key: cord-0056611-yyzuq5b9
authors: Mileto, Davide; Mancon, Alessandro; Staurenghi, Federica; Rizzo, Alberto; Econdi, Stefano; Gismondo, Maria Rita; Guidotti, Matteo
title: Inactivation of SARS-CoV-2 in the Liquid Phase: Are Aqueous Hydrogen Peroxide and Sodium Percarbonate Efficient Decontamination Agents?
date: 2021-02-16
journal: J Chem Health Saf
DOI: 10.1021/acs.chas.0c00095
sha: df8cf23ac1fada3165164e3154675aa515f8837c
doc_id: 56611
cord_uid: yyzuq5b9

[Image: see text] A diluted 3% w/w hydrogen peroxide solution acidified to pH 2.5 by adding citric acid inactivated SARS-CoV-2 virus by more than 4 orders of magnitude in 5 min. After a contact time of 15 min, no viral replication was detected. Aqueous solutions of sodium percarbonate inactivated coronavirus by >3 log(10) diminution in 15 min. Conversely, H(2)O(2) solutions with no additives displayed a scarce virucidal activity (1.1 log(10) diminution in 5 min), confirming that a pH-modifying ingredient is necessary to have a H(2)O(2)-based disinfectant active against the novel coronavirus.

The coronavirus disease 2019 , caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenged the world for effective strategies for infection prevention and control, mitigation and containment, against virus dissemination. 1 Preventive measures are currently the most effective strategy to limit pathogen diffusion, reduce the overload on the healthcare system, and isolate newly identified cases. Since person-to-person transmission mostly takes place through the emission of droplets from the upper respiratory tract during breathing, talking, sneezing, or coughing, it is essential to minimize the spread of such liquid particles, especially in confined environments, and to avoid the contamination of objects and surfaces likely to become a further source of infection. 2−4 Social distancing and mandatory face coverings prevent and mitigate interhuman transmission, especially when coupled with lock-down actions, quarantine, contact tracing, and massive testing of suspects. 5, 6 At the same time, special attention is paid to understanding coronavirus persistence on inanimate surfaces. 7 Several studies investigated the performance of simple chemical biocide agents for the safe inactivation and/or degradation of coronaviruses on surfaces and water. 8, 9 Such indications were the basis for national and international health agencies to define adequate procedures for cleaning, sanitization, and disinfection of indoor spaces, objects, furniture, and tools in healthcare facilities as well as in daily household practice. 1, 10 According to these guidelines, potentially contaminated surfaces are typically sprayed or wetted with large excess amounts of virucidal solution, and a few minutes of action at room temperature is necessary for a satisfactory viral inactivation (mainly, spanning from 1 to 15 min or until the sprayed solution evaporates). 10−12 To date, the most widely adopted methods to inactivate coronaviruses, in particular SARS-CoV-2, on surfaces primarily rely on highly concentrated aqueous alcohol solutions (>70% w/w), 13, 14 aqueous solutions of active chlorine-generating compounds, 15−17 and formulations containing quaternary ammonium cationic surfactants. 18 WHO's guidelines suggest ethanol and 2-propanol for hand sanitization because of their rapid activity, broad-spectrum microbicidal effectiveness, cheapness, and reasonable safety. 19−21 Alcohols act on enveloped viruses denaturing the envelope viral proteins and disrupting its phospholipidic double layer. 22 However, during the first months of the COVID-19 emergency, some southern European countries suffered from a shortage in the availability of ethanol and 2propanol, because of the sudden high demand on the local market, to be used in liquid and/or gel sanitizing formulations. 23 Also, the high percentage of highly flammable alcohols in disinfecting solutions (the flash point of a 70 vol % ethanol solution in water is 21°C 24 ) increased the risk of fires and explosions in facilities where large amounts of sanitizing solutions were stocked, and several accidents occurred in improvised warehouses within healthcare facilities. 25 Sodium hypochlorite-based aqueous solutions at concentrations 0.1−0.5% inactivate coronaviruses with a >3.0 log 10 reduction in viral titer within 1 min of contact time. 13, 26 Undissociated hypochlorous acid, HOCl, as well as highly active oxidizing free radicals formed during the dissolution of these agents in water play indeed a key role in the antimicrobial activity. Hypochlorite-containing diluted aqueous solutions are nonflammable and safe, but they can generate chlorine-containing products, which pose a threat in terms of long-term toxicity to humans and the environment. Indeed, several concerns exist (in terms of negative environmental impact on biota in surface waters or disruption of wastewater treatment unit operations) on the extensive use of chlorinebased biocidal agents, especially for large-scale sanitizations. 18, 27, 28 Then, various biocides containing quaternary ammonium salts (also known as quats 29 ) are included in the EPA's list N recommendation list: benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, or cetylpyridinium chloride. 30 However, research led to controversial results in coronavirus inactivation, 26 and their specificity on SARS-CoV-2 is still debatable. 31 Some authors, in fact, raised concerns on the environmental impact due to a massive use of quaternary ammonium agents, too. 32, 33 None of these virucidal formulations are free from drawbacks. Thus, a biocidal agent that is fast-acting, chlorine-free, intrinsically safe (in terms of flammability and toxicity to humans and environment), chemically stable, and cheap would attract major attention as a reliable countermeasure against the SARS-CoV-2 virus. Aqueous hydrogen peroxide is a suitable candidate for such formulations, as it is safe, especially at very low concentrations, does not give rise to hazardous or polluting byproducts (H 2 O 2 breaks down over time into molecular oxygen and water), and is easily available as pharmaceutical grade solutions at 3% w/w concentration (also known as 10 volume hydrogen peroxide). 34 In the present work, we evaluated the effectiveness of aqueous hydrogen peroxide solutions in the inactivation of SARS-CoV-2, controlling concentration, pH, and additives. A concentrated liquid suspension of the virus was treated with comparable volumes of diluted inactivating agent for a few minutes, in order to mimic the typical mode of action of a sanitizing solution on small amounts of infected body fluids. Two different aqueous hydrogen peroxide concentrations were tested, namely, ca. 3% w/w, as found in conventional commercial household disinfectants, and ca. 0.5% w/w, as described and studied in previous reports. 35, 36 The commercially available adduct sodium carbonate−hydrogen peroxide, as the so-called sodium percarbonate (Na 2 CO 3 ·1.5H 2 O 2 ), was also tested for its high stability and robustness over time, when in dry form, being suitable to efficiently replace unstable aqueous H 2 O 2 solutions.

