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Effects of in vivo exposure to tritium: a multi-biomarker approach using the fathead minnow, Pimephales promelas

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Tritium (3H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). However, tritium can also be incorporated into organic molecules, forming organically bound tritium (OBT). The present study characterized the effects of tritium on the health of the fathead minnow, Pimephales promelas. Fish were exposed to a gradient of HTO (activity concentrations of 12,000, 25,000, and 180,000 Bq/L) and OBT using food spiked with tritiated amino acids (OBT only, with an activity concentration of 27,000 Bq/L). A combined exposure condition where fish were placed in 25,000 Bq/L water and received OBT through feed was also studied. Fish were exposed for 60 days, followed by a 60-day depuration period. A battery of health biomarkers were measured in fish tissues at seven time points throughout the 120 days required to complete the exposure and depuration phases. HTO and OBT were also measured in fish tissues at the same time points. Results showed effects of increasing tritium activity concentrations in water after 60 days of exposure. The internal dose rates of tritium, estimated from the tissue free-water tritium (TFWT) and OBT activity concentrations, reached a maximum of 0.65 μGy/h, which is relatively low considering background levels. No effects were observed on survival, fish condition, and metabolic indices (gonado-, hepato-, and spleno-somatic indexes (GSI, HSI, SSI), RNA/DNA and proteins/DNA ratios). Multivariate analyses showed that several biomarkers (DNA damages, micronucleus frequency, brain acetylcholinesterase, lysosomal membrane integrity, phagocytosis activity, and reactive oxygen species production) were exclusively correlated with fish tritium internal dose rate, showing that tritium induced genotoxicity, as well as neural and immune responses. The results were compared with another study on the same fish species where fish were exposed to tritium and other contaminants in natural environments. Together with the field study, the present work provides useful data to identify biomarkers for tritium exposure and better understand modes of action of tritium on the fathead minnow.

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  1. Adam-Guillermin C, Pereira S, Della-Vedova C, Hinton T, Garnier-Laplace J (2012) Genotoxic and reprotoxic effects of tritium and external gamma irradiation on aquatic animals. Rev Environ Contam Toxicol 220:67–103

  2. Amara R, Selleslagh J, Billon G, Minier C (2009) Growth and condition of 0-group European flounder, Platichthys flesus as indicator of estuarine habitat quality. Hydrobiologia 627:87–98

  3. ASN (2010): Livre blanc du tritium—The tritium white paper.

  4. Audette-Stuart M, Kim SB, McMullin D, Festarini A, Yankovich TL, Carr J, Mulpuru S (2011) Adaptive response in frogs chronically exposed to low doses of ionizing radiation in the environment. J Environ Radioact 102:566–573

  5. Audette-Stuart M, Yankovich T (2011) Bystander effects in bullfrog tadpoles. Radioprotection 46:S497–S502

  6. Audette-Stuart M, Ferreri C, Festarini A, Carr J (2012) Fatty acid composition of muscle tissue measured in amphibians living in radiologically contaminated and non-contaminated environments. Radiat Res 178:173–181

  7. Bado-Nilles A, Betoulle S, Geffard A, Porcher JM, Gagnaire B, Sanchez W (2013) Flow cytometry detection of lysosomal presence and lysosomal membrane integrity in the three-spined stickleback (Gasterosteus aculeatus L.) immune cells: applications in environmental aquatic immunotoxicology. Environ Sci Pollut Res 20:2692–2704

  8. Beaugelin-Seiller K (2016) Effects of soil water content on the external exposure of fauna to radioactive isotopes. J Environ Radioact 151:204–208

  9. Bogen D, Welford G, White C (1979) Tritium distribution in man and his environment. IAEA-SM-232 75:567–574

  10. CNSC (2008) Standards and guidelines for tritium in drinking water. CNSC, Ontario, p 88

  11. Environment_Canada (2011) Biological test method: test of larval growth and survival using fathead minnows

  12. Erickson RC (1971): Effects of chronic irradiation by tritiated water on Poecilia reticulata, the guppy. Radionuclides in ecosystems, vol 2. National Technical Information Service, US Department of Commerce, Springfield

  13. Etoh H, Hyodo-Taguchi Y (1983) Effects of tritiated water on germ cells in medaka embryos. Radiat Res 93:332–339

  14. European_Union (2013) COUNCIL DIRECTIVE 2013/51/EURATOM of 22 October 2013 laying down requirements for the protection of the health of the general public with regard to radioactive substances in water intended for human consumption

  15. Festarini A, Shultz C, Stuart M, Kim SB, Ferreri C (2016) Cellular responses to tritium exposure in rainbow trout: HTO- and OBT-spiked feed exposure experiments. CNL Nuclear Rev 5:155–172

  16. Fournier M, Cyr D, Blakley B, Boermans H, Brousseau P (2000) Phagocytosis as a biomarker of immunotoxicity in wildlife species exposed to environmental xenobiotics. Am Zool 40:412–420

  17. Gagnaire B, Bado-Nilles A, Sanchez W (2014) Depleted uranium disturbs immune parameters in zebrafish, Danio rerio: an ex vivo/in vivo experiment. Arch Environ Contam Toxicol 67:426–435

