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Chronic exposure effects of silver nanoparticles on stream microbial decomposer communities and ecosystem functions Ahmed Tlili, Jérémy Jabiol, Renata Behra, Carmen Gil-Allué, and Mark O Gessner Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05508 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017
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Chronic exposure effects of silver nanoparticles on
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stream microbial decomposer communities and
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ecosystem functions
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Ahmed Tlili,*,†,‡ Jérémy Jabiol,†,# Renata Behra,‡ Carmen Gil-Allué,‡ and Mark O. Gessner†,§
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† Department of Experimental Limnology, Leibniz Institute of Freshwater Ecology and Inland
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Fisheries (IGB), Stechlin, Germany.
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‡ Department of Environmental Toxicology, Eawag: Swiss Federal Institute of Aquatic Science and
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Technology, Dübendorf, Switzerland.
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§Berlin Institute of Technology (TU Berlin), Department of Ecology, Berlin, Germany
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KEYWORDS: fungi, bacteria, leaf-litter decomposition, engineered nanoparticles, silver toxicity.
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ABSTRACT. With the accelerated use of silver nanoparticles (AgNP) in commercial products,
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streams will increasingly serve as recipients of, and repositories for, AgNP. This raises concerns about
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the potential toxicity of these nanomaterials in the environment. Here we aimed to assess the impacts
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of chronic AgNP exposure on the metabolic activities and community structure of fungal and bacterial
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plant litter decomposers as central players in stream ecosystems. Minimal variation in the size and
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surface charge of AgNP indicated that nanoparticles were rather stable during the experiment. Five
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days of exposure to 0.05 and 0.5 µM AgNP in microcosms shifted bacterial community structure but
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had no effect on a suite of microbial metabolic activities, despite silver accumulation in the
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decomposing leaf litter. After 25 days, however, a broad range of microbial endpoints, as well as rates 1
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of litter decomposition, were strongly affected. Declines matched with the total silver concentration in
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the leaves and were accompanied by changes in fungal and bacterial community structure. These
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results highlight a distinct sensitivity of litter-associated microbial communities in streams to chronic
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AgNP exposure, with effects on both microbial functions and community structure resulting in notable
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ecosystem consequences through impacts on litter decomposition and further biogeochemical
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processes.
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INTRODUCTION
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The rapidly accelerating production and widespread use of silver nanoparticles (AgNP) increases
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the likelihood of nanoparticle release into fresh waters and raises concern about potential toxicity in
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natural environments.1 Microorganisms and the processes they drive are prime candidates for being
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adversely affected, because AgNP are deliberately designed and applied to exert antimicrobial
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action.2,3 Not surprisingly, toxic effects of AgNP on a range of freshwater organisms have been
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reported.4-6 However, as is often the case in ecotoxicology, information about nanoparticle effects
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typically relies on simplistic laboratory tests in highly standardized settings,7,8 whereas assessments on
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levels of ecological organization above the individual are particularly scarce.7 Furthermore, with few
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exceptions,9-13 studies on AgNP effects have focused on short-term exposure scenarios, without
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considering effects in the long term that involve shifts in community structure. Consequently,
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approaches to evaluate risks posed by AgNP need to consider the complex biodiversity, functions and
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long-term response dynamics in ecosystems.2,7,14
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Being composed of many species performing multiple functions and displaying different
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sensitivities to stressors, microbial communities reflect much of the biological variability and
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complexity characterizing natural communities and ecosystems.2,15 Heterotrophic microbial
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communities composed of fungi and bacteria play key roles in fresh waters by colonizing and
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decomposing plant litter derived from terrestrial vegetation, a key resource in forest streams and other
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shallow aquatic ecosystems.16 These microorganisms degrade leaf litter enzymatically and also
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promote decomposition indirectly by making leaves more attractive to detritivorous 2
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macroinvertebrates.17 This dual role18,19 and the high microbial diversity comprising contrasting life-
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styles (e.g. filamentous fungi and single-celled bacteria)19 and sensitivities to contaminants20 suggest
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that the microbes associated with decomposing leaf litter in streams are a suitable model to assess
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contaminant effects on complex ecological systems.7,21 Although ionic metals, including Ag+, have
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been repeatedly reported to affect litter-associated microbial communities and decomposition in
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streams,22,23 and acute effects of AgNP at elevated concentrations have also been observed,22 the
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potential impacts of chronic exposure to AgNP remain poorly known.12 This is an important gap given
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that disruption of decomposer community structure and activities by chronic exposure to AgNP could
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have large ecosystem consequences, including on food webs, nutrient cycling and whole-ecosystem
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metabolism.
