GUAZATINE JMPR 1978
IDENTITY
Chemical Name
bis(8-guanidino-octyl)amine
Synonyms
bis(guanidino-8-octyl) amine guanidated
9-aza-1,17-diaminoheptadecane
1,1-[iminobis(octamethylene)]diguanidine
The following names refer to guazatine triacetate: EM
379; MC25; PN25; P3290; E116335.
Panoctine (R); Panolil(R), Pastulat(R).
Structural formula
NH NH
" "
H2N-C-NH-(CH2)8-NH(CH2)8-NH-C-NH2
C18H41N7
Other information on identity and properties
Guazatine is used as the triacetate. The commercial product,
which has been in production since about 1974, consists of a
standardised mixture of the acetates of guazatine and related
diguandines including guanidated 1,8-diamino-octane and higher
oligomers such an guanidated trioctylenetetramine. 82-84% of the
amino functions present are converted to guazatine groups. It is
not possible to isolate the oligomers or to produce the pure
guazatine by industrial processes but all oligomers have comparable
biological activit, and are considered together as active
ingredients (KenoGard 1978).
The following properties refer to the acetate(s).
Molecular Weight: 535 (range for components of technical material
260-760)
State: As water solution: milky yellowish brown,
somewhat viscous
Density: 1.060 g/cc
pH: as 2% solution in water 6 ± 1
Flash point: >100°C
Volatility: Not volatile
Melting point: No precise melting point owing to the presence of
various guanidines, but melts gradually
starting at approximately 95°C
Boiling point: Decomposes above 120°
Solubility (g/100g at 25°C):
water >300
dimethylformamide approx 50
dimethyl sulphoxide approx 100
ethanol approx 200
methanol >300
xylene very low
hydrocarbons very low
storage stability: formulations stable at normal temperature for more
than 2 years
Impurities in the technical material
Information was provided on the quality and composition of the
technical material which has been standardised by the only
manufacturer. The standardised technical material was used in all
toxicological metabolism and residue studies (KenoGard 1978).
Formulations
(1) Seed dressings containing 20-35% guazatine alone and in
combination with other seed treatment fungicides.
(2) Sugar cane dipping preparation containing 60% guazatine.
(3) Seed potatoes spray containing 60% guazatine.
(4) Citrus spray/dip containing 60% guazatine.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOCHEMICAL ASPECTS
Absorption, distribution and excretion
A male rat was orally treated with guazatine, labelled with
both 3H in the octyl moiety and 14C in the guanidino moiety.
Within 72 hours about 15% of the administered 14C radioactivity
were excreted in the urine and about 65% in the faeces, whereas
approximately 42% and 39% of the 3H activity were eliminated in
the urine and faeces respectively. After 3 days the body still
contained about 4% of the 14C-dose and 12.5% of the 3H-dose. The
14C/3H ratios in urine and faeces indicate that the compound
undergoes biotransformation. The TLC analysis resulted in one major
and a few minor components in urine and two major components in
faeces. None of the metabolites were identified (Leegwater, 1975).
Effects on other biochemical aspects
The acute pharmacodynamic effects on the vascular system were
studied in cats treated with single or repeated doses of EM 379
(composition of the formulation unknown) by the intravenous and
intraduodenal route. The application produced marked dose-related
depression of blood pressure when applied in i.v. doses of 0.5-8
mg/kg. The fall in blood pressure was accompanied by an increase in
heart and respiratory rate and doses of 2 mg/kg and above caused
additionally pupillary dilation, urination and hind limb movement.
Cumulative i.v. doses of 8-16 mg/kg led to death of the
experimental animal. Similar effects were obtained after
intraduodenal application of the test compound (Davis et al.,
1969).
TOXICOLOGICAL STUDIES
Special studies on reproduction and teratogenicity
Rat
Guazatine techn. grade; (54.8% w/w ai.i. in water) was fed to
groups of 10 male and 20 female rats at dietary levels of 0, 60 and
200 ppm for three successive generations (2 litters per
generation).
A teratogenicity study was carried out at the same time with
F3C and F3b-litters. Additionally a 4-week feeding study was
conducted using F3b-litters. The reproduction study revealed no
abnormal findings with respect to general condition, behaviour,
body weights and fertility of the dame or birth weight and survival
rate of the pups. No malformations were observed in any of the
groups.
