phytoremediation of soil contaminated with used motor oil: ii. greenhouse studies

ENVIRONMENTAL ENGINEERING SCIENCEVolume 21, Number 2, 2004© Mary Ann Liebert, Inc.

Phytoremediation of Soil Contaminated with Used Motor Oil:II. Greenhouse Studies

Elena Dominguez-Rosado1 and John Pichtel2,*

Ball State UniversityNatural Resources and Environmental Management

Muncie, IN 47306

ABSTRACT

The decomposition of used motor oil in soil as influenced by plant treatment was monitored in a green-house study. Soil contaminated with used motor oil (1.5% w/w) was seeded with soybean (Glycinemax)/green bean (Phaseolus vulgaris); sunflower (Helianthus annus)/Indian mustard (Brassica juncea);mixed grasses/maize (Zea mays); and mixed clover (red clover, Trifolium pratense/ladino clover, Trifoliumrepens) and incubated. Soxhlet-extractable oil and grease remaining in the soil was monitored after 100and 150 days. After 150 days in the clover treatment, the added oil was no longer detected. A total of67% of the oil was removed in sunflower/mustard, and with addition of NPK fertilizer, the oil was com-pletely removed. The grass/maize treatment resulted in a 38% oil reduction, which increased to 67% withfertilizer application. The control treatment reduced oil in soil by 82% when fertilizer was added. At 150days the sunflower/mustard and wheat/oats treatments produced the greatest biomass in the presence ofused oil. Gas chromatography/mass spectroscopy (GC/MS) spectra of oil/grease extracts revealed the pres-ence of new peaks associated with hydrocarbon decomposition. The presence of new hydrocarbons wascorroborated by changes in Fourier-transformed infrared spectrometry (FTIR) spectra. Fertilizer additionsto treatments resulted in negligible changes to FTIR bands. Based on oil/grease residues and biomass re-sults, the clover and sunflower/mustard treatments are considered superior to the other plant treatments interms of overall phytodegradation of used oil hydrocarbons.

Key words: hydrocarbons; micro-organisms; phytoremediation; rhizosphere; used motor oil

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*Corresponding author: Ball State University, Natural Resources and Environmental Management, Muncie, IN 47306. Phone:765-285-2182; Fax: 765-285-2606; E-mail: [email protected]

INTRODUCTION

“USED OIL” is defined by the U.S. EnvironmentalProtection Agency (40 CFR Part 279) as “any oil

that has been refined from crude oil or any synthetic oilthat has been used and as a result of such use is con-taminated by physical or chemical impurities.” Used mo-

tor oil is managed as an auxiliary fuel, rerefined, incor-porated into asphalt, and disposed in landfills. In theUnited States, approximately 800 million gallons of usedmotor oil are recycled annually (U.S. EPA, 2001); how-ever, significant volumes continue to be discharged im-properly into local environments. Millions of gallons aredisposed in trash, on land or into sewers, with the po-

tential for contaminating soil, groundwater and surfacewater (Indiana Department of Environmental Manage-ment, 2002).

A number of innovative physical and chemical tech-nologies are available to remediate soil contaminatedwith hydrocarbon pollutants. For example, soil washing,vapor extraction, encapsulation, and solidification/stabilization have been successful (Cole, 1994). Thesemethods, however, are expensive, and may only be partlyeffective. In addition, public pressures may restrict thefield utilization of such intensive techniques.

In recent years, bioremediation has emerged as an ef-fective technology for treatment of hydrocarbon conta-minants in soil. A diverse consortium of micro-organismsis capable of degrading a wide range of hydrocarbon mol-ecules; however, biodegradation is often limited by ex-tremes in pH, inadequate concentration of oxygen andnutrients, and high levels of contaminants such as met-als. Additions of fertilizer and other amendments may ac-celerate the degradation rate (Bollag et al., 1994). Recentstudies indicate that plant roots provide a beneficial habi-tat for hydrocarbon-degrading microbes. The use of veg-etation to enhance microbial populations and activity istermed phytoremediation (Cunningham et al., 1996; U.S.EPA, 2000).

