effective and environmentally friendly disinfection

Despite its rather ancient origins, the germ theory of disease, which states that many diseases are the result of the activity of various microorganisms, has undergone many changes in the course of human history. At one time (relatively recently, in fact), it was considered nonsense, and many scientists rejected it. However, now we know very well that this theory is true and should be listened to. That is why various disinfectants have appeared, aimed at combating bacteria, viruses and fungi. One of the most common of these is chloroxylenol. It is effective, but extremely environmentally unfriendly due to its strong chemical stability and mass consumption. Scientists from the Hong Kong University of Science and Technology conducted research, during which they managed to find an even more effective disinfectant, which does not harm the environment. What does this miracle product consist of, what are its properties, and will it be able to replace the chloroxylenol that dominates the market? We will find the answers to these questions in the scientists' report.

Research basis

Naturally, the adoption of the germ theory of disease has radically changed the human approach to, roughly speaking, cleanliness, especially within the walls of medical institutions. Among recent events, the COVID-19 pandemic has become another impetus for rethinking and even tightening personal and environmental disinfection measures to prevent the transmission of diseases. For example, according to Reckitt Benckiser (a manufacturer of household chemicals), sales of disinfectants have increased by almost 50% compared to pre-pandemic figures, amounting to $5.45 billion per year.

Since chemical disinfectants can enter the natural aquatic environment through wastewater and surface runoff, there are concerns that intensive disinfection may cause environmental problems in aquatic ecosystems. Therefore, environmentally friendly yet equally effective disinfectants are urgently needed.

Chloroxylenol, also known as para-chloro-meta-xylenol (PCMX from para-chloro-meta-xylenol), is a halogenated phenolic disinfectant. As with phenol, the mechanisms of phenolic disinfectants to inactivate pathogens include disruption of the cell membrane and disruption of microbial enzyme systems, while halogenated phenol derivatives generally have better antimicrobial activity than phenol. Since its introduction in the 1920s, PCMX has been widely used as a broad-spectrum antimicrobial agent. The use of PCMX has further increased in recent years. PCMX was found in 16.9% of antiseptic detergents in the US alone, 20.7% of household cleaners in the UK, and 56.3% of household disinfectants and 33.9% of hand sanitizers in China.

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Due to its widespread use and relatively high chemical stability, PCMX has been frequently detected in aquatic environments, such as 0.1–1.2 μg/L in Indonesian river water, 0.2–10.6 μg/L in Hong Kong river water, 1.62–9.57 μg/L in mainland China river water, and 0.06–0.79 μg/L in Kuwaiti seawater. Although PCMX is generally considered safe for humans, the United States Environmental Protection Agency (USEPA) has listed PCMX as moderately toxic to aquatic invertebrates and highly toxic to freshwater fish. Toxicology studies have reported adverse effects of PCMX on aquatic organisms, including endocrine disruption, embryonic mortality, and malformations. Chronic exposure to PCMX at environmental concentrations (~4.2 µg/L) can induce gene regulation and morphological changes in rainbow trout. Recently, ~120 halogenated disinfection by-products (DBPs) have been detected and identified disinfection by product) in disinfected drinking water and wastewater. Scientists have noticed that most of the newly identified DBPs are halophenolic compounds that are structurally similar to PCMX, such as halophenols, halonitrophenols, halohydroquinones, halohydroxybenzaldehydes, halohydroxybenzoic acids, and halosalicylic acids (Figure 1).

Research groups have also identified some novel halophenolic DBPs, including halobenzenetriols and halohydroxybenzonitriles. The structural properties of halophenolic DBPs appear to provide their antimicrobial activity as disinfectants. Furthermore, transformation studies of the newly identified DBPs have shown that some halophenolic DBPs can be detoxified by solar photolysis through dehalogenation and further ring cleavage to aliphatic compounds in seawater. This implies that the use of halophenolic DBPs as disinfectants may mitigate the environmental problems caused by disinfectants. However, the efficacy of halophenolic DBPs in inactivating pathogens, the degradability of halophenolic DBPs in aquatic environments without sunlight, and the associated toxicity variation remain unknown.

In the paper reviewed today, the scientists examined the efficacy of various DBPs against common pathogens in comparison to PCMX. By coupling disinfection efficacy with degradation and detoxification kinetics in seawater, a potential disinfectant was selected from these DBPs. The scientists found that the selected DBP demonstrated significantly higher antimicrobial efficacy than PCMX, and that its concentration and associated toxicity in the presence of seawater decreased rapidly, even in the dark. The results of the study demonstrate the potential of this DBP as an effective broad-spectrum disinfectant that is rapidly degraded and detoxified in seawater.

