Efficacy of frontline chemical biocides and disinfection approaches for inactivating SARS-CoV-2 variants of concern that cause coronavirus disease with the emergence of opportunities for green eco-solutions

The emergence of severe acute respiratory disease (SARS-CoV-2) variants that cause coronavirus disease is of global concern. Severe acute respiratory disease variants of concern (VOC) exhibiting greater transmissibility, and potentially increased risk of hospitalization, severity and mortality, are attributed to molecular mutations in outer viral surface spike proteins. Thus, there is a reliance on using appropriate counter-disease measures, including non-pharmaceutical interventions and vaccination. The best evidence suggests that the use of frontline biocides effectively inactivate coronavirus similarly, including VOC, such as 202012/01, 501Y.V2 and P.1 that have rapidly replaced the wild-type variant in the United Kingdom, South Africa and Brazil, respectively. However, this review highlights that efficacy of VOC-disinfection will depend on the type of biocide and the parameters governing the activity. VOC are likely to be similar in size to the wild-type strain, thus implying that existing guidelines for use and re-use of face masks post disinfection remain relevant. Monitoring to avoid injudicious use of biocides during the coronavirus disease era is required as prolonged and excessive biocide usage may negatively impact our receiving environments; thus, highlighting the potential for alternative more environmental-friendly sustainable biocide solutions. Traditional biocides may promote cross-antimicrobial resistance to antibiotics in problematical bacteria. The existing filtration efficacy of face masks is likely to perform similarly for VOC due to similar viral size; however, advances in face mask manufacturing by way incorporating new anti-viral materials will potentially enhance their design and functionality for existing and potential future pandemics.


Introductioncoronavirus and its implications for maintaining healthcare provision
The coronavirus disease (COVID-19) pandemic, caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2), has imposed tremendous challenges on healthcare systems globally [1e3]. At the time of writing (30th March, 2021), there have been 127,628,928 cases of COVID-19 worldwide, including 2,791,055 deaths [4]. COVID-19 elicits a broad infection spectrum ranging from very mild, non-respiratory symptoms to severe acute respiratory illness, sepsis with organ dysfunction and death; however, some infected people can be asymptomatic [1]. Evidence highlighting the contributions of super-spreaders of infectious airborne viral particles, including the more transmissible SARS-CoV-2 variants of concern (VOC), has also contributed to the occurrence of third and fourth waves of COVID-19 infections [5e7]. Addressing the ongoing COVID-19 pandemic has created unprecedented logistical challenges to maintain critical supplies of single-use personal and protective equipment (PPE) [8,9], where reuse and disinfection occurred under emergency use authorization. At the outset of COVID-19, there was a void in knowledge to effectively counter this disease; however, there is an increasing understanding of the potential role of different strategies to address COVID-19, including adopting non-pharmaceutical interventions (NPIs, such as correct wearing of face masks, hand hygiene, use PPE, maintaining social distancing, detection testing, contact tracing), along with delivering new vaccines [8,9]. Data generated from predictive mathematical modelling of multiple-contributing factors influencing the occurrence of COVID-19, and commensurate efficacy of disease counter-measures, is increasing, such information is translated to calculating risk probability to monitor and manage the basic reproduction number R 0 in the following [9]. It is challenging to appreciate the actual efficacy of specific COVID-19 disease interventions in real-time given the swiftly moving pace of this pandemic. This current opinion focuses on understanding the efficacy of frontline biocides and disinfection approaches against SARS-CoV-2 variants of concern.
Coronaviruses and implications for meeting personal and protective equipment supply chain shortage and disinfection reprocessing SARS-CoV-2 is a large positive-stranded RNA virus with an outer lipid envelope containing glycoprotein spikes ( Figure 1) [10]. In general, enveloped viruses, such as coronaviruses, are more sensitive to environmental deleterious stresses, such as chemical biocides, than similarly-treated naked viruses, due to the presence of a lipid membrane [9, 11,12]. Coronaviruses range from 60 to 140 nm in size, which is below the 300 nm pore diameter used in multiple layers of material used to make face masks. However, the use of multiple layers in single-use plastic face mask reduces the probable risk of penetration and transmission by acting as a barrier to respiratory droplets [13,14], which is likely to apply similarly to mitigating against VOC transmission. The effectiveness of single-use plastic filtering-face piece respirators face masks varies based on type and certification that is defined across three levels of protection depending upon leakage of particles into the interior of the mask that are 22% (FFP1, such as medical and procedural masks), and 8% (FFP2, such as N95-type respirators), and 2% for non-disposable FFP3-type respirators [2]. Use of non-thermal biocidal and disinfection approaches, such as vaporized hydrogen peroxide (30e 35% VH2O2) and moist heat (60e65 C for 30 min), and ultraviolet light at 254 nm (or fluence at 2000 mJ/cm 2 ), has been applied for reprocessing FFP1 and FFP2 type respirators, such as under emergency use authorization [2,25e27]. Non-thermal disinfection approaches of FFPs have been selected to enable retention of filtration performance, material compatibility, comfort fit, and pressure drop. In addition, there has been increased usage of alternative, cost-effective, home-made, cloth or fabric face coverings by the general public, where particle penetration efficacy was improved by using more than one layer of cotton-polypropylene and by introducing pleats when compared to testing using a fitted Structural components of SARS-COV-2 (left) and effective biocidal agents known to deactivate the virus (right).
N95 FFP2-type control [15e17]. Combining similar mild heat conditions, along with the use of detergent, for reuse of face coverings is theoretically plausible for coronavirus-disinfection; however, there remains a lack of information on disinfection and efficacy for informing the frequency of reuse that maintains filtration functionality [2,9]. Post COVID-19, it is likely that the changes in medical practice will drive sustaining or increasing high demand for PPE.

