| Copper oxide nanoparticles(CuO NPs)are widely used in industry and healthcare products,for environment remediation,agricultural production,and medical treatment,as well as in food industry.There is a lack of attention attributed to the fact that they are eventually released to the environment during manufacture,transport,use and disposal,posing threat to ecosystem and human health.The biological characteristics of vegetables vary in plant species,and subsequently affect the absorption,accumulation of CuO NPs in different plants,therefore,the biotoxicity of CuO NPs in vegetables differs species-specificly.Further investigation needs to be applied to understand the toxicity and mechanism of CuO NPs in different vegetable systems,providing theoretical basis for the environmental risk assessment and agricultural product safety regulations.In this study,two different types of vegetable crops,dicotyledonous lettuce and monocotyledonous garlic,were used to investigate the effects of CuO NPs on soil micriobial changes in different soil-plant system.This study aims to:Determine the toxicity of CuO NPs on soil microbial under different soil-plant system,specificly,the microbial activity and community composition changes in bulk and rhizosphere soil;Reveal the toxicity and mechanism of CuO NPs in two vegetable crops by investigating and comparing the changes of antioxidant system,apoplast barrier development and photosynthetic system of lettuce and garlic under CuO NPs exposure;Investigate the cytotoxicity of CuO NPs on human intestinal cells and liver cells through food delivery by culturing the cells with vegetable digestate.The findings are as follows:1.The effects of CuO NPs on soil microbiota were studied using pot experiments.Compared with Cu2+,CuO NPs showed its biotoxicity in soil microbiota.The changes of microbial biomass carbon,enzyme activity and microbial community structure of rhizosphere and bulk soil of lettuce and garlic were investigated.Results showed that the increase in CuO NPs significantly decreased the microbial biomass carbon in bulk soils of lettuce and garlic.The decrease in fluvo-aquic soil(2.2-29.5%)was smaller than that in paddy soil(4.8-38.1%),but there was no significant difference between lettuce and garlic plants.In rhizosphere,low exposure level of CuO NPs has increased soil microbial biomass carbon(8.5-12.0%for lettuce and 6.4-9.4%for garlic),while middle and high exposure levels of CuO NPs significantly decreased soil microbial biomass carbon.Differently from the changes in microbial biomass carbon,changes in soil enzyme activity were influenced by CuO NPs concentration,soil type,and plant type.In paddy soil,the activities of soil dehydrogenase,acid phosphatase and urease decreased significantly with increasing CuO NPs concentrations in both types of bulk soil,and the decrease in fluvo-aquic soil was less than that in paddy soil.Soil enzyme activities in rhizosphere were significantly affected by plant type.In both types of soil,low exposure level of CuO NPs had no effect on enzyme activity in lettuce rhizosphere soil,whereas soil enzyme activity in garlic rhizosphere was significantly reduced only at high exposure level of CuO NPs.Results of high-throughput sequencing indicated that CuO NPs could induce changes in soil microbial community composition,and the relative abundance of ?-and ? proteobacteria in garlic rhizosphere soil was higher than that in lettuce.The abundance of rhizosphere soil microbiota,of which in function for nitrogen fixation and resistance to heavy metal stress,was higher in garlic than in lettuce,so that the growth of garlic was less affected under CuO NPs exposure,whereas lettuce was more negatively affected.2.Hydroponics was used to cultivate lettuce and garlic plants in this section.The effects of CuO NPs on root growth of lettuce and garlic were studied by comparing with CuO BPs,and result of TEM-EDS showed that CuO NPs were absorbed by lettuce and garlic roots.Treatment with CuO BPs showed no effect on root elongation,but CuO NPs exposure significantly inhibited the root elongation of lettuce(decreased by 14.8-38.9%)and garlic(decreased by 13.2-25%).Under the exposure of CuO NPs,POD activities in roots of both vegetables decreased with the prolongation of treatment time,notably,the POD activity of garlic roots was higher than that of lettuce at the same treatment level.Meanwhile,the activity of SOD in lettuce roots increased with the prolongation of treatment time,and it reached the highest at 14 days(1.83,3.01 