Surfactant modified zeolites––new efficient adsorbents for mycotoxins

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Microporous and Mesoporous Materials 61 (2003) 173–180

Surfactant modified zeolites––new efficientadsorbents for mycotoxins

Magdalena Toma�ssevi�cc-�CCanovi�cc a, Aleksandra Dakovi�cc a,*,George Rottinghaus b, Sr �dan Matija�ssevi�cc a, Mirjana Ðuri�cci�cc a

a Institute for Technology of Nuclear and Other Mineral Raw Materials, P.O. Box 390, 11000 Belgrade, Yugoslaviab College Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA

Received 22 July 2002; received in revised form 6 January 2003; accepted 7 January 2003

Abstract

The in vitro mycotoxins adsorption by natural clinoptilolite––heulandite rich tuff modified with different amounts of

octadecyldimethyl benzyl ammonium chloride (Do) and dioctadecyldimethyl ammonium chloride (Pr) was investigated.

Two methods of preparation of the organo-zeolites (OZs) were used––wet process (activation in suspension) and dry

process (tribochemical process). The chemical stability of the two organically modified zeolites (OZs) obtained by wet

and dry processes was investigated after an electrolyte treatment at pH 1, 7 and 10 by thermal (DTA/DDTA/TG)

analysis and IR spectroscopy. Results of IR and thermal analysis, after electrolyte treatment, confirm that OZs ob-

tained by wet and dry processes are completely stable in the investigated pH region. Mycotoxin binding studies showed

that all the OZs effectively adsorbed aflatoxin B1, zearalenone, ochratoxin A and the ergopeptine alkaloids. The method

of preparation of the OZs had no influence on adsorption of mycotoxins. The non-linear shape of the mycotoxin

adsorption isotherms of OZs suggests that modification of zeolitic tuff with long chain organic cation Pr, by both wet

and dry processes of preparation, formed the same specific active sites which are relevant for adsorption of aflatoxin B1,

zearalenone and ochratoxin A by OZs.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Organo-zeolites; Modification; Activation in suspension; Tribochemical process; Mycotoxins

1. Introduction

Mycotoxins are toxic secondary metabolitesproduced by certain fungi in a number of agri-

cultural products. It has been estimated that my-

cotoxin contamination may affect as much as 25%

* Corresponding author. Tel.: +381-11-3691-722; fax: +381-

11-3691-583.

E-mail address: [email protected] (A. Dakovi�cc).

1387-1811/03/$ - see front matter � 2003 Elsevier Inc. All rights rese

doi:10.1016/S1387-1811(03)00365-2

of the world�s food crops each year [1]. The con-

sumption of mycotoxin contaminated diet may

induce acute and long term chronic effects such asteratogenic, carcinogenic, estrogenic and immu-

nosuppressive effects not only in animals but also

in humans. The most common mycotoxins are the

aflatoxins, ochratoxin A, zearalenone, the tri-

chothecenes, the fumonisins and the ergot alka-

loids.

Mycotoxins are complex organic compounds

that contain different functional groups. For

rved.

mail to: [email protected]

174 M. Toma�ssevi�cc-�CCanovi�cc et al. / Microporous and Mesoporous Materials 61 (2003) 173–180

example, zearalenon is a diphenolic compound,

ochratoxin A in its structure possesses a carboxylic

and phenolic group, while aflatoxin B1 contains a

b-keto-lactone functional group [2].

A practical approach to the prevention of

mycotoxicosis in livestock is the dietary inclu-sion of selective mineral adsorbents that tightly

bind mycotoxins in the gastrointestinal tract

and thereby decrease their bioavailability. Many

effective adsorbents based on the clays––mont-

morillonite and zeolite––clinoptlolite minerals

adsorbed mycotoxins that contain polar func-

tional groups, such as the aflatoxins [3]. The less

polar mycotoxins––zearalenone and ochratoxinA, however are not effectively adsorbed on the

hydrophilic negatively charged surfaces of these

unmodified minerals [4,5]. The modification of

surface properties of negatively charged minerals

and the control of hydrophobicity are possible by

cation exchange of natural charge-balance cations

(Naþ, Kþ, Ca2þ or Mg2þ) with high molecular

weight quaternary ammonium ions. The aminesexchange these cations only on the external sur-

faces of the zeolites whereas in bentonite all ex-

changeable positions are equally available for

reaction with quaternary ammonium ions [6].

