Food and Chemical Toxicology 48 (2010) 611–619

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate / foodchemtox

Health risk assessment of heavy metals via dietary intake of foodstuffsfrom 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, Indiab SPRU, Freeman Centre, University of Sussex, Brighton BN1 9QE, United Kingdom

a r t i c l e i n f o

Article history:Received 25 August 2009Accepted 19 November 2009

Keywords:Sewage waterHeavy metalsVegetablesCerealsMetal pollution indexHealth risk

0278-6915/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.fct.2009.11.041

* Corresponding author. Tel.: +91 542 2368156; faxE-mail address: madhoo58@yahoo.com (M. Agraw

a b s t r a c t

The present study was conducted to assess the risk to human health by heavy metals (Cd, Cu, Pb, Zn, Niand Cr) through the intake of locally grown vegetables, cereal crops and milk from wastewater irrigatedsite. Milk is not directly contaminated due to wastewater irrigation, but is an important route of foodchain transfer of heavy metals from grass to animals. Heavy metal concentrations were several foldhigher 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 veg-etables and cereals, but within the permissible limits in milk samples. The higher values of metal pollu-tion index and health risk index indicated heavy metal contamination in the wastewater irrigated sitethat presented a significant threat of negative impact on human health. Rice and wheat grains containedless heavy metals as compared to the vegetables, but health risk was greater due to higher contribution ofcereals in the diet. The study suggests that wastewater irrigation led to accumulation of heavy metals infood stuff causing potential health risks to consumers.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The growing problem of water scarcity has significant negativeinfluence on economic development, human livelihoods, and envi-ronmental quality throughout the world. Rapid urbanization andindustrialization releases enormous volumes of wastewater, whichis increasingly utilized as a valuable resource for irrigation in urbanand peri-urban agriculture. It drives significant economic activity,supports countless livelihoods particularly those of poor farmers,and substantially changes the water quality of natural waterbodies (Marshall et al., 2007). Wastewater may contain variousheavy metals including Zn, Cu, Pb, Mn, Ni, Cr, Cd, depending uponthe type of activities it is associated with. Continuous irrigation ofagricultural land with sewage and industrial wastewater maycause heavy metal accumulation in the soil and vegetables (Singhet al., 2004; Sharma et al., 2007; Marshall et al., 2007).

Heavy metals are generally not removed even after the treat-ment of wastewater at sewage treatment plants, and thus causerisk of heavy metal contamination of the soil and subsequentlyto the food chain (Fytianos et al., 2001). Intake of heavy metalsthrough the food chain by human populations has been widelyreported throughout the world (Muchuweti et al., 2006). Due to

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: +91 542 2368174.al).

the non-biodegradable and persistent nature, heavy metals areaccumulated in vital organs in the human body such as thekidneys, bones and liver and are associated with numerous serioushealth disorders (Duruibe et al., 2007). Individual metals exhibitspecific signs of their toxicity. Lead, As, Hg, Zn, Cu and Al poisoninghave been implicated with gastrointestinal (GI) disorders,diarrhoea, stomatitis, tremor, hemoglobinuria causing a rust-redcolour to stool, ataxia, paralysis, vomiting and convulsion, depres-sion, and pneumonia (McCluggage, 1991). The nature of effects canbe toxic (acute, chronic or sub-chronic), neurotoxic, carcinogenic,mutagenic or teratogenic (European Union, 2002).