A brief description of materials and methods is reported here. Further details can be found in the Supporting Information.

Two mother solutions of hydrogen peroxide at 6.64% ± 0.04 (w/w) and 1.02% ± 0.02 (w/w) were prepared diluting a 35.1% stabilized H 2 O 2 solution with sterile ultrapure deionized water and stored at +4°C until use. H 2 O 2 content was determined by volumetric iodometric titration.

A sodium percarbonate (SP) aqueous solution was freshly prepared by dissolving Na 2 CO 3 ·1.5H 2 O 2 in sterile ultrapure deionized water. The obtained mother solution at 1.00 ± 0.02% (w/w) was immediately used after measuring its H 2 O 2 content.

The peroxide-containing aqueous solutions were mixed in a 1:1 ratio with the virus suspension, so that the final concentration of the biocidal agent was 50% of the pristine concentration. Thus, in the solutions named HP-3, HP-0.5, and SP, the peroxide contents were 3.32% w/w, 0.51% w/w, and 0.51−0.53% w/w, respectively. Uncertainties for these values were around ±0.02% w/w. A SARS-CoV-2 viral suspension was mixed with sterile distilled water (1:1 ratio) and used as the positive control of untreated virus.

Aqueous solutions of 0.187 M sodium carbonate decahydrate, 1.04 M citric acid, and 3.12 M acetic acid were prepared with sterile ultrapure deionized water and used to adjust the test media pH to the desired value.

HP-3 and HP-0.5 solutions at pH 7.3 and SP solution at pH 10.5 were used as such, without any addition of further pHadjusting agents.

HP-3-C at pH 2.5 and HP-3-A at pH 3.6 were obtained by adding citric acid or acetic acid, respectively, to the HP-3 formulation. The potential presence of peroxoacids in HP-3-C and HP-3-A solutions was checked by differential ceriometric/ iodometric titration, as described in ref 37 and by 13 C NMR analysis.

