Field Dissipation of Metamitron in Soil and Sugar Beet Crop

Abstract

Bioaccumulation of herbicides in plant produce may cause ailing effect on animals and human beings through food chain contamination. Thus oblige the investigation of newer herbicide metamitron for its persistence and degradation in sugar beet crop and soil. Metamitron persist in plant up to 15 days while up to 30 days in soil. Its dissipation followed first order reaction kinetics. On day 90, metamitron was detected in the soil at 7.0 kg a.i. ha⁻¹ treated plot only. It would be concluded that metamitron at 3.5 kg a.i. ha⁻¹ can be safely applied to the sugar beet crop for weed control.

Herbicides are used extensively in Indian Agriculture nowadays to control or kill the non target plants and to have timely weed management. Though they are formulated to be biologically degradable, few pesticides was frequently reported to cause ground and surface water pollution (Kolpin et al. 1998; Planas et al. 1997) which may cause some degree of risk to humans, animals, or the environment. Hence, the monitoring of herbicide residues allows controlling the contamination of soils and agricultural products. Metamitron is one such herbicide used to control grass and broad leaved weeds in sugar beet, onion, bean etc. Metamitron [4-amino-3-methyl-6-phenyl-1,2,4-triazin-5(4H)-one) belonging to a triazinone group; it is a contact herbicide and readily absorbed by the roots. It acts as an inhibitor of photo system II and induces chlorotic and necrotic symptoms in leaves.

Photochemical and microbial deamination is the major degradation pathway of metamitron in soil, water and plant system (Cox et al. 1996; Engelhardt and Wallnofer 1978). Metamitron decomposition on soil surfaces and in water is very rapid (Cox et al. 1996) and degraded into deamino-metamitron (Engelhardt and Wallnofer 1978) within few days of its application. Its dissipation followed first order kinetics (Vischetti et al. 1999; EFSA 2008) in soil and the half life is highly variable (Mamy et al. 2005) ranged from 3.4 to 49.5 days (EFSA 2008). While soil properties like temperature, texture and moisture conditions have significant influence on metamitron degradation in soil (Fuhr and Mittelstaedt 1979; Allen and Walker 2006), the organic matter and pH of the matrices has little importance (Franco et al. 1997). Though few reports on metamitron dynamics in soil under laboratory conditions was published, little is known about the bioavailability of metamitron in soil and crop produce under field conditions in India and abroad. Similarly the impact of split application on its dissipation behavior is also absent. Hence the present study was carried out to determine the degradation of metamitron in soil and sugar beet plant at two application rates under split application. The use of two doses might help to recommend the suitable dose in terms of possible risk for the contamination of soil, crop produce and phytotoxicity to sensitive crops by metamitron residues.

Materials and Methods

A supervised field experiment was conducted at the experimental farm of Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. Sugar beet was grown as a test crop during rabi, 2008–2009 (Var. Cauvery) in a randomized block design with three replications. Nine plots each with a size of 30 m² were prepared and all sides of the plots were protected with soil boundaries raised to a level of approximately 35 cm height and 25 cm width. Sowing of the crop was taken up as per the standard agricultural practices suggested for sugar beet crop. Two different doses of metamitron @ 3.5 (standard dose) and 7.0 (double the standard dose) kg a.i. ha⁻¹ were applied in three equal splits (viz., 2–3, 4–6 and 8–10 weed leaf stages) by knap-sack sprayer using flat fan nozzle with the spray volume of 400 L ha⁻¹. A further three replicates of plot were sprayed with water alone and maintained as control. Soil samples were collected at 0 (2 h), 1, 7, 15, 30 and 90 days after last application of metamitron and at the time of sugar beet root lifting. About 3 kg of five-soil cores each were randomly taken from each treated and untreated plot avoiding the outer 20 cm fringes of the plots. Samples were taken using a soil auger up to a depth of 15 cm from the surface. Pebbles and other unwanted materials were removed manually. The cores were bulked together from each plot, well mixed and stored in polythene bags at −10°C until sample extraction. Samples from the control plots were collected before the herbicide treated plots for residue analysis. The experimental soil was sandy clay loam in texture (clay 29.1 %, silt 26.7 %, and sand 44.0 %), low in available nitrogen (152 kg ha⁻¹), medium in available phosphorus (18.0 kg ha⁻¹) and high in available potassium (432 kg ha⁻¹) with organic carbon 0.38 %, EC 0.49 dS m⁻¹ and pH 8.02. Plant samples were collected at 0 (2 h), 1, 7, 15, 30 and 90 days after last application of metamitron and at the time of sugar beet root lifting. About 500 g of representative plant samples were collected from each treated and untreated plots. The plant samples were cut into small pieces, then ground on mechanical grinder and used for residue analysis. Plant samples were stored at −20°C, until processed for residue extraction.

