Reproductive Consequences of Exposure to Waterborne Phytoestrogens in Male Fighting Fish Betta splendens

  • Louise M. Stevenson
  • Alexandria C. Brown
  • Tracy M. Montgomery
  • Ethan D. ClotfelterEmail author


Phytoestrogens are plant compounds that can act as endocrine disruptors in vertebrates. Biologically active levels of phytoestrogens have been found in aquatic habitats near wood pulp and paper mills, biofuel manufacturing plants, sewage-treatment plants, and agricultural fields. Phytoestrogens are known to cause hormonal and gonadal changes in male fish, but few studies have connected these effects to outcomes relevant to reproductive success. In one experiment, we exposed sexually mature male fighting fish Betta splendens to environmentally relevant (1 μg L−1) and pharmacological concentrations (1000 μg L−1) of the phytoestrogen genistein as well as to a positive control of waterborne 17β-estradiol (E2; 1 μg L−1), and a negative control of untreated water. In a second experiment, we exposed male B. splendens to environmentally relevant concentrations (1 μg L−1) of genistein and β-sitosterol singly and in combination as well as to the positive and negative controls. All exposures were 21 days in duration. We measured sex-steroid hormone levels, gonadosomatic index (GSI), sperm concentration and motility, and fertilization success in these fish. We found that exposure to genistein did not affect circulating levels of the androgen 11-ketotestosterone or the estrogen E2 relative to negative-control fish. We also found that neither of the compounds nor their mixture affected GSI, sperm concentration or motility, or fertilization success in exposed fish relative to negative-control fish. However, fish exposed to phytoestrogens showed some evidence of fewer but more motile sperm than fish exposed to the positive control E2. We conclude that sexually mature male B. splendens are relatively immune to reproductive impairments from short-term exposure to waterborne phytoestrogens.


Genistein Sperm Motility Paper Mill Sperm Concentration Fertilization Success 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank the following people for assistance in the laboratory and for the collection of pilot data that led to this manuscript: Tatiana Butler, Julian Damashek, Genelle Diaz-Silveira, Natalie Ferraiolo, Heather Leonard, Kathy Nieves-Puigdoller, and David Westwood. Thanks to Dante Vargas and M. Monica Giusti for conducting the HPLC analyses of our genistein water samples. Renae Brodie, Beth Jakob, Denise Pope, and two anonymous reviewers offered useful comments on an earlier version of this manuscript. Funding for this research was provided by the Webster Fund of the Department of Biology at Amherst College, the H. Axel Schupf ‘57 Fund for Intellectual Life at Amherst College, HHMI grant 52005107 to Amherst College, and National Science Foundation grant IOS-0725186 to E. D. C.

Supplementary material

244_2010_9561_MOESM1_ESM.docx (23 kb)
Supplementary material 1 (DOCX 22 kb)


  1. Bayley M, Junge M, Baatrup E (2002) Exposure of juvenile guppies to three antiandrogens causes demasculinization and a reduced sperm count in adult males. Aquat Toxicol 56:227–239CrossRefGoogle Scholar
  2. Bennetau-Pelissero C, Breton B, Bennetau B, Corraze G, Le Menn F, Davail-Cuisset B et al (2001) Effect of genistein-enriched diets on the endocrine process of gametogenesis and on reproduction efficiency of the rainbow trout Oncorhynchus mykiss. Gen Comp Endocrinol 121:173–187CrossRefGoogle Scholar
  3. Bistodeau TJ, Barber LB, Bartell SE, Cediel RA, Grove KJ, Klaustermeier J et al (2006) Larval exposure to environmentally relevant mixtures of alkylphenolethoxylates reduces reproductive competence in male fathead minnows. Aquat Toxicol 79:268–277CrossRefGoogle Scholar
