Effect of differential addition of water on mobility of fluopyram in two different soils of Kerala

ABSTRACT Fluopyram, a pyridyl ethyl benzamide broad-spectrum fungicide and nematicide, used for the management of plant parasitic nematodes and soil-borne fungi, is recommended for use in crops like tomato, potato and tobacco. Fluopyram being a pesticide used for drenching soil, there is every possibility of it getting leached down with water, ultimately leading to contamination of waterbodies. An experiment was conducted to study the downward movement of fluopyram in laterite and red loam soils in Kerala, India. Fluopyram and fluopyram benzamide, each at150 µg, were loaded separately on top of the respective soil column and were eluted separately with 20, 40, 80 and 160 mL each of water equivalent to 50, 100, 200 and 400 mm rainfall. It was observed that 76.84% and 74.85% of fluopyram and 73.48% and 64.24% of fluopyram benzamide were confined to the top 0–10 cm soil with application of 20 mL water in laterite and red loam soil, respectively. More than 50% of fluopyram and fluopyram benzamide were detected in the top 0–10 cm layer of soil with the application of 40 and 80 mL water. With the application of 160 mL water, considerable reduction in the concentration of both compounds was observed and the residues moved beyond 30 cm. Leaching of residues was more in laterite soil than in red loam soil, and they are in direct relation to the volume of water applied and the residues moved beyond 30 cm depth with 160 mL water.


Introduction
The primary challenge for modern farming is to increase food production quality and quantity while minimising the negative impact on the environment or natural resources.Agrochemicals are an inevitable component in eradicating pests and diseases in sustainable agriculture, but their excessive use can harm the ecosystem by polluting water, air and soil either directly or unintentionally [1,2].Pesticides have the potential to leach into waterbodies, volatilise and get sorbed onto soil particles.The major temporary reservoir of pesticide residue accumulation is soil.Adsorption, leaching and degradation are the main procedures responsible for the fate of a pesticide when it appears in the soil [3,4].In water and soil, the persistence of residues of hazardous pesticides affects the quality of soil, aquatic organisms and groundwater.Application of pesticides for the purpose of irrigation results in pollution of groundwater, leading to contamination of food chain [2,5].In India, organochlorine pesticides, mainly isomers of hexachlorohexane, dichlorodiphenyltrichloroethane, endosulphan, endrin, aldrin, dieldrin and heptachlor, were identified from drinking water samples [6].Evaluation of pesticide fate in soil and movement into the deeper soil layers, eventually into groundwater, is necessary for risk assessment [7][8][9][10].
Fluopyram, N-[2-[3-chloro-5-(trifluoromethyl)-2-pyridyl] ethyl]-α, α,α-trifluoro-orthotoluamide, is a new pyridinyl ethyl benzamide pesticide introduced by Bayer Crop Science in 2010 [11].It is effective against root-knot nematodes, cyst nematodes, other soil nematodes [12], white mould, early blight, powdery mildew and Alternaria brown spot in many crops, viz., cabbage, potato and tomato [13].In India, the registered formulation for fluopyram is 34.48% w/w SC according to the Central Insecticide Board and Registration Committee (CIB & RC), Government of India, and it is recommended for the use in tomato as a nematicide to control root-knot nematode (Meloidogyne incognita).Fluopyram is a soil-applied pesticide mainly used for soil drenching in nematode management.Soil drenching of pesticide also leads to elevated residue levels in soil, and higher levels of fluopyram may result in rapid fluopyram mobility in soil, posing a danger to the environment and other soil organisms [14,15].Fluopyram has a very low vapour pressure of 3.10 × 10 6 Paat 25°C [15], and this leads to the generation of more soil residues [16].The groundwater ubiquity score (GUS) of fluopyram is 3.23 (high leachability) [17], indicating the high potential of fluopyram to percolate down through soil to an aquifer and transport through runoff to surface waterbodies [9,10].In soil, fluopyram forms a major metabolite fluopyram benzamide [15,[18][19][20].Chawla et al. [15] reported that the fluopyram contents in soil after 15 days of application at 250 (standard dose) and 500 (double dose) g a.i.ha −1 were 0.19 and 0.59 mg kg −1 and residues of its metabolite fluopyram benzamide were not detected.Fluopyram benzamide was detected in the bell pepper fruits and leaves, and it constituted 0.5-4.4% of the parent compound, but it was not detected in the soil [20].Matadha et al. [21] also reported that fluopyram benzamide was not detected in soil but was detected in pomegranate leaves.Scanty data are available on the environmental fate of fluopyram and its dissipation and leaching under Indian soils.Hence, an attempt was made to study the leaching behaviour of fluopyram in laterite (Ultisol) and red loam (Oxisol) soils, which represented the major crop-growing areas of Kerala, India, under different rainfall circumstances.

