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Phytoremediation Efficiency of a PCPContaminated Soil using Four Plant Species as Mono- and Mixed Cultures Nejla Hechmi

a b

Naceur Jedidi

a

b

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, Nadhira Ben Aissa , Hassen Abdenaceur &

a

Laboratory of Wastewater Treatment , Water Research and Technologies Centre (CERTE), Ecopark of Borj Cedria BP , Soliman , Tunisia b

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National Agronomic Institute of Tunisia , Avenue Charles Nicolle City of Mahrajene, Tunisia Accepted author version posted online: 12 Aug 2013.Published online: 10 Mar 2014.

To cite this article: Nejla Hechmi , Nadhira Ben Aissa , Hassen Abdenaceur & Naceur Jedidi (2014) Phytoremediation Efficiency of a PCP-Contaminated Soil using Four Plant Species as Mono- and Mixed Cultures, International Journal of Phytoremediation, 16:12, 1241-1256, DOI: 10.1080/15226514.2013.828009 To link to this article: http://dx.doi.org/10.1080/15226514.2013.828009

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International Journal of Phytoremediation, 16:1241–1256, 2014 C Taylor & Francis Group, LLC Copyright  ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2013.828009

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PHYTOREMEDIATION EFFICIENCY OF A PCP-CONTAMINATED SOIL USING FOUR PLANT SPECIES AS MONO- AND MIXED CULTURES Nejla Hechmi,1,2 Nadhira Ben Aissa,2 Hassen Abdenaceur,1 and Naceur Jedidi1 1

Laboratory of Wastewater Treatment, Water Research and Technologies Centre (CERTE), Ecopark of Borj Cedria BP, Soliman, Tunisia 2 National Agronomic Institute of Tunisia, Avenue Charles Nicolle City of Mahrajene, Tunisia Bioremediation of soil polluted by pentachlorophenol (PCP) is of great importance due to the persistence and carcinogenic properties of PCP. Phytoremediation has long been recognized as a promising approach for removal of PCP from soil. The present study was conducted to investigate the capability of four plant species; white clover, ryegrass, alfalfa, and rapeseed grown alone and in combination to remediate pentachlorophenol contaminated soil. After 60 days cultivation, white clover, raygrass, alfalfa, and rapeseed all significantly enhanced the degradation of PCP in soils. Alfalfa showed highest efficiency for the removal of PCP in single cropping flowed by rapeseed and ryegrass. Mixed cropping significantly enhanced the remediation efficiencies as compared to single cropping; about 89.84% of PCP was removed by mixed cropping of rapeseed and alfalfa, and 72.01% of PCP by mixed cropping of rape and white clover. Mixed cropping of rapeseed with alfalfa was however far better for the remediation of soil PCP than single cropping. An evaluation of soil biological activities as a monitoring mechanism for the bioremediation process of a PCP-contaminated soil was made using measurements of microbial counts and dehydrogenase activity. KEY WORDS: Mixed cropping, phytoremediation, organic pollutant, pentachlorophenol

INTRODUCTION Pentachlorophenol (PCP), a class of persistent organochlorine pollutants (POPs), is widely disseminated in the environment especially in soils. Some genotoxic effects of this chemical have been proclaimed (Galil and Novak 1994; Sun et al. 2011c). As a pesticide, herbicide, and antiseptic, it was once used worldwide. The chemical and biocidale nature of PCP renders it resistant to microbial degradation at even low concentrations. (Walter et al. 2007). Due to its higher toxicity, recalcitrance, bioaccumulation and persistence in the environment and suspected carcinogen and mutagen to the living, PCP poses serious ecological problems as the environmental pollutants. PCP has been designated as a priority pollutant by the USEPA (USEPA 2004). Therefore, the remediation of soil PCP pollution is of great importance.

Address correspondence to Nejla Hechmi, Laboratory of Wastewater Treatment, Water Research and Technologies Centre (CERTE), Technopole Borj Cedria BP, Soliman, TUNISIA. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bijp. 1241

