Interactive Effects of Copper Pipe, Stagnation, Corrosion Control, and Disinfectant Residual Influenced Reduction of Legionella pneumophila during Simulations of the Flint Water Crisis

Flint, MI experienced two outbreaks of Legionnaires’ Disease (LD) during the summers of 2014 and 2015, coinciding with use of Flint River as a drinking water source without corrosion control. Using simulated distribution systems (SDSs) followed by stagnant simulated premise (i.e., building) plumbing reactors (SPPRs) containing cross-linked polyethylene (PEX) or copper pipe, we reproduced trends in water chemistry and Legionella proliferation observed in the field when Flint River versus Detroit water were used before, during, and after the outbreak. Specifically, due to high chlorine demand in the SDSs, SPPRs with treated Flint River water were chlorine deficient and had elevated L. pneumophila numbers in the PEX condition. SPPRs with Detroit water, which had lower chlorine demand and higher residual chlorine, lost all culturable L. pneumophila within two months. L. pneumophila also diminished more rapidly with time in Flint River SPPRs with copper pipe, presumably due to the bacteriostatic properties of elevated copper concentrations caused by lack of corrosion control and stagnation. This study confirms hypothesized mechanisms by which the switch in water chemistry, pipe materials, and different flow patterns in Flint premise plumbing may have contributed to observed LD outbreak patterns.


Introduction
The Flint Water Crisis began when the City of Flint, Michigan switched from purchasing its long-term Detroit municipal water supply (sourced from Lake Huron) to the local Flint River in April 2014. Although the Flint River water was predictably much more corrosive than Detroit water, no federally-mandated corrosion control program was implemented, resulting in rampant corrosion of lead plumbing and iron water mains, low chlorine residuals, elevated bacteria, and high levels of lead [1][2][3][4]. During the summers of 2014 and 2015, Flint also experienced two outbreaks of Legionnaires' Disease (LD), with 91 cases and 12 deaths documented in Genesee County (the county which Flint is located), compared to the 6-13 cases per year and no deaths during 2009-2013 while on Detroit water [1,3,5,6]. The period of Flint River water use was characterized by high levels of LD incidence [6] and L. pneumophila gene marker levels [1] associated with large buildings. However, lower LD incidence associated with residential exposure was noted from August 2015 onwards and our sampling during this period revealed undetectable or very low levels of L. pneumophila in residential plumbing [1,3].  Figure 1. Experimental framework and specific hypotheses for this study. Corrosivity of the municipal water supply influences levels of chlorine and iron in the water delivered to the premise plumbing. Premise plumbing materials, cross-linked polyethylene (PEX) or copper (Cu), further alter the water chemistry and overall propensity for Legionella to be controlled or to survive. Corrosion of copper and iron will consume free chlorine, whereas plastic materials have little or no chlorine demand. Corrosive water will also release soluble copper ions from copper pipe, especially in stagnant premise plumbing conditions. Elevated levels of either copper or chlorine can control Legionella.

Source Water Treatment
Raw water was directly collected from the Flint River at GPS coordinates 43.018230, −83.693944. Lake Huron-sourced drinking water (Detroit tap water) was collected after > 5 min flushing from the tap of a residential Flint home. Raw Flint River water and Detroit tap water were both collected on 18  Water treatments applied to raw Flint River water during the crisis were simulated in the laboratory. These included 56 mg/L ferric chloride for coagulation, 10 min of stirring for flocculation, 159 mg/L lime as Ca(OH)2 for softening, followed by another 15 min of flocculation. The water was subsequently settled for 4 h and filtered through a column of glass wool to simulate sand filtration. Water treated in this manner was designated as "treated Flint River" water ( Figure 2). Working stocks (10-20 L) of treated Flint River water and Detroit tap water were stored at room temperature (23 °C) until the supply was exhausted. Figure 1. Experimental framework and specific hypotheses for this study. Corrosivity of the municipal water supply influences levels of chlorine and iron in the water delivered to the premise plumbing. Premise plumbing materials, cross-linked polyethylene (PEX) or copper (Cu), further alter the water chemistry and overall propensity for Legionella to be controlled or to survive. Corrosion of copper and iron will consume free chlorine, whereas plastic materials have little or no chlorine demand. Corrosive water will also release soluble copper ions from copper pipe, especially in stagnant premise plumbing conditions. Elevated levels of either copper or chlorine can control Legionella.

