Toxicology of Insecticide –

Metabolic Inhibitors
Metabolic inhibitors

 Respiratory metabolic inhibitors

 Carbohydrate metabolism inhibitors
 Mixed function oxidase inhibitors
 Amine metabolism inhibitors

 Insect hormones
 Chitin synthesis inhibitors
Respiration
ATP synthesis at F1 results from repetitive
comformational changes as γ rotates


Rotates 1/3 turn- energy for ATP release
Insecticides affecting respiration

1. Arsenicals
2. Dinitrophenols
3. Fluorine compounds
4. Rotenones and rotenoids
1. Arsenicals

 Element – non toxic, compounds are toxic

Toxicity depends on the % metallic arsenic & water solubility ( more
solubility – more toxicity)
Two types
i) White arsenic/ arsenious oxide / arsenic trioxide (As2 O3)– anhydrides of
arsenious acid
ii) Arsenic oxide / arsenic pentoxide (As2O5) – anhydrides of arsenic acid

Arsenites – less stable, more toxic to plants & insects

Arsenates – less toxic than arsenites, more stable, safer to plants
1.Paris Green ( Copper acetoarsenite - (CH2COO)2Cu.3Cu(AsO2)2

o Brilliant green in color

o First compound developed against Colorado potato beetle in 1865
o Contains 33-39% metallic arsenic, 2-3% of which is water soluble
o Phytotoxic to plants
o Stomach poison to insects, used as mosquito larvicide

2.Sodium arsenite (NaAsO2and Na2 HAsO3)

o Highly water soluble, highly phytotoxic, sold as liquid formulation

o Used as poison bait, weed killer
o Arsenic content up to 44-57%
o Has residue problem
3. Calcium arsenate (Mixture of tricalcium arsenate Ca3(AsO3) and acid calcium
arsenateCaHAsO4)

☻Used as early as 1907

☻Basic calcium arsenate [Ca3 (AsO4)2 ]3 is the principal constituent of ‘safe’
calcium arsenates.
☻Arsenic content is upto 37%, (Kilmag) contains 25% arsenic
☻Soluble in water to 0.4 – 0.5%
☻LD50 35 mg/kg.
☻Commercial form 25D, 15% bait and 70% WP and
☻Used against cotton boll weevil, insects in orchards and garden crops.
☻LD50 (µ g/g) Pieris rapae 740, Trichoplusia ni 500, L. decemlineata 70 – 140.
4. Lead arsenate (PbHAsO4) (= acid lead arsenate).

☻Contains 20% arsenic and 0.25% soluble in water

☻Commercial formulation - acid orthoarsenate PbHAsO4 and basic

orthoarsenate Pb4 (PbOH) (AsO4)3

☻Used as insecticide in Massachusetts (USA) in 1892.

☻Commercial preparation contains 14% arsenic, hence less active than

acid lead arsenate.

☻Least phytotoxic to plants

☻LD50 100 mg/kg

☻ LD50 (µ g/g) B. moori 90, Leptinotarsa decemlineata 140-240

5.Arsenious oxide As2O3 (= white arsenic, arsenic trioxide).

☻Highly toxic, cheap contains 75% arsenic.

☻Solubility 1.2. g /100 cubic cm @ 200C, hence phytotoxic.
☻It is used as poison baits for grasshoppers, armyworms, ants and
cockroaches.
☻LD50 60-150 mg/kg for chicken, 10-30 for rabbit.

6.Basic copper arsenate (Cu (CuOH) AsO4)

☻26% arsenic, 56% copper oxide and water solubility is 0.1%.

☻used against chewing insects.
Toxicity

Man and other mammals

☻Abdominal pain, vomiting, a precipitious fall in BP (increased dialation and

permeability of capillaries) leading to a state of stock.
☻In lethal case, death may be delayed even upto 14 days and proceeded by
vomiting and profuse diarrhea due to direct effect on the alimentary tract.

☻ In sublethal case, polyneuritis with pain and tenderness in the limbs.

☻High altitude increases arsenite toxicity

☻A dose normally kills 5% of mice, kills 70% at attitudes of 2000 ft.

Insects

☻Stops eating, regurgitation (lepidoptera) activity steadily reduced and

☻An inevitability of increasing completeness, terminates in death.

