B(a)Py em solo

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Application of organic wastes on a benzo(a)pyrene polluted soil. Responseof soil biochemical properties and role of Eisenia fetida

Manuel Tejada a,n, Grazia Masciandaro b

a Department of Crystallography, Mineralogy and AgroChemistry, Crta de Utrera Km1, University of Seville, E-41013 Seville, Spainb Institute for the Ecosystem Studies, National Research Council (CNR), Via Moruzzi 1, 56124 Pisa, Italy

a r t i c l e i n f o

Article history:

Received 16 June 2010Received in revised form

3 September 2010

Accepted 7 October 2010Available online 26 November 2010

Keywords:

Benzo(a)pyrene

Bioremediation

Organic wastes

Eisenia fetida

a b s t r a c t

In this paper we studied the bioremediation effects of a soil artificially contaminated by benzo( a)pyrene

with and withouttwo organicwastes(organic municipalsolid waste, MSW, andpoultry manure,PM) andwith andwithout worms (Eisenia fetida) over 90 days. Forthe organictreatments,soil samplesweremixed

with MSW at a rate of 10% or PM at a rate of 7.6%, in order to apply the same amount of organic matter to

the soil. An unamended and non-polluted soil was used as control. Cellulase and glutathione-

S-transferase activities in worms andthe earthworms weight were measured at four different incubation

times (3, 15, 60 and 90 days). Cocoon numbers, average weight per cocoon and number of juveniles per

cocoon were measured 30 days after the benzo(a)pyrene exposure. Extractable benzo(a)pyrene in soils

and E. fetida was determinedduringthe incubation period. To observe theeffects of bioremediation of the

contaminated soil, ATP, urease and phosphatase activities were measured. At the end of the incubation

period and when compared with the polluted soil without worms and organic matter, the extractable

benzo(a)pyrenedecreasedby 41.2% forthe unamended polluted soil andwithout worms, by 45.8% forthe

organic-PM polluted soil and without worms, 48.3% for the organic-MSW polluted soil and without

worms, 55.4% for the organic-PM polluted soil and with worms, and 66.3% for the organic-MSW polluted

soil and with worms. This meant thatworm hydrocarbon absorptionwas lowest in the contaminated soil

amended with MSW and with worms, causing an increase in catabolic activity of the soil. These results

suggested thatthe co-application of organic wastes and E. fetidafor the bioremediation of benzo(a)pyrenepolluted soil is potentially advantageous.

& 2010 Elsevier Inc. All rights reserved.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) have been recognized

as a potential health risk due to their intrinsic chemical stability,

high recalcitrance to different types of degradation and high

toxicity for living microorganisms (Alexander, 1999; Andreoni

et al., 2004; Eibes et al., 2006).

Bioremediation of PAH-contaminated soil by indigenous micro-

flora can be stimulated by adding organic material and nutrients

(Wilson and Bouwer, 1997; Contreras-Ramos et al., 2007).Recently, the addition of horticultural compost (Maliszewska-

Kordybach et al., 2000), straw (Kucharski et al., 2000) and manure

(Coover andSims, 1987) tosoilshas been foundto immobilize PAHs

and reduce their negative effects on soil microbial populations and

enzymeactivities.This is probably dueto the role of organic matter

for sorption processes of organic pollutants (Kleineidam et al.,

1999; Guanasekara and Xing, 2003).

Furthermore, the organic matter plays an important role in PAH

biodegradation through contribution of soil nutrients as a result of

their mineralization and by stimulating microbial activity. Numerous

experimental studies have shown that amendment of nutrients can

result in the enhanced biodegradation of PAHs (Head and Swannell,

1999; Xu and Obbard, 2003; Xu et al., 2003). However, amendment of

nutrients to PAH-contaminated soils is often impracticable as water-

solublenutrients canbe rapidlydilutedand leached(LeeandDeMora,

1999). Organic matter mineralization releases nutrients continuously

or intermittently over a period of time and therefore has been appliedto PAH-contaminated soils to stimulate and maintain indigenous

biodegradation rates. However, the influence of organic matter on a

soils biological and biochemical properties depends on the amount,

type, and size of the added organic materials (Tejada et al., 2007). In

turn, the effect of each organic material on soil biological properties

depends on its dominant component.

