This article was downloaded by: [University of Winnipeg]On: 05 September 2014, At: 09:35Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Click for updates

International Journal ofPhytoremediationPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/bijp20

Comparing Anthracene and FluoreneDegradation in Anthracene andFluorene-Contaminated Soil by Singleand Mixed Plant CultivationKhanitta Somtrakoon a , Waraporn Chouychai b & Hung Lee ca Department of Biology, Faculty of Science , MahasarakhamUniversity , Kantharawichai , Mahasarakham , Thailandb Biology Program, Faculty of Science and Technology , NakhonsawanRajabhat University , Nakhonsawan , Thailandc School of Environmental Sciences , University of Guelph , Guelph ,Ontario , CanadaAccepted author version posted online: 18 Jun 2013.Publishedonline: 07 Oct 2013.

To cite this article: Khanitta Somtrakoon , Waraporn Chouychai & Hung Lee (2014) ComparingAnthracene and Fluorene Degradation in Anthracene and Fluorene-Contaminated Soil by Singleand Mixed Plant Cultivation, International Journal of Phytoremediation, 16:4, 415-428, DOI:10.1080/15226514.2013.803024

To link to this article: http://dx.doi.org/10.1080/15226514.2013.803024

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,

systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

International Journal of Phytoremediation, 16:415–428, 2014Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2013.803024

COMPARING ANTHRACENE AND FLUORENEDEGRADATION IN ANTHRACENE ANDFLUORENE-CONTAMINATED SOIL BY SINGLEAND MIXED PLANT CULTIVATION

Khanitta Somtrakoon,1 Waraporn Chouychai,2 and Hung Lee3

1Department of Biology, Faculty of Science, Mahasarakham University,Kantharawichai, Mahasarakham, Thailand2Biology Program, Faculty of Science and Technology, Nakhonsawan RajabhatUniversity, Nakhonsawan, Thailand3School of Environmental Sciences, University of Guelph, Guelph,Ontario, Canada

The ability of three plant species (sweet corn, cucumber, and winged bean) to remediatesoil spiked with 138.9 and 95.9 mg of anthracene and fluorene per kg of dry soil, respec-tively, by single and double plant co-cultivation was investigated. After 15 and 30 days oftransplantation, plant elongation, plant weight, chlorophyll content, and the content of eachPAH in soil and plant tissues were determined. Based on PAH removal and plant health,winged bean was the most effective plant for phytoremediation when grown alone; percent-age of fluorene and anthracene remaining in the rhizospheric soil after 30 days were 7.8%and 24.2%, respectively. The most effective combination of plants for phytoremediation wascorn and winged bean; on day 30, amounts of fluorene and anthracene remaining in thewinged bean rhizospheric soil were 3.4% and 14.3%, respectively; amounts of fluorene andanthracene remaining in the sweet corn rhizospheric soil were 4.1% and 8.8%, respectively.Co-cultivation of sweet corn and cucumber could remove fluorene to a higher extent thananthracene from soil within 15 days, but these plants did not survive and died before day30. The amounts of fluorene remaining in the rhizospheric soil of corn and cucumber wereonly 14% and 17.3%, respectively, on day 15. No PAHs were detected in plant tissues. Thissuggests that phytostimulation of microbial degradation in the rhizosphere was most likelythe mechanism by which the PAHs were removed from the spiked soil. The results show thatco-cultivation of plants has merit in the phytoremediation of PAH-spiked soil.

KEY WORDS: anthracene, fluorene, mixed plant cultivation, phytoremediation, polycyclic aromatic hydrocar-bons

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are hazardous organic pollutants due totheir carcinogenic and mutagenic potential. These compounds are recalcitrant and may be

Address correspondence to Khanitta Somtrakoon, Department of Biology, Faculty of Science,Mahasarakham University, Kantharawichai, Mahasarakham, Thailand, 44150. E-mail: khanitta.s@msu.ac.th;skhanitta@hotmail.com

415

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

416 K. SOMTRAKOON ET AL.

bioaccumulated up the food chain. The incomplete combustion of fossil fuels, accidentalspillage of crude oil from shipwrecks, oil wells, coking plants, domestic heating fuel,aircraft and car exhaust, forest fires and open field burning of vegetation residues allcontribute to PAH contamination in soil and sediment (Gadde et al. 2009; Hao et al. 2007;Reynoso-Cuevas et al. 2008; Zehetner et al. 2009). PAH contamination in agricultural soilhas been reported to reduce crop yield due to their adverse effects on plant growth throughreduced biomass accumulation, reduced seed germination rate, photosynthesis inhibitionand disruption of mineral transport by plant root (Huang et al. 1996; Kumervora et al. 2006;Kumervora et al. 2008). Restoration of PAH contaminated soil is recognized as essentialto diminish their toxic effects on plants and other organisms.

Phytoremediation has been suggested as an attractive choice to clean up PAHs fromcontaminated sites (Chouychai et al. 2009, 2012; Merkl et al. 2006). Plants in the Poaceae,Cucurbitaceae, and Fabaceae families have often been used in phytoremediation due toseveral advantages (Banks et al. 2003; Fan et al. 2008). The taproot system of legumesextends deeper in soil and these plants in symbiosis with Rhizobium can produce a nitrogensource (Kirk et al. 2002; Mehmannavaz et al. 2002). Legumes such as Trifolium repens,Astragalus membranaceus, and Aeschynomene indica have been used to remediate soilcontaminated with petroleum hydrocarbons and PAHs (Lee et al. 2008; Xu et al. 2006).Grasses, such as corn and sorghum, with extensive fibrous root systems have been used toremediate crude oil contaminated soil (Banks et al. 2003; Chouychai et al. 2009; 2012).Cucurbitaceae such as zucchini, pumpkin and Lagenaria siceraria have also been reportedto remediate soils contaminated with organochlorine pesticides such as PCBs, heptachlorand DDT, although the mechanism is thought to occur by bioaccumulation of these organicpollutants rather than rhizodegradation (Whitfield-Åslund et al. 2007; 2010; Campbell et al.2009; Inui et al. 2008).

Phytoremediation studies typically use single plant species. A few studies used morethan one type of plants to remediate PAH-contaminated soil, and this has been shown toincrease the rate and extent of PAH removal over single plant cultivation. For example,mixed cultures of corn and ryegrass or corn and white clover could remove phenanthreneand pyrene more efficiently than single culture of each plant (Xu et al. 2006). In anotherstudy, mixed culture of Brachiaria serrata and Eleusine coracana removed greater amountsof naphthalene and fluorene from soil than each plant alone (Maila et al. 2005).

