Combined Toxicological Effects And Mechanisms Of Modified Nano-carbon Black-Cd On Earthworms

With the promulgation and implementation of the"Action Plan on Prevention and Control of Soil Pollution"and the"Soil Pollution Control Law",Chinese soil pollution control has entered a new period.In light of the characteristics of slight and mild heavy metals pollution of soil in China,in-situ passivation remediation technology is booming.Nanomaterials have been widely used to immobilization in-situ of heavy metals in soils due to their superior adsorption capabilities.However,the nanomaterials applied in remediation of heavy metal-contaminated soil are obviously different from those that enter the soil via other ways.First,their concentration in the soil is high;and second,they selectively adsorb and passivate a large amount of heavy metals,altering their surface properties.In a soil environment,the long-term accumulation of nanomaterials especially that applied to immobilize heavy metals,is worrying.“Strengthening soil pollution control and remediation and effectively preventing risks”requires a clear understanding of the biotoxicological effects of nanomaterials entering the soil and the nanomaterials that will be used for contaminated soil remediation in future,which is essential for the soil pollution control.In this study,the nano-carbon black was used as the core material for research.First,the toxicity of three allomorphic carbon nanomaterials?CNs?—nano-carbon black?CB?,reduced graphene oxide?RGO?,single-walled carbon nanotube?SWCNT?—to earthworm?Eisenia fetida?were evaluated using the simulation test,the soil culture test and the in vitro tests based on the characterization of their morphology,surface charge,chemical composition,etc.The response of macro-and microscopic biomarkers of earthworms such as the mortality,the body weight,and the avoidance rate,and the antioxidant biomarkers,and the intestinal microbial composition and structure,and the non-targeted metabolomics based on UPLC-MS were determined.Second,the effects of surface modification on the toxicity of CB were further explored using filter paper contact test,culture test and comet assay and so on.Third,the combined toxicity of modified nano-carbon black?MCB?and cadmium?Cd?on earthworms were elucidated using in vitro test,culture test and MIXTOX model analysis.This study reveals the toxicological mechanisms that earthworms respond to three CNs with similar properties but very different shapes,CBs before and after surface modification,and MCB-Cd complex.The practical implication of this study is to provide a theoretical basis for the safe application of MCB in the passivation and remediation of the soil polluted by heavy metals.The main findings of this thesis are as follows:?1?CB was nearly spherical with all three dimensions at the nano-level.RGO was flake-like stacked by single or few layers of graphene with thickness at the nano-level but the lateral size at the micron or submicron scale;SWCNT was single walled-long tubular structure with a nanometer diameter and a micrometer length.The aspect ratios of three CNs as well as the average hydrodynamic diameter?D_H?could be ordered as follows:SWCNT?5000-30000?>RGO?134-5456?>CB?close to 1?.The specific surface area and Zeta potential of the three CNs were similar.The primary functional groups on the particles surface were C-C,C-O,C=C,O-H.CB had more aromatic ring structures,and SWCNT had a typical carbon skeleton structure.The oxygen content of RGO was 2.83-fold that of SWCNT and 7.50-fold that of CB.CB had the most defects,while SWCNT had more complete crystal forms and fewer defects.The iron?Fe?was the primary of heavy metal-impurities in the three CNs and its content in three CNs followed a sequence order:SWCNT>CB>RGO.?2?At a concentration lower than 15.7?g/cm² filter paper and 1000 mg/kg soil,three CNs had no significant effect on the survival of earthworms,but remarkably inhibited the growth of earthworms depended on the CNs type.After 28 days culture in soils,only earthworms in the CB treatment showed a positive growth;i.e.,in the RGO and the SWCNT treatments showed a negative growth.The oxidative stress level of earthworms induced by three CNs after filter paper contact 48 hours were obviously different.The overall trend was that the superoxide dismutase?SOD?activity of earthworms was promoted,and catalase?CAT?was not affected as obvious as the SOD,and the malondialdehyde?MDA?content was no significant change?P>0.05?.The inhibition of three CNs on the viability of earthworm coelomocyte increased with increasing concentration.At the concentration of 1 mg/L,the CNs had no significant effect on the viability of earthworm coelomocytes?P>0.05?.At 100 mg/L,the CB,RGO,and SWCNT all significantly inhibited the cell viability and the data were 76.2%,64.1%,and 53.9%of the control,respectively.For the intestinal microbiome,a total of 17.3%OTUs were common to all CNs treatments.Actinobacteria was the dominant phylum,followed by Proteobacteria and Firmicutes.The increase of Shannon diversity index was negatively correlated with the logarithm of the aspect ratio of the CNs.The main amino acids in earthworm metabolites were leucine,phenylalanine,and valine.The marker metabolite was choline.The intensity of choline in earthworm decreased by 14.0%,35.1%,and 46.8%in the group treated with 0.01%-CB,-RGO,and-SWCNT,respectively,and decreased by 38.8%,43.1%,and 64.8%at the