Immobilization of potentially toxic metals using different soil amendments

Immobilization of potentially toxic metals using different soil amendments

Chemosphere 85 (2011) 577–583 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Immobiliz...

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Chemosphere 85 (2011) 577–583

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Immobilization of potentially toxic metals using different soil amendments D. Tica, M. Udovic ⇑, D. Lestan Agronomy Department, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Received 11 March 2011 Received in revised form 20 June 2011 Accepted 21 June 2011 Available online 20 July 2011 Keywords: Bioavailability Potentially toxic metals (PTMs) Soil functionality Soil remediation Stabilization

a b s t r a c t The in situ stabilization of potentially toxic metals (PTMs), using various easily available amendments, is a cost-effective remediation method for contaminated soils. In the present study, we investigated the effectiveness of apatite and a commercial mixture of dolomite, diatomite, smectite basaltic tuff, bentonite, alginite and zeolite (Slovakite) on Pb, Zn, Cu and Cd stabilization by means of decreasing their bioavailability in contaminated soil from an old lead and zinc smelter site in Arnoldstein, Austria. We also investigated the impact of 5% (w/w) apatite and Slovakite applications on soil functionality and quality, as assessed by glucose-induced soil respiration, dehydrogenase, acid and alkaline phosphatase and b-glucosidase activity. Both amendments resulted in increased soil pH and decreased PTM potential bioavailability assessed by diethylenetriamine pentaacetic acid extraction and by sequential extractions in the water-soluble and exchangeable fractions. The efficiency of stabilization was reflected in the soil respiration rate and in enzymatic activity. The b-glucosidase activity assay was the most responsive of them. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Potentially toxic metals (PTMs) in soil are not biodegraded by natural processes, as organic contaminants are, and they therefore persist in soil for a long time after their introduction, leading to long term (chronic) effects in the environment (Bolan et al., 2003). To a certain extent, soils have a natural capacity to attenuate the bioavailability and mobility of PTMs by means of various mechanisms, such as precipitation, adsorption processes and redox reactions. However, when their concentrations become too high for the soils to limit their potential effect, PTMs can become mobilized, leading to soil and groundwater contamination and increasing the possibility of entering the human body, whether by accidental soil ingestion, by breathing contaminated soil dust particles or by the ingestion of polluted drinking water or food produced on contaminated soil (Mulligan et al., 2001). Choosing an adequate remediation technique in accordance with the site characteristics, types and concentration of pollutants and the further use of the contaminated soil is therefore necessary to limit the risk of polluted soils to organisms and for reclamation of the contaminated site. Conventional techniques of soil remediation, such as excavation, transport and landfilling of contaminated soils and wastes are effective but very expensive. Research is therefore focused on

⇑ Corresponding author. Address: Center for Soil and Environmental Science, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. Tel.: +386 1 320 32 05; fax: +386 1 423 10 88. E-mail address: [email protected] (M. Udovic). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.06.085

alternative remediation techniques, of which stabilization and immobilization of metals in soil are very promising because of their simplicity but high effectiveness, their in situ applicability and their low cost (Guo et al., 2005). PTM stabilization is a remediation technology based on adding easily available amendments to polluted soil (e.g. cement, apatite, zeolites, lime), in order to reduce the mobility and bioavailability of metals in the soil without altering their total concentration (Friesl-Hanl et al., 2009; Lee et al., 2009). It has been reported that the addition of phosphates and carbonates to soil increases soil alkalinity and consequently decreases the mobility and bioavailability of PTMs (Hettiarachchi and Pierzynski, 2004). For the purpose of our study, we used apatite Ca10(PO4)6(OH, F, Cl2), an effective phosphate stabilization agent, which in general acts through ionic substitution and replacement of Ca2+ by Ba2+, Sr2+, Cd2+ and Pb2+ in its crystalline structure (Srinivasan et al., 2006) and through precipitation of pyromorphite-type minerals (Kumpiene et al., 2008). Slovakite is an inorganic sorbent mixture of natural raw materials (dolomite, diatomite, smectite, basaltic tuff, bentonite, alginite, and zeolite) with a high sorption affinity for Pb, Zn, Cu, and Ni ions, developed for the remediation of PTM polluted soils (product disclaimer). Its carbonate compound can also immobilize PTMs in soil by supplying alkalinity to the soil, causing the precipitation of PTM-bearing insoluble soil constituents or directly by promoting PTM complexation with soil particles. PTMs occur in various soil ‘pools’ of different solubilities, chemical characteristics and, consequently, different functions (Ure, 1995). It is known that PTM is mandatory for predicting their mobility and bioavailability, as well as the efficiency of soil amendments. Various selective non-exhaustive extraction techniques are

