toxins

Article
A Novel Adsorbent Magnetic Graphene Oxide
Modified with Chitosan for the Simultaneous
Reduction of Mycotoxins
Atena Abbasi Pirouz 1,2 , Roghayeh Abedi Karjiban 3 , Fatimah Abu Bakar 1 and
Jinap Selamat 1,2, * ID
1 Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang,
43400 Selangor, Malaysia; atenapirouz.upm@gmail.com (A.A.P.); Fatim@upm.edu.my (F.A.B.)
2 Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Serdang,
43400 Selangor, Malaysia
3 Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, Serdang, 43400 Selangor, Malaysia;
roghayeh@upm.edu.my
* Correspondence: jinapselamat@gmail.com or jinap@upm.edu.my; Tel.: +601-9351-3140




Received: 20 June 2018; Accepted: 24 August 2018; Published: 6 September 2018


Abstract: A novel magnetic graphene oxide modified with chitosan (MGO-CTS) was synthesised
as an adsorbent aimed to examine the simultaneous removal of mycotoxins. The composite
was characterised by various procedures, namely Fourier-transform infrared spectroscopy (FTIR),
X-ray diffraction (XRD) and a scanning electron microscope (SEM). The adsorption evaluation
was considered via pH effects, initial mycotoxin concentration, adsorption time and temperature.
Adsorption isotherm data and kinetics experiments were acquired at the optimum pH 5 fit Freundlich
isotherm as well as pseudo-second-order kinetic models. The thermodynamic results indicated that
the adsorption of the mycotoxins was spontaneous, endothermic and favourable.

Keywords: adsorbent; reduction of mycotoxins; isotherms; kinetics; thermodynamics

Key Contribution: This study presents a novel adsorbent to reduce mycotoxins and an adsorption
study of three mycotoxins in palm kernel cake.

1. Introduction
Mycotoxins are produced as the secondary metabolites of fungal species and can exert toxic effects
on animals and humans [1]. The global contamination of foods and feeds by mycotoxins has become a
great challenge for researchers. Up to now, more than 400 different mycotoxins have been identified;
the foremost categories of mycotoxins influencing feedstuffs are aflatoxinB1 (AFB1 ), ochratoxinA (OTA)
and zearalenone (ZEA) [2]. This was reported from surveillance for detecting mycotoxins in cereals
and animal feeds, whereby more than one toxin can occur in the same commodity. Occurrences of
mycotoxins have synergistic or additive effects [3]. The most common combination of AFB1 , OTA
and ZEA is associated with a variety of toxic effects, chronic and acute in animals and humans.
Therefore, their presence is one of the most relevant and worrisome problems among feedstuffs [4].
Mycotoxin contamination has created a major concern, and the reduction of mycotoxins from feedstuffs
is necessary. Many attempts have been made to detoxify mycotoxins, including biological methods,
chemical treatments, and adsorption techniques. Among the methods used for treatment, adsorption
is the most effective [5]. Several studies have shown the applicability of adsorbents, namely clay [6],
activated charcoal (AC) [7], hydrated sodium calcium aluminosilicates (HSCAS) [8], and polymers for
the binding of mycotoxins [9]; however, in some cases, a single adsorbent has been shown to be effective

