Feasibility of using humic substances from compost to remove heavy metals (Cd, Cu, Ni, Pb, Zn) from contaminated soil aged for different periods of time

Accepted Manuscript Title: Feasibility of using humic substances from compost to remove heavy metals (Cd, Cu, Ni, Pb, Zn) from contaminated soil aged for different periods of time Author: Dorota Kulikowska Zygmunt Mariusz Gusiatin Katarzyna Bułkowska Barbara Klik PII: DOI: Reference:

S0304-3894(15)30014-5 http://dx.doi.org/doi:10.1016/j.jhazmat.2015.08.022 HAZMAT 17032

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

18-5-2015 9-7-2015 13-8-2015

Please cite this article as: Dorota Kulikowska, Zygmunt Mariusz Gusiatin, Katarzyna Bulkowska, Barbara Klik, Feasibility of using humic substances from compost to remove heavy metals (Cd, Cu, Ni, Pb, Zn) from contaminated soil aged for different periods of time, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.08.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Feasibility of using humic substances from compost to remove heavy metals (Cd, Cu, Ni, Pb, Zn) from contaminated soil aged for different periods of time   

Dorota Kulikowska* [email protected], Zygmunt Mariusz Gusiatin, Katarzyna Bułkowska, Barbara Klik  Department of Environmental Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland  *

Corresponding author.

 

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Highlights   Humic substances (HS) from compost showed good surfactant properties    Soil aging changed the distribution of Zn, Ni and Cu but not that of Pb and Cd   Cu, Ni and Zn removal with HS was greater in soils that had been aged less    Pb and Cd removal with HS was not affected by soil aging 

 

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Abstract  There is a need for inexpensive, readily-available and environmentally-friendly soil washing agents to remediate polluted soils. Thus, batch washing experiments were performed to evaluate the feasibility of using a solution of humic substances (HS) extracted from compost as a washing agent for simultaneous removal of Cu, Cd, Zn, Pb and Ni from artificially contaminated soils aged for 1 month, 12 months and 24 months. The efficiency of metal removal in single and multiple washings and kinetic constants (equilibrium metal concentration qe and rate constant k from the second-order kinetic equation) were determined. On average, triple washing removed twice as much metal as that removed with a single washing. At pH 7 and a HS concentration of 2.2 g C L-1, metal removal from all soils decreased in this order: Cd (79.1-82.6%) > Cu (51.5-71.8%) > Pb (44.8-47.6%) > Ni (35.446.1%) > Zn (27.9-35.8%). However, based on qe (mg kg-1), metal removal was in this order: Pb > Zn  Cu > Ni > Cd. This difference was due to different concentrations of metals, which is typical for multi-metal contaminated soils. Regardless of washing mode, removal of Cd and Pb was not affected by soil age, whereas removal of Cu, Ni and Zn was higher in soils that had been aged for a shorter time.  These results indicate that HS are suitable for remediating soil contaminated with multiple heavy metals in extremely high concentrations.  

Keywords: heavy metals; soil aging; soil washing; humic substances; biosurfactant

 

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1. Introduction Soil contamination with heavy metals is an important environmental problem that requires the development of suitable remediation technologies. One of these is soil washing, which allows removal of not only mobile but also more stable metals from soil. For soil washing, a variety of washing agents have been tested, i.e. inorganic acids, organic acids, chelating agents and natural biosurfactants. Although synthetic agents have

substantial capacity as washing agents for heavy-metal polluted soils, they cause environmental problems, which limits their use. Therefore, biosurfactants appear to be a more attractive choice, and microbial rhamnolipids [1] or plant-derived biosurfactants, such as saponin and tannic acid [2, 3, 4] have been shown to be suitable for metal removal. However, due to the relatively high cost of commercially available biosurfactants, current investigations are directed at searching for natural surfactants derived from waste or composted waste. Biosurfactants derived from these sources could have a considerable impact on the development of "green engineering" technologies. For example, the structural characteristics of humic substances (HS) from urban green waste led to predictions that these compounds would have good properties as complexing agents, ion-exchangers and surfactants [5]. This is because these substances are amphiphilic, with both hydrophobic and hydrophilic components. Humic -COOH and phenolic -OH groups are responsible for the formation of metal-humic complexes [6], and many studies indicate that the fulvic fraction (FF) has a higher capacity for metal binding than humic acids (HAs) because the FF has a higher number of reactive groups. For example, Borůvka and Drábek [7] showed that in heavily polluted soils of Fluvisol type, 98, 82, and 96% of organically bound Cd, Pb, and Zn, respectively, were in the FF. Donisa et al. [8] reported that 11 heavy metals were more abundant in the FF than in HAs in natural soils (andosol, podzol, cambisol). Fulvic acids (FAs) have a lower molecular weight and a higher content of functional groups than HAs, so they are assumed to

 

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form more soluble, mobile and bioavailable metal complexes than HAs, which means that FAs can act as metal carriers. These facts have encouraged industry and the scientific community to consider different sources of HS for soil washing, such as agricultural waste, municipal solid wastes and compost [9]. This is a promising trend because FAs constitute a substantial share of HS from waste sources. HS isolated from the litter layers of beech and Norway spruce forests have shown promise for removal of metal mixtures from contaminated soil [10]. However, the properties of HS depend mostly on their origin, and the use of HS as natural surfactants in soil remediation still needs research. When assessing the effectiveness of HS for soil washing, the age of contaminated soil also needs to be taken into account. Aging is considered an important factor in the availability and toxicity of metal in soil because metal bioavailability decreases with time [11]. Metal availability is highest immediately after soil contamination, then with time, reactions between metal ions and soils transform highly soluble forms of metal into less soluble ones. These reactions mainly include complexation, surface adsorption, exchange reactions, chelation, and precipitation of metal ions in the soil particle surface or diffusion into the mesopores and macropores of soil [12]. Therefore, the remediation efficiency of the same metal may vary with soil age. Despite this fact, to the authors’ knowledge, no data are available on the use of humic substances to simultaneously remove metal mixtures from soils aged for different periods of time after contamination. Therefore, this study tested the effectiveness of HS from sewage sludge compost for simultaneous removal of Cd, Cu, Ni, Pb and Zn from highly contaminated loam. Because soil aging affects metal distribution, which in turn may affect metal removal efficiency, the suitability of HAs was tested with loam aged for 1, 12 and 24 months after contamination. By using HS, the research follows the recommendations of the European Thematic Strategy for

 

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Soil Protection, particularly with regard to reducing soil pollution by using natural washing agents.