In this study, we examined the ability of mixed plant cultivation as compared tosingle cultures of sweet corn (Zea mays), winged bean (Psophocarpus tetragonolobus), andcucumber (Cucumis sativus) to remediate soil contaminated with anthracene and fluorene.These plants are commonly found in Thailand and are representative of the plant familiesPoaceae, Fabaceae, and Cucurbitaceae, respectively. The results showed mixed plants havegood potential for remediating PAH-contaminated sites in Thai soil.

MATERIALS AND METHODS

Soil Preparation

Soil with no previous history of anthracene and fluorene contamination was obtainedfrom grounds of the Kookaew Temple, Kantharawichai District, Mahasarakham Province,Thailand. The soil was air dried at room temperature (29 ± 1◦C) for at least 72 h to constantweight before use. A sample of this soil was sent to the Central Laboratory (Thailand) Co.,Ltd., Khonkan, Thailand for physical and chemical characterization. The analysis showedthe soil was of clay texture, with a pH of 8.1 and an electrical conductivity of 109.5 μs

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

PHYTOREMEDIATION OF ANTHRACENE AND FLUORENE 417

cm−1 (0.11 ds m−1). It contained 2.4% (dry) organic matter content, 0.29% total nitrogen,and 58.38 mg kg−1 available phosphorus. The soil did not contain any anthracene, fluoreneand fluoranthene as verified by GC-MS analysis following Soxhlet extraction (describedbelow).

An aliquot of the soil (about 20%) was spiked with a mixture of anthracene (Fluka,USA, purity 98%) and fluorene (Sigma-Aldrich, Germany, purity 98%) dissolved indichloromethane. The solvent was allowed to evaporate inside a fume hood at room tem-perature (29 ± 1◦C) for 48 h after thorough mixing. To the spiked soil was then added theremaining 80% of unspiked soil and mixed thoroughly before seed planting. The initialconcentrations of anthracene and fluorene in the mixed spiked soil were 138.9 and 95.9 mgkg−1 dry soil, respectively. Adequate mixing of soil was verified by taking five randomsamples (1 g of dried soil each) and processing them for anthracene and fluorene analysisby GC-MS (described below) before subdividing the mixed spiked soil to each pot.

Seedling Preparation

Seeds of sweet corn (Z. mays L.var sacharata Bailey) (commercial seeds ofChokkasikorn Seed Ltd., Nonthaburi province, Thailand), winged bean (P. tetragonolobus)(commercial seeds of Chuayongseng Ltd., Bangkok, Thailand), and cucumber (C. sativus)(commercial seeds of East West Seed Ltd., Nonthaburi province, Thailand) were rinsedwith sterile distilled water and then immersed in distilled water for 3 h. Seeds of sweetcorn, winged bean and cucumber were germinated in moist soil spiked with anthraceneand fluorene in experimental pots and kept at (29 ± 1◦C) in a plant nursery which receivednatural sunlight. After 10 days, healthy seedlings of plants with comparable size werepicked and transplanted into the experiment pots. The seedling transplantation date wasconsidered the start date (day 0) of the experiment.

Experimental Design

Pot experiments were performed in a plant nursery from April to May, 2012. In singleplant cultivation, each cylindrical pot containing 1 kg dried soil spiked with anthracene andfluorene was planted separately with 10-day old seedlings of sweet corn, winged bean orcucumber. For mixed plant cultivation, seedlings of sweet corn and winged bean, sweet cornand cucumber or winged bean and cucumber were grown together in one pot containing2 kg dried soil spiked with anthracene and fluorene. Pots set up in the same way with eithersingle or mixed plant seedlings, but without any PAHs being spiked into the soil servedas the controls. Another control was soil spiked with anthracene and fluorene, but withoutany plants. Three independent replicates of each treatment were prepared in a completelyrandomized design.

Soil in all pots was supplemented periodically with sterile distilled water to maintainthe water holding capacity of the soil at approximately 60% during the 30-day experiment.The placement of pots in the nursery was changed randomly every week.

One gram (dry weight) each of rhizospheric soil and bulk soil from planted soils werecarefully collected on days 15 and 30 for analysis of anthracene and fluorene concentrationsby GC-MS. Soils from unplanted control treatment were also collected and analyzed.

Plant Growth

The plants from single and mixed cultivation pots were sampled on days 15 and 30 todetermine the shoot length, root length, fresh weights and dried weights of shoot and root,

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

418 K. SOMTRAKOON ET AL.

as well as chlorophyll a, chlorophyll b and total chlorophyll content. Chlorophyll contentswere analyzed according Huang et al. (2004). Briefly, about 200 mg of plant leaves werecut and incubated in 80% acetone for 24 h at 4◦C in the dark. Absorbance of the solutionwas measured with a spectrophotometer at 645 and 663 nm. Chlorophyll concentrations(mg ml−1) were calculated using the following equations:

[Chl a] = [12.7 × A663] − [2.69 × A645]

[Chl b] = [22.9 × A645] − [4.68 × A663]

[Total Chl] = [8.02 × A663] + [20.2 × A645]

PAH Extraction from Soil Samples and Analysis

Soil samples were subjected to Soxhlet extraction as described in Chouychai et al.(2009). One gram dry weight of soil was mixed with anhydrous sodium sulfate powderin a 1:1 ratio. Fluoranthene (Sigma-Aldrich, USA, purity 99%; 50 μl of a 100 mg l−1

fluoranthene stock solution prepared in dichloromethane) was added as an internal standard.The soil sample was extracted with 180 ml of dichloromethane for 8 h at approximately 3–4cycles per h. The extracts were transferred to 250-mL pear-shaped flasks and evaporated tonear dryness under reduced pressure in a 60◦C water bath using a rotary evaporator (Buchi,Germany). The efficiency of Soxhlet extraction for the recovery of anthracene and fluorenefrom soil samples was over 80%, as judged by the amount of anthracene or fluorene andinternal standard recovered from soil samples to which was added known amounts of thesecompounds prior to extraction. Anthracene and fluorene in plant shoot and root sampleswere extracted in the same way as done for their extraction from soil samples.

Anthracene and fluorene concentrations in the dichloromethane extracts were mea-sured using a gas chromatograph (Shimadzu GC AOC-5000) equipped with a mass spec-troscopic detector (Shimadzu MS-QP2010). Separation was achieved using a Rtx R©-5MScapillary column (30 m × 25 mm, I.D. = 25 μm). The helium carrier gas flow rate was0.57 ml min−1 with a pressure of 45.0 kPa under split 30:1 ratio conditions. The oventemperature was programmed at 150◦C for 1 min, followed by a linear increase of 15◦Cmin−1 to 250◦C and held for 5 min at 250◦C. The injector and detector temperatures weremaintained at 250◦C.

