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Removal of Pyrene from Contaminated Soils by White Clover*1
Pedosphere 19(2): 265–272, 2009 ISSN 1002-0160/CN 32-1315/P c 2009 Soil Science Society of China Published by Elsevier Limited and Science Press
Removal of Pyrene from Contaminated Soils by White Clover∗1 XU Sheng-You1,2,3 , CHEN Ying-Xu1,∗2 , LIN Kuang-Fei3 , CHEN Xin-Cai1 , LIN Qi1 , LI Feng1 and WANG Zhao-Wei1 1 Department of Environmental Engineering, Zhejiang University, Hangzhou 310029 (China). E-mail: shengyouxu22 @yahoo.com.cn 2 College of Life and Environment, Huangshan University, Huangshan 245041 (China) 3 School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237 (China)
(Received May 16, 2008; revised December 8, 2008)
ABSTRACT Phytoremediation has been used as an emerging technology for remediation of soil contamination with polycyclic aromatic hydrocarbons (PAHs), ubiquitous persistent environmental pollutants derived from natural and anthropogenic processes, in the last decade. In this study, a pot experiment was conducted to investigate the potential of phytoremediation of pyrene from spiked soils planted with white clover (Trifolium repens) in the greenhouse with a series of pyrene concentrations ranging from 4.22 to 365.38 mg kg−1 . The results showed that growth of white clover on pyrenecontaminated soils was not affected. The removal of pyrene from the spiked soils planted with white clover was obviously higher than that from the unplanted soils. At the end of the experiment (60 d), the average removal ratio of pyrene in the spiked soils with white clover was 77%, which was 31% and 57% higher than those of the controls with or without micobes, respectively. Both roots and shoots of white clover took up pyrene from the spiked soils and pyrene uptake increased with the soil pyrene concentration. However, the plant-enhanced dissipation of soil pyrene may be the result of plant-promoted microbial degradation and direct uptake and accumulation of pyrene by white clover were only a small part of the pyrene dissipation. Bioconcentration factors of pyrene (BCFs, ratio of pyrene, on a dry weight basis, in the plant to that in the soil) tended to decrease with increase in the residual soil pyrene concentration. Therefore, removal of pyrene in the contaminated soils was feasible using white clove. Key Words:
microbial degradation, phytoremediation, plant uptake and accumulation, pyrene, white clover
Citation: Xu, S. Y., Chen, Y. X., Lin, K. F., Chen, X. C., Lin, Q., Li, F. and Wang, Z. W. 2009. Removal of pyrene from contaminated soils by white clover. Pedosphere. 19(2): 265–272.
INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous persistent environmental pollutants derived from natural and anthropogenic processes (Freeman and Cattell, 1990; Binet et al., 2000; Fabbri et al., 2004; Xu et al., 2005). In recent years, anthropogenic practices such as industrial processing, petroleum spills, and incomplete combustion of fossil fuel lead to the accumulation of PAHs in the environment. This has caused increasing environmental concerns (Haeseler et al., 1999; Joner and Leyval, 2003; Xu et al., 2006a). Soils as reservoirs receive a large amount of PAHs and some of them are carcinogenic and/or mutagenic and may pose threats to human health (Fujikawa et al., 1993; Joner and Leyval, 2003; Yang et al., 2005). Pyrene, a four-ring PAH that has a low biodegradability and high persistence in the environment, is one of the PAHs on the United States Environmental Protection Agency (US EPA) priority pollutant list (Binet et al., 2000; Yan et al., 2004). ∗1 Project
supported by the National Natural Science Foundation of China (Nos. 40432004 and 20677015), the Postdoctoral Science Foundation of China (No. 20070420094), the Postdoctoral Science Foundation of Shanghai Municipality, China (No. 08R214116), the Science and Technology Commission of Shanghai Municipality, China (No. 0752nm025), and the National High-Tech Research and Development Program (No. 2007AA06Z331). ∗2 Corresponding author. E-mail: [email protected].
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Phytoremediation, the use of vegetation for in-situ treatment of contaminated soils and sediments, has been used as an emerging technology for remediation of PAH-contaminated soils in the last decade. It also appears to have great potential for the treatment of soils contaminated with residual levels of PAHs (Reilley et al., 1996; Banks et al., 1999; Noble et al., 2003; Glick, 2003). Plants provide a robust, solar-powered system that has little or no maintenance requirement. With their copious root systems, plants can scavenge large areas and volumes of soil PAHs. The rhizosphere soil has microbial population orders of magnitude greater than those of the bulk soil (Haeseler et al., 1999; Alkorta and Garbisu, 2001; Sung et al., 2001; Chen et al., 2003). Laboratory and pot experiments have demonstrated that plants can enhance dissipation of PAHs when compared to unplanted controls (Banks et al., 1999; Yoshitomi and Shann,, 2001; Joner and Leyval, 2003; Xu et al., 2006b). Phytoremediation field trials have resulted in accelerated reduction of PAHs and other petroleum hydrocarbons in the rhizosphere. It was found that pyrene in spiked soils disappeared rapidly in the first four weeks with tall fescue and alfalfa (Schwab and Banks, 1994; Joner and Leyval, 2003; Tao et al., 2004). Liste and Alexander (2000) reported that the degradation of pyrene could be promoted by nine different plant species and up to 74% pyrene disappeared from the vegetated soils compared to 40% or less from the unplanted soils within eight weeks. During the last decade, there has been considerable interest in understanding the uptake of PAHs by plants (Kipopoulou et al., 1999; Mattina et al., 2003; Vervaeke et al., 2003; Xu et al., 2006b). However, results usually are not in good agreement. Some found direct relationships between soil and plant PAH concentrations, while others found no such relationship (Liste and Alexander, 2000; Gao and Zhu, 2004; Xu et al., 2005). Translocation of pyrene from roots to shoots is still ambiguous and the impact of these processes has not been clearly elucidated. Therefore, information about pyrene distribution and concentration of plants is important in predicting the effectiveness of phytoremediation operations. The objective of our study was to investigate the relationship between pyrene accumulated in the inner plant tissue and its concentration in the environment, in order to advance our understanding of the phytoremediation mechanisms of PAHs. MATERIALS AND METHODS Pyrene was obtained from the Sigma Chemical Co. with a purity of 99.9%. A loam soil (pH 6.12 and 23.6 g kg−1 organic matter) with no detectable PAHs was used in this study. The soil samples collected in the upper horizon (0–20 cm) of an agricultural field near Hangzhou City, Zhejiang Province, China, were air-dried and passed through a 2-mm sieve. Part of the soil samples were spiked with pyrene of high purity in acetone. After acetone had evaporated, the spiked soils were mixed thoroughly with the uncontaminated soil. The final concentrations of pyrene in the treated soils were measured by high performance liquid chromatography (HPLC) as the initial concentrations of