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I. .1-0 ‘IJ I‘QIV: {‘5‘ k b I ‘I \ if“! I ‘9‘: ilfo‘ ‘Ml‘th‘o ;. 14 99001.0": w...\ln.~a '5. I. - I! . k1...§... .18"! 1.3:...an- n ,I‘Nu‘hfl? ;....2 .51”. 2. ‘slwf ”an \ ......!2. Y . ‘ -. . rt 0‘ I ll) Ill L L I ll . s > I'I » r f k pf}; .n'pl‘. II ‘ _ . v» ‘P . r-. a); a n This is to certify that the thesis entitled SUBACUTE AND CHRONIC DIETARY TOXICITY 0F ETHALFLURALIN To MALLARDS AND BOBWHITES presented by William J. Breslin has been accepted towards fulfillment of the requirements for M.S. degreein Animal SCience ((Eéihfiiu7ezf4llgl4lgg3/ 77 Major professor Date Max 20, 1982 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution A; MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ___.._‘...A ml-I-IZJEI mil- SUBACUTE AND CHRONIC DIETARY TOXICITY OF ETHALFLURALIN TO MALLARDS AND BOBWHITES by William J. Breslin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1981 ABSTRACT SUBACUTE AND CHRONIC DIETARY TOXICITY OF ETHALFLURALIN TO MALLARDS AND BOBWHITES Ethalfluralin was evaluated for its subacute and chronic reproductive dietary toxicity to mallards (Anas platyrhynchos) and bobwhites (Colinus virginianus). Mallards and bobwhites fed ethalfluralin in the diet at 30, 100, 300, 1,000, 3,000, or 10,000 ppm for two and three week periods, respectively, showed no signs of intoxication, morbidity, increased mortality, or weight loss. Feed con- sumption of mallards fed ethalfluralin up to 10,000 ppm in the diet was not significantly reduced. Bobwhites on 10,000 ppm ethalflurlin diets significantly decreased feed consump- tion one week after the administration of treated feed, but feed consumption returned to normal the following week. Administration of ethalfluralin to mallards and bobwhites at 100, 300, or 1,000 ppm for 28 and 24 week periods, respectively, did not significantly affect behavior, mortality of adults, egg production, fertility, embryo survival, hatchability, egg shell thickness, or duckling and chick survivability. Ethalfluralin did not produce pathological tissue alterations when fed at these concentrations. ACKNOWLEDGEMENTS I would like to thank Dr. Robert K. Ringer, Dr. Richard J. Aulerich, and Dr. Harold Prince for their guidance throughout the duration of my studies. I would also like to thank my parents for their phycho- logical and financial support, and my wife for her love and encouragement. Special thanks go to Ms. Christine Flaga for her techni- cal assistance, and Ms. Kathy Breslin for reading the thesis. ii TABLE OF CONTENTS Page Acknowledgements ........................ . ............ ii List of Tables ....................................... v List of Figures ..... . ........ . ....................... vii Introduction ....... . ................................. 1 Literature Review ... ......... . ....................... 2 Physical and Chemical Properties .................. 3 Efficacy .......................................... 7 Mode of Action .................................... 9 Absorption, Translocation, and Metabolism in Plants .... ........................ ll Persistence . ....... . .............................. 12 Degradation ................ ....................... 13 Leaching and Water Movement ....................... 18 Toxicity ..... ....... .. ............................ l9 Experiment I ......................................... 23 Procedure ......................................... 23 Chronology of Study ............................ 23 Animals ........................................ 23 Diets and Treatment Level ....... .. ....... ...... 24 Facilities ................. . ......... .......... 24 Testing .. ........ . ........................ ..... 25 Statistical Analysis ... ...... . ................. 25 Results .......... ............ . .................... 25 Body Weights .... .......................... ..... 25 Food Consumption ............................... 27 Mortality ...................................... 34 Discussion ........................................ 34 Experiment II . ....................................... 39 Procedure .. ....................................... 39 Chronology of Study . ........................... 39 Animals ......... ..... . ......................... 4O Diets .... ...................................... 40 Facilities ................... .................. 41 Testing ...... ..... . ............................ 42 Egg Collection, Storage, and Incubation ................................... 42 Eggshell Thickness ........... . ................. 44 Statistical Analysis .............. . ..... ....... 45 Results .............. ..... ...... .................. 45 Body Weight . ................................... 45 Feed Consumption .......... ........ . ........ .... 48 Reproduction ....... ........ ... ................. 53 Clinical Signs of Toxicity ........... .......... 60 Mortality ... ................................... 60 iii Terminal Necropsy and Histopathology .................................. 62 Discussion ........................................... 64 Conclusion . .......................................... 68 Literature Cited ........................................ 69 Appendix ................................................ 74 Appendix A. Composition of duck breeder- layer diet ........................................ 74 Appendix B. Nutritional analysis of duck breeder-layer diet ........................... 75 Appendix C. Composition of quail breeder-layer diet ................................ 76 Appendix D. Nutritional analysis of quail breeder-layer diet .......................... 77 Appendix E. Composition of duck starter diet ...................................... 78 Appendix F. Analysis of duck starter diet .............................................. 79 Appendix G. Composition of quail starter ........................................... 80 Appendix H. Nutritional analysis of quail starter ..................................... 81 iv LIST OF TABLES Page Table 1. Chemical and physical properties of the dinitroaniline herbicides ethal- fluralin, trifluralin, and benefin ........ 6 Table 2. Annual grasses and broadleaf weeds for which ethalfluralin provides commer— cially acceptable control ................. 8 Table 3. Acute and subacute toxicity of ethalfluralin and trifluralin ............. 20 Table 4. Mean body weights of mallards on palatability test fed ethalfluralin ....... 26 Table 5. Mean body weights of bobwhites on palatability test fed ethal- fluralin .................................. 28 Table 6. Feed consumption of mallards on palatability test fed ethal- fluralin .................................. 29 Table 7. Feed consumption of bobwhite on palatability test fed ethal- fluralin .................................. 31 Table 8. Mallard and bobwhite dietary composition of ethalfluralin .............. 35 Table 9. Mean body weights of adult mallards fed ethalfluralin ........ ................. 46-47 Table 10. Mean body weights of adult bobwhite fed ethalfluralin ......................... 49-50 Table 11. Feed consumption of adult mallards fed ethalfluralin ......................... 51-52 Table 12. Feed consumption of adult bobwhite fed ethalfluralin ...... . .................. 54-55 Table 13. The effects of feeding ethalfluralin on reproductive parameters in mallards .... 56—57 Table 14. The effects of feeding ethalfluralin on reproductive parameters in bob- white ..................................... 58-59 Table 15. Table 16. Interim deaths for mallards fed ethalfluralin .............................. Interim deaths for bobwhite fed ethalfluralin .............................. vi LIST OF FIGURES Page Figure 1. Structure of 2,6-dinitroani1ine, ethalfluralin, and trifluralin . ............ 5 Figure 2. Degradation pathways of trifluralin ........ 17 Figure 3. Mean feed consumption of bobwhite on palatability test fed ethal- fluralin at various concentrations ......... 33 vii INTRODUCTION Ethalfluralin is a experimental dinitroaniline selective herbicide for the pre-emergent control of certain annual grasses and broadleaf weeds. It was developed by Eli Lilly and Company in the late 1960's as a variation of the sub- stituted 2,6-dinitroaniline herbicide trifluralin. Ethal- fluralin was synthesized in an attempt to develop a product with shorter soil residual properties and improved control of black nightshade (Solanum nigrum). In fulfillment of one of the Environmental Protection Agency's requirements for registering pesticides in the United States, Eli Lilly and Company awarded a contract to Michigan State University for the purpose of testing the effects of ethalfluralin on chronic avian reproduction. The two test species used were the bobwhite (Colinus virginianus) and the mallard (Anas platyrhynchos). Under the authority of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) the Environmental Protection Agency is required to publish guidelines stating the kinds of information that will be required to support the registra- tion of a pesticide (Federal Register, 1975a). This required information can be categorized into four broad sections: product performances, label development, chemistry, and hazard evaluation (Federal Register, 1975b). At present these guidelines, which are published in the Federal Register, are not final, and information required on a specific pesticide is determined on a case-by-case basis (Federal Register, 1975b). The chronic avian reproduction test is required if any of the following conditions exist: (1) "The pesticide or any of its major metabolites or degradation products is persistent in the environment to the extent that toxic amounts in avian feed could be expected to become present under normal pesti- cide use"; (2) "The pesticide, or any of its major metabolites or degradation products, is stored or accumulated in plant or animal tissues"; (3) "The pesticide product is intended for use under conditions where birds may be subjected to repeated or continued exposure to the pesticide or any of its major metabolites or degradation products"; and (4) "Any other test information which indicates that reproduction of birds may be adversely affected by a pesticide or by product use" (Federal Register, 1978). Condition three may exist with normal use of ethalfluralin. This herbicide has the potential to be used in a way birds may be subjected to repeated or continuous exposure. Bobwhites and mallards were selected as test animals because they are endemic to the continental United States, represent upland and aquatic forms of avian wildlife, and are readily available for toxicological testing. LITERATURE REVIEW The majority of literature available on the dinitro— anilines is directed towards trifluralin. Since ethalfluralin is still in the experimental stage of development and little literature has been released, this review will focus on the closely related substituted 2,6-dinitroani1ine herbicides in general. Data on ethalfluralin will be presented when avail- able. Physical and Chemical Properties Ethalfluralin (N-ethyl-N-(2 methyl-2—propeny1)-2,6- dinitro—4-(trifluromethyl) benzenamine) is produced by Eli Lilly and Company (Elanco) under the product name Solalan®. Its molecular formula is C13H14F3N3O4, has a molecular weight of 333.3 g/M, and a specific gravity of 1.32 g/ml (see Figure 1 for the structural formula). Ethulfluralin is a yellow- orange crystalline solid with a melting point of 55-56°C and a vapor pressure of 8.2 x 10‘5 mmHg at 25°C. Its thermal decomposition temperature is 256°C. The solubility in water at pH 7 and 25°C is 0.3 ppm. The chemical is readily soluble (> 500 mg/ml) in organic solvents such as acetone, aceton— itrile, benzene, chloroform, methylene chloride, and xylene. Its solubility in methanol is 80-100 mg/ml with a partition coefficient of 5.11. This herbicide is susceptible to decompo- sition from ultraviolet light (Elanco, 1977, 1979). Table 1 lists the physical and chemical properties of three major dinitroaniline herbicides. Most of the dini- troaniline herbicides, especially the substituted 2,6—dini- troanilines, have very similar physical and chemical properties. The first member in the class of herbicides labeled as substituted 2,6-dinitroani1ines was introduced by Lilly Figure 1. Structure of 2,6-dinitroaniline, ethalfluralin, and trifluralin. u.o|U_z_._.mO>z_Ezm nxu 0Iu-0:».z 05.0.01» cuz- .20» 3 m41cz 01w: OIMuOIn-_.