Environ Sci Pollut Res DOI 10.1007/s11356-015-4678-1
RESEARCH ARTICLE
The effectiveness of various treatments in changing the nutrient status and bioavailability of risk elements in multi-element contaminated soil Mercedes García-Sánchez 1 & Inmaculada García-Romera 2 & Jiřina Száková 1 & Lukáš Kaplan 1 & Pavel Tlustoš 1
Received: 13 February 2015 / Accepted: 7 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Potential changes in the mobility and bioavailability of risk and essential macro- and micro-elements achieved by adding various ameliorative materials were evaluated in a model pot experiment. Spring wheat (Triticum aestivum L.) was cultivated under controlled condition for 60 days in two soils, uncontaminated Chernozem and multi-element contaminated Fluvisol containing 4900 ± 200 mg/kg Zn, 35.4 ± 3.6 mg/kg Cd, and 3035±26 mg/kg Pb. The treatments were all contained the same amount of sulfur and were as follows: (i) digestate from the anaerobic fermentation of biowaste, (ii) fly ash from wood chip combustion, and (iii) ammonium sulfate. Macro- and micro-nutrients Ca, Mg, K, Fe, Mn, Cu, P, and S, and risk elements Cd, Cr, Pb, and Zn were assayed in soil extracts with 0.11 mol/l solution of CH3COOH and in roots, shoots, and grain of wheat after 30 and 60 days of cultivation. Both digestate and fly ash increased levels of macro- and micro-nutrients as well as risk elements (especially Cd and Zn; the mobility of Pb decreased after 30 days of cultivation). The changes in element mobility in ammonium sulfate-treated soils appear to be due to both changes in soil pH level and inter-element interactions. Ammonium sulfate tended to be the most effective measure for increasing nutrient
Responsible editor: Zhihong Xu * Jiřina Száková [email protected] 1
Department of Agro-Environmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamýcká 129, Prague 6, Suchdol, Czech Republic
2
Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Prof. Albareda 1, 18008 Granada, Spain
uptake by plants in Chernozem but with opposite pattern in Fluvisol. Changes in plant yield and element uptake in treated plants may have been associated with the higher proline content of wheat shoots cultivated in both soils compared to control. None of the treatments decreased uptake of risk elements by wheat plants in the extremely contaminated Fluvisol, and their accumulation in wheat grains significantly exceeded maximum permissible levels; these treatments cannot be used to enable cereal and other crop production in such soils. However, the combination of increased plant growth alongside unchanged element content in plant biomass in pots treated with digestate and fly ash suggests that these treatments have a beneficial impact on yield and may be effective treatments in crops grown for phytoremediation. Keywords Risk elements . Nutrients . Proline . Fly ash . Digestate . Spring wheat
Introduction Liming and application of organic manure are traditional agricultural practices which are effective in limiting levels of risk element levels in crops growing in contaminated soil (Madejón et al. 2011; Tlustoš et al. 2006). The effectiveness of natural and synthetic zeolites in immobilizing cadmium, zinc, copper, iron, lead, manganese, etc. has described and verified in several studies (Madrid et al. 2006; Nissen et al. 2000; Száková et al. 2007). In many cases, increasing soil pH helps to decrease soil solution concentrations of Cd and Pb and decrease of the levels of these elements in crop biomass (Lee et al. 2004; Hong et al. 2007). However, the application of alkaline-stabilized biosolid materials results in increased Mo mobility in soil and excessive Mo uptake by plants (Stehouwer and Macneal 2004), indicating the importance of
Environ Sci Pollut Res
