TISSUE RESIDUES OF STEERS G R A Z E D ON W H E A T PASTURE T R E A T E D WITH I N D A R SYSTEMIC F U N G I C I D E 1 G. W. Horn, F. P. Horn 2, W. R. Lyman a and D. G. Wagner
Summary
Residues in tissues (skeletal muscle, fat, liver and kidney, and fecal and urine grab samples of steers that grazed an 1NDAR fungicide-treated wheat pasture for 4 weeks, and after 1- or 3-week with&awl periods of grazing a nontreated wheat pasture were measured. INDAR parent compound (4-butyl-4H-1,2,4-triazote) concentrations ranged from 35 to 71 ppm of forage dry matter during the initial 4-week period and were higher than those that would be expected with normally recommended applications of INDAR fungicide. No effects of INDAR fungicide on steer grazing behavior or general alertness were observed. Total residue concentrations (INDAR fungicide and its metabolites or degradation products having the 1,2,4-triazole ring) in skeletal muscle, fat, liver and kidney samples averaged .50, .17, .84 and .57 ppm, respectively, at the end of the initial 4-week period on the treated wheat pasture. Tissue residues after a 3-week with&awl period,
and fecal and urine residues after 1- or 3-week withdrawal periods were not different (P>.05) from those of steers that grazed the nontreated pasture during the initial 4-week period. The data show that tissue residues of INDAR fungicide declined rapidly after stocker cattle are removed from the fungicide-treated pasture and would be expected to be extremely low in cattle from wheat pastures that received normal applications of the fungicide. (Key Words: INDAR, Wheat Pasture, Stocker Tissue Residues.) Introduction
The fungicide, INDAR 4 (4-butyl-4H1,2,4-triazole), is a systemic compound for the control of wheat leaf rust, Puccinia recondita (Von Meyer et al., 1970) by root uptake and (or) foliar spray. The effect of leaf rust on wheat grain yields has been the primary objective of many studies. However, reduced tillering (Hendrix and Fuchs, 1970; Williams, 1973), deterioration of roots (Johnston and Miller, 1934; Hendrix and Martin, 1972; Williams, 1973), increased water requirement (Weiss, 1924; Johnston and Miller, 1934; Williams, Journal Article 3532 of the Agricultural Experi- 1973) and reduced straw yields (Mains, 1930; ment Station, Oklahoma State University, Stillwater, Caldwell et al., 1934; Johnston and Miller, 74074. This paper reports the results of research only. Mention of a fungicide in this paper does not consti- 1934; Hendrix and Fuchs, 1970) of leaf rust infected wheat have been reported. tute a recommendation for use by the US Department of Agriculture nor does it imply registration under In years favorable to the growth of winter FIFRA as amended. Mention of a trade name, pro- wheat, over 1.5 million stocker cattle are grazed prietary product, or specific equipment does not constitute a guarantee or warranty by the US Depart- on Oklahoma wheat pastures. Effects of leaf ment of Agriculture and does not imply its approval rust on wheat forage production are therefore to the exclusion of other products that may be suit- of concern. In growth chamber experiments, able. Williams (1973) found that a severe infection of 2Southwestern Livestock and Forage Research leaf rust reduced oven-dry forage production Station, USDA, SEA, E1 Reno OK 73036. 47%; and in field experiments, oven-dry forage aAgricultural Products Research (Fungicides and production of resistant cultivars was 41% Biocides), Research Laboratories, Rohm and Haas Co., Spring House, PA 19477. greater when early fall rust infection severities 4Trade mark of Rohm and Haas Company, Spring were as high as 60% on susceptible wheat House, PA 19477. The word "triazbutit" has been cultivars. accepted as the American National Standard Common As of January, 1979 INDAR fungicide has name for 4-butyl-4H-1,2,4-triazole. The designation not been cleared by the Environmental ProtecRH-124 has also been used for this compound. 318 JOURNAL OF ANIMAL SCIENCE, Vol. 49, No. 2, 1975
Downloaded from https://academic.oup.com/jas/article-abstract/49/2/318/4700549 by Iowa State University user on 19 January 2019
Oklahoma Agricultural Experiment Station, Stillwater 74074
INDAR FUNGI('IDE-'I'I~,I.;ATI';DWtlEAT PASTURE
Experimental Procedure
