EFFECTS OF TRICYCLAZOLE ON SELECTED REPRODUCTIVE PARAMETERS IN MALLARDS AND BOBWHITE FOLLOWING CHRONIC DIETARY EXPOSURE By John Richard Hochstein A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1984 ABSTRACT EFFECTS OF TRICYCLAZOLE ON SELECTED REPRODUCTIVE PARAMETERS IN MALLARDS AND BOBWHITE FOLLOWING CHRONIC DIETARY EXPOSURE By John Richard Hochstein Tricyclazole was administered to mallards (Anas platyrhynchos) and bobwhite (Colinus virginianus) for 21 and 22 weeks, respectively, at dietary concentrations of O, 30, 100, and 300 ppm to evaluate chronic reproductive toxicity. Food consumption, body weight gain, and selected reproductive parameters were monitored. Chronic dietary administration of tricyclazole did not signifi- cantly affect food consumption, body weight gain, mortality, hatchling survival, or the reproductive parameters measured, including egg production, eggshell thickness, fertility, embryo survival, and hatchability. Based on the parameters measured the no-observed effect level for tricyclazole in the diet of mallards and bobwhites is 300 ppm, the highest level evaluated. ACKNOWLEDGMENTS My appreciation is extended to Dr. Robert K. Ringer and my other committee members; Dr. Richard Aulerich, Dr. Steven Bursian and' Dr. Harold Prince. I would like to thank my parents and family for their support and good natured harassment during the course of my studies. I would like to express my deepest gratitude to my future wife, Judi, for her constant encouragement and understanding. TABLE OF CONTENTS List Of TableSOOOOOCOOOOOOOOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOOOCOO w List Of Figures.’IOOOOIOOOOOOIOOOOOOOOO0.0...OOOOOOOOOOOOOOOOOOOO v IntrOdUCtionooooooooooooooo.00000000000000.000.000.00.00ooooooooo 1 Review of Literature............................................. 3 TriCYCIaZOIeOcoo-o...00000000009000.0900...cocoa-00.00.0000. 3 Mode of Action and Efficacy................................. 5 Bioavailability............................................. 6 Toxicology.................................................. 7 Materials and Methods............................................ 11 Chronology.................................................. 11 Animals..................................................... 13 Treatment Levels............................................ 15 Diets....................................................... 15 Facilities.................................................. 16 Testing..................................................... 17 Egg Collection, Storage, and Incubation..................... 13 Eggshell Thickness.......................................... 21 Statistical Analysis........................................ 21 PathOIOSYOOOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOOO00......O Results.O00......OOOIOOOOIOOOOIOOOCOOIOOIO...OOOOOOOOOOOOOCOOOOOO Room Variation-Mallard Study................................ 23 Feed Consumption............................................ 23 Body Weight................................................. 26 Reproduction................................................ 26 Clinical Observations....................................... 26 Intest Mortality............................................ 31 Pathology................................................... 31 Discussion....................................................... 35 Conclusion....................................................... 42 AppendiceSCOOOOOO00.0.0.0...0.0.0.0...OOOOOOCOOOOOOOOOOOI00...... Appendix A. Solubility of tricyclazole in organic solvents....................................... 43 Appendix B. Toxicology of tricyclazole..................... 44 Appendix C. Environmental toxicology of tricyclazole....... 45 Appendix D. Hazard evaluation of tricyclazole.............. 46 Appendix E. Subacute and Chronic toxicity of tricyclazole.. 47 Appenidx F. Nutrient requirement of mallards............... 48 Appendix G. Composition of duck breeder-layer diet......... 49 Appendix H. Calculated analysis of duck breeder-layer diet. 50 Appendix I. Composition of duck starter diet............... 51 ii Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix J. K. L. M. N. O. P. Q. R. S. T. Calculated analysis of duck starter diet....... Nutrient requirement of bobwhite............... Composition of quail breeder—layer diet........ Calculated analysis of quail breeder-layer diet........................................... Composition of quail starter diet.............. Calculated analysis of quail starter diet...... Stability and homogeneity of tricyclazole in duck and quail diets........................... Assays of diets that contained tricyclazole fed to mallards................................ Assays of diets that contained tricyclazole fed to bobwhite................................ Summary of pathologic diagnosis for mallards fed tricyclazole............................... Summary of pathologic diagnosis for bobwhite fed tricyclazole............................... BibliograthOOOOOO...OOOOOOOOOOOOOOOOOOOOOOOIOCOOOIOOOOOOOOOOO0.0 iii Page 52 53 54 55 56 57 58 S9 60 61 62 63 Table Table Table Table Table Table Table Table LIST OF TABLES Food consumption of adult mallards fed tricyclazole... Food consumption of adult bobwhite fed tricyclazole... Body weights of adult mallards fed tricyclazole....... Body weights of adult bobwhite fed tricyclazole....... Effect of feeding tricyclazole on reproductive parameters in mallardSOOOOOOO00......OOOOOOOOOOOOOOOOO Effect of feeding tricyclazole on reproductive parameters in bObWhiteooooooooooooooooooooooooooooonoo Clinical observations of mallards fed tricyclazole.... Interim deaths of mallards fed tricyclazole........... iv Page 24 25 27 28 29 3O 32 33 LIST OF FIGURES Page Figure 1. Structure of tricyclazole............................. 4 Figure 2. Protonation of tricyclazole........................... 8 Figure 3. Chronology of study for mallards fed triCYCIaZOIeIOO00.0.00...OOOOOOOOOOOOO0.00.00.00.00... 12 Figure 4. Chronology of study for bobwhite fed triCYCIaZOIeooo0000000000000000000.00.000000000000000. 14 INTRODUCTION Rice blast is one of the most serious and difficult to control of all rice diseases (Weber, 1982; Anonymous, 1982) and is gaining a substantial foothold in the rice producing areas of the United States' Gulf Coast. The ubiquitous organism Pyricularia oryzae is the infectiOus agent, but high soil nitrogen and organic matter levels as well as late planting, compound the problem for unknown reasons. Hot, humid weather also accentuates the problem. P;_ggyg§g.more easily infects young, developing plant tissue, but rice is susceptible to the fungus at all stages of growth, so timely fungicide applications and coverage are critically important in achieving optimum disease control (Weber, 1982; Anonymous, 1982). Tricyclazole is a new class of compound developed to serve as a fungicide for control of rice blast as a result of L $17.33 infestation. Tricyclazole is being developed by Eli Lilly and Company in an effort to effectively provide prolonged control of rice blast with increased efficiency due to reduced frequency of application with the added benefit of less labor and equipment usage. At the initiation of the study in September, 1980, chronic avian reproduction studies were required by the Environmental Protection Agency under authority of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) if any of the following conditions existed: 1) if toxic amounts of a pesticide or its metabolite(s) are expected to accumulate on avian feed under normal use conditions. 2) if storage or accumulation of the pesticide or its metabolite(s) in plant or animal tissues is possible 3) if the pesticide is used where birds may be subjected to continued or repeated exposure to the pesticide or its metabolite(s), especially preceeding or during the breeding season. 4) if any other test information indicates that avian reproduction may be adversely affected. (EPA, 1978) The purpose of this study was to determine the effects of chronic dietary exposure of the fungicide tricyclazole on reproduction in two avian species. Mallards (Ag§§_platyrhynchos) and bobwhite (Colinus virginianus) were the indicated species (EPA, 1978) due to their ubiquitous distribution and their representation as wild waterfowl and upland game species, respectively. LITERATURE‘REXIEE Tricyclazole Tricyclazole (S-methyl-l,2,4,-triazolo [3,4-b] benzothiazole) (Figure l) is a new systemic fungicide (Anonymous, 1982; Weber, 1982) developed by Elanco Products Company, Eli Lilly and Company for the control of rice blast disease caused by Pyricularia oryzae. It is at this time produced under the trade name Beamg’under the authority of an EPA experimental use permit and is for experimental use only. Tricyclazole is an odorless, crystalline solid with a molecular weight of 189.24 and its molecular formula is C9H7N3S. It has a melting point of 187-188°C and is thermally stable up to this point. Tricyclazole has a vapor pressure of 2 x 10'7mm Hg at 25°C. It is not readily degraded by water or sunlight. The solubility of tricyclazole in water at 25°C is 0.7 mg/ml. It is readily soluble in chloroform (>’500.0 mg/ml) but less soluble in other organic solvents (Appendix A) (Elanco, 1976; 1981). Gas chromatographic or high-performance liquid chromatography