Removal and degradation of β-lactam antibiotics in water using didodecyldimethylammonium bromide-modified montmorillonite organoclay

Removal and degradation of β-lactam antibiotics in water using didodecyldimethylammonium bromide-modified montmorillonite organoclay

Accepted Manuscript Title: Removal and degradation of ␤-lactam antibiotics in water using didodecyldimethylammonium bromide-modified montmorillonite o...

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Accepted Manuscript Title: Removal and degradation of ␤-lactam antibiotics in water using didodecyldimethylammonium bromide-modified montmorillonite organoclay Author: Tohru Saitoh Takayoshi Shibayama PII: DOI: Reference:

S0304-3894(16)30558-1 http://dx.doi.org/doi:10.1016/j.jhazmat.2016.06.003 HAZMAT 17792

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

14-10-2015 14-5-2016 3-6-2016

Please cite this article as: Tohru Saitoh, Takayoshi Shibayama, Removal and degradation of ␤-lactam antibiotics in water using didodecyldimethylammonium bromide-modified montmorillonite organoclay, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2016.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Removal and degradation of β-lactam antibiotics in water using didodecyldimethylammonium bromide-modified montmorillonite organoclay

Tohru Saitoha *, Takayoshi Shibayamab

a

Department of Biotechnology and Environmental Chemistry, Kitami Institute of

Technology, Koen-cho 165, Kitami 090-8507, Japan, bGraduate School of Engineering, Molecular Design and Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan,

*Corresponding author. Tohru Saitoh Telephone +81 157 26 9387, Fax +81 157 24 7719 E-mail address: [email protected]

Postal address: Department of Biotechnology and Environmental Chemistry, Kitami Institute of Technology, Koen-cho 165, Kitami 090-8507, Japan

1

H N O

O

Organoclay

S N

Penicillin G

O

-

O

Sorption

Degradation

H N O

Organoclay sorption method allowed rapid removal and eco-friendly degradation of β-lactam antibiotics.

O

S HN OH

-

O

Penicilloic acid

O

Highlights ♦ Dialkylated surfactant-modified organoclay was used for wastewater treatment. ♦ β-lactam antibiotics were rapidly collected from water to organoclay. ♦ Penicillin G in organoclay degraded into penicilloic acid missing β-lactam ring. ♦ Formation of hydrophobic region was essential for collection and degradation.

ABSTRACT β-Lactam antibiotics including penicillin G, nafcillin, cefazolin, cefotaxime, and oxacilline in water were rapidly removed and degraded by using didodecyldimethylammonium bromide (DDAB)-montmorillonite (MT) organoclay.

Removal of antibiotics increased

with increasing the amount of organoclay added and the amount of DDAB sorbed on MT. Extents of organoclay sorption of antibiotics were represented by the binding constants to DDAB molecules and correlated to the aqueous-octanol distribution coefficients. The degradation rate of β-lactam antibiotics was found to significantly increase by the organoclay sorption. Even under the mild conditions (25°C and pH 7), penicillin G (m/z = 335) nearly completely (>98%) degraded into penicilloic acid (m/z = 353) missing β-lactam ring within 2 h.

The first-order reaction rate of the primary degradation

increased with increasing in temperature.

The activation energy estimated from the

Arrhenius plot was 49 kJ mol-1 and lower than the value (83.5 kJ mol-1) in water, strongly suggesting catalytic activity of DDAB-MT organoclay.

The applicability to wastewater

2

treatment was demonstrated by using secondary effluents of municipal sewage treatment plants and synthesized hospital wastewaters.

Keywords: β-Lactam antibiotics; Organoclay; Rapid removal; Catalytic degradation; Hospital wastewater treatment

1. Introduction β-Lactam antibiotics are a broad class of antibiotics contains a β-lactam ring in their molecular structures.

They work by inhibiting cell wall biosynthesis in bacterial

organisms and are the most widely used group of antibiotics.

