Chapter 2
Pest Toxicology: The Primary Mechanisms
of Pesticide Action
John E. Casida
University of California, Berkeley, California
2.1 Introduction
Insects, plants, and fungi are our friends until they endan-
ger our health and compete with us for food, at which time
they become pests. Three millennia have past since Homer
mentioned “pest-averting sulfur” and more than a century
since dinitro-o-cresol became the first synthetic organic
insecticide. With the introduction of the insecticide DDT,
the herbicide 2,4-D, and the fungicide thiram in the 1930s
and 1940s, the golden age of pesticide research (Casida
and Quistad, 1998) and the chemical era of pest control
were underway. The study of pesticides started largely as
just “spray and count” for evaluating effectiveness. This
era was quickly followed by curiosity and then a critical
need for understanding what they do to people, crops, and
the environment (pesticide toxicology and environmen-
tal toxicology) and how they work on the pest. The field
of insect toxicology started by Hoskins in 1928 (Casida
and Quistad, 2001) was soon expanded to include weeds
and fungi. A new field was born to study how the pesti-
cide works on the pest – that is, pest toxicology. To place
pest toxicology in perspective as an aspect of pesticide
science, policy, and management, the reader is referred to
Tomlin (2006), Ware and Whitacre (2004), Stephenson and
Solomon (2007), and Krieger (2009).
Pesticides must be effective, selective, and safe. The
benefits of pest management have to outweigh the eco-
nomic, health, and environmental costs. Insecticides should
be selectively toxic to pest insects compared with people
and even relative to insect pollinators and beneficial arthro-
pods. Herbicides designed to kill weeds must not harm
closely related crops. Fungicides should control the grape
disease fungus yet not interfere with the yeast fermentation
to produce wine. The first generation of synthetic
organic pesticides was generally used at 1–10 pounds per
Hayes’ Handbook of Pesticide Toxicology
Copyright © 2009 American Chemical Society
acre. The effective doses for new compounds dropped
10- to 100-fold within the past half century. Pesticides are
not only increasingly more potent but also of higher organ-
ismal specificity. The coupling of high potency with safety
is achieved by utilizing unique differences at the target site
level. Nature provides an amazing diversity of mechanisms
for both pesticidal activity and selectivity. Species specific-
ity is also sometimes dependent on pesticide metabolism
(both activation and detoxification).
2.2 Primary targets
Pesticides are intended to disrupt a primary target in the
pest so it is no longer harmful. The pesticide per se or
as its bioactivated form binds to or interacts with a spe-
cific enzyme, receptor, protein, or membrane, initiating
a series of events that is deleterious or lethal to the pest.
Insecticides and herbicides have between four and six pri-
mary targets that make up three-quarters of world sales
(Figure 2.1). There are a few similar targets for the various
pesticide types but they are usually very different. Most
insecticides quickly disrupt neurotransmission to alter
insect behavior or survival. Rapid action is usually required
because insects cause economically important damage
within a few hours or days. Insecticides can be practical
with only a limited biological range like aphids or caterpil-
lars. Herbicides generally inhibit plant-specific pathways,
blocking amino acid or fatty acid biosynthesis or photosyn-
thesis to “starve” the weed over several days. Fungicides
act on many basic cellular functions important to hyphal
tip growth (Figure 2.1). To be economically feasible they
must control several diseases. Fungi are evolutionarily far
more diverse than insects or weeds. They include not only
the true fungi but also the Oomycetes having motile stages
103
104 Hayes’ Handbook of Pesticide Toxicology

insecticide (mostly neurotransmission)

other targets 7 8 9
6 10 11 12
1 chitin biosynthesis 5
2 glutamate(chloride) receptor 4
3 acetyl-CoA carboxylase 3
4 ATP synthase 2
5 ecdysone receptor 1 acetyl-
6 uncoupler cholinesterase
7 GABAA receptor
Bt toxin
8 NADH dehydrogenase acetylcholine
9 succinic dehydrogenase receptor Voltage-gated
10 octopamine receptor sodium channel
11 unspecific
12 unknown

herbicide (mostly plant specific pathways)

other targets 9
4 5 6 7 8
1 tubulin 3
2 photosystem I 2
3 protoporphyrinogen IX oxidase 1 EPSP synthase
4 4-hydroxyphenyl pyruvate dehydrogenase acetyl-CoA
5 phytoene desaturase carboxylase acetolactate
6 glutamine synthase auxin synthase
7 others receptor
8 unknown fatty acid
Photosystem II
elongases
9 unspecific

fungicide (mostly basic cellular functions)

