Pesticide analysis

PRESENTED BY-
SANTHOSH KUMAR T S
M.PHARM
1ST YEAR
PRESENTATION ON :
“PESTICIDE ANALYSIS ”
KARNATAKA COLLEGE OF PHARMACY
BANGALORE
FACILITATED TO-
MRS. AKKAMMA H.G
ASST. PROFESSOR
(PH. ANALYSIS)

CONTENTS
1. Introduction
2. Pesticide cycle
3. Organophosphorous pesticides analysis
4. Organo chlorine pesticides analysis
5. Determination of pesticidal residues in grains, vegetables, milk
and milk products

What are pesticides?
These are different forms of chemicals that are used in agriculture to kill pests and
protect crops. Many people call them poisons. Others call pesticides agrochemicals,
crop or plant protection products. The primary aim of pesticides is to control
diseases, insects and weeds in agricultural crops. But these pesticides are also
harmful to humans.
What are agricultural pests?
There are different pests that destroy crops in agriculture such as:
• Weeds or unwanted plants
• Insects
• Rodents
• Fungi.

Types of pesticides
Many different types of pesticides are used on farms. They are grouped (classified)
according to their use” or pests they intend to kill. The following are the most common
types used in South Africa:
• Herbicides - are meant to kill weeds ( ex: borax, nitrogen )
• Insecticides - are meant to kill insects ( ex: DDT, BHC )
• Fungicides- are meant to kill fungi (ex: bordeaux mixture )
• Rodenticides- are meant to kill rats, mice and other pest species of rodents.
These different pesticides are applied in agricultural activities such as weed killing, crop
protection, stock protection, soil fumigation, stock disinfection, etc. (ex: warfarin )
• Nematocides- (ex: DPCB, phorate )
• Molluscides- (ex: sodium pentachloridephenate)
• Algaecides- (ex: copper sulphate )

Forms of pesticides
There are three main forms of pesticides.
• Liquids – usually mixed with water and then sprayed
• Solids - usually they are pastes, pellets, dust and powder and may be applied directly
as solids. They may also be turned into other forms
• Gases- usually they are tiny particles
Effects of pesticides
• Damages the nerves
• Damages the lungs
• Cancer
• Sterility
• Birth defects
• Allergic reactions
• Fatal poisoning

PARTS OF PESTICIDE CYCLE :
• Adsorption
• Volatilization
• Spray drift
• Run off
• Leaching
• Absorption
• Degradation

ADSORPTION
•Adsorptionisthebindingofpesticidestosoilparticles.
•Theamountapesticideisadsorbedtothesoilvarieswiththetypeof pesticide,soil
moisture, soilpHandsoiltexture.
•Pesticidesarestronglyadsorbedtosoilsthatarehighinclayororganicmatter.
•Theyarenotstronglyadsorbedtosandysoils.
•Mostsoil-boundpesticidesarelesslikelytogiveoffvapoursorleachthrough thesoil.

Volatilization
• Volatilizationistheprocessofconvertingsolidsorliquidsintoagas,which can
moveawayfromtheinitialapplication site.
• Pesticidesvolatizemostreadilyfromsandandwetsoils.
• Hot,dryor windyweatherand small
spraydrops increasevolatilization.
• Incorporationofthepesticideinto the
soilcanhelpreduce volatilization.

SPRAY DRIFT
Spraydriftisthe airborne movementof spraydropletsawayfrom a
treatment site duringapplication
Spraydriftisaffectedby:
• Droplet size–smaller- more likely they will drift.
• Windspeed–stronger- more pesticidespraywilldrift.
• Distancebetween nozzleandtarget plant orground.
• Drift mayalsohazardto people,domestic animals,pollinating insects.
• Drift cancontaminate water bodieslike ponds,streams,and ditches;harm
to the fishor other aquaticplantsandanimals

RUN OFF
• Runoffisthemovementofpesticidesinwateroveraslopingsurface
• Thepesticidesareeithermixedinthewaterorboundtoerodingsoil
• Runoffcanalsooccurwhenwaterisaddedtoafieldfasterthanitcanbeabsorbedintothe
soil
• Runofffromareastreatedwithpesticidescanpollutestreams,ponds,lakes,andwell
• Pesticideresiduesin surfacewater canharm animalsandcontaminategroundwater

LEACHING
•Leachingisthemovementofpesticidesinwaterthroughthesoil
•Leachingoccursdownward orsideways
•Groundwatermaybecontaminatedifpesticidesleachfromtreatedfields,mixing
sites, washing sites,or wastedisposalareas

ABSORPTION
• Uptakeofpesticidesandotherchemicalsintoplantsormicroorganisms
• Pesticideresiduesmaybebrokendownorremaininsidetheplantoranimal,when the
animaldiesorastheplantdecaysreleasedback
• Somepesticidesstayinthesoillongenoughtobeabsorbedbyplantsgrowninafield years
later
• Theymaydamageorleaveresiduesinfuturecrops.

