Environmental Science and Pollution Research

, Volume 21, Issue 24, pp 13720–13731 | Cite as

Developmental toxicity of PAH mixtures in fish early life stages. Part I: adverse effects in rainbow trout

  • Florane Le Bihanic
  • Bénédicte Morin
  • Xavier Cousin
  • Karyn Le Menach
  • Hélène Budzinski
  • Jérôme Cachot
PAHs and fish – Exposure monitoring and adverse effects – from molecular to individual level


A new gravel-contact assay using rainbow trout, Oncorhynchus mykiss, embryos was developed to assess the toxicity of polycyclic aromatic hydrocarbons (PAHs) and other hydrophobic compounds. Environmentally realistic exposure conditions were mimicked with a direct exposure of eyed rainbow trout embryos incubated onto chemical-spiked gravels until hatching at 10 °C. Several endpoints were recorded including survival, hatching delay, hatching success, biometry, developmental abnormalities, and DNA damage (comet and micronucleus assays). This bioassay was firstly tested with two model PAHs, fluoranthene and benzo[a]pyrene. Then, the method was applied to compare the toxicity of three PAH complex mixtures characterized by different PAH compositions: a pyrolytic extract from a PAH-contaminated sediment (Seine estuary, France) and two petrogenic extracts from Arabian Light and Erika oils, at two environmental concentrations, 3 and 10 μg g−1 sum of PAHs. The degree and spectrum of toxicity were different according to the extract considered. Acute effects including embryo mortality and decreased hatching success were observed only for Erika oil extract. Arabian Light and pyrolytic extracts induced mainly sublethal effects including reduced larvae size and hemorrhages. Arabian Light and Erika extracts both induced repairable DNA damage as revealed by the comet assay versus the micronucleus assay. The concentration and proportion of methylphenanthrenes and methylanthracenes appeared to drive the toxicity of the three PAH fractions tested, featuring a toxic gradient as follows: pyrolytic < Arabian Light < Erika. The minimal concentration causing developmental defects was as low as 0.7 μg g−1 sum of PAHs, indicating the high sensitivity of the assay and validating its use for toxicity assessment of particle-bound pollutants.


PAH mixture Oil extract Pyrolytic extract Embryotoxicity Developmental defects Genotoxicity 



