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Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters


Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29 days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.

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  1. Ahrens L, Felizeter S, Sturm R, Xie Z, Ebinghaus R (2009) Polyfluorinated compounds in waste water treatment plant effluents and surface waters along the River Elbe, Germany. Mar Pollut Bull 58:1326–1333

  2. Akaishi FM, St-Jean SD, Bishay F, Clarke J, da Rabitto IS, de Oliveira Ribeiro CA (2007) Immunological responses, histopathological finding and disease resistance of blue mussel (Mytilus edulis) exposed to treated and untreated municipal wastewater. Aquat Toxicol 82:1–14

  3. Allam B, Paillard C, Auffret M (2000) Alterations in hemolymph and extrapallial fluid parameters in the Manila clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tapetis. J Invertebr Pathol 76:63–69

  4. Aminot Y, Le Menach K, Pardon P, Etcheber H, Budzinski H (2016) Inputs and seasonal removal of pharmaceuticals in the estuarine Garonne River. Mar Chem 185:3–11

  5. Auffret M, Mujdzic N, Corporeau C, Moraga D (2002) Xenobiotic-induced immunomodulation in the European flat oyster, Ostrea edulis. Mar Environ Res 54:585–589

  6. Auffret M, Duchemin M, Rousseau S, Boutet I, Tanguy A, Moraga D, Marhic A (2004) Monitoring of immunotoxic responses in oysters reared in areas contaminated by the “Erika” oil spill. Aquat Living Resour 17:297–302

  7. Auffret M, Rousseau S, Boutet I, Tanguy A, Baron J, Moraga D, Duchemin M (2006) A multiparametric approach for monitoring immunotoxic responses in mussels from contaminated sites in Western Mediterranea. Ecotoxicol Environ Saf 63:393–405

  8. Bates, D., M. Mächler, B. Bolker, and S. Walker. 2014. Fitting linear mixed-effects models using lme4. arXiv preprint arXiv:1406.5823

  9. Besse J-P, Garric J (2008) Human pharmaceuticals in surface waters: implementation of a prioritization methodology and application to the French situation. Toxicol Lett 176:104–123

  10. Besse J-P, Latour J-F, Garric J (2012) Anticancer drugs in surface waters: what can we say about the occurrence and environmental significance of cytotoxic, cytostatic and endocrine therapy drugs? Environ Int 39:73–86

  11. Bianchi VA, Castro JM, Rocchetta I, Nahabedian DE, Conforti V, Luquet CM (2015) Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli. Fish Shellfish Immunol 42:367–378

  12. Boisseaux, P., M. Gust, S. Betoulle, and J. Garric. 2014. Short-term immunotoxic effects of an anti-cancer drug (Etoposide) on the freshwater pondsnail Lymnaea stagnalis. Journal of Xenobiotics 4

  13. Boisseaux P, Delignette-Muller M-L, Abbaci K, Thomas H, Garric J (2016a) Analysis of hemocytes in Lymnaea stagnalis: characterization and effects of repeated hemolymph collections. Fish Shellfish Immunol 57:116–126

  14. Boisseaux, P., P. Noury, M. L. Delignette-Muller, H. Thomas, and J. Garric. 2016b. Recommendations for the analysis of hemocyte-related immunocompetent oxidative activity in the freshwater snail Lymnaea stagnalis. Journal of Xenobiotics 6

  15. Brion F, Tyler CR, Palazzi X, Laillet B, Porcher J-M, Garric J, Flammarion P (2004) Impacts of 17β-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile-and adult-life stages in zebrafish (Danio rerio). Aquat Toxicol 68:193–217

  16. Canesi L, Ciacci C, Betti M, Fabbri R, Canonico B, Fantinati A, Marcomini A, Pojana G (2008) Immunotoxicity of carbon black nanoparticles to blue mussel hemocytes. Environ Int 34:1114–1119

  17. Canesi L, Ciacci C, Fabbri R, Marcomini A, Pojana G, Gallo G (2012) Bivalve molluscs as a unique target group for nanoparticle toxicity. Mar Environ Res 76:16–21

  18. Cédat B, de Brauer C, Métivier H, Dumont N, Tutundjan R (2016) Are UV photolysis and UV/H 2 O 2 process efficient to treat estrogens in waters? Chemical and biological assessment at pilot scale. Water Res 100:357–366

  19. Coles JA, Farley SR, Pipe RK (1994) Effects of fluoranthene on the immunocompetence of the common marine mussel, Mytilus edulis. Aquat Toxicol 30:367–379

  20. Dagnino S, Gomez E, Picot B, Cavaillès V, Casellas C, Balaguer P, Fenet H (2010) Estrogenic and AhR activities in dissolved phase and suspended solids from wastewater treatment plants. Sci Total Environ 408:2608–2615

  21. Ducrot V, Askem C, Azam D, Brettschneider D, Brown R, Charles S, Coke M, Collinet M, Delignette-Muller M-L, Forfait-Dubuc C (2014) Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda). Regul Toxicol Pharmacol 70:605–614

