Exposure Of Graphene To Rice Plants:Bioaccumulation,Distribution,Depuration And Transformation

Since its inception,graphene has rapidly attracted the attention of the scientific community and industry.More and more graphene-based scientific research has been reported.Also,increasing quantity of graphene has been applied into commodities for human production and livelihood.As a result,graphene inevitably enters the environment via multiple pathways,affecting various organisms in the ecosystem.Therefore,ecological risk assessment of graphene is needed to ensure its safety.Among the previous study of graphene on terrestrial plants,a considerable amount of research results have refreshed our knowledge,but mainly focusing on toxicity studies.Bioaccumulation and distribution assessment about graphene on plants is rarely reported,which is the another important part of the ecological risk assessment of pollutants exposure assessment.The reason for this is mainly that graphene cannot be accurately quantified in biological samples,so we developed a method for labeling graphene with 14C,which can quantify the amount of graphene in plants very accurately to reach the purpose of tracing the change of graphene in the plant body.Based on this method,this study further explored the regularity of the enrichment,distribution,purification,and transformation of FLG with time in typical crop plants.The main research contents and results of this article are as follows:(1)Rice plants were subjected to hydroponic exposure of 14C-labeled graphene(14C-FLG).Three groups of experiments were conducted:FLG exposed group(50,100,250,500μg/L);FLG-NOM exposed group(50,100,250,500μg/L FLG;10 mg/L NOM);control group(pure nutrient solution;10 mg/L NOM).The exposure time is 21 d.The results showed that FLG can be ingested into the roots of rice and transferred to stems and leaves.The root concentration of FLG reached a maximum of approximately 700 μg/g at 7 d and then began to decline,decreasing to 50%at 21 d.On the contrary,when the NOM was involved,the root concentration was always on the rise and accounted for only 1/7 that of the FLG exposure group at 7 d.According to the EPM detection of the nutrient solution,it was found that FLG-NOM carries 2 times negative charge more than FLG.Since the root is also negatively charged,it was considered that the electrostatic force makes FLG-NOM more difficult to interact with the root.The accumulating pattern of FLG in shoots wa also similar to that of roots.The value of peak at 7 d was 53.7 μg/g,which is 35 times that of the NOM exposure group at the same time.The transfer rate of FLG from roots to shoots was also faster as translocation factor(TF)revealed.However,TF value was less than 0.2 under both treatments.In the absence of NOM,the total amount of FLG accumulated in roots was still the highest at 7 d,but the total amount of shoots reached its maximum at 14 d,indicating that the rice plants had clearance mechanisms for FLG.(2)This paper further explores the distribution patterns of FLG at the subcellular level in rice plants based on the uptake data.Shoots were separated into five subcellular components of F1 cell wall,F2 chloroplast,F3 cell nucleus,F4 mitochondrion,and F5 soluble fraction by differential centrifugation.As the uptake experiment,rice plantswere exposed to 250μg/L FLG for 21 days.It turns out that FLG was mainly found in F1 cell wall and F2 chloroplast,and FLG decreased with time in the chloroplast,but increased in the presence of NOM.In order to evaluate the possible effects of centrifugation on organelles or FLG in differential centrifugation,relevant control experiments were set up.First,it was found that FLG did not affect the subcellular fractionation during centrifugation.Secondly,the calculated amount of adsorption of organelles by FLG was 2.4-5.4%,comparable to the actual amount of adsorption(2.5-4.9%).It demonstrated that differential centrifugation is feasible in assessing the distribution of FLG on the subcellular level of shoots.Therefore,32.4%,43.8%,and 17.5%of FLG existed in the cell wall,chloroplast,and cell nucleus at 7 d.Considering the adsorption behavior occurring in the method,it could be assumed that the bioaccumulated FLG would be detained in the cell wall and enter into the chloroplasts and nucleus.To further corroborate the presence of FLG in the above subcellular components,we used TEM and Raman spectroscopy to characterize FLG in the cell wall(F1)and chloroplasts(F2).As a result,six-fold symmetry typical of graphene particles was observed during TEM SADP analysis.D and G bands of FLG were found during Raman spectra analysis.Through the above studies,we believe that FLG excreted or transformed in the tissue of rice,causing it decreased with time.(3)In this paper,we realized that the depuration behavior of FLG may have occurred in the rice plants.So we designed depuration experiments,experiments for collecting plant transformation products and simulating transformation experiments in vitro.The depuration experiments included hydroponic depuration and soil depuration.The hydroponic depuration was conducted as follows:the rice plants were exposed for 7 days and then purified with nutrient solution.After 14 days of purification,the FLG accumulation in the plants decreased with time,FLG was detected in the nutrient solution,but FLG decrease in the plant was 6%higher than the increase in the nutrient solution.It can be concluded that the FLG undergone transformation.In order to detect the transformation products,the exposure device with plants were placed together inside a closed box for a period of time.The gas in the box after 14 days was characterized by GC,showing abnormally high CO2 content,and the control experiment proved that the conversion process did not occur in the roots.After quantifying the gas in the box,it was found that the total 14CO2 amount account for 9%of bioaccumulated FLG in plants,basically consistent with ’6%FLG disappeared’ in the water purification test.Hence,it can be considered that FLG is degraded into CO2 in rice leaves.It is worth mentioning that NOM consistently plays the role of ’inhibitor’.Subsequent in vitro photochemical experiments revealed the mechanism of FLG degradation,demonstrating that FLG-enriched leaves produce free-radical OH·altered FLG,while NOM inhibited OH·production.In the final soil depuration experiment,a similar pattern to hydroponic depuration results was also observed—FLG content in shoots dropped rapidly,and it decreased to zero in 30 days;some FLGs were also detected in soil;more ’disappeared FLG’.The decrease of FLG content slowed down when NOM participated.In addition,FLG was not detected when quantifying the seeds harvested from maturing rice in FLG or FLG-NOM exposed groups,indicating that FLG would not be transmitted between generations in rice.