Micro- (M-BC) and nano-biochar (N-BC) particles are ready to be disintegrated from biochar (BC), which is extensively applied to remediate heavy metal-contaminated soil. Still, its effects on the remediation efficiency remain poorly understood. This study investigated the interactions between the BC particles (M-BC and N-BC) and Eisenia foetida (E. foetida) in cadmium (Cd)-contaminated soils. Results indicated that M-BC has weak negative effect on E. foetida with survival rates of ≥85% as it is failed to be internalized. The interactive effects between M-BC/N-BC and Cd reduced the mobility of Cd, leading to low avoidance behavior of E. foetida. The synergistic effect of 0.1% M-BC and E. foetida caused pH regulation, BC diffusion and alternation of soil microbial community in the soil. This favored the remediation of Cd-contaminated soils with 56.2% Cd fixation efficiency identified. Conversely, internalization of Cd-loaded N-BC by E. foetida was recorded when 0.1% N-BC was amended in the soil. This triggered DNA damage, antioxidant suppression, antiapoptotic inhibition, digestion impairment, reproductive decline, and survival rates reduction (55%) in E. foetida, indicating the essential role of E. foetida in the soil is likely to be depressed. These findings are helpful to understand the potential negative effects of BC application in soil remediation.

Thallium (Tl), though not essential for biological systems, is widely used in industrial activities, resulting in soil pollution and adverse effects on soil biota. Systematic toxicological studies on Tl, especially concerning soil organisms, are relatively rare. This research evaluates the toxic effects of Tl on earthworms by measuring oxidative stress biomarkers, such as superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG), and by assessing the expression of functional genes, such as heat shock protein 70 (Hsp70), metallothionein (MT), and annetocin (ANN). Additionally, this study employs the Biomarker Response Index (BRI) and two-way ANOVA to comprehensively assess the cumulative toxicity of Tl in earthworms. The findings indicate that Tl exposure significantly exacerbates oxidative stress and cellular damage in earthworms, particularly under conditions of high concentration and prolonged exposure. BRI results demonstrate a continuous decline in the physiological state of earthworms with increasing Tl concentration and exposure duration. Two-way ANOVA reveals significant dose-responsive increases in SOD and CAT activities, as well as in ANN gene expression. Apart from GST activity, other biomarkers significantly increased over time, and the changes in biomarkers such as SOD, CAT, MDA, and 8-OHdG were significantly influenced by dose and time. LSD post hoc tests show significant effects of dose, time, and their interactions on all biomarkers except for GST. These findings are valuable for gaining a deeper understanding of the ecological risks of Tl in soil environments and its potential threats to soil biota, aiding in the management of ecological risks associated with Tl-contaminated soils.

The ecological risks of sewage sludge biochar (SSB) after land use is still not truly reflected. Herein, the ecological risks of SSB prepared at different temperature were investigated using the earthworm E. fetida as a model organism from the perspectives of organismal, tissue, cellular, and molecular level. The findings revealed that the ecological risk associated with low-temperature SSB (SSB300) was more pronounced compared to medium- and high-temperature SSB (SSB500 and SSB700), and the ecological risk intensified with increasing SSB addition rates, as revealed by an increase in the integrated biomarker response v2 (IBRv2) value by 2.59-25.41 compared to those of SSB500 and SSB700. Among them, 10% SSB300 application caused significant oxidative stress and neurotoxicity in earthworms compared to CK (p < 0.001). The weight growth rate and cocoon production rate of earthworms were observed to decrease by 25.06% and 69.29%, respectively, while the mortality rate exhibited a significant increase of 33.34% following a 10% SSB300 application, as compared to the CK. Moreover, 10% SSB300 application also resulted in extensive stratum corneum injury and significant longitudinal muscle damage in earthworms, while also inducing severe collapse of intestinal epithelial cells and disruption of intestinal integrity. In addition, 10% SSB300 caused abnormal expression of earthworm detoxification and cocoon production genes (p < 0.001). These results may improve our understanding of the ecotoxicity of biochar, especially in the long term application, and contribute to providing the guidelines for applying biochar as a soil amendment.