Statistical Analysis

Percent of anthracene and fluorene remaining was expressed as the mean ± SD. One-way ANOVA was used to test for statistical significance among experiments. Subsequentmultiple comparisons of means were performed using the Tukey comparison method.Statistical significance was accepted at P < 0.05.

RESULTS

General Plant Health in PAH-Contaminated Soil

In control un-spiked soil, all tested plants grew normally either alone or together for30 days. The ability of plants to grow in fluorene- and anthracene-spiked soil varied greatlydepending on plant species. Winged bean grew normally in fluorene- and anthracene-spiked

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

PHYTOREMEDIATION OF ANTHRACENE AND FLUORENE 419

soil alone and did not exhibit apparent signs of stress during the 30-day experiment. Sweetcorn and cucumber grown in fluorene- and anthracene-spiked soil exhibited symptoms ofchlorosis on leaves. In addition, cucumber shoots appeared uncharacteristically short andundersized when compared with those grown in non-contaminated soil. When sweet cornand cucumber were grown together, they were more sensitive to fluorene and anthracenethan when each was grown alone and they died between 20–25 and 25–30 days, respectively.The reason for this observation is not known. Neither plant died when each was grownalone in PAH-contaminated soil. Cucumber also grew less well when planted together withwinged bean in PAH-contaminated soil. Leaves of cucumber exhibited chlorosis and theelongation of the shoots was stunted.

Effect of Fluorene and Anthracene on Shoot and Root Growth

The presence of fluorene and anthracene in soils did not significantly decrease shootlength, shoot fresh weight and shoot dried weight of sweet corn over 30 days irrespectiveof whether the plant was grown alone or together with winged bean (Table 1). In contrast,the presence of fluorene and anthracene affected the root of sweet corn, especially on rootlength at 15 days after transplantation (Table 2). On day 15, the root lengths of sweet corn

Table 1 Shoot length, shoot fresh weight and shoot dried weight of three plants (sweet corn, cucumber, wingedbean) grown in fluorene- and anthracene-contaminated or non-contaminated soil for 30 days.

Day 15 Day 30

Length Fresh Dried Length Fresh DriedPlant (cm) weight (mg) weight (mg) (cm) weight (mg) weight (mg)

Sweet Corn (SC)SC 19.0 ± 4.8a 558 ± 86a 115 ± 4a 24.7 ± 8.4a 1023 ± 57a 107 ± 55aSC + CU 28.0 ± 7.1 764 ± 108a 130 ± 21a 33.5 ± 8.4a 773 ± 4a 159 ± 7aSC + WB 27.1 ± 0.7a 674 ± 277a 130 ± 26a 23.5 ± 0.4a 775 ± 1a 164 ± 0aSC + PAHs 27.4 ± 2.7a 486 ± 184a 88 ± 25a 22.5 ± 2.4a 1101 ± 312a 220 ± 69aSC + CU + PAHs 25.7 ± 7.4a 610 ± 397a 130 ± 55 NA NA NASC + WB+ PAHs 30.5 ± 3.9a 436 ± 40a 119 ± 12a 26.4 ± 4.9a 951 ± 156a 196 ± 40a

Cucumber (CU)CU 23.3 ± 1.5a 1037 ± 25ab 100 ± 25a 38.0 ± 7.3a 747 ± 625b 120 ± 0aCU + SC 20.9 ± 3.0a 1102 ± 31a 99 ± 23a 24.2 ± 1.9b 1376 ± 301ab 168 ± 13aCU + WB 21.7 ± 3.3a 1103 ± 147a 148 ± 34a 30.9 ± 2.9ab 1832 ± 340a 156 ± 18aCU + PAHs 12.7 ± 2.1b 688 ± 136b 109 ± 23a 18.3 ± 1.4bc 1047 ± 137ab 152 ± 28aCU + SC + PAHs 8.0 ± 2.1b 406 ± 160b 93 ± 23a NA NA NACU + WB + PAHs 12.0 ± 1.7b 522 ± 40b 97 ± 3a 9.6 ± 0.1c 876 ± 1ab 149 ± 8a

Winged Bean (WB)WB 24.7 ± 1.5a 1383 ± 170a 217 ± 50a 38.5 ± 14.7a 217 ± 50a 163 ± 92aWB + SC 37.9 ± 9.9a 1151 ± 575a 219 ± 85a 61.8 ± 10.7a 219 ± 85a 297 ± 60aWB + CU 25.1 ± 2.1a 1152 ± 259a 225 ± 61a 54.7 ± 21.8a 225 ± 61a 229 ± 70aWB + PAHs 23.2 ± 4.1a 960 ± 42a 183 ± 5a 24.5 ± 14.7a 183 ± 5b 253 ± 140aWB +SC + PAHs 41.9 ± 22.6a 489 ± 338a 152 ± 82a 29.6 ± 16.1a 152 ± 82a 219 ± 33aWB + CU + PAHs 22.6 ± 8.7a 1476 ± 442a 275 ± 67a 52.9 ± 6.2a 275 ± 67a 343 ± 114a

Values are the Mean ± SD (n = 3).Different lower case letter denote significant difference (P < 0.05) between the same plant in the same column.

Abbreviations: SC = sweet corn; CU = cucumber; WB = winged bean.NA = not available because the plant died.

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

420 K. SOMTRAKOON ET AL.

Table 2 Root length, root fresh weight and root dried weight of three plants (sweet corn, cucumber, wingedbean) grown in fluorene- and anthracene-contaminated or non-contaminated soil for 30 days.