Source Water Treatment
Raw water was directly collected from the Flint River at GPS coordinates 43.018230, −83.693944. Lake Huron-sourced drinking water (Detroit tap water) was collected after > 5 min flushing from the tap of a residential Flint home. Raw Flint River water and Detroit tap water were both collected on 18  Water treatments applied to raw Flint River water during the crisis were simulated in the laboratory. These included 56 mg/L ferric chloride for coagulation, 10 min of stirring for flocculation, 159 mg/L lime as Ca(OH) 2 for softening, followed by another 15 min of flocculation. The water was subsequently settled for 4 h and filtered through a column of glass wool to simulate sand filtration. Water treated in this manner was designated as "treated Flint River" water ( Figure 2). Working stocks (10-20 L) of treated Flint River water and Detroit tap water were stored at room temperature (23 • C) until the supply was exhausted. Experimental design from source water to simulated distribution systems (SDSs) to simulated premise plumbing reactors (SPPRs). Source water was treated and stored in 10-30 L batches until fed to SDSs. Each SDS condition was set up in a flask containing 330 mL of source water, an iron wire (except for FR-no Fe), a stir bar, and 3.0 or 3.5 mg/L chlorine. Bold conditions were designed to replicate scenarios found before (DET-Cold), during (FR), or after the Flint Water Crisis (DET-Enhanced). Conditions in italics were designed to simulate hypothetical scenarios if corrosion control had been implemented or if water had not flowed through unlined iron pipe. After the SDSs simulation was completed, the water was fed to corresponding SPPRs containing either PEX (n = 3) or copper (n = 3). The total number of SPPRs was 36.

Simulated Distribution Systems: Chlorination and Corrosion
Six SDS conditions served to reproduce distributed waters that either occurred (conditions designated in bold font) under conditions relevant to the Flint Water Crisis or its aftermath or hypothetical scenarios if corrosion control had been implemented or if water had not flowed through unlined iron pipe (conditions designated in italics) ( Figure 2). In five of the six conditions, the practical influence of unlined iron distribution system pipe was simulated by addition of an iron wire to flasks mixing each water for 3 h. Treated Flint River water conditions included a condition with the omission of phosphate corrosion control (as was the case during the crisis) (FR), a hypothetical condition if 1 mg/L as PO4-P orthophosphate corrosion control had been implemented (FR-CC), and a condition without any phosphate corrosion control or iron corrosion (i.e., no iron wire) (FR-NoFe) representing some sections of Flint served by newer concrete lined iron or PVC distribution system pipe during the crisis. Detroit tap water conditions examined the pre-crisis effect of Lake Huron-sourced water with lower distribution system temperature (DET-Cold) containing 2.5 mg/L orthophosphate PO4-P, the post-crisis water with enhanced doses of chlorine and additional phosphate (3.5 mg/L chlorine and 4.0 mg/L orthophosphate) to assist with system recovery once Flint switched back to Detroit-sourced water (DET-Enhanced), and a hypothetical condition if normal Detroit distribution water with 2.5 mg/L orthophosphate had been as warm as treated Flint River water during summer months (DET).

General SDSs Water Preparation
Sodium hypochlorite (10% diluted Clorox™ bleach, the Clorox Company, Oakland, CA, USA) was added to 330 mL of each water condition until an initial stable target of 3 mg/L free chlorine residual was obtained (the only exception being a higher residual of 3.5 mg/L in DET-Enhanced), followed by the SDSs in 500 mL glass flasks containing magnetic stir bars and mixing 400 rpm for three hours. In all conditions, except FR-NoFe, the presence of iron pipe was simulated in the SDS with a 12 cm length of 99% 2 mm Figure 2. Experimental design from source water to simulated distribution systems (SDSs) to simulated premise plumbing reactors (SPPRs). Source water was treated and stored in 10-30 L batches until fed to SDSs. Each SDS condition was set up in a flask containing 330 mL of source water, an iron wire (except for FR-no Fe), a stir bar, and 3.0 or 3.5 mg/L chlorine. Bold conditions were designed to replicate scenarios found before (DET-Cold), during (FR), or after the Flint Water Crisis (DET-Enhanced). Conditions in italics were designed to simulate hypothetical scenarios if corrosion control had been implemented or if water had not flowed through unlined iron pipe. After the SDSs simulation was completed, the water was fed to corresponding SPPRs containing either PEX (n = 3) or copper (n = 3). The total number of SPPRs was 36.