other targets
11 12 13
1 succinic dehydrogenase 9 10 14 15
protein His kinase (osmo sensor) 8 16
2
RNA polymerase 7
3
4 scytalone dehydratase 6
sterol ∆14 reductase 5
5 unspecific
6 uncoupler 4
chemical reactives
7 methionine biosynthesis 3
8 protein kinase (osmo sensing) 2
9 phospholipid biosynthesis 1
tubulin sterol
10 protein biosynthesis (ribosomes) c14α-demethylase
cytochrome c
11 sterol 3-keto reductase reductase
12 ATP synthase
13 chitin biosynthesis
14 dihydroorotate dehydrogenase
15 inositol biosynthesis
16 others, unknown
Figure 2.1 Insecticide, herbicide, and fungicide targets as percent of world sales shown as pie-shaped proportion of the total for 2003 (revised from
Tietjen, 2003). Numbers designate minor targets in order of decreasing sales. “Others” include unspecific and unknown targets.
Chapter | 2 Pest Toxicology: The Primary Mechanisms of Pesticide Action
and controlled by oomyceticides. There are a broad vari-
ety of fungicide targets which vary in their importance for
survival. As primitive microorganisms fungi are able to
endure situations of energy depletion with the fungicide
acting more as a fungistat while the disease is actually ter-
minated by plant immune defense mechanisms.
2.3 Secondary targets
The primary interaction usually occurs with the pesticide at
picomolar or nanomolar levels and secondary interactions at
higher concentrations. However, this is not always the case.
There may be several targets of similar sensitivity but not
equal importance. As an example, chlorpyrifos oxon inhib-
its not only acetylcholinesterase (AChE) but also several
other serine hydrolases (Casida and Quistad, 2004), which
although equally or more sensitive are not necessarily signif-
icant secondary targets. In addition, when studies are made
in vitro at micromolar or millimolar levels, other second-
ary targets may become apparent although they are usually
not toxicologically relevant in vivo compared to the primary
effects. In yet another sense, lifetime feeding studies in rats,
mice, and dogs at maximum tolerated doses, a critical part
of the toxicology investigations for registration and estab-
lishing tolerance values, reveal biochemical and pathological
changes not related to the primary target. These secondary
targets in nonpest species play a major role in evaluating
safety but are not considered further here where the focus is
on the primary targets in the pest (i.e., pest toxicology).
2.4 Common target for
structurally diverse pesticides
The discovery of each new type of pesticide is followed
by structural optimization for the highest possible potency
involving sequential modification of each substituent to best
fit the target site. This is often followed by a surge of activ-
ity by competitors to discover, develop, and patent an ana-
log suitable to capture a portion of the potential market. The
analog may have an advantage in cost-effectiveness, avail-
ability of intermediates, persistence, ease of metabolism, or
safety. This is a predictable course of events. The surprise
comes when a very different type of compound is found
to work in the same way. Quite independent discoveries of
the highly effective sulfonylurea and imidazolidinone her-
bicides were followed by the realization that they have the
same primary target despite their very different structures.
Some primary targets are highly specific in the ligands that
bind while others have a broader scope for molecular rec-
ognition. Multiple classes of pesticides sometimes act at the
same target as shown by competitive binding assays [e.g.
respiratory inhibitors acting at the PSST site of Complex I
(Schuler et al., 1999) and cyclodienes, fiproles, and picro-
toxinin in blocking the -aminobutyric acid (GABA)-gated
105
chloride channel (Chen et al., 2006)]. The implications of
the common target become particularly apparent relative to
cross-resistance.
2.5 Resistance as a limiting factor
Houseflies became resistant to DDT when continued use
selected a strain with a less sensitive target site. The resis-
tance to DDT extended to the pyrethrins and the synthetic
pyrethroids. Cross-resistance was recognized as potentially
a major problem. Some organophosphate (OP)-selected
insects were resistant to both OPs and methylcarbamates
(MCs). Weed resistance to atrazine conferred cross-resis-
tance to some but not all herbicides acting at photosystem
II (PSII). Fungi with target site resistance to triadime-
fon were not effectively controlled by some other C14-
demethylase inhibitors. The doses were elevated but this
gave only a brief respite. All of the investment in devel-
oping a potent and safe pesticide can be lost without great
care in managing the selection pressure during use. The
severity of the problem was exacerbated by optimizing for
target site potency, low doses, and specificity conferring
safety, which ultimately favor the development of resis-
tant strains based on a less sensitive primary target and
more rapid metabolism of the exceedingly small amount
of pesticide. Resistance conferred by detoxification has a
completely different cross-resistance spectrum determined
by metabolizable functional groups rather than target site
insensitivity based on a common binding site.
Pesticide management is a major aspect of pest con-
trol (i.e., reducing the selection pressure to slow resistance
development). The rate of resistance development is depen-
dent on the number of generations of pesticide selection per
season which is normally 1 for plants compared to possi-
bly 1–3 for insects and 12–25 for fungi. Resistance can be
“disruptive” or “shifting” with very different consequences.
Disruptive resistance with a factor of perhaps 1000 causes
no fitness penalty for the pest. The pesticide does not
work anymore and the resistant pest spreads and becomes
established to eventually displace the wild type with con-
tinuing chemical use. By contrast, shifting resistance
has a factor of maybe 2–10 and is connected with a fitness
penalty. A higher dose of pesticide is required but at the
end of pesticide use the population shifts back to the wild
type. When resistance appears to one target site or mode of
action, the pesticide is replaced by another one with a differ-
ent mode of action or resistance group. The importance of
pesticide management led to the formation of the Insecticide
Resistance Action Committee (IRAC) (2008), the Herbicide
Resistance Action Committee (HRAC) (2005), and the
Fungicide Resistance Action Committee (FRAC) (2007).
The very knowledgeable experts on these committees are
mostly from industries involved in pesticide research and
development. Their compilations defining resistance groups
106 Hayes’ Handbook of Pesticide Toxicology