Soils, sediments, and groundwater contaminated withoily wastes have been targeted in phytoremediation stud-ies (Bossert and Bartha, 1984; Lee and Banks, 1993; Cun-ningham et al., 1996; Hinchman et al., 1998; Bailey andMcGill, 1999; Banks et al., 1999; Frick et al., 1999a,1999b; Karthikeyan et al., 2000). Legumes such as whiteclover (Trifolium repens) and alfalfa (Medicago sativa)were effective for petroleum degradation in soil (Frick etal., 1999b). Likewise, ryegrass (Lolium perenne L. ) ac-celerated the disappearance of aliphatic hydrocarbons insoil (Gunther et al., 1996), and Verde Kleingrass removedPAHs from the rhizosphere in contaminated clay soils byvirtue of its deep and dense root system (Qiu et al., 1997).Prairie Buffalograss was also used to remediate PAHs inclay soil (Qiu et al., 1997). Mixtures of tall fescue/west-ern wheatgrass and tall fescue/red clover, and birdsfoottrefoil and yellow sweet clover were used for remedia-tion of oily sediments in a vehicle wash facility in Kansas(Karthikeyan et al., 2000). The grass treatments markedlydecreased concentrations of PAHs after 2 years. In agreenhouse experiment, tall fescue (Festuca arundinaceaSchreber) degraded benzo[a]pyrene in soil contaminatedwith crude oil, and resulted in an increase in microbialpopulations (Banks et al., 1999).

Extraction of oil from soil has posed its share of analytical challenges. Schwab et al. (1999) found thatmechanical shaking of soil with a sequence of dichloro-methane or acetone treatments was equivalent to Soxh-

let extraction for total petroleum hydrocarbon and PAHdetermination. Mechanical shaking of diesel-contami-nated soil with hexane was found to be effective for ex-traction and analysis (Pichtel and Liskanen, 2001). Ad-ditionally, analytical characterization of oily substancesis hindered by the complex chemical structure of oil.Characterization by solubility provides information re-garding its chemical nature, but not chemical structure(Moschopedis and Hawkins, 1981). Techniques usingmass spectroscopy (MS) have been more successful inoil characterization (Dilts, 1998). Grimmer et al. (1981)used glass capillary GC-MS for PAH inventories of lu-bricating oils.

The focus of the reported study is to evaluate the degra-dation of used motor oil in the rhizosphere of selectedplants grown in the greenhouse. Specifically, plantspecies with or without fertilizer additions were assessedas a potential remediation technology. Trends of hydro-carbon degradation were assessed over time. GC/MS andFTIR were utilized to elucidate the degradation of motoroil in the rhizosphere.

EXPERIMENTAL METHODS

Soil collection and greenhouse setup

Soil material was collected from agricultural fields ineast-central Indiana. The soil, a Glynwood loam (fine, il-litic mesic Aquic Hapludalf), was transported to the lab-oratory and air dried, finely ground with a mortar andpestle, and sieved through a 2-mm mesh sieve. Used mo-tor oil was collected from several automobile crankcases.Chemical and physical analysis of the used motor oil andrecipient soil are described in Dominguez-Rosado et al.(2004).

Ceramic pots were prepared, containing 1 kg of soil andamended with 1.5% (w/w) used motor oil. The used oilwas thoroughly mixed with soil using a stainless steel stir-ring rod. Pots were seeded with the following species mix-tures: Soybean (Glycine max)/green bean (Phaseolus vul-garis); sunflower (Helianthus annus)/Indian mustard(Brassica juncea); mixed grasses (creeping red fescue,Festuca rubra; fawn tall fescue, F. arundinacea; perennialryegrass, L. perenne)/maize (Zea mays); and clover (redclover, Trifolium pratense; ladino clover, T. repens). Thecontrol treatment received oil but was not seeded. Mix-tures of plants were chosen based on similarities in physiology and success in earlier studies (Pichtel andLiskanen, 2001; Palmroth et al., 2002; Dominguez-Rosadoet al., 2004). Half the pots in each treatment received apreplant incorporation of a commercial 10-10-10 fertilizer.

