Direct and indirect effects of silver nanoparticles on freshwater and marine microalgae (Chlamydomonas reinhardtii and Phaeodactylum tricornutum)

Accepted Manuscript Direct and indirect effects of silver nanoparticles on freshwater and marine microalgae (Chlamydomonas reinhardtii and Phaeodactylum tricornutum) M. Sendra, M.P. Yeste, J.M. Gatica, I. Moreno-Garrido, J. Blasco PII:

S0045-6535(17)30507-6

DOI:

10.1016/j.chemosphere.2017.03.123

Reference:

CHEM 19041

To appear in:

ECSN

Received Date: 2 January 2017 Revised Date:

22 March 2017

Accepted Date: 28 March 2017

Please cite this article as: Sendra, M., Yeste, M.P., Gatica, J.M., Moreno-Garrido, I., Blasco, J., Direct and indirect effects of silver nanoparticles on freshwater and marine microalgae (Chlamydomonas reinhardtii and Phaeodactylum tricornutum), Chemosphere (2017), doi: 10.1016/ j.chemosphere.2017.03.123. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Direct and indirect effects of silver nanoparticles on freshwater and

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marine microalgae (Chlamydomonas reinhardtii and Phaeodactylum

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tricornutum)

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Sendra, M.1*; Yeste, M. P. 2; Gatica, J.M.2; Moreno-Garrido I.1and Blasco,

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J1.

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Andalusia (CSIC).Campus Río S. Pedro.11510, Puerto Real, Cádiz, Spain.

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Faculty of Sciences, University of Cadiz, E-11510 Puerto Real, Cádiz, Spain.

Department of Ecology and Coastal Management, Institute of Marine Sciences of

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Department of Material Science, Metallurgical Engineering and Inorganic Chemistry,

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Phone: +34956832612. Fax: +34956834701

Author for correspondence: Marta Sendra Vega. email: [email protected]

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Direct and indirect effects of silver nanoparticles on freshwater and

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marine microalgae (Chlamydomonas reinhardtii and Phaeodactylum

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tricornutum)

18 The last decade has seen a considerable increase in the use of silver nanoparticles

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(AgNPs), which are found in many every-day consumer products including

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textiles, plastics, cosmetics, household sprays and paints. The release of those

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AgNPs into aquatic environments could be causing ecological damage. In this

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study we assess the toxicity of AgNPs of different sizes to two species of

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microalgae, from freshwater and marine environment (Chlamydomonas

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reinhardtii and Phaeodactylum tricornutum respectively). Dissolution processes

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affect the form and concentration of AgNPs in both environments. Dissolution of

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Ag from AgNPs was around 25 times higher in marine water. Nevertheless,

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dissolution of AgNPs in both culture media seems to be related to the small size

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and higher surface area of NPs. In marine water, the main chemical species were

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AgCl2- (53.7 %) and AgCl3-2 (45.2 %). In contrast, for freshwater, the main

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chemical species were Ag+ (26.7 %) and AgCl- (4.3 %). The assessment of

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toxicological responses, specifically growth, cell size, cell complexity,

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chlorophyll a, reactive oxygen species, cell membrane damage and effective

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quantum yield of PSII, corroborated the existence of different toxicity

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mechanisms for microalgae. Indirect effects, notably dissolved Ag ions, seem to

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control toxicity to freshwater microalgae, whereas direct effects, notably

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attachment onto the cell surface and the internalization of AgNPs inside cells,

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seem to determine toxicity to the marine species studied. This research

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contributes to knowledge on the role of intrinsic and extrinsic factors in determining the behavior of NPs in different aquatic environments and the interaction with microalgae.

Keywords: toxicity; silver nanoparticles; freshwater; seawater; microalgae

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Introduction

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As a result of the rapid development of nanotechnology in the last decade, the

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production of engineered nanomaterials (ENMs) has increased in the USA, Europe and

ACCEPTED MANUSCRIPT East Asia (Wiesner et al., 2006, Vance et al., 2015a). This increased ENM production

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for use in products marketed globally is largely thanks to the special physico-chemical

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properties of ENMs in comparison with those of the same materials in conventional

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bulk form (Auffan et al., 2009). In the case of silver nanoparticles (AgNPs), their

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production and application in consumer products is primarily related to the well-known

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antibacterial property of the silver ion (Ag+) released from the particle’s surface. This

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property also makes them useful for many other applications in products as diverse as

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electrical devices, detergents, textiles, cosmetics, outdoor paints and agricultural

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products, and in water treatment (Luoma, 2008, Rai et al., 2009, Wijnhoven et al., 2009,

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Marambio-Jones and Hoek, 2010). AgNPs are the most popular advertised nanomaterial

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in the consumer products inventory (CPI); they are present in 438 products (24% of the

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total listed in the CPI updated in 2015 by Vance and colleagues), although AgNPs

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account for less than 2% of total production of all NPs (Vance et al., 2015b). In Europe,

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annual production of AgNPs ranges between 110 and 230 tons (Blaser et al., 2008).

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Quantitative data on the release of AgNPs into the aquatic environment and

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measurements of environmental concentrations of AgNPs are not currently available.

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Consequently, the amount of AgNP input to the aquatic environment can only be

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predicted by models, where recently the predicted environmental concentration was

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0.76 ng·L-1 in surface water (Blaser et al., 2008, Mueller and Nowack, 2008, Gottschalk

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et al., 2009, Gottschalk and Nowack, 2011, Gottschalk et al., 2015). However, there is

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some evidence, in studies of wastewater and sediment, that silver is released from silver

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nanoparticles in aqueous media (Brar et al., 2010, Colman et al., 2014, Hedberg et al.,

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2014).

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Nevertheless more analysis of environmental measurements is required to determine in

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detail the sources and fate of those AgNPs.

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ACCEPTED MANUSCRIPT 73 The most relevant processes that govern the stability and mobility of AgNPs in

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the aquatic environment are agglomeration, aggregation, dispersion, sedimentation and

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dissolution (Handy et al., 2008, Navarro et al., 2008a, Fabrega et al., 2011). The

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dissolution process, in particular, is affected by external conditions, such as

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temperature, pH, ionic strength, as well as the presence of organic and inorganic

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molecules in the medium (Liu and Hurt, 2010).

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Concerning the toxicity of AgNPs, the direct effect of the AgNPs and the indirect effect

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mediated by the Ag+ released from their surface need to be differentiated (Wijnhoven et

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al., 2009). Although data are available about the toxicity of AgNPs to microalgae, the

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question of which factors (intrinsic and/or extrinsic) are the source of the effect and

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mechanisms regarding the toxicity of AgNPs, is still under debate.

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AgNPs or ions that reach the cell wall could damage the cell membrane and cause loss of membrane integrity and cell lysis (Sondi and Salopek-Sondi, 2004,

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Karlsson et al., 2008, Navarro et al., 2008b, Oukarroum et al., 2012, Oukarroum et al.,

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2013). Oxidative stress is also associated with exposure to nanoparticulate and ionic Ag.

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The high reactivity of NPs and the occurrence of oxygen can result in the formation of

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free oxygen radicals at the NP surface (Nel et al., 2006). Other effects are associated

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directly with the behavior of AgNPs, including physical adhesion, response to shading

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and the internalization of AgNPs by cells (Navarro et al., 2008b, Miao et al., 2009,

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Huang et al., 2016, Wang et al., 2016).

