2
Vindicating the Impact of Heavy Metal Residues in Spinach
Through Household chemicals
3
4
Rai Muhammad Amir1*, Faqir Muhammad Anjum1, Muhammad Atif Randhawa1,
Muhammad Sarwar2
5
6
1
7
8
2
9
*
1
National Institute of Food Science and Technology, University of Agriculture, Faisalabad,
Pakistan
Institute of Animal Nutrition and Feed Technology, University of Agriculture, Faisalabad,
Pakistan
For correspondence: [email protected]
10
Abstract
11
Heavy metals are major category of globally distributed pollutants and natural elements that have
12
been extracted from earth and harnessed for human industry and products. Food production and
13
its safety is the major issue around the world. Metals are notable for their wide environmental
14
dispersion from such activity, their tendency to accumulate in selective tissues of the human
15
body and their overall potential to be toxic even at relatively minor levels of exposure. The
16
intake of heavy metals through diet leads to several disorders such as kidney damage, nervous
17
disorder, bone disease and tubular growth. The present study was conducted to mitigate the
18
residues of heavy metals Pb, As, Zn and Hg in spinach collected from self-grown supervised
19
field by using different washing techniques. The amount of trace metals were measured in fresh
20
and chemically washed spinach samples with the help of Atomic Absorption Spectrophotometer
21
(AAS). The results obtained in recent study indicated heavy metal residues reduced drastically
22
when spinach samples were subjected to washing treatment irrespective to type of washing
23
declined the mercury residues varying from 7 to 23 %, lead 7 to 28 %, zinc 15 to 54 % and
24
arsenic 6 to 22%. More reduction was found in zinc followed lead, mercury and arsenic,
25
respectively.
1
1
Keywords: Heavy metals, Spinach, Wastewater, Washing Treatments
2
Introduction
3
Pakistan is an agricultural country sanctified with plenteous natural possessions like productive
4
soil, irrigation water and miscellany of climate ranging from tropical to temperate. The use of
5
sewage water for agricultural purpose has tremendously increased during last few years due to
6
shortage of water availability, especially in semiarid region of Pakistan. The vegetables plays an
7
important role for the protection of health and prevention of various ailments, repair the organ of
8
body, maintaining alkaline reserve of the body and possess high carbohydrate, vitamins and
9
minerals contents (1).
10
The contamination of heavy metals mostly occurs due the application of fertilizers, waste water
11
irrigation, use of metal-based pesticides, industrial emissions and transportation on crops (2).
12
The contamination of soil is becoming environmental problem in Pakistan due to increasing
13
heavy metals (3). The wastewater irrigation is the major contributor of heavy metal contents to
14
the soil and ultimately vegetables becoming a very serious issue in Pakistan because these
15
effluents are heavily loaded with harmful metals and metallic compounds (4, 5, 6, 7). The
16
traditional washing methods are used to get rid of waste and filth prior to use vegetables because
17
these are considered significant for the reduction of residues of heavy metals. A series of
18
washing solutions such as ozonated water, bleach solution and strong acid has been found
19
successful in removing heavy metals, pesticides and chemical residues. The vegetable samples
20
washing with tap water for 2–3 times result the reduction of residues of heavy metals drastically
21
and the contaminated load was eliminated best for lead and cadmium from 75–100% than those
22
for copper and zinc 27– 55% (8).
2
1
The residues of heavy metal and other particles deposited on the surface of vegetables removes
2
significantly by simple washing (9). In spinach, heavy metal reduction was found 21%, 21%,
3
13% and 26% for Pb, Cd, Cr and Zn respectively but almost unaffected for Ni in unwashed and
4
washed samples. In coriander the concentration of Cd, Pb, Cr, Ni and Zn reduced by 11%, 31%,
5
11%, 5% and 6% and for methi reduction pattern 14%, 13%, 15%, 17% and 13% for unwashed
6
and washed samples, respectively (10).