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used to assess the potential bioavailability of metals in soil. Sequential extractions are used to assess different operationally defined PTM fractions in soil, discriminating PTMs according to their physico-chemical associations with other soil components, mobility and bioavailability (Mulligan et al., 2001). The basis of chelant extraction with diethylenetriamine pentaacetic acid (DTPA) (Lindsay and Norvell, 1978) and EDTA (Kosson et al., 2002) used in the study is PTM complexation with a ligand supplied by a metal ligand salt, allowing the desorption of PTMs from the organically bound phases and partially from the oxides and clay minerals (Ure, 1995). Neutral salt-based extraction with CaCl2 (Novozamsky et al., 1993) and inorganic soft-acid based extraction with ammonium acetate (Kalra and Maynard, 1991) are based on the removal of PTMs from the soil solid phase into solution by desorbing cations (McLaughlin et al., 2000). While several studies have focused on the ability of various inorganic amendments to reduce the bioavailability of PTMs in polluted soils as the primary measure of soil remediation effectiveness, the impact of such amendments on soil quality and functional recovery has only recently been gaining more systematic attention. Soil quality is usually defined as the capacity of a soil to fulfill its unique ecosystem functions (Wang et al., 2006). Soil microbial activity has an important role in soil organic matter turnover and element cycling, thus affecting all organisms in the soil. PTMs can affect microbial and, consequently, enzymatic activity in soil, either inhibiting or enhancing such activity (Kandeler et al., 1996; Zhang et al., 2010). This can therefore be used as a means of monitoring soil functional degradation due to pollution and soil functional recovery/restoration after a remediation intervention, in addition to monitoring soil physical and chemical characteristics (Wang et al., 2006; Garau et al., 2007; Lee et al., 2009). Considering that soil enzymes catalyze specific reactions, which can depend on various factors (e.g., soil pH, temperature, inhibitors, etc.), it is difficult to obtain an overall picture of soil status from a single enzymatic activity value (Pascual et al., 2000). We therefore used a set of commonly used enzymatic assays and compared them, in order to identify the most suitable one for monitoring the effect of stabilization amendments on polluted soil functional recovery. The first objective of this study was to investigate the effect of increasing application rates of apatite and Slovakite on PTM (Pb, Zn, Cu, Cd) stabilization resulting in reduced bioavailability. The second objective was to assess the impact of the most effective PTM stabilization amendment on soil functional recovery and to identify the most suitable enzymatic assay and chemical extraction test for monitoring the impact of the amendment applied to the polluted soil.

2. Materials and methods 2.1. Soil and soil analyses Lead and zinc smelting activity in Arnoldstein (Austria), which caused the dispersion of PTMs in the environment, ceased in 1992. The smelter had been active for more than 500 yr and, during that period, Pb, Zn and Cd, and to a lesser extent Cu and As were emitted. These contaminants were dispersed over the surrounding area, which has been used for housing (playgrounds), horticulture, forestry and agriculture (Friesl-Hanl et al., 2009). Soil samples were collected from the upper 30 cm surface layer (November 2009). For standard pedological analyses, non-amended and amended soils were air-dried and sieved to 2 mm. Soil pH was measured in a 1/2.5 (w/v) ratio of soil and 0.01 M CaCl2 suspension. Soil samples were analyzed for organic matter by modified Walkley–Black titrations (ISO 14235, 1998), cation exchange capacity (CEC) by the

ammonium acetate method (Rhoades, 1982), soil texture by the pipette method (Fiedler et al., 1964) and total N content after dry combustion (ISO 13878, 1995), carbonates manometrically after soil reaction with HCl (ISO 10693, 1995) and easily extractable P (P2O5) colorimetrically according to the Olsen method (Kalra and Maynard, 1991). Pedological analyses were performed in triplicate. 2.2. Experimental design The study was conducted in two parts. In the first part, we tested the effect of the addition of various amounts of Slovakite and apatite on Pb, Zn, Cu and Cd potential bioavailability. The following characteristics were assessed for the amendments: the CEC was 91.5 and 7.2 mmol C+ 100 g 1 for Slovakite and apatite, respectively, and the pH was 8.7 and 7.8, respectively. The carbonate content of Slovakite was 47%. We added 1%, 2.5% and 5% (w/w) of Slovakite (IPRES, Bratislava, Slovak Republic), sieved to 250 lm, and apatite (tri-calcium phosphate, Riedel-de Haën) to sieved (5 mm) fresh soil sampled in Arnoldstein. The percentage of the added amendment refers to dry soil weight. Equal amounts of non-amended soil and well mixed amended soils were placed in 300 mL plastic flasks, watered to 100% field capacity and incubated in the dark at 14 °C for 1 month. The soil moisture was regularly checked and adjusted as necessary. The experiments were performed in triplicate. At the end of the incubation time, the potential bioavailability of Pb, Zn, Cu and Cd in non-amended and amended soils was assessed by means of widely used selective non-exhaustive chemical extractions, as described below: extraction with DTPA, with calcium chloride, with ammonium acetate and with ethylenediamine tetraacetic acid (EDTA). The most effective amendment in terms of reducing the potential bioavailability of Pb, Zn, Cu and Cd, as assessed on the basis of single extraction tests (i.e. extractions with DTPA, CaCl2, NH4OAc and EDTA), was chosen for the second part of the study. In the second part, the same amendment procedure was repeated with a 5% (w/w) addition of Slovakite and apatite, respectively. The corresponding 7.5 kg of dry non-amended and amended soils were evenly distributed in large plastic columns, (40 cm height and 16 cm diameter) and incubated at 100% field capacity in the dark at 14 °C for 1 month. 2.3. Six-step sequential extraction A modified Tessier’s sequential extraction procedure (Lestan et al., 2003) was used to investigate the distribution of Pb, Zn, Cu and Cd among different soil fractions in non-amended and amended soil. The fraction soluble in the soil solution was extracted for 1 h in 10 mL of deionized water from 1 g of nonamended and amended air-dried soil, sieved to 250 lm. The exchangeable fraction from soil colloids was extracted from the residual soil sample with 10 mL of 1 M MgNO3 for 2 h. The fraction bound to soil carbonates was extracted after shaking in 10 mL of 1 M NH4OAc (pH 5) for 5 h. The fraction bound to Fe and Mn oxides was extracted with 20 mL of 0.1 M NH2OHHCl (pH 2) for 12 h. The fraction bound to organic matter was obtained after heating the soil suspension in 3 mL of 0.02 M HNO3 and 5 mL of 30% H2O2 for 3 h at 85 °C, followed by extraction with 15 mL of 1 M NH4OAc for 3 h. The final, residual fraction was obtained after digestion of the residual samples with aqua regia. Three determinations of Pb, Zn, Cu and Cd concentration were performed for each fractionation sequence. The final fractional recovery of Pb, Zn, Cu and Cd was calculated by comparing the sum of their concentration in all six fractions with their pseudototal concentration (assessed by aqua regia digestion) in corresponding non-amended and amended soils. Fractional recoveries were 108%, 111% and 108% for Pb, 104%,

