Comparative biomonitoring of leachates from hazardous solid

waste of two industries using Allium test

Saurabh Chandra
a
, L.K.S. Chauhan
a
, R.C. Murthy
b
, P.N. Saxena
a
,
P.N. Pande
c
, S.K. Gupta
a,
T
a
Cell Biology Section, Industrial Toxicology Research Centre, P.O. Box 80, M. G. Marg, Lucknow 226 001, India
b
Metal Analysis Section, Industrial Toxicology Research Centre, P.O. Box 80, M. G. Marg, Lucknow 226 001, India
c
Department of Zoology, K.S.S. Postgraduate College, Dr. R.M.L. Avadh University, Faizabad 224 001, India
Received 24 June 2004; received in revised form 1 December 2004; accepted 2 January 2005
Available online 9 February 2005
Abstract
Hazardous industrial wastes are inevitable source of environmental pollution. Leachates from these wastes might
contaminate the origins of potable water and affect human health. The study was carried out to determine the possible genotoxic
effects of leachates from solid waste of a metal and dye industry using the Allium cepa chromosome aberrations assay. The 10%
leachates were prepared from solid wastes obtained from both the industries and examined for the presence of heavy metal
content and genotoxicity. To simulate the field and laboratory conditions, A. cepa bulbs were exposed through soil and aqueous
medium for 48 h to 2.510% leachates. The results revealed that both metal waste leachate (MWL) and dye waste leachate
(DWL) contained high concentrations of chromium, nickel and iron that significantly induced cytogenetic alterations.
Significant inhibition of mitotic index (MI), inductions of chromosomal/mitotic aberrations (CA/MA) and micronuclei (MN)
formation were found in all experimental groups exposed to MWL and DWL. The effects observed were concentration
dependent and the frequency of aberrations was higher with treatment of MWL than DWL. The MI was severely inhibited at
10% aqueous exposure it was 4.59F0.69 ( Pb0.001) in MWL and almost half to that induced by DWL that was 8.62F0.69
( Pb0.05). Significant frequency of CA/MA and MN induced by MWL was 14.21 ( Pb0.001) and 0.33 ( Pb0.001) whereas CA/
MA and MN induced by DWL was 7.81 ( Pb0.001) and 0.13 ( Pb0.05) in the aqueous medium. The investigations inferred that
abnormalities caused by MWL were higher than DWL both in soil and aqueous media. These toxic responses may have relied
on raised heavy metal concentrations of metal-based than dye industrial wastes.
D 2005 Published by Elsevier B.V.
Keywords: Genotoxicity; Hazardous solid waste; Leachates; Allium test
0048-9697/$ - see front matter D 2005 Published by Elsevier B.V.
doi:10.1016/j.scitotenv.2005.01.002
T Corresponding author.
E-mail address: skgitrc@rediffmail.com (S.K. Gupta).
Science of the Total Environment 347 (2005) 4652
www.elsevier.com/locate/scitotenv
1. Introduction
Industries though responsible for environmental
pollution are indispensable components of nations
economic development. In India, there has been a
considerable increase in number of industrial units
including tanneries, metal, cyanides, dyes, paint etc.
They have a major potential for generating hazardous
waste, of which 5.2 million tonnes of waste are still
disposed on land (TERI, 2003). During the past
several years wastewater from industrial effluents,
sewage and agricultural soils of India have shown
ascending trend of environmental pollution caused
by extensive disposal of wastes containing pesticide
residue and heavy metals (Aleem and Malik, 2003).
The industrial wastewaters usually contain complex
mixtures of chemicals that substantially contaminate
ground and surface water reservoirs used for drink-
ing purposes (Mumtaz, 1995; Dewhurst et al., 2002),
and might become hazardous to human health by
inducing genetic alterations, as heavy metals are the
most common toxicants of wastewater sludge (Rank
and Nielsen, 1998). The clastogenicity of chemicals
found at several hazardous industrial waste sites
were observed using Tradescantia bioassay (Sandhu
et al., 1989), and Daphnia species have been
consistently used to assess the toxicity of leachates
from various industrial wastes (Seco et al., 2003).
Reduced birth weight in infants of the population
living near hazardous waste dumping sites showed
that leachate contaminated drinking water not only
affected adults but also their progeny (Berry and
Bove, 1997).