Vegetables, cereals and milk are major components of humandiet, being sources of essential nutrients, antioxidants and metab-olites in food items. In the present study, the concentrations ofheavy metals in locally produced vegetables, cereals and milk werequantified throughout a year at a suburban area of Varanasi city ofIndia, where treated and untreated wastewater has been used as asource of irrigation water for about 20 years. The contaminationlevels in soil and vegetable/cereal crops were evaluated with re-spect to the prescribed safe limits of different heavy metals set un-der national and international norms. Milk is not directlycontaminated by wastewater irrigation, but provides insight intothe food chain transfer of heavy metals from fodder grass to themilk of animals. A number of standard measures were used to as-sess the health risks associated with the measured levels of heavymetal 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) situ-ated at a suburban area in the north east of Varanasi (25�18’ N latitude 83�01’ E lon-gitude and 76.19 m above the sea level) city in eastern Gangetic plains of Indiaduring March 2006 to February 2007. Large-scale vegetable production is con-ducted in this area, largely to supply markets in the city. Dinapur sewage treatmentplant of 80 million liters per day (MLD) capacity was installed in 1986. Effluentsfrom various small scale industries situated in the city are also discharged alongwith sewage for treatment at DSTP. These industries include fabric painting, batter-ies, dye, plastic recycling and metal surface treatment. A large area around DSTP hasno access to clean water resources, so farmers use treated and untreated wastewa-ter for irrigation. Two major sites were demarcated in Dinapur having different irri-gation practices. At the wastewater irrigated (WWI) site, treated wastewater fromDSTP has been used for irrigating the fields for about 20 years. Some times due topower failure, the sewage treatment plant does not work and untreated wastewateris used for irrigation. Clean water from bore wells has been used for irrigating theagricultural 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 2006to February 2007. Soil samples were collected in triplicate by digging out a mono-lith of 10 � 10 � 15 cm3 size, from 10 sub sites of both clean (CWI) and wastewaterirrigated sites (WWI). Soil samples were air dried, crushed and passed through2 mm mesh size sieve and stored at ambient temperature before analysis. Bothclean and wastewater samples (100 ml) used for irrigation were collected in tripli-cate in a pre acid washed polypropylene bottle and 1 ml of concentrated HNO3 wasadded in the water sample to avoid the microbial activity. These samples werebrought 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, eitherfor home consumption or sale, were collected. The details of different plants sampledduring the experiment are given in Table 1. An area of 5 � 5 m2 was randomly markedat 10 subsites in triplicate and the edible portion of test vegetables were collectedfrom both CWI and WWI sites. Samples were brought back to the laboratory andwashed with clean tap water to remove the soil particles adhered to the surface of

EF ¼ Concentration of metal in edible part at WWI site=concentration of metal in soil at WWIConcentration of metal in edible part at CWI site=concentration of metal in soil at CWI site

:

the vegetables. After removing the extra water from the surface of vegetables withblotting paper, samples were cut into pieces, packed into separate bags, and kept inan oven until a constant weight was achieved. For cereal crops, plots of 5 � 5 m2 sizeswere marked in triplicate at 10 subsites at both CWI and WWI sites, and ears wereharvested upon maturity. Grains were separated and kept in an oven for drying, untilconstant weight was achieved. The dried samples were grinded and passed through asieve 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 washedpolypropylene bottles, at both CWI and WWI sites, and stored at 4 �C prior to diges-tion for heavy metal analysis.

2.5. Digestion of samples

2.5.1. Soil and plantSoil and plant samples (1 g) were digested after adding 15 ml of tri-acid mixture

(HNO3, H2SO4, and HClO4 in 5:1:1 ratio) at 80 �C until a transparent solution wasobtained (Allen et al., 1986). After cooling, the digested sample was filtered usingWhatman No. 42 filter paper and the filtrate was finally maintained to 50 ml withdistilled water.

2.5.2. Irrigation waterThe irrigation water sample (50 ml) was digested with 10 ml of concentrated

HNO3 at 80 �C until the solution became transparent (APHA, 2005). The solutionwas filtered through Whatman No. 42 filter paper and the total volume was main-tained to 50 ml with distilled water.