  18. Gagnaire B, Bado-Nilles A, Betoulle S, Amara R, Camilleri V, Cavalié I, Chadili E, Delahaut L, Kerambrun E, Orjollet D, Palluel O, Sanchez W (2015a) Former uranium mine-induced effects in caged roach: a multiparametric approach for the evaluation of in situ metal toxicity. Ecotoxicology 24:215–231

  19. Gagnaire B, Cavalié I, Pereira S, Floriani M, Dubourg N, Camilleri V, Adam-Guillermin C (2015b) External gamma irradiation-induced effects in early-life stages of zebrafish, Danio rerio. Aquat Toxicol 169:69–78

  20. Gagnaire B, Adam-Guillermin C, Festarini A, Cavalié I, Della-Vedova C, Shultz C, Kim SB, Ikert H, Dubois C, Walsh S, Farrow F, Beaton D, Tan E, Wen K, Stuart M (2017) Effects of in situ exposure to tritiated natural environments: a multi-biomarker approach using the fathead minnow, Pimephales promelas. Sci Total Environ 599-600:597–611

  21. Garnier-Laplace J, Della-Vedova C, Andersson P, Copplestone D, Cailes C, Beresford NA, Howard BJ, Howe P, Whitehouse P (2010) A multi-criteria weight of evidence approach for deriving ecological benchmarks for radioactive substances. J Radiol Prot 30:215–233

  22. Hagger JA, Atienzar FA, Jha AN (2005) Genotoxic, cytotoxic, developmental and survival effects of tritiated water in the early life stages of the marine mollusc, Mytilus edulis. Aquat Toxicol 74:205–217

  23. HPA (2007): Review on risks from tritium. Report from the independent advisory group on ionizing radiations. Documents of the Health Protection Agency radiation, chemical and environmental hazards, RCE-4

  24. Hunt J, Bailey T, Reese A (2009) The human body retention time of environmental organically bound tritium. J Radiol Prot 29:23–36

  25. Hyodo-Taguchi Y, Etoh H (1986) Effects of tritiated water on germ cells in medaka. II. Diminished reproductive capacity following embryonic exposure. Radiat Res 106:321–330

  26. Hyodo-Taguchi Y, Etoh H (1993) Vertebral malformations in medaka (teleost fish) after exposure to tritiated water in the embryonic stage. Radiat Res 135:400–404

  27. Hyodo Taguchi Y, Egami N (1977) Damage to spermatogenic cells in fish kept in tritiated water. Radiat Res 71:641–652

  28. Ichikawa R, Suyama I (1974) Effects of tritiated water on the embryonic development of two marine teleosts. Bull Jpn Soc Sci Fish 40:819–824

  29. ICRP (2008) ICRP publication 108. Environmental protection: the concept and use of reference animals and plants. Ann ICRP 37:1–242

  30. Jaeschke BC, Millward GE, Moody AJ, Jha AN (2011) Tissue-specific incorporation and genotoxicity of different forms of tritium in the marine mussel, Mytilus edulis. Environ Pollut 159:274–280

  31. Jha AN, Dogra Y, Turner A, Millward GE (2005) Impact of low doses of tritium on the marine mussel, Mytilus edulis: genotoxic effects and tissue-specific bioconcentration. Mutat Res Genet Toxicol Environ Mutagen 586:47–57

  32. Jha AN, Dogra Y, Turner A, Millward GE (2006) Are low doses of tritium genotoxic to Mytilus edulis? Mar Environ Res 62:S297–S300

  33. Kerambrun E, Henry F, Courcot L, Gevaert F, Amara R (2012) Biological responses of caged juvenile sea bass (Dicentrarchus labrax) and turbot (Scophtalmus maximus) in a polluted harbour. Ecol Indic 19:161–171

  34. Kim SB, Shultz C, Stuart M, McNamara E, Festarini A, Bureau DP (2013) Organically bound tritium (OBT) formation in rainbow trout (Oncorhynchus mykiss): HTO and OBT-spiked food exposure experiments. Appl Radiat Isot 72:114–122

  35. Kim SB, Shultz C, Stuart M, Festarini A (2015) Tritium uptake in rainbow trout (Oncorhynchus mykiss): HTO and OBT-spiked feed exposures simultaneously. Appl Radiat Isot 98:96–102

  36. Le Guernic A, Sanchez W, Bado-Nilles A, Palluel O, Turies C, Chadili E, Cavalié I, Delahaut L, Adam-Guillermin C, Porcher JM, Geffard A, Betoulle S, Gagnaire B (2016) In situ effects of metal contamination from former uranium mining sites on the health of the three-spined stickleback (Gasterosteus aculeatus, L.). Ecotoxicology 25:1234–1259

  37. Melintescu A, Galeriu D (2011) Dynamic model for tritium transfer in an aquatic food chain. Radiat Environ Biophys 50:459–473