2. Materials and methods 2.1. Soil contamination and aging Samples of unpolluted surface soil (depth 0-30 cm) were collected from an agricultural area in north-eastern Poland. Soil was spiked with a mixture of Cd, Cu, Ni, Pb and Zn. After spiking, the soil was aged at room temperature for 1 month (soil 1), 12 months (soil 2) or 24 months (soil 3). Detailed procedure is given in Supplementary material. 2.2. Properties of aged soil The soil was classified as loam in texture. The aged soils had slightly acidic pH and medium content of organic matter (Tab. 1). Organic matter (OM) content fluctuated somewhat, indicating some decomposition of native organic matter over time. Although cation exchange capacity in all soils stayed on a similar level with soil aging, hydrolytic acidity decreased, and concentration of exchangeable bases increased. In soil, Pb occurred at the highest concentration, whereas Cd at the lowest. 2.3. Characteristic of compost - source of HS  The source of HS was compost from sewage sludge (feedstock composition: 60% sewage sludge, 5% inoculation, 15% wood chips, 15% rape straw, 5% grass) that had been composted for 170 days. The compost had neutral pH, contained 48.2% organic matter and 164.5 g C kg-1 OM of HS. Concentrations of heavy metals in compost were far below the limits given in the Polish law [14, 15] (in mg kg-1 d.w.): 0.89 Cd, 49.7 Cr, 46.5 Cu, 9.78 Fe, 0.032 Hg, 278 Mn, 22.3 Ni, 19.8 Pb, 255 Zn.

 

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HS were extracted from compost according to the methodology described in Jouraiphy et al. [16] and Amir et al. [17]. Detailed extraction procedure of HS from compost is given in Supplementary material.  2.5. Optimization of metal removal from aged soil by HS Metal removal efficiency was analyzed in dependence on following parameters: 1/40 (w/v) ratio of soil mass to volume of washing solution; pH values of HS solution: 3, 5, 7, 9, 11 and 13; times of extraction: 5, 10, 30, 60, 90, 120, 180, 240, 300, 360, 720 and 1440 min. Detailed description is given in Supplementary material. 2.6. Analytical methods  Typical analytical methods were used. A more detailed section for analytical methods, kinetic modeling equations and statistical analysis was given in Supplementary material.  3. Results and discussion  3.1. HS solution  The concentration of HS extracted from compost was 2170 mg C L-1. In HS, HAs were 58%, and the rest was fulvic fraction (FF) (Tab. 2). This proportion is typical in HS extracted from compost because the humification increases the content of HAs and decreases that of FF [16, 20, 21]. HA contained more phenolic OH than carboxylic groups.  The HS solution had a strongly alkaline pH because it was extracted with NaOH. Because the concentrations of heavy metals in the HS solutions were negligible compared to those in the extracts after washing, the metal content in the HS was disregarded in further experiments in this study. HS more effectively decreased surface tension at pH 7 than at pH 13 (Fig. 1). This indicates that their surface active properties are dependent on pH. Previous studies have also found that the surface tension of HS solutions is affected by changes in pH. Surface tension decreases as pH is lowered from 9 to 4 in solutions of HAs those were obtained from rivers

 

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and soil [22].  This behavior may be related to acidic group neutralization, a phenomenon that generates intramolecular electrostatic interactions that affects the physical shape of the acid groups. At high pH, acid groups are fully ionized and there is a strong repulsion between charged groups causing the molecule to expand [23].  At pH 7 in the present study, surface tension decreased as HS concentration was increased to 1101 mg L-1, after which it remained relatively constant (40.6 ±1.0 mN m-1); at pH 13, surface tension decreased as HS concentration was increased to 1215 mg L-1, then remained at 51.7 ±0.4 mN m-1 (Fig. 1). Based on changes in surface tension, it can be assumed that these HS started to aggregate into micelles around 1100 mg L-1. HA obtained from lignocellulosic waste compost decreased surface tension between 40.8 and 50.4 mN m-1 [24].  Comparing the values of critical micelle concentration (CMC) for HS with literature values is quite difficult because usually HAs are characterized instead of HS. HAs from sewage sludge composts have a lower CMC than vermicompost HAs [25]. The CMC values for HAs of different samples obtained from composts of lignocellulosic wastes range widely from 471 to 4040 mg L-1 [24]. Although CMC is generally considered an important parameter for removal of organic pollutants (i.e. PAHs), high concentrations of biosurfactants (above CMC) are needed when using them for metal removal from soil [2, 4, 26]. Because HS revealed some surface-active properties (i.e. decreasing of surface tension) and formed micellar aggregates, they can increase metal mobility in a similar way as surfactants. The washing process with the use of surfactant above the CMC enables interactions between the surfactant hydrophilic anions and metal cations. In the present study, HS were used at concentration above CMC and at pH 7, which could reduce sorption of HS on soil and make them available for metal bonding. According to Wang and Mulligan [27],

 

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HAs might enhance the mobilization of heavy metals from mine tailings mainly through formation aqueous heavy metal-humate complexes. For example, stability of HAs and metal complexes changed in the order of Cu > Pb > Zn and the complexing capacity order was Pb > Cu > Zn [28]. This is attributed to dissociation of functional groups such as carboxylic and phenolic. Carboxyl groups dissociate at acidic or neutral pH, whereas phenolic groups at alkaline. This suggests that in the present study carboxyl groups were more responsible for metal complexation than phenolic groups, because soil washing was performed at pH 7. In addition, decreasing of surface tension may help to transfer theses complexes from soil into solution. 3.2. Metal transformations in soil aged for different periods of time  The results of metal fractionation are given in Fig. 2. In soil 1, Cd and Zn prevailed in exchangeable and acid soluble fraction (F1) followed by reducible fraction (F2), whereas metal content in the most stable fractions was only 2.4% and 9.6% of the total concentration, respectively. Similarly, Zhang et al. [29] found that Zn prevailed in carbonate and reducible fractions in soil. The low content of Cd in most stable fractions results from its low constant of ion adsorption to organic matter. Cd does not appear to form a strong complex with organic matter [30]. There was a relatively high Ni content in F1 fraction (42.4%). The second, most abundant metal concentration was F2 fraction. In contrast to Cd and Zn, Ni showed higher percentage content in fractions F3 and F4 (Fig. 2c). Using artificially contaminated soil of similar texture and pH as in the present study, Kuziemska et al. [31] found that regardless of initial Ni concentration (50 or 100 mg kg-1), about 70% was in exchangeable and acid soluble fraction and 14% in reducible fraction. However, this soil contained less organic matter. In F1 fraction, the percentage content of Cu was lower than Cd, Zn, Ni, but higher in the reducible fraction. Among all metals, percentage content of Cu in F3 and F4 fractions was the highest. Pb prevailed in reducible fraction (83%, Fig. 2d). Lead can either coprecipitate with metal

