Impact of Sewage Water on Radish (Raphanus

sativus L.) and Soil With Respect to Heavy

Metals in Tarlai, Islamabad

By
Muhammad Zeeshan

Thesis submitted to the Faculty of Engineering at PIEAS in partial

fulfillment of requirements for the Degree of M.S. Nuclear Engineering

Department of Nuclear Engineering

Pakistan Institute of Engineering & Applied Sciences
Nilore, Islamabad, Pakistan
October, 2017
ii
 iii

Department of Nuclear Engineering,

Pakistan Institute of Engineering and Applied Sciences (PIEAS)
Nilore. Islamabad 45650, Pakistan

Declaration of Originality

I hereby declare that the work contained in this thesis and the intellectual content of
this thesis are the product of my own work. This thesis has not been previously
published in any form nor does it contain any verbatim of the published resources
which could be treated as infringement of the international copyright law.

I also declare that I do understand the terms copyright and plagiarism, and
that in case of any copyright violation or plagiarism found in this work, I will be held
fully responsible of the consequences of any such violation.

Signature: _______________________________

Name: __________________________________

Date: ____________________

Place: ____________________
 iv

Certificate of Approval

This is to certify that the work contained in this thesis entitled

Impact of sewage water on vegetables and soils with respect

to heavy metals in Tarlai, Islamabad
was carried out by
Muhammad Zeeshan
Under our supervision and that in our opinion, it is fully adequate, in scope
and quality, for the degree of M.S. Nuclear Engineering from Pakistan
Institute of Engineering and Applied Sciences (PIEAS).

Approved By:
Signature:________________________
Supervisor: Dr. Samreen Shehzadi

Signature: ________________________
PIEAS Co-Supervisor: Dr. Masroor Ahmad

Verified By:
Signature: ________________________
Head, Department of Nuclear Engineering
Stamp:
 v

Dedication

I It is with our deepest gratitude and warmest affection

that I dedicate this report
to our teacher
Dr. Samreen Shehzadi
who has been a constant source
of knowledge and inspiration.
 vi

Acknowledgement
In the name of Allah, who is the most gracious and the most merciful. All praises to
Allah for His blessing which enable me in completing this term project. I extend my
sincere gratitude to my Supervisor, Dr. Samreen Shehzadi, who has supported me
throughout my project with her patience and knowledge. I attribute my Masters
degree to her encouragement and effort.

I would like to thanks my Co-Supervisor Dr. Masroor Ahmed for his guidance and
assistance in completion of my project.

I would like to thank Dr. Arif Raza Zaidi, Head CAFD, for providing me the facility
of research to at CAFD to complete my project and secondly I would like to thank Dr.
Kashif Naeem, Lab incharge of Atomoc Absorption Lab for his guidance about the
instruments.

Most importantly, without his assistance in lab I could not be able to complete my
project, is Mr. Tanveer Ahmad, Lab Analyst, who helped a lot. Also pay special
thanks to Mr. Junaid for his helping at the sampling site as well as in the lab.

At the end I will say thanks to each and every person who helped me in any way
throughout my life.

Muhammad Zeeshan
 vii

Table of Contents
1 INTRODUCTION ...................................................................................................................... 11

1.1 HEAVY METALS ....................................................................................................................... 12

1.1.1 Mechanism of Heavy Metal Toxicity .............................................................................. 13
1.1.2 Lead ................................................................................................................................ 14
1.1.3 Arsenic ............................................................................................................................ 15
1.1.4 Cadmium ........................................................................................................................ 15
1.2 ATOMIC ABSORPTION SPECTROSCOPY...................................................................................... 16
1.2.1 Principle ......................................................................................................................... 16
1.2.2 Hollow Cathode Lamp .................................................................................................... 16
1.2.3 Atomizer.......................................................................................................................... 17
1.2.4 Diffraction Grating ......................................................................................................... 19
1.2.5 Photomultiplier Tube (PMT) .......................................................................................... 19
1.3 LIMITS OF DETECTION .............................................................................................................. 20

2 LITERATURE REVIEW .......................................................................................................... 21

3 MATERIALS AND METHODS ............................................................................................... 25

3.1 SAMPLE COLLECTION ............................................................................................................... 25

3.1.1 Sewage Water Sampling ................................................................................................. 26
3.1.2 Soil Sampling .................................................................................................................. 26
3.1.3 Radish Plant Sampling ................................................................................................... 27
3.2 PLANT SAMPLE PREPARATION .................................................................................................. 27
3.2.1 Plant Digestion ............................................................................................................... 28
3.3 SOIL SAMPLE PREPARATION ..................................................................................................... 28
3.3.1 Soil Digestion ................................................................................................................. 28
3.3.2 Quality of the Chemicals ................................................................................................ 28

4 REFERENCES ........................................................................................................................... 38
 viii

