Adsorption and detoxification of ochratoxin A using hydrophobic bentonite clay

Abstract

Mycotoxins such as ochratoxin A (OTA) are hazardous chemicals produced from fungi that frequently contaminate feed ingredients, and this contamination can result in significant economic loss, disease and death. Given the persistent nature of OTA, this study was designed to provide effective and potentially scalable strategies to mitigate acute OTA toxicity using organoclay. The study explores the adsorption behavior of OTA on active binding surfaces of four organoclay composites that were synthesized by modifying sodium bentonite clay surfaces with different amounts of a hydrophobic quaternary ammonium compound (QAC). This modification serves to immobilize and stabilize the QAC. The study evaluates key thermodynamics, kinetics and binding markers including the Gibbs free energy (ΔG), adsorption capacity, enthalpy (ΔH), and binding affinity of these clays for OTA. Additionally, the protective role of these organoclays was evaluated using a sensitive ecotoxicology model. At gastric pH (pH 2), the organoclays demonstrated high adsorption capacities for OTA ranging between 0.34 and 0.51 mol kg⁻¹ versus parent clay (0.20 mol kg⁻¹). At intestinal pH (pH 6), the organoclay binding capacities were higher than the parent clay indicating that the modification of parent clay with QAC played a critical role in improving its adsorptive performance for OTA. Thermodynamic parameters indicated that the binding reaction was exothermic and thermodynamically favorable with ΔG values between −23.40 and −28.07 kJ mol⁻¹ and ΔH values ranging from −13.76 kJ mol⁻¹ to −24.31 kJ mol⁻¹. Importantly, organoclay treatment protected hydra from OTA lethality by 95% to 100%. These findings established the proof-of-concept that OTA interactions with organoclay resulted in the prevention of OTA toxicity in hydra. Based on these findings, further studies are warranted to justify the applications and efficacy of these organoclays for short-term treatment during acute outbreaks of ochratoxicosis.

1. Introduction

The Food and Agriculture Organization (FAO) has reported that mycotoxin contamination affects at least 25% of crops each year, and annual agricultural yields have shown a declining trend (Eskola et al., 2019). On a global scale, mycotoxin contaminants could result in economic losses in the agricultural and industrial sectors worth tens of billions of dollars (Tianpei et al., 2011). Large numbers of mycotoxins that can compromise the quality and safety of animal feed (and human food) have been discovered and characterized. Of these, special attention has been given to toxic mycotoxins produced by Fusarium, Penicillium, and Aspergillus species (Chilaka et al., 2022; Zhang et al., 2024; Poroşnicu et al., 2022). For ochratoxin, there are approximately 20 toxic congeners. Of these, ochratoxin A (OTA) is the most studied and has been shown to be produced by Aspergillus and Penicillium species (Alsalabi et al., 2023; Barborakova et al., 2023; Chuaysrinule et al., 2024).

Ochratoxin A (OTA) is distinguished by a dihydro-isocoumarin moiety that is bonded to phenylalanine by an amide bond (Fig. S1). Its IUPAC nomenclature is L-phenylalanine-N-[(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl) carbonyl]-(R)-isocoumarin. Besides animal feed, a variety of food items, such as spices, dried fruits, chocolate, coffee, cereals, wine, and beer, are regularly contaminated by OTA (Benites et al., 2017; Echodu et al., 2019; Wajih ul Hassan et al., 2020; Torovic et al., 2020; Costa da Silva et al., 2021). Numerous detrimental health consequences have been linked to OTA including nephrotoxicity, immunotoxicity, genotoxicity, carcinogenicity, neurotoxicity, teratogenicity, and hepatotoxicity (Bondy et al., 2021; Stoev et al., 2022; Wieckowska et al., 2023; Hassan et al., 2022; Obafemi et al., 2023; Perugino et al., 2024; Oladele et al., 2024). Importantly, OTA has also been identified as a human carcinogen (Group 2B) and has been reported to adversely affect the kidneys in animals and humans.

