*4.1. Water Stagnation*

Water age is defined as the time it takes water to move from one point to another in the system, which may influence OP growth through a variety of mechanisms. This includes the time from when it is freshly produced at the treatment plant and travels to the service line, as well as the time from when it first enters the building's plumbing to the point of use [71]. High water age in buildings is increased by: (1) existence of dead ends/legs and stagnation in plumbing systems [182,236]; (2) use of low flow devices or presence of large storage tanks such as those used for solar water heating or onsite rainwater collection [39]; and (3) using low volumes of water in a building or at a particular outlet [192]. Stagnation and infrequent water use may concentrate and enhance release of organic matter in water in plastic pipes and metals in metallic pipes [181,237–240]. Zhang et al. [241] found a four-fold increase in bulk water TOC in unplasticized PVC pipes between 24 h and 72 h of stagnation. Fixtures in a green building with the fewest water use events (most stagnation) also had greater organic carbon, bacteria counts, and heavy metal (Zn, Fe, Pb) concentrations [192,242].

Stagnation and high water age also increases the likelihood and rate of disinfectant decay. High consumption of chlorine and chloramine during stagnant periods of 24–72 h have been observed for synthetic pipes (0.4 and 0.6 mg/<sup>L</sup> of chlorine loss, respectively), and stagnant periods of 2–8 h in metallic pipes (3 and 4 mg/<sup>L</sup> chlorine loss, 1.5 and 3.5 mg/<sup>L</sup> chloramine loss, respectively) [54,241]. In a green building study, six-hour stagnation almost fully eliminated monochloramine (>99%) within pipes [71].

Such water quality changes have been related to increased levels of OPs in premise plumbing systems [39,243–245]. In a field sampling study of main water distribution system, 120 water samples were taken throughout a drinking water distribution system. Only four samples were positive for cultivable *L. pneumophila* and all four samples were taken from dead end points at the end of streets with no chlorine residual remaining [246]. Another field study identified their most frequently *Legionella* positive sites as being located at the end of the distribution system and having the highest turbidity, iron, TOC, and water age, as well as the lowest flow [247]. The association between OPs and stagnation has created interest in strategies to reduce building water stagnation effects such as removing dead-legs, flushing, maintaining the hot water system, and shock disinfection [248–251]. The effectiveness of these strategies should be evaluated within the context of the specific pipe materials that are present.

#### *4.2. Hot Water Recirculation Lines*

Some plumbing codes require or sugges<sup>t</sup> the use of recirculating hot water lines for water/energy conservation, convenience and comfort [1,252–254]. In these systems, water is circulated continuously between the water heater and the point of use, preventing cooling of the distal lines and allowing for nearly instant delivery of hot water at the point of use [255]. There are many important differences between hot water recirculating systems and conventional systems, which are stagnant during periods of disuse that can affect OP growth. The constantly flowing water can deliver more nutrients to biofilm and hypothetically increase OP growth [230]. On the other hand, continuous flow can deliver more disinfectants and more hot water, which are critical control measures for OPs [256,257]. The net effect depends on which of these factors is dominant.

Continuously recirculating water could also increase release of metals, increase deposition corrosion of anodes by constantly recirculating water through copper pipe, and result in greater accumulation of sediments and H2 gas. One study showed that recirculating systems with copper piping had 3–13 times more aluminum and copper, 4–6 times more hydrogen in effluent water, and 9% more aluminum anode weight loss, compared with standard (non-recirculating) systems [222]. Recirculating systems can also accumulate 3–20 times more sediments [222] arising from corrosion of metallic pipe material and the anode rods [157,232–234,258]. These sediments, which also collect at the bottom of hot water tanks, may serve as an important growth niche within warm regions of hot water tanks where influent cold water depresses temperatures, and there are also relatively low levels of disinfectant and high levels of nutrients for *Legionella*, heterotrophs, and host organisms [17,259].

## *4.3. Pipe Aging*

New plastic and copper pipes behave di fferently than older pipes. Specifically, corrosion and release of metals is strongly influenced by pipe age, with corrosion rates and metal release tending to decrease as thicker and more passivating pipe scales form. Aging can dramatically reduce levels of metal leaching from copper and other pipes [157,260,261]. The rate of aging, and whether it decreases release of pipe constituents at all, is highly a ffected by water chemistry and water use patterns [157]. Likewise, leaching of organics from plastic pipe may attenuate 50% to >99% after aging for a period of a few weeks with hot water exposure [51,170], but in other cases has been sustained for months [262] or even over a year [263]. Pipe aging is an important factor to consider when comparing PEX to copper's capacity for *Legionella* growth. One study showed that the *Legionella* numbers in bulk water of both PEX and copper pipes in a simulated warm water system were the same after two years [140]. We speculate that one possible cause for this convergence is that, as plastic pipes age, organic carbon migration to water decreases, whereas levels of antimicrobial copper released from copper pipe also tends to decrease. Hence, in some situations, it is expected that in very old copper and plastic pipe systems there would be little di fference between these pipe materials.

