Sludge Watch ==> Heavy Metals Accumulate in Earthworms in Muncipal Compost

[Maureen Reilly]

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Effect of Heavy Metals on Earthworm Activities During Vermicomposting of 
Municipal Solid Waste
Posted on: Sunday, 24 February 2008, 03:00 CST

By Kumar, Sunil Sharma, Vishal; Bhoyar, R V; Bhattacharyya, J K; 
Chakrabarti, Tapan

ABSTRACT: The effect of heavy metals on the activities of earthworm species 
Eudrillus eugineae was studied during vermicomposting of municipal solid 
waste (MSW) spiked with heavy metals. The activities of earthworms, in terms 
of growth and biomass production and number of cocoons produced, were 
monitored periodically, and the concentration of heavy metals in earthworms 
and substrates was determined at definite intervals. Laboratory- scale 
experiments were performed by mixing individual heavy metals in MSW. Copper, 
cadmium, chromium, lead, and zinc were selected for the study. The study 
concludes that heavy metals tend to accumulate in the body of earthworms; 
hence, the inherent concentration of heavy metals in the substrate before 
vermicomposting must be considered in view of composting of MSW and its 
application to soil. It was observed that copper and cadmium were toxic for 
the worms at 1.5 and 0.1 g/kg of the waste, respectively. The studies also 
suggest that earthworms are susceptible to the free form of heavy metals. 
Cadmium is the most toxic metal, followed by copper. Based on the 
investigation and observation, it was also found that earthworms should be 
separated from castings before the use of castings in soil amendments. Water 
Environ. Res., 80, 154 (2008).

KEYWORDS: earthworms, cocoons, contaminants, absorption, castings, biomass.

doi:10.2175/106143007X220824

Introduction

Rapid urbanization and a growing number of industries coupled with 
increasing population have resulted in a quantum increase of municipal solid 
waste (MSW) generation, and this production is expected to increase in 
subsequent years. Although large numbers of technologies are available for 
managing MSW, their applications are limited because of their high costs, 
infrastructure, and energy requirements. Options such as landfilling have 
become limited, as a result of the paucity of land for disposal, and 
incineration is not publicly acceptable or feasible or is very expensive, as 
a result of the low calorific value of MSW generated in India (NEERI Report, 
1996).

Management of MSW in an environmentally compatible manner adopting 
principles of economy, aesthetics, energy, and conservation is needed. 
Biotechnological treatments, such as composting and vermicomposting, have 
emerged as feasible options for converting MSW into nutrient-rich compost or 
organic biofertilizer.

Vermicomposting is a process in which earthworms play a major role with 
microbes in the conversion of organic solid waste into more stabilized and 
nutrient-rich compost that is rich in major and micronutrients, such as 
nitrogen, phosphorus, potassium, magnesium, sodium, and zinc (Benitez et 
al., 2002). Vermicomposting seems to provide a good quality end product 
without odor and is also aesthetic in appearance. Moreover, it does not 
require skilled personnel and mechanization. The MSW contains an appreciable 
amount of nutrients that can provide ample nutrition if applied as a manure. 
The use of these materials is possible only when they are converted to 
harmless substrates, such as by a bioprocessing approach (Beyer et al., 
1982).

However, the process is influenced by various physicochemical factors, among 
which, the toxic heavy metals are of much concern. A review of research on 
the use of earthworms in MSW processing and application of vermicasts in 
land has raised con- , cerns over the concentration of heavy metals in MSW. 
These metals may adversely affect earthworm activities and the overall 
vermicomposting process. Earthworms are known to accumulate heavy metals 
(Amoji et al., 2000; Ash and Lee, .1980; Hopkin, 1989). Vermicompost from 
solid waste invariably will contain these elements, although at low 
concentrations. Heavy metals selected for this study are copper, cadmium, 
chromium, lead, and zinc.

Metals enter the MSW stream from a variety of sources. Industrial 
discharges, pharmaceutical waste, insecticides, batteries, motor oils, paint 
chips, consumer electronics and electricals, house dust, lead foils, 
vehicular emissions, and other allied activities introduce metals to the 
solid waste stream. Heavy metals have relevance in the following two ways:

(1) They may be lethal to worms, thereby affecting vermiconversion; and

(2) They may enter the food chain through the application of MSW or MSW 
compost to soil.