  4. Björkblom C, Högfors E, Salste L, Bergelin E, Olsson P, Katsiadaki I et al (2009) Estrogenic and androgenic effects of municipal wastewater effluent on reproductive endpoint biomarkers in three-spined stickleback (Gasterosteus aculeatus). Environ Toxicol Chem 28:1063–1071CrossRefGoogle Scholar
  5. Borg B (1994) Androgens in teleost fishes. Comp Biochem Physiol C 109:219–245CrossRefGoogle Scholar
  6. Brucker-Davis F (1998) Effects of environmental synthetic chemicals on thyroid function. Thyroid 8:827–856CrossRefGoogle Scholar
  7. Burnison BK, Hartmann A, Lister A, Servos MR, Ternes T, Van Der Kraak G (2003) A toxicity identification evaluation approach to studying estrogenic substances in hog manure and agricultural runoff. Environ Toxicol Chem 22:2243–2250CrossRefGoogle Scholar
  8. Christianson-Heiska I, Smeds P, Granholm N, Bergelin E, Isomaa B (2007) Endocrine modulating actions of a phytosterol mixture and its oxidation products in zebrafish (Danio rerio). Comp Biochem Physiol C 145:518–527Google Scholar
  9. Clotfelter ED, Rodriguez AC (2006) Behavioral changes in fish exposed to phytoestrogens. Environ Pollut 144:833–839CrossRefGoogle Scholar
  10. Clotfelter ED, Bell AM, Levering KR (2004) The role of animal behaviour in the study of endocrine-disrupting chemicals. Anim Behav 68:465–476CrossRefGoogle Scholar
  11. Clotfelter ED, Curren LJ, Murphy CE (2006) Mate choice and spawning success in the fighting fish Betta splendens: the importance of body size, display behavior and nest size. Ethology 112:1170–1178CrossRefGoogle Scholar
  12. Clotfelter ED, McNitt MM, Carpenter RE, Summers CH (2010) Modulation of monoamine neurotransmitters in fighting fish Betta splendens exposed to waterborne phytoestrogens. Fish Physiol BiochemGoogle Scholar
  13. Couillard CM, Williams PJ, Courtenay SC, Rawn GP (1999) Histopathological evaluation of Atlantic tomcod (Microgadus tomcod) collected at estuarine sites receiving pulp and paper mill effluent. Aquat Toxicol 44:263–278CrossRefGoogle Scholar
  14. Dixon RA (2004) Phytoestrogens. Annu Rev Plant Biol 55:225–261CrossRefGoogle Scholar
  15. Dzieweczynski TL, Eklund AC, Rowland WJ (2006) Male 11-ketotestosterone levels change as a result of being watched in Siamese fighting fish, Betta splendens. Gen Comp Endocrinol 147:184–189CrossRefGoogle Scholar
  16. Earley RL, Edwards JT, Aseem O, Felton K, Blumer LS, Karom M et al (2006) Social interactions tune aggression and stress responsiveness in a territorial cichlid fish (Archocentrus nigrofasciatus). Physiol Behav 88:353–363CrossRefGoogle Scholar
  17. Ellis T, James JD, Stewart C, Scott AP (2004) A non-invasive stress assay based upon measurement of free cortisol released into the water by rainbow trout. J Fish Biol 65:1233–1252CrossRefGoogle Scholar
  18. Fitzpatrick JL, Craig PM, Bucking C, Balshine S, Wood CM, McClelland GB (2009) Sperm performance under hypoxic conditions in the intertidal fish Porichthys notatus. Can J Zool 87:464–469CrossRefGoogle Scholar
  19. Foberg A (2003) The Siamese fighting fish (Betta splendens): an alternative fish species to use in evaluating the impact of endocrine disrupting chemicals with focus on aggressive performance. Master’s thesis, Uppsala, Sweden, Uppsala UniversityGoogle Scholar
  20. Green C, Kelly A (2008) Effect of the exogenous soyabean phyto-oestrogen genistein on sperm quality, ATP content and fertilization rates in channel catfish Ictalurus punctatus (Rafinesque) and walleye Sander vitreus (Mitchill). J Fish Biol 72:2485–2499CrossRefGoogle Scholar
