A small-volume bioassay for quantification of the esterase inhibiting potency of mixtures of organophosphate and carbamate insecticides in rainwater: Development and optimization

The goal of this study was to develop a sensitive in vitro bioassay for quantification of the total esterase inhibiting potency of low concentrations of organophosphate and carbamate insecticides in relatively small rainwater samples. Purified acetylcholinesterase (AChE) from electric eel (Electrophorus electricus) and carboxylesterases from a homogenate of honeybee heads (Apis mellifera) were used as esterases, each having different affinities for the substrates S-acetylthiocholine-iodide (ATC) and N-methylindoxylacetate (MIA). MIA hydrolysis by honeybee homogenate was more sensitive to inhibition by organophosphate insecticides than ATC hydrolysis by purified AChE, although the latter parameter is often used for in vitro monitoring of esterase inhibitors. The higher sensitivity of carboxylesterases is attributed to the instant formation of a reversible Michaelis-Menten complex with the inhibitor, which competes with MIA for the active sites of the free enzymes. This dose-dependent instant inhibition can be quantified with kinetics for competitive inhibition at dichlorvos concentrations < 16 nM. At similar concentrations, purified AChE was not instantly inhibited, whereas both AChE and carboxylesterases were irreversibly and progressively inhibited at higher dichlorvos concentrations (IC50(10min) <greater than or equal to> 0.1 muM). Honeybee homogenate mediated MIA hydrolysis was applied as the most sensitive enzyme-substrate combination for experiments with fractionated extracts of 4 rainwater samples collected in a natural conservation area. Most esterase inhibiting potency was found in the polar methanol fraction, with recalculated concentrations equivalent to 12-125 ng dichlorvos per liter rainwater.