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April 2006, 27(2) 311-316 (2006) For personal use only Commercial distribution of this copy is illegal
Phytoremediation of soil contaminated with cadmium and/or 2,4,6-Trinitrotoluene Kyung-Hwa Baek1, Joo-Yun Chang1, Yoon-Young Chang2, Bum-Han Bae3, Jaisoo Kim1 and In-Sook Lee1 1Department of Life Science, Ewha Womans University, Seoul, Korea 2Department of Environmental Engineering, Kwangwoon University,Seoul, Korea 3Department of Civiland Environmental Engineering, Kyungwon University, Sung-Nam, Korea (Received: 23 January, 2004 ; Accepted: 21 March, 2005)
Abstract: Phytotoxicity, microbial activity, plant uptake and microbial degradation were examined using Rumex crispus in TNT and/or cadmium contaminated columns (TNT: 100 mg/kg of soil and Cd: 10 mg/kg of soil). The growth of plants was significantly inhibited by TNT, but not by Cd. The microbial activity was highly increased by plant root growth, decreased by Cd, and slightly reduced by TNT. The plant uptake of Cd was relatively well in Cd-contaminated column, but lowered by TNT in TNT+Cdcontaminated column. The microbial degradation of TNT occurred much faster in planted columns than in unplanted columns with minor effect of Cd (less 2-ADNT was produced). Therefore, it may be effective to treat TNT first and then Cd using phytoremediation in the TNT plus Cd contaminated sites. Key words: Cadmium, Microbial activity, Rumex crispus, 2,4,6-trinitrotoluene (TNT), Phytotoxicity. Introduction Phytoremediation, defined as the use of green plants to remove pollutants from the environment or to render them harmless (Cunningham and Berti, 1993), is considered a new highly promising technology for the remediation of polluted sites. This technology can be applied to both organic and inorganic pollutants present in soil, water or the air (Salt et al., 1998). The contamination of soil and groundwater by toxic chemicals is potentially a serious environmental and public health problem around military bases and ammunition manufacturing facilities throughout the world. Especially, at shooting ranges, heavy metals and explosive organic chemicals are contaminating the soil together (Lee et al., 2002) Among heavy metals, cadmium (Cd) is one of the most toxic heavy metals present in the environment (Wagner, 1993). It is easily taken up by roots and translocated to different plant parts (Baker et al., 1994). High accumulation generally causes growth inhibition and even plant death (Khan and Khan, 1983). Cd is of particular concern to human health, because it accumulates in the body with a half-life exceeding 10 years and has been linked with renal tubular dysfunction (Buchet et al., 1990), pulmonary emphysema (Ryan et al., 1982), and possibly osteoporosis (Bhattacharyya et al., 1988). Of the contaminants in explosives produced during the past 90 years, 2,4,6trinitrotoluene (TNT) is the most widespread and persistent. This compound is toxic to aquatic organisms (Smock et al., 1976) and terrestrial plants (Palazzo and Leggett, 1986), animals (Lemberg and Callaghan, 1944) and humans (Hathaway, 1977). An effective and affordable solution is therefore needed to TNT and Cd accumulation in soil and groundwater. Rumex crispus (curly dock) has been known as a plant species to remove large amount of Cd in wastewater (Cha, 1992) and soil (Kang et al., 2000). However, it has not known that this
species can still remove Cd well with TNT in soil and has any removal effect on TNT as well. The objective of this study was to investigate how Cd and TNT influence each other, especially with Rumex crispus, in terms of phytotoxicity, microbial activity, plant uptake, and, microbial degradation. Materials and Methods Plant and soil: Seed of Rumex crispus (obtained from the South Korean Rural Development Administration) were sown in commercial potting mixture (B.P.#2, Hungnong Seeds Company, South Korea), and then grown for 10 days in pots. Seedlings of similar height and fresh weight were selected and transplanted to a soil column. The soil, sandy loam (5.6% organic matter, pH 8.6) comprising 5.7% clay, 13.7% silt and 80.6% sand, was collected from an agricultural site at Seoul, South Korea. The soil was passed through a 0.4 cm sieve, airdried, and then artificially contaminated with 10 mg of CdSC>4 and/or 100 mg of TNT per kilogram of soil. Contaminated soils were allowed to equilibrate for 7 days at field moisture levels before the introduction of R. crispus. Uncontaminated soil was used as a control. Experimental design: Soil columns were constructed for 15 cm diameter and 100 cm lengths of polyvinyl chloride (PVC) pipe. Each column was filled to a depth of 70 cm with unsieved and uncontaminated soil, followed by 20 cm of contaminated soil on top. The bottom of each column had layers of plastic mesh netting to prevent the loss of tailings into the leachate. Three seedlings of similar height and fresh weight were transplanted into each column. Immediately after transplanting, the plants were placed in a green house with natural light, temperature, and humidity to mimic as closely as possible the conditions in the field. The plants were fertilized daily with Hoagland's solution for 4 weeks and then every 2 days until harvest. The plants were grown for 150 days.
