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Effect of Root-Induced Chemical Changes on Dynamics and Plant Uptake of Heavy Metals in Rhizosphere Soils
Pedosphere 20(4): 494–504, 2010 ISSN 1002-0160/CN 32-1315/P c 2010 Soil Science Society of China Published by Elsevier Limited and Science Press
Effect of Root-Induced Chemical Changes on Dynamics and Plant Uptake of Heavy Metals in Rhizosphere Soils∗1 K. R. KIM1,∗2 , G. OWENS2 and R. NAIDU2,3 1 Climate Change & Agroecology Division, Department of Agricultural Environment, National Academy of Agricultural Science, Suwon 441 857 (Republic of Korea) 2 Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095 (Australia) 3 Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Mawson Lakes, SA 5095 (Australia)
(Received September 24, 2009; revised March 15, 2010)
ABSTRACT It is increasingly recognized that metal bioavailability is a better indicator of the potential for phytoremediation than the total metal concentration in soils; therefore, an understanding of the influence of phytoremediation plants on metal dynamics at the soil-root interface is increasingly vital for the successful implementation of this remediation technique. In this study, we investigated the heavy metal and soil solution chemical changes at field moisture, after growth of either Indian mustard (Brassica juncea) or sunflower (Helianthus annuus L.), in long-term contaminated soils and the subsequent metal uptake by the selected plants. In addition, the fractions of free metal ions in soil solution were determined using the Donnan membrane technique. After plant growth soil solution pH increased by 0.2–1.4 units and dissolved organic carbon (DOC) increased by 1–99 mg L−1 in all soils examined. Soluble Cd and Zn decreased after Indian mustard growth in all soils examined, and this was attributed to increases in soil solution pH (by 0.9 units) after plant growth. Concentrations of soluble Cu and Pb decreased in acidic soils but increased in alkaline soils. This discrepancy was likely due to a competitive effect between plant-induced pH and DOC changes on the magnitude of metal solubility. The fractions of free Cd and Zn ranged from 7.2% to 32% and 6.4% to 73%, respectively, and they generally decreased as pH and DOC increased after plant growth. Metal uptake by plants was dependant on the soil solution metal concentration, which was governed by changes in pH and DOC induced by plant exudates, rather than on the total metal concentrations. Although plant uptake also varied with metal and soil types, overall soluble metal concentrations in the rhizosphere were mainly influenced by root-induced changes in pH and DOC which subsequently affected the metal uptake by plants. Key Words:
bioavailability, phytoremediation, speciation
Citation: Kim, K. R., Owens, G. and Naidu, R. 2010. Effect of root-induced chemical changes on dynamics and plant uptake of heavy metals in rhizosphere soils. Pedosphere. 20(4): 494–504.
Contamination of soils by metal(loid)s through anthropogenic activities is a widespread and serious problem confronting society, scientists, and regulators worldwide. For this reason, a lot of research has been conducted over the last 50 years to assess and remediate potentially hazardous sites. Phytoremediation is a developing technology that uses plants to ameliorate contamination of soils and is often perceived as a greener alternative to the traditional remediation technologies (Reeves et al., 2000). The basic principles of phytoremediation rely on an understanding of the factors that either control plant accumulation or contribute to immobilization of metal contaminants. The concentrations of bioavailable and soluble metals are considered more important than total metal concentrations because these parameters govern the form and accessibility of metals to plants and determine the fraction to be transferred to aboveground tissues (Ernst, 1996; Naidu et al., 2003). In the context of metal bioavailability, metal speciation in soil solution is important given that metal uptake by plants is often dictated by the nature ∗1 Supported
by the University of South Australia, Australia. author. E-mail: [email protected].
∗2 Corresponding
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of the metal species. Metal uptake by plants can also be dictated by kinetic parameters. Zhang et al. (2001) showed that for copper the kinetically labile pool played an important role in plant uptake. Plants have the potential to transform metal species for easier uptake, or to detoxify metals through root exudation or pH changes in the rhizosphere (Awad et al., 1994; Luo et al., 2000; Hinsinger, 2001; Whiting et al., 2001; Wang et al., 2002; Lin et al., 2004). Following the observation that the measurement of free metal ion activities in soil solution before plant growth did not provide the best prediction of metal supply to the plant (Nolan et al., 2005), it remains a controversial issue whether plants predominantly absorb free metal ions from soil solution (Hamon et al., 1995; Sauv´e et al., 1996). For this reason, determining metal speciation in the rhizosphere and understanding subsequent metal uptake by plants are currently subjects of much research. The present study investigated the effect of growing two plants commonly recommended for phytoremediation, Indian mustard (Brassica juncea) and sunflower (Helianthus annuus L.) (Dushenkov and Kapulnik, 2000; Reeves and Baker, 2000; Schmidt, 2003), on heavy metal dynamics and soil solution chemistry in two contrasting heavy metal contaminated soils and on the subsequent metal uptake. These two plant species may potentially differ in the mechanism for metal uptake since Indian mustard is best known as metal hyperaccumulator while sunflower is better known as a metal stabilizer. MATERIALS AND METHODS Soils and plants Soils were either collected from the vicinity of a Pb and Zn smelter operating in Port Pirie, South Australia (soils PP02 and PP03), or from a military shooting range in South Korea (soils Ko01 and Ko02). The soils collected from Australia were alkaline (pH > 8) and highly contaminated with multiple metals, while the Korean soils were slightly acidic (pH < 6.5) and either moderately contaminated (Ko01) or non-contaminated soils (Ko02). The physicochemical properties of the soils are summarised in Table I. TABLE I Selected physicochemical properties of the soils studied Soil No.
pH
Organic matter kg−1
PP02 PP03 Ko01 Ko02
9.2 8.2 6.2 6.4
g 3 17 6 11
Dissolved organic carbon mg 16 613 15 58
L−1
Clay
Cd
Cu
kg−1
g 120 130 180 70
Pb mg
17.8 3.6 7.9 0.1
446 630 87 3
Zn
kg−1 2 427 625 380 24
6 472 9 759 55 18
Indian mustard, a known hyperaccumulator for Zn, Cd, and Pb, was selected as representative of plants exhibiting the hyperaccumulation mechanism (Reeves and Baker, 2000; Schmidt, 2003), while sunflower was selected as an example of a plant exhibiting the heavy metal stabilization mechanism (Dushenkov and Kapulnik, 2000). Both of these plant species have the potential to be used for remediation of heavy metals in soils in the root zone by phytoextraction or phytostabilization, and may have significantly different mechanisms for interacting with metals. Plant growth experiment A plant growth experiment was conducted under greenhouse conditions. Air dried soil (500 g, < 2 mm) was weighed into 600 mL plastic pots and saturated with a basal fertilizer nutrient solution consisting of 150 mg kg−1 of N (as NH4 NO3 ), 75 mg kg−1 of P (as KH2 PO4 ), and 95 mg kg−1 of K (as KH2 PO4 ) (Quartacci et al., 2006). The nutrient solution was supplied to the plants via a saucer beneath the pots and the uptake was through a hole in the base of the pots. This method prevented the leaching
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of heavy metals. Soils were allowed to equilibrate for one week, and then additional water was added in the same manner described above, to compensate for any moisture loss due to evaporation. The pots were equilibrated for an additional 48 h prior to sowing seeds. During watering, it is unlikely that reducing conditions occurred because at no time were the pots fully saturated for any extended period. Pareuil et al. (2008) has previously shown that reducing conditions could not be induced within 21 days using water alone. Indian mustard and sunflower seeds were sown directly into the soils and the pots were covered with transparent plastic bags to maintain the moisture content. Twenty seeds for Indian mustard and twelve for sunflower were sown. Three days after germination the plastic bags were removed and one week after this the number of seedlings was thinned to 15 for Indian mustard and 7 for sunflower. Watering was conducted in the same way as the initial water saturation method every 3 days for 2 weeks and subsequently, as the plants developed, watering continued every two days until harvest. Plants were harvested 6 weeks after germination and plant tissue and soil solution samples were prepared for analysis using the methods described below. The experiment was conducted in triplicate for each soil and included control soils into which no plants were sown, but which were otherwise treated exactly as all other soils studied. It was assumed that the soils in the pots where plants were grown were examples of rhizosphere soils while the control soil, without plants, was considered to be bulk soil. Even though a rhizosphere soil