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101% and 101% for Zn, 105%, 103% and 101% for Cu and 134%, 134% and 131% Cd, for non-amended soil and soils amended with apatite and Slovakite, respectively.

cation were 0.1, 0.01, 0.03 and 0.02 mg L 1 for Pb, Zn, Cu and Cd, respectively. Reagent blank and analytical duplicates were used where appropriate to ensure accuracy and precision in the analysis.

2.4. PTMs potential bioavailability

2.6. Glucose-induced soil respiration

2.4.1. Extraction with DTPA This single-step extraction test was developed by Lindsay and Norvell (1978) to identify near-neutral and calcareous soil with insufficient plant available Zn, Fe, Mn and Cu, and later adopted to assess PTM phytoavailability and ecotoxicity (Conder et al., 2001), as well as being a measure of PTM bioavailability (Owojori et al., 2010). A DTPA extraction solution was prepared containing 0.005 M DTPA, 0.01 M CaCl2 and 0.1 M triethanolamine, and buffered at pH 7.30 ± 0.05. Ten g of non-amended and amended soils sieved to 2 mm were shaken in 20 mL of DTPA extraction solution for 2 h on a horizontal shaker at about 120 cycles min 1. After extraction, the suspensions were filtered (Whatman, pore size 2.5 lm) and the filtrates analyzed for Pb, Zn, Cu and Cd. The extractions were conducted in triplicate.

Glucose-induced respiration in moist (calculated for 100 g dry weight soil) non-amended soils and soils amended with 5% (w/ w) Slovakite and apatite, respectively, was measured manometrically (OxiTop, WTW, Weilheim) after 1 month of incubation, by alkali (25% NaOH) absorption of the CO2 produced during the 24 h incubation period at 90% of field capacity at 20 °C (ZimakowskaGnoinska and Bech, 2000). Glucose-induced respiration was expressed in mmol CO2 produced by microorganisms in 1 g of soil after 24 h of incubation at 20 °C. Four replicates of each treatment were performed.

2.4.2. Extraction with 0.01 M CaCl2 Five grams of air-dried and sieved (2 mm) non-amended and amended soils were extracted in 50 mL of 0.01 M CaCl2 solution at pH 5.30–6.00 on an orbital shaker for 3 h at about 120 cycles min 1 (Novozamsky et al., 1993). After extraction, the suspensions were centrifuged (10 min at 3000 rpm) and the supernatant analyzed for Pb, Zn, Cu and Cd. The extractions were conducted in triplicate. 2.4.3. Extraction with 0.05 M EDTA The mobility of PTMs in soil was assessed with 0.05 M EDTA extraction at pH 7.50 ± 0.05 (Kosson et al., 2002). Eighty mL of EDTA extraction solution was mixed with 0.8 g of air-dried sieved (2 mm) non-amended and amended soil and the suspension pH adjusted to 7.50 ± 0.05. After 48 h extraction on a head-over shaker at 20 cycles min 1, the samples were vacuum filtered (Whatman, pore size 2.5 lm) and the filtrates analyzed for Pb, Zn, Cu and Cd. The extractions were conducted in triplicate. 2.4.4. Extraction with 1 M ammonium acetate The exchangeable PTMs in non-amended and amended soil were determined by 1 M ammonium acetate extraction (Kalra and Maynard, 1991). Five g of each air-dried and sieved (2 mm) soil sample was extracted with 50 mL of 1 M ammonium acetate at pH 7 on a rotatory shaker at 150 cycles min 1 for 1 h. The suspension was vacuum filtered (Whatman, pore size 2.5 lm) and the filtrates analyzed for Pb, Zn, Cu and Cd. The extractions were conducted in triplicate. 2.5. PTM determination Air-dried samples (0.5 g) of non-amended soils and soils remediated with 5% (w/w) Slovakite and apatite, respectively, were ground in an agate mill, sieved (160 lm) and digested in 12 mL of aqua regia, consisting of HCl and HNO3 in a 3:1 ratio (v/v). After microwave digestion (Mars Xpress, CEM MDS-2000), the samples were filtered (Whatman, pore size 2 lm), diluted with deionized water up to 50 mL, and Pb, Zn, Cu and Cd analyzed by flame (acetylene/air) AAS (Varian AA240FS). Pb, Zn, Cu and Cd in the extracts (solutions from sequential extractions, DTPA, CaCl2, EDTA and ammonium acetate extractions) were determined by AAS directly. A standard reference material used in inter-laboratory comparisons (WEPAL 2003.1.1 and Wepal 2005.1.1) from HBLFA Raumberg-Gumpenstein, Irdning, Austria, was used in the digestion and analyses as part of the QA/QC protocol. The limits of quantifi-

