Environmental fate of glyphosate in citrus production soils of Florida 2

Email: rkanissery@ufl.edu 14 Phone: (239)658-3455 15 Fax: (239)658-3403 16 17 ABSTRACT: Chemical weed control using herbicide glyphosate to manage emerged weeds is 18 an important production practice in Florida citrus. Despite the extensive use of glyphosate in 19 citrus orchards, very limited information is available on its environmental fate and behavior in 20 Florida soils that are predominantly sandy in nature. Hence, the study's objective was to 21 understand the adsorption-desorption, dissipation dynamics, and vertical movement or leaching 22 of glyphosate in sandy soils in citrus orchards. Laboratory, field, and greenhouse experiments 23 were conducted at Southwest Florida Research and Education Center in Immokalee, Florida. The 24 adsorption-desorption behavior of glyphosate in the soils from three major citrus production 25 areas in Florida was studied utilizing a batch equilibrium method. The dissipation of glyphosate 26 was tracked in the field following its application at the rate of 4.20 kg ae ha. Soil leaching 27 columns in greenhouse conditions were used to study the vertical movement of glyphosate. The 28 results suggest that glyphosate has a relatively lower range of adsorption or binding (Kads = 14.28 29 to 30.88) in the tested soil types. The field dissipation half-life (DT50) of glyphosate from surface 30 soil was found to be 26 days. Glyphosate moved vertically or leached into the soil profile, up to 31 40 cm in the soil column, when analyzed 40 days after herbicide application. The primary 32 degradation product of glyphosate, i.e., aminomethyl phosphonic acid (AMPA), was also 33 detected up to the depth of 30 cm below the soil surface, indicating the presence of microbial 34 metabolism of glyphosate in the soil. 35 36


INTRODUCTION 41
Glyphosate is considered a relatively environmentally safe herbicide because it is quickly 42 inactivated in the soil by adsorption and degradation (Quinn et al., 1988). However, prior reports 43 have indicated that improper application practices and excessive use of this herbicide may 44 potentially contribute to its non-target movement and consequent effects in aquatic and terrestrial 45 environments (Borggaard & Gimsing, 2008;Hanke et al., 2010). Besides the direct sprays, 46 glyphosate's exposure routes in the environment may also include decaying plant residues and 47 exudation from roots of sprayed plants (Neumann et al., 2006;Mamy et al., 2016). Its non-48 judicious use in crop production has raised environmental and crop safety implications 49 worldwide (Kanissery, 2019). The most significant among these is its persistence and movement 50 in the soil and effects on non-target components of the crop ecosystem. 51 The major pathway for glyphosate's dissipation from the soil is microbial-mediated 52 transformation or biodegradation (Sprankle et al., 1975;Torstensson, 1985;Gimsing et al., 53 2004). Glyphosate may also be transported to groundwater, surface water, and water-sediment by 54 processes like surface runoff, erosion, drift, and leaching (Lupi et  plays an important role in predicting the availability; persistence and movement of the herbicide 61 from the top layer (15 cm depth) from each location. Following the collection, a composite 85 sample for each location was prepared by mixing all the subsamples and was stored at -20 o C 86 until the experiment. The soil samples were air-dried, sieved through a 2-mm mesh before use in 87 experiments. The relevant physico-chemical properties of the soils are provided in Table 1  (1000 mg L -1 ) was procured from Restek (Bellefonte, PA, USA). Five different concentrations 99 (5, 10, 25, 50, and 100 mg L -1 ) of glyphosate were prepared in 0.1 molar potassium chloride 100 (KCl) solution in water (Gimsing & Borggaard, 2001 The HPLC-MS conditions for analysis of glyphosate in an aqueous solution are provided as 117 supplemental information (Table S1). The limit of detection for glyphosate in the analysis was 118 each concentration and soil type were also included in the study for calibration and background 120 correction process. The amount of herbicide adsorbed to the soil was determined by calculating 121 the difference between herbicide concentration in the aqueous soil solution at equilibrium and 122 initial concentration in the shaking solution.  The method detection limit for glyphosate was 50 µg kg -1 soil. 164 Column preparation. Soil leaching columns (130 cm in length, 10 cm in diameter) made 168 of polyvinyl chloride (PVC) pipes were set up in the greenhouse at SWFREC. The columns were 169 filled with pre-collected soil from the incremental depths (up to 120 cm) from a site (without 170 prior glyphosate application history) adjacent to a citrus orchard in Immokalee to mimic the soil 171 depth profile of the orchard (Fig. 1a). The column was fitted with a PVC cap at the bottom, 172 equipped with a nylon screen and Whatman no. 4 filter paper to collect the leachate, if any. The 173 columns were secured upright in a specially prepared wooden stand (Fig. 1b,c), and each column 174 was watered to field capacity and allowed to drain for 24 hr.