  22. Farcy E, Gagné F, Martel L, Fortier M, Trépanier S, Brousseau P, Fournier M (2011) Short-term physiological effects of a xenobiotic mixture on the freshwater mussel Elliptio complanata exposed to municipal effluents. Environ Res 111:1096–1106

  23. Fent K, Kunz PY, Gomez E (2008) UV filters in the aquatic environment induce hormonal effects and affect fertility and reproduction in fish. CHIMIA Int J Chem 62:368–375

  24. Ferrari B t, Paxeus N, Giudice RL, Pollio A, Garric J (2003) Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac. Ecotoxicol Environ Saf 55:359–370

  25. Ferrari B, Mons R, Vollat B, Fraysse B, Paxēaus N, Giudice RL, Pollio A, Garric J (2004) Environmental risk assessment of six human pharmaceuticals: are the current environmental risk assessment procedures sufficient for the protection of the aquatic environment? Environ Toxicol Chem 23:1344–1354

  26. Fournier M, Pellerin J, Clermont Y, Morin Y, Brousseau P (2001) Effects of in vivo exposure of Mya arenaria to organic and inorganic mercury on phagocytic activity of hemocytes. Toxicology 161:201–211

  27. Gagnaire B, Gay M, Huvet A, Daniel J-Y, Saulnier D, Renault T (2007) Combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg). Aquat Toxicol 84:92–102

  28. Gagné F, Blaise C, Aoyama I, Luo R, Gagnon C, Couillard Y, Campbell P, Salazar M (2002) Biomarker study of a municipal effluent dispersion plume in two species of freshwater mussels. Environ Toxicol 17:149–159

  29. Gagné F, André C, Cejka P, Hausler R, Fournier M, Blaise C (2008) Immunotoxic effects on freshwater mussels of a primary-treated wastewater before and after ozonation: a pilot plant study. Ecotoxicol Environ Saf 69:366–373

  30. Gagnon C, Turcotte P, Trépanier S, Gagné F, Cejka PJ (2014) Impacts of municipal wastewater oxidative treatments: changes in metal physical speciation and bioavailability. Chemosphere 97:86–91

  31. Galloway TS, Depledge MH (2001) Immunotoxicity in invertebrates: measurement and ecotoxicological relevance. Ecotoxicology 10:5–23

  32. Gerecke AC, Schärer M, Singer HP, Müller SR, Schwarzenbach RP, Sägesser M, Ochsenbein U, Popow G (2002) Sources of pesticides in surface waters in Switzerland: pesticide load through waste water treatment plants––current situation and reduction potential. Chemosphere 48:307–315

  33. Gust M, Buronfosse T, Geffard O, Mons R, Queau H, Mouthon J, Garric J (2010) In situ biomonitoring of freshwater quality using the New Zealand mudsnail Potamopyrgus antipodarum (Gray) exposed to waste water treatment plant (WWTP) effluent discharges. Water Res 44:4517–4528

  34. Gust M, Fortier M, Garric J, Fournier M, Gagné F (2013a) Effects of short-term exposure to environmentally relevant concentrations of different pharmaceutical mixtures on the immune response of the pond snail Lymnaea stagnalis. Sci Total Environ 445:210–218

  35. Gust M, Fortier M, Garric J, Fournier M, Gagné F (2013b) Immunotoxicity of surface waters contaminated by municipal effluents to the snail Lymnaea stagnalis. Aquat Toxicol 126:393–403

  36. Gust M, Gagné F, Berlioz-Barbier A, Besse JP, Buronfosse T, Tournier M, Tutundjian R, Garric J, Cren-Olivé C (2014) Caged mudsnail Potamopyrgus antipodarum (Gray) as an integrated field biomonitoring tool: exposure assessment and reprotoxic effects of water column contamination. Water Res 54:222–236

  37. Knaap WPW, Meuleman EA, Sminia T (1987) Alterations in the internal defence system of the pond snail Lymnaea stagnalis induced by infection with the schistosome Trichobilharzia ocellata. Parasitol Res 73:57–65

  38. Köck-Schulmeyer M, Villagrasa M, de Alda ML, Céspedes-Sánchez R, Ventura F, Barceló D (2013) Occurrence and behavior of pesticides in wastewater treatment plants and their environmental impact. Sci Total Environ 458:466–476

  39. Lacaze E, Devaux A, Bony S, Bruneau A, André C, Pelletier M, Gagné F (2013) Genotoxic impact of a municipal effluent dispersion plume in the freshwater mussel Elliptio complanata: an in situ study. J Xenobiotics 3:6

  40. Lammel T, Boisseaux P, Fernández-Cruz M-L, Navas JM (2013) Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2. Part Fibre Toxicol 10:27

  41. Lammel T, Boisseaux P, Navas JM (2015) Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1. Environ Toxicol 30:1192–1204

  42. Le Guernic A, Sanchez W, Palluel O, Bado-Nilles A, Floriani M, Turies C, Chadili E, Della Vedova C, Cavalié I, Adam-Guillermin C (2016) Acclimation capacity of the three-spined stickleback (Gasterosteus aculeatus, L.) to a sudden biological stress following a polymetallic exposure. Ecotoxicology 25:1478–1499

