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In vitro adsorption study of zearalenone by Thai bentonite and mineral clays

S Wongtangtintan, N Silaratana, U Tengjaroenkul, K Pimpukdee and B Tengjaroenkul

Khon Kaen University, Khon Kaen 40002, Thailand


In vitro adsorptions of zearalenone (ZEN) onto Thai bentonite (TB) and mineral clays (MC) at different temperatures (25C, 37C and 45C) and pH levels (7.0 and 8.5) were studied. Results demonstrated that adsorptions for ZEN on TB and three MC including acid-activated montmorillonite (AAM), Ca-montmorillonite (CM) and clinoptilolite (CL) having a distinct structure at pH 7.0 and pH 8.5 ranged from 9.04-17.66 and 7.38-14.47 mg ZEN/g adsorbent, respectively. The highest adsorption capacities of the toxin to all adsorbents were demonstrated at pH 7.0 and 45C followed the order of CM (9.400.45 mg/g)<CL (10.200.57 mg/g)<AAM (13.160.33 mg/g)<TB (17.661.67 mg/g), respectively. The results indicated that TB was more effective than the MC on adsorbing ZEN. It is important to rigorously test an efficacy of mycotoxin binding agent in vivo in animal based on an in vitro study.

Keywords: adsorbent, clinoptilolite, montmorillonite, mycotoxin, toxin binder


Zearalenone (ZEN) is an important mycotoxin in temperate rain region of the world. The toxin is an estrogenic substance produced by Fusarium sp. ZEN usually detected in grains, such as maize, wheat, rice, barley and sorghum (Miller 1995, EFSA 2007, Juan et al 2012). The toxin produces potent hyperestrogenic response in susceptible animal species, particularly swine (Marlin et al, 2010). Symptoms of ZEN poisoning include retention of corpus lutea, anestrous, precocious sexual development, uterine enlargement, swollen vulva and mammae, pseudopregnancy, reduction in litter size, embryo loss, inflammation of prepuce, decrease size of testicles and epididymis, loss of libido and cessation of spermatogenesis (Long and Diekman 1986, Casteel and Rottinghouse 2000, Zinedine et al 2007, Minervini and Dell’Aquila 2008, Doll and Danicke 2011).

Methods for detoxification of mycotoxins in contaminated feedstuffs are critically needed. Recently, a reduction in the bioavailability of ZEN by adding adsorbents in contaminated feedstuffs has been considered as a cost-effective and practical strategy for industrial livestock production (Abbes et al 2007, Pasha et al 2007, Khadem et al 2012, Wang et al 2012, Neeff et al 2013). Several adsorbents including clays and modified clays are able to detoxify the toxins (Alegakis et al 1999, Huwig et al 2001, Pimpukdee et al 2004, Jiang et al 2010). Our preliminary study found that Thai bentonite from Lopburi province was capable of sequestering aflatoxin B1, and was not significantly different from several commercial available toxin binders using a modified Freundlich isotherm model analysis (Tengjaroenkul et al 2011). However, to date, study on efficacy of the TB on ZEN adsorption in vitro is very limited. The objective of this study was, therefore, to evaluate the ability of TB and three MC to adsorb ZEN from aqueous solution in laboratory at three different temperatures and two different pH levels.

Materials and Methods

Adsorbents, chemicals and reagents

Zearalenonewas purchased from Sigma Chemical Co. (St. Louis, MO). Thai bentonite (TB) was collected from Lopburi province, Thailand (Tengjaroenkul et al 2011), whereas acid-activated montmorillonite (AAM) was obtained from Kaiser Aluminum (Pleasanton, CA). Ca-montmorillonite (CM) was obtained from Source Clay Minerals (Columbia, MO), whereas clinoptilolite (CL) was obtained from Engelhard Co. (Cleveland, OH). All chemicals and reagents were of the highest purity commercially available. The high-purity water used in this study was prepared by processing deionized water through a Milli-Quf+ system.

