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CHAPTER- II

MATERIALS AND METHODS

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CHAPTER II

MATERIALS AND METHODS

2.1 STUDY AREA

The present study area is in Mysore city belongs to Karnataka state, India.

Karnataka State is in the south-western part of India. It is mainly a tableland and an

extension of Deccan plateau. The state extends to 805 km from north to south and to

about 283 km from east to west. The total area of the state is 192,493 sq. km. Mysore

District is an administrative district located in the southern part of the state of

Karnataka, India. The district is bounded by Mandya district to the northeast,

Chamarajanagar district to the southeast, Kerala state to the south, Kodagu district to

the west, and Hassan district to the northwest.

2.2 TOPOGRAPHY OF MYSORE CITY

Mysore city is one of the largest districts of Karnataka, Mysore is the former

capital of the kingdom of Mysore. Mysore is located at 770m above sea level at

12.180 N and 76.42

0E and is 135km away from Bangalore, the state capital.

The study area Mysore is having more than 9 lakh populations. The climate of

the city is moderated throughout the year with temperature during summer ranging

from 300 to 340C. The rainy season is from May to October. The winter season is

from November to February. For domestic and industrial purposes, the main source of

water is mainly from the Cauvery River and ground water.

Mysore is one of the growing citiy of Karnataka and it is so largely due to the

presence of Industrial resources and a well developed communication network.

Mysore has a rich and vibrant history and heritage. Hence it attracts a huge number of

tourists. Also, Mysore is now active center for production and industrialization. The

city has been growing as a magnet to Bangalore with large presence of software

companies and the population is growing at a faster rate due to the influx of many

industrial and commercial activities.

In recent years industrialization has become main cause of city’s growth.

There is diversity in industrial landscape of Mysore with haphazard distribution. The

industrial areas are distributed all over the city and it is surroundings with lack of

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order and regulation in industrial location. A large number of small medium and large

scale industries exists in and around the Mysore city, including Engineering,

Chemical, Pharmaceutical, Food, Brewery, Distillery, Textile, Steel and Smelting.

2.2.1 Climate:

Temperature influences considerably the socio-economic activities of the

people in a region. The district in general enjoys cool and equable temperatures. In the

period from March to May, there is a continuous rise in temperature. April is the

hottest month with the mean daily maximum temperature at 34.5°C and the daily

minimum at 21.1°C. On normal days, the day temperatures during summer may

exceed 39°C. There is welcome relief from the heat when thunder showers occur

during April and May. Mysore has a warm and cool climate throughout the year. The

climate of Mysore is moderate. The weather in winter is cool and summer is bearable.

There are three main seasons namely summer from March to May followed by

monsoon from June to October and winter from November to February. After mid-

November, both day and night temperatures decrease progressively. January is the

coldest month with mean daily maximum at 11°C. On some days during the period

November to January, the minimum temperature may go below 11°C.

2.2.2 Agro climatic conditions

The climatic conditions of the district are favourable to crops like paddy,

jowar, ragi, pulses, sugarcane and tobacco. The district can be divided into two major

agro-climatic zones like the Southern Dry Zone comprising of 4 taluks namely,

Nanjangud, T. Narasipur, Mysore and K.R. Nagar and Southern Transition Zone

consisting of H. D. Kote, Hunsur and Periyapatna taluks. Soil is red sandy loam in

most of the areas of the district. The annual rainfall ranges from 670 mm to 888.6 mm

in dry zones and from about 612 mm to 1054 mm in the transition zone. The average

annual rainfall of the district is 782 mm. The temperature ranges from 11°C to 38°C.

Thus the climate of Mysore district is temperate with moderate variations in

temperature in different seasons.

2.2.3 Rainfall

The variation in the annual rainfall from year to year is not large during the 85

years from 1901 to 1985, the highest annual rainfall amounting to 156 percent of the

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annual rainfall that occurred in 1903 and the lowest occurred in 1918. In the same 85

year period, the annual rainfall was less than 80 percent of the normal rainfall in 7

years, none of them consecutive, considering the rainfall at the individual stations.

