Oral Estrogen Masculinizes Female Zebra Finch Song System

Hormones and Behavior 41, 236 –241 (2002) doi:10.1006/hbeh.2001.1752, available online at http://www.idealibrary.com on

Oral Estrogen Masculinizes Female Zebra Finch Song System Allison E. Quaglino, 1 Christina B. Craig-Veit, Mark R. Viant, 2 Andrea L. Erichsen, D. Michael Fry, and James R. Millam 3 Department of Animal Science, University of California, One Shields Avenue, Davis, California 95616 Received July 20, 2001; revised August 18, 2001; accepted September 27, 2001

It is well established that parenteral treatment of female zebra finch chicks with estradiol masculinizes their song control nuclei and that as adults they are capable of song. Concern over the widespread use of putative environmental estrogens caused us to ask whether oral exposure to estrogens (a natural route of exposure) could produce similar effects. We dosed chicks orally with estradiol benzoate (EB; 1, 10, 100, and 1000 nmol/g of body mass per day, days 5–11 posthatch), the nonionic surfactant octylphenol (100 and 1000 nmol/g), or the pesticides methoxychlor (100 and 1000 nmol/g) and dicofol (100 nmol/g) and measured their song control nuclei as adults. EB treatment produced increases in song nuclei comparable to that induced by parenteral administration of estrogens. This is the first study of which we are aware to use an oral route of administration, which simulates the natural process of parent birds feeding their nestlings. We conclude that oral exposure to estradiol alters song control nuclei and we report in a related paper (Millam et al., 2001) that such exposure severely disrupts reproductive performance. Although we detected no influence of xenobiotics on induction of song control nuclei the possibility remains that oral exposure to xenoestrogens in high enough doses could affect development. © 2002 Elsevier Science (USA) Key Words: zebra finch; environmental estrogens; xenoestrogens; octylphenol; methoxychlor; dicofol; estradiol benzoate; sexual differentiation; birdsong; area X; nucleus robustus archistriatum; HVC.

1 Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA 90089-0191. 2 Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, CA 95616. 3 To whom correspondence and reprint requests should be addressed. Fax: 530.752.0175. E-mail:[email protected].

236

Extreme dimorphism of the song control system of the zebra finch brain provides an excellent system in which to study sexual differentiation. Four song control nuclei, area X of the paraolfactory lobe, the HVC, the robust nucleus of the archistriatum (RA), and the lateral magnocellular nucleus of the anterior neostriatum (LMAN) are highly developed in the male zebra finch, whereas HVC, RA, and LMAN are greatly reduced in the female brain, and females have no detectable area X. The size of these song control nuclei correlates with singing behavior; male zebra finches sing and females do not (Nottebohm and Arnold 1976). It is well established that administration of estradiol early in life masculinizes the female song control nuclei (e.g., Gurney and Konishi, 1980; Gurney, 1982; Adkins-Regan et al., 1994), although gonadal steroids are probably not involved in the normal masculinization of male brain (Arnold, 1997). More likely, masculinization of male song nuclei results from steroids synthesized de novo in male brain tissue (Schlinger and Arnold, 1993; Halloway and Clayton, 2001). Regardless, females treated with estradiol as chicks develop an area X and show enlargement of the song control regions HVC and RA. Such estradiol-treated females produce song when given testosterone as adults. Recurring reports of sexual disruption by environmental contaminants (Colborn, 1995) prompted us to ask whether oral exposure to putative xenobiotic estrogens in the environment might disrupt normal sexual differentiation of the song system in zebra finches, as does parenteral administration of estradiol in the laboratory. Masculinization of the female brain could potentially alter reproductive behavior, with unknown consequences on reproductive performance and populations.

