Bioresource Technology 146 (2013) 649–655

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Enhancement stabilization of heavy metals (Zn, Pb, Cr and Cu) duringvermifiltration of liquid-state sludge

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.07.144

⇑ Corresponding author. Tel./fax: +86 21 65984275.E-mail addresses: xmy5000@163.com, xingmeiyan@tongji.edu.cn (M. Xing).

Jian Yang, Chunhui Zhao, Meiyan Xing ⇑, Yanan LinKey Laboratory of Yangtze River Water Environment, Ministry of Education, State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Scienceand Engineering, Tongji University, Shanghai 200092, China

h i g h l i g h t s

� Stabilization of heavy metals in sludge treated by vermifilter has not been reported.� Variation of heavy metal concentrations was mainly due to organics degradation.� Heavy metals were transformed into stable fractions after vermifilter treatment.� Heavy metal accumulation by earthworms mainly depended on their chemical speciation.� Vermifiltration technology relieved the potential risk of heavy metals in sludge.

a r t i c l e i n f o

Article history:Received 8 May 2013Received in revised form 17 July 2013Accepted 20 July 2013Available online 6 August 2013

Keywords:EarthwormSewage sludgeVermifiltrationStabilizationHeavy metal

a b s t r a c t

This paper illustrated the potential effect of earthworms on heavy metal stabilization after vermifiltrationof liquid-state sludge. Significant enhancement of organics degradation in sludge caused an increase ofheavy metal concentrations in VF effluent sludge. However, the analysis of heavy metal chemical speci-ation indicated earthworms made unstable fractions of heavy metals transformed into stable fractions.Further investigation using principal component analysis revealed that transformations of heavy metalfractions were mainly due to the changes in sludge physico-chemical properties of pH, soluble chemicaloxygen demand and available phosphorus. The bioassay of earthworms indicated that only zinc wasaccumulated by earthworms because the unstable fraction was its main chemical speciation. Further-more, risk analysis demonstrated that earthworm activities weakened heavy metal risk due to the forma-tion of stable fractions although their total concentrations increased. These results indicated thatearthworms in vermifilter had a positive role in stabilizing heavy metals in sewage sludge.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

With the rapid urbanization in the last decades, much moremunicipal wastewater treatment plants (WWTPs) have been builtin China. Accordingly, large amounts of sewage sludge are pro-duced in WWTPs. The sewage sludge contains a variety of pollu-tants, such as biodegradable organic matter, heavy metals andpathogens, and the arbitrary discharge of the sludge would bringheavy pollution to the environment (Lasheen and Ammar, 2009).Specially, the heavy metals in sludge have drawn more and moreattentions because it can be accumulated along the food chainsand create potential risks to animals and humans. Total heavy me-tal concentration is an important indicator for their potential riskson the environment. However, the chemical speciation of heavymetals involves different fractions, and each fraction has dissimilar

potential impact on the environment. Thus, the specific chemicalspeciation of heavy metals is another key factor in determiningtheir eco-toxicity (Flyhammar, 1998). For the purpose of relievingthe potential risk of heavy metals, researchers are searching foran efficacious technique, which could realize the synchronousenhancement stabilization of heavy metals and degradation of mu-nicipal sewage sludge (Peruzzi et al., 2011).

The inoculation of earthworms in conventional biofilter (BF),termed vermifilter (VF), has been widely used to treat municipalsewage (Tomar and Suthar, 2011; Wang et al., 2011). Also, recentstudies found that VF technology was feasible to reduce and stabi-lize liquid-state sewage sludge under optimal conditions. Xinget al. (2012) found that the average volatile suspended solids(VSS) reduction of sludge treated by VF was 16.6% higher than thatby BF at the organic load of 1.12 kg-VSS/(m3d). Liu et al. (2012) re-ported that sludge reduction (VSS reduction) increased to 43.17%after VF treatment, which could achieve the level of 40% sludge sta-bilization for anaerobic and aerobic digestion (SEPA, 2002a). The

650 J. Yang et al. / Bioresource Technology 146 (2013) 649–655

capacity of VF technology to treat sewage sludge can be attributedto the earthworm ingestion of sludge and the interactions of earth-worm-microorganism in the VF reactor (Xing et al., 2012). For in-stance, cooperating with microbes, earthworms could increasethe degradation of organics, circulation of carbon, nitrogen andphosphorus, and enhance the sludge fertility (Liu et al., 2005).

Recently, studies investigating the potential effect of earth-worm activities on the availability of heavy metals have been con-ducted. For example, Spurgeon et al. (1994) reported thatearthworm activities made more Cu contained in soil was boundto the organics and sulfide, while less to the carbonates, and thebioavailability of Cu was consequently decreased. Liu et al.(2005) also found the bioavailability of Cd was reduced by earth-worms during vermicomposting process, and the change of the soilcharacteristics was its main reason. These studies indicated thatearthworm activities could change the availability of metals inmedium (soil, compost sludge) by changing its characteristics orheavy metal speciation. Nevertheless, given the pressure of excesssludge treatment, previous studies have primarily focused on theapplication of VF technology to enhance sludge degradation effi-ciency. There were few studies concern about the change andtransformation of heavy metals during vermifiltration of liquid-state sludge.