would strictly be that soil intimately associated with the root zone of the plant, the term rhizosphere soil was used here to easily distinguish between a soil cultivated with plants and the bulk soil where plants were not cultivated. Analysis Plant. The aboveground plant tissues (shoots) were cut and rinsed twice with Milli-Q water while the belowground tissues (roots) were rinsed with tap water after removing the soil particles clinging to the roots with vigorous shaking and washed using a 5% (v/v) aqueous detergent solution (Decon 90). Subsequently the roots were thoroughly rinsed three times with Milli-Q water. Additional washing using an ultrasonic bath was not conducted, as this had been shown to have little improvement on washing efficiency (Wang et al., 2003). Washed shoots and roots were dried at 65 ◦ C in a fanforced oven and periodically weighed until there was no change in dry mass. The dry plant tissues were finely ground using a commercial electronic grinder and stored in ziplock plastic bags prior to acidic digestion. The dried and ground plant tissues (0.5 g) were digested with concentrated HNO3 (5 mL) using a commercially available temperature-controlled digestion block (AIM 500, A. I. Scientific, Australia). Blanks, duplicates, and spiked samples were included at a rate > 5% to ensure quality control during plant digestion. Total heavy metal concentrations in digest solutions of plant tissues were determined using inductively coupled plasma mass spectrometry (ICP-MS). Routine soil laboratory analyses. Soil pH and electrical conductivity (EC) were determined in a 1:5 soil:water (w/v) suspension using a combination pH/EC meter (smartCHEM-LAB, TPS, Australia) after 1 h equilibration. Soil organic matter content was determined using the Walkley-Black method (Nelson and Sommers, 1996) and total dissolved organic carbon (DOC) was analysed using an automated total organic carbon analyser (Model 1010, O.I. Analytical, USA) after extraction of soil solution. Soil texture was determined using a micro-pipette method (Miller and Miller, 1987). Heavy metal contents in the soils were determined by ICP-MS after microwave (MARS5, CEM, USA)-assisted aqua regia digestion in accordance with USEPA (1997) method 3051H. Each microwave digestion batch included a standard reference material (Montana Soil SRM2711, certificated by the National Institute of Standards & Technology, USA) and a blank to validate the digestion operation. Soil solution extraction and heavy metal speciation. After plant harvest, sub-samples of soils from each pot were air dried and subsequently rewetted to allow soil solution extraction at 70% maximum water holding capacity (MWHC). Soil (20 g, < 2 mm) was weighed into 20 mL disposable plastic syringes having the outlet plugged with acid-washed glass wool. The syringe was inserted into a 50 mL
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centrifuge tube separated from the base by a polyvinyl chloride (PVC) spacer (Thibault and Sheppard, 1992). Water was added to the soil to obtain 70% MWHC and the soil was allowed to equilibrate for 24 h. The resulting soil paste was centrifuged at 2 500 r min−1 for 25 min and the isolated soil solutions were filtered through 0.45 μm cellulose acetate disposable filters (MillexTM , Millipore, USA) and stored for further analysis. In order to secure enough soil solution (100 mL) for metal speciation, 25 replicates of syringe extractions for each pot were required. Solutions from individual replicates were isolated and combined to obtain at least 100 mL of solution after filtration. Metal speciation was commenced within 2 h after collecting the soil solutions using the Donnan membrane technique (DMT). The DMT system used here was similar to that used by Temminghoff et al. (2000), including a negatively charged membrane (No. 55165 2U, BDH, UK). However, while Temminghoff et al. (2000) had used the conditions of 500 mL donor and 17 mL acceptor, in this study 100 mL of donor and 8 mL of acceptor solution were used. These adjusted conditions allowed for the limitation of collecting large volumes of soil solution by extraction at 70% MWHC. Of the four heavy metals studied (Cd, Cu, Pb, and Zn), only Cd and Zn were amenable to speciation by DMT because Cu and Pb existed predominantly as metal-organic complexes. Statistical analysis. All data were statistically analysed using SPSS 12.0.1 (SPSS Inc.) to examine interrelations between changes in metal concentrations in soil solution of the rhizosphere soil and key soil solution properties. Simple calculations were performed using Microsoft ExcelTM and the graphs were drawn using both SPSS 12.0.1 and Sigmaplot 10 (Systat Software Inc.). For regression analysis, individual data points were used instead of averages of triplicates. RESULTS AND DISCUSSION Effect of plant roots on rhizosphere soil pH and DOC Considerable controversy exists in the literature regarding the effect of plant growth on the pH at the soil-root interface. For instance, hyperaccumulator plants have been shown to either acidify rhizosphere soils and subsequently increase the dissolved concentrations of heavy metals (McGrath et al., 1997; Sas et al., 2001) or increase soil pH after plant growth (Knight et al., 1997; Luo et al., 2000). In the present study, both Indian mustard and sunflower increased the soil solution pH in all soils studied relative to the controls (Table II). TABLE II Soil solution pH and dissolved organic carbon (DOC) concentrations after plant growth Soil No.
pH Mustard
DOC Sunflower
Control
Mustard
Sunflower
Control
L−1
PP02 PP03 Ko01 Ko02
8.1±0.2a) 8.2±0.1 6.4±0.1 6.6±0.1
a) Mean±standard
8.1±0.0 8.1±0.1 5.8±0.2 5.8±0.2
7.9±0.1 7.7±0.1 5.5±0.2 5.2±0.1
45±5 290±10 32±1 52±1
mg 120±20 283±9 40±2 55±6
23±4 240±10 23±3 51±1
deviation.
Indian mustard increased pH in the rhizosphere soils more than sunflower, and a higher pH increase was observed in the acidic soils (Ko01 and Ko02) compared to the alkaline soils (PP02 and PP03). For instance, Indian mustard growth increased pH by 0.9 and 1.4 units in the soils Ko01 and Ko02, respectively, relative to the pH of the corresponding controls, 5.5 and 5.2, respectively. Sunflower growth increased pH by 0.3 and 0.6 units in the same soils (Table II). The concentrations of DOC in the rhizosphere soils increased irrespective of plant species due to organic exudates from plant roots (Table II). However, the magnitude of increase in DOC concentration differed with plant species and soil type.
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For example, a larger increase in DOC was observed in the highly contaminated alkaline soils, PP02 and PP03, relative to the acidic soils. This higher DOC probably contributed to both higher pH and heavy metal concentrations. These changes in soil pH and DOC concentrations were expected to significantly modify the chemical behaviours of all four heavy metals (Cd, Cu, Pb, and Zn) in the plant rhizosphere. Heavy metal dynamics in the rhizosphere soils Cadmium. Irrespective of plant species and soil type, plant growth resulted in a significant decrease in the soil solution concentration of Cd (Fig. 1a). Average soluble Cd decreased by more than 35% relative to the control soil (without plants) after both Indian mustard and sunflower growth in the alkaline soils, PP02 and PP03, which were highly contaminated with a number of metals. In the acidic soils, Ko01 and Ko02, which contained considerably high concentrations of soluble Cd fractions relative to total Cd, the effect of plant growth on soil solution Cd was even more marked. As shown in Fig. 1a, after Indian mustard growth the soil solution Cd decreased by 67% in Ko01 and 84% in Ko02. This decrease in soluble Cd in both soil types was attributed to an increase in soil solution pH due to plant growth as demonstrated by the observation of lower concentrations of Cd in solution as rhizosphere pH increased after plant growth (Fig. 2). Plant uptake of Cd from a soluble pool could also be potentially responsible for the observed decrease in soluble Cd if resupply from the soil was a limiting factor. However, linear regression analysis did not show any relationship between Cd uptake by plant and the extent of soluble Cd reduction. In contrast, the magnitude of the metal concentration reduction in soil solution was more strongly correlated with pH increase rather than the metal uptake by plants, which is strong evidence for the dominance of pH over other confounding factors. In particular, the influence of an increase in pH on Cd concentration in soil solution was greater in acidic soils than alkaline soils (Fig. 1). Naidu et al. (1994) also demonstrated that small increases in solution pH below pH 6 resulted in large increases in Cd sorption onto soils, while there was lower increase in Cd sorption with increase in solution pH above pH 8. This implied that soil solution Cd was more sensitive to changes in pH after plant growth in acidic soils. The fraction of free Cd in soils ranged from 7.2% to 32% of the total Cd in the control soils with the highest fraction being observed in the acidic soil Ko01 (Fig. 1b). After plant
Fig. 1 Cd (a), Cu (c), Pb (d), and Zn (e) concentrations and free Cd (b) and Zn (f) fractions in the solutions of two alkaline soils (PP02 and PP03) and two acidic soils (Ko01 and Ko02) after plant growth. Values are means of three replicates with standard deviations shown by vertical bars.
