Because ovotestes and Müllerian duct malformations are sensitive measures of endocrine disruption from in ovo exposure and are likely to persist in 14 day old chick, these tissues should be collected from at least one of the weekly batches of the F1 and F2 surviving chicks. Weights of the gonadal tissue and its appearance at necropsy should be recorded. The tissue should be preserved for histological examination and the gonadal tissues from control chicks and chicks of the high dosed parents evaluated histologically. Additional dose groups can be examined if warranted by the presence of abnormal gonadal tissue in the high dose chicks.

Having information on the dose and embryonic/ chick histological response to the chemical would provide exposure information and comparison for field studies or cases in which egg samples of wild birds can be collected.

7.1.3 Histopathology in Sexually Mature Individuals Histological methods are sensitive indicators of gonadal function and pathological processes in animals and can provide information on the site of action of a xenobiotic. However, usefulness of these observer based methods to quantify effects in avian reproduction toxicity tests is limited by lack of a uniform classification system and codification of evaluation criteria. Standardization of structural measurements, such as tubule lumens, randomization of selected structures, such as tubules, for measurement, and scoring criteria would provide repeatable interpretation of tissue changes induced by xenobiotics and endogenous hormones in birds that can be verified by different investigators. For example, routine histological evaluations detect testicular damage only when there is severe depletion of the seminiferous epithelium, obvious cellular degeneration, or sloughed cells in the seminiferous tubule lumen. Less prominent, though reproductively important lesions could be identified by morphometric measurements of the testicular structures. Of particular importance in the evaluation of testicular injury is the use of seminiferous tubule stages ( Creasy 1997; Lin and Jones 1993). Quantification of cell staging involves examining cross sections of seminiferous tubules to determine the frequency distribution of distinct, sequential associations of developing germ cells, which could be identified as stages of proliferation and differentiation. In the Japanese quail, these stages have been well characterized and are arranged in a helix that extends along the length of the seminiferous tubule ( Lin and Jones 1990). The duration of the stages is a time specific process that ranges from 2.5 h to 15.5 h in Japanese quail ( Lin et al. 1990). Therefore, the abundance of any given stage is directly proportional to the duration of the stage. Quantitative and semiquantitative methods devised for the assessment of the effects of xenobiotics on testicular Battelle Draft 77 April 23, 2003 germ cells involve measurement of the cross sectional area of semiferous tubules and the ratio of the germ cell to the Sertoli cell, and counting the number of spermatids per seminferous tubule ( Creasy 1997).

In addition to the codification of lesions or structural measurements, components of histological examinations that need to be standardized to avoid artifacts that can interfere with the evaluations are 1) handling of unfixed reproductive tissues; 2) the tissue embedding and sectioning processes; 3) selection of the chemical fixative protocol and stains; 4) sectioning procedures; and 5) the codification of lesions or structural measurements. Handling artifacts in testes are common and result from dropping testes on weighing scales or from excessive manipulation of the tissue with forceps ( Foley 2001). The common fixatives used in histology of reproductive tissues are 10% neutral buffered formalin and Bouin's solution. Tissue shrinkage and staining properties are different for these two fixatives. Formalin fixation, though routinely used, results in artifacts in the germinal epithelium of the testis when used prior to embedding in paraffin. Bouin's fixative provides superior preservation of cellular detail in tissues such as the testis ( Foley 2001). There are a number of disadvantages of using Bouin's fixative: critical timing of tissue trimming and processing, and storage of fixed tissue, for example. Therefore, protocols using a combination of initial fixation of the testes in formalin followed by immersion of trimmed sections in Bouin's solution have been developed to minimize the use of Bouin's fixative and retain the ease of collecting tissues in formalin   a single, multipurpose fixative ( Foley 2001). For the most precise staging of the seminferous epithelium in the testis, the germ cell acrosome should be stained using periodic acid Schiff's method ( Clermont 1972). Formalin fixation can be used to examine the seminferous epithelium if it is then embedded in plastic and stained with touluidine blue ( Russel and Frank 1978; Ulvick et al. 1982). Hematoxylin and eosin or touluidine blue are common stains applied to gonadal tissue. Each provides a different view of tissue structure. Use of plastic embedding provides greater support of the tissue and thinner sections. Samples for histopathological examinations should be collected from the same area of each organ and serially sectioned along the long axis of the gonad. Samples should be examined " blind," without knowledge of the treatment.

Because of their large volume of yolk, mature oocytes and ova have been difficult to preserve for microscopic examination. Immersion fixation of follicular oocytes in paraformaldehyde and glutaraldehyde fixative followed by a second immersion in fresh fixative to assure penetration and adequate preservation provides the best fixation of the ovarian follicles for light microscopy. For electron microscopy, tissues are trimmed further and placed in osmium tetroxide before sectioning ( Bakst 1993).

Preparation of specimens from the oviduct or excurrent duct for light microscopy are routine. However, the secretory function of the oviduct appears to develop sooner than the organs gross differentiation ( Reddy et al. 1992); therefore, use of histochemical or immunocytochemical techniques to detect oviductal secretion of fibrous albumen proteins could be used to measure the maturation of the organ. These oviductal secretions can be problematic when one is examining the tissue with scanning electron microscopy, because they can obstruct the luminal surface. Similarly, a clot of sperm can obscure the luminal surface in the excurrent ducts of the testis. Battelle Draft 78 April 23, 2003 Procedures are available that reduce the incidence of these interferences ( Bakst and Howarth 1975; Kirby et al. 1990).

Lectin probes have been used to distinguish the stages of avian spermatogenesis, by binding to a terminal saccharide of a glycoconjugate expressed on either a spermatogonia, spermtocyte, spermatid, or Serotoli cell ( Bakst and Cecil 1988). However, fixatives can mask this binding. Unfortunately, the fixative did not mask lectin binding, but provided adequate tissue preservation contains merucuric chloride ( sublimate formalin). Substitution of less hazardous zinc chloride could be possible, but has not been evaluated.

There are fewer professional pathologists with experience in evaluating avian tissues than of those with experience in mammalian histology. The level of quanitification and codified description of reproductive organs for GLP evaluations that has developed over the years for mammalian toxicity testing is lacking in avian applications. In particular, familiarity with the stages and cycle of the seminiferous epithelium of the testis that is essential to injury assessment has not been applied to avian toxicity studies. However, the stages of the germ cells in the Japanese quail and poultry are well described and used to evaluate fertility and the impact of endocrine moderating chemicals on spermatogenesis. Histological examination by professional pathologist in the USA costs from $ 8 to $ 16 per tissue, when conducted under GLP criteria for mammalian studies.

7.1.4 Histopathology and Organ Weights of Nonreproductive Tissues The thyroid, adrenal gland, and brain are potentially affected by a number of EDCs. They can be analyzed by classical histological techniques for evidence of abnormal structure that generally results in dysfunction. However, histopathological examination of tissues from birds exposed to potential reproductive toxins in avian reproduction toxicity tests has been limited ( Mineau et al. 1994). New immunological methods can provide more objective and precise information of changes in morphology and function in tissues. A review of mammalian reproduction studies indicates that the adrenal gland is the most susceptible of the endocrine glands to chemical induced lesions ( Ribelin 1984). About 40% of the studies reporting histological effects in endocrine tissue were compounds that damaged adrenal glands. The thyroid was affected by15% of the compounds. As noted above, methods of tissue handling, fixation, staining and lesion scoring should be clearly established to help standardize evaluations.

<IP_ADDRESS> Brain. In recent years, several research groups have been investigating the effect of steroids on the innervation of the septopreoptic region of the hypothalmus of birds. The medial preoptic nucleus and lateral septum are sexually dimorphic and highly involved in the activation of adult sexual behavior of Japanese quail ( Halldin et al. 1999; Balthazart and Surelemont 1990 a, 1990 b; Panzica et al. 1996). Hormonal manipulation of juveniles and adults results in profound morphological changes in these areas of the brain, including changes in the density of vasotocin fibers and the number and size of aromatase containing neurons ( Panzica et al. 1994). These changes occur in response to photostimulation and aging, but also as a result of exogenous steroid exposure ( Panzica et al. 1996). Although examination of the septopreoptic tissue can provide information on the underlying mechanisms of reproductive depression, measures of the Battelle Draft 79 April 23, 2003 behaviors activated by this area are highly sensitive to changes in steroid concentration and more economical to perform. Fixation of the tissue and the immunochemistry on serial sections of the septopreoptic region of the hypothalamus are relatively lengthy processes. The specific antibody for the immune reaction is not commercially available, and quantitative determination of the density of the immunoreactive structures requires computerized image analysis.

<IP_ADDRESS> Thyroid. The close link between secretory function of endocrine tissue, such as the thyroid, and proliferative activity agrees with the frequent observation in toxicity studies that a common response to excessive stimulation is endocrine hyperplasia neoplasia ( Pawlikowski 1982). Generally, therefore, organ weights should be obtained prior to histological examination of these organs. However, excising and trimming firmly bound organs such as the thyroid gland for organ weight determination is not advisable because of the risk of disruption of histological sections. The thyroid is a small, firmly attached organ deeply embedded in tissue and is difficult to excise and trim. Not only does this introduce variation in organ weight measurements, but the degree of handling required can so strongly affect the quality and orientation of sections as to offset the benefit of organ weight data. Therefore, thyroid tissue should not be excised for organ weight, but removed with minimal handling for histological preparation ( Greaves 1999). A means of obtaining thyroid weights is to excise the organ within the surrounding tissues and fix it in preservative prior to trimming. Subsequently, trimming and weighing the preserved gland will minimize damage to the gland and allow for more consistent weights. Thyroid hypertrophy can then be quantified histologically. Enlarged thyroids may reflect either a hyper or hypofunctioning gland ( Wentworth and Ringer 1986). Initially, examination of the thyroid should be completed on the controls and high dosed groups. Histopathology of the thyroid can be conducted on other groups if warranted by the detection of lesions in the thyroid tissue of the high dosed birds. Immunocytochemical techniques have superseded trandioal tinctorial stanis for the demonstration of poylpetide hormones ( Greaves 1999).

<IP_ADDRESS> Adrenal Gland. As noted above, the adrenal gland appears to be most susceptible of the endocrine organs to chemically induced structural change. Susceptibility appears to be due to the accumulation of lipophilic toxins in the lipid stores of the cortical cells and the metabolic conversion of chemicals to reactive toxic compounds ( Capen 2001). In addition, such changes appear to be frequently associated with alterations in reproductive organ function and structure ( Maronpot 1987). Even so, the range of histological responses in the adrenal gland is small ( Tucker 1998). The most commonly induced morphological change is hypertrophy with increased vacuolar degeneration ( Ribelin 1984). Cortical ( interrenal) hypertrophy resulting from impaired steroidogenesis and the accumulation of precursors, such as neutral fats, in the cortical cells is found in animals treated with either endogenous or exogenous steroids. The hypertrophy can be so great that it impairs cellular function. Administration of adrenocorticotropic hormone ( ACTH) will also cause hypertrophy of the cortex from increased size of the cells. In contrast, exogenous steroid treatment causing depressed ACTH levels results in atrophy of the cortex in mice. Cortical hyperplasia may also result from hormonal disruption, and propylthiouracil has been shown to cause proliferation in chromaffin cells medulla of mammals ( Tucker 1998; Ribelin 1984). Battelle Draft 80 April 23, 2003 Because of adhering fat, adrenal glands can be difficult to excise cleanly for organ weight measurements. To avoid variation in adrenal weights, a standardized necropsy protocol for the tissue should be established.

7.1.5 Japanese Quail Male Cloacal Gland Measures The cloacal gland is a secondary sex characteristic of male Japanese quail that develops in response to testosterone and has been used to measure androgen status in the male during sexual maturation ( Ottinger and Brinkley 1979a, 1979b). Typically, the length and width of the gland is measured with calipers, and the area is recorded as the product of longest length and greatest width of the gland. Coefficients of variation are typically low ( 4 to 10) for this measure. Measurement of just the length of the cloacal gland has also been used successfully to demonstrate the effect of hormone treatment on maleness in Japanese quail ( Hutchison 1978) and cloacal gland volume in cubic centimeters has also been used to assess reproductive activity ( CV of 4%) using the following formula 4/ 3Bab2 ( 2)

where a is half of the length of the long axis and b is half of the length of the short axis ( Chaturvedi et al. 1993).

Treatment with xenoestrogens in ovo has been shown to significantly decrease the area of the gland in the sexually mature male ( Halldin et al. 1999), and cloacal gland swelling has been used as a reliable indicator of androgenic activity in females treated with testosterone ( Phillips et al. 1997). Treatment of males with estrogen results in reduced gland area and deformities in the cloacal orifice ( Phillips et al. 1997; Halldin et al. 1999). As a measure that can be taken repeatedly during the course of a reproductive test, it is a useful endpoint for monitoring maturation and androgen status in P1 and F1 parent populations. The amount of foam in the cloacal glands is also used to determine male reproductive fitness. The cloacal gland is palpated and the proteinaceous foam measured with calipers and reported in units of square millimeters ( Sachs 1967; Hutchison 1978). However, foam measurements can be highly variable. Date of actual foam formation is used as a maturation endpoint for the male Japanese quail. This measure is applicable to the P1 ( if a prematuration exposure is used) and F1 chicks as they approach breeding.

