Spent bleaching earth (SBE) is the largest waste produced by the palm oil industry. However, according to several studies, SBE and its recovery product DBE have the potential as filler materials in NPK fertilizers. This study examines the influence of NPK fertilizer with SBE and DBE as filler materials on soybean plants’ biochemical and agronomic properties. The field-based experiment was done in a single-factor randomized complete block design with 4 replicates. We tested fertilizers of 10% bentonite clay mineral using NPK on a filler basis (control), 5% bentonite clay mineral with 5% SBE of NPK on a filler basis, and 5% bentonite clay mineral with 5% DBE using NPK on a filler basis. The variables observed include soil chemical properties after applying fertilizer, which involves the concentrations of several heavy metals. Biochemical characteristics, including the content of hydrogen peroxide (H₂O₂) and peroxidase (POD), superoxide dismutase (SOD) activity, malondialdehyde (MDA), relative electrolyte leakage (REL), total phenolic content, and proline content. The agronomic characteristics of soybean plants, including root and shoot dry weight. The data were analyzed using ANOVA and tested using the least significant difference test at a 95% confidence interval. The results indicated that materials of SBE and DBE could partially substitute the filler elements in bentonite clay mineral of NPK fertilizer on a filler basis, and they had the same influence in SOD activity, H₂O₂ content, POD, MDA, REL, total phenolic, proline and root dry weight and shoot of soybean plants.

• Alexieva, V., Sergiev, I., Mapelli, S., & Karanov, E. (2001). The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell and Environment, 24(12), 1337-1344. https://doi.org/10.1046/j.1365-3040.2001.00778.x

• Alia, Mohanty, P., & Matysik, J. (2001). Effect of proline on the production of singlet oxygen. Amino Acids, 21(2), 195-200. https://doi.org/10.1007/s007260170026

• Alloway, B. J. (1995). Heavy metals in soils (2nd ed.). Blackie Academic & Professional.

• Anugrah, C., Indradewa, D., & Putra, E. T. S. (2020). Biochemical response of hybrid maize (Zea mays L.) to NPK fertilization based on spent bleaching earth in field scale. E3S Web of Conferences, 142, 1-9. https://doi.org/10.1051/e3sconf/202014201004

• Bates, L. S., Waldren, R. P., & Teare, I. D. (1973). Rapid determination of free proline for water-stress studies. Plant and Soil, 39, 205-207.

• Békésiová, B., Hraška, Š., Libantová, J., Moravčíková, J., & Matušíková, I. (2008). Heavy-metal stress induced accumulation of chitinase isoforms in plants. Molecular Biology Reports, 35(4), 579-588. https://doi.org/10.1007/s11033-007-9127-x

• Bertrand, M., & Guary, J. C. (2002). Handbook of plant and crop physiology. In M. Pessarakli (Ed.), How Plants Adopt their Physiology to an Excess of Metals (2nd ed., pp. 751-761). Marcel Dekker.

• Brown, J. E., Khodr, H., Hider, R. C., & Rice-evans, C. A. (1998). Structural dependence of flavonoid interactions with Cu2+ ions: Implications for their antioxidant properties. Biochemical Journal, 330(3), 1173-1178. https://doi.org/10.1042/bj3301173

• Bücker-Neto, L., Paiva, A. L. S., Machado, R. D., Arenhart, R. A., & Margis-Pinheiro, M. (2017). Interactions between plant hormones and heavy metals responses. Genetics and Molecular Biology, 40(1), 373-386. https://doi.org/10.1590/1678-4685-gmb-2016-0087

• 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(3), 463-468. https://doi.org/10.1111/j.1399-3054.1991.tb00121.x

• Chun, O. K., Kim, D. O., & Lee, C. Y. (2003). Superoxide radical scavenging activity of the major polyphenols in fresh plums. Journal of Agricultural and Food Chemistry, 51(27), 8067-8072. https://doi.org/10.1021/jf034740d

• Cuypers, A., Remans, T., Weyens, N., Colpaert, J., Vassilev, A., & Vangronsveld, J. (2013). Soil-plant relationships of heavy metals and metalloids. In B. J. Alloway (Ed.), Heavy Metals in Soils: Trace Metals and Metalloids in Soils and Their Bioavailability (pp. 161-193). Springer Science+Business Media.

