Bioresource Technology 146 (2013) 649–655
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Enhancement stabilization of heavy metals (Zn, Pb, Cr and Cu) during vermiﬁltration of liquid-state sludge Jian Yang, Chunhui Zhao, Meiyan Xing ⇑, Yanan Lin Key Laboratory of Yangtze River Water Environment, Ministry of Education, State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and 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
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Article history: Received 8 May 2013 Received in revised form 17 July 2013 Accepted 20 July 2013 Available online 6 August 2013 Keywords: Earthworm Sewage sludge Vermiﬁltration Stabilization Heavy metal
a b s t r a c t This paper illustrated the potential effect of earthworms on heavy metal stabilization after vermiﬁltration of liquid-state sludge. Signiﬁcant enhancement of organics degradation in sludge caused an increase of heavy metal concentrations in VF efﬂuent sludge. However, the analysis of heavy metal chemical speciation indicated earthworms made unstable fractions of heavy metals transformed into stable fractions. Further investigation using principal component analysis revealed that transformations of heavy metal fractions were mainly due to the changes in sludge physico-chemical properties of pH, soluble chemical oxygen demand and available phosphorus. The bioassay of earthworms indicated that only zinc was accumulated by earthworms because the unstable fraction was its main chemical speciation. Furthermore, risk analysis demonstrated that earthworm activities weakened heavy metal risk due to the formation of stable fractions although their total concentrations increased. These results indicated that earthworms in vermiﬁlter had a positive role in stabilizing heavy metals in sewage sludge. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction With the rapid urbanization in the last decades, much more municipal wastewater treatment plants (WWTPs) have been built in China. Accordingly, large amounts of sewage sludge are produced in WWTPs. The sewage sludge contains a variety of pollutants, such as biodegradable organic matter, heavy metals and pathogens, and the arbitrary discharge of the sludge would bring heavy pollution to the environment (Lasheen and Ammar, 2009). Specially, the heavy metals in sludge have drawn more and more attentions because it can be accumulated along the food chains and create potential risks to animals and humans. Total heavy metal concentration is an important indicator for their potential risks on the environment. However, the chemical speciation of heavy metals involves different fractions, and each fraction has dissimilar ⇑ Corresponding author. Tel./fax: +86 21 65984275. E-mail addresses: [email protected]
, [email protected]
(M. Xing). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.144
potential impact on the environment. Thus, the speciﬁc chemical speciation of heavy metals is another key factor in determining their eco-toxicity (Flyhammar, 1998). For the purpose of relieving the potential risk of heavy metals, researchers are searching for an efﬁcacious technique, which could realize the synchronous enhancement stabilization of heavy metals and degradation of municipal sewage sludge (Peruzzi et al., 2011). The inoculation of earthworms in conventional bioﬁlter (BF), termed vermiﬁlter (VF), has been widely used to treat municipal sewage (Tomar and Suthar, 2011; Wang et al., 2011). Also, recent studies found that VF technology was feasible to reduce and stabilize liquid-state sewage sludge under optimal conditions. Xing et al. (2012) found that the average volatile suspended solids (VSS) reduction of sludge treated by VF was 16.6% higher than that by BF at the organic load of 1.12 kg-VSS/(m3d). Liu et al. (2012) reported that sludge reduction (VSS reduction) increased to 43.17% after VF treatment, which could achieve the level of 40% sludge stabilization for anaerobic and aerobic digestion (SEPA, 2002a). The
J. Yang et al. / Bioresource Technology 146 (2013) 649–655
capacity of VF technology to treat sewage sludge can be attributed to the earthworm ingestion of sludge and the interactions of earthworm-microorganism in the VF reactor (Xing et al., 2012). For instance, cooperating with microbes, earthworms could increase the degradation of organics, circulation of carbon, nitrogen and phosphorus, and enhance the sludge fertility (Liu et al., 2005). Recently, studies investigating the potential effect of earthworm activities on the availability of heavy metals have been conducted. For example, Spurgeon et al. (1994) reported that earthworm activities made more Cu contained in soil was bound to the organics and sulﬁde, while less to the carbonates, and the bioavailability of Cu was consequently decreased. Liu et al. (2005) also found the bioavailability of Cd was reduced by earthworms during vermicomposting process, and the change of the soil characteristics was its main reason. These studies indicated that earthworm activities could change the availability of metals in medium (soil, compost sludge) by changing its characteristics or heavy metal speciation. Nevertheless, given the pressure of excess sludge treatment, previous studies have primarily focused on the application of VF technology