7.2 Sexual Differentiation ( Including Time of Onset of Egg laying)

As described above in Section 7.1.2, in ovo exposure to estrogenic compounds will modify sex organ differentiation in birds, feminizing male sex organs and causing Müllerian duct abnormalities in genetic females. Failure of male gonadal differentiation in the presence of ecoestrogens may not persist from embryonic exposure into adulthood, but abnormalities are conserved in females ( Rissman et al. 1984; Adkins Regan et al. 1995). Ovotestes regress after hatch and disappear by sexual maturation ( Scheib and Reyss Brios 1979). Reorganization of the POM in the hypothalmus as a result of exposure to extrogenic compounds can result in apparently normal males that lack sexual behavioral differentiation ( Berg et al. 1999). Battelle Draft 81 April 23, 2003 Sexual differentiation endpoints include an array of morphological and behavioral landmarks that are easily recorded during the course of a reproductive toxicity test. The sequence of behavioral and morphological landmarks of sexual differentiation in male Japanese quail has been described ( Ottinger and Brinkley 1979a, 1979b). Their onset and maximum expression are highly correlated in time with serum testosterone levels. Table 7 1 lists these landmarks in sequence of their appearance during sexual maturation. Also listed are estrogen dependent landmarks that include appearance of dimorphic plumage and the onset of egg laying in females. Age at sexual maturity was among the most sensitive measures of impaired reproduction in the female birds exposed to lead and was detected at dietary concentrations that caused no body weight loss or overt signs of toxicity ( Edens et al. 1976). Onset of egg laying was delayed by up to 2 weeks in lead treated birds. In turn, peak egg production was also delayed and in most of the treatment groups. Similar delays in onset of egg laying have been observed in female quail in a restricted feeding program ( Zelenka et al. 1984). Timing of the transition from somatic growth to gonadal growth ( sexual maturity) is influenced by accumulation of energy reserves in the juvenile stage. Therefore, chemicals such as OP pesticides that alter growth rate will influence the onset of sexual maturity, especially in the female ( Zelenka et al. 1984).

7.3 Secondary Sex Characteristics Development of secondary sexual characteristics in males and females are predominantly determined by differences in steroid secretion of the gonads of each sex. Perturbations in the development of these characteristics can affect the reproductive performance of the animal ( Bortone et al. 1989; Bortone and Davis 1994). Because secondary sex characteristics in quail are hormonally controlled, they may be useful endpoints in endocrine disruption detection. Major secondary sex characteristics of Japanese quail include body size, distinctive plumage dimorphism, and the development of a foam producing cloacal gland and sternotracheal ( syringeal) muscles in males ( Adkins 1975; Balthazart et al. 1983; Schumacher and Balthazart 1984). Formation of medualary bone in females generally begins 10 days before egg laying under the influence of estrogen and testosterone, and could be considered a secondary sexual characteristic of female quail. It is readily induced in males by exposure to estrogens ( Johnson 2000). Classic radiographic techniques or X ray bone densiometry can be used to detect changes in medulary bone formation ( Schreiweis et al. 2001). Initial equipment cost range from about $ 14,000 to $ 35,000 for digital formats. Some hand held systems are available at lower cost that have had use in in vivo measurements of humerus and ulna radiographic density evalautions for poultry growth traits; however, these systems use radioisotopes with relatively short half lives that must be replaced as often as every 6 months at $ 1300 per replacement, and image quality diminishes as the isotope decays.

In New World quail such as the bobwhite, plumage dichromatism is thyroid estrogen based: that is, when the bird is adequately saturated with thyroid hormones and estrogen, it induces the development of female like plumage. Lack of estrogen results in male phenotype ( Hagelin and Ligon 2001). Low thyroid hormone concentrations result in asexual, juvenile type feathers. In the past, it was generally thought that male plumage was under androgen control and was the primary criterion for female birds' mating decisions. However, it is now recognized that traits such as testosterone mediated breeding displays and body size reflect male condition and are Battelle Draft 82 April 23, 2003 used by females of some species to select mates ( Hagelin and Ligon 2001). It is possible for very high testosterone exposure to result in female plumage in males due to the aromatization of testosterone to estrogen that would superimpose the estrogen dependent female type plumage on the male. Also, posthatch treatment of male Japanese quail with estradiol monobenzoate has been shown to result in plumage that resembled that of female Japanese quail ( Hutchison 1978). Changes in plumage characteristics has been reported as number of individuals per group displaying spotted ( female phenotype) or rufous colored ( male phenotype) breast feathers. The width of the brown spots covering the breast region has also been used to quantify degree of feminization in males ( Hutchison 1978).

The cloacal gland ( Section 7.1.5) is a secondary sex characteristics that is used to determine maturation of male Japanese quail. Bobwhite males lack this gland. Maturation is determined by size and morphological features of the gland and by it secretory activity ( Wada et al. 1992). The gland begins to enlarge as the bird approaches sexual maturation and takes on a red colored appearance. A proteinaceous foam is also produced in response to circulating androgen and secretion of foam, but not fluid, is a sign of maturation. Foam quantity has also been measured as an additional correlated variable to plasma testosterone, but the measure is subject to collection variability. These endpoints have also been used to provide qualitative information that could be used to support evidence of endocrine disruption ( Wada et al 1992; Halldin et al. 1999). Sternotracheal muscle weight ( combined left and right muscles) has been used occasionally to evaluate the effect of estrogenic treatment in male Japanese quail ( Hutchison 1978).

Most of the measures for these secondary sexual characteristics are relatively simple, though somewhat subjective, and can aid in interpretation of reproductive results.

7.4 Sex Ratio Direct exposure of chicks and adults or material transfer of an endocrine active compound and/ or its transformation products to the egg can produce and inappropriate estrogen signal that can cause permanent sex reversal of behavior in male birds and transient feminization of gonadal tissue, if exposure is discontinued ( Scheib and Reyss Brios 1979; Berg et al. 1999). Female quail exposed to antiestrogens in in ovo display masculine behaviors as adults ( Schlinger and Arnold 1995; Ottinger and Abdelnabi 1997). Sex ratios skewed toward a high number of females have been found in populations of birds exposed to estrogenic contaminants in the Great Lakes ( Feyk and Giesy 1998). Although the ovotestis is usually discernable from a normal testis, at high doses of estrogenic contaminants, it can be difficult to distinguish an ovary from an ovotestis, and alternate method of sex determination will need to be used. Sex ratio should be determined for a representative group of the F1 and F2 14 day survivors. To assure that the chemical has had opportunity to transfer to egg, hatchlings from one of the later hatches, for example, at Week 4, should be genetically sexed at hatch. When the chicks reach 14 days of age, they should be necropsied, the presence of abnormal gonad recorded, and the relative amount of ovarian tissue determined in ovo testes for the males only. Gonadal weights obtained, presence of oviduct on right side, and the steroid status should be determined for all chicks. Battelle Draft 83 April 23, 2003 Available laboratory techniques for determining sex include chromosomal ( karyotyping) and DNA probe   that is, the Western Dot Blot and polymerase chain reaction ( PCR) methods. Of these methods, DNA sexing is preferred for monomorphic or immature birds. Karyotyping involves growing the sampled tissue, such as feather pulp, in culture for 7 to 9 days and microscopically identifying the Z and W chromosomes in a stained preparation based on morphological features. Commercial laboratories charge between $ 35 and $ 100 per sample. However, karyotyping is not considered to be as reliable as the new DNA probe methods. DNA probes are labeled DNA fragments cloned from chromosomes. DNA probes have recently been developed that can accurately distinguish the DNA in the sex chromosomes of male and female birds. In Western Dot Blot assays, each DNA sample is placed onto a nylon membrane using a dot blot apparatus. The membrane is then hybridized with a chemoluminescent, sex specific DNA probe and exposed to an X ray film to illuminate the differences between male and female samples. This method is very reliable when sufficient amounts of high molecular weight genomic DNA can be collected.

PCR methods use a single set of primers to simultaneously amplify homologous segments of the CHD W and related Z linked gene, CHD Z ( Griffiths and Kom 1997). Because the two CHD products are of the same size, a restriction enzyme is employed to cut a fragment of the CHD Z product before gel electrophoresis ( Ellegren 1996; Griffiths et al. 1996). This assay has been recently modified ( Griffiths et al. 1998) to use two PCR primers that anneal to conserved regions of the gene and amplify across an intron that differs in length between in the CHD W and CHD Z genes ( Khan et al. 1998). Thus, the PCR products are of different sizes, and a restriction enzyme step is not required to differentiate between the sexes. Both dot blot and PCR methods are extremely accurate and can make use of blood or feather samples. Because only a drop of blood is needed, claw clipping will provide a sufficient sample if the blood spots are simply collected on filter paper. One feather, freshly plucked in a manner that obtains cells from the calamus, is an adequate sample ( Taberlet and Bouvet 1991). Blood and feather samples for DNA sexing require no special storage and can be archived for years. Experience is vital to perform these assays.

Costs at commercial laboratories for sexing birds using DNA probes range from about $ 17 to $ 20, and $ 22 to $ 25 per sample for blood and feather samples, respectively. Equipment cost to outfit a laboratory for dot blot assays can be as low as $ 2000 to $ 3000. For PCR methods, initial equipment cost can be $ 5000 $ 10,000 for manual methods and as much as $ 75,000$ 100,000 for highly automated, high through put fluorescent TaqMan analysis. If manual methods are Battelle Draft 84 April 23, 2003 compared, 400 samples could be completed in one day by dot blot, but it would require 3 days to complete the same number by PCR.

7.5 Biochemical Measures There are many hormonal actions of compounds that are categorized as EDCs. Endocrine disruption includes estrogenic, antiestrogenic, androgenic, and antiandrogenic effects, growth factor modulation, cytokine modulation, modulation of hormone metabolism, and many other activities ( Jimenez 1997). The primary function of estrogenic substances is control of ovulation, and hence, reproduction. Secondary functions of estrogenic substances include gender determination, development of secondary sex characteristics, regulation of mating and breeding behaviors, and regulation of calcium and water homeostasis. The major differences between the effects of estrogen in mammals and egg layers are the production of the egg yolk protein VTG in the liver, and eggshell formation in oviparous species. In birds, the primary sites of estrogen production are the gonads, but the brain also contains significant aromatase activity, suggesting that the brain is another major source of estrogen.

Estrogen production is regulated through a negative feedback loop by the pituitary peptide hormones, GnRH, leutinizing hormone ( LH), and FSH, a system that has been studied in the Japanese quail primarily within the context of reproductive aging ( reviewed by Ottinger et al. 1997). From the current understanding of reproductive biology, almost any hormone involved in the reproductive cycle is a potential biochemical marker for endocrine disruption. The transport of steroid hormones through the body, however, depends on the presence of various serum transporter proteins, which may differ across vertebrate classes. Steroid hormones readily pass through cell membranes and interact with receptors either in the cytosol or in the nucleus. Steroid hormones are metabolized primarily by the liver, where enzymes make them biologically inactive and water soluble. The water soluble conjugates are released into the blood and excreted in the urine or bile. Consequently, measures of liver enzyme activity or steroid byproducts in excrement are also potential biochemical measures of endocrine disruption. GnRh is currently being investigated as a potential endpoint for detecting endocrine disruption in avian reproduction toxicity tests by Mary Ann Ottinger and colleagues. To date, GnRH system appears to be robust, and therefore, the GnRH endpoint does not appear to be a sensitive indicator of endocrine effects.

Endocrine disruption is only one mechanism by which a chemical can interfere with reproduction and development. Thus, there is continued debate about whether endocrine disruption should be narrowly defined to pertain only to subcellular/ cellular effects associated with the nuclear hormone receptor signaling pathway, or to be more inclusive of all aspects of production, release, transport, metabolism, binding action, and elimination. Others argue that endocrine activity of xenobiotic compounds is meaningful only if changes occur to the whole organism, and therefore testing should include methods for measuring effects at all levels of biological organization. In practice, the test methods to assess biochemical endpoints vary considerably, and include production of gene products, cell proliferation assays, tissue responses, VTG induction, hormone assays, egg production and fertility studies, and development of secondary sex characteristics ( Jimenez 1997; Fairbrother 2000). Specific test methods that have Battelle Draft 85 April 23, 2003 been performed on birds include subcellular measures of receptor binding, gene activation and protein; DNA interactions; tissue endpoints of hormone concentration or enzymatic activity; and whole organisms studies with a focus on blood plasma concentrations of analyte ( Fairbrother 2000).