 

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oxides or can be adsorbed at the metal oxide surface [32]. Although, in the present study, metal-spiked soil was used, similar Pb distribution was observed in soils near Zvecan smelter in Kosovo. Pb concentration in the reducible fraction ranged from 32.1 to 3496 mg kg-1 (55 to 86% of its total content) [33].  Soil aging affected metal distribution in individual fractions, but a degree of redistribution was dependent on metal type. Redistribution of heavy metals in soils is characterized by initial fast retention and subsequent slow transformations. Soil aging favors metal adsorption, complexation and chemical reaction with soil components like oxides, organic matter and clay minerals. However, these reactions can depend on the chemical nature of the elements, their concentration and soil properties [34]. With the increase of aging time from 1 to 24 months, the content of F1 fraction for Cu, Ni, Pb and Zn gradually decreased (p < 0.05). For Cd, after 24 months of aging, there was a decrease of F1 and increase of F2. This means that Cd remained highly mobile for a long time. Ni and Zn were redistributed into reducible and oxidizable fractions. Therefore, the distribution pattern of these two metals in soil aged for short and long period was different. The content of Pb in F2 fraction did not change significantly with time, but it could be redistributed among different oxides. According to BCR procedure, metal in F2 fraction is associated with amorphous Fe and Mn (hydro)oxides. These oxides strongly sorb metals. Initially, these are in exchangeable forms, but increasingly with time they are transformed to less mobile, specifically adsorbed forms [35]. Probably due to strong bonding of Pb with Fe and Mn oxides and its high concentration in the F2 fraction (83% of total concentration, on average), Pb was difficult to redistribute into more stable fractions, i.e. the oxidizable and residual fractions, despite 24-months of incubation. Cu redistributed mainly among F1 and F3 fractions. In soil aged for 24 months, content of F2 decreased with p < 0.05 compared to soil

 

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aged for 1 and 12 months (Fig. 2b). According to Han et al. [34], metal at low concentration can be more effectively redistributed in soil than metal at high concentration. For individual metals in soils of different aging, the percentage content of F1 fraction (Fig. 2), corresponds to mobility factor (MF). Thus, metals were categorized as very highly mobile (Cd), highly mobile (Ni, Zn), medium mobile (Cu) and low mobile (Pb). Regardless of soil aging, Cd mobility was above 50%. Similarly, for Pb there was no effect of aging on MF values; however these values for Pb were 5-fold lower (8-9%). The higher decrease in MF with soil aging was observed for Zn (from 48 to 31%), Ni (from 42 to 29%) and Cu (from 19 to 13%). 3.3. The effect of pH on the efficiency of heavy metal removal from soils aged for different periods of time When the HS were extracted, the solution pH was about 13. However, because metal solubility depends on pH, soil washing was performed using a fixed concentration of HS (2170 mg C L-1) at pHs ranging from 3-13. With all metals in soils 1-3, removal efficiency depended on pH (Fig. 3).  Cd and Zn removal efficiency was highest at pH 3, ranging from 73-63% (soil 1-3) for Cd and 34-22% (soil 1-3) for Zn (Fig. 3). With both metals, efficiency gradually decreased as pH increased, and was lowest at pH 13. Greater removal of Cd and Zn at low pH is rather a typical phenomenon. Hong et al. [2], with saponin, showed that Cd was not removed from different soils at pH > 6; at pH 3, however, 90-100% of Cd was removed, and 85-98% of Zn. In contrast to removal of Cd and Zn, the efficiency of Cu removal with HS increased as pH increased and was highest at pH 13 (72% with soil 1 and 58% with soil 3). At pH 3, Cu removal efficiency was only 23% (soil 3) and 29% (soil 1). Other authors have reported similar findings for removal of Cu and have suggested that improved Cu removal at higher pH is due to increased formation of Cu-humate

 

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complexes. These complexes do not form at pH 2 because of strong competition from H+ for COOH binding sites [36]. HS greatly increase the mobility of Cu both in alkaline conditions and at the pH range 5-9, commonly found in the natural environment [37]. Many organicmatter molecules dissociate protons in strongly basic conditions and become negatively charged, which makes it easier for them to complex and remove Cu2+ ions. Because HS contain functional groups as dissolved organic matter, it is reasonable to suppose that they remove Cu via the same mechanism. The presence of HAs in the washing solution reduced metal precipitation at high pH, probably due to the formation of soluble metal-humate species, and improved mobilization of As, Zn, Pb and Cu [38]. This is because phenolic hydroxyl (OH) groups tend to dissociate at pH 8-13 and can react with metal cations, which is a highly probable mechanism for Cu removal at high pH. In the present study, it is also likely that Cu removal in alkaline conditions was increased by reactions between dissociated phenolic OH and Cu cations because the HS from sewage sludge compost contained more HAs than FF, and the HAs contained more phenolic OH than carboxylic groups. However, because the HS were extracted from compost with 0.1 M NaOH, it is also possible that sodium ions linked as counterions to the HS molecules were responsible for removal of Cu from the soil by ion exchange [39]. It is likely that this also occurred in the present study because 13-19% of total Cu in aged soil was in F1 fraction (Fig. 2). With Ni and Pb, the changes in removal efficiency with pH differed from those mentioned above. Ni removal was least affected by pH: at pH 3, it was 36-24% (soil 1-3); at pH 13, 26-17% (soil 1-3). Pb removal was highest at pH 7 (43% soil 1, 32% soil 3). Pb removal from kaolinite with EDTA and EDDS was very high in the pH from 4 to 10 (> 80%) and that it decreased significantly at pH below 4 [40]. In contrast, Pb removal efficiency with citric acid was approximately 50% at pH 4 and decreased with increasing pH.