pyrene (on a dry weight basis) in the soils of Treatments T0, T1, T2, T3, and T4; they were 0, 4.32 ± 2.56, 24.02 ± 0.52, 169.06 ± 3.24, and 365.38 ± 12.37 mg kg−1 , respectively. The treated soils (500 g dry weight soil per pot) were then packed into 15 cm diameter plastic pots lined with a 0.1 mm sieve. These pots were kept in a greenhouse and equilibrated for 7 d at field moisture before introduction of plants. Control A consisted of unplanted spiked soils with microbes; Control B consists of unplanted spiked soils with 2 g kg−1 NaN3 added to inhibit the microbial activity. Seeds of white clover (Trifolium repens) were grown for 15 d in vermiculite before being transferred. Twenty seedlings were transplanted to each designated greenhouse pot about 5 d after emergence. The pots containing plants and controls in triplicate were placed randomly in a growth chamber maintained at 25 ◦ C in the daytime (16 h) and at 19 ◦ C during the night (8 h). All the pots were watered as needed and fertilized once a week with an inorganic salt solution. The seeding date was considered as day 0. Both planted and unplanted soils were destructively sampled after 60 d. At 60 d, white clover was harvested, rinsed with tap water and distilled water, and separated into shoot and root components. All samples (soils, shoots, and roots) were freeze-dried, bagged, and stored at 4 ◦ C before analysis. The
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dry weights of shoots and roots were determined. The procedure used to extract PAHs from the soils was a modification of the methods in Simonich and Hites (1994) and Kipopoulou et al. (1999). Soil samples to be analyzed were spiked with known amounts of pyrene. The freeze-dried soil samples 2 g each were transferred into an Erlenmeyer flask with 10 mL of dichloromethane and the flask was placed into an ultrasonic bath and sonicated for 1 h followed by centrifugation. Then 3 mL of supernatant was filtered through 2 g of silica gel column eluted with 10 mL 1:1 (v/v) hexane and dichloromethane. The solvent was then evaporated to dryness and the solid was re-dissolved in 2 mL methanol. Pyrene in roots and shoots was extracted in a similar way to that used for soil pyrene. The freezedried plant samples 100 mg each were transferred into an Erlenmeyer flask with an extraction solvent of hexane and acetone (3:2, v/v). The Erlenmeyer flask was placed into an ultrasonic bath and sonicated for 30 min. The solvent was then decanted, collected and replenished. This process was repeated three times. The solvents were evaporated to dryness and re-dissolved in 2 mL of methanol. All methanol extracts from soil and plant extractions were filtered prior to analysis with a 0.45-μm Teflon syringe filter and analyzed by high performance liquid chromatography (Agilent, USA) with an ultraviolet (UV) detector and an automatic injector, fitted with a 4.6 mm × 150 mm Agilent reverse phase C18 column using methanol and water (90:10, v/v) as the mobile phase at a flow rate of 1 mL min−1 . Pyrene was detected by the absorbance at 250 nm. Replicate analyses gave an error in the range of 5%–10%. All samples were subjected to strict quality control procedures. The reproducibility and recovery of the extraction for the spiked soil samples were 97% ± 9.5% (n = 7). The recovery of pyrene in the spiked plant samples was 90% ± 5% (n = 7). Statistical significance was evaluated using SPSS version 10.0 with one-way analysis of variance (ANOVA) and least significant difference (LSD) tests for comparison of treatment means with P < 0.05. RESULTS AND DISCUSSION Plant growth The root and shoot yields of white clover on a dry weight basis are shown in Fig. 1. The results indicated that the plants showed no signs of stress and produced a similar biomass between pots with all the spiked soils and the controls. The dry weights of the roots changed at different treatment levels but the changes were not significant (P > 0.05). There was no apparent change in the shoot weights between the treated and control pots. As compared with the control pots, the roots in the spiked pots were also not changed and formed a dense fibrous root system in all soils regardless of treatment.
Fig. 1
Shoot and root biomass of white clover after 60 d on variously spiked soils (on a dry weight basis).
Fig. 2
Residual soil pyrene concentrations with white clover planted and unplanted after 60 d.
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PAHs had been described as physiological toxic pollutants. However, very few studies had been undertaken to evaluate the toxic effects of PAHs on plant growth, especially terrestrial dicotyledons (Ren et al., 1996) and findings from limited studies showed that different plant species had different responses to PAH contamination. Chen et al. (2003) pointed out that no significant detrimental effects were caused by the presence of PAHs on seed germination and root radicle elongation of five prairie grasses even at high concentrations of PAHs (> 300 mg kg−1 ). The low solubility of PAHs and their adsorption on soil particles would limit the physiological toxicity of PAHs in rooted plants. In the present study, growth of white clover on pyrene-contaminated soils was comparable to the control without any pyrene addition and also no differences in growth were observed during the whole experiment (Fig. 1). The results demonstrated that PAH contamination would not produce any significant toxic effects to white clover growing in the spiked soils. Removal of pyrene from spiked soils by white clover In this experiment, Control B, the removal of pyrene from the unplanted spiked soils with NaN3 added indicated the abiotic removal of this compound, including chemical degradation and physical sorption. The loss of pyrene in the unplanted and planted spiked soils may be considered as the microbial degradation and plant-promoted removal, respectively. Fig. 2 shows the measured concentrations of pyrene remaining in the soils at the time of harvesting for white clover. The results showed that the residual pyrene concentrations in the contaminated soils were much lower than the initial values. Pyrene remaining in the vegetated soils was significantly lower than that in the non-vegetated soils. The removal ratios (R) of pyrene in the treated pots were calculated as: R = (C0 − Ct ) × 100/Ct , where C0 is the initial soil concentration of pyrene and Ct the residual concentration of the compound. At the end of the 60-d experiment, the average removal ratio of pyrene in the spiked soils (T1–T4) with white clover was 77%, which was 31% and 57% higher than those of the unplanted Control A with microbes and Control B without microbes, respectively (Fig. 3). This indicated that the removal of pyrene in the spiked soils was clearly promoted by white clover. Removal ratios with lower concentrations of pyrene were significantly higher than those of the corresponding treatments with higher concentrations of pyrene (Fig. 3). At the lower treatment concentrations of pyrene, the removal ratios of pyrene clearly increased with the increasing concentration of pyrene. However, after the concentration of pyrene was elevated to 25 mg kg−1 , the removal ratios of pyrene slightly decreased with increase in pyrene concentration in both planted and unplanted soils.
