Health risk assessment of heavy metals via dietary intake of ...

Health risk assessment of heavy metals via dietary intake of foodstuffs
from the wastewater irrigated site of a dry tropical area of India
Anita Singh a , Rajesh Kumar Sharma a , Madhoolika Agrawal a, *, Fiona M. Marshall b
a Ecology Research Laboratory, Department of Botany, Banaras Hindu University, Varanasi 221005, India
b SPRU, Freeman Centre, University of Sussex, Brighton BN1 9QE, United Kingdom
article info
Article history:
Received 25 August 2009
Accepted 19 November 2009
Keywords:
Sewage water
Heavy metals
Vegetables
Cereals
Metal pollution index
Health risk
1. Introduction
abstract
The growing problem of water scarcity has significant negative
influence on economic development, human livelihoods, and environmental
quality throughout the world. Rapid urbanization and
industrialization releases enormous volumes of wastewater, which
is increasingly utilized as a valuable resource for irrigation in urban
and peri-urban agriculture. It drives significant economic activity,
supports countless livelihoods particularly those of poor farmers,
and substantially changes the water quality of natural water
bodies (Marshall et al., 2007). Wastewater may contain various
heavy metals including Zn, Cu, Pb, Mn, Ni, Cr, Cd, depending upon
the type of activities it is associated with. Continuous irrigation of
agricultural land with sewage and industrial wastewater may
cause heavy metal accumulation in the soil and vegetables (Singh
et al., 2004; Sharma et al., 2007; Marshall et al., 2007).
Heavy metals are generally not removed even after the treatment
of wastewater at sewage treatment plants, and thus cause
risk of heavy metal contamination of the soil and subsequently
to the food chain (Fytianos et al., 2001). Intake of heavy metals
through the food chain by human populations has been widely
reported throughout the world (Muchuweti et al., 2006). Due to
* Corresponding author. Tel.: +91 542 2368156; fax: +91 542 2368174.
E-mail address: madhoo58@yahoo.com (M. Agrawal).
0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2009.11.041
Food and Chemical Toxicology 48 (2010) 611–619
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
The present study was conducted to assess the risk to human health by heavy metals (Cd, Cu, Pb, Zn, Ni
and Cr) through the intake of locally grown vegetables, cereal crops and milk from wastewater irrigated
site. Milk is not directly contaminated due to wastewater irrigation, but is an important route of food
chain transfer of heavy metals from grass to animals. Heavy metal concentrations were several fold
higher in all the collected samples from wastewater irrigated site compared to clean water irrigated ones.
Cd, Pb and Ni concentrations were above the ‘safe’ limits of Indian and WHO/FAO standards in all the vegetables
and cereals, but within the permissible limits in milk samples. The higher values of metal pollution
index and health risk index indicated heavy metal contamination in the wastewater irrigated site
that presented a significant threat of negative impact on human health. Rice and wheat grains contained
less heavy metals as compared to the vegetables, but health risk was greater due to higher contribution of
cereals in the diet. The study suggests that wastewater irrigation led to accumulation of heavy metals in
food stuff causing potential health risks to consumers.
Ó 2009 Elsevier Ltd. All rights reserved.
the non-biodegradable and persistent nature, heavy metals are
accumulated in vital organs in the human body such as the
kidneys, bones and liver and are associated with numerous serious
health disorders (Duruibe et al., 2007). Individual metals exhibit
specific signs of their toxicity. Lead, As, Hg, Zn, Cu and Al poisoning
have been implicated with gastrointestinal (GI) disorders,
diarrhoea, stomatitis, tremor, hemoglobinuria causing a rust-red
colour to stool, ataxia, paralysis, vomiting and convulsion, depression,
and pneumonia (McCluggage, 1991). The nature of effects can
be toxic (acute, chronic or sub-chronic), neurotoxic, carcinogenic,
mutagenic or teratogenic (European Union, 2002).
Vegetables, cereals and milk are major components of human
diet, being sources of essential nutrients, antioxidants and metabolites
in food items. In the present study, the concentrations of
heavy metals in locally produced vegetables, cereals and milk were
quantified throughout a year at a suburban area of Varanasi city of
India, where treated and untreated wastewater has been used as a
source of irrigation water for about 20 years. The contamination
levels in soil and vegetable/cereal crops were evaluated with respect
to the prescribed safe limits of different heavy metals set under
national and international norms. Milk is not directly
contaminated by wastewater irrigation, but provides insight into
the food chain transfer of heavy metals from fodder grass to the
milk of animals. A number of standard measures were used to assess
the health risks associated with the measured levels of heavy
metal contamination at the study sites.

