3. MATERIALS AND METHODS

The present investigation was carried out to findout the

effect of different concentrations of lead acetate in soil on seed

germination, growth, yield responses, biochemical and mineral contents

of groundnut, and also the effect of suitable soil amendments in

ameliorating the lead toxicity in soil and enhance the overall responses

of ground.

The materials used and the methods followed in the

present investigation to meet the objectives of the present study are

described below.

3.1. MATERIALS

3.1.1. Seed and plant materials

Groundnut seeds were obtained from the “Oil Seed

Regional Research Station”, Virudhachalam, Tamil Nadu, India. Healthy

seeds were chosen and used for both laboratory and field experiments.

The experimental field was setup in B. Mutlur, Chidambaram, Cuddalore.

The vermicompost was obtained from the Department of

Zoology, Annamalai University, and the farmyard manure (FYM) was

obtained from local farmers and they were used for soil amendments.

3.2. PREPARATION OF LEAD ACETATE CONCENTRATIONS

Different concentrations (10, 25, 50, 75, 100 and 200 ppm)

of lead acetate solution were prepared freshly by using tap water and

they were used for treatments.

3.3. GERMINATION STUDY

The healthy seeds of groundnut were surface sterilized with

0.1 per cent Mercuric chloride for 2 min and washed thoroughly with

tap water followed by distilled water. Seeds of groundnut were arranged

equispacially in petridisc with filter paper. They were irrigated uniformly

with equal volume (10 mL) of different concentrations (10, 25, 50, 75,

100 and 200 ppm) of lead acetate. The seeds irrigated with tap water

were treated as control. They were allowed to grow for a week. Three

replications were maintained for this experiment.

3.3.1. Germination percentage

The number of seeds germinated in each treatment was

counted on each and every day and it was last upto 7th day after sowing.

The total germination percentage was calculated by using the

following formula:

Germination percentage = 100 sownseedsofnumberTotal

germinated seeds of number Total

3.4. FIELD EXPERIMENT

Experimental period : January 2010 to April 2010

Experimental design : Randomized block design (RBD)

Plot size : 1 m 1 m

Treatments : Control Lead acetate treatments

Replications : 3

Lead concentrations : 10, 25, 50, 75, 100 and 200 ppm

Sampling days : 30, 60, 90 and 105 days

Parameters studied

Morphological Parameters : Plant height, total leaf area, total

fresh weight, dry weight and yield

components

Biochemical Parameters : Chlorophylls, carotenoid, protein,

aminoacid, sugars, starch, proline,

leghemoglobin.

Enzymes : Catalase, proxidase, polyphenol

oxidase, -amylase, -amylase,

ascorbic acid.

Minerals : pH, EC, micro and macro nutrients

3.5. FIELD PREPARATION

The field was thoroughly ploughed three times before sowing.

The entire field was irrigated for two days before sowing. Groundnut

seeds were sown with a spacing of 15 15 cm.

3.6. IRRIGATION SCHEDULE

The water irrigated experimental plots were kept as control.

First irrigation was done to all plots two days before sowing. The

different concentrations (10, 25, 50, 75, 100 and 200 ppm) of lead

acetate were prepared with tap water. Equal volume of tap water as

well as diluted lead was irrigated.

3.7. MORPHOLOGICAL PARAMETERS

Plant samples were collected randomly at various stages

(30, 60, 90 and 105 DAS) of its growth and used for recording

morphometrical observations like shoot length, root length, total leaf

area, fresh weight, dry weight and yield components of study crop.

Five plants were selected from each concentration including control

for recording the various morphological parameters.

3.7.1. Shoot length and root length

Five seedlings were collected on 30, 60, 90 and 105 DAS.

Their heights were measured by using cm scale and recorded. In another

experiment, shoot length and root length were measured and recorded.

3.7.2. Total leaf area

The leaf area was calculated by measuring the length and

width of the leaf as described by Yoshida et al. (1972).

