Induction of Reactive Oxygen Species and Algal Growth Inhibition by Tritiated Water with or Without Copper C. Re ´ ty, 1 R. Gilbin, 1 E. Gomez 2 1 IRSN, Laboratory of Radioecology and Ecotoxicology, 13115 Saint Paul Lez Durance Cedex, France 2 UMR Hydrosciences, Montpellier 1 University, Montpellier, France Received 12 January 2010; revised 20 April 2010; accepted 24 April 2010 ABSTRACT: Tritium ( 3 H) is a radioactive element of ecological concern because of its release into aquatic ecosystems from nuclear power plants. However, the acute and chronic effects of tritiated water (HTO) on aquatic organisms are poorly documented, as are its effects on oxidative stress. In addition, the effects of HTO in combination with other contaminants remain largely unexamined. Herein, we document the effect of HTO on a primary aquatic producer (Chlamydomonas reinhardtii) by measuring growth and oxidative stress using fluorimetric (H 2 DCF-DA) determination of Reactive Oxygen Species (ROS) production. The maximum cell density of the alga (1.65 3 10 6 cells mL -1 ) was reduced by 23% (1.27 3 10 6 cells mL -1 ) at the highest exposure tested (59 MBq mL -1 HTO), whereas cells exposed to 0.9 MBq mL -1 showed a signif- icantly enhanced maximum cell density of 1.90 3 10 6 cells mL -1 , an increase of 15%. With regard to oxi- dative stress, exposure to HTO (0.04, 0.16, and 2.8 MBq mL -1 ) induced an early dose-dependent peak in ROS production after 14–15 min of exposure, followed by a slow decrease in ROS which stabilized after 60 min. Moreover, this study showed that the presence of HTO may influence the impact of other conven- tional, nonradioactive contaminants, such as copper, a well known oxidizing trace metal for aquatic organisms. A significant synergic effect of copper and HTO on ROS production was observed. This syner- gic effect on oxidative stress was shown to be linked to an enhanced copper uptake rate measured in the presence of HTO ( [ 4 times). We conclude that HTO should be considered as a sensitizer when in a mix- ture with other contaminants, especially through interactions on the antioxidant system of algae. # 2010 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2010. Keywords: tritiated water; reactive oxygen species; copper uptake; algae; chemical mixtures INTRODUCTION With the development of fusion energy, tritium ( 3 H) will definitely become an element of concern for ecological risk assessments in aquatic ecosystems. The International Ther- monuclear Experimental Reactor (ITER, a prototype of the fusion reactor) will release 10 14 Bq of 3 H each year into a single river (Gue ´tat et al., 2008). By comparison, current annual liquid releases of 3 H from all French Nuclear Power Plants (NPP) combined are 10 15 Bq year 21 (EDF, 2007). In terms of radioactivity (as Bq), 3 H constitutes the main component of radioactive releases from French NPPs into aquatic ecosystems, significantly more than other radionu- clides (10 11 and 10 10 Bq year 21 , respectively for 14 C and other radionuclides: 60 Co, 58 Co, 110m Ag, 63 Ni, 125 Sb, 124 Sb, 137 Cs, 123m Te, 54 Mn, 134 Cs, 131 I, ...). Tritium, a low energy b-emitter (mean energy 5 5.707 eV), naturally occurs in surface water at activities ranging from 1 3 10 24 to 9 3 10 24 Bq mL 21 (Gue ´tat et al., 2008). This background level is due to cosmic neutrons interacting Correspondence to: R. Gilbin; e-mail: rodolphe.gilbin@irsn.fr Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.20626  C 2010 Wiley Periodicals, Inc. 1