• Deswati, D., Khairiyah, K., Safni, S., Yusuf, Y., Refinel, R., & Pardi, H. (2020). Environmental detoxification of heavy metals in flood & drain aquaponic system based on biofloc technology. International Journal of Environmental Analytical Chemistry, 102(18), 7155-7164. https://doi.org/10.1080/03067319.2020.1826463

• Dhurhania, C. E., & Novianto, A. (2019). Uji kandungan fenolik total dan pengaruhnya terhadap aktivitas antioksidan dari berbagai bentuk sediaan sarang semut (Myrmecodia pendens) [Test of total phenolic content and its effect on antioxidant activity of various forms ant nest plant (Myrmecodia pendens)]. Jurnal Farmasi dan Ilmu Kefarmasian Indonesia, 5(2), 62-68. https://doi.org/10.20473/jfiki.v5i22018.62-68

• Díaz, J., Bernal, A., Pomar, F., & Merino, F. (2001). Induction of shikimate dehydrogenase and peroxidase in pepper (Capsicum annuum L.) seedlings in response to copper stress and its relation to lignification. Plant Science, 161(1), 179-188. https://doi.org/10.1016/S0168-9452(01)00410-1

• Dionisio-Sese, M. L., & Tobita, S. (1998). Antioxidant responses of rice seedlings to salinity stress. Plant Science, 135(1), 1-9. https://doi.org/10.1016/S0168-9452(98)00025-9

• Ehlert, B., & Hincha, D. K. (2008). Chlorophyll fluorescence imaging accurately quantifies freezing damage and cold acclimation responses in Arabidopsis leaves. Plant Methods, 4(1), 1-7. https://doi.org/10.1186/1746-4811-4-12

• Erfandy, D., & Juarsah, I. (2014). Teknologi pengendalian pencemaran logam berat [Control technology for heavy metal pollution]. In Konservasi Tanah Menghadapi Perubahan Iklim (pp. 159-186). Balai Penelitian Tanah.

• Eviati, & Sulaeman. (2009). Analisis Kimia Tanah, Tanaman, Air, dan Pupuk [Chemical analysis of soil, plant, water, and fertilizer]. Balai Penelitian Tanah.

• Fageria, N. K., Baligar, V. C., & Jones, C. A. (1997). Growth and mineral nutrition of field crop. Marcel Dekker. Inc.

• Farago, M. E., & Mullen, W. A. (1979). Plants which accumulate metals. Part IV. A possible copper-proline complex from the roots of Armeria maritima. Inorganica Chimica Acta, 32, 93-94. https://doi.org/10.1016/S0020-1693(00)91627-X

• Fargasova, A. (2001). Phytotoxic effects of Cd, Zn, Pb, Cu and Fe on Sinapis alba L. seedlings and their accumulation in roots and shoots. Biologia Plantarum, 44(3), 471-473. https://doi.org/10.1023/A:1012456507827

• Fitriani, R. N., Budiyanto, S., & Sukarjo, S. (2019). Respon tanaman kedelai (Glycine max L. Merill) pada berbagai konsentrasi cemaran ion logam Ni2+ dengan pemberian biokompos [Response of soybean (Glycine max L.) at various contamination concentrations ion of nickel metal (Ni2+) with biocompost application]. Journal of Agro Complex, 3(3), 184-193. https://doi.org/10.14710/joac.3.3.184-193

• Handayanto, E., Nuraini, Y., Muddarisna, N., Netty, S., & Fiqri, A. (2017). Fitoremediasi dan phytomining logam berat pencemar tanah [Phytoremediation and phytomining of heavy metal soil contaminants]. UB Press.

• Hanin, N. N. F., & Pratiwi, R. (2017). Kandungan fenolik, flavonoid dan aktivitas antioksidan ekstrak daun paku laut (Acrostichum aureum L.) fertil dan steril di kawasan mangrove Kulon Progo, Yogyakarta [Phenolic content, flavonoids and antioxidant activity of fertile and sterile sea fern (Acrostichum aureum L.) leaf extracts]. Journal of Tropical Biodiversity and Biotechnology, 2(2017), 51-56. https://doi.org/10.22146/jtbb.29819

• Harmiwati, H., Salmariza, S., Kurniawati, D., Lestari, I., Munaf, E., Desmiarti, R., & Zein, R. (2015). Biosorption of cadmium ion from aqueous solutions by low-cost soybean waste (Glycine max). Journal of Chemical and Pharmaceutical Research, 7(9), 94-100.

