N-(1,3-dimethylbutyl)-N '-phenyl-p-phenylenediamine quinone (6PPD-Q), a highly toxic environmental transformation product of the tire antioxidant N-(1,3-dimethylbutyl)-N '-phenyl-p-phenylenediamine (6PPD), persistently accumulates in soil via tire-wear particles, posing a potential threat to soil ecosystems. Therefore, in the present study, Eisenia fetida was selected as the test organism to conduct a 28-day exposure experiment to 6PPDQ. Three exposure concentrations of 6PPD-Q (10, 100, and 1000 mu g/kg) were set. Combining organismal, physiological, biochemical, molecular and transcriptomic approaches, we systematically investigated the ecotoxicity of 6PPD-Q in soil and its underlying mechanisms. The results demonstrated that 6PPD-Q exposure inhibited the growth, development and reproductive capacity of earthworms. Specifically, the growth inhibition rate and reduction rate of juvenile earthworms in the high-concentration group exceeded 60%. Meanwhile, the expression levels of related functional genes were downregulated. 6PPD-Q exposure induced the excessive accumulation of reactive oxygen species (ROS), thereby triggering a series of changes in the activities of antioxidant enzymes. The results of related functional gene assays and molecular docking (6PPD-Q with SOD and TCTP) further verified the aforementioned phenotypic changes and oxidative stress responses. In addition, oxidative stress further caused intestinal tissue damage and pathological changes in earthworms. Transcriptomic analysis identified 405 differentially expressed genes, revealing considerable perturbations in pathways related to xenobiotic metabolism, detoxification defense, lipid homeostasis, and DNA protection. Overall, this study provides multi-level data and scientific evidence, which supports the ecological risk assessment and environmental regulation of 6PPD-Q in soil ecosystems.

Di(2-ethylhexyl) phthalate (DEHP) is pervasive in agroecosystems; however, species-specific toxic mechanisms in soil detritivores remain unresolved. Here, we investigated cross-level mechanisms of DEHP toxicity in two earthworm’s species, Eisenia foetida and Metaphire guillelmi, using an integrative framework combining histopathology, residue analysis, transcriptomics and metabolomics. DEHP induced significant tissue injury in both species, with more pronounced lesions observed in M. guillelmi. Although M. guillelmi exhibited slightly lower DEHP residues than E. foetida, it showed stronger molecular disturbances, suggesting higher sensitivity independent of body burden. These differences likely reflect distinct ecological strategies influencing bioavailability and physiological responses. Transcriptomic analysis identified 2673 differentially expressed genes in M. guillelmi compared with only 138 in E. foetida, demonstrating markedly divergent transcriptional plasticity between the two species. Metabolomic profiling similarly revealed broader metabolic disruption in M. guillelmi (138 differentially expressed metabolites, (DMEs)) compared with E. foetida (30 DEMs). Integrated pathway analysis indicated that DEHP perturbed arachidonic acid metabolism and KEGG-annotated oxytocin signaling in E. foetida, whereas lipid, amino-acid, and purine/pyrimidine metabolism were primarily affected in M. guillelmi. These alterations were consistent with oxidative stress, immune imbalance, and disrupted energy metabolism. Convergent evidence from omics and histological analyses supports species-specific mechanisms underlying tissue injury, with M. guillelmi being more susceptible at the molecular level. Overall, these findings provide mechanistic biomarkers and adverse outcome pathway-based evidence for effect-driven soil health assessment and species-sensitive ecological risk evaluation within a One Health context.

Widespread use of the novel chiral triazole fungicide mefentrifluconazole (MFZ) poses a threat to soil ecological health. While previous studies have implicated energy supply disruption as a key mechanism underlying MFZ-induced toxicity in earthworms (Eisenia fetida), the precise effects of MFZ enantiomers on energy metabolism remain unclear. Here, we utilized transcriptomics, targeted energy metabolomics, and bioenergetic indicators to investigate the impact of 4 mg/kg R(-)-MFZ and S-(+)-MFZ soil exposure on E. fetida energy metabolism. Based on the previous finding that MFZ enantiomers inhibit oxidative phosphorylation (OXPHOS), further analyses showed that the TCA cycle is dysfunctional, characterized by reduced activities of the key enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. In response to energetic stress, metabolic reprogramming of OXPHOS to glycolysis transition was observed for the first time from E. fetida, as evidenced by elevated levels of key glycolytic intermediates and increased phosphofructokinase activity. Additionally, MFZ enantiomers induced enhanced fatty acid beta-oxidation by increasing carnitine palmitoyltransferase transcript levels and enzyme activity. Notably, S-(+)-MFZ exhibited a more pronounced effect on these parameters, highlighting its greater toxicity. These findings provide compelling evidence that MFZ enantiomers reprogram energy metabolism in E. fetida. The enantioselective nature of these effects underscores the necessity of incorporating chiral isomer considerations into ecological risk assessments to comprehensively evaluate potential health risks to ecosystems.