Leaf area (cm2) = K length breadth

Where K = Kemp’s constant (for dicot leaves 0.66)

3.7.3. Fresh and dry weight

The plant samples taken for morphometric studies were

used for determination of fresh weight and dry weight. They were dried

in a hot air oven at 80 C for 24 hrs and their dry weight was determined

by using electrical single pan balance.

3.8. YIELD AND YIELD COMPONENTS

Five plants were used for recording the various yield

parameters like number of pods per plant, number of seeds per plant,

seed weight and total yield.

3.9. BIOCHEMICAL ANALYSES

The photosynthetic pigments such as chlorophyll and

carotenoid and the biochemical contents such as sugars, starch,

amino acids and protein were estimated in the seventh day old

seedlings of groundnut grown in laboratory.

3.9.1. Chlorophyll (Arnon, 1949)

Five hundred mg of fresh leaf material was ground with a

mortar and pestle using 10 mL of 80 per cent acetone. The homogenate

was centrifuged at 800 rpm for 15 min. The supernatant was saved.

The residue was re-extracted with 10 mL of 80 per cent acetone.

The supernatant was saved and the absorbance values were read at

645 and 663 nm in a UV-Spectrophotometer (Hitachi). The chlorophyll ‘a’,

chlorophyll ‘b’ and total chlorophyll contents were estimated and

expressed in mg/g on fresh weight basis.

Chlorophyll ‘a’ = (0.0127) (O.D 663) – (0.00269) (O.D 645)

Chlorophyll ‘b’ = (0.0229) (O.D 645) – (0.00488) (O.D 663)

Total chlorophyll = (0.0202) (O.D 645) + (0.00802) (O.D 663)

3.9.2. Carotenoid (Kirk and Allen, 1965)

The same extract was used for chlorophyll and carotenoid

estimation. The acetone extract was read at 480 nm in a UV-

Spectrophotometer. The carotenoid content was calculated by using

the following formula and it is expressed in mg/g fresh weight.

Carotenoid = (O.D 480) – (0.114) (O.D 663) – (0.638) (O.D 645)

3.9.3. Estimation of protein (Lowry et al., 1951)

Five hundred mg of plant material was weighed and macerated

in a pestle and mortar with 10 mL of 20 per cent trichloroacetic acid.

The homogenate was centrifuged for 15 min at 600 rpm. The

supernatant was discarded. To the pellet, 5 mL of 0.1 N NaOH was

added and again centrifuged for 5 min. The supernatant was saved

and made upto 10 mL with 0.1 N NaOH. This extract was used for the

estimation of protein.

One mL of the extract was taken in a 10 mL test tube and

5 mL of reagent ‘C’ was added. The solution was mixed and kept in

darkness for 10 min. Later, 0.5 mL of folin-phenol reagent was added

and the mixture was kept in dark for 30 min. The sample was read

at 660 nm in the UV-Spectrophotometer. The protein contents were

expressed in mg/g fresh weight.

Reagent A: 0.4 g of sodium hydroxide was dissolved in 100 mL of

distilled water. To this solution, 2 g of sodium carbonate was added.

Reagent B: One per cent of copper sulphate was mixed with equal

volume of 2 per cent sodium potassium tartarate.

Reagent C: 50 mL of reagent A and 1 mL of reagent B were taken and

mixed and it was prepared freshly at the time of experiment.

Folin-phenol reagent: One mL of folin-phenol reagent was diluted

with 2 mL of distilled water.

3.9.4. Estimation of amino acids (Moore and Stein, 1948)

Five hundred mg of plant material was ground with 10 mL

of 80 per cent ethanol in pestle and mortar. The homogenate was

centrifuged for 10 min at 800 rpm. The supernatant was saved and

used for the estimation of amino acids.

One mL of the extract was pipetted out into a test tube.