procedures for determination of tricyclazole residues are discussed by Day ggngl, (1980) along with a discussion of biological, chemical and physical properties. Thin layer chromatography can also be used to identify tricyclazole in rice tissue and soil through a bioautographic assay technique using Cladosporium cucumerinum. In this assay, the fungus and compound interact to form a red zone of coloration (Elanco, 1976). The urinary metabolites of tricyclazole in the rat have been identified by thin-layer chromatography and gas chromatograph-mass spectrometry as the alcohol (1,2,4 triazolo [3,4-5]benzothiazole-S— methanol) and acid (1,2,4-triazolo[3.4-5]benzothiazole-S-carboxylic acid) derivatives of tricyclazole (Pierson and Howard, 1978). @001 \ N Figure 1. Structure of tricyclazole mgmfl Efficacy Tricyclazole is a systemic fungicide which is readily absorbed by the plant's roots and leaves, translocated to the leaf tips and provides residual rice blast control after a single soil or foliar application (Weber, 1982, Elanco, 1981, Froyd g£_§l,, 1976). This is in contrast to other fungicides which provide protection only for areas exposed to the fungicide (Anonymous, 1982). It also provides control regardless of the weather, cultural practices, or severity of disease. Tricyclazole does not prevent spore germination nor reduce appressorial formation on the rice plant surface. However, the compound does prevent formation of penetration hyphae from the appressoria which invade the epidermal walls of an untreated plant (Kubo gghgl,, 1982; Shiba and Nagata, 1981; Tokousbalides and Sisler 1978b; Chrysayi, 1976). Tricyclazole inhibits melanization by 1:; mg mycelia and appressoria without affecting fungal growth . (Woloshuk, g£_§l,, 1982, Yamaguchi, g£_§1,, 1982). Melanization in the appressoria appears to be a critical requirement for successful epidermal penetration (Woloshuk ggngl, 1983). Tokousbalides and Sisler (1978a) have demonstrated that tricyclazole blocks certain aspects of melanin synthesis which leads to a decrease in pigmen- tation. Several studies have indicated that tricyclazole blocks the reduction of 1,3,8-trihydroxynaphthalene to vermelone through inhibition of 1,3,8-trihydroxynapthalene reductase (Woloshuk §£_§l,, 1981; Woloshuk g£_§l, 1980; Tokousbalides and Sisler, 1979; Tokousbalides, 1978) and Tokousbalides (1978) suggest that this reductase may be an unusual type of enzyme peculiar to secondary metabolism in fungi. This reductase inhibition by tricyclazole has also been observed in other fungal species; Alternaria spp., Cladospgrium cucumerinum, Cochliobolus carbonum,rDiplodia natalensis, Duechsclera sorokiniana,, Macrophomina phaseoli, Rhizoctonia leguminicola, Sclerotinia 21222; Thielaviopsis basicola, §2g_Verticillium pp. However, tricyclazole is specific only in preventing rice blast caused by P;_g£15§g.and is not know to control any diseases associated with any of the above listed fungal species (Kubo §£_§l,, 1982; Wheeler, 1982; Elanco, 1981; Stipanovic and Wheeler, 1980; Tokousbalides and Sisler, 1979; Wheeler and Stipanovic, 1979a, b). It has been demonstrated that tricyclazole provides better protection more efficiently (reduced frequency of application) than other fungicides now being used for rice blast control. This is true whether tricyclazole is applied as a soil drench, root treatment or foliar spray (Hasawa g£_§l,, 1982; Froyd §£_al, 1978; Tsai, 1977; Froyd g;_a_l_., 1976; Hwang 3331., 1976). Hwang _e_t__a_l. (1976) have demonstrated that tricyclazole is more efficient when applied before inoculation than after inoculation. Tricyclazole has been shown to result in increased yields when compared to other fungicides (Sakaoka, 1982; Neto g; 31,, 1981; Yang, 1980). Bioavailability Weber (1982) discussed some of the factors that affect the bioavailability of tricyclazole. It was determined that aqueous solutions of tricyclazole have a pKa of 1.6. Tricyclazole is absorbed to a greater extent by clay and sandy loam soils than by soil organic matter. Absorption increases greatly as pH decreases. It is postulated that as pH decreases tricyclazole becomes protonated (Figure 2) and at pH levels of 0.5 or lower only protonated species are present while only molecular species are present at pH 3.0 and above (Weber, 1982). Water is effective in desorbing bound tricyclazole from soil organic matter and sandy loam soils but is relatively ineffective in desorbing the fungicide from clay soils. Desorption is greatly increased with buffered salts (Weber, 1982). High absorption of tricyclazole by soil organic matter, sandy loam and clay soils, effects of buffered salts and the pH dependence of absorption-desorption mechanisms have a significant impact on the bioavailability of the fungicide in soil systems and in aquatic systems containing soil particulate matter (Weber, 1982). Toxicology Pierson and Howard (1978) studied the disposition of tricyclazole in rats by using [14C] tricyclazole (uniformly labeled in the benzene ring) at 60 mg/kg administered by gavage. They found that the disappearance of radioactivity was biphasic, with a primary half-life (t5) of 2.5 hr was followed by a more prolonged decay. Peak plasma concentration occurred one hour post-oral administration at a level of 5.8 g/ml. Radioactivity was rapidly distributed to all organs with highest concentrations occurring in the liver and kidney. There was no accumulation of radioactivity in any tissue following 7 daily doses of 60 mg/kg. Radioactivity was completely eliminated (98.22) within 4 days, with urinary excretion being the major route of elimination (65.6); however, of elimination during the first 24 hours, biliary excretion accounted for 75.62 of the orally administered dose of tricyclazole. moaumo OdoumHO>OHuu oaoumao>owuu mo cowum:0uoum .N mummfim maonmaomowuu mzu a. + __ LN _ / z/z\ m \ A chronic toxicity-oncogenic study was conducted by Howard ggngl. (1978) in rats. Results showed a decrease in body weight at 620 and 1600 ppm of tricyclazole in the diet. However, at all dietary levels (up to 1600 ppm) of tricyclazole tested, there was no effect on survival, hematology, serum chemistry or urinalysis determinations. Terminal organ weights indicated a dose-related increase in mean relative weights (grams/100 grams body weight) for liver, kidney, and heart at the 620 and 1600 ppm dietary levels. This relative increase in organ weights parallels the decrease in body weights for these dose levels. Although there was an increase in mean relative weights, mean absolute organ weights were not significantly different from those of the control. Also gross and histopathologic examination of tissues and organs indicate that tricyclazole in the diets of rats, at all dietary levels tested, had no adverse effects (Howard g£_gl,, 1978). In a three month subchronic study in mice, 400 ppm of tricyclazole increased liver weights in female mice. Doses of 1000—3600 ppm resulted in mortality, decreased body weight, increased liver weight and serum glutamic pyruvic transaminase, and caused small bile duct proliferation (Howard g£_§l,, 1979). A two year chronic toxicity- oncogenic study in mice (doses of 50, 140, or 400 ppm of tricyclazole) resulted in no significant variation in survival, body weight, water consumption, organ weights, hematology, clinical chemistry, gross and histopathologic observations, or in the incidence of neoplasms. A no effect level of 400 ppm was concluded by Howard and his colleagues (1979). Other studies have demonstrated that tricyclazole has no effect on survival, growth and reproductive performance in mice and rats when 10 exposed to dietary concentrations up to 275 ppm for three generations. Also, tricyclazole has been shown not to be teratogenic in rabbits at daily oral doses of 50 mg/kg body weight. Teratogenicity does not occur in the rat or mouse at concentrations of 275 ppm of tricyclazole in the diet (Elanco, 1981). Moriya and his colleagues (1983) performed mutagenicity studies on pesticides by use of bacterial reVersion assay systems and concluded that triyclazole is not mutagenic. Elanco (1981) has compiled information from their own studies and those of others concerning toxicology, environmental toxicity, hazard evaluation, and grade tricyclazole, and subacute and chronic toxicity of tricyclazole. This information is summarized in Appendices B-E, respectively. MATERIALS AND METHODS Chronology Mallards The mallards (Anas platyrhynchos) used for the study were received on July 29, 1980 and were immediately placed into quarantine until September 21, 1980. The quarantine period was followed by a 15 day acclimation period, September 22, 1980 through October 6, 1980. During this time the mallards were in their appropriate testing pens and were fed the untreated basal diet. The mallards first received their assigned dietary level of tricyclazole on October 7, 1980, which was the beginning of the pre-production phase. This phase ended on December 9, 1980 when the photoperiod was increased from 7 hours light:l7 hours dark per day to 17 hours light:7 hours dark, initiating the egg production phase. The egg production phase ended on March 3, 1981. The last set (set 12) of two-week—old hatchlings was terminated on April 14, 1981 (Figure 3). Bobwhite The bobwhite (Colinus virginianus) used for the study