• Harmoni, K. (2014). Analisis persebaran iklim Klasifikasi Oldeman di Provinsi Daerah Istimewa Yogyakarta [Analysis of Oldeman climate clasification spreading in Daerah Istimewa Yogyakarta]. [Undergraduate Thesis]. Universitas Muhammadiyah Surakarta, Indonesia.

• Howlett, N. G., & Avery, S. V. (1997). Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation. Applied and Environmental Microbiology, 63(8), 2971-2976. https://doi.org/10.1128/aem.63.8.2971-2976.1997

• Janoušková, M., Pavlíková, D., & Vosátka, M. (2006). Potential contribution of arbuscular mycorrhiza to cadmium immobilisation in soil. Chemosphere, 65(11), 1959-1965. https://doi.org/10.1016/j.chemosphere.2006.07.007

• Jung, C., Maeder, V., Funk, F., Frey, B., Sticher, H., & Frossard, E. (2003). Release of phenols from Lupinus albus L. roots exposed to Cu and their possible role in Cu detoxification. Plant and Soil, 252(2), 301-312. https://doi.org/10.1023/A:1024775803759

• Kocheva, K. V., Georgiev, G. I., & Kochev, V. K. (2005). A diffusion approach to the electrolyte leakage from plant tissues. Physiologia Plantarum, 125(1), 1-9. https://doi.org/10.1111/j.1399-3054.2005.00533.x

• Kumalaningsih, S. (2007). Antioksidan Alami Penangkal Radikal Bebas [Natural Free Radical Scavengers]. Trubus Agrisarana.

• Li, C., Dang, F., Li, M., Zhu, M., Zhong, H., Hintelmann, H., & Zhou, D. (2017). Effects of exposure pathways on the accumulation and phytotoxicity of silver nanoparticles in soybean and rice. Nanotoxicology, 11(5), 699-709. https://doi.org/10.1080/17435390.2017.1344740

• Löffler, G., & Petrides, P. E. (1988). Physiologische chemie (4th ed.). Springer.

• Loh, S. K., Cheong, K. Y., Choo, Y. M., & Salimon, J. (2015). Formulation and optimisation of spent bleaching earth-based bio organic fertiliser. Journal of Oil Palm Research, 27(1), 57-66.

• Malecka, A., Piechalak, A., Zielińska, B., Kutrowska, A., & Tomaszewska, B. (2014). Response of the pea roots defense systems to the two-element combinations of metals (Cu, Zn, Cd, Pb). Acta Biochimica Polonica, 61(1), 23-28. https://doi.org/10.18388/abp.2014_1918

• Marciniak, A., Brzeszczyńska, J., Gwoździński, K., & Jegier, A. (2009). Antioxidant capacity and physical exercise. Biology of Sport, 26(3), 197-213. https://doi.org/10.5604/20831862.894649

• Marklund, S., & Marklund, G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry, 47(3), 469-474. https://doi.org/10.1111/j.1432-1033.1974.tb03714.x

• Mehta, S. K., & Gaur, J. P. (1999). Heavy metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris. New Phytologist, 143(2), 253-259. https://doi.org/10.1046/j.1469-8137.1999.00447.x

• Michalak, A. (2006). Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Polish Journal of Environmental Studies, 15(4), 523-530.

• Milić, B. L., Djilas, S. M., & Čanadanović-Brunet, J. M. (1998). Antioxidative activity of phenolic compounds on the metal-ion breakdown of lipid peroxidation system. Food Chemistry, 61(4), 443-447. https://doi.org/10.1016/S0308-8146(97)00126-X

• Mishra, S., & Dubey, R. S. (2006). Heavy metal uptake and detoxification mechanisms in plants. International Journal of Agricultural Research, 1(2), 122-141. https://doi.org/10.3923/ijar.2006.122.141

• Nair, P. M. G., & Chung, I. M. (2014). A mechanistic study on the toxic effect of copper oxide nanoparticles in soybean (Glycine max L.) root development and lignification of root cells. Biological Trace Element Research, 162(1-3), 342-352. https://doi.org/10.1007/s12011-014-0106-5