  43. Leclercq M, Mathieu O, Gomez E, Casellas C, Fenet H, Hillaire-Buys D (2009) Presence and fate of carbamazepine, oxcarbazepine, and seven of their metabolites at wastewater treatment plants. Arch Environ Contam Toxicol 56:408–415

  44. Mailler R, Gasperi J, Coquet Y, Deshayes S, Zedek S, Cren-Olivé C, Cartiser N, Eudes V, Bressy A, Caupos E (2015) Study of a large scale powdered activated carbon pilot: removals of a wide range of emerging and priority micropollutants from wastewater treatment plant effluents. Water Res 72:315–330

  45. Miege C, Choubert JM, Ribeiro L, Eusèbe M, Coquery M (2009a) Fate of pharmaceuticals and personal care products in wastewater treatment plants–conception of a database and first results. Environ Pollut 157:1721–1726

  46. Miege C, Gabet V, Coquery M, Karolak S, Jugan ML, Oziol L, Levi Y, Chevreuil M (2009b) Evaluation of estrogenic disrupting potency in aquatic environments and urban wastewaters by combining chemical and biological analysis. TrAC Trends Anal Chem 28:186–195

  47. Mitta G, Vandenbulcke F, Hubert F, Roch P (1999) Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge. J Cell Sci 112:4233–4242

  48. Müller C, Ruby S, Brousseau P, Cyr D, Fournier M, Gagné F (2009) Immunotoxicological effects of an activated-sludge-treated effluent on rainbow trout (Oncorhynchus mykiss). Comp Biochem Physiol Part C: Toxicol Pharmacol 150:390–394

  49. Oubella R, Maes P, Paillard C, Auffret M (1993) Experimentally induced variation in hemocyte density for Ruditapes philippinarum and R. decussatus (Mollusca, Bivalvia). Dis Aquat Org 15:193–197

  50. Ozaki N, Takamura Y, Kojima K, Kindaichi T (2015) Loading and removal of PAHs in a wastewater treatment plant in a separated sewer system. Water Res 80:337–345

  51. Renault T (2015) Immunotoxicological effects of environmental contaminants on marine bivalves. Fish Shellfish Immunol 46:88–93

  52. Russo J, Lagadic L (2004) Effects of environmental concentrations of atrazine on hemocyte density and phagocytic activity in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata). Environ Pollut 127:303–311

  53. Russo J, Madec L, Brehélin M (2009) Haemocyte lysosomal fragility facing an environmental reality: a toxicological perspective with atrazine and Lymnaea stagnalis (Gastropoda, Pulmonata) as a test case. Ecotoxicol Environ Saf 72:1719–1726

  54. Salo HM, Hébert N, Dautremepuits C, Cejka P, Cyr DG, Fournier M (2007) Effects of Montreal municipal sewage effluents on immune responses of juvenile female rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 84:406–414

  55. Schröder HF (1993) Surfactants: non-biodegradable, significant pollutants in sewage treatment plant effluents: separation, identification and quantification by liquid chromatography, flow-injection analysis—mass spectrometry and tandem mass spectrometry. J Chromatogr A 647:219–234

  56. Segner H, Wenger M, Möller AM, Köllner B, Casanova-Nakayama A (2012) Immunotoxic effects of environmental toxicants in fish—how to assess them? Environ Sci Pollut Res 19:2465–2476

  57. Seppälä O, Leicht K (2013) Activation of the immune defence of the freshwater snail Lymnaea stagnalis by different immune elicitors. J Exp Biol 216:2902–2907

  58. Team, R. C. 2016. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2015, URL h ttp. www. R-project. org

  59. Therneau, T. M. 2015. A package for survival analysis in S 2015. Version 2.38

  60. Urbaniak M, Kiedrzyńska E (2015) Concentrations and toxic equivalency of polychlorinated biphenyls in Polish wastewater treatment plant effluents. Bull Environ Contam Toxicol 95:530–535

  61. Vijayavel K, Gopalakrishnan S, Thiagarajan R, Thilagam H (2009) Immunotoxic effects of nickel in the mud crab Scylla serrata. Fish Shellfish Immunol 26:133–139

  62. Wigh A, Devaux A, Brosselin V, Gonzalez-Ospina A, Domenjoud B, Aït-Aïssa S, Creusot N, Gosset A, Bazin C, Bony S (2016) Proposal to optimize ecotoxicological evaluation of wastewater treated by conventional biological and ozonation processes. Environ Sci Pollut Res 23:3008–3017

  63. Yu J, Hu J, Tanaka S, Fujii S (2009) Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in sewage treatment plants. Water Res 43:2399–2408

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The Rhône-Alpes region provided financial support to Paul Boisseaux in a PhD fellowship program (ARC3 Environnement).

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Correspondence to Jeanne Garric.

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Boisseaux, P., Noury, P., Delorme, N. et al. Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters. Environ Sci Pollut Res 25, 16720–16728 (2018). https://doi.org/10.1007/s11356-018-1790-z

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  • Lymnaea stagnalis
  • Wastewater
  • Immunocompetence
  • Ecotoxicology