PESTICIDE RESIDUES IN FOOD - 1980
Sponsored jointly by FAO and WHO
EVALUATIONS 1980
Joint meeting of the
FAO Panel of Experts on Pesticide Residues
in Food and the Environment
and the
WHO Expert Group on Pesticide Residues
Rome, 6-15 October 1980
DITHIOCARBAMATES
Explanation
Dithiocarbamate fungicides were reviewed by the Joint Meeting in
1965, 1967, 1970, 1974, and 1977 (FAO/WHO, 1966, 1967, 1971 and
1975; FAO, 1978). The class of dithiocarbamates has been
considered in the past as a single group predominantly because of
the analytical procedures utilised in routine pesticide residue
chemistry programmes. The method of choice for regulatory residue
determinations has been a non-specific analytical assay incapable
of distinguishing individual dithiocarbamates.
The 1977 meeting divided the dithiocarbamates into the three major
classes: Dimethyl dithiocarbamates, ethylenebisdithiocarbamates and
propylenebisdithiocarbamates.
In previous toxicological evaluations of dithiocarbamates, data
were requested on carcinogenic effects, effects on thyroid
function, effects on reticuloendothelial and haematopoietic systems
and on reproductive physiology. Additionally in 1977, the U.S. EPA
issued a Rebuttable Presumption Against Registration (RPAR) of
pesticide products containing EBDC's. The health aspect of this
RPAR notice specified two major areas of concern: (1) oncogenicity
and (2) teratogenicity. The two areas of concern with respect to
health considered toxicological data on both EBDC's and
ethylenethiourea (ETU) (the major breakdown product). New data made
available to WHO have been reviewed by the Meeting. These data
include special studies on teratogenicity and short-term dietary
studies on specific EBDC fungicides and/or their major breakdown
products, ethylenethiourea (ETU) and ethylenebisisothiocyanate
pulphide (EBIS). In addition, the problems discussed in the RPAR
programme were considered by the Meeting.
At the time of the last full review in 1977 the following
requirements for further work or information on residues in food
were established.
Dithiocarbamates in general
1. A comprehensive survey of the use patterns of thiram, ferbam and
ziram.
2. Data on residues of thiram, ferbam and ziram from supervised
trials.
3. Data on residues of dithiocarbamates from crops grown under
glass.
Ethylenebisdithiocarbamates
1. Further data on the conversion of EBDC to ETU in various food
processing procedures.
2. Studies of procedures to minimise the formation of ETU during
food processing.
Propineb
Information regarding the fate of residues during food processing,
including cooking.
This monograph addendum includes a summary of the relevant
information that has been made available in response to these
requests.
DATA CONSIDERED FOR DERIVATION OF ACCEPTABLE DAILY INTAKE
TOXICOLOGICAL STUDIES
Special studies on teratogenicity
Groups of rats (26-27 pregnant CD-1 rats/group) were administered
zineb (Dithane(R) Z-78) at dosage levels of 0, 200, 632, or 2000
mg/kg/day for 14 treatments from day 6 through 19 of gestation.
Analytical chemical determinations on the purity of the zineb
showed 85.5% EBDC containing 0.35% ETU. The equivalent dosage
levels were 0, 170, 543, and 1710 mg/kg bw/day for EBDC and 0, 0.7,
2.2 or 7.0 mg/kg bw for ETU.
Maternal body weight data and food consumption data were recorded.
Pregnant rats were sacrificed at day 20 and a laparotomy was
performed. Foetal data included live, dead and resorbed foetuses
as well as somatic and skeletal abnormalities.
There was no maternal mortality but a substantial weight loss was
noted at the highest dosage level. Foetuses from the mothers
administered 2000 mg/kg also showed a reduced body weight. Foetal
mortality was not noted and there were no significant anomalies
noted on gross external examination. A higher incidence of
abnormalities of the tail were noted at the highest dosage level
(short or kinky tails).
Teratogenic events were recorded in foetuses at the highest dosage
level. A significant increase in lateral hydrocephalus and
hydrocephalus of the third ventricle was noted at 2O00 mg/kg. In
addition, at this highest dose level, there was an increased
incidence of skeletal anomalies (enlarged frontal fontanelle,
enlarged occipital fontanelle, split centra and incomplete
ossification of the supraoccipital). The abnormalities were not
noted at 632 and were suspected of being due to the presence of
ETU, which would occur both in the formulation (up to 7 mg/kg/day
was directly administered) and as a result of metabolism of zineb.
It was concluded that teratogenic anomalies were produced in rats
by zineb at doses that were extraordinarily high and were
maternally toxic. The abnormalities may have been due, in part, to
ETU known to be present in the formulation (Short et al., 1980).
In a study identical with that reported above for rats, mice (CD-1)
were administered zineb daily for 11 days of gestation (days 6-16)
and were sacrificed on day 18. Gross examination for maternal
well-being and foetal anomalies (both somatic and skeletal) failed
to show the occurrence of significant teratogenic events. There
were no teratogenic anomalies in mice as a result of administration
of zineb at dosage levels of up to and including 2000 mg/kg bw
(1710 mg/kg of zinc-EBDC and 7 mg/kg ETU) (Short et al., 1980).