‘Q._u|OIn-OIu .OMZ - -76» Gnu fiance‘s—.2 cowuqmoaeoooc Ou ofinaumwomom mmlmm oomA Oblmm ocmA oomA ommA oomA .55 A . o mm.m 00mm an czae mnOH x m.m Uooonmw Uflaom mcwafiuum>uo 30~H®> vomzmmodzmfio cmflmm mcprZHCu :QIOuuwcwplo .NIOLSAwfluu .:.=.:IH>:u0a:n~>u:nlz o.me~ ~.H n m :d o.~ u o 2; o.m u m :m cofluwmanOUOU Cu wanfludmomsm ommlmmn comA oomA oomA oomA oomA oomA Ema m .o no.m o.nm on 7:55 muod x m.mH Oomvtmv C«_Om mew-mum>uo owcmuo v0fi2mbo~2m~v cofiwmua mcfloflsaouumu~>aohmflplc .cncwuudcflcle.Nichsfiwfluunz.5.a m am e :g m :o o.~ o.m mxoo3 m.m cofiudmomeooop Cu wflnfluaoomsm ceauom oomA oodlom oomA oomA oomA oomA Elm m.o HH.m o.m~ um 7:25 m-o~ x ~.m o.sm-mm odaom ocfl-0umxuo 30-®> vameVH:MHU cmamcom OCfiEG ncvucvnlAa>cu0E0psflcfiuuvIvuouufisdp o .NIAH>c9eoLm ~|H>zuoe mvucn~>cumlz ccqodmsiacovp H1EL::% Owaduwdc; DotELxCLLL< Aoomm ..LCUE w33937: wca :fi 521 m.ov mflm>_:rssz cs snaizassm uncfiq uoqoa>tsodz :L >ufl~H2oow L.c:cafdmz o:c~>w ozcxv: ELCLCBOHLU o:3~:c: OHALDHCCDQU< Dadaou< #E\OE Auommv >oaaaozaom Deru>dxom “Jfi~:utsno Augmm ccn n say Lauc3 :« >uHHwosfiom uzoqoqwcvso coaufluuna 9L:mmopm LOQ0> D:L01 Tcflu~02 aboum Hmowmhzm nfizas2w Hsofluflmau can: moose was: fisoweocu cfimmcm: :HacL:~L_Le cusarssczcgsm .cwuwzon was .cqaduzdwquu .:LHmu:~w~mzuv mafiwoflnsmz vcfl~fizc0uoflcflt mzu we mwauu®QCLa doofim>;; 1:0 Loodaozu .H vance Research Laboratories in 1960 (Alder et a1., 1960). There are now at least 14 dinitroanilines on the market or in product development. The development started with the discovery of 2,6-dinitroani1ine (Figure 1), an intermediate in the produc- tion of other herbicides. 2,6-dinitroaniline was tested and found to have good non-specific herbicidal activity. Further investigations showed that the two nitro groups on the ring had a major role in the herbicide's activity. When the nitro groups were in the two and six positions the activity was enhanced. Substituting groups on the amine portion of the molecule also contributed to the activity of the herbicides. When R1 and R2 were both alkyls the products were more specific pre-emergence herbicides. Alkyls with three to eight carbons gave the best activity (Parka et 31., 1976). The most recent major discovery in the development of the 2,6-dinitroanilines came with the addition of a CF3 group in the fourth position of the ring. Adding CF3 gave many of the dinitroanilines maximal activity (Parka et 31., 1976). Efficacy Ethalfluralin has been shown to control many weed species as they germinate, with little or no damage to crops for which the herbicide was intended [soybeans (Glycune max), peanut (Arachis hypogaea), cotton (Gossypium Sp.), peas (Pisum Sp.), edible beans, and curcurbits]. Table 2 lists the annual grasses and broadleaf weed for which ethalfluralin provides commercially acceptable control (Elanco, 1977). In AHme QOmHmmv A.mm msnvcmumeav A.Qm Escmaomv AESQHM Esflpomocmnuv Amaummoom wflnoomv AonH50H>m Escomwaomv Amadmoflxmamfim EDHEMQV Asswflunmm asfiwomcmcmv Amunmom mapumnoflmv Amoflcoo mcwaflmv Ammomumao momHspHomv AMAUmE mHHmHHmumv Amumfiflflofiuum> omsHHozv mmaumflnp swammsm momemHm mmemnmunmflz mumpumsqmnfimq manoox pmmBuocm panama poemmmoow wmamsm opHHon hawnoumo HMHOQOU mamamusm coEEou pmmzxosno GOEEOU UmmBummHmu Amsumw mcm>¢v Amfimcmcmflaflo mflumoummumv AmHEHOLHHHm moHnooummqv Awsvnmocfl mounocwuv A.mm ESQflcmmv AESCOHOO MOHnooafinomv Ammcmmmams Esnmcomv AEDHOHMHDHDE ESHHOQV Amoflwfi maflmsmfimv Ade wflumummv A.mm aflumufimflov A.mm mDEoumv Aflaammlmsuo moH£oocH£omv Amsccm momv umo UHHE mmmuo xcflum QODmeamumm HMQCQMm mESHOHamm moaumamssh mmmum somCQOb mmmum mam cmflamuH mmmum mmoow mHHmaxom mommmnm amuo mommmnm mfioum mmmum Unmwcumm mmmummSHQ Hmsccfi mama ommaucmmom mama coEEou momm3 mmmapmoun Hmscc< mama oamapcmflom @EMC COEEOU mommmum amazed .Houucoo manmummoom waamflonmeaoo mmpfl>oum cfiamuoamamnpm QUHSB MOM mommB mmmaomoun 0cm mmmmmum Hmscq¢ .N magma cucurbit studies Humphreys gt gt. (1978) reported excellent control of large crabgrass (Digitaria sanguinalis), goosegrass (Eleusine indica), foxtail millet (Setario italica), and red- root pigweed (Amaranthos retroflexos) on soils ranging from sands of south and middle Florida to sandy clay loam in Texas, silt loam in Mississippi, and loams sands of Georgia and North Carolina at rates of 0.5 to 1.25 lbs/A, the recommended rate of application. All cucurbits showed good tolerance at these rates with no significant reduction in yields over the control. Other studies by Murphy and Marrow (1978) and Ilnicki gt gt. (1977) on potatoes (Solanum tuberosum) and Kenof (Hibiscus cannabinus) reported that ethalfluralin gave good weed control on a variety of weeds with little or no damage to the crop. Humphreys gt gt. (1978) reported that ethalfluralin, at higher rates than recommended (1.0 to 1.75 lbs/A) for most weeds, gave improved control of black nightshade as compared to other dinitroani- line herbicides. Mode of Action The dinitroanilines are classified as mitotic poisons. It is widely recognized that the major morphological effect of these herbicides in plants is swelling of the root tip. This swelling can be attributed to the inhibition or cessation of cell division with a continuation of radial expansion (Bayer gt gt., 1967). These herbicides interrupt both cortical and spindle microtubules in root cells (Parka and Soper, 1977). 10 The mitotic process is interrupted in metaphase, anaphase, and telophase, those phases which require the presence of micro- tubules. When microtubules are absent, the cells begin normal division with prophase, but fail to align along the metaphase plate, resulting in aberrant mitotic figures. When microtubules are present prior to cell division the mitotic process is arrested at the stage that had been reached when the disappearance had occurred (Parka and Soper, 1977). The dinitroaniline’s effect on chromosomes resembles the effects produced by colchicine, a known cortical and spindle microtubule inhibitor. While these herbicides may act similar to colchicine in plants their actions are different in animals. EE,XEE£9 polymerization studies with pig brain microtubules showed that trifluralin, orizaline and propham, all dinitroani- lines, did not inhibit polymerization of microtubular protein while colchicine did. Colchicine binds to a different site on the tubulin molecule than the herbicides (Bartels and Hilton, 1973). Moreland gt gt. (1972) reported the effects of 12 sub- stituted 2,6—dinitroani1ine herbicides in isolated spinach (Spinacia oleracca) chloroplasts, and mungbean (Phascolus ourcus) mitochondria. Both photoreduction. and coupled photophosphorylation were inhibited in chloroplasts by all the herbicides. In mitochondria, 11 of the dinitroanilines inhibited phosphorylating electron transport with malate as a substrate. Most were also strong inhibitors of NADH and succinate oxidation. Moreland gt gt. demonstrated that if 11 these herbicides became incorporated into the organelles, photosynthesis and respiration were inhibited. Since plants depend biochemically and physiolocially on oxidation and phosphorylation for energy, inhibition of these pathways may also be a major mechanism of the dinitroaniline's phytotoxicity. Some dinitroanilines are also known to inhibit the proteo— 1ytic enzymes, phytase and dipeptidase. The combined reduction of these enzymes is sufficient to influence seedling growth (Parka and Soper, 1977). Absorption, Translocation and Metabolism in Plants It is currently thought that the major site of absorption of the dinitroaniline herbicides is through the shoot of monocots and the hypocotyl of dicots (Nishimoto and Warren, 1971). Other studies conducted on grain crops have supported these areas as the site of uptake (Parka and Soper, 1977). The translocation of the dinitroanilines is concentrated in the root areas of the plant, although some acropetal and basipetal movement has been reported when the herbicides were applied in a topical fashion (Kecherside gt gt., 1969). When soybeans and cotton were treated with l4C-trifluralin the majority of radioactivity was found on the root surface and in the root walls of the xylem vessels. There was little movement out of the roots. Futher examination found that the majority of radioactivity was absorbed or bound to the root epidermis or cuticle. Research on peanuts, tobacco (Nicotiong tabacum) and tomato (Lycoporsicon esculentum) supports the conclusion 12 that the dinitroanilines are either absorbed by or absorbed to the roots with minimal translocation into the stems and leaves (Probst gt gt., 1967). Plant metabolism studies with trifluralin and nitralin indicate that the major residues are the parent compound. Identifiable metabolites represent 5% or less of the recovered compounds (Colab gt gt., 1967). These plant metabolites have also been found in soil. It has not been determined if the plants are absorbing the metabolites from the soil or if they have been transformed from the parent compound within the plant. Persistence Under most field conditions ethalfluralin and the other dinitroanilines will degrade in one season, allowing rotational crops to be seeded four to five months after application with- out toxic effects (Elanco, 1979). The persistence of these herbicides is greatly affected by the soil temperature and ion exchange capacity. Temperature may increase or decrease the persistence of the herbicides in the soil by altering volatilization rates. High temperatures will cause rapid volatilization of most dinitroanilines. In hot climates this increased loss reduces the waiting period before rotational crops can be safely planted. In contrast, colder soils, such as those found in Saskatchewan, may take in excess of one growing season before soil herbicide levels drop low enough to allow safe planting of rotational crops (Rahman and Ashford, 1973). 