Biodecolorization of azo dye Acid Blue 92 (AB92) by Ceratophyllum demersum L.: process optimization using Taguchi method and toxicity assessment


  • Zahra EFTEKHARI University of Guilan
  • Akbar NORASTEHNIA University of Guilan
  • Zahra MASOUDIAN University of Guilan



Azo dye, Acid Blue 92, Bioremediation, Ceratophyllum demersum, Oxidative stress


This study evaluated the ability of the submerged aquatic plant Ceratophyllum demersum to remove the Acid Blue 92 (AB92) dye. The effect of some operational parameters such as the reaction time, initial dye concentration, initial plant biomass, and pH, on dye removal efficiency was studied. Based on Taguchi’s results, the optimized conditions for dye removal were time 7 days, dye concentration 20 mg l-1, initial plant biomass 4 g, and initial pH 5. Fourier-transform infrared spectroscopy (FTIR) results confirmed the interaction between dye molecules and plants. Based on the results of this study, C. demersum had a reusability to remove the dye, this fact confirming the mechanism of biodegradation in the dye removal process. Also, the effect of AB92 on the physiological responses of C. demersum was investigated. Minimum relative growth rate, tolerance index, chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoids at a concentration of 20 mg l-1 of AB92 were observed. The concentration of cyanidin glycoside, lipid peroxidation, and antioxidant activity increased in both concentrations of 10 and 20 mg l-1. It can be concluded that both concentrations of AB92 induced antioxidant activity and the risk of oxidative stress for Ceratophyllum.

Author Biographies

Zahra EFTEKHARI, University of Guilan

MSc Student

Akbar NORASTEHNIA, University of Guilan

Department of Biology, Ph.D., Associate Prof.

Zahra MASOUDIAN , University of Guilan

Ph. D in Plant Physiology


Aravindhan, R., Rao, J. R., & Nair, B. U. (2007). Removal of basic yellow dye from aqueous solution by sorption on green alga Caulerpa scalpelliformis. Journal of Hazardous Materials, 142(1-2), 68-76.

Bhat, S. A., Bashir, O., Haq, S. A. U., Amin, T., Rafiq, A., Ali, M., . . . Sher, F. (2022). Phytoremediation of heavy metals in soil and water: An eco-friendly, sustainable and multidisciplinary approach. Chemosphere, 303, 134788.

Chiu, K.-H., & Wu, T.-M. (2017). Glutathione biosynthesis plays an important role against 4-tert-octylphenol-induced oxidative stress in Ceratophyl-lum demersum. Chemosphere, 183, 565-573.

Chorom, M., Parnian, A., & Jaafarzadeh, N. (2012). Nickel removal by the aquatic plant (Ceratophyllum demersum L.). International Journal of Envi-ronmental Science and Development, 3(4), 372.

Daneshvar, N., Khataee, A., Rasoulifard, M., & Pourhassan, M. (2007). Biodegradation of dye solution containing Malachite Green: Optimization of effective parameters using Taguchi method. Journal of Hazardous Materials, 143(1-2), 214-219.

Dhote, S., & Dixit, S. (2009). Water quality improvement through macrophytes—a review. Environmental Monitoring and Assessment, 152, 149-153.

Duman, F., & Koca, F. D. (2014). Single and combined effects of exposure concentration and duration on biological responses of Ceratophyllum de-mersum L. exposed to Cr species. International Journal of Phytoremediation, 16(12), 1192-1208.

El-Alfy, A. T., Ahmed, A. A., & Fatani, A. J. (2005). Protective effect of red grape seeds proanthocyanidins against induction of diabetes by alloxan in rats. Pharmacological Research, 52(3), 264-270.

Ena, A., Carlozzi, P., Pushparaj, B., Paperi, R., Carnevale, S., & Sacchi, A. (2007). Eficacia del helecho de agua” Azolla” para reducir la demanda química de oxígeno y los polifenoles del alpechín. Grasas y Aceites, 58(1), 34-39.

Ewadh, H. M. (2020). Removal of methylene blue by coontail (Ceratophyllum demersum) using phytoremediation concept. Plant Archives, 20(1), 2677-2681.

Forni, C., Chen, J., Tancioni, L., & Caiola, M. G. (2001). Evaluation of the fern Azolla for growth, nitrogen, and phosphorus removal from wastewater. Water Research, 35(6), 1592-1598.

Gałczyńska, M., Mańkowska, N., Milke, J., & Buśko, M. (2019). Possibilities and limitations of using Lemna minor, Hydrocharis morsus-ranae, and Ceratophyllum demersum in removing metals with contaminated water. Journal of Water and Land Development.

Hak, K., Ritchie, R. J., & Dummee, V. (2020). Bioaccumulation and physiological responses of the Coontail, Ceratophyllum demersum exposed to copper, zinc, and in combination. Ecotoxicology and Environmental Safety, 189, 110049.

Heath, R. L., & Packer, L. (1968). Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Bio-chemistry and Biophysics, 125(1), 189-198.

Hoagland, D. R., & Arnon, D. I. (1950). The water-culture method for growing plants without soil. Circular. California Agricultural Experiment Station, 347(2nd edit).