Maneb was administered to rats at dosage levels of 0, 400, 770 or
1420 mg/kg by gavage as a single dose on day 11 of gestation. Rats
were sacrificed on day 18 of gestation and foetuses were examined
for reproductive and teratogenic abnormalities.
A substantially increased resorption rate was noted at 770 mg/kg.
Gross malformations occurred in all surviving animals at 770 and
1420 mg/kg. (No malformations were observed in the single litter
of the low dose group). These abnormalities included: cleft
palate, hydrocephaly, and other serious defects. In another
experiment, maternal administration of zinc acetate (made in an
attempt to relieve the incidence of teratogenic events) was
somewhat therapeutic at a lower (750 mg/kg) dose but at a higher
dose (1380 mg/kg), the frequency and type of malformations were
unchanged (Larsson, et al., 1976).
Mancozeb was administered to rats at dosage levels of O, 380, 730,
or 1320 mg/kg on day 11 of gestation in a study similar to that
reported above with maneb. Again, a substantial increase in
malformations was observed at the highest dosage groups. There
were no effects noted at 730 mg/kg and below. Similar foetal
malformations were observed as reported for maneb (Larsson, et
al., 1976).
Propineb was administered to rats at dosage levels of 0, 400, 760,
or 2300 mg/kg by gavage on day 11 of gestation. The dams were
sacrificed and foetuses examined for gross external and internal
malformations on day 18 of pregnancy.
Maternal toxicity was observed at all dosage levels. At the
highest dosage, propineb was foetotoxic and induced a variety of
malformations in the surviving foetuses. At 760 mg/kg propineb was
slightly foetotoxic but did not induce malformations in surviving
foetuses. The pattern of foetal abnormalities was qualitatively
similar to that noted in the maneb- and mancozeb-treated rats
(Larsson et al., 1976).
Maneb and two EBDC metabolites, ETU and EBIS, were examined in two
rodent species for their potential to induce perinatal toxicity.
Additionally, ETU was administered to guinea pigs and golden
hamsters to evaluate the teratogenic potential in these species.
All compounds were administered during organogenesis by gavage.
The following is a summary of prenatal treatments:
Compound Species Dosages Treatment
(mg/kg) (gestation day)
Maneb Rat 0,120, 240 7-16
& 480
Mouse 0, 375, 750 7-16
& 1500
ETU Rat 0, 5, 10, 20, 7-21
30, 40, & 80
Mouse 0, 100 & 200 7-16
Hamster 0, 75, 150 5-10
& 300
Guinea-pig 0, 50 & 100 7-25
EBIS Rat 0, 7.5, 25 7-21
& 30
Mouse 0, 50, 100 7-16
& 200
In the prenatal teratology studies, rats were sacrificed on day 21,
mice on day 18, hamsters on day 15 and guinea pigs on day 35 and
foetuses examined for malformations (both somatic and skeletal).
Additional postnatal studies were performed with rats using
extended treatment periods.
Compounds Dosage
Maneb 0, 240 & 480
ETU 0, 20, 25 & 30
EBIS 0, 15 & 30
The treatment schedule included continuous exposure from day 7 of
gestation through parturition to day 15 of lactation. The pups
were weaned normally and postnatal studies were performed at 6
weeks for open-field behaviour.
All three compounds were maternally toxic at high dose levels in
the rat, less so in the mouse and (with ETU) the guinea pig and
hamster.
Maneb reduced rat maternal weight gain and increased the liver/body
weight ratios at all dose levels. A peripheral paralysis was
induced in maternal rats at high dose levels. Foetal data were
affected only at the highest dose level (reduced foetal weight,
reduced caudal ossification and hydrocephalus were noted). In the
mouse, maneb also affected the maternal liver and retarded
development, causing a decrease in foetal caudal ossification
centres (at all dosage levels).
EBIS reduced maternal growth, increased the liver/body weight ratio
and induced a peripheral paralysis at the highest dosage in rats.
While mouse maternal growth was unaffected, the liver was enlarged
at the highest dose level. EBIS was not teratogenic as it did not
affect foetal parameters.
ETU induced maternal toxicity and reduced growth in the rat at 80
mg/kg and was teratogenic to the rat, inducing substantial foetal
effects at all dosage levels above 10 mg/kg. Gross defects were
seen in the skeletal system and the central nervous system. Cleft
palate was noted, predominantly at the highest dosage level. The
defects appeared to follow a dose relationship decreasing until at
20 and 30 mg/kg an increased incidence of hydrocephalus was the
only defect noted. The maternal and foetal toxicity of ETU on the
mouse, guinea pig, and hamster was substantially less severe than
noted with the rat. In mice, at the highest dose, an increased
maternal liver weight and an increase in foetal supernumerary ribs
were noted. No effects were noted with other species.
Postnatal studies with maneb and EBIS were uneventful with respect
to the reproductive parameters and behaviourial studies performed.
ETU induced a variety of post-natal effects. Maternal milk
production was reduced or absent causing pup mortality at doses of
30 mg/kg and above. No significant dose-related behaviourial
abnormalities were observed with ETU-exposed pups.
