Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv Toxicity of pharmaceuticals in binary mixtures: Assessment by additive and non-additive toxicity models Š. Ukić a,∗ , M. Sigurnjak a , M. Cvetnić a , M. Markić a , M. Novak Stankov a , M. Rogošić a , B. Rasulev b , A. Lončarić Božić a , H. Kušić a , T. Bolanča a a University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10000, Zagreb, Croatia b North Dakota State University, Department of Coatings and Polymeric Materials, Fargo, ND, 58102, USA ARTICLE INFO Keywords: Toxicity Pharmaceuticals Binary mixtures Concentration addition Independent action ABSTRACT Current risk assessment in many countries, including European Union, is still placing focus on single substances rather than their mixtures, although mixtures are commonly found in the environment. To overcome this pro- blem and gain new insights, six pharmaceuticals, namely: azithromycin (AZM), erythromycin (ERM), carba- mazepine (CBA), oxytetracycline (OTC), dexamethasone (DXM), and diclofenac (DCF), were selected in order to analyze their combined toxicity in binary mixtures. Overall, 45 binary mixtures were analyzed. Single compo- nent toxicities were determined as well, for modelling purpose. Two most common mathematical models for the description of mixture toxicities were applied: concentration addition (CA) and independent action (IA) model. Comparison of the predicted and experimentally obtained toxicities provided information about the modes of toxicity action in the mixtures. OTC–DCF binary mixture indicated synergism with respect to additive behavior (CA model). All other binary combinations containing OTC or DCF were acting very similarly: the synergism with respect to additive behavior was observed for OTC–CBA and DCF-CBA combinations, while OTC–AZM, OTC–ERM, DCF–AZM and DCF–ERM exhibited antagonistic behavior with respect to CA model. All the re- maining binary mixtures indicated additive behavior. The applicability of IA model as a proof of independent toxic action of the components was confirmed in cases of DCF–AZM, DCF–ERM, and OTC–AZM mixtures. 1. Introduction Pharmaceuticals are chemical compounds designed to prevent, cure and treat diseases and thus improve health of living beings (Jelic et al., 2011). Their use and consumption are continuously increasing, mostly as a consequence of 1) rapid expansion of world population, 2) new drugs discovery and 3) rapidly increasing share of elderly people in highly developed countries (Daughton, 2003). For a long time, phar- maceuticals were exclusively considered as beneficial compounds while their adverse environmental effect was not recognized. The rapid growth of world population resulted in a global awareness about the importance of fresh-water resources and in increased efforts in monitoring the environment. First reports about the presence of pharmaceuticals in the environment (especially ground waters) started to come out in 1980s (Daughton, 2003), but the idea that these com- ponents might be a potential environmental risk emerged ten years later (Daughton and Ternes, 1999; Küster and Adler, 2014). Un- fortunately, it seems that the awareness about the true importance of the problem is growing very slowly. In most cases the concentration levels of pharmaceuticals in the environment are not considered fatal for humans and, accordingly, their determination is very often per- formed due to regulatory demands only. Nevertheless, many of those compounds, although present at very low concentrations, could po- tentially produce cumulative adverse effects (chronic effects), meaning that the real risk is hidden in the long-term exposure (Gracia-Lor et al., 2012). Therefore, “harmless” environmental levels of potentially ha- zardous substances like pharmaceuticals cannot be truly defined (Küster and Adler, 2014). A considerable risk can be attributed to the antibiotics in the environment, since microorganisms are very sensitive to pharmaceuticals with antibiotic activities and they proved to be very efficient in developing antibiotic resistance (Kümmerer, 2004). There are various pathways through which pharmaceuticals are released to the environment but the principal one is human or animal excretion following therapeutic use (Daughton, 2003; Sorell, 2016). Significant amount of pharmaceuticals in original (unmetabolized) form are excreted into raw sewage and waste-water treatment systems. https://doi.org/10.1016/j.ecoenv.2019.109696 Received 18 July 2019; Received in revised form 16 September 2019; Accepted 17 September 2019 ∗ Corresponding author. University of Zagreb, Faculty of Chemical Engineering and Technology, Department of Analytical Chemistry, Marulićev trg 19, 10000, Zagreb, Croatia. E-mail address: sukic@fkit.hr (Š. Ukić). Ecotoxicology and Environmental Safety 185 (2019) 109696 0147-6513/ © 2019 Published by Elsevier Inc. T