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2EARTH SCIENCES
MEASURES OF PREVENTION OF THE IMPACT OF HAZARDOUS FACTORS ON THE ECOLOGICAL STATE OF WATER BODIES
Savvova O. V.
Doctor of Engineering Science, Associate Professor, Senior Research Associate of the Department of the
Ceramics, Refractories, Glass and Enamel Technology National Technical University «Kharkiv Polytechnic Institute»
Babich O. V.
Candidate of Engineering Science, Research Associate of the Department of the Ceramics, Refractories,
Glass and Enamel Technology National Technical University «Kharkiv Polytechnic Institute»
Tsytlishvili K.O.
Research Associate, Graduate Student "Ukrainian Scientific Research Institute of Ecological Problems" (USRIEP)
Abstract
Ecologically hazardous factors of microbiological environmental contamination were analyzed. The current state of the biocidal materials development was investigated and the perspective environmentally-friendly methods for protection against pathogenic microorganisms were established. Effective oligodynamic antibacterial agents based on heavy metal oxides and the mechanism of their action were determined. The biocidal properties of glass coatings with a certain content of zinc oxide and its toxicity were investigated. It had been established that the effective preventing method of microbiological contamination of the ecosystem was the use of non-toxic zinc-containing glass coatings.
Keywords: ecosystem, pathogenic microorganisms, antibacterial agents, biocidal properties, zinc oxide, glass coatings.
Introduction
The determining factor for the biotic integrity preserving is the stable functioning of the ecological system (ecosystem). Technogenic activity, which leads to changes in ecosystems, can negatively affect the rebuilding of microbial communities and contribute to the artificial evolution of pathogens of infectious diseases that cause an increase in the activity of many foci of dangerous diseases [1].
Microorganisms are an important component of any ecosystem. Qualitative and quantitative changes of this component are very significant for characterizing ecosystems and the environment in general. The sources of biological ecologically hazardous factors are living organisms and products of their life activity. Biological contamination is both introduction of organisms alien to these communities into natural ecosystems as a result of anthropogenic activities and the spread of nutrients in those territories and / or water areas where they have not been previously observed. In the first case, when an unusually large number of microorganisms appear in the environment, associated with their mass reproduction on anthropogenic substrates or media, altered in the course of human economic activity, as well as the acquisition of pathogenic properties by the saprophytic or conditionally innocuous bacterial form, it is customary to speak of microbiological (bacteriological) contamination. In the second, when the indirect effect of organisms on ecosystems is observed, that is, through the substances synthesized during the functioning of these organisms or the degradation of the latter, it is to speak of biotic (biogenic) contamination.
Microbiological monitoring allows to assess not only the sanitary and epidemiological, but also the general ecological situation, determine the degree of danger of the spread of infectious diseases, and also predict the intensity and direction of influence of the exogenous factors of physical and / or chemical nature on this process in real conditions of chemical and physical contamination of various objects of the environment.
The issue of microbial contamination of water is a particular interest. A survey of 30 treatment plants in the Kharkiv region and sewage treatment plants in Ukraine, which were conducted by the laboratory of municipal and industrial sewage of the Research Institution "Ukrainian Scientific Research Institute of Ecological Problems" (USRIEP), showed that the waste water after treatment contains a significant number of microorganisms. Due to the fact that almost all wastewater treatment plants receive domestic and industrial wastewater, after the treatment facilities, the treated wastewater contains a pathogenic microflora. This contributes to the spread of such dangerous infectious diseases as Asian cholera and typhoid fever, dysentery and viral hepatitis [2].
Such pathogenic microorganisms as Escherichia coli, Pseudomonas aeruginosa, Ent Erococcus faecium and others with rainwater from public areas - hospitals (operating and resuscitation blocks, maternity and infectious departments, medical and pharmaceutical laboratories), public canteens (food blocks, refrigeration equipment, storage tanks and water heaters), underground (walls and details of architectural buildings of stations) and other objects fall into ecological systems in an amount that can exceed the epidemiological
threshold. This can lead to a potentially dangerous condition. All these microorganisms can survive depending on the degree of their resistance to various pH media, temperature changes and UV radiation.
