Over the past decade, the escalating prevalence of copper (Cu) pollution in soil has raised significant concerns due to its potential detrimental impacts on soil quality, microbial communities, plant health, food security, and land degradation. Despite extensive research, the response mechanisms, threshold levels, and reliable indicators of Cu pollution remain debated. Therefore, comprehensive studies are needed to gain a better understanding of these dynamics. This study address these gaps by: (1) evaluating Cu toxicity effects on soil biological, biochemical, barley germination, growth, biomass, and physiological parameters, and (2) identifying robust indicators for early assessment of Cu-associated risks. Soil was amended with CuSO4 at concentrations ranging from 0 to 210 mg kg-1. Factors exacerbating Cu toxicity included Cu concentration, pH levels, and the duration of Cu accumulation within the soil ecosystem. Consequently, at the highest Cu concentration a significant reduction in soil biological, biochemical, barley germination, growth, biomass, and physiological parameters was observed towards the end of the experiment. Simultaneously, there was a substantial increase in the levels of antioxidant enzymes, malondialdehyde (MDA), reactive oxygen species (ROS), and electrolyte leakage (EL) triggered by Cu presence. Correlation analyses highlighted bacterial populations, microbial biomass carbon (MBC), dehydrogenase activity, respiration rates, pH levels, seedling fresh biomass and height, chlorophyll content, photosynthetic activity, protein content, superoxide dismutase (SOD) activity, ROS levels, and MDA as sensitive indicators of Cu stress. As a result, these parameters are proposed as reliable indicators for predicting Cu toxicity thresholds, excessive accumulation, and associated risks within soil ecosystems. These indicators have implications not only for land degradation but also for food security considerations.

Czech University of Life Sciences Prague Faculty of Environmental Science Kamýcká 129 16500 Prague Czech Republic

Department of Agronomy University of Agriculture Faisalabad Faisalabad 38040 Pakistan

Department of Biochemistry College of Science King Saud University P O Box 2455 11451 Riyadh Saudi Arabia

Department of Soil and Environmental Sciences Muhammad Nawaz Shareef University of Agriculture Multan Pakistan

Department of Soil and Water Systems University of Idaho 875 Perimeter Drive MS 2060 Moscow ID USA

EPCRS Excellence Center Plant Pathology and Biotechnology Lab ; Agric Botany Dept ; Fac Agric Kafrelsheikh University Kafrelsheikh 33516 Egypt

Institute of Molecular Biology and Biotechnology The University of Lahore Lahore 54590 Pakistan

Institute of Surface Earth System Science School of Earth System Science Tianjin University Tianjin 300072 China

Islamic International Medical College Trust Pakistan Railway Hospital Rawalpindi Punjab Pakistan

Key Laboratory of Mountain Surface Processes and Ecological Regulation Institute of Mountain Hazards and Environment Chinese Academy of Sciences Chengdu 610041 China

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Alshaharni, M.O. (2024). Physiological and morphological responses of wheat (Triticum aestivum L.) and tomato (Solanum lycopersicum L.) to seed priming with Indole acetic acid as plant growth regulator. International Journal of Agriculture and Biosciences, 13(2), 118–127.

An, T., Gao, Y., Kuang, Q., Wu, Y., Zaman, Q. U., Zhang, Y., Xu, B., & Chen, Y. (2022). Effect of silicon on morpho-physiological attributes, yield and cadmium accumulation in two maize genotypes with contrasting root system size and health risk assessment. Plant and Soil, 477, 117–134.

Aponte, H., Meli, P., Butler, B., Paolini, J., Matus, F., Merino, C., Cornejo, P., & Kuzyakov, Y. (2020). Meta-analysis of heavy metal effects on soil enzyme activities. Science of the Total Environment, 737, 139744.

Arias, J. A. (2009). Symbiotic effects of the fungus Glomus sp. on chromium (III), chromium (VI), and lead (II) uptake by mesquite (Prosopis sp.): A novel method to remediate heavy metals. The University of Texas at El Paso.

Azarin, K., Usatov, A., Minkina, T., Alliluev, I., Duplii, N., Mandzhieva, S., Singh, A., Rajput, V. D., Kumar, S., Fakhr, M. A., & Elshikh, M. S. (2024). Impact nano-and micro-form of CdO on barley growth and oxidative stress response. Journal of King Saud University-Science, 36, 103493.

