Abstract:

The contaminants in the dynamic mutational environment potentiate the demand for safe, sustainable, noncorrosive and bio-degradable disinfectant. Health of mankind as well as livestock rests on the capabilities of an effective disinfectant. Chemical disinfectants have their own limitations and are loaded with side effects. The present study was aimed at establishing an essential oil based green disinfectant as an effective disinfectant for environmental contaminants. The samples taken from a manufacturing unit were assessed for various environmental contaminants post disinfection by the green disinfectant. The results obtained were compared with the results of post chemical disinfectant. It was observed that the green disinfectant was 99% effective against the environmental contaminants and was effective till 48 hours post disinfection. It may be inferred that green disinfectant could be the future tool for all bio security procedures.

Introduction

With the global advancement, technology transfer, extensive mining and excavations due to rapid urbanization and mass movement of migratory birds, animals as well as humans has resurfaced various infectious zoonotic agents, leading to outbreaks ranging from sporadics to pandemics. The present pandemic is a perfect example of this. If such episodes are not managed in a prudent way,the whole human race will be at stake. Henceforth its imperative to safeguard the mankind from zoonotic/infectious microbes. The one and only solution to prevent this contagious dissemination is to strengthen bio security.

Bio security may be defined as a holistic concept, where disease prevention strategies are implemented to reduce the risk of introduction and spread of disease agents. As the human health and the global economy both are intrinsically linked to animal health and environment, biosecurity requires a very focussed and deliberate operating procedure. The primary step of biosecurity involves disinfection and cleaning. Significance of disinfectants lies in providing a healthy pathogen free environment with reduced microbial load to avoid disease conditions in farms, sheds, poultry houses, hatcheries, animal barns, equipments, feeders & drinkers as well as working surfaces. Cleaning refers to the physical act of removing organic matter and solid debris (dirt, grease, feces, body fluids, etc.) and must always precede disinfection(Craig J-G. Hunt, 2019).It improves the penetration power of disinfecting agents and ensures quality disinfection. The phenomenon of cleaning and disinfection (C&D) involves the use of physical and chemical methods aiming towards reducing, removing, inactivating, immobilising or destroying pathogenic microorganisms. Thus, disinfection plays a vital role in clearing of or transfer of microorganisms and acts as a barrier in transmission of pathogens from animals to humans or vice-versa. However, the chemical disinfectants, despite having a multifactorial role and potential, have their own limitations too. Alcohol, Chlorine compounds, Formaldehydes etc may cause skin or eye irritation and allergic dermatitis. Chemical disinfectants are also known to be inflammable, toxic, corrosive as well as carcinogenic. They are known to posses residual effects and are non compatible with plastic/steel feeding troughs. Moreover indiscriminate and unmonitored usage of chemical disinfectants may also lead to microbial resistence amongst environmental contaminants.

Considering all the limitations of chemical disinfectants, this study was envisaged to evaluate the potential of a herbal disinfectant, which is equally potent to chemical agents but completely safe for use, provides sustainable disinfection and completely ecofriendly because of its biodegradable nature.

Material and Methods

The present study was undertaken to evaluate the disinfection potential of a herbal biodegradable disinfectant,NBD-99(Nutricare life sciences ltd.).The environmental contaminants were exposed to different concentrations of NBD-99 to assess its effectiveness. The study for alternate approach was divided in to three parts –

(A) Air/environmental organic contaminants were taken using a sterile sponge swab from various sensitive points which included water source, drains, areas in and around manufacturing plant, lab appliances as well as working desks. To check the environmental microbial contaminants, the swab was rubbed and rolled firmly several times across the sampling area or exposed to the work environment for a period of 15 minutes. The samples were carried in triplicates to avoid any discrepancies. The exposed swabs were thereafter streaked on a nutrient agar petridish and incubated for a period of 24-48 hours at 35-37 0C to check for colony forming units.

(B)Part B, the target area and critical check points were sanitized and disinfected with the help of NBD-99 at different concentrations and samples were again taken using a sterile sponge swab, incubated and checked for microbial growth at 0,6,12,24 & 48 hour to check the growth of environmental microbes.

The exposure of swabs was grouped as-

I) Control-swab streaked on nutrient agar and incubated without any disinfectant applied

II) NBD-99 (1%)-swab exposed to surfaces and environment post disinfected by NBD-99(1%)streaked incubated at 0,6,12,24 and 48 hours post disinfection.

III) NBD-99 (2%)-swab exposed to surfaces and environment post disinfected by NBD-99(2%), streaked and incubated at 0, 6, 12 ,24 and 48 hours post disinfection.

IV) NBD-99 (3%)-swab exposed to surfaces and environment post disinfected byNBD-99(3%),streaked and incubated at 0,6,12,24 and 48 hours post disinfection

V) Conventional disinfectant- swab exposed to surfaces and environment post disinfected by conventional disinfectant, streaked and incubated at 0,6,12,24 and 48 hours post disinfection.

Though, the disinfectant posseses activity much beyond 48 hours(as per field trials), but owing to lab/test limitations, we worked on 48 hours only.

