Effects of Pinus densiflora on soil chemical and microbial properties in Pb-contaminated forest soil
Effects of Pinus densiflora on soil chemical and microbial properties in Pb-contaminated forest soil
Journal of Ecology and Environment. 2011. Sep, 34(3): 315-322
Copyright ©2011, The Ecological Society of Korea
This is an Open Access article distributed under the terms of the CreativeCommons Attribution Non-Commercial License( permits unrestricted non-commercial use,distribution, and reproduction in any medium, provided the original workis properly cited.
  • Received : June 21, 2011
  • Accepted : July 08, 2011
  • Published : September 01, 2011
Export by style
Cited by
About the Authors
Sunghyun, Kim
School of Civil and Environmental Engineering, Yonsei University, Seoul 120-749, Korea
Insook, Lee
Division of EcoScience, Ewha Womans University, Seoul 120-750, Korea
Hojeong, Kang
School of Civil and Environmental Engineering, Yonsei University, Seoul 120-749, Korea

We investigated the effect of Pb uptake by Pinus densiflora and the Pb fraction in forest soil. We also investigated the change in soil physicochemical characteristics, microbial activity, and root exudates of Pinus densiflora in Pb-contaminated soils. Three-year-old pine seedlings were exposed to 500 mg/kg Pb for 12 months. The metal fractions were measured using sequential extraction procedures. Additionally, factors that affect solubility (three soil enzyme activities and amino acids of root exudate compounds) were also determined. The results showed that Pb contamination significantly decreased enzyme activities due to soil characteristics. In addition, organic matter, nitrate content, and Pb concentration were time dependent. The results indicate that changes in the Pb fraction affected Pb uptake by pine trees due to an increase in the exchangeable Pb fraction. The concentrations of organic acids were higher in Pb-spiked soil than those in control soil. Higher rhizosphere concentrations of oxalic acid resulted in increased Pb uptake from the soil. These results suggest that pine trees can change soil properties using root exudates due to differences in the metal fraction.
In Korea, base metal mines producing Cu-Pb-Zn ores are distributed almost over all of the country and were actively operated until the early 1980s. However, since then, base metal production has declined and most mines closed mainly due to economic reasons (Lee and Lee 2001). Upon closing of the mines, improperly disposed of mineral waste piles and untreated mine drainage have become important sources of heavy metals in the environment. In particular, lead (Pb) contamination increased substantially with industrialization of the forest(Shotyk et al. 1998). Forest ecosystems are particularly sensitive to Pb pollution from atmospheric deposition, as their canopies have a high capacity to intercept aerosols, which are either washed down by rain or reach the soil through litter fall (Bringmark et al. 1998, Ettler et al. 2005).This explains why forests often contain high Pb concentrations in their topsoils. Due to its toxicity, Pb can have a strong adverse impact on soil microorganisms, and their nutrient cycle functions (Nannipieri et al. 2003). Heavy metal stress generally induces soil microbial communities to employ more energy to maintain metabolic functions and detoxification processes, thereby permitting less energy expenditure for growth. Pb stress has also been reported to reduce plant biomass, respiration rates,and enzymatic activities (Frey et al. 2006).
Heavy metal-contaminated forest soil is recycled using organic matter and native woody plants for remediation. Enzymatic activities are frequently used for determining the influence of heavy metal pollutants on soil microbiological quality (Shen et al. 2005). Heavy metals inhibit enzymatic activity by interacting with the enzyme substrate complexes and denaturing the enzyme protein (Vig et al. 2003). Therefore, soil microbial activities are important to understand the mechanisms and changes in forest ecosystems following metal contamination.
However, total concentrations alone may not convey the entire picture of metal reactivity and its potential toxicity to soil biota. Although the solubility of Pb is generally very low, a considerable fraction of total Pb in soil can exist in soluble forms (Wang and Benoit 1996). The behavior of metals and their availability strictly depends on their chemical form and, thus, on their speciation. Therefore, determining chemical form factors or associations with different mineral phases is of great importance to better understand Pb toxicity in forest soils. An estimate of metal availability is more valuable, as it is related to specific bioavailability, reactivity, mobility, and uptake by plants (McBride 1994). To date, it has generally been accepted that the most appropriate methods for evaluating solid speciation are selective sequential extraction procedures. In fact, a large number of sequential extraction methods have been studied and reported, many of which are variants of the Tessier procedure (Tessier et al. 1979). The mechanisms of metal accumulation in soil lead to five major geochemical forms: (i) exchangeable, (ii) bound to the carbonate phase; (iii) bound to iron-manganese oxides; (iv) bound to organic matter; and (v) a residual metal phase. These metal fractions exhibit remarkable differences in mobility, biological availability and chemical behavior in soil.
