Bioaccumulation of Cobalt and Lead by Earthworm Eisenia fetida from Sewage sludge with different cattle dung during Vermicomposting


Keshav Singh1*, Deepak Kumar Bhartiya2, Harendra Kumar Chauhan2, Rahul Rai2,

Ram Nayan Singh3

1Vermiculture Research Laboratory, Department of Zoology, 

D.D.U. Gorakhpur University, Gorakhpur, 273009, UP, India

2Research Scholar, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur- 273009, UP, India

3Assistant Professor, Department of Zoology, K. N. I. P. S. S., Sultanpur-228118, UP, India

*Corresponding Author E-mail:



The aim of the present study was to investigate the heavy metals accumulated by earthworm Eisenia fetida from sewage sludge (SS) with different animal’s dung during vermicomposting. The cobalt (Co) and lead (Pb) were significantly decrease in vermicompost from all the combination of SS; maximum decreased Co (84.83%), and Pb (83.69%) from the SS as combination of BD+SS  (in ratio 1:1) and HD+SS (in ratio 1:3) respectively. The Co and Pb was significantly increase (4.34% and 7.14%) in earthworm body (10.127±0.003 and 14.319±0.005 mg/kg) when treated in the combination of PD control and BD+SS in 1:3 ratio respectively. The Pb was also observed in the earthworm body after treatment of SS with different combination of animal dung. The Co and Pb conc. were observed in earthworm body before inoculation at the level of (9.687±0.003 mg/kg) and 13.296 ±0.011 mg/kg respectively. These metals concentration were decreased in vermicompost resulted from as significantly increase in earthworm body as zooaccumulation during vermicomposting. The earthworm Eisenia fetida also important macro-organism was responsible for the reduction of heavy metals due to the accumulation in their body tissue.


KEYWORDS: Bioaccumulation, Eisenia fetida, sewage sludge, heavy metals and vermicomposting.



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.



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).



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.




Heavy metal concentration in mg/kg





% Change



% Change



0.294 ±0.003



0.924 ±0.005

0.237 ±0.003*




0.152 ±0.004



0.658 ±0.002

0.312 ±0.005*




0.178 ±0.003

0.027 ±0.003


1.004 ±0.002

0.265 ±0.004*



0.245 ±0.003

0.089 ±0.002*



0.346 ±0.003*



0.310 ±0.004

0.107 ±0.004*







0.068 ±0.005








0.153 ±0.004

0.042 ±0.003*



0.231 ±0.002*



0.217 ±0.004

0.063 ±0.004*



0.247 ±0.004*



0.269 ±0.003

0.079 ±0.004*



0.258 ±0.004*




0.745 ±0.003

0.209 ±0.003*



0.545 ±0.005*





 0.286 ±0.005



0.529 ±0.006*




0.137 ±0.003*



0.311 ±0.002*




0.109 ±0.003*



0.156 ±0.005*




0.081 ±0.002

0.024 ±0.005*


1.546 ±0.003

0.317 ±0.003*




0.156 ±0.004

0.065 ±0.004*


0.819 ±0.002

0.258 ±0.004*



0.214 ±0.004

0.074 ±0.005*


0.632 ±0.003

0.154 ±0.003*



0.236 ±0.003

0.102 ±0.003*


0.511 ±0.003

0.087 ±0.002*


* 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.




Heavy metal concentration in earthworm Eisenia fetida body  (mg/kg)



Before Inoculation

After Vermicomposting

%  Change

Before Inoculation

After Vermicomposting

%  Change



9.687± 0.003



13.296 ±0.011








13.296 ±0.011








13.296 ±0.011







13.296 ±0.011







13.296 ±0.011








13.296 ±0.011








13.296 ±0.011







13.296 ±0.011







13.296 ±0.011








13.296 ±0.011








13.296 ±0.011







13.296 ±0.011







13.296 ±0.011








13.296 ±0.011








13.296 ±0.011







13.296 ±0.011







13.296 ±0.011



* 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).



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.  



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.



Anastasi, A., Varese, G.C. and Marchisio, V.F., 2005. Isolation and identifi cation of fungal communities in compost and vermicompost. Mycologia, 97(1). 33-44.

Barrera, L. and Andres, P., 2001. Sewage sludge application on soils: effects on two earthworms species. Water, Air and Soil Pollution, 12. 319-32.

Benifez, E., Nogales, R., Elvira, C., Masciandaro, G. and Ceccanti, B., 1999. Enzyme and earthworm activities during vermicomposting of carbaryl–trated sewage sludge. Journal of Environmental Quality, 28. 1099-1104.

