Influence of liberated silver from silver nanoparticles on nitrification inhibition of Nitrosomonas europaea Tyler S. Radniecki a,⇑ , Dylan P. Stankus a , Arianne Neigh b , Jeffrey A. Nason a , Lewis Semprini a a School of Chemical, Biological and Environmental Engineering, 102 Gleeson Hall, Oregon State University, Corvallis, OR 97331, United States b nanoComposix, Inc., 4878 Ronson Court, STE K, San Diego, CA 92111, United States article info Article history: Received 22 February 2011 Received in revised form 24 May 2011 Accepted 5 June 2011 Available online 14 July 2011 Keywords: Nitrosomonas europaea Silver nanoparticles Silver dissolution Nitrification inhibition abstract The ecotoxicity of silver nanoparticles (Ag-NPs) to wastewater biota, including ammonia oxidizing bac- teria (AOB), is gaining increasing interest as the number of products containing Ag-NPs continues to rise exponentially and they are expected to accumulate in wastewater treatment plants. This research dem- onstrated that the addition order of Ag-NP and the media constituents had a profound influence on the stability of the Ag-NP suspension and the corresponding repeatability of results and sensitivity of Nitros- omonas europaea. N. europaea, a model AOB, was found to be extremely sensitive to ionic silver (Ag + ) and two sizes of Ag-NPs (20 and 80 nm). Ag + exposures resulted in the highest level of toxicity with smaller Ag-NPs (20 nm) being more toxic than larger Ag-NPs (80 nm). The increased sensitivity of N. europaea to smaller Ag-NPs was caused by their higher rates of dissolved silver (dAg) release, via dissolution, due to a greater surface area to volume ratio. dAg was shown to be responsible for the vast majority of the observed Ag-NP toxicity, as determined by abiotic Ag-NP dissolution tests. For the sizes of Ag-NP studied (20 and 80 nm), there appears to be a negligible nanoparticle-specific toxicity. This was further supported by similarities in inhibition mechanisms between Ag + and Ag-NP, with both causing decreases in AMO activity and destabilization of the outer-membrane of N. europaea. Finally, equal concentrations of total silver were found to be tightly associated to both Ag + and Ag-NP-exposed cells despite Ag-NP concentra- tions being five times greater, by mass, than Ag + concentrations. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Silver has been used since times of antiquity as a broad-spec- trum antimicrobial agent in a wide range of applications, particu- larly in the medical field (e.g. bandages and medical tubing) (Klasen, 2000; Simon, 2003). Recent advances in nanotechnology have enhanced the use of silver even further through the creation of silver nanoparticles (Ag-NPs), particles of elemental silver with a diameter of 100 nm or less. Ag-NPs have antimicrobial properties proposed to be due to the release of dissolved silver (dAg), which may be in the form of ionic silver (Ag + ) and other silver species (e.g. AgCl 2 or AgðNH 3 Þ þ 2 ) depending on the aquatic chemistry of the solution, and possibly due to the unique properties of the nano- particles themselves (e.g. being a catalyst for reactive oxygen spe- cies generation) (Kim et al., 2007; Marambio-Jones and Hoek, 2010). Because of their anti-microbial properties, Ag-NPs are the most widely used engineered nanomaterials in consumer products (259 out of 483 nanoproducts surveyed in 2009) and are used in a wide variety of materials including nanotextiles, food containers, clothes washers and dishwashers (Trusts, 2009; Wijnhoven et al., 2009). The use of these products can lead to the direct release (e.g. re- lease from clothes and dishwashers) or indirect release (e.g. leach- ing from nanotextiles and food containers) of dAg and/or Ag-NP into commercial and residential wastewater and wastewater treat- ment plants (WWTPs) (Benn and Westerhoff, 2008; Blaser et al., 2008; Geranio et al., 2009). Ag + have been reported to accumulate in WWTP biosolids going from influent concentrations ranging from 4 ppb to 1 ppm to WWTP biosolids concentrations ranging from 2 to 195 ppm (Schildkraut et al., 1998; Shafer et al., 1998; USEPA, 2009). Batch adsorption experiments have demonstrated that Ag-NP would also likely accumulate in WWTP biosolids in a manner similar to that of Ag + (Benn and Westerhoff, 2008). With the use of Ag-NP in consumer products continuously on the rise, the accumulation of dAg and/or Ag-NP in WWTP may lead to the disruption of important biological processes within the WWTP, including nitrogen removal (Mueller and Nowack, 2008; Kim et al., 2010). The removal of nitrogen from wastewater is a two-step process in which ammonia (NH 3 ), the most common form of nitrogen in a WWTP, is aerobically oxidized to nitrate (NO  3 ), via ammonia oxi- dizing bacteria (AOB) and nitrite oxidizing bacteria, before being anaerobically reduced to nitrogen gas, via denitrifying bacteria. 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.06.039 ⇑ Corresponding author. Tel.: +1 541 737 6895; fax: +1 541 737 3052. E-mail addresses: tyler.radniecki@oregonstate.edu (T.S. Radniecki), stankusd@ onid.orst.edu (D.P. Stankus), neigh@nanocomposix.com (A. Neigh), jeff.nason@ oregonstate.edu (J.A. Nason), lewis.semprini@oregonstate.edu (L. Semprini). Chemosphere 85 (2011) 43–49 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere