For nearly a decade, researchers have debated the mechanisms by which AgNPs exert toxicity to bacteria and other organisms. The most elusive question has been whether the AgNPs exert direct "particle-specific" effects beyond the known antimicrobial activity of released silver ions (Ag(+)). Here, we infer that Ag(+) is the definitive molecular toxicant. We rule out direct particle-specific biological effects by showing the lack of toxicity of AgNPs when synthesized and tested under strictly anaerobic conditions that preclude Ag(0) oxidation and Ag(+) release. Furthermore, we demonstrate that the toxicity of various AgNPs (PEG- or PVP- coated, of three different sizes each) accurately follows the dose-response pattern of E. coli exposed to Ag(+) (added as AgNO(3)). Surprisingly, E. coli survival was stimulated by relatively low (sublethal) concentration of all tested AgNPs and AgNO(3) (at 3-8 μg/L Ag(+), or 12-31% of the minimum lethal concentration (MLC)), suggesting a hormetic response that would be counterproductive to antimicrobial applications. Overall, this work suggests that AgNP morphological properties known to affect antimicrobial activity are indirect effectors that primarily influence Ag(+) release. Accordingly, antibacterial activity could be controlled (and environmental impacts could be mitigated) by modulating Ag(+) release, possibly through manipulation of oxygen availability, particle size, shape, and/or type of coating.

The antibacterial activity of silver nanoparticles (AgNPs) is partially due to the release of Ag(+), although discerning the contribution of AgNPs vs Ag(+) is challenging due to their common co-occurrence. We discerned the toxicity of Ag(+) versus a commercially available AgNP (35.4 ± 5.1 nm, coated with amorphous carbon) by conducting antibacterial assays under anaerobic conditions that preclude Ag₀ oxidation, which is a prerequisite for Ag(+) release. These AgNPs were 20× less toxic to E. coli than Ag(+) (EC₅₀: 2.04 ± 0.07 vs 0.10 ± 0.01 mg/L), and their toxicity increased 2.3-fold after exposure to air for 0.5 h (EC₅₀: 0.87 ± 0.03 mg/L) which promoted Ag(+) release. No significant difference in Ag(+) toxicity was observed between anaerobic and aerobic conditions, which rules out oxidative stress by ROS as an important antibacterial mechanism for Ag(+). The toxicity of Ag(+) (2.94 μmol/L) was eliminated by equivalent cysteine or sulfide; the latter exceeded the solubility product equilibrium constant (K(sp)), which is conducive to silver precipitation. Equivalent chloride and phosphate concentrations also reduced Ag(+) toxicity without exceeding K(sp). Thus, some common ligands can hinder the bioavailability and mitigate the toxicity of Ag(+) at relatively low concentrations that do not induce silver precipitation. Furthermore, low concentrations of chloride (0.1 mg/L) mitigated the toxicity of Ag(+) but not that of AgNPs, suggesting that previous reports of higher AgNPs toxicity than their equivalent Ag(+) concentration might be due to the presence of common ligands that preferentially decrease the bioavailability and toxicity of Ag(+). Overall, these results show that the presence of O₂ or common ligands can differentially affect the toxicity of AgNPs vs Ag(+), and underscore the importance of water chemistry in the mode of action of AgNPs.

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The widespread use of silver nanoparticles (AgNPs) raises the potential for environmental releases that could impact microbial ecosystem services. In the present study, the authors address how the AgNPs and Ag(+) that they release may impact nitrogen-cycling bacteria. The authors studied the cellular and transcriptional response of the denitrifier Pseudomonas stutzeri, the nitrogen fixer Azotobacter vinelandii, and the nitrifier Nitrosomonas europaea exposed to 35 nm (carbon-coated) AgNPs or to Ag(+) (added as AgNO3 ). Based on minimum inhibitory concentrations (MICs), Ag(+) was 20 times to 48 times more toxic to the tested strains than AgNPs (including Ag(+) released during exposure). Exposure to sublethal concentrations of AgNPs or Ag(+) (representing 10% of the respective MIC for AgNO3 ) resulted in no significant effect on the expression of the denitrifying genes narG, napB, nirH, and norB in P. stutzeri or the nitrogen-fixing genes nifD, nifH, vnfD, and anfD in A. vinelandii, whereas nitrifying genes (amoA1 and amoC2) in N. europaea were upregulated (2.1- to 3.3-fold). This stimulatory effect disappeared at higher silver concentrations (60% of the Ag(+) MIC), and toxicity was exerted at concentrations higher than 60% of the Ag(+) MIC. The MIC for N. europaea was 8 times to 24 times lower than for the other strains, indicating higher susceptibility to AgNPs. This was corroborated by the lower half-lethal concentration for N. europaea (87 µg/L) compared with P. stutzeri (124 µg/L) and A. vinelandii (>250 µg/L) when cells were exposed with Ag(+) for 24 h in 1 mM bicarbonate buffer. This suggests that ammonia oxidation would be the most vulnerable nitrogen-cycling process in wastewater treatment plants receiving AgNPs and in agricultural soils amended with biosolids that concentrate them.

Nanoscale zerovalent iron (NZVI) can be used to dechlorinate trichloroethylene (TCE) in contaminated aquifers. Dehalococcoides spp. is the only microbial genus known to dechlorinate TCE to ethene as a respiratory process. However, little is known about how NZVI affects the expression of genes coding for reductive dechlorination. We examined a high-rate TCE-dechlorinating mixed culture which contains organisms similar to known Dehalococcoides to study the effects of NZVI on the expression of two model genes coding for reductive dehalogenases (tceA and vcrA). A novel pretreatment approach, relying on magnetic separation of NZVI prior to reverse transcription qPCR (to avoid RNA adsorption by NZVI), was developed and used with relative quantification (relative to 16S rRNA as endogenous housekeeping gene) to quantify reductive dehalogenase gene expression. Both tceA and vcrA were significantly down-regulated (97- and 137-fold, respectively) relative to baseline (time 0) conditions after 72-h exposure to chlorinated ethenes (0.12 ± 0.03 mg/L cis-DCE, 0.69 ± 0.11 mg/L t-DCE, and 0.54 ± 0.16 mg/L VC) and bare-NZVI (1 g-NZVI/L). However, coating NZVI with an olefin maleic acid copolymer (a common approach to enhance its mobility in aquifers) overcame this significant inhibitory effect, and both tceA and vcrA were up-regulated (3.0- and 3.5-fold, respectively) after 48-h exposure. Thus, NZVI coating might enhance the expression of dechlorinating genes and the concurrent or sequential participation of Dehalococcoides spp. in the remediation process.

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