Many studies on genotoxicity of industrial waste-
water, sludge/effluents, and soil in short-term bio-
assays and gene mutations in Salmonella strains
have already been reviewed (Houk, 1992; Watanabe
and Hirayama, 2001). Bioluminescence inhibition
assays have demonstrated the cytotoxic impact on
Vibrio fischeri by wastewaters and Photobacterium
phosphoerum by small and large industrial dis-
charges (Microtox and ToxAlert tests) (Castillo et
al., 2001; Hao et al., 1996), and notable genotoxic
alterations were reported through in vivo tests on
aquatic amphibian larvae Xenopus laevis exposed to
aqueous samples of industrial waste (Bekaert et al.,
2002). Plant bioassays provide meaningful parame-
ters to assess the toxicity of complex mixtures like
industrial wastes even without knowledge of its
chemical composition. Previously many workers
have successfully employed plant bioassays viz.
Allium cepa, Vicia faba and Tradescantia paludosa
as sensitive and rapid bio-tools for genotoxicity
screening of environmental contaminants in soil,
surface and ground waters, landfill leachates and
wastewater/sludge etc. (Odeigah et al., 1997; Stein-
kellner et al., 1999; Cotelle et al., 1999; Cabrera and
Rodriguez, 1999).
The Allium test is a simple, sensitive and rapid
bioassay that has been widely used as a standard
for biomonitoring of environmental contaminants
using various genotoxicity parameters (Grant,
1982). Allium test generally provides useful esti-
mate of the total toxic effect resulting from the
treatment of root tip cells by mixture of wastes
(DeMarini, 1991; Fiskesjo, 1993). Earlier we have
reported significant genotoxic effects in somatic
cells of V. faba and A. cepa exposed to leachates of
tannery wastes (Chandra and Gupta, 2002; Chandra
et al., 2004). The aim of the present study was to
evaluate and compare the genotoxic potential of
leachates of two types of industrial solid wastes,
one from metal-based industry and the other from a
dye-based industry by employing root meristem
cells of A. cepa.
2. Materials and methods
2.1. Sampling and leachate preparation
Five random samples of solid waste were
collected from different places of open waste
disposal sites of metal-based industry that manufac-
tures flashlight components and of a dye industry-
manufacturing chrome pigments both situated in
urban areas of Lucknow city (India). The samples
were pooled, mixed well, air dried, finely ground
with a pestle mortar and sieved through a 63 Am
(pore size) sieve to get a homogenous mixture. This
mixture was used for all the studies. The aqueous
extraction was carried out to obtain a 10% leachate
of solid waste (Ferrari et al., 1999). Briefly, 100 g of
solid waste of both metal or dye waste was added to
1000 ml of demineralized water (w/v) and the
mixture was shaken continuously for 24 h at room
S. Chandra et al. / Science of the Total Environment 347 (2005) 4652 47
temperature (25F2 8C) and the undissolved materials
from the mixture were removed by centrifuging the
leachates at 3000 rpm for 15 min. These leachates
were used throughout the study.
2.2. Physicochemical and metal analysis
The physicochemical parameters like TDS (Total
dissolved solids), salinity and conductivity were
carried out in the leachates by water analysis kit
(Century-CK711, India) whereas, chloride, nitrate,
sodium, potassium and ammonium were measured
using ion selective electrode (Orion-960, U.S.A.). The
leachates were processed for metal analysis by the
standard methods (APHA, 1998) while the solid
wastes were digested by the EPA method 3050-B
(EPA, 1986). The processed samples were analyzed
for cadmium, chromium, copper, iron, manganese,
nickel, lead and zinc on Inductively Coupled Plasma
Emission Spectrometry (Labtam Plasmalab-8440,
Australia).
2.3. Test model and growth conditions
Healthy onion bulbs of A. cepa (2n=16) about 10
15 g were procured from the local market and their
outer scales were peeled and kept under running tap
water for half an hour prior to tests. The clean bulbs
were rooted over test soil or aqueous leachates in a
B.O.D. incubator (Indian Equipment Corporation,
Bombay) at 20F1 8C for 48 h.
2.4. Test concentrations and exposure schedule
The procedure previously used by Chandra et al.
(2004) was followed for the preparation of three test
concentrations of aqueous leachates (2.5%, 5% and
10%) and five bulbs each of A. cepa were exposed to
two exposure media for 48 h along with the negative
control. In the first group the bulbs were directly
exposed to different concentrations of aqueous leach-
ates in glass tubes while for the second group they
were rooted in garden soil mixed with leachates kept
in plastic containers (Dash et al., 1988).