The traditional methods used for wastewater treatment can be divided into three main groups: mechanical, physico-chemical (chemical), biological. Mechanical treatment of wastewater is used primarily as a preliminary. Mechanical cleaning provides removal of suspended solids from domestic sewage by 60-65 %, and from some industrial wastewater by 90-95 %. The goal of mechanical cleaning is to prepare water for physico-chemical and biological treatment [3].
Wastewater treatment by physico-chemical methods in combination with biological methods provides a high degree of purification and disinfection. Wastewater before biological treatment is subjected to mechanical cleaning, and after it chlorination with liquid chlorine or chlorine lime to remove pathogenic bacteria and chemical purification. For disinfection, other physico-chemical methods (ultrasound, electrolysis, ozonization, etc.) are also used.
Biological wastewater treatment is based on the use of special bacteria that decompose organic compounds into substances that are safe for human life and health. It should be noted that biological treatment of wastewater is the most universal, and due to this method of disposal of contaminants the greatest possible degree of purification is achieved [4].
The goal of the third stage of wastewater treatment - decontamination - is to destroy pathogenic bacteria and viruses that are or may be in wastewater. Methods of disinfection of waste water are divided into two groups: reactant and nonchemical or chemical, when the bactericidal action is carried out by chemical substances and physical, when microorganisms die due to the action of physical factors.
The wide distribution of chlorine in water treatment technologies was facilitated by its effectiveness in the disinfection of natural waters and the ability to preserve already purified water for a long time. In addition, preliminary chlorination of water can reduce the color of water, eliminate its odor and smack, reduce the consumption of coagulants, and maintain a satisfactory sanitary condition of treatment facilities of water treatment stations [5].
However, chlorine as a main water treatment reagent has significant drawbacks. For example, chlorine and chlorine-containing compounds have high toxicity, which requires strict adherence to safety requirements. Chlorine affects mainly the vegetative forms of microorganisms, while gram-positive strains of bacteria are more resistant to chlorine than gram-negative strains of microorganisms. Viruses, spores and cysts of protozoan and eggs of helminths are also highly resistant to the action of chlorine. The need to transport, store and use a significant amount of liquid chlorine at the waterworks, as well as discharges of this substance and its compounds into the environment, caused a high environmental hazard. In addition, chlorine has a high corrosive activity.
In connection with this, much attention was paid to other methods of disinfection, such as ozonization.
Ozonization provides disinfection of water with significant bacterial contamination of the water source, primarily in the presence of pathogenic microorganisms in water - enteroviruses and lamblia cysts, resistant to the action of chlorine-containing reagents. Organoleptic indicators of drinking water quality are improved, specific smells and tastes that appear at elevated plankton concentrations are eliminated. However, there are some drawbacks in this method. Firstly, its cost. Secondly, when water is ozonized, organic contaminants undergo destruction, as a result, the number of compounds that decompose biologically increases. In the water, the concentration of the so-called "assimilated organic carbon" increases, which is easily assimilated by microorganisms, contributing to their vital activity. This creates favorable conditions for the repeated bacterial contamination of purified water.
It should be noted that ozone is a toxic gas, therefore any use of it requires careful safety control.
The use of silver is widely known, however, it is effective only when treating water with a low content of salts and minimal organic contaminants. In addition, silver shows selective activity in relation to pathogens of various diseases. In connection with this, it is necessary to search for alternative antimicrobial agents that are characterized by high bactericidal and fungicidal activity against a wide range of pathogenic microorganisms and do not have a negative adverse effect on living organisms.
Therefore, an urgent problem is the development of effective environmentally safe measures to prevent microbiological contamination of biota, taking into account its integrity preserving. To do this, it is necessary to conduct an analytical review of the alternative use of antibacterial materials for decontamination at the stage of sewerage and under the conditions of the traditional scheme of biological sewage treatment.
