Rapid assessment of the toxicity of oil sands process-affected waters using fish cell lines

  • Bryan Sansom
  • Nguyen T. K. Vo
  • Richard Kavanagh
  • Robert Hanner
  • Michael MacKinnon
  • D. George Dixon
  • Lucy E. J. Lee


Rapid and reliable toxicity assessment of oil sands process-affected waters (OSPW) is needed to support oil sands reclamation projects. Conventional toxicity tests using whole animals are relatively slow, costly, and often subjective, while at the same time requiring the sacrifice of test organisms as is the case with lethal dosage/concentration assays. A nonlethal alternative, using fish cell lines, has been developed for its potential use in supporting oil sands reclamation planning and to help predict the viability of aquatic reclamation models such as end-pit lakes. This study employed six fish cell lines (WF-2, GFSk-S1, RTL-W1, RTgill-W1, FHML, FHMT) in 24 h viability assays for rapid fluorometric assessment of cellular integrity and functionality. Forty-nine test water samples collected from the surface of oil sands developments in the Athabasca Oil Sands deposit, north of Fort McMurray, Alberta, Canada, were evaluated in blind. Small subsample volumes (8 ml) were mixed with 2 ml of 5× concentrated exposure media and used for direct cell exposures. All cell line responses in terms of viability as measured by Alamar blue assay, correlated well with the naphthenic acids (NA) content in the samples (R2 between 0.4519 and 0.6171; p < 0.0001) when data comparisons were performed after the bioassays. NA or total acid-extractable organics group has been shown to be responsible for most of the acute toxicity of OSPW and our results further corroborate this. The multifish cell line bioassay provides a strong degree of reproducibility among tested cell lines and good relative sensitivity of the cell line bioassay as compared to available in vivo data that could lead to cost effective, high-throughput screening assays.


Fish cell lines Toxicity Naphthenic acids Alamar blue Oil sands 


  1. Allen E. W. Process water treatment in Canada’s oil sands industry: I. Target pollutants and treatment objectives. J. Environ. Eng. Sci. 7: 123–138; 2008.CrossRefGoogle Scholar
  2. Bols N. C.; Barlian A.; Chirino-Trejo M.; Caldwell S. J.; Goegan P.; Lee L. E. J. Development of a cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills. J. Fish Dis. 17: 601–611; 1994.CrossRefGoogle Scholar
  3. Bols N. C.; Dayeh V. R.; Lee L. E. J.; Schirmer K. Use of fish cells in the toxicology and ecotoxicology of fish. Biochemistry and molecular biology of fishes, vol. 6. Elsevier Science, Amsterdam, pp 43–84; 2005.Google Scholar
  4. Bols N. C.; Lee L. E. J. Cell lines: availability, propagation and isolation. In: Hochachka P. W.; Mommsen T. P. (eds) Biochemistry and molecular biology of fishes, vol. 3. Elsevier Science, Amsterdam, pp 145–149; 1994.Google Scholar
  5. Castaño A.; Bols N. C.; Braunbeck T.; Dierickx P.; Halder M.; Isomaa B.; Kawahara K.; Lee L. E. J.; Mothersill C.; Part P.; Repetto G.; Sintes J. R.; Rufli H.; Smith R.; Wood C.; Segner H. The use of fish cells in ecotoxicology. The report and recommendations of ECVAM workshop 47. ATLA Altern. Lab. Anim. 31: 317–351; 2003.Google Scholar
  6. Clemente J. S.; Fedorak P. M. A review of the occurrence, analyses, toxicity, and biodegradation of naphthenic acids. Chemosphere 60: 585–600; 2005.PubMedCrossRefGoogle Scholar
  7. Dayeh V. R.; Grominsky S.; DeWitte-Orr S. J.; Sotornik D.; Yeung C. R.; Lee L. E. J.; Lynn D. H.; Bols N. C. Comparing a ciliate and a fish cell line for their sensitivity to several classes of toxicants by the novel application of multiwall filter plates to Tetrahymena. Res. Microbiol. 156: 93–103; 2005a.PubMedCrossRefGoogle Scholar
  8. Dayeh V. R.; Schirmer K.; Bols N. C. Applying whole-water samples directly to fish cell cultures in order to evaluate the toxicity of industrial effluent. Water Res. 36: 3727–3738; 2002.PubMedCrossRefGoogle Scholar
  9. Dayeh V. R.; Schirmer K.; Lee L. E. J.; Bols N. C. Rainbow trout gill cell line microplate cytotoxicity test. In: Blaise C.; Ferard J. F. (eds) Toxicity test methods: small-scale freshwater toxicity investigations, vol. 1. Springer, Dordrecht, pp 473–503; 2005b.CrossRefGoogle Scholar
  10. Dokholyan B. K.; Magomedov A. K. Effect of sodium naphthenate on survival and some physiological-biochemical parameters of some fishes. J. Ichthyol. 23: 125–132; 1983.Google Scholar
  11. Dorn P. B. Case histories—the petroleum industry. In: Ford D. L. (ed) Toxicity reduction: valuation and control. Water quality management library, vol. 3. Technomic, Lancaster, pp 183–223; 1992.Google Scholar
  12. Dyer S.; Moorhouse J.; Laufenberg K.; Powell R. Undermining the environment: the oil sands report card. The Pembina Institute/WWF-Canada January 2008. 72pp http://www.strategywest.com/downloads/Undermining200801.pdf.
