Contents lists available at ScienceDirect Ecological Indicators journal homepage: www.elsevier.com/locate/ecolind Original Articles Integrative assessment of silver nanoparticles toxicity in Prochilodus lineatus fish Analía Ale a , Andrea Silvana Rossi a,b , Carla Bacchetta a , Susana Gervasio c , Fernando Román de la Torre d , Jimena Cazenave a,b, ⁎ a Instituto Nacional de Limnología, UNL, CONICET, Santa Fe, Argentina b Facultad de Humanidades y Ciencias, UNL, Santa Fe, Argentina c Instituto de Desarrollo Tecnológico para la Industria Química, UNL, CONICET, Santa Fe, Argentina d GECAP, Departamento de Ciencias Básicas e INEDES, UNLu, CONICET, Luján, Argentina ARTICLE INFO Keywords: Nanotoxicology Biomarkers Bacteria colony forming units Principal component analysis ABSTRACT The aim of this study was to evaluate simultaneously a battery of biomarkers in Prochilodus lineatus fish in order to obtain a holistic overview of silver nanoparticles (AgNP) toxicity. Juveniles were exposed to 2.5 and 25.0 μgAgNP L -1 for 5 and 15 days. We analyzed silver accumulation in tissues (liver, intestine and brain), morphometric and hematological parameters, oxidative stress markers (antioxidant enzymes, lipid peroxidation, and antioxidant capacity against peroxyl radicals-ACAP-), and metabolic responses in plasma (glucose, proteins, triglycerides and cholesterol), liver and muscle (glycogen, proteins, and lipids). Additionally, after 15-day ex- posure, the number of colony forming units (CFU) of bacteria living at the skin mucus secretion was analyzed to corroborate the AgNP biocide properties. Tissue Ag accumulation was concentration- and time-dependent: liver > intestine > brain. The hepatosomatic index increased at both periods of exposure at 25.0 μgAgNP L -1 . Hematological and plasma biochemical parameters increased after 5 days of exposure to the highest con- centration. When fish were exposed for 15 days to 25.0 μgAgNP L -1 , many changes were observed: glycogen content increased in liver and muscle, muscle protein concentration decreased, all antioxidant enzymes activities were enhanced and ACAP decreased in liver. The CFU decreased in the mucus of fish exposed to both AgNP treatments when compared to the control group. The multivariate analysis revealed a clear difference in phy- siological profiles of individuals exposed to the different treatments. Our results showed that AgNP in short and subchronic exposures threaten the health of fish. 1. Introduction There is a large body of evidence suggesting Nanotechnology as an expanding area of commerce due to the increasing number of products that contain nanoparticles (NP) (Mansoori et al., 2008). Scown et al. (2010) put emphasis on the vulnerability of aquatic environment as many of NP end up in it by intentional and accidental releases or via weathering of products that contain them. Particularly, the unique and efficient biocide properties of silver nanoparticles (AgNP) make them broadly used in health and fitness products (Lee et al., 2012). AgNP incorporated into spray, textiles, bandages and energetic applications have a higher release potential than those fully integrated into com- posites (Kennedy et al., 2014). According to the Nanodatabase (www. nanodb.dk) and The Project on Emerging Nanotechnologies (www. nanotechproject.org), a number of 378 and 442 commercial products containing AgNP were registered respectively in 2018. The scarce understanding of the harmful effects of NP on the en- vironment and their interaction with biotic and abiotic components has been raising concern over the last decades. AgNP can cause an effect called the Trojan horse, in which Ag + ions are released inside tissues or cells exerting responses different from those of the AgNP or the silver salt (Bermejo-Nogales et al., 2016). It has been suggested that the AgNP concentrations in the aquatic environment are in the order of μg L -1 or ng L -1 (Gottschalk et al., 2013), though these concentrations are still toxic to aquatic organisms (Garner et al., 2015; Sayed and Soliman, 2017). Unfortunately, precise estimates of emanations from silver- containing materials are hindered by the lack of information about the content and form of such metal in products (Geranio et al., 2009). Fish species have been widely used in studies for assessing the biological and biochemical impact of environmental contaminants (van der Oost et al., 2013). When AgNP come into contact with aquatic or- ganisms, these particles easily penetrate their tissues or are retained at https://doi.org/10.1016/j.ecolind.2018.06.023 Received 1 February 2018; Received in revised form 10 May 2018; Accepted 11 June 2018 ⁎ Corresponding author at: Instituto Nacional de Limnología, UNL, CONICET, Ciudad Universitaria, 3000 Santa Fe, Argentina. E-mail address: jcazenave@inali.unl.edu.ar (J. Cazenave). Ecological Indicators 93 (2018) 1190–1198 1470-160X/ © 2018 Published by Elsevier Ltd. T