CP 4

General Physiology and Toxicology

ALJUN P. PEREZ, MSc.

RENNIELYN F. CANALES, MSc.
Instructor
College of Agriculture and Agri-Industries
Caraga State University
Ampayon, Butuan City, Agusan del Norte, Ph
Chapter 3
Insecticide Research, Development and TOPIC OUTLINE
Bioassay

Research and Development Process

Data generation and their value of Safety
Economic and Legal Aspects of Insecticide Use
Bioassay and the Factors to Consider
Probit Analysis
Chapter 3

Toxicology
✔ Is the study of toxic compounds
✔ Is the study of the effects of certain compounds

Branches of Toxicology:
1. Pharmacological toxicology
✔ study on the possible effects of “would be drugs”
✔ Interested on the maximum effects of the drugs taken
in
✔ Consider the effect of drugs on man (either its
minimum or maximum effect)
 Chapter 3

2. Medical toxicology
✔ considers the mechanism by which the substances
(drugs) may be degraded or activated in the body.
✔ The metabolism of the compounds in the body is
considered here.
✔ Also interested on the usage of chemicals and the
harmful effects is determined by clinical diagnosis
and clinical treatment.
 Chapter 3

3. Industrial toxicology
✔ interested on the safety of industrial workers and other people who might be
accidentally exposed to the chemicals.
✔ Its main emphasis is on human and domestic animals.

Example: Minamata disease – a disease caused by mercury poisoning in Japan.

 Chapter 3

4. Veterinary toxicology
✔ considers the foreign substances that may affect
domestic animals and substances present on
animal products which may affect the
consumers.
 Chapter 3

5. Pesticide toxicology
✔ Study on the effect of toxicants
✔ Study on the mechanism (mode) of action of toxicants and the difference in their
inhibitory action and their metabolic fate in various organisms. Toxicologists
consider the tolerance level of pesticides to man by killing target pests without
affecting non-target ones.
✔ It is interested on the effects of occasional exposure to the toxicants
(contact/dermal, oral or inhalation).
 Chapter 3

5. Pesticide toxicology
✔ Therefore, before a toxicant could be marketed it must have the acute, chronic
and inhalation data.

Acute: - effect is immediately observed

Chronic: - effect is not immediately observed but there is minute
quantity of pesticide residues in the body
Inhalation data: - residues remain in lungs

Insecticide toxicology – deals with the study of a particular group of toxicant in

relation to different groups of organisms (insects).
 Chapter 3

6. Environmental toxicology
✔ concerned with the effects of toxins, whether purposely applied (such as
pesticides) or derived from industrial processes, on health and environment.

✔ It deals with the minute quantities

of pesticide in the environment
and on how the organisms react
to these.
Chapter 3

In 1985, the Asian and Pacific region accounted for 16% of the global pesticide
consumption, with average annual growth of 5-7%. Table 1 below shows the
percentages and values international market on the use of pesticides.
Table 1. The division of global pesticide use according to main groups, the percentage of regional share and value
of pesticide consumption in the Asian and Pacific region.

Class Global percentage Regional percentage Regional value ($US million)

Herbicides 44.2 13.4 338.00

Insecticides 31.9 75.8 1,921.90
Fungicides 17.5 8.4 213.30
Others 6.4 2.4 61.30
Total 100 100 2,534.50 10
Chapter 3

Rank of Pesticides according to

use in the USA:

✔ Insecticides
✔ Herbicides
✔ Fungicides
Chapter 3

Use of Pesticides in the Philippines is for:

✔ Crop Production
✔ Animal Health
✔ Public Health
✔ Stored product pests
✔ Structural pests etc..

