Ecotoxicology and Environmental Safety 109 (2014) 177–184

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Vermiremediation of heavy metals in wastewater sludge from paper and pulp industry using earthworm Eisenia fetida Surindra Suthar n, Poonam Sajwan, Kapil Kumar School of Environment & Natural Resources, Doon University, Dehradun-248001, India

ar t ic l e i nf o

a b s t r a c t

Article history: Received 2 April 2014 Received in revised form 8 July 2014 Accepted 24 July 2014 Available online 16 September 2014

This work presents the results of removing heavy metals from paper mill wastewater (PMS) sludge spiked with cow dung (CD) employing Eisenia fetida. A total of seven set-ups were prepared: CD (100 percent), PMS: CD (1:3), PMS:CD (1:2), PMS:CD (1:1), PMS (100 percent), PMS:CD (3:1) and PMS:CD (2:1) and changes in chemical parameters were observed for 60 days. Vermistabilization caused the significant decrease in the level of Cd (32–37 percent), Cr (47.3–80.9 percent), Cu (68.8–88.4 percent), and Pb (95.3–97.5 percent) and substantial increase in EC, total-N, available P and K at the end. At the end, the tissues of inoculated worms showed the high load (mg kg
1, dry biomass) of Pb (8.81–9.69), Cd (2.31–2.71), Cr (20.7–35.9) and Cu (9.94–11.6), respectively which indicated bioaccumulation of metals by worms. The PMS:CD (2:1 and/or 3:1) appeared to be suitable waste mixture in terms of high metal removal and earthworm growth rates. Bioaccumulation, as quantified using BCF, was in the order: Cd 4Cr4 Pb 4Cu. Results suggested vermiremediation as appropriate technology for bioremediation of heavy metals from PMS. & 2014 Elsevier Inc. All rights reserved.

Keywords: Paper mill Vermicompost Wastewater treatment Earthworm Soil nutrient mineralization

1. Introduction The land application of agro-industrial solid wastes and wastewater sludge is one of the options recommended for their safe disposal and reutilizations. The direct applications (without preprocessing/treatment) of such materials may cause adverse impact on soil environmental quality. Prior to land application, the stabilization of sludge can be an appropriate technique to reduce the risk of hazardous substances present in the industrial waste solids. The vermistabilization is the process of waste biodegradation, involving the joint action of earthworms and beneficial microbial communities (Suthar et al., 2012). In this process, the sludge is stabilized effectively because of many beneficial impacts of inoculated worms upon aerobic degradation mechanism in waste stabilization system. Although, microbes are responsible for biochemical degradation of organic matter, the earthworms are the important drivers of the process, conditioning the substrate and altering the biological activity (Aira et al., 2009; GomezBrandon et al., 2011). Earthworm promotes appropriate conditions in decomposing waste sub-systems, ingest organic solids, convert a portion of the organics to worm biomass and respiration products and expel worm-cast as partially stabilized matter with

n

Corresponding author. E-mail address: [email protected] (S. Suthar).

http://dx.doi.org/10.1016/j.ecoenv.2014.07.030 0147-6513/& 2014 Elsevier Inc. All rights reserved.

low contents of hazardous substances (Loehr et al., 1985, Dominguez and Edwards, 2011). The pulp and paper making industry is a very water-intensive industry. During the whole process a large quantity of the wastewater sludge and other solids are generated. The paper production generates around 45 percent wastewater sludge (Zambrano et al., 2003). The production rate of dry sludge is estimated around 4.3 percent of the final product, increasing to 20–40 percent in the case of recycled paper mills (World Bank, 2007). Due to chemical properties (high organic matter content, pH, buffer capacity, nitrogen and phosphorous level) the pulp mill sludge may represent a valuable resource for soil amendments (Zhang et al., 2004; Gallardo et al., 2012). But the heavy metals in wastewater sludge are of major concern from the ecotoxicological risk perspectives. Vermistabilization can be an appropriate technique to reduce the level of hazardous substances from wastewater sludge solids (Neuhauser et al., 1988; Negi and Suthar, 2013). There are evidences of utilizing humus-feeder earthworms in the stabilization of wastes/sludges originated from different industries: grape mark (Gomez-Brandon et al., 2011), paper mill sludge (Kaur et al., 2010; Negi and Suthar, 2013; Sonowal et al., 2013a,b), distillery (Suthar and Singh, 2008), leather processing industry sludge (Ravindran et al., 2008), milk processing industry (Suthar et al., 2012), food processing industry (Garg et al., 2012; Lim et al., 2014) etc. The recent studies have also indicated the potential of earthworms in