  21. Green C, Kelly A (2009) Effects of the estrogen mimic genistein as a dietary component on sex differentiation and ethoxyresorufin-O-deethylase (EROD) activity in channel catfish (Ictalurus punctatus). Fish Physiol Biochem 35:377–384CrossRefGoogle Scholar
  22. Hassanin A, Kuwahara S, Nurhidayat, Tsukamoto Y, Ogawa K, Hiramatsu K et al (2002) Gonadosomatic index and testis morphology of common carp (Cyprinus carpio) in rivers contaminated with estrogenic chemicals. J Vet Med Sci 64:921–926CrossRefGoogle Scholar
  23. Haubruge E, Petit F, Gage MJ (2000) Reduced sperm counts in guppies (Poecilia reticulata) following exposure to low levels of tributyltin and bisphenol A. Proc R Soc Lond B 267:2333–2337CrossRefGoogle Scholar
  24. Howell WM, Black D, Bortone S (1980) Abnormal expression of secondary sex characters in a population of mosquitofish, Gambusia affinis holbrooki: evidence for environmentally-induced masculinization. Copeia 4:676–681CrossRefGoogle Scholar
  25. Ishibashi H, Tachibana K, Tsuchimoto M, Soyano K, Tatarazako N, Matsumura N et al (2004) Effects of nonylphenol and phytoestrogen-enriched diet on plasma vitellogenin, steroid hormone, hepatic cytochrome P450 1A, and glutathione-S-transferase values in goldfish (Carassius auratus). Comp Med 54:54–62Google Scholar
  26. Jobling S, Tyler CR (2003) Endocrine disruption in wild freshwater fish. Pure Appl Chem 75:2219–2234CrossRefGoogle Scholar
  27. Karels AA, Manning S, Brouwer TH, Brouwer M (2003) Reproductive effects of estrogenic and antiestrogenic chemicals on sheepshead minnows (Cyprinodon variegatus). Environ Toxicol Chem 22:855–865Google Scholar
  28. Kavumpurath S, Pandian TJ (1993) Determination of labile period and critical dose for sex reversal by oral administration of estrogens in Betta splendens (Regan). Indian J Exp Biol 31:16–20Google Scholar
  29. Kime DE, Van Look KJW, McAllister BG, Huyskens G, Rurangwa E, Ollevier F (2001) Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish. Comp Biochem Physiol C 130:425–433Google Scholar
  30. Kiparissis Y, Hughes R, Metcalfe C (2001) Identification of the isoflavonoid genistein in bleached kraft mill effluent. Environ Sci Technol 35:2423–2427CrossRefGoogle Scholar
  31. Kiparissis Y, Balch G, Metcalfe T, Metcalfe C (2003) Effects of the isoflavones genistein and equol on the gonadal development of Japanese medaka (Oryzias latipes). Environ Health Perspect 111:1158–1163CrossRefGoogle Scholar
  32. Kristensen T, Baatrup E, Bayley M (2005) 17α-ethinylestradiol reduces the competitive reproductive fitness of the male guppy (Poecilia reticulata). Biol Reprod 72:150–156CrossRefGoogle Scholar
  33. Lahnsteiner F, Berger B, Kletzl M, Weismann T (2006) Effect of 17β-estradiol on gamete quality and maturation in two salmonid species. Aquat Toxicol 79:124–131CrossRefGoogle Scholar
  34. Latonnelle K, Fostier A, Le Menn F, Bennetau-Pelissero C (2002) Binding affinities of hepatic nuclear estrogen receptors for phytoestrogens in rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baeri). Gen Comp Endocrinol 129:69–79CrossRefGoogle Scholar
  35. Lehtinen KJ, Mattsson K, Tana J, Engström C, Lerche O, Hemming J (1999) Effects of wood-related sterols on the reproduction, egg survival, and offspring of brown trout (Salmo trutta lacustris L.). Ecotoxicol Environ Saf 42:40–49CrossRefGoogle Scholar
  36. Leusch FD, MacLatchy D (2003) In vivo implants of β-sitosterol cause reductions of reactive cholesterol pools in mitochondria isolated from gonads of male goldfish (Carassius auratus). Gen Comp Endocrinol 134:255–263CrossRefGoogle Scholar
  37. Lundgren MS, Novak PJ (2009) Quantification of phytoestrogens in industrial waste streams. Environ Toxicol Chem 28:2318–2323CrossRefGoogle Scholar