In recent years the use of earthworms, as an efficient methodto

support the bioremediation of a soil,has beenwidely experimented

(Ceccanti et al., 2006; Contreras-Ramos et al., 2006,2007).

Earthworms maintain aerobic conditions through the continuous

mixing of the soil (Kretzschmar, 1978; Schack-Kirchner and

Hildebrand, 1998). In addition, they ingestsoil andexpela partially

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/ecoenv

Ecotoxicology and Environmental Safety

0147-6513/$- see front matter& 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.ecoenv.2010.10.018

n Corresponding author. Fax: +34 954486436.

E-mail address: [email protected] (M. Tejada).

Ecotoxicology and Environmental Safety 74 (2011) 668674
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stabilizedproduct (casting). In this way theyensure the availability

of organic substrates for proliferation of the autochthonous micro-

organisms in the soil, thus increasing the microbiological and

biochemical soil activity (Xiao et al., 2006).

The use of biomarkers is a concept in earthworm toxicity testing.

Of the potential biomarkers, earthworm glutathione-S-transferase

and cellulalse enzymes are shown to respond to toxin exposure

(Xiao et al., 2006). Glutathione-S-transferase is an important

detoxification enzyme and its activity has been used as a potentialbioindicatorand biomarkerof earthworms forheavymetals(Lukkari

et al., 2004), pesticide (Booth et al., 2001; Xiao et al., 2006) and PAH

exposure (Zhang et al., 2009). Cellulase activity of earthworms

indicates their role in the decomposition of plant litter and other

cellulosic materials. It has been used as a biomarker of a pesticide

contamination on earthworms (Luo et al., 1999; Xiao et al., 2006).

On the other hand, soil enzymatic activities are responsible for

important cycles such as those of C, N, P and S. Also, enzyme

activities have often been used as indicators of microbial activity

and can also be useful to interpret the intensity of microbial

metabolism in soil (Ceccanti et al., 2006; Tejada et al., 2007).

Enzymes,in fact, are thecatalysts of important metabolic functions,

including the decomposition and the detoxification of contami-

nants (Nannipieri and Bollag, 1991).

Few studies have been reported using different organic matter

types and earthworms to remediate PAH-contaminated soil. For

this reason, the objective of this study was to investigate under

laboratory conditions the availability of benzo(a)pyrene in soils

amended with two organic wastes and with and without worms

(Eisenia fetida) andits influence on both soil biochemicalproperties

and the earthworms.

2. Materials and methods

2.1. Soil, organic wastes and PAH characteristics

Thesoil used in this experiment is a Plagic Anthrosol( FAO, 1989).The mainsoil

characteristics are shown in Table 1.Soil pH was determined in distilled water with a glass electrode (soil:H 2O ratio

1:2.5). Soil texture was determined by the Robinsons pipette method (SSEW, 1982)

and quantification and dominant clay types were determined by X-ray diffraction.

Total carbonates were measured by estimating the quantity of the CO2 produced

by HCl addition to the soil (MAPA,1986). Soil organic matter was determined by the

method ofYeomansand Bremner(1988). Humic and fulvic acids-like wereextracted

with 0.1 M sodium pyrophosphateand 0.1 M sodium hydroxideat pH13 (Kononova,

1966).Thesupernatant wasacidifiedtopH 2 withHCland allowed tostand for24 h at

room temperature. To separate humic acids-like from fulvic acids-like, the solution

was centrifuged and the precipitate containing humic acids-like was dissolved with

sodium hydroxide (Yeomans and Bremner, 1988). After the removal of humic acids-

like, the acidic filtrate containing the dissolved fulvic acid-like fraction was passed

througha column of XAD-8 resin. The adsorbed fulvic was then recovered by elution

with 0.1 M NaOH, desalted using Amberlyst 15-cation-exchange resin, and finally

freeze-dried. The carbon content of humic and fulvic acids-like were determined by

themethoddescribed.Total N wasdeterminedby theKjeldhal method (MAPA,1986).

Afternitric and perchloricaciddigestion,totalCa,Mg, Fe, Cu, Mn,Zn,Cd,Pb, Niand Cr

concentrations were determined by atomic absorption spectrometer and K was

determined by atomic emission spectrometer, according to MAPA methods (1986).