Bhartiya DK Singh K (2012b) Heavy metals remediation from maize (Zea mays) crop by the use of vermicomposts through vermicomposting by Eisenia fetida. American-Eurasian J Agric Environ Sci 12 (9):1215-1222

Bhartiya DK, Nath G, Singh K (2011) Vermicomposting; A tool for management of different wastes and self-employment for youth of B.P.L. and weaker sections. Proceeding of National Seminar on ‘Challenges for Biosciences in 21st Century’. Department of Zoology, S. P. P. G. College Shohratgarh, Siddharthnagar, p. 71-73

Bhartiya, D.K. and Singh, K., 2011. Accumulation of Heavy Metals by Eisenia foetida from Different animal dung and Kitchen wastes during Vermicomposting. International Journal of Life Science and Technology, 4(7). 47-52.

Bhartiya, D.K. and Singh, K., 2012a. Heavy Metals Accumulation from Municipal solid wastes with Different animal dung through Vermicomposting by Earthworm Eisenia foetida. World Applied Sciences Journal, 17 (1). 133-139.

Chauhan, H.K. 2013. Effect of different combinations of animal dung and agro wastes on the reproduction and development of earthworm Eisenia fetida. Ph.D. Thesis. Deen Dayal Upadhyay, Gorakhpur University, Gorakhpur U.P. India.

Chauhan, H.K. and Singh, K., 2012. Effect of binary combinations of buffalo, cow and goat dung with different agro wastes on reproduction and development of earthworm Eisenia fetida (Haplotoxida: Lumbricidae). World Journal of  Zoology, 7. 23-29.

Conder, J.M., Seals, L.D. and Lanno, R.P., 2003. Method for determining toxicologically relevant cadmium residues in the earthworm Eisenia foetida, Chemosphere, 49. 1-7.

CRI 1994. Report of the Soil and Plant Nutrient Division. Annual Report Coconut Research Institute, Lunuwila, Sri Lanka. p. 83-85.

Dei, J. and Becquer, T., 2004. Heavy metal accumulation by two earthworm species and its relationship to total and DTPA- extrctable metals in soil. Soil Biology and Biochemistry, 36. 91-98.

Devliegher, W. and Verstraete, W., 1996. Lumbricus terrestris in a soil core experiment: effect of nutrient-enrichment processes (NEP) and gut associated processes (GAP) on the availability of plant nutrients and heavy metals. Soil Biology and Biochemistry, 28. 489-496.

Dominguez, J. and Edwards, C.A., 1997. Effects of stocking rate and moisture content on the growth and maturation of E. anderi in pig manure. Soil Biology and Biochemistry, 29. 743-46.

Edrissbazrafshan, Zazouli, M.A., Bazrafshan, J. and Bandpei, A.M., 2005. Evaluation of Microbiological and chemical parameters during wastewater sludge and sawdust co-composting. Journal of Applied Sciences and Environmental Management, 10(2). 115-119.

Edwards, C.A. and Bohlen, P.J., 1996. Biology and ecology of earthworms. 3rd ed. Chapman and Hall, London, UK.

Elvira, C., Sampedro, L., Benitez, E. and Nogales R., 1998. Vermicomposting of sludge from paper mill and dairy industries with Eisenia andrei: A pilot scale study. Bioresource Technology, 63. 205-211.

Gajalakshmi, S., Ramasamy, E.V. and Abbsi, S.A., 2002. Vermicomposting of paper wastes with the anecic earthworm Lampito mauritii (Kingberg). Indian Journal of Chemical Technology, 9. 306-311.

Garg, V.K., Chand, S., Chhillar, A. and Yadav, A., 2005. Growth and reproduction of Esenia foetida in varios animal waste during vermicomposting. Applied Ecology and Environmental Resource, 3. 51-59.

Gupta, R. and Garg, V.K., 2008. Stabilization of primary sewage sludge during vermicomposting. Journal Hazard Material, 153. 1023-1030.

Handriks, A.J., Ma, W.C., Brounds, J.J., De Ruiter-Dijkman, E.M. and Gart, R., 1995. Modeling and monitoring organochloride and heavy metal accumulation in soils, Earthworms, and Shrews in Rhine-Delta floodplains. Archives of Environmental Contamination and Toxicology, 29. 115-127.

