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CHAPTER FOUR

RESULT AND DISCUSSION

4. Result and Discussion:


4.1. Biological Test Before Formulation:

Some biological tests are done to select the best combination of Albendazole and Mebendazole. The tests
are done on specific type of Earthworms (Lumbricus terrestris).

Six combinations are made with different combinations Albendazole and Mebendazole.

1.Combination 1- (400 mg Albendazole + 300 mg Mebendazole)

2. Combination 2- (350 mg Albendazole + 350 mg Mebendazole)

3. Combination 3- (500 mg Albendazole + 200 mg Mebendazole)

4. Combination 4- (300 mg Albendazole + 400 mg Mebendazole)

5. Combination 5- (700 mg Albendazole)

6. Combination 6- (700 mg Mebendazole)

Experiment 1:
Table 4.1: Different Combinations with Their Biological Activity.

Albendazole Albendazole Albendazole Albendazole Albendazole Mebendazole


400 mg + 350 mg + 500 mg + 300 mg + 700 mg 700 mg
Mebendazole Mebendazole Mebendazole Mebendazole
300 mg 350 mg 200 mg 400 mg

1.Sta Paraly 1.Start Paraly 1.Start 1.Start Paraly 1.Start 1.Start Paraly
rting sis ing sis ing ing sis ing ing sis
time- time- time- time- time- time- time- time- time- time-
2:08 3:oo 2:08 3:05 2:08 2:08 4:22 2:08 2:08 3:56
pm pm pm pm pm pm pm pm pm pm

Alive Alive

Death Death Death Death


time- time- time- time-
3:56 3:35 4:45 4:50
pm pm pm pm

2.Sta Paraly 2.Start 2.Start 2.Start 2.Start 2.Start


rting sis ing ing ing ing ing
time- time- time- time- time- time- time-
2:08 2:48 2:08 2:08 2:08 2:08 2:08
pm pm pm Alive pm Alive pm Alive pm Alive pm Alive
Death
time-
3:00
pm
3.Sta Paraly 3.Start 3.Start 3.Start 3.Start 3.Start
rting sis ing ing ing ing ing
time- time- time- time- time- time- time-
2:08 3:10 2:08 2:08 2:08 2:08 2:08
pm pm pm Alive pm Alive pm Alive pm Alive pm Alive
Death
time-
3:38
pm

Experiment 2:
Table 4.2: Different Combinations with Their Biological Activity.

Albendazole Albendazole Albendazole Albendazole Albendazole Mebendazole


400 mg + 350 mg + 500 mg + 300 mg + 700 mg 700 mg
Mebendazole Mebendazole Mebendazole Mebendazole
300 mg 350 mg 200 mg 400 mg

1.Star Paraly 1.Star Paraly 1.Start Paral 1.Star Paraly 1.Start Paraly 1.Start
ting sis ting sis ing ysis ting sis ing sis ing
time- time- time- time- time- time- time- time- time- time- time-
2:02 3:54 2:02 4:09 2:02 3:54 2:02 4:06 2:02 4:37 2:02
pm pm pm pm pm pm pm pm pm pm pm

Alive

Death Death Death Death Death


time- time- time- time- time-
4:00 4:44 4:o4 4:36 4:48
pm pm pm pm pm

2.Star Paraly 2.Star 2.Start Paral 2.Star Paraly 2.Start 2.Start


ting sis ting ing ysis ting sis ing ing
time- time- time- time- time- time- time- time- time-
2:02 3:58 2:02 2:02 4:02 2:02 4:06 2:02 2:02
pm pm pm pm pm pm pm pm pm
Death Death Death
time- Alive time- time- Alive Alive
4:32 4:35 4:39
pm pm pm

Experiment 3:
Table 4.3: Different Combinations with Their Biological Activity.

