Introduction

Heavy metal contamination has become a serious problem in the environment. Due to the non-biodegradable property of heavy metals in soils, their release to the environment should be restricted (Novais et al. 2011; Maity et al. 2008). In China, heavy metal pollution in urban soils, urban road dusts, and agricultural soils became serious with the rapid industrialization and urbanization during the last two decades (Wei and Yang 2010), and the potential public health risk associated with heavy metal contamination is of current concern.

Routine chemical analyses of metals cannot take into account issues such as mixture toxicity and the environmental conditions (such as soil structure and chemical absorption, temperature, pH, etc.) determining chemical bioavailability (Gastaldi et al. 2007). For the purpose of an adequate assessment of soil pollution, the use of bioassays to rapidly screen exposure and to demonstrate potential effects of pollutants can provide an ideal means to determine the complex effects of chemical mixtures (Xiao et al. 2006c). Among soil organisms, earthworms play an important role in the soil ecosystem and appear to be one of the best organisms for use in soil toxicity evaluation. Thus, they are included in a group of key indicators for ecotoxicological testing of industrial chemicals determined by the Organisation for Economic Co-operation and Development and the European Economic Community (EEC) (Capowiez et al. 2003). The use of molecular biomarkers can be a complementary approach to standard toxicity tests (mortality and reproduction rates) because it provides more information about the organism's stress response to individual toxicants and mixtures (Gastaldi et al. 2007; Calisi et al. 2011; Hankard et al. 2004; Svendsen et al. 2004).

The activities of certain enzymes in earthworms are regarded as fast and prognostic indices of individual reaction to the environmental stress (Łaszczyca et al. 2004). Superoxide dismutase (SOD), one of the antioxidant enzymes, has been considered a good molecular bioindicator for contaminant-mediated oxidative stress to reflect the magnitude of responses in earthworms exposed to toxic metals and other xenobiotics (Novais et al. 2011; Xie et al. 2011). It has been reported that the activity of acetylcholinesterase (AChE) can be a promising parameter in evaluating the toxicity of heavy metals and pesticides (Chakra Reddy and Venkateswara Rao 2008; Ribera et al. 2001; Saint-Denis et al. 2001). Cellulase is another important enzyme in earthworms, and changes in its activity can directly influence the earthworm’s ability to decompose plant litter and other cellulosic materials (Luo et al. 2009; Shi et al. 2007).

The comet assay has become one of the standard methods for assessing DNA damage because of its simplicity, sensitivity, versatility, speed, and economy (Collins 2004). The comet assay applied to earthworms has been widely used to evaluate the genotoxicity of PAHs (Di Marzio et al. 2005), pesticides (Xiao et al. 2006a), multi-contaminated field soils (Bonnard et al. 2010; Qiao et al. 2007), and metals (Bigorgne et al. 2010; Li et al. 2009; Manerikar et al. 2008).

Many studies focus on the impact of metals in soil on earthworms, but most of these studies are performed in artificial soils or soils artificially contaminated by the addition of metal in solution, generally as a single metallic element (Nahmani et al. 2007b). Relatively few studies have dealt with the impact of field-contaminated soils with multiple contaminants (Alvarenga et al. 2008; Nahmani et al. 2007a; Berthelot et al. 2008; Bonnard et al. 2009). The present study aimed to evaluate biochemical responses and genotoxicity of earthworm (Eisenia fetida) exposed to two multi-metal-contaminated soils following long-term exposure in view of the identification of potential biomarkers for early warning of environmental health impacts. We investigated biochemical responses as measured by activities of SOD, AChE, and cellulase in earthworm, while the comet assay was applied to evaluate the genotoxicity of multi-contaminated field soils. To our knowledge, our study is the first attempt in integrating SOD, AChE, cellulase, and DNA damage to determine the effects of soil heavy metal contamination on earthworms.

Materials and methods

Experimental animal

E. fetida, one of the epigeic worm species, was adopted as the test species because it is widely available and easily reared in laboratory culture. Earthworms were obtained from a local market in Nanjing, China. Healthy adult earthworms weighing 200–300 mg and having a well-developed clitellum were used for all experiments. They were acclimatized for at least 1 week under laboratory conditions in culture pots. One hundred grams dairy manure which was dried at 100 °C and ground to pass at 2-mm sieve was incorporated into soil as food during the acclimation period. Prior to exposure, the earthworms were extracted from the culture media and placed in petri dishes for 24 h on moist filter paper at 20 ± 1 °C in the dark to void their gut contents. Petri dishes were closed using lids with air holes.

