Chemosphere 114 (2014) 35–39

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Chemosphere

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PAHs in decaying Quercus ilex leaf litter: Mutual effects on litterdecomposition and PAH dynamics

http://dx.doi.org/10.1016/j.chemosphere.2014.03.1150045-6535/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +39 0824 305115; fax: +39 0824 305147.E-mail address: fdenicol@unisannio.it (F. De Nicola).

F. De Nicola a,⇑, D. Baldantoni b, A. Alfani b

a Dipartimento di Scienze e Tecnologie, Università degli Studi del Sannio, Via Port’Arsa 11, 82100 Benevento, Italyb Dipartimento di Chimica e Biologia, Università degli Studi di Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy

h i g h l i g h t s

�Mutual influences of PAH dynamics and leaf litter decay have been demonstrated.� After 246 d of decomposition PAH accumulation in litter bags was observed.� No effect of air PAH contamination on soil PAH content via litterfall was showed.

a r t i c l e i n f o

Article history:Received 11 February 2014Received in revised form 28 March 2014Accepted 31 March 2014

Handling Editor: Myrto Petreas

Keywords:Litter PAH contaminationDecompositionC/N ratioLitter bagsHolm oak

a b s t r a c t

The investigation of the relationships between litter decomposition and polycyclic aromatic hydrocar-bons (PAHs) is important to shed light not only on the effects of these pollutants on fundamental ecosys-tem processes, such as litter decomposition, but also on the degradation of these pollutants by soilmicrobial community. This allows to understand the effect of atmospheric PAH contamination on soilPAH content via litterfall. At this aim, we studied mass and PAH dynamics of Quercus ilex leaf litters col-lected from urban, industrial and remote sites, incubated in mesocosms under controlled conditions for361 d. The results highlighted a litter decomposition rate of leaves sampled in urban > industrial > remotesites; the faster decomposition of litter of the urban site is also related to the low C/N ratio of the leaves.The PAHs showed concentrations at the beginning of the incubation of 887, 650 and 143 ng g�1 d.w.,respectively in leaf litters from urban, industrial and remote sites. The PAHs in litter decreased alongthe time, with the same trend observed for mass litter, showing the highest decrease at 361 d for theurban leaf litter. Anyway, PAH dynamics in all the litters exhibited two phases of loss, separated by aPAH increase observed at 246 d and mainly linked to benzo[e]pyrene.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs), consisting of two ormore fused aromatic rings, share most of persistent organic pollu-tant (POP) characteristics (Belis et al., 2011). Urbanization andindustrial activities concur to widespread emissions in atmosphereof PAHs that are transported along a wide range and distributedthrough the global environment. In terrestrial ecosystems, airPAH depositions affect both plants and soil, which represents along-term sink. However, it is not clear if atmospheric PAH con-tamination necessarily leads to soil PAH accumulation. Vegetationcanopies play an important role in cycling and fate of PAHs,enhancing their downward fluxes (Horstmann and McLachlan,1996) with a consequent higher load in woodland than in

non-woodland soils due to filter effect of the canopy (Matzner,1984; McLachlan and Horstmann, 1998). The PAHs deposited andretained by tree leaves can also be delivered to soil via litterfall(Simonich and Hites, 1994), accumulating in soil surface organiclayers. Sometimes, however, low PAH concentrations in urban soilhave been observed, notwithstanding the high PAH accumulationsin the leaves of overlaying canopies (Maisto et al., 2004). In addi-tion, the PAHs from the soil, due to its high storage capacity, canreturn to the atmosphere only with difficulty, as discussed withinthe framework of fugacity approach (Wania, 1999; Wania andMcLachlan, 2001; Belis et al., 2011). Although a wide literatureon atmospheric transport and deposition of PAHs is available, theirfate in terrestrial ecosystems has been rarely explored (Howsamet al., 2001). To date, the whole PAH cycling in the environmentand the effects of these pollutants on litter decomposition, a keystep in matter cycle, affecting the overall ecosystem functionality,are poorly understood.