The development of highly efficient and environmentally benign catalysts for peroxymonosulfate (PMS) activation to degrade organic pollutants has become a central focus in advanced oxidation processes. In this study, tannic acid (TA), a naturally occurring polyphenolic compound, was employed to engineer the surface structure of Bi2Fe4O9, resulting in the construction of a novel PMS activation system (BTA/PMS) capable of efficiently degrading dimethyl phthalate (DMP) while effectively managing its toxicity. The introduction of 0.7 mol% TA was shown to refine both the crystalline architecture and surface chemical environment of the material, significantly enhancing its electron transfer capability. A DMP removal efficiency of 96.5% was achieved within 60 min, with 78.5% total organic carbon removal observed, surpassing the performance of common cobalt-based catalysts under identical experimental conditions. The rapid degradation of DMP (0.16 min-1) was achieved through a composite advanced oxidation strategy. A synergistically tailored surface, rich in hydroxyl species and oxygen vacancies, was engineered on the BTA material, which collectively enabled highly efficient PMS activation. Moreover, the BTA material exhibited excellent recyclability and the capacity for continuous wastewater treatment. Toxicity predictions using the T.E.S.T. model and phytotoxicity assessments confirmed the environmental safety of the treated effluent, with no significant adverse effects observed on rice seedlings. This work proposes an innovative strategy for the concerted modulation of metal oxide catalysts through the addition of a small amount of TA, providing mechanistic insights and a design paradigm for efficient PMS activation.

Fluensulfone (FSF) is a highly effective nematicide increasingly utilized in agricultural applications. In this study, we exposed earthworms (Eisenia fetida) to environmentally relevant concentrations of FSF and assessed the toxicological responses via an integrated approach combining non-targeted metabolomics, enzyme activity assays, and reverse transcription-quantitative PCR. Exposure to FSF disrupted core energy metabolism by suppressing glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. This disruption was reflected by decreased levels of central metabolites (e.g., pyruvate and α-ketoglutarate), inhibition of rate-limiting enzymes (including pyruvate kinase and α-ketoglutarate dehydrogenase), and the downregulation of key mitochondrial genes (CYTB, COX1, and ATP6). These findings collectively point to mitochondrial dysfunction as a central event. Concurrently, the downregulation of reproduction-associated gene ANN and marked declines in cocoon production and juvenile numbers were observed, suggesting a potential connection between systemic energy deficiency and reproductive impairment. Taken together, our study highlights energy metabolism as a sensitive and conserved target, underscoring its relevance to regulatory frameworks for FSF and potentially other next-generation nematicides.

To address the persistent challenge of refractory antibiotic contamination, a copper‑iron bimetallic catalyst anchored on nitrogen-doped spent coffee grounds biochar (CFC) was rationally designed for efficient peroxymonosulfate (PMS) activation toward ciprofloxacin (CIP) degradation. The CFC/PMS system achieves a high CIP removal efficiency of 97.7% within 10 min. Combined experimental characterizations and density functional theory (DFT) calculations reveal that the internal Cu0/Cu+ species function as an electron pump, creating a localized electron-rich microenvironment that thermodynamically drives the reduction of Fe(III) to Fe(II), thereby overcoming the kinetic bottleneck associated with sluggish Fe redox cycling. Consequently, this bimetallic electronic synergy promotes a transition from conventional radical pathways to a highly efficient non-radical process dominated by singlet oxygen (1O2) and direct electron transfer. The CFC catalyst exhibits robust structural stability and maintains excellent performance across various real water matrices, with metal leaching well below drinking water standards. Liquid chromatography-mass spectrometry (LC-MS) analysis identifies three major degradation pathways, in good agreement with the reactive sites predicted by DFT calculations. Furthermore, quantitative structure–activity relationship (QSAR) analysis combined with rice seedling assays confirms the effective detoxification of degradation intermediates. Ultimately, this work provides deep mechanistic insights into bimetallic synergistic PMS activation and demonstrates a sustainable strategy for antibiotic removal based on waste-derived catalysts.

