Toxic Effects Of TiO₂ Nanoparticles On Caenorhabditis Elegans In Soil

Titanium dioxide nanoparticles（nTiO₂）are one type of the most abundantly produced and used nanomaterials（NMs）,and their environmental behavior and bioeffects have become researcj hotspots in the field of environmental science.Soil is an important sink for NMs with their continuously entering into the environment.It is of great practical significance to study the toxic effects of nTiO₂ on soil organisms,especially the long-term effects.However,because of the complexity of soil constitution and the large number of influencing factors,there are many difficulties in the separation of test organisms and the analysis of environmental factors,thus most of the related studies are conducted in aqueous media.In this study,Caenorhabditis elegans,a soil model organism,was used as the research object,and an isolation method of C.elegans from soil was developed.The toxic effects of nTiO₂ on nematodes in five kinds of soils were studied,and the effects of physical/chemical properties of soils on the nanotoxicity were analyzed.Furthermore,the multigenerational effects and toxic mechanisms of nTiO₂ with three different crystal forms were studied in a paddy soil.The existing forms of nTiO₂ in the soil along with the biological responses of nematodes at physiological,biochemical,and molecular levels were investigated.The obtained results were as follows:（1）Based on comparative analysis of the efficiencies of different isolation methods for C.elegans from soil,a new isolation method was developed by attracting and collecting them from soil with food.This method was simple,less destructive,and had a significant higher recovery efficiency（92.5±5.0%）than the Bellman funnel method（30.0±1.6%）,shallow disk method（15.0±1.4%）,and ISO international standard colloid suspension method（62.0±4.3%）.In the developed method,the effect of soil impurities on the separation efficiency was avoided,and large quantities of nematodes with biological activity could be easily obtained.（2）The toxic effects of nTiO₂ on C.elegans in five kinds ofwere compared,and the correlation between the basic properties of soils including p H,conductivity,redox potential,Zeta potential,and total organic carbon content（TOC）and physiological indexes of C.elegans was analyzed after nTiO₂ exposure.The results showed that the survival rate,body length,and locomotion of C.elegans were reduced to varying degrees in the soil samples with 5000 mg/kg nTiO₂.Moreover,the correlation analysis results showed that soil p H was significantly positively-correlated with the relative survival rate,and TOC was significantly positively-correlated with the relative body length and relative headtrash frequency of C.elegans.Other soil basic properties and toxic indicators had no significant correlations.（3）We further investigated how multigenerational exposure to different crystal forms（anatase,rutile,and their mixture）of nTiO₂ affected the survival,behavior,physiological and biochemical traits,and lifespan of nematodes（C.elegans）in a paddy soil.The concentration of bioavailable form of nTiO₂ in the soil was low,and the toxicities of three nTiO₂ forms were largely comparable.The nTiO₂ exposure adversely influenced the survival and locomotion of nematodes,and increased intracellular reactive oxygen species（ROS）generation in a dose-dependent pattern.However,the toxic effect gradually attenuated and the lifespan of survived nematodes increased from P0 to F3 generation.The lethal effect and the increased oxidative stress may continuously screen out offspring possessing stronger anti-stress capabilities.Moreover,key genes（daf-2,age-1,and skn-1）in the insulin/IGF-like signaling（IIS）pathway actively responded to the nTiO₂exposure,which increased the stress resistance and longevity of survived nematodes,and reduced the toxicity of nTiO₂ across successive generations.