Degradation of phenanthrene and pyrene in rhizosphere of grasses and legumes

Available online at www.sciencedirect.com

Journal of Hazardous Materials 153 (2008) 892–898

Degradation of phenanthrene and pyrene in rhizosphere of grasses and legumes Sang-Hwan Lee a , Won-Seok Lee b , Chang-Ho Lee c , Jeong-Gyu Kim c,∗ a

Office of Environmental Geology, Korea Rural Community & Agriculture Corp., Uiwang 430-600, Republic of Korea b National Institute of Environmental Research, Incheon 404-708, Republic of Korea c Division of Environmental Science & Ecological Engineering, Korea Univisity, Seoul 136-701, Republic of Korea Received 13 March 2007; accepted 13 September 2007 Available online 16 September 2007

Abstract Phytoremediation is an emerging technology for the remediation of organic soil pollutants such as phenanthrene and pyrene (polycyclic aromatic hydrocarbons, PAHs). The PAH degradation ability of four native Korean plant species (Panicum bisulcatum, Echinogalus crus-galli, Astragalus membranaceus, and Aeschynomene indica) was compared in the greenhouse. During the 80-day experiment, soil samples were collected and analyzed periodically to determine the residual PAH content and microbial activity. More PAHs were dissipated in planted soil (i.e., with a rhizosphere) than in unplanted soil, and there were more obvious effects of plants on pyrene dissipation than on phenanthrene dissipation. After 80 days, >99 and 77–94% of phenanthrene and pyrene, respectively, had been degraded in planted soil, whereas 99% and 69% had been degraded in unplanted soil. This enhanced dissipation of PAHs in planted soils might be derived from increased microbial activity and plant-released enzymes. During the experimental period, a relatively large amount of phenolic compounds, high microbial activity, and high peroxidase activity were detected in planted soils. © 2007 Elsevier B.V. All rights reserved. Keywords: Microbial activity; Phytoremediation; Polycyclic aromatic hydrocarbons; Rhizosphere; Soil enzymes

1. Introduction Phytoremediation, i.e., the use of plants and their associated microorganisms for restoring and/or recovering polluted soils, is a new field of environmental remediation that has entered the active development phase during last decade [1]. Phytoremediation is likely to be effective based on studies documenting an increase in the disappearance of xenobiotics from planted soils [2–6]. However, phytoremediation efficacy varies greatly among plant species [7,8] and depends on soil and environmental conditions [9] and the physico-chemical nature of the contaminant [10,11]. Plants may contribute to the dissipation of organic contaminants through an increase in the number of microbes, improvement of physical and chemical soil conditions, increased

∗

Corresponding author. Tel.: +82 2 3290 3024; fax: +82 2 921 7628. E-mail address: [email protected] (J.-G. Kim).

0304-3894/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2007.09.041

humification, and adsorption of pollutants in the rhizosphere, but the impact of each process has not been clearly elucidated [11,12]. Several studies investigated the effect of plant–microbe interactions on the degradation of organic contaminants, based on the hypothesis that root exudates increase the rhizosphere microbial community. Walton et al. [13] speculated that when chemical stress occurs in soil, a plant may respond by increasing or changing its exudation to the rhizosphere, which then modifies the microfloral composition or activity of the rhizosphere. As a result, the microbial community might increase the transformation rates of toxicants. Because the release of compounds or enzymes from roots is presumed to be associated with rhizosphere biodegradation and plant types vary in the nature and quantity of compounds released, it follows that the plant species used could be a significant factor influencing the efficacy of phytoremediation. Plants (and plant types) vary widely with respect to root parameters such as morphology, root exudation [14], fine root turnover [15], root decomposition [16], and associated microbial communities [17]. If the dominant mechanism of

S.-H. Lee et al. / Journal of Hazardous Materials 153 (2008) 892–898

polycyclic aromatic hydrocarbon (PAH) dissipation in planted soil is associated with rhizosphere microbial activity, then the remediation potential should vary across plant species and ecotypes. For instance, a grass had the best and clover the worse PAH dissipation performance in one soil type [18], whereas grass inhibited PAH mineralization in another soil type [19]. Thus, the choice of plant species is critically important to the success of phytoremediation. To achieve maximum PAH reduction and successfully establish stable vegetation cover, various criteria must be considered. Plants should be chosen carefully so that they provide a maximum root surface area [2]. They should be native to the area in which they are being used and should be tolerant to local soil conditions. Because cost is an important factor, plants that require little attention (e.g., fertilizers) are preferable. Due to the usually poor nutrient availability in contaminated soils [20], much research has been conducted on the use of legumes, which fix nitrogen [6,21]. We screened four native Korean plant species for their tolerance to PAH contamination, as well as for the potential of their rhizospheres to enhance the degradation of PAHs. During the experiments, plant growth parameters, the number of microorganisms, root exudates (phenolic compounds), and extracellular enzymes (dehydrogenase and peroxidase) were monitored to select the most promising plant species for phytoremediation.