A drop of methyl red indicator was added. The sample was neutralized

with 1 mL of 0.1 N sodium hydroxide. To this, 1 mL of ninhydrin

reagent was added and mixed thoroughly. The whole content in the

test tube was heated for 20 min in a boiling water bath and with that

five mL of diluted solution was added and heated in water bath for

10 min. The tubes were cooled under the running tap water and the

contents were mixed thoroughly. Blank was prepared without extract.

The absorbance was read at 570 nm in a UV-Spectrophotometer. The

amino acid content was expressed in mg/g fresh weight.

Eight hundred g of hydrated stannous chloride was dissolved

in 500 mL of citrate buffer with pH 5.0 and 20 g of recrystallized

ninhydrin was dissolved in 500 mL of methyl cellosolve. Then these

two solutions were mixed together.

3.9.5. Estimation of sugars (Nelson, 1944)

Five hundred mg of plant material was ground with 10 mL

of 80 per cent ethanol in a pestle and mortar. The homogenate was

centrifuged for 10 min at 800 rpm. The supernatant was saved. Then,

the ethanol was evaporated in a water bath at 50 C. The net content

was made upto 20 mL with distilled water and the extract was used

for the estimation of reducing sugar.

One mL of extract was taken in a 25 mL test tube. 1 mL of

reagent ‘C’ was added. Then, the mixture was heated for 20 min at

100 C in a boiling water bath, cooled and 1 mL of arsenomolybdate

reagent was added. The solution was thoroughly mixed and diluted to

25 mL with distilled water. The sample was read in a UV-Spectrophotometer

at 520 nm. The sugar contents were expressed in mg/g fresh weight.

Reagent A: Twenty five gram of anhydrous sodium carbonate, 25 g of

sodium potassium tartarate, 20 g of sodium bicarbonate and 200 g of

anhydrous sodium sulphate were dissolved in 800 mL of distilled

water and made upto 1000 mL. Then, it was filtered and stored in a

glass stoppered brown bottle.

Reagent B: Fifteen per cent copper sulphate containing 1 or 2 drops

of concentrated sulphuric acid.

Reagent C: Fifty mL of reagent A and one mL of reagent B were mixed

well and it was prepared freshly at the time of experiment.

Arsenomolybdate reagent: To 450 mL of distilled water, 25 g of

ammonium molybdate, 21 mL of concentrated sulphuric acid were

added and 3 g of sodium arsenate was dissolved in 25 mL of distilled

water. The mixture was kept in a water bath at 37 C for 24 to 48 hrs.

The reagent was stored in a glass stoppered brown bottle.

3.9.6. Non-reducing sugars (Nelson, 1944)

Non-reducing sugars present in the ethanol extracts

(extraction as in reducing sugars) were hydrolysed with sulphuric acid

to reducing sugars. Reducing sugars present in the hydrolysates were

estimated following Nelson’s method. The differences between the total

sugars and the reducing sugars correspond to the non-reducing sugars.

One mL of extract was taken in a test tube and evaporated

to dryness in a water bath for 15 min. To the residue, 1 mL of distilled

water and 1 mL of 0.1 N sulphuric acid were added. The mixture was

hydrolysed by incubating at 49 C for 30 min in a thermostat. The

solution was neutralized with 0.1 N NaOH (5 mL) and the methyl red

as indicator. To this, 1 mL of reagent C (copper reagent) was added

and heated for 20 min, cooled and 1 mL of arsenomolybdate reagent

was added. The content was made upto 25 mL and the absorbance

was read at 495 nm in a UV-Spectrophotometer. The reducing sugar

contents were expressed in mg/g fresh weight. Blank was prepared

with 1 mL of distilled water.

