Dissipation of polycyclic aromatic hydrocarbons from soil added with manure or vermicompost

www.elsevier.com/locate/chemosphere

Chemosphere 65 (2006) 1642–1651

Dissipation of polycyclic aromatic hydrocarbons from soil addedwith manure or vermicompost

D. Alvarez-Bernal, E.L. Garcıa-Dıaz, S.M. Contreras-Ramos, L. Dendooven *

Laboratory of Soil Ecology, Department Biotechnology and Bioengineering, Cinvestav, Av. Instituto Politecnico Nacional 2508,

CP 07000 Mexico, DF, Mexico

Received 26 August 2005; received in revised form 14 February 2006; accepted 17 February 2006Available online 31 March 2006

Abstract

The dissipation of three PAHs, i.e., 500 mg phenanthrene kg�1 soil, 350 mg anthracene kg�1 soil and 150 mg benzo(a)pyrene kg�1

soil, was investigated in soil from Acolman (Mexico) added with cow manure or vermicompost while production of CO2 and inorganicN was monitored. At day 0, recovery of added phenanthrene was 95%, anthracene 96% and benzo(a)pyrene 100% in sterilized soil andconcentrations did not change significantly in sterilized soil over time. Application of organic material did not affect the concentration ofphenanthrene and anthracene, which decreased sharply in the unsterilized soil in the first weeks of the incubation. Less than 3% of theadded phenanthrene was detected after 100 days and less than 8.5% of the added anthracene (mean of the two experiments). The decreasein concentration of benzo(a)pyrene (BaP) was not fast as that of phenathrene and anthracene, and 22% was extractable from soil stillafter 100 days. It was concluded that addition of farm yard manure (FYM) and vermicompost only had an effect on the initial dissipationof phenanthrene, anthracene and benzo(a)pyrene in soil of Acolman.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Manure; Polycyclic aromatic hydrocarbons; Dissipation; Vermicompost

1. Introduction

Throughout the 20th century there has been a rapidincrease in contamination of soil with oil and its derivativesdue to petroleum spills, industrial wastes, and transportand storage accidents (Harrison et al., 2000). Accordingto PROFEPA (2002), an average of 550 environmentalemergencies from different sources of contamination occurin Mexico each year and 40% is due to crude oil.

Petroleum hydrocarbons belong to the most widespreadcontaminants of water and soil. The biodegradation ofmany components of petroleum hydrocarbons has beenreported in a variety of terrestrial and marine systems. Bio-remediation of hydrocarbon-contaminated soil by indi-genous microflora can be stimulated by adding organicmaterial and nutrients (Sims et al., 1990; Stegman et al.,

0045-6535/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.chemosphere.2006.02.028

* Corresponding author. Tel.: +52 5 7477000; fax: +52 5 7477002.E-mail address: [email protected] (L. Dendooven).

1991; Wilson and Bouwer, 1997). Readily available organicresidues, such as manure, municipal waste water, municipalsolid wastes, compost and biosolids have all been added tocontaminated soil, but stimulation of microbial degrada-tion activity is still an emerging biotechnology (Wilsonand Jones, 1993; Banerje et al., 1997; Wilson and Bouwer,1997; Namkoong et al., 2002). Kastner and Mahro (1996)reported a degradation of polycyclic aromatic hydrocar-bons (PAHs) by microorganisms of compost and this de-gradation was not caused by sorption to organic matter.The supply of macronutrients, notably N and P, hasenhanced PAHs degradation in some cases (Carmichaeland Pfaender, 1997; Liebeg and Cutright, 1999; Phillipset al., 2000). Raymond et al. (1976) studied oil biodegrada-tion in soil and found greater degradation in soils receivingfertilizer application and rototilling than in untreated soils.Hence the addition of N and P containing fertilizers can beused to stimulate microbial hydrocarbon degradation(Atlas, 1981).

D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651 1643

Biosolids are known to be effective in stimulating bio-degradation of soil contaminants, because they containlarge concentrations of inorganic nitrogen, available phos-phorous and organic material (Korboulewsky et al., 2002;Namkoong et al., 2002; Rivera-Espinoza and Dendooven,2004). They could thus be used to remediate hydrocarbon-contaminated soil (Eiceman et al., 1989; Gutierrez-Ruızet al., 1995; Benton and Wester, 1998). Most biosolids inMexico are low in heavy metal concentrations, but con-tain large concentrations of pathogens. The biosolid mustthus be treated before it could be used to remediate soil(Franco-Hernandez et al., 2003). Conventional treatmentof biosolid consist in adding lime to pH 12 (Thomas,1996). However, vermicomposting or composting withearthworms reduces pathogens within a shorter time thanin traditional composting and accelerates degradation oforganic material while maintaining amounts of nutrients.Vermicompost would be ideal to remediate soils (Pierreet al., 1982; Sinha et al., 2002; Field et al., 2004).

We are not aware of any studies that used vermicompostto stimulate remediation of hydrocarbon-contaminatedsoil. The aim of this work was to investigate the additionof vermicompost obtained from biosolid and the earth-worm Eisenia fetida and how this affected the degradationof PAHs. Soil was contaminated with phenanthrene,anthracene and BaP. Production of CO2, dynamics of inor-ganic N (NHþ4 , NO�2 and NO�3 ) and concentrations of thePAHs were monitored in an aerobic incubation at 22 ±2 �C for 100 days. FYM, partly used to generate the vermi-compost, was also added to the hydrocarbon-contaminatedsoil and its effect on remediation was studied.

2. Material and methods

2.1. Chemical products

Hydrocarbons were obtained from Sigma (USA) withpurity of >96% for phenanthrene, 99% anthracene and97% benzo(a)pyrene. Acetone was obtained from J.T.Baker (USA) with purity 99.7%. It would have been inter-esting to use 14C labelled PAHs to study mineralization,but they cannot be imported into Mexico for securityreasons.

2.2. Characteristics of the manure and vermicompost

Manure was collected from a cowshed and had anorganic C content of 432 g C kg�1, total phosphorous12 g P kg�1, available phosphorous 0.87 g P kg�1 andtotal nitrogen content 21.9 g N kg�1 on a dry matter base.E. fetida was cultivated in biosolid obtained from a waste-water treatment plant at Lerma, Edo. de Mexico. Details ofthe biosolid used and the vermicompost obtained can befound in Contreras-Ramos et al. (2005). Briefly, the vermi-compost was obtained from a mixture of 1800 g biosolidand 800 g manure at 70% water content added with 40E. fetida and conditioned for three months. The vermicom-

post with the best stability and maturity and a weight lossof 18% was obtained with 1800 g biosolid, no straw and800 g manure at 70% water content. This vermicomposthad the following properties: pH 7.9; organic C contentof 117 g kg�1; an electrolytic conductivity of 11 mS cm�1;a humic-to-fulvic acid ratio of 0.5 (HA/FA); total N con-tent of 9 g N kg�1; water soluble C (Cw) less than 0.5%; cat-ion exchange capacity of 41 cmolc kg�1; a respiration rateof 188 mg CO2–C kg�1 compost-C day�1; a NO�3 =CO2

ratio greater than 8; and a NHþ4 =NO�3 ratio lower than0.16. The vermicompost gave a germination index for cress(Lepidium sativum) of 80% after two months while theearthworm production increased 1.2-fold and volatilesolids decreased five-times. In addition, the vermicompostcontained less than three CFU g�1 Salmonella spp., nofaecal coliforms and Shigella spp. and no eggs of hel-minthes. Concentration of sodium was 152 mg kg�1 drycompost, while concentrations of chromium, copper, zincand lead were below the limits established by USEPA(1995).