were received on July 17, 1980 and were immediately placed into quarantine until September 22, 1980. -The quarantine period was followed by a 15 day acclimation period, September 23, 1980 through October 7, 1980. During this time the bobwhite were in their appropriate testing pens and were fed the untreated basal diet. The bobwhite first received their assigned dietary level of tricyclazole on October 8, 1980, which was the beginning of the pre—production phase. This phase ended on December 10, 1980 when the photoperiod was increased from 7 hours 11 12 Figure 3. Chronology of study for mallards fed tricyclazole. Birds received at testing facility July 29, 1980 * J, Quarantine period July 29-September 21, 1980 1 Acclimation period September 22-October 6, 1980 \L I Pre—production period (7 hours light:l7 hours dark) October 7-December 8, 1980 NI! Production and egg collection period (17 hours light:7 hours dark) December 9, 1980-March 3, 1981 Terminal kill and necropsy of adults March 3, 1981 \L Terminal kill of two-week old hatchlings from Set No. 12 April 14, 1981 13 light:17 hours dark per day to 17 hours light:7 hours dark, initiating the egg production phase. The egg production phase ended on March 11, 1981. The last set (set 10) of two-week-old hatchlings was terminated on April 24, 1981 (Figure 4). Animals Mallards Mallards were purchased from Whistling Wings, Hanover, IL. The mallards were hatched on June 16, 1980 and arrived at the test facility on July 29, 1980. The mallards were 14 weeks old at the start of the acclimation phase of the study, and the mean body weights (:S.D.) were 1242 :83 g and 1014 :93 g for males and females, respectively. At the start of the acclimation period, all mallards appeared to be in good health. Each mallard was banded with its own unique leg band and was randomly assigned to a pen (1 male and 3 females per pen). Random assignment of dietary level to pens was accomplished by the use of a random numbers table. Two pens per room were randomly eliminated at the end of the acclimation period to maintain equal replication of doses of four pens per treatment per room. Bobwhite Bobwhite were purchased from Illinois State Game Farm, Mt. Vernon, IL. The bobwhite were hatched on July 1, 1980 and arrived at the test facility on July 17, 1980. The bobwhite were 12 weeks old at the start of the acclimation phase of the study, and the mean body weights (: S.D.) were 177 i 12 g and 172 i 13 g for males and females, respectively. At the start of the acclimation period, all bobwhite appeared to be in good health. Each bobwhite was banded with its own 14 Figure 4. Chronology of study for bobwhite fed tricyclazole. . Birds received at testing facility July 17, 1980 l Quarantine period July l7-September 22, 1980 l Acclimation period September 23-October 7, 1980 l Pre-production period (7 hours light:17 hours dark) October 8-December 9, 1980 l Production and egg collection period (17 hours light:7 hours dark) December 10, 1980-March 11, 1981 J Terminal kill and necropsy of adults March 11, 1981 Terminal kill of two-week old hatchlings from Set No. 12 April 24, 1981 15 unique leg band and was randomly assigned to a pen (1 male and 1 female per pen). Random assignment of dietary levels to pens was accomplished by the use of a random numbers table. Treatment Levels The test compound used in both the mallard and bobwhite studies was tricyclazole (S-methyl—l,2,4,—triazolo [3,4-b] benzothiazole). Selection of dietary concentrations were based on previously conducted 5-day LC50 studies, mammalian reproduction studies, mallard and bob- white palatability studies and environmental residue studies. The four dietary levels selected for both mallard and bobwhite studies were 0.02 (control), 0.003% (30 ppm), 0.01% (100 ppm), and 0.03% (300 ppm) tricyclazole. The 300 ppm treatment level exceeds by 60 fold the maximum field residue values of 5 ppm in rough rice (Cochrane, 1980). For the mallard study there were 8 pens in each of the control and treatment groups. The bobwhite study had 15 cages in the control and 0.003 ppm treatment group, 13 in the 0.01 ppm treatment group, and 14 in the 0.03 ppm treatment group. Diets The test diets were prepared by Eli Lilly and Company, Green- field, Indiana using Lilly Mash. Homogeneity of mix and 8-week stability in feed were determined prior to the initiation of the study. Before the treated diets were shipped to the test facility, samples were removed for determination of the test chemical. Additionally, each dietary concentration was sampled when it was replaced with a freshly prepared diet to provide information on stability of tricyclazole during the 4-5 week period each lot was fed 16 to the mallards and bobwhites. Hatchlings were fed a starter diet for the two-week period they were raised. Both adults and hatchlings were given food and water 2g.libitum throughout the study. (See Appendices F through R for composition, analysis, dietary requirements, stability, homogeneity, and assays for tricyclazole.) Facilities Mallards The mallards were housed on the Michigan State University Poultry Research and Teaching Center in House 4, Rooms 4A and 4B. The rooms were entirely enclosed and measured 10.97 m x 12.19 m x 2.44 m (W x L x H). Three rows of six pens were suspended from the ceiling by chains. Each pen measured 1.47 m x 1.55 m x 0.70 m with no top. The pens were constructed of l" x 1" plastic coated wire mesh. The floor of each pen was sloped from the center downward to the two outer sides allowing the eggs to roll toward the aisles facilitating the collection of and the prevention of damage to the eggs. The pens were labeled with pen number, study number, compound number, dietary level, and individual bird numbers. Each pen had its own individually ident- ified feed container and scoop so that feed consumption could be accurately measured. Photoperiod was controlled by an automatic timer that was appropriately set for the current phase of the study. Light was provided by four 100 watt light bulbs equally spaced throughout the room. A thermometer was located on the wall inside the testing rooms. Room temperature was checked daily and, when necessary, gas heaters were lit to maintain temperatures at 10°C (50°F). 17 Bobwhites The bobwhite were housed on the Michigan State University Poultry Research and Teaching Center in House 4, Room E. The room was entirely enclosed and measured 5 m x 9.3 m x 2.4 m. (W x L x H). Two groups of pens were suspended from the ceiling, each consisting of two rows of 15 pens aligned back to back. Each pen was labeled with a cage number, compound number, study number, dietary level, and bird identification number. The dimensions of each cage were 40 cm wide x 43 cm long x 44.5 cm deep. The wire mesh dimensions were 2.6 x 9.8 cm, with the floor being 1.27 x 1.27 cm and plastic coated to protect the birds' feet. The floor of all the pens sloped toward the aisles which allowed the eggs to roll into the collecting area. Each pen was provided with an individual feed hopper (5.1 cm x 12.5 cm x 5.1 cm) and water cup. Also, each pen had its own individually identified feed container and scoop so that feed consumption could be accurately measured. Photoperiod was controlled by an automatic timer that was appropriately set for the current phase of the-study. Light was provided by four 100 watt light bulbs equally spaced throughout the room. A thermometer was located on the wall inside the testing room. Room temperature was checked daily and, when necessary, the gas heaters were lit to maintain temperatures at 15.6°C (60°F). Testing Food consumption was measured biweekly during the acclimation and dietary exposure periods for both studies. Adult body weights were measured on weeks 0, 2, 4, 6, 7, 9, 11, and at termination of the adult exposure phase of both studies. Body weights were not measured 18 during the egg production phase to avoid adverse effects on egg pro- duction due to handling stress. Egg production, mortality, morbidity, behavior, and any observable clinical signs of intoxication were recorded daily. All birds that died during the study were subjected to gross necropsy. If any abnormalities were noted, samples were taken for histopathology. All birds surviving to the planned termination date were killed by C02 asphyxiation and necropsied. At termination, tissues were collected for histopathology from at least 5 animals of each sex per treatment level. Egg_Collection, StorageI and Incubation Mallards Egg production began on December 12, 1980. Eggs were collected daily and each egg was marked in pencil with its corresponding pen number and date. Eggs that were soft-shelled, lacking shells, or extremely undersized were recorded as abnormal and discarded. Eggs were stored at 15.6°C (l4.4-16.8°C). At weekly intervals, all eggs were candled and those with cracks were recorded and discarded. The remaining eggs were placed in an incubator (Jamesway, single stage, Model 252) for 24 days. The temperature and relative humidity settings were 37.8°C (37.2-38.2°C) and 57% (56-57.2Z), respectively. The eggs were candled on day 14 of incubation and the numbers of fertile eggs, infertile eggs, and early embryonic deaths were recorded. On day 21, the eggs were candled again to determine embryo mortality that had occurred after day 14. On day 