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Phytoremediation, the use of plants to remediate contaminated soil, has been described as a promising approach to remediate soils contaminated with persistent organic pollutants such as PCP (He et al. 2005, 2009; Meade and D’Angelo 2005; Mills et al. 2006; Lin et al. 2006; Zhao et al. 2011; Hayat et al. 2011). Plants can help alleviate environmental pollution by PCP through a range of mechanisms: besides uptake from soil (phytoextraction) (Lin et al. 2008), plants are capable of enzymatic transformation of PCP (phytotransformation) (Takashi et al. 2004); by releasing a variety of secondary metabolites, plants also enhance the microbial activity in the root zone, improving biodegradation of PCPs (rhizoremediation) (Yan et al. 2005; Dams et al. 2007). Plants can modify the geochemical environment in the rhizosphere, furnishing ideal conditions for bacteria and fungi to grow and degrade organic contaminants. Plant roots penetrate the soil, providing zones of aeration and stimulate biodegradation (Sridhar et al. 2002). These applications have the potential for providing the most cost-effective and resource-conservative approach for remediating sites contaminated with a variety of hazardous chemicals (Sridhar et al. 2002). Laboratory and pot experiments had demonstrated that plants have enhanced dissipation of PCP when compared to unplanted controls (Dams et al. 2006, 2007). In addition spiking of PCP would provide results that are useful as reported by (Meade and D’Angelo 2005; Lin et al. 2006). Recently, Hayat et al. 2011 investigated the dissipation behavior of PCP in the aerobic-anaerobic interfaces established by the rhizosphere of rice root, a glasshouse experiment was conducted using a specially designed rhizobox with spiked soil (20 and 45 mg of PCP kg soil). During the last few decades, numerous plant species including ryegrass (Lolium perenne L), radish (Raphanus sativus), willow (Salix sp.), poplar (Populus sp) and sunflower (Helianthus annuus) have been found to be promising candidates for phytoremediation of PCP (Lin et al. 2006; Mills et al. 2006; Wang et al. 2009). Lin et al. (2006) reported that After 12 weeks’ incubation, the removal efficiency of PCP ranged from 62% to 96% in the ryegrass planted soil and 45% to 94% in the radish planted soil, depending on the contamination level of PCP and Cu. Mills et al. (2006) showed that poplar had greater tolerance of PCP than willow. And both species are able to survive, but not thrive, at concentrations of 200 mg kg−1 PCP. However, willows were unable to grow in soil contaminated with 300 mg kg−1 PCP or more. He et al. (2009) studied PCP degradation ability of ryegrass and reported that within 90 days experiment as much as 85% and 83% of the added PCP (20 and 50 mg kg−1) was depleted in the soil planted to ryegrass at the two different PCP rates. Zhao et al. (2011) noted that PCP removal was significantly enhanced in the phytoremediated sediments in comparison with the control sediments after 90 days treatment, and the removal rates of PCP in the sediments planted with P. communis Trin, T. orientalis and S. validus Vahl were 90.35 ± 0.03, 99.23 ± 0.02 and 99.33 ± 0.01%, respectively, while the rate was 29.87 ± 0.05% in the control sediments. Potential of three species of aquatic macrophytes to remediate pentachlorophenol (PCP)-contaminated sediments starting with initial concentration of 2,000 μg kg−1 dw (dry weight) was investigated by Zhao et al. (2011). He et al. (2005) found that PCP accumulation in the roots of 80 ryegrasses was 6 and 9 μg at the soil PCP concentrations of 8.7 ± 0.5 and 18 ± 0.5 mg kg−1, respectively, which represents 1.5% of the initial amounts of PCP added. These results were based on monoculture and little information is available on phytoremediation by combined plants cultivation in soils contaminated with PCP (Zhao et al. 2011). In the rhizosphere, processes determining transport and bioavailability of PCP are more complex than in unvegetated soils. In addition, when plants grown as a multi-species