36 F. De Nicola et al. / Chemosphere 114 (2014) 35–39

The decomposition process is influenced by many environmen-tal factors (Gholz et al., 2000), litter chemical composition(Coûteaux et al., 1995) and decomposer community characteristics(Strickland et al., 2009). Bacterial and fungal communities are ofteninvestigated in relation to their ability to produce extracellularnon-specific enzymes, able to decompose aromatic polymers aslignin and consequently also PAHs (Bogan and Lamar, 1996;Baldrian, 2006). Although specialized microbial community candegrade PAHs via either metabolism or co-metabolism, a lot ofthese pollutants can be degraded slowly (Fernández-Luqueñoet al., 2011). PAHs are recalcitrant compounds because their aro-matic structure and their low water solubility limit their bioavail-ability and degradation rate. PAH bioavailability can be altered alsoby invertebrates feeding on decaying materials (Uffindell et al.,2005; Dendooven et al., 2011). Although several microbial speciesare able to degrade low molecular weight PAHs, relatively fewmicroorganisms have been observed to degrade high molecularweight PAHs (Acevedo et al., 2011).

Conversely, PAHs can also affect decomposition and mattercycle by influencing health and survival of detritivorous microin-vertebrates, by altering their physical and trophic niches (Blakelyet al., 2002) and, in turn, affecting the activity and the structureof microbial communities by microphagy (Uffindell et al., 2005;Muckian et al., 2007).

Goal of this work is to relate the litter decomposition rate andpollution degree of leaf litters from sites affected by different airPAH loads (De Nicola et al., 2011b). This with the aim to highlightthe mutual influences of PAHs on litter decomposition and theeffects of litter decay on PAH dynamics. At this purpose, leaves ofQuercus ilex L., the climax species of the Mediterranean basin, werecollected from urban, industrial and remote sites, incubated in lit-ter bags in mesocosms under controlled conditions and monitoredfor PAH content and litter mass.

2. Materials and methods

2.1. Samplings

Mature one-year leaves were collected from holm oaks (Q. ilexL.) located at three sampling sites in Campania region, southernItaly: an urban square characterized by high vehicular traffic flow(M: 40�510N, 14�130E, 180 m a.s.l.), an industrial site with an activefoundry (F: 40�450N, 14�460E, 195 m a.s.l.) and a remote site farfrom direct emission sources of pollutants (V: 40�490N, 14�230E,400 m a.s.l.). The three sites were previously described and charac-terized for their air PAH loads (De Nicola et al., 2011b). The studyarea is characterized by a Mediterranean climate, with dry andhot summer and wet and cold winter. Mean annual temperatureis 17.4 �C and total amount of precipitation is 865.6 mm, accordingto temperatures and precipitations over the last 30 years (Mazza-rella, http://www.meteo.unina.it/clima-di-napoli).

The fresh leaves, collected from the outer part of the canopy of 8trees from each site, were pooled and deprived of petioles. A por-tion was oven-dried (75 �C) to constant weight, grounded into afine powder by an agate mortar and pestle (Fritsch Analysette Pul-verisette 0), and characterized for C and N concentrations by gaschromatography (CHNS-O analyzer, Thermo Flash EA 1112). Theremaining portion was air-dried to constant weight and used to lit-ter bags set up. For this purpose we chose to use fresh one-yearleaves, as often made (see Baldantoni et al., 2013 and referencestherein), in relation to the difficulty to collect older leaves, due totheir scant presence in the canopies of trees of urban environment(De Nicola et al., 2011a,b). However, we also analyzed PAH concen-trations in both fresh and brown senescent leaves just before thenatural fall from branches. Since PAH concentrations in fresh and

senescent leaves showed comparable values (data not shown),we were confident to use fresh leaves for litter bags set up andfor PAH dynamic study.

2.2. Litter bag and mesocosm preparation

A known amount of air-dried leaves (approximately 3.5 g) plusan identification label were placed in each nylon litter bag(120 � 150 mm and 1 mm-mesh in order to allow the migrationof small detritivores, minimizing the loss of small litter pieces).