Thus, maneb and EBIS were maternally toxic (inducing a peripheral
paralysis) to the rat but not the mouse. Maneb and ETU were
teratogenic to the rat but not the mouse (or hamster and guinea pig
with ETU). EBIS did not induce a teratogenic response in any
species tested (Chernoff et al., 1979).
Groups of pregnant rats were administered ETU at dosage levels of
0, 15, 30 or 45 mg/kg bw on day 15 of gestation and subjected to a
variety of test conditions to evaluate pre- and post-natal effects.
Mortality occurred postnatally at dosage levels above 15 mg/kg in
pups from treated mothers or pups cross-fostered to evaluate
lactation exposure. All pups from mothers treated with 45 mg/kg
died within 4 weeks of birth. A high incidence of hydrocephalus
and microphthalmia was observed in pups of mothers treated at a
dosage of 30 mg/kg. These pups died within 6 weeks of birth.
Motor defects were observed in some surviving (16/65) offspring of
the 30 mg/kg group. This was pathologically shown to be a direct
result of the hydrocephalic condition, which was accompanied by
atrophy of the cerebral cortex and subcortical white matter. The
defects were observed to be a direct result of in utero
administration of LTU and not from exposure during lactation.
(Cross-fostered pups showed the same effects as pups weaned from
treated dams).
All female offspring of rats administered 30 mg/kg, when mated to
normal male rats, gave birth to normal offspring. The F2
generation
was not impaired although some of the parents had neurological
defects. In these experiments no effects were observed on the
parameters examined at 15 mg/kg bw (Khera and Tryphonas, 1977).
When oral doses of ETU were administered in combination with sodium
nitrite, multiple foetal anomalies were produced in the mouse, a
species generally refractory to treatment with either chemical
alone (Teramoto et al., 1976).
Special studies on reproduction
Groups of rats (16 male and 16 female, ChR-CD rats/group) were fed
maneb in the diet for 3 months at levels of 0, 125 or 250 mg/kg and
mated in standard 3-generation, 2-litter per generation,
reproduction study. Groups of males and females from the F1b and
F2b litters were fed maneb for three months after weaning and
mated to become parents of the succeeding generation.
There were no effects on reproduction reported over the entire
study. The major reproduction indices were unaffected by maneb at
dietary levels up to and including 250 mg/kg. There was no
histologic evidence of congenital anomalies in a variety of tissues
and organs of the male and female rats (10 each group) of the F3b
litter subjected to histopathologic examination (Sherman and Zapp,
1966).
Special studies on mutagenicity
ETU was examined for its mutagenic potential in a series of
microbial and mammalian bioassays.
Microbial studies
A reversion assay ('Ames test') was carried out with 5 strains of
Salmonella and 2 strains of E. coli in the presence and
absence of a male rat metabolic activation system. Positive
mutagenicity indications were obtained with TA 1535 and TA 100 at
the highest concentrations (1 mg/plate) tested in the absence of a
metabolic activation system. ETU was suggested to have induced a
weak mutagenic effect in certain microbial tester strains (Teramoto
et al., 1977). Additional in vitro reversion studies on
different histidine-requiring Salmonella typhimurium strains
were performed in a semi-quantitative plate test. With strain his
G46 at doses of 20, 50, 80 or 200 mg/plate, ETU data were not
significantly different from that of the control. However, a rise
in the number of revertants per surviving cells was found with the
repair-deficient strain TA 1530. The increase over the spontaneous
reversion frequency was by a factor of 7.1 at 20 mg/plate, 9.1 at
40 mg/plate, 11.7 at 80 mg/plate and 11.1 at 200 mg/plate. No
induction of revertants could be observed in the frameshift mutants
TA 1531, TA 1532, and TA 1964. Thus, ETU-induced mutations were of
the base-pair substitution type. A large fraction of the induced
lesions were eliminated by excision repair (Schupbach and Hummler,
1977).
In a host-mediated assay, a weak but significant increase of the
reversion frequency (by a factor of 2.4 at a dose of 6000 mg/kg
body weight) could be detected with strain TA 1530 used as the
indicator organism in the mouse. No mutation induction was
observed at doses of 2000 mg/kg or below (Schupbach and Hummler,
1977).
Combinations of ETU and sodium nitrite have been reported to induce
a mutagenic response in the 'Ames' assay in E. coli and a
host-mediated assay (Shirasu et al., 1977)
Mammalian studies
A Chinese hamster cell line was treated with ETU, cultured and
examined for chromosomal abnormalities in an in vitro
cytogenetic assay. In addition, an in vivo cytogenetic assay
on bone marrow cells was performed following treatment of rats
orally (a single dose of 200 or 400 mg/kg or 2 to 5 consecutive
24-hour doses of 50-400 mg/kg).
Other than a severe cytotoxic effect noted at 3200 µg ETU/ml in the
in vitro cytogenetic assay, there were no cytogenetic effects
noted with ETU. Rat bone marrow cells were unaffected by ETU at
any dose tested. In an evaluation of aneuploidy, the frequency of
numerically aberrant cells, including aneuploid and polyploid
cells, was somewhat high although not significantly higher than
control values of 3-week old rats.
In a micronucleus test, Swiss albino mice were treated twice within
24 hours with 25, 700, 1860 or 6000 mg/kg ETU. A total of 2000
erythrocytes of the bone marrow of each animal was scored. No
increase in the number of erythrocytes containing micronuclei was
found in treated animals (Schupbach and Hummler, 1977).
