, Volume 23, Issue 7, pp 1326–1335 | Cite as

Optimization of NRU assay in primary cultures of Eisenia fetida for metal toxicity assessment

  • Amaia Irizar
  • Daniel Duarte
  • Lucia Guilhermino
  • Ionan Marigómez
  • Manu Soto


Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 105 seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 μg/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals.


Eisenia fetida Coelomocyte Primary cultures In vitro exposure Metal 



This research was supported by the Basque Government (ETORTEK IE10-273; SAIOTEK S-PC13UN028; and Grant to Consolidated Research Group, GIC07/26-IT-393-07; IT-810-13) and the University of the Basque Country (Research & Formation Unit in Ecosystem Health Protection, UFI 11/37). A.I has been recipient of a predoctoral fellowship from the Basque Government.

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Amaia Irizar
    • 1
  • Daniel Duarte
    • 2
  • Lucia Guilhermino
    • 2
  • Ionan Marigómez
    • 1
  • Manu Soto
    • 1
  1. 1.Cell Biology & Environmental Toxicology Research Group, Research Centre for Experimental Marine Biology & Biotechnology (PIE) & Zoology & Animal Cell Biology Department (Faculty of Science & Technology)University of the Basque CountryBilbaoSpain
  2. 2.Laboratory of Ecotoxicology, Institute of Biomedical Sciences of Abel Salazar (ICBAS)University of PortoPortoPortugal

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