7
The increasing concentration of heavy metal residues in water, soil and foodstuff is an alarming
8
phenomenon around the world. The pollution of water and soil directly linked with cross
9
contamination of food chain through irrigation. The United States and European Union has
10
already set up strict limits for most of the heavy metals in water but current food legislation does
11
not have legal limits for a number of heavy metals in food and this fact causes considerable
12
health hazards to babies, children, pregnant women and all other consumers. Keeping in view the
13
persistent nature and cumulative behavior as well potential toxic effects of heavy metal residues
14
as a result of consumption of vegetables, the present study has been designed to determine the
15
heavy metals in spinach to explore the efficiency of chemical and biological solutions to reduce
16
the residues of heavy metals.
17
Materials and Methods
18
Vegetable sample collection
19
The samples of spinach were collected from self grown, supervised field. One kg sample of
20
spinach was obtained at the point of optimum maturity. The samples were instantly shifted to the
21
laboratory of National Institute of Food Science and Technology, for subsequent analysis of
22
samples.
3
1
Sample Preparation
2
The collected vegetable samples were reduced to appropriate size by using knife to facilitate the
3
analysis. The study was designed with different washing treatments by using tap water
4
and following concentrations of chemical solutions in water along with one unwashed sample.
5
Table 1: Different Washing Treatments
Reagents
Unwashed
Tap water
Reddish extract
Sodium Carbonate
Hydrogen Peroxide
Lemon extract
Citric Acid
Concentrations
Washing Treatments
5%
10%
5%
10%
5%
10%
5%
10%
5%
10%
Treatments
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
6
7
After solution preparation vegetable sample were soaked in solution for ten minutes for washing
8
purpose. The washed vegetable was blended by using commercial blender to make
9
homogeneous paste of pulp and juice. The vegetable samples (three replicates of each
10
washing treatment) were kept in hot air oven at 70 -80oC till complete dryness. The dried
11
vegetable samples of different parts of vegetables were ground into a fine powder (80 mesh)
12
using a commercial blender and stored in polyethylene bags for analysis.
13
Sample Digestion
14
The powdered vegetable sample (0.5-1.0 g) was taken into a Pyrex beaker; 10 ml of concentrated
15
HNO3 was added to it and kept overnight without heating. It was then heated on a hotplate and
4
1
after evaporation near to dryness, the sample was cooled and 5 ml HClO4 was added and heated
2
again. After digestion was complete, the sample was filtered into a clean volumetric flask and
3
diluted to 50 ml with double de -ionized water (11).
4
Preparation of Standards
5
The standard solutions (5, 10, 15 and 20 ppm) of all the metals (Hg, Pb Zn and As) were
6
prepared from the stock standard solutions containing 1000 ppm in distilled water.
7
Determination of Metals
8
The heavy metals Hg, Pb Zn and As in unwashed, tape water, chemically washed, dried
9
and digested vegetable samples was determined by using Varian GTA 120 AA 240
10
Graphite Atomic Absorption Spectrophotometer (12). The data obtained was analyzed and
11
represented using standard statistical procedures i.e. completely randomized design (CRD) as
12
described by (13)
13
Results
14
The concentrations of mercury, lead, zinc and arsenic were analyzed in different samples of
15
spinach collected from supervised field at the stage of optimum maturity. The results regarding
16
these heavy metals concentration in spinach showed significant variation and linear reduction of
17
heavy metals as presented in Table 2, 3, 4 and 5, respectively. The concentrations of heavy
18
metals in unwashed spinach samples were higher than samples subjected to different washing
19
treatments. The result regarding mercury showed progressive increase in reduction of residues of
20
mercury with the increase of concentration of treatments (Table 2). Out of all the washing
21
treatments, it was observed that the minimum reduction of mercury residues 7% was recorded
22
whenT1 i.e. tap was applied to spinach. The spinach treated with T12 i.e 10% citric acid yielded
23
the maximum reduction of mercury which was 23% followed by lemon extract 22%, sodium
5
1
carbonate 17%, reddish extract 15% and hydrogen peroxide 12%. Furthermore, the maximum
2
reduction of lead residues 28% was observed when T12 i.e. 10% citric acid was applied to
3
spinach (Table3). Similarly unwashed samples of spinach were found to be heavily contaminated
4
with zinc and arsenic. The tap water reduces the lead, zinc and arsenic contents by 7, 15 and 6%
5
in spinach and T12 i.e. 10% citric acid was found to be more effective in all the spinach samples
6
to reduce lead, zinc and arsenic contents as shown in the Table 3, 4 and 5.