However, two or three consecutive years of good rainfall occurred once or twice at

fifty two out of sixty five rain gauge stations. It is observed that the average annual

rainfall in the district was between 600 mm and 900 mm in 66 years out of the 85

years.

2.2.4 Geology

Geologically, the district is mainly composed of igneous and metamorphic

rocks of Pre-Cambrian age either exposed at the surface or covered with a thin mantle

of residual and transported soils. The rock formation in the district falls into two

groups, charnockite series and granite genesis and gneissic granite. The soils of the

districts can be broadly classified as the laterite, red loam, sandy loam, red clay and

black cotton soils. The laterite soil occurs mostly in the western part of the district

while the red loam is found in the northwest. These two account for nearly half the

area of the district. The black cotton soil is found mostly in the northeastern parts of

the district. The red sandy loam soils are derived from the granites and gneisses. The

western taluks of Periyapatna, H D Kote and Hunsur are covered with hilly terrain

and contain red, shallow gravelly soils. In the taluks of T. Narasipura and

Nanjanagud, there is deep red loam occasionally interspersed with black soils. The red

soils are shallow to deep well drained and do not contain lime nodules. The black

soils are 1 to 1.5 metre in bases with good water holding capacity for a longer time.

2.2.5 Temperature

Temperature influences considerably the socio-economic activities of the

people in a region. The district in general enjoys cool and equable temperatures. In the

period from March to May, there is a continuous rise in temperature. April is the

hottest month with the mean daily maximum temperature at 34.5°C and the daily

minimum at 21.1°C. On normal days, the day temperatures during summer may

exceed 39°C. There is welcome relief from the heat when thunder showers occur

during April and May. With the advance of the southwest monsoon about the

beginning of June, the day temperatures drop appreciably and throughout the

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southwest monsoon period, the weather is pleasant. After mid-November, both day

and night temperatures decrease progressively.

The temperature remains nearly the same for several months but begins to rise

in February and touches the peak in either April or May, in both maximum and

minimum. Minimum is near about 20° C and the maximum is near about 30° C for

several months.

2.2.6 Soil

The soils of the districts can be broadly classified as the laterite, red loam,

sandy loam, red clay and black cotton soils. The laterite soil occurs mostly in the

western part of the district while the red loam is found in the north-west. These two

account for nearly half the area of the district. The black cotton soil is found mostly in

the north-eastern parts of the district. The red sandy loam soils are derived from the

granites and gneisses. The western taluks of Periyapatna, H D Kote and Hunsur are

covered with hilly terrain and contain red, shallow gravelly soils. In the taluks of

T.Narasipura and Nanjanagud there is deep red loam occasionally interspersed with

black soil. The red soils are shallow to deep well drained and do not contain lime

nodules. The black soils are 1 to 1.5 metre in bases with good water holding capacity

for a longer time.

2.2.7 Natural Vegetation

The area covered by forest is 4,126.45 sq. km, 34.52 per cent of the total area,

of which 3,875.6 sq. km, are reserved forest, and 250.9 sq. km. are classified as

forests. Mysore has two types of forests and they are moist deciduous where the

rainfall is 900-1100 mm and dry deciduous where the rainfall is 700-900mm. Mysore

district is the third richest in forest wealth in the State. The forest belt in the district

begins from the western part of Hunsur taluk, spreads along the border of Kerala and

Tamil Nadu into the south and east. The thickest and richest forest areas are in H D

Kote. The Principal species of trees in the forests are teak, honne, rosewood, dindiga,

eucalyptus and sandalwood.