Simulated Distribution Systems: Chlorination and Corrosion
Six SDS conditions served to reproduce distributed waters that either occurred (conditions designated in bold font) under conditions relevant to the Flint Water Crisis or its aftermath or hypothetical scenarios if corrosion control had been implemented or if water had not flowed through unlined iron pipe (conditions designated in italics) ( Figure 2). In five of the six conditions, the practical influence of unlined iron distribution system pipe was simulated by addition of an iron wire to flasks mixing each water for 3 h. Treated Flint River water conditions included a condition with the omission of phosphate corrosion control (as was the case during the crisis) (FR), a hypothetical condition if 1 mg/L as PO 4 -P orthophosphate corrosion control had been implemented (FR-CC), and a condition without any phosphate corrosion control or iron corrosion (i.e., no iron wire) (FR-NoFe) representing some sections of Flint served by newer concrete lined iron or PVC distribution system pipe during the crisis. Detroit tap water conditions examined the pre-crisis effect of Lake Huron-sourced water with lower distribution system temperature (DET-Cold) containing 2.5 mg/L orthophosphate PO 4 -P, the post-crisis water with enhanced doses of chlorine and additional phosphate (3.5 mg/L chlorine and 4.0 mg/L orthophosphate) to assist with system recovery once Flint switched back to Detroit-sourced water (DET-Enhanced), and a hypothetical condition if normal Detroit distribution water with 2.5 mg/L orthophosphate had been as warm as treated Flint River water during summer months (DET).

General SDSs Water Preparation
Sodium hypochlorite (10% diluted Clorox™ bleach, the Clorox Company, Oakland, CA, USA) was added to 330 mL of each water condition until an initial stable target of 3 mg/L free chlorine residual was obtained (the only exception being a higher residual of 3.5 mg/L in DET-Enhanced), followed by the SDSs in 500 mL glass flasks containing magnetic stir bars and mixing 400 rpm for three hours. In all conditions, except FR-NoFe, the presence of iron pipe was simulated in the SDS with a 12 cm length of 99% 2 mm diameter iron wire (approximately, 7.6 cm 2 Fe surface per liter of water) and orthophosphate was added to achieve corrosion control targets of 1 (FR-CC), 2.5 (DET, DET-Cold), or 4.0 mg/L (DET-Enhanced).

Simulated Premise Plumbing Reactors (SPPRs)
Following the SDS step, waters were transferred to 100 mL borosilicate glass bottles (36 total) designed to simulate changes occurring in water as it ages in premise plumbing (SPPR, Figure 2). Each SPPR was equipped with either eight pieces of 20 mm × 10 mm cross-sectional PEX (n = 18) or solid copper (n = 18) pipe material. Pipe coupons had been aged in the bottles for six years in prior experiments, described elsewhere [8][9][10]27], which provided a benefit of well-aged premise plumbing pipe materials and mature biofilms at the start of the experiment.

Initializing the SPPRs
All 36 SPPRs were conditioned prior to the experiment, by dosing a homogeneous aliquot of reactor effluents according to pipe material, followed by an acclimation phase of 50% water volume changes with treated Flint River water every three days for 101 days. This water change frequency and volume simulated a low use, high-stagnation condition considered to be conducive to Legionella growth in premise plumbing [8]. On Day 14, the SPPRs were inoculated with three environmental L. pneumophila isolates from Flint, MI buildings at a total concentration of 10,000 colony forming units per milliliter (CFU/mL). The inoculum was composed of a mixture of L. pneumophila serogroup 1 as well as two non-serogroup 1 isolates. Inoculum concentration was determined by optical density readings of L. pneumophila colonies scraped from agar plates, resuspended in Nanopure water, and measured at 600 nm using a 4500 HACH spectrophotometer (Hach Company, Loveland, CO, USA).
To avoid introduction of Legionella spp. that may have been present in the water shipments once the experiment was in progress, effluent SDS waters were monitored for survival of culturable Legionella prior to their addition to corresponding SPPRs. In no case was detectable culturable Legionella present after chlorination of the water and incubation in the SDSs.

Water Changes with SDS Conditions
Following inoculation and a 101-day conditioning period with treated Flint River water, 50% water changes were performed every 3-4 days for 175 days. SPPRs were reproducibly inverted five times for each water change to resuspend any settled material, and 50% of the volume was decanted and replaced with water from one of the six SDS conditions ( Figure 2). Each of the six SDS conditions were tested in triplicate copper or PEX SPPRs. Reactors were incubated under static conditions at 37 • C between water changes. Thus, the experimental design included 6 SDS conditions × 2 pipe materials × 3 replicates = 36 total SPPRs. Culturable L. pneumophila were enumerated as colony forming units per deciliter (CFU/mL) on Buffered Charcoal Yeast Extract (BYCE) agar (Remel, Lenexa, KS, USA) supplemented with 3 g/L glycine, 0.4 g/L L-cysteine, 80,000 units/L of polymyxin B sulfate, 0.001 g/L vancomycin, and 0.08 g/L cycloheximide. Initially, water was directly taken from SPPRs and plated onto BYCE; however, once CFUs dropped below detection of direct plating of 1 mL, 50 mL of effluent SPPR water was filter concentrated using 0.22 µM pore size mixed-cellulose ester membranes (Millipore, Billerica, MA, USA) and resuspended in 5 mL of Nanopure water prior to plating (1 mL). Water from each reactor was plated in triplicate. Plates were incubated at 37 • C for 5 days, after which L. pneumophila colonies were counted and CFU/mL were calculated. Direct plates with 0.02 CFU/mL were considered below detection. When no L. pneumophila colonies were detected from 50 mL concentrates, counts were considered below detection, resulting in a detection limit of 0.001 CFU/mL.

