Contents lists available at ScienceDirect Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat Responses of focculated activated sludge to bimetallic Ag-Fe nanoparticles toxicity: Performance, activity enzymatic, and bacterial community shift Ahmad Reza Yazdanbakhsh a , Mohammad Rafee a , Hasti Daraei a, ⁎ , Mohammad Ali Amoozegar b a Department of Environmental Health Engineering, School of Public Health and Safety, Shahid Beheshti University of Medical Sciences, Tehran, Iran b Extremophiles Lab., Dept. of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran ARTICLE INFO Keywords: Ag NPs ZVI NPs Activated sludge Metagenomics Enzymes activity ROS LDH ABSTRACT Ever-increasing production and use of nanoparticles (NPs) have aroused overarching concerns for their toxic efects on the environment and human. In the present study, the toxic efects of Silver (Ag) and Iron (Fe) NPs on the performance of activated sludge were investigated under continuous aerobic/anoxic/anaerobic conditions in laboratory-scale sequencing batch reactors (SBRs).Activated sludge was exposed to various concentrations (5–100 mg/L) of Ag-Fe NPs for 60 days and its response was assessed through the enzymatic activity, COD, nitrogen (TN) and phosphorus (TP) removal, toxicity tests, as well as variations in bacterial community. Compared with the pristine control sample, the exposure to NPs suppressed TN and TP removal efciencies. Indeed, the respiration rate and biomass concentration were signifcantly afected by the NPs. Although the simultaneous exposure to Ag-Fe NPs did afect the integrity of cell membrane (LDH) and key enzymes activities, the higher concentration induced an increased generation of reactive oxygen species (ROS). The metagenome analysis revealed a marked shift in the microbial community structure suggesting that both heterotrophic and autotrophic communities were afected by the presence of Ag-Fe NPs. Our results provide some evidence for compounded efects of NPs in their simultaneous presence, and generate new leads for future research eforts. 1. Introduction An increasing variety of engineered nanoscale materials are already being manufactured in large scales and incorporated into industrial and household products where their unique characteristics hold even greater promise for a broad range of future applications [1]. Nano- particles indeed show interest in environmental applications. This is due mainly to their larger permeability and specifc surface areas as compared to the bulk of materials, which confer them more reactive sites allowing for more rapid degradation of contaminants [2]. Parti- cularly, silver nanoparticles (AgNPs) and nano zero-valent metals have been pioneers in the feld of nanotechnology [3]. AgNPs are currently exploited in personal care and industrial products including water fl- ters, surface coatings, washing machines, and so on [4]. Among zero- valent metals, nanoscale zero valent iron (nZVI) has been the most widely studied NPs in remediation trials [5]. The widely and rapidly expanding production and use of NPs, notably silver and iron NPs, will inevitably increase the potentiality for their release into the environ- ment, such as wastewater treatment plants (WWTPs) [6] as well as natural aqueous and terrestrial environments [7]. WWTPs can also play a protective function in view of discharging nanoparticles into aqueous environments as these nano-scale particles could be absorbed onto the activated sludge and bacterial surfaces [8]. The literature is limited and somewhat contradictory with respect to the concentration in waste- water. The annual consumption of silver nanoparticles in industries has been reported in the range of 10 and 200 tons. Xu et al. [9] in their study reported the nanoparticles levels in wastewater in the range of μg/L to mg/L. The silver NPs concentration was estimated about 105–350 μg/L at the WWTP efuent [10,11]. Also, an analysis of the data available in the literature already reveals that about 1–49 g/L of nZVI are needed daily to groundwater purifcation, which is a large amount [12,13]. Ortega-Calvo et al. [14] in their study demonstrated that the environmentally relevant concentrations of nZVI were between 10 μg/L to 1 mg/L [11]. The release of toxic substances from particles (e.g. free Ag + ions from silver particles) into exposure media, pro- duction of toxic substances (e.g. reactive oxygen species) through sur- face interactions with media, and direct interaction of particles or their surfaces with biological targets (e.g. membranes or DNA) may poten- tially illustrate the mechanisms underlying the toxic action of en- gineered NPs, as proposed by Brunner et al. [15]. The Biological https://doi.org/10.1016/j.jhazmat.2018.11.098 Received 28 September 2018; Received in revised form 25 November 2018; Accepted 26 November 2018 ⁎ Corresponding author. E-mail addresses: yazdanbakhsh@sbmu.ac.ir (A.R. Yazdanbakhsh), rafee@sbmu.ac.ir (M. Rafee), ha.daraei@sbmu.ac.ir (H. Daraei), amoozegar@ut.ac.ir (M.A. Amoozegar). Journal of Hazardous Materials 366 (2019) 114–123 Available online 27 November 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved. T