Heavy metal contamination presents a profound threat to terrestrial biodiversity, yet the genetic adaptation and evolution of field organisms under persistent stress are poorly understood. In this study, the Cd-resistant earthworms Metaphire californica collected from the control (Meihua, MHC) and elevated-pollution (Lupu, LPC) pairwise sites were used to elucidate the underlying genetic mechanism. A 48-h acute test showed that LPC worms exhibited 2.34 times higher LC50 (50% lethal concentration values) compared to MHC ones. The Cd bioaccumulation, metallothionein (MT) protein contents, and MT gene expression of LPC M.californica were all significantly higher than those of MHC worms. The well-known MT gene of M.californica was successfully cloned and identified, however, the encoding nucleotide and amino acids displayed non-observable mutations and the phylogenetic tree also revealed that different populations clustered together. Additionally, the results of transcriptomics sequencing demonstrated 173 differentially expressed genes between LPC and MHC worms, primarily involved in stress-response and detoxification pathways, including signal transduction, material metabolism, and protein exports. The above results confirmed that the crucial MT gene did not undergo genetic mutations but rather exhibited global mRNA regulation responsible for the Cd resistance of M.californica. The current study partially disclosed the stress adaptation and evolution of organisms under long-term in situ contamination, which provides insights into maintaining biodiversity under adverse environment.

Soil contamination with micropollutants is an important global problem and the impact of these pollutants on living organisms cannot be underestimated. The effects of diclofenac (DCF) and sulfamethoxazole (SMX), their mixture (MIX), and wastewater containing these drugs on the mortality and reproduction of Eisenia fetida were investigated. The impact on the activities of antioxidant enzymes in earthworm cells was also assessed. Furthermore, the influence of the following parameters of the vertical flow constructed wetlands on wastewater toxicity was investigated: the dosing system, the presence of pharmaceuticals and the plants Miscanthus giganteus. The compounds and their mixture significantly affected the reproduction and mortality of earthworms. The calculated values of LC50,28 days values were 3.4 ± 0.3 mg kg-1 for DCF, 1.6 ± 0.3 mg kg-1 for SMX, and 0.9 ± 0.1 mg kg-1 for MIX. The EC50 (reproduction assay) for DCF was 1.2 ± 0.2 mg kg-1, whereas for SMX, it was 0.4 ± 0.1 mg kg-1, and for MIX, it was 0.3 ± 0.1 mg kg-1, respectively. The mixture toxicity index (MTI) was calculated to determine drug interactions. For both E. fetida mortality (MTI = 3.29) and reproduction (MTI = 3.41), the index was greater than 1, suggesting a synergistic effect of the mixture. We also observed a negative effect of wastewater (raw and treated) on mortality (32% for raw and 8% for treated wastewater) and fertility (66% and 39%, respectively) of E. fetida. It is extremely important to analyze the harmfulness of microcontaminants to organisms inhabiting natural environments, especially in the case of wastewater for irrigation of agricultural fields.

Cyantraniliprole, the second generation of diamide insecticides, is widely used to control various pests, which will certainly result in adverse effects on earthworms in soil. In this study, after exposure with six doses of cyantraniliprole (0, 0.5, 1, 2.5, 5, and 10 mg kg^-1) by artificial soil method, six biomarkers, four functional genes, and histopathological changes of Eisenia fetida were measured on the 7th, 14th, 21st, and 28th days. The comprehensive toxicity was assessed by the IBR version 2 (IBRv2) method. The results showed that the reactive oxygen species (ROS) level was induced significantly. The superoxide dismutase (SOD) activity was activated in 7-28 days. The catalase (CAT) and glutathione S-transferases (GST) activities were also activated in the initial 14 days. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) contents in the high treatment increased until the late stage of exposure. On the 28th day, the metallothionein (MT) and calreticulin (CRT) genes were up-regulated, the transcriptionally controlled tumor protein (TCTP) gene was down-regulated. The SOD gene showed a good correlation with SOD activity. Extensive histopathological damage was found in the endoderm and ectoderm of E. fetida. The 5 and 10 mg kg^-1 treatments showed higher comprehensive toxicity than the 0.5, 1, and 2.5 mg kg^-1 treatments on the 28th day. These results suggest that cyantraniliprole exerted certain subchronic toxic effects of oxidative stress, DNA damage, and histopathological changes to E. fetida, which provided theoretical basis for rational use of cyantraniliprole and evaluation of its safety to soil environment.