(C)To test the efficacy of the disinfectant, the disinfectant efficacy test SOP was followed. The subculture of Steptococcus, Enterococcus, Pseudomonas aeroginosa, Vibrio, Bacillus subtils and Aspergillus were taken from stalk culture on media plates and challenged with the herbal disinfectants. The tests were performed as per guidelines of USP-37.The incubation temperature used 30-35◦C for bacterial cultures for 24 hours and 20-25◦C for 48 hours for fungal cultures.

Results and Discussion

Table 1: Inhibition of bacterial growth at various concentration

+ Indicates positive growth observed on culture plates

- Indicates negative growth observed on culture plates

This study revealed that green disinfectant NBD-99 alleviated the environmental contaminants at 2% and 3% dilution. It was equally effective in disinfecting contaminated nonporous inanimate surfaces. Of all the samples taken from different critical points of manufacturing unit, lab, working desk and water outlets, this herbal disinfectant showed same potential for microbes at 0, 6, 12, 24 and 48 hour of exposure. The effectiveness of surface disinfectant was even evident at 48 hours post disinfection. This proves the stability of the herbal liquid, which despite being exposed to various environmental factors, was able to neutralize the microbial contaminants. The results revealed that though the standard conventional disinfectant worked well even at 0.5% and 1%, yet the efficacy of the green disinfectant at 2 and 3% was not only comparable to it, but also did not show any kind of residual or side effects.

Part B of the study involved examining the efficacy of the disinfectant against specific microbes. The cell lines were exposed to 3% concentrations of the herbal disinfectant. The results are depicted in Table 2. It was interesting to note that the specific target of neutralizing the most commonly occurring environmental microbes was achieved by the green disinfectant.

Table 2: Efficacy of NBD-99 against bacterial and fungal cultures

The constituents of green disinfectant such as Thyme, Pine and Euclaptyus oils are known for their antimicrobial effects (Tariq S et al., 2019; Dhakad AK et al.,2018 ;Chouhan S et al.,2017;Bachir and Benali,2012).Infact, in vitro disinfectant activity of essential oils against broad range of microbial, fungal, and viral species has been well studied and demonstrated (Cáceres, M et al.,2020; Brochot, A et al., 2017; Prabuseenivasan S et al.,2006.)

The antimicrobial activity of disinfectants may be classified in two different groups namely, (a)Growth inhibitor and(b) Microbicidal. The effectiveness of this essential oil based disinfectant can be attributed to the actives present in essential oils which acted synergistically to destabilize the cell membrane, increased cell permeability as well as inhibited bacterial cell division(de Aguiar et al.2018; Swamy et al.,2016;Nazzaro et al., 2013;Celikel and Kavas, 2008;Martinez & Baquero,2000).Swamy et al.,(2016) specified the mode of action and mentioned that essential oils disrupt the cell membrane of the targeted pathogens by increasing membrane permeability, inducing leakage of vital intracellular constituents, and interrupting the cellular metabolism and enzyme kinetics of the targeted pathogens. An acceptable activity of essential oils against gram‐positive and gram‐negative bacteria has been observed by de Aguiar et al.(2018).The antifungal effect of the liquid disinfectant may be justified by the action of its actives, which inhibited fungal growth and spore formation and destabilized the cell organelle causing cell lysis (Tariq S et al.,2019).The antiviral effect of the disinfectant may be due to of inhibition of viral protein translation as well. Bansod and Rai(2008) in their study justified the use of mixed essential oils in case of mycotic infections. Similarly, disinfecting activity of the tea tree essential oil showed effective inactivation of captured fungal and bacterial aerosol on the filter surface (Huang et al. 2010; Pyankov et al. 2008). In yet another study, Pyankov et al.,(2012)concluded that essential oils could be successfully used as effective disinfectant in bio-aerosol filtration applications.

The green disinfectant results may be correlated to the findings of Hendry et al.,(2012)who demonstrated that essential oil formulations posses antimicrobial efficacy for potential disinfection and elimination of microbial biofilms from hard surfaces. They further suggested that ,essential oils may be a useful adjunct to current infection control strategies .Besides this, according to a recent research result, the disinfectant spray made of the α-Terpineol effected the skin for 1 minutes could kill 99.996% Staphylococcus aureus, 99.998% Pseudomonas aeruginosa, 100% Escherichia coli and 100% Candida albicans. Exposure of same spray for 2 minutes on the skin could kill the 98.46% bacteria. (Center, B., & Cleaning, P. 2020).

Conclusion

NBD -99 proved to be a competent disinfectant for the environmental organic contaminants. At 2-3% dilution, it exhibited a >99% efficacy which lasted for 48 hours post disinfection. The product also worked well in eliminating the microbial growth in culture media with an efficacy rate of +98%.We may say that essential oil based disinfectant has a great potential in the field of decontaminants and can be potential product to enhance biosecurity procedures. The green disinfectant is the ecofriendly alternative approach, which is non-toxic, non-corrosive and readily usable on nonporous hard surfaces as well as farm organic material.

EFFICACY OF GREEN DISINFECTANT AGAINST COMMON ENVIRONMENTAL CONTAMINANTS