Mode of action

As the arsenic and phosphorus are in the same position in the periodic table of
elements, outermost electronic shells are more reactive.

Hence, arsenic can enter into e- transport chain and cause arsenolysis similar to
phosphorolysis.

P occupies a unique place in ATP, P-O-P bond and the arsenic can partially
substitute for ‘P’ resulting in arsenolysis with the loss of ATP.

Arsenates

☻Interfere with both substrate level phosphorylation (in glycolysis) and

☻oxidative phosphorylation (e- transport chain) as uncouplers ( as a substance

that do not allow the energy released by down hill transfer of e- in the e-
transport chain to couple ADP to change to ATP – resulting in non-production of
ATP).
Arsenites

Combines with SH groups of enzymes viz., pyruvate dehydrogenase

(glycolysis) and α - keto – glutarate dehydogenase (krebs cycle) and results
in arsenolysis.

SH + NADP + PI → SP + NADPH2
ADP Phosphorylation
ATP + S
SH + NADP + As(OH)3 → SAs (OH)2 +
NADPH2
+ H2O Arsenolysis
S + A3 (OH)3
 The evidence for the inhibition are

i.
 inhibition of the enzymes in in vitro
ii.  the two enzymes accumulates in arsenites treated
iii.  in living insects, arsenites results in respiratory failure
iv.  no nerve poisoning symptoms are mimicked by arsenites but
they resemble the symptoms of rotenone.

 leactic dehydrogenase, α - glycerophosphate and cytochrome

oxidase are also susceptible to arsenicals.
2. Fluorine compounds

 Fluorides - substitutes for arsenates.

 Highly active and do not leave residues on food crops like arsenates.
 Stomach poisons and toxicity depends on the fluorine content.
 Destroy the gut epithelium
 Inhibit number of metalloenzymes to the respiratory pathways and elsewhere.

 Rspiratory enzymes inhibited are phosphoglucomutase enolase, succinic

dehyehogenase and cytochrome oxidase.
 The non-respiratory enzymes inhibited are phosphatases, peroxidases
catalases etc
Inorganic fluorines
i.Sodium fluoride (NaF)

Discovered in 1915.
 Pure mineral is white powder but insecticidal product is always green to avoid
confusion with flours / medicines
soluble in water to the extent of 4.3% at ordinary temperatures.
used for the control of cockroaches, ants, other household pests and biting
lice (malophaga) of poultry.
Because of their phytotoxicity not used on plants.
Toxic to all forms of life and used as insecticide, rodenticide, herbicide and
wood preservative.
 LD50 rate (oral) 180-200 mg/kg.
Death of human beings @ 57 mg/kg.
ii.Sodium hexafluorosilicate (Na2SiF6)
(Disodium hexafluorosilicate; sodium fluorosilicate ‘Prodan’. )

white odourless and granular powder.

Used as poison baits for grass hoppers, crickets, cutworms, weevils and also
as moth proofing agent.
 Highly phytotoxic to plants.
LD50 rat (oral) 125 mg/kg.

iii. Sodium fluoaluminate / Sodium aminofluoride / cryolite (Na3AlF6) ‘Kryocide’

Natural mineral in greenland as well as synthetic form.

 white crystal discovered in 1929.
Effective against chewing insects like, codling moth of apple, pumpkin beetles etc.,
 LD50 rat >7500 mg/kg.
Organic fluoride compounds
i.Sodium fluoroacetate (FCH2COONa)

Colorless hygroscopic powder, soluble in water and insoluble in solvents.

High mammalian toxicity prevented the large scale use. used as rodenticide.
In 1930 it was reported to be systemic and was effective against bean weevil.
During World War II Germans were searching for warfare agents for which no
antidote was available. One such compound was fluoroacetatic acid
from South African plant Dichapetalum cymnosum or gifblaar.
The toxicity was due to lethal synthesis.
Formulated as aqueous solution containing 0.5% dye as warning color
Rapidly absorbed by gastrointestinal tract and not by intact skin but absorption
is faster when there is dermatitis or skin injury
 LD50 - 0.22 mg/kg.
2. Fluoacetamide (FCH2CONH2) : (2, fluoroacetamide) ‘Compound 1081’.

