INTRODUCTION:

The natural sources of heavy metals are erosion of rocks, volcanic activity and forest fire where as majorly by artificially as anthropogenic activity from industries, paper mills, vehicles etc. which can release large quantities to directly affect the flora, fauna as well as human being (Martin and Griswold 2009). These heavy metals entered in the human body by ingestion of contaminated foodstuff specially grains, cereals and leafy vegetables. It can cause several respiratory irritation lung disease, cancers and kidney problem (Ye et al., 2000). The large amount of heavy metals in sewage sludge caused potential risks for human health and environmental problems (Hernandez et al., 1991). Industrialization of the developing countries in Asia for rapid economic growth has created serious problem of waste disposal (ISWA and UNEP, 2002).

Industrial sewage sludge has also a serious problem for the society (Elvira et al., 1998, Gajalakshmi et al., 2002) that contains toxic heavy metals which added in soil to food chain and finally entered to human body (Hernandez et al., 1991; CRI 1994; Hu 2002). Heavy metals had adversely affect to the animal health and human being due to accumulation in specific organs i.e. kidney and liver (Longerwerff, 1972).

A possible way to utilize industrial wastes and live excreta by vermibiotechnology (Benifez et al., 1999; Mills 2006; Bhartiya and Singh, 2011; Chauhan and Singh, 2012; Rai and Singh, 2012). Tiquia et al. (2002) stated that sewage sludge can use as organic fertilizer and soil amendment, but its odors, heavy metal content, toxic organic compounds, and pathogens show the necessity of treatment and stabilization before application to farm lands. The large amount of organic residues including sewage sludge, animal wastes, crop residues and industrial refuse are converted into vermicomposts to the use of earthworms (Dominguez and Edwards 1997, Kale, 1998, Chauhan and Singh, 2012). Earthworms are important link in the food chain and they can accumulate the hazardous elements from the soil (Handriks et al., 1995; Spurgeon and Hopkin, 1996; Bhartiya and Singh, 2012a). The experimental earthworm Eisenia fetida an important epigeic worm, short life cycle, high growth and reproduction rate that produces stable humus and nutriments available for the plants from organic matter (Garg et al., 2005; Chauhan, 2013). There is a broad spectrum of organic waste in the diet of the worm that comes from animals (Bhartiya et al., 2011), vegetables, alimentary, textile, winemaking industries and sewage sludge (Bhartiya and Singh, 2011).

The earthworm Eisenia fetida have to accumulate the heavy metals in their bodies from soil as well as different biological wastes during vermicomposting (Sexena and Chauhan, 1998; Morgan and Morgan, 1999; Leonard and Dolfing, 2001; Dei and Becquer, 2004; Jain and Singh, 2004; Bhartiya and Singh, 2012a,b). Sellanduria et al. (2009) reported that from the municipal solid wastes the E. fetida and Eudrilus eugeniae are effective in reducing of the metal toxicity. Anastasi et al. (2005) reported that the use of redworm for processing sewage sludge increases the content of nutrients easily assimilable for plants in a vermicompost, which affects better quality of plant biomass and management of some bacterial and fungal diseases. Jain and Singh (2004) reported that the system of decomposition and excretion of organic wastes through the metabolic system of earthworms is vermicomposting; its simple and low-cost technique can be used in the removal of toxic metals and the breakdown of complex chemicals to non-toxic forms.

MATERIALS AND METHODS:

Collection of Different Wastes

The different animal wastes buffalo, horse, pig and sheep dung were collected from different farm houses of the Gorakhpur district and sewage sludge were collected from different part of Gorakhpur city and exposed to sun light for 5 to 10 days for removal of various harmful organism and noxious gases.

Collection of Earthworm

Earthworm Eisenia fetida an epigeic species have cultured in Vermiculture Research Center, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur. The collected earthworms will be cultured in laboratory condition, temperature (20 ⁰C to 30 ⁰C) and aeration, moisture have maintained up to 40% to 60% for proper growth and survival of earthworms (Chauhan, 2013).