Exposure procedure

The metal-contaminated soil samples were collected from the surface layer (0–20 cm) of a steel industry park in Nanjing, China. Topsoil (0–20 cm) from an uncultivated and unpolluted field in Zijin Mountain (a mountain located in Nanjing) was used as reference soil. Before E. fetida exposure, the soil was air-dried and sieved though a 2-mm mesh net. The soil samples were subjected to chemical characterization and total main heavy metal quantification (Cd, Cr, Cu, Ni, Pb, and Zn) by inductively coupled plasma-atomic emission spectrometry (OPTIMA 5300 DV, Perkin-Elmer, USA) after digestion. The main characteristics and total metal content of the soil samples are presented in Table 1.

Table 1 Main characteristics and total metal content of the soil samples
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The experiments were conducted in wide-mouth bottles (1 L) with 700 g dry soil, and the soil moisture content was adjusted to 35 % of the water-holding capacity (WHC) with deionized water. E. fetida were introduced in bottles, and the experiments ran at 20 ± 1 °C with 16:8 light/dark photoperiod for 28 days. Each treatment contained 3 replicates, and each bottle kept 12 earthworms. The bottles were covered with plastic film that had been punched with small holes. Water was sprayed into the room of the bottle regularly to keep the air humidity at 80 % during the experimental period. No food was added to the bottles throughout the period of exposure. Three earthworms were collected from each replicate bottle on the 2nd, 7th, 14th, and 28th day for analytical procedure. No mortality was observed throughout the experimental period.

Analytical procedure

Prior to biochemical assays, each gut-cleaned earthworm (three for each group) was cut into pieces and mixed with ice-cold 0.85 % NaCl at 1/9 w/v ratio individually. The mixture was homogenized using XO-150 ultrasonic cell disrupter system (Xian’ou Instrument Corp., Nanjing, China) and then centrifuged at 3,000 rpm for 10 min at 4 °C. The resulting supernatants were used in the determination of SOD, AChE, and cellulase levels.

SOD activity was assayed by its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT), as described by Dhindsa et al. (1981). One unit of enzyme activity was defined as the amount of the enzyme exhibiting 50 % inhibition of the auto-oxidation rate of 0.1 mM NBT in a 1 mL solution at 25 °C (U/mg protein). AChE activity was spectrophotometrically determined according to Ellman (1961) by measuring the increase in absorbance of the sample at 412 nm in the presence of 1 mM acetylthiocholine as substrate and 0.1 mM 5,5-dithiobis-2-dinitrobenzoic acid. AChE activity was expressed as nanomoles of product developed per milligram of proteins per minute (nmol/min mg protein). Cellulase activity was determined according to Zhang (1991). A small volume (0.5 mL) of enzyme preparation was added into a test tube containing 1.0 mL preheated sodium carboxyl methyl cellulose, and the mixture was incubated at 50 °C for 30 min. The concentration of glucose was determined by adding 3, 5-dinitrosalicylic acid and measured spectrophotometrically (WFZ UV-2000, Unico Instrument Corp., China) at 530 nm. Enzyme activities were expressed in milligram of glucose per milligram of protein per hour (U/h mg protein). Protein content was measured according to Bradford (1976).

For the comet assay, three earthworms were used for each group after exposure. Their coelomocytes were obtained according to the non-invasive extrusion method described by Eyambe et al. (1991). Individual earthworm was initially rinsed in the extrusion medium, which consisted of 5 % ethanol, 95 % saline, 2.5 mg/mL Na2-EDTA, and 10 mg/mL guaiacol glyceryl ether (pH 7.3) to spontaneously secrete the coelomocytes in the medium. Then, the obtained coelomocytes were washed twice with phosphate-buffered saline. The cells were collected by centrifugation (3,000 rpm, 3 min) and kept at 4 °C before analysis using the comet assay. The comet assay was performed as described by Singh et al. (1988), Tice et al. (2000), and Hu et al. (2010) with some modifications. Ethidium bromide-stained nuclei were examined with a fluorescent microscope (BX41, Olympus, Japan). Three slides per group were examined, and at least 50 cells were analyzed for each slide. Images were analyzed according to the method of Collins et al. (1995) using the comet assay software project (CASP 1.2.2). Although the software reports several parameters, Olive tail moment (OTM), which is the product of the tail length and fraction of total DNA in the tail, and the percentage of DNA in the comet tail (tail DNA%) are two of the most commonly used parameters. Tail DNA% has a better linearity with dose of damage over a reasonable range and is considered to be the most reliable parameter (Collins 2004). They are presented here as measures of single-strand DNA breaks/alkali-labile sites to evaluate DNA damage of E. fetida treated with different soils.