2.5. Cytogenetic assay
The A. cepa root tips were randomly sampled,
overnight fixed in cold Carnoys, hydrolyzed in 1 N
HCl for 5 min at 60 8C before staining with
hemotoxylin. The five slide replicates were blindly
and randomly coded for each test concentration before
cytogenetic analysis (Fiskesjo, 1985). The slides were
scored for mitotic index (MI), chromosomal/mitotic
aberrations (CA/MA) and micronuclei (MN). For MI
the dividing cells were counted from 40005000
interphase cells along with the scoring for micro-
nucleated cells while for aberrations 400500 dividing
cells were analyzed from five replicates of each test
group.
2.6. Statistical analysis
The data of mitotic index was taken as meanFSE
and their significance was determined by Students t
test. The chromosomal and mitotic aberrations
Table 1
Physicochemical analysis of leachates obtained from solid waste of
metal and dye industry
Parameters Metal Dye
pH 5.1 6.7
Chemical oxygen demand 21.7 24.7
Conductivity 2.3 1.3
Salinity 11.9 6.7
Total nitrogen 4.4 90.7
Total organic carbon 384.1 138.4
Total dissolved solids 309.0 372.0
Chloride 600.0 64.2
Nitrate 1120.0 870.0
Sodium 414.0 53.3
Potassium 11.7 3.6
Ammonium ND ND
All values are in mg/l except conductivity in M mho/cm.
ND (Not detected).
Table 2
Presence of heavy metals in solid wastes and leachates of metal and
dye industry
Heavy metals
(ppm)
Metal industry Dye industry
Solid waste Leachates Solid waste Leachates
Cadmium 1.3 0.01 b0.001 b0.001
Chromium 32,980.0 282.2 16,280.0 11.2
Copper 603.4 1.0 3559.9 1.6
Iron 3270.9 21.0 1360.9 4.2
Manganese 136.0 1.0 21.0 0.02
Nickel 1993.7 9.8 20,379.7 3.6
Lead 122.4 1.1 198.6 0.5
Zinc 459.9 2.1 751.9 1.2
S. Chandra et al. / Science of the Total Environment 347 (2005) 4652 48
observed were represented in percentage, and their
level of significance was calculated by Chi square test.
3. Results
Physicochemical analysis of leachates of metal and
dye industry showed that metal leachate was more
acidic (pH 5.1) than the dye leachate (pH 6.7).
Parameters like conductivity, salinity, total organic
carbon, chloride, nitrate, sodium, potassium of metal
leachate was high in comparison to dye leachate
whereas ammonium was not detected in any of the
leachate samples (Table 1).
Table 2 shows the concentrations of heavy metals in
solid waste and leachates of metal and dye industry
which revealed considerable concentrations of chro-
mium, copper, iron, manganese, nickel, lead and zinc in
the wastes that were finally disposed. Chromium, iron
and nickel were extractable in notable concentrations.
Cadmium levels were almost negligible both in solid
wastes as well as in the leachates of both the industries.
Further, the MWL had higher concentrations of
chromium, iron and nickel as compared to DWL.
Table 3 depicts the effects of leachates of metal and
dye industry on the mitotic index (MI) of A. cepa root
tip cells exposed through soil or aqueous medium for
48 h. All the test concentrations of leachates (2.5
10%) affected MI in a dose-dependent manner. On the
other hand decline of MI was more prevalent in MWL
than DWL treated A. cepa cells. For example it was
4.59F0.69 ( Pb0.001) in 10% and 7.43F0.84
( Pb0.05) in 5% MWL whereas it was 8.62F0.69
( Pb0.05) in 10% and 9.50F0.28 ( Pb0.05) in 5%
DWL. Corresponding concentrations in soil medium
Table 3
Effect of leachates on the mitotic index of the root meristem cells of
Allium cepa exposed the MWL and DWL for 48 h
Industries and
Concentrations (%)
Mitotic Index
a
Soil medium Aqueous medium
Metal Industry
Control 11.24F0.71 10.82F1.04
2.5 10.55F0.86 8.72F1.00
5 9.16F0.68T 7.43F0.84T
10 6.71F1.00TT 4.59F0.69TT
Dye Industry
Control 11.55F0.76 11.34F0.88
2.5 11.19F0.97 10.98F0.70
5 10.79F0.80 9.50F0.28T
10 9.12F0.62T 8.62F0.69T
(Significance level calculated by Students t test).
a
Data obtained from 40005000 cells and expressed as mean-
FSE from five replicates.
T Pb0.05.
TT Pb0.001.