The aims of the study were to investigate the effect of earth-worms on heavy metal (Zn, Pb, Cr and Cu) stabilization during ver-mifiltration of liquid-state sludge. The sequential extractionprocedure was employed to fractionize the chemical speciationof heavy metals in sewage sludge and to explore the transforma-tion of these fractions. Additionally, principal component analysis(PCA) was used to establish a relationship between the specificchemical speciation of heavy metal and the physico-chemicalproperties of sludge. A comprehensive environmental risk analysisof heavy metals for the effluent sludge was also studied.

2. Methods

2.1. Vermifilter setup and operation

Two sets (each has three parallel reactors) of cylindrical filterswere set up, one set was the vermifilters (Fig. 1) with an initialearthworm density of 32 g/L (fresh weigh basis) as suggested byZhao et al. (2010), while the conventional biofilters (BF) withoutearthworms were used as the control. Each filter (diameter of30 cm and depth of 90 cm, made of perspex) had a work volumeof 49.5 L and packed with ceramsites (10–13 mm in diameter). Alayer of plastic fiber was placed on the top of the filter bed to avoidthe direct hydraulic influence on the earthworms and ensure aneven influent sludge distribution. The earthworms, Eisenia fetida,used in this study were purchased from a farm in Yancheng city,China. The influent sludge was withdrawn from the secondary sed-imentation tank of a municipal WWTP in Shanghai, China. Thehydraulic load of the two sets of filters was kept at 4 m/d, andthe organic load of the influent sludge was at the range of 1.38–1.51 kg-VSS/(m3d). After passing through the filter bed, the sludgeentered into a sedimentation tank. These filters were operated con-tinuously for 8 months to investigate their performances on heavymetal stabilization after about 30 days of acclimation.

2.2. Sampling and physico-chemical analysis

The sludge samples and earthworm casts (EC) were sampled inthe middle of each month during the experiment period. The influ-ent sludge (IS), the BF effluent sludge (BES) and the VF effluentsludge (VES) were respectively collected and centrifuged at6000 rpm for 15 min, and then the residues were freeze-dried. Cer-

tain amounts of healthy earthworms were randomly selected fromthe VF reactor and kept in dark for 24 h to collect enough EC forfurther analysis. Samples of about 50 g earthworms, purchasedfrom the farm and the final earthworms survived in the VF reactor,were respectively kept in dark for 24 h to empty their gut, and thenusing deionized water to clear their excrement prior to freeze-dried. All samples were grounded to powder, and then sievedthrough 0.15 mm mesh before further analysis.

The suspended solids (SS) and VSS of the IS, BES and VES weredetermined according to standard methods (SEPA, 2002b). Solublechemical oxygen demand (SCOD) was measured using a NOVA60COD meter (Merck, Germany). Available phosphorus (AP) was ex-tracted by sodium bicarbonate and colorimetrically measured withthe molybdate acid procedure. The pH value was measured by pHmeter (WTW, Germany).

2.3. Determination of heavy metal concentrations

In order to measure the heavy metal concentrations of the IS,BES, VES, EC, and earthworm tissues, the samples were digestedprior to heavy metal concentration determination according tothe reference (Nemati et al., 2010). Freeze-dried samples werefirstly weighed into each microwave TFM vessel, and then a min-eral acid mixture of 6 ml HNO3, 2 ml HCl and 2 ml HF were added.These samples were digested in a microwave manufacturer (Mile-stone, Italy) with the procedure consisted of a 10 min gradual tem-perature increase to 200 �C (1000 W, 106 Pa), a 15 min step of200 �C and a ventilated cooling stage. After cooling down to roomtemperature, the digested samples were heated on a hot plate(120 �C) until they became near dry. These dry samples were fixedinto the flask with deionized water, and then filtered through a0.45 lm mixed cellulose ester membrane prior to inductively cou-pled plasma optical emission spectrometry (ICP-OES, PerkinElmerOptima 2100 DV, USA) analysis.

2.4. Sequential extraction of heavy metals

The chemical speciation of heavy metals is usually divided intothe following five fractions: the exchangeable fraction (F1), the car-bonate binding fraction (F2), Fe–Mn oxides binding fraction (F3),organic and sulfide binding fraction (F4) and the residual fraction(F5) (Peruzzi et al., 2011; Tessier et al., 1979). A five-step sequen-tial extraction procedure was used to determine the portion ofeach fraction of the investigated heavy metals in the IS, BES, VESand EC. The detailed sequential extraction procedure was listedin Table 1. All reagents were ultrahigh purity, and all the laboratoryglassware and polyethylene bottles were pre-cleaned by 4–10%chemical pure HNO3 and rinsed with deionized water prior to eachextraction procedure. Each fraction concentration of these heavymetals was also determined by ICP-OES.

