Antimicrobial effect of leather treated at the stage of fatliquoring
When assessing antimicrobial effect of leather fatliquored with oregano oil (3%) after one month, a very good antimicrobial activity of the leather was confirmed by considerable zones of growth inhibition for mould Scopulariopsis brevicaulis (>32 mm), yeasts Candida albicans (16 mm), bacteria Staphylococcus aureus and Staphylococcus epidermidis (10–11 mm) and the strain of Escherichia coli (9 mm)34. After 6 months of fatliquoring, a good antimicrobial effect against all the microorganisms in the test was still observed. The sizes of the zones of growth inhibition varied between 8–10 mm for Staphylococcus epidermidis up to>32 mm for Scopulariopsis brevicualis32.
In an experiment performed after 12 months of storage of the samples, the antifungal activity of leather with oregano oil (3%) was reduced with regard to filamentous fungi Scopulariopsis brevicaulis. In this case, the growth inhibition zone was equal to 0. For the other microorganisms, zones of considerable sizes were still observed (in the range between 4 mm and 15–17 mm), which demonstrated the strong antimicrobial effect34. For the control samples prepared without using the essential oil, no grow inhibition zones were observed around the leather disks either after 1, 6 or 12 months of storage. Under the samples, zero, slight or moderate growth of the reference strains was observed32,34.
The results presented allowed to conclude that lining leather fatliquored with the addition of oregano oil at a concentration of 3% by leather mass is characterised by good and durable antimicrobial effect persisting even for 12 months. The persistence of biostatic properties of the leather samples for such a long time resulted from the fact that the decrease in emission of carvacrol, i.e. the active component of the oil, was almost two times lower in comparison with the decreases in emissions of all the volatile organic compounds during the storage of the samples. This was corroborated in earlier experiments using the method of gas chromatography with mass spectrometry33.
Antimicrobial effect of leather treated by surface spraying
The results of antimicrobial activity tests for the leather sprayed with oregano oil are presented in Table 3. At 5 days after spraying considerable inhibition zones of microbial growth were observed both around the leather specimens sprayed on the grain side and on the flesh side. For specimens treated on the grain side the inhibition zones of the following size were noted: Staphylococcus aureus −18 mm (Fig. 1Ia), for Escherichia coli −25 mm (Fig. 1Ib), Candida albicans >32 mm (Fig. 1Ic). Weaker, but noticeable effect of leather microbial activity against Pseudomonas aeruginosa, which are resistant to antimicrobial agents, was also recorded in the form of a 1 mm inhibition zone (Fig. 1Id). For specimens sprayed with oil on the flesh side, the antimicrobial effect achieved was slightly weaker for Staphylococcus aureus (8–11 mm) (Fig. 1IIa) and Escherichia coli (6 mm) (Fig. 1IIb), and for Pseudomonas aeruginosa (Fig. 1IId), where no inhibition zone was observed. Equally strong (as on the grain side) antimicrobial effect was observed for yeast Candida albicans, where the inhibition zone was >32 mm (Fig. 1IIc). No microbial growth under the specimens was observed.
The leather sprayed with oregano oil on the grain side (I row) and flesh side (II row), after 5 days of storage, growth inhibition zones of Staphylococcus aureus (a), Escherichia coli (b), Candida albicans (c), Pseudomonas aeruginosa (d).
After 30 days the antimicrobial effect of the leather was attenuated (Table 3). For samples sprayed with oil on the grain side, the growth inhibition zones observed were of the following sizes: for Staphylococcus aureus – 6–10 mm (Fig. 2Ia); for Escherichia coli – 2–6 mm (Fig. 2Ib); for Candida albicans – 11–13 mm (Fig. 2Ic). For the bacteria Pseudomonas aeruginosa, the growth inhibition zone was reduced to 0 mm (Fig. 2Id).
The leather sprayed with oregano oil on the grain side (I row) and flesh side (II row), after 30 days of storage, growth inhibition zones of Staphylococcus aureus (a), Escherichia coli (b), Candida albicans (c), Pseudomonas aeruginosa (d).
When analysing the results obtained for samples sprayed on the flesh side, a reduction in the antimicrobial activity of the leather was also observed. The largest growth inhibition zone was observed for the yeast Candida albicans (15–18 mm) (Fig. 2IIc). For the bacteria Staphylococcus aureus, a zone with a diameter of 1–3 mm (Fig. 2IIa) was observed; and for Escherichia coli it was 0–1 mm (Fig. 2IIb). The antimicrobial effect of leather on the Gram-negative bacterium Pseudomonas aeruginosa was attenuated to such an extent that slight bacterial growth occurred under the sample (Fig. 2IId), which was not observed for any other sample tested after 30 days of spraying.
In the tests performed for the control group of samples (Table 4) no microbial activity was observed. No typical growth inhibition zones occurred around the samples (Fig. 3) and zero, slight or moderate microbial growth occurred under the samples.
