an analysis of heavy metals in the tannery effluent
TRANSCRIPT
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AN ANALYSIS OF HEAVY METALS IN THE TANNERY EFFLUENT
POLLUTED WATER BODY AND IN RICINUS COMMUNIS USING
ATOMIC ABSORPTION SPECTROSCOPY
Ramesh Krishna M. and *Sheela S.
Department of Plant Biology and Biotechnology, Loyola College (Autonomous), Chennai - 600034.
ABSTRACT
Tannery industries around Chennai do not treat the effluent properly
before releasing them into the water bodies which results in the severe
pollution of water bodies and hence affects the plants, animals and
have adverse effect on our environment. It is well known and studied
that tannery effluent may contain heavy metals which could be
absorbed by plants. This results in the heavy metal accumulation in
plants. The accumulated heavy metals have effects in plants
physiological and histological nature. The study aims to analysis the
presence of heavy metals in the tannery effluent polluted water body
and to analysis the accumulation of heavy metals in the plants that
grow along the water bodies. And to analyse the heavy metals
distribution in plant‟s root and shoot to estimate the difference in the heavy metal
concentration distributed with in a plant. The plant used in the study was Ricinus communis
and the location of the study i.e., the water sample and plant sample were collected from
Thirunnermalai, an outskirt of Chennai and a town near Chrompet which is known for tannery
industries.
KEYWORDS:- Water Pollution - Tannery Effluent - Heavy Metals - Ricinus communis
Heavy metal Accumulation in Plants - Atomic Absorption Spectroscopy.
1. INTRODUCTION
Water pollution is the major reason for the scarcity of suitable water for drinking and
agriculture in many developing countries. Water ecosystem is heavily affected by the
discharge of industrial effluents into the water bodies which in turn affects the environment.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.632
Volume 10, Issue 12, 1033-1044 Research Article ISSN 2278 – 4357
*Corresponding Author
Sheela S.
Department of Plant Biology
and Biotechnology, Loyola
College (Autonomous),
Chennai – 600034.
Article Received on
27 Sept. 2021,
Revised on 17 October 2021,
Accepted on 07 Nov. 2021
DOI: 10.20959/wjpps202112-20208
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Water bodies have been continuously polluted by the discharging of untreated waste
contaminants from industries. This is mainly due to the increased industrialization along with
rapid urbanization.[1]
Among all the contaminants, tannery effluents with heavy metals
possess a serious environmental concern because of their toxicity, persistent nature and non
degradability and their ability to accumulate in living organisms and affecting the food
chain[2]
In tannery industries, chemicals like chromium sulphate, sodium sulphate, sodium
chloride, organic dyes, acids, alkali and salts of calcium and ammonium are used to process
the leather. Tannery industries have become an emerging environmental concern because of
the disposal of contaminated waste water with heavy metals and other inorganic substances
into the water bodies.[3]
Water pollution is acute when tannery industries are clustered in a
small area along the river, in this study, tannery industries around Chennai Chrompet, along
the river Adayar. Chennai is the major leather processing city in South-India with the total
contribution of 50% leather export from India. Water is added in the tanning industry at
multiple stages of leather processing to allow reactions between the chemicals and the
skin/hide.
This generates an estimated 145 billion gallons of effluent per year with high concentration of
pollutant[4]
Agricultural activity and ground water quality are affected by the sludge
generated from tannery industries.[5]
According to the reports of the World Health
Organization (WHO), in India, the heavy metal concentration in industrial areas is found to
be much higher than the permissible levels, exposing the workers to severe occupational
hazards.[6]
Chronic exposure to heavy metals persisting in the environment could be a real
threat to the living organisms.[7]
Contamination of aquatic and terrestrial ecosystems has
raised global concern. The presence of higher levels of heavy metals in the biota of aquatic
animals can harm their ecological health and this may contribute to the decline in their
population.[8]
The rising impact on the ecological and human health can be correlated to the
negligence shown on the discharge of untreated industrial effluents, and the failure to adhere
to the strict regulations passed out by the government against environmental pollution. Heavy
metals tolerance in plants is the ability to survive virulent soil and its manifested by
interaction between the genotype and the atmosphere.[9]
Plants which have the ability to
actively uptake heavy metals from the environment is termed as Hyper-accumulators.
