Article pubs.acs.org/est
Changes in Bacterial Community Structure after Exposure to Silver Nanoparticles in Natural Waters Pranab Das,† Clayton J. Williams,‡ Roberta R. Fulthorpe,# Md Ehsanul Hoque,§ Chris D. Metcalfe,§ and Marguerite A. Xenopoulos*,‡ †
Environmental and Life Sciences Graduate Program, Trent University, Peterborough, ON, Canada Department of Biology, Trent University, Peterborough, ON, Canada # Department of Physical and Environmental Sciences, University of Toronto, ON, Canada § Water Quality Centre, Trent University, Peterborough, ON, Canada ‡
S Supporting Information *
ABSTRACT: Silver nanoparticles (AgNPs) are widely used in commercial products as antibacterial agents, but AgNPs might be hazardous to the environment and natural aquatic bacterial communities. Our recent research demonstrated that AgNPs rapidly but temporarily inhibited natural bacterioplankton production. The current study investigates the mechanism for the observed bacterial reaction to AgNPs by examining how AgNPs impact bacterial abundance, metabolic activity (5-cyano-2,3-ditolyl tetrazolium chloride (CTC+) cells), and 16S rRNA community composition. Natural bacterioplankton communities were dosed with carboxy-functionalized AgNPs at four concentrations (0.01−1 mg-Ag/L), incubated in triplicate, and monitored over 5 days. Ionic silver (AgNO3) and Milli-Q water treatments were used as a positive and negative control, respectively. Four general AgNP exposure responses, relative to the negative control, were observed: (1) intolerant, (2) impacted but recovering, (3) tolerant, and (4) stimulated phylotypes. Relationships between cell activity indicators and bacterial phylotypes, suggested that tolerant and recovering bacteria contributed the most to the community’s productivity and rare bacteria phylotypes stimulated by AgNPs did not appear to contribute much to cell activity. Overall, natural bacterial communities tolerated single, low level AgNP doses and had similar activity levels to the negative control within five days of exposure, but bacterial community composition was different from that of the control.
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INTRODUCTION Silver nanoparticles (AgNPs), due to their broad-spectrum antimicrobial, antifungal, and antiviral properties, are currently the largest (∼24%) and fastest growing segment of the commercial market for engineered nanomaterials.1 AgNPs are used widely in a variety of domestic, medical, textile, industrial and environmental products.2−4 Given their broad use, AgNPs have the potential to be released directly or through discharges of municipal wastewater into the aquatic environment.5,6 Once AgNPs are released into the environment, it is logical to suspect that aquatic bacteria will be negatively affected due to AgNPs antimicrobial properties. Disruption of natural bacterial communities and their activities by nanoparticles could have widespread consequences for nutrient cycles, aquatic metabolism, and ecosystem health.7 Recently, we reported that natural bacterioplankton activity was negatively affected by acute exposures to AgNPs (0.01−2.0 mg-Ag/L), but in the treatments at low AgNP concentrations (0.01−0.02 mg-Ag/ L), bacterial production returned to or exceeded control rates within 48 h of exposure.8 These production changes might indicate a shift in the bacterial community structure toward silver-tolerant species. For example, AgNP exposure (0.02−2.0 © 2012 American Chemical Society
mg/L) negatively impacted marine microbial biofilm development and bacterial species composition.9 In another study, AgNPs (0.025−1.0 mg/L) had negligible impacts on bacterial community diversity.10 The effects of AgNP exposure to aquatic bacterioplankton diversity, however, remain to be studied. In our previous study,8 bacterial production could have recovered through gains in the most active/respiring members of the bacterial community or through recruitment of formerly metabolically inactive bacteria, with the latter comprising an important fraction of natural bacterioplankton communities.11−16 Hence, AgNP exposure might impact the active bacterial pool and trigger recolonization from this larger, low to inactive bacterial community. On the other hand, the active members of the bacterioplankton may only suffer short-term toxicity from AgNPs and recover to pre-exposure levels, with or without any concomitant changes in actual community structure. Received: Revised: Accepted: Published: 9120
May 17, 2012 July 20, 2012 July 26, 2012 July 26, 2012 dx.doi.org/10.1021/es3019918 | Environ. Sci. Technol. 2012, 46, 9120−9128
Environmental Science & Technology
Article
In the present study, we used molecular fingerprinting techniques to determine the influence of AgNPs on bacterial community structure. We used two methods: the semiquantitative denaturing gradient gel electrophoresis (DGGE)17 to determine if AgNPs treated actually changed the composition of the bacterial community rather than just its metabolic activity. We then used terminal restriction fragment length polymorphism (T-RFLP)18 analyses to examine community composition changes in more detail. We also examined changes to cell specific bacterial production, bacterial respiration chain activity, and bacterial abundance. Water samples collected from a stream and stormwater management pond (SWMP) were used in bench scale experiments to determine the natural bacterial community responses to exposure over 5 days to AgNPs (0.01−1 mg-Ag/L) and AgNO3 (0.01 mg-Ag/L). We hypothesize that AgNPs hinder the growth rate of dominant bacterioplankton enough to alter bacterioplankton community compositions toward silver tolerant strains over time.
diameter,8,20 which is consistent with the manufacturer’s description. A working suspension of 30 mg-Ag/L of the stock concentration was prepared by diluting (1:10) AgNPs stock suspension in Milli-Q water. In addition, a working solution of Ag ions (10 mg-Ag/L) was prepared by dissolving AgNO3 crystals (Fisher Scientific) in Milli-Q water. Experimental AgNP exposure incubations were carried out in triplicate for each site at four AgNP concentration treatments along with positive (Ag+ addition) and negative control (Milli-Q water, no AgNPs or Ag+ addition) treatments (n = 18 per site). For each treatment replicate, 3.2 L of