 Crystalline solid freely soluble in water and moderately soluble in acetone and
little soluble in chloroform.
 Systemic insecticide - scales aphids and mites in fruit trees. used as rodenticide.
 Animals show restlessness, irritability, clonic convulsion and irregular respiration.
 Absorbed by skin.
 Less toxic than fluoroacetate - slow conversion to fluoroacetate in the body.
 Acetamide and L-cysteine hydrochloride - antidotes if given within 20-60 min.
after poisoning depending on species.
 LD50 13 mg/kg.
Symptoms
 Nausea, and vomiting
 Cardiac irregularities, cyanosis, convulsions. and death from ventricular
fibrillation and respiratory failure.
 Lethal dose for man 2-10 mg/kg, oral LD50 rat 0.22 mg/kg.
 Monoacetin injected intramuscularly as well as treatment with acetannide
10% solution intravenously as in glucose afford protection against man.
 Commercially aailable monoacetin (60% glycerol monoacetate) was
effective against mice, rats, rabbits, dogs etc.,
 Barbiturate help in reducing conventions.
Mode of action

Fluoroacetate has no effect on all enzymes tested in vitro.

Its toxicity was due to in vivo conversion to fluorocitric acid in the body just as
pyruvic acid incorporated into TCA cycle to form citric acid.
With the mediation of acetyl Co-A and condensation with oxaloacetate,
fluoroacetate → fluorocitrate which is termed as ‘lethal synthesis’.
Because of the structural similarity of fluorocitric acid to citric acid, fluoro citric
acid competes with the target enzyme of citric acid, aconitase and there
by blocks enzyme action.
 Evidence for this is:
i. Accumulation of citrate in fluoroacetate treated insects

ii. Fluoroacetate → fluorocitrate which is more toxic

iii. Aconitase powerfully inhibited by fluorocitrate in vitro

3. Dinitrophenols

They are used as insecticide, ovicides, herbicides and fungicides.

They were first used in 1890s.
They are yellow, relatively odorless solids, slightly soluble in water but readily
soluble in organic solvents.
Their acidic properties make them combine with bases and readily form water
soluble ammonium, Na, K and Ca salts.
These water soluble compounds are extremely toxic and mainly used as
herbicides.
For use against insects and mites, they are formulated with petroleum oils and
used as dormant sprays and ovicides.
i. DNOC (4,6 dinitro-o-cresol 2,methyl – 4,6 dinitrophenol) ‘Trifanex’, ‘Trifocide’,
‘Trifrina’
Yellow odorless solid, soluble in water to 0.014% at 150C meeting point 850C.
Readily forms salts with organic and inorganic bases and the
Ammonium, Na, K, Ca and barium salts are water soluble.
 Insecticide and acaricide with stomach and contact action.
In 1892 marketed as ‘Antinonnin’ for the control of nun moths.
 It was also used as contact herbicide.
It was in use 1930-1940 because of ovicidal action. LD50 25-40 mg/kg.

ii. Dinex or dinex-diclexine (2-cyclohaxyl – 4,6-dinitrophenol – 4,6 dinitro – o –

cydoexyl phenol, DINOCHP) ‘ Dynone I’

This is a methyl analogue of DNOC. M.P. 1060C.

Yellowish-white, odorless crystalline solid, forms salts with bases.
 Water solubility is 1.8 mg/lit @ pH 1.0; 15 mg @ pH 6.5.
Few cases used as herbicides.
 Used to control locusts, ovicidal and used against Tetranychus mites in fruit trees.
iii. Dinoseb : (1,2, sec – butyl – 4,6 – dinitrophenol; 4,6 dinitro – o – sec –
butylphenol = DNOSBP)

It is used as herbicide, fungicide and insecticide - as triethanolamine salt.

