Introduction

Vermicomposting is a very promising, non-expensive, and rapid eco-technique that uses earthworms for biodegradation of wastes such as animal dung, crop residues, as well as many different industrial wastes such as textile waste or sewage sludge (Garg et al. 2006; Singh et al. 2011; Wang et al. 2009). Currently, this biotechnology is more suitable because of high-quality obtained vermicompost and seems applicable in small- and medium-sized wastewater treatment plants. The possible application of the final product depends on its quality assessed using quantification of chosen chemical, physical, and biological parameters. The first experiments using Eisenia fetida species for bioconvertion of wastes were performed by Mitchell et al. (1977) in the USA. Later, sewage sludge was proposed as a substrate for vermicomposting reaction (Hernstein 1981; Mitchell et al. 1980). Composting and vermicomposting of sewage sludge as methods of aerobic biodegradation of organic wastes contribute powerfully to the nutrient recycling strategy.

Among approximately 8300 oligochaeta species (Reynolds and Wetzel 2004), only a few earthworm species can be successfully employed in vermicomposting process. In Europe, works concentrate on three epigeic species: E. fetida, Eisenia andrei, and Dendrobaena veneta. Organisms belonging to this group exhibited short life cycles and high reproduction rates (Dominguez and Edwards 2011; Rorat et al. 2014). Earthworms constitute very important element in food chain, being considered as soil ecosystem engineers and good candidates for biomonitoring of ecosystems (Edwards 1988; Lavelle and Spain 2001). Indeed, some Annelida oligochaeta are well known as sentinel species in ecotoxicological, biological, and genetic studies (see Pauwels et al. 2013), with Eisenia sp. recommended for OECD toxicity test (OECD 1984, 2007). As shown before, worms can inhabit contaminated areas (Morgan and Morgan 1998; Olchawa et al. 2006) where they are capable to accumulate pollutants, including metallic trace elements (MTEs). MTE’s accumulation in earthworms’ bodies is described as nonlinear, characterized by higher bioaccumulation factors (BAFs) at lower soil concentrations (Neuhauser et al. 1994). The level of accumulation of selected elements is a reflection of earthworms’ metabolic pathways, food selectivity, and detoxification mechanisms (Morgan and Morgan 1992), so it can be considered as species-specific. Many studies have shown that earthworms can remediate polluted wastes and transform them into valuable product called vermicompost. Removal of heavy metals is related, inter alia, to their accumulation in earthworms’ bodies (Edwards and Bater 1992; Pattnaik and Reddy 2011; Suthar and Singh 2009). Accumulation of certain metals can be strictly correlated to some physical and chemical parameters of substratum, like pH and organic matter (Peijnenburg et al. 1999). Thus, bioaccumulation is related to physiology of earthworms, physico-chemical parameters of soil, and to individual metals and metalloids properties (Mohee and Soobhany 2014) and can be expressed as BAFs (also referred as bioconcentration factors, concentration factors, or uptake factors). BAF is the ratio of the concentration of particular heavy metal in earthworm’s body to its concentration in the matrix. Therefore, although that many authors claimed the positive impact of earthworms on heavy metals removal (Gogoi et al. 2015; Suthar and Singh 2008), it was also found that due to progressive mineralization, the total metal concentration may increase during the process. The complex problem of MTE’s accumulation in earthworms’ bodies and its possible incorporation in higher levels of food chain (Roodbergen et al. 2008) is a big challenge while assessing the efficiency of vermicomposting process. Indeed, earthworms, as preys, can transfer metal fractions from cellular compartments and from ingested contents in gut into terrestrial food chains.

Although composting earthworm species are tolerant to many stress factors, their immune system is influenced by several contaminants present in the substrate. Among the others, heavy metals can cause a complex defense reaction manifested by high impact on earthworms’ immune cells (Rorat et al. 2013), expression of particular genes involved in detoxification process (Homa et al. 2015), or riboflavin content (Plytycz et al. 2009). Riboflavin, vitamin B2, is involved in immune responses in vertebrates and was recently found in eleocytes of earthworms (Koziol et al. 2006). The variation of riboflavin content depending on media pollution was previously recorded in Allolobophora chlorotica, E. andrei, D. veneta (Plytycz et al. 2011), or Dendrodrilus rubidus (Plytycz et al. 2009). Therefore, riboflavin may be considered as a biomarker useful for biomonitoring of vermicomposting process.