According to recent

literatures, more than 60% of commercially available antibiotics using in the world are β-lactam compounds [1]. They are not chemically stable in water and therefore almost degrade during conventional activated sludge processes [2-4]. However, the degree of degradation is largely dependent on the kinds of antibiotics and often insufficient [4]. These antibiotics can give damages to microbial community in sewage systems and aquatic environment [5,6] as well as induce the occurrence of antibiotic-resistant bacteria [7,8]. Therefore, simple and efficient non-microbial methods have to be developed for eliminating β-lactam antibiotics from wastewaters.

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Several physical and chemical processes have been developed for the removal or degradation of β-lactam antibiotics in environmental waters and wastewaters [1]. Photo-catalytic methods such as photo-Fenton reaction and semiconductor photocatalytic oxidation have been reported as the rapid degradation of β-lactams [9-10], but continuous ultra-violet irradiation likely boosts the cost of wastewater treatment.

Requirements of

high oxidant dosage and pH adjustment (typically pH 2~4) are additional expense for large scale treatment.

Activated carbon adsorption has been extensively used in practical water

and wastewater treatment processes [11,12] and applied to the removal of a wide range of pharmaceuticals [13-15].

However, the extent of removal for β-lactam antibiotics is

largely dependent on the kind of antibiotics and often insufficient because of their low hydrophobicity (log Kow = 0.9~2.9 [16]) and negative charge in neutral pH region (pKa ~ 2.7 [16]) [1,15]. An attractive alternative may be a sorption method using surfactant-modified clay minerals namely organoclay.

The organoclay can be readily prepared by mixing

appropriate surfactants and clay minerals in the aqueous media [17-19].

Hydrophobic

organic pollutants in water are incorporated into surfactant aggregates formed between the layers of clay minerals [17-22].

Rather polar and ionizable compounds such as phenols,

pesticides, and pharmaceuticals can also sorb on the organoclay [23-34].

Moreover,

recent reports about the sorption of a selected antibiotic, amoxicillin [28,33], strongly

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suggest the potential usefulness of the organoclay sorption method for the treatment of hospital wastewaters containing different antibiotics. In our preliminary study, a β-lactam antibiotic, penicillin G, was collected from the aqueous solution to surfactant-modified silica gels or surfactant-modified clay minerals. Moreover, we found that it rapidly degraded in these materials even at room temperature and neutral pH.

Such phenomenon has not been reported in previous literatures about

organoclay sorption of a β-lactam antibiotic [28,33], in which the degree of removal might be estimated as the sum of removal and degradation.

However, bifunctional property

strongly suggests the usefulness of organoclay sorption method not only for the removal of β-lactam antibiotics but for their low-cost or eco-friendly degradation.

In the present

study, we studied removal and degradation of different β-lactam antibiotics using dialkylated cationic surfactant-modified montmorillonite.

Factors and mechanisms for

the removal and degradation of antibiotics were investigated.

Furthermore, the

applicability to wastewater treatment was examined by using secondary effluents of municipal sewage treatment plants and synthesized hospital wastewaters.

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2. Materials and methods

2.1. Chemicals Montmorillonite K-30 (MT, surface area: 330 m2 g-1, cation exchange capacity: 0.5 meq g-1 [35]), penicillin G potassium salt, nafcillin sodium salt monohydrate, oxacillin sodium salt monohydrate, cloxacillin sodium salt monohydrate, cefazolin sodium salt, and cefotaxime sodium salt were obtained from Sigma-Aldrich (St. Louis, MO, USA).

A

standard material of (4S)-2-[carboxy[(phenylacetyl)amino]-methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acid) was obtained from LGC Standards (Teddington, UK). Cationic surfactants; cetyltrimethylammonium chloride (CTAC), didodecyldimethylammonium bromide (DDAB), ditetradecyldimethylammonium bromide (DTAB), and dihexadecyldimethylammonium bromide (DHAB), were purchased from Tokyo Chemical Industry (Tokyo, Japan).