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The goal of this study was to determine impacts of AgNP on stream microbial communities and
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metabolic activities and to assess the consequences of these effects at the ecosystem level. We
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hypothesized that exposure to AgNP long enough to induce shifts in the community structure of fungi
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and bacteria (i.e. long-term or chronic exposure) also affects microbial functional capacities and
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ultimately litter decomposition as a central process driving organic matter dynamics in streams. Thus,
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our specific questions were: (i) do AgNP affect microbial communities structure, including the relative
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and absolute importance of fungi and bacteria in decomposing leaf litter; (ii) what are the
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consequences of chronic exposure to AgNP on fungal and bacterial metabolic activities; and (iii) do
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any effects of AgNP on microbial community structure or function have consequences for litter
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decomposition? To answer these questions, we selected a suite of functional and structural endpoints
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specifically targeting fungi, bacteria or both groups of microorganisms growing on naturally colonized
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leaf litter in stream microcosms.
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MATERIAL AND METHODS
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Materials
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Citrate-coated AgNP were purchased from NanoSys GmbH (Wolfhalden, Switzerland) as an
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aqueous suspension with a nominal silver concentration of 1 g L-1 or 9.27 mM (size 25±13nm; zeta
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potential -36.6±3.2 mV in nanopure water). All other chemicals were purchased from Sigma-Aldrich
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(Seelze, Germany).
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Populus nigra (L.) leaves were collected just after abscission, air-dried and stored before cutting
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batches of thirty 14-mm leaf discs, which were subsequently enclosed in fine (0.5-mm mesh size)
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nylon mesh bags, 10x10 cm in size. A total of 100 litter bags were submerged in a first-order
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hardwater stream located in the north-eastern lowlands of Germany (53°06’46” N, 13°08’43” E). All
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litter bags were retrieved after four weeks, placed into a cool box with stream water and returned to
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the laboratory.
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Experimental design
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The microcosms used in the study consisted of cubic polypropylene vessels with an edge length of
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17 cm. They were filled with 300 mL of stream water passed through a 0.5-mm screen and diluted
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with deionized water at a ratio of 1:3 to ensure AgNP stability. To ensure identical physicochemical
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characteristics of the water throughout the study, the diluted stream water was stored at -20°C until
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use. Colonized leaf discs from the litter bags were pooled, rinsed with chlorine-free tap water and 40
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of them were randomly allocated to each microcosm. Exposure to AgNP and AgNO3 started 24 hours
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after distributing the discs to allow prior acclimation of the microbial communities.
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The experimental design involved three levels of AgNP addition (0, 0.05, and 0.5 µM) and one level
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of AgNO3 addition (0.5 µM) as a positive control for Ag+. All treatments were replicated five times.
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The tested AgNP concentrations corresponded to EC10 or EC20 of various functional endpoints
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determined in short-term AgNP exposure experiments22. Microcosms were incubated at 15 °C with
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shaking and a 12 h/12 h light/dark cycle. The water was renewed every five days (including AgNO3 or
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AgNP). Leaf litter and the associated microbial communities were sampled immediately and after 5
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and 25 days. Water samples were taken simultaneously to determine dissolved nutrient concentrations
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and other physico-chemical conditions in the microcosms (Table S1).
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Nanoparticle characterization and silver quantification
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The particle diameter of suspended AgNP was measured by nanoparticle tracking analysis (NTA)
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using a NanoSight LM10 (NanoSight Ltd., Wiltshire, UK). The surface charge (ζ-potential) was
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measured by dynamic light scattering (DLS) using a Zetasizer (Nano ZS, Malvern Instruments Ltd.,
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Worcestershire, UK). Suspensions of 0.05 and 0.5 µM AgNP were characterized both in fresh water
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before filling the microcosms and in conditioned water from the same microcosms 5 days later.
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Conditioned water for these analyses was collected twice from the control microcosms, the first time
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before first water renewal after 5 days and the second time at the very end of the experiment, 5 days
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after the last water renewal. This sampling was intended to account for effects of any substances
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released by the decomposing leaves or microbial communities developing during the study. AgNP
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were added to the collected water and analyzed immediately (i.e. within 15 min) for particle size, zeta
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potential and silver dissolution and then again after 5 days.
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The total Ag concentration in the AgNP suspensions and sets of three leaf discs that had been pre-
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rinsed with deionized water were measured by ICP-MS (Element 2 High Resolution Sector Field ICP-
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MS; Thermo Finnigan, Bremen, Germany) after digestion with 3 mL 65% HNO3 and 1 mL of H2O2 in
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a high-performance microwave digestion unit (MLS 1200 mega, MLS GmbH, Leutkirch, Germany;
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maximum temperature of 195 °C). To differentiate between total Ag and the fraction strongly
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associated with leaf litter, or taken up by organisms, a washing procedure was applied on sets of three
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leaf discs, which have been pre-rinsed with deionized water, and involved immersion for 5 min in 0.5
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mM cysteine as a strong chelating ligand that complexes Ag+,24 followed by three washing cycles in
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deionized water.
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Concentrations of dissolved Ag(I), which includes dissolved Ag+ and other dissolved oxidized silver species such as AgCln (aq) and AgOH (aq), were measured in AgNP suspensions by ICP-MS after
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centrifugal ultrafiltration (30 min at 3220 g) using Amicon Ultra-15 centrifugal filter units (Merck
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Millipore, Darmstadt, Germany; molecular cut-off of 3 kDa, corresponding to an estimated pore size
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