choosing appropriate immobilization measures according to the particular risk elements for which soil remediation is required. In the case of arsenic, studies have tested whether application of iron oxides and/or sulfates decreases the proportion of soil As which is bioavailable as well as decreasing As concentration in plants (Komárek et al. 2013; Warren and Alloway 2003) since the decrease in soil pH after application of iron sulfate resulted in increase of mobile macro- and micro-nutrients in soils. The risk element immobilization potential of industrial by-products rich in iron oxy-hydroxides such as water treatment sludge or red mud and red gypsum (by-products of the Al oxide and Ti oxide processing factories) has also been tested for use against copper and, to a lesser extent, arsenic (Lombi et al. 2004), cadmium, lead, and zinc (Friesl-Hanl et al. 2009; Lee et al. 2009). The effectiveness of organic waste materials such as sewage sludge or composts in risk element immobilization measures has been described (Juwarkar et al. 2008; Khan and Jones 2008), and these materials can also be used as slow-release nutrient sources (Guo et al. 2006). Wood fly ash, the ash remaining after combustion of biomass, contains several minerals essential for agricultural soils, and it would be beneficial if adding it to soils increased the proportion of nutrients in the soil as well as in plants. The pH increase associated with adding this ash to soils has been shown to cause a decrease in risk element mobility, limiting their uptake by plants (Ochecová et al. 2014); it can have a more sustained neutralizing effect than lime (Kumpiene et al. 2008). Digestate, biowaste from biogas production plants, has considerable potential as a fertilizer value due to its organic carbon content (Vaneeckhaute et al. 2013) and levels of N, P, and K and various micro-nutrients (Alburquerque et al. 2012). Walsh et al. (2012) recommended replacing inorganic fertilizer with digestate to maintain grassland productivity, although there are differences between the two treatments in terms of plant growth response and suppressed growth of root biomass following application of digestate has been observed (Andruschkewitsch et al. 2013). Johansen et al. (2013) demonstrated that fertilizing with anaerobically digested materials increased the soil concentrations of NO3 by 30–40 % compared with the application of raw cattle slurry. Digestate application has also been reported to result in better nitrogen recovery and lower N losses compared to untreated variants (Andruschkewitsch et al. 2013; Frøseth et al. 2014; Stinner et al. 2008). Fernández-Delgado Juárez et al. (2013) recommended combined use of wood ash and biogas digestate as an efficient method of disposing of and recycling both residues, as they produced no changes in soil health indicators such as microbial activity. Minor changes in microbial community composition (Johansen et al. 2013) and earthworm populations (Frøseth et al. 2014) in digestate-treated soil have been observed. Fly ash and stabilized sewage sludge can prevent the leaching of trace elements and their uptake by plants (for review, see Kumpiene et al. 2008).
Our previous investigation (García-Sánchez et al. 2014) showed that digestate application was an effective method of rapidly reducing levels of mobile mercury (Hg) in artificially contaminated soils. The decrease in the proportion of mercury that was mobile was followed by improvement in the nutrient status of the soils. In contrast, application of fly ash from wood chip combustion had no significant effect. Both digestate and wood fly ash are sulfur-rich materials, and S application has been shown to decrease element phytotoxicity (Gilabel et al. 2014). The presence of S is also of particular interest, as it is a component of amino acids such as cysteine and methionine, which are precursors of S-containing components such as proline which are capable of increasing plants’ tolerance of risk elements (Gill and Tetuja 2011). To separate out the effects of organic matter and S in the soil, it is necessary to study the effects of application of inorganic sources of S, such as ammonium sulfate, (NH4)2SO4. This study investigated the effects of both organic and inorganic treatments on mobility of certain risk elements in contaminated soil. The main objectives were as follows: (i) to assess the effect of the individual treatments (digestate, fly ash, and inorganic source of S) on the mobility and bioavailability of risk elements and essential macro- and micro-elements in a model pot experiment with soil contaminated by mining and smelting activities, (ii) to assess the effect of the treatments on growth, element uptake, and biochemical response of spring wheat (Triticum aestivum L.) plants, and (iii) to estimate the potential merit of the treatments if included into various soil remediation measures. We used proline activity as an indicator of oxidative stress in the plants.
