Journal of Hazardous Materials 271 (2014) 65–72

Contents lists available at ScienceDirect

Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat

Modified clay minerals efficiency against chemical and biological warfare agents for civil human protection Daniela Plachá a,c,∗ , Kateˇrina Rosenbergová b , Jiˇrí Slabotínsky´ b , d ˇ ˇ Studentová Kateˇrina Mamulová Kutláková a , Sona , Graˇzyna Simha Martynková a Nanotechnology Centre, VSˇ B-Technical University of Ostrava, 17. listopadu 15, Poruba, Ostrava 708 33, Czech Republic National Institute for NBC Protection, Kamenná 71, Milín 262 31, Czech Republic c Energy Units for Utilization of non Traditional Energy Sources, VSˇ B-Technical University of Ostrava, 17. listopadu 15, Poruba, Ostrava 708 33, Czech Republic d Regional Materials Science and Technology Centre, VSˇ B-Technical University of Ostrava, 17. listopadu 15, Poruba, Ostrava 708 33, Czech Republic a

b

h i g h l i g h t s • • • •

The efficient composite materials against chemical warfare agents such as yperite. The efficient materials against biological warfare agents such as Yersinia pestis. Efficient alternative material for protective clothing or filtration equipment. Material efficiency can be tailored according to the needs.

a r t i c l e

i n f o

Article history: Received 28 August 2013 Received in revised form 23 January 2014 Accepted 25 January 2014 Available online 12 February 2014 Keywords: Yperite Mustard gas Antibacterial activity Permeation Clay mineral

a b s t r a c t Sorption efficiencies of modified montmorillonite and vermiculite of their mono ionic Na and organic HDTMA and HDP forms were studied against chemical and biological warfare agents such as yperite and selected bacterial strains. Yperite interactions with modified clay minerals were observed through its capture in low-density polyethylene foil-modified clay composites by measuring yperite gas permeation with using chemical indication and gas chromatography methods. The antibacterial activities of synthetized organoclays were tested against selected Gram-positive and Gram-negative bacterial species in minimum inhibitory concentration tests. The obtained results showed a positive influence of modified clay minerals on the significant yperite breakthrough-time increase. The most effective material was the polyethylene-Na form montmorillonite, while the polyethylene-Na form vermiculite showed the lowest efficiency. With increasing organic cations loading in the interlayer space the montmorillonite efficiency decreased, and in the case of vermiculite an opposite effect was observed. Generally the modified montmorillonites were more effective than modified vermiculites. The HDP cations seem to be more effective compare to the HDTMA. The antibacterial activity tests confirmed efficiency of all organically modified clay minerals against Gram-positive bacteria. The confirmation of antibacterial activity against Y. pestis, plague bacteria, is the most interesting result of this part of the study. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +420 597 321 575; fax: +420 597 321 640. E-mail addresses: [email protected] (D. Plachá), [email protected] ´ (K. Rosenbergová), [email protected] (J. Slabotínsky), [email protected] (K.M. Kutláková), [email protected] (S. ˇ Studentová), [email protected] (G.S. Martynková). http://dx.doi.org/10.1016/j.jhazmat.2014.01.059 0304-3894/© 2014 Elsevier B.V. All rights reserved.

Chemical (CWA) and biological (BWA) warfare agents are perpetually a low-probability but high-impact risk to the military and the civilian population [1,2]. This fact was confirmed in the 1990s, when the proliferation of these agents to the terrorist groups was a warning. The globalisation and escalations of terrorist attacks in recent times induced the necessity to include these threats to national and international emergency and risk management plans

66

D. Plachá et al. / Journal of Hazardous Materials 271 (2014) 65–72

[1]. Due this fact, ongoing research aimed to human protection against CWA and BWA is still of great importance. There are many research studies, manuals as well as patents aimed at capturing and disposing of CWA and BWA. These methods are based on sorption, catalysis or chemical reactions [3–12]. However, decontamination and disinfection only deal with reduction of the effects of agent use. An important part of the research should be focused on prevention of injuries; for instance to develop materials which are able to effectively capture pervasive CWA or BWA and simultaneously prevent secondary contamination due to toxic compound release from material. Such material can be consequently used as personal protection clothing. One of the possibilities is polymer-clay hybrid microcomposites or nanocomposites that are known to have improved physical and mechanical characteristics including decreased gas permeability [13–15]. Yperite as one of the most significant CWA representative is often used for testing of resistance of polymeric barrier materials such as rubber, foils and protective materials because it easily penetrates through them [9]. Yperite or mustard gas (bis[2-chloroethyl]sulphide) is well known vesicant agent. This sulphur-containing organic substance is a highly reactive bifunctional compound with antimitotic, mutagenic, teratogenic, cytotoxic and carcinogenic properties. It is characterized by high toxicity, extreme multiple effects, high boiling point, high density and vapour pressure, high chemical stability, lipophility and penetration capacity [16]. It is an alkylating agent that, when absorbed, causes chemical reactions with cellular components resulting in cytotoxic effects [17]. Several studies after World War I and the Iraq–Iran conflict provided much detailed information about the long-term effects of yperite. The first symptoms can occur immediately or from several hours to days after exposure. Severe damages to the eyes, respiratory system, internal organs and skin are caused; the effects are related to dose and time of exposure [17,18]. It persists for a long period in temperate climates. There is no effective treatment against yperite intoxication. One of the possibilities is the wearing of protective clothing during decontamination or other contact [17]. Biological agents can be microorganism and toxins which are intended for use in military operations in order to kill, seriously injure or incapacitate exposed individuals by exerting their physiological effects [1]. A large number of biological agents have been investigated for their potential utilization as biological agents. Several bacterial strain such as Bacillus anthracis (Anthrax), Y. pestis (Plague), Francisella tularensis (Tularemia), Brucella spp. (Brucellosis) and Malleomyces pseudomallei (Melioidosis) have been ranged among most serious hazards. Modified clay minerals are well known to have the ability to adsorb natural and anthropogenic toxic compounds [19–22]. Montmorillonite and vermiculite are two important clay mineral representatives. Both are ranged into phyllosilicates 2:1 characterized with layered structures and negative charge on the