7.5.1 Vitellogenin and Other Biomarkers of Hepatic Metabolic Changes All oviparous species produce VTG, a protein absent in mammals. Both male and female livers may produce VTG, given sufficient estrogenic stimulation. VTG production is stimulated primarily by 17$ estradiol; estrone is only 5% to 10% as effective. VTG is released from the liver into the bloodstream, where it binds with free calcium ions, resulting in elevated total plasma calcium, as more ions are released from bone storage sites. Induction of vitellogenesis in Japanese quail leads to increases in the plasma concentrations of organically bound phosphorus, bound calcium, and total protein ( Robinson and Gibbins 1984). In female birds, oocytes take up the VTG and hydrolyze it to produce the yolk proteins, phosvitin, and lipovitellin. The synthesis of 1,25 hydroxycholecaciferal in the kidney is also affected indirectly by estrogen, where increased levels of 1,25 hydroxycholecaciferal increase absorption of calcium through the gastrointestinal tract. Exposure to environmental estrogens such as OC pesticides and phytoestrogens ( Robinson and Gibbins 1984) induces this egg forming system and is easily assessed in birds by measuring their VTG plasma levels. However, VTG is present in the plasma of female birds before ovulation. Therefore, it cannot be used as a biochemical marker of endocrine disruption in reproductively active females, because the natural production of VTG will obscure the contribution of EDCs.

The VTG structure varies among species and vertebrate classes. Wang and Williams ( 1980), for example, identified two distinct VTGs from white leghorn roosters based on amino acid, phosphorus composition, peptide maps, immunological reactivity, and relationship to the yolk lipovitellins. The authors concluded from these studies that the two proteins are distinct gene products that serve as precursors to different lipovitellin polypeptides. Similar findings were reported for the Japanese quail ( Gibbins and Templeton 1982). These results have implications for the development and/ or use of nucleic acid markers of endocrine disruption, such as messenger ribonucleic acid ( mRNA), in that a mechanistic understanding of VTG production will be required to select the most appropriate mRNA target for study and detection. Heppell et al. ( 1995) attempted to develop a universal VTG assay by developing mono and polyclonal antibodies against a conserved region of fish VTG sequences. The monoclonal antibodies did react with chicken plasma as measured by enzyme linked immunosorbent ( ELISA) and Western Blot assays, but not with the same sensitivity or specificity as it did with the different fish species. More importantly, however, there was no conclusive evidence that the monoclonal antibodies specifically react to VTG in the plasma, especially in the chicken, and no data were shown relative to the reaction of the so called universal polyclonal antibody with bird plasma. Thus, VTG assays continue to be species specific and difficult to compare across species or vertebrate classes.

Current methods for detecting VTG levels in oviparous animals principally rely upon tissue endpoints and immunological techniques such as ELISA and radioimmunoassay ( RIA). Battelle Draft 86 April 23, 2003 Induction of vitellogenesis has been measured indirectly in quail by determining plasma levels of protein bound phosphorus, total calcium, and total protein by conventional methods ( Robinson and Gibbins 1984). Sodium dodecyl sulfate polyacrylamide electrophoresis ( SDS PAGE) and rocket immunoelectrophoresis have also been described for quail plasma ( Robinson and Gibbins 1984). VTG was successfully detected and quantified in plasma from male Japanese quail injected with estrogen analogs or zearlenone by the rocket electrophoretic technique, but the method was unable to detect VTG in male birds injected with the weakly estrogenic compounds o, p1 DDT, chlordecone, or methoxycholor. ELISA and RIA systems are based on the highly specific reversible reaction between an antigen   VTG, in this case   and a specific antibody. Of the two methods, RIA can be more sensitive because it does not require dilution of samples to reduce the interferences encountered in ELISA techniques. However, ELISA methods do not require the use of unstable reagents or radioisotopes and are easier to set up. Both ELISA and RIA assays are readily adapted to production as commercial kits or to routine use in commercial laboratories, once specific antibodies have been developed. Cost per sample for VTG analysis in fish by these methods range between $ 15 to $ 25.

As noted above, there is no universal VTG antibody available. Until very recently, the necessary specific and highly reactive antibody for quail VTG had not been produced, and no specific adaptation of either method for avian VTG is described in the current literature. However, Masaru Wada ( College of Arts and Sciences, Tokyo Medial and Dental University) has recently developed a reportedly highly sensitive quail VTG ELISA assay kit in collaboration with Transgenic, Ltd., in Kumamoto Prefecture, Japan. The assay was developed using a rabbit anti Japanese quail lipovitellin antibody and is being evaluated for cross reactivity with other species. This method will be presented at the European Union SETAC 2002 Invited Symposium on Avian Endocrine Disruption. Wada has also developed an assay for circulating very lowdensity lipoprotein, another estrogen sensitive yolk lipoprotein precursor in quail. Very lowdensity lipoprotein is separated from the plasma matrix in this method by using an automated lipoprotein assay system. It is then purified using high pressure liquid chromatography, and is detected enzymatically. No data on the sensitivity and validation procedures for these assays are available at this time.

Tissue endpoints, such as VTG induction or enzyme activity measures, continue to be the preferred biochemical markers of endocrine disruption. As noted above, however, there is a lack of standard protocols and most assays require species specific antibodies due to structural differences in the VTG peptides in different vertebrate classes. The VTG test alone may also necessarily miss other potential estrogenic effects, and the relative sensitivity of the VTG process compared with other estrogen induced peptide and protein responses has not yet been established. Until such time, it should not be assumed that the VTG induction test would be a sensitive predictor of estrogenicity of xenobiotics in all oviparous species ( Fairbrother 2000). Major limitations with in vivo assays are that they use highly complex responses that can be modulated through mechanisms that do not directly involve steroid receptors, and therefore are not necessarily selective for substances that act through these receptors. Nonetheless, in vivo studies are essential for examining endocrine activity, because they account for pharmacodynamic and pharmocokinetic interactions. Battelle Draft 87 April 23, 2003 No in vitro assay for avian VTG has been developed, as exists for fish. However, avian hepatocytes have been cultured for use in chemical toxicity assays ( Hugla et al. 1996; Kennedy et al. 1996) indicating that there is potential for the development of an in vitro screening assay for VTG in birds.

Not only is the liver the major site of VTG synthesis in birds, but it is also the site of many other enzymatic processes that directly or indirectly contribute to reproductive fitness. For example, birds that consume petroleum contaminated foods develop an increased ability to metabolize the circulating contaminants through the action of a substrate inducible mixed function oxidase system. Although the primary function of the system is to rid the organism of the contaminants, it may also accelerate the turnover of some endogenous substrates, such as steroid hormones. Indeed, studies conducted with halogenated aromatic hydrocarbons ( HAHs) have shown a link between HAH exposure, liver enzyme function and health effects, such as developmental toxicity, hepatotoxicity, endocrine disruption, immunotoxicity, and death. Although the exact role of cytochrome P450 in HAH toxicity is not clear, both the toxic and biochemical responses, such as cytochrome P450 induction, appear to be mediated by a shared mechanism that involves changes in gene expression initiated by binding of HAH to the aryl hydrocarbon receptor. Cytochrome P450 induction in cultured cells therefore serves as a sensitive marker of the activation of aryl hydrocarbon receptor dependent pathways, and measures of liver enzyme activity could be useful indicators of endocrine disruption.

Liver enzyme activities in birds have been studied most thoroughly with respect to HAH and polychlorinated biphenyl ( PCB) exposures. The principal biomarker continues to be cytochrome P450 1A induction, assessed by measuring 7 ethoxyresorufin O deethylase ( EROD) activity using 7 ethoxy resorufin as substrate and a fluorescent plate reader. Cytochrome P450 1A concentration is typically quantified via immunological techniques, such as RIA, ELISA, or Western Blot. Kennedy et al. ( 1996), for example, determined concentration dependent effects of HAHs in primary hepatocyte cultures prepared from the embryos of four breeds of chicken, pheasants, turkeys, three breeds of duck, and herring gulls. Results from this study, and comparison with prior in vivo or in ovo studies, suggest that it could be possible to predict the sensitivity of a species to in ovo lethality by HAHs from the relative sensitivity of hepatocyte cultures to EROD induction. A practical implication of this research is that hepatocyte cultures could be used to estimate the sensitivity of rare or endangered wild bird species.

Brunstrom and Halldin ( 1998) also assessed EROD induction in chicken embryo livers to examine whether there are similar interspecific differences in the EROD inducing potencies of aryl hydrocarbon receptor agonists compared with embryo toxicities. The EROD assay was also extended to hen, turkey, domestic duck, Japanese quail, eider duck, and common tern eggs. The authors found good agreement between EROD inducing potency and embryo toxicity, measured as lethality or malformations, which supports the notion that species differences in ERODinduction reflect differences in an aryl hydrocarbon receptor mediated enhancement of Cytochrome P450 1A levels. The authors note, however, that differences in chemical injection method, carrier solution, and embryo age are known to affect the extent of EROD induction or activity. Further, the relative potencies of HAHs or PCBs can vary considerably among different Battelle Draft 88 April 23, 2003 avian species; the chicken appears to be the most sensitive of the birds. Freeman and McNabb ( 1991) pointed out that many enzyme activity assays have never been validated to specifically measure initial velocity conditions, such that many published estimates of enzyme activity could actually underestimate the true enzymatic activity of the cells or tissue. Assay standardization and validation are therefore prerequisite to the use of enzyme activity as a biomarker for endocrine disruption.

Other enzyme activities measured in hepatic tissues or cell cultures include naphthalene metabolizing activity in mallard ducks, using a radio labeled ( I125) substrate to measure specific activity as nanomoles naphthalene/ minute/ milligram microsomal protein ( Gorsline and Holmes 1981). A 5' deiodinase activity has been used as a biomarker in the Japanese quail; it is an assay that is based on initial velocity conditions ( Freeman and McNabb 1991). There, initial velocity conditions were defined as those where the enzyme activity increases proportionally with increasing enzyme concentration and increases linearly and proportionally with increasing incubation time. In this study, the authors measured the release of I125 from rT3 in liver homogenate and several other developing tissues from 12 h to 2 days of age. Of particular import to other enzyme activity measures was a demonstration that different sets of assay conditions could be selected for making quantitative measurements under initial velocity conditions, and that a validated assay can then be used to compare the effects of different endocrine disruptor treatments.

The estrogen synthesizing enzyme aromatase, a member of the cytochrome P450 family, has been colocalized with estrogen receptors in the brain of Japanese quail ( Dellovade et al. 1995) by immunocytochemical techniques and has been assayed in other contexts ( Balthazart et al. 1996; George and Wilson 1982; Schlinger and Arnold 1992), but not specifically as a biochemical measure of endocrine disruption. In birds, the enzyme plays an important role in determining sexual characteristics and reproductive behavior ( Elbrechet and Smith 1992; Schlinger and Arnold 1992). Ottinger and coworkers at the University of Maryland are currently conducting a preliminary application of this technique in a one generation reproductive test using northern bobwhite and Japanese quail challenged with methoxychlor, a weak estrogenic compound. If it proves to be a sensitive indicator of endocrine disruption in the various life stages of the birds, modifications to the histoimmunochemical technique to convert it to an ELISA would be useful to provide a more routine procedure for endpoint detection.

Although nucleic acid assays are typically conducted within the framework of deducing metabolic pathways and mechanisms, measuring mRNA or DNA protein interactions can also be used to assess hepatic metabolic changes. For example, Gordon et al. ( 1988) studied the effects of estrogen on the stability of mRNAs that code for the yolk precursor proteins apolipoprotein and VTG II in white leghorn cockerels. Total RNA was isolated from liver tissue and used for Northern Dot Blot analyses. Solution hybridization assays were also performed to measure mRNA decay constants. Gupta and Kanungo ( 1996) used total RNA isolated from the liver for Northern Blots and gel mobility shift to study the transcriptional regulation of the VTG operon. Edinger et al. ( 1997) used nucleic acid analysis to address the concept of hepatic memory in chickens, a concept that stems from observations that after the initial activation of the Battelle Draft 89 April 23, 2003 egg yolk protein genes by estrogen, subsequent responses to estrogen occur more rapidly. In this paper, the enzyme deoxyribonuclease ( DNAse) I footprinting was performed to identify DNA: protein complexes in the estrogen response element. Run on transcription assays were also performed to determine the extent of or delay in accumulation of apolipoprotein mRNA during transcription. Shimada et al. ( 1996) used Northern Blot and slot blots to study the induction of cytochrome P450 mRNA. Although the statistical power of nucleic acid techniques such as these to interrogate metabolic changes in mRNA production is widely known in the literature, they have yet to be applied as endpoint measures of endocrine disruption.