2.5. Statistical analysis

Principal component analysis (PCA), uses an orthogonal trans-formation to convert a set of observations of possibly correlatedvariables into a set of values of linearly uncorrelated variables,could simplify the analysis and visualization of multidimensionaldata sets and reveal important variables that are difficult to dis-cover (Pardo et al., 2004; Raychaudhuri et al., 2000). In this paper,PCA was used to investigate the correlations between the chemicalspeciation (F1–F5) of heavy metals and physico-chemical proper-ties (pH, SCOD and AP) of sludge.

Analysis of variance (ANOVA) was applied to evaluate the sig-nificance of results and p < 0.05 was considered to be statisticallysignificant. Statistical analysis was performed using the softwareSPSS 17.0.

Fig. 1. Schematic diagram of the vermifilter (with earthworms).

Table 1Reagents and tessier sequential extraction procedure for each fraction of heavymetals.

Fraction Reagents and conditions*

Exchangeable (F1) 16 ml 1 M MgCl2, pH 7.0, 25 �C, shaking for 2 hCarbonates binding

(F2)16 ml 1 M NaAc, adjusted to pH 5.0 with HAc, 25 �C,shaking for 2 h

Fe–Mn oxide binding(F3)

16 ml 0.04 M NH4OCl in 25% HAc, 85 �C in water bath,shaking for 2 h

Organic and sulfidebinding (F4)

8 ml 30% H2O2, adjusted to pH 2.0 with HNO3, shakingfor 5 h, then adding 10 ml 3.2 M NH4Ac in 20% HNO3,shaking for 0.5 h

Residual (F5) Performed with microwave as described of totalheavy metal concentrations digestion

* The extract was centrifuged at 9000 rpm for 15 min. The supernatant presentseach fraction, and the residue is used in the next extraction procedure.

Table 2Treatment performances of sludge by BF and VF, and the corresponding physico-chemical properties of the IS, BES and VES.

Samples VSSreduction(%)

VSS/SS pH SCOD (mg/L)

AP (mg/L)

IS 0.71 ± 0.03 7.21 ± 0.03 38.4 ± 7.4 3.53 ± 0.1BES 34.3 ± 3.0 0.67 ± 0.03 6.84 ± 0.01 31.3 ± 4.1 3.83 ± 0.4VES 48.5 ± 3.9 0.62 ± 0.03 6.57 ± 0.01 56.6 ± 10.0 5.31 ± 0.3

J. Yang et al. / Bioresource Technology 146 (2013) 649–655 651

3. Results and discussion

3.1. Sludge stabilization

Both the BF and VF reactors were operated steadily withoutclogging during the experimental period, and their performanceson sludge reduction were showed in Table 2. It was observed thatthe average VSS reduction increased to 48.5 ± 3.9% after VF treat-ment, while it was 34.3 ± 3.0% after BF treatment. It means thatsludge reduction was enhanced by 14.2% owing to the presenceof earthworms in the VF reactor. Nevertheless, such sludge reduc-tion is higher than that previously reported for VF systems treatingsewage sludge (Liu et al., 2012). This might be due to the fact thatthe larger size of reactor in this study improved the treating effi-ciency. Moreover, the degree of the sludge stabilization also couldbe assessed by the ratio of VSS/SS (Zhao et al., 2010), and a lowerVSS/SS ratio represents a higher organics degradation. As shownin Table 2, the average VSS/SS ratio was decreased to 0.62 in theVES from 0.71 in the IS. Although a decrease of VSS/SS ratio inthe BES was observed, it was not as significant as that in VES. Thus,these results indicated that earthworm activities promoted the

degradation of sewage sludge. Other researchers reported the sim-ilar results and suggested that the ingestion of earthworm and theearthworm-microorganism interactions played the important rolein sludge stabilization in the VF (Zhao et al., 2010). The physico-chemical properties of sludge, such as pH, SCOD and AP, were alsoobserved to be change after the BF and VF treatments (Table 2), andit will be discussed in the following text.

3.2. Heavy metal concentrations

The above study has shown that the earthworms promoted theorganics degradation in sewage sludge, however, the variations ofheavy metals in sludge before and after VF treatment were still un-known. Table 3 illustrates the total concentrations of the commonheavy metals (Zn, Pb, Cr and Cu) in the IS, BES and VES. Their cor-responding discharge standards (GB18918-2002) for land applica-tion of the excess sludge are also given in Table 3 (SEPA, 2002a).