There were five replicates of each plant treatment. Potswere watered to maintain the soil moisture content at ap-

170 DOMINGUEZ-ROSADO AND PICHTEL

proximately field capacity. Plants were grown for 150days under natural lighting in the greenhouse.

Analysis of used oil in the root zone

To monitor trends of used oil degradation in the rhi-zosphere, ultrasonic extraction, mechanical shaking, andSoxhlet extraction were assessed for degree of hydrocar-bon extraction from soil.

Ultrasonic extraction. Fifteen grams of soil weremixed with used motor oil, 30 g anhydrous Na2SO4, and50 mL hexane in a glass centrifuge tube. The tubes wereshaken in a touch mixer for 1 min and then transferredto an ultrasonic bath for 1 h. Tubes were centrifuged at1,000 3 g for 3 min, and the supernatant was decanted.An equivalent volume of hexane was added to the re-sulting soil pellet and the extraction was repeated. Su-pernatants from all extractions were combined and ana-lyzed via GC/MS as described below (U.S. EPA, 1996b).

Mechanical shaking. Two grams of contaminated soiland 3 mL of hexane were added to a glass centrifuge tubeand shaken in a mechanical shaker (120 osc./min) for 1 h. After centrifugation at 1,000 3 g for 3 min, the su-pernatant was retained and 3 mL hexane added to the soilpellet for a second extraction (Schwab et al., 1999; Pich-tel and Liskanen, 2001; Palmroth et al., 2002). Super-natants were combined and analyzed via GC/MS.

Soxhlet extraction. Sodium sulfate was purified by dry-ing overnight in an oven at 150°C. Round Soxhlet flaskswere dried at 105°C for 30 min. After cooling, the weightof the round flask and boiling chips was recorded. Tengrams of contaminated soil were mixed with 10 g dryNa2SO4 and placed in a cellulose extraction thimble (24mm outer diameter 3 65 mm length). Glass chips wereplaced at the surface to prevent the soil from escapingfrom the thimble during extraction. Three hundred mil-liliters of hexane was added to the flask and extracted for5 h at 70°C (U.S. EPA, 1996a).

Oil/grease content. Residual oil and grease was deter-mined in the Soxhlet-extracted soils. After evaporationof the hexane in a hot water bath, the round flask was al-lowed to cool and weighed again (Martin et al., 1991;U.S. EPA, 1998). Residual oil/grease content in the soilwas calculated as follows:

Gain in weight of flask (mg) 5 (weight of flask,boiling chips and residue after evaporation of

hexane) 2 (weight of round flask and boiling chips)

Oil and grease fraction (mg/kg) 5 Gain in weight offlask (mg) 3 1000/weight of the solid (g)

When oil only (i.e., no soil) was Soxhlet-extracted,oil/grease recovery was 88.3%; however, when oil plussoil was extracted, oil/grease recovery was lower. Theextraction data was nevertheless highly reproducible(standard deviation 5 0.005 g; n 5 3); therefore, the con-trol values of Soxhlet-extracted oil and grease were es-tablished as the baseline value for phytoremediation success.

The oil/grease obtained from Soxhlet extractions at 0,50, 100, and 150 days was recovered for further analy-sis by dissolving in 9 mL hexane. Solutions were analyzed by gas chromatography/mass spectroscopy(GC/MS) and Fourier-transformed infrared spectrometry(FTIR). Operating parameters for both instruments ap-pear in Dominguez-Rosado et al. (2004). All glasswarewas cleaned via acid washing, and, where appropriate,by CH3Cl to remove oily residues.

Plant biomass

At the completion of the incubations (150 days), plantshoots were cut at the soil surface using a stainless steelblade. Plant tissue was washed with deionized H2O, driedat 105°C for 48 h and weighed.