The organic wastes applied were the organic fraction of a municipal solid waste

(MSW) andpoultry manure.The generalpropertiesof theorganicwastesare shown in

Table 1. Organic matter was determined by dry combustion, according to the official

methodsof theSpanish Ministryof Agriculture( MAPA, 1986). Humic andfulvicacids-

likewere extracted, separated and determined by the methods previously described.

Total N was determined by the Kjeldhal method (MAPA, 1986). After nitric and

perchloric aciddigestion,total Ca,Mg, Fe,Cu, Mn,Zn, Cd,Pb, Ni andCr concentrations

were determined by atomic absorption spectrometer and K was determined by

atomic emission spectrometer, according to MAPA methods (1986).

Table 2 shows the acidic functional group contents of HAs isolated from both

organic wastes. The carboxyl groupcontent was estimated by directpotentiometric

titration at pH 8, the phenolic hydroxyl group content was estimated as two times

thechangein chargebetweenpH 8 andpH 10,andthetotalaciditywascalculatedby

addition (Ritchie and Perdue, 2003).

The hydrocarbon utilized was benzo(a)pyrene. This hydrocarbon was used

because is recalcitrant and therefore it is very difficult to degrade (Betancur-Galvis

et al., 2006). Benzo(a)pyrene was dissolved in acetone and added to soil at a

concentration of 50 mg kg1 soil.Thisconcentration wasusedbecauseit isnot toxic

for E. fetida earthworms (Contreras-Ramos et al., 2006).

2.2. Incubation procedure

Seven hundred grams of soil was pre-incubated at 25 1C for 7 days at 3040%of

their water-holding capacity, according to Tejada (2009), prior to the treatments.

After this pre-incubation period, the incubation treatments are detailed as follows:

1. C1, control soil, soil non-polluted, non-organic amended and without

earthworms

2. C2, soil non-polluted, non-organic amended and with earthworms

3. C3, soil polluted with benzo(a)pyrene, non-organic amended and without

earthworms

4. C4, soil polluted with benzo(a)pyrene and non-organic amended and with

earthworms

5. MSW1, soil non-polluted and amended with MSW and without earthworms

6. MSW2, soil polluted with benzo(a)pyrene, amended with MSW and without

earthworms

7. MSW3, soil polluted with benzo(a)pyrene, amended with MSW and withearthworms

8. PM1, soil non-polluted and amended with PM and without earthworms

9. PM2, soil polluted with benzo(a)pyrene, amended with PM and without

earthworms

10. PM3, soil polluted with benzo(a)pyrene, amended with PM and with

earthworms

Table 3 shows in simplified form the incubation treatments.

Triplicate treatments were kept in semi-closed microcosms at 2072 1C for 3,

15, 60 and 90 days.

For organic treatments, soil samples were mixed with MSW at a rate of 10% or

PM at a rate of 7.6%, respectively, in order to apply the same amount of organic

matter (32.8 g) to the soil.

Twenty two earthworms of the species E. fetida (approximately 210 mg fresh

weight) were included in the microcosm. The microcosm was covered with fine

nylon mesh to prevent soil loss andto keep theearthworms from escaping. E. fetida

were bred in laboratory cultures on organic waste materials, principally

Table 1

Characteristics of the experimental soil and organic wastes (means7standard

error, n4).

Soil PM MSW

pH (H2O) 8.670.2 7.170.3 6.270.3

CO32 (g kg1) 203712

Fine sand (g kg1) 142735

Coarse sand (g kg1) 387726

Silt (g kg1) 242719

Clay (g kg1) 229710

Clay types Smectite: 66%

Kaolinite: 20%

Illite: 14%

Organic matter (g kg1) 1.170.8 614726 469715

Humic acid-C (mg kg1) 18.572.4 67271.4 1030717

Fulvic acid-C (mg kg1) 9.871.1 715710 711710

Total N (g kg1) 0.470.1 38.872.9 17.371.3

Fe (mg kg1) 35.873.7 180722 815738

Cu (mg kg1) 9.771.3 1.670.3 82.679.8

Mn (mg kg1) 11.372.1 4.270.9 75.678.1

Zn (mg kg1) 8.171.5 3.370.8 134713

Cd (mg kg1) 6.571.2 0.470.1 1.170.3

Pb (mg kg1) 0.470.1 0.970.1 82.473.6

Ni (mg kg1) 2.970.7 1.370.2 13.671.5

Cr (mg kg1) 5.370.6 0.170.02 19.471.7

Table 2

Acidic functional group contents (means7standard error, n3) of humic acids

(HAs) isolated from MSW and PM. Data are the means of four samples.