It can be seen from Table 3 that Zn was the predominant metal(>1300 mg/kg-SS) in the IS, which accounted for more than 70% oftotal heavy metal content. After VF treatment, the concentration ofZn in the VES significantly increased to 1861 mg/kg-SS from1316 mg/kg-SS in the IS, whereas it merely increased to1745 mg/kg-SS after BF treatment. Similarly, the concentrationsof Pb, Cr and Cu were observed to increase slightly. It was obviousthat the degradation of the organics was the main reason for theincrease concentrations of heavy metal, because these heavy met-als were non-degradable. The higher organics degradation by VF

Table 3Total concentrations of heavy metals (Zn, Pb, Cr and Cu) in the IS, BES and VES andtheir permitted values in agriculture application.*

Heavy metals (mg/kg-SS)

Zn Pb Cr Cu

IS 1315.8 ± 59.4 130.2 ± 4.7 108.3 ± 6.5 311.0 ± 24.8BES 1745.0 ± 64.6 180.8 ± 7.1 149.2 ± 9.8 435.3 ± 23.2VES 1861.3 ± 94.6 199.4 ± 8.3 165.3 ± 9.9 481.2 ± 19.8

Threshold valueSoil pH < 6.5 2000 300 600 800Soil pH P 6.5 3000 1000 1000 1500

* The data reported are the averages ± their standard deviations for 8 samples.

652 J. Yang et al. / Bioresource Technology 146 (2013) 649–655

treatment than that by BF resulted in the higher concentrations ofheavy metals in the VES than BES. However, the decreases of heavymetal concentrations were reported during vermicomposting pro-cess, because metallic cations were observed to be release into theleachate (Suthar, 2009). In this study, no obvious variance of heavymetal concentrations were detected in the supernatants of the BESand VES, indicating the release of heavy metals to the supernatantscould be ignored.

3.3. Heavy metal chemical speciation

The increases of heavy metal concentrations in the BES and VES,especially for Zn, seem to heighten their potential risk to the envi-ronment; however, Table 3 showed that all heavy metal concentra-tions (Zn, Pb, Cr and Cu) obtained in both the BES and VES werewell below the threshold value requested in China for land applica-tion (SEPA, 2002a). Besides total concentrations of heavy metals,the chemical speciation is also another important factor for assess-ing their environmental risk (Zheng et al., 2007). Therefore, thespecific chemical speciation of heavy metal (precipitated with min-erals, complexed with organic ligands and so on) was investigatedin the following text to comprehensively evaluate the potential riskof these heavy metals included in the VES.

3.3.1. Transformation of heavy metal fractionsThe specific chemical speciation of heavy metals was fraction-

ized into exchangeable fraction (F1), carbonate precipitated frac-tion (F2), Fe–Mn oxides binding fraction occludes in amorphous(F3), organic and sulfide binding fraction (F4), and residual fraction(F5) (Tessier et al., 1979). Such specific classification of heavy met-als is critical for researchers to accurately assess their environmen-tal risk (Li et al., 1995; Thornton et al., 2008). Fig. 2 represented thepercentage of each fraction (F1–F5) to the total heavy metals (Zn,Pb, Cr and Cu) in the IS, BES, VES and EC. For all the heavy metals,recovery rates between the sum of the five fractional metal con-tents and total contents were generally acceptable (92.2–110.3%),indicating that the determination of the heavy metal chemical spe-ciation was reliable.

It can be seen from Fig. 2 that over 70% of Zn in the IS distrib-uted in F3, indicating Zn of the influent sludge was primarily oc-cluded in amorphous and weakly crystalline iron and manganeseoxides. After BF and VF treatment, F3 of Zn in the BES and VES de-creased from 73.5% (IS) to 61.2% and 41.4%, respectively, and the F4percentage of Zn in the VES was significantly increased while thatin the BES was slightly increased. A higher F5 percentage of Zn inthe EC was observed, thus led to its higher percentage in theVES. As regards the other three investigated heavy metals (Pb, Crand Cu), they predominantly distributed in the last two fractions(F4 and F5), and the sum of F4 and F5 in the IS accounted for71.8%, 71.3% and 85.3%, respectively (Fig. 2). The specific chemicalspeciation variations of Pb and Cr were nearly similar with those ofZn before and after treatments; the percentages of F1, F4, and F5

were increased, whereas F2 and F3 were observed to be decreased.However, different variations were seen in the chemical speciationof Cu; F2 was decreased, and F4 was increased, while the otherthree fractions (F1, F3, and F5) changed slightly (p > 0.05).

3.3.2. Mechanism of transformations among heavy metal fractionsThe above study has shown that after filtration (BF and VF)

treatment both the physico-chemical properties of sludge andthe specific fraction of heavy metals in sludge varied greatly. More-over, the specific fraction of heavy metals in sludge was observedto be depended on the pH, number and accessibility of adsorptionsites and metal affinity for solid components (Alvarez et al., 2002).It is speculated that some relationship between them could beestablished. Thus, PCA was used in this study to interpret the da-tum such as heavy metal fractions (F1–F5) and physico-chemicalproperties (pH, SCOD and AP) of the IS, BES and VES correlations.The results of PCA revealed that the presence of two principal com-ponents (PCs) explained a total variance of 77.3% (Fig. 3a). Themost significant PCs are the first PC (PC1) which explained 62.6%of data variability and the second PC (PC2) which had an explainvariance of 14.7%.