3.9.7. Extraction and estimation of starch (Dubois et al., 1956)

Five hundred mg of plant material was macerated in a

pestle and mortar with 10 mL of 80% ethanol. The sample was

centrifuged at 6000 rpm for 15 min. The supernatant was removed

and the pellets was dissolved with 52% perchloric acid for 30 min

at 0 C. The extract was centrifuged and supernatant was diluted upto

15 times. One mL of diluted sample was mixed with 2 mL of cold

anthrone reagent in ice bath and it was boiled for 10 min at 100 C in

a water bath. The content was cooled and the absorbance was read at

630 nm in a UV-Spectrophotometer. The starch was calculated by

multiplying with 0.9 to the values obtained from standard curve. The

starch contents were expressed in mg/g fresh weight.

3.9.8. Amylases

Amylase activities were estimated following the method of

Tarrago and Nicolas (1976).

One gram of plant material was homogenized in a

prechilled pestle and mortar with 10 mL of cold distilled water at 4 C.

The homogenate was centrifuged at 15,000 g for 30 minutes in a

cooling centrifuge at 4 C. The supernatant was saved and used as

enzyme source for estimating - and -amylase activities.

3.9.8.1. -amylase (1, 4, D-glucanglucanohydrolase EC: 3.2.1.1)

3 mM calcium chloride was added to 5 mL of enzyme extract

and heated for 5 minutes at 70 C to inactivate -amylase activity.

-amylase is stable during heating. This heated extract was used to

estimate the -amylase activity.

To 1 mL of 0.1 M citrate buffer (pH 5.0) and 0.5 mL of

2 per cent soluble starch (freshly prepared) solution was added with

0.5 mL of the heated enzyme extract. The enzyme was allowed to react

for 5 minutes at 30 C. The reaction was terminated at the end of

5 minutes by adding 2 mL of colour reagent. The mixture was heated

for 5 minutes in a water bath at 50 C. After cooling, the final volume

of the solution was made up to 10 mL with distilled water. The

absorbance was read at 540 nm in a spectrophotometer. A calibration

curve was established with maltose and used to estimate the maltose

released during the reaction.

3.9.8.2. -amylase (1, 4, D-glucanmaltohydrolase EC: 3. 2. 1. 2)

-amylase activity was estimated at low pH and with 0.1 M

EDTA to inactivate -amylase (Tarrago and Nicolas, 1976).

To one mL of 0.1 M citrate buffer (pH 3.4), 0.5 mL of 2 per cent

soluble starch was added with 0.5 mL of EDTA treated enzyme extract.

The reaction was allowed for 5 minutes, after the addition of starch at

30 C. After 5 minutes, the reaction was stopped by adding 2 mL of

colour reagent. The mixture was heated for 5 minutes in a water bath

at 50 C. After cooling, the final volume was made up to 10 mL with

distilled water. The absorbance was read at 540 nm in a spectrophotometer.

-amylase and -amylase activities are expressed in terms of

units = micrograms of maltose liberated per min per mg enzyme protein.

Colour reagent

One gram of 3, 5-dinitrosalicylic acid, 200 mg of crystalline

phenol and 50 mg of sodium sulphate were dissolved in 20 mL of

2 per cent sodium hydroxide and 40 grams of potassium tartrate. The

final volume was made up to 100 mL with distilled water and filtered,

freshly prepared reagent was used.

3.10. DETERMINATION OF PROLINE CONTENT

Proline was extracted and estimated by following the

method of Bates et al. (1973).

Five hundred milligram of fresh plant material was homogenized

in a mortar and pestle with 10 mL of 3% aqueous sulfosalicyclic acid.

Then the homogenate was filtered through Whatman No.1 filter paper.

The residue was re-extracted and pooled and the filtrates were made

upto 20 mL with aqueous sulfosalicylic acid and this extract was used

for the estimation of proline.

To 2 mL of proline extract, 2 mL of acid ninhydrin and

2 mL of glacial acetic acid were added. The mixture was incubated for

an hour at 100 C in a boiling water bath. Then the test tubes were

transferred to an ice bath to terminate the reaction. Then 4 mL of

toluene was added and mixed vigorously using a test tube stirrer for

20 seconds and the toluene containing the chromophore was separated

from the aqueous phase with the help of a separating funnel and the

absorbance was measured at 520 nm in a spectrophotometer using a

reagent blank. The proline content was determined from a standard

curve with proline and the results were expressed in milligrams per

gram dry weight.