Table 4
Chromosomal and mitotic aberrations in the root meristem cells of Allium cepa exposed to metal waste leachate for 48 h
Mediums and
Concentrations
(%)
Types of aberrations % Aberrant
Cells
a
% Micro-
nucleated
cells
b
Breaks
c
Aberrant
Anaphase
Stickiness Multipolar Laggard Bridges Unequal
Soil medium
Control ND ND 2 1 1 ND ND 0.70 0.01
2.5 ND ND 4 2 ND 1 ND 1.29 ND
5 5 ND 8 3 2 1 ND 3.68TT 0.05
10 11 2 12 5 5 4 1 8.29TT 0.14T
Aqueous medium
Control 1 ND 2 1 2 ND ND 1.17 0.01
2.5 2 ND 5 2 2 1 ND 2.52TT 0.05
5 8 1 8 3 4 4 ND 6.27TT 0.18TT
10 15 3 18 6 7 8 2 14.21TT 0.33**
(Significance level calculated by Chi square test).
a
Data represent percent frequency of aberrations from 400500 cells.
b
Data represent percent frequency of micronucleated cells from 4000-5000 cells.
c
Chromatid breaks and fragments, ND (Not Detected).
T Pb0.05.
TT Pb0.001.
S. Chandra et al. / Science of the Total Environment 347 (2005) 4652 49
revealed lesser inhibition of MI than that of exposed
through aqueous media. The MI in the root meristem
cells exposed through soil medium was about
6.71F1.00 ( Pb0.001) in 10% MWL whereas it was
9.12F0.62 ( Pb0.05) with 10% DWL exposure.
Tables 4 and 5 summarize the percentage of
chromosomal/mitotic aberrations (CA/MA) and
micronucleated cells (MN) in root meristem cells of
A. cepa exposed to 2.510% of metal and dye waste
leachates through soil and aqueous mediums for 48 h.
The induction of chromosomal/mitotic aberrations
was observed in a concentration dependent pattern.
Statistically significant ( Pb0.05 or Pb0.001) frequen-
cies of aberrations CA/MAwere recorded with 510%
leachates of both of MWL and DWL leachates
irrespective of exposure through soil or aqueous
medium. Further, the types of chromosomal as well
as mitotic aberrations were similar in both MWL and
DWL exposed A. cepa meristem cells. Chromatid
breaks and fragments were frequent CA whereas
multipolar arrangements, laggards, bridges and
unequal separation of chromosomes at anaphase stage
of cell division were found to be frequent MA.
However, percentage of aberrations was higher in
MWL than in DWL exposed A. cepa root tip cells.
The frequency of aberrations was 14.21% ( Pb0.001)
in aqueous medium and 8.29% ( Pb0.001) in soil
medium with 10% MWL whereas it was 7.81%
( Pb0.001) in aqueous medium and 4.76% ( Pb0.05)
in soil medium with 10% DWL. Both the industrial
leachates significantly ( Pb0.05 or Pb0.001) induced
micronucleated cells in both the exposure media
especially at 10% test concentration. The frequency
of micronucleated cells was relatively higher in MWL
treated cells (0.33 ( Pb0.001) in 10% aqueous and
0.14 ( Pb0.05) in 10% soil medium) while DWL was
only able to induce 0.13 ( Pb0.05) micronuclei in 10%
aqueous exposure.
4. Discussion
The findings of the present study indicate that both
the industrial leachates influence MI and induce CA/
MA in A. cepa root meristem cells whether exposed
through soil or aqueous medium. The inhibition of MI
by the leachates from both MWL and DWL reflects
cytotoxicity that directly affects root growth and
elongation. The decline of mitotic index below 22%
in comparison to negative control can cause lethal
effects on the organism (Antonsiewicz, 1990). The
wastes from metal and dye manufacturing industry
contain heavy metals and many undesirable cytotoxic
compounds that may cause cell death, which may
Table 5
Chromosomal and mitotic aberrations in the root meristem cells of Allium cepa exposed to dye waste leachate for 48 h
Mediums and
Concentrations
(%)
Types of aberrations % Aberrant
cells
a
% Micro-
nucleated
cells
b
Breaks
c
Aberrant
anaphase
Stickiness Multipolar Laggard Bridges Unequal
Soil medium
Control ND ND 2 ND ND ND ND 0.36 ND
2.5 ND ND 4 ND ND 1 ND 0.96 ND
5 2 ND 5 1 1 2 ND 2.25T 0.04
10 5 ND 8 2 3 4 ND 4.76T 0.07
Aqueous medium
Control ND ND 3 1 ND 1 ND 0.94 0.01
2.5 2 ND 5 2 1 2 ND 2.33T 0.01
5 3 ND 7 2 2 3 ND 3.65TT 0.06
10 8 ND 10 4 5 6 1 7.81TT 0.13T
(Significance level calculated by Chi square test).
a
Data represent percent frequency of aberrations from 400500 cells.
b
Data represent percent frequency of micronucleated cells from 40005000 cells.
c
Chromatid breaks and fragments, ND (Not Detected).