7
Table 2: Mean±S.D value and percent reduction of mercury in spinach
Treatments
T1 Unwashed
T2 Tap water washed
T3 Reddish extract 5 %
T4 Reddish extract 10 %
T5 Sodium Carbonate 5 %
T6 Sodium Carbonate 10 %
T7 Hydrogen Peroxide 5%
T8 Hydrogen Peroxide 10%
T9 Lemon extract 5 %
T10 Lemon extract 10 %
T11 Citric Acid 5 %
T12 Citric Acid 10 %
Vegetable
Spinach
Mean+S.D mg/kg
0.0481±0.00081a
0.0447±0.0023b
0.0436±0.0016bc
0.0408±0.0007de
0.0429±0.0013bcd
0.0399±0.0008ef
0.0446±0.0013b
0.0422±0.0005cd
0.0429±0.0017bcd
0.0374±0.0019f
0.0410±0.0016de
0.0370±0.0017f
Percent Reduction (%)
7
9
15
11
17
7
12
11
22
15
23
8
9
Table 3: Mean±S.D value and percent reduction of lead in spinach
Treatments
T1 Unwashed
T2 Tap water washed
T3 Reddish extract 5 %
T4 Reddish extract 10 %
T5 Sodium Carbonate 5 %
T6 Sodium Carbonate 10 %
T7 Hydrogen Peroxide 5%
T8 Hydrogen Peroxide 10%
T9 Lemon extract 5 %
T10 Lemon extract 10 %
Vegetable
Spinach
Mean+S.D mg/kg
0.9838±0.0055a
0.9127±0.0017b
0.8638±0.0039d
0.8034±0.0029i
0.8541±0.0043e
0.8235±0.0032g
0.9035±0.0037c
0.8548±0.0012e
0.8340±0.0038f
0.7267±0.0029j
6
Percent Reduction (%)
7
12
18
13
16
8
13
15
26
T11 Citric Acid 5 %
T12 Citric Acid 10 %
0.8121±0.0105h
0.7067±0.0032k
17
28
1
2
Table 4: Mean±S.D value and percent reduction of zinc in spinach
Treatments
T1 Unwashed
T2 Tap water washed
T3 Reddish extract 5 %
T4 Reddish extract 10 %
T5 Sodium Carbonate 5 %
T6 Sodium Carbonate 10 %
T7 Hydrogen Peroxide 5%
T8 Hydrogen Peroxide 10%
T9 Lemon extract 5 %
T10 Lemon extract 10 %
T11 Citric Acid 5 %
T12 Citric Acid 10 %
Vegetable
Spinach
Mean+S.D mg/kg
7.4747±0.0227a
6.3577±0.0220b
5.8498±0.0164d
5.0962±0.0992ef
5.9077±0.0099cd
4.4114±0.3785h
6.0669±0.0296c
5.0091±0.0003fg
5.2344±0.0181e
3.5816±0.0362i
4.8476±0.0811g
3.4316±0.0274i
Percent Reduction (%)
15
22
32
21
41
19
33
30
52
35
54
3
4
Table 5: Mean±S.D value and percent reduction of arsenic in spinach
Treatments
T1 Unwashed
T2 Tap water washed
T3 Reddish extract 5 %
T4 Reddish extract 10 %
T5 Sodium Carbonate 5 %
T6 Sodium Carbonate 10%
T7 Hydrogen Peroxide 5%
T8 Hydrogen Peroxide10%
T9 Lemon extract 5 %
T10 Lemon extract 10 %
T11 Citric Acid 5 %
T12 Citric Acid 10 %
Vegetable
Spinach
Mean+S.D mg/kg
0.1048±0.0105a
0.0987±0.0013b
0.0965±0.0006bcd
0.0913±0.0007def
0.0976±0.0005bc
0.0928±0.0005cdef
0.0976±0.0014bc
0.0945±0.0015bcde
0.0943±0.0006bcde
0.0889±0.0008f
0.0903±0.0009ef
0.0818±0.0011g
5
6
7
7
Percent Reduction (%)
6
8
13
7
11
7
10
10
15
14
22
1
Discussion
2
The results pertaining from the present study that the heavy metal residues reduced drastically
3
when spinach samples were subjected to washing treatment irrespective to type of washing,
4
different treatments declined the mercury residues varying from 7 to 23 %, lead 7 to 28 %, zinc
5
15 to 54 % and arsenic 6 to 22% in spinach. More reduction was found in zinc followed by lead,
6
mercury and arsenic, respectively. Zinc showed more reduction due to their physiochemical
7
properties and more dissolving power as compared to other heavy metals. The variation in
8
reduction of heavy metal residues during washing operation may be due to differences in the
9
behavior and nature of chemicals using in washing treatments. The washing of spinach with tap
10
water removes most of the heavy metal residues adhered to the surface of the spinach as well as
11