In this study, we analyzed (1) the influence of two different earthworm species on the changes of selected MTEs content in substratum during vermicomposting process, (2) the possible accumulation of MTEs in earthworm’s body, and (3) their impact on riboflavin content in worms. Application of three different substrates and two different earthworm species allowed analyzing the full profile of MTEs accumulation in worms during sewage sludge vermicomposting processes.

Materials and methods

Earthworms

Adult earthworms of E. fetida (mean body weight 0.35 g) and E. andrei (mean body weight 0.29 g) species were collected from genetically defined colonies maintained in Lille 1 University, where they were reared in controlled conditions (22 ± 2 °C, in the dark, moisture around 60 % by mass) and fed ad libitum with cow manure.

Municipal sewage sludge

Dewatered sewage sludge was taken from three different wastewater treatment plants in Nord-Pas de Calais region, France and transported to Laboratoire Génie Civil et géoEnvironnement (LGCgE)-Lille 1 where experiment started immediately. Sludges were classified according to their ascending content of heavy metal into W1, W2, and W3 (Table 1).

Table 1 Physical and chemical characteristics of three sewage sludge selected for experiments (W1, W2, and W3)
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Experimental scheme

Experiments were performed in Lille1 University, in France, under controlled laboratory conditions (22 ± 2 °C, in the dark, moisture around 60 %). Three municipal sewage sludges W1, W2, and W3 were separately mixed with 75 % commercial soil by mass. After 1 month of precomposting with aeration and mixing, mixtures were placed into plastic boxes with perforated lids (33 × 18.5 × 12 cm), 2200 g per box. Individuals of E. fetida and E. andrei were placed in boxes with W1, W2, or W3 (40 worms per each) or mixed (20 of each species per box) (Fig. 1). In control conditions, worms stayed in a mixture containing cow manure and organic compost soil (25–75 % m/m). Analysis of worms and mixtures were performed on day 0 and after 3, 6, and 9 weeks of exposure.

Fig. 1
figure 1

Experimental design; W1–3 selected sewage sludge, M1–3 mixtures of sewage sludge and commercial soil, Ef E. fetida, Ea E. andrei

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Soil and soil/sludge mixtures analysis

At day 0 and after 3, 6, or 9 weeks, the following parameters were determined for mixtures: moisture content, pH, volatile solids (VS), ash content, total carbon, total Kjeldahl nitrogen, and total metal content for Cu, Cd, Ni, Zn, and Pb. These analyses were carried out immediately after the samples were obtained. A total of 100 g was collected from each container using multi-point sampling conducted at random. The samples were dried at 60 °C in a ventilated oven for 48 h and then homogenized by grinding into small pieces using IKA® A11 basic analytical mill. Total Kjeldahl nitrogen was analyzed according to Polish standard methods (PN-ISO 11261:2002). The VS and ash content were determined by the loss of weight after ignition of sample at 550 °C for 2 h (APHA 1999). The pH was measured using digital pH meter (Cole Parmer Model No. 59002–00) in 1/10 (w/v) suspension of the sample in deionized water and in 1 M KCl after 1 day of equilibration. Total carbon was measured using a Multi N/C 2100 Analityk Jena (PN-ISO 10694:2002). Total phosphorous was measured according to ISO 6878:2004; before analysis, the samples were digested using a Berghof microwave digestion system (speed wave MWS-2-Microwave pressure digestion). Total heavy metals were measured by inductively coupled plasma optical emission spectrometry (ICP-OES; Thermo apparatus) after digesting of 300 mg of sample with 7 mL of concentrated HNO3. Samples were digested using microwave digestion. As an environmental matrix, the following reference materials were used: LGCQC3004 (soil material) and BCR-146R (sewage sludge material).