Therefore, to achieve the same surface properties

for zeolites less quaternary ammonium ions are

needed than for bentonites [7]. This is important

because the practical use of these chemicallymodified mineral adsorbents in animal feed is

limited by type and concentration of organic

modifier required.

Traditionally, preparation of organo-zeolites

(OZs) and organo-bentonites were done in water

suspensions [8,9], thus the aim of this study was

to compare chemical modification of natural

Ca-clinoptilolite rich zeolitic tuff by octadecyl-dimethylbenzyl ammonium chloride (Do) and

dioctadecyldimethyl ammonium chloride (Pr),

obtained by wet and dry processes. Both processes

were done in a turbo mixer and the obtained

products were used for in vitro adsorption studies

of mycotoxins. The ultimate usefulness of surfac-

tant-modified zeolites depends not only upon their

efficiency for adsorption of mycotoxins but alsoupon their chemical stability in an electrolyte at

different pH values (from pH 1 up to pH 10).

2. Experimental

2.1. Starting material

Sample 1 was clinoptilolite-heulandite zeoliticrich tuff from the Zlatokop deposit in Yugoslavia.

Raw zeolitic tuff was sieved to yield particles <100

lm. The basic characteristics of this zeolite were: atotal cation exchange capacity (CEC)––145 meq/

100 g and an external cation exchange capacity

(ECEC)––10 meq/100 g [10]. Calcium was the

dominant ion in an exchangeable position in this

tuff.Do and Pr supplied by Hoechst AG were used

for preparation of OZs.

2.2. Organo-zeolite preparation

Two different processes were applied for surface

modification of zeolitic tuff: Wet process was per-

formed by treating 100 g of natural zeolitic tuffwith 1000 ml distilled water containing different

amounts of each organic cation (OC). This process

was performed under controlled conditions: tem-

perature )50 �C, mixing speed––5000 rpm, and

reaction time––10 min. When the reaction was

complete, the suspensions were filtrated, washed

and dried at 80 �C. The drying process was per-

formed by treating 100 g of natural zeolitic tuffwith certain amounts of each OC in a beater mill at

9000 rpm for 3 min.

The type of OC, amount in OZ and method of

preparation is summarized in Table 1.

It can be seen from Table 1, that each OC was

added to the starting zeolitic tuff in quantities

below the ECEC value (10 meq/100 g). According

to literature data, when the OC is added inamounts6ECEC, quantitative ion exchange is

observed [6]. Our results are consistent with these

results when the wet process of preparation is

applied. In the case of the drying process, the same

reaction is observed when material is in the contact

with some water solution.

2.3. Organo-zeolite stability

Investigations of the chemical stability of OZs

at different pH values were done on samples 6

Table 1

Properties of the examinated samples (for explanations, see

text)

Sample Z or

OZ

OC Amount of OC

(meq/100 g)

Preparation

process

1 Z – – –

2 OZ Do 2 Wet

3 OZ Do 5 Wet

4 OZ Do 2 Dry

5 OZ Pr 2 Dry

6 OZ Pr 5 Dry

7 OZ Pr 5 Wet

M. Toma�ssevi�cc-�CCanovi�cc et al. / Microporous and Mesoporous Materials 61 (2003) 173–180 175

and 7. The samples (5 g) were placed into 100 ml

electrolyte solution (pH 1, 7 and 10), shaken at 20

�C for 60 min, filtrated and the OZs were dried at

80 �C. No free amines were detected in superna-

tants [11]. The changes in the OZ were followed bythermal (DTA, DDTA and TG) and IR analyses.

Thermal analysis was performed on a Netzsch

STA 409 EP. Samples were heated (20–800 �C) inan air atmosphere, with a heating rate of 10

�Cmin�1. IR spectra were obtained on the Hewlett

Packard IR spectrometer, in the range of 4000–250

cm�1, using KBr pellets.

