Introduction

Oil pollution in the earth’s biosphere can be caused either by natural phenomena or by human activity. Petroleum pollution has the potential to damage the ecosystem. It poses risks to the characteristics of ground water, air and soil and could also harm human health (Schaefer and Juliane 2007; Lopez et al. 2008). Even at low levels of contamination, carcinogenic and mutagenic crude oil compounds could cause severe chromosomal damage due to their persistence and biological toxicity (Aguilera et al. 2010; Robertson and Hansen 2015).

The production, transportation, storage, and exploration of oil have led to contamination of soil by Petroleum hydrocarbons (Bento et al. 2005). Crude oil is a variable mixture of thousands of compounds; alkanes, aromatics, resins, and asphaltenes being the four main fractions. Due to crude oil structural complexity, the measurements of petroleum from environmental samples are recorded as total petroleum hydrocarbons (TPHs) (Faustorilla et al. 2017).

.The presence of oil in soil may reduce aeration and water permeability by filling the soil pore spaces (Jia et al. 2017). It results in changes in soil physicochemical properties such as moisture content, pH value, total organic carbon, electric conductivity, and soil mineral nutrients, thereby indirectly affecting the development of microorganisms and plants (Wang et al. 2013; Devatha et al. 2019).

Following oil leakage to the soil, several physical and chemical techniques can be applied to reduce the level of pollution. However, these procedures are expensive, require high energy, and cause secondary contamination (Mrozik and Piotrowska-Seget 2010). On the other hand, bioremediation has been reported as an economic and environmentally friendly method by which microorganisms with enzymatic activity are involved in the degradation of petroleum hydrocarbons into less toxic products (Wang et al. 2019; Nneji et al. 2016).

The application of earthworm in a method known as vermiremediation in order to clean polluted soil is one of the most effective bioremediation technologies. Earthworms may reduce, or contribute to the reduction of various inorganic and organic contaminants such as heavy metals, polycyclic aromatic hydrocarbons (PAHs), crude oil, and pesticides from the soil (Schaefer and Juliane 2007; Hickman and Reid 2008). Earthworms could also improve the quality of the soil they inhibit by adding vermicast to the soil after reprocessing the polluted soil in the gut of the earthworm (Drake and Horn 2007).

Several studies have shown the possible use of earthworm to enhance soil depletion of hydrocarbons. By improving microbial soil activity, earthworms have increased the elimination of PAHs: phenanthrene, benzo(a)pyrene, and anthracene from polluted soil (Contreras-Ramos et al. 2008 and 2009). Earthworms also have improved microbial biomass in oil-contaminated soil, while TPH decreased by 28 day compared to controls (Schaefer et al. 2005; Schaefer and Filser 2007). Chachina et al. (2015) reported that the use of earthworms decreased by approximately 99% of hydrocarbons content from petroleum- and diesel-contaminated soil of 20–60 g/kg, after 22 weeks.

Although it appears clear that earthworms could accelerate and increase the rate of TPH degradation, a number of variables also need to be assessed in order to establish vermiremediation as a possible technique. Further studies at higher scales are required to demonstrate the efficacy of earthworm in the degradation of crude oil fractions. Hydrocarbon exposure to earthworms is also a major challenge to the continuation of the remediation process, as light crude oil has been reported to be more harmful to earthworms than heavy crude oil (Martinkosky et al. 2016).

The need to identify and assess the effect of contamination on the quality of the environment has led to the development of biological markers. Theoretically, biomarkers have significant advantages in ecotoxicological studies. They are used as measures of adverse effects in organisms below the individual level (molecular or cellular) (Van Gestel and Van Brummelen 1996). In earthworm ecotoxicology, there are a variety of biomarkers of toxic compounds such as detoxification cation enzymes, metallothioneins, cholinesterases, oxidative stress parameters, and others (Novais et al. 2011). Examination of novel and efficient biomarkers in earthworms is advancing in soil pollutant impact assessment phase.

Allopophora caliginosais earthworm (Savigny, 1826) is the dominant local type in Egypt (El-Duweini and Ghabbour 1965). Allopophora caliginosa is an endogeic species, living in topsoil mineral layers in horizontal burrows. Endogeic worms consume large amounts of soil during burrowing; this feeding pattern could increase the contact of soil particles with the worm gut and result in more TPH degradation. Several studies on bioremediation using other species of earthworms have been reported. However, research on the bioremediation of crude oil using A. caliginosa was limited. The purposes of this study were to (1) investigate the potential of A. caliginosa to remove TPH from crude oil-contaminated soils, in parallel with the measurement of variations in soil physicochemical properties. (2) study the impacts of crude oil on earthworms during remediation and to critically evaluate their utility as a bioindicator for crude oil toxicity in the soil; this has been carried out based on two directions; first, determine the concentration of hydrocarbons in earthworm tissues. Second, investigate biochemical and hematological changes in the tissues of the earthworm.