List of Figures

FIGURE 1-1: PRODUCTION OF LEAD, COPPER AND ZINC IN ALL OVER THE WORLD FROM 1850 TO 1990 [7].
...................................................................................................................................................... 13
FIGURE 1-2: HEAVY METAL ATTACK ON CELL AND AS RESULT PRODUCTION OF REACTIVE OXIDATIVE
SPECIES AND NEUTRALIZATION THE EFFECT BY ANTIOXIDANT [6]. ................................................ 13

FIGURE 1-3: SOURCES OF THE LEAD [6]................................................................................................... 14

FIGURE 1-4: SCHEMATIC DIAGRAM OF HOLLOW CATHODE LAMP [8]....................................................... 16
FIGURE 1-5: SCHEMATIC OVERVIEW OF GRAPHITE FURNACE [8]. ............................................................ 17
FIGURE 1-6: SCHEMATIC OF DIFFRACTION GRATING ............................................................................... 19
FIGURE 1-7; SCHEMATIC DIAGRAM OF PMT WITH NINE APPLICATION DYNODES [8]. .............................. 20
FIGURE 3-1: MAP OF TARLAI FARMS. ..................................................................................................... 25
FIGURE 3-1; SEWAGE WATER CANAL PASSING THROUGH TARLAI. .......................................................... 26
FIGURE 3-2; RADISH CROP FIELD ............................................................................................................ 27
FIGURE 3-3: FLOW DIAGRAM OF THE PROCEDURES FOLLOWED AFTER SAMPLING. .................................. 27
 ix

List of Tables

TABLE 1-1: TYPES OF ATOMIZER USED IN AAS [9]. ................................................................................ 17

TABLE 1-2: GF-AAS STAGES AND SCHEME OF TEMPERATURE INCREASE FOR LEAD MEASUREMENT. ..... 18
TABLE 1-3: DETECTION LIMIT OF HITACHI Z-8000 AAS......................................................................... 20
TABLE 3-1: SAMPLE COLLECTION SCHEDULE ......................................................................................... 25
TABLE 3-2: QUALITY OF CHEMICALS THAT WAS USED FOR DIGESTION ................................................... 28
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Abstract

With increasing population and urbanization, the sewage water generated by domestic,
industrial and commercial sources has increased. In the Urban areas, sewage water is
used as a source of irrigation water as it contains organic and inorganic elements
essential for plant growth. However, sewage water may also contain heavy metals
which contaminate the soil and toxic for plants which is a potential health risk to
consumers. For this purpose, a study was conducted at Tarlai farms, Islamabad,
Pakistan in 2016 to observe the impact of sewage water on heavy metals (Pb, Cd, As)
content in soil and its accumulation in radish. The sewage water, soil and radish
samples were collected at 7, 14, 20, 40 and 60 days after sowing (DAS) from farmers
field where use of sewage water for irrigation purpose is a regular practice for last many
years. The samples were analyzed using Atomic Absorption Spectrometry (AAS) and
Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). The physico-
chemical analyses of experimental soil at 0-15 cm depth indicated that it was sandy clay
loam in texture, alkaline in reaction, non saline with 0.08% total nitrogen and 1.8%
organic matter. The results revealed that the concentration of heavy metals in sewage
water and soil were in safe limits. However, in radish, the concentrations of heavy
metals (Pb, Cd) were above than the permissible levels set by the World Health
Organization (WHO). The highest concentrations of Pb ( 1.0 mg kg-1) and Cd (0.65 mg
kg-1) in radish were found at 7 DAS while maximum level of As (0.38 mg kg-1) was
found at 40 DAS. The levels of heavy metals in radish plant was found in the order of
Pb > Cd > As.
 11

1 Introduction

Water resources are limited and may not meet the requirements of increasing
population. Water is mainly required for drinking purpose, beside this it is also
demanded in a large quantity for irrigation. Normally, fresh water (rivers, lakes,
canals) and underground water (tube wells, submersible pumps) are used where these
sources are unavailable or limited the farmers also used sewage water. The
application of sewage water for irrigation purpose is very common practice in urban
areas of the under developing countries like Pakistan. The other cause of sewage
water is economy as it contains some essential nutrients which increase the fertility of
agriculture soil. Farmers also prefer to reduce the quantity of chemical fertilizer
application. However it also contains heavy metals, pathogens and hazardous
chemicals eliminated by industries. Heavy metals are first accumulated in soil due to
continuous use of it for irrigation and the migrated to plant and edible part and
become the part of the food [1,2].

Vegetables are rich in nutrients which are essential for health like potassium, dietry
fiber, folate (folic acid), vitamin A and vitamin C. Each nutrient has a great
importance in maintaining overall health. Naturally growing vegetables protect
against many diseases like heart disease (heart attack and stroke), obesity and type-2
diabetes. Vegetables containing potassium are more important for lowering blood
pressure, minimizes the formation of kidney stones and bone loss [3].