Rare earth elements inevitably release into the soil due to their widespread application. However, it is unclear how they affect the soil animals. The study surveyed the growth and physiological responses of earthworm (Eisenia fetida) exposed into artificial soils spiked with La, Ce, and their mixture, and actual mine soil collected from an abandoned La-Ce mining area (Mianning, Sichuan). The results showed that the 1000-1200 mg/kg combined exposure in two soils induced significant histopathological and phenotypic changes of earthworms. Concentration significantly affected the superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), and protein of E. fetida and the effects differentiated with the prolonging duration. These indicators were negatively affected under the La stress ≥800 mg/kg (SOD, POD, and protein), the 1200 mg/kg (SOD), Ce stress ≥1000 mg/kg (protein), and the combination ≥800 mg/kg (SOD, POD) and ≥1000 mg/kg (protein). Artificial combination had -15.04% (SOD), 8.87% (POD), 5.64% (MDA), and -8.34% (protein) difference compared with the contamination soil, respectively. Overall, E. fetida respond sensitively under the La and Ce stress, the antioxidant defense system and the lipid peroxidation were stimulated, and the artificial soil might overestimate eco-toxicological effect.

Deciphering the potential mechanism of chemical-induced toxicity enables us to alleviate the cellular and organismal dysfunction. The environmental presence of nonylphenol (endocrine disruptor) has a major health concern due to its widespread usage in our day-to-day life. The current study establishes a novel functional link among nonylphenol-induced oxidative stress, Heat shock protein 27 (Hsp27, member of stress protein family), and Ecdysone receptor (EcR, a nuclear receptor), which eventually coordinates the nonylphenol-induced sub-cellular and organismal level toxicity in a genetically tractable model Drosophila melanogaster. Drosophila larvae exposed to nonylphenol (0.05, 0.5 and 5.0 μg/mL) showed a significant decrease in Hsp27 and EcR mRNA levels in the midgut. In concurrence, reactive oxygen species (ROS) levels were increased with a corresponding decline in glutathione (GSH) level and Thioredoxin reductase (TrxR) activity. Increased lipid peroxidation (LPO), protein carbonyl (PC) contents, and cell death were also observed in a correlation with the nonylphenol concentrations. Sub-cellular toxicity poses a negative organismal response, which was evident by delayed larval development and reduced Drosophila emergence. Subsequently, a positive genetic correlation (p < 0.001) between EcR and Hsp27 revealed that nonylphenol-dependent EcR reduction is a possible link for the downregulation of Hsp27. Further, Hsp27 overexpression in midgut cells showed a reduction in nonylphenol-induced intracellular ROS, LPO, PC content, and cell death through the TrxR mediated regenerative pathway and reduced GSH level improving the organismal response to the nonylphenol exposure. Altogether, the study elucidates the potential EcR-Hsp27 molecular interactions in mitigating the nonylphenol-induced cellular and organismal toxicity.

Natural ecosystems are frequently exposed to complex mixtures of different chemicals. However, the environmental risk assessment is mainly based on data from individual substances. In this study, the individual and combined effects on the terrestrial earthworm E. fetida exposed to the anionic surfactant sodium lauryl ether sulphate (SLES) and the pesticides chlorpyrifos (CPF) and imidacloprid (IMI) were investigated, by using the avoidance behaviour as endpoint. Earthworms were exposed to a soil artificially contaminated with five sub-lethal concentrations of each contaminant, both as single substances and in combination of binary and ternary mixtures. Overall results showed that IMI provoked the highest avoidance effect on earthworms, with a concentration value that induced an avoidance rate of 50% of treated organisms (AC50) of 1.30 mg/kg, followed by CPF (AC50 75.26 mg/kg) and SLES (AC50 139.67 mg/kg). The application of the Combination Index (CI) method, indicated that a deviation from the additive response occurred for most of the tested chemical mixtures, leading to synergistic or antagonistic avoidance responses. Synergistic effects were produced by the exposure to the two lowest concentrations of the CPF+IMI mixture, and by the highest concentrations of SLES+CPF and SLES+CPF+IMI mixtures. On the contrary, antagonistic effects were observed at the lowest concentrations of the binary mixtures containing the SLES and at almost all the tested concentrations of the SLES+CPF+IMI mixture (with the exception of the highest tested concentration). These results show that the avoidance test is suitable to assess the detrimental effects exerted on earthworms by chemical mixtures in soil ecosystems and the use of behavioural endpoints can increase the ecological significance of environmental risk assessment procedures.

