SOIL BIOREMEDIATION: COMBINATION OF EARTHWORMS AND COMPOST FOR THE ECOLOGICAL REMEDIATION OF A HYDROCARBON POLLUTED SOIL

BRUNELLO CECCANTI1 , GRAZIA MASCIANDARO1, , CARLOS GARCIA2 , CRISTINA MACCI1 and SERENA DONI1
CNR-Istituto per lo Studio degli Ecosistemi (ISE)Sezione di Chimica del Suolo, Area della Ricerca, Via Moruzzi, 156124 Pisa (Italy); 2 CSIC-Centro de Edafologia y Biologia Aplicada del Segura, Campus de EspinardoMurcia (Spain) ( author for correspondence, e-mail: grazia.masciandaro@ise.cnr.it)
1

(Received 1 February 2006; accepted 14 May 2006)

Abstract. The present investigation, carried out in laboratory microcosms, regards the effects of some bioremediation treatments of a polluted soil and the use of specic parameters to study the evolution of biochemical processes which take place in the soil decontamination. The bioremediation treatments were the following: (1) a mixture of microorganisms-enzymesnutrients (MEN); (2); compost alone (C); (3) compost with earthworms (Eisenia fetida) (CL) and (4) control soil (without treatment) (BN). Chemical, physico-chemical, biological and biochemical parameters were determined to study the soil metabolic processes in order to assess the efciency of the bioremediation process involved in the degradation of hydrocarbons. The study showed an intense microbiological activity expressed as carbon dioxide evolution during the time, with a tendency to stabilize at the end of the experiments. The highest carbon dioxide release was found in the two compost treatments, showing the availability of organic substrate characterising the compost. The organic substrate reduction during the time caused a decrease of hydrolytic enzyme activities representative of Carbon (-glucosidase), Nitrogen (protease) and Phosphorus (phosphatase) cycles. However, the enzyme activities showed their highest values in the treatments with compost. Finally, the greatest reduction of hydrocarbons was found in the treatments with compost, in particular with earthworms that also contributed to regulate the biochemical equilibrium of the soil. Keywords: bioremediation, earthworms, enzyme activities, environmental pollution, hydrocarbons

1. Introduction The oil renery sludges resulting from depuration processes have a high content of petroleum-derived hydrocarbons which can have serious enviromental consequences and represent a risk for human health (Baheri and Meysami, 2001). Biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and hydrocarbon pollutants can be eliminated from the environment (Leahy and Colwell, 1990). Bioremediation could be an interesting biological technology to recover polluted environments while ensuring
Water, Air, and Soil Pollution (2006) 177: 383397 DOI: 10.1007/s11270-006-9180-4
C

Springer 2006

384

B. CECCANTI ET AL.

the conservation of the ecosystems biophysical properties. In addition, bioremediation is dened by Microbiology American Academy as The use of living organisms to reduce or eliminate environmental hazard resulting from accumulation of toxic chemicals and other hazardous wastes (Adriano et al., 1999). Bioremediation techniques accelerate the naturally occurring biodegradation processes by optimising the chemical-physical conditions (such as aeration, addition of nutrients, and control of pH and temperature, Atlas and Bartha, 1992; Morgan and Watkinson, 1989) in order to activate soil microorganisms. In addition to the stimulation of autochthonous microorganisms (bioenhancement), a bioremediation technique can also involve the introduction of microorganisms with specic degradative abilities (bioaugmentation). In recent years the use of earthworms, as an efcient method to support the bioremediation of a soil has been experimented (Singer et al., 2001). 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 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 (Wolters, 2000). In a previoous study, eathworms L. Terrestris and E. foetida were shown to induce higher microbial activity and oil degradation (Schaefer et al., 2005). In addition, the earthworms Eisenia foetida have been used as test organism for different contaminants (OECD, 2000). Several reports indicate that E. foetida tolerates 1.5% crude oil while Lumbricus terrestris did not survive 0.5% of it (Safwat et al., 2002) and the use of Eisenia foetida increases the PAH removal (Contreras-Ramos et al., 2006). Microbial activity represents one of the primary biological mechanisms to remove organic contaminants, such as petroleum products, aliphatic and aromatic hydrocarbon, industrial solvents (benzene, phenols, etc. . .) and other pollutants from soil. 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 (Schinner et al., 1996; van Beelen and Doelman, 1997). Nannipieri (1990) showed that the measurement of the activity of several enzymes in soil may be a good method for estimating the overall microbial activity and its response to widespread pollution. Enzymes, in fact, are the catalysts of important metabolic functions, including the decomposition and the detoxication of contaminants (Nannipieri and Bollag, 1991). We propose different bioremediation methods to stimulate soil microbial activity in order to create optimal conditions for hydrocarbon degradation. The aim of this study was to compare and evaluate the efciency of three different bioremediation treatments planned with the purpose of recovering the biological quality of a hydrocarbon polluted soil.

