Environmental Toxicology and Pharmacology 23 (2007) 279–285 Evaluation of the acute toxicity of perfluorinated carboxylic acids using eukaryotic cell lines, bacteria and enzymatic assays E. Mulkiewicz a , B. Jastorff b , A.C. Skladanowski c , K. Kleszczy ´ nski c , P. Stepnowski a,∗ a Faculty of Chemistry, University of Gda´ nsk, Sobieskiego 18, PL-80-952 Gda´ nsk, Poland b Centre for Environmental Research and Technology (UFT), University of Bremen, D-28359 Bremen, Leobener Str., Germany c Intercollegiate Faculty of Biotechnology, Medical University of Gda´ nsk & University of Gda ´ nsk, PL 80-211 Gda ´ nsk, ul. D˛ ebinki 1, Poland Received 9 August 2006; received in revised form 30 October 2006; accepted 7 November 2006 Available online 12 November 2006 Abstract The acute biological activity of a homologous series of perfluorinated carboxylic acids – perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) – was studied. To analyze the potential risk of the perfluorinated acids to humans and the environment, different in vitro toxicity test systems were employed. The cytotoxicity of the chemicals towards two different types of mammalian cell lines and one marine bacteria was investigated. The viability of cells from the promyelocytic leukemia rat cell line (IPC-81) and the rat glioma cell line (C6) was assayed calorimetrically with WST-1 reagent. The evaluation was combined with the Vibrio fischeri acute bioluminescence inhibition assay. The biological activity of the compounds was also determined at the molecular level with acetylcholinesterase and glutathione reductase inhibition assays. This is the first report of the effects of perfluorinated acids on the activity of purified enzymes. The results show these compounds have a very low acute biological activity. The observed effective concentrations lie in the millimole range, which is well above probable intracellular concentrations. A relationship was found between the toxicity of the perfluorinated carboxylic acids and the perfluorocarbon chain length: in every test system applied, the longer the perfluorocarbon chain, the more toxic was the acid. The lowest effective concentrations were thus recorded for perfluorononanoic and perfluorodecanoic acids. © 2006 Elsevier B.V. All rights reserved. Keywords: Toxicity; Perfluorinated acids; IPC-81; C6; Vibrio fischeri; AChE; GR 1. Introduction In recent years, growing concern has been expressed about perfluorinated compounds, the global production of which has increased since the 1970s. With their unique physicochemical properties, they have a broad spectrum of applications as sur- factants, refrigerants and polymers, and also as components of pharmaceuticals, fire retardants, lubricants, adhesives, paints, cosmetics, agrochemicals and food packaging (Key et al., 1997). Owing to the presence of high-energy carbon-fluorine bonds (the strongest of all covalent bonds), perfluorochemicals are stable and persistent in the environment (Banks et al., 1994). They do not undergo photolysis, hydrolysis, defluorination or phase II metabolism (Kudo and Kawashima, 2003). It is common knowl- edge that biodegradation is restricted to the non-perfluorinated ∗ Corresponding author. Tel.: +48 58 523 5448; fax: +48 58 523 5472. E-mail address: sox@chem.univ.gda.pl (P. Stepnowski). part of the molecules (Hagen et al., 1981). Furthermore, dur- ing microbial degradation, perfluorochemicals tend to be slowly converted to more bioaccumulative and more toxic products (Dimitrov et al., 2004). Perfluorochemicals have been detected not only in the phys- ical environment, but also in humans and wildlife. These contaminants have been found in oceanic waters (from several thousands pg/L in coastal waters to few tens of pg/L in the cen- tral Pacific Ocean) (Yamashita et al., 2005). Several studies have reported the presence of perfluorinated chemicals in a variety of wildlife species, including freshwater and marine mammals, fish, birds and shellfish (Giesy and Kannan, 2001; Kannan et al., 2001, 2002a,b). These investigators and others (Bossia et al., 2005; Martin et al., 2003) suggested that the chemicals undergo biomagnification at the top levels of the food chain. Increas- ing concentrations of perfluorochemicals have been observed in animal tissues (Giesy and Kannan, 2001; Kannan et al., 2002a). Human contamination by perfluorinated compounds has been reported, mostly in blood samples collected in the United States, 1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.11.002