Understanding coronaviruses and the role biocides in breaking cycle of COVID-19 infection
Coronaviruses are typically inactivated on different surfaces within 4e5 days at ambient room temperatures on different surfaces, such as tissue, wood, glass, plastic, stainless steel, surgical masks, and paper, that can be influenced by humidity, viral load, presence of organic matter [9,18]; for example, colder conditions, such as refrigeration (4 C), may extend SARS-COV-2 viability on surfaces beyond 14 days [19,20]. It took 14 days at 20 C to reduce SARS-CoV-2 on nitrile gloves by 5 log orders using simulated typical infectious body fluids from infective patients; however, viral persistence was evident up to 21 days on plastic face shields, N100 FFPs, and polyethylene overalls [21]. These observations imply that the colder conditions associated with winter may support longer survival of SARS-CoV-2 on contact surfaces and when suspended in aerosols. There is a pressing need to understand the role of different interventions in breaking the cycle of SARS-CoV-2 infection ( Figure 2) to protect frontline healthcare workers and patients [9]. This includes generating data over a longitudinal period to evaluate and harmonize deployment of these interventions (singly or combined) to prevent or reduce the risk of COVID-19 with a focus on infections agent (SARS-CoV-2 and co-infections), reservoir, portal of exit, mode of transmission, portal of entry, and susceptible host ( Figure 2). The efficacy of such data will be informed by mathematical modelling and randomized control studies [9]. The use of disinfectants or chemical biocides and disinfection approaches will feature strongly as key disease countermeasures in breaking the cycle of infection (Figure 2). Factors that influence the efficacy and performance of different types of biocides are varied and complex [22,23]; however, the parameters governing selection and performance of different biocide types are generally well-established, where the degree of application depends upon categories of risk to patients aligned with a commensurate level of treatment required to be achieved on contact surfaces and in the environment (Table 1). 'COVID-19 fatigue' of citizens is likely to play a contributing role in meeting compliance with deploying disease counter-measures to effectively manage new cases to protect frontline healthcare workers [24].

SARS-CoV-2 VOC
The World Health Organization monitors public health events associated with SARS-CoV-2 VOC [3]. Key features for these VOC are presented in Table 2. VOC 202012/01, 501Y.V2, and P.1 have commonly demonstrated an increase in transmissibility compared to wildtype (non-VOC) variants and have demonstrated a propensity for rapidly replacing other circulating SARS-CoV-2 strains. Variants 202012/01, 501Y.V2, and P.1 rapidly replaced the wild-type variant in the United Kingdom, South Africa, and Brazil, respectively. However, it is highly likely that more transmissible and pathogenic VOCs will emerge during this pandemic as the virus has ample opportunity to mutate given the high numbers of infected hosts globally. However, in terms of existing VOC, the WHO deploys 'a logistic model of competitive growth that highlighted additive increases in the effective reproduction number (Rt) relative to the wild-type variant that was estimated at 41% (95% CI: 41e42%) for 202012/01, 36% (95% CI: 32e40%) for 501Y.V2, and 11% (95% CI: 7e16%) for P.1' [3]. The transmissibility of P.1 is such that it is rapidly replacing the wild-type variant at a local level. Recent studies have shown VOC 202012/01 may be associated with an increased risk of hospitalization, severity, and mortality. There is a growing body of evidence on vaccine-induced neutralizing antibody activity against VOCs ( Table 2). The findings support that neutralizing activity is largely sustained against this variant. However, these findings highlight the importance of using combinational approaches, including the use of biocides for surface disinfection, as important to limit transmission of VOCs. Key mutations affect viral nonstructural proteins that are unlikely to affect the efficacy of frontline biocides described in Tables 3 and 4. There is an increasing interest in the future proof design of face masks by also incorporating potentially new antiviral materials with the provision for more environmentally friendly non-metal nanomaterials [66].