 

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The optimal pH for leaching metal varies depending on metal type [41; this study], type of washing agent [40] and its concentration [42; this study]. Tsang et al. [42] found that three different humic acids (leonardite humic acid, standard humic acid from IHSS and Aldrich humic acid) at two concentrations (20 mg L-1 and 100 mg L-1) and pH 4 had no effect on Cu, Cr and As extraction because of substantial adsorption of HAs. In an acidic environment, HAs may physically or chemically immobilize heavy metals rather than enhance their mobilization. The findings of the present study differ from those of Tsang et al. [42]. This may be due to the fact that the HS concentration was 2170 mg L-1 in this study (of which 58% was HAs, i.e. 1259 mg C L-1), but the HAs concentration was only 100 or 20 mg L-1 in Tsang et al. [42]. Taking into account the fact that various pHs were best for removing the different metals in this study (e.g. Cu, Cd, Pb), and considering the possible negative impact of extreme pH on soil properties, pH 7 was selected for further research.  3.4. Kinetics of heavy metal removal with HS  The study analyzed the kinetics of heavy metals removal (data not shown). Regardless of soil age, metal removal proceeded relatively quickly. Short extraction times during soil washing to remove heavy metals are rather typical, even with other washing agents. For example, Cu and Ni removal using chitosan and EDTA reaches a relatively high level at 2 h, when 38.72% of Cu and 51.25% of Ni were removed [43]. In this study, the removal of each heavy metal followed second-order kinetics. With all soils, the equilibrium concentration of Pb that was removed from the soil was higher than the individual concentrations of the other metals that were removed, the concentration of Cd was lower than those of the other metals, and the concentrations of Cu and Zn were similar. Interestingly, the k value was highest for Cd and lowest for Pb, regardless of soil age (Tab. 3). This indicates that Cd was the most mobile in soil, and Pb, the least mobile. The relative sizes

 

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of the rate constants were as follows: Cd > Ni > Zn ≈ Cu > Pb. In contrast, when saponin is used instead of HS for removal of heavy metals from sandy clay loam, clay loam and clay, removal follows first order kinetics and the relative sizes of the rate constants (Zn > Cd > Cu > Pb) differ from those in our study, with the highest rate constant being for removal from sandy clay loam [2].  It is worth emphasizing that in this study, although the rate constants for removal of the various metals differed by as much as an order of magnitude (Cd, Pb), the equilibrium concentration for all metals was obtained after 3 hours of extraction. This is because different k values affect mainly the first several minutes of extraction. Thus, from a practical point of view, the time needed to obtain equilibrium conditions is more useful than the k value. 3.5. Efficiency of heavy metal removal in dependence of soil aging in single-soil washing with HS  Heavy metals removal with HS was tested with soil aged 1 month (soil 1), 12 months (soil 2) and 24 months (soil 3). Whereas removal of Cd and Pb was not affected by soil age, removal of Cu, Ni and Zn was lower in soils that had been aged longer (Fig. 4). Cd was removed most efficiently (44%), and its removal was significantly more efficient than that of Pb (24-25%). This difference in efficiency is due to the fact that the metals were distributed differently. In all soils, Cd was present mainly in the F1 fraction, with only 2.4% of Cd in the most stable fractions (F3 and F4). The MF of Cd was ca. 60%, while that of Pb was six times lower (ca. 10%). Because soil aging (1, 12 and 24 months) did not influence the distribution of Cd and Pb, the removal efficiency by single washing of individual metals from soils 1-3 was similar. The efficiency of Cu removal from soil 1 was high (42%), but the efficiency of its removal from soil 3 was almost 1.7 times lower. The efficiency of Zn and Ni removal also decreased with greater soil age: from 23% to 12% (Zn), and from 26% to 17% (Ni). There

 

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was related to decrease in the MF of each metal with increased soil age (Cu, from 19% to 13%; Zn, from 48% to 31%; Ni, from 42% to 29%). Metal mobility could be crucial factor influencing process efficiency during first washing. Initial faster desorption of heavy metals from soil indicated the release of these metals from the water-soluble fraction and also from the adsorption sites of lower bonding energy (exchangeable fraction). Slower desorption of metals indicated release of metals from sites of relatively higher bonding energy than the exchangeable form [44]. Notably, the efficiency of Cu removal was higher than would be expected from the MF values. This indicates that not F1 but also F2 form are amenable to removal by a single soil washing with HS. In this study, the efficiency of heavy metal removal from loam with HS differed according to soil age but was generally in the following sequence: Cd > Cu > Pb > Ni > Zn. When comparing this result with those of other studies, it should be remembered that the efficiency of soil washing depends on the washing agent used, the type of soil and the operational conditions. When removing heavy metals from sandy loam, the efficiency of the washing agents was ethylenediaminetetraacetic acid > citric acid > tartaric acid, and metal extraction yields were typically in the following sequence: Cu > Ni > Zn > Cd > Pb [45]. Borggaard et al. [10] showed that HS extracted up to 45% and 54% of Cd and Cu from calcareous soils but only 4% of total Pb and 17% of Ni. In contrast, extraction of both Ni and Pb was about 30% with EDTA and NTA. This led to conclude that HS can replace synthetic washing agents to remove cadmium, copper, and optionally, nickel from contaminated soil, but that HS seem unsuited for Pb removal. Here, Pb removal by a single washing with HS (26-17% for soil S1-S3) was, however, higher than in Borggaard et al. [10], which could be because of the difference in soil type (slightly acidic loam rather than calcareous soil). This

 