Antimony (Sb) contamination has brought great environmental problems to the surrounding soils. However, few studies focused on the response of bacterial communities in earthworm gut to Sb. Eisenia fetida was cultured in four soils with Sb contents (5,25,50,100 mg•kg^-1) to investigate the distribution of Sb species in earthworm gut and the response mechanism of bacterial communities to Sb contamination. The results showed that Sb accumulated in the gut and tissues of earthworms, and the mortality of earthworms showed a dose-response relationship with the increase of Sb content. Sb(III) and Sb_exe were the major species in gut, whereas Sb(V) and Sb_srp were predominant in surrounding soil. There were significant differences in bacterial diversity between earthworm gut and soil, but there was no significant between the two with different Sb content. The network constructed by gut bacterial community of earthworm was less stable and more sensitive to Sb species than that in soil. Sb(III) had the greatest influence on the gut bacterial community of earthworm, which not only directly affected the community through Xanthomonadaceae, Rhodomicrobiaceae and Anaerolineaceae, but also indirectly influenced through Chthoniobacteraceae. This study fills a research gap on the effect of Sb contamination on the gut bacterial community of earthworm.

In this study, we investigated the bioavailability and toxicity of metals (Cu and Ni) in the soil containing polyethylene microplastics (PE-MPs). The bioavailability of the metals determined by the five-step chemical sequential extraction method increased with the addition of MPs (0.1%, 1%, 10%) in the soil, which was confirmed by the adsorption-desorption characteristics. To further examine the bioavailability and toxicity of metals, earthworms (Eisenia fetida) were exposed to soil containing Cu²⁺ (100 mg/kg) or Ni²⁺ (40 mg/kg) with different amounts (0.01%, 0.05%, and 0.1%) of PE-MPs for 21 days. The highest concentrations of Cu²⁺ and Ni²⁺ in earthworms reached to 73.3 and 36.3 mg/kg, respectively. Moreover, metal concentrations in earthworms increased with MP contents in the soil, which was consistent with the bioavailability measured by the sequential extraction method. Furthermore, changes in biomarkers including peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) activity, malondialdehyde (MDA) contents, and related gene expression levels in earthworms suggested that the pollutants caused toxicity to earthworms. Overall, MPs increased the bioavailability of metals in the soil and the toxic effects to earthworms. These findings provide insights regarding the impacts of MPs on the bioavailability of metals and the combined toxic effects of these two kinds of pollutants on terrestrial animals.

Earthworms are known to reclaim soil contamination and maintain soil health. In the present study, the concentration of DTPA extractable heavy metals, Cd, Cu, Cr, Pb, and Zn in vermicasts and tissues of the earthworms (anecic: Lampito mauritii; epigeic: Drawida sulcata) collected from the soils of four different industrial sites, Site-I (Sago industry), Site-II (Chemplast industry), Site-III (Dairy industry) and Site-IV (Dye industry) have been studied. The heavy metals in industrial soils recorded were 0.01-326.42 mg kg^-1 with higher Cu, Cr, and Zn contents while the vermicasts showed lower heavy metal loads with improved physicochemical properties and elevated humic substances. The higher humic substances dramatically decreased the heavy metals in the soil. The bioaccumulation factors of heavy metals (mg kg^-1) are in the order: Zn (54.50) > Cu (17.43) > Cr (4.54) > Pb (2.24) > Cd (2.12). The greatest amount of metallothionein protein (nmol g^-1) was recorded in earthworms from Site-IV (386.76) followed by Site-III (322.14), Site-II (245.82), and Site-I (232.21). Drawida sulcata can produce a considerable amount of metallothionein protein than Lampito mauritii as the metallothionein production is dependent upon the presence of pollutants. The molecular docking analysis indicates a binding score of 980 for Cd, Cr and Cu, and 372 for Zn. Pb may bind with a non-metallothionein protein of earthworms and bio-accumulated in the internal chloragogenous tissues. Metallothionein neutralizes the metal toxicity and controls the ingestion of essential elements.