layers, which is compensated by inorganic cations such as Na+ , K+ , Ca2+ , Mg2+ in the interlayer space. In their natural form they are hydrophilic. Due to simple ion exchange of inorganic cations for organic cations they become hydrophobic and can interact with organic compounds. The utilization of hexadecyltrimethylammonium (HDTMA) and hexadecylpyridinium (HDP) cations for this ion exchange is well known. Both cations are members of the quaternary ammonium salt group, which share a positively charged hydrophilic ammonium moiety and long hydrocarbon hydrophobic chain. The compounds have bacteriostatic and bactericidal activity, which is due to their abilities to alter the permeability of cellular membranes allowing intracellular low molecular-weight components to diffuse out [19,22]. This process results in cellular death. While natural montmorillonite and vermiculite or their Na form showed no antibacterial effects, organically modified montmorillonites and vermiculite are efficient materials against selected bacteria such Escherichia coli, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enteritidis as was described in several research works [19,22,23]. The aim of the study was to prepare an effective barrier that will have a sufficient breakthrough detection time for a CWA such as yperite, and will simultaneously prevent secondary contamination as a consequence of yperite release from the barrier. The prepared material could be used as a protective cloth material. The effective barrier was prepared from a low-density polyethylene foil (LDPE) with modified clay minerals as fillers using montmorillonite and vermiculite in their monoionic Na and organically modified HDTMA and HDP forms. The breakthrough detection time (BT), permeation rates (F), an estimation of yperite capture in material (A) and total quantities of yperite penetration through the barrier systems (Qp ) were determined for all prepared materials by use of chemical indication tests and dynamic gas chromatographic methods. Modified clay minerals were tested on antibacterial activities against potential biological agents such as Y. pestis and B. anthracis and other pathogenic bacteria Staphylococcus aureus, Streptococcus agalactiae, E. coli and P. aeruginosa which could be disposed on a protective clothing surface. 2. Experimental 2.1. Materials Natural Ca-montmorillonite from a deposit in Ivanˇcice (Czech Republic) and Mg-vermiculite from Brazil (supplied from Grena a.s., Czech Republic) with particle size smaller than 40 ␮m were used for preparation of modified clay minerals. Their crystallochemical formulas (Si7.96 Al0.04 ) (Al2.52 FeMg0.90 Ti0.04 ) O20 (OH)4 (Ca0.24 K0.06 Na0.09 Mg0.10 ) and (Si6.32 Al1.58 Ti0.1 ) (Mg4.75 Ca0.34 Fe0. 91 ) O20 (OH)4 (Ca0,04 K0.38 ) were calculated on the basis of the elemental analysis using X-ray Fluorescence Spectrometry methods. The cation exchange capacities (CEC) of montmorillonite and vermiculite were 86 and 122 cmol(+)/kg, respectively, both

Fig. 1. Reaction scheme of a) N-chloro-N-(2-tolyl) benzamide preparation, b) yperite interaction with N-chloro-N-(2-tolyl) benzamide [25].