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Fig. 2 Relationships between ΔpH and Cd concentration in solutions of the alkaline and acidic soils after plant growth. ΔpH = pH after plant growth − pH without plant.
growth both the fractions of free Cd and the total soil solution Cd decreased with increases in soil pH and DOC (Fig. 1) induced by root exudates. In particular, there was a strongly significant relationship (r2 = 0.85, P < 0.05) between DOC increase and the free Cd fraction decrease. Significantly less free Cd ions were found with the increases in DOC after plant growth. This suggested that the DOC exuded from the plant roots formed stable metal-DOC complexes, resulting in the observed significant reduction in free Cd in soil solution. Copper. The soil solution Cu concentrations increased by 7 folds after Indian mustard and 10 folds after sunflower growth in the highly contaminated soil, PP02, relative to the control (Fig. 1c). The increased concentration of Cu in soil solution was attributed to root exudation and the consequent formation of soluble Cu complexes with DOC induced by plant roots. Also soil solution Cu tended to decrease in the moderately contaminated acidic soil, Ko01, while there was no effect of plant growth on soil solution Cu in the non-contaminated acidic soil, Ko02 (Fig. 1c). Linear regression analysis showed a significant positive relationship between DOC and Cu concentration in soil solutions from both the highly contaminated alkaline soil (r2 = 0.51, P < 0.05) and the moderately contaminated acidic soil (r2 = 0.5, P < 0.05) after plant growth. Thus soluble Cu concentration increased with increasing DOC after plant growth in alkaline soils. No free Cu was detected in soil solution, demonstrating the conversion of free Cu to Cu organic complexes due to elevated plant root exudates. Lead. As with Cu, soil solution Pb increased in the highly contaminated soil, PP02, and decreased in the less contaminated soil, Ko01, after plant growth (Fig. 1d). Soil solution Pb in the rhizosphere of Ko01 decreased from 319 μg L−1 in the control to 29 μg L−1 after Indian mustard growth and to 162 μg L−1 after sunflower growth. This decrease was likely due to the increase in soil solution pH in Ko01 after plant growth. Correlation analysis using data from all soils showed a significant inverse exponential relationship between soil solution pH and soluble Pb (r2 = 0.75, P < 0.001). This demonstrated a strong influence of plant-induced pH changes on the chemistry of Pb in the rhizosphere. Further analysis using the data from the alkaline soils, PP02 and PP03, showed significant influence of DOC on Pb concentrations in solution. These demonstrated that for the acid soils, Ko01 and Ko02, solution Pb was influenced by pH, whereas both DOC and pH played a significant role in the high pH soils, PP02 and PP03. As with Cu, free Pb was not detected, which was likely due to formation of Pb organic complexes in soil solution. Zinc. In all the soils studied, soluble Zn decreased after plant growth, with the highest decrease observed after Indian mustard growth in the acidic soil, Ko01 (Fig. 1e). This decrease in soil solution Zn may be attributed to the competing effect of changes in pH and DOC after plant growth on metal chemistry. The increase in soil solution pH resulted in a marked exponential decrease in soil solution Zn (r2 = 0.75, P < 0.001) and the magnitude of the decrease in dissolved Zn increased as the soil solution pH increased (r2 = 0.29). In contrast, increases in DOC concentration up to 40 mg L−1 increased soil
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solution Zn, but Zn concentrations did not increase further, remaining stable at about 400 μg L−1 as DOC increased above 40 mg L−1 (Fig. 3). This implied that the DOC increases, due to root exudation, retarded the magnitude of the decrease in Zn solubility induced by pH increase after plant growth. The fraction of free Zn ranged from 6.4% to 73% of the total Zn concentration in soil solution (Fig. 1f). The lowest free Zn fraction was detected in PP03 where the soil pH and OM contents were higher than those of the other soils. The free Zn fraction after plant growth increased in Ko01 and Ko02 even though soil solution pH and DOC increased, whereas it decreased in PP02 and PP03. This is surprising given that pH increases generally enhance metal-ligand complexation and lead to subsequent decreases in free metal ions in soil solution. However, Indian mustard growth increased free Zn from 70% to 88% in Ko01 and 38% to 64% in Ko02. A similar increase in free Zn concentration was recorded in Ko02 after sunflower growth. This is attributed to the presence of other competing metal ions such as Pb and Cu which have greater selectivity for metal organic complexation in the soil solution. In our previous study we demonstrated that soluble Zn could be increased as soluble Pb and Cu concentrations increased (Kim et al., 2009). Fotovat and Naidu (1998) showed that for some Australian soils while Cu exists as Cu-DOC complex in both acidic and alkaline soils, Zn is present as an ionic form in acidic soils but largely as Zn-DOC complex in alkaline soils. Likewise, in the present study, Zn-DOC complexes were predominant in one of the alkaline soils, PP03, but free Zn was predominant in soil PP02 as shown in Fig. 1. The higher free Zn in PP02 despite higher pH was probably due to much higher concentration of Pb in this soil (Table I) compared to PP03 and resulted from competition by Pb with Zn for available binding sites. In the presence of high concentration of soluble Pb, it is likely that uncomplexed dissolved organic compounds are limiting for Zn organic complexation, explaining the presence of free Zn in the high pH soil, PP02.
Fig. 3 Relationships between ΔDOC and Zn in soil solution after plant growth. ΔDOC = DOC concentrations in soil solution after plant growth – DOC concentrations in soil solution without plant.
Heavy metal uptake by plant As discussed above, plant growth increased DOC and pH in all soils examined and these two factors influenced plant metal uptake through changes in the chemical conditions in the soil solution and consequently heavy metal speciation. Given that the magnitude of changes in DOC and pH varied with soil type, the extent of plant metal uptake also varied with soil type, plant species, and metal type. Overall, increases in pH after plant growth were expected to result in a negative effect on plant metal uptake due to reduced metal concentrations in solution. However, increases in DOC from root exudation may potentially counteract this mechanism and enhance metal uptake by increasing total dissolved metal concentrations. Cadmium. The Cd content ranged from 1.2 to 51 mg kg−1 in shoots and from 2.1 to 73 mg kg−1 in roots of Indian mustard. The ranges were from 1.5 to 70 mg kg−1 in shoots and from 2.2 to 114 mg kg−1 in roots of sunflower (Fig. 4a, b). Cadmium uptake by plants was more closely related to the soil
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solution Cd concentration (Fig. 5) rather than the total metal concentration in the soils. For instance, Cd uptake by plants grown in Ko01 which had the highest soil solution Cd were the highest despite the total Cd concentration being 2.5 times higher in PP02 than in Ko01. In addition, Indian mustard and sunflower accumulated similar amounts of Cd in their shoots from PP03 (1.7 and 1.2 mg kg−1 ) and Ko02 (2.0 and 1.5 mg kg−1 ) even though the total Cd was much higher in PP03. This indicated that plant Cd uptake and translocation was dependent on soil solution Cd and independent of total soil Cd concentration, implying the dependence of metal uptake by plants on the change in dissolved Cd concentration induced by pH increase after plant growth. There existed a negative relationship between increased pH and Cd uptake by plants in the alkaline soils (r2 = 0.52, P < 0.05). The translocation coefficients (Kt ) of Cd, defined as Kt =
MS MR
where MS and MR are the dry weight concentrations (mg kg−1 ) in shoots and roots, respectively, were lower than 1 (Fig. 6), which indicated higher Cd adsorption into the roots, probably due to formation of Cd-DOC complexes resulting from DOC increase in the rhizosphere, and less translocation of Cd to the shoots.
Fig. 4 Concentrations of Cd (a and b), Cu (c and d), Pb (e and f), and Zn (g and h) in the roots and shoots of Indian mustard and sunflower on two alkaline soils (PP02 and PP03) and two acidic soils (Ko01 and Ko02). Values are means of three replicates with standard deviations shown by vertical bars.