Metamitron was extracted from the soil and plant samples with methanol, filtered and evaporated at 40°C to about 10 mL. The contents were liquid partitioned with dichloromethane using 1 % aqueous NaCl and passed through anhydrous sodium sulfate. Eluted lower layer was concentrated on rotary vacuum evaporator at 60°C to moistened level and the residue was re-dissolved in acetonitrile for HPLC analysis. Metamitron reference analytical standard (99.0 % purity) was obtained from Punjab Chemicals and Crop Protection Ltd., Mumbai. All other chemicals and solvents used in the study were HPLC grade or analytical grade reagents of SD. Fine Chemicals, Mumbai, India. Metamitron residue was analyzed by using an Agilent Technologies HPLC (model 1200 series) equipped with photo diode array detector and auto sampler. Computer enabled Ez Chrome software was used for the acquisition of data. The separation of compounds was done using an Agilent XDB-C18 (5 μm, 4.6 × 150 mm) column and acetonitrile and water (3:7, v/v) as mobile phase with a flow rate of 0.5 mL min⁻¹. The detection wavelength was set at 306 nm and the volume of sample injected for detection was 5 μL. The approximate retention time of metamitron was 5.6 min. The residue was calculated by comparing the peak areas of the samples with its standards run under same HPLC conditions.

Different known concentrations of metamitron (1.0, 0.5 and 0.1 mg mL⁻¹) were prepared in acetonitrile by diluting the stock solution (1,000 mg mL⁻¹). Twenty microliters of standard solution were injected into HPLC and the peak area measured. Validation of method was performed in terms of recovery studies before the analysis of unknown samples. Known weight of plant and soil samples were taken and added into extraction flasks. 1 mL of standard solution of 1 or 0.1 mg mL⁻¹ metamitron was added uniformly on the surface of the matrix and mixed before adding extraction solvent. The extraction and cleanup processes were then performed as described in the methodology. Quantification of metamitron residues was accomplished by comparing the peak response for samples with peak area of the standards. Results have been reported without applying any correction factor.

Results and Discussion

The average recovery of metamitron in plant and soil was shown in Table 1. The instrumental detection limit and estimated method detection limit (EMDL) for metamitron was estimated as described by Sondhia (2008). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.01 and 0.05 μg g⁻¹, respectively and the signal to noise ratio was 3:1. Detector showed good sensitivity for the metamitron residues up to 0.001 μg mL⁻¹ but did not follow the linearity.

Table 1 Recovery of metamitron in sugar beet plant and soil

The metamitron recovery varied from 85 %–92 % to 87 %–99 % for sugar beet plant and soil samples fortified with 0.1 and 0.5 μg g⁻¹ of metamitron respectively. The recoveries of metamitron from soil and plant at different concentration levels were satisfactory being within the range 85 %–99 %, confirmed a good repeatability of the method (Table 1). The soil and plant blanks did not exhibit any peak interfering with the retention time of metamitron. The equations of analytical calibration graphs, obtained by plotting peak areas in y-axis against concentrations of metamitron in x-axis within the range of 2–0.01 μg mL⁻¹ was, ${\text{y}} = 10 4 4 9 {\text{x}} + 2 7 5 7$, and the correlation coefficient was 0.998.