Toxins 2018, 10, 361; doi:10.3390/toxins10090361 www.mdpi.com/journal/toxins

Toxins 2018, 10, 361 2 of 13

against one or two specific mycotoxins. The efficacy of adsorption depends on the functional groups
on the surface, the physical qualities of the adsorbent, and the physical and chemical characteristics of
the mycotoxins [10]. The scientific and health interest in the mentioned issues has grown during the
last few years, resulting in new research on the efficiency and simultaneous use of adsorbents used for
the removal of mycotoxins.
Chitosan (CTS) is a rich biopolymer attained from alkaline N-deacetylation of chitin, which
is considered the second most copious natural biopolymer, with no harmful byproducts [11].
Its hydrophilicity, biocompatibility and high effectivity have led to a diverse range of applications
in different industries [12]. CTS has shown high adsorption capacities resulting from its exceptional
properties, specifically the presence of amino and carboxyl functional groups, which create very
effective points for the anionic binding of polluted compounds in acidic solutions [13,14]. Previous
studies [15–17] have discussed the applications, particularly the efficacy and specificity of the
adsorption process, of CTS for mycotoxin detoxification.
Magnetic graphene oxide (MGO) nanocomposite has arisen as one of the next generation of
resources with versatile applications for the removal of various toxic elements and compounds
(organic and inorganic) [18]. The separation of MGO under a peripheral magnetic field can expedite
the extraction of adsorbents. Magnetic separation requires less energy but achieves improved
separation (particularly adsorbents with insignificant particle sizes) compared to traditional approaches
such as filtration, centrifugation and gravitational separation [19]. Alteration of MGO by chemical
techniques—for instance, addition of new functional groups and polymer grafting—can significantly
improve the adsorption capacities of MGO. In fact, a literature study shows that no survey has been
performed concerning the modification of MGO to increase the adsorption capacity of mycotoxins.
The favourable adsorption properties of MGO and the inherent properties of CTS suggest the
possibility of a MGO-CTS composite as an adsorbent. The carboxyl group of MGO can chemically
react with the amine group of CTS through chemical bonding between MGO and CTS [20].
The aim of this study was to investigate the effects of pH, initial mycotoxin concentrations, time,
and temperature on mycotoxins adsorption. To the best of our knowledge, this is the first report of the
detoxification of mycotoxins with MGO-CTS.

2. Results and Discussion

2.1. Characterisation of MGO-CTS

Numerous characterisation methods including FTIR, XRD and SEM were used to understand the
behaviour and properties of MGO-CTS as a new adsorbent by examining its physical, chemical and
structural properties.
FTIR is one of the primary and commonly used analytical procedures to obtain information
about the accessibility of certain functional groups on the surface of MGO-CTS and determine the
binding mechanism of pollutants (both organic and inorganic) [21]. The FTIR spectra of all the
studied compounds are shown in Figure 1. The MGO high points are located at 1664 cm−1 and
589 cm−1 , related to epoxy groups, and indicate the symmetric stretching of the iron oxide (Fe3 O4 )
nanoparticles [22]. The wide band at 3294 cm−1 in the FTIR spectra of CTS included the stretching
vibration of the hydroxyl group and N–H bond, while the peaks at 1673 cm−1 and 1018 cm−1 are
attributed to C=O of the –NH=C=O bond stretching, and the C–OH bond stretching, respectively [21].
The synergistic effect in the MGO nanocomposite by adding CTS was detected from the characteristic
adsorption bands centred at 1644–1560 cm−1 related to the amide I (C=O stretching), amide II (Schiff’s
base), and –C-O stretching of the primary alcohol group in CTS, respectively. Furthermore, a peak
at 573 cm−1 can be observed in the FTIR spectra of MGO-CTS nanocomposite, and the existence of
ferrous ferric oxide was confirmed with no noticeable change [23].
Toxins 2018, 10, 361 3 of 13

Figure 1. FTIR spectra of Magnetic graphene oxide (MGO), Chitosan (CTS) and MGO modified with
chitosan MGO-CTS.

XRD is an effective technique to determine the crystal structure of MGO-CTS by providing

valuable data about the physical as well as chemical forms of MGO nanoparticles rooted in the CTS
matrix. The XRD spectrograms of CTS, MGO, and MGO-CTS composites are presented in Figure 2.
The pure Fe3 O4 particles with spinel structures produced four distinguishing peaks for Fe3 O4 (2θ = 35.5,
45.3, 50.4, and 52.2) in both samples [24]. Both the patterns of CTS and MGO-CTS (Figure 2) exhibited
a broad peak at 2θ = 21.3◦ owing to the amorphous state of CTS, signifying that the amorphous-like
structure of CTS was not altered by introduction of MGO in the nanocomposite. The results showed
that magnetic composites were achieved successfully and it was not changed during the synthesis
process [25].

