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.
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