T Pb0.05.
TT Pb0.001.
S. Chandra et al. / Science of the Total Environment 347 (2005) 4652 50
appear as decline in the MI. The inhibition of mitotic
index can also be attributed to be the effect of
environmental chemicals on DNA/protein synthesis
of the biological system (Chauhan et al., 1998). These
results are in agreement with our earlier studies
conducted on A. cepa and V. faba exposed to tannery
waste leachates that inferred chromium and nickel
compounds jointly affect amino acids and/or DNA
biomolecule which led to significant decrease of
mitosis and root growth (Chandra and Gupta, 2002;
Chandra et al., 2004).
The significant and concentration dependent induc-
tion of chromosomal/mitotic aberrations including
micronucleus formation, in root tip cells exposed to
leachates containing chromium, nickel and iron
indicates the genotoxic potential of both industrial
wastes. The aberrant mitotic stages may have been the
outcome of spindle poisoning that causes chromo-
some disturbances during mitotic cell division. Vari-
ous heavy metals are known to induce chromosome
breaks, fragments and micronucleus formation in
plants and mammalian test systems (Knasmuller et
al., 1998), and their effects were emphasized to be the
result of formation of DNADNA and DNAprotein
cross-links (DeFlora et al., 1990; Costa, 1991; Costa
et al., 1994). Chromium and nickel have been reported
to affect the mitotic spindles thereby causing aberrant
mitotic stages (Anderson, 1985), while the additive
effects of nickel, in chromium induced genotoxicity
have been reported to induce sister chromatid
exchanges (SCE) in exposed welders (IARC, 1990).
The induction of micronuclei is usually the outcome
of chromosome breaks/fragments or spindle poisoning
which is an anomalous disjunction of chromosomes at
anaphase stage of cell cycle (Grover and Kaur, 1999).
Any essential element for plant and humans may
synergies adversely when collectively found with
toxic metals like chromium and nickel as iron in the
case of test leachates. Evidence already exists that
combined effects of iron with chromium and zinc
induce high percentage of micronucleated RBC and
other genotoxic effects in newt larvae tests (Godet et
al., 1996). Predominantly, there are two ways by
which the toxic metals can induce genotoxicityone
is the cross-linking with the DNA and/or protein and
the other is generation of reactive oxygen species. The
DNA damage may be due to the occurrence of nickel
present in the leachates, which may be irreversible, as
nickel is known to inhibit DNA repair process
(Hartwig, 2000). Nickel has been detected in both
waste leachates besides high concentrations of chro-
mium that could have additively or synergistically
induced genotoxicity in exposed cells. There is always
a risk that heavy metals may become the part of
trophic food chain and interfere with the genetic
material of individuals. The presence of heavy metals
and other pollutants in agricultural soils have lead to
bioaccumulation of various toxicants in food crops
(Sukreeyapongse et al., 2002). Hence the interactions
among metals or with other elements even at lower
levels present in leachates can have synergistic or
additive actions. The elevated concentrations of
nitrate, chloride, sodium and potassium in leachate
samples may also produce deleterious effects as a
whole, if not alone. There are reports of ground water
samples comprising nitrate, nitrite, atrazine, trichloro-
ethylene etc. have shown increased micronucleated
cells in Tradescantia bioassay (Steinkellner et al.,
1999). The industrial wastes can become hazardous
when nontoxic chemicals change to toxic forms and
induce toxicity as soil microflora may convert various
nongenotoxic compounds to their genotoxic derivates
(Watanabe and Hirayama, 2001).
It may be concluded that industrial waste/sludge
containing complex mixtures of chemicals of
unknown specificity, dumped in unsecured open
landfills may cause health hazards, so treatment of
wastes is essential prior to disposal. Further, the use of
sensitive plant bioassays may be an amenable tool to
screen the genotoxicity of aqueous leachates including
various other test systems.
Acknowledgements
The authors thank Prof. Y.K. Gupta, Director,
I.T.R.C. for encouragement and keen interest in the
study. Authors are thankful to Dr. Neeraj Mathur for
statistical analysis. The technical assistance of Mr.
V.B. Singh is also acknowledged.
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