Toxicological assessment of indium nitrate on aquatic organisms and investigation of the effects on the PLHC-1 fish cell line Jorge L. Zurita a , Angeles Jos b , Ana del Peso a , Manuel Salguero a , Ana M. Cameán b , Miguel López-Artíguez a , Guillermo Repetto a,b, ⁎ a National Institute of Toxicology and Forensic Sciences, Av. Dr Fedriani s/n, 41009, Seville, Spain b Area of Toxicology, University of Seville, Prof. García González 2, 41012, Seville, Spain Received 24 May 2007; received in revised form 18 July 2007; accepted 19 July 2007 Available online 5 September 2007 Abstract Indium nitrate is mainly used as a semiconductor in batteries, for plating and other chemical and medical applications. There is a lack of available information about the adverse effects of indium compounds on aquatic organisms. Therefore, the toxic effects on systems from four trophic levels of the aquatic ecosystem were investigated. Firstly, the bacterium Vibrio fischeri, the alga Chlorella vulgaris and the cladoceran Daphnia magna were used in the toxicological evaluation of indium nitrate. The most sensitive model was V. fischeri, with a NOAEL of 0.02 and an EC 50 of 0.04 mM at 15 min. Although indium nitrate should be classified as harmful to aquatic organisms, it is not expected to represent acute risk to the aquatic biota. Secondly, PLHC-1 fish cell line was employed to investigate the effects and mechanisms of toxicity. Although protein content, neutral red uptake, methylthiazol metabolization, lysosomal function and acetylcholinesterase activity were reduced in cells, stimulations were observed for metallothionein levels and succinate dehydrogenase and glucose-6-phosphate dehydrogenase activities. No changes were observed in ethoxyresorufin-O-deethylase activity. To clarify the main events in PLHC-1 cell death induced by indium nitrate, nine modulators were applied. They were related to oxidative stress (α-tocopherol succinate, mannitol and sodium benzoate), disruption of calcium homeostasis (BAPTA-AM and EGTA), thiol protection (1,4-dithiotreitol), iron chelation (deferoxiamine) or regulation of glutathione levels (2-oxothiazolidine-4-carboxylic acid and malic acid diethyl ester). The main morphological alterations were hydropic degeneration and loss of cells. At least, in partly, toxicity seems to be mediated by oxidative stress, and particularly by NADPH-dependent lipid peroxidation. © 2007 Elsevier B.V. All rights reserved. Keywords: Indium nitrate; Cytotoxicity; Alternative methods; Aquatic environment; Fish cell lines 1. Introduction Indium is a rare, lustrous silver-white metal with atomic number 49. It is widely distributed in the earth's crust in minute quantities (0.1 ppm) and it is also found in the hydrosphere. Indium belongs to group IIIA in the periodic table. Indium resembles tin in its physical and chemical properties and to some extent in its Science of the Total Environment 387 (2007) 155 – 165 www.elsevier.com/locate/scitotenv Abbreviations: AChE, Acetylcholinesterase; EC 50 , Mean effective concentration; EROD, Ethoxyresorufin-O-deethylase; G6PDH, glu- cose-6-phosphate dehydrogenase; MTS, 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt; NOAEL, Non-observed adverse effect level; SDH, succinate dehydrogenase. ⁎ Corresponding author. National Institute of Toxicology and Forensic Sciences, Av. Dr Fedriani s/n, 41009, Seville, Spain. Tel.: +34 954371233; fax: +34 954370262. E-mail address: repetto@us.es (G. Repetto). 0048-9697/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2007.07.057