Culture Confirmation
To gain insight into the types of Legionella that persisted through the experiment, colonies visually determined as Legionella and non-Legionella species were picked from plates after 5 days of incubation at 37 • C for polymerase chain reaction confirmation. Polymerase chain reaction was used Pathogens 2020, 9,730 6 of 17 to confirm Legionella spp. (i.e., genus), L. pneumophila, and serogroup 1 using established primers and protocols [28,29].

Water Quality Analyses
Influent SDSs, effluent SDSs (influent SPPRs), and effluent SPPR waters were analyzed on Days 0, 9, 20, 72, 87, 126, 131, and 153. Inorganics, including dissolved and particulate iron and copper, were measured by inductively coupled plasma-mass spectrometry (ICP-MS) following 2% acidification with nitric acid. Total organic carbon (TOC) was measured according to standard method 5310 C using a persulfate-ultraviolet detection by a Sievers Model 5300 C (General Electric Company, Boston, MA, USA). pH was measured using an Oakton 110 series meter (Cole Parmer, Count Vernon Hills, IL, USA).
Free chlorine was measured using a 4500 HACH spectrophotometer (Loveland, CO) according to 4500-Cl standard method. To examine the kinetics of chlorine in the various water conditions used in this study, chlorine decay tests were performed on source waters (treated Flint River water and Detroit tap water) in non-reactive glass containers, on SDS water conditions with iron wire according to the experimental design ( Figure 2), and after the SDSs waters were added to the SPPRs.

Data Analysis
Statistical tests were performed using R Studio (Version 1.0.153). A Shapiro-Wilk normality test was performed and none of the data were normally distributed. Arithmetic means were calculated for displaying results due to the high proportion of non-detect values in the dataset. Wilcoxon rank sum and Kruskal-Wallis rank sum tests with post-hoc Tukey tests were performed to determine statistical correlations. Wilcoxon tests were used for Legionella culture data (log transformed), pipe material, iron, and copper data, whereas the Kruskal-Wallis test was used to determine significance of chlorine data between SPPRs. Significance was set at a p value ≤ 0.05.

Treated Source Waters Employed in this Experiment
To recreate water quality conditions in Flint, influent water conditions were simulated by treating raw Flint River water in the lab and collecting Lake Huron-sourced water from a well-flushed tap in Flint post crisis (Detroit tap water). The unaltered pH of treated Flint River water ranged between 7.84 and 8.57, while Detroit tap water ranged from 7.96 to 8.06, which recreated the stable pH observed when Flint was using Detroit water and the more variable pH when using Flint River water in 2014 [3,12].
The source water was added in 300-mL aliquots to six glass flasks (3 with Detroit tap water, 3 with treated Flint River water) with iron wire and mixed for 3 h to simulate six different conditions in distribution systems (SDSs). Just prior to being added to the SDSs, the source waters (treated Flint River water and Detroit tap water) were chlorinated, achieving an initial disinfectant residual of 3.10 mg/L Cl 2 (Table 1 section B). Additional chlorine was added to only the DET-Enhanced SDS condition to achieve a higher average initial residual of 3.80 ± 0.19 mg/L (Table 1 section B). The possible short-term role of cooler temperature during distribution while on Detroit tap water was tested in this work with the DET-Cold SDS condition, held at an average of 18.3 ± 1.4 • C compared to an average 21.8 ± 1.3 • C of the other five SDS conditions (FR, FR-NoFe, FR-CC, DET, DET-Enhanced) ( Table 1 section B,C). This~3 • C difference served to recreate the reported average summer water temperature of 19.9 ± 2.24 • C (pre-crisis, Detroit) and 22.6 ± 2.14 • C (during crisis, Flint River) ( Table 1 section A) [3].   2017), chlorine data are from monitoring station 6, iron data are from citizen science sampling of flushed water from the same 150 homes in August 2015 (crisis) and August 2017 (post-crisis), and copper water crisis data are from a subset of first draw samples from homes that records indicate had at least partial copper service lines (n = 79). UNK = unknown.ˆSection (B): Same parameters as in Section (A) were measured in the influent to the SDSs mean (5th-95th percentile) (n = 10 samples over a 10-month period) (Ambient laboratory set point reported for temperature data). Bold conditions SDSs were designed to simulate actual conditions found during the crisis. Conditions in italics were designed to simulate hypothetical scenarios. "DET" conditions received Detroit tap water influent. "FR" conditions received treated Flint River influent. + Section (C): Mean and standard deviation of temperature and chlorine and mean (5th-95th percentile) of iron effluent from SDSs (i.e., influent to the SPPRs) and mean (5th-95th percentile) of copper in the effluent of copper pipe SPPRs.