Bis-Tris (Dojindo Molecular

Technologies, Kumamoto, Japan) was used as a buffer component.

Orange II (Wako Pure

Chemical Industries, Osaka, Japan) was employed as a colorimetric agent for cationic surfactants.

A molecular probe, N-phenyl-1-naphthylamine (PN, AccuStandard Inc.,

New Haven, CT, USA), was used as 1.0 mM ethanol solution. were analytical or HPLC grade agents.

Other chemicals used

Water was purified with a Milli-Q Integral Water

Purification System (Merck Millipore, Billerica, MA, USA) having a UV irradiation unit.

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2.2. Preparation and characterization of organoclays MT (typically 40 mg) was placed in a 15 mL polypropylene conical tube and rinsed with 10 mL of 50 mM Bis-Tris buffer solution (pH 7) for 1 h.

After removing the buffer

solution, 10 mL of 5 mM Bis-Tris (pH 7) solution containing prescribed amount of cationic surfactant (16 mg of DDAB for degradation study) was added.

The resulting solution

was mildly (60 rpm) shaken for 2 h to prepare surfactant-modified MT organoclay.

The

formed organoclay was washed three times with water before use. Formation of organoclay was confirmed based on the XRD and FT-IR spectra of surfactant-modified MT and unmodified one.

A Rigaku RINT-2000 XRD system (Tokyo,

Japan), [CuKα radiation (λ =1.54056 Ǻ) at 40 kV and 20 mA, divergent slit 1/2°, scatter slit 1/2°, receiving slit 0.30 mm, scan step 0.004°] was used for measuring the XRD spectra of organoclay and unmodified MT in wet conditions.

A Jasco FT/IR-4200

infrared spectrometer (Hachioji, Japan) was employed for measuring IR spectra of freeze-dried clays.

The samples were prepared by well mixing with KBr (1:100) and

pressing into the tablets. The amount of surfactant sorption was estimated from the difference in the initial and residual concentrations in the aqueous solution. The cationic surfactant was determined by an Orange II method composing of ion-pair extraction to chloroform phase

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with an anionic dyestuff, Orange II, and its spectrophotometric determination at 485 nm [36].

For evaluating hydrophobicity of organoclay, 10 µL of PN solution was added to 10

mL of aqueous solution containing cationic surfactant and finely dispersive fraction of MT. The fluorescence spectra were measured by using a PerkinElmer LS-50B luminescence spectrometer (Waltham, MA, USA) with a 1-cm quartz cell.

The emission wavelength

was 340 nm, while width of the excitation and mission wavelength were 2 nm.

2.3. Removal of and degradation of β-lactam antibiotics A 10 mL-portion of aqueous solution (1 mM Bis-Tris, pH 7) containing 10 mg L-1 of antibiotic was added into a 15 mL polypropylene conical tube in which organoclay composing of 40 mg of MT and prescribed amount of DDAB was placed. organoclay was prepared as described above. mixed for 1 min.

The

The resulting solution was gently (60 rpm)

Then, the bulk aqueous solution was sampled using a Hamilton

Gastight® microsyringe (1 mL, Reno, NV, USA) attaching an Advantec Dismic® syringe filter (hydrophilic PTFE, filter size: 13 mm, pore size: 0.45 µm, Tokyo, Japan) in order to eliminate organoclay.

A 20-µL aliquot of the filtrated solution was introduced into a

JASCO HPLC 2000 system (Hachioji, Japan) with an InertSustain® C18 column (length: 150 mm, inner diameter: 3.0 mm, particle size: 5 µm, GL Sciences, Tokyo, Japan) for the separation and determination of antibiotics.

The flow rate of mobile phase was 0.5 mL

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min-1. The composition of mobile phase was 20%(v/v) acetonitrile containing 0.27 mM formic acid and 4.73 mM ammonia (pH 5) for the separation of penicillin G and nafcillin. For separating cefazolin and cefotaxime, 20%(v/v) methanol instead of acetonitrile was employed.