#### *4.4. Possible Mixed Material Interactions*

Building plumbing is typically comprised of multiple pipe materials, either in the original design or after partial retrofits or renovations. It is anticipated that there are sometimes synergistic and other times antagonistic interactions between pipe materials that would influence growth of OPs. Copper deposition accelerating the evolution of H2 from aluminum, zinc, magnesium and iron corrosion, as discussed in Section 3.2.3, is an important exemplar. Copper is also known to catalyze degradation of plastic pipes [264–268], and the presence of copper pipe upstream of plastic pipe might enhance organic carbon release [268], surface roughness for biofilm growth [264], and perhaps even disinfectant consumption due to copper in the scale. Iron pipes upstream of copper may produce mixed Fe-Cu oxides, which can be extraordinary catalysts for free chlorine decay [269]. Similarly, copper released upstream of iron pipes could increase iron release [270]. Any galvanic coupling between two metals in plumbing materials (copper/brass-lead [271,272], copper/iron [270,273,274] iron/zinc [275,276], copper/aluminum [277,278], copper/zinc [271,279], copper/magnesium [280]) has the potential to enhance corrosion and cause changes to water quality parameters relevant to corrosion and OP growth [235,281], dissolved oxygen (DO) [273], metal concentrations [271,272], and disinfectant residual concentration. These reactions also create microenvironments of very high or very low pH [235,238]. Given that in the 2017 American Housing Survey 10% of households that reported any home improvement projects also reported adding or replacing an interior water pipe [282], understanding the e ffects of mixing pipe materials during renovation appears to be a valuable research area as antiquated premise plumbing is increasingly replaced.

#### **5. Mediating Role of Microbiome and other Microbiological Considerations**

#### *5.1. The Role of Pipe Material in Shaping the Premise Plumbing Microbiome and Resident Amoeba Host Organisms*

Interactions between OPs and the microbial communities surrounding them are key to OP proliferation and are likely influenced by pipe materials. OPs can be parasitic to free-living amoebae that first prey upon them in drinking water biofilms, before they reproduce inside and eventually kill the host organism [24]. In fact, there is some doubt that *Legionella* actually reproduces significantly in drinking water outside of an amoeba host [283]. Amoebae can also protect OPs from disinfectants and provide access to nutrients. For example, *Legionella* exclusively use amino acids, which are abundant in amoeba vacuoles, as a carbon source [210–214,284,285]. Thus, although poorly studied, any factor altering growth of key host amoebae (including *Acanthamoeba*, *Vermamoeba*, and *Naegleria)* is expected to indirectly a ffect growth of OPs, including *L. pneumophila, P. aeruginosa,*

and NTM [122,210–214,225,257,286,287]. In one experiment, copper coupons were found to host more *Acanthamoeba polyphaga* than PVC coupons [288], possibly because copper hosts less diverse eukaryotic communities [64,289] and limits competition for *A. polyphaga*. As a result, *L. pneumophila* grew and shed to the bulk waters in higher numbers on these copper coupons than on PVC coupons if co-inoculated with *A. polyphaga* [122].

Interbacterial interactions may also influence the growth of OPs. Broadly speaking, OPs benefit from the biofilm community through access to nutrients and protection from disinfectants [24,35,36,290]. Some studies have identified correlations between specific taxa and OPs in premise plumbing [291], cooling towers [292] and drinking water distribution systems [293]. However, the significance of these correlations to premise plumbing material selection is not well understood, as most studies examining differences in bacterial communities focus on very broad measures of community structure [48,59,64,216,289,294–296]. Certain waterborne bacteria are known to produce toxins that inhibit *L. pneumophila* growth [216,297] or exude other compounds that have secondary bacteriostatic effects on *Legionella* [298]. Intra-bacterial inhibition also may be mediated through amoebae by reducing host uptake [299,300] or killing the host population [134,301,302]. More research is needed to elucidate how the broad ecological differences resulting from pipe material influence these interactions. Integration of metagenomic or meta-transcriptomic analyses targeting the production of bacteriocins or other toxins with known effects on OPs could elucidate the ecological effects of taxonomic shifts resulting from pipe material. Interrupting OP-amoeba endosymbiosis through the enrichment of preferential non-OP amoeba prey [299,300] has been suggested as a probiotic means of controlling OPs [303], and pipe material could be explored as a means of enrichment of these taxa.