Naturally, most of the plant species exhibit basic tolerance to heavy metals. This is because
plants possess a complex system with the mechanism of uptake (efflux), transport
(sequestration), and chelation.[10]
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Major steps involved in the hyper-accumulation of involves the following steps namely (i)
transport of metals across the plasma membrane of roots cells, (ii) xylem loading and
translocation, (iii) detoxification and sequestration of metals at the whole plants cellular level.
Members of Brassicaceae and Fabaceae are the first known hyper-accumulators.[11]
The
exact mechanism of hyper-accumulation is not understood. Usually the accumulation of
heavy metals is based on the uptake capacity and intracellular transformation in plant‟s
tissue.[12]
Ricinus communis is a plant adapted to a wide range of climates and can be found
in most tropical and subtropical parts of the world. Ricinus communis, Family:
Euphorbiaceae popularly known as "Castor Plant'' and commonly known as 'Palm of
Christ', Aamanaku (Tamil), Jada (Oriya), Verenda (Bengali), Endi (Hindi). The plant is
widespread throughout tropical regions as ornamental plants. This plant is common and
quite wild in the jungles in India and it is cultivated throughout India, chiefly in the states
of Tamil Nadu, West Bengal and Maharashtra. Two varieties of R.communis are known as
perennial bushy plants with large fruits and large red seeds which yields about 40 PC of
oil and a much smaller annual shrub with small grey seeds having brown spots and yielding
37% oil.[13]
The castor oil is a fast-growing, suckering perennial shrub or occasionally a soft
wooded small tree up to 6 meters or more, but it is not hardy in nature. These plants were
cultivated for leaves and flower colors and oil production. Leaves are green or reddish in
color and about 30-60 cm in diameter. The stems are varying in pigmentation. The flowers
are monoecious and about 30-60 cm long (The Wealth of India, 1972). The fruit is a three-
celled thorny capsule. The capsule of fruit covered with differences in size and colour.
They are oval, compressed, 8-18mm and 4-12mm broad. Castor seeds have a warty
appendage called the caruncle, which present usually at one end from which runs the
raphe to terminate in a slightly raised chalaza at the opposite end of the seed.[14]
Atomic Absorption Spectroscopy (AAS) is a very common and efficient technique for
detecting metals in the environmental samples. Atomic absorption spectroscopy is an
analytical technique that measures the concentrations of elements qualitatively and
quantitatively. If light of just the right l impinges on a free, ground state atom, the atom
absorbs the light as it enters an excited state in a process known as atomic adsorption.[15]
It
depends on the Beer-Lambert law standard in which atomic absorption techniques measure
the vitality as photons of light that are consumed by the sample.[16]
The tannery effluent
polluted water body collected from the site of pollution and the plant samples were analyzed
for the presence of the following heavy metals Cadmium (Cd), Chromium (Cr), Iron (Fe),
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Lead (Pb) and Zinc (Zn) using Atomic Absorption Spectroscopy (AAS).
2. MATERIALS AND METHODS
2.1 Sample collection
The water sample required for the study was taken from the polluted water canal located near
Thiruneermalai, outskirt of Chennai. And the plant samples of Ricinus communis were
collected from the same location.
2.2 Water analysis
Water analysis was performed to derive basic physico-chemical parameters of the water used
for the study. Parameters like pH, Total Suspended Solids (TDS), Total Hardness, Total
Alkalinity, Total Acidity were established from the analysis of the collected water sample.
The testing methods adopted for the study were based on 'Methods of analysis of soils, plants,
water, fertilizers and organic manure' by HLS Tandon (FDCO) 2009 and 'Methods for
chemical analysis of water and wastes' EPA, US (1983).