1. Literature data analysis and a problem statement
An important direction in the development of methods for protecting against pathogenic microorganisms is the creation of alternative ecological means of their inhibition, which will provide a long-term antibacterial protection. Currently, to protect against the microbiological contamination, much attention has been paid to the creation and use of antibacterial materials: polymer, composite, metal, glass-ceramic and vitreous enamel coatings [6]. To ensure a prolonged antibacterial action of coatings the heavy metal ions are added into the initial composition of glass. The mechanism of their action as denaturing agents is based on: interaction with biologically important substances of microorganism cells (cellular metabolites), decrease in the enzyme activity; disturbance of the structure and functioning of biomembranes and cell walls.
Consider the modern antibacterial materials.
1.1 Antibacterial polymer materials
The use of polymers containing silver ions for the manufacture of medical devices with antibacterial properties allows the serial production of instruments, patient-care items, special utensils and various types of packages for medicines and medical equipment [7],
which, in comparison with analogues of metals and glass, are more affordable.
The polymer coating AgION with the content of silver ions is used to protect the internal surface of refrigerators in the production of household appliances Bosch (Germany). The modern technology of the Ag-Nano Silver nanocoating applying, developed by the laboratory of LG Electronics (South Korea), is used in the manufacture of washing machines [8].
Antibacterial coatings are also used in computer equipment and portable electrical devices (cellular phones), etc. Microspheres, used as a filler for polymer paints, are characterized by high antibacterial effect against E. Coli bacteria group. The microspheres are produced by sol-gel method from compositions with a molar ratio of Si: Al: Ag = 1.00: 0.01: 0.01 (herewith
— = 1 to 3.3) [9].
Ag
1.2 Antibacterial coatings on metals
The Kobe Steel (Japan) is actively engaged in the production of antibacterial steel coatings called KENIFINE [10], which are obtained by the method of electrochemical deposition of nickel on steel and used in the manufacture of construction and furniture fittings for public places, as well as in devices (scales, air cleaners), etc.
Antibacterial coatings that are obtained when applying silver to stainless steel by the plasma method are widely used in orthopedic surgery [11]. The use of antibacterial materials and coatings with content of silver,
Japanese scientists [14] conducted a study of the antibacterial activity of CaO, MgO, ZnO ceramic powders by an indirect conductimetric assay. Candida albicansNBRC1060, Saccharomyces cerevisiae NBRC1950, Aspergillus niger NBRC4067 Ta Rhizopus stolonifer NBRC4781 fungi were usedfor the tests. CaO, MgO powders showed a significant bactericidal effect against the action of the above-mentioned fungi. ZnO powder also inhibited fungal growth, however, this effect required its substantial amount of more than 100 ^g ml-1.
1.4.Antibacterial glass materials
For today, antibacterial glasses of KJ1000 and KJ2000 Glaverbel series produced by AGC Flat Glass Europe, which eliminate microorganisms from the beginning of contact with the glass surface, are known. The antimicrobial action of glass is based on the bactericidal properties of silver [15]. For instance, the German scientists F. Oberlis and G. Pollman thoroughly examined the question of the effect of microorganisms on glass and developed the antibacterial glasses. Metal
titanium and nickel metal nanoparticles as bactericidal components is explained by their relatively low cost and manufacturability. On the other hand, the use of Ag+ and Ni2+ ions is limited by their toxic effect and negative impact on the environment. Therefore, an obligatory condition for the application of these materials in the sewerage treatment plant system is compliance with the regulatory requirements for MPC for sewage.
According to the data of the authors [12], a film with TiO2 on stainless steel is characterized by a sufficiently high antibacterial effect against the Bacillus pu-milus bacteria group.
1.3 Biocidal ceramic materials
Antibacterial ceramic powders, in particular for protection from the bio-contamination, have found wide application as inhibiting agents in various types of materials. The promising use of antibacterial ceramic powders as biocidal agents is determined by their exceptional long-term action against a wide range of pathogenic microorganisms.