are actually the outlines and listings of primary target sites
in pest toxicology. The discussion below of comparative
biochemistry or molecular toxicology is based largely on
the IRAC, HRAC, and FRAC reports considered by primary
target sites rather than type of pesticides.
Target site resistance can be a major limiting factor for
insecticide action at a common nerve target – that is, the
OPs and MCs at AChE, the pyrethroids and DDT at the
sodium channel, and the cyclodienes and phenylpyrazoles
at the GABA-gated chloride channel. Some relief is pro-
vided by indoxacarb and the avermectins.

2.6 Nerve (Table 2.1, Figure 2.2)

Most insecticides by number, amount, and market value
act on the nervous system at the synapse or the axon. The
cholinergic system is the principal insecticide target with
OP and MC compounds inhibiting AChE to prolong the
excitatory action of acetylcholine (ACh). The nicotinic
ACh receptor (nAChR) is the target for the neonicotinoids
as competitive agonists for ACh, spinosad as an alloste-
ric modulator, and cartap as a noncompetitive antagonist.
The axonal voltage-gated sodium channel is the target of
DDT, pyrethrins, and pyrethroids acting as modulators
and indoxacarb as a blocker. Synaptic neurotransmission
at the GABA-gated chloride channel is the target for the
noncompetitive antagonists and blockers endosulfan and
fipronil and the GABA/glutamate-gated chloride channel
is stimulated by the avermectins. The G protein, coupled Figure 2.2 Most insecticides act on the nervous system at several
octopamine receptor is the target for the agonist amitraz. synaptic and axonal sites.

Table 2.1 Nerve Targets for Insecticides

System Chemical type Compounds

Example Number

A. Cholinergic
1. Acetylcholinesterase Organophosphate Chlorpyrifos 64
Methylcarbamate Carbaryl 26
2. Nicotinic acetylcholine receptor

   a. Competitive agonist Neonicotinoid Imidacloprid 7

Botanical alkaloid Nicotine 1
   b. Allosteric agonist Spinosyn Spinosad 2
   c. Antagonist Nereistoxin analog Cartap 4
B. Sodium channel
1. Modulator Pyrethroid Pyrethrins 31
DDT analog DDT 2
2. Voltage-gated sodium channel blocker Oxadiazine Indoxacarb 1
Semicarbazone Metaflumizone 1
C. Chloride channel
1. GABA-gated chloride channel antagonist Cyclodiene Endosulfan 2
Phenylpyrazole Fipronil 2
2. Glutamate-gated chloride channel activator Avermectin Abamectin 3
D. Octopamine receptor agonist Formamidine Amitraz 1
Chapter | 2 Pest Toxicology: The Primary Mechanisms of Pesticide Action 107

2.7 Photosynthesis and pigment
synthesis (Table 2.2, Figure 2.3)
Green plant pigments absorb light and with the coupled sys-
tems of chloroplasts convert light energy to the chemical
energy of adenosine triphosphate (ATP). Herbicides disrupting
these processes unique to plants are usually of low toxi­
city to mammals which lack analogous targets. PSII was
an early target for herbicides and is still highly important,
being acted upon by 50 commercial compounds. More than
one target is involved since resistance to one PSII inhibitor
does not confer cross-resistance to all others denoted
here as the triazine, urea, and nitrile “sites.” The photo-
system I (PSI) electron pathway is diverted by bipyri-
dilium herbicides with paraquat as the principal example.
Protoporphyrinogen IX oxidase is the target for 26 herbicides
of many chemical types. Carotenoids protect chlorophylls
from overactivation and destruction by light. These yellow/
orange pigments must be present to protect the green pig-
ments. Inhibition of four herbicide targets leads to bleach-
ing and weed death. Phytoene desaturase is highly sensitive
to seven herbicides mostly with m-trifluoromethylphenyl

Table 2.2 Photosynthesis and Pigment Synthesis Targets for Herbicides

System Chemical type Compounds

Example Number

A. Photosynthesis
1. PSII

   a. Triazine site Triazine Atrazine 14

Triazinone Metribuzin 3
Uracil Bromacil 3
Phenylcarbamate Desmedipham 2
Triazolinone Amicarbazone 1
Pyridazinone Pyrazon 1
   b. Urea site Urea Diuron 18
   c. Nitrile site Amide Propanil 2
Nitrile Bromoxynil 3
Phenylpyridazine Pyridate 2
Benzothiadiazinone Bentazon 1
2. PSI electron diversion Bipyridylium Paraquat 2
3. Protoporphyrinogen Diphenylether Acifluorfen-Na 8
IX oxidase N-phenylphthalimide Flumiclorac-pentyl 3
Triazolinone Azafenidin 3
Phenylpyrazole Fluazolate 2
Thiadiazole Fluthiacet-methyl 2
Oxadiazole Oxadiazon 2
Pyrimidindione Benzfendizone 2
Oxazolidinedione Pentoxazone 1
Other Pyrafluazol 3
B. Pigment synthesis (bleaching)
1. Phytoene desaturase Pyridinecarboxamide Diflufenican 2
Pyridazinone Norflurazon 1
Other Fluridone 4
2. Lycopene cyclase Triazole Amitrole 1
3. 4-Hydroxyphenyl pyruvate Pyrazole Benzofenap 3
dehydrogenase Triketone Mesotrione 2
Isoxazole Isoxachlortole 2
Other Benzobicyclon 1