612 A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619
2. Materials and methods
2.1. Study sites
The study was conducted around Dinapur sewage treatment plant (DSTP) situated
at a suburban area in the north east of Varanasi (25°18’ N latitude 83°01’ E longitude
and 76.19 m above the sea level) city in eastern Gangetic plains of India
during March 2006 to February 2007. Large-scale vegetable production is conducted
in this area, largely to supply markets in the city. Dinapur sewage treatment
plant of 80 million liters per day (MLD) capacity was installed in 1986. Effluents
from various small scale industries situated in the city are also discharged along
with sewage for treatment at DSTP. These industries include fabric painting, batteries,
dye, plastic recycling and metal surface treatment. A large area around DSTP has
no access to clean water resources, so farmers use treated and untreated wastewater
for irrigation. Two major sites were demarcated in Dinapur having different irrigation
practices. At the wastewater irrigated (WWI) site, treated wastewater from
DSTP has been used for irrigating the fields for about 20 years. Some times due to
power failure, the sewage treatment plant does not work and untreated wastewater
is used for irrigation. Clean water from bore wells has been used for irrigating the
agricultural fields at the clean water irrigated site (CWI) for a similar period of time.
2.2. Soil and water sampling
Soil and water samples were collected at fortnightly interval from March 2006
to February 2007. Soil samples were collected in triplicate by digging out a monolith
of 10 10 15 cm 3 size, from 10 sub sites of both clean (CWI) and wastewater
irrigated sites (WWI). Soil samples were air dried, crushed and passed through
2 mm mesh size sieve and stored at ambient temperature before analysis. Both
clean and wastewater samples (100 ml) used for irrigation were collected in triplicate
in a pre acid washed polypropylene bottle and 1 ml of concentrated HNO3 was
added in the water sample to avoid the microbial activity. These samples were
brought back to the laboratory and kept in a refrigerator before digestion.
2.3. Plant sampling
All the major vegetables and cereal crops grown in the experimental area, either
for home consumption or sale, were collected. The details of different plants sampled
during the experiment are given in Table 1.Anareaof5 5m 2 was randomly marked
at 10 subsites in triplicate and the edible portion of test vegetables were collected
from both CWI and WWI sites. Samples were brought back to the laboratory and
washed with clean tap water to remove the soil particles adhered to the surface of
the vegetables. After removing the extra water from the surface of vegetables with
blotting paper, samples were cut into pieces, packed into separate bags, and kept in
an oven until a constant weight was achieved. For cereal crops, plots of 5 5m 2 sizes
were marked in triplicate at 10 subsites at both CWI and WWI sites, and ears were
harvested upon maturity. Grains were separated and kept in an oven for drying, until
constant weight was achieved. The dried samples were grinded and passed through a
sieve of 2 mm size and then kept at room temperature for further analysis.
2.4. Milk sampling
Fresh milk (250 ml) was collected from 10 different buffalos in pre acid washed
polypropylene bottles, at both CWI and WWI sites, and stored at 4 °C prior to digestion
for heavy metal analysis.
2.5. Digestion of samples
2.5.1. Soil and plant
Soil and plant samples (1 g) were digested after adding 15 ml of tri-acid mixture
(HNO 3,H 2SO 4, and HClO 4 in 5:1:1 ratio) at 80 °C until a transparent solution was
obtained (Allen et al., 1986). After cooling, the digested sample was filtered using
Whatman No. 42 filter paper and the filtrate was finally maintained to 50 ml with
distilled water.
2.5.2. Irrigation water
The irrigation water sample (50 ml) was digested with 10 ml of concentrated
HNO 3 at 80 °C until the solution became transparent (APHA, 2005). The solution
was filtered through Whatman No. 42 filter paper and the total volume was maintained
to 50 ml with distilled water.
2.5.3. Milk
For digestion of milk, the method given by Crounse (1983) was followed. Milk
sample (50 ml) was taken in a beaker and heated on hot plate to reduce the water
content (without boiling). When the mass became syrupy, it was cooled and 10 ml
of HNO3 (70% V/V) was added. The mixture was warmed until the evolution of
brown fumes of NO 2 ceased and a colourless solution was obtained. About 2.5 ml
of HClO 4 was added and again heated for complete digestion. The extract after filtration
was diluted with distilled water to 25 ml.
2.6. Analysis of heavy metals
Concentrations of Cd, Cu, Pb, Zn, Ni and Cr in the filtrate of digested soil, water,
plant and milk samples were estimated by using an atomic absorption spectrophotometer
(Model 2380, Perkin Elmer, Inc., Norwalk, CT, USA). The instrument was fitted
with specific lamp of particular metal. The instrument was calibrated using
manually prepared standard solution of respective heavy metals as well as drift
blanks. Standard stock solution of 1000 ppm for all the metals were obtained from
Sisco Research Laboratories Pvt. Ltd., India. These solution were diluted for desired
concentrations to calibrate the instrument. Acetylene gas was used as the fuel and
air as the support. An oxidising flame was used in all cases.
