INTRODUCTION
Fungicides are agents
that are used to prevent or eradicate fungal infections from plants or seeds.
Numerous substances having widely varying chemical constituents are used as
fungicides (Gupta, 1988; Gupta, 2016).Fungicides have been classified according
to chemical structures or have been categorized agriculturally and
horticulturally according to the mode of action (Ballantyne, 2004). According
to the mode of application, fungicides are grouped as foliar, soil and dressing
fungicides. Foliar fungicides are applied as liquids or powders to the aerial
green parts of plants, producing a protective barrier on the cuticular surface
and systemic toxicity in the developing fungus. Soil fungicides are applied as
liquids, dry powders, or granules, acting either through the vapor phase or by
systemic properties. Dressing fungicides are applied to the postharvest crop as
liquids or dry powders to prevent fungal infestation of the crop, particularly
if stored under less than optimum conditions of temperature and humidity. Thus,
effective fungicides must be protective, curative, or eradicative and should
possess the following properties: (1) low toxicity to the plant/animal
but high toxicity to the particular fungus; (2) activity per se or the ability
to convert themselves (by plant or fungal enzymes) into toxic intermediates;
(3) the ability to penetrate fungal spores or the developing mycelium to reach
the site of action; (4) low ecotoxicity; and (5) the ability to form a
protective, tenacious deposit on the plant surface that will be resistant to
weathering by sunlight, rain and wind (Phillips, 2001). With a few exceptions,
most of the newly developed chemicals have a low order of toxicity to mammals.
Public concern has focused on the positive mutagenicity tests obtained with
many fungicides and the predictive possibility of both teratogenic and
carcinogenic potential. The quantity of fungicides used on major crops is
estimated to have increased 2.3-fold between 1964 and 1997. Use of inorganics
(primarily copper compound) and dithiocarbamates has declined since the 1960s,
but captan, chlorothalonils and other organic materials account for 90% of
fungicide use. Newer groups, such as benzimidazoles, conazoles, dicarboximides
and metal organic compounds, account for approximately 10% of fungicide use
(Osteen and Padgitt, 2002). In this chapter, fungicides are discussed using a
chemical classification system.
Background
The earliest
fungicides were inorganic materials such as sulfur, lime, copper and mercury
compounds. The use of element sulfur as a fungicide was recommended as early as
1803. It has become an important component of integrated pest management
systems because it can be used in “organic farming.” There are an increasing
number of instances of dermatitis in human farmworkers and diseases in
ruminants caused by exposure to high levels of sulfur (Gammon et al.,
2010). The mercury-containing fungicides have been responsible for many deaths
or permanent neurological disability. Some of the earlier inorganic metallic
fungicides have been withdrawn in many countries because of their toxicity and
adverse environmental effects (Ballantyne, 2004). Another compound,
hexachlorobenzene (HCB), was extensively used from the 1940s through the 1950s
as a fungicidal dressing applied to seed grains as a dry powder. Between 1955
and 1959, an epidemic of poisoning occurred in Turkey and resulted in a
syndrome called black sore and caused more than 4000 deaths. Although this agent
has largely fallen by the wayside, it is still being used in developing
countries. It is a highly toxic compound and can lead to severe skin
manifestations including hypersensitivity (Hayes, 1982; Gupta, 2010a). Since
that time, many compounds have been developed and used to control fungal
diseases in plants, seeds and produce. Carbamic acid derivatives, including
ethylenebisdithiocarbamates (EBDCs), are a group of fungicides that have been
used widely throughout the world since the 1940s. The important members of this
class include mancozeb, maneb, metiram, zineb and nabam. All the members have
an EBDC backbone, with different metals associated with the individual
compounds. Captan, folpet and captafol have been in use for more than 55 years.
These compounds belong to the chloroalkylthiodicarboximide class of fungicides
due to the presence of chlorine, carbon and sulfur in the side chain. Related
compounds associated with this fungicide class are dichlofluanid and
tolylfluanid. These two compounds have a fluorine atom substituted for one of
the terminal chlorine atoms. Another compound, chlorothalonil, which is a
halogenated benzonitrile fungicide, was first registered for use as an
agrochemical in the United States in 1966. Chlorothalonil also has wider biocidal
applications, including use in paints and lubricant fluids. The benzimidazole
fungicides, benomyl and carbendazim, have been in use for more than 40 years,
whereas anilinopyrimidines, a new class of fungicides (cyprodinil, mepanipyrim
and pyrimethanil), were introduced in 1993 (cyprodinil by Ciba in France) for
application on cereal grains (Ollinger et al., 2010).
