Earthworm Coelomocytes Research Articles

Novel mechanistic insights into Cr(VI) and Cr(III) induced discrepancies of cellular toxicity and oxidative injury events in Eisenia fetida

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 β-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.

Earthworm coelomocytes as a soil health assessment tool

Different pollutants can disrupt earthworm coelomocytes integrity and functions, and their responses can be applied as biomarkers of sublethal contaminant exposure. In this context, the aim of this study was to develop an in vitro protocol for coelomocyte extraction, maintenance and analysis with regard to soil health status and chemical toxicity profile assessments. The extrusion technique was first tested comparing previously depurated (purged stomach content) and non-depurated and resampled earthworms. After testing, earthworms were exposed to different 2,4D and chloroacetamide concentrations for methodology validation. The values of viability were not affected by food restriction since no statistical difference was observed between non-depurated (sample A) and depurated (sample B) organisms. Regarding to cell density, a significant (p<0;05) reduction of 22% was observed between non-depurated and depurated organisms, indicating that food restriction may affect cell density. However, the non-depurated resampling did not show a significant reduction, indicating that this assessment may not be affect by resampling of the same organism. For both chemical compounds, no change in cell viability was observed at all assessed concentrations and exposure times. However, for cell density, a mainly time-dependent effect was observed for organisms exposed to chloroacetamide, and concentration-dependent effect for organisms exposed to 2,4D. The proportion of immune system cells was altered, mainly after 24 h, with the increasing of granular amoebocytes proportion. The difference in the proportion of granular amoebocytes in earthworms exposed to 2,4D can be explained by the existence of recognition and elimination mechanisms for this chemical substance. Thus, assessments of pollutant responses with in vitro coelomocytes seem to be a powerful tool for ecotoxicological studies.

The combined effects of polystyrene nanoplastics with nickel on oxidative stress and related toxic effects to earthworms from individual and cellular perspectives

Nanoplastics may adsorb other pollutants in the environment due to their high specific surface area and small size. We used earthworms as experimental organisms to evaluate the ecotoxicity of NPs and Ni combined pollution at the individual and cellular levels. The results showed that when only 20 mg/L Ni2+ was added to the combined pollution system, the antioxidant system of earthworm coelomocytes was destroyed to a certain extent, the ROS level increased, the cell viability decreased significantly, and the redox balance was destroyed. With the introduction of PS-NPs and the increase of concentration, the oxidative damage in the coelomocytes of earthworms gradually increased, and finally tended to be stable when the maximum concentration of 50 mg/L PS-NPs and Ni were exposed together. At the animal level, the activities of CAT and SOD decreased within 28 days of exposure, and the combined pollution showed a synergistic effect. At the same time, it promoted the synthesis of GST in earthworms, improved their detoxification ability and reduced oxidative damage. The changes of T-AOC and MDA showed that the combined pollution caused the accumulation of ROS and caused more serious toxicological effects. With the increase of exposure time, the antioxidant system of earthworms was continuously destroyed, and the oxidative damage was serious, which induced more serious lipid peroxidation and caused the damage of earthworm body wall structure.

Earthworm Coelomocyte Internalization of MoS2 Nanosheets: Multiplexed Imaging, Molecular Profiling, and Computational Modeling.