0018-506X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

237

Oral Estrogen and Song System Differentiation

Previous experiments have used parenteral routes of administration in studying mechanisms of sexual differentiation of the song system. However, as chicks in the wild would likely face dietary exposure to xenobiotics, e.g., by being fed insects or seeds tainted with pesticides, we administered test substances orally. Additionally, we used equimolar amounts of putative xenobiotics to permit the characterization of estrogenicity in comparison to a prototypical estrogen, estradiol benzoate (EB). As the sensitive period for masculinization of the female brain occurs during a period of rapid growth (approximately days 5 through 11 posthatch) we dosed chicks daily on a per body mass basis to reflect the changing amounts of feed consumed during development. Using this approach, we tested the following: the organochlorine pesticide methoxychlor (MXC), which is used on a variety of food crops, particularly apples, and is also licensed for veterinary use to control parasites on some livestock species (Bulger et al., 1978; Gray et al., 1985; Shelby et al., 1997; Roy et al., 1997); another organochlorine, dicofol (D), which is used on a variety of food and fiber crops, particularly fruits and cotton (Roy et al., 1997; Fry and Toone, 1981); and octylphenol (OP), a non-ionic surfactant used in a variety of industrial and agricultural applications (Roy et al., 1997; Staples et al., 1998; Servos, 1999; Nimrod and Benson, 1996). OP is found in occasional abundance in such substrates as sewage treatment plant sludge, which is used as a soil amendment (Bennie, 1999). All of these substances have shown estrogenic activity in other test systems (White et al., 1994; Desbrow et al., 1998; Purdom et al., 1994; Jobling and Sumpter, 1993; Naylor, 1992).

hatch nestlings were weighed and down feathers were color marked with food coloring. Chicks were weighed daily until 11 days of age and on days 15 and 20. Beginning at 5 days of age, and continuing through day 11, chicks were orally dosed with 1 ␮l/g of body mass of one of the following substances: 1, 10, 100, or 1000 mM estradiol benzoate (equivalent to 1, 10, 100, or 1000 nmol/g of body mass, respectively), 100 or 1000 mM OP, 100 or 1000 mM MXC, 100 mM D, or canola oil (control). Canola oil (Spectrum Naturals, pure pressed, Spectrum Naturals, Inc., Petaluma, CA) was used as the delivery vehicle for the substances and as a vehicle control. Treatments were delivered orally using positive-displacement disposable micropipettes (WiretrolII, Drummond Scientific Co., Broomall, PA). Chicks typically “begged” for food if the micropipettes contacted their beak region. An additional control group consisted of birds in which no treatment was delivered (no vehicle control). This no vehicle group was compared to the canola oil control group to assure that the canola oil vehicle exerted no estrogenic effects. Sibling chicks were used both within and among treatment groups, although most chicks within each treatment group were unrelated. Fledglings were removed from breeding cages at 45 to 60 days of age and placed in mixed-sex cages, where they were allowed to reach full somatic maturity, i.e., the capability to reproduce with full potential. After 130 days of age testosterone propionate (TP)containing silastic ropes (1.5 mg; ⬃4 – 6 mm in length) were implanted in the breast muscle or subcutaneously, adjacent to the breast muscle (Gurney, 1980, 1982; Simpson and Vicario, 1991a,b; Adkins-Regan et al., 1994) 1 day prior to behavioral testing employed in other experiments (Erichsen et al., 1999).

METHODS Histology Housing and Treatment Zebra finches were housed in a closed colony with 16 h of light and 8 h of darkness. “Finch super-vitamin enriched” diet (Volkman Seed, Ceres, CA) and water were given ad libitum. The diet for breeding pairs and their young was supplemented with hard-boiled egg and shell (approximately 15 g per pair) twice a week. Pairs were housed in individual breeding cages (46 ⫻ 46 ⫻ 41 cm) equipped with a metal nest box (15 ⫻ 13 ⫻ 13 cm) mounted opposite the cage door. The nest boxes opened at the top to allow easy access to chicks and were monitored daily for eggs and chicks. Eggs were date marked as they were discovered. At day of

Approximately 2 weeks after behavioral testing (at 4 to 11 months of age) birds were deeply anesthetized with equithesin [chloral hydrate:sodium pentobarbital (4:1)] and perfused with 0.1 M sodium phosphatebuffered saline, pH 7.4, solution until perfusate ran clear (approximately 7 min), followed by 2% paraformaldehyde in 0.1 M sodium phosphate, pH 8.0, solution containing 0.03% glutaraldehyde (approximately 15 min). Brains were removed, preserved in a 2% paraformaldehyde solution without glutaraldehyde, and stored at 4°C until sectioning. Brains were sliced into 50-␮m sections using a Vibratome, Model 1000 (Technical Products Interna-