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mixture, the interaction of roots modifies the root physiology, root colonization, root surface properties and properties of the rhizosphere (Hauggaard and Jensen 2001; Joner and Leyval 2003). Combinations of root types and exudate patterns are assumed to allow greater infiltration of and stimulation of microbial communities, with a net positive stimulation of microbial catabolic potential (Phillips et al. 2006; Wang and Oyaizu 2009). The underlying assumption is that the effects of mixed plant populations will be proportionally cumulative, with the positive benefits of each individual plant species summing to a greater whole (Xu et al. 2006) how reported that combined plants cultivation enhanced dissipation of phenanthrene and pyrene in spiked soils. The aim of the present work was to investigate the potential of PCP removal by different plant species alone and when they were grown in a combined culture and to examine whether phytoremediation using the combined culture is an effective treatment option to reduce PCP levels in contaminated soils by the overall degradation of PCP by the plants and associated changes in microbial activities. White clover (Trifolium repens), ryegrass (L. perenne), alfalfa (M. sativa), and rapeseed (B. napus) representing fine variety of root, morphological and physiological characters, were selected for this study. Greenhouse experiments were designed to compare the potential of four plant species to enhance PCP degradation in soil, and screen suitable plants for phytoremediation. MATERIALS AND METHODS Chemicals PCP of chromatographic grade (purity > 98%) was purchased from Aldrich Chemical. Chromatographic reagents were used for gas chromatograph analysis. All other chemicals were of analytical grade and of the highest purity available. Double deionised water was used in all aqueous solutions and dilutions through the experiment. Preparation of Contaminated Soil From an experimental field of national institute of agronomic in Tunis, Tunisia, soils were collected from surface horizon (0–20 cm depth) were air-dried, sieved through a 2 mm mesh. Selected physical and chemical properties, which were measured following standard procedures (Sparks et al. 1996), were Soil pH was 7.31 ± 0.1 (1:2.5 soil/water), organic matter 22.3 g kg−1, available nitrogen 128.6 mg kg−1, extractable P 24.6 mg kg−1, available potassium 106.3 mg kg−1 and water-holding capacity 38.4%. The particle size distribution (50% sand, 39% silt, and 11% clay) hint the soil as a sandy loam soil. Soil was spiked with a concentration of high purity of pentachlorophenol (PCP) 100 mg kg−1 in acetone. To achieve homogeneity, the spiked soil was mixed and sieved again through a 2 mm mesh. After aging, for five weeks, the soil was fertilized with 1.64 g of KH2 PO3 and 2.28 g of NH4 NO3 per kilogram dry weight (dw) of soil (Shen et al. 2009) and again sieved to get homogeneity. The treated soils were packed into ceramic pots (1 kg dry weigh soil per pot). The temperature was maintained at 18–25◦ C in the greenhouse, and soil moisture was kept at 60% of the water-holding capacity (WHC) (Zhang et al. 2009). Plant Materials and Experimental Design Three forage species white clover (Trifolium repens), ryegrass (L. perenne), alfalfa (M. sativa), widely cultivated in Tunisia and rapeseed (B. napus) were chosen as the plant

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species in our experiment. Height (8) treatments were established (as: S0 = no plant, S1 = white clover, S2 = ryegrass, S3 = rapeseed, S4 = alfalfa, S5 = S1 + S3, S6 = S2 + S3, S7 = S3 + S4. Seeds of each plant were germinated and grown for 7 days in moist perlite plates and seedlings of uniform size were transplanted to the designated pots. Ten seedlings of white clover or ryegrass or alfalfa and six seedlings of rapeseed in single plant cultivation pots were used. Five seedlings of white clover or alfalfa and three seedlings of rapeseed were grown in combined cultivation pots. Three replicates of each treatment were prepared in a completely randomized manner. Seedling transplanting date was considered the starting time of experiment. The water content was checked and adjusted regularly with sterilized water to maintain about 60% WHC and fertilized every 2 weeks. The experiment ran for 2 months. After 60 days of plant growth, the soils and plants were sampled. The planted and unplanted soils were carefully collected, homogenized and divided into two sets, one for chemical analysis and other for biological analysis. Soil samples were stored at 4◦ C before analysis. PCP Extraction and Quantification in Soil and Plants Residual of pentachlorophenol was measured with HPLC-MS. According to K¨ahk¨onen et al. (2007), soil (2 g) was sonicated for 30 min in 5 ml methanol, then centrifuged for 10 min in 3000 g. 3,5-Dichlorophenol (DCP) was used as an internal standard. Supernatant was removed and supernatant was washed with 5 ml methanol. Sonication and centrifugation were repeated and supernatants were combined. The extracts were concentrated under N2 and dissolved in methanol. Content of PCP was measured with HPLC-MS (Agilent 1100 Series LC/ MSD Trap XCT Plus, Agilent Technologies, Inc., Santa Clara, Ca USA). DCP was used as an external standard. The HPLC was equipped with Zorbax SB-C18 column (2.1 by 70 mm, Agilent Technologies). The initial eluent was 80% water (1% acetic acid) and 20% methanol and increased during 10 min to 100% methanol, and then it remained for 5 min as 80% water (1% acetic acid) and 20% methanol. In full scan mode the range was from 50 to 650 m/z in the negative ion mode. PCP concentrations were calculated on the basis of peak area measurements by comparison with an external standard of known concentrations of PCP prepared in methanol. After 60 d growth period, all harvested plants were cut into roots and aboveground parts, washed with tap water and then rinsed with deionized water two times and blotted with clean tissue paper in order to remove excess water. The fresh weights of roots and aboveground parts were weighed and then subjected to oven-dried at 50◦ C for overnight and their oven-dried weights were determined. The procedure for PCP extraction in plant tissues was almost the same as the procedure for soils except that ultrasonic extraction step was performed twice. Extraction method show a recovery rates ranged from 83 to 95% with the relative standard deviation (RSD) less than 2.2% (n = 3) for the PCP contaminated soil and plants. Microbial Assays Microbial counts. Rhizosphere heterotrophic microbial numbers were measured after 60 days of growth. The microbial population was extracted from the soil which firmly attached to the roots (located 3–10 mm from the plant root) and was defined as “rhizosphere soil”. Direct plate counting method was used to enumerate the viable microbial population. The enumeration method was carried out using a 10 g-sample of soil diluted in 100 ml of

