Bioresource Technology 96 (2005) 1867–1871

Bioremediation of municipal sludge by vermitechnologyand toxicity assessment by Allium cepa

Richa Srivastava a, Dinesh Kumar b, S.K. Gupta a,*

a Cell Biology Section, Industrial Toxicology Research Centre, P.O. Box 80, M.G. Marg, Lucknow 226 001, Indiab Department of Botany, University of Lucknow, Lucknow 226 007, India

Received 1 October 2004; received in revised form 20 January 2005; accepted 29 January 2005

Available online 31 May 2005

Abstract

The aim of this study was to evaluate municipal sludge (MS) for its toxic potential by Allium cepa and also to understand the

effect of vermicomposting on the reduction of toxicity, if any. Municipal sludge (MS) and vermicomposted sludge (VS) were eval-

uated. Elemental analysis of MS showed the presence of heavy metals. Morphological studies of A. cepa roots indicated coiled and

wavy roots on exposure to MS but no root abnormality was reported in VS. Under genotoxic studies, inhibition in mitotic index was

concentration dependent and the control values of 11.76 gradually reduced to 5.40 at 10% MS leachate whereas mitotic index was

increased to 9.48 at 10% VS leachate. Exposure of leachate induced chromosomal aberrations, micronucleus formation and binu-

cleate cells in a dose dependent manner. However, mitotic aberrations were observed significant at 10% MS leachate but they were

insignificant at 10% VS leachate. The wet and dry weight of roots, root elongation and chlorophyll contents were reduced as the

concentration of leachate increased but VS leachate did not produce considerable reduction. The wet and dry weight of A. cepa roots

were 20.312 g and 3.250 g respectively and they were reduced to 10.82 g and 1.68 g respectively at 10% MS leachate but VS leachate

showed an increase to 18.127 g and 2.53 g respectively. Total chlorophyll in control, 10% MS leachate and 10% VS leachate were

0.245 g, 0.162 g and 0.214 g respectively. It could be concluded that the MS was toxic to a remarkable extent but vermicomposting

of sludge might be beneficial for bioremediation and recommended before land filling.

� 2005 Elsevier Ltd. All rights reserved.

Keywords: Municipal sludge leachate; Allium cepa; Eisenia foetida; Metals; Chromosomal abnormalities; Vermicomposting

1. Introduction

Leachates from active and closed municipal solid

waste (MSW) landfills can be a major source of contam-

ination to groundwater and surface waters (Hancock

et al., 1995; Flyhammer, 1997; Ding et al., 2001). Gener-ally industrial solid waste/sludge is also mixed to MSW

and used for landfills, which also potentiate the

toxicity of MSW due to various pollutants. Landfill

0960-8524/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2005.01.029

* Corresponding author.

E-mail address: itrcskg@rediffmail.com (S.K. Gupta).

leachate is generated by excess rainwater percolating

through the waste layers in a landfill (Christensen and

Tjell, 1984). The leachates of various hazardous constit-

uents of the waste contaminates soil as well as water

bodies. Toxicity of leachate was reported using multiple

bioassays (Cameron et al., 1982; Plotkin and Ram, 1984;Calleja et al., 1986). Genotoxicity is one of the most

dreaded effects of leachates for future generation. Muni-

cipal sludge may contain heavy metals and organic pol-

lutants, which are potentially harmful to crops and

microorganisms in soil. Contaminated crops reduce

quality of the feed and food (McLaughlin et al., 1999;

Giller et al., 1998; Das et al., 1997; McBride, 1995).

The utility of plant bioassay to evaluate the genotoxicity

1868 R. Srivastava et al. / Bioresource Technology 96 (2005) 1867–1871

of heavy metal contaminated soils and water is well

accepted (Knasmuller et al., 1998). Allium cepa, a sensi-

tive, reliable and cost effective test system is routinely

used for environmental biomonitoring (Grant, 1982;

Fiskesjo, 1985). Eisenia foetida were used to vermicom-

post woodchip and sewage sludge that are produced aswaste product by platinum mines (Maboeta and Rens-

burg, 2003). In the present communication, Allium cepa

was used to evaluate the genotoxicity of the leachate of

MSW and the leachate of postvermicomposted sludge

and earthworm E. foetida was used for vermicompos-

ting.

2. Methods

2.1. Sample collection

The Municipal sludge was collected from a disposal

site situated on Lucknow–Kanpur highway in the state

of Uttar Pradesh (India) having one automobile indus-

try in the vicinity. The semisolid sludge was air driedat room temperature to get dried sludge.