The results of the teratogenicity study showed no
abnormalities as regards the dams. Implantation losses and
resorptions did not significantly differ in the various groups. At
200 ppm foetal weight was slightly reduced in the F4a-litters. No
visceral or skeletal anomalies were found, but in the F3c
generation a tendency to retardation in bone development was
observed.
The 4-week feeding study revealed no treatment-related
abnormal findings as regards the general health, growth and food
consumption. A dose-related increase of the kidney weights was
found in both sexes. The relative thymus weights were also
increased in all treated animals, dose-related in females, without
dose-relationship in males. These changes in organ weight were not
accompanied by gross or microscopic morphological alterations (Til
et al., 1976a).
Special study on carcinogenicity
Rat
See under long term studies.
Acute toxicity
Species Sex Route LD50 References
Rat M F oral 227 Anonymous, 1973a
Rat M F i.p. 53 De Groot, 1976a
Rat inhalation 11 mg/m3 Kruysse and Immel, 1976
(LC504h)
Cat M F oral 382 De Groot, 1976b
Rabbit M F dermal (24h) 2800 van Beek et al., 1976
Fish (carp) 2.9 ul/l Spanjers and Til, 1974a
(LC50 96h)
(guppy) 0.54 ul/l Spanjers and Til, 1974b
(LC50 96h)
Short term studies
Rat
Groups of 10 male and 10 female rats were maintained on a diet
containing 0, 60 and 200 ppm guazatine (techn. grade; 54.8% w/w
a.i. in water) for a period of 90 days. The treatment did not
affect behaviour, mortality, body weight gain and food consumption.
The values of haematology, clinical chemistry and urine analysis
were within normal limits. No distinct differences in the relative
organ weights existed between the various groups. The gross and
microscopic examination revealed no pathological findings which
could be attributed to the ingestion of the test compound
(Sinkeldam and van der Heiyden, 1974).
Groups of 10 male and 10 female rats were fed with guazatine
(techn. grade; 54.8% w/w a.i. in water) at dietary levels of 0 and
800 ppm. After 6 weeks of feeding the guazatine level was increased
to 1200 ppm for the last 7 weeks of the study. The treatment had no
effect on general condition, behaviour and survival rate. The
haematological and biochemical examinations revealed no abnormal
values. Slight reduced body weights were observed in both sexes
accompanied by slightly diminished food consumption in the treated
male rats. Besides a lower specific gravity of the urine in the
treated females no abnormal findings were observed. The relative
organ weights of the testicles and adrenals were increased in males
compared to the control animals. No abnormal pathological gross
autopsy or microscopic findings were observed, except for one type
of thyroid lesion found in two females (out of total 10) in the
test group, the abnormal thyroids were increased in weight and
contained small follicles (Til and Feron, 1975).
A 13-week feeding study was performed with groups of 10 male
and 10 female rats maintained on a diet supplemented with 0 and
1500 ppm guazatine (techn. grade; 40% w/w a.i. in water) for the
first four weeks of the study and 2000 ppm for the last nine weeks
of the experiment. General health, behaviour and mortality was not
affected by the treatment, whereas the treated animals showed
reduction in body weight and food consumption. Results of
haematological clinical biochemistry and urine examinations were
within normal limits. The kidney function test revealed a slight
decrease in specific gravity of urine in test group. At 1500/2000
ppm increased relative organ weights of the kidney, liver and heart
(females only) were found, whereas; the mean relative thyroid
weights were reduced in both sexes. Gross autopsy examination did
not reveal pathological changes, the histopathological examination
however showed hyperplastic, epithelium of the excretory ducts of
the parotid salivary glands, often accompanied by infiltrates of
inflammatory cells. 6 out of 10 male rats and 6 out of 10 female
rats showed these changes compared to none in the control group
(Til and Hendriksen, 1976b).
