Long-term fate of the herbicide mefenacet in a rice-grown lysimeter over a period of 6 consecutive years

Abstract

To show the long-term fate of the herbicide mefenacet in rice paddies, [aniline-¹⁴C]mefenacet was applied to a lysimeter of loamy soil with a depth of 1 m, and rice plants were cultivated for 6 consecutive years according to the conventional methods in Korea. Mineralization of [¹⁴C]mefenacet to ¹⁴CO₂ and volatilization from the soil surface were 12.01 and 0.02 %, respectively, of the originally applied amount during the first 23 weeks following application. Throughout the 6-year period, the total proportion of ¹⁴C-radioactivity that leached through the lysimeter soil was 0.778 % of the original radioactivity. The total ¹⁴C-radioactivity absorbed and translocated by rice plants throughout the 6 years was 2.46 % of the applied ¹⁴C. Measurement of the ¹⁴C-radioactivity distributed in each part of the rice plant (the straw, the ears without rice grain, the chaff, and the brown rice grain) indicated that the amount of ¹⁴C in straw was 25.87 times higher than that in brown rice grain over the 6 years. The ¹⁴C-radioactivity remaining in the soil layer after 6 years was 44.58 % of that applied, 91.45 % of which was distributed in the upper 0–20-cm layer. These results strongly indicate that mefenacet moved downward very slowly and more than half of the herbicide applied was released into the air, mainly through mineralization to CO₂ in soil during the experimental period.

Introduction

Mefenacet (2-(1,3-benzothiazol-2-yloxy)-N-methylacetanilide) introduced by Schmidt et al. (1984) is a selective herbicide used as pre-emergence and early post-emergence herbicide for transplanted rice in Korea. It has been reported to be effective on annual monocotyledonous and some dicotyledonous weeds in rice paddies. Fedtke (1991) and Hess et al. (1990) reported that the herbicide inhibited cell division and cell enlargement in green alga and oat. Morphological studies on Echinochloa spp. treated with mefenacet have been conducted (Ito et al. 1989), and an investigation on the morphological and anatomical effects on barnyard grass was published by Ito et al. (2000). Krauskopf et al. (1989) reported uptake and translocation studies of mefenacet in rice and Echinochloa crus-galli in pots for a short period of time under greenhouse conditions. Führ and Mittelstadt (1982) and Lee et al. (1989) reported a substantial difference in uptake and translocation of mefenacet between micro-ecosystem (pot) and lysimeter experiments; in the pot experiments, uptake was about 50 (Führ and Mittelstadt 1982) and 17 (Lee et al. 1989) times higher than in the lysimeter experiment. They attributed differences in the homogeneity of pesticides in soil, the ratio of roots to soil, the supplemental period of water, and climatic conditions at the experimental sites (Führ and Mittelstadt 1982). For these reasons, it is essential to use outdoor lysimeters with undisturbed soil profiles, and radio-labeled compounds to more accurately show the fate of pesticides applied to agricultural fields.

Many researchers have reported that outdoor lysimeters with undisturbed soil structure reflect field conditions (Scheunert et al. 1983; Kubiak et al. 1988; Stork et al. 1994; Schroll et al. 2008). Also, the use of radio-labeled chemicals can significantly increase the accuracy of research on the fate and behavior of polycyclic aromatic hydrocarbons (PAHs) by establishing a mass balance (Abbott et al. 1992). As mentioned above, the use of lysimeters, which can be used to simulate paddy fields with radiotracers, is an ideal technique for the study of fate of pesticides, including the study of uptake and translocation by crops, metabolism in soil–crop–water systems, movement and leaching in the soil column, and mineralization (CO₂ evolution) during crop cultivation. Thus, a large number of results from outdoor lysimeter experiments have been reported using ¹⁴C-labeled chemicals to show their fate in the agroenvironment (Kubiak et al. 1988; Stork et al. 1994; Lee et al. 1994, 2003; Schroll and Kühn 2004; Lee et al. 2005; Schroll et al. 2008). The present investigation is aimed at showing the long-term fate of mefenacet used for the treatment of rice paddy soil using a ¹⁴C-radiotracer in a rice-grown lysimeter, simulating the rice paddy conditions over 6 consecutive years.

Materials and methods

Chemical composition of mefenacet

The [aniline-¹⁴C]mefenacet used in this experiment was purchased from International Isotopes Inc. (Münich, Germany). As can be seen in Fig. 1, the position labeled with ¹⁴C in the chemical structure of mefenacet is the benzene ring carbons of the aniline moiety. The specific radioactivity was 3.4 MBq/mg and the radiochemical purity was greater than 98 %.