D. Plachá et al. / Journal of Hazardous Materials 271 (2014) 65–72

determined by the Cd2+ exchange method using Absorption Atomic Spectrometry as found in Refs. [15,20,21,24]. Sodium chloride (NaCl), hexadecyltrimethylammonium bromide (HDTMA) C19 H42 N+ Br− and hexadecylpyridinium chloride monohydrate (HDP) C21 H38 N+ Cl− ·H2 O were used for natural montmorillonite and vermiculite modifications. All solutions were prepared with analytical grade chemicals and ultra-pure water (Milli-Q Academic system). All chemicals were supplied by Sigma–Aldrich. Test substance was bis(2-chloroethyl) sulphide (mustard gas, yperite or HD, ≤96%). The chemical indicator for the BT indication was prepared by impregnation of fragrance-free paper tissue (Melitrade a.s.) by aqueous solution of congo red (Sigma–Aldrich), consequently dried and impregnated by N-chloro-N-(2-tolyl) benzamide. Sensitivity for yperite vapour was determined to be 5 ␮g cm−2 [25]. N-chloro-N-(2-tolyl) benzamide was prepared in the laboratory by benzoylation of o-toluidine and a subsequent chlorination of benzoyl-o-toluidine (Fig. 1a), and Sigma–Aldrich supplied all chemicals used. The reaction scheme of yperite with N-chloro-N-(2-tolyl) benzamide is presented in Fig. 1b. Antibacterial effects of modified clay minerals were tested on bacterial strains that were obtained from the Czech Collection of Microorganisms (CCM) of Masaryk University (Brno, Czech Republic). The Gram-positive bacteria strains (G+) Staphylococcus aureus (S. aureus, CCM 299) and Streptococcus agalactiae (Str. agalactiae, CCM6187) and the Gram-negative bacteria strains (G−) Escherichia coli (E. coli, CCM 3954) and Pseudomonas aeruginosa (P. aeruginosa, CCM 1960) were used together with G+ bacteria B. anthracis (B. anthracis, vaccine strain Antraxen, Bioveta Czech Republic) and G− bacteria Y. pestis (Y. pestis, NCTC 5923, National Collection of Type Cultures, United Kingdom). The bacterial strains were selected to compare results presented in previous studies, and the last two are well-known biological warfare agents, originators of Anthrax and Plague. Chemical activity (permeation) tests were performed using the low-density polyethylene foils (LDPE), thickness 0.08 mm (Granitol Beroun, Czech Republic) and textile from polyacrylonitrile (PAN) spun bonded fabrics, an average weight of 1.0 g m−2 , and average fiber diameters of 200–300 nm. 2.2. Methods 2.2.1. Preparation of modified clay minerals Firstly, monoionic Na forms of the montmorillonite and vermiculite were obtained by saturation of their natural forms repeatedly with 2 M NaCl aqueous solutions. Consequently the Namontmorillonite and Na-vermiculite were intercalated with either HDTMA or HDP cations in aqueous solutions. Two different organic cation concentrations were used for the both clay mineral intercalations, which correspond to 20% and 50% of the vermiculite CEC (0.2 and 0.5 CEC) to prepare organoclays with approximately equal organic cation loadings. The lower levels of intercalations were used in accordance with our previous research [20,21]. To obtain the defined composition of all materials the same procedure was used for treatment of both clay minerals as described in [20,21]. The Na forms of clay minerals were denoted as M0 and V0, organically modified clay minerals M1–M4 and V1–V4, see Table 1. The synthetized modified clay minerals were dried at 50 ◦ C and kept dry as possible before tests in a dark place for characterization and subsequent permeation and antibacterial activity tests. 2.2.2. Characterization of modified clay minerals The structural changes evoked by the intercalations in modified montmorillonite and vermiculite in interlayer space were confirmed by XRD analysis and analysis of organic carbon (TOC),

67

Table 1 Modified clay mineral parameters. Indication M0 M1 M2 M3 M4 V0 V1 V2 V3 V4

Clay mineral Montmorillonite Montmorillonite Montmorillonite Montmorillonite Montmorillonite Vermiculite Vermiculite Vermiculite Vermiculite Vermiculite