Baghaie, A. H., & Mirzaee, R. (2020). Multi-walled carbon nanotubes, zeolite and arbuscular mycorrhizal fungi can affect plant Ni concentration in a Ni-polluted soil that was naturally polluted with diesel fuel. Journal of Water and Environmental Nanotechnology, 5, 157–167.

Balaram, V. (2019). Rare earth elements: A review of applications, occurrence, exploration, analysis, recycling, and environmental impact. Geoscience Frontiers, 10, 1285–1303.

Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry, 44, 276–287.

Berger, L. J. (2018). Katharina M. Keiblinger, Martin Schneider, Markus Gorfer, Melanie Paumann, Evi Deltedesco. Harald. Ecotoxicology, 27, 217–233.

Bhardwaj, I., & Garg, N. (2023). Phytohormones and arbuscular mycorrhizal Rhizoglomus intraradices together modulate defense mechanisms in mungbean to reduce Ni toxicity. Rhizosphere, 27, 100723.

Bolan, N., Kunhikrishnan, A., Thangarajan, R., Kumpiene, J., Park, J., Makino, T., Kirkham, M. B., & Scheckel, K. (2014). Remediation of heavy metal (loid) s contaminated soils–to mobilize or to immobilize? Journal of Hazardous Materials, 266, 141–166.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

Briffa, J., Sinagra, E., Blundell, R. (2020). Heavy metal pollution in the environment and their toxicological effects on humans. Heliyon, 6, e04691.

Cakmak, I., & Horst, W. J. (1991). Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiologia Plantarum, 83, 463–468.

Chen, X., Chen, J., Yu, X., Sanganyado, E., Wang, L., Li, P., & Liu, W. (2022). Effects of norfloxacin, copper, and their interactions on microbial communities in estuarine sediment. Environmental Research, 212, 113506.

Chrysargyris, A., Papakyriakou, E., Petropoulos, S. A., & Tzortzakis, N. (2019). The combined and single effect of salinity and copper stress on growth and quality of Mentha spicata plants. Journal of Hazardous Materials, 368, 584–593.

Chuong, N. V., & Tri, T. L. K. (2024). Enhancing soil fertilizer and peanut output by utilizing endophytic bacteria and vermicompost on arsenic-contaminated soil. International Journal of Agriculture and Biosciences, 13, 596–602.

Dhaliwal, S. S., Singh, J., Taneja, P. K., & Mandal, A. (2020). Remediation techniques for removal of heavy metals from the soil contaminated through different sources: A review. Environmental Science and Pollution Research, 27, 1319–1333.

Dong, B., Zhang, R., Gan, Y., Cai, L., Freidenreich, A., Wang, K., Guo, T., & Wang, H. (2019). Multiple methods for the identification of heavy metal sources in cropland soils from a resource-based region. Science of the Total Environment, 651, 3127–3138.

Fagnano, M., Agrelli, D., Pascale, A., Adamo, P., Fiorentino, N., Rocco, C., Pepe, O., & Ventorino, V. (2020). Copper accumulation in agricultural soils: Risks for the food chain and soil microbial populations. Science of the Total Environment, 734, 139434.

Georgiadou, E. C., Kowalska, E., Patla, K., Kulbat, K., Smolińska, B., Leszczyńska, J., & Fotopoulos, V. (2018). Influence of heavy metals (Ni, Cu, and Zn) on nitro-oxidative stress responses, proteome regulation and allergen production in basil (Ocimum basilicum L.) plants. Frontiers in Plant Science, 9, 862.

Ghazaryan, K., Agrawal, S., Margaryan, G., Harutyunyan, A., Rajput, P., Movsesyan, H., Rajput, V. D., Singh, R. K., Minkina, T., Elshikh, M. S., & Alwahibi, M. S. (2024). Soil pollution: An agricultural and environmental problem with nanotechnological remediation opportunities and challenges. Discover Sustainability, 5, 1–33.

Gupta, S. D., Agarwal, A., & Pradhan, S. (2018). Phytostimulatory effect of silver nanoparticles (AgNPs) on rice seedling growth: An insight from antioxidative enzyme activities and gene expression patterns. Ecotoxicology and Environmental Safety, 161, 624–633.

