It describes about different steps and methods of pesticide residue analysis.

1. PESTICIDE RESIDUE ANALYSIS
Presented By: Manish Joshi
Department of Entomology, SDAU, SK Nagar (Gujarat)
Submitted To: Dr. P. S. Patel (Assoc. Prof.)
Department of Entomology, SDAU, SK Nagar (Gujarat)

2. Pesticide
Pest- “Pest is any harmful, destructive, or
troublesome animal, plant or microorganism.”
Pesticide- “Pesticide is any substance or mixture
of substances intended for preventing, destroying,
repelling, or mitigating any pest.”
• US Environmental Protection Agency (EPA)

3. WHY DO WE NEED PESTICIDES ?
1. Protect Crop losses/ yield reduction
2. Protection during Storage
3. Vector Disease Control
India produces 16% of the world’s food grain but using less than 2% pesticides
used world-wide. Hence there is a good potential of growth.

4. Pesticide : Side effects on human body
Agrochemicals and Security: Homeland Security and Pesticides Pest-09
Acute exposure
Chronic exposure
Inhalation
Absorption through skin
Through food or water
How pesticides get in
Exposure levels
Central nervous system
Skin irritation
Eye irritation
Health effects
(depends on specific pesticide)
Hormone imbalance
Cancer
Liver damage
Reproductive effects

5. 5
Factors Responsible for Increase Pesticide Residues

6. Label
Level of
toxicity
LD50 Listed chemicals
Extremely toxic 1-50
Monocrotophos, Zinc
phosphide, Ethyl mercury
acetate
Highly toxic 51-500
Endosulfan, Carbaryl,
Quinalphos
Moderately
toxic
501-5000 Malathion, Thiram, Glyphosate
Slightly toxic More than 5000
Mancozeb, Oxyfluorfen,
mosquito repellant oils and
liquids, and most other
household insecticides.
Color code of different Pesticides

7. New Colour code submitted by CIB

9.  Pesticide Residues means any specified
substances in food, agricultural commodities,
or animal feed resulting from the use of a
pesticide.
 The term includes any derivatives of a
pesticide, such as conversion products,
metabolites, reaction products and
impurities considered to be of toxicological
significance.
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What is Pesticide Residue?

10. Terminology
 Residue : When pesticides is applied on the commodity, its
parent compound or metabolism having pharmacological
property and/or toxicological nature, together termed as residue.
 Maximum residual limit (MRL) : The limit beyond which
pesticide levels are not legally permitted.
 Acceptable daily intake (ADI) : It is the daily dose of chemical
which during an entire life time, appears to be without
appreciable risk on the basis of all facts known at that time.
 Waiting period (TMRL) : The determination of the minimum
interval required between application of pesticide and harvesting
of crop.
 RL50 or T½ (Half-life value) : It is the time in days required to
reduce the pesticide residues to half of its initial deposits.
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11. Persistence and Degradation
Persistence – The chemical that remain active for a
long period of time after application.
Degradation – The breakdown of a substance into
simpler molecular or atomic components through
chemical reaction(s) either in a plant or animal
(metabolic degradation) or in the environment
(environmental degradation)
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12. Deposit: The amount of initially laid down
pesticide on the surface is called deposit.
Dissipation and persistence: In nature,
disappearance of residues takes place in two
steps.
The first step is the initial phase in which the
disappearance of the residue is fast. This phase is
called "Dissipation".
The second phase, in which there is a slow
decrease in the amount of residue, is known as
"Persistence".
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13. Workflow for analysis of pesticide
residues
sampling
Sample
preparation
Sample
Analysis
Data
evaluation
And Reporting

14. STEPS
IN
PESTICIDE RESIDUE ANALYSIS
• SAMPLING
• SAMPLE PREPARATION
• EXTRACTION
• CLEANUP
• QUANTIFICATION
• CONFIRMATION
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15. 15
SAMPLING PROCEDURES
Sample collection is the process of taking
portion(s) of a food or other substance, for
laboratory analysis, so that resulting analytical
data and conclusions can be applied to the
original food or substance sampled.