24, the live eggs were transferred to pedigree baskets and separated according to pen 19 number. The eggs were placed in the hatchery incubator (Jamesway, single stage, Model 252) which had temperature and relative humidity settings of 37.3°C (36.9-38.2°C) and 66% (63.5-682), respectively. On day 27 (or on day 28 if the hatch was poor), all pipped (dead and live), unpipped (dead and live), and hatchlings (dead and live), were recorded. All hatchlings were individually weighed, wing banded, and recorded by parental pen number. The hatchlings were placed in cardboard chick boxes for the 5 mile transfer to the MSU Poultry Research and Teaching Center. Precautions were taken to prevent exposure of the hatchlings to cold temperatures. The hatchlings were transferred quickly from the incubation room to a pre—warmed transporting vehicle, and from the vehicle to House 3 of the research facility. The hatchlings were immediately placed in a Petersime 250- 24 battery brooder according to dietary level of parental stock. Temperatures were maintained at 37.5°C and the hatchlings were given both starter diet and water §g_libitum. The hatchlings were observed daily for two weeks and all mortalities were recorded. At the end of the two-week period, all surviving hatchlings were killed by exposure to chloroform and individual weights recorded. Necropsies were not performed on hatchlings that died during the 2 week survival period, or on those that were killed at termination of the survival period. Bobwhite Egg production began on January 6, 1980. Eggs were collected daily and each egg was marked in pencil with its corresponding pen number and date. Eggs that were soft-shelled, lacking shells, or extremely undersized were recorded as abnormal and discarded. Eggs were stored at 15.6°C (14.4-16.8°C). At weekly intervals all eggs 20 were candled and those with cracks were recorded and discarded. The remaining eggs were placed in an incubator (Jamesway, single stage, Model 252) for 21 days. The temperature and relative humidity settings were 37.8°C (37.2-38.2°C) and 57% (56-57.2%), respectively. The eggs were candled on day 11 of incubation and the numbers of fertile eggs, infertile eggs, and early embryonic deaths were recorded. On day 18, the eggs were candled again to determine embryo mortality that had occurred after day 11. On day 21, the live eggs were transferred to pedigree baskets, and separated according to pen number. The eggs were placed in the hatchery incubator (Jamesway, single stage, Model 252) which had temperature and relative humidity settings of 37.3°C (36.9-38.2°C) and 662 (63.5-682), respectively. On day 24 (or on day 25 if the hatch was poor), all pipped (dead and live), unpipped (dead and live), and hatchlings (dead and live) were recorded. All hatchlings were individually weighed, wing banded, and recorded by parental pen number. The hatchlings were placed in cardboard chick-boxes and transported to the broader room. Precautions were taken to prevent exposure of the hatchlings to cold temperatures. They were immediately placed in a Petersime 250-24 battery brooder according to dietary level of parental stock. Temperatures were maintained at 37.5°C and the hatchlings were given both starter diet and water gg.1ibitum. The hatchlings were observed daily for two weeks and all mortalities were recorded. At the end of the two-week period, all surviving hatchlings were killed by exposure to chloroform and individual weights recorded. Necropsies were not performed on hatchlings that died during the 2 week survival period, or on those that were killed at termination of the survival period. 21 Eggshell Thickness For the mallard study, eggs were collected on days 2, 16, 30, 44, and 58 from each pen (day l was the first day an egg was laid in each individual pen). In the bobwhite study, the 7th, 14th, and let egg laid from each pen was collected. Every egg was marked with collection date and pen number. The eggs were cracked open at the girth, contents removed, and shells washed with tap water to remove the albumen. The shells were left to air dry on paper at room temperature for at least 48 hours. Eggshell thickness was measured using an Ames Thickness Measure (Model 25MB). The shell thickness (including shell membranes) was measured to the nearest 0.01 mm at four approximately equidistant points around the circumference of the shell. An average shell thickness was calculated using these four data points. Statistical Analysis Treatment means of the following parameters were compared using Dunnett's two-tailed "t"-test; average number eggs laid/hen/day, X egg fertile, 2 eggs hatched, Z hatchling survival and average eggshell thickness (Gill, 1978). Sample units were the individual pens within each experimental group. Adjustments were made in survivability when live embryos or hatchlings were determined to have suffered an accidental death unrelated to the nature of the study. Prior to "t"-test for the mallard study, which conducting Dunnett's two-tailed was conducted using two separate rooms, a Student's "t"-test was used to compare room treatment means within a parameter to determine if room diffences existed (Gill, 1978). 22 Pathology All animals were necropsied following death. The necropsy was a systematic examination of the animal's general physical condition, body orifices, external and internal organs and tissues. The following organs and tissues were collected from at least five mallards and five bobwhite of each sex from each dose, placed in a fixative and processed for histopathologic evaluation: kidney, liver, heart, lung, spleen, thymus, pancreas, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, colon, ovary, magnum, shell gland, testis, skin, pectoral muscle and leg muscle. Selected tissues were also collected from birds with grossly observed lesions, but no tissues were collected from the remaining grossly normal birds. Histologic preparations of the tissue specimens collected at necropsy were examined microscopically by a certified veterinary pathologist, with experience in evaluating avian tissues, at the Lilly Research Laboratories, Greenfield, Indiana. Particular attention was directed to the identification of treatment-related lesions. RESULTS ‘Rggm,Variation-Mallard Study There were no differences in the means (Student t, P1< 0.05) between Rooms 4A and 4B in the mallard study for the reproductive parameters measured. Thus, control and treatment group data from Rooms 4A and 4B were combined for comparison by Dunnett's "t"-test. Eggg.Consumption There was no apparent effect in either study on food consumption between the birds in the control groups and the birds fed dietary concentrations of tricyclazole (Tables 1 and 2). The biweekly measurements varied, but this variation was consistent across the control and treatment groups. Food consumption plateaued until the two-week period prior to the increase in photoperiod. During this two week period, the control and treatment groups uniformly dropped in food consumption. Following the increase in photoperiod the mallards' food consumption returned to the earlier levels and continued to increase as reproduction commenced. However, the bobwhite maintained this decreased food consumption for an additional two week period after- which they increased to levels similar to those prior to the decrease. The bobwhite then continued increasing food consumption throughout the duration of the study, unlike the mallards which experienced a decline in food consumption during the final two-week period of the production phase. Mallard food consumption decline paralleled the decline in mallard egg production during the same final two-week period. 23 24 .ummmmcue— newsmaouoga m .AauAetAAAAEMLa A .m u z A A. «A m. a a a e m A . o. A A e. . um AAA AAA mAN AOA Am. CA. mm mm. mm_. AAA AAA «a .m pamm com «A AA AA «A a A AA a. AA A m . m mm mm. AAA sou AA. AA. oAA ma am. As. oAA me. Am .A pumam 8-. AA A. m. GA o. a a A o. AA A m um SAA. as. as. mm. Ae— AAA mA AmA omA AAA me— sa .m . pawn on m. «A AA A a m A m a 4A m m mm. mop ¢o~ mm. mm. mm. mm. AA mm. Am. on. mm. 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There was no apparent effect on mean body weights in either study between birds in the control groups and the birds fed dietary concentrations of tricyclazole (Tables 3 and 4). Mean body weights increased slightly in all treatment groups prior to the onset of the production phase, however, a slight decrease occurred during the two- week period prior to the increase in the photoperiod. This decrease in mean body weights corresponded to the deCrease in food consumption during the same period. At the termination of the studies, mean body weights were slightly greater than the pre-egg production levels. Reproduction Chronic dietary administration of tricyclazole had no effect on the reproduction in mallards and bobwhite (Tables 5 and 6). There were no significant differences between the means of the control groups and the means from the treatment groups in the following para- meters: number of eggs/hen/day, mean eggshell thickness, mean 2 fertility, mean 2 hatchability, and mean Z survivability. Also, tricyclazole in the diets of mallard and bobwhite breeders had no effect on eggshell thickness of either species. Clinical Observations No clinical signs could be attributed to feeding dietary concentrations of tricyclazole to either mallards or bobwhite. In mallards, the clinical signs observed were the result of increasing the photoperiod which resulted in excessive aggression or treading. The limping observed in some birds could also be attributed to injuries caused by the pen flooring. Hyperpnea was observed in one female 27 42.9.3 mm H R A .AA u z A .A u z m eozuoezerA e .comoocuc. cOAAoavogs m .mczmoa acoEAAoAA N .mczmoa newton cowuos..uu< . AA A SA. .A .