Radish is a root vegetable. It is from Brassicaceae family and biological name is

Raphanus Sativus. Different types of radish are found in the world; daikon, black
spanished radish, green radish and watermelon radish. Daikon (Asian type) has long
white root and cultivated mostly in Southeast and East Asia. It is winter crop and
duration of crop is 60 to 70 days after sowing. It is commonly used in salad and also
cooked and pickled. The main health benefits described by researcher are respiratory
health, digestive health, detoxify the body, cancer prevention, activate immune
system, bone health, weight loss and skin health [3].
 12

Besides all these benefits vegetables may be contaminated with heavy metals and
have potential risk on human health. Vegetables are considered one of the dominant
route for heavy metals to added in the human body through food chain. Like other
nutrients vegetables uptake heavy metals through their roots. Agricultural soils have
the property of accumulating and translocating the heavy metal in the soil
environment R. Soils can accumulate the heavy metals for many years. Normally
heavy metals are present in the soil below the threshold value in agriculture soils it is
considered that in most cases the sewage water, sewage sludge or pesticides are the
major sources of heavy metals [4,5].

A research study was conducted with the aim to investigate the level of heavy metals
mainly lead, cadmium, arsenic and chromium in sewage water, agriculture soil and
radish in Tarlai, Islamabad.

1.1 Heavy Metals

Heavy metals are defined the substances with specific density more than 5 g/cm3.
They show high electrical conductivity, luster and malleability. When concentration
of these substances in the environment overshoot certain threshold value then they
affect negatively [6]. Lead, cadmium, mercury and arsenic exposure are considered
most dangerous for human body. It is admitted that heavy metals cause several serious
health effects and remain accumulated for many years in the body and their poisoning
creates problems related to nutritionally and environmentally. However still their
exposure continues in many parts of the world. Figure 1-1 confirms the production
and emission of lead, copper and zinc was on increasing trend from 1850 to 1990.
However started to decrease from 1990 in developed countries [7].

Heavy metals enters into the environment both from natural and anthropogenic
activities, which include urban runoff industrial effluents, sewage discharge, soil
erosion, natural weathering of earths crust, mining, pesticide application and many
others [6]. So these hazardous substances may come into human body through air,
food, water or soil [7]. The present study includes sources and health effects of only
three heavy metals, lead, cadmium and arsenic.
 13

Figure 1-1: Production of lead, copper and zinc in all over the world from 1850 to 1990 [7].

1.1.1 Mechanism of Heavy Metal Toxicity

Figure 1-2 gives the schematic view that describes the survival of cell against lead and
other heavy metal attack. In cell of both human and plant there is equilibrium in the
production of antioxidants (substances that inhibit oxidation) and free radicals. The
antioxidants are very important to reduce the effect of reactive oxidative species

Figure 1-2: Heavy metal attack on cell and as result production of reactive oxidative species and
neutralization the effect by antioxidant [6].
 14

(ROS) and maintains the cell structure, e.g. gluthione protect the cell from free radical
attack. However in the influence of heavy metals the balance is disturbed between
free radicals (ROS) and antioxidants [6]. Gluthone is found in both reduced (GSH)
and oxidized (GSSH) form. In the reduced state the thiol group of cysteine is able to
donate a reducing equivalent (H+ + e-) to other molecules, such as ROS to detoxify
them. With donating electron gluthione itself reacts with another reactive gluthione to
form gluthione disulfide (GSSH). The oxidative stress resulting from heavy metals
exposure shown in figure 1-2 increases the GSSH concentration and the ratio of GSH
and GSSH reaches to 10:90. The other effects of oxidative stress are lipid
peroxidation and high level of ROS produce stress condition at cellular level [6].

1.1.2 Lead
Lead is a bright silvery metal, slightly bluish in dry atmosphere and widely used
throughout the world in different processes [6]. During last century lead was mainly
entered into the environment through exhaust of the motor vehicle as lead compound
were used as antiknocking agent in gasoline but now due to free lead fuel lead
evolvement has been decreased [7]. Figure 1-2 shows the sources of lead emission to
the environment. The major sources of lead pollution are industrial processes,

Figure 1-3: Sources of the lead [6]

 15

smoking, domestic sources, gasoline, house paint, storage batteries and faucets.
Unlike other micronutrients (Zn, Cu, Mn and Fe) lead has no role in any biological
activity [6].

Lead has the ability to replace other bivalent metal ions like Ca2+, Mg2+, Fe2+ and
monovalent cations like Na+. The ionic mechanism of lead poisoning adversely affect
the biological function like cell-adhesion, protein folding, apoptosis, intra- and inter-
cellular signaling, ionic transportation, enzyme regulation and release of
neurotransmitter [6]. Even at Pico molar level lead has the capability to replace
calcium which results bad effects on protein kinase C that regulates neural excitation
and memory storage [6].

1.1.3 Arsenic
Arsenic has metalloid properties and is the 20th most abundant element on the earth.
Arsenic compounds are found both in organic and inorganic forms. The major sources
of arsenic are water, smelting of metals, arsenical pesticides, preservatives for wood,
and plastic stabilizer [7]. In the body inorganic arsenic (iAs) through biological
reaction is methylated and as result produces monomethylarsonic acid (MMA) and
dimethylarsinic acid (DMA).
() () () () ()
The product MMA(iii) remain accumulated in the body and does not excreted and
gives exposures to the body [6].