Modifier +

Na HDTMA HDTMA HDP HDP Na+ HDTMA HDTMA HDP HDP

foc (%) 0.01 7.40 12.44 4.63 13.17 0.04 5.24 13.46 5.57 12.21

SBET (m2 g−1 ) 75 16 7 19 5 21 9 4 7 4

d0 0 1 (nm) 1.29 1.80 2.03, 2.77 1.75 2.16 1.19 1.21 1.37, 2.72 1.21 2.52, 3.43

where the amount of organic carbon was denoted as a percentage of organic carbon (foc ) in the sample. The specific surface area (SBET ) of modified clay minerals was also determined. The XRPD patterns were recorded under CoK␣ irradiation ( = 1.789 Å) using the Bruker D8 Advance diffractometer (Bruker AXS) equipped with a fast position sensitive detector VÅNTEC 1. Measurements were carried out in the reflection mode, and powder samples were pressed in a rotational holder. Phase composition was evaluated using database PDF 2 Release 2004 (International Centre for Diffraction Data). The quantities of organic matter were determined by TOC analysis with a TOC analyzer MULTI N/C 3100 Carl Zeiss Jena. The values of SBET (m2 g−1 ) of each modified clay mineral were determined by use of a Sorptomatic 1990 (Thermo Scientific). 2.2.3. Chemical activity tests The chemical activity tests were performed with using a composite system consisting of two LDPE layers with textile from nonwoven fleece embedded between layers. Inter-fibre spaces of the fleece were filled with modified clay minerals with a defined mass weight (0.1 g). The LDPE foils ensure that only yperite vapours permeate through the tested material, and they restrict its permeation rate. The fleece was used for bulk filler fixation and for its uniform spreading. One of outer layers of the composite system was wetted with yperite for the duration of the experiments. The permeation to the reverse bottom side was detected in two ways: 1) with chemical indication and 2) with gas chromatography. For comparison one model of the composite material was prepared in the identical way where NaCl was used as filler due to its negligible sorption properties instead of modified clay minerals (marked in followed text as PE-NaCl) and one model of tested material consisted only of two LDPE layers with fleece (2xPE). For evaluation of the chemical indication test an indication paper was attached to the bottom side of the LDPE composite. An impermeable circular annulus (area of 4.5 cm2 ) was delimited on the outer top layer of LDPE composite. This annulus was wetted with yperite for the duration of the experiment. The entire system was enclosed between two transparent glasses and kept at 25 ± 1 ◦ C. The first variation in colour of the indication paper was registered as the breakthrough detection time (BTCHI ) from the beginning of yperite contamination [25]. For the breakthrough detection time (BTD ) evaluation by use of gas chromatography the testing composite system was closed between two metal parts of a measuring cup (internal circular area of 4.9 cm2 ). Yperite vapours that permeated through the composite system were pulled away with clean air (with a flow of 100 cm3 min−1 ) that was blown under the bottom side of the LDPE foils. The yperite vapours were diverted to a sampling loop (volume of 1 cm3 ) and injected with helium directly to the chromatographic column. The analysis was performed using a gas chromatograph DANI 1000 with flame ionization detector (GC/FID), equipped with column Restek MXT® −1301, 15 m × 0.53 mm × 3.00 ␮m. The

68

D. Plachá et al. / Journal of Hazardous Materials 271 (2014) 65–72

described in Ref. [23]. The selected bacterial cultures were inoculated (1.0 × 109 CFU ml−1 ) to all the prepared suspensions in test tubes. The test tubes were agitated at room temperature; and aliquots of the final suspensions were after the elapse of 30, 60, 90, 120, 180, 240 and 300 min, and further 6 days always in 24 h intervals taken and inoculated on blood agar plates for the bacterial viability assessment. The bacterial incubation on blood agar plates was performed for 24 h at 37 ◦ C. The minimum inhibitory concentrations (MIC) of the prepared modified clay minerals were determined as the lowest concentrations that caused full inhibition of bacterial growth.

3. Results and discussion 3.1. Characterization of modified clay minerals

Fig. 2. XRD patterns for modified clay minerals a) montmorillonite, b) vermiculite.

analyses were carried out with isothermal temperature program of 150 ◦ C. The limit of detection for yperite vapours is 6 mg dm−3 . Both given methods are commonly used for yperite permeation tests in the National Institute for NBC Protection, Milín, Czech Republic. They are accredited by the Czech Accreditation Institute and are in accordance with the standards ISO/IEC 17025:2005 [25–27]. 2.2.4. Antibacterial activity tests Ten dispersions of 10% (w/v) of the modified clay minerals in twice distilled water were prepared for antibacterial activity testing. The dispersions were consequently diluted by a three-fold dilution method in glucose stock to obtain samples with concentrations of 3.33%, 1.12%, 0.37%, 0.12%, 0.041% and 0.014% (w/v) as

The results of the characterization of all synthesized modified clay minerals are given in Table 1. Only traces of organic carbons in the M0 and V0 samples were determined by the TOC analysis. The samples were characterized by the highest external specific surface area values. Montmorillonite showed a 3.5 times higher value than vermiculite, and the values are in accordance with data reported in the literature [27]. The XRPD patterns showed intensive basal reflections (d0 0 1 ) of 1.29 nm for the Na form of montmorillonite and 1.19 nm for the Na form of vermiculite. The patterns correspond to well-ordered material and confirmed the mono ionic form of both clay minerals. The M1, M3, V1 and V3 samples were prepared to reach the lower level of either HDTMA or HDP cations loadings. They were synthetized by the treatment of M0 and V0 samples with the organic cation aqueous solutions, with concentrations corresponding to 0.2 CEC of the vermiculite in order to ensure similar organic cations loadings in the interlayer space of the both clay. The realized cation exchange can be deduced from the results of TOC analyses, which verified almost similar contents of organic carbon (foc ) in M3, V1 and V3 samples. The higher organic carbon content was obtained in the M1 sample probably due to the less complex structure of the HDTMA cation, which can penetrate into the interlayer space more easily than the HDP. The HDTMA cations can be arranged in interlayer space in a more well-ordered way than the HDP cation, as was shown in our previous studies [20,21]. The XRD patterns (Fig. 2) confirmed an intercalation of both organic cations to the interlayer space in all analysed samples when the basal spacing values were shifted towards higher values. In the vermiculite galleries with a higher negative layer charge only a slight change was observed (d0 0 1 = 1.21 nm), and the hexadecylcarbon chains correspond to the internal arrangement

Fig. 3. Yperite BTCHI as determined by chemical indication tests. Two arrows (M0, M3) indicate that no BTCHI was reached after 6 h of observation.