2.5.3. MilkFor 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 watercontent (without boiling). When the mass became syrupy, it was cooled and 10 mlof HNO3 (70% V/V) was added. The mixture was warmed until the evolution ofbrown fumes of NO2 ceased and a colourless solution was obtained. About 2.5 mlof HClO4 was added and again heated for complete digestion. The extract after fil-tration 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 spectropho-tometer (Model 2380, Perkin Elmer, Inc., Norwalk, CT, USA). The instrument was fit-ted with specific lamp of particular metal. The instrument was calibrated usingmanually prepared standard solution of respective heavy metals as well as driftblanks. Standard stock solution of 1000 ppm for all the metals were obtained fromSisco Research Laboratories Pvt. Ltd., India. These solution were diluted for desiredconcentrations to calibrate the instrument. Acetylene gas was used as the fuel andair 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 ofsamples against National Institute of standard and technology, Standard ReferenceMaterial (SRM 1570) for all the heavy metals. The results were found within ±2% ofthe certified value. Quality control measures were taken to asses contamination andreliability of data. Blank and drift standards (Sisco Research Laboratories Pvt. Ltd.,India) were run after five determination to calibrate the instrument. The coeffi-cients of variation of replicate analysis were determined for different determina-tions for precision of analysis and variations below 10% were considered correct.

2.8. Data analyses

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 be-tween the sites, the enrichment factor (EF) was calculated by using the formula gi-ven 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 indexwas obtained by calculating the geometrical mean of concentrations of all the met-als in the vegetables, cereals and milk (Usero et al., 1997).

MPIðlg g�1Þ ¼ ðCf1 � Cf2 � � � � � CfnÞ1=n

where Cfn = 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 were4 � 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 bytheir 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

Baverage weight

where Cmetal, Dfood intake and Baverage weight represent the heavy metal concentrationsin plants (lg g�1), daily intake of vegetables and average body weight, respectively.The average daily vegetable intake rate was calculated by conducting a survey where100 people having average body weight of 60 kg were asked for their daily intake ofparticular vegetable from the experimental area in each month of sampling (Ge,1992; Wang et al., 2005).

Table 1Plant samples collected from the experimental sites.

Edible part of vegetable/cereal crops Common name Botanical name Family

Leaf Palak Beta vulgaris L. ChenopodiaceaeLeaf Amaranthus Amaranthus caudatus L. AmaranthaceaeLeaf Cabbage Brasssica oleracea L. var capitata L. BrassicaceaeInflorescence Cauliflower Brassica oleracea L. var. Botrytis L. BrassicaceaeFruit Lady’s finger Abelmoschus esculentus L. MalvaceaeFruit Brinjal Solanum melongena L. SolanaceaeFruit Tomato Lycopersicon esculentum L. SolanaceaeFruit Bottle gourd Lagenaria siceraria Mol. CucurbitaceaeFruit Sponge gourd Luffa cylindrica L. CucurbitaceaeFruit Bitter gourd Momordica charantia L. CucurbitaceaeFruit Pumpkin Cucurbita maxima Duch. CucurbitaceaeFruit Pointed gourd Tricosanthes dioica Roxb. CucurbitaceaeRoot Radish Raphanus sativus L. BrassicaceaeGrain Wheat Triticum aestivum L. PoaceaeGrain Rice Oryza sativa L. Poaceae

