Lethal and sub lethal effects of the biocide chlorhexidine on aquatic organisms Fa ´tima T. Jesus • Rhaul Oliveira • Andreia Silva • Ana L. Catarino • Amadeu M. V. M. Soares • Anto ´nio J. A. Nogueira • Ine ˆs Domingues Accepted: 27 August 2013 / Published online: 13 September 2013 Ó Springer Science+Business Media New York 2013 Abstract Chlorhexidine is among the most used biocides in Europe, however its toxicity to aquatic organisms is scarcely known. The main objective of this study was to assess the lethal and sub lethal effects of chlorhexidine digluconate (ChD) on four aquatic model organisms: the bacteria Vibrio fischeri, the algae Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the embryos of the fish Danio rerio. ChD was very toxic to algae and crustaceans, with a 72 h-EC50 of 62.2 lg/l and a 48 h-EC50 of 45.0 lg/l, respectively. Toxicity to fish embryos and the bacteria was lower, with a 96 h-EC50 of 804.0 lg/l and a 15 min-EC50 of 1,694.0 lg/l, respec- tively. Concerning sub lethal effects on D. magna (feeding inhibition) a 6 h-EC50 of 503.7 lg/l was obtained. In fish, ChD caused developmental abnormalities, namely altera- tions in the amniotic fluid (48 h-EC20 of 753.6 lg/l) and early hatching. Moreover, enzymatic biomarkers on fish embryos showed an induction of cholinesterase activity in all ChD tested concentrations (80–900 lg/l). The catalase activity was also induced at the highest concentration tes- ted (900 lg/l) whereas no changes were observed for glutathione-S-transferase and lactate dehydrogenase activ- ities. The toxicity of ChD to the algae and crustacean raises concerns about its potential effects in aquatic food webs, since these organisms are in the base of trophic chains, and highlights the need for further studies on ChD toxicity to aquatic organisms. Keywords Zebrafish embryos  Daphnia  Algae  Bacteria  Biomarkers  Feeding inhibition Introduction Biocides are used worldwide in daily activities to control organisms that are harmful to human or animal health or that cause damage to materials (e.g. biofouling). None- theless, they can pose risks to humans, animals and the environment due to both their intrinsic biocidal properties and associated use patterns (European Council 2012). Facing the increasing use of biocides, the European Commission recently implemented new rules on biocidal products, which concern their commercialization, in order to improve consumers and environmental safety (European Council 2012). Chlorhexidine and its salts (acetate, digluconate, and hydrochloride) are among the most used biocides in Europe (Boxall et al. 2006; Garvey et al. 2001). Chlorhexidine digluconate (ChD) is a disinfectant with antimicrobial activity against Gram-positive bacteria and, to a less extent, to Gram-negative bacteria, fungi and certain types of virus (Fiorentino et al. 2010). ChD leads to cell wall/membrane disruption, causing the loss of lipid components (Cheung et al. 2012). Due to its high antimicrobial activity, ChD is used in hospital, veterinary and cosmetic products (Fio- rentino et al. 2010; Hebert et al. 2003). In particular, ChD can be used in surgical scrubs, preoperative skin prepara- tions, skin antiseptics and cleansers, wound protectors, Fa ´tima T. Jesus and Rhaul Oliveira have contributed equally to this study. Electronic supplementary material The online version of this article (doi:10.1007/s10646-013-1121-6) contains supplementary material, which is available to authorized users. F. T. Jesus  R. Oliveira (&)  A. Silva  A. L. Catarino  A. M. V. M. Soares  A. J. A. Nogueira  I. Domingues Department of Biology and CESAM, Universidade de Aveiro, Campus Universita ´rio de Santiago, 3810-193 Aveiro, Portugal e-mail: rhaul.oliveira@gmail.com; rhaul.oliveira@ua.pt 123 Ecotoxicology (2013) 22:1348–1358 DOI 10.1007/s10646-013-1121-6