Effects of Soil Amendments and EDTA on Lead ...

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Effects of Soil Amendments and EDTA on Lead Uptake by Chromolaena Odorata: Greenhouse and Field Trial Experiments a

a

Phanwimol Tanhan , Prayad Pokethitiyook , Maleeya Kruatrachue a b

c

, Rattanawat Chaiyarat & Suchart Upatham

d

a

Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand b

Mahidol University International College, Nakornpathom, Thailand

c

Faculty of Environment and Resource Studies, Mahidol University, Nakornpathom, Thailand d

Burapha University, Tambon Saensook, Amphur Muang, Chonburi, Thailand Available online: 05 Apr 2011

To cite this article: Phanwimol Tanhan, Prayad Pokethitiyook, Maleeya Kruatrachue, Rattanawat Chaiyarat & Suchart Upatham (2011): Effects of Soil Amendments and EDTA on Lead Uptake by Chromolaena Odorata: Greenhouse and Field Trial Experiments, International Journal of Phytoremediation, 13:9, 897-911 To link to this article: http://dx.doi.org/10.1080/15226514.2010.525556

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International Journal of Phytoremediation, 13:897–911, 2011 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2010.525556


EFFECTS OF SOIL AMENDMENTS AND EDTA ON LEAD UPTAKE BY CHROMOLAENA ODORATA: GREENHOUSE AND FIELD TRIAL EXPERIMENTS Phanwimol Tanhan,1 Prayad Pokethitiyook,1 Maleeya Kruatrachue,1,2 Rattanawat Chaiyarat,3 and Suchart Upatham4 1

Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand Mahidol University International College, Nakornpathom, Thailand 3 Faculty of Environment and Resource Studies, Mahidol University, Nakornpathom, Thailand 4 Burapha University, Tambon Saensook, Amphur Muang, Chonburi, Thailand 2

Greenhouse and field trial experiments were performed to evaluate the use of Chromolaena odorata with various soil amendments for phytoextraction of Pb contaminated soil. Pb mine soils contain low amount of nutrients, so the additions of organic (cow manure) and inorganic (Osmocote and NH4 NO3 and KCl) fertilizers with EDTA were used to enhance plant growth and Pb accumulation. Greenhouse study showed that cow manure decreased available Pb concentrations and resulted in the highest Pb concentration in roots (4660 mg kg−1) and shoots (389.2 mg kg−1). EDTA increased Pb accumulation in shoots (17-fold) and roots (11-fold) in plants grown in soil with Osmocote with Pb uptake up to 203.5 mg plant−1. Application of all fertilizers had no significant effects on relative growth rates of C. odorata. Field trial study showed that C. odorata grown in soil with 99545 mg kg−1 total Pb accumulated up to 3730.2 and 6698.2 mg kg−1 in shoots and roots, respectively, with the highest phytoextraction coefficient (1.25) and translocation factor (1.18). These results indicated that C. odorata could be used for phytoextraction of Pb contaminated soil. In addition, more effective Pb accumulation could be enhanced by Osmocote fertilizer. However, the use of EDTA in the field should be concerned with their leaching problems. KEY WORDS Chromolaena odorata, Pb, greenhouse experiment, field trial experiment, soil amendments

INTRODUCTION There has been an increasing interest in phytoremediation as a plant-based alternative for cost-effective and environmentally sound clean up of heavy metal-contaminated soils (Cunningham et al. 1995; Raskin et al. 1997; Salt et al. 1998). The success of phytoextraction is dependent upon the ability of plants to absorb a substantial amount of metal into their roots and preferentially translocate the metal into the harvestable above-ground biomass (Kumar et al. 1995), the high biomass yield of plants, the plant’s tolerance of toxic metals,

Address correspondence to Prayad Pokethitiyook, Department of Biology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. E-mail: [email protected] 897


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P. TANHAN ET AL.

