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Unravelling the complex behavior of AgNPs driving NP-cell interactions and toxicity to algal cells Anzhela Malysheva, Nicolas H. Voelcker, Peter E. Holm, and Enzo Lombi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03470 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 13, 2016
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Environmental Science & Technology
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Unravelling the complex behavior of AgNPs
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driving NP-cell interactions and toxicity to algal
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cells
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Anzhela Malyshevaǂ*, Nicolas Voelckerǂ, Peter E. Holm#, Enzo Lombiǂ*
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ǂ Future Industries Institute, University of South Australia, Mawson Lakes Campus, SA 5095,
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Australia
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# Department of Plant and Environmental Sciences, Faculty of Sciences, University of
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Copenhagen, Denmark
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*Phone: 618 830 26267; e-mail: Enzo
[email protected] ,Phone: 618 830 25504; e-
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mail:
[email protected] 12 13
TOC
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ABSTRACT: While the importance of nanoparticle (NP) characterization under relevant test
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conditions is widely recognized in nanotoxicology, few studies monitor NPs behavior in the
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presence of exposed organisms. Here we studied the behavior of nine types of silver
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nanoparticles (AgNPs) during the 48h algal toxicity test. In particular, we investigated NP
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aggregation and dissolution by time-resolved inductively-coupled plasma mass spectrometry
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and ultrafiltration, and performed mass balance measurements to study the distribution of Ag
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in the test system. We also determined the amount of extra-and intra-cellular Ag by
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chemically etching AgNPs on the surface of algal cells and used dark field microscopy for
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their imaging. We observed that positively charged branched polyethilenimine (bPEI)-coated
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AgNPs tend to aggregate in the presence of algae and interact with test vessels and algal cells,
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while citrate–coated AgNPs have a tendency to dissolve. On the other hand, with large
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variation of half maximum effective concentration (EC50) across tested NPs (5.4 to 300 ngAg
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ml-1), Ag internalized by the algal cells at EC50 was similar (0.8 to 3.6 ngAg ml-1) for all
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AgNP types. These data shows that while sorption to the vessels, dissolution and aggregation
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impact on the distribution of AgNPs in the test system and on interactions with algal cells,
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AgNP toxicity is strongly correlated with the NP-cell surface interaction, and internalization
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of Ag.
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INTRODUCTION
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The classical approach to assessing the environmental risk of a contaminant is to expose
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relevant organisms to increasing doses of said contaminant and observe at which point the
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negative effect (toxicity) appears; then compare the effect concentration to the environmental
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concentration of the contaminant of interest.
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Neither of these two steps is easy to achieve in the case of nanoparticles (NPs). To detect and
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characterize them in the environment we face low concentrations at the limit of current
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analytical techniques, complex media and dynamic transformations1. To assess biological
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effects, we deal with complex behaviors of NPs and organisms that influence each other
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during the exposure. NPs may dissolve, sorb to surfaces, re-precipitate forming insoluble
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complexes, form homo- and hetero-agglomerates and, finally, interact with the organism to
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exert (or not) a negative effect. These processes are specific to the test conditions and NP
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characteristics including concentration, size and surface functionality. Levard et al.2
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previously reviewed in detail the impact of silver NPs (AgNP) transformations on their
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behavior and toxicity observed in various studies. To solve the problem of AgNP “aging”,
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shorter endpoints were used by Navarro et al.3, (2h photosynthetic inhibition) and Sørensen et
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al.4 (2h C-14 assimilation test). However, transformations occur rapidly, and the NP
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concentration and size distribution at the end of the test may differ from the original.
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As suggested by the OECD guidelines for environmental risk assessment of manufactured
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nanomaterials, NPs are to be characterized and their concentrations measured - throughout the
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test duration - to account for transformations and possible losses, and correctly estimate the
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exposure dose. This information is also necessary to link specific characteristics of NPs to
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observed negative effects and gain an understanding of toxicity mechanisms.5 It is well
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established that products of NP dissolution (i.e. metal ions) play an important role in
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toxicity.6,
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mechanisms of combined AgNP and ion toxicity remains a focus point for toxicologists.8-10
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Internalization and sub-cellular fate of NPs also need more research, to elucidate whether
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cellular uptake or the surface interaction is the key determinant of toxicity.11, 12
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To investigate how the transformations of AgNPs in contact with algal cells vary with
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different NPs’ sizes and surface properties, and influence their toxicity, we used a library of 9
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AgNPs of various surface functionality and size: polyethylene glycol (PEG), citrate and
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branched polyethylenimine (bPEI) coated 10, 30 and 70 nm AgNPs. We chose the OECD
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algal growth inhibition test as an established aquatic toxicology assay. Many techniques are
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currently available for characterization of nanomaterials, including high-resolution
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microscopy techniques, classic light scattering methods, and nano-specific elemental analysis.