Hasanuzzaman, M., Bhuyan, M. B., Zulfiqar, F., Raza, A., Mohsin, S. M., Mahmud, J. A., Fujita, M., & Fotopoulos, V. (2020). Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator. Antioxidants, 9, 681.

Hassan, W., Bano, R., Bashir, S., & Aslam, Z. (2016a). Cadmium toxicity and soil biological index under potato (Solanum tuberosum L.) cultivation. Soil Research, 54, 460–468.

Hassan, W., Bashir, S., Ali, F., Ijaz, M., Hussain, M., & David, J. (2016b). Role of ACC-deaminase and/or nitrogen fixing rhizobacteria in growth promotion of wheat (Triticum aestivum L.) under cadmium pollution. Environmental Earth Sciences, 75, 1–14.

Hassan, A., Amjad, S. F., Saleem, M. H., Yasmin, H., Imran, M., Riaz, M., Ali, Q., Joyia, F. A., Ahmed, S., & Ali, S. (2021). Foliar application of ascorbic acid enhances salinity stress tolerance in barley (Hordeum vulgare L.) through modulation of morpho-physio-biochemical attributes, ions uptake, osmo-protectants and stress response genes expression. Saudi Journal of Biological Sciences, 28, 4276–4290.

Ivashchenko, K. V., Korneykova, M. V., Sazonova, O. I., Vetrova, A. A., Ermakova, A. O., Konstantinov, P. I., Sotnikova, Y. L., Soshina, A. S., Vasileva, M. N., & Vasenev, V. I. (2022). Phylloplane biodiversity and activity in the city at different distances from the traffic pollution source. Plants, 11, 402.

Jäggi, W. (1976). Die Bestimmung der CO2-Bildung als Mass der bodenbiologischen Aktivität. Schweizer Landwirtschaftliche Forschung, 15, 371–380.

Jamir, E., Kangabam, R. D., Borah, K., Tamuly, A., Deka Boruah, H., & Silla, Y. (2019). Role of soil microbiome and enzyme activities in plant growth nutrition and ecological restoration of soil health. Microbes and Enzymes in Soil Health and Bioremediation, 1, 99–132.

Kalu, C. M., Ogugua, U. V., Udeh, E. L., Adeosun, W. B., Kanu, S. A., Ntushelo, K., Loots, D. T., Adriaanse, P., & Tekere, M. (2024). Plant-microbes’ interactions and their roles in bioremediation: A case study of Phragmites australis in acid mine condition. International Journal of Agriculture and Biosciences, 13, 669–682.

Keiblinger, K. M., Schneider, M., Gorfer, M., Paumann, M., Deltedesco, E., Berger, H., Jöchlinger, L., Mentler, A., Zechmeister-Boltenstern, S., & Soja, G. (2018). Assessment of Cu applications in two contrasting soils—effects on soil microbial activity and the fungal community structure. Ecotoxicology, 27, 217–233.

Koç, E., & Karayiğit, B. (2022). Assessment of biofortification approaches used to improve micronutrient-dense plants that are a sustainable solution to combat hidden hunger. Journal of Soil Science and Plant Nutrition, 22, 475–500.

Kumar, V., Pandita, S., Sidhu, G. P. S., Sharma, A., Khanna, K., Kaur, P., Bali, A. S., & Setia, R. (2021). Copper bioavailability, uptake, toxicity and tolerance in plants: A comprehensive review. Chemosphere, 262, 127810.

Mahatthanaphatcharakun, P., & Taratima, W. (2025). Comparative effect of drought stress on growth and physiological performance of three different rice cultivars. International Journal of Agriculture and Biosciences, 14, 84–93.

Lawrence, R. A., Parkhill, L. K., & Burk, R. F. (1978). Hepatic cytosolic non selenium-dependent glutathione peroxidase activity: Its nature and the effect of selenium deficiency. The Journal of Nutrition, 108, 981–987.

Li, C., Zhou, K., Qin, W., Tian, C., Qi, M., Yan, X., & Han, W. (2019). A review on heavy metals contamination in soil: Effects, sources, and remediation techniques. Soil and Sediment Contamination: An International Journal, 28, 380–394.

Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology, 148, 350–382.