Mouse
A 13-week comparative study with guazatine (techn. grade., 40%
w/w a.i. in water) and the structurally related compound
guanethidine, a pharmaceutical against hypertension was carried out
with groups of 30 male and 30 female mice. The diet was
supplemented with either 0 and 50 ppm guanethidine or 0 and 500 ppm
guazatine. The feeding did not affect the health condition,
behaviour and the weight gain of the test animals. A significant
decrease in blood pressure was produced in the guanethidine fed
males after 4 weeks of experiment, whereas no marked changes in
blood pressure could be observed after guazatine treatment of mice
(Feron and Mullink, 1977).
Hen
In a 18-day feeding experiment with egg-laying hens the
animals were fed ad libitum with seed that had been treated with
2 ml 40% panoctine/kg seed (normal use level) (40% w/v a.i. in
water) and 4 ml 40% panoctine/kg seed. The test compound showed a
marked repellent effect on poultry causing weight loss and
reduction of egg-production (Kivimae 1973). Similar results were
obtained in a feeding study with pigeons and pheasants (Anonymous,
1973b).
Dog
Groups of 4 male and 4 female dogs were fed with a diet
supplemented with 0, 60, 200 and 300 ppm guazatine (techn. grade,
54.8% w/w a.i. in water) for two years. 26 weeks after the
beginning of the study the high dietary level was increased to 600
ppm until the end of the experimental period. The feeding did not
influence the general condition, behaviour, growth rate and food
consumption. The results of the haematologic and clinical chemistry
determinations fall within normal limits, except for a slight
decrease in the white blood cell counts at most stages of the
experiment in the 300/600 ppm group compared to the control. The
urinary findings did not suggest any changes due to treatment. In
the kidney function and liver function tests normal values were
obtained. The only alteration in relative organ weight was found in
the 300/600 ppm group where female animals showed increased
relative ovary weights. No gross pathological or histopathological
findings were discovered (Reuzel et al., 1976).
Long term studies
Rat
Groups of 60 male and 60 female rats were maintained on a diet
containing 0, 69 20, 60 and 200 ppm guazatine (techn. grade; 54.8%
w/w a.i. in water) for two years. After a few months of feeding the
6 ppm group was discarded. The treatment did not adversely affect
the general condition survival rate and food intake of the test
animals. In females treated with dietary levels of 20-200 ppm a
reduced body weight of 10-20% was noted during month 22 and 24.
Since this effect showed no dose dependency and was noted only in
females at the end of the experiment it was not considered to be
related to the treatment. No dose-related alterations in the
haematological and biochemical blood parameters were found, nor did
the urine analysis and kidney nor function test show abnormal
results. Some differences in the relative organ weights compared to
control were observed in the treated animals but no
dose-relationship was evident. The macroscopic and microscopic
examination of the control and the 200 ppm-group did not reveal
organ lesions that could be attributed to treatment in
histopathological alterations of the parotidasalivary lands were
found. The tumour incidence was similar in the control and treated
groups (Til et al., 1976).
COMMENTS
Radioactive labelled guazatine, when applied as a single oral
dose to rats is excreted at about 80% within 72 hours after
application. There is no indication of tissue accumulation.
Although no experimental studies were available to determine the
exact metabolic pathway it can be assumed that the absorbed
components of low molecular weight may be degraded further to
normal body constituents.
Results of short-term and 2-year toxicity studies were
available. These studies showed a no effect level of 200 ppm in
rats and dogs. In a three generation study no effects on
reproduction and on teratogenicity were observed. There were no
differences between control and treated groups with respect to
tumour incidence.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effect
Rat: 200 ppm, (54.8% active ingredient as acetate in water)
equivalent to 5 mg/kg body weight of the active
ingredient
Dog: 200 ppm (54.8% active ingredient as acetate in water) in
the diet equivalent to 3 mg/kg body weight of the active
ingredient
Estimate of acceptable daily intake for man
0 - 0.03 mg active ingredient as acetate/kg body weight
RESIDUES IN FOOD AND THEIR EVALUATION
USE PATTERN
Guazatine is a fungicide developed in about 1968 for the
control of most cereal seed diseases, including those in rice, rice
blast, pineapple disease of sugar cane and a number of disease in
seed potatoes.
In recent years it has been shown to be particularly effective
against fungal diseases of citrus especially Penicillium, Diploida,
Alternaria and Geotrichium which cause serious wastage of citrus
fruit during transport, storage and marketing (Tugwell, 1977,
CascoGard 1976, Hartill et al., 1977).
