phytoremediation of subarctic soil contaminated with diesel fuel

Phytoremediation of subarctic soil contaminated with diesel fuel

Marja R.T. Palmroth a,*, John Pichtel b, Jaakko A. Puhakka a

a Institute of Environmental Engineering and Biotechnology, Tampere University of Technology, P.O. Box 541, 33101 Tampere, Finlandb Natural Resources and Environmental Management, Ball State University, Muncie, IN 47306-0495, USA

Received 25 January 2002; received in revised form 21 February 2002; accepted 25 February 2002

Abstract

The effects of several plant species, native to northern latitudes, and different soil amendments, on diesel fuel removal from soil

were studied. Plant treatments included Scots Pine (Pinus sylvestris), Poplar (Populus deltoides�Wettsteinii), a grass mixture (Red

fescue, Festuca rubra; Smooth meadowgrass, Poa pratensis and Perennial ryegrass, Lolium perenne) and a legume mixture (White

clover, Trifolium repens and Pea, Pisum sativum). Soil amendments included NPK fertiliser, a compost extract and a microbial

enrichment culture. Diesel fuel disappeared more rapidly in the legume treatment than in other plant treatments. The presence of

poplar and pine enhanced removal of diesel fuel, but removal under grass was similar to that with no vegetation. Soil amendments

did not enhance diesel fuel removal significantly. Grass roots accumulated diesel-range compounds. This study showed that utili-

sation of selected plants accelerates removal of diesel fuel in soil and may serve as a viable, low-cost remedial technology for diesel-

contaminated soils in subarctic regions. � 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Diesel fuel; Grasses; Hydrocarbons; Legume; Phytoremediation; Pine; Poplar; Soil amendments; Subarctic soil

1. Introduction

Hydrocarbons comprise the most common categoryof environmental contaminants in industrialised coun-tries. There are urgent needs to find effective, low-costtechnologies to clean up contaminated soils. Phyto-remediation uses green plants to remove, contain orrender harmless, environmental contaminants. Phyto-remediation can be an alternative to harsher cleanuptechnologies, which drastically alter the chemical andphysical properties of a soil, creating a relatively inertmaterial.

Plants or plant-associated microflora are known totransform certain pollutants to non-toxic forms (Cunn-ingham and Berti, 1993; Cunningham et al., 1995). Inaddition, plants exude organic and inorganic substancesto the soil environment during normal metabolism(Anderson et al., 1993). Root exudates help to degradetoxic organic chemicals and act as substrates for soilmicro-organisms. Certain plants have been found todecompose a limited range of fuel hydrocarbon com-pounds in soil. Planting soil with alfalfa and horseradish

reduced concentration of kerosene-based jet fuel by 57–90% in five months (Karthikeyan et al., 1999). Chaııneauet al. (2000) grew maize (Zea mays L.) in soil micro-cosms and liquid cultures containing 3300 mg/kg fueloil. They observed that degradation of saturated andaromatic hydrocarbons was faster in the presence ofmaize. Wiltse et al. (1998) found that various strains ofalfalfa (Medico sativa L.) were capable of reducing crudeoil contamination in the rhizosphere by 33–56% com-pared to control treatments, while 80% of diesel fueldecomposed after eight weeks with an alfalfa treatment(Komisar and Park, 1997) and 46% of crude oil wasremoved in 12 weeks with broad bean (Vicia faba),compared to 33% with no plants (Radwan et al., 2000).Introduction of fertilisers into oil-contaminated, non-vegetated soil has been shown to enhance the degrada-tion of hydrocarbons via biostimulation (Venosa et al.,1996).

Many studies have shown that microbial counts in-crease in soil contaminated with hydrocarbons andvegetation increases the effect. Lee and Banks (1993)found that viable counts in soil spiked with polyaro-matic and aliphatic hydrocarbons (final concentration100 ppm) were greater in the presence of alfalfa than insoil without plants. Radwan et al. (1995) found morehydrocarbon-utilising micro-organisms in the root zone

Bioresource Technology 84 (2002) 221–228

*Corresponding author. Tel.: +358-3-3115-2687; fax: +358-3-3115-

2869.

E-mail address: [email protected] (M.R.T. Palmroth).

0960-8524/02/$ - see front matter � 2002 Elsevier Science Ltd. All rights reserved.

PII: S0960-8524 (02 )00055-X

than in non-rhizosphere samples. Komisar and Park(1997) observed high microbial counts in diesel-con-taminated soil, and removal was more rapid in vegetated(alfalfa) soils.

In the reported study several plant species, native tonorthern latitudes, and different soil amendments wereevaluated for their ability to contribute to hydrocarbonremoval. The purpose was to screen plant species andsoil amendments for use in field and full-scale appli-cations. Furthermore, diesel fuel removal mechanismswere investigated to determine if phytoaccumulationand phytodegradation in addition to rhizodegradationplayed a role in diesel fuel removal. Possible toxicity ofdiesel fuel to plants was assessed to determine appro-priate concentrations for the plant species under study.

2. Methods

Soil material used in a growth chamber study wascollected from A and B horizons of a mixed coniferousforest in Tampere, Finland. The soil was mixed andsieved through a 3-mm mesh sieve. Soil pH was mea-sured in a 1:2 (w:v) soil:deionized water slurry. Organiccarbon content of the soils was determined by loss onignition at 600 �C (Nelson and Sommers, 1982) andtotal P was determined using the Bray-1 method (Olsenand Sommers, 1982). Cation exchange capacity wasdetermined by Ba replacement (Rhoades, 1982). Theconcentrations of soluble NO�

3 , PO3�4 and SO2�

4 weremeasured on a 10:1 soil:water extract using a Dionexmodel 2000i ion chromatograph. Metals were analysedby EPA method 3051. Soil particle size analysis wasdone by dry and wet sieving and additionally by sedi-graph.

Plant treatments included Scots pine; Poplar; a grassmixture; and a legume mixture (Table 1). In addition to

plants, four different soil amendments were utilised(Table 1), including a commercial mixed nitrogen–phosphorus–potassium (NPK) fertiliser (Biolan, Eura,Finland), compost extract, microbial enrichment cul-ture, and no amendments. The compost extract wasprepared from matured biowaste compost obtainedfrom the Tarastenj€aarvi landfill, Tampere. Four hundredgrams of sieved compost and 1 l of distilled water wasmixed in a water bath at 40 �C for 5 h and the decantedliquid was applied. Total organic carbon of the filtered(0.45 lm) extract was 700 mg/l. For the microbial en-richment culture treatment, a total of 1:6� 1010 cells(DAPI total cell count) was applied per pot. The mi-crobial enrichment medium was prepared from DSM465 mineral medium (DSMZ, German collection ofmicro-organisms and cell cultures), in which magnesiumchloride was replaced with magnesium sulphate. 0.6% ofdiesel fuel was used as sole carbon source and 0.02%of the same Tampere soil material was used as inoculum.

Commercial diesel fuel (Neste Futura) was mixedwith the soil at an initial concentration of 0.5% (w/w),and 500 g of the field-moist soil was placed into plasticpots. The soil–diesel mixture was allowed to stabilise forfive days before planting. Four replicates of each planttreatment/soil treatment were used. The study was con-ducted at room temperature, approximately 20–22 �C.Watering of pots with tap water was maintained atmoderate levels, so that no leachate was produced.Control treatments consisted of pots of each plant treat-ment without diesel or amendment application. Addi-tional control pots were mixed with 0.5% diesel, andtreated initially and at 50 days, with 0.5% NaN3 to de-termine non-biological losses of diesel compounds.A total of 100 pots was used in this study.

The physical appearance of the plants was monitoredthroughout the experiment. At the termination of theexperiment needle and leaf length, yearly growth of

Table 1

Design of phytoremediation experiments

Plant treatment Addition Details

Design of plant treatments

Scots pine One year old seedlings (Pinus sylvestris)

Poplar One year old seedlings (Populus deltoids�Wettsteinii)

Grass mixture Seeds 60% red fescue (Festuca rubra)

30% smooth meadow-grass (Poa pratensis)

10% perennial rye-grass (Lolium perenne)

Legume mixture Seeds White clover (Trifolium repens)

Green pea (Pisum sativum)

Soil treatment Addition to 500 g of soil Details

Design of soil treatments

NPK fertiliser 25 mg 16.6% N, 4% P and 25.3% K

Compost extract 5 ml Biowaste compost extract, TOC 700 mg/l

Microbial enrichment culture 1:6� 1010 cells (DAPI total cell count) 0.6% of diesel fuel and 0.02% sieved soil added in DSM 465

mineral medium, incubated two weeks on a rotary shaker (150

rpm) at 20–22 �C

222 M.R.T. Palmroth et al. / Bioresource Technology 84 (2002) 221–228

pines and possible toxicity/deficiency symptoms of thetrees were recorded. Average needle lengths, yearlygrowth and symptoms were divided into three classes(0 ¼ healthy, 1 ¼ some symptoms, 2 ¼ symptoms in>50% of needles/leaves). The symptoms recorded werechlorosis, necrosis and burns as well as abnormalgrowth.

Four replicate samples were collected from each plantand soil treatment combination every 30 days. Samplesfrom pots with trees were collected until day 60.Thereafter, the trees were transferred outside for thewinter. The effect of recontamination of soil with 2%diesel fuel was studied during the following summer asa short-term, one-month experiment for the trees only.Other treatments continued until 180 days. In the short-term experiment, control pots without vegetation, steri-lised and not poisoned, were used to separate the effectsof the plant on decomposition of diesel compounds. Soilwas sterilised by autoclaving twice to remove spores andby a subsequent addition of 0.5% NaN3.

To assess hydrocarbon removal, 2 g of soil samplewas extracted twice with 3 ml of HPLC-quality n-hexane(Merck, Darmstadt, Germany) in an ultrasonic bath,and the extracts were combined. The extracts wereanalysed for hydrocarbons using gas chromatographywith a mass-selective detector (GC/MSD) HP-6890 inscan mode. The GC was equipped with cross-linked5% phenyl methyl siloxane capillary column, HP-5MS.Helium was used as carrier gas. The temperature pro-gram was started at 40 �C and raised by 10 �C/min until300 �C, which was maintained for 8 min. In the studywith two-year-old trees, the temperature program wasstarted at 60 �C, and continued as mentioned above.Fourteen aliphatic hydrocarbons ranging from nonaneto hexatriacontane were analysed (aliphatic hydrocar-bons calibration mix DRH-007, Accustandard, NewHaven, CT, USA). The detection limit was 4 mg/l, whichcorresponded to 12 mg/kg of soil. External standardswere prepared by dissolving commercial diesel fuel inn-hexane. Diesel fuel concentrations were measured asarea of total ion chromatogram (TIC) from retentiontime of nonane to retention time of octacosane. Thedetection limit was 50 mg/l, which corresponds to about130 mg/kg dry soil. Extraction efficiency was determinedby extracting 16 samples of freshly spiked soil and wasfound to be 93� 12%. Soil moisture content was de-termined by measuring weight loss after oven-dryingat 105 �C and the diesel fuel concentrations in soil arereported on a dry weight basis.

At the completion of the experiment, plant dry matterwas analysed after heating at 40 �C for 12 h. Roots wererinsed at least five times with Milli-Q� H2O beforedrying. The grass and legume roots were extracted todetermine possible hydrocarbon uptake. Root tissue wasextracted twice (6 and 3 ml of n-hexane) in an ultrasonicbath. The combined extract was allowed to evaporate to

dryness under a fume hood and was concentrated to 2ml. In the experiment with two-year-old trees, rootswere extracted twice and extracts were analysed sepa-rately to determine whether the hydrocarbons were ad-sorbed to surfaces or accumulated within tissue. First,the roots were dried at 40 �C for 12 h and cut into smallpieces with a disposable surgical blade. The first ex-traction involved rinsing the tissue with hexane/acetone(1:2) for 1 min and during the second extraction tissuewas sonicated with a 1:1 hexane/acetone mixture for 30min. All root extracts were analysed with the sameprocedure as for soil.

One-way analysis of variance (ANOVA) was used toevaluate if plant/soil treatments accelerated removalof diesel fuel. Kolmogorov–Smirnov and Ryan–Joinertests were used to determine if the results were normallydistributed. MINITAB software was used in the statis-tical analyses. Plant properties were compared usingone-way ANOVA and Tukey’s test.

3. Results and discussion

3.1. Soil characterization

Total (HNO3) extractable metal concentrations werebelow Finnish guideline values for contaminated soil(Ministry of Environment, Finland, 1994) (data notshown). The soil material was characterized as shown inTable 2. It was acidic and was classified as sand. The soilcontained considerable nitrogen compared to fertiliserN added in soil amendments (Table 1).