13 The dinitroanilines are quickly and strongly absorbed to soil particles. Soils that are high in organic matter and clay have a higher ion exchange capacity and absorb the herbicides more readily. High ion exchange capacity soils bind the dinitroanilines tightly, increasing their persistence while rendering them ineffective in weed control. Ethalfluralin is not recommended for soils with an organic matter content above 10% (Elanco, 1977). T ’- ‘I)‘ ‘l Degradation The major mechanisms of degradation and removal of most dinitroanilines from the soil are volatilization, photochemical instability, and microbial decomposition. These three pro- cesses are influenced by soil moisture, temperature, texture, and rate of application. Most dinitroanilines are considered to be slightly volatile. This is the major route of loss from the soil. Parochetti and Hein (1973) reported a 24.5% and 12.5% loss of trifluralin and benefin, respectively, over a three hour period from a Lakeland loam soil. Trifluralin and benefin, which have a similar vapor pressure to ethalfluralin, showed an increase in volatilization when moisture levels were increased from air—dry to saturation. These authors also reported a significant increase in volatilization when the soil temperature was increased from 30°C to 40°C. Kennedy and Talbert (1977) reported a 34%, 25%, and 18% loss of profluralin, benefin, and trifluralin, respectively, over a 24-hour period, due to volatilization from a Toloka silt loam. l4 Delayed soil incorporation of several dinitroanilines (nitralin, butralin, dinitramine, fluchloralin, penaxalin, trifluralin, profluralin, benefin, isopropalin, and orizalin) for up to three days caused a dramatic increase in herbicide loss. Only orizalin concentrations were not greatly reduced over the three-day period (Kennedy and Talbert, 1977). Orizalin has a very low vapor pressure (1 x 10-7 mmHg at 25°C). Without soil incorporation, especially in moist warm soils, most of the dinitroanilines will volatilize, reducing their concentration and effectiveness. Thus, when the herbicides are used as post-emergance (surface applied), the recommended application rates are increased to assure that adequate amounts remain in the soil. The dinitroanilines are susceptible to photodecomposition. Kennedy and Talbert (1976) reported oryzalin, nitralin, butralin, dinitramine, fluchloralin, peuoxalin, trifluralin, profluralin, benefin, and isopropalin underwent some degree of photodecomposition under ultraviolet light over a 24-hour period. When comparing treated thin-layer soil plates to treated unsoiled glass plates, the soil greatly reduced photodecomposition. In another study, Wright and Warren (1965) also reported reduced photodecomposition when a dinitroaniline was applied to soil. Trifluralin on glass completely lost its biological activity when exposed to six hours of sunlight. This reduced degradation has been attributed to the soil's nonuniform surface. Unlike the glass plate's smooth surface, which exposes most of the herbicide to light, the soil allows 15 the herbicide to penetrate the exposed surface and escape the light's effect. Parechetti and Dec (1978) studied the decomposition of eleven dinitroanilines in soil exposed to unfiltered sunlight for 7 days. Photodecomposition was noted for all of the herbicides tested. The losses ranged from 8.2% for isopro- palin to 73.3% for dinitramine. The average was 25.8%. The major photochemical reactions appeared to involve oxidative dealkylation, nitro reduction, and cyclization (Letis and Crosby, 1974; Plimmer and Klingebiel, 1974; and Newsom and Woods, 1973). The biological breakdown of the dinitroanilines accounts for only a fraction of the loss in soils. Messersmith gt gt. (1971) reported that the conversion of l4C-trifluralin to 14C02 was only 3 to 5% of the loss of 14C trifluralin actitity. Probst gt gt. (1967) used autoclaved and nonautoclaved soil to determine if microorganisms played an important role in the breakdown of trifluralin. Their results showed that the nonautoclaved soil degraded trifluralin more rapidly than did the autoclaved soil. Although they failed to find a buildup of any specific organism, Hamdi and Tewifik (1969) isolated some species of bacteria Pseudomonas sp. capable of degrading trifluralin. Helling (1976) stated that dinitroanilines degrade in soil via oxidation and reductive pathways, involving oxidative dealkylation of carbon chains, reduction of the nitro groups, and oxidative cyclization (see Figure 2 for the postulated major pathways of degradation of trifluralin). 16 Figure 2. Degradation pathways of trifluralin. l7 Trifluralin ”7°3'N'03H7 -NH2 I CF3 3 Q. C 9. ‘5' 3 I CF3 1 reduction L H'N'H H703'N-H “703'"‘03H7 ”2* / "2 ”2* H2 "2"' «H2 \ tfilmllwlation Fdealkylation I éFa éFa CFa 18 Leachingtand Water Movement Since the dinitroanilines are strongly absorbed to soil and have low water solubilities, they are among the least mobile herbicides. Many studies indicate a relatively minute amount of leaching occurs. Gagnon and Hamilton (1973) reported no detectable leaching of butralin, dinitramine, nitralin, profluralin, and trifluralin on sandy loam plots during a one-year study in which 145 cm of rain and irrigation water entered the soil. Trifluralin was found to be immobile in fourteen soils ranging from sand loam to silty clay (Helling, 1971). The degree of leaching can be affected by soil moisture levels. Koren (1972) showed that leaching of trifluralin and oryzalin increased when the soil was initially moist rather than dry. This may be due to a reduction in soil-herbicide binding. Increasing the water content of the soil can reduce the availability of binding sites by acting as a barrier or competitor to the herbicide. In either case, moist or dry, leaching was minute. Runoff water from various crop fields have also shown little or no movement of the dinitroanilines. Davis and Rahn (1970) found no trifluralin in surface water from a BOO-acre lima bean field on a loamy sand soil when applied at 0.75 lbs/A. In addition the researchers found no trifluralin in a drainage ditch next to a 20-acre soybean field that had been treated. Sheets gt gt. (1973) studied the movement of trifluralin in surface runoff water and found that less than 1% of the 19 total herbicide applied could be recovered. The highest concentration detected in the water was 24 ppm. The filtered sediments of the runoff contained an average of 84% of the detectable herbicide, indicating most of the herbicide was carried from the field soil-bound. Toxicity Technical grade ethalfluralin and the dinitroanilines, as a group, are not hazardous to mammals and birds but are toxic to fish and other aquatic organisms (Elanco, 1979). Acute toxicity date (Table 3) indicate that both ethalfluralin and trifluralin are slightly or probably non-toxic to mice, rats, dogs, cats, rabbits, chickens, bobwhite, and mallards. Subacute, dermal, and ocular data (Table 3) also indicate ethalfluralin is relatively non-toxic to rats, bobwhite, and mallards. Commercial herbicides containing ethalfluralin or trifluralin produce greater toxicities in mammals and birds. This increased sensitivity can be attributed to the solvent vehicles (Elanco, 1977; 1979). Unlike mammals and birds, aquatic organisms are very sensitive to the dinitroanilines. Low concentrations of ethalfluralin are toxic to fish (Table 3). Although most dinitroanilines are extremely toxic to fish they do not represent a threat unless dumped directly into a water source or spilled in quantities large enough to overcome the soil's binding capabilities. Parka and Worth (1965) reported that using trifluralin under field conditions and recommended 20 Table 3. Acute and subacute toxicity of ethalfluralin and trifluralinl. Ethalfluralin Trifluralin LD ——1%at >10,0002 >10,000 Bobwhite >2,000 --- Dog >200 >2,000 Cat >200 --- Rabbit --- >2,000 Rabbit (dermal) >2,000 --- Chicken --— >2,000 LDSQ Mouse >10,000 >5,000 Rat >10,000 >10,000 Mallard --- >2,000 Blugill 0.032 0.047 Rainbow trout 0.32 0.01-0.09 Goldfish 0.26 --- LCSQ Rat >5,000 --- Bobwhite >5,000 --- Mallard >5,000 --- Daphnia --— 0.56 Rabbit (ocular) Slight irritant Slight irritant l Elanco, 1979. Parts per million. 21 application rates, 2 inches of soil from 45.6 acres of Princeton fine sand would have to be moved into a one—acre pond with an average depth of three feet to produce the same affect as an LCSO on bluegills (Lepomis macrochirus). Emmerson and Anderson (1966) reported that orally administered l4C—trifluralin was excreted during the first 24 hours after dosing. About 80% of the activity was recovered in the feces while the remaining 20% was removed through the urine. Of the total amount excreted in the feces, 8% was recovered as unchanged trifluralin. The major metabolite in the feces was a compound resulting from the reduction of one nitro group into an amine, a metabolite similar to one that occurs in the soil. The authors suggested that even though trifluralin was quickly absorbed into fats it had a low order of absorption from the gut. Bile excretion accounted for only 11-18% of the activity. Also, similar structurally related compounds such as halogenated nitrobenzenes are known to be poorly absorbed from the gut when administered orally. Ten urinary metabolites were found but only three could be isolated and identified. One metabolite was formed by the complete dealkylation of trifluralin. The other two were formed by the reduction of one nitro group and/or the removal of both propyl groups (dealkylation). Emmerson and Anderson (1966) reported that the fate of trifluralin in the dog was similar to that of the rat. When dairy cows were administered oral doses of 14C- trifluralin, 99% of the ingested activity was recovered after 22 a six-day period, 18% in the urine and 81% in the feces. After six-days, no radioactivity could be found in the blood or milk (Golab gt gt., 1969). Golab gt gt. (1970) also reported similar results when using l4C-benefin. EXPERIMENT l. The effects of ethalfluralin on feed palat- ability in the mallard and bobwhite. PROCEDURE Chronology of Study Adult mallards and bobwhite were obtained in mid May, 1979 and were immediately placed in quarantine for one week. After this period, both species went through a two-week acclimation period. During the acclimation period the birds were in their appropriate pens and were fed an untreated diet (control). The mallard study began on June 5, 1979 and continued through July 3, 1979. From June 5 through June 18, 1979 the birds were fed untreated diets. On June 19, ethalfluralin- treated feed was administered for the remaining two-week period. The bobwhite study began on June 4, 1979 and continued through July 10, 1979. From June 4 through June 18, 1979 the quial were fed untreated diets. On June 19, the birds received ethalfluralin-treated diets for the remaining three- week period. Animals Male mallards were obtained from Whistling Wings Hatchery, Hanover, Illinois. The birds were leg banded and randomly assigned, three to a pen. Male bobwhites were obtained from Michigan State Univer- sity Poultry Science Research and Teaching Center in East Lansing, Michigan. The birds were wing banded and randomly assigned, one animal per pen. 24 A11 birds selected for the study appeared to be in good health. Diets and Treatment Level The test diets were prepared by Lilly Research Labora- tories in Greenfield, Indiana, using Lilly Mash (see Appendix A for composition and analysis). The dietary levels mixed were 0.001%, 0.003%, 0.010%, 0.030%, 0.100%, 0.300%, and 1.000% ethalfluralin. Random assignments of S. dietary ethalfluralin levels to pens and cages was accomp- lished with the use of a random numbers table. Two mallard pens and six quail cages were assigned to each dietary level. Facilities All birds were housed at the Michigan State University Poultry Research and Teaching Center. The rooms were entirely enclosed. Each pen and cage was numbered and labeled with dietary level and bird identification numbers. Each pen and cage also had its own individually identifiable feed container and scoop so that feed consumption could be accurately measured. Photoperiods were controlled by an automatic timer (Paragon model 41005—0). The mallard rooms measured 11.0 m x 12.2 m x 2.4 m (W x L x H). There were three rows of six pens suspended from the ceiling by chains. Each pen measured 1.5 m x 1.6 m x 0.7 m (W x L x H) with no top. Pens were constructed of plastic coated 2.5 cm x 2.5 cm steel mesh wire. The bobwhite rooms measured 5.0 m x 9.3 m x 2.4 m (W x L x H). Two groups of cages hung from the ceiling, each 25 consisting of 15 cages aligned back to back. The dimensions of each cage were as follows: 40.0 cm x 43.0 cm x 44.5 cm (W x L x H). The size of the mesh of the wire caging was 2.6 cm x 9.8 cm and the flooring was 1.3 cm x 1.3 cm. The flooring was plastic coated wire. Testing From the beginning of the tests, feed consumption was measured weekly throughout the duration of the study. Body weights were measured at day 0, 7, 14, 21, and 28 for the mallards and day 0, 7, 14, 21, 28, and 35 for the bobwhite. Mortality, morbidity, behavior, and any signs of intoxica- tion were recorded daily. Statistical Analysis Body weight and food consumption were evaluated with the use of a Dunnett's two-tailed test (Gill, 1978). Sampling units were individual pens. RESULTS Body Weights Body weights of mallards fed ethalfluralin at 0.003%, 0.010%, 0.030%, 0.100%, 0.300%, and 1.000% of the diet were not significantly different from those of the control birds throughout the length of the experiment (Table 4). Body weights between all dietary treatments at any given time were fairly uniform. The largest difference in weights between concentrations was 11% which occurred in the second week prior to the administration of treated feed. There was a 26 .amo.o A av aouucoo map anw ucmammme mapcmonchHm Ho: mum HQHHomQSm 0800 map nuHB CESHOU 0600 map CH mammz H mo.a H mmma moa H mmma mom H mmma mam H mmma amm H mmma ooo.a mma H mmma maa H omma o.m H mmma mmm H mama «gm H mmma oom.o mva H mama mo.a H aama mo.m H Noqa o.m H mama wo.m H mmma ooa.o 80.4 H mmma m.ma H mmma 0.0 H mmma o.m H moma mo.o H qvma omo.o mom H vmma 8mm H amma mmm H vmma mom H mama mma H mmaa oao.o mma H vama mma H mmga o.m H mmma mom H mmma mom H omma moo.o m.ma H mmma mma H mmma mma H mmma «cm H woma mom H aama ooo.o v m m a armams m xmmz xmmz ammz xmmz amauaaa caamasamamsum ucmEHmmae ucmEpmmaulmam mo Hm>mH %H0HmHQ .GHHMHSHMHmnum pom ummu muHHHnmumamm co mUHmHHmE mo A.m.m H my muanmB moon :00: .