Acid-ninhydrin reagent

To 1.25 gms of ninhydrin, 30 mL warm glacial acetic acid,

20 mL of 6 M phosphoric acid were added with agitation.

3.11. ESTIMATION OF LEGHAEMOGLOBIN CONTENT OF ROOT NODULES

(Sciffman and Lobel, 1970)

Tris-acetic acid buffer: 0.1 N acetic acid (6.0 g/l) is adjusted to

pH 4.0 with 0.2 tris M (hydroxy methane) methylamine (24.38 g/l).

Benzidine reagent: One hundred mg of benzidine was added to 0.5 mL

of hydrogen peroxide and the volume was made upto 50 mL.

The nodules were washed and weighed. They were crushed

in tris-acetic acid buffer. The extract was centrifuged at 3000 g for

20 min. and 0.1 to 1.0 mL of supernatant was taken and it was made

upto final volume of 4.0 mL by tris-acetic acid buffer so as to get an

absorbance reading between 0.2 and 0.4. Later, 2 mL of freshly prepared

benzidine reagent was added. The rate of colour formation was noted by

observing the change in optical density by using UV-spectrophotometer

at 540 nm. A standard graph was prepared by plotting the absorbance at

the end of 30 second against different concentrations (0.8 to 1.5 g per mL)

of the ox-blood haemoglobin. The leghaemoglobin content of the test

sample was calculated from the standard graph and expressed in

mg g of nodules on fresh weight basis.

3.12. ENZYMES

3.12.1. Catalase activity (CAT, EC: 1.11.1.6)

Catalase activity was assayed as described by Chandlee

and Scandalios (1984).

Five hundred milligram of frozen material was homogenized

in 5 mL of ice-cold 50 mM sodium phosphate buffer (pH 7.5) containing

1mM PMSF. The extract was centrifuged at 4 C for 20 minutes at

12,500 rpm. The supernatant was used for enzyme assay.

The activity of enzyme catalase was measured using the

method of Chandlee and Scandalios (1984) with modification. The

assay mixture contained 2.6 of 50 mL of 50 mM potassium phosphate

buffer (pH 7.0) 0.4 mL, 15 mM H2O2 and 0.04 mL of enzyme extract.

The decomposition of H2O2 was followed by the decline in absorbance

at 240 nm. The enzyme activity is expressed in units 1 mM of H2O2

reduction per minute per mg protein.

3.12.2. Peroxidase activity (POX, EC: 1.11.1.7)

Peroxidase was assayed by the method of Kumar and Khan (1982).

Assay mixture of Peroxidase contained 2 mL of 0.1 M phosphate buffer

(pH 6.8), 1 mL of 0.01 M pyrogallol, 1 mL of 0.005 M H2O2 and 0.5 mL

of enzyme extract. The solution was incubated for 5 min at 25 C after

which the reaction was terminated by adding 1 mL of 2.5 N H2SO4.

The amount of purpurogallin formed was determined by measuring

the absorbance at 420 nm against a blank prepared by adding the

extract after the addition of 2.5 N H2SO4 at zero time. The activity is

expressed in unit mg-1 protein. One unit is defined as the change in

the absorbance by 0.1 min-1 mg-1 protein.

3.12.3. Polyphenol oxidase (o-diphenol: O2 oxidoreductase, EC. 1.10.3.1)

Polyphenol oxidase activity was assayed by the method of

Kumar and Khan (1982).

Assay mixture for polyphenol oxidase-contained 2 mL of

0.1 M phosphate buffer (pH 6.0), 1 mL of 0.1 M catechol and 0.5 mL of

enzyme extract. This was incubated for 5 minutes at 25 C, after which

the reaction was stopped by adding 1 mL of 2.5 N sulphuric acid.

