Poor sanitation is one of the leading causative factors of infectious diseases such as cholera, diarrhea, dysentery, hepatitis A, typhoid, and polio. 1 Being an important facility for sanitation, the purpose of a toilet is to provide a sanitation fixture for the storage or disposal of human wastes, including feces and urine, to improve hygienic conditions. However, the toilet and its immediate environment are recognized to be hot-spots of bio-contamination 2,3 and diverse types of bacteria have been detected in public restrooms. 2, 4 Toilet plume is a major contributor to the transmission of gastroenteric diseases. 5 Flushing of toilets generates numerous fine droplets. The risk of infection of microorganisms in toilets is primarily related to infective doses of pathogens. For certain enteric pathogens, such as norovirus and enterohemorrhagic Escherichia coli (EHEC) in which low doses (less than 50 cells) suffice in infection, the likelihood of transmission is high. 6,7 Depending on the toilet design and other environmental factors such as flushing pressure, a single toilet flushing generates a large number of potentially infectious aerosols ranging between hundreds of thousands and millions. 8, 9 In the literature, a high fecal viral load of >10 9 viral particles copies per g of feces has been detected in patients infected with norovirus. 10, 11 If feces containing pathogenic organisms are shed into the toilet bowl and the toilet is flushed, water will be atomized and copious pathogen-laden aerosol droplets are produced. 2,3, 12, 13 Small droplets would usually become airborne and could travel very long distances while coarse droplets would settle near the toilet bowl. Also, these pathogen-laden droplets would result in two routes of exposure or transmission of infectious airborne pathogens due to toilet flushing, namely inhalation and contact modes. 2,3,5, 14 Through airborne pathway infections occur by direct inhalation of pathogenic airborne droplets. 15, 16 In extant studies, bioaerosols were detected even at a few tens of centimeters above the toilet seat persisting up to an hour after flushing. 2,5, 12 In light of this observation, it is not unreasonable to imply that enteric viruses in the air present a potential risk of infection via inhalation among toilet users. 2, 5, 17 For the contact mode of infection, the fine and coarse pathogen-laden droplets would lead to surface or fomite contamination. A toilet user will inevitably touch various surfaces inside the cubicle. Surface contamination studies have identified microbial contamination of washroom surfaces,
This article is protected by copyright. All rights reserved including doors, toilet seats, sinks, and floors. 2,4,13,18 Contact exposures are no doubt an important risk, as toilet users may become infected whenever they touch surfaces already contaminated by rapidly falling fecal microbes. This source of contamination is also a major public health concern because hand contact with contaminated surfaces can result in self-inoculation through touching of the eyes, nose, or mouth. 13 One matter further complicating the issue is that the microorganisms from the fecal waste may remain on the bowl surfaces even after multiple flushing. Studies have observed that residual microorganisms on the bowl might be aerosolized later. 13,19 A recent field measurement 12 in a hospital also confirmed the collection of airborne microorganisms, even by simply flushing without fecal waste. The observation has important implications for public hygiene control.
Besides, recent studies have found SARS-CoV-2, the causative pathogen of COVID-19 in toilet areas. 20, 21 This evidence suggests that COVID-19 can be potentially generated through toilet flushing.
To reduce the risk of airborne transmission of pathogens due to flushing, the control cannot only be effective for episodic events. The use of mechanical ventilation to remove odor and pollutants is a conventional approach and can be very costly. Recently, some disinfection units, which can be mounted on walls or ceilings are becoming more popular for commercial buildings. Most of them utilize UV for the disinfection of pathogens. More importantly, the devices are always mounted at the washing basins, which means that the "disinfection action" would not take place until the pathogens are well-mixed within the restroom. To date, no studies have reported the quantitative disinfection performance of those devices in field settings.