Clay preparation

TB and MC were prepared by washing with deionized water (100 ml/g clay) under agitation for 24 hr. The washed clays were dried at 25C for 72 hr, then ground and sieved through a 325-mesh sieve to obtain particles that were less than 60 m.

Adsorption capacity

A stock solution of ZENwas prepared by dissolving the pure crystals in acetonitrile. A volume of the stock solution was injected into two different pH levels (7.00, 8.50) to yield 4 mg/ml solution of ZEN. The concentration was then checked with a recording UV-visible spectrophotometer at a wavelength of 236 nm (UV-1601 PC, Shimadzu, Shimadzu Scientific Instrument). The adsorption study was conducted in three replication for each adsorbent sample. Approximately 10 mg of each adsorbent (TB or MC) was weighed in a 16 x 125 ml disposable borosilicate test tube. Purified water was then added to the adsorbent to make a 2 mg/ml suspension. The suspension of adsorbent was vortexed to achieve homogeneity. From this suspension, 50 ml (containing 100 g of adsorbent) was added to each 5 ml solution of ZEN (4 ppm). There were three controls for each tested adsorbent: a purified water control, toxin control containing 4 g/ml of ZEN without adsorbent, and an adsorbent control containing 5 ml of two different pH Tris buffer solutions (7.00, 8.50) to and 100 mg of each adsorbent. The samples and the controls were capped and placed on an electric shaker (IKA-VIBRAX-VXR, Bacter, McGraw, IL) at 1000 rpm for 24 hr in an incubator at three different temperatures (25C, 37C and 45C). After shaking, the samples were centrifuged (International Centrifuge, Model UV, International Equipment Co., Needham, MA) at 2000 rpm for 30 min to separate the adsorbent from the supernatant. The supernatant from samples was analyzed of absorbent at a wavelength of 236 nm to determine concentrations of ZEN remaining in the solution.

Statistical analysis

ZEN adsorption data were represented as the mean SD of three replicates per sample. Means showing significant differences in ANOVA were compared using Duncan’s multiple range testin Proc.GLM of SAS program (Ott 1993, SAS 1998). All statements of differences were based on a significance of p<0.05.


TB and MC were evaluated for their abilities to adsorb ZEN from aqueous solution under equilibrium conditions. Adsorptions for ZEN on TB and MC were significantly different ranged from 7.38 to 17.66 mg ZEN/g adsorbent (Table 1). The highest adsorption capacities of the toxin to all adsorbents were demonstrated at pH 7.0 and 45C followed the order of CM (9.400.45 mg/g) <CL (10.200.57 mg/g) <AAM (13.160.33 mg/g) <TB (17.661.67 mg/g), respectively, whereas AAM, CL and CM demonstrated the lowest adsorption capacity at pH 8.5 and 25C followed the order of CM (9.04 0.52 mg/g) <CL (9.350.54 mg/g) <AAM (12.060.33 mg/g) <TB (16.171.78 mg/g), respectively (Table 1). As temperature increased, the adsorption capacity for ZEN of all adsorbents also increased (Table 1, Figure 1-2). However, when the pH levels increased, the adsorption capacity for ZEN of all adsorbents decreased (Table 1, Figure 1-2).

Table 1. Presenting in vitro adsorption capacities for ZEN by Thai bentonite and three mineral clays at different temperatures and pH levels
Adsorbent Adsorption Capacity for ZEN (SD)(mg ZEN/ g adsorbent)
              25C               37C               45C
7.00 8.50 7.00 8.50 7.00 8.50
TB 16.171.78a 13.461.11a 16.331.67a 13.801.21a 17.661.67a 14.471.35a
AAM 12.060.21b 10.000.12b 12.490.26b 10.220.14b 13.160.33b 10.590.19b
CM 9.040.52c 7.380.31c 9.220.38c 7.660.28c 9.400.45c 8.040.33c
CL 9.350.54c 7.620.38c 9.600.48c 7.750.37c 10.200.57c 8.080.38c
TB: Thai bentonite, AAM: Acid-activated montmorillonite, CM: Ca-montmorillonite, CL: Clinoptilolite abc Means in the same column with different superscripts are significantly different (p<0.05)