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2.3 Industrial zone of Mysore

In Mysore the Karnataka Industrial Areas Development Board(KIADB) have

developed Industrial Areas in the Mysore Zonal Office jurisdiction comprising of four

districts namely:

1. Mysore District.

2. Mandya District.

3. Chamarajanagar District.

4. Madikeri District.

2.3.1 Mysore District Industrial Area

Mysore Industrial Area comprises of 6 Industrial Areas namely:

a. Metagally

b. Hebbal (General and Hebbal Electronic City)

c. Hootagally

d. Belavadi

e. Koorgally – Mysore III Phase

Locations within radius of 7 kms from Mysore City the above Industrial Areas have

been developed with the following details:

a. Metagally

i. Land acquired : 519.00 Acres

ii. Area formed : 519.00 Acres

iii. No. of Plots formed : 162 Nos.

iv. No. of Units allotted : 113 Nos.

v. Length of roads (All roads are asphalted) : 6.50 kms.

vi. Civic Amenities (KIADB Office Complex) : 3.00 Acres

b. Hebbal




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v. Length of roads : 20.00 Kms.

vi. Civic Amenities, Water Supply,

Pump house, Quarters, etc.

(including Housing Area) : 22.00 Acres

c. Hootagally





v. Length of roads : 3.80 Kms. (18 Mts. wide)


Pump house, Quarters, etc., : 2.00 Acres

d. Belavadi





v. Length of roads : 1.00 Kms. (18 Mts. wide)


Pump house, Quarters, etc., : 0.75 Acres

e. Koorgally industrial area – Mysore III Phase:


ii. Area of land developed : 500.00 Acres

iii. No. of plots formed : 40 Nos.

iv. No. of units allotted : 45 Nos.

v. Water supply Capacity : 1MGD

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Figure 2.1 LOCATION OF STUDY AREA.

In the present study, the study area which are comprising more in industries

(Fig 1). In recent year’s industrialization has become main cause of city’s growth.

There is diversity in industrial landscape of Mysore with haphazard distribution. The

industrial areas are distributed all over the city and its surroundings with lack of order

and regulation in industrial location. A large number of small and medium scale

industries exist in and around the Mysore city. Most of all medium scale industries

are engineering, chemical, pharmaceutical, food, brewery, textile, steel and metal

smelting industries.

In Hebbal industrial area small scale industries and medium scale industries

are more in number compared to large scale industries like electrical appliances

industries, textile industry metal product industries.

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Hootagalli industrial area is smaller in its size as compared to metagalli and

Hebbal industrial areas. Here the industries like textile, heavy earth movers

manufacturing industry and very few small scale industries are situated. In the present

study sample locations were widely distributed in the study area and nine

representative samples were collected.

2.4 Sampling Location

Karnataka Industrial Areas Development Board (KIADB) has established four

industrial areas in Mysore city to encourage Industrial Development of the city. These

are located at,

1. Hootagalli industrial area.

2. Hebbal (Electronic City) industrial area.

3. Metagalli industrial area.

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Figure 2.2 Land use plan of Mysore city showing the study area

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Source : KIADB

Figure 2.3 SAMPLING LOCATION OF STUDY AREA.

In the present investigation interest was focused to know the nature and

composition of soil from industrial zone, with special interest to know the

concentration of heavy metal and bioavailability. In this process distinction of impact

of heavy metal in soil/sediment and uptake by biota is of special interest. The

sampling stations in the present study are selected on these criteria. The work

involves sampling of 10 soil samples from industrial zone of Mysore city. Table 2.1

shows the sampling stations.

Table 2.1 List of sampling locations

Sl. No Station code Location Industrial Area

1 P1 Automotive excel Hootagahalli

2 P2 Chamundi Textiles Hootagahalli

3 P3 BEML Hootagahalli

4 P4 Wipro lightings Hebbalu

5 P5 Rane madras Hebbalu

6 P6 VikranthTyres Metagalli

7 P7 Falcon Tyres Metagalli

8 P8 Bhoruka Aluminium Metagalli

9 P9 Triveni Gears. Metagalli

10 P10 Shimoga steels Hebbalu

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2.5 Sample Collection

Assessment of pollution is largely depending upon the systematic monitoring

and evaluation of pollutants. Similarly the success of a monitoring study depends

upon the planning made prior to sampling. The plan should include not only the

selection of sampling sites but also parameters to be analyzed, total number of

samples, size of each samples, frequency of samples collection, date and time of

sample collection, is very much important and to achieve the objectives the field study

has been planned. Selection of sampling sites, numbers of samples, sampling

frequency were also carried out as per limitation such as approachability.