3.2. Plant response to diesel additions

The appearance of plants was monitored throughoutthe experiment to determine whether the application ofdiesel fuel inhibited growth and germination. In diesel-contaminated treatments, seeds germinated at a similarrate to control pots (data not shown). Initial growth of

Table 2

Selected chemical and physical properties of the study soil

Parameter Measured value

pH 5.9

Total N (%) 0.3

NO�3 (mg/kg) 65

Total P (mg/kg) 381

PO3�4 Not detected

SO2�4 (mg/kg) 160

Total C (%) 0.91

Cation exchange capacity (cmol/kg) 4.0

Sand (%) 73

Silt (%) 25

Clay (%) 2

M.R.T. Palmroth et al. / Bioresource Technology 84 (2002) 221–228 223

white clover was partially inhibited by diesel fuel, al-though plants survived the duration of the experiment,180 days. In a study by Kulakow et al. (2000), all thelegumes tested, including white clover, died within 60days when grown on weathered hydrocarbon-conta-minated sediment. In a study with two fuel oils, the re-sistance of seeds to oil contamination followed thedecreasing order: sunflower > bean > wheat > clover >maize > barley > lettuce (Chaııneau et al., 1997).

Total biomass of aboveground plant tissue was col-lected after the experiment and dry mass was measuredto determine the effect of diesel addition on plant growth(Table 3). Lower total grass and legume biomass wasproduced in contaminated soil than in the vegetatedcontrol soil (43% and 64% of the biomass compared tothe control treatment, respectively), and legumes andgrasses grew taller in uncontaminated soil (data notshown). Phytotoxicity could account for part of theobserved poor growth (Anderson et al., 1993; Vouilla-moz and Milke, 2001). Phytotoxicity was highest for lowmolecular weight and aromatic hydrocarbons (Chaııneauet al., 1997). However, additions of less volatile hydro-carbons, such as heavy and medium crude oil or oilywaste organics to soil were reported to increase plantgrowth by up to 70% (Salanitro et al., 1997; Mendozaet al., 1995).

Growth and visual symptoms of control trees andtrees grown in diesel fuel-contaminated soil were com-pared to determine the phytotoxicity of diesel fuel.Numerous poplar leaves suffered from drying, leaf burnsand necrosis. Burns were most prominent at leaf edges,but they sometimes covered the entire leaf, and some-times burns started at the leaf tips. Trapp et al. (2001)found Populus nigra to be more sensitive to diesel fuelthan two willows, Salix, but some poplars still survivedat 10 000 mg/kg. In the present experiment, all poplarssuffered from leaf drying in the second diesel applica-tion, but they developed new leaves. None of the poplarsdied during the first year experiment, but mortality rate

was higher during the second experiment, possibly dueto exposure to the higher diesel fuel concentration.

Physical appearance of pines in the treated soil didnot differ from the control plants. All 30 pines, apartfrom one dead, belonged to either class 1 (some symp-toms) or to class 2 (symptoms in >50% of needles).Yearly growth, needle length and symptoms did notdiffer significantly (P < 0:05).

3.3. Removal of diesel fuel

The removal of diesel fuel in combinations of planttreatment (pine, poplar, legume, grass and no vegeta-tion) and soil amendment was studied and the results arediscussed below.

3.3.1. Removal of diesel fuel in poplar treatmentAt 30 days in the poplar treatment, NPK fertilizer-

amended soil resulted in highest removal of diesel fuel.Results of day 30, with an average of 60% diesel fuelremoval, were almost identical, regardless of treatment,when standard deviation of diesel fuel concentration(approximately �1600 mg/kg) was taken into account.Diesel fuel removals at 330 days ranged from 94% withcompost extract to 97% with NPK fertilizer, which in-dicates that the soil amendments no longer accelerateddiesel removal at that time. When 95% confidence limitsof these results are compared, i.e. about �2000 mg/kg at30 days and 100–500 mg/kg at 330 days depending ontreatment, the values of each sampling are very close.Statistical analysis of results showed significant(P < 0:05) differences only when diesel concentrationsin NPK fertilised soil at 60 days were compared to thoseof compost extract-amended and non-fertilised soil(P ¼ 0:011).

In the second diesel fuel application to the trees,diesel was removed considerably faster than in the firstyear. Removal after 14 days was 50–80% depending onsoil treatment. After four weeks, 80–90% of added diesel

Table 3

Effect of soil amendment and diesel fuel addition on plant growth

Soil amendment Plant Diesel fuel added Mass of dry shoot tissue (g) Control biomass (%)

NPK-fertiliser Legume Yes 1.23 33

Compost extract Legume Yes 2.55 68

Microbial enrichment Legume Yes 3.33 89

No additions Legume Yes 2.51 67

Average of all soil treatments Legume Yes 64

No additions Legume Control 3.75 100

NPK-fertiliser Grass Yes 1.74 56

Compost extract Grass Yes 1.08 35

Microbial enrichment Grass Yes 1.13 36

No additions Grass Yes 1.46 47

Average of all soil treatments Grass Yes 43

No additions Grass Control 3.13 100

Mass of dry shoots determined by oven-drying at 105 �C.


fuel had been removed. Higher removal rate could in-dicate that rhizosphere micro-organisms utilized dieselfuel without any lag period. The effect of soil treatmentwas insignificant (P > 0:05). When pine and poplartreatments were compared, poplar treatment clearlyenhanced diesel removal more than pine treatment(Table 4).

