Legionella is the causative agent of Legionnaires\' disease, and its prevalence in potable water is a significant public health issue. Water stagnation within buildings increases the risk of Legionella. However, there are limited studies investigating how stagnation arising through intermittent usage affects Legionella proliferation and the studies that are available do not consider viable but non culturable (VBNC) Legionella. This study used a model plumbing system to examine how intermittent water stagnation affects both VBNC and culturable Legionella. The model plumbing system contained a water tank supplying two biofilm reactors. The model was initially left stagnant for ≈5 months (147 days), after which one reactor was flushed daily, and the other weekly. Biofilm coupons, and water samples were collected for analysis at days 0, 14 and 28. These samples were analysed for culturable and VBNC Legionella, free-living amoebae, and heterotrophic bacteria. After 28 days, once-a-day flushing significantly (p < 0.001) reduced the amount of biofilm-associated culturable Legionella (1.5 log10 reduction) compared with weekly flushing. However, higher counts of biofilm-associated VBNC Legionella (1 log10 higher) were recovered from the reactor with once-a-day flushing compared with weekly flushing. Likewise, once-a-day flushing increased the population of biofilm-associated Vermamoeba vermiformis (approximately 3 log10 higher) compared with weekly flushing, which indicated a positive relationship between VBNC Legionella and V. vermiformis. This is the first study to investigate the influence of stagnation on VBNC Legionella under environmental conditions. Overall, this study showed that a reduction in water stagnation decreased culturable Legionella but not VBNC Legionella.

In recent years, the frequency of nosocomial infections has increased. Hospital water systems support the growth of microbes, especially opportunistic premise plumbing pathogens. In this study, planktonic prokaryotic communities present in water samples taken from hospital showers and hand basins, collected over three different sampling phases, were characterized by 16S rRNA gene amplicon sequencing. Significant differences in the abundance of various prokaryotic taxa were found through univariate and multivariate analysis. Overall, the prokaryotic communities of hospital water were taxonomically diverse and dominated by biofilm forming, corrosion causing, and potentially pathogenic bacteria. The phyla Proteobacteria, Actinobacteriota, Bacteroidota, Planctomycetota, Firmicutes, and Cyanobacteria made up 96% of the relative abundance. The α-diversity measurements of prokaryotic communities showed no difference in taxa evenness and richness based on sampling sites (shower or hand basins), sampling phases (months), and presence or absence of Vermamoeba vermiformis. However, β-diversity measurements showed significant clustering of prokaryotic communities based on sampling phases, with the greatest difference observed between the samples collected in phase 1 vs phase 2/3. Importantly, significant difference was observed in prokaryotic communities based on flow dynamics of the incoming water. The Pielou\'s evenness diversity index revealed a significant difference (Kruskal Wallis, p < 0.05) and showed higher species richness in low flow regime (< 13 minutes water flushing per week and ≤ 765 flushing events per six months). Similarly, Bray-Curtis dissimilarity index found significant differences (PERMANOVA, p < 0.05) in the prokaryotic communities of low vs medium/high flow regimes. Furthermore, linear discriminant analysis effect size showed that several biofilm forming (e.g., Pseudomonadales), corrosion causing (e.g., Desulfobacterales), extremely environmental stress resistant (e.g., Deinococcales), and potentially pathogenic (e.g., Pseudomonas) bacterial taxa were in higher amounts under low flow regime conditions. This study demonstrated that a hospital building water system consists of a complex microbiome that is shaped by incoming water quality and the building flow dynamics arising through usage.

Secondary water supply systems (SWSSs) are widely used to supply water to high-rise households in urban residential buildings. A special mode of double tanks with one used while another was spared was noted in SWSSs, which would facilitate microbial growth due to longer water stagnation in the spare tank. There are limited studies on the microbial risk of water samples in such SWSSs. In this study, the input water valves of the operational SWSSs consisting of double tanks were artificially closed and opened on time. Propidium monoazide-qPCR and high-throughput sequencing were performed to systematically investigate the microbial risks in water samples. After closing the tank input water valve, it may take several weeks to replace the bulk water in the spare tank. The residual chlorine concentration in the spare tank decreased by up to 85 % within 2-3 days compared with that in the input water. The microbial communities in the spare and used tank water samples clustered separately. High bacterial 16S rRNA gene abundance and pathogens-like sequences were detected in the spare tanks. Most antibiotic-resistant genes (11/15) in the spare tanks showed an increase in their relative abundance. Moreover, when both tanks within one SWSS were in use, the water quality of the used tank water samples deteriorated to varying degrees. Overall, running SWSSs with double tanks will reduce the replacement rate of water in one storage tank, and consumers who use taps served by the presented SWSSs may have a higher microbial risk.