The detection wavelength was 230 nm for penicillin G and nafcillin or 280

nm for cefazolin and cefotaxime. For studying the degradation of antibiotic, the organoclay involving the antibiotic, which was prepared by above described manner, was collected on an OmniporeTM membrane filter (hydrophilic PTFE, filter size: 25 mm, pore size: 0.45 µm, Merck Millipore). The filter had been placed on a stainless supporting mesh setting in a glass holder having 16 mm of inner diameter. The collection was performed passing organoclay slurry with suction but further suction was avoided in order to prevent dryness of the organoclay.

The filter was detached from the glass holder and placed in a glass vial

which had preheated or precooled in an incubator setting the temperature (15, 25, 40, 50, or 60°C) with an EYELA CTP-1000 water circulator (Tokyo, Japan).

After the

incubation, the organoclay was repeatedly (4-times) washed with 2 mL of 95% (v/v) ethanol for completely eluting antibiotics and degraded products.

The eluting solution

was passed through another OmniporeTM membrane filter to remove dispersive organoclay particles and placed into a 10 mL volumetric flask to fix the volume. A 20-µL portion of the resulting solution was introduced into the HPLC system as above described.

To

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identify penicillin G and its degraded products, 5-µL of the solution was introduced into a Waters LC/MS system composing of an Alliance e2695 separation module and a 3100 mass spectrometric detector (Milford, MA, USA).

The separation column, mobile phase,

and flow rate were the same as those in normal HPLC separation.

Ionization potential

and souse temperature were 30 V and 350ºC, respectively. Note that the experimental scale for the removal and degradation of antibiotics was 10 mL being extremely smaller than that of real wastewater treatment plants. This scale was selected for easy handling of the solution, rapid separation of organoclay, and rapid temperature setting which were essential to obtain informative results with minimizing experimental errors.

3. Results and discussion

3.1. Formation and stability of DDAB-MT organoclay Figure 1(A) shows the XRD spectra of unmodified montmorillonite (MT), cetyltrimethylammonium chloride-sorbed montmorillonite (CTAC-MT), and didodecyldimethylammonium bromide-sorbed montmorillonite (DDAB-MT) under wet conditions. The 2θ value of unmodified MT (4.32°) decreased into 2.58° for CTAC-MT and 2.51° for DDAB-MT This observation indicates the incorporation of surfactant

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aggregates between the layers of MT and comparable to the results in literatures about formation and structures of organoclays [18,19, 22].

The modification with DDAB was

also confirmed based on a broad absorbance (νN-H: ~3400 cm-1) and sharp peaks (νC-H: 2960, 2850 cm-1) in the FT-IR spectra of freeze-dried samples (Fig. 1(B)). Figure 2(A) shows the sorption of different cationic surfactants on 1.0 g of MT as a function of the amount of the surfactant added.

Up to ca. 200 mg of mono-alkylated

cationic surfactant, CTAC, nearly completely (>99%) sorbed.

However, the amount of

sorption was limited to ca. 300 mg, probably because of the saturation of cation-exchange site on MT surfaces.

On the other hand, such limitation was not observed in the sorption

of a dialkylated cationic surfactant; DDAB, up to at least 800 mg.

This can be ascribed to

strong interaction of DDAB vesicles with layers of clay minerals [18,22]. Similar tendency was also observed in other dialkylated cationic surfactant; DTAB and DHAB. In the present study, DDAB was selected because of the nearly complete (>99.9%) sorption up to 400 mg, being the greatest amount of stable sorption among the surfactants examined in this study. Stability of DDAB-MT organoclay (composing of 400 mg DDAB and 1.0 g MT) was studied by repeatedly washing with water and compared with that of CTAC-MT (200 mg of CTAC and 1.0 g of MT).

Although there have been extensive studies about the

isotherm of surfactant sorption on clay minerals and the structure of different organoclays

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[17-19,32,34], low-level leaks of these surfactants had rarely been reported.

