Soil Metabolism of [¹⁴C]MRC-04 under Aerobic and Aerobic Flooded and Anaerobic Conditions

Chemicals

The radiolabeled test compound, [benzyl-¹⁴C]MRC-04 (Figure 1), was synthesized at Korea Radiochemicals Center (Suwon, Korea). The radiochemical purity was >99%, and specific activity was 6.87MBq/mg. The compound was used without further purification. HPLC-grade acetonitrile, ethyl acetate, methanol and water were purchased from Duksan Co. (Ansan, Korea). All the other reagents and common chemicals were analytical grades and commercially available.

Fig. 1.

Chemical structure of MRC-04 and 14C-labeled position.

Test soil

A sandy loam soil was sampled from top 10 cm of a drained upland field (402, Hadae-ri, Gyeryong-myeon, Gongju-si, Chungcheongnam-do, Korea). The soil was air-dried and sieved at room temperature for 24 h then was stored at -20°C to maintain microbial activity prior to use (NIAST, 1988a; SSSA, 1996; SSSA, 1986; Rueppel et al., 1997). Soil characterization results were shown in Table 1.

Table 1.

Characterization of the test soils

Radioassay

Radioactivity of all liquid samples was measured by a liquid scintillation counter (LSC; Tri-Carb 2900TR, PerkinElmer, USA). Radioactivity in gross amounts of less than twice the background was considered to be the limit of determination accuracy. Ultima Gold^® scintillation cocktail (4 mL) was used for aquatic and organic samples and Insta-Gel Plus^® (4mL) was used for CO₂ trapping. The non-extractable soil residue (0.2 g) was combusted by the sample oxidizer (Model 307, PerkinElmer, USA). The [¹⁴C]carbon dioxide produced was absorbed in Carbo-Sorb E^® (5 mL) and mixed with PermaFluor E+^® ( 1 0mL) scintillator for LSC counting. The efficiency of the oxidizer was determined using aliquots of Spec-Chec-¹⁴C check source for the automatic sample oxidizers, and was greater than 95%. Measurement of radio-activity was corrected by the oxidizer efficiency.

Chromatography

Identification of MRC-04 and its metabolites, and determination of radioactivity were performed using a PerkinElmer series 200 HPLC system (PerkinElmer, USA) equipped with an UV/vis detector and flow scintillation analyzer (Radiomatic 610TR, PerkinElmer, USA) by cochromatography with the authentic compounds. A reverse phase C₁₈ column (Luna 5 μ PFP(2) 100A, 250 × 4.6 mm i.d., Phenomenex, USA) was used. The detection wavelength was 254 nm. The elution solvents were acetonitrile and water (containing 0.1% trifluoroacetic acid). A linear gradient (from 30% acetonitrile to 90% for 27 min) using a flow rate of 1.0 ml/min was adopted to separate the peaks of MRC-04 and the metabolites produced. The flow rate of the scintillation cocktail (Ultima-Flo M^®) was 3 mL/min.

Extraction Efficiency

Soil samples (50 g, air-dry weight) were added with 5 mL of distilled water to adjust to 60% the field moisture holding capacity for the aerobic and anaerobic conditions, or 70 mL to form 2 cm water depth on the soil surface for the aerobic flooded condition. The [benzyl-¹⁴C]MRC-04 stock solution was prepared in 400 mg/L in acetonitrile, then 150 μL of the stock solution containing 60 μg of [benzyl-¹⁴C]MRC-04 was added to the soil sample. MRC-04 concentration in the soil (1.2 mg/kg) was equivalent to 0.3 kg/ha. Following the treatment, the soil was thoroughly mixed. After 30 min, the samples were extracted three times sequentially with acetonitrile/water (9:1, v/v, 100 mL), acetonitrile/water (1:1, v/v, 100 mL), and methanol/water/36% hydrochloric acid (9:1:0.1, v/v/v, 100 mL). Triplicate aliquots (0.5mL) of the soil extracts were analyzed by LSC to measure the radioactivity.

Soil Incubation

For the aerobic condition, the soil (50 g, air-dry weight) was weighed into the 100 mL incubation flask and added with 5 mL of the sterilized water. For the aerobic flooded condition, the soil (70 g, air-dry weight) in the incubation flask was flooded with 70 mL of the sterilized water to form >2 cm water depth from the soil surface (OECD, 2002; EPA, 2008a; EPA, 2008b; Chang et al., 2007). In case of the anaerobic condition, the soil (50 g, air-dry weight) was weighed into the 100 mL incubation flask and added with 5 mL of the sterilized water and maintained at the aerobic condition for 30 days, then the samples were added with 40 mL of N₂ gas-purged sterilized water and maintained at the anaerobic condition by flowing a N₂ gas.