1978 - pesticides attained its peak of maximum

importation in the Philippines.
1986 - IPM: core of crop protection policy
✔ In May 1986, through a presidential
pronouncement of then President Corazon C.
Aquino, the Philippines adopted IPM as the core of
crop protection policy in agriculture. Since then IPM
has evolved into a more dynamic, practical and
farmer-driven activity.
 Chapter 3

Application of pesticides by farmers is influenced by:

✔ Availability and cost of pesticides

✔ Kind of crops produced
✔ Availability of financing

Application of Pesticides in Plantations is influenced by:

✔ Residual tolerance limit

✔ Established by the consumers
✔ Established by mother companies
 Chapter 3

FPA- Fertilizer & Pesticide Authority

✔ Issues rules and regulations governing the

importation, manufacture, repacking, distribution,
delivery, sale, storage, and use of pesticides.
✔ It has jurisdiction over all handlers of pesticides
✔ It issues registration and license to pesticide
handlers
✔ It issue a stop sale, stop use, removal and hold order
of pesticides included on the restricted list.
Chapter 3

Symptoms of Poisoning exhibited by A latent period is usually

ORGANISMS observed before the appearance
of the first symptom.
- During this period the following
are usually observed:
a) Cleaning movement
b) Curling and telescoping
movements

Exciting period is manifested by:

1) Frantic running or flying
a) Excitation movements
b) Convulsion 2) Restlessness
c) Paralysis
d) Death
Chapter 3

Research and Development Process

Chapter 3

Research and Development Process

Recent studies indicate that major agrochemical companies spend an average

of 7.5% of their annual sales on research and development of new crop
protection products and on improving the activity or safety of existing ones
(CropLife, 2005; Phillips McDougall, 2005).

The development of a new product from discovery to first sales typically takes
8-9 years and costs roughly US$200 million. Of this, ca. US$67 million is
spent on chemistry related research, ca. US$80 million on biological studies,
and ca. US$53 million on toxicological and environmental fate studies.
Chapter 3

Research and Development Process

Need for new compounds

The main driving forces behind the research for new pesticides are as follows:

a) improvements over or replacement of existing products by new compounds that

are more environment-friendly and less toxic to nontarget organisms;

b) resistance towards currently used control agents;

c) new pests and diseases, including invasive species, necessitating novel control
methods;

d) economic incentives.
Chapter 3

Research and Development Process

FRAC, 2005
IRAC, 2005

Thus, resistance management

has become an essential part of
crop protection and one of the
promising ways of decreasing
the risk of its occurrence is the
Heap, 2006; HRAC,2005 use of novel compounds, ideally
with a different mode of action.
Chapter 3

Research and Development Process

Ways to find new bioactive compounds

It has recently been estimated that some 100,000 compounds have to be tested to
bring a new pest control agent to market. These new substances could be obtained by:

a) natural product screening;

b) screening of randomly prepared synthetic compounds, e.g., in-house or acquired

compound libraries;

c) analogue design using structural modifications of natural products or compounds

developed by other companies;
Chapter 3

Research and Development Process

Ways to find new bioactive compounds

d) enzyme inhibitor design based on vital biochemical reaction mechanisms;

e) computer aided molecular design using the three-dimensional (3D) structure of the
target site;

f) serendipity.
 Chapter 3

Research and Development Process

Important things to consider in order to obtain a qualitative response:

1. Experimental method
✔ it should be standardized
✔ Test insects should be uniform (size, age, sex)
✔ Extrinsic factors should be uniform
2. Number of insects used
✔ The choice of number should be governed by practical considerations.
✔ Greater accuracy will be obtained if there is a larger number/replicate, however, it should
not be over 50 but not less than 15.
✔ Population should be homogenous
✔ Randomize the allocation of insects to replicates or batches
3. Dosage
Dosage should be in geometric manner.
Chapter 3

Bioassay and the Factors to Consider

Bioassay

✔ (commonly used shorthand for biological assay), or biological

standardization is a type of scientific experiment. Bioassays

✔ are 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. Both are procedures by which the
potency or the nature of a substance is estimated by studying its effects on
living matter.
Chapter 3

Bioassay and the Factors to Consider

Factors Affecting Bioassay

1) Extrinsic factors 2. Intrinsic factors(inherent)

a) Temperature a) Specificity
b) Relative humidity b) Stage of the insect
c) Nutrition c) Instar
d) Population density d) Age
e) Light e) Size
f) Rainfall and dew f) Sex
g) Wind
h) Handling of insects
Chapter 3

Bioassay and the Factors to Consider

Extrinsic factors

A. Temperature - most insecticides are effective and most insects react normally at
ordinary temperature. In this case temperature should be kept the same.