2.4. Mycotoxin adsorption

For mycotoxin adsorption studies 100 mg of

each zeolite and OZ was added to 10 ml of pH 3

buffered solution containing either aflatoxin B1 (2

ppm), zearalenone (2 ppm), ochratoxin A (2 ppm),

vomitoxin (4 ppm) or ergopeptine alkaloids (0.5

ppm) in duplicate. Samples were placed on a ro-

Table 2

Adsorption of mycotoxins by natural zeolitic tuff (Z) and OZs (OZ)

Adsorption index (%)

Sample 1 Sample 2 Sample 3

Aflatoxin B1 99 99 88

Zeralenone 5 94 98

Vomitoxin 1 <1 <1

Ochratoxin A 40 96 99

Ergosine 92 96 69

Ergotamine 94 97 83

Ergocornine 82 92 60

Ergocryptine 87 98 80

Ergocristine 94 98 87

tator shaker for 30 min, centrifuged and the su-

pernatant analyzed against the original buffered

solution containing each mycotoxin, by HPLC.

Adsorption isotherms were prepared for aflatoxin

B1, ochratoxin A and zearalenone using samples 6

and 7, with solutions buffered at pH 7. OZ (2 mg/ml) was added to test tubes containing different

amounts (1, 2, 3 and 4 lg/ml) of each mycotoxin

and shaken for 30 min. The suspensions were

centrifuged and the supernatants analyzed for

mycotoxins. The amount of adsorbed mycotoxin

was calculated from the difference between initial

and final concentration. Toxin controls were also

prepared containing each mycotoxin without ad-dition of adsorbents. HPLC analyses were per-

formed on a Perkin Elmer 250 Isocratic LC Pump

equipped with a Perkin Elmer ISS 100 auto sam-

pler. The experimental conditions––columns, mo-

bile phases, wavelengths are described in detail in

Ref. [12]. The limit of detection by this method is

20 pg and the error of measure is ±5%.

3. Results and discussion

The summary of mycotoxin adsorption by

seven samples is presented in Table 2.

The results presented in Table 2 showed that

all the OZs effectively adsorbed aflatoxin B1,

zearalenone, ochratoxin A and the ergopeptinealkaloids. The method of preparation of the OZs

had little influence on adsorption of mycotoxins.

Organic modification of the zeolitic tuff by

both processes (wet and dry) changed its surface

Sample 4 Sample 5 Sample 6 Sample 7

100 100 100 97

96 90 98 99

9 6 9 3

97 90 96 99

97 98 96 79

99 99 99 90

94 94 91 65

87 97 96 78

99 99 99 89

Fig. 1. IR spectra for sample 6 before and after electrolyte

treatment at pH 1 (6/1), 7 (6/7) and 10 (6/10).


properties and improved its binding properties for

the less polar mycotoxins such as zearalenone and

ochratoxin A. The presence of long chain OCs on

the zeolitic surface significantly improved the ad-

sorption of ochratoxin A and zearalenone, sug-

gesting that an increase in hydrophobicity of thezeolitic surface probably plays a role in adsorption

of these mycotoxins. There were no differences in

adsorption of zearalenone (2 ppm) and ochratoxin

A (2 ppm) on OZs containing 2 and 5 meq/100 g of

both OCs. On the other hand, unmodified zeolitic

tuff effectively bound only alfatoxin B1 (2 ppm)

and the ergopeptine alkaloids (0.5 ppm). Grant

et al. [13] showed that interaction of aflatoxin B1with Ca2þ ions in the interlamellar regions of a

montmorillonite clay––hydrated sodium calcium

alumosilicate (HSCAS)––may be responsible for

the majority of the adsorption by HSCAS. Con-

sidering that Ca2þ is the main cation at the zeolitic

surface of unmodified tuff suggests that the same

interactions are responsible for adsorption of

aflatoxin B1 on unmodified tuff. Surface modifi-cation of zeolitic tuff with Pr and Do does not

significantly change the adsorption of aflatoxin B1.

It can be seen that adsorption of aflatoxin B1 on

OZs modified with 2 and 5 meq/100 g of Pr was

100%, while 5 meq/100 g of Do decreased the ad-

sorption of aflatoxin B1 as well as the ergopeptine

alkaloids. Vomitoxin was not effectively bound by

either unmodified zeolitic tuff or OZs.The chemical stability of the OZs was investi-

gated by treatment of samples with an electrolyte

at pH 1, 7 and 10. After filtration and drying, the

samples were analyzed by IR spectroscopy and

thermal methods. Two samples containing the

same amount (5 meq/100 g) of OC––Pr were used:

sample 6 is OZ obtained by the dry process and

sample 7 is OZ obtained by the wet process (seeTable 1).