Material and method

Collection of soil and acclimatization of earthworm

Soil samples were obtained from an agricultural farm in Zagazig, Sharkia Governorate, Egypt, at a depth of 10–12 cm. The soil was dark brown in color; soil texture was clay loam with 34.6% sand, 30.5% silt, and 34.9% clay (according to Gee and Bauder 1986) and with water holding capacity (WHC) 79%. The background TPH concentration was 1963 ± 14 mg/kg. The collected soil was sundried by spreading it for 48 h on a flat, clean board surface. The dried soil was sieved using a 5-mm mesh plastic filter to remove debris and large stones.

Adult Allolobophora caliginosa earthworms (About 90 individuals) weighing 1.5–2.8 g and with length 10–12 were also collected from the same farm by digging and hand sorting with hand trowel. The collected specimens were transported to the laboratory within an hour accompanied by the moist soil and acclimatized for 7 days in holding rectangle containers covered with netting wire and fed under controlled laboratory conditions. The experimental animals were maintained at 20 °C ± 2 °C and relative humidity 70–80%. Only adult earthworms with well-developed clitella were used in this study.

Crude oil

The crude oil used in this experiment was a light crude oil with specific gravity 0.84 and API gravity 37. The oil obtained from Misr petroleum company, Cairo, Egypt, and its characterization was performed using the standard test method of analysis ASTM (2003). The concentrations used were 5 and 7.5 ml. The concentrations used in this study were analogous to other ecotoxicological studies, such as Ekperusi and Aigbodion (2015).

Experimental design

One kilogram of sun-dried soil was weighed and placed into nine rectangular plastic boxes (1 kg in each box) (18 × 9 × 10 cm). In addition to the control, three replicates of each concentration (5 and 7.5 ml) were prepared. Using a 10-ml glass beaker, 5 and 7.5 ml of crude oil were added and extensively mixed into each of the six boxes. Treatments with crude oil-contaminated soil were extensively mixed and remained exposed to the elements in the laboratory for 7 days. After 7 days, cow dung was freshly collected by the traditional ways from the campus, and about 25 g cow dung weighed was thoroughly mixed with both controlled and contaminated soils in the boxes. Immediately after the addition of cow dung, earthworms were removed from the holding containers, washed with clean water, and then measured and weighed. Earthworms were inoculated into the crude oil polluted soils and controlled soils (10 earthworms in each container). Distilled water was added to the boxes twice a week to keep the soil moistened, with water content between 69 and 82%.

Preparation of earthworm tissues

According to the experimental plan, on day 60, earthworms from control and contaminated breeding boxes have been removed from the soil and washed. The blood samples were collected directly into K3 EDTA tubes prior to other procedures. The earthworms were placed on wet filter paper in a petri dish to get rid of their gut contents (Dalby et al. 1996). After which, they were killed rapidly by putting them on ice. A set of procedures involving the weighing and dissecting of earthworms have been executed. One gram of tissue was homogenized in phosphate-buffered saline. The homogenates were centrifuged at 8000 rpm at 5 °C and the supernatants were stored at −80 °C for biochemical analysis. In parallel, 5 g of tissue was used for determination of TPH by GC-MS system.

Determination of concentration of total petroleum hydrocarbon in soil and earthworm tissues

The TPH content was extracted twice from the treated and untreated soil samples and earthworm tissues using the method outlined by Njoku et al. (2016). Five grams of each sample were weighed, and 10 ml of the mixed solution of hexane and dichloromethane was added as a solvent with a ratio of 3:7. The mixture was shaken for 30 min using a mechanical shaker. The filtrate was purified by a pasture pipette packed with anhydrous sodium sulfate. Then, it was filtered, and the filtrate was pooled together and allowed to concentrate to 1 ml. The concentration of total petroleum hydrocarbons in soil and earthworm tissues was determined by the GC-MS system (Agilent Technologies) and was equipped with gas chromatograph (7890B) and mass spectrometer detector (5977A) at Central Laboratories Network, National Research Centre, Cairo, Egypt. The GC was equipped with HP-5MS column (30 m × 0.25 mm internal diameter and 0.25-μm film thickness). Analyses were performed using helium as a carrier gas at a flow rate of 1.0 ml/min at a splitless, injection volume of 2 μl, and the following temperature program: 50 °C for 10 min; rising at 8 °C/min to 300 °C and held for 10 min. The injector and detector were held at 280 °C and 220 °C, respectively. The data were estimated by integration of the area under the resolved chromatographic profiles using the TotalChrom software (Villas-Bôas et al. 2006).