2.5.1. Sample Preservation and Storage

Samples can change very rapidly. However, no single preservation method

will serve for all samples and constituents, so the purpose of sample preservation is to

minimize any physical, chemical, and/or biological changes that may take place in a

sample from the time of sample collection to the time of sample analysis.

Three approaches (i.e., refrigeration, use of proper sample container, and

addition of preserving chemicals) are generally used to minimize such changes,

refrigeration (including freezing) is a universally applicable method to slow down all

loss processes. The only exception that refrigeration does not help water samples are

preserved for metal analysis, (Spellman, 2008). Cold storage will adversely reduce

metal solubility and enhance precipitation in the solution. The proper selection of

containers (material type and headspace) is critical to reduce losses through several

physical processes, such as volatilization, adsorption, absorption, and diffusion.

Colored (amber) bottles help preserve photosensitive chemicals such as PAHs.

The addition of chemicals is essential to some parameters for their losses due

to chemical reaction and bacterial degradation. Chemical addition or pH change can

also be effective to reduce metal adsorption to glass container walls.

2.6 Soil sampling:

• Divide the study area into sampling points so that each sample represents an area

of not more than 6 acres.

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• Fix sampling spots to represent the study area.

• Scrape away the surface litter, stones etc. and collect samples in to a bucket from

each spot up to the required depth by making a ‘V’ shaped cut by using a spade.

Take a slice of soil from both the sides. Collect the same quantity from each of

sampling spots. Place the sample on a plastic sheet and mix by discarding stones,

roots etc. and take out the required quantity of soil in to polythene bag.

2.7 Pre-treatment of the soil sample

The soil samples were collected at different points of the industrial zone of

Mysore city, India. The soil samples are collected during 2010 to 2012. Soil samples

were dried with the help of oven in the laboratory and then ground in an agate mortar

and pestle to pass through a 0.5mm stainless steel sieve. Then they were stored in

polythene covers at room temperature. The soil samples were analyzed for physico-

chemical properties using standard analytical methods (APHA 1998).

2.8 Soil Analysis

The collected soil samples were analyzed for various physico-chemical

parameters such as pH, Electrical Conductivity, Lime content, organic carbon, organic

matter, Calcium, Magnesium, Sodium, Potassium.

2.8.1 pH:

The soil pH is the negative logarithm of the active hydrogen ion (H+)

concentration in the soil solution. It is the measure of soil sodicity, acidity or

neutrality. It is a simple but very important estimation for soils, since soil pH

influences to a great extent the availability of nutrients to crops. It also affects

microbial population in soils. Most nutrient elements are available in the pH range of

5.5 to 6.5.

A 25gm suspension of air dried soil was prepared in double distilled water. It

was then allowed to settle for 1 hour and the pH for all the soil filtrates was checked

using a calibrated pH meter.

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2.8.2 Electrical Conductivity (EC):

The electrical conductivity (EC) is a measure of the ionic transport in a

solution between the anode and cathode. This means, the EC is normally considered

to be a measurement of the dissolved salts in a solution.

The measurement of EC will give the concentration of soluble salts in the soil

at any particular temperature. EC measured in 1:2 or 1:5 soil-water suspension with

the help of conductivity meter. Calibrate EC meter using standard KCl solution and

determine the EC of suspension used in pH determination.

2.8.3 Organic Carbon :

A known volume of soil sample is treated with an excess volume of standard

potassium dichromate solution in the presence of con.H2SO4. The soil is digested by

the heat of dilution of sulphuric acid and organic carbon in the soil is thus oxidize to

carbon dioxide. The excess of potassium dichromate , unused in oxidation is titrated

against standard solution of ferrous ammonium sulphate in the presence of fluoride or

phosphoric acid and diphenylamine solution indicator. The organic carbon content of

soil is calculated using the relationship of 1ml of 1N K2Cr2O7 = 0.003g of organic

carbon. The organic carbon in the sample is oxidized with 1N potassium dichromate

and sulphuric acid. The excess potassium dichromate is titrated against 0.5N ferrous

ammonium sulphate.

