title: Measurements of cholinesterase activity in the tropical freshwater cladoceran Pseudosida ramosa and its standardization as a biomarker.
abstract: 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 0.02M. The measurements of the reaction rates for the different substrate concentrations showed that the maximum reaction rate (32mODmin(-1)) was achieved by the final concentration of 2mM 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 250μL of buffer, 20 individuals (7 days-old) in 250μL of buffer and 15 individuals (both 14 and 21 days-old) in 300μL of buffer. The limits of quantitation obtained were 1.419mODmin(-1) (≤72h-old), 1.670mODmin(-1) (7 days-old), 0.943mODmin(-1) (14 days-old) and 0.797mODmin(-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.
authors: Post-Graduate Program of Ecology and Natural Resources, Department of Ecology and Evolutionary Biology, Federal University of São Carlos, Rodovia Washington Luis, km 235, CEP 13565-905 São Carlos, SP, Brazil. Electronic address: emanuelacfreitas@gmail.com.
authors: Emanuela Cristina
authors: EC
authors: Freitas
authors: Special Bureau for the Environment, Federal University of São Carlos, Rodovia Washington Luis, km 235, CEP 13565-905 São Carlos, SP, Brazil. Electronic address: liane@ufscar.br.
authors: Liane Biehl
authors: LB
authors: Printes
authors: Department of Physiological Sciences, Federal University of São Carlos, Rodovia Washington Luis, km 235, CEP 13565-905 São Carlos, SP, Brazil. Electronic address: dmnf@ufscar.br.
authors: Marisa Narciso
authors: MN
authors: Fernandes
authors: Department of Ecology and Evolutionary Biology, Federal University of São Carlos, Rodovia Washington Luis, km 235, CEP 13565-905 São Carlos, SP, Brazil. Electronic address: doro@ufscar.br.
authors: Odete
authors: O
authors: Rocha
mesh_terms: D000818:Animals; D015415:Biomarkers; D003445:Crustacea / Q000201:enzymology*; D054750:Ecotoxicology / Q000379:methods*; D005618:Fresh Water; D006868:Hydrolysis; D012015:Reference Standards; D014329:Tropical Climate
chemical_list: D015415:Biomarkers
keywords: Biochemical biomarkers; Cholinesterases; Native species; Pseudosida ramosa; Tropical ecotoxicology