MATERIALS  

AND  
METHODS 

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 MATERIALS AND METHODS

Chemicals: Acetone, Mercuric chloride (Hgcl2), Potassium hydroxide (KOH), Sodium

chloride (NaCl), Sulphuric acid (H2So4), Sodium hydroxide (NaoH), Cupric sulphate,
Arsenomolybdate, Sodium arsenate, Ammonium molybdate, Ethanol (C2H5OH),
Hydrochloric acid (HCl), Ninhydrin, Proponal, Pyridine, 1,1,3,3-tetraehoxy propane, NBT,
Ethylene diame tetra acetate (EDTA), Trichloro acetic acid (TCA).

Oryza sativa L. and Triticum vulgare L. seeds were obtained from Nagarjuna Ranga
agricultural regional research station Tirupati, Andhra Pradesh, India. Seeds were surface
sterilized with 0.1% Hgcl2 solution for 10 min and rinsed with double distilled water. Seeds
were sown in earth pots (30 cm x 25 cm, diameter and deep) containing red soil with Pharma
and battery industrial effluent. The industrial effluent samples Pharma and Battery were
collected near the Amaraja Battery industry as well as Gajulamandyam of Renigunta. The
pots were kept under natural photo radiation and each pot contained 20 seedlings. The pot
culture of rice was analyzed the Pigmental and biochemical changes of growth and yielding
of three interval of 10th, 20th and 30th day experimental period.

Pot culture experiments

Rice (Oryza sativa L.) and Wheat (Triticum vulgaries L.) cultivar plants were grown
in pots in untreated soil (control) and in soil to which industrial effluents had been applied.

Biochemical analysis

Chlorophyll ‘a’, chlorophyll ‘b’, total chlorophyll, carotene, total sugars, starch,
amino acids, proline and protein contents in plant samples were estimated by the following
methods.

Estimation of chlorophyll (Arnon, 1949):

Chlorophylls: The chlorophyll content was estimated according to the method of Arnon
(1949). About 1 gr of leaf sample was cut in to small pieces and homogenized in a pre-cooled
mortar and pestle using 80% (V/V) acetone. A pinch of calcium carbonate was added while
grinding. The extract was centrifuged at 3000 rpm for 15 min and made up to 25 ml with
80% (V/V) acetone. The clear solution was transferred to a colorimeter tube and the optical
density was measured at 645 nm and 663 nm, against an 80% acetone blank in shimadzu

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Double Beam spectrophotometer (UV 240).The levels of chlorophyll ‘a’ and chlorophyll ‘b’
were determined using the equation given below:

Chlorophyll ‘a’ (µ/g/ml) = (12.7 x O.D. at 663 nm) – (2.69 x O.D. at 645 nm)

Chlorophyll ‘b’ (µ/g/ml) = (22.9 x O.D. at 645 nm) - (4.08 x O.D. at 663 nm)

Total chlorophyll (µ/g/ml) = (20.2 x O.D. at 645 nm) + (8.02 x O.D. at 663 nm)

The chlorophyll content was expressed as mg chlorophyll per gram fresh weight of the leaf.

Carotenoids: Carotinoids were determined as per the method of Kirk and Allen, 1965. One
gram of freshly harvested leaf material made into small pieces was macerated with acetone in
a mortar. The extract was centrifuged and residue thus obtained was again reextracted
followed by centrifugation. The procedure was repeated until no more pigments were
extracted. The supernatants were pooled up and the acetone was removed in vacuo. The
residue after completed removal of acetone was dissolved in a small volume of 10% (UV)
methanolic KOH solution (10 ml final solutions for each mg of pigment). The mixture was
allowed to stand at room temperature for two hours and then transferred to a separating
funnel. The reaction was stopped by the addition of 5% (W/V) aqueous NaCl solution. After
separation, the aqueous phase was extracted thrice with petroleum ether and the combined
petroleum to remove methanol.

Estimation: the residue was dissolved in a known volume of acetone and O.D. was measured
at 445 nm in shimadzu double beam spectrophotometer (UV 240). The Carotinoids content
was calculated using the following equation.

C = d.v.f.10/2500

Where C = Carotinoids in mg

d = Optical density at the middle main maximum.

v = total volume in ml.

f = dilution factor for the sample actually measured.