D. Plachá et al. / Journal of Hazardous Materials 271 (2014) 65–72

in lateral monolayers [29]. The interlayer space of the montmorillonite galleries (with lower negative layer charge) increased more (d0 0 1 = 1.75 and 1.80 nm), which corresponds to the fact that arrangement of the intercalated organic cations depends on the layer charge and long carbon chains are probably arranged in bilayers [29]. The organic cation presence was confirmed with lower values of SBET for all four organoclays. The results are given in Table 1. The M2, M4, V2 and V4 samples were prepared to have higher organic cations loading. In our previous studies we found that the complete cation exchange is not necessary for sorption processes due to reservation of sufficient space in the interlayer space for accepting other molecules. Thus, the exchanged organic cation quantities corresponded to 0.5 CEC of the vermiculite. The TOC analyse confirmed higher organic carbon content in all four samples; the results were almost similar (122.1–134.6 g kg−1 ). The XRD patterns of montmorillonite samples showed wellordered structures where basal spacing values increased to 2.16 nm (HDP-montmorillonite; M4) and 2.03 and 2.77 nm (HDTMAmontmorillonite; M2). The XRD patterns of vermiculites showed a less regular arrangement of their structure, especially in HDPvermiculite (V4) where several domains were formed with various basal spacing values of (3.43, 2.52, 1.91 and 1.24 nm). In the HDTMA-vermiculite (V2) only two main domains were significant, with basal spacing of 2.72 and 1.37 nm (Fig. 2). The V2 structure is better arranged than the V4. The hexadecylcarbon chains form a pseudotrimolecular or paraffin-type arrangement in the interlayer space of both modified clay minerals [29]. Observed differences between organically modified montmorillonite and vermiculite structures can be explained by two premises: 1) the higher negative charge on the vermiculite layers makes exchange of inorganic cations for organic more difficult than in montmorillonite structure, 2) simultaneously the HDP cations due to their molecular structure can be located more disorderly in the interlayer space of a clay gallery structure than the HDTMA [20,21]. The SBET values for M2, M4, V2, V4 are ranged in Table 1, the values are the lowest from Na forms, organic cation forms with lower and higher organic carbon content. It corresponds to a higher quantity of organic cations that are located not only in the interlayer space but also on the external surfaces of both clay minerals.

69

3.2. Test of chemical activities Synthetized materials efficiency against yperite vapours was evaluated on the basis of: 1) comparison of breakthrough detection time (BT), which is time immediately preceding that at which the tested chemical is firstly detected, determined by chemical indication (BTCHI ) and gas chromatography tests (BTD ) and 2) yperite vapour permeation rate (F) through each modified clay mineral based composite material on the basis of the obtained gas chromatography results. The two LDPE foils served as a restrictor of the permeation rate, with which the initial vapour concentrations reached the clay mineral layer embedded between foils. The compared BT values for two LDPE layers filled only with unwoven fleece (2xPE) were determined as: 1) 55 min with the chemical indication, 2) 91 min with the gas chromatographic determination. This variance is done by different analytical method sensitivity when the chemical indication method is able to immediately indicate a local vapour penetration through the weakest site, since the used indicator is in direct contact with the LDPE foil bottom side. Utilization of the dynamic gas chromatographic method is accompanied by dilution of permeating yperite vapours with carrier gas flow, thus value of limit of detection is higher. The study of BT determination of yperite vapours through LDPE-modified clay mineral composite by the chemical indication method showed the important influence of clay minerals in their mono ionic and modified forms on BTCHI values (Fig. 3). The analyses were compared with model PE-NaCl. All tested LDPEmodified clay mineral composites showed higher BTCHI values than PE-NaCl. M0 was the most efficient material for yperite vapour capture when the determined BTCHI value was determined as higher than 361 min, while V0 was the least, only 101 min. Regardless; the V0 BTCHI value was nearly twice higher than PE-NaCl. In case of montmorillonite composite the sorption efficiency declined with growing content of organic cations in the interlayer space of montmorillonite. Conversely, the BTCHI values increased in modified vermiculite forms, where no significant differences were observed in dependency of the organic cation loading (Fig. 3).

Fig. 4. Yperite vapour permeation rate (F) through LDPE-modified clay mineral composites. FN is normalized permeation rate, FN = 0.1 ␮g cm−2 min−1 .

70

D. Plachá et al. / Journal of Hazardous Materials 271 (2014) 65–72

Table 2 Parameters of yperite capture determined by dynamic gas chromatographic method. Composite M0 M1 M2 M3 M4 V0 V1 V2 V3 V4