Contact times of 5, 10, and 15 min were chosen to simulate surface sanitizing conditions in common household practice. Each virucidal solution was tested in triplicate. At the end of contact time, a stoichiometric excess of catalase suspended in 50 mM potassium phosphate buffer at pH 7.0 was added to each test solution to decompose any residual H 2 O 2 and stop the test. Either sodium carbonate or citric acid was added to adjust pH to a neutral level before the addition of catalase. The efficacy of the catalase action was confirmed by iodometric titration, and in all cases, no detectable amounts of residual H 2 O 2 were found.

SARS-CoV-2 virus stock was prepared propagating the virus isolate (GenBank accession number: MW000351) in Vero E6 cells culture (see the Supporting Information).

The virus titer decay was evaluated using a plaque assay on Vero E6 cells for each experimental setting, and the results were compared with the positive control (see further details in the Supporting Information). 38 The same experimental protocol was used to investigate the possible cytopathic effect of catalase, citric acid, acetic acid, PC, and HP solutions, as single ingredients and in a mixture.

A quantitative real-time polymerase chain reaction, qRT-PCR, was performed to estimate viral RNA concentration of the untreated viral suspension and in samples with a significant after-treatment viral titer reduction. The final RNA concentration was expressed as copies per milliliter. Figure S3 ). Such plaques can be then counted to quantify virus titer, 38 the difference between before and after treatment titers being proportional to the virucidal activity. A virucidal solution is here considered effective against SARS-CoV-2 when at least a >3 log 10 (>99.9%) diminution in the number of active viral units is recorded at the end of the treatment. 39, 40 Diluted aqueous solutions of hydrogen peroxide were first tested under conditions as close as possible to the ones reported in the previous literature, 26, 35 namely, at concentrations of 0.5% and 3.0% as well as short treatment times of 5 min (Table 1) . At the end of the testing time, the excess H 2 O 2 was quenched by the addition of catalase (see the Supporting Information). A specific blank test was carried out to evaluate whether the presence of organic compounds in the culture media (DMEM medium with fetal bovine serum and penicillin−streptomycin additive) is able to lead to a diminution in the initial amount of H 2 O 2 due to undesired redox reactions. Under the conditions tested, a maximum 2.5% decrease in hydrogen peroxide with respect to the expected initial content was observed, and the actual H 2 O 2 concentrations reported in Tables 1 and 2 already take into account this intrinsic diminution.