Dissolvability, adsorption, and the fraction of heavy metals in soils relate to low molecular weight root exudates, such as organic and amino acids (Kuang et al. 2003). McGrath et al. (1997) reported that Zn bioavailability in soil increases as a result of root exudates. Mench and Fargues (1994) found that the root exudates released from Avena sativa L. increase the bioavailability of soil Zn, Cu, and Ni by dissolving ferriferous oxides. Additionally, Kim et al. (2010) reported that the bioavailable metal fraction in soils increased with the addition of root exudates. However, understanding the mechanism controlling nutrient availability in soil requires a comprehensive knowledge of the qualitative and quantitative composition of root exudates (Schilling et al. 1998).
The pine tree has been widely studied as a bio-indicator for measuring heavy metal toxicity (Poykio and Torvera 2001, Yilmaz 2002). Because pine trees are one of the dominant trees species in eastern Asia, we believe that information on the effects of Pb on pine trees is highly valuable. We report here on the impact of Pb contamination on accumulation and root exudates of Pinus densiflora , emphasizing the interdependency of soil chemistry and microbiology.
The purpose of this study was to investigate the toxicity of P. densiflora, soil properties, and soil microorganisms in a forest. We also investigated changes in Pb bioavailability that might affect the microbial activity and root exudates of P. densiflora .
- Experimental design
Natural soil was sampled from the campus of Ewha Woman’s University, which is located in Seoul, Korea. We collected surface soils from a pine tree ( Pinus koraiensis S. et Z .) dominant forest. The physicochemical properties of the soil are shown in Table 1 . The soil was passed through a 2 mm sieve and then air-dried. For each experiment, 32 kg soil aliquots were artificially contaminated with 500 mg/kg Pb, added as an aqueous solution of PbSO 4 . The soils were then thoroughly mixed to ensure uniformity and then aged 1 week to stabilize. Contaminated soils were measured 24 h prior to the start of each test. Soils were divided into two groups (uncontaminated soils and contaminated soils) in a total of 32 pots. Three-year old pine seedlings ( P. densiflora ), which were obtained from the Korean Forest Service, were planted in both uncontaminated soils and Pb-contaminated soils in 1 kg test pots (1.5 L cylindrical plastic pots; diameter, 10 cm). Thirty mL of water and 20 mL of 1/2 Hoagland solution (Hoagland and Arnon 1950) were added to the soil weekly. The plants were incubated for 12 months in a greenhouse. Every 4 months, the shoots, roots, and soil in each pot were harvested. After the harvest, plants were washed with water to remove soil particles. To determine the amount of Pb in the plants, the roots and shoots were further separated with scissors and then dried in an oven at 70°C for 24-h. Plants samples were digested in concentrated HNO 3 in an automatic microwave digester (MDS-2000; CEM, Matthews, NC, USA) and allowed to digest for 1-h at full power (630 w). Pb content was then determined with an atomic absorption spectrometer 100 graphite-furnace (AAS analysis 100; Perkin Elmer, Kundenunterstützung, Germany).
For quality control, a certified reference sample of total metal content in plants was analyzed using the methods described above. Observed concentrations for the certified SRM 1575 (pine needles; National Institute of Standards Technology) were measured. The percentage recovery for the metal of interest was 92.6%.
- Soil characteristics and enzyme activities
Soil pH was determined by adding soil to water at a ratio of 1:5 (w:v). Soil organic matter content was determined by loss-on-ignition at 700°C in a furnace (MAS 7000; CEM) (Saxena and Bartha, 1983). Soil cation-exchange capacity (CEC) was determined by EPA 9081 methods (US Environmental Protection Agency 1986). Soil nitrate (NO 3 -) content was determined by extracting soil with deionized water and then measuring NO 3 - content in the liquid phase using an NO 3 - electrode (Gelderman and Beegle 1998).
Dehydrogenase activity was measured with an was 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride assay (Tabatabai 1982). The mixtures (fresh soil 3 g) were incubated for 2-h at 37°C. Reaction products were detected using a spectrophotometer (DR/3000 Spectrophotometer; HACH, Loveland, CO, USA) at 485 nm.