Even in very

low levels, surfactant leakage can be a fatal problem in the practical wastewater treatment, because most synthetic cationic surfactants are highly toxic to aquatic organisms [38,39]. Figure 2(B) shows the amounts of surfactants leaked from DDAB-MT or CTAC-MT organoclay by repeatedly washing with 50 mL each of water.

The leakage of CTAC

gradually decreased but still remarkably observed even by washing with 400 mL (50 mL × 8) of water, meaning that the use of CTAC-MT organoclay is not adequate to the practical purposes.

On the other hand, the elution of DDAB became to be undetectable levels

(<0.02 mg/L) after washing with 50 mL of water.

Strong stability of DDAB-MT

organoclay can be ascribed to the highly stable sorption of DDAB vesicles. stability was also observed in DTAB-MT and DHAB-MT organoclays.

Similar

However, the

washing of these organoclays involving more than 350 mg (g MT)-1 of surfactant resulted in small but detectable leakage of the surfactant.

As already described above, DDAB-MT

organoclay was selected because of the greatest amount of stable surfactant sorption. Next, we evaluated hydrophobic property of DDAB-MT organoclay because hydrophobic interaction plays important role for organoclay sorption [17,21].

For this

purpose, a fluorescent molecule, PN, was used because it has successfully been employed for evaluating hydrophobicity of different surfactant assemblies [40-42]. Figure 3 shows the emission spectra of PN in the aqueous systems of MT and DDAB-MT.

In the

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presence of DDAB, the emission of PN was significantly enhanced and the emission wavelength shifted to lower region, apparently indicating the formation of hydrophobic region.

A box in Fig. 3 shows the correlation between the relative permittivity of

different solvents and wavelength at the maximum emission (λmax) of PN. indicates λmax value (~408 nm) for DDAB-MT organoclay.

An arrow

By comparing with the values

for different solvents, the organoclay had hydrophobicity like rather polar organic solvents corresponding to 1-octanol or ethyl acetate.

3.2. Removal of β-lactam antibiotics The effect of penicillin concentration on the equilibrium sorption, qe [mg (g dry MT)-1], is shown in Fig. 4(A), in which the concentration is represented by the residual or equilibrium concentration, Cres [mg L-1].

As described in section 3.3, penicillin G

gradually degraded in the organoclay media. The degradation was found to be first-order reaction. Therefore, the removal (%) was compensated by extrapolating the values at different mixing times (e.g. 20 ~ 60 s) to the value at time of zero. The sorption profile of penicillin G was similar to that of different class of organic pollutants, in which the sorption curves were fitted by Langmuir- or Freundlich-type adsorption isotherm [24,32,34].

From the intercept of the linear curve (1/qe = 0.581/Cres + 0.0435, inserted

figure), the sorption capacity of penicillin G estimated was 23.0 ± 0.8 mg (g dry

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DDAB-MT)-1.

Similarly, the capacities of nafcillin (25.4 ± 1.2 mg g-1) and cefotamime

(26.2 ± 1.0 g g-1) were estimated.

However, the reliable value for cefazolin was not

obtained, probably because of the fast degradation rate as well as the requirement of longer time for the equilibrium sorption in high concentration range. Percentage removal (%) of an antibiotic can be calculated from the difference in the initial concentration (C0 [mg L-1]) and the residual one. Removal (%) = (C0 - Cres)/C0 × 100 Figure 4(B) shows that the removal of four β-lactam antibiotics; penicillin G, nafcillin, cefazolin, and cefotaxime, increased with increasing the amount of DDAB-MT organoclay. When DDAB-MT organoclay composing of 40 mg MT and 16 mg DDAB (4.0 g L-1 of MT and 1.6 g L-1 of DDAB) was used for 10 mL solution, the extents of removal were 92 ± 1% for penicillin G, 99 ± 1% for nafcillin, 88 ± 2% for cefazolin, 63 ± 4% for cefotaxime. These values became almost constant by contacting the solution with organoclay for 30 s, indicating the ability of organoclay sorption method for the rapid collection of these antibiotics.