BTD (min) 1180 830 826 1130 791 171 175 196 413 448

t (min) 1089 739 735 1039 700 80 84 105 322 357

A (␮g cm−2 ) 762 517 514 727 490 56 59 74 225 250

The observed results were confirmed by the BTD values determined by the dynamic gas chromatographic method. The BTD values were derived from time dependence of yperite vapour permeation rate F through LDPE-modified clay mineral composites (Fig. 4). Normalized permeation rate FN = 0.1 ␮g cm−2 min−1 was used as a limit value for the BTD determination according to ISO standard ISO 6529 [30]. Found BTD values for the LDPE composites are listed in Table 2. Unambiguously, the highest sorption efficiency of M0 is confirmed, followed by M3, M1, M2, and M4. The least effective composites were V0, V1 and V2, and the V3 and V4 samples showed a nearly twice-higher ability for the yperite capture. We can suppose stronger interactions of yperite molecules with pyridinium rings as consequence of different electron density distribution. Table 2 contains experimental and calculated values of captured yperite vapours on the basis of steady-state permeation rate of yperite vapour FPE∞ through LDPE layers of a defined thickness at a defined temperature. This permeation rate for yperite permeation through LDPE foils was estimated as FPE∞ = 0.7 ␮g cm−2 min−1 and corresponds to the value of the constant permeation rate that occurs after breakthrough when the chemical contact is continuous and all forces affecting permeation have reached equilibrium [31,32]. It is expected that yperite will permeate from LDPE into the porous composite at the same rate, and before reaching BTD a quantity of yperite A will be captured by the composite material during the time from contamination beginning at BTD . The A can be calculated as A = FPE∞ × t (␮g cm−2 ), where t = BTD (composite)

-BTD (2xPE). BTD (2xPE) is breakthrough detection time of yperite LDPE foils permeation, and corresponds to 91 min in this study [27]. The results of determined t and A are given in Table 2. The total quantity of yperite (Qp ) penetrating the prepared composites at 25 ◦ C in time is presented in Fig. 5, the red line indicates the limit value of 0.005 mg cm−2 [27]. The breakthrough time (BTD ) can be also determined as the intersection of the curve with this limit value line. All commented results confirmed our previous observation that montmorillonite is more efficient yperite sorbent than vermiculite. Consequently, we observed that while the sorption efficiency of the montmorillonite decreases with organic cations modifications; the vermiculite sorption efficiency increases with organic cations loadings. The significant distinctions in M0 and V0 yperite capture efficiencies can be explain with different structural properties of montmorillonite and vermiculite. While penetration of organic molecules to the montmorillonite gallery is generally effortless, it is more difficult for them to penetrate to the vermiculite gallery due to higher negative charge on the vermiculite internal layers [28,29]. Thus we can expect smaller interactions of yperite molecules with internal surfaces of the vermiculite in Na-form and consequently faster permeation rate through LDPE-vermiculite composite in comparison to the montmorillonite in Na-form. Modification of interlayer space by organic cations increased basal spacing values and it opens up the interlayer space of the both clay minerals [15,20,21,28,29]. The yperite molecules can more easily enter to the vermiculite interlayer space and interact with Na+ ions localised there or with internal surface of the vermiculite, which was demonstrated by higher BT values. The organic cations in the montmorillonite galleries seem to obstruct a part of the internal surface and thus decrease yperite interactions, which is accompanied by a shortening of BT values. It is also necessary to note that if the BTCHI value for M0 was >361 and for M3 >342 min and permeation rate was not detected, it does not mean breakthrough has not occurred. It means that permeation was not detected after an observation time of six hours. Permeation may have occurred, but at a rate less than the minimum detectable permeation rate. From calculated A values (Table 2) it is concluded that M0 and M3 are the most efficient fillers in LDPE-modified clay mineral

Fig. 5. Total quantity of yperite (Qp ) penetrating composites in time at 25 ◦ C.

D. Plachá et al. / Journal of Hazardous Materials 271 (2014) 65–72

71

Fig. 6. Antibacterial activity for three bacterial strains.

composites. M1, M2 and M4 reached a comparable level of yperite vapour capture. The V0, V1 and V2 are fillers with the poorest capturing properties. The efficiency of V3 and V4 is otherwise higher but it is nearly half of the M1, M2 and M4 efficiencies.

3.3. Antibacterial effects Antibacterial effects of tested clay minerals in relation to the resistance of selected bacterial strains are different. The Na-forms of both clay minerals showed no bacterial activity against all tested bacterial strains, which corresponds to previous studies [19,23,33,34]. The good inhibition activity against the bacteria G+ S. aureus and Str. agalactiae and also for bacteria G− Y. pestis was observed for all samples of organically modified clay. Generally, the inhibition activity increased with organic cation loadings, see Fig. 6. All organically modified samples showed good antibacterial activity to Str. agalactitae. There was practically no difference between the clay minerals used. The V1 and V3 samples showed better results for S. aureus than M1 and M3. Zero antibacterial effects were observed in case of G− bacteria E. coli, P. aeruginosa and spores of B. anthracis, even after 6 days of testing. For that reason, the modified clay minerals cannot be considered as universal antibacterial materials in human protection against biological warfare, and their effects must be evaluated before targeted usage. The inhibition of bacteria Y. pestis is a significant result in respect to the protection against BWA and pathogenic organisms. Y. pestis is the originator of plague, one of the oldest diseases, that still remains endemic in many natural foci around the world. It still occurs in the tropics and subtropics and in warmer areas of countries. It is accompanied by high mortality, although rapidly diagnosis and prompt treatment with antibiotics leads to a successfully reduction [35]. Thus the organically modified clay minerals can be considered as an alternative to existing disinfection remedies against this bacterial strain. On the other hand no antibacterial activity was

