Mechanical and Spectroscopic Analysis

Mechanical and Spectroscopic Analysis Investigation of Antibacterial Efficacy of Biocidal Product-Additive Erasers Against Escherichia Coli and Staphylococcus Aureus Bacteria and Spectroscopic Analyses

1. Abstract

Today, antibacterial materials have many application areas. In many different fields such as medical devices, dental fillings, and the food sector, these materials are needed to eliminate microorganisms and prevent their spread. Heavy metal ions, particularly silver (Ag), zinc (Zn), copper (Cu), mercury (Hg), and titanium (Ti), exhibit high antibacterial activity. These ions inactivate bacterial enzymes, preventing bacteria from carrying out enzyme reactions and shortening their lifespan [1]. Although antibacterial products have always attracted consumer interest, they have become especially important following the Covid 19 pandemic. In this study, the objective was to impart antibacterial properties to erasers, which are used in many areas such as schools, offices, and homes and are particularly prone to hand-to-hand transmission and bacterial spread, while ensuring that the product poses no chemical risk.

2. Introduction

Microorganisms such as bacteria and fungi, though invisible to the naked eye, live in all environments, and some may be pathogenic harmful bacteria. Bacterial proliferation can occur even under normal humidity and temperature conditions, and such bacteria easily create living spaces on commonly used objects, including plastics, causing negative effects on hygiene, health, and aesthetics. On objects where they multiply, bacteria can cause surface damage and cracking, discoloration, slime formation, and unpleasant odors. Additionally, they can create environments and conditions that lead to contagious diseases. Antimicrobial compounds are widely used in various industrial processes, drinking water, waste management, cosmetics used daily, furniture, and antibacterial drugs. Antimicrobial compounds are commonly used to control the growth of pathogenic microorganisms or to eliminate them from inanimate objects, surfaces, or intact skin [2]. Products with antimicrobial surfaces are highly desirable. These surfaces preferably exhibit antibacterial effects against both Gram-positive bacteria (for example, Staphylococcus aureus) and Gram-negative bacteria (for example, E. coli) [3]. Antimicrobial products provide broad protection: they inhibit the growth of bacteria, fungi, and viruses. These products, when integrated into various plastic items, provide antibacterial protection properties especially on the surface of the item. The eraser is one of the tools commonly used for educational purposes and applications (writing, designing, modifying). The use of erasers is not limited only to erasing; it can be applied to create highlights, textures, and even deep visual effects. People (for example, students, teachers, and other professionals) are in frequent contact with such products. Therefore, their contents are subject to certain chemical limitations. The presence of antibacterial properties in such widely used tools plays a critical role in people, especially those with sensitive health conditions. As Adel, we have imparted antibacterial functionality to our newly developed erasers using Avient masterbatch listed in the 'Ministry of Health Biocidal Inventory Registration Products' list and antimicrobial technology.

3. Material and Method

3.1. Performing the Antibacterial Test 3.1.1. Preparation of Test Inoculum: From a culture incubated for 16-24 hours in PCA, using a sterile loop, transfer to 1/500 NB and adjust density to 6x10⁸ cfu/ml. Ensure the bacteria is completely dispersed. To adjust bacterial concentration, dilute again with 1/500 NB. Bacterial concentration should be between 2.5x10⁵ and 10x10⁵ cfu/ml. This prepared suspension is used in the test inoculum.

3.1.2. Sample Preparation

Six untreated and three treated test samples are used. Treated or untreated test samples are cut to 50x50 mm ±2 flat. Samples should not be thicker than 10 mm. Surface area should be between 400 mm² and 1600 mm². For each sample, a 40×40 mm sterile film is cut. When preparing samples, avoid contamination from microorganisms and organic debris. Surface cleaning is performed using 70% ethanol.