There are different detoxification strategies that have been developed to mitigate OTA toxicity, however, sorbent-based toxin removal technology demonstrated significant advantages over them. While a number of chemical processes, including alkalization and oxidation, have been proposed; they could be linked to obvious toxicity and possible environmental problems (Ding et al., 2023). Similarly, thermal methods are energy-intensive and often fail to completely decompose OTA even at temperatures as high as 250 °C (Ding et al., 2023). Moreover, radiation-based detoxification is limited by the potential highly reactive radicals that damage essential nutrients such as vitamins and proteins (WHO, 1999; Di Stefano et al., 2014). However, the application of advanced synthetic adsorbents and biosorbents offers reusable, efficient alternatives that avoids the accumulation of harmful chemical residues (Belajová et al., 2006; Solis-Cruz et al., 2017).

Effective detoxification, and/or removal of OTA from animal feed during outbreaks of ochratoxicosis, has been limited due to its chemical stability and its moderately high lipophilicity (log P 4.74). However, one promising strategy is feed supplements containing a composite material containing bentonite clay and QAC (quaternary ammonium compound) to reduce OTA’s exposure. Clay materials (including organoclays) are promising adsorbents for OTA due to their layered structures, favorable charge properties, and high surface area (Phillips, 1999; Quintela et al., 2012; Oladele et al., 2024). Furthermore, clay-based adsorbents are classified as GRAS (generally-recognized-as-safe), have a broad range of applications, are recyclable in nature and inexpensive (Oladele et al., 2024). Recent advances from our group have involved the synthesis of green organoclays enriched with chlorophyllin and chlorophyll, and these have been shown to bind OTA (Oladele et al., 2024). This current study is a continuation of our organoclay work and investigates four sodium clays amended with a hydrophobic quaternary ammonium compound. This study explores the capacity of these composite materials to bind and detoxify OTA using in vitro and in vivo approaches. These approaches include (a) establish isothermal adsorption models to examine OTA binding behavior at active binding surfaces of the organoclays, (b) investigate the kinetics and thermodynamics governing the adsorption of OTA to these organoclays and (c) assess the detoxification efficacy of the organoclays using Hydra vulgaris as a living ecotoxicological model system.

2. Materials and methods

2.1. Clays and chemicals

The four organoclay composites and parent clay are products of Bentonite Performance Minerals LLC, (Houston, TX). The sodium bentonite clay is from Wyoming with an estimated cation exchange capacity equal to 75 cmol kg⁻¹, external surface area of approximately 70 m² g⁻¹ and an average total surface area as high as 850 m² g⁻¹. The generic formula for these clays is (Na,Ca)_0.3(Al,Mg)₂Si₄O₁₀(OH)₂·nH₂O. The four organoclays are modified sodium bentonites manufactured by mixing the clay with different levels of a quaternary amine resulting in hydrophobic materials including: ENVIRO-TROL (ENT); AGRI-TROL B (ATB); AGRI-TROL M (ATM); ORGANO-TROL (OT2). These organoclays are proprietary products of Bentonite Performance Minerals LLC. Table 1 gives a summary of the physical properties of these organoclays. OTA was obtained from Sigma Aldrich (Saint Louis, MO, USA). Ultra-pure deionized water (18.2 MΩ) used in this study was produced using an Elga^™ automated filtration system (Woodridge, IL). High-pressure liquid chromatography (HPLC)- grade acetonitrile, simulated gastric fluid, and pH calibration buffers (4.0, 7.0, and 10.0) were sourced from VWR (Atlanta, GA, USA).

Table 1.

Organoclay specifications.

	ENVIRO-TROL	AGRI-TROL B	AGRI-TROLM	ORGANO-TROL
Appearance	Gray to tan	Gray to tan	Gray to tan	Gray to tan
Bulk Density, lb/ft3	57	57	57	57
LOI %	34–40	34–40	34–40	34–40
Screen analysis % > 200 mesh	84% Min	84% Min	84% Min	84% Min
Specific Gravity	1.7	1.7	1.7	1.7

(Data collected from product information sheets).