Figure 2. X-ray diffraction XRD patterns of the CTS, MGO and MGO-CTS.

The morphologies and structures of CTS, MGO and MGO-CTS composites are depicted in Figure 3.
As shown in Figure 3a, the SEM image of MGO illustrated the formation of magnetic nanoparticle
(with regular particle size of approximately 9 nm). The typical SEM of CTS (Figure 2b) showed a tight
fracture and smooth surface. The addition of MGO was successfully detected on the surface of CTS,
which produced a high surface, a high density and enhanced adsorption of the active sites (Figure 3c).
Toxins 2018, 10, 361 4 of 13

Figure 3. SEM images of (a) MGO; (b) CTS; and (c) MGO-CTS.

2.2. Effect of pH on Adsorption

The effect of aqueous solution pH is a key parameter in overall adsorption procedure and
impacts—the surface charge of the adsorbents as well as the speciation of the adsorbents. In the case
of mycotoxins sorption behaviour, the range of pH for the batch experiments was 2–6. The adsorption
increased from pH 2–5 and later displayed a sharp decrease, which might be the result of an
increasing pH of the solution, as shown in Figure 4. This is normally observed because of weakened
forces and, subsequently, the decrease in attraction and interaction among mycotoxin molecules and
MGO-CTS [26]. In addition, the number of protonated NH2 groups decreased and extreme hydroxyl
ions of MGO-CTS might strive with the mycotoxin anions at a pH above 5; hence, obvious reductions
were observed [20]. Therefore, it can be suggested that the most optimal pH value for the decrease of
most anion ions by MGO-CTS was at pH 5.0. Most of the MGO-CTS contained numerous functional
groups, for instance –NH2, –NH, –COOH, –OH, Fe+2 , and Fe+3 , which change according to the pH
of the solution. At a low pH, the majority of functional groups in MGO-CTS are protonated and
positively charged. Thus, electrostatic attraction, hydrogen bonding, and π–π interactions amongst the
mycotoxin molecules and the MGO-CTS surface could be the predominant adsorption mechanism.

Figure 4. Effect of pH on the adsorption of aflatoxinB1 (AFB1 ), ochratoxinA (OTA) and zearalenone
(ZEA) by using 0.03 g absorbent with initial concentration (50 ng/L) at 30 ◦ C.

2.3. Adsorption Kinetics

Adsorption is a procedure concerning liquid/solid interactions and mass transfer from the liquid
surface. Adsorption kinetics can assist in predication of adsorption rates and offers valuable evidence
about the mechanism of adsorption [27].
Toxins 2018, 10, 361 5 of 13

The experimental data obtained in this study were analysed by two common adsorption kinetics,
namely pseudo-first-order and pseudo-second-order representations, to find the best model to describe
the adsorption of the mycotoxins in PKC [28]. Towards the evaluation suitability of the diverse models,
it is needed to present the correlation coefficient, R2 close or equal to 1. A higher R2 value specifies a
further relevant model to the kinetics of the mycotoxins adsorption [29]. The capabilities of pseudo-first
and pseudo-second order kinetic representations were examined at pH 5 in the initial concentration of
the mycotoxins (50 µg/L) at a given temperature of 30 ◦ C.
The linearised models of the pseudo-first-order and pseudo-second-order kinetic models of
Lagergren are often expressed based on the following equations:

ln(qe − qt ) = ln(qe ) − k1 t (1)

