Effects Of Silver Nanoparticles On Dissimilatory Nitrate Reduction In Estuarine And Tidal Sediments

Due to the excessive input of reactive nitrogen,the nitrogen cycle in estuarine and coastal areas is seriously disturbed,resulting in frequent occurrence of a series of ecological and environmental problems such as severe eutrophication,harmful algal blooms and low-oxygen areas.Thus,nitrate transformation processes have been paid more attention in recent decades.Dissimilatory nitrate reduction processes,including denitrification、anaerobic ammonium oxidation（Anammox）and dissimilatory nitrate reduction to ammonium（DNRA）,plays an important role in the biogeochemical cycle of nitrogen.Both denitrification and Anammox removes nitrate from aquatic ecosystems by conversion to gaseous nitrogen,which is an important reactive nitrogen reduction pathway in the estuarine coastal ecosystem.In contrast,DNRA reduces nitrate to ammonium,as a biologically-available form,still being retained in estuarine and coastal areas.Silver nanoparticles（AgNPs）are metallic silver elements with diameter between1 and 100 nm.AgNPs are characterized by unique chemo-physical virtues and biological properties,leading to ubiquitous applications in optoelectronics,catalysis system,medical fields and personal healthcare,and becoming the most common group of metal engineered nanomaterials.The increasing uses of AgNPs in consumer products have increased their release to the environment in the processes of their manufacture,use and disposal,and thus the ecotoxicity of AgNPs has been paid more attentions recently.Estuarines are the main channel linking both land and coastal sea,and play an important role in the global nitrogen cycle.Human activity can cause accumulation of AgNPs in estuarine and coastal environments,however the effects of accumulated AgNPs on nitrogen transformation remain unclear.This study investigated the effects of AgNPs on denitrification,Anammox and DNRA rates in the Yangtze estuarine and intertidal sediments,through exposure experiments of different sizes of AgNPs（10nm,30 nm and 100 nm）and different AgNPs concentrations（0 mg/L,0.1 mg/L,5mg/L and 10 mg/L）,and the abundance of denitrifiers nirS gene,nirK gene and Anammox 16S rRNA gene was also determined by real-time QPCR.These results are of great significance to understanding the influences of emerging pollutants on estuarine nitrogen cycle.The main findings of this study are as follows:（1）Using isotope-tracing techniques combined with AgNPs–treated incubation experiments,we studied the effects of AgNPs on denitrification、Anammox and DNRA rates.AgNPs inhibited the denitrification,Anammox and DNRA rates in the sediments with different salinity levels,but the inhibition did not increase significantly with time.AgNPs inhibited the denitrification and anammox rates up to73.5%and 72.5%,respectively.The inhibition of AgNPs on DNRA rate was up to33.2%,which was lower than that on denitrification and Anammox.（2）AgNPs size,concentration and sediment salinity were important factors affecting the toxicity of AgNPs.Under lower AgNPs concentration,the inhibition of10 nm AgNPs on denitrification,Anammox and DNRA rates was greater than that of30 nm and 100 nm AgNPs;However,when the concentration of AgNPs was further increased,the inhibition of 30 nm and 100 nm AgNPs on denitrification,Anammox and DNRA rates were significantly increased,which was larger than that of 10 nm AgNPs.For denitrification,Anammox and DNRA processes,the AgNPs toxicity among different sizes were changed at 5 mg/L,10 mg/L and 10 mg/L,respectively.Although the patterns of released Ag⁺concentration differed across the estuarine salinity,the difference of denitrification,Anammox and DNRA rates among different salinity treatments was not significant（P>0.05）.There was no significant correlation between Ag⁺concentration and the inhibition of AgNPs on denitrification,Anammox and DNRA rates（P>0.05）,indicating that the inhibitory effect of AgNPs on dissimilatory nitrate reduction processes could not be fully explained by the toxicity of Ag⁺.（3）The effects of AgNPs on denitrification and Anammox-related gene abundances were explored based on nirS gene,nirK gene and Anammox 16S rRNA gene.The abundances of the nirS gene,nirK gene and Anammox 16S rRNA gene in the blank group were 0.11×10⁹-0.77×10⁹ copies g^-1,3.27×10⁷-10.52×10⁷ copies g^-1 and 0.16×10⁷-2.04×10⁷ copies g^-1,respectively.The abundance of nirK gene was lower than that of nirS gene in this study area,and the following was Anammox 16S rRNA gene.In the AgNPs treatments,the nirS gene abundances,nirK gene abundances and Anammox 16S rRNA gene abundances were 0.09×10⁹-0.9×10⁹copies g^-1,0.27×10⁷-10.32×10⁷ copies g^-1 and 0.25×10⁷-2.38×10⁷ copies g^-1,respectively.Compared with controls,the abundance of nirS gene and anammox 16S rRNA gene increased in AgNPs treatments（up to 37.1%and 6.1%,respectively）.In addition,AgNPs significantly inhibited the abundance of nirK gene（up to 80.2%）.There was no significant correlation between the abundance of nirS gene and denitrification rate（P>0.05）,and the abundance of nirK gene was significantly correlated with denitrification rate（P<0.05）,suggesting that the nirK gene is more sensitive to AgNPs than nirS gene,and further suppressed the denitrification rate.