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Fig. 1: Dehydrogenase acivity (DHA) in different soil columns. Table – 1: Growth of Rumex crispus in different soil columns. Treatment Control Cd TNT Cd+TNT
0 day 0.50 ±0.01 0.50 ±0.01 0.50 ±0.01 0.50 ±0.01
30 days 7.12 ±0.32 7.94 ±0.22 2.14 ±0.21 0.54 ±0.01
fresh weight/plant (g) 60 days 90 days 14.37 15.20 ±1.32 ±3.21 19.67 25.25 ±2.12 ±2.53 6.98 10.09 ±1.56 ±1.21 4.07 9.97 ±1.05 ±1.65
Plant and soil analysis: Randomly selected columns were pulled out of the green house for destructive analysis. Plants were harvested by gently removing them from soil. Prior to analysis, plants were washed with water to remove soil deposits. For determining the amount of Cd in the plants, roots and shoots were further separated with scissors and then dried in an oven at 70°C for 3 days. The dried samples were
120 days 26.72 ±3.45 22.63 ±2.65 15.05 ±2.01 18.67 ±2.11
150 days 24.58 ±3.54 23.01 ±3.86 19.90 ±3.04 14.27 ±2.55
digested with concentrated nitric acid, and subjected to flame atomic absorption spectrophotometery (Flame-AAS; Analyst 100 spectrophotometer, Perkin-Elmer, USA). The PVC columns were cut using a saw into the fallowing lengths starting from the top; 0-20, 20-40, 40-60, 60-80 and 80-100 cm. After the columns were cuts, the entire soil sample was pushed put into a zip-lag bag and saved for analysis. Soil samples were dried at
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Fig. 2: Relationship between dehydrogenase activity and root density in different soil columns (p< 0.001). Table – 2: Concentration of TNT and metabolites in different soil columns on 30 days of transplantation. Treatment TNT, plant TNT, no plant TNT+Cd, plant TNT+Cd, no plant
TNT (mg/kg) 0.0 ± 0.0 10.3 ±3.5 0.0 ±0.0 12.2 ±2.1
room temperature and analyzed for water-soluble metals by equilibrating 1 g of soil with 20 ml of water for 2 hr. The amount of exchangeable Cd in soil was estimated by extracting 1 g of the soil with IN ammonium acetate for 1 hr. Soil Cd was analyzed with AAS. The detection limits of Cd were 0.05 mg/kg for soil and plant. TNT and derivatives [including 2-amino-4, 6dinitrotoluene (2ADNT), 4-amino-2, 6-dinitrotoluene (4ADNT)] in plants were extracted in methanol using solid-phase extraction with C8. Also, TNT and its derivatives in soil were extracted in acetonitrile for 18 hr using an ultrasonicator according to the U.S EPA SW-846, method 8330. The extract
2-ADNT (mg/kg) 4.8 ±0.8 2.9 ±1.2 3.1 ±0.2 1.9 ±0.1
4-ADNT (mg/kg) 11.7 ±2.3 9.0 ±1.8 11.8±0.8 8.8 ±0.14
solution was filtered to obtain and analyzed by HPLC-UV (Shimazdu 10A, Japan) using Xterra RP!8 column (4.6 x 250 mm, 5 µm; Waters, USA). The UV detector was set at 230 nm. A 20-µl aliquot of anlyte was injected into the mobile phase, which was a mixture of methanol and water (50:50, v/v) with a flow rate of 0.8 ml/min. The detection limit of TNT was 0.3 mg/kg for soil. Dehydrogenase activity: Dehydrogenase activity was measured with spectrophotometer at 480 nm by using iodonitrotetrazolium chloride as the substrate (Trevor et al., 1982).
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Baek et al. time due to removal of Cd and/or TNT. Also, Cd rather than TNT influenced dehydrogenase activity because it was low at the bottom of Cd and TNT+Cd columns, where Cd concentration was high due to leaching. As shown in Fig. 2, root density and dehydrogenase activity was highly correlated (both were high in the upper soil). These correlation slopes implied that the root density influenced microbial activity more in highly contaminated soil (TNT+Cd > Cd > TNT > Control).