Copper. As with Cd, plant uptake of Cu was higher in the acidic soils Ko01 and Ko02 even though the total Cu was several times greater in the alkaline soils PP02 and PP03 (Fig. 4c, d). For instance, total Cu was the highest in PP02 and PP03, but Indian mustard and sunflower accumulated the highest amounts of Cu in shoots from Ko01, which resulted in the highest translocation coefficients of 0.09 for Indian mustard and 0.07 for sunflower. In addition, even though there was a marked difference in total and soluble Cu between PP03 and Ko02, the amounts of Cu accumulated in both Indian mustard and sunflower shoots were similar, which could not be attributed to either pH or DOC changes in soil solution. This could be explained by the effective concentration (CE ) concept, defined as the metal
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Fig. 5
Relationships between Cd concentrations in soil solution after plant growth and Cd in the plant shoots.
Fig. 6 Relationships between ΔDOC and translocation coefficient (Kt ) of Cd. ΔDOC = dissolved organic carbon (DOC) concentrations in soil solution after plant growth – DOC concentrations in soil solution without plant.
concentration that would have to be present in the soil solution to supply an equivalent mass of metal to that accumulated by diffusive gradients in thin films (DGT) (Zhang et al., 2001). The CE value includes the soil solution concentration as well as the concentration resupplied from the solid phase. Zhang et al. (2001) found that Cu contents in plant shoots were more closely related to CE than any other Cu measures (free Cu activity, EDTA extraction, or soil solution concentration). Based on this concept, it is likely that Cu uptake relies on the ability of the soil to resupply Cu to the soil solution rapidly enough for plant absorption. The higher plant Cu uptake observed in the acidic soils was probably due to faster resupply of Cu in these soils as it became depleted. Lead. As with Cd, Pb in plant shoots, which ranged from 0.3 to 34 mg kg−1 for Indian mustard and 0.3 to 40 mg kg−1 for sunflower (Fig. 4e), was positively related to the soluble fraction of Pb in the soils (r2 = 0.76, P < 0.01). The content of Pb in plant roots was also linearly related to Pb concentration in rhizosphere soil solution (r2 = 0.85, P < 0.001). This implied that pH and DOC increases after plant growth could affect Pb uptake by changing dissolved Pb concentrations. Linear regression analysis showed that the Pb content in plant roots significantly increased as DOC concentration increased after plant growth (r2 = 0.73, P < 0.001), and this was assumed to be the reason that less Pb was translocated to the shoots. Indian mustard accumulated the highest amounts of Pb from PP02 and Ko01. It seemed that the replenishing rate for Pb in the soils was high with more Pb being released into soil solution as the Pb became depleted by plant uptake. Zinc. The shoot Zn contents ranged from 34 to 448 mg kg−1 in Indian mustard and from 47 −1 in sunflower. The highest shoot Zn content was observed in plants grown in PP02 to 297 mg kg (Fig. 4g). The root Zn contents ranged from 71 to 1 393 mg kg−1 in Indian mustard and from 88 to 1 368 mg kg−1 in sunflower (Fig. 4h). Like Cd and Pb, Zn content in plant shoots was linearly related with soluble Zn concentration in the rhizosphere after plant growth (r2 = 0.82, P < 0.001). For instance, in the control in which plants were not grown, soluble Zn was higher in Ko01 than in PP02 and PP03. However, after plant growth plant uptake of Zn was significantly less from Ko01 than from both PP02 and PP03. This may be due to the reduction in the soluble Zn caused by an increase in pH of the soil Ko01 after plant growth and insignificant resupply from the soil to its solution because of relatively low total Zn in this soil. Increased DOC complexation with Zn caused a lower translocation coefficient (Kt ) of Zn as evidenced by regression analysis. The translocation coefficient (Kt ) of Zn decreased exponentially as DOC increased (r2 = 0.48, P < 0.05). CONCLUSIONS Metal concentrations in the rhizosphere soil solution were considerably influenced by pH and DOC
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concentrations which, in turn, were influenced by plant growth and rhizosphere effects. Increased pH and DOC interacted antagonistically with regard to increased metal concentrations in solution. In the acidic soils (pH < 6.5), the effect of pH increases was stronger than that of DOC increases, resulting in an overall decrease in dissolved metal concentrations in these soils. In contrast, the increased DOC after plant growth increased dissolved metal concentrations in the alkaline soils. Chemical changes in the rhizosphere also played an important role in controlling the speciation of metals in soil solution. Plant growth resulted in reduction of free Cd through formation of Cd-DOC complexes and also through pH increases. In contrast to Cd, free Zn concentrations were probably affected by the co-existence of other heavy metals forming more stable complexes with dissolved organic compounds from root exudates and soil binding sites. In the acidic soils, the proportion of free Zn increased while soluble Zn decreased considerably, possibly due to the higher affinity of DOC for metals other than Zn, consequently leaving most of the Zn in soil solution as free ions. Changes in dissolved metal concentrations and species greatly influenced metal uptake by plants. Plant uptake was primarily related to the concentrations of metals in the soil solution rather than total metal concentrations of the soil. REFERENCES Awad, F., Romheld, V. and Marschner, H. 1994. Effect of root exudates on mobilization in the rhizosphere and uptake of iron by wheat plants. Plant Soil. 165: 213–218. Dushenkov, S. and Kapulnik, Y. 2000. Phytofiltration of metals. In Ilya, R. and Ensley, B. D. (eds.) Phytoremediation of Toxic Metals. John Wiley & Sons, Inc., New York. pp. 89–106. Ernst, W. H. O. 1996. Bioavailability of heavy metals and decontamination of soils by plants. Appl. Geochem. 11(1–2): 163–167. Fotovat, A. and Naidu, R. 1998. Changes in composition of soil aqueous phase influence chemistry of indigenous heavy metals in alkaline sodic and acidic soils. Geoderma. 84: 213–234. Hamon, R. E., Lorenz, S. E., Holm, P. E., Christensen, T. H. and McGrath, S. P. 1995. Changes in trace metal species and other components of the rhizosphere during growth of radish. Plant Cell Environ. 18: 749–756. Hinsinger, P. 2001. Bioavailability of trace elements as related to root-induced chemical changes in the rhizosphere. In Gobran, G. R., Wenzel, W. W. and Lombi, E. (eds.) Trace Elements in the Rhizosphere. CRC Press, Boca Raton. pp. 25–40. Kim, K. R., Owens, G. and Naidu, R. 2009. Heavy metal distribution, bioaccessibility and phytoavailability in long-term contaminated soils from Lake Macquarie, Australia. Aust. J. Soil Res. 47(2): 166–176. Knight, B., Zhao, F. J., McGrath, S. P. and Shen, Z. G. 1997. Zinc and cadmium uptake by the hyperaccumulator Thlaspi caerulescens in contaminated soils and its effects on the concentration and chemical speciation of metals in soil solution. Plant Soil. 197: 71–78. Lin, Q., Chen, Y. X., He, Y. F. and Tian, G. M. 2004. Root-induced changes of lead availability in the rhizosphere of Oryza sativa L. Agr. Ecosyst. Environ. 104(3): 605–613. Luo, Y. M., Christie, P. and Baker, A. J. M. 2000. Soil solution Zn and pH dynamics in non-rhizosphere soil and in the rhizosphere of Thlaspi caerulescens grown in a Zn/Cd-contaminated soil. Chemosphere. 41(1–2): 161–164. McGrath, S. P., Shen, Z. G. and Zhao, F. J. 1997. Heavy metal uptake and chemical changes in the rhizosphere of Thlaspi caerulescens and Thlaspi ochroleucum grown in contaminated soils. Plant Soil. 188: 153–159. Miller, W. P. and Miller, D. M. 1987. A micro pipette method for soil mechanical analysis. Commun. Soil Sci. Plant Anal. 18: 1–15. Naidu, R., Bolan, N. S., Kookana, R. S. and Tiller, K. G. 1994. Ionic-strength and pH effects on the sorption of cadmium and the surface charge of soils. Eur. J. Soil Sci. 45: 419–429. Naidu, R., Rogers, R., Gupta, V. V. S. R., Kookana, R. S., Bolan, N. S. and Adriano, D. C. 2003. Bioavailability of metals in the soil plant environment and its potential role in risk assessment. In Naidu, R., Rogers, R., Gupta, V. V. S. R., Kookana, R. S., Bolan, N. S. and Adriano, D.C. (eds.) Bioavailability, Toxicity and Risk Relationships in Ecosystems. Science Publishers, Inc., New Hampshire. pp. 21–58. National Environment Protection Council (NEPM) (ed.). 1999. Schedule B(1) Guideline on the Investigation Levels for Soil and Groundwater, Australia. Available online at http://www.ephc.gov.au/sites/default/files/ASC NEPMsch 01 Investigation Levels 199912.pdf (verified on March 12, 2010). Nelson, D. W. and Sommers, L. E. 1996. Total carbon, organic carbon, and organic matter. In Page, A. S. (ed.) Methods of Soil Analysis. Part 3. Chemical Methods. Soil Sci. Soc. Am. (SSSA) Book Series No. 5. SSSA, Madison. pp. 961–1110. Nolan, A. L., Zhang, H. and McLaughlin, M. J. 2005. Prediction of zinc, cadmium, lead, and copper availability to wheat in contaminated soils using chemical speciation, diffusive gradients in thin films, extraction, and isotopic dilution techniques. J. Environ. Qual. 34: 496–507.