Statistical analysis

Data was treated statistically by analysis of variance(ANOVA) and Tukey’s test (Chaîneau et al., 1997) us-ing a Datadesk® software package.

RESULTS AND DISCUSSION

Oil extraction from soil

Using ultrasonic extraction at several concentrations ofused oil, no useful chromatographic peaks were observedin any GC-MS scans. With multiple mechanical shakingsof used oil, a limited number of peaks was observed (e.g.,1,19,10-(methoxy-methylidyne) tris-benzene at 27.1 min,and a,a-dipheny-benzene methanol at 27.5 min). Limi-tations with the use of GC for oil characterization includepoor results caused by interferences, low recovery due tothe standard selected, and petroleum changes caused byvolatility (Restek, 1994). The ASTDR (1999) states thatthere are inherent inaccuracies in GC methods for pe-troleum analysis. Several recent reports have detailed theproblems with certain GC methods for oil analysis(George, 1992; Rhodes et al., 1995/1996).

Given the poor recovery via these extraction methods,combined with GC source fouling presumably resultingfrom the high metal concentrations in the used oil (seeDominguez-Rosado et al., 2004), an alternate means ofassessing the progress of phytoremediation was deemed

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ENVIRON ENG SCI, VOL. 21, NO. 2, 2004

necessary. Soxhlet extraction, to measure the oil andgrease content, was ultimately chosen to extract hydro-carbons from the contaminated soil.

Decomposition of oil/grease by plant treatment

In the nonfertilized treatments, the clover performedoptimally in reducing the oil/grease content in soil [Fig.1(a)]. At 50 days, 42% of the initial oil/grease contentwas removed, at 100 days the residue decreased to 50%and at 150 days the extractable oil disappeared com-

pletely, presumably due to the action of rhizospheric micro-organisms. In the wheat/oats treatment, 64% of the initial oil had disappeared after 100 days, and at 150days the oil was reduced by 77%. The sunflower/mus-tard and grass/maize treatments substantially decreasedthe oil/grease content over the study period. When fertilizer was not applied, the clover, wheat/oats, and sun-flower/mustard contained lower levels of oil/grease after150 days, that is, 100, 77 and 67% reductions, respec-tively, compared with the control (47% reduction). Dieselfuel disappeared more rapidly in a contaminated soil


Figure 1. Oil/grease content remaining in the soil: (a) at 0, 100, and 150 days, no fertilizer; and (b) at 0 and 150 days, fertil-izer added.

a

b

grown to mixed clovers compared with mixed grasses,poplar and Scots Pine (Palmroth et al., 2002).

When fertilizer was added to the treatments, the re-sidual oil/grease typically disappeared more rapidly [Fig.

1(b)]. Vegetated treatments at 100 days performed betterthan the control (35% oil/grease reduction) except forwheat/oats (27% reduction). After 100 days, the sun-flower/mustard reduced the oil/grease content by 68%



Figure 2. Plant biomass: (a) after 100 days incubation; and (b) after 150 days incubation.

a

b

and at 150 days all extractable oil/grease was removed.The grass/maize treatment reduced oil/grease by 56% at100 days and 68% at 150 days. In a greenhouse experi-ment, tall fescue (Festuca arundinacea Schreber) de-graded benzo[a]pyrene in soil contaminated with crudeoil, and microbial populations increased in vegetated soils(Banks et al., 1999). In the present study, soybean/beanswas among the weakest of the plant treatments in termsof reducing oil/grease content [Fig. 1(b)], with a 52% re-duction at 100 days and no change by 150 days. At 100days the control reduced oil/grease to 35%, and at 150days it was reduced to 18% of the initial value.

Used motor oil is a nonvolatile substance; however,volatilization of short-chain hydrocarbons was inferred

from gravimetric data. A mean volatilization loss of 13%was detected over the study period. Pichtel and Liskanen(2001) and Chaineau et al. (1997) conducted phyto-remediation studies on hydrocarbon-contaminated soilswhich had experienced similar volatilization losses.