There are serious healths effects due to long term arsenic exposure, some of these are
hypertension and cardiovascular disease, neurological effects, cancer etc [7].

1.1.4 Cadmium
The Agency for Toxic Substances and Disease Registry (ATSDR) in USA categorizes
cadmium as 7th most toxic metal in the environment. Once it accumulates in the body,
remains there for whole life. Now a days cadmium is widely used in alkaline
rechargeable batteries. Other sources are its uses in coating, pigments, plating and
plastic stabilizer also present in phosphate fertilizer and tobacco smoking. ASTDR
reported that in each year 500,000 workers in USA are exposed to toxic cadmium [6].
 16

The mechanism of cadmium toxicity is less clear but it was observed that Cd can
replace iron and zinc and in liver it is accumulated in renal tissue. Cd has been
classified as carcinogen, its exposure may cause kidney failure, tubular damage and
renal failure.

1.2 Atomic Absorption Spectroscopy

This technique is used to measure the element in the sample solution. Actually atoms
of element are responding to absorption of specific wavelength.
1.2.1 Principle
The atoms of the analyte in the sample is converted into free gaseous atomic state
through flame or electric heating that absorb the characteristic wavelength of light
coming from line source and get excited. The amount of absorption is used for analyte
concentration [8].

The main parts of the AAS are given below

I. Hollow cathode lamp (HCL)
II. Atomizer
III. Grating
IV. Photomultiplier tube (PMT)
1.2.2 Hollow Cathode Lamp
HCL is used as source of light in AAS. It is a line source and emits the
electromagnetic radiation of that specific wavelength from which metal is made.
Schematic overview of the hollow cathode lamp is shown in figure 1-4. It consists on
hollow cathode of specific metal element, anode, housed in a glass and filled with
inert gas like He or Ne. When potential is applied, electrons from cathode strike with

Figure 1-4: Schematic diagram of hollow cathode lamp [8].

 17

inert gas and ionized it. The positive ions (He+) migrate toward cathode with high
velocity and dislodge the atoms from surface, when de-excited emit light of specific
wavelength [9].
1.2.3 Atomizer
In the atomizer the sample is aspirated and converted to gaseous atoms. Table 1-1 lists
the details of atomizer and temperature that is used in AAS. Moreover on the basis of
Table 1-1: Types of atomizer used in AAS [9].

Type of Atomizer Atomization Temperature

Flame 1700-3150
Electrothermal vaporization 1200-3000

atomization AAS is further classified into three categories, Flame Atomic Absorption
Spectroscopy FAAS, Graphite Furnace-Atomic Absorption Spectroscopy (GF- AAS)
and Chemical Vapor or Hydride Generation Atomic Absorption Spectroscopy (HG-
AAS). GF-AAS and HG-AAS have been used for analyses and will be discussed in
detail.
Graphite Furnace; Sensitivity of AAS is increased by electrical heating the sample
in graphite furnace. The main benefit is least sample requirement (1-100 l) and stage
heating. The sample is injected in the hole of graphite tube as shown in figure 1-5. It
has four stages, in first stage temperature is raised to1000C evaporate the solvent, is
called drying stage, in 2nd stage which is called ashing temperature is further increased
300- 1200 0C and organic matter is converted to H2O and CO2, the inert gas flow is

Figure 1-5: Schematic overview of graphite furnace [8].

 18

maintained to remove volatile material. In next stage the gas flow is stopped and the
temperature raises 1700-3000 0C to convert the sample into free atomic state and
measurement is taken, ashing stage. At last cleaning is done by passing the inert gas
through the tube, known as cleaning stage. The GF-AAS is preferred over flame as
the whole sample gets same temperature at same time and analyte reside for larger
time in atomization step. Therefore detection limit is increased to 1000 time the flame
measurement [8].

The four stages name with temperature and sample residence time for lead analyses in
GF-AAS is shown in the table 1-2.
Table 1-2: GF-AAS stages and scheme of temperature increase for lead measurement.

Stages Unit Temp/ Time

0
C 80 120
Drying
Sec 60
0
C 400 400
Ashing
Sec 30
0
C 2100 2100
Atomization
Sec 10
0
C 3000 3000
Cleaning
Sec 3

Hydride Generation; Several element are very toxic at even ppb level like Hg, As,
Se etc so there detection at lower level is desired. In this technique hydride of As, Se,
Te, Sb and Bi are generated by adding a reductant which is normally sodium
borohydride (NaBH4) in acid media (HCl). The reaction of the reagents with analyte
are shown in the chemical equation below
3BH - (aq) + 3H+ (aq) + 4H3AsO3 (aq) 3H3BO3 (aq) + 4AsH3 (g) + 3H2O (l)
The volatile hydride are transferred to the burner slot made of quartz, for heating,
through continuous supply of inert gas (argon) where hydride is decomposed and
analyte is atomized. In this way selectively analyte is atomized in the path of light
coming from hollow cathode lamp and sensitivity of the technique increased. The
detection limit through HG-AAS is enhanced 10 to 100 times [9].
 19

1.2.4 Diffraction Grating

In this device light is diffracted into its component when strikes with its grooves on its
surface. Very fine grooves are made either by laser (holographic) or diamond wire
(master grating) on the surface of grating as shown in the figure 1-6. The bending of
light is known as diffraction. And from which specific wavelength of light is imparted
on the detector [8]. It works on the principle of diffraction grating of which equation
is
2 =
Where n = 1, 2, 3
D = distance between two grooves
= angle of incidence and angle of reflection
= wavelength of light incident light.

