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Ecotoxicology and Environmental Safety 74 (2011) 668674

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Ecotoxicology and Environmental Safety


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Application of organic wastes on a benzo(a)pyrene polluted soil. Response of soil biochemical properties and role of Eisenia fetida
Manuel Tejada a,n, Grazia Masciandaro b
a b

Department of Crystallography, Mineralogy and AgroChemistry, Crta de Utrera Km1, University of Seville, E-41013 Seville, Spain 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 2010 Received in revised form 3 September 2010 Accepted 7 October 2010 Available 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 articially contaminated by benzo(a)pyrene with and without two organic wastes (organic municipal solid waste, MSW, and poultry manure, PM) and with and without worms (Eisenia fetida) over 90 days. For the organic treatments, soil samples were mixed 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 glutathioneS-transferase activities in worms and the 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 determined during the incubation period. To observe the effects 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)pyrene decreased by 41.2% for the unamended polluted soil and without worms, by 45.8% for the 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 that worm hydrocarbon absorption was lowest in the contaminated soil amended with MSW and with worms, causing an increase in catabolic activity of the soil. These results suggested that the co-application of organic wastes and E. fetida for the bioremediation of benzo(a)pyrene polluted 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 microora can be stimulated by adding organic material and nutrients (Wilson and Bouwer, 1997; Contreras-Ramos et al., 2007). Recently, the addition of horticultural compost (MaliszewskaKordybach et al., 2000), straw (Kucharski et al., 2000) and manure (Coover and Sims, 1987) to soils has been found to immobilize PAHs and reduce their negative effects on soil microbial populations and enzyme activities. This is probably due to the role of organic matter for sorption processes of organic pollutants (Kleineidam et al., 1999; Guanasekara and Xing, 2003).

Corresponding author. Fax: + 34 954486436. E-mail address: mtmoral@us.es (M. Tejada).

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 watersoluble nutrients can be rapidly diluted and leached (Lee and De Mora, 1999). Organic matter mineralization releases nutrients continuously or intermittently over a period of time and therefore has been applied to PAH-contaminated soils to stimulate and maintain indigenous biodegradation rates. However, the inuence 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 efcient method to support the bioremediation of a soil, has been widely 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 ingest soil and expel a partially

0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.10.018

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stabilized product (casting). In this way they ensure the availability of organic substrates for proliferation of the autochthonous microorganisms 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 detoxication enzyme and its activity has been used as a potential bioindicator and biomarker of earthworms for heavy metals (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 the catalysts of important metabolic functions, including the decomposition and the detoxication of contaminants (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) and its inuence on both soil biochemical properties and the earthworms.

Table 2 Acidic functional group contents (means 7 standard error, n 3) of humic acids (HAs) isolated from MSW and PM. Data are the means of four samples. Total acidity PM MSW 4.0 7 0.1 4.3 7 0.1 COOH (mol kg 1) 3.0 7 0.1 3.2 7 0.1 Phenolic OH 1.0 7 0.1 1.1 7 0.1

2. Materials and methods 2.1. Soil, organic wastes and PAH characteristics The soil used in this experiment is a Plagic Anthrosol (FAO, 1989). The main soil characteristics are shown in Table 1. Soil pH was determined in distilled water with a glass electrode (soil:H2O ratio 1:2.5). Soil texture was determined by the Robinsons pipette method (SSEW, 1982) and quantication 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 of Yeomans and Bremner (1988). Humic and fulvic acids-like were extracted with 0.1 M sodium pyrophosphate and 0.1 M sodium hydroxide at pH 13 (Kononova, 1966). The supernatant was acidied to pH 2 with HCl and allowed to stand for 24 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 acidslike, the acidic ltrate containing the dissolved fulvic acid-like fraction was passed through a 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 nally freeze-dried. The carbon content of humic and fulvic acids-like were determined by the method described. Total N was determined by the Kjeldhal method (MAPA, 1986). After nitric and perchloric acid digestion, total Ca, Mg, Fe, Cu, Mn, Zn, Cd, Pb, Ni and 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) and poultry manure. The general properties of the organic wastes are shown in Table 1. Organic matter was determined by dry combustion, according to the ofcial methods of the Spanish Ministry of Agriculture (MAPA, 1986). Humic and fulvic acidslike were extracted, separated and determined by the methods previously described. Total N was determined by the Kjeldhal method (MAPA, 1986). After nitric and perchloric acid digestion, total Ca, Mg, Fe, Cu, Mn, Zn, Cd, Pb, Ni and Cr 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 group content was estimated by direct potentiometric titration at pH 8, the phenolic hydroxyl group content was estimated as two times the change in charge between pH 8 and pH 10, and the total acidity was calculated by addition (Ritchie and Perdue, 2003). The hydrocarbon utilized was benzo(a)pyrene. This hydrocarbon was used because is recalcitrant and therefore it is very difcult to degrade (Betancur-Galvis et al., 2006). Benzo(a)pyrene was dissolved in acetone and added to soil at a concentration of 50 mg kg 1 soil. This concentration was used because it is not 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 with earthworms 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 simplied form the incubation treatments. Triplicate treatments were kept in semi-closed microcosms at 20 7 2 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 ne nylon mesh to prevent soil loss and to keep the earthworms from escaping. E. fetida were bred in laboratory cultures on organic waste materials, principally