Pastures and Forage Samples. Two pastures of Triumph 64 wheat of approximately 3.6 and 4.9 ha each were used. The smaller pasture was not treated with fungicide, and the larger pasture was sprayed with 439 ml of INDAR 70 5+Cs per ha (maximum labcl rate) with ;i tractor-drawn sprayer. Water was used as thc diluent for fungicide application. Wheat forage, consisting of hand-clipped samples from at least 10 randomly selccted locations within each pasture, was taken weekly throughout the trial. The samples wcrc composited within weck, and dry matter content of the samples was determined by drying the samples to constant weights in a forced-air oven at 65 C. The samplcs were stored dry until analyzed for residues. Tissue, Fecal and Urine Samples. Twelve ltereford steer calves weighing 525 kg + 4.3 (SEM) were randomly assigncd to four groups the day (January 13, 1976) immediatcty after the pasture was sprayed with the fungicide. Group one was placed on the nontreated pasture, and groups two, three and four were placed on thc fungicide-treated pasturc. The steers grazed their respective pastures for a 4-week period, after which steers in groups one and two were killed for tissuc rcsidue analyscs; those in groups three and four were moved to the control pasture for withdrawal periods of one and 3 weeks, respectively. Tissues obtained for total residue analyses
s307 ml 4-butyl-4H-l,2,4-triazolc. l.ot number SW75/0313. 64-Butyl-4H-l,2,4-triazole and inetabolites or degradation products having the 1,2,4-triazole ring. 7Kontes Glass Co., Vindand, NJ 08360.
were: skeletal muscle (Iongksimus, semimembra,wsus, biceps J>moris), fat (kidney,, brisket and caul), liver and kidney. 1\11 tissue samples were immediately frozen anti stored until analyzed. Urine and fecal grab samples wcrc taken from all steers at the end of the secomt and fourth wcck of the trial, and from the remaining steers aftcr 5- and 3-weck withdrawal periods for residue analyses. Urinc samples wcrc obtained by induced micturition (Turner et al., 5930) and fecal samples werc obtained from the rectum. "1"hc samples wcrc stored frozen until analyzed. Initial and final shrunk weights (overnight stand without feed of water) werc measurcd, and nonshrunk weights were measured at weckly intervals throughout the trial. All stcers were observed daily throughout the trial for an}" advcrsc or faw~rable effects that might have been due to tNDAR fungicidc (e.g., refusal to graze, gcneral alertness and toxic signs). Residue A;m/yses. Tissuc, urine, fecal and wheat forage samples were analyzed for total residues 6. Samples wcrc extracted with mcthanol in cellulose cxtraction thimbles for 16 hr in a Soxhlet extraction apparatus. The methanol extracts were acidified with 1 N HC1 in roundbottom flasks and concentrated by rotary vacuum evaporation in a water bath at 70 to 75 C. The acidic methanol extracts were partitioned in 500 ml scparatory funnels against methylene chloride anti thcn ethyl acetate to remove substrate components that produce foaming and spattering in the subsequent sulfuric acid digestion, ek,a aliquot of the acidic aqueous extracts was withdrawn and the water removcd. Residues wcrc digestcd with boiling sulfuric acid and then sodium hydroxide (50% w/v) was added dropwise until the mixtures were basic to a I lydrion test strip. "rriazolc was extracted from the neutralized digestion mixtures into cthyl acctate in a liquid/liquid extractor 7. After concentration of the ethyl acetatc, the samples were separated from interfering substances by silicic acid column chromatography, concentrated and reacted with 2,6dinitro-4-(trifluoromethyl) fluorobenzene to form l-(2,6-dinitro-4-trifluoromcthylphenyl)11 I-1,2,4-triazolc. Following another concentration step and further cleanup by silicic acid chromatography and conccntration again, the samples were dissolved in toluene-acetone, 100-5 (v/v) and the triazole derivative (representing thc total residues) was determined with a'l'racor Model 222 gas liquid chromatograph 8 equipped
Downloaded from https://academic.oup.com/jas/article-abstract/49/2/318/4700549 by Iowa State University user on 19 January 2019
tion Agency for use in the control of wheat leaf rust. The objective of this study was to measure residue concentrations in tissues and fecal and urine grab samples of stccrs that grazed an 5NDAR fungicide-treated wheat pasture and to measure the decline in residuc conccntrations after steers were placed on a nontreated wheat pasture. Forage residue concentrations were intentionally increased, above those that would be expected with normal applications of 5NDAR fungicide, in order to produce measureable residue concentrations in tissues and so that the rate of decline in residue concentrations could be measured.