Albendazole Albendazole Albendazole Albendazole Albendazole Mebendazole


400 mg + 350 mg + 500 mg + 300 mg + 700 mg 700 mg
Mebendazole Mebendazole Mebendazole Mebendazole
300 mg 350 mg 200 mg 400 mg

1.Sta Paraly 1.Start Paraly 1.Star Paraly 1.Star Paraly 1.Star 1.Start Paraly
rting sis ing sis ting sis ting sis ting ing sis
time- time- time- time- time- time- time- time- time- time- time-
12:31 1:35 12:31 2:00 12:31 2:00 12:31 2:22 12:31 12:31 3:05
pm pm pm pm pm pm pm pm pm pm pm

Alive

Death Death Death Death Death


time- time- time- time- time-
2:22 3:03 2:33 3:05 3:50
pm pm pm pm pm
2.Sta Paraly 2.Start Paraly 2.Star Paraly 2.Star Paraly 2.Star 2.Start
rting sis ing sis ting sis ting sis ting ing
time- time- time- time- time- time- time- time- time- time-
12:31 1:45 12:31 3:05 12:31 2:10 12:31 3:14 12:31 12:31
pm pm pm pm pm pm pm pm pm Alive pm Alive

Death Death Death Death


time- time- time- time-
2:25 3:36 2:41 3:40
pm pm pm pm
3.Sta Paraly 3.Start 3.Star 3.Star 3.Star 3.Start
rting sis ing ting ting ting ing
time- time- time- time- time- time- time-
12:31 2:16 12:31 12:31 12:31 12:31 12:31
pm pm pm Alive pm Alive pm Alive pm Alive pm Alive
Death
time-
2:38
pm

Discussion: Among all the combinations only Combination 1 gives the best result. So we have to work
on that combination.
4.2. Carr’s Index:

The Carr’s index is frequently used in pharmaceutics as an indication of the flowability.In a free-flowing
powder, the bulk density and tapped density would be close in value, therefore, the Carr’ index would be
small. On the other hand, in a poor-flowing powder where there are greater interparticle interactions, the
difference between the bulk and tapped density observed would be greater, therefore, the Carr index
would be bigger. A Carr index greater than 25 is considered to be an indication of poor flowability, and
below 15, of good flowability.

Criteria for Carr’s Index

Figure 4.1: Carr’s Index

Formulation A:

Table 4.4: Carr’s Index of Formulation A

Serial No Carr’r Index Criteria

1 23.77% Poor

2 28.12% Poor

3 27.53% Poor

4 35.33% Very poor

Formulation B:

Table 4.5: Carr’s Index of Formulation B


Serial No Carr’s Index Criteria

1 51.20% Extremely poor

2 42.33% Extremely poor

3 44.49% Extremely poor

4 56.37% Extremely poor

Formulation C:

Table 4.6: Carr’s Index of Formulation C

Serial No Carr’s Index Criteria

1 37.75% Very poor

2 33.54% Poor

3 42.42% Extremely poor

4 35.73% Very poor

Formulation D:

Table 4.7: Carr’s Index of Formulation D

Serial No Carr’s Index Criteria

1 18.32% Satisfactory

2 22.69% Satisfactory

3 19.11% Satisfactory

4 30.67% Poor

Formulation E:
Table 4.8: Carr’s Index of Formulation E

Serial No Carr’s Index Criteria

1 33.38% Poor

2 29.59% Poor

3 28.81% Poor

4 25.66% Poor

Formulation F:

Table 4.9: Carr’s Index of Formulation F

Serial No Carr’s Index Criteria

1 23.34% Poor

2 20.77% Satisfactory

3 21.39% Satisfactory

4 21.87% Satisfactory

Discussion: Among all the formulations only Formulation D and Formulation F give the satisfactory
result.

4.3. Hausner Ratio:

Bulk density is the volume of powder per gram of weight in a cylinder, after 50 mechanical taps.  Tap
density is measured in a tapping machine containing a graduated cylinder that moves up and down.
Powdered material is introduced into the cylinder. The tapping begins. The mark of the graduated
cylinder is noted before tapping (V0) and after 2, 4, 6, 8, 10, 15, 20, 30, and 50 taps (V50). The column
height as indicated by the mark in the graduated cylinder is related to the volume measured, and the
diameter of the volume is fixed. Volume may be calculated accordingly. The powder content ( W) is
weighed, and the bulk density is calculated as W/V50 g/ml. Bulk density may be used as an indication of
flow properties. The ratio of tapped density W/V50 to fluffy density (W/V0 g/ml) is known as the Hausner
ratio. A good flow is indicated by a Hausner ratio greater than 1.25, and a poor flow may have a value of
1.5.

