Persistence of metribuzin in aridisols as affected by various abiotic factors and its effect on soil enzymes

ABSTRACT Herbicide degradation is one of the key processes determining whether the use of herbicide will have any impact on environmental quality, and it is dependent on various biotic and abiotic factors. The present study evaluates the impact of various abiotic factors such as soil type, application rate, incubation temperature, soil pH, soil microorganisms and sunlight on persistence of metribuzin and its effect on soil enzymes. Half-lives (DT50) varied significantly with application rate of metribuzin and physicochemical properties of soil and ranged from 15.17 to 46.59 days. Metribuzin degraded 2.2 to 6.5 times slower in sterilised soil (DT50 = 35.21 to 115.60 days) as compared to unsterilised soil, indicating microbial degradation is one the most predominant contributors towards degradation of metribuzin in soil. Increase in degradation rate on exposure to sunlight indicated photodegradation also contributes significantly to the degradation of metribuzin. Deaminometribuzin, diketometribuzin and deaminodiketometribuzin metabolites of metribuzin were detected in all treatments. Dehydrogenase and alkaline phosphatase activities were significantly affected by application of metribuzin while urease activity remained unaffected. The physicochemical properties viz. pH, organic matter content and temperature should be considered along with application rate of metribuzin in order to achieve satisfactory weed control and reduce environmental risk associated with the use of metribuzin in different crops.


Introduction
To satisfy the increasing demands of ever-growing population, crop production needs to be evaluated.For this, any hindrance that retards the pace of increasing crop production needs to be tackled [1].One such factor that lowers the crop quality and quantity is weed emergence as these compete with the main crop for essential nutrients, water and sunlight [2].Array of pre-and post-emergence herbicides have been recommended to improve the quality and yield of crops.Despite the efficiency of herbicides in enhancing and stabilising agricultural output by control of obnoxious weeds, these chemicals also affect the non-target organisms and contaminate soil ecosystem [3].Active ingredients of various herbicides also hinder the biochemical processes and impede the soil microbial activity [4][5][6].Modifications in the microbial activity can cause risk to balanced equilibrium between diverse groups of microorganisms playing a significant role in nitrification, mineralisation and nutrient recycling [7].Numerous studies have shown that the level of deterioration of soil biochemical properties depend upon the herbicide persistence in the environment and its toxicity [4].To enable the rational use of herbicides, a comprehensive knowledge of their persistence, fate and mobility in the environment under different conditions is of paramount importance [8].