Polycyclic aromatic hydrocarbon


Aryl hydrocarbon receptor


Degree day


Dry weight






Early life stage






Arabian Light oil


Erika heavy oil


  1. Amat A, Burgeot T, Castegnaro M, Pfohl-Leszkowicz A (2006) DNA adducts in fish following an oil spill exposure. Environ Chem Lett 4(2):93–99CrossRefGoogle Scholar
  2. Baars B-J (2002) The wreckage of the oil tanker “Erika”—human health risk assessment of beach cleaning, sunbathing and swimming. Toxicol Lett 128(1–3):55–68. doi:10.1016/S0378-4274(01)00533-1 CrossRefGoogle Scholar
  3. Barbee GC, Barich J, Duncan B, Bickham JW, Matson CW, Hintze CJ, Autenrieth RL, Zhou G-D, McDonald TJ, Cizmas L, Norton D, Donnelly KC (2008) In situ biomonitoring of PAH-contaminated sediments using juvenile coho salmon (Oncorhynchus kisutch). Ecotoxicol Environ Saf 71(2):454–464CrossRefGoogle Scholar
  4. Barron MG, Carls MG, Short JW, Rice SD (2003) Photoenhanced toxicity of aqueous phase and chemically dispersed weathered Alaska North Slope crude oil to pacific herring eggs and larvae. Environ Toxicol Chem 22(3):650–660CrossRefGoogle Scholar
  5. Barron MG, Carls MG, Heintz R, Rice SD (2004) Evaluation of fish early life-stage toxicity models of chronic embryonic exposures to complex polycyclic aromatic hydrocarbon mixtures. Toxicol Sci 78(1):60–67. doi:10.1093/toxsci/kfh051 CrossRefGoogle Scholar
  6. Baršienė J, Dedonytė V, Rybakovas A, Andreikėnaitė L, Andersen OK (2006) Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil. Aquat Toxicol 78(0):S99–S104. doi:10.1016/j.aquatox.2006.02.022 Google Scholar
  7. Baumard P, Budzinski H, Garrigues P (1998) PAHs in Arcachon Bay, France: origin and biomonitoring with caged organisms. Mar Pollut Bull 36(8):577–586. doi:10.1016/S0025-326X(98)00014-9 CrossRefGoogle Scholar
  8. Belanger SE, Balon EK, Rawlings JM (2010) Saltatory ontogeny of fishes and sensitive early life stages for ecotoxicology tests. Aquat Toxicol 97(2):88–95CrossRefGoogle Scholar
  9. Billiard SM, Meyer JN, Wassenberg DM, Hodson PV, Di Giulio RT (2008) Nonadditive effects of PAHs on early vertebrate development: mechanisms and implications for risk assessment. Toxicol Sci 105(1):5–23. doi:10.1093/toxsci/kfm303 CrossRefGoogle Scholar
  10. Brinkmann M, Hudjetz S, Kammann U, Hennig M, Kuckelkorn J, Chinoraks M, Cofalla C, Wiseman S, Giesy JP, Schäffer A, Hecker M, Wölz J, Schüttrumpf H, Hollert H (2013) How flood events affect rainbow trout: evidence of a biomarker cascade in rainbow trout after exposure to PAH contaminated sediment suspensions. Aquat Toxicol 128–129(0):13–24. doi:10.1016/j.aquatox.2012.11.010 CrossRefGoogle Scholar
  11. Brinkworth LC, Hodson PV, Tabash S, Lee P (2003) CYP1A induction and blue sac disease in early developmental stages of rainbow trout (Oncoryhchus mykiss) exposed to retene. J Toxicol Environ Health A 66:627–646CrossRefGoogle Scholar
  12. Budzinski H, Letellier M, Thompson S, Le Menach K, Garrigues P (2000) Combined protocol for the analysis of polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs) from sediments using focussed microwave assisted (FMW) extraction at atmospheric pressure. Fresenius J Anal Chem 367(2):165–171. doi:10.1007/s002160051618 CrossRefGoogle Scholar
  13. Burczynski ME, Penning TM (2000) Genotoxic polycyclic aromatic hydrocarbon ortho-quinones generated by aldo-keto reductases induce CYP1A1 via nuclear translocation of the aryl hydrocarbon receptor. Cancer Res 60(4):908–915Google Scholar
  14. Cachot J, Geffard O, Augagneur S, Lacroix S, Le Menach K, Peluhet L, Couteau J, Denier X, Devier MH, Pottier D, Budzinski H (2006) Evidence of genotoxicity related to high PAH content of sediments in the upper part of the Seine estuary (Normandy, France). Aquat Toxicol 79(3):257–267CrossRefGoogle Scholar