Table 1 Characteristics of the experimental soil and organic wastes (means 7 standard error, n 4). Soil pH (H2O) CO3 2 (g kg 1) Fine sand (g kg 1) Coarse sand (g kg 1) Silt (g kg 1) Clay (g kg 1) Clay types 8.6 7 0.2 203 7 12 142 7 35 387 7 26 242 7 19 229 7 10 Smectite: 66% Kaolinite: 20% Illite: 14% 1.1 7 0.8 18.5 7 2.4 9.8 7 1.1 0.4 7 0.1 35.8 7 3.7 9.7 7 1.3 11.3 7 2.1 8.1 7 1.5 6.5 7 1.2 0.4 7 0.1 2.9 7 0.7 5.3 7 0.6 PM 7.1 7 0.3 MSW 6.2 7 0.3

Organic matter (g kg 1) Humic acid-C (mg kg 1) Fulvic acid-C (mg kg 1) Total N (g kg 1) Fe (mg kg 1) Cu (mg kg 1) Mn (mg kg 1) Zn (mg kg 1) Cd (mg kg 1) Pb (mg kg 1) Ni (mg kg 1) Cr (mg kg 1)

614 7 26 672 7 1.4 715 7 10 38.8 7 2.9 180 7 22 1.6 7 0.3 4.2 7 0.9 3.3 7 0.8 0.4 7 0.1 0.9 7 0.1 1.3 7 0.2 0.1 7 0.02

469 7 15 1030 7 17 711 7 10 17.3 7 1.3 815 7 38 82.6 7 9.8 75.6 7 8.1 134 7 13 1.1 7 0.3 82.4 7 3.6 13.6 7 1.5 19.4 7 1.7

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Table 3 Scheme of the incubation treatments performed. Treatments Pollution with benzo(a)pyrene C1 C2 C3 C4 MSW1 MSW2 MSW3 PM1 PM2 PM3 (+ ): Yes. ( ): No. vermicomposts. The substrates used to obtain the vermicomposts and vermicomposting process are detailed in (Tejada et al., 2010). () () (+ ) (+ ) () (+ ) (+ ) () (+ ) (+ ) Organically amended () () () () (+ ) (+ ) (+ ) (+ ) (+ ) (+) Earthworms addition () (+ ) () (+ ) () () (+ ) () () (+ )

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 signicance level of P o 0.05 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 signicantly 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 signicant differences between these treatments. The benzopyrene content in earthworms increased gradually during the incubation period (Fig. 1b). At the end of the experiment, the highest contents occurred in the C4 treatment. Benzo(a)pyrene content decreased by 15.9% for the PM3 treatment and 21.3% for the MSW3 treatment. However, the ANOVA indicates no signicant 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 and when compared with the C2 treatment, the weight of earthworms decreased by 19.8%, 22.4% and 38.2% for the MSW3, PM3 and C2 treatments, respectively. At the end of the incubation period, the cellulase and glutathione-S-transferase activities in earthworms decreased signicantly (p o 0.05) for the C2 treatment (Fig. 2). In the contaminated and organically amended soils, both enzymatic activities decreased less sharply. Also and for both organically amended soils, there was 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 signicant) between the contaminated and amended soils, observing the highest cocoon numbers 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 signicantly (p o 0.05) 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 At the end of the incubation period, the highest soil ATP contents were observed for uncontaminated and organically amended soils (Table 5). In this respect, the highest values were observed for the