  13. Energy Resources Conservation Board. ST98-2012: Alberta’s energy reserves 2011 and supply/demand outlook 2012–2021. http://www.ercb.ca/sts/ST98/ST98-2012.pdf.
  14. Frank R. A.; Kavanagh R.; Burnison B. K.; Arsenault G.; Headley J. V.; Peru K. M.; Van der Kraak G. V.; Solomon K. R. Toxicity assessment of collected fractions from an extracted naphthenic acid mixture. Chemosphere 72: 1309–1314; 2008.PubMedCrossRefGoogle Scholar
  15. Frank R. A.; Kavanagh R.; Burnison B. K.; Headley J. V.; Peru K. M.; Van Der Kraak G. V.; Solomon K. R. Diethylaminoethyl-cellulose clean-up of a large volume naphthenic acid extract. Chemosphere 64: 1346–1352; 2006.PubMedCrossRefGoogle Scholar
  16. Gagne F.; Douville M.; Andre C.; Debenest T.; Talbot A.; Sherry J.; Hewitt L. M.; Frank R. A.; McMaster M. E.; Parrott J.; Bickerton G. Differential changes in gene expression in rainbow trout hepatocytes exposed to extracts of oil sands process-affected water and the Athabasca River. Comp. Biochem. Physiol. C 155: 551–559; 2012.Google Scholar
  17. Garcia-Garcia E.; Pun J.; Perez-Estrada L. A.; Din M. G.; Smith D. W.; Martin J. W.; Belosevic M. Commercial naphthenic acids and the organic fraction of oil sands process water downregulate proinflammatory gene expression and macrophage antimicrobial responses. Toxicol. Lett. 203: 62–73; 2011.PubMedCrossRefGoogle Scholar
  18. Glawdel T.; Elbuken C.; Lee L. E. J.; Ren C. Microfluidic system with integrated electroosmotic pumps, concentration gradient generator and fish cell line (RTgill-W1)—towards water toxicity testing. Lab Chip 9: 3243–3250; 2009.PubMedCrossRefGoogle Scholar
  19. Goodfellow W. L.; Ausley L. W.; Burton D. T.; Denton D. L.; Dorn P. B.; Grothe D. R.; Heber M. A.; Norberg-King T. J.; Rogers Jr. J. H. Major ion toxicity in effluents: a review with permitting recommendations. Environ. Toxicol. Chem. 19: 175–182; 2000.CrossRefGoogle Scholar
  20. Grewer D.; Rozlyn F.; Young R.; Whittal R.; Fedorak P. Naphthenic acids and other acid extractables in water samples from Alberta: what is being measured? Sci. Total. Environ. 408: 5997–6010; 2010.PubMedCrossRefGoogle Scholar
  21. Han X.; MacKinnon M. D.; Martin J. W. Estimating the in situ biodegradation of naphthenic acids in oil sands process waters by HPLC/HRMS. Chemosphere 76: 63–70; 2009.PubMedCrossRefGoogle Scholar
  22. He Y.; Wiseman S. B.; Zhang X.; Hecker M.; Jones P. D.; El-Din M. G.; Martin J. W.; Giesy J. P. Ozonation attenuates the steroidogenic disruptive effects of sediment free oil sands process water in the H295R cell line. Chemosphere 80: 578–584; 2010.PubMedCrossRefGoogle Scholar
  23. Headley J. V.; McMartin D. A review of the occurrence and fate of naphthenic acids in aquatic environments. Environ. Sci. Health A 39: 1989–2010; 2004.Google Scholar
  24. Holowenko F. M.; MacKinnon M. D.; Fedorak P. M. Characterization of naphthenic acids in oil sands wastewaters by gas chromatography–mass spectrometry. Water Res. 36: 2843–2855; 2002.PubMedCrossRefGoogle Scholar
  25. Holroyd P.; Simieritsch T. In: Fauth L. (ed) The water that binds us. The Pembina Institute, Drayton Valley; 2009. 49pp.Google Scholar
  26. Hrovat M.; Segner H.; Jeram S. Variability of in vivo fish acute toxicity data. Regul. Toxicol. Pharmacol. 54: 294–300; 2009.PubMedCrossRefGoogle Scholar
  27. Ivanova N. V.; Zemlak T. S.; Hanner R. H.; Hebert P. D. N. Universal primer cocktails for fish DNA barcoding. Mol. Ecol. Notes 7: 544–548; 2007.CrossRefGoogle Scholar