The absorbance of the purpurogallin formed was read at 495 nm.

To the blank 2.5 N sulphuric acid was added at the zero time of the

same assay mixture. The enzyme activity was expressed in units = 0.1

absorbance per minute per mg protein.

3.12.4. Ascorbic acid activity

Ascorbic acid was extracted and estimated by the method

of Omaye et al. (1979).

One gram of plant tissue was homogenized in a pestle and

mortar with 5 mL of 10 per cent trichloroacetic acid (TCA), and centrifuged

at 3500 g for 20 minutes. The pellet was re-extracted twice with

10 per cent TCA and supernatant was made to 10 mL and used as extract.

To one mL of dinitrophenylhydrazine; thiourea and copper

sulphate reagents were added to 0.5 mL of extract and mixed thoroughly.

Then the tubes were incubated at 37 C for 3 hours and to this 0.75 mL of

ice cold 65 per cent sulphuric acid was added. The tubes were then

allowed to stand at 30ûC for 30 minutes. The resulting colour was read

at 520 nm in a Spectrophotometer (U-2001– Hitachi). The ascorbic

acid content was determined using a standard curve prepared with

ascorbic acid and the results are expressed in mg per gram dry weight.

DTC reagent

To 3 gram of 2,4-dinitrophenylhydrazine (DNPH), 0.4 grams

of thiourea and 0.05 gram of copper sulphate in100 mL of 9N

sulphuric acid. Standard solution 10 mg/100 mL 10 per cent TCA.

3.13. MINERALS

Both macronutrients (N, P, K, Ca and Mg) and micronutrients

(Zn, Cu and Fe, Mn, Pb) were analysed and recorded on 30, 60, 90

and 105 DAS. They were estimated by following the methods mentioned

earlier in germination studies.

3.13.1. Total nitrogen (Jackson, 1958 quoted by Yoshida et al., 1972)

Two hundred mg of dried and powered plant sample was

taken in a 100 mL Kjeldahl flask. Two hundred mg of salt mixture

(potassium sulphate, cupric sulphate and selenium powder mixed in

the ratio of 50:10:1) and 3 mL of concentrated sulphuric acid was

added. After digestion, 10 mL of distilled water was added and cooled.

The diluted sample was decanted into the micro-Kjeldahl

distillation flask. To that, 10 mL of 40 per cent sodium hydroxide was

added and distilled. The distillate was collected in a conical flask

containing 10 mL of 4 per cent boric acid and 3 drops of mixed

indicator (0.3 g bromocresol green and 0.2 g methyl red in 400 mL of

90 per cent ethanol). This solution was titrated against 0.05 N HCl.

Nitrogen content was estimated using the following formula,

1000 weight Sample

10014 HCl of N litre)blank - titrate (Sample (%) Nitrogen

3.13.2. Phosphorus (Black, 1965 quoted by Yoshida et al., 1972)

One gram of dried and powdered plant material was digested

with 10 mL of acid mixture (nitric acid 750 mL, sulphuric acid 150 mL

and perchloric acid 300 mL). The digest was cooled and made upto

50 mL and filtered. One mL of the digest was mixed with 2 mL of

2 N nitric acid and diluted to 8 mL. One mL of molybdovanadate

reagent (25 g of ammonium molybdate in 500 mL of water, 1.25 g of

ammonium vanadate in 500 mL of 1 N nitric acid, both were mixed in

equal volume) was added, shaken and the absorbance was measured

at 420 nm in UV-Spectrophotometer after 20 min. Calibration curve

was prepared using potassium dihydrogen phosphate as standard.