3 19
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HORN ET AL.
8Tracor Instrument Co., 6500 Tracor Lane, Austin, TX 78721. 9Supelco, Inc., Supelco Park, Bellefonte, PA 16823.
which were also discarded. The aqueous phase was made basic by adding 5 g of NaItC03 and 4-butyl-4H-1,2,4-triazole was partitioned into chloroform by three 100-ml extractions. After each partition, the lower chloroform phase was drawn off and allowed to percolate through anhydrous NazS04 into a round-bottom flask. The combined chloroform extracts were concentrated to 25 ml with a rotary evaporator at 75 to 80 C under gentle vacuum. Interfering substances were then further removed by silicic acid and aluminum oxide column chromatography. The silicic acid column eluate was evaporated to dryness in a rotary flash evaporator at 60 C under gentle vacuum and redissolved in 25 ml ethyl acetate before chromatography on aluminum oxide. 4-butyl-4H- 1,2,4-triazole was quantitated with a Tracor Model MT-220 gas liquid chromatograph 8 equipped with a Coulson electrolytic conductivity detector 8 with a four-port rotary valve operated in the reductive nitrogen mode with a nickel wire catalyst. A .9 m pyrex glass, 2 mm I.D. column packed with 5% Carbowax 20M 9 on 80/100 mesh Chromosorb W HP 9 was used. Carrier gas was helium at a flow rate of 85 ml/minute. Recoveries through the drying step were not measured. Analytical recoveries of fortifications to dried forage samples averaged 76% for parent compound and 50% for total residues. Fortifications, in each case, were parent compound, 4-butyl-4H- 1,2,4-triazole. Statistical Analysis. Steer weight gains and tissue, fecal and urine residue concentrations were analyzed by analysis of variance, single criterion of classification. Weight gains were compared across treatments, at weekly intervals, during the initial 4-week period. Possible effects of time on fecal and urine residue concentrations were not included in the analysis of variance, where sequential measurements were made On the same animal, since all control steers were killed at the end of the initial 4-week grazing period. The specific treatments that were included in the analysis of variance are indicated as footnotes to each of the tables. Where F values were significant (P
Summary
Residues in tissues (skeletal muscle, fat, liver and kidney, and fecal and urine grab samples of steers that grazed an 1NDAR fungicide-treated wheat pasture for 4 weeks, and after 1- or 3-week with&awl periods of grazing a nontreated wheat pasture were measured. INDAR parent compound (4-butyl-4H-1,2,4-triazote) concentrations ranged from 35 to 71 ppm of forage dry matter during the initial 4-week period and were higher than those that would be expected with normally recommended applications of INDAR fungicide. No effects of INDAR fungicide on steer grazing behavior or general alertness were observed. Total residue concentrations (INDAR fungicide and its metabolites or degradation products having the 1,2,4-triazole ring) in skeletal muscle, fat, liver and kidney samples averaged .50, .17, .84 and .57 ppm, respectively, at the end of the initial 4-week period on the treated wheat pasture. Tissue residues after a 3-week with&awl period,
and fecal and urine residues after 1- or 3-week withdrawal periods were not different (P>.05) from those of steers that grazed the nontreated pasture during the initial 4-week period. The data show that tissue residues of INDAR fungicide declined rapidly after stocker cattle are removed from the fungicide-treated pasture and would be expected to be extremely low in cattle from wheat pastures that received normal applications of the fungicide. (Key Words: INDAR, Wheat Pasture, Stocker Tissue Residues.) Introduction