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 et al. (1995). A sample of 1.5 g of soil was put in a 15-ml Pyrex tube and 10 ml acetone was 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 supernatant was added to 20 ml glass asks and the acetone used to extract PAHs was left to evaporate. The same procedure was repeated again twice and the extracts were added to a 20-ml ask. The extracts were passed through a 0.45-mm syringe lter. The ltered extracts were concentrated to 1 ml and then analyzed by GC. The earthworms were placed on wet lter paper for in Petri dishes for a period of 24 h to allow the depuration of their gut contents. They were then cleaned and frozen in liquid nitrogen. The frozen earthworms were ground with a pestle and mortar and mixed with 1.5 times their wet weight of Na2SO4. The mixture was extracted with 10 ml acetone by sonication at 60 1C for 20 min, centrifuged at 13 700g for 15 min and decanted. The same procedure was repeated again twice. The extracts were pooled and passed through a 0.45-mm syringe lter. The ltered extracts 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 was equipped with an FID detector set at 310 1C tted with an HP-5 capillary column (15 m by 0.53 mm, lm thickness 1.5 mm); carrier gas was helium; the oven temperature was increased from 140 to 170 1C at 5 1C min 1 and from 170 to 280 1C at 30 1C min 1. 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 25 7 1 1C covered with three moist lter papers. According to Xiao et al. (2006), the lter papers in these dishes were changed every three days to prevent bacterial growth. At the end of the test (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 worms were selected and placed on wet lter paper in Petri dishes for 24 h to clear 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-transferase activity was measured according 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 asks were shaken in a cool bath. Then the mixture was ltered through Whatman paper and an aliquot was used to measure the ATP content by means of 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.

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60 50
Benzo (a) pyrene (mg kg-1)

b b

b b

C3 b b b b b b

C4 b

MSW2 b

MSW3 b

PM2

PM3

40 30 20 10 0

ab ab ab a a a ab

ab

15

60

90

6 b C4 5
Benzo (a) pyrene (mg kg-1)

MSW3

PM3 ab b b ab ab a

4 ab

2 a 1 a a

15

60

90

Fig. 1. Extractable benzo(a)pyrene (mean 7 standard error, n 3) in soils (a) and Eisenia fetida (b) during the experimental period. Columns (mean 7 S.E.) followed by the same letter(s) are not signicantly different (p 4 0.05).

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 with worms, 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 and worms. At the end of the experimental period and 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 signicant differences (p o 0.05) 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 soil decreased this 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 reproduction of E. fetida over a period of 11 weeks. However, the application of the worms in polluted soil decreased the soil hydrocarbon concentration and therefore, improved soil biochemical properties. Earthworms burrow through the soil, turning it over continuously, maintaining its fertility and structure, and improving aeration and water inltration capacity (Edwards, 1998). These animals come into close contact with PAHs by moving around in the soil (Johnsen et al., 2005). This activity contributes to the biodegradation of organic contaminants, such as the microorganisms in the earthworm 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 degrading 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.

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240 220 200 Weight (mg) 180 160 140 120 100 700 650 Cellulase activity (mg glucose mg protein h-1) 600 550 500 450 400 350 130 120 110 100 90

C2 b b

C4 b

MSW3 b

PM3 b ab ab

ab a ab a a a

3 C2 C4 MSW3 PM3 b

15

60

90

b ab b ab ab ab a a ab

3 C2 b ab C4 b MSW3 ab ab PM3 b

15 b b b

60 b b b

90 b b

Glutathione-S-transferase activity (nmol mg protein min-1)

a 80 70

15

60

90

Fig. 2. Weight and cellulase and glutathione-S-transferase activities (mean 7 standard error, n 3) in Eisenia fetida exposed to benzo(a)pyrene during the experimental period. Columns (mean 7 S.E.) followed by the same letter(s) are not signicantly different (p 4 0.05).

There are two major uptake routes by which PAHs can be accumulated by earthworms. The rst is simple diffusion across 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) found that in soils contaminated with pesticides or 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 gluthatione-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

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Table 4 Cocoon production, average weight of cocoons (mg) and number of juveniles per cocoon (mean 7 standard error, n 3) of Eisenia fetida exposed to benzo(a)pyrene. Cocoon numbers Average weigh of cocoon (mg) 8.8ba 7 0.5 5.8a 7 0.9 7.2b 7 0.7 7.0ab 7 0.7 Number of juveniles per cocoon 3.1ba 7 0.4 2.5a 7 0.6 2.7ab 7 0.4 2.6a 7 0.4

Table 5 ATP and urease and phosphatase activities (mean 7 standard error, n 3) in soils exposed to benzo(a)pyrene. Incubation days 3 15 60 90

C2 C4 MSW3 PM3
a

2.9ba 7 0000.4 1.8a 7 0.2 2.4b 7 0.4 2.3ab 7 0.6

Different letters following the numbers indicate a signicant difference at P o 0.05.