In small amounts, trace elements, such as copper, zinc, and chromium, are 
essential for the growth of plants or animals. Cadmium, lead, and chromium 
are of prime concern as contaminants because of their potential to harm soil 
organisms, animals, and humans, and these should receive close scrutiny in 
relation to the application of MSW to agricultural land and to the overall 
vermicomposting process (Atiyeh et al., 2000; Collins and Stotzky, 1989). 
Information on vermicomposting of MSW, in terms of heavy metal concentration 
and its effect on earthworms, are scanty.

The present study envisages primarily how these heavy metals affect 
earthworm activities, in terms of their growth, reproduction, and cocoon 
production, and the overall process of vermicomposting. In the present 
study, the valence states of heavy metals in the waste during composting are 
not considered.

Experimental Studies

Laboratory-scale experiments were conducted, in triplicate, to determine the 
effect of heavy metals on the activities of earthworms, in terms of their 
growth, population, and cocoon formation and the effects on the process of 
vermicomposting. Municipal solid waste containing an inherent concentration 
of heavy metals was used as a control during various studies. To avoid the 
influence of decomposition products, such as volatile fatty acids and 
gaseous ammonia, and the effects of increased temperature in the waste mass, 
the waste was allowed to predecompose for approximately 15 days (Amoji et 
al., 2000). The predecomposed MSW (approximately 2 kg dry wt.) was placed in 
earthen pots and used as a substrate for conducting experiments. The 
experiments were conducted for 90 days.

Materials. All of the chemicals and reagents used in the study were of 
analytical grade. The Merck inductively coupled plasma (ICP) multi-element 
standard solution IV (23 elements) (Merck KgaA, 64271, Darmstadt, Germany) 
was used as a standard for plotting calibration curves and for determination 
of the concentration of heavy metals by inductively coupled plasma atomic 
emission spectrometry (ICP-AES). The ICP-AES (Perkin-Elmer, Wellesley, 
Massachusetts) was used to analyze heavy metals in earthworms and substrate 
samples (Beyer et al., 1982).

The metals, such as copper, cadmium, chromium, lead, and zinc, in the form 
of metallic salts, were used in the study. The MSW was spiked with these 
metals in the concentration range 0.5 to 3.5 g/kg of the waste. Similarly, a 
10-mg/L solution of individual metals was prepared in double-distilled water 
to assess the effect of the free form of metals.

The MSW used as a substrate in the experimental studies was obtained from 
Municipal Corporation, Nagpur, India, which is a second capital of 
Maharashtra State, with an approximate population of 2.5 million. The 
physical composition of the waste is presented in Table 1.

Earthworms of the species Eudrillus eugineae were obtained from Go-vigyan 
Kendra, Nagpur, India. Cow dung, known to be one of the best natural feeds 
for earthworms, was used as a medium for culturing earthworms.

A microwave digestion system (Ethos 900, Milestone, Italy) was used for 
digesting earthworms and substrate samples (Booth et al., 2001).

Methods. The laboratory-scale investigations were carried out in the 
following three parts:

(1) Part I: Effect of heavy metals on earthworms in distilled water,

(2) Part II: Effect of inherent concentration of heavy metals in MSW, and

(3) Part III: Vermicomposting of MSW spiked with individual heavy metals.

The toxic effects of heavy metals vary, depending on the state of heavy 
metals (i.e., free forms), concentration, reactivity, complex formation, and 
stability (Nieboer and Fletcher, 1996). Studies were conducted to determine 
the effect of the free or organically bound form of heavy metals.

Effects of Heavy Metals in Distilled Water. To assess the effect of the free 
form of heavy metals, the individual metal solution of 10 mg/L copper, 
cadmium, chromium, lead, and zinc were prepared in double-distilled water. 
Double-distilled water was taken as a control for the study. Five adult 
earthworms were introduced to each metal solution, and preparations were 
made to monitor their activities. Similarly, various permutation 
combinations of metal solutions were prepared by mixing 10 mg/L aqueous 
solutions, and earthworms (5 per 500 mL) were added to the mixed metal 
solution. Table 2 presents finding of the individual and mixed heavy metals. 
The heavy metal concentration in the control MSW is presented in Table 3.