177
Experimental design and treatments. The experiment was set up in a completely 178 randomized design (CRD) with three replications. The treatments consisted of glyphosate 179 application (4.20 kg ae ha -1 ) and untreated control (water spray). Control was included for 180 background correction purposes. Glyphosate (Roundup Custom TM ) was mixed thoroughly in 181 deionized water (carrier volume = 280 L ha -1 ) to prepare the herbicide solutions and applied with 182 a handheld CO2 backpack sprayer to the soil surface in the soil column. The frequency and 183 volume of water being added to a citrus orchard per unit area through irrigation were calculated 184 and applied to the column regularly (two times a week) to simulate field irrigation. The soil was removed, and the columns were split longitudinally by removing the duct tape from one 188 side. The soil in the columns was collected in an interval of 10 cm depths for the column's entire 189 length. The collected soil samples were analyzed for parent herbicide, glyphosate, and its 190 primary metabolite aminomethylphosphonic acid (AMPA) content using HPLC-MS in a 191 commercial laboratory (Waters Agricultural Laboratories, Camilla, GA). The method detection 192 limit for glyphosate and AMPA were 50 and 120 µg kg -1 soil, respectively. 193

Data Analysis 194
The adsorption and desorption isotherms were fitted using the transformed Freundlich 195 isotherm equation: log Cs = log Kf + 1/n log Ce, where 'Cs' is the concentration of glyphosate 196 adsorbed in the soil (mg kg -1 ), Kf is the equilibrium constant of Freundlich reflecting the binding 197 affinity of the soil for the herbicide, 'Ce' is the concentration of glyphosate in the solution (mg L -198 1 ) at equilibrium, and 1/n is the degree of linearity of the isotherm. The K (intercept) and 1/n 199 (slope) were calculated using regression analysis in the adsorption and desorption isotherms. for the analysis of variance (ANOVA), and mean separation was achieved using Tukey's HSD 208 test at p ≤ 0.05. 209

RESULTS AND DISCUSSION 210
Adsorption-desorption study 211 For the range of herbicide concentrations (5 to 100 mg L -1 ) and soils, adsorption data 212 from the current experiment were well-fitted into the Freundlich isotherm model (R 2 > 0.91). 213 herbicide. The soil from Lake Alfred location showed a significantly higher (p ≤ 0.05) adsorption 219 coefficient than the soils from Immokalee and Fort Pierce. The adsorption coefficients for 220 Immokalee and Fort Pierce soil were found to be similar (Table 2).   The results obtained from the adsorption-desorption study are in the lower range of Kads 271 and 1/n values obtained for glyphosate from different soil types and crop production systems. 272 There is a large variation in the literature values, with Kads ranging from 0.6 to 5.0 x 10 5 and 1/n 273 ranging from 0.26 to 1.26 (Vereecken, 2005). This variability in glyphosate adsorption is of soils tend to affect glyphosate adsorption positively (Vereecken, 2005). The citrus growing 278 soils in Florida are predominantly sandy (>90% sand) and low in organic matter (Obreza & 279 Also, it has to be noted that adsorption was comparatively higher in soil from Lake 282 Alfred compared to the Immokalee and Fort Pierce soils. Lake Alfred soil has more silt and clay, 283 and phosphorus (P) than the other two soils tested in this study (Table 1) higher amounts of phosphorus observed in the Lake Alfred soil (Table 1)

could be an indication 293
of better availability of adsorption sites in that soil, owing to the presence of coated sands, and 294 could be a probable reason behind the higher adsorption of glyphosate in this particular soil. 295 The value of slope (1/n) < 0.5 in the adsorption model (Table 2) (Table 3). Higher desorption or release of glyphosate from Immokalee and Fort Pierce 304 soils suggests that the herbicide's mobility for leaching losses or its availability for 305 biodegradation and plant uptake in these soils will be higher. The relatively lower desorption in 306 the Lake Alfred soil suggests that glyphosate will be less mobile and more bound to this soil than 307 the other two citrus production soils tested. Generally, glyphosate is non-bioavailable and 308 inactive when bound or adsorbed to soils (Sorensen et al., 2006). 309