in the skin of the vegetable due to its dissolving power because that is considered a universal
12
solvent. The present study results also indicated that citric acid showed maximum reduction of
13
heavy metal residues due its working ability as a chelating agent and resulting made the heavy
14
metal residues unavailable, salt solution sodium carbonate also reduced significant amount of
15
heavy metal residues and biological extract lemon showed more reduction of heavy metal
16
residues as compared to reddish extract. The acidic detergent solution was more effective in the
17
elimination of the zinc, lead, mercury and arsenic under investigation than salt solution and
18
biological extracts. The vegetables play vital role in maintaining the nutritional status of humans.
19
The accumulation of heavy metals in vegetables uptake from soil, water and atmosphere.
20
The intake of heavy metals through diet leads to several disorders such as kidney damage,
21
nervous disorder, bone disease and tubular growth. The uptake of metals in vegetables from
22
contaminated soil by absorbing them as well as from deposits on the vegetable parts exposed to
23
the air from polluted environments.
8
1
The results of the present study are in conformity with the previous findings of (14) who found
2
that the residues of different heavy metals lead and cadmium were declined considerably during
3
washing of vegetables. The results of the present studies are also fairly in agreement with the
4
findings of (8). All vegetable samples were found to be heavily contaminated with different
5
metals. The excessive intake of toxic metals can cause severe complications in humans and
6
animals. There are already published works related to the incidence of cancer of the pancreas,
7
urinary bladder or prostate and gastrointestinal cancer (15). Lead, mercury and chromium were
8
found to be accumulated in the shoot and roots of plants at low, medium or high levels (16). The
9
leafy vegetables have the tendency to uptake toxic metals more as compare to other vegetables
10
because of their more exposure to environmental pollution (17) also reported that leafy
11
vegetables were more contaminated with cadmium (0.09μgg-1) and the minimum concentration
12
(0.002μgg-1) was in cucurbit vegetables (Indian squash). Results also showed that maximum
13
concentration (0.15μgg-1) of lead was found in leafy vegetables (coriander) and the minimum
14
concentration (0.001μgg-1) in root/ tuberous vegetables (sugar beet). The results of the current
15
study are very close to the achievement of (18) who estimated the concentration of cadmium in
16
unwashed vegetables. The cadmium reduction pattern in this study was parallel to the
17
investigation that was carried out by (8) to estimate the heavy metal load of unwashed and
18
washed vegetables in peri-urban, Delhi. In addition, (10) also reported the same reduction pattern
19
of cadmium in spinach, okra and brinjal, exposed to different degrees of pollution in Agra, by
20
washing with tape water. The arsenic is a toxic element and the humans may get exposure to
21
arsenic through drinking water obtained by wells bored into arsenic contaminated areas or
22
through contaminated water by agro-chemical waste or industrial effluents. (19) also reported the
9
1
arsenic reduction by washing of vegetable with tape water but that was slightly high than in
2
present study.