COMBINATION OF EARTHWORMS AND COMPOST

385

2. Materials and Methods 2.1. EXPERIMENTAL

LAYOUT

The characteristics of the soil used for the laboratory experiments, carried out in triplicate, are reported in Table Ia. The soil was contaminated by oil renery wastes and came from the south-east of Spain (Murcia). The characteristics of the oil renery sludge are reported in Table Ib. 1 kg of polluted soil was placed in plastic containers (microcosm). All containers were covered with perforated lids and maintained under controlled temperature and humidity for three months. The oxygen was assured by a continuous ow of air (scheme 1). Chemical and biochemical parameters were determined monthly and carbon dioxide every two days. The bioremediation treatments were the following: (1) a mixture of microorganisms-enzymes-nutrients (MEN) (Table II); (2) compost (C); (3) compost with earthworms (Eisenia fetida) (CL); 4) soil without treatment (control soil) (BN). In the earthworm treatment, 10 adult earthworms were used as agents of bioremediation and introduced after 15 days of experiment. This was

Scheme 1. The microcosm used in the experiment.

386

B. CECCANTI ET AL.

TABLE Ia Chemical, physical and biological parameters in the soil and compost used for the experiments Soil E.C. (1/10) (dS m ) pH (1/10) SO4 2 (mg kg1 ) Cl (mg kg1 ) NO3 (mg kg1 ) TOC (mg g1 ) WSC (mg g1 ) N-tot. (mg g1 ) NH3 (mg kg1 ) P-tot. (mg kg1 ) P-ass. (mg kg1 ) Sand (%) Silt (%) Clay (%) ATP (ng ATP g1 ) TPH (mg g1 )
1

Compost 3.5 0.17 7.9 0.31 180 10.8 2.23 0.07 8.33 0.52 40 0.8 1999 60 50.3 4.03

4.0 0.2 7.3 0.44 15891 476 351 14 153 4.6 42.3 1.3 0.85 0.03 2.0 0.08 19.2 0.95 394 19.1 3.32 0.13 14.2 0.85 82.7 5.7 3.15 0.12 764 43.2 11.27 0.56

E.C., Electrical Conductivity; Pass, available phosphorus; Ptot, total phosphorus; TOC, Total Organic Carbon; TPH, Total Petroleum hydrocarbons; WSC, Water-Soluble Carbon. TABLE Ib Chemical parameters of the oil renery sludge used in the experiments Oil renery TOC (mg g1 ) NH3 (mg kg1 ) NO3 (mg kg1 ) Cl (mg kg1 ) SO4 2 (mg kg1 ) F (mg kg1 ) Phenol (mg kg1 ) Pb (mg kg1 ) Cd (mg kg1 ) Zn (mg kg1 ) Cu (mg kg1 ) Cr (mg kg1 ) TPH (mg g1 ) 192 14.2 8.21 0.66 22 2.1 1200 121 22500 1524 1.26 0.09 534 32.5 105 8.3 <1.2 0.08 237 13.6 48 4.1 154 12.8 221 16.8

TOC, Total Organic Carbon; TPH, Total Petroleum hydro-carbons.