SDSs Chlorine
The effluent water collected following the 3-h SDSs reaction time ( Figure 2) successfully replicated known trends in chlorine residuals observed in the Flint water distribution system before, during, and after the water crisis. To assess inherent chlorine demand prior to the SDSs step, treated Flint River and Detroit tap waters were aliquoted to non-reactive glass containers without iron. The chlorine residual in treated Flint River water dropped from~3 to~1 mg/L in 180 min, presumably due to relatively high levels of organic matter (5.2 ± 0.03 mg/L TOC), whereas there was little to no decay occurred in the Detroit tap water (1.2 ± 0.03 mg/L TOC) over the same time period ( Figure 3A). The addition of iron wire to simulate unlined iron pipe corrosion during distribution further reduced chlorine residuals in conditions with both treated Flint River water and Detroit tap water as influents ( Figure 3B). However, while some residual was consistently detected in the Detroit tap water effluents after simulated distribution (DET, DET-cold, DET-Enhanced; 0.5-1 mg/L Cl 2 after 180-min exposure), treated Flint River water conditions (FR, FR-CC, FR-no Fe) generally had no detectable residual ( Figure 3B). Condition FR-NoFe is not shown in Figure 3B because no iron wire was added to the SDSs for that condition.

SDSs Chlorine
The effluent water collected following the 3-h SDSs reaction time ( Figure 2) successfully replicated known trends in chlorine residuals observed in the Flint water distribution system before, during, and after the water crisis. To assess inherent chlorine demand prior to the SDSs step, treated Flint River and Detroit tap waters were aliquoted to non-reactive glass containers without iron. The chlorine residual in treated Flint River water dropped from ~3 to ~1 mg/L in 180 min, presumably due to relatively high levels of organic matter (5.2 ± 0.03 mg/L TOC), whereas there was little to no decay occurred in the Detroit tap water (1.2 ± 0.03 mg/L TOC) over the same time period ( Figure 3A). The addition of iron wire to simulate unlined iron pipe corrosion during distribution further reduced chlorine residuals in conditions with both treated Flint River water and Detroit tap water as influents ( Figure 3B). However, while some residual was consistently detected in the Detroit tap water effluents after simulated distribution (DET, DET-cold, DET-Enhanced; 0.5-1 mg/L Cl2 after 180-min exposure), treated Flint River water conditions (FR, FR-CC, FR-no Fe) generally had no detectable residual ( Figure 3B). Condition FR-NoFe is not shown in Figure 3B because no iron wire was added to the SDSs for that condition.  While there was variability due to seasonal changes in the source water and variable iron wire corrosion rates throughout the experiment, the mean chlorine concentration (n = 43) after incubation in the SDSs exhibited a general trend of (lowest to highest): FR-CC ≈ FR < FR-NoFe ≈ DET ≈ DET-Cold < textbfDET-Enhanced (Table 1). Based on a Kruskal-Wallis rank sum test, the mean chlorine concentrations across the SDS conditions were significantly different (p value < 2 × 10 −16 ), while a pairwise post-hoc Tukey test further confirmed specific differences between conditions indicated by a "<" sign in the above trend analysis (all p values ≤ 0.009).
Overall, key expectations were also recreated with respect to known trends resulting from water chemistry and corresponding chlorine residual in SDSs effluent. Specifically, the SDSs successfully reproduced chlorine residuals comparable to those during the crisis of 0.28 ± 0.24 mg/L (at Flint city monitoring station 6) [30], compared to levels of 0.26 ± 0.23 mg/L in our treated Flint River water simulation (FR condition, Table 1 section C). SDS conditions also successfully simulated pre-crisis (DET-Cold) and post-crisis (DET-Enhanced) high chlorine, with actual values only 1 mg/L higher than measured during the pre-or post-crisis conditions ( Table 1 section C). Both conditions with treated Flint River water and iron present (FR and FR-CC) occasionally had undetectable chlorine residuals under the conditions tested, whereas FR-NoFe and all conditions with Detroit tap water consistently had a measurable chlorine disinfectant residual following simulated distribution, as hypothesized ( Figure 1). Iron has been shown to decay chlorine residual in typical drinking waters [31], but the chlorine decay observed in the SDS step was accelerated beyond what is typical due to the corrosivity of the treated Flint River water and lack of corrosion control.