Figure 1-6: Schematic of diffraction grating

1.2.5 Photomultiplier Tube (PMT)

PMT is commonly used detector in the AAS. A schematic diagram of PMT is given in
the figure 1-7, which provide the idea about its working. Basically it works on the
principle of photoelectric effect. It consists on a cathode, an anode and numbers of
dynodes. Light falls on the cathode and electrons are emitted due to photoelectric
effect. These electrons are made fall on dynodes. Each dynode is made positive than
previous dynode. Dynodes are conductors which multiply the number of electrons and
hence at the anode, the signal enhances. The PMT is housed in glass and evacuated.
During the measurements PMT is not allow to sense the light other then HCL [9].
 20

Figure 1-7; Schematic diagram of PMT with nine application dynodes [8].

1.3 Limits of Detection

The minimum concentration of specific analyte which can be measured through an
instrument is known as Detection Limit. Every instrument has different detection limit
for same analyte [9]. Table 1-2 enlist the detection limit of elements through AAS Z-
8000 Hitachi.
Table 1-3: Detection limit of Hitachi Z-8000 AAS.

Elements Detection Limits

 21

2 Literature Review

Khan et al [10] mentioned after his study that waste water contained essential nutrient
but also contained heavy metals which had a potential health risks. A study was
conducted to investigate the heavy metal content in soil, sewage water and vegetables.
The samples were collected from peri-urban areas of Lahore District and analyzed for
heavy metals (Zn, Mn, Cu, Fe, Pb, Cd and Ni). The result showed that Cu, Mn, Ni,
and Cd content were found above the safe limit in sewage water samples while Pb, Fe
and Zn were within the permissible limit. DTPA extractable heavy metal content were
below threshold value in soil samples. It was concluded that the heavy metal
concentration of all leaf and fruit samples were found higher the maximum allowable
limit set by World Health Organization (WHO).

Jaishankar et al [6] mentioned that heavy metals pose potential health risks and
remain in the body for long time. It was concluded that heavy metals enhanced the
oxidation stress and replace the element (ionic replacement) which led to cell damage.
The heavy metals toxicity depended on exposure to human being, which is the
function of concentration of heavy metals and contact time. It was concluded that
heavy metals toxicity through any route is significantly dangerous for the human
body.

Chang et al [4] conducted a research study to determine the level of heavy metals
(Hg, Cd, Pb, Cr and As) in leafy vegetables at south china. They extended the study to
find the Bioconcentration factors (BCF) of heavy metals from soil to vegetables and
potential health risks were estimated. The result showed that migration of Cd from
soil to vegetable was maximum the rest of the heavy metals. It was concluded that
sewage water irrigation and fertilizer application were major cause of their
contamination. As the levels of heavy metals were below the safe limit described by
the World Health Organization (WHO), they mentioned that their consumption did
not cause serious health issues.
 22

Balkhair et al [1] conducted a research study at Western Region of Saudi Arabia to

find the concentration of heavy metals (Cd, Cr, Cu, Pb and Zn) in Okra (lady finger)
vegetable irrigated by sewage water. The statistical analysis was also performed on
the data and result showed that Pollution Load Index was higher. Moreover Ni, Pb,
Cd and Cr concentration were higher the maximum allowable limit in 90, 28, 83 and
63% of samples respectively. Health Risk Index (HRI) and Enrichment Factor (EF)
revealed that Okra was unsafe for its consumption. It was also concluded that sewage
water applied for irrigation polluted the soil and increased the food contamination
with heavy metals.

Ye et al [11] also investigated the soil and vegetables samples collected from
Zhejiang Province, China for heavy metal content. The samples were analyzed for Pb,
Cd, As, Hg and Cr level. The average value of Cd, Pb and Cr concentration were
0.020, 0,048 and 0.043 mg/kg respectively whereas As and Hg were found below the
detection limit which were 0.005 and 0.002 mg/kg respectively. Total 68 out of 202
agriculture soil samples were found higher level than the safe limit. Correlation
analysis showed that that migration of heavy metals from soil to vegetables were
significant. At the end it was concluded that Cd was significantly enriching in
vegetables as compared to Pb and Cr.

Nazemi et al [12] investigated eight vegetables including radish for heavy metals
content in Shahroud, Iran. Atomic Absorption spectroscopy (AAS) were used to
determine the level of Pb, As, Cr, Cd and Zn. The result showed that Pb and Cd were
more uptaken by radish the rest of the vegetables. He concluded that the consumption
of these vegetables were unsafe especially those in which heavy metals were found in
higher concentration as settled by international organization like WHO.