238

tional, Inc., St. Louis, MO). Alternate sections were used for Nissl staining or, occasionally, for immunohistochemistry, as a validation for Nissl definition of song nuclei. Thus, Nissl-stained sections were 100 ␮m apart. After dehydration in a graded ethanol series (70 to 100%), sections for Nissl staining were mounted on gelatin-coated slides, Nissl-stained using thionin (Weiss and Greep, 1977), and coverslipped for viewing. Morphometry Song control nuclei were outlined by an experimentally blind observer and nuclear volumes were calculated after digital reconstruction of serial sections using Neurolucida morphometric software (Microbrightfield, Colchester, VT) and an Olympus BX-60 microscope. In a subsample of brains, no differences were detected between the right and left nuclei for each area (Nottebohm and Arnold, 1976), so only one side was used to measure each of the song control regions. For a select number of brains, measurements, as determined by Nissl staining, were compared to corresponding measurements as determined by methionine-enkephalin (MENK) immunohistochemistry, using the methods of Millam et al. (1993), and antiMENK (Diasorin, Inc., Stillwater, MN) at 1:5–10,000 dilution. The size of areas measured by these two processes matched, thus confirming the song control region boundaries as defined by Nissl staining. Statistical Analysis Measurements of nuclear area volumes were logtransformed and then subjected to analysis of variance [ANOVA; SAS, GLM, Cary, NC; a constant was added to each raw value to permit transformation of areas that measured zero (area X was absent in all control group females and several xenobiotic-treated females)]. The Fisher-corrected, least significance difference method was used to compare means. Occasionally, one or more of the song control nuclei of an individual bird were excluded from the data set because they were unmeasurable due to poor tissue quality.

RESULTS No significant changes in any song nuclear volumes were detected in brains of treated males. LMAN volume was highly variable, and since its role in song learning and production and its developmental pat-

Quaglino et al.

tern are poorly understood, we omitted LMAN from the following analysis. No significant differences were found in the volumes of any of the song control nuclei between the no-vehicle and canola oil-treated control groups (Fig. 1). Thus, canola oil appears to show no appreciable estrogenicity. In contrast, EB treatment produced increases in area X, RA, and HVC. All doses of EB treatment elevated the nuclear volume of area X, while only the three highest doses (10, 100, and 100 nmol/g of body mass) significantly increased the nuclear area of RA and HVC. The 1 nmol/g of body mass treatment group had the lowest N of the EB-treated groups. More animals in this dose group may have produced statistical significance for growth of RA and HVC, as well as area X. The greatest increase in song control nuclear volume was observed in the 100 nmol/g of body mass EB dose group. None of the xenobiotics induced significant growth of song control areas at the doses tested.

DISCUSSION There are few existing data on the oral dosing of estradiol in the avian literature. Previous birdsong studies have used either silastic implants or intramuscular or subcutaneous injections to administer steroids (e.g., Gurney, 1982; Nordeen et al., 1986; AdkinsRegan et al., 1994; Jacobs et al., 1994; Castro and Ball, 1996). To our knowledge, this is the first study in songbirds to report the use of an oral route of administration to alter sexual differentiation. This route of administration provides a naturalistic method for evaluating the exposure risk of chicks to environmental estrogens that might be found in contaminated seeds, insects, worms, and phytoestrogens fed to nestling birds. At application rates at which D and MXC are used in the environment, Kenaga’s nomogram (Hoerger and Kenaga, 1972), as modified by Fletcher et al. (1994), estimates that chicks fed 1 to 8 g of insects or seeds per day would ingest from 7 to 135 mg of D or MXC per gram of feed ingested, for a maximum likely daily intake of up to 1080 ␮g [calculated on a 1 pound per acre application rate, which is a conservative estimate, as typical application rates in the United States are 2 to 3 pounds per acre (2.25–3.36 kg/hectare) or more]. The 100 nmol/g of body mass doses used in this study amount to a daily dose of D of 29 ␮g/g of body mass or 101.5 to 261 ␮g per day during the period of rapid weight gain (approximately 3.5 to 9 g

Oral Estrogen and Song System Differentiation

FIG. 1. Volumes (mm 3; mean ⫾ SE; log plot; N noted on bar of each treatment group) of female zebra finch song nuclei area X, HVC, and nucleus robustus of the archistriatum (RA). Finches were treated as chicks (days of age 5 through 11) orally with the substances denoted under each bar. Canola was used as a control and as a vehicle for estradiol benzoate (EB), octylphenol (OP), methoxychlor (MXC), and dicofol (D). Another control group received no treatment (No vehicle). Asterisks denote groups that are significantly greater than canola-treated controls. Area X was not detectable in groups that lack bars.