As shown in Fig. 3a, F1 of the four heavy metals located in thefirst, second and fourth quadrant, and their irregular distributionon the biplot of loadings of PCA might be due to the low percent-ages of heavy metal fractions (less than 5%) in the sludge samples.F2 of Pb, Cr, Cu, F3 of Zn, Pb, and pH clustered in the left of PC1,indicating a correlation between those fractions of heavy metalsand pH. In addition, the data in Table 2 has shown that the pH ofthe BES and VES was decreased from 7.21 (IS) to 6.84 and 6.57,respectively, which was due to the bioconversion of the organicsinto organic acids, and the transformation of organic nitrogenand phosphorus to nitrites/nitrates and orthophosphates duringsludge degradation (Ndegwa et al., 2000). Under such weak acidconditions, percentages of F2 and F3 in heavy metals were ob-served to be decreased (Fig. 2), suggesting they were redistributedinto other fractions. The lower F3 percentage of heavy metals inVES than BES was due to the effect of earthworms, in view of thesignificant decrease of F3 percentage in the EC. Previous studyhas found that the pH of EC would be decreased as long as foodtransit through the earthworm guts (Li et al., 2009).

Previous studies had found that earthworms in the VF were ableto transform organic materials from insoluble to soluble forms andcaused the increase of SCOD in the VES. The data in Fig. 3a showedthat F4 of the four heavy metals and SCOD of sludge located in thefourth quadrant, and the dissolved organics could complex withsoluble metal ions into stable fraction (F4) (Antoniadis and Allo-way, 2002; Ashworth and Alloway, 2004), indicating that therewas a moderate positive loadings between F4 and SCOD on PC1.These were consistent with the above observation that F4 of Zn,Pb, Cr, Cu were significantly increased after VF treatment (Fig. 2),while much more organics produced in the reactor of VF than BFdue to the earthworm activities (Table 2). Furthermore, Xinget al. (2011) repored that the interactions of earthworms andmicroorganisms in the VF could lead to rapid humification and pro-mote the formation of –COOH groups. Moreover, researchers havefound the formation of F4 was resulted from –COOH group of or-ganic acids complexation with metal ions (Cheng and Wong,2002; Ndegwa et al., 2000). Similar changes were seen betweenF5 of Zn, Pb, Cr and AP of sludge in Fig. 3a, and they were stronglypositive with PC2 (distributed in the first quadrant). Heavy metalsdistributed in F5 were mainly immobilized by insoluble salt suchas phosphates. This was also consistent with the observed increaseof F5 in the VES when more AP (5.31 mg/L) was released after VFtreatment compared with AP concentration in the BES (3.83 mg/L)(Table 2). The gut transit of earthworms was the main reason forthe higher AP concentration caused by earthworms (Buck et al.,

0

10

20

30

40

50

60

70

80

Chemical speciation of heavy metals

Perc

enta

ge o

f ea

ch f

ract

ion

to th

e to

tal (

%)

IS BES VES EC

Zn

0

10

20

30

40

50Pb

F1 F2 F3 F4 F50

10

20

30

40

50

Cr

F1 F2 F3 F4 F50

10

20

30

40

50

Cu

Fig. 2. Comparisons of the chemical speciation (F1, F2, F3, F4 and F5) of heavy metals (Zn, Pb, Cr, Cu) in the IS, BES, VES and EC. Error bars represent standard deviations for 8samples.

Fig. 3. Principal component analysis (PCA) of heavy metal fractions (F1, F2, F3, F4 and F5) and the physico-chemical properties (pH, SCOD and AP) of the IS, BES and VES.(a) Represents of PC1 versus PC2 loadings; (b) Plots of PC1 versus PC2 scores.

J. Yang et al. / Bioresource Technology 146 (2013) 649–655 653

1999). It further demonstrated that the earthworm activities couldimmobilize metals and transform them into F5 (Walker et al.,2003).

Loading plots were used to depict a group of original variablesbased on PCs, and score plots were used to show the projectionof original data onto new biplot according to the loadings (Felip-e-Sotelo et al., 2008). It was found from score plots (Fig. 3b) thatsamples (IS, BES and VES) were clustered in different groups onthe biplot based on the total variance. Both the IS and BES distrib-uted in the left section of the biplot (high negative scores of PC1),mainly associated with F2 and F3 of heavy metals. In contrast, VES

located in the right quadrant (positive loadings on PC1), indicatinga correlative relationship with F4 and F5. This led to the conclusionthat earthworms in the VF reactor played an important role inredistributing metal chemical speciation into F4 and F5.