In order to set up mesocosms reproducing a soil ecosystem, 15plastic containers (35 � 65 � 20 cm) were filled with the soil col-lected from the top layer (0–20 cm) in a natural holm oak forest(40�410N, 14�450E, 235 m a.s.l.), without altering soil profile neitherremoving litter layer. Soil PAH concentration was on average140 ng g�1 d.w. We set up 6 mesocosms for leaf litter from M site,6 for F and 3 for V. Twelve litter bags were placed on the soil sur-face of each mesocosm. All the mesocosms were incubated in anunlighted and climatized chamber (27 �C, RH 70%) and the litterswere irrigated once a week with deionized water for all the dura-tion of the experiment. The same origin of the soils used in themesocosms (characterized by the same chemical and biologicalproperties), and the same incubation conditions, allow attributingthe possible variations in decomposition rates among different leaflitters uniquely to their provenances.

During one year, from the start of the incubation (0 d) and after39, 105, 177, 246, 303 and 361 d, at each time 5 litter bags wererandomly sampled from both M and F mesocosms, whereas 3 litterbags were randomly collected from V mesocosms, to determine themass loss. Simultaneously, other litter bags were randomly col-lected for PAH analyses: 6 litter bags at each time from 0 to303 d and 12 litter bags at 361 d of incubation, for both M and Fmesocosms, and 4 litter bags at each time for V mesocosms. No lit-ter bags from V mesocosms were collected after 361 d for mass losseither PAH analyses.

2.2.1. Mass loss and PAH analysesAt each sampling, M, F and V leaf litters were cleaned with

tweezers to remove soil particles, fungal hyphae, invertebrate eggsand ejecta, as carefully as possible.

The mass loss was determined gravimetrically, after leaf litteroven-drying at 75 �C until constant weight, and expressed asremaining mass as percentage of the initial value. To calculatethe decomposition rate, the exponential decay model was appliedand the k constant calculated using Olson (1963) equation:

lnðMt=M0Þ ¼ �kt

where Mt is the litter mass at time t (d) and M0 is the initial littermass.

For PAH quantification, the material from two litter bags waspooled to reach a quantity of sample suitable as a replicate forPAH extraction. PAH analyses were carried out in triplicates forM and F and in duplicate for V. Leaf litter samples, mixed to anhy-drous sodium sulphate and added to deuterated PAHs (d10 ace-naphthene, d10 phenanthrene, d12 chrysene, d12 perylene), wereextracted by 3 sonications (Misonix, XL2020) in a mix dichloro-methane–acetone. The final extracts were vacuum rotary evapo-rated, filtered and gently dried under a N2 stream. Consecutively,the dried residues were re-dissolved in 4 mL of cyclohexane andanalyzed by GC-MSD (HP 5890-HP 5971 with HP-5MS capillarycolumn 30 m � 0.25 mm i.d. � 0.25 lm film thickness). Heliumwas used as carrier gas (constant flow rate of 1.11 mL min�1).The oven temperature program started at 70 �C and increased, withramp rate 20 �C min�1, to 280 �C and held for 24 min. The selectedion monitoring acquisition mode was used. Acenaphthylene (Acy),

Table 1Mean concentrations of C and N (% d.w.) in Q. ilex fresh leaves sampled at the urban(M), industrial (F) and remote (V) sites. Values of C/N ratio are also reported. Inbrackets the standard errors, expressed as percentage of the mean concentrations, arereported. Different letters indicate statistically significant differences (a = 0.05).

M F V

C 44.39 (4%)a 46.28 (2%)ab 48.92 (6%)b

N 1.55 (4%)a 1.36 (7%)b 1.49 (6%)ab

C/N 28.69a 34.20b 32.83b

39 105 177 246 303 3610

20

40

60

80

100M (urban)F (industrial)V (remote)

Days

L

itter

mas

s (%

initi

al v

alue

)

Fig. 1. Remaining mass, as percent of the initial value, of Quercus ilex leaf littersfrom M (urban), F (industrial) and V (remote) sites, during decomposition. Verticalbars represent standard errors.

F. De Nicola et al. / Chemosphere 114 (2014) 35–39 37

acenaphthene (Ace), fluorene (Flu), phenanthrene (Phen), anthra-cene (Ant), fluoranthene (Flt), pyrene (Pyr), benzo[a]anthracene(B[a]A), chrysene (Crys), benzo[b + k]fluoranthene (B[b + k]F), ben-zo[e]pyrene (B[e]P), benzo[a]pyrene (B[a]P), perylene (Per),indeno[1,2,3-c,d]pyrene (IP), dibenzo[a,h]anthracene (DB[a,h]A)and benzo[g,h,i]perylene (B[g,h,i]P) were measured after a calibra-tion curve has been performed for each compound. The percentrecovery of deuterated PAHs, ranging from 66% to 72%, was usedto correct the quantification of all the investigated PAHs.