to enhance sludge degradation efﬁciency. There were few studies concern about the change and transformation of heavy metals during vermiﬁltration of liquidstate sludge. The aims of the study were to investigate the effect of earthworms on heavy metal (Zn, Pb, Cr and Cu) stabilization during vermiﬁltration of liquid-state sludge. The sequential extraction procedure was employed to fractionize the chemical speciation of heavy metals in sewage sludge and to explore the transformation of these fractions. Additionally, principal component analysis (PCA) was used to establish a relationship between the speciﬁc chemical speciation of heavy metal and the physico-chemical properties of sludge. A comprehensive environmental risk analysis of 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 ﬁlters were set up, one set was the vermiﬁlters (Fig. 1) with an initial earthworm density of 32 g/L (fresh weigh basis) as suggested by Zhao et al. (2010), while the conventional bioﬁlters (BF) without earthworms were used as the control. Each ﬁlter (diameter of 30 cm and depth of 90 cm, made of perspex) had a work volume of 49.5 L and packed with ceramsites (10–13 mm in diameter). A layer of plastic ﬁber was placed on the top of the ﬁlter bed to avoid the direct hydraulic inﬂuence on the earthworms and ensure an even 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 sedimentation tank of a municipal WWTP in Shanghai, China. The hydraulic load of the two sets of ﬁlters was kept at 4 m/d, and the 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 sludge entered into a sedimentation tank. These ﬁlters were operated continuously for 8 months to investigate their performances on heavy metal stabilization after about 30 days of acclimation. 2.2. Sampling and physico-chemical analysis The sludge samples and earthworm casts (EC) were sampled in the middle of each month during the experiment period. The inﬂuent sludge (IS), the BF efﬂuent sludge (BES) and the VF efﬂuent sludge (VES) were respectively collected and centrifuged at 6000 rpm for 15 min, and then the residues were freeze-dried. Cer-
tain amounts of healthy earthworms were randomly selected from the VF reactor and kept in dark for 24 h to collect enough EC for further analysis. Samples of about 50 g earthworms, purchased from the farm and the ﬁnal earthworms survived in the VF reactor, were respectively kept in dark for 24 h to empty their gut, and then using deionized water to clear their excrement prior to freezedried. All samples were grounded to powder, and then sieved through 0.15 mm mesh before further analysis. The suspended solids (SS) and VSS of the IS, BES and VES were determined according to standard methods (SEPA, 2002b). Soluble chemical oxygen demand (SCOD) was measured using a NOVA60 COD meter (Merck, Germany). Available phosphorus (AP) was extracted by sodium bicarbonate and colorimetrically measured with the molybdate acid procedure. The pH value was measured by pH meter (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 digested prior to heavy metal concentration determination according to the reference (Nemati et al., 2010). Freeze-dried samples were ﬁrstly weighed into each microwave TFM vessel, and then a mineral acid mixture of 6 ml HNO3, 2 ml HCl and 2 ml HF were added. These samples were digested in a microwave manufacturer (Milestone, Italy) with the procedure consisted of a 10 min gradual temperature increase to 200 °C (1000 W, 106 Pa), a 15 min step of 200 °C and a ventilated cooling stage. After cooling down to room temperature, the digested samples were heated on a hot plate (120 °C) until they became near dry. These dry samples were ﬁxed into the ﬂask with deionized water, and then ﬁltered through a 0.45 lm mixed cellulose ester membrane prior to inductively coupled plasma optical emission spectrometry (ICP-OES, PerkinElmer Optima 2100 DV, USA) analysis. 2.4. Sequential extraction of heavy metals The chemical speciation of heavy metals is usually divided into the following ﬁve fractions: the exchangeable fraction (F1), the carbonate 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 sequential extraction procedure was used to determine the portion of each fraction of the investigated heavy metals in the IS, BES, VES and EC. The detailed sequential extraction procedure was listed in Table 1. All reagents were ultrahigh purity, and all the laboratory glassware and polyethylene bottles were pre-cleaned by 4–10% chemical pure HNO3 and rinsed with deionized water prior to each extraction procedure. Each fraction concentration of these heavy metals was also determined by ICP-OES. 2.5. Statistical analysis Principal component analysis (PCA), uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables, could simplify the analysis and visualization of multidimensional data sets and reveal important variables that are difﬁcult to discover (Pardo et al., 2004; Raychaudhuri et al., 2000). In this paper, PCA was used to investigate the correlations between the chemical speciation (F1–F5) of heavy metals and physico-chemical properties (pH, SCOD and AP) of sludge. Analysis of variance (ANOVA) was applied to evaluate the signiﬁcance of results and p < 0.05 was considered to be statistically signiﬁcant. Statistical analysis was performed using the software SPSS 17.0.
