New approaches on the use of tunicates (Ciona robusta) for toxicity assessments


After the accidental release of crude oil in marine environment, dispersants are applied on sea surface transferring oil into the water column where it can be broken down by biodegradation, thereby reducing potential pollution to coastal areas. Before they can be used in the wild, the ecotoxicity of such dispersants is usually evaluated with toxicity assays using algae, crustacean and fishes. Nowadays, there is a need to find alternative species to reduce the use of vertebrates both for ethical considerations and for reducing the cost of bioassays. Ciona robusta is a solitary ascidian that inhabits shallow waters and marine coastal areas. This species has been recently adopted as valuable biological model for ecotoxicity studies, thanks to its rapid embryonic and larval development, resemblance to vertebrates, and low risk of ethical issues. On this ground, the lethal and sublethal toxicity of two dispersants has been evaluated on Ciona juveniles. At this stage, the organisms become filter-feeders and the morphological alterations of the organs can be easily observed. The median lethal concentrations at 96 h (96hLC50) for Dispersant 1 (non-ionic surfactant) and for Dispersant 2 (mixture of non-ionic surfactants and anionic surfactants) are 41.6 mg/L (38.6–44.9) and 92.5 mg/L (87.7–97.5), respectively. The Ciona juvenile model was compared to Dicentrarchus labrax fish juveniles test, and it showed increased sensitivity for Ciona to these compounds. These results suggest that 96 h mortality test bioassay could be a good alternative method to 96 h mortality assay with D. labrax, limiting the use of vertebrates for dispersant toxicity.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3


  1. Anderson HE, Christiaen L (2016) Ciona as a simple chordate model for heart development and regeneration. J Cardiovasc Dev Dis 3(3):1–20

    CAS  Google Scholar 

  2. Berrill NJ (1947) The development and growth of Ciona. J Mar Biol Assoc U K 26:616–625.

    CAS  Article  Google Scholar 

  3. Chiba S, Sasaki A, Nakayama A, Takamura K, Satoh N (2004) Development of Ciona intestinalis juveniles (through 2nd ascidian stage). Zool Sci 21:285–298.

    Article  Google Scholar 

  4. Conti D, Balzamo S, Paina A, Martone C, Raso E, Cadoni F, Savorelli F, Croppo M, Bellaria V, Pati A (2015) European sea bass (Dicentrarchus labrax L. 1758) as a sentinel species in Europe to study the effects of contaminants. Annu Res Rev Biol 8(4):1–13

    Article  Google Scholar 

  5. Delsuc F, Brinkmann H, Chourrout D, Philippe H (2006) Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439(7079):965–968

    CAS  Article  Google Scholar 

  6. Dybern, B. I. (1965). The life cycle of Ciona intestinalis (L) f. typical; in relation to environmental temperature Oikos(16), 109–131

  7. Halder ME, Kienzler A, Whelan M, Worth A (2014) EURL ECVAM Strategy to replace, reduce and refine the use of fish in aquatic toxicity and bioaccumulation testing. Publications Office of the European Union.

  8. IMO/UNEP (2011) Guidelines for the use of dispersants for combating oil pollution at sea in the Mediterranean region. Part II: Basic information on dispersants and their application.

  9. Lambert CC, Brandt CL (1967) The effect of light on the spawning of Ciona intestinalis. Biol Bull 132:222–228

    Article  Google Scholar 

  10. Lillicrap A, Belanger S, Burden N, Du Pasquier D, Embry MR, Halder M, Lampi MA, Lee L, Norberg-King T, Rattner BA, Schirmer K, Thomas P (2016) Alternative approaches to vertebrate ecotoxicity tests in the 21st century: a review of developments over the last 2 decades and current status. Environ Toxicol Chem 35(11):2637–2646.

    CAS  Article  Google Scholar 

  11. Manfra L, Tornambè A, Guyomarch J, Le Guerrogue P, Kerambrun L, Rotini A, Savorelli F, Onorati F, Magaletti E (2017) Dispersant approval procedures in France and Italy: a comparative ecotoxicity study. Ecotoxicol Environ Saf 143:180–185

    CAS  Article  Google Scholar 

  12. Manfra L, Tornambè A, Guyomarch J, Duboscq K, Faraponova O, Sebbio C (2019) Could a harmonized tiered approach assess dispersant toxicity in Italy and France? Environ Sci Pollut Res.