Fully understanding the cellular uptake and intracellular localization of MoS2 nanosheets (NSMoS2) is a prerequisite for their safe applications. Here, we characterized the uptake profile of NSMoS2 by functional coelomocytes of the earthworm Eisenia fetida. Considering that vacancy engineering is widely applied to enhance the NSMoS2 performance, we assessed the potential role of such atomic vacancies in regulating cellular uptake processes. Coelomocyte internalization and lysosomal accumulation of NSMoS2 were tracked by fluorescent labeling imaging. Cellular uptake inhibitors, proteomics, and transcriptomics helped to mechanistically distinguish vacancy-mediated endocytosis pathways. Specifically, Mo ions activated transmembrane transporter and ion-binding pathways, entering the coelomocyte through assisted diffusion. Unlike molybdate, pristine NSMoS2 (P-NSMoS2) induced protein polymerization and upregulated gene expression related to actin filament binding, which phenotypically initiated actin-mediated endocytosis. Conversely, vacancy-rich NSMoS2 (V-NSMoS2) were internalized by coelomocytes through a vesicle-mediated and energy-dependent pathway. Mechanistically, atomic vacancies inhibited mitochondrial transport gene expression and likely induced membrane stress, significantly enhancing endocytosis (20.3%, p < 0.001). Molecular dynamics modeling revealed structural and conformational damage of cytoskeletal protein caused by P-NSMoS2, as well as the rapid response of transport protein to V-NSMoS2. These findings demonstrate that earthworm functional coelomocytes can accumulate NSMoS2 and directly mediate cytotoxicity and that atomic vacancies can alter the endocytic pathway and enhance cellular uptake by reprogramming protein response and gene expression patterns. This study provides an important mechanistic understanding of the ecological risks of NSMoS2.

Molecular interaction mechanisms and cellular response of superoxide dismutase and catalase to fluoranthene.

As an important raw material and intermediate product of the petrochemical industry, fluoranthene (Fla) can be emitted with industrial activities and has become a typical polycyclic aromatic hydrocarbon enriched in the Chinese topsoil layer, posing a significant threat to sensitive soil biota. Here, multispectral tools and molecular simulation techniques were integrated to elucidate the molecular mechanism of Fla interaction with key antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) at the molecular level. Meanwhile, we further revealed the cellular responses of SOD and CAT and the associated redox states in earthworm (Eisenia fetida) coelomocytes based on the molecular-level results. Our results showed that the exposure to Fla affected the backbone structure of SOD and CAT molecules and resulted in the formation of Fla-SOD polymers as well as an overall reduction in the size of the Fla-CAT binding system. Fla altered the microenvironment around Tyr residues in the SOD molecule and quenched the endogenous fluorescence of Tyr within the CAT molecule. In earthworm coelomocytes, Fla at 60 and 80 μM resulted in a significant elevation of CAT and SOD activities by 114% (p = 0.032) and 6.09% (p = 0.013), respectively. Molecular simulation results suggested that Fla-induced changes in the structure and conformation of SOD and CAT may be the key reason for their altered activities. The related redox homeostasis detection in earthworm coelomocytes indicated that high concentrations (80 μM) of Fla led to a significant accumulation of intracellular ROS (p = 0.018) and resulted in the development of lipid peroxidation. Our work contributes to an in-depth understanding of the biological effect of Fla to sensitive soil fauna, thus providing new ideas for Fla ecological risk prevention and control.

Coupled Lipidomics and Digital Pathology as an Effective Strategy to Identify Novel Adverse Outcome Pathways in Eisenia fetida Exposed to MoS2 Nanosheets and Ionic Mo.

Molybdenum disulfide (MoS2) nanosheets are increasingly applied in several fields, but effective and accurate strategies to fully characterize potential risks to soil ecosystems are lacking. We introduce a coelomocyte-based in vivo exposure strategy to identify novel adverse outcome pathways (AOPs) and molecular endpoints from nontransformed (NTMoS2) and ultraviolet-transformed (UTMoS2) MoS2 nanosheets (10 and 100 mg Mo/L) on the earthworm Eisenia fetida using nontargeted lipidomics integrated with transcriptomics. Machine learning-based digital pathology analysis coupled with phenotypic monitoring was further used to establish the correlation between lipid profiling and whole organism effects. As an ionic control, Na2MoO4 exposure significantly reduced (61.2-79.5%) the cellular contents of membrane-associated lipids (glycerophospholipids) in earthworm coelomocytes. Downregulation of the unsaturated fatty acid synthesis pathway and leakage of lactate dehydrogenase (LDH) verified the Na2MoO4-induced membrane stress. Compared to conventional molybdate, NTMoS2 inhibited genes related to transmembrane transport and caused the differential upregulation of phospholipid content. Unlike NTMoS2, UTMoS2 specifically upregulated the glyceride metabolism (10.3-179%) and lipid peroxidation degree (50.4-69.4%). Consequently, lipolytic pathways were activated to compensate for the potential energy deprivation. With pathology image quantification, we report that UTMoS2 caused more severe epithelial damage and intestinal steatosis than NTMoS2, which is attributed to the edge effect and higher Mo release upon UV irradiation. Our results reveal differential AOPs involving soil sentinel organisms exposed to different Mo forms, demonstrating the potential of liposome analysis to identify novel AOPs and furthermore accurate soil risk assessment strategies for emerging contaminants.