Reports have confirmed the efficacy of guazatine against a
number of fungal diseases of bananas, coffee, soybeans and flower
bulbs. A list of uses is given in Table 1. The application rates
are those currently recommended or under investigation (CascoGard
1976).
RESIDUES RESULTING FROM SUPERVISED TRIALS
A limited amount of information was available from trials
designed to determine the level and fate of guazatine residues in
raw agricultural commodities following treatment.
Thornberg (1977) reported the results of analysis of 39
samples of raw cereals including barley (13), oats (5), rye (9) and
wheat (12) from field trials in Sweden, Germany and South Africa.
These represented grain collected from crops grown from seed
treated with guazatine acetate at the rate of 60-120g/100 kg of
seed. The grain was collected 4-10 months after the seed was sown.
The analytical method used had a limit of determination of 0.1 mg/kg.
No guazatine was found in any of the samples, nor was any residue
detected in 20 samples of straw from the same trials.
Analysis of potato tubers from crops grown from seed potatoes
treated with guazatine 5 months previously revealed no guazatine
(< 0.05 mg/kg).(Kemanord AB, 1976).
Pineapple fruit, shells and leaves from plants grown from
seedpieces that had been dipped in guazatine solution 21 months
previously were found to contain no detectable residues (< 0.1
mg/kg) (Thornberg 1976). Sugar cane, bagasse, molasses and raw
sugar from a crop or sugar cane grown from setts (planting
cuttings) treated with guazatine at a concentration of 100 and 250
mg/l contained no detectable residues (Kemanobel Research
Laboratory 1976).
Citrus
The Israeli Ministry of Agriculture (Resnick and Greenberg,
1977) reports the results of a trial in which "Shamouti" oranges
were dipped experimentally in aqueous solutions and in wax
emulsions containing guazatine at concentrations ranging from 5 to
40 g/l. This is up to 10 times higher than is recommended for
post-harvest dips. It is recognized that fungicides applied in wax
are much less effective than when applied from a water-dip prior to
waxing.
The residues were determined by the method of Mori (1975). The
results are indicated in Table 2.
Fruit from two of the treatments (the lowest and highest
concentrations) were examined 1, 10 and 20 days after treatment to
determine the degree of chemical breakdown. As indicated in Table
3 there was no loss during 20 days (the temperature of storage was
not given).
In two trials carried out in Japan (More, 1975) mandarin
oranges ("Satsuma" variety) were sprayed on the trees with
guazatine solution (500 mg/l) at the rate of 1000 l/ha yielding the
residue data reported in Table 4. The concentration in the flesh or
juice was less than 1/10 of that in the peel.
In a trial in which radio-labelled guazatine was applied to
Jaffa oranges from Israel, Bodin (1978) studied the uptake,
penetration and persistence of guazatine residues on oranges stored
up to 50 days at 4°C. Separate lots of oranges were drenched with
aqueous solutions of guazatine acetate labelled with both 14C and
3H at concentrations of 0.1% and 0.2% at 12-18°C for 30-60
seconds. The dipped fruit was dried and some was subjected to
degreening with ethylene gas. All fruit was then waxed with a
commercial citrus wax containing thiabendazole before being dried
and stored at 4°C. Duplicate samples were analysed
TABLE 1. Use pattern of guazatine.
Crop Pest Concentration Method of application
1. Wheat, oat, Fusarium spp. 60-80 g/100 kg Seed dressing machines
barley, rice, Tilletia spp. of seed Seed dusters
rye Septoria nodorum
2. Sugar cane Pineapple disease 100 mg/l (hot) Dipping tank
(Ceratocystis 250 mg/l (cold)
paradoxa)
3. Potatoes Rhizoctonia solani 15-30 g/tonne Spray at time of
Phoma spp. sorting
4. Citrus Penicillium spp. 100-500 mg/l Dipping tank
Diploida
Alternaria spp. 1-2 g/1 Wax-treatment spray
Geotrichium
5. Rice Foliar diseases ? Broadcast spray
by liquid scintillation measurement after 0, 1, 8, 24 and 50 days
storage. Before analysis each orange was separated into yellow peel,
white and pulp. The average weight of each fruit was 220 g and the
proportion of peel, white and pulp was 25%, 14% and 61% respectively.
