Pesticide analysis

SYED RASHED FAIZAN MEHDI
ROLL NO: 13451R0051
M.PHARMACY ANALYSIS

TOPICS COVERED
• INTRODUCTION
• METHODS OF PEST CONTROL
• CLASSIFICATION OF PESTICIDES
BASED ON CHEMICAL COMPOSITION
BASED ON ACTION
• PESTICIDES CYCLE
• ANALYSISOFORGANOPHOSPHORUS AND
ORGANOCHLORINE PESTICIDES ANALYSIS
• DETECTION OF PESTICIDES RESIDUES.

What is a pesticides?
Pests:
Pest is any animal , plant or microorganism that
causes trouble, injuries (Economic damage) or
destruction to plants or crops.
The common pests includes insects, bacteria,
fungi, rats (rodents) and weeds.
Pesticides:
• A pesticide is any substance that is
used to prevent, destroy, or repel pests
or reduce the damage pests cause.
• Pesticides can target insects
(insecticides), rats and mice
(rodenticides), weeds (herbicides) and
fungi (fungicides).
• Pesticides even include common
household disinfectants like bleach or
bathroom cleaner that kill bacteria.

METHODS OF PEST CONTROL
There are two methods available for the control of pest.
1. Natural control 2. Artificial control
Topographical influence of the
seasons changes, changing
temperatures, rainfall, soil,
atmospheric humidity and other
natural factors also shows their
effect on insects and their hosts.
Artificial control of pest have been
developed by man.
These methods can be categorized as
Mechanical, Agricultural, Chemical and
Biological methods

METHODS OF PEST CONTROL (ARTIFICIAL CONTROL)
1. Mechanical control 2. Agriculture control 3. chemical control 4. biological control
By using manual labor as
well as mechanical
devices for collection or
destruction of pest.
Like hand picking, burning
, trapping are employed
for the destruction of
eggs, larvae and adult
insects.
It is the oldest method
and includes deep
ploughing for eradication
of weeds and early stages
of insects. Alternative
crop rotation of changing
environmental conditions
are some methods which
lead to obstruction of the
life cycle of pests
Chemical agents are
used for killing pests or
for protecting crops,
animals or other
properties against the
attack of the pest.
Like DDT, BHC.
Biological pest control
uses the natural enemies
of the pests to destroy
them.
Like keeping cats in
granaries and mills.

CLASSIFICATION OF PESTICIDES
Based on the chemical composition
• Chlorinated hydrocarbons and related pesticides:
Dichlorodiphenyltrichloroethane (DDT) Hexachlorocyclohexane: Gamma benzene
• Chlorinated phenoxyalkanoic acid herbicides:
2,4-Dichlorophenoxyacetic acid or (2,4-D) 2,4,5-Trichlorophenoxyacetic acid

• Organophosphorus pesticides:
Parathion(O,O-Diethyl O-(4-nitrophenyl)
phosphorothioate)
• Carbamate insecticides:
Carbaryl (1-naphthyl methylcarbamate)
Mesurol(3,5-Dimethyl-4-(methylthio)phenyl
methylcarbamate)
Methyl parathion(O,O-Dimethyl-O-p-
nitrophenylphosphorothioate)

• Inorganic pesticides:
Calcium arsenate
Ca3(AsO4)2
• Miscellaneous insecticides:
Ethylene oxide Methyl bromide
Lead arsenate
PbHAsO4

• Pesticides of plant origin:
pyrethrum Nicotine( 3-(1-methyl-2-
pyrrolidinyl)pyridine)

Based on the Action
1)Fungicides: They are used to prevent or control the plant diseases caused by phytopathogenic fungi
and mostly are used as seed or soil fungicidal disinfectant.
They can be of different types based upon their use
a)Disinfectant for seeds
b) Disinfectant for soil
c) Leaf fungicides
2)Herbicides: They are weed killers, which are used for destroying the unwanted plant or to prevent
their growth.
3)Insecticides: Insecticides can only be used when the materials to be protected are already infested
with insects at various stages of development. They cannot be used previously as insecticides before
the infection.

Based on the Action
4)Acaricides:
The substance contains both fungicides and insecticides, they are used to control infestation caused by
mites especially those belong to arthropod family.
5)Nematocides: they are used for the control of nematodes like thread-worms living-free in soil and also
plants.
6)Rodenticides: They are agents used to prevent the infestation of the rodents in the cultivated crops.
7)Bactericides: these are the agents which are mostly used for the control of bacterial infection.
Other agents such as molluscides, bird deterrents, rodent deterrents are also used for
protection of plants.

Pesticide cycle:
• Adsorption
• Volatilization
• Spray drift
• Runoff
• Leaching
• Absorption
• Degradation
(Breakdown
Processes)

• Adsorption is the binding of pesticides to soil particles.
• The amount a pesticide is adsorbed to the soil varies with the type of
pesticide, soil moisture, soil pH and soil texture.
• Pesticides are strongly adsorbed to soils that are high in clay or organic matter.
• They are not strongly adsorbed to sandy soils.
• Most soil-bound pesticides are less likely to give off vapours or leach through
the soil.
ADSORPTION

• Volatilization is the process of converting solids or liquids into a gas, which
can move away from the initial application site.
• Pesticides volatize most readily from sandy and wet soils.
Volatilization
• Hot, dry or windy weather and small
spray drops increase volatilization.
• Incorporation of the pesticide into
the soil can help reduce volatilization.