  38. Melintescu A, Galeriu D, Kim SB (2011) Tritium dynamics in large fish—a model test. Radioprotection 46:S431–S436

  39. Mothersill C, Smith RW, Heier LS, Teien HC, Land OC, Seymour CB, Oughton D, Salbu B (2014) Radiation-induced bystander effects in the Atlantic salmon (Salmo salar L.) following mixed exposure to copper and aluminum combined with low-dose gamma radiation. Radiat Environ Biophys 53:103–114

  40. Nawar WW (1973) The effects of ionizing radiation on lipids. Progress in the Chemistry of Fats and Other Lipids 13:89–118

  41. O'Neill-Mehlenbacher A, Kilemade M, Elliott A, Mothersill C, Seymour C (2007) Comparison of direct and bystander effects induced by ionizing radiation in eight fish cell lines. Int J Radiat Biol 83:593–602

  42. OPG (2017): ONTARIO POWER GENERATION. Environmental emissions data for Darlington nuclear. OPG, pp. 7

  43. Paquet F, Harrison J (2018) ICRP task group 95: internal dose coefficients. Ann ICRP 47:63–74

  44. Parisot F, Bourdineaud JP, Plaire D, Adam-Guillermin C, Alonzo F (2015) DNA alterations and effects on growth and reproduction in Daphnia magna during chronic exposure to gamma radiation over three successive generations. Aquat Toxicol 163:27–36

  45. R_Core_Team (2017): R: A language and environment for statistical computing., Vienna, Austria

  46. Real A, Sundell-Bergman S, Knowles JF, Woodhead DS, Zinger I (2004) Effects of ionising radiation exposure on plants, fish and mammals: relevant data for environmental radiation protection. J Radiol Prot 24:A123–A137

  47. Richetti SK, Rosemberg DB, Ventura-Lima J, Monserrat JM, Bogo MR, Bonan CD (2011) Acetylcholinesterase activity and antioxidant capacity of zebrafish brain is altered by heavy metal exposure. NeuroToxicol 32:116–122

  48. R Studio_Team (2015): RStudio: Integrated Development for R. RStudio, Inc., Boston, MA URL

  49. Sanchez W, Palluel O, Meunier L, Coquery M, Porcher JM, Aït-Aïssa S (2005) Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels. Environ Toxicol Pharmacol 19:177–183

  50. Sanchez W, Porcher JM (2009) Fish biomarkers for environmental monitoring within the water framework Directive of the European Union. TrAC - Trends in Analytical Chem 28:150–158

  51. Sazykina TG, Kryshev AI (2003) EPIC database on the effects of chronic radiation in fish: Russian/FSU data. J Environ Radioact 68:65–87

  52. Smith JT, Bowes MJ, Denison FH (2006) Modelling the dispersion of radionuclides following short duration releases to rivers: part 1. Water and sediment. Sci Total Environ 368:485–501

  53. Smith RW, Moccia RD (2001) The radiation induced bystander effect: is there relevance for aquaculture? Ann Aquac Res 3:1026–1029

  54. Soldatov AA (2005) Peculiarities of organization and functioning of the fish red blood system. J Evol Biochem Physiol 41:272–281

  55. Strand JA, Fujihara MP, Poston TM, Abernethy CS (1982) Permanence of suppression of the primary immune response in rainbow trout, Salmo gairdneri, sublethally exposed to tritiated water during embryogenesis. Radiat Res 91:533–541

  56. Stuart M, Festarini A, Schleicher K, Tan E, Kim SB, Wen K, Gawlik J, Ulsh B (2016) Biological effects of tritium on fish cells in the concentration range of international drinking water standards. Int J Radiat Biol 92:563–571

  57. Suyama I, Etoh H, Maruyama T, Kato Y, Ichikawa R (1981) Effects of ionizing radiation on the early development of Oryzias eggs. J Radiat Res 22:125–133

  58. Whyte SK (2007) The innate immune response of finfish—a review of current knowledge. Fish Shellfish Immunol 23:1127–1151

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The authors are grateful for Jerry Piekarski at Lipid Analytical (University of Guelph) for performing the lipid analysis. The authors also want to thank Matt Bond, Jennifer Olfert, and Joanne Ball (CNL) for carefully reviewing the manuscript.

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Correspondence to Béatrice Gagnaire.

Additional information


– Following a previously published field investigation, this study aims to characterize tritium effects on fish health under controlled conditions in a laboratory setting.

– Fathead minnows were exposed to tritium activity concentrations up to 180,000 Bq/L.

– At the highest levels of exposure, tritium increased DNA damage and modulated the immune responses.

– Other markers were affected, including the neural system, oxidative stress, and fatty acid composition.

– No effects are reported on the measured health indices and antioxidant activities.

Responsible editor: Philippe Garrigues

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Gagnaire, B., Gosselin, I., Festarini, A. et al. Effects of in vivo exposure to tritium: a multi-biomarker approach using the fathead minnow, Pimephales promelas. Environ Sci Pollut Res 27, 3612–3623 (2020).

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  • Fathead minnow, Pimephales promelas
  • Tritium internal dose rate
  • In vivo exposure
  • Genotoxicity
  • Immune system response
  • Oxidative stress response
  • Neural response
  • Fatty acid composition