7.5.2. Plasma and Fecal/ Urate Hormone Concentrations; Estrogen, Testosterone, GnRH, T3/ T4, TSH, Corticosterone The hepatic enzyme measurements described above require test animals to be sacrificed, a practical consideration that is not conducive to profiling changes in endocrine status in test animals. Thus, assays have been developed to measure many of the hormones and proteins in blood plasma and urine. With the advent of RIA systems in the 1960s and 1970s, circulating hormones could be easily and accurately quantified. For example, in a study with quail, the minimum detectable dose of estrogen was 3.8 pg / tube, the concentration at 50% binding was 188 pg/ mL, and the interassay CV was 7.6% ( Soh and Koga 1994). Androgen assay by single antibody RIA kit has been validated for use in Japanese quail plasma by Ottinger and Mahlke ( 1984). Intra assay CV was 5%. Corticosterone concentrations were measured by a double antibody RIA by the same authors. Originally developed to measure the circulating titers of sex steroid hormones in mammals, RIA assays were readily adapted for use in birds because of the similarity of steroid hormones among phyla ( Wingfield and Farner 1975). Serum thyroid hormones have also been quantified in Japanese quail treated with endocrine active compounds, such as DDT, PCBs, and thiouracil, by RIA using commercial mammalian kits ( e. g., Grässle and Biessmann 1982). RIA methods for gonadotropins are sufficiently accurate to detect changes of 20% in mean blood hormone levels with group sizes of 20 or more ( Thorell and Larson 1978). RIA continues to be the main technique for hormonal analysis in plasma and other sample sources ( Ottinger et al. 2001; Jones and Satterlee 1996; Watson et al. 1990; Jones et al. 1992; Adkins Regan et al. 1995; Schlinger and Arnold 1992; Wingfield and Farner 1975; Nisbet et al. 1999; Millam et al. 1998; Abdelnabi et al. 2000; Marai et al. 2000; Bluhm et al. 1984) due in part to the ease with which specific antibodies can be developed, and the sensitivity of radioactive assays. However, fluorescent immunoassays such as ELISA are becoming more prevalent, thus eliminating the hazards of radiolabeled substrates ( Tell and Lasley 1991; Millam et al. 1998; Alston Mills et al. 1989; Otani et al. 1993). Despite the number of studies on circulating hormones in birds, there are relatively few studies concerning the relationship between circulating hormone levels and endocrine disruption. Gorsline and Holmes ( 1982) investigated the mechanism whereby petroleum contaminants alter adrenocortical function by studying the distribution, metabolic clearance rate, and estimated in vivo secretory rate of corticosterone in mallard ducks. Results showed that lowering of the plasma corticosterone concentration in petroleum exposed birds is associated with a decline in corticosterone secretory rate. Biessmann ( 1982) studied the effects and mode of action of PCBs on gonads and sex hormone balance in juvenile quail during sexual maturation by measuring plasma concentrations of 17$ estradiol, testosterone, 5" dihydrotestosterone, and progesterone. 17$ estradiol and Battelle Draft 90 April 23, 2003 calcium were unaffected by PCB. Cavanaugh and Holmes ( 1982) studied plasma estrogen and progesterone concentrations to determine whether impaired reproductive measures in mallard ducks exposed to petroleum are due to an endocrine dysfunction in the ovary. None of their present evidence suggested that circulating petroleum hydrocarbons can stimulate estrogenic change by binding to estrogen receptors. Some hydrocarbons seem to impair reproductive cyclicity in a manner suggesting impairment of gonadal steroid hormone synthesis. The authors suggest this second type of effect was probably responsible for the disturbances observed during the study. A subsequent study ( Cavanaugh and Holmes 1987) suggested a high probability that petroleum products accumulate preferentially in steroidogenic cells in the developing ovaries, such that petroleum compounds can interfere directly with gonadotropin dependent steroid hormone synthesis.

Unfortunately, frightening or painful stimuli can cause instantaneous changes in endocrine systems, particularly the hypothalamic pituitary adrenal axis. Disturbances such as movement of the cage or handling of the animal can alter some circulating steroid concentrations. Therefore, blood sampling methods that subject the animal to minimal stress will result in more accurate concentrations of hormone concentrations in the circulation. Application of fecal/ urate sampling to reproduction toxicity testing in birds is a means of sampling for steroids with minimal disturbance to the birds.

Measures of the steroid or steroid metabolite content of feces have been used in conservation biology for over a decade. These measures have been shown to reliably detect hormone status and adrenal activity in a wide array of mammalian species ( Wasser et al. 2000) and avian species ( Tell and Lasley 1991; Wasser et al. 1997). It is currently being adapted for use in monitoring endocrine changes in birds ( Brewer et al. 2002a, 2002b; J. Clark, A. Faribrother, L. Brewer and R. Bennett, personal communication, 2002, Effects of exogenous estrogen on mate selection by female house finches [ Carpodacus mexicanus], submitted paper). The method involves collecting fecal samples, or fecal/ urate samples in the case of birds, extracting the steroid or metabolites, and analyzing the extract by RIA. Extraction methods, originally involving labor intensive sequences of extraction and chromatography steps, have been replaced by a rapid method validated by Wasser et al. ( 1991). Current fecal extraction methods are based on modifications by Brown et al. ( 1994) and Wasser et al. ( 1994). The estrogen and testosterone content of the extracts are analyzed by double antibody I125 RIA using commercially available kits. Recovery of 17$ estradiol from spiked fecal samples has been reported at 99.2% ± 9.1%. Assay sensitivity for estrogen was 1.25 pg/ tube, and the interassay CV was reported to be 6.7%. In the testosterone assays, recovery of testosterone was also 99.2% ± 9.1%, the assay sensitivity was 0.1 ng/ mL and the interassay CV was 11.1% to 14.2% ( Wasser et al. 1991). Similar recovery and CV are obtained for progesterone analyzed by single antibody I125 RIA that cross reacts with progesterone metabolites ( Brown et al. 1994; Wasser et al. 1994). The fecal steroid values are comparable to serum steroid secretory profiles measured in a number of wild species ( Wasser et al. 1991, 1995, 2000). Development of an assay to measure glucocorticoid stress hormone in fecal material has only recently been developed. Assays for these hormones were difficult to develop because they undergo extensive and rapid metabolism prior to excretion that is both species specific and subject to further transformation by gut flora ( Erikson 1971; Palme et al. 1997; Bahr et al. 2000). Using a commercial I125 corticosterone RIA assay ( ICN 2 Field preservation techniques have been developed.

Battelle Draft 91 April 23, 2003 Pharmaceuticals, Costa Mesa, California), Wasser et al. ( 2000) directly compared serum and fecal concentrations of corticosterone in the spotted owl ( Strix occidentalis caurina) and showed that the metabolites measured in the feces accurately reflected the adrenal stress response induced by a natural stress ( removal to a new location) and challenge by ACTH. The interassay CV was 5.5% for a high sample ( 27% bound) and 3.0% for a low sample ( 62% bound). Intra assay CV was 5.18% for 30 pairs.

Because fecal/ urate measures of steroid hormones are noninvasive, they can provide both more cost effective methods for determining hormone status in test birds and much needed endpoints that can be correlated to field investigations. 2 Sampling blood is an invasive procedure that involves capture and withdrawal of blood; because it stresses the birds, it should be avoided during egg laying. This limits sampling for steroid hormones to prematuration periods and the termination of the study. Sampling of blood, as noted above, requires a large expenditure of labor and can be difficult to accomplish within an appropriate period of a circadian cycle. With fecal sampling, samples can be collected over the entire exposure period with minimal effort. Typically, validation involves direct comparison between blood and fecal levels, and the hormones of interest over one or more reproductive cycles for reproductive steroids, and ACTH challenge studies for glucocorticoids. A study correlating fecal/ urate steroid concentrations with house finch nesting behavior was recently completed, and a second study comparing excreted steroid concentrations with plasma levels during the reproductive cycle of Japanese quail is currently in progress under funding from the American Chemistry Council ( L. Brewer, personal communication, 2002). An indirect validation for life cycle studies is to track fecal hormone concentrations over time in the species of concern, comparing the monitored values with expected biologically relevant profiles. An evaluation of the effect on assay results of potential interferences such as phytoestrogens that may be present in diet and in excrement is needed. Cost per sample from noncommercial laboratories ( e. g., university laboratories) in the U. S. range from $ 10 to $ 15 up to about $ 25. Fecal steroid analysis was extensively researched at San Diego Zoo as a noninvasive means of sexing monomorphic avian species, but as of yet is not currently commercially available.

The use of feces for noninvasive monitoring of thyroid hormones has not been reported; however, there is a high probability that similar assays can be adapted to these hormones because they are primarily excreted through the bile. There is growing interest among conservation biologist to develop a thyroid assay from feces owing to the importance of thyroid hormones as an index of starvation ( S. Wasser, personal communication, 2002).

Regardless of the assay method or sample matrix, thyroid measures should be viewed with caution. Plasma T3 levels are of doubtful value for diagnosing hypothyroidism because of a common syndrome that evolves in animals experiencing various nonthyroidal illnesses, stress, or starvation. This syndrome, called Sick Euthyroid Syndrome, or Low T3 Syndrome, could develop to protect the body from catabolic processes that accompany these conditions ( Davison et al. 1985). Although T4 would then appear to be the better choice for evaluating Battelle Draft 92 April 23, 2003 hypothyroidism in birds, even plasma T4 concentrations can be influenced by handling, bleeding ( Williamson and Davison 1985a), food intake ( Williamson and Davison 1985b), and increased corticosterone concentration ( Davison et al. 1985). Because of the short half life of T3 and T4 in avian blood, plasma fluctuations of T3 and T4 render it difficult to document hypo or hyperthyroidism from a single sample ( Lumeij 1994; Oglesbee et al. 1997), such as would be taken at necropsy. Here the ability to monitor thyroid hormones over a period of time via sampling the feces could enhance the interpretive power of thyroid assays.

Whereas these prior studies demonstrate that circulating hormones and peptides are readily measured in birds, the obvious lack of mechanistic understanding currently precludes their effective use as biochemical markers of endocrine disruption. Current methods for assessing wildlife health effects are generally targeted at detecting effects rather than mechanisms, and may not adequately evaluate effects on the endocrine system. This is particularly true for exposures that occur during critical developmental periods ( Jimenez 1997). In this regard, previously highlighted methods and approaches for assessing endocrine disruption must be considered within the mechanistic black box that defines risk assessment. For estrogen or an estrogenic substance to exert its effects, for example, it must bind to an estrogen receptor with sufficient affinity and specificity to elicit the downstream production of species appropriate proteins. Thus, similarities among species hormones or estrogen receptor structures could be important in determining whether or not a chemical elicits an estrogenic or antiestrogenic response. Further, receptor binding assays are limited because they only measure the ability of a chemical to bind to the estrogen receptor and do not include any measure of its ability to pass through the cell membranes and contact the nuclear receptors, nor do they reveal whether the receptor binding initiates mRNA transcription. Receptor binding assays also suffer from limitations imposed by the artificial situation whereby substances are tested for their ability to compete with small amounts of radiolabeled tracer estrogen for binding sites. This is different from the in vivo situation, where substances compete with greater amounts of natural hormones that generally have a much higher affinity for the estrogen receptor ( Fairbrother 2000).

8.0 RESPONSE TO ESTROGEN AGONISTS AND ANTAGONISTS The potential for environmental chemicals to mimic the effects of estrogen in birds was first reviewed by Rattner et al. ( 1984). The potential for such interactions to result in feminization of males ( both in wildlife and in humans) focused attention on the endocrine disrupting mode of action of reproductive toxicants ( Colborn et al. 1996.). As a result, a large amount of work has been done in the past decade to develop sensitive assays for detection of estrogenic effects of xenobiotics, mostly focusing on mammalian systems, but more recently assessing applications to Battelle Draft 93 April 23, 2003 fish and birds ( Ankley et al. 1998; DiGuilio and Tillett 1999), and invertebrates ( DeFur 1999). Initial concerns focused on estrogen mimics, primarily due to binding to the estrogen receptor and stimulating down stream responses; however, scant attention has been paid to detection of antiestrogenic effects of chemicals, such as receptor blockers.

8.1 Sexually Mature Life Stages All vertebrate classes share some similarities of response to estrogenic stimulation, whereas certain aspects of avian physiology respond differently ( Fairbrother 2000). Common responses are development and expression of secondary sex characteristics, reproductive behaviors, control of follicular growth and maturation, and calcium regulation. Adult oviparous species, such as birds, herpetofauna, and many fishes, also require estrogen for VTG synthesis, oviduct development and maturation, and shell gland function. Estrogen may regulate seasonal reproductive cycles through stimulation of the pineal gland to produce melatonin.

8.1.1 Sensitivity to 17$ estradiol or Synthetic Estrogen Exposure Estrogen increases production of VTG by increasing the number of copies of VTG mRNA in hepatocytes. VTG production can be under the control of one gene, for example in the chicken, or of more than one gene, as in quail. Alternatively, different forms of vitellogenic peptides may result from posttranslational processes. Differential effects of estrogen on species with the various production modes have not yet been determined. VTG production is stimulated primarily by 17$ estradiol ( see Section 7.5.1); potency of synthetic estrogens, such as diethylstilbesterol, on VTG production varies, but generally is less than that of estradiol. Estrogen also plays a role in passerine male singing behavior and on copulatory behaviors of both sexes ( reviewed by Fairbrother 2000). Distinct estrogen receptive neurons have been located in the brain, using [ H3] 17$ estradiol.