Field dissipation study 310
The dissipation parameters of glyphosate obtained from the field study conducted in 311 Immokalee, FL, are presented in Table 4. The dissipation of glyphosate from the surface soil (top 312 15 cm) over time (Fig. 3) adequately fitted into a first-order kinetic model with an R 2 value of 313 0.88. The first-order rate constant (k) value was 0.026 day -1 . The dissipation half-life (DT50 : 314 time for dissipating 50% of initially applied herbicide from the soil) of glyphosate from the 315 surface soil was 25.9 days. The time for dissipating 90% of initial herbicide (DT90) was 86 days. 316 The cumulative amount of precipitation received in the experimental site during the dissipation 317 study duration is shown in the supplemental materials (Fig. S1).   The relatively lower adsorption affinity of glyphosate to sandy soils in citrus orchards in Florida 362 observed from the current study could be potentially implied as a reason behind the quick 363 dissipation of glyphosate, owing to its enhanced mobility from the soil surface. 364 Soil column leaching study 365 glyphosate in the soil columns (recovery from various depths accounts for 81% of the initially 381 applied amount) in the current study could be attributed to the herbicide's microbial 382 transformation. The primary metabolite AMPA was detected at various soil depths in the 383 columns (Fig. 4). Most citrus-producing soils in Florida, including the Immokalee soil used in 384 this column study, have low soil organic matter (SOM) ( Table 1). As SOM is closely linked to 385 soil microorganisms, the potential for microbial transformation of herbicides is expected to be 386 relatively low in these soils. However, the detection of AMPA in our soil columns, the most 387 common product of glyphosate's microbial metabolism (Dick and Quinn 1995), indicates the 388 presence of microbial transformation as a contributing mechanism for glyphosate dissipation and 389 loss from the citrus orchards. 390 Only 35% of the applied glyphosate stayed in the top 10 cm in the column, followed by 391 relatively lower concentrations in the subsequent soil depths. The vertical movement of 392 glyphosate through the soil profile could be associated with its hydrophilicity and high-water 393 solubility. The soil type utilized for this column study (Immokalee soil) is notably very sandy 394 (>90% sand) with low clay content characterized by lower herbicide retention. Therefore, this 395 may have promoted glyphosate leaching from the surface soils, especially when coupled with 396 simulated citrus tree irrigation in the soil columns. On the other hand, the soils from this location 397 have poor drainage properties due to a subsurface organic hardpan (Obreza and Collins 2000)  Glyphosate was found to have low adsorption or binding affinity to the soils from all three citrus-405 producing locations in Florida. Among the various production areas, soil from Lake Alfred in 406 the central area had higher adsorption for glyphosate than the other two areas, Immokalee in the 407 southwest and Fort Pierce in the southeast. Field dissipation of glyphosate from surface soils in 408 citrus orchard was relatively fast, with a calculated DT50 of ~26 days. The herbicide moved 409 vertically from the surface soils up to 40 cm in soil profile columns within 40 days after 410 herbicide application. The current study is among the first attempts to evaluate glyphosate's 411 interaction with F.L. sandy soils under citrus production to the best of our knowledge. The 412 information generated in this study could be utilized to better understand glyphosate's fate and 413 behavior in citrus and other similar production systems in sandy soils. Additionally, these 414 observations may be used to improve glyphosate's crop-safe application while minimizing the 415 non-target effects and other environmental implications. 416

SUPPLEMENTAL MATERIAL 417
The supplemental material contains additional detail on HPLC-MS conditions utilized during the 418 analysis of glyphosate and information on the rainfall received during the field dissipation study 419 (includes Table S1 and Fig. S1) 420 421 422 Table S1. High-performance liquid chromatography-mass spectrometry (HPLC-MS) conditions 579 used for the analysis of glyphosate in aqueous solution.