2.3. Experimental site and soil sampling

The experimental site is located near the ex-convent ofAcolman in the State of Mexico (Northern Latitude 19�38 0 Western Longitude 98� 55 0). Its average altitude is2250 m above sea level and characterized by a sub-humidtemperate climate with a mean annual temperature of14.9 �C and average annual precipitation of 624 mm mainlyfrom June through August (http://www.inegi. gob.mx). Thesoil in this area is mainly cultivated with maize and oat andthat for >20 years, receiving a minimum amount of inor-ganic fertilizer without being irrigated (http://www.inegi.gob.mx). Soil for the first experiment was sampled in springand for the second one in summer. The sandy loam soil(Gee and Bauder, 1986, USDA modified soil texture trian-gle) with water holding capacity (WHC) 68.25%, pH inwater 6.58 (Thomas, 1996), organic C content 18 g kg�1 soil(Amato, 1983), inorganic C 0.7 g kg�1 soil (Nelson andSommers, 1996), and total N 0.84 g kg�1 soil (Bremner,1996) contained 220 g clay kg�1, 140 g silt kg�1and 640 gsand kg�1 (Gee and Bauder, 1986). Three plots of ca.400 m2 were sampled at random from an agricultural fieldof 1 ha. Thirty samples were collected by augering the top0–15 mm layer of each plot.

2.4. Addition of PAHs

In experiment one in which FYM was added to soil, 1 mlacetone was used to add 500 mg phenanthrene kg�1, 350 mganthracene kg�1 and 150 mg benzo(a)pyrene kg�1 together.The soil was left to stand for 30 min to allow evaporation ofthe acetone and thoroughly mixed. However, it appearedthat not all acetone evaporated as the production of CO2

was larger in soil to which PAHs were added as comparedto untreated soil. The CO2 can be generated by the degrada-tion of residual acetone, PAHs or/and killed biomass.

1644 D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651

Therefore a different protocol was applied in experimenttwo. Five gram soil was added to a 120 ml glass flask andthe acetone solution with PAHs added. This procedurewas used by Leyval and Binet (1998) as acetone might killmicroorganisms in soil, while the microorganisms in theunamended 5 g soil will re-inoculate the contaminated 5 gsoil (Brinch et al., 2002). The flask was placed under vac-uum in a desiccator for 10 min, removed and 20 g soil wasadded to each flask. The soil was mixed thoroughly andadjusted to 40% WHC by adding distilled water.

2.5. Treatments and experimental design

The soil was taken to the laboratory and treated as fol-lows. The soil from each field was passed separatelythrough a 5 mm sieve, adjusted to 40% of water holdingcapacity (WHC) by adding distilled water (H2O) and con-ditioned at 22 ± 2 �C for 7 days in drums containing a bea-ker with 100 ml 1 M sodium hydroxide (NaOH) to trapcarbon dioxide (CO2) evolved and a beaker with 100 mldistilled H2O to avoid desiccation of the soil.

In experiment one, 147 sub-samples of 25 g soil fromeach of the three sampled fields were added to 120 ml glassflask. Twenty one flasks were used for each of the seventreatments combining the application of 46.3 mg FYM or1 g C kg�1 soil and a mixture of 500 mg phenanthrenekg�1 soil, 350 mg anthracene kg�1 soil and 150 mg BaPkg�1 in acetone to sterilized or unsterilized soil (Fig. 1).

Soil collected from the 0-20 cm layer of three fie

and for the second experimen

Soil from each field sieved separately five

Experiment 1

Soil

Soil + Acetone

Soil + PAHs

Soil + FYM

Soil + FYM + PAHs

Soil + PAHs

Soil + FYM + PAHs

Incubation at 22±2oC and dynamics of C, inorgan

56, 70 and 100 days. Samples wer

Fig. 1. Experimental plan to investigate dynamics of C, N and hy

As such, 2.5 · 103 kg FYM ha�1 was added consideringthe top 10 cm layer with a density of 1.4 kg dm�3. An una-mended soil served as a control and soil treated withacetone allowed to investigate a possible effect of the ‘‘car-rier’’. Sterilization of soil was done three times for 30 minwith an interval of a day with pressurized steam at 121 �Csupplied by an autoclave (Wolf and Skipper, 1996). Theacetone with PAHs was added to previously sterilized soilunder sterile conditions to measure abiotic factors affectingthe dynamics of PAHs, the acetone was left to evaporateunder sterile conditions for 10 min and the flasks were thenair-tight sealed. Three flasks were chosen at random fromeach treatment and each field, and 20 g soil was extractedfor inorganic N with 80 ml 0.5 M K2SO4 solution, shakenfor 30 min, filtered through Whatman No 42 filter paper�

and analyzed. A sub-sample of 1.5 g soil was extracted forPAHs with acetone. These provided zero-time samples.The remaining flasks were placed in 945 ml glass jars con-taining 10 ml distilled H2O, and a vessel with 20 ml 1 MNaOH solution to trap CO2 evolved. The jars were sealedand stored in the dark for 100 days at 22 ± 1 �C. Additional18 jars containing a vessel with 10 ml distilled H2O and onewith 20 ml 1 M NaOH were sealed and served as controls.After 7, 14, 28, 56, 70 and 100 days, three jars were selectedat random from each treatment, opened and the vessels con-taining NaOH solution were removed, sealed air-tight andstored until analyzed. The soil was analyzed for inorganicN and PAHs as mentioned before. The remaining flasks

lds in Acolman (Mexico) on 23 April 2003

t on 13 September 2004

-mm and conditioned for seven days

Experiment 2

Soil

Soil + PAHs

Soil + Vermicompost

Soil + Vermicompost + PAHs

Soil + PAHs

Soil + Vermicompost + PAHs

ic N and PAHs monitored after 0, 7, 14, 28,

e aired to avoid anaerobicity

drocarbons in soil amended with manure and vermicompost.

D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651 1645

were opened and aired for 10 min to avoid anaerobicity,sealed and further incubated.

The FYM had no effect on the degradation rate ofPAHs in soil, so in experiment two a larger concentrationof a substrate richer in nutrients was added to soil. Vermi-compost might contain large amounts of microorganisms,i.e., bacteria and fungi >106 (Singleton et al., 2003; Morganand Burrows, 1982) and some of them are known todegrade hydrocarbons (Cerniglia, 1993; Juhasz and Naidu,2000; Johnsen et al., 2005).