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sterile distilled water. The mixture was blended and tenfold dilution series were prepared. Afterwards, 0.1 ml of the suspensions were aliquoted onto three replicate plates per dilution of agar-nutrient (Acumedia) media for bacteria (pH 7.0) and Martin agar media for fungi (pH 5.0). Plates were incubated for 7 days at 25◦ C after overnight incubation at 37◦ C as recommended by (Zhang et al. 2011). Colonies were enumerated and microorganisms expressed as colony forming units (CFU) per gram of dry Soil. Dehydrogenase activity. It has been reported to be a suitable measure of microbial activity in soil (Taylor et al. 2002). Soil dehydrogenase activity was measured by the reduction of 2,3,5-triphenyl tetrazolium chloride (TTC) to 1,3,5-triphenyl formazan (TPF) using a modification of the methods of (Mills et al. 2006). Five gram soil samples, collected from each pot, were individually placed in 30 mL plastic vials with 0.1 g of CaCO3 , and 3 mL of TTC solution (5 g l−1 in 0.2 M Tris–HCl buffer, pH 7.4) and were incubated for 24 h at 37◦ C. After incubation, 20 ml of methanol was added, the sample shaken and then filtered through Whatman 41 filter paper to extract the triphenylformazan (TPF). Following extraction, the absorbance of the filtered solutions was measured at 485 nm using a spectrophotometer (Beckman DU 640).

Data Calculation 1. BCF or bioconcentration factor; dry weight ratio of PCP concentration in plant to the soil. BCF = [PCP] plant / [PCP] soil 2. TF or translocation factor; ratio of shoot BCF to root BCF. TF = BCFshoot /BCFroot 3. The dissipation rate (R) of PCP in different treated pots after 60 days plantation of crops was calculated by the following formula R = (C0 −Ct ) ×100% /C0 where C0 was the initial soil concentrations of PCP and Ct the residual concentrations of this pollutant at day t.

Statistic Analyses All values presented are the means of three replicates. Least significant differences were calculated at a level of p = 0.05 using the software Statistical Package for Social Sciences (SPSS 16.0 for Windows).

RESULTS PCP Effects on Different Plant Biomass The shoot and root biomasses of plants on a dry weight basis grown in the soil contaminated with pentachlorophenol shown in Table 1. Different plant species displayed different responses to the presence of PCP in the soil. At the end of 60 days experiment, root and shoot yields of all plants were significantly lower in PCP-treated soils than in control soils. The greatest reduction in biomass was observed in alfalfa, which produced approximately 35% of the biomass of control. Rape seed was the most resistant to the presence of PCP. Results also reveal that root/shoot ratios of the plant species under study decreased as

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Table 1 Plant biomass (g dry weight pot−1) of shoots and roots and root shoot ratio (R/S) of plants in control and PCP-treated soils after 60 days cultivation. Wroot Treatment

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S1 S2 S3 S4

Wsoot

R/S

PCP−

PCP+

PCP−

PCP+

PCP−

PCP+

1.07 ± 0.12a 1.95 ± 0.66a 1.63 ± 0.41a 1.40 ± 0.26a

0.80 ± 0.07b 1.02 ± 0.11b 0.52 ± 0.12b 0.86 ± 0.19b

6.35 ± 0.34a 9.47 ± 1.68a 6.42 ± 1.11a 7.87 ± 0.90a

4.02 ± 0.65b 7.16 ± 1.08b 2.63 ± 0.75b 5.74 ± 1.28b

0.17 0.205 0.254 0.178

0.19 0.141 0.197 0.150

Values in each column followed with different lowercase letters (a and b) indicated significant (p ≤ 0.05) difference between plant biomass grown in control and PCP-treated soils. S1 = white clover, S2 = ryegrass, S3 = rapeseed, S4 = alfalfa.