3
The level of lead is higher than the recommended (0.2 μgg-1) in humans, cause bones,
4
pancreases, gum, liver, nervous system, teeth and blood diseases. Lead toxicity results in
5
reduction of hemoglobin synthesis, joints pain, kidney damage, defects in reproductive,
6
cardiovascular and nervous systems. The results of the present study regarding the lead
7
concentration in unwashed vegetables are the same as described by (20) to estimate
8
concentration of different heavy metals in vegetables. Lead reduction by tap water washing
9
follows the same pattern as reported by the (8) to estimate the heavy metal load of unwashed and
10
washed vegetables in peri urban, Delhi. In past, higher level (0.02μgg-1) of mercury was found in
11
fenugreek/methi i.e. leafy vegetables and the lower contents (0.001μgg-1) were found in
12
root/tuberous vegetables and fruity vegetables (okra) was found to be heavily contaminated
13
with arsenic (0.083 μgg-1) whereas the minimum level (0.014μgg-1) was detected in cauliflower
14
(21).
15
Conclusions
16
The spinach samples were contaminated with different residues of heavy metals. Wastewater
17
irrigation is the single largest and main source of heavy metals contamination in soil as well as in
18
vegetables. The spinach shows higher accumulation of different metals because of its large
19
surface area and the washing of spinach with tap water and household chemical solutions not
20
only remove the dirt and dust particles but also reduced the heavy metals significantly. The
21
washing treatments mechanically remove the heavy metals deposited on the surface of
22
the vegetables. On the basis of present study, it is strongly recommended that vegetables must
10
1
not be grown with sewerage water and at household level the vegetables must be washed with
2
tap water as well as washing treatments like citric acids, hydrogen peroxide, sodium carbonate
3
and biological extracts lemon and radish in order to reduce the residues of heavy metals. These
4
methods are very simple because they can easily perform at local conditions and beneficial due
5
their working ability to mitigate the metal residues.
6
Conflict of Interest
7
The authors declare that there is no conflict of interests regarding the publication of this paper.
8
References
9
1 Marwat, S.K., M. A. Khan, M. A. Khan, M. Ahmad, M. Zafar, F. Rehman and S. Sultana,
10
2009. Vegetables Mentioned in Holy Quran and Aadith and their Ethnomedicinal
11
Studies in Dera Ismail Khan, N.W.F.P., Pakistan. Pak. J. Nutri., 8:530-538.
12
2 Agrawal, M., and F.M. Marshall, 2003. Heavy metals contamination in vegetables grown in
13
wastewater irrigation areas of Varanasi, India, Bull. Environ. Contam. Toxicol., 77:
14
311-318.
15
3 Bhutto, M.A., P. Zahida, S.I. Riazuddin, A. Mubarik and N. Sahar, 2009. Monitoring of
16
heavy and essential trace metals in wheat procured form various countries. Int. J.
17
Biol. Biotechnol., 6:247-256.
18
4 Singh, K.P., D. Mohon, S. Sinha and R. Dalwani, 2004. Impact assessment of treated/untreated
19
wastewater toxicants discharge by sewage treatment plants on health, agricultural and
20
environmental quality in waste water disposal area. Chemosphere., 55: 227-255.
21
5 Mapanda, F., E.N. Mangwayana, J. Nyamangara and K.E. Giller, 2005. The effect of long-
22
term irrigation using wastewater on heavy metal contents of soils under vegetables
23
in Harare, Zimbabwe. J. Agri. Ecosys. Environ., 107: 151-165.