It is always reasonable and effective to control exposure at the spot of emission if condition permits. That is the concept of localized disinfection. Very recently, the authors have tested the concept of localized disinfection by utilizing low-pressure mercury UVC lamps. It might be the first study that systematically tested the disinfection performance of UVC for toilet-flushing generated pathogens. The results were compared with measurements from a conventional upperroom ultraviolet germicidal irradiation (UR-UVGI) device and higher disinfection efficacy was observed for the localized UV disinfection device. For airborne tests involving E. coli, the disinfection efficacy was 51% for the UR-UVGI device, and 97% for the localized device. As the irradiation level for the UR-UVGI device, when measured at the toilet seat level, was virtually zero, it was not surprising to observe that the surface disinfection efficacy was also very low, approximately 17% while for the localized device, it is about 60%. 22
This article is protected by copyright. All rights reserved Even though the mercury-type localized UVC device has been shown to reduce the bioburden of toilets more significantly, the prototype is bulky and cannot be used in practice. In this study, we utilized Light-emitting diodes (LED) to design a localized disinfection device for the toilet seat.
The use of LEDs to produce UV radiation presents many advantages. LEDs require no warm-up time, less energy to operate, exhibit a very long operational lifespan up to 100,000 hours. 23 Also, LEDs are environmentally friendly as they do not contain toxic materials or pollutants.
Furthermore, another strength of LEDs over lamps is that their disposal treatment is very easy.
Most importantly, they are very compact.
The authors have utilized UVC-LED for upper-room installation. 24 A very high disinfection performance was observed. However, the indoor environment and the toilet bowl have very different emission characteristics. Flushing-generated airborne pathogens are intense, flowing around for a short period (~ seconds) in the vicinity of the toilet, followed by mixing inside the toilet cubicle and entire restroom. From the pollutant control perspective, it is recognized that for very high efficacy, it is better to remove pathogens in the vicinity of the source. However, this approach has not been applied to commercial products. The major challenge of the localized device is how to ensure that sufficient UV dose is absorbed by the pathogens since the emission period during flushing and the exposure time are very short.
This study is the first time to investigate the use of UVC-LED to disinfect the toilet bowl. The results would not only be useful for developed countries but also third world counties because toilet hygiene is still extremely poor in many countries. Therefore, the development of a compact, portable toilet disinfection technology can be of significant benefits to global public hygiene. In this work, we aim to test the efficacy of the novel toilet disinfection system for airborne and settled infectious pathogens; factors affecting the efficacy, i.e. number of LEDs and different bacteria species.
The experimental chamber consists of a custom-made pre-existing toilet rig. The facility was equipped with an American standard wash-down type water closet (WC), one 50-liters volume water tank, and a flushometer. Also, the toilet facility was equipped with a clean water supply.
The UVC-LED has a UVC output of less than 20 mW (www.cisuvc.com) with a rated current of 400 mA. Each LED was soldered on a printed circuit board (PCB) and each PCB was fixed onto a small aluminum plate -12 mm x 18 mm -for mounting on a tailor-made aluminum ring (see
This article is protected by copyright. All rights reserved Figure 1 ). In this study, the ring, fitted with LEDs, was put on the top of the water closet (W.C.)
for disinfection of airborne pathogens due to toilet flushing.
Different configurations of LEDs were designed and tested in this study: (1) Evenly distributed configuration involving 3 LEDs, 5 LEDs, and 8 LEDs (see Figure 2a -c) (2) Concentrated configuration involving two-sided 5-LEDs ( Figure 2d ). The purpose of the first arrangement was to achieve uniform irradiance distribution in the toilet bowl. On the contrary, the second arrangement was designed to mimic a non-uniform irradiance distribution scenario in the toilet bowl.
Depending on the number of the LEDs used for the particular set of experiments, the small aluminum plate (with LED) was mounted to the aluminum ring (see Figure 2a -d). During toilet flushing, water splashing was anticipated, and to prevent the LEDs or the PCB from the ingress of water, a thin transparent film was used to wrap the PCB.
To correlate the UV irradiance level and disinfection performance, the measurement of UV irradiance was required. A UV-VIS fiber-optic spectrometer (AvaSpec-ULS3648) was used to measure the irradiance. Two sets of measurements were taken, one for individual LED for different distance, and the other for different configurations inside the toilet bowl. For individual LED measurement, a UVC-LED irradiance was measured at different distances from the source up to seven centimeters. Five different LEDs were measured in this set.