2.7. Quality control analysis
Precision and accuracy of analysis was assured through repeated analysis of
samples against National Institute of standard and technology, Standard Reference
Material (SRM 1570) for all the heavy metals. The results were found within ±2% of
the certified value. Quality control measures were taken to asses contamination and
reliability of data. Blank and drift standards (Sisco Research Laboratories Pvt. Ltd.,
India) were run after five determination to calibrate the instrument. The coefficients
of variation of replicate analysis were determined for different determinations
for precision of analysis and variations below 10% were considered correct.
2.8. Data analyses
Concentration of metal in edible part at WWI site=concentration of metal in soil at WWI
EF ¼
Concentration of metal in edible part at CWI site=concentration of metal in soil at CWI site :
2.8.1. Enrichment factor (EF)
To examine the translocation of heavy metals from the soil to the edible portion
of test plants, and to show the difference in metal concentrations in the plants between
the sites, the enrichment factor (EF) was calculated by using the formula given
by Buat-Menard and Chesselet (1979):
2.8.2. Metal pollution index (MPI)
To examine the overall heavy metal concentrations in all crops analysed in the
wastewater irrigated site, metal pollution index (MPI) was computed. This index
was obtained by calculating the geometrical mean of concentrations of all the metals
in the vegetables, cereals and milk (Usero et al., 1997).
MPIðlgg 1 Þ¼ðCf1 Cf2 CfnÞ 1=n
where Cf n = concentration of metal n in the sample.
2.8.3. Health risk index (HRI)
The health risk index was calculated as the ratio of estimated exposure of test
crops and oral reference dose (Cui et al., 2004). Oral reference doses were
4 10 2 , 0.3 and 1 10 3 mg kg 1 day 1 for Cu, Zn and Cd, respectively (USEPA,
2002) and 0.004, 0.02 and 1.5 mg kg 1 day 1 for Pb, Ni and Cr, respectively (USEPA,
1997). Estimated exposure is obtained by dividing daily intake of heavy metals by
their safe limits. An index more than 1 is considered as not safe for human health
(USEPA, 2002).
Daily intake was calculated by the following equation:
Daily intake of metal ðDIMÞ ¼ Cmetal Dfood intake
B average weight
where Cmetal, Dfood intake and Baverage weight represent the heavy metal concentrations
in plants (lgg 1 ), daily intake of vegetables and average body weight, respectively.
The average daily vegetable intake rate was calculated by conducting a survey where
100 people having average body weight of 60 kg were asked for their daily intake of
particular vegetable from the experimental area in each month of sampling (Ge,
1992; Wang et al., 2005).

Table 1
Plant samples collected from the experimental sites.
2.9. Statistical analysis
The significance of differences between the concentrations of heavy metals in
soil at wastewater (WWI) and clean water irrigated (CWI) sites were shown by
using Student’s t-test. The data of heavy metal concentrations in the plants at different
sites were subjected to two way analysis of variance (ANOVA) test for assessing
the significance of differences in heavy metal concentrations due to different
irrigation practices. All the statistical tests were performed using SPSS software
(SPSS Ins., version 11).
