3. MATERIALS AND METHODS
3.1 Construction of cement tanks
Brick work cement tanks were constructed under a tiled animal house at Govt Arts
College (Autonomous), Kumbakonam, for this vermiculture and vermicomposting study.
3.1.1 Large cement tanks
Six rectangular brick tanks were constructed using cement and sand (1:5 ratio). The
size is 180 x 75 x 90cm. These tanks were used for the partial decomposition of organic
materials selected for this study.
3.1.2 Small cement tanks
Similarly, 18 brick tanks of size, 8 x 52 x 36cm were also constructed for keeping the
stock adult earthworms.
3.2 Procurement of earthworms
The specimens of anecic adult live earthworm, L.mauritii with a body size of 10 to 14
cm in length and 700 to 900 mg in weight were collected from a cow dung pit at
Chettimandabam, Kumbakonam. Adult specimens of another epigeic earthworm,
P.excavatus having a body size of 10 to 12 cm in length and 600 to 800 mg in weight were
purchased from the vermiculture unit of Periyar Maniyammai University, Thanjavur, Tamil
Nadu.
3.2.1 Maintenance of adult earthworms
The above specimens were kept separately in tanks with substrate medium containing
50 % cow dung and 50 % soil. They were maintained at room temperature 28±5°C (medium
temperature 25±2°C) for the entire study period as suggested by Martin (1982). The tanks
were covered with cotton clothes to protect these earthworms from their predators. Sufficient
water was added in these tanks to ensure optimum growth and to maintain a safe moisture
condition (60-70 %).
27
3.3 Collection of organic materials
3.3.1 Water hyacinth
About 1000 kg of fresh water hyacinth were collected from the Chettimandabam pond
located at the junction of Kumbakonam bypass and Mayiladudurai – Kumbakonam NH road.
The plants were cut into smaller pieces (size : < 7 cm) for easy decomposition.
3.3.2 Paddy wastes
About 400 kg of dry paddy waste materials were collected from a paddy harvesting
site at Chettimandabam, Kumbakonam.
3.3.3 Fresh cow dung
Fresh cow dung of about 500 kg was collected from a local dairy farm at Autonagar
adjacent to Govt. Arts College (Autonomous), Kumbakonam.
3.4 Collection of soil
Air dried alluvium soil collected from the Cauvery riverbank adjacent to Govt. Arts
College (Autonomous), Kumbakonam, was manually powdered using stone mortar. It was
kept in polythene bags after passing through a iron sieve (size:1mm x 1mm) to obtain soil
media with particle size less than 1mm as suggested by Reinecke and Venter (1985).
3.5 Partial decomposition of organic materials
3.5.1 Water hyacinth
The first, second and third tanks were filled with cut pieces of water hyacinth. After
adding sufficient water, these tanks were covered with polythene sheets to avoid water
evaporation and a possible release of foul smell during decomposition. Water was regularly
added in these tanks after removing the polythene sheets for proper decomposition. Once in
six days, the decomposing materials were thoroughly mixed using a spade to ensure uniform
decomposition. Ideal semi decomposed water hyacinth can be available only after 90 days of
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decomposition. About 150 kg of dry (0 % moisture) semi decomposed water hyacinth can be
obtained during this process.
3.5.2 Paddy waste
The fourth and fifth tanks were filled with wetted paddy wastes for partial
decomposition. These tanks were covered with polythene sheets and maintained as per the
procedures followed in partial decomposition of water hyacinth. Ideal semi decomposed
paddy waste can be obtained only after 45 days of decomposition. About 150 kg of dry (0 %
moisture) partly decomposed paddy waste can be collected during this process.
3.5.3 Cow dung
The last tank was filled with fresh cow dung. It was covered with polythene sheets
for proper decomposition. At weekly intervals water was added if needed in the
decomposing tank after removing the polythene sheets. The cow dung in the tank was
thoroughly mixed using a spade to ensure uniform decomposition. Ideal semi decomposed
cow dung can be available only after 20 days of decomposition. About 150 kg of dry (0 %
moisture) semi decomposed cow dung can be collected from this process.
3.6 Separation of core particles
The partly decomposed organic materials collected were separately powdered using
thick wooden rod and sieved through a iron net (size:1mm x 1mm) to obtain medium with
particle size less then 1mm as suggested by Reinecke and Venter (1985).They were stored
separately in polythene bags for vermiculture and plant cultivation study.