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Pareuil, P., P´ enilla, S., Ozkan, N., Bordas, F. and Bollinger, J. C. 2008. Influence of reducing conditions of metallic elements released from various contaminated soil samples. Environ. Sci. Technol. 42: 7615–7621. Quartacci, M. F., Argilla, A., Baker, A. J. M. and Navari-Izzo, F. 2006. Phytoextraction of metals from a multiply contaminated soil by Indian mustard. Chemosphere. 63(6): 918–925. Reeves, R. D. and Baker, A. J. M. 2000. Metal-accumulating plants. In Raskin, I. and Ensley, B. D. (eds.) Phytoremediation of Toxic Metals. Wiley-Interscience, New York. pp. 193–230. Sas, L., Rengel, Z. and Tang, C. 2001. Excess cation uptake, and extrusion of protons and organic acid anions by Lupinus albus under phosphorus deficiency. Plant Sci. 160: 1191–1198. Sauv´ e, S., Cook, N., Hendershot, W. H. and McBride, M. B. 1996. Linking plant tissue concentrations and soil copper pools in urban contaminated soils. Environ. Pollut. 94: 153–157. Schmidt, U. 2003. Enhancing phytoextraction: the effect of chemical soil manipulation on mobility plant accumulation, and leaching of heavy metals. J. Environ. Qual. 32: 1939–1954. Temminghoff, E. J. M., Plette, A. C. C., Eck, R. V. and Riemsdijk, W. H. V. 2000. Determination of the chemical speciation of trace metals in aqueous systems by the Wageningen Donnan Membrane Technique. Anal. Chim. Acta. 417: 149–157. Thibault, D. H. and Sheppard, M. I. 1992. A disposable system for soil pore-water extraction by centrifugation. Commun. Soil Sci. Plant Anal. 23(13&14): 1629–1641. U.S. Environmental Protection Agency (USEPA). 1997. Method 3051a: Microwave Assisted Acid Dissolution of Sediments, Sludges, Soils, and Oils. 2nd ed. USEPA, U.S. Government Printing Office, Washington, DC. Wang, W. S., Shan, X. Q., Wen, B. and Zhang, S. Z. 2003. Relationship between the extractable metals from soils and metals taken up by maize roots and shoots. Chemosphere. 53(5): 523–530. Wang, Z., Shan, X. Q. and Zhang, S. 2002. Comparison between fractionation and bioavailability of trace elements in rhizosphere and bulk soils. Chemosphere. 46(8): 1163–1171. Whiting, S. N., Leake, J. R., McGrath, S. P. and Baker, A. J. M. 2001. Assessment of Zn mobilization in the rhizosphere of Thlaspi caerulescens by bioassay with non-accumulator plants and soil extraction. Plant Soil. 237: 147–156. Zhang, H., Zhao, F., Sun, B., Davison, W. and McGrath, S. P. 2001. A new method to measure effective soil solution concentration predicts copper availability to plants. Environ. Sci. Technol. 35: 2602–2607.
Effect of Root-Induced Chemical Changes on Dynamics and Plant Uptake of Heavy Metals in Rhizosphere Soils∗1 K. R. KIM1,∗2 , G. OWENS2 and R. NAIDU2,3 1 Climate Change & Agroecology Division, Department of Agricultural Environment, National Academy of Agricultural Science, Suwon 441 857 (Republic of Korea) 2 Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095 (Australia) 3 Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Mawson Lakes, SA 5095 (Australia)
(Received September 24, 2009; revised March 15, 2010)
ABSTRACT It is increasingly recognized that metal bioavailability is a better indicator of the potential for phytoremediation than the total metal concentration in soils; therefore, an understanding of the influence of phytoremediation plants on metal dynamics at the soil-root interface is increasingly vital for the successful implementation of this remediation technique. In this study, we investigated the heavy metal and soil solution chemical changes at field moisture, after growth of either Indian mustard (Brassica juncea) or sunflower (Helianthus annuus L.), in long-term contaminated soils and the subsequent metal uptake by the selected plants. In addition, the fractions of free metal ions in soil solution were determined using the Donnan membrane technique. After plant growth soil solution pH increased by 0.2–1.4 units and dissolved organic carbon (DOC) increased by 1–99 mg L−1 in all soils examined. Soluble Cd and Zn decreased after Indian mustard growth in all soils examined, and this was attributed to increases in soil solution pH (by 0.9 units) after plant growth. Concentrations of soluble Cu and Pb decreased in acidic soils but increased in alkaline soils. This discrepancy was likely due to a competitive effect between plant-induced pH and DOC changes on the magnitude of metal solubility. The fractions of free Cd and Zn ranged from 7.2% to 32% and 6.4% to 73%, respectively, and they generally decreased as pH and DOC increased after plant growth. Metal uptake by plants was dependant on the soil solution metal concentration, which was governed by changes in pH and DOC induced by plant exudates, rather than on the total metal concentrations. Although plant uptake also varied with metal and soil types, overall soluble metal concentrations in the rhizosphere were mainly influenced by root-induced changes in pH and DOC which subsequently affected the metal uptake by plants. Key Words:
bioavailability, phytoremediation, speciation
Citation: Kim, K. R., Owens, G. and Naidu, R. 2010. Effect of root-induced chemical changes on dynamics and plant uptake of heavy metals in rhizosphere soils. Pedosphere. 20(4): 494–504.
Contamination of soils by metal(loid)s through anthropogenic activities is a widespread and serious problem confronting society, scientists, and regulators worldwide. For this reason, a lot of research has been conducted over the last 50 years to assess and remediate potentially hazardous sites. Phytoremediation is a developing technology that uses plants to ameliorate contamination of soils and is often perceived as a greener alternative to the traditional remediation technologies (Reeves et al., 2000). The basic principles of phytoremediation rely on an understanding of the factors that either control plant accumulation or contribute to immobilization of metal contaminants. The concentrations of bioavailable and soluble metals are considered more important than total metal concentrations because these parameters govern the form and accessibility of metals to plants and determine the fraction to be transferred to aboveground tissues (Ernst, 1996; Naidu et al., 2003). In the context of metal bioavailability, metal speciation in soil solution is important given that metal uptake by plants is often dictated by the nature ∗1 Supported
by the University of South Australia, Australia. author. E-mail: [email protected].
