Toxicology of Insecticide –Toxicology of Insecticide –

Metabolic InhibitorsMetabolic 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 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 (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 (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 - keto – glutarate dehydogenase (krebs cycle) and results in arsenolysis.

SH + NADP + PSH + NADP + PII SP + NADPHSP + NADPH

22

ADPADP PhosphorylationPhosphorylation

ATP + SATP + S

SH + NADP + ASH + NADP + Ass(OH)(OH)

33 SAs (OH)SAs (OH)22 + +

NADPHNADPH22

+ H+ H22OO ArsenolysisArsenolysis

S + AS + A33 (OH) (OH)

33

i.i.

The evidence for the inhibition are

inhibition of the enzymes in in vitro

ii.ii. the two enzymes accumulates in arsenites treated

iii.iii. in living insects, arsenites results in respiratory failure

iv.iv. no nerve poisoning symptoms are mimicked by arsenites but they resemble the symptoms of rotenone.

leactic dehydrogenase, - 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:Evidence for this is:

i.i. Accumulation of citrate in fluoroacetate treated insectsAccumulation of citrate in fluoroacetate treated insects

ii.ii. Fluoroacetate Fluoroacetate fluorocitrate which is more toxic fluorocitrate which is more toxic

iii.iii. Aconitase powerfully inhibited by fluorocitrate Aconitase powerfully inhibited by fluorocitrate in vitroin 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 (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 (C23H22O6) 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:

1.1. Derris ellipticaDerris ellipticaD. malaccensisD. malaccensis

- - MalayaMalaya -- Toeba / tuba rootToeba / tuba root

2.2. Lonchocarpus utilisLonchocarpus utilisL. urucuL. urucu

-- South AfricaSouth Africa -- ‘‘Cube’ rootCube’ root

3.3. TephrosiaTephrosia spp. spp. -- East AfricaEast Africa

A number of related compounds of rotenone, known as ‘rotenoids’ have been isolated. They are :

NameName Structural formulaStructural formula Melting pointMelting point

RotenoneRotenone CC2323HH2222OO66 163 (1a)163 (1a)

DeguelinDeguelin CC2323HH2222OO66 171 (2a)171 (2a)

ElliptoneElliptone CC2020HH1616OO66 179 (3a)179 (3a)

SumatolSumatol CC2323HH2222OO77 200 (1b)200 (1b)

ToxicarolToxicarol CC2323HH2222OO77 127 (2b)127 (2b)

MalaceolMalaceol CC2020HH1616OO77 227 (3b)227 (3b)

MunduseroneMunduserone CC1919 H H1212OO66 162 (4)162 (4)

PachyrrhizonePachyrrhizone CC2020HH1414OO77 272 (5a)272 (5a)

DolineoneDolineone CC1616HH22OO66 235 (5b)235 (5b)

ErosoneErosone CC2020HH1616OO66 218 (6)218 (6)

AmorphinAmorphin CC3434HH4040OO2626 155 (7)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

O2 Substrate

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

• Oxidisable alternate substrates

• Compete for binding site (Cytochrome P450)

• Act on the ETC

• Interfere with activation of P450

MOA