3.7 Procurement of earthen pots
Three hundred earthen pots (size: 25 cm diameter and 24 cm height) were purchased
from Ammachatram, Kumbakonam, to carry out the vermiculture and plant cultivation study.
29
3.8 Preparation and maintenance of vermiculture media
An organic mixture of 1:1:1 ratio (volume / volume) was prepared using the above
three partly decomposed organic materials for the culture practices of earthworms along with
other organic materials. Six sets of seven doses, 100, 75, 50, 40, 30, 20 and10 percent
substrate ratios (PSR) were prepared from partly decomposed water hyacinth / paddy waste /
cow dung / organic mixture using dry soil with volume by volume basis. Four litres of each
substrate (each PSR dose) were taken in an earthen pot and sufficient water was added into it
to ensure optimum moisture condition (60 – 70 %) as suggested by Martin (1982). To assess
the rate of cocoon production in the above media, 12 adult earthworms of either L.mauritii or
P.excavatus were introduced into each pot. Six sets of control (soil alone as substrate)
experiment with 12 adult earthworms (L.mauritii or P.excavatus) were also maintained
simultaneously along with these media. In order to provide optimum moisture condition to
the experimental earthworms the media were regularly added with water. All these pots were
covered with cotton clothes to protect the earthworms from their predators (frog, ant, rat,
centipedes, millipedes and termites).
3.8.1 Collection of cocoons
Cocoons produced by earthworms in each organic matter were collected once in six
days and recorded for a period of 30 days. The body weight and survival of earthworms were
also measured / observed along with cocoon collection. Rate of cocoon production per day
was calculated.
3.8.2 Collection of vermicomposts
At the end of vermiculture study, the culture media used by earthworms were collected
as vermicomposts and stored separately in polythene bags. They were used for macro and
micronutrients analysis and for chilly plant cultivation.
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3.8.3 Maintenance of cocoons
The cocoons collected from different PSR media were placed in separate plastic cups
having the same PSR media. The cocoons kept in the plastic cups were covered with cotton
clothes. Sufficient water was added in the plastic cups to maintain the optimum moisture
condition (60 -70 %) for normal development of embryos in the cocoons .The hatching time
of cocoon was recorded individually.
3.9 Culture of F1 hatchlings
Twelve hatchlings in each PSR dose were taken immediately after hatching and
placed in the earthen pots containing the same PSR media.
Measurements of length and weight of growing hatchlings were made at 10 days
intervals until they developed into a clitellate stage.
To assess the efficiency of cocoon production by F1 earthworms cultured in the above
media, the mature F1 worms were allowed to reproduce for a further period of one month
after renewing fresh substrate. Maintenance of culture medium, protection of F1 earthworms
and collection of cocoons were followed as per the procedure adapted in the culture practices
of parent earthworms.
3.10 Calculation
Data collected from F0 and F1 earthworms were used to calculate the following
growth and reproductive parameters.
3.10.1 Percent Weight Change (PWC)
Final body weight - Initial body weight
PWC
=
––––––––––––––––––––––––––––––– × 100
Initial body weight
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3.10.2 Growth Rate (GR)
Final body weight (mg) - Initial body weight (mg)
GR (mg/day)
=
––––––––––––––––––––––––––––––––––––––––
Growth period (days)
3.10.3 Cocoon Production Rate (CPR)
Total cocoons produced ÷ No. of worms used
CPR (C/W/D) =
––––––––––––––––––––––––––––––––––––––
Period of cocoon collection (days)
3.10.4 Hatchling Production Rate (HPR)
Total hatchlings emerged from the cocoons
HPR (H/C)
=
––––––––––––––––––––––––––––––––––––
Total cocoons incubated
3.10.5 Percent Hatching Success (PHS)
No. of hatched cocoons
PHS
=
–––––––––––––––––––– × 100
(or)
Total cocoons incubated
Total cocoons incubated - Unhatched cocoons
PHS
= –––––––––––––––––––––––––––––––––––––– × 100
Total cocoons incubated
3.11 Physic - chemical analysis
The levels of pH, electrical conductivity (EC), macro nutrients (organic carbon, total
nitrogen, total phosphorus, total potassium, total sodium and total calcium) and micro
nutrients (iron, manganese, zinc and copper) present in the samples obtained from soil, partly
32
decomposed and vermicomposted organic materials (water hyacinth, paddy waste, cow dung
and a mixture of all the three organic materials) were measured / determined.