Parveen et al [13] analyzed the sewage water and subsequent soil and vegetable
samples to investigate the concentration of heavy metals. For this reason forty
samples of each sewage water, soil and vegetable were collected from Peshawar,
Pakistan. The result showed that water samples are alkaline and electrical
conductivity was also high. Moreover Ni, Pb and Cd were also found above the safe
limit in some of the samples. The soil samples were digested through AB-DTPA and
result showed that the heavy metals (Ni, Pb, Cd, and Cr) were in safe limits. In
 23

vegetables samples it was found that Ni, Cd and Pb were higher than the safe limit in
90, 10 and 78% of the samples respectively. The correlation of Ni, Pb, Cd and Cr
between sewage water and soil were calculated positive significant. Correlation
between soil and leaves for Cd was positive non-significant while for Ni, Pb and Cr
was positive significant. In edible part Ni, Pb, Cd and Cr were found higher the
permissible limit as set by WHO and it was stated that the consumption of these
vegetables were pose health at potential health risk.

Farzana et al [2] performed field experiments on soils and vegetable samples (green
chili and gourd) collected from six different sites near Lahore, Pakistan irrigated by
sewage water for a long time. Also a reference site which was irrigated with canal
water was also studied. The different extraction was performed for the digestion of
soil samples and the result showed that all metal were available in the residual phase.
Correlation analyses were performed to understand the transfer of metal from soil to
vegetables. Positive correlations were found from soil to vegetable which indicate the
bioavailability of metals fraction to plant.

Parveen et al [14] was also performed the similar study about food contamination
with heavy metals. She investigated the level of heavy metals (Zn, Ni, Mn, Cu and
Cd) in turnip (Brassica rapa) irrigated by treated municipal waste water. The plant
were sown in different pots and irrigated with 50 and 100% wastewater separately.
The result revealed that the level of heavy metal in sewage water were below safe
limit but were above in vegetable plant. The results further showed that metal
concentration were higher at 40 and 55 days after sowing (DAS). The metals
concentration were found in following order: Fe> Zn> Ni> Mn> Cu> Cd.

Hassan et al [15] conducted the field experiments on five vegetables (turnip, radish,
coriander, spinach and brassica) irrigated by sewage water and fresh water at sub-
urban areas of Lahore, Pakistan. Atomic Absorption Spectrophotometer was used for
analysis of cadmium and mercury. The result showed that Cd concentration was
greater in vegetables irrigated with sewage water as compared to fresh water. Cd
concentration in sewage, river and tube well irrigated water was ranged between 0.03-
0.61, 0.03-0.53 and 0.03- 0.32 mg/kg respectively. In two vegetable sample Cd were
 24

found higher than the safe limit while Hg were found within safe limit recommended
by World Health Organization (WHO) in all vegetables.
Safi et al [16] studied the microbial and heavy metal poisoning in irrigation water, soil
and marketed vegetables of Kabul, Afghanistan. The results revealed that sewage
water was contaminated with fecal pathogens Ascaris (coliform bacteria, salmonella,
shigella, entamobia and Pinworms [oxyuris vermiculairs syn, enterobius
virmicularis]) while vgetable was also contaminated with pathogens. The result
further showed that coliform in vegetables was ranged between 0.5- 2 E7 clls per
100g, which was above the permissible limit. The possible reason of present
contamination suggested by them were traffic, residues of war and use of untreated
sewage water.

Khan et al [17] described that human being were getting exposed to heavy metals by
eating vegetables grown on wastewater irrigated soil. The study was performed in
Beijing, China. The result showed that the level of heavy metals was above the
maximum permissible value set by the both State Environmental Protection Agency
(SEPA) and WHO in plants. It was concluding by calculating the health risk index
that these plants consumption could be serious concerned about health.

Parashar et al [18] studied the impact of sewage water irrigation on vegetables

(Spinach, Radish, Cabbage, Beetroot, Okra, Tomato and Cucumber) with respect to
heavy metals. The samples of sewage water, soil, and vegetables were collected from
Agra District, India and analyzed for Fe, Cd, Cu, Zn and Pb. It was found that
concentration of heavy metals were in safe limit in both sewage water and soil as set
by WHO but was higher in vegetables. The correlation analysis indicated that
concentration of Cd and Pb was higher in untreated sites as compared to treated sites.
It was concluded that this food is highly contaminated and consumption of it was
declared unsafe.
 25

3 Materials and Methods

The samples of soil and vegetable (Raphanus stavius L.) were collected from Tarlai
Farms that are located on 330 38 north and 730 08 east. These farms are located near a
town named Trameri. These farms are named as Tarlai Poultry & Vegetable Scheme
-1. These farms are irrigated by sewage water canal named Gumrah Kas. Sewage
water of Islamabad Territory comes to this canal. Figure 3-1 is the map of Tarlai
Farms and sewage water canal. The canal sewage water is commonly used for
irrigation in that region Weather specifications of the site are
Average annual rainfall = 80 mm
Average temperature = 21.2 0C

Figure 3-1: Map of Tarlai Farms.