3. Results and discussion
3.1. Levels of heavy metals in water samples
The concentrations (lgml 1 ) of heavy metals in the irrigation
water at WWI site ranged between 0.00–0.02 for Cd, 0.02–0.07
for Cu, 0.07–0.13 for Pb, 0.05–0.18 for Zn, 0.02–0.08 for Ni and
0.03–0.08 for Cr during March 2006 to February 2007, whereas
at CWI site, heavy metal concentrations in irrigation water were
very low or below the detectable limits (Fig. 1). Among all the heavy
metals, Cd concentration exceeded the permissible limit set by
FAO (1985). Heavy metals in the sewage water are associated with
small scale industries such as colouring, electroplating, metal surface
treatments, fabric printing, battery and paints, releasing Cd,
Cu, Pb, Zn, Ni and other heavy metals into water channels, which
are accessed for irrigation. As compared to the present concentration
of heavy metals in the wastewater, Singh et al. (2004) have
reported lower ranges of Cd (0.00–0.006 lgml 1 ), Cr (0.00–
0.049 lgml 1 ) and Pb (0.012–0.088 lgml 1 ), but higher ranges
of Cu (0.00–0.203 lgml 1 ), Ni (0.01–0.22 lgml 1 ) and Zn
(0.023–0.18 lgml 1 ) were reported in the water samples of Dinapur
area of Varanasi receiving treated and untreated sewage water
for irrigating the agricultural fields. Sharma et al. (2007) reported
similar ranges of Cd, Ni and Zn in irrigation water of DSTP, but
Cu, Pb and Cr were twofold higher during the present study. The
comparison of the concentrations of heavy metals in treated
wastewater of Dinapur STP from STP of Titagarh, West Bengal, India
showed that Cd (0.01 lgml 1 ) and Cr (0.04 lgml 1 ) were similar,
but Zn (1.17 lgml 1 ), Ni (0.39 lgml 1 ), Pb (3.54 lgml 1 ) and
Cu (0.98 lgml 1 ) were lower during the present study (Gupta
et al., 2008). Among the heavy metals, the mean concentration
was maximum for Zn (0.151 mg l 1 ) and minimum for Cd
(0.02 mg l 1 ) in the irrigation water from DSTP (Fig. 1). The lower
concentrations of heavy metals in the irrigation water may be
due to dilution of heavy metals in the water medium, but the continuous
application of these treated and untreated wastewater for
irrigation resulted into accumulation of heavy metals into the soil.
A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619 613
Edible part of vegetable/cereal crops Common name Botanical name Family
Leaf Palak Beta vulgaris L. Chenopodiaceae
Leaf Amaranthus Amaranthus caudatus L. Amaranthaceae
Leaf Cabbage Brasssica oleracea L. var capitata L. Brassicaceae
Inflorescence Cauliflower Brassica oleracea L. var. Botrytis L. Brassicaceae
Fruit Lady’s finger Abelmoschus esculentus L. Malvaceae
Fruit Brinjal Solanum melongena L. Solanaceae
Fruit Tomato Lycopersicon esculentum L. Solanaceae
Fruit Bottle gourd Lagenaria siceraria Mol. Cucurbitaceae
Fruit Sponge gourd Luffa cylindrica L. Cucurbitaceae
Fruit Bitter gourd Momordica charantia L. Cucurbitaceae
Fruit Pumpkin Cucurbita maxima Duch. Cucurbitaceae
Fruit Pointed gourd Tricosanthes dioica Roxb. Cucurbitaceae
Root Radish Raphanus sativus L. Brassicaceae
Grain Wheat Triticum aestivum L. Poaceae
Grain Rice Oryza sativa L. Poaceae
3.2. Levels of heavy metals in the soil
Elevated levels of heavy metals in irrigation water led to significantly
higher concentrations of heavy metals in the soil at WWI
site as compared to those obtained from clean water irrigated site
(Table 2). The heavy metal concentrations were, however, below
the safe limits of Indian (Awashthi, 2000) and EU standard (European
Union, 2002) at WWI site (Table 2). The lower concentrations
of heavy metals than the safe limits at WWI site may be due to the
continuous removal of heavy metals by the vegetables and cereals
grown in this area and also due to leaching of heavy metals into the
deeper layer of the soil. The increments in heavy metal concentrations
in the soil were 109% for Cd, 151% for Cu, 162% for Pb, 32% for
Zn, 161% for Ni and 112% for Cr at WWI site as compared to CWI
site in the present study (Table 2). Singh et al. (2004) have also reported
increments of 40.29% for Cu, 2.05% for Pb, 41.42% for Zn and
15.7% for Cr in soil of Dinapur area irrigated by treated wastewater
as compared to the site irrigated by clean water. In the present
study, Zn (58.1 lgg 1 ), Pb (21.4 lgg 1 ), Ni (23.6 lgg 1 ) and Cu
(21.1 lgg 1 ) concentrations were higher and Cr (19.1 lgg 1 ) concentration
was lower than the mean concentrations of 2.80, 20.35,
15.57, 43.56, 13.37 and 30.67 lgg 1 for Cd, Cu, Pb, Zn, Ni and Cr,
respectively, reported by Sharma et al. (2007) in the soil of wastewater
irrigated area of Dinapur. Zn concentration in soil was highest
and Cd was lowest at both CWI and WWI sites. Highest
concentration of Zn was also reported by Singh and Kumar
(2006) in the soil of Najafgarh, Delhi where the main sources of
contamination were sewage water irrigation and by Singh et al.
(2004) and Sharma et al. (2007) from Dinapur area.