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reducing concentration and bioavailability of metals/trace elements in industrial waste solids (Suthar and Singh, 2008; Kaur et al., 2010; Azizi et al., 2013; Wang et al., 2013a, 2013b; Fernández-Gómez et al., 2013; Singh and Kalamdhad, 2013). Nonetheless, the impact of earthworm on metals in sludge solids is contradictory in published literature. Few earlier researchers have reported (Wang et al., 2013a, 2013b; Hait and Tare, 2012; Azizi et al., 2011) decrease in water-soluble fractions of heavy metals and increase in total metal load during the vermistabilization process while, others (e.g. Suthar, 2008; Suthar and Singh, 2008) have reported the significant reduction in heavy metal concentration in same process. Singh and Kalamdhad (2013) have also reported the reduction in the exchangeable and carbonate fractions of Cu, Ni, and Cr in all trials of water hyacinth vermicomposting. Few studies have reported an interesting fact that the duration of exposure to sludge mainly decides the burden of heavy metals in worm-tissues and worm worked materials (see Azizi et al. 2013). However, Sizmur and Hodson (2009) in their review concluded that earthworms increase metal mobility and availability in soils and sludge solids but more studies are required to determine the precise mechanism for this. Earlier reports have presented interesting results on vermicomposting of paper mill sludge (Elvira et al., 1998; Kaur et al., 2010, Sonowal et al., 2013a, 2013b; Negi and Suthar, 2013) but, changes in metal concentration during the process is not well addressed. Assessing the metal load and bioavailability in wormworked materials can be sound option to measure the ecotoxicity of the end products in the environment (Suthar and Singh, 2008; Singh and Kalamdhad, 2013). Keeping this in view, the present experiment was designed on vermiremediation of heavy metals in wastewater sludge from a pulp and paper industry employing Eisenia fetida. The changes in chemical characteristics of sludge mixture and earthworm biological parameters were measured during the experimentation. The heavy metal loads in worm tissues were monitored in order to see the heavy metal bioaccumulative efficacy of inoculated worms.

2. Methodology 2.1. Eisenia fetida, paper mill waste water sludge, cow dung The culture of E. fetida was procured from vermiculture laboratory, Indian Veterinary Research Institute (IVRI), Izatnagar, India. For stock, earthworms were cultured in contamination-free substrate decomposing litter biomass, under laboratory conditions. For experimentation, the second generation of earthworms was used to avoid any contamination history. For that freshlydeposited cocoons were segregated and then cultured in metalfree bedding separately. The sludge was collected form wastewater treatment unit of a paper mill—Sri Bardari Kedar Papers Pvt., Nazibabad, Utter Pradesh, India. The wastewater treatment unit consists of primary treatment and secondary treatment set-ups. The sludge was obtained from operation units of primary treatment set-ups. The paper mill sludge (containing 65–74 percent moisture) in form of thick liquid/wet— sludge was collected in plastic circular containers (20 L) and then brought to the laboratory. Prior to use sludge in experimentation preprocessing was done. The wet-sludge was dried on polythene begs in shed to evaporate excess water. The material was turned daily to reduce characteristics smell of putrescible and biotoxic substances. The dried sludge cake solids were then shredded and homogenized and then used to prepare waste mixture for vermistabilization trial. The physic-chemical characteristics of paper mill wastewater sludge were: pH, 7.9970.07; EC, 0.2570.004 (mS); TOC, 64470.2 (g kg
1);

TKN, 5.5970.02 (g kg
1); K, 0.2470.004 (g kg
1); available P, 3.1970.003; (g kg
1) and C:N ratio, 114.271.20. In composting/vermicomposting, the bulky material is required to accelerate microbial degradation process. Cow dung was used as bulky agent to dilute the paper mill sludge substrate for vermistabilization operation. Freshly deposit cow dung (CD) was procured from a local cowshed, Mothrawala, Dehradun. CD was partially dried in shed and used for further experimentations. The main characteristic of CD were: pH, 9.0170.04; EC, 0.1370.01 (mS); TOC, 59570.02 (g kg
1); TKN, 5.9270.004 (g kg
1); K, 0.2870.002 (g kg
1); available P, 3.1470.01 (g kg
1) and C:N ratio, 101.170.25. 2.2. Preparation of experimental set-up For laboratory trial, a total of seven different combinations (dry weight proportion) of paper mill sludge (PMS) and CD were prepared: T1—CD (100 percent). T2—PMS: CD (1:3). T3—PMS: CD (1:2). T4—PMS: CD (1:1). T5—PMS (100 percent). T6—PMS: CD (3:1). T7—PMS: CD (2:1). The waste mixture acted as bedding and feeding material for worms. For experimentation, 400 g (dry weight basis) waste mixtures for each mixture was filled in plastic circular containers of 1.5 L capacity. Each treatment was kept in triplicate. The bedding material was moistened with distilled water to maintain appropriate moisture level (65–70 percent). The sludge mixtures were kept (1-week) for initiation of microbial degradation and softening of waste mixtures prior to inoculation of worms. For verminremediation trial, fifteen earthworms (4 to 5 week old) with live weights in the rages of 496–516 mg were released into each test containers. Experimental set-ups were incubated in a humid and shady place at ambient temperature (27–28 1C). The changes in physico-chemical characteristics of sludge mixture were measured at 0, 10, 20, 30, 40, 50, and 60 days. For that, the homogenized samples of sludge substrate were drawn from each set-up. The segregated samples were oven dried (at 60 1C), and stored in sterilized plastic airtight containers for further analysis. The biological parameters—weight changes, earthworm death rate, cocoon production and earthworm population were observed in all experimental set-ups using standards methodology as described by Negi and Suthar (2013). 2.3. Analytical procedures The pH was measured through digital pH meter (Systronics made) in 1:10 (w/v) aqueous solution (deionized water). Digital conductivity meter was used to measure EC. The total organic carbon (TOC), total Kjeldahl nitrogen (TKN), available phosphorous (AP) and total potassium (K) content was measured by following methods as described in Carter and Gregorich (2008). The heavy metals were analyzed by following the methodology as described by Pedersen and van Gestel, 2001). For estimation of heavy metals, 1 g of sludge sample was digested with mixture of HNO3 and HClO4. Digested samples were diluted with Millipore water and filtered with Whatman no.42 filter paper. Then heavy metal concentration was estimated using Atomic Absorption Spectrophotometer (Thermo Fisher. Model iCE 3000 Series AA System). During analysis appropriate quality control was maintained, including preparation of blanks, spike sand standard reference material etc. for quality assurance.