Day 15 Day 30

Length Fresh Dried Length Fresh DriedPlant (cm) weight (mg) weight (mg) (cm) weight (mg) weight (mg)

Sweet Corn (SC)SC 7.1 ± 0.8a 84 ± 20a 11 ± 2a 4.4 ± 1.9ab 153 ± 50a 10 ± 1aSC + CU 6.4 ± 0.1a 82 ± 2a 8 ± 1a 4.5 ± 0.1ab 94 ± 20ab 9 ± 1aSC + WB 7.5 ± 0.7a 93 ± 16a 13 ± 2a 5.6 ± 1.5a 108 ± 0ab 10 ± 0aSC + PAHs 3.1 ± 1.5b 61 ± 38a 7 ± 7a 2.5 ± 1.4ab 47 ± 49b 6 ± 4aSC + CU + PAHs 3.6 ± 0.5b 67 ± 25a 11 ± 5a NA NA NASC + WB+ PAHs 2.9 ± 1.5b 51 ± 14a 10 ± 3a 1.1 ± 0.2b 43 ± 11b 5 ± 1a

Cucumber (CU)CU 6.0 ± 1.4ab 30 ± 4a 3 ± 0a 4.8 ± 1.0a 19 ± 16ab 2 ± 1aCU + SC 4.2 ± 0.2b 23 ± 5a 3 ± 0a 4.1 ± 2.0a 31 ± 3a 3 ± 1aCU + WB 7.7 ± 1.5a 36 ± 8a 3 ± 0a 5.8 ± 2.2a 42 ± 6a 3 ± 1aCU + PAHs 3.5 ± 1.5bc 16 ± 2a 3 ± 1a 3.4 ± 1.9a 24 ± 7ab 2 ± 1aCU + SC + PAHs 0.9 ± 0.3c 19 ± 13a 4 ± 1a NA NA NACU + WB + PAHs 2.8 ± 1.0bc 26 ± 5a 4 ± 1a 0.6 ± 0.1b 4 ± 1b BD

Wing Bean (WB)WB 13.0 ± 1.8a 167 ± 21b 33 ± 5a 16.5 ± 6.5ab 333 ± 180a 20 ± 14aWB + SC 11.9 ± 2.2a 89 ± 4b 19 ± 2b 12.5 ± 4.9ab 690 ± 289a 51 ± 7aWB + CU 11.2 ± 2.5a 169 ± 22b 33 ± 6a 22.6 ± 5.3a 912 ± 367a 39 ± 6aWB + PAHs 6.4 ± 3.3a 90 ± 25b 11 ± 1b 7.4 ± 5.2b 121 ± 60a 29 ± 19aWB + SC + PAHs 6.0 ± 3.5a 68 ± 20b 19 ± 2b 19.1 ± 7.3ab 320 ± 180a 28 ± 13aWB + CU + PAHs 8.1 ± 7.1a 304 ± 21a 28 ± 6a 31.5 ± 12.5a 644 ± 340a 56 ± 19a

Values are the Mean ± SD (n = 3).Different lower case letter denote significant difference (P<0.05) between the same plant in the same column.

Abbreviations: SC = sweet corn; CU = cucumber; WB = winged bean.NA = not available because the plant died.BD = below detection limit.

were only 3.1 ± 1.5, 2.9 ± 1.5, and 3.6 ± 0.5 cm when cultivated alone or in combinationwith winged bean and cucumber, respectively, on PAH-spiked soil. In comparison, the rootlength of sweet corn grown alone or in combination with another plant in un-spiked soilranged from 6.4–7.5 cm. On day 30, the root length of sweet corn was further reduced to1.1 cm when cultivated with winged bean in PAH-contaminated soil. The root fresh weightand dried weight of sweet corn grown alone or in combination with winged bean were notsignificantly different from each other in PAH-contaminated soil on both days 15 and 30after transplantation (Table 2).

The shoot length of cucumber was shortened when the plant was cultivated eitheralone or together with another plant in fluorene- and anthracene-contaminated soil ascompared to that of cucumber grown in un-spiked soil. At 30 days, the shoot lengths ofcucumber were only 18.3 ± 1.4 and 9.6 ± 0.1 cm when cultivated alone or with winged bean,respectively, in PAH-spiked soil (Table 1). In comparison, the shoot lengths of cucumberwere 38.0 ± 7.3 and 30.9 ± 2.9 cm when it was cultivated alone or together with wingedbean, respectively, in un-spiked soil after 30 days (Table 1). In contrast to shoot length,the shoot dry weight of cucumber grown alone or together with winged bean in fluorene-and anthracene-spiked soil were not statistically different (Table 1). The toxic effects of thePAHs were also observed in cucumber root. On day 30, the root lengths of cucumber grown

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

PHYTOREMEDIATION OF ANTHRACENE AND FLUORENE 421

in fluorene- and anthracene-spiked soils were 0.6 ± 0.1 and 3.4 ± 1.9 cm when grownwith winged bean and alone, respectively (Table 2). The root fresh weight of cucumbercultivated with winged bean in fluorene- and anthracene-spiked soils were lower than thatcultivated in un-spiked soil. At 30 days, the root fresh weight of cucumber was only 4 ±1 mg when the plant was grown together with winged bean, while the root dried weightwas below the detection limit of 0.1 mg (Table 2).

The presence of fluorene and anthracene in soil did not significantly affect the shootand root growth of winged bean. Cultivation of winged bean either alone or with anotherplant resulted in similar shoot (Table 1) and root lengths and weights (Table 2) in bothPAH-contaminated or non-contaminated soils.

Chlorophyll Content of Plants Grown in Fluorene- and Anthracene

Contaminated Soils

When sweet corn was grown with cucumber in fluorene- and anthracene-spiked soilsfor 15 days, the chlorophyll a, chlorophyll b and total chlorophyll contents in leaves were3.2 ± 0.0, 2.1 ± 0.0, and 5.5 ± 0.0 mg ml−1, respectively (Table 3). These values were very

Table 3 Chorophyll a, chlorophyll b and total chlorophyll of three plants (sweet corn, cucumber, winged bean)grown in fluorene- and anthracene-contaminated or non-contaminated soil for 30 days.

Day 15 Day 30

Total TotalChlorophyll Chlorophyll chlorophyll Chlorophyll Chlorophyll chlorophyll

Plant a (mg ml−1) b (mg ml−1) (mg ml−1) a (mg ml−1) b (mg ml−1) (mg ml−1)

Sweet Corn (SC)SC 8.4 ± 4.8c∗ 6.58 ± 0.3c 15.4 ± 0.4c 21.8 ± 0.8a 26.1 ± 11.3a 49.1 ± 10.6aSC + CU 21.8 ± 0.1a 18.5 ± 0.4a 41.4 ± 0.4a 21.6 ± 0.0a 20.1 ± 0.0a 42.9 ± 0.0aSC + WB 16.1 ± 0.6b 14.2 ± 0.4b 31.2 ± 0.4b 21.6 ± 0.0a 20.1 ± 0.0a 42.9 ± 0.0aSC + PAHs 15.7 ± 0.2b 14.1 ± 0.2b 30.7 ± 0.0b 20.9 ± 0.8a 18.7 ± 5.2a 40.7 ± 5.8aSC + CU + PAHs 3.2 ± 0.0d 2.1 ± 0.0c 5.5 ± 0.0d NA NA NASC + WB + PAHs 15.7 ± 0.2b 14.3 ± 0.4b 30.8 ± 0.3b 21.3 ± 0.6a 16.0 ± 4.5a 38.4 ± 5.2a