IR spectra of sample 6, before and after treat-

ment at pH 1 (6/1), pH 7 (6/7) and pH 10 (6/10) are

presented in Fig. 1. It can be seen that there is no

difference between the IR spectra, which suggests

that this OZ is stable over a pH range of 1–10.

Sample 7 was also analyzed in a similar manner.

IR spectra for both starting samples (6 and 7),together with spectra after electrolyte treatment at

pH 1, 7 and 10 are presented in Fig. 2.

As can be seen in Figs. 1 and 2, both OZs, be-fore electrolyte treatment, had two intense bands

around 2800 and 3000 cm�1 which were assigned

to asymmetric (vas) and symmetric (vs) stretching

vibration of C–CH2 of the alkyl chain, respectively

[14,15]. IR spectra of OZs obtained by wet and dry

processes were identical, suggesting that in both

cases OCs were present at the zeolite surface.

Following treatment of OZs with electrolytes atpH 1, 7 and 10 there were no changes in peak

positions and their intensities. These results clearly

showed that OZs obtained by the wet and dry

process were stable over the pH region of 1–10.

Similar data were obtained from thermal ana-

lysis. TG, DTA and DDTA curves of OZs 6 and 7,

before and after treatment with electrolyte (pH 1, 7

and 10) are presented in Fig. 3.Before electrolyte treatment, the broad OC ox-

idation exothermic peak can be distinguished on

DTA curves for both samples 6 (341 �C) and 7

(328 �C) (Fig. 3a). That is in accordance with one

derivative peak at 286 �C for sample 6 and 287 �Cfor sample 7 on the DDTA curves (Fig. 3b). Ac-

cording to literature data, the temperature of this

Fig. 2. IR spectra for samples 6 and 7 before and after elec-

trolyte treatment at pH 1, 7 and 10.


exothermic peak is an indication that the OC is

strongly bound to the surface of the zeolitic tuff

[16,17].

It can be observed from DTA and DDTA

curves of the samples treated at pH 1, 7 and 10that exotherm peaks of OC oxidation remain un-

changed regarding intensity and temperature

range. The slope of TG curves of samples 6 and 7

(Fig. 3c), treated with electrolytes, are also similar.

The results of the thermal analysis confirm that

OZs obtained by wet and dry processes are stable

over the pH range of 1–10.

Adsorption of aflatoxin B1, ochratoxin A andzearalenone was studied in detail for two OZ

samples, 6 and 7, at pH 7. The adsorption curves

obtained by plotting the amounts of adsorbed

mycotoxin (lmol/g) against the equilibrium my-

cotoxin concentrations in solution (lmol/l) are

presented in Figs. 4–6.

As can be seen from Figs. 4–6, adsorption of the

three mycotoxins is represented by non-linear ad-

sorption isotherms. Generally, adsorption of these

mycotoxins was high by samples 6 and 7. From the

adsorption curves it can be observed that for

aflatoxin B1 and ochratoxin A there were smalldifferences in their adsorption on samples 6 and 7.

The adsorption of aflatoxin B1 was higher for

sample 7 (Fig. 4), while adsorption of ochratoxin

A was higher for sample 6 (Fig. 5). However, the

maximum amount of aflatoxin B1 adsorbed by

sample 6 was 4.48 and 4.81 lmol/g by sample 7.

The maximum adsorbed amount of ochratoxin A

was 3.66 lmol/g by sample 6 and 3.22 lmol/g bysample 7. The maximum amount of zearalenone

adsorbed was 5.09 and 5.21 lmol/g by samples 6

and 7, respectively. From the maximum amounts

of each mycotoxin adsorbed by samples 6 and 7 it

can be concluded that the method of preparation

of OZ had no influence on the mycotoxin ad-

sorption.