  15. Cachot J, Law M, Pottier D, Peluhet L, Norris M, Budzinski H, Winn R (2007) Characterization of toxic effects of sediment-associated organic pollutants using the λ transgenic medaka. Environ Sci Technol 41(22):7830–7836CrossRefGoogle Scholar
  16. Carls MG, Meador JP (2009) A perspective on the toxicity of petrogenic PAHs to developing fish embryos related to environmental chemistry. Hum Ecol Risk Assess 15(6):1084–1098CrossRefGoogle Scholar
  17. Carls MG, Thedinga JF (2010) Exposure of pink salmon embryos to dissolved polynuclear aromatic hydrocarbons delays development, prolonging vulnerability to mechanical damage. Mar Environ Res 69(5):318–325CrossRefGoogle Scholar
  18. Carls MG, Rice SD, Hose JE (1999) Sensitivity of fish embryos to weathered crude oil: Part I. Low-level exposure during incubation causes malformations, genetic damage, and mortality in larval pacific herring (Clupea pallasi). Environ Toxicol Chem 18(3):481–493. doi:10.1002/etc.5620180317 CrossRefGoogle Scholar
  19. Cook PM, Robbins JA, Endicott DD, Lodge KB, Guiney PD, Walker MK, Zabel EW, Peterson RE (2003) Effects of aryl hydrocarbon receptor-mediated early life stage toxicity on lake trout populations in Lake Ontario during the 20th century. Environ Sci Technol 37(17):3864–3877. doi:10.1021/es034045m CrossRefGoogle Scholar
  20. Couillard CM (2002) A microscale test to measure petroleum oil toxicity to mummichog embryos. Environ Toxicol 17(3):195–202. doi:10.1002/tox.10049 CrossRefGoogle Scholar
  21. De Andrade VM, De Freitas TRO, Da Silva J (2004) Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment. Mutat Res Genet Toxicol Environ Mutagen 560(1):57–67CrossRefGoogle Scholar
  22. Di Toro DM, Zarba CS, Hansen DJ, Berry WJ, Swartz RC, Cowan CE, Pavlou SP, Allen HE, Thomas NA, Paquin PR (1991) Technical basis for establishing sediment quality criteria for nonionic organic chemicals using equilibrium partitioning. Environ Toxicol Chem 10(12):1541–1583. doi:10.1002/etc.5620101203 CrossRefGoogle Scholar
  23. EC (2010) Directive 2010/63/EU of the european parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes. vol L276. Official Journal of the European Union,Google Scholar
  24. Embry MR, Belanger SE, Braunbeck TA, Galay-Burgos M, Halder M, Hinton DE, Léonard MA, Lillicrap A, Norberg-King T, Whale G (2010) The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat Toxicol 97(2):79–87CrossRefGoogle Scholar
  25. Escarti E, Porte C (1999) Biomonitoring of PAH pollution in high-altitude mountain lakes through the analysis of fish bile, vol 33. American Chemical Society, Washington, DC, ETATS-UNISGoogle Scholar
  26. Fallahtafti S, Rantanen T, Brown RS, Snieckus V, Hodson PV (2012) Toxicity of hydroxylated alkyl-phenanthrenes to the early life stages of Japanese medaka (Oryzias latipes). Aquat Toxicol 106–107(0):56–64. doi:10.1016/j.aquatox.2011.10.007 CrossRefGoogle Scholar
  27. Frenzilli G, Nigro M, Lyons BP (2009) The Comet assay for the evaluation of genotoxic impact in aquatic environments (review). Mutat Res Rev Mutat Res 681(1):80–92CrossRefGoogle Scholar
  28. Fujiwara A, Nishida-Umehara C, Sakamoto T, Okamoto N, Nakayama I, Abe S (2001) Improved fish lymphocyte culture for chromosome preparation. Genetica 111:77–89CrossRefGoogle Scholar
  29. Geffard O, Geffard A, His E, Budzinski H (2003) Assessment of the bioavailability and toxicity of sediment-associated polycyclic aromatic hydrocarbons and heavy metals applied to Crassostrea gigas embryos and larvae. Mar Pollut Bull 46(4):481–49CrossRefGoogle Scholar