observed against spores of the other very dangerous bacteria, B. anthracis. The antibacterial activities of the HDTMA and HDP modified clay minerals against E. coli and P. aeruginosa were not observed in this study as compared with antibacterial activity of vermiculitechlorhexidine complex, where effects were confirmed, especially for E.coli, in all range of tested intercalates [23]. The efficiency of the materials described in [23] against P. aeruginosa appeared when chlorhexidine loading in the vermiculite gallery reached 2.0, 3.0 and 4.0 CEC. Antibacterial activity of HDP and HDTMA modified montmorillonite against E.coli is also identified in other studies [22]. The organic cation loadings in the modified clay minerals in our study were relatively low with respect to exchanged interlayer cations, which can be the reason why antibacterial activities against E. coli and P. aeruginosa were not detected.

4. Conclusion CWA and BWA have still been a serious threat for populations as a consequence of unexpected use. Protective personal clothes on the basis of materials with low permeation abilities is one possibility of protection of humans before undesirable contacts with these toxic substances. Monoionic and modified clay minerals show good barrier properties for yperite permeation, and could be considered as fillers for microcomposite or nanocomposite polymer materials convenient for preparation of protective cloths or filtration equipments. Montmorillonite in monoionic Na form showed the best barrier properties followed by organically modified montmorillonites. Their BT values significantly exceed the BT of vermiculite derivatives. Related to achieved results, and results referred to in several research studies, the HDTMA and HDP montmorillonites and vermiculites can be used as materials with proven antibacterial effects, and can be both considered as efficient remedies against pathogenic organisms. Particularly they can be classified as

72

D. Plachá et al. / Journal of Hazardous Materials 271 (2014) 65–72

important antibacterial materials due to their growth inhibition of Y. pestis and S. aureus. While monoionic montmorillonite exhibited the best barrier properties with respect to yperite vapour permeation, organically modified clay minerals showed two activities: good barrier properties and antibacterial effects which is their advantage, if we consider the potential of attacks at sites with unknown contamination where a dispersion of chemical or biological agents is expected. Due to this fact they can be designed for utilization in bifunction protective devices, such as filters or in the protective clothing or masks. Materials simultaneously will prevent a permeation of chemicals to the body of person wearing and bioagents will be eliminated on the protective clothing surface. They can be used as functional adsorbent materials with antibacterial properties for air or water source decontamination. However, several disadvantages have to be considered, for example: 1) elimination of the used materials has to be solved, 2) materials with antibacterial properties are not versatile and they have to be tested before use, 3) efficiency against BWA such as viruses, funghi, toxins and spores are not confirmed so there are only limited BWA that can be eliminated.

[12]

[13] [14]

[15]

[16] [17] [18]

[19]

[20]

[21]

Acknowledgements [22]

This work was supported by the projects MSMT CZ.1.07/2.3.00/20.0074 and Energy Units for Utilisation of NonTraditional Energy Sources, reg. no. CZ.1.05/2.1.00/03.0069. K.R. and J.S. were supported by the project VG 20102014049 (Ministry ˇ by the project MSMT of the Interior of the Czech Republic) and S.S. CZ.1.05/2.1.00/01.0040. References [1] L. Szinicz, History of chemical and biological warfare agents, Toxicology 214 (2005) 167–181. [2] H. Vogel, Weapons of mass destruction, WMD, Eur. J. Radiol. 63 (2007) 205–213. [3] J. Ali, A. Dwyer, J. Eldridge, F.A. Lewis, W.C.I.I.I. Patrick, F.R. Sidell, Jane’s Chemical-Biological Defense Guidebook, second sub ed., Jane’s Information Group, Virginia, 2001. [4] B. Singh, G.K. Prasad, K.S. Pandey, R.K. Danikhel, R. Vijayaraghavan, Decontamination of chemical warfare agents, Def. Sci. J. 60 (2010) 428–441. [5] D. Thavaselrem, R. Vijayaraghavan, Biological warfare agents, J. Pharm. Bioall. Sci. 2 (2010) 179–188. [6] M. Pohanka, K. Kuˇca, Biological warfare agents, Exp. Suppl. 100 (2010) 559–578. [7] E. Raber, A. Jin, K. Noonan, R. McGuire, Decontamination issues for chemical and biological warfare agents: How clean is clean enough? Int. J. Environ. Health. Res. 11 (2001) 128–148. [8] R.M.A. Roque-Malherbe, Adsorption and Diffusion in Nanoporous Materials, CRC Press, Boca Raton, 2007. [9] D. Rivin, W.J. Shuely, F. Palya, R.S. Lindsay, A. Rodriguez, P.W. Bartram, Estimating the permeation resistance of nonporous barrier polymers to sulfur mustard (HD) and sarin (GB) chemical warfare agents using liquid simulants, DHHS (NIOSH) (2008) 2008–2141. [10] United States Patent No: 8 084 662, Method for degrading chemical warfare agents using Mn (VII) oxide with and without solid support, (2011). [11] A.A. Fatah, A.K. Judd, L.E. O‘Connor, H. Ch. Lattin, C.Y. Wells, Guide for the Selection of Chemical, Biological, Radiological and Nuclear Decontamination