• Paleg, L. G., Stewart, G. R., & Bradbeer, J. W. (1984). Proline and glycine betaine influence protein solvation. Plant Physiology, 75(4), 974-978. https://doi.org/10.1104/pp.75.4.974

• Pasaribu, K. F., & Sukandar, S. (2017). Analisis manfaat biaya pengelolaan limbah spent bleaching earth melalui pemanfaatan dan penimbunan dengan memperhitungkan nilai gas rumah kaca [Benefit-cost analysis of spent bleaching earth management through recovery and landfilling by using greenhouse gases as externality]. Jurnal Tehnik Lingkungan, 23(2), 33-42. https://doi.org/10.5614/j.tl.2017.23.2.4

• Pratap, A., Gupta, S. K., Kumar, J., & Solanki, R. K. (2012). Soybean. In S. K. Gupta (Ed.), Technological Innovations in Major World Oil Crops, Volume 1: Breeding (pp. 1-405). Springer Science+Business Media. https://doi.org/10.1007/978-1-4614-0356-2

• Purba, R. S., Irwan, S. N. R., & Putra, E. T. S. (2020). The effect of spent bleaching earth filler-based NPK fertilization on proline, growth and yield of maize. Caraka Tani: Journal of Sustainable Agriculture, 35(1), 44-53. https://doi.org/10.20961/carakatani.v35i1.34166

• Rusnam, R., Asmiwarti, A., Efrizal, E., & Sofyani, A. (2013). The influence of water hyacinth to decrease the heavy metals mercury (Hg) concentration for irrigation. International Journal on Advanced Science Engineering and Information Technology, 3(6), Article 368. https://doi.org/10.18517/ijaseit.3.6.350

• Rusnam, R., Puari, A. T., Yanti, N. R., & Efrizal, E. (2022). Utilisation of exhausted coffee husk as low-cost bio-sorbent for adsorption of Pb2+. Tropical Life Sciences Research, 33(3), 229-252. https://doi.org/10.21315/tlsr2022.33.3.12

• Shah, F. U. R., Ahmad, N., Masood, K. R., Peralta-Videa, J. R., & Ahmad, F. D. (2010). Heavy metal toxicity in plants. In M. Ashraf (Ed.), Plant Adaptation and Phytoremediation (pp. 1-481). Springer Science+Business Media. https://doi.org/10.1007/978-90-481-9370-7

• Sharma, P., & Dubey, R. S. (2004). Ascorbate peroxidase from rice seedlings: Properties of enzyme isoforms, effects of stresses and protective roles of osmolytes. Plant Science, 167(3), 541-550. https://doi.org/10.1016/j.plantsci.2004.04.028

• Sharma, P., Jha, A. B., Dubey, R. S., & Pessarakli, M. (2012). Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of Botany, 2012, 1-26. https://doi.org/10.1155/2012/217037

• Sharma, P. N., Kumar, N., & Bisht, S. S. (1994). Effect of zinc deficiency on chlorophyll contents, photosyntehsis, and water relations of cauliflower plants. Photosyntehtica, 30, 353-359.

• Shofi, M. (2017). Daya hambat perak nitrat (AgNO3) pada perkecambahan biji kacang hijau (Vigna radiata) [Inhibition of silver nitrate (AgNO3) to seed germination of mung bean (Vigna radiata)]. Al-Kauniyah: Jurnal Biologi, 10(2), 98-104. https://doi.org/10.15408/kauniyah.v10i2.4869

• Siripornadulsil, S., Traina, S., Verma, D. P. S., & Sayre, R. T. (2002). Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae. Plant Cell, 14(11), 2837-2847. https://doi.org/10.1105/tpc.004853

• Smeets, K., Opdenakker, K., Remans, T., Van Sanden, S., Van Belleghem, F., Semane, B., Horemans, N., Guisez, Y., Vangronsveld, J., & Cuypers, A. (2009). Oxidative stress-related responses at transcriptional and enzymatic levels after exposure to Cd or Cu in a multipollution context. Journal of Plant Physiology, 166(18), 1982-1992. https://doi.org/10.1016/j.jplph.2009.06.014

• Smeets, K., Ruytinx, J., Van Belleghem, F., Semane, B., Lin, D., Vangronsveld, J., & Cuypers, A. (2008). Critical evaluation and statistical validation of a hydroponic culture system for Arabidopsis thaliana. Plant Physiology and Biochemistry, 46(2), 212-218. https://doi.org/10.1016/j.plaphy.2007.09.014