16. 16
COLLECTION
OF
WATER SAMPLES

17. 17
Water
• Underground water
• Do not take a sample from tank
• Purge for 10 minutes
• Collect water in washed glass bottle
• Collect 2.5 L water to represent one sample
• Transport sample to the laboratory at 40C

18. 18
SAMPLE SIZE
• Samples of 2.5 to 5 liter / sampling site
• These are reduced with through mixing and
by filling samples in to 1 to 2 bottles

19. 19
SAMPLING PROTOCOL
• Label it immediately
• Also record :
Geography depth & diameter
Exact location
Date and time of sampling
Water volume
Water and air temperature
Weather parameter
Sampling personal

20. 20
COLLECTION
OF
SOIL SAMPLES

21. 21
SAMPLING
• Sample taken with auger
• In grass land – 10 cm
• In forest land – 20 cm
• In orchard – 0 to 25 & 25 to 50 cm
• Sampling site spread evenly
• Sample should not taken from border strip or
places where pesticides have been transferred
from container to tank or where spray have
been mixed
• In large field, the sampling site should be 5 m
away from field periphery or above site

22. 22
SAMPLE SIZE
• In small field : 3 x 3 grid
• In medium size field : 4 x 4 grid
• In large size field : 5 x 5 grid
• Same in untreated plots

23. 23
Sample size
Type
Unit
weight (g)
Minimum amount
to be collected
Small or light units
e.g. Berries, peas etc.
25 g or less 1 kg
Medium sized units
e.g. Apple, orange, carrot, potato
25 - 250 g
1 kg
(At least 10 units)
Large sized units
e.g. Cabbage, cauliflower, melons
>250 g
2 kg
(At least 5 units)
Dairy products
e.g. Butter, Milk cream, Cheese
0.5 kg
Poultry products e.g. egg 0.5 kg
Animal tissue e.g. Meat, Chicken, Fish and
their products
0.5 kg
Oil and fats e.g. Cotton seed 0.25 kg
Spices e.g. Cumin, Fennel 0.5 kg
Cereals e.g. Wheat, Rice 1.0 kg

24. Sample preparation
• Reduce the sample – sub sampling
• Store reserve portion of the sample
• Homogenize the sub sample by :-
Chopping
Grinding
Blending
• For this, knives, grinder, warning blender are used.
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25. Extraction process
• There are several extraction approaches
employed such as:
• Pressurized liquid extraction(PLE)
• Supercritical fluid extraction (SFE)
• Aqueous extraction
• Microwave-assisted extraction(MAE)
• Solid-phase extraction (SPE)
• Solid-phase microextraction (SPME)
• Matrix solid-phase dispersion (MSPD)
This is a
fundamental
process in
pesticide residue
analysis because
no in-situ
technique has
yet been
invented.
Extraction is the process by which toxicant is transferred from the
treated bulky biological material in to a solvent.

26. Pressurized liquid extraction(PLE)
PLE Is an accelerated solvent extraction.
Uses liquid for extraction.
Samples are loaded into vessels, which require a dispersant/drying agent to
aid solvent flow.
Extraction occur at elevated temperature I.e room temp to 40-200 and
pressure 1000-3000 psi to improve recoveries.
Static extraction mode is used to equilibrate the sample to extraction
conditions and dissolve the analytes in liquid.
Vessel is flushed with solvent rapidly, rather than a controlled dynamic flow in
Superficial Fluid Extraction using a restrictor thus making PLE faster than SFE.
Any liquid can be used provided it can be pumped through sample and its
pH is between 2-12.

27. Supercritical fluid extraction (SFE)
SFE uses supercritical fluid as an extraction solvent.
CO2 is common solvent used in SFE, due to its safe nature,
uncreativeness, ready availability and inexpensiveness.
The rest of the process is similar to that of Pressurized
Liquid Extraction.

28. Solid-phase Extraction
Solid-phase extraction is a separation process by which compounds that
are dissolved or suspended in a liquid mixture are separated from other
compounds in the mixture according to their physical and chemical
properties.
SPE consists of four steps: column preparation (prewash), sample loading
(retention or sorption), column post wash, and sample desorption (elution
or desorption).
compounds of interest are retained on the sorbent while interferences are
washed away.
Analytes are recovered via an elution solvent.

29. Clean-up
Clean-up is done to obtain uncontaminated products for analysis.
The clean-up process is vital especially in analysis of fatty foods.
Liquid-liquid and liquid-solid partitioning in which separation is based on polarity is the
primary mean of clean-up in residue analysis.
Separations based on molecular weight, gel-permeation or size exclusion chromatography is
common clean-up technique.
Other techniques used include precipitation, centrifugation, ultrafiltration, dialysis and
immunochemical.
Isolation of toxicant from interfering substances or solvents.