In general, neuroanatomimical distribution in the brain of the steroid receptive cells is similar across species. Enstrom et al. ( 1997) conducted a study of mate choice among dark eyed juncos ( Junco hyemalis), in which estradiol levels were manipulated. Treatment with estradiol enhanced female sexual behavior and promoted precopulatory displays, although estradioltreated females had significantly smaller ovaries than control females.

Turner and Eliel ( 1978) studied the ability of DDT and its metabolites ( o, p' DDT, p, p' DDT, o, p' DDE, and o, p' DDD) to compete with H3 estradiol for binding to the estrogen receptor on Japanese quail oviduct cells. Only o, p' DDT competed significantly for binding sites, but a large molar excess ( 20,000 X) was required. This suggests that o, p' DDT has a very weak affinity for the estrogen receptor and likely will not elicit a true hormone specific response in these cells.

Another chlorinated insecticide, chlordecone, also known as kepone, has been shown to have estrogenic activity at doses lower than that which causes systemic toxicity ( McFarland and Lacy 1969). The greatest effect occurs when the pituitary is intact, because chlordecone stimulates the Battelle Draft 94 April 23, 2003 hypothalamic pituitary axis to release FSH. It also is possible that the pituitary trophic hormones increased the responsiveness of the oviduct to direct action by the chemical. Chlordecone also may block the release of LH, and prolong the release of FSH and ovarian estrogen secretion. It binds to nuclear estrogen receptors in the magnum and shell gland regions of the oviduct, and mimics estrogen effects in these locations, although its potency is only about 1/ 1000 of that of 17$ estradiol ( Rattner et al. 1984). In males, depressed cloacal gland activity following chlordecone exposure could be due to inadequate LH release and associated depressed testosterone secretion.

8.1.2 Antiestrogens There are no reports in the literature of effects of avian exposures to antiestrogenic chemicals. Pharmaceuticals such as raloxofene, tamoxifen, and danazol have been developed for use in women. Raoloxofene and tomoxofen are selective antagonists blocking the estrogen receptors on the uterus and breast tissue, but are agonists in other tissues, such as those in bone. Danazol also binds to and blocks numerous steroid receptors, including those for estrogen and testosterone, but its primary use is through the inhibition of production of FSH and LH by the pituitary. Tamoxifen has been used as an antagonist in estrogen research in poultry and exotic bird medicine ( Lupu 2000), in which it is effective in controlling egg laying. Recent investigations in sex specific neural development have shown that tamoxifen is effective in inhibiting song circuit development in the male zebra finch ( Holloway and Clayton 2001).

8.1.3 Relevance, Sensitivity, Use History, Uncertainty Study of the potential for excess estrogen to affect behavior and reproduction of adult birds has been conducted primarily though the administration of the endogenous hormone 17$ estradiol. Although this regime definitely mimics the natural action of estrogen, it has been criticized by some because its efficacy could be affected by endogenous negative feedback cycles. Further, it does not provide a means of determining the consequences of effects on synthetic or degradative or metabolic pathways, such as cytochrome P450 systems, for estrogen. Use of synthetic estrogens such as ethinyletradiol ( EE2), which is a widely used contraceptive, or DES also provides a reasonable model in the avian system, but is susceptible to the same shortcomings as use of 17$ estradiol. There is a lack of suitable chemicals to serve as known antiestrogenic controls for bird studies. However, measurement of hormonally driven behaviors such as singing, copulatory receptivity, and nest building, and concentrations of circulating or excreted hormones such as estradiol and estrone will provided the necessary information for determination of general mode of action of reproductively active chemicals.

8.2 Juvenile Life Stages 8.2.1 Sensitivity to 17$ estradiol or Synthetic Estrogen Exposure Sexual dimorphism in terms of behavior and neuroendocrinology has been well studied in the Japanese quail. Female quail are demasculinized by endogenous estrogens ( Hutchison 1978), Battelle Draft 95 April 23, 2003 and embryonic treatment with estrogen during the critical period of brain development at Days 4 12 of embryogenesis results in an irreversibly depressed response of copulatory behaviors in the adult male to the activating effects of testosterone ( Halldin et al. 1999). Likewise, estrogen suppresses development of the singing center in brains of embryonic passerines.

Berg et al. ( 1999) clearly showed that the synthetic estrogens EE2 and DES exhibit significant estrogenic activity during embryonic maturation of Japanese quail, resulting in feminization of male embryos and malformation of the Müellerian ducts in females. EE2 was 3 to 10 times more potent than DES, as indicated by a dose dependent increase in the frequency of male embryos exhibiting an ovotestis. Halldin et al. ( 1999) then studied the effects of embryonic exposure to these same synthetic estrogens on sexual behavior in adult Japanese quail. Sexual behaviors were significantly depressed following in ovo treatment by either estrogen. Testis weight asymmetry was significantly increased by EE2 but not by DES. The cloacal gland area was significantly reduced by treatment with DES. However, plasma testosterone concentrations did not differ significantly from the control values and were not correlated with frequency of sexual behaviors. This suggests that the in ovo exposure resulted in altered sensitivity of the brain to the activating effects of testosterone, rather than affecting the ability of the testes to produce the hormone. Taken together, these two studies indicate that EE2 is more potent than DES during embryonic exposures.

Schumaker et al. ( 1989) identified Day 9 of incubation to be the time when embryonic development of Japanese quail is most sensitive to estrogenic effects. Estradiol benzoate ( EB) treatment demasculinized sexual behavior and cloacal gland growth of males. However, some dependent variables, such as plasma levels of luteinizing hormone and crowing, were still affected by EB treatment on Day 14. A 5 : g dose of EB totally suppressed the capacity of the adult male to show copulatory behavior in response to testosterone, if it was present in the egg on Day 9 of incubation.

Treatment of embryonic Japanese quail and chickens by egg injection on Day 3 of incubation with the plastic monomere, bisphenol A, resulted in malformed Müllerian ducts at 2 days prior to hatch in female quail and ovotestes feminization in male chickens ( Berg et al. 2001). Effective doses of bisphenol A were embryolethal in chickens, but not in quail. DES used as a positive control showed the same species specific effects on Müllerian duct and ovotestes formation. DES appears to be five times more potent than bisphenol A in the quail, but only three times more potent in the chicken. Thus, target organs, gender effects, and sensitivity to xenobiotics appear to be species specific. The flame retardant, tetrabromobisphenol A, had no sublethal effects on the developing embryo of either species. Both of these compounds are readily excreted in the bile, and the authors acknowledged that it is possible that female birds do not deposit sufficiently high levels in the egg to result in the same effects observed in the laboratory egg injection studies.

Sexual differentiation is not a unitary process in quail: various responses differentiate at embryonic ages between Days 9 and 12, and require different levels of estrogen. The process Battelle Draft 96 April 23, 2003 also is progressive, and some aspects of behavior and morphology are not complete until the posthatching period. Yoshimura et al. ( 2000) documented changes in gonadal growth and histology, age of sexual maturation, and ovarian weights of Japanese quail exposed to DES during the posthatch period , Day 0 to Day 53. Age of sexual maturation was delayed in males as a result of slower testicular development. Semen production and copulation activity appeared to be unaffected, as measured by egg fertility rates. In females, ovarian development also was delayed during early posthatch lifestages, although the age of sexual maturation was no different from controls. A greater number of ovarian interstitial cells were found in DES treated birds than in controls. The authors suggest that these effects may be due to binding of DES to the estrogen receptor, stimulating downstream activities.

Feminization of male gull embryos occurred following injections of DDT at concentrations similar to those found in gull eggs from southern California ( cited in Halldin et al. 1999), suggesting that environmental contaminants may have similar effects on embryonic development as the estrogens reviewed above. However, relative potency of the various chemicals is not known.

8.2.2 Antiestrogens Very little is known about antiestrogenic properties of xenobiotics administered to birds. McMurry and Dickerson ( 2001) and others showed that 2,3,7,8 tetrachlorodibenzo p dioxin ( TCDD) is both antiestrogenic and antiandrogenic in northern bobwhite quail. TCDD is approximately as potent as EE2 and decreases eggshell gland weight in a dose response fashion when injected into eggs prior to the start of incubation. Indole 3 carbinol is a naturally occurring, phytoantiestrogen, although it causes minimal effects   decreased weight gain   in developing bobwhite embryos ( McMurry and Dickerson 2001). The result of the inhibition of aromatase in 5 day old chicken embryos by 5( p cyanophenyl) 5,6,7,8 tetrahydroimidaxo [ 1,5"] pyridine hydrochloride was that all hatchlings developed as phenotypic males ( Elbrecht and Smith 1992). Sex reversed females developed bilateral testes that were capable of complete spermatogenesis and had the physical appearance and behavior of normal males. Similar effects occurred after exposure to ergosterol biosynthesis inhibiting fungicides that also inhibit aromatase P450 enzymes ( Dawson 2000).

8.2.3 Relevance, Sensitivity, Use History, Uncertainty In birds, the developing embryo is the most sensitive life stage to the activity of estrogens or their antagonists. Changes that occur during either in ovo or posthatch exposure will persist throughout the life of the bird and influence adult reproduction, even if exposure occurs only in the early life stages. Because birds develop in hard shelled eggs, they are most likely to be exposed to such substances throughout embryogenesis, including the most sensitive stage of development, which is Day 9 in the quail. Some xenobiotics   OC pesticides, for example   require metabolic degradation into secondary metabolites prior to exerting estrogenic effects. It is not clear whether such degradation can occur in the egg, or whether the secondary products must be Battelle Draft 97 April 23, 2003 deposited by the hen during egg formation. Regardless, once present in the egg, these products cannot be excreted as readily as they are in mammals, even though clearance of metabolic wastes by the bile does occur. Therefore, they may continue to pose a risk throughout the incubation period. Hatchlings can continue to be exposed for several days immediately posthatch, because they rely on the resorption of the yolk sac as an energy source during that time. It is clear that sexual development continues during the posthatch period and can be significantly influenced by increased levels of estrogen during that time. Both the natural and synthetic estrogens are reasonable models for use in demonstrating estrogenic effects during these life stages. Further studies of estrogenic xenobiotics that require metabolic activation will determine whether or not this occurs in the embryo, and if so, its relationship to the sensitive developmental period.

9.0 RESPONSE TO ANDROGEN AGONISTS AND ANTAGONISTS Birds appear to be affected by androgenic or antiandrogenic substances to a much lesser extent than they are affected by estrogens and antiestrogens. Because males are the homozygous sex in birds, sexual dimorphism results from a demasculinization of female embryos under the influence of estrogen; lack of estrogen results in production of phenotypic males ( see Section 3.2). In contrast, excess or insufficient testosterone is unlikely to affect female birds, unless levels are so low that there is no substrate for the action of aromatase, and therefore no production of estrogens. Adult male and female birds have equivalent levels of circulating plasma testosterone. It has been shown that 17$ estradiol administered to the adult male quail induces receptivity, but females treated with testosterone fail to show male sexual behaviors ( reviewed by Balthazart et al. 1983). Consequently, less research has been focused on understanding potential effects on testosterone active xenobiotics in birds than on estrogen related substances.

9.1 Sexually Mature Life Stages 9.1.1 Sensitivity to Androgenic Steroid Exposure Testosterone exerts its influence through binding to cell receptors, entering the target cell, and then undergoing conversion into a number of metabolites. These metabolites bind to nuclear receptors and stimulate DNA to produce appropriate physiological responses. For example, the 5" reduced metabolites ( 5" dihydrotestosterone [ 5" DHT] and 5" androstane 3", 17$ estradiol [ 5", 3" diol]) activate strutting, crowing, and cloacal gland development. The production of the 5" reduced metabolites is much higher in male cloacal glands than in females ( Balthazart et al. 1983). However, in the brain of birds, a specific enzyme, 5$ reductase, reduces testosterone to 5$ dihyrotestosterone ( 5$ DHT) which then is converted to 5$ androstane 3", 17  estradiol( 5B, 3" diol). These $ metabolites are inactive; it has been shown that injections of 5$ DHT do not activate sexual behavior or development of secondary sex characteristics. The pituitary gland and syringeal muscles used in singing of females produced more of the 5$ reduced metabolites than was generated in these locations by males ( Balthazart et al. 1983). Because circulating plasma levels of testosterone do not differ significantly between males and females in photostimulated Japanese quail, it is likely that sexual dimorphism is a result of the Battelle Draft 98 April 23, 2003 differences in metabolic activation/ deactivation reactions rather than in the capacity of ovarian or testicular tissues to produce testosterone ( Balthazart et al. 1983).