The disinfection of pathogens depends on the dose absorbed. The latter set of measurment was to measure the total irradiance for each configuration inside the toilet bowl. It is important to measure the total irradiance by different configurations for relative comparison, thus in this regard, the selection of the depth of the sensing probe was arbitrary. The water in the toilet bowl was drained and the probe was put at a preset depth below the seating level in the middle of the bowl for measuring the total irradiance. The setup illustrated in Figure 3 was used to measure the incident irradiance distribution. The actual design of the aluminum ring could allow the mounting of ten LEDs, labeled "A" to "J" as shown in Figure 4 . For instance, for 3 LEDs configuration,
This article is protected by copyright. All rights reserved "A", "E" and "F" LEDs were mounted on the ring and the intensity was measured. The mounting positions of LEDs for other configurations are reported in section 5.2.
The criteria for the selection of micro-organisms include biosafety issues and pathogenic
properties. In this study, three species of pathogenic bacteria were selected including Escherichia 
Before seeding the toilet bowl with bacteria, the toilet bowl and the cistern were thoroughly cleaned with a measure of 100 mL of commercially available Clorox (chlorine) bleach and a toilet brush and then flushed three times, to completely remove residues of the cleaning compound and any micro-organisms present in flushing water. A solution of 12 mL of sodium thiosulphate was then added to inactivate any bleach chemicals present in the water. Finally, water was again used to wash the bowl and cistern in the same manner as previously described. The water used for this study was public utility water and had been filtered to remove suspended solids or microbes. This cleaning process was repeated before each experiment. After thoroughly cleaning the system, the tank was filled with water. During the cleaning process, the air in the chamber was simultaneously disinfected using an upper-room ultraviolet germicidal irradiation system which was installed at the upper part of one of the chamber walls. The UR-UVGI device was turned off when the cleaning of the toilet bowl and cistern was completed. It is worth noting that for safety reasons, appropriate protective kits were worn.
At the end of the cleaning task, the LEDs were fixed to the rim of the W.C. Subsequently, three air sampling components were installed at carefully selected locations to mimic the inhalation of Control experiments were also conducted in the same environmental conditions without exposure to UVC-LED irradiation for equal time points and on the same day as the treatment experiment.
During the experiments, the environmental conditions such as relative humidity and temperature were closely monitored and were kept the same between control and treatment.
Each inactivation experiment was repeated in triplicate. Each data bar represents the arithmetic mean of the three replicates and the standard deviation of the three trials was used as the error bar.
The disinfection efficiency ( ) was estimated using equation (1).  Figure 7 shows the emission spectrum of UV light produced by a typical LED. The spectrum was measured at 8 V and a distance of 1 cm. The LED exhibited a peak emission wavelength at 269.60 nm with a corresponding intensity of 98.63 Wcm -2 and full widths at half maximum (FWHM) spectral bandwidth (SBW) of 12.14 nm. Figure 8 shows the averaged irradiance and standard deviations of the sum of 5 individual LED versus distance starting from the LEDs. The UVC-LED irradiance was measured from the source up to seven centimeters, to estimate the effect of distance on irradiance changes, specifically for future modeling and estimating UV dose for the prototype unit. The mean LED irradiance varied from 98.63±14.72 to 0 µWcm -2 when the distance increased within 1 to 7 cm. High uncertainly was found for the measurements nearest to the UV source. At the location with such a high intensity, even a very small deviation may cause a large difference in the reading. Also, it is observed that the intensity drops very rapidly and reaches close to zero when it is just 4 cm away from the LED.
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The decrease in irradiance with such a small distance could have implications for system designs.
It suggests a potential scenario. It could indicate that the bacteria disinfection reported in this study occurred at a short emission distance within the locality of the UVC-LEDs, that is, almost immediately the flushing was activated.
The results for the total irradiance measured inside the toilet bowl for different LED configurations are reported in Table 1 . The total UV irradiance increases with the numbers of the LEDs tested. For the 3-LEDs, 5-LEDs, and 8-LEDs well-distributed configurations, the measured irradiances were 0.86, 1.15, and 3.07 μWcm -2 , respectively. Likewise, the irradiance of the 5-LEDs two-sided non-distributed configuration was 1.57 μWcm -2 . Due to the arrangement of the LEDs, it can be observed that the total irradiance was not linearly proportional to the number of LEDs used. Besides, the two-sided 5-LEDs gave a higher irradiance than the well-distributed 5-LEDs.
