Kinetics of glyphosate and aminomethylphosphonic acid sorption onto montmorillonite clays in soil and their translocation to genetically modified corn

Abstract

The co-occurrence of glyphosate (GLP) and aminomethylphosphonic acid (AMPA) in contaminated water, soil, sediment and plants is a cause for concern due to potential threats to the ecosystem and human health. A major route of exposure is through contact with contaminated soil and consumption of crops containing GLP and AMPA residues. However, clay-based sorption strategies for mixtures of GLP and AMPA in soil, plants and garden produce have been very limited. In this study, in vitro soil and in vivo genetically modified corn models were used to establish the proof of concept that the inclusion of clay sorbents in contaminated soils will reduce the bioavailability of GLP and AMPA in soils and their adverse effects on plant growth. Effects of chemical concentration (1–10 mg/kg), sorbent dose (0.5%−3% in soil and 0.5%−1% in plants) and duration (up to 28 days) on sorption kinetics were studied. The time course results showed a continuous GLP degradation to AMPA. The inclusion of calcium montmorillonite (CM) and acid processed montmorillonite (APM) clays at all doses significantly and consistently reduced the bioavailability of both chemicals from soils to plant roots and leaves in a dose- and time-dependent manner without detectable dissociation. Plants treated with 0.5% and 1% APM inclusion showed the highest growth rate (p ≤ 0.05) and lowest chemical bioavailability with up to 76% reduction in roots and 57% reduction in leaves. Results indicated that montmorillonite clays could be added as soil supplements to reduce hazardous mixtures of GLP and AMPA in soils and plants.

Introduction

Glyphosate (GLP), the active ingredient in Roundup^®, has become one of the most prevalent organophosphorus herbicides worldwide, with almost 1 million tons used annually (Maggi et al., 2020). It is the most applied pesticide in the US (40%) and worldwide (72%) (Battaglin et al., 2014; Bento et al., 2016; Singh et al., 2020). One factor contributing to the dominant and increasing use of GLP is the availability of genetically modified (GM), GLP-resistant crops, which make up more than 50% of global GLP applications (Mamy and Barriuso, 2005; Kells and Tharp, 2002). GLP is degraded by microorganisms in soil primarily through C–N cleavage (Ogbourne and Bai, 2016; Sun et al., 2019a) and metabolized in plant tissue through glyphosate oxidase activity (Gomes et al., 2016) to form its major metabolite aminomethylphosphonic acid (AMPA), which is also classified as a toxicologically significant compound (Harmon and Chamkasem, 2016).

Soil plays a key role in the environmental fate of herbicides since large amounts of applied herbicides reach the soil from direct application and/or after foliage wash off (Mamy and Barriuso, 2005). In soil, factors such as organic matter, pH, phosphate content and the presence of iron and aluminum oxides may potentially influence glyphosate sorption (Hall et al., 2018). A study found that GLP and/or AMPA were present in 45% of the agricultural topsoils collected, with a maximum concentration of 2 mg/kg (Silva et al., 2018). Importantly, GLP and AMPA are usually detected as mixtures in the environment, and only 2.3% of GLP has been detected without AMPA in water and sediment samples in the US (Battaglin et al., 2014; Gomes et al., 2014). Furthermore, the bioavailable fractions of GLP and AMPA in soil, with little or no clay in their composition, can translocate to edible plants such as vegetable crops, resulting in potential exposures for humans and animals through food consumption. Both chemicals have been detected in GM crops as well as non-target plants, and adverse effects have been observed in plants (Ding et al., 2011; Gomes et al., 2016; Reddy et al., 2004; Tong et al., 2017). Studies suggested that the acute toxicities of both compounds were relatively low, but their chronic health effects on humans and animals have been recently discovered (Ogbourne and Bai, 2016; Mesnage et al., 2015). The risk of occupational exposure to these chemicals is increased in agricultural workers, and GLP has been detected in blood and urine samples from those who are highly exposed (Jauhiainen et al., 1991). Therefore, remediation strategies that are field-practical and economically feasible are needed to: (1) reduce the bioavailability of mixtures of GLP and AMPA from soil through skin contact, inhalation of soil particles, and hand-to-mouth ingestion, which is estimated at 50–200 mg soil/day (Petruzzelli et al., 2020); (2) reduce the consumption of mixtures of GLP and AMPA residues in contaminated food products, and (3) prevent potential phytotoxicity and growth stunting from chemical uptake in plants.