2.2.1 Preparation of the water sample for heavy metals analysis
The collected water sample was filtered through normal filter paper followed by whatman
paper of grade 1.This filtered sample was acid digested to proceed with the metals analysis.
The filtered water sample of 50 ml was transferred to a 100ml beaker and 5ml of
concentrated nitric acid was added and heated until the mixture was reduced to the half the
volume. The digested sample was stored in the refrigerator overnight. Then the digested
sample was heated in a boiling water bath and cooled at room temperature. Finally, the total
volume was made upto 50ml using double distilled water. Metal concentration in the
digested water was using Atomic Absorption Spectroscopy (AAS). The standard solutions for
heavy metals to be analyzed are prepared based on 'Method 7000B, FAAS, EPA (20027)'
2.3 Estimation of chlorophyll content
Heavy metals are known to interact with the plant's photosynthetic pigment, Chlorophyll.
The chlorophyll estimation was done by Arnon's method (1949).
1. 1gram of fresh leaf samples of Ricinus communis were homogenized in a pre- cooled
mortar and pestle using 80% acetone.
2. A pinch of CaCo3 was added while grinding to prevent premature acidification and
pheophytin formation during the assay.
3. The resulting homogenate was centrifuged at 10,000 rpm for 15 minutes.
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Following the centrifugation, the supernatant was filtered into glass beakers. The absorbance
was measured at 645nm and 663nm.
The amount of Chlorophyll a, Chlorophyll b and the Total chlorophyll content were
determined using the prescribed formula.
Chlorophyll a = 12.7 (A663) – 2.69 (A645)
Chlorophyll b = 22.9 (A645) – 4.08 (A663)
Total Chlorophyll = 20.2 (A645) + 8.02 (A663)
2.4 Processing of plant samples for metal analysis
The collected plant samples were dried at 80 degree celsius in hot air over for 24 hours. The
dried plants were separated into their plant parts: roots, stem, leaves. Each plant part was
ground using mortar and pestle. The ground plant parts were stored in individually labeled
ziploc bags for further analysis. Phytoextraction properties of the accumulating plants cause
the plants to extract the metals and translocate them to various plant parts, and to
store/degrade them within the plant tissues. Hence, categorizing the plants into individual
parts allows the estimation of metal accumulated in the respective plant part, and to conduct a
comparative study.
2.4.1 Acid digestion of plant sample for AAS analysis
Estimation of metal concentration in the plant sample was done by Atomic Absorption
Spectroscopy (AAS) method. The plant samples were acid digested prior to AAS analysis
by adopting the Wet acid digestion method. The acid digestion was done using a mixture of
concentrated nitric acid and concentrated perchloric acid in the ratio of 3:1. The ground
plant samples were transferred into 50ml beakers and digested using 12 ml of the mixture of
concentrated nitric acid and concentrated perchloric acid. This mixture was heated on a hot
plate until the fumes reduced and the digest became clear. The digestion was performed
inside the fume hood as the acid mixture can release toxic fumes. Following the digestion,
the mixture was allowed to cool and incubated at room temperature for 24 hours. The
digested mixture was then filtered through Whatman filter paper of Grade 1 into a 25ml
Standard Measuring Flask. The final volume was made up to 50 ml using double distilled
water. Metal concentrations in the digested samples were determined by Atomic Absorption
Spectroscopy (AAS) analysis.
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Fig. 2.1: Acid digested water sample. Fig. 2.2: Acid digested plant samples.
3. RESULTS AND DISCUSSION
3.1. Physicochemical parameters of the water sample
The physicochemical properties of the collected water from the contaminated site for the
study were established through water analysis. The results for the water analysis are shown in
Table 5.1. The established physicochemical parameters include pH, total dissolved solids,
total hardness, acidity and alkalinity of the polluted water sample.
Table 3.1: Physicochemical parameters of the water sample.