Indication of biocide efficacy against coronavirus
Biocides encompass chemicals with antiseptic, disinfectant, and/or preservative activity (Table 3). Biocides are used for a broad range of purposes, 'usually with inanimate objectives (hard surface disinfectants), externally on the skin (antiseptics and topical antimicrobials), to prevent or limit microbial infection for preoperative skin infection or incorporated (preservatives) into pharmaceutical, cosmetic, or other types of products to prevent microbial contamination' [28]. Desirable properties of biocides include virucidal within the time that can allow for it to be in contact with materials to be treated; effectiveness not diminished under conditions of disinfection; does not damage material treated, has a suitable spectrum of activity; low toxicity and resistance to it has not emerged; inexpensive. Biocidal efficacy is influenced by several, and sometimes, inter-related factors d notably concentration, period of contact, pH, temperature, presence of organic or other interfering or enhancing materials or compounds, nature, numbers (dose), location (planktonic, biofilm), and condition of microorganisms (recalcitrant endospores vs sensitive enveloped viruses) (Table 3) whereas those with low h-values (such as QACs, chlorhexidine, orthopthalaldehyde) retain considerable activity on dilution. This difference is highly relevant when considering both lethal disinfection activity and potential implications on receiving environment, where the potentially deleterious impact of biocide residues must be considered [28]. In addition, many frontline biocides have optimal pH activity, such as hypochlorite and phenolics are most effective at acid pH, whereas glutaraldehyde and cationic biocides (e.g. QACS) are most potent at alkaline pH. Several researchers have reported that biocide activity can be influenced by interaction with organic matter (e.g. dirt, blood, serum, vomit, the presence of biofilm), and non-ionic surfaces, and adsorption on containers and other contact surfaces (Table 3).
Coronaviruses are incapable of supporting independent life; thus, biocide disinfection is determined by using in vitro bioassays, where reduction of cytopathic effects in tissue culture monolayers are observed that is attributed to a reduction in viral infectivity compared with untreated controls. Surviving viral fractions are typically expressed through Log 10 reductions enumerated either by determining the 50% titration reduction endpoint for infectivity (known as tissue culture infectious dose 50%, Table 1 Factors governing anti-viral efficacy of biocides.

Factors a Comments
Relevance and implication for usage in practice  Table 2 Synopsis of key information on SARS-CoV-2 variants of concern, as reported by World Health Organization on 23rd March, 2021.
Emerging information a Variant of concern (VOC)

Disinfection of SARS-CoV-2
As an enveloped virus, SARS-CoV-2 is susceptible to commercial disinfectant chemicals, technologies, and physical disinfection methods [33,34] (Table 4). Recent studies have detected SARS-CoV-2 RNA on surfaces in isolation wards 28-days following exposure via RT-PCR methods [35], where the infectivity ability of viral RNA is unknown. However, it is unlikely that undamaged viral RNA realized on treated surfaces would remain a significant risk because of its inability to enter human lung cells as RNA only. Determination of biocidal efficacy against SARS-CoV-2 is not always feasible as the virus requires biosafety level 3 or higher; therefore, fewer pathogenic viruses as surrogate indicators of infectivity are frequently used [36]. Virucidal efficacy is determined by quantitative suspension tests, namely EN 14476 requiring 4 log reduction using surrogate enveloped species such as polio, adenovirus and murine norovirus, where efficacy against SARS-CoV-2 has yet to be elucidated experimentally. The framework of the European Committee for Standardization outlines surrogate species suitable for disinfection studies for many microorganisms. For virucidal activity against enveloped viruses, including SARS-CoV-2, the vaccinia virus has been specified as the relevant test organism according to this framework [37]. In clinical settings, SARS-CoV-2 has been detected on surfaces in intensive care units (4.4e5.2 log 10 ), in isolation rooms, and on general wards (2.8e4.0 log 10 ) [38]. While the viral load of SARS-CoV-2 on fomites directly following contact with infected persons is currently unknown, it is known that the virus remains infectious on surfaces for up to 9 days [39,40] depending on the surface material, pH, temperature, and humidity [40]. The suitability of suspension tests to determine efficacy on surfaces is unknown, particularly where the organic matter may be present such as in healthcare settings. ISO 18184 is a surface carrier test for virucidal activity; at present, no studies have demonstrated biocidal efficacy against SARS-CoV-2 using this method. The disinfection of surfaces and hand sanitation is of paramount importance in controlling viral transmission as recommended by the WHO. Disinfectant efficacy is affected by viral type, organic matter, viral titre, pH, viral clumping, biocide contact time and concentration. As such, cleaning is an essential prerequisite for disinfection to remove contaminating organic matter. In healthcare settings, disinfection agents in use include high-pressure steam sterilization, dry heat, UV-light, ethylene oxide (EtO) gas, hydrogen peroxide gas plasma, and biocidal chemicals [41] (  (Table 4). A concentration of 6% acetic acid reduced human coronavirus (hCo-V) viability by 3.5 log 10 after 1 min contact time [43] on surfaces. 60e70% ethanol reduced surface viral load by >3 log 10 after 1 min exposure in healthcare settings [44]. For the inactivation of SARS-CoV-2, the most common disinfectants used are 62e70% ethanol, 0.5% H 2 O 2 , and 0.1% sodium hypochlorite, which are effective with 1 min exposure via oxidative reactions [42]. Pulsed plasma gas discharge has also produced biocidal water comprising short-lived oxygenated free radicals that has potential contact surface disinfection leaving no unwanted chemical residues [45]. For hand disinfection, the WHO recommends the use of 75% IPA or 80% ethanol hand rubs for 30 s to inactivate SARS-CoV-2 [46]. However, there is increasing opportunities to exploit advances in digitization and modelling to inform the future efficacy of disinfectants, including combining biocidal approaches and to hurdle limitations for broad applications [9,47]. Material compatability and functionality are important factors to consider when using new eco-alternatives to conventional biocides that includes combinational treatments [64].