4. Unknown Isoxazolidinone Clomazone 1

Urea Fluometuron 1
Diphenylether Aclonifen 1
108 Hayes’ Handbook of Pesticide Toxicology

substituents. Lycopene cyclase is inhibited by amitrole.

4-Hydroxyphenyl pyruvate dehydrogenase inhibition by
eight herbicides leads to bleaching by an entirely different
sequence of reactions. There are also three other bleachers
of different chemical types and unknown target.

Figure 2.3 Photosystem II is inhibited by a great variety of herbi-
cides and the photosystem I electron pathway is diverted by bipyridilium
compounds.
2.8 Biosynthesis
2.8.1 Herbicides (Table 2.3, Figure 2.4)
Plants synthesize their own amino acids whereas animals
do not and require in their diet the essential amino acids

Table 2.3 Biosynthesis Targets for Herbicides

Target Chemical type Compounds

Example Number

A. Amino acid
1. EPSP synthase Glycine derivative Glyphosate 2
2. AHAS or ALS Sulfonylurea Chlorsulfuron 31
Imidazolinone Imazapyr 6
Triazolopyrimidine Flumetsulam 6
Pyrimidinyloxybenzoate Bispyribac-Na 5
Sulfonylaminocarbonyl-triazolinone Flucarbazone-Na 2
3. Glutamine synthase Phosphinic acid Bialaphos 2
B. Microtubule and cell division
1. Microtubule assembly Dinitroaniline Trifluralin 7
Phosphoroamidate pyridine Butamiphos 2
Benzamide Dithiopyr 2
Benzoic acid Propyzamide 2
Chlorthal-dimethyl 1
2. Mitosis/microtubule organization Carbamate Chlorpropham 3
3. DHP synthase Carbamate Asulam 1
C. Fatty acid synthesis
1. Acetyl-CoA carboxylase (ACCase) Cyclohexanedione Sethoxydim 8
Aryloxyphenoxy-propionate Diclofop-methyl 8
Phenylpyrazoline Pinoxaden 1
2. Not ACCase Thiocarbamate Molinate 14
Chlorocarbonic acid Dalapon 3
Benzofuran Benfuresate 2
Phosphorodithioate Bensulide 1
3. Very long chain fatty acid synthesis Chloroacetamide Acetochlor 12
Acetamide Diphenamid 3
Oxyacetamide Flufenacet 2
Tetrazolinone Fentrazamide 1
Other Anilofos 3
D. Cell wall (cellulose) Nitrile Dichlobenil 2
Benzamide Isoxaben 1
Triazolocarboxamide Flupoxam 1
Chapter | 2 Pest Toxicology: The Primary Mechanisms of Pesticide Action
that they cannot make. Amino acid biosynthesis is there-
fore a preferred target for herbicides since there are no
corresponding systems in mammals. Three major targets
are involved: enolpyruvylshikimate 3-phosphate synthase
(EPSP) for glyphosate and one other herbicide; acetohy-
droxy acid synthase (AHAS), also known as acetolactate
synthase (ALS), for 31 sulfonylureas, six imidazolinones,
and 13 chemicals of other types; and glutamine synthase for
glufosinate and one other compound. Overexpressed EPSP
synthase and glutamine synthase as low sensitivity targets
are normally coupled with overexpressed herbicide detoxi-
fication systems for enhancing crop tolerance. The micro-
tubule system and cell division have three herbicide targets.
The largest number of compounds (17) of varied chemical
type including trifluralin alter the microtubule assembly
process. Three carbamates inhibit mitosis/microtubule
organization and another dihydropteroate (DHP) synthase.
Figure 2.4 Plant amino acid biosynthesis is inhibited at the most
important EPSP synthase and AHAS sites and two other enzymes by
many herbicides.
Table 2.4 Biosynthesis Targets for Fungicides (f) and Insecticides (i)
Target Chemical type
A. Sterol (f )
1. C14-demethylase Triazole
Imidazole
Pyrimidine
Piperazine
Pyridine
2. 14-reductase and 8→7-isomerase Morpholine
Piperidine
Spiroketal-amine
3. 3-keto reductase, C4-demethylation Hydroxyanilide
4. Squalene-epoxidase Allylamine
Thiocarbamate
109
Fatty acid synthesis is a favored target as evident from the
58 herbicides acting in this way, some on acetyl-CoA car-
boxylase (ACCase) and others at diverse sites altering very
long chain fatty acid synthesis. Cell wall biosynthesis is
inhibited by four herbicides including dichlobenil.
2.8.2 Fungicides and Insecticides (Table 2.4,
Figure 2.5)
The fungal sterol is ergosterol versus the mammalian
cholesterol with critical differences in their biosynthetic
pathways that allow selective inhibition. Four sterol tar-
gets are involved in fungicide action. The most important
is the C14-demethylase inhibited by 26 triazoles includ-
ing triadimefon, five imidazoles, and four other fungicides.
This site is highly sensitive and very prone to target site
insensitivity, but fortunately the selection is counteracted
somewhat by the fitness penalty resulting in only shifting
resistance. The 14-reductase and 8→7-isomerase are
inhibited by four morpholines and some structurally unre-
lated compounds and two other steps by diverse chemicals.
Four targets of fungicides in nucleic acid biosynthesis are
RNA polymerase I (six fungicides, including benalaxyl),
adenosine-deaminase (three fungicides), DNA/RNA syn-
thesis (two fungicides), and DNA topoisomerase type II
(gyrase) (one compound). Four antibiotic fungicides such
as streptomycin block protein synthesis. Antitubulin fun-
gicides mostly affect -tubulin assembly in mitosis (eight
compounds of diverse types with benomyl as an example)
while others block cell division or delocalize spectrin-like
proteins (two compounds). Phospholipid biosynthesis is
blocked by four fungicides inhibiting the methyl transfer-
ase while isoprothiolane and five others disrupt cell wall
Compounds
Example Number
Triadimefon 26
Imazalil 5
Fenarimol 2
Triforine 1
Pyrifenox 1
Adimorph 4
Fenpropidin 2
Spiroxamine 1
Fenhexamid 1
Naftitine 2
Pyributicarb 1
(Continued)
110 Hayes’ Handbook of Pesticide Toxicology