Research results

Antimicrobial properties of DBP

Scientists initially screened halophenolic DBPs for potential disinfectants primarily based on their acute toxicity and degradability. Nitrogenous DBPs such as halonitrophenols and halohydroxybenzonitriles were excluded because they tend to have higher toxicity and greater stability than carbonaceous DBPs. Among the newly identified halophenolic DBPs, 5-bromosalicylic acid and 2,5-dibromohydroquinone showed the shortest half-lives under solar irradiation. 2,4-dihalophenols showed relatively high photodegradation rate constants and low toxicity among the halophenols. Recent advances in DBP research have shown that iodinated DBPs are generally more toxic but less stable than their brominated and chlorinated counterparts. Therefore, chlorinated, brominated, and iodinated species of three DBP groups (i.e., 2,4-dihalophenols, 2,5-dihalohydroquinones, and 5-halosalicylic acids) were included.

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First, the scientists studied the antimicrobial activity of the selected DBP and PCMX against Escherichia coli (gram-negative bacteria), which is a commonly accepted indicator of pathogenic contamination and has been identified as a driver of colorectal cancer. Following the USEPA recommended time range of 0.25–10 minutes for surface disinfection, a contact time of 5 minutes was selected. Disinfection efficacy against E. coli was in descending order: 2,4-dihalophenols > 2,5-dihalohydroquinones > 5-halosalicylic acids (pH 7.2; 2a).

Survival curves E. coli followed the delayed Chick-Watson law, with 2,4-diiodophenol (0.79 L/mg/h) having a higher inactivation rate constant than PCMX (0.66 L/mg/h), which in turn had a higher inactivation rate constant than the other DBPs.

Considering that disinfectants are usually diluted with ordinary household water (pH 6.5–8.5) before use, inactivation E. Coli DBP was studied under different pH conditions. With increasing pH, the disinfection efficiency of 2,4-dihalophenols remained the same, while that of 2,5-dihalohydroquinones increased significantly by 1.8–3.2 times (2b). Since 5-halosalicylic acids have been found to be ineffective in inactivating E. coli at the tested concentrations and pH values ​​(2a, 2b), they were excluded from further testing. To test the broad-spectrum antimicrobial potential of DBPs, the scientists examined their effectiveness in inactivating Staphylococcus aureus (gram-positive bacteria), Candida albicans (fungi) and bacteriophage MS2 (viruses) at pH 7.2 and a contact time of 5 minutes. Ranking order of the effectiveness of PCMX and DBP in inactivating S. aureus, C. albicans or MS2 was basically the same as in inactivation E. coliwhile 2,4-diiodophenol showed the greatest antimicrobial effect at the same dose (2c).

DBP Toxicity

Seawater is the immediate or final destination for municipal wastewater and urban runoff. Municipal wastewater has been damaging marine ecosystems for decades. Platynereis dumerilii is a ubiquitous marine polychaete in coastal waters where it feeds on seaweeds and plays a vital role at the base of the food pyramid in marine ecosystems. It has been widely used as a model organism to assess marine ecotoxicity. To assess the risk of using DBP as disinfectants for embryonic development P. dumeriliithe scientists looked at both the use case and toxicity of PCMX and DBP. Taking the dose for a 3-log (99.9%) reduction E. coli As the concentration used, scientists defined the dilution factor (DF from dilution factor) this concentration at which 50% of embryos developed normally compared to the control sample (EC_50,DF; 2d).

Disinfectant with higher EC_50,DF requires a higher dilution factor to attenuate its toxicity, indicating a higher risk. EC_50,DF was in descending order 2,5-dihalohydroquinones > 2,4-dihalophenols > PCMX. In the presence of seawater, PCMX and DBP degraded according to pseudo-first-order kinetics upon exposure to sunlight or in the dark.

Solar photolysis of 2,4-dihalophenols (half-life 35–83 hours; 2e) was faster than PCMX (half-life 257 hours). In the absence of sunlight, both 2,4-dihalophenols and PCMX degraded slightly (less than 5% in 120 hours; 2f). 2,4-Dihalophenols showed 1.9–2.1 times higher EC_50,DF and 3.1–7.3 times greater solar decay constant than PCMX, suggesting that 2,4-dihalophenols may be potential disinfectants.