Cucumber (CU)CU 19.3 ± 3.2a 24.0 ± 9.2a 44.4 ± 9.2ab 24.0 ± 1.7a 26.7 ± 17.4a 52.0 ± 15.8aCU + SC 20.8 ± 0.1a 32.8 ± 0.1a 54.7 ± 0.0a 21.3 ± 0.2b 32.3 ± 2.6a 54.8 ± 2.5aCU + WB 18.5 ± 5.2a 15.6 ± 1.7a 35.2 ± 3.8b 21.2 ± 0.1b 28.4 ± 3.1a 50.8 ± 3.0aCU + PAHs 11.6 ± 0.2b 19.8 ± 1.0b 32.0 ± 0.9c 21.3 ± 0.1b 20.3 ± 12.7a 51.6 ± 0.6aCU + SC + PAHs 21.2 ± 0.3a 21.6 ± 0.1a 43.9 ± 0.4ab NA NA NACU + WB + PAHs 21.4 ± 0.1a 21.4 ± 0.1a 44.0 ± 0.1ab 2.8 ± 0.0c 1.2 ± 0.0b 4.2 ± 0.0b

Winged Bean (WB)WB 21.4 ± 0.4a 21.5 ± 9.4a 44.2 ± 9.8a 21.0 ± 0.2a 31.3 ± 1.6a 53.4 ± 1.4aWB + SC 22.4 ± 0.4a 31.5 ± 0.5a 55.2 ± 0.7a 21.4 ± 0.1a 22.9 ± 0.6a 43.2 ± 0.5aWB + CU 21.3 ± 0.1a 16.1 ± 0.1a 38.6 ± 0.1b 20.9 ± 0.1a 30.2 ± 0.6a 52.2 ± 0.5aWB + PAHs 9.5 ± 0.0b 19.8 ± 1.0a 16.3 ± 0.5c 18.6 ± 4.7a 29.1 ± 0.6a 39.9 ± 17.6aWB + SC + PAHs 22.0 ± 0.2a 20.0 ± 0.0a 39.9 ± 17.6a 21.2 ± 0.4a 27.2 ± 9.4a 49.6 ± 9.0aWB + CU + PAHs 21.4 ± 0.0a 18.2 ± 0.4a 40.7 ± 0.4a 20.4 ± 2.7a 14.4 ± 10.0a 35.8 ± 15.3a

Values are the Mean ± SD (n = 3).Different lower case letter denote significant difference (P < 0.05) between the same plant in the same column.

Abbreviations: SC = sweet corn; CU = cucumber; WB = winged bean.NA = not available because the plant died.

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

422 K. SOMTRAKOON ET AL.

low compared to the chlorophyll contents in leaves of sweet corn grown under all otherconditions. Further underscoring their poor health, sweet corn grown with cucumber diedaround 25–30 days after transplantation. In contrast, the chlorophyll a, chlorophyll b andtotal chlorophyll contents in leaves of sweet corn cultivated alone or with winged bean influorene- and anthracene-spiked soils were not significantly different from those of plantsgrown in un-spiked soil at 30 days (Table 3).

The chlorophyll a and chlorophyll b contents in leaves of cucumber grown alone influorene- and anthracene-spiked soil were significantly lower than those of plants grown inun-spiked soil at 15 days after transplantation. On day 30, chlorophyll b and total chlorophyllcontent in leaves of cucumber grown alone in fluorene- and anthracene-spiked soil was notsignificantly different from that grown alone and grown together with another plant inun-spiked soils. However, chlorophyll a content in leaves of cucumber grown alone in un-spiked soils was higher than those of cucumber grown alone in fluorene- and anthracene-spiked soil and grown together with another plant in spiked and un-spiked soil on day 30.In fluorene- and anthracene-contaminated soils, the chlorophyll a, chlorophyll b and totalchlorophyll contents of leaves of cucumber cultivated with winged bean for 30 days were2.8 ± 0.0, 1.2 ± 0.0, and 4.2 ± 0.0 mg ml−1, respectively. These values are significantlylower than those of leaves of cucumber grown alone [(the chlorophyll a, chlorophyll band total chlorophyll contents were 21.3 ± 0.1, 20.3 ± 12.7, and 51.6 ± 0.6 mg ml−1,respectively]. Moreover, the cucumber plant appeared unhealthy with the shoots wiltingand leaves exhibiting chlorosis; they died around 20–25 days after transplantation whenco-cultivated with corn (Table 3). In contrast, the chlorophyll a, chlorophyll b and totalchlorophyll contents of leaves of winged bean cultivated alone or together with anotherplant in fluorene- or anthracene-spiked soils were not significantly different from those ofleaves of winged bean grown in un-spiked soils at 30 days after transplantation (Table 3).

Removal of Fluorene and Anthracene from Soil in Single Plant Pots

Generally, fluorene and anthracene were removed to a greater extent from soil inpots grown with sweet corn, winged bean or cucumber alone as compared to unplantedcontrol pots. For example, fluorene and anthracene concentrations were reduced to 13.8and 8.8% in rhizospheric soil of sweet corn grown alone on day 30. In contrast, the amountof fluorene and anthracene remaining were 60.8 and 76.1%, respectively, in unplantedcontrols on day 30. In sweet corn grown pots, fluorene was removed to a greater extentin the bulk soil (2.0% remaining) than rhizospheric soils (13.8% remaining) on day 30(Table 4). In contrast, anthracene was removed to a greater extent in the rhizospheric soil(8.8% remaining) than bulk soil (49.7% remaining) on day 30 (Table 4).

The removal of both fluorene and anthracene in rhizospheric soil of cucumber wasgreater than in bulk soil. Only 4.6% and 34.3% of fluorene remained in rhizospheric andbulk soil, respectively, of cucumber on day 30 after transplantation (Table 4). In contrast, theremoval of anthracene was similar in rhizospheric and bulk soil of cucumber; the amountsof anthracene remaining were 9.5% and 12.0% in rhizospheric and bulk soil, respectively,on day 30 (Table 4).

In pots grown with winged bean, the removal of fluorene and anthracene from soilwas slow and in general more hydrocarbons were removed in rhizospheric soil than bulksoil (Table 4). About 7.8% of fluorene remained in rhizospheric soil of winged bean on day30. Winged bean was less efficient at removing anthracene from soil compared to the other

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

PHYTOREMEDIATION OF ANTHRACENE AND FLUORENE 423

Table 4 Percentage of fluorene and anthracene remaining in soil grown with sweet corn, winged bean andcucumber grown alone in spiked soil. The initial concentrations of fluorene and anthracene were 95.9 and138.9 mg/kg, respectively.