and the availability of the toxic metal in the soil for plant uptake (Begonia et al. 2002b). Two approaches have been generally proposed for the phytoextraction of heavy metals: the use of natural hyperaccumulator plants with exceptional metal-accumulating capacity, and the utilization of high biomass plants in combination with a chemically enhanced method of phytoextraction. However, adverse factors such as acidity, nutrient deficiency, toxic heavy metal ions, and their interactions with most contaminated soils inhibit plant establishment and growth on these soils (Pichtel and Salt 1998). In general, amendments such as applications of organic materials or inorganic fertilizer are necessary for establishment of plants on contaminated soils. Therefore, it is desirable to increase heavy metal solubility, and thus to enhance plant uptake and translocation by means of soil-applied amendments which provide both cations capable of exchanging soil-absorbed heavy metals and ligands that combine with the metals to form soluble complexes (Xiong and Feng 2001). Lead (Pb), as one of the most widespread metal pollutants in soil, has both limited solubility in soil solution and bioavailability due to complexation with organic and inorganic soil colloids, sorption on oxides and clays and precipitation as carbonate, hydroxides and phosphates (Rudy et al. 1999). Chelates such as ehtylenediaminetriacetic acid (EDTA) have been used to increase the solubility of Pb in soil and nutrient solutions and were reported to have significant effects on Pb accumulation in high biomass crop plants such as peas (Huang et al. 1997), sunflowers (Meers et al. 2005; Liphadzi and Kirkham 2006), corn (Huang et al. 1997), Brassica species (Grˇcman et al. 2001; Blaylock et al. 1997), wheat and other Pb accumulators such as Sesbania species (Begonia et al. 2002a, 2002b; Begonia et al. 2004), and Brachiaria decumbens (Santos et al. 2006). These plants have been shown to accumulate significant amounts of Pb in their shoots when induced through the addition of EDTA. It has been proposed that the Pb-EDTA complex is most likely taken up by the plant roots and then translocated to the shoots using transpiration as the main driving force (McGrath et al. 2002). Organic amendments such as the addition of organic wastes such as manure compost, sewage sludge, and refuse can improve plant growth because they serve as a slow release nutrient source (Wong 2003) and also improve the physical characteristics and nutrient states of mine waste (Bradshaw and Chadwick 1980). They can also decrease heavy metal bioavailability depending on the types of metal and soil, degree of humification in soil, and soil pH (Alm˚as et al. 1999; Ross 1994; Shuman 1999; Walker et al. 2003). In general, it is commonly recognized that manure compost has positive effects on crop production, although the undesirable substances such as heavy metals may impose health hazards if edibles are grown. Chromolaena odorata (L.) (King & Robinson) is a perennial shrub of the Asteraceae family. It is native to North America but has been introduced to Africa and Asia. It is sometimes grown as a medicinal plant to treat skin wounds (Phan et al. 1996). In Thailand, vegetation on metalliferous degraded sites is often dominated by C. odorata. This perennial shrub has already been identified in several metal contaminated sites. Rotkittikhun et al. (2006) reported that C. odorata was one of the pioneer shrub species in the metalliferous soils at Bo Ngam Pb mine in Kanchanaburi province, Thailand, where soil Pb concentration was up to 100000 mg kg−1. C. odorata accumulated 3500 mg kg−1 Pb in shoots and 9800 mg kg−1 in roots. Pharenark et al. (2009) reported that C. odorata could grow around Zn mine area with very high soil Cd and Zn concentration (Cd 1266 mg kg−1, Zn 41216.5 mg kg−1) and accumulated 166 mg kg−1 and 110.3 mg kg−1 of Cd in shoots and roots, respectively. Plants also accumulated 1773.3 mg kg−1 of Zn in shoots and 1494.8 mg kg−1in roots (Phaenark et al. 2009). This pseudometallophyte fulfills many of the requirements of a


EFFECTS OF SOIL AMENDMENTS AND EDTA ON LEAD UPTAKE BY C. ODORATA

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potential plant for phytoremediation, i.e., it concentrates high levels of metals in its tissues, it is fast growing and has a very high biomass. The use of C. odorata in phytoremediation technology is not widely recognized. Nevertheless, C. odorata was used as the biomarker in assessing heavy metals including Pb in traffic and solid waste polluted areas (Asumbiade and Fawale 2009). Singh et al. (2009) showed that C. odorata is the potential plant for phytoremediation of Cs. Our previous study revealed that under hydroponic culture (10 mg L−1 of Pb solution), C. odorata accumulated up to 1700 mg kg−1 and 60000 mg kg−1 in shoots and roots, respectively (Tanhan et al. 2007). Since most studies were performed on liquid media, it would be of great interest to determine whether the results from hydroponics were similar to those from soil study. Zabludowska et al. (2009) suggested that the characteristics of species that were discovered in the course of hydroponic studies might not be found when they are grown in soil. The objectives of this study were to evaluate Pb accumulation in C. odorata grown in Pb-contaminated soils under greenhouse conditions. In addition, the effects of different soil amendments and EDTA on the uptake and translocation of Pb in C. odorata were investigated. Moreover, a field trial experiment was performed to evaluate the possible use of C. odorata for remediation of Pb-contaminated soil. MATERIALS AND METHODS General Physico-Chemical Properties of Soils The physico-chemical parameters of soils were assessed by the Department of Soil Science, Ministry of Agriculture and Cooperatives, Bangkok, Thailand. Soil pH was measured using a glass electrode pH meter. The EC (electrical conductivity) was determined by an EC meter. Organic matter was determined using the method described by Walkley and Black (1934), total N using the Kjedahal method (Blake 1965), available P using the Bray II method (Bray and Kurtz 1945), available K using an atomic absorption spectrophotometer after extraction with NH4 OAc (ICARDA 2001). Soil texture was analyzed using the hydrometer method (Allen et al. 1974). Total concentrations of Pb were determined by HNO3 digestion (APHA, AWWA, WEF 1998). The DTPA extraction was performed using ammonium bicarbonate (NH4 HCO3 ) and diethylenetriamine pentaacetic acid (DTPA) at pH 7.6 for 15 min (ICARDA 2001). Then Pb concentrations were determined by a flame atomic absorption spectrophotometer (Variance; SpectrAA 55B, Australia). Plant Materials Stem cuttings of C. odorata used in both greenhouse and field trial experiments were obtained from plants grown in a non-contaminated area at Sai Yok district, Kanchanaburi province, Thailand. They were grown in agricultural soil under greenhouse conditions (relative humidity 66%; temperature 29◦ C; light intensity 20000 lux) for 3 months. Uniform plants with the same height and weight were then selected for further experiments. Pot Experiment Pb-contaminated soil (14898.0 mg kg−1) was collected from Bo Ngam Pb mine area, Kanchanaburi province (0–10 cm in depth). It was air-dried, gently ground to pass through a 2-mm nylon sieve, homogenized and stored dry. Prior to the experiment, the sieved soil was