Biocera (USA) developed antibacterial ceramic powders based on calcium phosphates, alumina and silica with silver ions [13]. The product has the FDA safety certificate, as a non-toxic material, that characterized by a high antibacterial efficacy (99%) and long-term action, since it is stabilized under the high temperature.
Table 1 shows the characteristics of antibacterial materials: inorganic (Biocera A Series) and organic (Biocera MB) nature.
cations which, in increasing antibacterial effect, are arranged in the following order: Ag+> Hg2+> Cd2+> Cu2+> Au3+> Ni2+ > Zn2+, find application in production of the mentioned glasses. In addition to the above ions, the magnesium, tin, iron, cobalt and cerium ionscan be used to prepare antibacterial coatings with the sol-gel method [16]. Glasses, that contained the following oligodynamic oxides, wt. %: Ag2O - 0,25; CuO - 2,0; CdO < 8,0; AS2O3 - 0,9; TiO2 - 3,0; ZnO < 17,0; PbO -8,5, were characterized by the maximum resistance to infection with mold fungi.
An antimicrobial coating based on TiO2 and AgO-TiO2for glass microscope slides is known [17]. These photocatalytically active coatings showed bactericidal action against Staphylococcus aureus, Escherichia Coli and Bacillus cereus.
These coatings are effective due to their robustness, stability to cleaning, reuse, and excellent antimicrobial response to all organisms and can be used for surfaces in a hospital environment.
Table1
Characteristics of antibacterial materials
Type Biocera A Series Biocera MB Antibacterial Master Batch
Features Antibacterial, deodorization, antifungal Antibacterial, emission of far infrared rays, thermal resistance of plastic products
Components CaO, MgO, P2O5, Al2Os, SiO2, ZnO and Ag+ PP, PE, ABS, GPPS, PVC etc.
Pack size Fine white powder with particle size3-10^m Pellet
To date, photocatalytic technologies with the use of TiO2 for the decomposition of organic contaminants, destruction of microorganisms, viruses and malignant cells, deodorization are intensively developed [20]. Recently, much attention has been paid to the TiO2use for the antibacterial protection of heterogeneous photocatalysis, the application of which allows solving important social and environmental problems. For example, antibacterial nano-sized films based on the titanium or zinc dioxide on silica glass using the photocatalysis method can be used for water purification. Photocatalysis is the process of oxidation or reduction of organic compounds or inorganic ions, which takes place in the presence of a photo-catalyst under the light. The formation of free charge carriers-electrons and holes when the surface of the photo-catalyst (TiO2, ZnO, SnO2) is irradiated with the wavelength light of less than 390 nm playes the main role in the process of splitting of substances on the surface of the photocatalyst under the light.The use of this method in combination with effective methods of biological purification will solve the urgent problem of simultaneous prevention and purification from microbiological contamination of water bodies.
2. Aim of work
The aim of the given work is the establishment of effective environmentally safe methods for preventing microbiological contamination of biota. To achieve this aim, the following objectives were set:
- to analyze ecologically hazardous factors of microbiological contamination of the environment;
The toxicity of the experimental materials was estimated by the change in the dehydrogenase activity (DHA) of the biotest cultures. The DHA method is based on monitoring the activity of the enzymatic system of biotests when they contact with experimental samples. Determination of DHA biotests is based on the ability of dehydrogenases to reduce to formazan (triphenylformazan), which has a dark red color, by de-hydrogenation of a substrate of colorless triphenylte-trazolium chloride (TTC). The amount of formed form-azan (the indicator of the color intensity) is proportional to the activity of the dehydrogenases: the greater the enzyme of the dehydrogenase, the more intense the red color of the test sample. The amount of reduced dehy-drogenases of TTC microorganisms were determined from the optical density of the solution by colorimetry and the DHA value was calculated using the calibration curve.