The degrees of removal were also estimated by extrapolating the values

obtained at different immersing times for minimizing the effect of degradation. As shown in Fig. 5(A), unmodified MT hardly collected β-lactam antibiotics, while the collection or removal increased with increasing the amount of DDAB involving the organoclay.

The effect of DDAB on the removal efficiency of a certain antibiotic can be

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represented by the binding constant, Kb, of the antibiotic to DDAB. Kb = X V / q (100 – X) Here, X denotes the difference in the removal (%) of the antibiotic with unmodified MT and that using DDAB-MT organoclay, V is solution volume (L), and q is the amount of DDAB (kg) sorbed on MT.

The correlation between logarithmic binding constants (log

Kb) and logarithmic aqueous-octanol distribution coefficients (log Kow) was shown in Fig. 5(B), in which the values for two other β-lactams; oxacillin and cloxacillin, are also indicated.

A fairly good relationship indicates the significance of hydrophobic

interaction for organoclay sorption.

3.3. Degradation of β-lactam antibiotics Figure 6(A) shows the chromatograms of penicillin G found in the eluting solutions from DDAB-MT organoclay after immersing it in a thermostat chamber (25.0 ± 0.2°C). Peak area or height of penicillin G (retention time: tR = 9.8) decreased with increasing the immersing time, while another peak (tR = 4.1) occurred and the area increased. According to the mass spectra of the respective compounds (Fig. 6(B)) and the retention times of standard materials, penicillin G (m/z = 335) was found to degrade into the hydrolyzed form, penicilloic acid (m/z = 355) having no β-lactam ring that is essential for antibiotic activity.

Same degradation feature is also encountered in conventional

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wastewater treatment plants and environmental water [43], where the degradation is induced by bacteria having β-lactamase. Time-dependent degradation profiles in different media are indicated in Fig. 6(C), in which the extent of degradation is represented by the ratio of the concentration in initial solution to that in eluting solution (C/C0).

Although penicillin G is not so stable in water

[34], its degradation was negligible in this experimental period (2 h). shows the result in 10 mM DDAB solution.

Figure 6(C) also

The DDAB solution is slightly turbid

because of the formation of micelles or vesicles.

Catalytic activity of surfactant

assemblies (namely micellar catalysis) has extensively been studied for several reactions (particularly for hydrolysis of esters) [44].

In the DDAB solution, however, distinct

degradation was not observed in this experimental period. degradation is not owing to micellar catalysis.

This fact indicates that rapid

Significant degradation was observed only

by the presence of both of DDAB and MT. The sorption on DDAB-MT organoclay for 2 h allowed nearly complete (>98%) degradation of penicillin G. No other chemicals or energies were required.

This result strongly suggests potential applicability of organoclay

sorption method as an eco-friendly process for the degradation of antibiotics in wastewaters. The first-order reaction can be represented by, ln (C/C0) = - k t,

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where k [min-1] is rate constant, and t [min] is the time of organoclay sorption.

Figure

7(A) indicated first-order reaction curves for the degradation of penicillin G in DDAB-MT organoclay at different temperatures. The rate constant, k, estimated from the slope of linear curve increased with increasing the temperature.

Logarithmic rate constant, ln k,

is represented by an Arrhenius equation and the plot is depicted in Fig. 7(B). ln k = -Ea/RT + ln A Here, Ea [J mol-1] is activation energy, R [J K-1 mol-1] is gas constant, T [K] is temperature, and A is frequency factor.

Activation energy, Ea, was successfully estimated from the

slope of the Arrhenius plot and was 48 ± 1 kJ mol-1, being far lower than the reported value (83.5 kJ mol-1 [45]) in water.