Calculation

(B-S) × 0.003 × mcf

Organic Carbon (%) = W

Where

B = ml of ferrous ammonium sulphate solution used for blank.

S = ml of ferrous ammonium sulphate solution used for sample.

mcf = moisture correction factor.

W = sample weight (g).

0.003 = conversion factor (including a correction factor for a supposed 70%

oxidation of organic carbon.)

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2.8.4 Lime content:

Weigh g of soil sample to a conical flask. Add mL distilled water, g of CaSO4

powder, mL of N aluminum chloride and drops of each of bromothymol blue and

bromocresol green indicators. Boil the contents for minutes by placing the flask on a

hot plate. If the solution becomes green, CaCO3 is present. If it is turns golden

yellow, CaCO3 is absent. Titrate the green suspension against 0.5 N H2SO4 till it turns

yellow. Bring it back to a boil, if it changes to green continue boiling and titration till

a permanent golden yellow color is obtained.

Calculations.

Titartion value X N of H2SO4x 0.05x 100

Lime content =

Weight of soil sample.

2.8.5 Potassium: Flame photometric method

The atoms or ions present in solution gets energy from a flame, they get

excited and results in the emission of spectrum. The energy absorbed by electrons,

shifts them to position more distant from the atomic nucleus. As the electrons regain

their state, the previously absorbed energy is remitted as electromagnetic radiations.

The wavelengths of which correspond to the quantity of energy involved in the

respective electron shifts and the quantity of radiation is directly proportion to the

amount of the element emitting the rays.

Weigh 5g of soil sample into a conical flask. Add 25mL of neutral normal

ammonium acetate solution. Shake the contents of the flask on the clectric shaker for

5minutes and filter through Whatsman No.1 paper. Feed the filtrate in to

flamephotometer which has been adjusted to 100 with 40ppm standard solution of K

and note down the reading.

Preparation of standard curve:

Dissolve 1.91g of KCl in distilled water and make up the volume to 1L to give

1000ppm solution of K. from this stock solution prepare the 100ppm working solution

from which 10.20.30 and 40ppm solutions can be prepared. Adjust the

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flamephotometer to read 100 with 40ppm. Plot the curve shoeing the relationship

between of K and flamephotometer readings.

2.8.6 Sodium : Flame photometric method

A known weight of soil is extracted with 50mL of neutral ammonium acetate.

The ammonium ions exchange with the exchangeable sodium ions of the soil. The

sodium content in the equilibrium solution is estimated with a flamephotometer .

Pipette 0,5,10,15,20 mL of 100ppm of Na into a series of 50mL volumetric flask and

makeup the volume with distilled water or ammonium acetate to get the concentration

of working solutions of 0,10,20,30 and 40ppm of Na. Adjust the flamephotometer to

read 100 with 40ppm of Na. Feed different solutions having increasing concentrations

of Na into flamephotometer and note the readings. Plot the flamephotometer readings

verses sodium concentration to get standard curve. Feed the unknown sample to a

flamephotometer and record the readings. In case concentration is high dilute the

sample.

Calculation

C × 25 × mcf

Available Na, K (mg/kg) = Sample weight (g)

Where,

C = Concentration of potassium in filtrate.

mcf = Moisture correction factor.

25 = Volume of Ammonium acetate.

2.8.7 Determination of exchangeable Calcium and Magnesium :

• Calcium + Magnesium

Transfer 5-10mL of ammonium acetate extract in to conical flask. Add 5-10mL of

buffer complex to the contents to attain the pH to 10. Add 10 drops of EBT indicator

and titrate against Std.EDTA solution taken in the burette till the color changes from

pink to blue.

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• Calcium:

Transfer 5-10mL of the ammonium acetate extract into conical flask. Add 5mL of

10% NaOH solution to attain pH12. Add a pinch of mureoxide indicator and titrate

with std.EDTA solution till the color changes from pink to blue.

