Environ. Sci. Technol. 2001, 35, 2917-2925
Reproductive Effects of Long-Term Exposure to Bisphenol A in the Fathead Minnow (Pimephales promelas) P. J. T. R. J.
S O H O N I , † C . R . T Y L E R , * ,‡ K . H U R D , § CAUNTER,§ M. HETHERIDGE,§ WILLIAMS,§ C. WOODS,§ M. EVANS,§ TOY,| M. GARGAS,⊥ AND P. SUMPTER†
Department of Biological Sciences, Brunel University, Uxbridge, UB8 3PH, U.K., School of Biological Sciences, Hatherly Laboratories, University of Exeter, The Prince of Wales Road, Exeter, EX4 4PS, U.K., Brixham Environmental Laboratory, AstraZeneca, Freshwater Quarry, Devon, TQ5 8BA, U.K., Shell Chemicals Ltd., London, SE1 7NA, U.K., and The Society of the Plastics Industry Inc., 1801K Street NW, Suite 600K, Washington, D.C.
Bisphenol A (BPA), a high-volume chemical used to make polycarbonate plastic, epoxy resins, and other chemicals has been reported to be weakly estrogenic. To investigate the effects of long-term exposure to Bisphenol A, a multigeneration study was conducted in which fathead minnows (Pimephales promelas) were exposed to water concentrations of BPA in the range from 1 to 1280 µg/L. In this paper, we report the growth and reproductive effects of BPA on sexually mature adults in the F0 generation (after 43, 71, and 164 d of exposure) and the effects on hatchability in the F1 generation. Mean measured concentrations of BPA in the water for all doses, over a 164-d exposure period, were between 70% and 96% of nominal. An inhibitory effect of BPA on somatic growth (length and weight) occurred in adult male fish exposed to 640 and 1280 µg/L (after 71 and 164 d). BPA induced vitellogenin synthesis (VTG; a biomarker for estrogen exposure) in males at concentrations of 640 and 1280 µg/L after 43 d and 160 µg/L after 71 d. In females, plasma VTG concentrations were elevated above controls only after 164-d exposure to 640 µg/L. Inhibition of gonadal growth (as measured by the gonadosomatic index) occurred in both males and females at concentrations of 640 and 1280 µg/L after 164 d. In males, a concentration of 16 µg/L altered the proportion of sex cell types in the testis, suggesting inhibition of spermatogenesis. Concentrations of BPA that induced VTG synthesis and affected gonadal development were lower than those that resulted in discernible effects on reproductive output. Egg production was inhibited at a BPA concentration of 1280 µg/L, and hatchability in the F1 generation was reduced at a BPA concentration of 640 µg/L (there were not enough eggs spawned in the 1280 µg/L group for hatchability studies to be conducted). The results demonstrate that BPA acts as a weak estrogen to fish when administered via the water, with effects on breeding at and above 640 µg/L. 10.1021/es000198n CCC: $20.00 Published on Web 06/01/2001
2001 American Chemical Society
Introduction Concern has been expressed about the presence of chemicals in the environment that mimic hormones (reviewed in refs 1 and 2). Most of the hormone mimics identified to date are estrogenic in nature. They include natural and synthetic estrogens (e.g., estradiol and ethinyl estradiol; 3-5); phytoand myco-estrogens (6) derived from plants and fungi; and a wide variety of man-made industrial compounds, such as some phthalate plasticizers (7), degradation products of some surfactants (8), and food additives and pesticides (9-11). Interactions of these compounds with the endocrine systems of mammals (including humans) have been suggested to cause or at least contribute to altered reproductive capacity, infertility, endometriosis, and cancers of the breast, uterus, and prostate (12-14). Most of the evidence for endocrine disruption, however, has come from studies on wildlife, especially organisms living in or closely associated with the aquatic environment (15-17). Freshwater and marine environments receive large volume discharges of domestic and industrial wastes that can contain complex cocktails of estrogens (and other endocrine mimics, e.g., androgen agonists and antagonists). Adverse reproductive effects that are indicative of exposure to environmental estrogens have been demonstrated in wild fish in both the riverine environment (18) and in the marine environment (19, 20). Furthermore, laboratory studies on fish have shown that some of the chemicals of concern are present in the aquatic environment at concentrations sufficient to cause adverse reproductive effects (21). Bisphenol A (BPA), a high production volume chemical used to make polycarbonate plastic, epoxy resins, and other chemicals, has been reported to be estrogenic (22, 23). BPA binds to the estrogen receptor with an affinity 2000 times less than the affinity of estradiol-17β (24). In vivo studies in mammals have indicated BPA is only weakly estrogenic, however, and some endocrine-mediated effects could not be replicated. In the original work by Nagel et al. (25), maternal exposure to low doses of BPA was reported to reduce both daily sperm production and seminal vesicle weight and resulted in an increase in prostate weight of male mouse pups. These results, however, could not be reproduced by Cagen and colleagues (26). Recent studies have reported no persistent adverse effects of neonatal exposure to BPA on the reproductive tract (27, 28), but effects of postnatal