Fig. 1

Structural formula and labeled position (asterisk) of mefenacet (2-(1,3-benzothiazol-2-yloxy)-N-methylacetanilide)

Lysimeter and preparation of the soil core

The lysimeter used in this investigation was manufactured with stainless steel (8 mm in thickness). The surface area was 0.25 m², the height was 1.1 m, and the inner diameter was 0.564 m. The undisturbed soil core was obtained from rice paddy fields by pressing the lysimeter down into a rice paddy located at Bokdae-dong, Cheongju, Korea, with the aid of a fork crane. Table 1 presents the properties of the soil.

Table 1 Physicochemical properties of the lysimeter soil layers

Application of [¹⁴C]mefenacet and cultivation of rice plants

Before transplanting, the lysimeter soil was fertilized with N–P–K at the ratio of 150–90–110 kg/ha; 80 % of the total nitrogen fertilizer was applied just before transplanting and the rest at the earing stage. Rice seedlings (Oryza sativa cv. Akibare, 33–39 days old) were transplanted every year into the lysimeter soil, at the rate of nine hills of three seedlings per hill. [¹⁴C]Mefenacet was applied to the lysimeter soil 70 days after transplantation as a mixture of the commercial granular formulation, Mannyang^® (3.5 % mefenacet + 0.07 % pyrazosulfuron-ethyl + 96.43 % adjuvant), at the recommended application rate of 1.05 kg a.i. mefenacet/ha in Korea. The commercial granular formulation was macerated to powder in a mortar before mixing to ensure homogeneous application and convenience in mixing with soil used as the diluent. [¹⁴C]Mefenacet (10.561 MBq, equivalent to 3.106 mg a.i.) dissolved in 10 mL methanol and 0.657 g (equivalent to 23.00 mg a.i. mefenacet) of the commercial granular formulation were added to about 200 g of soil. After methanol was completely evaporated, the soil was mixed well and scattered over the lysimeter soil surface. The leachates from the lysimeter were collected in a 2 L plastic container, and the volume and radioactivity measured biweekly. Rice plants were grown for 136 days until harvest, according to the conventional farming methods in Korea. Throughout the cultivation period, the soil was flooded as the rice paddies would be; in Korea, rice crops are usually grown under flooded conditions almost until harvest. Accordingly, during the dry season, tap water was supplied for additional irrigation. To prevent the run-off of [¹⁴C]mefenacet from flooded soils by heavy rain, the lysimeter was roofed with vinyl film during the rice-growing period. After harvest each year, the lysimeter was kept open until the following cultivation, and the leachate was collected continuously. A diagram of the lysimeter illustrating the growing of rice plants and the collection of leachate is given in Fig. 2 (Lee et al. 1994).

Fig. 2

Diagram of the lysimeter showing the growth of rice plants and the collection of leachate (Lee et al. 1994)

Mineralization and volatilization of mefenacet during rice cultivation

The ¹⁴CO₂ and volatile substances that evolved from the mefenacet-treated soil surface were trapped with a special device made of Pyrex^® glass (Lee et al. 1994). Four devices were placed on the surface of the soil, each covered an area of 50.24 cm² representing about 1/50 of the lysimeter surface area. The ¹⁴CO₂ and volatile substances were trapped in 1 N NaOH and 0.1 N H₂SO₄, respectively, and the ¹⁴C-radioactivity was measured biweekly.

Soil and rice plant sampling

After harvest, a soil sample from each 5 cm layer was collected to a depth of 40 cm in the first year, to 50 cm in the second year, to 45 cm in the fourth year, and to 90 cm in the sixth year using a soil core sampler attached to a stainless steel core of 5.05 cm diameter, 5 cm length, and 100 cm³ volume. The samples were taken randomly from three spots, and those of each layer were combined, air dried, and ground in a mortar for analysis. The harvested rice plants were separated into straw, ears without rice grain, chaff, and brown rice grain; freeze-dried with a freeze drier (Chem Lab. Instruments LTD., SB4, England); and pulverized with a blender (Philips model HR 2094, China) to measure the radioactivity by combustion. Brown rice grain was obtained by removing chaff.