  28. Jivraj M.; MacKinnon M. D.; Fung B. Naphthenic acid extraction and quantitative analysis with FT-IR Spectroscopy. In: Syncrude analytical manuals, 4th ed. Syncrude Canada Ltd., Edmonton AB, 12pp; 1995.Google Scholar
  29. Johnson E. A.; Miyanishi K. Creating new landscapes and ecosystems—the Alberta oil sands. Ann. NY Acad. Sci. 1134: 120–145; 2008.PubMedCrossRefGoogle Scholar
  30. Kannel P. R.; Gan T. Y. Naphthenic acids degradation and toxicity mitigation in tailings wastewater systems and aquatic environmentts: a review. J. Environ. Sci. Health A 47: 1–21; 2012.Google Scholar
  31. Kavanagh R. J.; Frank R. A.; Burnison B. K.; Young R. F.; Fedorak P. M.; MacKinnon M. D.; Solomon K. R.; Van Der Kraak G. V. Fathead minnow (Pimephales promelas) reproduction is impaired when exposed to a naphthenic acid extract. Aquat. Toxicol. 116–117: 34–42; 2012.PubMedCrossRefGoogle Scholar
  32. Kavanagh R. J.; Frank R. A.; Oakes K. D.; Servos M. R.; Young R. F.; Fedorak P. M.; MacKinnon M. D.; Solomon K. R.; Dixon D. G.; Van Der Kraak G. V. Fathead minnow (Pimephales promelas) reproduction is impaired in aged oil sands process-affected waters. Aquat. Toxicol. 101: 214–220; 2011.PubMedCrossRefGoogle Scholar
  33. Kelly E.; Schindler D.; Hodson P.; Short J.; Radmanovich R.; Nielsen C. Oil sands development contributes elements toxic at low concentrations to the Athabasca River and its tributaries. Proc. Natl. Acad. Sci. 107: 16178–16183; 2010.PubMedCrossRefGoogle Scholar
  34. Lee L. E. J.; Bufalino M. R.; Christie A. E.; Frischer M. E.; Soin T.; Tsui C. K. M.; Hanner R. H.; Smagghe G. Misidentification of OLGA-PH-J/92, believed to be the only crustacean cell line. In Vitro Cell. Dev. Biol. – Anim. 47: 665–674; 2011.PubMedCrossRefGoogle Scholar
  35. Lee L. E. J.; Caldwell S. J.; Gibbons J. Development of a cell line from skin of goldfish, Carassius auratus, and effects of ascorbic acid on collagen deposition. Histochem. J. 29: 31–43; 1997.PubMedCrossRefGoogle Scholar
  36. Lee L. E. J.; Clemons J. H.; Bechtel D. G.; Caldwell S. J.; Han K. B.; Pasitschniak-Arts M.; Mosser D. D.; Bols N. C. Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity. Cell Biol. Toxicol. 9: 279–294; 1993.PubMedCrossRefGoogle Scholar
  37. Lee L. E. J.; Dayeh V.; Schirmer K.; Bols N. C. Fish cell lines as rapid and inexpensive screening tools for whole effluent testing. Integr. Environ. Assess. Manag. 4: 372–374; 2008.PubMedCrossRefGoogle Scholar
  38. Lee L. E. J.; Vo N.; Werner J.; Weil R.; Denslow N. D.; Law R. D. Development of a liver cell line from fathead minnow, Pimephales promelas, and their molecular and biochemical characterisation. In Vitro Cell Dev. Biol.—Anim. 45(Suppl 1): A-2020; 2009.Google Scholar
  39. Lemphers N.; Dyer S.; Grant J. In: Lines R. (ed) Toxic liability: how Albertans could end up paying for oil sands mine reclamation. Report 2075. The Pembina Institute, Drayton Valley; 2010. 58pp.Google Scholar
  40. Leung S. S.; MacKinnon M. D.; Smith R. E. H. Aquatic reclamation in the Athabasca, Canada, oil sands: naphthenate and salt effects on phytoplankton communities. Environ. Toxicol. Chem. 20: 1532–1543; 2001.PubMedCrossRefGoogle Scholar
  41. Leung S. S.; MacKinnon M. D.; Smith R. E. H. The ecological effects of naphthenic acids and salts on phytoplankton from the Athabasca oil sands region. Aquat. Toxicol. 62: 11–26; 2003.PubMedCrossRefGoogle Scholar
  42. MacKinnon M. D.; Boerger H. Description of two treatment methods for detoxifying oil sands tailings pond water. Water. Pollut. Res. J. Can. 21: 496–512; 1986.Google Scholar
