Livestock Research for Rural Development 23 (11) 2011 Guide for preparation of papers LRRD Newsletter

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Study on efficacy of natural Thai clays for adsorption of aflatoxin B1

Bundit Tengjaroenkul, Urai Tengjaroenkul* and Sawitree Wongtangtintharn**

Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen 40002, Thailand,
* Faculty of Science, Chiang Mai University, Chiang Mai 53200, Thailand
** Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand


Natural Thai clays from different areas and commercial adsorbents were investigated in relation to their adsorption capacities of aflatoxin B1 (AFB1) in vitro. The Thai clays were capable of sequestering AFB1 from aqueous solution differently. The experimental data were fitted to modified Freundlich model for investigating adsorption capacities and affinity constant. The S-shaped isotherms studies to measure capacity and affinity of toxin adsorption were observed for these clays as well as the commercial adsorbents.

Results of X-ray diffraction spectrometry demonstrated that several Thai clays contained mainly montmorillonite. These results may be useful for predicting the efficacy of adsorbents in vivo prior to include in animal feeds.

Keywords: adsorbent, contamination, feed, fungi, toxin


One encouraging approach in solving the mycotoxin problems is by addition of adsorptive clay to feedstuffs (Phillips et al 1988). This effort could be considered as practical and cost-effective method. Several in vitro studies have shown that various adsorbents, such as bentonite, zeolites and phyllosilicates are capable of binding aflatoxin (Kubena et al 1991; Phillips 1999; Pimpukdee et al 2004). Earlier studies have utilized modified methods of equilibrium isothermal analysis to characterize the adsorption of aflatoxin onto the surfaces of various adsorbents (Grant and Phillips 1998; Pimpukdee et al 2000). These methods have provided an insight into the molecular mechanisms of action of adsorbents and comparison of critical similarities and differences. To best of our knowledge, studies on the efficacy of natural Thai clays in adsorption of AFB1 have not been reported. Thus, this work aimed to investigate the adsorption capacity and affinity of different Thai clays of AFB1 by utilizing isothermal adsorption models. 

Materials and Methods

Determination of adsorption capacity (Phillips et al 1995; Pimpukdee et al 2000)

Three bentonites (BN) and four commercial toxin binders (COM) were obtained from suppliers. Natural Thai clays (S) with size less than 60 m were obtained from 14 provinces: Lop Buri (S1), Nakhon Ratchasima (S2), Buriram (S3), Phetchabun (S4), Phitsanulok (S5);  Kanchana Buri (S6), Phet Buri (S7), Lampang (S8), Lamphun (S9), Ratch Buri (S10), Suphan Buri (S11), Nakorn Srithammarat (S12), Prachuab Khirikhan (S13) and Chon Buri (S14). Purified water without toxin, AFB1 without adsorbent, and a mixture containing purified water and adsorbent were set as control treatments. After mixing AFB1 solution (Sigma Chemical Co., USA) into clays, the samples were centrifuged. The supernatants were analyzed to determine the concentrations of AFB1 using UV-visible spectrophotometer (Perkin Elmer, MA) at λ 362 nm.

 Study of isothermal adsorption (Grant and Phillips 1998; Pimpukdee et al 2000)

A series of AFB1 solutions having the concentrations from 0.5 to 8.0 g/ml were prepared.  Each isotherm study consisted of 4.0 ml AFB1 solution at different concentration. The equilibrating condition, separation procedure and determination of the AFB1 concentrations were the same as described above. The UV/Visible absorption data were entered into an Excel spreadsheet (Microsoft, Redmond, WA) to calculate the amount of AFB1 left in solution, and the amount AFB1 adsorbed (q) for each data point. The AFB1 concentrations left in the supernatants (Ce) and the amount AFB1 adsorbed (q) were calculated from the calibration curves obtained from the external standard method obeyed Beer’s law. The adsorption data were then transported to Table Curve 2D (Andel Scientific, USA) fit isotherm equations. The Langmuir and modified Freundlich isothermal adsorption models were entered, and the maximum adsorption capacity (Qmax) and distribution coefficient (Kd) were calculated and compared for each adsorbent.

Analyses of clay composition and structure

Composition of the clay minerals was analyzed using inductive couple plasma optical emission spectrophotometry (ICP-OES; Optima 3000, Perkin-Elmer, USA). High adsorptive capacities for aflatoxin B1 of the clay samples were analyzed their structures using X-ray diffraction spectrometry (XRD; X’Pert Pro MRD, PANalytical, B.V., The Netherlands).

