Influence of liberated silver from silver nanoparticles on nitrification inhibition of Nitrosomonas europaea

► Addition order of test media and Ag-NP influences the repeatability of assays. ► Nitrosomonas europaea is most sensitive to Ag+, followed by 20nm Ag-NP then 80nm Ag-NP. ► Dissolved Ag (dAg) was found to be responsible for Ag-NP toxicity. ► dAg release was inversely proportional to the surface area to volume ratio. ► Aggregation of Ag-NP resulted in decreased dAg release and decreased toxicity. The ecotoxicity of silver nanoparticles (Ag-NPs) to wastewater biota, including ammonia oxidizing bacteria (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 demonstrated 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 Nitrosomonas europaea. N. europaea, a model AOB, was found to be extremely sensitive to ionic silver (Ag+) and two sizes of Ag-NPs (20 and 80nm). Ag+ exposures resulted in the highest level of toxicity with smaller Ag-NPs (20nm) being more toxic than larger Ag-NPs (80nm). 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 80nm), 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 concentrations being five times greater, by mass, than Ag+ concentrations.