Vermicomposting of Municipal Solid Waste with an Inherent Concentration of 
Heavy Metals. The study was conducted to provide a larger and more 
comprehensive examination of vermicomposting of MSW, with respect to the 
concentration of heavy metals. The laboratory- scale studies (in triplicate) 
were performed in earthen pots. Adult earthworms (10 per kg of waste) were 
added to each pot. The temperature was maintained between 27 and 
29[degrees]C. Water was maintained between 45 and 50 % substrate by spraying 
water at a regular interval during the studies. The experimental pots were 
aerated by turning at definite intervals. Earthworms and substrate samples 
were obtained periodically and analyzed to determine die concentration and 
status of heavy metals. Similarly, the growth of earthworms, in terms of 
their population, reproduction, and cocoon formation, was also assessed 
periodically (i.e., in 30,60, and 90 days). The findings are discussed in 
the Results and Discussion section.

Vermicomposting of Municipal Solid Waste Spiked with Individual Heavy 
Metals. The toxicity of heavy metals to organisms depends on the valence 
state of the metals, contact time with the organism, and their concentration 
(Holm et al., 1995). The laboratory-scale experiments were performed in 
triplicate in earthen pots. Copper, cadmium, chromium, lead, and zinc were 
selected for the study. For every individual metal, experiments were 
conducted separately using different concentrations of heavy metals (0.5 to 
3.5 g/kg of waste). Simultaneously, experiments were conducted with MSW 
having an inherent concentration of heavy metals. Predecomposed MSW was 
mixed with individual heavy metals of the above-stated different 
concentrations. The metals were uniformly mixed; each earthen pot was 
labeled accordingly. Adult earthworms (10 per kg of waste) were added at the 
start of the experiment. The activities of the earthworms were monitored, in 
terms of their growth in numbers, biomass production, and cocoon production. 
Samples of earthworms and substrates were collected periodically and 
analyzed. The changes in chemical composition of the substrate, in terms of 
metal concentration (initial and final), were also determined. The pots were 
watered periodically, and the contents were mixed for aeration. The 
temperature was controlled to remain between 27 and 29[degrees]C. During the 
experiment, no new substrate was added. The results are depicted in Tables 4 
to 7 and Figures 1 to 3.

Sampling and Analysis. The concentrations of heavy metals were determined in 
earthworms and substrates samples and castings. Earthworms were lyopholized 
at - 30[degrees]C, dried at 100 +- 5[degrees]C, weighed, and digested with 
concentrated nitric acid (analytical-grade) in a microwave digestion system 
(Ethos 900), as per the methodology described by Knight et al. (1998) and 
Marquenie (1988). Similarly, substrate samples (0.5 g) were weighed, mixed 
with 10 mL concentrated nitric acid (analyticalgrade), and digested by a 
microwave digestion system. A preset microwave program of 15 minutes was 
adjusted in three steps, as follows: 5 minutes at 500 W, followed by 7 
minutes at 400 W, followed by 3 minutes at 250 W. The samples were filtered 
and analyzed for heavy metals. Estimation of heavy metals was carried out as 
described by Marinussen et al. (1997). Extraction of organically bound and 
exchangeable forms of heavy metals in substrates was carried out as per the 
method described by Emmerich et al. (1982).

The portions (both for the organically bound and exchangeable form of metal) 
were filtered by a Millipore filter assembly (Milhpore, Bedford, 
Massachusetts) with glass microfiber filter paper (pore size of 0.45 
micron), and the total volume was increased to 100 mL using 1 M nitric acid.

The concentrations of total heavy metal content in earthworms, substrates, 
and castings, and organically and exchangeable forms in substrates were 
measured by ICP-AES, as per the methodology described by Knight et al. 
(1998).

Results and Discussion

Solubility is an important determinant in metal toxicity (Nieboer and 
Fletcher, 1996). The extent of toxic agent that the animal body tolerates 
determines the reactivity. To obtain information on the effect of the free 
form of heavy metals on earthworms, the aqueous extract of metals was tested 
separately and in combination.