Compared with advanced remediation processes, adsorption technology has the advantages of low treatment cost, easy operation, and excellent results (Sun et al., 2019b; Yu et al., 2021). Adsorption studies have mostly focused on carbonaceous materials, with very limited studies on the development of clay-based sorbents (Hagner et al., 2015; Hall et al., 2018; Junqueira et al., 2020; Yu et al., 2021). Also, kinetic and isothermal evidence is lacking for GLP remediation in soil (Yasid et al., 2019). Although carbons have shown promising results for binding GLP, factors such as carbon aging, interference with GLP degradation rates, structural diversity, and performance differences may limit their utilization for GLP and AMPA remediation purposes (Hall et al., 2018; Zhelezova et al., 2017). Whereas, montmorillonite clay has been shown to bind GLP mostly at interlayer surfaces with high affinity and capacity (Flores et al., 2018; Hearon et al., 2021; Orr et al., 2021; Wang et al., 2020a, 2019a, 2019b). Additionally, our previous in vitro work using adsorption and desorption isotherms, thermodynamics, computational modeling, and sensitive ecotox models (Hydra vulgaris, Caenorhabditis elegans, and Lemna minor) (Wang et al., 2021b) demonstrated high affinity binding of montmorillonite clays for GLP and AMPA. The molecular mechanisms involved electrostatic interactions and hydrogen bonding onto active surfaces within interlamellar spaces of calcium montmorillonite clays (Wang et al., 2022). Based on this preliminary work, the objective of this study was to establish the proof of concept in vivo and to validate that our clay-based sorbents can tightly bind mixtures of GLP and AMPA, reducing their bioavailability from soil, their uptake in corn, and their adverse effect on plant growth.

We expect montmorillonite clays including calcium montmorillonite (CM) and acid-processed montmorillonite (APM) (Hearon et al., 2020; Wang et al., 2021a, 2019c; Wang and Phillips, 2019, 2020) will bind GLP and AMPA in soil and reduce their translocation to GM corn plants. Effects of chemical concentration, sorbent dose, and treatment duration on sorption kinetics and chemical bioavailability from soil were investigated. Montmorillonites are naturally present in soils and their effects on blank soils and plant growth were also tested. Only GLP was spiked in soils, but GLP and AMPA mixtures were detected and quantified in soils and plants.

1. Materials and methods

1.1. Characterization of clay-based materials

Calcium montmorillonite (CM) clay was obtained from BASF (Ludwigshafen, Germany) with a generic formula of (Na,Ca)_0.3(Al,Mg)₂Si₄O₁₀(OH)₂·nH₂O and a cation exchange capacity equal to 89.2 cmol/kg (Grant and Phillips, 1998; Phillips et al., 2019). Acid processed montmorillonite (APM) clay was synthesized based on a previously described method (Phillips and Wang, 2018). The molecular structure of APM has been previously reported as a mixture of delaminated parent clay aggregates and amorphous and cross-linked silica (Wang et al., 2019c). Clays were sieved at 100 mesh to achieve a uniform particle size equal to, or less than, 150 μm. Quality control of montmorillonite clays has been routinely performed. Composition and particle size were consistent from lot to lot, and representative samples were tested for environmental contaminants including polychlorinated dibenzo-p-dioxins/furans (PCDDs/PCDFs) and heavy metals (e.g., As, Cd, Hg, and Pb) following standard USEPA protocols (e.g., Methods 6010B and 7471A) to ensure compliance with federal and international regulations (Afriyie-Gyawu et al., 2008; Wang et al., 2005).

Important physicochemical properties of these clay-based sorbents were characterized in this study. The density was calculated based on the difference between pycnometer weight (with and without 2 mL of the sample) divided by the sample volume (Cefali et al., 2019). The surface hydrophobicity of sorbents was assessed by measuring the mass ratio of their absorption of n-heptane/water vapor for 24 hr at ambient conditions (Dos Reis et al., 2016). Their coefficient of linear expansibility in water (COLE) was measured by the volume of sorbents following thorough equilibrium hydration and swelling for 24 hr in water versus the initial volume (2 mL) (Wang, 2017). A higher ratio indicated greater hydration and expansion of the sample. The X-ray fractionation (XRF) spectroscopy of sorbents has been previously reported (Wang et al., 2020b). The metal analysis of aluminum, calcium, sodium, and lead in clays was conducted by the ALS-Environmental-Kelso Laboratory (Kelso, WA).