3.3.2. Removal of diesel fuel in pine treatmentIn the pine treatment, the highest removal of diesel

fuel at 30 days, 60%, occurred in NPK-fertilised soil, butthe differences between treatments could be explained bythe standard deviation of diesel concentration (�1000mg/kg dry soil). At 60 days the no-fertiliser treatmentwas slightly more effective than the other treatments.Combination of pine with soil treatments resulted inremoval of at least 88% of original diesel fuel by 330days. One-way ANOVA showed that differences be-tween soil treatments were not significant (P < 0:05) atany of the samplings.

The second diesel application to the pine treatmentresulted in a 20–70% removal of added diesel fuel over28 days. The NPK fertilised treatment resulted in lowestfinal diesel concentrations, but the differences betweentreatments were minor.

3.3.3. Removal of diesel fuel in grass treatmentIn the grass treatment all the amendments, except

microbial enrichment, resulted in removal of 40% ofdiesel fuel in 60 days. In the microbial enrichmentamended soil, most of the diesel fuel, up to 98%, re-mained at 60 days. Similar trends continued throughoutthe experiment. At the end of the grass experiment (180days), 83–84% of diesel fuel had been removed, exceptwith the microbial enrichment, in which only 58% ofdiesel had been removed. One-way ANOVA of micro-bial enrichment vs. all other treatments showed that theaddition of the microbial enrichment significantly in-hibited the removal of diesel at 30 days (P ¼ 0:017) andat 180 days (P ¼ 0:031). Soil amendments did not sig-nificantly (P < 0:05) enhance diesel removal. The grasstreatment did not accelerate the removal of diesel fuel

as did the other plant treatments. This result may havebeen due to nutrient requirements of grasses vs. nitro-gen-fixing legumes. Furthermore, plants grew very closeto each other. Vouillamoz and Milke (2001) also foundplant overcrowding not beneficial.

3.3.4. Removal of diesel fuel in legume treatmentIn the legume treatment, soil amendments enhanced

the removal of diesel fuel at 30 days, resulting inremoval of 30% in treatment without fertilisers and 55–68% with amendments. At 60 days the equivalent re-moval rates were 61% and 67–74%. Later the differencedecreased, but the remaining diesel concentration washighest at 180 days in soil without additions. The dif-ference, however, was only 3% and removals rangedfrom 96% to 99%. Diesel concentrations in amendedsoils differed significantly from non-amended soil at 30days only (P ¼ 0:002). This indicates that soil amend-ments enhanced diesel removal only in the early stagesof experiment.

3.3.5. Removal of diesel fuel in non-vegetated controlsIn the non-vegetated treatment with no soil additions,

diesel concentrations decreased more slowly than withother soil amendments/no vegetation. At 150 days, 70%of added diesel fuel remained in the soil. By 150 daysthis difference was significant (P ¼ 0:009), when thetreatment with no additions was compared to compostextract and NPK treatments. However, this effect dis-appeared by 180 days. Addition of 0.5% sodium azide(NaN3) did not effectively sterilise the soil; diesel re-moval rates were even higher than without poisoning(Fig. 1). Wang and Jones (1994) found that a concen-tration of 1% sodium azide affected the evaporation ofchlorobenzene in sludge-amended soil and the structureof the soil. Similar soil aggregation was observed in thecurrent study in poisoned treatments.

In the second diesel fuel application, diesel was re-moved in non-vegetated soil and poisoned, non-vege-tated soil controls at equal rates, resulting in about 70%removal by 28 days compared to 80–90% in poplartreatment by 28 days.

Table 4

Effect of plant treatment on diesel fuel removal one-way analysis of variance was performed at each sampling

Response at experiment Sampling day P-value Significantly different from other plant treatments

1 30 0.003 Poplar from grass

1 60 0.000 Legume from sodium azide treated, pine and grass

1 90 0.000 Legume from all and grass from all (except from no vegetation)

1 120 0.000 Legume from all

1 150 0.047 Grass treatment from no vegetation

1 180 0.000 Legume from no vegetation and grass

1 330 0.312 No significant differences between poplar and pine

2 14 0.255 No significant differences between poplar and pine

2 28 0.038 Significant difference between poplar and pine

Sixteen replicate samples of each plant treatment, except 4 of sodium azide treatment. Results of experiment 1 presented in Fig. 1.