a result of PCP amendment of the soil. The decrease in the root/shoot ratios of plants can be attributed to the more negative effect of PCP on the plants root than shoot. Plant Uptake and Accumulation of PCP In our study all plants accumulated pentachlorophenol in the plant parts grown in the PCP spiked soils. As seen from Figure 1, great variations of root and shoot PCP concentrations were observed among different plant species. For all species, the concentrations of PCP in roots were considerably higher than those in shoots. At 60th day of plant growth, the maximum PCP amounts in roots of the four plant species attained 1.60 ± 0.21, 2.30 ± 0.36, 5.33 ± 0.53, and 8.17 ± 0.89 mg kg−1 for white clover (S1), ryegrass (S2), rapeseed (S3), and alfalfa (S4), respectively. Alfalfa exhibited the greatest accumulation capacity among different plant species. Thus, the disparity of root and shoot uptake of these PCP

Figure 1 PCP concentrations in plant tissue (roots and shoots) under different planting patterns; S1 = white clover, S2 = ryegrass, S3 = rapeseed and S4 = alfalfa, S5 = S1 + S3, S6 = S2+ S3 and S7 = S3 + S4. Bars (means ± SE, n = 3) with different letters are significantly different based on LSD (p ≤ 0.05).

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would come from plant properties. As evident, PCP concentrations in all plants under mixed cropping were significantly lower than those under mono-cropping (Fig. 1). Bioconcentration factor (BCF), expressed as the dry weight PCP concentration ratio of plant tissue to that in the soils ([PCP] root or shoot/ [PCP] soil), was used to compare the relative abilities of different species to take up and translocate PCP in the shoots and roots. The average BCFs was varied from 0.12 (S2 = ryegrass) to 0.49 (S4 = alfalfa) for the shoots and from 0.38 (S1 = white clover) to 1.46 (S4 = alfalfa) for the roots (Fig. 2a). This suggests that plant species obviously affected plant uptake and translocation of PCP due to different plant properties. Translocation factor (TF), calculated as the PCP concentration ratio of the shoots to the roots, is an interest value denoting PCP transfer from the roots to the shoots. Fig. 2b showed that the calculated TF for the plant species were lower than 1.0 with great variation among the different species, but no significant differences were observed (P > 0.05) (Fig. 2b). This designated that most of the PCP absorbed were maintained in the roots while small amount of PCP were translocated into the shoots, resulting in concentration in the roots. Determination of PCP accumulated in plant tissue (roots and shoots) (Fig. 1) indicated

Figure 2 Bioconcentration factor (BCF) (a) and translocation factor (TF) (b) for PCP in different plant species after 60 days of plant growth. S1 = white clover, S2 = ryegrass, S3 = rapeseed and S4 = alfalfa. Bars (means ± SE, n = 3) with different letters are significantly different based on LSD (p ≤ 0.05).

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Figure 3 Residual concentrations (mg kg−1) and Percent removal (%) of PCP in treated soils after 60 days of plant growth. S1 = white clover, S2 = ryegrass, S3 = rapeseed, S4 = alfalfa, S5 = S1 + S3, S6 = S2 + S3 and S7 = S3 + S4. Bars (means ± SE, n = 3) with different letters are significantly different based on LSD (p ≤ 0.05).

that cropping patterns evidently affect the potential ability of plants to accumulate PCP: mixed cropping significantly reduced the accumulation of PCP in plant tissues comparing to single cropping at the same initial level of soil PCP. Plant uptake was a minor plant contribution to remove PCP, accounting for less than 1.0 plant-enhanced loss under multispecies cultivation (Fig. 2b)

Residual Concentrations and the Dissipation Rate of PCP in Spiked Soil Pentachlorophenol concentration remaining in variously treated soils after 60 days are shown in Figure 3. Comparing with the respective initial concentration, the residual PCP in the soils was all significantly decreased, either in the planted or unplanted soils after 60 days of experiment. But a more marked rate of loss was evident in the presence of plants. However, the residual concentrations of soil PCP at the presence of plants were apparently lower than that of the control (S0 ), the removal rates of PCP planted with white clover, ryegrass, rapeseed and alfalfa were 59.40%, 71.97%, 69.4, and 72.9% respectively. Among single plant treatments, alfalfa displayed the highest PCP degradation rate (72.9%). As compared with monocultures, PCP concentration and absolute accumulation amount in plants and enhanced biodegradation in soils all increased in multispecies mixtures. As evident in Figure 3, 84.5% and 89.84% of PCP were removed by combined plant

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S6 and S7 , respectively. Removal rates of PCP varied evidently among different combinations of plant. As revealed in Figure 3, treatment S7 , i.e., rapeseed + alfalfa, was most efficacious in PCP removal (89.84%). This suggests that planting rapeseed and alfalfa in mixture played an important role in the remediation of the PCP contaminated soil.

