, Volume 19, Issue 7, pp 1312–1321 | Cite as

Development of partial life-cycle experiments to assess the effects of endocrine disruptors on the freshwater gastropod Lymnaea stagnalis: a case-study with vinclozolin

  • Virginie Ducrot
  • Mickaël Teixeira-Alves
  • Christelle Lopes
  • Marie-Laure Delignette-Muller
  • Sandrine Charles
  • Laurent Lagadic


Long-term effects of endocrine disruptors (EDs) on aquatic invertebrates remain difficult to assess, mainly due to the lack of appropriate sensitive toxicity test methods and relevant data analysis procedures. This study aimed at identifying windows of sensitivity to EDs along the life-cycle of the freshwater snail Lymnaea stagnalis, a candidate species for the development of forthcoming test guidelines. Juveniles, sub-adults, young adults and adults were exposed for 21 days to the fungicide vinclozolin (VZ). Survival, growth, onset of reproduction, fertility and fecundity were monitored weekly. Data were analyzed using standard statistical analysis procedures and mixed-effect models. No deleterious effect on survival and growth occurred in snails exposed to VZ at environmentally relevant concentrations. A significant impairment of the male function occurred in young adults, leading to infertility at concentrations exceeding 0.025 μg/L. Furthermore, fecundity was impaired in adults exposed to concentrations exceeding 25 μg/L. Biological responses depended on VZ concentration, exposure duration and on their interaction, leading to complex response patterns. The use of a standard statistical approach to analyze those data led to underestimation of VZ effects on reproduction, whereas effects could reliably be analyzed by mixed-effect models. L. stagnalis may be among the most sensitive invertebrate species to VZ, a 21-day reproduction test allowing the detection of deleterious effects at environmentally relevant concentrations of the fungicide. These results thus reinforce the relevance of L. stagnalis as a good candidate species for the development of guidelines devoted to the risk assessment of EDs.


Partial life-cycle tests Anti-androgens Ecological risk assessment Reproduction Mixed-effect models Gastropods 



The authors thank Micheline Heydorff for her technical assistance, Jean-Pierre Cravedi and Lennart Weltje for providing complementary information on vinclozolin fate and effects, and the INRA for funding this study. We also thank the two anonymous reviewers for their careful reading and relevant comments, which helped us to improve the quality of this publication.


