Enzyme Inhibition-Based Assay to Estimate the Contribution of Formulants to the Effect of Commercial Pesticide Formulations

Abstract

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.

Keywords: bioluminescent assay; conjugated enzyme reactions; enzyme inhibition-based assay; formulants; luminous bacteria; pesticides.

Figure 1

Absorption spectra of the deltamethrin solutions (solid lines) and commercial pesticide formulation Delcid with similar deltamethrin content (dashed lines) obtained with 1 cm optical path length. Acetonitrile was used as the solvent. Vertical dotted lines refer to the wavelengths used for measuring the activity of the enzymes. Bioluminescence intensity was measured in the 400–600 nm range. The absorption of the commercial pesticide formulation demonstrates higher optical density and indicates the need to take into account the inner filter effect when measuring enzyme activities, especially for the assay system with trypsin.

Figure 2

The effect of glyphosate as AIa on single-enzyme assay systems: (a) BChE; (b) ADH; (c) ALP; (d) trypsin. The assay systems differ in their sensitivity to the same AIa (glyphosate); the IC₅₀ values for BCh, ADH, ALP, and trypsin are 0.035, 5.14, 1.08, and 0.96 μg/L, respectively.

Figure 3

Relationship between residual activity of enzyme assay systems and concentration of deltamethrin as AIa: (a) ADH; (b) Red + Luc; (c) ADH + Red + Luc. The inhibitory effects of deltamethrin as AIa were enhanced by elongation of the chain of conjugated enzyme reactions.

Figure 4

The effects of (a) the Tornado Extra commercial pesticide formulation (with glyphosate as the active ingredient) and (b) glyphosate as AIa on the Red + Luc coupled enzyme system. Glyphosate as AIf had a much stronger inhibitory effect on the Red + Luc assay system compared with glyphosate as AIa; the values of IC₅₀ for glyphosate as AIf and AIa were 1.8 and 288 mg/L, respectively.

Figure 5

The effects of (a) the Sempay commercial pesticide formulation (with fenvalerate as the active ingredient) and (b) fenvalerate as AIa on the Red + Luc coupled enzyme system. The same as for glyphosate, the inhibitory effect of fenvalerate as AIf on the Red + Luc system was stronger than the effect of fenvalerate as AIa; the values of IC₅₀ for fenvalerate as AIf and AIa were 0.0014 and 4.8 mg/L, respectively.

Figure 6

Comparison of the effects of the Briz commercial pesticide formulation (with cypermethrin as the active ingredient) and cypermethrin as AIa on enzyme assay systems with different lengths of the enzyme conjugation chain. Cypermethrin concentration was 2 mg/L. The inhibitory effects of cypermethrin as AIf were enhanced by elongation of the chain of conjugated enzyme reactions. No such dependence, though, was found for cypermethrin as AIa.

Figure 7

Circular dichroism spectra of ADH without additives (black) and in the presence of ethanol (blue) and the Muravyed commercial pesticide formulation (red). ADH concentration was 0.1 mg/mL, diazinon concentration in Muravyed was 0.02 mg/mL. Neither ethanol nor Myravyed alters the secondary structure of ADH.

Figure 8

Fluorescence spectra of LDH (a,b) and BChE (c–f) under 290 nm excitation in the presence (dashed lines) and in the absence (solid lines) of commercial pesticide formulations Muravyed (a,b), Tornado Extra (c,d), and Biotlin (e,f). Normalized (b,d,f) and not normalized (a,c,e) spectra are shown. Protein concentrations were 0.4 mg/mL of LDH, 0.25 mg/mL of BChE. Concentrations of diazinon in Muravyed, glyphosate in Tornado Extra, and imidacloprid in Biotlin were 1.25 mg/L, 2.4 mg/mL, and 0.02 mg/mL, respectively. For LDH, no effect of Muravyed on protein fluorescence was observed. The components of Tornado Extra and Biotlin formulations cause the blue shift of the BChE fluorescence.

Figure 8

Fluorescence spectra of LDH (a,b) and BChE (c–f) under 290 nm excitation in the presence (dashed lines) and in the absence (solid lines) of commercial pesticide formulations Muravyed (a,b), Tornado Extra (c,d), and Biotlin (e,f). Normalized (b,d,f) and not normalized (a,c,e) spectra are shown. Protein concentrations were 0.4 mg/mL of LDH, 0.25 mg/mL of BChE. Concentrations of diazinon in Muravyed, glyphosate in Tornado Extra, and imidacloprid in Biotlin were 1.25 mg/L, 2.4 mg/mL, and 0.02 mg/mL, respectively. For LDH, no effect of Muravyed on protein fluorescence was observed. The components of Tornado Extra and Biotlin formulations cause the blue shift of the BChE fluorescence.