Simultaneous adsorption of aflatoxin B1 and zearalenone by mono- and di-alkyl cationic surfactants modified montmorillonites

Accepted Manuscript Simultaneous adsorption of aflatoxin B1 and zearalenone by mono- and di-alkyl cationic surfactants modified montmorillonites Gaofeng Wang, Yushan Miao, Zhiming Sun, Shuilin Zheng PII: DOI: Reference:

S0021-9797(17)31105-0 https://doi.org/10.1016/j.jcis.2017.09.074 YJCIS 22826

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

20 August 2017 15 September 2017 20 September 2017

Please cite this article as: G. Wang, Y. Miao, Z. Sun, S. Zheng, Simultaneous adsorption of aflatoxin B1 and zearalenone by mono- and di-alkyl cationic surfactants modified montmorillonites, Journal of Colloid and Interface Science (2017), doi: https://doi.org/10.1016/j.jcis.2017.09.074

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Simultaneous adsorption of aflatoxin B1 and zearalenone by mono- and di-alkyl cationic surfactants modified montmorillonites Gaofeng Wang, Yushan Miao, Zhiming Sun*, Shuilin Zheng**

School of Chemical and Environmental Engineering, China University of Mining and Technology (Beijing), Beijing 100083, China

*Corresponding authors. Fax: +86 10 62339920 (Z. Sun); +86 10 62331345 (S. Zheng) E-mail address: [email protected] (Z. Sun); [email protected] (S. Zheng) 1

Abstract. Organo-montmorillonites (OMts) modified with mono- and di-alkyl cationic surfactants were prepared to remove polar mycotoxin aflatoxin B1 (AFB1) and weak polar, hydrophobic mycotoxin zearalenone (ZER) simultaneously. The structural and surface properties of the prepared OMts were investigated. In vitro adsorption experiments were carried out to simulate the in vivo conditions of gastrointestinal tract of animals by a batch mode. The adsorption of AFB1 and ZER in both single and binary-contaminate systems were investigated systematically. Both OMts showed super enhanced adsorption capacities towards AFB1 and ZER whenever in single and binary-contaminate systems compared with raw Mt, indicating the effectiveness of the prepared OMts acted as mycotoxins adsorbents. DODAC-Mt showed a higher adsorption capacity towards AFB1 and ZER than OTAB-Mt. The equilibrium data of AFB1 on OMts were fitted satisfactorily with Freundlich and Linear models, suggesting the co-existence of different adsorption mechanism which were proposed to be ion-dipole interactions (between surfactant cations and carbonyl groups of AFB 1) and adsorption/partition mechanisms. The adsorption isotherms of OMts to ZER matched best with Linear models, implying the adsorption/partition mechanism. For simultaneous adsorption, the adsorption process of one mycotoxin was slightly affected by the presence of the other mycotoxin due to the requirement of partial same sorption sites. In addition, the solution pH had negligible influence on the adsorption process of OMts, meaning no desorption occurred when the adsorbents pass through from stomach to intestine as animal feed.

Keywords:

Simultaneous

adsorption;

Organo-montmorillonite

2

Aflatoxin

B1 ;

Zearalenone;

1.

Introduction In recent years, contamination of cereal grains and animal feeds by mycotoxins

has become a serious concern in the world. It has been estimated that 25% of food crops over the world are contaminated with mycotoxins each year [1]. Mycotoxins are a group of structurally diverse secondary fungal metabolites including aflatoxins, zearalenone, ochratoxins, trichothecenes, fumonisins and ergot alkaloids. Many of these mycotoxins can cause adverse health effects or even death in humans and animals. For example, aflatoxin, a common mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus, was reported to cause serious biochemical and structural alterations in different organs, including liver, lungs, kidneys and heart [2, 3]. It was highly toxic to humans as well as to several animal species such as pigs and poultry [4-6] due to their mutagenicity, and carcinogenicity [7, 8]. Consequently, effective removal of mycotoxins which are highly toxic to human and animals is becoming one of the most urgent challenges. At present, the most promising and economical approach to detoxifying mycotoxins contaminated animal feedstuffs is addition of nutritionally inert mineral adsorbents to the diet to decrease the bioavailability of mycotoxins in the gastrointestinal tract [9, 10]. Previous studies have demonstrated that natural phyllosilicate minerals especially montmorillonite showed effectiveness in binding polar mycotoxin aflatoxins and reducing their toxicity [11-16]. However, the hydrophilic negatively charged surfaces of montmorillonites were less effective in binding low polar and hydrophobic mycotoxins such as zearalenone and ochratoxin [17, 18]. These mycotoxins are also found to be carcinogenic, genotoxic, and immunotoxic in livestock and humans as well. In order to improve the adsorption capacity of the natural adsorbents for low polar and hydrophobic mycotoxins, organic modification of aluminosilicate clay minerals with organic surfactants such as cetylpyridinium and octadecyldimethylbenzylammonium has been proposed [19, 20]. It resulted in increased hydrophobicity of the mineral surface, providing a high affinity for in vitro adsorption of hydrophobic mycotoxins. However, much work so far has just focused on the removal of single mycotoxin, 3