3.3.6. Effect of soil amendments on diesel fuel removalThe effect of soil amendments on diesel fuel removal,

all plant treatments combined, was studied. The NPKfertiliser treatment accelerated diesel fuel mineralisationin the beginning of the experiment (at 60 days, P ¼0:009), but at later samplings the effect of soil amend-ment was not significant (P > 0:05). According toVenosa et al. (1996), additions of fertilisers may be un-necessary if nutrients exist in soil at background levelsappropriate to remediation. Their conclusion is sup-ported by this study, because fertilisers did not signifi-cantly enhance diesel fuel removal in most planttreatments. Fertiliser concentrations were smaller thanthose applied in previous studies, e.g., Chaııneau et al.(2000) and Vouillamoz and Milke (2001). In this study,the addition of microbial inoculum or the compost ex-tract did not significantly (P < 0:05) enhance the re-moval of diesel fuel, indicating that the appropriatetypes and amounts of hydrocarbon-degrading microbialpopulations were already present in the soil. Compostaddition combined with phytoremediation aided theremoval of diesel fuel in the study of Vouillamoz andMilke (2001). One way ANOVA of diesel fuel concen-tration versus soil treatment/plant treatment showedthat soil treatment was a less significant factor thanplant treatment in removal of diesel fuel.

3.3.7. Effect of plant treatments on diesel fuel removalWith all soil treatments combined, the legume treat-

ment removed diesel fuel most efficiently, but data fortrees was almost similar (Fig. 1). Plants accelerate re-moval of hydrocarbons, but in a few months, theremoval rates become similar in non-vegetated andvegetated soils (Chaııneau et al., 2000; Komisar andPark, 1997). Soil treated with some alfalfa genotypes

degraded less hydrocarbons than a non-vegetated con-trol (Wiltse et al., 1998). In a study by Angehrn et al.(1999), vegetation (white clover or grasses) did notenhance the removal of residual hydrocarbons fromremediated soil. In the current study, plants (exceptgrasses) improved diesel removal throughout the ex-periment and the legume treatment provided the highestremoval rates. The effect of plant treatment on hydro-carbon removal was found to be significant at P < 0:05at each sampling (Table 4), except at day 330 of the firstexperiment and at the beginning of the second tree ex-periment. Results of all samplings were normally dis-tributed, when the Kolmogorov–Smirnov normality testwas applied.

3.4. Removal of aliphatic compounds in diesel fuel

Fourteen aliphatic hydrocarbons ranging from non-ane to hexatriacontane were analysed in soil samples todetermine the affect of the chain length of hydrocarbonson their removal from soil. Eleven of the standardcompounds could be quantified in the initial sampleextracts (Table 5). By day 30, the most volatile hy-drocarbons, i.e., nonane to dodecane, were no longerdetected. None of these eleven straight-chained hydro-carbons were detected in samples collected later than 60days. According to Chaııneau et al. (1995) hydrocarbonsbelow C14 (e.g. tetradecane) are lost by evaporationwithin a few days. More branched hydrocarbons withinthe retention time range of C10 (decane) were still pre-sent at later samplings, indicating that short-chainedlinear alkanes were removed first. Linear alkanes areknown to be easily biodegradable (e.g. Atlas, 1981). Anunresolved complex mixture of diesel fuel was still pre-sent after 180 days and one year in all samples. More ofthe short-chained hydrocarbons were removed in thelegume treatment than other treatments (Table 6).

Fig. 1. The effect of plant (pine, poplar, legume, grass) or control

treatments (no vegetation or sodium azide treated, no vegetation) on

mean total diesel fuel concentrations (mg/kg dry soil). Results shown

as averages of 16 replicates, all soil amendments combined.

Table 5

Standard aliphatics, their carbon numbers and concentration range

found in the initial extracts of soil contaminated with 0.5 wt.% of diesel

fuel

Aliphatic Number of carbon

(C) atoms

Concentration

(mg/kg dry soil)

Nonane 9 9� 3

Decane 10 21� 8

Dodecane 12 52� 18

Tetradecane 14 78� 20

Hexadecane 16 30� 9

Octadecane 18 89� 7

Nonadecane 19 57� 43

Eicosane 20 92� 21

Docosane 22 45� 15

Tetracosane 24 15� 7

Hexacosane 26 5� 2

Four replicates were analysed and their standard deviation is given.


