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Article
Effects of differently coated silver nanoparticles on the photosynthesis of Chlamydomonas reinhardtii Enrique Navarro, Bettina Wagner, Niksa Odzak, Laura Sigg, and Renata Behra Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01089 • Publication Date (Web): 27 May 2015 Downloaded from http://pubs.acs.org on June 5, 2015
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Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Environmental Science & Technology
Ag+ Ag+
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abiotic conditions
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algal cells-AgNP interactions
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ACS Paragon Plusreinhardtii Environment Chlamydomonas
Environmental Science & Technology
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Effects of differently coated silver nanoparticles on the photosynthesis of Chlamydomonas reinhardtii
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Enrique Navarro†*, Bettina Wagner‡, Niksa Odzak‡, Laura Sigg‡ and Renata Behra‡
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†. CSIC, Pyrenean Institute of Ecology, Av. Montañana 1005, Apdo. 13034, 50059 Zaragoza,
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Spain.
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‡.Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, P.O.
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Box 611, 8600 Dübendorf, Switzerland
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KEYWORDS: Chlamydomonas reinhardtii, silver nanoparticles, dissolved silver, bioavailability,
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coatings.
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* Corresponding author Name: Address: Phone: Fax: E-mail address:
Enrique Navarro CSIC, Pyrenean Institute of Ecology, Av. Montañana 1005, Apdo. 13034, 50059 Zaragoza, Spain. +34 976 369 393 +34 976 719 019
[email protected] Page 1 of 23 ACS Paragon Plus Environment
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ABSTRACT
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Various factors have been invoked to explain the toxicity of silver nanoparticles (AgNP) to
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microorganisms including particle size, the nature of stabilizing coatings as well as the amount of
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dissolved silver occurring in AgNP suspensions. In this study we have assessed the effects of nine
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differently coated AgNP (chitosan, lactate, polyvinyl pyrrolidone, polyetheleneglycol, gelatin,
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sodium-dodecyl-benzenesulfonate, citrate, dexpanthenol and carbonate) and AgNO3 on the
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photosynthesis of the freshwater algae Chlamydomonas reinhardtii. We have thus examined how
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AgNP effects to algae relate to particle size, measured dissolved silver (Agd) and bioavailable
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silver (Agbioav). Agbioav was indirectly estimated in toxicity experiments by cysteine-silver
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complexation at the EC50. The EC50 calculated as a function of measured Agd concentrations
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showed for some coatings similar values to that of dissolved Ag, while other coated AgNP
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displayed lower EC50. In all cases, excess cysteine completely prevented effects on photosynthetic
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yield, confirming the role of Agd as a cause of the observed effect on the photosynthesis. Toxicity
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was neither related to particle size nor to the coatings. For all differently coated AgNP
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suspensions, the EC50 values calculated as a function of Agbioav were comparable to the value of
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AgNO3. Depending on the coatings Agbioav was comparable or higher than measured Agd.
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INTRODUCTION
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Because of their bactericidal properties [1], silver nanoparticles (AgNP) are present in numerous
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consumer products. During recent years, an increasing number of studies have demonstrated
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toxicity of AgNP to different microorganisms such as bacteria
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AgNP to microorganisms has been suggested to result from both the release of silver ions (Ag+)
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and interactions between AgNP and cell membrane
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explaining the observed toxicity of AgNP to microorganisms is experimentally supported by the
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fact that complexation of Ag+ by thiol ligands, as well as anaerobic conditions prevent toxicity of
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AgNP [5, 7, 11-15]. Thus, risk assessments of AgNP need careful consideration of the contribution of
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Ag+ to toxicity. Ionic silver might occur in AgNP suspensions, both as residual from particle
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synthesis and formed upon particle oxidation. Major factors expected to influence AgNP
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dissolution in aqueous solutions include pH, ionic strength, and the presence of ligands [16, 17].
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[2-4]
or algae
[5-7]
. The toxicity of
[2, 8-10]
. The determining role of Ag+ in
AgNP are typically synthesized with different surface coatings to minimize particle [1]
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agglomeration in aqueous systems
. Depending on their chemical nature, coatings might by
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complexation influence the release of Ag+ ions from particles
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particles and indirectly, their toxicity to aquatic organisms
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of coatings might contribute to observed effects of AgNP
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systematically examined the contribution of size, dissolved silver and of several coatings on AgNP
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toxicity in a common approach.