but simultaneous removal of multiple mycotoxins with different physical and chemical properties receives little attention. Multiple mycotoxins such as aflatoxins, zearalenone, ochratoxin and fumonisins always co-contaminate crops and foods intended for both animal and human consumption [21]. On one hand, the presence of a mixture of these mycotoxins may present a problem in terms of determining clinical symptoms of an individual mycotoxicosis. On the other hand, concomitant exposure to mycotoxins has been associated with higher teratogenic, mutagenic, estrogenic, neurogenic and immunotoxic risks than individual one. Thus, simultaneous removal of multiple mycotoxins with different biotoxicity is more meaningful to practical application. Moreover, comparative study between mono- and di-alkyl cationic surfactants modified montmorillonite and their adsorption properties for multiple mycotoxins has not been studied before. In this work, organo-montmorillonites (OMts) modified with mono- and di-alkyl cationic surfactants were prepared through ion exchange. XRD, TOC, FT-IR, hydrophobicity and BET were employed for the characterization of the products. The polar mycotoxin aflatoxins B1 (AFB1) and low polar, hydrophobic mycotoxin zearalenone (ZER) whose chemical structures and basic information were presented in Table 1 were selected as the target pollutants to evaluate the adsorption performance of the prepared OMts. The mono- and di-alkyl surfactants on the structures, surface properties and adsorption performances for AFB1 and ZER were compared systematically. Besides, the adsorption process of AFB 1 and ZER were investigated in both single and binary-contaminant systems, and the adsorption mechanisms involved in the adsorption process of AFB1 and ZER by the OMts were discussed as well. Table 1 Chemical structures and basic information of aflatoxin B1 and zearalenone Name

Aflatoxin B1

4

Zearalenone

Structure formula

2.

Molecular formula

C17H12O6

C18H22O5

Molecular weight (g/mol)

312.3

318.36

Solubility (mg/L)

10–30

max 20

Dipol moment (D)

9.5

2.2

Materials and methods

2.1 Materials The Ca-montmorillonite used in this study is supported by Baifubang Mining Fertilizer Co. Ltd. from Neimenggu province, China. The cation exchange capacity (CEC)

of

Mt

is

68.29

meq/100g.

The

mono-alkyl

cationic

surfactant

octadecyltrimethylammonium bromide (OTAB) and di-alkyl cationic surfactant dioctadecyldimethylammonium chloride (DODAC) with a purity of 99% were obtained from Aladdin Industrial Co., China. Aflatoxins B1 (AFB1) and zearalenone (ZER) were purchased from Fermentek Co. Ltd. Other chemical reagent such as phosphoric acid, potassium phosphate monobasic, dipotassium phosphate, acetonitrile and methyl alcohol were all purchased from Beijing Reagent Co. (Beijing, China). Acetonitrile and methyl alcohol are chromatographic grade, and other reagents are analytical grade and used without further purification.

2.2 Preparation of the OMts A stoichiometric amount of surfactants were firstly dissolved in 200 mL of deionized water at 50 °C under stirring for 0.5 h, and then a 2 g of Mt was slowly added into the surfactant solution. The amounts of surfactants added was 0.5, 1.0, 1.5 and 2.0 CEC of Mt and the mass ratio of water/clay mineral was 100:1. The mixture was firstly treated on an ultrasonic bath with 120Hz for 1 h at 60 °C and then stirred 5

for 24 h at 60 °C with a Kexi magnetic stirrer. In the following step, the prepared OMts were washed with deionized water, and then dried in an oven at 60 °C for 24 h. The products prepared by OTAB and DODAC at a concentration of 0.5 CEC were denoted as “0.5 CEC-OTAB” and “0.5 CEC-DODAC”, respectively. The others were denoted in a similar way.