Statistical analysis

The values of biochemical responses are mean ± SD (n = 3), while the values of comet assay are mean ± SE (n = 3). Statistical significances of differences between control and treated samples were determined by the use of one-way analysis of variance (ANOVA). ANOVA was also performed for differences between times of exposure, using SPSS 17.0 statistical software, taking p < 0.05 as significance level according to the Tukey’s post hoc test.

Results

Soil properties and metal content

Table 1 presents the soil properties and metal content in all sites. The soils were neutral, and soil from site III presented a slightly lower content in organic C than the other soils. Of the metals analyzed, the detection limits for Cd, Cr, Cu, Ni, Pb, and Zn were 0.3, 0.2, 0.2, 0.9, 3, and 0.2 mg/kg, respectively. Cd levels were below detection limit in all soil samples, and the total contents for Cr, Cu, Ni, Pb, and Zn in soils from sites II and III were high and largely exceeded the contents in reference soil. Compared with site II, soil from site III had a higher content of Cu, Pb, and Zn, which were 466, 394, and 1,729 mg/kg, respectively. However, soil from site II was characterized by a higher Cr content of 289 mg/kg.

Biochemical assays

Changes of all tested enzyme activities are summarized in Table 2, and the duration of exposure had a significant effect on all the biochemical responses studied. In some cases, significant differences (p < 0.05) were also found between controls from different time points, like, for example, in SOD activity, where a significant difference was found between controls from all four times of exposure. In general, SOD activity increased with the duration of exposure at all sites, including the controls. In contrast, cellulase activity in controls decreased over time. Similarly, AChE activity in controls decreased substantially with the duration of exposure, except on day 14.

Table 2 Biochemical responses of E. fetida exposed to metal-contaminated soil
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In comparison with reference site I, a significant decrease (p < 0.05) in SOD activity was observed in site III soil for 2 days after exposure. On day 7, the SOD activity in E. fetida exposed to site II and III soil increased 40.5 and 28.2 % compared to the reference soil, respectively. However, a significant inhibition (p < 0.05) of SOD activity was found at both contaminated soils on day 14 and day 28.

A significant inhibition (p < 0.05) of AChE activity was found at both contaminated soils after 2 days of exposure. After 7 days of exposure, the AChE activity at site III soil increased significantly compared to the reference soil, while the site II soil still inhibited AChE activity. On day 14, the AChE activity increased (p < 0.05) at site III soil compared to the control. Moreover, the AChE activity of treated earthworms was markedly induced by contaminated soils at site II and site III on day 28, with increased rates of 25.5 and 36.1 % compared to the control, respectively.

The contaminated soils (site II and site III) exerted a significant inhibition effect on cellulase activity during the entire exposed time (28 days) except on day 14. On day 14, a significant increase was observed at site III. But this effect was transient and inconsistent. By 28 days of exposure, the cellulase activity at site II and site III both decreased compared to the reference soil.

Comet assay

OTM, the product of the distance between the center of gravity of the head and the center of the gravity of the tail and percent tail DNA, was chosen to express the comet assay results. The results of the OTM presented in Fig. 1 show that the DNA damage of earthworm E. fetida exposed to metal-contaminated soils was always significantly higher than those exposed to reference soil through the entire experimental period. In comparison with the control, OTM of E. fetida increased 56.5 and 552.0 % on day 2 after exposure to site II and III soil, respectively. DNA damages of earthworms exposed to site III and reference soils decreased with the time of exposure during the early and middle exposed times (2, 7, and 14 days). Although the OTM of E. fetida exposed to site II soil had an elevation on day 7, all OTM, including the control, reached the lowest level on day 14. However, on day 28, there was an elevation of DNA damage in all groups after they reached the lowest level.

Fig. 1
figure 1

DNA damages of earthworm coelomocytes (represented as Olive tail moments) exposed to soils for 28 days. The values are mean ± SE; *significant difference as compared to the reference soil (p < 0.05)

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DNA damages of earthworm coelomocytes represented by tail DNA% are presented in Fig. 2. The results of tail DNA% were similar to the results of OTM during the exposure period. Photos of control assays show that no damage (Fig. 3a), contaminated soil exposure caused damage (Fig. 3b).

