Penthiopyrad, a chiral fungicide with a riskier S-enantiomer, exhibited poorly understood stereoselective behaviors in mammals. This study investigated the enantioselective accumulation and metabolism of penthiopyrad enantiomers in mammals using in vivo, in vitro, and in silico approaches. Penthiopyrad accumulated in the liver and kidneys at concentrations over 10-fold higher than those in the blood. S-penthiopyrad showed preferential absorption with 1.6-fold higher bioavailability than the R-enantiomer. Twenty-three metabolites (12 novel) were identified, with CYP-mediated N-demethylation as the dominant pathway. S-penthiopyrad was preferentially metabolized in mouse liver microsomes with a 1.5-fold shorter half-life, while human liver microsomes favored the R-enantiomer. CYP2E1, CYP2C (mouse), and CYP3A4 (human) mediated stereoselective metabolism, with the preferential metabolism of the R-enantiomer by human CYP3A4 attributed to its low binding energy and favorable conformation. This study comprehensively clarified the stereoselective behavior and metabolic pathway of penthiopyrad in mammals, providing valuable metabolic insights for further toxicological evaluation of penthiopyrad.

A dual-signals fluorescent and colorimetric peptide-based probe FHSH with 5-carboxy fluorescein (5-FAM) fluorophore modified tripeptide (His-Ser-His-NH2) was successfully synthesized. FHSH exhibited remarkably high selectivity for Cu2+ and Hg2+ based on the corresponding fluorescence quenching, and the limit of detections (LODs) of 27.9 nM for Cu2+and 19.2 nM for Hg2+, respectively. The FSH-Cu2+ ensemble and FHSH-Hg2+ ensemble responded to S2- with notable selectivity through fluorescence enhancement effect and colorimetric changes, and the LODs for S2- were 14.1 nM (FHSH-Cu2+ensemble) and 7.6 nM (FHSH-Hg2+ensemble). FHSH not only successfully achieved fluorescence imaging of Cu2+, Hg2+ and S2- in living cells and zebrafish larvae, but also obtained practical application in real water samples with satisfactory results. In addition, we have developed test strips with convenient and low cost for the visual detection under a 365 nm UV light. Meanwhile, an ultra-sensitive molecular logic gate was designed based on the different changes in FHSH fluorescence. Likewise, the smartphone colour recognizer was used to read the original colour of the solutions and convert to G/B value for semi-quantitative analysis. Finally, the applications of FHSH-Cu2+ ensemble and FHSH-Hg2+ ensemble for "naked-eye" monitoring of S2- production during food spoilage in realtime under 365 nm UV light.

Sulfasalazine (SAS) has a certain inhibitory effect on cancer, but its therapeutic effect on intracranial metastasis of hepatocellular carcinoma (HCC) remains unclear. The purpose of this study was to explore whether SAS has a beneficial effect on intracranial metastasis of HCC and reveal its potential molecular mechanism. HCC cells were treated with SAS, and PIAS3/JAK1/STAT3 was regulated by siRNA, pcDNA 3.1 overexpression vector, and JAK inhibitors. The regulatory effects of SAS on the PIAS3/JAK1/STAT3 axis were evaluated by loss- and gain-of-function and rescue experiments. Cancer cells were evaluated by C11-BODIPY probe, MDA detection, iron ion detection, and LPO detection. Proliferation was assessed by clonal formation assay, apoptosis by flow cytometry, invasion and migration by Transwell assay, and EMT-associated protein expression by western blot. The ability of cancer cells to metastasize was evaluated by blood–brain barrier model and xenografted mouse model. SAS effectively hampered HCC cell proliferation, invasion, and migration and EMT process, induced apoptosis and ferroptosis of HCC cells, and prevented brain metastases (BMS) of HCC cells. SAS inhibited JAK1/STAT3 pathway activation in HCC cells by upregulating PIAS3. JAK1 inhibitor enhanced the impact of SAS on HCC, while PIAS3 had the opposite action. SAS blocks JAK1/STAT3 pathway by upregulating PIAS3, thereby inducing ferroptosis and inhibiting BMS in HCC. Combined administration of SAS and JAK inhibitors may be a potential strategy for HCC with brain metastases.