2.3 Characterization of adsorbents Powder X-ray diffraction (XRD) patterns were recorded between 2° and 12° on a Bruker D8 diffractometer with Cu Kα radiation (λ=0.15406 nm) operating at 40 kV and 40 mA at a step size of 0.02. Elemental analysis of C and H was conducted on a Germany Vario Elementar cube. Fourier transform infrared (FT-IR) spectroscopy was observed in the wavenumber region of 4000–700 cm-1 with a Nicolet IS10 spectrometer using KBr pellets. The hydrophobicity of OMts were deduced from the moisture adsorption experiments under 20 ℃ with the relative humidity of 33%. The experiments were operated in a programmble temperature and humidity test chamber. Pore structure parameters such as surface area, pore volume and average pore size were measured from N2 adsorption–desorption isotherms using a Micromeritics ASAP2020 instrument. The samples were degassed at 105 ℃ for 12 hours prior to N2 adsorption.

2.4 In vitro adsorption experiments The in vitro adsorption experiments of AFB1 and ZER simulated the in vivo conditions of the gastrointestinal tract of animals and were performed at 37 ℃ by a batch mode. Initially, a primary AFB1 (36.4 mg/L) and ZER stock solution (100 mg/L) was prepared in chromatographic grade methyl alcohol. Then, 10 mg of raw Mt and the prepared OMts were added into 10 ml aliquots of phosphate-buffered solution containing AFB1 and ZER at different initial concentrations. The mixture was placed on a rotary shaker for 60 min, centrifuged for 5 min at 10,000 rpm, and 1 ml of the aqueous supernatant was filtered with 0.22 μm filter membrane for HPLC analysis. Moreover, a control treatment without adsorbent was carried out for each experiment 6

in case of any possible nonspecific binding of AFB1 and ZER. The adsorption experiments included two different systems: (1) adsorption in single system and (2) simultaneous adsorption. For system (1), the adsorption contained only a single contaminant with an initial concentration of 1.000-2.000 mg/L for AFB1 and 10.000-20.000 mg/L for ZER, respectively. The pH of buffer solution was adjusted to 3.5 by adding small amounts of 0.1 M H3PO4. For system (2), the adsorption contained two different contaminants simultaneously. The initial concentration of one contaminant was set as the same as that in system (1), and the other one was kept as a constant of 1.456 mg/L for AFB1 and 12.000 mg/L for ZER, respectively. Similarly, the effect of solution pH on the adsorption of AFB1 and ZER was studied by adjusting the pH of phosphate-buffered solution to 3.5, 6.5 and 9, respectively. All adsorption experiments in this paper were carried out in triplicate and the averaged values were presented. HPLC analyses were performed on an Agilent 1200 bin pump with a G1329A autosampler and a 4.6 × 250 mm XB-C18 column (welchrom, China). Isocratic elution was performed with a fluorescence detector at an excitation of 360 nm and an emission of 440 nm for AFB1, an excitation of 274 nm and an emission of 440 nm for ZER. The mobile phase water and acetonitrile (v/v=50:50) was pumped at a flow rate of 1.0 mL/min. Calibration curves were achieved using eight standards over the concentration range of the sorption samples and repeated for three times. The adsorbed amount (%) of AFB1 and ZER was calculated from the difference of the peak area between stock solution and equilibrium supernatant:

 A  R= 1  1   100% A0  

(1)

where A0 and A1 are the peak area of stock solution and equilibrium supernatant analyzed by HPLC method.

3.

Results and discussion

3.1 Characterizations 7

The XRD patterns of raw Mt and OMts prepared with OTAB and DODAC at different surfactant loadings were presented in Fig. 1. The basal spacing of original Mt was 14.10 Å (Fig. 1a), indicating a typical 001 plane of calcium montmorillonite. The expansion of interlayer spacing of OMts could be observed obviously. For OTAB modified OMts, the d-spacings expanded to 14.52 Å, 20.53 Å and 22.52 Å, respectively at 0.5, 1.0 and 1.5 CEC level. However, when the surfactant loading was increased to 2.0 CEC, two d (001) spacings of 30.22 and 21.12 Å were obtained. The little expansion of 14.52 Å at 0.5 CEC suggested a 4.92 Å of interlayer spacing calculated by subtracting the thickness of Mt unit (9.6 Å). The value was close to the size of the “nail-head” of the cationic surfactant [22, 23], which implied a lateral monolayer arrangement of surfactant molecules in the interlayer space of Mt. For 1.0 CEC-OTAB, a basal spacing at 20.53 Å showed the transition status varying from lateral monolayer to lateral bilayer arrangements. The interlayer spacing of 22.52 Å at 1.5 CEC reflected a paraffin-type monomolecular arrangement. Taking the length of surfactant size and the thickness of TOT layer into account, it can be calculated that the angle (α) between the alkyl chain and basal surface is approximately 29.64°. Finally, the basal spacing of 30.22 Å with a shoulder of 21.12 Å at 2.0 CEC implied a paraffin-type monomolecular arrangement companied with a lateral bilayer arrangement. Compared with the exchange of OTAB, the modification of Mt by DODAC results in larger expansions. The 0.5 CEC-DODAC resulted in expansion to 14.43 Å, which was also attributed to the lateral monolayer arrangement. The 1.0 CEC-DODAC had two d (001) spacings of 26.91 and 14.11 Å, and the small shoulder was considered to the lateral monolayer arrangement due to the same d-value with 0.5 CEC-DODAC, but the basal spacing of 26.91 Å was corresponding to the paraffin-type monomolecular arrangement. At higher surfactant loadings (1.5 and 2.0 CEC), three basal spacings of OMts were observed. The peaks at 29.83 Å of 1.5 CEC-DODAC and 33.96 Å of 2.0 CEC-DODAC were postulated to the paraffin-type monolayer arrangement, while the peaks at 20.82 Å and 17.66 Å corresponded to the 8