Fig. 2
figure 2

DNA damages of earthworm coelomocytes (represented as tail DNA%) exposed to soils for 28 days. The values are mean ± SE; *significant difference as compared to the reference soil (p < 0.05)

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Fig. 3
figure 3

Typical comet image of earthworm E. fetida cells in control (no obvious damage) (a) and exposed to contaminated soil (significant damage) (b)

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Discussion

The results demonstrate that in E. fetida, exposure to multi-metal-contaminated soils induced significant changes in all the studied biomarkers. However, some biochemical assays (SOD activity) were less sensitive to these particular types of soil contamination, whereas DNA damage and the activities of AChE and cellulase were much more sensitive.

Some reports suggest that in some cases, organisms primarily respond to certain soil attributes rather than to the pollutant concentration (Chang et al. 1997). Soil pH and organic C have been claimed to be important factors for affecting metal bioavailability to ecological receptors (Dayton et al. 2006; Peijnenburg and Jager 2003; Basta et al. 2005; Spurgeon and Hopkin 1996). In our studies, soil pH remained in the neutral to slightly alkaline range (i.e., 7.55, 7.80, and 7.95 for site I, II, and III soil, respectively), and organic C values in all soils were similar. Compared with the soil environmental quality standards (Table 3), the metal contents in contaminated soils largely exceeded the maximum permissible concentration in soils defined by many countries. Therefore, the heavy metal pollution in soils accounts for the observed toxicological responses to earthworms.

Table 3 Soil environmental quality standards of China and other countries (mg/kg)
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Many studies have reported the metal toxicity to earthworms tested in the artificial and field soil (Neuhauser et al. 1985; Spurgeon and Hopkin 1995; Spurgeon et al. 1994). The LC50 values of Cu, Zn, Ni, and Pb reported by Neuhauser et al. (1985) were 643 (549–753) mg/kg, 662 (574–674) mg/kg, 757 (661–867) mg/kg, and 5941 (5,292–6,670) mg/kg in artificial soil, respectively. The Zn content in the soils from site II (685 mg/kg) and site III (1,729 mg/kg) exceed the LC50 value of Zn, but no mortality was observed in our study. This confirms that toxic effects of metals were less severe in field soils (Spurgeon and Hopkin 1995). As reported by many studies, Cu and Zn were considered more toxic than other metals such as Ni and Pb (Neuhauser et al. 1985; Spurgeon and Hopkin 1995); we assumed that the contamination in site III was more severe than site II. On the other hand, it should be noted that simultaneous exposure to several metals can also lead to antagonistic, not necessarily to additive or synergistic, effects, as observed by Khalil et al. (1996).

SOD activity increased significantly at 7 days in metal-contaminated soils compared with reference soil. But the inhibition observed at 14 and 28 days was somewhat unexpected. Honsi et al. (1999) concluded that antioxidant enzymes such as SOD and catalase (CAT) in two Eisenia species were not inducible and hence not suitable as biomarkers of metal-induced oxidative stress. The results of the present study also showed that SOD is not a suitable biomarker for metal-contaminated soils in our case. In contrast, Berthelot et al. (2008) found that SOD responded well to contamination when the earthworms were exposed to metal- and energetic compound-contaminated soils in Gagetown. The toxicity mechanisms that account for these differences remain unknown. However, as Łaszczyca et al. (2004) stated that though no simple specific and unequivocal signal should be expected, activity of antioxidative enzymes (such as CAT and SOD) and glutathione S-transferase antioxidant activities are promising earthworm biomarkers of exposure in earthworms.

AChE is a critical enzyme in the nervous system of vertebrates and invertebrates and is the functional target of several xenobiotics. It is well established that AChE inhibition is a useful biomarker for organophosphate and carbamate pesticides both in in vivo and in vitro conditions (Rao and Kavitha 2004; Ribera et al. 2001; Venkateswara Rao et al. 2003; Key and Fulton 2002). However, the effect of the metal ion on the activity of AChE revealed some contradictory results as the activation observed in some studies does not coincide with the inactivation reported in other investigations (Beauvais et al. 2001; Romani et al. 2003; Frasco et al. 2005; Sarkarati et al. 1999; Zatta et al. 2002).

In our study, AChE activities in contaminated soils were inhibited after 2 days of exposure, but they increased at the 28th day of exposure. The reason for these fluctuations remains unclear. But the elevation observed on day 28 indicated an activating effect of metals on AChE and led to an improved catalytic efficiency of AChE in earthworms. Such an activating effect of metals on AChE has been described in many studies. For example, Romani et al. (2003) demonstrated an increase in AChE activity (V m/K m) after Sparus auratus was exposed to sublethal copper concentrations. Zatta et al. (2002) also found from in vitro studies that aluminum chloride had an activation of mouse brain AChE. Because of the complexity of different metal effects, it is necessary to consider the effects of metals on AChE when using this enzyme as an environmental biomarker, particularly in environments polluted with several classes of chemicals (Frasco et al. 2005).