The activities of β-glucosidase and acid phosphatase were measured using the methylumbelliferone (MUF)-substrate method (Freeman et al. 1996). The concentration of the MUF-β-glucoside substrate solution was 400 μM (Sigma, St. Louis, MO, USA), whereas the concentration of the MUF-phosphate substrate solution was 800 μM (Sigma). The enzymatic activities in the soil and substrate solution (1:5, w/v) were measured using a fluorometer.
Soil physicochemical characteristicsValues represent means of three replicates.Significant after t-test at *P ≺ 0.05, **P ≺ 0.01, ***P ≺ 0.001 (vs. control group).CEC, cation exchange capacity.
Lager Image
Soil physicochemical characteristics Values represent means of three replicates. Significant after t-test at *P ≺ 0.05, **P ≺ 0.01, ***P ≺ 0.001 (vs. control group). CEC, cation exchange capacity.
- Sequential soil extraction procedures
To determine the amount of Pb in the soils, Pb was fractionated by the sequential extraction procedure of Tessier et al. (1979) in triplicate. Extractions were conducted in 50 mL polypropylene centrifuge tubes. Between each extraction, the supernatant was centrifuged at 6,000 rpm for 15 min and filtered. The five fraction methods indicated below used 1 g of dry soil.
(i) Exchangeable: The soil was extracted at room temperature for 1-h with 8 mL of magnesium chloride solution (1M MgCl 2 , pH 7.0);
(ii) Bound to the carbonate phase: The residue from (i) was leached at room temperature with 8 mL of 1 M NaOAc adjusted to a pH of 5.0 with acetic acid (HOAc). Continuous agitation was maintained, and the time necessary for complete extraction was evaluated;
(iii) Bound to iron-manganese oxides: The residue from (ii) was extracted with 20 mL 0.4 M NH 2 OH HCl in 25% (v/v) HOAc. The latter experiments were performed at 96 ± 3°C with occasional agitation, and the time needed for complete dissolution of the free iron oxides was evaluated;
(iv) Bound to organic matter: To the residue from (iii), 3 mL of 0.02M HNO 3 and 5 mL of 30% H 2 O 2 were added and adjusted to a pH of 2 with HNO 3 , and the mixture was heated to 85 ± 2°C for 2-h with occasional agitation.
A second 3 mL aliquot of 30% (v) residual metal phase: The residue from (iv) was digested with a HF-HClO 4 mixture according to the procedure described below for total metal analysis.
(vi) Total: Fresh sample (1.0 g) was digested with 8 mL of aqua regia (HCl and HNO 3 , 3 + 1, v/v) for 2-h at 120ºC.
To verify the reliability of our sequential extraction procedures, recovery (%) of the sum of the Pb concentrations in individual fractions to the total Pb concentration in soils was calculated and found to be 102.5%.
- Analysis of root exudates
Plants were harvested by gently removing them from the soil. Prior to analysis, the plants were washed with water to remove soil particles. The plants were sampled and the extracts were prepared as described by Yun and Kil (1992). Fresh roots (100 g) were immersed in 1 L of distilled water for 48-h, after which the solutions were filtered through a 0.45 ㎛ syringe filter and used undiluted.
Organic acids were analyzed with an ion chromatography unit (DX-500; Dionex, Sunnyvale, CA, USA) equipped with an ED40 electro-chemical detector, an IonPac AS11 (4 × 250 mm) column, and an ASRS-Ultra II Anion self-regenerating suppressor (CA). The total concentration of the root organic acids was calculated as the sum of the concentrations of individual organic acids.
- Statistical analysis
Comparison of enzyme activities in rhizosphere soilsValues represent means of three replicates.Significant after t-test at *P ≺ 0.05, **P ≺ 0.01, ***P ≺ 0.001 (vs. control group).
Lager Image
Comparison of enzyme activities in rhizosphere soils Values represent means of three replicates. Significant after t-test at *P ≺ 0.05, **P ≺ 0.01, ***P ≺ 0.001 (vs. control group).