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Microbial Enumeration At the end of experiment, the total numbers of bacteria and fungi were counted in the soils with different treatments Figure 4. The tabulated data revealed that total number of microorganisms in soils after 60 days of cultivation was significantly influenced by the PCP contamination. PCP had stimulatory effect on microbial counts. This effect was obvious in rhizosphere of all plants as well as in unplanted control. Bacterial numbers in mixed cropping alfalfa and rapeseed (S7 ) were significantly the highest, 2 times more control soil (S0). The Fungal counts also showed the similar trend like bacterial counts. The Response of Dehydrogenase Activity dehydrogenase activity was monitored at the end of the phytoremediation experiment to determine effect of different plant species single and in combination on microbial activity under PCP contamination. Figure 4 shows that the dehydrogenase activities were much higher in planted soils compared to unplanted control irrespective to the presence of PCP in soil. After 60 days of plant growth, the dehydrogenase activities were 1.2–59.8 and 2.8–105 μg TPF g−1 dry soil in uncontaminated and contaminated soils, respectively. Among mixed cropping alfalfa and rapeseed S7 had the highest dehydrogenase activity whereas lowest values were observed in white clover single cropping. These results accords well the degradation data of PCP. In the present study, the stimulation of dehydrogenase activity matches well with the enhanced microbial and fungal counts. DISCUSSIONS Phytoremediation has been demonstrated to be a promising technology for the cleanup of sites contaminated with pentachlorophenol (He et al. 2005; Zhao et al. 2011). In the process, plants could be used to uptake, detoxify, and/or sequester toxic pollutants from soil (Child et al. 2007). This study explores plant-mediated dissipation of PCP under diverse cropping patterns, implying that the presence of vegetation appreciably enhanced the dissipation of pentachloropheol in the soil environments, which was in accord with other reports (Mills et al. 2006; Lin et al. 2006). However, plant species tested (S1 = white clover, S2 = ryegrass, S3 = rapeseed, S4 = alfalfa,) did not exhibit obvious signs of toxicity, and it appears that vegetation establishment with these plants in PCP-contaminated soil is feasible. The results of Zhao et al. (2011) indicated that the three species of aquatic macrophytes (S. validus Vahl, T. orientalis, and P. Communis Trin) could adapt effortlessly to PCP in sediments up to 2,000 μg kg−1 PCP level. Similar results were observed on ryegrass under lower concentration (8,000 μg kg−1) of PCP (Yan et al. 2005). Furthermore, unlike the previous findings, PCP concentrations above certain levels (200 and 250 mg kg−1) decreased dry weight of two plant species willow and poplar and the two plants could not survive at PCP concentrations above 250 mg kg−1 in soil (Mills et al. 2006). Concentrations of PCP in shoots were less than in roots (Fig. 1). This result concurs with the findings from He et al. (2005). Who reported the concentration of PCP in the roots

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Figure 4 Rhizospheric bacteria (a), fungus (b) and dehydrogenase activity (c) after 60 days of plant growth. Bars (means ± SE, n = 3) with different letters are significantly different based on LSD (p ≤ 0.05). Capital letters (A and B) indicated significant (p ≤ 0.05) differences difference between control (PCP−) and PCP-treated (PCP+) soils, and in each row followed with different lowercase letters (a–c) indicated significant (p ≤ 0.05) difference between different treatments. Where S0 = no plant, S1 = white clover, S2 = ryegrass, S3 = rapeseed, S4 = alfalfa, S5 = S1 + S3, S6 = S2 + S3 and S7 = S3 + S4. PCP− and PCP+ were 0 and 100 mg kg−1 of pentachlorophenol.