Groups of male mice were administered ETU orally for 5 consecutive
days at dosage levels of 0, 300 or 600 mg/kg and mated to begin a
5-week dominant-lethal study. There were no indications of a
dominant-lethal effect at either dosage level tested (a positive
control, EMS, reflected the susceptibility of the strain to
inducing such effects as evidenced by reduced implantations and
live embryos at weeks 1 and 2 of testing) (Teramoto et al.,
1977).
In another dominant-lethal test, male mice received single doses of
500, 1000 or 3500 mg/kg respectively. Although a slightly reduced
fertility rate was observed in the highest dose group, no
correlation between dose and incidence of dominant lethals was
found (Schupbach and Hummler, 1977).
Short-term studies
Rat - ETU
Groups of rats (20 male and 20 female Sprague-Dawley strain
rats/group; 24 of each sex were used as controls) were fed ETU in
the diet for 90 days at dosage levels of 0, 1, 5, 25, 125, or 625
mg/kg. Additional groups of rats of both sexes were fed amitrole
(50 mg/kg) and propylthiourea (PTU) (125 mg/kg) as a positive
control group. Growth and food consumption were recorded weekly and
daily observations were made for behaviourial changes and
mortality. At 30, 60 and 90 days, groups of 10 rats of each sex
were sacrificed for examination of thyroid function tests (T-3,
T-4, TBG, TSH, and 125I uptake). Gross and microscopic analyses of
tissues and organs were performed at these intervals.
Clinical signs of poisoning, mortality, and growth reduction were
observed at the highest dose level. No adverse clinical signs were
noted at lower ETU doses or in the amitrole or PTU animals.
Biochemical changes reflecting effects on thyroid function were
noted at dosages of ETU exceeding 25 mg/kg. There were decreases
in T3 and T4, an increase in TSH, and a decrease in iodine
uptake. These changes were also observed with the two positive
controls groups. Based on biochemical indicators, the most
sensitive parameters for evaluating thyroid function, a no-effect
dietary level for this 90-day study is 25 mg/kg. Gross and
microscopic examinations showed substantial effects on a variety of
organs and tissues at the highest ETU dose group. At 125 mg/kg,
thyroid hyperplasia was observed. No effects were noted at 25
mg/kg equivalent to an ETU uptake of 1.78 mg/kg bw (Freudenthal et
al., 1977)
Rat - EBIS
Groups of rats (60 male and 60 female Sprague-Dawley rats/group; 30
of each sex ware used as controls) were fed EBIS in the diet at
dosage levels of 0, 1, 10, 100 or 1000 mg/kg for 90 days.
[Positive controls for thyroid function tests were utilised to
assure quality of the clinical procedures. These controls were
administered just prior to sacrifice and clinical assay and were
not necessarily included in the feeding trial. Positive controls
included aminotriazole (administered to groups of 6 rats of each
sex daily for 4 days by gavage at a dose of 4000 mg/kg) and
methimazole (administered to groups of 6 rats of each sex twice
daily for 4 days by gavage at a dose of 0.6 mg/kg)]. Growth was
measured at weekly intervals over the 12-week trial. Groups of 20
of each sex were sacrificed monthly for thyroid function tests and
gross and microscopic pathology.
EBIS was toxic at 1000 mg/kg inducing a reversible paralysis of the
hind legs followed by death if once affected the animals were
continued on the dosing regimen. Animals removed from the EBIS
diet recovered from the paralysis. However, they became
reafflicted when exposed to EBIS at the high dosage level. No
histological lesion could be identified with the motor paralysis.
Thyroid function was substantially affected by EBIS at 1000 mg/kg
as was growth in both males and females. Thyroxine (T-4) levels
were significantly reduced as was iodine uptake. At 100 mg/kg, no
adverse effects of EBIS were noted on clinical parameters or on
gross or histopathologic examinations. A no-effect level in this
study was 100 mg/kg in the diet based on data observed with
clinical and histopathologic studies (Freudenthal et al., 1977).
OBSERVATIONS IN MAN
Epidemiological studies were conducted on workers in the rubber
industry by Parkes (1974) and Smith (1976). Parkes (1974) examined
the records relating to the national or regional incidence of
thyroid cancer, to evaluate what proportion of such cases might
have originated amongst men employed in the rubber industry where
ETU is used extensively. Parkes concluded that, under the
conditions in which ETU has been used in the past, there is no risk
of man contracting thyroid cancer as a result of industrial
exposure to ethylenethiourea.
Smith (1976) conducted a detailed study involving 1,929 workers in
rubber compounding plants in the Birmingham, England area. No
thyroid cancers were found in the health records of these workers.
Smith concluded that this study does not demonstrate any risk of
thyroid cancer from the normal industrial use of ETU.
Bruerman et al. (1980) studied a population of workers
manufacturing Dithane(R), employing a battery of thyroid function
tests as well as other parameters to evaluate their health status
and to detect subtle thyroid function abnormalities. Based on all
data available, there were no abnormalities associated with the
exposure to the EBDC's during their manufacture.