lateral bilayer arrangements. The small peaks at 12.41 Å and 12.03 Å were ascribed to the lateral monolayer arrangement. The co-existence of various molecular structure configuration in the interlayer space of Mt indicated that the steric effect of DODAC has taken into effect [24]. At the same surfactant loadings, only 2.0 CEC-OTAB had two types of molecular arrangements, but all DODAC modified OMts excepted for 0.5 CEC-DODAC had more than one molecular arrangement. This may lead to the properties difference between mono- and di-alkyl cationic surfactant modified OMts such as organic carbon content, packing density, surface hydrophobicity and porous structure nature which would influence the adsorption performance of the OMts [25].

Fig.1. XRD patterns of Ca-montmorillonite and the prepared OMts intercalated with (a) OTAB and (b) DODAC.

The organic carbon contents of OMts were obtained from elemental analysis of C, H and N, which was summarized in Table 2. It can be seen from Table 2 that the contents of C, H and N increased with increasing the surfactant loadings (confirmed further by FT-IR spectra illustrated in Fig. S1 and Fig. S2 in supplementary material), and the C content of OMts reached to 24.39% and 32.01% at 2.0 CEC, respectively. The high organic carbon content can function as a partition medium for the adsorption 9

of non-polar and hydrophobic contaminants. In addition, DODAC modified OMts had higher C and H content than OTAB modified OMts at the same surfactant loadings, which was ascribed to the presence of double carbon chains in DODAC. The genuine surfactant loading intercalated into the interlayer and/or adsorbed on the external surface of Mt were calculated based on the N content. It was observed that less DODAC combined with Mt than OTAB when adding the same surfactant loadings resulted from the steric effect of DODAC. The 1.0 CEC-OTAB and 1.0 CEC-DODAC had 0.97 CEC of OTAB and 0.81 CEC of DODAC, implying the very little residue of exchangeable cations. The hydrophobicity of OMts were deduced from the moisture adsorption abilities of OMts, carried out under 20 ℃ with the relative humidity of 33%, and the result was presented in Fig. S3 in supplementary material and summarized in Table 2. It was obvious that the OMts adsorbed less moisture than raw Mt, indicating the increase of hydrophobicity of OMts. The 1.0 CEC-OTAB owned slightly better hydrophobicity than 1.0 CEC-DODAC, which may be due to more remaining exchangeable cations in 1.0 CEC-DODAC. The increased hydrophobicity of OMts is inclined to capture the hydrophobic molecules.

Table 2 Elemental analysis and hydrophobicity of Mt and the prepared OMts Sample ID

C (wt.%)

H (wt.%)

N (wt.%)

GSL (CEC)

Ma (%)

Mt

—

—

—

0.00

10.08

0.5 CEC-OTAB

8.80

2.60

0.62

0.50

6.94

1.0 CEC-OTAB

15.01

3.45

0.93

0.97

5.52

1.5 CEC-OTAB

20.38

4.29

1.23

1.29

5.54

2.0 CEC-OTAB

24.39

5.01

1.43

1.50

5.66

0.5 CEC-DODAC

10.0

3.01

0.48

0.50

10.55

1.0 CEC-DODAC

19.44

4.44

0.77

0.81

7.21

1.5 CEC-DODAC

27.21

5.46

1.01

1.06

4.10

2.0 CEC-DODAC

32.01

6.16

1.16

1.21

4.44

10

GSL: Genuine surfactant loading, calculated from N analysis; Ma: moisture adsorption ability, carried out under 20 ℃ with the relative humidity of 33%.

