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Pesticides can affect the health of individual organisms and the function of the entire ecosystem. Therefore, thorough assessment of the risks associated with the use of pesticides is a high-priority task. An enzyme inhibition-based assay is used in this study as a convenient and quick tool to study the effects of pesticides at the molecular level. The contribution of formulants to toxicological properties of the pesticide formulations has been studied by analyzing effects of 7 active ingredients of pesticides (AIas) and 10 commercial formulations based on them (AIfs) on the function of a wide range of enzyme assay systems differing in complexity (single-, coupled, and three-enzyme assay systems). Results have been compared with the effects of AIas and AIfs on bioluminescence of the luminous bacterium Photobacterium phosphoreum. Mostly, AIfs produce a considerably stronger inhibitory effect on the activity of enzyme assay systems and bioluminescence of the luminous bacterium than AIas, which confirms the contribution of formulants to toxicological properties of the pesticide formulation. Results of the current study demonstrate that “inert” ingredients are not ecotoxicologically safe and can considerably augment the inhibitory effect of pesticide formulations; therefore, their use should be controlled more strictly. Circular dichroism and fluorescence spectra of the enzymes used for assays do not show any changes in the protein structure in the presence of commercial pesticide formulations during the assay procedure. This finding suggests that pesticides produce the inhibitory effect on enzymes through other mechanisms.

Pesticides are commonly used in agriculture and are an important factor of food security for humankind. However, the overuse of pesticides can harm non-target organisms, and, thus, it is vital to comprehensively study their effects on the different metabolic pathways of living organisms. In the present study, enzyme-inhibition-based assays have been used to investigate the effects of commercial pesticide formulations on the key enzymes of the organisms, which catalyze a wide variety of metabolic reactions (protein catabolism, lactic acid fermentation, alcohol metabolism, the conduction of nerve impulses, etc.). Assay conditions have been optimized, and the limitations of the methods used in the study, which are related to the choice of the solvent for commercial pesticide formulations and optical effects occurring when commercial pesticide formulations are mixed with solutions of enzymes and substrates of assay systems, have been revealed. The effects of commercial pesticide formulations on simple chemoenzymatic assay systems (single-enzyme reactions) have been compared to their effects on complex multicomponent molecular systems (multi-enzyme reactions) and organisms (luminescent bacterium). The in vitro assay systems have shown higher sensitivity to pesticide exposure than the in vivo assay system. The sensitivity of the in vitro assay systems increases with the elongation of the chain of conjugated chemoenzymatic reactions. The effects exerted by commercial pesticide formulations with the same active ingredient but produced by different manufacturers on assay system functions have been found to differ from each other.

We formulated the principles of designing bioluminescent enzyme tests for assessing the quality of complex media which consist in providing the maximum sensitivity to potentially toxic chemicals at a minimal impact of uncontaminated complex media. The developed principles served as a basis for designing a new bioluminescent method for an integrated rapid assessment of chemical safety of fruits and vegetables which is based on using the luminescent bacterium enzymes (NAD(P)H:FMN oxidoreductase and luciferase) as a test system.

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