3.4. Bioaccumulation of heavy metals by earthworms

As we known, the ingestion effect of earthworms was the mainreason for sludge reduction (Xing et al., 2012), and it might causethe bioaccumulation of heavy metals in their tissues. Thus, the hea-vy metal concentrations of both the initial purchased earthworms

0

20

40

60

80

100

**

**

*

VESBESIS

Perc

enta

ge (

%)

Unstable fraction Stable fraction

VESBESISPb

VESBESISCr

VESBESISCuZn

*

Fig. 4. Comparisons of the unstable (the sum of F1, F2 and F3) and stable (the sumof F4 and F5) fractions of heavy metals in the IS, BES and VES. Asterisks indicatestatistical differences (p < 0.05) from the IS.

654 J. Yang et al. / Bioresource Technology 146 (2013) 649–655

and the earthworms collected from the VF reactor were measuredand listed in Table 4. The concentration of Cr was not detected inthe two types of earthworms (the initial and final earthwormsrespectively withdrawn from farm and the VF reactor), whichmight ascribe to the species-specific metal physiology of earth-worms (Wang et al., 2013). Compared with the initial earthworms,a statistical increase of Zn concentration (p < 0.05) was observed inearthworm tissues from the VF reactor; however, the variations ofconcentrations between Pb and Cu was not significant (p > 0.05).The chemical speciation of heavy metals could affect their inges-tion by earthworms. For instance, F3 of heavy metals is readily tobe assimilated by plants and animals and finally accumulates inthe food chain (Hare et al., 2003). Thus, the increase of Zn concen-tration in earthworm tissues was consistent with the above obser-vation that F3 was the main chemical speciation in the IS. Withregard to Pb and Cu, they mainly distributed in F4 and F5, whichwas hardly accumulated in food chain. Therefore, the enrichmentsof Pb and Cu in earthworm tissues were insignificant (p > 0.05).

3.5. Risk analysis of heavy metals

It is well known that both total concentrations and chemicalspeciation of heavy metals in excess sludge are the key factorsfor assessing their potential risk to the environment. Table 3 hasshown that the concentration of each heavy metal was still far be-low the discharge standards of pollutants for municipal wastewa-ter treatment plant; however, the investigation of heavy metalchemical speciation (F1–F5) in sludge before and after filter treat-ment revealed that those fractions transformed. Different fractionsof heavy metals behave dissimilar potential risk to the environ-ment. For instance, F1 presents readily bioavailable proportion ofmetal content in environment and it can be stated that this fractionis toxic (Yuan et al., 2011); F2 and F3 are sensitive to pH and easilytransformed into F1 under weak acid condition (Li et al., 2009; Tan-dy et al., 2009). Thus, these three fractions (F1, F2 and F3) could beidentified as unstable and direct toxic. F4 is considered to be po-tential effect and less toxic to the environment, because it will bereleased into environment only under the conditions of strong acidand oxidizing agent (Chen et al., 2008); F5 is regarded as themostly stable fraction and no eco-toxic to the environment (Walk-er et al., 2003). The two fractions (F4 and F5) are considered to bethe stable and no eco-toxic. Therefore, the comparisons of theunstable (the sum of F1, F2 and F3) and stable (the sum of F4and F5) fractions of heavy metals in the IS, BES and VES were inves-tigated and showed in Fig. 4.

As seen in Fig. 4, the stable fractions of heavy metals (Zn, Pb,and Cr) in sludge were increased after filter treatment. Especiallyfor Zn, the percentage of unstable fraction was the highest(79.0%) among the four heavy metals in the IS, which showing ahigh bioavailability and eco-toxicity to the environment; however,after VF treatment, the unstable percentage decreased by 32.3%,indicating the potential toxicity of Zn in the VES were decreased.

Table 4Total heavy metal concentrations in the earthworm tissue respectively withdrawnfrom the farm and VF reactor.a

Heavy metals (mg/kg-tissue)

Zn Pb Cr Cu

Initial earthwormsb 153.7 ± 5.29 4.7 ± 0.28 – 20.1 ± 1.16Final earthwormsc 166.4 ± 6.15 4.5 ± 0.29 – 19.7 ± 1.81

– Represents Cr was not detected.a The data reported are the averages and their standard deviations for 3 samples.b Indicates the initial earthworms purchased from the farm.c Indicates the earthworms collected from the VF reactor at the end of the

experiment.

Besides, only a decrease of 7.3% was observed in the BES. These re-sults further supported that earthworm activities could enhancethe stabilization of heavy metals during sludge treatment. Similarvariations of the unstable fractions for the other two heavy metals(Pb and Cr) were observed during sludge treatment. As for Cu,85.3% of the chemical speciation distributed in F4 and F5 in theIS, showing a lower eco-toxicity than the other three heavy metals,therefore the increase of stable Cu was insignificant. These resultsindicated that earthworm activities played a positive role in reliev-ing the potential risk of heavy metals by transforming the mobileand available fractions into low or no eco-toxic fractions.