3.5. Plant uptake of diesel hydrocarbons

Hydrocarbon concentrations in shoot and root tissuewere analysed to determine if phytoaccumulation andphytodegradation played a role in diesel fuel removal.Grass roots accumulated diesel-range compounds in therange of 10 g/kg dry plant tissue, but legume roots didnot accumulate diesel at detectable levels. No plantcompounds were found in control root extracts of le-gumes and grasses. Chaııneau et al. (1997) observedno uptake of petrogenic hydrocarbons at contaminationlevels not inhibiting maize (Zea mays L.) growth. Inheavily contaminated soil, however, adsorption of hy-drocarbons to leaves and stems was rapid and maizeplants died quickly. Radwan et al. (2000) found thatlong-chain hydrocarbons accumulated in broad bean(Vicia faba), grown in oily soil. The accumulation, es-pecially into seeds, was thought to pose a risk to humanor animal nutrition. In the current study, hydrocarbonswere found in poplar and pine root extracts. Differen-tiation of diesel fuel compounds from plant compoundscannot be clearly made, because retention times overlap.

These results indicate that phytoaccumulation was not asignificant diesel removal mechanism.

4. Conclusions

All plant species tested tolerated the applied dieselfuel concentration (0.5% w/w). Accumulation of dieselfuel to plant biomass was not significant. The growthchamber study showed that the presence of plants ac-celerates removal of diesel fuel in soil. Addition of soilamendments did not enhance this process. Treatment ofdiesel fuel-contaminated soil with legume species wasshown to be the most effective method for diesel fuelremediation. Treatment with pine and poplar also en-hanced diesel fuel removal. Therefore, these plants aresuitable candidates for diesel fuel phytoremediationunder subarctic conditions.

Acknowledgements

The study was funded by The Neste Foundation(Espoo, Finland). The authors are grateful to Eeva-LiisaViskari for help in interpreting plant phytotoxicity re-sults, to Kirsi-Maarit Lehto for help with GC/MSDoperation, and to Pasi Niskanen for soil particle analysisand Chiara Truccolo for technical assistance.

References

Anderson, T.A., Guthrie, E.A., Walton, B.T., 1993. Bioremediation in

the rhizosphere. Environ. Sci. Technol. 27, 2630–2636.

Angehrn, D., Schluep, M., G€aalli, R., Zeyer, J., 1999. Movement and

fate of residual mineral oil contaminants in bioremediated soil.

Environ. Toxicol. Chem. 18, 2225–2231.

Atlas, R.M., 1981. Microbial degradation of petroleum hydrocarbons:

an environmental perspective. Microbiol. Rev. 45, 180–209.

Chaııneau, C.H., Morel, J.L., Oudot, J., 1995. Microbial degradation

in soil microcosms of fuel oil hydrocarbons from drilling cuttings.

Environ. Sci. Technol. 29, 1615–1621.

Chaııneau, C.H., Morel, J.L., Oudot, J., 1997. Phytotoxicity and plant

uptake of fuel oil hydrocarbons. J. Environ. Qual. 26, 1478–1483.

Chaııneau, C.H., Morel, J.L., Oudot, J., 2000. Biodegradation of fuel

oil hydrocarbons in the rhizosphere of maize. J. Environ. Qual. 29,

569–578.

Cunningham, S.D., Berti, W.R., 1993. The remediation of contami-

nated soils with green plants: an overview. In Vitro Cell. Dev. Biol.

29P, 207–212.

Cunningham, S.D., Berti, W.R., Huang, J.W., 1995. Phytoremediation

of contaminated soils. Trends Biotechnol. 13, 393–397.

Karthikeyan, R., Davis, L.C., Mankin, K.R., Erickson, L.E., Kula-

kow, P.A., 1999. Biodegradation of Jet Fuel (JP-8) in the presence

of vegetation. In: Proceedings of the 1999 conference on hazardous

waste research, St. Louis, Missouri, May 24–27, pp. 243–256.

Komisar, S.J., Park, J., 1997. Phytoremediation of diesel-contaminated

soil using alfalfa. In: Fourth International In Situ and On-Site

Bioremediation Symposium, April 28–May 1, 1997, New Orleans,

LA, vol. 3, pp. 331–336.