¢ mHQMB 27 trend at all dietary levels towards increasing body weights over the four-week test period. All concentrations had greater body weights at termination than at the onset of the experiment. Average body weights did not decrease after the introduction of treated feed at any treatment concentra- tion. Body weights of bobwhite fed ethalfluralin were also not significantly different from the control birds (Table 5). There was a trend for decreasing body weight as dietary concentration of ethalfluralin increased, but the trend was present prior to the administration of treated feed and is considered to be a random occurrence. Body weights within treatments showed an increasing trend over the five-week duration of the study. Only the birds that received ethal— fluralin at 1.000% decreased in weight one week after the administration of treated feed. The weight loss was not maintained and the birds gained in the following week. Final body weights for all dietary levels were higher than the original weights. Food Consumption There was no significant difference in food consumption of either mallards fed the various concentrations of ethal- fluralin (Table 6). No uniform trend in mallard food consumption could be established. Drakes fed dietary concentrations of ethalfluralin at 0.000%, 0.003%, and 0.010% increased or maintained their consumption rates during the first week the treated diets were introduced. Mallards fed 28 map ana acmamaaao maucmoHMHcmHm poo mam HmHaomnsm @800 may SHHB QEDHOO mEmm mnu 0H mammz .amo.o A my aouucoo a mm.m H mma a.m H ama m.m H oma am.v H mma mm.m H mma mm.m H mma ooo.a av.m H ama 6¢.m H mma ww.v H mma ma.m H mma no.4 H mma mmm H mma oom.o am.v H com ag.v H mma wo.¢ H «ma mm.m H mma mm.m H mma am.m H mma ooa.o mo.m H mma am.m H mma mm.m H mma mo.m H oma mo.m H oma am.m H mma omo.o am.m H mom wo.m H mma am.m H mma wa.m H mma mm.m H mma mg.m H mma oao.o am.¢ H mom mm.v H aom ma.¢ H aom mm.4 H com no.4 H mom am.m H com moo.o am.m H oam mm.m H mom am.m H mom mo.m H aom mo.m H aom Mm.m H mma 000.0 m m m m a unmams amv :aamHSaa x003 xmmz x003 x003 x003 HMHHHQH Iamnpm mo pcmaummae pcmEpmeulmam COHumnucmocoo mampmHo 1111/1 .IIIIII/I .GHHmusamamnum pom pmmH huHHHnmumHmm so mmuHSBQon moa.m.m H mo musmHmB moon cams . m wHQME 29 anm Hcmamwpr wawcmonHcmHm Ho: mam umHaoszm 060m 050 LHHB CEDHOO 0800 may 0H mammz .Amo.o A my Hoaucoo map a 00.0 H mma mm.a H mma 00.a H mma 00.0 H 00a 000.a 00.0 H mma m0.0a H mma m~.ma H mma ma.m H mma 00m.0 00.00 H mma 00.0a H mma 00.00 H 00a 00.0a H ama 00a.0 mm.0 H 0aa 00.0 H 00a m.a.0a H mma m.m.aa H 00a 0m0.0 mm.m H 00a 00.0 H 0aa mm.0a H maa 0m.m H 00a 0a0.0 mm.0 H 0aa m0.a H mma 00.0 H mma 0a.m H 0ma m00.0 00.ma H mma mm.ma H mma 0mm H 00a H0.0 H mma 000.0 0 m m a m xmmz x663 xmmz ammz caamusamamaum usmEummHB ucmEummaulmam mo Hm>wH >HmumHo .caamusamamaam ama ummu MHHHHQmumHMQ co mpamHHwE mo a.m.m H x .>00\UHHQ\mEmumv COHHQEDmCOU pooh .w manms 30 dietary concentrations of 0.030%, 0.100%, 0.300%, and 1.000%, ethalfluralin decreased their consumption during this period. Final mallard feed consumption values for most concentrations, including the controls, declined from the previous week but were not significantly different from their initial values. Treating the feed with ethalfluralin at these levels did not significantly decrease mallard feed consumption at any time during the study. Similarly, there was no significant difference in the bobwhite's initial or final feed consumption values (Table 7). However, birds fed ethalfluralin at 1.000% had a significantly lower consumption rate than the controls one week after the administration of the treated diets (Figure 3). Feed con- sumption for this dietary concentration increased in the following weeks but never achieved pre-treatment concentra- tions. Food consumption between and within levels varied and little uniform trends could be established. All groups of birds, including the controls, either maintained or decreased their consumption after the onset of the treatment phase. Final feed consumption rates were lower than the initial rates in dietary concentrations of 0.003%, 0.030%, 0.100%, and 1.000% while the 0.000%, 0.010%, and 0.300% concentrations had final consumption rates greater than their initial rates. Mallard and bobwhite dietary consumption of ethalfluralin was correlated to the concentration of the diet. Dietary concentrations were increased roughly 300% over the previous lower dose. Since feed consumption was not effected by the 31 .amo.o A 00 Hoaucoo man Eouw pcmammme mawcmoHMHcmHm no: mam umHuomQSm 0500 0:0 QHHB mammz a 000.0 H 0.0a 0a0.a H 0.0a 00a.0 H 0.aa 0am.a H 0.0a 000.a H 0.0a 0 000.a 000.0 H 0.0a 0a0.0 H 0.0a 000.0 H 0.0a 00m.a H 0.0a 0ma.m H 0.0a 0 00m.0 0m0.0 H 0.0a 000.0 H 0.0a 00m.0 H 0.0a 0m0.0 H 0.0a 000.0 H 0.0a 0 00a.0 0a0.0 H 0.0a 000.a H 0.0a 0m0.a H 0.0a 000.a H 0.0a 0m0.a H 0.0a 0 0m0.0 000.a H 0.0a 00a.a H 0.0a 00a.a H 0.0a 00m.a H 0.00 000.0 H 0.0a 0 0a0.0 000.0 H m.0a 000.0 H 0.0a 0m0.0 H 0.0a 000.0 H m.0a 000.a H 0.0a 0 m00.0 0m0.a H 0.0a 0am.a H 0.0a 000.0 H 0.0a 0a0.0 H 0.00 M00.0 H 0.0a 0 000.0 m 0 m m a 0 0 0003 0002 0003 0003 x002 00aa0usa0a0000 000800009 ucmEpmmHulmHm mo Hm>mH wumumHo .caa0usa0a0000 00m 0000 poHHQmumamm co mHHSBQOQ mo a.m.m H x .wmo\pan\mEmumv COHumEsmcoo pmmm .0 manme 32 Figure 3. Mean feed consumption of bobwhite on palatibility test fed ethalfluralin at various concentrations. 33 00000 0000 COOP 000 000 00 ... [ o ' Tenn: .93.. 5205 200:0 0x003 pr NP 0w @— mp or hr or m.. ON PN (Rep/puq/swelfi) uoudumsuog paag ueaw 34 herbicide, ethalfluralin intake also increased 300% from one dose level to the next highest level (Table 8). Mallard and bobwhite consumed a maximum of 1,365 and 163 grams/bird/day, respectively. Mortality No mortality occurred in the mallard or bobwhites studied over the 28 or 35 day test period, respectively. No obser- able signs of intoxication or morbidity were observed in any animal in either study throughout the duration of the treat- ment period. All animals on examination appeared to be in good health at the study termination. DISCUSSION Comparing pesticide levels in feed tolerated by birds in the laboratory to those levels found or expected in the environment can be useful in estimating the potential hazard of pesticides to birds (Kenaga, 1973). Low levels of highly toxic pesticides such as endrin produce toxicity and death in laboratory animals (Omer, 1970; Hill gt gt., 1975). One could reason that similar or higher levels of this same chemical contaminant, found in the environment on feedstuff specific to an organism, would also produce toxicities to those organisms consuming the contaminated feed. This rela- tionship has been shown to occur in a variety of animals (Prince gt gt., 1971; Murphy, 1980; Hornshaw, 1981). Conversely, high levels of other pesticides such as triflura— lin are relatively nontoxic to laboratory animals (mammals .m.m H m 35 a 0.00 H 000 a.00 H 000 0.mma H 000 0.0 H m0a 0.0a H a0a 0.m0 H 0aa 0 000.a 0.0a H 000 0.0a H 000 m.0a H 000 a.0 H 00 0.0 H 00 0.0 H 00 0 00m.0 0.0 H m0 0.0 H 00 0.a H m0 0.0 H 0a 0.0 H 0a 0.0 H 0a 0 00a.0 0.0 H m0 0.0 H 00 0.0 H 00 0.0 H 0 0.0 H 0 0.0 H 0 0 000.0 0.0 H 0 0.0 H 0a 0.0 H 0a 0.0 H 0 a.0 H 0 a.0 H 0 0 0a0.0 a.0 H m 0.0 H m a.0 H 0 0.0 H a 0.0 H a 0.0 H a 0 m00.0 mmmmmmmm 0.00 H a00 0.0m H 000 0.00 H 000.a 0.00 H 0am.a 0 000.a m.aa H 000 0.am H 00m 0.0a H 0mm 0.00 H 000 0 00m.0 0.m0 H 00 0.00 H 00 0.0m H 0ma 0.00 H 00a 0 00a.0 m.0 H 00 0.a H 00 0.0 H mm a.0 H mm 0 0m0.0 0.0 H 0 0.0 H 0 0.0 H 0a m.a H 0a 0 0a0.0 a.0 H 0 a.0 H m a.0 H 0 aa.0 H 0 0 m00.0 000aa0z m 0 a m 0 a 0 0 0003 0003 0002 0003 0002 0003 caa000a0a0000 000\00a0\00\0s 000\00an\0e mo a0>0a wumuwHo .0000\0uan\00\020 paw A>MU\©HHQ\UEV GHHMHDHMHmnuw mo QOHHQEDmcoo mHMHmHU muH£3noa paw UHMHHMZ .0 0H309 36 and birds) resulting in few reported incidences of poisoning due to environmental exposure (Worth and Anderson, 1965). Ethalfluralin, which is similar structurally, physically, and chemically to trifluralin, was shown to cause little or no observable signs of toxicity to mallards and bobwhite in these subacute studies. Overall behavior, body weight, and food consumption values for these two species were similar to those reported by Flaga (1980) and Jones (1977). Increasing body weight trends in the mallard and bobwhite over the duration of the studies can be attributed to normal growth and development of the animals. All dietary levels for both the mallard and the bobwhite, with the exception of the bobwhite on the 10,000 ppm ethalfluralin diet, continued to show an increase in body weight after the administration of treated feed, suggesting that ethalfluralin had no detrimental effect on body growth. Feed consumption in the mallard and bobwhite showed no trend with increasing dietary levels of ethalfluralin, indicating ethalfluralin was tolerated at the concentrations administered. The only sign of feed rejection was with bobwhites on the 10,000 ppm ethal- fluralin diet. These birds temporarily reduced their feed intake resulting in a small, short term loss of body weight. Environmental levels of ethalfluralin should be predict- able when considering the data available on the herbicide frequency and rate of application, rate of degradation, and solubilities. Recommended application rates of 1.5 gallons per acre of Sonalan® would result in approximately 0.505 37 grams of technical grade ethalfluralin per square meter of soil and vegetation (surface applied). With volitilization rates ranging from four to five percent in a three-hour period (Parochetti gt gt., 1976) to 50 percent in a 24-hour period (Helling, 1975) for trifluralin, which volatilizes at a similar rate to ethalfluralin, the amount of ethalfluralin expected to be remaining in the field would be reduced signi- ficantly in a short period of time. Photodecomposition would further reduce the residue levels on exposed plant and soil surfaces. Kenaga (1973) states that field residue levels of most pesticides decline rapidly with most levels falling below 1 ppm within one week after application. Even the more persistant chlorinated hydrocarbons have been reported to drop below 10 ppm one week after application. In addition, residue