Material and methods Soils and ameliorative materials The contaminated Fluvisol was sampled next to Trhové Dušníky village in the mining and smelting district of Příbram, Czech Republic. A detailed description of the location including a detailed map of the area is available elsewhere (Vanek et al. 2005). This area is known for its Pb-Ag-Zn poly-metallic mineral deposits, which were mined and processed from the Middle Ages until the 1970s (Ettler et al. 2007). Emissions from primary and secondary Pb smelters are responsible for the high concentrations of metallic contaminants (Pb, Cd, and Zn) in soils. At the time of this study, soil pH was 7.0, the oxidizable carbon content (Cox) was 3.24, and the cation exchange capacity (CEC) was 175 mmol/kg. Uncontaminated Chernozem with a CEC=230 mmol/kg, Cox=2.31 %, and soil pH 8.5 was obtained from Suchdol, near Prague in the Czech Republic. Soils were sampled at a depth of 20 cm and immediately homogenized, sieved through 5-mm-diameter
Environ Sci Pollut Res
mesh and then stored at room temperature until the start of the experiment. The fly ash (pH 12.1) was produced by combustion of wood ash in two reactors (1.8 and 0.6 MW). The digestate sample (pH 8.2) originated from a biogas station (1732 kW/h), where the digested material consisted of sugar beet pulp (50 %), marc of fruit (42 %), and maize silage (8 %). The physicochemical parameters of both ameliorative materials and the methods used to determine macro- and micronutrient levels have been described and discussed elsewhere (García-Sánchez et al. 2014). The total As, Cd, and Pb contents of the ameliorative materials were determined using the same methods. Solid particles of (NH4)2SO4 (Fisher Scientific) were used as an inorganic ameliorative. Experimental design and setup Spring wheat was cultivated in a fully randomized pot experiment with four replicates per treatment under controlled conditions (75±5 % relative humidity, 25-15 °C temperature, 16:8-h photoperiod, and 460 μmol/m2/s irradiance). The experimental design was a 2×4 design with days of cultivation (30 and 60 days) as within-subjects factor and treatment (control, digestate, fly ash, and (NH4)2SO4) as a between-subjects factor. The experiment was carried out in 0.3-l pots filled with 300 g of Chernozem or Fluvisol. Digestate and fly ash were added to achieve levels of 11.6 and 15 g/kg, respectively. (NH4)2SO4 was applied at a rate of 2.5 g/kg. The dose of each ameliorative was based on their sulfur content and preliminary studies (García-Sánchez et al. 2014). Wheat seeds that were germinated in plastic trays contain a 5-cm layer of vermiculite, to which 500 ml 1:1 Hewitt solution (Hewitt 1966) was added every other day. Seedlings were germinated under controlled conditions; after 15 days growing time (when plants had three fully expanded leaves), they were transferred to pots (one plant per pot). Plants were cultivated for 2 months, during which soil moisture was maintained at 60 % of field capacity. After 30 and 60 days of plant growth, a portion of soil was collected, sieved (2-mm mesh), and dried at room temperature for analysis. Wheat plants were harvested and divided into two subsamples for dry biomass and biochemical analysis in the same way. The biomass of wheat plants was estimated after drying shoots, roots, and grains in an oven for 48 h at 72 °C; they were then homogenized prior to chemical analysis.
determined by decomposing the soil samples using pressurized wet ashing as follows: aliquots (∼0.5 g) of air-dried samples were decomposed in a digestion vessel containing 10 ml of Aqua regia (1:3 mixture of nitric acid and hydrochloric acid). The mixture was heated in an Ethos 1 (MLS, Germany) microwave-assisted wet digestion system for 33 min at 210 °C. The proportion of element content which was bioavailable was determined by adding 0.5 g of each sample to 10 ml of 0.11 mol/l solution of CH3COOH; this mixture was then shaken overnight (Quevauviller et al. 1993). Each extraction was carried out in triplicate. Extracts were centrifuged in a Hettich Universal 30 RF (Germany) at 3000 rpm (i.e., 460 g) for 10 min at the end of each extraction procedure, and the supernatants were stored at 6 °C prior to analysis. To determine the element contents of plant biomass samples, an aliquot (∼0.5 g of dry matter) of the plant sample was weighed in a digestion vessel. Concentrated HNO3 (8.0 ml) (Analytika Ltd., Czech Republic) and 30 % H2O2 (2.0 ml) (Analytika Ltd., Czech Republic) were added, and the mixture was heated in an Ethos 1 (MLS GmbH, Germany) microwave-assisted wet digestion system for 30 min at 220 °C. After cooling, the digest was quantitatively transferred to a 20-ml glass tube which was filled up with deionized water. Inductively coupled plasma-atomic emission spectrometry (ICP-OES) using Agilent 720 (Agilent Technologies Inc., USA) equipped with a two-channel peristaltic pump, a Sturman-Masters spray chamber, and a V-groove pneumatic nebulizer made of inert material was used to determine the As, Cd, Cr, Cu, Fe, Mn, Pb, Zn, P, and S contents of soil and plant digests and soil extracts (spectrometry parameters were power, 1.2 kW; plasma flow, 15.0 l/min; auxiliary flow, 0.75 l/min; nebulizer flow, 0.9 l/min). Flame atomic absorption spectrometry using a Varian 280FS (F-AAS, Varian, Australia) was used to determine the Ca, Mg, and K contents of the solutions; the parameters were as follows: air flow, 13.5 l/min; acetylene flow, 2.2 l/min; burner height, 13.5 ml; and nebulizer uptake rate, 5 ml/min. Proline content was assayed in samples of ground wheat leaves in liquid nitrogen following the method described by Bates et al. (1973). One milliliter of leaf extract was transferred to a glass tube and mixed with 5 ml of ninhydrin solution and 5 ml of glacial acetic acid. The reaction mixture was placed in a 100 °C water bath for 1 h and then placed in ice for 10 min. Then, 10 ml of toluene was added in the tube and the proline concentration was read in a spectrophotometer at 520 nm.