  30. González-Doncel M, González L, Fernández-Torija C, Navas JM, Tarazona JV (2008) Toxic effects of an oil spill on fish early life stages may not be exclusively associated to PAHs: studies with Prestige oil and medaka (Oryzias latipes). Aquat Toxicol 87(4):280–288. doi:10.1016/j.aquatox.2008.02.013 CrossRefGoogle Scholar
  31. Guasch H, Ginebreda A, Geiszinger A, Akkanen J, Slootweg T, Mäenpää K, Leppänen M, Agbo S, Gallampois C, Kukkonen JK (2012) Bioavailability of organic contaminants in freshwater environments. In: Emerging and priority pollutants in rivers. The handbook of environmental chemistry. Springer, Heidelberg, pp 25–53. doi:10.1007/978-3-642-25722-3_2 CrossRefGoogle Scholar
  32. Hatlen K, Sloan CA, Burrows DG, Collier TK, Scholz NL, Incardona JP (2010) Natural sunlight and residual fuel oils are an acutely lethal combination for fish embryos. Aquat Toxicol 99(1):56–64CrossRefGoogle Scholar
  33. Hawkins SA, Billiard SM, Tabash SP, Brown RS, Hodson PV (2002) Altering cytochrome P4501A activity affects polycyclic aromatic hydrocarbon metabolism and toxicity in rainbow trout (Oncorhynchus mykiss). Environ Toxicol Chem 21(9):1845–1853. doi:10.1002/etc.5620210912 CrossRefGoogle Scholar
  34. Hayashi M, Ueda T, Uyeno K, Wada K, Kinae N, Saotome K, Tanaka N, Takai A, Sasaki YF, Asano N, Sofuni T, Ojima Y (1998) Development of genotoxicity assay systems that use aquatic organisms. Mutat Res Fundam Mol Mech Mutagen 399(2):125–133CrossRefGoogle Scholar
  35. Heintz RA, Short JW, Rice SD (1999) Sensitivity of fish embryos to weathered crude oil: Part II. Increased mortality of pink salmon (Oncorhynchus gorbuscha) embryos incubating downstream from weathered Exxon valdez crude oil. Environ Toxicol Chem 18(3):494–503. doi:10.1002/etc.5620180318 CrossRefGoogle Scholar
  36. Hicken CE, Linbo TL, Baldwin DH, Willis ML, Myers MS, Holland L, Larsen M, Stekoll MS, Rice SD, Collier TK, Scholz NL, Incardona JP (2011) Sublethal exposure to crude oil during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish. Proc Natl Acad Sci 108(17):7086–7090. doi:10.1073/pnas.1019031108 CrossRefGoogle Scholar
  37. Hodson PV, Qureshi K, Noble CAJ, Akhtar P, Brown RS (2007) Inhibition of CYP1A enzymes by β-naphthoflavone causes both synergism and antagonism of retene toxicity to rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 81(3):275–285. doi:10.1016/j.aquatox.2006.12.012 CrossRefGoogle Scholar
  38. Incardona JP, Collier TK, Scholz NL (2004) Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol Appl Pharmacol 196(2):191–205CrossRefGoogle Scholar
  39. Incardona JP, Carls MG, Teraoka H, Sloan CA, Collier TK, Scholz NL (2005) Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish development. Environ Health Perspect 113(12):1755–1762CrossRefGoogle Scholar
  40. Incardona JP, Carls MG, Day HL, Sloan CA, Bolton JL, Collier TK, Scholz NL (2009) Cardiac arrhythmia is the primary response of embryonic pacific herring (Clupea pallasi) exposed to crude oil during weathering. Environ Sci Technol 43(1):201–207. doi:10.1021/es802270t CrossRefGoogle Scholar
  41. Karlsson J, Sundberg H, Åkerman G, Grunder K, Eklund B, Breitholtz M (2008) Hazard identification of contaminated sites—ranking potential toxicity of organic sediment extracts in crustacean and fish. J Soils Sed 8(4):263–274. doi:10.1007/s11368-008-0015-3 CrossRefGoogle Scholar
  42. Kocan RM, Matta MB, Salazar SM (1996) Toxicity of weathered coal tar for shortnose sturgeon (Acipenser brevirostrum) embryos and larvae. Arch Environ Contam Toxicol 31:161–165CrossRefGoogle Scholar