Biomass production

Plant tissue production at 100 days decreased or wasunchanged with oil application [Fig. 2(a)]; for example,grass/maize biomass declined by 28% when oil was ap-plied. Sunflower/mustard biomass was essentially un-changed compared with the no-oil treatment at 100 days.With fertilizer addition, wheat/oats, soybean/bean, andsunflower/mustard treatments increased in biomass. Inwheat/oats, biomass increased 2.8-fold (100 days) whenfertilizer was applied.

At 150 days biomass increased for most treatments[Fig. 2(b)]. The biomass of soybean/bean (oil, not fertil-ized; oil plus fertilizer) at 150 days was similar to thatproduced at 100 days, however. Sunflower/mustard bio-mass increased by 16% with fertilizer compared to thecorresponding control treatment without oil. The sun-flower/mustard and grass/maize treatments reduced theextractable oil/grease content in the soil at 150 days [Fig.1(b)]. Although biomass production was affected by theoil, these treatments nevertheless produced some of thehighest biomass by 150 days. Wheat/oats increased inbiomass and reduced the residual oil/grease in the soil by150 days; however, higher concentrations of oil remainedin the soil as compared to the sunflower/mustard andgrass/maize treatments [Fig. 1(b)]. In an early study soy-beans actually grew better with small additions (0.75%)


Figure 3. Plant biomass plotted vs. oil/grease content in soil,fertilizer added, after 150 days.

Figure 4. Leaves of soybean grown on used oil-contaminated soil, showing chlorosis and necrosis.

of oil (Carr, 1919). Giddens (1976) showed that in a soilcontaminated with used motor oil, soybean, maize (Zeamays L.) and peanuts (Archis hypogeae L.) grew betterwhen fertilizer, particularly N-based, was applied. Othercommercial plant species can tolerate significant amountsof petroleum hydrocarbons; for example, tomatoes, kale,and leaf lettuce (Schwendinger, 1968).

Associations of oil/grease vs. biomass resulted in anegative slope at 100 and 150 days, with or without fer-tilizer. Oil/grease vs. biomass at 150 days when fertilizerwas applied showed the strongest overall association(r2 5 0.62, p , 0.1, Fig. 3). The downward trend inoil/grease concentrations could be correlated with exten-sive development of above-ground tissue and with ap-plications of fertilizer; in other words, a healthy plant sys-tem combined with a healthy microbial consortium mayhave accelerated oil decomposition.

All plants appeared healthy in the presence of the usedoil with the exception of the soybeans, which experienced

leaf chlorosis and necrosis (Fig. 4). In a study by Ray-mond et al. (1975), beans grown in oil-contaminated soildeveloped deformations in leaves and experienced sig-nificant stunting.

GC/MS and FTIR of oily residues

GC/MS spectra of oil/grease recovered from sun-flower/Indian mustard at 100 days (Fig. 5) revealed newaliphatic and aromatic peaks associated with nascent hy-drocarbons, presumably formed by the biodegradation ofused oil in the rhizosphere. At 50 days, methyl ester do-decanoic acid and methyl tetradecanoate were detected.At 100 days, both components were identified with a 94%match. Products such as long chain alcohols, aldehydes,and organic acids are common products of the so-called“beta-oxidation” sequence. This is promising data, show-ing that potentially toxic oily wastes are being metabo-lized by native microbial populations. The presence of



Figure 5. GC/MS spectrum of oil/grease from sunflower/mustard treatment at 100 days.

certain plant species (i.e., sunflower/mustard, grasses) isfurther conducive to such decomposition.

In the clover treatment (Fig. 6), several unidentifiablecompounds occurred in the soil extracts. Based on pre-vious studies, these may be branched chains (seeChaîneau et al., 1997). Additional studies are needed tofurther elucidate this so-called “unrecognized complexmatter.” The GC-MS spectrum for the control treatmentshowed few biogenic or other hydrocarbon peaks (Fig. 7).

