HA + Zn(II) EEWPmax: 340/438

MSWC-HA + Cu(II) EEWPmax: 335/436

MSWC-HA + Pb(II) EEWPmax: 340/436

MSWC-HA + Cd(II) EEWPmax: 340/438

MSWC0-HA EEWPmax: 455/513

MSWC0-HA + Zn(II) EEWPmax: 455/511

MSWC0-HA + Cu(II) EEWPmax: 325/445

MSWC0-HA + Pb(II) EEWPmax: 440/509

MSWC0-HA + Cd(II) EEWPmax: 455/511

MSWC40-HA EEWPmax: 455/512

MSWC40-HA + Zn(II) EEWPmax: 455/510

MSWC40-HA + Cu(II) EEWPmax: 330/440

MSWC40-HA + Pb(II) EEWPmax: 440/509

MSWC40-HA + Cd(II) EEWPmax: 440/508

Anal Bioanal Chem (2006) 386:2133–2140 2137

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MSCW-HA + Zn(II)

MSWC-HA + Cu(II)

MSWC-HA + Pb(II)

MSWC-HA + Cd(II)

MSWC0-HA + Zn(II)

MSWC0-HA + Cu(II)

MSWC0-HA + Pb(II)

MSWC0-HA + Cd(II)

MSWC40-HA + Zn(II)

MSWC40-HA + Cu(II)

MSWC40-HA + Pb(II)

MSWC40-HA + Cd(II)

Fig. 2 Experimentally-determined values (dots) and fits with the Ryan-Weber model (lines) of the fluorescence intensity (I) of the main peaksin fluorescence spectra of humic acids (HAs) isolated from municipalsolid waste compost (MSWC), soil amended with MSWC at a rate of

40 t ha−1 yr−1 (MSWC40), and the corresponding unamended controlsoil (MSWC0), as a function of increasing total concentration (CM) ofCu(II), Zn(II), Cd(II), and Pb(II) ions

2138 Anal Bioanal Chem (2006) 386:2133–2140

of all HAs, especially soil HAs, is quenched by addition ofZn(II) and Cd(II), and, especially, Cu(II) and Pb(II); this isin agreement with previous findings on similar systems [31,41]. The main fluorescence peaks of soil HAs also tend toshift slightly to shorter excitation and emission wavelengths(i.e. blue shift) on interacting with metal ions, especiallywith Cu(II) and Pb(II), whereas the maximum fluorescencewavelengths of MSWC-HA remain almost constant. Anapparent decrease of the emission wavelength is alsoobserved for the secondary peaks (i.e. less intense fluoro-phores) of soil HAs when Cu(II) and Pb(II) ions are added,most probably because of the large quenching effect ofthese metal ions on HA fluorescence at long emissionwavelengths. These results are indicative of markedmodification of the electronic structure of the HAs,especially soil HAs, on interaction with Zn(II), Cd(II),and, especially, Cu(II) and Pb(II). The different extents ofthe modifications observed can be ascribed to the differentstrength of bonding between Cu(II), Zn(II), Cd(II), or Pb(II)and the various HAs, and to conformational changes of theHAs as the complexing process occurs [41].

Metal-ion-binding properties of humic acids–stabilityconstants and complexing capacities

The intensities of the main peaks in the fluorescence EEMspectra of each HA as a function of metal ion concen-trations are shown in Fig. 2. The metal-ion-binding datacalculated by nonlinear fitting of the Ryan-Weber model tothe experimental fluorescence datasets (i.e. the fluorescenceintensities of metal ion-saturated HA complexes, thecorrelation coefficients for predicted and measured fluores-cence intensity, the stability constants of metal ion-HAcomplexes, and the metal ion complexing capacities) arelisted in Table 1. The curves calculated for the HAs usingthese predicted data (Fig. 2) fit the observed fluorescence

data very well (dots in Fig. 2), as has been demonstrated inprevious work on similar systems [23, 27, 28, 30–32].

For any HA examined, the stability of metal ion-HAcomplexes, as indicated by the log KM values, and themetal-ion-binding capacity follow the same order-Pb(II)>Cu(II)>Cd(II)>Zn(II). These results are in agreement withthe Irving-Williams [42] series for the binding strength ofdivalent metal ion complexes, irrespective of the natureof the ligand, and are also in agreement with the findingsof other authors for metal ion complexes with HS ofdifferent origin and nature [24, 25, 27, 31, 32]. For anymetal species, the stability constants of metal ion-HAcomplexes and the metal ion complexing capacities followthe order MSWC-HA<MSWC40-HA<MSWC0-HA.

