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Impacts of silver nanoparticles on a natural estuarine plankton community Mafalda Saraiva Baptista, Robert J. Miller, Elisa R Halewood, Shannon Kareem Hanna, C. Marisa R. Almeida, Vitor M. Vasconcelos, Arturo A. Keller, and Hunter S Lenihan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03285 • Publication Date (Web): 07 Oct 2015 Downloaded from http://pubs.acs.org on October 8, 2015
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Environmental Science & Technology
Impacts of silver nanoparticles on a natural estuarine plankton community
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Mafalda S. Baptista,*,† Robert J. Miller, § Elisa R. Halewood, § Shannon K. Hanna, ǂ C. Marisa
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R. Almeida, † Vitor M. Vasconcelos, †,‡ Arturo A. Keller, ǂ Hunter S. Lenihanǂ
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†
CIIMAR/CIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade
do Porto, Porto, Portugal §
ǂ
Marine Science Institute, University of California, Santa Barbara, California, USA
Bren School of Environmental Science and Management, University of California, Santa
Barbara, California, USA ‡
Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal
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* Corresponding author:
[email protected] 14
CIIMAR/CIMAR - Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos
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Bragas 289, 4050-123 Porto, Portugal
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Phone: +351 220402579; fax: +351 220402659
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Abstract
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Potential effects of metal nanoparticles on aquatic organisms and food webs are hard to predict
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from results of single-species tests under controlled laboratory conditions, and more realistic
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exposure experiments are rarely conducted. We tested whether silver nanoparticles (Ag NPs)
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had impact on zooplankton grazing on their prey, specifically phytoplankton and
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bacterioplankton populations. If Ag NPs directly reduced the abundance of preys, thereby
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causing the overall rate of grazing of their predators to decrease, a cascading effect on a
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planktonic estuarine food web would be seen. Our results show that the growth rates of both
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phytoplankton and bacterioplankton populations were significantly reduced by Ag NPs at
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concentrations ≥ 500 µg L-1. At the same time, grazing rates on these populations tended to
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decline with exposure to Ag NPs. Therefore, Ag NPs did not cause a cascade of effects through
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the food web but impacted a specific trophic level. Photosynthetic efficiency of the
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phytoplankton was significantly reduced at Ag NPs concentrations ≥ 500 µg L-1. These effects
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did not occur at relatively low concentrations of Ag that are often toxic to single species of
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bacteria and other organisms, suggesting that impacts of Ag NP exposure may not be apparent
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at environmentally relevant concentrations due to compensatory processes at the community
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level.
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Keywords: Bacterioplankton, chlorophyll fluorescence, dilution method, ecotoxicity, metallic
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nanoparticles, phytoplankton
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Environmental Science & Technology
INTRODUCTION
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The impact of nanoparticles (NPs) on natural ecosystems is a growing concern in environmental
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science and management.1 Silver nanoparticles (Ag NPs) are one of the most widely used NPs
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in consumer and industrial products, mainly because of their anti-bacterial properties, with
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applications in cosmetics and fabrics,2 and medicine and hygiene.3 Nanomaterials discharged
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into the environment find their way through waste disposal and other routes, and ultimately into
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estuaries and nearshore marine environments.4 Predicted environmental concentrations (PEC) in
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surface waters are in the range of ng L-1 (part per trillion) but are expected to continue
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increasing.5 The PEC of Ag NPs in surface waters is higher than other metal oxide
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nanomaterials including ZnO, TiO2, fullerenes or carbon nanotubes, and may pose risks to
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aquatic organisms.5 Our knowledge of nanomaterial toxicity largely comes from in vitro or
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single-species assays that may not reflect responses of natural ecosystems and associated food
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webs. Impacts of anthropogenic contaminants on aquatic food webs can cause complex
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ecological effects: grazers or predators can be reduced through toxicity, thereby releasing their
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prey from predation and increasing the prey’s abundance. If the prey also acts as predators on
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smaller species, then increasing the numbers of these midlevel predators can cause decreases in
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the smaller prey species. This combination of direct and indirect interactions, in a food web with
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three levels or more, is called a trophic cascade. At the same time, toxicants can directly affect
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prey populations. Pollutants can cause trophic cascades6 but such cascades have not been
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observed for nanomaterials. Research to examine the direct and indirect ecological effects of
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nanomaterials in aquatic ecosystems is important to develop a more realistic understanding of
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the environmental implications of nanotechnology.7
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In vitro toxic effects of Ag NPs have been documented for organisms in practically every
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major taxonomic group, including mammals,8 insects,9 snails,10 plants,11 algae,12 and bacteria.13
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In the aquatic environment, Ag NPs negatively affect prokaryotes, invertebrates, and fish.14,15
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Toxicity mechanisms have not yet been well defined for most NPs including Ag, but can
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include destabilization of outer-membrane integrity,16 disruption of membrane potential,13
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cytotoxicity,17 genotoxicity,18 interruption of energy transduction,19 and formation of reactive
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oxygen species.20 Due to their small size, NPs can penetrate biological systems in novel ways.21
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Aggregated nanoparticles of varying sizes are expected to result from discharges of NPs into
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natural waters and may mitigate some such effects.22 However, even aggregated particles are
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likely to eventually dissolve, releasing toxic Ag+ ions.23,24
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Plankton is a polyphyletic group of aquatic organisms, including nearly 25,000
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morphologically defined species distributed among eight major divisions or phyla.25 In oceans
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and estuaries, phytoplankton and bacterioplankton provide many ecological services, including
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especially biogeochemical cycling of carbon, nutrients, and trace metals.26 Coastal marine
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phytoplankton are major primary producers,27 and changes to the structure of these communities
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has potential to impact higher trophic levels including fisheries species.28 Plankton communities
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are highly dynamic, and their productivity is influenced by nutrient availability, water quality
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(including toxicants), and grazing, defined as consumption of phytoplankton and
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bacterioplankton cells by other heterotrophic (or mixotrophic) plankton species. Protozoan or
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zooplankton grazers can regulate the trophic transfer of energy from planktonic production to
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higher trophic levels, as smaller grazers are consumed by larger organisms.