PAH concentrations (Pij) were expressed as percentages of theirinitial values, according to the equation reported in Baldantoniet al. (2013):

Pij ¼Cij �Wi

C0j �W0� 100

where C0j is the initial concentration of the j-th PAH analyzed, W0

the initial litter weight, Cij the concentration of the j-th PAH atthe i-th sampling and Wi the litter weight at the i-th sampling.

In this study, PAHs refer to the sum of the concentrations of the17 compounds, the LMW (low molecular weight) PAHs to the sumof PAHs from Acy to Flu, the MMW (medium molecular weight)PAHs to the sum of PAHs from Phen to Crys, and the HMW (highmolecular weight) PAHs to the sum of PAHs from B[b + k]F toB[g,h,i]P.

2.3. Statistical analyses

The significance of differences among sites for C and N concen-trations in fresh leaves, as well as for PAH concentrations in leaf lit-ters at the start of the incubation (0 d), was assayed by one-wayanalysis of variance (ANOVA), followed by Newman–Keuls posthoc test (with a = 0.05). Normality was assayed by Kolmogorov–Smirnov test. The analyses were performed with the Prism Vs 5(GraphPad Software).

The significance of the differences in the mass and in PAHdynamics among M, F and V leaf litters were evaluated by one-way analysis of covariance (ANCOVA) with the site as fixed factorand the time as covariate, followed by the Tukey post hoc test.Non-metric Multidimensional Scaling (NMDS), a non-parametricordination technique (Legendre and Legendre, 1998), based onthe Euclidean distance and with the superimposition of the tempo-ral gradient, was performed in order to evaluate the variations indynamics of PAH profiles. To highlight the differences among thesites and the PAH molecular weight classes, the confidence ellipses(for a = 0.05) were also superimposed. The analyses were per-formed with the R programming language (R Core Team) withthe functions of the ‘‘stats’’ (R Core Team, 2013) and ‘‘vegan’’(Oksanen et al., 2013) packages.

39 105 177 246 303 3610

50

100

150

300350 M (urban)

F (industrial)V (remote)

Days

PAH

s (%

initi

al v

alue

)

Fig. 2. PAH dynamics, as percent of the initial value, of Quercus ilex leaf litters fromM (urban), F (industrial) and V (remote) sites, during decomposition. Vertical barsrepresent standard errors.

3. Results

3.1. Decomposition dynamics

The characterization of Q. ilex fresh leaves showed significantdifferences among sites for carbon (F = 4.638, P < 0.05), nitrogen(F = 6.069, P < 0.01) and C/N ratio (F = 16.050, P < 0.001). Carbonconcentration in leaves from the remote site (V) was significantlyhigher than in leaves from the urban site (M), the last ones show-ing nitrogen concentration significantly higher than that measuredin leaves from the industrial site (F). Thereby, for M fresh leaves thesignificantly lowest C/N ratio was obtained (Table 1).

The litter decomposition dynamics highlighted for each of thethree sites a fast mass loss from 0 to 246 d, when M, F and V litterslost respectively 68%, 59% and 50% of their initial weight (Fig. 1).Then, at the last sampling time, the mass decreased at a slower

rate, reaching values of remaining mass equal to 33%, 44% and49% for M, F and V litters, respectively (Fig. 1). Overall, the decom-position dynamics significantly (F = 22.363, P < 0.001) differedamong leaf litters, with values of remaining mass higher(P < 0.001) in V than in both F and M leaf litters along the time.The daily decomposition constants (k day�1), calculated at 303 d,were 0.00354 ± 0.00018 for M, 0.00301 ± 0.00017 for F and0.00237 ± 0.00017 for V leaf litters and the variances (R2) explainedby the three curves were 0.7892, 0.7204 and 0.7574, respectively.

3.2. PAH dynamics

At the start of the incubation (0 d), PAH concentrations in leaflitters from the three sites significantly (F = 106.700, P < 0.001) dif-fered: litters from M and F sites showed PAH concentrations (887and 650 ng g�1 d.w., respectively) 6- and 4-fold higher than litterfrom V site (143 ng g�1 d.w.).