of cocoon and the number of juveniles per cocoon decreased in the experiment. For this reason, it is very probable that in these adverse conditions, the total biomass of worms decreases. The decrease in the soil hydrocarbon content inuenced the improvement of soil biochemical properties. Also, it is widely recognized that earthworms can signicantly 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 and moisture 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 and deposition of cast excreta are stimulators for soil microorganisms (Hickman and Reid, 2008). Thus, within the eld of bioremediation, the use of earthworms to improve soil conditions and to subsequently promote microbial numbers, diversity and activity should bring about benets for levels of catabolic activity and subsequent enhancement 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 that the adsorption capacity of organic matter is more important than the absorptive capacity of the hydrocarbon by the worm. However, differences were found depending on the chemical composition of organic matter applied to soil. 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 content increases in the organic matter, compared to the fulvic acid-like content, probably due to the fact that humic acid-like substances are characterized by a higher number of carboxylic groups than fulvic acidlike substances (Tejada et al., 2007; 2008). Therefore, and similar 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 be responsible for the increase in the soil biochemical properties. These results are in agreement with those of Murphy et al. (1990), KollistSiigur et al. (2001) and Tejada et al. (2008), who suggested that humic acids had greater binding afnity for PAHs than fulvic acids. The combined addition of earthworms and organic matter to contaminated soil resulted in the greatest enhancement in terms of catabolic activity. It is suggested that the earthworm presence sufciently altered (biologically, chemically and physically) the nature of the substrate so as to determine a signicant 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 both the organic matter and the contaminated soils catabolically active microorganisms.

soil) ATP (ng ATP g C1 304aba 7 23 C2 306ab 7 20 C3 276a 7 13 C4 288a 7 22 MSW1 326ab 7 18 MSW2 299ab 7 17 MSW3 303ab 7 30 PM1 323ab 7 26 PM2 293ab 7 14 PM3 301ab 7 20

304ab 7 18 308 7 22 249a 7 15 260a 7 20 331ab 7 19 289a 7 14 302ab 7 11 313ab 7 16 277a 7 24 294ab 7 22

296ab 7 20 314ab 7 25 235a 7 21 248a 7 12 366b 7 16 270a 7 14 290a 7 24 338 7 28 259a 7 10 283a 7 19

295ab 7 26 312ab 7 21 221a 7 23 236a 7 30 410b 7 28 259a 7 22 284a 7 17 388b 7 25 248a 7 14 270a 7 19

+ -1 -1 Urease activity (mg NH4 g h ) C1 1.9ba 7 0.2 C2 2.0b 7 0.4 C3 1.2a 7 0.3 C4 1.3a 7 0.2 MSW1 2.3bc 7 0.3 MSW2 1.6b 7 0.3 MSW3 1.8b 7 0.5 PM1 2.1b 7 0.4 PM2 1.4b 7 0.2 PM3 1.6b 7 0.2

1.8b 7 0.2 2.1b 7 0.3 0.9a 7 0.1 1.0a 7 0.2 2.5c 7 0.3 1.2a 7 0.1 1.6b 7 0.3 2.3c 7 0.5 1.1a 7 0.2 1.4b 7 0.2

1.7b 7 0.3 2.2b 7 0.4 0.8a 7 0.1 0.8a 7 0.2 2.9c 7 0.3 1.1a 7 0.2 1.5b 7 0.2 2.6c 7 0.3 1.0a 7 0.2 1.4b 7 0.1 4.6ab 7 1.6 4.8ab 7 1.5 3.1a 7 1.7 3.3a 7 1.2 6.4b 7 1.8 3.7a 7 1.1 4.3a 7 0.9 6.0b 7 1.6 3.5a 7 1.1 3.9a 7 1.0

1.6b 7 0.3 2.4bc 7 0.4 0.7a 7 0.1 0.7a 7 0.1 3.3 7 0.5 0.94a 7 0.19 1.3b 7 0.2 3.0c 7 0.3 0.9a 7 0.2 1.2b 7 0.2 4.2ab 7 1.3 4.9ab 7 1.4 2.8a 7 1.1 3.0a 7 1.5 7.6c 7 1.8 3.4a 7 1.0 3.8a 7 1.0 7.1c 7 1.9 3.2a 7 0.7 3.6a 7 1.1

Phosphatase C1 C2 C3 C4 MSW1 MSW2 MSW3 PM1 PM2 PM3

activity (mmol PNP g 1 h 1) 4.9ab 7 1.5 4.8ab 7 1.4 4.9ab 7 2.0 5.0ab 7 1.6 4.0a 7 1.1 3.5a 7 1.2 4.0a 7 1.8 3.6a 7 1.0 5.3b 7 1.5 5.6b 7 1.5 4.4a 7 1.1 4.1a 7 1.3 4.7ab 7 1.3 4.5ab 7 1.6 5.2b 7 1.8 5.4b 7 1.9 4.1a 7 1.1 3.8a 7 1.2 4.6ab 7 1.2 4.2a 7 1.4

PNP: p-nitrophenol.
a

Different letters following the numbers indicate a signicant difference at P o 0.05.

Furthermore, it is emphasized that the organic matter type inuenced this process, highlighting the lower values of hydrocarbon 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 potentially advantageous for the bioremediation of hydrocarbon-contaminated soil. However, the organic matter type applied to the soil play a very important role. The application of organic matter rich 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 Tejada thanks the Spanish Ministry of Education for the nancial support of the scholarship of university teaching staff mobility.

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

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