3.3. Levels of heavy metals in the plants
Heavy metal concentrations showed variations among different
vegetables/cereals collected from CWI and WWI irrigated sites
(Figs. 2 and 3). Results of two way ANOVA test showed that variations
in the heavy metal concentrations were significant due to
site, plant and site plant interaction (Table 3). The variations in
heavy metal concentrations in vegetables/cereals of the same site
may be ascribed to the differences in their morphology and physiology
for heavy metal uptake, exclusion, accumulation and retention
(Carlton-Smith and Davis, 1983; Kumar et al., 2009). Several
fold higher concentrations of all the heavy metals were observed
in all the vegetables and cereal at WWI site as compared to CWI
site. The use of contaminated irrigation water at WWI site increased
the uptake and accumulation of the heavy metals in the
plants. This is consistent with reports of higher concentrations of
heavy metals in vegetables from sewage water irrigated areas as

614 A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619
Fig. 1. Monthly variations in heavy metal concentrations of water at WWI and CWI sites.
Table 2
The range and mean concentrations (lg g 1 ) of heavy metals in soil of wastewater (WWI) and clean water irrigated (CWI) sites.
Heavy metals CWI WWI Safe limits
Range Mean Range Mean Indian (Awashthi, 2000) International (European Union, 2002)
Cd 0.81–2.62 1.49 1.92–4.53 3.12 **
Cu 6.30–11.56 8.39 18.36–25.50 21.13 ***
Pb 7.73–8.60 8.15 14.26–24.10 21.39 ***
Zn 38.96–50.18 44.19 53.43–64.56 58.13 ***
Ni 7.16–11.91 9.06 19.93–28.18 23.64 ***
Cr 7.35–11.17 9.07 17.92–21.18 19.21 ***
Student’s t-test was done for mean value of heavy metal concentrations between CWI and WWI site.
**
Level of significance: p 6 0.01.
***
Level of significance: p 6 0.001.
compared to the tubewell water irrigated areas of Ludhiana city of
Punjab (Kawatra and Bakhetia, 2008).
Among leafy vegetables (palak, amaranthus and cabbage) at
WW1 site, Ni (20.19 lgg 1 ) concentration was highest in palak
3–6 3.0
135–270 140
250–500 300
300–600 300
75–150 75
– 150
(Fig. 3). Sharma et al. (2007) have reported much lower concentration
of Ni in palak grown in the area irrigated with treated sewage
water. This difference may be ascribed to samples collected during
two specific periods in the year i.e. winter (December to January)

and summer (April to May) season by Sharma et al. (2007),
whereas in the present study sampling was conducted fortnightly
for a year. For other vegetables, Zn concentration was highest in lady’s
finger (68.54 lgg 1 ). The observed value of Zn during the
present study was lower than the value (130.14 lgg 1 ) recorded
by Sharma et al. (2006) in lady’s finger collected from the agricultural
field of Dinapur area irrigated with treated wastewater.
Among all the vegetables the concentration of Cu was maximum
(17.95 lgg 1 ) in tomato. Liu et al. (2006) found 12-fold higher
Cu concentration (201.75 lgg 1 ) in tomato collected from the
wastewater irrigated area of Zhengzhou city, China than clean
water irrigated area. In radish, the mean concentrations of Cd
(2.19 lgg 1 ), Pb (12.20 lgg 1 ), Cr (3.69 lgg 1 ) and Cu
(13.75 lgg 1 ) were higher than the values (0.082 lgg 1 for Cd,
0.47 lgg 1 for Pb, 0.38 lgg 1 for Cr, 8.65 lgg 1 for Cu) obtained
from a suburban area of Zhengzhou city, Henan Province, China
(Liu et al., 2006), but were lower than the concentrations
(17.79 lgg 1 for Cd, 57. 63 lgg 1 for Pb, 78.02 lgg 1 for Cr and
28.08 lgg 1 for Cu) reported in radish collected from treated
wastewater irrigated suburban area of Titagarh (Gupta et al.,
2008). Cauliflower also had lower concentrations of all the heavy
metals during the present study as compared to the values by Gupta
et al. (2008). When the concentrations of Cu and Zn in radish,
A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619 615
Fig. 2. Mean concentration of heavy metals in plant samples collected from CWI sites.
brinjal and cauliflower grown at wastewater irrigated sites of
Rajasthan, India (Arora et al., 2008) were compared, Zn was similar
and Cu was higher in all the three vegetables during the present
study.
Mean concentrations of all the heavy metals were lower in the
cereals (wheat and rice) as compared with the vegetable crops
(Fig. 3). Sinha et al. (2006) have also found lower concentrations
of the heavy metals in cereal crops as compared to leafy and non
leafy vegetables grown in wastewater irrigated areas. Among all
the heavy metals, Zn showed maximum and Cd showed minimum
concentration in all the vegetables and cereals. Sharma et al.