The differences found in the stability constants might beexplained by the chelating effect of the acidic functionalgroups in the HAs examined. An aromatic carboxyl groupand adjacent phenolic OH group, or two adjacent aromaticcarboxyl groups, are known to form highly-stable salicy-late-like and phthalate-like ring structures with metal ions[10, 11]. The lower content of acidic functional groups andthe lower degree of aromatic polycondensation of MSWC-HA compared with soil HAs suggest the presence of feweraromatic carboxyl and phenolic OH groups in the former,which is expected to result in a smaller chelating effect. Incontrast, the relatively large stability constants measured forMSWC0-HA may be attributed to the high content of acidicfunctional groups and marked aromatic character. Theintermediate values of the stability constants of metal ion-MSWC40-HA complexes confirm the partial modificationwhich occurred in amended soil HA as a result of partialincorporation of MSWC-HA structures into native soil HA.

The binding capacities of HAs examined seem to bestrongly related to their acidic functional group content, bothcarboxyl and phenolic OH groups, which are usually believedto be mainly responsible for metal-ion binding to HS [10, 11].

Table 1 Resultsa from fitting to the Ryan-Weber model

Origin of HA sample Property Origin of HA sample

MSWC MSWC0 MSWC40 MSWC MSWC0 MSWC40

ICuL 38.4 3.4 7.7 ICdL 160.7 145.9 120.6rCu 0.9994b 0.9997b 0.9997b rCd 0.9996b 0.9998b 0.9997b

log KCu 4.97 5.53 5.43 log KCd 4.35 4.60 4.49CCCu 0.87 1.21 1.04 CCCd 0.59 0.78 0.70IZnL 160.1 143.2 121.9 IPbL 93.3 3.2 2.7rZn 0.9997b 0.9999b 0.9997b rPb 0.9995b 0.9997b 0.9998b

log KZn 4.23 4.44 4.37 log KPb 5.09 5.76 5.56CCZn 0.53 0.68 0.65 CCPb 1.10 1.80 1.59

a IML (arbitrary units) is the fluorescence intensity of the metal ion-saturated complex, rM the correlation coefficient for predicted and measuredfluorescence intensity, log KM the stability constant, and CCM (mmol g−1 ) the complexing capacity for binding of Cu(II), Zn(II), Cd(II),and Pb(II) to humic acids (HAs) isolated from municipal solid waste compost (MSWC), soil amended with MSWC at a rate of 40 t ha−1 yr−1

(MSWC40), and the corresponding unamended control soil (MSWC0)b Statistical significance or probability value (P value)<0.001

Anal Bioanal Chem (2006) 386:2133–2140 2139

In agreement with data obtained previously for HAs isolatedfrom different types of organic amendment and from amendedand the corresponding unamended soils, however [31, 32], theCCM values much smaller than the corresponding acidicfunctional group content. This may be ascribed to:

1. the involvement of bidentate salicylic and phthalic-typebinding sites;

2. the formation of 2:1 complexes, with the metal ionbridging two HA molecules; and/or

3. the presence of several carboxyl and phenolic hydroxylgroups that are unavailable for metal-ion bindingbecause of steric hindrance, proton competition, orelectrostatic effects (e.g. metal ion complexation at onesite can reduce the capacity of neighboring functionalgroups to complex other metal ions).

Conclusions

The free-metal-ion concentration is a measure of thetendency of the metal ion to be involved in other types ofinteraction, so the general effect of metal-ion binding to HAis to attenuate metal ion reactivity, including its participa-tion in other chemical reactions and uptake by biota. Theresults described above indicate that binding capacities andaffinities of MSWC-amended soil HA are smaller thanthose of unamended soil HA, which indirectly suggests thatapplication of MSWC may increase metal bioavailability,mobilization, and transport in soil. Addition of MSWC tosoil has the potential to substantially increase organicmatter and HA content, however, so the metal-ion-bindingcapacity of HA on a total mass basis may be increased inMSWC-amended soil. As a consequence, application ofMSWC may reduce the environmental risk of pollution ofsoil and groundwater by metals.

Acknowledgements The authors are grateful to Professor C.E.Clapp, Department of Soil, Water and Climate, University ofMinnesota, St Paul, MN, USA, for providing the MSWC and soilsamples used in this work. C. Plaza is a recipient of a Ramón y Cajalcontract funded by the Spanish Ministry of Education and Science.