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NPs have been shown to depress phytoplankton population growth rates.29-31 However, NPs may also impact grazers such as relatively large copepods (>500 µm),32 and protozoans (5-15
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µm),33 potentially changing the outcome of toxicity across the community. In this study, we
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tested whether and how Ag NPs affect a community composed of zooplankton predators and
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bacterioplankton and phytoplankton prey in a shallow estuarine lagoon. We predicted that Ag
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NPs would be toxic phytoplankton and bacterioplankton, reducing their abundance and the
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overall rate of grazing of their predators, specifically zooplankton, triggering a trophic cascade.
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Our alternative hypothesis was that Ag NPs would not cause a cascading effect but instead
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impact specific trophic levels. To evaluate these effects, we conducted dilution experiments34 to
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simultaneously estimate phytoplankton and bacterioplankton growth rates and zooplankton
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grazing rates, and the effect of exposure to Ag NPs on these critical ecological processes.
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MATERIALS AND METHODS
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Study site and sampling
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The study was conducted at an estuarine lagoon on the University of California Santa Barbara
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campus. The shallow lagoon (~2 m depth) covers 0.125 km2. Salt water is pumped into the
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lagoon from the ocean and freshwater is input from runoff.35 Water samples were collected in
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June 2011, when the average air temperature was 16 ºC and salinity of the lagoon was 33 ‰.
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Dilution experiments
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Grazing rates were assessed in serial dilution experiments.34 Samples were collected from a
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depth of 0.5 m, using 20 L carboys. A portion of the water was filtered through 0.45 μm
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nitrocellulose filters (Millipore). Dilutions were established using 100%, 75%, 50%, and 25%
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lagoon water diluted with 0.45 μm filtered lagoon water, and were executed in triplicate, in 0.5
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L polycarbonate bottles. The carboys and bottles were soaked overnight in a 5% (v/v) HCl bath
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and rinsed with deionized water filtered with a 30 Da filter (nanopure water) prior to use.
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Nutrient (phosphate and nitrogen) concentrations were measured spectrophotometrically, in
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duplicate, in 50 mL of water filtered by 0.45 μm nitrocellulose filters (Millipore), and were in
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the millimolar range (PO43- = 969 ± 2, NOx = 671 ± 13, NH4+ = 292 ± 70 mmol L-1); therefore
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nutrients were not supplemented. All the bottles were incubated for 24 h, partially submerged in
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an outside holding tank plumbed with circulating lagoon water to maintain natural temperature
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and light levels.
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Exposure to Ag NPs
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To assess the effect of Ag NPs to the lagoon plankton community, triplicate bottles were
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exposed to 0.1, 0.5 and 1 mg L-1 Ag NPs. The same serial dilution experiments described above
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were employed for the bottles with Ag NPs. Concentrations were chosen based on previous
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experiments with aquatic organisms.15
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Ag NPs > 99.9% trace metal basis were obtained from Sigma-Aldrich, and were
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characterized by the University of California Center for Environmental Implications of
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Nanotechnology (UC CEIN). The Ag NPs were semispherical, and averaged 57 ± 20 nm in
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primary size as measured by TEM. Hydrodynamic diameter was 143 ± 9 nm (DLS). Purity was
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measured as 100% (XRD). Zeta potential ζ was -44.7 ± 1.6 nV, and electrophoretic mobility
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(EPM) was -3.36 ± 0.12 10-8 m2 V-1 s-1 (ZetaPALS in DI water). To produce 100 mg L-1 stock
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dispersions, 10 mg of Ag NPs were added to 1 mL of deionized water, sonicated for 30 min,
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vortexed for 30 seconds, diluted into filtered (0.2 µm Millipore) natural seawater containing 10
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mg L-1 of alginate, and again vortexed for 30 seconds.
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Community composition
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Water samples were preserved with 2.5% paraformaldehyde,36 frozen at -80 ºC for 24 h, and
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analyzed using a BD™ LSR II flow cytometer (Biosciences, USA) equipped with a 488-nm
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excitation laser and standard filter set. For counts of autofluorescent cells the samples were
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diluted 1:5 with nanopure water, and analyzed with a flow rate of 2 µL s-1 (total volume 200
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µL). For counts of total cells, the samples were diluted 1:100 with nanopure water, incubated
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for 20 minutes with 1% SYBR Green I stain (Sigma-Aldrich), and analyzed with a flow rate of
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0.5 µL s-1 (total volume of 45 µL). Dilutions were chosen36 in order to keep the counting rate