COMBINATION OF EARTHWORMS AND COMPOST

387

TABLE II Composition of the mixture microorganisms-enzymes-nutrients

(MEN)

MEN composition Nutrients Commercial product: Bacterial cells (NH4 )H2 PO4 NH4 NO3 Bacillus subtilis Bacillus megaterim Bacillus thuringiensis Saccaromices Oxidase

Yeast Enzymes

done to avoid exposure of the worms to high pollution. In the compost treatment, the compost derived from the organic fraction of urban residues and previously subjected to a stabilization period of 3 months (Table I), was added in the ratio of 10% on weight (1 kg of soil : 100 g of compost). In the last treatment with microorganisms, enzymes and nutrients, 4 g of a commercial product containing a mixture of microorganisms and enzymes was added to 1 kg of soil, while the nutrients were added in the ratio 10:1:0,2 of carbon, nitrogen and phosphorus, respectively (E.N. Drake et al., 1995). 2.2. CHEMICAL
PARAMETERS

Electrical conductivity (EC) and pH were measured in 1/10 (w/v) acqueous solution. Total and soluble organic carbon (WSC), soil: water ratio 1:10 w/v, (Garcia et al., 1990) were determined by dichromate oxidation (Yeomans and Bremner, 1988), Nitrogen by the method of Kjeldahl (Jackson, 1960) and phosphorous by the Murphy and Riley method (1962). Nitrate and NH+ were determined in a 1:10 4 (w/v) water extract, by ionic chromatography using a DIONEX chromatograph and by an ammonia-selective electrode (ORION, mod 95-12), respectively. 2.3. BIOCHEMICAL
PARAMETERS

The methods used to assay hydrolase activities (-glucosidase, BAA-hydrolyzing protease, urease, and phosphatase) are described by Garcia et al. (1993). To determine -glucosidase activity, 0.05 M 4-nitro-phenyl--D-glucanopyranoside (PNG) was used as substrate (Hayano and Tubai, 1985), while 0.115 M -nitrophenyl phosphate (PNPP) was used as substrate to measure the Phosphatase activity . The -nitrophenol (PNP) released as product by -glucosidase and phosphatase activity was extracted and determined spectrophotometrically at 398 nm (Tabatabai and Bremner, 1969).

388

B. CECCANTI ET AL.

To determine the BAA-hydrolysing protease and urease activities, 0.03 M N-benzoyl-L-argininamide (BAA) and 6.4% urea, respectively, were used as substrates (Nannipieri et al., 1980). The ammonium released by the hydrolytic reactions was measured by an ammonium selective electrode (ORION, mod. 95-12). ATP was determined on the basis of the light emitted by the luciferin-luciferase system, according to the method of Ciardi and Nannipieri (1990). The respirometry activity was determined with a tritimetric method measuring the CO2 produced by microbial biomass (Parr and Smith, 1969). The CO2 was collected under a stream of air in a vial containing 40ml of NaOH 2N solution. The carbonate produced was precipitated with bariumcloride and the excess of NaOH solution was titrated with a standard HCl 1N solution, using phenolphthalein as indicator of the end point of reaction. The values of CO2 for each sample were obtained by difference from a control sample without soil. The results are expressed as mg of carbon produced as CO2 per day, per Kg of soil. 2.4. TOTAL
HEAVY HYDROCARBONS ANALYSIS

The total heavy hydrocarbons were determined by the gravimetric method 1664 (EPA, 1983; APHA, 1992) using n-pentane instead of n-hexane, as modied by Filareto (2002). The soil samples (1g) were air-dried and mixed with Na2 SO4 to remove the residual water. Total hydrocarbons were extracted for three times with 5ml of pentane in an ultrasound bath for 15 minutes. Total hydrocarbon content is estimated by weighing the dry residue after solvent evaporation under nitrogen ow. The content of hydrocarbons was determined in the soil before (Table I) and at the end (T3, Tables III and IV) of the experiments. 2.5. STATISTICAL
ANALYSES

Statistical analyses were performed on soil chemical, biochemical and microbiological parameters. All results reported in the text are the means of determinations made on three replicates. The means were compared by using least signicant differences calculated at P < 0.05 (Tukeys test). The principal components analysis (PCA) procedure of STATISTICA was used to help isolate the principal components (PC) and the contained variables, which are responsible for most of the global data variance (Reyment and Joreskog, 1993). The PCs were extracted by applying the principal or main axis method. Only component loadings >0.5 were considered for interpretation of the principal components (Backhaus et al., 1996). The statistical analyses selected are methods of data classication that are able to evaluate for each response the relative importance of the type of treatment. In addition, PCA analysis gives information about groups of correlations, which can also be clearly presented graphically.

