BENOMYL JMPR 1975
Benomyl was considered by the Joint Meeting in 1973 (FAO/WHO,
1974), but tolerances could not be established owing to lack of
toxicological information. However, as a considerable amount of
information on use patterns, mode of action, actual residue levels,
etc. was available, a number of guideline levels reflecting good
application practices were recommended. Further, some requests were
made for desirable additional information.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
Absorption, distribution and excretion
A male rat was fed a basal diet supplemented with 2500 ppm
benomyl for 12 days and then given a single dose of 2C14 benomyl by
gastric intubation. After 24 hours, 78.9% of the radioactivity was
detected in the urine and 8.7% in the faeces. A total of 91.8% of the
calculated C14 dose was accounted for, of which 99.5% was found in
the urine and faeces by the end of the 72-hour test period. The major
metabolite was methyl-5 hydroxy 2-benzimidazolecarbamate (5 HBC) which
was present in the urine as the glucuronide and/or sulfate. Very
little of the parent compound or methyl 2 benzimidazolecarbamate (MBC)
was present in the urine (<5%) (Gardiner et al,, 1974),
A male dog was fed a diet containing 2500 ppm benomyl. After
seven weeks he received a single dose of C14 benomyl by gelatin
capsule. Again, the bulk of the radioactivity (>99%) was eliminated
from the animal in 72 hours. The major route of excretion, in this
case, was by way of the faeces where benomyl and/or MBC and 5-HBC were
detected (Gardiner et al., 1974).
Groups of two dairy cows were fed 0, 2, 10 and 50 ppm benomyl for
32 days. Total amounts of benomyl ingested were 0.88, 4.36 and 21.8 g.
One cow at each level was killed at 32 days, the other after a
one-week withdrawal period. No apparent adverse effects on the animals
or on milk production were observed. No residues of benomyl and/or MBC
were found in milk, urine or faeces. 5 HBC and
methyl-4-hydroxy-2-benzimidazole carbamate (4 HBC) were not detected
in milk at dietary levels of 2 ppm. At higher dietary levels constant
levels of metabolites were present in milk within 24 hours. No
residues in milk (less than 0.01 ppm) were detected 48 hours after
benomyl was removed from the diet. No residues were found in the
animal tissues (liver, kidney, subcutaneous fat, lean muscle).
Detectable amounts of 5 HBC and 4 HBC in urine and of 5 HBC in faeces
were observed (Gardiner et al., 1974).
Groups of eight chickens were fed 0, 5 and 25 ppm benomyl for
four weeks. Composite samples of eggs were taken the last two days of
each week. At four weeks composite samples of faeces were obtained and
half the birds in each group sacrificed. Liver, fat and muscle samples
were taken. Remaining birds were placed on a control diet for one week
prior to sacrifice. No adverse effects on body weight gain, feed
consumption or egg production were noted. No residues of benomyl or
its degradation products were detected in the tissue samples. Only 5
NBC (0.03-0.06 ppm) was found in the high level egg samples. Residue
was not detectable (<0.02 ppm) in eggs one week after hens were
placed on untreated diet (Gardiner et al., 1974).
2-C14 benomyl was administered by gastric intubation to a male
rat at a dosage level of 900 mg/kg. After one hour blood was taken and
analysed for benomyl and MBC by thin-layer chromatography and C14
radioscanning. The radioactivity in the blood consisted of 68% 2-C14
MBC and less than 15% intact benomyl (Sherman et al., 1975).
A male ChR-CD rat was fed a diet containing 2500 ppm MBC for 14
days. The animal was then given 2-C14 MBC by intragastric intubation.
Urine and faeces were collected. Animal was sacrificed after 72 hours.
Bulk of the C14 activity was eliminated in the first 24-hour interval
(83.5% in urine, 9.6% in faeces). One major and two minor C14
labelled compounds were detected in the urine. After enzymatic
hydrolysis and thin-layer chromatography, 85% of the activity was
found in a single spot corresponding to 5 HBC. No residual C14 MBC
was detected (<5%) (Gardiner et al., 1974).