SPRAY DRIFT
Spray drift is the airborne movement of spray droplets away from a
treatment site during application
Spray drift is affected by:
1.Droplet size – smaller- more likely they will drift.
2.Wind speed – stronger- more pesticide spray will drift.
3.Distance between nozzle and target plant or ground.
• Drift may also hazard to people, domestic animals, pollinating
insects.
• Drift can contaminate water bodies like ponds, streams, and
ditches; harm to the fish or other aquatic plants and animals

RUN OFF
• Runoff is the movement of pesticides in water over a sloping surface
• The pesticides are either mixed in the water or bound to eroding soil
• Runoff can also occur when water is added to a field faster than it can be absorbed into the
soil
• Runoff from areas treated with pesticides can pollute streams, ponds, lakes, and wells
Pesticide residues in surface water can harm animals and contaminate groundwater

LEACHING
• Leaching is the movement of pesticides in water through the soil
• Leaching occurs downward or sideways
• Groundwater may be contaminated if pesticides leach from treated fields, mixing sites,
washing sites, or waste disposal areas

ABSORPTION
• Uptake of pesticides and other chemicals into plants or microorganisms
• Pesticide residues may be broken down or remain inside the plant or animal, when
the animal dies or as the plant decays released back
• Some pesticides stay in the soil long enough to be absorbed by plants grown in a field
years later
• They may damage or leave residues in future crops.

Degradation or Breakdown Processes
• Microbial breakdown is the breakdown of chemicals by microorganisms such as
fungi and bacteria.
• Chemical breakdown is the breakdown of pesticides by chemical reactions in the
soil.
• Photo degradation or Photolysis is the breakdown of pesticides by sunlight. All
pesticides are susceptible to photo degradation to some extent.
• Hydrolysis: Water also degrades pesticides by dividing large molecules into
smaller ones.

Types of analytical methods for pesticide residues
There are several approaches which vary in their degree of complexity; in the time, effort and
analytical instrumentation required to complete them.
• Multiresidue methods(MRMs):
It has been designed to detect band measure a multiplicity of residues in a range of foods.
multistep contains:
Out of 10 MRMs currently used by FDA and USDA, *8 based on gas chromatography and the *2 based on
HPLC.
However, none of these MRMs procedures can detect all the residues on all crop types.
In practice, they represent a compromise among the number of residues that can be detected, the range
of food types that can be handled, and the levels of residues that can be measured.
The principal advantage resides in the number of different residues that they can detected and
determined.

Types of analytical methods for pesticide residues.
• Single residue methods(MRMs):
It has been designed to measure a single analyte and, often, its principal metabolites and
transformation products of toxicological importance.
multistep contains:
Each step is optimized for the analyte of interest.
Generally, they are less time consuming to perform and often provide lower limits of detection than
MRMs.
However, they do vary in the level of validation to which they have been subjected Volume 2 of the
Pesticides Analytical Manual(PAM 2) consists solely of SRMs.
In PAM 2, those methods that have received EPA review are listed with Romain numerals, whereas
methods that have not been reviewed are lettered.

• Semiquantitative and Qualitative methods:
Semiquantitative and qualitative methods range widely in their abilities to estimate the level of a
particular pesticides residues in a sample.
In general, they are capable of detecting a limited number of somewhat similar pesticides.
Also called as Screening methods as they are capable of assaying a large number of samples for the
presence of limited number of pesticides residues in relatively short time
Additionally, they are generally robust in character(i.e. less sensitive to small changes in the purity of
reagents, quantities of reagent, time, temperature and environmental conditions).
Semiquantitative methods provide an estimate of the concentration ranges for detected residues,
Qualitative methods will detect the pesticides if present above some predetermined level.
The principal benefit of these methods are their low cost, relative speed, and simplicity.
These methods use TLC, Enzyme inhibition, and Immunoassay.

• Quantitative methods:
The basic steps of a quantitative analytical method for pesticides residues include the following:
1)Sample preparation: the plant parts are separated into edible and non edible fractions followed by
chopping , grinding, or macerating of the sample.
2)Extraction: Pesticides residues are removed from most of the samples other constituents by
solubilizing them in a suitable solvent. This steps often involves blending the chopped sample with
solvent in a homogenizer, followed by a filtration.
3)Clean up: The crude extract is purifies further by removing those co-extractives that can be
interfere in the subsequent determination steps.
4)Separation: The components of the purifies extract are further separated by a differential
partitioning between a mobile phase (liquid or gas) and a stationary phase.
5)Detection and quantitation: A physical parameter of the separated components in the mobile
phase is measured as they passthrough a detector, this signal is then related to the quantity of
analyte via a quantitation step.