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The heavy metals in tissues of inoculated worms were measured to quantify the bioaccumulation of heavy metals in worm tissues. For that, the adult specimens of worms were separated from each treatment containers at the end. The worms were washed in deionized distilled water to remove adhering materials. To clear the gut the worms were placed in sterilized glass Petri dishes with one filter paper (Whatman No. 1) and a few drops of distilled water to maintain them moist. The worms were kept in the dark for 3 days and filter paper was changed daily. The earthworms of approximately equal weight were separated from Petri dishes subsequently sacrificed through deep freezing method. The alimentary contents of worms were removed by dissection. They were then oven dried (48 h at 70 1C) to constant weight. The dried worm biomass was tested for metal contents by following method as described above. The metal accumulation in worm tissues was estimated using the bioconcentration factor (BCF). The bioconcentration factor (BCF) is defined as Mountouris et al. (2002): BCF ¼

C biota ; C substrate

where Cbiota and Csubstrate were the total concentrations in taxa (earthworm) and substrate (used for vermicomposting experiments), respectively in mg kg
1. 2.4. Statistical analysis The statistical analysis of data sets was performed using SPSSs statistical package (Window Version 13.0). The statistically difference among treatments was calculated using One-way ANOVA followed Tukey’s t-test. The relations between individual parameters/interdependent parameters were evaluated with the help of regression equation. The cluster analysis was done in order to identify the statistically significant difference among different waste mixtures for metal removal rate. The results of cluster analysis were presented in the form of dendrogram. All statements reported in this study are at the p o0.05 and p o0.001 levels.

3. Results and discussions 3.1. Heavy metal content in vermistabilized sludge The vermiremediation caused the significant impact on the concentration of heavy metals in sludge substrate at the end. There was statistically significant difference among treatments set-ups for the level of Cd (ANOVA; F¼24.67, p o0.001), Cr (ANOVA; F¼379.01, p o0.001), Cu (ANOVA; F ¼262.49, p o0.005) and Pb (ANOVA; F¼7.702, po0.001). The concentration of heavy metals in end material (vermistabilized sludge solids) ranged from 23.22 70.62 to 65.59 70.58 mg kg
1 substrate for Cr, 7.89 70.002 to 7.91 70.002 mg kg
1 substrate for Cd, 1.41 70.24 to 2.0 7 0.46 mg kg
1 substrate for Pb and 16.79 70.43 to 36.06 7 0.23 mg kg
1 substrate for Cu (Table 1). The vermistabilization caused significant reduction in concentration of metals: Cd (32–37 percent), Cr (47.3–80.9 percent), Cu (68.8–88.4 percent), and Pb (95.3–97.5 percent), at the end. In terms of removal rate, the sludge mixtures can be arranged in the order: T2 4T7 4T6 4 T3 4 T5 4T4 4T1 for Cr, T1 4 T2 4T3 4 T4 4T5 4 T6 ¼ T7 for Cd, T5 4T4 4T3 4 T1 4T2 4T6 ¼T7 for Pb and T5 4T7 4 T6 4 T4 4T1 4T3 4T2 for Cu. The comparison of metal removal in different sludge mixtures are presented in Fig. 1S. The increasing proportion of paper mill wastewater sludge resulted in the high removal of metals during the process especially for metals: Cu and Pb. During vermistabilization the organic matter is decomposed/mineralized by inoculated earthworms-microbes