  1. Baatrup E, Junge L (2001) Antiandrogenic pesticides disrupt sexual characteristics in the adult male guppy (Poecilia reticulata). Environ Health Perspect 109:1063–1070Google Scholar
  2. Bayley M, Larsen PF, Bækgaard H, Baatrup E (2003) The effects of vinclozolin on male guppy secondary sex characters and reproductive success. Biol Reprod 69:1951–1956CrossRefGoogle Scholar
  3. Berrie AD (1965) On the life cycle of Lymnaea stagnalis (L.) in the West of Scotland. Proc Malacol Soc Lond 36:283–295Google Scholar
  4. Bryan GW, Gibbs PE (1991) Impact of low concentrations of tributyltin (TBT) on marine organisms: a review. In: Newman MC, McIntosh AW (eds) Metal ecotoxicology: concepts and applications. Lewis, Ann Arbor, pp 323–361Google Scholar
  5. Bryan GW, Gibbs PE, Burt GR, Hummerstone LG (1987) The effects of tributyltin (TBT) accumulation on adult dogwhelks, Nucella lapillus: long-term field and laboratory experiments. J Mar Biol Assoc UK 67:525–544CrossRefGoogle Scholar
  6. Casey D, Pascoe D, Tattersfield L, Hutchinson TH (2005) A comparison of freshwater pulmonate and prosobranch mollusc responses to steroidal estrogens. In: Abstracts of the Society of Environmental Chemistry and Toxicology (SETAC) 15th Annual Meeting, Lille, France, 22–26 May 2005Google Scholar
  7. Chen CW (2001) Assessment of endocrine disruptors: approaches, issues, and uncertainties. Folia Histochem Cytobiol 39(Suppl 2):20–23Google Scholar
  8. Colborn T, vom Saal FS, Soto AM (1993) Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ Health Perspect 101:378–384CrossRefGoogle Scholar
  9. Coutellec M-A, Lagadic L (2006) Effects of self-fertilization, environmental stress and exposure to xenobiotics on fitness-related traits of the freshwater snail Lymnaea stagnalis. Ecotoxicology 15:199–213CrossRefGoogle Scholar
  10. Czech P, Weber K, Dietrich DR (2001) Effects of endocrine modulating substances on reproduction in the hermaphroditic snail Lymnaea stagnalis L. Aquat Toxicol 53:103–114CrossRefGoogle Scholar
  11. DeFur PL, Crane M, Ingersoll C, Tattersfiels L (eds) (1999) Endocrine disruption in invertebrates, testing and assessment. SETAC Press, PensacolaGoogle Scholar
  12. Ducrot V, Péry ARR, Lagadic L (2010) Modelling long-term effects of diquat under realistic patterns of exposure in genetically differentiated populations of the freshwater gastropod Lymnaea stagnalis. Philos Trans R Soc Lond B, 365 (in press)Google Scholar
  13. Flynn KM, Delclos KB, Newbold RR, Ferguson SA (2001) Behavioral responses of rats exposed to long-term dietary vinclozolin. J Agric Food Chem 49:1658–1665CrossRefGoogle Scholar
  14. FOOTPRINT (2006), FOOTPRINT PPDB database. In the EU-project FOOTPRINT (FP6-SSP-022704). Accessed 23 Feb 2010
  15. Fretter V, Graham A (1962) British Prosobranch Molluscs. Their functional anatomy and ecology. Ray Society, LondonGoogle Scholar
  16. Geraerts WPM, Joosse J (1984) Freshwater snails (Basommatophora). In: Tompa AS, Verdonk NH, van den Biggelar JAM (eds) The mollusca reproduction, vol 7. London Academic Press, London, pp 141–207Google Scholar
  17. Gist GL (1998) National Environmental Health Association position on endocrine disrupters—adopted July 2, 1997. J Environ Health 60:21–23Google Scholar
  18. Gourmelon A, Ahtiainen J (2007) Developing test guidelines on invertebrate development and reproduction for the assessment of chemicals, including potential endocrine active substances—the OECD perspective. Ecotoxicology 16:161–167CrossRefGoogle Scholar
  19. Gray LE, Ostby JS, Kelce WR (1994) Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicol Appl Pharmacol 129:46–52CrossRefGoogle Scholar
  20. Gray LE, Ostby JS, Monosson E, Kelce WR (1999) Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol Ind Health 15:48–64CrossRefGoogle Scholar
  21. Haeba MH, Hilscherová K, MazurováE, Bláha L (2008) Selected endocrine disrupting compounds (vinclozolin, flutamide, ketoconazole and dicofol): effects on survival, occurrence of males, growth, molting and reproduction of Daphnia magna. Environ Sci Pollut Res 15:222–227Google Scholar