2.7. Determination of soil enzymes activity Selected enzymatic activity assays were performed on nonamended soils and on soils amended with 5% (w/w) Slovakite and apatite, respectively, after 1 month of incubation. 2.7.1. b-Glucosidase activity Determination of b-glucosidase activity in the soil was based on p-nitrophenol (PNP) formation after incubation of the soil with pnitrophenyl glucoside (PNG) as substrate for 1 h at 37 °C (Eivazi and Tabatabai, 1988). One g of field moist soil was incubated with 0.25 mL of toluene, 4 mL of modified universal buffer (MUB, pH 6.5) and 1 mL of PNG for 1 h at 37 °C. One mL of 0.5 M CaCl2 and 4 mL of Tris buffer (pH 12) were then added and the suspension filtered (Whatman 2V). The developed p-nitrophenol was measured spectrophotometrically at 400 nm against the blank. All measurements were carried out in five repetitions. Data are expressed as developed PNP (lg) g 1 of dry soil h 1. 2.7.2. Acid and alkaline phosphatase activity Acid and alkaline phosphatase activity in the soil was determined according to Tabatabai and Bremner (1969). One gram of field moist soil was incubated with 0.25 mL of toluene, 4 mL of modified universal buffer (MUB at pH 6.5 for acid and MUB at pH 11 for alkaline phosphatase activity) and 1 mL of 0.115 M PNP phosphate solution on a rotatory shaker at 37 °C. After 1 h, the reaction was stopped by adding 1 mL of 0.5 M CaCl2. The PNP released was extracted with 4 mL of 0.5 M NaOH, the suspension filtered (Whatman 2V) and the PNP in the filtrate measured at 400 nm. Analyses were conducted in five replicates. Results are expressed as developed PNP (lg) g 1 of dry soil h 1. 2.7.3. Dehydrogenase activity Dehydrogenase activity was determined according to the method described by Thalmann (1968), based on the reduction of tryphenyltetrazolium chloride (TTC) to triphenylformazan (TPF). Five grams of field moist soil were incubated with 5 mL of TTC in Tris buffer for 24 h in darkness at 30 °C. The developed TPF was extracted by adding 40 mL of acetone and incubating at 30 °C for 2 h. The soil suspensions were filtered (Whatman 2V) and TPF in the filtrate measured spectrophotometrically at 546 nm. The results are expressed as developed TPF (lg) g 1 of dry soil. 2.8. Data analyses The differences between Pb, Zn, Cd and Cu concentrations in extracts of non-amended soils and soils amended with 5% (w/w) Slovakite and apatite, respectively, (DTPA and sequential extraction)

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D. Tica et al. / Chemosphere 85 (2011) 577–583 Table 1 Selected pedological properties and pseudototal metal concentrations in nonamended soil and soils amended with 5% (w/w) apatite and Slovakite, respectively. The results are presented as means of three replicates ± SD. The letters a, b, c denote significant differences between the results before and after remediation (Duncan, p < 0.01).

Physical and chemical soil properties pH Organic matter (%) CEC (mmol C+ 100 g 1) C/N P2O5 (mg 100 g 1) Sand (%) Silt (%) Clay (%) Soil texture Pseudototal metal concentration Pb (mg kg 1) Zn (mg kg 1) Cu (mg kg 1) Cd (mg kg 1)

Fig. 1. Pb, Zn, Cd and Cu potential bioavailability in non-amended (NA) soil and soils amended with apatite and Slovakite (1%, 2.5% and 5% w/w, respectively), assessed by diethylenediamine pentaacetic acid (DTPA) and ammonium acetate (NH4OAc) extractions. The results are presented as means of three replicates ± SD.

were determined with Duncan’s multiple range test (p < 0.05) using Statgraphics 4.0 for Windows. 3. Results and discussion 3.1. Soil amendment We investigated the effectiveness of two inorganic amendments, apatite and Slovakite, to reduce the availability of PTMs in soil. Among the three different shares of apatite and Slovakite used to amend the soil in the first part of the study, the most effective in terms of reducing the potential bioavailability of Pb, Zn, Cd and Cu were 5% (w/w) of apatite and 5% (w/w) of Slovakite (Fig. 1, only the results for DTPA and ammonium acetate extraction are shown).