Results and Discussion

Means and standard deviations of the binding capacities of these adsorbents are reported in Figure 1. Our results are in as agreement with those reported by Pimpukdee and Tengjaroenkul (2004) indicating that commercial toxin binder COM1 provides the highest adsorption capacity for AFB1. The bentonite clays had moderate adsorption capacities. Bentonite 1 was not significantly different from bentonite 2, but significantly different from bentonite 3. For the Thai clays, the highest adsorption capacity was found for S9. However, there were some other interesting clays, such as S1, S2, S6, S8, S11 and S12, had average capacity greater than 4 x 10-3 mol/kg. Furthermore, clay S12 presented the highest distribution constant. Isotherm shapes are categorized into four types designated as H, L, C, and S which represents different adsorption mechanisms (Phillips 1999; Pimpukdee et al 2000). In this experiment, isothermal adsorption study of AFB1 on different adsorbents at 25C using a series of different concentrations of AFB1 was performed. Similar S-shaped isotherms were observed for all adsorbents in which the amount adsorbed increases as a semi-linear function of concentration (Figure 1). The S isotherm is observed when a molecule does not have a strong affinity for the surface, until there is a significant amount adsorbed, then the slope increases as the affinity for the surface increases (Phillips 1999; Pimpukdee et al 2000). This occurs because the solute molecule has modified the surface, or has begun to bind to previously adsorbed molecules. The results also indicated that each sorbent adsorbed toxin differently especially at high toxin concentration. 

Figure 1. The S-shaped isotherm plots of clay S1 for aflatoxin B1

Figure 1 reports an example of isotherm plots of the clay S1. The results obtained in this study showed that each sorbent could adsorb AFB1, especially at high toxin concentration. Since Langmuir isotherm model (q = Qmax[KdCe/(1+ KdCe)]) is the most appropriate for monolayer adsorption and the modified Freundlich isotherm model (q = Qmax[KdCe]n) is usually applied to multiple-site or multilayer adsorption. The Ce and Kd were selected to fit the data. With user defined functions, the experimental data indicated that all adsorptive materials fitted the modified Freundlich isotherm better than the Langmuir model. The modified Freundlich model showed high correlation coefficients (r2) for all adsorbents in the range of 0.80-0.99 and allowed a quantitative comparison of Qmax and Kd for adsorbents. The fitting result for MFM showed that the clay S1 had the highest average maximum adsorption capacity, whereas the clay S12 has the highest the distribution constant. This study indicated that several Thai clays were capable of sequestering AFB1 from aqueous and the extent of adsorption could depend upon their physicochemical properties and the experimental conditions. Normally, adsorption of AFB1 could occur at original edge sites, external surface, interlayer surface, interlayer exchangeable cations, or previously adsorbed molecules. Small particle size with large active surface as well as internal area are capable to enhance AFB1 adsorption (Kinniburgh 1986; Phillips 1988; Hinz 2001). Previous studies (Phillips et al 1995; Pimpukdee et al 2004) showed that smectite and montmorillonite clays exhibit high adsorption capacity for AFB1 due to their high CEC, high cohesion and adhesion, high equilibrium capacity, high selectivity and extremely large specific surface area. In contrast, some clays (kaolin, mica, talc, pyrophyllite, kaolinite, illite, chlorite) showed little AFB1 adsorption because of their low CEC and lack of internal surface area (Phillips 1988). Thus, the fact that commercial adsorbents showed binding AFB1 differently, this could be due to their various components or mixtures.

The XRD result for a commercial clay BN1 presented montmorillonite as a major component having value of deviated peak of the beam at angle 2q was approximately 6.4. The three highest adsorption capacity of the natural Thai clay (S1, S8 and S9), to adsorb AFB1 also demonstrated that they contained mainly montmorillonite. The clay S1 collected from Lobburi contained montmorillonite greater than that of S8 and S9; however, it also contained critobalite of which the value of deviated peak at angle 2q was approximately 22.2 (Figure 2).

Our findings are important as they suggest that several natural Thai clays have potential to protect animals from the deleterious effects of aflatoxicosis. We proposed that the selection of adsorbents useful for adsorption or detoxification of AFB1 should be based on: high capacity, high affinity with toxin, high selectivity only for the toxin, and not produce nor leave toxic residues in animals.  

Figure 2. The EDX spectrogram of clay S1 for aflatoxin B1

This study indicates that several natural Thai clays could be effective for detoxification of AFB1. However, further research of clay should be verified by in vivo studies.   


This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission.


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Received 15 September 2011; Accepted 19 September 2011; Published 4 November 2011

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