  22. Hommen U, Ashauer R, van den Brink P, Caquet T, Ducrot V, Lagadic L, Ratte HT (2010) Extrapolation methods in aquatic effect assessment of time-variable exposures to pesticides. In: Brock TCM, Alix A, Brown C, Capri E, Gottesbüren BFF, Heimbach F, Lythgo CM, Schultz R, Streloke M (eds) Linking aquatic exposure & effects in the risk assessment of pesticides. SETAC, Brussels, pp 211–242Google Scholar
  23. Jarne P, Vianey-Liaud M, Delay B (1993) Selfing and outcrossing in hermaphrodite freshwater gastropods (Basommatophora): where, when and why. Biol J Linn Soc 49:99–125CrossRefGoogle Scholar
  24. Kelce WR, Wilson EM (1997) Environmental antiandrogens: developmental effects, molecular mechanisms, and clinical implications. J Mol Med 75:198–207CrossRefGoogle Scholar
  25. Kelce WR, Monosson E, Gamcsik MP, Laws SC, Gray LE (1994) Environmental hormone disruptors: evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites. Toxicol Appl Pharmacol 126:276–285CrossRefGoogle Scholar
  26. Kiparissis Y, Metcalfe TL, Balch GC, Metcalfe CD (2003) Effects of the antiandrogens vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes). Aquat Toxicol 63:391–403CrossRefGoogle Scholar
  27. Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K (2004) Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect 112:524–531CrossRefGoogle Scholar
  28. Kolodziejczyk A, Martynuska A (1980) Lymnaea stagnalis (L): Feeding habits and production of faeces. Ekol Pol 28:201–217Google Scholar
  29. Kubota K, Ohsako S, Kurosawa S, Takeda K, Qing W, Sakaue M, Kawakami T, Ishimura R, Tohyama C (2003) Effects of vinclozolin administration on sperm production and testosterone biosynthetic pathway in adult male rat. J Reprod Dev 49:409–412CrossRefGoogle Scholar
  30. Lagadic L, Bursztyka J, Baradat M, Lorber S, Heydorff M, Azam D, Quemeneur A, Cravedi J-P (2005) Influence of the complexity of aquatic test systems on the fate of vinclozolin and its reprotoxic effects in Lymnaea stagnalis. In: Abstracts of the SETAC Europe 15th Annual Meeting, Lille, France, 22-26 May 2005, p 105Google Scholar
  31. Lagadic L, Coutellec M-A, Caquet T (2007) Endocrine disruption in aquatic pulmonate molluscs: few evidences, many challenges. Ecotoxicology 16:45–59CrossRefGoogle Scholar
  32. Makynen EA, Kahl MD, Jensen KM, Tietge JE, Wells KL, Van der Kraak G, Ankley GT (2000) Effects of the mammalian antiandrogen vinclozolin on development and reproduction of the fathead minnow (Pimephales promelas). Aquat Toxicol 48:461–475CrossRefGoogle Scholar
  33. Martinović D, Blake LS, Durhan EJ, Greene KJ, Kahl MD, Jensen KM, Makynen EA, Villeneuve DL, Ankley GT (2008) Reproductive toxicity of vinclozolin in the fathead minnow: confirming an anti-androgenic mode of action. Environ Toxicol Chem 27:478–488CrossRefGoogle Scholar
  34. Matthiessen P (2003) Historical perspective on endocrine disruption in wildlife. Pure Appl Chem 75:2197–2206CrossRefGoogle Scholar
  35. Matthiessen P (2008) An assessment of endocrine disruption in molluscs, and the potential for developing internationally-standardised mollusc life cycle test guidelines. Integr Environ Assess Manag 4:274–284CrossRefGoogle Scholar
  36. Matthiessen P, Johnson I (2007) Implications of research on endocrine disruption for the environmental risk assessment, regulation and monitoring of chemicals in the European Union. Environ Pollut 146:9–18CrossRefGoogle Scholar
  37. Mercadier C, Vega D, Bastide J (1998) Chemical and biological transformation of the fungicide vinclozolin. J Agric Food Chem 46:3817–3822CrossRefGoogle Scholar
  38. Myers JP, Zoeller RT, vom Saal FS (2009) A clash of old and new scientific concepts in toxicity, with important implications for public health. Environ Health Perspect 17:1652–1655Google Scholar
  39. NIEHS (2001) Endocrine disruptors low-dose peer review. In: National Toxicology Program’s Report. U.S. Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 487 ppGoogle Scholar
  40. Nilsson EE, Anway MD, Stanfield J, Skinner MK (2008) Transgenerational epigenetic effects of the endocrine disruptor vinclozolin on pregnancies and female adult onset disease. Reproduction 135:713–721CrossRefGoogle Scholar