2.2. In vitro isothermal sorption

A stock solution of OTA was prepared by dissolving its high-purity crystals in HPLC-grade acetonitrile to achieve a final concentration of 1250 ppm (μg/mL). OTA detection was carried out using UV/Vis spectrophotometry as earlier described by Oladele et al. (2024). The cuvette path length is 1 cm and acetonitrile is used as the reference solvent for background correction. The analytical method was validated prior to sample analysis. Calibration curves were prepared and exhibited good linearity within the range of 0.30 μg/mL and 10 μg/mL with the regression equation y = 0.1117x + 0.0723 and a correlation coefficient (r²) of 0.9951. The limit of detection (LOD) and limit of quantification (LOQ) were 0.28 μg/mL and 0.86 μg/mL. Recovery was assessed by spiking at concentrations between 1.0 and 8.0 μg/mL resulting to mean recoveries between ranges 84% and 97%. A determined aliquot of the 1250 μg/mL OTA stock solution was accurately measured using a glass syringe and dissolved in either pH 2 or pH 6 water to simulate gastric acidity and small intestinal pH conditions respectively (Fallingborg, 1999). The resulting 1 μg/mL of OTA solution was used for each isothermal adsorption experiment.

To assess the binding capacity of the organoclays, 6 μL of 0.5 mg/mL of each clay suspension was mixed with a gradient concentration of 1 μg/mL OTA ranging from 10% to 100% in 1.5 mL solution. The reaction is incubated at 37 °C for 2 h with constant shaking at 1000 rpm using an IKA^® electric shaker (VIBRAX VXR basic, Werke, Germany). All samples were subsequently centrifuged at 3000×g for 5 min to separate the clay-OTA complex from the solution, and the resulting supernatants were transferred into clean tubes. The absorbance for OTA was taken at 212.2 nm using a SHIMADZU UV-1800 spectrophotometer (SHIMADZU Corporation, Japan) (Oladele et al., 2024).

2.3. Adsorption kinetics

In this study, adsorption kinetics were used to evaluate the binding efficiency of the organoclays with OTA over a total contact period of 24 h. Each organoclay was individually mixed with 1 μg/mL OTA in pH 2 water at 37 °C and agitated continuously at 1000 rpm. At 30 min intervals, samples were taken and analyzed for residual OTA concentration. To characterize the adsorption behavior, kinetic data were fitted to pseudo-first order, pseudo-second order and Elovich models for detailed assessment of the interaction mechanisms and rate-limiting steps governing OTA-organoclay binding.

2.4. Thermodynamic assessment of the adsorption

For the thermodynamic evaluation of the binding of OTA to the organoclays, the mixture of each clay and 1 μg/mL OTA were incubated at three temperatures: 4 °C, 18 °C, and 37 °C for 2 h with constant shaking at 1000 rpm. This allows for temperature-dependent binding behavior assessment and calculation of thermodynamic endpoints including Gibbs free energy and enthalpy. After incubation, the mixtures were subsequently centrifuged at 3000×g for 5 min to separate the clay-OTA complex from the solution. The concentration of the free OTA remaining in the supernatants was quantified by at 212.2 nm which corresponds to the absorbance under the tested conditions (Oladele et al., 2024).

2.5. Ecotoxicological bioassay (Hydra vulgaris)

The Hydra vulgaris model reported by Oladele et al. (2024, 2025) has been well-established in our laboratory as a highly sensitive assay for mycotoxins and environmental chemicals. This model was used to investigate the detoxification efficacy of the organoclays in this study. Briefly, Hydra vulgaris were obtained from Environmental Canada in Montreal and kept under controlled laboratory conditions at 18 °C. A detailed morphological scoring system on a scale of 1 to 10 (Table S1) was used to evaluate the various degrees of toxicity resulting from OTA exposure. Hydra vulgaris were exposed to 1 μg/mL of OTA and organoclays (individually) at four inclusion rates (0.05%, 0.1%, 0.2% and 0.5%). Each experiment was carried out in three independent replicates of two hydra colonies. Morphological scores were recorded at intervals of 0, 4, 20, 28, 44, 68, and 92 h to monitor the progression of OTA-mediated toxicity and to evaluate the protective effect offered by each of the organoclays.

Morphological scores were between media control (score 10) and test groups. The t-value was calculated (N = 3) and compared in a p-value table to determine the statistical significance. Results were considered significant at p < 0.05.

2.6. Data analysis and model fittings

The kinetic behavior of OTA adsorption at binding surfaces of the organoclays was analyzed using nonlinear models.

The pseudo-first order model in nonlinear form is expressed as:

$${\text{q}}_{\text{t}}={\text{q}}_{\text{e}}\times \left[1-\text{exp}\left(-{\text{K}}_1\times \text{t}\right)\right]$$ (1)

Where qe and qt (mg kg⁻¹) represent the adsorbed amounts at equilibrium and at time t (min), respectively, and K₁ (min⁻¹) is the first-order rate constant.