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suggests that HS are more appropriate for removing Pb from acidic than from calcareous soils. However, it must be remembered that the efficiency of soil washing can also be affected by co-existing metals. In particular, this has been shown in the case of chelating agents, which are nonspecific and can remove different ions from soil. Zhang et al. [46] found that at low EDTA concentration (below stoichiometric requirements), Pb was removed from soil much more efficiently than Zn. When EDTA concentration was greater, Pb and Zn removal efficiencies were similar. Use of an excess of chelating agent may minimize the possible competition effects between target heavy metals and interfering cations (e.g. Al, Ca, Fe, Mg, Mn) during soil washing [47]. It was found that Pb extraction from soil at a pH above 6 decreased its removal due to competition between Pb and Ca or Mg for the EDTA coordination sites [48]. Gusiatin et al. [49] found that during simultaneous metal removal from soil, saponin, a plant biosurfactant, removed more Cu than Pb and Zn. Due to excessive Cu concentration in soil, it could outcompete other metals, especially Pb, for dissociated carboxylic groups on saponin. Based on the qe values (Tab. 3), the relative amounts of metal removed from all soils were as follows: Pb > Zn  Cu > Ni > Cd. However, the efficiency of metal removal was in the following sequence: Cd > Cu > Pb > Ni > Zn. Thus, to assess the suitability of washing agents for remediation of multi-metal contaminated soil, two criteria should be considered: the equilibrium concentrations of metals in the washing solution after washing (qe) and metal removal efficiency (E). The qe value allows determination of the amount of metal removed at equilibrium, which is crucial to assess the feasibility of using a washing agent to remove different metals. In contrast, E shows how removal of a metal depends on operational conditions (i.e., single or multiple washing, and pH) and soil aging. Therefore, in this study, E

 

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was used to assess the effectiveness of HS for removing metals from aged soils, and to evaluate the effects of multiple washings on the removal of individual metals by HS. 3.6. Removal of metals and their fractions from soils during multiple soil washing with HS During removal of all metals, process efficiency significantly increased with an increased number of washes (p < 0.05) (Fig. 5). With Cd, triple washing gave the highest efficiency of removal for any single metal and was 1.8 times more effective than single washing. The high efficiency of Cd removal was due to its release from the F1 fraction, which contained the largest amount of Cd in all soils. Thus, the distribution pattern of Cd in treated soil changed substantially due to its removal from the F1 fraction (Figs. 2a and 5a). Similar to Cd removal, triple washing with HS nearly doubled Pb removal (from 2425% to 47-45%), regardless of soil aging. Although the efficiency of Pb removal was lower than that of Cd removal, its total concentration decreased by about 1800-1900 mg kg-1, which demonstrates that HS can remove a substantial amount of Pb. Because Pb was mostly in F2 fraction (Fig. 2d), its distribution pattern did not change after triple washing, although total Pb decreased in all soils (Fig. 5d). A large decrease in Pb concentration after washing is not typical, because usually Pb is difficult to remove from soils or other solid materials. For example, despite 10 consecutive washings with rhamnolipids, only 15% of Pb was removed from mine (3780 mg Pb kg-1) and ore contaminated (23 900 mg Pb kg-1) soils. The content of Pb in the residual fraction was mainly responsible for limiting Pb removal from both soils [50]. In the case of Cu, removal efficiency after triple washing decreased with longer soil aging after contamination: 72% (soil 1) > 64% (soil 2) > 52% (soil 3) (Fig. 5b). For comparison, triple washing with saponin removed 86% of Cu from loam soil aged for 3 months after spiking [3]. Therefore, HS remove Cu slightly less efficiently than commercial biosurfactant. HS were able to remove Cu from all fractions, even the most stable. Although

 

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the total efficiency of Cu removal from older soils was less, the relative efficiency of its removal from the individual fractions followed the same order (Fig. 5b). In soil 1, Cu was removed from the F1, F2, F3 and F4 fractions with efficiencies of 92, 77, 47 and 37%, respectively; whereas in soil 3, it was removed from these fractions with efficiencies of 86, 60, 26 and 22%, respectively. After washing, the distribution pattern of Cu was different from that before washing (Fig. 2b). Zn and Ni had comparable distribution patterns in the polluted soils, and they were removed in a similar manner (Fig. 5). Both metals were effectively removed from the F1, but less effectively from the F2 fraction. Although the amounts of these metals that were in the F1 and F2 fractions depended on the aging process, both metals were predominately in the F2 fraction after washing (Fig. 5c, e). The present study indicates that HS from sewage sludge compost are a promising washing agent for reducing soil pollution in slightly acidic and heavily polluted soils. Even with three-step soil washing, single extractants may not be sufficient to remove metals from soils heavily contaminated with multiple metals. Thus, as with other washing agents, sequential extraction using extractants in addition to HS may be needed to remove multiple contaminants from loam. 4. Conclusions In multi-metal contaminated soil, the aging process affected the mobility of Zn, Ni and Cu but not that of Cd and Pb. The efficiency of Cu, Ni and Zn removal with HS from sewage sludge compost decreased with greater soil aging, while that of Pb and Cd removal did not depend on soil aging. Based on equilibrium metal concentrations (qe, mg kg-1) metals were removed in this order: Pb > Zn  Cu > Ni > Cd. Multiple washing with HS significantly improved removal of all metals and affected the distribution pattern of all except Pb. HS from sewage

 

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sludge compost are a cheap, promising agent for reducing soil pollution in slightly acidic and heavily polluted soil.  Acknowledgement  The study was supported by a Ministry of Science and Higher Education in Poland (Statutory Research, 528-0809-0801).

 

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References  [1] C.N. Mulligan, Recent advances in the environmental applications of biosurfactants, Curr. Opin. Colloid Interface Sci. 14 (2009) 372-378. [2] K.-J. Hong, S. Tokunaga, T. Kajiuchi, Evaluation of remediation process with plantderived biosurfactant for recovery of heavy metals from ontaminated soils, Chemosphere 49 (2002) 379-387. [3] Z.M. Gusiatin, E. Klimiuk, Metal (Cu, Cd and Zn) removal and stabilization during multiple soil washing by saponin, Chemosphere 86 (2012) 383-391. [4] Z.M. Gusiatin, Tannic acid and saponin for removing arsenic from brownfield soils: Mobilization, distribution and speciation. J. Environ. Sci. (China) 26 (2014) 855-864. [5] E. Montoneri, V. Boffa, P. Savarino, D.G. Perrone, G. Musso, R. Mendichi, M.R. Chierotti, R. Gobetto, Biosurfactants from urban green waste, ChemSusChem. 2 (2009) 239247. [6] K.M. Spark, J.D. Wells, B.B. Johnson, The interaction of a humic acid with heavy metals, Aust. J. Soil Res. 35(1997) 89-101. [7] L. Borůvka L, O. Drábek, Heavy metal distribution between fractions of humic substances in heavily polluted soils, Plant Soil Environ. 50 (2004) 339-345. [8] C. Donisa, R. Mocanub, E. Steinnes, Distribution of some major and minor elements between fulvic and humic acid fraction in natural soils, Geoderma 111 (2003) 75-84. [9] X. Mao, R. Jiang, W. Xiao, J. Yu, Use of surfactants for the remediation of contaminated soils: A review, J. Hazard. Mater. 285 (2015) 419-435.