Experimental setup for Vermicomposting

The vermicomposting conducted on cemented earth surface. The different combinations of animal dung with sewage sludge in 1:1, 1:2 and 1:3 ratios were used for preparation of vermibeds. The size of each vermibed is 3m × 1m × 9cm. After the preparation of vermibed were inoculated 2kg of cultured Eisenia fetida in each bed. The beds were covered with jute pockets and moisten the beds daily up to 40 to 50 days for maintaining the moisture content. After one week interval, each vermibed were manually turned over up to 3 weeks. After 60 days granular tea like vermicompost appear on the upper surface of each bed. The prepare vermicomposts and inoculated earthworm were used for chemical analysis in experiments.

Analysis of Heavy Metals in Initial feed mixture and Final Vermicompost

The heavy metal content of the initial feed mixture and final vermicompost were measured by the method of Maboeta (2003). About 1 gm of initial feed mixture and final vermicompost required the samples. These sample will be subjected to digestion by adding excess of nitric acid (1:1) and were placed on hot plate and heated for 4 hours at 90 ⁰C to 100 ⁰C. It will be take care to ensure that simple did not dry out during digestion. After digestion sample will be poured into 100 ml flask through Whatman No 41 filter paper and injected into flame atomic absorption for determination of the heavy metal concentration.

Analysis of Heavy metals in Earthworm Body

The heavy metals in the earthworm body tissue will be digested using by the method of Katz and Jenneis (1983). Earthworm will be individually dried, ground and burned to ash at high temperature. Afterwards the ash will be placed in test tube about 10 to 15 ml of 55% nitric acid will be added in it. The solution will be left for 12 hrs at room temperature. After that the sample will heated at 40 ⁰C to 60 ⁰C for 2 hrs and then at a temperature of 120 ⁰C to 130 ⁰C for one hrs solution will be called at room temperature. Reheated the sample at 120 ⁰C to 130 ⁰C and 1 ml of 70% perchloric acid will be added. The sample will be allowed to cooling before adding 5 ml of distilled water. Samples will be again reheated up to 130 ⁰C until white fumes emitted. The sample will be allowed to cool finally before being micro filtered. The solution will be filtered through Whatman No 41 filter paper in to 100 ml flasks and will be measured. The heavy metals content in earthworm body by flame atomic absorption.

Statistical Analysis

All the data are mean ± SD of 6 replicates. Students ‘t’ test was applied to determine the significant (P<0.05) difference between combination of buffalo, horse, pig and sheep dung with sewage sludge of initial, final vermicompost, before inoculation and final earthworm E. fetida body (Sokal and Rohlf, 1973).

RESULTS AND DISCUSSION:

The significant decreased concentration of heavy metals (Co, Cr, Pb, Ni, Cd and As) were observed in final vermicompost of buffalo, horse, pig and sheep dung with sewage sludge (SS) than initial feed mixture. The cobalt (Co) was observed significantly change in vermicompost of buffalo and horse dung control in the level of BDL which showed the significant decrease 99.934 and 99.853% during vermicomposting respectively. The Lead (Pb) was observed 0.601 to 1.546 mg/kg in the initial feed material of different combination of SS with livestock while after vermicomposting decrease 52.58 to 83.69%. The minimum conc. of Pb (0.087±0.005 mg/kg) was observed in vermicompost of HD+SS (1:3) (Table 1-2).

Table 1. Concentration of heavy metals (mg/kg) in combination of different animal dung with sewage sludge in initial feed mixture of vermibed.