Criteria for Hausner ratio:

Figure 4.2: Hausner Ratio

Formulation A:

Table 4.10: Hausner Ratio of Formulation A

Serial No Hausner ratio Criteria

1 1.44 Poor

2 1.49 Very poor

3 1.37 Poor

4 1.41 Poor

Formulation B:
Table 4.11: Hausner Ratio of Formulation B

Serial No Hausner ratio Criteria

1 2.05 Very, very poor

2 2.12 Very, very poor

3 1.93 Very, very poor

4 2.09 Very, very poor

Formulation C:

Table 4.12: Hausner Ratio of Formulation C

Serial No Hausner ratio Criteria

1 1.62 Very, very poor

2 1.75 Very, very poor

3 1.69 Very, very poor

4 1.83 Very, very poor

Formulation D:

Table 4.13: Hausner Ratio of Formulation D

Serial No Hausner ratio Criteria

1 1.30 Passable

2 1.33 Passable

3 1.21 Fair

4 1.32 Passable

Formulation E:

Table 4.14: Hausner Ratio of Formulation E


Serial No Hausner ratio Critera

1 1.32 Passable

2 1.27 Passable

3 1.34 Passable

4 1.24 Fair

Formulation F:

Table 4.15: Hausner Ratio of Formulation F

Serial No Hausner ratio Criteria

1 1.30 Passable

2 1.34 Passable

3 1.23 Fair

4 1.29 Passable

Discussion: Among all the formulations Formulation D, Formulation E and Formulation F give fair
results.

4.4. Angle of Repose:

There are numerous methods for measuring angle of repose and each produces slightly different results.
Results are also sensitive to the exact methodology of the experimenter. As a result, data from different
labs are not always comparable. One method is the triaxial shear test, another is the direct shear test.

If the coefficient of static friction is known of a material, then a good approximation of the angle of
repose can be made with the following function. This function is somewhat accurate for piles where
individual objects in the pile are minuscule and piled in random order.
Methods in determining the angle of repose
The measured angle of repose may vary with the method used.

Tilting box method

This method is appropriate for fine-grained, non-cohesive materials with individual particle size less than
10 mm. The material is placed within a box with a transparent side to observe the granular test material. It
should initially be level and parallel to the base of the box. The box is slowly tilted until the material
begins to slide in bulk, and the angle of the tilt is measured.

Fixed funnel method

The material is poured through a funnel to form a cone. The tip of the funnel should be held close to the
growing cone and slowly raised as the pile grows, to minimize the impact of falling particles. Stop
pouring the material when the pile reaches a predetermined height or the base a predetermined width.
Rather than attempt to measure the angle of the resulting cone directly, divide the height by half the width
of the base of the cone. The inverse tangent of this ratio is the angle of repose.

Revolving cylinder method

The material is placed within a cylinder with at least one transparent end. The cylinder is rotated at a
fixed speed and the observer watches the material moving within the rotating cylinder. The effect is
similar to watching clothes tumble over one another in a slowly rotating clothes dryer. The granular
material will assume a certain angle as it flows within the rotating cylinder. This method is recommended
for obtaining the dynamic angle of repose, and may vary from the static angle of repose measured by
other methods.

Figure 4.3: Angle of Repose

Formulation A:

Table 4.16: Angle of Repose of Formulation A


Serial No Angle of Repose Criteria

1 51.34° Poor

2 50.19° Poor

3 53.77° Poor

4 48.90° Poor

Formulation B:

Table 4.17: Angle of Repose of Formulation B

Serial No Angle of Repose Criteria

1 45.00° Passable

2 41.67° Passable

3 44.26° Passable

4 41.94° Passable

Formulation C:

Table 4.18: Angle of Repose of Formulation C

Serial No Angle of Repose Criteria

1 41.19° Passable

2 40.00° Fair

3 41.55° Passable

4 42.21° Passable

Formulation D:

Table 4.19: Angle of Repose of Formulation D


Serial No Angle of Repose Criteria

1 33.72° Good

2 39.19° Fair

3 40.74° Fair

4 31.48° Good

Formulation E:

Table 4.20: Angle of Repose of Formulation E

Serial No Angle of Repose Criteria

1 36.86° Fair

2 39.22° Fair

3 37.14° Fair

4 40.28° Fair

Formulation F:

Table 4.21: Angle of Repose of Formulation F

Serial No Angle of Repose Criteria

1 35.51° Good

2 32.69° Good

3 38.17° Fair

4 34.53° Good

Discussion: Among all the formulations Formulation D and Formulation F give good results.
4.5. Hardness Test:

Hardness: Resistance of a material to deformation, indentation, or penetration by means such as


abrasion, drilling, impact, scratching, and/or wear, measured by hardness tests. The unit of hardness given
by the test is known as the Vickers Pyramid Number (HV) or Diamond Pyramid Hardness (DPH).