t 1 t
= + , (2)
qt k2 q2e qe
where qt and qe (mg/g) = the amount of solute adsorbed at time (min) and at equilibrium, respectively.
k1 and k2 (min−1 ) = the pseudo-first-order rate constants for the kinetic model. The parameters are
calculated from the slope and intercept of the linear plots of ln (qe − qt ) vs. t and (t/qt ) vs. t, respectively,
as summarised in Table 1 [30].
According to Table 1, the correlation coefficients of the pseudo-second-order model, 0.989–0.996,
are considerably higher when compared to pseudo-first-order, which was in the range of 0.661–0.945.
Adsorption capacities for AFB1 , OTA and ZEA were 3.85, 3.19 and 3.33 ng/g, respectively. Thus, it can
clearly be stated that the pseudo-second-order model, suggesting a chemisorption process, can explain
the interaction amongst the adsorbate and adsorbent accurately. Therefore, it can be concluded that
the process obeyed chemisorption and involved valency forces through the allocation or exchange
of electrons between the hydrophilic sites of adsorbent and mycotoxins. This process is irreversible
because the chemically adsorbed molecules are not able to move on the surface [31].

Table 1. Kinetics parameters for mycotoxins adsorption onto MGO-CTS.

Mycotoxin Pseudo-First-Order Pseudo-Second-Order

h  i
Qt = Qe 1 − e−k1t Qt = Qe 1 − 1+K12 Q t


e

k1 (1/min) qe,cal (ng/g) R2 k2 (g/ng min) qe,cal (ng/g) R2

AFB1 0.002 2.45 0.928 0.002 3.85 0.989
ZEA 0.002 1.95 0.945 0.005 3.19 0.999
OTA 0.0008 1.59 0.661 0.03 3.33 0.996
AFB1 : aflatoxinB1 ; OTA: ochratoxinA; ZEA: zearalenone.

2.4. Adsorption Isotherms

The adsorption isotherm is vital for describing the surface properties of the adsorbent, the
capacity of an adsorption and the design of adsorption systems. Numerous other studies have been
done to define the equilibrium of adsorption systems. We used two of the most common models
(Langmuir and Freundlich) employed to define the adsorption behaviour of mycotoxins at different
initial concentrations (5–100 ng/g).
The Langmuir isotherm is an empirical model for building a monolayer on the surface that
contains a finite quantity of localised sites; all the adsorption sites on the adsorbent take place at precise
homogeneous sites within the material [32].
The general form of the Langmuir isotherm is presented by the equation given below:

qmax K L Ce
qe = (3)
1 + K L Ce
Toxins 2018, 10, 361 6 of 13

Based on this isotherm equation, once a template inhabits a site, no additional sorption can occur
at that site. Hence, all sites are dynamically alike and no interactions amongst the molecules adsorbed
on neighbouring sites occur [33].
Equation (4) can be represented in a linear form as well:

Ce Ce 1
= + , (4)
qe qmax K L qmax

where qe is the number of mycotoxins per unit weight of adsorbent (ng/g), qmax is the maximum
adsorption capacity (ng/g), Ce is the equilibrium concentration in solution, and K L is a constant
relating to the energy of sorption (L/ng). The values of qmax and K L can be evaluated from the linear
plot of Ce /qe against Ce . This design provides a straight line with the slope and intercept equal to
1/qmax and (1/K L qmax ), respectively [34].
The empirical Freundlich model applies to multilayer adsorption by non-uniform circulation
of adsorption temperature and attractions over the heterogeneous surface. The Freundlich
isotherm represents the proper sorption data on heterogeneous surfaces at low and intermediate
concentrations [35], which is stated by the following equation:

qe = K F Ce 1/n (5)

Equation (6) can be expressed in linear form as well:

1
ln qe = ln K F + ln Ce , (6)
n
where KF and n are the Freundlich constants, indicating the adsorption capacity and the degree of
heterogeneity of the sorbent surface, respectively. A plot of ln qe versus Ln Ce provides a straight
line with a slope of 1/n and an intercept equal to Ln KF , as reported in Figure 5 and Table 2 [34].
Progressively larger KF values indicate larger adsorption capacity [36]. The value of n is not only the
amount of deviation from linearity; nevertheless, it informs us about the heterogeneity degree of the
sorption sites. If n lies below 1 it indicates a favourable sorption process. Additionally, the isotherms
with n < 1 are categorised as isotherms with high attraction amongst the adsorbate and the adsorbent,
which is considered an indication of chemisorption and proof of an increase in hydrophobic surface
properties subsequent monolayer [36,37].