Occurrence of cellulase in the earthworms’ gut indicates their role in the decomposition of plant litter and other cellulosic materials (Shi et al. 2007), and it has been presumed as a bioindicator for pollution by insecticides. Many pesticides (e.g., imidacloprid and acetochlor) were found to inhibit the cellulase activity of earthworms (Xiao et al. 2006b; Luo et al. 1999). Additionally, Hu et al. (2010) also found that TiO2 and ZnO nanoparticles in soil decreased the cellulase activity in earthworms. The results of our study showed that metal-contaminated soil exposure may have a negative effect on the biochemical metabolism of earthworms, thus inhibiting cellulase activity.

To explore the potential for using the biochemical responses of earthworms as biomarkers for monitoring contamination in soils, knowledge of time- and dose-dependent relationships of the responses is needed in order to use them as bioindicators (Ribera et al. 2001). Activities of SOD, AChE, and cellulase in controls were strongly affected by the duration of exposure. Such variations have been reported in previous studies (Saint-Denis et al. 1999; Saint-Denis et al. 2001; Novais et al. 2011; Shi et al. 2007). We cannot explain these variations at this moment, and further investigation is needed to examine this effect when working on biomarkers.

The comet assay is capable of examining DNA strand breaks (DSB) in individual eukaryotic cells after in vivo or in vitro exposure and is considered to be a sensitive biomarker for identification and quantification of genotoxicity (Faust 2004). Recently, the comet assay has been widely used to evaluate the genotoxicity of field soils contaminated by metals and organic pollutants (Xiao et al. 2006c; Lourenço et al. 2011). In our case, DNA integrity of earthworms was significantly affected by the exposure to the metal-contaminated soils since the damages in the DNA of coelomocytes were always significantly higher in organisms exposed to contaminated soils than in those exposed to reference soil. According to Barillet et al., some metals could induce the production and intracellular accumulation of reactive oxygen species (ROS), which yield DNA damages (Barillet et al. 2005). Many other studies have revealed the genotoxicity of heavy metals like Ni and Cr (S. A. Reinecke AJR 2004; Manerikar et al. 2008; Bigorgne et al. 2010). The constant low DSB levels in the controls are assumed to be background values derived from endogenous and unavoidable exogenous sources (Lutz 1998).

Noticeably, the DSB levels in all groups declined after the 14-day exposure, which could be attributed to either activation of antioxidant systems against ROS or activation of DNA repair mechanisms or compartmentalization of metals in different earthworm tissues leading to the reduced bioavailable metals in circulation. According to Ching et al., DNA repair systems might be activated after the invertebrate tissue has accumulated sufficient toxicant above a threshold level (Ching et al. 2001). This may explain why earthworms exposed to less contaminated soils of site II were slower in repairing DNA and still showed increased DNA damage on day 7. In contrast, earthworms exposed to heavy contaminated soils of site III recovered faster. However, Ching et al. (2001) also suggested that prolonged exposure, even at low levels, may have results in subsequent levels exceeding the threshold, thus resulting in an activation of the repair system. This is in coincidence with our results that earthworms exposed to soils of site II repaired DNA from day 7 to day 14. We speculate that elevation of DSBs levels on day 28 may be due to the lack of nutrients.

Moreover, it should be mentioned that organic pollutants may also contribute to the responses. The contaminated soils in the present study were collected from a steel industry park, and heavy metals were considered to be the critical pollutants in this area. But more extensive investigation is required to determine the interaction of the heavy metals and organic pollutants in the future.

Conclusion

The present study showed clearly that exposure to metal-contaminated soils affected SOD, AChE, and cellulase activities in earthworm E. fetida. It can be concluded that heavy metals exhibit inhibition on cellulase activity, while long-time exposure (28 days) showed an activating effect of AChE. Due to the complexity of different enzymes, further investigation is needed to explain the particular mechanisms. A clear evidence of DNA damage and repair process was found, and this indicated that metal-contaminated soils were genotoxic for E. fetida. Based on the present work, we confirmed that the comet assay is a useful technique to evaluate the toxicity of metal-contaminated soils, and DNA damages of earthworm could be used as early biomarker. As for the biochemical assay, our results showed that cellulase was more sensitive to soil contamination and it is also a promising biomarker of exposure effects.