Hernandez, T., Moreno, J.I. and Costa, F., 1991. Influence of sewage sludge application on crop yields and heavy metal availability. Soil Science Plant Nutrition, 37. 201-210.

Hu, H., 2002. Human health and heavy metal exposure. The environmental and Human Health, (pp. 1-13). Michael Mc Callyed., MIT press.

International Solid Wastes Association and United National Environment Programme (ISWA and UNEP) (2002). Wastes Management, Industry as a partner for sustainable development, 92-807-2194-2.

Jain, K., and Singh, J., 2004. Modulation of fly ash induced genotoxicity in vicia faba by vermicomposting. Ecotoxicology and Environmental Safety, 59. 89-94.

Kale, R.D., 1998. Earthworms: natures gift for utilization of organic wastes. Earthworms Ecology. CRC Press, Boca Raton FL. 355-77.

Katz, S.A., and Jenneis, S.W., 1983. Regulatory compliance monitory by atomic absorption spectroscopy.

Leonard, A.O., and Dolfing, J., 2001. Cadmium uptake by earthworms as related to the availability in the soil in the intestine. Environ. Contam. Toxicol., 20. 1786-1791.

Longerwerff, J.V., 1972. Lead, Mercury and Cadmium as Environmental contaminants. In Micronutrients in Agricultur. Soil Science Society of America, Tnc., Madison, Wisconsin. pp: 596-636.

Maboeta, M., 2003. Vermicomposting of industrially produced wood chips and sewage sludge.utilizing E. foetida. Ecotoxicology and Environmental Safety, 56. 265-270.

Martin, S., and Griswold, W., 2009. Human Health Effect of Heavy Metals. Environ Science and Technology Briefs for Citizens, 15, 1-6.

Mills, T., 2006. Composting cafeteria residuals with earthworm. Biocycle. 47. 55-55.

Morgan, J.E., and Morgan, A.J., 1999. The accumulation of metals (Cd, Cu, Pb, Zn and Ca) By two ecologically contrasting earthworm species. Applied Soil Ecology, 13. 9-20.

Nath, G., Singh, K., and Singh, D.K., 2009. Chemical analysis of Vermicomposts/Vermiwash of different combinations of animal, agro and kitchen wastes. Australian Journal of Basic and Applied Science, 3(4). 3672-3676.

Rai R, Singh K (2012) Physico–chemical analysis and Management of different combinations of sugar mill and distillery effluents with different animal dungs during vermicomposting by earthworm Eisenia fetida. J Bio Agri Health 2(11):21-28

Saxena, M., and Chauhan, A., 1998. Flyash vermicomposting from non-organic wastes. Pollution Research, 17. 5-11.

Sellanduria, G., Ambusaravanan, N., Shyam, K.P., Palanivel, K. and  Kadalmani, B., 2009. Biomanagement of municipal sludge using epigenic earthworms Eudrilus eugeniae and Eisenia foetida. Advances in Environmental Biology, 3(3). 278-284.

Sokal, R. and Rohlf, F.J., 1973. Introduction to Biostatistics. W.H. Freeman and Co.San Fransisco.

Spurgeon, D.J. and Hopkin, S.P., 1996. Risk assessment of the threat of secondary poisoning by metals to predators of earthworms in the vicinity of a primary smelting works. Science of Total Environment 7. 167-183.

Suthar S, Singh S, Dhawan S (2008) Earthworm as bioindicators of metals (Zn, Fe, Mn, Cu, Pb and Cd) in soils: Is metal bioaccumulation affected by their ecological categories. Ecol Eng 32: 99-107

Suthar, S. and Singh, S., 2008. Biococentrations of metals (Fe, Cu, Zn, Pb) in Earthworm (Eisenia foetida), Inoculation in Municipal Sewage Sludge: Do Earthworms Pose a Possible Risk of Terrestrial Food Chain Contamination?. Environmental Toxicology, 24. 25-32.

Tiquia, S.M., Wan, J.H.C. and Tam, N.F.Y. 2002. Microbial population dynamics and enzyme activities during composting. Compost Science and Utilization, pp. 150–161.

Ye, J.S., Wang, M., Barger, V. and Shi Castranava, X., 2000. Activation of androgen response element by cadmium; a potential mechanism for a carcinogenic effect of cadmium in the prostate. Journal of Environmental Pathology, Toxicology and Oncology, 19. 275-80.



Received on 27.04.2015       Modified on 20.05.2015

Accepted on 25.05.2015      ©A&V Publications All right reserved

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

DOI: 10.5958/2349-2988.2015.00025.X