3.11.1 Determination of pH
The pH was determined by Potentiometric method of Jackson (1973).The term pH is
a notation used to express the acidity or alkalinity of the given sample.
Procedure
Twenty gm of organic sample was taken in a 100 ml beaker and added 50 ml water to
make the sample -water ratio as 1:2.5. The suspension was stirred well with rubber tipped
glass rod at regular intervals for 30 minutes. After warming up for 15-20 minutes, the pH
meter was set at zero with the help of zero set knobs. The electrodes were dipped into a
buffer solution of known pH. The pH reading was adjusted with the help of buffer set knob.
The electrodes were then dipped into the sample water suspension and recorded the pH.
Ratings of pH level in soil
Acidic: < 6.5
Normal: 6.5 – 7.5
Saline/calcareous: 7.5 – 8.5
Alkaline: > 8.5
3.11.2 Determination of EC
The EC was determined by Conductometric method of Jackson (1973). It is
expressed as reciprocal ohms per cm. As the values of EC for organic samples and the most
irrigation waters are very small, EC was expressed as deci Semiens per meter (dSm-1).
Procedure
Twenty gm of organic sample was transferred into a 100 ml beaker and added 50 ml
water. The suspension was thoroughly mixed and allowed to stand for half an hour. The
instrument was checked with saturated CaSO4 (EC 2.2 dSm-1) or 0.01 N KCl solutions (EC
33
1.41 dSm-1) before proceeding with the sample. The electrodes were washed with distilled
water and immersed into the sample suspension. The specific conductivity of the sample
solution was read after balancing the galvanometer
Ratings of EC level in soil
Harmless: 0.0-1.0 dSm-1
Injurious: 1.0-3.0 dSm-1
Critical : >3 dSm-1
3.11.3 Macronutrients analysis
3.11.3.1 Estimation of organic carbon (OC)
OC was determined by chromic acid wet digestion method of Walkley and
Black (1934).
Principle
OC in the sample was oxidized by chromic acid (formed during the reaction of
K2Cr2O7 with H2SO4) thereby releasing CO2. The unreduced chromic acid in this reaction
was determined by back titration with ferrous ammonium sulphate or ferrous sulphate
solution.
4Cr6+ + 3C
→
4Cr3+ + 3C4+
2H2Cr2O7 + 3C + 6H2SO4
→
2Cr2 (SO4)8 +3CO2 + 8H2O
2H2Cr2O7 + 6H2 + 6H2SO4
→
2Cr2 (SO4)3 + 14H2O
Procedure
Five hundred mg of finely powdered sample (after passing through 0.5 mm × 0.5 mm
sieve) was taken in a 500 ml conical flask and added 10 ml of 1 N potassium dichromate
solution and 20 ml of conc. H2SO4. The contents were thoroughly mixed and set aside on an
asbestos pad for half an hour. At the end of 30 minutes, 200 ml of distilled water, 10 ml of
phosphoric acid and 1 ml of diphenylamine indicator were also added. The blue colour
34
developed in the flask was titrated against 0.5 N ferrous ammonium sulphate solution until
the contents turn to green colour. Titration with reagent blank (without any sample) was also
carried out simultaneously.
Calculation
Volume of 1 N K2Cr2O7 taken
= 10 ml
Volume of 0.5 N ferrous ammonium
sulphate used in the blank titration
= X ml
Volume of 0.5 N ferrous ammonium
sulphate used in the sample titration
= Y ml
Volume of 1 N K2Cr2O7 required for
oxidizing the organic carbon
= (X-Y)/X × 10
1 ml of 1 N K2Cr2O7
= (0.003 gm of C)
Organic carbon in the sample (%)
= (X-Y)/X ×10×0.003×100/0.5
Percentage of organic matter
= % of organic carbon × 1.724
Ratings of OC level in soil
Low: < 0.5 %
Medium: 0.5 - 0.75 %
High: > 0.75 %
3.11.3.2 Estimation of total nitrogen (TN)
The levels of TN present in the samples were determined using Kjeldahl method of
Jackson (1973). This method was carried out by two steps (1) digestion of the given sample
to convert N compound to NH4+ form and (2) distillation and determination of NH4+ in the
digest.
