General

Physiology and
Toxicology
Ms. Gellie G. Casas
Course Instructor
 PART II

Bioassay of
Insecticides

AG-MC312
 Outline

1 2 3 4
Dose Methods of
Evaluation
Definition Response Toxicity
Of Toxicity evaluation
curve

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AG-MC312
 Introduction

▪ Recent advances in research and

technology has renewed interest in this
subject and resistance risk assessments
▪ Thus, it is important to analyze not only
the technical and formulated materials
but also minute quantities of their
residues on/in plant and animal tissues.

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 What is Bioassay

▪ Bios -life & assay- determination.

▪ Thus, bioassay stands for

determination of relative toxicity
of insecticides by studying and
examining their effects on living
organisms
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 Bioassay

▪ According to Finney (1952) the term biological assay should

be understood to mean “the measurement of the potency of
any stimulus physical, chemical or biological, physiological
or psychological by means of the reactions which it produces
in living matter”

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 Bioassay

▪ It measures the efficacy of insecticides on insect pests; determine the impact

on non-target organism; assessment of resistance

▪ It is typically conducted to measure the effects of a substance on a living

organism and are essential in the development of new drugs and in
monitoring environmental pollutants

▪ It is a procedure for the determination of the concentration of a particular

constitution of a mixture.
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 Principles of Bioassay

▪ It studies to evaluate the toxicity of insecticides with diverse

mode of action to the same species under the same test
conditions.
▪ In an effective bioassay, the indicator species should be
sufficiently sensitive to detect even small amounts of
insecticides and should express the response with increasing
concentrations.
▪ The toxic interactions of an insecticide with a biological system
8 are dose dependent.
 Effect of Dose

Relative toxicity of a substance is often expressed in the dose-

response relationship.

▪ Dose- quantity administered

▫ expressed as dose per organism weight, or mg/kg(where mg
represent toxicant dose and kg expresses animal weight)

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 Evaluation of toxicity in
insecticides
▪ LD50 (lethal dose) - the dose required to kill
50% of a test population.
▪ This value represents the lethal dose of the
poison per unit weight which will kill 50 per
cent population of test animals.
▫ It is expressed as milligrams per kilogram of body
weight
 The higher the LD50 value, the lower the
toxicity.
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 Examples
Substances LD50 (mg/kg)
Sugar >10,000
Caffeine 100
Nicotine 1
Rattlesnake venom 0.1
Botulism toxin (Clostridium botulinum) 0.0001

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 Other terms

▪ LC50 (lethal concentration)- is used to express the

concentration of the insecticides in the external media that will
kill half of the test population because the exact dose initially
given to the insect cannot be determined.
▪ LT50 (lethal time)- it represent the time required to kill 50% of
the test population at a certain dose or concentration
▪ ED50 & EC50 (effective dose & effective concentration)- used
to express 50% effective dose and 50% effective concentration.
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 Dose-response curves

 Are generated for various agents by plotting percent of population

affected vs. dose (mg/kg).

NOEL(no observable effect limit)- is the daily dosage of a pesticide that,

when administered over a long period, produces no observable
symptoms or pathological effects in experimental animals
- safety level of pesticides in humans
LOEL(lowest observed effect level) - as a point of departure for
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determining a safe dose.
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 Toxicity

▪ Ability of a chemical to bring about changes in the biological

system of the target animal.

▫ Acute toxicity – It is the acute stage of poisoning due to

the application of a single dose.
▫ Chronic toxicity- It is the condition of toxicity which lasts
for the entire life of the target animal and has the
accumulating effect of small repeated doses
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 Hazard Vs. Risk
▪ Hazard- It is the probability of
being harmed due to the use/
exposure /handling of the toxic
substance.
▪ Risk- It is the degree of physical,
biochemical and histochemical
changes acceptable in terms of
usefulness of a chemical and its
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possible effects on public health.
 Sources of Variability in dose-
response experiments