All the soil samples were maintained in a flow-through metabolism chamber, and were pre-incubated at 25 ± 1°C for 2 weeks in the dark prior to the treatment. Each gas (air for aerobic and the aerobic flooded conditions; N₂ for the anaerobic condition) was passed through the system at a flow rate of 20 mL/min through a 1.0 N sodium hydroxide solution to remove carbon dioxide, followed by distilled water to humidify. After pre-incubation, an aliquot of the application for [benzyl-¹⁴C]MRC-04 was applied to the soil for the aerobic and anaerobic conditions and onto the water surface for the aerobic flooded condition at a rate of 1.2 mg/kg as describe above. The soil samples treated were then again incubated in the flow-through system at 25 ± 1°C. The effluent gas from the chamber was passed through two ethylene glycol traps (40 mL) and two sodium hydroxide solution traps (1.0 N, 40 mL) in sequence to trap volatile compounds and [¹⁴C]carbon dioxide, respectively. The treated soil was sampled at 0, 3, 7, 14, 30, 60, 90 and 120 days for the soil under the aerobic condition, at 0, 3, 5, 14, 30 and 60 days for the soil under the aerobic flooded condition and 30, 0, 7, 14, 30, 60, 90 and 120 days for the soil under the anaerobic condition. Readjustment of the soil moisture content and sampling of [¹⁴C]carbon dioxide and volatile products were carried out every two weeks. Triplicate aliquots (1m L) of ethylene glycol and sodium hydroxide solution from the traps were counted by LSC for total radioactivity.

Extraction and Analysis of Soil

At each soil sampling date, three flasks per treatment were taken, and the soil samples (including water) were extracted with acetonitrile/water (9:1, v/v, 100 mL) by sonication (15 min) and shaking (30 min). The extract was centrifuged at 5000 rpm for 15 min, and then the supernatant was taken. The remaining soil was then sequentially extracted with 100 mL of acetonitrile/water (1:1, v/v) and 100 mL of methanol/water/36% hydrochloric acid (9:1:0.1, v/v/v). In each procedure, the same extraction was repeated two times, and analysis was done by LSC. Each extract from the soil was combined, acetonitrile of the pooled extract was removed under a reduced pressure (35°C), 0.05 mL of 36% hydrochloric acid solution was added to the aqueous solution, and the remainder was extracted twice with 100 mL of ethyl acetate. The ethyl acetate solution was concentrated in vacuo at 35°C and redissolved in 2 mL of acetonitrile. An aliquot of the solution was analyzed by Radio-HPLC to determine the concentration of the parent and its metabolites. All the post-extracted soil samples were air-dried, homogenized and weighed. Triplicate portions (0.2 g) were combusted with the oxidizer before LSC analysis.

Table 2.

Extractability of [14C]MRC-04 with different solvent systems

Distribution of Non-extractable Radioactivity in Soil

The non-extractable residue by the extraction solvent system was fractionated with a strong base and acid into three fractions of humin, humic acid, and fulvic acid (Kim et al., 1998; Parsons, 1988). Ten grams (dry weight equivalent) of the non-extractable soil residue were extracted with 30 mL of 0.5 N sodium hydroxide solution by shaking for 24 h at room temperature. The extract was separated by centrifugation at 5000 rpm for 15 min and the supernatant (fulvic and humic acid) was collected. This procedure was repeated until the radioactivity of the extract reached the background level, and the extract were combined. Triplicate subsamples (0.2 g) of the precipitate (humin fraction) were combusted to determine the radioactivity content. The combined supernatant was added with concentrated hydrochloric acid to adjust to pH 1, and maintained at ambient temperature for 24 h. The mixture was then centrifuged at 5000 rpm for 15 min, and the supernatant (fulvic acid fraction) was collected. The resulting precipitate (humic acid fraction) was washed with 10 mL of 1 N hydrochloric acid solution and centrifuged (15 min, 5000 rpm). The supernatants were combined with the fulvic acid fraction. The volume of the combined solution was measured and 0.5 ml of the solution was analyzed by LSC. The humic acid precipitate was redissolved in 20 mL of 0.5 N sodium hydroxide solution, and the volume of the mixture was measured and then triplicate aliquots (0.1m L) were analyzed by LSC.