COMBINATION OF EARTHWORMS AND COMPOST

389

TABLE III Chemical parameters in soil (BN) and soil with addition of microorganisms, enzymes and nutrients (MEN) after 1 month (T1), 2 months (T2) and 3 months (T3) of incubation BN T1 pH E.C. (dS m1 ) TOC (mg g1 ) WSC (mg g1 ) N-tot (mg g1 ) NH3 (mg kg1 ) P-tot. (mg kg1 ) P-ass. (mg kg1 ) NO3 (mg kg1 ) Cl (mg kg1 ) SO4 2 (mg kg1 ) C/N TPH (mg g1 ) 7.61 b 3.88 a 42.7 a 0.33 a 2.06 a 20.4 a 399 a 3.41 a 130 b 390 c 11885 a 20.7 T2 7.93 a 3.41 a 40.3 b 0.30 a 2.12 a 18.0 b 391 a 3.30 a 154 a 538 a 11839 a 19.0 T3 6.94 c 3.16 a 31.8 c 0.30 a 2.08 a 15.5 c 383 a 3.12 a 136 b 460 b 12053 a 15.3 9.47 0.28 T1 7.44 b 5.57 a 43.3 a 0.34 a 3.51 a 41.3 a 753 a 30.1 a 6044 b 390 c 13940 a 12.3 MEN T2 7.80 a 6.06 a 42.0 a 0.29 b 3.33 b 24.5 b 674 a 25.0 a 6720 a 538 a 12897 b 12.6 T3 6.98 c 5.91 a 28.3 b 0.27 b 3.26 c 4.0 a 567 b 19.5 b 7149 a 460 b 11956 b 8.7 0.17

For each treatment different letters indicate statistically different values (P < 0.05) E.C., Electrical Conductivity; Pass, available phosphorus; Ptot, total phosphorus; TOC, Total Organic Carbon; TPH, Total Petroleum hydrocarbons; WSC, Water-Soluble Carbon.

3. Results and Discussion The results of chemical parameters (Tables III and IV) showed a decrease in the concentration of total carbon, total phosphorous, and the available forms of C and N (WSC, ammonia) in all the treatments. This suggests a progressive degradation of organic compounds, probably including the pollutants, especially when new microorganisms were added and/or authoctonous microorganisms were stimulated with the addition of organic substrates, such as compost and earthworm casting. The high TOC reduction in MEN treatment suggested a sort of priming effect of native soil organic matter induced by the addition of mineral nutrients, as already found in mineral fertilization of agricultural soils (Marinari et al., 2000). Total N (Tables III and IV) in the organic treatments remained quite unchanged, meaning that the compost and earthworm casting regularly released nitrogen compounds. Special account has to be taken of nitrate concentration (Tables III and IV) which, as expected, was highest in MEN due to the addition of mineral nutrients. However, at the end of the experiment, nitrate increased in all treatments, but more in that with the addition of earthworms, as has already been reported in studies on in situ vermicomposting of biological sludges (Masciandaro et al., 2000). The earth-

390

B. CECCANTI ET AL.

TABLE IV Chemical parameters in the soil with addition of compost (C) and soil with addition of compost and earthworms (CL) after 1 month (T1), 2 months (T2) and 3 months (T3) of incubation. C and CL characteristics C T1 pH E.C. (dS m1 ) TOC (mg g1 ) WSC (mg g1 ) N-tot (mg g1 ) NH3 (mg kg1 ) P-tot (mg kg1 ) P-ass. (mg kg1 ) NO3 (mg kg1 ) Cl (mg kg1 ) SO4 2 (mg kg1 ) C/N TPH (mg g1 ) 7.72 b 4.52 b 54.1 a 0.55 a 2.85 a 21.9 a 599 a 12.0 a 642 b 738 a 15036 a 19.0 T2 8.10 a 4.76 b 48.5 b 0.48 b 2.60 a 4.2 b 534 a 9.1 a 710 a 785 a 14788 a 18.7 T3 6.88 c 5.18 a 45.1 b 0.44 b 2.54 a 4.5 b 520 a 4.9 b 758 a 589 b 14456 a -0,953z 17.8 6.82 0.16 T1 7.77 b 4.42 a 55.4 a 0.55 a 2.99 a 13.1 a 584 a 13.2 a 694 c 708 a 15588 a 18.5 T2 8.15 a 4.53 a 54.6 a 0.54 a 2.54 a 5.0 b 557 a 5.8 b 810 b 510 b 14973 b 21.4 CL T3 6.84 c 4.44 a 42.1 b 0.49 b 2.49 a 4.4 b 533 a 5.0 c 950 a 570 b 14560 b 16.9 5.60 0.21

