Stress Response Of Earthworm To Arsenic Pollution And Its Effects On Soil Physical And Chemical Properties

Mineral development and smelting,pesticide,and industrial wastewater caused large increased arsenic discharge and thus resulted in different levels of arsenic pollution.Sodium arsenite is a typical trivalent arsenic compound,which has been widely used in insecticides and wood preservatives,and used in other modern industries.At present,the research about the soil arsenic pollution mainly facus on the arsenic forms,environmental behavior and the remediation technology.Only a few studies focused on the ecological toxicology of arsenic in soil,and the basic data and theory of arsenic pollution control are still insufficient.Earthworm is one of the main animal groups in the soil and is known as a soil ecological engineer.Earthworms can reconstruct the surface soil,improve the physical and chemical properties of soil and increase crop yields by burrow.The ecological toxicology of arsenic on earthworms mainly studied the effect of arsenic on some common physiological and biochemical indicators in earthworms,but there is no report on the effect of earthworms’burrowing and the mechanism of oxidative stress.Based on the above reasons,earthworms were selected as experimental animals to study the effects of sodium arsenite on earthworms’burrow behavior and some typical molecular toxicological indicators,trying to explore the molecular mechanism of earthworm behavior toxicity;and then we studied the effects of earthworms’burrow behavior on soil physical and chemical properties,in order to provide a basis for monitoring,evaluation and management of soil arsenic pollution.The main results are as follows:1)Effect of arsenic on the earthworms’burrowing behaviorFirstly,the median lethal concentration(LC₅₀)of sodium arsenite against strontium was determined by filter paper method and artificial soil method,and then a series of concentration gradients were set according to the determined LC₅₀ for subsequent research.The results showed that the artificial soil method LC₅₀ was 122 mg.kg^-1 dry soil.The result of the avoidance experiment indicated that the earthworms have some tolerance to the arsenite.Based on this result,we investigated the effects of early arsenic contamination(5,10,20,40,80 mg.As kg^-1)on the burrow behavior of two kinds earthworms（Metaphire californica and Eisenia foetida）,long-term arsenic stress(5,20,80 mg.As kg^-1)and the arsenite exposure(20 mg.kg^-1)in transparent soil on the burrow behavior of E.foetida.Since this method has been established not for long time,the changes of earthworm behavior under arsenic stress of 20 mg.kg^-1 were preliminarily studied in this paper.The results showed that the cumulative burrowing distance increased with time,but the burrowing ability was different in different arsenic-contaminated soils.The cumulative burrow length increased with the increase of arsenic concentration in soil,but it was opposite in aging arsenic-contaminated soil.In the early stage of the arsenic pollution soil,the cumulative burrow length of M.californica is significantly higher than that of E.foetida.The daily burrow length of M.californica is also hingher than that of E.foetida.The maximum burrow depth of of M.californica is larger than that of E.foetida.This may be related to their sensitivity to arsenic.Under the long-term arsenic stress and the transparent soil with arsenic,the burrow activity of earthworms was inhibited.2)Oxidative stress response of earthworm to sodium arseniteAdded different amounts of sodium arsenite(in terms of arsenic,concentrations of 0,5,10,20,40,80 mg.kg^-1)in natural soil.Samples were taken at 0,3,7,14,21,28 d to determine the response of oxidative stress indicators in earthworm.The results showed that after exposurement,the arsenic in the earthworm could be acumulate from 3.05 mg.kg^-1 to 330mg.kg^-1,and the bioconcentration factors was 0.79-3.8.The content of reactive oxygen species（ROS）in the earthworm increased with the arsenic concentration increased;the activities of superoxide dismutase（SOD）and catalase（CAT）declined with the arsenic increased;The amount of malondialdehyde（MDA）produced in the earthworm increased with arsenic concentration increasing.In order to investigate the oxidative stress process in earthworm,this paper studied the changes of Nuclear factor（erythroid-derived 2）-like 2（Nrf2）signaling pathway.The results showed that the characteristic protein Nrf2 and the downstream protein heme oxygenase 1（HO-1）of this Nrf2 signaling pathway up-regulated with the increase of arsenic concentration and exposure time;ROS production,SOD activity,CAT enzyme activity and the amount of MDA produced was significantly correlated with soil arsenic concentration,exposure time,and the interaction between the two factors.The expression of Nrf2 and HO-1 was significantly correlated with exposure time,exposure concentration and the interaction between the two factors.These results indicate that the antioxidant balance system is destroyed after the arsenic accumulated in the earthworm,causing certain oxidative damage to the body,which affect the behavior of the earthworms.3)Effect of arsenic stress on the expression of total proteinTwo-dimensional electrophoresis was used to study the effects of different concentrations of sodium arsenite(0,5,10,20,40,80 mg.kg^-1)on the expression of total protein.The results show that there were a total of 36protein spots in all the groups,the significant difference was more than 2times,of which 24 protein spots were successfully identified.The identified proteins are divided into seven major components according to their functions:（1）metabolic related proteins,such as ATP-binding proteins,metal-binding proteins and lumbrokinases,accounting for 33%of the total;（2）stress-responsive proteins,such as heat shock protein（HSP）,etc.,accounting for 8%of the total;（3）transport-related proteins,such as protein transport and lipid transport of transferrin and serum albumin,accounting for 13%of the total;（4）signal transduction proteins,such as intermediate silk protein,accounting for 6%of the total;（5）regulatory proteins,such as cingulate protein,accounting for 13%of the total;（6）predicted and hypothetical protein,accounting for 8%of the total;（7）other,such as dystrophin,etc.,accounting for 13%of the total.We found that these differential proteins are closely related to changes in earthworms’behavior.Actin may be directly related to the movement of earthworm,and the expression of ATP-binding kinase is down-regulated,indicating that ATP synthesis is decreased under arsenic stress,causing weight loss.The expression changes of dystrophin may be related to the morphological changes of earthworms under arsenic stress.4)Effects of earthworm activity on soil physical and chemical properties in arsenic contaminated soilThe soil contaminated with different arsenic concentrations(0,5,20,80 mg.kg^-1)was aged for one month and inoculated with earthworms for 60days.Then measure the changes of soil physical and chemical properties before and after inoculation.The results show that the proportion of arsenic forms changed after aging,the residual arsenic（Re-As）content was the highest,the composition of loose-bound arsenic（Av-As）was the lowest.After 60 days of earthworm inoculation,the proportion of Av-As increased,the corresponding proportion of Re-As decreased.After the earthworms’incubation,the conversion rate of soil arsenic morphology in the treatments with arsenite was lower than that of the control groupn.After aging,the soil pH value of arsenic-contaminated soil was significantly lower than that of control group;soil conductivity and nitrate nitrogen content significantly increased;soil available potassium content significantly decreased as the arsenite increasing.After 60 days inoculation with earthworms,the content of ammonia nitrogen in soil decreased significantly;the content of nitrate nitrogen in soil increased significantly,and the content of nitrate nitrogen in soil increased with the increasing of arsenic concentration,reached the maximum value in 80 mg.kg^-1 treatment,which was significantly higher than other treatments.After earthworms’activity,the content of soil available phosphorus increased rapidly,which was significantly higher than that of the aging soil.The content of available potassium in soil was significantly higher than that of aging soil in the same treatment group.The results show that earthworm activities could affect the physical and chemical properties of arsenic contaminated soil.