The porous nature plays a vital role on the adsorption capacities of OMts as well. Brunauer-Emmett-Teller (BET) gas sorption measurements were conducted to examine the porous nature of the prepared OMts. Fig. 2 showed the nitrogen adsorption–desorption isotherms of raw Mt and the prepared OMts modified with OTAB and DODAC, respectively. The BET surface area, pore volume, as well as the average pore size are summarized in Table 3. It showed that all the isotherms exhibited Type II sorption behaviors according to the BDDT classification [26]. The hysteresis loops of all the isotherms followed type H5 loop according to the IUPAC classification, which suggested that raw Mt and OMts were mesoporous material. Raw Mt has a specific surface area of 50.941 m2/g with a pore volume of 0.101 cm3/g and average pore size of 7.180 nm obtained from Table 3. However, the BET surface area and pore volume of OMts were lower than raw Mt and decreased with increasing surfactant loadings. At higher surfactant loadings, more sorption sites existed in the interlayer space of clay mineral which leaded to an increase in adsorption and desorption pore diameter and decrease in both the surface area and pore volume [27, 28]. Compared with OTAB modified OMts, the surface area and pore volume of DODAC modified OMts declined significantly at 0.5 and 1.0 CEC surfactant loadings. However, the values kept almost constant when the surfactant loadings surpassed 1.0 CEC level, indicating that the sorption sites for N 2 didn’t increase with surfactant loadings. It may be due to the di-alkyl surfactant DODAC has two carbon chains, the N2 sorption sites in the interlayer space and external surface were occupied totally when the surfactant loadings were over 1.0 CEC. Continuing increasing the surfactant loadings of DODAC only resulted in changes of surfactant molecular arrangements decided from XRD analysis. The difference of porous structure nature between monoand di-alkyl surfactant suggested a different adsorption property of OMts.

11

Fig.2. Nitrogen adsorption–desorption isotherms of Ca-montmorillonite and the prepared OMts intercalated with (a) OTAB and (b) DODAC.

Table 3 Surface parameters of Mt and the prepared OMts Sample ID

SBET (m2/g)

VPa (cm3/g)

Dpb (nm)

Mt

50.941

0.101

7.180

0.5 CEC-OTAB

22.153

0.075

6.025

1.0 CEC-OTAB

19.250

0.062

6.059

1.5 CEC-OTAB

18.844

0.050

6.471

2.0 CEC-OTAB

13.062

0.036

6.470

0.5 CEC-DODAC

27.103

0.069

5.687

1.0 CEC- DODAC

16.822

0.049

6.123

1.5 CEC- DODAC

16.450

0.045

6.185

2.0 CEC- DODAC

15.775

0.042

6.274

a BJH desorption cumulative pore volume of pores between 1.7 and 300 nm in diameter. b Adsorption average pore diameter (4V/A by BET).

3.2 In vitro adsorption in single system 12

In order to investigate the adsorption characteristics of the prepared OMts modified with different surfactants, 1.0 CEC-OTAB and 1.0 CEC-DODAC, denoted as OTAB-Mt and DODAC-Mt respectively, were selected as the representatives to carry out the in vitro adsorption experiments. The experiments simulate the in vivo conditions of the gastrointestinal tract of animals, at pH 3.5 which is equivalent to stomach and at pH 6.5 which is equivalent to intestine conditions [29]. Besides, adsorption in alkaline condition (pH=9.0) was also conducted. Two mycotoxins investigated are polar and weak polar molecules, which have different structures and chemical properties. All the adsorption isotherms of OTAB-Mt, DODAC-Mt as well as the original Mt for AFB1 were determined and the results were displayed below. 3.2.1 Adsorption of AFB1 Fig.3 showed the adsorption isotherms of raw Mt, OTAB-Mt and DODAC-Mt for AFB1. The experimental data were fitted with the Langmuir, Freundlich and Linear isotherm models. It is assumed by Langmuir isotherm model that the adsorption process is monolayer, which means no further adsorption occurs once adsorbate takes place at specific sites in the adsorbent. The relationship is expressed as follows: ce 1 1  ce  qe qm qm KL

(1)

Where qe is the equilibrium adsorption capacity (mg/g), ce represents the equilibrium concentration in solution (mg/L), KL is the Langmuir constant (L/mg) and qm represents the maximum adsorption capacity of the adsorbent (mg/g). Experimental values of qm and KL are calculated from the slope and intercept of the linear plot of ce/qe against ce. On the other hand, the Freundlich model is an empirical expression, which assumed that a multilayer adsorption occurs on the heterogeneous surface or surface supporting sites of various affinities [30]. The equation is described as follows:

ln q e 

1 ln ce  ln K F n

(2) 13

Where KF and 1/n are the Freundlich constants which represent the adsorption capacity and adsorption strength, respectively. The magnitude of 1/n quantifies the degree of heterogeneity of the adsorbent surface and the favorability of adsorption [31]. KF and n can be obtained from the intercept and slope of the linear plot of ln qe versus ln ce. The linear isotherm model can be expressed as:

qe  K dce

(3)