893

2.2. Greenhouse study The soil was passed through a 4-mm sieve. A mixture of two types of PAH was prepared using pure PAHs (phenanthrene and pyrene >96% purity; Aldrich). The soil was spread in a thin layer, and PAHs (dissolved in acetone) were added to the soil. The soil was mixed thoroughly and then left for 7 days to ensure the evaporation of the acetone. Treated soils (1000 g dry weight soil per pot) were then packed into greenhouse pots (Ø 15 cm × H 15 cm) that were lined with sand and a 0.1-mm screen at the bottom to avoid soil loss and allowed to equilibrate in the greenhouse for 7 days at field moisture conditions before the introduction of plants. Seeds of each plant were germinated in moist sand, and seedlings were transplanted to greenhouse pots 10–20 days after emergence. For comparison, seedlings were also transplanted into control soil (i.e., PAH-free soil). Four replicates of each treatment were prepared, and the pots were completely randomized in the greenhouse. Seedling transplantation was considered the start of the experiment. The water content was checked and adjusted regularly with sterilized water to maintain about 70% of the water holding capacity. The greenhouse was maintained at 20–25 ◦ C during the day and 10–15 ◦ C at night. During the experiment, soil samples were destructively sampled every 20 days and analyzed for PAH content and microbial activity. 2.3. Analytical methods

2. Materials and methods 2.1. Soils and plants The soil used for this experiment was obtained from Dukso, Korea. This soil belongs to the Sachon series, has been classified as a grayish brown soil, and has no previous history of exposure to PAHs or other contaminants. The particle size distribution (22.9% sand, 55.9% silt, and 21.2% clay) identified the soil as a silty clay loam. The organic carbon content was 1.97% and the pH was 6.7. The cation exchange capacity (CEC) was 15.2 cmol kg−1 . The nutrient levels were 28.0 mg kg−1 NO3 -N, 22.8 mg kg−1 NH4 -N and 172 mg kg−1 available phosphorus. Two grasses (Panicum bisulcatum Thunb. and Echinochloa crus-galli) and two legumes (Astragalus membranaceus and Aeschynomene indica) were used for the phytoremediation experiment. These plants are easy to establish and manage, grow under a wide variety of soil conditions, and are widely distributed in Korea. Grass species have frequently have been suggested as effective plants for treating hydrocarbon-contaminated soils due to their fibrous root systems, which have a large surface area per unit volume near the soil surface [2,22]. A larger root surface area should stimulate higher microbial populations, possibly enhancing bioremediation [2]. A possible advantage of using legumes is their close association with nitrogen-fixing microorganisms, which make them especially promising due to their nitrogen independence; this is important for hydrocarboncontaminated soils typically characterized by high C/N ratios [23].

2.3.1. Biological parameters 2.3.1.1. Plant biomass. The dry biomass produced by plants at each sampling time was measured separately for shoots and roots. The dry weight of the plant was determined after oven drying at 105 ◦ C for 24 h. 2.3.1.2. Number of microorganisms. To enumerate the viable microbial population, aqueous extracts of 1-g soil samples were serially diluted and spread on nutrient agar. Plates were incubated for 3–5 days at 28 ◦ C prior to counting the numbers of colony forming units (CFU). 2.3.1.3. Water-soluble phenols. Water-soluble phenols were quantified colorimetrically [24]. Soils were extracted with 10 mL of distilled water for 4 h with shaking, and the samples were then centrifuged at 3000 × g for 15 min. A 10-mL aliquot of extract or standard was placed in a 200 mm × 25-mm test tube, and then 3 mL of Na2 CO3 solution and 1 mL of Folin-Ciocalteau reagent were added. The solution was mixed well and allowed to stand for 1 h at room temperature. Light absorbance by the blue complex was read at 750 nm. Vanillic acid was used as the standard, and the amount of phenolic compounds is expressed as vanillic acid equivalents. 2.3.1.4. Dehydrogenase. Dehydrogenase activity was determined by the reduction of 2,3,5-triphenylterazolium chloride (TTC) to triphenyl formazane (TPF) [25]. A 1-mL aliquot of TTC solution (1%) and 1.5 mL of distilled water were added to 10 g of soil mixed with 0.1 g of CaCO3 . After 24 h