The indiscriminate use of veterinary antibiotics (VAs) poses a potential risk to soil ecosystems. However, a systematic understanding of their toxic mechanisms toward soil organisms is still lacking. We evaluated the toxic effects of three typical VAs tylosin (TYL), enrofloxacin (ENR), and chlortetracycline (CTC) on earthworms from multiple systemic levels. The three VAs inhibited earthworm growth and reproduction, induced oxidative stress, and caused dysregulation of the expression of stress-related genes. Histopathological analysis revealed severe lesions in the epidermal and intestinal structures of earthworms. These responses triggered digestive dysfunction, which may be attributed to prolonged oxidative damage and downregulation of key gene expression. In addition, VAs disrupted the gut microbiota homeostasis in earthworms, suppressed of key functional taxa, and increased the abundance of potential pathogens. Concurrently, the reshaping of the microbial network increased the risk of antibiotic resistance gene (ARG) proliferation. ARGs were enriched in earthworm guts and soil, with an enhanced potential for horizontal transfer. Together, these findings indicate that the multifaceted toxicity of VAs poses a significant threat to the soil ecosystem and public health. This study provides a scientific basis and data support for the risk assessment and management of soil ecological health under VA pollution.

Per- and polyfluoroalkyl substances (PFAS) are a class of persistent environmental pollutants that pose significant threats to plant health and ecosystem stability. However, the molecular mechanisms underlying their toxicity, particularly how carbon chain length influences biological effects, remain poorly understood. This study employed an integrated multi-omics approach to systematically investigate the toxicity mechanisms of PFAS in the soil-plant system. We find that PFAS exposure inhibits tomato seedling growth, inducing reactive oxygen species accumulation and membrane lipid peroxidation damage. Molecular docking revealed that perfluorooctanoic acid (PFOA) exhibited stronger binding affinity to superoxide dismutase (SOD, -7.6 kcal/mol) and glutathione (GSH, -7.8 kcal/mol) compared to perfluorobutanoic acid (PFBA, -5.2 and -6.0 kcal/mol). Rhizosphere microbial analysis demonstrated PFAS-driven community restructuring, with PFOA treatments increasing Pseudomonadota abundance by 157% and Bacillota by 185%. Metabolomics identified 704 differentially expressed metabolites, showing PFAS suppressed fatty acid metabolism while activating ABC transporters and antioxidant biosynthesis. Random forest identified 15 key biomarkers for oxidative damage, including microbial genera (Methylibium, Thauera) and plant metabolites (Tomatine). This research elucidates the chain length-dependent toxicity mechanisms of PFAS across molecular, microbial, and metabolic dimensions, providing critical biomarkers for ecological risk assessment and remediation strategies.

Widespread application of veterinary antibiotics is contaminating soil via animal feces, leading to uptake by plants and environmental damage. Currently, research on the toxicological mechanisms associated with various classes of antibiotics remains inadequate. Therefore, this study utilized tomato as the test species and selected three representative antibiotics-chlortetracycline (CTC), enrofloxacin (ENR), and tylosin (TYL)-to systematically evaluate their differential toxicity and associated metabolic mechanisms through 14 and 28 days exposure experiments. At the individual level, antibiotics significantly suppressed biomass accumulation and photosynthesis in tomato seedlings, the ENR exhibited maximum inhibition rates of 37.4% for fresh weight and 26.7% for plant height. In contrast, the CTC recorded peak values of 28% for leaf area and 25.1% for SPAD measurements. Furthermore, exposure to antibiotics induced oxidative stress in tomato seedlings, with SOD demonstrating its highest activation rate of 18.3% in the TYL. Within the rhizosphere microenvironment, there was a notable decrease in the abundance of the dominant phylum Bryobacter, which was accompanied by alterations in bacterial community structure, an increase in network complexity, and a reduction in modularity. Under antibiotic stress, microbial communities demonstrated distinct metabolic responses: enhanced lipid metabolism in CTC, elevated carbohydrate metabolism with ENR, and activated nucleotide metabolism associated with TYL. In summary, antibiotics present global ecological risks by inhibiting plant growth and disrupting the rhizosphere microbiome. The class-specific toxicity of these substances necessitates the implementation of targeted risk management strategies.