Chemicals
Certified reference material of fluopyram (99.4%) and its metabolite fluopyram benzamide (99.4%) was obtained from Bayer Crop Science, India.All the chemicals and organic solvents used in the study were of analytical grade or HPLC grade.Adsorbents were activated before use.

Experimental details
The mobility of fluopyram and fluopyram benzamide in the laterite and red loam soil columns was assessed by analysing the residues at different depths after loading 3 mL, 150 µg of each fluopyram and fluopyram benzamide prepared in methanol and the subsequent elution with different levels of water, viz., 20, 40, 80 and 160 mL.Representative soils for the study were collected from the identified locations for laterite and red loam soils in Thiruvananthapuram, Kerala.The required soils were collected from the surface layers (0-15 cm depth).The soil was tested for any pesticide residues and confirmed the absence of fluopyrambenzamide.The physico-chemical characteristics of these have been measured using conventional techniques as shown in Table 1.Soils were analysed using typical analytical methods, and Table 1 shows the estimated parameters of experimental soil.
PVC columns of length 50 cm and internal diameter 2.50 cm were taken to which 400 g soil sample was packed to a height of 30 cm maintaining the bulk density and simulating the field compaction level and were fixed firmly to the burette stands.The lower end of the columns was firmly fastened using a muslin cloth so as to retain the soil as per the desired bulk density and immersed in a beaker containing distilled water for 12 h for attaining saturation.On the next day, the saturated soil column was top loaded with 5 g soil containing 3 mL of 150 µg of fluopyrambenzamide and placed in a dry conical flask for collecting the leachate.Here, both laterite and red loam soil was filled in four separate columns each.The columns were eluted by controlled addition of water at 20, 40, 80 and 160 mL, respectively, using drip system at a steady flow in accordance with the hydraulic conductivity of both soils (0.4 mL min −1 ) obtained from the analysis.The water used for elution corresponds to 50, 100, 200 and 400 mm rainfall and is used for the prediction of relative mobility along with water under field condition.Conical flasks were kept below each soil column in which the leachates were collected.After the completion of leaching (after 24 h), the leachates as well as the soil were analysed for residues.For the soil sample analysis, the pipe was cut into six portions of 5 cm each, viz., 0-5, 5-10, 10-15, 15-20, 20-25 and 25-30 cm, and the soil sample was collected from each 5 cm fractions.The residues present in each of the vertical soil fractions were extracted and quantified.

Method validation
The analytical method used for estimation of fluopyram and fluopyram benzamide in laterite soil, red loam soil and water was validated by calculating and assessing various performance parameters such as analyte recoveries, limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy, precision, repeatability and reproducibility.

Recovery experiment
Recovery studies were conducted by spiking different concentrations (0.01, 0.05, 0.1, 0.5 and 1.00 mg kg −1 ) of analytical standards of fluopyram and fluopyram benzamide in untreated laterite and red loam soil samples.Five replicates were analysed at each spiking level.Accuracy of analytical methods was determined based on repeatability and relative standard deviation, which is generally considered satisfactory for residue quantification.

Extraction of pesticide residues from soil and leachate
The residues of fluopyram benzamide were extracted from the soil samples by the modified QuEChERS method [22].The soil (10 g) in triplicate was extracted with 20 mL acetonitrile.The extract was concentrated and made up to 1 mL using methanol, filtered through 0.22 µm polyvinylidene fluoride syringe filter and collected in an LC vial and was analysed using LC-MS/MS.The leachates were separately collected from the columns, and from each sample, a 10 mL subsample was transferred to a separatory funnel to which 90 mL distilled water was added.The reagents required for 100 mL were calculated, and 20 g sodium chloride and 10 mL dichloromethane were added followed by mechanical shaking for 5 min to cause layer separation, and the lower layer was collected in the round bottom flask.The extraction was repeated with dichloromethane and again extracted with n-hexane, and the upper layer separated was collected.The extracts were concentrated and were made up to 1 mL using methanol [22].