3.1.3. Inoculation of Samples

Three treated and six untreated samples are placed separately in sterile petri dishes. The test surface should face upward. From the prepared test inoculum, 0.4 ml is pipetted onto each sample. A sterile film 40mm x 40mm is placed over the inoculum and gently pressed down. After the sample is covered with film, the petri dish lid is closed. Three treated and three untreated samples are placed in the incubator (these samples are considered as the 24-hour contact time). Incubate at 35±1°C for 24±1 hours. Relative humidity in the incubator should not be less than 90%. The other three untreated samples are considered as the 0-hour contact time. Washing is applied immediately after inoculation. After the sample is covered with sterile film, there should be no air bubbles or air in the inoculum.

3.1.4. Recovery of Bacteria from Test Sample

Immediately After Inoculation Immediately after inoculation, 10 ml of SCDLP solution (soybean casein digest medium containing lecithin and polyoxyethylene sorbitan monooleate) is added directly to the remaining three untreated samples, the petri dish lid is closed, and mixed gently. Ensure the sample is completely wetted with neutralization solution. Using a pipette, the solution is drawn from the petri dish at least 4 times and pipetted back onto the sample. If less than 10 ml needs to be used depending on the shape and size of the sample, the volume used is recorded in the report.

3.1.5. Method for Finding Bacterial Numbers Using the Pour Plate Method

After washing, 1 ml of suspension is taken and pipetted into 9 ml phosphate buffer (1/10 dilution). From this prepared solution, 1 ml is taken again and pipetted into 9 ml phosphate buffer. In this way, a 1/100 diluted solution is prepared. From each dilution, 1 ml is placed into 2 separate petri dishes. From PCA (Plate count agar) cooled to 46-48°C, 15-20 ml of bacterial suspension is poured onto the petri dish. Mix gently and allow to cool at room temperature. After the agar solidifies, incubate at 35±1°C for 40-48 hours. After incubation, the bacteria grown in serially diluted petri dishes are counted. 30 to 300 colonies are observed. If the number of colonies grown is <30, an additional 1 ml from this solution is inoculated onto agar. If there is no growth at all on the agar, it is reported as <1.

3.1.6. Finding Bacterial Numbers

For each sample, the number of bacteria grown is calculated using the following formula: N= (100×C×D×V) / A N: Bacterial number C: Average colony count in duplicate petri dishes D: Dilution factor V: Volume of SCDLP solution used (ml) A: Film surface area (mm²) If no bacterial colonies grow on the agar, 3.1.7. Validation of the Test For the test to be valid, the following three conditions must be met. a) (Lmax - Lmin) / (Lmean) ≤ 0.2 Log values of bacterial numbers in untreated samples are taken after inoculation. b) The average bacterial number in untreated samples immediately after inoculation should be between 6.2x10³ cells/cm² and 2.5x10⁴ cells/cm². c) The bacterial number obtained after 24-hour incubation of untreated samples should not be less than 6.2x10¹ cells/cm² [4]. Lmax: Logarithm of the maximum cfu count found on untreated sample replicates Lmin: Logarithm of the minimum cfu count found on untreated sample replicates Lmean: Logarithm of the average cfu count of untreated sample replicates

3.1.8. Calculation of Antibacterial Activity

R= (Ut – U0) – (At – U0) = Ut – At R: Antibacterial activity U0: Logarithmic value of untreated sample immediately after inoculation Ut: Logarithmic value of untreated sample immediately after 24-hour incubation At: Logarithmic value of treated sample after 24-hour incubation For results to be considered effective, R ≥ 2 must be satisfied [5].

3.2. Physical and Spectroscopic Characterization of Erasers

3.2.1. Mechanical Properties Density, hardness, tensile strength at breaking, elongation at breaking, and modulus at 100% were examined for test samples containing antibacterial agent and those without antibacterial agent. Density value was determined according to ASTM D792, hardness value according to ASTM D2240, and other mechanical values according to ASTM D412 standards.

3.2.2. Chemical Analysis Using Spectroscopic Methods

3.2.2.1. Determination of Polycyclic Aromatic Hydrocarbons (PAH) To 500 mg eraser sample, 20 ml of a mixture of toluene and internal standard is added and extracted in an ultrasonic bath at 60°C for 1 hour. After 1 hour, 1 ml of the solution cooled to room temperature is taken into a vial and polycyclic aromatic hydrocarbons are determined using the AfPS GS 2019:01 PAH method in a GC-MS instrument [6].