3.1 Sample Collection

Radish crop duration is 60 to 70 days and total five samples of sewage water, soil and
Raphanus stavius L. were collected in crop duration, from sowing to the harvesting.
Table 3-1 gives the schedule of sampling from experimental field. The Table 3-1
shows the proper schedule of sampling.
Table 3-1: Sample Collection Schedule

Sr. No. Sampling Date DAS

 26

1 26/10/2016 7
2 2/11/2016 14
3 9/11/2016 21
4 26/11/2016 ~ 40
5 16/12/2016 ~ 60

3.1.1 Sewage Water Sampling

Sampling requires special attention as a little mistake may lead to different condition.
International standards are followed for genuine sampling. Gloves are worn to avoid
contamination as sewage water contains pathogens. The Figure 3-1 shows that typical
sewage water canal from where sampling has been done.

At first grab samples has been collected and then combined to get a composite sample
and is sealed in plastic bottles and properly labeled.

Figure 3-2; Sewage water canal passing through Tarlai.

3.1.2 Soil Sampling

Auger has been used for soil sampling. The depth of soil collected was 0-15 cm. In
the diagonal way the grab samples has been collected from the crop farms and
combined to get composite sample. Then composite samples packed in polythene
bags and safely transported to lab.
 27

Figure 3-3; Radish Crop field

3.1.3 Radish Plant Sampling

Along the diagonal way of the field the grab (spot) samples have been collected and
packed in polythene bags. The removal requires care as the plant should completely
(with roots) be collected. Safely transported to lab. The samples were washed with
distilled water to remove the debris and air dried.

3.2 Plant Sample Preparation

The plant samples that were collected gently washed with running tap water to
remove the soil particles attached to the plant surfaces and, finally rinsed with
distilled water. Three parts of plants were made (leaves, stems and roots) Then
samples were air dried. Dried samples were crushed with mortar and pestle, sieved
and stored.

Figure 3-3 shows the series of steps that are followed after sampling to the digestion
procedure.

Crushing to Storage
Washing Air Drying Powder

Figure 3-4: Flow diagram of the procedures followed after sampling.

 28

3.2.1 Plant Digestion

1.0 g of dry plant sample was taken in the flask, added 5 ml of conc. nitric acid
(HNO3), well mixed and kept overnight. Next day 12 ml of HNO3 and perchloric acid
(HClO4) in 3:1 was added and kept on hot plate for heating and remained there until
volume of solution reduces to 2 ml. The samples color turned from brown to clear
form at the end. The dissolved samples were cooled, filtered and diluted with distilled
water to make the volume 25 ml.

3.3 Soil Sample Preparation

Soil samples were oven dried, ground, and passed through 0.25 mm sieve and then
stored.
3.3.1 Soil Digestion
0.5g of soil were taken in Teflon beaker and added in few drops of water. Perchloric
acid and hydrochloric acid each 5ml were added separately and left it for 16 hours.
Next day it was heated (60-70 0C) on the hot plate till dryness (1-2ml). Again added
HClO4 and HF 5ml each and heated till dryness. To completely evolve HF, mixed
2.5ml of HCLO4 and heated till dryness. 10ml of distilled water and then 2.5ml of
HCl were added to completely dissolve the residues. After that distilled water was
added to make the total volume 25ml.

3.3.2 Quality of the Chemicals

Table shows the concentration of the acid that was used for digestion.
Table 3-2: Quality of chemicals that was used for digestion

Chemicals Molarity (mole/dm3) Percentage

Nitric acid 14.54 65%
Perchloric acid 11.87 71%
Hydrochloric acid 12.07 37
Hydrofluoric acid
 29

4 Results and Discussions

Table 4-1: Physicochemical Characteristics of Experimental Soil (0-15cm)

Properties Units Values

Sand % 56
Silt % 22
Clay % 22
Textural Class --- Sandy Clay Loam
pH (1:2.5) --- 7.46
ECe (1: 2.5) dSm-1 0.7
Moisture % 17
Total organic carbon % 1.0
Organic Matter % 1.80
Total Nitrogen % 0.08
Extractable Phosphorus g g-1 soil 10
Extractable Potassium g g-1 soil 400

Element Value LOD FAO Safe Limits

Analyses by AAS technique
ng/ml or ppb g/ml
Pb ND 8.76 5000
Cd ND 0.35 10
Cr 5.6 2.59 100
Cu ND 14.66 200
Hg ND 0.52
As 2.17 0.69 100
Se ND 0.816

Analyses by ICP-OES technique

g/ml
Na 35.60
Ca 34.38
K 6.32
Mg 23.25

1Adapted from National Academy of Sciences (1972) and Pratt (1972).