studies with mammals report that similar dinitroani- lines do not readily accumulate in animal tissues, and are completely excreted seven days after exposure. Considering the high doses of ethalfluralin tolerated during past acute and subacute studies with mallards and bobwhite, and this palatability study, it is unlikely that any acute environmental exposure due to normal use of this pesticide would produce an immediate threat to these species. The mallard and bobwhite are capable of ingesting 1,365 mg/bird/day (mallard and 163 mg/bird/day (bobwhite) of ethalfluralin for up to a two and three week period, respec- tively, without any apparent detrimental effects. If concentrations in the environment should rise above 10,000 ppm, such as in a spill, data presented indicates that the 38 bobwhite will reject feed when high levels of ethalfluralin are present. Although the mallards did not reject feed at the 10,000 ppm level, ethalfluralin did not produce toxicity. In addition, high levels of ethalfluralin in the environment would be short lived due to the herbicides physical and chemical properties. EXPERIMENT 2. The effects of ethalfluralin on mallard and bobwhite reproduction. PROCEDURE Chronology of Stugy The mallards used for this study were received on August 23, 1979 and were immediately placed into quarantine until September 3, 1979. Bobhwites were received on August 29, 1979 and placed in quarantine until September 4, 1979. Both species were then allowed a two-week acclimation period in which the birds were placed in their appropriate testing cages and were fed untreated basal diets. The mallards and bobwhites first received their assigned dietary level of ethalfluralin on September 18 and 19, respectively, which was the beginning of the pre-production phase. The pre-produc- tion phase lasted eight weeks, at which time the photoperiods were increased from seven hours 1ight:17 hours dark to 16 hours 1ight:eight hours dark. This phase of increased photo- period, the production phase, lasted throughout the remainder of the study. The adult terminal kill for mallards and bob- whites was on March 26, and March 5, 1980, respectively. The mallard egg collection period started on January 4, 1980 and ended on March 13, 1980. The bobwhite egg collection period was from December 17, 1979 through March 2, 1980. The final groups of ducklings and chicks hatched from eggs provided by the treated breeders were terminated on April 24, 1980 and May 1, 1980, respectively. 39 40 Animals Mallards were purchased from the Max McGraw Foundation, Elgin, Illinois. The birds were hatched on June 19, 1979 and were received at the test facility on August 23, 1979. The birds were 13 weeks old at the start of the pre—production phase of the study. Bobwhites were purchased from Barrett's Quail Farm, Houston, Texas. The quail were hatched on May 26, 1979 and were received at the test facility on August 29, 1979. The birds were 16.5 weeks old at the start of the pre-production phase. All birds of both species appeared to be in good health upon arrival. All birds were randomly assigned to cages, two males and five females per mallard pen and one male and one female per quail cage. Random assignment of dietary concentration to cage was accomplished by the use of a random numbers table. The test diets were prepared by Lilly Research Laboratory, Greenfield, Indiana, using Lilly Mash (see Appendix A through I for composition and analysis). The dietary concentrations mixed for adult birds were 0.00% (control), 0.01%, 0.03%, and 0.10% of ethalfluralin. Before the rations were shipped to the test facility, samples were removed for determination of the presence of the test chemical at the desired concentra- tion. When the initial lots were prepared, homogeneity of mix and eight week stability in feed were determined. 41 Facilities All birds were housed at the Michigan State University Poultry Science Research and Teaching Center. All rooms were entirely enclosed. Photoperiod was controlled by an automatic timer that was appropriately set for the current phase of the study. Electric and gas heaters were used to maintain a temperature of 10°C. Mallard rooms measured 11.0 m x 12.2 m x 2.4 m (W x L x H). There were three rows of six pens that 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 plastic coated l" x 1" steel mesh wire. The floor of each pen was sloped downward from the center to the outer sides allowing the eggs to roll from the center of the pen towards the aisles facilitating egg collection. Bobwhite rooms measured 5.0 m x 9.3 m x 2.4 m (W x L x H). Two groups of cages hung from the ceiling, each consisting of two rows of 15 cages aligned back to back. The dimmensions of each cage were as follows: 40 cm wide x 43 cm long x 45 cm deep. The size of the mesh wire caging equaled 2.6 x 9.8 cm and the flooring was 1.27 cm x 1.27 cm. The flooring wire was plastic coated. The floor of all cages sloped toward the aisles which allowed the eggs to roll down to the collecting area. Each pen and cage was provided with an individual feeder and watering cup. Pens and cages were numbered and labeled with study number, dietary level, and individual bird numbers. 42 Individually identifiable feed containers and scoops were used for each pen or cage. Testing Beginning with the initiation of the test, feed consump- tion was measured biweekly throughout the duration of the study. Body weights were measured at 0, 2, 4, 6, and 8 weeks and at termination. In order to avoid any adverse effects on egg production, body weights were not measured during the production phase. 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 study termination were killed by C02 asphyxiation and necropsied. At this date, tissues were collected for histopathology from at least five animals of each sex per treatment level. The following tissues were taken: kidney, liver, heart, lung, spleen, pancreas, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, colon, testes, ovary, magnum, shell gland, pectoral muscle, leg muscle, thymus, sciatic nerve, and skin. Egg Collection, Storgge, and Incubation Egg collection began when approximately 50% egg produc- tion was reached. Prior to this time, all eggs laid were discarded. Once 50% production was achieved eggs were collected daily and each was marked in pencil with the 43 corresponding cage number and date. Eggs that were soft- shelled or lacking shells were recorded and discarded. Eggs were stored at 15.6°C (14.4-16.7°C) until one week's collec- tion had accumulated. At that time, all eggs were candled and those with cracks were recorded and discarded. The remaining eggs were organized according to pen number and were then placed in an incubator (Jamesway, single stage, Model 242) for 23 days. The dry and wet bulb temeprature readings were 37.5°c (37.2-37.8°C) and 30°C (29.4-30.6°C), respectively. On day 14 of incubation, mallard eggs were candled to determine the number of fertile and infertile eggs, and the number of early dead embryos. The eggs were candled again on day 21 to check for embryo deaths that had occurred beyond day 14. On day 23 the eggs were transferred to pedigree baskets in which all eggs were separated according to pen number. The eggs were then placed in the hatchery incubator (Jamesway, single stage, Model 252) which had dry and wet bulb temperature readings of 37.2°C (36.9—37.5°C) and 31.7°C (31.1-32.2°C), respectively. On day 27, all pipped (dead and live), unpipped (dead and live), and hatched duck- lings were recorded. All hatchlings were individually wing banded and recorded according to parent stock number. The hatchlings were placed in cardboard chick boxes and were transported the five mile distance to the MSU Poultry Research and Teaching Center. Precautions were taken to prevent exposure of the ducklings to cold temperatures. The ducklings were moved quickly from the incubation room to the vehicle and 44 and from the vehicle to Building 3 of the research facility. The vehicle was warmed prior to transportation. At the research facility, the ducklings were placed in Petersime 250-24 battery brooders according to dietary level of parent stock. Temperatures were maintained at 35°C (34.4- 35.5°C) and the ducklings were given both feed (starter diet) and water gg libitum. The ducklings were observed daily for two weeks and all mortality was recorded. At the end of the two-week period, all surviving ducklings were killed by exposure to chloroform. Bobwhite eggs and chicks were handled identically, except for duration of incubation and candling dates. Bobwhite eggs were candled on day 11 and day 18. The eggs were transferred into the hatcher on day 21 and the hatchlings removed on day 24. Eggshell Thickness On Wednesday of every other week, eggs were collected for determination of eggshell thickness. The eggs were marked with collection date and pen number. The eggs were cracked in half, contents removed, and shells washed with tap water to remove the albumin. The shells were then left to air dry at room temperature 21.1°C (20.0—22.2°C) on paper for at least 48 hours. Eggshell thickness was measured using an Ames Thickness Gauge (model 25ME). Four equidistant points around the shell circumference were approximated, and thickness was measured (shell plus eggshell membranes) to the nearest 0.01 mm. From these four data points, an average was calculated. 45 Statistical Analysis The following parameters were measured and recorded throughout the study: body weight, food consumption, number eggs laid/hen/day, percent eggs cracked, number of eggs set, percent eggs fertile, number live embryos at the second candling, number live pipped, number dead pipped, number dead not pipped, percent hatched, percent hatchlings surviving 14 days, and eggshell thickness. Treatment means were compared by use of Dunnett's two—tail test (Gill, 1978). Adjustments were made in survivability whenever dead ducklings were found without wing bands. In such cases, the numbers of bandless, dead ducklings were apportioned among the stock pen numbers within their treatment concentration for that particular hatch number. Adult mortality was analysed by the chi squared proce- dure (Gill, 1978). RESULTS Body Weight Ethalfluralin at dietary concentrations of 0.0%, 0.010%, 0.030% and 0.10% had no effect on mallard body weight (Table 9). All groups of birds increased in mean body weight in the first two-week period immediately following the administra- tion of treated diets, and continued to increase in weight until the onset of the production phase. Mallard weights in all groups were lower at the study termination than at the onset of the production phase, but remained higher than their initial weights. Male body weights were consistently higher than female weights. 