 

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[10] O.K. Borggaard, P.E. Holm, J.K. Jensen, M. Soleimani, B.W. Strobel, Cleaning heavy metal contaminated soil with soluble humic substances instead of synthetic polycarboxylic acids, Acta Agr. Scand. B-S. P. 61 (2011) 577-581. [11] M.A. Wijayawardena, R. Naidu, M. Megharaj, D. Lamb, P. Thavamani, T. Kuchel, Influence of ageing on lead bioavailability in soils: a swine study, Environ. Sci. Pollut. Res. 22 (2015) 8979-8988. [12] M. Jalali, Z.V. Khanlari, Effect of aging process on the fractionation of heavy metals in some calcareous soils of Iran, Geoderma 143 (2008) 26-40. [13] OME, Ordinance of the Minister of Environment on soil and ground quality standards, J. Law. 165 (2002) 10561-10564. [14] OMARD, Ordinance of the Minister of Agriculture and Rural Development on fertilizers and fertilization, J. Law. 236 (2004) 16834-16839 (in Polish). [15] OMARD, Ordinance of the Minister of Agriculture and Rural Development on fertilizers and fertilization, J. Law. 119 (2008) 6515-6520 (in Polish). [16] A. Jouraiphy, S. Amir, M. Gharous, J.C. Revel, M. Hafidi, Chemical and spectroscopic analysis of organic matter transformation during composting of sewage sludge and green plant waste, Int. Biodeter. Biodegr. 56 (2005) 101-108. [17] S. Amir, M. Hafidi, J.R. Bailly, J.C. Revel, Structural characterization of humic acids, extracted

from

sewage

sludge

during

composting,

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chromatography–mass spectrometry, Process Biochem. 41 (2006) 410-422. [18] М.М. Kononova, Soil Organic Matter, PWRiL, Warsaw, Poland, 1968.

 

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[19] M. Pueyo, J. Mateu, A. Rigol, M. Vidal, J.F. Lopez-Sanchez, G. Rauret, Use of the modified BCR three-step sequential extraction procedure for the study of trace element dynamics in contaminated soils, Environ. Pollut. 152 (2008) 330-341. [20] C. Paredes, M.P. Bernal, J. Cagarra, A. Roig, Biodegradation of olive mill wastewater sludge by its co-composting with agricultural wastes, Bioresour. Technol. 85 (2002) 1-8. [21] D. Kulikowska, E. Klimiuk, Organic matter transformations and kinetics during sewage sludge composting in a two-stage system, Bioresour. Technol. 102 (2011) 10951-10958. [22] L.M. Yates, R. von Wadruszka, Effects of pH and metals on the surface tension of aqueous humic materials, Soil Sci. Soc. 63 (1999) 1645-164. [23] E. Tombácz, Colloidal properties of humic acids and spontaneous changes of their colloidal state under variable solution conditions, Soil Sci. 164 (1999) 814-824. [24] G. Quadri, X. Chen, J.W. Jawitz, F. Tambone, P. Genevini, F. Faoro, F. Adani. Biobased surfactant-like molecules from organic wastes: The effect of waste composition and composting process on surfactant properties and on the ability to solubilize tetrachloroethene (PCE), Environ. Sci. Technol. 42 (2008) 2618-2623. [25] V.A. Ramírez Coutiño, L.G. Torres Bustillos, L.A. Godínez Mora Tovar, R.J. Guerra Sánchez, F.J. Rodríguez Valadez, pH effect on surfactant properties and supramolecular structure of humic substances obtained from sewage sludge composting, Rev. Int. Contam. Ambie. 29 (2013) 191-199. [26] S. Mukhopadhyay, M.A. Hashim, J.N. Sahu, I. Yusoff, B.S. Gupta, Comparison of a plant based natural surfactant with SDS for washing of As(V) from Fe rich soil, J. Environ. Sci. (China) 25 (2013) 2247-2256.

 

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[27] S. Wang, C.N. Mulligan, Enhanced mobilization of arsenic and heavy metals from mine tailings by humic acid, Chemosphere 74 (2009) 274-279. [28] G. Abate, J.C. Masini, Acid-base and complexation properties of a sedimentary humic acid, A study on the Barra Bonita Reservoir of Tiete River, Sao Paulo State, Brazil, J. Brazil. Chem. Soc. 12 (2001) 109-116. [29] W. Zhang, H. Huang, F. Tan, H. Wang, R. Qiu, Influence of EDTA washing on the species and mobility of heavy metals residual in soils, J. Hazard. Mater. 173 (2010) 369-376. [30] M.A. Kashem, B.R. Singh, T. Kondo, S.M.I. Hug, S. Kawai, Comparison of extractability of Cd, Cu, Pb, and Zn with sequential extraction in contaminated and noncontaminated soils, Int. J. Environ. Sci. Te. 4 (2007) 169-176. [31] B. Kuziemska, S. Kalembasa, W. Wieremiej, Distribution of nickel in fractions extracted with the BCR procedure from nickel-contaminated soil, J. Elem. 3 (2014) 697-708. [32] Q.M. Jaradat, A.M. Massadeh, M.A. Zaitoun, B.M. Maitah, Fractionation and sequential extraction of heavy metals in the soils of scrap yard of discarded vehicles, Environ. Monit. Assess. 112 (2006) 197-210. [33] F. Nannoni, G. Protano, F. Riccobono, Fractionation and geochemical mobility of heavy elements in soils of a mining area in northern Kosovo, Geoderma 161 (2011) 63-73. [34] F.X. Han, A. Banin, W.L. Kingery, G.B. Triplett, L.X. Zhou, S.J. Zheng, W.X. Ding, New approach to studies of heavy metal redistribution in soil, Adv. Environ. Res. 8 (2003) 113-120. [35] O. Kaplan, M. Yaman, G. Kaya, Distribution of nickel in different phases of soil samples and plant parts taken from serpentine and copper mining area, Asian J. Chem. 21 (2009) 5757-5767.  