  41. OECD (1998) Guideline for testing of chemicals No 211. Daphnia magna reproduction test, adopted September 1998. Organisation for Economic Cooperation and Development, Paris, 21 ppGoogle Scholar
  42. OECD (2006) OECD Environment Health and Safety Publications—series on testing and assessment N° 54. Current approaches in the statistical analysis of ecotoxicity data: a guidance to application. Organisation for Economic Cooperation and Development, Paris, 214 ppGoogle Scholar
  43. Oehlmann J, Di Benedetto P, Tillmann M, Duft M, Oetken M, Schulte-Oehlmann U (2007) Endocrine disruption in prosobranch molluscs: evidence and ecological relevance. Ecotoxicology 16:29–43CrossRefGoogle Scholar
  44. R Development Core Team (2007) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, AustriaGoogle Scholar
  45. Sanderson JT, Boerma J, Lansbergen GWA, van den Berg M (2002) Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol Appl Pharmacol 182:44–54CrossRefGoogle Scholar
  46. Segner H, Caroll K, Fenske M et al (2003) Identification of endocrine-disrupting effects in aquatic vertebrates and invertebrates: report from the European IDEA project. Ecotoxicol Environ Saf 54:302–314CrossRefGoogle Scholar
  47. Sharpe RM, Irvine DS (2004) How strong is the evidence of a link between environmental chemicals and adverse effects on human reproductive health? Br Med J 328:447–451CrossRefGoogle Scholar
  48. Sternberg RM, Gooding MP, Hotchkiss AK, LeBlanc GA (2010) Environmental-endocrine control of reproductive maturation in gastropods: implications for the mechanism of tributyltin-induced imposex in prosobranchs. Ecotoxicology 19:4–23CrossRefGoogle Scholar
  49. Tillmann M, Schulte-Oehlmann U, Duft M, Markert B, Oehlmann J (2001) Effects of endocrine disruptors on prosobranch snails (mollusca: Gastropoda) in the laboratory—part III: cyproterone acetate and vinclozolin as antiandrogens. Ecotoxicology 10:373–388CrossRefGoogle Scholar
  50. Toppari J, Larsen JC, Christiansen P et al (1996) Male reproductive health and environmental xenoestrogens. Environ Health Perspect 104:741–803CrossRefGoogle Scholar
  51. US-EPA (2000) Registration eligibility decision facts—vinclozolin, Report N°EPA-738F00021. United States Environmental Protection Agency, Washington DC, 114 ppGoogle Scholar
  52. Vom Saal FS (1995) Environmental estrogenic chemicals: their impact on embryonic development. Hum Ecol Risk Assess 1:3–15Google Scholar
  53. Vreugdenhil E, Geraerts WPM, Jackson JF, Joosse J (1985) The molecular basis of the neuro-endocrine control of egg-laying behaviour in Lymnaea. Peptides 6(Suppl 3):465–470Google Scholar
  54. Weltje L, Scholz C, Stark C, Ziebart S, van Doornmalen J, Oehlmann J (2003). Endocrine disruption in the freshwater pond snail Lymnaea stagnalis L. In: Abstracts of the SETAC Europe 13th Annual Meeting. Hamburg, Germany, 27 April–1 May 2005Google Scholar
  55. Weltje L, vom Saal FS, Oehlmann J (2005) Reproductive stimulation by low doses of xenoestrogens contrasts with the view of hormesis as an adaptive response. Hum Exp Toxicol 24:431–437CrossRefGoogle Scholar
  56. Wong C, Kelce WR, Sar M, Wilson EM (1995) Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J Biol Chem 34:19998–20003Google Scholar
  57. Zonneveld (1992) Animal energy budgets: a dynamic approach. Dissertation, Vrije Universiteit of AmsterdamGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Virginie Ducrot
    • 1
  • Mickaël Teixeira-Alves
    • 2
    • 3
  • Christelle Lopes
    • 4
  • Marie-Laure Delignette-Muller
    • 2
    • 5
  • Sandrine Charles
    • 2
    • 3
  • Laurent Lagadic
    • 1
  1. 1.INRA, Équipe Écotoxicologie et Qualité des Milieux Aquatiques, UMR985 Ecologie et Santé des EcosystèmesRennesFrance
  2. 2.Université de LyonLyonFrance
  3. 3.Laboratoire de Biométrie et Biologie EvolutiveUniversité Lyon 1, CNRS, UMR5558VilleurbanneFrance
  4. 4.Cemagref, UR MALYLyonFrance
  5. 5.VetAgro Sup Campus Vétérinaire de LyonMarcy l’EtoileFrance

Personalised recommendations