Additionally, the fracture surfaces of sorbents were sputter-coated with a thin layer (5 nm) of Pt prior to imaging with a field emission scanning electron microscope (SEM) (JSM-7500F, JEOL) operating at 5 kV. The crystal structures of the sorbents were investigated using XRD with a Bruker D8 Endeavor with Cu radiation (λ = 1.5406 Å) operating at 40 kV and 25 mA. Data collection was automated with a COMMANDER program by employing a DQL file and analyzed by the program EVA. Fourier transform-infrared (FT-IR) spectra were recorded on an IR Prestige 21 system, equipped with a diamond attenuated total reflection (ATR) lens (Shimadzu Corp., Japan), and analyzed using IRsolution v.1.40 software.

1.2. Chemical analysis

Analytical standards of GLP and AMPA were purchased from Sigma-Aldrich (St. Louis, MO) with purity > 99.9%. GLP and AMPA were analyzed using a Waters Acquity^® Ultra Performance Liquid Chromatography (UPLC)-Tandem Mass Spectrometer (MS)/MS. A Hypersil BDS C18 column (50 × 2.1 mm, 5 μm) was used for separation due to its increased retention for polar chemicals via dipole-dipole interactions. Nitrogen gas was used as the collision gas and curtain gas, and argon gas was used as the nebulizer gas and heater gas (Pereira, 2006). Separation was obtained using a mobile phase of water with 0.1% formic acid (eluate A) and acetonitrile with 0.1% formic acid (eluate B) (5%–100% of eluate B in 10 min) at 0.3 mL/min. Injection volume was 40 μL and a negative electrospray ionization mode was set at 4.5 kV spray voltage. The source temperature was held at 225°C for GLP and 450°C for AMPA. The MS was operated under multiple reaction monitoring (MRM) mode and the monitored precursor and product ions were 168 and 63/81 for GLP and 110 and 63/79 for AMPA. The unit mass resolution was used for the ion mass analyzer. Empower analyst software was used to control the system and acquire the data. To ensure consistency of the detection methods and linearity of peak concentrations for GLP and AMPA, standard solutions of the analytes were prepared in distilled water at concentrations between 10 and 0.05 μg/g to measure standard curves for each set of samples. The standard curves for GLP and AMPA were linear (r² > 0.99) with a limit of detection for both analytes at 50 μg/kg.

1.3. Soil studies

Garden soil (Scotts Miracle-Gro Co, Marysville, OH) and compost (Foxfarm Soil & Fertilizer Co, Humboldt, CA) at equal mixtures were used in the soil and plant studies. The composition of garden soil including compost, processed forest products, sphagnum peat moss, a wetting agent, and fertilizer containing 0.09% total nitrogen, 0.05% available phosphate, and 0.07% soluble potash. The composition of the compost consisted of aged forest products, sphagnum peat moss, perlite, sandy loam, and fertilizer containing 0.30% total nitrogen, 0.45% available phosphate, and 0.05% soluble potash, and 1.00% calcium derived from fish emulsion, crab meal, shrimp meal, earthworm castings, bat guano, kelp meal and oyster shell (Hearon et al., 2022). No presence of bacteria was reported from the manufacturers. The physicochemical properties of soils were also characterized as mentioned above.

Soil was air-dried and sieved through a 1 mm mesh before use. Soil samples (1 g) in a disposable culture tube were first spiked with 2 mL of water solution containing varying GLP concentrations at 1, 4, 8, and 10 μg/g and thoroughly mixed to ensure even distribution. Only 1 μg/g GLP was used in the rest of the soil studies as an environmentally relevant concentration (Gunarathna et al., 2018; Helander et al., 2019). Spiked soils were left uncapped in a fume hood for 7 days allowing the metabolism of GLP and evaporation of water (Giesy et al., 2000). Individual sorbents including CM and APM were then added at 0.5%, 1%, 2%, and 3% w/w to soil samples based on our previous in vitro adsorption studies in water (Wang et al., 2022). Control groups included blank soil, soil spiked with GLP, and soil with sorbent inclusions. Soil samples, with and without GLP and sorbents, were hydrated by adding 4 mL of distilled water and slowly agitated at 200 r/min for 1 day. In the time course study, soils containing 1 μg/g GLP and 0.5% sorbents (minimum effective dose) were agitated for 1, 5, 10, 14, and 21 days to measure the adsorption kinetics and potential dissociation.