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The objective of this work was to assess the intrinsic and extrinsic factors that

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affect the toxicity of AgNPs to freshwater microalgae, Chlamydomonas reinhardtii

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(P.A. Dang) (CHLOROPHYCEAE) and to marine microalgae, Phaeodactylum

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tricornutum (Bohlin) (BACILLARIOPHYCEAE), as model organisms in both

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freshwater and marine environments. The effect of intrinsic characteristics of NPs (such as size and zeta potential) and extrinsic characteristics of culture media (such as

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different pH and ionic strength,) on AgNPs dissolution, agglomeration, interaction

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between NPs and cells and their effect on toxic response are also investigated.

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According to the observed behavior of AgNPs in the culture media tested, a second

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objective of this work was to determine the direct and indirect mechanisms of the

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toxicity of AgNPs to microalgae.

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Material and methods

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Reagents: AgNP1 (<2 nm; US7130), AgNP2 (<15 nm; US7140), both in aqueous

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suspension, and AgNP3 (30-50 nm US1036), in the form of nanopowder, were obtained

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from US Research Nanomaterials, Inc. DCFH-DA (2’-7’-dichlorofluorescein diacetate),

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and propidium iodide were supplied by Sigma Aldrich. All glassware was washed with

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diluted nitric acid (10%) and rinsed several times with de-ionized water (Milli-Q) before

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use.

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Suspensions of AgNPs.

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Stocks of AgNPs were prepared in ultrapure water immediately before the experiments.

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Suspensions were sonicated with a tip sonicator (UP 200S Dr. Hielscher GmbH) for 10

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min with cycle of 0.5 and frequency of 50.

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Characterization of AgNPs (in 3 particle size groups: NP1, NP2 and NP3).

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Particle size distributions and images revealing particle shape and nano-structure of the

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samples were obtained by means of Transmission Electron Microscopy (TEM). The

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microscope used was a JEOL2010F model, working at 200 kV. This instrument has a

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structural resolution of 0.19 nm. The number of images recorded allowed the

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measurement of 130 particles to determine average and mode size.

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Textural features were characterized by measuring the physisorption of N2 at 196 °C, employing an Autosorb Quantachrome automatic device. Before measurements

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were made, samples were subjected to a surface cleaning pre-treatment under high

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vacuum at 200 °C during 2 hours. The isotherms obtained were used to calculate the

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specific surface area (SBET) of the powdered sample. In the case of the AgNP1 and

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AgNP2 samples, an estimate of total surface area was obtained from the TEM particle

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size distributions.

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The initial particle size of AgNPs were analyzed in ultrapure water, freshwater and

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artificial marine water by Dynamic Light Scattering (Zetasizer Nano ZS90, Malvern

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Instruments, software version 7.10) at 1 mg·L-1. Furthermore, agglomeration was

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assessed with the Zetasizer Nano ZS90 after 0, 0.5, 3, 6 and 24 hours. The zeta potential

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of NP can be indirectly detected with DLS based on the electrophoretic mobility of

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particles, and calculated by application of the Henry equation that expresses the

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electrophoretic mobility of a particle as a function of its zeta potential, and of the

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dielectric constant and viscosity of the medium (Malvern Instruments Ltd., 2007).

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Speciation of dissolved Ag in artificial freshwater and marine culture media.

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The speciation of silver at 1 mg·L-1 in artificial freshwater and marine water was

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calculated with the Visual Minteq 3.1 software, which is a chemical equilibrium

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computer program that possesses an extensive thermodynamic database enabling users

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to calculate the speciation, solubility, and equilibrium of solid and dissolved phases of

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minerals in an aqueous solution (Gustafsson, 2006).

ACCEPTED MANUSCRIPT Dissolution of AgNPs in different culture media.

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In order to quantify dissolved Ag originating from 1 mg·L-1 of AgNPs in artificial

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freshwater and marine water, samples were filtered by ultra-filtration (3000 MWCO)

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and subsequently measured by a single quadrupole (SQ) Inductively Coupled Plasma-

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Mass Spectrometer (ICP-MS) (Thermo Fisher ScientificTMiCAPTM RQ ICP-MS) in

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KED mode. Samples were quantified against an Ag standard curve in 0.1 M HNO3. The

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accuracy of the measurement was established through blanks and spike recovery

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experiments (100±7 % recovery). The isotope analyzed was 107Ag. Sampling times were

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0, 3, 24 and 48 hours. Dissolution experiments were conducted in triplicate. The limit of

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detection (LOD) was 0.95 ppt.

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Test organisms.

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Two microalgae species were selected, one from freshwater environments

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(Chlamydomonas reinhardtii (Dangeard, 1888), CHLOROPHYCEAE) and the other

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from marine environments (Phaeodactylum tricornutum (Bohlin, 1897),

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BACILLARIOPHYCEAE), both obtained from the ICMAN Marine Microalgae Culture

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Collection (IMMCC). Cells were grown in filtered (0.2 µm) freshwater (pH: 7.2) and

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marine culture medium (pH: 8.2) and F/2 marine medium without EDTA for two weeks

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prior to the experiment (Guillard and Ryther, 1962, Fábregas et al., 2000). Synthetic

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marine water used was the Substitute Ocean Water D1141-75 from ASTM (ASTM,

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1975). The recipes of the culture media are provided in supplementary information.

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Toxicity bioassays

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Bioassays were carried out with AgNPs at the following concentrations 10, 40, 75, 150

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and 300 µg·L-1. Experiments were performed under continuous visible light (300 µE-2s-

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). Growth inhibition bioassays followed the OECD procedure (OECD, 1994), in order

ACCEPTED MANUSCRIPT to determine the effective concentration. Values for 50% inhibition (EC50%) after 72

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hours were obtained for microalgae cellular concentration (initial cell density, 104 cells

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mL-1). Cells were counted by flow cytometry (BD Accuri C6). All treatments and

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controls were carried out in triplicate. EC50% growth inhibition values were calculated

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using TSK 1.5 software (Trimmed Spearman-Karber method).

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Sampling for flow cytometry (BD Accuri C6) was carried out at 6, 24, 48 and 72 h, for

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AgNP1, AgNP2 and AgNP3 treatments.

Because forward light scatter (FSC) is correlated with the size or volume of a

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cell or particle, this detector was calibrated with the SPH-PPS-6K kit from Spherotech,

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Inc, and showed an r2 value of 0.98. The sphere diameters were in the following ranges:

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2.0-2.4, 3.0-3.4, 5.0-5.9, 7.0-7.9, 8.0-12.9 and 13.0-17.9 µm. Side light scatter (SSC) is

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correlated with the intracellular complexity (Shapiro, 2005).

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From 1 mL of each sample a volume of 100 µL was taken for analysis by flow cytometry to study the alterations in the cell size and intracellular complexity of the

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microalgae selected. For cell complexity, microalgae populations were washed three

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times with PBS and 0.02 M of EDTA to prevent interference from NPs adsorbed on the

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cell walls. SSC signal should provide reliable data related to the degree of internal cell

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complexity. In the same way, autofluorescence (FL3 670 nm) was assessed by flow

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cytometry to study potential changes in the chlorophyll a content of microalgae.

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The effective quantum yield of photosynthetic energy conversion in PSII in

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darkness was measured fluorometrically using a Phyto-PAM instrument (Heinz Walz

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GmbH) equipped with an ED-101 US/MP Optical Unit. This parameter measures the

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efficiency of the photochemical energy conversion process (Schreiber et al., 1995).