Nano zinc oxide induced lipid peroxidation, oxidative stress, genotoxicity, phagocytic alteration, and detoxification response in the coelomocytes of an anecic earthworm of India

Zinc oxide nanoparticle (ZnO NP) is an emerging soil pollutant of industrial origin. The unique physicochemical properties of ZnO NP viz. the bioavailability and subsequent ecotoxicological effects on organisms have made it an ecological concern. Lampito mauritii (Kinberg, 1866) is a common anecic earthworm and is considered as a keystone species of soil ecosystems in India. They are continuously exposed to various environmental toxins during their feeding and other activities. The current study aimed to estimate the immunotoxicological effects of ZnO NP in the coelomocytes, the chief immunoeffector cells of L. mauritii. The results of the study indicated decreased total count, phagocytic activity, antioxidative, and detoxifying potential of the coelomocytes in L. mauritii at 7 and 14 days of exposure. ZnO NP exposure resulted in the generation of oxidative molecules, lipid peroxidation, and DNA damage within coelomocytes. The higher degree of cytotoxicity on ZnO NP exposure was suggestive of immunocompromisation in L. mauritii. An integrated biomarker response (IBR) guided methodology was also utilized in estimating the comprehensive effects of ZnO NP on the health of L. mauritii. The present study would provide useful information on the ZnO NP induced chemical stress in L. mauritii, with the selected endpoints having the potential to function as markers for ZnO NP toxicity.

Nanoplastics induces oxidative stress and triggers lysosome-associated immune-defensive cell death in the earthworm Eisenia fetida

Nanoplastics (NPs) are increasingly perceived as an emerging threat to terrestrial environments, but the adverse impacts of NPs on soil fauna and the mechanisms behind these negative outcomes remain elusive. Here, a risk assessment of NPs was conducted on model organism (earthworm) from tissue to cell. Using palladium-doped polystyrene NPs, we quantitatively measured nanoplastic accumulation in earthworm and investigated its toxic effects by combining physiological assessment with RNA-Seq transcriptomic analyses. After a 42-day exposure, earthworm accumulated up to 15.9 and 143.3 mg kg−1 of NPs for the low (0.3 mg kg−1) and high (3 mg kg−1) dose groups, respectively. NPs retention led to the decrease of antioxidant enzyme activity and the accumulation of reactive oxygen species (O2− and H2O2), which reduced growth rate by 21.3 %–50.8 % and caused pathological abnormalities. These adverse effects were enhanced by the positively charged NPs. Furthermore, we observed that irrespective of surface charge, after 2 h of exposure, NPs were gradually internalized by earthworm coelomocytes (∼0.12 μg per cell) and mainly amassed at lysosomes. Those agglomerations stimulated lysosomal membranes to lose stability and even rupture, resulting in impeded autophagy process and cellular clearance, and eventually coelomocyte death. In comparison with negatively charged nanoplastics, the positively charged NPs exerted 83 % higher cytotoxicity. Our findings provide a better understanding of how NPs posed harmful effects on soil fauna and have important implications for evaluating the ecological risk of NPs.

Cytotoxicity and Oxidative Stress Effects of Indene on Coelomocytes of Earthworm (Eisenia foetida): Combined Analysis at Cellular and Molecular Levels.