For pseudo-second order model, the equation applied is:

$$q_t=\frac{k_2\times q{e}^2\times t}{1+qe\times k_2\times t}$$ (2)

Where K₂ (mg kg⁻¹ min⁻¹) is the second-order rate constant. The Elovich expression is as follows:

$${\text{q}}_{\text{t}}=1/\beta \text{ln}\alpha \beta +1/\beta \text{ln}\text{t}$$ (3)

Where α (mg kg⁻¹ min) is the initial adsorption rate and β (mg kg⁻¹) relates to the surface coverage and activation energy. The parameters were estimated via regression, using an optimization computational approach to minimize the sum of squared deviation and coefficient of determination between the experimental data and predicted values.

OTA concentration (mol kg⁻¹) was determined by comparing the values of treated and control groups using isothermal plots with TableCurve 2D v.5.01 (Systat software, Palo Alto, CA). Model fitting was performed using maximum likelihood estimation, standard errors and 95% confidence intervals were calculated (Yan et al., 2015; Gong and shiny, 2020).

For adsorption isotherms, the Langmuir model was used to characterize monolayer adsorption onto the organoclay surface with homogeneous energy sites:

$$L\mathit{\text{angmuir}}\mathit{\text{model}}q=Q_{\mathit{\text{max}}}\frac{{\text{K}}_{\text{d}}{\text{C}}_{\text{w}}}{1+{\text{K}}_{\text{d}}{\text{C}}_{\text{w}}}$$ (4)

K_d represents the Langmuir affinity constant, C_w is the equilibrium concentration of OTA in mol L⁻¹, Q_max is the maximum adsorption capacity in mol kg⁻¹, and q is the quantity of OTA adsorbed at equilibrium in mol kg⁻¹.

The following equation was applied to determine Ke^∘ from K_d:

$${K_e}^{\circ }=\frac{{\text{K}}_{\text{d}}{\left[\text{adsorbate}\right]}^{\circ }}{\gamma }$$ (5)

Here, [adsorbate]^∘ was the standard concentration of the adsorbate = 1 mol/L, K_e^∘ was the thermodynamic equilibrium constant, and γ was the coefficient of activity.

The free energy (ΔG^∘) and enthalpy (ΔH) for the adsorption process were calculated by using the van’t Hoff and Gibbs free energy equations:

$$\Delta H=\frac{-\text{RIn}\left(\frac{{\text{K}}_{\text{d}2}}{{\text{K}}_{\text{d}1}}\right)}{\left(\frac{1}{{\text{T}}_2}\right)-\left(\frac{1}{{\text{T}}_1}\right)}$$ (6)

$$\Delta \text{G}=\Delta {\text{G}}^{\circ }+\text{RTInK}{\text{e}}^{\circ }$$ (7)

where R (gas constant) = 8.314 J/mol/K, T (absolute temperature) = 273 + t (°C). When ΔG is negative, it indicates that the adsorption process is spontaneous and enhanced thermodynamically, thus proceeding in the forward direction (Ke^∘ > 1). However, when it is positive, this suggests the adsorption process is not energetically favorable (Ke^∘ < 1). ΔG will be zero for an adsorption system at equilibrium.

3. Results

3.1. Isothermal adsorption of OTA at active binding surfaces of the organoclays in pH 2 and simulated gastric fluid

Fig. 1 A and B depict the isothermal adsorption behavior of OTA on active binding sites of the organoclays at pH 2 and simulated gastric fluid (SGF) respectively (37 °C). The adsorption of OTA was best described by the Langmuir model with high correlation coefficients ranging from 0.90 to 0.96 (Table 2). This suggests a saturable binding process characterized by high binding affinity (Kd) interactions ranging between 1.06 × 10⁵ and 1.95 × 10⁵. The adsorption capacity (Qmax) for the parent clay (sodium bentonite clay) was 0.20 mol kg⁻¹ while all organoclays demonstrated significantly higher adsorption capacities ranging between 0.34 and 0.51 mol kg⁻¹. AGRI-TROL B showed the highest binding capacity of 0.51 mol kg-1 followed by ENT (0.46 mol kg⁻¹), while ATM and OT2 were 0.38 and 0.34 mol kg⁻¹ respectively (Table 2). The Gibbs free energy (ΔG) values for OTA adsorption ranged between −25.87 and −27.59 kJ mol⁻¹, which was consistent with a chemisorption mechanism. Under simulated gastric conditions, the organoclays displayed higher binding capacities (0.21 to 0.39 mol kg⁻¹) for OTA versus the parent clay (0.15 mol kg⁻¹). The Qmax in the fluid with enzyme pepsin (SGF) was lower than pH 2 without enzyme, indicating that enzymes could interfere with OTA binding. However, they have high binding affinity (Kd) interactions at magnitudes of 10⁵.

