Joint action of combinations of pollutants on the acetylcholinesterase activity of several marine species.

: Acetylcholinesterase (AChE) is used as a specific biomarker of the effects of organophosphorous (OP) and carbamate (C) insecticides on the coastal marine environment. Studies of mixtures (by pairs) of five of these substances showed cumulative, synergistic inhibitory effects in all cases. The strongest synergy was observed in organophosphate-carbamate mixtures (OP-C) and the least in mixtures of substances of the same type (OP-OP, C-C). The intensity of the synergistic effect was directly related to the length of time the enzyme was incubated with the inhibitory mixtures. Among the major organic contaminants of the marine environment, DDT and lindane (organochlorines), as well as atrazine and isoproturon, are not AChE inhibitors and had no effect on the inhibitory action of the OP and C insecticides tested. Among contaminants of metallic origin, zinc chloride, cadmium chloride, tributyltin chloride and methylmercury did not inhibit AChE at the concentrations measured in the different marine compartments (water, sediment, living matter). Mercuric chloride and arsenite had a weak inhibitory action in certain organisms. Zinc chloride, cadmium chloride and arsenic enhanced the inhibitory effects of some OP and C insecticides. The dragonet (Callionymus lyra) proved to be a particularly sensitive target species for monitoring pollutant effects.

Simultaneous and iterative weighted regression analysis of toxicity tests using a microplate reader.

A system is described for determination of LC50 or IC50 by an iterative process based on data obtained from a plate reader using a marine unicellular alga as a target species. The esterase activity of Tetraselmis suesica on fluorescein diacetate as a substrate was measured using a fluorescence titerplate. Simultaneous analysis of results was performed using an iterative process adopting the sigmoid function Y = y/1 (dose of toxicant/IC50)slope for dose-response relationships. IC50 (+/- SEM) was estimated (P less than 0.05). An application with phosalone as a toxicant is presented.

Routine determination of enzyme kinetics using plate reader.

A method is described for the routine determination of kinetics parameters from colorimetric enzyme reactions using a 96-well microtiter plate reader commonly used in immunoassay. A program in Turbo Pascal have been developed for the analysis of results. The system performs the description of plate format, the definition of conditions of assays, the measurement of enzyme reactions and collection of data, analysis, visualization of results and modification of data. Measurement of K(m) and V(max) is made with different methods, including iterative determination, Lineweaver-Burk transformation, and Hofster transformation, to give the final values of kinetic parameters with their associated standards errors. This approach should be applicable to routine determination of kinetic parameters.