RESIDUES IN FOODS
ETHYLENETHIOUREA IN WINE
Carbon-14 labelled zineb and ethylenethiourea (ETU) have been used
to study the behaviour of the compounds during the wine-making
processes. At the start of wine-making, zineb remains absorbed on
the solid parts of the grapes where it undergoes extensive
degradation to form some slightly soluble compounds, including ETU,
ethyleneurea (EU), hydantoin and ethylenediamine. Of the zineb
present in the liquid portion of the must, 18% passes into the wine
as metabolites, but of these ETU and EU are at levels well below
0.01 mg/kg. ETU added to the must reacts very rapidly with the
natural components and remains in the solid part of the must.
There is thus little probability that significant amounts of ETU
would be present in wines prepared from grapes that had been
treated with zineb (Santi et al., 1980).
Improved analytical methods involving direct GLC of ETU, without
derivatisation, using a flame photometric detector have enabled a
detection limit of 0.01 mg/kg for ETU to be achieved in wines, with
mean recoveries of about 71% at levels between 0.05 and 0.10 mg/kg.
Together with an additional procedure to confirm identity, direct
GLC has been used to examine wines from several countries, randomly
purchased in France, ltaly and Federal Republic of Germany. In
none of the 39 samples reported was ETU observed at above the limit
of detection of 0.01 mg/kg. Any propylenethiourea that might have
been present would also have been detected (Fabbrini et al.,
1980).
Both of these papers stress the need to obtain positive
confirmation of identity for any 'apparent' ETU that may seem to be
present.
RESIDUES IN COMMERCIAL FOODSTUFFS
A 'market basket' study of EBDC and ETU residues in food has
recently been reported by Gowers and Gordon (1980). Over 500
samples of 34 foods were analysed, plus 26 samples of drinking
water. The water samples contained no residues, 53 of the food
samples showed EBDC residues above the limit of detection of the
method, but all were below the USA maximum limits while only two
results were confirmed as positive for ETU, both around 0.01 mg/kg.
In a second study, 203 samples of tomato products were examined;
none contained ETU but 19% contained EBDC residues in the range 0.2
to 0.5 mg/kg. On the basis of these analyses, estimates of daily
dietary intake were 6.6 × 10-4 mg/kg bw for EBDC and 2.3 × 10-4
mg/kg bw for ETU.
Holt (1977) has also reported the results of a market basket survey
for ETU and EBDC residues in food items purchased in 9 states of
the USA (California, Colorado, Oregon, Texas, Illinois, Florida,
North Carolina, New York and Massachusetts). The foods studied
were canned tomatoes, tomato juice, tomato paste, catsup, canned
potatoes, instant potatoes, canned green beans, applesauce, carrots
and pickles. No EBDC residues were observed in any sample (<0.2
mg/kg as maneb). ETU was not observed in tomatoes, green beans,
applesauce or pickles (<0.01 mg/kg) and only random trace positive
results were found in the other foods. Of the 189 samples
analysed, 144 showed no ETU residue (<0.01 mg/kg) and none had
values greater than 0.05 mg/kg. Positive ETU results were
confirmed by mass spectrometry.
Typical food processing procedures, such as washing, scrubbing,
trimming and peeling, remove about 87% of EBDC residues. A 'table
top' study in which whole prepared meals were analysed for ETU has
also been described by Gowers and Gordon (1980). Of 60
home-prepared and 40 restaurant-prepared meals, only one showed any
apparent ETU, which could not be confirmed as to identity. Of the
87 meals examined for EBDC, 11 were positive and the levels found
averaged 0.3 mg/kg. A second study of another group of 100 meals
showed no ETU present while only four of the meals contained EBDC
in the range 0.2 to 0.4 mg/kg. If the single result for ETU is
taken to be real, the average daily intake for the group of 200
meals would be 1.35 × 10-6 mg/kg bw; that
for EBDC is 7.5 × 10-4 mg/kg bw based on the overall average of
0.03 mg/kg of EBDC in the meals. Hydrolysis of EBDC residues
during cooking can be a source of ETU but not always a dietary
source since discarding the cooking water, as is frequently done,
removes both the ETU and the EBDC residues. Up to 10 - 15% of the
EBDC residues present can be converted to ETU, which is not the
only decomposition product.
The stability of ETU and maneb residues in canned vegetables has
been studied by Han (1977) using 14C-labelled material. Samples
of commercial canned tomato sauce and spinach were fortified
separately and then re-sterilized by simulated commercial
procedures. Residues of intact ETU were found to diminish rapidly
with time, dropping to 0.2% of the amount added to tomato sauce and
to 10% of the amount added to spinach after 4 weeks storage at room
temperature. Similar results were found after addition of labelled
maneb to the same vegetables. Ethyleneurea and more polar
materials accounted for the bulk of the 14C-residue. These polar
degradation products were shown to be incapable of releasing ETU on
acidic or basic hydrolysis, thus ruling out the possibility that
they are present as complexes or simple conjugates of ETU. This
rapid degradation of ETU is probably the most important factor
involved in explaining why very little or no ETU is found in
commercially processed crops, which may contain maneb residues at
harvest.