In this work, we propose to evaluate the effect of agriculture intensification under greenhouses on the biochemical and transcriptomic responses of the earthworms Eisenia andrei. This work was conducted on two sites in Téboulba and Sahline (Monastir governorate) and a control site in an experimental plot that is undergoing organic farming. For this purpose, the earthworms Eisenia andrei were exposed to the soils during 7 and 14 days. The physicochemical properties of the soils were analyzed. The biochemical biomarkers of metallothioneins (MTs) and malondialdehyde (MDA) accumulations were also assessed. Moreover, the gene expression level of the MTs was analyzed. The results of our study revealed a significant trace element accumulation accompanied by a high level of MDA and MT proteins. Moreover, a significant expression of the MT gene was observed in earthworms exposed to the soils from Sahline and Téboulba. Hence, this work reveals that intensive agriculture can affect the biological responses of earthworms and consequently, the soil's biofertility.

It is widely acknowledged that a simplified and robust approach to evaluating thecombined effects of chemical mixtures is critical for ecological risk assessment (ERA) of contaminated soil. The earthworm (Eisenia fetida) was used as a model to study the combined effects of polymetallic contamination and the herbicide siduron in field soil using a microcosm experiment. The responses of multiple biomarkers, including the activities of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and acetylcholine esterase (AChE), the concentrations of glycogen, soluble protein (SP), malonaldehyde (MDA), and metallothionein (MT), and the neutral red uptake test (NRU), were investigated. Multivariate analysis, Principal Component Analysis (PCA) and Spearman's Rank Correlations analysis (BVSTEP) revealed that the activities of AChE and CAT and the NRU content were the prognostic biomarkers capturing the minimum data set of all the variables. Internal Cd (tissue Cd) in earthworms was closely related to the health status of worms under combined contamination of heavy metals and siduron. The integrated effect (E_mix) calculated based on the activities of AChE and CAT and NRU content using the stress index method had significantly linear regression with internal Cd (p<0.01). E_mix(10), E_mix(20), and E_mix(50) were then calculated, at 1.27, 1.63 and 2.71 mg/kg dry weight, respectively. It could be concluded that a bioassay-based approach incorporating multivariate analysis and internal dose was pragmatic and applicable to evaluating combined effects of chemical mixtures in soils under the guidance of the top-down evaluation concept of combined toxicity.

Sulfur has been shown to mitigate the toxic effects of metals on soil organisms. Here we report the effects of sulfur on cadmium toxicity to the collembolan Folsomia candida in soil, including its effects on glutathione (GSH) level, catalase (CAT) activity and metallothionein (MT) content. Following sulfur treatment, catalase, glutathione and metallothionein activities were all significantly increased in cadmium-contaminated soil, and as the cadmium concentration increased, the activities decreased. In addition, because of the reducing effects of pH and organic matter on cadmium bioavailability, the bioavailable cadmium varied among soils of different pH values and organic matter contents, causing the catalase activity, glutathione content and metallothionein levels of F. candida to vary among soils. Our study suggests that sulfur can affect the toxicity of certain concentrations of cadmium and that soil properties are very important to consider. This study provides insight into the effects of sulfur application on soil animals.

Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.