and 2.92 times of that at 3 days),and subsequently decreased.However,the activity of SOD in garlic roots continued to increase in 21 days(4.22,4.64 and 3.27 times of that in 3 days).The results showed that garlic roots could maintain higher antioxidant enzyme activity and reduce the damage of reactive oxygen species caused by CuO NPs.In addition,garlic plants can alleviate the stress of CuO NPs exposure through the thickening of cell wall and the development of suberin lamellae in root tip,so as to reduce the inhibition of CuO NPs on root growth.3.Hydroponics was used to cultivate lettuce and garlic plants in this section.Further investigaitons were applied to evaluate the effect of CuO NPs on the photosynthesis of lettuce and garlic plants.The results showed that CuO NPs significantly inhibited the growth of the two plants.The aboveground biomass of lettuce decreased by 24.9-51.2%,while that of garlic decreased by 15.5%and 20.2%at middle and high exposure concentrations.CuO NPs affected the absorption of magnesium and inhibited the synthesis of chlorophyll.The chlorophyll content of the two plants decreased with the increase of CuO NPs concentration,while the decline of lettuce was more significant than that of garlic.The results of CF Imager showed that the Fv/Fm(14.4-18.6%)and ?PS?(9.1-12.9%)of lettuce decreased with the growing exposure of CuO NPs,while the data of garlic decreased slightly under CuO NPs exposure and showed no significant difference among all concentrations.In addition,CuO NPs treatment significantly reduced the Rubisco activity(29.3-38.4%,lettuce;54.5-72.7%,garlic),and the reduction of lettuce was significantly greater than that of garlic.Generally,the light harvesting efficiency of lettuce and garlic decreased under CuO NPs exposure,and the photosynthetic system of lettuce and garlic were damaged to varying degrees,but the damage mechanism was different.The results showed that the activities of enzymes in the photosynthetic system of garlic were decreased by the exposure of CuO NPs,which inhibited the cycle of C5 to C3 in chloroplast,reduced the energy transfer,and led to the decline of photosynthetic rate.While the chloroplast development of lettuce was damaged due to more ROS stress,which led to the damage of photosynthetic system and chloroplast development,and thus reduced the photosynthetic rate and inhibited the plant growth.4.Caco-2 cell model was used to evaluate the bioavailability and biotoxicity of CuO NPs in lettuce to human intestinal cells through food delivery.The results showed that CuO NPs could transfer to human intestinal cells through the edible part of lettuce,and the bioavailability of CuO NPs increased with the increase of concentration.Lettuce exposed to low concentrations of CuO NPs significantly affected the viability of Caco-2 cells(72%higher than that of CK),and increased the level of ROS,resulting in vacuoles in mitochondria,while lettuce treated with middle and high concentrations of CuO NPs could cause cell death(cell survival rate was 59.3%and 35.4%).Besides,the growth of intestinal cell microvilli were inhibited under middle and high CuO NPs exposure,resulting in the up-regulated response to pro-inflammatory related genes.These results indicated that the consumption of CuO NPs exposed lettuce could lead to ROS damage and intestinal inflammation in intestinal cells,and it also could decreae the intake of nutrients.In addition,compared with ZnO NPs and CeO2 NPs,it was found that,the toxicity of NPs is related with ion release,and the toxicity of CuO NPs was between ZnO NPs and CeO2 NPs.5.Human liver cell line(HL-7702)were cultured with the transport digestate of Caco-2 model to study the effect of CuO NPs on liver through intestinal absorption.Cell viability(MTT)and lactate dehydrogenase(LDH)release assays showed no significant effect of the transfer solution on cell viability and cell morphology of human liver cells after intestinal cells absoption.However,potential risk has occurred under the transport digestate exposure.ROS level showed that the transfer digestate under CuO NPs exposure was able to cause increase in the ROS level of liver cells at middle and high exposure levels(104.9%and 108.6%).The mitochondrial membrane potential results showed that the high concentration treatment of CuO NPs exposed to the transfer digestate was able to increase the mitochondrial membrane potential of human liver cells,resulting in ROS damage in mitochondria and DNA. |
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