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Production of intracellular ROS (reactive oxygen species: superoxide, hydroxyl and hydrogen peroxide) was quantified using the 2’-7’-dichlorofluorescein diacetate

ACCEPTED MANUSCRIPT (DCFH-DA) method (He et al., 2002, Stachowski-Haberkorn et al., 2013). ROS

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measurements were made at 6, 24, 48, and 72 h. For this probe a negative control

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(without treatment) and a positive control with 0.1mM of H2O2 were employed. A

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quantity of 80 µM DCFH-DA (0.8% DMSO) was added to 1 mL of fresh sample. After

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thirty minutes in darkness, at room temperature conditions, ROS were measured by

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green fluorescence (FL1, 533/30 nm) of stained cells using a flow cytometer. Finally,

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the extent of oxidative stress was calculated from the number of cells that fluoresce

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green (stained with DCFH-DA), as a percentage of the total number of cells counted by

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the flow cytometer.

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Membrane integrity was also quantified by the Propidium Iodide (PI) method (Xiao et

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al., 2011). This endpoint was measured at 6, 24, 48 and 72 h. PI cannot cross the

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membrane and intercalate with nucleic acids inside the cell if the membrane is intact,

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but penetrates the cells if the integrity of the membrane is altered. PI stock solution

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(0.15 mM) was prepared by dissolving PI into PBS buffer (pH 7.2) and stored at 4 ºC

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until use. Samples were incubated for 20 min at the PI dosage (10 µg·mL-1 for 2·106

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cells). Fluorescence of PI inside the cells was detected with an FL2 detector by flow

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cytometry (the same equipment as was used for the ROS measurement). The proportion

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of cells with membrane damage was calculated as the total number of cells that

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fluoresce orange (stained with PI), as a percentage of the total number of cells counted.

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Cell suspensions were incubated with the appropriate fluorochrome at room temperature

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and darkness for the necessary time. The lowest fluorochrome concentration and the

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shortest incubation time were chosen in order to obtain significant and stable staining of

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cells without toxicity taking place.

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ACCEPTED MANUSCRIPT Statistical Analysis

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All bioassays were carried out in triplicate. Data are shown as average ± standard

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deviation between replicates. Statistical analyses were carried out using the IBM SPSS,

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Statistics 23 program. To assess all the responses (cell growth, cell size, cell

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complexity, chlorophyll a, effective quantum yield, % of ROS and % of cells with

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membrane damage) two General Linear Models (GLM; SPSS, 2005) were developed

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(McCullagh and Nelder, 1989, SPSS., 2005). The first GLM is a general model in

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which all the factors are in competition, and the second GLM is segmented by medium

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(freshwater and marine species). The fixed factors are medium (freshwater and marine

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water), treatments (AgNP1, AgNP2 and AgNP3), concentration (10, 40, 75, 150 and

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300 µg·L-1) and exposure time (6, 24, 48, 72 h).

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Data were checked for homogeneity of variance (Levene test); a one-way Anova test

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and a Dunnett post hoc test at p<0.05 were also applied.

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Results

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Characterization and behavior of AgNPs in different culture media.

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Figure 1 and Table S1 give the main results corresponding to the nano-structure and

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physical characterization of the three types of AgNPs investigated in this research. The

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TEM study enabled us to detail the particle size distribution of each sample beyond the

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nominal size values provided by the supplier. The range of particle sizes, as well as the

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mean size value, for each sample varies significantly (4.5±2.3, 16.7±6.2 and 46.7±14.6

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nm for AgNP1, AgNP2 and AgNP3, respectively). As confirmed by the textural

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measurements, sample AgNP1 has the largest specific surface area, 71 m2g-1, followed

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by 27 m2g-1 for AgNP2, and 10 m2g-1 for AgNP3.

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ACCEPTED MANUSCRIPT Zeta potential was measured in the two different media. Higher zeta potential was

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reported in artificial marine water than in freshwater media (Table S1). Figure S1 shows

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the agglomeration of the three AgNPs employed over 24 h in the assayed culture media.

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AgNP2 presented the smallest size of agglomerates in both culture media (fresh and

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marine water) after 24 h of experiment. However for the agglomerates of the other NPs

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there were differences with respect to size. In freshwater, the sequence of relative

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agglomerate size was AgNP2 < AgNP3 < AgNP1, while in marine water it was AgNP2

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< AgNP1 ~ AgNP3.

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Ag dissolution from AgNPs also showed differences between the three NP treatments and between the two culture media. It was the AgNP1 that showed the

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highest dissolution with respect to the other two NPs (AgNP2 dissolution was lower

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than that of AgNP3) and in both culture media (Figure 2). These differences observed

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among NPs seem to be related to the size of the NPs (checked by TEM). The dissolution

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of Ag NPs showed differences in behavior between the two culture media. In artificial

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freshwater, the percentage of Ag dissolved from 0 h to 48 h ranged between 1.8 and 4%

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for AgNP1, between 0.3 and 1.4% for AgNP2, and < 0.1% for AgNP3. However, in

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artificial marine water the percentage of Ag dissolved ranged between 35 and 95% for

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AgNP1, between 14 and 23% for AgNP2, and between 0.38 and 1.25% for AgNP3

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(Figure 2).

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Considering the various chemical species formed from the dissolved Ag in each

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media, these were: AgCl(aq) (64.3%) and Ag+ (26.7%) in freshwater media; and AgCl-,

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AgCl2- (53.7%) and AgCl3-2 (45.2%) in marine media (Table 1 and Table S3).

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ACCEPTED MANUSCRIPT Toxicological responses of microalgae exposed to AgNPs

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Cell density

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The freshwater microalgae, C. reinhardtii, showed greater sensitivity to AgNP1 and

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AgNP2 than to AgNP3. The lowest concentration tested (µg·L-1) caused growth

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inhibition of more than 50% for AgNP1 and AgNP2, although AgNP1 seems to be more

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toxic. For AgNP3, cell density did not show inhibition but there was a hormetic

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response at the lower concentrations (Figure 3). Thus, the EC50% concentration after

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72h for C. reinhardtii was < 10 µg·L-1 with both AgNP1 and AgNP2, and >300 µg·L-1

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with AgNP3 (Table S5).

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In the case of the marine microalgae tested, P. tricornutum, it was AgNP2 that

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provoked the greatest toxic response, with an EC50% concentration after 72h of 143 -

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184 µg·L-1. However for this species, neither the AgNP1 nor AgNP3 exposure reached

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the EC50% threshold at any of the concentrations assayed (Figure 3 and Table S5).

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Cell size

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For C. reinhardtii exposure to AgNP1 and AgNP2 resulted in increased cell size at

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concentrations of 40 µg·L-1 and higher (p<0.05). After 72 h of experiment, the cell size

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of the controls was 5 ± 0.15 µm and 8 ±0.6 µm for AgNP1 and AgNP2 at 300 µg·L-1,

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respectively. However, differences in cell size were not found for exposure to AgNP3

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(Figure 4). In the case of the marine microalgae, P. tricornutum, exposure to AgNP2

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was the only treatment which produced changes significantly different with respect to

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the controls (p<0.05) at 150 and 300 µg·L-1: controls had a cell size of 4 ± 0.2 µm,

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while for the population exposed to AgNP2 the cell size increased to 6 ± 0.5 µm (Figure

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4).

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ACCEPTED MANUSCRIPT Cell complexity.

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C. reinhardtii showed increased cell complexity when exposed to AgNP1 (after 48h of

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experiment) and AgNP2 (at all exposure times) with respect to the controls (p<0.05).