For each treatment different letters indicate statistically different values ( P < 0.05) E.C., Electrical Conductivity; Pass, available phosphorus; Ptot, total phosphorus; TOC, Total Organic Carbon; TPH, Total Petroleum hydrocarbons; WSC, Water-Soluble Carbon.

worms increase the availability of NO3, which is an electron-acceptor, thus helping to degrade the organic matter. Degradation of the organic matter and the immobilization of mineral nutrients, suggested by the reduction in assimilable phosphorous and ammonia, created the conditions to increase total microbial biomass, as supposed for the organic treatments with the highest WSC and CO2 values (Tables III and IV). CO2 evolution (Figure 1), which represents microbial catabolic metabolism, showed the highest concentrations for both compost treatments in which available substrates stimulated soil microbial activity. This was also conrmed by the ATP values (Figure 2) measured at the end of the experiments (T3). ATP is considered an indicator of microbial activity due to the linear relationship between the ATP content and soil microbial biomass. In addition, from a biochemical point of view, as it is a phosphoric substrate, ATP degradation is related to the phosphatase enzymes (Gil-Sotres et al., 1992; Nannipieri et al., 1996), which hydrolyse organic phosphoric esters, giving phosphate as a product. The enzymatic metabolic picture was followed by testing hydrolitic enzyme activities linked to the C (b-glucosidase), N (urease, protease), and P (phosphatase) cycles. All the enzymes showed, as seen for CO2 and ATP, highest activities in

COMBINATION OF EARTHWORMS AND COMPOST

391

Figure 1. CO2 release in control soil (BN), soil with addition of microorganisms, enzymes and nutrients (MEN), soil with addition of compost alone (C) and soil with addition of compost and earthworms (CL) during the experimental period.

Figure 2. ATP values in control soil (BN), soil with addition of microorganisms, enzymes and nutrients (MEN), soil with addition of compost alone (C) and soil with addition of compost and earthworms (CL) at the end of the experimental period (T3).

the organic treatments with compost. However, for each treatment, the enzyme activities showed different trends on the basis of the utilization/release of the substrates/products controlling the enzyme synthesis and activity. In fact, b-glucosidase (which catalyses the hydrolysis of cellobiose to glucose) decreased during the time (Figure 3) due to the mineralization of the organic substrates, probably including also the pollutants, as illustrated by the reduction of TOC and WSC. On the other hand, the reduction in P compounds, especially in available P, reected an increase in the phosphatase enzyme (Figure 3), which is inhibited by its product (phosphate), as has been widely reported (Nannipieri et al., 1990). The increase in phosphatase activity could also be due to the stimulation of microbial biomass in all the bioremediation experiments, both adding mineral nutrients and organic substrates, as suggested before by the ATP concentrations. The N cycle was studied through protease, hydrolising the BAA substrate with NH3 as a product, and urease, hydrolising urea into CO2 and NH3 enzymes

392

B. CECCANTI ET AL.

Figure 3. Total -Glucosidase and Phosphatase enzyme activities in control soil (BN), soil with addition of microorganisms, enzymes and nutrients (MEN), soil with addition of compost alone (C) and soil with addition of compost and earthworms (CL) after 1 month (T1), 2 months (T2) and 3 months (T3) of incubation.

