J. Agric. Food Chem., Vol. 26,No. 1, 1978
Fate of Diflubenzuron
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Fate of Diflubenzuron in Cattle and Sheep G. Wayne Ivie The fate of a radiolabeled preparation of the insect growth regulator diflubenzuron (Dimilin, TH-6040,
N-[ [ (4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide) was determined after oral administration to a lactating cow and castrate male sheep, in vitro incubation with ovine and bovine digestive tract fluids, and dermal application to cattle. Orally administered diflubenzuron was absorbed, extensively metabolized, and almost totally excreted by cattle and sheep. Only very small amounts of carbon-14 were secreted into the milk of a cow fed [14C]diflubenzuron. Studies with bile-duct cannulated sheep showed that bile is of greater importance than urine in the elimination of diflubenzuron metabolites from the body of ruminants. Major metabolites of diflubenzuron excreted by the cow and sheep resulted from hydroxylation on the difluorobenzoyl and chlorophenyl rings and by cleavage between the carbonyl and amide groups to give metabolites that were excreted either free or as conjugates. Diflubenzuron was not metabolized when incubated with digestive tract fluids from these ruminants. When applied dermally to cattle held outdoors in an unprotected pasture, diflubenzuron residues disappeared rapidly, but the compound was not chemically degraded or absorbed through the skin to any detectable degree. The major hydroxylated diflubenzuron metabolite in cow milk when fed to white rats was rapidly and quantitatively excreted with little further biotransformation. residual behavior. In a companion report (hie and Wright, The insect growth regulator diflubenzuron (Dimilin, TH-6040, N-[ [ (4-chlorophenyl)amino]carbonyl]-2,6-di- 1978), the fate of [14C]diflubenzuron in the stablefly (Stomoxys calcitrans) and housefly (Musca domestica) is fluorobenzamide) is very toxic to the larval stages of a considered. Certain aspects of both of these studies have previously been reported in preliminary form (Ivie, 1977).
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number of insect species and is highly selective due to its unique mode of action. Diflubenzuron reduces chitin deposition into insect cuticle, which disrupts normal moulting and development processes (Ishaaya and Casida, 1974; Mulder and Gijswijt, 1973; Post et al., 1974). The compound has shown excellent potential for controlling the larval stages of mosquitoes (Mulla et al., 1974; Schaeffer et al., 1975) and other Diptera (Miller, 1974; Miller et al., 1975; Wright, 1974; Wright and Harris, 1976; Wright and Spates, 1976), certain Lepidoptera (Granett and Dunbar, 1975; Tamaki and Turner, 1974), and Coleoptera (Moore and Taft, 1975; Neal, 1974). The high degree of toxicity exhibited by diflubenzuron toward many destructive insects and its extremely low mammalian toxicity (Ferrell, 1977) indicate that the compound may be extensively used for insect control. A thorough evaluation of the environmental fate of diflubenzuron is therefore needed. Studies by Metcalf et al. (1975) showed that diflubenzuron was moderately stable in a model ecosystem, but the compound was not highly concentrated through food chains or by absorption from water. Diflubenzuron was not metabolized by the salt marsh caterpillar Estigmene acrea (Metcalf et al., 1975), the boll weevil Anthonomous grandis (Still and Leopold, 1975), or Pieris brassicae larvae (Verloop and Ferrell, 1977). Diflubenzuron was degraded only to a very limited extent by sheep liver microsomes (Metcalf et al., 1975),but was extensively metabolized after oral administration to laboratory rats (Verloop and Ferrell, 1977). The investigations reported here were initiated in an effort to provide additional information on the fate of diflubenzuron in nontarget species. Radiolabeled diflubenzuron was orally or dermally administered to cattle and sheep for an evaluation of the compounds metabolic and Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas 77840.