Repeated measures analysis of variance (ANOVA) for Pb treatments with increasing timeVariables include the physicochemical characteristics and soil enzyme activities (dehydrogenase, acid phosphatase, and β-glucosidase).MC, moisture content; OM, organic matter; CEC, cation exchange capacity; DHA, dehydrogenase; APA, acid phosphatase; BGO, β-glucosidase
Lager Image
Repeated measures analysis of variance (ANOVA) for Pb treatments with increasing time Variables include the physicochemical characteristics and soil enzyme activities (dehydrogenase, acid phosphatase, and β-glucosidase). MC, moisture content; OM, organic matter; CEC, cation exchange capacity; DHA, dehydrogenase; APA, acid phosphatase; BGO, β-glucosidase
We used a repeated-measures analysis of variance (ANOVA) to determine the effects of time and Pb treatment on the physical and chemical characteristics of the soil and on soil enzyme activity using SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA). The relationship between metal uptake and the metal fraction was determined using a classic multivariate linear regression model on Microsoft Excel.
- Soil characteristics and enzyme activity
The soil characteristics are shown in Table 1 . The physicochemical properties of the soil were as follows: Soil texture, loamy sand (54.1% sand, 30.9% silt, and 15.0% clay); total organic matter, 2.8%; total moisture content, 10%; CEC, 10.3 mmol/kg. Soil pH was in the weakly acidic range (5.6-6.2).
Table 2 describes the soil enzyme activity. Dehydrogenase, acid phosphatase, and β-glucosidase decreased remarkably with increasing Pb (P < 0.01) ( Table 2 ). Relative to the controls, dehydrogenase, acid phosphatase, and β-glucosidase activity in the Pb treatment group decreased 33%, 32%, and 12%, respectively. However, in Pb-contaminated soils, dehydrogenase and β-glucosidase increased with time. In contrast, acid phosphatase activity decreased slightly over the same time period.
The pH decreased slightly in Pb-contaminated soils due to the use of the PbSO 4 solution, although it did not significantly change over time. Organic matter and nitrate were greatly influenced by both Pb and time. Additionally, there were Pb-X time interactions, as indicated by the repeated-measures ANOVA ( Table 3 ). Organic matter increased with time and Pb treatment. Nitrate decreased with Pb treatment but increased with time.
- Changes in the soil Pb fraction and uptake by Pinus densiflora
The soil Pb binding forms are presented in Fig. 1 . Most of the Pb soil fraction was bound to iron-manganese oxides. The percentage present as exchangeable (F1) and Pb-bound to carbonate (F2) fractions and bioavailability decreased over time, although the F3 and F4 fractions increased over time. The amount of Pb bound to Fe-Mn oxides (F3) and bound to organic matter (F4) increased consistently with increasing incubation time. After 8 months of incubation, the Pb concentration in the Fe-Mn oxide-bound fraction in soil reached 358 mg/kg. Pb concentrations were 62 mg/kg in the organic matter fraction.
Fig. 2 shows the correlations between soil exchangeable Pb concentrations and pine tree uptake of Pb. Pb uptake by roots and shoots was positively correlated to an increase in exchangeable Pb in the soil. Absorption and accumulation of Pb in the roots and shoots increased with time. The Pb content bound to roots increased to nearly 90% upon exposure to Pb.
- Organic acids in root exudates
Table 4 shows the organic acid concentrations of pine root exudates, which affected growth rate and heavy metal uptake. The primary organic acid in the pine exudates was succinic acid. Furthermore, small amounts of oxalic acid, acetic acid, and citric acid were detected in root exudates. Specifically, the amount of oxalic acid was 4-fold greater in the roots of pine in the pots treated with Pb after 12 months, although succinic acid decreased during the incubation period. Acetic acid was only detected in
Lager Image
The concentration of soil Pb in fractions using a sequential extraction procedure. F1 exchangeable; F2 bound to carbonates; F3 bound to Fe-Mn oxides; F4 bound to organic matter; F5 residual; M months.
Lager Image
Correlation between soil exchangeable Pb concentrations and shoot Pb concentration (a) and root Pb concentration (b).
The concentrations (μg/g) of organic acids in pine root exudatesM, months; ND, not detectable.
Lager Image
The concentrations (μg/g) of organic acids in pine root exudates M, months; ND, not detectable.
Pb-contaminated soils.