The guazatine content of the separate tractions, calculated from the
scintillation counts, is shown in Tables 5 and 6, which show the range
and mean residues in the three fractions following treatment at the
two concentrations. The mean was derived from separate values
determined at 0, 1, 8, 24 or 50 days after treatment.
TABLE 2. Guazatine residues in shamouti oranges dipped post-harvest.
a.i. in water or wax, Date of Residue, mg/kg
mg/kg treatment
20,000 (wax) 11.1.77 11.0 9.0
40,000 (wax) 11.1.77 13.0 12.0
5,000 (water) 11.2.77 1.5
5,000 (wax) 11.2.77 1.5
10,000 (wax) 11.2.77 7.0
20,000 (wax) 11.2.77 8.0
2,500 (water) 24.2.77
5,000 (wax) 24.2.77 1.5
5,000 (wax) 24.2.77 3.0
10,000 (wax) 24.2.77 6.0
20,000 (wax) 24.2.77 9.0
It will be apparent that closely comparable results were obtained
from the measurement of the 14C and 3H labels, indicating that
minimal decomposition or metabolism has occurred. From the 250
separate analyses in the original data there is great variation
between fruits. The variation could well be greater in fruit treated
in commercial packing houses and therefore further studies under
packing house conditions are required. The degreening treatment had no
significant influence on the residue content. There was a tendency for
a slight decrease in the residue concentration in the peel during 50
days at 40°C, indicating some diffusion into the pulp.
Preliminary results were available from trials now in progress in
Australia. When Valencia oranges were dipped under laboratory
conditions in guazatine solutions ranging from 140 mg/l to 500 mg/l
the residue in the whole fruit was as follows (Ahmed, 1978):
Dip Residue
Concentration (mg/kg)
(mg/l)
140 0.22
260 0.45
290 0.5
500 0.9
TABLE 3. Stability of guazatine residues in stored shamouti oranges.
a.i., mg/kg 1 day after 10 days after 20 days after
treatment treatment treatment
5,000 1,2; 1,2; 1,5 - -
20,000 11; 9.0; 10.0 12.0; 9.0; 10.0 11.0; 8.0; 10.0
Fruit from commercial trials where guazatine had been applied by
(a) dipping and (b) flooding were found to contain guazatine residues
as shown in Table 7.
FATE OF RESIDUES
In animals
The fate of guazatine in rats has been described under the
Section "Biochemical Aspects" (Leegwater 1975). No other information
was available to demonstrate the level and fate of residues in
livestock or foods of animal origin following the feeding of
guazatine-treated seed or dried pulp from guazatine-treated citrus.
Kivimäe (1973) reported an extensive and complex trial carried
out at the Royal Agricultural College of Sweden in which hens and
cocks were fed a mixture of wheat and barley that had been treated
with guazatine seed dressing at normal and double dosage. The trial
was designed to determine feed consumption and effect on weight,
egg-laying, fertilization and hatchability of the eggs. Many of the
chickens starved to death rather than eat the treated seed. After a
period of 18 days on the treated feed the chickens were given a choice
of treated or untreated grain and they avoided the treated seed
completely. The effect on weight and other parameters was directly
related to the starvation. No attempt was made to determine whether
the chickens accumulated any guazatine from the small amount of seed
consumed but post-mortem examinations were carried out to determine
the cause of death of those that did not survive the experiment.
TABLE 4. Residues of guazatine in satsunya mandarin fruit (Japan).
LOCATION Concn Rate No. of Pre-harvest Residues (mg/kg)
mg/l l/ha sprays intervals flesh peel juice
days
0 0 0 - < 0.05 < 0.05 < 0.05
AICHI 500 500 1 3 0.18 2.7 -
PREFECTURE 500 500 1 8 0.11 1.1 0.06
500 500 1 13 0.07 1.1 -
500 500 1 18 < 0.05 0.5 -
0 0 0 - < 0.05 < 0.05 < 0.05
SHIZUCKA 500 500 1 3 0.06 1.5 -
PREFECTURE 500 500 1 8 0.05 2.1 0.05
1 13 0.05 1.5 -
1 18 < 0.05 1.1 -
TABLE 5. Guazatine residues in stored oranges (normal moisture content) not degreened.