Determination of physicochemical parameter of soil

Soil samples were taken at the three randomly replicated treatments at different exposure periods (t0, t22, t60) to determine physicochemical parameters such as pH, electric conductivity (EC), nitrate (N), phosphate (P), sodium (Na) potassium (K), calcium (Ca), magnesium (Mg), and organic matter (OM). These parameters have been determined using procedures by Sparks (1996).

Antioxidant biomarkers

Malondialdehyde (MDA) was determined according to methods of Uchiyama and Mihara (1978), catalase (CAT) was measured according to Aebi (1974), total antioxidant capacity (TAC) was estimated according to Nemec et al. (2000), and glutathione (GSH) was carried out according to the method Beutler et al. (1963) according to the corresponding assay kit protocol (Sat 450, AMS, Italy). Total proteins were measured using total proteins kit according to Biuret method.

Hematological parameter

The hematological parameters; hemoglobin concentration (Hgb), red blood cell count (RBCs), hematocrit (Hct), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), platelet (Plt), and white blood cell count (WBCs) were measured according to the methods of Brown (1993). These parameters were obtained at once for each blood sample using an automated hematology analyzer (Celltac α/MEK-6420K).

Statistical analysis

Data were analyzed by using the SPSS version 20 (SPSS, Richmond, VA, USA). Two-way ANOVA was used to determine the impact of crude oil concentration and time exposure on TPH concentrations in soil. One-way ANOVA was used to compare the effects of crude oil concentrations on TPH accumulation, hematological parameters, and biochemical biomarkers in earthworms. Significant differences at P < 0.05 among treatments were conducted using Duncan test as a post hoc test.

Results

Earthworm mortality

After exposure to 7.5 ml of crude oil, the percentage of dead earthworm was 40% after 60 days (at the end of the experiment). No mortality was recorded after 60 days of exposure to 0.0 (control) or 5 ml of crude oil.

Effect of earthworm A. caliginosa on concentration of TPH in crude oil-contaminated soil

As shown in Table 1, the concentration of TPH was significantly reduced in soil samples with earthworm at t22 and t60 compared to the treatments without worms at t0 (P < 0.05). The concentration TPH was significantly affected by crude oil concentrations and duration of exposure. In addition, the TPH concentration showed time and dose-dependent patterns. After treatment with 5 ml of crude oil, TPH decreased by 33.56 and 54.98% after 22 and 60 days of exposure. On the other hand, TPH decreased by 32.24 and 71.04 % after treatment with 7.5 ml.

Table 1 Concentrations of total petroleum hydrocarbon (mg/kg) in oil-contaminated soil samples with the earthworm A. caliginosa at different exposure periods
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Effect of earthworm A. caliginosa on physicochemical parameters of crude oil-contaminated soil

Results in Table 2 showed that physicochemical soil parameters decreased after crude oil treatment after 22 and 60 days of the experiment. Statistical analysis showed that there was no significant difference (P > 0.05) between pH, EC, Na, K, Ca, Mg, and organic matter at t0 (soil without earthworm) and the treatments (soils with earthworms) at different concentrations. On the other side, there was a significant difference (P < 0.05) between nitrate, phosphate, and humidity at t0 (soil without earthworm) and the treatments (soils with earthworms) with 5 and 7.5 ml crude oil.

Table 2 Physicochemical parameters of oil-contaminated soil with earthworm A. caliginosa through 60 days
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Bioaccumulation of TPH in the tissues of earthworm A. caliginosa

The concentrations of TPH in tissues of crude oil exposed earthworms for 60 days have been shown in Fig. 1. Total petroleum hydrocarbon accumulation was significantly affected by crude oil concentrations. The highest concentrations of TPH were detected in earthworms after 60 days of exposure to 7.5 ml crude oil (216800 mg/kg) compared to control. The lowest oil residues were retained in earthworm after 60 days of exposure to 5 ml crude oil were (175,800 mg/kg) compared to control (16,400 mg/kg).