Sl. no. Parameters Unit Results
1 pH - 4.5
2 Total Dissolved Solids mg/L 960
3 Total Hardness mg/L 375
4 Total Acidity mg/L 1,240
5 Total Alkalinity mg/L 241
The tannery effluent polluted water sample taken for the analysis of heavy metals had
unfavourable parameters and was completely unsuitable for domestic usage. The pH of the
collected water sample was acidic with a pH of 4.5 against the WHO prescribed 7.4 for river
water. The total hardness of the collected water sample was found to be 960 mg/L which is
considered as “very hard” for consumption. On the other hand, the total dissolved solids
(TDS) was calculated as 375 ppm per litre which is poor according to the WHO‟s standard for
water quality parameters. The collected water sample‟s total acidity and alkalinity were found
to be 1240 mg/L and 241 mg/L respectively. All these parameters clearly explain that the
tannery effluent polluted water sample taken for the presence of heavy metals is not at all
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recommended for any types of domestic usage.
3.2.Concentration of heavy metals in the water sample
Table 3.2: The concentration of heavy metals in the water sample.
Sl. no. Heavy Metals
analyzed
Metal concentration
(ppm)
WHO Permissible
limit (ppm)
1 Cadmium 1.17 0.005
2 Chromium 0.36 0.1
3 Copper 1.01 1.0
4 Iron 5.41 0.1
5 Lead 1.48 0.05
6 Zinc 0.83 5.0
Graph 3.1: The Concentration of Heavy Metals compared with WHO’s Permissible
limits in ppm.
The Atomic Absorption Spectroscopy (AAS) technique was used for the analysis of
Cadmium (Cd), Chromium (Cr), Copper (Cu), Iron (Fe), Lead (Pb) and Zinc (Zn) in the
tannery effluent polluted water body. And the results of AAS confirmed the presence of the
heavy metals. The obtained results were compared with the WHO‟s permissible limit of
heavy metals concentration in water and it is found that except Zinc (Zn), heavy metals such
as Cadmium (Cd), Chromium (Cr), Copper (Cu), Iron (Fe) and Lead (Pb) were present in
extremely high concentration than the permissible limits prescribed by WHO in the water
sample taken for analysis from the tannery effluent polluted site. Even though the
concentration of Cd, Cr, Cu, Fe and Pb were alarming, the only good thing was the metal
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Zinc (Zn) was less than the permissible level.
Estimation of chlorophyll content from the ricinus communis leaves
The influence of the heavy metals on the photosynthetic pigment, Chlorophyll, in plants was
determined by following the Chlorophyll extraction method prescribed by Arnon (1949). The
absorbance values of the extracted chlorophyll were read at 645 nm and 663 nm. The
estimated readings are presented in Tables 3.3. and 3.4.
Table 3.3: Chlorophyll Estimation - Absorbance reading at 645 and 663 nm.
OD (nm) Absorbance
A645 0.680
A663 0.640
Table 3.4: Chlorophyll Estimation - Determination of Chlorophyll a, Chlorophyll b &
Total Chlorophyll (mg/g).
Chlorophyll type Chlorophyll content (mg/g)
Chlorophyll a 6.2988
Chlorophyll b 12.9608
Total Chlorophyll 18.8688
Plants like other living organisms possess homeostatic mechanisms that are recruited to
maintain the right concentrations of metal ions in the plants, and also to prevent the
accumulation of such metals to toxic levels. Either deficient or in excess, heavy metals can
cause disorders in plant growth and development affecting physiological processes of the
plant system. The concentration of heavy metals should be maintained at low levels since
the element is extremely toxic concerning its high redox properties. This might be the
reason for the reduced amount of Chlorophyll „a‟ content in the leaves extract.
Normally, Chlorophyll „a‟ content are present in two to three times more in number than
Chlorophyll „b‟ content. Heavy metals when present at a concentration greater than the
required optimal metal concentrations, heavy metals are known to interfere with the growth
and important physiological processes of plants like photosynthesis and respiration.[17,18]
3.3. Metal concentration in Root and Shoot samples
The Ricinus communis plant samples were collected from the tannery effluent contaminated
site, cleaned, dried and separated into their respective plant parts and digested using aqua
regia to determine the concentrations of heavy metals in individual plant parts using Atomic
Absorption Spectroscopic (AAS) analysis. The Atomic Absorption Spectroscopy results are
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presented in Table 3.5.