3.2.2.2. Determination of Phthalates

To 50 mg eraser sample, 5 ml tetrahydrofuran (THF) solution is added and left for 30 minutes in a shaker mixer for dissolution. After 30 minutes, 10 ml acetonitrile or hexane is added for every 5 ml THF and shaken for 5 minutes. After shaking is complete, the solution is filtered through a 45 μm membrane filter into a vial and phthalates are determined using the CPSCCH-C1001-09.4 method in a GC-MS instrument [7].

3.2.2.3. Determination of Cadmium

0.5 gram eraser sample is weighed into a Kjeldal tube. The tube is placed on a heater, 10 ml sulfuric acid is added, and heated to high temperature to remove organic compounds; heating is continued for 15 minutes after white fumes form, then the tube is removed from the heater and cooled for 10 minutes. Using a funnel, 5 ml hydrogen peroxide solution is slowly added into the tube and heated to high temperature, then cooled to room temperature, 5 ml hydrogen peroxide is added again, and heated to high temperature once more. This hydrogen peroxide addition process is repeated 4 times with 5 ml additions. The solution cooled to room temperature is transferred to a 100 ml volumetric flask and made up to volume with distilled water. A 1 ml sample from the solution is taken through a membrane filter into a vial and cadmium is determined using the BS EN 1122:2001 method in an ICP-MS instrument [8].

3.2.2.4. Determination of Lead

150 mg eraser sample is weighed into a microwave tube. 5 ml concentrated nitric acid is added to the tube at room temperature. The tube is capped and heated in a microwave device at 200°C for 20 minutes. The sample is allowed to cool for 5 minutes before removal from the microwave. The microwave vessel is opened in a fume hood and the solution inside is transferred to a 50 ml volumetric flask and made up to 50 ml with distilled water. From the solution made up to 50 ml, 1 ml is taken into a vial and lead is determined using the CPSC-CH-E1002 method in an ICP-MS instrument [9].

4. Results

As a result of the studies conducted, the antibacterial activity (R) value of the eraser sample containing antimicrobial additive, tested according to ISO 22196:2011 standard, against E. Coli and S. Aureus microorganisms was found to be greater than 2, and the %Antibacterial Activity value was 99.99%. The antibacterial activities of our product against infectious bacteria harmful to humans, such as E. Coli and S. Aureus, have been demonstrated. The following results were obtained in the antibacterial testing conducted. Control of standard characteristics, such as mechanical properties, is important in the materials industry for ensuring product quality. For this reason, an additive agent should allow the enhancement of performance without impairing the original character of the polymeric composition [5]. The mechanical test results for standard erasers without antibacterial agent and erasers containing antibacterial agent are shown in the table below. Table 3 presents mechanical test results for eraser samples with and without antibacterial agent additives. According to the data, it was observed that the density values of both samples were the same. While tensile strength at breaking and elastic modulus values at 100% were quite close to each other, it was observed that the antibacterial agent increased the hardness of the finished product by approximately 4 units. Along with the increase in hardness, a decrease in the elongation value of the material containing antibacterial agent was observed. This increase in hardness value and the decrease observed in elongation at breaking are within the desired limit values of the standard eraser sample. As a result of spectroscopic analysis of the eraser sample, phthalate types were controlled with a GC-MS instrument and none of the phthalate types listed below (Table 4) were detected. According to Turkey's KKDIK (Registration, Evaluation, Authorisation and Restriction of Chemicals) regulations, the use of these phthalates at concentrations higher than 0.1% by weight in plastic material is prohibited [10]. Based on analysis of our eraser, it was found to comply with the relevant regulatory values. PAH types of the eraser sample were controlled with a GC-MS instrument and none of the PAH types listed below (Table 5) were detected. According to Turkey's KKDIK regulations, some of the following PAH types (*) cannot be present in amounts greater than 0.5 mg/kg in plastic or rubber materials that may be in prolonged contact with human skin [10]. Based on analysis of our eraser, it was found to comply with the relevant regulatory values. The lead and cadmium content of the eraser sample was controlled using an ICP-MS instrument; lead (Table 6) and cadmium (Table 7) compounds were not detected. According to KKDIK regulations, if lead concentration in items or item parts is equal to or higher than 0.05% by weight during normal and foreseeable use, the items cannot be used by the public, cannot be put in the mouths of children, and cannot be placed on the market. It is stated that mixtures and items produced from plastic materials cannot be placed on the market if they contain cadmium at a concentration equal to or greater than 0.01% by weight of the plastic material [10]. Based on analysis of our eraser, the lead and cadmium values were found to comply with the relevant regulatory limit values.