Indene (IND) is a kind of important aromatic hydrocarbon that is extracted from coal tar and has important applications in industry and biology. In the process of production and utilization, it is easy to enter the soil and produce toxic effects on the soil or organisms. The earthworm is an important organism in the soil. The toxicity of indene on earthworm coelomocytes is rarely studied, and the oxidative stress effects of IND on earthworm coelomocytes remain unclear. In this study, coelomocytes from earthworms and antioxidant enzymes were selected as the research targets. In addition, IND caused oxidative stress, and its related toxic effects and mechanisms were systematically studied and evaluated at the cellular and molecular levels. The results showed that IND destroyed the redox balance in earthworm coelomocytes, and the large accumulation of reactive oxygen species (ROS) significantly inhibited the activities of the antioxidant system, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), and caused lipid peroxidation and membrane permeability changes, resulting in a decrease in cell viability to 74.5% of the control group. At the molecular level, IND was bound to SOD by the arene-H bond, and the binding constant was 4.95 × 103. IND changed the secondary structure of the SOD and led to a loosening of the structure of the SOD peptide chain. Meanwhile, IND caused SOD fluorescence sensitization, and molecular simulation showed that IND was mainly bound to the junction of SOD subunits. We hypothesized that the changes in SOD structure led to the increase in SOD activity. This research can provide a scientific basis for IND toxicity evaluation.

Distinct Uptake Routes Participate in Silver Nanoparticle Engulfment by Earthworm and Human Immune Cells.

The consequences of engineered silver nanoparticle (AgNP) exposure and cellular interaction with the immune system are poorly understood. The immunocytes of the Eisenia andrei earthworm are frequently applied in ecotoxicological studies and possess functional similarity to vertebrate macrophages. Hence, we characterized and compared the endocytosis mechanisms for the uptake of 75 nm AgNPs by earthworm coelomocytes, human THP-1 monocytes, and differentiated THP-1 (macrophage-like) cells. Our results indicate that microtubule-dependent, scavenger–receptor, and PI3K signaling-mediated macropinocytosis are utilized during AgNP engulfment by human THP-1 and differentiated THP-1 cells. However, earthworm coelomocytes employ actin-dependent phagocytosis during AgNPs uptake. In both human and earthworm immunocytes, AgNPs were located in the cytoplasm, within the endo-/lysosomes. We detected that the internalization of AgNPs is TLR/MyD88-dependent, also involving the bactericidal/permeability-increasing protein (BPI) in the case of human immunocytes. The exposure led to decreased mitochondrial respiration in human immunocytes; however, in coelomocytes, it enhanced respiratory parameters. Our findings provide more data about NP trafficking as nano-carriers in the nanomedicine field, as well as contribute to an understanding of the ecotoxicological consequences of nanoparticle exposure.

A comparative account of coelomocyte of earthworm ecotypes with reference to its morphology, morphometry, density, phagocytosis, autofluorescence, and oxidative status.

Earthworms inhabit different strata of moist soil. Epigeic and endogeic earthworms prefer superficial and inner stratum of soil respectively, whereas, semiaquatic species are distributed around hydrated soil near ponds and lakes. Coelomocytes, the chief immunoeffector cells of coelomic origin, perform diverse physiological functions like phagocytosis, maintenance of cellular homeostasis, and acid-base balance of coelomic fluid, graft rejection, elicitation of cytotoxic, and oxidative responses under the challenges of pathogens and toxins. The present study aims to analyze selected morphological and functional parameters in three differentially adapted Indian earthworms of nonsimilar habitats. Coelomocytes of Glyphidrilus tuberosus (Stephenson, 1916) (semiaquatic), Perionyx excavatus (Perrier, 1872) (epigeic), and Eutyphoeus orientalis (Beddard, 1883) (endogeic) were isolated for morphological and morphometric analyses and subjected to determination of phagocytic, oxidative, and cytotoxic responses. Activities of phenoloxidase, pro, and antioxidant enzymes, and autofluorescence were determined in the extruded coelomocytes of earthworms of three contrasting habitats. The differential result may be correlated with species-specific responses and variation in habitat preference and related adaptation.