46 00 0 OH 0 ma 0 ma 0 z m.mm 0.00 N.Nm 0.a 0.0m 0.0 0.0m 0.mm mm mmma omoa Hmma m0oa momH OOOH whaH NHOH M 500 000a 0H 0 0H 0 0H 0 ma 0 z o.H0 0.HN 0.mm m.00 m.om 0.0a 0.am 0.0 mm mmNH 0HOH Nmma 000 mmHH 000 mmHH M00 M 5mm 000 ma 0 ma 0 ma 0 ma 0 z m.0m 0.0m 0.0m 0.0H m.am o.HH m.aa 0.0m mm mHmH 0moa mHmH mmm mmHH 00m mmHH mum M Emm 00H ma 0 0H m 0H 0 ma 0 z o.mm m.0a 0.00 H.0H 0.0m m.0a m.00 0.MH mm mmHH smoa mmHH mmoa mHHH 0mm mvaa mom M Emm o m 2 m 2 m 2 m E H0>0H 0 0003 N 0003 o 0003 NI x003 0H000Ho 000Eum0me 000Eum0uul0um .CHHMHDHMH0500 00m moumaame waspm mo muan03 0000 0002 -m magma 47 0a 0 0a 0 0a 0 z 0.00 a.00 a.00 a.00 0.0m 0.0a mm 000a 00aa 00ma 00aa m00a 00aa m 500 000a 0a 0 0a 0 0a 0 z 0.00 0.mm a.00 0.am 0.00 a.00 mm 000a 000a 0mma 00aa 000a a00a m 500 000 0a 0 0a 0 0a 0 z 0.00 0.a0 0.m0 0.am 0.0m 0.00 mm 000a 000a 000a 0aaa m00a 000a m 000 00a 0a 0 0a 0 0a 0 z 0.00 0.aa 0.m0 0.00 0.00 0.a0 mm 000a 000a 000a 0aaa 0a0a 000a m Emm o m z m z m z H0>0H SOHumcHEH0B m 0003 m x003 wumu0HQ #:0500009 .00.0oov .0 0an0e 48 Similarly, ethalfluralin had no effect on the body weights of bobwhite (Tablellfl. No significant difference could be established when comparing the weights of the control birds with the weights of the birds on treated diets. Birds in all treatment levels maintained or increased in body weight from the onset of the study until the beginning of the production phase. Terminal body weights for female birds continued to show an increase over the previous measurement while most males had a reduced body weight at termination. Only the control males continued to increase in weight through the final measurement. Feed Consumption No significant difference in feed consumption between control mallards and any treatment level mallards occurred throughout the 30-week experiment (Table 11). Consumption in all concentrations increased during the first four weeks after the pre-treatment phase. During weeks five and six consumption dropped in all concentrations from the previous week but not below the initial pre-treatment phase. Feed consumption again increased during the seventh and eighth weeks but then sharply declined during weeks 9 and 10, the start of the production phase when the photOperiod was increased to stimulate reproduction. Following this decline consumption increased steadily in all levels for an eight-week period. During the remaining weeks feed consumption in all groups tended to level, then decline, until the termination of the study. Variation between treatments was relatively small 49 NH NH NH NH NH NH NH NH 2 m.0 0.0 0.0 0.0 0.0 H.o m.0 o.m mm mmH NmH mmH 00H 00H omH HOH 00H M HNMImmmH NH NH NH NH NH NH NH NH z 0.0 0.0 m.m m.0 m.0 m.0 m.0 0.m mm mmH HmH mmH omH NmH HmH HnH HnH m ledloolm MH MH mH mH MH mH MH mH z 0.0 0.0 0.0 0.0 m.0 m.0 0.m H.m mm 00H 00H mmH 00H NmH 00H HmH NNH m IHulamloolH mH mH mH mH mH mH mH mH z 0.0 N.m 0.0 m.0 o.m 0.m m.0 0.0 mm HON OQN 00H 00H 00H 00H mnH me m lemmlm .m 2 .m E m E .m 2 H0>0H 0 0003 N 0003 o 0003 NI 0003 mumu0Ho 0:0800008 us0Eum0nul0um .00a000a0a0000 000 00H03£00 pHspm mo muamH03 0003 :00: .OH 0HQMB 50 0a 0a 0a 0a 0a 0a 2 0.0a 0.0 0.0 0.0 0.0 0.0 mm 000 m00 000 000 000 a00 m 500 000a 0a 0a 0a 0a 0a 0a 2 0.0 0.0 0.0 0.0 0.0 a.0 mm 000 00a 000 000 00a 00a 0 200 00m ma ma ma ma ma ma 2 0.0 a.0 0.0 m.0 m.0 0.0 mm a00 000 0a0 000 000 a00 m 500 00a 0a 0a 0a 0a 0a 0a 2 a.0 0.0 0.0 0.0 0.0 0.0 mm a00 aa0 ma0 0a0 aa0 000 m Emm 0 m 2 m 2 m 2 H0>0H COH000HEH09 m 0003 m 0003 >0000HQ 000800009 .00.coov .0a 0an00 51 .00H0 m0H08 0000 .000 000 mo 0oH000H8HH0 mo 0H0000 000 0H 0oH000HH000 H00000D 0 .000.0 A 00 maou0 I000 0>H000mm00 0H000 8000 0000000H0 0H0000H0H00Hm 000 000 :0: 0QH000000 00H3 00002 N .>0©\©0H0\08000 H 0 0 0 0 0 0 0 0 z H.m o.NH m.a H.HH m.a 9.0 0.0 N.0 mm 000a 0a0a 000 000a 000a 0m0a 0a0a 00.00a m 00H.o 0 0 0 0 0 0 0 0 z m.m m.m 0.mH m.0 m.0 0.9 o.m H.v mm 000a 00ma 000 0ama 000a 00ma 0m0a 0a.00a m 0m0.0 m m m m 0 0 0 m0 2 0.0 m.H N.@ 0.0 N.m m.mH m.m m.H mm 000a 000a 000 000a 00ma 000a 000a M0.00a m 0Ho.o 0 0 0 0 0 0 0 v z 0.0 N.NH 0.0 0.0 0.0 0.0H H.mH N.9 mm 00H 0MH Hm mmH ONH mNH omH Ho.mOH M woo.o 0H NH OH 0 o v N NI 0003 H0>0H 0003 0003 0003 0003 0003 0003 0003 000800000 00000H0 000800009 I000 .0HH005H0H0000 U00 0000HH08 0HDU0 mo 0OHOQ800000 U000 .HH 0H009 52 0 0 0 0 0 0 0 z 0.0 0.0 0.0a 0.0 0.0 0.0 0.0a mm 0a0a 000a 000a 00a0 00a0 00a0 000a 0 00a.0 0 0 0 0 0 0 0 z 0.0a 0.0a 0.0a m.0a 0.0a 0.0a 0.0a 00 000a 000a 00a0 0000 00m0 00m0 000a 0 0m0.0 m m m m 0 m m z 0.0a m.00 0.0a 0.0a 0.0a 0.0 0.0 00 000a 000a 0000 00a0 00a0 00a0 000a 0 0a0.0 0 0 0 0 0 0 0 z a.0a 0.0a 0.0a 0.0a 0.0a 0.0a 0.aa 00 00a 00a 00a 00a 000 000 00a 0 000.0 00 00 00 00 00 0a 0a a0>0a 0003 0003 0003 0003 0003 0003 0003 m0000H0 000800009 .00.0oov .aa 0an00 53 with no group of birds consuming greater than 20% more feed than another. All levels followed very similar increasing and decreasing trends over time. Feed consumption of bobwhites fed the treated diets was not significantly different than the control throughout the duration of the experiment (Table 12). The biweekly measure- ments varied, but this variation was consistent across all treatment levels, including control. All treatment groups showed a trend for increasing food consumption throughout the study. This increase can be attributed to the nutritional needs for normal growth and development of the birds as evident by the increases in body weight. All feed consumption values are estimates; wastage was not measured. Reproduction Feeding ethalfluralin in the diet had no detectable effect on the reproductive performance of the mallard or bobwhite (Table 13 and 14) breeders. No significant difference between the controls and any treatment level could be established for the following parameters: eggs per bird per day, percent of eggs fertile, l4-day embryo survival (mallard), 21-day embryo survival (mallard), ll-day embryo survival (bobwhite), 18- day embryo survival (bobwhite), percent of eggs pipped alive, percent of eggs pipped dead, percent of eggs unpipped alive, percent of eggs unpipped dead, percent of eggs hatching, and eggshell thickness. Similarly, no significant difference in duckling or chick survivability occurred between the control and any treatment level during the test period. 54 0>H000mm00 0H000 8000 0000000H0 0H0000H0H00Hm 000 000 :0: 0QH000000 00H3 00002 .Amo.o A my H000000 0 .000\00H0\08000 H 0a 0a 0a 0a 0a 0a 0a 0a 2 00.0 a0.0 00.0 0m.0 am.0 0m.0 0m.0 a0.0 00 00.00 0m.a0 00.0a 00.0a 00.0a 00.0a 00.0a 00.0a m 00a.0 0a 0a 0a 0a 0a 0a 0a 0a 2 00.0 00.0 00.0 00.0 00.0 00.0 0m.0 mm.0 mm 00.00 00.a0 00.00 00.0a 0m.0a 00.0a 00.0a 00.0a m 0m0.0 ma 0a ma ma ma ma ma ma 2 00.0 00.0 00.0 00.0 00.0 am.0 a0.0 0m.0 mm 00.m0 00.a0 00.0a 00.0a 00.0a 00.0a 0a.0a M0.0a m 0a0.0 0a 0a 0a 0a 0a 0a 0a 0a 2 00.0 00.0 m0.0 0m.0 0m.0 0m.0 00.0 00.0 mm m.m0 0.a0 0.00 0.00 0.0a m.0a 0.0a a0.0a 0 000.0 0a 0a 0a 0 0 0 0 0003 a0>0a 0003 0003 0003 0003 0003 0003 0003 000800000 >0000HQ . , 000800009 I000 .00a000a0a0000 000 000003000 0a000 00 00H0m800000 0000 .NH 0HQ09 55 0a 0a 0a 0a 0a 2 0m.a 0m.a 00.a 0m.a 00.a mm 00.00 00.00 00.00 00.00 00.m0 m 00a.0 0a 0a 0a 0a 0a 2 00.a 00.a 00.a a0.0 00.0 mm 0m.00 0a.00 00.00 0a.00 0a.00 m mmmdm ma ma ma ma ma 2 0a.a 00.a 00.a a0.0 00.0 mm 0m.00 00.00 00.00 00.00 00.00 m Hqum 0a 0a 0a 0a 0a 2 00.0 00.0 00.0 00.0 00.a 00 0.00 0.00 0.00 0.00 0.m0 0 000.0 00 00 00 0a 0a a0>0a 0003 0003 0003 0003 0003 >0000Ho 000800009 .3003 .NH 0HQ09 56 Table 13. The effects of feeding ethalfluralin on reproductive parameters in mallards. Dietary 14 day 21 day concentration Eggs embryo embryo Percent of set per Percent sur— sur- pipped ethalfluralin E/F/Dl hen fertile2 vival3 vival4 alive5 0.00% 2 0.393 27a 88a 96a 93a 2.2a SE 0.060 4.2 3.6 1.7 3.8 0.22 N 4 4 4 4 4 4 0.01% Y 0.35a 25a 86a 98a 96a 3.3a SE 0.044 3.1 3.5 0.7 1.6 1.96 N 3* 3 3 3 3 3 0.03% Y 0.61a 43a 86a 97a 95a 5.2a SE 0.050 3.5 5.2 0.7 0.9 0.74 N 4 4 4 4 4 4 0.10% i 0.38a 27a 74a 97a 96a 6.2a SE 0.073 5.1 9.0 2.2 2.1 3.38 N 4 4 4 4 4 4 l Eggs/female/day. 2 No. eggs fertile x 100/no. eggs set. 3 No. embryos alive at day 14 x 100/no. eggs fertile. 4 No. embryos alive at day 21 x lOO/no. eggs fertile. : No. of eggs pipped alive x 100/no. eggs fertile. Means in the same column with the same subscript are not significantly different from the control (P 2 0.05). Unequal replication is the result of the elimination of pens due to bird deaths. 10 11 Table 13. (con't). Dietary Egg concentration Percent Percent Percent Per- Percent shell of pipped unpipped unpip ed cent surviv- thickness ethalfluralin dead7 alive dead hatchlo ability (mm) 0.00% X 4.0a 0.5a 17a 70a 88a 0.45a SE 1.62 0.25 1.3 3.7 4.5 0.002 N 4 4 4 4 4 4 0.01% i 3.7a 1.0a 23a 66a 93a 0.43a SE 1.36 0.47 3.6 5.6 2.2 0.014 N 3 3 3 3 3 3 0.03% X 4.0a 0.8a 25a 61a 96a 0.44a SE 0.35 0.65 2.2 0.7 0.4 0.002 N 4 4 4 4 4 4 0.10% X 2.0a O-Oa 26a 61a 84a 0.44a SE 0.61 0.00 4.6 6.3 1.9 0.004 N 4 4 4 4 4 4 7 No. of eggs pipped dead x lOO/no. eggs fertile. 8 No. of eggs unpipped alive x 100/no. eggs fertile. 9 No. of eggs unpipped dead x 100/no. eggs fertile. No. of eggs hatched x 100/n0. eggs fertile. No. of ducklings surviving 14 days x lOO/no. ducklings hatched. 58 Table 14. The effects of feeding ethalfluralin on reproductive parameters in adult bobwhite. Dietary Eggs Per- Per- Per- concentration set cent cent cent Percent of per fer- sur- sur- pipped ethalfluralin E/F/Dl hen tile2 vival vival alive 0.00% 2 0.483 30a 63a 81a 77a 28a SE 0.070 19.2 8.7 8.8 8.5 5.3 N 15 15 15 15 15 15 0.01% X 0.52a 32a 70a 90a 87a 40a SE 0.062 15.6 7.4 4.9 4.9 4.0 N 13* 13 13 13 13 13 0.03% X 0-44a 35a 76a 92a 89a 39a SE 0.062 13.7 6.8 3.5 4.0 6.1 N 12 10 10 10 10 10 0.10% X 0.29a 18a 62a 80a 80a 36a SE 0.068 14.0 8.3 6.2 6.1 6.6 N 12 11 ll 10 10 10 1 Eggs/female/day. 2 No. eggs fertile x lOO/no. eggs set. 3 No. embryos alive at day 11 x 100/no. eggs fertile. 4 No. embryos alive at day 18 x 100/no. eggs fertile. 5 No. of eggs pipped alive x 100/no. eggs fertile. 6 Means in the same column with the same subscript are not significantly different from the control (P > 0.05). Unequal replication is the result of the elimination of pens due to bird deaths and disqualification (i.e. 0% because 0 fertile). 59 Table 14. (con't). Dietary Per- Per- Per- Percent Egg shell concentration cent cent cent Per- sur- thick- of pipped unpipped unpipped cent vivabi— ness ethalfluralin dead alive dead9 hatch10 lityll (mm) 0.00% X 0.53a 3.9a 11.9a 34a 64a 0.23a SE 0.399 2.28 5.74 7.8 7.9 0.005 N 15 15 15 15 9 14 0.01% X 0.92a 5.3a 2.8a 38a 62a 0.23a SE 0.810 2.18 1.86 6.5 6.1 0.005 N 13 13 13 13 ll 12 0.03% i 0.80a 1.1a 3.3a 43a 70a 0.23a SE 0.506 1.04 1.78 4.4 5.4 0.007 N 10 10 10 10 9 7 0.10% X 1.90a 7.4a 1.8a 33a 68a 0.22a SE 0.754 3.22 0.91 8.4 11.9 0.011 N 10 10 10 10 9 6 7 No. of eggs pipped dead x lOO/no. eggs fertile. 8 No. of eggs unpipped alive x lOO/no. eggs fertile. No. of eggs unpipped dead x lOO/no. eggs fertile. 10 11 No. of eggs hatched x lOO/no. eggs fertile. No. of chicks surviving 14 days x 100/no. chicks hatched. 