The hardness number can be converted into units of pascals, but should not be confused with pressure,
which uses the same units.

There are two types of water hardness, temporary and permanent. Temporary Hardness is due to the
bicarbonate ion, HCO3-, being present in the water.

 hardness is important from an engineering standpoint because resistance to wear by either friction or


erosion by steam, oil, and water generally increases with hardness.

The application of hardness testing enables you to evaluate a material's properties, such as strength,
ductility and wear resistance, and so helps you determine whether a material or material treatment is
suitable for the purpose one requires.

Different types of hardness test:

 Brinell Hardness Test.


 Rockwell Hardness Test.
 Knoop Hardness Test.
 Vickers Hardness Test.

Oral tablets have the hardness of 4-10 kg.

Formulation A:

Table 4.22: Hardness of Formulation A


Serial No Hardness(kg)

1 8.33

2 7.96

3 7.52

4 7.91

Formulation B:

Table 4.23: Hardness of Formulation B

Serial No Hardness(kg)

1 7.16

2 7.02

3 8.12

4 7.68

Formulation C:

Table 4.24: Hardness of Formulation C

Serial No Hardness(kg)

1 7.24

2 7.09

3 6.79

4 6.94

Formulation D:

Table 4.25: Hardness of Formulation D


Serial No Hardness(kg)

1 6.47

2 6.99

3 6.81

4 6.38

Formulation E:

Table 4.26: Hardness of Formulation E

Serial No Hardness(kg)

1 6.33

2 \ 6.17

3 5.26

4 5.73

Formulation F:

Table 4.27: Hardness of Formulation F

Serial No Hardness(kg)

1 6.63

2 6.51

3 6.80

4 5.47

Discussion: All the formulations are within suitable range.

4.6. Dissolution Test:


Dissolution testing measures the extent and rate of solution formation from a dosage form, such as tablet,
capsule, ointment, etc. The dissolution of a drug is important for its bioavailability and therapeutic
effectiveness. Dissolution and drug release are terms used interchangeably.

Figure 4.4: Dissolution Test of Formulation A for Albendazole

Figure 4.5: Dissolution Test of Formulation B for Albendazole


Figure 4.6: Dissolution Test of Formulation C for Albendazole

Figure 4.7: Dissolution Test of Formulation D for Albendazole


Figure 4.8: Dissolution Test of Formulation E for Albendazole

Figure 4.9: Dissolution Test of Formulation F for Albendazole


Figure 4.10: Dissolution Test of Formulation A for Mebendazole

Figure 4.11: Dissolution Test of Formulation B for Mebendazole


Figure 4.12: Dissolution Test of Formulation C for Mebendazole

Figure 4.13: Dissolution Test of Formulation D for Mebendazole


Figure 4.14: Dissolution Test of Formulation E for Mebendazole

Figure 4.15: Dissolution Test of Formulation F for Mebendazole

Discussion:For Albendazole, Formulation F gives the best result. But other formulations also give the
satisfactory results.

For Mebendazole, Formulation A gives the best result. But other formulations also give the satisfactory
results.
4.7. Disintegration Test:

Disintegration testing accurately measures, under standard conditions, the ability of a sample to break into
smaller particles. This testing is typically performed on tablets, capsules and enteric coated tablets.

Formulation A:

Table 28: Disintegration Time for Formulation A

Serial No Disintegration time


1 7.30 seconds
2 9.22 seconds
3 7.59 seconds
4 7.43 seconds

Formulation B:

Table 29: Disintegration Time for Formulation B

Serial No Disintegration time


1 9.20 seconds
2 8.44 seconds
3 8.53 seconds
4 8.36 seconds

Formulation C:

Table 30: Disintegration Time for Formulation C

Serial No Disintegration time


1 11.28 seconds
2 9.31 seconds
3 11.55 seconds
4 12.39 seconds
Formulation D:

Table 31: Disintegration Time for Formulation D

Serial No Disintegration time

1 2.30 seconds

2 2.47 seconds
3 3.00 seconds

4 3.36 seconds

Formulation E:

Table 32: Disintegration Time for Formulation E

Serial No Disintegration time

1 10.52 seconds

2 10.39 seconds

3 11.32 seconds

4 10.27 seconds

Formulation F:

Table 33: Disintegration Time for Formulation F

Serial No Disintegration time

1 12.48 seconds

2 12.33 seconds

3 12.41 seconds

4 12.22 seconds

Discussion: Among all the formulations Formulation D gives the shortest disintegration time.
4.8. Friability Test:

Friability testing is a laboratory technique used by the pharmaceutical industry to test the durability of
tablets during transit. This testing involves repeatedly dropping a sample of tablets over a fixed time,

using a rotating wheel with a baffle.

