AN ANALYSIS OF HEAVY METALS IN THE TANNERY EFFLUENT

www.wjpps.com │ Vol 10, Issue 12, 2021. │ ISO 9001:2015 Certified Journal │

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Krishna et al. World Journal of Pharmacy and Pharmaceutical Science

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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AN ANALYSIS OF HEAVY METALS IN THE TANNERY EFFLUENT