Ihsanullah, I., Jamal, A., Ilyas, M., Zubair, M., Khan, G., & Atieh, M. A. (2020). Bioremediation of dyes: Current status and prospects. Journal of Water Process Engineering, 38, 101680.

Keskinkan, O., & Lugal Göksu, M. (2007). Assessment of the dye removal capability of submersed aquatic plants in a laboratory-scale wetland system using ANOVA. Brazilian Journal of Chemical Engineering, 24, 193-202.

Khataee, A., Dehghan, G., Ebadi, A., Zarei, M., & Pourhassan, M. (2010). Biological treatment of a dye solution by Macroalgae Chara sp.: Effect of operational parameters, intermediates identification and artificial neural network modeling. Bioresource Technology, 101(7), 2252-2258.

Khataee, A., Movafeghi, A., Torbati, S., Lisar, S. S., & Zarei, M. (2012). Phytoremediation potential of duckweed (Lemna minor L.) in degradation of CI Acid Blue 92: Artificial neural network modeling. Ecotoxicology and Environmental Safety, 80, 291-298.

Khataee, A., Movafeghi, A., Vafaei, F., Salehi Lisar, S., & Zarei, M. (2013). Potential of the aquatic fern Azolla filiculoides in biodegradation of an azo dye: modeling of experimental results by artificial neural networks. International Journal of Phytoremediation, 15(8), 729-742.

Kong, J.-M., Chia, L.-S., Goh, N.-K., Chia, T.-F., & Brouillard, R. (2003). Analysis and biological activities of anthocyanins. Phytochemistry, 64(5), 923-933.

Krems, P., Rajfur, M., Wacławek, M., & Kłos, A. (2013). The use of water plants in biomonitoring and phytoremediation of waters polluted with heavy metals. Ecological Chemistry and Engineering S, 20(2), 353-370.

Kvesitadze, G., Khatisashvili, G., Sadunishvili, T., & Ramsden, J. J. (2006). Biochemical mechanisms of detoxification in higher plants: basis of phy-toremediation. Springer Science & Business Media.

Lang, W., Sirisansaneeyakul, S., Ngiwsara, L., Mendes, S., Martins, L. O., Okuyama, M., & Kimura, A. (2013). Characterization of a new oxygen-insensitive azoreductase from Brevibacillus laterosporus TISTR1911: Toward dye decolorization using a packed-bed metal affinity reactor. Biore-source Technology, 150, 298-306.

Li, Z., Wakao, S., Fischer, B. B., & Niyogi, K. K. (2009). Sensing and responding to excess light. Annual Review of Plant Biology, 60, 239-260.

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

Liu, J., & Wang, Y. (2023). Research on Improved MOF Materials Modified by Functional Groups for Purification of Water. Molecules, 28(5), 2141.

Mahmoodi, N. M., Arami, M., Bahrami, H., & Khorramfar, S. (2010). Novel biosorbent (Canola hull): Surface characterization and dye removal ability at different cationic dye concentrations. Desalination, 264(1-2), 134-142.

Masoudian, Z., Salehi-Lisar, S. Y., & Norastehnia, A. (2020). Phytoremediation potential of Azolla filiculoides for sodium dodecyl benzene sulfonate (SDBS) surfactant considering some physiological responses, effects of operational parameters, and biodegradation of surfactant. Environmental Science and Pollution Research, 27, 20358-20369.

Masoudian, Z., Salehi-Lisar, S. Y., Norastehnia, A., & Tarigholizadeh, S. (2022). Duckweed potential for the phytoremediation of linear alkylbenzene sulfonate (LAS): identification of some intermediate biodegradation products and evaluation of antioxidant system. Bulletin of Environmental Contamination and Toxicology, 109(2), 364-372.

Mishra, S., Srivastava, S., Tripathi, R., Dwivedi, S., & Shukla, M. (2008). Response of antioxidant enzymes in coontail (Ceratophyllum demersum L.) plants under cadmium stress. Environmental Toxicology: An International Journal, 23(3), 294-301.

Mishra, S., Srivastava, S., Tripathi, R., Kumar, R., Seth, C., & Gupta, D. (2006). Lead detoxification by coontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidant system in response to its accumulation. Chemosphere, 65(6), 1027-1039.

Mohan, J., Shakya, N. B., Shukla, N. K., & Kumar, V. (2017). Studies on phytoremediation of municipal wastewater with reference to aquatic plant Ceratophyllum demersum. Annals of Plant Sciences, 6(12), 1918-1921.

Movafeghi, A., Khataee, A., Moradi, Z., & Vafaei, F. (2016). Biodegradation of direct blue 129 diazo dye by Spirodela polyrrhiza: an artificial neural networks modeling. International Journal of Phytoremediation, 18(4), 337-347.

Nabi, M. (2021). Heavy metals accumulation in aquatic macrophytes from an urban lake in Kashmir Himalaya, India. Environmental Nanotechnology, Monitoring & Management, 16, 100509.