A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619 613

2.9. Statistical analysis

The significance of differences between the concentrations of heavy metals insoil at wastewater (WWI) and clean water irrigated (CWI) sites were shown byusing Student’s t-test. The data of heavy metal concentrations in the plants at dif-ferent sites were subjected to two way analysis of variance (ANOVA) test for assess-ing the significance of differences in heavy metal concentrations due to differentirrigation 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 (lg ml�1) of heavy metals in the irrigationwater at WWI site ranged between 0.00–0.02 for Cd, 0.02–0.07for Cu, 0.07–0.13 for Pb, 0.05–0.18 for Zn, 0.02–0.08 for Ni and0.03–0.08 for Cr during March 2006 to February 2007, whereasat CWI site, heavy metal concentrations in irrigation water werevery low or below the detectable limits (Fig. 1). Among all the hea-vy metals, Cd concentration exceeded the permissible limit set byFAO (1985). Heavy metals in the sewage water are associated withsmall scale industries such as colouring, electroplating, metal sur-face treatments, fabric printing, battery and paints, releasing Cd,Cu, Pb, Zn, Ni and other heavy metals into water channels, whichare accessed for irrigation. As compared to the present concentra-tion of heavy metals in the wastewater, Singh et al. (2004) havereported lower ranges of Cd (0.00–0.006 lg ml�1), Cr (0.00–0.049 lg ml�1) and Pb (0.012–0.088 lg ml�1), but higher rangesof Cu (0.00–0.203 lg ml�1), Ni (0.01–0.22 lg ml�1) and Zn(0.023–0.18 lg ml�1) were reported in the water samples of Dina-pur area of Varanasi receiving treated and untreated sewage waterfor irrigating the agricultural fields. Sharma et al. (2007) reportedsimilar ranges of Cd, Ni and Zn in irrigation water of DSTP, butCu, Pb and Cr were twofold higher during the present study. Thecomparison of the concentrations of heavy metals in treatedwastewater of Dinapur STP from STP of Titagarh, West Bengal, In-dia showed that Cd (0.01 lg ml�1) and Cr (0.04 lg ml�1) were sim-ilar, but Zn (1.17 lg ml�1), Ni (0.39 lg ml�1), Pb (3.54 lg ml�1) andCu (0.98 lg ml�1) were lower during the present study (Guptaet al., 2008). Among the heavy metals, the mean concentrationwas 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 lowerconcentrations of heavy metals in the irrigation water may bedue to dilution of heavy metals in the water medium, but the con-tinuous application of these treated and untreated wastewater forirrigation resulted into accumulation of heavy metals into the soil.

3.2. Levels of heavy metals in the soil

Elevated levels of heavy metals in irrigation water led to signif-icantly higher concentrations of heavy metals in the soil at WWIsite as compared to those obtained from clean water irrigated site(Table 2). The heavy metal concentrations were, however, belowthe safe limits of Indian (Awashthi, 2000) and EU standard (Euro-pean Union, 2002) at WWI site (Table 2). The lower concentrationsof heavy metals than the safe limits at WWI site may be due to thecontinuous removal of heavy metals by the vegetables and cerealsgrown in this area and also due to leaching of heavy metals into thedeeper layer of the soil. The increments in heavy metal concentra-tions in the soil were 109% for Cd, 151% for Cu, 162% for Pb, 32% forZn, 161% for Ni and 112% for Cr at WWI site as compared to CWIsite in the present study (Table 2). Singh et al. (2004) have also re-ported increments of 40.29% for Cu, 2.05% for Pb, 41.42% for Zn and15.7% for Cr in soil of Dinapur area irrigated by treated wastewateras compared to the site irrigated by clean water. In the presentstudy, Zn (58.1 lg g�1), Pb (21.4 lg g�1), Ni (23.6 lg g�1) and Cu(21.1 lg g�1) concentrations were higher and Cr (19.1 lg g�1) con-centration was lower than the mean concentrations of 2.80, 20.35,15.57, 43.56, 13.37 and 30.67 lg g�1 for Cd, Cu, Pb, Zn, Ni and Cr,respectively, reported by Sharma et al. (2007) in the soil of waste-water irrigated area of Dinapur. Zn concentration in soil was high-est and Cd was lowest at both CWI and WWI sites. Highestconcentration of Zn was also reported by Singh and Kumar(2006) in the soil of Najafgarh, Delhi where the main sources ofcontamination 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 differentvegetables/cereals collected from CWI and WWI irrigated sites(Figs. 2 and 3). Results of two way ANOVA test showed that varia-tions in the heavy metal concentrations were significant due tosite, plant and site � plant interaction (Table 3). The variations inheavy metal concentrations in vegetables/cereals of the same sitemay be ascribed to the differences in their morphology and phys-iology for heavy metal uptake, exclusion, accumulation and reten-tion (Carlton-Smith and Davis, 1983; Kumar et al., 2009). Severalfold higher concentrations of all the heavy metals were observedin all the vegetables and cereal at WWI site as compared to CWIsite. The use of contaminated irrigation water at WWI site in-creased the uptake and accumulation of the heavy metals in theplants. This is consistent with reports of higher concentrations ofheavy metals in vegetables from sewage water irrigated areas as

Fig. 1. Monthly variations in heavy metal concentrations of water at WWI and CWI sites.