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 Analysis of Heavy metals in Aqua regia extraction

The aqua regia extraction (1995) was based on the procedure recommended by the
International Organization for Standardization (ISO). In this procedure the soil sample intake
was of 0.1 gr and palnt sample was 0.5gr, which were placed in 250 ml Pyrex digestion tubes.
First, the pre-digestion step was done at room temperature for 16 h with 28 ml of 37% HCl:
◦
70% HNO3 (3:1) mixture. Then, the suspension was digested at 130 C for 2 h, in a reflux

condenser. The obtained suspension was then filtered through an ash less Whatman 41 filter,

diluted to 100 ml with0.5 moll−1 HNO3, and stored in polyethylene bottle sat 4◦C for

analyses.

Fig-3: Heavy metal analysis in soil and plant samples by ICP –OES Instruments

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 Fig-4: Seed germination of Oryza sativa L.

Fig-5: Control plant of Oryza sativa L.

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 th
10  Day 
th
20 Day

th
30  Day 

Fig-6: Pot culture experiments of Oryza sativa L.

Fig-7: Seed germination of Triticum vulgare L.

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 Fig-8: Control plant of Triticum vulgare L.

th th th
10  Day  20 Day 30  Day 

Fig-9: Pot culture experiment of Triticum vulgare L.

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Estimation of total sugars (Nelson, 1944):

A plant sample was treated with 80 per cent boiling ethanol for taking extractions (5
ml extract representing 1 gr of tissue). Five readings for each sample were taken. One ml of
ethanol extract taken in the test tubes was evaporated in a water bath. To the residue, 1 ml of
distilled water and 1 ml of 1 N sulphuric acid were added and incubated at 49 oC for 30 min.
The solution was neutralised with 1 N sodium hydroxide using methyl red indicator. One ml
of Nelson’s reagent was added to each test tube prepared by mixing reagent A and reagent B
in 25:1 ratio (Reagent A: 25 gr sodium carbonate, 25 gr sodium potassium tartarate, 20 gr
sodium bicarbonate and 200 gr anhydrous sodium sulphate in 1000 ml; Reagent B: 15 gr
cupric sulphate in 100 ml of distilled water with 2 drops of concentrated sulphuric acid). The
test tubes were heated for 20 min in a boiling water bath, cooled and 1 ml of arsenomolybdate
reagent (25 gr ammonium molybdate, 21 ml concentrated sulphuric acid, 5 gr sodium
arsenate dissolved in 475 ml of distilled water and incubated at 37oC in a water bath for 48 h)
was added. The solution was thoroughly mixed and diluted to 25 ml and measured at 495 nm
in a spectrophotometer. The reducing sugar contents of unknown samples were calculated
from glucose standard.

Estimation of starch (Summner and Somers, 1949):

The ethanol insoluble residues taken from ethanol extraction were dried at 60oC for
48 h in an oven. Three ml of 6 N HCl was added to 200 mg of the powdered residue and
autoclaved at 100oC for an hour. The flask was cooled and the volume was raised to 25 ml
with distilled water. One ml of aliquot was withdrawn, neutralized with 1 N NaoH and sugar
was estimated by Nelson’s (1944) method. The amount of starch was arrived by multiplying
the sugar by the factor 0.9.

Estimation of total free amino acids (Moore and Stein, 1948):

One ml ethanol extract was taken in 25 ml test tubes and neutralized with 0.1 N
sodium hydroxide using methyl red. One ml of ninhydrin reagent was added (800 mg
stannous chloride in 500 ml citrate buffer, PH 5.0, 20 gr ninhydrin in 500 ml methyl
cellosolve; both solutions were mixed). The contents were boiled in a water bath for 20 min,
5 ml of diluent solution (distilled water and n-propanol mixed in equal volume) was added,

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cooled and diluted to 25 ml with distilled water. The absorbance was measured at 570 nm in a
spectrophotometer. The standard graph was prepared using leucine.

Proline estimation

Proline content in leaves was determined according to Bates et al., (1973). Fresh
leaves (1gr) were extracted with 0.1 mol/L sulfosalicylic acid and centrifuged at 5000 × gr for
30 min. The proline content of the supernatant was measured at 520 nm, calculated from the
standard curve and expressed as μg/gr fresh weight. Free amino acids were extracted from
fresh leaves with 80% ethanol and determined using a standard ninhydrin assay. Quantitative
estimation of free amino acids was made according to Lee and Takahashi, (1966). H2O2
content was determined at 570 nm using 5% K2Cr2O7 and glacial acetic acid and expressed as
μmol/g fresh weight (Sinha, 1972).

