This document describes a study that aimed to identify a fungal strain that can degrade the pesticide chlorpyrifos and optimize the concentration for degradation. The fungal strain was identified as Fusarium sp. based on morphological analysis under a microscope. The strain was grown in media containing different concentrations of chlorpyrifos and it was found to grow best at a 1.2% concentration, demonstrating this is the optimum level for degradation. The study concludes Fusarium sp. has potential for bioremediation of chlorpyrifos contamination.

1. Morphological Identification &
Concentration Optimization of
Pesticide Degrading Fungal
Strain
Minor Project
Under The Supervision of
Dr. Jyoti Saxena
2016-2017
By
Neha Mishra
Jyotsna Mehra

2. Morphological Identification and Concentration
Optimization of Pesticide Degrading Fungal Strain
 INTRODUCTION________________3-4
 Pesticide_______________________4
 Effects of Pesticides______________5
 Classification _________________6-7
 Bioremediation__________________8
 Material and Methods_________9-10
 Result and Discussion _________1-14
 Conclusion ____________________15

3. Pesticide
• Chemical or biological substance designed to kill or retard the growth of
pests that damage or interfere with the growth of crops, shrubs, trees,
timber and other vegetation desired by humans are called pesticides.
• Goal: to stop or limit pest occurrence
• Benefits: Improve quality and quantity of our food supply.
 Used in timber, horticulture, aquatic, and structural pest control industries.
 Homeowners and home gardeners often use pesticides in their homes,
yards, and on pets.
• Types:
– Insecticides – kill insects
– Herbicides – kill weeds
– Fungicides – suppress or kill fungi

4. What Happens after Application?
• When applied to the goal they will remain in the target area for long enough
time to control a specific pest.
• Pesticides are mobile in the environment (air, soil, water).
• This movement can be beneficial (moving pesticide to target area, such as
roots) but can also reduce the effect on the target pest and injure non target
plants and animals.
• World wide use of pesticide all over the world is 1 billion tons per year.
• An average of 23 deaths occur each year with pesticide.
• Farmers who use pesticides have a ‘significantly high rate of cancer
incidence’ then non farmers.

5. Hazards = Exposer X Toxicity
Ways of exposer:
• Dermal (skin)
• Oral (mouth) • Inhalation (lungs)
• Eyes contact

6. Classification of Pesticide
1. Carbamates: derived from carbamic acid and
kill insects. Interferes with cholinesterase causes
acute poisoning.
2. Organochlorine: are chlorinated hydrocarbons
I. Used extensively in agriculture and
mosquito control.
II. lead to environmental pollution and
accumulation in mammals, resulting in
cumulative poisoning or damage.
3. Organophosphates (OP): highly toxic, interferes
with an important nervous system enzyme
“cholinesterase”. Large exposures can cause acute
poisoning.
Chlorpyrifos in market
Chemical structure of Chlorpyrifos

7. Chlorpyrifos: A type of organophosphorus pesticide and its chemical name is O,
O-diethyl O- (3, 5, 6-trichloro-2-pyridyl) phosphorothioate.
 A widely used insecticide and effective against a broad spectrum of insect
pests of economically important crops.
 Also used for the control of mosquitoes (larvae and adults), flies, various
soil pests, many foliar crop pests and household pests.
 Classified by the World Health Organization as class II, moderately
hazardous pesticide (WHO, 1997).
 Affect the central nervous system, the cardiovascular system, and the
respiratory system. Also a skin and eye irritant.
 Skin which has come in contact with this material should be washed
immediately with soap and water and all contaminated clothing should be
removed.
Chlorpyrifos

8. Bioremediation
The degradation of noxious waste from the environment using microorganisms is
called as bioremediation. Microorganisms like bacteria, fungi, algae etc. take part
in bioremediation. Forms of bioremediation are given as bioleaching, bio-venting,
phyto-remediation, land-farming, composting, rhizo-filtration, bio-absorption,
bio-augmentation and myco-remediation.
• Applications of bioremediation
 Bioremediation protects us from the use of chemicals and artificial
mechanisms that are to be used for the cleaning processes.
 It has no side effects.
 Pollution free environment is the result of bioremediation Processes like
incineration requires a lot of energy where as bioremediation is energy
independent process