Fig. 1
figure 1

Accumulation of TPH (mg/kg) inside body of the earthworm A. caliginosa exposed to different concentrations of crude oil for 60 days

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Evaluation of the effect of different concentrations of crude oil on earthworm A. caliginosa

Biochemical biomarkers in earthworm A. caliginosa exposed to different concentrations of crude oil

Table 3 shows that the levels of MDA, GSH, and TP in earthworm treated with 5 and 7.5 ml of crude oil were significantly higher than that of the control group. On the other hand, levels of CAT and TAC were significantly decreased (P < 0. 05) in earthworm after 60 days of exposure. After exposure to 5 and 7.5 ml of crude oil, MDA, GSH, and TP were increased by 29.4–131.3%, 53.9–129.5%, and 8.9–12.7%, respectively, while CAT levels were reduced to −16.3% and –34.9% after treatment with 5 and 7.5 ml, respectively.

Table 3 Biochemical biomarkers in gut of earthworm A. caliginosa treated with different concentrations of crude oil for 60 days
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Hematological parameters

Table 4 illustrates hematological indices in earthworm A. caliginosa after exposure for 60 days to 5 and 7.5 ml of crude oil. Hgb, RBCS, Hct, MCV, MCH, platelet count, and WBCs were significantly decreased (P < 0. 05) in earthworm exposed to 5 and 7.5 ml crude oil. On the other hand, MCHC value was significantly increased after exposure to 7.5 ml crude oil. The result showed that earthworm treated with 7.5 ml crude oil was more effective on hematological indices than those treated with 5 ml oil.

Table 4 Hematological parameters of earthworm A. caliginosa treated with different concentrations of crude oil for 60 days
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Discussion

Earthworms are reported to either biotransform or biodegrade the inorganic and organic pollutants in the soil, rendering them nontoxic to their tissues and to the ecosystem (Sinha et al. 2010). At the end of the present study, it was found that TPH concentrations were significantly decreased by 54.98 and 71.05%, respectively, in soil samples contaminated with 5 and 7.5 ml of crude oil. These findings revealed that Allolobophora caliginosa inoculation has an effect on the rate of degradation of petroleum hydrocarbons in contaminated soils. This was consistent with previous research using different species of earthworm. After 28 days, the analysis of crude oil polluted soils that inhibited by three different species of earthworms: Allolobophora chlorotica, Lumbricus terrestris, and Eisenia fetida showed that TPH decreased between 17 and 42% (Schaefer et al. 2005). However, the activities of Eudrilus eugeniae and L. terrestris led to 88.50 and 76.42% TPH losses, respectively, from the soil polluted with 3 ml crude oil after treatment for 30 days (Njoku et al. 2016). In another study by Almutairi (2019) upon inoculation of E. eugeniae earthworm into crude oil-contaminated soil of 30,000 mg/kg, TPH dropped by 42% after 150 days. Chachina et al. (2015) reported that, in crude oil-contaminated soil with petroleum concentration of 20–60 g/kg and in the presence of Eisenia fetida, 99% of the hydrocarbon content had been removed after 22 weeks. However, in diesel polluted soils with a concentration of 1014 μL kg−1, the hydrocarbons decreased by 43–52% after 90 days (Fernández et al. 2011). The reduction of hydrocarbons from contaminated soil may be attributed to the mineralization of petroleum products with the aid of earthworms (Contreras-Ramos et al. 2008). However, Dabke (2013) informed that increasing the activity of microorganisms in the digestive tract of earthworms may lead to clean up of hydrocarbons. Another possible mechanism for hydrocarbon degradation was the stimulation of soil microbe activity as a result of earthworm activity (Schaefer et al. 2005). In this study, the loss of TPH in soil treatments could be due to any or a combination of such mechanisms.