of body mass during the administration period of 5 through 11 days of age) for a total 7-day dose of 1.14 mg. Amounts of MXC are similar. Thus the 100 and

239

1000 nmol/g of body mass doses of pesticides used in this study are well within predicted exposures in the wild. The 100 nmol/g of body mass dose of OP represents a daily intake of about 100 to 200 ␮g of OP for finches between 3.5 and 9 g of body mass (5 to 11 days of age). Bennie (1999) summarized several studies reporting OP concentrations of 200 to 5000 ␮g/g of OP in such substrates as raw sewage, sewage treatment plant final effluent, and chemical plant effluent. Servos (1999) reported bioaccumulation and/or bioconcentration factors for OP and other related alklyphenols and alkylphenol polyethoxylates from one to several hundred in a variety of organisms, reaching several thousand in certain marine animals. While trophic studies are needed to better assess exposure threats in terrestrial environments, nestlings fed biota grown in such highly contaminated environments could likely encounter the loads used in the present study. The oral doses we used were based on daily body mass changes to approximate likely dosing exposure in the wild, as level of exposure would likely increase with growth and increased food intake. Thus, the oral dosing protocol mimics both the mode and the amount of environmental estrogens birds might be exposed to in contaminated areas. The vehicle used to dissolve and/or suspend substances at higher doses appears to be nonestrogenic, as no significant differences were detected between no-dose and canola oiltreated controls. Chicks tolerated oral dosing very well. In preliminary trials using either subcutaneously or intramuscularly administered estradiol benzoate, we experienced considerable chick mortality. Indeed, in addition to mortality, it can be difficult to be confident of the consistency in the site of injection in chicks that weigh only a few grams, even when using a fine (31-gauge) needle. We also occasionally observed that injected substances leaked out of the injection site after withdrawal of the needle. In contrast, oral dosing reduced chick mortality to background levels (in our laboratory, approximately 1% between days 5 and 11) and, particularly at stages of development when chicks beg aggressively, delivery of correct dosage was assured. The dose–response relationship we observed can be compared to that earlier studies using parenteral routes of administration. Gurney (1982) found that 100-␮g implants of estradiol increased the size of RA and HVC in female birds. Similarly, HVC, RA, and area X were significantly enlarged in female finches injected with 20 ␮g of EB daily for the first 3 weeks of

240

life (Adkins-Regan et al., 1994). In absolute terms, the increases we observed in song nuclear size were comparable to those found using parenteral routes of adminstration (e.g., Adkins-Regan et al., 1994; Gurney, 1982; MacDougall-Shackleton et al., 1998). We estimate that our 100 nmol/g of body mass EB dose is roughly equivalent to 20-␮g daily injections used in other studies (e.g., Adkins-Regan et al., 1994); that is, it is the minimum dose sufficient to elicit maximum growth of the song nuclear areas. A 100 nmol/g of body mass treatment of EB per day is equivalent to 37.65 mg/kg/day or 132 to 339 ␮g of EB per day for chicks weighing from 3.5 to 9 g. This suggests that oral dosing requires approximately 11 times the amount of EB as a parenteral dose to achieve the same effect on the growth of song control nuclei. Oral estradiol and xenoestrogen dosing in rodents is approximately 40 –50 times less sensitive than parenteral routes of administration (Odum et al., 1997). However, it is difficult to determine how our 7-day regimen of 100 nmol/g of body mass EB compares to a 20-␮g daily dose, if the latter is not adjusted for the changing body mass of the animal. A fixed daily dose of 20 ␮g could vary by three- to fivefold in hormone per unit of body mass in rapidly growing individuals. Our adjustment for daily increase in body mass over the 7 days of treatment allows for a more accurate assessment of treatment– effect per unit of test substance. Further, our dosing protocol permits equimolar comparisons of the estrogenicity of putative xenoestrogens to a prototypical estrogen, EB. The doses of EB that we tested clearly exert effects beyond the song control system. In related work reported elsewhere (Millam et al., 2001), both 10 and 100 nmol/g of body mass EB significantly impaired reproductive performance of males and females in sexspecific ways, leading to complete reproductive failure of pairs in which both males and females were treated as chicks with 100 nmol/g of body mass EB. However, reproductive performance of pairs treated with 100 nmol/g of body mass OP did not produce detectable effects comparable to those with EB-treated birds in egg production, candled fertility, or incidence of broken eggs (Millam et al., 2001). These results demonstrate that posthatch exposure to oral estrogen masculinizes song control nuclei of female zebra finches in a manner reminiscent of that in numerous reports using parenteral routes of estradiol administration. This suggests that wild birds could be masculinized by exposure to man-made xenoestrogens in the wild, although we did not detect a significant effect of octylphenol, methoxychlor, or dicofol