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of incubation at 25 ◦ C, the reaction product (i.e., TPF) was extracted with methanol, and the absorbance was measured at 485 nm.

Table 1 Biomass (g dry weight plant−1 ) of shoots and roots as a function of time for different plant species Plant species

2.3.1.5. Peroxidase. Soil peroxidase (EC 1.11.1.7) was extracted using 0.05 M phosphate buffer (pH 6.0). The peroxidase assay was conducted as follows: 0.3 mL of 0.06% H2 O2 in 0.05 M phosphate buffer (pH 6.0), 0.05 mL of 0.5% o-dianisidine in methanol, and 1 mL of soil extract were combined. The ingredients were mixed and the increase in optical density was recorded continuously at 460 nm. As an enzyme standard, horseradish peroxidase Type II, 192 purpurogallin units/mg, was used [26]. 2.3.2. PAH analysis Soil samples were stored at 4 ◦ C until extraction. Soil samples were homogenized and then 10 g (fresh weight) of soil was placed in a 50-mL solvent-resistant centrifuge tube and shaken with 25 mL of acetone for 24 h on a horizontal shaker (200 rpm) at room temperature. The tubes were then centrifuged, decanted, and the supernatant was stored in glass vials at 4 ◦ C until analysis. Extracts were quantified using a Hewlett Packard 5890A gas chromatograph equipped with a DB-5 capillary column (30 m length, 0.25 mm i.d., 0.25 ␮m film thickness). The oven temperature program for PAH analysis went from 80 ◦ C (initial time 2 min) to 300 ◦ C at a rate of 10 ◦ C min−1 , then held at 300 ◦ C for 6 min. Helium was used as carrier gas at a flow rate of 1.5 mL min−1 . 2.4. Statistical analyses All values presented for the chemical and biological analyses of soil are the means of four replicates. The means were compared using least significant differences calculated at a significance level of α = 0.05 using SAS (SAS Institute, 1989). Correlations between the parameters were analyzed using Pearson product moment correlations. 3. Results and discussion 3.1. Plant biomass Plant biomass was measured at each sample time to verify that vegetation could survive in the contaminated soils and to monitor plant development (Table 1). The different plant species displayed different responses to the presence of PAHs in the soil. Overall, legumes were better than grasses at withstanding the adverse effects of the contaminants. In most cases, the root and shoot yields of all plants were consistently lower in PAHtreated soils than in control soils during the initial periods of the experiment; the greatest reduction in biomass was observed in P. bisulcatum, with approximately 70% lower biomass production. In A. indica, biomass was higher in PAH-treated soil than in control soil. The reduction in plant biomass that was observed for most species during the initial periods might result from the toxicity of PAHs. Plants, especially seedlings, are sensitive to low-molecular-weight volatile hydrocarbons, which

Grasses

Panicum bisulcatum

Echinochlora crus-galli

Legumes

Astragalus membranaceus

Aeschynomene indica

Time (days)

Shoot

Root

PAH+

PAH−

PAH+

PAH−

20 40 60 80 20 40 60 80

1.63b 3.34b 3.20b 2.51b 3.32b 8.81b 7.93b 7.35b

5.33a 10.22a 9.77a 6.95a 6.37a 11.80a 12.21a 9.12a

0.13b 0.33b 0.34b 0.25b 0.59b 1.56b 0.70b 2.15a

0.28a 0.44a 0.74a 0.70a 1.09a 2.12a 1.07a 2.49a

20 40 60 80 20 40 60 80

1.00b 1.90a 6.41a 13.60a 1.51a 4.62a 8.72a 9.15a

2.04a 1.53a 6.37a 11.99b 1.17a 3.52b 9.80a 9.97a

0.38b 0.68a 2.21a 5.27a 0.62a 3.96a 5.68a 8.60a

0.84a 0.48a 2.29a 4.65a 0.49a 3.2b 5.12a 9.2a

Value for a given time period followed by the same letter are not significantly different (P < 0.05).