exposure to BPA in rat uterotrophic assays (29) and a depression in plasma testosterone in males (30) do suggest that BPA has endocrine activity in vivo, albeit only when administered at very high doses (31). For aquatic animals, like fish, major routes of exposure to environmental chemicals include uptake directly from the water across the skin and/or gill surfaces, which may result in a very different biological potency for endocrine disrupting chemicals (EDCs) as compared with exposure via the diet. Data available for exposure of animals to 4-tertnonylphenol (4-NP) would appear to support this hypothesis; very high oral doses of 4-NP are needed to induce an estrogenic effect in rodents (tens of milligrams per kilogram of body weight per day for several days; 32), whereas only * Corresponding author telephone: 01392 264450; fax: 01392 263700; e-mail:
[email protected]. † Brunel University. ‡ University of Exeter. § AstraZeneca. | Shell Chemicals Ltd. ⊥ The Society of the Plastics Industry Inc. VOL. 35, NO. 14, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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microgram per liter concentrations induce vitellogenin (VTG, a biomarker for estrogen exposure) synthesis in fish (33). For example, exposure of fathead minnows (Pimephales promelas) to 57.7 ( 3 µg of 4-NP/L via water completely inhibits reproduction (34). The explanation for this apparent pronounced difference in the sensitivities of mammals and fish to EDCs are likely, at least in part, to lie in the quite different pharmacokinetics associated with the two test systems. In the case of exposure of mammals to EDCs using the oral route, the test chemical will probably undergo some digestion within the alimentary system, and any remaining chemical that is absorbed will travel first from the intestine to the liver, the major site of metabolism of xenobiotics, before any reaches target organs such as the reproductive organs. In contrast, in fish (and probably many other aquatic organisms), water-borne chemicals probably enter the bloodstream via the gills, and from there they would go directly to the target organs without first passing to the liver. These factors emphasize the need to more fully establish the effects of long-term exposures to low concentrations of weak EDCs, like BPA, administered via the water to aquatic organisms. Little is known about long-term (continuous) exposure of fish, or any aquatic organism, to environmental estrogens. Indeed, to date, despite the potential developmental and reproductive impacts of estrogens (either steroid estrogens or estrogen mimics), there have been no reported multigeneration studies to investigate these chemicals in fish. To investigate the effects of long-term exposure to BPA, a multigeneration study was set up in which fathead minnows (one of the most widely used fish species in ecotoxicology) were exposed to a wide range of concentrations of BPA ranging from 1 up to 1280 µg/L. The low end of this range included concentrations of BPA that have been measured in effluents in Europe and the United States (up to 25 µg/L; 35-37). In this study, we report the growth and reproductive effects of exposure to BPA in sexually mature adults (exposed for up to 164 d) and the effects on hatching in the F1 generation.
Materials and Methods Fish/Embryo Maintenance and Culture. Fathead minnow (P. promelas) were derived from breeding stocks maintained at Brixham Environmental Laboratory, AstraZeneca UK Limited. Fish were fed Promin (Promin Ltd., Hampshire, U.K.) at a regularly adjusted rate to provide a daily ration of approximately 2% of body weight, supplemented with frozen Artemia. Dechlorinated tap water was used to culture all embryos and fish. In the test solutions, dissolved oxygen was between 5.6 and 8.6 mg/L, pH was between 7.1 and 7.8, and water temperature was between 24.1 and 25.8 °C. During the course of the study, the conductivity of the laboratory water supply ranged between 197 and 323 mS/cm; total ammonia was from 640 µg/L) and reproductive output (egg production, 1280 µg/L) in the F0 adults and on hatchability in the F1 generation (> 640 µg/L) were only 7-fold below the 96-h LC50 for BPA in fish. As such, these effects (with the exception of VTG induction) may have been a result of sublethal toxicity rather than necessarily an effect mediated via the endocrine axis alone. The concentrations of BPA required to inhibit somatic growth and reproductive performance (as defined by egg production and hatchability of the subsequent offspring) were at least 3 orders of magnitude higher than concentrations measured in most aquatic environments (less than 1 µg/L; 5, 50). This information, together with the knowledge that BPA has a low potential to bioaccumulate in fish (it has a bioconcentration factor of between 5 and 68; 37), would suggest that BPA is unlikely to pose a major environmental problem for fish in most aquatic environments.
Acknowledgments A.S. was supported by a grant from The Society of the Plastics Industry.
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Received for review August 29, 2000. Revised manuscript received April 10, 2001. Accepted April 16, 2001. ES000198N
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