Fig. 3
Removal ratios of pyrene in the variously spiked soils after 60 d.
Pyrene accumulation in roots and shoots Concentrations of pyrene in white clover shoots and roots after 60 d under different pyrene levels in soil are shown in Fig. 4. With the increase of soil pyrene concentration, pyrene concentration of the roots
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significantly increased and more pyrene was transferred to the upper parts of the plant, which eventually accumulated in the shoots. The concentration of pyrene in the roots was higher than that of the shoots (Fig. 4). It was notable that the pyrene concentrations of roots in the unspiked soils were undetectable; however, the pyrene concentration of shoots in the unspiked soils was 1.02 mg kg−1 , implying that the uptake of pyrene by shoots from the ambient air, possibly originally volatilized from the soils, was an important pathway for the pyrene taken up by the above-ground parts of the plants.
Fig. 4
Concentrations of pyrene in roots and shoots of white clover.
Fig. 5
Root and shoot bioconcentration factors (BCF) of pyrene.
Relationships between pyrene in white clover and its residues in soil Bioconcentration factors (BCFs), the ratio of pyrene concentration in the root or shoot tissues of white clover to the residue in soil, are shown in Fig. 5. The average residual soil pyrene concentration after harvest of white clover was used for the estimation of BCFs. Although many uncertainties should be acknowledged, the results implied that pyrene found in white clover plants was just taken up from the corresponding pot in which they grew. BCFs of pyrene tended to decrease with the increase of the residual soil concentrations (Fig. 5). Root BCFs of pyrene were 1.0–1.8, showing that the root samples had a higher content of pyrene than the soils. Root BCFs of pyrene were much higher than shoot BCFs of pyrene in the treatments with the higher residual soil pyrene concentrations, which was contrary to the situation at the lower residual soil pyrene concentrations. Pathways of pyrene removal Removal of pyrene from the spiked soils was obviously promoted by white clover. At the end of the experiment, with white clover, the loss of pyrene in the spiked soils with the highest level of pyrene was 86% of the initial soil pyrene, which was 30% and 68% larger than the losses in soils of the controls A and B, respectively (Table I). Similar results showed that more PAH contaminants in soil were degraded in the presence of various plant species (Reilley et al., 1996; Sung et al., 2001; Xu et al., 2006a). The enhanced degradation of PAHs in the planted soil could be due to a higher density and greater activity of microorganisms in the rhizosphere than the non-vegetated soil as the root exudates and plant litter could enhance the bioavailability of the contaminant, providing more substrates for co-metabolic degradation and modifying the soil environment to become more suitable for microbial transformation (Cunningham and Ow, 1996; Reilley et al., 1996; Xu et al., 2006b). It was notable that pyrene concentrations of shoots in the controls were detectable, implying that shoot uptake of pyrene from the ambient air, possibly originally volatilized from the soils, was an important pathway for the pyrene taken up into the white clover above-ground parts. Gao and Zhu (2004) found that the shoots of 12 plant species grown on the unspiked control soils apparently accumulated pyrene from the air. Table I suggests that if there was no pyrene in the soil, there might still be some pyrene in the plant, which also proved the atmospheric
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contamination route. However, they also found that the concentrations of phenanthrene and pyrene in the air samplers located 5 or 15 cm above the surface of greenhouse pots showed no differences and volatilized concentrations of phenanthrene and pyrene were very low. TABLE I Mass balance of pyrene in the spiked soils with and without white clover planteda) Item
Treatment T0
Input Plant uptake Remained in soil Planted Control A Control B a) Control
T1
T2
T3
T4
0 0.06
2.16 1.03
mg 12.01 1.98
84.53 9.62
182.09 15.68
0 0 0
0.81 1.47 1.75
1.70 5.39 10.79
17.44 42.72 76.91
33.15 99.86 159.16
A = unplanted spiked soil with microbes; Control B = unplanted spike soil without microbes.