Guazatine acetate, mg/kg
Drench PEEL WHITE PULP
Concentration
14C Range 3.00 - 9.35 0.19 - 0.70 0.0019 - 0.00739
0.1% Mean = 6.35 = 0.38 = 0.0336
14C Range 12.0 -21.5 0.25 - 3.54 0.0307 - 0.3398
0.2% Mean = 15.9 = 1.21 = 0.1306
TABLE 5. (Cont'd)
Guazatine acetate, mg/kg
Drench PEEL WHITE PULP
Concentration
3H, Range 3.68 - 8.99 0.25 - 0.89 0.0106 - 0.0902
0.1% Mean = 6.15 = 0.49 = 0.0346
3H, Range 11.48-21.74 0.59 - 4.61 0.0259 - 0.4272
0.2% Mean = 17.29 = 1.48 = 0.1315
Untreated Range 0.0068 - 0.0068 0.0061 - 0.0081 0.0038 - 0.0038
Mean = 0.0068 = 0.0071 = 0.0038
TABLE 6. Guazatine residues in stored oranges (normal moisture content) degreened.
Guazatine acetate, mg/kg
Drench PEEL WHITE Pulp
Concentration
14C Range 4.27 - 7.05 0.08 - 0.24 0.0202 - 0.0643
0.1% Mean = 5.18 = 0.15 = 0.0422
14C Range 12.15 - 18.80 0.27 - 0.98 0.0528 - 0.4118
0.2% Mean = 14.44 = 0.59 = 0.1411
3H Range 3.75 - 6.17 0.09 - 0.18 0.0067 - 0.0460
0.1% Mean = 4.47 = 0.14 = 0.0240
3H Range 12.34 - 22.55 0.35 - 1.99 0.0374 - 0.5242
0.2% Mean = 14.92 = 0.89 = 0.1430
TABLE 7. Guazatine residues in dipped or flooded citrus fruit
Fruit Concentration Method of Guazatine residues
of bath, Application (mg/kg)
mg/l Peel Whole fruit
Valencia orange 250 dip - 0.42
Lemon 250 dip - 0.48
Tangerine 250 dip - 0.48
Valencia orange 500 dip 3.6 0.51
Lemon 500 dip 3.3 0.90
Tangerine 500 dip 5.5 0.83
Valencia orange 500 flood - 0.671/
Lemon 500 flood - 0.761/
Tangerine 500 flood - 0.931/
1/ Held 7 days at 7.5°C before analysis.
Svensson (1975) carried out trials to determine the effect on
domestic poultry of seed dressings containing imazalil alone and in
combination with guazatine. The fungicides were incorporated into a
balanced layer-ration apparently at a level corresponding to the
incorporation of treated seed as one of the components. The
concentration was 600 mg of guazatine and 40 mg of imazalil per kg of
feed. Imazalil had no effect on feed consumption, weight gain, egg
production or hatchability. The group which received the ration
containing guazatine refused to eat, lost weight heavily and within
two weeks stopped laying. When they were returned to the base ration
without guazatine they recovered weight and egg production. Lund
(1973) carried out an experiment to investigate the degree of
repellency of guazatine-treated seed to seed-eating birds. The
experiment showed that guazatine had a strong repellent effect on
pigeons and pheasants but no attempt was made to determine i,tether
any residues resulted from the consumption of the seed which was
eaten.
In plants
Bodin (1975) studied the distribution of radioactivity in wheat
plants grown from seed that had been treated with radio-labelled
guazatine. The plants were grown in pots in the glasshouse. At the end
of 8.5 months the whole plants were harvested and divided into root,
leaf, ear and stem. The radioactivity was measured by the method
described by Peterson et al., (1969) in which the 14C and 3H
activity can be counted separately.
A total of less than 1% of the 14C radioactivity applied to the
seed was found in the whole wheat plant. About 8% of the 3H activity
was found in the plant but virtually all in the roots. The absence of
14C-activity in the ears at a detection limit of 0.01 mg/kg means
that neither guazatine nor its metabolites is transferred to grain.