Where qe is the solid-phase concentration of the sorbate per unit mass of the sorbent (mg/g), ce is the equilibrium solute concentration (mg/L), and Kd is the linear sorption (partition) coefficient (L/g). It can be seen from Fig. 3 that Freundlich model matched best with the experimental data among all the three isotherm models. The result can be confirmed by the relevant parameters of isotherm models (Table 4) as well, based on the correlation coefficients (R2). The superior result obtained from Freundlich isotherm model suggested that either the presence of a heterogeneous sorbent surface or the co-existence of different adsorption mechanism, or both phenomena at the same time [32]. For raw Mt, the adsorption of AFB1 showed an S-shaped isotherm curve (Fig. 3a), suggesting that a multiple layer adsorption occurred on external surface [33]. Our calculation based on the geometric sizes of the nearly-planar AFB1 molecule reported by Phillips et al [34] suggests that one adsorbed AFB1 molecule occupied about 1.38 nm2 surface area. The 0.51, 3.00 and 3.35 mg/g adsorption capacity of Mt, OTAB-Mt and DODAC-Mt required a specific surface area of 1.36, 8.00 and 8.93 m2/g at monolayer adsorption, respectively. Raw Mt has a specific surface area of 50.941 m2/g as presented in Table 3 which is dramatically larger than the required specific surface area, indicating that raw Mt has little sorption sites for AFB 1. Adsorption of AFB1 to natural Mt has been generally considered as ion-dipole interactions and coordination between exchangeable cations and carbonyl groups of AFB 1 (illustrated in Fig. 4①) [8, 33, 35]. After being modified with OTAB and DODAC, the exchangeable cations of Mt 14

were mostly replaced by cations of surfactants, and very little exchangeable cations of Mt remained as calculated above. On one hand, the ion-dipole interactions could also take place between surfactants’ cations and carbonyl groups of AFB1 (illustrated in Fig. 4②). On the other hand, the experimental data of OMts also fitted well with linear isotherm model (Fig. 3b, c), implying the presence of another adsorption mechanism. The required sorption sites of OMts increased to at most 8.00 and 8.93 m2/g after modification with OTAB and DODAC, which demonstrated that the intercalation of OTAB and DODAC into the interlayer of Mt played an important role in the enhancement of the sorption sites for AFB1. It should be noted that the required sorption sites were about half of the specific surface areas of OMts. Considering that OTAB and DODAC were mainly intercalated into the interlayer of Mt, the adsorption of AFB1 by OMts probably mainly occurred in the interlayer of OMts. At that situations, the sandwiched molecules would require twice of the molecule area which equals to the specific surface areas of OMts [34]. Thus, we assumed that the other adsorption mechanism between AFB1 and OMts followed the adsorption/partition model (Fig. 4③). The adsorption of AFB1 occurred in the interlayer of OMts with the surfactants, and the long carbon chains intercalated into the interlayer of Mt functioned as a partition medium. The adsorption affinity between sorbate AFB1 and different sorbents can be observed from the values of dimensionless parameter 1/n. The value of 1/n reflects the shape of isotherm as either S-type (1/n > 1), C-type (1/n = 1) or L-type (0 <1/n < 1). The S-type isotherm indicates that the sorbate–sorbent combination is weak, while L-type isotherm means that the adsorption is easy to perform [8]. As presented in Table 4, the calculated 1/n values of Mt, OTAB-Mt and DODAC-Mt were found to be 5.7078, 0.8863 and 0.7874, respectively. Thus, the adsorption affinity between AFB1 and Mt is weak (1/n > 1), while the adsorption affinity between AFB1 and the two OMts is strong. The result clearly demonstrated that the modification of Mt enhanced the adsorption affinity of OMts to AFB1.

15

Fig.3. Adsorption isotherms of (a) Mt, (b) OTAB-Mt and (c) DODAC-Mt to AFB1.