Figure 1. Average percent adsorption capacity for ZEN by Thai bentonite and three mineral clays
at different temperatures at pH 7.00. TB: Thai bentonite, AAM: Acid-activated
montmorillonite, CM: Ca-montmorillonite, CL: Clinoptilolite

Figure 2. Average percent adsorption capacity for ZEN by Thai bentonite and three mineral clays
at different temperatures at pH 8.50. TB: Thai bentonite, AAM: Acid-activated
montmorillonite, CM: Ca-montmorillonite, CL: Clinoptilolite


The TB and MC with different structures were not all equal in their abilities to adsorb ZEN in vitro. TB was more effective than all three MC on adsorbing ZEN with the highest binding at pH 7.0 and 45C, whereas the MC demonstrated the lowest adsorption capacity at pH 8.5 and 25C, respectively. The extent of sorption could be dependent on the chemical and physical characteristics of the clays (Sprynskyy et al 2012). Among tested clays, maximal binding of ZENwas observed on TB that may possess high surface area, high cation exchange capacity, and its accessible to bind into the interlayer region. Bentonite (B) is an alumino silicate clay consisting of between 8 and 145 minerals, and being categorized in the smectite family. There are different types of B, each named after the major element, such as sodium, calcium and potassium. The B consists of three sandwich-arranged layers: a central octahedral alumina (Al2O3) layer, and two tetrahedral silica (SiO2) layers. The silicon and aluminium ions often undergo isomorphous substitutions by lower valence metals, such as Ca, Mg and Fe. These substitutions lead to a charge imbalance, compensated by exchangeable cations, especially Ca2+, Mg2+ and Na+ ions as well as positive electrical charges of the mycotoxin contaminated in the feedstuffs (EFSA 2007, Segad et al 2010, Bergaya and Lagaly 2013).

The results in the present study were also supported by several in vitro studies by Ramos et al (1996) who found that B, montmorillonite, sepiolite and magnesium trisilicate differently adsorbed ZEN from buffer solution. Bueno et al (2005) demonstrated that B, calcium sulfate and talc were less efficient than activated carbon but still could bind ZEN.Bočarov-Stančić (2011) presented that B had greater adsorption index for ZEN than zeolite and diatomite in the toxin solution at pH 3.0 and 6.9. Santos et al (2011) reported that activated B was capable to adsorb ZEN in single layer sorption equilibrium using Langmuir and Redlich-Peterson equations, and the B had adsorption capacity more than 96% on 500 ppb of ZEN at various pH levels as expected in digestive tract of single stomach animal. Besides mycotoxin binding characteristic, B was accepted as a safe feed additive by the European Food Safety Authority (EFSA 2007) for the absence of its chemical disadvantages and genotoxic evidences.

Montmorillonite (M) is the most sub family of the smectite mineral. It composes of a dioctahedralsmectite that isomorphous substitution in M occurs in the octahedral sheet rather than in the tetrahedral sheet. The large cation exchange capacity of smectites arises from substitution of Al3+ for Si4+ in tetrahedral sheets and substitution of divalent cations such as Mg2+, Ca2+ and Fe2+ for trivalent cations such as Al3+ and Fe2+ in octahedral sheets. M swell in water, and the extent of the swelling is influenced by the hydration of the interlayer cations (Segad et al 2010, Bergaya and Lagaly 2013). CM, for example, can swell when immersed in water to give a basal spacing corresponding to the inclusion of three to four layers of water. Na-montmorillonite is greater swelling than CM when immersed in water, and in many instances the mean layer separations of these Na-exchanged clays can amount to hundreds of units. Molecular sorption on M may involve chemical as well as physical effects. The large internal surface area of M (up to 8 x 105 m² kg¹) provides most of the adsorption surface. In addition, the external surface area of M is generally greater than that of other clay minerals due to its small particle size. The most important chemical properties of M include high cation exchange capacity, ion selectivity and molecular sorption. The most important physical properties of M are expansion, retention of large quantities of water, high cohesion and adhesion, small particle size and extremely large specific surface area (Barrer 1978, Sapalidis et al 2011, Bergaya and Lagaly 2013). Our result indicates that AAM and CM are different in adsorption ability of ZEN. It is probably because AAM activated in acids with high amorphous SiO2 content having a new microstructure and pores of greater adsorption properties. The results in the present study were supported by several studies by Feng et al (2008) reported that M and modified M composites adsorbed and reduced ZEN levels from solution in thelaboratory. Kolossova et al (2011) demonstrated that B and M nanoparticle also removed ZEN in the in vitro trial. Li et al (2014) presented that organo-modified M has greater potential than non-modified M, and can be a high-performance material to control ZEN contamination.

