(145d) Silver Nanoparticle Inhibition of the Ammonia Oxidizing Bacterium, Nitrosomonas Europaea: Influence of Aquatic Chemistry On Bioavailability and Exposure

The use of silver nanoparticles (Ag-NP) as a broad spectrum biocide in a wide range of consumer goods has grown exponentially since 2006 (1), which may result in an increased release of Ag-NP into wastewater streams and ultimately the receiving bodies of water.  Ammonia oxidizing bacteria (AOB) play a critical role in the removal of nitrogen during wastewater treatment through the oxidation of ammonia (NH₃) to nitrite (NO₂^-) and  are widely considered to be the most sensitive fauna in wastewater treatment plants (WWTP) (2) being readily inhibited by many wastewater contaminants, including Ag-NP (3).  This research used physiological and genomic techniques in combination with physical/chemical assays to characterize the inhibition of Nitrosomonas europaea (N. europaea), the model AOB, by silver ions (Ag⁺), 20 nm Ag-NP and 80 nm Ag-NP under a variety of aqueous chemistries.  In addition, the stability of Ag-NP suspensions was examined under a variety of aqueous chemistries including various pH values, ionic strengths and NH₃ concentrations. 

Batch experiments were conducted to test the stability of phosphate stabilized 20 nm Ag-NP (nanoComposix, Inc., San Diego, CA) in a simple test medium consisting of 2.5 mM (NH₄)₂SO₄ and 30 mM HEPES buffer (pH 7.8).  The 20 nm Ag-NP immediately aggregated and achieved a hydrodynamic diameter of 500 nm after 1 h of exposure to the test media.  However, if the 20 nm Ag-NP were first added and dispersed in distilled and deionized (DDI) water and then had the (NH₄)₂SO₄ and HEPES added to it, no aggregation of the Ag-NP was observed over the course of the 3 h test.  We hypothesize that the increased stability is due to a reduction in the localized particle concentration effect going from 3,630 ppm Ag-NP (stock concentration) to 1 ppm (test concentration) reducing the predicted aggregation kinetics by 8 orders of magnitude (from seconds to days).

Using the stable Ag-NP/test media suspensions, N. europaea was found to be extremely sensitive to Ag⁺, 20 nm Ag-NP and 80 nm Ag-NP with concentrations of 0.1, 0.5 and 1.5 ppm, respectively, resulting in a 50% decrease in nitrification rates.  The inhibition was correlated with the amount of Ag⁺ released into solution with 25% (mass based) of 20 nm Ag-NP and 6% of 80 nm Ag-NP being released as Ag⁺.  It is suspected that the inhibition observed from Ag-NP exposure is caused by the liberated Ag⁺. 

This hypothesis is further supported from an examination of the inhibition of the ammonia oxidation pathway in which the ammonia monooxygenase enzyme (AMO) oxidizes NH₃ to hydroxylamine (NH₂OH) which is further oxidized to NO₂^- by the hydroxylamine oxidoreductase enzyme (HAO).  Exposure to either Ag⁺, 20 nm Ag-NP or 80 nm Ag-NP resulted in identical inhibition patterns with AMO activity being severely inhibited, while HAO activity was more mildly inhibited. 

The aquatic chemistry of the test media was found to have a profound influence on the stability of Ag-NP suspensions with Ag-NP dissolution correlating most strongly with pH and does not appear to correlate well with the ionic strength of the test media.  The presence of divalent cations (e.g. Ca²⁺ or Mg²⁺) resulted in the rapid aggregation of Ag-NP leading to a decrease in Ag⁺ liberation and thus a decrease in N. europaea inhibition.  Interestingly, the presence of divalent cations also decreased N. europaea inhibition caused by aqueous Ag⁺.  This protection is hypothesized to result from a competition between the divalent cations and Ag⁺ for entrance into the cell.  Thus, divalent cations are thought to protect N. europaea from Ag-NP through a dual-mode mechanism of decreasing Ag⁺ liberation from aggregated Ag-NP and by competing with liberated Ag⁺ for entrance into the cell.   

 Whole-genome microarray experiments are being conducted to determine if differences between Ag⁺ and Ag-NP inhibition can be observed through N. europaea's gene expression patterns.  Additionally, these experiments will identify genes that up-regulated in response to Ag⁺ and/or Ag-NP.  The expression of these Ag⁺/Ag-NP-indicator genes will be used to help determine the bioavailability of Ag⁺ and Ag-NP under a wide variety of complex aqueous chemistries commonly found in WWTP, including activated sludge environments.

We would like to thank nanoComposix, Inc. for donating the Ag-NPs.  This research was funded through an Oregon Nanoscience and Microtechnologies Institute/United State Air Force Research Labs grant # 235271B, Amend. No. 5.

(1) Wijnhoven, S.; Peijnenburg, W.; Herberts, C.; Hagens, W.; Werner, I.; Oomen, A.; Heugens, E.; Roszek, B.; Bisschops, J.; Gosens, I.; van de Meent, D.; Kekkers, S.; de Jong, W.; van Zijverden, M.; Sips, A.; Geertsma, R., Nano-silver - a review of available data and knowledge gaps in human and environmental risk assessment. Nanotoxicology 2009 1:1-30.

(2) U.S.EPA Process Design Manual: Nitrogen Control; 625/R-93/010; U.S. Environmental Protection Agency: Washington, D.C., 1993.

(3) Choi, O.; Cleuenger, T. E.; Deng, B. L.; Surampalli, R. Y.; Ross, L.; Hu, Z. Q., Role of sulfide and ligand strength in controlling nanosilver toxicity. Water Research 2009 43(7):1879-1886.