Adkins Regan and Ottinger ( 1988) demonstrated a pronounced diurnal rhythm in rate of clearance of injection induced elevations in plasma testosterone levels in Japanese quail, with faster clearance rates occurring during the day than at night. In addition, many birds follow a cranial cycle as well. They are seasonal breeders, responding to increasing length of photostimulation by enlargement of the gonads and onset of copulatory behaviors. Some birds molt just prior to onset of reproductive capacity, and most begin their molt shortly after completing the breeding and/ or brood rearing phase of the reproductive cycle. At the onset of short day lengths, gonads are receptive to the inhibitory effects of reduced amount of light. Eventually, the gonads become insensitive to the gonadosuppressive effect of the short photoperiod, and reproductive activity increases. The onset and length of the period of sensitivity of the gonads to photosuppression varies by species. Treatment of day old Japanese quail chicks with high levels of testosterone proprionate resulted in the development of testes that were always sensitive to the effects of short day length, and therefore never were stimulated to return to breeding condition. The same testosterone treatment in female chicks resulted in suppressed ovarian development.

Stoehr and Hill ( 2001) addressed the question of interference of hormones on molting and plumage coloration. They found that male birds exposed to exogenous testosterone close to their molt were likely to molt into plumage that was duller than that which they previously displayed. Males that had been treated with testosterone earlier in the year did not show as marked an effect.

9.1.2 Antiandrogens Inhibitors of the enzyme that reduces testosterone to its active 5" metabolites have been used as testosterone antagonists for the treatment of prostate cancer. However, it is not clear whether inhibitors of 5$ reductase, such as finasteride, will have any physiological effect on birds. The level of circulating testosterone will increase, at least transiently, which may have an effect on the amount of testosterone that is changed to the 5" metabolites. In humans, finasteride is marketed under the name Proscar by Merck & Co., Inc., for the treatment of enlarged prostates; it shrinks the size of the organ. Treatment with the 5$ reduced metabolites does not result in any antiandrogenic effects.

Cyproterone acetate ( CyA) is an antiandrogenic substance that has been shown to suppress nestsoliciting behavior and ovarian development of ring doves ( Columba palumbus), copulatory behavior and external morphology of Japanese quail, and courtship behavior of zebra finches ( Taeniopygia guttata) ( Suresh and Chaturvedi 1987). The interaction of CyA with lengthening photoperiod, which is a reproductive stimulator, in the red headed bunting ( Emberiza bruniceps) was studied by Suresh and Chaturvedi ( 1987). Under normal day lengths, CyA inhibited testicular growth and reduced body weight. It partially arrested the activity of the developing gonad during the photostimulatory period of lengthening days, although these effects were Battelle Draft 99 April 23, 2003 completely and quickly reversed at the cessation of treatment. Effects of CyA were partially offset by simultaneous treatment with testosterone proprionate.

Another pesticide, DDT, and its degradative p, p' metabolites, are weakly antiandrogenic, and TCDD is both antiandrogenic and antiestrogenic in northern bobwhite quail, depending upon dose and tissue ( McMurray and Dickerson 2001). Neither of these OC compounds influences the levels of circulating hormone. Their mode of action is similar to that of vinclozolin, acting as androgen receptor antagonists.

9.1.3 Relevance, Sensitivity, Use History, Uncertainty Administration of excess testosterone or its active metabolites appears to have little effect in the adult bird. The one notable exception is the lack of response of birds to increasing day length for the spring reproductive season, for example. Nevertheless, several chemicals have been investigated to determine whether a xenobiotic substance will introduce such a response. Antiandrogenic effects appear to be somewhat more pronounced, although none was of the same magnitude as those induced by estrogenic substances. Most of the notable changes in birds exposed to antiandrogenic substances occurred in reproductive behaviors. This lack of responsiveness in the adult bird to testosterone manipulations is not surprising, because sufficient hormonal differentiation occurs in ovo and during the first two posthatch weeks to maintain gender appropriate behaviors ( Hutchinson 1978). Altered male behaviors in birds result from the lack of testosterone, not elevated levels of androgens.

9.2 Juvenile Life Stages 9.2.1 Sensitivity to Androgenic Steroid Exposure The inactive testosterone metabolite, 5$ dihydrotestosterone, had no demasculinizing effects if administered on Day 9 of embryonic development ( Schumacher et al. 1989), indicating that it has no estrogenic activity. Similarly, no behavioral effects occurred in the adult bird as a result of embryonic exposure to 5$ DHT. Thus, this hormone appears to be inactive in quail. Furthermore, it is not capable of being acted upon by aromatase to form estrogens. Embryonic quail have high 5$ reductase activity in the hypothalamus, suggesting that they transform testosterone into this inactive metabolite so it cannot be aromatized into estrogen during embryonic development. Thus, although 5$ DHT is categorized as a testosterone, it has no activity, and therefore is neither an agonist nor antagonist. TCDD increases testosterone hydroxylation in herons, with the position on the ring that is hydroxylated dependent upon age and sex ( Dawson 2000). However, treatment with TCDD sufficient to cause P450 induction had no effect on circulating testosterone or estradiol concentrations, suggesting that changes in hydroxylase activity were compensated for by endogenous feedback mechanisms.

Plants produce androgenic substances as well as phytoestrogens. The ketone fraction of both wheat germ oil and gibberellic acid ( a plant hormone) is androgenic, as evidenced by a positive response, increased weight, in the chick comb bioassay in male chickens treated by injection Battelle Draft 100 April 23, 2003 from 1 to 35 days of age ( Gawienowski et al. 1997). Similar treatment with these two substances had no effect on female chickens, suggesting that their potency is relatively weak.

Hutchison ( 1978) showed that exposure of 6 to 12 h old Japanese quail hatchlings to elevated testosterone through implants of testosterone proprionate had no effect on the differentiation of sexual behavior in intact males or females. In gonadectomized birds, 57% of the testosterone treated females displayed male sexual behaviors, male vocalizations, and cloacal gland development. Gonadectomized birds that had initially been treated with estradiol benzoate were later treated with testosterone proprionate, and vice versa. Evidence suggests that a critical period during embryonic development occurs when estrogen eliminates the development of male behaviors, which normally occurs only in females. However, estrogen must continue to be present in the posthatch period for such behaviors to become established in females as adults. Estrogen given in adulthood to males castrated at hatching induces both male and female behaviors, whereas estrogen given embryonically demasculinizes males.

9.2.2 Antiandrogens Juvenile Japanese quail exposed to aflatoxins prior to and during sexual maturation developed testes approximately half the size of testes from untreated birds. They had delayed testicular development and reduced production of testosterone ( Doer and Ottinger 1980). Vinclozolin (( R. S.) 3 ( 3,5 dichlorophenyl) 5 methyl 5 vinyl 1,3 oxazolidine 2,4 dione), a dicarboximide fungicide applied on crops and ornamental plants, has known antiandrogenic properties in mammals. Similar studies were conducted with Japanese quail ( McGary et al. 2001). Embryos exposed on Day 4 of incubation metabolized vinclozolin to its metabolically active form, which significantly altered GnRH levels in male hatchlings, but not in females. The onset of male reproductive behaviors seemed to be delayed, with the number of mounts and cloacal contacts significantly lower at the beginning of the reproduction period. There were no measurable effects on GnRH levels in adults, plasma steroid levels in either hatchlings or adults, proctodeal foam gland growth during maturation, or relative testicular weight at 7 weeks of age. The authors speculated that observed effects were due to vinclozolin acting as an androgen receptor antagonist in the hypophyseal region of the brain.

9.2.3 Relevance, Sensitivity, Use History, Uncertainty There has been little interest among researchers to study the potential consequences of exposing avian embryos or hatchlings to elevated testosterone levels. Hutchison ( 1978) and others clearly established that estrogen is the active hormone during embryogenesis, with testosterone levels remaining similar in both sexes. However, because testosterone is responsible for inducing development of singing centers in the brain, male copulatory behaviors, and the cloacal and foam glands, chemicals that might inhibit the action of testosterone should be of more concern. Unfortunately, there are few studies of the effects of antiandrogens on the developing embryo or hatchling. McGary et al. ( 2001) produced the most definitive study conducted to date ( see Section 9.2.2). Further work needs to be done to investigate whether or not reduced levels of the 5" metabolites are more likely to cause adverse effects through blockage of the reductase Battelle Draft 101 April 23, 2003 enzymes than is reduction of the parent compound. However, there likely will continue to be less interest in understanding the potential for xenobiotics to influence the androgen system in birds than there is for the estrogen dependent responses, due to the requirement of estrogen for demasculinization of genotypic females. This is significantly different from effects in mammals, where masculinization of the female fetus is of much greater concern, due to the requirement for elevated testosterone for genotypic males to develop normally; females will be masculinized under these conditions regardless of endogenous estrogen production. Thus, results from mammalian studies designed to determine endocrine disrupting effects are likely not directly applicable to the avian model.

10.0 RESPONSE TO THYROID AGONISTS AND ANTAGONISTS Relatively little work has been done to assess the effects of xenobiotics on thyroid activity in birds; only the potential for thyroid inhibition has been studied. However, hyperthyroidism can result after the cessation of treatment by thyroid inhibitors ( Peebles and Marks 1991). Thyroid function can be affected as a result of interference with pituitary production of TSH, iodination and coupling of tyrosine residues to form T4, or conversion of T4 into T3 through enzymatic activation in the liver. Metabolism and excretion of T4 and T3 also could be affected, and it is possible that chemicals bind to T3 cellular receptors, resulting in either agonistic or antagonistic effects.

10.1 Sensitivity to Thyroid Stimulation Various crude oils were shown to affect thyroid function, as measured by reduced body weights, thyroid hypertrophy, and increased plasma thyroxine levels in several species of seabirds: herring gulls, black guillemots ( Cepphus grylle) and adult Leach's petrels ( Oceanodroma leucorhoa) ( Peakall et al. 1981). Several other hormones, such as circulating corticosterone, and corticotrophic hormone, also were elevated. The authors noted that the effects of thyroxine on regulating basal metabolism of birds are equivocal. Growth in several bird species following thyroxine administration ranged from none to moderate, with some studies indicating significant increases in body weight at low administered doses, and reduced body weight at high levels. Peakall et al. ( 1981) suggested that the observed effect of oil on circulating thyroid hormones most likely is a compensatory response to changes in osmoregulation induced by alterations in the intestinal mucosa due to oil ingestion, which affects both osmotic balance and nutrient uptake. This indicates that crude oil would not be classified as an EDC, because there was no direct effect of the substance on the thyroid or steroid reproductive hormone systems.

There were no other reports found in the literature documenting effects of thyroid stimulation in birds, either through direct exposure to thyroid hormone treatment or through exogenous thyroid agonists. Battelle Draft 102 April 23, 2003 10.2 Inhibition of Thyroid Function It has been suggested that maternal thyroid activity influences functional eggshell properties in birds, playing a significant role in egg hatchability ( Peebles and Marks 1991). Exposure of high growth rate Japanese quail to low amounts ( 0.2%) of dietary propylthiouracil, a known thyroid inhibitor, had no effect on embryonic growth, but resulted in significantly reduced body water due to changes in eggshell permeability ( Peebles and Marks 1991). Although molt and plumage characteristics are thought to be primarily controlled by testosterone ( Stoehr and Hill 2001), thyroid hormone may play a role as well. Propylthiouracil treatment of American kestrels ( Falco sparverius) resulted in changes in the width of subterminal bands on tail feathers, feather reflectance, and duration of the molt ( Quinn et al. 2002). However, the potential for xenobiotics to result in similar responses is unclear. PCBs appear to have a bimodal response on the thyroid system: low levels are stimulatory, and higher concentrations result in inhibition ( Quinn et al. 2002). Nevertheless, when kestrels were fed Aroclor 1242 at concentrations that significantly decreased circulating thyroid hormone levels, no measurable change in plumage characteristics resulted ( Quinn et al. 2002). High dietary concentrations of DDT, toxaphene, and PCBs all resulted in enlarged thyroid glands and reduced body weights of adult bobwhite quail after 4 months of continuous exposure ( Hurst et al. 1974). Low doses of PCBs reduced the size of the thyroid, whereas similar doses of DDT and toxaphene had no effect. The authors suggested that DDT and toxaphene could decrease the effective level of T3, thus enhancing TSH secretion, causing the thyroid to grow in size. DDT could affect T3 through changes in liver metabolic enzymes or through direct action on pituitary secretion of TSH. The authors provided no explanation for the bimodal response to PCB exposure. Grässle and Biessmann ( 1982) also studied the effects of DDT and PCBs on quail thyroid systems. They showed that Japanese quail exposed to either the OC, DDT, or the PCB, Aroclor 1254, for up to 120 days had significantly decreased T4 levels and altered thyroid histology suggestive of glandular inactivity, but little to no change in circulating T3 levels. The authors suggested that PCBs have a different mode of action than does propylthiouracil, possibly acting on the hypothalamus pituitary thyroid axis rather than at the glandular level. Furthermore, there was no relationship between T4 or T3 levels and eggshell breaking strength for either OC or propylthiouracil exposed birds, supporting the conclusion that thyroid hormone activity does not play a significant role in reducing eggshell quality.