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P. TANHAN ET AL. Table 1 Experimental design for pot experiments (n = 9)

Abbreviation


S SE SO SOE SM SME SMH SMHE SNK SNKE

Treatment

Unamended soil (Control) Unamended soil (Control) + 5 mmole/kg dry soil EDTA Unamended soil + 0.1% (w/w) commercially available Osmocote fertilizer (14-14-14) Unamended soil + 0.1% (w/w) commercially available Osmocote fertilizer (14-14-14) + 5 mmole/kg dry soil EDTA Unamended soil + 33.3% (v/v) cow manure Unamended soil + 33.3% (v/v) cow manure + 5 mmole/kg dry soil EDTA Unamended soil + 28.6% (v/v) cow manure + 14.3% (v/v) rice husk Unamended soil + 28.6% (v/v) cow manure + 14.3% (v/v) rice husk + 5 mmole/kg dry soil EDTA Unamended soil + 0.1% (w/w) NK fertilizer in form of NH4 NO3 and KCl Unamended soil + 0.1% (w/w) NK fertilizer in form of NH4 NO3 and KCl + 5 mmole/kg dry soil EDTA

allowed to equilibrate for 2 months in the greenhouse undergoing three cycles of saturation with water and air drying, before being remixed and plated according to Blaylock et al. (1997). The effects of various types of soil amendment with the application of EDTA on growth and Pb phytoextraction were evaluated. Four soil mixtures were prepared by mixing contaminated soil with different fertilizers while unamended soil served as a control (Table 1). Sieved soils, cow manure, rice husks and fertilizers at the predetermined ratios were thoroughly mixed in plastic pots (18 cm in diameter, 10 cm in height, 2 kg per pot) and allowed to equilibrate under greenhouse conditions for 2 weeks prior to planting. C. odorata of uniform size were planted into the pots (1 plant per pot). Each pot was placed in a separate dish to collect the excess soil moisture drained from perforations. Pots were placed in the greenhouse with randomized complete block design with 9 replications (1 pot/replicate) and the plants were grown for 3 months. They were watered every other day depending on the evaporation demand. Once per week plants were fertilized with 100 mL of full strength modified Hoagland’s solution with very low phosphate (0.05 mM KH2 PO4 ; 5 mM KNO3 ; 5 mM Ca(NO3 )2 ; 2 mM MgSO4 ; micronutrients and 5 ppb FeCl3 ·6H2 O, pH 5.5). EDTA was applied as 100 mL of aqueous solution equal to 5 mmole kg−1 dry soil by surface irrigation 1 week prior to the harvest at 3 cm radius from plant roots. After harvest, roots were carefully removed from soil and plants were washed with running tap water and distilled water twice before separating into shoots and roots. Then they were oven dried at 70◦ C for 2 d and their dry weights were determined before they were prepared for heavy metal analysis. Field Trial Experiment The field trial experiment was conducted at Bo Ngam Pb mine, Kanchanaburi province, Thailand. The climate is tropical monsoon with the annual mean temperature of 28.8◦ C, the annual rainfall of 1045.8 m3, and the mean relative humidity of 70.1%. Pb in this area is in the form of cerussite (PbCO3 ). Three independent locations at Bo Ngam Pb mine with different soil Pb concentrations were selected for the field trial experiment: location A at natural pond area (14◦ 56 56.1 N, 98◦ 55 22.5 E) with the lowest soil Pb