In experiment two, 126 sub-samples of 25 g soil fromeach of the three sampled fields were added to 120 ml glassflask. Twenty one flasks were used for each of the six treat-ments combining the application of 308 mg vermicompostin 25 g of soil or 2 g C kg�1 soil and a mixture of 500 mgphenanthrene kg�1 soil, 350 mg anthracene kg�1 soil and150 mg benzo(a)pyrene kg�1 soil to sterilized or unsteril-ized soil (Fig. 1). As such, 17.3 · 103 kg ha�1 vermicompostwas added considering the top 10 cm layer with a density of1.4 kg dm�3. The incubation was repeated as described inexperiment one.

The experimental protocol was complex (Fig. 1) as itinvolved two experiments. In experiment one, the soil con-taminated with PAHs was added with farmyard manurewhile in experiment two with vermicompost. Sterilized con-trols were used to distinguish between biotic and abioticfactors that might affect dissipation of PAHs from soil.

2.6. Extractions of hydrocarbons from soil

The concentrations of phenanthrene, anthracene andbenzo(a)pyrene were measured as described by Songet al. (1995). A sub-sample of 1.5 g soil was weighted intoa Pyrex tube and 10 ml acetone were added, the tubes wereplaced in a sonicating bath at 35–40 �C for 20 min, vor-texed for 15 s, and sonicated again for 20 min, and thenthe extracts were separated from the soil by centrifugationat 3500 · rpm for 15 min. The supernatant was added to aglass flask, the acetone evaporated, and the same procedurewas repeated twice. The extracts were pooled, passedthrough a 0.45 lm syringe filter; filtrates were concentratedto 1 ml and analyzed by gas chromatography.

2.7. Chemical analyses

pH was measured in 1:2.5 soil or biosolids/H2O suspen-sion using a 716 DMS Titrino pH meter (Metrohm Ltd.CH.-901, Herisau, Switzerland) fitted with a glass electrode(Thomas, 1996). Total C was determined by oxidation withpotassium dichromate (K2Cr2O7) and titration of excessdichromate with ammonium ferrosulfate [(NH4)2FeSO4](Kalembasa and Jenkinson, 1973), and inorganic C by add-ing 5 ml 1 M hydrochloric chloride (HCl) solution to 1 gair-dried soil and trapping CO2 evolved in 20 ml 1 MNaOH. Total N was measured by the Kjeldhal methodusing concentrated H2SO4, K2SO4 and HgO to digest thesample (Bremner, 1996), soil particle size distribution by

the hydrometer method as described by Gee and Bauder(1986) and cation exchange capacity (CEC) with the bar-ium acetate method (Jackson et al., 1986). Total P wasmeasured by aqua regia digestion with sodium carbonatefusion (Crosland et al., 1995). Available P was determinedby the antimony–potassium–tartrate method (Watenabeand Olsen, 1965). The CO2 in the 1 M NaOH was deter-mined by titration with 0.1 M HCl (Jenkinson andPowlson, 1976). Water holding capacity (WHC) was mea-sured on soil samples water-saturated in a funnel and leftto stand overnight to drain freely and defined by differencesof weight. Ammonium (NHþ4 ), nitrite (NO�2 ) and nitrate(NO�3 in the K2SO4 extract was measured colourimetrically(Mulvaney, 1996).

The Agilent 4890D gas chromatograph was equippedwith a FID detector set at 310 �C and with the carriergas helium flowing at 7 ml min�1. A HP-5 capillary column(15 m by 0.53 mm, film thickness 1.5 lm) with the oventemperature increasing from 140 �C to 170 �C at 5 �C min�1

and from 170 �C to 280 �C at 30 �C min�1 was used toseparate phenanthrene, anthracene and benzo(a)pyrene.The injector temperature was 280 �C. Quantification ofthe three PAHs was done from integrated peak areas usingstandard curves.

2.8. Statistical analysis

CO2 production was regressed on elapsed time using alinear regression model, which was forced to pass throughthe origin, but allowed different slopes (production rates)for each treatment. This approach was based upon theassumption that no CO2 was produced at time zero andthat the CO2 in the atmosphere was accounted for.

Concentrations of inorganic N (NHþ4 , NO�2 and NO�3 ),phenanthrene, anthracene and benzo(a)pyrene, and CO2

production rates were subjected to one-way analysis of var-iance to test the significant differences between the treat-ments. All analyses were performed using SAS statisticalanalysis (SAS Institute, 1989).

3. Results

3.1. First experiment: addition of FYM and PAHs

The cumulative production of CO2 was lowest in theunamended soil and largest when PAHs were added(Fig. 2a). Addition of acetone and FYM increased produc-tion rate of CO2 significantly 1.9-times and 1.7-times,respectively, compared to the unamended soil, while addi-tion of PAHs and PAHs plus FYM increased it 2.8-times(P < 0.05).

Concentrations of NHþ4 showed a small decrease at theonset of the incubation and remained below 2 mg NHþ4 –Nkg�1 soil thereafter (Fig. 3a). Mean concentrations of NO�2were significantly lower in the soil added with acetone com-pared to the unamended soil and soil added with FYM, butsignificantly larger than in soil added with PAHs (Fig. 3b)

Time (days)

Cum

ulat

ive

prod

uctio

n of

CO

2 (m

g C

kg-1

soi

l)

0

1000

2000

3000

4000

5000

0 20 40 60 80 100

Con PAHs PAHs+Ver Ver

0

1000

2000

3000

4000

5000

0 20 40 60 80 100

Ace Con FYM FYM+PAHs PAHs

(b)

(a)

Fig. 2. (a) Cumulative CO2 production rate (mg C kg�1 soil) fromunamended soil (Con) and from soil amended with polycyclic aromatichydrocarbons (PAHs), farmyard manure (FYM), polycyclic aromatichydrocarbons plus farmyard manure (FYM + PAHs) or acetone (Ace)(Experiment 1) and (b) from unamended soil (Con) and from soil amendedwith polycyclic aromatic hydrocarbons (PAHs), vermicompost (Ver) orvermicompost plus polycyclic aromatic hydrocarbons (PAHs + Ver)(Experiment 2). Bars are the least significant difference between thetreatments for each measurement in time (P < 0.05).

(mg

N k

g-1 s

oil)

Time (days)

0.00.20.40.60.81.0

0 20 40 60 80 100

Ace Con FYM FYM+PAHs PAHs

0

10

20

30

40

0 20 40 60 80 100

0

5

10

15

0 20 40 60 80 100

(a)

(b)

(c)

Fig. 3. (a) Concentration of NHþ4 , (b) NO�2 and (c) NO�3 (mg N kg�1 soil)in unamended soil (Con), and in soil amended with polycyclic aromatichydrocarbons (PAHs), manure (FYM) or with polycyclic aromatichydrocarbons plus manure (FYM + PAHs). Bars are the least significantdifference between the treatments for each measurement in time (P < 0.05).

