Measurements of cholinesterase activity in the tropical freshwater cladoceran Pseudosida ramosa and its standardization as a biomarker

摘要

The activity of cholinesterases (ChE) has been recognized as a useful tool for assessing the toxicity in the environmental assessment programs. Nevertheless, the prior optimization of the experimental conditions for the appropriate measuring of the ChE activity enables us to get reliable results. Thus, the main objective of this study was to adapt and optimize a microplate assay for measuring the activity of ChE in the tropical cladoceran Pseudosida ramosa. The best readings for the reaction rates were obtained with buffers of pH 8.0 and molarity of 0.02 M. The measurements of the reaction rates for the different substrate concentrations showed that the maximum reaction rate (32 mOD min~(-1)) was achieved by the final concentration of 2 mM of substrate. In relation to the enzyme concentration, reaction rates were directly proportional to the protein concentration, which confirmed the linear kinetics for a maximum reaction rate. On the basis of the results of the assays for the effect of the number of individuals and homogenate dilution on the reaction rate of substrate hydrolysis and ChE activity, we recommend using of 30 individuals (3 days-old) in 250 μL of buffer, 20 individuals (7 days-old) in 250 μL of buffer and 15 individuals (both 14 and 21 days-old) in 300 μL of buffer. The limits of quantitation obtained were 1.419 mOD min~(-1) (≤72h-old), 1.670 mOD min~(-1) (7 days-old), 0.943 mOD min~(-1) (14 days-old) and 0.797 mOD min~(-1) (21 days-old). In conclusion, it was possible to measure the ChE activity in P. ramosa with the methodology adapted, thus contributing to the implementation of a biochemical biomarker in freshwater toxicity assessments in tropical regions.