Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul Investigation of the toxic efects of diferent polystyrene micro-and nanoplastics on microalgae Chlorella vulgaris by analysis of cell viability, pigment content, oxidative stress and ultrastructural changes Layla J. Hazeem a, ⁎ , Gamze Yesilay b , Mohamed Bououdina c , Simone Perna a , Demet Cetin d , Zekiye Suludere e , Alexandre Barras f , Rabah Boukherroub f a Department of Biology, College of Science, University of Bahrain, 3203, Bahrain b Molecular Biology and Genetics Department, Hamidiye Institute of Health Sciences, University of Health Sciences, Istanbul 34668, Turkey c Department of Physics, College of Science, University of Bahrain, 3203, Bahrain d Department of Mathematics and Science Education, Gazi Faculty of Education, Gazi University, 06500 Ankara, Turkey e Department of Biology, Faculty of Science, Gazi University, 06500 Ankara, Turkey f Univ. Lille, CNRS, Central Lille, ISEN, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France ARTICLE INFO Keywords: Microplastics Nanoplastics Chlorella vulgaris Cytotoxicity test Ultrastructural changes ABSTRACT Plastics of diferent sizes (micro- and nano-sized) are often identifed in aquatic environments. Nevertheless, their infuence on marine organisms has not been widely investigated. In this study, the responses of the mi- croalga Chlorella vulgaris to micro- and nanoplastics exposure were examined using long term toxicity test. The plastics tested were carboxyl-functionalized and non-functionalized polystyrene of 20, 50 and 500 nm in dia- meter. A reduction in algal cell viability and chlorophyll a concentration has been observed after exposure to the small sizes (20 and 50 nm) of plastics. Lactate dehydrogenase activity and reactive oxygen species concentra- tion/production were signifcantly higher after exposure to the 20 nm nanoplastics than that of control con- frming the stress condition. Fourier transform infrared (FTIR) spectroscopy analysis proved the attachment of nanoplastics to microalgae and rearrangement of extracellular polymeric substances. The cellular stress ap- peared as increased cell size, deformed cell wall and increased volume of starch grains. 1. Introduction Recently, plastics of diferent sizes are being used in many appli- cations. Microplastics (MPs) and nanoplastics (NPs) are widely em- ployed in numerous applications such as wastewater treatment, en- vironmental bioremediation, and in medical feld (Koelmans et al., 2015; Bergami et al., 2017; Ko et al., 2018). Cosmetic products, ex- foliating scrubs and skin cleansers were all found to contain plastic materials. MPs and NPs can be released either intentionally into the environment due to human activities (Guzzetti et al., 2018; Walkinshaw et al., 2020) through many consumer products (Besseling et al., 2014) or unintentionally through degradation of delicate, weathered plastic manufactured goods (Lambert and Wagner, 2016; Auta et al., 2017). Due to their potential hazards on aquatic organisms, MPs and NPs gained more attention (Horton et al., 2017). Plastic wastes have been universally recognized as a threat for marine ecosystems (Andrady, 2011). They make a large percentage of marine litter (Ruiz- Orejόn et al., 2016). Nevertheless, their infuence on marine organisms, phy- toplankton for instance, has not been widely studied (Mao et al., 2018). Marine life at diferent trophic levels are being exposed and afected by plastics of various sizes. Hence, it is crucial to evaluate and comprehend how marine organisms are afected by this type of pollutants (Chae et al., 2019). Polyethylene (PE), polystyrene (PS) and polypropylene (PP) were the mutual forms of plastics that can be found in aquatic environments (Cincineli et al., 2017; Pitt et al., 2018). MPs are plastic particles < 5 mm in diameter according to U.S. National Oceanic and Atmospheric Administration (Barnes et al., 2009). NPs, on the other hand, are generally < 100 nm in diameter (Bergami et al., 2016; Pitt et al., 2018). The assessment of the efect of the small fraction of plastic debris, defned as NPs, is one of the least explored areas, and the sig- nifcance of this evolving risk on the marine environment should be seriously investigated. https://doi.org/10.1016/j.marpolbul.2020.111278 Received 10 March 2020; Received in revised form 12 May 2020; Accepted 12 May 2020 ⁎ Corresponding author at: University of Bahrain, P.O. Box 32038, Bahrain. E-mail addresses: lhazeem@uob.edu.bh (L.J. Hazeem), sperna@uob.edu.bh (S. Perna), demetcetin@gazi.edu.tr (D. Cetin), zekiyes@gazi.edu.tr (Z. Suludere), alexandre.barras@univ-lille.fr (A. Barras), rabah.boukherroub@univ-lille.fr (R. Boukherroub). Marine Pollution Bulletin 156 (2020) 111278 Available online 22 May 2020 0025-326X/ © 2020 Elsevier Ltd. All rights reserved. T