The effect of exchangeable cations in clinoptilolite and montmorillonite on the adsorption of aflatoxin B1

Author/s :
Magdalena Tomaševic-Canovic* - Aleksandra Dakovic - Vesna Markovic - Dragan Stojšic1

Institute for Technology of Nuclear and Other Mineral Raw Materials, P. O. Box 390, YU-11001,
Belgrade and 1 VMA - Institute for Hygiene, YU-11000, Belgrade, Yugoslavia


(Received 25 January 2001)

The adsorption of aflatoxin B 1 (AFB1) by cation-exchanged clinoptilolite
zeolitic tuff and montmorillonite was investigated at 37 ºC and pH 3.8 from an aqueous
electrolyte having a composition similar to that of gastric juices of animals. Both minerals
were exchanged from the natural form to the sodium form and then to the Cu 2+ ,Zn 2+
and Co 2+ -rich forms. The cation exchange was different for the different cations, but in
all cases the exchanges were larger on montmorillonite than on clinoptilolite. The degree
of exchange on montmorillonite was 76 % for copper (from a total of CEC 0.95
meq/g, Cu 2+–0.73 meq/g) and 85 % for zinc and cobalt. Under the same conditions
(concentration, temperature, pH, contact time), the degree of exchange on zeolitic tuff
was 12 % for Cu 2+ (from a total CEC of 1.46 meq/g, Cu 2+ –0.17 meq/g), 8 % for Zn 2+
and 10 % for Co 2+ . Both groups of mineral adsorbents showed high AFB1 chemisorption
indexes (ca). For the montmorillonite forms, ca ranged from 0.75 for the
Cu-exchanged montmorillonite to 0.89 for the natural Ca-form, 0.90 for the
Zn-exchanged form and 0.93 for the Co-exchanged montmorillonite. The adsorption of
AFB1 on the different exchanged forms of clinoptilolite gave similar values of ca for the
Cu and Ca forms (0.90) and values of 0.94 and 0.95 for the Zn- and Co-exchanged form.
The impact of the mineral adsorbents on the reduction of essential nutrients present in
animal feed (Cu, Zn, Mn and Co) showed that the Ca-rich montmorillonite had a higher
capability for the reduction of the microelements than the Ca-rich clinoptilolite.

Keywords: clinoptilolite, montmorillonite, cation exchange, adsorption, aflatoxins.


INTRODUCTION


Aflatoxins comprise a diverse group of pervasive, naturally occurring, fungal
elaborated poisons that have been strongly implicated in animal diseases. Aflatoxin B 1
(AFB1) is the most toxic and cancerogenic of the aflatoxins.1

Phillips et al.2-4 and Tomaševic-Canovic,et al.5 have shown that certain natural
aluminosilicates (bentonite and zeolite) may be used, in animal diets, to prevent signs of certain mycotoxicoces. These mineral materials bind aflatoxins, forming highly stable
complexes. Hydration of the exchangeable cations creates a hydrophilic environment
on the surface of zeolites and on the surface and in the interlayer region of montmorillonite.
This parameter has an influence on the adsorption of different organic molecules,
including mycotoxins, on zeolite and montmorillonite particles and on the stability of
the adsorbed complexes.4,6,7 A proposed mechanism of aflatoxin chemisorption by
mineral adsorbents involves the rapid formation of a complex between a ligand and the
mineral.8

Although these mineral adsorbents are added to animal rations to prevent the neg-ative
effects of aflatoxins, their impact on other components of the feed (vitamins,
aminoacids, and microelements) is not well known. Chung and Baker 9 reported effects
of montmorillonite on phosphorous and Chung et al.10 on Zn, Mn, vitamin Aand ribo-flavin.
They noted no reductive effects on phosphorous, Mn, vitamin A, and only a
slight reduction in Zn utilization. The addition of 0.2–0.5 %clinoptilolite to basal diets
did not impair the utilization of tryptophane, phenylalanine, vitamin A, D and E.11 The
in vitro adsorption of vitamin B6 on different mineral adsorbents showed that the Ca and
H form of clinoptilolite adsorbed this vitamin to about 20 %. On the contrary, vitamin
B6 was tightly bound to hydrate sodium calcium aluminosilicate (HSCAS)-mineral adsorbent
based on montmorillonite (98 %).12