Instrumentation
The standards as well as samples were analysed on LC-MS/MS system (Thermo Scientific, Dionex Ultimate 3000 UHPLC equipped with TSQ Quantiva Mass Spectrometry).The Dionex Ultimate 3000 UHPLC system was used for chromatographic separation of fluopyram and its metabolite fluopyram benzamide, using Accucore aQ column (100 × 2.1, 2.6 micron particle size) placed in a column oven temperature maintained at 30℃.Elution was done using two elutents (solvent mixtures), viz., [A]: 0.10% formic acid + 5 mM ammonium formate in water; [B]: 0.10% formic acid + 5 mM ammonium formate in methanol.The flow rate was maintained at 0.30 mL min −1 and 10 min run time.Then, the effluent from LC was introduced into Thermo Scientific TSQ Quantiva mass spectrometer.The source parameters were as follows: ion source type is H-ESI (heated electrospray ionisation); sheath gas, 60.00 (arbitrary units); aux gas, 5.00 (arbitrary units); and sweep gas, 1.00 (arbitrary units), with ion transfer tube temperature of 320℃ and ion spray voltage source of 3500 V (positive ion).The vaporisation temperature is 450℃.The residues were quantified in MS/MS system.The polarity of fluopyram and fluopyram benzamide was positive, and retention time (in minute) was 6.28 and 6.15, respectively.Other compound-dependent parameters used are shown in Table 2.

Statistical analysis
The mobility of fluopyram and fluopyram benzamide as affected by different factors, viz., soil, depth of soil column and levels of water, was statistically analysed using threefactorial experiments using statistical software GRAPES (General R-shiny based Analysis Platform Empowered by Statistics) developed by Kerala Agricultural University.

Recovery experiments
Results revealed that the mean recovery in soils ranged from 78.31% to 93.79% and 78.87% to 97.73%, respectively, for laterite and red loam soils.Corresponding values for distilled water ranged from 83.82% to 118.57%.The relative standard deviation (RSD) values obtained ranged from 0.25 to 7.09 for soils and 0.67 to 5.35 for distilled water (Table 3).Similarly, the recovery of fluopyram benzamide in laterite soils, red loam soils and water was 79.31-92.46%,78.21-94.37%and 78.87-98.04%,respectively.The RSD remained in the range of 0.71-2.02for both soils and 1.19−2.42for distilled water (Table 3).The results obtained for recovery and precision were within the acceptance validation criteria of 70-120% with relative standard deviation below 20 as per the European Union guideline SANTE [23].The retention time for fluopyram and fluopyram benzamide was 6.28 and 6.15 min, respectively.