Dawson ( 2000) reviewed the effects of various OCs on growth and other thyroid functions of young birds. Such effects were first investigated in the late 1960s and early 1970s in pigeons exposed to DDT and dieldrin, then in gulls ( Larus sp.) and pigeon guillemots ( Cepphus columba) exposed to PCBs. Changes in mass and histology of the thyroid were measured, but were not sufficiently severe to result in gross physiological effects. Mallards ( Anas platyrhynchos) exposed to PCBs for 5 weeks in a repeated dose exposure showed a slight increase in T3 at doses 1000 times that used in rodent studies ( Fowles et al. 1997). Three week old herring gulls ( Larus argentatus) exposed to tetrachlorobenzene showed no changes in T4, but significantly decreased levels of T3. Conversely, in ovo exposure of chickens to tetrachlorobenzene showed no change in T3 and an increase in T4. In ovo exposure of chickens, pigeons, and great blue herons ( Ardea herodias) to TCDD resulted in no effects on T3 or T4 levels, even when EROD and P450 enzymes were substantially induced. Field studies of Battelle Draft 103 April 23, 2003 thyroid related effects due to exposure of birds to PAHs were also inconclusive, because eggs were contaminated with both PCBs and polychlorinated dibenzofurans. Nevertheless, no significant changes in circulating levels of T3 or T4 were documented.

10.3 Relevance, Sensitivity, Use History, Uncertainty There has been relatively little interest in studying the potential effects of xenobiotic chemicals on the avian thyroid system. Early studies were conducted to determine whether the thyroid played a significant role in eggshell quality, but as it became clear that other hormones or cellular mediators , such as prostaglandins, were more directly related to eggshell synthesis, interest waned. Effects of thyroid function on metabolic rate, such as weight gain in embryos or juveniles, was studied briefly in the poultry science field, but again did not appear to play as significant a role as did other dietary parameters. More recently, the role of thyroid hormone in plumage development was investigated, but also shown to be equivocal. It may be that the thyroid system is relatively robust and not particularly sensitive to xenobiotic effects. Generally, there is an excess of circulating T4, and approximately 30% to 40% of normal T4 levels are sufficient to produce an adequate amount of T3. Therefore, unless a chemical acts directly on conversion of T4 to T3, or binds to T3 receptors, sufficiently high doses would be required that systemic toxicity is likely to result prior to the onset of signs of thyroid dysfunction. To date, only propylthiouracil, which is a known thyroid inhibitor that is used pharmacologically to control Graves disease in humans, is known to inhibit the conversion of T4 to T3. Propylthiouracil, along with several other pharmacological agents, also inhibits the action of thyroid peroxidase, thereby reducing the amount of T4 produced.

Measurement of thyroid specific endpoints is relatively simple, because T4 and T3 hormone structures are well conserved across species, and therefore circulating levels are easily measured with commercially available ELISA kits. Thyroid weights also can be measured at necropsy, and body weights   especially weight gain in juveniles   are routinely measured in all avian studies. Therefore, identification of thyroidogenic modes of action of xenobiotics can readily be detected during the avian two generation test. It is likely, however, that thyroid related fitness endpoints will be much less sensitive than other hormone related effects and can occur at exposure concentrations that also result in generalized systemic toxicity.

11.0 CANDIDATE PROTOCOLS Candidate protocols for the development of an avian two generation toxicity test guidelines include established life cycle (" one generation") reproduction tests ( ASTM, OECD, and EPA) and a proposed short term life cycle test using proven breeders ( OECD 2000). Two principal designs under consideration for an avian two generation reproduction test are a proven breeder design and a pre egg laying exposure design, based on a draft guideline proposed by OECD ( 1999) and suggested modifications of the EPA OPPTS 850.2300 one generation avian Battelle Draft 104 April 23, 2003 reproduction test guideline ( EPA 1996) proposed by EDSTAC ( EPA 1998) for the development of a two generation guideline with endocrine endpoints from which the OECD ( 1999) proposed guideline, in part, evolved. Each of these existing and proposed guidelines is reviewed and their strengths and weaknesses discussed and contrasted. Table 11 1 compares these guidelines by test species, husbandry, test procedures, test design, endpoints, and reportable results.

Detailed reviews of the one generation avian reproductive test guidelines and their strengths and weakness were presented by Bennett and Ganio ( 1991) and OECD ( 1996). The advantages and disadvantages of design and endpoint components of the draft OECD guideline for a two generation avian reproduction test were reviewed in detail by Bennett et al. ( 2001). One generation reproduction tests are conducted with bobwhite and mallard ducks. ASTM also permits other nonpasserine species, and OECD allows the use of the Japanese quail. The proposed two generation guidelines limit the tests to the two quail species. Battelle Draft 105 April 23, 2003 Table 11 1. Comparison of Avian Reproductive Toxicity Tests PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

TEST ANIMALS Species JQ, BW Bobwhite ( BW( b)), mallard duck ( MD( c)), other nonpasserines Japanese quail ( JQ( d)), BW,

MD BW, MD JQ Age Approaching first breeding season; Successful fertilization should have taken place before start of pretreatment BW: $ 6 months old at onset of lay Birds of similar age; age in first season must be ± 10%

of mean age of group; if proven breeders used,

must be same age in years BW: 20 24 weeks ± 1 week JQ: proven breeders ± ½ week MD: 9 12 months ± 2 week $ 7 months old; birds approaching first breeding season; same age ± 1 month Approaching first breeding season ( 4 wk old)

Criteria for use Birds should appear healthy and free of abnormalities or injury that could affect test results Birds should not receive medication beginning 1 week prior to start of acclimation Successful fertilization demonstrated prior to test Parental mortality during last 2 weeks of acclimation should not exceed 3%

All birds should be from same hatch Birds from one source and strain. Birds in poor physical condition,

deformed, or with plumage that differs from that of wild birds must not be used Birds from the same population of well known parentage Free of disease and injury Population should not be used if > 3% of either sex die or become debilitated during acclimation period Birds from same source and strain with known breeding histories, lighting regimes, disease record,

drug or medication administered Not selected for resistance to toxic substances Not used in a previous test Phenotypically indistinguishable from wild stock Birds should appear healthy and free of abnormalities or injury that could affect test results Birds should not receive medication beginning 1 week prior to start of acclimation Onset of egg laying should have taken place before start of treatment Parental mortality during last 2 weeks of acclimation should not exceed 3%

All birds should be from same hatch PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 106 April 23, 2003 One Generation Test Guidelines HOUSING Location Adults Young indoors in pairs ( 1M: 1F)

To minimize injury, etc., from aggression, males and females may be housed separately; pairs placed together for $ 1 H/ day for 5 days/ week to maintain fertility In groups preferably by treatment group Indoors; outdoors for some species In pairs ( 1M: 1F) or in groups containing no more than 1M Not specified Indoors preferred; outdoors permissible In pairs ( 1M: 1F) or in groups:

BW/ JQ: 1M/ 2F MD: 1M: 3F In groups by pen of origin Together if birds individually marked indoors BW: in pairs ( 1M: 1F) or in groups ( 1M: 2F)

MD: in pairs ( 1M: 1F) or in groups ( 1M: 3F)

In groups by pen of origin Together if birds individually marked indoors in pairs ( 1M: 1F) by lineage,

males from one line paired with females from another line To minimize injury, etc., from aggression, males and females may be housed separately; and house together long enough to maintain fertility In groups preferably by treatment group F1 cohort for breeding paired ( 1M: F) at 4 wk old PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 107 April 23, 2003 Pens Size ( cm2/ bird)

Construction BW: 625 JQ: 1 week old: 50 2 week old: 75 3 4 week old: 100 > 4 week old: 625 Stainless or galvanized steel or other inert materials. Wire pens with slanting floors and egg catchers recommended for adults. Food troughs should be covered with wire grid to minimize food spillage Adequate size From literature:

BW: > 760 JQ: > 200 MD: > 5000 Stainless steel, galvanized steel, and material coated with perfluourocarbon plastics preferred. Any nontoxic material not capable of excessive sorption of test substance,

not dissolved by water or loosened by pecking are acceptable BW: 1250 JQ: 750 MD: 5000 Not specified BW: > 5000 MD: > 10,000 Galvanized or stainless steel sheeting for common walls and ceilings, and wire mesh for floors and external walls. Material coated with perfluorocarbon plastics also acceptable JQ 1 week old: 50 2 week old: 75 3 4 week old: 100 > 4 week old: 625 Stainless or galvanized steel or other inert materials. Wire pens with slanting floors and egg catchers recommended for adults. Measures should be taken to reduce food spillage ( e. g., cover food troughs with wire grid)

ENVIRONMENT Temperature ( ° C)

Adult Egg Storage Hatching Brooding 16 27 13 16 37 37.5 35 38 first week 30 35 second week 23 27 third week 21 12 16 39 Upper brooder temperature is species specific; provide gradient within brooder down to 21 22 ± 5 BW/ JQ: 15 16 MD: 14 16 37.5 BW/ JQ: 35 38 first week,

decrease by 4 5/ week MD: 32 35 first week,

decrease by 3 4/ week 21 16 37.5 35 to 22 temperature gradient within brooder measured at 2.5 4 cm above floor 16 27 13 16 37 37.5 35 38 first week 30 35 second week 23 27 third week 16 27 fourth week on PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 108 April 23, 2003 Percentage RH (%)

Adult Egg storage Incubation Hatching Young 40 80 55 75 50 70 70 75 40 80 45 70, higher for MD at high elevations 65 70 45 70, higher for MD 50 75 BW: 50 65 JQ: 50   70 MD: 60 65 BW/ JQ: 70 75 MD: 75 85 BW/ JQ: 50 75 MD: 60 75 BW/ JQ: 55 75 MD: 60 85 55 55 80 70 45 70, higher for MD 40 80 55 75 50 70 70 75 40 80 Lighting Intensity ( Lux)

Adult ( h of light)

Young ( h of light) Sunlight spectrum $ 10 16 17 JQ: 16 17 Sunlight spectrum 65 8 prior to photostimulation ( 8 weeks)

17 thereafter 14 7 8 for 8 weeks 16 18 thereafter Transition dawn/ dusk 65 7 8 for 6 8 weeks 16 17 thereafter or increased by 15 min/ day from 17 14; 15 30 min transition at dawn/ dusk Daylight visual spectrum automatically controlled $ 10 at level of feeder 16 17 JQ: 16 17 Ventilation 1 to 15 changes/ h recommended As in CCAC ( 1984, 1993) Good ventilation Good ventilation ( rate: 4 changes/ h in winter; 15/ h in summer suggested) 8 to 15 changes/ h recommended Egg turning Storage: optional Incubation: yes Hatching: no Storage: optional Storage: optional Incubation: yes Hatching: no Storage: yes daily Incubation: yes Storage: optional Incubation: yes Hatching: no PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 109 April 23, 2003 Feed Ad libitum Must meet nutrient requirements of the species Caloric and water content of diet must be reported Extra calcium added to adult diet if same diet used for chicks and adults Ad libitum Any unmedicated commercial diet that meets minimum nutritional standards Description of basal diet:

analyze for contaminants Ad libitum Avoid use of chemicals or medication Contaminant free as possible ( no pesticides,

heavy metals) Ad libitum Must meet nutrient requirements of JQ Caloric and water content of diet must be reported\

Sufficient space for feeding must be provided during first week after hatching so weak birds have access Extra calcium added to adult diet if same diet used for chicks and adults Water Ad libitum Sufficient space for drinking must be provided during first week after hatching so weak birds have access Ad libitum Ad libitum Ad libitum Water bottles or automatic water devices recommended Bacitracin or one of its forms can be added to drinking water of young birds if necessary Ad libitum Sufficient space for drinking must be provided during first week after hatching so weak birds have access TEST PROCEDURES Quarantine at least 2 weeks Acclimation $ 2 weeks BW: photostimulation 6 weeks prior to acclimation period $ 1 week Incompatible birds rearranged or replaced $ 2 weeks $ 2 weeks, can coincide with quarantine $ 2 weeks If necessary photostimulate birds during acclimation ( onset of laying can occur during acclimation) PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 110 April 23, 2003 Test Substance Mix to obtain homogenous distribution in diet. Use of premix is advisable.

Frequency of diet preparation chosen so degradation and volatilization of test substances $ 80% of initial concentration. Frequency of diet renewal # 1X/ day and $ 1X/ week. Can keep frozen until use; Stability,

homogeneity tests required Physical, chemical,

biological properties Analyses to determine stability in diet, frequency of diet preparation,

homogeneity in diet Chemical identification data;

water solubility; vapor pressure; structural formula;

purity; chemical stability in water, light, and diet;

octanol/ water partition coefficient; biodegradability Cannot use guideline for highly volatile or unstable substances Must have characteristics that allow uniform mixing in diet Chemical name, source,

composition: major ingredients and percentage of each Impurity, known physical and chemical properties ( solubility, volatility,

degradation rate)

Use technical grade Mix to obtain homogenous distribution in diet. Use of premix is advisable.