Liu, Y., Zhang, J., Yang, W., Wu, F., Xu, Z., Tan, B., Zhang, L., He, X., & Guo, L. (2018). Canopy gaps accelerate soil organic carbon retention by soil microbial biomass in the organic horizon in a subalpine fir forest. Applied Soil Ecology, 125, 169–176.

Lladó, S., López-Mondéjar, R., & Baldrian, P. (2017). Forest soil bacteria: Diversity, involvement in ecosystem processes, and response to global change. Microbiology and Molecular Biology Reviews, 81, e00063-e16.

Loreto, F., & Velikova, V. (2001). Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiology, 127, 1781–1787.

Lutts, S., Kinet, J., & Bouharmont, J. (1996). NaCl-induced senescence in leaves of rice (Oryza sativaL.) cultivars differing in salinity resistance. Annals of Botany, 78, 389–398.

Lysenko, E. A., Klaus, A. A., Kartashov, A. V., & Kusnetsov, V. V. (2020). Specificity of Cd, Cu, and Fe effects on barley growth, metal contents in leaves and chloroplasts, and activities of photosystem I and photosystem II. Plant Physiology and Biochemistry, 147, 191–204.

Mohy-Ud-Din, W., Akhtar, M. J., Bashir, S., Asghar, H. N., Nawaz, M. F., & Chen, F. (2023). Isolation of Glyphosate-Resistant Bacterial Strains to Improve the Growth of Maize and Degrade Glyphosate under Axenic Condition. Agriculture, 13, 886.

Nabi, A., Naeem, M., Aftab, T., Khan, M. M. A., & Ahmad, P. (2021). A comprehensive review of adaptations in plants under arsenic toxicity: Physiological, metabolic and molecular interventions. Environmental Pollution, 290, 118029.

Nakano, Y., & Asada, K. (1987). Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant and Cell Physiology, 28, 131–140.

Naz, M., Dai, Z., Hussain, S., Tariq, M., Danish, S., Khan, I. U., Qi, S., & Du, D. (2022). The soil pH and heavy metals revealed their impact on soil microbial community. Journal of Environmental Management, 321, 115770.

Nkwunonwo, U. C., Odika, P. O., & Onyia, N. I. (2020). A review of the health implications of heavy metals in food chain in Nigeria. The Scientific World Journal, 2020, 6594109.

Ozfidan, C., Turkan, I., Sekmen, A., & Seckin, B. (2012). Abscisic acid-regulated responses of aba2-1 under osmotic stress: The abscisic acid-inducible antioxidant defence system and reactive oxygen species production. Plant Biology, 14, 337–346.

Page, A., Miller, R., & Keeney, D. (1982). Methods of soil analysis. Part 2. American Society of Agronomy. Soil Science Society of America, Madison, WI, USA, 4, 167–179.

Papaioannou, D., Koukoulakis, P., Lambropoulou, D., Papageorgiou, M., & Kalavrouziotis, I. (2019). The dynamics of the pharmaceutical and personal care product interactive capacity under the effect of artificial enrichment of soil with heavy metals and of wastewater reuse. Science of the Total Environment, 662, 537–546.

Qin, X., Chai, M., Ju, D., & Hamamoto, O. (2018). Investigation of plating wastewater treatment technology for chromium, nickel and copper. IOP Conference Series: Earth and Environmental Science, 176(1), 012006.

Rehman, M., Liu, L., Wang, Q., Saleem, M. H., Bashir, S., Ullah, S., & Peng, D. (2019a). Copper environmental toxicology, recent advances, and future outlook: A review. Environmental Science and Pollution Research, 26, 18003–18016.

Rehman, M., Maqbool, Z., Peng, D., & Liu, L. (2019b). Morpho-physiological traits, antioxidant capacity and phytoextraction of copper by ramie (Boehmeria nivea L.) grown as fodder in copper-contaminated soil. Environmental Science and Pollution Research, 26, 5851–5861.

Rizwan, M., Ali, S., Abbas, F., Adrees, M., Zia‐ur‐Rehman, M., Farid, M., Gill, R.A., & Ali, B. (2017). Role of organic and inorganic amendments in alleviating heavy metal stress in oilseed crops. Oilseed Crops: Yield and Adaptations under Environmental Stress, 1, 224–235.