This paper found that Allolobophora caliginosa earthworms have a potential for decomposition of total petroleum hydrocarbons (TPHs) in crude oil-contaminated soils ranging from 45,200 to 62,410 mg/kg. It was noted that the TPH of 45,200 mg/kg was not harmful to earthworm survival, but TPH of 62,410 mg/kg reduced their survival to 40% after 60 days. This means that they can tolerate high concentrations of hydrocarbons in contaminated soils compared to other species mentioned in previous studies. According to Hentati et al. (2013), earthworm Eisenia andrei reported a 67% mortality rate after 14 days when exposed to a concentration of 1000 mg/kg of oil-contaminated soil. Another research also concluded that 0.5% of the oil content had no toxic impact on the survival of Eisenia fetida earthworm, but when the concentration was increased to 1.5%, the survival rate decreased by 40% after 7 days (Shakir Hanna and Weaver 2002). Thirty percent of Eisenia fetida died after exposure to 40,000 mg kg−1 concentration of diesel after 14 days (Chachina et al. 2016). Another study with the same earthworm, contaminant, and exposure time showed that there was no earthworm survived at 30,000 mg kg−1 (Moon et al. (2013). The lower mortality rate in this study could be due to the physiological adaptation of earthworms to the pollutants and conditions.

In this study, all measured soil physicochemical parameters: PH, OM, EC, and soil mineral nutrients decreased after crude oil treatment, and this decrease continued overtime for all treatments. This could mean that, due to the increase in soil worm activity in the contaminated soil to improve remediation process, the soil properties tend to decrease. This was supported by the results of Ceccanti et al. (2006) which detected a decrease in EC, total carbon, and phosphorous of soil polluted by hydrocarbon, indicating a progressive decomposition of the contaminants. However, the use of organic carbon as a source of energy by earthworms and microorganisms has led to a reduction in organic carbon in polluted soils (Azizi et al. 2013).

Earthworms perform different functions in soil. They recover soil structure and contribute to the decomposition of organic matter and nutrient cycling. They also enhance the biological, physical, and chemical characteristics of the soil in order to improve its fertility (Richardson et al. 2009). According to Ekperusi and Aigbodion (2015), it was found that earthworms Hyperiodrilus africanus significantly changed the physicochemical characteristics of the crude oil-contaminated soil after 12 weeks. This was not in accordance with our observations, as by the end of 60 days, there were no signs of improvement in the soil, and the results are still so far from the standard levels of control soil. Also, there was a significant decrease in nitrate and phosphate concentrations which was proportional to the crude oil concentration. This finding proved the presence of a correlation between the nutrient level in the soils and their petroleum hydrocarbon content. The TPH in the soil could increase the carbon concentration which in turn could influence the nutrients equilibrium in the soil (Devatha et al. 2019). However, Njoku et al. (2016) reported that the more take-up of nutrients by earthworms, the more influence they will have on soil pollutant content and consequently more remediation. Phosphorus is an essential micronutrient for soil fauna; the reduction in soil levels may be attributed to the soil microbes that could utilize large amounts of AP during the degradation of hydrocarbons (Wang et al. 2009). However, reduction in soil nitrate in soils contaminated with petroleum hydrocarbons could be due to earthworms, because they absorb considerable quantities of nitrogen for digestion in order to maintain their biological function (Iordache and Borza 2012; Sandor and Schrader 2012).

Earthworms are known as good bioindicators of soil quality. Endogenic species can tolerate a wide range of soil contaminants than epigeic ones (Paoletti 1999). Epigeic earthworms are known to be able to degrade or accumulate hydrocarbons (Contreras-Ramos et al. 2009; Parrish et al. 2006). The results of this study further confirmed the suggestion that endogenic species would be useful to the same extent or to a greater extent than epigeal species (OECD 2010). Significant amounts of TPH were detected in tissues of A. caliginosa at the end of the experiment when compared to the control. This point suggested that the accumulation of petroleum hydrocarbons in earthworm tissues could be another possible mechanism for accelerating TPH degradation in the soil.

Hydrocarbon accumulation was found to be higher in soil polluted with 7.5 ml of crude oil than in soil polluted with 5 ml; this may be due to body wall or intestinal absorption of soil hydrocarbons. According to Belfroid et al. (1996), the earthworms accumulate several lipophilic organic compounds from their surrounding soil by passive absorption via the body wall and even by intestinal absorption during the passage of the soil through the intestine. This deposition increased as the surrounding soil content of the pollutant increased. The results of this study were in accordance with Ekperusi and Aigbodion (2015), who detected elevated amounts of TPH and BTEX in earthworm tissues after 12 weeks from exposure to crude oil.

Earthworms through their body walls as well as by intestinal surfaces are always exposed to pollutants in the soil. Thus, they have a consistent detoxification system for their survival (Kilic 2011). Biochemical examinations were used to evaluate the possible mode of action of stressors and the ability of the organism to adapt to the external environment (Abdel–Tawwab, M. 2016). Many biochemical parameters are estimated to be used as biomarkers in the present study. These include total protein, antioxidant enzyme, and hematological parameters.