Antibiotic pollution poses a serious threat to aquatic ecosystems and human health. Developing efficient adsorbent materials is crucial to tackle this issue. In this study, the BiPO4/MIL-88A(Fe) (BM-7) composite material with good adsorption effect on tetracycline hydrochloride (TC) and ciprofloxacin hydrochloride (CIP) and potential for environmental application was synthesized by the green solvothermal method for the first time. The removal efficiencies of TC and CIP were significantly affected by pH (3-10) and dose, whereas the effects of interfering ions and humic acid were less than 5 % and negligible. The adsorptive removal efficiency of TC and CIP by BM-7 was 98.15 % and 93.57 %, respectively, which exceeded most of the reported metal-organic framework (MOF)-based materials. 0.01 M HCl can effectively elute contaminants, and the adsorption efficiency remains above 85.14 % after 5 cycles. Combined with XPS and FTIR analyses, the adsorption of TC is mainly determined by the bidentate chemical coordination of Fe-O clusters, whereas the adsorption of CIP is dependent on the it-it stacking effect and hydrogen bonding of BM-7 for CIP. In real wastewater, BM-7 removes over 82.66 % of TC and 78.40 % of CIP. The leaching concentrations of Fe3 + and Bi3+ in BM-7 were lower than 0.270 mg L- 1 and 0.026 mg L- 1, respectively. Hydroponic experiments with rice further validate its effectiveness in alleviating CIP's inhibitory effects on plant growth. The BM-7 had achieved the integrated function of "adsorption-regeneration-ecological restoration".

The increasing agricultural use of fluensulfone, an efficient nematicide, raises significant concerns about its potential environment risk. This study presents a comprehensive assessment of fluensulfone-induced soil ecotoxicity by integrating phenotypic and molecular responses in the earthworm Eisenia fetida, a key indicator of soil health. Significant adverse effects were revealed, including substantial biomass reduction (up to 22.49 % at 10 mg/kg) and dysregulation of genes associated with development and regeneration. RNA-seq profiling demonstrated 4963 differentially expressed genes, with significant enrichment in pathways related to cell cycle, energy metabolism, and oxidative stress. These findings were corroborated by qRT-PCR validation of antioxidant gene dysregulation, elevated levels of oxidative damage markers, and histopathological evidence. Notably, integrated biomarker response analysis identified glutathione S-transferase activity as a sensitive biomarker for fluensulfone exposure, further supported by molecular docking simulations. In summary, this study elucidates the multi-level toxicity of fluensulfone in earthworm and establishes a scientific basis for assessing its ecotoxicological effects on non-target organisms.

The extensive application of plasticizers has led to significant environmental issues. This study focused on the ecotoxic effects on earthworms of the traditional plasticizer di(2-ethylhexyl) phthalate (DEHP) and non-phthalate plasticizers di(ethylhexyl) terephthalate (DEHT) and acetyltributyl citrate (ATBC). At an environmentally relevant concentration (50 mg/kg), significant accumulation of ROS was observed in earthworms, with a trend of DEHP > DEHT > ATBC, inducing oxidative stress and lipid peroxidation. DEHP, DEHT, and ATBC impaired the energy metabolism in earthworms, as evidenced by a sharp reduction in ATP content ranging from 43.2 % to 75.8 %, which was attributed to the disruption of glycolysis and the TCA cycle. Concurrently, the numbers of cocoons and juvenile earthworms decreased by 23.3 %-76.7 % and 24.2 %-75.8 %, respectively, indicating a significant decline in reproductive capacity. Using qPCR, AlphaFold2, and molecular docking techniques, this study is the first to report that because of their similar molecular structures, the alternatives to DEHP exhibit estrogen-like effects in earthworms, which may be a key mechanism of reproductive toxicity. These results provide valuable references and profound insights for the development of novel plasticizer alternatives and the assessment of their impact on soil ecosystems.