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concentration (179.9 mg kg−1), location B at an open pit area (14◦ 59 49.4 N, 98◦ 57 29.4 E) with a moderate soil Pb concentration (5477 mg kg−1), and location C at a stockpile area for PbCO3 (14◦ 59 48.9 N, 98◦ 57 25.3 E) with the highest soil Pb concentration (99544.6 mg kg−1). The distance between each location was approximately 2–3 km. The experiments were conducted during March–August 2008. Each plot occupied an area of 4 m × 4 m, and contained four subplots (4 m × 1 m) representing four replications of 4 plants each, with spacing 1 m between subplot. There were 2 treatments: treatment 1, plants were not supplied with Osmocote fertilizer, and treatment 2, plants were supplied with Osmocote fertilizer. In treatment 2, at the transplanting, 12 g of Osmocote fertilizer was applied at the soil surface to each plant and again on the third month after planting. Plots were irrigated when rainfall was insufficient during summer (March–May, 2008) through a sprinkling system. Plants were grown for 6 months. During the experiment, four plants from each plot were randomly harvested at 2month intervals. Plants were carefully removed from the soil and thoroughly washed in running tap water for 5 min; with a solution of phosphate-free detergent for 15 s; and then with tap water for another 15 s before being carefully rinsed twice with deionized water (Rotkittikhun et al. 2006). The roots and shoots were separated and oven dried at 70◦ C for 2 d to maintain a constant dry weight before heavy metal analysis. Heavy Metal Analysis Dry plant samples were weighed and milled with a mortar, while the dry soil samples were ground to pass through 2-mm nylon sieve before they were digested with conc. HNO3 (70%: BDH, England) according to APHA, AWWA, and WEF (1998). The final solution was diluted with deionized water to 25 mL of total volume and analyzed with a flame atomic absorption spectrophotometer. Standard solution of Pb [Pb(NO3 )2 : Merck, Germany] was used to generate calibration curve to convert absorbance reading to concentrations. Data Analysis The phytoextraction coefficient (PC) was calculated by determining the ratio of mg kg−1 dry weight of metal in the plant to the mg kg−1 quantity of total metal concentration in the dry soil in which the plant was grown (Kumar et al. 1995). The remediation factor (RF) was calculated in order to evaluate the efficiency of the phytoextraction process after treatments with several amendments in pot experiment. It was calculated from the percentage ratio of the amount of Pb in plant (mg) to the amount of Pb in soil (mg) (Vyslouˇzilová et al. 2003). The translocation factor (TF) indicated the plant’s ability to translocate heavy metals from the roots to the harvestable aerial parts (Mattina et al. 2003). It was calculated on a dry weight basis by dividing the metal concentration in shoots by the metal concentration in roots. The relative growth rate (RGR) was calculated according to Hunt (1982). RGR = [ln(w2 ) – ln(w1 )]/(t2 − t1 ); w1 and w2 are plant fresh weight (g) at time t1 and t2 . Statistical Analysis The data of plant dry biomass, Pb accumulation in plant, under different treatments in pot and field trial experiments were analyzed using a SPSS statistical package (version

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P. TANHAN ET AL. Table 2 Physico-chemical properties of soils used in the pot experiments


Parameter Soil texture Sand (%) Silt (%) Clay (%) pH OM (%) EC (dS m−1) Total N (%) Available K (ppm) P (ppm) Ca (ppm)

Cow manure

8.4 4.92 1.59

0.22 0.57

S

SO

SM

SMH

SNK

Loam 49 38 13 7.5 0.4 0.21 0.04

Loam 49 38 13 7.6 0.4 0.42 0.03

Sandy loam 57 34 9 7.2 1.6 1.8 0.08

Sandy loam 61 34 5 7.9 2 2.7 0.10

Sandy loam 71 24 5 7.6 0.4 0.28 0.03

20 4 1800

20 7 1800

474 196 1279

736 736 1563

10 2 1600

O, Osmocote; M, cow manure; H, rice husk; N, NH4 NO3 ; K, KCl; OM = organic matter content; EC = electrical conductivity.