Toxicokinetics
Toxicokinetic studies
provide important data on the amount of toxicant delivered to a target as well
as species-specific metabolism. Animals are exposed to fungicides through
ingestion or they are absorbed through the skin or the respiratory system.
Different factors regulate their absorption, distribution, metabolism and
excretion. In general, the liver is the primary site for biotransformation and
may include detoxification as well as activation reactions (Gupta, 1986). Some
fungicides do not undergo any metabolism and bind with other active binding
sites. The aryl organomercurials methyl- and ethylmercury chloride are poorly
excreted and tend to accumulate in muscle, brain and other tissues, whereas the
aryl organomercurial phenylmercury is more readily excreted via the kidney and
less likely to accumulate in brain and muscles. Similarly, HCB possesses all
the properties of chemical stability, slow degradation and biotransformation,
environmental persistence and bioaccumulation in adipose tissue and organs
containing a high content of lipid membranes (Costa, 2008). The newly
introduced class of fungicides are rapidly absorbed, metabolized and excreted
and do not accumulate in tissues, but some of them are partially absorbed from
the gastrointestinal (GI) tract. For example, absorption of chlorothalonil from
the GI tract is on the order of 30–32% of the administered dose. At least 80%
of the administered dose is excreted in feces within 96 h. The highest
concentrations are observed in the kidneys – approximately 0.1% of the dose. In
this case, gut microflora plays a role in the disposition and metabolism in
rats. Glutathione conjugation plays a central role in the metabolism and
subsequent complex metabolic processing of these conjugates, resulting in
selective renal uptake and urinary excretion of thiol-derived metabolites.
Hepatic glutathione levels are decreased, and renal glutathione levels are
elevated. The depletion of hepatic glutathione is considered a direct
consequence of glutathione conjugation within the liver utilizing tissue
resources. The increase in renal glutathione content is more difficult to
explain, but it may be a consequence of urinary excretion of glutathione
conjugates (Parsons, 2010).
The captan is
rapidly degraded to 1,2,3,6-tetrahydrophthalimide (THPI) and thiophosgene (via
thiocarbonyl chloride) in the stomach before reaching the duodenum. THPI has a
half-life of 1–4 s, and thiophosgene is detoxified by reaction with cysteine or
glutathione and is rapidly excreted. No captan is detected in blood or urine.
It is therefore unlikely that these compounds or even thiophosgene would
survive long enough to reach systemic targets such as the liver, uterus, or
testes. Due to rapid elimination, meat, milk, or eggs from livestock/poultry
would be devoid of the parent materials. Humans appear to metabolize captan in
a similar manner to other mammals (Krieger and Thongsinthusak, 1993; JMPR,
2004; Gordon, 2010).
Cyprodinil, an
anilinopyrimidine class fungicide, is rapidly absorbed from the GI tract into
systemic circulation in rats. Approximately 48–68% of the administered dose is
excreted in the urine, whereas 29–47% is found in the feces. Total excretions
reach 92–97% of the administered dose within 48 h. Cyprodinil is almost
completely metabolized. No unchanged parent molecule is found in urine, whereas
minor amounts of unchanged cyprodinil are found in feces. Most of the
administered cyprodinil is metabolized by sequential oxidation of the phenyl
and pyrimidine ring (Figure 48.1). In urine and feces, there is no difference
in the metabolite patterns of the phenyl or pyrimidyl labeled cyprodinil. Seven
urinary, two biliary and two fecal metabolites have been identified, which in
total account for 65–80% of the administered dose. Cyprodinil is absorbed in
goats to a lesser extent and more slowly than in rats. The major route of
excretion is in urine and feces, whereas excretion via milk is minimal. In
laying hens, cyprodinil is rapidly and completely eliminated. Residues in eggs
and edible tissues are very low. The metabolic pathways of cyprodinil in
lactating goats and laying hens are similar to those observed in rats (Waechter
et al., 2010).