MATERIALS AND METHODS
Chemicals. ['4C]Diflubenzuron was supplied by Thompson-Hayward Chemical Co., Kansas City, Kans. The radiochemical was either technical crystalline material or was formulated as a 25% wettable powder (WP, 2-5 pm particle size). Both samples were uniformly and essentially equally labeled in the two rings. The difluorobenzoyl ring contained 51.1% of the total radioactivity and the chlorophenyl ring 48.9%. The sp act. was 17.4 mCi/mM. The radiochemical purity, as determined by thin-layer chromatographic (TLC) analysis followed by radioautography and liquid scintillation counting (lsc) was 299.0'70 in both samples. Unlabeled preparations of diflubenzuron and certain compounds considered as likely degradation products were also supplied by Thompson-Hayward. These compounds included 4-chlorophenylurea, 2,6-difluorobenzoic acid, 2,6-difluorobenzamide, 4-chloroaniline, N-[ [ (4-chloro-2-hydroxyphenyl)amino] carbonyl]-2,6-difluorobenzamide, and N-[ [ (4-chloro-3-hydroxypheny1)amino]carbonyl]-2,6-difluorobenzamide.The two hydroxylated diflubenzuron analogues were designated 4chloro-2-hydroxydiflubenzuronand 4-chloro-3-hydroxydiflubenzuron. In addition, the followng compounds were obtained commercially: 4-chloroacetanilide and 4chlorophenol (Aldrich, Milwaukee, Wis.), 4-chloro-Nmethylaniline (Calbiochem, San Diego, Calif.), and 4chloro-N,N-dimethylaniline(Adams Chemical Co., Round Lake, Ill.). Lactating Cow. A 360-kg lactating Jersey cow obtained from a local dairy was placed in a metabolism stall and catheterized (Foley retention catheter, size 28FR) to allow separate collection of urine and feces. The animal was provided water and coastal bermudagrass hay ad libitum and was fed a commercial dairy ration twice daily. Although milk production had averaged about 10 kg daily, production dropped to about 7 kg daily during the course of the study. This drop was probably caused by stress on the animal as a result of unfamiliar surroundings, constant retention within the stanchion, and catheterization. For treatment, the ['4C]diflubenzur~n25 W P formulation (in water) was diluted with unlabeled diflubenzuron
This paper is in the public domain. Published 1978 American Chemical Society
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J. Agric. Food Chem., Vol. 26, No. 1, 1978
25 W P such that the final treatment mixture contained 3.6 g of diflubenzuron active ingredient and a total of 0.65 mCi of radiocarbon, or a sp act. of 400 dpm/pg of diflubenzuron. The treatment mixture, as a slurry in 500 mL of water, was administered as a single oral dose to the animal via a stomach tube and was equivalent to 10 mg of diflubenzuron/kg of body weight. After treatment, total urine and feces samples were collected at 24-h intervals, and the animal was machine milked every 12 h. Aliquots of fresh milk and urine (0.2 mL) were assayed for radiocarbon by lsc. Parts of the remaining samples were frozen for later analysis. Feces samples were mixed thoroughly, and small parts (0.5-1.0 g) were removed and air-dried for quantitation of radiocarbon residues by oxygen combustion and lsc. Parts of the remaining samples were frozen for subsequent study. Seven days after treatment, the cow was killed, and numerous tissue samples were collected and frozen after small samples (99% partitioned into ethyl acetate in all samples), and TLC showed that unaltered diflubenzuron comprised 199% of the radiocarbon in all extracts. A very minor radioactive component of lower TLC Rf than diflubenzuron was detected in the extracts of certain samples of sheep digestive tract fluids, but attempts to reproduce these findings using fluids obtained from additional sheep were not successful. This product was also detected in some incubations in which the fluids had been previously boiled; thus, the unidentified compound likely arose through nonenzymatic reactions. Fate of 2,6-Difluoro-3-hydroxydiflubenzuroni n Rats. The major hydroxylated diflubenzuron metabolite occurring in milk was rapidly excreted after oral administration to laboratory rats. About 23% of the administered 14C was excreted in the urine within 3 days after treatment, and >85% of the urinary excretion occurred during the first day after treatment. Seventy-one percent of the 14Cwas eliminated through the feces of the treated rats. Radiocarbon elimination in the feces was almost complete within 2 days after treatment, and less than 2% of the administered 14Cwas recovered in the 3-day feces sample. Analysis of tissues collected when the animals were killed 3 days after treatment confirmed that excretion of radiocarbon was essentially complete. Samples of brain, liver, kidney, muscle, and omental fat from each animal contained no detectable radiocarbon residues. Ethyl acetate extraction of acidified samples of urine and feces gave >95% partitioning of radiocarbon into the organic phase from each sample. Two-dimensional TLC of the extracts indicated that the metabolite underwent little additional degradation in the rat before excretion. In the feces, >90% of the radiocarbon was recovered as unmetabolized 2,6-difluoro-3-hydroxydiflubenzuron. None of the four other radioactive components in the feces extracts cochromatographed with any of the available compounds of known structure. TLC analysis of the radioactive components in the urine extracts revealed more evidence of metabolism than was seen in feces. About 76% of the radiocarbon in