13.0, SPSS Inc., Chicago, USA). One way analysis of variance (ANOVA) was carried out to compare the means of different treatments where significant F value was obtained. Differences between individual means were tested using Duncan’s Multiple Range Test at 0.05 significance level. RESULTS AND DISCUSSION Pot Experiment General physico-chemical properties of soils. The physico-chemical properties of soils used in the pot experiment are shown in Table 2. The pH of the tested soils ranged from 7.2–7.9. The organic matter, EC, and available K, P, and Ca in soil with cow manure (SM) and soil with cow manure and rice husk (SMH) were higher than those in unamended soil. However, their clay content was lower than in unamended soil. The differences in EC among soils (0.21–2.70 dS m−1) could imply that the addition of cow manure and husk resulted in a stronger adsorption potential of heavy metals than in unamended soil and soils amended with inorganic fertilizers. Effects of soil amendments and EDTA on growth performance. The addition of cow manure and rice husks significantly decreased the available Pb concentrations in soils (P < 0.05) (Table 3), whereas the application of inorganic fertilizers including Osmocote fertilizer (SO) and NH4 NO3 with KCl (SNK) did not significantly decrease available Pb concentrations (P > 0.05). C. odorata grew normally (with 100% survival rate) in all soil treatments (Table 4). The application of fertilizers had no significant effect on RGR of C. odorata as compared with control plants (S; unamended soil) (P > 0.05) (Table 4). However, without EDTA, the application of Osmocote fertilizer and NH4 NO3 and KCl, resulted in higher RGR than those grown in unamended soil. In addition, the RGRs of C. odorata decreased in the order of: SO (0.91 g month−1) > SNK (0.73 g month−1) > S (0.57 g month−1) > SMH (0.46 g month−1) > SM (0.33 g month−1). In general, the application of EDTA resulted in the decrease of dry biomass production and RGRs of C. odorata except for plants grown on cow manure with rice husk treatment. After 3 months, the dry biomass production decreased in the following


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Table 3 Total and extractable Pb concentration of Pb-contaminated soils with various amendments and EDTA Pb concentration in soil (mg kg−1)


Soil mixture S SE SO SOE SM SME SMH SMHE SNK SNKE

Total Pb

Extractable Pb

14898.0 ± 158.4 13243.5 ± 129.8 12826.2 ± 212.5 15323.8 ± 313.8 19449.5 ± 7355.5 14142.4 ± 1508.4 13220.2 ± 130.9 13367.8 ± 171.0 15057.5 ± 456.3 12975.2 ± 252.2

1443.5 ± 38.5 2720.9 ± 54.1 1266.6 ± 10.4 2406.8 ± 39.6 963.4 ± 182.5 1254.3 ± 791.2 713.8 ± 10.8 1460.0 ± 56.4 1232.3 ± 21.8 3098.0 ± 23.1

S, unamended soil; O, Osmocote fertilizer; M, cow manure; H, rice husk; N, NH4 NO3 ; K, KCl; E, EDTA.

order: SOE (22.6 g per pot) > SMHE (15.2 g per pot) > SNKE (7.2 g per pot) > SE (5.6 g per pot) > SME (3.3 g per pot). In the third month, without EDTA, plants grown in SO mixture had the highest dry biomass production (30.5 g per pot), followed by SMH (13.4 g per pot), S (12.7 g per pot), SM (10.2 g per pot), SNK (8.9 g per pot). Moreover, visual toxicity symptoms (necrosis on older leaves) from Pb or EDTA were observed in plants grown in soils supplied with EDTA. In this study, the application of fertilizers resulted in the increase in biomass production of C. odorata (the biomass of plants grown with Osmocote fertilizer was 2.5 times that of plants grown on unamended soil). Manure is a more easily mineralizable organic matter source, in which nutrients are more readily available to plants (Walker et al. 2003). Recent studies reported the improved growth of plants grown in Pb-contaminated soil with organic wastes including Agropyron elongatum and Trifoliums repens (Ye et al. 1999), Vetiveria zizanioides (Chiu et al. 2006; Rotkittikhun et al. 2007), Thysanolaena maxima (Rotkittikhun et al. 2007), and Phragmites australis (Chiu et al. 2006). This may explain the higher biomass yield obtained when this amendment was applied. In addition, the improvement of the soil physical properties, such as density, compactness, and water infiltration, were also promoted by the addition of organic amendments (Stewart et al. 2000). The decrease in the biomass production of C. odorata after the application of EDTA was due to the combined toxicity of Pb and EDTA in soil. The effects of chelators were dose- and time-dependent. The application of chelators alone resulted in the removal of essential metal nutrients from soil, leading to deficiencies in the plants (Ruley et al. 2006). Grˇcman et al. (2003) found that in all chelate treatments including EDTA, necrotic lesions were observed on cabbage leaves and were more prominent on the older leaves. Pb accumulation. In general, roots accumulated much higher Pb concentrations than shoots (Table 4). Without EDTA, the addition of cow manure (SM) resulted in the highest Pb concentration in roots (4660 mg kg−1) and control plants had the highest shoot Pb concentration (389.2 mg kg−1). Other types of soil amendments (SO, SMH, SNK) did not have any significant effect on Pb concentration in C. odorata compared with the unamended soil (P > 0.05). EDTA significantly increased the concentration of Pb in shoots and roots of C. odorata relative to the plants grown in Pb with fertilizer only (Table 4). In SO treatment with EDTA,