It is urgent to develop highly sensitive detection technologies to achieve precise detection of mercury ions (Hg2+) and sulfur ions (S2- ) as they pose a serious threat to human health and the ecological environment. In this work, a novel peptide-based fluorescent probe TPE-DAH with tetrastyrene (TPE) derivative and tripeptide backbone (Asp-Ala-His-NH2) was precisely designed and successfully synthesized, which exhibited the highly emitting characteristic of aggregation-induced emission (AIE) effect. As design, TPE-DAH exhibited excellent selectivity and high sensitivity for Hg2+ with the limit of detection (LOD) as low as 8.4 nM. Importantly, the complex TPEDAH-Hg2+was investigated by fluorescence technique which clearly established the high selectivity towards S2- based on fluorescence "off-on" response, and the LOD of S2- was 98.9 nM. Notably, TPE-DAH not only successfully achieved fluorescence imaging in living cells and zebrafish larvae with low toxicity and excellent permeability, but also successfully applied to detection of Hg2+ and S2- in real water samples with good recovery and accuracy. Advantageously, TPE-DAH was fabricated into a portable fluorescence test strips and was used for visual detection of Hg2+ and S2- , demonstrating that its potential for multi-functional applications. Remarkably, an ultrasensitive logic gate with TPE-DAH was constructed by using sequential detection strategy. Furthermore, we ingeniously combined the smartphone App analysis device with the colour change of TPE-DAH solutions to construct a visual analysis device, thereby achieving semi-quantitative determination.

Glyphosate is widely used in agriculture and often co-applied with urea. However, the impact of urea on glyphosate degradation and its potential soil toxicity remains unclear. This study investigated the effects of urea co-application (240 kg pure nitrogen per hectare) on glyphosate degradation and related ecological risks through laboratory soil experiments. Results showed that urea extended the half-life of glyphosate by 1.99 times. Co-application altered soil available nitrogen levels, reduced the abundance of microbes including Pseudomonas and Bacillus, and significantly decreased the relative abundance of glyphosate degradation genes such as goxB. Partial least squares structural equation modeling (PLS-SEM) confirmed that soil available nitrogen content was the main factor affecting glyphosate residues. Co-application also reduced β-D-cellobiosidase activity, hindered organic carbon decomposition, and disrupted the coupling of soil carbon and nitrogen cycles. Urease and leucine aminopeptidase activities were inhibited, nitrogen-cycling microbes such as Methylomirabilota declined, and nitrogen availability decreased. Microbial network complexity increased under co-application, but resilience declined, indicating reduced soil ecological stability. This study reveals the mechanisms by which urea affects glyphosate degradation and provides theoretical support for the rational application of urea and glyphosate.

The green α,β-C(sp3)-H difunctionalization of saturated cyclic amines and ethers was developed through FeCl3-catalyzed dehydrogenative [4 + 2] cycloaddition under air, enabling the construction of structurally novel spirooxindole scaffolds. This method featured earth-abundant Fe catalysis, benign oxidant (air), along with atom- and step-economical formal triple C(sp3)-H functionalization. Also, the protocol offers operational simplicity, acceptable yields, and good functional group compatibility.

Heparin is a highly sulfated linear glycosaminoglycan with anticoagulant properties, and its excessive use may lead to many diseases. Therefore, developing sensitive detection techniques for precise detection of heparin is of utmost importance. Herein, we reported a novel peptide-based fluorescent probe (TPE-GRGRG) based on tetraphenylethylene (TPE)-labeled pentapeptide (Gly-Arg-Gly-Arg-Gly-NH2) for the highly selective and outstanding sensitive detection of heparin. TPE-GRGRG exhibited a large Stokes shift (132 nm) and typical aggregation-induced emission (AIE) characteristics in DMSO/H2O binary mixtures. TPE-GRGRG demonstrated a significant enhancement of fluorescence intensity in the presence of heparin with a response time of under 30 s and the limit of detection (LOD) as low as 0.30 nM. A series of characterizations revealed that TPE-GRGRG and heparin formed nanoaggregates via electrostatic interactions, including fluorescence spectroscopy, UV-Vis, FTIR, CD, zeta potential, DLS measurements and fluorescence lifetime analyses, which in turn restricted the intramolecular rotation and triggered the fluorescence enhancement. TPE-GRGRG enabled heparin detection over a wide pH range and exhibited good biocompatibility, and was successfully applied to image heparin in living cells and zebrafish larvae. Furthermore, this study established detection platforms based on swab tests for visual qualitative analysis and smartphone RGB analysis, thereby achieving portable and visual heparin monitoring with the LOD of 0.93 μM. Finally, heparin detection was achieved in 0.1% fetal bovine serum with the LOD of 1.46 nM. TPE-GRGRG offers a reliable strategy for point-of-care testing of heparin, holding potential application value in clinical diagnosis and biosensing.