J. Yang et al. / Bioresource Technology 146 (2013) 649–655
Fig. 1. Schematic diagram of the vermiﬁlter (with earthworms).
Table 1 Reagents and tessier sequential extraction procedure for each fraction of heavy metals. Fraction
Reagents and conditions*
Exchangeable (F1) Carbonates binding (F2) Fe–Mn oxide binding (F3) Organic and sulﬁde binding (F4)
16 ml 1 M MgCl2, pH 7.0, 25 °C, shaking for 2 h 16 ml 1 M NaAc, adjusted to pH 5.0 with HAc, 25 °C, shaking for 2 h 16 ml 0.04 M NH4OCl in 25% HAc, 85 °C in water bath, shaking for 2 h 8 ml 30% H2O2, adjusted to pH 2.0 with HNO3, shaking for 5 h, then adding 10 ml 3.2 M NH4Ac in 20% HNO3, shaking for 0.5 h Performed with microwave as described of total heavy metal concentrations digestion
* The extract was centrifuged at 9000 rpm for 15 min. The supernatant presents each fraction, and the residue is used in the next extraction procedure.
3. Results and discussion 3.1. Sludge stabilization Both the BF and VF reactors were operated steadily without clogging during the experimental period, and their performances on sludge reduction were showed in Table 2. It was observed that the average VSS reduction increased to 48.5 ± 3.9% after VF treatment, while it was 34.3 ± 3.0% after BF treatment. It means that sludge reduction was enhanced by 14.2% owing to the presence of earthworms in the VF reactor. Nevertheless, such sludge reduction is higher than that previously reported for VF systems treating sewage sludge (Liu et al., 2012). This might be due to the fact that the larger size of reactor in this study improved the treating efﬁciency. Moreover, the degree of the sludge stabilization also could be assessed by the ratio of VSS/SS (Zhao et al., 2010), and a lower VSS/SS ratio represents a higher organics degradation. As shown in Table 2, the average VSS/SS ratio was decreased to 0.62 in the VES from 0.71 in the IS. Although a decrease of VSS/SS ratio in the 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 similar results and suggested that the ingestion of earthworm and the earthworm-microorganism interactions played the important role in sludge stabilization in the VF (Zhao et al., 2010). The physicochemical properties of sludge, such as pH, SCOD and AP, were also observed to be change after the BF and VF treatments (Table 2), and it will be discussed in the following text. 3.2. Heavy metal concentrations The above study has shown that the earthworms promoted the organics degradation in sewage sludge, however, the variations of heavy metals in sludge before and after VF treatment were still unknown. Table 3 illustrates the total concentrations of the common heavy metals (Zn, Pb, Cr and Cu) in the IS, BES and VES. Their corresponding discharge standards (GB18918-2002) for land application 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% of total heavy metal content. After VF treatment, the concentration of Zn in the VES signiﬁcantly increased to 1861 mg/kg-SS from 1316 mg/kg-SS in the IS, whereas it merely increased to 1745 mg/kg-SS after BF treatment. Similarly, the concentrations of Pb, Cr and Cu were observed to increase slightly. It was obvious that the degradation of the organics was the main reason for the increase concentrations of heavy metal, because these heavy metals were non-degradable. The higher organics degradation by VF
Table 2 Treatment performances of sludge by BF and VF, and the corresponding physicochemical properties of the IS, BES and VES. Samples
IS BES VES
VSS reduction (%) 34.3 ± 3.0 48.5 ± 3.9
SCOD (mg/ L)
AP (mg/ L)
0.71 ± 0.03 0.67 ± 0.03 0.62 ± 0.03
7.21 ± 0.03 6.84 ± 0.01 6.57 ± 0.01
38.4 ± 7.4 31.3 ± 4.1 56.6 ± 10.0
3.53 ± 0.1 3.83 ± 0.4 5.31 ± 0.3
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Table 3 Total concentrations of heavy metals (Zn, Pb, Cr and Cu) in the IS, BES and VES and their permitted values in agriculture application.* Heavy metals (mg/kg-SS)
IS BES VES Threshold value Soil pH < 6.5 Soil pH P 6.5 *
1315.8 ± 59.4 1745.0 ± 64.6 1861.3 ± 94.6
130.2 ± 4.7 180.8 ± 7.1 199.4 ± 8.3
108.3 ± 6.5 149.2 ± 9.8 165.3 ± 9.9
311.0 ± 24.8 435.3 ± 23.2 481.2 ± 19.8
The data reported are the averages ± their standard deviations for 8 samples.