References

1. McKinney J, Rogers R (1992) Environ Sci Technol 26:1298–12992. Wong SC, Li XD, Zhang G, Qi SH, Min YS (2002) Environ

Pollut 119:33–443. Liu HY, Probst A, Liao BH (2005) Environ Sci Technol 339:153–1664. Parat C, Chaussod R, Lévêque J, Andreux F (2005) Soil Biol

Biochem 37:673–6795. Wise DL, Trantolo DJ, Cichon EJ, Inyang HI, Stottmeister U

(2000) Remediation engineering of contaminated soils. MarcelDekker, New York

6. Ochoa-Loza FJ, Artiola JF, Maier RM (2001) J Environ Qual30:479–485

7. Sposito G (1994) Chemical equilibria and kinetics in soils. OxfordUniversity Press, New York

8. Sparks DL (2002) Environmental Soil Chemistry, 2nd edn.Academic Press, San Diego

9. Sauve S, Hendershot W, Allen HE (2000) Environ Sci Technol34:1125–1131

10. Tipping E (2002) Cation binding by humic substances. CambridgeUniversity Press, New York

11. Senesi N, Loffredo E (2005) In: Sparks DL, Tabatabai MA (eds)Chemical processes in soil. SSSA, Madison, WI

12. Senesi N, Plaza C, Brunetti G, Polo A (2006) Soil Biol Biochem,(in press)

13. De Bertoldi M, Sequi P, Lemmes B, Papi T (1996) The science ofcomposting. Chapman and Hall, London

14. Kiikkilä O, Perkiömäki J, Barnette M, Derome J, Pennanen T,Tulisalo E, Fritze H (2001) J Environ Qual 30:1134–1143

15. Bolan NS, Adriano DC, Natesan R, Koo BJ (2003) J EnvironQual 32:120–128

16. Veeken A, Hamelers B (2002) Sci Total Environ 300:87–9817. Senesi N, Miano TM, Provenzano MR, Brunetti G (1991) Soil Sci

152:259–27118. Mobed JJ, Hemmingsen SL, Autry JL, McGown LB (1996)

Environ Sci Technol 30:3061–306519. Chen J, LeBoeuf EJ, Dai S, Gu B (2003) Chemosphere 50:639–64720. Bertoncini EI, D’Orazio V, Senesi N, Mattiazzo ME (2005) Anal

Bioanal Chem 381:1281–128821. Ryan DK, Weber JH (1982) Anal Chem 54:986–99022. Cabaniss SE (1992) Environ Sci Technol 26:1133–113923. Luster J, Lloyd T, Sposito G (1996) Environ Sci Technol

30:1565–157424. Gao K, Pearce J, Jones J, Taylor C, Taylor C (1999) Environ

Geochem Health 21:13–2625. Evangelou VP, Marsi M (2001) Plant Soil 229:13–2426. Elkins KM, Nelson DJ (2001) J Inorg Biochem 87:81–9627. Elkins KM, Nelson DJ (2002) Coord Chem Rev 228:205–22528. Ghatak H, Mukhopadhyay SK, Jana TK, Sen BK, Sen S (2004)

Wetlands Ecol Manage 12:145–15529. Provenzano MR, D’Orazio V, Jerzykiewicz M, Senesi N (2004)

Chemosphere 55:885–89230. Plaza C, D’Orazio V, Senesi N (2005) Geoderma 125:177–18631. Plaza C, Brunetti G, Senesi N, Polo A (2006) Environ Sci Technol

40:917–92332. Hernández D, Plaza C, Senesi N, Polo A (2006) Environ Pollut

DOI 101016/jenvpol20051103833. Miikki V, Senesi N, Hänninen K (1997) Chemosphere 34:1639–165134. Rivero C, Chirenje T, Ma LQ, Martinez G (2004) Geoderma

123:355–36135. Provenzano MR, De Oliveira SC, Santiago Silva MR, Senesi N

(2001) J Agric Food Chem 49:5874–587936. Provenzano MR, Albuzio A, D’Orazio V (2005) J Agric Food

Chem 53:374–38237. Brunetti G, Plaza C, Clapp CE, Senesi N (2006) Soil Biol

Biochem, (in press)38. Schnitzer M (1982) In Page BL, Miller RH, Keeney DR (eds)

Methods of soil analysis, part 2, chemical and microbiologicalproperties, 2nd edn. Agronomy Monograph No 9, SSSA,Madison, WI

39. Plaza C, Brunetti G, Senesi N, Polo A (2005) Environ Sci Technol39:6692–6697

40. Cory RM, McKnight DM (2005) Environ Sci Technol 39:8142–814941. Wu FC, Mills RB, Evans RD, Dillon PJ (2004) Anal Chem

76:10–11342. Irving HMNH, Williams RJP (1948) Nature 162:746–747

2140 Anal Bioanal Chem (2006) 386:2133–2140