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of 80 ryegrasses. PCP was also undetectable in the aboveground part of the macrophytes S. validus Vahl, T. orientalis, and P. Communis Trin (Zhao et al. 2011). Our data showed that pentachlorophenol degradation in the treated soils was significantly greater in soils with plants than with no plants (S0 ) which was in accordance with other reports (Meade and D’Angelo 2005; Mills et al. 2006). The enhanced PCP degradation in the planted soil could be due to a higher density and activity of microorganisms in the rhizosphere than the unplanted soil (Wang et al. 2008; Zhang et al. 2009) with the root exudates enhancing PCP bioavailability (He et al. 2005). Root exudates also provide the substrates for co-metabolic degradation and modify the soil environment to be more suitable for microbial transformation (Reza et al. 2002). The mechanisms of phytoremediation mainly include the direct plant uptake of organic pollutants, degradation by plant-derived degradative enzymes, and stimulated biodegradation in plant rhizosphere (Gao and Zhu 2003). Remediation effectiveness varied greatly among four tested plant species, and the potential of white clover (S1 ), was lower than other species in promoting the degradation of PCP in contaminated soil. Similar results were observed phytoremediation for soils contaminated by polycyclic aromatic hydrocarbon Wei and Pan (2010). The obvious interspecies differences of plants on phytoremediation were possibly due variation in their root characteristics and exudate compounds (as carbon source and biosurfactant), which influence microbial degradation, soil properties, and pollutant mobilization (Wei and Pan 2010). Furthermore, plants also differ in their ability to take up and metabolize organic pollutant from the soil via root adsorption, transpiration, and enzymatic pathways (Harvey et al. 2002). Consequently, Plant choice is a main parameter to optimize for the phytoremediation of PCP-contaminated soils. As mentioned previously, the apparent interspecies dissimilarity of plants on phytoremediation were due to the difference in structures of root, exudates, and consequently rhizosphere microbial communities (Wei and Pan 2010). Combined cropping of rapeseed with alfalfa (S7 ) or ryegrass (S6 ) illustrated clearly higher remediation of soil PCP contamination than single cropping (S1 , S2 , S3 , or S3 ), which indicates that plant diversity can improve PCP degradation in contaminated soils suggesting an ‘bio-augmentation’ effect among plant species (Wei and Pan 2010). Similar findings by Meng et al. (2011) how have reported that using multispecies cultivation appeared to be a more efficient approach of phytoremediation to remove PAHs from industrial soils as compared with monoculture cropping. The enhanced degradation of PCP in soils by combined cropping was possibly due to the improved soil physiochemical and biological (Lewis et al. 2001; Wei and Pan 2010). The enhanced degradation of PCP in mixed plantation treatments compared to single plantation might be the result of different roots interaction constituents. Which may have two possible explanations: (1) roots interaction modified root physiology (enzyme activity, exudation) in a manner that stimulates PCP degradation, either by root derived enzymes or by rhizosphere organisms (He et al. 2007), (2) interaction roots colonization affected root surface properties or rhizosphere soil properties (Hauggaard and Jensen 2001; Joner and Leyval 2003; Xu et al. 2006). The combined plant cultivation has potential to enhance remediation process (Cheema et al. 2010). Meng et al. (2011) confirmed that using multispecies cultivation appeared to be a more effective approach of phytoremediation to remove PAHs from industrial soils as compared with monoculture cropping. On the other hand, it has been well considered that multispecies combination can improve the efficiency of heavy metal phytoremediation relative to monoculture, because it provides a more beneficial rhizosphere

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condition for improving biodegradation and bioavailability of pollutants (Wenzel 2009). By comparison, the contribution rates of plant-microbial interactions on dissipation of PCP is the greatest among two pathways, its average contribution rates on dissipation of PCP by mixed group indicating that the predominant pathway responsible for the degradation of soil PCP under mixed cropping pattern was the plant microbial interactions, and the plant uptake and accumulation (TF and BF) of these compounds was negligible. This result is very consistent with the finding reported by Sun et al. (2011a) investigated in situ phytoremediation of PAH-contaminated soil by intercropping alfalfa with tall fescue and revealed that although plants have taken up certain quantities of PAHs, the absolute amount of PAHs stored in plant compartments makes little contribution to the removal of total soil PAHs. Maximum plant uptake in all plants in mixed cropping indicated that phytoextraction removed less than 1% of the total PAH mass in the soil, which was consistent with Meng et al. (2011) and Wei and Pan (2010) that plants take up less than 2% of total soil PAHs. Microbiological parameters such as enzyme activities and the diversity of soil microbial communities may serve as important indices of the impact of pollution on soil health (Tu et al. 2011). As mentioned by Pascula et al. (2000). Soil microbial activity serves as a biomarker of degradation and remediation processes. Dehydrogenase is an important oxidoreductase in soils, which is the catalyst for important metabolic processes, including the decomposition of organic inputs and the detoxification of xenobiotics (Margesin et al. 2000; Parrish et al. 2005). Dehydrogenase activity can be used as an indirect indicator of microbially-mediated remediation of soil because biological oxidation of organic compounds typically involves a dehydrogenation catalyzed by dehydrogenase enzymes (Balba et al. 1998). Our results demonstrate high dehydrogenase activity in the PCP contaminated soil which might be attributed to the increased microbial activity as a result of enhanced root exudation consequence of PCP toxicity to plants. The enhanced biodegradation performance for PCP observed might be due to an increase in microbial activities and bioavailable PCP in soils caused by combined effects of plants. Lee et al. (2008) also reported 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. Walton et al. (1994) 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. Dehydrogenase activities increased in planted treatments compared with unplanted control. The results were in line with those from Sun et al. (2011a). Enhancement of dehydrogenase activity was associated to the plant root systems. Dehydrogenase increases with root growth and rooting density (Drury et al. 1991). On the other hand, the biodegradability of the contaminants also depends on extracellular enzymes in soils (e.g., dehydrogenase and peroxidase), which are released into rhizosphere of plant by cells (Shen-wang et al. 2008). Moreover, soil enzyme activities augmented in the planting alfalfa in mixture than alfalfa and rapeseed in monoculture. Based on these observations, it could be concluded that planting alfalfa and rapeseed in mixture enhanced the degradation of soil PCP by stimulating microbial activities in the soil. A synergistic action by both rhizosphere microorganisms that leads to increased availability of hydrophobic compounds, and plants that leads to their removal and/or degradation, may overcome many of the limitations, and thus provide a useful basis for enhancing remediation of contaminated environments (Chaudhry et al. 2005). Intercropping alfalfa with rapeseed may be a promising practice for the phytoremediation of PCP-contaminated soil with at least partial restoration of the microbial functioning of the contaminated soil. Similar results have also