Leaching of fluopyram and fluopyram benzamide
The results of distribution of fluopyram and fluopyram benzamide in laterite and red loam soils and in leachate are given in Tables 4-8.Different levels of water application in the soil column experiment showed a significant effect in the leaching of fluopyram and fluopyram benzamide.The soil column eluted with 20 and 40 mL water showed lower leaching of fluopyram and fluopyram benzamide, while 160 mL showed the highest leaching with the residue distributed in all the 5 cm fractions of soil columns.This result is similar to the findings of Osman and Cemile [24] wherein increased rainfall can result in groundwater pollution by leaching of the pesticides.This result was also similar to the reports of Sarkar [25] that mobility of fluopyram was increased with increased amount of water (40-160 mL).Similar results were reported for other chemicals in laterite soil [26], clay loam (Entisol) and sandy loam (Inceptisol) [9] leached with varying amount of water.mong the soils used for the study, residue of fluopyram and fluopyram benzamide in laterite soil was found to be significantly higher than that in red loam soil.The results suggested that fluopyram and fluopyram benzamide had significantly higher leachability in laterite soil compared to red loam soil and the leachability increased with increased amount of water.The mean value of fluopyram and fluopyram benzamide residue obtained in the laterite soil were significantly higher than those in red loam soil which can be presumably predominance due to the of macropores and higher content of organic matter in the red loam soil than in the laterite soil.Soil organic matter content and chemical composition are the major factors influencing pesticide adsorption and leaching [27].Comparison of fluopyram and fluopyram benzamide detected at different depths of the soil column showed significantly higher values in the top 0-5 cm followed by 5-10 cm and the lowest value was observed at 25-30 cm depth.The effect of soils and the water used for elution and the interaction effect of all the factors, viz., soil, water level and depth on fluopyram and fluopyram benzamide were also found to be significant.
With the addition of 20 mL water (lowest level), fluopyram migrated up to 25 cm depth in laterite soil with 76.84% confined to the top 0-10 cm layer soil, while in the case of red loam soil, fluopyram leached up to 20 cm with recovery of 74.85% in the upper 0-10 cm depth.The average residue of fluopyram detected in the laterite soil (22.20 µg) was found to be significantly higher than that of red loam soil (20.89 µg).Fluopyram benzamide leached to the bottom in both laterite and red loam soil column eluted with 20 mL water.A higher concentration of fluopyram benzamide was confined to the top 0-10 cm in laterite (73.48%) and red loam (64.24%) soils.However, no residue of fluopyram and fluopyram benzamide was detected in the leachate collected from both soils.When the  water used for elution was increased to 40 mL, fluopyram was leached up to 25 cm and 56.90% was recovered in the top 0-10 cm depth in laterite soil, and similarly, more than half of the applied fluopyram recovered (56.14%) at 0-10 cm depth in red loam soil with the residues leaching only up to 20 cm.High mobility was observed for fluopyram benzamide with 54.19% and 50.47% recovery in the top 0-10 cm depth of laterite and red loam soils, respectively.Fluopyram benzamide leached up to 30 cm depth, and 1.05 and 1.07 µg of fluopyram benzamide were detected in the leachate of laterite and red loam soils, respectively.When the water used for elution was 80 mL, more downward movement was detected for both fluopyram and fluopyram benzamide, and they leached up to the bottom of the column (30 cm) in both soils.The quantity of fluopyram in the top layer (0-5 cm) was reduced to 53.12 µg (35.41%) in laterite soil, which was on par with red loam soil (53.94 µg).On elution with 80 mL water, fluopyram was detected in the leachate collected from the laterite soil column (0.08 µg) though it was only 0.05% of the applied pesticide, and it was not detected in the leachate obtained from red loam soil.In laterite soil, fluopyram benzamide was detected in all the 5 cm fractions with 54.02% confined to the top 0-10 cm layer and 27.11% and 10.94% in the middle (10-20 cm) and bottom (20-30 cm), respectively, and it was also detected in the leachate (3.14 µg).Similarly, in red loam soil column eluted with 80 mL water, mobility of fluopyram benzamide increased and only 46.52% was detected in the top 0-10 cm layer, and the residue detected in the leachate was 2.79 µg.The mobility of fluopyram and fluopyram benzamide was distributed more to the lower layer with the addition of 160 mL water in laterite and red loam soils.Only, 42.12% of the applied chemical was detected in the top 0-10 cm layer and 42.17% in the 10-20 cm layer in laterite soil.Likewise, red loam soil also showed a recovery of 42.97% in the top 0-10 cm layer and 38.48% in the middle 10-20 cm.Fluopyram was detected in the leachate collected from laterite (1.56 µg) and red loam (0.78 µg) soils.Mobility of fluopyram benzamide was also increased significantly with the addition of more volume of water (160 mL) with 39.85%, 37.64% and 16.03% in 0-10 cm, 10-20 cm and 20-30 cm depth, respectively, in laterite soil and 33.43%, 39.05% and 19.61% in the 0-10 cm, 10-20 cm and 20-30 cm depth, respectively, in red loam soil.Fluopyram benzamide entered the leachate, and 5.64 µg of residue was detected in laterite soil, which was higher than that in red loam soil (3.27 µg).
Most of the applied chemicals in the soil were found to be distributed in the top 0-10 cm of soil column, a small amount was found at 10-20 cm depth and only a little amount was detected at soil depths of 20-30 cm with higher levels of water application.Similar results were shown by Zhou et al. [28].Faske and Brown [29] also found that nematode-toxic levels of soil-applied fluopyram were not detected past 5 cm depth in sandy loam soil and not more than 10 cm depth in sandy soil.Mobility of a substance in soil is assessed by Kd and Koc.Koc is the soil organic carbon water partitioning coefficient, and Kd is the distribution coefficient between the two phases [30,31].A higher value indicates that the substance is strongly adsorbed onto soil and organic matter and does not move through the soil and is highly mobile in soil if the value is lower [32].Koc is a very important input parameter for estimating environmental distribution and environmental exposure level of a chemical substance.It can be inferred from our observations that fluopyram possesses high water solubility (16 mg L −1 ) due to which it moved down with percolating water and at the same time possesses a high adsorption to soil with soil organic matter adsorption coefficient (K oc ) of 266-460 in soil [15,25], suggesting moderate mobility for fluopyram in soil.Greater amount of pesticide was adsorbed in the upper layer of soil when applied with 20 and 40 mL of water.When eluted with a higher amount of water (80 or 160 mL), it moved beyond 30 cm depth.This result was also similar to the reports of Sarkar [25] that mobility of fluopyram was increased with increased amount of water (40-160 mL), and higher leaching was found in Ultisol and minimum in Entisols, and most of the residue was confined to the top 0-10 cm depth.
The physico-chemical properties of the pesticides and different soil properties, viz., texture, clay content, OM and permeability, play a critical role in the leaching process [33].Pesticides' leaching potential is generally favoured by their high water solubility, long persistence and low adsorption in soil [4].According to the GUS score index, fluopyram is a member of a high mobility group compound [34].Fluopyram benzamide is more mobile than fluopyram, but it is considered relatively safe to the environment [15,20,35].The result of our study is similar to the findings of Zhou et al. [28] that fluopyram exhibits high adsorption and low leachability in different banana planting soils.In their study, alluvial soil has the highest adsorption capacity and lowest leachability for fluopyram, whereas sandy soil had the highest leachability.Fluopyram was not detected in the leaching water of the three soil columns.Most of the residues were found in the top 0-10 cm of the soil column, low amount was found at 10-20 cm depth and only a little found at soil depths of 20-30 cm.