1646 D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651

(P < 0.05). Concentrations of NO�3 decreased at the onsetof the incubation, increased again and did not show signif-icant differences after 28 days. Treatment had no significanteffect on concentrations of NO�3 (Fig. 3c).

Recovery of phenanthrene was 95%, anthracene 96%and benzo(a)pyrene 100% in sterilized soil at day 0(Fig. 4a–c). Concentration of phenanthrene, anthraceneand benzo(a)pyrene did not change significantly in the steri-lized soil (Fig. 4a). Addition of FYM had no significanteffect on the concentration of PAHs in sterilized or unsteri-lized soil. Concentration of phenanthrene decreased shar-ply in unsterilized soil and 36% was removed from thesoil after 7 days (mean of soil added with or withoutFYM) and 97% after 100 days. The decrease in concentra-tion of anthracene in unsterilized soil was similar to that ofphenathrene and 30% was removed from the soil after7 days. After 100 days, however, 17% was still detectable.At the onset of the incubation, concentration of benzo(a)-pyrene in unsterilized soil decreased more sharply thanphenathrene and anthracene. Only 26% of the added ben-zo(a)pyrene was detectable in the unsterilized soil after7 days. Concentrations of benzo(a)pyrene in unsterilizedsoil, however, did not change significantly thereafter.

3.2. Second experiment: addition of vermicompost

and PAHs

The cumulative production of CO2 was lowest in theunamended soil and largest when vermicompost was added(Fig. 2b). Addition of vermicompost increased productionrate of CO2 significantly and 3.6-times compared to theunamended soil while addition of PAHs had no significanteffect (P < 0.05).

Concentrations of NHþ4 decreased at the onset of theincubation in each of the treatments and remained below2 mg NHþ4 –N kg�1 soil after 7 days (Fig. 5a). Mean con-centrations of NO�2 were not significantly different betweenthe treatments (Fig. 5b).

Increase in concentrations of NO�3 was significantly and2.5-times larger in vermicompost amended soil comparedto unamended soil, the addition of vermicompost to PAHsamended soil increased production rate of NO�3 1.4-times(Fig. 5c) (P < 0.05).

Recovery of phenanthrene was 95%, anthracene 96%and benzo(a)pyrene 100% in sterilized soil at day 0(Fig. 6a–c). Concentrations of phenanthrene, anthraceneand benzo(a)pyrene in the sterilized soil added with orwithout vermicompost did not change significantly overtime (Fig. 6a). Concentration of phenanthrene decreasedsharply in the unsterilized soil in the first weeks of the incu-bation and the decrease was significantly faster in vermi-compost amended soil compared to the unamended soil

(mg

C k

g-1 s

oil)

Time (days)

0

200

400

600

800

1000

0 20 40 60 80 100

0

150

300

450

600

0 20 40 60 80 100

0

60

120

180

240

300

0 20 40 60 80 100

FYM+PAHs PAHs sFYM+PAHs sPAHs

(a)

(b)

(c)

Fig. 4. (a) Concentration of phenanthrene, (b) anthracene and (c)benzo(a)pyrene (mg C kg�1 soil) in sterilized soil amended with polycyclicaromatic hydrocarbons (sPAHs) or polycyclic aromatic hydrocarbonsplus manure (sFYM + PAHs) and in unsterilized soil amended withpolycyclic aromatic hydrocarbons (PAHs) or polycyclic aromatic hydro-carbons plus manure (FYM + PAHs). Bars are the least significantdifference between the treatments for each measurement in time (P < 0.05).

(mg

N k

g-1 s

oil)

Time (days)

0

1

2

3

4

5

050

100150200250

0

5

10

15

0 20 40 60 80 100

0 20 40 60 80 100

0 20 40 60 80 100

Con PAHs PAHs+Ver Ver

(a)

(b)

(c)

Fig. 5. (a) Concentration of NHþ4 , (b) NO�2 and (c) NO�3 (mg N kg�1 soil)in unamended soil (Con), and soil amended with polycyclic aromatichydrocarbons (PAHs), vermicompost (Ver) or polycyclic aromatic hydro-carbons plus vermicompost (PAHs + Ver). Bars are the least significantdifference between the treatments for each measurement in time (P < 0.05).

D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651 1647

at day 7 and 14, but similar thereafter (Fig. 6a). After7 days, 32% of the added phenanthrene was removed in soiladded with PAHs and 48% in soil added with vermicom-post and PAHs. Concentrations of anthracene followedthe same pattern as phenanthrene in the unsterilized soil,but the addition of vermicompost had no significant effect(Fig. 6b). After 100 days, no anthracene was detectable.The addition of vermicompost had no significant effecton the concentration of benzo(a)pyrene. The decrease inconcentration of benzo(a)pyrene was slower than that ofphenathrene and anthracene, and 77% was detectable after7 days. After 100 days, 83% was removed from soil withvermicompost while 24% remained in the unamended soil.

4. Discussion

4.1. C and N mineralization

The production of CO2 in the unamended soil of experi-ment one was lower than in experiment two. Soil for exper-iment one was sampled before the growing season while thesecond just after harvest. Organic residues were left on thefield so the concentration of easily decomposable soilorganic matter was high after harvest. A larger amount ofeasily decomposable substrate will increase cumulative pro-duction of CO2. Patra et al. (1990) showed that the evolution

of CO2 for experiments done under similar conditions variedmarkedly with sampling time and more C substrate wasavailable for decomposition in July than in November.

There are different explanations why acetone added tosoil increased production of CO2 in experiment one. First,although acetone is very volatile and most will evaporateupon application and mixing of the soil, some of it mightremain in soil and could be used as C substrate. Silleret al. (1996) isolated an acetone-degrading bacterium Para-coccus solventivorans that can use acetone as a C source forgrowth. Second, acetone is known to be lethal for somemicroorganisms, so the surviving microorganisms coulduse the killed microbial biomass as a C source for growth(Brinch et al., 2002). However, some microorganisms, suchas Mycobacterium sp., Rhodococcus erythropolis and Pseu-

domonas putida, can tolerate different solvents (DeCarvalho et al., 2004). Third, acetone has been used toenhance the solubility of non-polar organic compoundsof which some might be rendered available for decomposi-tion (Radtke et al., 2002; Chu and Kwan, 2003). The firstexplanation was the most likely for an increased produc-tion of CO2 in experiment one (Rivera-Espinoza, 2003).Acetone in experiment one served as a C substrate and thismight affect microbial activity and thus decomposition ofPAHs. Placing the soil under vacuum for 10 min, removedmost, if not all acetone from the soil in experiment two so apossible stimulating effect of acetone was eliminated.