(2009) have also found highest concentration of Zn as compared
to Cu, Cd and Pb in the vegetables collected from market as well
as production sites of Varanasi city, India. The variations in the metal
concentrations of vegetables may also be ascribed to the variability
in the absorption of metals in plants and their further
translocation within the plants (Vousta et al., 1996). Computation
of correlation coefficients showed that Cu and Cd concentrations in
palak and rice were positively and significantly correlated with
their respective concentrations in the soil (Table 4). In amaranthus,
palak and bitter gourd, only Cd showed positive relationship. In
case of radish, cauliflower, tomato and sponge gourd, the correlations
were significant for Cr. Positive correlations suggest that

616 A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619
Table 3
Results of two way ANOVA test for heavy metal concentrations in plants.
Metals Site Plants Site plant
Cd 417.89 ***
Cu 191.63 ***
Pb 604.43 ***
Zn 588.58 ***
Ni 934.22 ***
Cr 94.80 ***
*** Level of significance: p < 0.001.
the metals in plants were translocated efficiently from the soil
through root system. Significant negative correlations were found
for Pb in palak, Cd in bottle gourd and radish and Zn in palak, tomato,
pumpkin and pointed gourd. Sinha et al. (2006) have also
found positive and negative correlations between heavy metal concentrations
of plants and soil, which may be due to multiple interactions
among heavy metals for uptake in the plants (An et al.,
2004).
When the present concentrations of metals were compared
with permissible limits of Indian Standard (Awashthi, 2000) and
safe limits given by WHO/FAO (WHO/ FAO, 2007), then it was
found that at WWI site Cd, Pb and Ni concentrations were higher
Fig. 3. Mean concentration of heavy metals in plant samples collected from WWI sites.
7.17 ***
16.09 ***
10.01 ***
73.54 ***
2.70 ***
3.86 ***
7.44 ***
5.92 ***
10.36 ***
46.91 ***
2.52 ***
4.36 ***
in all the vegetables and cereal crops, whereas Zn concentration
was higher than both the safe limits in lady’s fingers and cabbage.
The present concentrations of Cd and Pb were higher in all the vegetables
and cereal crops when compared with safe limits given by
EU commission regulation (European Union, 2006). Cadmium and
Pb are nonessential metals causing adverse health effects even at
very low concentrations (Ikeda et al., 2000). Zhuang et al. (2009)
have also found higher than the maximum permissible levels of
Cd and Pb concentrations in vegetables collected from six sampling
sites around Dabaoshan mine located at Shaoguan city, Guangdong,
southern China.
3.4. Heavy metal concentrations in milk
Concentrations of all the heavy metals were higher in milk samples
collected from wastewater irrigated site as compared to the
samples from clean water irrigated site (Figs. 2 and 3). Heavy metal
concentration was highest for Zn followed by Cu > Pb > Cr > -
Ni > Cd. Concentrations of all the heavy metals were below the safe
limits (WHO/FAO, 2007).
Milk has property of retention of metals due to formation of
bioactive (lipophilic) complex (Leeuwen and Pinheiro, 2001;
Buechler et al., 2002). Milk samples collected from wastewater irrigated
site showed about three times higher concentrations of Cd

Table 4
Correlation coefficients (r 2 ) between heavy metal concentrations in the edible parts of the plants and metal concentrations in the soil.
Vegetables/cereals Cd Cu Pb Zn Ni Cr
Palak 0.86 **
Amaranthus 0.86 **
Cabbage 0.04 NS
Cauliflower 0.28 NS
Lady’s fingers 0.38 NS
Brinjal 0.29 NS
Tomato 0.39 NS
Bottle gourd 0.81 **
Sponge gourd 0.001 NS
Bitter gourd 0.79 **
Pumpkin 0.08 NS
Pointed gourd 0.48 NS
Radish 0.69 **
Wheat 0.12 NS
Rice 0.81 **
NS = not significant.
* Level of significance: p < 0.05.
** Level of significance: p < 0.01.
and Ni, five times of Cu, Pb, Zn and seven times of Cr as compared
to the respective heavy metal concentrations in the milk samples
collected from clean water irrigated site. In the present study, milk
was found to be the least responsible for causing health risk due to
heavy metal intake as the concentrations of all the heavy metals
were very low as compared to the vegetables/cereals. Zheng
et al. (2007) have also found lower concentrations of Cd and Cu
in milk samples as compared to other common foodstuff collected
from the industrial area of Huludao city, China where Hg, Pb, Cd, Zn
and Cu are added into the environment in large quantities through
atmospheric deposition, solid waste disposal, sludge application
and wastewater irrigation. The mean concentrations Cd, Pb, Zn
and Cu reported in milk samples of Huludao city, China were
respectively, 0.002, 0.011, 28.98 and 0.307 lgml 1 (Zheng et al.,
2007), whereas during the present study the concentrations of
respective metals were 0.003, 0.044, 0.290 and 0.0.055 lgml 1 .