- Changes in soil characteristics and enzyme activity
Enzyme activity significantly decreased in Pb-contaminated soils. Such a decrease in microbial activity was concomitant with a marked increase in exchangeable and soluble Pb. Effects exerted on microbial activity may be attained by lowering the soil pH with organic acid (Shotyk et al. 1998), and complexation with heavy metals (Wierzbicka 1999), clay minerals and metal oxides with organic ligands from bacterial exudation (Gadd 2000). The effects of soil pollution on enzyme activity are complex; the response of different enzymes to the same pollutant may vary greatly, and the same enzyme may respond differently to different pollutants (He et al. 2003). In this study, Pb inhibited all enzyme activity, whereas Pb only slightly inhibited organic C-acquiring enzyme activity (β-glucosidase). It has been reported that different metal ions exhibit different behavior in their ability to act as β -glucosidase inhibitors (Scigelova and Crout 1999).
However, dehydrogenase and β-glucosidase increased over time in the rhizosphere. Under the influence of root activity, Pb extracted from polluted soils increased, as pH decreased. Exchangeable Pb was much higher in the rhizosphere than that in bulk soil. Plants can modify metal speciation and behavior in the rhizosphere by producing exudates (Laperche et al. 1997), and these results were time dependent. The pine exudates may have been affected by an interaction with microbial activities and soil properties in the rhizosphere. Some researchers have studied the relationships between the pine rhizosphere and metal toxicity. Hartley-Whitaker et al. (2000) reported that Cd and Zn accumulation in P. sylvestris seedlings increases and reduces the effects of metal toxicity due to enhanced root microbes. The toxic effect of Scot pine seedlings is associated with their root fungi during multi metal contamination (Hartley et al. 1999). Also, a positive correlation exists between soil properties, such as cation exchange capacity and microbial processes under heavy metal stress (Baath 1989).
- Correlation between soil exchangeable Pb and shoot and root Pb concentrations
Fig. 1 shows the time-dependent changes in Pb concentration of each fraction. Cultivation time significantly affected the Pb fraction distribution. The newly added Pb existed mainly in the surface soil particles as an exchangeable fraction.
Fig. 2 presents the correlations between soil exchangeable Pb concentrations and pine tree uptake of Pb. Exchangeable metals in the soil are easily available for plant uptake (Kabata-Pendias 1993). Metal toxicity and accumulation in plants are related to the effects of synergetic and antagonistic metal interactions. These effects may vary depending on the physicochemical factors such as organic matter content, pH, redox potential, and ion speciation. These factors could influence metal mobility and bioavailability in soils (van Gestel 2008). For example, lower soil pH favors the release of metals into soil solution, allowing plants to take up more metals (Tang et al. 2003, Wu et al. 2009). In the present study, soil pH decreased slightly with the addition of Pb ( Table 1 ). Moreover, a high organic matter content in soil resulted in increased organic matter in the bound fractions (F3 and F4) over time ( Table 2 ). These results show that the metal fraction in soil was related to changes in the organic matter content due to microbial activity (Lock and Janssen 2001). Investigations relative to these questions should complement the present approach for a full understanding of the multifaceted issue of bioavailability in soil systems.
- Organic acids in root exudates
An increase in Pb uptake was associated with increases in root exudates, suggesting that the root exudates activate Pb by dissolution and chelation. Because pH is a key factor controlling heavy metal extractability and mobility, changes in organic acids in root exudates may modify Pb availability by changing the pH (Zhang et al. 2006). Other studies have shown the significance of root exudates in the bioavailability of heavy metals in soil (Chen et al. 2006, Zhang et al. 2006). High molecular weight organic acids, such as humic acid, can reduce the bioavailability and toxicity of heavy metals (Liao and Huang 2002), whereas low molecular weight organic acids and amino acids can increase the bioavailability and plant accumulation of heavy metals by decreasing the rhizosphere pH or chelating metals in the soil (Liao and Huang 2002).
The results of this study demonstrate that changes in the soil metal fraction are related to soil chemical characteristics, microbial activity, and root exudates. The exchangeable metal content increased in soil with a low pH and the presence of organic acids from root exudates. In addition, root exudates change microbial activity that use the main C-sources: d-malic acid, d-glucosaminic acid, and α-ketobutyric acid (data not shown). These findings indicate that organic acids from plant roots can combine with metal ions, which can then be taken up easily by microbes and plants.
This study was conducted with the support of NRF (2010-0028708), Eco River 21, Eco Star, and AEBRC.