+. 8: AA A 8A. A... A AA: AA .+. AA. 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AA.8 AHAS AHAS A:A$A A A AA. A A. AA. A .+. AAA A H AA. A A .AA A H AAA A H .AA A H AAA A goo: WAS. AHAE AHAE THE. Tug. AHAE AHAE AHA: A88: VHS. AHAE AHAB 7H2. THE. AHAS AHAE AHA: AASA VHS. AuAfl AHAE Tfla. THE. AHAE AHAE AHAE AASA AME. AHAS AHA2 THE. THE. AHAE AHAE ANAS AAASA A+A2 A+fi. A+2A AAA: A+A2 A+:. A+£. AA+8. 3 fig PA .5: 8m PF baa: RF A... PA. sun on m: ELAEucob h! xom .o>o. AgoquA .A.AAA.AAA.AA AAA AA.A;AAA A.=AA AA AAAAAA: AAAA .A A.AA. 29 oo.c A m N oA mo.c mm mm.c AA AA AA AA Am.c .m Am:m com Ac.c N A A A mo.o mm mm.o mm on AA AA Am.o .m Ammm co. 8A A A . A .AA.A AA mm.c AA AA AA om mm.o .m Aamm an oo.o N m N A Ao.o um mm.o mm AA AA om om.o .x AAAAAAou Aesv , AAAAAAA> ‘ AAAAAAA ongaso AuA>Auoo Aucxcon Ao>oA AmoezuAgp -A>A=A -gouo: o>AA aeoocoa \mmu Agoavo AAogmmmo ocoocom acoquA aeougoa acousoA (.mucoAAos AA.AcoaosAAAA.o>Auo=voAaoA :o voquoqusa cheoou Ac AquAm .m oAnoA 30 .AoAAagoA o omsouon ongneo o>AA c .o.Av :oAquAAAAoscAAu Ac AAAAoA A AA :oAuocAeAAo on ose AAo>oA AgoquN :AgaAz AoAuooAAaoA Anacoea "NAN: NA NA. NA NA NA NA A oc.o A N A m Ao.o um oN.o AA AA NA AN mm.c .x 5mm com A. AA NA NA NA N. A No.o m N o N NN.N mm AN.o AN AA AN AN NA.o .w mum ooA NA NA AA NA NA NA = co.c N A A A cm.o NA AN.o NA NA AN AN NA.o .x 3mm cm NA NA NA NA NA AA A oo.o A A A A Ao.o mm oN.c NN NA NA NN Am.c .x Accucou Asev AAAAAAA> AAA—AAA onsnso AAAAAquA AANAAo: Ao>oA AmoczuAgp uA>AAA -zuuos o>AA acoucoA \NNN AAAAoAN AAoAANNo acousoa acougom ueougoA Acousog . .ouAgznoA AA Acouoeogoq.o>Auu:quAoA co oAAAAAAAAAAA NcAvooA AA uquAN .N oAAAA 31 mallard in the control group, but she returned to normal within one day. Also, one female in the 0.032 treatment group suffered a prolapsed oviduct (Table 7). I In the bobwhite, the clinical signs observed were feather loss and head and leg lacerations. These lesions appeared to be caused by the wire caging, however, feather loss could have been the result of excessive treading. Intest Mortality Six female mallards died during the course of the study (Table 8). No mortalities occurred prior to increasing the photoperiod. Two females from the 30 ppm treatment level and 3 females from the 100 ppm treatment level died as a result of excessive treading or aggression between females. One female from the control group died from bacteriemia. In the bobwhite study, only one male, from the 300 ppm treatment group, died during the course of the study. The cause of death was not apparent and histopathology showed no substantive tissue alteration. No mortality could be attributed to the chronic admini— stration of dietary tricyclazole to either mallards or bobwhite. Pathology Pathological findings provided by the Eli Lilly Company are provided in the Appendix (Appendices S and T). Alterations that were present included gross and microscopic lesions of nodular and ulcerative dermatitis present on the feet and heads of some control and treated bobwhite. A few mild inflammatory and degenerative lesions were observed in ducks that died on test as well as in ducks killed at the termination of the study. Some birds experienced a 32 Table 72 Clinical observations of mallards fed tricyclazole. Observation Cause Feather loss Aggression Weight loss, lameness Injured leg Lameness . Injured leg Ataxia, weight loss Injured leg Hyperpnea (dose 0.00%) Unknown Lameness Injured foot Prolapse Parturition 33 Table 8. Interim deaths Of Mallards fed tricyclazole Pen #/Dietary Level Bird 1 Sex Date of Death Cause of Deathl 20 / ' 0.00% 23 9 ‘ 2-19-81 Bacteriemia 16 / 0.003% 70 9 1- 9-81 Trauma, Exces- . sive treading 19 / 0.003% 64 9 2-16-81 " 17 / 0.01% ’ 93 9 2-19-81 " 23 / 0.01% 75 9 1-24-81 " 23 / 0.01% ' 51 9 2—27-81 " 1? I Diagnosis provided by Lilly Research Laboratories. 34 slight degree of amyloidosis, however, this condition and the other pathological alterations that occurred were not considered to be related to the compound tricyclazole. DISCUSSION The Environmental Protection Agency (EPA) (1978) suggests, for mallard reproduction studies, that a minimum of 2 males and 5 females per pen replicated by four or more pens be used per treatment group. A minimum of ten pens per treatment group should be used if only one male and one female are to be used. The EPA (1978) suggests a minimum of a 12 pen replication for bobwhite studies, if one male and two females per pen are used. If only one male and one female per pen are used, then additional pens should be utilized. In mallard chronic reproduction studies where there was a 2 male:5 female ratio per pen, a majority of the deaths that occurred were due to trauma associated with excessive aggression and rape (Jones, 1977; Breslin, 1981; Flaga, 1981). In an effort to reduce this cause of death, the present study used only one male and three females per pen. As a result, there was a reduction in number of interim deaths. Only six female mallards died during the study and of these, five were attributed to excessive treading, rape, and aggression between females. The absence of male deaths in this study can be attributed to the lack of male — male aggression. Single males in each pen were unable to physically express their aggressive breeding behavior toward other males due to cage separation. In bobwhite chronic reproduction studies with similar design to the present study (1 male - 1 female per cage with a replication of 15), there was a comparatively higher mortality rate than in the present study in which only one male bobwhite died from the 300 ppm treatment group (Breslin, 1981; Flaga, 1981; Howell, 1981). Bobwhite 35 36 mortality, in all studies mentioned, were not attributed to any common cause and none were attributed to the test compounds. Of the deaths that were not attributed to aggression, one control female mallard died of bacteriemia. The death of the single male bobwhite was due to unknown causes. Histopathology revealed no sub- stantive tissue alteration and death was not considered to be treat- ment related. Bobwhite food consumption was similar to consumption rates seen by Breslin (1981), Flaga (1981), and Howell (1981). There was a continuous increase in food consumption throughout the entire study period. There was a slight decrease and then plateau in consumption . during the.2 two-week periods prior to and following the increase in the photoperiod. After this, food consumption returned to normal levels and then continued to increase throughout the remainder of the study. Mallard food consumption continuously increased from the onset of the study until the two-week period prior to the increase in the photoperiod, when a decrease in consumption occurred. A decrease in consumption would not be expected until after the increase in the photoperiod when hormonal flucuations stimulating reproduction would also result in increased aggressiveness which is characteristic of reproductively active birds (Sturkie, 1976; Welty, 1979). Decreased food consumption following an increased photoperiod was observed by Breslin (1981), Flaga (1981), and Jones (1977). Following the decline in food consumption during the two-week period prior to the increase in photoperiod, there was an increase throughout the remainder of the study. During the final two—week period, a slight decline in food 37 consumption again occurred. This decline paralleled a similar decline in egg production for the same period, which itself corresponds to normal decline in reproductive Performance following prolonged repro— ductive activity (Ringer, 1980). A breeding season for mallards normally lasts ten weeks (EPA, 1978) therefore the decline in food consumption and egg production was expected during the final weeks of ,the present study, especially since the study had a 12 week breeding season. Dietary tricyclazole did not adversely affect food consump- tion in either mallards or bobwhite. Body weight changes generally parallelled the fluctuations in food consumption. Body weights consistently increased during the pre- production phase until food consumption decreased at the time of photoperiod increase. Mallards experienced a decline in body weight in the two weeks prior to the increase in the photoperiod, while bobwhite showed an insignificant decline after the change in the photoperiod. The flucuations in body weights corresponded well with food consumption characteristics. This pattern of body weight fluc- tuations was also seen by Breslin (1981), Flaga (1981), Howell (1981), and Jones (1977). _ During the production phase, the birds were not weighed until termination in an effort to eliminate stress on reproduction due to handling. Thus, trends in body weights could not be thoroughly docu- mented during this phase of the Study, but it can be assumed that body weights continued to increase (perhaps at decreased rates), possibly with a slight decrease when food consumption declined during the final two-week period of the production phase. This decline occurred only with the mallards, Bobwhite continued to increase food consumption up 38 to termination of the study. A decline in mallard body weights did not neccessarily occur during the final weeks of the studies even though mallard food consumption declined. During this same period, reproductive activity was declining, allowing a higher percentage of energy intake to be utilized for body maintenanCe and growth. Chronic administration of tricyclazole in the diet of mallards and bobwhite did not affect body weight gain in either species. Normal values for mallard and bobwhite egg production during a ten week breeding season are 28 to 38 eggs per female (EPA, 1978). Egg production in the present studies agree well with the expected values, with a range of 45 to 48 (12 week production period) and 34 to 37 eggs per female for mallards and bobwhite, respectively. These values are similar to those obtained by Flaga (1981), Howell (1981), land Jones (1977). Bobwhite values are similar to those of Breslin (1981), but mallard values tended to be greater in the present study. The range of values for eggs cracked are 2.5 (0.03 ppm) to 4.0% (0.0032) for mallards and 7.0 (0.01 ppm) to 13.5% (control) for bobwhite. The values for the mallards are in good agreement with expected values (EPA, 1978), but are lower than those reported by Jones (1977). Bobwhite values are much greater than expected (EPA, 1978), with the control group having the highest percentage of cracked eggs. However, these values for cracked eggs in bobwhite are only slightly greater than those of Howell (1981). These extreme values are not considered to be dose related and may have been a result of cage design. The floor of the cages were slanted to allow the eggs to roll freely into a collection area, thus facilitating collection and preventing damage by the adult birds. Some floors developed upward 39 bows that sometimes prevented eggs from rolling into the collection area or trapped the eggs between the front wall of the cage and the floor itself. The eggs could then be trampled by the adult birds or cracked by the cage due to the slight springing action of the cage floor during bird activity. . Eggshell thickness for bobwhite correspond well with the expected values listed by the EPA (1978), ranging from 0.20 to 0.21 mm. Bobwhite eggshell thickness in this study was similar to those of Howell (1981), but tend to be slightly less than those of Flaga (1981). In contrast, mallard eggshell thickness, for all treatment levels, averaged 0.39 mm, which is greater than expected (EPA, 1978), but are similar to Jones (1977). Breslin (1981) and Flaga (1981) both obtained greater values for shell thickness in mallards than the present study. Values for the reproductive parameters; fertility, embryo survival (days 18 and 21 of incubation for bobwhite and mallards, respectively), and hatchability are within the normal ranges expected (EPA, 1978) for the bobwhite. The bobwhite did have lower embryo survival in the 30 and 300 ppm treatment levels. The mallards, however, have values below the normal range for fertility and embryo survival, with hatchability being low but still within the expected range. Studies by Breslin (1981) and Flaga (1981) reported lower values in fertility, embryo survival, and hatchability for bobwhite while Howell (1981) reported similar hatchability but greater fertility than did the present study. For the same reproductive parameters in mallards, Breslin (1981), Flaga (1981), and Jones (1977) 40 reported higher values than did this study, except Breslin (1981) reported a decrease in embryo survival. Though the results for the reproductive parameters discussed exhibited a tendency for values below what is to be expected, none of the treatment groups, from either the mallard or the bobwhite studies, differed significantly from their respective controls for any of the reproductive parameters evaluated. I Survivability of mallard and bobwhite hatchlings were within the normal range (EPA, 1978). Breslin (1981) and Flaga (1981) experienced high mortality rates in their studies and suspect it was a result of exposure to cold environmental temperatures. When young animals be— come chilled, the lack of ability to adequately control body temp- erature through thermoregulation greatly decreases the chance of sur- vival. To prevent inadequate thermoregulation, attempts were initiated to avoid adverse environmental conditions. Bobwhite hatchlings were reared in the same building they were hatched in and mallard hatchlings were transported to their respective brooding batteries in prewarmed vehicles only. These precautions seemed suc- cessful as survival rates were much greater than those of Breslin (1981) and Flaga (1981). Pathologically, no mortality or lesion in either the mallard or the bobwhite studies could be attributed to the fungicide, tricyclazole. Mortality, feather loss, dermititis, and other lesions about the head and feet were considered due to aggression, rape, and caging. The single case of bacteriemia suffered by a control female mallard was due to the presence of microorganisms or their poisonous products in the blood stream (Thomas, 1981). Though some birds 41 experienced a slight degree of amyloidosis the condition was not considered to be dose related. CONCLUSION Chronic dietary administration of tricyclazole did not affect the food consumption or body weight gain of adult mallards or bobwhite. Similarly, tricyclazole did not adversely influence the manifestation of degenerative alterations in tissue or organ morphology, nor did it increase mortality in either species. Evaluation of the reproductive parameters of egg production, fertility, embryo survival, hatchability, hatchling survival, and eggshell thickness revealed no negative relationship between dietary tricyclazole exposure and the a forementioned parameters. In conclusion, chronic dietary administration of tricyclazole did not result in toxicity to mallards or bobwhite at concentrations up to 300 ppm, the highest level tested, which is a 60 fold increase over the maximum environmental residue value of 5 ppm in rough rice. 42 APPENDICES 43 Appendix A. Solubility of tricyclazole in organic solvents]. Solvent Solubility (mg/ml)2 Chloroform >500.0 Methylene chloride 33.0 Ethyl alcohol 25.0 Methyl alcohol ’ 25.0 Acetone 10.4 Acetonitrile 10.4 Cyclohexanone 10.0 Benzene 4.2 Xylene . 2.1 Water 0. 7 Hexane <0.l ‘ Courtesy of Eli Lilly and Co., Greenfield, IN, 1931. ' 2 25°c. 44 Appendix B. Toxicology of Tricyclazole1 Species ' Sex LDo2 . LDso2 Albino ICR mouse (Mus musculus) M,F 245 Albino Wistar rat (Rattus noruegjcus) M,F 314 Beagle dog (Canis familiaris) M,F > so Bobwhite (Colinus virginianus) M,F >2000 Mallard (Anas platyrhynchos) M,F > 100 1Courtesy of Eli Lilly and CO., Greenfield, Indiana 1981. 2flu/kg. 45 .Awm— .Ncwwtcm .Upwv$comsw ..ou tam AAAAA rpm mo ammugaoup AAAAN NV quc A. AEAA NNNNAV AN.vomoz Amogocxdcxqum Aec AscAAoNv zuAgo AAozc oAAgznoN AA: Nev 5AA e.cN omuu Aocmoe AAAAQANV uAccamo .AAAA A.A ..0A A. AAA AA. eAeA AAAAAA oz AA cm AAA AAA LSEA... AA AA AAA AAA tAA..AAe AAv AA. AA cm .1: AAA L.323... AAv A.. AA om AAA AAA LAA..AAe AAv A.A AA om ALA AAA LSEA... A.A. AA om AAA AAA L3:3... A.A AA AAA AAA LSEA... A.A. AA AA cm om om U: u: u: ALA.AAA L52.3... A.A 1.1; AAA LAAAAAAA A.A. AAA AAA LA...AAe A.A. AA AAA AAA eSEA... A.A. AA AAAAquAAou AsoAAAesAv Eeozgueom AAAAAAAA AouoocAAAAuv euro oaAN .AA.A..AAA AAAA AAAA LAAAA.A AAAAAANAA> AoAAoAAAeuv AooAAo :AoAAAN Asscmeoau Azoocoav AeAeeA zAAA AAoAAAAA AAANAANV ozmuosAz AAAAAmoAAA> coeocAeaxuv soccAe cmogAAoosm Amauauocam AAAAAAAAAV AAAAAAA Aoeeozu AAcoachAm oeAANV «sore roacAAz AAAAAgquer AAEAQNAV :AAAAAA AAANoAAN AAAAAAAA AAAAAAAANV :AAANAON AoAmqu AAAAAmme ALAN if I voququAeA Ac AuAquoA AoueoseoeA>cm .N xAucoAA< 46 1 Appendix D. Hazard Evaluation of Tricyclazole Species Rate of Exposure Dose Results Rat Oral -- L050 = 354 mg/kg2 Rat Inbaiation2 3.71 mg/liter No toxicity Inhalation3 0.146 mg/liter No toxicity Rabbit ~Dermalz’3 2 g/kg No irritation or systemic toxicity Rabbit Ocuiar2 41 mg Corneal dullness, mild iritis and slight conjuncti- vitis which cleared in 96 hr Ocular3 78 mg Slight iritis and conjuncti- vitis which cleared in 72 hr Guinea Pig Dermal No sensitization produced during 5 week challenge 1 Courtesy of Eli Lilly and Co., Greenfield, Indiana 1981. 2Beam(R) (tricyclazole formulated as a 75% wettable powder) 3Technical tricyclazole. 47 Appendix E. Subacute and chronic toxicity of tricyclazole]. No-effect Species Route Duration level Dog Oral 3 months 7.5 mg/kg/day Chicken Diet 1 month 600 ppm Mouse Diet 2 years 400 ppm Rat Diet 2 years 275 ppm 1 Courtesy of Eli Lilly and Co., Greenfield, IN, 198l. 48 Appendix F. Nutrient Requirements of Mallards (in Percentage or Amount Per Kilogram of Feed) Nutrient Growing Breeding Ducks Ducks Metabolizable energy (kcal/kg) 2,900 2,900 Protein (%) l6 l5 Lysine (%) 0.9 0.7 Methionine + cystine (%) 0.8 0.55 Vitamin A (m) 4,000' 4,000 Vitamin D (ICU) 220 500 Riboflavin_(mg) f 4 4 Pantothenic acid (mg) ll l0 Niacin (mg) - 55 » 40 Pyridoxine (mg) 2.6 3 Calcium (%) 0.6. 