(mg

C k

g-1 s

oil)

Time (days)

0

200

400

600

800

1000

0 20 40 60 80 100

sPAHs sPAHs+Ver PAHs PAH+Ver

0

150

300

450

600

0 20 40 60 80 100

0

60

120

180

240

300

0 20 40 60 80 100

(b)

(a)

(c)

Fig. 6. (a) Concentration of phenanthrene, (b) anthracene and (c)benzo(a)pyrene (mg C kg�1 soil) in sterilized soil amended with polycyclicaromatic hydrocarbons (sPAHs) or polycyclic aromatic hydrocarbonsplus vermicompost (sPAHs + Ver) and in unsterilized soil amended withpolycyclic aromatic hydrocarbons (PAHs) or polycyclic aromatic hydro-carbons plus vermicompost (PAHs + Ver). Bars are the least significantdifference between the treatments for each measurement in time (P < 0.05).

1648 D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651

N mineralization in experiment one was lower than thatin experiment two, confirming that the easily decomposableorganic matter was lower in soil upon the first samplingcompared to the second sampling. Concentrations ofNO�3 in experiment two were lower for soil added withPAHs and vermicompost plus PAHs compared to soiladded with vermicompost. It has often been reported thatduring the decomposition of organic material NO�3 canbe immobilized (Zagal and Persson, 1994), especially whenorganic material with a large C:N ratio is added to soil, asin experiment two. The C:N ratio of the PAHs and PAHsplus vermicompost added was 22.6 and 22, respectively.Van Veen (1977) reported that addition of organic materialwith a C:N ratio between 20 and 30 generally tips the bal-ance towards N immobilization.

4.2. The dynamics of PAHs

Different physical, chemical, biological and environmen-tal factors affect the rate and extent of PAHs degradation,adsorption or covalent attachment of PAHs to natural sol-ids, such as clays and humic material and this can affectbioavailability, transport, biological activity and degrada-tion of the compound in the environment (Pignatello andXing, 1996).

The extraction efficiency of PAHs from soils is influ-enced by several factors, such as soil moisture content,the polar properties of solvents used, PAH content in sam-ples, aged time and the texture of soils (Fischer et al., 1994;Noordkamp et al., 1997; Letellier et al., 1999). These char-acteristics were similar in both experiments, because theextraction efficiency for the different PAHs added was sim-ilar in both experiments, i.e., 95% for phenanthrene, 96%for anthracene and 100% for benzo(a)pyrene. It appearsthat the technique used in this experiment, although timeconsuming, i.e., two sonications repeated three times, wasvery efficient (Song et al., 1995).

No sequestration of PAHs took place in soil of Acolmanas no significant changes in concentration of phenanthrene,anthracene and benzo(a)pyrene were found in sterilized soiland addition of organic material with FYM and vermicom-post neither affected characteristics of PAHs. Organic mat-ter and clay content, which are though to be the mostimportant factors in sequestration of PAHs, were low,i.e., 3% for soil organic matter and 22% for clay.

Chung and Alexander (1998) found that in soils with 4–7% organic matter and 45–60% clay content sequestrationoccurred. Additionally, the experiment might have beentoo short to detect sequestration of PAHs. Morrisonet al. (2000) reported that hydrocarbons that were addedto soil a long time ago are far less available than thosefreshly added. It has to be reminded that autoclaving mightdisrupt soil physical properties, but it might also stabilizethe soil by forming soil aggregates, thus affecting sequestra-tion of PAHs (Lotrario et al., 1995). Additionally, the soilorganic matter might change due to autoclaving whichmight further affect sequestration of PAHs (McNamaraet al., 2003; Egli et al., 2006). Sterilization with gamma rayshas usually been used as a method for sterilization (e.g.,Liste and Alexander, 2002), but it might also inducechanges in soil properties and soil organic matter (Sandoliet al., 1996).

Loser et al. (1999) reported a lag phase of 4 days whendifferent concentrations of phenanthrene (1000–5500 mg kg�1

soil) were added to soil followed by fast microbial degrada-tion within the next 4 days. The rate of degradation thenslowed down and stopped nearly completely despite oftenlarge residual concentrations of phenanthrene. The concen-tration of PAHs in both experiments reported here fol-lowed the same pattern as described by Loser et al. (1999).

Phenanthrene was rapidly degraded in both experimentsin spite of its high initial concentration. Hwang andCutright (2002) found that approximately 5% phenan-threne remained in soil after 32 days as reported in thisexperiment. The degradation of anthracene was lower thanthe degradation of phenanthrene. This slower degradationof anthracene can be attributed to its low solubility inaqueous systems (0.07 mg l�1) compared to phenanthrene(1.29 mg l�1), which renders it only slowly available formicrobial attack (Juhasz and Naidu, 2000; Ataganaet al., 2003). BaP has a lower aqueous solubility (3.8 lg l�1)than anthracene, a higher tendency to sorb onto the

D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651 1649

organic soil fraction with a logKow 6.04 compared to4.46 for phenanthrene, and a more complex structure(Pignatello and Xing, 1996; Juhasz and Naidu, 2000), sothat its residual concentrations were larger than that ofanthracene and phenanthrene. Furthermore, BaP mightbe toxic to bacteria, fungi or algae when added in largeconcentrations further reducing its removal from soil(Juhasz and Naidu, 2000).

The biodegradation of hydrocarbons is also defined bybiological characteristics of the soil environment. There isa great diversity of organisms capable of degrading thelow molecular weight PAHs (Juhasz and Naidu, 2000). Areview of the literature on the biodegradability of represen-tative hydrocarbons in petroleum indicates that microbes(bacteria and fungi) isolated from soil, sediments, and bio-solids can readily metabolize compounds of chain lengthsup to C30–C44 (Salanitro, 2001), and even complexesPAHs, such as BaP. Liquid culture experiments haveshown that bacteria can degrade BaP when grown on analternative carbon source, such as phenanthrene (Chenand Aitken, 1999; Juhasz and Naidu, 2000). However,Rafin et al. (2000) showed that the fungus Fusarium solaniis able to grow in liquid medium with BaP as sole carbonsource. Leys et al. (2004) isolated bacterial strains of thegenus Sphingomonas from contaminated soils through theirability to use PAHs as the sole source of carbon andenergy.

There are several studies in which different kinds oforganic material such as plant crop residues and manureswere added to soil to accelerate bioremediation (Rhykerdet al., 1999). FYM and vermicompost contain large amountof organic material, nutrients such as N and P, and micro-organisms, that might accelerate removal of PAHs (Beffaet al., 1995; Marinari et al., 2000). Wellman et al. (2001)found that degradation of petroleum hydrocarbons wasmuch faster and more complete in manure-amended soils.Up to 81% of the petroleum hydrocarbons in the 20% man-ure-treated soil were removed by day 41. The addition oforganic waste material, such as poultry litter and coir pith,facilitates aeration through small pores and increases thewater holding capacity of the soil, enhancing bioremedia-tion (Amadi and Ue Bari, 1992).