In this study, a thermally-assisted Bi2Fe4O9 activated permonosulfate (PMS) system was deemed the optimal solution for achieving efficient degradation of dimethyl phthalate (DMP) in water, after compared with the efficaciousness of other auxiliary methods. The degradation efficiency of this system (96.6%) markedly surpasses that of other BixFeyOz materials or a homogeneous activation system of iron ion. Optimal conditions and influencing factors for this novel approach were systematically determined. Electron paramagnetic resonance and scavenging experiments of free radicals identified the predominant reactive species. The mechanism of PMS activation by Fe and Bi sites was speculated by XPS, PMS decomposition rate and electrochemical test. Mild thermal assistance (50 degrees C) was found to expedite the decomposition of PMS and facilitate generation of active species, which appears to be a pivotal factor in enhancing the degradation of DMP. Twelve degradation intermediates including monomethyl phthalate were detected by liquid chromatography-mass spectrometry, and plausible degradation pathways were postulated and corroborated through density functional theory calculations. The Bi2Fe4O9 exhibited commendable stability and recyclability under thermal conditions, with negligible metal leaching (0.19 mg/L). Finally, the toxicity of the degradation solution was preliminarily studied by using the actual growth index of wheat seedlings. Collectively, this study furnishes robust data supporting the efficacy of heat-assisted transition metal-activated PMS systems for pollutant degradation.

Phthalate esters (PAEs) are typically released from agricultural plastic films and veterinary antibiotics introduced through livestock manure. They often accumulate in agricultural soils, posing complex ecological risks and severe biological effects that are not yet fully understood. Therefore, this study investigated the ecotoxicity and risks of di(2-ethylhexyl) phthalate (DEHP), sulfadiazine (SDZ), and their co-exposure on earthworms. DEHP, SDZ, and their co-exposure was found to significantly impair earthworm growth and reproduction, induced oxidative stress, and altered the expression of functional genes (tctp, ann, sod, cat, hsp70, er). Both DEHP and SDZ strongly bound to key earthworm proteins (SOD and TCTP), further supporting the evidence of oxidative stress and adverse effects on growth and development. Risk assessment revealed that DEHP exacerbated the reproductive and oxidative stress compared to SDZ and the co-exposure. Furthermore, histopathological and flow cytometric results suggested antagonistic interactions between DEHP and SDZ during co-exposure. Transcriptomics data demonstrated that SDZ activated pathways related to oxidative stress repair (peroxisome pathways) and detoxification (glutathione metabolism) in earthworms, which explains the relatively lower toxicity of co-exposure. Overall, these findings provide multi-level insights into the antagonistic effects of compound pollution in soil ecosystems and support the ecological risk assessment of PAEs and antibiotics.

Advanced oxidation processes involving the activation of persulfate offer an effective approach for mitigating environmental pollution caused by dimethyl phthalate (DMP). In this study, a heterogeneous catalyst enriched with oxygen vacancies for peroxymonosulfate (PMS) activation was prepared by simultaneously subjecting Bi2Fe4O9 to acid etching and light irradiation. The resultant material, designated as BFO-LA, was optimized by varying the acid etching concentrations to achieve the most efficacious degradation of DMP (10 mg/L). Comprehensive characterization techniques revealed the structural characteristics and presence of oxygen vacancies in BFO-LA. Results showed that the Fe2+ sites in BFO-LA activate PMS to generate active species, with singlet oxygen and free radicals playing key roles in DMP degradation. In the system of 0.3 g/L BFO-LA and 1 mM PMS, it can degrade 94.7 % of DMP in 60 min, with the degradation kinetic constant of 0.0542 min-1. Oxygen vacancies enhanced electron transfer and the Fe3+/Fe2+ redox cycle, improving PMS activation efficiency from 9.7 % to 88.3 %, compared to unmodified Bi2Fe4O9. Notably, the BFO-LA catalyst retained its superior stability and catalytic activity even upon repeated use. After 6 cycles of experiments, the degradation efficiency of DMP was still as high as 94.1 %. Toxicity evaluations utilizing the T.E.S.T. software and rice seedlings demonstrated that the degradation process effectively mitigated the toxicity associated with DMP, alleviating the adverse effects on seed germination and seedling growth. Collectively, this study highlights the significance of oxygen vacancies in BFO-LA, establishing it as an efficient and stable PMS activation catalyst for DMP degradation.

Di(2-ethylhexyl) phthalate (DEHP) is a widespread distributed and merging contaminant in soil, causing extensive concern about micro-ecological risks. However, the ecological response strategies of soil microorganisms to DEHP in saline environments and their carbon cycling effects are still poorly understood. To address this, we used high-throughput sequencing technology, RT-qPCR, enzyme assays, and their coupled techniques to assess the ecotoxic effects of DEHP on microorganisms and carbon cycling in saline soils. DEHP interfered with carbon cycle processes by regulating soil enzyme activities and altering bacterial community structure, with these effects being simultaneously regulated by salinity and time. DEHP stimulated the enrichment of phthalate-degrading bacteria, promoted the expression of carbon degradation genes (cbhI and abfA), and enhanced soil carbon degradation. DEHP did not exacerbate its impact on soil microorganisms under salinity stress, but the microbial response to DEHP was delayed. Molecular ecological network analysis further revealed that bacterial communities adopted distinct interaction strategies under different salinization levels to cope with DEHP-induced disturbances. Overall, soil salinity affects microbial ecological responses to DEHP. These findings provide new insights into the environmental risks of phthalate contamination in saline soils.