[23]

[24]

[25] [26]

[27]

[28] [29]

[30]

[31]

[32] [33]

[34]

[35]

Equipment for Emergency First Responders, US. Department of Homeland Security, Washington, 2007. K.L. Koenig, C.J. Boatright, J.A. Hancock, F.J. Denny, D.S. Teeter, A. Ch. Kahn, C.H. Schultz, Health care facility-based decontamination of victims exposed to chemical, biological, and radiological materials, Am. J. Emerg. Med. 26 (2008) 71–80. T.J. Pinnavaia, G.W. Beall (Eds.), Polymer-Clay Nanocomposites, John Wiley &Sons, Chichester, 2000. M.C. Corobea, V. Uricanu, D. Donescu, C. Radovici, S. Serban, S. Garea, H. Iovu, Poly(vinyl acetate)-clay hybrids prepared via emulsion polymerization assisted by a nonionic surfactant, Mater. Chem. Phys. 103 (2007) 118–126. ˇ G. Simha Martynková, M. Valáˇsková, P. áapkovˇs, V. MatCjka, Structural ordering of organovermiculite: experiments and modeling, J. Colloid Interface Sci. 313 (2007) 281–287. R.C. Malhotra, K. Ganesan, K. Sugendran, R.V. Swamy, Chemistry and toxicology of sulphur mustard—a rewiev, Def. Sci. J. 42 (1999) 97–114. M. Naguib, The pharmacology of commonly used chemical agents, Perioper. Med. Pain 22 (2003) 230–238. M.H. Ashmore, C.P. Nathanail, A critical evaluation of the implications for risk based land management of the environmental chemistry of sulphur mustard, Environ. Int. 34 (2008) 1192–1203. P. Herrera, R.C. Burghardt, T.D. Phillips, Adsorption of Salmonella enteritidis by cetylpyridinium-exchanged montmorillonite clays, Vet. Microbiol. 74 (2000) 259–272. D. Plachá, G. Simha Martynková, M.H. Rümmeli, Preparation of organovermiculites using HDTMA: structure and sorptive properties using naphthalene, J. Colloid Interface Sci. 327 (2008) 341–347. D. Plachá, G. Simha Martynková, M.H. Rümmeli, Variations in the sorptive properties of organovermiculites modified with hexadecyltrimethylammonium and hexadecylpyridinium cations, J. Sci. Conf. Proceed. 2 (2010) 36–41. K. Malachová, P. Praus, Z. Pavlíˇcková, M. Turicová, Activity of antibacterial compounds immobilised on montmorillonite, Appl. Clay Sci. 43 (2009) 364–368. S. Holeˇsová, M. Valaˇsková, E. Plevová, E. Pazdziora, K. Matˇejová, Preparation of novel organovermiculites with antibacterial activity using chlorhexidine diacetate, J. Colloid Interface Sci. 342 (2010) 593–597. M.F. Brigatti, E. Galan, B.K.G. Theng, Structures and mineralogy of clay minerals, F. Bergaya, B.K.G. Theng, G. Lagaly (Eds.), Handbook of Clay Science, Vol. 1, Elsevier, Oxford, 2006, pp. 19–86. H. Ort, S. Florus, Protective properties of laboratory gloves against chemical warfare agents, Adv. Military Technol. 2 (2007) 85–102. MAZL 03–95, Estimation of breakthrough time of mustard drops through protective materials at static conditions, National Institute for NBC Protection, Milín, (1995). MAZL 39–10, Determination of resistence foil material to penetration of mustard by gas chromatography, National Institute for NBC Protection, Milín, (2010). S. Yariv, H. Cross (Eds.), Organo-Clay Complexes and Interactions, Marcel Dekker, New York, 2002. G. Lagaly, M. Ogawa, I. Dékány, Clay mineral organic interactions, F. Bergaya, B.K.G. Theng, G. Lagaly (Eds.), Handbook of Clay Science, Elsevier, Oxford, 2008, pp. 309–377. ISO 6529:2001, Protective clothing-Protection against chemicalsDetermination of resistance of protective clothing materials to permeation by liquid and gases, (2001). Chemical permeation, DuPont Permeation Guide (On line). Available on http://www.dpp-europe.com/chemical/inc/lang/flyer en.pdf (accessed: 07.09.3013). F.L. Cussler, Diffusion Mass Transfer Fluid Systems, Second ed., Cambridge University Press, New York, 2005. G, Özdemir, M.H. Limoncu, S. Yapar, The antibacterial effect of heavy metal and cetylpridinium-exchanged montmorillonites, Appl. Clay Sci. 48 (2010) 319–323. N. Meng, N.L. Zhou, S.Q. Zhang, J. Shen, Controlled release and antibacterial activity chlorhexidine acetate (CA) intercalated in montmorillonite, Int. J. Pharm. 382 (2009) 45–49. Plague, WHO (On line). Available on http://www.who.int/csr/disease/plague/en/ (accessed: 07.13.13).