Total aci di ty COOH (mol kg1) Phenolic OH

PM 4.070.1 3.070.1 1.070.1

MSW 4.370.1 3.270.1 1.170.1

M. Tejada, G. Masciandaro / Ecotoxicology and Environmental Safety 74 (2011) 668674 669


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vermicomposts. The substrates used to obtain the vermicomposts and vermicom-

posting process are detailed in (Tejada et al., 2010).

2.3. Benzo(a)pyrene extraction in soil and earthworms

The amount of benzo(a)pyrene in the soil was measured as described by Song

etal. (1995). A sampleof 1.5 g ofsoilwas put ina 15-mlPyrex tubeand 10 mlacetonewas added, shaken on a vortex and sonicated for 20 min. The PAHs extracted with

acetone were separated from the soil by centrifugation at 13 700g for 15 min, the

supernatantwas added to 20 ml glass flasks andthe acetone used to extractPAHs was

leftto evaporate. Thesame procedure wasrepeated againtwiceand theextracts were

added to a 20-ml flask. The extracts were passed through a 0.45-mm syringe filter.

The filtered extracts were concentrated to 1 ml and then analyzed by GC.

The earthworms were placedon wet filterpaper for in Petri dishes for a period of

24 h to allowthe depuration of theirgut contents. They were then cleaned and frozen

in liquid nitrogen. The frozen earthworms weregroundwith a pestle and mortar and

mixed with 1.5 times their wet weight of Na2SO4. The mixture was extracted with

10 ml acetoneby sonication at 60 1C for20 min, centrifuged at 13 700gfor15 minand

decanted. The same procedure was repeated again twice. The extracts were pooled

and passed through a 0.45-mm syringe filter. The filteredextracts were cleaned up on

a 10% deactivated alumina column and eluted with acetone. The eluted sample was

concentrated to 1 ml and analyzed on an Agilent 4890D gas chromatograph, which

wasequipped with an FID detectorset at 3101C fitted withan HP-5capillary column

(15 m by 0.53 mm, film thickness 1.5 mm); carrier gas was helium; the oventemperature was increased from 140 to 1701C at 5 1C min1 and from 170 to

280 1C at 30 1C min1. The injector temperature was 280 1C.

2.4. Earthworm analysis

Cocoon production of the worms was determined after 30 days of exposure.

Cocoons were collected by hand sorting and weighed, and then incubated for four

additional weeks, as described by Maboeta et al. (1999). Cocoons were cultured in

Petri dishes at 2571 1C covered with three moist filter papers. According to Xiao

et al. (2006), the filter papers in these dishes were changed every three days to

prevent bacterialgrowth. At theend of thetest(30 days),the weights of cocoon and

number of juveniles per cocoon were determined.

For 3, 15, 60 and 90 days of the incubation period and for each treatment, three

wormswere selectedandplaced onwetfilterpaperinPetri dishesfor24 h toclear gut

contents, and their weights were recorded after blotting them dry on paper towels.

Earthworms were digested in the 1:1 nitricperchloric extract after digestion at

450 1C for 6 h. Cellulase activity was measured as described by Mishra and Dash(1980), and glutathione-S-transferaseactivity was measuredaccording to the method

described by Habig et al. (1974) and Saint-Denis et al. (1998).

2.5. Soil analysis

Adenosine triphosphate (ATP) was extracted from soil using the Webster et al.

(1984) procedure and measured as recommended by Ciardi and Nannipieri (1990).

20 ml of a phosphoric acid extractant was added to 1 g of soil, and the closed flasks

were shaken in a cool bath. Then the mixture was filtered through Whatman paper

andan aliquot was usedto measure theATP content by meansof luciferinluciferase

assay in a luminometer (Optocomp 1, MGM Instruments, Inc.). Soil urease activity

was determined by the method of Kandeler and Gerber (1988) using urea as

substrate. Phosphatase activity was measured using p-nitrophenyl phosphate as

substrate (Tabatabai and Bremner, 1969).