The objective of the present study was to evaluate the affinity of different cation-
exhanged forms of clinoptilolite and montmorillonite for aflatoxin B1 in vitro.In
addition, the impact of natural Ca-rich clinoptilolite and Ca-rich montmorillonite on essential
microelements (Cu, Zn, Co and Mn) present in animal feed were examined.


EXPERIMENTAL

Preparation of the different forms of the adsorbents

The natural zeolite sample used in this study was a clinoptilolite-rich tuff from the Zlatokop
deposit (Vranjska Banja, Yugoslavia). From X-ray diffraction analysis, the content of clinoptilolite
was = 90 %. The chemical composition of this sample was as follows (wt. %): SiO2 – 64.21, Al2O3
11.48, Fe2O3 – 0.88, CaO – 4.55, MgO – 1.45, Na2 O –1.71,K2 O – 1.29, L.I. – 14.0. The cation exchange capacity (CEC) was 1.46 meq/g. The sample was crushed and sieved, and the fraction <63 µm was selected for study.

The Ca-rich clinoptilolite tuff (10 %suspension) was reacted with2MNaClsolution to obtain
a Na-rich clinoptilolite tuff. The suspension was continuously stirred for 24 h at room temperature,
then the supernatant was decanted off and the residue washed with deionized water until Cl- ions were no longer delectable. The obtained sample was air dried at 105 ºC.

A natural Ca-rich bentonite from the [ipovo deposit in Bosnia with average particle size < 5 µm was used. It contained about 90 % montmorillonite and a small amount of quartz and calcite
(X-ray diffraction analysis). The chemical composition was as follows (wt. %): SiO2 –55.36, Al2O3
–22.94, Fe2O3 –3.65, CaO –3.58, MgO –3.27, Na2O –0.11, K2 O –0.32, L.I. –12.31. The total CEC
was 0.95 meq/g.

The Na-exchanged form of montmorillonite was obtained by passing a 5 %suspension of Ca-rich bentonite through a glass column filled with Woffatit KPS resin.

Cation exchange of both clinoptilolite and montmorillonite was carried out in an aqucons suspension after the addition of inorganic salts: CuSO4 . 5H2 O, ZnSO4 . 7H2 O and CoCl2 . 6H2 O (supplied by Merck). The initial concentration of the metal ions before ion exchange was 1.50 meq/g for
clinoptilolite and 1.00 meq/g for montmorillonite. Those concentrations satisfied the CEC of the
starting zeolitic tuff and of Ca-rich montmorillonite. The contents of exchangeable cations in all the
mineral adsorbents used for AFB1 adsorption are listed in Table I.

TABLE I. The contents of exchangeable cations in different forms of clinoptilolites and montmorillonites

Mineral adsorbent

Exchangeable cations/(meq/g)
Ca2+
Mg2+
Na+
K+
Cu2+
Zn2+
Co2+

Ca-rich (natural) clinoptilolite

0.95
0.13
0.22
0.16
Na-exchanged clinoptilolite
0.38
0.14
0.81
0.16
Cu-exchanged clinoptilolite
0.39
0.13
0.59
0.16
0.17
Zn-exchanged cilinoptilolite
0.39
0.14
0.65
0.16
0.12
Co-exchanged clinoptilolite
0.37
0.14
0.65
0.16
0.15
Ca-rich montmorillonite
0.89
0.04
0.006
0.01
Cu-exchanged montmorillonite
0.08
0.04
0.046
0.01
0.73
Zn-exchanged montmorillonite
0.08
0.04
0.016
0.01
0.81
Co-exchanged montmorillonite
0.08
0.04
0.006
0.01
0.81