These results support the assumption that DDAB-MT

organoclay has catalytic activity for the degradation of penicillin G. Figure 8 indicates the effects of solution pH (A) and the amount of DDAB sorption (B) on the k value.

The k value increased with the decrease in the solution pH which

resulted in the increase in protonated moieties such as Si-OH and Al-OH on MT surfaces [46]. These hydroxyl groups may act as acidic points of catalysis for the hydrolysis reaction of penicillin G. amount of DDAB sorption.

On the other hand, the k value increased with increasing the Occurrence of hydrophobic region was requisite for the

incorporation and degradation of penicillin G. The catalytic activity for hydrolysis reaction has also been reported in a bifunctional

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organoclay modified with a cationic surfactant, (N-decyl-N,N-dimethyl-N-(2-aminoethyl) ammonium [47,48].

Organoclay-catalyzed hydrolysis of an organophosphate insecticide,

methyl parathion, occurred in alkaline conditions, because of the basic functionality of the surfactant.

On the other hand, DDAB does not have such basic functionality. Moreover,

the catalytic ability for hydrolysis reaction increased in not alkaline conditions but weekly acidic conditions.

Similar catalytic activity of organoclay may only be found in the

micelle-catalyzed hydrolysis reaction of an organophosphate insecticide, fenitrothion, in the presence of clay mineral [49], where the surfactant-modified clay or organoclay can form and potentially act as the catalyst. Figure 9(A) shows time-dependent degradation profiles of four β-lactam antibiotics. Penicillin G and nafcillin are penicillin-class antibiotics whose β-lactam fuses to saturated five-membered ring, while cefazolin and cefotaxime are cephalosporin antibiotics having a β-lactam ring fusing to unsaturated six-membered ring.

The degradation rate was largely

dependent on the kind of β-lactam antibiotics but seemed to be independent of the class of β-lactams.

Of these antibiotics, cefazolin was the most degradable, while the lowest

degradability was found in another cephalosporin-type antibiotic; cefotaxime.

The extent

of organoclay sorption or the accessibility to MT surfaces may influence the degradability. Figure 9(B) shows the percentage of remaining antibiotics in the solution as a function of time contacting with DDAB-MT organoclay.

Different from the case of Fig.

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9(A), degradation occurs accompanying with the distribution between bulk aqueous phase and organoclay.

The degradation reduces the activity of antibiotic and thus shifts the

distribution equilibrium from the aqueous phase to organoclay.

Even cefotaxime having

the lowest removal and degradability was nearly completely (>99%) eliminated from water by the organoclay contacting for 3 h.

3.4. Application to wastewater Finally, applicability to wastewater treatment was studied by using different wastewater samples.

Table 1 lists water qualities such as pH, conductivity, and chemical

oxygen demand (CODMn) of secondary effluents from conventional activated-sludge municipal sewage treatment plants (samples A and B) and synthesized hospital wastewaters (samples C, D, and E) containing different amounts of a protein (bovine serum albumin). The kinds and amounts of ingredient added were selected based on the characteristics of hospital effluents in reference to a literature [50] and are listed at a footnote in Table 1.

Although the concentrations of most antibiotics in hospital effluents

are normally far below mg L-1-levels [1,5,51], model wastewaters containing 10 mg L-1 of penicillin G were used to clarify the applicability to highly contaminated effluents.

As

also listed in Table 1, ca. 90% or greater extent of penicillin G was eliminated from these wastewater samples.

Existence of highly concentrated (1000 mg L-1) protein (in sample

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E) did not significantly interfere with the penicillin removal.

This fact may be ascribable

to the high stability of DDAB-MT organoclay and strongly suggests the practical applicability to different wastewaters containing large amounts of dissolved organic components. Use of matrix-immobilized organoclays or matrix-organoclay mixtures as column or fixed-bed systems have extensively been studied for removing different organic pollutants from water [52-54].

For continuous wastewater treatment, DDAB-MT organoclay may

be also used as column system. The application to hospital effluents or wastewaters from pharmaceutical industries would reduce environmental impact of antibiotics.