The fungicide, INDAR 4 (4-butyl-4H1,2,4-triazole), is a systemic compound for the control of wheat leaf rust, Puccinia recondita (Von Meyer et al., 1970) by root uptake and (or) foliar spray. The effect of leaf rust on wheat grain yields has been the primary objective of many studies. However, reduced tillering (Hendrix and Fuchs, 1970; Williams, 1973), deterioration of roots (Johnston and Miller, 1934; Hendrix and Martin, 1972; Williams, 1973), increased water requirement (Weiss, 1924; Johnston and Miller, 1934; Williams, Journal Article 3532 of the Agricultural Experi- 1973) and reduced straw yields (Mains, 1930; ment Station, Oklahoma State University, Stillwater, Caldwell et al., 1934; Johnston and Miller, 74074. This paper reports the results of research only. Mention of a fungicide in this paper does not consti- 1934; Hendrix and Fuchs, 1970) of leaf rust infected wheat have been reported. tute a recommendation for use by the US Department of Agriculture nor does it imply registration under In years favorable to the growth of winter FIFRA as amended. Mention of a trade name, pro- wheat, over 1.5 million stocker cattle are grazed prietary product, or specific equipment does not constitute a guarantee or warranty by the US Depart- on Oklahoma wheat pastures. Effects of leaf ment of Agriculture and does not imply its approval rust on wheat forage production are therefore to the exclusion of other products that may be suit- of concern. In growth chamber experiments, able. Williams (1973) found that a severe infection of 2Southwestern Livestock and Forage Research leaf rust reduced oven-dry forage production Station, USDA, SEA, E1 Reno OK 73036. 47%; and in field experiments, oven-dry forage aAgricultural Products Research (Fungicides and production of resistant cultivars was 41% Biocides), Research Laboratories, Rohm and Haas Co., Spring House, PA 19477. greater when early fall rust infection severities 4Trade mark of Rohm and Haas Company, Spring were as high as 60% on susceptible wheat House, PA 19477. The word "triazbutit" has been cultivars. accepted as the American National Standard Common As of January, 1979 INDAR fungicide has name for 4-butyl-4H-1,2,4-triazole. The designation not been cleared by the Environmental ProtecRH-124 has also been used for this compound. 318 JOURNAL OF ANIMAL SCIENCE, Vol. 49, No. 2, 1975
Downloaded from https://academic.oup.com/jas/article-abstract/49/2/318/4700549 by Iowa State University user on 19 January 2019
Oklahoma Agricultural Experiment Station, Stillwater 74074
INDAR FUNGI('IDE-'I'I~,I.;ATI';DWtlEAT PASTURE
Experimental Procedure
Pastures and Forage Samples. Two pastures of Triumph 64 wheat of approximately 3.6 and 4.9 ha each were used. The smaller pasture was not treated with fungicide, and the larger pasture was sprayed with 439 ml of INDAR 70 5+Cs per ha (maximum labcl rate) with ;i tractor-drawn sprayer. Water was used as thc diluent for fungicide application. Wheat forage, consisting of hand-clipped samples from at least 10 randomly selccted locations within each pasture, was taken weekly throughout the trial. The samples wcrc composited within weck, and dry matter content of the samples was determined by drying the samples to constant weights in a forced-air oven at 65 C. The samplcs were stored dry until analyzed for residues. Tissue, Fecal and Urine Samples. Twelve ltereford steer calves weighing 525 kg + 4.3 (SEM) were randomly assigncd to four groups the day (January 13, 1976) immediatcty after the pasture was sprayed with the fungicide. Group one was placed on the nontreated pasture, and groups two, three and four were placed on thc fungicide-treated pasturc. The steers grazed their respective pastures for a 4-week period, after which steers in groups one and two were killed for tissuc rcsidue analyscs; those in groups three and four were moved to the control pasture for withdrawal periods of one and 3 weeks, respectively. Tissues obtained for total residue analyses
s307 ml 4-butyl-4H-l,2,4-triazolc. l.ot number SW75/0313. 64-Butyl-4H-l,2,4-triazole and inetabolites or degradation products having the 1,2,4-triazole ring. 7Kontes Glass Co., Vindand, NJ 08360.