Table 2The 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** 3–6 3.0Cu 6.30–11.56 8.39 18.36–25.50 21.13*** 135–270 140Pb 7.73–8.60 8.15 14.26–24.10 21.39*** 250–500 300Zn 38.96–50.18 44.19 53.43–64.56 58.13*** 300–600 300Ni 7.16–11.91 9.06 19.93–28.18 23.64*** 75–150 75Cr 7.35–11.17 9.07 17.92–21.18 19.21*** – 150

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.

614 A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619

compared to the tubewell water irrigated areas of Ludhiana city ofPunjab (Kawatra and Bakhetia, 2008).

Among leafy vegetables (palak, amaranthus and cabbage) atWW1 site, Ni (20.19 lg g�1) concentration was highest in palak

(Fig. 3). Sharma et al. (2007) have reported much lower concentra-tion of Ni in palak grown in the area irrigated with treated sewagewater. This difference may be ascribed to samples collected duringtwo specific periods in the year i.e. winter (December to January)

Fig. 2. Mean concentration of heavy metals in plant samples collected from CWI sites.

A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619 615

and summer (April to May) season by Sharma et al. (2007),whereas in the present study sampling was conducted fortnightlyfor a year. For other vegetables, Zn concentration was highest in la-dy’s finger (68.54 lg g�1). The observed value of Zn during thepresent study was lower than the value (130.14 lg g�1) recordedby Sharma et al. (2006) in lady’s finger collected from the agricul-tural field of Dinapur area irrigated with treated wastewater.

Among all the vegetables the concentration of Cu was maxi-mum (17.95 lg g�1) in tomato. Liu et al. (2006) found 12-fold high-er Cu concentration (201.75 lg g�1) in tomato collected from thewastewater irrigated area of Zhengzhou city, China than cleanwater irrigated area. In radish, the mean concentrations of Cd(2.19 lg g�1), Pb (12.20 lg g�1), Cr (3.69 lg g�1) and Cu(13.75 lg g�1) were higher than the values (0.082 lg g�1 for Cd,0.47 lg g�1 for Pb, 0.38 lg g�1 for Cr, 8.65 lg g�1 for Cu) obtainedfrom a suburban area of Zhengzhou city, Henan Province, China(Liu et al., 2006), but were lower than the concentrations(17.79 lg g�1 for Cd, 57. 63 lg g�1 for Pb, 78.02 lg g�1 for Cr and28.08 lg g�1 for Cu) reported in radish collected from treatedwastewater irrigated suburban area of Titagarh (Gupta et al.,2008). Cauliflower also had lower concentrations of all the heavymetals during the present study as compared to the values by Gup-ta et al. (2008). When the concentrations of Cu and Zn in radish,

brinjal and cauliflower grown at wastewater irrigated sites ofRajasthan, India (Arora et al., 2008) were compared, Zn was similarand Cu was higher in all the three vegetables during the presentstudy.

Mean concentrations of all the heavy metals were lower in thecereals (wheat and rice) as compared with the vegetable crops(Fig. 3). Sinha et al. (2006) have also found lower concentrationsof the heavy metals in cereal crops as compared to leafy and nonleafy vegetables grown in wastewater irrigated areas. Among allthe heavy metals, Zn showed maximum and Cd showed minimumconcentration in all the vegetables and cereals. Sharma et al.(2009) have also found highest concentration of Zn as comparedto Cu, Cd and Pb in the vegetables collected from market as wellas production sites of Varanasi city, India. The variations in the me-tal concentrations of vegetables may also be ascribed to the vari-ability in the absorption of metals in plants and their furthertranslocation within the plants (Vousta et al., 1996). Computationof correlation coefficients showed that Cu and Cd concentrations inpalak and rice were positively and significantly correlated withtheir respective concentrations in the soil (Table 4). In amaranthus,palak and bitter gourd, only Cd showed positive relationship. Incase of radish, cauliflower, tomato and sponge gourd, the correla-tions were significant for Cr. Positive correlations suggest that

Fig. 3. Mean concentration of heavy metals in plant samples collected from WWI sites.