After the reaction, a soil extraction method (Chamkasem and Harmon, 2016; Hearon et al., 2021) with modification was used to separate both GLP and AMPA from soil samples. Briefly, 4 mL of 50 mmol/L acetic acid and 10 mmol/L Na₂-EDTA were added to 1 g of soil samples before agitating at 1000 r/min for 1 hr. Samples were centrifuged at 2000 ×g for 20 min and the supernatants were passed through Strata C18-E (55 μm, 70A) columns (Phenomenex, Torrance, CA) that had been preconditioned with 2 mL methanol and 2 mL water. The SPE columns with loaded supernatants were eluted with 5% methanol: 95% water (Zaller et al., 2021). This extraction was repeated 3 times and the collected eluates were resuspended in 0.1% formic acid for analysis using LC-MS/MS as described above. Peak areas of GLP and AMPA from samples with sorbent inclusions were compared to the chemical controls with the same concentration and duration to calculate percent reduction in soil. Mass balance of the chemical mixtures was used to calculate the recovery percentage in the soil.

1.4. Plant uptake studies

Hybrid Seed Corn (217–76STX) was obtained from Channel Bio (St. Louis, MO). The same soil and compost mixture was used in the plant study. The soil matrix was first spiked with 1, 2, 4, 8, and 10 μg/g GLP to determine the recovery percentage and efficacy of chemical translocation to plants. For sorbent treatment, sorbents at 0.5% and 1% (W/W) were individually included in soils exposed to 1 μg/g GLP aqueous solution. After 7 days of pre-treatment with GLP and sorbents, 10 g of soil was transferred to each planter containing 1 corn seed and hydrated with 3 mL of water per day for 7 days. In each control and test group, at least 3 planters were included to ensure an adequate number of viable plants. On day 7, planters were moved to a hydroponic system with nutrient solutions containing the same GLP and sorbent compositions as in the soil (Hearon et al., 2021). GM corn roots were allowed to grow and extend into the nutrient solution. This hydroponic system was used to simulate flood scenarios during which roots can be soaked and exposed to mobilized water-soluble pesticides in flood waters. After a total exposure period in soil and the hydroponic system for 9, 14, 21, and 28 days, roots and leaves were separated, washed thoroughly with deionized water to remove remaining soils, and air-dried before weights of roots and leaves and lengths of leaves were measured. Weighted root and leaf samples were then homogenized by a 150 Homogenizer and 7 mm Generator Probe (Fisher Scientific, Waltham, MA) to a powder-like texture in 5 mL of 50 mmol/L acetic acid with 10 mmol/L Na₂-EDTA (Hearon et al., 2021). This plant extraction solution was then sonicated for 30 min and centrifuged at 2000 ×g for 20 min. This process was repeated 3 times, and the supernatants were passed through Strata C18-E (55 μm, 70A) columns and analyzed on LC-MS/MS as described above to calculate concentration and uptake rate of GLP and AMPA in roots and stems.

1.5. Statistical analysis

A one-tailed t-test was used to determine statistical significance. Each experiment was conducted in triplicate in all groups for: (1) concentrations of GLP and AMPA in soils and plants, (2) root weight, and (3) leaf length and weight. The derived means and standard deviations were compared using a Tukey test for t-values to determine the p-value. Results were considered significant at p ≤ 0.05.

2. Results and discussion

2.1. Sorbent characterization

The bulk density and moisture percentage of montmorillonites were within the common range for bentonite clays (Table 1). The acid process resulted in an increase in surface acidity as shown by the pH, and an increase in surface area up to 1213 m²/g. It was higher than coconut shell activated carbons with an average area of 1100 m²/g and the parent montmorillonite clay with an average area of 850 m²/g. The process also produced permanent mesoporosity in the clay structure by leaching metal ions from the interlayers (Ca and Na) and clay framework (Al), which partially delaminated the clay. APM reduced the COLE ratio, possibly due to reduced metal ions that have hydration energy and attract water molecules.

Table 1 –

Important physicochemical properties of montmorillonite sorbents.

	Calcium montmorillonite	Acid processed montmorillonite
Appearance	Off-white to greyish-green powder	Off-white powder
pH	8.2	3
Particle size	5% +100 mesh	100% −100 mesh
	18% +200 mesh
	60% −325 mesh
Bulk density (loose)	640.74 kg/m 3	571.8 kg/m3
Moisture	9.0%	8.8%
Hydrophobicity	0.57	1.31
Surface area	823.4 m2/g	1213.4 m2/g
COLE	2.0	1.2
Metal analysis	Al 7730 mg/kg	Al 5760 mg/kg
	Ca 20700 mg/kg	Ca 3120 mg/kg
	Na 149 mg/kg	Na 40 mg/kg
	Pb 11.7 mg/kg	Pb 12.0 mg/kg

COLE: coefficient of linear expansibility in water.