60 Clinical Signs of Toxcity No clinical signs of toxicity could be directly attributed to feeding ethalfluralin to adult mallards or bobwhite at 0.01%, 0.03%, or 0.10% in the diet. The only clinical signs observed in mallards were feather loss, ataxia, and limping. Feather loss and ataxia occurred equally in all treatment groups including the control. These two signs were attributed to increasing the photoperiod which induced excessive aggression, primarily among the drakes. Ataxia occurred prior to death in all cases. Limping was attributed to injuries caused by the caging. No other abnormalities were observed in the mallards. Abnormalities observed in the quail were limping, swollen legs, ataxia, and weight loss. Limping and swollen legs were again attributed to injuries caused by the wire caging. The loss of weight and ataxia that was observed in one bird was believed to be the result of starvation. The cause of starva- tion was attributed to ulcerative enteritis found at necropsy. Mortality Thirteen mallards died during the course of the study: one female from the control diet, three males and one female from the 0.01% level, two males and two females from the 0.03% level, and two males and two females from the 0.10% level (Table 15). No mortality occurred prior to increasing the photoperiod. Following the increased photoperiod, the two males in each pen became excessively agressive resulting in the deaths of seven males. Excessive breeding and aggression between females caused the loss of six female birds. Tissue 61 Table 15. Interim deaths for mallards fed ethalfluralin. Dietary Date Cause concentration Sex of death of death 0.00% F February 6 General trauma 0.01% F February 6 General trauma 0.01% M January 20 General trauma 0.01% M January 21 Spleen amyloidosis 0.01% M January 12 General trauma 0.03% M November 22 General trauma 0.03% F December 30 General trauma 0.03% F January 10 General trauma 0.03% M January 31 General trauma 0.10% M November 25 General trauma 0.10% M January 2 General trauma 0.10% F January 31 General trauma 0.10% F January 3 General trauma 62 samples were taken for histopathology from all animals; one male fed the 0.01% diet showed signs of splenic amyloidosis. Eight bobwhite died during the course of the study (Table 16): one male and one female from the 0.01% diet, two males and one female from the 0.03% diet, and one male and two females from the 0.10% diet. The causes of death were acci— dental strangulation in two males, ulcerative enteritis in two females, emaciation in one male, impacted oviduct in one female, fractured tibia in one female, and an unknown cause in one male. No distinct signs of chemical toxicity were observed. Terminal Necropsy and Histopathology Terminal necropsy and histopathology indicated that very little substantive tissue alterations occurred in either species. Mallard abnormalities included magnum or shell gland obstruction in one female fed the control diet and one female fed the 0.10% diet, thymic candidiasis in one female on the 0.01% diet and hepatic microgranulomas in one female, ulcera— tive enteritis of the jejumum in a 0.01% female, crop mycotic ingluvitis accompanied by emaciation and mild intestinal enteritis in a 0.01% male, focal hemosiderosis of the heart and duodenum in a 0.03% male and air sacculitis in a 0.10% female. No consistent grossly abnormal observations or patho- logical alterations could be attributed to feeding ethalflura- lin in the diet. 63 Table 16. Interim deaths for bobwhite fed ethalfluralin. Dietary Date Cause concentration Sex of death of death 0.01% M September 9 Accidental strangula- tion 0.01% F November 8 Ulcerative enteritis 0.03% F February 14 Fractured tibia 0.03% M February 26 Emaciation 0.03% M January 12 Unknown 0.10% M October 4 Accidental strangula— tion 0.10% F October 22 Ulcerated enteritis 0.10% F February 18 Impacted oviduct DISCUSSION Of the 13 mallards which died during the course of the study, general trauma accounted for 12 deaths. The trauma was induced by aggressive behavior assodiatedImith the normal activities of reproduction. No mallards died prior to the onset of the reproductive phase. Because of the abnormally high concentration of breeding birds held together without a means of escape, normal conflicts which usually end in one duck fleeing resulted in prolonged aggression and death. Competition between males resulted in seven dead drakes while excessive tredding, rape, and female to female aggres- sion resulted in six female deaths. Similar behavior and mortality were reported by Hochstein and Ringer (1982), Flaga (1980), and Jones (1977) in reproductive feeding trials with mallards. The single interm death caused by splenic amyloidosis which occurred in a male on the 0.01% ethalfluralin diet could not be attributed to feeding ethalfluralin. No mallards or bobwhite on the 0.03 or 0.10% ethalfluralin diet showed similar signs of amyloidosis. Although all eight deaths in the bobwhite groups occurred on the ethalfluralin treated diets, no significant difference in mortality between the control and any other treatment level could be established. None of the deaths were considered to be chemically induced. The two incidences of strangulation were caused by the birds becoming caught between the cage bottom and the forward side of the cage, which allows the eggs 64 65 to roll out into the collecting area. One incident of a fractured tibia was also attributed to injuries caused by the caging. The two cases of ulcerative enteritis were not considered abnormal in a study of this size. Bobwhite are known to be extremely susceptable to the bacterium (Clostridium colinum), which causes this disease, when kept in captivity (Schwartz, 1977). Impaction of the oviduct occurs with muscle paralysis or partial twisting of the oviduct, (Schwartz, 1977) which is commonly found in large populations of laying hens. The cause of emaciation could not be determined. Pre—reproductive body weights for mallards and bobwhite were similar to weights reported by Jones (1977), Flaga (1981), and Hochstein and Ringer (1982) in similar reproductive studies. Trends for increasing body weights over the pre- reproductive phase occurred in both species. Hochstein and Ringer and Flaga also showed increasing weights over time while Jones reported a trend for slightly lower body weights in mallards over the same period. Body weights for male and female mallards did not continue to increase over the repro- ductive phase. Terminal weights were similar or lower than the final pre-reproductive weights. This decrease is in contrast to the body weights of reproductively active mallards reported by Jones (1977) and Hochstein and Ringer (1982). Hochstein and Ringer, and Jones reported average increases of 140 and 90 grams, respectively, during the reproductive phase, while the mallards of this study declined in weight an average of 50 grams over a similar period. Bobwhite weights at the 66 study termination were consistant with those reported by Flaga (1981) and Hochstein and Ringer (1982). Feed consumption values for mallards and bobwhite were similar to figures reported by Jones (1977), Flaga (l981),and Hochstein and Ringer (1982). Both species showed trends for increased consumption over time which corresponded to increases in body weights. A sharp decline (33%) in mallard feed consumption occurred during the tenth week of the study. This reduction corresponded to the increase in photoperiod which stimulated aggressive behavior between males. Once dominance was established in the pens, feed consumption rates returned to normal. No significant difference in reproductive parameters occurred between any treatment groups. Total number of eggs laid per bird per day for the mallards and bobwhite ranged from 25 to 43 and from 18 to 35, respectively. Mallard egg production in all dietary levels was in general agreement with results reported by Jones (1977), Federal Register (1978), and Flaga (1982). Bobwhite egg production with the exception of the 1000 ppm group, was also in agreement with these data sources. Bobwhite fed the 1000 ppm diet averaged only 18 eggs per bird over the 10 week laying period. The poor performance of this group was due to three females failing to produce eggs. Most of the remaining birds in the 1000 ppm group produced at rates equivalent to birds on the 300, 100, and 0 ppm diet. Fertility for mallards and bobwhite was consistant with data reported by Jones (1977), Flaga (1981), and Hochstein and 67 Ringer (1982). Fertility on control bobwhites and mallards fed 1000 ppm diets was lower than that reported as typical for these species. Embryo survival and the number of eggs pipped were also within the range provided by these sources. Hatchability for mallards and bobwhite was consistant with results reported by Elaga (1981). Hatchability values reported in the Federal Register (1978) for the bobwhite were higher than what was obtained in this study, while Hochstein and Ringer (1982) reported mallard hatchability below mallards on ethalfluralin. Mallard 14 day survival was lower in the control and 1000 ppm group than results reported by Hochstein and Ringer (1982), Federal Register (l978),and Jones (1977). All bobwhite groups had lower survival rates than reported by these sources. Lower survival rates in the bobwhite and possibly in the mallards may have been attributed to cold exposure. The birds were transported in cold weather from the hatchery to the testing facility. Although precautions were taken to prevent chill, the birds may have been exposed to the cold for short periods. The one-day old bobwhites' small volume to surface area ratio and inability to thermoregulate leaves it suscept- ible to rapid cooling. Young birds incapable of thermoregula- tion show decreased survivability when their body temperatures drop too low from cold exposure (Ringer, 1981). Eggshell thickness for both species was consistant with data published by Hochstein and Ringer (1982), Flaga (1981), and Jones (1977). Mallard eggshell thickness was considerably _ . 00.00-:4 68 greater than the values reported in the Federal Register (1978). No significant chemically induced pathological altera- tions occurred in the mallards or bobwhite. A few inflammatory, bacterial, and fungal lesions occurred in both species, but were not considered abnormal for a population of this magni- tude. One mallard showed signs of splenic amyloidosis. Amyloidosis is characteristic of tuberculosis, osteomyelitis, adn carcinoma (Thomas, 1981). None of these diseases were diagnosed in this case. CONCLUSION Ethalfluralin did not produce toxicity when fed to mallards and bobwhite at concentrations up to 1000 ppm in the diet. Ethalfluralin caused no observable chemically induced alterations in normal behavior, reproductive behavior, reproductive functionwmr performance, tissue function, or tissue morphology. Adult survivability, food consumption, body weight, egg production, and fertility were not altered significantly by ethalfluralin. Similarly, eggshell thick- ness, embryo survivability, hatchability, and 14 day chick and duckling survivability were not significantly effected by this herbicide. —— ___—._ -_. .~ 0..., LITERATURE CITED Alder, E.F., W.L. Weight, and Q.F. Soper, 1960. Control of seedling grasses in turf with diphenylacatonitrile and a substituted dinitroaniline. Proc. N. Cent. Weed Contr. Conf. 17:23-24. Barrentine, W.L. and G.F. Warren, 1971. Shoot zone uptake and translocation of soil applied herbicides. Weed Sci. 19:156-161. Bartels, P.G. and J.L. Hilton, 1973. Comparison of triflura- lin, oryzalin, pronaminide, propham, and colchicine treatments on microtubules. Pest. Biochem. Physiol. 3:462-472. Bayer, D.E., C.L. Foy, T.E. Mallroy, and E.G. Culter, 1967. Morphological and histological effects of trifluralin on root development. Am. J. Bot. 54:945-952. Davis, W.A. and E.M. Rahn, 1970. Atrazine, trifluralin, and bromacil in surface water from selected agriculture and industrial sites. Proc. Northeast Weed Contr. Conf. 24:283. Elanco, 1977. Technical report on Sonalan® a selective, soil-encorporated, preemergence herbicide. Lilly Research Laboratories, a division of Eli Lilly and Co., Indianapolis Indiana 46206. pp. 4. Elanco, 1979. Herbicide Handbook of the Weed Science Society of America. Ed.‘W.R,.Mulleson, 4th Edition. pp. 200-203. Emmerson, J.L. and R.C. Anderson, 1966. Metabolism of tri- fluralin in the rat and dog. Toxicol. Appl. Pharmacol. 9(1):84-97. Federal Register, July 3, 1975a. U.S. Environmental Protection Agency. Vol. 40, No. 129. pp. 28281-28284. Federal Register, July 3, 1975b. U.S. Environmental Protection Agency. Vol. 40, No. 129. pp. 28260-28261. Federal Register, July 10, 1978. U.S. Environmental Protection Agency. Vol. 43, No. 132. pp. 29729-29731. Flaga, C., 1981. The effect of dietary administration of fluridone on selected reproductive parameters of bobwhites and mallards. M.S. Thesis, Michigan State University. Gagnon, S.A. and K.C. Hamilton, 1973. Persistence of various dinitroanilines under irrigated and desert fallow condi- tions. Proc. W. Soc. Weed Sci. 26:24—25. 