PAH dynamics in leaf litters (Fig. 2) were significantly(F = 9.147, P < 0.001) different among the M, F and V sites. Signifi-cantly lower values were found along the time in M litter (P < 0.001in respect to both V and F litters), whereas higher (P < 0.001) valueswere observed in V litter in respect to F and M litters.

After the first sampling time (39 d), a decrease of 10% in PAHvalue was observed only in M litter, whereas V litter showed anincrease of 50% in respect to the initial value (Fig. 2). At 177 d ofincubation, PAHs reached the lowest values showing, respectivelyfor M and F litters, a decrease of 85% and 66% in respect to the

Fig. 3. Non-metric Multidimensional Scaling (NMDS) biplot of PAHs, as percent oftheir initial values, in Quercus ilex leaf litter from M (urban), F (industrial) and V(remote) sites. PAHs are indicated by their abbreviations, reported in the Materialsand Methods section, while black dots indicate the observations. The temporalgradient (grey lines) and the confidence ellipses (a = 0.05) for the sites (solid lines)and the PAH molecular weight classes (LMW, MMW and HMW PAHs, dashed lines)are also shown. Confidence ellipses are labelled according to the groups to whichthey refer.

38 F. De Nicola et al. / Chemosphere 114 (2014) 35–39

initial values, and in V litter a decrease of 38% (Fig. 2). At 246 d,PAHs increased in all the three leaf litters, reaching in V litter thehighest value (304%), with a following decrease until the last sam-pling time (Fig. 2). In fact, at 303 d, V litter reached PAH value equalto 158% than that measured at the start of the incubation. At 361 d,M litter lost 85% and F litter lost 50% of their initial PAHs (Fig. 2).

NMDS analysis (Fig. 3) showed a clear separation among thethree sites in relation to PAH dynamics. In particular, V is betterdifferentiated from the others by the higher value of Phen, whereasM and F were distinguished, although with slightly overlappingconfidence ellipses, for the higher values of DB[a,h]A and B[e]P,respectively. MMW PAHs showed different dynamics as comparedwith LMW and HMW PAHs, being higher between 0 and 220 d,whereas HMW and LMW PAHs were higher between 220 and280 d of decomposition. Moreover, MMW PAHs were higher in Vlitter, whereas HMW and LMW PAHs were higher in M and F lit-ters. The analyzed PAHs clustered into three main groups in rela-tion to their dynamics: Phen, Ant, DB[a,h]A, B[a]A, Pyr and Fltwere associated to the earlier stages of decomposition (0–220 d),whereas B[g,h,i]P and IP to the latter (280 d to the end) and allthe others to an intermediate stage (220–280 d).

4. Discussion

The carbon and nitrogen concentrations (and consequently C/Nratio) of the investigated holm oak leaves, although differingamong the three sites, fell in the range reported in other studies(Alfani et al., 2000; Baldantoni et al., 2011,2013). The lowest C/Nratio in leaves from the urban site, due to the lowest carbon andhighest nitrogen concentration, can be affected by the foliar uptakeof nitrogen oxides (Alfani et al., 2000). In leaves from the urbansite, the lowest C/N ratio, a fundamental factor influencing litterdecomposition rate, could have determined the fastest decomposi-tion with a mass loss of 67% at 361 d of incubation. The decompo-sition rate of the three litters (urban > industrial > remote) showeddaily decomposition constants within the range of values

previously calculated for Q. ilex leaf litter decaying (Baldantoniet al., 2011). A faster decay for urban than remote litter was previ-ously reported for European aspen leaves (Nikula et al., 2010) andattributed to litter quality (nitrogen, lignin and polyphenols). Thedifferences in leaf palatability and toughness as well as in nutrientcontents (Nikula et al., 2010) cannot be excluded to explain the dif-ferent decomposition rates in litter from urban, industrial andremote sites.

The higher PAH concentrations, at the start of incubation, in lit-ter from the urban and industrial sites than from the remote site,reflected the spatial trend of PAH depositions widely reported inour previous biomonitoring studies (De Nicola et al., 2011b;Baldantoni et al., 2014). Because of the low PAH concentrations(140 ng g�1 d.w.) in the soil used to set up the mesocoms, a contri-bution to litter PAH content is to rule out.