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[36] R.M. Town, H.K.J. Powell, Ion-selective electrode potentiometric studies on the complexation of copper (II) by soil-derived humic acids, Anal. Chim. Acta 279 (1993) 221233. [37] J. Wu, L.J. West, D.I. Stewart, Effect of humic substances on Cu(II) solubility in kaolinsand soil, J. Hazard. Mater. 94 (2002) 223-238. [38] S. Wang, C.N. Mulligan, Enhanced mobilization of arsenic and heavy metals from mine tailings by humic acid, Chemosphere 74 (2009) 274-279. [39] C.M. Mulligan, R.N. Yong, B.F. Gibbs, S. James, H.P.J. Bennett, Metal removal from contaminated soil and sediments by the biosurfactant surfactin, Environ. Sci. Technol. 33 (1999) 3812-3820. [40] M. Niinae, K. Nishigaki, K. Aoki, Removal of lead from contaminated soils with chelating agents, Mater. Trans. 49 (2008) 2377-2382. [41] Z. Zou, R. Qiu, W. Zhang, H. Dong, Z. Zhao, T. Zhang, X. Wei, X. Cai, The study of operating variables in soil washing with EDTA, Environ. Pollut. 157 (2009) 229-236. [42] D.C.W. Tsang, W.E. Olds, P. Weber, Residual leachability of CCA-contaminated soil after treatment with biodegradable chelating agents and lignite-derived humic substances, J. Soils Sediments 13 (2013) 895-905. [43] W. Jiang, T. Tao, Z. Liao, Removal of heavy metal from contaminated soil with chelating agents, OJSS 1 (2011) 70-76. [44] G. Kandpal, P.C. Srivastava, B. Ram, Kinetics of desorption of heavy metals from polluted soils: influence of soil type and metal source, Water Air Soil Poll. 161 (2005) 353363.

 

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[45] R.A. Wuana, F.E. Okieimen, J.A. Imborvungu, Removal of heavy metals from a contaminated soil using organic chelating acids, Int. J. Environ. Sci. Tech. 7 (2010) 485-496. [46] W.H. Zhang, I.M.C. Lo, EDTA-enhanced washing for remediation of Pb-and/or Znontaminated soils, J. Environ. Eng. ASCE 132 (2006) 1282-1288. [47] Z.A. Begum, I.M.M. Rahman, Y. Tate, S. Sawai, T. Maki, H. Hasegawa, Remediation of toxic metal contaminated soil by washing with biodegradable aminopolycarboxylate chelants, Chemosphere 87 (2012) 1161-1170. [48] R.W. Peters 1999. Chelant extraction of heavy metals from contaminated soils, J. Hazard. Mater. 66 (1999) 151-210. [49] Z.M. Gusiatin, K. Bułkowska, T. Pokój, Tannic acid as a cost-effective substitute for saponin in soil remediation, Environ. Biotech. 10 (2014) 66-72. [50] J.W. Neilson, J.F. Artiola, R.M. Maier, Characterization of lead removal from contaminated soils by nontoxic soil-washing agents, J. Environ. Qual. 32 (2003) 899-908.

25

Figure Captions 75

pH 7

70

pH 13

-1  (mN m )

65 CMC = 1215 mg L-1

60 55 50 45 40 35

CMC = 1101 mg L-1

30 0

500

1000

1500

2000

2500

-1

CSH (mg L )

Fig. 1. Changes of surface tension of the humic substances depending on their concentration and critical micelle concentration (CMC) (n = 3).

26

a)

b) F1

100

F1

100

F2

F2 F3

80 a

F4

40

b

a

a

60

b

20 a

a

soil 1 (48.1)

a

b

40

20 a

0

a

%

a

%

60

F3

80

F4

a

b

soil 2 (46.2)

b

a

a

a

b b

c b

a

c

0

soil 3 (48.4)

soil 1 (992.3)

soil 2 (951.3)

soil 3 (995.9)

d)

c) F1

100

a

F3

80

a

F2

a

F3

80

F4

F4

60 a

40

b a

%

60 %

F1

100

F2

b

b

40

c c

20

a

20

b

a

b

c

a

b

a

a

a

a

0

0 soil 1 (523.3)

soil 2 (503.9)

soil 1 (4043.9)

soil 3 (519.3)

soil 2 (4048.6)

c

b b

soil 3 (4088.0)

e) F1

100

F2 F3

80

soil 2 - aged for 12 months

F4

soil 3 - aged for 24 months

60 a

%

soil 1 - aged for 1 month

c

b a

b

40

c

20

c a

b

a

b

b

0 soil 1 (2062.1)

soil 2 (1954.3)

soil 3 (2074.0)

Figure 2. Percentage content of metal in individual fractions in aged soil: a) Cd, b) Cu, c) Ni, d) Pb, e) Zn. The sum of metal concentration (in mg kg-1) in individual fractions is given in brackets. For a given fraction in soil 1-3 means followed by the same letter are not significantly different at p < 0.05, using the Tukey HSD test.

27

b) 80

80

60

60

E (%)

E (%)

a)

40 20

40 20

0

0 0

2

4

6

8

10

12

14

0

pH soil 1

2

4

6

8

10

12

14

pH

soil 2

soil 3

soil 1

soil 2

soil 3

d) 80

80

60

60

E (%)

E (%)

c)

40

40 20

20

0

0 0

2

4

6

8

10

12

14

0

2

4

6

soil 1

soil 2

soil 1

soil 3

8

10

12

14

pH

pH soil 2

soil 3

E (%)

e) 80

soil 1 - aged for 1 month

60

soil 2 - aged for 12 months

40

soil 3 - aged for 24 months

20 0 0

2

4

6

8

10

12

14

pH soil 1

soil 2

soil 3

Fig. 3. The efficiency (E) of washing with HS at different pH in removing metals from soils that were aged for different periods of time: a) Cd, b) Cu, c) Ni, d) Pb, e) Zn (n = 3).

28

a)

b) 50

a

a

a

50

a

E (%)

E (%)

40 30

30

20

20

10

10

0

b

40

c

0

soil 1

soil 2

soil 3

soil 2

soil 3

a

a

a

soil 1

soil 2

soil 3

d) 50

50

40

40

30

E (%)

E (%)

c)

soil 1

a b

20

c

30 20

10

10

0

0

soil 1

soil 2

soil 3

e) 50

soil 1 - aged for 1 month

E (%)

40 30

soil 2 - aged for 12 months a

soil 3 - aged for 24 months

b

20

c 10 0

soil 1

soil 2

soil 3

Figure 4. The efficiency (E) of a single washing with HS in removing metals from soils that were aged for different periods of time: a) Cd, b) Cu, c) Ni, d) Pb, e) Zn. For a given metal in soil 1-3, means followed by the same letter are not significantly different at p < 0.05, using the Tukey HSD test.