As shown by SEM, parent CM showed the typical layer-lattice structures (Appendix A Fig. S1a and b). However, with acid treatment, a mixture of layer-lattice structures (Appendix A Fig. S1c) with increased pore volumes (Appendix A Fig. S1d) was observed, and this finding supports the increase in pore size distribution for most acid treated clays as reported in the scientific literature (Amari et al., 2018).

The XRD patterns of montmorillonites, before and after acid treatment, are displayed in Appendix A Fig. S2. Most of the aggregates showed broken edges and fractured surfaces, which might be due to grinding. Montmorillonite was indicated as the main compound by the characteristic XRD peaks (2θ = 5.9, 19.9, 27.5, 35.1°; or d = 14.5, 4.5, 3.2, 2.6 Å) (Motshekga et al., 2013). The data also showed the presence of trace levels of impurities by the small peaks indicative of quartz (2θ = 20.9° and 26.7°) and calcite (2θ = 29.5°) (Julinawati et al., 2019; Nirwan et al., 2020). The d₀₀₁ value of montmorillonite was 14.5 Å, indicating a calcium-rich montmorillonite as the major clay mineral (Alver et al., 2016). Acid treatment also modified the structure based on changes in XRD patterns. With acid treatment, the basal spacing (15.0 Å) was similar to the parent montmorillonite clay, suggesting that hydronium cations can intercalate into the interlayer spacing of montmorillonite by the mechanism of cation exchange, without the loss of layer structure (Bieseki et al., 2013). The significant reduction in the intensity and increase in the width of the d₀₀₁ peak indicated that the crystallinity of CM was decreased by acid activation, and that the process favored the production of an amorphous silica phase by decomposing montmorillonite crystalline structure (Angaji et al., 2013; Amrani, 2014). Reduction in intensity of most peaks while the increased intensity at 26.7° 2θ (d = 3.3 Å) showed partial degradation of montmorillonite structure with the formation of the amorphous silica phase (Sharma and Sarasan, 2017).

The FT-IR spectra and the assigned bands of parent CM and APM are shown in Appendix A Fig. S3 and Table S1. The characteristic band at 3620 and 918 cm⁻¹ for both CM and APM showed that the hydroxyl group was coordinated with octahedral cations (Al³⁺) and Al-Al-OH bending vibration in the montmorillonite clay. This indicated that the layered structure of the original clay remained upon acid treatment, aligning with the SEM and XRD results above. However, the FT-IR spectra also reflected the structural degradation of the clay components and formation of an amorphous silica phase. For example, leaching caused the reduced intensity of the structural −OH bands at 3620 cm⁻¹, −OH stretching vibrations at 3395 cm⁻¹ and bending vibrations at 1628 cm⁻¹ of water molecules upon the acid treatment, which was an indication of loss of physically absorbed water and partial destruction of the structure as previously reported (Alver et al., 2016; Temuujin et al., 2004). Also, Si-O group out-of-plane and inplane stretching vibrations were seen at 1065 and 995 cm⁻¹ respectively for CM, but the bands were reduced or shifted after acid treatment. The pair of bands at 548 and 424 cm⁻¹ for both CM and APM was due to Al-O-Si and Si-O-Si bending (Sharma and Sarasan, 2017). The molecular modeling of CM and APM was previously performed using Hyperchem 8.0 (Hypercube, Inc., Gainesville, FL) and CHARMM (Orr et al., 2020, 2021; Wang et al., 2019c).

2.2. Remediation in soils (in vitro)

The composition of matter in soils was mentioned in the method and important physicochemical properties were characterized and reported in Table 2. An equal ratio of garden soil and compost representing a complex base matrix in the environment was hydrated with 1, 4, 8 and 10 μg/g GLP for 7 days (Battaglin et al., 2014; Duke et al., 2012; Helander et al., 2019; Silva et al., 2018). The results show that the mass of extracted GLP from 1 g soil (Fig. 1a, orange bars) was lower than the mass of AMPA (Fig. 1b, blue bars) at all spiked concentrations. This was consistent with the literature that AMPA was found at higher concentrations in hydrated soil than GLP, possibly due to the high degradation rate of GLP, higher persistence of AMPA in the soil, and higher potential for GLP degradation in hydrologic settings (Battaglin et al., 2014; Bento et al., 2016; Okada et al., 2018). The mass of extracted GLP (r² = 0.95) and AMPA (r² = 0.93) in soil was linearly related to spiked GLP levels, suggesting the degradation of GLP was at a constant rate within 7 days. The recovery percentage of GLP was up to 12%, which was within the recovery range in soil from the literature (Druart et al., 2011), but lower than that in sandy loam, possibly due to negligible binding of GLP in sandy soils (without organic matter and a clay fraction) that resulted in higher extraction and recovery rates.