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and this mutual workout causes the releasing of soluble fraction of heavy metals from sludge solids (Liu et al., 2007; Suthar and Singh, 2008). Probably, the metal reduction could be attributed to the assimilation of free forms of metals by inoculated earthworms (Wang et al., 2013a). In case of Cd the maximum removal was in T1 while other set-ups have shown relatively the slow rate of removals. The removal trend of Cr in different treatments was opposite to Cd (Fig. 1S) removal pattern. A variety of factors like: pH, metal speciation, equilibrium between soluble and bound forms of metals, organic matter contents, chemical-interaction among cations and anions affect the rate of heavy metal removal in vermistabilization sub-system (Morgan and Morgan, 1999; Suthar and Singh, 2008; Wang et al., 2013a). However, each metal has a specific chemical property that fix the availability/solubility of metal in waste system during vermistabilization process. On average the metal removal rate was in the order: Pb 4Cu 4Cr4Cd, in the present study. The dendrogram of removal patterns of different metals in vermiremediation system is described in Fig. 1. The heavy metals grouped into two statistically significant clusters. Cluster 1 consists of Cd and cluster 2 consists of rest of metals (Pb, Cr and Cu). Cluster 2 further subcategorized into two clusters: sub-cluster 1 consist of Cr and subcluster 2 consist of Pb and Cu. The cluster clearly indicates the statistically significant difference in heavy metal removal rate during the vermistabilization process. The trend of heavy metal removal during the experimental duration is illustrated in Fig. 2. For Pb a trend of rapid loss was observed upto 40 days thereafter; the removal rate was slow. In Cr straight trend of loss was observed till the end, except in T1 and T2. Similarly, Cd also had different pattern of loss during vermistabilization process (Fig. 2). However, in contrast to our findings, few earlier researchers (e.g. Hartensein and Hartenstein, 1981; Azizi et al., 2011) have reported the enhanced metal load in worm-worked waste/feed materials as a result of organic matter mineralization (releasing of more tightly bound fractions of metals). Azizi et al. (2011) have reported 0.25–11.6-fold high concentration of metals in wormworked materials than the initial materials. They claimed the excretion of non-accumulated heavy metals in waste stuff as primary mechanism for sludge metal enrichment during vermiremediation process. When organic matter or waste stuff passes through the gut of worms then a proportion of that is digested and a considerable amount of soluble-fractions of metal is released. However, the concentration of metals in worm-worked materials is depends upon the quantity of assimilated and excreted (nonaccumulated) contents of metals during vermistabilization process. The possible mechanisms of metal remediation in vermistabilization process could be as: (1) metal accumulation by inoculated worms—bioaccumulation (Hsu et al., 2006; Suthar and Singh, 2008), (2) leaching or precipitation of soluble fractions of metal from sludge during vermistabilization and, (3) adsorption of metals on the surface of waste fractions (Wang et al., 2013a). Also, Hobbelen et al. (2006) reported that after 54 days of vermicomposting durations, in spite of high availability of heavy metal concentrations in earthworms, Cu and Zn concentrations in vermicompost decreased. Pb showed a very high removal (95–99 percent) removal but worm tissue-metal was not in that proportion, although tissue-metal load indicates the extractable or bioavailable metal contents. Probably, leaching or precipitation or adsorption could be responsible for Pb loss in our case. The sludge mixture was of alkaline nature and that might have affected the metal removal process. The alkaline substances increase the rate of adsorption of heavy metals through increasing negative charges on the surface of the bedding stuff (Wang et al., 2013b). However, the proportion of paper mill sludge in experimental waste mixtures had shown the direct impact on metal removal

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Table 1 Heavy metal concentration changes during vermistabilization of sludge mixtures (mean 7SD, n ¼3). Parameters

Cr

Cd

Pb

Cu

Initial substrate (wastewater sludge) T1—CD (100%) T2—PMS: CD (1:3) T3—PMS: CD (1:2) T4—PMS: CD (1:1) T5—PMS (100%) T6—PMS: CD (3:1) T7—PMS: CD (2:1)

124.4 70.3 121.4 70.4 114.2 70.6 128.3 70.6 187.3 70.4 175.3 71.2 158.7 71.8

9.29 7 0.01 8.777 0.01 8.75 7 0.01 8.717 0.02 8.75 7 0.04 8.677 0.03 8.647 0.02

59.6 70.3 45.6 70.5 42.3 70.8 49.7 70.4 62.5 70.5 42.8 70.5 35.470.6

110.5 70.5 115.5 70.6 114.5 70.5 129.5 70.6 144.4 70.3 142.3 70.7 123.5 71.1

Vermistabilized wastewater sludge T1—CD (100%) T2—PMS: CD (1:3) T3—PMS: CD (1:2) T4—PMS: CD (1:1) T5—PMS (100%) T6—PMS: CD (3:1) T7—PMS: CD (2:1) a 2 R

65.6 7 0.6 e 23.2 7 0.7 a 25.6 7 0.9 a 32.3 7 0.7 b 43.6 7 0.9 d 39.3 7 0.4 c 32.0 7 1.0 b 0.08

7.917 0.002 d 7.89 7 0.001 ab 7.90 7 0.001 cd 7.89 7 0.003 bc 7.90 7 0.002 d 7.917 0.001 d 7.89 7 0.002 a 0.99

2.0 7 0.041 b 2.0 7 0.021 b 1.41 70.24 a 1.53 70.001 a 1.54 70.02 a 2.0 7 0.05 b 1.667 0.03 ab 0.55

23.75 7 0.52 b 36.06 70.23 d 25.95 7 0.41 c 26.127 0.12 c 16.79 7 0.43 a 23.50 7 0.43 b 1s8.647 0.41 a 0.51

0.7–10 39 r5 7.89–7.91

70–1000 300 r100 1.4–2

210–4000 600 r300 16.8–36.1

The permissible limit of heavy metals in composted biosolids 70–200 EU limit rangeb (mg kg
1) 1200 US biosolids limitc (mg kg
1) r50 Indian limitd (mg kg
1) PMS (this study) (mg kg
1) 32–65.6

SD ¼ Standard deviation, mean value followed by different letters is statistically different (ANOVA; Tukey’s t-test, p o 0.05). a

Regression analysis from calculation of removal (%) as a function of concentration in substrate. Limit set for compost in United States and European Countries. FIA (2007).

b,c d

Fig. 1. Dendrogram showing cluster of heavy metals removal rates on the basis of similarity.