Metribuzin (4-amino-6-tert-butyl-4-dihydro-3-methylthio-1,2,4-triazin-5-one) is a selective, systemic and symmetrical triazine herbicide that is applied extensively for pre-and post-emergence control of broad-leaved weeds, annual grasses and sedges in potatoes, tomato, sugarcane, soya bean and wheat [9].The primary mechanism of action of metribuzin is to hamper the photosynthetic electron transport between primary and secondary acceptors of photosystem II [10].Metribuzin was first launched in 1970 and is an active ingredient in number of formulations.Its consumption has increased from 2 tons in 2005-2006 to 195 tons in 2009-2010, and amongst the different states of India, Punjab is a major consumer of metribuzin [11].Additionally, Phalaris Minor, a major weed of wheat crop, has developed resistance to isoproturon, and the alternative recommended herbicides like fenoxaprop, clodinafop, pinoxaden, sulfosulfuron etc. are also not providing satisfactory control to this weed.To combat this, metribuzin and its formulation with fenoxaprop, clodinafop, flufenacet, sulfosulfuron, trifluralin and metolachlor are getting popularity due to their versatility in managing weeds of broad spectrum [11].
It has moderate-to-high persistence in soils with half-lives varying from 16 to 329 days [12][13][14].It is classified as a weakly adsorbed and easily desorbed molecule with very high potential to get rapidly transported from terrestrial to aquatic environment due to high mobility (organic carbon adsorption coefficient (K OC ) of 24.3-106 mL g −1 ) and high water solubility (10,700 mg L −1 at 20°C) [15].The residues of metribuzin have been detected in most of the groundwater samples and different water bodies located in sandy soil areas of Wisconsin [16].It poses a serious risk to aquatic flora, fauna, human and terrestrial life.Metribuzin has deleterious impact on aquatic organisms with lethal dose (LD 50 ) of 74.6 and 49 mg kg −1 for Oncorhynnchus mykiss and Daphnia magna, respectively.At single high doses, it causes suppression of central nervous system while at repeated doses it affects the thyroid gland and stimulates metabolic enzymes of liver [17].It also exhibits toxicity to aquatic plants and algae with effective concentration (EC 50 ) of 0.008 and 0.02 mg kg −1 in Lemna gibba and Scendesmus subsicapitata.
Based on the environmental impacts and increased use, it is of utmost importance to study the degradation behaviour of metribuzin in soil under different scenarios so as to predict its residual levels and impact on soil biochemical properties.It has been investigated that physicochemical characteristics of soils significantly affects the degradation behaviour of metribuzin [12,[18][19][20][21][22].It has low persistence in Lebanese alfisol soils [23,24] of mid hill zone due to higher organic matter (OM) content while it has high persistence in soils of Tylstrup fields of Denmark owing to its low pH, which results in more adsorption and consequently slower degradation [12].The information of dissipation of metribuzin in these provinces cannot be generalised to aridisol of subtropical humid regions of India due to different abiotic situations and soil physicochemical properties.Hence, the degradation behaviour of metribuzin in soils of subtropical humid region of India needs to be scrutinised.The present investigation evaluates the influence of several abiotic factors such as soil physicochemical characteristics, herbicide application rate, temperature, microorganisms, pH and sunlight on degradation behaviour of metribuzin in aridisol of subtropical humid regions of India under laboratory conditions.Although there are numerous studies on the effect of triazines on soil enzymatic activity [10,25,26], the effect of metribuzin on soil biological activity remains insufficiently researched.In view of this context, the present study also investigates the effect of metribuzin application on enzymatic activity of soil.