69 7O Gill, J.L., 1978. Design and Analysis of Experiments in the Animal and Medical Sciences. Vol. 1. Iowa State University Press, Ames, Iowa. pp. 409. Golab, T., R.J. Herberg, S.J. Parka, and J.B. Tepe, 1967. Metabolism of carbon-l4 trifluralin in carrots. J. Agric. Food Chem. 15:638-641. Golab, T., R.J. Herberg, E.W. Day, A.P. Raun, F.J. Helzer, and G.W. Probst, 1969. Fate of carbon—l4 trifluralin in artificial rumen fluid and in runinant animals. J. AGric. Food Chem. 17(3):576-580. Golab, T., R.J. Herberg, J.V. Gramlich, A.P. Raun, and G.W. Probst, 1970. Rate of benefin in soils, plants, artifi- cial rumen fluid and the ruminant animal. J. Agric. Food Chem. 18:838-844. Hamdi, Y.A. and M.S. Tewifik, 1969. Decomposition of the herbicide trifluralin by a pseudomonad. Acta. Microbiol. Pol. Ser. B. 1(2):83-84. Helling, C.S., 1971. Pesticide mobility in soils. III. Influence of soil properties. Soil. Sci. Soc. Am. Proc. 35:743-748. Helling, C.S., 1976. Dinitroaniline herbicides in soils. J. Environ. Qual. 5:1-15. Hill, E.F., R.G. Heath, J.W. Spann, and J.W. Williams, 1975. Leathal dietary toxicities of environmental pollutants to birds. U.S.D.I., U.S. Fish and Wildlife Service, Special Scientific Report-Wildlife No. 191, Washington, D.C. p. 61. Hochstein, J.R. and R.K. Ringer, 1982. One generation reproductive study in mallards and bobwhite with Lilly compound 48828. Research Report to Eli Lilly and Co., pp. 56. Hornshaw, T.C., 1981. Renewed use of underutilized species of Great Lakes fish for animal feed. M.S. Thesis, Michigan State University. Humphreys, W.H., D.A. Addison, R.D. Hicks, K.E. McNeill, J. F. Nicholson, L.B. Rowland, and H.L. Webster, 1978. Ethalfluralin for weed control in cucurbits. Proc. South Weed Sci. Soc. 31:159-166. Ilnicki, R.D., J.R. Justin, and R.W. Michieka, 1977. The effects of some dinitroaniline herbicides on kenaf. Proc. Northeast Weed Sci. Soc. 31:93-97. 71 Jones, R.E., Jr., 1977. Toxicity of diisoprophlmethylphos- phonate and dicyclopentadiene on the mallard (Anas platyrhynchos). M.S. Thesis, Michigan State University. Kecherside, M.D., T.E. Boswell, and M.G. Merkle, 1969. Uptake and translocation of substituted aniline herbicides in peanut seedlings. Agron. J. 61:185-187. Kenaga, E.E., 1973. Factors to be considered in the evaluation of the toxicity of pesticides to birds in their environment. Env. Qual. Safe. 2:166-181. Kennedy, J.M. and R.E. Talbert, 1976. Comparative persistence of dinitroanilines on the soil surface. Proc. South Weed Sci. Soc. 27:321. Kennedy, J.M. and R.E. Talbert, 1977. Comparative persistence of dinitroaniline type herbicides on the soil surface. Weed Sci. 25(5):373—381. Koren, E., 1972. Leaching of triflurolin and oryzalin in soil with three surfactants. Weed Sci. 20:230-232. Leitis, E. and D.G. Crosby, 1974. Photodecomposition of tri— fluralin. J. Agric. Food Chem. 22:842-848. Messersmith, C.G., O.C. Burnside, and T.L. Lavy, 1971. Biological and nonbiological dissipation of trifluralin in soil. Weed Sci. 19:285-290. Moreland, D.E., F.S. Farmer, and G.G. Hussy, 1972. Inhibition of photosynthesis and respiration by substituted 2,6- dinitroaniline herbicides. Murphy, H.J. and L.S. Morrow, 1978. An evaluation of several herbicides for weed control in Katahid potatoes. Proc. Northeast. Weed Sci. Soc. 32:157-160. Murphy, S.D., 1980. "Pesticides". In: Casarett and DouZZ's Toxicology: The Basic Science of Poisons. Ed. Doull, J., C.D. Klaassen, and M.O. Amdur. 2nd edition. MacMillan Publishing Co., Inc., pp. 778. Newsom, H.C. and W.G. Woods, 1973. Photolysis of the herbicide dinitramine (N3,N3-diethyl-2,4-dinitro-6-trifluoromethyl- m-phenyl-enediamine). J. Agric. Food Chem. 21:598-601. Nishimoto, R.K. and G.F. Warren, 1971. Shoot zone uptake and translocation of soil applied herbicides. Weed Sci. 19: 156-161. Omer, V.V. St., 1970. Chronic and acute toxicity of the chlorinated hydrocarbon insecticides to mammals and birds. Can. Vet. J. 11(11):215-226. 72 Parka, S.J. and H.M. Worth, 1965. The effects of trifluralin to fish. Proc. South. Weed Conf. 18:469-474. Parka, S.J., Q.F. Soper, and J.R. Beck, 1976. History of development and physiology of dinitroaniline herbicides. Presented as an invitational paper at the Weed Science Society of America Meeting, Denver , Colorado. Parka, S.J. and Q.F. Soper, 1977. The physiology and mode of action of the dinitroaniline herbicides. Weed Sci. 25: 79-87. Parochetti, J.V. and E.R. Hein, 1973. Volatility and photo- decomposition of trifluralin, benefin, and nitralin. Weed Sci. 21:469-473. Parochetti, J.V., G.W. Dec, Jr., and G.W. Burt, 1976. Volatility of eleven dinitroaniline herbicides. Weed Sci. 24(6):529-532. Parochetti, J.V. and G.W. Dec, 1978. Photodecomposition of eleven dinitroaniline herbicides. Weed Sci. 26:153-156. Plimmer, J.K. and V.I. Klingebiel, 1974. Photodecomposition of N-sec-butyl-4-tect-butyl-2,6-dinitroaniline. J. Agric. Food Chem. 22:689-693. Prince, M.A., R.D. Radeleff, S.E. Kunz, and R.E. Everett, 1971. Toxicity of soil application of dursban to bobwhite quail. Tex. Agric. Exp. Stn. Prog. Rep. 3000. pp. 1-3. Probst, G.W., T. Golab, R.J. Herberg, F.J. Holzen, S.J. Parka, C Van der Scans, and J.B. Tepe, 1967. Fate of triflura- lin in soils and plants. J. Agric. Food Chem. 15:592- 599. Rahman, A. and R. Ashford, 1973. Persistance of trifluralin under field conditions in Saskatchewan. Can. J. Plant Sci. 53(2):421-423. Ringer, R.K., 1981. Personal Communication. Sheets, T.J., J.R. Bradley, Jr., and M.D. Jackson, 1973. Movement of trifluralin in surface water. Proc. South. Weed Sci. Soc. 26:376. Schwartz, L.D., 1977. Poultry Health Handbook. 2nd edition. College of Agriculture, The Pennsylvania State University. pp. 221. Thomas, C.L., 1981. Taber's Cyclopedic Medical Dictionary. 14th edition. F.A. Davis Co., Philadelphia, PA. pp. 1818. 73 Worth, H.M. and R.C. Anderson, 1965. The toxicity of tri- fluralin, Treflan®, an herbicide to mammals and chickens. Proc. Southern Weed Conf. 18:711-712. Worth, H.M., 1968. The toxicologic evaluation of benifin and trifluralin. Ind. Med. Surg. 37:545. Wright, W.L. and G.F. Warren, 1965. Photochemical decomposi- tion of trifluralin. Weed Sci. 13:319-331. APPENDIX A Composition of duck breeder-layer diet. Ingredients Percent lbs/ton Corn, yellow ground 62.42 1248.4 Soybean meal, solent extracted, dehulled (49%) 19.58 391.6 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 18.6 Dicalcium phosphate, feed grade 0.65 13.0 Limestone 6.82 136.4 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 4.0 100.00 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 B12, 100 mcg of biotin, and 125 mg of ethoxyquin per kg of complete feed. 74 no: mucmflomumcfl xumuwflo umcuo mo ucmucoo >HMpmHo mo pcmoumm m mm pmmmmumxm moflom OGHEM map ucmmmumwu mammnucmumm cfl mosaw> .Umwsaocfl uxHEmum CHEmuH> Eouw omHHmQSm m4 m .cflmuoum H AVN.HV Aoo.vv AHV.HV Amm.mv Avm.mv A¢N.mv HAhm.mv 75 «m.a w .oflom camaocflq mm.o w .cmzmowawua «v.0 moflom ocHEm Homasm Hmuoe mm.0 w .mcflummo mv.o w .mcflcoflnumz OH.H w .mcnosao mm.0 w .mCHmmq mN.H w .mcHCHmH4 mum mx\moe .anuon boa mx\moE .mam CHEmuH> Nmoa mx\moE .Uflom oaaom m.m mx\mE .mcHEMHSB m.m mxxas .cH>mHmonnm o.n mx\mE .mm CHEmuH> m.oH mx\mE oflom 0Hcm£uoucmm m.mm mx.mE .GHUMHZ voma mx\mE .mCHHOQU m.0 mmx\mE .M CHEMHH> oom N.om omom H Hmwa comm NhHN boa m.mm m.a m.moa 0.am mv.o mm.0 mN.m hm.oa mm.m om.m AH.HnV mmmm om.ma mx\ooH .o cHsmpH> mx\mE .m CHEmuH> mx\oH .« cflsmufl> mx\mE .mcflpoH mx\ms .Esfloom mx\mE .Esammmuom mx\mE .Esflmmcmmz mx\moe .ESHGmHmm mx\mE .UCHN mx\mE .Hmmmou mx\mE .couH mx\mE .mmocmmcmz .msuozmmonm magmafim>< w .msuosmmozm w .EDHOHMU w .sm4 w .umnflm w .umm oaumu m\wz mx\amom .mmumcw .umz w .cflwuoum .gwflo umhmHlumommHQ xUSC mo mflmhamcm HocOHUHHpsz m XHQmemd APPENDIX C Composition of quail breeder-layer diet. Ingredient Percent lbs/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 D, 40 mg of vitamin E, 0.7 mg of vitamin K, 1000 mg of choline, 70 mg of niacin, 4 mg of panthothenic of riboflavin, 100 mcg of vitamin B12, 100 mcg of and 125 mg of ethoxyquin per kg of complete feed. Trace mineral premix provides 75 mg of manganese, zinc, 25 mg of iron, and 1 mg of iodine per kg of feed. 76 acid, 4 mg biotin, 50 mg of complete 77 mumumflp mo usmonmm m mm ommmmumxm moflom ocHEm map ucmmmummu mflmmzpcmumm cflcwwwmmm a m.mm mx\ma .m anamufl> com mx\ooH .a anamun> mm.a m .ofiom oflmdosflq mmov mx\oH .< cfismpn> 10¢.Hv hmm.o w .canOpmsHe H mxxme .mcflon Amo.vv mmm.o mason omen mx\me .esnwom ocHEm HDMHSm Hmuoe mm.0 w .Eofimmmuom An¢.HV mmm.o w .mcflumso «cam mx\ma .ssflmmcmmz Amo.mv mmm.o w .mcflcoflspmz HNH mx\moe .echmHmm Aom.mv mqm.a w .mcnomaw «m mx\ms .ocHN Amm.mv omm.H w .wsflqu ma mx\mE .Hmmooo Ama.nv 5H5.H w .mcflcflmum oHH mx\ma .couH Hom mx\moE .CHpOHm mm mx\mE .mmmcmmcmz moa mx\mE .mHm CHEmpH> mm.0 w .msuonmmocm .Hfim>¢ m.a mx\ma .eflom onaom 00.a w .msuosmmonm ~.m mx\me .mcflamflna 0m.a w .aafloamo m.o mx\me .cfl>mamonflm 0m.a m .zm< m.n mx\ms .mm anam0H> mm.m w .umnflm m.ma mx\mE .pflom oacmspowcmm mm.m w .umm no mx\ma .afiomnz Amo.mmv ms.mHH oflumu m\mz mmam mx\me .mcflaono momm mx\amom .smumcm .002 m.0 mx\me .m cflsmpfl> oo.em w .cflmuoum .umflo mommHuuwommun Hamsv mo mammamcm HMGOHUansz Q XHDmem< APPENDIX E 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, feed grade 0.64 12.8 Limestone 0.34 6.8 Trace mineral premix TK—Ol (1.02)1 0.10 2.0 Salt 0.30 6.0 Vitamin premix TK—01 (1.03)2 0.50 10.0 Methionine hydroxy analog 0.20 4.0 100.00 2000.0 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. 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 mg of choline, 70 mg of niacin, 4 mg of pantothenic acid, 4 mg of riboflavin, 100 mcg of vitamin B12, 100 mcg of biotin, and 125 mg of ethoxyquin per kg of complete feed. 78 79 .cwaoum wnmume mo #cmoumm m mm pmmmmumxm mpHom OQHEm map ucmmmumou mHmmnpcmumm CH mmsHm> uoc mucmemnmcH humumHo Hmzpo mo ucmucoo uxHEmum CHEmuH> Eonw omHHmmom m4 m .pmosHosH H Amm.Hv Hom.mv Amv.Hv AHv.mv AHm.mV Aom.mv Amo.hv NN.H mm.0 mm.0 mm.0 mm.0 om.H HN.H mm.H 0mm 50H mva m.m m.m m.h m.HH m.mm NmmH m.0 w .oHom UHmHocHH w .cmsmoummue oHom ocHEm wdesm Hmuoe m .mcHummu w .wchoHnumz w .mcHomHo w .mchmq mw .mchHmufl mx\moE .chon mx\moE .NHm GHEwuH> mx\moE .pHom OHHom mx\mE .mcHEMHSB ar\ae .ea>mamoeam mx\mE .wm CHEMDH> mx\mE .pHom UHcmcuoucma mx\mE .GHosz ar\ae .aeaaoro me\mE .M GHEmuH> com H.mm vmmv H ommH omHm mvHN NHH m.mh m.mH m.mm o.hm mv.o 05.0 0m.0 mH.m vu.m vh.v Am.mov m.mMH whom Ho.mm mx\ooH .o casmua> mx\mE .m CHEmuH> mxon .< casmue> mx\mE .mcHooH mx\mE .EsHoom mx\mE .ESHmmmpom mx\mE .Eonmcmmz mx\moE .EchmHmm mx\mE .UCHN mx\ma .Hmmmou mx\mE .GOHH mx\mE .mmmcmmcmz w .msuonmmonm .HHm>¢ w .msuosmmonm w .EsHonU w .£m< w .HmQHm w .umm Oeumu m\mz mx\HmoM .wmumcm .umz w .chuoum m xHQmemfl .umHo umuumum xoso mo mHmmHmcm APPENDIX G Composition of quail starter. Ingredient Percent lbs/160 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 2.25 100.00 750.00 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 B12, 100 mcg of biotin, and 125 mg of ethoxyquin per kg of complete feed. 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. 80 81 .chpoum mumuoHo wo ucwoumm m mm ommmmumxm mpHom ocHEm map pcmmoummn mHmmzucmumm CH mmsHm> H m.vm mx\m8 .m cHEmuH> com mr\ooH .o seamua> eH.H m .eaom 0H0Hocaq mmme ox\DH .< casmua> Aom.ov meH.o m .cmemouesue H mx\ms .maaeoH Amo.av aNH.H m .meaom mare mrxms .esaeom ocHEm HowHom Hmuoe MH.H w .EsHmmmuom Amv.Hv mHv.o w .mchmmu mem mx\mE .EsHmmcmmz Aem.mv oae.o m .mcaeoHsuwz mma ox\moa .Esaamamm Amm.mv mmv.H w .mcHomHo mm mx.mE .ocHN Amm.mv meo.H m .meaqu om mr\ms .ummdoo AHH.>V Ham.H w .wchHmum mmH mx\mE .conH «mm mx\moE .chon mm mx\mE .mmocmmcmz >0H mx\moE .mHm GHEmuH> mm.0 w .mouonmmosm .HHm>¢ m.a mr\me .eaom oaaoe mm.0 m .mseoeemoem m.m mxxme .meaamane oe.o w .asaoemo m.a mr\me .aa>mamoeam am.m a .emm m.e mr\me .em ea2m0a> ae.m a .emeam m.ae mr\me .eaom oaemepoucmm ea.m m .umm NOH mr\me .eaomaz Aea.eec ~N.Noa oaeme e\mz ommm mx\mE .wcHHonU Hmmm ox\Hmom .hmumcw .umz m.0 mx\me .x casmua> mm.e~ m .camuonm .Hmuumum HHmsv mo mHmmHmcm HmcoHpHHusz m XHQZMQQé slnimmifliigifiiiiii"