11
1
6 Sinha, S., and R. Dalwani, 2005. Impact assessment of treated/ untreated wastewater
2
toxicants discharged by sewage treatment plants on health, agricultural and
3
environmental quality in the wastewater disposal area. J. Chem., 55: 227-255.
4
7 Sharma, O.P., B. Bangar, K.S. Rajesh Jain and P.K. Sharma, 2006. Heavy metals
5
accumulation in soils irrigated by municipal and industrial effluent. J. Environ. Sci.
6
Engi., 46: 65-73.
7
8
9
8 Singh, S., and M. Kumar, 2006. Heavy metal load of soil,water and vegetables in peri-urban
delhi. J. Environ. Monit. Assess., 120: 79-91.
9 Sharma, R.K., M. Agarwal and F.M. Marshal, 2008. Contamination of vegetables in urban
10
India, a case study in Varanasi. J. Environ. Poll.,154: 254-263.
11
10 Suruchi, K., and A. Jilani, 2011. Assessment of heavy metal concentration in washed and
12
unwashed vegetables exposed to different degrees of pollution in agra, india.
13
Electronic J. Environ. Agri. Food Chem., 2700-2710.
14
11 Nwajei, G.E., 2009. Trace Elements in Soils and Vegetation in the Vicinity of Shell
15
Petroleum
Development Company Operating Area in Ughelli, Delta State of Nigeria.
16
J. Sustain.
Agri., 3: 574- 578.
17
18
19
20
21
22
23
24
25
26
12 AOAC. 2006. Official Methods of Analysis of Association of Official Analytical Chemists
International. In: Horwitz, W. (Ed.), 18th ed. AOAC Press, Arlington, VA, USA.
13. Steel, R.G.D.; Torrie, J.H.; Dickey, D.A. 1997. Principles and Procedures of Statistics: A
Biometrical Approach. 3rd Ed. McGraw Hill Book Co. Inc., New York, USA,
14 Krol, W.J., T.L. Arsenault, H.J. Pylypiw and M.J. Incorvia, 2000. Reduction of pesticide
residues on produce by rinsing. J. Agric. Food Chem., 48: 4666-4670.
15 Waalkes, M.P., and S. Rehm, 1994.Cadmium and prostate cancer, J. Toxicol. Environ.
Health. 43: 251-269.
16 Adeyeye, E.I. 2005. Trace metals in soils and plants from Fadama farms in Ekiti State,
Nigeria. Bull.Chem. Soc. Ethopia, 19: 23-24.
12
1
17 Sharma, R.K., Agrawal, M. and F.M. Marshal, 2009. Heavy metals in vegetables collected
2
from production and market sites of a tropical urban area of India. J. Food Chem.
3
Toxicol., 47: 583-590.
4
18 Perveen, Z., M.I. Khuhro and N. Rafiq, 2003. Market basket survey for lead, cadmium,
5
chromium and zinc in fruits and vegetables. Bull. Environ. Contam. Toxicol., 71:
6
1260-1271.
7
19 Nadine, A., H.E. Johan, J.O. Pierre and A. Samer, 2009. Measurement of levels of heavy
8
metals contamination in vegetables grown and sold in selected areas in Lebanon.
9
Jordan J. chem., 4:303-315.
10
20 Naser, H. M., N.C. Shil, N.U. Mahmud, M. Rashid and K.M. Hossain, 2009 .lead, cadmium
11
and nickel contents of vegetables grown in industrially polluted and non-polluted
12
areas of Bangladesh. Bangladesh J. Agri. Res., 34(4): 545-554.
13
21 Nergus, Y., Ahmed, S.I. and M. Sharif, 2005. Impact of contaminated vegetables, fruits and
14
fodders on human health by Malir river farms Karachi. J. Chemical Soc. Pak., 6:
15
561-574.
13