There was clear evidence of at least partial metabolism of guazatine
prior to incorporation into the plant tissue. Hydrolysis can give rise
to urea or carbon dioxide. However, one study with radio-labelled
material applied to oranges (described earlier, Bodin 1978) indicates
that deposits on oranges remain virtually unchanged for 50 days at
4°C.
In soil
Leegwater (1975) reported a study in which guazatine labelled
with 3H in the octyl moiety and 14C in the guanidino moiety was used
to observe the fate in a sample of sandy loam. An aliquot of
radio-labelled guazatine was mixed with loam containing 20% water; the
pH of an aqueous extract was 5.4 and the organic matter content was 7
of the dry weight. The preparation was kept at room temperature (max
28°C) for 7 days. At the end of this period about 83% of the 14C and
about 50% of the 3H appeared to be tightly bound to the soil,
particularly to the humus. About 8% of the 14C was extractable with
water and about 2% was recovered as carbon dioxide. About 30% of the
3H was extractable with water and about 13% evaporated from the
sample and could be trapped in water. It was presumed to be water
formed by degradation.
The Royal Institute of Technology, Stockholm (Anon. 1978) reports
a study carried out in accordance with the German Biologische
Bundesanstalt "Merkblatt 36" in 1978 in which the fate of guazatine in
standard soil was measured by means of radiometry. This showed that
the guanidino carbon is converted to and released as carbon dioxide.
No intermediate could be detected. This suggests a cleavage of the
guanidino group producing amine and urea, followed by a further
decomposition of the urea by urease. Deguanidation seems to be the
rate-determining step. The study indicated that when guazatine was
incorporated in soil at a concentration of 500 mg/kg, 50%
decomposition occurred in 4 weeks at 20°C. it was concluded that
guazatine was degraded extensively in soil.
In processing and cooking
The work of Bodin (1978) who treated oranges with radio-labelled
guazatine indicated that the bulk of the deposit remains as a residue
in the yellow peel and white pith of the orange. Only 2% of the
deposit on the whole fruit is to be found in the pulp. It therefore
follows that in the preparation of orange juice the bulk of the
residue will remain in the skin and be discarded.
No other information was available on the effect of processing or
cooking.
METHODS OF RESIDUE ANALYSIS
Mori (1975) developed a method of determining guazatine residues
in mandarin oranges. It is based on extraction with butanol of the
substrate which has been treated with picric acid to form guazatine
picrate. The extract is hydrolysed with alkali at 160°C to form the
corresponding triamine (di(8-amino-octyl)amine). The triamine is
measured by gas chromatography using a nitrogen-thermionic detector.
The limit of determination is said to be 0.05 mg/kg. The recovery of
guazatine from oranges fortified at the 2 mg/kg level ranged from 87
to 97%.
Resnick and Greenberg (1977) used the same method to measure
guazatine residues in organes treated with a wax emulsion containing
guazatine. They report the recovery to be only 50% and the limit of
determination 1-2 mg/kg. Two Australian laboratories attempted to use
this method without success (Snelson, 1978). The method has been
modified by Thornberg (1978). The main modification appears to be that
the triamine formed by alkaline hydrolysis at 160°C (autoclave) is
extracted into benzene and converted to trifluoroacetamide by reaction
with trifluoroacetic anhydride for determination by GC-MS using
multiple-ion detection.
Guazatine residues are usually firmly adsorbed to the substrate
and care is needed to ensure complete extraction. The temperature of
hydrolysis (160°C) is critical, as is the time of heating. The
standard solution, which consists of a solution of the standardised
technical material, has recently been shown to be unstable, the
concentration decreasing when dilute aqueous solutions are stored in
glass. Fresh solutions and silanised glassware should be used. After
trifluoro-acetylation, the derivative is stable and readily handled or
stored. The original author reports trouble with the
gas-chromatographic column owing to excessive bleeding and difficulty
in obtaining good quality glass capillary columns. The amide is
distinctly polar and tailing occurs during the chromatographic step.