Soils
Sandy loam (SL), loamy sand (LS) and clay loam (CL) soils with no history of herbicide application were collected from 0 to 20 cm deep plough layer.Air-dried soil samples were sieved through 2-mm sieve, stored in labelled polythene bags and were used within 1 month to avoid exhaustion of biological activity of soil [27].The physicochemical properties and geographical locations of the studied soils are given in Table 1.

Chemicals and reagents
Certified reference standard of metribuzin (purity 98%) was purchased from Sigma Aldrich, Germany.It has a vapour pressure of 0.121 mPa (25°C), water solubility of 10,700 mg/L at 20°C, octanol-water partition coefficient (K ow ) of 5.62 × 101 and pKa of 1.3 at 25°C.All other chemicals and solvents were purchased from Hi Media Laboratories Pvt, Ltd., Mumbai (India), and Sisco Research Laboratories Pvt, Ltd (SRL)., Mumbai (India).Commercial formulation (70 WP) of metribuzin brought from local market was used for dissipation study.

Experimental setup
The experimental study was arranged in a complete randomised block design with three replications of every treatment.For each soil, 63 pots (10 cm x 8.5 cm) were filled and each pot has 500 g of soil.The pots were divided into three sets (Set 1, Set 2 and Set 3) having 21 pots each.Set 1, 2 and 3 were incubated at 25 ± 2, 35 ± 2 and 45 ± 2°C, respectively.The soil moisture was maintained at 80% field capacity by adding distilled water periodically throughout the experimentation period.After equilibration in every set, three pots each were treated with metribuzin at 0.0875 and 0.175 µg g −1 corresponding to the recommended and double the recommended rate of 175 and 350 g/ha in wheat crop; 0.263 and 0.525 µg g −1 corresponding to the recommended and double the recommended rate of 525 and 1050 g/ha in tomato, potato and soya bean; 0.7 and 1.4 µg g −1 corresponding to the recommended and double the recommended rate of 1400 and 2800 g/ha in sugarcane [28].Remaining three pots in each set served as the control.
To study the effect of pH, loam 1 and loam 2 soils having pH 5.0 and 9.0 were treated with metribuzin at application rates of 175, 350, 525, 1050, 1400 and 2800 g ha −1 at 25 ± 2°C.
To study the effect of light on degradation, a set of pots of LS, SL and CL soils in triplicates were treated with metribuzin at 175, 525 and 1400 g ha −1 .These pots were kept in sunlight along with the control pots.The effect of soil microorganisms on degradation of metribuzin was studied by autoclaving SL, LS and CL soils at 120°C and 15 atm pressure for 60 min twice in sterile conical flasks.The sterile soil was treated with herbicide at application rates of 175, 525 and 1400 g ha −1 under laminar flow and incubated in biological oxygen demand (BOD) incubator at 30°C.Autoclaved distilled water was sprayed at regular intervals to maintain the moisture to 80% field capacity.Control treatment was also maintained under similar conditions.Soil sample (20 g) were withdrawn from each pot at 0 (5 h), 3, 7, 15, 30, 45, 60 and 90 days after treatment (DAT) and analysed for residues of metribuzin.

Extraction and analysis of herbicides
Metribuzin from soil was extracted by the matrix solid phase dispersion (MSPD).Briefly, 10 grams of soil sample was blended finely with 5 g of pre-activated silica.After homogenisation, it was packed into a glass column containing 3 mg charcoal and 2 g sodium sulphate.The column was eluted with 80 mL of acetone.The collected solvent was evaporated to dryness using rotary vacuum evaporator, and the residues were redissolved in 2 mL of acetonitrile and analysed using LC-MS/MS.

Soil biochemical assay
The effect of metribuzin on enzymatic activity was evaluated in LS, SL and CL soils at 25 ± 2, 35 ± 2 and 45 ± 2°C.The soil samples from the dissipation experiment were collected at 0 (5 h), 15, 30 and 60 DAA of metribuzin for the estimation of enzymatic activity.Dehydrogenase activity (DHA), alkaline phosphatase (APA) and urease activity were determined by a method reported by Tabatabai [29], Tabatabai and Bremner [30] and Douglas and Bremner [31].

Data analysis
Dissipation parameters for metribuzin were estimated using first-order kinetics equation: Where C 0 is the initial concentration of metribuzin and C t is the concentration of metribuzin at time t (days) in μg g −1 and k (day −1 ) is the rate constant.Half-life (DT 50 ) was determined as: Complete randomised designs (CRDs) with significance level (CD value) less than 5% were used for statistical analysis.

Method Validation
The mean percent recovery of metribuzin varied from 80.21 to 97.37% (Table 2).The intraday precision (%RSD r ) and interday precision (RSD R %) was evaluated by analysing the samples spiked with 1.0, 0.5, 0.1, 0.05 and 0.01 µg g −1 of metribuzin three times a day and 2.1 a Each value is mean ± relative standard deviation (RSD%) of three replicates three times for three consecutive days, respectively.%RSD r and %RSD R were less than 10% for all the soils.The limit of detection (LOD) and limit of quantification (LOQ) were 0.002 and 0.006 μg g −1 , respectively.