% of PAH remaining

Fluorene Anthracene

Plant Day 15 Day 30 Day 15 Day 30

Rhizospheric soil of SC 11.3 ± 9.0b∗ 13.8 ± 20.0b∗ 14.3 ± 4.6d∗ 8.8 ± 2.3c∗Bulk soil of SC 103.7 ± 15.6a 2.0 ± 1.4bc∗ 98.8 ± 3.7a 49.7 ± 15.3ab∗Rhizospheric soil of CU 6.2 ± 4.6b∗ 4.6 ± 4.0bc∗ 38.3 ± 0.7cd∗ 9.5 ± 10.1c∗Bulk soil of CU 99.4 ± 11.4a 34.3 ± 13.9ab∗ 54.2 ± 5.0c∗ 12.0 ± 15.9c∗Rhizospheric soil of WB 89.6 ± 23.0a 7.8 ± 1.1c∗ 63.0 ± 30.5d∗ 24.2 ± 12.0bc∗Bulk soil of WB 103.1 ± 17.8a 37.2 ± 33.8ab∗ 26.6 ± 9.8bc∗ 39.6 ± 6.6bc∗No Plant (Control) 87.5 ± 11.8a 60.8 ± 18.1a∗ 85.4 ± 17.8ab 76.1 ± 21.1a

Values are the Mean ± SD (n = 3).Different lower case letter showed significant difference (P < 0.05) between treatment on the same day.∗showed significant difference between the amount of PAHs remaining compared to the values on day 0 for

each treatment. Abbreviations: SC = sweet corn; CU = cucumber; WB = winged bean.

plants. On day 30, 24.2% and 39.6% of anthracene remained in rhizospheric and bulk soils,respectively (Table 4).

Removal of Fluorene and Anthracene from Soil in Pots with Two Plants

In pots grown with sweet corn and cucumber, good removal of fluorene from soilwas seen, even though both plants died before the end of the 30-day experiment. About14.0% and 17.3% of fluorene remained in rhizospheric soils of sweet corn and cucumber,respectively, on day 15 (Table 5).

Removal of anthracene was slower than fluorene in pots grown with both sweet cornand cucumber. The amounts of anthracene remaining were 37.7% and 52.9% in rhizosphericsoil of sweet corn and cucumber, respectively, on day 15 (Table 5). However, anthraceneseemed to be removed at a greater rate by sweet corn or cucumber grown alone than whenthey were grown together; only 14.3% and 38.3% of anthracene remained in rhizosphericsoil of sweet corn and cucumber, respectively, grown alone for 15 days (Table 4).

Co-cultivation of sweet corn and winged bean stimulated fluorene and anthraceneremoval from soil to a greater extent than co-cultivation of sweet corn and cucumberor cucumber and winged bean. Only 4.1%, 3.4%, and 18.8% of fluorene remained inrhizospheric soil of sweet corn and winged bean and bulk soil, respectively, on day 30(Table 5). However, both plants also removed fluorene from soil effectively when each wasgrown alone (Table 4).

Anthracene was removed to a greater extent from soil when sweet corn and wingedbean were grown together as compared to when each was grown alone. Only 8.8% and14.3% of anthracene remained in rhizospheric soil of sweet corn and winged bean growntogether, respectively, on day 30 (Table 5). On the other hand, the removal of anthracenefrom soils was limited when winged bean was grown alone; the amount of anthraceneremaining was 24.2% in rhizospheric soil of winged beans grown alone on day 30 (Table 4).However, a greater amount of anthracene was removed from the bulk soil by sweet corn and

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

424 K. SOMTRAKOON ET AL.

Table 5 Percentage of fluorene and anthracene remaining in soil grown with sweet corn, winged bean andcucumber in pairs in spiked soil. The initial concentration of fluorene and anthracene were 95.9 and 138.9 mg/kg,respectively.

% of PAH remaining

Fluorene Anthracene

Plant Day 15 Day 30 Day 15 Day 30

SC + CURhizospheric soil of SC 14.0 ± 2.3b∗ NA 37.7 ± 7.9c∗ NARhizospheric soil of CU 17.3 ± 3.5b∗ NA 52.9 ± 14.5c∗ NABulk soil of SC + CU 33.0 ± 25.8b∗ NA 73.7 ± 29.4ab NA

SC + WBRhizospheric soil of SC 74.4 ± 1.6a 4.1 ± 0.6bc∗ 70.1 ± 35.3ab∗ 8.8 ± 5.5b∗Rhizospheric soil of WB 4.0 ± 4.5b∗ 3.4 ± 1.1c∗ 61.6 ± 25.9ab∗ 14.3 ± 18.2b∗Bulk soil of SC + WB 85.7 ± 27.2a 18.8 ± 4.9bc∗ 52.0 ± 14.9c∗ 12.2 ± 1.4b∗

CU + WBRhizospheric soil of CU 6.15 ± 1.7b∗ BD 75.1 ± 29.7ab BDRhizospheric soil of WB 100.4 ± 5.0a 37.8 ± 7.9ab∗ 54.6 ± 8.4bc∗ 25.3 ± 5.2b∗Bulk soil of CU + WB 101.8 ± 21.3a 28.0 ± 23.1bc∗ 83.4 ± 10.1a 21.0 ± 18.9b∗

No Plant (Control) 87.5 ± 11.8a 60.8 ± 18.1a∗ 85.4 ± 17.8a 76.1 ± 21.1a

Values are the Mean ± SD (n = 3).Different lower case letter showed significant difference (P < 0.05) between treatment on the same day.∗showed significant difference between the amount of PAHs remaining compared to the values on day 0 for

each treatment. Abbreviations: SC = sweet corn; CU = cucumber; WB = winged bean; NA = not availablebecause the plant died; BD = below detection limit.

winged bean grown together as compared to when each plant was grown alone. Only 12.2%of anthracene remained in bulk soil of pots grown with both plants on day 30 (Table 5).

On day 15, the amount of fluorene remaining was only 6.2% in rhizospheric soil ofcucumber when it was cultivated with winged bean (Table 5). Single cultivation of wingedbean was more effective at removing fluorene from soil than co-cultivation of winged beanwith cucumber. The amounts of fluorene remaining on day 30 were 37.8% and 7.8% inrhizospheric soil of winged bean grown together with cucumber (Table 5) or alone (Table 4),respectively. Less anthracene was removed by co-cultivation of cucumber and winged beanthan when each plant was grown alone. On day 30, the amount of anthracene remainingin rhizospheric soil of winged bean was 25.3% when it was cultivated with cucumber. Thecucumber plant died before day 30, thus no rhizospheric soil was collected for analysis.However, cucumber did not promote anthracene removal from soil before it died; about75.1% of anthracene remained on day 15 in rhizospheric soil of cucumber when it wascultivated with winged bean (Table 5).