Groups of male mice, rabbits and sheep were dosed orally with 0.1
g benomyl/kg body weight. Urine and faeces were collected for 96 hours
at intervals of 24 hours. The metabolism of benomyl was also studied
in vitro using various tissue homogenates as well as blood and sheep
rumen fluid as enzyme sources. All three species showed similar
patterns of metabolites both in vivo and in vitro. Detoxification
proceeded primarily through two major routes, hydroxylation and
hydrolysis. Two metabolites were formed by hydroxylation. and two by
ester hydrolysis. Approximately 20% of the administered dose was
excreted as the sulfate and glucuronide conjugates of hydroxylated
metabolites. No unchanged benomyl was found in either urine or faeces.
Urine contained 41-71% and faeces 21-46% of the excreted metabolites.
The liver was found to be the major site of hydroxylation, other
tissues forming only small amounts of hydroxylated metabolites (Douch,
Special studies on mutagenicity
Mutants of Neurospora crassa resistant to benomyl were isolated
following ultra violet irradiation of conidia. Genetic analysis of 15
of the mutant strains revealed that the resistance was due to a single
allele. Tests of heterokaryons containing both resistant and sensitive
alleles indicated that the resistance was dominant. The mutants did
not possess the same degree of resistance to the fungicide (Borck et
Forward mutations were induced in Fusarium oxysporm f.sp. melonis
when incubated with a culture medium containing benomyl (5 µg/ml).
This effect was shown only with growing cells. In another study roots
of young Allium cepa which were dipped in benomyl (0, 50 and 100
µg/ml) and incubated for up to 12 hours, did not reveal any
chromosomal aberrations (Dassenay et al., 1973).
In a dominant lethal study four groups of 10 male ChR-CD rats
received diets containing 0, 250, 1250 and 2500 ppm benomyl for seven
days. Each male was then mated with three ChR-CD females. After seven
days, males were removed and mated with three additional females. This
procedure was followed for six weeks. In the control animals the
mating index was significantly lower and the pre-implantation losses
and early resorptions consistently higher than in the test groups. Due
to these effects the calculation of the dominant lethal mutation rates
for benomyl based on either post implantation loss or on pre- and
post-implantation losses were negative (Sherman et al., 1975).
Special studies on reproduction
In a three generation reproduction study, four groups of rats (6
male and 6 females/group - 1st generation; 12 male and 12 females -
2nd generation; 20 male and 20 females - 3rd generation) were fed 0,
100, 500 and 2500 ppm benomyl in their diet. Two litters were produced
in each of the 1st and 2nd generations with three litters being
produced in 3rd generation. When pups of the F3B generation were
weaned, two males and two females were selected from each of five
litters from each group and subjected to gross pathological
evaluation. Tissues from the control and 2500 ppm, group were
evaluated histopathologically. At weaning, animals of the F3C
generation were kept on their respective diets for nine weeks and then
placed on the control diet for an additional six weeks. Reproductive
performance in all groups was satisfactory. No significant effects
were observed in the number of pups born alive, fertility, gestation
or lactation indices. No compound related gross or microscopic
pathologic changes were observed. Slightly lower weanling weights in
F2B, F3A, F3B and F3C litters at 500 and 2500 ppm were noted.
However, the absence of a change in the growth curves when the F3C
test animals were placed on the control diet. demonstrated that the
lower weanling body weights were not compound related (Sherman et al.,
Special studies on teratogenicity
Pregnant ChR-CD rats were given 0, 100, 500, 2500 and 5000 ppm
benomyl in their diet from day 6 through day 15 of gestation. Animals
were killed on day 20 of gestation and foetuses removed by caesarean
section. No clinical signs of toxicity were observed in any of the
pregnant females. No significant effects were observed in the number
of implantation sites per pregnant female, number of resorption sites,
number of live foetuses, mean foetus weight, sex ratio or crown-rump
length. Incidence of skeletal and visceral abnormalities in the
treated groups was comparable to those of the control group. The
number and type of anomalies were also found to be comparable with
standard values established for this strain of rat (Sherman et al.,
When benomyl was administered by gavage to rats from day 1 to day
20 of gestation, 100% post-implantation deaths were observed at 500
mg/kg bw. In a similar study, carbendazim was shown to be
approximately half as embryotoxic as benomyl (Stenberg et al., 1975).