Conclusion
Variant strains of SARS-CoV-2 are constantly emerging due to innate mutagenic changes in the viral genome, primarily altering structural components such as spike proteins. Although the efficacy of vaccinations program against these variants is unknown, such structural changes are unlikely to confer disinfection resistance as non-specific destruction of proteins and lipids of the viral capsid occur. Frontline biocides appear to be affective against VOC, but factors governing usage needs careful consideration. Vigilance is needed to protect our environment during the COVID-19 era, particularly by avoiding injudicious use of biocide that may negatively impact our agroecosystems [48]. Prolonged and excessive biocide use may give rise to situations that potentially promote cross-antimicrobial resistance in problematical bacteria to frontline antibiotics [49]. Adaptive resistance to frontline biocides has been reported since the early 1990s such as against bisphenol, triclosan, glutaraldehyde, and oxidising agents [22]. Paul et al. [48] highlighted that extensive application of biocides affects microbial flora that may lead to a decrease in the number and diversity of beneficial microbes that may directly affect the functioning of nutrient cycles; thus, careful considerations should be given to biocide neutralization, environmental management and sustainability [50,51]. Understanding these factors is important for the training of end-users, (e.g. healthcare, industry and community), to ensure the efficacy of biocidal product is maintained and effectively neutralized, along with monitoring policy for effective and responsible deployment of biocides. This current opinion supports Article 18 of the European Union's biocidal products regulation that directs the European Commission to issue a report on how the biocidal products regulation contributes to the sustainable use of biocidal products to reduce the risks posed to human health, animal health, and the environment by biocidal products. The aforementioned also recommends a series of actions to be completed by 2024, including investing additional resources in enforcement activities; developing the best available techniques reference documents that can be relevant for biocidal products used in industrial processes, and encouraging the development and implementation of standards that could contribute to the sustainable use of biocidal products and alternatives to biocidal products.

Funding
No funding.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References
Papers of particular interest, published within the period of review, have been highlighted as:  CoV-2 by commonly used disinfection products and methods. Sci Rep 2021, 11:2418, https://doi.org/10.1038/ s41598-021-82148-w. Reports on the investigation of disinfection products on SARS-CoV-2 inactivation using standard quantitative suspension tests. Optimal exposure times and concentrations for varying disinfection agents are elucidated providing key information in preventing disease transmission. The use of SARS-CoV-2 instead of a surrogate species makes this experimental data highly relevant and topical. Infectivity was determined via TCID50 assay using Vero-E6 cells which are derived from the kidney of an African green monkey and are used for the culture of viral species of medical importance.
hand sanitizers are described in terms of their anti-viral activity where studies conclude SARAS-CoV-2 has similar sensitivity to the standard test viral surrogate vaccinia as used in EN 14476 or DVV/RKI testing guidelines. The study is of significance as it elucidates the suitability of the recommended surrogate vaccinia to represent SARS-CoV-2 in disinfection studies.