Calculations

Ca + Mg (m.eq per 100g) = TV1 X N of EDTA X Vol.made X 100

Weight of the soil X Aliquot taken.

Calcium (m.eq per 100g) = TV2 X N of EDTA X Vol.Made X 100 Weight of the soil X Aliquot taken.

Magnesium (m.eq per 100g) = m.eq of (Ca+Mg) – m.eq of Ca.

2.9 Instrumental Methods to determine heavy metal analysis

2.9.1. Atomic Absorption Spectroscopy (AAS)

Heavy metals analysis was performed on an Atomic Absorption

Spectrophotometer (GBC Avanta version 1.31) using acetylene gas as fuel (at 8 psi)

and air as an oxidizer. Operational conditions were adjusted to yield optimal

determination. The calibration curves were prepared separately for all the metals by

running suitable concentrations of the standard solutions. Digested samples were

aspirated into the fuel rich air-acetylene flame and the concentrations of the metals

were determined from the calibration curves. Average values of three replicates were

taken for each determination. Suitable blanks were also prepared and analysed in the

same manner. The detection limits for iron (Fe), zinc (Zn), copper (Cu), nickel (Ni),

chromium (Cr), lead (Pb) and cadmium (Cd) were 0.05, 0.008, 0.025, 0.04, 0.05, 0.06

and 0.009 ppm respectively.

2.9.2. Inductively Coupled Plasma (ICP- AES)

Inductively Coupled Plasma Atomic Emission Spectroscopy techniques (ICP-

AES) also referred to as inductively coupled plasma optical emission spectrometry

(ICP-OES) is an analytical technique used for the detection of trace metals. It is so

called "wet" sampling methods whereby samples are introduced in liquid form for

analysis. In ICP a sample solution is introduced into the core of inductively coupled

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argon plasma (ICP), which generates temperature of approximately 6000-8000°C. At

this temperature all elements become thermally excited and emit light at their

characteristic wavelengths. This light is collected by the spectrometer and passes

through a diffraction grating that serves to resolve the light into a spectrum of its

constituent wavelengths. Within the spectrometer, this diffracted light is then

collected by wavelength and amplified to yield an intensity measurement that can be

converted to an elemental concentration by comparison with calibration standards,

(Figure 2-2). ICP-AES instruments allow determinations of multiple metals

simultaneously from a single sample solution. These highly sensitive instruments can

be configured with a variety of detectors, depending upon the desired application.

2.10. Methodology for determination of Heavy metal in Soil.

Most metals are of geological origin, but contamination with them may be due

to industrial, mining, agricultural, waste handling or other activity. Often a mixture of

such metals occurs. The most common contaminants are Cadmium, Chromium,

Copper, Lead, Nickel and Zinc. In contrast to organic contaminants, metals cannot be

degraded by microbes or plants. Thus the bioremediation strategy is based on the

movement of metals, e.g., from soil to plants as in photo remediation, or on

bioleaching. Some metals can undergo microbial oxidation–reduction or become

methylated.

Figure-2.4 The ICP–AES (JY, 2000) manufactured by Horiba JobinYvon.

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2.10.1. Soil/Sediment samples

The total metal concentration estimation is carried out by digesting the

samples with aqua-regia. One gram of the pre-treated samples was taken in a conical

flask to which 8 mL of aqua-regia and 50 mL of distilled water were added. Then the

sample was evaporated near to dryness on hot plate using sand bath dissolved by

adding 5-10 mL of dilute nitric acid to the conical flask. After cooling to room

temperature, sample was filtered through Whatman No.41 filter paper and filtrate was

made up to 50 mL with distilled water. Total heavy-metal concentration of Cadmium,

Chromium, Copper, Iron, Lead, Nickel and Zinc are analyzed by ICP-AES.

2.10.2. Fodder Samples (Greens)

The fodder samples were thoroughly washed to remove all adhered soil

particles. Samples were cut into small pieces, air dried for 2 days and finally dried at

100° C ± 1° C inhot air oven for two hours. In warm condition, the samples were

ground and passed through 1 mm sieve. The fine powder samples (2 g/50 mL distilled

water) were subjected to acid digestion by adding 10 mL concentrated nitric acid on

hot plate and filtrate was diluted up to 50 mL with distilled water. Total heavy-metal

concentration is analysed by using ICP-AES.