 30

Table 4.3 Analyses of sewage water used as irrigation for Radish at Tarlai
Farms, Islamabad.
Parameters Units Value 1FAO Safe Limits

pH 6.6 7
Conductivity s/cm 765 - 914
TDS mg/l 420 - 500 450
TSS mg/l 4 -17 50
Ammonia mg/l 0.03- 0.46
Alkalinity mg/l 335 - 419
Sulfide mg/l 0 - 4.1
Flouride mg/l 0.5 - 0.95 1.0
Chloride mg/l 4.7 - 24.1
Sulphate g/ml 30.59
Total Coliforms CFU/100ml 89
COD mg/l 40
BOD
Table 4.4 Concentration of Arsenic, lead and cadmium in sewage water used for
irrigation
at different time interval during experiment

Sr. no. DAS Pb As Cd

ng ml-1
Mean + S.D
1 7 DAS ND 2.16 + 0.08 ND
2 14DAS ND 3.81 +0.07 ND
3 20 DAS ND 2.23 + 0.12 ND
4 40 DAS ND 5.12 + 0.07 ND
5 60 DAS ND 2.43 + 0.07 ND
LOD 8.76 0.69 0.35
*Permissible 5000 100 10
Limit (g/ml)
Where DAS= days after sowing; ND= Not Detected; LOD =Limit of Detection
* Source (WHO, 1989) page 30

Table 4.5 Concentration of Arsenic, Lead and Cadmium in soil (0-15 cm) at
different time
 31

interval during experiment

Sr. no. DAS Pb As Cd

mg kg1
Mean + S.D
1 7 DAS 1.76 + 0.07 2.69 + 0.15 0.31+ 0.04
2 14 DAS 2.28 + 0.12 2.43 + 0.09 0.13+ 0.04
3 20 DAS 2.56 + 0.12 2.47 + 0.05 0.14+ 0.04
4 40 DAS 3.10 + 0.18 3.14 + 0.13 0.14+
5 60 DAS 3.34+ 0.2 3.04 + 0.04 0.11+ 0.07
LOD 0.43 0.036 0.017
*Permissible 500 30 1.0
Limit (mg kg-1)
Where DAS= days after sowing; LOD =Limit of Detection
*Source (Alloway (1990), page 32

Table 4.6 Concentration of major nutrients in Sewage water during the

experiment using ICP-OES technique

Major Sewage water LOD

Nutrients
g/ml
Ca 34.4 - 84.0 0.002
 32

K 6.3 - 17 0.165
Mg 23.2 - 34.0 0.001
Na 35.6 - 86 0.198
Zn ND 0.005
So4 30.6 47.0 0.05

Table 4.7 Concentration of Arsenic, Lead and Cadmium in radish at different

time
interval during experiment

Sr. no. DAS Pb As Cd

mg kg-1
Mean + S.D
1 7 DAS 1.02 0.01 0.65
2 14 DAS 0.30 ND 0.44
3 20 DAS 0.09 ND 0.18
4 40 DAS 0.62 0.38 0.25
5 60 DAS 0.23 0.14 0.09
LOD
*Permissible 0.30 0.43 0.20
Limit (mg kg-1)
Where DAS= days after sowing; LOD =Limit of Detection
Source: (Weigert (1991), FAO/WHO (2001), pp33

Exponential
 33

Table 4.8 Comparison of measured values with certified values (g g -1 ) of

standard reference materials

Standard Reference
Elements Material Measured value Certified value
g g-1
AS CL IAEA-359 0.10 0.10
PL -1547 0.06 0.06
IAEA Soil-7 11.86 13.4
Cd IAEA Soil-7 1.4 1.3

Where CL= Cabbage Leaves, PL= Peach Leaves

Table 4.9 Concentration of heavy metals (As, Pb, Cd) in the soils (0-15 cm)
without sewage
water use at Tarlai farms, Islamabad

Soil -A Soil-B
Location Farm No.43, Tarlai farms, Tarlai farms, Islamabad
Islamabad
Source of irrigation Tube well
Pb (mg kg-1)
As ( mg kg-1)
Cd (mg kg-1)
 34

LOD

0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
7 DAS 14 DAS 20 DAS 40 DAS 60 DAS

Fig 4.1 Concentration of As (mg kg-1) in radish at different time interval of

experiment
 35

1.2

0.8

0.6

0.4

0.2

0
7 DAS 14DAS 20 DAS 40 DAS 60 DAS

Fig 4.2 Concentration of Pb (mg kg-1) in radish at different time interval

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0
7 DAS 14DAS 20 DAS 40 DAS 60 DAS

Fig 4.3 Concentration of Cd (mg kg-1) in radish at different time interval

 36

0.25

0.2

0.15
Root
0.1 Leaves

0.05

0
20 DAS 40 DAS 60 DAS

Fig 4.4 Concentration of As (mg kg-1) in roots and leaves of radish at 20, 40, 60
DAS

0.8

0.7

0.6

0.5

0.4 Root
Leaves
0.3

0.2

0.1

0
20 DAS 40 DAS 60 DAS

Fig 4.5 Concentration of Pb (mg kg-1) in roots and leaves of radish at 20, 40, 60
DAS
 37

0.6

0.5

0.4

0.3 Root
Leaves
0.2

0.1

0
20 DAS 40 DAS 60 DAS

Fig 4.6 Concentration of Cd (mg kg-1) in roots and leaves of radish at 20, 40, 60
DAS
 38

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