∗2 Corresponding
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of the metal species. Metal uptake by plants can also be dictated by kinetic parameters. Zhang et al. (2001) showed that for copper the kinetically labile pool played an important role in plant uptake. Plants have the potential to transform metal species for easier uptake, or to detoxify metals through root exudation or pH changes in the rhizosphere (Awad et al., 1994; Luo et al., 2000; Hinsinger, 2001; Whiting et al., 2001; Wang et al., 2002; Lin et al., 2004). Following the observation that the measurement of free metal ion activities in soil solution before plant growth did not provide the best prediction of metal supply to the plant (Nolan et al., 2005), it remains a controversial issue whether plants predominantly absorb free metal ions from soil solution (Hamon et al., 1995; Sauv´e et al., 1996). For this reason, determining metal speciation in the rhizosphere and understanding subsequent metal uptake by plants are currently subjects of much research. The present study investigated the effect of growing two plants commonly recommended for phytoremediation, Indian mustard (Brassica juncea) and sunflower (Helianthus annuus L.) (Dushenkov and Kapulnik, 2000; Reeves and Baker, 2000; Schmidt, 2003), on heavy metal dynamics and soil solution chemistry in two contrasting heavy metal contaminated soils and on the subsequent metal uptake. These two plant species may potentially differ in the mechanism for metal uptake since Indian mustard is best known as metal hyperaccumulator while sunflower is better known as a metal stabilizer. MATERIALS AND METHODS Soils and plants Soils were either collected from the vicinity of a Pb and Zn smelter operating in Port Pirie, South Australia (soils PP02 and PP03), or from a military shooting range in South Korea (soils Ko01 and Ko02). The soils collected from Australia were alkaline (pH > 8) and highly contaminated with multiple metals, while the Korean soils were slightly acidic (pH < 6.5) and either moderately contaminated (Ko01) or non-contaminated soils (Ko02). The physicochemical properties of the soils are summarised in Table I. TABLE I Selected physicochemical properties of the soils studied Soil No.
pH
Organic matter kg−1
PP02 PP03 Ko01 Ko02
9.2 8.2 6.2 6.4
g 3 17 6 11
Dissolved organic carbon mg 16 613 15 58
L−1
Clay
Cd
Cu
kg−1
g 120 130 180 70
Pb mg
17.8 3.6 7.9 0.1
446 630 87 3
Zn
kg−1 2 427 625 380 24
6 472 9 759 55 18
Indian mustard, a known hyperaccumulator for Zn, Cd, and Pb, was selected as representative of plants exhibiting the hyperaccumulation mechanism (Reeves and Baker, 2000; Schmidt, 2003), while sunflower was selected as an example of a plant exhibiting the heavy metal stabilization mechanism (Dushenkov and Kapulnik, 2000). Both of these plant species have the potential to be used for remediation of heavy metals in soils in the root zone by phytoextraction or phytostabilization, and may have significantly different mechanisms for interacting with metals. Plant growth experiment A plant growth experiment was conducted under greenhouse conditions. Air dried soil (500 g, < 2 mm) was weighed into 600 mL plastic pots and saturated with a basal fertilizer nutrient solution consisting of 150 mg kg−1 of N (as NH4 NO3 ), 75 mg kg−1 of P (as KH2 PO4 ), and 95 mg kg−1 of K (as KH2 PO4 ) (Quartacci et al., 2006). The nutrient solution was supplied to the plants via a saucer beneath the pots and the uptake was through a hole in the base of the pots. This method prevented the leaching
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of heavy metals. Soils were allowed to equilibrate for one week, and then additional water was added in the same manner described above, to compensate for any moisture loss due to evaporation. The pots were equilibrated for an additional 48 h prior to sowing seeds. During watering, it is unlikely that reducing conditions occurred because at no time were the pots fully saturated for any extended period. Pareuil et al. (2008) has previously shown that reducing conditions could not be induced within 21 days using water alone. Indian mustard and sunflower seeds were sown directly into the soils and the pots were covered with transparent plastic bags to maintain the moisture content. Twenty seeds for Indian mustard and twelve for sunflower were sown. Three days after germination the plastic bags were removed and one week after this the number of seedlings was thinned to 15 for Indian mustard and 7 for sunflower. Watering was conducted in the same way as the initial water saturation method every 3 days for 2 weeks and subsequently, as the plants developed, watering continued every two days until harvest. Plants were harvested 6 weeks after germination and plant tissue and soil solution samples were prepared for analysis using the methods described below. The experiment was conducted in triplicate for each soil and included control soils into which no plants were sown, but which were otherwise treated exactly as all other soils studied. It was assumed that the soils in the pots where plants were grown were examples of rhizosphere soils while the control soil, without plants, was considered to be bulk soil. Even though a rhizosphere soil would strictly be that soil intimately associated with the root zone of the plant, the term rhizosphere soil was used here to easily distinguish between a soil cultivated with plants and the bulk soil where plants were not cultivated. Analysis Plant. The aboveground plant tissues (shoots) were cut and rinsed twice with Milli-Q water while the belowground tissues (roots) were rinsed with tap water after removing the soil particles clinging to the roots with vigorous shaking and washed using a 5% (v/v) aqueous detergent solution (Decon 90). Subsequently the roots were thoroughly rinsed three times with Milli-Q water. Additional washing using an ultrasonic bath was not conducted, as this had been shown to have little improvement on washing efficiency (Wang et al., 2003). Washed shoots and roots were dried at 65 ◦ C in a fanforced oven and periodically weighed until there was no change in dry mass. The dry plant tissues were finely ground using a commercial electronic grinder and stored in ziplock plastic bags prior to acidic digestion. The dried and ground plant tissues (0.5 g) were digested with concentrated HNO3 (5 mL) using a commercially available temperature-controlled digestion block (AIM 500, A. I. Scientific, Australia). Blanks, duplicates, and spiked samples were included at a rate > 5% to ensure quality control during plant digestion. Total heavy metal concentrations in digest solutions of plant tissues were determined using inductively coupled plasma mass spectrometry (ICP-MS). Routine soil laboratory analyses. Soil pH and electrical conductivity (EC) were determined in a 1:5 soil:water (w/v) suspension using a combination pH/EC meter (smartCHEM-LAB, TPS, Australia) after 1 h equilibration. Soil organic matter content was determined using the Walkley-Black method (Nelson and Sommers, 1996) and total dissolved organic carbon (DOC) was analysed using an automated total organic carbon analyser (Model 1010, O.I. Analytical, USA) after extraction of soil solution. Soil texture was determined using a micro-pipette method (Miller and Miller, 1987). Heavy metal contents in the soils were determined by ICP-MS after microwave (MARS5, CEM, USA)-assisted aqua regia digestion in accordance with USEPA (1997) method 3051H. Each microwave digestion batch included a standard reference material (Montana Soil SRM2711, certificated by the National Institute of Standards & Technology, USA) and a blank to validate the digestion operation. Soil solution extraction and heavy metal speciation. After plant harvest, sub-samples of soils from each pot were air dried and subsequently rewetted to allow soil solution extraction at 70% maximum water holding capacity (MWHC). Soil (20 g, < 2 mm) was weighed into 20 mL disposable plastic syringes having the outlet plugged with acid-washed glass wool. The syringe was inserted into a 50 mL
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centrifuge tube separated from the base by a polyvinyl chloride (PVC) spacer (Thibault and Sheppard, 1992). Water was added to the soil to obtain 70% MWHC and the soil was allowed to equilibrate for 24 h. The resulting soil paste was centrifuged at 2 500 r min−1 for 25 min and the isolated soil solutions were filtered through 0.45 μm cellulose acetate disposable filters (MillexTM , Millipore, USA) and stored for further analysis. In order to secure enough soil solution (100 mL) for metal speciation, 25 replicates of syringe extractions for each pot were required. Solutions from individual replicates were isolated and combined to obtain at least 100 mL of solution after filtration. Metal speciation was commenced within 2 h after collecting the soil solutions using the Donnan membrane technique (DMT). The DMT system used here was similar to that used by Temminghoff et al. (2000), including a negatively charged membrane (No. 55165 2U, BDH, UK). However, while Temminghoff et al. (2000) had used the conditions of 500 mL donor and 17 mL acceptor, in this study 100 mL of donor and 8 mL of acceptor solution were used. These adjusted conditions allowed for the limitation of collecting large volumes of soil solution by extraction at 70% MWHC. Of the four heavy metals studied (Cd, Cu, Pb, and Zn), only Cd and Zn were amenable to speciation by DMT because Cu and Pb existed predominantly as metal-organic complexes. Statistical analysis. All data were statistically analysed using SPSS 12.0.1 (SPSS Inc.) to examine interrelations between changes in metal concentrations in soil solution of the rhizosphere soil and key soil solution properties. Simple calculations were performed using Microsoft ExcelTM and the graphs were drawn using both SPSS 12.0.1 and Sigmaplot 10 (Systat Software Inc.). For regression analysis, individual data points were used instead of averages of triplicates. RESULTS AND DISCUSSION Effect of plant roots on rhizosphere soil pH and DOC Considerable controversy exists in the literature regarding the effect of plant growth on the pH at the soil-root interface. For instance, hyperaccumulator plants have been shown to either acidify rhizosphere soils and subsequently increase the dissolved concentrations of heavy metals (McGrath et al., 1997; Sas et al., 2001) or increase soil pH after plant growth (Knight et al., 1997; Luo et al., 2000). In the present study, both Indian mustard and sunflower increased the soil solution pH in all soils studied relative to the controls (Table II). TABLE II Soil solution pH and dissolved organic carbon (DOC) concentrations after plant growth Soil No.
pH Mustard
DOC Sunflower
Control
Mustard
Sunflower
Control
L−1
PP02 PP03 Ko01 Ko02
8.1±0.2a) 8.2±0.1 6.4±0.1 6.6±0.1
a) Mean±standard
8.1±0.0 8.1±0.1 5.8±0.2 5.8±0.2
7.9±0.1 7.7±0.1 5.5±0.2 5.2±0.1
45±5 290±10 32±1 52±1
mg 120±20 283±9 40±2 55±6
23±4 240±10 23±3 51±1
deviation.