All biological parameters were measured in triplicate at 3, 15, 60 and 90 days

and for each treatment.

2.6. Statistical analysis

Analysis of variance (ANOVA) was performed using the Statgraphics Plus 2.1.

The means were separated using Tukeys test, considering a significance level of

Po0.05 throughout the study. For the ANOVA, triplicate data were used for each

treatment and every incubation day.

3. Results

3.1. Extractable form of benzo(a)pyrene in soils and earthworms

At the end of the incubation period, the highest contents of

extractable benzo(a)pyrene in soil were in the C3 treatment

(Fig. 1a). For the other treatments studied, this hydrocarbon

decreased. Compared with the C3 treatment, this decrease was

significantly higher for the MSW3 treatment (66.3%), followed by

the PM3 treatment (55.4%) and then the C4 treatment (41.2%).

Although the extractable content of benzopyrene also decreased

for the MSW2 and PM2 treatments (28.2% and 22.1%) compared

with the C4 treatment, the statistical analysis indicated no sig-

nificant differences between these treatments.

The benzopyrene content in earthworms increased gradually

during the incubation period (Fig.1b).At the endof theexperiment,the highest contents occurredin the C4 treatment. Benzo(a)pyrene

content decreasedby 15.9%for thePM3treatment and 21.3%for the

MSW3 treatment. However, the ANOVA indicates no significant

differences between these treatments at the end of the incubation

period.

3.2. Effect of benzo(a)pyrene in E. fetida

During the incubation period the weight of earthworms

decreased in contaminated soils (Fig. 2). However, this decrease

was lower for the organically amended soils. At the end of the

experiment andwhencompared with the C2 treatment, the weight

of earthworms decreased by 19.8%, 22.4% and 38.2% for theMSW3,

PM3 and C2 treatments, respectively.At the end of the incubation period, the cellulase and glu-

tathione-S-transferase activities in earthworms decreased signifi-

cantly (po0.05) for the C2 treatment (Fig. 2). In the contaminated

and organically amended soils,both enzymaticactivities decreased

less sharply. Also andfor both organically amended soils, therewas

a smaller decrease in cellulase and glutathione-S-transferase

activities in soils amended with MSW than for PM.

The cocoon numbers were higher in uncontaminated soil

(Table 4). For the contaminated soils, the lowest cocoon number

was observed for the organically amended soils. There were also

differences (though not statistically significant) between the con-

taminated and amended soils, observing the highest cocoon num-

bers for the MSW3 treatment than for the PM3 treatment (4.9%).

The average weight per cocoon was also lowest in polluted soils(Table 4). However, and compared with uncontaminated soil, this

decrease was lowest for the organically amended soils. Again, this

decrease was lowest for the MSW3 than for the PM3 treatment.

Compared with the C2 treatment, the number of juveniles per

cocoon decreased significantly (po0.05) for the other treatments

(Table 4). And also compared with the C2 treatment, this decrease

was lowest for the contaminated and organically amended soils

(11.9% for the MSW3 treatment and 16.5% for the PM3 treatment).

3.3. Soil biochemical analysis

Atthe end ofthe incubation period, the highest soil ATPcontents

were observed for uncontaminated and organically amended soils

(Table 5). In this respect, the highest values were observed for the

Table 3

Scheme of the incubation treatments performed.

Treatments Pollution with

benzo(a)pyrene

Organically

amended

Earthworms

addition

C1 () () ()

C2 () () ( + )

C3 ( + ) () ()

C4 ( + ) () ( + )

MSW1 () (+ ) ()MSW2 ( + ) (+ ) ()

MSW3 ( + ) (+ ) ( + )

PM1 () (+ ) ()

PM2 ( + ) (+ ) ()

PM3 ( + ) (+ ) ( + )

(+ ): Yes.

(): No.

M. Tejada, G. Masciandaro / Ecotoxicology and Environmental Safety 74 (2011) 668674670


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MSW1 treatment followed by PM1. When benzo(a)pyrene was

applied to the soil, the ATP content decreased. However, this

decrease was lowest in organically amended soils,and withworms,

followed by the organically amended soils without worms and

non-organically amended soil without worms.

The results obtained for soil urease activity showed a decrease

in the treatments where benzo(a)pyrene was applied (Table 5).