Categories of reaction to temperature:

✔ Most chlorinated cyclodiene insecticides (chlordane, aldrin, dieldrin, heptachlor),

some organophosphates (diazinon) and toxaphene are more toxic at high
temperature (positive temperature coefficient).
✔ Gamma BHC gave variable results
✔ Pyrethrins and carbamates showed a slighty greater toxicity at low temperature
✔ DDT is more toxic at low temperature (negative temp.coefficient).
Chapter 3

Bioassay and the Factors to Consider

B. Relative Humidity (RH)

1. Pre-test effect – relative humidity may affect the physiological process and
alter the susceptibility of insects.

2. Exposure effect

✔ Contact poisons- minor effect

✔ Fumigants- reaction varies with species and type of fumigant used
✔ Residual film- reaction vary with insecticides and insect species used
e.g DDT is more toxic to P. xyllostella(diamond backmoth)at high & low RH
 Chapter 3

Bioassay and the Factors to Consider

C. Nutrition

✔ Quantity and quality of the rearing media affect the size and survival
capacity of an organism
✔ There is a difference in tolerance between recently fed individuals and those
starved for various periods.
✔ Diet affects the susceptibility of an organism

e.g Weevils reared on corn are more susceptible to pirimphos methyl than
those reared on sorghum.
Red spider mites are less susceptible to Solegnium acaricide when
reared on roses than on beans.
Chapter 3

Bioassay and the Factors to Consider

C. Nutrition
1. Effect of starvation
○ Starvation before a test ensures greater uniformity of the metabolic
processes and thus give more constant results. Feeding decreases
the susceptibility of an insect.
○ Starvation after the test
2. Toxicity of the stomach poison is decreased by
feeding after test which dilute the poison and hasten excretion.
3. Prolonged starvation after test decreases the vitality and body weight of
insects.
 Chapter 3

Bioassay and the Factors to Consider

D. Population density

✔ overcrowding will result to lower tolerance due to smaller size and higher metabolic
rate.
E. Illumination

✔ affects the tolerance of an insect through its metabolic rate

✔ affects the movement of insects
• Insects moving closer will contaminate each other. In some insects toxicity is enhanced
by high illumination.
e.g houseflies- more sensitive to DDT residues in light than in darkness.
Carbofuran- more toxic at 300 FC(footcandle) than at 450FC
• Under field condition sunlight may degrade the insecticides rapidly. Thus Carbofuran
remains for 15 days only due to sunlight degradation.
Tropical: 15-30 days Temperate >30days
Chapter 3

Bioassay and the Factors to Consider

F. Rainfall and dew

✔ surface moisture on foliage aids retention of dust.
✔ heavy rain may remove insecticidal deposition on the plant surface.

G. Wind
✔ Strong wind may result to uneven distribution, loss by evaporation and
mechanical removal of insecticides thus reducing toxicity

H. Handling of insects
✔ each species should be handled carefully according to their own habit and
mode of life.
✔ the cages should be well aerated, with uniform lighting and always kept clean
or thoroughly sanitized.
Chapter 3

Bioassay and the Factors to Consider

Intrinsic factors

A. Specificity

Group specificity- can be between classes of arthropods or within class insect

Insects will show different reactions to poisons

e.g Carbaryl- bees are more susceptible to this insecticide than houseflies.
Chapter 3

Bioassay and the Factors to Consider

Intrinsic factors

A. Specificity
Species specificity
Toxicity of insecticides differ in different insect species may be due to:

i. Uptake of insecticides (refers to the quantity taken in by the insect. It is

dependent on the following:
Hunger of the insect
Physical state of the poison
Manner of incorporation with the food
Repellency or attractiveness of the food
Chapter 3

Bioassay and the Factors to Consider

Intrinsic factors

ii. Penetration of insecticides

(stomach) poisons action would depend on the quantity of insecticides
penetrating the gut wall;mouth,oral
(contact) action depends on the quantity of insecticides contaminating and
penetrating the cuticle; bodywall
(fumigant) action depends on the quantity penetrating the cuticle and the spiracle.