35
Principle
Organic form of nitrogen was converted into ammonium sulphate by digestion with
conc. H2SO4. When this was distilled with excess alkali, ammonia was liberated. It was
absorbed in known excess of standard acid. The unused acid was determined by back
titration with standard alkali.
Procedure
Digestion of the sample
Depending upon the N content present in the organic material, 0.5 to 1.0 gm of sample
was taken into a Kjeldahl flask and added 30 ml of conc. H2SO4 containing 1.0 gm of
salicylic acid. The contents were thoroughly mixed and kept as such for 30 minutes. After 30
minutes, 5.0 gm of sodium thio sulphate was added and allowed to stand for 5 minutes. Then
it was gently heated and added 10 gm of potassium sulphate and 1.0 gm of copper sulphate.
The contents were digested till they turned into apple green colour.
Distillation
The contents in the digestion flask were transferred into a distillation flask without
any loss of solution. The Kjeldahl flask was rinsed with water three or four times and
transferred the washing also into the distillation flask. The volume of the contents in the
distillation flask, was made upto 300 ml and a few porcelain and zinc bits were added to it.
Then 120 to 150 ml of 40% NaOH and about 10 ml of 10% sodium sulphide were added into
it. After placing a beaker containing a known excess of 0.1 N H2SO4 with 2 to 3 drops of
methyl red, distillation process was started. After distillation, the content in the beaker was
back titrated the excess acid against 0.1 N KOH till the pink colour changed to straw yellow.
Nitrogen content was calculated from the volume of acid consumed.
36
Calculation
Weight of organic matter taken
= 1.0 gm
Volume of 0.1 N H2SO4 taken in
the beaker to absorb ammonia
= A ml
Volume of 0.1 N KOH consumed
in the back titration
= B ml
Actual volume of 0.1 N H2SO4
consumed
= (A-B) ml
1 ml of 0.1 N H2SO4
= 0.0014 gm of N
(A-B) ml 0.1 N H2SO4
= 0.0014 × (A-B) gm of N
1.0 gm of organic matter contains
= 0.0014 × (A-B) gm of N
3.11.3.3 Estimation of total phosphorus (TP)
The levels of TP present in the organic samples were determined using ammonium
phospho - molybdate method of Pemberton (1945).
Principle
Phosphorus was extracted by digestion with conc. HCl and HNO3. It was precipitated
as ammonium phospho-molybdate in nitric acid medium. The acid precipitate was filtered
and washed with water. After washing, the precipitate was dissolved in a known excess of
alkali. The excess alkali was back titrated against nitric acid using phenolphthalein as
indicator. From the volume of alkali consumed, total phosphorus was determined.
Reactions
The following equations showed the reaction between ammonium phospho molybdate and alkali.
Na2HPO4 +12 NH4MoO3 +23 HNO3 → (NH4)3 PO4.12 MoO3 +12 H2O +
23 NH4NO3 + 2NaNO3
37
2(NH4)3 PO4 12 MoO3 + 46 KOH
→ 23 K2MoO3 + (NH4)2 MoO4+
2 (NH4)2 HPO4 + 22 H2O
2 (NH4)2 HPO4
→ P2O5 + 4NH3 + 3 H2O
Procedure
a. Digestion and extraction of the sample
Two gm of organic sample was taken into a 250 ml conical flask and added 30 ml of
1:1 HCl and 3 ml of conc. HNO3. The contents were digested on a sand bath for about an
hour. The contents were diluted with 100 ml distilled water and filtered through Whatman
No.40 filter paper. The filtrate was collected in a 250 ml volumetric flask. Again the residue
was washed with hot water till the filtrate reached to 240 ml. The filtrate was cooled and
made up to 250 ml with water.
b. Precipitation
Ten ml of the above extract was transferred into a 250 ml beaker and added 20 ml
distilled water. The solution was made to alkaline by adding NH4OH. Then the same was
made to acidic using dilute HNO3. About 2 gm of solid NH4NO3 was added and the contents
were warmed to 70° C by keeping the beaker in a hot water bath. A precipitant mixture was
prepared in a 100 ml beaker by using 10 ml of 20% ammonium molybdate and 10 ml of 7:3
HNO3 and water. From this mixture, 20 ml was taken in a 100 ml beaker and added slowly to
the aliquot of phosphate solution. The contents were gently stirred and kept at 65° C for 30
minutes to settle the canary yellow precipitate.
c. Filtration and titration
After precipitation, filtration was done using Whatman No. 40 filter paper and the
precipitate was collected in a beaker. Then the precipitate was dissolved in known excess of
0.1619 N KOH. After adding 2 or 3 drops of phenolphthalein, the solution was titrated
38
against 0.1619 N HNO3. From the actual amount of KOH required to react with the
precipitate, the percentage of P2O5 was calculated.