Degradation or BreakdownProcesses
•Microbialbreakdownisthebreakdownofchemicalsbymicroorganismssuchas fungi
andbacteria.
•Chemicalbreakdownisthebreakdownofpesticidesbychemicalreactionsinthe soil.
•PhotodegradationorPhotolysisisthebreakdownofpesticidesbysunlight.All pesticides
aresusceptibletophotodegradationtosomeextent.
•Hydrolysis:Wateralsodegradespesticidesbydividinglargemoleculesinto smaller
ones.

• Organochlorines pesticides are organic compounds with five or more chlorine
atoms.
• Organochlorines were the first synthetic organic pesticides to be used in
agriculture and in public health.
• Organochlorine insecticides act as nervous system disruptors leading to
convulsions and paralysis of the insect and its eventual death.
• Some of the commonly used representative examples of organochlorine
pesticides are DDT, lindane, endosulfan, aldrin, dieldrin and chlordane and their
chemical structures are presented here under.
Organochlorine pesticides

• Organochlorine pesticides are insecticides composed primarily of
carbon, hydrogen, and chlorine.
• They break down slowly and can remain in the environment long after
application and in organisms long after exposure.
Organochlorines uses
Specific uses take a wide range of forms, from pellet application in
field crops to sprays for seed coating and grain storage. Some
organochlorines are applied to surfaces to kill insects that land there.

Examples:
Lindane
Alachlor
Endrin
Aldrin
Dieldrin

Q: How do organochlorines affect our health?
A: Organochlorines contribute to many acute and chronic illnesses.
Acute illness can include:
o headache
o dermal irritation
o respiratory problems
o dizziness
o nausea
o seizures
Chronic illness include:
o various types of cancer
o neurological damage
o Parkinson's disease
o birth defects
o respiratory illness
o abnormal immune system function.

PRACTICAL USES
CHEMICALS PESTS CONTROLLED
Endosulfan Ants, grasshoppers, lepidopteran larva
Chlordane Aphids, grasshoppers. Used in homes to
control household insects, termites,
wireworms and other soil insects
Dieldrin Surface treatment of soil, seed dressing,
root dipping. Also used in public health to
control cockroaches, fleas and tsetse
DDT Bollworms and aphids on cotton Also
control mosquitoes
Methoxychlor Flea beetles, Colorado beetle
Aldrin Bollworms, cutworms, dipteran larva,
mealybug, Ceratitis capitata, white grubs,
termite

METHODS OF ANALYSIS
Method involves the following steps
A. Determination by GC.
B. Sodium Biphenyl Reduction Method.
C. Radioactive Tracer Method

ALACHLOR
Alachlor is an herbicide from the chloroacetanilide family.
It is an odorless, white solid.
The greatest use of alachlor is for control of annual grasses
and broadleaf weeds in crops.
Use of alachlor is illegal in the European Union and no
products containing alachlor are currently registered in
the United States.

A. DETERMINATION OF ALACHLOR BY GC
Principle:
• Test portion is dissolved in acetone containing di-N-pentyl phthalate as
internal standard, analyzed by GC with flame ionisation detector and
quantited by comparison with internal standard.
Apparatus:
• Gas chromatography - FID and on column injection port
• Temperatures:

Reagents
Acetone
Di-N-pentyl phthalate-internal standard
Alachlor
Chromatographic conditions
Column oven- 230°C
Injection port-250°C
Detector-260°C
Injection volume- 1micro lit
Run time- 15mins
Column- glass column
Pre condition- over night at 250°C before use
Retention times for alachlor and internal standard are 5.5 and 11.2
respectively

PROCEDURE
• Weigh test portion containing 0.2g alachlor into small flask
• 30 ml internal std solution and shake well to extract alachlor
• Make replicate 1micro lit injections of alachlor standards and measure response
ratios for each injection
• Make duplicate injections of test solution and determine average R
CALCULATION
Alachlor percentage =(R/R’)*(W’/W)*P
Where, R and R’= avg response ratios for test and standard solution.
W and W’ = weight in gms of test portion and standard.
P= % purity of standard.

B. SODIUM BIPHENYL REDUCTION METHOD
Principle:
This is applicable to aldrin, diendrin and endrin.
Organic halogen compounds are decomposed by sodium biphenyl and liberated halide ion is
titrated by volhard method after extraction with water from reaction medium.
Reagents:
a.Dilute nitric acid
b.Sodium biphenyl reagent
c.Toluene
Preparation of sample:
0.1g of sample was weighed into 125ml separator containing 25-30 ml toluene.
Add 10-14g of sodium biphenyl reagent
Mix by swirling and allow to stand for 5mins.