treatment than that by BF resulted in the higher concentrations of heavy metals in the VES than BES. However, the decreases of heavy metal concentrations were reported during vermicomposting process, because metallic cations were observed to be release into the leachate (Suthar, 2009). In this study, no obvious variance of heavy metal concentrations were detected in the supernatants of the BES and VES, indicating the release of heavy metals to the supernatants could 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 environment; however, Table 3 showed that all heavy metal concentrations (Zn, Pb, Cr and Cu) obtained in both the BES and VES were well below the threshold value requested in China for land application (SEPA, 2002a). Besides total concentrations of heavy metals, the chemical speciation is also another important factor for assessing their environmental risk (Zheng et al., 2007). Therefore, the speciﬁc chemical speciation of heavy metal (precipitated with minerals, complexed with organic ligands and so on) was investigated in the following text to comprehensively evaluate the potential risk of these heavy metals included in the VES. 3.3.1. Transformation of heavy metal fractions The speciﬁc chemical speciation of heavy metals was fractionized into exchangeable fraction (F1), carbonate precipitated fraction (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 metals is critical for researchers to accurately assess their environmental risk (Li et al., 1995; Thornton et al., 2008). Fig. 2 represented the percentage 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 contents and total contents were generally acceptable (92.2–110.3%), indicating that the determination of the heavy metal chemical speciation was reliable. It can be seen from Fig. 2 that over 70% of Zn in the IS distributed in F3, indicating Zn of the inﬂuent sludge was primarily occluded in amorphous and weakly crystalline iron and manganese oxides. After BF and VF treatment, F3 of Zn in the BES and VES decreased from 73.5% (IS) to 61.2% and 41.4%, respectively, and the F4 percentage of Zn in the VES was signiﬁcantly increased while that in the BES was slightly increased. A higher F5 percentage of Zn in the EC was observed, thus led to its higher percentage in the VES. As regards the other three investigated heavy metals (Pb, Cr and Cu), they predominantly distributed in the last two fractions (F4 and F5), and the sum of F4 and F5 in the IS accounted for 71.8%, 71.3% and 85.3%, respectively (Fig. 2). The speciﬁc chemical speciation variations of Pb and Cr were nearly similar with those of Zn 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 speciation of Cu; F2 was decreased, and F4 was increased, while the other three fractions (F1, F3, and F5) changed slightly (p > 0.05). 3.3.2. Mechanism of transformations among heavy metal fractions The above study has shown that after ﬁltration (BF and VF) treatment both the physico-chemical properties of sludge and the speciﬁc fraction of heavy metals in sludge varied greatly. Moreover, the speciﬁc fraction of heavy metals in sludge was observed to be depended on the pH, number and accessibility of adsorption sites and metal afﬁnity for solid components (Alvarez et al., 2002). It is speculated that some relationship between them could be established. Thus, PCA was used in this study to interpret the datum such as heavy metal fractions (F1–F5) and physico-chemical properties (pH, SCOD and AP) of the IS, BES and VES correlations. The results of PCA revealed that the presence of two principal components (PCs) explained a total variance of 77.3% (Fig. 3a). The most signiﬁcant PCs are the ﬁrst PC (PC1) which explained 62.6% of data variability and the second PC (PC2) which had an explain variance of 14.7%. As shown in Fig. 3a, F1 of the four heavy metals located in the ﬁrst, second and fourth quadrant, and their irregular distribution on the biplot of loadings of PCA might be due to the low percentages 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 metals and pH. In addition, the data in Table 2 has shown that the pH of the BES and VES was decreased from 7.21 (IS) to 6.84 and 6.57, respectively, which was due to the bioconversion of the organics into organic acids, and the transformation of organic nitrogen and phosphorus to nitrites/nitrates and orthophosphates during sludge degradation (Ndegwa et al., 2000). Under