Marti-Cid et al. (2009) have also found lower concentration of
Mn in milk sample as compared to the cereal and vegetable crops
collected from localities of Tarragona Country (Catalonia, Spain),
near a hazardous waste incinerator.
3.5. Enrichment factor
0.69 **
0.56 NS
0.85 NS
0.06 NS
0.15 NS
0.39 *
0.75 *
0.33 NS
0.47 NS
0.49 NS
0.43 NS
0.22 NS
0.26 NS
0.29 NS
0.76 **
Higher values of enrichment factor (EF) suggest poor retention
of metals in soil and/or more translocation in plants. Within the
Table 5
Enrichment factor of heavy metals in collected foodstuffs from the experimental site.
Foodstuffs Cd Cu Pb Zn Ni Cr
Palak 2.63 0.94 23.08 1.63 18.47 3.47
Amaranthus 2.74 0.72 24.39 1.03 20.69 8.75
Cabbage 10.88 0.59 51.92 3.08 17.22 5.47
Cauliflower 2.30 1.71 28.33 1.42 18.71 1.66
Lady’s fingers 8.04 0.42 31.38 6.14 10.45 14.35
Brinjal 3.92 0.84 30.63 1.37 20.60 8.96
Tomato 3.77 1.12 21.61 1.34 9.68 5.25
Bottle gourd 1.41 0.29 11.48 0.67 3.50 2.00
Sponge gourd 4.22 0.61 16.34 1.08 16.23 2.78
Bitter gourd nf nf nf nf nf nf
Pumpkin 3.43 0.77 52.12 1.43 18.17 4.67
Pointed gourd 0.37 0.18 3.79 0.30 4.10 0.27
Radish 2.17 1.15 14.02 1.36 15.28 4.32
Wheat 4.57 1.54 34.98 2.42 15.26 0.58
Rice 6.87 1.23 31.59 1.52 7.98 31.51
nf = Not found at clean water irrigated site during sampling period.
A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619 617
0.56 **
0.70 *
0.10 NS
0.38 NS
0.95 **
0.22 NS
0.44 NS
0.26 NS
0.27 NS
0.001 NS
0.57 NS
0.79 *
0.15 NS
0.37 NS
0.31 NS
0.86 **
0.44 NS
0.59 NS
0.28 NS
0.45 NS
0.59 NS
0.76 **
0.08 NS
0.33 NS
0.08 NS
0.68 *
0.69 *
0.43 NS
0.03 NS
0.27 NS
0.08 NS
0.16 NS
0.99 **
0.33 NS
0.01 NS
0.11 NS
0.62 *
0.92 **
0.44 NS
0.85 **
0.24 NS
0.86 **
0.13 NS
0.23 NS
0.79 **
plants, cabbage (leafy vegetable) showed highest EF value (10.88)
for Cd and amaranthus for Ni (Table 5). Fytianos et al. (2001) have
reported higher enrichment factor for Cd through leafy vegetables.
Sridhara Chary et al. (2008) also reported highest enrichment factor
for heavy metals through leafy vegetables. Enrichment factor of
other metals like Pb, Zn and Cr was highest in pumpkin, lady’s fingers
and rice, respectively (Table 5). Enrichment factor of heavy
metals depends upon bioavailability of metals, which in turn depends
upon its concentration in the soil, their chemical forms, difference
in uptake capability and growth rate of different plant
species (Tinker, 1981). The higher uptake of heavy metals in leafy
vegetables may be due to higher transpiration rate to maintain the
growth and moisture content of these plants (Tani and Barrington,
2005).
3.6. Metal pollution index and health risk assessment
0.34 NS
0.43 NS
0.62 NS
0.98 **
0.41 NS
0.07 NS
0.91 **
0.66 *
0.69 **
0.65 *
0.53 NS
0.50
0.89 **
0.06 NS
0.72 *
Metal pollution index (MPI) is suggested to be a reliable and
precise method for metal pollution monitoring of wastewater irrigated
areas (Usero et al., 1997). Among different vegetables, cabbage
showed highest value of MPI followed by palak. As
compared to the vegetables, wheat and rice showed lower metal
pollution index (Fig. 4). Higher MPI of cabbage, palak, brinjal and
lady’s finger suggests that these vegetables may cause more human
health risk due to higher accumulation of heavy metals in
the edible portion.