Aqueous H 2 O 2 failed to effectively inactivate the virus, the best result being a ca. 0.70 log 10 reduction in 5 min, with a 3.07% w/w solution, which is comparable to common H 2 O 2 household formulations found on the market. A 0.5% w/w hydrogen peroxide aqueous solution led to a negligible diminution of the virus titer in 5 min (1.2 × 10 6 vs 1.4 × 10 6 ). Such a result seems to be in contradiction with the efficacy data reported in some previous works, where a >4 log 10 abatement was claimed after a contact time of 1 min. 26, 35 In that case, an acidic formulation (pH 3.0) of hydrogen peroxide aqueous solution, specifically designed for the disinfection of hard surfaces, 41 showed an optimal virucidal activity against human coronavirus at concentrations as low as 0.5% w/w. 35 However, such a solution is a patented "accelerated" version of H 2 O 2 in which proprietary additives (stabilizers, complexing agents, orthophosphoric acid derivatives, and pH buffers) are mixed to the active peroxide ingredient. 40, 41 These additional ingredients play a relevant synergistic role in the inactivation of the coronavirus and positively affect the intrinsic virucidal capability of H 2 O 2 itself. 40 Conversely, the results collected here are in line with previous observations, in which hydrogen peroxide solutions (pharmaceutical grade at 3.0% and 6.0% v/v concentrations) showed minimal virucidal activity for very short contact times (15 and 30 s), in particular when they are used as oral rinsing liquids. In that previous literature work, the authors observed a maximum 1.8 log 10 virus titer reduction. 42 With regard to human coronaviruses, then, a very good virucidal activity (>4 log 10 reduction) on surfaces was observed only for longer contact times (2−3 h) by application of H 2 O 2 vapors, therefore, under far more drastic conditions 43 or in the presence of peroxyacetic acid as an additive. 44 The modest virucidal activity of diluted H 2 O 2 was shown on other target viruses too, and very long contact times or higher concentrations are typically used to achieve satisfying virucidal activities. 45−48 Hydrogen peroxide displays in many cases a good biocidal activity, which is attributed to the in situ generation of hydroxyl radicals and other oxidizing oxygenated species reacting with lipids, proteins, and nucleic acids via several parallel mechanisms and hence disrupting the structure and the function of the biological pathogen. 49 Nevertheless, Citric acid was chosen as an additive to lower the pH of the aqueous solution as it is a cheap, biologically compatible, and environmentally friendly acidifying agent. In addition, aqueous solutions of polycarboxylic acids, such as citric, malic, fumaric, malonic, and succinic acids, exhibited a high virucidal activity against enveloped viruses on their own, in the absence of other biocidal agents, 50 and have found practical applications as an additional ingredient in disinfectant solutions. 51, 52 On the other hand, in order to have a basic solution, the sodium carbonate−hydrogen peroxide adduct was chosen as a starting material, since it is a cheap and stable product that contains, at the same time, the active peroxide ingredient and the alkaline modifier. Its use as a biocidal or virucidal agent has been scarcely studied, 53−55 and only three products listed in US EPA's List N contain Na 2 CO 3 ·1.5H 2 O 2 as an active ingredient. 30 Sodium percarbonate can be considered a more robust alternative to diluted hydrogen peroxide, as it can be stored for longer times and transported more easily without any significant loss in active ingredient. It finds applications in dry powder formulations for the oxidative decontamination and abatement of hazardous biological or toxic materials. 56−58 In the second set of tests (Table 2) , the presence of residual amounts of potentially virucidal agents (H 2 O 2 , citric acid, or sodium carbonate) was quenched by adding dosed amounts of catalase, sodium carbonate, or citric acid, respectively. Control tests were performed to confirm that in situ produced sodium citrate (pH 6.8 in the final mixture, after neutralization) did not show any virucidal activity on its own against SARS-CoV-2 or cytotoxic effects on Vero cells.

The initial 1:1 diluted untreated SARS-CoV-2 viral suspension showed a virus titer of 3 × 10 6 pfu mL −1 , with an RNA concentration of 1.5 × 10 11 copies mL −1 ; both virus and RNA values were high enough to appreciate a potential post-treatment decay ( Table 2 ). The initial virus stock contains a virus titer far higher than the one found in biological fluid samples of an average infected patient. 59 The 1:1 mixture of the original virus suspension with the virucidal solution thus represents more drastic conditions than the ones found in real sanitization examples of surfaces contaminated by viruscontaining droplets. In these cases, a large excess of disinfecting liquid is typically used over tiny amounts of deposited body fluids. Contact times in the range 5−15 min were then chosen, as they mimic the standard action time for most peroxide-containing virucidal formulations on objects.

A clear contact time-dependent viral titer reduction was observed for all aqueous solutions. With 3.32% aqueous H 2 O 2 , HP-3, at buffered 7.3 pH value, a gradual 1 log pfu mL −1 diminution was observed every 5 min of exposure to the disinfectant solution. This result is fully in line with the data reported in Table 1 for HP-3. A relatively long contact time (15 min) was thus necessary to obtain a ca. 2.9 log 10 reduction of the viral titer in the absence of additives or accelerating coformulants.