Only one sample of tomato crops contained ETU (0.03 mg/kg) after
spraying maneb and mancozeb 5 to 8 times at intervals of 3-4 days
(von Stryk and Jarvis, 1980). However, in canned juice made from
these whole fruits a mean ETU level of 0.02 mg/kg was found. In
canned peeled fruit, residues were lower (mean 0.01 mg/kg). In a
greenhouse experiment ETU residues (up to 0.08 mg/kg, mean 0.0097)
were detected in more samples than under field conditions; mancozeb
residues ranged up to 2.8 mg/kg with a mean of 0.7 mg/kg.
Ripley et al (1978) studied residues of mancozeb and ETU on
grapes and grape products. Mean values of 6.8 mg/kg EBDC and 0.03
mg/kg ETU were found immediately after application and these
residues declined by 50% in the next 15-20 days. Wine prepared
from the treated grapes contained 0.037 mg/kg ETU but no residues
of EBDC were detected. Heat treatment of the harvested grapes
demonstrated conversion of EBDC to ETU (18%). Most commercial
grape products analysed showed less than 0.02 mg/kg ETU except some
concentrates that contained 0.06 mg/kg; EBDC was found in only one
of these samples.
Spraying tomatoes with maneb or mancozeb at rates of 2.7 kg/ha at
intervals of 7-12 days resulted in levels of <0.05 mg/kg ETU in
the fruits after 7 applications, whereas ETU residues in tomato
juice and whole pack products prepared from these treated fruits
sampled in the first 3 days after the 6th application of fungicides
ranged from not detectable (<0.01) to 0.17 mg/kg. Boiling of
fruit increased ETU residues by up to 800% (from 0.04 to 0.36
mg/kg). Commercial tomato products contained ETU residues of
<0.03 mg/kg, most containing <0.01 mg/kg (Ripley and Cox,
1978).
Ripley and Simpson (1977) monitored ETU residues on pears after
spraying with zineb (5 kg/ha). ETU residues ranged from 0.02-0.01
mg/kg over the 21-day trial. Four out of six samples of pear baby
food contained residues of ETU in the range of 0.01-0.05 mg/kg.
Boiling pears containing a zineb residue converted 3-6% of zineb
into ETU. Higher percentages of EBDC conversion into ETU (9-25%)
have been found by Casanova and Dachaud (1977). Spinach leaves
containing residues of maneb, zineb and mancozeb were boiled and
the increase in ETU residue observed. A considerable portion of
the ETU formed was present in the boiled spinach leaves.
In 1975, Ross et al (1978) studied residues of EBDC and ETU in
apples and apple products. In fresh apples harvested 42 days after
the last of nine sprays each of either mancozeb or metiram the EBDC
residues were 1.7 and 0.5 mg/kg respectively; residues of ETU were
not found above the limit of determination of 0.01 mg/kg. Canned
apple juice and sauce prepared from apples treated with mancozeb
both contained 0.05 mg/kg of ETU while the level in the pomace was
0.17 mg/kg. Apples that had been treated with metiram showed
similar levels. Similar trials in 1976 yielded EBDC residues of
less than 1 mg/kg on fresh fruit that had received one, two, three
or four cover sprays of the compounds, while ETU was below the
limit of determination in all cases.
A very limited amount of data on residues of dithiocarbamate
fungicides on crops grown in greenhouses was received from member
countries. It was, however, inadequate in extent and presentation
for any conclusions regarding estimated maximum residue levels to
be drawn. The need remains for a clear presentation of residue
data on glasshouse-grown crops, especially leafy vegetables and
fruiting vegetables.
REMOVAL OF RESIDUES
Marshall and Jarvis (1979) have described methods claimed to be
effective for the removal of residues of
ethylenebisdithiocarbamates (EBDC) and ETU from field-treated
tomatoes and hence in decreasing the residue levels in the derived
fruit juices. The preferred procedure involves a wash in dilute
sodium hypochlorite followed by a dip in dilute sodium sulphite,
the resulting residue levels then being below the detection limits.
Details of the mechanisms involved in the oxidative destruction of
ethylenethiourea by alkaline hypochlorite have been given by
Marshall and Singh (1977) and by Marshall (1978).
METHOD OF ANALYSIS
Lesage (1980) has drawn attention to the fact that copper can
interfere in the analysis of residues of
ethylenebisdithiocarbamates by carbon disulphide evolution methods,
low results being obtained.
Formulations
A comparison of gas and liquid chromatographic procedures for the
determination of ETU in maneb has shown a 5-fold bias in the gas
chromatographic values when methanol extracts of six formulations
were simultaneously analysed by both techniques (Fisher, 1977).
Hence, liquid chromatography is the preferred procedure. Use of
procedure on 35 samples of maneb produced at various locations in
USA showed the average ETU content to be 813 mg/kg, with a range of
249-1408 mg/kg (Keeler, 1977).
Residues
A procedure for the determination of those dithiocarbamates that
can be regarded as ETU-precursors has been suggested by Greve and
Hogendoorn (1978). Acid hydrolysis of ethylenebisdithiocarbamates
in the presence of tin (II) chloride yields ethylenediamine which
is then allowed to react with pentafluorobenzylchloride to form
1,2-bis(pentafluorobenzamide)ethane. The detection limit of the
method is about 0.1 mg/kg on endive and leek, recoveries of maneb
and zineb being in the range 75-95%.