4. Conclusions

The inoculation of earthworms to biofilters enhanced organicdegradation in sewage sludge, which led to the increase of heavymetal concentrations. Analysis of heavy metal chemical speciationshowed that some unstable fractions were transformed into stablefractions due to earthworm activities. Further analysis indicatedtransformations among these fractions were due to the changesof sludge physico-chemical properties. The bioassay of earthwormdemonstrated that only Zn was accumulated, indicating theenrichment of heavy metals by earthworms depended on theirchemical speciation. At last, risk analysis further supported thatearthworms weakened environmental risk of heavy metals aftervermifiltration of sludge.

Acknowledgements

This work was partially supported by the National Natural Sci-ence Foundation of China (NSFC, No:51109161), the PhD ProgramsFoundation of Ministry of Education of China (20110072120029),the Fundamental Research Funds for The Central Universities(0400219187), the Open Analysis Fund for Large Apparatus andEquipments of Tongji University (No. 2012055), the National SparkProgram of China (2010GA680004). The authors also thank theanonymous reviewers for their helpful suggestions.

References

Alvarez, E.A., Mochon, M.C., Sanchez, J.C.J., Rodriguez, M.T., 2002. Heavy metalextractable forms in sludge from wastewater treatment plants. Chemosphere47 (7), 765–775.

Antoniadis, V., Alloway, B.J., 2002. The role of dissolved organic carbon in themobility of Cd, Ni and Zn in sewage sludge-amended soils. Environ. Pollut. 117(3), 515–521.

J. Yang et al. / Bioresource Technology 146 (2013) 649–655 655

Ashworth, D.J., Alloway, B.J., 2004. Soil mobility of sewage sludge-derived dissolvedorganic matter, copper, nickel and zinc. Environ. Pollut. 127 (1), 137–144.

Buck, C., Langmaack, M., Schrader, S., 1999. Nutrient content of earthworm castsinfluenced by different mulch types. Eur. J. Soil Biol. 35 (1), 23–30.

Chen, M., Li, X., Yang, Q., Zeng, G., Zhang, Y., Liao, D., Liu, J., Hu, J., Guo, L., 2008. Totalconcentrations and speciation of heavy metals in municipal sludge fromChangsha, Zhuzhou and Xiangtan in middle-south region of China. J. Hazard.Mater. 160 (2–3), 324–329.

Cheng, J., Wong, M., 2002. Effects of earthworms on Zn fractionation in soils. Biol.Fertil. Soil 36 (1), 72–78.

Felipe-Sotelo, M., Tauler, R., Vives, I., Grimalt, J.O., 2008. Assessment of theenvironmental and physiological processes determining the accumulation oforganochlorine compounds in European mountain lake fish throughmultivariate analysis (PCA and PLS). Sci. Total. Environ. 404 (1), 148–161.

Flyhammar, P., 1998. Use of sequential extraction on anaerobically degradedmunicipal solid waste. Sci. Total. Environ. 212 (2–3), 203–215.

Hare, L., Tessier, A., Borgmann, U., 2003. Metal sources for freshwater invertebrates:pertinence for risk assessment. Hum. Ecol. Risk Assess. 9 (4), 779–793.

Lasheen, M.R., Ammar, N.S., 2009. Assessment of metals speciation in sewage sludgeand stabilized sludge from different wastewater treatment plants, greater Cairo.Egypt. J. Hazard. Mater. 164 (2–3), 740–749.

Li, X., Coles, B.J., Ramsey, M.H., Thornton, I., 1995. Sequential extraction of soils formultielement analysis by Icp-Aes. Chem. Geol. 124 (1–2), 109–123.

Li, L., Wu, J., Tian, G., Xu, Z., 2009. Effect of the transit through the gut of earthworm(Eisenia fetida) on fractionation of Cu and Zn in pig manure. J. Hazard. Mater.167 (1–3), 634–640.

Liu, X., Hu, C., Zhang, S., 2005. Effects of earthworm activity on fertility and heavymetal bioavailability in sewage sludge. Environ. Int. 31 (6), 874–879.

Liu, J., Lu, Z., Yang, J., Xing, M., Yu, F., Guo, M., 2012. Effect of earthworms on theperformance and microbial communities of excess sludge treatment process invermifilter. Bioresour. Technol. 117, 214–221.

Ndegwa, P.M., Thompson, S.A., Das, K.C., 2000. Effects of stocking density andfeeding rate on vermicomposting of biosolids. Bioresour. Technol. 71 (1), 5–12.

Nemati, K., Abu Bakar, N.K., Bin Abas, M.R., Sobhanzadeh, E., Low, K.H., 2010.Comparative study on open system digestion and microwave assisted digestionmethods for metal determination in shrimp sludge compost. J. Hazard. Mater.182 (1–3), 453–459.