[18-21]
, or affect the stability of
[8, 22-24]
. Moreover, intrinsic properties
[25]
. So far, no studies have
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In this study we have assessed the toxicity of differently coated AgNP and of dissolved Ag
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(provided as AgNO3) on the freshwater algae Chlamydomonas reinhardtii and examined how
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effects related to particle size, dissolved silver (Agd) and bioavailable silver (Agbioav) which is the
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concentration that provokes toxicity. Considered coatings are representative of different chemical
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families (Table 1) and were selected considering their differences in Ag binding properties and
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molecular size.
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We first assessed short-term effects on photosynthesis and measured Agd in experimental media
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used for toxicity testing. The concentration of Agbioav was assessed indirectly in toxicity
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experiments by modifying the speciation of Agd with cysteine. This use of the cysteine is
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supported by recent studies showing that the uptake of Ag in presence of cysteine is strongly
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reduced
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were compared to infer the influence of coatings on bioavailability and toxicity of AgNP.
[15, 26]
. Therefore effective concentrations on the base of dissolved and bioavailable silver
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MATERIALS AND METHODS
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AgNP characterization. AgNP were provided by NanoSys GmbH (Wolfhalden, Switzerland)
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as aqueous suspensions of 1 g/L total Ag (nominal concentrations). Original AgNP suspensions
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(see Table 1 for bulk concentrations) were kept in the dark. Experimental concentrations were
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prepared using 10 mM MOPS (3-morpholine propanesulfonic acid, buffered at pH 7.5). For
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cysteine experiments, fresh stock solutions of cysteine were prepared in nanopure water, and kept
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on ice to prevent oxidation. For exposure studies, AgNO3 solutions and AgNP suspensions were
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freshly prepared before use. Unless otherwise indicated, all chemicals were purchased from
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Sigma-Aldrich. AgNP were characterized under experimental conditions (10 mM MOPS, pH 7.5)
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for size and ζ –zeta– potential by Dynamic Light Scattering (DLS) using a Zeta Sizer (Nano ZS,
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Malvern Instruments).
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ICP-MS measurements. The silver concentration (isotope
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Ag) was measured in acidified
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solutions (0.1 M HNO3) by ICP-MS (Element 2 High Resolution Sector Field ICP-MS; Thermo
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Finnigan). Reliability of the measurements was controlled using a water reference solution
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(Standard Reference Material 1643e, National Institute of Standards & Technology, USA).
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For total Ag measurements AgNP suspensions were digested with HNO3 in a microwave oven
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(MLS 1200 Mega, Microwave Laboratory Systems). Recovery of Ag was 88 – 95 % of the
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nominal concentrations.
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Dissolved silver present in AgNP suspensions was examined by centrifugal ultrafiltration
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(Millipore Amicon Ultra-4 3K) through a membrane with a nominal molecular weight limit of 3
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kDa. Recovery measurements using AgNO3 and the same experimental media were done, showing
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that between 90-99% of the silver was recovered after the Ultrafiltration process. Suspensions
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(5µM AgNP in 0.01 M MOPS at pH 7.5) were centrifuged for 30 min at 3000×g (Megafuge 1.0R,
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Heraeus Instruments). Concentration of dissolved Ag (Agd
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Information -SI- Table S1) in the filtrate was expressed as % of the total Ag concentration after 1
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or 2 h in the experimental media without algae (Table 1). Measurement of Agd in the AgNP
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suspensions were confirmed using diffusive gradients in thin films (DGT)
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ultracentrifugation. Bioavailable Ag (Agbioav) was on the basis of cysteine-silver complexation
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experiments (see below).
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mostly Ag+, see at Supporting
[5, 27]
, and by
Speciation calculations. Calculations of the Agd speciation upon cysteine addition to AgNO3 or [28]
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AgNP were done using the software ChemEQL 3.0
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constants, as described in Navarro et al. 2008
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constants and appropriate references are available in SI Tables S1 and S2 respectively.