Fig. 1.

Adsorption isotherms of OTA at the binding surfaces of the organoclays in (A) pH 2 and (B) simulated gastric fluid at 37 °C.

ENT: ENVIRO-TROL; ATB: AGRI-TROL B; ATM: AGRI-TROL M; OT2: ORGANO-TROL; SB: sodium bentonite clay; SGF: simulated gastric fluid. The solid lines represent the adsorption isotherm plots based on the Langmuir model, while the dashed lines represent the 95% confidence band.

Table 2.

Parameters and correlation coefficients of adsorption isotherms of OTA at binding surfaces of the organoclays.

Clays	Adsorption parameters @ pH2				Adsorption parameters in SGF				Adsorption parameters @ pH6
	Qmax	Kd	ΔG	r2	Qmax	Kd	ΔG	r2	Qmax	Kd	ΔG	r2
ENT	0.46	1.60 × 105	−27.40	0.96	0.22	3.59 × 105	−26.59	0.95	0.15	2.36 × 105	−24.65	0.92
ATB	0.51	1.57 × 105	−27.59	0.94	0.39	1.61 × 105	−27.09	0.94	0.16	4.71 × 105	−25.55	0.91
ATM	0.38	1.46 × 105	−26.47	0.90	0.21	2.28 × 105	−25.99	0.96	0.32	2.28 × 105	−27.02	0.93
OT2	0.34	1.06 × 105	−25.87	0.95	0.22	1.52 × 105	−25.21	0.95	0.34	1.72 × 105	−26.52	0.95
SB	0.20	1.95 × 105	−25.87	0.93	0.15	2.40 × 105	−24.73	0.96	0.08	5.52 × 105	−24.06	0.91

ENT: ENVIRO-TROL; ATB: AGRI-TROL B; ATM: AGRI-TROL M; OT2: ORGANO-TROL; SB: sodium bentonite clay. ΔG: Gibbs free energy (kJ/mol); K_d: binding affinity; Q_max: binding capacity (mol/kg); r²: correlation coefficient.

3.2. Isothermal adsorption of OTA at the binding surfaces of the organoclays at pH 6

At pH 6 (intestinal pH) and 37 °C, the adsorption of OTA onto the organoclays followed the Langmuir model as indicated by high correlation coefficients ranging from 0.91 to 0.95 and characteristic curvature (Fig. 2) (see Fig. 1). There was a marked decline in the adsorption capacity of all the organoclays at pH 6 when compared with those at pH 2. For instance, Qmax for the parent clay (sodium bentonite clay) was 0.08 mol kg⁻¹ while that of the organoclays ranged between 0.15 and 0.34 mol kg⁻¹, except for ORGANO-TROL organoclay which remained unchanged by change in pH. Despite the shift from acidic to near-neutral pH, the binding capacities for the organoclays were still higher than the parent clay indicating that the functionalization of the parent clay played a critical role in improving their adsorptive performance for lipophilic OTA. All the organoclays displayed high binding affinities in the 10⁵ magnitude range which reflected strong interactions with OTA. A detailed profile of these adsorption parameters is presented in Table 2.

Fig. 2.

Adsorption isotherms of OTA at the binding surfaces of the organoclays at pH 6 and 37 °C.

ENT: ENVIRO-TROL; ATB: AGRI-TROL B; ATM: AGRI-TROL M; OT2: ORGANO-TROL; SB: sodium bentonite clay. The solid lines represent the adsorption isotherm plots based on the Langmuir model, while the dashed lines represent the 95% confidence band.