Table 2.4 (Continued)

Target Chemical type Compounds

Example Number

B. Nucleic acid (f )

1. RNA polymerase I Acylalanine Benalaxyl 4

Oxazolidinone Oxadixyl 1
Butyrolactone Ofurace 1
2. Adenosine-deaminase Hydroxy-(2-amino) pyrimidine Buprimate 3
3. DNA/RNA synthesis (proposed) Isoxazole Hymexazole 1
Isothiazolone Octhilinone 1
4. DNA topoisomerase type II (gyrase) Carboxylic acid Oxolinic acid 1
C. Protein (f ) Antibiotic Blasticidin-S 1
Kasugamycin 1
Streptomycin 1
Oxytetracycline 1
D. Mitosis and cell division (f )

1. -tubulin assembly in mitosis Benzimidazole Benomyl 4

Thiophanate Thiophanate-methyl 2

2. -Tubulin assembly in mitosis N-phenylcarbamate Diethofencarb 1

3. -Tubulin assembly in mitosis Toluamide Zoxamide 1

4. Cell division (proposed) Phenylurea Pencycuron 1

5. Delocalization of spectrin-like proteins Pyridinylmethylbenzamide Fluopicolide 1
E. Lipid
1. Lipid and phospholipid biosynthesis

   a. P
 hospholipid biosynthesis methyl Phosphothiolate Iprobenfos 3
transferase (f ) Dithiolanes Isoprothiolane 1
   b. Phospholipid biosynthesis and Carboxylic acid amide Dimethorph 6
cell wall deposition (f )
   c. Lipid biosynthesis (i) Tetronic acid Spiromesifen 2
2. Lipid peroxidation (f ) Amino/nitrobenzene Dicloran 3
Benzene Biphenyl 2
Thiophosphate Tolclofos- 1
Thiadiazole methyl 1
Etridiazole
3. Cell membrane permeability, fatty acid (f ) Amides and thioamides Prothiocarb 3
F. Glucans (f)
1. Trehalase and inositol Glucopyranosyl antibiotic Validamycin 1
2. Chitin synthase Peptidyl pyrimidine nucleoside Polyoxin 1
G. Methionine biosynthesis (f ) Aniline-pyrimidines Cyprodinil 3
H. Cell wall (melanin) (f )
1. Reductase Various Tricyclazole 3
2. Dehydratase Carboxamides Carpropamid 3
Chapter | 2 Pest Toxicology: The Primary Mechanisms of Pesticide Action 111

deposition. Lipid peroxidation is initiated by seven fungi-

cides and cell membrane permeability is disrupted by three
others. Biosynthesis of glucans is inhibited by two fungi-
cides (including polyoxin for chitin synthase), of methio-
nine by cyprodinil and two others, and of melanin by six
fungicides. Insecticidal activity is achieved with tetronic
acids, such as spiromesifen, inhibiting lipid biosynthesis.