Considering that the UV component of sunlight can only penetrate into the uppermost meters of seawater and that the degradation of 2,4-dihalophenols occurs mainly through solar photolysis in the surface fraction of seawater during sunny daytime, 2,4-dihalophenols are still not ideal enough for use as disinfectants. In comparison, 2,5-dihalohydroquinones were degraded extremely rapidly under light and dark conditions (2e, 2f) with half-lives of 0.12–0.16 hours. Due to rapid degradation, EC values_50,DF The chlorinated, brominated and iodinated 2,5-dihalohydroquinones were calculated to be lower than those of PCMX after 0.92, 0.94 and 0.71 hours, respectively, in seawater in the dark. However, the extreme instability in water (pH 6.2–8.2) and high concentrations when used in disinfection made 2,5-dihalohydroquinones unsuitable as disinfectants.

However, scientists note that with increasing pH, the disinfection efficiency of 2,5-dihalohydroquinones increased sharply (2b). Hydroquinone is susceptible to oxidation in alkaline solutions. This led scientists to hypothesize that dihalobenzoquinones (oxidation products of dihalohydroquinones) may be more effective in inactivating pathogens.

Antimicrobial properties of 2,6-dichlorobenzoquinone (2,6-DCQ)

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To test this hypothesis, the scientists examined the antimicrobial properties of 2,6-dichlorobenzoquinone (2,6-DCQ), which is also a newly identified DBP and was found to be slightly more effective in inactivating E. colithan its isomer 2,5-DCQ. 2,6-DCQ demonstrated significantly higher disinfection efficacy against all four pathogens tested than PCMX (3a). The inactivation rate constants of 2,6-DCQ and PCMX were determined against E. coli, S. aureus, C. albicans And MS2. With a fixed contact time of 5 minutes to achieve 3-log inactivation E. coli, S. Aureus, C. albicans And MS2 (3a) 2,6-DCQ required doses of 12.8, 28.6, 35.4, and 46.1 mg/L, respectively. The corresponding doses required for PCMX were 188, 308, 320, and 1010 mg/L, which are 9.0–22 times higher than the doses required for 2,6-DCQ.

To gain insight into the mechanism of 2,6-DCQ disinfection, the scientists stained E. Coli propidium iodide, a membrane-impermeable dye, and analyzed by fluorescence microscopy. Disinfection with 2,6-DCQ resulted in a significant amount of E. colistained with propidium iodide, indicating that the cells E. coli lost membrane integrity after exposure to 2,6-DCQ. Higher fluorescence intensity indicates a higher number of cells with damaged membrane. The increase in fluorescence intensity (3.5–59.2 times compared to the control sample) with increasing 2,6-DCQ dose indicates that cell membrane disruption plays an important role in disinfection by 2,6-DCQ.

Characteristics of 2,6-DCQ degradation and detoxification in seawater

To address the potential environmental concerns associated with the use of the proposed disinfectant, the scientists studied the degradation of 2,6-DCQ in seawater (pH 8.2). It followed pseudo-first-order kinetics (3b) with and without solar irradiation. Interestingly, the degradation rate of 2,6-DCQ in the dark was comparable to that in sunlight. In seawater, the half-life of 2,6-DCQ under sunlight (1.33 hours) was 193 times shorter than that of PCMX (257 hours), and the half-life of 2,6-DCQ in the dark (1.74 hours) was 3000 times shorter than that of PCMX (~240 days).

To characterize the rapid degradation of 2,6-DCQ in seawater, the scientists next investigated the effect of pH on the degradation kinetics of 2,6-DCQ (3b). When seawater was adjusted to pH 6.2 and 7.2, the half-lives of 2,6-DCQ under solar radiation were 4.13 and 3.20 hours, respectively; the half-lives of 2,6-DCQ in the dark were 76.2 and 13.4 hours, respectively.

The results show that sunlight is effective in promoting the degradation of 2,6-DCQ, with the effect being more pronounced at lower pH. The enhanced degradation by sunlight may be due to photonucleophilic substitution and reactions with generated reactive oxygen species (e.g., hydroxyl radicals). In addition, the degradation of 2,6-DCQ was dramatically accelerated with increasing pH, indicating an important role of hydrolysis in the degradation of 2,6-DCQ, especially in the dark.