Figure 5. Freundlich isotherms for the adsorption of AFB1, OTA and ZEA with MGO-CTS at 50 ◦ C.
Toxins 2018, 10, 361 7 of 13

The applicability of the isotherm equation is equated by finding R2 from the Langmuir and
Freundlich adsorption constants. The corresponding R2 values are listed in Table 2, in which the R2
obtained using the Freundlich equation were higher than those calculated using Langmuir. On the
other hand, the value of n < 1 indicates a favourable and stronger interaction between the mycotoxins
and MGO-CTS. The values of n for all three mycotoxins were below 1 at 50 ◦ C, thus indicating more
interaction between the mycotoxins and MGO-CTS (Table 2).

Table 2. Langmuir and Freundlich isotherm factors for AFB1, OTA and ZEA on MGO-CTS (pH = 5,
t = 6 h).

Mycotoxin Langmuir Equation Freundlich Equation

T qmax KL R2 KF n R2
(◦ C) (ng/g) (L/ng) (L/ng)
30 9.62 0.04 0.89 1.39 1.35 0.993
AFB1 40 7.35 0.1 0.95 2.26 1.27 0.985
50 10.64 0.05 0.97 6.57 0.74 0.99
30 7.25 0.02 0.99 8.08 1.25 0.994
ZEA 40 5 0.02 0.99 5.31 1.28 0.994
50 23.26 0.53 0.945 10.93 0.66 0.988
30 72.46 0.005 0.915 3.32 1.03 0.993
OTA 40 11.11 0.05 0.951 1.58 1.87 0.995
50 27.02 0.01 0.912 4.58 0.94 0.993

A literature survey reported that the maximum amounts of AFB1, OTA and ZEA adsorbed by
cross-linked chitosan and grape pomace were (5.67, 24.8 and 9.18 mg/g) and (15.0, 6.3 and 8.6 mg/g),
respectively [37,38]. On the other hand, studies by Merdivan et al. and Trentin et al. have shown that
the adsorption capacity of AFB1 and OTA was 0.73 and 2.66 mg/g, respectively [15,39],. Moreover,
our team investigated the Qmax of ZEA = 5.4 mg/g using MGO [40]. The current study has shown
that MGO-CTS for the first time is used as an adsorbent for the reduction of AFB1, OTA and ZEA.
The predicted maximum adsorption capacity (Qmax ) was 31.1, 75.4 and 27.7 mg/g, respectively.
In comparison, MGO-CTS had higher adsorption capacity for three toxins. The results indicated
that MGO-CTS would be a very promising adsorbent due to the large surface area and different
functional groups.

Thermodynamics
Thermodynamic adsorption research offers additional data concerning the practicability and
spontaneity of the adsorption procedure. The thermodynamic factors for the adsorption of mycotoxins,
for instance the modification in Gibbs free energy (∆G◦ , kJ/mol), enthalpy (∆H ◦ , kJ/mol) and entropy
(∆S◦ , J/mol K), were estimated at various temperatures using the following equations:

∆G ◦ = − RT ln Kc (7)

∆G◦ = ∆H ◦ − T∆S◦ (8)

By combining Equations (7) and (8) we obtain:

qe ∆S ∆H
ln( )= − , (9)
Ce R RT

where R (8.3145 J/mol K) is the universal gas constant, T is the absolute temperature (in Kelvin) and
Kc (qe /Ce ) is the adsorption affinity. Referring to Van’t Hoff in Equation (9), the values of ∆H ◦ and ∆S◦
evaluated from plotting ln Kc against 1/T provide a straight line with a slope and intercept equal to
Toxins 2018, 10, 361 8 of 13

(−∆H ◦ /R) and (∆S◦ /R), respectively (Figure 6). ∆G◦ was calculated from Equation (8) a procedure
conferring to Equation (8) [8].