Clinoptilolite is a species of zeolite minerals having non-expandable property. Zeolite is a type of hydrous aluminosilicate belonging to tectosilicates in which the SiO4 tetrahedra form a 3-dimensional cage-like framework. In the zeolite structure, some Si4+ ions are replaced by Al3+, which results in a net negative charge that needs to be balanced by exchangeable cations. The approximate chemical formula of CL is (Ca, Na, K) 6Al6Si30O72•24H2O (Li 1999, Kowalczyk 2006). CL’s framework consists of four channels. Three channels are formed of 8-membered rings of oxygen and one channel of 10 membered rings of oxygen. The dimensions of the open channels in CL are 0.89 x 0.35 nm² for the 10-member ring and exchangeable cation to enter and exit freely but too small for ZEN sorption (Ramesh et al 2011, Inglezakis and Zorpas 2012). Thus, the sorption of ZEN on CL is only limited to the external surface. The previous reports could be explained to our finding that CL was relatively low in its ability to adsorb ZEN when compared to TB and AAM probably due to its structural and cation exchange properties. Furthermore, Dakovic et al (2007) reported that natural zeolites with a rich in CL could effectively adsorb ZEN as non linear isotherm pattern. Pasinli and Henden (2013) presented that organo CL modified with octadecyltrimethyl ammonium bromide had a higher adsorption percentage than unmodified CL and the modified CL with tetramethylammonium bromide on the ZEN. Papaioannou et al (2004) evaluated efficacy of CL on ZEN in swine. They found that reproductive performances of sow and gilt were enhanced when combined the CL into the diets.

From Figure 1, it was found that the adsorbed amount of ZEN slightly increased when temperature increased from 25 C to 45C. The changes of temperatures had slightly affected the adsorption behaviors of the toxin on the adsorbents. This implied that the high degree of physisorption interaction may occur for these adsorbents, i.e., it might mean that physisorption interaction may be more sensitive to the temperature changes than of the chemisorptions. From Table 1 and Figure 1, the pH value increased (become less acid), the adsorption capacity for ZEN of all adsorbents decreased. This may be when the experimental adsorbents immersed in alkali solution, which led to high negative electric charges on the surface of the adsorbent. Moreover, in the alkali pH, ZEN is mostly in an anion form, therefore, basophilic ZEN likely less interacts on the negatively or alkali electric charges surface of the adsorbents. The decreasing interaction reflects to the lower average percent of adsorption capacity for ZEN as the clays are in the alkali pH or basic solution.


The TB and MC with different structures are not all equal in their ability to adsorb ZEN. TB was more effective than the other three mineral clays (AAM, CL, CM) on adsorbing ZEN. It is important to rigorously test and thoroughly characterize potential mycotoxin binding agents in vitro to provide understanding the effectiveness as well as mechanism of adsorption of the clays in order to prevent ZEN induced disease in animals.


Financial and technical supports were provided by the Higher Education Research Promotion and National Research University Project of Thailand and the Research Group on Toxic Substances in Livestock and Aquatic Animals, Khon Kaen University. The authors thank Prof. Frank F. Mallory for reviewing the manuscript.


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Received 26 March 2015; Accepted 4 August 2015; Published 1 September 2015

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