• If the solution is not dark green, add 10-14g of reagent. (Dieldrin and endrin required,
15min after final addition of reagent.)
Destroy excess reagent by drop wise addition of water, shaking frequently between
additions, untill green color is completely removed.
Add 25ml dil.HNO₃.
Avoid vigorous shaking.
• Rinse stopper and walls of seperator with H₂O and drain aqueous phase into 250ml
Erlenmeyer flask.
• Re-extract with two 25ml portions of dil HNO₃
• Shake vigorously.
• Add acid aqueous extracts to Erlenmeyer flask.

Determination:
Colourless solutions:
• To acid aqueous solution add 30ml H₂O, 10ml nitrobenzene, 3ml ferric indicator,
from burette add 0.4-0.6ml of potassium thiocyanate.
• Swirling constantly, titrate with 0.1N AgNO₃ until red is discharged.
• Shake vigorously for 15 sec, without refilling burette titrate slowly with 0.05N KSCN
until end point approaches (distinct reddish colour)

Coloured solutions or chlorides in presence of bromine/ iodide:
• To acid aqueous solution add 30ml H₂O, transfer to 400ml beaker, adjust volume to
250ml, add 0.5g Ba(NO₃)₂.
• Titrate with 0.1N AgNO₃ potentiometrically, with stirring, using cell system of either
glass reference electrode and Ag indicating electrode or Ag-AgCl electrode system,
electronic voltmeter, and 10ml burette.
• Blank determination: Determine blank on all reagents by adding 10-14g sodium
biphenyl reagent to 25ml toluene and follow the above procedure.
Calculations :
• %Aldrin =(net ml AgNO₃ - ml blank) *normality * 35.45 * F/(10*g sample)

C. RADIOACTIVE TRACER METHOD
Principle:
Method is based on addition of pure γ isomer labeled with radioactive 36Cl to
sample of BHC containing unknown amount of γ isomer. Determination of
decrease in radioactivity from standard level to diluted level, on pure
weighable γ fraction recovered from mixture is measure of γ isomer content
of sample. Isolation of pure γ material need to be quant.

Counting:
 Use thin-wall, glass, liquid-jacketed counting tube for solution counting.
 Take background count before counting sample.
 Substract this value from both sample and standard value.

ORGANOPHOSPHATE PESTICIDES:
An organophosphate or phosphate ester is the general name
for esters of phosphoric acid. Organophosphates are the basis
of many insecticides, herbicides, and nerve agents.
• Controls a broad spectrum of pests.
• They are nerve poisons; used as stomach poison and also as
contact poison and fumigant.
• Organophosphate are biodegradable, cause minimum
environmental pollution and slow pest resistance.
Ex: Temephos and Fenitrothion

• Parathion
• Methyl parathion
• Fensulfothion
• Chlorpyrifos
• Sulprofos
• Temefos
• TEPP
• Isofenphos
• Glyphosphate
• Formothion
• Ethyl parathion
• Ethion disulfoton
• disulfoton
• Malathion
• Diazinon
• Fenithion
• DDVP
• Azinphos methyl
Examples for Organophosphates:

Mechanism
• organophosphate acts on the enzyme acetylcholinesterase
• They irreversibly inactivate acetylcholinesterase, which is essential to nerve function
in insects, humans, and many other animals.
• Organophosphate pesticides affect this enzyme in various ways, and thus increases
their potential for poisoning.
• Ex: parathion, one of the first OP is more potent than malathion, an insecticide used
in combatting the Mediterranean fruit fly (Med-fly) and West Nile Virus-transmitting
mosquitoes.

Organophosphorus pesticides analysis
Gas chromatography = 40.00%
Liquid chromatography = 26%
Biosensors = 18.00%
Other = 6.00%
Electrochemistry = 5.00%
Immunochemistry = 5.00%

GC method for parathion and methyl parathion

Parathion
A) Standard solution:
i) Dipentyl phthalate internal standard solution: 2.0 ± 0.1g of dipentyl
phthalate was dissolved in CS2 diluted to 500 ml with CS2.
ii) Parathion standard solution: 125mg of parathion in 50ml volumetric
flask+25ml of internal standard solution and mix.
B) Sample preparation:
125mg of sample containing parathion in 50ml volumetric flask+ 25ml of
internal standard solution and mix

Methyl parathion:
A) Standard solution:
i) p,p’ DDE internal standard solution: 5.0+0.1g of 2,2’-bis[p-chlorophenyl]-1,1-
dichloroethylene in CS2 and dilute to 1l with CS2.
ii) Methyl parathion standard solution: 125mg of methyl parathion into 50ml
volumetric flask + 25ml of internal standard solution and mix.
B) Sample solution:
Liquid sample: 125 mg of sample containing methyl parathion+ 25ml of internal
standard solution and mix.