Analytical methods Statistics Soil pH was determined using deionized water. CEC was calculated as the sum of Ca, Mg, K, Na, Fe, Mn, and Al extractable in 0.1 mol/l BaCl2 (w/v=1+20 for 2 h) (ISO 1994). Pseudo-total contents of the various elements were
One-way analysis of variance was used with α=0.05 as the criterion for significance. Analyses were carried out using the Statistica 12 program (StatSoft, USA).
Environ Sci Pollut Res 10.0
Results and discussion
c 9.0
Pseudo-total and mobile element contents in soils, the effect of the ameliorative materials
bc
d
A bc
b
c a
8.0
As Table 1 shows, the ameliorative materials differed in terms of nutrient and risk element content. As expected, the levels of risk elements (Cd, Cr, Pb) in the digestate were relatively low as were the levels of Fe, Mn, and Zn. Levels of Cu and Mg were similar to those of the soils, and levels of Ca, K, P, and S were significantly higher than in the soils. Low levels of risk elements in digestates from agricultural residues have been previously documented (Demirel et al. 2013; Vaneeckhaute et al. 2013). In general, levels of risk elements were higher in fly ash than in digestate (Table 1); one would therefore expect these two treatments to have different effects on soil nutrient status; the potential for risk element contamination with fly ash should be taken into account when this treatment is used. The level of Pb was even higher in Fluvisol than in fly ash. In the Czech Republic, maximum permissible soil levels of certain elements are given by public notice (Anonymous 1994); the pseudo-total element concentrations have been set at 1.0, 200, 100, 140, and 200 mg/kg for Cd, Cr, Cu, Pb, and Zn, respectively. No maximum permissible soil level has been set for other elements. Our analysis (Table 1) indicated that the pseudo-total levels of risk elements in Chernozem did not exceed these limits, but levels of Cd, Pb, and Zn exceeded the permissible limit in Fluvisol. Fluvisol also had higher levels of Fe, Mn, and S than Chernozem, whereas levels of Ca, K, Mg, and P were lower in Fluvisol than in Chernozem. As mentioned above, the ameliorative materials used in this study are characterized by high pH and their application significantly increased pH levels in both soils (Fig. 1). This pH increase was more apparent in the neutral Fluvisol, where it was seen after both 30 and 60 days of cultivation; in Table 1 Nutrient and risk element contents in the dry matter of ameliorative materials and soils (n=3) Element
Fly ash
Digestate
Chernozem
Fluvisol
P (%) K (%) Mg (%) Ca (%) S (%) Fe (%) Mn (%) Zn (%) Cd (mg/kg) Cr (mg/kg) Cu (mg/kg) Pb (mg/kg)
1.29±0.01 7.74±0.02 1.44±0.02 13.4±0.1 4.07±0.01 2.79±0.01 1.29±0.01 3.58±0.08 64.5±1.3 219±2 644±45 650±40
1.20±0.01 2.12±0.01 0.49±0.02 3.15±0.01 0.60±0.01 0.18±0.01 0.02±0.00 0.03±0.00 0.16±0.01 3.52±0.15 43.6±0.8 1.32±0.29
0.08±0.00 0.88±0.06 0.56±0.03 1.16±0.05 0.03±0.00 2.60±0.07 0.06±0.00 0.01±0.00 0.26±0.01 44.4±1.3 25.2±0.4 27.0±0.5
0.04±0.00 0.62±0.07 0.31±0.00 0.26±0.03 0.07±0.01 3.4±0.01 0.32±0.01 0.49±0.02 35.4±3.6 32.9±1.0 56.8±4.6 3035±26
a
pH 7.0 6.0 5.0
Control
Digestate
Ash
Ammonium sulfate
Treatment 30 days 60 days 10.0
B 9.0 b
8.0 a
pH
c
b
b a
7.0
a
6.0 5.0
c
Control
Digestate
Ash
Ammonium sulfate
Treatment 30 days 60 days
Fig. 1 The effect of digestate, fly ash, and ammonium sulfate application on soil pH: a Chernozem and b Fluvisol. The bars marked by the same letter did not significantly differ at P
RESEARCH ARTICLE
The effectiveness of various treatments in changing the nutrient status and bioavailability of risk elements in multi-element contaminated soil Mercedes García-Sánchez 1 & Inmaculada García-Romera 2 & Jiřina Száková 1 & Lukáš Kaplan 1 & Pavel Tlustoš 1