In the experiments reported here, however, addition oforganic material had only a limited and transient effecton the dissipation of PAHs in soil. Addition of FYMand vermicompost increased microbial activity as indicatedby the increase in cumulatively produced CO2, but this hadonly a small effect on dissipation of PAHs. Concentrationsof inorganic N increased in soil, but this did not increasedissipation of PAHs. It is questionable if added microor-ganisms will affect degradation of PAHs as their survivalrate in soil is low (Martin-Laurent et al., 2001).

Kastner and Mahro (1996) showed that addition of com-post facilitated the degradation of PAHs within 25 days,but it was not due to the microorganisms found in thecompost. Although the addition of vermicompost signifi-cantly reduced concentration of phenanthrene after 7 and

14 days, the addition of FYM had no lasting significanteffect on the concentration of PAHs. Manure has been usedin several studies to successfully remediate hydrocarbon-polluted soil, but application rates were so high that theyare not applicable in the field: a restriction we applied tothe amount of organic material added in the experimentsreported here. For instance, Wellman et al. (2001) added20% manure or 286 · 103 kg ha�1 (considering the top10 cm soil layer with density 1.4 kg dm3) for remediationof a soil with 5 g kg�1of hydrocarbons with a removal up81%, while Wong et al. (2002) added 25% pig manure(357 · 103 kg ha�1) as a co-composting for remediation ofsoil spiked with 0.3 g kg�1with 90% removal.

It was concluded that addition of FYM and vermicom-post increased the initial dissipation of phenanthrene,anthracene and benzo(a)pyrene in soil of Acolman, butremaining amounts were similar. This accelerated decreaseof PAHs after biostimulation, might reduce possible fur-ther contamination although removal of the remaininghydrocarbons has to be obtained by other means, e.g., phy-toremediation. It is well known, however, that applicationrates of both the contaminant and the organic material, theincubation conditions and soil characteristics will affectdissipation of PAHs so further experiments are necessaryto study those factors and their effects on remediation ofsoil.

Acknowledgements

We thank M. Luna-Guido and J.M. Ceballos-Ramirezfor technical assistance. The research was funded by theDepartment of Biotechnology and Bioengineering, Centrode Investigacion y de Estudios Avanzados del IPN (Cinve-stav). D.A.-B. and S M.C.-R. received grant-aided supportfrom Consejo Nacional de Ciencia y Tecnologıa (CONA-CyT), Mexico and E.L.G.-D. from Sistema Nacional deInvestigadores (SNI), Mexico.

References

Amadi, A., Ue Bari, Y., 1992. Use of poultry manure for amendment ofoil-polluted soils in relation to growth of maize (Zea mays L.).Environ. Int. 18, 521–527.

Amato, M., 1983. Determination of carbon 12C and 14C in plant and soil.Soil Biol. Biochem. 15, 107–114.

Atagana, H.I., Haynes, R.J., Wallis, F.M., 2003. Optimization of soilphysical and chemical conditions for the bioremediation of creosote-contaminated soil. Biodegradation 14, 297–307.

Atlas, R.M., 1981. Microbial degradation of petroleum hydrocarbons: anenvironmental perspective. Microbiol. Rev. 45, 180–209.

Banerje, M.R., Burton, D.L., Depoe, S., 1997. Impact of biosolidapplication on soil biological characteristics. Agr. Ecosyst. Environ.66, 241–249.

Beffa, T., Blanc, M., Marilley, L., Lott Fisher, J., Lyon, P.F., Aragno, M.,1995. Taxonomic and metabolic microbial diversity during compo-sting. In: De Bertoldi, M., Sequi, P., Lemmes, B., Papi, T. (Eds.), TheScience of Composting, pp. 149–161.

Benton, M.W., Wester, D.B., 1998. Biosolids effects on tobosograss andalkali sacatonin a chihuahuan desert grassland. J. Environ. Qual. 27,199–208.

1650 D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651

Brinch, U.C., Ekelund, F., Jacobsen, C.S., 2002. Method for spiking soilsamples with organic compounds. Appl. Environ. Microb. 68, 1808–1816.

Bremner, J.M., 1996. Total nitrogen. In: Sparks, D.L. (Ed.), Methods ofSoil Analysis Chemical Methods Part 3. Soil Science Society ofAmerica Inc., American Society of Agronomy, Inc., Madison,Wisconsin, USA, pp. 1085–1122.

Carmichael, L., Pfaender, F., 1997. The effect of inorganic and organicsupplements on the microbial degradation of phenanthrene and pyrenein soils. Biodegradation 8, 1–13.

Cerniglia, C., 1993. Biodegradation of polyciclic aromatic hydrocarbons.Biodegradation 3, 351–368.

Chen, S.H., Aitken, M.D., 1999. Salicylate stimulates the degradation ofhigh molecular weight polycyclic aromatic hydrocarbons by Pseudo-

monas saccharophila P15. Environ. Sci. Technol. 33, 435–439.Chu, W., Kwan, C.Y., 2003. Remediation of contaminated soil by a

solvent/surfactant system. Chemosphere 53, 9–15.Chung, N., Alexander, M., 1998. Differences in sequestration and

bioavailability of organic compounds aged in dissimilar soils. Environ.Sci. Technol. 32, 855–860.

Contreras-Ramos, S.M., Escamilla-Silva, E.M., Dendooven, L., 2005.Vermicomposting of biosolids with cow manure and oat straw. Biol.Fert. Soils 41, 190–198.

Crosland, A.R., Zhao, F.J., McGrath, S.P., Lane, P.W., 1995. Compar-ison of aqua regia digestion with sodium carbonate fusion for thedetermination of total phosphorus in soil by inductively coupledplasma atomic emission spectroscopy (ICP). Commun. Soil Sci. Plan.26, 1357–1368.

De Carvalho, C.C.C.R., Da Cruz, A.A.R.L., Pons, M.N., Pinheiro,H.M.R.V., Cabral, J.M.S., Da Fonseca, M.M.R., Ferreira, B.S.,Fernandes, P., 2004. Mycobacterium sp., Rhodococcus erythropolis,and Pseudomonas putida behavior in the presence of organic solvents.Microsc. Res. Techniq. 64, 215–222.

Eiceman, G.A., Gradea-Torresdey, J.L., O_Connor, G.A., Urquhart,N.S., 1989. Sources of error in analysis of municipal sludges andsludge-amended soils for di(2-ethylhexyl)phthalate. J. Environ. Qual.18, 374–379.

Egli, M., Mirabella, A., Kagi, R., Tomasone, R., Colorio, G., 2006.Influence of steam sterilisation on soil chemical characteristics, tracemetals and clay mineralogy. Geoderma 131, 123–142.

Field, S.G., Kurtz, J., Cooper, E.L., Michiels, N.K., 2004. Evaluation ofan innate immune reaction to parasites in earthworms. J. Invertebr.Pathol. 86, 45–49.