5. Conclusions and Discussion

In this study, we proceeded with a thermoplastic elastomer-containing polymer and Avient antimicrobial solution in eraser production. Antimicrobial actives move within the polymer matrix and spread on the material surface, providing a protective barrier in antimicrobial effectiveness. Organic antimicrobials are effective at low concentrations and offer a suitable cost/performance ratio in polymers processed at temperatures not exceeding 250°C. The antibacterial effect test results are provided in the tables above (Tables 1 and 2). In the tested samples, according to ISO 22196:2011 standards, it was observed that Escherichia coli and Staphylococcus aureus bacteria decreased by 5.63 and 4.35 log, respectively, after 24 hours. This shows that the antibacterial activity R is greater than 2 and is 99.99% in percentage terms. These erasers, which demonstrate high antibacterial activity, have become a product that consumers can use safely for extended periods. According to the study conducted, the hardness value of the eraser at 61 Shore A increased to 65 Shore A with the addition of the antibacterial agent. This value, which is within standards, is suitable for use. As a result of all antibacterial, physical, and chemical tests conducted, it was found that our eraser complies with the limit values specified in the studied standards and regulations, and additionally possesses antibacterial effectiveness. 6. References [1] Oz, Elif. Photo-"Click" Reaction Prepared Silver Nanoparticle Containing Thermoset Polymers Industrial Applications. Master's Thesis, Yalova University, 2016. [2] US 10,314,312 B2, Vineet Sharma, Ashutosh Agarwal, 'Synergistic antimicrobial composition of zinc pyrithione', 2018-10-31, assigned to Jubilant Life Sciences Ltd [3] US 2012/0276040 A1, Vincent T. Chuang, 'Antimicrobial compositions and methods of making same', 2012-11-01 [4] ISO 22196:2011 Measurement Of Antibacterial Activity On Plastics And Other Non-Porous Surfaces [5] Michele Pittola *, Daiane Tomacheskia,b, Douglas Naue Simõesa,b, Vanda Ferreira Ribeiroa,b, Ruth Marlene Campomanes Santanab (2017) 'Antimicrobial Performance of Thermoplastic Elastomers Containing Zinc Pyrithione and Silver Nanoparticles. [6] Product Safety Commission (AfPS) GS Specification Testing and assessment of Polycyclic Aromatic Hydrocarbons (PAHs) AfPS GS 2019:01 PAK [7] CPSC-CH-C1001-09.4 Standard Operating Procedure for Determination of Phthalates January 17, 2018. [8] BS EN 1122:2001 Plastics - Determination of Cadmium - Wet Decomposition Method [9] Test Method: CPSC-CH-E1002-08.3 Standard Operating Procedure for Determining Total Lead (Pb) in Nonmetal Children's Products. [10] Ministry of Environment and Urbanization Regulation No. 30105 on the Registration, Evaluation, Authorisation and Restriction of Chemicals Elif Öz Çetin Research and Development Specialist Chemical Engineer Adel Kalemcilik Ticaret ve Sanayi A.Ş. Dr. Fatma Seda Güreli Feridun Product Safety and Regulation Specialist Chemical Doctor Adel Kalemcilik Ticaret ve Sanayi A.Ş.