Received: 13 February 2015 / Accepted: 7 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Potential changes in the mobility and bioavailability of risk and essential macro- and micro-elements achieved by adding various ameliorative materials were evaluated in a model pot experiment. Spring wheat (Triticum aestivum L.) was cultivated under controlled condition for 60 days in two soils, uncontaminated Chernozem and multi-element contaminated Fluvisol containing 4900 ± 200 mg/kg Zn, 35.4 ± 3.6 mg/kg Cd, and 3035±26 mg/kg Pb. The treatments were all contained the same amount of sulfur and were as follows: (i) digestate from the anaerobic fermentation of biowaste, (ii) fly ash from wood chip combustion, and (iii) ammonium sulfate. Macro- and micro-nutrients Ca, Mg, K, Fe, Mn, Cu, P, and S, and risk elements Cd, Cr, Pb, and Zn were assayed in soil extracts with 0.11 mol/l solution of CH3COOH and in roots, shoots, and grain of wheat after 30 and 60 days of cultivation. Both digestate and fly ash increased levels of macro- and micro-nutrients as well as risk elements (especially Cd and Zn; the mobility of Pb decreased after 30 days of cultivation). The changes in element mobility in ammonium sulfate-treated soils appear to be due to both changes in soil pH level and inter-element interactions. Ammonium sulfate tended to be the most effective measure for increasing nutrient
Responsible editor: Zhihong Xu * Jiřina Száková [email protected] 1
Department of Agro-Environmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamýcká 129, Prague 6, Suchdol, Czech Republic
2
Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Prof. Albareda 1, 18008 Granada, Spain
uptake by plants in Chernozem but with opposite pattern in Fluvisol. Changes in plant yield and element uptake in treated plants may have been associated with the higher proline content of wheat shoots cultivated in both soils compared to control. None of the treatments decreased uptake of risk elements by wheat plants in the extremely contaminated Fluvisol, and their accumulation in wheat grains significantly exceeded maximum permissible levels; these treatments cannot be used to enable cereal and other crop production in such soils. However, the combination of increased plant growth alongside unchanged element content in plant biomass in pots treated with digestate and fly ash suggests that these treatments have a beneficial impact on yield and may be effective treatments in crops grown for phytoremediation. Keywords Risk elements . Nutrients . Proline . Fly ash . Digestate . Spring wheat
Introduction Liming and application of organic manure are traditional agricultural practices which are effective in limiting levels of risk element levels in crops growing in contaminated soil (Madejón et al. 2011; Tlustoš et al. 2006). The effectiveness of natural and synthetic zeolites in immobilizing cadmium, zinc, copper, iron, lead, manganese, etc. has described and verified in several studies (Madrid et al. 2006; Nissen et al. 2000; Száková et al. 2007). In many cases, increasing soil pH helps to decrease soil solution concentrations of Cd and Pb and decrease of the levels of these elements in crop biomass (Lee et al. 2004; Hong et al. 2007). However, the application of alkaline-stabilized biosolid materials results in increased Mo mobility in soil and excessive Mo uptake by plants (Stehouwer and Macneal 2004), indicating the importance of
Environ Sci Pollut Res
choosing appropriate immobilization measures according to the particular risk elements for which soil remediation is required. In the case of arsenic, studies have tested whether application of iron oxides and/or sulfates decreases the proportion of soil As which is bioavailable as well as decreasing As concentration in plants (Komárek et al. 2013; Warren and Alloway 2003) since the decrease in soil pH after application of iron sulfate resulted in increase of mobile macro- and micro-nutrients in soils. The risk element immobilization potential of industrial by-products rich in iron oxy-hydroxides such as water treatment sludge or red mud and red gypsum (by-products of the Al oxide and Ti oxide processing factories) has also been tested for use against copper and, to a lesser extent, arsenic (Lombi et al. 2004), cadmium, lead, and zinc (Friesl-Hanl et al. 2009; Lee et al. 2009). The effectiveness of organic