Earthworm activity increases pesticide-sensitive esterases in soil

摘要

Carboxylesterases (CbEs) are serine hydrolases involved in the detoxification of anticholinesterase (organophosphorus and methylcarbamate) pesticides. Past studies have documented the occurrence of these esterases in soil, but little is known about their origin, function, and particularly, their reactivity against agrochemicals. In this study, it was compared the potential CbE activity in earthworm-treated and control (earthworm-free) soils by enzyme kinetics with multiple carboxylic esters and native polyacrylamide gel electrophoresis. After 12 weeks of inoculation, CbE activity was between two- and four-fold higher in the earthworm-treated soils (a-naphthyl acetate = 4.85 +/- 1.58 mu mol h(-1) g(-1) dry soil, alpha-naphthyl butyrate = 2.93 +/- 1.60, alpha-naphthyl valerate = 2.64 +/- 1.27, 4-nitrophenyl acetate = 1.41 +/- 0.37, 4-nitrophenyl butyrate = 0.87 +/- 0.15 and 4-nitrophenyl valerate = 0.89 +/- 0.11; values are presented as mean +/- standard deviation) than in controls. Although this enhanced esterase activity remained unchanged for 1 month following earthworm removal, it decreased under soil desiccation (31%-60%) or thermal denaturing (43%-82%). The potential sources for enhanced soil CbE activity were also examined through plate-count of microorganisms and zymographic techniques. The earthworm gut microenvironment was a significant source of soil CbE activity, and the casts were found to be the main contributors to the esterase activity analyzed. Soil CbE activity was strongly inhibited by organophosphorus (chlorpyrifos-oxon, paraoxon-ethyl and paraoxon-methyl) and, at less extent, by methylcarbamates (carbaryl and carbofuran). In vitro inhibition kinetics showed a biphasic curve that revealed at least two sensitive esterases and a resistant fraction; the latter varied widely depending on the enzyme substrate (7-68% of control activity). Likewise, spiking of earthworm-treated soils with 4 mg kg(-1) (wet weight) of chlorpyrifosoxon led to a significant inhibition of CbE activity 2 (40-72% inhibition) and 6 days (37-53%) after its application. Current results suggest that the soil-dwelling earthworm Lumbricus terrestris may be used as a promoter of soil enzyme activities with a direct benefit for pesticide bioremediation