Baath E 1989 Effects of heavy metals in soil on microbial processes and populations Water Air Soil Pollut 47 335 - 379    DOI : 10.1007/BF00279331
Bringmark L , Bringmark E , Samuelsson B 1998 Effects on mor layer respiration by small experimental additions of mercury and lead Sci Total Environ 213 115 - 119    DOI : 10.1016/S0048-9697(98)00082-5
Chen RF , Shen RF , Gu P , Dong XY , Du CW , Ma JF 2006 Response of rice (Oryza sativa) with root surface iron plaque under aluminium stress Ann Bot 98 389 - 395    DOI : 10.1093/aob/mcl110
Ettler V , Van?k A , Mihaljevic M , Bezdi?ka P 2005 Contrasting lead speciation in forest and tilled soils heavily polluted by lead metallurgy Chemosphere 58 1449 - 1459    DOI : 10.1016/j.chemosphere.2004.09.084
Freeman C , Liska G , Ostle NJ , Lock MA , Reynolds B , Hudson J 1996 Microbial activity and enzymic decomposition processes following peatland water table drawdown Plant Soil 180 121 - 127    DOI : 10.1007/BF00015418
Frey B , Stemmer M , Widmer F , Luster J , Sperisen C 2006 Microbial activity and community structure of a soil after heavy metal contamination in a model forest ecosystem Soil Biol Biochem 38 1745 - 1756    DOI : 10.1016/j.soilbio.2005.11.032
Gadd GM 2000 Bioremedial potential of microbial mechanisms of metal mobilization and immobilization Curr Opin Biotechnol 11 271 - 279    DOI : 10.1016/S0958-1669(00)00095-1
Gelderman RH , Beegle D , Brown JR 1998 Nitrate-nitrogen. In: Recommended Chemical Soil Test Procedures for the North Central Region University of Missouri-Columbia Columbia 17 - 20
Hartley J , Cairney JWG , Freestone P , Woods C , Meharg AA 1999 The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings Environ Pollut 106 413 - 424    DOI : 10.1016/S0269-7491(99)00095-0
Hartley-Whitaker J , Cairney JWG , Meharg AA 2000 Sensitivity to Cd or Zn of host and symbiont of ectomycorrhizalPinus sylvestrisL. (Scots pine) seedlings Plant Soil 218 31 - 42    DOI : 10.1023/A:1014989422241
He ZL , Yang XE , Baligar VC , Calvert DV 2003 Microbiological and biochemical indexing systems for assessing quality of acid soils Adv Agron 78 89 - 138    DOI : 10.1016/S0065-2113(02)78003-6
Hoagland DR , Arnon DI 1950 The Water Culture Method for Growing Plants without Soil University of California Agricultural Experiment Station Berkley
Kabata-Pendias A 1993 Behavioural properties of trace metals in soils Appl Geochem 8 (Suppl 2) 3 - 9    DOI : 10.1016/S0883-2927(09)80002-4
Kim S , Lim H , Lee I 2010 Enhanced heavy metal phytoextraction byEchinochloa crus-galliusing root exudates J Biosci Bioeng 109 47 - 50    DOI : 10.1016/j.jbiosc.2009.06.018
Kuang YW , Wen DZ , Zhong CW , Zhou GY 2003 Root exudates and their roles in phytoremediation Acta Phytoecol Sin 27 709 - 717
Laperche V , Logan TJ , Gaddam P , Traina SJ 1997 Effect of apatite amendments on plant uptake of lead from contaminated soil Environ Sci Technol 31 2745 - 2753    DOI : 10.1021/es961011o
Lee CH , Lee HK 2001 Hydrochemical monitoring and heavy metal contaminations at the Narim Mine Creek in the Sulcheon District Republic of Korea Environ Geochem Health 23 343 - 368    DOI : 10.1023/A:1012248603765
Liao M , Huang C 2002 Effects of organic acids on the toxicity of cadmium during ryegrass growth Chin J Appl Ecol 13 109 - 112
Lock K , Janssen CR 2001 Modeling zinc toxicity for terrestrial invertebrates Environ Toxicol Chem 20 1901 - 1908    DOI : 10.1002/etc.5620200907
McBride MB 1994 Environmental Chemistry of Soils Oxford University Press New York 31 - 62
McGrath SP , Shen ZG , Zhao FJ 1997 Heavy metal uptake and chemical changes in the rhizosphere ofThlaspi caerulescensandThlaspi ochroleucumgrowon in contaminated soils Plant Soil 188 153 - 159    DOI : 10.1023/A:1004248123948