Carbamic acid
derivative fungicides, such as EBDCs, are only partially absorbed, and then
they are rapidly metabolized and excreted with no evidence of long-term
bioaccumulation. Absorption of oral doses is rapid, and doses are excreted
within 24 h with approximately half eliminated in the urine and half in the
feces. Their common metabolite is ethylenethiourea (ETU). Only low-level
residues are found in tissues, particularly the thyroid. Another compound in
this class, propamocarb, is rapidly and nearly completely absorbed and
distributed with a concentration reaching peak levels within 1 h. Elimination
from tissues is rapid; with a half-life ranging from 11 to 26 h, urine is the
main route of excretion (~75–91% within 24 h). Up to 6% of the administered
dose is excreted in feces. Propamocarb is extensively metabolized, and only
small quantities are unchanged in urine. Metabolism involves aliphatic
oxidation of the propyl chain (to form hydroxyl propamocarb) and N-oxidation
and N-demethylation of the tertiary amine resulting in propamocarb N-oxide
and mono demethyl propamocarb, respectively. Both benomyl and carbendazim are
well absorbed after oral exposure (80–85%) but poorly absorbed after dermal
exposure (1 or 2%) in rats, mice, dogs and hamsters. The major pathway of
clearance is urinary elimination in rats and mice, but in dogs the majority of
the dose (83.4%) is eliminated via feces, with only 16.2% of the dose
eliminated in the urine after 72h of dosing. In animals, benomyl is converted
into carbendazim through the loss of the n-butylcarbamyl side chain
prior to further metabolism. In dogs and rats, carbendazim undergoes aryl
hydroxylation–oxidation at the 5 and 6 positions of the benzimidazole ring,
followed by sulfate or glucuronide conjugation before elimination. The urinary
excretion half-life of carbendazim in both male and female rats is
approximately 12 h. Benomyl or carbendazim or their metabolites are cleared
rapidly from blood and exhibit minimal potential for bioaccumulation in rats
exposed orally or intravenously (Gardiner et al., 1974; JMPR, 2005).
Similarly, amide
fungicides are rapidly absorbed and eliminated. Metalaxyl-M and metalaxyl can
lead to stimulation of hepatic and renal cytochrome P450 and some other drug metabolizing
enzymes. Tolylfluanid is rapidly and extensively absorbed, followed by rapid
metabolism and almost complete excretion, mainly in the urine and to a lesser
extent in the bile, within 48 h. High tissue concentration has been seen soon
after dosing in the kidney and liver, with lower concentrations in the
perirenal fat, brain, gonads and thyroid. In most species, the concentration of
fluoride in the bone and teeth increases in a dose-related manner (JMPR, 2002,
2005).
After oral dose,
conazole fungicides such as triadimenol and triadimefon are rapidly absorbed
and widely distributed in liver and kidney. Excretion and metabolism is rapid
and extensive, predominantly through oxidation of the t-butyl methyl
group. Propiconazole indicates rapid and extensive absorption (80% of the
administered dose) and is widely distributed, having the highest concentration
in liver and kidney. Excretion is more than 95% in the urine and feces within
48 h. There is extensive enterohepatic recirculation. The compound is extensively
metabolized with oxidation of propyl side chain, hydroxylation of phenyl and
triazole rings, and conjugation. The cleavage of dioxolane is significantly
different according to species and sex (JMPR, 2004). The other compound,
fludioxonil, is rapidly and extensively (80%) absorbed, widely distributed,
extensively metabolized and rapidly excreted, primarily in feces (80%), with a
small amount being excreted in the urine (20%). The maximum blood concentration
is reached within 1 h after administration. Elimination is biphasic, with
half-lives of between 2 and 5 h for the first phase and between 30 and 60 h for
the second phase. The compound is extensively metabolized, involving primarily
oxidation of the pyrrole ring (57% of the administered dose) and a minor
oxo-pyrrole metabolite (4% of the administered dose), followed by glucuronyl
and sulfate conjugation. There is no potential of accumulation in the tissues.
Trifloxystrobin is rapidly absorbed (66%) in 48 h and is widely distributed,
with highest concentrations in blood, liver and kidney. Within 72 h, 72–96% of
the administered dose is eliminated in the urine and feces. Metabolism is
extensive, and the compound undergoes hydroxylation, O-demethylation,
oxidation, conjugation, chain shortening and cleavage between glyoxylphenyl and
trifluoromethyl moieties (JMPR, 2004).
Mechanism of action
There are a series of
biochemical changes or free radical-mediated processes; some may also be
produced by other mechanisms that have been used to assess tissue injury. This
is exemplified by the phenomenon of lipid peroxidation, which has been invoked
as a toxic mechanism in many situations and also occurs subsequent to cell
death and membrane lysis. However, in most situations, it is difficult to
identify the exact mechanism of action. For example, in fungicides containing
mercury, the mercury ions inhibit the sulfhydryl group of enzymes involved in
the transfer of amino acids across the blood–brain barrier and then interfere
with protein synthesis. Organomercurials can also release some mercury ions in
the body, but their toxicity is not believed to be a primary action of mercury
ions (Sandhu and Brar, 2009). There are several theories regarding the
mechanism by which sulfur produces its toxic action. The oxidized sulfur theory
attributes toxicity to its oxidation products, such as sulfur dioxide, sulfur
trioxide, thiosulfuric acid, or pentathionic acid. The reduced sulfur theory
ascribes toxicity to hydrogen sulfide. Direct action theory suggests toxicity
due to crosslinking of proteins, formation of other cellular components by free
radicals of sulfur or polysulfides, or extensive oxidation of thiol groups
leading to loss of function or structural integrity of proteins.