Table 3Results of two way ANOVA test for heavy metal concentrations in plants.

Metals Site Plants Site � plant

Cd 417.89*** 7.17*** 7.44***

Cu 191.63*** 16.09*** 5.92***

Pb 604.43*** 10.01*** 10.36***

Zn 588.58*** 73.54*** 46.91***

Ni 934.22*** 2.70*** 2.52***

Cr 94.80*** 3.86*** 4.36***

*** Level of significance: p < 0.001.

616 A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619

the metals in plants were translocated efficiently from the soilthrough root system. Significant negative correlations were foundfor Pb in palak, Cd in bottle gourd and radish and Zn in palak, to-mato, pumpkin and pointed gourd. Sinha et al. (2006) have alsofound positive and negative correlations between heavy metal con-centrations of plants and soil, which may be due to multiple inter-actions among heavy metals for uptake in the plants (An et al.,2004).

When the present concentrations of metals were comparedwith permissible limits of Indian Standard (Awashthi, 2000) andsafe limits given by WHO/FAO (WHO/ FAO, 2007), then it wasfound that at WWI site Cd, Pb and Ni concentrations were higher

in all the vegetables and cereal crops, whereas Zn concentrationwas higher than both the safe limits in lady’s fingers and cabbage.The present concentrations of Cd and Pb were higher in all the veg-etables and cereal crops when compared with safe limits given byEU commission regulation (European Union, 2006). Cadmium andPb are nonessential metals causing adverse health effects even atvery low concentrations (Ikeda et al., 2000). Zhuang et al. (2009)have also found higher than the maximum permissible levels ofCd and Pb concentrations in vegetables collected from six samplingsites around Dabaoshan mine located at Shaoguan city, Guang-dong, southern China.

3.4. Heavy metal concentrations in milk

Concentrations of all the heavy metals were higher in milk sam-ples collected from wastewater irrigated site as compared to thesamples from clean water irrigated site (Figs. 2 and 3). Heavy metalconcentration was highest for Zn followed by Cu > Pb > Cr > -Ni > Cd. Concentrations of all the heavy metals were below the safelimits (WHO/FAO, 2007).

Milk has property of retention of metals due to formation ofbioactive (lipophilic) complex (Leeuwen and Pinheiro, 2001;Buechler et al., 2002). Milk samples collected from wastewater irri-gated site showed about three times higher concentrations of Cd

Table 4Correlation coefficients (r2) 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** 0.69** �0.56** � 0.86** 0.08NS �0.34NS

Amaranthus 0.86** 0.56NS �0.70* 0.44NS 0.16NS 0.43NS

Cabbage 0.04NS 0.85NS �0.10NS �0.59NS �0.99** 0.62NS

Cauliflower �0.28NS 0.06NS 0.38NS 0.28NS 0.33NS 0.98**

Lady’s fingers �0.38NS �0.15NS 0.95** 0.45NS 0.01NS �0.41NS

Brinjal 0.29NS 0.39* 0.22NS �0.59NS 0.11NS 0.07NS

Tomato 0.39NS 0.75* 0.44NS �0.76** 0.62* 0.91**

Bottle gourd �0.81** 0.33NS 0.26NS 0.08NS 0.92** �0.66*

Sponge gourd 0.001NS �0.47NS 0.27NS 0.33NS 0.44NS 0.69**

Bitter gourd 0.79** �0.49NS 0.001NS 0.08NS �0.85** �0.65*

Pumpkin 0.08NS �0.43NS 0.57NS �0.68* 0.24NS 0.53NS

Pointed gourd �0.48NS 0.22NS �0.79* �0.69* �0.86** �0.50Radish �0.69** 0.26NS 0.15NS 0.43NS �0.13NS 0.89**

Wheat 0.12NS 0.29NS 0.37NS 0.03NS 0.23NS 0.06NS

Rice 0.81** 0.76** 0.31NS �0.27NS �0.79** �0.72*

NS = not significant.* Level of significance: p < 0.05.** Level of significance: p < 0.01.