such weak acid conditions, percentages of F2 and F3 in heavy metals were observed to be decreased (Fig. 2), suggesting they were redistributed into other fractions. The lower F3 percentage of heavy metals in VES than BES was due to the effect of earthworms, in view of the signiﬁcant decrease of F3 percentage in the EC. Previous study has found that the pH of EC would be decreased as long as food transit through the earthworm guts (Li et al., 2009). Previous studies had found that earthworms in the VF were able to transform organic materials from insoluble to soluble forms and caused the increase of SCOD in the VES. The data in Fig. 3a showed that F4 of the four heavy metals and SCOD of sludge located in the fourth quadrant, and the dissolved organics could complex with soluble metal ions into stable fraction (F4) (Antoniadis and Alloway, 2002; Ashworth and Alloway, 2004), indicating that there was 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 BF due to the earthworm activities (Table 2). Furthermore, Xing et al. (2011) repored that the interactions of earthworms and microorganisms in the VF could lead to rapid humiﬁcation and promote the formation of –COOH groups. Moreover, researchers have found the formation of F4 was resulted from –COOH group of organic acids complexation with metal ions (Cheng and Wong, 2002; Ndegwa et al., 2000). Similar changes were seen between F5 of Zn, Pb, Cr and AP of sludge in Fig. 3a, and they were strongly positive with PC2 (distributed in the ﬁrst quadrant). Heavy metals distributed in F5 were mainly immobilized by insoluble salt such as phosphates. This was also consistent with the observed increase of F5 in the VES when more AP (5.31 mg/L) was released after VF treatment compared with AP concentration in the BES (3.83 mg/L) (Table 2). The gut transit of earthworms was the main reason for the higher AP concentration caused by earthworms (Buck et al.,
J. Yang et al. / Bioresource Technology 146 (2013) 649–655
Percentage of each fraction to the total (%)
20 10 10 0
Chemical speciation of heavy metals 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 8 samples.
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.
1999). It further demonstrated that the earthworm activities could immobilize metals and transform them into F5 (Walker et al., 2003). Loading plots were used to depict a group of original variables based on PCs, and score plots were used to show the projection of original data onto new biplot according to the loadings (Felipe-Sotelo et al., 2008). It was found from score plots (Fig. 3b) that samples (IS, BES and VES) were clustered in different groups on the biplot based on the total variance. Both the IS and BES distributed 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), indicating a correlative relationship with F4 and F5. This led to the conclusion that earthworms in the VF reactor played an important role in redistributing 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 main reason for sludge reduction (Xing et al., 2012), and it might cause the bioaccumulation of heavy metals in their tissues. Thus, the heavy metal concentrations of both the initial purchased earthworms
J. Yang et al. / Bioresource Technology 146 (2013) 649–655
and the earthworms collected from the VF reactor were measured and listed in Table 4. The concentration of Cr was not detected in the two types of earthworms (the initial and ﬁnal earthworms respectively withdrawn from farm and the VF reactor), which might ascribe to the species-speciﬁc metal physiology of earthworms (Wang et al., 2013). Compared with the initial earthworms, a statistical increase of Zn concentration (p < 0.05) was observed in earthworm tissues from the VF reactor; however, the variations of concentrations between Pb and Cu was not signiﬁcant (p > 0.05). The chemical speciation of heavy metals could affect their ingestion by earthworms. For instance, F3 of heavy metals is readily to be assimilated by plants and animals and ﬁnally accumulates in the food chain (Hare et al., 2003). Thus, the increase of Zn concentration in earthworm tissues was consistent with the above observation that F3 was the main chemical speciation in the IS. With regard to Pb and Cu, they mainly distributed in F4 and F5, which was hardly accumulated in food chain. Therefore, the enrichments of Pb and Cu in earthworm tissues were insigniﬁcant (p > 0.05).