Water quality deterioration often occurs in secondary water supply systems (SWSSs), and increased heavy metal concentrations can be a serious problem. In this survey, twelve residential neighborhoods were selected to investigate the influence of SWSSs on the seasonal changes in heavy metal concentrations from input water to tank and tap water. The concentrations of nine evaluated heavy metals in all groups of water samples were found to be far below the specified standard levels in China. The concentrations of Fe, Mn, and Zn increased significantly from the input water samples to the tank and tap water samples in spring and summer (p < 0.05), especially for the water samples that had been stagnant for a long time. Negative correlations were found between most of the heavy metals and residual chlorine (Fe, Cu, Zn, and As, r = -0.186 to -0.519, p < 0.05). In particular, a high negative correlation was observed between Fe and residual chlorine (r = -0.489 to -0.519, p < 0.01) in spring and summer. Fe and Mn displayed positive correlations with turbidity (r = 0.672 and 0.328, respectively; p < 0.05). In addition, Cr and As were found to be positively associated with some nutrients (NO3-, TN, and SO42-; r = 0.420-0.786, p < 0.01). The material of the storage tanks had little influence on the difference in heavy metal concentrations. Overall, this survey illustrated that SWSSs may pose a chronic threat to water quality and could provide useful information for practitioners.

In premise plumbing, microbial water quality may deteriorate under certain conditions, such as stagnation. Stagnation results in a loss of disinfectant residual, which may lead to the regrowth of microorganisms, including opportunistic pathogens. In the present study, microbial regrowth was investigated at eight faucets in a building over four seasons in one year. Water samples were obtained before and after 24 h of stagnation. In the first 100‍ ‍mL after stagnation, total cell counts measured by flow cytometry increased 14- to 220-fold with a simultaneous decrease in free chlorine from 0.17-0.36‍ ‍mg L-1 to <0.02‍ ‍mg L-1. After stagnation, total cell counts were not significantly different among seasons; however, the composition of the microbial community varied seasonally. The relative abundance of Pseudomonas spp. was dominant in winter, whereas Sphingomonas spp. were dominant in most faucets after stagnation in other seasons. Opportunistic pathogens, such as Legionella pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, and Acanthamoeba spp., were below the quantification limit for real-time quantitative PCR in all samples. However, sequences related to other opportunistic pathogens, including L. feeleii, L. maceachernii, L. micdadei, M. paragordonae, M. gordonae, and M. haemophilum, were detected. These results indicate that health risks may increase after stagnation due to the regrowth of opportunistic pathogens.

Secondary water supply systems (SWSSs) are characterized by long water stagnation and low levels of chlorine residuals, which may pose a high microbial risk to terminal users. In this study, the SWSSs of 12 residential neighborhoods in a metropolitan area of 5 million people in southeastern China were seasonally investigated to assess their microbial risks by determining more than 30 physicochemical and biological parameters. Although the microbiological quality of SWSS water met the requirements of the standards for drinking water quality of China, it did deteriorate in various aspects. The heterotrophic plate counts with R2A media were high (> 100 CFU/mL) in some SWSS tank and tap water samples. Propidium monoazide (PMA)-qPCR revealed a one magnitude higher abundance of viable bacteria in the tank and tap water samples (average 103.63±1.10 and 103.65±1.25 gene copies/mL, respectively) compared with the input water samples, and Enterococcus, Acanthamoeba, and Hartmannella vermiformis were only detected in the tanks. In particular, the high detection frequency of Legionella in 35% tank and 21% tap water samples suggested it is a supplementary microbial safety indicator in SWSSs. The microbial regrowth potential was more obvious in summer, and Illumina sequencing also demonstrated distinct seasonal changes in the relative abundance of bacterial gene sequences at the genus level. Turbidity and residual chlorine were closely connected with total bacterial biomass, and the latter seemed responsible for microbial community structure alteration. The extremely low chlorine residuals associated with a high abundance of total bacteria (as high as 106.48 gene copies/mL) and Legionella (as high as 106.71 gene copies/100 mL) in the closed valve tanks highlighted the high microbial risk increased by mishandling the operation of SWSSs. This study found that SWSSs possessed a higher microbial risk than the drinking water network, which suggested that the frequency and scope of monitoring the microbial risk of SWSSs in megacities should be strengthened for the purpose of waterborne epidemic disease prevention and control.