The relationship between decomposition and PAH contamina-tion is an important issue because it sheds light both on the eco-system functioning and the biodegradation of these pollutants.All leaf litters studied showed at 177 d of incubation a decreaseof PAHs, likely attributable to microbial community that can usebenzenic molecules (in our study mainly medium molecularweight PAHs) as carbon source (Uyttebroek et al., 2006). FromPAH polluted soil, some PAH-degrading bacteria of genera Pseudo-monas, Burkholderia, Sphingomonas and Mycobacterium were iso-lated (Wick et al., 2001; Wattiau et al., 2002). Moreover, anactive chemotactic transport induced by PAHs themselves mayincrease the rate of pollutant dissipation by microbial community(Ortega-Calvo et al., 2003).

The PAH dynamics for all the leaf litters showed an astonishingPAH increase at 246 d, highest for litter from remote site, mainlylinked to benzo[e]pyrene. Analyzing these data together with thoseof litter mass loss, we can note that at 246 d not only a PAHincrease occurs, but the litter has lost all the more easily decom-posable compounds, showing the maximum values of mass loss.The concomitance of these data can do us to suppose a biodegrada-tion by fungal metabolism of complex aromatic polymers such aslignin (the last in decomposition order to be attacked) in otherlower molecular weight aromatic compounds (Song, 2009) andtheir rearranging. However, several pieces of evidence are presentfor a biological formation of PAHs, from soil microorganisms, underanaerobic conditions, and fungi and bacteria linked to termites (seeBandowe et al., 2009 and references therein). Moreover, the finaldecrease in PAHs may also be due to ligninolytic enzymes involvedin lignin degradation that happens in the last stage of leaf litterdecomposition. These enzymes can also be involved in the degra-dation of a wide range of organopollutants such as PAHs(Hammel et al., 1986; Kästner, 2000; Pointing, 2001). PAH degrada-tion is reported to need the cooperation between white rot fungiand bacteria: the first can oxidize PAHs increasing their bioavail-ability that in turn can increase their mineralization by bacteria(Baldrian, 2008). The high fungal colonization of the littersobserved during the last samplings (data not shown) can supportthis hypothesis for PAH decrease.

Besides the role of decomposition on PAH degradation, alsosome effects of PAHs on litter decomposition were previouslyreported. Qasemian et al. (2012) reported that anthracene favorsfungal community, less sensitive than the bacterial community tothis pollutant, and that the addition of anthracene onto the litterincreases fungal ligno-cellulolytic activities, thus enhancing the lit-ter decomposition. So the higher PAH loads in urban and industriallitters could have favored not only decomposition of the leaf litterfrom these sites respect to litter from remote one, but also a fasterdegradation of PAHs. This hypothesis is consistent with previousdata (Maisto et al., 2004) that highlighted low PAH concentrationsin urban soil notwithstanding high PAH accumulation in leaves ofoverlaying holm oak canopies.

F. De Nicola et al. / Chemosphere 114 (2014) 35–39 39

5. Conclusions

The chemical characteristics of Q. ilex leaves, related to theirexposition to different PAH loads, are responsible for a decomposi-tion rate in urban > industrial > remote leaf litters. Moreover, dif-ferent molecular weight PAHs are associated to different timesalong decomposition and a mutual influence between PAH dynam-ics and leaf litter decay has been demonstrated. Anyway, to betterunderstand the influences on a fundamental process, such usdecomposition, in terrestrial ecosystems affected by PAHs, itappears important to analyze not only the effect of these contam-inants on litter decay dynamics, but also their impact on the com-munities of the degradative succession in decaying litter. Theresults obtained press us to carry out further studies to select fun-gal strains able to degrade PAHs, suitable in bioremediation of soilsaffected by persistent organic pollutants.

Acknowledgments

We wish to thank Dr. Claudia Lancellotti for her precious assis-tance in the field and laboratory work during the Ph.D. and Dr.Alessandro Bellino for his help in statistical analyses. This workwas financially supported by Ministry of Education, Universitiesand Research (MIUR), Italy (PRIN 2004 – PROT_2004057727_004).

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