Earthworm analyses

Total metal concentration analysis

Earthworms were maintained 48 h with 1 % agarose in order to remove gut content. Then worms were lyophilised, crushed in liquid nitrogen, and then stored at room temperature. Total metal contents were determined after acid digestion as described before (Demuynck et al. 2007). Briefly, 100 mg of dry mass was digested for 12 h at room temperature with 0.5 mL of 65 % nitric acid and then progressively heated at 120 °C in order to reduce the half of the volume and remove nitrous vapor. Subsequently, samples were heated at 180 °C with 1 mL of a solution of nitric (10 V), sulphuric (2 V), and perchloric acids (3 V). Then, samples were filtered and volume was adjusted to 20 mL with ultrapure Milli-Q water. Metal concentrations were analyzed using ICP (ICP-OES IRIS Interpid II XSP Thermo, Thermo Scientific, Whatman, MA, USA). Bioaccumulation factors (BAFs) were calculated according to Eq. (1).

$$ {\mathrm{BAF}}_{Me}\kern0.5em =\kern0.75em \frac{c_{Me}\ \mathrm{earthworm}}{c_{Me}\ \mathrm{soil}} $$
(1)

where c Me earthworm is a total concentration of selected metal in earthworm’s body (mg g−1) and c Me soil is a total concentration of the same metal in substratum (mg g−1).

Harvesting coelomocytes

Earthworms were stimulated for 30 s with a 4.5 V electric voltage to expel coelomic fluid with suspended coelomocytes through the dorsal pores. Briefly, earthworms were placed individually in Petri dishes containing 3 mL of extrusion buffer (phosphate-buffered saline, PBS, supplemented with 2.5 g/L ethylenediamine tetra-acetic acid, EDTA, Sigma-Aldrich). Freshly prepared 2 mL suspensions were used for spectrofluorimetry. Each worm was used for coelomocyte harvesting only once.

Spectrofluorimetric measurements and analysis

The spectrofluorimetric measurements were performed on coelomocyte suspension lysates (2 % Triton; Sigma-Aldrich) using PerkinElmer Spectrofluorimeter LS50B. Excitation spectra were recorded between 300 and 520 nm (lambda at 525 nm), while emission spectra were recorded between 380 and 700 nm (lambda at 370 nm). The spectrofluorimetric signatures of unbound riboflavin are two excitation maxima (370 and 450 nm) and one emission maximum (525 nm). Arbitrary units (AU) of fluorescence were recorded using Microsoft Excel v. 2010.

Statistical analysis

Results were expressed as means + standard errors. Differences between means were determined by one-way ANOVA with post hoc Tukey’s t test (Microsoft Excel 2010) with the level of significance established at p < 0.05. Linear regressions were used to test correlation between variables.

Results

Individual earthworm body weights and riboflavin contents

Body weights of both E. fetida and E. andrei significantly increased during 3 weeks after exposure to control conditions as well as soil amended with sewage sludge slightly, moderately, or strongly contaminated (M1, M2, or M3, respectively), and then they gradually decreased during subsequent 3-week periods of exposure reaching the initial level only in a case of E. fetida in M1 substratum (Fig. 2a).

Fig. 2
figure 2

Changes in individual body weight IBW (a), riboflavin content RF (arbitrary units, AU) (b), and riboflavin content in relation to body weight RF/BW (c) in coelomocyte lysates during vermicomposting process. C control conditions (commercial soil mixed with cow manure), M1–3 mixtures of sewage sludge and commercial soil, Ef E. fetida, Ea E. andrei. Results shown as means ± SE. n = 8 Means not sharing the same latter are statistically different

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In contrast, the total riboflavin content in lysates on coelomocytes retrieved from earthworms was relatively stable, but always slightly declined after exposure to sewage sludge-amended soil and then come back to the initial level. Nevertheless, this decrease was statistically significant only in a case of E. fetida in highly polluted M3 mixture (Fig. 2b).

However, when riboflavin content was expressed as a function of body weight (RF/BW), it turned out that this is significantly decreased in both species of earthworms during the third or sixth week of exposure to all the mixtures of soil (Fig. 2c). Nevertheless, the same tendency was observed in control and experimental conditions.

Selected chemical and physical parameters

Table 2 shows selected parameters analyzed in mixtures before (group 0w) and after the experiment (9w). Mixtures M1, M2, and M3 are similar according to analyzed parameters and differ significantly only in TN concentration. Total organic carbon (TOC) decreased slightly throughout the experiment, and N fluctuates slightly around initial values, which results in small decrease in C/N ratio in M1 and M2. Surprisingly, in M3, E. fetida and mixture of earthworms caused important decrease in N content, so at the end of exposure, C/N ratio increased significantly. None of applied earthworm species caused changes in TP and VS content in all analyzed mixtures. TS percentage increased in all conditions, with statistical importance in M2 and M3 mixture. Moreover, pH decreased in all conditions.