Calculation
Weight of sample taken
= 2 gm
Volume of the extract made up after
digestion
= 250 ml
Aliquot pipette out
= 10 ml
Known excess of 0.1619 N KOH
added to dissolve the precipitate
= X ml
Volume of 0.1619 N HNO3 used for
back titration
= Y ml
Actual volume of 0.1619 N KOH
utilized to dissolve the precipitate
= (X-Y) ml
1 ml of 0.1619 N KOH
= 0.0005 gm P2O5
Percentage of P2O5 in the sample
= (X-Y) (0.0005)
(250/10) (100/2)
3.11.3.4 Estimation of total potassium (TK) and total sodium (TNa)
The levels of TK and TNa present in the organic samples were determined by Flame
photometry method of Stanford and English (1949).
Principle
Certain elements when excited in flame emitted radiations. The excitation caused the
electrode of natural atom to jump to an outer orbit of higher energy level and the atom
returned to lower energy, light of characteristic wave length was emitted. The intensity of
light measured in the flame photometer was proportional to the concentration of the element
present in the sample.
39
Procedure
a. Preparation of sample solution
One gm of organic sample was accurately weighed and transferred into a 1000 ml
volumetric flask. It was dissolved with water and made up to 1000 ml. From this solution, 10
ml was transferred into a 250 ml volumetric flask and made up to the mark with water. The
content in the flask was thoroughly mixed and the concentrations of K and Na present in the
samples were measured using a flame photometer.
b. Preparation of standard solution for K and Na
Exactly 1.907 gm of KCl and 2.338 gm of NaCl was taken and separately transferred
into 1000 ml volumetric flasks. They were dissolved with distilled water and made up to
1000 ml. These solutions were equivalent to 1000 ppm of K or Na. From these stock
solutions, various standard solutions ranging from 10 to 100 ppm K or Na were prepared.
c. Recording the readings
The galvanometer was adjusted to 0 for blank and 100 for 100 ppm K or Na solution.
Galvanometer readings were recorded for all the standard solutions of K and Na. Separate
standard graphs were constructed using the readings recorded for various standard solutions
of K and Na.
About 5 to 10 ml of sample solution was transferred into a vial and inserted into the
aspirator. The galvanometer reading of the sample was noted. The concentration of
potassium and sodium present in the unknown sample solution was determined from the
standard graph.
Calculation
Weight of sample taken
= 1gm
Volume make up in first dilution
= 1000ml
Volume of aliquot taken for second
40
dilution
= 10 ml
Volume made up in second dilution
= 250 ml
Concentration of K or Na in the solution
as deduced from the standard curve
= X ppm
Therefore percentage of K or Na in the
given sample of KCl/K2SO4 or NaCl
= (X/1000000) (250/10)
(1000)(100)
3.11.3.5 Estimation of total calcium (TCa)
The levels of TCa present in the organic samples were determined by adopting the
EDTA or Versenate method of Jackson (1973).
Principle
The most widely used salt for EDTA is the disodium salt with the formula Na2 H2 Y.
2H2O where Y is the tetravalent anion of EDTA. When Ca2+ treated with H2Y-2 a very stable
complex was formed.
Ca2+ + H2Y-2 → CaY-2 + 2H+
Procedure
Ten gm of organic sample was taken in a 500 ml beaker and saturated with neutral
normal ammonium acetate. The sample solution was filtered in a buchner funnel under
suction and washed continuously with ammonium acetate solution. The sample solution was
made up to 500 ml with distilled water.
Ten ml of aliquot was pipette out into a 100 ml dry clean conical flask and added 5
ml of 10% NaOH solution. After adding a pinch of murexide powder, it was titrated against
0.02 N EDTA till the red colour changed into violet. A titration with blank was also carried
out with 10 ml of water instead of calcium solution.
