2.5. Determination of Chlorpyrifos Decomposition in Soil Samples

In order to determine the concentrations of chlorpyrifos the pesticide was extracted from 3 g of soil with three portions of ethyl acetate (5 mL). The extracts were mixed, evaporated and dissolved in acetonitrile. Chlorpyrifos extracts were analyzed using HPLC (Knauer Smartline Pump 1000, Knauer Smartline UV Detector 2600, Berlin, Germany) and a Diasfer 110-C18 5 μm, 4.0 × 250 mm reverse-phase chromatography column (Biochemmack CT, Moscow, Russia) with a spectrophotometric detector (274 nm) and isocratic elution. Acetonitrile-water mixture (60:40) was applied as the eluent. The retention time for chlorpyrifos was 31 min. The eluent flow rate was 1 mL·min⁻¹, and the detector cell temperature was 25 °C. The sample volume was 20 μL.

The concentration of chlorpyrifos degraded by enzyme preparation was calculated by the formula Ce = Cc − Cd [42], where Ce is the actual concentration of pesticide degraded by enzyme in soil, Cc is the pesticide concentration in soil without immobilized or non-immobilized enzyme (control), Cd is the determined pesticide concentration in soil with immobilized enzyme preparation.