Fischer, R., Kreuzig, R., Bahadir, M., 1994. Extraction behavior ofpolycyclic aromatic hydrocarbons adsorbed on waste incinerator flyash. Chemosphere 29, 311–317.

Franco-Hernandez, O., Mckelligan-Gonzalez, A.N., Lopez-Olguin, A.M.,Espinosa-Ceron, F., Escamilla-Silva, E., Dendooven, L., 2003.Dynamics of carbon, nitrogen and phosphorus in soil amended withirradiated, pasteurized and limed biosolids. Bioresour. Technol. 87,93–102.

Gee, G.W., Bauder, J.W., 1986. Particle size analysis. In: Klute, A. (Ed.),Methods of Soil Analysis, Part 1 Physical and Mineralogical Methods,second ed. Soil Science Society of America Inc., American Society ofAgronomy, Inc., Madison, Wisconsin, USA, pp. 383–411.

Gutierrez-Ruız, M.E., Siebe, Ch., Cifuentes, E., Sommer, I., 1995.Environmental aspects of land application of wastewater from MexicoCity metropolitan area: a bibliographical review and analysis ofimplications. Environ. Rev. 3, 318–330.

Harrison, A.B., Ripley, M.B., Dart, R.K., Betts, W.B., Wilson, A.J., 2000.Effect of protein hydrolisate on the degradation of diesel fuel in soil.Enzyme Microb. Technol. 26, 5–6.

Hwang, S., Cutright, J.T., 2002. Biodegradability of aged pyrene andphenanthrene in a natural soil. Chemosphere 42, 891–899.

Jackson, M.L., Lim, C.H., Zelanzny, L.W., 1986. Oxides, hydroxides, andaluminosilicates. In: Klute, A. (Ed.), Methods of Soil Analysis, Part 1Physical and Mineralogical Methods. Soil Science Society of America

Inc., American Society of Agronomy, Madison, WI, USA, pp. 101–150.

Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatmentson metabolism in soil. A method for measuring soil biomass. Soil Biol.Biochem. 8, 209–213.

Johnsen, A.R., Wick, L.Y., Harms, H., 2005. Principles of microbialPAH-degradation in soil. Environ. Pollut. 133, 71–84.

Juhasz, A., Naidu, R., 2000. Bioremediation of high molecular weightpolycyclic aromatic hydrocarbons: a review of the microbial degrada-tion of benzo(a)pyrene. Int. Biodeter. Biodegr. 45, 57–88.

Kalembasa, S.J., Jenkinson, D.S., 1973. A comparative study of titrimetricand gravimetric methods for the determination of organic carbon insoil. J. Sci. Food Agri. 24, 1085–1090.

Kastner, M., Mahro, B., 1996. Microbial degradation of polycyclicaromatic hydrocarbons in soils affected by the organic matrix ofcompost. Appl. Microbiol. Biot. 44, 668–675.

Korboulewsky, N., Bonin, G., Massiani, C., 2002. Biological andecophysiological reaction of white wall rocket (Diplotaxis erucoides

L.) grown on biosolid compost. Environ. Pollut. 117, 365–370.Letellier, H., Budzinski, J., Bellocq, J., Connan, J., 1999. Focused

microwave-assisted extraction of polycyclic aromatic hydrocarbonsand alkanes from sediments and source rocks. Org. Geochem. 30,1353–1365.

Liebeg, E., Cutright, T., 1999. The investigation of enhanced bioremedi-ation through the addition of macro and micro nutrients in a PAHcontaminated soil. Inter. Biodeter. Biodegr. 44, 55–64.

Liste, H., Alexander, M., 2002. Butanol extraction to predict bioavail-ability of PAHs in soil. Chemosphere 46, 1011–1017.

Loser, C., Seidel, H., Hoffmann, P., Zehnsdorf, A., 1999. Bioavailabilityof hydrocarbons during microbial remediation of a sandy soil. Appl.Microbiol. Biot. 51, 105–111.

Lotrario, J.B., Stuart, B.J., Lam, T., Arands, R.R., O’Connor, O.A.,Kosson, D.S., 1995. Effects of sterilization methods on the physicalcharacteristics of soil: implications for sorption isotherms analyses.Bull. Environ. Contam. Toxicol 54, 668–675.

Leys, N.M.E.J., Ryngaert, A., Bastiaens, L., Verstraete, W., Top, E.M.,Springael, D., 2004. Occurrence and phylogenetic diversity of Sphingo-

monas strains in soils contaminated with polycyclic aromatic hydro-carbons. Appl. Environ. Microbiol. 70, 1944–1955.

Leyval, C., Binet, P., 1998. Effect of polyaromatics hydrocarbons in soilon arbuscular mycorrhizal plants. J. Environ. Qual. 27, 402–407.

Marinari, S., Masciandaro, G., Ceccanti, B., Grego, S., 2000. Influence oforganic and mineral fertilisers on soil biological and physical proper-ties. Bioresour. Technol. 72, 9–17.

Martin-Laurent, F., Philippot, L., Hallet, S., Chaussod, R., Germon, J.C.,Soulas, G., Catroux, G., 2001. DNA extraction from soils: old bias fornew microbial diversity analysis methods. Appl. Environ. Microbiol. 7,2354–2359.

McNamara, N.P., Black, H.I.J., Beresford, N.A., Parekh, N.R., 2003.Effects of acute gamma irradiation on chemical, physical and biolog-ical properties of soils. Appl. Soil Ecol. 24, 117–132.

Morgan, M., Burrows, I., 1982. Earthworm/microorganisms interactions.Rothamsted Exp. Stn. Rep.

Morrison, D.E., Robertson, B., Alexander, M., 2000. Bioavailability toearthworms of aged DDT, DDE, DDD, and dieldrin in soil. Environ.Sci. Technol. 34, 709–713.

Mulvaney, R.L., 1996. Nitrogen—inorganic forms. In: Sparks, D.L. (Ed.),Methods of Soil Analysis: Chemical Methods Part 3. Soil ScienceSociety of America Inc., American Society of Agronomy, Inc.,Madison, Wisconsin, USA, pp. 1123–1184.

Namkoong, W., Hwang, E.Y., Park, J.S., Choi, J.J., 2002. Bioremediationof diesel contaminated soil with composting. Environ. Pollut. 119, 23–31.

Nelson, D.W., Sommers, L.E., 1996. Carbon and inorganic matters. In:Sparks, D.L. (Ed.), Methods of Soil Analysis: Chemical Methods Part3. Soil Science Society of America Inc., American Society ofAgronomy, Inc., Madison, Wisconsin, USA, pp. 961–1010.

D. Alvarez-Bernal et al. / Chemosphere 65 (2006) 1642–1651 1651

Noordkamp, E.R., Grotenhuis, J.T.C., Rulkens, W.H., 1997. Selection ofan efficient extraction method for the determination of polycyclicaromatic hydrocarbons in contaminated soil and sediment. Chemo-sphere 35, 1907–1917.