Quaglino et al.

treatment on the masculinization of female song nuclei. We have shown that oral exposure to EB masculinizes zebra finch brain, and in a related study, we have shown that this treatment disrupts reproductive performance (Millam et al., 2001). It is not a question of whether posthatch exposure to oral estrogens can impair normal sexual development, it is rather a question of dose. Whether environmental estrogens exert comparable effects in wild populations remains to be determined.

ACKNOWLEDGMENT This work was supported by EPA Grant R-855294-01 to D.M.F. and J.R.M.

REFERENCES Adkins-Regan, E., Mansukhani, V., Seiwert, C., and Thompson, R. (1994). Sexual differentiation of brain and behavior in the zebra finch: Critical periods for effect of early treatment. J. Neurobiol. 25, 865– 877. Arnold, A. P. (1997). Sexual differentiation of the zebra finch song system: Positive evidence, negative evidence, null hypothesis, and paradigm shift. J. Neurobiol. 33, 572–584. Bennie, D. T. (1999). Review of the environmental occurrence of alkylphenols and alkylphenol ethoxylates. Water Qual. Res. J. Can. 34, 79 –122. Bulger, W. H., Muccitelli, R. M., and Kupfer, D. (1978). Studies on the in vivo and in vitro estrogenic activities of methoxychlor and its metabolites. Role of hepatic mono-oxygenase in methoxychlor activation. Biochem. Pharmacol. 27, 2417–2423. Castro, J. M., and Ball, G. F. (1996). Early administration of 17␤estradiol partially masculinizes song control regions and ␣2-adrenergic receptor distribution in european starlings (Sturnus vulgaris). Horm. Behav. 30, 387– 406. Colborn, T. (1995). Environmental estrogens: Health implications for humans and wildlife. Environ. Health Perspect. 103(Suppl. 7), 135–136. Desbrow, C., Routledge, E. J., Brighty, G. C., Sumpter, J. P., and Waldock, M. (1998). Identification of estrogenic chemicals in STW effluent. Chemical fractionation and in vitro biological screening. Environ. Sci. Techol. 32, 1549 –1558. Erichsen, A. L., Craig-Veit, C. B., Viant, M. R., Quaglino, A., Fry, D. M., and Millam, J. R. (1999). Evaluation of the effects of xenoestrogens on sexual differentiation and reproductive performance in an altricial songbird model, Zebra Finch (Tanaepygia guttata). Keystone Symposium on Endocrine Disruptors B5: 41. Fletcher, J. S., Nellessen, J. E., and Pfleeger, T. G. (1994). Literature review and evaluation of the EPA food-chain (Kenaga) nomogram, an instrument for estimating pesticide residues on plants. Environ. Toxicol. Chem. 13, 1383–1391. Fry, D. M., and Toone, C. K. (1981). DDT-induced feminization of gull embryos. Science 213, 922–924. Gray, L. E., Jr., Ferrell, J. M., and Ostby, J. S. (1985). Alteration of behavioral sex differentiation by exposure to estrogenic com-