SDSs Iron and Corrosion Control
Known benefits of corrosion control (FR vs. FR-CC; FR vs. DET) in terms of hindered iron release and maintenance of higher chlorine residuals in the actual Flint distribution system (Table 1) were not achieved in these simplistic simulations. Based on a prior study [2], the addition of phosphate corrosion control to treated Flint River water reduced iron weight loss by 5.1 times compared to that observed in treated Flint River water without phosphate, while also reducing chlorine decay rates. Further, iron corrosion rates were 8.6 times lower in Detroit tap water with corrosion control versus treated Flint River water without corrosion control, a trend confirmed by our citizen science field sampling throughout Flint in August 2015 versus August 2017 ( Figure 1) [3,5]. However, the corrosion control simulation applied to the SDSs in this study did not produce known significant differences in mean effluent iron (i.e., FR, FR-CC, and DET; Table 1 section C). The only condition with relatively low iron in this work was treated Flint River water without any iron present (FR-NoFe), in which mean iron was 15.4 ± 19.4 µg/L compared to the 60.5 ± 212 µg/L observed in August 2017 flushed water samples collected in Flint (Table 1).
We were aware that the simple approach applied here would not effectively replicate impacts of iron corrosion control, given that phosphate inhibition of iron corrosion and associated chlorine decay can sometimes require 6-12 months to produce expected benefits even under continuous-flow conditions in water mains, and even longer under more stagnant conditions [32,33]. In this seven-month simulation, the iron was only exposed to the water approximately 6 h each week, which translates into seven days total exposure of iron to the target water over the entire study. Thus, the analysis that follows considers that this particular aspect of the simulation is not representative of what occurred in the field.

SPPRs Chlorine
After the effluents from the SDSs were transferred to the SPPRs, the 50% water change produced an immediate dilution of chlorine. Beyond dilution, there is an immediate chlorine demand from the combination of aged pipe material, pipe surface area, biofilm, and water within each reactor [24,26]. Notably, all SDS conditions, except DET-Enhanced, experienced an immediate chlorine demand within the first 10 min, which exceeded the 50% loss expected from dilution. FR and FR-CC never received any initial chlorine residual flowing into the SPPRs (Figure 4), whereas FR-NoFe retained a low, but detectable, chlorine residual (>0.1 mg/L) for a period of 60-120 min in the PEX SPPRs and 1-10 min in the copper SPPRs (data not shown). Chlorine was reduced in the Detroit tap water SPPRs to below 0.1 mg/L within 30-60 min in both PEX and copper SPPRs, while chlorine was maintained above 0.1 mg/L for up to 120 min in DET-Cold with PEX ( Figure 4) versus just 10-30 min in the corresponding copper SPPRs. Chlorine residuals in the DET-Enhanced conditions after 120 min were 0.92 and 0.38 mg/L in the PEX and copper SPPRs, respectively (Figure 3). In some instances, chlorine was still detectable in DET-Enhanced SPPRs after 24 h. As a general rule, when detectable chlorine residual was present in the influent to the SPPRs, levels were higher in the system with PEX after 10 min than in the equivalent system with copper, consistent with the overall hypothesis of this work and our prior research [25] (Figure 1).  120 min after the effluent from the SDSs were fed to the simulated premise plumbing reactors (SPPRs) (50% fresh SDSs water with 50% remaining SDSs following incubation in the SPPRs the previous cycle). Dashed lines indicate the calculated initial chlorine level added for each water or reactor type. Bars represent the maximum and minimum, the upper and lower bounds of the box are the first and third quartiles, and the median is indicated by the internal dash. The detection limit was 0.02 mg/L.

SPPRs Copper
Mean total copper in influent water (Table 1B) to all SPPRs was consistently < 15 g/L and mean effluent copper from PEX reactors was consistently < 100 g/L, in accordance with the assumption that the only source of copper was traces from plumbing used to collect well-flushed raw water samples in the field. However, SPPRs containing copper pipe consistently produced effluent with total mean copper concentrations > 700 g/L (Table 1 section C) and were statistically higher than the copper concentrations from the PEX SPPRs effluent (p value = 2  10 −16 ). Further, effluents from SPPRs receiving treated Flint River water, simulating Flint water during the crisis (FR), contained higher total copper than each of the and (B) 120 min after the effluent from the SDSs were fed to the simulated premise plumbing reactors (SPPRs) (50% fresh SDSs water with 50% remaining SDSs following incubation in the SPPRs the previous cycle). Dashed lines indicate the calculated initial chlorine level added for each water or reactor type. Bars represent the maximum and minimum, the upper and lower bounds of the box are the first and third quartiles, and the median is indicated by the internal dash. The detection limit was 0.02 mg/L.
Overall, these results illustrate quick and drastic decay of the chlorine disinfectant residual in premise plumbing systems (Figure 4) that added to decay in the distribution systems ( Figure 3). The U.S. Environmental Protection Agency (EPA) recommends that a free chlorine disinfectant residual be detectable (often, >0.1 or >0.2 mg/L) in 95% of distribution system samples [34], which has previously been acknowledged not to be adequate for the reduction of Legionella in large buildings, single-family homes, or small buildings [35]. The results from the Detroit tap water SPPRs (DET, DET-Cold, DET-Enhanced) demonstrate that the residual was detectable (>0.1 mg/L Cl 2 ) after 120 min in the SPPRs only when the disinfectant residual entering much higher than 0.2 mg/L Cl 2 (Figure 4).