The direct determination of alkylenebisdithiocarbamates by gel
permeation chromatography with UV absorption detection has been
reported by Pflugmacher and Ebing (1980). The method has been
tested for maneb, zineb, propineb, nabam and mancozeb added to
apples, lettuce, potatoes, carrots, tomatoes, beans and cucumbers.
Recoveries ranged from 78 to 105% at levels from 0.25 to 2 mg/kg.
The GLC method of Otto et al (1977) for ETU residues has been
subjected to a collaborative study (GIFAP, 1979). Amounts of ETU
in the range 0.02-0.1 mg/kg were added to apples, tomatoes and
grapes, recoveries averaging greater than 80%. The conversion of
maneb to ETU during the analysis was also studied; apart from one
experiment in which 2.7% conversion was found, all determinations
showed less than 1% conversion. Results from four to five
laboratories involved in the collaborative study (on samples of
maneb-treated tomatoes, tomato juice, beer, and celery) were in
good agreement at 0.01 to 0.1 mg/kg level.
A method for the gas-chromatographic determination of ETU in
apples, green beans, potatoes and tomatoes has been described by
King (1977). Following derivatisation with trifluoromethylbenzyl
chloride the determination is carried out by electron capture gas
chromatography.
A sensitive method for the analysis of ETU residues has been
described by Hirvi, Pyysalo, and Savolainen (1979) in which amounts
of more than 0.01 ng ETY can be detected quantitatively by gas
chromatography with glass capillaries and without derivatisation.
FFAP, Carbowax 20M, OV-17, and OV-101 were successful as liquid
phases.
According to Newsome and Panopio (1978), residues of 2-imidazoline,
a product of the degradation of ETU, can be determined by high
pressure liquid chromatography. At first the residues are absorbed
to a cation exchange resin and then they are treated with
p-nitrobenzoyl chloride and the derivative is detected by UV
absorption.
Singh et al (1979) developed a one-step extraction and
derivatisation method for ETU determination. ETU is derivatised
with dichloroacetic anhydride and partitioned into CH2Cl2 using
acetonitrile as a phase transfer agent. GLC was performed on
OV-330 or OV-17 columns with EC detection. The method was used as
a rapid screening procedure for the presence of ETU in water
samples at the 0.01-0.05 mg/kg level.
A collaborative study of a GLC headspace CS2 procedure for the
determination of residues of dithiocarbamate fungicides, organised
by the UK Committee for Analytical Methods for Residues has been
completed and a report on the work is in course of preparation for
publication (Abbott, 1980). As a result of the study a method of
analysis for lettuce was recommended which has been used, and can
be further adapted for other crops.
SPECIFICATIONS
A booklet of FAO specifications for maneb, nabam, ferbam, ziram,
thiram and metham-sodium is now available (FAO, 1979); a
'tentative' specification for mancozeb has been published (FAO,
1980).
EVALUATION
COMMENT AND APPRAISAL
The Joint Meeting in 1977 considered the dimethyldithiocarbamates
to be sufficiently similar in chemical structure, metabolism and
toxicology to be grouped together. Ferbam and ziram were allocated
an ADI of 0-0.02 mg/kg bw/day, while thiram was allocated a
temporary ADI of 0-0.005 mg/kg bw/day. As further information was
not received, no evaluation was made of the
dimethyldithiocarbamates and the previous evaluation was
reaffirmed.
The ethylenebisdithiocarbamates, maneb, zineb and mancozeb, were
also considered as a group by the 1977 Meeting. These compounds
are in part degraded or metabolised to ethylene thiourea(ETU). ETU
is a decomposition product, metabolite and contaminant of EBDC
fungicides. It has been shown to be considerably more toxic than
the parent molecule and has, in the past, given cause for concern
because of its thyroid toxicity and tumorigenicity. The 1977
Meeting was aware of further work in progress and the temporary ADI
of 0-0.005 mg/kg bw/day was reaffirmed for the
ethylenebisdithiocarbamates.
Propineb, a propylenebisdithiocarbamate, is degraded to
propylenethiourea and was considered separately from other groups
at the 1977 Meeting when a full review of available data was made.
A temporary ADI of 0.005 mg/kg/day was reaffirmed.
Newly-submitted results derived from studies on the short-term
toxicity and teratogenicity as well as the effect on reproduction
of EBDC pesticides, including some of their major metabolites and
breakdown products, have been evaluated. Three EBDC pesticides,
maneb, zineb and mancozeb, were maternally toxic at high doses in
the rat, less so in mice, hamsters and guinea pigs, but were not
teratogenic. ETU was maternally toxic in the rat at 80 mg/kg bw,
was teratogenic at doses higher than 10 mg/kg bw and also produced
a variety of postnatal effects. In short-term feeding studies (90
days) ETU showed adverse effects on thyroid function at doses
greater than 25 mg/kg (1.8 mg/kg bw). EBIS also induced thyroid
dysfunction in the rat. The no-effect level based on thyroid
function is 100 mg/kg (4.4 mg/kg bw). ETU was weakly mutagenic in
the Ames test (base-pair substitution) but with mammalian
mutagenicity systems it proved to be negative both in vitro and
in vivo.