The aim of this study was to develop a new method, using a vibration sensor, to address the drawbacks of preexisting methods for monitoring soil ecological toxicity. A novel method was designed by inspiration from seismometers, which record signals originating from the ground motion caused by earthquake events. Similarly, the newly developed method using a vibration sensor detects the signals generated by earthworm activity, which reflects the soil ecological toxicity. To establish the new method, a stepwise approach was adopted: (1) the effects of operational conditions on the overall performance of the system were evaluated, and (2) the feasibility of the method was tested by an application study. A number of crucial factors influencing the overall performance of the method were evaluated. These were categorized based on three features: soil, tested organism, and instrumentation. The soil properties evaluated included soil type (artificial and natural), moisture content, and bulk density. In terms of the organism, the effect of the number of earthworms was investigated. Finally, with regard to instrumentation, appropriate soil chamber specifications and monitoring duration were identified. The most effective conditions for each factor were determined based on a comparative evaluation of changes in the activity levels and body weights of the earthworms. After the first step of the study, an application study was carried out to demonstrate the feasibility of the proposed method. Zinc (Zn)-contaminated soils were tested under the most efficient operational conditions identified in the preceding study. The results of the study confirm that the method is applicable to natural soils, and the best performance was achieved under soil conditions of 50-60% maximum water holding capacity and 0.95 g/cm³ bulk density. Furthermore, the optimal number of earthworms was found to be five, which corresponds 19.84 g soil per earthworm. With respect to the instrumental conditions, the most efficient specification was a cylindrical soil chamber with a diameter of 94 mm and height of 54 mm. Additionally, the most relevant monitoring duration was found to be 7 days. The results indicate that the method can shorten the testing period, reduce the soil amount and earthworm number required, and facilitate the real-time monitoring of mortality. Based on the results of the application study, we validated the proposed vibration sensor-based method for characterizing earthworm behavior in terms of its feasibility for monitoring the ecological toxicity of soil. The results indicate that dermal contact and feeding activity of earthworms decreased significantly with increasing Zn concentrations in the soil. The EC₅₀ value of Zn calculated based on the earthworm behavior was 340.97 mg/kg. Based on the results, it is concluded that the proposed method cannot only overcome the shortcomings of traditional test methods using earthworms, but also enable real-time ecotoxicity in soil environments.

Assessing the ecological risk of combined pollution, especially from a holistic perspective with the consideration of the overarching functions of soil ecosystem, is crucial and beneficial to the improvement of ecological risk assessment (ERA) framework. In this study, four soils with similar physicochemical properties but contrasting heavy metals contamination levels were selected to explore changes in the integrated functional sensitivity (MSI), resistance (MRS) and resilience (MRL) of soil microbial communities subjected to herbicide siduron, based on which the ecological risk of the accumulation of siduron in the four studied soils were evaluated. The results suggested that the microbial biomass carbon, activity of denitrification enzyme and nitrogenase were indicative of MSI and MRS, and the same three parameters plus soil basal respiration were indicative of MRL. Significant dose-effect relationships between siduron residues in soils and MSI, MRS and MRL under combined pollution were observed. Heavy metal polluted soils showed higher sensitivity and lower resistance to the additional disturbance of herbicide siduron due to the lower microbial biomass, while the resilience of heavy metal polluted soils was much higher due to the pre-adaption to the chemical stresses. The quantifiable indicator microbial functional stability was incorporated in the framework of ERA and the results showed that the accumulation of siduron in the studied soils could exhibit potential harm to the integrated functional stability of soil microbial community. Thus, this work provides insights into the application of integrated function of soil microbial community into the framework of ERA.

Tolclofos-methyl (TM) is an organophosphorus fungicide and widely utilized to control soil-borne diseases. However, toxic effects of TM on terrestrial invertebrates are still unknown. Here we measured the bioaccumulation of TM in earthworms (Eisenia fetida) to assess its environmental bioavailability. Mortality, weight change, and oxidative damage of earthworms were determined to investigate the toxicological bioavailability of TM. ROS, SOD and MDA in highest concentration treatment group significantly increased compared to the control group, suggesting that hazardous effects of TM to earthworms were caused by the oxidative stress. To further examine its toxicological bioavailability, cytotoxicity test was carried out by using extracted earthworm coelomocytes. The biomarkers, e.g., intracellular ROS, extracellular LDH, and cell viability showed correlation with TM in the culture media, demonstrating that cytotoxicity test could be employed to reflect the toxicological bioavailability of pollutants to earthworms or other organisms.

