Biodegradation of imidacloprid by an isolated soil microorganism

Abstract

Imidacloprid (1-[(6-chloro-3-pyridinyl)-methyl]-N-nitro-2-imidazolidinimine), a chloronicotinyl insecticide used to control biting and sucking insects, is very persistent in the soil with a half-life often greater than 100 days. Although a few soil metabolites have been reported in the literature, there are no reports of imidacloprid-degrading soil microorganisms. Our objectives were to discover, isolate, and characterize microorganisms capable of degrading imidacloprid in soil. Two soil-free stable enrichment cultures in N-limited media were obtained that degraded 19 mg L^{− 1} (43%) and 11 mg L^{− 1} (16%) of the applied imidacloprid, and produced about 19 mg L^{− 1} 6-chloronicotinic acid in three weeks. Enrichment media without microorganisms had no loss of imidacloprid. Strain PC-21, obtained from the enrichment cultures, degraded 37% to 58% of 25 mg L^{− 1} imidacloprid in tryptic soy broth containing 1 g L^{− 1} succinate and D-glucose at 27°C incubation over a period of three weeks. Trace amounts of NO₃ ⁻/NO₂ ⁻were produced and six metabolites were characterized by high performance liquid chromatography (HPLC) using ¹⁴C-methylene-imidacloprid and liquid chromatograph-electrospray-mass spectrometer (LC-MS). Two of the metabolites were identified as imidacloprid-guanidine and imidacloprid-urea by HPLC standards and LC-MS. During the experiment, 6-chloronicotinic acid was not produced. Less than 1% of the applied ¹⁴C was incorporated into the microbial biomass and no ¹⁴CO₂ was detected. Strain PC-21, identified as a species of Leifsonia by PCR amplification of a 500 bp sequence of 16s rRNA, cometabolized imidacloprid.

Keywords::

• Biodegradation
• imidacloprid
• insecticide
• metabolites
• Leifsonia
• soil microorganism

Acknowledgments

Funding for this research was provided by Bayer Corporation, Stilwell, KS. We thank Beth Douglass for her laboratory assistance. We also thank Tracey Pepper of the Bessey Microscope Facility for carrying out the scanning electron microscope (SEM) work.

Notes

^a Zorbax™ C₈ column.

^b Symmetry™ C₁₈ column.

^a BD = below detection limit of 0.4 mg L^{− 1}

^b14C-4,5-imidazolidin-imidaclopridwas applied to these samples.