2.2. Preparation of leachates

Ten percent leachate of solid waste was prepared

according to the method described by French Standard

method (Ferrari et al., 1999). In brief, 100 g of MS/VS

was added to 1000 ml of distilled water, which was thenkept on a rotary shaker at 180 rpm at 30 ± 1 �C for 24 hfor continuous shaking. The suspension was first coarse

filtered by glass wool, and then by Whatman filter paper

No. 42. To remove the fine suspended particles it was

centrifuged at 3000 rpm for 15 min and the supernatant

was used after making test dilutions (2.5%, 5.0% and

10%) with double distilled water.

pH of MS and VS leachates was taken with the helpof pH meter. To determine the pH of sludge, 1:2

(sludge:deionised water) was thoroughly mixed with

the help of a magnetic stirrer for 3 h. After settling down

the particulate matter, the fluid was decanted separately

and the pH was determined. To determine the pH of

leachate, the 10% leachate was directly used.

2.3. Vermicomposting

MS was mixed with the cow dung manure in equal

parts (1:1, w/w) and mixed homogeneously in a mechan-

ical mixer. Sufficient moisture (30% approx.) was main-

tained by adding tap water. One hundred healthy and

clitellate earthworms (E. foetida) were inoculated in

the 10 kg mixture for vermicomposting. After 30 days,

the earthworm population was separated from the ver-micompost. Only 10% leachate of VS was prepared as

described earlier for further studies.

2.4. Test concentrations of leachates

Three test concentrations prepared as above were

used to study the effect of leachate of MS but only one

concentration of leachate (10%) was used in the case

of VS.

2.5. Metal analysis

The sludge and plant samples were air dried and

digested in a mixture of perchloric acid:nitric acid (1:6)

until white residue remained at the bottom of the flask.

The residue was dissolved in 0.1 N HNO3 (Berman,

1980). The concentrations of metals in MS leachateand A. cepa were determined by Inductively Coupled

Plasma Emission Spectrometry (ICP, Labtam Plasma-

lab 8440).

2.6. Genotoxicity studies

Healthy onion bulbs of A. cepa were purchased from

local market and submerged in tap water for 2 h. Outerscales were peeled off and kept on the mouth of test

tubes in such a way so that the root primordia touched

the water surface. The bulbs were not disturbed for 2–

3 days to get the roots of 2–3 cm long. The bulbs were

taken out and placed on test tubes containing different

test concentrations of MS leachate viz. 2.5%, 5.0% and

10% for 24 h exposures of leachates (in the case of VS,

only 10% leachate was evaluated).

2.7. Fixation, slide preparation and scoring

After treatment, the root tips were exposed and fixed

in freshly prepared mixture of acetic acid and ethanol

(1:3 v/v) for 24 h. Thereafter, root tips were stained in

hematoxylin to study the frequency of cell division and

chromosomal abnormalities at different concentra-tions.For fixation and staining of the root tips the

method recommended by Chauhan et al. (1986) was

adopted.

2.8. Phytotoxicity studies

Under this study, Root elongation test (Kapustka

and Reporter, 1993) was conducted. The root elonga-tion was observed at above three concentrations of the

MS leachate. The sand and soil (1:1) mixture was pre-

pared with 3.0% organic material (cow dung manure)

and it was spiked with different concentrations of leach-

ate. Twenty bulbs were planted in each spiked and con-

trol soil and kept at 25 ± 1 �C. Root elongation in A.cepa bulbs was measured only after 15 days. Dry and

wet weight of A. cepa roots were observed. The rootswere dried in oven at 60 ± 1 �C till the constant weightwas gained. Chlorophyll estimation (Arnon, 1949) in

Table 2

Effect of leachates on the mitotic index of the root meristem cells of

Allium cepa exposed for 24 h

Concentrations Mitotic index

Control 11.76 ± 4.08

2.5% 9.29 ± 2.01

5% 7.20 ± 0.32*

10% (Pure) 5.40 ± 0.38**

10% (VS leachate) 9.48 ± 1.84

From each group 4000–5000 cells were scored to determine mitotic

index (MI). The significance level was determined by Student �t� test.* P < 0.05.** P < 0.01.

R. Srivastava et al. / Bioresource Technology 96 (2005) 1867–1871 1869

the leaves of A. cepa was observed to assess the impact

of MS on ecosystem. Chlorophyll contents were mea-

sured in the leaves of A. cepa after exposure to various

test concentrations of the leachate.

