Natural Organic Matter Alters Biofilm Tolerance to Silver Nanoparticles and Dissolved Silver Stacy M. Wirth, † Gregory V. Lowry, †,‡ and Robert D. Tilton* ,†,§ † Center for the Environmental Implications of Nanotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ‡ Department of Civil and Environmental Engineering, and § Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States * S Supporting Information ABSTRACT: Motivated by the need to understand environ- mental risks posed by potentially biocidal engineered nano- particles, the effects of silver nanoparticle (AgNP) exposure on viability in single species Pseudomonas fluorescens biofilms were determined via dye staining methods. AgNP dispersions, containing both particles and dissolved silver originating from the particles, negatively impacted biofilm viability in a dose- dependent manner. No silver treatments (up to 100 ppm AgNPs) resulted in 100% biofilm viability loss, even though these same concentrations caused complete viability loss in planktonic culture, suggesting some biofilm tolerance to AgNP toxicity. Colloidally stable AgNP suspensions exhibited greater toxicity to biofilms than corresponding particle-free supernatants containing only dissolved silver released from the particles. This distinct nanoparticle-specific toxicity was not observed for less stable, highly aggregated particles, suggesting that biofilms were protected against nanoparticle aggregate toxicity. In both the stable and highly aggregated dispersions, dissolved silver made a significant contribution to overall toxicity. Therefore, despite increased colloidal stability when humic acid adsorbed to AgNPs, the presence of humic acid mitigated the toxicity of AgNP suspensions because it bound to silver ions in solution. ■ INTRODUCTION The growing use of silver nanoparticles (AgNPs) in commercial and consumer products makes their release to waste streams and the environment inevitable. This has raised concern, since the broad spectrum biocidal properties that motivate AgNP use in consumer and medical applications could have detrimental consequences for environmental micro-organisms that are crucial to normal ecosystem function. 1 Microbial toxicity of AgNPs and Ag + is well documented for laboratory-grown planktonic bacterial cultures, where cells are suspended in nutrient-rich culture media. 2-9 In most natural environments, however, surface attached biofilms, adherent bacterial communities surrounded by a self-produced matrix of extracellular polymeric substance (EPS), are the prevailing microbial community structure. 10,11 Biofilms can be up to 1000 times more resistant to toxicants than their planktonic counterparts, 12 and data obtained from planktonic cultures cannot be used to predict antibiotic effects in biofilms. 10 Additionally, biofilms may be sinks for nanoparticles and their aggregates as they sediment in the environment. 13,14 Thus, studies of nanoparticle effects on biofilms are necessary in order to predict potential environmental consequences of nano- particle release, though few studies have addressed this topic. 15-21 Another critical feature when considering nanoparticles released into the environment is the presence of naturally occurring macromolecules that can adsorb to their surfaces. Humic acids, which comprise a major fraction of the ubiquitous natural organic matter (NOM) in most environments, are likely to interact with AgNPs. Increased colloidal stability following humic acid adsorption has been demonstrated for several nanoparticles, 22-26 including silver. 27,28 Therefore, the compo- nent of interest for environmental relevance is not simply the nanoparticle itself but the nanoparticle-macromolecule com- plex that forms in multicomponent environmental media. The adsorbed layer will affect colloidal stability and adherence to soil surfaces. 29,30 This in turn will affect nanoparticle mobility in the environment, likely including transport in the biofilm matrix, and affect subsequent interactions with other environ- mental colloids, biomolecules, and cells, with probable implications for toxicity. 31 Additionally, molecules that chelate silver ion may decrease its toxicity, 32,33 so NOM may indirectly Received: April 17, 2012 Revised: October 19, 2012 Accepted: October 30, 2012 Published: October 30, 2012 Article pubs.acs.org/est © 2012 American Chemical Society 12687 dx.doi.org/10.1021/es301521p | Environ. Sci. Technol. 2012, 46, 12687-12696