waste materials such as sewage sludge or composts in risk element immobilization measures has been described (Juwarkar et al. 2008; Khan and Jones 2008), and these materials can also be used as slow-release nutrient sources (Guo et al. 2006). Wood fly ash, the ash remaining after combustion of biomass, contains several minerals essential for agricultural soils, and it would be beneficial if adding it to soils increased the proportion of nutrients in the soil as well as in plants. The pH increase associated with adding this ash to soils has been shown to cause a decrease in risk element mobility, limiting their uptake by plants (Ochecová et al. 2014); it can have a more sustained neutralizing effect than lime (Kumpiene et al. 2008). Digestate, biowaste from biogas production plants, has considerable potential as a fertilizer value due to its organic carbon content (Vaneeckhaute et al. 2013) and levels of N, P, and K and various micro-nutrients (Alburquerque et al. 2012). Walsh et al. (2012) recommended replacing inorganic fertilizer with digestate to maintain grassland productivity, although there are differences between the two treatments in terms of plant growth response and suppressed growth of root biomass following application of digestate has been observed (Andruschkewitsch et al. 2013). Johansen et al. (2013) demonstrated that fertilizing with anaerobically digested materials increased the soil concentrations of NO3 by 30–40 % compared with the application of raw cattle slurry. Digestate application has also been reported to result in better nitrogen recovery and lower N losses compared to untreated variants (Andruschkewitsch et al. 2013; Frøseth et al. 2014; Stinner et al. 2008). Fernández-Delgado Juárez et al. (2013) recommended combined use of wood ash and biogas digestate as an efficient method of disposing of and recycling both residues, as they produced no changes in soil health indicators such as microbial activity. Minor changes in microbial community composition (Johansen et al. 2013) and earthworm populations (Frøseth et al. 2014) in digestate-treated soil have been observed. Fly ash and stabilized sewage sludge can prevent the leaching of trace elements and their uptake by plants (for review, see Kumpiene et al. 2008).
Our previous investigation (García-Sánchez et al. 2014) showed that digestate application was an effective method of rapidly reducing levels of mobile mercury (Hg) in artificially contaminated soils. The decrease in the proportion of mercury that was mobile was followed by improvement in the nutrient status of the soils. In contrast, application of fly ash from wood chip combustion had no significant effect. Both digestate and wood fly ash are sulfur-rich materials, and S application has been shown to decrease element phytotoxicity (Gilabel et al. 2014). The presence of S is also of particular interest, as it is a component of amino acids such as cysteine and methionine, which are precursors of S-containing components such as proline which are capable of increasing plants’ tolerance of risk elements (Gill and Tetuja 2011). To separate out the effects of organic matter and S in the soil, it is necessary to study the effects of application of inorganic sources of S, such as ammonium sulfate, (NH4)2SO4. This study investigated the effects of both organic and inorganic treatments on mobility of certain risk elements in contaminated soil. The main objectives were as follows: (i) to assess the effect of the individual treatments (digestate, fly ash, and inorganic source of S) on the mobility and bioavailability of risk elements and essential macro- and micro-elements in a model pot experiment with soil contaminated by mining and smelting activities, (ii) to assess the effect of the treatments on growth, element uptake, and biochemical response of spring wheat (Triticum aestivum L.) plants, and (iii) to estimate the potential merit of the treatments if included into various soil remediation measures. We used proline activity as an indicator of oxidative stress in the plants.