Figure 4. Total Urease and Protease enzyme activities in control soil (BN), soil with addition of microorganisms, enzymes and nutrients (MEN), soil with addition of compost alone (C) and soil with addition of compost and earthworms (CL) after 1 month (T1), 2 months (T2) and 3 months (T3) of incubation.

(Figure 4). Both enzymes followed the trend of the other enzymes for the control and MEN treatments, which decreased during the time, as expected, due to the degradation of substrates which are not replaced in these treatments. The reduction in hydrolitic enzyme activities during the time has also been observed in other studies on the bioremediation experiments using mineral nutrients (Margesin et al., 2000). In the organic treatments with compost, protease and urease activities increased or remained quite unchanged, probably due to the constant release of nitrogen compounds acting as substrate for protease and urease enzymes. In compost treatments, the activation of a metabolic pattern due to the assistance in the supply of organic substrates could effectively be active in the degradation of pollutants (hydrocarbons). Hydrocarbon concentration was measured at the end of the experiment (T3) to evaluate the efciency of different bioremediation methods intended to stimulate soil microbial biomass in the degradation of hydrocarbons, without

COMBINATION OF EARTHWORMS AND COMPOST

393

Figure 5. Total Petroleum Hydrocarbons at the end of the experimental period (T3).

Figure 6. Principal Component Analysis (PCA) factor scores of bioremediation treatments and their assignment to the 1st and 2nd principal components (PC) at the end of the experimental period (T3). E.C., Electrical Conductivity; Pass, available phosphorus; Ptot, total phosphorus; TOC, Total Organic Carbon; TPH, Total Petroleum hydrocarbons; WSC, Water-Soluble Carbon.

altering the biochemical quality of the soil (Figure 5). The greatest reduction was for the treatment with earthworms and compost followed by compost alone, clearly underlying the combined action of compost and earthworms in the degradation of each type of organic matter, including the pollutant organic substrates. This action could be a direct degradation of the organic substrates by earthworms, and/or an indirect effect through the stimulation of soil microbial metabolism by available substrate from compost and earthworm casting. The statistical analyses explain more clearly the analytical chemical and biochemical results; PCA analysis was used to statistically elaborate experimental data. PCA analysis isolated two principal components (PC) covering variables related to chemical and biochemical parameters of the nal sampling (T3) (Figure 6). The highest loadings of the 1st PC (66.4% of the total variance) included chemical parameters expressing the organic matter (TOC and WSC), microbiological and biochemical parameters (CO2, ATP, enzyme activities), and the total hydrocarbons

394

B. CECCANTI ET AL.

TABLE V Principal components (PC) and component loadings extracted from treatments and chemical and biochemical parameters Principal components Variables pH E.C. TOC WSC N-tot NH3 P-tot P-ass. NO3 Cl SO4 2 C/N UREASE PROTEASE -GLUCOSIDASE PHOSPHATASE TPH ATP CO2 Var. Sp. Prop.Tot.

1 0.938 0.1241 0.965 0.983 0.184 0.545 0.369 0.481 0.489 0.988 0.999 0.746 0.926 0.997 0.954 0.935 0.953 0.949 0.950 12.611 0.664

2 0.272 0.968 0.168 0.123 0.983 0.838 0.925 0.874 0.868 0.008 0.026 0.648 0.356 0.069 0.162 0.175 0.172 0.315 0.313 5.931 0.312

Variables with component loadings used to interpret the PC E.C., Electrical Conductivity; Pass, available phosphorus; Ptot, total phosphorus; TOC, Total Organic Carbon; TPH, Total Petroleum hydrocarbons; WSC, Water-Soluble Carbon.

(Table V). The bioremediation processes is practically summarised by this PC, indicating that the organic matter is crucial to sustain microbial activity, thus promoting the degradation of hydrocarboons. The 2nd PC (31.2% of the total variance) included electrical conductivity and nutrients of N and P, both in their total and available forms, suggesting a contribution of these nutrients to the soil salinity. The XY-ordination of the factor scores of the two PCs reected the distribution of the treatments on the basis of the considered parameters (Figure 6). Organic treatments with compost alone and compost plus earthworms were in the area corresponding to the metabolic parameters linked to the biochemical and microbiological processes

COMBINATION OF EARTHWORMS AND COMPOST

395

involved in the degradation of hydrocarbons. The MEN treatment fell in the area characterised by mineral N and P nutrients and electrical conductivity (EC). The control soil did not seem to be inuenced by the activation of metabolic processes aimed at extra-degrading the organic substrates.