  43. Magwood S.; George S. In vitro alternatives to whole animal testing. Comparative cytotoxicity studies of divalent metals in established cell lines derived from tropical and temperate water fish species in a neutral red assay. Mar. Environ. Res. 42: 37–40; 1996.CrossRefGoogle Scholar
  44. Mikula R. J.; Kasperski K. L.; Burns R.; MacKinnon M. D. The nature and fate of oil sands fine tailings. In: Schramm L. L. (ed) Suspensions: fundamentals and applications in the petroleum industry, vol. 251. ACS, Washington, D.C., pp 677–723; 1996.CrossRefGoogle Scholar
  45. Mount D. R.; Gulley J. M.; Hockett J. R.; Garrison T. D.; Evans J. M. Statistical models to predict the toxicity of major ions to Ceriodaphnia dubia, Daphnia magna, and fathead minnows (Pimephales promelas). Environ. Toxicol. Chem. 16: 2009–2019; 1997.Google Scholar
  46. Nero V.; Farwell A.; Lee L. E. J.; Van Meer T.; MacKinnon M. D.; Dixon D. G. The effects of salinity on naphthenic acid toxicity to yellow perch: gill and liver histopathology. Ecotoxicol. Environ. Saf. 65: 252–264; 2006.PubMedCrossRefGoogle Scholar
  47. O’Connor S.; McNamara L.; Swerdin M.; Van Buskirk R. G. Multifluorescent assays reveal mechanisms underlying cytotoxicity—phase I. CFTA compounds. In Vitro Toxicol. 4: 197–206; 1991.Google Scholar
  48. OECD. Organization for Economic Cooperation and Development Guideline for Testing of Chemicals. Test No. 203: Fish, Acute Toxicity Test, pp 1–3; 1992.Google Scholar
  49. OECD. OECD Test Guideline 401 was deleted in 2002: A major step in animal welfare: OECD reached agreement on the abolishment of the LD50 acute toxicity test. http://www.oecd.org/env/chemicalsafetyandbiosafety/testingofchemicals/oecdtestguideline401wasdeletedin2002amajorstepinanimalwelfareoecdreachedagreementontheabolishmentoftheld50acutetoxicitytest.htm; 2002.
  50. OECD. Fish testing framework. Series on testing and assessment. No 171. http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=ENV/JM/MONO(2012)16&doclanguage=en; 2012.
  51. Peters L. E.; MacKinnon M. D.; Van Meer T.; van den Heuvel M. R.; Dixon D. G. Effects of oil sands process-affected waters and naphthenic acids on yellow perch (Perca flavescens) and Japanese medaka (Orizias latipes) embryonic development. Chemosphere 67: 2177–2183; 2007.PubMedCrossRefGoogle Scholar
  52. Rogers V.; Wickstrom M.; Liber K.; MacKinnon M. Acute and subchronic mammalian toxicity of naphthenic acids from oil sands tailings. Toxicol. Sci. 66: 347–355; 2002.PubMedCrossRefGoogle Scholar
  53. Saito H.; Koyasu T.; Shigeoka T.; Tomita I. Cytotoxicity of chlorophenols to goldfish GFS cells with the MTT and the LDH assays. Toxicol. In Vitro 8: 1107–1112; 1994.PubMedCrossRefGoogle Scholar
  54. Schirmer K. Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicology 224: 163–183; 2006.PubMedCrossRefGoogle Scholar
  55. Schirmer K.; Chan A. G. J.; Greenberg B. M.; Dixon D. G.; Bols N. C. Methodology for demonstrating and measuring the photocytotoxicity of fluoranthene to fish cells in culture. Toxicol. In Vitro 11: 107–119; 1997.PubMedCrossRefGoogle Scholar
  56. Schirmer K.; Ganassin R. C.; Brubacher J. L.; Bols N. C. A DNA fluorometric assay for measuring fish cell proliferation in microplates with different well sizes. J. Tissue Cult. Methods 16: 133–142; 1994.CrossRefGoogle Scholar
  57. Schirmer K.; Tanneberger K.; Kramer N. I.; Volker D.; Scholz S.; Hafner C.; Lee L. E. J.; Bols N. C.; Hermens J. L. M. Developing a list of reference chemicals for testing alternatives to whole fish toxicity tests. Aquat. Toxicol. 90: 128–137; 2008.PubMedCrossRefGoogle Scholar