SPPRs Copper
Mean total copper in influent water (Table 1B) to all SPPRs was consistently < 15 µg/L and mean effluent copper from PEX reactors was consistently < 100 µg/L, in accordance with the assumption that the only source of copper was traces from plumbing used to collect well-flushed raw water samples in the field. However, SPPRs containing copper pipe consistently produced effluent with total mean copper concentrations > 700 µg/L ( Table 1 section C) and were statistically higher than the copper concentrations from the PEX SPPRs effluent (p value = 2 × 10 −16 ). Further, effluents from SPPRs receiving treated Flint River water, simulating Flint water during the crisis (FR), contained higher total copper than each of the corresponding conditions representing Detroit water (DET-Cold, DET-Enhanced, p values < 4 × 10 −5 ), consistent with the lack of copper corrosion control during this time period. Thus, the laboratory simulation successfully reproduced the trends in copper levels characteristic of pre-/during and post-crisis conditions in Flint, where mean, 5th, and 95th percentiles of first draw copper during the crisis were approximately three times higher than post-crisis (Table 1 section A,C).

Legionella pneumophila Response to Simulation of Water Chemistry and Premise Plumbing Material
After 101 days of SPPRs acclimation to the SDSs water and the cross-inoculation period, culturable L. pneumophila numbers were greatest in SPPRs receiving treated Flint River waters, particularly the PEX condition ( Figure 5A). Among all SPPRs containing PEX material, L. pneumophila CFU/mL were significantly higher in treated Flint River water-sourced ( Figure 5A) compared to Detroit-sourced (Days 25-175; p < 0.05; Figure 5C) water. This demonstrated the main hypothesized effect of treated Flint River water being more conducive than Detroit tap water to maintaining viable L. pneumophila, at least in the absence of copper pipe (Figure 1).
Throughout the study, L. pneumophila persisted at low numbers in the copper SPPRs fed with treated Flint River waters, but at levels significantly lower than in the PEX SPPRs (p value = 0.03). In particular, the copper SPPRs receiving FR-NoFe influent water sustained little to no culturable L. pneumophila beyond 75 days ( Figure 5B). During our field sampling at the height of the summer 2015 LD outbreak, the pH was 7.0 in Flint homes, in which case the higher acidity likely caused much higher levels of Cu +2 in premise plumbing [3,12,14] than in this study at a pH of 7.8. Together, the results from the treated Flint River water copper SPPRs at a pH 7.8 suggest that, under conditions of corrosive influent water (including the FR-CC water as evidenced by chlorine decay tests; Figure 3B), the elevated copper concentrations can enhance reduction of L. pneumophila, consistent with the overarching hypotheses of this study (Figure 1).
The Detroit tap water conditions provide a simulation of what occurred before the city of Flint switched to the treated Flint River water (April 2014), and after they switched treated Flint River water back to the Detroit municipal water supply on 16 October 2015. Initial culturable counts of L. pneumophila declined under all Detroit tap water conditions within the first month ( Figure 5C,D), which was consistent with our field data [1,3] and the corresponding drop in LD incidence after switching back to Detroit water [5]. The loss of culturable L. pneumophila was greatest in PEX SPPRs for all three Detroit tap water SDS conditions, with culturable L. pneumophila falling below detection by Day 25, with a single exception ( Figure 5C). Interestingly, L. pneumophila fared better in Detroit SPPRs containing copper pipe relative to those containing PEX pipe material, consistent with a previous study in the same Detroit tap water PEX reactors with no chlorine and additional mixing [12]. Plate counts in Detroit SPPRs containing copper remained near 1 CFU/mL from Day 25 until the end of the experiment. This suggests the additional chlorine demand and reduced chlorine levels caused by the presence of copper can actually increase growth of Legionella as hypothesized (Table 1 and Figure 1). Notably, the colony counts in the DET-Enhanced condition dropped below detection after just 100 days ( Figure 5D), illustrating that extra chlorine can overcome the demand exerted by copper and more effectively control L. pneumophila. The presence of orthophosphate in Detroit tap water also would have reduced toxicity of the L. pneumophila to copper as reported in earlier studies [12,24,36]. 13 25-175; p < 0.05; Figure 5C) water. This demonstrated the main hypothesized effect of treated Flint River water being more conducive than Detroit tap water to maintaining viable L. pneumophila, at least in the absence of copper pipe (Figure 1). Throughout the study, L. pneumophila persisted at low numbers in the copper SPPRs fed with treated Flint River waters, but at levels significantly lower than in the PEX SPPRs (p value = 0.03). In particular, the copper SPPRs receiving FR-NoFe influent water sustained little to no culturable L. pneumophila beyond 75 days ( Figure 5B). During our field sampling at the height of the summer 2015 LD outbreak, the pH was 7.0 in Flint homes, in which case the higher acidity likely caused much higher levels of Cu +2 in premise plumbing [3,12,14] than in this study at a pH of 7.8. Together, the results from the treated Flint River water copper SPPRs at a pH 7.8 suggest that, under conditions of corrosive influent water (including the FR-CC water as evidenced by chlorine decay tests; Figure 3B), the elevated copper concentrations can enhance reduction of L. pneumophila, consistent with the overarching hypotheses of this study (Figure 1). Figure 5. The effects of water source, distribution conditions, and pipe material on culturable L. pneumophila in simulated premise plumbing reactors (SPPRs). Effluent log transformed average L. pneumophila numbers (CFU/mL) and standard deviations from SPPRs receiving simulated distribution system (SDS) water corresponding to: (A) treated Flint River water with PEX pipe coupons; (B) treated Flint River water with copper pipe coupons; (C) Detroit tap water with PEX pipe coupons; and (D) Detroit tap water with copper pipe coupons. All SPPRs influent waters were spiked to an initial target concentration of 3 mg Cl2/L and Figure 5. The effects of water source, distribution conditions, and pipe material on culturable L. pneumophila in simulated premise plumbing reactors (SPPRs). Effluent log transformed average L. pneumophila numbers (CFU/mL) and standard deviations from SPPRs receiving simulated distribution system (SDS) water corresponding to: (A) treated Flint River water with PEX pipe coupons; (B) treated Flint River water with copper pipe coupons; (C) Detroit tap water with PEX pipe coupons; and (D) Detroit tap water with copper pipe coupons. All SPPRs influent waters were spiked to an initial target concentration of 3 mg Cl 2 /L and aged three hours under completely-mixed conditions at 23 • C in the presence of an iron wire (SDS step), except FR-NoFe conditions, which had no iron wire, and DET-Cold condition, which was incubated at 17 • C. FR-CC additional orthophosphate corrosion control agent added at 1 mg/L, DET-Enhanced additional CL 2 added at 3.5 mg/L and orthophosphate at 2.5 mg/L.