are soluble in hydrophobic plant materials and can penetrate cell membranes [27,28]. Reilley et al. [29] suggested indirect adverse effects of PAHs; PAHs might reduce the ability of contaminated soil to provide water and nutrients to plants, leading to a decline in biomass production. Despite reduced biomass in some species, the plant species we tested did not exhibit signs of stress or toxicity, and it appears that vegetation establishment in PAH-contaminated soil is feasible with these plants. 3.2. Biological activity in soil The numbers of soil microbes fluctuated during the experiment (Table 2). In PAH-treated soils, the growth pattern was characterized by a substantial increase during the initial period, followed by a decline in the microbial population. The maximum number occurred in soil of E. crus-galli at 20 days, but there were no significant differences in the numbers of microbes between planted and unplanted soil. The initial marked increase in the number of microbes coincided with the rapid decrease in PAHs during the initial period of the experiment, apparently due to the consumption of bioavailable substrates. In both planted and unplanted soils, the number of microbes declined at the end of the experiment to about 3 × 107 cfu g−1 soil. Water-soluble phenolic compounds were monitored to evaluate the influence of plant species and growth stage on the amount of phenolic compounds to understand the significance of these factors in the bioremediation of PAHs. Plants that release high concentrations of phenol into the rhizosphere may selectively foster the growth of PCB-degrading bacteria [30]. Liste and Alexander [31] suggested that the exudation capacity of phenolic compounds be used as a screening method for the use of plant species in phytoremediation. Water-soluble phenolic com-

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Table 2 Changes in the number of microorganisms (cfu g−1 soil × 107 ) in the PAH-treated (PAH+) and control (PAH−) soils Plant species

Panicum bisulcatum Echinochlora crus-galli Astragalus membranaceus Aeschynomene indica Nonplanted

20 days

40 days

60 days

80 days

PAH+

PAH−

PAH+

PAH−

PAH+

PAH−

PAH+

PAH−

11.40Ba 22.10Aa 18.81ABa 21.73Aa 14.02AB

11.73Aa 11.49Ab 6.32Bb 10.46ABb

3.42Ca 5.11Ba 2.90Ca 6.28Ab 2.67C

2.20ABb 3.25Ab 1.47Bb 2.79ABb

3.16Ba 3.40Ba 3.49Ba 4.01Aa 1.89C

2.92Ab 2.56ABb 2.89Ab 2.26ABb

3.21Ca 5.76Aa 3.71Ba 5.50Aa 3.04C

2.85Cb 4.41Ab 3.59Bb 3.13Cb

Uppercase letters indicate significant differences among species (within columns); lowercase letters indicate significant differences between PAH+ and PAH− (within rows) at each sample time (P < 0.05).

pounds in PAH-treated soils peaked after 40–60 days of plant growth (Table 3). More phenolic compounds were observed in the rhizospheres of E. crus-galli and A. membranaceus than of the other species. According to Liste and Alexander [31], these two plant species might be useful for the phytoremediation of soils polluted with aromatic compounds. In PAH-treated soil, phenolic compounds derive from two sources. One source is the degradative intermediates of PAHs and the other is plant root exudates. This was confirmed by the detection of a significant amount of water-soluble phenols in unplanted, but PAH-treated, soil. Cerniglia [32] and Kraus et al. [33] also reported that a large portion of phenolic compounds found in PAH-contaminated soils was the result of the aromatic ring cleavage of PAHs. Water-soluble phenol concentration showed a wave in PAH-free planted control soil; only plants affect the rhizosphere phenolic content in this type of soil. The complex rise and fall in phenolic compound concentrations observed here may be related to plant growth conditions, as well as to microbial dynamics. Root turnover may have been a major contributor to the increase in rhizosphere phenolic compounds. The black, decaying appearance of roots at 60–80 days, compared to the bright yellow roots observed at earlier stages, strongly suggests that root turnover at the end of the growing season may release large quantities of cellular phenolics into the soil solution. This, therefore, may be the primary contributor to the increased amount of complex phenols in the rhizosphere [34]. Root turnover and decay might result in selective growth and long-term survival of certain soil microbes, a situation conducive to the enhancement of rhizosphere-facilitated degradation of recalcitrant pollutants such as PAHs [30]. The investigation of mature plants over long periods will be especially valuable in developing phytoremediation technology for