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After 72 h of exposure to AgNP2, the population of C. reinhardtii showed 4.6 times

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more intracellular complexity than the control population (Figure 5). In the case of the

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marine diatom P. tricornutum, cell complexity presented a trend similar to that of C.

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reinhardtii. The population exposed to AgNP1 showed changes only after exposure for

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48 h, while the population exposed to AgNP2 showed increased cell complexity at 48

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and 72 h (p<0.05). In this latter case, exposed cells showed twice as much intracellular

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complexity as the control populations (Figure 5).

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Chlorophyll a.

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Chl a was measured by autofluorescence using flow cytometry (a FL3 detector). Data

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showed changes in the Chl a concentration per cell in C. reinhardtii when cells were

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exposed to AgNP1 and AgNP2 (p<0.05). The Chl a concentration decreased at 24 h and

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48 h for exposure to AgNP1 (a decrease of 1.6 % with respect to the controls). For

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exposure to AgNP2, differences were found after 72 h (a decrease of 1.4 % with respect

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to the controls). Populations exposed to AgNP3 did not show significant fluctuations in

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Chl a concentrations. In the case of P. tricornutum, a decrease of 1.5% was observed

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with respect to the controls when exposed to AgNP1. In contrast, when the microalgae

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population was exposed to AgNP2, the Chl a signal showed increases, with respect to

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the controls, at 24 and 48 h (p<0.05). As with the other responses measured, the various

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concentrations of AgNP3 tested did not produce significant changes in the Chl a value

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(Figure 6).

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ACCEPTED MANUSCRIPT Effective quantum yield of PSII.

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Effective quantum yield of PSII showed a decrease in both microalgae when exposed to

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AgNP1 and AgNP2 after 72 h (p<0.05). With respect to the effects on effective

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quantum yield of PSII after exposure to AgNP3, C. reinhardtii did not show any

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significant changes over time. On the other hand, for P. tricornutum, differences were

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found after 24 and 48 h (p<0.05) (Figure 7).

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Intracellular ROS.

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For the freshwater species, intracellular ROS was higher with respect to the controls for

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exposure to both AgNP1 and AgNP2 (p<0.05). After exposure for 72 h at 300 µg·L-1 an

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increase of ROS of 22 and 45 % compared with controls was found for the population

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exposed to AgNP1 and AgNP2, respectively, In the case of P. tricornutum, a significant

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increase of ROS compared with controls (p<0.05) was recorded after exposure to

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AgNP1, AgNP2 and AgNP3 for 48 and 72 h. The controls showed an increase of 6.5 %

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in the level of intracellular ROS; for AgNP1 the increase was 31 %; for AgNP2, 53 %;

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and for AgNP3, 52 %) (Figure 8).

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Cells with membrane damage.

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The population of C. reinhardtii showed significant increases in the percentage of cells

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with membrane damage when exposed to AgNP1 and AgNP2 at the various

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concentrations tested (p<0.05), but not after exposure to AgNP3. With regard to P.

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tricornutum, exposure provoked an increase in the percentage of non-viable cells. Cells

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exposed to AgNP2 suffered an increase in cell membrane damage, compared with

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controls, after all exposure times assayed (p<0.05), whereas the population exposed to

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AgNP1 and AgNP3 showed differences with respect to the controls only after 48 h of

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exposure time (Figure 9).

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ACCEPTED MANUSCRIPT Discussion.

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The toxic effect of AgNPs, in three different size groups, on two microalgae species (C.

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reinhardtii and P. tricornutum), from two different environmental compartments

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(freshwater and marine water, respectively) have been investigated. The object is to

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understand better the role of intrinsic and extrinsic factors in the mechanisms of AgNP

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toxicity to microalgae. Intrinsic factors studied include the size of NPs, their specific

343

surface area (SBET), zeta potential, and porosity, among others; extrinsic factors include

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the behavior of the NP agglomeration process in different culture media. Previous

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research suggests that, in both media, the Ag that is dissolved from AgNPs over time,

346

and the speciation of the dissolved Ag, are important factors involved in the toxicity of

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metallic NPs in aquatic environments (Miao et al., 2009, Wijnhoven et al., 2009). In

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this research, the starting hypothesis is that AgNPs trigger both direct and indirect toxic

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effects on microalgae, depending on the environment to which the microorganism is

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exposed.

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It has been demonstrated in many studies (Ratte, 1999, Hiriart-Baer et al., 2006,

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Navarro et al., 2008b, Fabrega et al., 2011) that one of the main mechanisms of toxicity

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of AgNPs is related to Ag dissolved from AgNPs, which in both media has a

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relationship with the size and exposed surface of the AgNPs. The reactivity of small

356

NPs is potentially much greater than that of larger particles (Auffan et al., 2009, Ma et

357

al., 2011, Angel et al., 2013). In our case, for both culture media, freshwater and

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marine, the order in terms of the percentage of Ag dissolved is AgNP1 > AgNP2 >

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AgNP3. This finding is in good agreement with the TEM sizes and specific surface area

360

measured for these materials. Although differences in dissolution are found due to the

361

size of particles, these differences were more pronounced in relation to the culture

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ACCEPTED MANUSCRIPT media. In marine water we measured over 20 times more dissolved Ag than in our

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experiments in freshwater media. This result agrees closely with Oukarroum et al.,

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2012, who found almost 18 times more dissolved Ag in marine culture media than in

365

freshwater culture media (Oukarroum et al., 2012). The explanation for the much higher

366

dissolution of AgNPs in marine water than in freshwater is the high concentration (420

367

mM) of NaCl with respect to freshwater. The chloride compounds present in the

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freshwater culture media are Cl2Co·6H2O, CaCl2·2H2O, and MnCl2·4H2O. Sodium

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chloride catalyzes the dissolution of AgNPs, and so plays an important role in the

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behavior of AgNPs in saline water and biological media (Kent and Vikesland, 2012).

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Differences in the various compounds present in artificial fresh and marine

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water determine the formation of new species that contain Ag in their formulation.

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These species and their percentage concentration are very different in the two biological

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media, and the formation of new species of Ag can trigger an indirect toxic effect of

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AgNPs for microalgae. According to the literature, the toxicity of AgNPs is mainly due

377

to free Ag+ ions. In the present study, the main species formed in freshwater were Ag+

378

(26.7%) and AgCl-(aq) (64.3%). In contrast, the species formed in marine water were

379

AgCl2- (53.7%) and AgCl3-2 (45.2 %). In marine culture media the chemical species

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present are not as bioavailable to marine microalgae as in freshwater culture media. The

381

percentage concentration of total Ag+ in marine water was not very important in the

382

total percentage of all chemical species analyzed with Visual Minteq software: Ag+

383

showed a concentration in the marine culture medium of less than 1.9x10-10 µM, and in

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the ranking of chemical species by concentration, Ag+ was in sixth place after AgCl3-2,

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AgCl2-, AgCl (aq), AgBr (aq), and AgBr2-. However, the concentration of Ag+ in the

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freshwater medium was greater, at 2.4x10-6 µM, and in the ranking by concentration of

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ACCEPTED MANUSCRIPT all the chemical species formed, it was in second place after AgCl (aq) at 5.9x10-6 µM.

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Thus the concentration of Ag+ is four orders of magnitude greater in the freshwater than

389

in the marine culture medium. Considering the Ag dissolved from AgNP1 in each

390

medium (40 ppb for freshwater and 845 ppb for marine water) and using Visual Minteq

391

software, the concentration of Ag+ is, again, much higher in freshwater (9.88x10-8 µM)

392

than in marine water (1.59x10-10 µM).