Fig. 2. Trend of heavy metal loss from sludge mixtures during vermistabilization.

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rate in this study. In majority of cases, the bedding with PMS: CD (2:1) or PMS (100 percent) showed the maximum removal of heavy metals. The regression analysis, from calculation of removal (percent) as a function of initial metal load in substrate, showed close relationship between removal rate and metal load in substrate (Cd: R2 ¼0.99, Pb: R2 ¼ 0.55 and Cu: R2 ¼0.51), except in Cr (R2 ¼ 0.08). All experimental sludge mixtures were also taken for the cluster analysis to see the difference among set-ups for BCF for different metals. The result of cluster analysis is presented in Fig. 2S. In this dendrogram all the seven waste mixtures are grouped into two statistically significant clusters: cluster 1 – consist of T1 and cluster 2 – consists of T2, T3, T4, T5, T6 and T7. Cluster 2 further consists of two distinct sub-clusters: cluster 2A – T2 (PMS: CD, 1:3) and cluster 2B. Cluster 2B showed further sub categories (Fig. 2S). Cluster analysis clearly shows that difference between T6 and T7 and between T3 and T4 was not statistically significant. But group of T6–T7 showed significantly different rate of heavy metal removal than group T4–T5. The chemical characteristics of waste stuff affects the palatability of feed material for inoculated worms in vermicomposting system that results in different metal reduction rates in experimental set-ups. The ranges of heavy metals in ready vermicompost were significantly high than prescribed Indian limit for Cr (in T1) and Cd (for all) as decided by Fertilizer Association of India (FIA, 2007). The permissible limit of heavy metals in compost is high in European and US standards as compare to Indian standards. The Cd safe limit is o5 mg kg
1 as per FIA but value of vermstabilizated sludge was in the ranges of 7.89–7.91 (mg kg
1). But in terms of Pb and Cr contamination, the vermicomposted sludge stuff seems safe for further land application in agro-ecosystem. 3.2. Metal concentration in earthworm tissues and BCFs The bioavailability of metals in earthworms can be evaluated in terms of relative toxicity (as lethality) index and through bioaccumulation determinations, yielding bio-concentration factors (BCF) (Abdul Rida and Bouche, 1994). There was significant difference among the different sludge mixtures for level of Pb (ANOVA: F¼379.50, po0.05), Cd (ANOVA: F¼1771.18, po0.05), Cr (ANOVA; F¼75.77, po0.005) and Cu (ANOVA: F¼77.38, po0.05) in worm tissues. The concentration of Pb, Cd, Cr and Cu ranged from 8.8170.02 (T4) to 9.6970.02 (T1) mg kg
1 (dry biomass), 2.3170.002 (T7) to 2.7270.01 (T1)

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mg kg
1, 20.6770.8 (T3) to 35.970.8 (T5) mg kg
1 and 8.8470.004 (T3) to 11.5870.11 (T5) mg kg
1, respectively (Table 2). The bioaccumulation of heavy metals by inoculated earthworms in vermicomposting system is well documented in published literature (Kizilkaya, 2004; Hsu et al., 2006; Suthar and Singh, 2008; Suthar, 2008; Azizi et al., 2013). During the waste decomposition process the extractable or available fractions of metals are removed by the earthworm through the mechanism of gut/ skin absorption. The difference among experimental set-ups for worm-tissue metal concentration could be attributed to the chemical characteristics (organic matter, pH, food palatability etc.) of the waste feedstock used in vermistabilization. Lukkari et al. (2006) have stated that the binding of metals with organic matter (more tightly bound fractions) partly reduces the availability of metals to inoculated worms. Also, initial metal load in waste mixture is directly related to the worm-tissue metal concentrations. The relationship between the metal removal rate and worm-tissue metal level examined by a linear regression analysis (Table 3), resulted in a strong correlation between these parameters (Cu: R2 ¼0.0.51, Pb: R2 ¼ 0.64, Cr: R2 ¼0.45 and Cd: R2 ¼ 0.72). The assimilation of metals in earthworm tissues can be quantified using bioconcentration factor (BCF) (Mountouris et al., 2002). However, BCF is also used by few earlier workers (e.g., Wang et al., 2013a,b; Suthar and Singh, 2008) to trace the loss of metals from vermistabilized waste stuffs. There was statistically significant variations among different sludge mixtures for BCF calculated for Pb (ANOVA: F ¼147.19, p o0.05), Cd (ANOVA: F¼ 103.07, po0.05), Cr (ANOVA; F¼13.50, p o0.005) and Cu (ANOVA: F¼52.23, po 0.05). The BCFs varied from 0.15 70.001 (T5) to 0.25 70.004 (T7) for Pb, 0.28 70.0001 (T6) to 0.327 0.0006 (T3) for Cd, 0.16 70.005 (T7) to 0.21 70.004 (T4) for Cr and 0.080 70.001 (T5 and T6) to 0.0927 0.001 (T2) for Cu. BCF or accumulation rate was in the order: Cd 4Cr 4Pb 4Cu. Accumulation of metals, especially Cd, and Cu in earthworm is facilitated by metallothioneins (MTs), low molecular weighed protein-metal complexes. Hopkin (1989) suggested that earthworms have specific capacity to regulate metals, particularly trace metals and accumulation and regulation mechanisms could be a metal-specific. However, few authors (e.g. Wang et al., 2013a; Suthar and Singh, 2008) have used BCFs to quantify the metal loss from substrates through linear regression analysis. But in this study, the BCF have shown the strong but inverse relationship with metal removal rate examined by a linear