3.3. Thermodynamics of the binding of OTA at clay surfaces

The binding interactions between OTA and organoclay surfaces were further characterized by evaluating the thermodynamic parameters, specifically enthalpy and Gibbs free energy of binding at three different temperatures: 4 °C, 18 °C and 37 °C (Fig. 3). As shown in Table 3, the enthalpy change (ΔH) associated with the binding process ranged between −13.76 kJ mol⁻¹ and −24.31 kJ mol⁻¹ indicating an exothermic interaction (Table 3). The corresponding Gibbs free energy (ΔG) values were between −23.40 and −27.73 kJ mol⁻¹ at 4 °C and between −25.21 and −28.07 kJ mol⁻¹ at 18 °C. The negative ΔH and ΔG values across all temperatures confirmed that the adsorption was spontaneous in the forward direction for surface binding and was thermodynamically favorable.

Fig. 3.

Adsorption isotherms for OTA at binding surfaces of organoclays (A) 18 °C and (B) 4 °C. ENT: ENVIRO-TROL; ATB: AGRI-TROL B; ATM: AGRI-TROL M; OT2: ORGANO-TROL; SB: sodium bentonite clay. The solid lines represent the adsorption isotherm plots based on the Langmuir model, while the dashed lines represent the 95% confidence band.

Table 3.

Thermodynamic parameters for adsorption isotherms of OTA at binding surfaces of the organoclays.

Clays	Adsorption parameters @ 18 °C				Adsorption parameters @ 4 °C				Enthalpy
	Qmax	Kd	ΔG	r2	Qmax	Kd	ΔG	r2	ΔH
ENT	0.46	1.64 × 105	−27.32	0.92	0.31	2.41 × 105	−27.11	0.92	−13.76 ± 6.82
ATB	0.41	2.79 × 105	−27.90	0.94	0.35	2.82 × 105	−27.33	0.92	−17.74 ± 7.11
ATM	0.38	2.85 × 105	−28.07	0.94	0.34	2.82 × 105	−27.73	0.91	−20.43 ± 8.60
OT2	0.21	2.49 × 105	−25.99	0.95	0.17	2.10 × 105	−25.23	0.91	−24.31 ± 13.37
SB	0.17	1.93 × 105	−25.21	0.94	0.14	1.20 × 105	−23.40	0.91	−15.83 ± 8.67

ET: ENVIRO-TROL; ATB: AGRI-TROL B; ATM: AGRI-TROL M; ORT: ORGANO-TROL; SB: sodium bentonite clay. ΔG: Gibbs free energy (kJ/mol); K_d: binding affinity; Q_max: binding capacity (mol/kg); r²: correlation coefficient; ΔH: enthalpy.

3.4. Kinetics of OTA binding at clay surfaces

To elucidate the kinetics of OTA adsorption to the organoclays, nonlinear forms of the pseudo-first order, pseudo-second order and Elovich models were applied based on methods previously established in our laboratory. Among these models, the pseudo-second order model provided the best fit, as evidenced by the correlation coefficients and the close relationship between the experimentally observed equilibrium adsorption capacities (qe, exp) and calculated values (qe, cal) as shown in Fig. 4 and Table 4.

Fig. 4.

Adsorption kinetics of OTA at binding surfaces of the organoclays. ENT: ENVIRO-TROL; ATB: AGRI-TROL B; ATM: AGRI-TROL M; OT2: ORGANO-TROL; SB: sodium bentonite clay.

Table 4.

Adsorption kinetic parameters for OTA on the organoclays.

Clays	qe (exp mgkg−1)	qe (cal mgkg−1)	K2	r2	RMSE	AIC
ENT	0.11	0.07	3.91 × 10−1	0.94	0.025	−126.44
ATB	0.09	0.06	4.0 × 10−1	0.90	0.027	−141.01
ATM	0.05	0.03	2.25 × 103	0.91	0.010	−143.59
OT2	0.06	0.02	1.41 × 105	0.95	0.023	−140.46
SB	0.06	0.03	5.16 × 105	0.88	0.012	−129.23

ENT: ENVIRO-TROL; ATB: AGRI-TROL B; ATM: AGRI-TROL M; OT2: ORGANO-TROL; SB: sodium bentonite clay. K₂: rate constant of the second order; qe (exp): binding at equilibrium in experiment; qe (cal): binding at equilibrium calculated by pseudo-second order; r²: correlation coefficients; RMSE: root mean square error; AIC: Akaike information criterion.