 Also used against mites and weeds.
Orange crystalls, slightly soluble in water and partially soluble in organic
solvents
 LD50 , 58 mg/kg
toxic to bees.

iv. Binapacryl (2-sec-butyl – 4,6-dinitrophenyl –3-methyl but – 2 – enoate)

used as acaricide and fungicide

It is a colorless crystal, insoluble in water and partially soluble in organic

solvents.
 Hydrolysed by alkali and strong acid.
Non toxic to bees
 LD50 150-225 mg/kg..
Trade names : ‘Acricid’, ‘Endosan’ ‘Morocide’.
v. Dinocap (2,6-dinitro-4-octyphenyl crotonates and 2,4-dinitropheyl-6-
octyphenyl crotonates in which ‘octyl’

mixture of 1 – methylhephyl, 1-ethylhexyl and 1 – propylpentyl groups. Trade

names : ‘Karathane’ ‘Crotothane’ ‘Sialite’.
Dark brown liquid insoluble in water and soluble in organic solvents.
 Rapidly decomposed by light and hydrolysed by alkaline media.
Acaricide and fungicide
LD50 480 mg/kg for males and 1190 mg/kg for females non-toxic to bees.
Toxicity and symptoms

They are highly toxic to all forms of life.

 Oral LD50 (mg/kg) rats are : DNOC 25, dinoseb 58, dinex, 80, binapacryl 421,
dinocap 980.
Signs and symptoms of acute poisoning in man include nausea, gastric upset,
restlessness, sensation of heat, sweating, rapid respiration, tachycardia, fever,
cyanosis, and finally collapse and coma.
Blood levels of > 20 ppm produce toxic symptoms and >50 ppm is extremely
dangerous.
Treatments consists of ice bath to reduce fever, administration of oxygen and
electrolyte therapy (infusion of large quantities of isotonic solution) to replace less by
sweating.

Insects
Hypersensitivity, convulsions, paralysis and death mostly within an hour.
 Increase in oxygen consumption
LD50 (µ g/g). DNOC – B. moori 49 ; A. mellifera 20 .
Dinocap : B. moori >, H. armigera 87, L. decenlineats larvae 16.
Mode of action

 DNOC, the close coupling between the respiratory chain and

phosphonylation is lost, repiratory control is lost

 e- transport along the chain occurs at a maximal rate without ATP

formation.
It was also suggested that it bind to uncoupler binding site of
ATPase (close to sub unit of ATPase) which carries out
phosphonylation of ADP.
4. Rotenones and rotenoids

In 1912 Nagai, isolated a compound having melting point of 1630C from
Derris chinensis (“gyoto”) grown in formosa and called as ‘roten’ by natives.
 Names it as ‘rotenone’ as it showed the characters of ketone.
Correct molecular structure (C23 H22 O6) was proposed by Takei and Koide
(1929).
Over centuries, natives in far Eastern tropics had used various kinds of
plants to catch fish or aid in hunting. These were called as ‘toeba’ or ‘tuba’
in Malay and were found to be members of ‘Leguminosae’. The primary
sources are:

1. Derris elliptica - Malaya - Toeba / tuba root

D. malaccensis
2. Lonchocarpus utilis - South Africa - ‘Cube’ root
L. urucu
3. Tephrosia spp. - East Africa
A number of related compounds of rotenone, known as
‘rotenoids’ have been isolated. They are :

Name Structural formula Melting point

Rotenone C23 H22 O6 163 (1a)
Deguelin C23 H22 O6 171 (2a)
Elliptone C20 H16 O6 179 (3a)
Sumatol C23 H22 O7 200 (1b)
Toxicarol C23 H22 O7 127 (2b)
Malaceol C20 H16 O7 227 (3b)
Munduserone C19 -H12 O6 162 (4)
Pachyrrhizone C20 H14 O7 272 (5a)
Dolineone C16 H2O6 235 (5b)
Erosone C20 H16 O6 218 (6)
Amorphin C34 H40 O26 155 (7)
Rotenone : (2R, 6aS, 12aS) – 1,2,6,6a,12,12a – hexahydro – 2 – isopropenyl – 8,9 –
dimethoxychromeno (3,4-b) fluoro (2,3,-h) chromen-6-one). ‘

Chemfeet’, ‘Cube root’ ‘Prenfish’, ‘Synprenfish’.