Our results suggest that the pyrene concentrations of shoots on the spiked soils were positively correlated with their respective initial soil pyrene concentrations (Fig. 4). On average 96% of pyrene in shoots was translocated from root uptake; on the average only 4% of pyrene in shoots was from the atmosphere (Table I), implying that the concentrations of pyrene in shoots grown in the spiked soils were much larger than the shoot uptake and accumulation from the atmosphere. Similar results have been reported (Schroll et al., 1994; Wang and Jone, 1994). Wang and Jones (1994) reported that root or shoot accumulation of pyrene in the contaminated soils was elevated with the increase of their soil concentrations. Schroll et al. (1994) showed that about 32%–96% of pyrene in shoots was translocated from root uptake. Bioconcentration factors (BCFs) of pyrene generally tended to decrease with the increase of their residual soil concentrations. BCFs of pyrene by roots were much higher than those by shoots for the same treatment, which indicated that the transfer of pyrene from roots to shoots was considerably restricted. Similar results have been achieved by Gao and Zhu (2004) who showed that BCFs of phenanthrene and pyrene in roots for plants grown in contaminated soils were 0.05–0.67 and 0.23–4.44, respectively. Samsoe-Petersen et al. (2002) also found that the BCFs of PAHs in potatos were 0.020–0.100 and in most cases decreased with increasing concentrations of PAHs in soils. Besides, the relationships between BCFs and the physico-chemical properties of pollutants, such as lipophilicity, water solubility, vapour pressure, and Henry’s law constant, have been examined by several investigators (Simonich and Hites, 1994; Wang and Jones, 1994). Although some good correlations were obtained, additional studies are needed to investigate these relationships more thoroughly. The fates of PAHs in spiked soils include volatilization, leaching, photo-degradation (contaminated at the surface), plant uptake, biodegradation, and other abiotic losses. For pyrene, a four-ring PAH, volatilization and leaching were negligible due to its low solubility and low vapor pressure (Reilley et al., 1996; Binet et al., 2000). However, the amount of pyrene remaining in the soils as well as accumulated in plants could only account for a very small portion of the total pyrene input (Table I), suggesting that most of the added pyrene disappeared. The pyrene loss from the spiked soils could be due to biotransformation, biodegradation, and abiotic degradation. It was found that abiotic disappearance of pyrene was not a major pathway for the removal of pyrene in both vegetated and non-vegetated soils (Reilley et al., 1996). Our results also supported the argument that the abiotic loss of pyrene by volatilization and leaching was not significant. Microbial transformation and biodegradation were thus suggested as the principal processes for the successful removal and elimination of PAHs from the environment (Catallo and Portier, 1992; Wilson and Jones, 1993). Obviously, plants contributed to the dissipation of pyrene by the increase in microbial numbers. Binet et al. (2000) speculated that when a
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chemical stress was present in soil, a plant might respond by increasing or changing exudation to the rhizosphere, which modifies rhizospheric microflora composition or activity. In sum, enhanced removal of soil pyrene by white clover was the results of plant uptake, accumulation, and plant-promoted microbial degradation. As compared to the other pathways, the plant uptake and accumulation of pyrene was very low. This was consistent with the report that the contribution of plant off-take of PAHs to the total remediation enhancement in the presence of vegetation was less than 0.24% for pyrene and, by contrast, plant-promoted biodegradation was the predominant contribution to the remediation enhancement for soil pyrene (Gao and Zhu, 2004). CONCLUSIONS The present study explored the potential of phytoremediation with white clover to remove pyrene from the spiked soils. The growth of white clover in the pyrene-contaminated soils was comparable to the control with no significant differences, indicating that white clover was not affected by elevated pyrene levels in the soil. The removal of pyrene in the spiked soils planted with white clover was obviously higher than that from the unplanted soils. At the end of the 60-d experiment, the average removal ratio of pyrene in the spiked soils with white clover was 77%, which was 31% and 57% higher than those of Controls A and B, respectively. Residual pyrene declined quickly with sampling time. Both roots and shoots of white clover took up pyrene from the spiked soils and plant uptake generally increased with increasing initial soil concentrations. However, the plant-enhanced dissipation of soil pyrene may, predominantly, be the result of plant-promoted microbial degradation, whereas direct uptake and accumulation of pyrene by white clover was very small compared with the microbial degradation pathways. Bioconcentration factors of pyrene generally tended to decrease with the increase of their residual soil concentrations and root BCFs were much higher than shoots for the same treatment. The presence of white clover induced marked reduction of pyrene in the soils and the healthy growth of the plant on the variously spiked soils indicated that removal of pyrene from contaminated soils by using white clover is a feasible approach. REFERENCES Alkorta, I. and Garbisu, C. 2001. Phytoremediation of organic contaminants in soils. Bioresource Technol. 79: 273–276. Banks, M. K., Lee, E. and Schwab, A. P. 1999. Evaluation of dissipation mechanisms for enzo[a]pyrene in the rhizosphere of tall fescue. J. Environ. Qual. 28(1): 294–298. Binet, P., Portal, J. M. and Leyval, C. 2000. Dissipation of 3–6-ring polycyclic aromatic hydrocarbons in the rhizosphere of ryegrass. Soil Biol. Biochem. 32: 2 011–2 017. Cunningham, S. D. and Ow, D. W. 1996. Promises and prospects of phytoremediation. Plant Physiol. 110: 715–719. Chen, Y. C., Banks, M. K. and Schwab, A. P. 2003. Pyrene degradation in the rhizosphere of tall fescue (Festuca arundinacea) and switchgrass (Panicum virgatum L.). Environ. Sci. Technol. 37: 5 778–5 782. Fabbri, D., Vassura, I., Sun, C. G., Snape, C. E., McRae, C. and Fallick, A. E. 2004. Source apportionment of polycyclic aromatic hydrocarbons in a coastal lagoon by molecular and isotopic characterization. Mar. Chem. 84: 123–135. Freeman, D. J. and Cattell, F. C. R. 1990. Woodburning as a source of atmospheric polycyclic aromatic hydrocarbons. Environ. Sci. Technol. 24: 1 581–1 585. Fujikawa, K., Fort, F. L., Samejima, K. and Sakamoto, Y. 1993. Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test. Mutat. Res. 290: 175–182. Gao, Y. Z. and Zhu, L. Z. 2004. Plant uptake, accumulation and translocation of phenanthrene and pyrene in soils. Chemosphere. 55: 1 169–1 178. Glick, B. R. 2003. Phytoremediation: synergistic use of plants and bacteria to clean up the environment. Biotechnol. Adv. 21: 383–393. Haeseler, F., Blanchet, D., Druelle, V., Werner, P. and Vandecasteele, J. P. 1999. Ecotoxicological assessment of soils of former manufactured gas plant sites: Bioremediation potential and pollutant mobility. Environ. Sci. Technol. 33(24): 4 380–4 384. Joner, E. J. and Leyval, C. 2003. Rhizosphere gradients of polycyclic aromatic hydrocarbon (PAH) dissipation in two industrial soils and the impact of arbuscular mycorrhiza. Environ. Sci. Technol. 37: 2 371–2 375. Kipopoulou, A. M., Manoli, E. and Samara, C. 1999. Bioconcentration of polycyclic aromatic hydrocarbons in vegetables grown in an industrial area. Environ. Pollut. 106(3): 369–380. Liste, H. H. and Alexander, M. 2000. Plant-promoted pyrene degradation in soil. Chemosphere. 40: 7–10.