Human exposure to concurrent multiple pesticide residues is inevitable given the application of pesticides in modern agricultural, yet the associated health risks remain insufficiently characterized. A mixture of ten frequently detected pesticides was incorporated into the diet of male ICR mice at three dose levels (ADI, 100 ×ADI, and 10 ×NOAEL) for 17 weeks to evaluate intestinal risks. ADI exposure caused no significant colonic inflammation or injury, whereas 100 ×ADI and 10 ×NOAEL caused IBD, characterized by shortened colon length, epithelial injury, upregulated pro-inflammatory cytokines (IL-1β, IL-6, IL-22), and downregulated tight junIction proteins (ZO-1, occludin). In DSS-induced model, even ADI group developed evident IBD, and those at 100 ×ADI and 10 ×NOAEL group exhibited more exacerbated intestinal inflammatory responses and tissue injury, indicating that chronic ADI dose pesticide cocktail exposure markedly increased susceptibility to IBD. At 10 ×NOAEL dose, the relative abundances of Lactobacillus, Streptococcus, and Candidatus_Saccharimonas were significantly reduced, whereas Faecalibaculum and Akkermansia were significantly enriched, which was closely associated with increased pro-inflammatory cytokines (IL-6, IL-22) and upregulation of RORγt and IL-17a alongside reduced Foxp3 and IL-10, indicating a disrupted Th17/Treg balance linked to colonic inflammation and barrier dysfunction. FMT confirmed the critical role of the gut microbiota in mediating both pesticide-induced colonic inflammation and aggravation of DSS-induced colitis. Collectively, these findings demonstrate that chronic low-dose pesticide cocktail exposure disrupts gut microbial homeostasis, alters the Th17/Treg balance, upregulates pro-inflammatory cytokines, and compromises intestinal barrier integrity, thereby triggering colonic inflammation and exacerbating DSS-induced IBD. This work highlights the necessity of the evaluation of mixed pesticide residue.

Ethiprole, a phenylpyrazole insecticide widely used as a fipronil alternative, raises emerging ecological concerns due to its poorly characterized metabolic fate in paddy fields. This study systematically explored the environmental behavior, metabolic mechanisms, and metabolite-specific risks of ethiprole across a water-sediment-animal-plant (WSAP) continuum. Rice cultivation markedly accelerated ethiprole degradation by 4.33-fold (half-life of 2.22 days) compared to nonvegetated systems. A sulfide metabolite (E-R) was exclusively detected in plant-containing continuums and exhibited a 15-fold higher acute toxicity to benthic loach (LC50 of 0.07 mg/L). Crucially, E-R was endogenously generated and preferentially accumulated in rice roots, while oxidative metabolism in aerial tissues produced ethiprole sulfone (E-O). Root-derived E-R subsequently migrated to sediments, persisted for over 50 days, and posed extreme ecological risk (RQ >2000). This work reveals a previously unrecognized rice-driven pathway generating highly toxic metabolites and highlights the need for metabolite-specific risk assessment.

The frequent detection of oxytetracycline in agricultural soils may alter the environmental behavior and risks of coexisting pesticides. This study investigated the influence of oxytetracycline on the degradation and phytotoxicity of pendimethalin in a soil-soybean system. Results demonstrated that oxytetracycline at 50 mg/kg prolonged the half-life of pendimethalin in soil from 33.0 days to 40.8 days. Coexposure inhibited the activities of urease, dehydrogenase, and catalase in the soil. While pendimethalin alone showed no adverse effects on soybean growth, coexposure with oxytetracycline induced pronounced growth inhibition and enhanced oxidative stress. Ecological risk assessment confirmed significantly elevated risks to earthworms. Further analysis revealed suppressed CYP450 and GST activities in both soil and soybean plants, with more pronounced reductions observed in the plants. These findings indicate that the coexistence of antibiotic-herbicide can enhance herbicide persistence and increase environmental contamination risks.