904

100 100 100 100 100 100 100 100 100 100

S SE SO SOE SM SME SMH SMHE SNK SNKE

0.57 0.35 0.91 0.66 0.33 0.14 0.46 0.68 0.73 0.37

RGR (g month−1) Shoot 389.2 ± 26.3a 6672.4 ± 120.3c 184.3 ± 35.3a 4468.8 ± 190.8bc 322.6 ± 99.2a 1149.5 ± 437.8ab 242.1 ± 72.8a 1476.0 ± 812.2ab 295.2 ± 46.5a 4683.7 ± 256.0bc

12.7 ± 2.2 5.6 ± 1.0 30.5 ± 1.7 22.6 ± 0.3 10.2 ± 4.0 3.3 ± 1.5 13.4 ± 6.3 15.2 ± 8.8 8.9 ± 1.6 7.2 ± 2.7

1537.5 ± 7.4ab 6469.4 ± 484.3c 1157.8 ± 128.4a 4533.7 ± 369.3bc 4660.0 ± 712.8bc 9873.0 ± 229.7d 2702.5 ± 984.3ab 1041.2 ± 405.0a 1417.5 ± 79.6ab 6010.4 ± 152.0c

Root

Pb concentration (mg kg−1)

Dry biomass production (g pot−1) 24.47 73.60 40.93 203.46 50.82 36.37 39.44 38.26 15.24 77.00

Total Pb accumulation (mg plant−1)

RGR, relative growth rate; S, unamended soil; O, Osmocote fertilizer; M, cow manure; H, rice husk; N, NH4 NO3 ; K, KCl; E, EDTA. Data with different letters in the same column indicate a significant different at 5% level according to Duncan’s test.

Survival rate (%)

Soil mixture

0.13 0.99 0.10 0.59 0.26 0.78 0.22 0.19 0.11 0.82

PC

0.25 1.03 0.16 0.99 0.07 0.12 0.09 1.42 0.21 0.78

TF

8 28 16 66 13 13 15 14 5 30

RF (%)

Table 4 Growth performance, Pb concentration, total Pb accumulation, phytoextraction coefficient (PC), translocation factor (TF), and percentage remediation factor (RF) of C. odorata grown in Pb-contaminated soils with various amendments and EDTA for 3 months




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C. odorata attained higher Pb concentrations in both roots and shoots. There were 174-fold and 11-fold increases in shoot and root Pb concentration, respectively, compared with control plants. C. odorata accumulated Pb in the shoots greater than 1000 mg kg−1 with the concentration in plants under different fertilizers generally in the descending order of S (6672.4 mg kg−1) > SNK (4683.7 mg kg−1) > SO (4468.8 mg kg−1) > SMH (1476.0 mg kg−1) > SM (1149.5 mg kg−1). Without EDTA, C. odorata grown on soil with different fertilizers showed very low phytoextraction coefficients (PC 0.10–0.26) and translocation factors (TF 0.07–0.25; Table 4). The application of EDTA greatly increased the PC and TF values. The highest PC value was found in C. odorata grown in SE treatment (0.99). The TFs of C. odorata grown in unamended soil and SMHE treatment were more than 1 and the highest TF value was observed in plants grown in SMH treatment (1.42) (Table 4). Furthermore, when the amount of Pb in plants was calculated, it was shown that plants grown on SOE soil accumulated highest Pb in their tissues (203.5 mg/plant) and also the highest remediation factor (RF 0.66). The application of EDTA one week prior to harvest resulted in the increases of Pb concentration in shoots up to 46-fold (in the first month of exposure; data not shown). In many pot experiments EDTA has been shown to be effective in enhancing Pb accumulation and phytoextraction, for example, in Zea mays (27-fold), Phaseolus vulgaris (70-fold), Dianthus chinensis (15-fold), Sinapis alba (48-fold), Brassica juncea (2.8-fold), B. rapa (59.7-fold), B. oleracea (105-fold), Brachiaria decumbens (1.87-fold), and S. arvensis (6–18-fold in shoot, 4–13-fold in root) (Huang et al. 1997; Grˇcman et al. 2001, 2003; Kos et al. 2003; Wu et al. 2004; Lai and Chen 2005; Luo et al. 2005; Santos et al. 2006; Surat et al. 2008). In this study, EDTA enhanced Pb accumulation in C. odorata shoots 17 times in SE treatment (175 times in leaves; data not shown), and 16 times in SNKE treatment (133 times in leaves; data not shown) when compared with those of S and SNK treatments, respectively. Metal-EDTA complexes are absorbed by plants and transported to shoots via the xylem using transpiration as the main driving force (Epstein et al. 1999; Collin et al. 2001; McGrath et al. 2002). Pb-EDTA complex is expected to be the dominant metal-EDTA complex formed in most soil with pH between 5.2 and 7.7 (Sommers and Landsay 1979). However, the EDTA-metal complex is resistant to degradation by soil microorganisms. In addition, the application of chelates poses some risks of groundwater contamination, especially for soils with higher groundwater table or for sandy soils (Lombi et al. 2001; Wasay et al. 1998).