Figure 6. Van’t Hoff plot for assessment of thermodynamic parameters.

As shown in Table 3, the values of ∆G◦ for AFB1 , OTA, and ZEA at various temperatures
were negative. Henceforth, the adsorption of mycotoxins on MGO-CTS was spontaneous, while the
positive ∆S◦ changes produced increased randomness during the adsorption process. From the results,
the positive values of ∆H ◦ show that this process was endothermic. The relatively high values of
∆H ◦ may be due to the formation of strong chemical bonds between the mycotoxin molecules and
functional groups of the adsorbent surface.

Table 3. Thermodynamic factors for the adsorption of mycotoxins onto MGO-CTS.

Mycotoxin C0 T (◦ C) qe Kc ∆G◦ ∆H ◦ ∆S◦

100 30 6.28 0.09 −18.97 18.78 62.68
AFB1 100 40 6.34 0.26 −19.60
100 50 6.42 0.55 −20.22
100 30 56.63 1.29 −10.52 7.31 34.75
OTA 100 40 62.22 1.32 −10.87
100 50 81.35 1.47 −11.22
100 30 2.69 −3.10 −49.52 51.62 163.61
ZEA 100 40 3.67 −2.51 −57.43
100 50 4.71 −1.83 −59.06

3. Conclusions
MGO-CTS was successfully produced, characterised and used as a new adsorbent for the
simultaneous reduction of mycotoxins. Our results indicate that the adsorption of these mycotoxins
on the surface of MGO-CTS depends on several parameters such as pH, contact time, adsorbate
concentration and temperature. Analysis of adsorption mechanisms showed that the hydroxyl groups,
amine and iron ions (Fe+2 and Fe+3 ) of CTS and MGO are predominantly responsible for binding
mycotoxins. The adsorption isotherms and kinetics were calculated in detail. The equilibrium
data provide the best fit using the Freundlich model by illustrating multilayer adsorption.
The pseudo-second-order kinetic model successfully described the sorption reaction of the mycotoxins,
demonstrating that chemisorption is the rate-controlling procedure. According to the negative value
of ∆G◦ , the adsorption was spontaneous, while the positive values of ∆S◦ illustrated the amplified
randomness at the solid and liquid crossing point. The adsorption of AFB1 , OTA and ZEA onto
MGO-CTS increased with increasing temperature, suggesting that the increase of temperature from
30 to 50 ◦ C caused an increase in the adsorption capacity. Therefore, the adsorption process was
Toxins 2018, 10, 361 9 of 13

endothermic. MGO-CTS as an adsorbent has promising characteristics for future applications, such
as high sensitivity and simple and eco-friendly reduction of mycotoxins in environmental samples.
MGO-CTS has a potentially efficient capacity for AFB1 , OTA, and ZEA at 50 ◦ C and pH 5. It can be
concluded here that MGO-CTS can be reduced to AFB1 , OTA, and ZEA at 50 ◦ C and pH 5.

4. Materials and Methods

4.1. Chemicals
Palm kernel cakes (PKC) are attained from numerous local factories in diverse districts (Kelantan
and Shah Alam) of Malaysia. PKC was crushed to a sufficient powder and sieved with mesh size of
750 mm before being used for spiked with mycotoxins. Chitosan (CTS > 85% deacetylation), graphite
powder, sodium hydroxide (NaOH) and sodium chloride (NaCl) were provided by Sigma-Aldrich (St.
Louis, MO, USA). Analytical pure standards (AFB1 , OTA, and ZEA), AOZ immunoaffinity columns
(IAC), and the phosphate-buffered saline solution (PBS) were prepared by dissolving PBS in distilled
water provided by VICAM (Watertown, MA, USA). Glass microfibre filters (GF/A grade) and fluted
filter papers (24 cm) were acquired from Whatman (Maidstone, UK). Ferrous ammonium sulphate
[(NH4 )2 SO4 FeSO4. 6H2 O], ammonium ferric sulphate [NH4 Fe(SO4 )2 ·12H2 O], sodium nitrate (NaNO3 ),
sulfuric acid (H2 SO4 ) 98%, ethanol (C2 H6 O), nitric acid (HNO3 ), potassium permanganate (KMnO4 ),
hydrogen peroxide (H2 O2 ), methanol (CH3 OH) and acetic acid (CH3 COOH) (HPLC grade) were
purchased from Merck (Darmstadt, Germany).