29

a)

b) 1st washing

2nd washing

c

E (%)

80

3rd washing

b

2nd washing

c

a

40

3rd washing

c

b

a

a

1st washing

80

b

b

60

100

c

c

E (%)

100

60

b a

c b

a

40

a 20

20

0

0 soil 1

soil 2

soil 3

soil 1

c)

soil 2

soil 3

d) 100

1st washing

2nd washing

3rd washing

100

2nd washing

3rd washing

80

60

c

c

b

40

c

b

a

b

a

a

20

E (%)

E (%)

80

1st washing

60 40

c a

c

c

b

b

b a

a

20

0

0 soil 1

soil 2

soil 3

soil 1

soil 2

soil 3

e) 100

1st washing

2nd washing

3rd washing

soil 1 - aged for 1 month

E (%)

80

soil 2 - aged for 12 months

60

soil 3 - aged for 24 months

40

a

b

c a

20

b

c

b

b

a

0 soil 1

soil 2

soil 3

Fig. 5. The efficiency (E) of triple washing with HS in removing metal from soils that were aged for different periods of time after contamination: a) Cd, b) Cu, c) Ni, d) Pb, e) Zn. For a given metal in individual soil, means followed by the same letter are not significantly different at p < 0.05, using the Tukey HSD test.

30

a)

F1

100

F1

100

F2

F2

80

F3

b

F4

60 b

40 a

b

20

b a

a

soil 1 (10.3)

soil 2 (11.4)

soil 3 (10.2)

0

a

40

a

c)

a

a

b

soil 2 (339.0)

soil 3 (482.0)

F1

100 a

a

F2

a

F3

80

F4

F4

b

60

%

%

a

a

soil 1 (298.1)

F3

60

a

a

d)

F2

80

b b

a

a

0 F1

100

a

a

%

a

a

20

F3

80

F4

a

a

60

%

b)

40

40 a

a a

20

a

a a

a a

b

20

a

a a

a

a

a a

a

a

0

0 soil 1 (313.6)

soil 2 (357.3)

soil 1 (2036.7)

soil 3 (636.2)

e)

soil 2 (1993.2)

soil 3 (1987.9)

F1

100

F2 F3

80 a

b

a

F4

soil 1 - aged for 1 month

%

60

soil 2 - aged for 12 months 40 a 20

a a

a a

a

b

a

a

soil 3 - aged for 24 months

0 soil 1 (1201.8)

soil 2 (1336.7)

soil 3 (1423.5)

Fig. 6. Distribution patterns of heavy metals (in %) after triple washing with HS of soils that were aged for different periods of time after contamination: a) Cd, b) Cu, c) Ni, d) Pb, e) Zn. Residual metal concentrations (as the sum of the concentrations in individual fractions, in mg kg-1) are given in brackets. For a given metal, difference in percentage content of individual fraction in soil 1-3 followed by the same letter are not significantly different at p < 0.05, using the Tukey HSD test.

31

Tables Table 1. Characteristics of metal contaminated soil aged for different periods (tA) (mean ± standard deviation, n = 3). Mean values followed by the same letters do not differ significantly at p < 0.05.

Unit

Soil 1 (tA = 1 month)

Soil 2 (tA = 12 months)

Soil 3 (tA = 24 months)

pHKCl

–

5.8 (±0.1)a

6.01 (±0.1)a

6.3 (±0.1)b

OM

%

12.4 (±0.32)a

14.3 (±0.36)b

10.6 (±0.27)a

TOC

%

Characteristic

3.3 (±0.26)a

3.5 (±0.25)a

2.8 (±0.18)a

cmol(+) kg

-1

4.0 (±0.13)a

3.3 (±0.3)a

2.7 (±0.2)b

S

cmol(+) kg

-1

34.8 (±0.4)a

35.6 (±0.4)a

37.0 (±0.4)a

CEC

cmol(+) kg-1

Kh

38.8 (±1.2)a

38.9 (±1.6)a

40.1 (±2.2)a

mg kg

-1

46.0 (±1.4)a

46.0 (±1.2)a

47.0 (±0.4)a

Cu

mg kg

-1

1019 (±19)a

981 (±26)a

1020 (±16)a

Ni

mg kg-1

534 (±13)a

510 (±10)a

498 (±25)a

Pb

mg kg

-1

4133 (±105)a

3924 (±101)a

4094 (±62)a

mg kg

-1

2070 (±41)a

1952 (±81)a

2110 (±88)a

Cd

Zn

32

Table 2. Characteristics of HS extracted from sewage sludge compost (mean ± standard deviation, n = 3).

Characteristic Humic substances (HS)

Unit mg C L

Value -1

2170 (±20)

Humic acid, HA (as % of HS)

%

58.0 (±3.5)

Fulvic fraction (as % of HS)

%

42.0 (±2.4)

pH

–

12.7 (±0.2)

meq 100 g-1

862 (±10)

COOH groups

%

21.2 (±2.6)

OH groups

%

78.8 (±2.9)

Cd

mg L-1

0.015 (±0.0)

Cu

mg L-1

2.1 (±0.01)

Ni

-1

mg L

0.19 (±0.001)

Pb

mg L-1

0.1 (±0.02)

Zn

mg L-1

1.6 (±0.07)

Total acidity of HA

 

 

33

Table 3. Kinetic constants for removal of heavy metals from soil with HS.

Soil No.

qe (mg kg-1) Cu

Ni

Cd

Pb

Zn

Soil 1

380.4

140.8

16.4

1000

427.0

Soil 2

322.0

121.9

17.1

1000

333.0

Soil 3

302.5

96.4

16.9

959

244.0

-1.

-1

k (kg mg min ) -3

3.23·10

-3

8.10·10-3

1.92·10-4

2.45·10-3

Soil 1

2.27·10

Soil 2

3.57·10-4

5.92·10-4

5.42 ·10-3

9.80·10-5

3.27·10-4

Soil 3

2.33·10-4

3.65·10-4

3.87·10-3

1.61·10-4

1.93·10-4

       

 

34