Table 2 Chemical analysis of soil/sludge mixtures (M1, M2, and M3) at the beginning of experiments (0w) and after nine weeks (9w) with E. fetida (Ef) or E. andrei (Ea) or 1:1 mixture of those species (Ef + Ea)
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Metal content in soil/sewage sludge mixtures

Cd concentration decreased slightly in all vermicomposting conditions, but differences were statistically important only when the process was conducted by E. fetida in M1. The highest decrease was achieved in the less contaminated mixture. Similarly, Pb content diminished throughout the process, and for higher concentrations, this decrease was modest. Ni and Cu concentrations were slightly diminished after vermicomposting process. Zn concentration increased in M1 and M2 mixtures in all applied conditions, while it decreased slightly in M3 treated by E. andrei and mixture of both species (Table 3).

Table 3 Metal contents (mg kg−1) in M1, M2, and M3 mixtures at the beginning of experiments (0w) and after nine weeks (9w) with E. fetida (Ef) or E. andrei (Ea) or 1:1 mixture of those species (Ef + Ea
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Metal content in earthworms’ bodies and its relation to metal contents in substrates

Concentration of Cd and Cu in earthworm bodies increased with increasing concentration in soil for both species but E. andrei revealed higher tendency to accumulate those two metals than E. fetida. Zn body content differs slightly between mixtures, independently from the significantly increasing content in soil. Statistically important differences were observed in Ni body burdens, which is highest in M3 and its value is inversely proportional to the concentration in soil. The highest body content of Pb was achieved in the moderately contaminated mixture M2, and no important correlation with soil contents was observed (Figs. 3a, b and 4).

Fig 3
figure 3

Differences between selected metal concentration in mixtures of sewage sludge and soil M1, M2, or M3 at the beginning of experiment (0), and after 9 weeks after addition of earthworms (Ef, Ea) (a); differences in selected metal concentrations in earthworm bodies, baseline correspond to the metal content in non-exposed earthworms form breeding medium (b), and body accumulation factorsl (c) between M1, M2, and M3 mixtures after 9 weeks of exposure. Results shown as means ± SE. n = 3 Means not sharing the same latter are statistically different

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Fig 4
figure 4

Correlation between substrate and tissue metal content for selected metals

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Bioaccumulation factors

In both species, bioaccumulation factors (BAFs) were arranged as follows: Cd > Zn > Cu > Ni > Pb. BAF decreases significantly with increasing soil contamination for Cd, Zn, Cu, and Ni. Pb accumulation was higher in M2 mixture than in M1 or M3 mixtures (Fig. 3c).

Discussion

The main aim of this paper was to study the possible accumulation of MTEs in two earthworm species during vermicomposting of sewage sludge. Additionally, changes in one of immune system parameters, namely riboflavin, allowed us to assess the influence of pollutants present in sewage sludge on immune system of earthworms.

Animals used in this experiment come from genetically defined breeding cultures from Lille 1 University (Rorat et al. 2014). In the previous study (Rorat et al. 2013), we indicated that an addition of 25 % of sewage sludge to commercial soil was an optimum according to the influence of contaminants on the immune system of D. veneta earthworms. In the contrary, 50 % addition of sewage sludge strongly affected their immune cells and caused inhibition of fecundity, which can lead to mortality and interrupt vermicomposting process. In this study, 25 % addition of each of selected sewage sludge constituted a good source of nutrition for both species. It is noteworthy that earthworms succeeded in keeping the energy balance throughout the experiment by modulating their energy expenditure priorities. Sharp increase in body weight of earthworms was followed by a slight decrease, probably as the result of the trade-off mechanism between weight gain and reproduction (Brulle et al. 2007; Spurgeon and Hopkin 1996). Indeed, after 6 and 9 weeks of experiments, the top layer of substratum was rich in cocoons, which confirms that high-energy cost of reproduction is probably compensated by reduction in other expensive processes. Likewise, riboflavin, as a chosen biomarker, revealed time-depended fluctuations in the immune-system activity. Probably, the early decrease in riboflavin content is a reflection of immune system mobilization. This substance is considered as chemoattractant for earthworms’ immune cells (Mazur et al. 2011) that facilitates encapsulation of foreign bodies. It was shown that riboflavin level diminishes in D. rubidus transferred from unpolluted to polluted conditions (Plytycz et al. 2010; Plytycz et al. 2009). Similar changes have been observed in control earthworms and earthworms exposed to sewage sludge. The observed reaction can be explained as a reaction due to medium changes but not a toxic effect suggesting that sewage sludge was a good source of nutrients for earthworms in this study.

