Amphetamine exposure imbalanced antioxidant activity in the bivalve Dreissena polymorpha causing oxidative and genetic damage Marco Parolini a, * , Stefano Magni a , Sara Castiglioni b , Andrea Binelli a, ** a Department of Biosciences, University of Milan, Via Celoria 26, 20133, Milan, Italy b IRCCS e Istituto di Ricerche Farmacologiche “Mario Negri”, Department of Environmental Health Sciences, Via La Masa 19, 20156, Milan, Italy highlights  The chronic toxicity of amphetamine to non-target aquatic organisms is unknown.  We measured sub-lethal effects induced by amphetamine to the zebra mussel.  Low amphetamine concentration did not cause significant adverse effects.  Significant imbalance of enzymatic activity was noticed at 5000 ng/L.  Increases of protein carbonyl content and DNA fragmentation were noticed at 5000 ng/L. article info Article history: Received 12 March 2015 Received in revised form 18 July 2015 Accepted 4 August 2015 Available online xxx Keywords: Illicit drugs Amphetamine Oxidative and genetic biomarkers Dreissena polymorpha abstract Illicit drugs have been recognized as emerging aquatic pollutants due to their presence in aquatic eco- systems up to mg/L level. Among these, the synthetic psycho-stimulant drug amphetamine (AMPH) is commonly found in both surface and wastewaters worldwide. Even though the environmental occur- rence of AMPH is well-known, the information on its toxicity towards non-target freshwater organisms is completely lacking. This study investigated the imbalance of the oxidative status and both oxidative and genetic damage induced by a 14-day exposure to two concentrations (500 ng/L and 5000 ng/L) of AMPH on the freshwater bivalve Dreissena polymorpha by the application of a biomarker suite. We investigated the activity of antioxidant enzymes (SOD, CAT and GPx), the phase II detoxifying enzyme GST, the lipid peroxidation level (LPO) and protein carbonyl content (PCC), as well as primary (Single Cell Gel Elec- trophoresis assay) and fixed (DNA diffusion assay and Micronucleus test) genetic damage. Our results showed that a current realistic AMPH concentration (500 ng/L) did neither cause notable imbalances in enzymatic activities, nor oxidative and genetic damage to cellular macromolecules. In contrast, the bell- shaped trend of antioxidants showed at the highest tested concentration (5000 ng/L) suggested an overproduction of reactive oxygen species, leading to oxidative damage, as confirmed by the significant increase of protein carbonylation and DNA fragmentation. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Amphetamine-type substances (ATS) are psychoactive drugs extensively used worldwide because of their stimulant, euphoric, anorectic and, in some cases, empathogenic, entactogenic and hallucinogenic properties (Carvalho et al., 2012). Amphetamine- type substances, excluding ‘ecstasy’, constitute the second most commonly used group of illicit substances worldwide after cannabis, with reported 13.9 million to 54.8 million estimated users by the newest World Drug Report, accounting for the 0.7% of the global population aged 15e64 (UNODC, 2014). Despite the diffi- culties to quantify the global production of ATS, its market is globally in expansion, as shown by the increase in dismantling of ATS-manufacturing laboratories and in the number of seizures, as well as by the spreading of manufacture and the development of new markets (UNODC, 2014). For instance, the number of dismantled laboratories increased from12,571 in 2011 to 14,322 in 2012, while for the second year, ATS seizures reached an all-time high of 144 tons, of which approximately half of them were * Corresponding author. ** Corresponding author. E-mail address: marco.parolini@unimi.it (M. Parolini). Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere http://dx.doi.org/10.1016/j.chemosphere.2015.08.025 0045-6535/© 2015 Elsevier Ltd. All rights reserved. Chemosphere 144 (2016) 207e213