3. Substantiation of the oligodynamic components choice in the preparation of biocide ceramic nanopowders based on oxides
Current data on the toxicity of heavy metals [18] indicate that in this action of metal oxides and salts, the metal cation is of primary importance. However, the acid radical can change the biological and toxic effect
- to investigate the current state of development of biocidal materials and methods for preventing microbiological contamination of the ecosystem;
- to identify effective oligodynamic antibacterial agents based on heavy metal oxides and the mechanism of their action;
- to investigate the biocidal properties of glass coatings with inorganic powders and their toxicity;
- to determine effective methods for preventing microbiological contamination of biota;
- to investigate the possibility of using antibacterial materials for the wastewater decontamination of treatment plants.
Accordingly, the biocidal properties of the materials were determined by the following methods using dense, liquid and gaseous media:
The first method is diffusion (qualitative) one, which is based on the study of the inhibition zone formation of the test microbe around the test-sample using dense agar media. The suppression of growth at the site of sample contact with the agar nutrient medium depends on the degree of the antimicrobial agents diffusion into the layer of nutrient agar;
The second is quantitative method, which is based on counting the growth level of biotest microorganisms inoculated into liquid nutrient medium in the presence of test-samples and without them.
Biocidal properties of the materials were determined according to the above mentioned methods using the following scheme:
Preparation of inoculum
of salts, due to the specific action of an anion (for example, providing the potentiation effect), as well as the tendency to hydrolysis and dissociation.
To prevent a negative impact on the treatment plants PMQ for metals contained in oligodynamic components, should be regulated by the appropriate DBN V.2.5-75: 2013 "Sewage. External lines and constructions", have no toxic effect on the microflora of biological treatment of wastewater, and also do not affect the composition of sewage that is discharged into a water body after treatment [19].
Among the metals of group II-B, zinc and nano-components based on it have widely used as a bactericidal agent. It is known that ZnO inhibits the growth of microbes at a concentration of 100 mg/ml and E. Coli in a concentration of 1.4 - 2.3 mg/dm3. The promising use of ZnO is due to its selective toxicity to microorganisms and minimal effect on cells of living organisms. Antibacterial activity of ZnO is realized by the following mechanisms:
• irreversible inhibition of enzymatic activity by binding of the functional group of the enzyme molecule;
Study on biocidal properties
Choice of biotest cultures of microorganisms
Standardization of the biotest inoculum
• denaturation of proteins due to the formation of mercaptides, albuminates, disruption of the cell membranes permeability and connection with DNA cells by catalytic oxidation;
• oxidation and decomposition of organic components of bacteria by a photocatalytic method under the ultraviolet irradiation;
• formation of active oxygen as a result of contact of zinc with water, which oxidizes and destroys the DNA of microorganisms.
The implementation of the last two mechanisms, while ensuring the prevention of microbiological contamination through the use of nanomaterials based on zinc oxide, will solve the pressing problems of environmental protection of the environment.
4. Results of the study
To establish the safety of the use of zinc oxide as an antibacterial agent in the glass coating, its denaturing ability was studied by irreversible inhibition of the enzymatic activity of the enzyme molecule of a pathogenic microorganism.
Antibacterial properties of BPZ-1, BPZ-2, BPZ-3, BPZ-4 glass coatings with a content of 1.0, 2.0, 3.0 and 4.0 wt. % of zinc white per 100 wt. % of frit were evaluated by qualitative and quantitative methods in relation to E. Coli bacteria and C. Albicans fungi. For comparison, as a control, ESP-117 vitreous enamel on the basis of the cover titanium frit (Kcoatmg) was used.
Determination of biocidal activity in relation to E. Coli with diffusion method allowed to establish that all experimental coatings are characterized by the diameter of the zone of growth retardation around the biotest of 12 mm and was determined as bacteriostatic action on the biocidal activity evaluation scale. The fungicidal activity of the experimental samples in relation to C. Albicans was not established by this method.