Length- and polymer-dependent ecotoxicities of microfibers to the earthworm Eisenia andrei

Microfibers are widespread environmental pollutants introduced to the soil environment because of the increasing use of plastic polymers. However, research on the soil ecotoxicity of microfibers is limited, especially when compared to research on their aquatic toxicity. This study investigated the effects of sub-chronic microfiber exposure on the earthworm Eisenia andrei. We compared the effects of three types of microfibers: short lyocell microfibers (231 ± 126 μm long), short polypropylene microfibers (191 ± 107 μm long), and long polypropylene microfibers (891 ± 141 μm long). After exposure for 21 days, earthworm survival, coelomocyte viability, cast microbial viability, and gut microbial viability were assessed, and a histopathological examination of the digestive tract and reproductive tissues was conducted. In addition, long polypropylene microfibers egested by the earthworms were collected to explore the possibility of earthworm-driven biofragmentation. Results indicated that high exposure concentration (1000 mg/kg dry soil) negatively affected earthworm coelomocytes and intestinal tissue, gut, and cast microbiomes. Although all three microfiber types reduced earthworm survival, the short polypropylene microfibers were more toxic to the earthworms than the long polypropylene microfibers or short lyocell microfibers, which indicated that size-dependent soil ecotoxicity was induced. PP microfibers were found to more negatively affect cast microbial activity and intestinal tissue than lyocell microfibers, indicating polymer-dependent soil ecotoxicity potential against earthworm species. This study provides evidence that synthesized microfibers cause cytotoxicity and decrease gut microbiome viability in earthworms, and that they can be biofragmented by earthworms.

Characterisation of Lectin from Pisum sativum and Agaricus bisporus and their Immunomodulatory Effects on Earthworm Coelomocytes (Immunomodulatory studies)

Characterisation of Lectin from Pisum sativum and Agaricus bisporus and their Immunomodulatory Effects on Earthworm Coelomocytes (Immunomodulatory studies)

Metal accumulation and morphofunctional damage in coelomocytes of earthworm collected from industrially contaminated soil of Kolkata, India

The current study is aimed to assess the ecotoxicological effects of toxic metals and seasonal shift of the physicochemical characteristics of soil in an endogeic earthworm Metaphire posthuma of industrially contaminated soil of Calcutta leather complex. The accumulation of cadmium, chromium, lead and mercury was quantitated in whole earthworms and coelomocytes. The accumulation of metals was derived to be high in the coelomocytes than whole earthworms. Morphofunctional shift in coelomocytes indicated a high level of metal toxicity in soil inhabitants. The shift in differential coelomocyte count and cellular damage including intense cytoplasmic vacuolation and membrane blebbing of coelomocytes of M. posthuma of contaminated soil were suggestive to a state of immunocompromisation in the same species. Shift in the generation of nitric oxide and activity of inducible nitric oxide synthase indicated a possible immunosuppression in earthworm. Depletion in the acetylcholinesterase activity of coelomocytes indicated neurotoxicity of metals leached from the dumped wastes in Calcutta leather complex. Integrated biomarker response based analysis was carried out to assess the biomarker potential of experimental endpoints of M. posthuma to monitor metal toxicity in soil.

Response of earthworm coelomocytes and catalase to pentanone and hexanone: a revelation of the toxicity of conventional solvents at the cellular and molecular level.

Organic solvents like 2-pentanone and 2-hexanone which are widely used in industrial production make up a large proportion of the source of chemical pollution. What is worrisome is that the cellular and molecular toxicity of 2-pentanone and 2-hexanone has not been reported yet. Based on this, earthworms and catalase (CAT) were chosen as target receptors for the toxicity studies. The cytotoxicity of 2-pentanone and 2-hexanone was revealed by measuring the multiple intracellular indicators of oxidative stress. At the molecular level, changes in the structure and function of CAT were characterized in vitro by the spectroscopy and molecular docking. The results show that 2-pentanone and 2-hexanone that induced the accumulation of reactive oxygen species can eventually reduce coelomocytes viability, accompanying by the regular changes of antioxidant activity and lipid peroxidation level. In addition, the exposure of 2-pentanone and 2-hexanone can shrink the backbone structure of CAT, quench the fluorescence, and misfold the secondary structure. The decrease in enzyme activity should be attributed to the structural changes induced by surface binding. This study discussed the toxicological effects and mechanisms of conventional solvents at the cellular and molecular level, which creatively proposed a joint research method.