Table 3.5: The concentration of heavy metals in Root and Shoot samples.
Sl.
No.
Heavy metals Metal concentration in plant parts
(ppm)
WHO permissible level
(ppm)
Root Shoot Plants
1 Cadmium 1.29 0.78 0.02
2 Chromium 0.52 0.39 1.30
3 Copper 0.81 0.56 10
4 Iron 8.21 7.27 -
5 Lead 1.99 1.0 2.0
6 Zinc 1.39 1.02 0.60
Graph 3.2: The Concentration of Heavy Metals in Root and Shoot Samples.
Plants exhibit tolerance to heavy metals toxicity by adopting mechanisms on a cellular level
that includes preventing the accumulation of toxic concentrations of heavy metals at
sensitive sites within the cell preventing the damaging effects of the metals rather than
developing proteins that can resist the heavy metal effects.[17,18]
Organic acids excreted by
the plants can facilitate metal uptake but these molecules also tend to inhibit the uptake of
metals through the formation of a complex with it outside the root and thus preventing its
uptake. In a study performed by Elleuch, 2013, it was studied that the metal was primarily
sequestered within the root system. Despite the plant Ricinus communis used for the study
were collected from the heavily tannery effluent polluted site, castor plants were able to
thrive in that environment. Following the accumulation within the roots, subsequent metal
accumulation was noticed in the leaves of the plants. This accumulation had taken place
through the mechanism of translocation employing the xylem and phloem system,
transporting the metal from the roots to the leaves. Compared to the metal concentration
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observed in root and shoot, the shoot had reduced metal concentration, which also denotes
reduced accumulation of metal had taken in the shoot region. The castor plant has been
studied for its efficiency to withstand harsh conditions. The bio-availability of the metal is
a major factor that contributes to effective phytoremediation. Bio-availability determines how
much metal from the soil is available for the plants to take in. This study is focused only
for water analysis and limited for the soil analysis.
4. CONCLUSION
Heavy metals pollution is a serious concern to all living organisms and to the environment.
The effects of heavy metals contamination can directly or indirectly affect the food chain and
hence can disrupt the ecological balance of all the ecosystems. Tannery effluents are the
major anthropogenic source for heavy metals. The industrial effluents have been released
into the water bodies without proper effluent processing techniques. Many
physicochemical techniques to process tannery effluents have shown poor removal efficiency
of heavy metals. Plants that have grown near the tannery effluent contaminated sites are
known to uptake and remediate the heavy metals. Phytoremediation technique has gained
much attention with its eco- friendly approach to be able to restore a site contaminated with
toxic environmental pollutants, primarily from industrial sources such as tannery effluents.
Over hundreds of species including Ricinus communis have been identified with the potential
to uptake metals from their surrounding environment. The effectiveness of the
phytoremediation can vary between what is observed under laboratory conditions as in the
field, the nature of the soil, pollutant concentration and climatic factors. Heavy metals are
normally hazardous when their concentration is above the threshold levels. The World Health
Organization (WHO) has listed a permissible limits for heavy metals to determine the heavy
metal contamination in the environment.
5. ACKNOWLEDGEMENT
I extend my heartfelt gratitude to my mentor, Dr. S. Sheela for her guidance and
supervision, and for her encouragement throughout.
I deliver my sincere thanks to Dr. John Milton, Head, Department of Zoology, Loyola
College, and Mr. Magesh Daniel, Research Scholar, Department of Zoology, Loyola
College for providing the facility for Atomic Absorption Spectroscopy analysis at their
laboratory.
I thank Mr. Sridhar and Mr. Preetam Raj, Laboratory Assistants, Department of
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Biotechnology, Loyola College for providing me with all necessary laboratory
requirements on time, and for being supportive.
I thank my friends and all other faculty members of the Department of Biotechnology,
Loyola College, who have been helpful and supportive during the days of my project
work.
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