Table 2 Worm tissue metal load mg kg
1) in different sludge mixtures and BCF (mean 7SD, n ¼3). Parameters

Pb worm

Cd worm

Cr worm

Cu worm

T1—CD (100%) T2—PMS: CD (1:3) T3—PMS: CD (1:2) T4—PMS: CD (1:1) T5—PMS (100%) T6—PMS: CD (3:1) T7—PMS: CD (2:1)

9.69 7 0.02 e 8.90 7 0.02 b 8.96 7 0.01 b 8.81 7 0.02 a 9.58 7 0.02 d 9.167 0.02 c 8.87 7 0.01 ab

2.72 7 0.01 f 2.46 7 0.01 d 2.59 7 0.01 e 2.417 0.004 c 2.417 0.004 c 2.357 0.04 b 2.317 0.02 a

25.27 70.2 b 20.677 0.8 a 21.677 0.6 a 26.78 7 0.5 b 35.907 0.8 d 32.1 70.8 c 25.27 70.5 b

9.94 7 0.015 b 10.63 7 0.02 c 8.84 7 0.004 a 10.90 7 0.03cd 11.58 7 0.11e 11.39 7 0.25 de 10.43 7 0.03 bc

Bio concentration factors (BCF) for metals BCFPb

BCFCd

BCFCr

BCFCu

T1—CD (100%) T2—PMS: CD (1:3) T3—PMS: CD (1:2) T4 – PMS: CD (1:1) T5– PMS (100%) T6 – PMS: CD (3:1) T7 – PMS: CD (2:1)

0.317 0.003 e 0.30 7 0.001 d 0.32 7 0.001 f 0.30 7 0.002 c 0.30 7 0.002 c 0.28 7 0.002 b 0.29 7 0.001 a

0.20 7 0.002 cd 0.17 70.006 ab 0.197 0.004 bcd 0.217 0.004 d 0.197 0.004 bcd 0.187 0.006 bc 0.167 0.005 a

0.090 7 0.002 c 0.092 7 0.001 c 0.0777 0.001 a 0.084 7 0.002 b 0.080 7 0.001 a 0.080 7 0.001 a 0.084 7 0.001 b

0.167 0.001 a 0.20 7 0.002 c 0.217 0.004 d 0.18 7 0.001 b 0.157 0.001 a 0.217 0.003 d 0.25 7 0.004 e

(max wt worm
1 ay
) (cocoon worm
1) SD ¼ Standard deviation, mean value followed by different letters is statistically different (ANOVA; Tukey’s t-test, p o 0.05).

a

b

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regression analysis (results presented in Table 4). This suggests the adsorption or precipitation or leaching as the main mechanism of heavy metal loss rather than absorption by worms. Although, further detailed study is required to support the hypothesis. In order to see the effect of TOC loss, pH and C/N ratio of waste feedstock on metal loss rate (percent) and BCFs, a Person correlation coefficient (R) was calculated. As illustrated in Table 1S, the pH showed significant reserve relationship with Pb, Cr and Cu reduction in waste mixtures. The loss of Pb and Cd in waste mixtures also correlated significantly with C loss (Table 1S). The metal bioavailability in sludge mixtures in vermistabilization system showed the significant relationship with pH (for BCFCu, p o0.001), C reduction (for BCFCr, p o0.05; BCFCu, p o0.05 and BCFPb, p o0.001) and C/N ratio (
0.755, p o0.001). Results suggests that carbon mineralization in sludge mixture during vermicomposting system enhances the bioavailability of heavy metals in sludges. The results are in accordance with Wang et al. (2013a) and Hsu and Lo (2001) who have reported direct role of organic matter contents in metal mobility and availability in end material during vermicomposting/composting process. The TOC reduction leads to formation of intermediate metabolites and acids (humic acids) which in result reduce pH of the sludge mixtures. But a close relationship between pH and metal loss rate was recorded only for Pb and Cu (p o0.05 and p o0.01, respectively. The phenomena of metal reduction in vermicomposting process can be explore thorugh further studies on speciation of metals and their levels in vermibeds after completion of the composting/vermicomposting process (Hsu and Lo, 2001; Miaomiao et al., 2009; Singh and Kalamdhad, 2013; Wang et al., 2013a). In general, metal accumulation in tissues is a specific-specific phenomenon and each metal has a specific physiological mechanism of assimilation and/or