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In general the GLM for both species of microalgae and all of the responses

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measured (population growth, cell size, cell complexity, Chl a, ROS and cells with

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membrane damage), showed statistically significant differences with respect to the

396

culture media, the AgNP treatment, concentration, time of exposure and, finally, the

397

interaction between factors. The effective quantum yield of PSII, on the other hand, did

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not show statistically significant differences between the two species selected (Table 2).

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With regard to the toxicity of AgNPs to freshwater microalgae C. reinhardtii,

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the main toxicity seems to be related to indirect toxic mechanisms of AgNPs, such as

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the amount of Ag+ present in culture media, which is in turn associated with the particle

402

size measured by TEM. However, the percentages of Ag dissolution from AgNP1 and

403

AgNP2 are very different but the toxicity of each type of nanoparticle is not very

404

different. Therefore, other factors related to the direct effect of AgNPs must influence

405

toxicity. For instance, changes in the cell complexity are found after exposure to both

406

AgNP1 and AgNP2; however, exposure to AgNP2 produced greater differences in cells,

407

with respect to the controls, than exposure to AgNP1. Changes in cell complexity are

408

reported to be related to the possible internalization of NPs (Suzuki et al., 2007). After

409

72 hour of experiment, C. reinhardtii showed changes in its cell complexity but only

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when exposed to AgNP2. One reason for this higher uptake of NPs with AgNP2

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compared to AgNP1 could be the lower stage of agglomeration reached by AgNP2 after

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ACCEPTED MANUSCRIPT 24 h in freshwater media. Uptake of NPs by microalgae depends on the characteristics

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of the cell wall, the thickness of which ranges from 5 to 20 nm (and this determines its

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barrier properties) (Navarro et al., 2008a). The very small hydrodynamic size of AgNP2

415

in the agglomeration process after 24 h of experiment could explain why there was little

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difference in the growth inhibition triggered by AgNP1 and AgNP2.

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In relation to the toxicity of AgNPs to marine microalgae, P. tricornutum, the mechanisms of this toxicity could be also controlled by other factors (i.e. by direct

419

effect of AgNPs, not related to Ag dissolved from AgNPs). It was found that AgNP2

420

caused a stronger toxic response in P. tricornutum with an EC50% concentration for

421

growth inhibition of between 143 and 184 µg·L-1. This direct effect could be due to

422

internalization of NPs and adhesion of AgNPs onto the cell wall. Some of the responses

423

measured, such as cell complexity, cell size and effective quantum yield, showed bigger

424

changes with respect to the controls with exposure to AgNP2. The toxicity of AgNP2

425

could be related to its stage of agglomeration, hence to the internalization and

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interaction of small agglomerates with the cells, as AgNP2 showed a smaller

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hydrodynamic size than AgNP1 and AgNP3 in artificial marine water after 24 h of

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experiment.

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Changes in chlorophyll a (autofluorescence measured with FL3 detector) is an adequate means for assessing the physiological state of photosynthetic cells under

431

potential stress factors (González-Barreiro et al., 2004, Chalifour et al., 2009,

432

Hadjoudja et al., 2009). Some authors have stated that a decrease of chlorophyll a may

433

also be related to changes in environmental variables (Schoen et al., 1995, Tsiaka et al.,

434

2013). The loss of chlorophyll a found in this work is probably due to pollutants

435

interfering with pigments synthesis (Zhang et al., 2012), and is related to damage in

436

chloroplast ribosomes (S P Mayfield et al., 1995, Liu et al., 2012). Furthermore, a loss

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ACCEPTED MANUSCRIPT of chlorophyll a content may be due to inhibition in the electron transport chain in the

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donor center, whereas an increase of chlorophyll a content should be due to inhibition

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on the acceptor side. Both changes, an increase and a decrease of autofluorescence,

440

translate as the loss of effective quantum yield of PSII through the inactivation of some

441

PSII centers (Samson and Popovic, 1988, Cid et al., 1995).

442

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In both freshwater and marine microalgae, responses such as effective quantum yield, percentage of ROS and percentage of cells with membrane damage, were affected

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by type of AgNPs and level of concentration. Our results differ from those of other

445

studies (Morones et al., 2005, Oukarroum et al., 2012). According to those authors, the

446

toxicity of AgNPs is mainly related to direct effects such as uptake of AgNPs and

447

attachment of AgNPs onto cells, triggering membrane damage. However, free Ag+ ions

448

from dissolved AgNPs were not considered in those studies. Other researchers have

449

shown the toxicity of AgNPs as being due to indirect effects such as the dissolution of

450

Ag (Navarro et al., 2008b, Angel et al., 2013). The present work shows for the first time

451

both direct and indirect effects of AgNPs acting under two scenarios, on freshwater and

452

marine microalgae, with intrinsic and extrinsic factors that together determine the

453

toxicity of AgNPs.

454

Conclusions

455

Our results have shown the mechanisms of toxicity of AgNPs in freshwater and marine

456

microalgae. Indirect effects seem to govern the toxicity of AgNPs to freshwater

457

microalgae, C. reinhardtii, where an intrinsic factor, particularly the size of the NPs,

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determines the amount of Ag dissolved from the NPs; and extrinsic factors, like the

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composition of the culture medium, govern the chemical species formed from dissolved

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Ag, with a relatively large amount of Ag+1.

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ACCEPTED MANUSCRIPT On the other hand, direct effects seem to cause the toxicity of AgNPs to the marine

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microalgae, P. tricornutum, where extrinsic factors, such as the agglomeration of NPs,

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govern the toxicity to marine microalgae. The larger sizes of NP agglomerate that are

464

formed are not internalized by cells, while the smaller NP agglomerates are internalized

465

and have the effect of increasing cell complexity.

466

Acknowledgements

467

This research has been funded by the Junta de Andalucía (PE2011-RNM-7812 project

468

and FQM-110 group) and the Spanish National Research Plan (CTM2012-38720-C03-

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03 and Project MINECO/FEDER MAT2013-40823-R). We also thank the SC-ICYT of

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Cadiz University (UCA) for the use of its Electron Microscopy division facilities.

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References

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Angel, B.M., Batley, G.E., Jarolimek, C.V. & Rogers, N.J., 2013. The impact of size on the fate and toxicity of nanoparticulate silver in aquatic systems. Chemosphere, 93, 359-365. Astm, 1975. Standard specification for substitute ocean water. Designation D 1141-75 American standard for testing and materials. Auffan, M., Rose, J., Bottero, J.-Y., Lowry, G.V., Jolivet, J.-P. & Wiesner, M.R., 2009. Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. Nature nanotechnology, 4, 634-641. Blaser, S.A., Scheringer, M., Macleod, M. & Hungerbühler, K., 2008. Estimation of cumulative aquatic exposure and risk due to silver: contribution of nanofunctionalized plastics and textiles. Science of the total environment, 390, 396409. Bohlin, K.H., 1897. Zur morphologie und biologie einzelliger algen. Brar, S.K., Verma, M., Tyagi, R. & Surampalli, R., 2010. Engineered nanoparticles in wastewater and wastewater sludge–Evidence and impacts. Waste management, 30, 504-520. Cid, A., Herrero, C., Torres, E. & Abalde, J., 1995. Copper toxicity on the marine microalga Phaeodactylum tricornutum: effects on photosynthesis and related parameters. Aquatic Toxicology, 31, 165-174. Colman, B.P., Espinasse, B., Richardson, C.J., Matson, C.W., Lowry, G.V., Hunt, D.E., Wiesner, M.R. & Bernhardt, E.S., 2014. Emerging contaminant or an old toxin in disguise? Silver nanoparticle impacts on ecosystems. Environmental science & technology, 48, 5229-5236. Chalifour, A., Spear, P.A., Boily, M.H., Deblois, C., Giroux, I., Dassylva, N. & Juneau, P., 2009. Assessment of toxic effects of pesticide extracts on different green algal species by using chlorophyll a fluorescence. Toxicological & Environmental Chemistry, 91, 1315-1329. Dangeard, P.-A., 1888. Recherches sur les algues inférieures.

