The Ecological Toxicology Of Titanium Dioxide And It's Oxygen Deficient Phase To Microorganism In Estuarine Aquatic Environment

Titanium dioxide nanoparticles(TiO2-NPs)have been widely used in various consumer and industrial products,such as papermaking,cosmetics,wall paint and catalysts,and the production is increasing annually.Among three crystal structures of TiO2-NPs,rutile(Ru),anatase(An)and brookite are more widely used.P25 is a kind of commercial TiO2-NPs,mixed with anatase and rutile by 80:20.TiO2-NPs(An,Ru and P25)will lose oxygen atoms after annealing at high temperature and turn into oxygen deficient TiO2-NPs(TiO2-OD),which show higher activity and potential application prospect compared with their precursors.The extensive use of TiO2-NPs increases their potential release into aquatic environment.In addition,TiO2-NPs,which are stable and resistant to be dissolved in the environment,could be persistent pollutants in water and sediment,posing potential environmental risks to various organisms in aquatic environment.As the junctional area of salt water and fresh water,estuary environment is relatively complicated,so it is significantly important to study the toxicity of TiO2-NPs in estuary environment to protect estuarine ecological environment.Escherichia coli(E.coli)was chosen as the model microorganism in this study to investigate the phototoxicity of six kinds of TiO2-NPs with UVA irradiation.The results showed that,compared with no UVA irradiation,the survival rate of E.coli reduced significantly under UVA when exposed to P25,An,P25-OD and An-OD,and oxygen deficient materials exhibite d stronger toxicity than their precursors,while Ru and Ru-OD showed no obvious toxicity.The toxicities of six kinds of TiO2-NPs were An-OD>P25-OD>P25>An>Ru-OD?Ru.Comparing the photocatalytic performances of six kinds of TiO2-NPs on methylene blue(MB),we found that the degradation of MB obeys a kinetic equation,and the decomposition reaction rate constants of P25,P25-OD,An,An-OD,Ru and Ru-OD were KAn-OD>KP25-OD>KP25>KAn>KRu-OD>KRu.It is reported that TiO2-NPs can produce reactive oxygen species(ROS)under UV irradiation.By detecting the contents of ROS for different TiO2-NPs in different concentrations,we found that the contents of ROS for six kinds of TiO2-NPs showed significant differences,taking the concentration of 100 mg/L for example,after irradiated by UV for 30 min,the contents of ROS produced by P25,P25-OD,An,An-OD,Ru and Ru-OD were 11.90,18.61,8.31,21.04,2.99 and 3.97?mol/L,respectively.ROS has a significant negative correlation(r = 0.9385,P<0.05)with the survival rate of E.coli.Finally,we concluded that the production of ROS was the reason of photocatalytic ability and photoinduced toxicity of both TiO2-NPs and TiO2-OD.To further test the salinity effect on the cytotoxicity of TiO2-NPs and TiO2-OD,P25 and P25-OD were chosen for the tests under varying salinities from 0‰ to 30‰.The examined salinities are representatives of freshwater to seawater.The results indicated that TiO2-OD showed more cytotoxicity toward E.coli than the precursor TiO2-NPs at all tested salinities.The calculated 30 min-LC50 for P25 at salinity of 0,1,5,10,30‰ were 207,138,101,40.6,17.7 mg/L,respectively and 161,107,81.5,18.2,12.8 mg/L for P25-OD,respectively.We found the enhanced cytotoxicity of NPs at high salinity,whereas reduced concentrations of ROS produced by certain amount of NPs were determined in brackish water.High salinity would accelerate the aggregation of nanoparticles,and the phenomenon that hydrodynamic diameters of P25 and P25-OD increased with the increase of salinity confirmed the shortly decrease of ROS.In the present study,we have analyzed the osmoregulation related gene,including OmpR OmpF and OmpC in E.coli at different salinities.In general,all three genes showed down-regulated with increasing salinity,which indicated the inhibited activities of bacteria in brackish water.Therefore,we hypothesized that under osmotic stress,although bacterial cells survived,the cellular activity including the antioxidant capability had decreased in present study.We also investigated the expression of six antioxidant genes in E.coli at increasing salinity with and without UVA irradiation;with and without TiO2-NPs(P25 and P25-OD)for 30 mins.The results showed that under UVA irradiation,with and without TiO2-NPs,the expression of tested genes in general decreases with increasing salinity.However,compared to the group without TiO2-NPs,the expression of tested genes generally increased in the groups with P25 and P25-OD.It is reasonable because that bacterial cells could up-regulate the antioxidant genes to defense the ROS produced by TiO2-NPs under UVA irradiation.All above indicated that TiO2-NPs have no effect on the E.coli without UV irradiation and the down-regulate expression of genes were mainly caused by salinity.Overall,these results suggest that the antioxidant capability of bacteria decreases with an increasing salinity,and this is especially true for relatively high salinity.Therefore,when exposed to brinish water,the antioxidant capacity of E.coli could be deactivated,and a synergistic lethal effect could be triggered by additional ROS produced by TiO2 under UV light.This may explain the reason why an increasing mortality of E.coli was observed in water with increased salinity,although the ROS is reduced in brackish water.On the other hand,The TEM images of E.coli;E.coli and P25;E.coli and P25 at 10‰ salinity after exposed to UV showed that a large quantity of TiO2-NPs absorbed on the surface of E.coli.The high affinity of TiO2-NPs to E.coli may enhance the effect of ROS on bacteria directly,and further cause the visible deformation of E.coli cell.Three estuarine water samples(QYK,CY and DH)were taken with increasing salinity gradients and further ultrafiltered to verify the effect of salinity on the cytotoxicity of TiO2-NPs toward E.coli in the natural estuarine environment.Compared to the LC50 that acquired from the salinity effect experiment using SDW,the LC50 of both P25 and P25-OD in natural estuarine water were within the scope according to the synthetic brine solution(except that P25-OD in CY water showed slightly lower LC50 than that achieved in SDW at salinity of 5‰).Therefore,salinity could be a dominant factor that controls the cytotoxicity of NPs in estuarine aquatic systems.In order to further explore the effect of TiO2-NPs on E.coli genes,we analyzed the E.coli genome after treated by TiO2-NPs with whole genome sequencing technology.Six mutations were found,and one of them related with bacterial secretion system,two of them related with bacterial gene mutation induced by enzymes,these findings require subsequent experiments.