Colorless crystal, lightly soluble in water and soluble in organic solvents
 Decomposes on exposure to light and air.
Insecticide and acaricide with contact and systemic action.
Used for the control of sucking pests, fire ants, mosquito larvae and ectoparasites
of animals.
LD50 132 – 1500 mg/kg.
Rotenone content may be only a few per cent.
 Toxicarol upto 60%, deguelin ¼ th insecticidal activity
Surmatriol little/absent.
In some cases upto 15%.
Rotenone is thermolabile, photolabile and on oxidation to dehydrorotenone which is
non-insecticidal.
Except deguelin all are levorotatory.
Toxicity to mammals

Toxicity varies with type of formulation method of administration and animal

species.
Acute oral LD50 132 mg/kg for rat, 60 mg/kg for guinea pig and minimal lethal
dose is about 1500 mg/kg.
Rotenone poison in man is very rare, but local effects include conjuctivities
dermatitis, pharyngitis, and rhinitis.
On ingestion it produces gastro intestinal, irritation, nausea and vomiting.
Fatal oral dose (for 70 kg man) is 100-200g.
 Inhalation of dust is more hazardous and it can cause respiratory stimulation
followed by respiratory depression, in-coordination, clonic or tonic convulsions,
muscle tremors and death from respiratory failure.
Death is very slow following a oral fatal close and may take 2-10 days or more.
Toxicity to insects

Acts as contact and stomach poisons in insects.

Kills insects slowly but stops feeding immediately.
Activity is lost in 1-3 days if exposed to sun.
Some cases mouthparts become paralysed, stop feeding and die of saturation.
Poisoning symptoms are inactivity, locomotive instability, knockdown, paralysis
and slow death.
Death is mostly because of respiratory failure.
Silk worm may consume as much as 30 times lethal dose before stopping
feeding.
This also act as inhibitor of glutamate oxidation in the muscle of the beetle and
has the ability to block conduction in isolated nerve cards.
Mode of action

It inhibits the respiratory metabolism.

 Precisely it interferes with e- transport system between NADH
dehydrogenase and cytochrome b
 = piericidin A (isolated from Streptomyces mobarensis, inhibitor of
mitochondrial respiratory chain in beef heart mitochondria causing severe
reduction in oxygen consumption and not between NADH and flavoprotein.
Amine metabolic inhibitors
• Large no. of amine neurotransmittors - octapomine, 5HT,
adrenaline, nor-adrenaline

• Distict receptor for each amine, even for single amine several
receptors
Amine metabolic inhibitors
• Chlordimeform

• N-methyl chlordimeform

• Amitraz
Effective against
• Mites
• Rice stem borer
• Tick
• Fire fly
• Helicoverpa
• Spodoptera
• Sphinx moth
• Red spider mites
Symptoms
• Locomotor stimulant,eclosion inhibitor
• Insectistatisic
• Rice stem borer – CDM – glycogen exhausted,
no oviposition, anorexia, motionless
• Fire fly – flickering frequency affected
• Sphynx moth – mouth parts paralysed
• Spiders spin off from treated surface
• Dialation of pupil, Hyper excitation, motionless -
aninals
MOA
• At high doses – depressant, at low doses –excitant
• No effect on mono amine oxygenases
• Demethylated CDM – octapomine agonist
• Interfere with membrane bound Ca+
• Symptoms are similar to CNS effcet – no octopomine receptors
studied in insect CNS
• No report of octopomine receptors in lepidoptera
MFO inhibitors
• Microsomal MFO
• Microsomal mono-oxygenases
Liver
Fat bodies
Mid gut
Abdomen
Substrate
O2
H2O

S + O2 + H2X SO + X + H2O
Microsomal P450 system

-- microsome- abundant in liver = “small body”

-- complex of flavoprotein, cytochrome P450, FeS &

protein

-- hydroxylates foreign matter- detoxifies

a) hydroxylated metabolites are more solubilized and

better for excretion

b) also able to be conjugated for excretion

c) detoxifying system in some cases backfires

Reactions catalysed by MFO

• Hydroxylation
• Dealkylation
• Oxidation of thio esters
• Oxidation of phosphoro thionates
• Epoxidation
Analogoue synergists

• Methylene dioxyl phenyl compounds

• Aryloxy alkyl amines (SKF 525 A)
• Organo thiocyanates (benzyl thiocyanate)
• Oxime ethers (propynyl phosphonate)
• Benzothiodiazoles (1,2,3 benzothiodiazole)
• Inidazoles
MOA
• Oxidisable alternate substrates
• Compete for binding site (Cytochrome
P450)
• Act on the ETC
• Interfere with activation of P450