Cellular and molecular responses of earthworm coelomocytes and antioxidant enzymes to naphthalene and a major metabolite (1-naphthol)

Naphthalene exerts potential threats on the soil environment and soil-dwelling species. The toxic effects on soil organisms, the response of antioxidant enzymes and the underlying molecular mechanisms for naphthalene metabolites remain to be fully elucidated. Here we report the cytotoxicity and oxidative stress induced by naphthalene and a major metabolite (1-naphthol) on Eisenia fetida coelomocytes and the interaction mechanism between 1-naphthol and the critical antioxidant enzymes (CAT and SOD). Earthworm coelomocytes were more susceptible to 1-naphthol than to naphthalene. At the molecular level, 1-naphthol preferentially bound to the active site of CAT with catalytic residues (His 74, Tyr 357 and Asn147) and the surface of SOD via hydrophobic forces and hydrogen bonds. Direct exposure to 1-naphthol resulted in the unfolding of the CAT skeleton accompanied by secondary structure changes, though only changes in SOD skeleton were observed, without a change in the secondary structure. Static fluorescence quenching of CAT and SOD were initiated with 1-naphthol by the formation of a non-fluorescent complex. Direct 1-naphthol binding inhibited the activity of CAT but presented no adverse effects on the activity of SOD. This paper provides a theoretical basis for the elucidation and better understanding of the toxicity of naphthalene and its metabolites.

Toxic mechanism on phenanthrene-induced cytotoxicity, oxidative stress and activity changes of superoxide dismutase and catalase in earthworm (Eisenia foetida): A combined molecular and cellular study

Phenanthrene (PHE) is an important organic compound, which is widespread in the soil environment and exhibits potential threats to soil organisms. Toxic effects of PHE to earthworms have been extensively studied, but toxic mechanisms on PHE-induced cytotoxicity and oxidative stress at the molecular and cellular levels have not been reported yet. Therefore, we explored the cytotoxicity and oxidative stress caused by PHE in earthworm coelomocytes and the interaction mechanism between PHE and the major antioxidant enzymes SOD/CAT. It was shown that high-dose PHE exposure induced the intracellular reactive oxygen species (ROS) generation, mediated lipid peroxidation, reduced total antioxidant capacity (T-AOC) in coelomocytes, and triggered oxidative stress, thus resulted in a strong cytotoxicity at higher concentrations (0.6–1.0 mg/L). The intracellular SOD/CAT activity in cells after PHE exposure were congruent with that in molecular levels, which the activity of SOD enhanced and CAT inhibited. Spectroscopic studies showed the SOD/CAT protein skeleton and secondary structure, as well as the micro-environment of aromatic amino acids were changed after PHE binding. Molecular docking indicated PHE preferentially docked to the surface of SOD. However, the key residues Tyr 357, His 74, and Asn 147 for activity were in the binding pocket, indicating PHE more likely to dock to the active center of CAT. In addition, H-bonding and hydrophobic force were the primary driving force in the binding interaction between PHE and SOD/CAT. This study indicates that PHE can induce cytotoxicity and oxidative damage to coelomocytes and unearthes the potential effects of PHE on earthworms.

Post COVID-19 pandemic: Biofragmentation and soil ecotoxicological effects of microplastics derived from face masks

Because of the COVID-19 pandemic, used face masks have increasingly littered the environment and are causes for concern since they are commonly made of plastics such as polypropylene. Understanding production of microplastics from face masks is essential for predicting the post COVID-19 pandemic impact on the soil ecosystem. We investigated the generation of nanofibers from meltblown face mask filters (MB filters) and their adverse effects on soil species, particularly the earthworm and springtail. Results of MB filter soil bioassays at a high concentration (1000 mg/kg dry soil) suggest inhibited reproduction and stunted growth in springtails, decreased intracellular esterase activity in earthworm coelomocytes, and inhibited spermatogenesis in male earthworm reproductive tissues. Moreover, it was estimated that generation of nanofibers from microfibers and fragments of MB filters might occur in the soil ecosystem post COVID-19. This study does not oppose the use of face masks but aims to encourage appropriate disposal of the masks. Preservation of human health and the ecosystem should be prioritized even amidst the COVID-19 pandemic.