2.9. Statistical analysis

For the mitotic index, mean ± SE were calculated and

the level of significance was determined by Student�s �t�test. The chromosomal and mitotic aberrations were

represented in percentage and the significance level

was analyzed by Chi square test.

3. Results

3.1. Metals

The elemental composition in the leachate of MS and

VS and roots of A. cepa are summarized in Table 1. It

was evident from the results that all the metals (Cr,

Cu, Ni and Pb) were reduced after vermicomposting.

3.2. pH of leachates

The pH of MS leachate and VS leachate were found

7.61 and 6.84 respectively.

3.3. Morphological observations

Low concentrations (2.5% and 5.0%) of leachate did

not produce morphological abnormalities but 10.0%

concentration exerted major changes in the morphology

of A. cepa roots. All the roots became coiled or wavy

whereas the control roots were straight and normal.

3.4. Mitotic index

The concentration dependent inhibition in MI was

observed. The MI of control was 11.76 and it reduced

to 5.4 at 10% concentration of MS leachate. When the

onion bulbs were exposed to 10% concentration of VS

leachate, a hike in MI was observed and it increased

upto 9.48, which was very close to control values

(Table 2).

Table 1

Concentration of heavy metals in MS and VS leachate and Allium cepa

roots

Metals Leachate (lg/l) Root of Allium cepa (lg/g)

MS VS MS VS

Cr 0.37 ± 0.01 0.27 ± 0.05 17.1 ± 0.52 14.59 ± 0.66

Cu 0.38 ± 0.01 0.02 ± 0.01 23.3 ± 0.74 22.15 ± 0.45

Ni 3.99 ± 0.01 2.15 ± 0.06 7.57 ± 1.13 5.46 ± 1.05

Pb 2.11 ± 0.02 0.32 ± 0.02 20.62 ± 0.17 14.78 ± 0.84

Data were represented as mean ± SE.

3.5. Mitotic and chromosomal aberrations

Genotoxic effects ofMS leachate on somatic cells were

estimated on the basis of mitotic and chromosomal

abnormalities. The lowest concentration (2.5%) produced

least impact whereas 10% leachate produced maximum

abnormalities on A. cepa root meristem cells. Chromo-

somal bridges, fragments and gaps were prominent at10% concentration. Apart from this, micronuclei and

binucleated cell were observed in 10% leachate whereas

10% leachate of VS showed only mild frequency of aber-

rations. These abnormalities were probably the outcome

of the impact of MS on spindle apparatus (Table 3).

3.6. Phytotoxic studies

The plant weight decreased as the concentration of

sludge leachate increased (Table 4). The wet and dry

weight of control roots were found to be 20.312 g and

3.250 g respectively and a linear reduction in both types

of weight was observed after exposure of A. cepa bulbs

at various concentrations. But the weights in the VS

study were very close to the control values, indicating

no significant reduction in weight.The root elongation in the A. cepa was concentration

dependent and it was 11.2 cm in control whereas it was

decreased to 4.1 cm at 10% MS leachate but on the

exposure to VS leachate it was observed 10.3 cm.

The results of chlorophyll estimation are summarized

in Table 5. Control values were 0.1988 mg/g fresh

weights for chlorophyll-a, 0.0465 mg/g for chlorophyll-

b and total chlorophyll 0.2454 mg/g fresh wt. Both theforms of chlorophyll-a and b were reduced to 0.1355

and 0.0274 mg/g fresh wt, respectively as compared to

control. After vermicomposting, the values of chloro-

phyll-a and b were 0.1739 and 0.0415 mg/g fresh wt.

(Table 5).

4. Discussion

The present study indicated that the municipal sludge

was genotoxic as well as phytotoxic. The chromosomal

Table 3

Mitotic and chromosomal aberrations in the root meristem cells of Allium cepa exposed to leachates for 24 h

Medium

and

concentrations

Chromosomala aberrations Mitotic aberrationsb Micronucleated

cellscBi-nucleated

cellscBreak Fragment % Aberrations Stickiness Multipolar Laggard Bridge % Aberrations

cells

Control – – – – – – 0.25 0.25 ND ND

2.5% – 0.27 0.27 – – 0.54 – 0.54 0.02 0.02

5% 0.64 1.29 1.93 – – 0.64 2.26 2.90 0.02 0.03

10% 0.66 1.33 1.99 0.33 0.33 1.00 2.67 4.33** 0.03 0.05

10% (VS leachate) – 0.50 0.50 – – – 0.50 0.50 0.02 ND

ND: not detected. The significance level was determined by Chi square test, P < 0.01.a Data obtained from 250–300 cells.b Data obtained from 450–500 cells.c Data obtained from 4000–5000 cells.