Particulars	Ratios	Heavy metal concentration in mg/kg
		Co			Pb
		Initial	Final	% Change	Initial	Final	% Change
SS	-	0.294 ±0.003	0.128±0.002*	-56.463	0.924 ±0.005	0.237 ±0.003*	-74.351
BD	-	0.152 ±0.004	BDL *	99.934	0.658 ±0.002	0.312 ±0.005*	-52.584
BD+SS	1:1	0.178 ±0.003	0.027 ±0.003	-84.831	1.004 ±0.002	0.265 ±0.004*	-73.606
	1:2	0.245 ±0.003	0.089 ±0.002*	-63.673	1.231±0.005	0.346 ±0.003*	-71.893
	1:3	0.310 ±0.004	0.107 ±0.004*	-65.484	1.423±0.003	0.413±0.002*	-70.977
HD	-	0.068 ±0.005	BDL*	99.853	0.601±0.002	0.098±0.005*	-83.694
HD+SS	1:1	0.153 ±0.004	0.042 ±0.003*	-72.549	0.815±0.003	0.231 ±0.002*	-71.656
	1:2	0.217 ±0.004	0.063 ±0.004*	-70.968	0.969±0.003	0.247 ±0.004*	-74.510
	1:3	0.269 ±0.003	0.079 ±0.004*	-70.632	1.401±0.004	0.258 ±0.004*	-81.585
PD	-	0.745 ±0.003	0.209 ±0.003*	-71.946	1.412±0.005	0.545 ±0.005*	-61.402
PD+SS	1:1	0.635±0.002	0.286 ±0.005	-54.961	1.186±0.006	0.529 ±0.006*	-55.396
	1:2	0.589±0.003	0.137 ±0.003*	-76.740	0.992±0.006	0.311 ±0.002*	-68.649
	1:3	0.426±0.004	0.109 ±0.003*	-74.413	0.654±0.003	0.156 ±0.005*	-76.147
SD	-	0.081 ±0.002	0.024 ±0.005*	-70.370	1.546 ±0.003	0.317 ±0.003*	-79.495
SD+SS	1:1	0.156 ±0.004	0.065 ±0.004*	-58.333	0.819 ±0.002	0.258 ±0.004*	-68.498
	1:2	0.214 ±0.004	0.074 ±0.005*	-65.421	0.632 ±0.003	0.154 ±0.003*	-75.633
	1:3	0.236 ±0.003	0.102 ±0.003*	-56.780	0.511 ±0.003	0.087 ±0.002*	-82.975

* Significant P<0.05 “t” test between initial feed mixture and final vermicompost. Each value is the Mean ±SD of six replicates. BDL-Below detectible limit i.e. 0.0001 mg/kg, SS-sewage sludge, BD-buffalo dung, HD-horse dung, PD-pig dung, SD-sheep dung.

Table 2. Concentration of heavy metals (mg/kg) in combination of different animal dung with sewage sludge in final earthworm body after vermicomposting.

Particulars	Ratios	Heavy metal concentration in earthworm Eisenia fetida body (mg/kg)
		Co			Pb
		Before Inoculation	After Vermicomposting	% Change	Before Inoculation	After Vermicomposting	% Change
SS	-	9.687± 0.003	9.795±0.004*	1.103	13.296 ±0.011	13.852±0.002*	4.014
BD	-	9.687±0.003	9.762±0.006*	0.768	13.296 ±0.011	13.524±0.005*	1.686
BD+SS	1:1	9.687±0.003	9.781±0.003*	0.961	13.296 ±0.011	13.971±0.005*	4.831
	1:2	9.687±0.003	9.793±0.005*	1.082	13.296 ±0.011	14.106±0.003*	5.742
	1:3	9.687±0.003	9.827±0.005*	1.425	13.296 ±0.011	14.118±0.002*	5.822
HD	-	9.687±0.003	9.701±0.005*	0.144	13.296 ±0.011	13.714±0.003*	3.048
HD+SS	1:1	9.687±0.003	9.759±0.003*	0.738	13.296 ±0.011	13.805±0.002*	3.687
	1:2	9.687±0.003	9.833±0.005*	1.485	13.296 ±0.011	13.821±0.002*	3.799
	1:3	9.687±0.003	9.851±0.003*	1.665	13.296 ±0.011	14.178±0.003*	6.221
PD	-	9.687±0.003	10.127±0.003*	4.345	13.296 ±0.011	13.864±0.004*	4.097
PD+SS	1:1	9.687±0.003	10.039±0.004*	3.506	13.296 ±0.011	14.009±0.005*	5.090
	1:2	9.687±0.003	10.016±0.003*	3.285	13.296 ±0.011	13.719±0.003*	3.083
	1:3	9.687±0.003	9.958±0.002*	2.721	13.296 ±0.011	13.537±0.004*	1.780
SD	-	9.687±0.003	9.723±0.004*	0.370	13.296 ±0.011	14.319±0.005*	7.144
SD+SS	1:1	9.687±0.003	9.715±0.003*	0.288	13.296 ±0.011	13.722±0.005*	3.105
	1:2	9.687±0.003	9.803±0.005*	1.183	13.296 ±0.011	13.689±0.003*	2.871
	1:3	9.687±0.003	9.793±0.004*	1.082	13.296 ±0.011	13.671±0.006*	2.743