In mammals, benomyl is rapidly absorbed, metabolized primarily in
the liver and excreted. Metabolism apparently proceeds through two
major routes, by hydroxylation and hydrolysis. The metabolites are
excreted in the urine and faeces and do not appear to be stored in
body tissues. In the rat a large proportion of orally administered
benomyl is rapidly metabolized to methyl benzimidazol-2-yl carbamate
(MBC). The elimination pattern and the identity of the major
metabolite in the urine is identical after the administration of
either benomyl or MBC. This indicates the rat metabolizes the two
compounds in a similar manner. Thus, animals in toxicological studies
exposed to benomyl are also exposed to MBC.
A three generation reproduction study in the rat with dietary
levels up to 2500 ppm did not indicate significant effects. Benomyl
was not embryotoxic. Indications of a mutagenic action were found in a
fungal system. A dominant lethal study in the rat with dietary levels
up to 2500 ppm was negative. Benomyl showed no evidence of
teratogenesis when fed to rats at 5000 ppm (250 mg/kg body weight) in
the diet. However, a recent report from the USSR observed embryotoxic
effects (100% post-implantation deaths of the embryo) when a high dose
of benomyl (500 mg/kg/body weight) was administered to rats by gavage.
No information is available on long-term studies in any species
of animal. This precludes an estimation of an acceptable daily intake
No acceptable daily intake allocated.
RESIDUES IN FOOD AND THEIR EVALUATION
Benomyl is a systemic fungicide with many indications of a
widespread and still increasing use in the control of several species
of fungi. Simultaneously, however, critical studies have been
published of toxic effects in earthworms (Stringer and Lyons, 1974;
Krupka, 1974) residues resulting from supervised trials and of
adverse phytotoxic effects (Butt et al., 1973) in the form of
increased fruit russet and reduction of crop and fruit size in certain
apple species after foliar treatment in the early growing season.
RESIDUES RESULTING FROM SUPERVISED TRIALS
A substantial amount of new information on residues from
supervised trials and experimental work has been published or brought
to the attention of the Meeting since 1973 from the manufacturers as
well as from various governments. The majority of this, however, has
confirmed the ranges of residues and general patterns which were
described in the earlier monograph (FAO/WHO, 1974). The following is
therefore quoted with special emphasis on its supplementary nature.
Several trials on benomyl applications to greenhouse cultures,
especially lettuce, have been reported to the Meeting from some
European countries. The results with lettuce (Tables 1 and 2) indicate
that considerable amounts may be deposited on leaves or taken up
through the roots of this leafy plant.
In the winter season from four to six weeks may be required after
early foliar application to achieve residues below 5 mg/kg (calculated
as carbendazim) while for levels of 1-2 mg/kg more than two months
will be needed, in both cases requiring a further careful trimming of
outer leaves. The summer experiments indicate shorter periods of the
order of two to three weeks. Comparatively, soil treatment or early
watering around the plants seems to give rise to lower residues,
although the continued uptake results in residues which will require
practically the same preharvest intervals.
TABLE 1. Benomyl trials on greenhouse lettuce, Belgium1
Time after (No. of applications)
Treatment rate application 1× 2× 3×
Spraying 1.5 kg/ha 0 days - - 106
2 weeks - 36.2 33.1
3 weeks - - 21.0
4 weeks 6.2 10.5 11.6
5 weeks - 7.3 7.3
6 weeks 1.6 4.7 4.7
8 weeks 0.9 1.5 2.5
Spraying 3.0 kg/ha 0 day - - 190
2 weeks - 45.5 65
3 weeks - - 37.4
4 weeks 11.8 17.6 23.6
5 weeks - 13.9 14.8
6 weeks 5.5 11.6 12.8
8 weeks 1.6 3.8 5.1
1 From Henriet et al. (1975).
2 All results calculated and expressed as carbendazim.
TABLE 2. Benomyl trials on greenhouse lettuce, Denmark1 and Netherlands2
Residues (mg/kg)3 mean and range
Dosage Time after last
Treatment rate application, days May-June November-March
Spraying1 1.5 kg/ha 2 50 (47-53) 32 (26-38)
9 17 (16-19) 24 (23-25)
16 6.6 (5.2-8.0) 22 (22-22)
23 0.8 (0.6-1.0) 21 (20-21)
Watering1 3.75 kg/ha 2 16 (15-18) 16 (13-19)
9 6.3 (5.2-7.4) 15 (12-18)
16 2.3 (1.5-3.1) 12 (10-15)
23 0.5 (nd.-O.9) 8.9 (7.0-11)
treatment1 3.75 kg/ha 11 45 (39-56)
19 20 (14-28)
28 10 ( 8-11)
32/33 7.7 (3.8-18)
41 8.8 (4.6-12)
53 3.0 (1.8-4.0)