Pardo, R., Helena, B.A., Cazurro, C., Guerra, C., Deban, L., Guerra, C.M., Vega, M., 2004.Application of two- and three-way principal component analysis to theinterpretation of chemical fractionation results obtained by the use of theBCR procedure. Anal. Chim. Acta 523 (1), 125–132.

Peruzzi, E., Masciandaro, G., Macci, C., Doni, S., Ravelo, S.G.M., Peruzzi, P., Ceccanti,B., 2011. Heavy metal fractionation and organic matter stabilization in sewagesludge treatment wetlands. Ecol. Eng. 37 (5), 771–778.

Raychaudhuri, S., Stuart, J.M., Altman, R.B., 2000. Principal components analysis tosummarize microarray experiments: application to sporulation time series. Pac.Symp. Biocomput., 452–463, Hawaii, USA.

Spurgeon, D.J., Hopkin, S.P., Jones, D.T., 1994. Effects of cadmium, copper, lead andzinc on growth, reproduction and survival of the earthworm eisenia foetida

(savigny): assessing the environmental impact of point-source metalcontamination in terrestrial ecosystems. Environ. Pollut. 84 (2), 123–130.

State Environmental Protection Administration of China, 2002a. Discharge standardof pollutants for municipal wastewater (GB 18918–2002). China EnvironmentalScience Press, Beijing, China (in Chinese).

State Environment Protection Administration of China, 2002b. Standard methodsfor the examination of water and wastewater, fourth ed. China EnvironmentalScience Press, Beijing, China (in Chinese).

Suthar, S., 2009. Earthworm communities a bioindicator of arable land managementpractices: a case study in semiarid region of India. Ecol. Indic 9 (3), 588–594.

Tandy, S., Healey, J.R., Nason, M.A., Williamson, J.C., Jones, D.L., 2009. Heavy metalfractionation during the co-composting of biosolids, deinking paper fibre andgreen waste. Bioresour. Technol. 100 (18), 4220–4226.

Tessier, A., Campbell, P.G.C., Bisson, M., 1979. Sequential extraction procedure forthe speciation of particulate trace metals. Anal. Chem. 51 (7), 844–851.

Thornton, I., Farago, M.E., Thums, C.R., Parrish, R.R., McGill, R.A.R., Breward, N.,Fortey, N.J., Simpson, P., Young, S.D., Tye, A.M., Crout, N.M.J., Hough, R.L., Watt, J.,2008. Urban geochemistry: research strategies to assist risk assessment andremediation of brownfield sites in urban areas. Environ. Geochem. Health 30(6), 565–576.

Tomar, P., Suthar, S., 2011. Urban wastewater treatment using vermi-biofiltrationsystem. Desalination 282, 95–103.

Walker, D.J., Clemente, R., Roig, A., Bernal, M.P., 2003. The effects of soilamendments on heavy metal bioavailability in two contaminatedMediterranean soils. Environ. Pollut. 122 (2), 303–312.

Wang, L., Zheng, Z., Luo, X., Zhang, J., 2011. Performance and mechanisms of amicrobial-earthworm ecofilter for removing organic matter and nitrogen fromsynthetic domestic wastewater. J. Hazard. Mater. 195, 245–253.

Wang, L., Zheng, Z., Zhang, Y., Chao, J., Gao, Y., Luo, X., Zhang, J., 2013.Biostabilization enhancement of heavy metals during the vermiremediationof sewage sludge with passivant. J. Hazard. Mater. 244–245 (2013), 1–9.

Xing, M., Zhao, L., Yang, J., Huang, Z., Xu, Z., 2011. Distribution and transformation oforganic matter during liquid-state vermiconversion of activated sludge usingelemental analysis and spectroscopic evaluation. Environ. Eng. Sci. 28 (9), 619–626.

Xing, M., Li, X., Yang, J., Lv, B., Lu, Y., 2012. Performance and mechanism ofvermifiltration system for liquid-state sewage sludge treatment usingmolecular and stable isotopic techniques. Chem. Eng. J. 197, 143–150.

Yuan, X., Huang, H., Zeng, G., Li, H., Wang, J., Zhou, C., Zhu, H., Pei, X., Liu, Z., Liu, Z.,2011. Total concentrations and chemical speciation of heavy metals inliquefaction residues of sewage sludge. Bioresour. Technol. 102 (5), 4104–4110.

Zhao, L., Wang, Y., Yang, J., Xing, M., Li, X., Yi, D., Deng, D., 2010. Earthworm-microorganism interactions: a strategy to stabilize domestic wastewatersludge. Water Res. 44 (8), 2572–2582.

Zheng, G.D., Gao, D., Chen, T.B., Luo, W., 2007. Stabilization of nickel and chromiumin sewage sludge during aerobic composting. J. Hazard. Mater. 142 (1–2), 216–221.