AC C

EP

TE D

M AN U

SC

RI PT

461

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Fabrega, J., Luoma, S.N., Tyler, C.R., Galloway, T.S. & Lead, J.R., 2011. Silver nanoparticles: behaviour and effects in the aquatic environment. Environment international, 37, 517-531. Fábregas, J., Domínguez, A., Regueiro, M., Maseda, A. & Otero, A., 2000. Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis. Applied Microbiology and Biotechnology, 53, 530535. González-Barreiro, Ó., Rioboo, C., Cid, A. & Herrero, C., 2004. Atrazine-induced chlorosis in Synechococcus elongatus cells. Archives of environmental contamination and toxicology, 46, 301-307. Gottschalk, F., Lassen, C., Kjoelholt, J., Christensen, F. & Nowack, B., 2015. Modeling Flows and Concentrations of Nine Engineered Nanomaterials in the Danish Environment. International journal of environmental research and public health, 12, 5581-5602. Gottschalk, F. & Nowack, B., 2011. The release of engineered nanomaterials to the environment. Journal of Environmental Monitoring, 13, 1145-1155. Gottschalk, F., Sonderer, T., Scholz, R.W. & Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environmental science & technology, 43, 9216-9222. Guillard, R.R.L. & Ryther, J.H., 1962. Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Canadian Journal of Microbiology, 8, 229-239. Gustafsson, J.P., 2006. Visual minteq. Capturado em de, 26. Hadjoudja, S., Vignoles, C., Deluchat, V., Lenain, J.-F., Le Jeune, A.-H. & Baudu, M., 2009. Short term copper toxicity on Microcystis aeruginosa and Chlorella vulgaris using flow cytometry. Aquatic Toxicology, 94, 255-264. Handy, R., Owen, R. & Valsami-Jones, E., 2008. The ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs. Ecotoxicology, 17, 315-325. He, Y.Y., Klisch, M. & Häder, D.P., 2002. Adaptation of Cyanobacteria to UV B Stress Correlated with Oxidative Stress and Oxidative Damage¶. Photochemistry and photobiology, 76, 188-196. Hedberg, J., Skoglund, S., Karlsson, M.-E., Wold, S., Odnevall Wallinder, I. & Hedberg, Y., 2014. Sequential studies of silver released from silver nanoparticles in aqueous media simulating sweat, laundry detergent solutions and surface water. Environmental science & technology, 48, 7314-7322. Hiriart-Baer, V.P., Fortin, C., Lee, D.-Y. & Campbell, P.G., 2006. Toxicity of silver to two freshwater algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata, grown under continuous culture conditions: influence of thiosulphate. Aquatic toxicology, 78, 136-148. Huang, J., Cheng, J. & Yi, J., 2016. Impact of silver nanoparticles on marine diatom Skeletonema costatum. Journal of Applied Toxicology. Karlsson, H.L., Cronholm, P., Gustafsson, J. & Moller, L., 2008. Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes. Chemical research in toxicology, 21, 1726-1732. Kent, R.D. & Vikesland, P.J., 2012. Controlled evaluation of silver nanoparticle dissolution using atomic force microscopy. Environmental science & technology, 46, 6977-6984.

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ACCEPTED MANUSCRIPT

EP

TE D

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Liu, J. & Hurt, R.H., 2010. Ion release kinetics and particle persistence in aqueous nano-silver colloids. Environmental science & technology, 44, 2169-2175. Liu, W., Ming, Y., Huang, Z. & Li, P., 2012. Impacts of florfenicol on marine diatom Skeletonema costatum through photosynthesis inhibition and oxidative damages. Plant Physiology and Biochemistry, 60, 165-170. Luoma, S.N., 2008. Silver nanotechnologies and the environment. The Project on Emerging Nanotechnologies Report, 15. Ma, R., Levard, C., Marinakos, S.M., Cheng, Y., Liu, J., Michel, F.M., Brown Jr, G.E. & Lowry, G.V., 2011. Size-controlled dissolution of organic-coated silver nanoparticles. Environmental science & technology, 46, 752-759. Malvern Instruments Ltd. Zetasizer Nano User Manual, Worcestershire, U.K., 2007. Marambio-Jones, C. & Hoek, E.M., 2010. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. Journal of Nanoparticle Research, 12, 1531-1551. Mccullagh, P. & Nelder, J.A., 1989. Generalized linear models: CRC press. Miao, A.-J., Schwehr, K.A., Xu, C., Zhang, S.-J., Luo, Z., Quigg, A. & Santschi, P.H., 2009. The algal toxicity of silver engineered nanoparticles and detoxification by exopolymeric substances. Environmental Pollution, 157, 3034-3041. Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramírez, J.T. & Yacaman, M.J., 2005. The bactericidal effect of silver nanoparticles. Nanotechnology, 16, 2346. Mueller, N.C. & Nowack, B., 2008. Exposure Modeling of Engineered Nanoparticles in the Environment. Environmental Science & Technology, 42, 4447-4453. Navarro, E., Baun, A., Behra, R., Hartmann, N.B., Filser, J., Miao, A.-J., Quigg, A., Santschi, P.H. & Sigg, L., 2008a. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology, 17, 372-386. Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N., Sigg, L. & Behra, R., 2008b. Toxicity of silver nanoparticles to Chlamydomonas reinhardtii. Environmental science & technology, 42, 8959-8964. Nel, A., Xia, T., Mädler, L. & Li, N., 2006. Toxic potential of materials at the nanolevel. science, 311, 622-627. Oukarroum, A., Barhoumi, L., Pirastru, L. & Dewez, D., 2013. Silver nanoparticle toxicity effect on growth and cellular viability of the aquatic plant Lemna gibba. Environmental Toxicology and Chemistry, 32, 902-907. Oukarroum, A., Bras, S., Perreault, F. & Popovic, R., 2012. Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Ecotoxicology and environmental safety, 78, 80-85. Rai, M., Yadav, A. & Gade, A., 2009. Silver nanoparticles as a new generation of antimicrobials. Biotechnology advances, 27, 76-83. Ratte, H.T., 1999. Bioaccumulation and toxicity of silver compounds: a review. Environmental Toxicology and Chemistry, 18, 89-108. S P Mayfield, C B Yohn, A Cohen, A. & Danon, A., 1995. Regulation of Chloroplast Gene Expression. Annual Review of Plant Physiology and Plant Molecular Biology, 46, 147-166. Samson, G. & Popovic, R., 1988. Use of algal fluorescence for determination of phytotoxicity of heavy metals and pesticides as environmental pollutants. Ecotoxicology and Environmental Safety, 16, 272-278. Schoen, D.J., Henry, G. & Grime, J., 1995. Methods in Comparative Plant Ecology. JSTOR.