The toxicity of the poison will depend also on its retention in the gut or cuticle.
The passage of food will vary on different insect species and may affect the
poison.
 Chapter 3

Bioassay and the Factors to Consider

Intrinsic factors

iii. Degradation of insecticides

✔ Rapid degradation will affect toxicity.

e.g Spodoptera eridania is tolerant to pyrethrin, which is easily degraded in

the alimentary canal, but not to rotenone

iv. Inherent toxicity after penetration

✔ There are differences in the relative toxicity of different poisons to different
insects even in equal amount of insecticides were able to penetrate the gut of
cuticle.
 Chapter 3

Bioassay and the Factors to Consider

Intrinsic factors

B. Stage
✔ Egg is generally resistant to the effect of poisons due to lack of penetration
✔ Order or resistance of different stages:
Egg>Pupa>Larva>adult

Causes of differences:
1) Penetration difference
2) Greater sensitivity associated with higher metabolism or more numerous sense organs
3) Greater tolerance due to the buffing power of fat reserves
• In some cases larvae(newly molted) are more susceptible than the egg, pupa or adult
• Toxicity variation in the different stages with gradual metamorphosis is slight
 Chapter 3

Bioassay and the Factors to Consider

Intrinsic factors

C. Instars
✔ Differences in the susceptibility is associated with the growth and changes associated with molting.
✔ The concentration required to give a particular kill increase with the different instars. The ration of
increase vary with the instars.
✔ The rate of increase will also vary with the insecticide used.
e.g C. fatigans from first to fourth instar; organophosphates- 4 to 7 times increase; carbamates- 3 times
increase; DDT- no change

Changes in molting:
Cuticle thickness
Reduction of pore canals
Reduction in lipid content which may hinder most contact insecticides
Size
• Penetration is faster on early instars than the later instar
Chapter 3

Bioassay and the Factors to Consider

Intrinsic factors

D. Age

✔ Larvae are more sensitive after molting due to the thinness of the cuticle and pore
canal with protoplasmic processes.
✔ Tolerance rise and fall during an instar
✔ Eggs vary in size depending on species of insects
✔ Pupa is more tolerant on its middle age
✔ Newly emerged adults are more susceptible but become tolerant at later adult stage
 Chapter 3

Bioassay and the Factors to Consider

Intrinsic factors

E. Size
✔ In general, larger animals require bigger dose than smaller ones
✔ To minimize size effect, express dose in weight/ weight basis

F. Sex
✔ Females are more tolerant than males
✔ Male rats are more tolerant than female rats
Chapter 3

Probit Analysis
Probit analysis is a
specialized form of
regression analysis, which is
applied to binomial response
variables, i.e., variables with
only one of two possible
outcomes (positive/negative).
The procedure transforms a
concentration-response
curve to a straight line that
can then be analyzed by
either least squares or
maximum likelihood
regression
Chapter 3

Probit Analysis
Probit analysis is a
specialized form of
regression analysis, which is
applied to binomial response
variables, i.e., variables with
only one of two possible
outcomes (positive/negative).
The procedure transforms a
concentration-response
curve to a straight line that
can then be analyzed by
either least squares or
maximum likelihood
regression
Chapter 3 Abbott’s Formula = natural
allowance for natural mortality
Probit Analysis Net percent mortality =
A-B x 100
100- B

Where:
A = % mortality in
treatment
B = % mortality in control
Chapter 3

Probit Analysis

Data that is published using the method of probits should always be on the basis of
TRUE CONCENTRATION values as well as the log values if graphs are used.
THANK YOü!