Isolation and Identification of Metabolites

Isolation and identification of the metabolite produced from MRC-04 were conducted from the soil under the aerobic flooded, because the major metabolites were produced only in that soil condition. To obtain a large amount of metabolites, 2 grams of MRC-04 was treated into the 1.5 kg soil under the aerobic flooded condition. The soil was extracted using acetonitrile/water (9:1, v/v) and acetonitrile/water (1:1, v/v) solution at 170 days. The solvent was evaporated and then the sample was extracted with ethyl acetate twice (80 mL, 70 mL). The extract was evaporated until complete dryness and redissolved with 2 mL acetonitrile, and the solution was analyzed by radio-HPLC to determine the metabolites. The extracted metabolites were isolated using precoated silica gel 60 F₂₅₄ chromatoplate (20 × 20 cm, 0.5 mm layer thickness, Merck, Germany). The solvent system was n-hexane/ethyl acetate (6:1, v/v) and development was conducted twice. Finally, three purified metabolites were identified by nuclear magnetic resonance (NMR) spectrometer (Bruker, Germany) operating at 400 MHz for ¹H and ¹³C with deuterochloroform (99.8%, Merck) and tetradeuteromethanol (99.9%, Cambridge Isotope Laboratories, Andover, MA, USA) as a solvent, respectively. EI positive spectra were recorded on High Resolution Mass Spectrometer (HRMS, JMS-700, JEOL, Japan).

Calculation of Half-Life

A pseudo-first-order kinetics model was used to calculate the half-life (NIAST, 1988b; Wang et al., 2009b; Dictor et al., 2008). The rates of degradation of [¹⁴C]MRC-04 were calculated by non-linear regression analysis using SigmaPlot 12.0 software (SPSS Science, USA)

Extraction Efficiency

[Benzyl-¹⁴C]MRC-04 was recovered from the test soil with high yield (92.3-100.8%) using acetonitrile/water (9:1, v/v) extraction solution. A small amount (<2.7%) was detected in the acetonitrile/water (1:1, v/v) extraction solution. Although most of radioactivity was recovered in the acetonitrile/water fraction, methanol/water/36% HCl (9:1:0.1, v/v/v) solution was used to extract metabolites, assuming they were potentially more polar or acidic than the parent compound (Hwang et al., 2013; Kim et al., 2003).

Mass Balance and Distribution. Aerobic condition

Mass balance was calculated at every sampling date by determining the radioactivity recovered from the solvent extracts, [¹⁴C]carbon dioxide, volatile compounds, and non-extractable residues (Wang et al., 2009a). The average mass balance ranged 90.2-97.8% of the applied during the study (Figure 2, A). The extractable radioactivity decreased with time and accounted for 30.2% of the applied at 120 days. The rate of degradation was rapid until first 60 days and then slower up to 120 days. The non-extractable residue increased with time and reached up to 35.9% of the applied at 120 days. The increasing rate was fast until 60 days and then slower up to 120 days. [¹⁴C]Carbon dioxide increased steadily as the soil aged and accounted for 24.1% of the applied at 120 days. No volatile products from the soil were detected throughout the study. From these results, we could assume that about 70% of MRC-04 treated was metabolized, 36% of those were rapidly bound to the soil, and 24% of those were further completely decomposed to carbon dioxide by aerobic microbes in the soil. The radioactivity of the non-extractable was determined 8.4, 15.1 and 12.5% of the applied in fulvic acid, humic acid, and humin fractions at 120 days, respectively, indicating bound residue was associated relatively more with humic acid and humin (Figure 3, A).

Fig. 2.

Distribution of radioactivity in the soil treated with [benzyl-14C]MRC-04 under the aerobic (A), aerobic flooded (B) and anaerobic (C) conditions. Each data point represents means± SD.

Figure 3.

Distribution of non-extractable radioactivity in the soil treated with [benzyl-14C]MRC-04 under the aerobic (A), aerobic flooded (B) and anaerobic (C) conditions.

Degradation of [¹⁴C]MRC-04 and Formation of Metabolites

Degradation of [benzyl-¹⁴C]MRC-04 and formation of metabolites in the soil under the aerobic, aerobic flooded, and anaerobic conditions were shown in Figure 4. In the soil under the aerobic condition, MRC-04 decreased to 32.1% of the applied until 60 days, after then and further to 23.6% at 120 days (Figure 4, A). The estimated half-life was 63.0 days. Four minor metabolites were produced and the each ranged 0.2-5.4% of the applied, and the combined percentages for all the metabolites at each sampling date were from 4.9 to 8.1%, and peaked at 14 days (Figure 4, A).