1. Age
2. Sex
3. Rearing temperature
4. Food supply
5. Population density
6. Illumination
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 Synergism
▪ It occurs when the resultant toxicity of a mixture of chemicals
is greater than the additive total of the individual chemicals
▪ Example synergist are sesame oil, piperonyl butoxide, can be
used to
1. Reduce the necessary dose of insecticide
2. Reduce the degree of resistance in resistance insects
3. Permit the use of previously ineffective insecticides

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 Dose vs. Dosage

Dose- quantity of pesticide (ai)

delivered to target organism
Dosage- quantity of pesticide
delivered over an area

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 Qualitative and Quantitative Bioassay
a. Qualitative bioassays- are used for assessing the physical effects
of a substance that may not be quantified, such as abnormal
development.
b. Quantitative bioassays- involve estimation of the concentration
or potency of a substance by measurement of the biological
response that it produce
- are typically analyzed using the methods of biostatistics

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 Factors affecting
bioassay:
▪ Biological:
▫ It is true that the selection of test insect for bioassay depends
on their susceptibility to toxicant yet factors such as stage of
the insect, sex, size etc.
▪ Physical and Chemical:
▫ physically contamination with toxic or non-toxic material may
interfere with the process of bioassay.
▫ Chemically some insecticides are more volatile (viz.phosdrin
aldrin, lindane etc.) and get vaporised readily
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 Basic Criteria for Test
Insect in Bioassay
Availability of test insect:
▫ It is extremely difficult to have bioassay program on the
basis of insect trapped from field.
▫ Rearing of test insect is therefore, an essential pre-
requisite for any bioassay program.
▪ Food:
▫ On plant food limited number of test insect is obtained
around the year.
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▫ Artificial diet has overcome this difficulty.
 Basic Criteria for Test
Insect in Bioassay
▪ Sensitivity:
▫ The test insect should be sensitive enough to the
insecticidal response
▫ e.g. Drosophila, housefly, mosquito larvae.
▪ Easy handling:
▫ The test insect must be such which could be handled
easily

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 Methods of Evaluation

▪ Topical application
▫ Topical application on the test insect is by
means of topical applicator.
▫ Most commonly employed method of
exposure of insect
▫ Insecticides is first dissolved and known
volume of the test solution is applied to the
body surface using a micro syringe
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 Methods of Evaluation

▪ Injection method
▫ The toxicant is directly injected in the
body system of organism by
hypodermic needle
▫ Injection is made at the abdominal
sterna or the intersegmental regions

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 Methods of Evaluation

▪ Dipping method

▫ The insects are picked up with

forceps and dipped in the
insecticide preparation which
either a suspension or an emulsion

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 Methods of Evaluation

▪ Leaf Dipping method

▫ The leaf containing insects is

dipped in the insecticidal solution
of known strength.

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 Methods of Evaluation

▪ Feeding method

▫ This is used for testing insecticides against larvae

▫ Larvae are placed individually in a container and starved
for a few hours, they are then feed individually on a small
leaf disk that had been topically treated with various
concentration of insecticides
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 Methods of Evaluation

▪ Fumigation method

▫ For some stored grain pests the

grains are subjected to fumigation
by suitable preparation of the
insecticide for a specified duration

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 Why Bioassay is
necessary ?
▪ Bioassay helps in ascertaining the potency of the pesticide used.
▪ Relative toxicity of different pesticides can be ascertained.
▪ Insect resistance to different insecticides can be studied.
▪ Micro-quantities of insecticidal residues retained by the plants can be
detected.
▪ Formation of toxic metabolites produced from the insecticides can also be
assured.
▪ New and promising toxicants can be screened.
▪ New formulations can be developed.
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 Ways to estimate LD50

1. Graphical methods- simplest way

2. Standard computation using a desk calculator
3. Computer program of software (SAS, SPSS,
PoLoPlus software)- the faster and more
accurate way

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 Questions?

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 Hope you
Thank You learned
for Your something
Participation today!

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AG-MC312