From the results presented in Table I, it can be seen that the amounts of Cu, Zn and Co exchanged by montmorillonite were much higher than by clinoptilolite. The degree of ion exchange for each cation was calculated as (x/?) . 100 where x represents the amount of cation (Cu, Zn or Co) in an exchangeable position and ? is the sum of the exchangeable cations in the starting sample (clinoptilolite tuff or montmorillonite). For the different cationic forms of montmorillonite, the degree of ion exchange was 76 % for Cu and 85 % for Zn and Co. For the different forms of clinoptilolite, the degree of ion exchange was 12 %for Cu, 10 %for Co and 8 %for Zn. The obtained cation-exchanged clinoptilolites and montmorillonites were used for AFB1 adsorption.


Adsorption of aflatoxin B1

Aflatoxin B1 (AFB1) was obtained from Sigma Co. The solution from which the adsorption was to be examined was chosen to simulate the gastric juice of animals (electrolyte). It contained: 0.1
mol/dm3 HCl and 0.05 mol/dm3 NaCl. The content of AFB1 was determined in the AFB1 containing
electrolyte both without and with a mineral adsorbent. The experiment was carried out at pH 3.8 and at a temperature of 37 ºC. Acertain amount of AFB1(200 µg) was added to 100 cm3 lectrolyte and an aliquot (0.4 cm3 ) was taken for the determination of the total toxin concentration present in the solution (ct ). Then,1 g of mineral adsorbent was added to the contaminated electrolyte solution. At the end of the reaction time (2 h), the concentration of non-adsorbed AFB1 was determined in the supernatant (cf ). The total and non-adsorbed concentrations of AFB1 were determined, after chloroform extraction, by the HPLC method. The chromatographic analysis was performed on a Bio-Sil C18 HLColumn (250 x 4.6 mm; 5µm particle size) using a LKB Broma Chromatograph, Model 215 HPLC Pump with a RHEODINE 7125 injector.


Reduction of the microelements


To satisfy the physiological needs of the animals, cattle fodder must contain 10 ppm Cu, 100
ppm Zn, 80 ppm Mnand 0.8 ppm Co. At the same time, for the prevention of mycotoxicosis, about 0.5 % of a mineral adsorbent should be added. In this way, a certain ratio between mineral adsorbent– microelement is achieved requiring the determination of any possible reduction in the amounts of the available microelements present.

The following procedure was used: Acertain amount of each microelement: Cu–2 mg, Zn–20 mg, Mn–16 mg and Co–0.16 mg was added to 100 cm3 of the electrolyte and an aliquot was taken for the determination of the starting concentrations of the cations present in the solution (cg) Then, 1 g of mineral adsorbent was added to the electrolyte. After gentle shaking for 2 h, the concentrations of the non-exchanged microelements were determined in the supernatant (cf). The total and non-exchanged concentrations of the microelements were determined by atomic absorption spectrophotometry noing a Perkin Elmer, Model 703 instrument.


RESULTS AND DISCUSSION

The chromatograms of AFB1 (200 µg) determined in the electrolyte without (1) and with different ion exchanged clinoptilolites (2–6) are presented in Figure 1, while similar chromatograms without (1) and with different ion exchanged forms of mon-tmorillonites (2–5) are presented in Fig. 2.
The chemisorption index4 for the different mineral adsorbents was calculated as:
ct - cf
ca =
_______________
ct

where ct = total concentration of AFB1 and cf = the concentration of non-adsorbed
AFB1; ca = 1 represents total binding of AFB1.


