Effects of silver nanoparticles on nitrification and associated nitrous oxide production in aquatic environments

Abstract

Silver nanoparticles (AgNPs) are the most common materials in nanotechnology-based consumer products globally. Because of the wide application of AgNPs, their potential environmental impact is currently a highly topical focus of concern. Nitrification is one of the processes in the nitrogen cycle most susceptible to AgNPs but the specific effects of AgNPs on nitrification in aquatic environments are not well understood. We report the influence of AgNPs on nitrification and associated nitrous oxide (N₂O) production in estuarine sediments. AgNPs inhibited nitrification rates, which decreased exponentially with increasing AgNP concentrations. The response of nitrifier N₂O production to AgNPs exhibited low-dose stimulation (<534, 1476, and 2473 μg liter^-1 for 10-, 30-, and 100-nm AgNPs, respectively) and high-dose inhibition (hormesis effect). Compared with controls, N₂O production could be enhanced by >100% at low doses of AgNPs. This result was confirmed by metatranscriptome studies showing up-regulation of nitric oxide reductase (norQ) gene expression in the low-dose treatment. Isotopomer analysis revealed that hydroxylamine oxidation was the main N₂O production pathway, and its contribution to N₂O emission was enhanced when exposed to low-dose AgNPs. This study highlights the molecular underpinnings of the effects of AgNPs on nitrification activity and demonstrates that the release of AgNPs into the environment should be controlled because they interfere with nitrifying communities and stimulate N₂O emission.

Fig. 1. Percentage reduction of nitrification rate in AgNP or Ag⁺ treatments compared to the no-silver control (incubation time = 12 hours; n = 3).

EC₁₀ and EC₅₀ represent the concentrations that produced a 10 or 50% reduction in nitrification rate relative to the control, respectively. Nonlinear fitted curves (ExpDec1) and equations are given (P < 0.01).

Fig. 2. Effect of AgNPs or Ag⁺ on N₂O emission during nitrification (incubation time = 12 hours).

Data show the percentage changes of N₂O emission in the AgNP or Ag⁺ treatments compared to the no-silver control (n = 3). Nonlinear fitted curves (Gauss) and equations are given (P < 0.01).

Fig. 3. Identification of key N₂O production pathways.

(A) SP values. (B) Contribution of NH₂OH oxidation pathway to N₂O emission. (C) Isotopomer ratios at the central site of N₂O. (D) Isotopomer ratios at the end site of N₂O. Horizontal lines indicate the median, five-point stars show the mean, asterisks indicate outlier, the boxes give the 25th and 75th percentiles, and whiskers show range from the 5th to 95th percentile. Control group represents the incubation without silver. The 10 nm, 30 nm, 100 nm, and Ag⁺ in the “N₂O stimulation” area represent the incubations with AgNPs (100 μg liter⁻¹, 10 nm; 500 μg liter⁻¹, 30 nm; and 1000 μg liter⁻¹, 100 nm) and Ag⁺ (5 μg liter⁻¹), wherein 43.0, 84.1, 121.5, and 73.9% of N₂O emission enhancement were detected, respectively. The 10 nm, 30 nm, 100 nm, and Ag⁺ in the “N₂O inhibition” area represent the incubations with AgNPs (2000 μg liter⁻¹, 10 nm; 3000 μg liter⁻¹, 30 nm; and 3000 μg liter⁻¹, 100 nm) and Ag⁺ (500 μg liter⁻¹), wherein 89.9, 48.3, 19.0, and 59.3% of N₂O emission inhibition were detected, respectively. The incubation time was 12 hours.

Fig. 4. Response of nitrifying communities to AgNP exposure.

(A) Schematic model depicting the effects of AgNPs on the expression of gene families involved in nitrification. N₂O can be produced through NO₂⁻ reduction (the bold pink arrows) or incomplete NH₂OH oxidation (the bold blue arrows). Upward green arrows indicate that the gene expressions were up-regulated when exposed to AgNPs, the downward red arrows indicate that the gene expressions were down-regulated, and “N” denotes that gene expression was not affected by AgNP exposure. CusA, Cu(I)/Ag(I) efflux system membrane protein cusA; CusB, Cu(I)/Ag(I) efflux system periplasmic protein cusB; amo, ammonia monooxygenase; hao, hydroxylamine oxidase; Cyt554, cytochrome c554; mCyt552, cytochrome c_m552; nir, nitrite reductase (NO-forming) nirK; nor, nitric oxide reductase norQ; nxr, nitrite oxidoreductase; Cyt551, cytochrome c551; Cyt552, cytochrome c552; Cyt553, cytochrome c553; Q, ubiquinone; QH₂, ubiquinol. The roman numbers refer to the enzyme complex I (NADH-ubiquinone reductase), complex III (ubiquinol-cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F-type ATPase) in the respiratory chain (related gene expression regulations by AgNPs are shown in fig. S12). Colored proteins were detected in the cDNA libraries, whereas those in dark gray were not identified but are included in the model of electron transport for reference (74). Dotted blue arrows show the movement of electrons, and white arrows show movement of protons. The membrane was broken by dotted line, as nitrite oxidation did not often occur in the same organism with ammonia oxidation, with the exception of recently discovered comammox Nitrospira (55, 56). (B) Fold change (FC) of transcripts encoding proteins involved in heavy metal stress response, oxidative stress release, and the nitrogen transformation process of nitrifying organisms when exposed to 30-nm AgNP (500 μg liter⁻¹) for 12 hours. FC in relative gene expression was calculated by comparing AgNP-treated samples to the no-silver control. Gene expression levels were calculated on the basis of FPKM. (C) Contribution of different pathways to N₂O emission in the no-silver control and the 30-nm AgNP (500 μg liter⁻¹) treatment. (D) TEM image of the no-silver control at 12 hours. (E) TEM image of the 30-nm AgNP (500 μg liter⁻¹) treatment at 12 hours. No apparent physical damage to the cell surface was observed.