Metamitron persistence in plant as detected by HPLC was presented in Table 2. The concentration of metamitron determined in sugar beet plant on 0 day after last application was 0.235 and 0.513 μg g⁻¹ and on 7 days was 0.079 and 0.197 μg g⁻¹, respectively for 3.5 and 7.0 kg a.i. ha⁻¹, respectively for 3.5 and 7.0 kg a.i. ha⁻¹ application rates. Metamitron residue was decreased gradually with the passage of time and by 30 days, residues were found below the detection limit (<0.01 μg g⁻¹) at both the application rates. Dissipation of metamitron was faster during initial period with a dissipation rate of 33.4 % on 1 day (Table 2) irrespective of the dose applied. Similar result was reported by EFSA (2008) that when metamitron was applied at 1.74 kg a.i. ha⁻¹, only 74 mg kg⁻¹ of it was presented in the sugar beet foliage after 2 days of last application. On 15th day after its last application, more than 80 % of the metamitron dissipated from the plant and this could be the critical period of its dissipation in sugar beet plant. A straight line was found when the log of residue in sugar beet samples was plotted against time. The significant values of coefficient of determination (r² = 0.98) established that the dissipation process followed first order reaction kinetics (Table 3). Calculated mean half life of metamitron was 5.7 and 6.1 days for the treatments at 3.5 and 7.0 g a.i. ha⁻¹, respectively.

Table 2 Persistence of metamitron (μg g⁻¹) in sugar beet plant and field soil

Table 3 The regression equation, correlation coefficient and the half-lives (t_1/2) of metamitron in sugar beet plant and field soil

The initial concentration of metamitron in soil varied from 1.21 to 2.04 μg g⁻¹ across different doses with highest value in double dose (7.00 kg a.i. ha⁻¹) of application. Metamitron dissipation in soil was faster than in plant and more than 88 % of the applied metamitron disappeared from the soil on 15th day irrespective of the dose of application. Breakdown of metamitron into its major metabolite desamino-metamitron through photo transformation (EFSA 2008) might be the reason for such a faster dissipation from soil. This could further attributed to the enhanced microbial degradation by higher sunshine hours (2.8–8.5 h) and soil temperature (29.5–35.2°C) that prevailed during the crop growing period (Engelhardt and Wallnofer 1978; Parekh et al. 1994; Charnay et al. 2005). Residue of metamitron went below the maximum residue limit of 0.05 ppm (EFSA 2008; Kucharski 2008) on 45th day under standard dose and becomes below detectable level on 90th day under both the dose of application. This would be the result of leaching of metamitron from the soil easily by the high rainfall received (14.65 mm) during the period of herbicide application, and might have been further accelerated by the sandy clay loam nature of soil texture (Allen and Walker 2006). Metamitron dissipation in soil followed first order reaction kinetics (Vischetti et al. 1999; Macounova et al. 2001) at both the doses and a straight line was obtained in each case when log values of the residue was plotted against different time intervals. The significant correlation coefficient (r² = 0.983**) indicated statistical conformity of the dissipation data to first order reaction kinetics (Table 3). The increase in dose increased the DT₅₀ values and could be established that the fraction of the total herbicide content which was available in the soil solution influence the dissipation of it from soil (Allen and Walker 2006). Half life calculated was 4.9 and 5.4 days for metamitron from treatments at 3.5 and 7.0 kg a.i. ha⁻¹, respectively (Table 3). Similar result was reported by Vischetti et al. (1999), who observed a field half life of 4.4–8.0 days in sandy clay loam soil for metamitron.

An effort was made to study the uptake of metamtiron from the soil by bioassay using sensitive crops. After the harvest of sugar beet, the herbicide sensitive crops viz., pearl millet, cowpea and sunflower were grown in the experimental plots. These species were monitored for germination count and plant height and found that they were unaffected by the treatments. Whole plant samples of all species were analysed for metamitron residues 6 weeks later. The residues of metamitron were all non-detectable indicating that metamitron residue in soil were not taken up by the succeeding crops (Ramesh and Maheswari 2004) under experimental conditions described in this study.

On the basis of above findings it could be concluded that metamitron at 3.5 kg a.i. ha⁻¹ can be safely applied to the sugar beet crop as post emergence herbicide in three equal splits. The residue of metamitron dissipated faster in soil than in plant. Though the concentration of metamitron residues in crop produce were found below the maximum residue limits set by EFSA (0.05 μg g⁻¹), the biomagnification of metamitron residues in crop produce under continuous use should be investigated.