1 Green-Lauridsem et al. (1975).
2 The Netherlands (1975).
3 All results calculated and expressed as benomyl (in mg/kg).
Information on benomyl residues in cereal grains (wheat, barley,
oats and rice) has been collected from several countries and submitted
by Kirk (1974) as shown in Table 3. This supplements that which was
available earlier to the Meeting. It confirms the earlier recommended
guideline level of 0.5 mg/kg in raw cereals,* covering the four
mentioned species, and gives grounds for amending the guideline level
of 2 mg/kg for barley straw to include also straw from rice and wheat.
* The guideline level for raw cereals recommended by the 1973 Joint
Meeting was correctly recorded as 0.5 mg/kg in the Evaluations
(FAO/WHO, 1974b) but erroneously printed as 0.1 mg/kg in the Report
FATE OF RESIDUES
Previously reviewed studies an the fate of benomyl in animals
have been extended and published by Gardiner et al. (1974). These
confirm that single, oral doses of benomyl (C14-labelled) are
eliminated via the urine and faeces within 72 hours from both rat and
dog. From 83.4 to 88.0% of the ingested amount was recovered from the
urine and 11.3-16.2% from the faeces. The major metabolite was methyl
5-hydroxy-2-benzimidazolecarbamate (5-HBC) found as glucuronide and/or
sulfate conjugates in the urine. The studies showed that, on the
evidence of residue analyses, cows and chickens show the same route of
metabolism and elimination. Other possible degradation products in
animal systems include methyl 4-hydroxy-2-benzimidazolecarbamate
(4-HBC) and methyl 2-bensimidazolecarbamate (MBC). The residue data
from these studies, which include two-year chronic feeding tests with
dogs and rats (Table 4), demonstrate that benomyl and its metabolites
do not accumulate in animal tissues. Milk from dairy cows (FAO/WHO,
1974) and eggs from chickens (Table 5) contain detectable residues
(0.1 mg/kg or less) only at such high dietary levels of benomyl as
In an earlier quoted series of field trials on metabolism and
disappearance rates in different soils (Baude et al., 1974) it was
indicated that the leaching of benomyl and its degradation products
MBC and 2-amino benzimidazole (2-AB) from soils was negligible.
Additional studies by Rhodes and Long (1974) support this conclusion
through direct experiments under greenhouse and laboratory conditions.
Their experiments elucidate both run-off and leaching characteristics
of the compounds and show that benomyl, MBC and 2-AB are all immobile
in soil and do not leach or move significantly from the site of
TABLE 3. Benomyl residues in cereals
Number of interval Residues
Crop Treatment Dosage treatments (days) (mg/kg) Country
Wheat, barley, Seed
and oat treatment 200 g/100 kg 133-398 <0.4 Finland
g/100 kg 94-303 <0.1 United States
treatment 1 kg/ha 64-77 <0.1 Netherlands
600 g/ha 3 × 12-75 0.1-0.5 France
240 g/ha 105 0.0-0.2 West Germany
400 g/ha 4 × 30-32 <0.03 Belgium
Wheat straw 1 kg/ha 64-77 0.15-0.93 Netherlands
treatment 0.25-1 kg/ha 1-2× - 0.49 United States
(<0.05-2.8) of America
straw 0.25-1 kg/ha 1-2× - 3.52 United States
(<0.05-9.0) of America
TABLE 4. Residue data from benomyl chronic feeding studies in rat
and dog (Gardiner et al., 1974)
Residue1, mg/kg (male and female)
Tissue (mg/kg) carbendazim 5-HBC 4-HBC
Muscle 0 <0.05 <0.05 <0.1
2500 <0.05-0.06 0.51-0.52 <0.1
Fat 0 <0.05 <0.05 <0.1
2500 <0.05 <0.05-0.08 <0.1
Liver 0 <0.05 <0.05 <0.1
2500 0.20-0.61 1.7-2.5 <0.1
Kidney 0 <0.05 <0.05 <0.1
2500 0.20-1.4 2.8-22 <0.1-0.45
Muscle 0 <0.05 <0.05 <0.1
2500 <0.05 <0.05 <0.1
Fat 0 <0.05 <0.052 <0.1
2500 <0.05-0.15 <0.05-0.14 <0.1
Liver 0 <0.05 <0.05 <0.1
2500 <0.05 <0.05 <0.1
Kidney 0 <0.05 <0.05 <0.1
2500 <0.05 <0.05-0.12 <0.1
1 All results corrected for average recoveries (benomyl+MBC: 79%;
5-HBC: 65%; 4-HBC: 55%).