9. Enzymatic degradation of Chlorpyrifos

10. Material and Methods
1. Morphological analysis:
 10J the fungal strain was taken from the previously prepared petri
dishes.
 Identification of fungus was done by its staining procedure.
 Fungal isolates were spread on the glass slide followed by addition of a
drop of lacto phenol stain.
 Slide is visualized under microscope at 40 X magnification
 The fungus was identified after the morphological analysis

11.  The concentration of chlorpyrifos was optimized by growing the identified
fungal isolate at different concentrations of chlorpyrifos viz. 0.3%, 0.6%,
0.9%, 1.2%.
 Discs of selected fungal isolates were inoculated into 50 ml of CZM
broth at 6.5 pH, containing different concentrations of chlorpyrifos
 Broth was incubated at optimum temperature (28∓2⁰C) for 7 days.
 After the respective period, percentage of chlorpyrifos degradation and
growth of fungus (dry mass) was calculated and compared with that of
control
Optimization of concentration for the growth of
Chlorpyrifos tolerant fungal isolates

12. Result and discussion
1. Morphological analysis:
The fungal isolate was characterized by microscopic analysis using lacto phenol cotton
blue staining procedure. Hence it is concluded that the strain is Fusarium sp.
Microscopic image of Fusarium sp.

13.  The strains were inoculated in varied concentrations (0.3%,
0.6%, 0.9%, 1.2%) of chlorpyrifos.
 Maximum growth was observed at concentration 1.2 % of
Chlorpyrifos at 7 days incubation i.e. 1.040 mg in strain 10J.
 Minimum growth was observed at concentration 0.3% i.e. 0.815.
 As pesticide concentration was increased a gradual decrease in
growth was observed in both the strains after 7 days of incubation.
 Hence, the optimum pesticide concentration was found to be 1.2%
keeping incubation period constant.
Concentration Optimization

14. Table 1 : Dry Mycelial weight with chlorpyrifos concentration variation in
strain 10J after 7 days growth.
SN.
NO.
Chlorpyrifos
(v/v %)
Weight of filter paper
(mg)
Weight of filter paper
with mycelia after
drying
(mg)
Dry mycelial
biomass (mg)
01 0.3% 2.700 3.515 0.815
02 0.6% 2.694 3.720 1.026
04 0.9% 2.611 3.619 1.008
05 1.2% 2.748 3.788 1.040
Control 2.739 3.879 1.140
Dry Mycelial weight with Chlorpyrifos concentration
variation in strain 10J after 7 days growth.

15. 0
0.2
0.4
0.6
0.8
1
1.2
0.30% 0.60% 0.90% 1.20% control
1 2 3 4 5
Drymass(mg)
Concentration v/v%
Dry mass (mg)
0
0.4
0.6
0.8
1.0
1.2
0.2
Dry mass (mg)
Control
5
0.60%
2
0.90%
3
1.2%
4
0.30%
1
Concentration optimization Graphical representation
(Dry mass v/s Concentration)

16.  The use of chemical pesticides has brought benefits such as the increment
of agricultural production soil productivity.
 Only 10% of applied pesticides reach to the target organism,
 A high percentage impacts to non-target organism such wild life, besides
affecting public health.
 Due to the extensive pesticides use, currently there are polluted sites with
these compounds (mainly soils), so it is necessary to generate strategies for
waste treatment and/or for the bioremediation of polluted sites.
 The biological treatment is an important technology from an economical and
environmental point of view.
 On the basis of above mentioned results, after the morphological analysis
the strain has been identified as Fusarium sp. and the optimum
concentration for growth as well as Chlorpyrifos degradation for strain
Fusarium sp.is 1.2%.
Conclusion