2.9 Respiration (Table 2.5, Figure 2.6)

Figure 2.5 Fungal ergosterol biosynthesis is inhibited at the most
important CYP450 C14-demethylase and three other sites by a great
variety of fungicides.
The synthesis of ATP, the energy currency of the cell, is a
complex process carried out by the mitochondrial respi-
ratory electron transport chain involving a series of five
membrane-bound complexes (I–V). Pesticides disrupt
many sites by binding and inhibition (I–IV) or acting as

Table 2.5 Respiration Targets for Insecticides (i), Herbicides (h), and Fungicides (f)

Target Chemical type Compounds

Example Number

A. Electron transport
1. Complex I NADH oxidoreductase

   a. Coupling site I (i) Various Rotenone 7

   b. Other (f) Pyrimidinamine Diflumetorim 1
2. Complex II

   a. Succinic dehydrogenase (i,f ) Carboxamide Carboxin 1

Various Penthiopyrad 9
Acrylonitrile Cyenopyrafen 1
3. Complex III

   a. Coupling site 2 (i) Various Acequinocyl 3

   b. Ubiquinol oxidase at Qo site (f��� ��) Various Kresoxim- 10
Methoxyacrylate methyl 3
Azoxystrobin
   c. Cytochrome b Qo site (i) Carbazate Bifenazate 1
   d. Cytochrome bc1 at Qi site (f��� ��) Cyanoimidazole Cyazofamid 1
Sulfamoyltriazole Amisulbrom 1
4. Complex IV (i) Fumigant HCN, PH3 3
B. Oxidative phosphorylation
1. Uncouplers via disruption of Dinitrophenol Binapacryl 7
proton gradient (i, h, f��� ��) Liposoluble insecticide Chlorfenapyr 1
Dinitroaniline Fluazinam 1
Pyrimidinone-hydrazone Ferimzone 1

2. ATP synthase (i) Carbodiimide progenitor Diafenthiuron 1

3. ATP production (i, f��� ��) Triorganotin Cyhexatin 6
Thiophenecarboxamide Silthiofam 1
C. Aconitase (i) Haloaliphatic acid Fluoroacetate 1
D. Others (i) Various Propargite 2
112
Figure 2.6 The mitochondrial respiratory electron transport chain has pesticide inhibition sites in each of the five membrane-bound complexes
(modified from Schuler and Casida, 2001).
uncouplers to prevent oxidative phosphorylation and for-
mation of the proton gradient. Rotenone and a series of
miticides inhibit by binding to the PSST site in Complex I.
Carboxin and nine other fungicides and a recently reported
metabolite of the acaricide cyenopyrafen inhibit succinic
dehydrogenase in Complex II, and the strobilurins block
the quinol oxidation center of Complex III. The insec-
ticide acequinocyl inhibits Complex III coupling site 2,
bifenazate at cytochrome b Qo site, and two fungicides at
the cytochrome bc1 Qi site. Cyanide and phosphine block
Complex IV and carbodiimides and triorganotins the ATP
synthase of Complex V. The insecticide chlorfenapyr is
one of 10 pesticidal uncouplers of oxidative phosphoryla-
tion. Fluoroacetate is converted to fluorocitric acid which
inhibits aconitase of the tricarboxylic acid cycle. The steps
in electron transport are sufficiently conserved between
insects and mammals that it is difficult to achieve large
degrees of selectivity for inhibitors. In animals inhibition
of the respiratory chain leads to radical accumulation and
induces apoptosis, which is especially damaging in neuronal
cells. Inhibition of the respiratory chain in plants can be
irrelevant as long as photosynthesis supplies NADH and
ATP and in fungi it leads to a type of starvation.
2.10 Growth regulators (Table 2.6,
Figure 2.7)
Every organism follows a programmed course of growth
and development carefully synchronized for species prop-
agation and environmental integration. Compounds that
disrupt these delicate hormone-guided processes serve as
insect growth regulators (IGRs), plant growth regulators
(PGRs), and fungal growth regulators or host plant defense
inducers. Insect development is controlled by a balance
Hayes’ Handbook of Pesticide Toxicology
in time and amount of juvenile hormone to stay young,
and growth and differentiation hormone or ecdysone to
develop, molt, and become an adult. Juvenile hormone
mimics and analogs such as methoprene are very effective
and selective but provide slow control. Molting disruptors
include ecdysone receptor agonists, e.g. diacylhydrazines
such as tebufenozide that act at the ecdysone binding site.
Cryomazine for Diptera is also a molting disruptor as is the
natural azadirachtin by undefined mechanisms. Chitin bio-
synthesis inhibitors for Lepidoptera (benzoylphenyl ureas)
and Homoptera (buprofezin) act in very different ways.
The benzoylphenyl ureas block chitin synthesis in vivo but
not chitin synthase in vitro, so the mechanism is unsolved.
In comparison, cellulose biosynthesis-inhibiting herbi-
cides (dichlobenil) and cell wall biosynthesis-inhibiting
oomyceticides (iprovalicarb and mandipropamid) also do
not directly inhibit the sugar polymerase. Plant disease
development is regulated by auxins, ethylene, cytokinins,
and gibberellins, among others. Several types of carbox-
ylic acids such as 2,4-D serve as synthetic auxins and other
compounds including naptalam as auxin transport inhibi-
tors. Finally, fungal growth can be altered by host defense
inducers such as acibenzolar S-methyl and probenazole.
2.11 Unknown, nonspecific
and other targets (Table 2.7)
The endotoxins of the bacterium Bacillus thuringiensis (Bt)
disrupt insect midgut membranes with effectiveness depen-
dent on the strain of Bt and the particular pest. “Bt crops”
with expressed recombinant endotoxin such as Cry1Ab
play a critical role in control of lepidopterous larvae. The
newly introduced diamide ryanodine receptor modulators
offer great promise based on potency for lepidopterous
Chapter | 2 Pest Toxicology: The Primary Mechanisms of Pesticide Action 113