  38. MacLatchy D, Van Der Kraak G (1995) The phytoestrogen β-sitosterol alters the reproductive endocrine status of goldfish. Toxicol Appl Pharmacol 134:305–312CrossRefGoogle Scholar
  39. MacLatchy D, Peters L, Nickle J, Van Der Kraak G (1997) Exposure to β-sitosterol alters the endocrine status of goldfish differently than 17β-estradiol. Environ Toxicol Chem 16:1895–1904CrossRefGoogle Scholar
  40. Mahmood-Khan Z, Hall E (2003) Occurrence and removal of plant sterols in pulp and paper mill effluents. J Environ Eng Sci 2:17–26CrossRefGoogle Scholar
  41. Mahmood-Khan Z, Hall E (2008) Quantification of plant sterols in pulp and paper mill effluents. Water Qual Res J Can 43:173–181Google Scholar
  42. Martinović D, Hogarth WT, Jones RE, Sorensen PW (2007) Environmental estrogens suppress hormones, behavior, and reproductive fitness in male fathead minnows. Environ Toxicol Chem 26:271–278CrossRefGoogle Scholar
  43. Mattsson K, Tana J, Engstrom C, Hemming J, Lehtinen KJ (2001) Effects of wood-related sterols on the offspring of the viviparous blenny, Zoarces viviparus L. Ecotoxicol Environ Saf 49:122–130CrossRefGoogle Scholar
  44. Mellanen P, Petänen T, Lehtimäki J, Mäkelä S, Bylund G, Holmbom B et al (1996) Wood-derived estrogens: studies in vitro with breast cancer cell lines and in vivo in trout. Toxicol Appl Pharmacol 136:381–388CrossRefGoogle Scholar
  45. Munkittrick KR, Portt CB, Van der Kraak G, Smith IR, Rokosh DA (1991) Impact of bleached kraft mill effluent on population characteristics, liver MFO activity, and serum steroid-levels of a Lake Superior white sucker (Catostomus commersonii) population. Can J Fish Aquat Sci 48:1371–1380CrossRefGoogle Scholar
  46. Munkittrick KR, McMaster ME, McCarthy LH, Servos MR, Van Der Kraak GJ (1998) An overview of recent studies on the potential of pulp-mill effluents to alter reproductive parameters in fish. J Toxicol Environ Health 1:347–371CrossRefGoogle Scholar
  47. Organization for Economic Cooperation and Development (1992) Fish prolonged toxicity test: 14 day study. Guideline 204. Paris, FranceGoogle Scholar
  48. Örn S, Svenson A, Viktor T, Holbech H, Norrgren L (2006) Male-biased sex ratios and vitellogenin induction in zebrafish exposed to effluent water from a Swedish pulp mill. Arch Environ Contam Toxicol 51:445–451CrossRefGoogle Scholar
  49. Orrego R, Burgos A, Moraga-Cid G, Inzunza B, Gonzalez M, Valenzuela A et al (2006) Effects of pulp and paper mill discharges on caged rainbow trout (Oncorhynchus mykiss): biomarker responses along a pollution gradient in the Biobio River, Chile. Environ Toxicol Chem 25:2280–2287CrossRefGoogle Scholar
  50. Orrego R, Guchardi J, Hernandez V, Krause R, Roti L, Armour J et al (2009) Pulp and paper mill effluent treatments have differential endocrine-disrupting effects on rainbow trout. Environ Toxicol Chem 28:181–188CrossRefGoogle Scholar
  51. Pollack SJ, Ottinger MA, Sullivan CV, Woods LC (2003) The effects of the soy isoflavone genistein on the reproductive development of striped bass. North Am J Aquacult 65:226–234CrossRefGoogle Scholar
  52. Puglisi E, Nicelli M, Capri E, Trevisan M, Del Re AAM, Trevisan M (2003) Cholesterol, β-sitosterol, ergosterol, and coprostanol in agricultural soils. J Environ Qual 32:466–471CrossRefGoogle Scholar
  53. Rurangwa E, Kime DE, Ollevier F, Nash JP (2004) The measurement of sperm motility and factors affecting sperm quality in cultured fish. Aquaculture 234:1–28CrossRefGoogle Scholar
  54. Schoenfuss HL, Levitt JT, Rai R, Julius ML, Martinovic D (2009) Treated wastewater effluent reduces sperm motility along an osmolality gradient. Arch Environ Contam Toxicol 56:397–407CrossRefGoogle Scholar
  55. Scholz S, Gutzeit H (2001) Lasting effects of xeno- and phytoestrogens on sex differentiation and reproduction of fish. Environ Sci 8:57–73Google Scholar