A qualitative method for assessing biocidal activity is effective only for migrating compounds. For example, according to the data of the authors [21], when ZnO is used as a bactericidal agent in vitreous enamel coatings, the estimation of their biocidal properties by
the diffusion method is approximate, since ZnO practically does not dissolve in water (solubility at 20 °C is 0.00016 g per 100 g of water).
Based on the results of the quantitative tests, the increase in the colony-forming units (CFU) of E. coli for experimental coatings decreased with increasing content of ZnO in the coating composition. So for example, CFU of cells for the BPZ-4 coating after 24 and 48 hours increased by 3.6 and 25 times, respectively; this index for the Kculture increased by 18.8 and 250 times, respectively (Fig. 1, a). The CFU of E. Coli for the Kcoatmg and BPZ-1 coating were the highest and increased 17.8 and 130 times after 24 and 48 hours. Therefore, addition of ZnO 4 wt. % to the glass coating composition resulted the decreasing of CFU of E. Coli in relation to the microorganisms culture growth (Kcul-ture) in 10 times, which was about 80 % and was characterized as an excellent bactericidal effect. Consequently, the concentration of denatured E. Coli cells increased in proportion to the ZnO content of the experimental coatings composition.
The increase in the CFU of C. Albicans during the exposure time of 24 and 48 hours for all experimental coatings was almost the same. For instance, CFU of cells increased by 3.66 times for the BPZ-1 coating and Kcoating after 24 hours; this index for the Kculture increased by 3.4 - 3.7 times. The CFU of C. Albicans for experimental coatings during 48 hours did not change (Fig. 1, b). It was established that a proportional increase in the fungicidal effect on C. Albicans of glass coatings with a content of 4 wt. % of zinc oxide was observed during the one- and two-day exposure.
Thus, the optimal content of zinc white in the composition of glass coatings (4 wt. %), that allows to provide a biocidal effect in short-term conditions was established based on the results of the conducted studies.
To determine the effect of leaching of Zn2+ ions on the biocidal properties of experimental vitreous enamel coatings, samples were boiled for 48 hours in distilled water. It was found that the PMQ Zn2+ for experimental coatings is 0.016-0.018 mg/l, which is within the PMQ.
Fig.1 - The growth of CFU E. Coli with Cinitial = 5.0 • 104kl/ml (a) and C. Albicans Cinitial= 2.4105 kl/ml (b) under the action of coatings with zinc oxide and without it (Kculture)
As a result of contact of biotest cultureswith BPZ-4, the concentrations of formazan as an indicator of the E. Coli dehydrogenases activity in the medium with the experimental sample and the Cculture are close.
This fact shows that there is no toxic effect of the experimental BPZ-1 on the E. Coli bacteria cultures. The research results also made it possible to establish that the developed coating is not a nutrient medium for the growth of experimental biotests, which, along with nontoxicity, indicates its usefulness as an antibacterial material at the stage of the microbiological environmental contamination prevention.
Conclusions
The relevance of using alternative antimicrobial agents that are characterized by high bactericidal and fungicidal activity against a wide spectrum of pathogenic microorganisms and the absence of negative effect on living organisms were established.
Effective oligodynamic antibacterial agents based on heavy metal oxides for inhibiting pathogenic microflora were determined. It has been established that the addition of 4 wt. % zinc oxide to the glass coating composition allows to provide biocidal properties of glass coatings in relation to E. Coli and C. Albicans for 24 and 48 hours.
The absence of toxic effect of glass coating with the zinc oxide content on the E. Coli bacteria cultures was established. This indicates the expediency of its use as an antibacterial material. It has been established that the effective method of preventing microbiological contamination of biota is the use of non-toxic zinc-containing glass coatings.
To prevent the impact of the hazardous microbiological contamination factors on the ecological state of water bodies, the use of biocidal properties of glass coatings in the sewage treatment systems, in particular, at the stage of channelling of domestic and industrial wastewater was proposed.
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