It has not been known how immune responses in soil invertebrates occur against microplastics (MPs). This study aims to investigate the effects of MPs on endocytosis, including phagocytosis and pinocytosis, of immune cells of soil invertebrates in the soil ecosystem in the process of bacterial infection. We employed polystyrene micro- plastics (similar to 1 mu m PS MPs) to treat earthworm Eisenia andrei during the infection of Escherichia coli for in vitro (1, 5, 10, and 50 mg/L) and in vivo (1, 10, and 1000 mg/kg dry soil) assays. The results of in vitro migration assay revealed that MPs caused inhibitory effects on the phagocytosis, pinocytosis and oxidative stress in coelomocytes. Soil bioassay also confirmed that endocytosis of coelomocytes and mitochondrial damages in the intestinal epithelium were significantly altered in the polluted soil with MPs. Thus, MPs induced adverse effects to inhibit bacterial endocytosis, which may disturb the immune system of soil invertebrates. This study is the first report on the inhibition of phagocytosis in the soil invertebrates by MPs. These findings contribute to understanding the response of soil invertebrates, which play important roles in the soil food web with cellular level towards microplastic pollution in soil.

Chromium (Cr) poses a high ecological risk, however the toxic mechanisms of Cr in different valence states to soil organisms at cellular and molecular levels are not exactly. In this study, the Eisenia fetida coelomocytes and Cu/ Zn-superoxide dismutase (Cu/Zn-SOD) were chosen as the target subjects to investigate the effects and mechanisms of cellular toxicity induced by Cr(VI) and Cr(III). Results indicated that Cr(VI) and Cr(III) significantly reduced the coelomocytes viability. The level of reactive oxygen species (ROS) was markedly increased after Cr (VI) exposure, which finally reduced antioxidant defense abilities, and induced lipid peroxidation and cellular membrane damage in earthworm coelomocytes. However, Cr(III) induced lower levels of oxidative stress and cellular damage with respect to Cr(VI). From a molecular perspective, the binding of both Cr(VI) and Cr(III) with Cu/Zn-SOD resulted in protein backbone loosening and reduced beta-Sheet content. The Cu/Zn-SOD showed fluorescence enhancement with Cr(III), whereas Cr(VI) had no obvious effect. The activity of Cu/Zn-SOD continued to decrease with the exposure of Cr. Molecular docking indicated that Cr(III) interacted more readily with the active center of Cu/Zn-SOD. Our results illustrate that oxidative stress induced by Cr(VI) and Cr (III) plays an important role in the cytotoxic differences of Eisenia fetida coelomocytes and the binding of Cr with Cu/Zn-SOD can also affect the normal structures and functions of antioxidant defense-associated protein.

Copper (Cu) toxicity is a pressing concern for several soils, especially in organic viticulture. The objective of this work was to assess Cu toxicity on the non-target organism Eisenia fetida, employing both traditional and novel tools for early identification of Cu-induced damages. In addition to traditional tests like avoidance and reproductive toxicity experiments, other tests such as the single cell gel electrophoresis (SCGE) and gut microbiome analysis were evaluated to identify early and more sensitive pollution biomarkers. Four sub-lethal Cu concentrations were studied, and the results showed strong dose-dependent responses by the earthworm avoidance test and the exceeding of habitat threshold limit at the higher Cu doses. An inverse proportionality was observed between reproductive output and soil Cu concentration. Bioaccumulation was not detected in earthworms; soil concentrations of potentially bioavailable Cu were not affected by E. fetida presence or by time. On the contrary, the SCGE test revealed dose-dependent genotoxicity for the 'tail length' parameter already at the second day of Cu exposition. Gut microbiome analysis a modulation of microbial composition, with the most aboundant families being Pectobateriaceae, Comamonadaceae and Microscillaceae. Bacillaceae increased over time and showed adaptability to copper up to 165 mg/kg, while at the highest dose even the sensitive Acetobacteriaceae family was affected. The research provided new insights into the ecotoxicity of Cu sub-lethal doses highlighting both alterations at earthworms' cellular level and changes in their gut microbiota.

With the expansion of urban areas, the amount of sludge produced by sewage treatment plants is increasing, raising big problems regarding the reintroduction of this sludge into nature in order to fully solve the wastewater problem. The application of sludge to agricultural surfaces or degraded land is a controversial solution since, despite the well-known benefits, sludge can, in certain cases, represent a real threat to both human health and the environment, with long-term harmful effects. The present study evaluates the potential genotoxicity of sludge using the Comet Test and three cellular bioindicators (lymphocytes, coelomocytes, and Allium cepa L.) for its quantification. To perform the tests, the soluble fraction of the sludge was used at concentrations of 25%, 50%, 75%, and 100%, as well as a negative control (H2O) and a positive control (H2O2). The Comet test indicated an increase in DNA damage among cells exposed for 4 h in the following order: coelomocytes, lymphocytes, and Allium cepa L. cells. Our results indicate that Allium cepa L. nuclei are more sensitive, with genotoxic effects being evident at concentrations as low as 25%. In coelomocytes, we recorded nuclear damage starting at a concentration of 75%. These results indicate the necessity of using multiple genotoxicity tests, combined in a test battery, to achieve a greater level of relevance. The concentration of the soluble fraction of the sludge has an inverse relationship with the auxin content in leaves and roots, suggesting varying levels of stress. The results of this study can contribute to the creation of a genotoxic profile of sewage sludge, facilitating decisions related to reducing its negative impact.