30. Various method of estimation

31. Spectrophotometric Method
• Three kinds of spectrophotometric method used in pesticide
residue analysis.
• Ultraviolet, visible and infra-red methods.
• Measure the conc. of pesticide in solution by measuring the
amount of the light that is absorbed by the solution.
• Range 180nm to 400nm

32. Chromatography
• The chromatography consist in the unique distribution of the
components of a mixture between stationary phase and
mobile phase due to different affinity of a single compounds
between forward both phase.
• It involve passing a mixture dissolved in a “mobile phase”
through a stationary phase, which separates the analytes to
be measured from other molecules in the mixture based on
differential distribution coefficient and different net rate of
migration between the mobile and stationary phase.
Chromo means “colour” & Graphy means “ To write”

33. Various techniques of chromatography
• Paper Chromatography
• Thin layer
Chromatography
Planar
Chromatography
• Gas Chromatography
• Liquid Chromatography
Column
Chromatography

34. Planar Chromatography
• Planar chromatography is a separation technique
in which the stationary phase in or on a plane.
• The plane can be paper, serving as such or
impregnated by a substance as the stationary bed
(Paper Chromatography)
OR
• A layer of solid particles spread on a support such
asa glass plate ( Thin layer chromatography)

35. Column Chromatography
• Stationary phase is placed into glass or metal column.
• The mixture of analytes is then applied and the mobile
phase, commonly referred to as the Eluent passed
through column either gravity feed or by use of
pumping system or applied gas pressure.
• The stationary phase either coated on to discrete small
particles (Matrix) and packed in to column.
• As the Eluent flow through the column analyte
separate on the basis of their distribution coefficient
and emerge individually in the eluent as it leaves the
column.

36. Gas Chromatography(GC)
• Volatility and thermal stability.
• Carrier gas- Helium, Argon and Nitrogen: Which are
chemically inert.
• The stationary phase is adhered to the inside of a small-
diameter glass tube ( a capillary column) or a solid matrix
inside a larger metal tube ( a packed column).
• 80% of pesticide can be analysed.
• Organochlorines, Organophosphates, Carbamates

37. Pesticide detected using GC
Organochlorines e.g. Dieldrine, DDT, Lindane
Organophosphorus e.g. Diazinon, dichlorovos,
malathion
Pyrethroid e.g. cypermethrin, deltamethrin
Aromatic fungicides e.g. Chlorothalonil, quintozene
Carbamates, imidazole, benzoylureas, sulfonylureas

38. Liquid Chromatography (LC)
• It is a separation technique in which the mobile phase is a
liquid. Present day liquid chromatography that generally
utilizes very small packing particles and a relatively high
pressure is referred to as high performance liquid
chromatography (HPLC).
• In the HPLC techniques, the sample is forced through a
column that is packed with irregularity or spherically shaped
particles or a porous monolithic layer ( Stationary phase) by a
liquid (Mobile phase) at high pressure.

39.  Carbamates e.g. Carbendazim
 Conazoles e.g. Fenamidone
 Sulfonylureas e.g. triasulfuron
 Triazines e.g. Simazine
 Phenylureas e.g. linuron
 Strobilurins e.g. Azoxystrobin
 Neonicotinoids e.g. Acetamiprid
 All the above plus their metabolites can be analysed by liquid
chromatography
Pesticide detected using LC

40. QuEChERS- Multiresidue-Method
ADVANTAGES
Rapid (8 Samples in Less Than 30 min)
Simple (No Laborious Steps, Minimal Sources of
Errors)
Cheap
Low Solvent Consumption (10 ml Acetonitrile)
Practically no Glassware Needed
Wide Pesticide Range (Polar, pH-Dependent
Compounds)
Extract in Acetonitrile (GC- and LC-Amenable)

41.  Avoid un-judicial use of pesticides
 Use only recommended dose of pesticides
 Use pesticides only need base
 Follow safe harvesting period i.e. waiting period
 Avoid to use of pesticide near to ripening stage of crop
 Follow washing and cooking before use of vegetables
 Use botanicals / microbial insecticides for the control
of various crop pests
 Purchase pesticides only from authorized dealers
 Banned pesticides should not be used for pest control
 Get detailed information from the authorized people
before mixing of various pesticides.
 Follow IPM & GAP
Steps to overcome
pesticide residue problems
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42. “Technical development always follow
the way from the primitive via the
complicated to simple”