The contaminants, instead of single contaminants, are usually found as chemical mixtures in terrestrial environment. However, little information is now available about the joint effects of heavy metals and farm chemicals on the soil organism. Our current study aimed to assess the combined toxicity of cadmium (Cd) and four pesticides (atrazine, chlorpyrifos, lambda-cyhalothrin and abamectin) to the earthworm (Eisenia fetida) with binary, ternary, quaternary and quinquenary mixtures. Two distinct kinds of bioassay systems were applied in this study, including contact filter paper test and artificial soil test. Synergistic effects were observed from two quaternary mixtures (Cd-Atrazine-Chlorpyrifos-Lambda-cyhalothrin and Cd-Chlorpyrifos-Lambda-cyhalothrin-Abamectin) and one quinquenary mixture (Cd-Atrazine-Chlorpyrifos-Lambda-cyhalothrin-Abamectin) in both bioassay systems. Besides, two binary mixtures (Cd-Atrazine and Cd-Lambda-cyhalothrin) and three ternary mixtures (Cd-Atrazine-Chlorpyrifos, Cd-Atrazine-Lambda-cyhalothrin and Cd-Chlorpyrifos-Lambda-cyhalothrin) also exhibited synergistic effects on E. fetida by the soil toxicity test. It is more practical to evaluate the contaminant toxicities to earthworm by soil toxicity test. Therefore, the effects of chemical mixtures on soil invertebrates might be underestimated by data obtained from single toxicant. Our findings would offer a better understanding of the complex effects of chemical mixtures on non-target living creatures, and these findings provided valuable insights into the interplay of different chemicals in natural environment.

In this study, the acute and subchronic toxicity of 1-alkyl-3-methyl imidazole bromide ionic liquids (ILs) [C_nmim]Br (n = 2, 4, 6, 10, 12) was evaluated utilizing earthworms (Eisenia fetida) as a basis for their impact on terrestrial ecosystems. The filter paper tests and artificial soil tests were conducted as acute toxicity tests to investigate the LC₅₀ of ILs, while in subchronic toxicity tests, earthworms were exposed to ILs in artificial soil (5, 10, 20 and 40 mg kg^-1) for 28 d. Reactive oxygen species (ROS), antioxidant enzymes, detoxifying enzymes and oxidative damage were measured to determine subchronic effects of ILs on E. fetida. The results showed that when the earthworms were exposed to these five ILs in acute toxicity experiments, [C₂mim]Br had the lowest toxicity, as the alkyl length increased, the toxicity increased up to [C₁₀mim]Br: a "cut-off effect" (decreased toxicity) was observed at [C₁₂mim]Br. The results highlight the varying toxicity of ILs with different alkyl chains to E. fetida and provide valuable data for detailing the impact of ILs on ecological receptors.

As a neurotoxic insecticide, imidacloprid (IMI) has been widely used for crop protection. However, continuous application of such pesticide in the environment may damage the non-target organisms in soil. In the present study, we aimed to investigate the effects of IMI on earthworms in terms of survival, avoidance behavior, reproduction, detoxification enzyme activity and gene expression using a systematic experimental approach. The results showed that the 14-day LC₅₀ value of IMI was 2.26 (2.09-2.43) mg a.i. kg^-1, and the 2-day AC₅₀ value (concentration inducing an avoidance rate of 50%) of IMI was 1.34 (1.02-1.91) mg a.i. kg^-1 to E. fetida. For reproduction, the 56-day EC₅₀ value of IMI was 0.87 (0.66-1.33) mg a.i. kg^-1 to E. fetida, and there was a positive correlation between the growth rate of earthworms and the number of juveniles in IMI treatments. Activities of carboxylesterase (CarE) and glutathione-S-transferases (GST) in earthworms were disturbed by IMI exposure. Moreover, effects of IMI on the CarE activity in earthworms were more severe and sensitive compared with the GST activity. The expressions of annetocin (ann) and calreticulin (crt) at the transcriptional level were decreased upon IMI exposure, reaching the lowest levels of 0.09 fold and 0.16 fold on day 7 and day 14, respectively. Transcriptionally controlled tumor protein (tctp), heat shock protein 70 (hsp70) and gst exhibited relatively obvious variations (up-regulation or down-regulation) when the exposure duration was extended. Taken together, these results comprehensively contributed to further understandings of the impacts of IMI on earthworms.