the extracts of urine was unaltered 2,6-difluoro-3-hydroxydiflubenzuron, but at least five additional radioactive components were resolved from the mixture. However, none of these cochromatographed with any of the available metabolite standards, and no further attempts were made to determine their chemical nature. Fate of [ 14C]Diflubenzuron Applied Dermally to Cattle. [14C]Diflubenzuron residues were rapidly dissipated after the insecticide was applied dermally to cattle that were subsequently held in an unprotected pasture. Both W P and oil-based formulations were lost from the treated areas very rapidly-C4% of the applied radiocarbon remained after 1week and only 0.1 % of the applied dose remained after 4 weeks (Table VII). TLC examination of acetone rinses from the treated areas showed that diflubenzuron was the only detectable radioactive component in all samples. Analysis of numerous tissues taken when the animals were killed showed none to have detectable radiocarbon residues, other than hair and skin samples collected at or adjacent to the treated areas. There were no significant differences in persistence or residue patterns between the WP and oil based formulations. Although the radiochemical used to spike both formulations was in wettable powder form, the particle size of the [14C]diflubenzuron was the same as that of both formulations (2-5 pm) and the extremely minute amounts of WP
Ivie
formulation accompanying the radiochemical likely did not significantly alter the properties of the oil based-oil carrier mixture applied to the animals. The 4-week study was conducted during late summer, a period of mostly sunny and hot weather. A total of about 2 cm of rain was recorded during the study period, but rain clearly was not the cause of the rapid dissipation of diflubenzuron residue from the animals. The first rain did not fall until 1 day after the 1-week animal was killed for analysis. Studies with a stanchioned, catheterized cow indicated that diflubenzuron is not absorbed through the skin to any significant extent after dermal spraying of the insecticide. During a 3-day period after application of [14C]diflubenzuron W P formulation to the cow, no detectable residues were excreted in the urine. On the basis of studies involving oral dosing of cattle and sheep with diflubenzuron, radiocarbon excretion in the urine would have been expected if diflubenzuron had been absorbed through the skin. Although 2.1% of the applied radiocarbon was detected in the feces, this almost certainly represents contamination by rub-off or other external transfer processes. The feces from the animal were allowed to remain where deposited until collected at 24-h intervals, and some contamination was probably inevitable. After 3 days, 68% of the radioactivity applied to the stanchioned animal was recovered by clipping and extracting the treated hair and thoroughly washing the exposed skin with acetone. TLC of these fractions showed that diflubenzuron was the only detectable radioactive component. DISCUSSION
The studies reported here indicate that oral treatment of sheep and cattle with diflubenzuron is followed by absorption of the compound through the gastrointestinal tract, metabolism, and elimination of residues through the urine, feces, and to a very limited extent, milk. The fact that only a trace of unmetabolized diflubenzuron was secreted into milk and none was seen in urine or bile of the treated animals indicates that the absorbed diflubenzuron is almost completely metabolized to more polar products before excretion. Intact diflubenzuron was eliminated in the feces of orally dosed cattle and sheep, but considerable quantities of metabolites and unextractable radiocarbon were also detected in feces of all but the bile-duct cannulated animals. The radiocarbon eliminated in the feces of bile-duct cannulated sheep was almost totally extracted and consisted only of unmetabolized diflubenzuron. These observations, and the studies indicating that diflubenzuron is not metabolized in vitro by ruminant digestive tract fluids, seem conclusive evidence that this insecticide is not degraded within the digestive tract of ruminants to any significant degree. It follows that the levels of diflubenzuron metabolites detected in feces of ruminants are a direct indication of the extent of biliary excretion. Thus, even though no studies with bile-duct cannulated cattle were made, the data in Table VI indicate that >50% of the radiocarbon eliminated in the feces of a cow (that as metabolites or unextracted radiocarbon) likely came from biliary excretion. Estimates of biliary excretion based on the levels of diflubenzuron metabolites in feces are, if in error, likely to be low because of enterohepatic circulation. Sheep and cattle qualitatively metabolized diflubenzuron almost identically, but there were significant differences in the relative amounts of the metabolites generated. The major identified metabolic transformation in the cow was hydroxylation at the 3 position of the 2,6difluorobenzoyl ring, but in sheep the major identified
J. Agric. Food Chem., Vol. 26, No. 1, 1970
Fate of Diflubenzuron
Table VII. Persistence of [ 14C]Diflubenzuron o n the Hair and Skin after Dermal Application to Cattle Held in an Unprotected Pasture Weeks after treatment 1
2 4 1 2
4
Diflubenzuron residue, radio..IJP”’ carbon, % of applied Hair Skin Wettable powder formulation 85.1 0.4 3.8 19.7 0.1 1.7 2.9