Indian mustard increased pH in the rhizosphere soils more than sunflower, and a higher pH increase was observed in the acidic soils (Ko01 and Ko02) compared to the alkaline soils (PP02 and PP03). For instance, Indian mustard growth increased pH by 0.9 and 1.4 units in the soils Ko01 and Ko02, respectively, relative to the pH of the corresponding controls, 5.5 and 5.2, respectively. Sunflower growth increased pH by 0.3 and 0.6 units in the same soils (Table II). The concentrations of DOC in the rhizosphere soils increased irrespective of plant species due to organic exudates from plant roots (Table II). However, the magnitude of increase in DOC concentration differed with plant species and soil type.
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For example, a larger increase in DOC was observed in the highly contaminated alkaline soils, PP02 and PP03, relative to the acidic soils. This higher DOC probably contributed to both higher pH and heavy metal concentrations. These changes in soil pH and DOC concentrations were expected to significantly modify the chemical behaviours of all four heavy metals (Cd, Cu, Pb, and Zn) in the plant rhizosphere. Heavy metal dynamics in the rhizosphere soils Cadmium. Irrespective of plant species and soil type, plant growth resulted in a significant decrease in the soil solution concentration of Cd (Fig. 1a). Average soluble Cd decreased by more than 35% relative to the control soil (without plants) after both Indian mustard and sunflower growth in the alkaline soils, PP02 and PP03, which were highly contaminated with a number of metals. In the acidic soils, Ko01 and Ko02, which contained considerably high concentrations of soluble Cd fractions relative to total Cd, the effect of plant growth on soil solution Cd was even more marked. As shown in Fig. 1a, after Indian mustard growth the soil solution Cd decreased by 67% in Ko01 and 84% in Ko02. This decrease in soluble Cd in both soil types was attributed to an increase in soil solution pH due to plant growth as demonstrated by the observation of lower concentrations of Cd in solution as rhizosphere pH increased after plant growth (Fig. 2). Plant uptake of Cd from a soluble pool could also be potentially responsible for the observed decrease in soluble Cd if resupply from the soil was a limiting factor. However, linear regression analysis did not show any relationship between Cd uptake by plant and the extent of soluble Cd reduction. In contrast, the magnitude of the metal concentration reduction in soil solution was more strongly correlated with pH increase rather than the metal uptake by plants, which is strong evidence for the dominance of pH over other confounding factors. In particular, the influence of an increase in pH on Cd concentration in soil solution was greater in acidic soils than alkaline soils (Fig. 1). Naidu et al. (1994) also demonstrated that small increases in solution pH below pH 6 resulted in large increases in Cd sorption onto soils, while there was lower increase in Cd sorption with increase in solution pH above pH 8. This implied that soil solution Cd was more sensitive to changes in pH after plant growth in acidic soils. The fraction of free Cd in soils ranged from 7.2% to 32% of the total Cd in the control soils with the highest fraction being observed in the acidic soil Ko01 (Fig. 1b). After plant
Fig. 1 Cd (a), Cu (c), Pb (d), and Zn (e) concentrations and free Cd (b) and Zn (f) fractions in the solutions of two alkaline soils (PP02 and PP03) and two acidic soils (Ko01 and Ko02) after plant growth. Values are means of three replicates with standard deviations shown by vertical bars.
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Fig. 2 Relationships between ΔpH and Cd concentration in solutions of the alkaline and acidic soils after plant growth. ΔpH = pH after plant growth − pH without plant.
growth both the fractions of free Cd and the total soil solution Cd decreased with increases in soil pH and DOC (Fig. 1) induced by root exudates. In particular, there was a strongly significant relationship (r2 = 0.85, P < 0.05) between DOC increase and the free Cd fraction decrease. Significantly less free Cd ions were found with the increases in DOC after plant growth. This suggested that the DOC exuded from the plant roots formed stable metal-DOC complexes, resulting in the observed significant reduction in free Cd in soil solution. Copper. The soil solution Cu concentrations increased by 7 folds after Indian mustard and 10 folds after sunflower growth in the highly contaminated soil, PP02, relative to the control (Fig. 1c). The increased concentration of Cu in soil solution was attributed to root exudation and the consequent formation of soluble Cu complexes with DOC induced by plant roots. Also soil solution Cu tended to decrease in the moderately contaminated acidic soil, Ko01, while there was no effect of plant growth on soil solution Cu in the non-contaminated acidic soil, Ko02 (Fig. 1c). Linear regression analysis showed a significant positive relationship between DOC and Cu concentration in soil solutions from both the highly contaminated alkaline soil (r2 = 0.51, P < 0.05) and the moderately contaminated acidic soil (r2 = 0.5, P < 0.05) after plant growth. Thus soluble Cu concentration increased with increasing DOC after plant growth in alkaline soils. No free Cu was detected in soil solution, demonstrating the conversion of free Cu to Cu organic complexes due to elevated plant root exudates. Lead. As with Cu, soil solution Pb increased in the highly contaminated soil, PP02, and decreased in the less contaminated soil, Ko01, after plant growth (Fig. 1d). Soil solution Pb in the rhizosphere of Ko01 decreased from 319 μg L−1 in the control to 29 μg L−1 after Indian mustard growth and to 162 μg L−1 after sunflower growth. This decrease was likely due to the increase in soil solution pH in Ko01 after plant growth. Correlation analysis using data from all soils showed a significant inverse exponential relationship between soil solution pH and soluble Pb (r2 = 0.75, P < 0.001). This demonstrated a strong influence of plant-induced pH changes on the chemistry of Pb in the rhizosphere. Further analysis using the data from the alkaline soils, PP02 and PP03, showed significant influence of DOC on Pb concentrations in solution. These demonstrated that for the acid soils, Ko01 and Ko02, solution Pb was influenced by pH, whereas both DOC and pH played a significant role in the high pH soils, PP02 and PP03. As with Cu, free Pb was not detected, which was likely due to formation of Pb organic complexes in soil solution. Zinc. In all the soils studied, soluble Zn decreased after plant growth, with the highest decrease observed after Indian mustard growth in the acidic soil, Ko01 (Fig. 1e). This decrease in soil solution Zn may be attributed to the competing effect of changes in pH and DOC after plant growth on metal chemistry. The increase in soil solution pH resulted in a marked exponential decrease in soil solution Zn (r2 = 0.75, P < 0.001) and the magnitude of the decrease in dissolved Zn increased as the soil solution pH increased (r2 = 0.29). In contrast, increases in DOC concentration up to 40 mg L−1 increased soil
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solution Zn, but Zn concentrations did not increase further, remaining stable at about 400 μg L−1 as DOC increased above 40 mg L−1 (Fig. 3). This implied that the DOC increases, due to root exudation, retarded the magnitude of the decrease in Zn solubility induced by pH increase after plant growth. The fraction of free Zn ranged from 6.4% to 73% of the total Zn concentration in soil solution (Fig. 1f). The lowest free Zn fraction was detected in PP03 where the soil pH and OM contents were higher than those of the other soils. The free Zn fraction after plant growth increased in Ko01 and Ko02 even though soil solution pH and DOC increased, whereas it decreased in PP02 and PP03. This is surprising given that pH increases generally enhance metal-ligand complexation and lead to subsequent decreases in free metal ions in soil solution. However, Indian mustard growth increased free Zn from 70% to 88% in Ko01 and 38% to 64% in Ko02. A similar increase in free Zn concentration was recorded in Ko02 after sunflower growth. This is attributed to the presence of other competing metal ions such as Pb and Cu which have greater selectivity for metal organic complexation in the soil solution. In our previous study we demonstrated that soluble Zn could be increased as soluble Pb and Cu concentrations increased (Kim et al., 2009). Fotovat and Naidu (1998) showed that for some Australian soils while Cu exists as Cu-DOC complex in both acidic and alkaline soils, Zn is present as an ionic form in acidic soils but largely as Zn-DOC complex in alkaline soils. Likewise, in the present study, Zn-DOC complexes were predominant in one of the alkaline soils, PP03, but free Zn was predominant in soil PP02 as shown in Fig. 1. The higher free Zn in PP02 despite higher pH was probably due to much higher concentration of Pb in this soil (Table I) compared to PP03 and resulted from competition by Pb with Zn for available binding sites. In the presence of high concentration of soluble Pb, it is likely that uncomplexed dissolved organic compounds are limiting for Zn organic complexation, explaining the presence of free Zn in the high pH soil, PP02.