IS BES VES Zn
IS BES VES Pb
IS BESVES Cr
IS BESVES Cu
Fig. 4. Comparisons of the unstable (the sum of F1, F2 and F3) and stable (the sum of F4 and F5) fractions of heavy metals in the IS, BES and VES. Asterisks indicate statistical differences (p < 0.05) from the IS.
3.5. Risk analysis of heavy metals It is well known that both total concentrations and chemical speciation of heavy metals in excess sludge are the key factors for assessing their potential risk to the environment. Table 3 has shown that the concentration of each heavy metal was still far below the discharge standards of pollutants for municipal wastewater treatment plant; however, the investigation of heavy metal chemical speciation (F1–F5) in sludge before and after ﬁlter treatment revealed that those fractions transformed. Different fractions of heavy metals behave dissimilar potential risk to the environment. For instance, F1 presents readily bioavailable proportion of metal content in environment and it can be stated that this fraction is toxic (Yuan et al., 2011); F2 and F3 are sensitive to pH and easily transformed into F1 under weak acid condition (Li et al., 2009; Tandy et al., 2009). Thus, these three fractions (F1, F2 and F3) could be identiﬁed as unstable and direct toxic. F4 is considered to be potential effect and less toxic to the environment, because it will be released into environment only under the conditions of strong acid and oxidizing agent (Chen et al., 2008); F5 is regarded as the mostly stable fraction and no eco-toxic to the environment (Walker et al., 2003). The two fractions (F4 and F5) are considered to be the stable and no eco-toxic. Therefore, the comparisons of the unstable (the sum of F1, F2 and F3) and stable (the sum of F4 and F5) fractions of heavy metals in the IS, BES and VES were investigated 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. Especially for Zn, the percentage of unstable fraction was the highest (79.0%) among the four heavy metals in the IS, which showing a high 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 4 Total heavy metal concentrations in the earthworm tissue respectively withdrawn from the farm and VF reactor.a Heavy metals (mg/kg-tissue)
Initial earthwormsb Final earthwormsc
153.7 ± 5.29 166.4 ± 6.15
4.7 ± 0.28 4.5 ± 0.29
20.1 ± 1.16 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 results further supported that earthworm activities could enhance the stabilization of heavy metals during sludge treatment. Similar variations 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 the IS, showing a lower eco-toxicity than the other three heavy metals, therefore the increase of stable Cu was insigniﬁcant. These results indicated that earthworm activities played a positive role in relieving the potential risk of heavy metals by transforming the mobile and available fractions into low or no eco-toxic fractions. 4. Conclusions The inoculation of earthworms to bioﬁlters enhanced organic degradation in sewage sludge, which led to the increase of heavy metal concentrations. Analysis of heavy metal chemical speciation showed that some unstable fractions were transformed into stable fractions due to earthworm activities. Further analysis indicated transformations among these fractions were due to the changes of sludge physico-chemical properties. The bioassay of earthworm demonstrated that only Zn was accumulated, indicating the enrichment of heavy metals by earthworms depended on their chemical speciation. At last, risk analysis further supported that earthworms weakened environmental risk of heavy metals after vermiﬁltration of sludge. Acknowledgements This work was partially supported by the National Natural Science Foundation of China (NSFC, No:51109161), the PhD Programs Foundation of Ministry of Education of China (20110072120029), the Fundamental Research Funds for The Central Universities (0400219187), the Open Analysis Fund for Large Apparatus and Equipments of Tongji University (No. 2012055), the National Spark Program of China (2010GA680004). The authors also thank the anonymous reviewers for their helpful suggestions. References Alvarez, E.A., Mochon, M.C., Sanchez, J.C.J., Rodriguez, M.T., 2002. Heavy metal extractable forms in sludge from wastewater treatment plants. Chemosphere 47 (7), 765–775. Antoniadis, V., Alloway, B.J., 2002. The role of dissolved organic carbon in the mobility of Cd, Ni and Zn in sewage sludge-amended soils. Environ. Pollut. 117 (3), 515–521.
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