One of the factors that influence immune system of earthworms is essential and non-essential metals which are present in the environment in important quantities due to human activities. Living organisms that inhabit soil interact with metals and may accumulate them, increasing the risk of its incorporation to higher trophic levels (Fritsch et al. 2010).

In this study, bioaccumulation factors (BAF) were highest in less-polluted mixtures, what stays in agreement with other experimental results (Dai 2004; Domínguez-Crespo et al. 2011; Maboeta and Rensburg 2003; Neuhauser et al. 1994). Neuhauser et al. (1994) explained that fact by a higher elimination rate of metals by earthworms living in strongly contaminated soil. However, detoxification processes may be different from one metal to another. For example, it has been demonstrated that E. fetida was able to eliminate Pb but not Cd when exposed to contaminated soils (Bernard et al. 2010). The lower observed BAF could also be due to the low bioavailability of the metal in the studied mixture (Dai 2004). In Liu et al. (2005), authors pointed that while BAF is lower than 1, earthworms can only absorb, but not accumulate, metals, and they have shown that BAF gradually decreases with the increase of metals presence in soil. However, this phenomenon remains to be investigated. The recorded decrease in Cd concentration in SM1 mixture is most likely due to its observed accumulation in earthworms’ body. Spurgeon and Hopkin (1999) reported two different trends for essential and non-essential MTEs, concerning uptake and excretion kinetics (Spurgeon and Hopkin 1999). These authors described a fast initial uptake followed by equilibrium after few days for Cu and Zn (essential), and continuously increasing uptake and a very low excretion for Cd and Pb (non-essential). This fact and the probable reduction in the mass of the mixture due to the biodegradation process can explain the recorded increase of Zn content in the mixture. In the contrary, other researchers have shown an important decrease in Zn and Cu contamination while sewage sludge/cow dung mixtures were being vermicomposted using E. fetida during 75 days (Gogoi et al. 2015). Authors of that study have shown an important relation of pH and metal adsorption, with an optimum at <6 for Cu and 6 for Zn.

The highest Pb body content for earthworms introduced in M2 mixture is most likely due to a higher bioavailability in this mixture. This may be caused by the chemical properties of the soil which surely influences the bioaccumulation process in earthworm tissues, what is confirmed by the low correlation between total Pb soil content and the BAF (Domínguez-Crespo et al. 2011).

After 9-week experiment, decrease in pH was observed in all analyzed samples. The ideal range during vermicomposting is 6.8–7.4 (Manna et al. 2003) and is crucial for bioabsorption process and bioavailability of heavy metals. According to Elvira et al. (1998), decrease in pH may be caused by production of CO2 while metabolic activity of earthworms and bacteria. This decrease is also considered to be a result of mineralization of nitrogen and phosphorus that form nitrites/nitrates and orthophosphates and decomposition of organic matter into organic acids (Ndegwa and Thompson 2000; Yadav and Garg 2009). Similarly, total organic carbon (TOC) decreases as an effect of forming CO2 by earthworms and bacteria (Prakash and Karmegam 2010). Moreover, both mentioned organism groups use carbon as a source of energy and nitrogen as cell components. Decrease in C/N ratio and volatile solids is explained by decomposition of organic matter (Hait and Tare 2011).

Results obtained in this study confirmed the effectiveness of vermicomposting process using three different sewage sludge varying in MTE’s contents. Applied technique has led to valorization of sewage sludge, by changing some of their physical and chemical properties, but future studies are required to assess the environmental risk of using the obtained product as a fertilizer. Both species revealed similar metal accumulation tendency, but E. andrei has higher capabilities to accumulate some of metals. BAF’s order (Cd > Zn > Cu > Ni > Pb) stays in agreement with previous researches employing Eisenia sp. Nevertheless, different earthworms’ species may have different pathways of heavy metals detoxification. The BAF’s values were classified in the order Cd > Ni > Cu > Co > Cr > Zn when using Eudrilus eugeniae species to decompose municipal solid waste (Soobhany et al. 2015). Contaminants present in sewage sludge did not affect earthworms’ immune system, but future analysis should be performed to explain the mechanisms of accumulation and detoxification of particular metal fractions by earthworms and to assess the risk of possible incorporation of some MTE’s in higher trophic chains.