  58. Schramm L. L.; Stasiuk E. N.; MacKinnon M. D. Surfactants in Athabasca oil sands slurry conditioning, flotation recovery, and tailings processes. In: Schramm L. L. (ed) Surfactants: fundamentals and applications in the petroleum industry. Cambridge University Press, Cambridge, pp 365–430; 2000.CrossRefGoogle Scholar
  59. Scott A. C.; MacKinnon M. D.; Fedorak P. M. Naphthenic acids in Athabasca oil sands tailings waters are less biodegradable than commercial naphthenic acids. Environ. Sci. Technol. 39: 8388–8394; 2005.PubMedCrossRefGoogle Scholar
  60. Scott W. B.; Crossman E. J. Freshwater fishes of Canada. Fisheries Research Board of Canada Bulletin 184, Ottawa; 1973.Google Scholar
  61. Segner H. Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish. ATLA Altern. Lab. Anim. 32: 375–382; 2004.Google Scholar
  62. Tollefsen K. E.; Blikstad C.; Eikvar S.; Finne E. F.; Gregersen I. K. Cytotoxicity of alkylphenols and alkylated non-phenolics in a primary culture of rainbow trout (Onchorhynchus mykiss) hepatocytes. Ecotoxicol. Environ. Saf. 69: 64–73; 2008.PubMedCrossRefGoogle Scholar
  63. Tollefsen K. E.; Petersen K.; Rowland S. J. Toxicity of synthetic naphthenic acids and mixtures of these to fish liver cells. Environ. Sci. Technol. 46: 5143–5150; 2012.PubMedCrossRefGoogle Scholar
  64. US EPA. Short methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth edition. EPA-821-R-02-013. United States Environmental Protection Agency. Washington, DC. 335pp.; 2002.Google Scholar
  65. van den Heuvel M. R.; Power M.; MacKinnon M. D.; Dixon D. G. Effects of oil sands related aquatic reclamation on yellow perch (P. flavescens). II. Chemical and biochemical indicators of exposure to oil sands related waters. Can. J. Fish. Aquat. Sci. 56: 1226–1233; 1999.Google Scholar
  66. Vo N. T. K.; Sansom B.; Kozlowski E.; Bloch S.; Lee L. E. J. Development of a permanent cell line derived from fathead minnow testis and their applications in environmental toxicology. MDIBL Bull. 49: 83–86; 2010.Google Scholar
  67. Waymouth C. Osmolality of mammalian blood and of media for culture of mammalian cells. In Vitro 6: 109–127; 1970.PubMedCrossRefGoogle Scholar
  68. Wilensky C. S.; Bowser P. R. Growth characteristics of the WF-2 cell cultures. J. World Aquacult. Soc. 36: 538–541; 2005.CrossRefGoogle Scholar
  69. Winton J.; Batts W.; DeKinkelin P.; Leberre M.; Bremont M.; Fijan N. Current lineages of the epithelioma papulosum cyprini (EPC) cell line are contaminated with fathead minnow, Pimephales promelas, cells. J. Fish Dis. 33: 701–704; 2010.Google Scholar
  70. Wolf K.; Mann J. A. Poikilotherm vertebrate cell lines and viruses: a current listing for fishes. In Vitro 16: 168–179; 1980.PubMedCrossRefGoogle Scholar
  71. Wort D. J.; Patel K. M. Response of plants to naphthenic and cycloalkanecarboxylic acids. Agron. J. 62: 644–646; 1970.CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2012

Authors and Affiliations

  • Bryan Sansom
    • 1
    • 2
  • Nguyen T. K. Vo
    • 1
  • Richard Kavanagh
    • 3
  • Robert Hanner
    • 3
  • Michael MacKinnon
    • 4
  • D. George Dixon
    • 2
  • Lucy E. J. Lee
    • 1
    • 5
  1. 1.Department of BiologyWilfrid Laurier UniversityWaterlooCanada
  2. 2.Department of BiologyUniversity of WaterloWaterlooCanada
  3. 3.Department of Integrative BiologyUniversity of GuelphGuelphCanada
  4. 4.OSPM SolutionsHamiltonCanada
  5. 5.Faculty of ScienceUniversity of the Fraser ValleyAbbotsfordCanada

Personalised recommendations