Measurement of Fluorene and Anthracene in Plant Tissues

Fluorene and anthracene were not detected (detection limit was 0.2 mg kg−1 for eachcompound) in shoot and root samples of winged bean, sweet corn and cucumber irrespectiveof whether the plants were cultivated alone or in pairs. The tissue samples were collectedat either 15 or 30 days after transplantation. Thus, the PAHs were most likely degraded

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

PHYTOREMEDIATION OF ANTHRACENE AND FLUORENE 425

by indigenous microorganisms in rhizospheric and bulk soil. Potential metabolites of thesePAHs were not measured.

DISCUSSION

Several lines of evidence from this study show that winged bean was the best of the3 plants tested for use in phytoremediation of PAH-contaminated soils either grown aloneor in combination with other plants. First, the growth of winged bean in fluorene- andanthracene-spiked soil was not significantly different from that in un-spiked soil. Second,growth of winged bean was not adversely affected by co-cultivation with other plants.Third, fluorene was removed to a greater extent in the rhizospheric and bulk soil of wingedbean cultivated either alone or together with sweet corn and cucumber.

Phytostimulation has been suggested to be the main mechanism by which legumessuch as vetch (Vicia sativa L.), alfalfa (Medicago sativa), pea (Pisum sativum) and yellowlupine (Lupinus luteus) can remove PAHs from contaminated soils (Liste and Felgentreu2006; Liste and Prutz 2006). The plant rhizosphere is known to harbor competent microor-ganisms capable of degrading PAHs. The roots of legume could exude some compounds,such as flavonoids and phenolics, the structures of which resemble the aromatic ring ofPAHs. These compounds may induce the production of PAH-degrading enzymes by rhizo-spheric microorganisms to enhance PAH biodegradation (Hall et al. 2011). Several legumeswere reported to decrease PAHs from contaminated sites. For example, cultivation of al-falfa for 60 days decreased pyrene concentrations by 69.2% from an initial concentrationof 493 mg kg−1 soil (Fan et al. 2008). The presence of plant root enhanced pyrene re-moval more than in the non-rhizospheric soil by 6% (Fan et al. 2008). After 80 days,Aeschynomene indica was reported to decrease phenanthrene and pyrene concentrationsin soil from 87.56 and 98.62 mg kg−1 to 0.72 and 23.00 mg/kg, respectively. The amountof phenanthrene and pyrene remaining in unplanted soil were 1.19 and 31.29 mg kg−1,respectively, over the same time period (Lee et al. 2008).

In this study, planting of sweet corn and cucumber led to the highest extent of removalof anthracene from soil as compared to winged bean. Corn has been used in several PAHphytoremediation studies due to its ability to stimulate PAH degradation by microorganismsin the rhizosphere (Chouychai et al. 2009; Chouychai et al. 2012; Liste and Prutz 2006;Xu et al. 2006). Corn root exudates could stimulate microbial growth in the rhizosphereand this in turn enhanced the mineralization of pyrene in soil (Yoshitomi and Shann 2001).Unfortunately, in our current study, sweet corn was found not to tolerate the toxicity of amixture of fluorene and anthracene well. This limits its utility for phytoremediation of thistype of PAH contaminants.

Although cucumber could remove both fluorene and anthracene from soil well, itwas also judged to be unsuitable for phytoremediation of these PAHs because our resultsshowed that cucumber was the most sensitive to fluorene and anthracene contaminants insoil. Cucumber was less healthy when grown together with sweet corn or winged bean andthe cucumber plant died between 25–30 days after transplantation when grown togetherwith sweet corn.

In this study, single plant cultivation led to high efficiency of PAH removal fromsoils. However, contaminated sites typically contain multiple plants. Thus, co-cultivationof plants may provide information that is more representative of contaminated sites, asthey offer more realistic interaction between the root and root exudates of different plants.Several studies have reported on the benefits of plant co-cultivation to phytoremediation.

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

426 K. SOMTRAKOON ET AL.

In our study, co-cultivation of sweet corn and cucumber led to early death of both plants.Consistent with the poor health of these co-cultivated plants, their chlorophyll contentswere decreased on day 15 after transplantation. The reason for the poor health of theseplants when they are grown together is not known.

There have been recent reports of using co-cultivation of plants to remove PAHsfrom soil. For example, co-cultivation of rapeseed together with alfalfa could stimulatephenanthrene and pyrene degradation better than cultivation of alfalfa alone (Wei andPan 2010). In addition, co-cultivation of rapeseed and alfalfa reduced the accumulation ofphenanthrene and pyrene in root and shoot tissues (Wei and Pan 2010). In another study,cultivation of birch (Betula pendula) and mulberry (Morus rubra) along with ryegrass(Lolium perenne) for one year was found to increase the biodegradation of several PAHs inaged contaminated soils (Rezek et al. 2009). Here, fluoranthene and pyrene concentrationswere reduced from 103.5 and 83.3 mg kg−1 to 28 and 18 mg kg−1, respectively (Rezeket al. 2009). In the third study, co-cultivation of tall fescue (Festuca arundinacea) witheither alfalfa or rapeseed (Brassica napus) or rapeseed with alfalfa together decreased theamounts of phenanthrene and pyrene from contaminated soil as compared to single plantcultivation (Cheema et al. 2010). After 65 days, combined plant cultivation could reducephenanthrene and pyrene concentrations by 98.3–99.2% and 88.1–95.7%, respectively. Incomparison, single plant cultivation reduced phenanthrene and pyrene concentrations by97–98% and 79.8–86%, respectively (Cheema et al. 2010).

SUMMARY

In summary, the co-cultivation of corn and winged bean was the most efficientpair for phytoremediation of soil contaminated with anthracene and fluorene. The co-cultivation of corn and cucumber was the least efficient because of poor plant health,as both plants died before day 30. Among the three plants tested, winged bean was themost effective in promoting anthracene and fluorene removal from soil. There was noevidence of accumulation of these two PAHs in any of the plant tissues during the 30-dayexperiment in which 70–90% of fluorene and 70–80% of anthracene were removed fromsoil. Phytostimulation of microbial degradation in the rhizosphere was most likely themechanism by which the PAHs were removed from the contaminated soil.

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial support from Faculty of Science,Mahasarakham University (Grant no. MSU-SC 14/55).