Conclusion
The present findings conclude that the leaching of fluopyram in soil columns was higher in the case of laterite soil as compared to red loam soil under varying amount of water levels (rainfall) in Kerala condition.Based on this analysis, it can be stated that fluopyram may leach down faster in laterite soil than in red loam soil, and only a nominal amount of fluopyram and fluopyram benzamide reaches the water table on elution with higher water levels.The leaching of fluopyram increases with the increased amount of water.Fluopyram benzamide was detected in the leachate collected from the columns applied with 40, 80 and 160 mL water in both laterite and red loam soils.Fluopyram content was detected only in the leachate collected from the column eluted with 160 mL in red loam soil and with 80 and 160 mL in laterite soil.This indicates that there exists a possibility of groundwater pollution with the application of higher concentration of fluopyram in the high rainfall regions.Application of fluopyram on crops in laterite soil may cause a higher risk of groundwater contamination.
The higher amount of organic carbon in the case of red loam soil may account for reducing downward mobility of fluopyram in the soil column.The present findings may be used for modelling the environmental fate of fluopyram in laterite and red loam soil conditions.Along with this, varying amount of water application as equivalent to rainfall can be used to assess the leaching as an attempt to prevent the groundwater pollution.However, a comprehensive analysis under field conditions in an undisturbed column is recommended to gain better insight into fluopyram leaching in the environment and the impact of rainfall on the mobility behaviour of the pesticide through the soil profile.

Table 1 .
Physical and chemical properties of the soil.

Table 2 .
Mass parameters for fluopyram and its metabolite.

Table 3 .
Recovery of fluopyram and fluopyram benzamide in laterite soil, red loam soil and water at different fortification levels.

Table 4 .
Effect of varying amount of water, soil and depth of soil column on leaching of fluopyram (µg).
CD: critical difference; the values are significant at p < 0.05 level of significance; SEm (±): standard error of the mean.

Table 6 .
Interaction effect of soil and depth of soil column on leaching of fluopyram (µg).
CD: critical difference; the values are significant at p < 0.05 level of significance; SEm (±): standard error of the mean.

Table 5 .
Interaction effect of varying amount of water and depth of soil column on leaching of fluopyram (µg).

Table 7 .
Interaction effect of varying amount of water, soil and depth of soil column on leaching of fluopyram (µg).

Table 8 .
Residues of fluopyram (µg) and fluopyram benzamide (µg) in the leachate obtained from the soil column with varying amount of water.