Patra, D.D., Brookes, P.C., Coleman, K., Jenkinson, D.S., 1990. Seasonalchanges of soil microbial biomass in arable and grassland soil whichhave been under uniform management for many years. Soil Biol.Biochem. 22, 739–742.

Phillips, T., Seech, A., Liu, D., Lee, H., Trevors, J., 2000. Monitoringbiodegradation of creosote in soils using radiolabels, toxicity tests, andchemical analysis. Environ. Toxicol. 15, 99–106.

Pierre, V., Phillip, R., Margnerie, L., Pierrette, C., 1982. Anti-bacterialactivity of the haemolytic system from the earthworms Eisenia foetida

andrei. Invertebr. Pathol. 40, 21–27.Pignatello, J.J., Xing, B., 1996. Mechanisms of slow sorption of organic

chemicals to natural particles. Environ. Sci. Technol. 30, 1–11.PROFEPA, 2002. Procuradurıa Federal de Proteccion al Ambiente.

Direccion general de Inspeccion de Fuentes de Contaminacion,Mexico. Available from: <http://www.profepa.gob.mx>.

Radtke, C.W., Smith, D.M., Owen, G.S., Roberto, F.F., 2002. Fielddemonstration of acetone: pretreatment and composting of particu-late-TNT-contaminated soil. Biorem. J. 6, 191–204.

Rafin, C., Potin, O., Veignie, E., Lounes-Ha dj Sahraoui, A., Sancholle,M., 2000. Degradation of benzo[a]pyrene as sole carbon source by anon-white rot fungus Fusarium solani. Polycycl. Aromat. Compd. 21,311–329.

Raymond, R., Hudson, O., Jamison, V., 1976. Oil degradation in soil.Appl. Environ. Microbiol. 31, 522–535.

Rhykerd, R.L., Crews, B., McInnes, K.J., Weaver, R.W., 1999. Impact ofbulking agents, forced aeration and tillage on remediation of oil-contaminated soil. Bioresour. Technol. 67, 279–285.

Rivera-Espinoza, Y., 2003. Funcion biologica en la reutilizacion de sueloscontaminados con petroleo. Ph.D. Thesis. Centro de Investigacion y deEstudios Avanzados del IPN, Mexico, Mexico.

Rivera-Espinoza, Y., Dendooven, L., 2004. Dynamics of carbon, nitrogenand hydrocarbons in diesel-contaminated soil amended with biosolidsand maize. Chemosphere 54, 379–386.

Salanitro, J.P., 2001. Bioremediation of petroleum hydrocarbons in soil.Adv. Agron. 72, 53–105.

Sandoli, R.L., Ghiorse, W.C., Madsen, E.L., 1996. Regulation ofmicrobial phenanthrene mineralization in sediment samples by sor-bent–sorbate contact time, inocula and gamma irradiation-induced.Environ. Toxicol. Chem. 15, 1901–1907.

SAS Institute, A., 1989. Statistic Guide for Personal Computers. Version6.04, Edn. SAS Institute, Cary.

Siller, H., Rainey, F.A., Stackebrandt, E., Winter, J., 1996. Isolation andcharacterization of a new gram-negative, acetone-degrading, nitrate-reducing bacterium from soil, Paracoccus solventivorans sp. nov. Int. J.Syst. Bacteriol. 46, 1125–1130.

Sims, J.L., Sims, R.C., Matthews, J.E., 1990. Approach to bioremediationof contaminated soil. Hazard. Waste Hazard. Mater. 7, 117–149.

Sinha, R.K., Heart, S., Agarwal, S., Asadi, R., Carretero, E., 2002.Vermiculture and waste management: study of action of earthwormsEisenia foetida, Eudrilus euginae and Perionyx excavantus on biodeg-radation of some community waste in India and Australia. Environ-ment 22, 261–268.

Singleton, D.R., Hendrix, B.F., Coleman, D.C., Whitmann, W.B., 2003.Identification of uncultured bacteria tightly associated with theintestine of the earthworm Lumbricus rubellus (Lumbricidae; Oligo-chaeta). Soil Biol. Biochem. 35, 1547–1555.

Song, Y.F., Ou, Z.Q., Sun, T.H., Yediler, A., Lorinci, G., Kettrup, A.,1995. Analytical method for polycyclic aromatic hydrocarbons (PAHs)in soil and plants samples. Chin. J. Appl. Ecol. 6, 92–96.

Stegman, R., Lotter, S., Heerenklage, J., 1991. Biological treatment of oil-contaminated soils in bioreactors. In: Hinchee, R.E., Olfenbuttel, R.F.(Eds.), On Site Bioreclamation. Butterworth-Heinemann, Boston, pp.188–208.

Thomas, G.W., 1996. Soil pH and soil acidity. In: Sparks, D.L. (Ed.),Methods of Soil Analysis, Part 3. Chemical Methods. Soil ScienceSociety of America Inc., American Society of Agronomy, Inc.,Madison, WI, pp. 475–490.

USEPA, A., 1995. A guide to the biosolids risk assessment for the EPA Part503 Rule EPA/B32-B-93-005. United States Environmental ProtectionAgency Office of Wastewater Management, Washington, DC.

Van Veen, J.A., 1977. The behaviour of nitrogen in soil. Doctoraatswerk,Wageningen, Nederland, Landbouwhogeschool, 164 pp.

Watenabe, F.S., Olsen, S.R., 1965. Test of an ascorbic acid method fordetermining phosphorous in water and NaHCO3 extracts from soil.Soil Sci. Soc. Am. Proc. 29, 677–678.

Wellman, D.E., Ulery, A.L., Barcellona, M.P., Duerr-Auster, S., 2001.Animal waste-enhanced degradation of hydrocarbon-contaminatedsoil. Soil Sediment. Contam. 10, 511–523.

Wilson, L.P., Bouwer, E.J., 1997. Biodegradation of aromatic compoundsunder mixed oxygen/denitrifying conditions: a review. J. Ind. Micro-biol. Biot. 18, 116–130.

Wilson, S.C., Jones, K.C., 1993. Bioremediation of soil contaminated withpolynuclear aromatic hydrocarbons (PAHs): a review. Environ. Pollut.81, 229–249.

Wong, J.W.C., Wan, C.K., Fang, M., 2002. Pig manure as a co-composting material for biodegradation of PAH-contaminated soil.Environ. Technol. 23, 15–26.

Wolf, D.C., Skipper, H.D., 1996. Soil sterilization. In: Sparks, D.L. (Ed.),Methods of Soil Analysis. Part 2. Microbiological and BiochemicalProperties. Soil Science Society of America Inc., American Society ofAgronomy, Inc., Madison, Wisconsin, USA, pp. 1085–1122.

Zagal, E., Persson, J., 1994. Immobilization and remineralization ofnitrate during glucose decomposition at four rates of nitrogenaddition. Soil Biol. Biochem. 26, 13–1321.