3.13.3. Potassium (Williams and Twine, 1960)

Dried and powdered plant material of 0.5 g was digested in

100 mL Kjeldahl flask using 10 mL of concentrated nitric acid, 0.5 mL

of 60% perchloric acid and 0.5 mL of sulphuric acid. The inorganic

residue was cooled and diluted with 15 mL of distilled water and

filtered through Whatman No. 42 filter paper. The filterate was made

upto 50 mL with distilled water. The filtrate was used for potassium

estimation by Flame photometer and standards were prepared with

potassium chloride.

3.13.4. Calcium, manganese and magnesium (Yoshida et al., 1972)

Two mL of the filterate was mixed with 2 mL of 5 per cent

lanthanum chloride solution and diluted with 10 mL of 1 N hydrochloric

acid. The solution was fed into an atomic absorption spectrophotometer

at 211.9 nm for calcium and 285.4 nm for magnesium. Standard

curve was prepared by using calcium chloride/magnesium chloride.

3.13.5. Copper, iron and zinc (De Vries and Tiller, 1980)

One mL of sulphuric acid and 15 mL of double distilled

water were added to a Kjeldahl flask containing 0.5 g of dried and powdered

material was incubated at 80 C for overnight. After that 5 mL of acid

mixture (nitric acid and perchloric acid in the ratio of 3:1) was added

and then digested. The digested material was cooled, made upto 50 mL

and filtered through Whatman No. 42 filter paper. The sample was

aspirated to an Atomic Absorption Spectrophotometer with air/ acetylene

flame and the readings were taken for iron (568 nm), copper (324.6 nm)

and zinc (214 nm).

3.14. SOIL ANALYSES

The soil samples were collected from each plot before

sowing and after harvesting and labelled separately. Their physico-

chemical properties such as pH, electrical conductivity, nitrogen,

phosphorus, potassium, copper, iron, zinc, magnesium, manganese lead

and calcium were estimated and recorded.

3.14.1. pH

Twenty gram of soil sample was air-dried and 50 mL of

distilled water was added and mixed well. The solutions were taken in a

beaker and the pH of the soil sample was recorded by using a pH meter.

3.14.2. Electrical conductivity

Twenty gram of dried soil sample was taken and dissolved

in 50 mL of distilled water and mixed well. The solution was used to

measure the electrical conductivity of the sample soil with the help of

an electrical conductivity meter.

3.14.3. Available nitrogen (Subbiah and Asija, 1976)

Twenty gram of the soil sample was taken in a flask and

20 mL of distilled water, 100 mL of freshly prepared 0.32 per cent

potassium permanganate solution and 100 mL of 2.5 per cent sodium

hydroxide were added. The flask was heated and 30 mL of distillate

was collected in 50 mL of N/50 sulphuric acid. Excess acid was

titrated against N/50 NaOH solution using methyl red indicator. The

amount of available nitrogen per cent in the soil was calculated by

using the following formula:

Available nitrogen (mg kg-1) =

1000

gram in soil of Weight

0.28 consumed NaOH N 0.02 of VolumeSOH N 0.02 of Volume 42

3.14.4. Available phosphorus (Jackson, 1958)

One gram of soil was suspended in 200 mL of 0.002 N

sulphuric acid, shaken well and then filtered through Whatman No. 42

filter paper. To 10 mL of filtrate, three drops of 0.02 per cent

2,4-dinitrophenol indicator were added. Whenever, the solution

became yellow, 2 N sulphuric acid was added until the disappearance

of the yellow colour. If the solution was colourless after adding the

indicator, 4 N sodium carbonate was added till it became colourless.

To that solution, 2 mL of sulphomolybdic acid (ammonium molybdate

25 g in 200 mL; 275 mL con. H2SO4 diluted to 700 mL both were

cooled, mixed and made upto 1000 mL) and 0.5 mL of chlorostannous

acid (25 g SnCl2.2H2O in 50 mL of concentrated HCl diluted to 500 mL

with water and made upto one litre with 1.2 N HCl) were added

and made upto 50 mL. The solution was shaken well and read in a

UV-Spectrophotometer at 660 nm after 5 min. Standard graph was

prepared using potassium dihydrogen phosphate.