2.75 Phosphorus (%) 0.6 0.6 Sodium (%) 0.15 0.15 Manganese (mg) 40 25 Magnesium (mg) 500 500 From: Nutrient Requirements of Pofiltry.*1977. 49 Appendix G. Composition of Duck Breeder-Layer Diet Ingredients Percent lbs/ton Corn, yellow ground 62.42 l248.4 Soybean meal, solvent extracted, dehulled (49%) l9.58 391.6 Fish meal 2.00 40.0 Meat and bone meal 4.00 80.0 Oat groats, rolled 2.50 50.0 Beef tallow 0.93 l8.6 Dicalcium phosphate, feed grade 0.65 l3.0 Limestone 6.82 l36.4 Trace mineral premix TK-Ol (1.02)1 0.10 2.0 Salt 0.30 6.0 Vitamin premix TK-Ol (1.03)2 0.50 10.0 Methionine hydroxy analog 0.20 4 0 T0070’ ‘200630 1Trace mineral premix provides 75 mg of manganese, 50 mg 0f ZINC: 25 mg 0f iron and 1 mg of iodine per kg of complete feed. Vitain premix provides 3000 IU of vitamin A, 900 ICU of vitamin D, 40 mg of vitamin E, 0.7 mg of vitamin K, 1000 mg of choline, 70 mg of niacin, 4 mg of pantothenic acid, 4 mg of riboflavin, 100 mcg of vitamin 812, l00 mcg of biotin, and l25 mg of ethoxyquin per kg of complete feed. 2 50 Appendix H. Calculated Analysis of Duck Breeder-Layer Diet Protein, % 18.50 Met. Energy, Kcal/kg. 2893 ME/P Ratio 156.4 (71.1) Fat, % 3.80 Fiber, % 2.58 Ash, % , V 10.97 Calcium, % 3.25 Phosphorus, % 0.66 Available Phosphorus, % 0.46 Manganese, mg/kg 89.0 (Iron, mg/kg 105.7 Copper, mg/kg 9.7 Zinc, mg/kg 76.6 Selenium, mcg/kg 107 Magnesium, mg/kg 2172 Potassium, mg/kg 6706 Sodium, mg/kg 1321 Iodine, mg/kg 1 Vitamin A, IU/kg 5060 Vitamin D, ICU/kg 900 Vitamin K,mg/kg Choline, mg/kg Niacin, mg/kg Pantothenic acid,mg/kg Vitamin 85, mg/kg Riboflavin, mg/kg Thiamine, mg/kg Folic acid, mcg/kg Vitamin Biz, mcg/kg Biotin, mcg/kg Arginine, % Lysine, % Glycine, % Methionine, % Cystine, % Total sulfur amino acids TryptOphan, % Linoleic acid, % Vitamin E, mg/kg 1804 85. 10. 1082 107 228 1 56. £00100“) 0 C 0-0 .29(6.97) .97(5.24) .10(5.94) .48(2.59) .26(1.41) .74(4.00) .23(1.24) .24 2 values in parenthésis represent the aminb aCids expressed as a % of dietary protein. 51 Appendix I. Composition of duck starter diet. Ingredients . Percent lbs./ton Corn, yellow ground 60.43 1208.6 Soybean meal, solvent extracted, dehulled (49%) 27.09 541.8 Fish meal 2.00 40.0 Meat and bone meal 4.00 80.0 Oat groats, rolled 2.50 50.0 Beef tallow 1.90 38.0 Dicalcium phosphate, féed grade 0.64 12.8 Limestone 0.34 6.8 Trace mineral premix TK-O (1.02)1 0.10 2.0 Salt 0.30 6.0 Vitamin premix TK-Ol (1.03)2 0.50 10.0 . Methionine hydroxy analog _0_._2_0_ 4.0 100.00 2000.0 1 Trace mineral premix provides 75 mg of manganese, 50 mg of zinc, 25 mg of iron, and 1 mg of iodine per kg of complete feed. 2 Vitamin premix provides 3000 IU of vitamin A, 900 ICU of vitamin D, 40 mg of vitamin E, 0.7 mg of vitamin K, 1000 mg of choline, 70 mg of niacin, 4 mg of pantothenic acid, 4 mg of riboflavin, 100 mcg of vitamin 312. 100 mcg of biotin, and 125 mg of ethoxyquin per kg of complete feed. 52 Appendix J. Calculated analysis of duck starter diet. Protein, % 22.01 Vitamin K, mg/kg 0.7 Met. energy, Kcal/kg 3079 Choline, mg/kg 1932 ME/P ratio 139.9 (63.6) Niacin, mg/kg 89.7 Fat, 1 ' 4.74 Pantothenic acid, Fiber, 2 2,74 mg/kg 11.8 Ash, % 5.19 Vitamin 85, mg/kg 7.5 Calcium, % 0.80 Riboflavin, mg/kg 5.8 Phosphorus, % 0.70 Thiamine, m9/k9 2.8 Avail. phosphorus, % 0.46 Folic acid, mcg/kg 1345 Manganese, mg/kg 37,0 Vitamin B12. meg/kg 107 Iron, mg/kg 99,7 31°t109 mcg/kg 250 Copper, mg/kg 12,3 Arsinine. 51 1.56 (7.09) Zinc, mg/kg 73.9 Lysine, % 1.21 (5.50) Selenium, mcg/kg 112 Glycine, % 1-30 (5-91) Magnesium, mg/kg 2149 Mathi°"1"e’ % 0-53 (2-41) Potassium, mg/kg 8120 Cystine, % 0.32 (1.45) S°d1"m’ mglkg 182° T°§Zia§“'f“' amino 0.85 (3.86) I°di"e’ mg/kg ‘ Tryptophan, % 0.29 (1.32) Vitamin A” IU/kg 4994 Linoleic acid, 5 1.22 Vitamin D, ICU/k9 900 Vitamin E, mg/kg 56.1 1 Values in parenthesis represent the amino acids expressed as a Z of dietary protein. Appendix K. Nutrient requirements of bobwhite (in percentage or amount per kilogram of feed). Nutrient Growing Breeding Metabolizable energy (kcal/kg) 2,800 2,800 Protein (%) 28 24 Lysine (1) 1.4 0.7 Methionine + cystine (z) 0.9 0.6 Glycine + serine (%) 1.6 0.9 Vitamin A (IU) 3,000 3,000 Vitamin D (100) 900 ,900 Riboflavin (mg) 3.8 4.0 Pantothenic acid (mg) 12.6 15 Niacin (mg) 31 20 Choline (mg) 1,500 1,000 Linoleic acid (2) 1.0 1.0 Calcium (Z) 0.65 2.3 Chlorine (Z) 0.11 0.15 Phosphorus (%) 0.65 1.0 Sodium (%) 0.085 0.15 Iodine (mg) 0.30 0.30 Magnesium (mg) 600 400 Manganese (mg) 90 70 Zinc (mg) 50 50 FROM: Nutrient Requirements of Poultry (1977). 54 Appendix L. Composition of quail breeder-layer diet. Ingredients Percent 1bs./750 Corn, yellow ground 49.37 370.28 Soybean meal, solvent extracted dehulled (49%) 35.67 267.52 Fish meal, Menhaden 2.00 15.00 Corn distillers dried solubles 4.00 30.00 Beef tallow 1.00 7.50 Dicalcium phosphate, feed grade 2.88 21.60 Calcium carbonate (limestone) 3.88 29.10 Vitamin premix TK-Ol (1.03)1 0.50 3.75 Trace mineral premix TK-Ol (1.02)2 0.10 0.75 Salt . 0.30 2.25 Methionine hydroxy analog 0.30 2.25 100.00 ' 750.00 ‘ Vitamin premix provides 3000 IU of vitamin A, 900 ICU of vitamin 0, 40 mg of vitamin E, 0.7 mg of vitamin K, 1000 mg of choline, 70 mg of niacin, 4 mg of pantothenic acid, 4 mg of riboflavin, 100 mcg of of vitamin B12, 100 mcg of biotin, and 125 mg of ethoxyquin per kg of complete feed. 2 Trace mineral premix provides 75 mg of manganese, 50 mg of zinc, 25 mg of iron, and 1 mg of iodine per kg of complete feed.‘ Appendix M. Calculated analysis of quail breeder-layer diet. Protein, % Met. energy, Kcal/kg ME/P ratio Fat, 1 Fiber, % Ash, % Calcium, % Phosphorus, % Avail. phosphorus, % Manganese, mg/kg Iron, mg/kg Conner. m9/k9 Zinc, mg/kg Selenium, mcg/kg Magnesium, mg/kg Potassium, % Sodium, mg/kg Iodine, mg/kg Vitamin A, IU/kg Vitamin D, ICU/kg 24.00 2803 9 2 1 93 116 18 84 121 2104 0 1626 1 4929 900 116379 (53.09) 3. 2. .80 .30 .00 0. 32 68 72 .95 Vitamin K, mg/kg 0.7 Choline, mg/kg 2139 Niacin, mg/kg 97 Pantothenic acid, mg/kg 12.6 Vitamin 85, mg/kg 7.5 Riboflavin, mg/kg 6.2 Thiamine, mg/kg 2.7 Folic acid, mg/kg 1.6 Vitamin Biz, meg/kg 103 Biotin, mcg/kg 301 Arginine, % 1.717 (7.15)1 Lysine, % 1.380 (5.75) Glycine, 2 1.248 (5.20) Methionine, % 0.629 (2.62) Cystine, % 0.353 (1.47) Total sulfur amino acids, % 0.982 (4.09) Tryptophan, % 0.337 (1.40) Linoleic acid, % 1.22 Vitamin E, mg/kg 55.2 1 dietary protein. Values in parenthesis represent the amino acids expressed as a % of 56 Appendix N. Composition of quail starter. Ingredients' Percent - lbs./750 Corn, yellow ground 46.52 348.90 Soybean meal, solvent extracted dehulled (49%) 41.26 309.45 Fish meal, Menhaden - 4.00 30.00 Corn distillers dried solubles 4.00 30.00 - Dried whey 2.00 15.00 Dicalcium phosphate, feed grade 0.39 2.92 Calcium carbonate (limestone) 0.73 5.48 Vitamin premix TK-Ol (1.03)1 0.50 3.75 Trace mineral premix TK-Ol (1.02)2 0.10 0.75 Salt 0.20 1.50 Methionine hydroxy analog 0.30 2.25 100.00 100.00 ‘ Vitamin premix provides 3000 IU of vitamin A, 900 ICU of vitamin 0, 40 mg of vitamin E, 0.7 mg of vitamin K, 1000 mg of choline, 70 mg of niacin, 4 mg of pantothenic acid, 4 mg of riboflavin, 100 mcg of vitamin 812, 100 mcg of biotin, and 125 mg of ethoxyquin per kg of complete feed. 2 Trace mineral premix provides 75 mg of manganese, 50 mg of zinc, 25 mg of iron, and 1 mg of iodine per kg of complete feed. Appendix 0. Calculated analysis of quail starter diet. 99 .46) .46 .79 .54 .70 .65 .34 .13 Protein, % 27. Met. energy, Kcal/kg 2861 ME/P ratio 102.22 (46 Fat, % 2 Fiber, % 2 Ash, % 5 Calcium, % 0 Phosphorus, % 0 Avail. phosphorus, % O Manganese, mg/kg 95 Iron, mg/kg 123 Copper, mg/kg 20 Zinc, mg/kg 89 Selenium, mcg/kg 168 Magnesium, mg/kg 2146 Potassium, z 1 Sodium, mg/kg 1629. Iodine, mg/kg 1 Vitamin A, IU/kg 4535 Vitamin D, ICU/kg 900 Vitamin K, mg/kg Choline, mg/kg Niacin, mg/kg Pantothenic acid, m9/k9 Vitamin B5, mg/kg Riboflavin, mg/kg Thiamine, mg/kg Folic acid, mg/kg Vitamin 812, mcg/kg Biotin, mcg/kg Arginine, % Lysine, % Glycine, % Methionine, % Cystine, % Total sulfur amino acids, % TryptOphan, Z Linoleic acid, Z Vitamin E, mg/kg 2330 102 107 324 1.991 (7 1.675 (5 1.488 (5 0.413 (1 1.124 (4. .60) .17 0.169 (0 1 54. ho ho u: ho to .11) .98) .32) 0.710 (2. .48) 54) 02) 8 1 dietary protein. Values in parenthesis represent the amino acids expressed as a % of 58 Appendix P. Stability and homogenicity of tricyclazole in mallard and bobwhite diets . (Concentration (theoritical) tested: 0.001%) Stability of Tricyclazole §torage Assay result (2) Diet conditions Héek_0; *Neek 1 ‘Week 2 Week 4* Week 8* Duck 25°C 0.0009 .0.0009 0.0009 0.0012 0;0004 Duck 3730 0.0009 0.0009 0.0009 0.0006 0.0004 Quail 25°C 0.0009 0.0009 0.0010 0.0006 0.0006 Quail 37°C 0.0009 0.0010 0.0009 0.0006 0.0003 Homogenicity of Tricyclazole Assay result (2) 73amp1e Diet ’1’ 2* 3* 5 Duck 0.0009 0.0008 0.0010 0.0008 0.0008 Quail 0.0007 0.0007 0.0008 0.0009 1 .0.0008 Courtesy of Eli Lilly and Co., Greenfield, IN, 1981. 59 Appendix Q. Results of assays of diets that contained tricyclazole fed to mallards1. Nominal Tricyclazole activity Date dietary _____—_% of diet 9%:of nominal prepared conc. (%) fresh 4-week fre5h 4-week 9/27/80 0.0 Neg Neg -- -- 0.003 0.003 0.0029 100 97 0.01 0.01 0.01 100 100 0.03 0.028 0.027 93 90 10/23/80 0.0 Neg - Neg -- -- 0.003 0.0027 0.0029 90 97 0.01 0.01 0.0092 100 92 0.03 0.028 0.027 93 90 11/20/80 0.0 Neg Neg -- -- 0.003 0.0023 0.0024 77 80 0.01 0.0096 0.009 96 90 0.03 0.026 0.027 87 90 12/17/80a 0.0 Neg Neg -- -- 0.003 0.0032 0.0024 107 80 0.01 0.011 0.0082 110 82 0.03 0.032 0.026 107 87 1/14/81 0.0 Neg ‘ Neg -- -- 0.003 0.0027 0.0023 90 77 0.01 0.0099 0.0079 99 79 0.03 0.03 0.026 100 87 2/11/813 0.0 Neg Neg -- -- 0.003 0.0035 --- 117 -- 0.01 0.011 --- 110 -- 0.03 0.026 --- 87 -- 1 Courtesy of Eli Lilly and Co., Greenfirld, IN, 1982. 