Proteins play an essential role in metabolic pathways and are used as a diagnostic tool to assess the physiological status of the organism (Prasath and Arivoli 2008). The current study clearly showed that total proteins increased significantly in earthworm A. caliginosa treated with crude oil. This increase may be due to cellular defense mechanisms that are stimulated to resist these toxicants (El-Sayed et al. 2020).

Antioxidant enzyme activities are found broadly distributed in the tissues of invertebrate organisms (Livingstone 2001). Assessing antioxidant enzymes can demonstrate the antioxidant status of organisms by using them as biomarkers for contaminant-mediated oxidative stress. Furthermore, they are suitable biomarkers reflecting not only exposure to contaminants but also their toxicity (Valavanidis et al. 2006). Malondildhyde (MDA) is one of the final products of cell peroxidation of polyunsaturated fatty acids and is used as a biomarker for oxidative stress (Davey et al. 2005). This study showed a significant increase in MDA levels in A. caliginosa earthworm after exposure to crude oil. Similarly, Doherty et al. (2019) found that MDA levels increased in earthworm, Eudrilus eugeniae, after exposure to benzene, toluene, ethylbenzene, and xylene. This increase may be due to an increase in free radicals that cause MDA overproduction.

Catalase (CAT) provides the first line of protection against reactive oxygen species and is used as an oxidative stress biomarker (Van der Oost et al. 2003). In the present study, CAT activity was decreased in earthworm A. caliginosa after exposure to crude oil for 60 days. Such decreases in CAT activities may result from the accumulation of H2O2 levels. In the same study, Doherty et al. (2019) found that CAT activity decreased in the earthworm, Eudrilus eugeniae, after exposure to benzene, toluene, ethylbenzene, and xylene for 28 days. Total antioxidant capacity (TAC) is used to assess the antioxidant status of the organism and to evaluate the antioxidant response against the free radicals produced in the tissue sample (Richetti et al. 2011). The present study showed a decline in TAC levels for crude oil-exposed earthworms. This decrease may be due to its protective role against damages induced by free radicals. GSH maintains the redox status of the cell and protects cells from oxidative stress (Dickinson and Forman 2002). The GSH levels in this study have been significantly increased. These results were contrary to the findings of Doherty et al. (2019) who stated a decrease in GSH levels in the earthworm, Eudrilus eugeniae, after exposure to benzene, toluene, ethylbenzene, and xylene for 28 days. This increase in GSH levels can be attributed to their protective functions against oxidative stress.

Hematological changes are reported to be early warning indicators of the toxic effects of pollutants in vertebrates (Dauwe et al. 2006); however, they are not well studied in invertebrates and in earthworms for comparison. Earthworms have a circulatory system which is closed. Blood consists of hemoglobin, a large extracellular hemoprotein that flows through a closed circulatory system. Despite the fundamental function of the respiratory pigment in the physiology of the earthworm, little is known about its susceptibility to environmental contaminants. In this study, a significant decrease in the Hgb, RBCS, Hct, MCV, MCH, platelet count, and WBCs was detected in earthworms exposed to crude oil (5 and 7.5 ml) for 60 days when compared with the control. This was in agreement with the findings of Calisi et al. (2011) which showed that heavy metals including cadmium, mercury, and copper cause changes in hematological parameters in the earthworm Lumbricus terrestris. Finally, it can be said that the blood analysis can be suitable biomarker of exposure/effect to be involved in a multibiomarker strategy in earthworm in soil monitoring assessment.

The present study has shown that Allolobophora caliginosa earthworms along with their success in being a good bioremediator have been able to tolerate high concentrations of hydrocarbons in crude oil-contaminated soils compared to other species mentioned in previous studies. Additionally, the severe effects detected in antioxidant and hematological parameters in earthworms during the remediation process have not often been discussed in this type of subject in previous studies.

Conclusion

The results of this study have shown that the endogenic earthworms Allolobophora caliginosa have the potential to clean up petroleum hydrocarbons from crude oil polluted soils. Higher removal efficiency was found in soils containing 7.5 ml crude oil compared to 5 ml. In order to achieve a higher level of remediation and therefore restore the physical-chemical properties of contaminated soils, longer experiments would be needed as 60 days were not sufficient time for complete degradation. Also, it was found that the significant accumulation of petroleum hydrocarbons in earthworm tissues could lead to severe effects in their hematological and biochemical parameters.