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ACCEPTED MANUSCRIPT

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Schreiber, U., Bilger, W. & Neubauer, C., 1995. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. Ecophysiology of photosynthesis. Springer, 49-70. Shapiro, H.M., 2005. Practical flow cytometry: John Wiley & Sons. Sondi, I. & Salopek-Sondi, B., 2004. Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. Journal of colloid and interface science, 275, 177-182. Spss., 2005. Linear Mixed Effects Modeling in SPSS: An Introduction to the MIXED Procedure. Technical Report LMEMWP-0305. Stachowski-Haberkorn, S., Jérôme, M., Rouxel, J., Khelifi, C., Rincé, M. & Burgeot, T., 2013. Multigenerational exposure of the microalga Tetraselmis suecica to diuron leads to spontaneous long-term strain adaptation. Aquatic toxicology, 140, 380388. Suzuki, H., Toyooka, T. & Ibuki, Y., 2007. Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. Environmental science & technology, 41, 3018-3024. Tsiaka, P., Tsarpali, V., Ntaikou, I., Kostopoulou, M.N., Lyberatos, G. & Dailianis, S., 2013. Carbamazepine-mediated pro-oxidant effects on the unicellular marine algal species Dunaliella tertiolecta and the hemocytes of mussel Mytilus galloprovincialis. Ecotoxicology, 22, 1208-1220. Vance, M.E., Kuiken, T., Vejerano, E.P., Mcginnis, S.P., Hochella Jr, M.F., Rejeski, D. & Hull, M.S., 2015a. Nanotechnology in the real world: Redeveloping the nanomaterial consumer products inventory. Beilstein journal of nanotechnology, 6, 1769-1780. Vance, M.E., Kuiken, T., Vejerano, E.P., Mcginnis, S.P., Hochella, M.F., Jr., Rejeski, D. & Hull, M.S., 2015b. Nanotechnology in the real world: Redeveloping the nanomaterial consumer products inventory. Beilstein Journal of Nanotechnology, 6, 1769-1780. Wang, S., Lv, J., Ma, J. & Zhang, S., 2016. Cellular internalization and intracellular biotransformation of silver nanoparticles in Chlamydomonas reinhardtii. Nanotoxicology, 10, 1129-1135. Wiesner, M.R., Lowry, G.V., Alvarez, P., Dionysiou, D. & Biswas, P., 2006. Assessing the Risks of Manufactured Nanomaterials. Environmental Science & Technology, 40, 4336-4345. Wijnhoven, S.W., Peijnenburg, W.J., Herberts, C.A., Hagens, W.I., Oomen, A.G., Heugens, E.H., Roszek, B., Bisschops, J., Gosens, I. & Van De Meent, D., 2009. Nano-silver–a review of available data and knowledge gaps in human and environmental risk assessment. Nanotoxicology, 3, 109-138. Xiao, X., Han, Z.-Y., Chen, Y.-X., Liang, X.-Q., Li, H. & Qian, Y.-C., 2011. Optimization of FDA–PI method using flow cytometry to measure metabolic activity of the cyanobacteria, Microcystis aeruginosa. Physics and Chemistry of the Earth, Parts A/B/C, 36, 424-429. Zhang, W., Zhang, M., Lin, K., Sun, W., Xiong, B., Guo, M., Cui, X. & Fu, R., 2012. Eco-toxicological effect of Carbamazepine on Scenedesmus obliquus and Chlorella pyrenoidosa. Environmental Toxicology and Pharmacology, 33, 344352.

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Figure 1. Representative TEM Images and particle size distributions of AgNP1, AgNP2

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Figure 2. Dissolution of Ag from 1000 ppb of AgNPs over time. The error bars

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represent the standard deviation (SD).

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Figure 3. Cell density of C. reinhardtii and P. tricornutum when microalgae were

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exposed to five different concentrations of AgNPs. Asterisk (*) indicates p<0.05.

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Figure 5. Cell complexity of C. reinhardtii and P. tricornutum when microalgae were

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exposed to five different concentrations of AgNPs. Asterisk (*) indicates p<0.05.

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Figure 6. Autofluorescence (chlorophyll a) of C. reinhardtii and P. tricornutum when

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microalgae were exposed to five different concentrations of AgNPs. Asterisk (*)

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indicates p<0.05.

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Figure 7. Effective quantum yield of photosystem II (E.Q.Y.) of C. reinhardtii and P.

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tricornutum when microalgae were exposed to five different concentrations of AgNPs.

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Asterisk (*) indicates p<0.05.

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Figure 8. Percentage of ROS of C. reinhardtii and P. tricornutum when microalgae

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were exposed to five different concentrations of AgNPs. Asterisk (*) indicates p<0.05.

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Figure 9. Percentage of non-viable cells of C. reinhardtii and P. tricornutum when

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microalgae were exposed to five different concentrations of AgNPs. Asterisk (*)

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indicates p<0.05.

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Tables

741

Table1. Speciation of Ag in artificial freshwater and marine water.

742 % of total concentration Freshwater 26.7

AgCl (aq)

64.3

1

AgCl2-

8.6

53.7

AgCl3-2

0.02

45.2

AgSO4-

0.3

AgNO3 (aq)

0.06

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Ag+

AgBr (aq)

0.03

AgBr2-

0.03

743 744 745

Marine water

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Species name

Table 2. General Linear Model (for both microalgae species)

Growth

TE D

GLM Type III

FL3

Cell size

Cell

ROS

complexity

Interceptation

*

Media

cell

yield

damage

Sig.

Sig.

Sig.

Sig.

Sig.

Sig.

*

*

*

*

*

*

*

*

*

*

*

*

0.1

*

*

*

*

*

*

*

Concentration

*

*

*

*

*

*

*

Time

*

*

*

*

*

*

*

Media*Treatment

*

*

*

*

*

*

*

Media*Time

*

*

0.4

*

*

*

*

Treatment*Time

*

*

*

*

*

*

*

Treatment

746 747

EP

Sig.

AC C

Factor

Membrane Quantum

ACCEPTED MANUSCRIPT 748 749 750

AC C

EP

TE D

M AN U

SC

RI PT

751

ACCEPTED MANUSCRIPT

Supporting Information

area (m2/g)

AgNP1

4.5±2.8

71b

AgNP2

16.7±6.2

27b

AgNP3

46.7±14.6

4.9 c

Artificial

water

Freshwater

Marine water

Hydrodynamic radii (nm)

165.7

371.9

536.6

PDI: 0.37

PDI:0.62

PDI:0.79

189.3

313.8

271.9

PDI:0.37

PDI: 0.59

PDI:0.46

220.6

221

258.6

PDI: 0.4

PDI: 0.49

AC C

EP

PDI: 0.43

a

average data from particle size distribution obtained by TEM

b

estimate from particle size distribution data obtained by TEM

c

BET value obtained from nitrogen physisorption

PDI: polydispersity index

Ultrapure

Artificial

Artificial Marine

water

Freshwater

water

SC

(nm)

Artificial

M AN U

Specific surface

Ultrapure

TE D

TEMa

RI PT

Table S1. Characterization of AgNPs

ζ potential (mV)

-8.2.1±22.6

-17.5±23.4

-6.5±1

-9.93±22.5

-16.2±24.5

-5.6±2

-50.2±18.5

-21.7±21.9

-12.6±2

ACCEPTED MANUSCRIPT

AC C

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Table S2. Percentage of dissolved Ag from AgNPs over 48 hours of experimen