• Stadtman, E. R. (1992). Protein oxidation and aging. Science, 257(5074), 1220-1224. https://doi.org/10.1080/10715760600918142

• Stoyanova, Z., & Doncheva, S. (2002). The effect of zinc supply and succinate treatment on plant growth and mineral uptake in pea plant. Brazilian Journal of Plant Physiology, 14(2), 111-116. https://doi.org/10.1590/S1677-04202002000200005

• Taufiq, A., & Sundari, T. (2012). Respons tanaman kedelai terhadap lingkungan tumbuh [Soybean response to grow environment]. Buletin Palawija, 22, 13-26.

• Vangronsveld, J., & Clijsters, H. (1994). Toxic effects of metals. In Plants and the Chemical Elements. Biochemistry, Uptake, Tolerance and Toxicity (pp. 150-177). VCH Publishers.

• Vianello, A., Zancani, M., Peresson, C., Petrussa, E., Casolo, V., Krajňáková, J., Patui, S., Braidot, E., & Macrì, F. (2007). Plant mitochondrial pathway leading to programmed cell death. Physiologia Plantarum, 129(1), 242-252. https://doi.org/10.1111/j.1399-3054.2006.00767.x

• Vicuna, D., Malone, R. P., & Dix, P. J. (2011). Increased tolerance to abiotic stresses in tobacco plants expressing a barley cell wall peroxidase. Journal of Plant Sciences, 6(1), 1-13. https://doi.org/10.3923/jps.2011.1.13

• Wang, Z., Zhang, Y., Huang, Z., & Huang, L. (2008). Antioxidative response of metal-accumulator and non-accumulator plants under cadmium stress. Plant and Soil, 310(1-2), 137-149. https://doi.org/10.1007/s11104-008-9641-1

• Winkel-Shirley, B. (2002). Biosynthesis of flavonoids and effects of stress. Current Opinion in Plant Biology, 5(3), 218-223. https://doi.org/10.1016/S1369-5266(02)00256-X

• Wisnubroto, M. P., Putra, E. T. S., & Kurniasih, B. (2020). Tanggapan Biokemis, Fisiologis, dan Agronomis Kedelai (Glycine max L. Merrill) terhadap Pemupukan NPK Berperekat Spent dan Deoiled Bleaching Earth [Biochemical, physiological, and agronomic responses of soybean (Glycine max L. Merrill) to spent and deoiled bleaching earth filler-based NPK fertilization]. [Unpublished Master’s Thesis]. Universitas Gadjah Mada, Indonesia.

• Wisnubroto, M. P., Putra, E. T. S., & Kurniasih, B. (2021). Effects of spent and deoiled bleaching earth filler-based NPK Fertilization on the soil nutrient status and growth of soybean (Glycine max (L.) Merrill). Caraka Tani: Journal of Sustainable Agriculture, 36(2), Article 213. https://doi.org/10.20961/carakatani.v36i2.43847

• Wisnubroto, M. P., Putra, E. T. S., & Kurniasih, B. (2023). Agronomic responses of soybean (Glycine max L. Merrill) to spent and deoiled bleaching earth filler-based NPK fertilization. Jurnal Agronomi Tanaman Tropika (JUATIKA), 5(1), 172-184. https://doi.org/https://doi.org/10.36378/juatika.v5i1.2685

• Yruela, I. (2005). Copper in plants. Brazilian Journal of Plant Physiology, 17(1), 145-156. https://doi.org/10.1590/s1677-04202005000100012

• Zakiah, Z., Suliansyah, I., Bakhtiar, A., & Mansyurdin. (2017). Effect of crude extracts of six plants on vegetative growth of soybean (Glycine max Merr.). International Journal of Advances in Agricultural Science and Technology, 4(7), 1-12.

• Zhang, F. Q., Wang, Y. S., Lou, Z. P., & Dong, J. De. (2007). Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza). Chemosphere, 67(1), 44-50. https://doi.org/10.1016/j.chemosphere.2006.10.007

• Zhang, J., Cui, S., Li, J., Wei, J., & Kirkham, M. B. (1995). Protoplasmic factors, antioxidant responses and chilling resistance in maize. Plant Physiology Biochemistry, 33(5), 567-575.