Field Trial Experiment Soil. The physico-chemical properties of soils from three different locations used in the field trial experiment are shown in Table 5. The pHs of all soils were 7.0 to 7.1 while the EC value of soil from location B (open pit area) was the highest (2.1 dS m−1). Soil from location A (natural pond area) had the highest organic matter, N, K and P. The total Pb in soils from location C (99545 mg kg−1) was significantly higher than in those from location B (5477 mg kg−1) and A (180 mg kg−1). In addition, the available Pb concentration in soil from location B was the highest (2216 mg kg−1) followed by location C (1996 mg kg−1) and location A (28.7 mg kg−1). Growth performance. Under field conditions, C. odorata grew normally and had 100% survival rate. Plants grown at location A and B and supplied with Osmocote produced

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P. TANHAN ET AL.


Table 5 Physico-chemical properties of soils at different locations in Bo Ngam Pb mine in the field trial experiments Parameter

Location A

Location B

Location C

Soil texture Sand (%) Silt (%) Clay (%) pH OM (%) EC (dS m−1) Total N (%) Pb (ppm) Available K (ppm) P (ppm) Pb (ppm)

Clay loam 42 24 34 7.1 1 0.2

Loam 32 44 24 7.1 0.3 2.1

Loam 41 41 18 7.0 0.3 0.2

0.07 179.9

0.04 5477

0.09 99544.6

194 3 28.7

23 3 2216

120 2 1996

Location A, natural pond area; Location B, open pit area; Location C, stock pile area; OM, organic matter content; EC, electrical conductivity.

the highest biomass (163 g and 170 g, respectively) and the highest RGR (0.96 and 1.03, respectively; Table 6). Pb accumulation. In general, the increase in exposure time did not significantly affect Pb accumulation by C. odorata (P > 0.05). Increased soil Pb concentration resulted in a significant increase in Pb concentration (P < 0.05) (Table 6). The highest Pb accumulation was observed in C. odorata grown at location C after 4 months (shoots 3730.2 mg kg−1, roots 6698.2 mg kg−1). Pb mainly accumulated in root tissues of C. odorata grown at location A and B. However, the amount of Pb accumulations in plant were significantly increased when the exposure times were increased. The highest amount of Pb was uptaken in the plants grown at location C after 6 months (748 mg plant−1). As a consequence, the TF and PC values were not significantly different. However, plants grown at location C had TF values more than 1 or closer to 1 (0.90–1.18) indicating that at very high soil Pb concentration, C. odorata was able to translocate Pb from roots to shoots (Table 6). Laboratory based studies are important for providing an insight into some of the fundamental mechanisms of metal uptake in accumulator plants, but field trials are essential to give a measure of their performance in the real environment (McGrath et al. 2006). However, many factors of contaminated soils such as poor physical structure, low water and nutrient holding capacity, deficiency of major nutrients (N, P, K), acidity and alkalinity, water supply, toxic materials, salinity, stability, surface temperature are known to affect plant establishment on contaminated soils (Bradshaw and Chadwick 1980). The results of this study indicated that Pb mine soils contained high levels of total and extractable Pb, and low levels of major nutrients (N, P, K) and organic materials. These are major constraints for general plant establishment. In spite of this, C. odorata grew normally and was the predominant shrub species in Bo Ngam Pb mine area. Osmocote fertilizer was chosen for the application in the field trial experiment because it performed best in the pot experiment. In addition, it is easy to handle and apply and has no environmental impact on the site. Plant establishment and growth (6-month experiment)

907

0 2 4 6 0 2 4 6 0 2 4 6

Month

100 100 100 100 100 100 100 100 100 100 100 100

— 0.96 0.15 0.24 — 1.03 0.70 0.59 — 0.80 0.55 0.57

RGR (g month−1) 3.08 ± 1.2 22.7 ± 1.8 84.2 ± 2.0 163.6 ± 5.4 3.08 ± 1.2 22.0 ± 1.3 86.2 ± 7.6 169.5 ± 14.3 3.08 ± 1.2 21.2 ± 2.1 51.3 ± 4.5 110.1 ± 65.1