FTIR analysis of oil/grease residues at 0, 50, 100, and150 days in the sunflower/mustard treatment are shown inFig. 8. Bands related to C—H vibrations appear in therange of 2957 to 2850 cm21. An intense band occurs at1460 cm21 and a less intense band at about 1377 cm21,which could be produced by a mixture of compounds with

small chain lengths and branching vibrations from C—Hof the methylene (—CH2—) chains in used motor oil. At100 days a broad and intense band is shown at 3424 cm21 and a new band appears at 1645 cm21, whichare related to the O—H stretching band and the H—O—Hbending vibrations of water. A band at 1740 cm21 is as-sociated with carbonyl groups in ketones, aldehydes,and/or acids. At 100 and 150 days a band appears at 1707and 1709 cm21, respectively. These bands could be asso-ciated with new carbonyl groups arising from the micro-bial oxidation of used oil in the rhizosphere. Similar trendsin FTIR spectra were observed in the grass/maize treat-ment (Fig. 9). At 100 days, new bands occur at 1708 and1740 cm21, which could correspond to new carbonyl-based compounds, possibly ketones or aldehydes formedby microbial oxidation processes.


Figure 6. GC/MS spectrum of oil/grease from control treatment at 100 days.

The effect of fertilizer application on oil/grease residueswas compared across different plant species. There wereno marked differences in bands between different treat-ments.

SUMMARY AND CONCLUSIONS

Based on residual oil/grease content measurements ofthe contaminated soil, the clover, sunflower/mustard, andgrass/maize treatments decomposed hydrocarbons morerapidly than other plant treatments. Based on decrease inoil/grease content, fertilizer additions enhanced hydro-carbon decomposition in sunflower/mustard, grass/maize, and wheat/oats. At 150 days the sunflower/mus-tard and wheat/oats treatments produced the greatest bio-mass in the presence of used oil. A slight negative cor-relation (p , 0.1) was noted for biomass vs. oil/greasewhen plants were grown with added fertilizer, indicating

that healthy plant tissue and microbial activity could pos-sibly result in rapid hydrocarbon degradation.

GC/MS spectra of oil/grease extracts in plant treat-ments revealed new peaks related to hydrocarbon de-composition. Methyl tetradecanoate and methyl ester do-decanoic acid were identified as likely hydrocarboncompounds as well as mono (2-ethylhexyl) ester hexa-nedoic acid and methyl ester undecanoic acid. The pres-ence of these new hydrocarbons was also implied bychanges in chemical structure via FTIR. This data couldindicate new hydrocarbon formation due to microbial ac-tivity in rhizosphere. Fertilizer additions to soil resultedin negligible transformations in FTIR bands.

In the absence of added fertilizer, the clover treatmentwas superior in terms of removing extractable oil/greasefrom the affected soil. When fertilizer was added, the sun-flower/mustard treatment was equally superior in over-all phytodegradation of used oil hydrocarbons. Thesespecies would be expected to enhance microbial break-



Figure 7. GC/MS spectrum of oil/grease from clover treatment at 100 days.


Figure 8. FTIR spectra of oil/grease from sunflower/mustard treatment at: (a) 0 days (control); (b) 50 days; (c) 100 days; and(d) 150 days.

Figure 9. FTIR spectra of oil/grease from grass/maize treatment at: (a) 0 days (control); (b) 50 days; (c) 100 days; and (d)150 days.

down of used motor oil in field situations. The controltreatment was also partly successful in oil decompositionwhen fertilizer was added.

Some of the added used oil was presumably incorpo-rated into the soil organic matter. Studies are currentlyunderway to determine the fate of added oils in humicmaterials.

ACKNOWLEDGMENTS

Financial support from The Eppley Foundation for Re-search, the Indiana Academy of Science, and the BallState University Office of Academic Research and Spon-sored Programs is gratefully acknowledged. The authorsare grateful to Patti Lang and Bob Morris, Ball State Uni-versity Chemistry Department, for interpretations of an-alytical data.

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phytoremediation of soil contaminated with used motor oil: ii. greenhouse studies

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