4. Conclusions The bioremediation treatments were effective in the degradation of hydrocarbon polluted compounds. In particular, the use of just mineral nutrients to stimulate soil microbial activity caused a high decrease in total organic carbon, thus suggesting an attack of the native soil organic matter (priming effect). On the other hand, the addition of compost (especially with earthworms) had the double effect of adding nutrients and labile organic matter for the soil authoctonous microrganisms, but also of introducing new microrganisms in the soil system. Finally, the compost treatments, stimulating soil metabolic processes, achieved the best result in the hydrocarbon degradation preserving the biochemical quality of the soil.

References
Adriano, D.C., Bollag, J.M., Frankenberger, W.T.Jr. & Sims, R.C. (1999). Biodegradation of contaminated Soil, Agronomy Monograph 372, in Science Society of America, Madison, 772. APHA-American Public Health Association (1992). Standard Methods for the Examination of Water and Wastewater, in 18th Edition APHA, NW, Washington, D.C. 20005, Method 5520B. Atlas, A. S. & Bartha, R. (1992). Hydrocarbon biodegradation and oil spill bioremediation, Adv. Microbial Ecol. 12, 287338. Baheri, H. & Meysami (2001). Feasibility of fungi bioaugmentation in composting a are pit soil, J. Hazard. Mater., B 89, 279286. Backhaus, K., Erichson, B., Plinke, W. & Weiber, R. (1996). Multivariate Analysemethoden, in 8th ed. Springer Verlag, Berlin, Germany. Ciardi, C. & Nannipieri, P. (1990) A comparison of methods for measuring ATP in soil, Soil Biol. Biochem. 22, 725727. Contreras-Ramos, S.M., Alvarez-Bernal, D. & Dendooven, L. (2006). Eisenia foetida increased removal of polycyclic aromatic hydrocarbons from soil, Environmental Pollution 141, 396401. Drake, E.N., Stokely, K.E., Calcavecchio, P., Bare, R.E., Rothenburger, S.J., Douglas, G.S. & Prince, R.C. (1995). Nutrient stimulated biodegradation of aged renery hydrocarbons in soil, In E. Hinchee, G.S., Douglas, Say Kee Ong (eds.), Monitoring and Verication of Bioremediation, RBattelle Press, Columbus, Ohio 2938. EPA-Environmental Protection Agency (1983). Methods for Chemical Analysis of Water and Wastes in 3rd Edition, EPA, Environmental Monitoring Systems Laboratory-Cincinnati (EMSL-Ci), Cincinnati, Ohio 45268, EPA-600/4-79-020, Method 413.1. Filareto, A. (2002). Approccio metodologico per studiare i processi di decontaminazione dei suoli inquinati da idrocarburi (Bioremediation), Tesi di Laurea facolt` di Scienze matematiche, siche a e naturali, corso di laurea Biologia, Universit` di Pisa, 1119. a