Copper ions (Cu2+) and sulfide ions (S2-) are important markers in many physiologies processes. However, its excessive emissions have caused serious harm to biological systems and ecological environment. Herein, a new dual-mode fluorescent and colorimetric peptidyl probe NGHK was developed based on 7-Nitro-2,1,3-benzoxadiazole (NBD) fluorophore coupled tripeptide backbone (Gly-His-Lys-NH2). NGHK exhibited excellent selectivity (only Cu2+), high sensitivity (LOD = 74.5 nM), rapid response (within 50 s) and wider pH range (6.0-12.0) towards Cu2+ in 10.0 mM HEPES buffer at pH 7.4, with the non-fluorescence complex (NGHK-Cu2+) formation via the 1:1 binding mode. Interestingly, NGHK-Cu2+ ensemble exhibited dual-mode responses to S2- through obvious color changes and fluorescence enhancement responses, the limit of detection (LOD) for S2- was 36.9 nM. In addition, NGHK was used for fluorescence imaging in living cells and zebrafish larvae based on the satisfactory low toxicity. For practical applications, NGHK was not only capable of monitoring Cu2+ and S2- concentrations in real food and water samples with satisfactory results, but also could conduct the analysis of the surface of actual samples. Utilizing NGHK, we also developed the portable test strips for the visual detection of Cu2+ and S2-. Simultaneously, NGHK was successfully developed as an ultrasensitive logic gate with truth table.

A new dual-signals peptide-based probe FAHK was rationally designed and successfully synthesized for sequential detection of Cu2+ and glyphosate through fluorescence and colorimetric sensing method. The limit of detections (LODs) for Cu2+ and glyphosate were calculated to be 49.7 nM and 66.3 nM, respectively. Additionally, FAHK not only demonstrated excellent accuracy in the determination of real food, water and vegetables samples, but also was applied for fluorescence imaging Cu2+ and glyphosate in two biological systems. Besides, test strips experiments showed that FAHK has extraordinary potential in environmental monitoring and food analysis applications. Meanwhile, we explored the application of FAHK in molecular logic gates, and a visual portable sensing platform was established via the smartphone color picker App system, and successfully achieving semi-quantitative analysis of Cu2+ and glyphosate. More importantly, FAHK has successfully achieved the detection of glyphosate residues on six food surfaces and soil sample with satisfactory results.

Detection of hazardous substances in feed is important for ensuring human health. A method based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was developed and validated for the screening and confirmation of 420 hazardous substances, including pesticides, veterinary drugs, and mycotoxins commonly found in feed. The screening phase employed less stringent criteria to minimize false negatives caused by matrix effects. Subsequently, stricter identification criteria were applied for confirmation to avoid false positives from interfering compounds. The performance of the proposed method was verified by limit of detection (LOD, 5~500 μg/L), screening detection limits (SDL, 50~500 μg/L), matrix effect (ME, 36.12~121.16%), precision (0.02~14.98%), stability, and accuracy. The method was successfully applied to real feed samples, demonstrating its capability to detect the presence of the 420 target hazardous substances. We believe our method provides strong technical support for ensuring the quality and safety of feed.

Pyriproxyfen (PYR), as a sanitary insecticide, is widely used in agricultural applications and vector control programs for controlling pest breeding sites, and it inevitably enters aquatic ecosystems and transforms into a series of degradation products during applications. However, the effects of pyriproxyfen and its degradation products on non-target organisms in aquatic ecosystems have not been fully explored. In this study, zebrafish embryos (6-120 h post-fertilization) were used as a model to explore the acute toxicity and developmental toxicity of pyriproxyfen and its nine degradation products. Pyriproxyfen (PYR) caused moderate acute toxicity in zebrafish embryos, while its degradation products 4'-OH-PYR and 5″-OH-PYR showed higher toxicity; most other degradation products showed lower acute toxicity. During early development, pyriproxyfen degradation products reduced embryo hatching rates, induced malformations, disrupted antioxidant balance and promoted apoptosis. Based on the screening results, three representative degradation products (4'-OH-PYR, 4-OH-POP, and PYPAC) were selected for further investigation of neurotoxicity. These degradation products were found to impair larval locomotor capacity by suppressing acetylcholinesterase activity, altering neurotransmission, and disrupting neuromuscular development through downregulation of key genes. These findings enhance our understanding of the developmental effects of pyriproxyfen on non-target organisms in the environment carry substantial implications for the comprehensive evaluation of its ecological and health-related risk profiles.