concentration of 0.1%.In short,the toxic effects intensity of three CNs on earthworms followed a sequence order:SWCNT>RGO>CB.?3?The main changes in surface characteristics of CB subjected to acidic potassium permanganate oxidation,were an increase in oxygen-containing functional groups and a decrease in Zeta potential.The effects of modified nano-carbon black?MCB?on the survival,weight,and abnormality of earthworms were not significantly?P>0.05?different from those of CB,but the avoidance rate was significantly higher than CB?P<0.05?,and showed a dose-dependent relationship.At concentration of 1.5%,the avoidance rate in MCB group was up to 93.3%.The integrated biomarker response?IBR?was used to comprehensively understand the effects of CB and MCB on the physiological and biochemical indexes of earthworm.The result showed that the stress on earthworms imposed by MCB was higher than CB.At a high concentration,CB caused tissue and cell pathologies and inhibited the cell viability.After surface modification,MCB significantly aggravated cell death and DNA damage compared with CB.?4?The adsorption of Cd on MCB was better described by the Langmuir adsorption isotherm,which implied a monolayer adsorption with the maximum adsorption amount of 18.9mg/g.The adsorption kinetic showed the activation energy was 36.6 kJ/mol.The adsorption thermodynamic showed that it was a spontaneous reaction with enthalpy change of-98.4 kJ/mol.The mechanism of Cd adsorption on MCB was presumably chemical coordination and?-cation interaction.The EC50s of Cd and MCB to earthworm coelomocytes were 17.67 mg/L and 94.00mg/L,respectively.MCB-Cd could be phagocytosed by coelomocytes,causing cell mitochondrial damage,disturbing nuclear membrane stability,and leading to apoptosis.The MIXTOX model,combination index analysis,and Cd uptake test(2mg/L Cd²⁺+20 mg/L,10mg/L Cd²⁺+60 mg/L MCB)supported that low-dose mixtures exhibited antagonism,and high-dose mixtures exhibited synergism.After adding 1.5%MCB,the available-Cd content in the soil decreased by 33.3%.However,after 28 days of culture,the survival of earthworms in soils treated with Cd alone decreased by 29.6%while the treatment with MCB+Cd decreased by 51.9%compared with the control.Earthworms had a significant enrichment effect on Cd in the soil.The biological concentration factor?BCF?in Cd-contaminated soil was 9.25,and the BCF after adding MCB was 9.53.MCB+Cd significantly increased the GSSG content?P<0.05?,and decreased GSSG/GSH by 39%of the control.Cd significantly increased the activity of CAT and LDH,and MCB+Cd inhibited the activity of CAT and LDH.Therefore,Cd and MCB existed a combined toxicological effect.?5?The morphology of carbon nanomaterials was the most important factor leading to its toxicological effects on earthworms.Shorter SWCNT may enter the cell through endocytosis or penetration.Longer tubes could not completely enter the cell membrane,which seriously disrupts the stability of the cell membrane.RGO had sharp lateral sides and destroyed cell membranes presumably by"wrapping","inserting","extracting","nano-knife"and other forms.CB was nearly spherical with the smallest particle size,which contributed to easier to be swallowed and excreted,hence CB had the least damage to cell membranes among the three materials.The direct reason for the increased toxicity of MCB over CB was oxidative stress probably induced by the decrease in Zeta potential and the increase of oxygen-containing functional groups.Both the adsorption capacity and stability of Cd on the MCB were significantly increased compared with that on the CB.Under the reaction of chemisorption,MCB and Cd tightly bonded to form a"new compound?MCB-Cd?",which reduced the biotoxicity of the Cd.Inversely,the adsorption of Cd changed the surface properties and thus reduced the biotoxicity of the MCB.Therefore,MCB-Cd showed a toxicological antagonistic effect.The strong adsorption of Cd on MCB may facilitate the transport of Cd into the coelomocytes,triggering a series of toxic and side effects and leading to a synergistic toxicity.The MCB-Cd phagocytosed by earthworm coelomocytes perhaps resulted in antagonism to synergism switching in a dose-dependent manner.The toxic influence of adding MCB to Cd-contaminated soil on earthworms mainly occurred due to the body uptake.MCB loaded Cd into the intestinal tract of earthworms,and intestinal secretions including a large number of small molecules such as polysaccharides and amino acids complexed or chelated with adsorbed Cd,which promoted the activation of Cd in the intestine and in turn led to toxicological effects on earthworms.Most studies have confirmed that carbon nanomaterials have better adsorption performance for heavy metals,which can significantly reduce the bio-available/accessible content of heavy metals in the soil,reduce the migration of heavy metals to the plant shoots,and protect food safety.However,this study shows that due to the nano-scale particle size,morphology,and surface characteristics,carbon nanomaterials pose a bio-toxicological effect in some extents.In especial,when applying CNs to immobilize and remediate the soil contaminated by cadmium,the adsorbing and passivating of cadmium of the MCB does not apparently reduce the toxic effect of the heavy metal on earthworms,but make the ecological risk greater.Therefore,it is recommended to cautiously use immobilization agents at nano-level for repairing heavy metals-contaminated soils.