recalcitrant pollutants, where plant phenolics serve as cometabolites for the degradative properties of selected microbes [35]. Microbial extracellular soil enzyme activity was monitored during the phytoremediation experiment in soil contaminated with PAHs and in control soil to determine how PAHs and plants affect microbial activity. Dehydrogenase and peroxidase activities were much higher in planted than in unplanted soils in the PAH treatment (Tables 4 and 5). Overall microbial activity, as determined by dehydrogenase activity, was significantly depressed by the PAH treatment. The dehydrogenase activity in planted soils increased with time, whereas it remained relatively constant in unplanted soil. From the dehydrogenase activity data, the greatest stimulation of PAH-degrading microbes is expected in soils planted with E. crus-galli and A. membranaceus. This matches well the PAH degradation data. Dehydrogenase activity assays in soil have often been used to obtain correlative information on the biological activity of microbial populations in soil, i.e., as an index of total microbial activity [3]. Strong correlations between hydrocarbon removal and dehydrogenase activity are frequently observed [36,37]. The presence of plant roots had a significant effect on the amount of extractable peroxidase in soil. Peroxidase activity was consistently higher in planted soils than in unplanted soils (Table 5). In unplanted soil, minimal activity was detected in the range of 0.39–0.83 peroxidase units. Peroxidase activity increased continuously with time in planted soil, and the maximum activity was observed in the A. membranaceus rhizosphere at 80 days. Peroxidase activity might be stimulated by PAH treatment. Enhanced peroxidase activity in the rhizosphere may counteract the adverse effects of PAHs on plants and contribute to enhanced dissipation of PAHs [38]. Root contact with toxic

Table 3 Changes in the water-soluble concentration of phenolic compounds (␮g vanillic acid g−1 soil) in the PAH− treated (PAH+) and control (PAH−) soils Plant species

Panicum bisulcatum Echinochlora crus-galli Astragalus membranaceus Aeschynomene indica Nonplanted

20 days

40 days

60 days

80 days

PAH+

PAH−

PAH+

PAH−

PAH+

PAH−

PAH+

PAH−

1.63Ba 4.86Aa 2.84ABa 4.22Aa 2.73AB

1.07Bb 1.04Bb 1.64ABb 1.96Ab

3.23Ba 4.39ABa 2.943Ba 5.33Aa 2.78B

2.26ABb 2.38Ab 1.43Bb 2.07Ab

3.33BCa 5.16Aa 2.92Ca 4.92ABa 2.89C

2.92Ab 2.56ABb 2.89Ab 2.26Bb

4.30ABCa 4.93ABa 3.94BCa 5.20Aa 2.15C

3.29Cb 4.24Aa 3.59Bb 3.13Cb

Uppercase letters indicate significant differences among species (within columns); lowercase letters indicate significant differences between PAH+ and PAH− (within rows) at each sample time (P < 0.05).

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Table 4 Changes in the dehydrogenase activity (mg TPF g−1 soil) in the PAH− treated (PAH+) and control (PAH−) soils Plant species

Panicum bisulcatum Echinochlora crus-galli Astragalus membranaceus Aeschynomene indica Nonplanted

20 days

40 days

60 days

80 days

PAH+

PAH−

PAH+

PAH−

PAH+

PAH−

PAH+

PAH−

1.59Bb 1.82Bb 2.66Ab 2.02ABb 2.33AB

21.48Ba 22.97Ba 15.91Ca 40.53Aa

2.39BCb 2.73ABb 1.64Cb 3.50Ab 1.68C

35.02Aa 33.65Aa 16.86Ba 28.46Aa

2.81Bb 5.08Ab 2.53Bb 4.18Ab 2.29B

21.11ABa 24.87Aa 14.38Ba 26.80Aa

3.22BCb 5.28Ab 4.75ABb 3.74ABCb 2.22C

16.69Aa 17.54Aa 15.00Aa 17.09Aa

Uppercase letters indicate significant differences among species (within columns); lowercase letters indicate significant differences between PAH+ and PAH− (within rows) at each sample time (P < 0.05).