Copper constitutes an essential trace metal and hence was selected for 
investigation. Cadmium was selected because of its relatively higher 
toxicity, and chromium was selected because its toxicity depends on its 
oxidation state-Cr(IfI) or Cr(VI) (Kimbrough, 1999). Lead was chosen for 
study because it is deposited commonly from vehicular emission, lead foils, 
flue gases, and other sources, and zinc was selected for its high ambient 
concentration in MSW.

The result of the free form of heavy metals in the aqueous phase (Table 2) 
indicates a pronounced synergetic effect of the combination of metals in 
solution relative to the use of an individual metal in solution. The 
concentration of heavy metals was more in the integument than in the 
alimentary canal, suggesting integument absorption of metals in free form. 
The worms were dissected to separate the alimentary canal and integument. 
Copper and cadmium were highly toxic for worms, as the worms died within a 
few hours after introduction of the metals. Within hours of the introduction 
of free metals, the movements of earthworms became abnormal and later slowed 
down. In copper and cadmium solution, their clitella became swelled, and 
several ruptures in the body were seen also. It was observed that 
combinations of metals produced more pronounced toxicity than the individual 
metals (Olaniya and Bhide, 1990; Oste et al., 2001). The earthworms were 
presumed dead when they did not respond to external stimuli by touching or 
pricking. Earthworm mortality in aqueous metals solutions may result from 
ingestion and absorption through integument. Integumentary uptake of 
dissolved organics was reported by Sylvia and Arme (1982).

The concentrations of heavy metals inherently present in MSW obtained from 
Municipal Corporation, Nagpur, India, did not influence the activity of 
earthworms. Appreciable growth in the earthworm population was observed; the 
earthworms increased from an initial 20 to 400 in 90 days, and the number of 
cocoons was also observed to be in the hundreds. Analysis of the samples 
determined that the metals are more in organically bound forms. After 3 
months, the earthworms had turned the odorous and unaesthetic MSW into fine 
granular odorless and soft castings.

During aerobic degradation of the waste mass, the pH is in the alkaline 
range; therefore, solubilization of heavy metals is negligible. Under such 
conditions, the effect on the activity of the earthworm is a function of 
dose response (i.e., intake of feed substrate and concentration of heavy 
metals in it [Loehr et al., 1985; Raymond et al., 1985]). Hence, experiments 
were conducted to obtain a comprehensive feature of the vermicomposting 
process under different concentrations of heavy metals in MSW.

Studies were carried out by fortifying MSW with individual metals (copper, 
cadmium, chromium, lead, and zinc). Cadmium was the most toxic metal, 
followed by copper for earthworms, and hampered the overall vermicomposting 
process. Composting in the presence of cadmium at concentrations in excess 
of 0.1 g/kg was unfavorable for earthworm and vermicomposting, whereas 
copper was toxic at 1.5 g/ kg. However, at the concentrations provided, 
other metals, such as chromium, lead, and zinc did not affect the activities 
of earthworms; considerable growth, in terms of population and cocoon 
formation, was observed in experiments with these metals up to 2.5g/ kg. 
However, from 2.5 g/kg upwards, their concentration in waste became 
unfavorable for earthworms and the overall vermicomposting process. Tables 4 
and 8 and Figure 1 show the population of earthworms and production of 
cocoons during vermicomposting of MSW mixed with copper, chromium, lead, and 
zinc for 90 days. The studies with these metals are discussed below.

Studies with Copper. Copper is an essential trace metal for growth. Copper 
at 0.5 g/kg of waste was favorable for earthworms, as the percent increases 
in their population were 155, 610, and 800% for 30, 60, and 90 days, 
respectively. As the concentration of copper increased from 0.5 g/kg of 
waste, the population of earthworms and cocoons decreased and significantly 
decreased at the 1.5-g/kg concentration. Copper at 2.0 g/kg of waste became 
toxic for earthworms, as they were found dead with 15 days of the 
experiment. As the concentration of copper increased in the waste, the metal 
accumulated more in the earthworms (Figure 2).

Studies with Cadmium. Cadmium is well-known for its toxicity in organisms 
(Pearson et al., 2000). Cadmium at concentrations exceeding 0.1 g/kg of 
waste was lethal for earthworms, as they died within 1 week. The MSW spiked 
with cadmium at 0.1, 0.3, and 0.5 g/ kg of waste indicated that worms could 
tolerate cadmium concentrations up to 0.1 g/kg in the waste (Table 8).