Effect of application rate and soil type
The initial residues of metribuzin after 3 hrs of treatment varied according to soil type and ranged from 0.072 ± 0.01 to 0.092 ± 0.044 µg g −1 and 0.132 ± 0.011 to 0.1643 ± 0.009 µg g −1 at an application rate of 175 and 350 g ha −1 , respectively.However, the initial residues were higher at higher application rates and varied from 0.235 ± 0.016 to 1.569 ± 0.015, 0.203 ± 0.009 to 1.512 and 0.186 ± 0.012 to 1.416 ± 0.023 µg g −1 in LS, SL and CL soils, respectively, at application rates ranging from 525 to 2800 g ha −1 .The residues of metribuzin decreased successively as a function of time and ranged between 0.0185 ± 0.0011 and 0.402 ± 0.0628 µg g −1 90 DAT at all the application rates in studied soils (Figure 1).
Dissipation followed first-order kinetics and DT 50 varied significantly with application rate of metribuzin and physicochemical properties of soil.It ranged from 19.59 to 46.59, 17.89 to 41.17 and 15.17 to 35.68 days in LS, SL and CL soils, respectively.Slower dissipation in LS can be explicated on the basis of the adsorption-desorption behaviour of metribuzin in soil as it will affect the concentration of herbicide in soil solution, thereby affecting its availability for degradation.Metribuzin is weakly adsorbed (adsorption capacity (K ads ) ranging from 0.02 to 0.25 mL g −1 ) and readily desorbed (desorption capacity (K des ) ranging from 0.14 to 0.56 mL g −1 ) from alkaline soils [13,19,[32][33][34] due to weak hydrogen bonding between NH 2 group of metribuzin (Figure 2) and reactive sites of OM.This increases herbicide bioavailability to soil microorganisms for degradation and resulted in rapid degradation of metribuzin in soil having high OM content.
The metabolites of metribuzin were recognised in association with the literature data [20,35].Degradation of metribuzin could occur through two possible metabolic pathways.In one of the possible pathways, metribuzin undergoes reductive deamination to form deaminometribuzin (DA), while in other it undergoes oxidative desulfuration to form diketometribuzin (DK).Both of these primary metabolites metabolise to form secondary metabolite deaminodiketometribuzin (DADK) as shown in Figure 3. Metabolites so formed were recognised qualitatively through their molecular ion peaks.DA exhibited molecular ion peak at m/z 200.30 and its fragment ions at m/z 172 and 130. DK exhibited molecular ion peak at m/z 186.00 and its fragment ions at m/z 157 and 141, while DADK exhibited molecular ion peak at m/z 170.10 and its fragment ions at m/z 157.
The metabolites formed varied with the soil type.In SL and LS soils, metribuzin predominantly undergoes reductive deamination to form DA. It started appearing 3 DAT and persisted up to 7 DAT where secondary metabolite DADK started appearing.In CL soil, it undergoes oxidative desulfuration to form degradation product DK, which started appearing 3 DAT and persisted up to 15 DAT where DADK started appearing.The DADK formed persists up to 30 DAT in SL and LS soils and 45 DAT in CL soil.It ultimately gets mineralised to form CO 2 and NH 3 .The quantification of metabolites was not feasible due to non-availability of their analytical standards.Similarly, two different pathways (reductive deamination and oxidative desulfuration) followed by the degradation of metribuzin depending on physicochemical characteristics of soil have been reported by Locke and Harper [36].