C) Gas chromatography:
1.2*4 mm glass column packed with 1.5% SE-30 plus.
Temperature:
• Injector port: 210
• Column: 180±10
• FID detector: 250
• N2 or He carrier gas: 55-75ml/min
• Air and H2 as specified by manufacturer
• Injection volume: 1-2µl
• Retention time of methyl parathion: 3.5-5.5 min
• Retention time of parathion: 6-8 min
• Retention time of methyl p,p’-DDE: 6-8 min
• Retention time of dipentyl phthalate: 8-10 min

D) Determination: Inject aliquots of standard solution until peak height ratio of parathion/
methyl parathion varies ≤ 1% with dipentyl phthalate/ p,p’-DDE
Then duplicate injections of sample followed by duplicate of standard solutions
E) Calculation:
Parathion:
% parathion = [R/R’]*[W’/W]*P
Methyl parathion
% methyl parathion = [R/R’]*[W’/W]*F*P
W= mg of sample
W’= mg of standard
R’= average of 4 peak heights ( 2 sample and 2 standard)
R= average 2 peak heights of sample
F=1 for liquid
P = % purity of standard

A) Apparatus:
i) Operating conditions:
Temperature: Ambient
Flow rate of mobile phase: 0.8ml/min
Wavelength of detector: 230nm
Sample size: 10µl
ii) Column: Whatmann partisil PXS stainless steel 25cm*4.6mm
iii) filter: Teflon

B) Reagents:
i) Methanol
ii) 85% phosphoric acid
iii) Internal standard solution (250mg benzophenone/ml): 250mg of benzophenone in
1l volumetric flask and diluted to mark with methanol.
iv) Standard solution (0.3mg fensulfothion/ml): 150mg of fensulfothion flask + 100ml
methanol.
10ml of above solution + 40ml internal standard solution and mix.
v) Mobile phase: methanol: water (80:20) buffered to 0.0025M with phosphoric acid.
C) Sample solution:
Spray concentrate: 150mg of sample containing fensulfothion in flask + 100ml methanol.
10ml of above solution + 40ml internal standard solution.

D) Determination:
Adjust injection size and attenuation to give 60-80% peaks
Duplicate injections of sample followed by duplicate injections of standard.
Elution time of fensulfothion = 4-7 min
Elution time of benzophenone = 2-4 min
Calculation:
% fensulfothion= [R/R’]*[W’/W]*P
W= mg of sample
W’= mg of standard
R= average peak height ratios of sample
R’= average peak height ratios of standard
P= % purity of standard

Pesticide Detection Method Sample preparation
LOD
Chlorpyrifos
GC-MS
GC-ECD
LLSE
LLE
0.13 μg kg-1
14.0 μg kg-1
Malathion
GC/PFPD SPE 0.03 μg L-1
Parathion
HPLC-DAD UASEME
0.10 μg L-1
1.50 μg kg-1
Trichlorfon
GC-MS
GC-MS
SPME
ASE
0.07 μgL-1
5.1 μg kg-1
Phosmet
GC-MS
HPLC-DAD
ASE
UASEME
0.50 μg kg-1
0.10 μg L-1

• ASE: Accelerated Solvent Extraction
• LLE: Liquid-Liquid Extraction
• LLSE: Liquid-Liquid Solvent Extraction
• SPE: Solid Phase Extraction
• SPME: Solid-Phase Micro Extraction
• TOTAD: Through Oven Transfer Adsorption Desorption
• DSPE: Dispersive Solid Phase Extraction
• HS-SPME: Solid Phase Micro Extraction in mode HeadSpace
• UASEME: Ultrasound-Assisted Surfactant-enhanced
Emulsification Micro Extraction
• HPLC DAD: HPLC – diode array detector

Immunochemical methods for OP pesticides analysis
Enzyme-linked immunosorbent assay (ELISA) for the insecticide chlorpyrifos was
developed by synthesizing haptens for chlorpyrifos. Certain haptens were used as
immunogens after coupling to protein. Hapten protein complex were injected to
Rabbits to immunize them(produce antibodies). Blood was collected and serum was
separated. Serum was analyzed for antibodies and using them ELISA was developed.

Types of ELISA
• DAS (double antibody sandwich) ELISA
• DAC (direct antigen coating) ELISA
• PAC (protein A coating) ELISA
Haptens
Haptens are small molecules which are inactive as such but when complexed with
carrier protein posses antigen property

Haptens are either metabolites of chlorpyrifos or synthesized by chemical reaction.
Hapten protein complex was injected to rabbit to produce antibodies.
Hapten Carrier protein Immunogen / antigen

Pesticide Method LOD
Diazinon ELISA 46 μg L-1
Fenthion ELISA 10 μg L-1
Malathion ELISA 16 μg L-1
Immunochemical methods for OP pesticides
analysis

Pesticide analysis

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