Widespread use of the new chiral triazole fungicide mefentrifluconazole (MFZ) poses a threat to soil organisms. Although triazole fungicides have been reported to induce reproductive disorders in vertebrates, significant research gaps remain regarding their impact on the reproductive health of soil invertebrates. Here, reproduction-related toxicity end points were explored in earthworms (Eisenia fetida) after exposure for 28 d to soil containing 4 mg/kg racemic MFZ, R-(-)-MFZ, and S-(+)-MFZ. The S-(+)-MFZ treatment resulted in a more pronounced reduction in the number of cocoons and juveniles compared to R-(-)-MFZ treatment, and the expression of annetocin gene was significantly downregulated following exposure to both enantiomers. This reproductive toxicity has been attributed to the disruption of ovarian steroidogenesis at the transcriptional level. Further studies revealed that MFZ enantiomers were able to activate the estrogen receptor (ER). Indirect evidence for this estrogenic effect is provided by the introduction of 17 beta-estradiol, which also induces reproductive disorders through ER activation.

Continued application of new chiral fungicide mefentrifluconazole (MFZ) increases its risk to soil ecosystem. However, the toxicity of MFZ enantiomers to soil fauna and whether stereoselectivity exists remains poorly elucidated. Based on multilevel toxicity endpoints and transcriptomics, we investigated the negative effects of racemic, R-(-)-, and S-(+)-MFZ on Eisenia fetida. After exposure to S-(+) configuration at 4 mg/kg for 28 day, its reactive oxygen species levels were elevated by 15.4% compared to R-(-) configuration, inducing enantiospecific oxidative stress and transcriptional aberrations. The S-(+) isomer induced more severe cell membrane damage and apoptosis than the R-(-) isomer, and notably, the selectivity of apoptosis is probably dominated by the mitochondrial pathway. Mechanistically, differential mitochondrial stress lies in: S-(+) isomer specifically upregulated mitochondrial cellular component compared to R-(-) isomer and identified more serious mitochondrial fission. Furthermore, S-(+) conformation down -regulated biological processes associated with ATP synthesis and metabolism, with specific inhibition of mitochondrial respiratory electron transport chain complex I and IV activity resulting in more severe electron flow disturbances. These ultimately mediated enantioselective ontogenetic process disorders, which were supported at phenotypic (weight loss), genetic, and protein (reverse modulate TCTP and Sox2 expression) levels. Our findings offer an important reference for elucidating the enantioselective toxicological mechanism of MFZ in soil fauna.

Fosthiazate (FOS) is a widely used organophosphorus insecticide effective against soil root-knot nematodes. However, its ecotoxicity to non-target soil organisms, particularly in combination with microplastics (MPs), is unclear. This study explores the toxic-effects and molecular mechanisms of co-exposure to FOS and MPs on earthworms (Eisenia fetida) using multilevel toxicity endpoints and transcriptomics. Results showed that both FOS and MPs elevated the intracellular levels of reactive oxygen species (ROS), malondialdehyde (MDA), and 8-hydroxy-2-deoxyguanosine (8-OHdG) in earthworms' cells. The superoxide dismutase (SOD) and catalase (CAT) activities followed a similar trend in all treatments, with changes observed at 14 and 28 days, indicating that co-exposure to FOS and MPs increased DNA oxidative damage. Notably, the co-exposure more significantly inhibited Ca2+-ATPase activity and exacerbated neurotoxicity compared to individual treatments, closely associated with changes in intracellular ROS levels that mediate neuroinhibition and lead to neurotoxicity. KEGG enrichment analysis revealed that MPs and FOS disrupted pathways related to metabolism, immunity, and apoptosis, while co-exposure primarily impaired endocrine and receptor pathways, showing higher toxicity. Our study offers novel insights into the ecotoxicological effects and mechanisms of pesticides and microplastics on earthworms, providing valuable data for evaluating the soil environmental health risks associated with compound pollution.