2 An apparent contamination corresponding to 1.2 mg/kg noted in
one male dog.
EVIDENCE OF RESIDUES IN COMMERCE
Market sample surveys carried out in the Netherlands in 1973 on
(mainly domestically grown) fruit and vegetables showed that residues
from benzimidazole fungicides, among which benomyl would be included,
were regularly present in some crops (Table 6), In strawberries about
20% of the samples were positive, while in leafy vegetables, apples
and tomatoes taken together about 4-5% were positive. At the time of
the survey, the tolerances were 2 mg/kg for fruits and vegetables and
0.05 mg/kg for potatoes, expressed and calculated as carbendazim.
Similarly, Belgian surveys on strawberries carried out in 1972
and 1974 showed 26% of the samples to be positive (Dejonckheere et
al., 1975). A total of 188 market samples were analysed and none
contained residues exceeding 5 mg/kg (expressed as carbendazim).
METHODS OF RESIDUES ANALYSIS
Since the Joint Meeting in 1973, the greatest interest in the
analytical methodology of benomyl and other benzimidazoles has been
directed to two problems, namely the extraction efficiency from crops
and soils and the selective determination of benzimidazole compounds,
especially by means of high speed liquid chromatography (HSLC).
Austin and co-workers (1975) in a recent review deal with these
questions and offer a convenient extraction solvent for aged
carbendazim residues from soils consisting of 1 M NH4Cl-solution
(aqueous) mixed with an equal volume of acetone or methanol. Further,
they conclude that HSLC using UV-absorption detectors still shows
promise (FAO/WHO, 1974), but is not yet at a suitable stage of
development for routine purposes owing to some lack of selectivity and
The need for further work on the separate determination of
carbendazim and its precursors, which include benomyl, is still
Extensive new data on use patterns, mode of action, residue
levels etc. of benomyl have been published or otherwise come forward
since 1973. Much of this provides support for previously considered
information and confirms recommendations for already established
There are however additional data on residues in greenhouse grown
lettuce which permit a recommendation to be made for a guideline level
for this crop. Further information on cereals confirms the earlier
recommendation for a guideline level of 0.5 mg/kg in raw cereals.
TABLE 5. Residues in chickens' eggs after benomyl feeding at 5 and 25
mg/kg level (Gardiner et al., 1974)
5-hydroxy-MBC found, mg/kg
description Control 5mg/kg level 25 mg/kg level
7 days <0.02 <0.02 0.03
14 days <0.02 <0.02 0.06
28 days <0.02 <0.02 0.03
Breast, 28 days <0.02 <0.02 <0.02
Fat, 28 days <0.02 <0.02 <0.02
Liver, 28 days <0.1 <0.1 <0.1
Faeces, 28 days 0.161 1.6 7.5
TABLE 6. Residues of methylbenzimidazole (including benomyl) in
marketed samples, Netherlands
Number of samples containing ... mg/kg1
Crop <0.1 0.1-1.0 1.0-2.0 >2.0
Potatoes 10 2 -
Endive 29 - 12
Lettuce 57 2 -
Spinach 28 - -
Tomatoes 10 2 -
Apples (imported) 5 13
Strawberries 129 20 8 4
1 Expressed as carbendazim.
2 Contained 4 mg/kg.
3 Contained 15 mg/kg. Imported.
Further studies have been presented on residues in animal
products. They demonstrate that neither benomyl nor its metabolites
accumulate in animal tissues and that residues of the hydroxylated MBC
metabolites are present in barely detectable amounts in milk and eggs
only after feeding high dietary levels of benomyl.