Table 6

Retention time ranges of diesel fuel peaks found at each sampling

Sampling (days) Plant treatment Eluted between

retention times

0 No vegetation –C30a

30 Poplar C10–C28

30 Pine C9–C28

30 Legume C9–C28

30 Grass C9–C28

30 No vegetation C10–C28

30 Poisoned, no vegetation C10–C28

60 Poplar C12–C30

60 Pine C10–C28

60 Legume C10–C28

60 Grass C10–C28



90 Legume C12–C26

90 Grass C10–C28



120 Legume C14–C28

120 Grass C10–C28



150 Grass C10–C28



180 Legume C14–C26

180 Grass C10–C28



330 Poplar C12–C22

330 Pine C10–C24

Sample retention times compared to retention times of standard ali-

phatic mixture.aNonane found in sample.


Kulakow, P.A., Schwab, P., Banks, M.K., 2000. Screening plant

species for growth in weathered petroleum hydrocarbon-contam-

inated sediments. Int. J. Phytoremediation 2, 297–317.

Mendoza, R.E., Taboada, M.A., Rodriguez, D., Caso, O., Portal, R.,

1995. Use of oily waste organics as amendment to soils. In:

Hinchee, R.E., Kittel, J.A., Reisinger, H.J. (Eds.), Bioremediation

of Applied Bioremediation of Petroleum Hydrocarbons. Battelle

Press, Columbus, OH.

Ministry of Environment, 1994. Saastuneet maa-alueet ja niiden

k€aasittely Suomessa. Saastuneiden maa-alueiden selvitys- ja ku-

nnostusprojekti, loppuraportti. Ymp€aarist€ooministeri€oo, Ymp€aarist€oonsuo-

jeluosaston muistio 5/1994. Painatuskeskus Oy, Helsinki. Report in

Finnish.

Nelson, D.W., Sommers, L.E., 1982. Total carbon, organic carbon,

and organic matter. In: Page, A.L., Miller, R.H., Keeney, D.R.

(Eds.), Methods of Soil Analysis, Part 2: Agronomy Monograph 9.

American Society of Agronomy, Madison, WI, pp. 539–580.

Olsen, S.R., Sommers, L.E., 1982. Phosphorus. In: Page, A.L., Miller,

R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis. Part 2:

Agronomy Monograph 9. American Society of Agronomy, Mad-

ison, WI, pp. 539–580.

Radwan, S.S., Sorkhoh, N.A., El-Nemr, I., 1995. Oil biodegradation

around roots. Nature 376, 302.

Radwan, S.S., Al-Awadhi, H., El-Nemr, I.M., 2000. Cropping as a

phytoremediation practice for oily desert soil with reference to crop

safety as food. Int. J. Phytoremediation 2 (4), 383–396.

Rhoades, J.D., 1982. Cation exchange capacity. In: Page, A.L., Miller,

R.H., Keeney, D.R. (Eds.), Methods of Soil Analysis. Part 2:

Agronomy Monograph 9. American Society of Agronomy, Mad-

ison, WI, pp. 539–580.

Salanitro, J.P., Dorn, P.B., Huesemann, M.H., Moore, K.O., Rhodes,

I.A., Jackson, L.M., Vipond, T.E., Western, M.M., Wisniew-

ski, H.L., 1997. Crude oil hydrocarbon bioremediation and

soil ecotoxicity assessment. Environ. Sci. Technol. 31, 1769–

1776.

Trapp, S., K€oohler, A., Larsen, L.C., Zambrano, K.C., Karlson, U.,

2001. Phytotoxicity of fresh and weathered diesel and gasoline to

willow and poplar trees. J. Soils Sediments 1 (2), 71–76.

Venosa, A., Suidan, M., Wrenn, B., Strohmeier, K.L., Haines, J.R.,

Eberhart, B.L., King, D., Holder, E., 1996. Bioremediation of an

experimental oil spill on the shoreline of Delaware Bay. Environ.

Sci. Technol. 30, 1764–1775.

Vouillamoz, J., Milke, M.W., 2001. Effect of compost in phytoreme-

diation of diesel-contaminated soils. Wat. Sci. Technol. 43 (2), 291–

295.

Wang, M.-J., Jones, K.C., 1994. Behaviour and fate of chlorobenzenes

in spiked and sewage sludge-amended soil. Environ. Sci. Technol.

28, 1843–1852.

Wiltse, C.C., Rooney, W.L., Chen, Z., Schwab, A.P., Banks, M.K.,

1998. Greenhouse evaluation of agronomic and crude oil-phyto-

remediation potential among alfalfa genotypes. J. Environ. Qual.

27 (1), 169–173.


phytoremediation of subarctic soil contaminated with diesel fuel

Documents