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been found in other studies (Wei and Pan 2010; Sun et al. 2011a). Phillips et al. (2006) showed that alfalfa had a dominant effect on the structure of rhizosphere microbial communities in mixed plant treatment, stimulating relative increases in specific Bacteroidetes and Proteobacteria populations. These results revealed that phytoremediation with cultivated plant species stimulated rhizosphere microbial communities in organic contaminated soils. Binet et al. (2000) investigated the dissipation of a mixture of eight PAHs, ranging from three to six rings, in the rhizosphere of ryegrass. They concluded that the increased PAH dissipation in rhizospheric soil was associated with an enhancement of PAH degraders.

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CONCLUSION Phytoremediation appeared to have great potential for the remediation of PCP contaminated soil. The presence of vegetation significantly promoted the dissipation of PCP in the soil environment at 100 mg kg−1 as initial concentration. Remediation capacity varied greatly among plant species. Under monoculture, white clover (S1) had the lowest ability for the removal of PCP, while alfalfa (S4) showed highest ability for the remediation of PCP. Based on these results, alfalfa was selected for the further investigation of the phytoremediation of PCP–contaminated soil. As compared to single cropping, combined cultivar plants rapeseed and alfalfa (S7 ) or ryegrass (S6 ) illustrated clearly higher remediation of soil PCP contamination than mono-cultivar plants (S1 , S2 , S3 , S4 ). The difference was attributed not just to the effect of higher overall biomass yields but to different roots interaction constituents. Roots interaction modified root physiology (enzyme activity) in a manner that stimulates PCP degradation, either by root derived enzymes and associated microbial groups in the rhizosphere exerting different effects on soil PCP. Via expanded metabolic range of the rhizosphere bacterial community, promote enzyme activity, and thereby facilitate bioavailability of PCP and enhance pollutants dissipation. So, combined cultivar plants provided the desired result of increasing the effective depth of remediation by stimulating microbial activities in the soil. The establishment of selective combined vegetation might be a feasible remediation method for soils contaminated with PCP, and an efficient vegetation remediation choice could be better than many alternative clean-up technologies. Full elucidation of the potential applicability of this mixed-cropping method will require further studies in a range of contaminants. ACKNOWLEDGMENTS The authors acknowledge all staff of Sol Direction, Olive institute the specialized unit of Tunis in Tunisia for technical support. We also would like to thank Professor Andrew Hursthouse, PhD lain McLellan and all staff of School of Science, University of the West of Scotland for their great assistance in pollutant (PCP) extraction and quantification. REFERENCES Balba MT, Al-Awadhi N, Al-Daher R. 1998. Bioremediation of oil-contaminated soil: microbiological methods for feasibility assessment and field evaluation. J Microbiol Methods 32(2): 155–164. Binet P, Portal JM, Leyval C. 2000. Dissipation of 3–6-ring polycyclic aromatic hydrocarbons in the rhizosphere of ryegrass. Soil Biol. Biochem 32:2011–2017. Chaudhry Q, Blom-Zandstra M, Gupta S, Joner EJ. 2005. Utilizing the synergy between plants and rhizosphere microorganisms to enhance breakdown of organic pollutants in the environment. Environ Sci Pollut Res 12:34–48.

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