396

B. CECCANTI ET AL.

Garcia, C., Hernandez, M.T. & Costa, F. (1990). Study on water extract of sewage sludge compost, Soil Sci. Plant Nutr. 37, 399408. Garcia, C., Hernandez, T., Costa, F., Ceccanti, C., & Gianni, A. (1993). Hydrolases in the organic matter fractions of sewage sludge: Changes with compostine, Biores. Technol., 45, 47 52. Gil-Stores, F., Trasar-Cepeda, M.C., Ciardi C., Ceccanti, B. & Lerios, M.C. (1992). Biochemical characterization of biological activity in very young mine soils, Biol. Fert. Soils 13, 2530. Hayano, K., & Tubaki, K. (1985). Origin and properties of -glucosidase activity of a tomato-eld, Soil Biol. Biochem. 17, 553557. Jackson, M.L. (1960). Soil Chemical analysis, Prentice-Hall Inc, Englewood Cliffs. Kretzschmar, A. (1978). Quantication ecologique des galeries de lombriciens. Techniques et premieres estimations, Pedobiologia, 18, 3138. Leahy, J.G. and Cowell, R.R. (1990) Microbial degradation of hydrocarbons in the environment Microbiol. Rev, 54(3), 305315. Margesin, R., Zimmerbauer, A. & Schinner, F. (2000) Monitoring of bioremediation by soil biological activities, Chemosphere, 40, 339346. Masciandaro, G., Ceccanti, B. & Garcia, C. (2000). In situ vermicomposting of biological sludges and impacts on soil quality, Soil Biol. Biochem. 32, 10151024. Marinari, S., Masciandaro, G., Ceccanti, B. & Grego, S. (2000) Inuence of organic and mineral fertilizers on soil biological and physical properties, Bioresour. Technol, 72, 917. Morgan, P., & Watkinson, R. J. (1989). Hydrocarbon degradation in soil and methods for soil biotreatment. CRC Critical Reviews in Biotechnology, 8, 305333. Murphy, J. and Riley, H.P. (1962) A modied single solution method for the determination of phosphate in natural waters, Analytica Chimica ACTA, 27, 3136. Nannipieri, P., Ceccanti, B., Cervelli, S. & Matarrese, E. (1980). Extraction of phosphatase, urease, protease, organic carbon and nitrogen from soil, Soil Sci. Soc. Am. J., 44, 10111016. Nannipieri, P., Greco, S., Ceccanti, B. (1990). Ecological signicance of the biological activity in soil, in Bollag J.M., Stozky G. (eds), Soil Biochemistry, 6 Marcel Dekker, New York. Nannipieri, P. & Bollag, J.M. (1991). Use of enzymes to detoxify pesticide-contaminated soil and waters, J. Environ. Quality, 20, 510517. Nannipieri, P., Sastre, I., Landi, L., Lobo, M.C. & Pietramellara, G. (1996). Determination of extracellular neutral phosphomonoesterase activity in soil, Soil Biol. Biochem., 28, 107112. Organization for Economic Co-operation and Development (OECD) (2000). Guideline for Testing Organic Chemicals. Proposal for New Guideline: Earthworms Reproduction Test (Eisenia fetida/andrei). http://www.oecd.org. Parr, J.F. & Smith, S.S. (1969). A multi-purpose manifold assembly: use in evaluation microbial effects of pesticides, Soil Science, 107, 271276. Reyment, R. & Joreskog, K. G. (1993). Applied factor analysis in the natural science, Cambridge University Press, Cambridge, UK. Safwat, H., Hanna, S. & Weaver, R.W. (2002). Earthworm survival in oil contaminated soil, Plant and Soil, 240, 127132. Schack-Kirchner, H. & Hildebrand, E.E. (1998). Changes in soil structure and aeration due to liming and acid irrigation Plant Soil, 199, 167176. Schaefer, M., Petersen, S.O. & Filser, J. (2005). Effects of Lombricus terrestris, Allolobophora chlorotica and Eisenia fetida on microbial community dynamics in oil-contaminated soil, Soil Biol. Biochem., 37, 20652076. Schinner, F., Ohlinger, R., Kandeler, E. & Margesin, R. (1996). Methods in Soil Biology., Springer, Heidelberg. Singer, A.C., Jury, W., Luepromchai, E., Yahng, C.S. & Crowley, D.E. (2001). Contribution of earthworms to PCB bioremediation Soil Biol. Biochem., 33, 765776.

COMBINATION OF EARTHWORMS AND COMPOST

397

Tabatabai, M.A., & Bremner, J.M. (1969). Use of -nitrophenol phosphate in assay of soil phosphatase activity, Soil Biol. Biochem., 1, 301307. van Beelen, P. V. & Doelman, P. (1997). Signicance and application of microbial toxicity tests in assessing ecotoxicological risks of contaminants in soil and sediments, Chemosphere, 34, 455 499. Wolters, V. (2000). Invertebrate control of soil organic matter stability, Biol. Fert Soils, 31, 119. Yeomans, J.C. & Bremner, J.M. (1988). A rapid and precise method for routine determination of organic carbon in soil, Comm. Soil Sci. Plant Anal., 19, 14671476.