Work in Australia (Snelson, 1978) has led to the development of a
method which depends on the reaction of the amino group with Coomassie
Brilliant Blue G, a dyestuff normally used for dyeing wool and
polyamide fibres. This forms a stable blue complex which may be
measured in a spectrophotometer at 595 nm. The procedure involves
extraction with butanol in the presence of excess alkali using a
blender. The first extraction gives about 60% recovery and two
extractions greater than 90%. The butanol extract is recovered by
centrifuging and after dilution with hexane is washed with sodium
hydroxide before being partitioned into sulphuric acid. The sulphuric
acid solution is neutralized and re-extracted with butanol. The
extract is diluted with hexane and then partitioned into phosphoric
acid. The phosphoric acid solution is reacted with Coomassie Brilliant
Blue G and the coloured product determined by measurement at 595 nm.
Calibration is with a solution of the reaction product of the standard
and the dyestuff. Reproductable results have been obtained on three
classed of citrus (oranges, lemons and tangerines) and on cantaloups.
The outcome of this ongoing study is to be published.
NATIONAL MRLs REPORTED TO THE MEETING
The following MRLs were reported:
The Netherlands
Raw cereals ................... 0.1 mg/kg (at or about the limit
of determination)
Sweden
Citrus ........................ 6 mg/kg
APPRAISAL
Guazatine is a fungicide developed for the control of cereal seed
diseases and leaf diseases of cereals including rice blast. It is also
used against diseases of pineapple, sugar cane and of seed potatoes,
and for preventing diseases that cause severe post-harvest wastage of
citrus.
Technical material, which has been in production since about
1974, consists of a standardised mixture of the acetates of guazatine
and related diguanidines, including guanidated 1,8-diaminooctane and
higher oligomers such as guanidated trioctylenetetramine. 82-84% of
the amino groups present are converted to guanidine groups. It is not
possible to isolate the oligomers or to produce the pure guazatine by
industrial processes but all oligomers have comparable biological
activity and are considered together as active ingredients.
Only four residue trials on citrus have been reported, three of
which were carried out under atypical conditions so that the results
are of limited value in estimating the level and fate of residues on
various citrus varieties treated under a range of commercial
conditions. The residue remains in the peel and only negligible
quantities are transferred to pulp or juice.
Analysis of barley, oat, rye and wheat grain from crops grown
from guazatine-treated seed failed to reveal any guazatine residues
when analysed by a method with a limit of determination of 0.1 mg/kg.
Potatoes harvested from plants grown from guazatine-treated seed also
contained no detectable residues ( 0.05 mg/kg).
Pineapples, including pulp, skin and leaf, grown from
guazatine-treated seed pieces and sugar cane, bagasse, molasses and
(raw sugar from crops grown from guazatine-treated "setts" showed no
guazatine residues (0.1 mg/kg).
Radio-tracer studies on the fate of guazatine following its use
as a seed dressing indicates that none is transferred to the grain of
the resulting crop. Such amounts as find their way into the growing
plant appear to be incorporated into plant tissue after metabolism.
The product was strongly repellent to chickens, pheasants and
pigeons but there is no information on its fate in poultry or other
livestock. Some information on the fate in plants indicates extensive
metabolism. Following application to soil there is rapid degradation
with the residues being firmly bound to soil organic matter.
The only residue analytical method so far developed is apparently
not easy to adapt to local laboratory conditions, National tolerances
have been established in the Netherlands for raw cereals and in Sweden
for citrus.
RECOMMENDATIONS
The following maximum residue limits are recommended.
Commodity Limit, mg/kg
Cereals (raw) 0.1*
Pineapples 0.1*
Potatoes 0.1*
Sugar cane 0.1*
* at or about the limit of determination.
The following temporary maximum residue limit is recommended.
Citrus fruits 5
FURTHER WORK OR INFORMATION
Required (by 1980 and before the MRL for citrus can be confirmed)
1. Information on the level, distribution and fate of guazatine
residues when the material is applied to major citrus fruit varieties
in commercial practice.
2. Information on the level and fate of guazatine residues in meat
and milk following the feeding of citrus pulp containing residues of
guazatine to cattle.
3. An analytical method suitable for regulatory purposes.
Desirable
1. A teratogenicity study with higher dose levels.
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