Figure 4.

Degradation of [benzyl-14C]MRC-04 and formation of metabolites in the soil under the aerobic (A), aerobic flooded (B) and anaerobic (C) conditions.

In the soil under the aerobic flooded condition, MRC-04 was decreased slowly to 86.8% of the applied until 5 days, and rapidly to 11.7% at 60 days (Figure 4, B). The calculated half-life was 17.9 days. Three major metabolites were detected and identified as 2,6-difluorobenzoic acid (Met-1), 4-((2,6-difluorobenzyl)oxy)-3-hydroxy-3-methyl-1-(o-tolyl)butan-1-one (Met-2), and 1-((2,6-difluorobenzyl) oxy)propan-2-ol (Met-3) (Figure 4, B). Met-1 and Met-3 increased with time and peaked up to 25.5 and 24.4% of the applied at 30 days, thereafter decreased to 8.6 and 14.2% at 60 days, respectively. On the other hand, Met-2 increased steadily and accounted for 14.2% of the applied at 60 days. Beside the major metabolites, 6 minor metabolites were detected, and the amount of each metabolite was 0.3-2.5% of the applied. The combined minor metabolites ranged from 1.8 to 6.7% of the applied and were maximum detected at 60 days.

In the soil under the anaerobic condition, MRC-04 decreased rapidly and accounted for 71.8% for first 30 days under the aerobic condition; however, after converted to anaerobic condition only 7.4% of the applied was additionally degraded until 120 days (Figure 4, C). Therefore, the half-life in the anaerobic condition could not be calculated. Five minor metabolites were detected and the amount of each ranged from 0.2 to 2.5% of the applied. Maximum combined percentage for all minor metabolites was 6.7% of the applied at 60 days (Figure 4, C). Each representative chromatograms are shown in Figure 5.

Figure 5.

Representative radio-HPLC chromatograms of solvent extracts from the soil under the aerobic (A), aerobic flooded (B) and anaerobic (C) conditions after application of [benzyl-14C]MRC-04.

In summary, MRC-04 was rapidly degraded in the aerobic and aerobic flooded conditions, but not in the anaerobic condition. The major metabolites were detected only in the aerobic flooded condition. These results first suggested that MRC-04 metabolism in soil is mainly associated with aerobic microbes, when the aerobic and aerobic flooded condition were compound, CO₂ was produced more in the flooded condition, while more soil bound residue was measured in the non-flooded condition. These results indicate that the larger portion of metabolites produced from MRC-04 were further metabolized and bound to soil under the non-flooded condition, while the metabolites were completely decomposed to carbon dioxide rather than binding to soil under the aerobic flooded condition.

Identification of Isolated Metabolites

The NMR data and chemical structure of the identified metabolites, Met-1, Met-2 and Met-3, are shown in Table 3 and Figure 6, respectively.

Table 3.

1H and 13C NMR data for MRC-04, Met-1, Met-2 and Met-3

Figure 6.

Chemical structure of MRC-04 and the identified metabolites from the soil under the aerobic flooded condition.

Met-1

In the ¹H-NMR spectrum, three proton signals at δ 7.06 (1H), 7.52 (1H) and δ 7.06 (1H) at C19, 20 and 21 were indicated the presence of the difluorobenzene ring, respectively, but a hydroxyl proton peak of 15-1 or 16-1 was not detected. The signals in the ¹³C-NMR spectrum for Met-1 were assigned as follows: δ 163.13 (C16), 132.52 (C17), 161.44 (C18), 132.52 (C19), 158.92 (C20), 132.52 (C21), and 161.51 (C22). The signal of C16 was significantly shifted to the down field, and the signals in the benzene ring were slightly moved to the down field compared with those of MRC-04. The EI-MS spectrum showed a molecular mass in peak at m/z 158 and a fragment peak at m/z 141 and 113 (Figure 7, A). Met-1 was observed m/z 158.0175 by HRMS. On the basis of these results, Met-1 was identified as 2,6-difluorobenzoic acid (m/z 158.0179), and might be produced as the cleavage of ether bond between isoxazoline ring and difluorobenzene ring of MRC-04.

Figure 7.

EI-MS spectrums for metabolites ((A) Met-1, (B) Met-2, (C) Met-3) from the soil under aerobic flooded condition.