Pentachlorophenol (PCP), a halogenated substituted monocyclic aromatic, acts
cellularly to uncouple oxidative phosphorylation, with the target enzyme being
Na+/K+-ATPase. Oxygen consumption is increased, whereas
adenosine triphosphate (ATP) formation is decreased. The energy is lost as heat
instead of being stored as high-energy phosphate bonds. The electron transport
chain responds by using increasingly more available oxygen (increased oxygen
demand) in an effort to produce ATP, but much of the free energy is lost as
body heat. This leads to depletion of energy reserves (Eaton and Gallagher,
1997). Similarly, organotin compounds, particularly triethyltin, uncouple
oxidative phosphorylation, whereas other agents (e.g., sulfur) in the presence
of sulfiting agents such as sulfur dioxide uncouple oxidative phosphorylation.
Thiamine is cleaved into its constituent pyrimidine and thiazole moieties,
rendering it inactive.
Although the
biochemical and molecular mechanism(s) by which captan and its analogs exert
their cellular toxicity has not been fully established, captan is known to
react with cellular thiols to produce thiophosgene, a potent and unstable
chemical capable of reacting with sulfhydryl-, amino-, or hydroxyl-containing
enzymes (Cremlyn, 1978). Thiols reduce the potency of captan. A volatile product
of captan is responsible for mutagenic activity, the intermediate being
short-lived and formed more quickly at higher levels at an alkaline pH. There
are several other mechanisms by which these chemicals can induce cellular
toxicity. For example, mouse tumors develop with oral administration above a
threshold if maintained for at least 6 months. As shown in Figure 48.1,
epithelial cells that comprise the villi are damaged by exposure to captan and
sloughed off into the intestinal lumen at an increased rate. Second, the basal
cells in the crypt compartment that normally divide at a rate commensurate with
the normal loss of villi cells from the tips of the villi increase, resulting
in high cell proliferation, which is not carcinogenic per se but does play a
role in tumor development. However, there appears to be no treatment-related
duodenal tumor incidence of captan in rats or dogs. Some of the data have been
compiled in reviews, and a task force and framework have been evolving for
evaluation of the mode of toxicity and tumorogenicity findings in mice bioassay
and human relevance for risk assessment purposes (Arce et al., 2010;
Cohen et al., 2010; Gordon, 2010).
Chlorothalonil
fungicide is a reactive molecule toward thio (–SH) groups. It is a soft
electrophile with a preference for sulfur nucleophiles rather than
nitrogen/oxygen nucleophiles. Such chemicals tend to show reactivity toward
protein containing critical S electrophiles rather than toward DNA (containing
critical O and N nucleophiles). A mechanistic interpretation for the
carcinogenicity of chlorothalonil has been published by Wilkinson and Killeen
(1996). Repeated administration of chlorothalonil causes hyperplasia in the forestomach
of rats and mice. The data are consistent with a temporal sequence of events
starting with increased cell proliferation, multifocal ulceration and erosion
of the forestomach mucosa, regenerative hyperplasia and hyperkeratosis, and
ultimately progressing to the formation of gastric tumors within the
forestomach. In dogs, there is no evidence of either neoplastic development or
the occurrence of pre-neoplastic lesions in the kidney or stomach. The absence
of stomach lesions in dogs is attributable to the anatomical differences
between rodents and dogs – dogs do not possess a forestomach. Continued
administration of chlorothalonil leads to the development of a regenerative
hyperplasia within the renal proximal tubular epithelium. Continued regenerative
hyperplasia ultimately results in progression of the kidney lesion to tubular
adenoma and carcinoma. Initial cytotoxicity and regenerative hyperplasia within
the proximal tubular epithelium are essential prerequisites for subsequent
tumor development. The proposed mode of action for the induction of renal
toxicity in rodents is outlined in Figure 48.2.
Fig 48.2 here
(plz insert fig 55.3 from 2nd edition of Vety Toxicology)
For details read full article
Gupta PK, (2018) Toxicity of fungicides. In Veterinary Toxicology- Basic and Clinical Principals, Gupta RC (ed.) 3nd ed. Elsevier, USA pp 569-582.