The leather not sprayed with oregano oil (control group), the grain side (I row) and flesh side (II row), no growth inhibition zones of Staphylococcus aureus (a), Escherichia coli (b), Candida albicans (c), Pseudomonas aeruginosa (d).
When comparing antimicrobial activity of leather treated using the spraying method with the activity of leather fatliquored with oregano oil after 30 days of storage, it should be noted that it was possible to achieve a good effect of antimicrobial treatment by both methods. Although the samples refined by fatliquoring had stronger effect, microbial growth inhibition zones of considerable sizes were also observed for samples treated by the spraying method. The test results show that surface spraying with oregano oil is one of the possible methods that can be applied in order to refine sock lining leather and give it antimicrobial properties. Although the effect is attenuated over time, the experiment indicates that this effect is fully sufficient to protect the inside of the shoe against multiplication of microorganisms hazardous to human health for some time. These microorganisms may also contribute to decomposition of footwear materials. Reapplication of the oil finish by the user (e.g. by spraying with an atomizer) would allow maintaining antiseptic properties at the desired level for a longer time, thus eliminating the need to use chemical antimicrobial agents, e.g. foot and shoe deodorant sprays as well as biocides in raw material finishing. What is more, the proposed method of direct spraying can be used for shoe insoles, also those made of materials other than leather, e.g. textiles or leather-like materials. These shoe components treated with oregano oil and replaced by the user at appropriate intervals might enhance hygienic properties of shoes understood in the context of antimicrobial activity.
Estimation of the toxicity level of oregano oil and synthetic biocidal preparations used in the tanning industry
The results of toxicity assessment for oregano oil and commercial biocidal preparations used in tanning and marketed under specified trade names are presented in Table 5. These results are based on biocidal material safety data sheets (MSDS), containing the data that allow the acute toxicity estimate (ATE) values to be derived, and information on acute toxicity of these biocidal substances. This assessment included the four main areas of toxicity effects of dangerous substances including hazards resulting from oral, inhalation and dermal route of exposure as well as eye contact.
The use of substitutes for chemical substances (biocides) to meet the requirements of the Best Available Techniques for the tanning industry requires raw materials of lower toxicity and lower environmental impact to be used at the lowest possible concentration levels36. In industrial practice, special care should be taken when working with chemical compounds that pose the greatest hazard and are assigned to category 1 within any hazard group (or such contact should possibly be avoided).
With regard to the number of hazards and the categories of their toxicity Biocide 1 should be considered the most hazardous among the biocidal preparations used in the tanning industry, presented in Table 5. This is a dangerous product containing highly toxic substances and therefore its toxicity has been classified as category 1 in three areas. Among them one deserves special attention: 2-(thiocyanomethylthio)-benzothiazole (TCMTB), it is present in biocide 1 (the range of concentrations 30 to 50%), and also in biocide 2 (the range of concentrations 25 to 30%). The toxicity of this compound and the products of its degradation was assess by Nawrocki et al.37 in the acute test (48-hours), and the chronic test (7-days), carried out for model species Ceriodaphnia dubia. The EC50 (median effective concentration) value measured by authors for TCMTB were 15.3 µg/L and 9.64 µg/L, in the acute and in the chronic tests, respectively. Moreover Nawrocki et al.37 established that TCMTB was an order of magnitude more toxic than the products of its degradation. The value of LD defined for this compound for other tested organisms can differ. For example LD50 measured for rats directly fed (oral) with TCMTB ranges between 2538 mg/kg or even 679 mg/kg, depending on the data source38. The website of PubChem39 contains similar data set for acute effects of 4-chloro-3-methylphenol, which is present in biocide 4 (Table 5) at the range of concentrations 30 to 40%. The LD50 value after oral administration to rats was 1830 mg/kg, whereas to mice 600 mg/kg. Biocide 4 contained also 2-octyl-2H-isothiazol-3-one, its concentrations was 8 to 10%. LD50 value of this compound measured for rats (oral administration) was 550 mg/kg40.
Oregano oil containing 94% of carvacrol is the most environmentally friendly substance among all the compounds (Table 5) analysed. This oil ingredient known by the full chemical name 5-isopropyl-2-methylphenol has the lowest number of hazards, and in one area only, i.e. eye contact, so it was classified in toxicity category 1. According to research41, LD50 value of carvacrol which was orally administrated to rats was 810 mg/kg. Based on the data present in MSDS of carvacrol it can be concluded that none of the ingredients present in concentrations bigger than 0.1% was defined by International Agency for Research on Cancer (IARC) as probable, possible or confirmed carcinogen for humans. In addition, this substance does not contain components considered either persistent, bioaccumulative and toxic, or very persistent and very bioaccumulative (vPvB) at the level of concentration 0.1% or above.
Source: Ecology - nature.com