Friability is the tendency for a tablet to chip, crumble or break following compression. This tendency is
normally confined to uncoated tablets and surfaces during handling or subsequent storage.

Formulation A:

Table 34: Friability Test of Formulation A

Serial No Initial weight (per After rotation weight Difference


tablet) (per tablet)
1 1.121 gm 1.120 gm 0.001 gm

2 1.153 gm 1.152 gm 0.001 gm

3 1.123 gm 1.122 gm 0.001 gm

4 1.139 gm 1.138 gm 0.001 gm

Formulation B:

Table 35: Friability Test of Formulation B

Serial No Initial weight After rotation weight Difference

1 1.164 gm 1.162 gm 0.002 gm

2 1.137 gm 1.135 gm 0.002 gm

3 1.151 gm 1.149 gm 0.002 gm

4 1.149 gm 1.148 gm 0.001 gm

Formulation C:
Table 36: Friability Test of Formulation C

Serial No Initial weight After rotation weight Difference

1 1.187 gm 1.186 gm 0.001 gm

2 1.153 gm 1.151 gm 0.002 gm

3 1.171 gm 1.149 gm 0.002 gm

4 1.165 gm 1.164 gm 0.001 gm

Formulation D:

Table 37: Friability Test of Formulation D

Serial No Initial weight After rotation weight Difference

1 1.131 gm 1.131 gm 0.00 gm

2 1.164 gm 1.163 gm 0.001 gm

3 1.183 gm 1.183 gm 0.00 gm

4 1.139 gm 1.138 gm 0.001 gm

Formulation E:

Table 38: Friability Test of Formulation E

Serial No Initial weight After rotation weight Difference

1 1.108 gm 1.107 gm 0.001 gm

2 1.144 gm 1.142 gm 0.002 gm

3 1.167 gm 1.167 gm 0.00 gm

4 1.128 gm 1.126 gm 0.002 gm

Formulation F:
Table 39: Friability Test of Formulation F

Serial No Initial weight After rotation weight Difference

1 1.194 gm 1.193 gm 0.001 gm

2 1.168 gm 1.166 gm 0.002 gm

3 1.183 gm 1.183 gm 0.00 gm

4 1.138 gm 1.136 gm 0.002 gm

Discussion: Among all the formulations Formulation D gives the best result.

4.9. FTIR Analysis:

Fourier Transform Infrared Spectroscopy, also known as FTIR Analysis or FTIR Spectroscopy, is an


analytical technique used to identify organic, polymeric, and, in some cases, inorganic materials.
The FTIR analysis method uses infrared light to scan test samples and observe chemical properties.

Fourier Transform Infrared Spectroscopy (FTIR) identifies chemical bonds in a molecule by producing an
infrared absorption spectrum. The spectra produce a profile of the sample, a distinctive molecular
fingerprint that can be used to screen and scan samples for many different components.

FTIR analysis measures the range of wavelengths in the infrared region that are absorbed by a material. A
simple device called an interferometer is used to identify samples by producing an optical signal with all
the IR frequencies encoded into it. The signal can be measured quickly.

Identification of Albendazole:
Figure 4.16: FTIR spectrum for Albendazole

Identification of Mebendazole:
Figure 4.17: FTIR spetrum for Mebendazole

Identification of the Combination of Albendazole and Mebendazole:


Figure 4.18: FTIR spectrum for Albendazole and Mebendazole

Discussion: Here with the help of the peaks we can identify Albendazole, Mebendazole and the
combination of Albendazole and Mebendazole.

4.10. SEM Analysis:


SEM analysis is a powerful investigative tool which uses a focused beam of electrons to produce
complex, high magnification images of a sample's surface topography .