Non-amended soil

Amended soil Apatite (5%)

Slovakite (5%)

a

6.0–6.1 3.7 ± 0.2 a 14.5 ± 0.2 a 10.2 ± 0.6 a 3.8 ± 0.8 a 42.4 ± 0.1 a 44.3 ± 0.6 a 13.4 ± 0.6 Loam

b

6.2–6.3 3.6 ± 0.1 b 17 ± 0.1 ab 9.1 ± 0.4 b 572 ± 6.8 a 43.2 ± 1.5 a 42.2 ± 2.0 a 14.5 ± 0.8 Loam

c

a

a

b

a

b

b

a

b

b

790 ± 12 489 ± 6 a 60 ± 0.6 a 6±0

736 ± 9 456 ± 13 a 56.7 ± 1.7 a 7±0

7.4 3.1 ± 0.1 28.1 ± 0.4 b 8.3 ± 0.5 a 5.3 ± 0.3 a 44.0 ± 0.9 a 45.1 ± 0.6 b 10.9 ± 0.3 Loam c

724 ± 14 450 ± 6 a 56.4 ± 2.2 a 6±0

The second part of the study was therefore conducted with 5% (w/w) amendments with apatite and Slovakite, respectively. Overall, the properties of the amended soils assessed by standard pedological analyses did not significantly differ from the non-amended soil, with a few exceptions. The soil used in this study was loamy and slightly acidic (pH 6.0 ± 0.1). The addition of Slovakite and apatite caused a significant increase of soil pH, more evident when Slovakite was added. Adding apatite, i.e., phosphate minerals, resulted in an expected significant increase in plant-available phosphate, while the high specific surface of zeolites results in a high CEC (Querol et al., 2006; Shi et al., 2009), also apparent in our study, in which the soil CEC increased by a factor of four after amendment with Slovakite (Table 1). The amendments affected the fractionation of PTMs in soil. In non-amended soil, 1.5%, 11%, 1.6% and 33% of Pb, Zn, Cu and Cd, respectively, were present in the water-soluble and exchangeable soil fractions, which are considered to contain the most available metal forms (Lestan et al., 2008). In general, the application of apatite to the soil shifted the Pb, Zn and Cd distribution from the exchangeable, carbonate bound and Fe and Mn-oxide bound

Table 2 Fractionation of Pb, Zn, Cu and Cd in non-amended soil and soils amended with 5% (w/w) apatite and Slovakite, respectively. The results are presented as means of three replicates ± SD. The letters a, b, c denote significant differences between non-amended and amended soils (Duncan, p < 0.01). IA

II

III

IV

a

2.2 ± 0.3 1.6 ± 0.1 b 1.1 ± 0.2

a

10.3 ± 0.5 b 2.9 ± 0.1 c 4.6 ± 0.6

a

a

a

a

54.4 ± 0.8 b 6.0 ± 0.2 c 0.8 ± 0.2

a

V

VI

1

Pb (mg kg ) Non-amended soil Apatite 5% Slovakite 5% Zn (mg kg 1) Non-amended soil Apatite 5% Slovakite 5% Cu (mg kg 1) Non-amended soil Apatite 5% Slovakite 5% Cd (mg kg 1) Non-amended soil Apatite 5% Slovakite 5% A

b

2.7 ± 0.1 1.3 ± 0.5 b 0.7 ± 0.0

b

a

0.3 ± 0.0 0.3 ± 0.0 a 0.3 ± 0.0 a

163.9 ± 5.9 b 12.3 ± 0.4 a 168.0 ± 3.9 41.36 ± 2.1 b 21.0 ± 0.3 c 51.3 ± 1.5

a

a

a

b

0.7 ± 0.0 0.7 ± 0.0 b 0.9 ± 0.0

0.9 ± 0.0 0.7 ± 0.0 a 1.0 ± 0.0

a

a

a

a

b

b

0.5 ± 0.0 0.4 ± 0.3 a 0.4 ± 0.0

2.3 ± 0.0 1.0 ± 0.1 c 0.7 ± 0.0

1.9 ± 0.1 1.0 ± 0.0 c 2.7 ± 0.0

135.9 ± 24.2 b 1.7 ± 0.4 c 73.8 ± 8.5

a

a

a

b

b

55.9 ± 3.0 33.1 ± 1.4 a 59.7 ± 1.9 a

1.1 ± 0.3 0.6 ± 0.0 b 0.8 ± 0.1 b

114.5 ± 4.2 173.3 ± 13.3 a 118.9 ± 1.6 a

23.9 ± 0.4 23.6 ± 0.2 b 21.5 ± 0.2 a

a

a

b

b

1.6 ± 0.1 1.8 ± 0.1 b 1.8 ± 0.1

a

472.2 ± 27.6 748.2 ± 13.8 a 475.5 ± 14.8

65.6 ± 1.9 51.9±.2 a 61.5 ± 3.0

b

1.1 ± 0.1 3.7 ± 0.0 a 0.9 ± 0.1

I water extractable; II exchangeable; III bound to carbonates; IV bound to Fe and Mn-oxides; V bound to organic matter; VI residual fraction.