Table 4 The parameters of isotherm models for AFB1 and ZER adsorption. Langmuir model

Freundlich model

Henry/Liner model

AFB1 Qmax(mg/g)

KL(L/mg)

R2

KF(mg1-1/nL1/n/g)

1/n

R2

Kd

R2

Mt

0.05

2.3267

0.9713

100.74

5.7078

0.9946

4.0983

0.9511

OTAB-Mt

20.08

1.3037

0.8121

17.79

0.8863

0.9949

20.5440

0.9943

DODAC-Mt

10.83

3.0870

0.9605

15.60

0.7874

0.9977

20.2288

0.9962

Langmuir model

Freundlich model

Henry/Liner model

ZER Qmax(mg/g)

KL(L/mg)

R2

KF(mg1-1/nL1/n/g)

1/n

R2

Kd

R2

OTAB-Mt

48.97

5.8563

0.8679

41.49

0.0867

0.7814

6.0348

0.9947

DODAC-Mt

52.57

5.0728

0.9709

43.12

0.1360

0.8035

11.8598

0.9940

16

Fig.4.

Schematic mechanism of AFB1 and ZER adsorption on Mt and OMt.

Fig.5 exhibited the effect of solution pH on the adsorption process of Mt, OTAB-Mt and DODAC-Mt, respectively. The adsorption capacity of Mt increased with increasing solution pH, which could be mainly due to the variation of surface charge of Mt at different pH. Raw Mt has a negative charged surface resulted from the isomorphous substitution [36, 37]. Increasing solution pH would lead to more negative charged surface, and more exchangeable cations would adsorb in the interlayer of Mt, providing more sorption sites for AFB1. In the case of OMts, the adsorption capacities only slightly affected by pH, which could be due to that adsorption/partition model made main contribution to the adsorption of AFB1. Since pH did not affect the solubility of AFB1, it has slight influence on the adsorption capacity of OMts. It should be noticed that AFB1 inclines to disintegrate in alkaline condition (pH = 9 ~ 10), the increased removal rate of AFB 1 from pH value of 6.5 to 9.0 may partially due to the disintegration of AFB1. The pH results satisfied well to the proposed adsorption mechanisms of Mt and OMts.

17

Fig.5. Effect of pH on the adsorption process of raw Mt, OTAB-Mt and DODAC-Mt to AFB1.

3.2.2 Adsorption of ZER Fig.6 illustrated the adsorption isotherms of OMts for ZER in single system. Previous literatures have reported that the adsorption capacities of raw Mt toward ZER were very low [1, 20]. This work also found that adsorption of ZER to Mt was negligible. Thus, the adsorption isotherm of Mt was not plotted in Fig.6. From Fig.6a, b, linear isotherm model fitted best with the experimental data of OTAB-Mt and DODAC-Mt, indicating the adsorption/partition mechanism between ZER molecule and OMts (Fig. 4④). The modification of Mt with organic cationic surfactants converts Mt surface from hydrophilic to hydrophobic, which is in favor of the adsorption of low polar, hydrophobic contaminants [38]. When the initial concentration of ZER increased to a certain value, the maximum adsorption capacities of OMts appeared and kept constant with further increasing the initial concentration of ZER. The maximum adsorption capacities of OTAB-Mt and DODAC-Mt observed from Fig.6 were 47.25 and 50.92 mg/g, respectively. The values were extremely larger 18

than raw Mt (nearly no adsorption) and most other adsorbents. In addition, DODAC-Mt with higher organic carbon contents at the same surfactant loadings showed higher adsorption capacity than OTAB-Mt.

Fig.6. Adsorption isotherms of (a) OTAB-Mt and (b) DODAC-Mt to ZER.

The adsorption process of OTAB-Mt and DODAC-Mt to ZER were affected by solution pH obtained from Fig. 7. There was a slight decrease on the removal rate when solution pH increased from 3.5 to 6.5, but the removal rate decreased sharply at strong alkaline condition (pH=9). ZER is a diphenolic compound with an estimated pKα1= 7.62, suggesting that at pH 3.5 it is mainly in the neutral form, while at pH 6.5 the phenolate anion is present in solution and at pH 9, ZER is almost entirely in the anionic form. The solubility of ZER thus varies at different pH values, which is 4.51, 4.81 and 40.15 μg/ml at pH 2, 7 and 10, respectively according to the literature [18]. It was obvious that the adsorption capacities of both OMts for ZER at different pH values showed negative correlation with the solubility of ZER. The result supported the adsorption/partition mechanism between ZER molecule and OMts. Luckily, at pH=3.5 and 6.5 conditions, solution pH had negligible influence on the adsorption process, meaning no desorption occurred when the adsorbents pass through from stomach to intestine.

19

Fig.7. Effect of pH on the adsorption process of OTAB-Mt and DODAC-Mt to ZER.