#### *5.2. Variation in Copper Tolerance Among Species and Strains*

Strain-to-strain differences in intrinsic tolerance of copper, acclimation to copper concentrations with time through induction of the appropriate genes, or acquisition of copper resistance via mutation or horizontal gene transfer in premise plumbing might explain some of the discrepancies in variable outcomes of copper on OPs (Table 2). *Legionella* [155] and other OPs [58] may acclimate to high copper levels through the expression of copper detoxification or efflux systems. Bedard et al. [155] reported four-fold differences in the copper tolerance of environmentally-isolated *L. pneumophila* strains, noting that more resistant strains showed increased copper ATPase *copA* expression, speculating that their increased tolerance may also be a result of higher biofilm production. Strikingly, Williams et al. [58] showed that, during exposure to 95 mg/<sup>L</sup> of copper over 6 h in liquid culture, culturable *A. baumannii* levels (CFU/mL) could increase by 2-logs or decrease by 2-logs, depending on the strain. The authors identified putative copper detoxification and efflux systems within the genome of the most resistant isolate and identified specific genes that were upregulated in response to copper exposure. However, a majority of the less tolerant strains tested also possessed these genes, leading the authors to sugges<sup>t</sup> that further definition of the proteins involved in copper resistance is required. One recent study showed two environmentally-isolated *Legionella* strains reduced by less than one log in culturability, even after two weeks of exposure to 5 mg/<sup>L</sup> copper, which the authors attributed to adaptation to the high levels of copper (average 0.48 mg/<sup>L</sup> ) in the hot water system from which these isolates were collected [154]. A profile of *Fusarium* isolates revealed that tap water isolates were more copper-tolerant than soil isolates [303]. *P. aeruginosa* isolates isolated from a hospital with copper plumbing exhibited only slightly limited growth in the presence of 0.15 mg/<sup>L</sup> copper [129]. All of these strains were found to harbor GI-7, a mobile genetic element that confers copper resistance and that has also been identified in a *P. aeruginosa* strain associated with hospital outbreaks [304]. Limited data sugges<sup>t</sup> that *A. baumannii* and mycobacteria are more difficult to inactivate with copper than other OPs, while *P. aeruginosa* is more readily inactivated [91,92,98,108,109]. *L. pneumophila* has been found both at the more resistant [98] and less resistant [91,108,109] ends of this spectrum. The wide variability among OPs and even strains of OPs in their intrinsic tolerance of copper, ability to

acquire genetic resistance, and ability to acclimate to elevated levels of copper makes it difficult to precisely predict the efficacy of copper and other antimicrobials for OP control.

#### *5.3. Confounding E*ff*ects of VBNC Bacteria*

The discovery of VBNC bacteria has complicated prior understanding for all OP control strategies, including copper. Virtually all prior work relied on culture methods to determine copper's efficacy for killing OPs [62,63,83,91,92,98,108,109,120,137,153], but some microbes rendered not culturable might remain viable and still infect host amoebae or humans [74,76,305–307]. The existence of VBNC pathogens in premise plumbing has been demonstrated by comparing culture-based numbers with those enumerated via fluorescence (e.g., live/dead) and molecular-based (e.g., quantitative polymerase chain reaction) monitoring methods [308].

Bench-scale studies examining copper's antimicrobial efficacy have found discrepancies between culture-based and molecular-based numbers of *L. pneumophila* [121,122] that are also suggestive of a copper-induced VBNC state. Similar discrepancies have been noted for *P. aeruginosa*, *Stenotrophomonas maltophilia,* and *M. avium* [104,109,127,132,133]. Evidence of copper-induced VBNC activity is particularly strong in the case of *P. aeruginosa*, where one study applied multiple non-culture-based measures of viability [127,132]. Furthermore, VBNC *P. aeruginosa* have been shown to partially recover infectivity after removal of copper from solution [132,133]. To understand how VBNC bacteria contribute to OP infections, additional studies are needed to delineate the premise plumbing conditions more precisely that induce VBNC status and to confirm the range of functionality maintained in this state. A primary challenge in achieving this is that there are currently no reliable methods for confidently enumerating VBNC bacteria.