b

a

241.1 ± 6.9 223.2 ± 4.9 b 224.5 ± 2.6 b

a

36.0 ± 0.8 32.2 ± 0.9 b 32.9 ± 0.5 b

a

1.0 ± 0.1 0.9 ± 0.1 a 1.2 ± 0.2 a

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Table 3 Pb, Zn, Cu and Cd mobility (assessed by EDTA extraction), phytoavailability (assessed by DTPA and CaCl2 extraction) and exchangeability (assessed by NH4OAc extraction) in non-amended soil and soils amended with 5% (w/w) apatite and Slovakite, respectively. The results are presented as means of three replicates ± SD. The letters a, b, c denote significant differences between non-amended and amended soils (Duncan, p < 0.01). LOQ, below the limit of quantification. EDTA Pb (mg kg 1) Non-amended soil Apatite 5% Slovakite 5% Zn (mg kg 1) Non-amended soil Apatite 5% Slovakite 5% Cu (mg kg 1) Non-amended soil Apatite 5% Slovakite 5% Cd (mg kg 1) Non-amended soil Apatite 5% Slovakite 5%

a

546 ± 27.9 521 ± 12.2 a 532 ± 33.2 a

ab

130 ± 4.8 113 ± 6.0 b 140 ± 8.5 a

DTPA

CaCl2

a

229 ± 25.3 115 ± 13.1 b 105 ± 12.3

b

a

57.8 ± 4.9 31.1 ± 3.9 b 26.2 ± 4.1 b

a

NH4OAc

1.1 ± 0.2 LOQ LOQ

a

35.8 ± 0.5 c 2.0 ± 0.3 b 5.2 ± 0.1

17.4 ± 0.4 b 1.3 ± 0.0 c 0.3 ± 0.2

a

18.1 ± 2.8 b 3.7 ± 0.1 b 3.6 ± 0.1

a

a

a

a

0.6 ± 0.1 0.5 ± 0.0 a 0.6 ± 0.0

a

a

a

a

a

a

a

b

b

15.5 ± 0.7 15.4 ± 1.1 a 16.3 ± 0.2 a

2.1 ± 0.1 2.0 ± 0.1 b 2.3 ± 0.1 a

3.6 ± 0.3 3.2 ± 0.3 a 2.9 ± 0.3 2.0 ± 0.2 1.0 ± 0.1 b 1.2 ± 0.2

1.2 ± 0.0 0.2 ± 0.0 LOQ

0.8 ± 0.0 0.8 ± 0.2 a 1.0 ± 0.0 2.3 ± 0.0 0.4 ± 0.0 0.9 ± 0.0

c

b

fractions to less mobile and available chemical forms measured in the fraction bound to the soil organic component, i.e., by factors of 1.6, 1.5 and 3.4, respectively (Table 2). In the case of Pb, stable Pb phosphates, such as pyromorphite may have formed (Miretzky and Fernandez-Cirelli, 2008). It is probable that extraction with 1 M NH4OAc and 0.1 M NH2OHHCl at low pH values (e.g. pH 5 and pH 2, respectively), induced a progressive desorption of Pb from the soil constituents, increasing its available concentration for reactions in soil solution and leading to the formation and precipitation of pyromorphite (Kumpiene et al., 2008). The strong oxidative environment gained by extracting the residual soil sample in 30% H2O2 at 85 °C might be then able to dissolve not only the organic matter present in the soil but also the newly formed pyromorphite, resulting in a high Pb concentration in the fraction supposed to contain only PTMs associated to organic matter (Table 2) (McBride, personal communication). Cd was fairly equally distributed among all the soil fractions, although its concentration was higher in the exchangeable fraction. Apatite induced the conversion of Cd into the fractions bound to Fe and Mn oxides and to the organic matter, while the calcareous component of Slovakite, induced its precipitation as Cd carbonates, which were retrieved in the carbonate fraction (Table 2). On the other hand, the changes in Cu fractionation after amendment with apatite and Slovakite were minimal (Table 2). The majority of Cu was bound in semior non-labile chemical forms to the organic matter and residual soil fractions, where it is known to form more stable complexes than the other metals (Kizilkaya, 2004), thus reducing the efficiency of the amendments. In general, the reduced Pb, Zn and Cd concentrations in the most available soil fractions are congruent with the increase in soil pH (Table 1), since the solubility of metals is known to be inversely proportional to the soil pH (Kumpiene et al., 2008), as well as to the increase in CEC due to the zeolite constituents of Slovakite (Shi et al., 2009).

3.2. PTM potential bioavailability It is widely recognized that not all chemical forms of PTMs interact with living organisms in the same manner, and that their potential bioavailability has to be considered when assessing soil pollution. However, no single method is recognized universally. The extraction methods used in this study were initially developed for the assessment of PTM phytoavailability but are also used for

Fig. 2. Enzymatic (acid phosphatase, alkaline phosphatase, b-glucosidase and dehydrogenase) activity and substrate (glucose) induced respiration in nonamended soil (NA) and soils amended with 5% (w/w) apatite (A 5%) and Slovakite (SL 5%), respectively. The results are presented as means (n = 5) ± SD. Error bars represent standard deviation (n = 5). The letters a, b, c denote significant differences between the non-amended and amended soils (Duncan, p < 0.01).