* Significant P<0.05 “t” test between earthworm body of each vermibeds before inoculation and after vermicomposting. Each value is the Mean ±SD of six replicates. SS-sewage sludge, BD-buffalo dung, HD-horse dung, PD-pig dung, SD-sheep dung.

Edwards and Bohlen (1996) reported that the organic matter ingested by earthworm under goes different chemical and microbial changes during vermic activity, the part of organic matter is digested and pH of the microbial activity content increased. Suthar et al. (2008) stated that different metal content in final vermicompost are related to the different rate of physiological metabolism of earthworm. It is possible that vermic activity, growth and development of earthworm E. fetida are better in combination of kitchen waste with cow and sheep dung (Nath et al., 2009) and E. fetida are highly accumulate heavy metals in body from buffalo dung with kitchen waste (Bhartiya and Singh, 2011). It is clear that the reduction of heavy metals concentration of different animal dung with kitchen wastes was directly related to earthworm activity during wastes decomposition system (Devliegher and Verstraete, 1996).

The significantly increase the heavy metals concentration in E. fetida body after vermicomposting in different combinations of buffalo, horse, pig and sheep dung with sewage sludge after vermicomposting. The Co was significantly increase (4.34%) in earthworm body (10.127±0.003 mg/kg) when treated in the combination of PD control. The Pb was also observed in the earthworm body after treatment of SS with different combination of animal dung. The combination of BD+SS in 1:3 ratio showed the significant increase (7.14%) Pb in earthworm body at the level of (14.319±0.005 mg/kg) compare to 13.296 ±0.011 mg/kg in the body of earthworm before inoculation (Table 2). Morgan and Morgan (1999) reported that accumulation of metals (Cd, Cu, Pb, Zn and Ca) by earthworm species during vermicomposting. Earthworm E. fetida have ability to bioaccumulation the heavy metals in their body from municipal solid wastes (Conder et al., 2003; Suthar and Singh, 2008; Bhartiya and Singh, 2012). E. fetida have to accumulate the heavy metals in their bodies from soil as well as different biological wastes during vermicomposting (Sexena and Chauhan, 1998; Morgan and Morgan, 1999; Leonard and Dolfing, 2001; Dei and Becquer, 2004; Jain and Singh, 2004).

CONCLUSION:

There was significant increase of heavy metals in the body tissue of E. fetida due to zooaccumulation of these metals whereas decreased heavy metals level in final vermicompost of different animal dung with sewage sludge and the combination of horse dung with sewage sludge E. fetida have maximum accumulation of heavy metals in their body. The earthworm E. fetida play important role in management of heavy metals as well as sewage sludge with different combinations of animal dung. From present study it is clearly demonstrated that E. fetida accumulate heavy metals in their body and decreased heavy metals in the final vermicompost of all combination of animal dung with sewage sludge. Vermibiotechnology is useful process for the management of the heavy metals from different wastes and protect the human health and environment.

ACKNOWLEDGEMENT:

Authors are thankful to Prof. V. K. Garg, Associate Professor, Department of Environmental Science and Engineering, Guru Jambheshwar University, Hissar, Hariyana, India for technical support during this study.

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Received on 27.04.2015 Modified on 20.05.2015

Research J. Science and Tech. 7(3):July- Sept. 2015; Page 183-190

DOI: 10.5958/2349-2988.2015.00025.X