Effect of temperature
The initial residues varied from 0.072 ± 0.0045 to 1.569 ± 0.0023, 0.066 ± 0.0033 to 1.5089 ± 0.0054 and 0.052 ± 0.0032 to 1.420 ± 0.0029 at 25 ± 2, 35 ± 2 and 45 ± 2°C.Dissipation followed first-order kinetics, with DT 50 varying from 15.17 to 46.59, 13.95 to 42.58 and 10.60 to 36.21 days at 25 ± 2, 35 ± 2 and 45 ± 2 °C in studied soils (Table 3).The dissipation rate constant (k) varied from 0.0148 to 0.0456, 0.0162 to 0.049 and 0.0191 to 0.0653 at 25 ± 2, 35 ± 2 and 45 ± 2°C.It was observed that the degradation rate constant of metribuzin was highly temperature dependent, and an increase in the incubation temperature from 25 ± 2° to 35 ± 2° or from 35 ± 2° to 45 ± 2° increased the rate constant for dissipation of herbicide but not by same proportion.The nonproportional increase was determined by calculating Q 10 value, which is defined as the ratio of the rate constant at higher temperature to the rate constant at lower temperature [22].It is important to obtain these data for accurate determination of herbicide fate model in order to avoid overestimation or underestimation of the effect of temperature on degradation rate of herbicide.Q 10 values increased with increasing temperature and varied from 1.14 to 1.35.Enhanced rate of degradation with increase in temperature could be due to its positive effect on microbial growth and activity, increased water solubility of metribuzin and increased rates of abiotic reactions such as hydrolysis and oxidation [37].Similar enhanced degradation of metribuzin with increase in temperature has been reported by Lechon et al. [38], Henriksen et al. [12] and Walker [39].Lechon et al. [38], Henriksen et al. [12] and Walker [39] have observed that increase in temperature from 5 to 20°C increases the rate of degradation by 6 to 10 times, while in the present study, increase in temperature from 25 to 45°C enhances the rate of degradation by 1.09 to 1.17 folds.The effect of temperature was less important in experiments conducted by Hyzan and Zimdahl [18] and Smith and Walker [40].The variable results suggest that physicochemical properties of soil, soil depth, management and agronomic practices and subtle interplay between these days plays important role in determining the degradation of metribuzin.

Effect of pH
As pH can also influence the degradation of metribuzin in soil, the dissipation studies were conducted on loam 1 and loam 2 soils having pH 5.0 and 9.0.It was observed that the rate of degradation of metribuzin decreases with the decrease in pH, and DT 50 varied from 30.02 to 70.34 and 25.54 to 63.29 days in loam 1 and loam 2 soils having pH 5.0 and 9.0, respectively.The reduced rate of degradation of metribuzin with decrease in soil pH was due to the fact that pH of soil affects the ability of soil to adsorb and retain the metribuzin.As the soil pH is lowered, H + ions get associated with metribuzin to give it cationic characteristics, leading to its strong adsorption due to electrostatic attractive forces between negative charges of colloids in the soil and cationic metribuzin (Figure 2).This will decrease the concentration of metribuzin in soil solution, thereby decreasing the bioavailability of herbicide for soil microflora, resulting in slower dissipation.The higher persistence of metribuzin in low pH soil may cause carryover effect on succeeding sensitive crops and potential hazards associated with its residues in soil [41,42].4; Figure 4).Slower dissipation of metribuzin in sterile soil indicated that microbial activity plays a fundamental role in degradation process of metribuzin [35].DT 50 in the present study was notably shorter than that reported by Henriksen et al. [12] (>500 day) as in later study, soil was acidic in nature (5.50 to 5.98) and differential bonding interaction between metribuzin and soil strongly influences the rate of dissipation [19].