Table 4

Root weight of Allium cepa exposed to the sample of leachate

Concentrations Wet weight (g) Dry weight (g)

Control 20.312 ± 1.32 3.250 ± 0.65

2.5% 17.104 ± 1.05 2.859 ± 0.48

5% 14.416 ± 2.15 2.135 ± 0.21

10% 10.824 ± 1.94 1.687 ± 0.09

10% (VS leachate) 18.127 ± 0.99 2.531 ± 0.43

1870 R. Srivastava et al. / Bioresource Technology 96 (2005) 1867–1871

abnormalities in the form of bridges in A. cepa exposed

to industrial effluents have been observed (Joshi and

Singh, 1989; Dixit and Nerle, 1985). The decrease in

MI might be due to the presence of lead (Jiang and

Liu, 2000) but since the leachate could have many heavy

metals (the present study), genotoxicity as well as phyto-

toxicity could be the combined effect of heavy metals or

other organic compounds present in the MS. Chauhanet al. (1986) and Thangapandian et al. (1995) observed

binucleated condition in fishes exposed to insecticides

and industrial effluent. An environmental health survey

of drinking water has also shown the contamination of

water by leachate from a pesticide waste dump (Clark

et al., 1982). It is well understood that the earthworms

are capable of bioaccumulating heavy metals in their

body tissues specially chloragocytes and the intestinalmicroflora has the capacity to detoxify most of the pes-

Table 5

Chlorophyll estimation in A. cepa exposed to leachate

Concentration Chlorophyll-a mg/g

fresh weight

Average Chlorophyll-b

fresh weight

Control 0.1906 0.1988 0.0446

0.1832 0.0434

0.2228 0.0517

10.0% (MS Leachate) 0.1515 0.1355 0.0280

0.1406 0.0275

0.1145 0.0267

10% (VS leachate) 0.1712 0.1739 0.0420

0.1738 0.0374

0.1769 0.0450

ticides. From the present study it is evident that vermi-

composting could be an important tool to reduce the

toxicity of municipal sludge as evidenced by our results

of MI and aberrations. Thus, it can be concluded that

vermitechnology should be employed before using themunicipal sludge for landfills or dumping. It is known

that the most widely used acute phytotoxicity tests

involving vascular plants is root elongation test (Hilman

and Debra, 1986). In the present study, the toxicity of

municipal sludge was judged by plant wt. and root elon-

gation, to understand the impact on growth of A. cepa.

The reduction in the chlorophyll contents on exposure

to 10% leachate of sludge could be the cumulative effectof a number of toxicants present in the sludge, or due to

any single component. The reduction in the total chloro-

phyll content was reported in the presence of metalaxyl

and gaucho. Some of the possible reasons for the de-

crease in chlorophyll content may be due to the forma-

tion of enzyme chlorophyllase, which is responsible for

chlorophyll degradation.

The various concentrations of leachates exerted mod-erate to significant inhibition in the plant weight in a

concentration dependent manner. More than 50% de-

crease in plant weight in 10% leachate of municipal

sludge may be the result of toxicants present in the

sludge. The sludge also had many nutritive components

mg/g Average Chlorophyll-total mg/g

fresh weight

Average

0.0465 0.2352 0.2454

0.2266

0.2745

0.0274 0.1795 0.1629

0.1682

0.1412

0.0415 0.2132 0.2154

0.2112

0.2220

R. Srivastava et al. / Bioresource Technology 96 (2005) 1867–1871 1871

along with metals responsible for plant growth, which

increased the root wt of A. cepa after vermicomposting.

5. Conclusions

From the present study, it was evident that vermi-

composting could be an important tool to reduce the

toxicity of municipal sludge as evidenced by the results

of genotoxicity and phytotoxicity. Thus, vermitechnol-

ogy could be an excellent technique for recycling of mu-

nicipal sludge.

Acknowledgements

The authors are grateful to Prof. Y.K. Gupta, Direc-

tor, Industrial Toxicology Research Centre, Lucknow

for his keen interest in the present work. Thanks are also

due to Drs. L.K.S. Chauhan, P.N. Saxena and Mr. Sau-

rabh Chandra for constant help during present study.

I.T.R.C. Communication No. 2279.

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