Fluazinam is a widely used agricultural fungicide, but the effects of subchronic exposure remain unclear. This study investigated the liver and intestinal toxicity of fluazinam in mice exposed for 12 weeks at 0.01 and 1 mg/kg bw/day. Results showed that fluazinam caused weight loss, metabolic disorders, and liver toxicity. 16S rRNA sequencing revealed gut microbiota dysbiosis, which is associated with impaired intestinal barrier function and inflammation. Notably, the abundance of bile-acid-metabolizing bacteria (such as Lactobacillus, Lachnospiraceae, and Muribaculum) decreased. Mechanistically, this abnormality triggered a cascade reaction via the gut-liver axis, leading to decreased total bile acid levels, downregulated hepatic synthesis genes (Cyp7a1, Cyp8b1), and upregulated intestinal Fgf15. These changes impaired the farnesoid X receptor pathway and bile acid metabolism, exacerbating liver and intestinal damage. This study revealed that fluazinam mediated metabolic toxicity through the gut-liver axis, providing a novel perspective for its health risk assessment.

Near-infrared spectroscopy (NIRS) is a powerful tool for the rapid identification and quantitative analysis of dairy farm slurry components. Temperature variations significantly influence on the spectra measurements while their underlying spectral response mechanisms remain insufficiently understood. This study investigates the impact of temperature variations on the spectral characteristics of slurry to ascertain the availability of improving the NIRS-based analysis. Using a portable detection device of slurry nutrients, near-infrared diffuse reflectance spectra were acquired from slurry samples across temperatures ranging from 0 to 40 degrees C. Principal component analysis (PCA) and two-dimensional correlation spectroscopy (2D-COS) were applied to assess spectral response intensity, temporal sequence and correlations among functional groups. The results indicate that increasing temperature weakens hydrogen bonding strength, leading to intensity changes. In particular, hydrogen-bonded water partially dissociates, enhancing features associated with free O-H groups and indicating temperature-induced changes in intermolecular interactions. Additionally, elevated temperatures increase the susceptibility of specific absorption bands ((N-H), and (O-H)) to shift, affecting the stability of slurry composition predictions. 2D-COS analysis reveals the sequential evolution of absorption band changes of specific functional groups under thermal perturbation, offering insight into dynamic transformations. While 2D-COS has been previously applied to various agricultural materials, this study extends its application to temperatureinduced spectral analysis of dairy farm slurry, addressing a specific challenge in NIRS-based nutrient analysis where conventional temperature correction approaches have shown limitations. By identifying temperaturesensitive spectral regions and molecular transformations, this study provides a scientific basis for developing temperature-resilient NIRS models, which are crucial for facilitating real-time nutrient monitoring and precision slurry management on dairy farms.

Nitrogen fertilizers play a critical role in enhancing crop yields; however, excessive application has resulted in significant environmental challenges, including water contamination and increased greenhouse gas emissions. Therefore, improving nitrogen use efficiency is essential for sustainable agriculture. This review based on a systematic search of Web of Science and CNKI for peer-reviewed studies on maize nitrogen efficiency published between 1945 and 2024 (excluding conference abstracts), this review presents the first multiscale synthesis demonstrating how balanced nitrate–ammonium nutrition coordinates N–C metabolism and phytohormone signaling to boost nitrogen use efficiency and stimulate maize growth, with supporting evidence from other crops. By integrating results from hydroponic and field experiments, the review evaluates the influence of mixed nitrogen sources on nitrogen uptake, root morphology, photosynthesis, carbon metabolism, and hormone signaling. Findings indicate that optimal NO3−:NH4+ ratios improve nitrogen absorption through enhanced root development and activation of specific nitrogen transporters. Additionally, mixed nitrogen nutrition increases photosynthetic efficiency, promotes carbon assimilation, reduces energy expenditure, and stimulates auxin-mediated growth. This review shows that balanced nitrate–ammonium co-application synergistically enhances crop nitrogen-use efficiency and yield, provides a theoretical basis for high-efficiency nitrogen-fertilizer development, and helps alleviate environmental pressures, advance sustainable agriculture, and secure food and ecosystem safety. Its efficacy, however, is modulated by soil type, climate, and genotypic variation, necessitating systematic validation and application optimization in future research.