Dry biomass production (g plant−1) 0 27.1 ± 4.1a 38.8 ± 2.2a 45.7 ± 1.3a 0 99.5 ± 6.9a 156.3 ± 37.7a 96.5 ± 4.9a 0 2337.6 ± 178.3b 3730.2 ± 340.7b,∗ 2385.7 ± 537.8b

Shoot 0 134.1 ± 77.9a 118.3 ± 11.8a 179.5 ± 48.7a 0 722.5 ± 248.3a 924.9 ± 359.5a 516.3 ± 157.2a 0 2594.1 ± 494.5a 6698.2 ± 170.8b,∗ 2026.0 ± 518.1a

Root

Pb concentration (mg kg−1)

0 3.66 13.23 36.84 0 18.08 93.20 103.87 0 104.55 534.98 748.39

Total Pb accumulation (mg plant−1)

0 0.90 0.87 1.25 0 0.15 0.20 0.11 0 0.05 0.10 0.04

PC

— 0.20 0.33 0.25 — 0.14 0.17 0.19 — 0.90 0.56 1.18

TF

Location A, natural pond area; Location B, open pit area; Location C, stock pile area; RGR, relative growth rate. Data with different letters in the same column with the same location indicate a significant difference at 5% level according to Duncan test and (∗ ) indicate the significant difference for each other between locations in the same time.

C

B

A

Location

Survival rate (%)

Table 6 Growth performance, Pb concentration, total Pb accumulation, phytoextraction coefficient (PC) and translocation factor (TF) of C. odorata in the field trial experiments



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was maximal in both soils containing 1862.2 and 5477 mg kg−1 total Pb while slightly lower biomass production was obtained in plants grown in soil with 99544.6 mg kg−1 total Pb. The results confirmed the data from the pot experiments. C. odorata was well established and produced the highest biomass yield when grown in Pb-contaminated soil at 1000–5000 mg kg−1 (total Pb). The results from this study suggest that fertilizer application can significantly increase the amount of Pb extraction capacities of C. odorata by increasing plant biomass. The concentration of Pb in plants grown in field trial experiments may be lower than in the greenhouse experiments, possibly due to excessive soil drainage, fluctuations in temperature or other uncontrolled environmental variables (e.g., drought, heavy metal toxicity). However, according to Tanhan et al. (2007), C. odorata grown in the field trial experiment showed a similar trend in Pb accumulation roughly corresponding to Pb concentrations in soil. In the search for plants with phytoremediation potential of Pb and Cd-contaminated soils in the Pb mine and Zn mine area, C. odorata was found to be able to accumulate > 1000 mg kg−1 of Pb and > 100 mg kg−1 of Cd in its shoots (Tanhan et al., 2007; Phaenark et al., 2009). Hydroponic experiments were in line with the findings on metal accumulation (Tanhan et al. 2007). The pot experiment in the present study demonstrated quantitative difference in Pb uptake and translocation ability of C. odorata. Pb accumulations without the addition of EDTA were much lower than those observed in the hydroponics (Tanhan et al. 2007). This is due to greater Pb availability (PbNO3 ) in the solution (Xue et al. 2004). The low translocation and accumulation of Pb in shoots resulted in a low phytoextraction potential. The addition of fertilizers did not improve the extraction, only the growth. However, this study showed that phytoextraction potential of C. odorata was greatly enhanced with the addition of EDTA. The results of the field trial experiment confirmed those of the field sampling (Tanhan et al. 2007). C. odorata could tolerate soil Pb as high as 100,000 mg kg−1 and the addition of Osmocote fertilizer greatly improved the plant growth. Another similar observation was that C. odorata was able to translocate very high concentration of Pb from roots to shoots only at relatively high concentration of Pb in the soil. Our study demonstrated that the biomass production and accumulation of Pb by C. odorata were enhanced by the application of inorganic fertilizer including Osmocote fertilizer and NK fertilizer. EDTA can be applied to C. odorata several days before harvesting the aerial parts in order to enhance the translocation of Pb from roots to shoots. C. odorata was the suitable plant for chemically induced phytoextraction due to its fast growth rate, high tolerance to polluted soils, large biomass and high shoot Pb accumulation. However, the application of EDTA contains some major drawbacks and environmental concerns, limiting its acceptance for use in the field.

ACKNOWLEDGMENTS This research work was supported by the grant from the Center on Environmental Health, Toxicology and Management of Toxic Chemicals under Science & Technology Postgraduate Education and Research Development Office (PERDO) of the Ministry of Education, and Mahidol University, Bangkok, Thailand. We are grateful to Assist. Prof. Philip Round for editing the manuscript.


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