3.5. Toxic effects of OTA on Hydra vulgaris and the protective role of the organoclays

The outcomes of the in vivo assessment of OTA toxicity in Hydra vulgaris and the protection offered by the organoclays are presented in Fig. 5. OTA caused 10% morphological damage to the hydra colonies after 20 h of exposure and its toxicity increased to 55% damage at 44 h of exposure. The hydra colonies were completely disintegrated and dead after 68 h of exposure. However, inclusion of the organoclays demonstrated significant protection at the various inclusion rates (0.05% to 0.5%) used in this study (Fig. 5A–D). At only 0.05% inclusion, the organoclays resulted in 95% - 100% protection. Remarkably, all of the organoclays resulted in complete (100%) protection at the 0.1% inclusion rate. In contrast, the parent clay did not demonstrate any protection at lower inclusion rates, however, at higher inclusion, parent clay offered some protection (Fig. 5E).

Fig. 5.

Protective effect of organoclays on OTA-induced toxicity in Hydra vulgaris (A) ENT (B) ATB (C) ATM (D) OT2 and (E) SB clays. ENT: ENVIRO-TROL; ATB: AGRI-TROL B; ATM: AGRI-TROL M; OT2: ORGANO-TROL; SB: sodium bentonite clay.

4. Discussion

The adsorption behavior of OTA onto the active binding sites of base sodium montmorillonite clay and four hydrophobic quaternary ammonium-modified bentonite clays revealed significant differences in their sorption capacities for OTA as demonstrated by the higher Q_max and K_d values of the organoclays versus the base clays. The binding affinities, capacities, thermodynamics and kinetics of the reaction of organoclays with OTA suggest that they will bind strongly in the stomach and remain bound throughout the GI tract. The enhanced adsorption observed with the organoclays may be attributed to structural modifications imparted by the intercalation of quaternary ammonium cations, which increases surface hydrophobicity and expands the interlayer spacing of the bentonite matrix. The observed differences in the adsorption capacity among the organoclays indicates the variations in surface functionalization and organic modification density. This agrees with our earlier report on the hydrophobicity of the organoclays (Oladele and Phillips, 2026). These structural modifications enable stronger interactions with hydrophobic OTA molecules and increase efficient encapsulation of OTA within the clay interlayers. Similar findings from our group have been reported with chlorophyll-amended organoclays which exhibited higher sorption capacities for aflatoxin B1 and benzo(a)pyrene that the base clays through improved π-π stacking and hydrophobic interactions (Wang et al., 2021).

OTA adsorption by both the base clay and the organoclays revealed a pH-dependent sorption behavior with higher adsorption observed at pH 2 compared to pH 6. This could be due to the protonation state of clay surfaces and OTA. At lower pH, OTA will predominately exist in its neutral lipophilic form (Cagnasso et al., 2019) suggesting that it tightly binds through van der Waals attraction within the hydrophobic interlayers of the organoclays. The cationic sites on the organoclays are not influenced by pH and more likely engage in ion-exchange reactions in acidic states (Cruz-Guzmán et al., 2004), while at near-neutral pH, deprotonation of OTA can result in negatively charged COO- groups and negatively charged surfaces of the clay, thereby lowering the sorption efficiency (Perry et al., 2003; Cagnasso et al., 2019). This supports our findings in vitro at the higher pH. Based on our findings in this study, at pH 2, OTA binding may be assisted by electrostatic surface interactions. These interactions may be between positive OTA and negatively charged clay surfaces and hydrophobic quats in the organoclay resulting in tight binding. At pH 6, sorption was lower than at pH 2. This difference may be due to non-binding interactions between negatively charged OTA and negatively charged clay surfaces. The data for the adsorption was fit by the Langmuir model, indicating a monolayer adsorption process, where specific binding sites on the surfaces of the clay were saturated at high OTA concentration. The increased K_d values for the organoclays were comparable to the parent clay and also implied a higher affinity and more favorable sorption interactions (Oladele et al., 2025).

The thermodynamic data for the adsorption of OTA onto the surfaces of organoclays revealed sorption behavior at different temperatures used in this study. The observed enthalpy changes (ΔH) for the organoclays ranged between −10 kJ/mol and −25 kJ/mol, consistent with chemisorption but on the lower end of the spectrum (Adegbeye et al., 2020). Importantly, the negative values of ΔH confirmed that the adsorption was exothermic, which has been a distinct characteristic for mycotoxin adsorption onto clays (Phillips et al., 2019). The spontaneity of the adsorption was shown by the negative values of Gibbs free energy (ΔG) at the three temperatures in the experiments. This indicated a favorable sorption process driven by enthalpic contributions.

