Degradación de Imidacloprid por Cladosporium cladosporioides: cinética, vías metabólicas y toxicidad
; Pérez, Débora Jesabel ; Salvio, Carla ; Wolski, Erika Alejandra Otros responsables
Fecha
Publicador:
Consejo Nacional de Investigaciones Científicas y Técnicas
Fecha de depósito:
12/09/2025
Fecha de creación:
Clasificación temática
Palabras clave
Resumen
Imidacloprid is one of the most popular and widely used neonicotinoid insecticides, despite reports of its adverse effects on non-target organisms. It is persistent in soil, with a half-life of 100 days and due to its high solubility easily contaminates water bodies around the world. Therefore, it is crucial to remove imidacloprid from contaminated environments and identify alternatives for treating agricultural liquid waste. This study presents the first report of imidacloprid degradation by the native fungus Cladosporium cladosporioides. The isolate was identified by analysis of the ITS region sequences (ITS1 and ITS4) and based on morphological characteristics. Degradation kinetics were performed using concentrations of 20, 40, and 100 mg L?¹ of imidacloprid in the presence of glucose as a carbon source. After 10 days, the fungus degraded 71%, 56%, and 30% of the initial concentration, respectively. The maximum degradation rate was of 2.66 mg L?¹ d?¹ for 40 mg L?¹. Biomass development and laccase activity patterns were both coincident with the degradation kinetics. Transformation products as 5-hydroxyimidacloprid, imidacloprid olefin, and 6-chloronicotinic acid, were identified by LC-MS/MS and FTIR, suggesting a possible metabolic degradation pathway. These products exhibited lower toxicity, on Eisenia fetida avoidance tests, than imidacloprid. This research is the first step toward the future application of C. cladosporioides in the treatment of liquid effluents to prevent pollution and remediate contaminated environments.
DNA extraction: DNA extraction for each of the isolates was performed following the protocol of Mamani et al. (2014). The DNA was quantified using NanodropTM 2000 (Thermo Scientific) and stored at -20 °C. Polymerase Chain Reaction (PCR): For the amplification of the ITS1-5.8 S-ITS2 region, primers ITS1 (5‘-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5‘-TCCTCCGCTTATTGATATATGC-3’) were used. Reactions were performed in a total volume of 25 µL containing 2.5 µL of 10X Reaction Buffer, 1.5 µL of 25 mM MgCl2, 1.5 µL Mix dNTPs (10 mM), 1 ?L of each primer (10 ?M), 0.3 ?L of Taq DNA Polymerase High Fidelity (5 U µL-1) and 2 ?L of DNA (100 ng µL -1) from each isolate. Nuclease-free water replaced the template DNA in the negative controls. PCR cycling was as follows: 4 min at 94°C for DNA denaturation, then 35 cycles of 30s at 94°C, annealing at 45s at 54°C, 1 min at 72°C. All reactions were finished with a final extension of 10 min at 72°C. Sequencing: Polymerase Chain Reaction products were purified by the PuriPrep-GP Kit. DNA quality was verified by determining the ratio of A260/A280 and A260/A230 on a NanoDrop. Sequencing was carried out at Macrogen Inc. in Korea. The sequences obtained were analyzed using the Blast tool of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov). For phylogenetic analysis, the MEGA program (version 11) was used, applying the Maximum Likelihood (ML) method with the Tamura-Nei substitution model. Branch strength was evaluated using the bootstrap method with 1000 replicates.
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Cin_tica_de_degradaci_n_de_imidacloprid_a_concentraciones_de_20__40_y_100_mg_L___por_Cladosporium_cladosporioides.xlsx
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Cinetica_de_degradacion_de_imidacloprid_por_Cladosporium_cladosporioides_a_20_dias.xlsx
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Velocidad_de_degradacion.xlsx
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Valores_de_pHs.xlsx
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