  43. Liu JJ, Wang XC, Fan B (2011) Characteristics of PAHs adsorption on inorganic particles and activated sludge in domestic wastewater treatment. Bioresour Technol 102:5305–5311CrossRefGoogle Scholar
  44. Milinkovitch T, Kanan R, Thomas-Guyon H, Le Floch S (2011a) Effects of dispersed oil exposure on the bioaccumulation of polycyclic aromatic hydrocarbons and the mortality of juvenile Liza ramada. Sci Total Environ 409(9):1643–1650. doi:10.1016/j.scitotenv.2011.01.009 CrossRefGoogle Scholar
  45. Milinkovitch T, Ndiaye A, Sanchez W, Le Floch S, Thomas-Guyon H (2011b) Liver antioxidant and plasma immune responses in juvenile golden grey mullet (Liza aurata) exposed to dispersed crude oil. Aquat Toxicol 101(1):155–164. doi:10.1016/j.aquatox.2010.09.013 CrossRefGoogle Scholar
  46. Milinkovitch T, Imbert N, Sanchez W, Le Floch S, Thomas-Guyon H (2013) Toxicological effects of crude oil and oil dispersant: biomarkers in the heart of the juvenile golden grey mullet (Liza aurata). Ecotoxicol Environ Saf 88(0):1–8. doi:10.1016/j.ecoenv.2012.10.029 CrossRefGoogle Scholar
  47. Nahrgang J, Camus L, Carls MG, Gonzalez P, Jönsson M, Taban IC, Bechmann RK, Christiansen JS, Hop H (2010) Biomarker responses in polar cod (Boreogadus saida) exposed to the water soluble fraction of crude oil. Aquat Toxicol 97(3):234–242. doi:10.1016/j.aquatox.2009.11.003 CrossRefGoogle Scholar
  48. Olive PL, Banáth JP (1995) Sizing highly fragmented DNA in individual apoptotic cells using the Comet Assay and a DNA crosslinking agent. Exp Cell Res 221(1):19–26. doi:10.1006/excr.1995.1348 CrossRefGoogle Scholar
  49. Regoli F, Gorbi S, Frenzilli G, Nigro M, Corsi I, Focardi S, Winston GW (2002) Oxidative stress in ecotoxicology: from the analysis of individual antioxidants to a more integrated approach. Mar Environ Res 54:419–423. doi:10.1016/S0141-1136(02)00146-0 CrossRefGoogle Scholar
  50. Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1):184–191. doi:10.1016/0014-4827(88)90265-0 CrossRefGoogle Scholar
  51. Sundberg H, Ishaq R, Akerman G, Tjarnlund U, Zebuhr Y, Linderoth M, Broman D, Balk L (2005) A bio-effect directed fractionation study for toxicological and chemical characterization of organic compounds in bottom sediment. Toxicol Sci 84(1):63–72. doi:10.1093/toxsci/kfi067 CrossRefGoogle Scholar
  52. Udroiu I (2006) The micronucleus test in piscine erythrocytes. Aquat Toxicol 79:201–204Google Scholar
  53. Wang Z, Hollebone BP, Fingas M, Fieldhouse B, Sigouin L, Landriault M, Smith P, Noonan J, Thouin G (2003) Characteristics of spilled oils, fuels, and petroleum products: 1. Composition and properties of selected oils. US EPAGoogle Scholar
  54. Wang B, Liu Y, Chen X, Fan Z (2010) Amitosis-like nuclear division in erythrocytes of triploid rainbow trout Oncorhynchus mykiss. J Fish Biol 76:1205–1211. doi:10.1111/j.1095-8649.2010.02556.x CrossRefGoogle Scholar
  55. Wassenberg DM, Di Giulio RT (2004) Synergistic embryotoxicity of polycyclic aromatic hydrocarbon aryl hydrocarbon receptor agonists with cytochrome P4501A inhibitors in Fundulus heteroclitus. Environ Health Perspect 112(17):1658–1664CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Florane Le Bihanic
    • 1
  • Bénédicte Morin
    • 1
  • Xavier Cousin
    • 2
    • 3
  • Karyn Le Menach
    • 1
  • Hélène Budzinski
    • 1
  • Jérôme Cachot
    • 1
  1. 1.University of Bordeaux, EPOC, UMR CNRS 5805Talence CedexFrance
  2. 2.IFREMER, Ecotoxicology laboratoryL’HoumeauFrance
  3. 3.INRA LPGP, Campus de BeaulieuRennesFrance

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