ACCEPTED MANUSCRIPT Artificial freshwater

Artificial marine waters

AgNP1

1.8-4 %

35-95 %

AgNP2

0.3-1.4 %

14-23 %

AgNP3

<0.1 %

0.38-1.25 %

RI PT

From 0-48 hours

Table S3. Speciation of silver in artificial freshwater and artificial marine water. Chemical species formed in freshwater culture media Concentration (µM) 6.15E-16

Ag(SeO3)2-3

2.79E-17

SC

Ag (OH)2Ag+1

2.47E-06

Ag2MoO4 (aq)

3.53E-19

AgCl2

5.96E-06

M AN U

AgCl (aq) -

7.98E-07

AgCl3-2

1.56E-09

AgNO3 (aq)

5.33E-09

AgOH (aq)

3.40E-11

AgSeO3-1

TE D

AgSO4-

3.98E-11 2.42E-08 Concentration (µM)

Ag (OH)2-

4.63E-18

Ag+1 AgBr (aq) AgBr2

-

EP

Chemical species formed in marine culture media

1.93E-10 3.10E-09 2.44E-09 1.44E-11

AgBr4-3

1.50E-13

AgCl (aq)

9.74E-08

AgCl2-

4.97E-06

AgCl3-2

4.19E-06

AgF (aq)

6.37E-14

AgH2BO3 (aq)

7.50E-14

AgOH (aq)

1.93E-14

AgSO4-

1.57E-11

AC C

AgBr3-2

ACCEPTED MANUSCRIPT Table S4. GLM for short cases (by species) C.reinhardtii

GLM Type III Sig

Sig

Interceptation

*

*

*

*

*

*

*

Treatment

*

*

*

*

*

*

* *

*

*

*

Treatment*Time

*

*

*

Concentration*Time

*

*

*

Treatment*Time

*

*

*

*

*

*

*

*

*

* * * *

*

*

*

*

*

*

*

*

Sig

*

*

*

*

*

*

*

*

*

*

*

*

*

*

Growth

AC C

*

*

* *

Treatment*Concentration

*

*

Treatment*Time

*

*

Concentration*Time

*

Treatment*Time

*

* * *

*

*

0.3 * * *

Effective quantum yield

Sig

Membrane cell damage

Sig

ROS

Sig

Cell Complexity

Sig

EP

Treatment

Sig

Size (µm)

Interceptation

Sig

FL3

Factor

Time

*

*

*

GLM Type III

TE D

P.tricornutum

Concentration

*

*

*

M AN U

Treatment*Concentration

*

RI PT

*

*

ROS

Time

*

SC

Concentration

*

Effective quantum yield

Sig

Membrane cell damage

Sig

Cell Complexity

Sig

Size (µm)

Sig

FL3

Sig

Growth

Factor

0.2 * *

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

0.04

*

* *

ACCEPTED MANUSCRIPT Table S5. EC50% growth inhibition. Calculated with TSK software.

EC50% growth inhibition (µg L-1)

Species

AgNP2

AgNP3

Chlamydomonas reinhardtii

<10

<10

>300

Phaeodactylum tricornutum

>300

162.5 (143.4-184.0)

SC

RI PT

AgNP1

>300

AC C

EP

TE D

M AN U

Figure S1. Agglomeration of AgNPs in different culture media after 24H.

ACCEPTED MANUSCRIPT Recipe of culture media Freshwater (Preparation for 1 L) Stock A: KNO3

80.88 g

Na2HPO4

5.67 g

MgSO4 · 7H2O

RI PT

Stock B: 49.3 g

Stock C: CaCl2 · 2H2O

22.19 g

525.94 mg

Cl2Co · 6H2O

2.37 mg

SO4Cu · 5H2O

2.49 mg

Cr2O3

15.19 mg

MnCl2 · 4H2O

M AN U

C6H5FeO7 · 5H2O

SC

Stock D:

197.9 mg

Na2MoO4 · 2H2O

24.19 mg

SeO2

1.10 mg

B12

TE D

Vitamins 30.0 mg

Thiamine (B1)

50.0 mg

EP

Biotine

35.0 mg

Reference: Fábregas J, Domínguez A, Regueiro M, Maseda A, Otero A. Optimization

AC C

of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis. Applied Microbiology and Biotechnology 2000; 53: 530-535.

Artificial marine water: Standard Specification for SUBSTITUTE OCEAN WATER (Preparation for 1 L) Stock n° 1:

MgCl2·6H2O

555.6 g L-1

CaCl2

57.9 g L-1

SrCl2·6H2O

2.1 g L-1

ACCEPTED MANUSCRIPT

KCl

69.5 g L-1

NaHCO3

20.1 g L-1

KBr

10.0 g L-1

H3BO3

2.7 g L-1

NaF

2.1 g L-1

RI PT

Stock n° 2:

Preparation of Substitute Ocean Water: Dissolve in ultrapure water (± 500 mL) 24.534 g L-1

NaCl

4.094 g L-1

Na2SO4 2)

Add 20 mL L-1

M AN U

Stock n° 1

SC

1)

10 mL L-1

Stock n° 2 3)

Dilute to 1 L

4)

Adjust the pH to 8.2 with 0.1 N NaOH

Reference: ASTM (American Society for Testing and Materials) 1975.

TE D

Standard specification for Substitute Ocean Water. Designation D 1141-75. Medium f/2 with double concentration of nitrate and phosphate. Basic components of stock solution: 75 g

PO4H2Na·2H2O

5g

EP

NO3Na EDTA-Na2·2H2O

2.4 g

FeCl3·6H2O

1.6 g

AC C

Dissolve in almost 300 mL de-ionized water and autoclave. When cooled, add:

0.5 mL of trace metal solution (Sol. 1) 50 mL of vitamin solution (Sol. 2)

Extend to 500 mL with de-ionized water. Store at 4ºC in the dark. 1 mL of the solution must be added to one L of culture. Solution 1 (trace metals): CuSO4·5H20

0.10 g

ZnSO4·7H20

0.22 g

ACCEPTED MANUSCRIPT CoCl2·4H20

0.10 g

MnCl2·4H20

1.80 g

Na2MoO4·2H2O

0.06 g

Extend to a final volume of 10 mL and store in cold and dark as stock solution. Solution 2 (vitamins):

RI PT

Dissolve 50 mg of vit. B12 and 1 g of vit. B1 in 500 mL of de-ionized water, filter the solution through 0.22 µm, and store in cold and dark as stock solution. Silicate solution (for diatom cultures):

Dissolve 3.7 mL of silicate pure solution (in saturation*) in 100 mL of de-ionized water.

SC

Filter through 0.22 µm, and store in cold and dark. Add 1 mL of this solution to each L of culture.

* We use Silicate pure solution from PANREAC, code 2111714

M AN U

Reference: Guillard, R.R.L. & Ryther, J.H. 1962. Studies on marine planktonic diatoms, I. Cyclotella nana Hustedt and Detonula confervaceae (Cleve) Gran. Can J Microbiol

AC C

EP

TE D

8: 229-239.

ACCEPTED MANUSCRIPT Highlights: - Ionic strength and size of AgNPs govern the dissolution process. - Toxicity of AgNPs to freshwater microalgae seems to be determined by the presence of Ag+ in culture media (indirect effect). - Toxicity of AgNPs in marine microalgae seems to be ruled by direct effects of AgNPs, such as

AC C

EP

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adsorption to the cell surfaces and internalization mechanisms.