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Removal of Pyrene from Contaminated Soils by White Clover∗1 XU Sheng-You1,2,3 , CHEN Ying-Xu1,∗2 , LIN Kuang-Fei3 , CHEN Xin-Cai1 , LIN Qi1 , LI Feng1 and WANG Zhao-Wei1 1 Department of Environmental Engineering, Zhejiang University, Hangzhou 310029 (China). E-mail: shengyouxu22 @yahoo.com.cn 2 College of Life and Environment, Huangshan University, Huangshan 245041 (China) 3 School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237 (China)
(Received May 16, 2008; revised December 8, 2008)
ABSTRACT Phytoremediation has been used as an emerging technology for remediation of soil contamination with polycyclic aromatic hydrocarbons (PAHs), ubiquitous persistent environmental pollutants derived from natural and anthropogenic processes, in the last decade. In this study, a pot experiment was conducted to investigate the potential of phytoremediation of pyrene from spiked soils planted with white clover (Trifolium repens) in the greenhouse with a series of pyrene concentrations ranging from 4.22 to 365.38 mg kg−1 . The results showed that growth of white clover on pyrenecontaminated soils was not affected. The removal of pyrene from the spiked soils planted with white clover was obviously higher than that from the unplanted soils. At the end of the experiment (60 d), the average removal ratio of pyrene in the spiked soils with white clover was 77%, which was 31% and 57% higher than those of the controls with or without micobes, respectively. Both roots and shoots of white clover took up pyrene from the spiked soils and pyrene uptake increased with the soil pyrene concentration. However, the plant-enhanced dissipation of soil pyrene may be the result of plant-promoted microbial degradation and direct uptake and accumulation of pyrene by white clover were only a small part of the pyrene dissipation. Bioconcentration factors of pyrene (BCFs, ratio of pyrene, on a dry weight basis, in the plant to that in the soil) tended to decrease with increase in the residual soil pyrene concentration. Therefore, removal of pyrene in the contaminated soils was feasible using white clove. Key Words:
microbial degradation, phytoremediation, plant uptake and accumulation, pyrene, white clover
Citation: Xu, S. Y., Chen, Y. X., Lin, K. F., Chen, X. C., Lin, Q., Li, F. and Wang, Z. W. 2009. Removal of pyrene from contaminated soils by white clover. Pedosphere. 19(2): 265–272.
INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous persistent environmental pollutants derived from natural and anthropogenic processes (Freeman and Cattell, 1990; Binet et al., 2000; Fabbri et al., 2004; Xu et al., 2005). In recent years, anthropogenic practices such as industrial processing, petroleum spills, and incomplete combustion of fossil fuel lead to the accumulation of PAHs in the environment. This has caused increasing environmental concerns (Haeseler et al., 1999; Joner and Leyval, 2003; Xu et al., 2006a). Soils as reservoirs receive a large amount of PAHs and some of them are carcinogenic and/or mutagenic and may pose threats to human health (Fujikawa et al., 1993; Joner and Leyval, 2003; Yang et al., 2005). Pyrene, a four-ring PAH that has a low biodegradability and high persistence in the environment, is one of the PAHs on the United States Environmental Protection Agency (US EPA) priority pollutant list (Binet et al., 2000; Yan et al., 2004). ∗1 Project
supported by the National Natural Science Foundation of China (Nos. 40432004 and 20677015), the Postdoctoral Science Foundation of China (No. 20070420094), the Postdoctoral Science Foundation of Shanghai Municipality, China (No. 08R214116), the Science and Technology Commission of Shanghai Municipality, China (No. 0752nm025), and the National High-Tech Research and Development Program (No. 2007AA06Z331). ∗2 Corresponding author. E-mail: [email protected].
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Phytoremediation, the use of vegetation for in-situ treatment of contaminated soils and sediments, has been used as an emerging technology for remediation of PAH-contaminated soils in the last decade. It also appears to have great potential for the treatment of soils contaminated with residual levels of PAHs (Reilley et al., 1996; Banks et al., 1999; Noble et al., 2003; Glick, 2003). Plants provide a robust, solar-powered system that has little or no maintenance requirement. With their copious root systems, plants can scavenge large areas and volumes of soil PAHs. The rhizosphere soil has microbial population orders of magnitude greater than those of the bulk soil (Haeseler et al., 1999; Alkorta and Garbisu, 2001; Sung et al., 2001; Chen et al., 2003). Laboratory and pot experiments have demonstrated that plants can enhance dissipation of PAHs when compared to unplanted controls (Banks et al., 1999; Yoshitomi and Shann,, 2001; Joner and Leyval, 2003; Xu et al., 2006b). Phytoremediation field trials have resulted in accelerated reduction of PAHs and other petroleum hydrocarbons in the rhizosphere. It was found that pyrene in spiked soils disappeared rapidly in the first four weeks with tall fescue and alfalfa (Schwab and Banks, 1994; Joner and Leyval, 2003; Tao et al., 2004). Liste and Alexander (2000) reported that the degradation of pyrene could be promoted by nine different plant species and up to 74% pyrene disappeared from the vegetated soils compared to 40% or less from the unplanted soils within eight weeks. During the last decade, there has been considerable interest in understanding the uptake of PAHs by plants (Kipopoulou et al., 1999; Mattina et al., 2003; Vervaeke et al., 2003; Xu et al., 2006b). However, results usually are not in good agreement. Some found direct relationships between soil and plant PAH concentrations, while others found no such relationship (Liste and Alexander, 2000; Gao and Zhu, 2004; Xu et al., 2005). Translocation of pyrene from roots to shoots is still ambiguous and the impact of these processes has not been clearly elucidated. Therefore, information about pyrene distribution and concentration of plants is important in predicting the effectiveness of phytoremediation operations. The objective of our study was to investigate the relationship between pyrene accumulated in the inner plant tissue and its concentration in the environment, in order to advance our understanding of the phytoremediation mechanisms of PAHs. MATERIALS AND METHODS Pyrene was obtained from the Sigma Chemical Co. with a purity of 99.9%. A loam soil (pH 6.12 and 23.6 g kg−1 organic matter) with no detectable PAHs was used in this study. The soil samples collected in the upper horizon (0–20 cm) of an agricultural field near Hangzhou City, Zhejiang Province, China, were air-dried and passed through a 2-mm sieve. Part of the soil samples were spiked with pyrene of high purity in acetone. After acetone had evaporated, the spiked soils were mixed thoroughly with the uncontaminated soil. The final concentrations of pyrene in the treated soils were measured by high performance liquid chromatography (HPLC) as the initial concentrations of pyrene (on a dry weight basis) in the soils of Treatments T0, T1, T2, T3, and T4; they were 0, 4.32 ± 2.56, 