chemicals like PAHs also induces peroxidase activity, which may have an intracellular function as part of a defense mechanism and/or a direct effect on the degradation of aromatics [33]. The expression of perodixase is stimulated by anthracene treatment [39]. Although there is no direct evidence that plantderived peroxidase is related to PAH degradation, initial ring cleavage is accomplished by oxygenase enzymes such as peroxidase [40]. Oxidoreductive enzymes like peroxidases are capable of oxidizing aromatic compounds to free radicals or quinones and benzoquinone imines [41]. These oxidation products can couple with each other, resulting in the formation of waterinsoluble oligomers. The biotransformation to corresponding quinones, which are more available and can be easily degraded by microorganisms, has been documented, and this process could be associated with peroxidase activity [32,42]. Oxidative coupling reactions mediated by peroxidase can be exploited for the removal of phenols, anilines, and other aromatic compounds from aqueous solutions; this reaction has also been proposed for environmental decontamination [33,38,41]. 3.3. Dissipation of phenanthrene and pyrene in soil Although the soil used in this experiment was agricultural topsoil without any known contamination history, the indigenous microorganisms exhibited a remarkable capacity to degrade 3–4ring PAHs. The overall extent of PAH loss from soils was clearly compound-dependent; phenanthrene dissipated more rapidly and completely than pyrene (Table 5), which is in agreement with many other studies, suggesting that high-molecular-weight PAHs are more resistant to microbial attack than low-molecularweight PAHs [32,43].

The concentrations of phenanthrene decreased from 87.56 mg kg−1 to less than 20 mg kg−1 during the first 20 days in all treatments. As early as 40 days, the dissipation rate was not significantly faster in planted soil than unplanted soil, even though soil planted with some species had higher concentrations of phenanthrene. After 80 days, there were significant differences between planted and unplanted soils. At the end of experiment, the lowest concentration of phenanthrene was in soil with E. crus-galli, but it did not differ significantly from that in soils with other plants (Table 6). Pyrene was more slowly degraded than phenanthrene, but the effects of plants on pyrene degradation were more obvious than on phenanthrene degradation. Only 13–42% of the initial mass was reduced during the first 20 days. After 80 days, pyrene was degraded to less than 80% of the initial concentration in most planted soil, whereas approximately 70% of pyrene was degraded in unplanted soil. The highest degradation of pyrene was recorded in soil of A. membranaceus (Table 6). The enhanced effect of the rhizosphere was more evident after 60–80 days of growth, when the plants and their root systems were fully developed. Therefore, the very rapid decline in PAH concentrations during the early sampling periods was due to dissipation without much benefit from rhizosphere reactions. Previous experiments also reported very small differences between planted and unplanted soil in PAH removal if the plants were small because the root systems were not very active, and no major benefit was obtained from rhizosphere interactions [29,44]. The loss of PAHs from soil could be due to biotransformation, biodegradation, plant uptake, or abiotic dissipation, including leaching and volatilization [2,29,45]. Abiotic losses by leaching were insignificant because the water content of the soil was

Table 5 Changes in the peroxidase activity (␮mol H2 O2 g−1 soil × 104 ) in the PAH-treated (PAH+) and control (PAH−) soils Plant species

Panicum bisulcatum Echinochlora crus-galli Astragalus membranaceus Aeschynomene indica Nonplanted

20 days

40 days

60 days

80 days

PAH+

PAH−

PAH+

PAH−

PAH+

PAH−

PAH+

PAH−

0.49Ba 1.19Aa 1.05ABa 1.11Ab 0.83AB

0.76Aa 1.09Aa 0.47Ba 1.08Aa

3.89Ba 5.39ABa 6.91Ab 6.02Aa 0.66C

4.38ba 5.32ba 4.84ba 6.93Aa

8.25Ba 10.14Aa 6.61Bb 6.92Bb 0.43C

5.27Cb 10.02Ba 10.06Ba 17.96Aa

7.17Ca 11.40Ba 20.25Aa 7.63Cb 0.39D

5.00Bb 5.52Bb 15.52Bb 12.00Aa

Uppercase letters indicate significant differences among species (within columns); lowercase letters indicate significant differences between PAH+ and PAH− (within rows) at each sample time (P < 0.05).