REFERENCES

Banks MK, Kulakow P, Schwab AP, Chen Z, Rathbone K. 2003. Degradation of crude oil in therhizosphere of Sorghum bicolor. Int J Phytoremed 5(3): 225–234.

Campbell S, Arakaki AS, Li QX. 2009. Phytoremediaion of heptachlor and heptachlor epoxide insoil by Cucurbitaceae. Int J Phytoremed 11(1): 28–38.

Cheema SA, Khan MI, Shen C, Tang X, Farooq M, Chen L, Zhang C, Chen Y. 2010. Degradation ofphenanthrene and pyrene in spiked soils by single and combined plants cultivation. J HazardMat 177: 384–389.

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

PHYTOREMEDIATION OF ANTHRACENE AND FLUORENE 427

Chouychai W, Tongkukiatkul A, Upatham S, Lee H, Pokethitiyook P, Kruatrachue M. 2009. Plant-enhanced phenanthrene and pyrene biodegradation in acidic soil. J Environ Biol 30(1):139–144.

Chouychai W, Tongkukiatkul A, Upatham S, Pokethitiyook P, Kruatrachue M, Lee H. 2012. Effectof corn plant on survival and phenanthrene degradation capacity of Pseudomonas sp. UG14Lrin two soils. Int J Phytoremed 14(6): 585–595.

Fan S, Li P, Gong Z, Ren W, He N. 2008. Promotion of pyrene degradation in rhizosphere of alfalfa(Medicago sativa L.). Chemosphere 71: 1593–1598.

Gadde B, Bonnet S, Menke C, Gariviat S. 2009. Air pollutant emission from rice straw open fieldburning in India, Thailand, and the Philippines. Environ Pollut 157: 1554–1558.

Hall J, Soole K, Bentham R. 2011. Hydrocarbon phytoremediation in the family fabaceae—a review.Int J Phytoremed 13(4): 317–332.

Hao R, Wan H, Song Y, Jiang H, Peng S. 2007. Polycyclic aromatic hydrocarbons in agriculturalsoils of the southern subtropics, China. Pedosphere 17(5): 673–680.

Huang XD, Zeiler LF, Dixon DG, Greenberg BM. 1996. Photoinduced toxicity of PAHs to the foliarregions of Brassica napus (Canola) and Cucumbis sativus (Cucumber) in simulated solarradiation. Ecotoxicol Environ Saf 35: 190–197.

Inui H, Wakai T, Gion K, Kim YS, Eun H. 2008. Different uptake for dioxin-like compounds byzucchini subspecies. Chemosphere 73: 1602–1607.

Kirk JL, Klironomos JW, Lee H, Trevors JT. 2002. Phytotoxicity assay to assess plant species forphytoremediation of petroleum-contaminated soil. Bioremed J 61: 57–63.

Kummerova M, Krulova J, Zezulka S, Trıska J. 2006. Evaluation of fluoranthene phytotoxicity inpea plants by Hill reaction and chlorophyll fluorescence. Chemosphere 65: 489–496.

Kummerova M, Vanova L, Krulova J, Zezulka S. 2008. The use of physiological characteristics forcomparison of organic compounds phytotoxicity. Chemosphere 71: 2050–2059.

Lee SH, Lee WS, Lee CH, Kim JG. 2008. Degradation of phenanthrene and pyrene in rhizosphere ofgrasses and legumes. J Hazard Mat 153: 892–898.

Liste H, Felgentreu D. 2006. Crop growth, culturable bacteria, and degradation of petrol hydrocarbons(PHCs) in a long-term contaminated field soil. Appl Soil Ecol 31: 43–52.

Liste H, Prutz I. 2006. Plant performance, dioxygenase-expressing rhizosphere bacteria, and biodegra-dation of weathered hydrocarbons in contaminated soil. Chemosphere 62: 1411–1420.

Maila MP, Randima P, Cloete TE. 2005. Multispecies and monoculture rhizoremediation of poly-cyclic aromatic hydrocarbons (PAHs) from the soil. Int J Phytoremed 7(2): 87–98.

Mehmannavaz R, Prasher SO, Ahmad D. 2002. Rhizosphere effects of alfalfa on biotransformationof polychlorinated biphenyls in a contaminated soil augmented with Sinorhizobium meliloti.Process Biochem 37: 955–963.

Merkl N, Schultze-Kraft R, Arias M. 2006. Effect of the tropical grass Brachiaria brizantha (Hochst.ex A. rich.) stapf on microbial population and activity in petroleum-contaminated soil. Micro-biol Res 161: 80–91.

Reynoso-Cuevas L, Gallegos-Martınez ME, Cruz-Sosa F, Gutierrez-Rojas M. 2008. In vitro evalua-tion of germination and growth of five plant species on medium supplemented with hydrocar-bons associated with contaminated soils. Bioresources Technol 99: 6379–6385.

Rezek J, in der Wiesche C, Mackova M, Zadrazil F, Macek T. 2009. Biodegradation of PAHs inlong-term contaminated soil cultivated with European white birch (Betula pendula) and redmulberry (Morus rubra) tree. Int J Phytoremed 11(1): 65–80.

Wei S, Pan S. 2010. Phytoremediation for soils contaminated by phenanthrene and pyrene withmultiple plant species. J Soil Sediment 10: 886–894.

Whitfield Åslund ML, Zeeb BA, Rutter A, Reimer KJ. 2007. In situ Phytoextraction of polychlorinatedbiphenyl-(PCB) contaminated soil. Sci Total Environ 374: 1–12.

Whitfield Åslund ML, Lunnery AI, Rutter A, Zeeb BA. 2010. Effects of amendments on the uptakeand distribution of DDT in Cucurbita pepo ssp. pepo plants. Environ Pollut 158: 508–513.

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14

428 K. SOMTRAKOON ET AL.

Yoshitomi KJ, Shann JR. 2001. Corn (Zea may) root Exudates and their impact on 14C-pyrenemineralization. Soil Biol Biochem 33: 1769–1776.

Xu SY, Chen YX, Wu WX, Wang KX, Lin Q, Liang XQ. 2006. Enhanced dissipation of phenanthreneand pyrene in spiked soils by combined plants cultivation. Sci Total Environ 363: 206–215.

Zehetner F, Rosenfellner U, Mentler A, Gerzabek MH. 2009. Distribution of road salt residues, heavymetals and polycyclic aromatic hydrocarbons across a highway-forest interface. Water, Air,Soil Pollut 198: 125–132.

Dow

nloa

ded

by [

Uni

vers

ity o

f W

inni

peg]

at 0

9:35

05

Sept

embe

r 20

14