On the contrary, the 3.32% H 2 O 2 solution at pH 2.5, HP-3-C, showed the highest virucidal activity with more than 4.6 log 10 pfu mL −1 reduction after only 5 min and a total virus elimination right after 10 min, so well beyond the threshold limit suggested by WHO for the disinfection of surfaces. 39 This is, to the best of our knowledge, the best virucidal performance recorded for diluted aqueous H 2 O 2 solutions containing simple, nonpatented additives on viruses. The addition of citric acid to the disinfectant solution, not only modifies the pH of the final medium but also enhances the effect of hydrogen peroxide on the biological agent. Such a cooperative effect was previously observed in biocidal solutions, where short-chain carboxylic acids increase the biocidal action of the peroxide on various bacterial strains. 60 The in situ formation of monoperoxocitric acid could be hypothesized, under these conditions, 61 and such peroxoacid could play a key role in the inactivation of this coronavirus. 62 It was thus necessary to ascertain the presence or absence of peroxocitric species, in the final liquid medium. By means of 13 C NMR spectroscopy, no differences were observed in the spectra of aqueous solutions of citric acid in the absence and in the presence of hydrogen peroxide, under conditions as close as possible to those used in virus inactivation tests. In particular, no changes were detected in the signals at 176.7 and 173.4 ppm, attributed to the two types of carboxyl groups of citric acid before and after the treatment with H 2 O 2 (Supporting Information, Figures S1 and S2) . Moreover, the absence of peroxocitric acid was confirmed by differential ceriometric/iodometric titration. Actually, the hydrogen peroxide content revealed by ceriometric method (that titrates H 2 O 2 , but not peroxoacids) was practically the same as the peroxide content measured by iodometric titration (method that is able to detect the presence of both hydrogen peroxide and peroxoacids). 37 In order to establish whether carboxylic acids alone possess a virucidal activity against SARS-CoV-2, the viral suspension was mixed into pure solutions of citric acid (pH 2.5) and acetic acid (pH 3.6) ( Table 3) . Only a minimal diminution in viral titer was recorded (<0.8 log 10 ) in the presence of the acids alone. On the contrary, a remarkable decrease of 3.6 log 10 pfu mL −1 was observed in the presence of the solution HP-3-A, prepared by mixing hydrogen peroxide and acetic acid (in order to have pH 3.6). This virus inactivation value is comparable to the one observed with HP-3-C, with citric acid as an additive (Table 2) . Carboxylic acids are not able to satisfactorily inactivate the virus on their own. Rather, they are able to enhance the virucidal activity of H 2 O 2 by lowering the pH value in the medium. In addition, the in situ formation of active peroxocarboxylic acids can be excluded, under the tested conditions.

In the presence of Na 2 CO 3 ·1.5H 2 O 2 , SP, a gradual diminution of ca. 1 log 10 pfu mL −1 was observed every 5 min of contact time (Table 2) . Nevertheless, the results obtained with SP are remarkable if one considers that the content of peroxide in SP solutions is only 1/6 of the amount present in HP-3-C solutions. In fact, the amount of Na 2 CO 3 · 1.5H 2 O 2 was computed to have 0.51% w/w of H 2 O 2 equivalents in the final liquid mixture vs ca. 3.0% w/w in the HP-3 and HP-3-C aqueous solutions prepared by diluting concentrated H 2 O 2 . This means that the actual inactivation capability of SP is very high, and the goal of >3 log 10 of virus inactivation (in line with the WHO guidelines 39 ) was successfully achieved after 15 min, also in the presence of a relatively less concentrated formulation. Actually, aqueous solutions of SP with ca. 3.0% of H 2 O 2 equivalents cannot be prepared, as they are be beyond the solubility threshold of the solid in water. The good performance of aqueous SP solutions can be attributed not only to the oxidizing properties of H 2 O 2 released by SP when dissolved in water but also to the highly alkaline environment (pH 10.5), caused by the presence of relatively high concentrations of Na 2 CO 3 (0.683 M), which contribute in inactivating the virus by alkali-promoted degradation of its phospholipidic envelope. 63−65 However, the excellent virus inactivation recorded for HP-3-C and SP was not observed in terms of residual RNA concentration: none of them showed a significant viral RNA diminution compared to the initial untreated viral suspension. Such an outcome was not unforeseen, since in several previous examples, H 2 O 2 proved to be effective in virus inactivation, but with a modest or negligible degradation effect on viral RNA. The delicate interaction between the viral particle surface with its cellular receptor may easily be perturbed by a strong oxidant like H 2 O 2 . On the other hand, H 2 O 2 itself has a less marked effect on "bare" nucleic acids, and typically the addition of catalytic amounts of metal centers (such as Fe(II) species, via Fenton-like reactions) is necessary to promote the extensive degradation of these substrates. 66−68 Moreover, the oxidative capability of the tested chemical compounds could have been partially depleted and weakened during the virus inactivation, making it less effective on nucleic acids. It is therefore likely that H 2 O 2 -containing solutions have a detrimental effect on the coronavirus envelope, thus inhibiting the virus' replication capability, without having a strong degradation effect on its genetic material. Therefore, high qRT-PCR values for RNA levels obtained from the liquid media after treatment with HP or SP do not necessarily reflect a scarce inactivation of SARS-CoV-2 virus and a reduction in its ability to infect target cells. Rather, they indicate that a large number of incompletely damaged short RNA sequences are still present in the culture medium after the inactivation process.