the assessment of PTM release from soil and their consequent potential availability to all soil organisms (McLaughlin et al., 2000). Due to the increased CEC and pH measured in the amended soils, we expected less PTMs to be desorbed from the amended soil solid phase into the solution than from the non-amended soil (Querol

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Table 4 Correlation coefficients (r) of linear relationships between chemical (soil pH, soil cation exchange capacity (CEC) and PTM bioavailability assessed by diethylenediamine pentaacetic acid (DTPA), ethylenediamine tetraacetic acid (EDTA), ammonium acetate (NH4OAc), CaCl2 and sequential (sum of water soluble and exchangeable fractions) extractions and microbial soil glucose-induced respiration (S.I.R.), acid phosphatase (acid PA), alkaline phosphatase (alkaline PA), b-glucosidase (b-GLU) and dehydrogenase (DEH) activity) characteristics in non-amended soil and soils amended with 5% (w/w) apatite and Slovakite, respecitvely. S.I.R.

Acid PA

pH

0.38

0.52

CEC

0.38

0.48

0.13 0.21 0.06 0.44

Alkaline PA

b-GLU

DEH

0.68**

0.85***

0.01

0.63

0.88***

0.09 0.13 0.12 0.16

0.52 0.48 0.57 0.25

**

0.76 0.77** 0.69** 0.73**

0.09

0.72** 0.78** 0.59* 0.81***

DTPA

Pb Zn Cd Cu

EDTA

Pb Zn Cd Cu

0.34 0.43 0.51 0.84

0.29 0.21 0.25 0.73

0.50 0.54 0.49 0.25

0.35 0.08 0.15 0.27

0.19 0.60* 0.76** 0.49

NH4AOc

Pb Zn Cd Cu

0.01 0.05 0.09 0.23

0.02 0.04 0.06 0.14

0.55 0.52 0.61* 0.05

0.72** 0.74** 0.66* 0.13

0.62* 0.64* 0.50 0.64*

CaCl2

Pb Zn Cd Cu

LOQ 0.15 LOQ 0.07

LOQ 0.09 LOQ 0.10

LOQ 0.53 LOQ 0.15

LOQ 0.75** LOQ 0.03

LOQ 0.73** LOQ 0.03

Sequential

Pb Zn Cd Cu

0.14 0.82*** 0.69*** 0.73***

0.38 0.81*** 0.69** 0.83***

0.17 0.46* 0.49* 0.64**

0.11 0.89*** 0.82*** 0.88***

0.36 0.69*** 0.73*** 0.71***

*

p < 0.1. p < 0.05. *** p < 0.01. **

et al., 2006; Shi et al., 2009). DTPA, CaCl2 and ammonium acetate extractions showed that Pb, Zn and Cd bioavailability in general decreased equally after the two amendments compared to the non-amended soil (Table 3). The extent of the decrease in PTM extractability, however, differed among the tests used, as also observed in previous studies (e.g. Udovic et al., 2009), indicating the difficulty in choosing the most appropriate tool to assess the bioavailability of PTMs and the effectiveness of the remediation applied to polluted soil. In contrast to the results of Madrid et al. (2006), no significant change (p < 0.05) was measured in Pb, Zn, Cd and Cu EDTA-extractability after amendment of polluted urban soil with 5% Slovakite. However, the soils used in their study contained more sand than the soil from Arnoldstein and the PTMs may therefore be more accessible to EDTA-chelation. No differences in Cu availability were found after the amendments were applied to the soil (Table 3). This is congruent with the minimal changes in Cu fractionation after the amendments, due to the presence of the majority of Cu in already non-labile soil fractions, as shown in Table 2. 3.3. Soil microbial activity The effects of soil pollution on enzyme activities are complex. The responses of different enzymes to the same PTM can vary, and the same enzyme may respond differently to different PTMs (Zhang et al., 2010). The inhibition of microbial activity by PTMs can be caused indirectly through their effect on microbial populations or by their direct impact on extracellular enzyme functioning (Lee et al., 2009). Moreover, soil remediation actions, which can alter the enzymatic activity in soil, usually also affect its other physical and chemical characteristics (Lestan et al., 2008). Among them, pH and CEC have probably the most important role (Acosta-Martinez and Tabatabai, 2000; Wang et al., 2006). Enzymatic activity did not increase after the amendment in our study, as we would expect on the basis of the decreased potential bioavailability of PTMs (Ta-