Sairam, R., Shukla, D., & Saxena, D. (1997). Stress induced injury and antioxidant enzymes in relation to drought tolerance in wheat genotypes. Biologia Plantarum, 40, 357–364.

Samantara, K., Bohra, A., Mohapatra, S. R., Prihatini, R., Asibe, F., Singh, L., Reyes, V. P., Tiwari, A., Maurya, A. K., Croser, J. S., & Wani, S. H. (2022). Breeding more crops in less time: a perspective on speed breeding. Biology, 11(2), 275.

Saleem, M. H., Fahad, S., Khan, S. U., Din, M., Ullah, A., Sabagh, A. E., Hossain, A., Llanes, A., & Liu, L. (2020). Copper-induced oxidative stress, initiation of antioxidants and phytoremediation potential of flax (Linum usitatissimum L.) seedlings grown under the mixing of two different soils of China. Environmental Science and Pollution Research, 27, 5211–5221.

Sharma, P., & Hall, D. (1996). Effect of photoinhibition and temperature on carotenoids in sorghum leaves. Indian Journal of Biochemistry & Biophysics, 33, 471–477.

Shuaib, M., Azam, N., Bahadur, S., Romman, M., Yu, Q., & Xuexiu, C. (2021). Variation and succession of microbial communities under the conditions of persistent heavy metal and their survival mechanism. Microbial Pathogenesis, 150, 104713.

Silambarasan, S., Logeswari, P., Valentine, A., Cornejo, P., & Kannan, V. R. (2020). Pseudomonas citronellolis strain SLP6 enhances the phytoremediation efficiency of Helianthus annuus in copper contaminated soils under salinity stress. Plant and Soil, 457, 241–253.

Tehulie, N., & Eskezia, H. (2021). Effects of nitrogen fertilizer rates on growth, yield components and yield of food Barley (Hordeum vulgare L.): A Review. Journal of Plant Sciences and Agricultural Research, 5, 46.

Touceda-González, M., Prieto-Fernández, Á., Renella, G., Giagnoni, L., Sessitsch, A., Brader, G., Kumpiene, J., Dimitriou, I., Eriksson, J., & Friesl-Hanl, W. (2017). Microbial community structure and activity in trace element-contaminated soils phytomanaged by Gentle Remediation Options (GRO). Environmental Pollution, 231, 237–251.

Uchimiya, M., Bannon, D., Nakanishi, H., McBride, M. B., Williams, M. A., & Yoshihara, T. (2020). Chemical speciation, plant uptake, and toxicity of heavy metals in agricultural soils. Journal of Agricultural and Food Chemistry, 68, 12856–12869.

Vardhini, B. V. (2020). Brassinosteroids and salicylic acid as chemical agents to ameliorate diverse environmental stresses in plants. Protective Chemical Agents in the Amelioration of Plant Abiotic Stress: Biochemical and Molecular Perspectives, 1, 389–412.

Vardumyan, H., Singh, A., Rajput, V. D., Minkina, T., El-Ramady, H., & Ghazaryan, K. (2024). Additive-mediated phytoextraction of copper-contaminated soils using Medicago lupulina L. Egyptian Journal of Soil Science, 64, 599–618.

Vasilchenko, A. V., Galaktionova, L. V., Tretyakov, N. Y., Dyachkov, S. M., & Vasilchenko, A. S. (2023). Impact of agricultural land use on distribution of microbial biomass and activity within soil aggregates. Soil Use and Management, 39, 618–633.

Yadav, S., Modi, P., Dave, A., Vijapura, A., Patel, D., & Patel, M. (2020). Effect of abiotic stress on crops. Sustainable Crop Production, 3, 5–16.

Zhang, Q.-C., Shamsi, I. H., Xu, D.-T., Wang, G.-H., Lin, X.-Y., Jilani, G., Hussain, N., & Chaudhry, A. N. (2012). Chemical fertilizer and organic manure inputs in soil exhibit a vice versa pattern of microbial community structure. Applied Soil Ecology, 57, 1–8.

Zhao, X., Sun, Y., Huang, J., Wang, H., & Tang, D. (2020). Effects of soil heavy metal pollution on microbial activities and community diversity in different land use types in mining areas. Environmental Science and Pollution Research, 27, 20215–20226.