[5]
using the corresponding equilibrium
. Detailed calculations and the list of stability
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Algal culture and photosynthetic yield measurements. Chlamydomonas reinhardtii (wild
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type, CC-125 137c mt+) were cultured under controlled conditions (25 °C, 90 rpm, 120 µE) in [29]
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standard growth medium at pH 7.5
. Cell number was measured by an electronic particle
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counter (aperture 50 µm; Z2 Coulter Counter; Beckman Coulter, Fullerton, CA). While growth is
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a more sensitive endpoint towards silver with EC50 values of 12 nM [30], photosynthetic yield was
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chosen as a toxicity endpoint because effects are measurable upon short-term exposure thus
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avoiding accumulation of secreted biomolecules. The algal photosynthetic yield of the
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photosystem II in light was measured by fluorometry using a PHYTO-PAM (Heinz Walz GmbH)
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equipped with an Optical Unit ED-101US/MP. The yield reflects the efficiency of the
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photochemical energy conversion process
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light intensities than those used during growth and exposure (140 µmol photons m-2 s-1 PAR
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radiation).
[31]
. Fluorometry measurements were done at similar
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Effects of AgNO3 and AgNP on photosynthetic yield. Exponentially growing algae in culture
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media were first centrifuged (2000×g, 10 min) and then resuspended in 10 mM MOPS at pH 7.5
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to a final volume of 25 mL in glass Erlenmeyers and a density of 1.7x106 cells mL-1.
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Photosynthetic yield was not affected by the algal translocation into MOPS or by exposure to
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cysteine
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response experiments by exposing algae to increasing concentrations of AgNO3 and AgNP (see SI
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Table S3 for a comprehensive list of the experiments and concentrations used). At least 3
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concentration-response experiments per coating were done. AgNP and AgNO3 toxicity to the algal
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photosynthetic yield was measured after short-term exposure (1 and 2 h) in order to minimize
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accumulation of algal products in the exposure media, and thus changes in the silver speciation.
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The photosynthetic values of all concentration-response experiments were represented as
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percentage of the respective controls. Values were plotted as a function of the total Ag or Agd
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concentration measured in AgNP suspensions, in the absence of algae in the suitable concentration
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range. The Agd bound to the algae was estimated to be negligible in comparison with the EC50
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concentrations, as calculated based on previous studies (ref. 26), see calculations in SI. The Agd
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values were determined by centrifugal ultracentrifugation. Data were fitted to a four parameters
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logistic curve to determine EC50 values as described below in the Statistics section.
[5]
. Toxicity of AgNP and AgNO3 to photosynthesis was assessed by concentration-
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Cysteine-silver complexation experiments. Complexation experiments were carried out in
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order to determine the concentration of bioavailable silver (Agbioav). Based on our previous results
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[5]
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Ag+ and to decrease the free Ag+ concentrations to levels which show no effect to photosynthesis
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(see detailed speciation calculation at SI Table S1 and S2). Different cysteine concentrations were
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first added to one concentration of the various AgNP which were in the range of the respective
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EC50. After ten minutes pre-equilibration (time to reach equilibrium of cysteine-silver complexes)
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algae were added to the suspensions and their photosynthetic yield measured after 1 h of exposure.
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Up to 47 complexation experiments were done with various AgNP and cysteine concentrations (SI
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Table S3). The photosynthetic values were represented as percentage of the respective controls
, cysteine concentrations similar to or higher than Agd (mostly Ag+), were expected to complex
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and plotted as a function of cysteine concentration. Points were fitted to the best curve
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(maximizing R2 value), using Sigma Plot 12 (© Systat Software Inc.). The models were then used
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to calculate the lowest amount of cysteine required to completely prevent the toxicity to
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photosynthesis. For each AgNP, the concentration of bioavailable Agbioav at EC50 (1h) was
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estimated as the product of the EC50 values (based on Agd) by the cysteine/Agd ratio (Agbioav = Agd
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EC50 × Cys/Agd). This approach is detailed in the discussion.
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The potential effects of cysteine on the dissolution of Ag or the characteristics of AgNP were
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assessed by measuring dissolved Agd, as well as size and ζ- potential of AgNP in the presence of
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500 nM cysteine which was the highest concentration used in the silver complexation
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experiments.
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Statistics. Errors of measurements (total number of experiments and replicates are shown in SI
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Table S3) are expressed as standard deviation (SD). Concentration-response curves were fitted to
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a four parameters logistic curve using R and drc package
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values and standard error (SE). The same package was also used to perform comparison tests,
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using R “compPAR” function; the null hypothesis is that the ratio equals 1. The ratio was obtained
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by dividing EC50 value for AgNO3 by EC50 for each AgNP. If the ratio significantly differs from 1,
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the null hypothesis should be rejected, meaning that values are significantly different (p