2 Aged sample was 5-weeks. 3 No age diet sample was taken. 60 Appendix R. Results pf assays of diets that contained tricyclazole fed to bobwhite . Nominal ' Tricyclazole activity Date dietary __ Z of diet Y of nominal prepared conc. (%) fresh 4-weék Trash 4-week 9/28/80 0.0 Nega Neg -- -- 0.003 0.0033 0.0029 110 97 0.01 0.01 0.01 100 100 0.03 0.029 0.028 97 93 . 10/23/80 0.0 Neg Neg -- -- 0.003 0.0026 0.0029 87 . 97 0.01 0.0092 0.10 92 100 0.03 0.027 0.027 90 90 11/19/80 0.0 Neg Neg -- -- 0.003 0.0026 0.0026 87 87 0.01 0.009 0.0088 90 88 0.03 0.031 0.030 103 100 12/18/802 0.0 Neg Neg -- -- 0.003 0.0031 0.0029 103 97 0.01 0.0089 0.0012 89 120 0.03 0.028 0.031 93 103 1/15/81 0.0 Neg Neg -- -- 0.003 0.0031 0.0023 103 77 0.01 0.0094 0.0091 94 91 0.03 0.029 0.025 97 83 2/11/813 0.0 Neg --- -- -- 0.003 0.0033 --- 110 -- 0.01 0.01 --- 100 -- 0.03 0.027 --- 90 -- 1 2 3 Sample aged 5-weeks. No. aged sample was taken. Courtesy of Eli Lilly and Co., Greenfield, IN, 1982. 61 .A.AE.AA Ac Aogssz A .A voA A .Axmmz .N .AAAAAAAQ AAo.A .oAAoA mAA.AAAASABA A.AA.A AAAAAAAA "AAA. .2. .A.A..AAAAA ..AA AAA A...A ..A .o AAerAoA . m , N 395 . 25A: . . A.AAAAAAAA u.emumxm A. A. A A. A A. A . AA.AAeoA.A AAAAAA Amosm m>.u:AuAA:A A: ..A:.Esma um...x A A A A A A . A . AA.AAeoA.A oAAA.A A.AoumoAAAE m>.u=mumnzm o: ..A=.sgma um..ox A A . A AA.AAeoA.A msmm.u A.AAAAAAA.E A>Au=AAAA=A o: .ummu co AA.A AA AA A AA A AA AA 0 o n o n o. b AA.A .A.A AAA.A AA.A ..m.o~a.u»u.gu no. AAAA..AE so» Ammozmo.u u.mo.ogaaa AA Agasszm .m x.u:maa< 62 .A.Ae.=m mo AoAEAz .u voo .Axmmz mm .Ao.augzo m N "AA.A .oAAoA "AA-AAAA .AAAAA ".A.A. AAAeroA HAAA. .zA .A.A..AooAA ..AA AAA A...A ..A AA AAoAAAoA . P . . . m A.A.»AEAAA A>.AAAAA.: . . Amosm .A.Auoz A.Am m o. A N. m o. m A AA.AAAAA.A mamm.u Amogm A>PAAAAAAAA o: ..A=.Egma um...x A A m A m A m m AA.AAAAA.A «AAA.» . A.AAAAAAA.E A>.u=AuAAAA A: ..A:.scou um..ox . :o.»mema.a «AAA.» A.AAquAA.E A>AAAAAAAAA o: .umou co AA.A A. A. A.. A. A. A. A. AA. 0 b o n o O o n mo.o .A.A moo.o AA.A ..m.o~a.u>u.gu ump.ou.:znoa so» Ammocmm.v u.mo.oguoa Ac Agasssm .. x.u:oAA< BIBLIOGRAPHY Anonymous, 1982. Rice blast: still a mystery. The Rice J. Fall- Winter Issue, p. 7, l4. Breslin, W.J., 1981. Subacute and chronic dietary toxicity of ethal— fluralin to mallards and bobwhites. M.S. Thesis, Michigan State University. Chrysayi, M., 1976. Effects of tricyclazole on Pyricularia oryzae. M.S. Thesis, University of Maryland. Cochrane, R.L., 1980. Personal Communication. Day, E.W., Jr., J.R. Koons, 0.D. Decker, 1980. Anal. Methods. Pestic. Plant Growth Regul. 11:263—273. Elanco, 1976. Technical report on tricyclazole, an experimental fungicide for the control of rice blast (Piricularia oryzae). Lilly Research Laboratories, a division of Eli Lilly and Co., Indianapolis, Indiana 46206. pp. 4. Elanco, 1981. Technical report on Beamch a systemic fungicide for the control of rice blast (Pyricularia oryzae). Lilly Research Laboratories, a division of Eli Lilly and Co., Indianapolis, Indiana 46206. pp. 4. Environmental Protectionn Agency, July 10, 1978. Federal Register. Flaga, C., 1981. The effect of chronic dietary administration of fluridone on selected reproductive parameters of bobwhites and mallards. M.S. Thesis, Michigan State University. Froyd, J.D., C.J. Paget, L.R. Guse, B.A. Dreikorn, and J.L. Pafford, 1976. Tricyclazole: a new systemic fungicide for control of Pyricularia oryzae on rice. Phytopath. 66:1135-1139. Froyd, J.D., L.R. Guse, and Y. Kushiro, 1978. Methods of applying tricyclazole for control of Pyricularia orzzae on rice. Phytopath. 68:818-822. Gill, J.L., 1978. Design and Analysis 2£_Experiments‘in_thg_Animal gndDMedical Sciences. Vol. 1. Iowa State University Press, Ames, Iowa. pp. 409. 63 64 Hasama, W., T. Tomiku, S. Sato, and E. Yasuynki, 1982. Effect of various new fungicides on rice blast. Kyushu Byogaichu Kenkyukai Ho 28:12-14. Howard, L.C., H.M. Worth, N.V. Owen, S.S. Young, D.M. Morton, and L. Golberg, 1978. A two-year chronic toxicity study with tricyclazole, a rice blasticide, in rats. Toxicol. Appl. Pharm. 45:322. Howard, L.C., N.V. Owen, S.S. Young, D.M. Morton, and L. Goldberg, 1979. A two-year chronic toxicity study of tricyclazole in mice. Toxicol. Appl. Pharm. 48:Al94. Howell, K.S., 1981. Toxicity of dicyclopentadiene and diisopropyl methylphosphonate to the bobwhite quail (Colinus virginiannus). M.S. Thesis, Michigan State University. Hwang, B.K., E.J. Lee, C.S. Park, and K.H. Lee, 1976. The effectiveness of a new systemic fungicide EL—29l for the control of rice blast disease. Kor. J. P1. Prot. 15:57-60. Jones, R.E., Jr., 1977. Toxicity of diisopropyl methylphosphonate and dicyclopentadiene on the mallard (Anas platyrhynchos). M.S. Thesis, Michigan State University. Kubo, Y., K. Suzuki, I. Furusawa, and M. Yamamoto, 1982. Effects of tricyclazole on appressorial pigmentation and penetration from appressoria of Colletotrichum lagenarium. Phytopathology 72:1198—1200. Moriya, M., T. Ohta, K. Watanabe, T. Miyazawa, K. Kato, and Y. Shirasu, 1983. Further mutagenicity studies on pesticides in bacterial reversion assay systems. Mutation Research 116:185-216. National Research Council, 1977. Nutrient Requirements g£_Pou1try. National Academy of Sciences, Washington, D.C. pp. 62. Neto, F.B., P.R. Machado, P.R. Rolim, and D.A. Oliveira, 1981. Action of new fungicides in rice blast (Pyricularia oryzae, Cav.) control. Biologico 47(8):219-222. Pierson, C.L. and L.C. Howard, 1978. Disposition of tricyclazole, a rice blasticide, in the rat. Toxicol. Appl. Pharm. 45:322. Ringer, R.K., 1980. Personal Communication. Ringer, R.K., 1984. Personal Communication. Sakaoka, A., 1982. Control of rice blast by Beam powdery formulation. Noyaku Tsushin 116:41-42. Shiba, Y. and T. Nagata, 1981. The mode of action of tricyclazole in controlling rice blast. Ann. Phytopath. Soc. Japan 47:662—667. 65 Stipanovic, R.D. and M.H. Wheeler, 1980. Accumulation of 3,31-bif1aviolin, a melanin shunt product, by tricyclazole-treated Thielaviopsis basicola. Pest. Biochem. and Physiol. 13:198-201. Sturkie, P.D., 1976. Avian Physiology, 3rd Edition. Springer— Verlag New York Inc., New York. pp. 400. Thomas, C.L., 1981. Taber's Cyclopedic Medical Dictionary. 14th Edition. F.A. Davis Co., Philadelphia. pp. 1818. Tokousbalides, M.C., 1978. Site of inhibition by tricyclazole in the melanin biosynthetic pathway of Verticillium dahliae. Ph.D. Thesis, University of Maryland. Tokousbalides, M.C. and H.D. Sisler, 19783. Effect of tricyclazole on growth and secondary metabolism in Pyricularia oryzae. Pestic. Biochem. and Physiol. 8:26-32. Tokousbalides, M.C. and H.D. Sisler, 1978b. Effects of tricyclazole on Pyricularia oryzae. Proc. Am. Phytopathol. Soc. 4:220. Tokousbalides, M.C. and H.D. Sisler, 1979. Site of inhibition by tricyclazole in the melanin biosynthetic pathway of Verticillium dahliae. Pest. Biochem. and Physiol. 11:64-73. Tsai, W.H., 1977. Control of rice blast by tricyclazole 75WP. Inter. Rice Res. Newsletter 6/77:14. Weber, J.B., 1982. Ionization and absorption-desorption of tricyclazole by soil organic matter, montmorillonite clay and cape tear sandy loam soil. J. Agr. Food Chem. 30(3):584-588. Welty, J.C., 1979. Th; Life g£_Birds. 2nd Edition. Sanders Publishing Co., Philadelphia. pp. 623. Wheeler, M.H. and R.D. Stipanovic, 1979a. Melanin biosynthesis in Thielaviopsis basicola. Experimental Mycology 3:340-350. Wheeler, M.H. and R.D. Stipanovic, 1979b. The effects of tricyclazole on melanin biosynthesis in Thielaviopsis basicola. Proc. Annu. Meet. Electron Micros. Soc. Am. 37:268-269. Wheeler, M.H., 1982. Melanin biosynthesis in Verticillium dahliae: dehydration and reduction reactions in cell-free homogenates. Experimental Mycology 6:171—179. _Woloshuk, C.P., H.D. Sisler, M.C. Tokousbalides and S.R. Dutky, 1980. Melanin biosynthesis in Pyricularia oryzae: site of tricyclazole inhibition and pathogenicity of melanin-deficient mutants. Pest. Biochem. and Physiol. 14:256-264. 66 Woloshuk, C.P., P.M. Wolkow and H.D. Sisler, 1981. Effects of three fungicides specific for control of rice blast disease on growth and melanin biosynthesis by Pyricularia oryzae, Cav. Pesticide Sci. 12:86-90. Woloshuk, C.P. and H.D. Sisler, 1982. Tricyclazole, pyroguilon, tetrachlorophthalide, PCBA, coumarin and related compounds inhibit melanization and epidermal penetration by Pyricularia oryzae. J. Pest. Sci. 7:161-166. Woloshuk, C.P., H.D. Sisler and E.L. Virgil, 1983. Action of anti- penetrant tricyclazole on appressoria of Pyricularia oryzae. Physiol. Pl. Path. 22:245-259. Yamaguchi, I., S. Sekido and T. Misato, 1982. The effect of non- fungicidal anti-blast chemicals on the melanin biosynthesis and infection by Pyricularia oryzae. J. Pestic. Sci. 7(4): 523-530. Yang, Y.Z., 1980. Chemical control of rice panicle blast by low volume application. Taiwan Ag. Bimonthly l6(5):60—63. HICHIGRN STRTE UN 111111 1111111111 IV. LIBRRRIES l(IllIIU(IIINHIWIWIWI 293 7863700 * I I