Table 2.6 Growth Regulator Targets

Organism Chemical type Compounds

Example Number

A. Insect and IGRs

1. Juvenile hormone mimic Juvenile hormone analog Methoprene 3
Phenoxyphenoxy Ether Fenoxycarb 2
2. Molting disruptor

   a. Ecdysone agonist Diacylhydrazine Tebufenozide 4

   b. Dipteran Triazine Cyromazine 1
   c. Botanical Neem constituent Azadirachtin 1
3. Chitin biosynthesis

   a. Lepidopteran Benzoylurea Diflubenzuron 11

   b. Homopteran Buprofezin Buprofezin 1
B. Plant and PGRs
1. Synthetic auxin Phenoxy carboxylic acid 2,4-D 8
Pyridine carboxylic acid Picloram 4
Benzoic acid Chloramben 3
Quinoline carboxylic acid Quinclorac 2
Other Benazolin-ethyl 1
2. Auxin transport Semicarbazone Diflufenzopyr-Na 1
Pthalamate Naptalam 1
3. Ethylene generator Chloroethylphosphonic acid Ethephon 1
4. Cytokinin Adenine derivatives Benzyladenine 2
5. Gibberellin Diterpenoid acid Gibberellic acid 2
C. Fungi-host plant defense inducers
1. Salicylic acid pathway Benzothiadiazole BTH Acibenzolar S-methyl 1
2. Other Thiadiazole-carboxamide Tiadinil 2
Benzisothiazole Probenazole 1
Natural Laminarin 1

larvae and safety. One or both of the selective feeding
blockers (pymetrozine and flonicamid) may disrupt nerve
processes. Compounds that inhibit mite growth often dif-
fer in mode of action from those for insects but the mecha-
nisms remain unknown. Synergists act both as CYP450
monooxygenase inhibitors such as piperonyl butoxide and
esterase inhibitors with tribufos as an example. Methyl
bromide continues to be used as a major fumigant. There
are six other unknown targets for insecticides. The her-
bicide endothal inhibits protein phosphatase 2A, while
19 other herbicides act on unknown targets. Seven fungi-
cides disrupt signal transduction at two different sites. The
multisite fungicides have been extremely important for
many decades with dithiocarbamates, copper, and sulfur as
major examples. There are also 16 fungicides of unknown
mode of action.
2.12 Overview (Table 2.8)
A compilation of pesticides by targets (including differ-
ent binding sites in the same target) and numbers based on
Tables 2.1–2.7 is presented as an overview in Table 2.8.
It is no surprise that only the insecticides act on nerve
processes and only the herbicides on photosynthesis and
pigment synthesis light processes. Biosynthesis inhibi-
tors include major herbicides and fungicides in number
and variety of structures. Respiration inhibitors are largely
insecticides and fungicides and the growth regulators are
particularly important for insects and plants. There are a
large number of “other” targets, often with only a single
example. In this form of compilation, the numbers of tar-
gets are similar for insecticides, herbicides, and fungicides,
whereas the number of compounds is less for fungicides.
114 Hayes’ Handbook of Pesticide Toxicology

Figure 2.7 Insect growth regulators disrupt the molting process by acting as juvenile hormone mimics (e.g. methoprene), ecdysone receptor agonists
(e.g. tebufenozide), or chitin biosynthesis inhibitors (e.g. diflubenzuron).

Table 2.7 Unknown, Nonspecific and Other Targets

Chemical type Compounds

Example Number

A. Insecticide
1. M
 icrobial disruptor of insect Bacillus Cry1Ab 8
midgut membrane thuringiensis (Bt)
Crop proteins
2. Ryanodine receptor modulators Diamide Flubendiamide 2
3. Selective feeding blocker Various Pymetrozine 2
4. Mite growth inhibitor Various Clofentezine 3
5. Synergists

   a. Cyp450 monooxygenase Methylenedioxyphenyl Piperonyl butoxide 1

   b. Esterase OP Tribufos 1
6. Fumigant Alkyl halide Methyl bromide 3
7. Other Benzoximate Benzoximate 6
B. Herbicide
1. Protein phosphatase 2a Dicarboxylic acid Endothal 1
2. Unknown Arylaminopropionic acid Flamprop-M- 2
Organoarsenical methyl 2
Pyrazolium DMSA 1
Other Difenzoquat 14
Cinmethylin
Chapter | 2 Pest Toxicology: The Primary Mechanisms of Pesticide Action 115

Table 2.7 (Continued)