4.2. Instrumentation
The following instruments were used for data collection of the adsorbent: (i) distilled water
obtained from a Milli-Q purification system (Bedford, MA, USA); (ii) adjustable pipettes (Gilson
Pipetman L, Paris, France); (iii) a nitrogen evaporator with a heated block (N-EVAP, Organomation
Associates, Inc., Berlin, MA, USA); (iv) a Waters high-performance liquid chromatography (HPLC)
system (Milford, MA, USA); (v) an incubator shaker (New Brunswick Scientific C24, Edison, NJ, USA);
(vi) Centrifuge (Sigma 3–18s Darmstadt, Germany); (vii) a vortex mixer (Harmony, Tokyo, Japan).

4.3. Synthesis of Adsorbents

Three adsorbents were prepared: (i) graphite oxide (GO); (ii) MGO; and (iii) MGO modified with
CTS nanocomposite (MGO-CTS).
GO was prepared using a modified Hummers method [41]. GO was produced by vigorously
stirring 5 g of commercial graphite powder and 2.5 g of NaNO3 in 75 mL of concentrated H2 SO4 (98%)
in an ice bath at 0 ± 2 ◦ C. After stirring, 15 g of KMnO4 were gradually added, and the frequency
of addition was measured to avoid an unexpected rise in the temperature. Afterwards, the mixture
was preserved at 35 ◦ C for 30 min. Next, 230 mL of distilled water were gradually added to the
reaction container, keeping the temperature below 98 ± 2 ◦ C for 15 min. Then, 700 mL of distilled
water and a consequent addition of 2.5 mL of hydrogen peroxide (30 wt %) were added to the mixture.
The consequential combination was filtered and cleansed three times using ultrapure water and alcohol,
prior to drying at 70 ◦ C for 12 h in a vacuum oven.
MGO was formed by a chemical co-precipitation method [42]. The iron (III) oxide (Fe2 O3 )
nanoparticles were set by dissolving 5.8 g of (NH4 )2 SO4 FeSO4 ·6H2 O in 10.7 g of NH4 Fe(SO4 )2 ·12H2 O
in 100 mL of ultrapure water to produce a mixed iron salt solution under nitrogen gas. Then and
there, the chemical precipitation was attained by the addition of 75 mL NH4 OH solution (29.6%)
dropwise for 30 min at 25 ◦ C. A black precipitate instantly presented after the addition of the NH4 OH
solution. The precipitated particles are covalently bonded to carboxyl group of GO and Fe–O of iron
oxide nanoparticles. Typically, 1 g of dried GO was dissolved into 100 mL of ultrapure water with
ultra-sonication to produce a steady suspension. Lastly, the MGO solid was composed via a magnet,
Toxins 2018, 10, 361 10 of 13

washed three times with ultrapure water and anhydrous C2 H6 O, and dried at 70 ◦ C for 12 h in a
vacuum oven.
The MGO-CTS composite was prepared via the following procedure. The CTS solution was
arranged by dissolving 0.4 g of powder CTS (2% w/v) into 20 mL of a CH3 COOH solution (2% w/v)
and stirred for 2 h at room temperature, followed by the addition of 0.3 g of MGO to the prepared
solution. The mixed solution was stirred uninterruptedly for 90 min in a water bath at 50 ± 2 ◦ C.
The pH of the solution was adjusted to 9–10 by micro-additions of NaOH (0.1 mol/L) and it was kept
in a water bath for another 60 min at 80 ± 2 ◦ C. The black product was washed with C2 H6 O and
purified water until the pH reached approximately 7, and then it was dried in a vacuum oven at 50 ◦ C
to produce the concluding product, MGO-CTS. The arranged products (GO, MGO, and MGO-CTS)
were ground to a fine powder, sized 75–125 mm, after sieving.