The assessment of the disinfection efficiency on the surface of a toilet seat for the different LED configurations and the bacteria under evaluation is shown collectively in Figure 9 . The estimated mean efficiencies (mean ± SD) with 3-LEDs, 5 
This article is protected by copyright. All rights reserved plateau and, in this work, perhaps 5-LEDs are adequate for achieving the same performance.
Secondly, an avoidable higher energy cost may be incurred by using 8-LEDs instead.
The numbers of CFU at different levels (i.e. ASH, ASM, and ASL) were counted and summed up.
The results of the efficacy of airborne disinfection by the localized UVC-LED system are shown in Figure 10 . respectively. These results appear to be in almost the same order of efficiency as with the mercury vapor UV lamps reported in our previous study 22 without having any of the problems associated with this UV source (e.g., toxicity, fragility, high energy consumption, etc.).
In Figure 10 , disinfection efficacy increased with the number of LEDs used as clearly shown.
However, a distinct point is observed. It seems that the intensity did not affect S. epidermidis much, nonetheless, E. Coli was affected significantly. In the case of S. epidermidis, the cell structure is thick, resilient, and counteracts, thereby making its inactivation more difficult compared to other bacteria tested. This is, nevertheless, not unexpected and consistent with the observed behavior in the literature related to other disinfection approaches such as mercury vapor UV lamps 22 and ionizers. 25 Similarly, to further support this claim, it is worth noting that all published values of UVGI rate constants for all of the three test organisms have also indicated that the susceptibilities of E. Coli and S. typhimurium are considerably higher than those for S. epidermidis for air medium 27 . To further indirectly confirm the accuracy of the present results, the order of UVC susceptibility of these bacteria, as observed in our current study, is the same on both surfaces and in air disinfection: Higher for E. Coli, followed by S. typhimurium, and then S.
It is also worth noting that higher LED configuration, specifically 10-LEDs, was tried but no significant increase in the irradiance and disinfection efficacy was observed (data not shown).
Therefore, the 8-LEDs configuration is considered optimum for the current airborne disinfection application.
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Here, we further tested the influence of UVC-LEDs configurations on the efficacy of the localized disinfection system. We compared the germicidal results of two cases of UVC-LED configuration, both of which had 5-LEDs but one was evenly distributed and the other was concentrated at two opposing sides, against E. coli -being the most susceptible to UVC-LED among the three tested bacteria.
With the evenly distributed and concentrated (two-sided) irradiation, the mean disinfection efficiencies for airborne disinfection were 63.25±8.17% and 53.74±4.47%, respectively. Similarly, the estimated mean efficiency under surface disinfection was 72.03±9.02% for the evenly distributed irradiation and 36.83±7.47% for the concentrated (two-sided) irradiation ( Figure 11 ).
The results implied that the performance of the localized disinfection system with the evenly distributed irradiation was about 95.57% (surface) and 17.70% (airborne) higher than the concentrated (two-sided) irradiation.
An independent sample t-test (two-tail, α  0.05) was conducted and the result showed that the difference between the efficacy of the evenly distributed and the concentrated (two-sided) UVC-LED arrangements is statistically significant (p  .006) for surface disinfection. However, there is no statistically significant difference between the performance of these two cases for airborne
It is interesting to note that even though the numbers of the LEDs used were the same (the same irradiation output), the measured intensity was different. For evenly distributed pattern, the intensity was 1.15 µWcm -2 while for the 2-sided pattern, it was 1.57 µWcm -2 as noted earlier in section 5.2. Although the intensity was higher for the 2-sided configuration, the disinfection efficacy was lower. The rationale for this is that the aerosolization process occurs randomly in the bowl surface, hence the uniform intensity might give better results unless the design of the bowl is special to spread the water nozzles unevenly around the rim of the bowl.
Toilets are potential sources for the transmission of fecal-borne diseases, being unique locations in buildings for defecation and urination. The size of flushing-generated pathogens influences their role in airborne and contact transmissions. The total and size-resolved airborne droplet emissions per flush under different flushing conditions had been reported in our previously study 17 . In that study, we showed statistically significant differences in the generation of different sizes airborne droplets, with the average airborne concentrations decreasing as the particle size increased.