Fig. 3: Concentrations of soluble Cd in (A) Cd contaminated and (B) Cd + TNT contaminated soil columns. Results and Discussion Phytotoxicity: The growth of the plants in contaminated soil with TNT (100 mg/kg) and TNT+Cd (100 mg TNT/kg plus 10 mg Cd/kg) was greatly inhibited compared to plants grown in uncontaminated soil (Table 1). After 90 days, the roots of control plants and Cd-exposed plants extended well into the bottom of the column, whereas the fine root structures of the TNT-exposed plants and TNT+Cd exposed plants did not. After a further 90 days, the roots of control plants were almost unchanged, but the roots of the exposed plants extended into the bottom of the column. Therefore, TNT is the only factor to inhibit the growth of R. crispus in the TNT+Cd soil columns. Soil microbial activity: The soil microbial activity was assessed through an indirect measure, the dehydrogenase activity (Fig. 1). Soil dehydrogenase activity was higher in planted soils than in unplanted soils. Although the upper sections of columns were contaminated with Cd and/or TNT, microbial activity in this section increased with the passing of
Contaminant uptake by plant: Cd accumulation is generally higher in roots than in shoots (Salt et al., 1995), and absorbed Cd is mainly associated with cell walls (Hart et al., 1998) or sequestered in vacuoles (Li et al., 1997). At 150 days after transplantation, R. crispus grown in the soil contaminated with Cd had accumulated substantial amounts of Cd in their roots and shoots (297.2 µg and 57.0 µg of Cd per plant, respectively), whereas the roots and shoots grown in TNT + Cdcontaminated soil had accumulated considerably less Cd (76.2 µg and 36.0 µg of Cd per plant, respectively). TNT significantly affected Cd uptake by the plant due to may be influencing plant growth. Therefore, it is strongly recommended that TNT needs to be treated prior to Cd treatment in the TNT and Cd mixed contaminated sites if plants were applied for Cd clean-up. Contaminants disappearance from soil: Cd is analyzed with bioavailable Cd (Fig. 3) and exchangeable Cd (Fig. 4). In unplanted soils, bioavailable Cd moved near the bottom (>80 cm) of columns, whereas in planted soils bioavailable Cd moved to only 40-60 cm below the top of the columns. This result supports the theory that the build-up of organic carbon in the rhizosphere due to root necrosis and exudates retards the movement of Cd thorough the stabilization or adsorption of pollutants. The removal and movement of Cd in TNT+Cdexposed soils was significantly lower than Cd-exposed soils. Since soil particles alone will not effectively prevent mobility of contaminants, stabilization or adsorption on organic matter such as the well-developed roots of the plant is responsible for the majority of contaminant retardation in the planted soils. TNT in TNT and TNT + Cd-contaminated soils with plants were 86.10 ± 0.26 mgkg-1 and 85.85 ± 5.23 mgkg-1 at time zero, respectively. It was almost completely removed nearly at 30 days after transplantation, while remained about 10 mgkg-1 in unplanted columns (Table 2). In this study, the movements of TNT were not observed in planted and unplanted column. The amount of 2-ADNT in TNT and TNT + Cd soils with plants was much greater than that in unplanted soils as well as the amount of 4-ADNT. For all treatments, 4-ADNT than 2ADNT was more produced than 2-ADNT. These results suggest that the removal of TNT mainly occurs through microbial activity in contaminated soil rather than that by plant uptake because TNT and its metabolites were not much detected in plants. It has been reported that organic compounds such as TNT are mostly bound to the organic matrix of soil, mainly humic substances, making it difficult for plants to uptake (Anzhi et al., 1997). Therefore our findings suggest that R. crispus doesn't phytormediate through taking up TNT itself, but rather indirectly stimulates microbial remediation.
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Fig. 4: Concentrations of exchangeable Cd in (A) Cd contaminated and (B) Cd+TNT contaminated soil colums. In several studies, plants have been used successfully to recover metals from contaminated soils and waters and have been shown to accelerate the disappearance of several herbicides, PAHs, and chlorinated compounds (Cunningham et al., 1995). In this experiment, Rumex crispus had accumulated substantial amounts of Cd in their roots and shoots, but not TNT. The deep-rooted plants may assist in the remediation of recalcitrant organic compounds through the release of root exudates into the soil. These act as microbial substrates that increase soil microbial populations and activity in the vicinity of the organic contaminant. Although TNT was reduced more rapidly in planted soils than in unplanted soils, enhanced contaminant degradation by root effects may be minor. These observations suggest the potential of plants and associated microbial communities to promote TNT degradation and detoxification of metal in soil. Acknowledgments This work was supported by the Korea Science and Engineering Foundation grants No. 2000-2-30900-002-3.
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Correspondence to : Dr. In-Sook Lee Department of Life Science, Ewha Womans University, Seoul, 120-750, Korea E-mail:
[email protected] Fax: +82-2-3277-2375 Tel.: +82-2-3277-3285
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