Effect of light
Initial residues of metribuzin ranged from 0.072 to 0.7921 µg g −1 at 0 DAT.It degrades very rapidly under light conditions, and about 60.4 to 94.23% of metribuzin gets degraded over the period of 15 days as compared to 24.56 to 39.45% degradation under dark conditions (Table 4; Figure 4).This difference could be attributed to the fact that under dark conditions, degradation occurred as a result of microbial activity and abiotic reactions, while under sunlight, photodegradation also contributed significantly to degradation mechanism of metribuzin besides microbial and chemical degradation.The present study revealed that dissipation of metribuzin in aridisol followed firstorder kinetics.Most of the available literature deals with dissipation of metribuzin in acidic and neutral soil with pH ranging from 4.68 to 7.5, and dissipation of metribuzin in these studies were well described by the first-order kinetics [19,43].However, deviation from first-order kinetics has been reported.Maqueda et al. [44], Henriksen et al. [12] and Noshadi and Homaee [45] have reported that biexponential or twocompartment model was more accurate descriptor for dissipation of metribuzin.In two-compartment model, initial fast degradation was observed as herbicide was readily available in soil solution for microbial degradation, while in the later part, herbicide is sorbed on OM surface and diffused into intra aggregated pores.Both these processes depend on sorption properties of herbicide and soil and reduce herbicide concentration in soil solution, thus reducing its accessibility to microbes contributing to decreased degradation rate [12].Moreover, the activity and population of degrading microorganisms in soil decrease with time due to limited access to nutrient and hydrocarbon sources.Noshadi and Homaee [45] reported better fit of metribuzin degradation under field conditions to bi-exponential model or first-order kinetics depending on the method of application of herbicide.The deviation was probably because soil is a heterogenous substance characterised by spatial variation in vertical and horizontal directions, and seasonal variation in soil temperature and moisture can affect herbicide availability in soil and thus degradation, leading to deviation from first-order kinetics and transforming it into bi-phasic kinetic.Kempson-Jone and Hance [46] demonstrated that apparent order of dissipation of metribuzin varied between soils, samples of same soil taken from different depths, between samples incubated at different temperature, moisture content and also with method of incubation, suggesting large discrepancies from first-order kinetics may be due to results of artefacts from experimental technique.
In the present study, DT50 of metribuzin ranged from 15.17 to 115.60 days and increased with increase in OM content and pH of soil.Similar positive correlation with pH and OM has been reported by Henriksen et al. [12] and Allen and Walker [43], while Maqueda et al. [44] has reported greater influence of formulation type on dissipation of metribuzin and it was positively correlated with pH but independent of OM content of soil.The discrepancy between different studies was due to difference in physicochemical properties of soil and experimental conditions viz lab or field study, temperature, soil moisture and formulation type.Additionally, previous studies on degradation of metribuzin had reported highly variable DT 50 values.Henriksen et al. [12] reported its high persistence under lab conditions (DT 50 = 149.0days) due to low incubation temperature and pH of the studied soil while Khoury and his co-workers [20] observed faster degradation of metribuzin (DT 50 = 12.20 to 14.15) due to high OM of the soils.Rapid dissipation of metribuzin with DT 50 ranging from 13.7 to 18.8 days [20] has been observed in the field study under agro-climatic environment of sub-temperate mid-hill zone of Himachal Pradesh.Comparatively faster dissipation under field study was probably because under laboratory conditions, water content and temperature are usually held constant promoting stagnant conditions in soil pores, whereas under field conditions, water and oxygen are replenished by wetting and drying and heating and cooling cycle, promoting microbial growth.

Dehydrogenase and alkaline phosphatase activity
It was observed that DHA was most sensitive and decreased significantly 3 DAT in contaminated soils as compared to the control.This might be due to direct effect of metribuzin on dehydrogenase activity.Moreover, it was observed that percent DHA inhibition in treated soils decreased significantly with an increase in soil contamination levels from 175 to 1400 g ha −1 , and 2-50% inhibition in DHA (in comparison with control) was observed at metribuzin application rate of 175, 350, 700 g ha −1 , respectively.Further increase in the metribuzin application to 2800 g ha −1 resulted in slight increase in inhibition, indicating higher dose might have toxic influence on DHA and, in turn, the microbial activity in soil, while at lower doses, it might serve as carbon source for the survival of microorganisms (Figure 5).The soil DHA showed about 30.6 to 56.6% stimulation from 3rd to 30th day of incubation in all the studied soils due to increase in microbial community with the potential of using the herbicides as carbon source [47].DHA decreases thereafter from 30th to 45th DAT.The decrease might be due to the fact that the herbicide which served as carbon source for the survival of microorganisms decreased significantly at 45 DAT.It was also observed that DHA was highest in CL followed by SL and LS soils in both control and treated soils irrespective of the herbicide application due to high OM content, which acts as energy or food source for the soil microorganisms.Soil DHA was also affected by change in temperature.Incubation of both treated and control soils at higher temperature increased the DHA by 1.2-2.0times in studied soils.
The soil phosphatase activity is responsible for the conversion of organic phosphorus into inorganic phosphates (form available for plants and soil microorganisms).APA declined significantly 3 DAT in contaminated soils, and percent reduction ranged from 27.8, 36.5, 41.8, 46.42, 50.09 and 50.64% at 175, 350, 525, 700, 1400 g ha −1 , respectively.It increased significantly from 3rd to 45th DAT in all the studied soils (Figure 6).Initial inhibition in APA activity might be due to the lethal effect of metribuzin on membrane permeability of phosphate solubilizers, which are responsible for release of phosphatase [48,49].Incubation of both treated and control soils at higher temperature increased the APA by 1.08 to 1.5 times in studied soils.