2.11. Scanning Electron Microscopic and Energy Dispersion X-ray Spectroscopy

The purity of the mineral sorbents was checked by powder XRD analysis, but

scanning electron microscopy/energy dispersive spectrometry (SEM/EDXS) has

ability to obtain both morphological information and the elemental composition of the

particles. Recently, SEM/EDXS systems have become automated, making automated

GSR by computer- controlled SEM the method of choice for most laboratories

conducting this analysis. Morphology of soil was studied by Scanning Electron

Microscope with a device for Energy Dispersive X-ray Spectroscopy (SEM-EDX

System). In the present study, JEOL (JSM - 840 A) scanning electron microscope

(SEM) was used to study the morphology of the samples collected from the sampling

stations.

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2.12. X-ray diffraction

In order to identify qualitatively, the mineral composition, XRD spectra of soil

sample of each location were taken. These spectra indicate the type of minerals

present in the samples.

XRD Spectrograph (model MiniFlex™ II benchtop XRD system) was used for

the analysis and identification of various types of mineral in the soil samples. The

scanning was done in the range 10o to 80

o with copper target at 2 and 4 kilocycles per

second (kcs) speed.

2.13. Speciation of Heavy metal in Sediment/Soil samples

Analysis using acid digestion allows ascertaining the total content of heavy

metal contamination. It is insufficient to assess the environmental impact of the

contaminated sludge or sediment as the chemical form of the metal is not known. The

geochemical behaviour of trace metals and their chemical forms can be ascertained

with the help of Sequential extraction procedures.

In the light of the importance of metal speciation, it is vital to find the species

of metals in the soil and sediments collected from the study area. This will help to

understand their bioavaialibility and toxicity to aquatic environment. The study of

speciation of few heavy metals like Cadmium, Chromium, Copper, Zinc, Nickel,

Lead, Iron and in soil and sediment samples has been carried adopting Tessier

et.al.,(1979) procedure.

2.13.1 Multi-step sequential extraction.

The sequential extraction procedure used in this study is Tessier et al. (1979)

method. According to Tessier et al. heavy metals are associated with the fractions as

described as follows:

The exchangeable fraction (F1), which is likely to be affected by changes in

water ionic composition as well as sorption–desorption processes. The carbonate

fraction (F2), that is susceptible to changes in pH. The reducible fraction (F3), that

consists of iron and manganese oxides which are unstable under anoxic conditions.

The organic fraction (F4), that can be degraded leading to a release of soluble metals

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under oxidizing conditions and the residual fraction (F5) that contains mainly primary

and secondary minerals, which may hold metals within their structure. These metals

are not expected to be released in solution over a reasonable time span under the

conditions normally encountered in nature. This fraction was calculated as the

difference between the total metals and the sum of extracted metals.

The extraction was carried out progressively on an initial mass of 1.00 g of

sample of soil samples. The samples for sequential extraction were dried in an oven at

60◦C for 24 h in order to avoid, as far as possible, the transformation of some chemical

forms (exchangeable and carbonate). The selective extractions were conducted in 50

ml capacity centrifuge tubes. After each extraction step, the sample was subjected to

30 min of centrifugation at 4,000 rpm, the supernatant was separated from the residue

with a pipette and transferred into a 25-ml calibrated flask. The residue was

centrifugation and later washed thoroughly, the obtained second supernatant was

added to the flask, which was diluted to the desired volume. The extracts obtained

were acidified using aquaregia and stored in stopper polyethylene vessels until their

analysis by using inductively coupled plasma atomic emission spectroscopy

techniques (ICP-AES). The total content of metals was determined after digesting 0.4

g of sample with aquaregia. The concentration of particular heavy metals was

expressed per 1 kg of air dry sample. The content of heavy metals in the obtained

solution was determined by using ICP-AES.

