Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/watres Ecotoxicological effects of the antioxidant additive propyl gallate in five aquatic systems Jorge L. Zurita a ,A ´ ngeles Jos b , Ana del Peso a , Manuel Salguero a , Miguel Lo ´ pez-Artı ´guez a , Guillermo Repetto a,b,Ã a National Institute of Toxicology and Forensic Sciences, Av. Dr Fedriani s/n, 41009 Seville, Spain b Area of Toxicology, University of Seville, Prof. Garcı´a Gonza ´ lez 2, 41012 Seville, Spain article info Article history: Received 31 July 2006 Received in revised form 29 January 2007 Accepted 7 February 2007 Available online 23 March 2007 Keywords: Propyl gallate Ecotoxicity Cytotoxicity Alternative methods Aquatic environment abstract Propyl gallate is an antioxidant widely used in foods, cosmetics and pharmaceuticals. The occurrence and fate of additives in the aquatic environment is an emerging issue in environmental chemistry. To date, there is little available information about the adverse effects of propyl gallate on aquatic organisms. Therefore, the toxic effects were investigated, using five model systems from four trophic levels. The most sensitive system was the hepatoma fish cell line PLHC-1 according to total protein content, with an EC 50 of 10 mM and a NOAEL of 1 mM at 72 h, followed by the immobilization of Daphnia magna, the inhibition of bioluminescence of Vibrio fischeri, the salmonid fish cell line RTG-2 and the inhibition of the growth of Chlorella vulgaris. Although protein content, neutral red uptake, methylthiazol metabolization and acetylcholinesterase activity were reduced in PLHC-1 cells, stimulations were observed for lysosomal function, succinate dehydrogenase, glucose-6-phosphate dehydrogenase and ethoxyresorufin-O-deethylase activities. No changes were observed in metallothionein levels. The main morphological observations were the loss of cells and the induction of cell death mainly by necrosis but also by apoptosis. The protective and toxic effects of propyl gallate were evaluated. General antioxidants and calcium chelators did not modify the toxicity of propyl gallate, but an iron-dependent lipid peroxidation inhibitor gave 22% protection. The results also suggest that propyl gallate cytotoxicity is dependent on glutathione levels, which were modulated by malic acid diethyl ester and 2-oxothiazolidine-4-carboxylic acid. According to the results, propyl gallate should be classified as toxic to aquatic organisms. & 2007 Elsevier Ltd. All rights reserved. 1. Introduction Propyl gallate is an antioxidant widely used to preserve and stabilize the freshness, nutritional value, flavor and color of foods, fats and oils, and medicinal preparations (JEFCA, 1996). It is an additive authorised (INS No. 310) in the European Union and in many others countries, where it is listed as a common preservative, and is generally recognized as safe. It has been used since 1948 to stabilize cosmetic, and food- packaging materials, and foods containing fats, and as an ARTICLE IN PRESS 0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2007.02.003 Ã 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). Abbreviations: AChE, acetylcholinesterase; EC 50 , mean effective concentration; EROD, ethoxyresorufin-O-deethylase; G6PDH, glucose-6-phosphate dehydrogenase; MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium salt; NOAEL, nonobserved adverse effect level; SDH, succinate dehydrogenase WATER RESEARCH 41 (2007) 2599– 2611