This article is protected by copyright. All rights reserved Inferring from the results, the ratio of droplet concentration of 0.3 to 0.6 m to > 2-10 m can be as high as 124 times. These findings further provide justification for concerns that flushing toilets could play a role in both airborne and surface transmission of infectious disease through droplet bioaerosols. Once aerosols become airborne, they can settle near the toilet bowl, and the small aerosols can stay airborne for up to hours and may lead to surface contamination, which is believed to be a major route for transmission of infective diseases. Moreover, fecal bacteria get preserved in suspended droplet capsules, where they remain infectious and have greater potential to infect toilet users. Although this dynamic and complex phenomenon is also possible in toilet environments with saturated humidity, albeit slower than in dry environments, it is important to also note that some of the flushing-generated droplets would maintain their water content, be denser, and less likely to stay airborne, increasing the risk of surface transmission.
Since air-and surface-mediated transmissions are common for toilet acquired infections, good infection control methods must attempt to keep toilet microenvironments free from pathogens. In this regards, control of toilet infections must, therefore, involve the disinfection of both air and surfaces in the toilet microenvironment. Also, it is crucial to acknowledge here that fecal pathogens can persist and survive from hours to weeks on inanimate surfaces including the toilet seat. 29,30
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To reduce infection risk, engineering control strategies should be implemented. A conventional approach for reducing infection risk for toilets is through dilution of pathogen concentration. It can be realized by increasing ventilation rate, however, this approach is not sustainable. Another potential issue of increasing ventilation rate is that enhancing mixing inside toilets can increase the spatial spread of the airborne pathogens, which may lead to long range transport of pathogens induced by toilet flushing. Besides dilution, another active control strategy is inactivation.
It is worth noting that the inactivation of airborne indoor pathogens has generally been limited to the use of UR-UVGI systems. Although the UR-UVGI has been proven to be an excellent intervention technology for airborne infection, the use of such an approach is limited to the upper room, due to exposure concerns when used in real-time and does not perform source control function for reducing whole room dispersal of pathogens generated from toilet flushing. 22
In this study, we addressed these issues through the use of a novel localized UVC-LED 
This article is protected by copyright. All rights reserved necessary to consider the disinfection efficacy not only for those evaluated in this study but also for other bacteria that are transmitted through contaminated surfaces and air and become a burden in toilet infections such as shigella sp. Listeria monocytogenes, Clostridium difficile, Candida, and Cryptosporidium, et cetera.
This study is one of the first to demonstrate an intervention technology for inactivating enteropathogenic bacteria. To conclude, this study proposed an innovative design of utilizing very low levels of UVC-LED irradiance to achieve a significant reduction in the concentration levels of deposited and airborne pathogens in the laboratory environment and provides an alternative intervention strategy for air and surface disinfection in high-risk microenvironments such as toilets facilities. It was shown that the potential of LEDs to remove bacteria and prevent dispersal into the whole room during toilet flushing depends on the quantity of UVC-LED irradiance in the toilet bowl. It was observed that for higher UV irradiance, the disinfection efficiency increased, although in a non-linearly manner as expected. While disinfection performance increases with the numbers of LEDs, the plateau stage seems to occur consistently. Thus, it is necessary to test the irradiance intensity output before fixing the device current to avoid wasting energy.
One of the interesting features of LEDs is that they are environmentally friendly. This property confers a significant advantage of this source control approach described in our study to other traditional approaches using chemical disinfectants (e.g., chlorine), which have been linked to significant environmental implications. Moreover, other unique characteristics, which give this hazard-free novel method advantage over competitive disinfection technologies include a longer lifetime, very low-cost requirement (i.e., they are very cheap), and very low energy consumption.
Finally, we believe that the point-source nature of the localized UVC-LED emission paves the way toward the instantaneous removal of deadly fecal pathogens during toilet flushing before spreading.
A novel UVC-LED disinfection system was designed, and its disinfection efficacy was tested. We presented the concept of a source control method using UVC-LEDs to inactivate toilet-flushinggenerated bacteria thereby hampering their spread onto surfaces and in the air within the toilet space. The design was very compact and has the potential to make it portable.
The LED-based technology for the inactivation of fecal bacteria demonstrated promising results. 