24.02 ± 0.52, 169.06 ± 3.24, and 365.38 ± 12.37 mg kg−1 , respectively. The treated soils (500 g dry weight soil per pot) were then packed into 15 cm diameter plastic pots lined with a 0.1 mm sieve. These pots were kept in a greenhouse and equilibrated for 7 d at field moisture before introduction of plants. Control A consisted of unplanted spiked soils with microbes; Control B consists of unplanted spiked soils with 2 g kg−1 NaN3 added to inhibit the microbial activity. Seeds of white clover (Trifolium repens) were grown for 15 d in vermiculite before being transferred. Twenty seedlings were transplanted to each designated greenhouse pot about 5 d after emergence. The pots containing plants and controls in triplicate were placed randomly in a growth chamber maintained at 25 ◦ C in the daytime (16 h) and at 19 ◦ C during the night (8 h). All the pots were watered as needed and fertilized once a week with an inorganic salt solution. The seeding date was considered as day 0. Both planted and unplanted soils were destructively sampled after 60 d. At 60 d, white clover was harvested, rinsed with tap water and distilled water, and separated into shoot and root components. All samples (soils, shoots, and roots) were freeze-dried, bagged, and stored at 4 ◦ C before analysis. The
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dry weights of shoots and roots were determined. The procedure used to extract PAHs from the soils was a modification of the methods in Simonich and Hites (1994) and Kipopoulou et al. (1999). Soil samples to be analyzed were spiked with known amounts of pyrene. The freeze-dried soil samples 2 g each were transferred into an Erlenmeyer flask with 10 mL of dichloromethane and the flask was placed into an ultrasonic bath and sonicated for 1 h followed by centrifugation. Then 3 mL of supernatant was filtered through 2 g of silica gel column eluted with 10 mL 1:1 (v/v) hexane and dichloromethane. The solvent was then evaporated to dryness and the solid was re-dissolved in 2 mL methanol. Pyrene in roots and shoots was extracted in a similar way to that used for soil pyrene. The freezedried plant samples 100 mg each were transferred into an Erlenmeyer flask with an extraction solvent of hexane and acetone (3:2, v/v). The Erlenmeyer flask was placed into an ultrasonic bath and sonicated for 30 min. The solvent was then decanted, collected and replenished. This process was repeated three times. The solvents were evaporated to dryness and re-dissolved in 2 mL of methanol. All methanol extracts from soil and plant extractions were filtered prior to analysis with a 0.45-μm Teflon syringe filter and analyzed by high performance liquid chromatography (Agilent, USA) with an ultraviolet (UV) detector and an automatic injector, fitted with a 4.6 mm × 150 mm Agilent reverse phase C18 column using methanol and water (90:10, v/v) as the mobile phase at a flow rate of 1 mL min−1 . Pyrene was detected by the absorbance at 250 nm. Replicate analyses gave an error in the range of 5%–10%. All samples were subjected to strict quality control procedures. The reproducibility and recovery of the extraction for the spiked soil samples were 97% ± 9.5% (n = 7). The recovery of pyrene in the spiked plant samples was 90% ± 5% (n = 7). Statistical significance was evaluated using SPSS version 10.0 with one-way analysis of variance (ANOVA) and least significant difference (LSD) tests for comparison of treatment means with P < 0.05. RESULTS AND DISCUSSION Plant growth The root and shoot yields of white clover on a dry weight basis are shown in Fig. 1. The results indicated that the plants showed no signs of stress and produced a similar biomass between pots with all the spiked soils and the controls. The dry weights of the roots changed at different treatment levels but the changes were not significant (P > 0.05). There was no apparent change in the shoot weights between the treated and control pots. As compared with the control pots, the roots in the spiked pots were also not changed and formed a dense fibrous root system in all soils regardless of treatment.
Fig. 1
Shoot and root biomass of white clover after 60 d on variously spiked soils (on a dry weight basis).
Fig. 2
Residual soil pyrene concentrations with white clover planted and unplanted after 60 d.
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PAHs had been described as physiological toxic pollutants. However, very few studies had been undertaken to evaluate the toxic effects of PAHs on plant growth, especially terrestrial dicotyledons (Ren et al., 1996) and findings from limited studies showed that different plant species had different responses to PAH contamination. Chen et al. (2003) pointed out that no significant detrimental effects were caused by the presence of PAHs on seed germination and root radicle elongation of five prairie grasses even at high concentrations of PAHs (> 300 mg kg−1 ). The low solubility of PAHs and their adsorption on soil particles would limit the physiological toxicity of PAHs in rooted plants. In the present study, growth of white clover on pyrene-contaminated soils was comparable to the control without any pyrene addition and also no differences in growth were observed during the whole experiment (Fig. 1). The results demonstrated that PAH contamination would not produce any significant toxic effects to white clover growing in the spiked soils. Removal of pyrene from spiked soils by white clover In this experiment, Control B, the removal of pyrene from the unplanted spiked soils with NaN3 added indicated the abiotic removal of this compound, including chemical degradation and physical sorption. The loss of pyrene in the unplanted and planted spiked soils may be considered as the microbial degradation and plant-promoted removal, respectively. Fig. 2 shows the measured concentrations of pyrene remaining in the soils at the time of harvesting for white clover. The results showed that the residual pyrene concentrations in the contaminated soils were much lower than the initial values. Pyrene remaining in the vegetated soils was significantly lower than that in the non-vegetated soils. The removal ratios (R) of pyrene in the treated pots were calculated as: R = (C0 − Ct ) × 100/Ct , where C0 is the initial soil concentration of pyrene and Ct the residual concentration of the compound. At the end of the 60-d experiment, the average removal ratio of pyrene in the spiked soils (T1–T4) with white clover was 77%, which was 31% and 57% higher than those of the unplanted Control A with microbes and Control B without microbes, respectively (Fig. 3). This indicated that the removal of pyrene in the spiked soils was clearly promoted by white clover. Removal ratios with lower concentrations of pyrene were significantly higher than those of the corresponding treatments with higher concentrations of pyrene (Fig. 3). At the lower treatment concentrations of pyrene, the removal ratios of pyrene clearly increased with the increasing concentration of pyrene. However, after the concentration of pyrene was elevated to 25 mg kg−1 , the removal ratios of pyrene slightly decreased with increase in pyrene concentration in both planted and unplanted soils.