Fig. 3 Relationships between ΔDOC and Zn in soil solution after plant growth. ΔDOC = DOC concentrations in soil solution after plant growth – DOC concentrations in soil solution without plant.
Heavy metal uptake by plant As discussed above, plant growth increased DOC and pH in all soils examined and these two factors influenced plant metal uptake through changes in the chemical conditions in the soil solution and consequently heavy metal speciation. Given that the magnitude of changes in DOC and pH varied with soil type, the extent of plant metal uptake also varied with soil type, plant species, and metal type. Overall, increases in pH after plant growth were expected to result in a negative effect on plant metal uptake due to reduced metal concentrations in solution. However, increases in DOC from root exudation may potentially counteract this mechanism and enhance metal uptake by increasing total dissolved metal concentrations. Cadmium. The Cd content ranged from 1.2 to 51 mg kg−1 in shoots and from 2.1 to 73 mg kg−1 in roots of Indian mustard. The ranges were from 1.5 to 70 mg kg−1 in shoots and from 2.2 to 114 mg kg−1 in roots of sunflower (Fig. 4a, b). Cadmium uptake by plants was more closely related to the soil
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solution Cd concentration (Fig. 5) rather than the total metal concentration in the soils. For instance, Cd uptake by plants grown in Ko01 which had the highest soil solution Cd were the highest despite the total Cd concentration being 2.5 times higher in PP02 than in Ko01. In addition, Indian mustard and sunflower accumulated similar amounts of Cd in their shoots from PP03 (1.7 and 1.2 mg kg−1 ) and Ko02 (2.0 and 1.5 mg kg−1 ) even though the total Cd was much higher in PP03. This indicated that plant Cd uptake and translocation was dependent on soil solution Cd and independent of total soil Cd concentration, implying the dependence of metal uptake by plants on the change in dissolved Cd concentration induced by pH increase after plant growth. There existed a negative relationship between increased pH and Cd uptake by plants in the alkaline soils (r2 = 0.52, P < 0.05). The translocation coefficients (Kt ) of Cd, defined as Kt =
MS MR
where MS and MR are the dry weight concentrations (mg kg−1 ) in shoots and roots, respectively, were lower than 1 (Fig. 6), which indicated higher Cd adsorption into the roots, probably due to formation of Cd-DOC complexes resulting from DOC increase in the rhizosphere, and less translocation of Cd to the shoots.
Fig. 4 Concentrations of Cd (a and b), Cu (c and d), Pb (e and f), and Zn (g and h) in the roots and shoots of Indian mustard and sunflower on two alkaline soils (PP02 and PP03) and two acidic soils (Ko01 and Ko02). Values are means of three replicates with standard deviations shown by vertical bars.
Copper. As with Cd, plant uptake of Cu was higher in the acidic soils Ko01 and Ko02 even though the total Cu was several times greater in the alkaline soils PP02 and PP03 (Fig. 4c, d). For instance, total Cu was the highest in PP02 and PP03, but Indian mustard and sunflower accumulated the highest amounts of Cu in shoots from Ko01, which resulted in the highest translocation coefficients of 0.09 for Indian mustard and 0.07 for sunflower. In addition, even though there was a marked difference in total and soluble Cu between PP03 and Ko02, the amounts of Cu accumulated in both Indian mustard and sunflower shoots were similar, which could not be attributed to either pH or DOC changes in soil solution. This could be explained by the effective concentration (CE ) concept, defined as the metal
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Fig. 5
Relationships between Cd concentrations in soil solution after plant growth and Cd in the plant shoots.
Fig. 6 Relationships between ΔDOC and translocation coefficient (Kt ) of Cd. ΔDOC = dissolved organic carbon (DOC) concentrations in soil solution after plant growth – DOC concentrations in soil solution without plant.
concentration that would have to be present in the soil solution to supply an equivalent mass of metal to that accumulated by diffusive gradients in thin films (DGT) (Zhang et al., 2001). The CE value includes the soil solution concentration as well as the concentration resupplied from the solid phase. Zhang et al. (2001) found that Cu contents in plant shoots were more closely related to CE than any other Cu measures (free Cu activity, EDTA extraction, or soil solution concentration). Based on this concept, it is likely that Cu uptake relies on the ability of the soil to resupply Cu to the soil solution rapidly enough for plant absorption. The higher plant Cu uptake observed in the acidic soils was probably due to faster resupply of Cu in these soils as it became depleted. Lead. As with Cd, Pb in plant shoots, which ranged from 0.3 to 34 mg kg−1 for Indian mustard and 0.3 to 40 mg kg−1 for sunflower (Fig. 4e), was positively related to the soluble fraction of Pb in the soils (r2 = 0.76, P < 0.01). The content of Pb in plant roots was also linearly related to Pb concentration in rhizosphere soil solution (r2 = 0.85, P < 0.001). This implied that pH and DOC increases after plant growth could affect Pb uptake by changing dissolved Pb concentrations. Linear regression analysis showed that the Pb content in plant roots significantly increased as DOC concentration increased after plant growth (r2 = 0.73, P < 0.001), and this was assumed to be the reason that less Pb was translocated to the shoots. Indian mustard accumulated the highest amounts of Pb from PP02 and Ko01. It seemed that the replenishing rate for Pb in the soils was high with more Pb being released into soil solution as the Pb became depleted by plant uptake. Zinc. The shoot Zn contents ranged from 34 to 448 mg kg−1 in Indian mustard and from 47 −1 in sunflower. The highest shoot Zn content was observed in plants grown in PP02 to 297 mg kg (Fig. 4g). The root Zn contents ranged from 71 to 1 393 mg kg−1 in Indian mustard and from 88 to 1 368 mg kg−1 in sunflower (Fig. 4h). Like Cd and Pb, Zn content in plant shoots was linearly related with soluble Zn concentration in the rhizosphere after plant growth (r2 = 0.82, P < 0.001). For instance, in the control in which plants were not grown, soluble Zn was higher in Ko01 than in PP02 and PP03. However, after plant growth plant uptake of Zn was significantly less from Ko01 than from both PP02 and PP03. This may be due to the reduction in the soluble Zn caused by an increase in pH of the soil Ko01 after plant growth and insignificant resupply from the soil to its solution because of relatively low total Zn in this soil. Increased DOC complexation with Zn caused a lower translocation coefficient (Kt ) of Zn as evidenced by regression analysis. The translocation coefficient (Kt ) of Zn decreased exponentially as DOC increased (r2 = 0.48, P < 0.05). CONCLUSIONS Metal concentrations in the rhizosphere soil solution were considerably influenced by pH and DOC
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concentrations which, in turn, were influenced by plant growth and rhizosphere effects. Increased pH and DOC interacted antagonistically with regard to increased metal concentrations in solution. In the acidic soils (pH < 6.5), the effect of pH increases was stronger than that of DOC increases, resulting in an overall decrease in dissolved metal concentrations in these soils. In contrast, the increased DOC after plant growth increased dissolved metal concentrations in the alkaline soils. Chemical changes in the rhizosphere also played an important role in controlling the speciation of metals in soil solution. Plant growth resulted in reduction of free Cd through formation of Cd-DOC complexes and also through pH increases. In contrast to Cd, free Zn concentrations were probably affected by the co-existence of other heavy metals forming more stable complexes with dissolved organic compounds from root exudates and soil binding sites. 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