Mench MJ , Fargues S 1994 Metal uptake by iron-efficient and inefficient oats Plant Soil 165 227 - 233    DOI : 10.1007/BF00008066
Nannipieri P , Ascher J , Ceccherini MT , Landi L , Pietramellara G , Renella G 2003 Microbial diversity and soil functions Eur J Soil Sci 54 655 - 670    DOI : 10.1046/j.1351-0754.2003.0556.x
Poykio R , Torvera H 2001 Pine needles (Pinus Sylvestris) as a bio-indicator of sulphur and heavy metal deposition in the area around a pulp and paper mill complex at Kemi northern Finland Int J Environ Anal Chem 79 143 - 154    DOI : 10.1080/03067310108035906
Saxena A , Bartha R 1983 Microbial mineralization of humic acid-34-dichloroaniline complexes Soil Biol Biochem 15 59 - 62    DOI : 10.1016/0038-0717(83)90120-7
Schilling G , Gransee A , Deubel A , Lezovic G , Ruppel S 1998 Phosphorus availability root exudates and microbial activity in the rhizosphere J Plant Nutr Soil Sci 161 465 - 478
Scigelova M , Crout DHG 1999 Microbial β-N-acetylhexo-saminidases and their biotechnological applications Enzyme Microb Technol 25 3 - 14    DOI : 10.1016/S0141-0229(98)00171-9
Shen G , Lu Y , Zhou Q , Hong J 2005 Interaction of polycyclic aromatic hydrocarbons and heavy metals on soil enzyme Chemosphere 61 1175 - 1182    DOI : 10.1016/j.chemosphere.2005.02.074
Shotyk W , Weiss D , Appleby PG , Cheburkin AK , Frei R , Gloor M , Kramers JD , Reese S , Van Der Knaap WO 1998 History of atmospheric lead deposition since 1237014C yr BP from a Peat Bog Jura Mountains Switzerland Science 281 1635 - 1640    DOI : 10.1126/science.281.5383.1635
Tabatabai MA , Page AL 1982 Soil enzymes. In: Methods of Soil Analysis Part 2. Agronomy Monograph American Society of Agronomy Madison WI 903 - 904
Tang S , Xi L , Zheng J , Li H 2003 Response to elevated CO2of Indian mustard and sunflower growing on copper contaminated soil Bull Environ Contam Toxicol 71 988 - 997    DOI : 10.1007/s00128-003-0224-9
Tessier A , Campbell PGC , Bisson M 1979 Sequential extraction procedure for the speciation of particulate trace metals Anal Chem 51 844 - 851    DOI : 10.1021/ac50043a017
US Environmental Protection Agency 1986 Test Methods for Evaluating Solid Waste. SW-846 Method 9081 US Environmental Protection Agency Washington D.C. DC
van Gestel CAM 2008 Physico-chemical and biological parameters determine metal bioavailability in soils Sci Total Environ 406 385 - 395    DOI : 10.1016/j.scitotenv.2008.05.050
Vig K , Megharaj M , Sethunathan N , Naidu R 2003 Bioavailability and toxicity of cadmium to microorganisms and their activities in soil Adv Environ Res 8 121 - 135    DOI : 10.1016/S1093-0191(02)00135-1
Wang EX , Benoit G 1996 Mechanisms controlling the mobility of lead in the spodosols of a northern hardwood forest ecosystem Environ Sci Technol 30 2211 - 2219    DOI : 10.1021/es950590e
Wierzbicka M 1999 Comparison of lead tolerance inAllium cepawith other plant species Environ Pollut 104 41 - 52    DOI : 10.1016/S0269-7491(98)00156-0
Wu H , Tang S , Zhang X , Guo J , Song Z , Tian S , Smith DL 2009 Using elevated CO2 to increase the biomass of aSorghum vulgare × Sorghum vulgarevar.sudanensehybrid and Trifolium pratense L. and to trigger hyperaccumulation of cesium J Hazard Mater 170 861 - 870    DOI : 10.1016/j.jhazmat.2009.05.069
Yilmaz S 2002 Determination of optimal land use of Erzurum plain Ataturk Univ Agric Fac 32 485 - 498
Yun KW , Kil BS 1992 Assessment of allelopathic potential inArtemisia princepsvar.orientalisresidues J Chem Ecol 18 1933 - 1940    DOI : 10.1007/BF00981917
Zhang MK , He ZL , Calvert DV , Stoffella PJ 2006 Extractability and mobility of copper and zinc accumulated in sandy soils Pedosphere 16 43 - 49    DOI : 10.1016/S1002-0160(06)60024-6