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Table 6 Concentrations of phenanthrene and pyrene remaining in soil with different plant species as a function of time Plant species

20 days

Panicum bisulcatum Echinochlora crus-galli Astragalus membranaceus Aeschynomene indica Nonplanted

40 days

60 days

80 days

PHN

PYR

PHN

PYR

PHN

PYR

PHN

PYR

9.53B 7.19BC 14.71A 7.49BC 6.46C

83.53A 71.34BC 67.76BC 65.65C 75.25AB

4.78AB 4.05AB 4.68AB 3.78BC 6.56A

41.78BC 53.46B 34.16C 69.97A 70.14A

1.78AB 2.18AB 1.55B 2.11AB 2.46A

43.25B 42.27B 24.12C 60.69A 53.97A

0.58B 0.47B 0.58B 0.72B 1.19A

19.18BC 18.31C 6.71D 23.00B 31.29A

Initial concentration (at time zero) of phenanthrene and pyrene was 87.56 and 98.62 mg kg−1 , respectively. Letters indicate significant differences among species (within columns) for each compound at each sample time (P < 0.05).

maintained at about 70% of the water holding capacity; thus, no leachate was produced during the experiment. The non-ionic, non-polar structure of PAH compounds leads to its partitioning out of the polar water phase and onto hydrophobic surfaces in the soil matrix. Lipophilic soil organic matter acts as an adsorbent and immobilizes hydrophobic PAHs [46]. The losses of phenanthrene and pyrene via volatilization from soil are also unlikely to occur due to their low vapor pressure [29]: 10−1.00 and 10−2.05 Lat mmol−1 for phenanthrene and pyrene, respectively [47]. Although the amount of PAHs in plant tissues was not determined, the loss of PAHs from soil by plant uptake/accumulation can be assumed to be negligible, as reported in previous studies [29,44]. Dietz and Schnoor [48] demonstrated that hydrophobic compounds with log Kow > 4 are not readily taken up by plants through transpiration due to their hydrophobicity; log Kow for phenanthrene and pyrene is 4.17 and 5.13, respectively [47]. Our results suggest that the enhancement of PAH disappearance might be caused by increased rhizosphere microbe activity compared to unplanted soil due to phenomena such as increased microbial activity and degradation mediated by plant-secreted enzymes in the root zone. The increased dissipation of PAHs in the rhizosphere may also be due to the decreased extractability of PAHs with the formation of bound residues. The rhizosphere could stabilize pollutants by polymerization reactions such as humification [13,49]. The decreased extractability of the compound or increased degradation in the rhizosphere attributed to the oxidation of PAHs to quinone could be associated with peroxidase or lactase activity [29]. Table 7 Correlation matrix of PAHs decontamination and soil biological parameters Parametera

PHN PYR MN WSP DHN

The biological parameters measured during the experiment were significantly correlated with PAH concentration; the number of microbes (P < 0.01) and the water-soluble phenol content (P < 0.05) were positively correlated with PAH concentration, whereas dehydrogenase and peroxidase activities were strongly negatively correlated with PAH concentration (P < 0.01; Table 7). 4. Conclusions We examined the phytoremediation potential of four species of plants in PAH-contaminated soil. There were quite clear interspecific differences in response to the PAH treatment, although the species have ecologically similar characteristics. Our results suggest that the enhancement of PAH disappearance is caused by an increase in the rhizosphere microbe community and microbial activity compared to that in unplanted soil. Enhanced dissipation in planted versus unplanted soil was 0.47–0.72 mg for phenanthrene and 8.29–24.58 mg for pyrene. PAHs are considered to be serious health risks at very low concentrations; thus, even a small dissipation enhancement in the presence of plants is potentially important. The influence of plants on the degradation of contaminants probably results from unspecific influences of the vegetation on soil environmental conditions. Our results show that E. crus-galli and A. membranaceus are suitable candidates for the phytoremediation of soils contaminated with recalcitrant pollutants because they are capable of robust growth and efficient extracellular enzyme production in soil. Further laboratory and field investigations should elucidate the complex processes at the soil–plant interface. Acknowledgement

Correlation coefficients PYR

MN

WSP

DHN

PER

0.714**

0.657**

0.360*

−0.533**

0.572**

0.301* 0.203

−0.696** −0.765** −0.557** −0.433** 0.694**

−0.467** −0.345** −0.384**

Sixty-four samples were used in the analysis, i.e., four replicates for each treatment and sample time. a PHN, phenanthrene; PYR, pyrene; MN, number of microbes; WSP, watersoluble phenol; DHN, dehydrogenase; PER, peroxidase. * P < 0.05. ** P < 0.01.

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