Examination of pesticides residues and heavy metals
The vast number of pesticides, and the fact that it is usually not known whether, which and in
what quantity the plant grower has used pesticides, make it expedient to test for typical
representatives of major groups of pesticides
1)Examination for Chlorinated Hydrocarbon and Organo-Phosphoric Acid Esters.
2)Determination of Total Chlorine and Phosphorus.
3)Determination of chlorides.
4)Determination of phosphates.

A)Preparation
of sample:
Examination for Chlorinated Hydrocarbon and Organo-Phosphoric Acid Esters.
Contd..

B) Gas Chromatography
HP 5750 G gas chromatography with ECD-63Ni and FID.
Temperatures: Injector block 240℃, detector 290℃, furnace running
isothermally190 ℃, and in program 150-200℃.
Carrier gas: Purified helium, 30mlmin.
Flushing gas: Argonmethane 90:10, 100-130mlmin.
Advanced speed of paper: 6.35mmmin
Columns: 3.8% SE 30 on chromosorb W AW-DMCS(80-100 mesh), 1.8m glass
column.
The quantitative evaluation is made either on the area, of the height of the
peak, in comparison with comparative substance and internal or external
standards.

C) Determination by Gas Chromatography
(1) First separation system
Use vitreous silica column, 30m long with an internal diameter of 0.25mm, packed with a chemically bound
phase of 5% phenyl, 95% methyl-polysiloxane. Use the following temperature program:
• Heat at 60℃ for 0.5 minutes.
• Increase the temperature at a rate of 30℃ per minute to 160℃ and maintain this temperature for 2 minutes.
• Increase the temperature at a rate of 2℃ per minute to 250℃ and maintain this temperature for 5 minutes.
Use a “split/split-free” injector to inject the sample solution and maintain the injection port at a temperature of
240℃. Inject a volume of 1 microliter at a rate of 30 seconds(“split-free”). The detector temperature should be
at 300℃.
(2) Second separation system
Use a vitreous silica column, 15m long with an internal diameter of 0.25 mm, packed with a chemically bound
phase of 7% cyanopropyl, 7% phenyl, 86% methyl polysiloxane. Use the following temperature program:
• Heat at 60℃ for 0.2 minutes;
• Increase the temperature at a rate of 30℃ per minute to 180℃ and maintain this temperature for 1 minute.

Determination by Gas Chromatography
• Increase the temperature at a rate of 2℃ per minute to 250℃ and maintain this temperature for 5
minutes.
Use an on-column injector to inject a volume of 1 micro liter of sample solution. The detector temperature
should be 300℃.
Measure the peak height of the pesticides obtained in the chromatograms and calculate the concentration
of the residues in mgkg using the following formula:
Ht × 10/W × Wt/ht
Where
Ht= Peak height obtained for the test solution in mm.
W= Quantity of the sample in the purified extract (g).
Wt= Quantity of the pesticides in ng in the reference solution injected.
Ht= Peak height obtained for the reference solution in mm.

Examination for Total Chlorine and Phosphorus.
A) Preparation of Samples
As most of the pesticides
contain organically bound
chlorine and phosphorus, the
WHO has recommended the
following procedures for
estimation:

B)Preparation of
column
• Use Florisil grade 60/100 PR, activated at 650℃ as support.
• If this material is obtained in bulk, It is transfer to the glass jar with glass
stopper and Stored in dark.
• Before use, heat at not less than 130℃, cool in a desiccator to room
temperature
• Heat once again to130℃ after 2 days
• Prepare the column (external diameter, 22 mm) which contains, after
setting.
• 10cm of activated Florisil topped with about 1cm of anhydrous sodium
sulfate R.
• Pre-wet the column with 40-50 ml of light petroleum, place a graduated
flask under the column to receive the elute.

C) Method

Method
Cont.…

Determination of Chlorides.
Apparatus:
The determination is made with a
spectrophotometer capable of
measuring absorbance at 460 nm
using absorption cells with path
length of 2cm and 10cm
Method

Determination of Phosphates.
• The phosphomolybdate method is based on the reaction of the phosphate ion with
ammonium molybdate to form a molybdophosphate complex.
• Which is subsequently reduced to form a strongly blue-colored molybdenum complex,
the intensity of the blue color is measure spectrophotometrically.
• This method is applicable for the determination of any phosphates that have undergone a
prior separation procedure.
• Naturally occurring phosphates are present in most samples and are often not removed
during the clean-up procedure.
• In order to obtain background values, therefore, it is necessary to proceed with the
determination for all the samples even those with no phosphate containing pesticide
residues.
• Extracts of most uncontaminated materials contain about 0.05-0.1 mg/kg of phosphorus.
• Therefore, no contamination with organophosphate pesticides can be assumed for results
in this range.

Determination of Phosphates.
Apparatus:
The determination is made with a
spectrophotometer capable of
measuring absorbance at 820 nm
using absorption cells with path
length of 1cm.
Method

REFERENCES
1.A text book of Quality Control of Herbal Drugs by Dr.s.Ravi shankar,
pg no:210 to 245.

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