Context: Leaf senescence is the final stage of leaf development, which is often accompanied by nitrogen remobilization. And also, leaf senescence can be modulated by auxin. Objective: The objective was to advance in the knowledge of the auxin-mediated mechanisms of leaf senescence and nitrogen assimilation that regulated grain yield. Methods: Using maize variety Zhengdan 958 (ZD958) as material, a two-year field experiment was conducted to assess the leaf senescence and nitrogen assimilation-related physiological process regulated by Naphthalene acetic acid (NAA) (H2O, 0.1 mmol/L NAA) and nitrogen levels (N0: No N application, N240: Normal N). Results: The results showed that NAA significantly increased grain yield under low N conditions, which increased by 55.0 % in 2022 and 29.7 % in 2023, respectively. Auxin promotes grain filling mainly by extending the duration of filling days and increasing the average filling rate. In addition, auxin reduced nitrogen output from leaves, and significantly increased nitrogen accumulation in other organs. The total N accumulation of auxin treatment was increased by 34.7 % under low N condition, and increased by 17.5 % under normal N condition. Furthermore, NAA significantly increased the activities of NR (Nitrate Reductase), GS (Glutamine Synthetase) and GOGAT (Glutamate synthase), thus promoting the nitrogen assimilation ability of leaves. Conversely, auxin application reduced the content of abscisic acid and ethylene, increased the activity of antioxidant enzymes, and decreased the expression of senescence-related genes. This delayed leaf senescence, which was conducive to extending the grain-filling duration and the accumulation of grain starch and protein, subsequently improved the maize yield. Conclusions: Auxin increased nitrogen assimilation ability and delayed leaf senescence, increase protein and carbohydrate accumulation in the grain, and thus increased the maize yield by 9.3 % and 42.4 %, under normal and nitrogen-deficiency condition. Implications: This study highlights the impact of externally auxin application on N assimilation and senescence and its regulatory function in grain development.

Copper ions (Cu2+) and glyphosate (Glyp) pose serious threats to human health and the ecological environment, and it is of great significance to conduct efficient and sensitive detection of these substances. Herein, a simple and novel fluorescence "on-off-on" peptidyl probe NGGH with 7-Nitro-2,1,3-benzoxadiazole (NBD) conjugated tripeptide (Gly-Gly-His-NH2) was developed for relay detection of Cu2+ and glyphosate in 100 % aqueous solutions, and the limit of detections (LODs) reached up to 24.4 nM and 4.8 nM for Cu2+ and glyphosate, respectively. The binding stoichiometry of NGGH with Cu2+ was proved to be 1:1 based on Job's plot curve, fluorescence titration, ESI-HRMS and theoretical calculations. Notably, fluorescence imaging of Cu2+ and glyphosate revealed that NGGH was successfully applied on living cells and zebrafish larvae based on remarkable fluorescence behavior and low cytotoxicity. In addition, NGGH not only successfully determined Cu2+ and glyphosate in two real samples, but also applied to monitor the residues of Cu2+ and glyphosate in the surfaces of millet, kiwifruit and leaf. Furthermore, NGGH was fabricated into the fluorescence test strips and used for the visual detection of Cu2+ and glyphosate using naked eye under the 365 nm UV lamp. Finally, we constructed the sensitive INHIBIT logic gate based on the fluorescence "on-off-on" response effect characteristic.

The phytotoxicity risks of atrazine to crops have received widespread attention, but the toxic effects of its metabolites on plants have been largely overlooked. In this study, the contributions and mechanisms underlying phytotoxicity of the atrazine and its metabolites (DEA and DIA) were systematically investigated in soybean seedlings. Two dealkylation metabolites DEA and DIA caused growth suppression, inhibited photosynthesis, activated the antioxidant system, and induced changes in chloroplast ultrastructure in soybean seedlings. Integrated Biological Response (IBR) analysis indicated that at equivalent environmentally relevant concentrations, the toxicity indices of DEA and DIA were 73.60% and 34.00% of atrazine, respectively. Molecular docking analysis revealed that both DEA and DIA exhibited high binding energies with Photosystem II D1 protein, with their potential target protein in soybean plants being consistent with that of atrazine. Metabolomic analysis further confirmed that the metabolites DEA and DIA disrupt key metabolic pathways, including alpha-linolenic acid metabolism, consistent with the mode of action of atrazine. These effects are associated with the inhibition of the photosynthetic electron transport chain and ROS accumulation. By calculating the environmental risk quotient, the risk of metabolites DEA to succeeding crops is likely to exceed that posed by the parent atrazine. These findings suggested that dealkylation metabolites of atrazine are overlooked contributors to soybean phytotoxicity in atrazine residue, and the risks posed by herbicide metabolites to crops need to be addressed.