41
Calculation
Volume of 0.02 N EDTA used for
sample titration
= A ml
Volume of 0.02 N EDTA used for
blank titration
= B ml
Corrected titre value
= (A-B) ml
Meq. of Ca in V1 ml of aliquot
= (A-B) ×0.02
Meq. of Ca in V ml of the extract
= (A-B) ×0.02×V/V1
10 gm of sample contains
= (A-B) ×0.02×V/V1 Meq. Ca
100 gm of sample contains
= (A-B)×0.02×V/V1×100/10 Meq.Ca
3.11.3.6 C: N ratio
The ratio of the percentage of carbon to that of nitrogen (C/N ratio) was arrived at by
dividing the percentage of carbon with the percentage of nitrogen determined in the given
organic sample.
3.11.4 Micronutrients analysis
3.11.4.1 Estimation of micronutrients
The levels of micronutrients such as iron (Fe), manganese (Mn), zinc (Zn) and
copper (Cu) present in the organic samples were determined by Atomic Absorption
Spectrophotometry method of Lindsay and Norwell (1978).
Principle
Diethylene triamine penta acetic acid (DTPA) acts as a mild chelating agent who
extracts the easily soluble iron, manganese, zinc and copper. The extracting solution
prevented the dissolution of calcium carbonate and permitted the right amount of Fe, Mn, Zn
and Cu to be dissolved. The dissolved elements in the extract were then measured by the
Atomic Absorption Spectrophotometer (AAS) wherein the extracted sample was converted
42
first into an atomic vapour, usually by a flame and irradiated by the metal being sought. The
absorption of the light by the vaporization samples is related to the concentration of the
desired metal in it.
Procedure
Ten gm of organic sample was transferred into a 100 ml polythene bottle and added
20 ml of extracting solution. The content of the bottle was vigorously mixed for 2 hours. The
solution was filtered through Whatman No.42 filter paper. The filtrate was used for the
estimation of Fe, Mn, Zn and Cu using appropriate hallow cathode lamp in AAS. A separate
calibration curve for each element was constructed using standard solutions of varying
concentrations after setting the AAS with suitable hallow cathode lamp. Using the above
extract, the concentration of particular element present in the sample was determined.
3.12 Cultivation of chilly plant
3.12.1 Collection of seedlings
Seedlings of 30 days old chilly plant were purchased from the local seedling shop at
Kumbakonam. Twelve earthen pots for partly decomposed and a similar number for
vermicompost were taken and to each, 5 litres of following doses, 75, 50, 40, 30, 25, 20, 15,
10, 7.5, 5.0, 2.5 and 0 PSR doses of water hyacinth / paddy waste / cow dung / a mixture of
all the 3 organic matters were filled. Two sets of same experiment were also carried out
simultaneously using the same 12 PSR media in each organic matter for statistical
comparison.
One healthy seedling was transplanted in each pot at a depth of 4 cm from the top of
the substrate medium. Sufficient water was added in all the seedling pots. The pots were
regularly poured with sufficient water to ensure proper growth till all plants get harvested (up
to 90 days).
43
At weekly intervals the plants raised in earthen pots were counted their total leaves,
total flowers and total fruits and measured their shoot height.
The pot cultivation experiment was conducted at the terrace roof of PG and Research
Department of Zoology, Govt Arts College (Autonomous), Kumbakonam. Care was taken to
ensure the growing plants free from predation, if any, by way of erecting a wire mesh around
the earthen pots.
After 90 days of transplantation, the plants were uprooted. The total height, total
leaves, total fruits, fruit length, fruit perimeter and fruits dry weight were measured.
3.13 Statistical analysis
Basic statistics such as mean and standard deviation for various parameters were
calculated. Student‘t’ test was applied to test the statistical significance between the mean
values of macro and micro nutrients present in the partly decomposed and earthworms
exposed organic samples, length and body weight of adult F0 / F1 earthworms before and
after exposure to different doses of organic materials, and various plant parameters of control
and experimental chilly plants through window based statistical package (SPSS) at P < 0.01
and P < 0.05 levels. Data of plant parameters were also analyzed statistically by analysis of
variance (F- test) using one way classification and the level of significance at P < 0.01.