Finally, in order to avoid any misinterpretation of cell death due to undesired toxic effects by any of the ingredients of the disinfecting solutions, a control test was performed on a solution where H 2 O 2 was completely disproportionated by catalase, and pH was brought back to a physiological value by the addition of a neutralizing agent. The absence of residual H 2 O 2 and the final pH were confirmed by colorimetric cerium tests and pH spot measurements on the treated liquid solution. Interestingly, no cytotoxic effect was observed in any Vero cell culture treated with the peroxide-free and neutralized solutions. Therefore, in the present study, the cell monolayer disruption is solidly attributable to virus lytic ability rather than to a direct action of products or byproducts contained in the treated solutions.

Diluted aqueous solutions of hydrogen peroxide are often considered a poorly effective means of disinfection and biological decontamination. However, under acidic conditions and thanks to the addition of simple coformulants, H 2 O 2 can be considered an active ingredient for the inactivation of SARS-CoV-2 virus. The copresence of citric acid to obtain an acid pH in the solution (ca. 2.5) and a minimum contact time of 5 min was a good combination to achieve a very good virus depletion with >4 log 10 diminution. Such a result is the best performance recorded so far in the field of virus inactivation with diluted aqueous hydrogen peroxide liquid solutions.

Hydrogen peroxide with acetic acid, as an additive, proved to be an effective virucidal solution too, with a 3.6 log 10 viral titer reduction in 5 min. In none of these cases, however, can the virus inactivation capability be attributed to the in situ formation of peroxocarboxylic acids.

The solid sodium carbonate−hydrogen peroxide adduct showed a less rapid inactivation capability, but in 15 min, a satisfactory reduction of more than 3 orders of magnitude in virus titer was attained.

H 2 O 2 -containing aqueous solutions displayed a limited direct degradation of the nucleic acid of the pathogen, since qRT-PCR target RNA sequences were found after the virucidal treatment. However, thanks to the addition of coformulants acting on the pH of the disinfecting solutions, liquid suspensions of SARS-CoV-2, with a virus concentration that is approximately 3−5 orders of magnitude higher than the one found in real biological fluid samples, were satisfactorily inactivated.

The promising results using acidified aqueous H 2 O 2 diluted solution or with solid sodium percarbonate open the way to easily accessible, cheap, safe, robust, environmentally friendly, and efficient disinfecting agents to be applied in everyday-life sanitization practices and to be used by nonspecialized personnel too. Indeed, the use of diluted hydrogen peroxide modified, for instance, by the addition of common lemon juice, vinegar, or the use of sodium percarbonate for household cleaning purposes, may represent a viable and sustainable approach to obtain reliable virucidal solutions for a very broad range of the world population, also in countries with weaker economies. Further investigations are needed to show the effectiveness of these pH-modified aqueous H 2 O 2 disinfectant formulations against other pathogens. ■ ASSOCIATED CONTENT * sı Supporting Information

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The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission

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Inactivation of severe acute respiratory syndrome coronavirus 2 by WHO-recommended hand rub formulations and alcohols

Stability of SARS-CoV-2 in different environmental conditions

Study on the resistance of severe acute respiratory syndrome-associated coronavirus

Hypochlorous Acid: A Review

Biocides and Novel Antimicrobial Agents for the Mitigation of

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Testing of the World Health Organization-recommended formulations for surgical hand preparation and proposals for increased efficacy

Inactivation of surrogate coronaviruses on hard surfaces by health care germicides

Efficacy of ethanol against viruses in hand disinfection

Hand sanitisers amid CoViD-19: A critical review of alcohol-based products on the market and formulation approaches to respond to increasing demand

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Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents

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Observations on disinfectant performance

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