However,this decrease was less pronounced in soils amended with

organic material andworms. At theend of the experimental periodand compared with the control soil (C1), the urease activity

decreased by 18.8% for the MSW3 treatment, followed by the

PM3 (25%), MSW2 (42.2%) and PM2 (43.1%) treatments. At the end

of the incubation period the statistical analysis indicated signifi-

cant differences (po0.05) between the C1 treatment and MSW2

and PM2 treatments.

The results obtained for soil phosphatase activity were similar to

those obtained for urease activity (Table 5). Again, the application of

benzo(a)pyrene to soildecreasedthis enzyme activity. However, the

decrease in phosphatase activity was lower in soils amended with

organic matter and worms. It was also noted that there were

differences between the organic matter type applied to soil, noting

that application of MSW in contaminated soil largely decreased the

negative effect of the hydrocarbon in soil phosphatase activity.

4. Discussion

Our results indicated that benzo(a)pyrene induced negative

effects on soil biology (morphological, reproductive and enzymatic

activities on E. fetida and soil biochemical properties). These results

are in accordance with Contreras-Ramos et al. (2006, 2007), who

studied the toxicity of benzo(a)pyrene on the growth and repro-

duction of E. fetida over a period of 11 weeks.

However, the application of the worms in polluted soil decreasedthe soil hydrocarbon concentration and therefore, improved soil

biochemical properties. Earthworms burrow through the soil, turn-

ing it over continuously, maintaining its fertility and structure, and

improving aeration and water infiltration capacity (Edwards, 1998).

These animals come into close contact withPAHs by moving around

in the soil (Johnsen et al., 2005). This activity contributes to the

biodegradation of organic contaminants, such as the microorgan-

isms in theearthworm gut degrade contaminants. Some reports also

indicate that annelids can metabolize benzo(a)pyrene. E. fetida and

other annelids have cytrochrome-P450 enzymes capable of degrad-

ing this compound (Achazi et al., 1998). However, apart from

accelerating decomposition of PAHs, earthworms also accumulate

them. However, only small amounts of PAHs were found in the

tissues of the earthworms in this study.

0

10

20

30

40

50

60

9060153

Benzo(a)pyren

e(mgkg-1)

C3 C4 MSW2 MSW3 PM2 PM3bb

b

b

a

b

bb

b

b

b

bb

a

a

a

bb

b

ab ab

abab

ab

0

1

2

3

4

5

6

9060153

Benzo(a)pyrene(mgkg-1)

C4 MSW3 PM3

aa a

a

a

b

b

b

ab

ab

ab

ab

Fig.1. Extractablebenzo(a)pyrene (mean7standard error, n3)insoils (a) and Eisenia fetida(b) during theexperimental period. Columns (mean7S.E.)followedby thesame

letter(s) are not significantly different (p40.05).



5/7

There are two major uptake routes by which PAHs can be

accumulated by earthworms. The firstis simple diffusionacross the

earthworms dermis. This passive dermal uptake is facilitated by

the hydrophobic nature of the PAHs, resulting in a partitioning of

soil-associated PAHs in the lipid-rich earthworm tissues. Secondly,

when PAHs are ingested together with soil, this leads to the

diffusion of the contaminants through the gastrointestinal tract

and accumulation in the earthworm tissues (Krauss et al., 2000;

Stroo et al., 2000).

Due to the absorption of benzo(a)pyrene by the worm, the worm

weight decreased. This is different to that found for other pollutants

such as pesticides or heavy metals.In this sense, Ribeiro et al. (2001)

foundthat in soils contaminatedwith pesticidesor heavy metals, the

weight loss of worms is due to a situation of food inhibition,

decreasing the consumption rate and therefore affecting the

growth rate. The decrease of earthworm cellulase and glutha-

tione-S-transferase activities is possibly due to a physiological

adaptability in order to compensate for hydrocarbon stress. To

overcome the stress situation, animals require high energy, and this

energy demand may have led to protein catabolism (Mosleh et al.,

2003). Furthermore, this decrease in protein content might be a

result of mechanical lipoprotein formation, which is used to repair

damaged cells, tissues and organs (Ribeiro et al., 2001).

