| Arsenic（As）is a toxic metalloid element widely distributed in the world and a persistent bioaccumulation carcinogen.Earthworms are commonly used as indicator organisms to investigate the bioavailability of As in contaminated soils.However,the process of As accumulation in earthworms and the transformation pattern of As species in earthworms are still unclear.In this study,the earthworm Eisenia fetida was exposed to soil containing exogenous As（sodium arsenite and disodium hydrogen arsenate）in order to reveal the changes of As accumulation by earthworms in contaminated soil over time;to investigate the transformation pattern of six As species in the earthworms during the process of As accumulation;to explore the main sites of As accumulation in earthworms and the structural changes in the vital organs of earthworms;to analyze the changes in soil physicochemical properties and to explore the differences in microbial communities between soil and earthworms.The main conclusions of this paper are as follows:（1）Earthworms were exposed to sodium arsenite contaminated soil(0,20,80 mg kg-1 As)for 56 d.The total As content accumulated by earthworms in the different treatments increased linearly with time.A highly significant positive correlation（p<0.01）was found between the total As content in earthworms and the exposure time.At the end of the experiment,the total As content in earthworms exposed to high contamination of As pollution was the highest,reaching 772±20.7 mg kg-1,followed by 579±42.4 mg kg-1 in earthworms exposed to low As contamination,and the lowest in control earthworms,30.6±1.33 mg kg-1.At 56 d of the experiment,the proportion of trivalent As（As-III）increased to 90.0%-90.8%,the pentavalent As（As-V）decreased to 8.1%-9.9%.The four organic As species,including monomethylarsine（MMA）,dimethylarsine（DMA）,arsenical choline（As C）,and arsenobetaine（As B）,all increased with time from 1-28 d and then decreased with time.The total proportion of organic As was less than 1%.In addition,the As accumulated by earthworms in the contaminated environment may come from non-specifically absorbed As.（2）Earthworms were exposed to soil contaminated with disodium hydrogen arsenate(0,25,100 mg kg-1 As)for 70 d.The total As content accumulated by earthworms increased linearly with time in all treatments.A highly significant positive correlation（p<0.01）was found between the total As content in earthworms and the exposure time.At the end of the experiment,the highest total As content in earthworms was 1104±32.1 mg kg-1 under high As contamination,followed by 722±83.1 mg kg-1 under low As contamination,and the lowest total As content in control earthworms was 44.6±5.63 mg kg-1.In the experiment,the proportion of As-III in the earthworms increased with time,reaching a maximum of more than90%,the proportion of As-V decreased by 10%-19%.The proportion of the four organic As species（MMA,DMA,As B and As C）increased in the early stage of the experiment but then decreased,and their total proportions were less than 1%.In addition,the earthworms exposed to As contamination showed obvious intestinal damage at 70 d of the experiment,and the higher the As concentration,the more severe the intestinal damage.（3）Earthworms were exposed to different concentrations of sodium arsenite and disodium hydrogen arsenate contaminated soil for 28 d.A significant difference（p<0.01）was found in the bacterial communities between the gut of Eisenia fetida and the surrounding soil.The diversity and abundance of bacterial communities in the gut of the earthworms were lower than those in the corresponding soil.The experimental results showed that the gut microbiota of Eisenia fetida is dominated by Proteobacteria,Actinobacteriota,and Firmicutes.Arsenic contamination affected the abundance and diversity of the earthworm gut bacterial communities and could clearly alter the compositional structure of the earthworm gut bacterial communities,selectively stimulating or inhibiting the abundance of the bacterial communities.Exposure to As contamination altered the abundance of Proteobacteria,Actinobacteriota,and Bacteroidota in the gut of earthworms,which could cause imbalance in the earthworm gut microbiota and affect the degradation of organic matter and nutrient cycling in the body.In combination with the predicted functional analysis of the earthworm gut bacterial communities,it was found that the growth and reproduction of bacteria in earthworms exposed to As contamination were affected and cellular processes were weakened.（4）Earthworms were exposed to high concentrations of sodium arsenite and disodium hydrogen arsenate contaminated soil for 28 d.It was found that the total As content accumulated in the gut of earthworms was significantly higher（p<0.05）than the total As content in the whole earthworm and the epidermis.This indicated that the gut of earthworms was the main site of As accumulation.When earthworm samples were soaked in 80%ethanol and 4%paraformaldehyde fixative,it was found that 80%ethanol fixation was more effective than 4%paraformaldehyde fixation under 0.5 h of soaking.The morphology of earthworm tissues and organs was stable so that it was easy to dissect and collect.Moreover,the former method had less effect on the loss of total As content of earthworm gut and epidermis.The latter method reduced the total As content of earthworm gut and epidermis than the former method by 14.53%-51.70%.When earthworms exposed to As-contaminated soil were soaked with 4%paraformaldehyde fixation,the total As content leached from the solution showed an increasing trend with the prolongation of soaking time.The loss rate of total As in 4%paraformaldehyde fixation was 31.79%-48.91%after 32 h of soaking,and more than 98.9%of them were As-III.The loss rate of total As content in the gut and epidermis of earthworms was more than 80%.The results of this study improved the understanding of the accumulation and transformation mechanisms of As in earthworms in As-contaminated soils,which is an important reference for studying the toxicity mechanism of As to soil animals.The study demonstrated that exposure to As-contaminated soil interferes with the composition of the earthworm gut microbiota and cell growth reproduction,which provides valuable information for a deeper understanding of the role and function of gut microbiota in earthworms. |
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