Table 3 Results of linear regression analysis of reduction of percentage of metal concentration and worm-tissues metal level. Linear regression analysis

R2

p Value

Metal loss and worm-tissues metal level Curemoval ¼
39.95 þ 13.43Cuworm tissues Pbremoval ¼
154.44þ 21.99Pbworm tissues Crremoval ¼8.701 þ3.66Crworm tissues Cdremoval ¼0.98 þ 0.55Cdworm tissues

0.51 0.64 0.45 0.72

nnn

Metal loss and BCF Curemoval ¼- 258.46 þ 187CuBCF Pbremoval ¼100–2766.1PbBCF Crremoval ¼232.94–675.74BCF Cdremoval ¼0.22 þ 0.03BCF

0.35 0.93 0.18 0.32

nn

nnn nnn nnn

nnn nn nn

Significant effect. nn

At p o 0.05. At p o 0.001.

nnn

excretion during their metabolism in earthworm’s gut. The mechanism of metal loss can be quantified by using speciation of metals in waste feedstock.

3.3. Changes in pH, EC, TOC, TKN, TP, TK level and C/N ratio during vermistabilization The vermistabilized sludge mixture was rich in terms of plant nutrient quantity. The difference among the experimental set-ups was statistically significant: pH (ANOVA: F¼405.861, po0.05), EC (ANOVA: F¼1310.769, po0.05), TOC (ANOVA: F¼311.218, po0.005), TP (ANOVA: F¼0.840, po0.05), TKN (ANOVA: F¼30.811, po0.001), K (ANOVA: F¼13.895, po0.001), and C/N ratio (ANOVA: F¼298.051, po0.001). The vermistabilization caused the reduction in pH (1.01– 1.09-fold), TOC (1.2–1.4-fold) and C/N ratio (2.7–3.4-fold) and substantial increase in EC (7.8–21.4-fold), TKN (2.2–2.5-fold), TK (1.2–2fold) and AP (1.1–1.6-fold) (Table 2S). The vermistabilization is a complex process mainly driven by earthworms and associated microbial communities. Although microbes are mainly responsible for mineralization of waste mixtures but earthworm play a vital role in whole process by providing appropriate environmental for microbial degradations. The enhanced nutrient level in worm-worked material suggested the suitability of worm technology in industrial waste stabilization. The nutrient enrichment in worm-worked sludge mixture during vermistabilization is well documented by previous researchers (Suthar, 2008; Kaur et al., 2010; Hait and Tare, 2012; Wang et al., 2013b; Singh and Kalamdhad, 2013). The pH shifting during vermistabilization could be attributed to the formation of organic acids and other intermediate metabolic products of organic matter degradations (Lim et al., 2011). EC indicated the high mineralized nutrient stuff in worm-worked sludge mixtures. The increase of EC in all treatments after vermistabilization could be due to the loss of organic matter and release of minerals in the form of cations (Lim et al., 2011). TOC reduction in sludge is facilitated by worms and active microbes. Soil respiration and assimilation of easy digestible C-pools as worm biomass are the main cause of C loss from substrate. Paper mill wastewater sludge is considered N-deficient substances. The worm body secretion/ excreta and decaying worm body (in case of death) might be the main sources of N-enrichment of sludge during vermistabilization (Negi and Suthar, 2013). The paper mill wastewater sludge is rich in organic carbon/cellulosic components but deficient in other nutrients. The high AP in vermistabilized sludge was the results of mineralization of organic-P in waste mixtures. C/N ratio of sludge reduced significantly but it was higher than prescribed limit (C/N ratio ¼ below 20) of biosolids for agronomical purposes. Overall, vermistabilization converted the sludge into added-value product with agronomic applicability.

Table 4 Earthworm biological properties and worm tissue metal contents (mg kg
1) in different sludge mixtures (mean7 SD, n¼ 3). Parameters

T1—CD (100%) T2—PMS: CD (1:3) T3—PMS: CD (1:2) T4—PMS: CD (1:1) T5—PMS (100% ) T6—PMS: CD (3:1) T7—PMS: CD (2:1)

Individual biomass of earthworm (mg) Initial

Final

402.2 7 50 388.9 7 23 397.8 7 44 393.3 7 61 3717 39 415.6 7 57 413.3 7 77

612.7 731.6 a 506.9 736 b 594.7 785 ab 964.9 793 cd 760.8 7136 bc 1025 746 d 1112 7115 d

a

Biomass change

Growth rate

þ40% þ23% þ33% þ59% þ50% þ59% þ63.5

4.63 7 0.9a 3.93 7 1.4 a 5.377 2.7 ab 9.53 7 0.7 bcd 6.50 7 2.3 abc 10.2 7 1.3 cd 147 0.9 d