The scientists also investigated the effect of high chloride levels in seawater on the degradation of 2,6-DCQ in the dark. The results showed that chloride ions had no significant effect on the degradation of 2,6-DCQ. The rapid degradation of 2,6-DCQ in seawater was attributed to the slightly alkaline nature (pH 8.2) of seawater, where the hydroxide concentration is 15.8 times higher than at pH 7.0, and enhanced hydrolysis of 2,6-DCQ can occur.

The scientists then investigated the degradation pathway of 2,6-DCQ in seawater in the dark. Using high-performance liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry (UPLC/ESI-tqMS from ultra-performance liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry), the scientists detected three degradation products of 2,6-DCQ in seawater in the dark. The most significant degradation product, 3-hydroxyl-2,6-dichloro-1,4-benzoquinone (OH-DCQ), was synthesized and the purity of the synthesized compound was checked by UPLC/ESI-tqMS. With the rapid degradation of 2,6-DCQ (3c) OH-DCQ was rapidly formed via hydrolysis of 2,6-DCQ. The concentration of OH-DCQ reached its maximum after 5.5 h, corresponding to 41% of the initial molar concentration of 2,6-DCQ, and then remained stable. The molecular ion was also rapidly formed and was proposed to be a dimeric product of OH-DCQ related to the parent compound (3d). The coupling product can undergo hydrolysis to form 3-hydroxyl-6-monochloro-1,4-benzoquinone and OH-DCQ, and thus its peak area is maximized with a relatively short degradation time (3.75 h). The hydrolysis of the coupling product is confirmed by the stable peak area of ​​OH-DCQ after depletion of the parent compound.

Considering the doses required for 3-log inactivation E. coliscientists assessed the comparative risks of 2,6-DCQ and PCMX for embryo development P. Dumerilii (3e). At the point of release into seawater (with degradation time set at 0 hours), 2,6-DCQ showed slightly higher toxicity than PCMX. However, when 2,6-DCQ was rapidly degraded in seawater in the dark, the toxicity of 2,6-DCQ (essentially a mixture of 2,6-DCQ and its degradation products) decreased dramatically, with an EC value of_50,DF decreased from 21.7 to 0.28 in 48 hours. The scientists also assessed the toxicity of OH-DCQ, the main degradation product of 2,6-DCQ. Toxicity of 2,6-DCQ to Embryos P. dumerilii was 149 times higher than that of OH-DCQ. This indicates that the degradation of 2,6-DCQ in seawater is a rapid detoxification process. In contrast, the toxicity of PCMX remained unchanged from 0 to 48 h, consistent with the negligible degradation of PCMX in the dark. In particular, after 2 h of degradation, 2,6-DCQ and PCMX were comparable in toxicity with an EC_50,DF 8.48 and 8.58 respectively. As the degradation time increases, the EC value_50,DF 2,6-DCQ continued to decline and reached only 3.0% of EC_50,DF PCMX after 48 hours. In other words, 2,6-DCQ was 31 times less toxic than PCMX after 48 hours of degradation in the dark.

For a more detailed look at the nuances of the study, I recommend taking a look at scientists' report And additional materials to him.

Epilogue

In the work we have reviewed today, scientists have tried to find an alternative to the highly effective, but very environmentally unfriendly disinfectant PCMX (chloroxylenol). This substance has shown high efficiency in the fight against various bacteria, viruses and fungi, but its strong chemical stability and mass use cause a lot of environmental problems within the marine ecosystem. The fact is that PCMX is an extremely toxic and tenacious compound that negatively affects the development of many living organisms in the marine environment, among which the marine polychaete is indicative

Platynereis dumerilii

which plays an important role at the base of the trophic pyramid in marine ecosystems.

The disinfectant developed by the scientists (2,6-DCQ or 2,6-dichlorobenzoquinone) is a by-product of PCMX, but its effectiveness in combating microbes is much higher, as is its rate of decomposition, and its toxicity is much lower. An important role in such positive characteristics of 2,6-DCQ is played by the slightly alkaline nature of seawater, which reduces toxicity and accelerates decomposition.

According to the authors of the development, their work is not only an important milestone on the path to environmentally friendly and highly effective disinfectants, but also a striking example of the fact that the creation of environmentally friendly industrial products is not only potentially possible, but also quite real. For example, pesticides, pharmaceuticals and personal hygiene products can be developed taking into account the knowledge obtained in this study, as a result of which their new versions will not lose their effectiveness, but will become much less harmful to the environment.

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