The hostile environment, in which the worms develop, prompts

them to use a high amount of energy for survival rather than

reproduction. Therefore, the cocoon numbers, the average weight

Weight(mg)

C2 C4 MSW3 PM3

a

ab

a

a

a

a

bbb b

b b

b ab

abab

Cellulaseactivity

(mgglucosemgprote

inh-1)

C2 C4 MSW3 PM3

a

a

b

b

bb b

b b b

abab

ab

ab

a

ab

100

120

140

160

180

200

220

240

350

400

450

500

550

600

650

700

70

80

90

100

110

120

130

3

Glutathione-S-transferaseactivity

(nmolmgproteinmin-1)

C2 C4 MSW3 PM3

a

a

b

bb

bb

bb

b

b

b

b

abab

ab

15 60 90

3 15 60 90

3 15 60 90

Fig. 2. Weight and cellulase and glutathione-S-transferase activities (mean7standard error, n3) in Eisenia fetida exposed to benzo(a)pyrene during the experimental

period. Columns (mean7S.E.) followed by the same letter(s) are not significantly different (p40.05).



6/7

of cocoon and the number of juveniles per cocoon decreased in the

experiment. Forthisreason,it is very probable that in these adverse

conditions, the total biomass of worms decreases.

The decrease in the soil hydrocarbon content influenced the

improvement of soil biochemical properties. Also, it is widely recog-

nized that earthworms can significantly and positively affect the soil

environment in terms of soil organic matter dynamics and turnover,

improved soil structure, improved soil fertility (Kersante et al., 2006),

breakdown of soil particles, substrate aeration andmoisture retention

and drainage (Edwards and Bohlen, 1996). Many of these actions and

factors, such as the excretion of protein rich mucous, reworking and

fragmentation of carbon anddeposition of cast excreta arestimulators

for soil microorganisms (Hickman and Reid, 2008). Thus, within the

field of bioremediation, the use of earthworms to improve soil

conditions and to subsequently promote microbial numbers, diversity

and activity should bring about benefits for levels of catabolic activity

andsubsequentenhancement of organic contaminant biodegradation.

Indeed, recent works have shown that earthworms, through their

biological, chemical and physical actions on soils, can assist in

increased losses of PAHs (Contreras-Ramos et al., 2006), crude oils

(Schaefer and Filser, 2007) and PCBs (Tharaken et al., 2006).

The application of organic matter in the contaminated soil and

without worms decreased the toxic effect of benzo(a)pyrene on soil

biochemical properties. This aspect shows thatthe adsorption capacity

of organic matteris more importantthan the absorptive capacityof the

hydrocarbon by the worm. However, differences were found depend-ingon the chemicalcomposition of organic matterapplied tosoil. Thus,

there was a minor inhibitory effect on soil ATP content and urease and

phosphatase activities in soils amended with MSW.

Several studies of metal complexation with organic matter indicate

that the sorption of heavy metals increases when the humic acid-like

contentincreases in theorganic matter, comparedto thefulvic acid-like

content, probably due to the fact that humic acid-like substances are

characterizedby a highernumber of carboxylic groups than fulvicacid-

like substances (Tejadaet al., 2007; 2008). Therefore, andsimilar to the

heavy metal complexation, the sorption of benzo(a)pyrene increased

with the humic acid-like content in the organic waste applied to the

soil. The higher sorption probably caused a higher decrease of

hydrocarbon in the soil solution, and therefore, lower availability of

benzo(a)pyrene for soil microorganisms. This fact could probably beresponsible for the increase in the soil biochemical properties. These

results are in agreement with those of Murphy et al. (1990), Kollist-

Siigur et al. (2001) and Tejada et al. (2008), who suggested that humic

acids had greater binding affinity for PAHs than fulvic acids.

The combined addition of earthworms and organic matter to

contaminated soil resulted in the greatest enhancementin terms of

catabolic activity. It is suggested that the earthworm presence

sufficiently altered (biologically, chemically and physically) the

nature of the substrate so as to determine a significant increase in

catabolic activity. It follows that where hydrocarbon catabolic

activity was enhanced, biodegradation would also be enhanced.

Importantly, the earthworm effects would include the promotion

of boththe organic matter and the contaminated soils catabolically

active microorganisms.