In natural ecosystems, organisms are commonly exposed to chemical mixtures rather than individual compounds. However, environmental risk is traditionally assessed based on data of individual compounds. In the present study, we aimed to investigate the individual and combined effects of four pesticides [fenobucarb (FEN), chlorpyrifos (CPF), clothianidin (CLO), acetochlor (ACE)] and one heavy metal chromium [Cr(Ⅵ)] on the earthworm (Eisenia fetida) using avoidance behavior as an endpoint. Our results indicated that CLO had the highest toxicity to E. fetida, followed by Cr(Ⅵ), while FEN showed the least toxicity. Two mixtures of CPF+CLO and Cr(Ⅵ)+CPF+CLO+ACE exhibited synergistic effects on the earthworms. The other two quaternary mixtures of CLO+FEN+ACE+Cr(Ⅵ) and Cr(Ⅵ)+FEN+CPF+ACE at low concentrations also displayed synergistic effects on the earthworms. In contrast, the mixture of Cr(Ⅵ)+FEN had the strongest antagonistic effects on E. fetida. Besides, the quinquenary mixture of Cr(Ⅵ)+FEN+CPF+CLO+ACE also exerted antagonistic effects. These findings highlighted the importance to evaluate chemical mixtures. Moreover, our data strongly pointed out that the avoidance tests could be used to assess the effects of combined effects.

Siduron is a widely used herbicide in urban lawn and has been frequently detected in urban and suburban surface water. However, characteristics of its environmental behavior in soil are seldom reported. The combined pollution of heavy metals, especially for Cu, Pb, Cd, Zn and siduron would be common because of the widely existence of heavy metal pollution in urban soils. In this study, four soils with similar physicochemical properties but different levels of preexisting heavy metals were selected to investigate the adsorption and successive desorption of siduron using batch experiments. The results revealed a low sorption of siduron to all the tested soils. The organic carbon normalized distribution coefficient (K_oc) of siduron in the studied soils ranged from 117 to 137 L kg^-1 and was not significantly correlated to heavy metal levels. No apparent desorption hysteresis was observed with the hysteresis index (HI) ranging from 0.921 to 1.11. More than 50% of the sorbed siduron was readily released into soil solution. Results suggested that siduron was highly mobile and bioavailable in the studied soils. Significant correlation was found between adsorption/desorption parameters and soil organic carbon (SOC) in four soils. soil organic matter was thus considered as the dominant factor determining the adsorption and desorption of siduron in soils. Different from most of reported studies conducted by laboratory-amended soils, the influence of preexisting heavy metals on the adsorption-desorption of siduron was not significant in this work.

Combined pollution of agrichemicals and heavy metals in urban lawn soils were commonly observed throughout the world, and the co-existed two chemicals could interact with each other both in environment behavior and toxic effect. However, little has been reported on the ecological risk of their combined pollution, especially in field due to lack of systematic methodology. In this study, four soils (C, N1, N2, N3) from two public parks in Beijing, China, with similar properties but contrasting heavy metal contaminated level were chosen to assess the ecological risks of co-existed herbicide siduron and heavy metals. Environmental behaviors of siduron in studied soils were investigated with batch experiments in lab, based on which the environmental exposure level of siduron was simulated with HYDRUS-1D. Results suggested that soil organic matter (SOM) rather than the co-existed heavy metals was the dominant factor affecting the fate and the accumulation of siduron in soils. Soil N2 with the highest SOM, showed the strongest tendency to retain siduron among the studied soils. Significant joint effect of siduron and heavy metals on cucumber root elongation was observed through lab experiments. Thus, the joint toxicity of siduron and heavy metals were calculated based on single toxicology data of them using independent action (IA) and concentration addition (CA) model. Then, the predicted no effect concentration (PNEC_soil) of siduron was calculated with equilibrium partitioning method and extrapolation techniques. The PNEC_soil of siduron was the lowest in heaviest heavy metal contaminated soil N3. The risk characterization ratios (RCR) of siduron in four soils were all >1. The highest RCR of siduron in soil N3 suggested that it was the joint toxicity of siduron and heavy metals to organisms determining the ecological risks of siduron in combined polluted soils.