A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619 617

and Ni, five times of Cu, Pb, Zn and seven times of Cr as comparedto the respective heavy metal concentrations in the milk samplescollected from clean water irrigated site. In the present study, milkwas found to be the least responsible for causing health risk due toheavy metal intake as the concentrations of all the heavy metalswere very low as compared to the vegetables/cereals. Zhenget al. (2007) have also found lower concentrations of Cd and Cuin milk samples as compared to other common foodstuff collectedfrom the industrial area of Huludao city, China where Hg, Pb, Cd, Znand Cu are added into the environment in large quantities throughatmospheric deposition, solid waste disposal, sludge applicationand wastewater irrigation. The mean concentrations Cd, Pb, Znand Cu reported in milk samples of Huludao city, China wererespectively, 0.002, 0.011, 28.98 and 0.307 lg ml�1 (Zheng et al.,2007), whereas during the present study the concentrations ofrespective metals were 0.003, 0.044, 0.290 and 0.0.055 lg ml�1.Marti-Cid et al. (2009) have also found lower concentration ofMn in milk sample as compared to the cereal and vegetable cropscollected from localities of Tarragona Country (Catalonia, Spain),near a hazardous waste incinerator.

3.5. Enrichment factor

Higher values of enrichment factor (EF) suggest poor retentionof metals in soil and/or more translocation in plants. Within the

Table 5Enrichment 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 3.47Amaranthus 2.74 0.72 24.39 1.03 20.69 8.75Cabbage 10.88 0.59 51.92 3.08 17.22 5.47Cauliflower 2.30 1.71 28.33 1.42 18.71 1.66Lady’s fingers 8.04 0.42 31.38 6.14 10.45 14.35Brinjal 3.92 0.84 30.63 1.37 20.60 8.96Tomato 3.77 1.12 21.61 1.34 9.68 5.25Bottle gourd 1.41 0.29 11.48 0.67 3.50 2.00Sponge gourd 4.22 0.61 16.34 1.08 16.23 2.78Bitter gourd nf nf nf nf nf nfPumpkin 3.43 0.77 52.12 1.43 18.17 4.67Pointed gourd 0.37 0.18 3.79 0.30 4.10 0.27Radish 2.17 1.15 14.02 1.36 15.28 4.32Wheat 4.57 1.54 34.98 2.42 15.26 0.58Rice 6.87 1.23 31.59 1.52 7.98 31.51

nf = Not found at clean water irrigated site during sampling period.

plants, cabbage (leafy vegetable) showed highest EF value (10.88)for Cd and amaranthus for Ni (Table 5). Fytianos et al. (2001) havereported higher enrichment factor for Cd through leafy vegetables.Sridhara Chary et al. (2008) also reported highest enrichment fac-tor for heavy metals through leafy vegetables. Enrichment factor ofother metals like Pb, Zn and Cr was highest in pumpkin, lady’s fin-gers and rice, respectively (Table 5). Enrichment factor of heavymetals depends upon bioavailability of metals, which in turn de-pends upon its concentration in the soil, their chemical forms, dif-ference in uptake capability and growth rate of different plantspecies (Tinker, 1981). The higher uptake of heavy metals in leafyvegetables may be due to higher transpiration rate to maintain thegrowth and moisture content of these plants (Tani and Barrington,2005).

3.6. Metal pollution index and health risk assessment

Metal pollution index (MPI) is suggested to be a reliable andprecise method for metal pollution monitoring of wastewater irri-gated areas (Usero et al., 1997). Among different vegetables, cab-bage showed highest value of MPI followed by palak. Ascompared to the vegetables, wheat and rice showed lower metalpollution index (Fig. 4). Higher MPI of cabbage, palak, brinjal andlady’s finger suggests that these vegetables may cause more hu-man health risk due to higher accumulation of heavy metals inthe edible portion.