Total cocoons

Mortality (%)

Reproduction rate

55.0 7 2.1 56.3 7 2.2 69.0 7 2.5 60.0 7 0.6 60.7 7 2.4 54.3 7 5.0 57.37 6.8

31.17 2.2 28.9 7 4.4 24.5 7 2.2 20.0 7 0.01 17.8 7 2.2 13.3 7 3.9 11.17 2.2

5.4 7 0.4 5.3 7 0.3 6.17 0.4 5.0 7 0.1 4.9 7 0.1 4.2 7 0.5 4.3 7 0.6

SD ¼ Standard deviation, mean value followed by different letters is statistically different (ANOVA; Tukey’s t-test, p o 0.05). a b

(max wt worm
1 ay
). (cocoon worm
1).

b

S. Suthar et al. / Ecotoxicology and Environmental Safety 109 (2014) 177–184

3.4. Earthworm survival, biomass changes and cocoon production The earthworm’s mortality is an indicator of the suitability of waste mixtures for vermistabilization process. The earthworm mortality in set-ups ranged from 11.17 2.2 (T7) to 31.1 72.2 percent (T1). In terms of total mortality the experimental set-up can be arranged in the order: T1 4 T2 4T3 4T4 4T5 4T6 4T7. The difference among different experimental set-ups was statistically significant for total mortality (ANOVA: F¼ 7.307, p ¼0.001). The rate of earthworm mortality was high in beddings those contained relatively high proportion of paper mill sludge in experimental set-ups. Apart from the micro-environmental conditions the feed quality can directly affects the survival rate of worms in waste mixtures (Negi and Suthar, 2013; Shak et al., 2014). Probably, high alkaline pH scale in some waste mixture could cause worm mortality. The earthworm population built-up in waste mixture did not show statistically significant difference among different set-ups (ANOVA: F¼3.667, p ¼0.021) (Table 4). The total numbers of earthworms in experimental set-ups ranged from 10.0 70.58 (T1) to 14.3 72.52 (T6), at the end. The cocoon viability, hatchling success and survival rate of newborns juvenile etc. are important factors in population build-up in vermistabilized waste substrates. The individual live weight in earthworm was 23–64 percent high than initial level. The difference among different experimental set-ups was statistically significant for: final individual biomass (ANOVA: F¼ 22.92, p o0.001), weight gain/worm (ANOVA: F¼30.78, p o0.001) and maximum growth rate (mg wt worm
1 day
1) (ANOVA: F¼15.11, p o0.001). The biomass gain is attributed to the assimilation of absorbed nutrient stuff from waste mixtures to worm biomass. The earthworm biomass gain was in the order: T7 4T6 ¼T4 4 T5 4T1 4T3 4T2. The worm weight is directly related to the feed rate and assimilation of digested nutrients. Probably, the proportion of easily metabolizable organic matter, non-assimilated carbohydrates etc. in beddings with PMS:CD (3:1/2:1) supported the worm growth. Few authors have also supported the fact that the growth rate in earthworm is directly related to the microbial populations and availability of nutrient pools in vermibeds (Kaur et al., 2010; Gomez-Brandon et al., 2013). The biomass gain is again useful in metal removal mechanism as worm accumulates a considerable amount of heavy metals in their tissues. The cocoon production is an important biological indicator of waste suitability for worms. The maximum numbers of cocoons were recorded in T3 (69.0 72.5) followed by T5, T3, T7, T2, T1, and T6 (Table 4). The cocoon production in worm depends upon a variety of factors like waste mixture quality, metal/toxic stress, food palatability, stocking density etc. The difference among experimental set-ups was not statistically significant (ANOVA: F¼1.88, p ¼0.154). The high cocoon production was recorded in beddings those exhibited the mortality rate. This suggested stress as inducting agent in reproduction performance in worms. Few studies have attributed chemical/toxic stress as stimulating factor for enhanced cocoon production rate (Zhang et al., 2011; Suthar, 2013). The cocoon rate (cocoon worm
1) suggests overall reproduction performance of worms which is calculated as total cocoons in proportionate to the worm numbers in waste mixtures. The maximum reproduction rate was recorded in T3 followed by T1, T2, T4, T5, T7 and T6 (Table 4).

4. Conclusions The load of heavy metals in vermistabilized sludge mixture was lower than initial waste mixtures. The high concentration of heavy metals in worm-tissues indicates the transfer of metals from substrate to tissues of inoculated earthworms. The earthworm

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growth and cocoon production pattern acts as suitable bioindicator of suitability of waste mixtures for earthworm growth. In all waste mixtures, the earthworm showed the high growth and reproduction pattern but waste mixture with 2:1 or 3:1 (PMS: CD) showed the better earthworm growth performance. Results suggested that vermistabilization can be as appropriate technology for bioremediation of heavy metals from industrial sludge.

Acknowledgment Authors are highly thankful to four anonymous reviewers for critical comments and fruitful suggestions on earlier version of the manuscript.

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