Surface analysis of Albendazole-Mebendazole combination tablet:

Figure 4.19: Surface of tablet

Figure 4.20: Surface of tablet


Figure 4.21 : Surface of tablet

Figure 4.22: Surface of tablet

Discussion: The surfaces of the tablets analyzed by SEM Analysis are acceptable.
4.11. HPLC Analysis:

High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid


chromatography) is a technique in analytical chemistry used to separate, identify, and quantify each
component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample
mixture through a column filled with a solid adsorbent material. Each component in the sample interacts
slightly differently with the adsorbent material, causing different flow rates for the different components
and leading to the separation of the components as they flow out of the column.

HPLC has been used for manufacturing (e.g., during the production process of pharmaceutical and
biological products), legal (e.g., detecting performance enhancement drugs in urine), research (e.g.,
separating the components of a complex biological sample, or of similar synthetic chemicals from each
other), and medical (e.g., detecting vitamin D levels in blood serum) purposes. [1]

Chromatography can be described as a mass transfer process involvingadsorption. HPLC relies on pumps


to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the
separation of the sample components. The active component of the column, the adsorbent, is typically a
granular material made of solid particles (e.g., silica, polymers, etc.), 2–50 μm in size. The components of
the sample mixture are separated from each other due to their different degrees of interaction with the
adsorbent particles. The pressurized liquid is typically a mixture of solvents (e.g., water, acetonitrile
and/or methanol) and is referred to as a "mobile phase". Its composition and temperatureplay a major role
in the separation process by influencing the interactions taking place between sample components and
adsorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and
ionic, most often a combination.

HPLC is distinguished from traditional ("low pressure") liquid chromatographybecause operational


pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on
the force of gravity to pass the mobile phase through the column. Due to the small sample amount
separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm
length. Also HPLC columns are made with smaller adsorbent particles (2–50 μm in average particle size).
This gives HPLC superior resolving power (the ability to distinguish between compounds) when
separating mixtures, which makes it a popular chromatographic technique.
Blank

Figure 4.23: HPLC spectrum for Blank


Identification of Albendazole:

Figure 4.24: HPLC spectrum for Albendazole


Identification of Mebendazole:

Figure 4.25: HPLC spectrum for Mebendazole


Identification of Albendazole-Mebendazole combination powder:

Figure 4.26: HPLC spectrum for Albendazole-Mebendazole powder


Identification of Albendazole-Mebendazole combination tablet:

Figure 4.27: HPLC spectrum for Albendazole-Mebendazole combination tablet

Discussion: Here with the peaks we can identify Albendazole, Mebendazole, Albendazole-Mebnedazole
combination powder, Albendazole-Mebendazole combination tablet.
4.12. Biological Test After Formulation:

Table 40: Biological Activity Test

Formulation Formulation B Formulation C Formulation D Formulation Formulation F


A E
1.Sta Paraly 1.Start Paraly 1.Star Paraly 1.Sta Paraly 1.Star 1.Start Paraly
rting sis ing sis ting sis rting sis ting ing sis
time- time- time- time- time- time- time- time- time- time- time-
12:2 1:38 12:20 2:00 12:20 2:00 12:20 1:32 12:20 12:20 3:05
0 pm pm pm pm pm pm pm pm pm pm pm

Alive

Death Death Death Death Death


time- time- time- time- time-
2:22 3:03 2:33 2:04 3:50
pm pm pm pm pm
2.Sta Paraly 2.Start Paraly 2.Star Paraly 2.Sta Paraly 2.Star 2.Start
rting sis ing sis ting sis rting sis ting ing
time- time- time- time- time- time- time- time- time- time-
12:2 1:52 12:20 3:05 12:20 2:10 12:20 1:45 12:20 12:20
0 pm pm pm pm pm pm pm pm pm pm

Alive Alive

Death Death Death Death


time- time- time- time-
2:25 3:36 2:41 2:15
pm pm pm pm
3.Sta 3.Start 3.Star 3.Sta Paraly 3.Star 3.Start
rting ing ting rting sis ting ing
time- time- time- time- time- time- time-
12:2 12:20 12:20 12:20 1:55 12:20 12:20
0 pm pm pm pm pm pm pm
Alive Alive Alive Alive Alive

Death
time-
2:27
pm

Discussion: According to the test Formulation D gives the best result. Because with this formulation
earthworms die in a short time.
Overall Discussion: Considering the results, we can declare Formulation D is the best
formulation.

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