3.3 Simultaneous adsorption Fig.8 compared the adsorption isotherms of OTAB-Mt and DODAC-Mt to AFB1 and ZER in single and binary-contaminant systems. According to the comparative adsorption to AFB1 (Fig.8a, b), the adsorption capacities of both OMts to AFB 1 in the binary-contaminant system were lower than that in the single adsorption system. The finding indicated that the adsorption of AFB1 on OMts was affected by the existence of ZER, which may be due to the requirement of partial same adsorption sites for AFB1 and ZER. The decreased amount of AFB1 adsorbed in binary-contaminant system compared to that in single system were summarized in Table 5. It could be seen that the decreased amount of AFB1 by DODAC-Mt was obvious lower than that of OTAB-Mt, demonstrating that DODAC-Mt owned more stability in adsorbing AFB1. Additionally, the decrease seemed to become smaller when increasing the initial concentration of AFB1, which suggested that the adsorption process of AFB1 in high concentration was less affected by the existence of ZER. Similarly, the adsorption capacities of OMts to ZER also affected by the 20

existence of AFB1 obtained from Fig.8c, d. The DODAC-Mt also showed more stability in adsorbing ZER than OTAB-Mt and adsorption of ZER in high initial concentration was less influenced by AFB1 compared to that in low concentration. These findings indicated that the adsorption of one mycotoxin was affected by the existence of the other though the adsorption capacities of OMts to both AFB1 and ZER were comparatively high. The DODAC-Mt may have more stability when applying as mycotoxins adsorbents than OTAB-Mt, and increasing the initial concentration would reduce the impact.

Fig.8. Comparative adsorption of AFB1 and ZER on OTAB-Mt and DODAC-Mt in single and binary-contaminant adsorption system: (a) adsorption isotherm of OTAB-Mt to AFB1 ; (b) adsorption isotherm of DODAC-Mt to AFB1; (c) adsorption isotherm of OTAB-Mt to ZER; (d) adsorption isotherm of DODAC-Mt to ZER.

Table 5 21

The decreased amount of AFB1 and ZER in single and binary systems. AFB1

Decreased Amount (%)

ZER

Initial Concentration

Initial OTAB-Mt

DODAC-Mt

Concentration

OTAB-Mt

DODAC-Mt

（mg/L）

(mg/L)

4.

Decreased Amount (%)

0.68

26.12

3.15

12.00

25.50

12.38

0.72

24.77

2.92

13.00

21.32

11.36

0.77

20.25

5.68

14.00

18.80

10.37

0.88

14.73

2.84

15.00

25.30

10.09

0.93

15.14

4.82

16.00

23.26

9.49

mycotoxins

adsorbents

Conclusion Compared

to

the

previous

reported

such as

montmorillonite [1, 10, 20], zeolites [17], halloysite [39], talc and diatomite [40], this work has demonstrated that OMts modified with mono- and di-alkyl cationic surfactants could remove polar mycotoxin AFB1 and weak polar, hydrophobic mycotoxin ZER from simulated gastrointestinal tract of animals simultaneously, since previous literatures mainly focused on the adsorption of single mycotoxin. The intercalation of organic cationic surfactants expanded the interlayer of Mt and increased the organic carbon content, providing new sorption sites for AFB 1 and ZER. The modification converted the surface of Mt from hydrophily to hydrophobicity revealed by hydrophobicity measurement, which made the adsorption of ZER realizable. In vitro adsorption of AFB1 and ZER in both single and binary-contaminate systems as well as the comparison between two types of OMts were investigated systematically for the first time in this work. Both OMts showed super adsorption capacities to AFB1 and ZER compared to raw Mt and other mycotoxins adsorbents [18, 19, 41]. Moreover, no desorption occurred at pH=3.5 and 6.5 conditions suggesting the applicability of the prepared OMts as mycotoxins adsorbents. The adsorption isotherms of AFB1 revealed the ion-dipole interactions 22

mechanism

of

Mt

and

the

co-existence

of

ion-dipole

interactions

and

adsorption/partition mechanisms of OMts, which demonstrated larger adsorption capacities of OMts than Mt. The results of this work might provide solutions for removing polar and non-polar mycotoxins simultaneously.

Acknowledgements The authors gratefully acknowledge the financial support provided by the National Natural Science Foundation of China (Grant No. 51774294) and the Fundamental Research Funds for the Central Universities (2015QH01).

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25

Graphical Abstract Adsorption of AFB1 to natural Mt has been generally considered as ion-dipole interactions and coordination between exchangeable cations and carbonyl groups of AFB1, while the adsorption of AFB1 on OMts was proposed to be ion-dipole interactions (between surfactant cations and carbonyl groups of AFB 1) and adsorption/partition mechanisms. ZER molecules were captured on OMts through adsorption/partition model.

26