Table 2 –

Important physicochemical properties of soils.

	Garden soil	Compost
Appearance	Black grainy soil	Heterogenous loose powder
Particle size (max)	1 mm	1 mm
pH	7.29	5.39
Bulk density (loose)	1217.3 kg/m3	772.2 kg/m3
Moisture	0.9%	5.4%
Hydrophobicity	0.28	0.52
COLE	1.36	1.2

COLE: coefficient of linear expansibility in water.

Fig. 1 –

Mass of GLP (a) and AMPA (b) extracted from soil spiked with 1, 4, 8, and 10 μg/g GLP and inclusion of CM and APM at 0.5% for 1 day. The chemical uptake of GLP and AMPA was positively correlated with GLP contamination levels in soil, and montmorillonite clays consistently adsorbed and reduced both chemicals at all contamination levels.

Individual sorbents (CM and APM) were included at 0.5% in the soil to determine binding efficacy at varying GLP and AMPA concentrations. Treatment with 0.5% CM and APM similarly reduced the bioavailability of both chemicals from spiked soils by 11%–36% and 12%–28% for GLP, and 27%–52% and 26%–56% for AMPA, respectively. This result was consistent with previous studies using soil nematodes that both CM and APM can effectively adsorb chemicals and reduce their soil bioavailability (Wang et al., 2022). More specifically, the reduction percentages of AMPA on both clays were higher than GLP, which was supported by the higher binding capacity of AMPA versus GLP in the in vitro adsorption isotherm studies. The mechanisms of sorption were due to electrostatic interactions and hydrogen bonding mainly on the interlayers of the clay surfaces (Wang et al., 2022).

To investigate the binding kinetics and possible chemical dissociation, a time course study was conducted with soils spiked with 1 μg/g GLP for 7 days, and with the addition of 0.5% sorbent treatment for another 1, 5, 10, 14, and 21 days. Fig. 2 shows that the mass of extracted GLP was consistent for the first 14 days with a slight decrease on day 21, while the mass of extracted AMPA was consistent for the first 10 days and increased on days 14 and 21. Results suggested that GLP degradation to AMPA occurred fast within the first 8 days (7 days of pre-treatment and 1 day with clay treatment) and continued for a total of 28 days (7 days of pre-treatment and 21 days with clay treatment). The treatment with CM and APM consistently reduced GLP and AMPA for all time durations, with APM reducing more GLP than CM. Importantly, no detectable chemical dissociation was observed with both clay treatments; instead, more chemicals remained bound to sorbents with longer treatment duration (e.g. 21 days). This tight binding was supported by the previous thermodynamic results showing chemisorption of both chemicals to clay surfaces (Wang et al., 2022).

Fig. 2 –

Mass of GLP (a) and AMPA (b) extracted from the soil spiked with 1 μg/g GLP for 7 days and inclusion of CM and APM at 0.5% for 1, 5, 10, 14, and 21 days. The chemical reduction by montmorillonite clays occurred fast and was consistent throughout the treatment.

Previous soil remediation studies for GLP have used commercial sorbents containing carbons at high inclusion levels of up to 30% (Zhelezova et al., 2017). In this study, a dosimetry study with 0.5%, 1%, 2%, and 3% CM and APM was conducted to remediate 1 μg/g GLP in soil for 7 days. The results in Fig. 3 show that high sorbent inclusions resulted in a higher reduction of both GLP and AMPA. The highest inclusion level of CM and APM (i.e., 3%) reduced 41% and 50% GLP, and 86% and 73% AMPA, respectively. These results suggested that sorbent dose will depend on chemical concentrations at highly contaminated sites. The mining of montmorillonite clays and process of acid treatment have been previously established by industry and the cost was similar to commonly used activated carbon and biochar products (Hearon et al., 2021). The scaleup of APM is ongoing with our industrial partners to optimize cost efficiency. The reduction percentage of CM and APM was similar for AMPA, while APM reduced GLP more effectively ranging from 28% to 50%, compared to CM (17%–41%). This result is consistent with the above time course study, suggesting a high correlation using this soil model. Since AMPA-sorbent complexes were suggested to be stable in a soil environment and clays are natural components of many soil types, the inclusion of these materials as soil amendments at low levels should have negligible adverse environmental effects (Hearon et al., 2021).