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We relied on time-resolved inductively coupled mass spectrometry (TR-ICP-MS) to measure
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However, the negative effect of NPs per se has not been excluded and the
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NP concentration, size distribution and aggregation state in test suspensions and, for the first
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time, in the presence of organisms. TR-ICP-MS has been a groundbreaking tool for
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characterization of NPs in environmental matrices, but has been far less employed in
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toxicological studies.13 Finally, we used conventional ICP-MS measurements to obtain a
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complete mass distribution of Ag in the test systems. Using ultrafiltration we separated the
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dissolved fraction from nanoparticulate; performing adsorption test, we measured Ag present
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in test suspension and sorbed on the container; and distinguished Ag associated with the cell
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surface from internalized Ag by chemical etching. To the best of our knowledge, this etching
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protocol previously used to measure uptake of NPs into mammalian cells11, 14 has not been
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applied to study algal nano-toxicity. As a complementary technique, we used dark field
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microscopy to image AgNPs with different surface coatings associated with cells, before and
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after chemical etching.
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The primary objectives of this study were: 1) to assess the dissolution, aggregation of AgNPs
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and their sorption to the container walls in presence of algal cells; 2) to investigate
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interactions between cells and AgNPs and distinguish surface interactions from cell
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internalization; 3) to interpret the results of algal growth inhibition assay, considering the
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measured concentrations, transformations that had occurred, NP-cell interactions and uptake.
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EXPERIMENTAL SECTION
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AgNPs of 10, 30 and 70 nm nominal diameter, and different surface functionality (PEG-,
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bPEI- and citrate-coated AgNPs) were purchased from Nanocomposix and characterized in
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house by TR-ICP-MS (Agilent 8800) and DLS with ζ-potential measurements (Nicomp 380
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ZLS) for size and surface charge. Detailed methods and results are reported in the SI (Table
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S1).
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Algal growth inhibition test. Algal cultures of green freshwater algae Pseudokirchiniella
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subcapitata were obtained from CSIRO Algal supply service; cultures were maintained in
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stable growing conditions, and re-cultured weekly in fresh OECD media (composition in SI).
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The algal density was monitored daily by fluorescence measurements (Figure S1), and
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exponentially growing algae used as test inoculums. The standard OECD method for algal
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growth inhibition test was adapted to use 12-well microplates, following the protocol
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described by Paixão et al.15 with few modifications. The AgNPs exposure concentrations
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derived from preliminary “range finding” experiments (data not shown) were from 1.56 to
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400 ngAg ml-1 for 70 nm AgNPs and from 0.8 to 200 ngAg ml-1 for 10 and 30 nm AgNPs. To
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avoid the loss of AgNPs during dilution steps, suspensions containing the highest test
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concentrations were transferred onto the plates, and serial dilutions performed directly on the
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plates (the final volume was 2 ml in each well). Subsequently, each well was inoculated with
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0.2 ml of algal culture to achieve an algal density of approximately 104 cells ml-1. Each plate
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contained nine NP concentrations, a blank with OECD media and two control wells with
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algae but no Ag (SI Figure S2). The plates were incubated for 48 h under conditions identical
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to the stock cultures and OECD validity criteria were met for all tests (see SI). The
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experiment was repeated under identical conditions four times for citrate- and bPEI- coated
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NPs, and three times for PEG-coated NPs. Each repeated experiment consisted of a complete
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series of coatings and sizes and included two replicates of each tested AgNPs and one
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replicate of AgNO3 in the range of 0.67-10 ngAg ml-1 as a positive control. The fluorescence
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measurements were made at 0, 24 and 48 h with a fluorescence spectrophotometer plate
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reader (SynergyTM HT, Bio-Tek), using 430 nm/670 nm excitation and emission wavelengths,
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respectively.
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Mass balance and cell surface etching. Algal cells were exposed for 48 h to the library of
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nine AgNPs at concentrations equivalent to the respective EC50 values (Table 1) under
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OECD test conditions. In order to obtain enough material for a comprehensive mass balance,
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each test suspension of AgNPs and algal cells was produced in multiple replicates. After 48 h
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incubation, samples were collected to perform tests and measurements as summarized in
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Figure 1 and described below in detail.
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Figure 1. Schematic of the experimental work-flow. Alphabetically labelled are the samples
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collected at different stages to determine as follows: a) Concentration of Ag still in suspension
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after 48 h; b) Concentration of Ag sorbed on the surface of the test vessel; c) Concentration of
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Ag in suspension, non-associated with cells; d) Concentration of Ag in suspension, present in
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dissolved form (