Measurement of tissue lipid peroxides

The levels of lipid peroxides in terms of MDA were determined by the method of
Okhawa et al., 1979.

A 10% tissue homogenate was prepared in 1.15% of KCl for lipid peroxidation. To
0.1ml of the tissue homogenate, added 0.2 ml of 8.1% SDS and 1.5 ml of 0.8% TBA. The
total volume was made up to 4ml with distilled water and the tubes were kept at 95°C for 60
min, and then cooled. To this added 1ml of distilled water along with 5ml of n-butanol-
pyridine mixture (15:1 v/v) and the contents were mixed vigorously. Then the tubes were
centrifuged at 4000 rpm for 10min and the colour of the organic layer was measured at 532
nm using colorimeter. A standard curve was plotted by taking 1, 1, 3, 3-tetraethoxy propane
as standard and the values of the test samples were obtained from the standard curve based on
their optical density values.

Antioxidant enzyme activities in Rice and Wheat plants

In order to study the impact of industrial effluents on the antioxidant enzyme activity
of the Rice and Wheat plants, the antioxidant enzyme activities were checked in the presence
and absence of Pharma and Battery industrial effluents. For this purpose, all experiments
were carried out 10, 20, and 30 days after the Pharma and Battery industrial effluents
treatment conditions employed. The antioxidant enzyme activities were measured in roots
and stems Rice and Wheat plants.

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Superoxide dismutase assay

The activity of SOD was analyzed by measuring its ability to inhibit the
photochemical reduction of Nitro blue tetrazolium (NBT) using the method of Beauchamp
and Fridovich, (1982). Three milliliters of reaction mixture containing 50 mM phosphate
buffer at PH 7.8, 13 mM methionine, 75 μM NBT, 2 μM riboflavin, 1.0 mM EDTA and 20 μl
enzyme extraction. Riboflavin was added last and the reaction was initiated by placing the
tubes 30 cm below 15 W fluorescent lamps. The reaction was started by switching on the
light and was allowed to run for 10 min. Switching off the light stopped the reaction and the
tubes were covered with black cloth. Non-illuminated tubes served as control. The
absorbance at 560 nm was read. One unit of SOD is the amount of extracts that gives 50%
inhibition of the rate of NBT reduction (El-Beltagi et al., 2012).

Catalase activity assay

Catalase activity (CAT; EC 1.11.1.6) was determined by consumption of H2O2 using

the method of Dhindsa et al. (1981). The reaction mixture (3 ml) contained 50 mM potassium
phosphate buffer at PH 7.0, 15 mM H2O2 and 50 ml enzyme extraction. The reaction was
initiated by adding the H 2 O2 . The consumption of H2O2 was monitored
spectrophotometrically at 240 nm (extinction coefficient 39.4 mM-1 cm-1) for 3 min (El-
Beltagi et al., 2012).

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

Fresh tissue weighing 0.5 gr was macerated in 20 per cent trichloroacetic acid using
mortar and pestle. The homogenate was then centrifuged at 600 rpm for 30 min and the
supernatant was discarded. Five ml of 0.1 N NaOH was added to the pellet and it was
centrifuged for 30 min. The supernatant was saved for the estimation of protein. To 0.5 ml of
the extract, 5 ml of copper reagent ‘C’ was added (Reagent C: mixture of reagents A and B in
the 50:1 ratio; Reagent A: 2 per cent Na2CO3 in 0.1 N NaOH; Reagent B: equal volume of 1
per cent CuSO4 and 2 per cent sodium potassium tartrate). The tubes were shaken well and
allowed to stand in dark for 10 min at room temperature, 0.5 ml of properly diluted Folin-
Ciocalteau reagent was added to the solution and mixed thoroughly. The absorbance was read

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at 500 nm in a spectrophotometer against an appropriate blank. Bovin serum albumin was
used as the standard.

STATISTICAL ANALYSIS:

Data presented are the averages of three replicates, obtained from two independent
experiments. The experiments were performed in a factorial completely randomized design.
The data from the experiments were subjected to two-way ANOVA followed by separation
of means at Standard Devition of each mean was calculated to represent that on the tables and
graphs.

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