Urease activity
Urease plays a very significant function in the nitrogen cycle as it hydrolyses urea into ammonia (NH 3 ) and carbon dioxide (CO 2 ).Soil urease activity almost remained unaffected by the application of metribuzin except some reduction at very high application rates.This might be due to the reason that urease are extracellular enzymes strongly associated by organic and inorganic colloids hence are least affected by the changes in soil environment [50].There were no regular trends in urease activity as the time passed except a slight increase in the activity at 45 DAT (Figure 7).This can be explained by the fact that there is no significant function of urease in the metabolism of metribuzin, thus urease activity remains unaltered by its application during the initial stages.But the metabolites produced after degradation might have been used as specific substrates for urea hydrolysing enzyme, and in later stages, they reach at a concentration level enough to stimulate urea synthesis [51].Urease activity was also positively affected by increase in OM content and temperature.

Conclusion
Dissipation of metribuzin was significantly affected by application rate of herbicide, physicochemical characteristics of soil, temperature, pH and soil microorganisms.Degradation kinetics fitted well to first-order kinetics, with DT 50 varying from 15.17 to 46.59 days.The.The soil OM content, pH and temperature had positive influence on the degradation of metribuzin.Metribuzin was more persistent in sterile soils as compared to unsterilised soils.The metabolites deaminometribuzin, diketometribuzin and deaminodiketometribuzin were detected in all treatments.The dehydrogenase and alkaline phosphatase activities were considerably influenced by the application of metribuzin, while the urease activity followed no regular trends.The findings indicated that both physicochemical properties of soil and the application rate of metribuzin play an important role in achieving satisfactory weed control and reducing the environmental risk associated with its use in different crops.In present study, the persistence of metribuzin in soil at different application rates corroborated well with critical period of weed control, i.e. upto 57 days in wheat; 63 days in soya bean/tomato/potato and 120 days in sugarcane, indicating metribuzin will be effective for control of weeds in aridisols in these crops.Field experiments are in progress for the critical assessment of weed control efficacy and its impact on the environment under actual conditions.

Figure 4 .
Figure 4. Residues of metribuzin under sterile conditions in (a) LS, (b) SL and (c) CL; light conditions in (d) LS, (e) SL and (f) CL at studied application rates; LS: Loamy sand, SL: Sandy loam, CL: Clay loam.

Table 1 .
Physico-chemical properties and geographical locations of soils.

Table 2 .
Mean percent recoveries, inter and intraday reproducibility of metribuzin.

Table 3 .
Half-lives and other statistical parameters for metribuzin dissipation in studied soils.

4. Effect of soil microorganisms
In sterile soils, degradation of metribuzin was very slow and only 25.68 to 42.12% of the applied metribuzin degraded by 90th day as compared to 76.34 to 86.99% degradation of metribuzin in non-sterile soils at 25 ± 2°C.DT 50 of metribuzin in sterile soil varied from 35.21 to 115.60 days (Table a Rate constant in days −1 b Half-life in days *Half-life varied significantly with soil type, application rate, temperature and moisture content with CD less than 5% c Mean value of rate constant is average of rate constants of all the dose rates at a certain temperature.LS: Loamy sand, SL: Sandy loam, CL: Clay loam 3.2.

Table 4 .
Kinetics parameters of metribuzin dissipation under sterile conditions and direct sunlight at 25°C.