Extractable and non-extractable ¹⁴C-soil residues

Each 150 g of soil sample was collected from 0 to 5 and 5 to 10 cm depths from the lysimeter, and was exhaustively extracted with acetone and methanol consecutively. The combined extracts were then subjected to ¹⁴C-radioactivity measurement. Soils extracted previously with the solvents were combusted to determine the non-extractable bound residues; an additional 5 g of the solvent-extracted soils was extracted with 0.1 M Na₄P₂O₇. The combined extracts were divided into fulvic and humic acid by acidification (Lee et al. 1991), and then the ¹⁴C-radioactivity incorporated into fulvic and humic acid was measured. The remaining extracted soils were combusted for the ¹⁴C-radioactivity in the humin fraction.

Measurement of radioactivity

The radioactivities in the soil and rice plants were measured by combusting 0.3 g of soil and 0.2 g of rice plants that had been freeze-dried and using a biological oxidizer (OX-400, R. J. Harvey Instrument Corp., USA) to give ¹⁴CO₂. The flow rate of oxygen and nitrogen in the biological oxidizer was 300 mL/min. The temperatures of the catalyst zone and combustion zone were 700 and 900 °C, respectively, and the combustion time was 4 min. The ¹⁴CO₂ evolved was absorbed in 15 mL of Carbo Max™ Plus (Lumac LSC B. V., The Netherlands). Radioactivity was measured using a liquid scintillation counter (LSC, PW 4700, Philips) with automatic quench correction. The radioactivities dissolved in organic solvents were measured using 15 mL of Ready-organic (Packard, USA) after the solvents were evaporated and those of ¹⁴CO₂ absorbed in 1 N NaOH and volatile substances absorbed in 0.1 N H₂SO₄ were measured using Aquasol (Du Pont, NEN Research Products, USA) as a cocktail.

Microbial activity as measured by DMSO reduction and the number of microbial colonies in the lysimeter soil

The dimethyl sulfoxide (DMSO) reduction rate as a parameter of microbial activity in soil was measured by the method of Alef and Kleiner (1989). To count the number of microorganisms present in the soil, 1 g of soil was added to 10 mL of sterile distilled water and shaken for 2 h. The soil suspension was diluted adequately, 0.1 mL of which was spread on the nutrient broth medium (5 g peptone, 3 g beef extract, and 10 g agar in 1 L distilled water) and incubated at 30 °C for 48 h. The colonies were counted with a colony counter (Seeley et al. 1991).

Results and discussion

Mineralization and volatilization during the experimental period

Figure 3 shows that the total amount of ¹⁴CO₂ that evolved during the experimental period was 12 % of the originally applied ¹⁴C-radioactivity. After rice harvest, the soil was maintained under non-flooded dry conditions. However, the ¹⁴CO₂ evolution did not increase with time. Meanwhile, volatilization increased to 0.017 % under dry conditions following harvest. In contrast, in our previous investigations of the insecticide imidacloprid (Lee et al. 2003) and the herbicide azimsulfuron (Lee et al. 2005), ¹⁴CO₂ evolution increased drastically under non-flooded dry conditions following harvest. These results might be due to active microbial and photolytic degradation under these conditions.

Fig. 3

Mineralization of [¹⁴C]mefenacet to ¹⁴CO₂ and volatilization during the lysimeter experiment. ¹⁴C-Radioactivity applied = 100 %. 1st–17th week, flooded rice-growing period; 18th–23rd week, non-flooded dry period after harvest

Distribution of the ¹⁴C in rice plants

As shown in Table 2, the total amount of ¹⁴C in the plants was only 1.978 % of the originally applied ¹⁴C-radioactivity after harvest in the first year, most of it being detected in straw (1.855 %). The amount measured in brown rice grains was 0.037 % of the originally applied ¹⁴C. When this amount was calculated as the concentration of mefenacet equivalent, it corresponds to 0.089 µg/g. In the second year, the amount absorbed and translocated to the various parts of rice plants was 8.7 times lower than that in the first year. In subsequent years, the amount continued to decrease gradually. Krauskopf et al. (1989) reported that mefenacet was rapidly absorbed by leaves of rice and Echinochloa crus-galli, whereas root uptake was very slow, especially in rice. They also reported that in soil, mefenacet is strongly adsorbed and less than 10 % was found to be available to rice and Echinochloa. In this long-term experiment, the total ¹⁴C-radioactivity absorbed and translocated over 6 consecutive years was only 2.46 %. This amount may be small because mefenacet-bound residues remaining in soil were not readily available to rice plants transplanted and grown in the following years.