4.4. Characterisation Techniques

The XRD forms were recorded on an XRD diffractometer (model Richard Seifert 3003 TT,
Ahrensburg, Germany) with a CuKα radiation for the crystalline phase identification (k = 0.15405 nm
for CuKα). The FTIR spectrophotometer Nicolet 6700 (Thermo Nicolet Corp., Madison, WI, USA) was
used to attain the FTIR spectra of MGO, CTS and MGO-CTS. The SEM (LEO 1455 VPSEM, Kensington,
UK) was applied to produce an image of the material in order to find the surface morphology.

4.5. Adsorption Experiments

Batch adsorption of AFB1 , OTA and ZEA was carried out using MGO-CTS as the adsorbent.
For the purpose of kinetic studies, the same amount of mycotoxin was used and kept in contact with
the standard solution, which was diluted in water and methanol (50:50; v/v) at each time interval
(3, 4, 5, 6, 7 and 8 h) within the optimum pH and room temperature. To examine the adsorption
isotherm, 5 g of PKC were spiked with AFB1 , OTA and ZEA at three diverse concentrations (5.0,
25.0 and 100.0 ng/g). Each spike was done three times. The sampled spikes were stored in the dark
overnight to permit solvent evaporation. In the preliminary study, the total amount of MGO-CTS,
from 0.005 to 0.03 g, was studied; however, the decrease of mycotoxins after 0.03 g was insignificant.
Henceforth, consistent with this preliminary study, the sample was agitated with 0.03 g of MGO-CTS,
and an additional 20 mL distilled of methanol and water (80/20; v/v) were added. The suspensions
were shaken in an incubator shaker to keep the temperature constant at 25 ◦ C. The initial pH values
were attuned with small amounts of 0.1 M HNO3 . The agitation speed and incubation time were
160 rpm and 24 h, respectively. After the adsorption procedure, MGO-CTS was separated by magnetic
separation, the supernatant was analysed for residual mycotoxin by HPLC [43] and the adsorption
capacity of MGO-CTS for the mycotoxins was evaluated by the following equation:

(C0 − Ce )v
qe = , (10)
m
where C0 (µg/L) and Ce (µg/L) are the primary and equilibrium concentratis of the mycotoxins,
respectively; v (L) is the volume of the mycotoxins solution; and m (g) is the mass of adsorbent [44].
To study the thermodynamic adsorption, 5 g of PKC was spiked with mycotoxins at the highest
concentration (100.0 ng/g). Thus, the effect of temperature on equilibrium was investigated at
30–50 ◦ C.

Author Contributions: Conceptualisation, A.A.P. and J.S.; Methodology, A.A.P.; R.A.K. and J.S. Software, A.A.P.;
R.A.K.; Validation, F.A.B.; R.A.K. and J.S.; Formal Analysis, A.A.P.; Investigation, F.A.B.; R.A.K. and J.S.; Resources,
J.S.; Data Curation, A.A.P.; Writing—Original Draft Preparation, A.A.P.; Writing—Review & Editing, F.A.B.; R.A.K.
and J.S.; Visualisation, R.A.K. and J.S.; Supervision, F.A.B.; R.A.K. and J.S.; Project Administration, J.S.; Funding
Acquisition, J.S.
Funding: This research was funded by the Ministry of Education Malaysia (grant number LRGS 10214).
Toxins 2018, 10, 361 11 of 13

Acknowledgments: The authors gratefully acknowledge the Ministry of Education Malaysia for financial support
from its Long-term Research Grant Scheme (LRGS) [LRGS number10214].
Conflicts of Interest: In accordance with the Toxins Publishing Group, the authors declare no competing nor any
other interests that might be perceived as affecting the results of this study.

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