Fig. 3
Removal ratios of pyrene in the variously spiked soils after 60 d.
Pyrene accumulation in roots and shoots Concentrations of pyrene in white clover shoots and roots after 60 d under different pyrene levels in soil are shown in Fig. 4. With the increase of soil pyrene concentration, pyrene concentration of the roots
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significantly increased and more pyrene was transferred to the upper parts of the plant, which eventually accumulated in the shoots. The concentration of pyrene in the roots was higher than that of the shoots (Fig. 4). It was notable that the pyrene concentrations of roots in the unspiked soils were undetectable; however, the pyrene concentration of shoots in the unspiked soils was 1.02 mg kg−1 , implying that the uptake of pyrene by shoots from the ambient air, possibly originally volatilized from the soils, was an important pathway for the pyrene taken up by the above-ground parts of the plants.
Fig. 4
Concentrations of pyrene in roots and shoots of white clover.
Fig. 5
Root and shoot bioconcentration factors (BCF) of pyrene.
Relationships between pyrene in white clover and its residues in soil Bioconcentration factors (BCFs), the ratio of pyrene concentration in the root or shoot tissues of white clover to the residue in soil, are shown in Fig. 5. The average residual soil pyrene concentration after harvest of white clover was used for the estimation of BCFs. Although many uncertainties should be acknowledged, the results implied that pyrene found in white clover plants was just taken up from the corresponding pot in which they grew. BCFs of pyrene tended to decrease with the increase of the residual soil concentrations (Fig. 5). Root BCFs of pyrene were 1.0–1.8, showing that the root samples had a higher content of pyrene than the soils. Root BCFs of pyrene were much higher than shoot BCFs of pyrene in the treatments with the higher residual soil pyrene concentrations, which was contrary to the situation at the lower residual soil pyrene concentrations. Pathways of pyrene removal Removal of pyrene from the spiked soils was obviously promoted by white clover. At the end of the experiment, with white clover, the loss of pyrene in the spiked soils with the highest level of pyrene was 86% of the initial soil pyrene, which was 30% and 68% larger than the losses in soils of the controls A and B, respectively (Table I). Similar results showed that more PAH contaminants in soil were degraded in the presence of various plant species (Reilley et al., 1996; Sung et al., 2001; Xu et al., 2006a). The enhanced degradation of PAHs in the planted soil could be due to a higher density and greater activity of microorganisms in the rhizosphere than the non-vegetated soil as the root exudates and plant litter could enhance the bioavailability of the contaminant, providing more substrates for co-metabolic degradation and modifying the soil environment to become more suitable for microbial transformation (Cunningham and Ow, 1996; Reilley et al., 1996; Xu et al., 2006b). It was notable that pyrene concentrations of shoots in the controls were detectable, implying that shoot uptake of pyrene from the ambient air, possibly originally volatilized from the soils, was an important pathway for the pyrene taken up into the white clover above-ground parts. Gao and Zhu (2004) found that the shoots of 12 plant species grown on the unspiked control soils apparently accumulated pyrene from the air. Table I suggests that if there was no pyrene in the soil, there might still be some pyrene in the plant, which also proved the atmospheric
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contamination route. However, they also found that the concentrations of phenanthrene and pyrene in the air samplers located 5 or 15 cm above the surface of greenhouse pots showed no differences and volatilized concentrations of phenanthrene and pyrene were very low. TABLE I Mass balance of pyrene in the spiked soils with and without white clover planteda) Item
Treatment T0
Input Plant uptake Remained in soil Planted Control A Control B a) Control
T1
T2
T3
T4
0 0.06
2.16 1.03
mg 12.01 1.98
84.53 9.62
182.09 15.68
0 0 0
0.81 1.47 1.75
1.70 5.39 10.79
17.44 42.72 76.91
33.15 99.86 159.16
A = unplanted spiked soil with microbes; Control B = unplanted spike soil without microbes.