Table 2 Amounts of the ¹⁴C-radioactivity absorbed and translocated in each part of rice plants grown in the lysimeter soil treated with [¹⁴C]mefenacet. ¹⁴C-Radioactivity applied = 100 %

Leaching

Table 3 presents the total amount of leachate and ¹⁴C-radioactivity exuded from the lysimeter soil during the experimental period. As seen in this table, only 0.778 % of the originally applied ¹⁴C-radioactivity leached through the soil layer over 6 consecutive years. Previously, Schmidt et al. (1984) suggested low soil mobility of mefenacet. They showed that in a soil thick-layer chromatography study, despite following an application of water equivalent to 27 cm of precipitation over a 3–4-h period, the majority of herbicidal activity remained in the upper 5 cm of soil. In our long-term leaching experiment, which simulated the undisturbed soil layer for 6 years, it was confirmed that mefenacet has very low soil mobility.

Table 3 Amounts of ¹⁴C-radioactivity leached through the lysimeter soil treated with [¹⁴C]mefenacet for 6 consecutive years. ¹⁴C-Radioactivity applied = 100 %

Distribution of the ¹⁴C in soil layers

Table 4 presents the distribution of ¹⁴C in each lysimeter soil layer after long-term leaching. In the first year, the soil samples were taken down to a depth of 40 cm. The total amount of ¹⁴C-radioactivity remaining in the lysimeter soil layers was 82.24 % of the originally applied ¹⁴C-radioactivity. Considering the fact that the total amount of ¹⁴CO₂ that evolved during the first 23 weeks was 12 % of the originally applied ¹⁴C-radioactivity, much more ¹⁴CO₂ must have evolved during the rest of the experimental period. Because the ¹⁴C-radioactivity in the leachate collected in the first year was only 0.001 % (Table 3) and the ¹⁴C absorbed and translocated by rice plants was 1.978 % (Table 2), the rest of the ¹⁴C-radioactivity must have been lost by mineralization to ¹⁴CO₂. Even if the loss by volatilization was negligible, it would have increased during the non-flooded dry period following harvest (Fig. 3). In the second year, the soil samples were taken down to 50 cm. In this case, the total amount of the ¹⁴C remaining in the soil layers was 50.04 %. The ¹⁴C remaining in the 0–5-cm layer was 67.74 % in the first year and 34.17 % in the second year. This means that mefenacet moved downward slowly but steadily. The gradual decrease in the ¹⁴C in the soil layers indicates that more ¹⁴CO₂ might have evolved during the period, even if it was not observed in this study. The ¹⁴C detected in the leachate collected until the second year was only 0.011 % of the originally applied ¹⁴C (Table 3). This indicates that very small amounts of ¹⁴C are lost by leaching. To minimize the disturbance of the lysimeter soil profile, sampling was done biennially beginning the second year. In the fourth year, the soil samples were taken down to 45 cm. The total amount of the ¹⁴C remaining in the soil layers was only 49.1 % of the originally applied ¹⁴C; almost half of the ¹⁴C was therefore lost. In the final year, the soil samples were taken down to 90 cm. The total ¹⁴C remaining in the soil layers was 44.58 %; in these samples, more than half of the ¹⁴C-radioactivity applied was lost. In the sixth year, the ¹⁴C-radioactivity remaining in the 0–5-cm layer was only 14.63 %. This is 4.4 times lower than the ¹⁴C remaining in the same layer in the first year (64.74 % of the originally applied ¹⁴C). This trend indicates that even if most of the ¹⁴C-radioactivity was lost, ¹⁴C moved downward slowly.

Table 4 Amounts of ¹⁴C-radioactivity remaining at different depths of the lysimeter soils treated with [aniline-¹⁴C]mefenacet after harvest. ¹⁴C-Radioactivity applied = 100 %