Oral Estrogen and Song System Differentiation

pounds during a critical neonatal period: Effects of zearalenone, methoxychlor, and estradiol in hamsters. Toxicol. Appl. Pharmacol. 80, 127–136. Gurney, M. E. (1982). Behavioral correlates of sexual differentiation in the zebra finch song system. Brain Res. 231, 153–172. Gurney, M. E., and Konishi, M. (1980). Hormone-induced sexual differentiation of brain and behavior in zebra finches. Science 208, 1380 –1382. Holloway, C. C., and Clayton, D. F. (2001). Estrogen synthesis in the male brain triggers development of the avian song control pathway in vitro. Nature Neurosci. 4, 170 –175. Hoerger, F., and Kenaga, E. E. (1972). Pesticide residues on plants: Correlation of representative data as a basis for estimation of their magnitude in the environment. In F. Coulston and F. Korte (Eds.), Environmental Quality and Safety: Chemistry, Toxicology, and Technology, pp. 9 –28. Thieme, Stuttgart, West Germany. Jacobs, E. C., Grisham, W., and Arnold, A. P. (1994). Lack of a synergistic effect between estradiol and dihydrotestosterone in the masculinization of the zebra finch song system. J. Neurobiol. 27, 513. Jobling, S., and Sumpter, J. P. (1993). Detergent components in sewage effluent are weakly oestrogenic to fish: an in vitro study rainbow trout (Oncrhynchus mykiss) hepatocytes. Aquat. Toxicol. 27, 361–372. MacDougall-Shackleton, S. A., Hulse, S. H., and Ball, G. F. (1998). Neural correlates of singing behavior in male zebra finches (Taeniopygia guttata). J. Neurobiol. 36, 421– 430. Millam, J. R., Craig-Veit, C. B., Quaglino, A. E., Erichsen, A. L., Famula, T. R., and Fry, D. M. (2001). Post-hatch oral estrogen exposure impairs adult reproductive performance of zebra finch in a sex-specific manner. Horm. Behav. 40, 542–549. Millam, J. R., Faris, P. L., Youngren, O. M., El Halawani, M. E., and Hartman, B. K. (1993). Immunohistochemical localization of chicken gonadotropin-releasing hormones I and II (cGnRH I and II) in turkey hen brain. J. Comp. Neurol. 333, 68 – 82. Naylor, C. G. (1992). Environmental fate of alkylphenol ethoxylate. Soap Cosmet. Chem. Spec. 68, 27–72. Nimrod, A. C., and Benson, W. H. (1996). Environmental estrogenic effects of alkylphenol ethoxylates. Crit. Rev. Toxicol. 26, 335–364. Nordeen, K. W., Nordeen, E. J., and Arnold, A. P. (1986). Estrogen

241

establishes sex differences in androgen accumulation in zebra finch brain. J. Neurosci. 6, 734 –738. Nottebohm, F., and Arnold, A. P. (1976). Sexual dimorphism in vocal control areas of the songbird brain. Science 194, 211–213. Odum, J., Lefevre, P. A., Tittensor, S., Paton, D., Routledge, E. J., Beresford, N. A., Sumpter, J. P., and Ashby, J. (1997). The rodent uterotrophic assay: critical period features, studies with nonyl phenols, and comparison with a yeast estrogenicity assay. Reg. Toxicol. Pharmacol. 25, 176 –188. Purdom, C. E., Hardiman, P. A., Bye, V. J., Eno, N. C., Tyler, C. R., and Sumpter, J. P. (1994). Estrogenic effects of effluents from sewage treatment works. Chem. Ecol. 8, 275–285. Roy, D., Palangat, M., Chen, C., Thomas, R. D., Colerangle, J., Atkinson, A., and Yan, Z. (1997). Biochemical and molecular changes at the cellular level in response to exposure to environmental estrogen-like chemicals. J. Toxicol. Environ. Health 50, 1–29. Schlinger, B. A., and Arnold, A. P. (1993). Estrogen synthesis in vivo in the adult zebra finch: Additional evidence that circulating estrogens can originate in brain. Endocrinology 133, 2610 –2616. Servos, M. R. (1999). Review of the aquatic toxicology, estrogenic responses and bioaccumulation of alkylphenols and alkylphenol polyethoxylates. Water Qual. Res. J. Can. 34, 123–177. Shelby, M. D., Newbold, R. R., Tully, D. B., Chae, K., and Davis, V. L. (1997). Assessing environmental chemicals for oestrogenicity using a combination of in vitro and in vivo assays. Environ. Health Perspect. 104, 1296 –1300. Simpson, H. B., and Vicario, D. S. (1991a). Early estrogen treatment alone causes female zebra finches to produce learned, male-like vocalizations. J. Neurobiol. 22, 755–776. Simpson, H. B., and Vicario, D. S. (1991b). Early estrogen treatment of female zebra finches masculinizes the brain pathway for learned vocalizations. J. Neurobiol. 22, 777–793. Staples, C. A., Weeks, J., Hall, J. F., and Naylor, C. G. (1998). Evaluation of aquatic toxicity and bioaccumulation of C8- and C9alkylphenol ethoxylates. Environ. Toxicol. Chem. 17, 2470 –2480. Weiss, L., and Greep, R. O. (1977). Histology, 4th ed. McGraw Hill, New York. White, R., Jobling, S., Hoare, S. A., Sumpter J. P., and Parker, M. G. (1994). Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135, 175–182.