  13. Mansueto V, Cangialosi MV, Faqi AS (2011) Postembryonic development effect of Bisphenol a and Tributyltin effects in Ciona intestinalis. Caryologia 64(4):478–484

    Google Scholar 

  14. Mariani L, De Pascale D, Faraponova O, Tornambè A, Sarni A, Giuliani S, Ruggiero G, Onorati F, Magaletti E (2006) The use of a test battery in marine ecotoxicology: the acute toxicity of sodium dodecyl sulfate. Environ Toxicol 21:373–379

    CAS  Article  Google Scholar 

  15. Messinetti S, Mercurio S, Parolini M, Sugni M, Pennati R (2017) Effects of polystyrene microplastics on early stages of two marine invertebrates with different feeding strategies. Environ Pollut 237:1080–1087.

    CAS  Article  Google Scholar 

  16. Millar RH (1953) Ciona. In: L.M.B.C. Memoirs on Typical British Marine Plants and Animals. Liverpool University Press, Liverpool, pp 1–123

    Google Scholar 

  17. OECD/203 (1992) Test no. 203: fish, acute toxicity test, OECD guidelines for the testing of chemicals, Section 2. OECD Publishing Paris

  18. Okamura Y, Nishino A, Murata Y, Nakajo K, Iwasaki H, Ohtsuka Y, Tanaka-Kunishima M, Takahashi N, Hara Y, Yoshida T, Nishida M, Okado H, Watari H, Meinertzhagen IA, Satoh N, Takahashi K, Satou Y, Okada Y, Mori Y (2005) Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes. Physiol Genomics 22(3):269–282 Retrieved from

    CAS  Article  Google Scholar 

  19. Petersen JK, Schou O, Thor P (1995) Growth and energetics in the ascidian Ciona intestinalis. Mar Ecol Prog Ser 120:175–184.

    Article  Google Scholar 

  20. Ribelles A, Carrasco C, Rosety M, Aldana M (1995) A histochemical study of the biological effects of sodium dodecyl sulfate on the intestine of gilthead seabream, Sparus aurata. Ecotoxicol Environ Saf 32:131–138

    CAS  Article  Google Scholar 

  21. Rosety RM, Ordonez FJ, Rosety M, Rosety JM, Rosety I, Ribelles A, Carrasco C (2001) Morphohistochemical changes in the gills of turbot, Scophthalmus maximus L ., induced by sodium dodecyl sulfate. Ecotoxicol Environ Saf 3:223–228

    Google Scholar 

  22. Rotini A, Manfra L, Canepa S, Tornambè A, Migliore L (2015) Can Artemia hatching assay be a (sensitive) alternative tool to acute toxicity test. Bull Environ Contam Toxicol 95(6).

  23. Sasakura Y, Mita K, Ogura Y, Horie T (2012) Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis. Develop Growth Differ 54(3):420–437

    CAS  Article  Google Scholar 

  24. Sato Y, Terakado K, Morisawa M (1997) Test cell migration and tunic formation during post-hatching development of the larva of the ascidian, Ciona intestinalis. Develop Growth Differ 39:117–126

    CAS  Article  Google Scholar 

  25. Satoh N (1994) Developmental biology of ascidians. Cambridge University Press, New York

    Google Scholar 

  26. Scott MJ, Jones MN (2000) The biodegradation of surfactants in the environment. Biochim Biophys Acta (BBA) 1508(1–2):235–251

    CAS  Article  Google Scholar 

  27. Willey A (1893a) Studies on the Protochordata. I. on the origin of the branchial stigmata, praeoral lobe, endostyle, atrial cavities, etc. in Ciona intestinalis, Linn., with remarks on Clavelina lepadiformis. Q J Micr Sci 34:317–360

    Google Scholar 

  28. Willey A (1893b) Studies on the Protochordata. II. The development of the neuro-hypophyseal system in Ciona intestinalis and Clavelina lepadiformis, with an account of the origin of the sense organs in Ascidia mentula. Q J Micr Sci 35:295–316

    Google Scholar 

  29. Wise J, Wise JP (2011) A review of the toxicity of chemical dispersants. Rev Environ Health 26(4):281–300

    CAS  Article  Google Scholar 

  30. Yamamoto M, Okada T (1999) Origin of the gonad in the juvenile of a solitary ascidian, Ciona intestinalis. Dev Growth Differ 41:73–79

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Loredana Manfra.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Responsible Editor: Philippe Garrigues

Electronic supplementary material


(DOCX 28.2 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Eliso, M.C., Manfra, L., Savorelli, F. et al. New approaches on the use of tunicates (Ciona robusta) for toxicity assessments. Environ Sci Pollut Res (2020).

Download citation


  • Oil spill management
  • Dispersants
  • Toxicity
  • Alternative biological pattern
  • Ascidian