Our results suggest that the pyrene concentrations of shoots on the spiked soils were positively correlated with their respective initial soil pyrene concentrations (Fig. 4). On average 96% of pyrene in shoots was translocated from root uptake; on the average only 4% of pyrene in shoots was from the atmosphere (Table I), implying that the concentrations of pyrene in shoots grown in the spiked soils were much larger than the shoot uptake and accumulation from the atmosphere. Similar results have been reported (Schroll et al., 1994; Wang and Jone, 1994). Wang and Jones (1994) reported that root or shoot accumulation of pyrene in the contaminated soils was elevated with the increase of their soil concentrations. Schroll et al. (1994) showed that about 32%–96% of pyrene in shoots was translocated from root uptake. Bioconcentration factors (BCFs) of pyrene generally tended to decrease with the increase of their residual soil concentrations. BCFs of pyrene by roots were much higher than those by shoots for the same treatment, which indicated that the transfer of pyrene from roots to shoots was considerably restricted. Similar results have been achieved by Gao and Zhu (2004) who showed that BCFs of phenanthrene and pyrene in roots for plants grown in contaminated soils were 0.05–0.67 and 0.23–4.44, respectively. Samsoe-Petersen et al. (2002) also found that the BCFs of PAHs in potatos were 0.020–0.100 and in most cases decreased with increasing concentrations of PAHs in soils. Besides, the relationships between BCFs and the physico-chemical properties of pollutants, such as lipophilicity, water solubility, vapour pressure, and Henry’s law constant, have been examined by several investigators (Simonich and Hites, 1994; Wang and Jones, 1994). Although some good correlations were obtained, additional studies are needed to investigate these relationships more thoroughly. The fates of PAHs in spiked soils include volatilization, leaching, photo-degradation (contaminated at the surface), plant uptake, biodegradation, and other abiotic losses. For pyrene, a four-ring PAH, volatilization and leaching were negligible due to its low solubility and low vapor pressure (Reilley et al., 1996; Binet et al., 2000). However, the amount of pyrene remaining in the soils as well as accumulated in plants could only account for a very small portion of the total pyrene input (Table I), suggesting that most of the added pyrene disappeared. The pyrene loss from the spiked soils could be due to biotransformation, biodegradation, and abiotic degradation. It was found that abiotic disappearance of pyrene was not a major pathway for the removal of pyrene in both vegetated and non-vegetated soils (Reilley et al., 1996). Our results also supported the argument that the abiotic loss of pyrene by volatilization and leaching was not significant. Microbial transformation and biodegradation were thus suggested as the principal processes for the successful removal and elimination of PAHs from the environment (Catallo and Portier, 1992; Wilson and Jones, 1993). Obviously, plants contributed to the dissipation of pyrene by the increase in microbial numbers. Binet et al. (2000) speculated that when a
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chemical stress was present in soil, a plant might respond by increasing or changing exudation to the rhizosphere, which modifies rhizospheric microflora composition or activity. In sum, enhanced removal of soil pyrene by white clover was the results of plant uptake, accumulation, and plant-promoted microbial degradation. As compared to the other pathways, the plant uptake and accumulation of pyrene was very low. This was consistent with the report that the contribution of plant off-take of PAHs to the total remediation enhancement in the presence of vegetation was less than 0.24% for pyrene and, by contrast, plant-promoted biodegradation was the predominant contribution to the remediation enhancement for soil pyrene (Gao and Zhu, 2004). CONCLUSIONS The present study explored the potential of phytoremediation with white clover to remove pyrene from the spiked soils. The growth of white clover in the pyrene-contaminated soils was comparable to the control with no significant differences, indicating that white clover was not affected by elevated pyrene levels in the soil. The removal of pyrene in the spiked soils planted with white clover was obviously higher than that from the unplanted soils. At the end of the 60-d experiment, the average removal ratio of pyrene in the spiked soils with white clover was 77%, which was 31% and 57% higher than those of Controls A and B, respectively. Residual pyrene declined quickly with sampling time. Both roots and shoots of white clover took up pyrene from the spiked soils and plant uptake generally increased with increasing initial soil concentrations. However, the plant-enhanced dissipation of soil pyrene may, predominantly, be the result of plant-promoted microbial degradation, whereas direct uptake and accumulation of pyrene by white clover was very small compared with the microbial degradation pathways. Bioconcentration factors of pyrene generally tended to decrease with the increase of their residual soil concentrations and root BCFs were much higher than shoots for the same treatment. The presence of white clover induced marked reduction of pyrene in the soils and the healthy growth of the plant on the variously spiked soils indicated that removal of pyrene from contaminated soils by using white clover is a feasible approach. REFERENCES Alkorta, I. and Garbisu, C. 2001. Phytoremediation of organic contaminants in soils. Bioresource Technol. 79: 273–276. Banks, M. K., Lee, E. and Schwab, A. P. 1999. Evaluation of dissipation mechanisms for enzo[a]pyrene in the rhizosphere of tall fescue. J. Environ. Qual. 28(1): 294–298. Binet, P., Portal, J. M. and Leyval, C. 2000. Dissipation of 3–6-ring polycyclic aromatic hydrocarbons in the rhizosphere of ryegrass. Soil Biol. Biochem. 32: 2 011–2 017. Cunningham, S. D. and Ow, D. W. 1996. Promises and prospects of phytoremediation. Plant Physiol. 110: 715–719. Chen, Y. C., Banks, M. K. and Schwab, A. P. 2003. Pyrene degradation in the rhizosphere of tall fescue (Festuca arundinacea) and switchgrass (Panicum virgatum L.). Environ. Sci. Technol. 37: 5 778–5 782. Fabbri, D., Vassura, I., Sun, C. G., Snape, C. E., McRae, C. and Fallick, A. E. 2004. Source apportionment of polycyclic aromatic hydrocarbons in a coastal lagoon by molecular and isotopic characterization. Mar. Chem. 84: 123–135. Freeman, D. J. and Cattell, F. C. R. 1990. Woodburning as a source of atmospheric polycyclic aromatic hydrocarbons. Environ. Sci. Technol. 24: 1 581–1 585. Fujikawa, K., Fort, F. L., Samejima, K. and Sakamoto, Y. 1993. Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test. Mutat. Res. 290: 175–182. Gao, Y. Z. and Zhu, L. Z. 2004. Plant uptake, accumulation and translocation of phenanthrene and pyrene in soils. Chemosphere. 55: 1 169–1 178. Glick, B. R. 2003. Phytoremediation: synergistic use of plants and bacteria to clean up the environment. Biotechnol. Adv. 21: 383–393. Haeseler, F., Blanchet, D., Druelle, V., Werner, P. and Vandecasteele, J. P. 1999. Ecotoxicological assessment of soils of former manufactured gas plant sites: Bioremediation potential and pollutant mobility. Environ. Sci. Technol. 33(24): 4 380–4 384. Joner, E. J. and Leyval, C. 2003. Rhizosphere gradients of polycyclic aromatic hydrocarbon (PAH) dissipation in two industrial soils and the impact of arbuscular mycorrhiza. Environ. Sci. Technol. 37: 2 371–2 375. Kipopoulou, A. M., Manoli, E. and Samara, C. 1999. Bioconcentration of polycyclic aromatic hydrocarbons in vegetables grown in an industrial area. Environ. Pollut. 106(3): 369–380. Liste, H. H. and Alexander, M. 2000. Plant-promoted pyrene degradation in soil. Chemosphere. 40: 7–10.
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