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

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).