ble 2). Acid phosphatase activity decreased, which may be partially attributed to the 1.3-fold increase in soil pH after the application of Slovakite, as also reported by other authors (Acosta-Martinez and Tabatabai, 2000; Wang et al., 2006), while alkaline phosphatase and dehydrogenase activity did not significantly (p < 0.05) change (Fig. 2). Dehydrogenase activity was positively correlated with both soil pH and CEC (Table 4). In contrast, the activity of b-glucosidase, which is the rate-limiting enzyme in the microbial degradation of cellulose to glucose, significantly decreased after the soil was amended with apatite and Slovakite, by 18% and 50%, respectively (Fig. 2). Its activity was negatively correlated to soil pH (Table 4), which is in accordance with the results of Garau et al. (2007), who reported b-glucosidase activity to be inhibited by an increase in soil pH and CEC due to the amendment of acidic polluted soil with red mud and lime. Furthermore, b-glucosidase activity showed a general positive correlation with the potentially bioavailable fraction of PTMs in soil (Table 4). This may be a consequence of the fact that b-glucosidase is one of the crucial enzymes involved in the C-cycle in soils, which has a very important role especially in polluted soils, in which microorganisms have a higher C requirement for repair and maintenance (Kandeler et al., 1996). After the stabilization of soil with apatite and Slovakite, the bioavailable share of PTMs in soil decreased, thus improving the environmental conditions for soil microorganisms and decreasing the C requirement. In non-polluted agricultural soil, liming and the consequent rise in soil pH have an opposite effect, i.e. they promote bglucosidase activity (Acosta-Martinez and Tabatabai, 2000). Because dehydrogenases are not extracellular enzymes in soil, their activity is used to evaluate the metabolic activity of soil microorganisms, since they are a very sensitive indicator of the effect of PTMs on soil microbial communities (Nannipieri et al., 2002; Garau et al., 2007) Dehydrogenase activity, although not significantly different between the non-amended and amended soils, was negatively correlated with the bioavailability of PTMs and strongly correlated with the CEC and with the soil pH (Table 4),

D. Tica et al. / Chemosphere 85 (2011) 577–583

indicating metabolic recovery of the soil (Garau et al., 2007; Lee et al., 2009). Basal and substrate-induced soil respiration is widely used to estimate PTM toxicity for soil organisms (Brookes, 1995). The low glucose-induced respiration rate in the non-amended soil indicates a poor functional state of the soil, which was significantly (p < 0.05) improved by the amendments applied (Fig. 2). The up to 5-fold increase in the respiration rate was negatively correlated with Zn and Cd, and positively with Cu potential bioavailability (Table 4). Reports in the literature are often contradictory, thus stressing the variability of enzymatic responses to inter-dependent soil factors affected by PTM pollution Zhang et al. (2010), as well as by soil amendments (Acosta-Martinez and Tabatabai, 2000; Garau et al., 2007). 4. Conclusions Apatite and Slovakite amendments were both successful in lowering the potential bioavailability of PTMs, Slovakite being more effective at the same w/w share with the soil. The significant increase in soil pH after apatite and Slovakite amendment and the significant conversion of the most labile and hence potentially available PTM chemical forms into less labile soil fractions affected the soil microbial activity. b-Glucosidase activity and glucose-induced respiration in particular showed that the amendments had improved the functional state of the soil. Among the extraction tests used to assess the affects of the amendments on the potential bioavailability of PTMs in soil, the PTM water-soluble and exchangeable fractions, assessed with the sequential extraction, correlated the most with the microbial activities assays, indicating its suitability for assessing the effectiveness of PTM stabilization in highly polluted acidic loamy soils. Dehydrogenase and b-glucosidase were the most sensitive enzymes to pH, CEC and PTM potential bioavailability changes in the soil, demonstrating their potential as indicators of soil stress, health and quality, together with glucose-induced soil respiration. Acknowledgements This work was supported by the Slovenian Research Agency (Grant Z4-3671). The work of Dragana Tica was funded through a Grant provided by the Slovene Human Resources Development and Scholarship Fund. We are grateful to Dr. Wolfgang Friesl-Hanl for providing the experimental soil and to Dr. Murray McBride for constructive comments and suggestions. References Acosta-Martinez, V., Tabatabai, M.A., 2000. Enzyme activities in a limed agricultural soil. Biol. Fertil. Soils 31, 85–91. Bolan, N.S., Adriano, D.C., Duraisamy, P., Mani, A., Arulmozhiselvan, K., 2003. Immobilization and phytoavailability of cadmium in variable charge soils: I. Effect of phosphate addition. Plant Soil 250, 83–94. Brookes, P.C., 1995. The use of microbial parameters in monitoring soil pollution by heavy metals. Biol. Fertil. Soils 19, 269–279. Conder, J.M., Lanno, R.P., Basta, N.T., 2001. Assessment of metal availability in smelter soil using earthworms and chemical extractions. J. Environ. Qual. 30, 1231–1237. Eivazi, F., Tabatabai, M.A., 1988. Factor affecting glucosidase and galactosidase activities in soils. Soil Biol. Biochem. 22, 891–897. Fiedler, H.J., Hoffmann, F., Schmiedel, H., 1964. Die Untersuchung der Boden, Band first ed. Theodor Steinkopff, Dresden. Friesl-Hanl, W., Platzer, K., Horak, O., Gerzabek, M.H., 2009. Immobilising of Cd, Pb and Zn contaminated arable soils close to former Pb/Zn smelter: field study in Austria over 5 years. Environ. Geochem. Health 31, 581–594. Garau, G., Castaldi, P., Santona, L., Deiana, P., Melis, P., 2007. Influence of red mud, zeolite and lime on heavy metal immobilization, culturable heterotrophic microbial populations and enzyme activities in a contaminated soil. Geoderma 142, 47–57. Guo, G., Zhou, Q., Ma, L.Q., 2005. Availability and assessment of fixing additives for the in situ remediation of heavy metal contaminated soils: a review. Environ. Monit. Assess. 116, 513–528.

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