  56. Scholz S, Klüver N (2009) Effects of endocrine disrupters on sexual, gonadal development in fish. Sex Dev 3:136–151CrossRefGoogle Scholar
  57. Sepúlveda MS, Quinn BP, Denslow ND, Holm SE, Gross TS (2003) Effects of pulp and paper mill effluents on reproductive success of largemouth bass. Environ Toxicol Chem 22:205–213CrossRefGoogle Scholar
  58. Servos MR, Munkittrick K, Carey J, Van Der Kraak G (1995) Environmental fate and effects of pulp and paper mill effluents. St. Lucie Press, Boca Raton, FLGoogle Scholar
  59. Sharpe R, Drolet M, MacLatchy D (2006) Investigation of de novo cholesterol synthetic capacity in the gonads of goldfish (Carassius auratus) exposed to the phytosterol beta-sitosterol. Reprod Biol Endocrinol 4:60CrossRefGoogle Scholar
  60. Spengler P, Korner W, Metzger JW (2001) Substances with estrogenic activity in effluents of sewage treatment plants in southwestern Germany. 1. Chemical analysis. Environ Toxicol Chem 20:2133–2141CrossRefGoogle Scholar
  61. Sumpter JP, Jobling S (1995) Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ Health Perspect 103:173–178CrossRefGoogle Scholar
  62. Toft G, Edwards TM, Baatrup E, Guillette LJ (2003) Disturbed sexual characteristics in male mosquitofish (Gambusia holbrooki) from a lake contaminated with endocrine disruptors. Environ Health Perspect 111:695–701CrossRefGoogle Scholar
  63. Toft G, Baatrup E, Guillette LJ (2004) Altered social behavior and sexual characteristics in mosquitofish (Gambusia holbrooki) living downstream of a paper mill. Aquat Toxicol 70:213–222CrossRefGoogle Scholar
  64. Tremblay L, Van Der Kraak G (1998) Use of a series of homologous in vitro and in vivo assays to evaluate the endocrine modulating actions of β-sitosterol in rainbow trout. Aquat Toxicol 43:149–162CrossRefGoogle Scholar
  65. Tremblay L, Van Der Kraak G (1999) Comparison between the effects of the phytosterol β-sitosterol and pulp and paper mill effluents on sexually immature rainbow trout. Environ Toxicol Chem 18:329–336CrossRefGoogle Scholar
  66. van den Heuvel M, Ellis R (2002) Timing of exposure to a pulp and paper effluent influences the manifestation of reproductive effects in rainbow trout. Environ Toxicol Chem 21:2338–2347CrossRefGoogle Scholar
  67. Volckaert FA, Galbusera PH, Hellemans BA, Van den Haute C, Vanstaen D, Ollevier F (1994) Gynogenesis in the African catfish (Clarias gariepinus). I. Induction of meiogynogenesis with thermal and pressure shocks. Aquaculture 128:221–233CrossRefGoogle Scholar
  68. Wartman CA, Hogan NS, Hewitt LM, McMaster ME, Landman MJ, Taylor S et al (2009) Androgenic effects of a Canadian bleached kraft pulp and paper effluent as assessed using three-spine stickleback (Gasterosteus aculeatus). Aquat Toxicol 92:131–139CrossRefGoogle Scholar
  69. Wilson-Leedy JG, Ingermann RL (2007) Development of a novel CASA system based on open source software for characterization of zebrafish sperm motility parameters. Theriogenology 67:661–672CrossRefGoogle Scholar
  70. Zhang L, Khan IA, Foran CM (2002) Characterization of the estrogenic response to genistein in Japanese medaka (Oryzias latipes). Comp Biochem Physiol C 132:203–211CrossRefGoogle Scholar
  71. Zhang L, Khan IA, Willett KL, Foran CM (2003) In vivo effects of black cohosh and genistein on estrogenic activity and lipid peroxidation in Japanese medaka (Oryzias latipes). J Herb Pharmacother 3:33–50CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Louise M. Stevenson
    • 1
  • Alexandria C. Brown
    • 1
  • Tracy M. Montgomery
    • 1
  • Ethan D. Clotfelter
    • 1
    Email author
  1. 1.Department of BiologyAmherst CollegeAmherstUSA

Personalised recommendations