were: skeletal muscle (Iongksimus, semimembra,wsus, biceps J>moris), fat (kidney,, brisket and caul), liver and kidney. 1\11 tissue samples were immediately frozen anti stored until analyzed. Urine and fecal grab samples wcrc taken from all steers at the end of the secomt and fourth wcck of the trial, and from the remaining steers aftcr 5- and 3-weck withdrawal periods for residue analyses. Urinc samples wcrc obtained by induced micturition (Turner et al., 5930) and fecal samples werc obtained from the rectum. "1"hc samples wcrc stored frozen until analyzed. Initial and final shrunk weights (overnight stand without feed of water) werc measurcd, and nonshrunk weights were measured at weckly intervals throughout the trial. All stcers were observed daily throughout the trial for an}" advcrsc or faw~rable effects that might have been due to tNDAR fungicidc (e.g., refusal to graze, gcneral alertness and toxic signs). Residue A;m/yses. Tissuc, urine, fecal and wheat forage samples were analyzed for total residues 6. Samples wcrc extracted with mcthanol in cellulose cxtraction thimbles for 16 hr in a Soxhlet extraction apparatus. The methanol extracts were acidified with 1 N HC1 in roundbottom flasks and concentrated by rotary vacuum evaporation in a water bath at 70 to 75 C. The acidic methanol extracts were partitioned in 500 ml scparatory funnels against methylene chloride anti thcn ethyl acetate to remove substrate components that produce foaming and spattering in the subsequent sulfuric acid digestion, ek,a aliquot of the acidic aqueous extracts was withdrawn and the water removcd. Residues wcrc digestcd with boiling sulfuric acid and then sodium hydroxide (50% w/v) was added dropwise until the mixtures were basic to a I lydrion test strip. "rriazolc was extracted from the neutralized digestion mixtures into cthyl acctate in a liquid/liquid extractor 7. After concentration of the ethyl acetatc, the samples were separated from interfering substances by silicic acid column chromatography, concentrated and reacted with 2,6dinitro-4-(trifluoromethyl) fluorobenzene to form l-(2,6-dinitro-4-trifluoromcthylphenyl)11 I-1,2,4-triazolc. Following another concentration step and further cleanup by silicic acid chromatography and conccntration again, the samples were dissolved in toluene-acetone, 100-5 (v/v) and the triazole derivative (representing thc total residues) was determined with a'l'racor Model 222 gas liquid chromatograph 8 equipped
Downloaded from https://academic.oup.com/jas/article-abstract/49/2/318/4700549 by Iowa State University user on 19 January 2019
tion Agency for use in the control of wheat leaf rust. The objective of this study was to measure residue concentrations in tissues and fecal and urine grab samples of stccrs that grazed an 5NDAR fungicide-treated wheat pasture and to measure the decline in residuc conccntrations after steers were placed on a nontreated wheat pasture. Forage residue concentrations were intentionally increased, above those that would be expected with normal applications of 5NDAR fungicide, in order to produce measureable residue concentrations in tissues and so that the rate of decline in residue concentrations could be measured.
3 19
320
HORN ET AL.
8Tracor Instrument Co., 6500 Tracor Lane, Austin, TX 78721. 9Supelco, Inc., Supelco Park, Bellefonte, PA 16823.
which were also discarded. The aqueous phase was made basic by adding 5 g of NaItC03 and 4-butyl-4H-1,2,4-triazole was partitioned into chloroform by three 100-ml extractions. After each partition, the lower chloroform phase was drawn off and allowed to percolate through anhydrous NazS04 into a round-bottom flask. The combined chloroform extracts were concentrated to 25 ml with a rotary evaporator at 75 to 80 C under gentle vacuum. Interfering substances were then further removed by silicic acid and aluminum oxide column chromatography. The silicic acid column eluate was evaporated to dryness in a rotary flash evaporator at 60 C under gentle vacuum and redissolved in 25 ml ethyl acetate before chromatography on aluminum oxide. 4-butyl-4H- 1,2,4-triazole was quantitated with a Tracor Model MT-220 gas liquid chromatograph 8 equipped with a Coulson electrolytic conductivity detector 8 with a four-port rotary valve operated in the reductive nitrogen mode with a nickel wire catalyst. A .9 m pyrex glass, 2 mm I.D. column packed with 5% Carbowax 20M 9 on 80/100 mesh Chromosorb W HP 9 was used. Carrier gas was helium at a flow rate of 85 ml/minute. Recoveries through the drying step were not measured. Analytical recoveries of fortifications to dried forage samples averaged 76% for parent compound and 50% for total residues. Fortifications, in each case, were parent compound, 4-butyl-4H- 1,2,4-triazole. Statistical Analysis. Steer weight gains and tissue, fecal and urine residue concentrations were analyzed by analysis of variance, single criterion of classification. Weight gains were compared across treatments, at weekly intervals, during the initial 4-week period. Possible effects of time on fecal and urine residue concentrations were not included in the analysis of variance, where sequential measurements were made On the same animal, since all control steers were killed at the end of the initial 4-week grazing period. The specific treatments that were included in the analysis of variance are indicated as footnotes to each of the tables. Where F values were significant (P