The kinetic investigation of OTA adsorption onto the organoclays showed significant adsorption. Remarkably, adsorption kinetics were best described by a pseudo-second order model, as suggested by high correlation coefficients and excellent fits of the experimental data. This kinetic model indicated that chemisorption was the rate-limiting step, involving valency forces via exchange or sharing of electrons between OTA molecules and the binding sites on the organoclay surfaces (Foo and Hameed, 2010; Ho and McKay, 1999). Furthermore, the organoclays exhibited higher kinetic rate constants than the base clay. This implied faster binding of OTA, possibly due to increased availability of the hydrophobic interlayer and the functionalized quat sites on the clays that enhanced stronger interactions and hydrophobic attraction with the OTA molecules (Wang et al., 2021).

Quaternary ammonium compounds are cationic surfactants widely used for disinfectant and antimicrobial purposes in food-processing environments. They have been generally recognized to interact with cell membranes. In this study the organoclays showed no observable toxicity to Hydra vulgaris at doses up to 0.5% w/v. This could possibly be due to a low amount of QAC in these organoclays and/or minimal desorption of QACs. The European Food Safety Authority (EFSA) has proposed a level of 0.1 mg/kg as the dietary exposure limit (MRL) for QACs (EFSA, 2014; EFSA, 2023).

Although QACs have several uses and significance, their toxicity prevents them from being widely applied. The regulated release of QACs through the development of QAC composites (QACCs) to reduce their toxicity without losing their antibacterial efficacy has been the subject of many investigations (Liu et al., 2019, 2021; He, 2021). These are functionalized materials that act as carriers to reduce damage to healthy tissues (Ghanbar et al., 2018) and can be covalently attached to food packaging surfaces to reduce leakage (Rukmanikrishnan et al., 2021). As a result, the formulation of QACCs significantly expands the applications of QACs and provides a workable solution to problems related to the potential toxicity individual QACs. The organoclays tested in this study are similar to QACCs in that they use a carrier matrix of bentonite clay to attenuate the toxicity QAC while maintaining the hydrophobicity and activity of the composite.

The Hydra vulgaris model has been extensively used as an ecotoxicology model to: 1) confirm the toxicity of environmental chemicals including pesticides and mycotoxins, 2) validate in vitro results, and 3) confirm protective effects of adsorbent materials. In this study, hydra exposed to 1 ppm of OTA exhibited a range of toxic responses which led to severe irreversible morphological damage and ultimately death. This finding was consistent with previous reports by Oladele et al. (2024) and others linking OTA toxicity in hydra to mechanisms involving oxidative stress (Dash and Phillips, 2012). Importantly, the inclusion of the organoclays provided remarkable protection. In particular, the organoclays demonstrated complete protection from OTA mediated toxicity even at lower inclusion rates. This complete protection against OTA toxicity was offered over the 92-h exposure period used in this study. The hydra model provides valuable insight into how sorbent-OTA binding may persist in a lower organism over a 92-h period, supporting the potential of these organoclays to serve as toxin binders in animal feeds for short duration use during an outbreak of ochratoxicosis.

5. Conclusions

Overall, the findings from this study demonstrate that four commercially available organoclay composites can be used to significantly bind OTA with higher binding capacity than parent sodium bentonite clay. This supports the use of these organoclays as effective adsorbents for OTA. The proposed mechanism for the adsorption of lipophilic OTA at the surfaces of the organoclays is a strong interaction at organophilic binding sites containing hydrophobic moieties. The difference in the performance of the organoclays may be due to a variation in the level of QAC (and hydrophobicity). This study also established that the adsorption process follows a Langmuir model and was thermodynamically favorable in the forward direction. The organoclays remarkably reduced OTA toxicity in the Hydra vulgaris model and were not toxic to the hydra at concentrations used in this study suggesting that the composite material is active, but not overly toxic at levels tested. This offers a promising strategy for OTA mitigation. Future work to optimize the efficacy these organoclays as sorbents for OTA to protect animals against acute OTA toxicity is warranted.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.toxicon.2026.109066.

Data availability

Data will be made available on request.