Material and methods Soils and ameliorative materials The contaminated Fluvisol was sampled next to Trhové Dušníky village in the mining and smelting district of Příbram, Czech Republic. A detailed description of the location including a detailed map of the area is available elsewhere (Vanek et al. 2005). This area is known for its Pb-Ag-Zn poly-metallic mineral deposits, which were mined and processed from the Middle Ages until the 1970s (Ettler et al. 2007). Emissions from primary and secondary Pb smelters are responsible for the high concentrations of metallic contaminants (Pb, Cd, and Zn) in soils. At the time of this study, soil pH was 7.0, the oxidizable carbon content (Cox) was 3.24, and the cation exchange capacity (CEC) was 175 mmol/kg. Uncontaminated Chernozem with a CEC=230 mmol/kg, Cox=2.31 %, and soil pH 8.5 was obtained from Suchdol, near Prague in the Czech Republic. Soils were sampled at a depth of 20 cm and immediately homogenized, sieved through 5-mm-diameter
Environ Sci Pollut Res
mesh and then stored at room temperature until the start of the experiment. The fly ash (pH 12.1) was produced by combustion of wood ash in two reactors (1.8 and 0.6 MW). The digestate sample (pH 8.2) originated from a biogas station (1732 kW/h), where the digested material consisted of sugar beet pulp (50 %), marc of fruit (42 %), and maize silage (8 %). The physicochemical parameters of both ameliorative materials and the methods used to determine macro- and micronutrient levels have been described and discussed elsewhere (García-Sánchez et al. 2014). The total As, Cd, and Pb contents of the ameliorative materials were determined using the same methods. Solid particles of (NH4)2SO4 (Fisher Scientific) were used as an inorganic ameliorative. Experimental design and setup Spring wheat was cultivated in a fully randomized pot experiment with four replicates per treatment under controlled conditions (75±5 % relative humidity, 25-15 °C temperature, 16:8-h photoperiod, and 460 μmol/m2/s irradiance). The experimental design was a 2×4 design with days of cultivation (30 and 60 days) as within-subjects factor and treatment (control, digestate, fly ash, and (NH4)2SO4) as a between-subjects factor. The experiment was carried out in 0.3-l pots filled with 300 g of Chernozem or Fluvisol. Digestate and fly ash were added to achieve levels of 11.6 and 15 g/kg, respectively. (NH4)2SO4 was applied at a rate of 2.5 g/kg. The dose of each ameliorative was based on their sulfur content and preliminary studies (García-Sánchez et al. 2014). Wheat seeds that were germinated in plastic trays contain a 5-cm layer of vermiculite, to which 500 ml 1:1 Hewitt solution (Hewitt 1966) was added every other day. Seedlings were germinated under controlled conditions; after 15 days growing time (when plants had three fully expanded leaves), they were transferred to pots (one plant per pot). Plants were cultivated for 2 months, during which soil moisture was maintained at 60 % of field capacity. After 30 and 60 days of plant growth, a portion of soil was collected, sieved (2-mm mesh), and dried at room temperature for analysis. Wheat plants were harvested and divided into two subsamples for dry biomass and biochemical analysis in the same way. The biomass of wheat plants was estimated after drying shoots, roots, and grains in an oven for 48 h at 72 °C; they were then homogenized prior to chemical analysis.
determined by decomposing the soil samples using pressurized wet ashing as follows: aliquots (∼0.5 g) of air-dried samples were decomposed in a digestion vessel containing 10 ml of Aqua regia (1:3 mixture of nitric acid and hydrochloric acid). The mixture was heated in an Ethos 1 (MLS, Germany) microwave-assisted wet digestion system for 33 min at 210 °C. The proportion of element content which was bioavailable was determined by adding 0.5 g of each sample to 10 ml of 0.11 mol/l solution of CH3COOH; this mixture was then shaken overnight (Quevauviller et al. 1993). Each extraction was carried out in triplicate. Extracts were centrifuged in a Hettich Universal 30 RF (Germany) at 3000 rpm (i.e., 460 g) for 10 min at the end of each extraction procedure, and the supernatants were stored at 6 °C prior to analysis. To determine the element contents of plant biomass samples, an aliquot (∼0.5 g of dry matter) of the plant sample was weighed in a digestion vessel. Concentrated HNO3 (8.0 ml) (Analytika Ltd., Czech Republic) and 30 % H2O2 (2.0 ml) (Analytika Ltd., Czech Republic) were added, and the mixture was heated in an Ethos 1 (MLS GmbH, Germany) microwave-assisted wet digestion system for 30 min at 220 °C. After cooling, the digest was quantitatively transferred to a 20-ml glass tube which was filled up with deionized water. Inductively coupled plasma-atomic emission spectrometry (ICP-OES) using Agilent 720 (Agilent Technologies Inc., USA) equipped with a two-channel peristaltic pump, a Sturman-Masters spray chamber, and a V-groove pneumatic nebulizer made of inert material was used to determine the As, Cd, Cr, Cu, Fe, Mn, Pb, Zn, P, and S contents of soil and plant digests and soil extracts (spectrometry parameters were power, 1.2 kW; plasma flow, 15.0 l/min; auxiliary flow, 0.75 l/min; nebulizer flow, 0.9 l/min). Flame atomic absorption spectrometry using a Varian 280FS (F-AAS, Varian, Australia) was used to determine the Ca, Mg, and K contents of the solutions; the parameters were as follows: air flow, 13.5 l/min; acetylene flow, 2.2 l/min; burner height, 13.5 ml; and nebulizer uptake rate, 5 ml/min. Proline content was assayed in samples of ground wheat leaves in liquid nitrogen following the method described by Bates et al. (1973). One milliliter of leaf extract was transferred to a glass tube and mixed with 5 ml of ninhydrin solution and 5 ml of glacial acetic acid. The reaction mixture was placed in a 100 °C water bath for 1 h and then placed in ice for 10 min. Then, 10 ml of toluene was added in the tube and the proline concentration was read in a spectrophotometer at 520 nm.