Influences and mechanisms of nanoparticles on pentachloronitrobenzene accumulation by earthworms

Pesticides and nanoparticles may coexist in soil; however, influences of nanoparticles on accumulation of pesticides in terrestrial organisms are still unclear. This study aims to investigate the influences and mechanisms of metal oxide nanoparticles (nano ZnO and nano CuO) on accumulation of pentachloronitrobenzene (PCNB) in earthworms and their combined toxicity. The earthworms were cultivated in the soil spiked with nanoparticles (10, 50, 250 mg/kg) and PCNB (100 μg/kg) for 21 days. The concentrations of PCNB in earthworms in binary exposure treatments (PCNB + ZnO and PCNB + CuO) reached 2.47 and 3.13 times of that in individual PCNB exposure treatment, indicating that nanoparticles facilitated the accumulation of PCNB in earthworms. The contents of reactive oxygen species (ROS) in earthworms in treatments PCNB + ZnO 250 and PCNB + CuO 250 reached 379 and 316 fluorescence intensity/mg Protein, respectively, which were significantly higher than that in control group (183 fluorescence intensity/mg protein), indicating that nanoparticles would cause oxidative stress to earthworms. Earthworm coelomocytes were extracted from healthy earthworms and cultivated in culture media in cytotoxicity tests. Changes of intracellular ROS contents and cell viability suggested that PCNB and nanoparticles caused serious oxidative damage to earthworm coelomocytes, thus leading to the damage of cell membrane and cell death. In in vivo tests, changes of biomarkers (ROS and malondialdehyde) demonstrated that these pollutants injured the earthworms. Increased accumulation of PCNB in binary exposure treatments was due to the damage of body cavity caused by nanoparticles. This study provides a novel hypothesis for nanoparticles facilitating organic pollutants entering terrestrial organisms and determines whether nanoparticles would bring about greater environmental risks of other pollutants.

Effects of biochar and thermally treated biochar on Eisenia fetida survival, growth, lysosomal membrane stability and oxidative stress

Despite its known positive impacts when added to soil, the negative effects of biochar on earthworms are not fully understood. Here, we investigated the toxicity of nine biochars of three feedstock origins, animal (cow dung), plant (corncob) and microorganism (sewage sludge), produced at three pyrolysis temperatures (350 °C, 550 °C and 750 °C) on earthworms. Vermitoxicity was first assessed using acute toxicity test, neutral red retention time (NRRT) assay and oxidative stress response assay. Furthermore, we evaluated whether the thermal treatment of biochars could reduce their vermitoxicity using an acute toxicity assay. We found that, according to LC50 and earthworm weight loss, cow dung biochar was more toxic than corncob or sewage sludge biochar; thus, production feedstock is apparently important to biochar vermitoxicity. Furthermore, NRRTs indicated cow dung biochar disrupted lysosomal membrane stability in earthworm coelomocytes, providing further evidence for the toxicity of this biochar to earthworms. Disturbed antioxidant enzyme activities and elevated malondialdehyde content showed that earthworm suffer oxidative stress, also implying a potential vermitoxicity. However, thermal treatment of cow dung biochar substantially improved its LC50 and decreased earthworm weight loss, implying that the PAHs in this biochar might be damage factors and that heating could reduce the potential toxicity of biochar. Besides, NRRT assay was first used to evaluate the effects of biochar on earthworms and clear dose-effect relationships indicated that NRRT assay might be a useful tool for assessing the potential negative effects of biochar. Overall, given the different effects of various biochars, including toxicity, reported here, our findings will help improve understanding of biochar vermitoxicity mechanisms, serve to improve biochar ecological risk assessments and provide a reference for the proper application of biochar amendments.