Fig. 3 –

Reduction percentage of GLP (a) and AMPA (b) in the soil after treatment with CM and APM at 0.5%, 1%, 2% and 3% for 1 day. The clay reduction was dose-dependent and 3% inclusion reduced almost half of GLP in soil and 73%–86% of APMA in soil.

2.3. Remediation in plants (in vivo)

Genetically modified (GM) corn was used in the plant study due to the high rate of application of GLP (Arregui et al., 2004; Bento et al., 2016; Reddy et al., 2008). After a total of 14 days of exposure to GLP at 1, 2, 4, 8, 10 μg/g in soil and hydroponic solution, the dry weights of roots and leaves were measured in each group and showed no significant differences at all GLP exposure levels. This finding is possibly due to the high tolerance of GLP-resistant GM plants and accumulation during growth (Fig. 4).

Fig. 4 –

Dry weight of roots (a) and leaves (b) in GM corn plants after exposure to GLP at 1, 2, 4, 8, and 10 μg/g for 14 days. No statistical difference on growth was shown, which may be due to high tolerance of GM corn to GLP.

The chemical uptake results (mg/kg) were expressed as the mass of GLP and AMPA extracted from roots (Fig. 5a) and leaves (Fig. 5b). Both GLP and AMPA were detected in roots and leaves even at the lowest exposure level (1 μg/g GLP), with a linear relationship between exposure concentrations and the total mass of chemical uptake in roots (R² = 0.74) and leaves (R² = 0.72). Specifically, levels of AMPA in all groups were higher than GLP, which was consistent with findings in the soil studies and the literature (Battaglin et al., 2014; Hearon et al., 2021). This was because soils were pre-treated with GLP for 7 days before culturing plants for another 14 days, leading to more than half of GLP degraded. Additionally, higher chemical content was observed in roots than in leaves, which was supported by the literature that chemicals had higher bioavailability and uptake in roots than in vegetative parts (Kanissery et al., 2019; Tong et al., 2017). This result indicates that plant-available GLP could be continuously taken up by roots, metabolized and transported into the edible parts (USEPA, 1993). GLP uptake and translocation in GM corn during the 14-day cultural period ranged from 6.5%−40%, which was within the reported transfer rate in crops and weed species (Arregui et al., 2004). Our extraction results showed a linear association with varying GLP concentrations and high alignment with the literature on GLP uptake in plants.

Fig. 5 –

Mass of GLP and AMPA in roots (a) and leaves (b) in GM corn plants after exposure to GLP at 1, 2, 4, 8, and 10 μg/g for 14 days. Both chemicals were bioavailable to roots and leaves with the transfer rate at 6.5%−40% from soil.

A time course study was conducted to investigate the transfer of chemicals to different parts of plants, and sorbent binding with time. Plants were exposed to 1 μg/g GLP and 0.5% sorbents for 7 days in soil and transferred to a hydroponic system with the same treatment for another 2, 7, 14, and 21 days. Dry weight of roots and dry weight and length of leaves were measured after each time point and showed continued growth (Fig. 6). Specifically, the growth of roots was most significant in the first 9 days and the growth of leaves continued for 28 days. Importantly, the inclusion of 0.5% CM and APM showed similar results in these growth parameters with no adverse effect observed on the growth and appearance of the plants, supporting the relative safety of including montmorillonite clays at levels below 0.5% as soil supplements.

Fig. 6 –

Dry weight of roots (a), dry weight of leaves (b), and leaf length (c) in GM corn plants in a time course study after exposure to 1 μg/g GLP and 0.5% CM and APM for a total of 9, 14, 21, and 28 days. The inclusion of montmorillonite showed no interference in plant growth.