Parent, C., Capelli, N., & Dat, J. (2008). Reactive oxygen species, stress and cell death in plants. Comptes Rendus Biologies, 331(4), 255-261. doi: 10.1016/j.crvi.2008.02.001

Phillips, D., Human, L., & Adams, J. (2015). Wetland plants as indicators of heavy metal contamination. Marine Pollution Bulletin, 92(1-2), 227-232.

Pillai, H., Girish, K., & Agsar, D. (2014). Isolation, characterization, and screening of actinomycetes from textile industry effluent for dye degradation. International Journal of Current Microbiology and Applied Sciences, 3(11), 105-115.

Qadri, H., Uqab, B., Javeed, O., Dar, G. H., & Bhat, R. A. (2022). Ceratophyllum demersum-An accretion biotool for heavy metal remediation. Science of The Total Environment, 806, 150548.

Radić, S., Babić, M., Škobić, D., Roje, V., & Pevalek-Kozlina, B. (2010). Ecotoxicological effects of aluminum and zinc on growth and antioxidants in Lemna minor L.. Ecotoxicology and Environmental Safety, 73(3), 336-342.

Rezania, S., Taib, S. M., Din, M. F. M., Dahalan, F. A., & Kamyab, H. (2016). Comprehensive review on phytotechnology: heavy metals removal by di-verse aquatic plant species from wastewater. Journal of Hazardous Materials, 318, 587-599.

Sah, M. K., Edbey, K., EL-Hashani, A., Almshety, S., Mauro, L., Alomar, T. S., . . . Bhattarai, A. (2022). Exploring the biosorption of methylene blue dye onto agricultural products: A critical review. Separations, 9(9), 256.

Sampath, M., & Vasanthi, M. (2013). Isolation, structural elucidation of flavonoids from Polyalthia longifolia (Sonn.) Thwaites and evaluation of antibacterial, antioxidant, and anticancer potential. International Journal of Pharmacy and Pharmaceutical Sciences, 5(1), 336-341.

Santabarbara, S., Agostini, G., Casazza, A. P., Syme, C. D., Heathcote, P., Böhles, F., . . . Carbonera, D. (2007). Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1767(1), 88-105.

Sharma, S., Singh, B., & Manchanda, V. (2015). Phytoremediation: role of terrestrial plants and aquatic macrophytes in the remediation of radionu-clides and heavy metal contaminated soil and water. Environmental Science and Pollution Research, 22, 946-962. .

Silver, S. (1991). Bacterial heavy metal resistance systems and possibility of bioremediation. Paper presented at the Biotechnology: Bridging Research and Applications: Proceedings of the US-Israel Research Conference on Advances in Applied Biotechnology June 24–30, 1990; Haifa, Israel.

Singh, K., & Arora, S. (2011). Removal of synthetic textile dyes from wastewaters: a critical review on present treatment technologies. Critical Reviews in Environmental Science and Technology, 41(9), 807-878.

Solís, M., Solís, A., Pérez, H. I., Manjarrez, N., & Flores, M. (2012). Microbial decoloration of azo dyes: a review. Process Biochemistry, 47(12), 1723-1748.

Srinivasan, A., & Viraraghavan, T. (2010). Oil removal from water using biomaterials. Bioresource Technology, 101(17), 6594-6600.

Torbati, S., Movafeghi, A., & Khataee, A. (2015). Biodegradation of CI Acid Blue 92 by Nasturtium officinale: study of some physiological responses and metabolic fate of dye. International Journal of Phytoremediation, 17(4), 322-329.

Vafaei, F., Khataee, A., Movafeghi, A., Lisar, S. S., & Zarei, M. (2012). Bioremoval of an azo dye by Azolla filiculoides: Study of growth, photosynthetic pigments, and antioxidant enzymes status. International Biodeterioration & Biodegradation, 75, 194-200.

Varjani, S., Rakholiya, P., Shindhal, T., Shah, A. V., & Ngo, H. H. (2021). Trends in dye industry effluent treatment and recovery of value added prod-ucts. Journal of Water Process Engineering, 39, 101734.

Wagner, G. J. (1979). Content and vacuole/extra vacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts. Plant Physi-ology, 64(1), 88-93.

Weinberg, T., Lalazar, A., & Rubin, B. (2003). Effects of bleaching herbicides on field dodder (Cuscuta campestris). Weed Science, 51(5), 663-670.

Zhang, Z., O’Hara, I. M., Kent, G. A., & Doherty, W. O. (2013). Comparative study on adsorption of two cationic dyes by milled sugarcane bagasse. Industrial Crops and Products, 42, 41-49.

Zhou, J., Wu, Z., Yu, D., & Yang, L. (2020). Toxicity of the herbicide flurochloridone to the aquatic plants Ceratophyllum demersum and Lemna minor. Environmental Science and Pollution Research, 27, 3923-3932.




How to Cite

EFTEKHARI, Z., NORASTEHNIA, A., & MASOUDIAN , Z. (2023). Biodecolorization of azo dye Acid Blue 92 (AB92) by Ceratophyllum demersum L.: process optimization using Taguchi method and toxicity assessment. Acta Agriculturae Slovenica, 119(2), 1–13.



Original Scientific Article