This procedure is having five steps fractionization.

Fraction 1- Exchangeable Fraction

Samples (1g) of soil were extracted at room temperature for 1 hour with 16mL

of magnesium chloride solution (1M MgCl2) at pH 7. Soil and extraction solution

were thoroughly agitated throughout the extraction. This is mainly an adsorption-

desorption process. Metals extracted in the exchangeable fraction include weakly

adsorbed metals and can be released by ion-exchange process. Changes in the ionic

composition of the water would strongly influence the ionic exchange process of

metal ions with the major constituents of the samples like clays, hydrated oxides of

iron, and manganese. The extracted metals were then decanted from the residual soil.

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Fraction 2- Bound to Carbonates.

The metals bound to carbonate phase are affected by ion exchange and

changes of pH. The residue of Fraction 1 was extracted with 16 mL of 1 M sodium

acetate/acetic acid buffer at pH 5 for 5 hours at room temperature. Significant amount

of trace metals can be coprecipitated with carbonates at the appropriate pH. The

extracted metal solution was decanted from the residual soil. The residual soil was

used for the next extraction.

Fraction 3- Bound to Oxides:

The residue from fraction 2 was extracted under mild reducing conditions.

13.9g of hydroxyl amine hydrochloride (NH2OH·HCl) was dissolved in 500 mL of

distilled water to prepare 0.4M NH2OH·HCl. The residue was extracted with 20 mL

of 0.4M NH2OH·HCl in 25% (v/v) acetic acid with agitation at 96°C in a water bath

for 6 hours. Iron and manganese oxides which can be present between particles or

coatings on particles are excellent substrates with large surface areas for absorbing

trace metals. Under reducing conditions, Fe (III) and Mn (IV) could release adsorbed

trace metals. The extracted metal solution was decanted from the residual soil which

was used for the next extraction.

Fraction 4- Bound to organics:

The residue from fraction 3 was oxidized as follows: 3mL of 0.02 M

HNO3 and 5mL of 30% hydrogen peroxide, which has been adjusted to pH 2, was

added to the residue from fraction 3. The mixture was heated to 85°C in a water bath

for 2 hours with occasional agitation and allowed to cool down. Another 3mL of 30%

hydrogen peroxide, adjusted to pH 2 with HNO3, was then added. The mixture was

heated again at 85°C for 3h with occasional agitation and allowed to cool down. Then

5mL of 3.2M ammonium acetate in 20% (v/v) nitric acid was added, followed by

dilution to a final volume of 20mL with de-ionized water. Trace metals may be bound

by various forms of organic matter, living organisms, and coating on mineral particles

through complexation or bioaccumulation. These substances may be degraded by

oxidation leading to a release of soluble metals. The extracted metal solution was

decanted from the residual soil which was used for the next extraction.

61

Fraction 5- Residual or Inert fraction:

Residue from Fraction 4 was oven dried at 105°C. Digestion was carried out

with a mixture of 5 mL conc. HNO3 (HNO3, 70% w/w), 10 mL of hydrofluoric acid

(HF, 40% w/w) and 10 mL of perchloric acid (HClO4, 60% w/w) in Teflon beakers.

Fraction 5 largely consists of mineral compounds, where metals are firmly bonded

within crystal structure of the minerals comprising the soil. To validate the procedure,

the instrument was programmed and it carried out metal detection by displaying three

absorbance readings and what was reported was the average.

2.14 Statistical analysis

Statistical analysis was carried out to find out the correlation between

quantitative variables. The Pearson correlation coefficients are corresponds to the

classical linear correlation coefficient. This coefficient is well suited for continuous

data. Its value ranges from -1 to +1, and it measures the degree of linear correlation

between two variables. It gives the relation of the variability of a variables. The result

obtained from the physico-chemical analysis and total heavy metal concentrations

were tabulated. One way ANOVA application has been adopted to determine the

mean, average and significance between the metals. Pearson’s correlation matrix has

been followed to find out the correlation between the physico-chemical parameters

and total heavy metals.

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