Analytical methods Statistics Soil pH was determined using deionized water. CEC was calculated as the sum of Ca, Mg, K, Na, Fe, Mn, and Al extractable in 0.1 mol/l BaCl2 (w/v=1+20 for 2 h) (ISO 1994). Pseudo-total contents of the various elements were
One-way analysis of variance was used with α=0.05 as the criterion for significance. Analyses were carried out using the Statistica 12 program (StatSoft, USA).
Environ Sci Pollut Res 10.0
Results and discussion
c 9.0
Pseudo-total and mobile element contents in soils, the effect of the ameliorative materials
bc
d
A bc
b
c a
8.0
As Table 1 shows, the ameliorative materials differed in terms of nutrient and risk element content. As expected, the levels of risk elements (Cd, Cr, Pb) in the digestate were relatively low as were the levels of Fe, Mn, and Zn. Levels of Cu and Mg were similar to those of the soils, and levels of Ca, K, P, and S were significantly higher than in the soils. Low levels of risk elements in digestates from agricultural residues have been previously documented (Demirel et al. 2013; Vaneeckhaute et al. 2013). In general, levels of risk elements were higher in fly ash than in digestate (Table 1); one would therefore expect these two treatments to have different effects on soil nutrient status; the potential for risk element contamination with fly ash should be taken into account when this treatment is used. The level of Pb was even higher in Fluvisol than in fly ash. In the Czech Republic, maximum permissible soil levels of certain elements are given by public notice (Anonymous 1994); the pseudo-total element concentrations have been set at 1.0, 200, 100, 140, and 200 mg/kg for Cd, Cr, Cu, Pb, and Zn, respectively. No maximum permissible soil level has been set for other elements. Our analysis (Table 1) indicated that the pseudo-total levels of risk elements in Chernozem did not exceed these limits, but levels of Cd, Pb, and Zn exceeded the permissible limit in Fluvisol. Fluvisol also had higher levels of Fe, Mn, and S than Chernozem, whereas levels of Ca, K, Mg, and P were lower in Fluvisol than in Chernozem. As mentioned above, the ameliorative materials used in this study are characterized by high pH and their application significantly increased pH levels in both soils (Fig. 1). This pH increase was more apparent in the neutral Fluvisol, where it was seen after both 30 and 60 days of cultivation; in Table 1 Nutrient and risk element contents in the dry matter of ameliorative materials and soils (n=3) Element
Fly ash
Digestate
Chernozem
Fluvisol
P (%) K (%) Mg (%) Ca (%) S (%) Fe (%) Mn (%) Zn (%) Cd (mg/kg) Cr (mg/kg) Cu (mg/kg) Pb (mg/kg)
1.29±0.01 7.74±0.02 1.44±0.02 13.4±0.1 4.07±0.01 2.79±0.01 1.29±0.01 3.58±0.08 64.5±1.3 219±2 644±45 650±40
1.20±0.01 2.12±0.01 0.49±0.02 3.15±0.01 0.60±0.01 0.18±0.01 0.02±0.00 0.03±0.00 0.16±0.01 3.52±0.15 43.6±0.8 1.32±0.29
0.08±0.00 0.88±0.06 0.56±0.03 1.16±0.05 0.03±0.00 2.60±0.07 0.06±0.00 0.01±0.00 0.26±0.01 44.4±1.3 25.2±0.4 27.0±0.5
0.04±0.00 0.62±0.07 0.31±0.00 0.26±0.03 0.07±0.01 3.4±0.01 0.32±0.01 0.49±0.02 35.4±3.6 32.9±1.0 56.8±4.6 3035±26
a
pH 7.0 6.0 5.0
Control
Digestate
Ash
Ammonium sulfate
Treatment 30 days 60 days 10.0
B 9.0 b
8.0 a
pH
c
b
b a
7.0
a
6.0 5.0
c
Control
Digestate
Ash
Ammonium sulfate
Treatment 30 days 60 days
Fig. 1 The effect of digestate, fly ash, and ammonium sulfate application on soil pH: a Chernozem and b Fluvisol. The bars marked by the same letter did not significantly differ at P