To assess the health risk associated with heavy metal contamination
of plants grown locally, estimated exposure and risk index
were calculated. The results showed that Cd, Pb and Ni contamination
in plants had greatest potential to pose health risk to the consumers
(Table 6). Health risk index was more than 1 for Cd in all
the plants except radish, pointed gourd and tomato. For Pb, it
was higher in all the leafy vegetables, lady’s finger, brinjal, bottle
gourd and cereal crops, whereas for Ni, it was higher in cereal crops
and all the leafy vegetables except cabbage. Although cereal crops
(wheat and rice) have lesser concentrations of metals than vegetables,
but the health risk index was higher. This may be due to higher
proportion of cereals in diet, which consequently increased the
health risk index. In the present study, Cu, Zn and Cr were not
found to cause any risk to the local population. Cui et al. (2004)
have also reported that local residents of an area near a smelter
in Nanning, China have been exposed to Cd and Pb through consumption
of vegetables, but no risk was found due to Cu and Zn.

618 A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619
4. Conclusions
Irrigation of agricultural lands with treated and untreated sewage
wastewater led to the accumulation of heavy metals in the soil,
vegetables, cereals and milk samples. Variations in the heavy metal
concentrations between the test vegetables/cereal crops reflect the
differences in uptake capabilities and their further translocation to
the edible portion of the plants. Cadmium, Pb and Ni concentrations
were above the national and various international permissible
limits in all the vegetables and cereal crops. The metal
pollution index and health risk index of heavy metals also suggest
that Cd, Pb and Ni contamination in most of the test plants had potential
for human health risk due to consumption of plants grown
at waste water irrigated site. Milk is found to be least contaminated
by heavy metals as its metal pollution index and health risk
index were lower compared to other foodstuffs. The health risk index
of cereals was higher than vegetables due to higher proportion
of cereals in the diet. Consumption of foodstuff with elevated levels
of heavy metals may lead to high level of accumulation in the body
causing related health disorders. The study suggests that even
though there are low concentrations of heavy metals in irrigation
water, its long term use caused heavy metal contamination leading
to health risk of consumers. Thus urgent attention is needed to
Fig. 4. Metal pollution index in different foodstuffs from wastewater irrigated site.
Table 6
Health risk index (HRI) of heavy metals via intake of foodstuffs from wastewater irrigated sites.
HRI
Foodstuffs Cd Cu Pb Zn Ni Cr
Palak 4.62 2.4E 02 2.91 5.1E 03 1.31 2.4E 04
Amaranthus 5.32 1.6E 02 4.10 3.4E 03 1.61 4.2E 04
Cabbage 10.16 9.0E 03 1.68 8.8E 03 0.83 2.0E 04
Cauliflower 3.88 3.1E 03 7.49 7.4E 03 1.80 2.0E 04
Lady’s fingers 5.08 6.0E 03 1.52 5.7E 03 0.43 2.9E 04
Brinjal 1.48 8.0E 03 1.11 1.1E 03 0.47 8.0E 05
Tomato 0.49 5.0E 03 0.43 6.0E 04 0.18 5.0E 05
Bottle gourd 1.07 6.0E 03 0.82 8.0E 04 0.37 2.5E 04
Sponge gourd 2.10 9.0E 03 0.95 1.2E 03 0.54 2.3E 04
Bitter gourd 1.61 3.0E 03 1.09 1.0E 03 0.39 3.0E 04
Pumpkin 1.14 4.0E 03 0.96 1.0E 03 0.36 7.0E 05
Pointed gourd 0.76 4.0E 03 0.31 5.0E 04 0.21 1.3E 04
Radish 0.49 4.0E 03 0.41 7.0E 04 0.21 4.0E 04
Wheat 5.86 5.16E 02 4.37 4.81E 03 2.40 2.97E 04
Rice 9.15 4.39E 02 6.83 8.40E 03 1.32 2.17E 04
Milk 2.0E 05 1.0E 06 4.0E 04 2.0E 06 7.0E 04 1.0E 06
3devise and implement appropriate means of monitoring and regulating
industrial and domestic effluent, and providing appropriate
advice and support for the safe and productive use of wastewater
for irrigation.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
A. Singh is thankful to the Center for Advanced Study in Botany,
Banaras Hindu University and R.K. Sharma to CSIR, New Delhi for
providing Senior Research Fellowships. The present research work
is an out-put of collaborative research project entitled ‘Contaminated
irrigation water and food safety for the urban and peri-urban
poor: appropriate measure for monitoring and control from field
research in India and Zambia’ led by Fiona Marshall and funded
by Department for International Development (DFID), UK (EnKar
R8160, www.pollutionandfood.net).

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