Furthermore, it is emphasized that the organic matter type

influenced this process, highlighting the lower values of hydro-

carbon in soils with MSW and worms. Thus, soil biochemical

properties improved and the worms could use more energy for the

reproduction process. Compared with previous results, this aspect

leads to an increase in the total biomass of worms in polluted soil

treated with organic matter.

5. Conclusions

The co-application of organic matter and earthworms is poten-tially advantageous for the bioremediation of hydrocarbon-con-

taminated soil. However, the organic matter typeapplied to the soil

play a very importantrole. Theapplication of organic matterrich in

humic-like acids to polluted soil, increased the hydrocarbon

adsorption, thus decreasing the hydrocarbon availability. This,

together with hydrocarbon absorption by the worms resulted in

a decrease in the inhibition of soil biochemical properties.

Acknowledgment

Manuel Tejadathanks the Spanish Ministry of Education for the

financial support of the scholarship of university teaching staff

mobility.

Table 4

Cocoon production, average weight of cocoons (mg) and number of juveniles per

cocoon (mean7standard error, n3) ofEisenia fetida exposed to benzo(a)pyrene.

Cocoon numbers Average weigh of

cocoon (mg)

Number of juveniles

per cocoon

C2 2.9ba70000.4 8.8ba70.5 3.1ba70.4

C4 1.8a70.2 5.8a70.9 2.5a70.6

MSW3 2.4b70.4 7.2b70.7 2.7ab70.4

PM3 2.3ab70.6 7.0ab70.7 2.6a70.4

a Different lettersfollowing thenumbers indicate a significant difference at Po0.05.

Table 5

ATP and urease and phosphatase activities (mean7standard error, n3) in soils

exposed to benzo(a)pyrene.

Incubation days

3 15 60 90

ATP (ng ATP g1 soil)

C1 304aba723 304ab718 296ab720 295ab726

C2 306ab720 308722 314ab725 312ab721C3 276a713 249a715 235a721 221a723

C4 288a722 260a720 248a712 236a730

MSW1 326ab718 331ab719 366b716 410b728

MSW2 299ab717 289a714 270a714 259a722

MSW3 303ab730 302ab711 290a724 284a717

PM1 323ab726 313ab716 338728 388b725

PM2 293ab714 277a724 259a710 248a714

PM3 301ab720 294ab722 283a719 270a719

Urease activity (mg NH4+ g-1 h-1)

C1 1.9ba70.2 1.8b70.2 1.7b70.3 1.6b70.3

C2 2.0b70.4 2.1b70.3 2.2b70.4 2.4bc70.4

C3 1.2a70.3 0.9a70.1 0.8a70.1 0.7a70.1

C4 1.3a70.2 1.0a70.2 0.8a70.2 0.7a70.1

MSW1 2.3bc70.3 2.5c70.3 2.9c70.3 3.370.5

MSW2 1.6b70.3 1.2a70.1 1.1a70.2 0.94a70.19

MSW3 1.8b7

0.5 1.6b7

0.3 1.5b7

0.2 1.3b7

0.2PM1 2.1b70.4 2.3c70.5 2.6c70.3 3.0c70.3

PM2 1.4b70.2 1.1a70.2 1.0a70.2 0.9a70.2

PM3 1.6b70.2 1.4b70.2 1.4b70.1 1.2b70.2

Phosphatase activity (mmol PNP g1 h1)

C1 4.9ab71.5 4.8ab71.4 4.6ab71.6 4.2ab71.3

C2 4.9ab72.0 5.0ab71.6 4.8ab71.5 4.9ab71.4

C3 4.0a71.1 3.5a71.2 3.1a71.7 2.8a71.1

C4 4.0a71.8 3.6a71.0 3.3a71.2 3.0a71.5

MSW1 5.3b71.5 5.6b71.5 6.4b71.8 7.6c71.8

MSW2 4.4a71.1 4.1a71.3 3.7a71.1 3.4a71.0

MSW3 4.7ab71.3 4.5ab71.6 4.3a70.9 3.8a71.0

PM1 5.2b71.8 5.4b71.9 6.0b71.6 7.1c71.9

PM2 4.1a71.1 3.8a71.2 3.5a71.1 3.2a70.7

PM3 4.6ab71.2 4.2a71.4 3.9a71.0 3.6a71.1

PNP: p-nitrophenol.

a Different lettersfollowing thenumbers indicatea significant difference at Po0.05.



7/7

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