Organism Chemical type Compounds

Example Number
C. Fungicide
1. Signal transduction Quinoline Quinoxyfen 1
   a. G proteins in early cell signaling Dicarboximide Vinclozolin 4
   b. Map/histidine kinase Phenylpyrrole Fenpiclonil 2
2. Multisite Dithiocarbamate Maneb 8
Phthalimide Captan 3
Chloronitrile Chlorothalonil 1
Various organic Dodine 7
Various Inorganic Cu, S 2
3. Unknown Various Fosetyl-Al 12
Diverse Mineral oils 4

Table 2.8 Number of Pesticide Targets Including Different Binding Sites and Compounds Acting at Those Targets

Type of target Number of targetsa Number of compoundsb

Insect Herb Fung Total Insect Herb Fung Total

Nerve 9 0 0 9 147 0 0 160

Photosynthesis 0 9 0 9 0 97 0 97
and pigment
synthesis
Biosynthesis 1 10 23 34 2 134 103 239
Respiration 10 1 6 17 36 10 43 89
Growth 6 5 2 13 23 25 5 53
regulators
Others 7 2 4 13 26 20 44 88
Total 33 27 35 95 234 286 195 715
a
Includes unknown targets tabulated as 1 in each category.
b
The same compound may appear in two or three categories if it has multiple uses or actions such as pesticides affecting oxidative phosphorylation.

A totally different picture of pesticides is obtained on
evaluating by percent of world market value or amount
used. Glyphosate with glyphosate-resistant crops is the
most important by world market value and sulfur by
amount used in California. Several aspects of pest manage-
ment are not considered. These include insect pheromones,
nematicides, and rodenticides. This overview is dependent
primarily on the IRAC, HRAC, and FRAC sources used,
and secondarily on the approach in arranging and interpret-
ing the available information.
The author proposed about 20 years ago that there
may be a finite number of practical targets for pesticide
action (Casida, 1990). The frequency at which the new
compounds acted at the same old targets raised concerns
that we cannot indefinitely rely on discovering new tar-
gets. However, screening of natural products and synthetic
116
compounds has continued to come up with a diversity of
mechanisms and effective structures. There has also been
a concerted search for new types of pest control agents
that ultimately led to new targets. Clearly there are many
more yet to discover. However, despite many advances
in recent years, the number of targets with more than 5%
market share in 2003 was only four for insecticides, six for
herbicides, and four for fungicides (Figure 2.1) (see also
Tietjen et al., 2005).
Conclusion
Pests are currently controlled with about 715 pesticides
acting by perhaps 95 different mechanisms. Can we stop
research now and just continue to use the current com-
pounds effectively and safely? Do we really need more
pesticides? Advances in chemical pest control are depen-
dent on a thorough understanding of both pest and pesti-
cide toxicology. We must be ready when pests inevitably
develop resistance. With current pesticides, pests are gener-
ally much more sensitive than people and crops. However,
we can still achieve higher target site potency and lower
use levels, reduced environmental impact, and improved
safety. Pesticide legislation policies for risks and hazards
continue a trend toward ever-greater restrictions. We
must constantly maintain a pool of safe pesticides acting
on many targets to meet demands for increased food pro-
duction and improved human health. Continued success
in pest management depends on developing and using an
expanding knowledge base in comparative biochemistry
and molecular toxicology considering pests, people, and
crops. The time has arrived when advances in pesticide
toxicology are dependent on or coupled with discoveries in
pest toxicology.
Postscript
The following paragraph is a comment on “Why I do
Science” (Casida, 2008):
I find that crossword and jigsaw puzzles are not so much
fun because you know there is a solution. But try a mecha-
nism study on a chemical that works on a totally unknown
target—now you can really have fun. It may be easy, or it
may take 50 years before the background science is avail-
able. Alternatively, the new chemical and novel mode of
action serves as a probe to dissect a new area of science.
Pesticides—chemicals that affect the growth or survival of
a pest—are particularly intriguing. Hundreds of thousands
of compounds are sifted every year in the search for very
unusual effects, which are great fun to sort out. Each new
discovery, advance, or stage of understanding carries with
it the thrill of the moment, but they are really for all time
as they become part of our knowledge base and toolkit.
Hayes’ Handbook of Pesticide Toxicology
The approach of our laboratory is to take a new compound
and optimize its potency—meaning that you can use less of
it because it is designed to go to just the right place to do
the assigned job. When other strategies fail, we use tritium
to make the compound highly radioactive and then use this
radioligand to quantitate, assay, purify, isolate, and ulti-
mately identify the target. Genomics, proteomics, and all the
other “omics” really help us solve problems as never before.
Once you identify the mechanism, you can manipulate a life
process. Can you create a useful new pesticide, a new can-
cer drug, or a new way to measure and modulate a receptor
in the brain? Then the challenge is to find a way to use the
chemical without side effects while fitting the economic real-
ity of the marketplace. We leave that to the entrepreneurs.
Acknowledgments
The author gives special thanks to Alex Gulevich who
assisted with devotion and distinction in compiling and
presenting the information; to Motohiro Tomizawa, Ralf
Nauen, Dale Shaner, and Klaus Tietjen who provided help-
ful review comments; and to the University of California at
Berkeley William Muriece Hoskins Chair in Chemical and
Molecular Entomology for continuing support.
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