It is well known that binding of mycotoxins byadsorbents is dependent on the properties of the

mycotoxins, like polarity, solubility in water, size,

shape etc. [18]. Aflatoxin B1 is a hydrophobic

molecule that is only slightly soluble in water,

suggesting that aflatoxin B1 would partition on the

hydrophobic surfaces of these modified minerals

[13]. It was already shown that interaction of

aflatoxin B1 with Ca2þ ions at the zeolitic surfaceis essential for adsorption of this toxin on un-

modified tuff. On the other side, high aflatoxin B1

adsorption indexes were achieved on all modified

OZs (Table 2) demonstrating that partitioning

may occur. Partitioning of non-polar molecules by

adsorbents is usually represented by linear ad-

sorption isotherms [9,19]. However, the aflatoxin

B1 adsorption isotherms on two OZs––samples 6and 7 (Fig. 4) were non-linear suggesting that

partitioning is not the only one mechanism rele-

vant for aflatoxin B1 adsorption on OZs. Con-

sidering that the amount of OC on the surface of

OZ is much below the ECEC value, it is possible

that Ca2þ ions on an uncovered surface are addi-

tionaly responsible for aflatoxin B1 adsorption.

Zearalenone and ochratoxin A are also fairlynon-polar molecules, less soluble in water. The

shape of the zearalenone and ochratoxin A

Fig. 3. DTA (a), DDTA (b) and TG (c) curves for OZs before (6 and 7) and after electrolyte treatment at pH 1 (6/1 and 7/1), pH 7 (6/7

and 7/7) and pH 10 (6/10 and 7/10).

0

1

2

3

4

5

6

0 1 2 3 4 5

in solution aflatoxin B1, µmol/l

adso

rbed

afl

atox

in B

1, µ

mol

/g

7

6

Fig. 4. Aflatoxin B1 adsorption on OZs (samples 6 and 7, pH¼ 7).


adsorption isotherms on OZs (samples 6 and 7)

were also non-linear (Figs. 5 and 6). It has already

been shown that the presence of large OCs at the

surface greatly improved zearalenone and ochra-

toxin A adsorption by OZs (Table 2). These results

suggest that hydrophobicity plays a role in ad-

sorption of zearalenone and ochratoxin A. The

non-linear shape of the isotherms for ochratoxin A

0

1

2

3

4

5

6

0 1 2 3 4 5

in solution ochratoxin A, µmol/l

adso

rbed

och

rato

xin

A, µ

mol

/g

6

7

Fig. 5. Ochratoxin A adsorption on OZs (samples 6 and 7,

pH¼ 7).

0

1

2

3

4

5

6

0 2 3 4 51

in solution zearalenone, µmol/l

adso

rbed

zea

rale

none

, µm

ol/g

6

7

Fig. 6. Zearalenone adsorption on OZs (sample 6 and 7,

pH¼ 7).


and zearalenone adsorption on samples 6 and 7,

indicates that an additional mechanisms may be

involved.

However, the shape of non-linear adsorption

isotherms of three investigated mycotoxins is dif-ferent suggesting that the hydrophobic character,

shape of molecule, their size etc. may have an in-

fluence on the adsorption process.

It is possible that modification of zeolitic tuff

with long chain OC Pr by both wet and dry pro-

cesses of preparation formed the same specific

active sites, which are relevant for adsorption of

aflatoxin B1, zearalenone and ochratoxin A by

OZs.

4. Conclusions

The surface modification of clinoptilolite rich

tuff by wet and dry processes resulted in prepara-

tion of OZs. Results of IR and thermal analysis of

two OZs, after electrolyte treatment at pH 1, 7 and

10, confirm that OZs obtained by wet and dry

processes are completely stable over the pH region

from 1 to 10. From the results of mycotoxins ad-

sorption by modified zeolitic tuff, it can be con-cluded that addition of 2 meq/100 g of either Do or

Pr to zeolitic rich tuff maximized the adsorption of

mycotoxins. These results suggest that under these

circumstances, less than 1% of the organic phase in

the final product could be utilized as an additive

for animal feed if in vivo trials are successful.

Acknowledgements

This work is part of research activities which

are conducted throughout the Project ‘‘The phys-ical-chemical and biochemical base of production

and application of new materials for toxic sub-

stances’’, financed by the Science Fund of Serbia.

The mycotoxin in vitro binding studies were done

at the Fusarium/Poultry Research Laboratory,

Veterinary Medical Diagnostic Laboratory, Uni-

versity of Missouri, Columbia, Missouri, MO,

65211, USA. The authors wish to express specialthanks to Drs George Rottinghaus, David Ledoux

and Alex Bermudez for financial support of this

research.

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Surfactant modified zeolites––new efficient adsorbents for mycotoxins

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