4. Conclusions Didodecyldimethylammonium bromide (DDAB)-montmorillonite (MT) organoclay was a useful sorbent not only for the removal of β-lactam antibiotics from water but for their eco-friendly degradation.

DDAB hardly leaked from clay minerals by repeated

washing with water, strongly suggesting the availability of the organoclay for practical purposes.

The antibiotics incorporated into the organoclay were found to rapidly be

degraded to the hydrolyzed form missing a β-lactam ring.

The sorption method using

DDAB-MT organoclay would be useful for the treatment of hospital wastewaters containing highly concentrated β-lactam antibiotics.

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Acknowledgments This study was supported by a Grant-in-Aid for Science Research Japan (B) (15H03842). We thank Dr. Masataka Hiraide (emeritus professor of Nagoya University) for many valuable discussions and Dr. Takafumi Maeda (Kitami Institute of Technology) for helping the measurement of XRD spectra.

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29

Figure captions Fig. 1. XRD (A) and FT-IR spectra (B) of surfactant-modified and unmodified MT. DDAB (mg g-1): amount (mg) of DDAB sorbed on 1 g of MT.

30

31

Fig. 2. Sorption of CTAC (

), DDAB (

), DTAB (

), and DHAB (

) on MT

(A) and surfactant leakage from CTAC-MT or DDAB-MT organoclay by repeatedly washing with water (B).

32

33

Fig. 3. Emission spectra of PN in different media.

Δ indicates difference in the intensity

in DDAB-MT organoclay from that in unmodified MT (back ground).

A box shows the

correlation between relative permittivity of solvent and the wavelength (λmax) for the maximum emission of PN (1: water, 2: methanol. 3: ethanol, 4: 1-butanol, 5: 1-octanol, 6: ethyl acetate, 7: diethyl ether, 8: benzene).

34

Fig. 4. Sorption of different concentration of penicillin G on DDAB-MT organoclay (A) and removal of four β-lactam antibiotics (B) in which symbols are described.

35

36

Fig. 5.

Effect of the amount of DDAB sorbed on MT on the removal of β-lactam

antibiotics (A) and the correlation between logarithmic binding constant (log Kb) and logarithmic aqueous-octanol distribution coefficient (log Kow) (B).

Symbols in Fig. 5(A)

are the same as those used in Fig. 4(B).

37

38

Fig. 6. Chromatograms of penicillin G and its degradation product in the eluting solution from DDAB-MT organoclay (A), mass spectra for the respective peaks (B), and time-dependent degradation of penicillin G in different media (C).

39

40

Fig. 7.

First-order reaction curves for the degradation of penicillin G at different

temperatures (A) and Arrhenius plot (B).

41

42

Fig. 8. Effects of solution pH (A) and the amount of DDAB sorption (B) on k.

43

Fig. 9. Time-dependent degradation of penicillin G ( (

), and cefazoline (

), nafcillin (

), cefotaxime

) in DDAB-MT organoclay (A) and their time-dependent

elimination from water by continuously contacting the organoclay (B).

44

45

Table legend Table 1 Water quality and removal of penicillin G using DDAB-MT organoclay (1.6 g L-1 of DDAB and 4.0 g L-1 of MT).

Sample

A*

B*

C**

D**

E**

pH

7.3

6.8

6.8

6.9

6.8

CODMn (mg L-1)

2.1

14.1

570

676

Albumin (mg L-1)

-

-

1

Removal (%)

97 ± 2

89 ± 4

95 ± 2

50

95 ± 3

1620 1000

93 ± 5

*Effluent from an activated-sludge sewage treatment plant. **Ingredient (mg L-1): Urea 1500, Creatinine 50, D-Glucose 360, Starch 200, Propionic acid 100, Butyric acid 50, Ammonium acetate 110, NaCl 3000, KH2PO4 33, NaNO2 220..

46