In response to the request from an earlier Meeting, information
on market sample surveys made in the Netherlands and in Belgium has
been presented to the Meeting. The data show that residues of
benzimidazole fungicides, presumably including benomyl, may be present
in up to 20-26% of a number of fruits, vegetables and berries.
Residues in positive samples are generally low and only in occasional
individual samples approach or exceed earlier established guideline
The recommendations for guideline levels made in 1973 (FAO/WHO,
1974) are amended as follows. They refer to total residues of benomyl
carbendazim and 2-aminobenzimidazole, expressed as carbendazim.
Rice straw, wheat straw 2
Raw cereals (wheat, barley, rye, rice) 0.5*
Meat or poultry, eggs 0.1**
* Attention is drawn to the footnote on p. [text missing] of this monograph
** At or about limit of determination.
FURTHER WORK OR INFORMATION
REQUIRED (before an acceptable daily intake can be allocated)
1. Long-term studies in at least one mammalian species.
2. Short-term studies in several animal species including a
non-rodent mammalian species.
3. Acute oral studies in several animal species.
1. A supplementary carcinogenic study.
2. Observations in man.
3. Further development of analytical methods for the separate
determination of benomyl and carbendazim.
4. Further information on residues in food in commerce.
Austin, D. J., Briggs, G. G. and Lord, K. A. (1975) Problems in the
assay of residues of carbendazim and its precursors. Proc. 8th Brit.
Insec. Fund. Conf. Brighton. November
Baude, F. J., Pease, H. L. and Holt, R. F. (1974) Fate of benomyl on
field soil and turf. J. Agric. Food Chem., 22 (3), 413-418
Borck, K. and Braymer, H. D. (1974) The genetic analysis of resistance
to benomyl in Neurospora crassa. Journal of General Microbiology,
Butt, D. J., Kirby, A. H. M. and Williamson, C. J. (1973) Fungitoxic
and phytotoxic effects of fungicides controlling powdery mildew on
apple. Ann. appl. Biol., 75, 217-228
Dassenay, B. and Meyer, J. A. (1973) Mutagenic effect of benomyl on
Fusarium oxysporum. Mutation Research, 21, 119
Dejonckheere, W., Steurbaut, W. and Kips, R. H. (1975) Report received
from Rijksnoviversiteit Gent, Laboratorium voor Fytofarmacie, Gent. 11
Douch, P. G. C. (1973) The metabolism of benomyl fungicide in mammals.
Xenobiotica, 3, 367-380
Gardiner, J. A., Kirkland, J. J., Klopping, H. L. and Sherman, H.
(1974) Fate of benomyl in animals. Journal Agriculture Food Chemistry,
Green-Lauridsen, M. (1975) Prepublication data submitted from National
Food Institute, Soborg, Denmark, November
Henriet, J., Meens, P., Baelus, F. and Valange, B. (1975)
Beschouwingen over het gebruik van benomyl in glassla en aanbevelingen
voor slakwekers. Technische Nota 9/18 from Rijksstation voor
Phytopharmacie, Gembloux, Belgium
Kirk, W. F. (1974) Information on Benlate treatments on cereals
covering seed treatments and foliar applications submitted by DuPont
de Nemours International, Geneva, 3 April (Unpublished report)
Krupka, R. M. (1974) On the anti-cholinesterase activity of benomyl.
Pesticide Science, 5, 211-216
Netherlands. (1975) Information of the Netherlands on pesticides to be
considered by the JMPR 1975 November
Rhodes, R. C. and Long, J. D. (1974) Run-off and leaching studies on
benomyl in soils and turf. Bull. Environ. Contamin. Toxicol., 12,
Sherman, H., Culik, R. and Jackson, R. A. (1975) Reproduction,
teratogenic and mutagenic studies with benomyl. Toxicology and Applied
Pharmacology, 32, 305-315
Stenberg, A. I., Orlova, N. V. and Torchinskii, A. M. (1975) Action of
pesticides of different chemical structure on the gonads and
embryogenesis of experimental animals. Gig. Sanit. (8), 16-20 (Russ.)
Stringer, A, and Lyons, C. H. (1974) The effect of benomyl and
thiophanatemethyl on earth-worm populations in apple orchards.
Pesticide Science, 5, 189-196