To assess the health risk associated with heavy metal contami-nation of plants grown locally, estimated exposure and risk indexwere calculated. The results showed that Cd, Pb and Ni contamina-tion in plants had greatest potential to pose health risk to the con-sumers (Table 6). Health risk index was more than 1 for Cd in allthe plants except radish, pointed gourd and tomato. For Pb, itwas higher in all the leafy vegetables, lady’s finger, brinjal, bottlegourd and cereal crops, whereas for Ni, it was higher in cereal cropsand all the leafy vegetables except cabbage. Although cereal crops(wheat and rice) have lesser concentrations of metals than vegeta-bles, but the health risk index was higher. This may be due to high-er proportion of cereals in diet, which consequently increased thehealth risk index. In the present study, Cu, Zn and Cr were notfound to cause any risk to the local population. Cui et al. (2004)have also reported that local residents of an area near a smelterin Nanning, China have been exposed to Cd and Pb through con-sumption of vegetables, but no risk was found due to Cu and Zn.

Fig. 4. Metal pollution index in different foodstuffs from wastewater irrigated site.

Table 6Health 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�04Amaranthus 5.32 1.6E�02 4.10 3.4E�03 1.61 4.2E�04Cabbage 10.16 9.0E�03 1.68 8.8E�03 0.83 2.0E�04Cauliflower 3.88 3.1E�03 7.49 7.4E�03 1.80 2.0E�04Lady’s fingers 5.08 6.0E�03 1.52 5.7E�03 0.43 2.9E�04Brinjal 1.48 8.0E�03 1.11 1.1E�03 0.47 8.0E�05Tomato 0.49 5.0E�03 0.43 6.0E�04 0.18 5.0E�05Bottle gourd 1.07 6.0E�03 0.82 8.0E�04 0.37 2.5E�04Sponge gourd 2.10 9.0E�03 0.95 1.2E�03 0.54 2.3E�04Bitter gourd 1.61 3.0E�03 1.09 1.0E�03 0.39 3.0E�04Pumpkin 1.14 4.0E�03 0.96 1.0E�03 0.36 7.0E�05Pointed gourd 0.76 4.0E�03 0.31 5.0E�04 0.21 1.3E�04Radish 0.49 4.0E�03 0.41 7.0E�04 0.21 4.0E�04Wheat 5.86 5.16E�02 4.37 4.81E�03 2.40 2.97E�04Rice 9.15 4.39E�02 6.83 8.40E�03 1.32 2.17E�04Milk 2.0E�05 1.0E�06 4.0E�04 2.0E�06 7.0E�04 1.0E�06

618 A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619

4. Conclusions

Irrigation of agricultural lands with treated and untreated sew-age wastewater led to the accumulation of heavy metals in the soil,vegetables, cereals and milk samples. Variations in the heavy metalconcentrations between the test vegetables/cereal crops reflect thedifferences in uptake capabilities and their further translocation tothe edible portion of the plants. Cadmium, Pb and Ni concentra-tions were above the national and various international permissi-ble limits in all the vegetables and cereal crops. The metalpollution index and health risk index of heavy metals also suggestthat Cd, Pb and Ni contamination in most of the test plants had po-tential for human health risk due to consumption of plants grownat waste water irrigated site. Milk is found to be least contami-nated by heavy metals as its metal pollution index and health riskindex were lower compared to other foodstuffs. The health risk in-dex of cereals was higher than vegetables due to higher proportionof cereals in the diet. Consumption of foodstuff with elevated levelsof heavy metals may lead to high level of accumulation in the bodycausing related health disorders. The study suggests that eventhough there are low concentrations of heavy metals in irrigationwater, its long term use caused heavy metal contamination leadingto health risk of consumers. Thus urgent attention is needed to

3devise and implement appropriate means of monitoring and reg-ulating industrial and domestic effluent, and providing appropriateadvice and support for the safe and productive use of wastewaterfor 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 forproviding Senior Research Fellowships. The present research workis an out-put of collaborative research project entitled ‘Contami-nated irrigation water and food safety for the urban and peri-urbanpoor: appropriate measure for monitoring and control from fieldresearch in India and Zambia’ led by Fiona Marshall and fundedby Department for International Development (DFID), UK (EnKarR8160, www.pollutionandfood.net).

A. Singh et al. / Food and Chemical Toxicology 48 (2010) 611–619 619

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