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. REFERENCES Ahmed, M. Biological and Chemical Research Institute, Rydalmere (1978) NSW Australia. Submitted for publication. Pesticide Science. Anonymous Determination of the acute oral toxicity of Panoctine (1973a) E-116335 in rats. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, CIVO-TNO 3x submitted by Keno Gard. Anonymous Feeding experiment with Panoctine-treated seed on pigeons (1973b) and pheasants with respect to the grade of repellency. Unpublished report from the Stensoffa Ecologiska-Station, Lund, submitted by Keno Gard. Anonymous Soil analysis. Royal Institute of Technology, Stockholm, (1978) Sweden. Bodin, S. Distribution of radioactivity in wheat plants treated with (1975) 14C and 3H labelled Panoctine. Keno Gard Report 5/9/75. Bodin, S. Residual analysis of radio-labelled guazatine acetate (1978) in oranges. Keno Gard report dated 78-08-01. Casco Gard Panoctine, new broad spectrum fungicide. Technical (1976) Bulletin. Casco Gard AB, Stockholm, Sweden. Davis, B., Boorman, G.C. and Mathew, R. Acute pharmacodynamic activity (1969) of EM 379 in cats. Unpublished report from Glaxo Research Ltd. No. 69 PG 1. De Groot, A.P. Determination of the intraperitoneal toxicity of (1976a) Panoctine R 42 in rats. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. CIV0-NTO 21-1-76, submitted by Keno Gard. De Groot, A.P. Determination of the acute oral toxicity of (1976b) Panoctine 42 in cats. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, submitted by Keno Gard. Feron, V.J. and Mullink, J.W.M.A. The effect of guazatine on blood (1977) pressure: a 13-week study in mice. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. R 5379, submitted by Keno Gard. Hartill, W.F.T., Canter-Visscher, T.W. and Sutton, P.G. An (1977) alternative fungicide to benomyl for the control of green mould in citrus. N.Z. J. Exp. Agric. 5: 291-292. Kemanord, A.B. Residue Analysis of Guazatine in Potatoes from (1976a) Sweden. 1976 Report of Kemanord AB - Analyscentrum. Kemanord, A.B. Research Laboratory - Residue Analysis of cane and (1976b) products from cane. Havali, 1976. KenoGard Basic data for determination of tolerance for Panoctine in (1978) citrus. Submission to FAO 78-08-01. Kivimäe, A. Feeding experiment with Panoctine-treated grain on (1973) egg-laying hens. Unpublished report of the Poultry Department, The Royal Agricultural College of Sweden, submitted by Keno Gard. Kruysse, A. and Immel, H.R. Acute inhalation toxicity study with (1976) Panoctine 42 in rats. Unpublished report from the Central Institute tor Nutrition and Food Research Zeist, No. R 4956, submitted by Keno Gard. Leegwater, D.C. Study on the fate of (14C, 3H) Guazatine (1975) preparation in the rat and in sandy loam, Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. 4823, submitted by Keno Gard. Lund Feeding experiment with Panoctine - treated seed on pigeons and (1973) pheasants with respect to the grade of repellency. Report of Stensoffa Ecologiska Station - Sweden. Mori, Y. A method for the determination of guazatine residues (1975) in mandarine orange. Report of Dainipon Ink and Chemicals Inc,, Japan. Petersen et al., Anal. Biochem., 31: 189 and 204. (1969) Reuzel, P.G.J., Til, H.P. and Köllen, C.H. Long term (2-year) (1976) toxicity study with Guazatine in beagle dogs. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. R 4983, submitted by Keno Gard. Reznich, H. and Greenberg, R. Panoctine residues in oranges. Report of (1977) Plant. Protection Institute, Israeli Ministry of Agriculture, July 1977. Sinkeldam, E.J. and van der Heijden, C.A. Sub-chronic (90-day) (1974) toxicity study with guazatine in albino rats (final report). Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. R 4354, submitted by Keno Gard. Snelson, J.T. Personal communication to FAO. (1978) Spanjer, M. Th. and Til, H.P. Determination of the acute toxicity (1974a) (LC50 -96 hours) of Guazatine in carps (Cyprinus carpio). Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. R 4523, submitted by Keno Gard. Spanjer, M.Th. and Til, H.P. Determination of the acute toxicity (1974b) (LC50 - 96 hours) of Guazatine in guppies. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. R 4399, submitted by Keno Gard. Svensson, S.A. Feeding experiment with fodder treated with (1975) Imazalil plus Panoctine to domestic hens. Report of Institute of Animal Husbandry of the Agricultural College, Uppsala, Sweden. Thornberg, O. Residue analysis of panoctine in pineapple from (1976) Hawaii 1976. Report of Kemanord Analyscentrum. Thornberg, O. Residue analysis of guazatine in cereals from (1977) Sweden 1974. Kemanord AB Report 1/2/77. Thornberg, O. Proposed method for determination of residues of (1978) panoctine in citrus fruit. KemaNord Analyscentrum method 780124. Til, H. P. and Feron V.J. Feeding study with guazatine in rats for 13 weeks. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. R 48709 submitted by Keno Gard. Til, H.P., Köeter, H.B.W.M., Immel, H.R. and van der Heijden, (1976a) C.A. Multigeneration study with guazatine in rats. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. R 5078, submitted by Keno Gard. Til, H.P. and Hendriksen, C.F.M. Toxicity study with panoctine (1976b) 42 (guazatine) in rats for 13 weeks. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. R 5062, submitted by Keno Gard. Til H.P., Hendriksen, C.F.M. and van der Heijden, C.A. (1976c) Combined chronic toxicity and carcinogenicity study with guazatine in rats (final report). Unpublished report from the central Institute for Nutrition and Food Research Zeist, No. R 4985, submitted by Keno Gard. Tugwell, B.L. A system of post-harvest mould control of citrus (1977) fruits in South Australia. Australian Citrus News, August 1977 P. 11. van Beek, R. Eye irritation test with panoctine 42 in albino (1974) rabbits. Unpublished report from the Central Institute for Nutrition and Food Research Zeist, No. 4363, submitted by Keno Gard. van Beek, R., van Oostrum, E.C.M. and Immel, H.R. Acute dermal (1976) toxicity study with panoctine 42 in albino rabbits. Unpublished report from the Central Insititute for Nutrition and Food Research Zeist, No. R 4921, submitted by Keno Card.
See Also: Toxicological Abbreviations Fenthion (ICSC) Fenthion (WHO Pesticide Residues Series 1) Fenthion (WHO Pesticide Residues Series 5) Fenthion (Pesticide residues in food: 1977 evaluations) Fenthion (Pesticide residues in food: 1979 evaluations) Fenthion (Pesticide residues in food: 1980 evaluations) Fenthion (Pesticide residues in food: 1983 evaluations) Fenthion (Pesticide residues in food: 1995 evaluations Part II Toxicological & Environmental) Fenthion (Pesticide residues in food: 1995 evaluations Part II Toxicological & Environmental) Fenthion (Pesticide residues in food: 1997 evaluations Part II Toxicological & Environmental)