Frequency of diet preparation chosen so degradation and volatilization of test substances $ 80% of initial concentration. Frequency of diet renewal # 1X/ day and $ 1X/ week. Can keep frozen until use; Stability analyses under conditions on test prior to start or during rangefinding , verify during main test. Check stability at end of 1st feeding period and at end of last Homogeneity analysis prior to test of at 1st mix for study Administration Route Carrier/ diluent Maximum carrier in feed (%) In the diet A vehicle of negligible toxicity,

such as food or corn oil, water Acetone can be used if allowed to completely evaporate prior to feeding < 2 A constant amount should be added to each test group and control diet to keep caloric value and moisture content equal between dosage groups In the diet Solvent or other material Determine concentration,

stability, homogeneity of test substance in diet Stability, volatility determine frequency of diet preparation and storage method < 2 In the diet Water, corn oil Should not interfere with toxicity of test substance < 2 In the diet Distilled water preferred. If not water soluble, could dissolve in reagent grade evaporative diluent such as acetone, methylene chloride. Solvent should be completely evaporated prior to feeding Other carriers: corn oil,

propylene glycol, gum arabic ( arcacia)

< 2 Equivalent amount of diluent should be added to control diet In the feed or water A vehicle of negligible toxicity, such as food or corn oil, water Acetone can be used if allowed to completely evaporate prior to feeding < 2 A constant amount should be added to each test group and control diet to keep caloric value and moisture content equal between dosage groups PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 111 April 23, 2003 Number of dietary concentrations/ choice of concentration One or multiple If multiple, then at least three plus control. Highest concentration should be level expected to reveal significant effects on adult health or reproductive parameters.

Highest dose should not cause mortality or other severe signs of parental toxicity. If no significant reproduction effects expected at lower test concentration, then highest should be the expected concentration of the chemical in the environment.

If this concentration is at lower than 1000 mg/ kg diet, no need to test above 1000 mg/ kg.

Lowest concentration should not impact adult health or reproductive parameters Intermediate concentration should be geometrically spaced between the highest and lowest doses. 2 methods to establish treatment levels: control + 3 levels; geometrically spaced control + 1, or more expected or known environmental concentration If nonstatistical, $ 1 concentration must produce effect or contain 0.1% of test material, or be 100X the highest field or measured concentration At least 3 + control Results of a dietary LC50 test ( TG 205)

Highest concentration should approximate ½ of LC 10.

Lower concentrations should be geometrically spaced at fractions of highest dose ( e. g. 1/ 6 and 1/ 36 of the highest dose) 3 + control Higher 2 treatment concentrations will be multiples such as 5 of the lowest treatment level. The highest treatment levels usually will be below lethal levels. Highest nonlethal level is estimated from a dietary LC50. Concentrations should include an actual or expected field residue exposure level. Not specified Choice on basis of toxicological data from range finding test, prior avian tests,

and/ or tests with mammalsHighest concentration should be level expected to reveal significant effects on adult health or reproductive parameters.

Highest dose should not cause mortality or other severe signs of parental toxicity. If no significant reproduction effects expected at lower test concentration, then highest should be the expected concentration of the chemical in the environment with addition of 5X safety factor.

Lowest concentration should not impact adult health or reproductive parametersIntermediate concentration should be geometrically spaced between the highest and lowest doses.

Minimum number of pens per concentration Sufficient pairs, such as 20, to ensure 16 breeding pairs in the control groups during egglaying Penmates can be replaced during pretreatment If statistic approach used,

enough to give detection level of 25% at "= 5,

power= 0.8 Nonstatistical approach,

use $ 16 12 pairs or BW/ JQ: 12 groups MD: 8 groups BW: 12 pairs or groups, 20 pairs in control group MD: 12 pairs or 8 groups Sufficient pairs, such as 20,

to ensure 16 breeding pairs in the control groups to end of treatement period.

Penmates can be replaced during pretreatment PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 112 April 23, 2003 Treatment period prior to egg laying ( weeks) no treatment prior to egglaying $ 10 for persistent compounds < 10 for nonpersistent;

sometime during egg laying 10 12 8 12 no treatment prior to egglaying P1: 6 weeks after laying established F1 breeders from hatch through 6 wk post fertility F1 chicks from hatch to termination at 14 days F2 not treated Post egg laying treatment period ( weeks) 6 ( 8) MD/ BW: may be unnecessary to collect more eggs than would be laid in the wild with 2 clutches Terminate when control pens produce 25 eggs, or 6 weeks after 50% of control hens have laid 1 egg 8 10 8 12 At least 6 Egg collection At least once daily for 6 weeks Daily until control pens produce 25 eggs, or 6 weeks after 50% of control hens have laid 1 egg Daily for last 10 weeks of treatment Daily for last 8 10 weeks At least once daily for 6 weeks Eggs hatched Entire pretreatment and treatment period Entire egg collection period Entire egg collection period Entire egg collection period Entire pretreatment and treatment period Candling Day 0 for cracks Fertility/ early embryo viability:

BW: Day 11 JQ: Day 8 Embryo survival:

BW: Day 20 21 JQ: Day 15 16 Day 0 for cracks At 1 and 2 weeks of age for fertility Prior to incubation to detect cracks Day 0 for cracks Fertility/ early embryo viability:

BW: Day 11 MD: Day 14 Embryo survival:

BW: Day 18 MD: Day 21 Day 0 for cracks Fertility/ early embryo viabilityDay 8 Embryo survival: Day 15 16 PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 113 April 23, 2003 Storage period Maximum 1 week Weekly Weekly or biweekly BW: Weekly or biweekly MD: biweekly Maximum 1 week Incubation period/ hatch BW:

Day 20 21/ Day 24 25 JQ: Day 15 16; Day 17 18 BW: Day 21/ Day 23 24 JQ: Day 16/ Day16 17 MD: Day 23/ Day 25 27 BW: Day 21/ Day 24 : Day 15 16/ Day 17 18 Rearing period ( days) 14 14 14 14 14 Brood from 6th wk used to establish F1 breeding pairs ENDPOINTS PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 114 April 23, 2003 Recommended additional endocrine endpoints Gross morphology and histology Size/ weight of gonads, brain,

thyroid, adrenals Histology of thyroid, brain,

adrenals, gonads Testicular spermatid counts and morphology Gross abnormality of genital tract Cloacal gland area Plasma and fecal/ urate hormones Steroid hormones, estradiol,

testosterone, corticosterone,

VTG ( males), thyroid hormones, TSH Brain chemistry: GnRH,

catecholamine, aromatase,

foam gland test Fresh egg weight Growth curve for young Behavior of young and adults Effects on multiple generations Genetic sex ratio at hatchinge For 12 genetic males and 12 genetic females/ group at 14 days of age: presence at necropsy of structures on right side, histologically determined relative amount of cortex and medulla, and development of oocytes,

serum sex steroids Both sexes at 14 days old:

organ weights including brain, body weights, wing and bone length, thyroid weight, skeletal x ray; if differences appear, then thyroid histopathology should be performed on all groups, otherwise, on highdose and control groups only All surviving chicks at 14 days of age: subjected to visual cliff test, challenged with cold stress test, and nest attentiveness test Reproductive capability of offspring Gross morphology and histology Size/ weight oftestes Sex of F1 chicks PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 115 April 23, 2003 RESULTS Validity of test ( quality criteria) Test substance concentration in diet is satisfactorily maintained and reported Losses of < 20% initial concentrations acceptable.

Higher loss rates must be investigated, explained $ 16breeding pairs of control birds that have produced eggs must survive to end of treatment period All control group mortalities should be explained In controls, hatching success for incubated eggs laid during fifth and sixth week of exposure should be $ 50%

Rate of viability should remain $ 80%

$ 10 breeding pairs of controls should survive until end of test BW chick/ MD duckling productivity in control groups does not average 12 or 10, respectively, 14

day old survivors per pen over a 10 week period Average eggshell thickness in control groups is BW: < 0.19 mm MD: < 0.34 mm > 10% of adult control birds die Test substance concentration in diet is satisfactorily maintained and reported Losses of < 20% initial concentrations acceptable.

Higher loss rates must be investigated, explained $ 16breeding pairs of control birds that have produced eggs must survive to end of treatment period All control group mortalities should be explained Parental mortality during last two weeks of acclimation should exceed 3%

If control reproductive parameters do not meet typical values ( provided in Annex 2), procedure and husbandry conditions should be checked for problems PARAMETER OECD Revised Draft Guideline, April 2000 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite Short Term Life Cycle Test ( Proven Breeders) ASTM E1062 86 Standard Practice for Conducting Reproductive Studies with Avian Species( a)

Life Cycle Test OECD 206 Avian Reproduction Test Life Cycle Test EPA OPPTS 850.2300 Avian Reproductive Test e Life Cycle Test OECD First Draft Guideline,

December 1999 Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail Two Generation Test ( P1 Proven Breeders)

Battelle Draft 116 April 23, 2003 One Generation Test Guidelines Treatment of data Numerical data should be presented in tabular form,

separating clearly the pretreatment and treatment data Treated groups compared with control group by methods in MacLeod ( 1994)

Methods that compare individual performance before and during exposure using covariate analysis can be used If there appears to be delayed toxic responses after 1 2 gamete cycles, results should be evaluated in 1 2 week increments to avoid reduction in the power of the test to detect effects when responses from all treatment weeks are averaged together NOAEL ( mg/ kg body weight/ day) should be determined for all health and reproductive parameters evaluated Analyze continuous variables ( body weight,

eggshell thickness) by ANOVA or general linear models Separate means by multiple comparison procedures ( e. g., Dunnett's test)

Discrete variables such as count of eggs laid, cracked eggs, or 14 day old survivors can be analyzed by contingency tables   chisquare analysis Derived variables such as 14 day old survivors as percentage of eggs laid can be compared by ANOVA percentages should be arcsin transformed and weighted ANOVA performed Test groups individually compared with control group by statistical procedure designed in study plan ( e. g.,

ANOVA, NOAEL) Experimental groups compared with controls by ANOVA Regression analysis is highly desirable if the data and number of dose levels allow Sample units are individual pens Numerical data should be presented in tabular form All adult health data should be recorded per individual bird ( food consumption per pair). Since P1 parental pair is primary statistical unit and F1 parental pair is secondary unit, all reproductive data should be related by lineage.

Raw data should be reported by pen. Measurement of endpoints made on adult birds will be evaluated by comparing values obtained from birds from treated groups with values obtained from control birds. An NOEC expressed in mg/ kg diet and mg/ kg body weight per day should be determined for all health and reproductive parameters evaluated a) Reapproved in 1991; discontinued in 2000.

b) BW Bobwhite ( Colinus virginianus).

c) MD Mallard duck ( Anas platyrhynchos).

d) JQ Japanese quail ( Coturnix japonica).

e) EDSTAC ( EPA 1998) recommended extension for a two generation avian reproduction test using the Japanese quail and a pre breeding exposure for the P1 generation. The additional endpoints recommended by EDSTAC for a two generation test a summarized in bold under the " Recommended additional endocrine endpoint" section of this table in the OPPTS 850.2300 column. Battelle Draft 117 April 23, 2003 11.1 Life Cycle (" One Generation") Reproduction Tests The three one generation avian reproduction toxicity test guidelines considered in this discussion are ASTM E1062 86, OECD 206, and EPA OPPTS 850.2300. The last guideline, OPPTS 850.2300 ( EPA 1996), represents a harmonization of previous test guidelines developed by the Office of Prevention, Pesticides and Toxic Substances, EPA. ASTM E1062 86 was established in 1986, reapproved in 1991, and discontinued in 2000. No replacement protocol has been proposed. OECD 206 is similar to the OPPTS 850.2300 guideline. A proposed short term life cycle ( proven breeder) guide ( OECD Revised Draft Guideline, April 2000, Proposal for a New Avian Reproduction Toxicity Test in Japanese Quail or Northern Bobwhite) is also discussed.

All of the life cycle tests were designed to be first line screening tests for identifying potential reproductive effects in birds exposed to environmental chemicals. All use a similar array of measurement endpoints to evaluate effects of chemicals on components of reproduction that reflect the reproductive success of the female and prerecruitment survival of young. The endpoints are also selected to aid in identifying cause and effect relationships.

11.1.1 OPPTS 850.2300 The test simulates a chronic dietary exposure for the purpose of detecting potential long term effects of persistent chemicals or those chemicals that are repeatedly or continuously applied to the environment. It takes into account the measured or estimated residues in the environment with the goal of determining both the highest and the lowest dietary concentration that produces an observable adverse effect on an array of reproduction measurements.