Studies with Chromium, Lead, and Zinc The activities of earthworms were 
normal with Cr(III) from 0.5 to 2.5 g/kg of waste. The percent increases in 
their population were 295,500, and 1000% for 30, 60, and 90 days, 
respectively. The concentration of chromium was higher in castings than in 
earthworms. Chromium at 2.5 g/kg upwards was unfavorable for 
vermicomposting, as the population of earthworms decreased. Similarly, 
percent increases of 5, 80, and 355% for 30, 60, and 90 days, respectively, 
in the earthworm population occurred with lead concentrations between 0.5 
and 2.0 g/ kg wastes. Lead at 2.5 to 3.5 g/kg of waste became unfavorable 
for earthworms. Zinc also favored the process, until 0.5 to 2.5 mg/kg of the 
waste, as the percent increase in earthworm population was found to be 145, 
525, and 1375% for 30, 60, and 90 days, respectively; however, from 2.5 g/kg 
upwards, zinc also showed a pattern of marginal decrease in the population 
of worms, as reported by Spurgeon et al. (2000).

Tables 6 and 7 summarize the data on accumulation of heavy metals in 
earthworms and castings during 90 days of vermicomposting. It has been 
observed that heavy metals, such as copper and zinc, are accumulated more in 
earthworms than in castings, whereas lead and chromium accumulated more in 
castings, as reported by Rao and Rao (2001). Integument absorption of metals 
was more than absorption by the alimentary canal. Figures 2 and 3 present 
findings for the accumulation of heavy metals in earthworms and castings. 
Medium Difference and Bioconcentration Factor. When earthworms cultured in 
medium with a high concentration of heavy metals were transferred to a 
medium having comparatively lower heavy metals concentration (i.e., soil), 
the earthworms tended to release heavy metals from their body into the 
surrounding medium in which they are transferred, which can be concluded 
from Table 9.

The bioconcentration factor was calculated for copper, chromium, lead, and 
zinc, as per David and Hopkin (1996). The bioconcentration factor for 
copper, chromium, lead, and zinc was observed to range from 0.57 to 0.92, 
0.25 to 1.49, 0.11 to 0.55, and 0.84 to 1.47, respectively.

Conclusions

After comparing the data on earthworm population and cocoon formation, it is 
concluded that cadmium was the most toxic metal and hampered the process, 
even at 0.1 g/kg of waste. Copper up to a concentration of 1.5 g/kg of waste 
was favorable for earthworm populations and thereafter became unfavorable. 
Similarly, the activities of earthworms, such as growth, reproduction, and 
cocoon formation, were normal for chromium, zinc, and lead at concentrations 
between 0.5 and 2.5 g/kg of waste. However, from 2.5 g/kg upwards, their 
concentration in waste also became unfavorable for vermicomposting. 
Vermicomposting with lead at all concentrations showed an increase in 
population growth and cocoon formation, though at a reduced rate compared 
with chromium and zinc.

Credits

This manuscript has been prepared based on the findings of the project 
sponsored by the Department of Biotechnology, Government of India, New 
Delhi. The authors are thankful to the advisor and director of the 
Department of Biotechnology, Government of India, for their guidance and 
support in completing the project activities. The authors are very thankful 
to Sukumar Devotta, National Environmental Engineering Research Institute 
(Nagpur, India), for permission to release the manuscript.

Submitted for publication September 2, 2006; revised manuscript submitted 
June 14,2007; accepted for publication July 16, 2007.

The deadline to submit Discussions of this paper is May 15, 2008.

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Sunil Kumar*, Vishal Sharma, R. V. Bhoyar, J. K. Bhattacharyya, Tapan 
Chakrabarti

National Environmental Engineering Research Institute, Nehru Marg, Nagpur, 
India.

* National Environmental Engineering Research Institute, Kolkata Zonal 
Laboratory, 1-8, Sector 'C, East Kolkata, P.O. East Kolkata Township, 
Kolkata-700 107, West Bengal, India; e-mail: s_kumar at neeri.res.in, 
sunil_neeri at yahoo.co.in.