3.14.5. Available potassium (Jackson, 1958)

Ten gram of soil was taken in 250 mL conical flask and

100 mL of 1 N ammonium acetate was added to it. The flask was

stoppered, shaken intermittently for 10 min and filtered by suction.

Ammonium acetate was poured to the soil to get a volume of 250 mL

and then evaporated to dry. Dried samples were ashed in a muffle

furnace at 700-800 C for 20-30 min. To the residue, 50 mL of 0.1 N HCl

was added and warmed gently and the extract was fed to Flame

photometer. Potassium chloride was used to prepare the standard solution.

3.14.6. Available calcium (Yoshida et al., 1972)

Five gram of soil was extracted with 50 mL of 1 N

ammonium acetate. Two mL of the extract was mixed with 2 mL of

5 per cent lanthanum oxide solution and diluted with 10 mL of

1 N HCl. The solution was fed into an atomic absorption spectrophotometer

at 211.9 nm. Standard solution was prepared using calcium chloride.

3.14.7. Available magnesium (Jackson, 1958)

Ten gram of soil sample was extracted with 50 mL of 1 N

ammonium acetate and the extract was filtered and used for the

determination of magnesium. The determination procedure was

adopted as in the case of calcium. The amount of magnesium was

estimated by using Atomic Absorption Spectrophotometer. Magnesium

chloride was used for the standard preparation.

3.14.8. Copper, iron and zinc, lead (Piper, 1966)

Fifty gram of soil was extracted with 100 mL of extraction

solution (diethylenetriaminepentaacetic acid – DPTA) and shaken thoroughly

for 2 hrs. The solution was filtered through Whatman No. 42 filter

paper. The filtrate was read at 568 nm for iron, 324.6 nm for copper

and 214 nm for zinc by using the appropriate hollow cathode lamps in

Atomic Absorption Spectrophotometer.

3.14.9. Estimation of manganese (De Vries and Tiller, 1980)

One mL of sulphuric acid and 15 mL of double distilled

water were added to a Kjeldahl flask containing 0.5 g of dried and

powdered material and incubated at 80 C for over night. After that

5 mL of acid mixture (nitric acid, 3: perchloric acid, 1) was added and

digested until the nitric acid and perchloric acid were driven off.

The digest was cooled, diluted, filtered through Whatman No.42 filter

paper and made up to 50 mL.

The solution was directly, aspirated to an Atomic Absorption

Spectrophotometer (Perkin – Elmer – 2280), with air/acetylene flame

for estimating for manganese.

3.15. REMEDIATION BY SOIL AMENDMENTS

3.15.1. Pot culture experiment

Pot culture experiments were conducted with groundnut to

find out the suitability of various soil amendments for getting higher

yield of crop grown under lead acetate polluted soil. Five kg of polluted

soil was taken in an earthen pot. The polluted soil mixed with some

soil amendments such as farmyard manure and vermicompost. The

following soil amendments were given to the polluted soil in pot

culture experiments.

T1 – Garden soil (Control)

T2 – 10 ppm + Lead acetate + Vermicompost

T3 – 25 ppm + Lead acetate + Vermicompost

T4 – 50 ppm + Lead acetate + Vermicompost

T5 – 75 ppm + Lead acetate + Vermicompost

T6 – 100 ppm + Lead acetate + Vermicompost

T7 – 200 ppm + Lead acetate + Vermicompost

The seeds of groundnut were sown in earthen pots. The

pots were irrigated once in a week. Three replicates were maintained for

this experiment. Five plant samples were randomly selected from each

and every treatment and they were used for recording various morphological,

growth and yield parameters as stated earlier in this section.

3.16. STATISTICAL ANALYSIS

The statistical analyses (standard deviation) of experimental

results were carried out as per the procedure given by Sockal and

Rohalf (1973).