At the end of each exposure duration, plants were extracted and GLP and AMPA were measured in roots and leaves. Over the 28-day exposure period, the total mass of chemical uptake increased and accumulated in roots (Fig. 7a) and transferred to leaves (Fig. 7b). Moreover, GLP degraded to AMPA with time, as shown by the increased proportion of AMPA in roots. We expect that this chemical uptake and accumulation would continue if the plants were cultured for a longer period. Similar to the results in the above plant study with varying chemical concentrations, levels of AMPA were higher than GLP in both compartments for all durations, and chemical levels in roots were higher than in leaves, which have been shown in the literature (Hearon et al., 2021; Kanissery et al., 2019; Tong et al., 2017). With sorbent inclusion at 0.5% in the soil and hydroponic solution, the uptake of GLP decreased by up to 62% in roots and 82% in leaves (Fig. 7c). A similar reduction is shown for AMPA by up to 84% in roots and 86% in leaves (Fig. 7d). This reduction in chemical uptake occurred quickly within 9 days and was not time-dependent. Importantly, the inclusion of APM clay reduced both chemicals more effectively than CM with the same treatment duration. The overall results in the time course study showed: (1) a continuous uptake and accumulation of chemicals by the plants (roots and leaves), (2) the inclusion of CM and APM clays did not interfere with plant growth within 28 days, and (3) sorbents effectively and consistently decreased both chemical uptake in plant compartments for all treatment periods.

Fig. 7 –

Mass of GLP and AMPA in roots (a) and leaves (b) in GM corn plants after exposure to 1 μg/g GLP for 9, 14, 21, and 28 days, showing continuous accumulation of chemicals. Reduction of GLP (c) and AMPA (d) in roots and leaves after treatment with 0.5% CM and APM clays. Montmorillonite clays consistently reduced both chemicals in roots and leaves with up to 82% GLP and 86% AMPA reduction by APM.

Since APM showed higher reductions of GLP and AMPA, APM was included at both 0.5 and 1% and compared to 0.5% CM. Root and leaf dry weights and leaf lengths in Fig. 8 show no difference in plants growing in blank soil and sorbent control groups. Compared to the chemical control group, the inclusion of APM clay at 0.5 and 1% significantly increased leaf dry weight (p ≤ 0.05). This increase in growth can be attributed to the binding ability of APM clay, as supported by the reduction in the chemical mass in Fig. 9. Specifically, 1% APM inclusion delivered the highest reduction of both chemicals by 76.6% in roots and 57.4% in leaves, followed by the reduction of 61.7% in roots and 34% in leaves with 0.5% APM. The inclusion of CM also reduced the uptake of both chemicals, with a lower reduction percentage than the APM clay. This study suggested that sorbent inclusion at higher inclusion levels in soil and nutrient solution may deliver a higher reduction in chemical uptake. CM and especially APM clays can significantly reduce both GLP and AMPA uptake in roots and leaves, even at levels as low as 0.5% inclusion.

Fig. 8 –

Dry weight of roots (a), dry weight of leaves (b), and leaf length (c) in GM corn plants after exposure to 1 μg/g GLP and treatment with 0.5% CM and 0.5 and 1% APM for 14 days (*p ≤ 0.05, **p ≤ 0.01 compared to soil blank; ^#p ≤ 0.05 compared to chemical control). APM significantly increased leaf dry weight (b) compared to the GLP exposure group.

Fig. 9 –

Mass of GLP and AMPA in roots (a) and leaves (b) in GM corn plants after exposure to 1 μg/g GLP and inclusion of 0.5% CM and 0.5 and 1% APM for 14 days. The inclusion of 1% APM clay was the most effective and reduced both chemicals by 76.6% in roots and 57.4% in leaves.

3. Conclusions

In this study, we have characterized the ability of calcium montmorillonite and acid processed montmorillonite clays to sorb a mixture of GLP and AMPA in soils and plants. In vitro soil studies using varying chemical concentrations and doses of sorbents for different treatment durations showed that CM and APM clays significantly and consistently adsorbed both chemicals and reduced their bioavailability in the soil matrix in a dose- and time-dependent manner. In vivo plant studies using genetically modified corn exposed to varying concentrations of chemicals and doses of sorbents for different durations showed a continuous uptake and accumulation of both GLP and AMPA in the roots and leaves. CM and APM clay inclusion in the soil and nutrient solution increased plant growth and reduced the mass of GLP and AMPA detected in plants in a dose- and time-dependent manner. The results established the proof of concept for the potential application of montmorillonite clays (CM and APM) as soil supplements for remediating mixtures of GLP and AMPA in soil, reducing chemical uptake from soil by corn, and protecting plant growth from contamination toxicities. Further studies on the long-term implementation of these sorbents for in situ soil remediation in community gardens, lawns, and recreational areas, with comparison to commercial products (especially carbonaceous materials), are warranted for real-life applications.