Formation of bound residues and change in the polarity of ¹⁴C in soil extracts

The formation of non-extractable bound residues was examined 136 days following [¹⁴C]mefenacet treatment. Based on our previous investigations (Lee et al. 1991, 1988), the period of 136 days was sufficiently long for the formation of non-extractable bound residues of mefenacet. As shown in Table 5, about 95–97 % of the applied [¹⁴C]mefenacet was strongly adsorbed in the upper 0–10-cm layer to form non-extractable bound residues. This soil layer (0–10 cm) contained 3.1 % organic matter, which is a higher proportion than is present in the deeper layers (Table 1). Nakamura et al. (1996) investigated the influence of organic matter content on adsorption of mefenacet to soil and the phytotoxic activity of mefenacet in relation to its concentration in soil water varying in organic matter content. They demonstrated that the phytotoxic activity of mefenacet applied to soils containing various amounts of organic matter decreased as the organic carbon content increased, and the adsorption in the solid phase decreased with a decrease in organic carbon content. In this study, the ¹⁴C extracted with acetone followed by methanol was only 5.01 and 3.46 % from the 0–5 and 5–10-cm layers, respectively. The differences in the amount extracted could be due to the differences in the organic carbon content, even if it was not differentiated between the 0–5 and 5–10-cm layers in our soil analysis. In the soil-bound residues of [¹⁴C]mefenacet, more than half of the non-extractable bound residues were characterized in the humin fraction (52–66 %), which was essentially inactive biologically 136 days after [¹⁴C]mefenacet treatment (Table 6; Nam and Kim, 2002).

Table 5 Formation of non-extractable bound residues of [¹⁴C]mefenacet in the lysimeter soil^a

Table 6 Distribution of the non-extractable [¹⁴C]soil-bound residues in humic substances of the lysimeter soil

Microorganisms in soil as affected by rice cultivation

Alef and Kleiner (1989) presented a method for the determination of microbial activities in soils and soil aggregates, based on the enzymatic reduction of DMSO to dimethyl sulfide (DMS). Using their method, in this study, the number of microbial colonies in the topsoil (0–10-cm depth) and their activity were observed to have increased greatly after rice cultivation (Table 7); the number of colonies was about 45 times higher and the microbial activity was about 1.4 times higher following rice cultivation. This could be ascribed to the fact that various kinds of exudates accumulated in the rhizosphere (Rovira 1965; Kimura et al. 1977; Curl 1986). A review by Rovira (1965) shows the wide array of substances that have been found in root exudates, including carbohydrates, amino acids, organic acids, and enzymes. Kimura et al. (1977) also found that various kinds of sugars, amino acids, and organic acids had accumulated in the rhizosphere of rice plants. Microorganisms in the rhizosphere could use these substances as their nutrients.

Table 7 Comparison of the number of microbial colonies and the microbial activities as indicated by the DMSO reduction in the topsoil (0–10-cm depth) of the lysimeter soil before and after the cultivation of rice plants^a

Fate of [¹⁴C]mefenacet in a rice plant-grown lysimeter

Table 8 summarizes the fate of [¹⁴C]mefenacet used as a one-time treatment on the lysimeter soil containing transplanted rice plants and grown every year for 6 consecutive years. In the first year, the recovery accounted for 96.2 % of the originally applied ¹⁴C-radioactivity. As time went on, the recovery dropped sharply, and was 64.3 % in the second year. This result indicates that most of the [¹⁴C]mefenacet residue in soil was lost by the evolution of ¹⁴CO₂ during the second year. This finding is supported by the fact that after the harvest of rice plants in the first year, most of the residues of [¹⁴C]mefenacet and its degradation products (64.74 %) remained in the top 0–5-cm of soil. These residues on the surface of soil are vulnerable to aerobic microbial degradation, photolysis, and many other environmental factors. It is important to note that the lysimeter soil was totally exposed to the natural conditions from October through May of the following year, until transplantation of the rice plants in the following season. Accordingly, this period is not flooded so that conditions are not suitable for microbial degradation and other dissipating factors. Even if the ¹⁴C in rice plant roots could be measured, the possibility of finding a significant amount of the ¹⁴C-radioactivity would be very low, because uptake of mefenacet by rice roots has been reported to be very slow (Krauskopf et al. 1989). Furthermore, this argument is based on the fact that only 1.978 % of the originally applied [¹⁴C]mefenacet was absorbed and translocated into the various parts of the rice plants in the first year. The total amount of the ¹⁴C-radioactivity leached over the 6-year period was only 0.778 %, indicating that the downward movement of mefenacet in soil is very slow, but steady. In conclusion, the strong adsorption of [¹⁴C]mefenacet onto soil organic matter, the formation of large amounts of non-extractable bound residues in the upper soil layer (0–10 cm), and the low detection of the chemical in rice grains observed in this study indicate that exposure of groundwater and crops to [¹⁴C]mefenacet does not necessarily pose a health risk.

Table 8 Fate of [¹⁴C]mefenacet after treatment onto rice plant-grown lysimeter soil during the experimental period of 6 consecutive years. ¹⁴C-Radioactivity applied = 100 %