The concern of the Meeting regarding the toxicity of ETU has been
alleviated by studies demonstrating no-effect levels for its
teratogenicity and thyroid toxicity in the rat; the oral no-effect
levels for ETU were established at 15 mg/kg bw and 1.8 mg/kg bw,
respectively.
Further residues results reported to this Meeting showed that there
was little likelihood of significant amounts of ETU being present
in wine or processed foods prepared from EBCD-treated produce.
Information is still lacking on use patterns of thiram, ferbam and
ziram and on their residue from supervised trials, as is adequate
data on residues of dithiocarbamates from crops grown under glass.
The new data available did not enable any amendments to be made to
the recommendations for limits made in 1977.
For sake of clarity, the limits for dithiocarbamates (and guideline
levels for ethylenethiourea (ETU) recorded in 1977 are repeated in
Annex 1. It was confirmed that the levels apply to residues,
determined and expressed as CS2, arising from the use of the
dimethyldithiocarbamates, the ethylenebisdithiocarbamates, and
propineb as separate groups. In those cases in which a member or
members of more than one group may be present in a commodity, the
MRLs should be regarded as additive.
However, some analytical methods are now available that enable the
EBDC compounds, which are ETU precursors, to be distinguished from
the other dithiocarbamate fungicides, including propineb. Further
work to validate these methods is required, together with use of
these procedures to obtain confirmatory residues data on some
crops. Comparison with results obtained by a validated head-space
GLC procedure for CS2 is also desirable.
No-effect levels and ADI
Dimethyldithiocarbamates
Ferbam
Level causing no toxicological effect
Rat: 250 mg/kg in the diet equivalent to 12.5 mg/kg bw/day
Dog: 5 mg/kg bw/day
Estimate of acceptable daily intake for man
0-0.02 mg/kg bw
This applies to ferbam and ziram individually or as the sum of the
two.
Ziram
Level causing no toxicological effect
Rat: 250 mg/kg in the diet equivalent to 12.5 mg/kg bw/day
Dog: 5 mg/kg bw/day
Estimate of acceptable daily intake for man
0-0.02 mg/kg bw
This applies to ferbam and ziram individually or as the sum of the
two.
Thiram
Level causing no toxicological effect
Rat: 48 mg/kg in the diet equivalent to 2.5 mg/kg bw/day
Dog: 5 mg/kg bw/day
Estimate of temporary acceptable daily intake for man
0-0.005 mg/kg bw.
Propylenebisdithiocarbamates
Propineb
This was considered separately from the EBDC fungicides and
although no further data was provided on this compound, or on its
breakdown product propylenethiourea the data provided on ETU were
taken into account considering its potential for thyrotoxicity and
tumorigenicity (see FAO/WHO 1978a, p. 30). The meeting decided to
postpone a further evaluation of propineb until further data are
available.
Level causing no toxicological effect
Rat: 10 mg/kg in the diet equivalent to 0.5 mg/kg bw/day
Dog: 3000 mg/kg in the diet equivalent to 75 mg/kg bw/day.
Estimate of temporary acceptable daily intake for man
0-0.005 mg/kg bw
Ethylenebisdithiocarbamates
Maneb
Level causing no toxicological effect
Rat: 250 mg/kg in the diet equivalent to 12.5 mg/kg bw/day
Estimate of acceptable daily intake for man
0-0.05 mg/kg bw, of which not more than 0.002 mg/kg bw may be
present as ETU.
This ADI applies to maneb, mancozeb, and zineb individually or the
sum of any combination of them.
Mancozeb
Level causing no toxicological effect
Rat: 100 mg/kg in the diet equivalent to 5 mg/kg bw/day.
Estimate of acceptable daily intake for man
0-0.05 mg/kg, of which not more than 0.002 mg/kg bw may be present
as ETU.
This ADI applies to maneb, mancozeb, and zineb individually or the
sum of any combination of them.
Zineb
Level causing no toxicological effect
Rat: not determined (but less than 500 mg/kg)
Dog: 2000 mg/kg in the diet equivalent to 50 mg/kg bw/day
Estimate of acceptable daily intake for man
0-0.05 mg/kg bw, of which not more than 0.002 mg/kg bw may be
present as ETU.
This ADI applies to maneb, mancozeb, and zinab individually or as
the sum of any combination of them.
FURTHER WORK OR INFORMATION
Required (by 1983)
1. Further development and validation of the methods of analysis
for the separate determination of ethylenebisdithiocarbamates which
are ETU precursors.
2. Residue data on a few crops obtained by use of a method specific
for the ETU precursors as compared with use of a validated
head-space CS2 GLC procedure.
3. Information regarding the current use pattern of thiram, ferbam,
ziram, and propineb together with residue data from supervised
trials.
4. Data on residues of dithiocarbamate fungicides from crops grown
under glass.
5. Studies to resolve the effects of thiram-inducing anaemia (as
reported to the 1977 Meeting in abstract form only).
6. Full evaluation of the teratogenic potential of thiram.
Desirable
1. Further studies to establish a no-effect level with zineb.
2. Further studies on other EBDC fungicides such as Polyram(R) and
metiram (and others that may give rise to ETU as a metabolite).
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