Small-scale distributed water purifiers (SSDWPs), providing better quality drinking water, are popularly used both in homes and in the public domain. Non-continuous operation leads to water stagnation and ultimately induces microbial contamination. However, information related to such contamination in these purifiers is reported scarcely. In the present study, an SSDWP, consisting of sand filtration (SF), granular activated carbon (GAC), and ultrafiltration (UF) processes, was established to explore microbial changes induced by water stagnation, based on the aspects of bacterial count, microbial size, microbiome and pathogenic communities. Our results primary showed that: first, compared with drinking water distribution system (DWDS), bacterial counts increased more rapidly in SSDWPs, growing to > 500 cfu/mL after 2.5 h stagnation. The proportion of intact cells also increased with stagnation time. Conversely, microbial size decreased with stagnation time according to changes in forward scatter detected using flow cytometry. Second, microbiome evolution followed the isolated island model, while in stagnated DWDS, microbiome evolved according to the continent island model, and the former had higher abundance of biodiversity. Furthermore, stagnation evidently caused microbiome changes in each unit, and spatial differences contributed to microbiome dissimilarity more significantly than temporal differences. Third, Mycobacterium was the dominant pathogenic genus in the SF and GAC units while Acinetobacter was the most abundant in the UF unit. Pathogenic risks increased with water stagnation time and lower nutrients level contributed to pathogenic community richness. Therefore, terminal disinfection of SSDWPs is strongly advised.

This paper studied stagnation-induced changes of disinfectant and bacteria using an orthogonal test and kinetic analysis, and then proposed a disinfection strategy. Tap water from a drinking water distribution system and ultrafiltrated water were collected and disinfected with four disinfectants (concentrations were set 0.2-1 mg/L as Cl2. The study had several findings. First, disinfectants expanded lag phases and shortened generation times of the microbiome. Reduction in culturability, substrate responsiveness, respiratory activity, membrane potential and integrity subsequently occurred with increasing disinfection concentration. Second, the disinfectant decay rate decreased with initial disinfection concentration, and the effective disinfection phase (heterotrophic plate count (HPC) was less than 100 cfu/mL) was longer in water samples with lower organic matter. Moreover, the disinfection process was divided into an effective phase and an invalid phase (HPC>100 cfu/mL). Then a disinfection efficiency model was built and the regulation of disinfection by-products (DBPs) production was studied in chlorinated water samples, which provides a general method for other disinfectant studies. The average trihalomethanes (THMs) production during the effective phase (marked as THM/th) and THMs production during the invalid phase (marked as ΔTHM) were proposed to evaluate the DBPs production. The level of THM/th and ΔTHM were lower in ultrafiltrated water than those in tap water. THM/th were negatively correlated with initial chlorine concentration while ΔTHM were positively correlated with initial chlorine concentration. Finally, for the purpose of raising disinfection efficiency and decreasing DBPs, we propose periodic pulse disinfection.

Drinking water is a potential source of exposure to lead (Pb), which can pose risk to humans. The regulatory agencies often monitor Pb in water treatment plants (WTP) and/or water distribution systems (WDS). However, people are exposed to tap water inside the house while water may stay in the plumbing premise for several hours prior to reaching the tap. Depending on stagnation period and plumbing premise, concentrations of Pb in tap water can be significantly higher than the WDS leading to higher intake of Pb than the values from WDS or WTP. In this study, concentrations of Pb and water quality parameters were investigated in WDS, plumbing pipe (PP) and hot water tanks (HWT) for 7months. The samples were collected and analyzed on bi-weekly basis for 7 times a day. Several linear, non-linear and neural network models were developed for predicting Pb in PP and HWT. The models were validated using the additional data, which were not used for model development. The concentrations of Pb in PP and HWT were 1-1.17 and 1-1.21 times the Pb in WDS respectively. Concentrations of Pb were higher in summer than winter. The models showed moderate to excellent performance (R2=0.85-0.99) in predicting Pb in PP and HWT. The correlation coefficients (r) with the validation data were in the ranges of 0.76-0.90 and 0.97-0.99 for PP and HWT respectively. The models can be used for predicting Pb in tap water, which can assist to better protect the humans.

The drinking water quality changes during the transport through distribution systems. Domestic drinking water systems (DDWSs), which include the plumbing between the water meter and consumer\'s taps, are the most critical points in which water quality may be affected. In distribution networks, the drinking water temperature and water residence time are regarded as indicators of the drinking water quality. This paper describes an experimental research on the influence of stagnation time and temperature change on drinking water quality in a full-scale DDWS. Two sets of stagnation experiments, during winter and summer months, with various stagnation intervals (up to 168 h of stagnation) were carried out. Water and biofilms were sampled at two different taps, a kitchen and a shower tap. Results from this study indicate that temperature and water stagnation affect both chemical and microbial quality in DDWSs, whereas microbial parameters in stagnant water appear to be driven by the temperature of fresh water. Biofilm formed in the shower pipe contained more total and intact cells than the kitchen pipe biofilm. Alphaproteobacteria were found to dominate in the shower biofilm (78% of all Proteobacteria), while in the kitchen tap biofilm Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria were evenly distributed.