The determined AFB1 chemisorption indexes (ca) for the different ion exchanged clinoptilolites and montmorillonites are presented in Table II.

ca
Clinoptilolite
Montmorillonite

Ca-rich (natural)

0.90
0.89
Na-exchanged
0.93
Cu-exchanged
0.90
0.75
Zn-exchanged
0.94
0.90
Co-exchanged
0.95
0.93


For all the used ion exchanged mineral adsorbents, high adsorption indexes were found. These results show that both groups of minerals have a high efficiency for in vitro binding of AFB1. The chemisorption indexes for different cation exchanged montmorillonites were between 0.75 for the Cu-exchanged form and as high as 0.93 for the Co-exchanged montmorillonite. All the clinoptilolite materials had greater adsorption values (ca = 0.90) than the different forms of montmorillonite. The Ca-rich and Cu-exchanged clinoptilolite showed chemisorption indexes of 0.90, the Na-exchanged clinoptilolite 0.93 and Co-exchanged clinoptilolite 0.95.

The Cu-exchanged form of montmorillonite had the lowest binding affinity for AFB1 (ca = 0.75), but the Co-forms of both minerals were the best (ca from 0.93 to 0.95).

The presented in vitro data clearly demonstrate that all of the used mineral adsorbents greatly diminished the toxicity of AFB1 at a concentration of 200 µg per g of adsorbent. This value corresponds to 1 mg/kg of AFB1 in the diet, if the adsorbent is incorporated into the diet at a level of 0.5 %. Considering the toxicity of AFB1, most countries allow a concentration in animal feed of no more than 20 ppb (0.020mg/kg).

The results of the reduction of the concentrations of the microelements (Cu, Zn, Co and Mn) by cation-exchange on Ca-rich clinoptilolite and Ca-rich montmorillonite, at pH 3.8 (the same electrolyte as for the AFB1 adsorption) are presented in Table III.
The initial microelement concentration (milligram per gram of adsorbent) (ct) was obtained based on the addition of an adsorbent to the diet at the 5 g/kg level.



TABLE III. The chemisorption indexes (ca) of the microelements (Cu, Zn, Mn, Co) on Ca-rich
clinoptilolite and Ca-rich montmorillonite

Micro element
ct (mg/g)
ca
Ca-rich clinoptilolite
Ca-rich montmorillonite
Cu
2
0.23
0.80
Zn
20
0
0.18
Co
0.16
0.10
0.87
Mn
16
0.03
0.15

The results presented in Table III suggest that Cu and Co showed high chemisorption indexes, ca = 0.80, for Ca-rich montmorillonite, but for Ca-rich clinoptilolite tuff ca = 0.20. The chemisorption indexes for Zn and Mn on both minerals were = 0.20.

Examination of the impact of the mineral materials on the microelements clearly show a greater reduction of the concentrations of the microelements by montmorillonite materials than by clinoptilolite materials. The results indicate that Cu and Co are highly adsorbed by Ca-rich montmorillonite (ca = 0.80), whereas on Ca-rich clinoptilolite, copper has a chemisorption index of 0.23 and cobalt a value of 0.10. Zinc and
manganese were not bound as strongly on montmorillonite as Cu and Co (ca = 0.20).
Also, Zn and Mn showed negligible adsorption by clinoptilolite materials; for Mn ca = 0.03. Clearly Ca-rich montmorillonite has a greater capability of reducing the amounts of microelements in animal feed than Ca-rich clinoptilolite.


CONCLUSION

The obtained results show that different exchanged forms of clinoptilolites and montmorillonites adsorbed substantial amount of aflatoxin B1 . Generally, the different exchanged forms of clinoptilolite have higher aflatoxin B1 chemisorption indexes than the same forms of montmorillonite. For both minerals, the Co-exchanged forms showed the greatest adsorption and the Cu-exchanged form of montmorillonite the
lowest. The results of microelement reduction suggest that the Ca-rich montmorillonite material would greatly reduce the amount of micronutrient present in the animal feed, compared with Ca-rich clinoptilolite.


REFERENCES

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