Isolate Analysis
To gain insight into whether a single strain or mixture of strains of Legionella persisted under the various conditions, 56 representative isolates collected from the SPPRs on Days 0, 47, 82, 175, and 210 were subject to genotypic screening by PCR. Interestingly, it was observed that L. pneumophila survived through the end of the experiment across all simulations, except DET-Enhanced (Table S1). Of the L. pneumophila strains recovered from SPPRs fed with treated Flint River water by Day 150, 56/56 were characterized as serogroup 1. By Day 210, FR and all other water conditions supporting Legionella were confirmed to contain a mixture of serogroup 1 and non-serogroup 1 L. pneumophila, based on PCR detection of the wzm gene. The ability to multiple serogroups of L. pneumophila to persist under the various conditions of this experiment suggests that the trends observed in response to the water conditions employed in this study were robust across multiple serogroups.

Chlorine Disinfectant
Chlorine is by far the most widely implemented and relied upon secondary disinfectant residual applied in the U.S. and around the world. Previous studies have indicated that concentrations >0.5 mg/L are sometimes sufficient for limiting detectable levels of Legionella in large building plumbing water systems [37,38]. As demonstrated above, once added to SPPRs, chlorine concentrations immediately decreased and were often no longer detectable after 60 min following a water change. Further, in all SPPRs, except those receiving DET-Enhanced water, chlorine concentrations decreased below 0.5 mg/L within the first 10 min. The persistence of culturable Legionella in all conditions, except the DET-Enhanced, essentially proves that adequate disinfection was not achieved for the other 10 conditions

Pipe Material: PEX and Copper
Comparing copper versus PEX pipe materials further illustrated the potential for premise plumbing conditions to mediate the effects of the distribution system water chemistry. Copper is of particular interest because of its known antimicrobial properties towards L. pneumophila [28,[39][40][41]. Ironically, the lack of corrosion control, which triggered higher iron and lower chlorine residuals and exacerbated Legionella problems in some portions of the Flint distribution system during the crisis [3], also released high levels of copper that might have helped to control Legionella growth in some buildings and homes. In this study, regardless of influent water sources, copper SPPRs displayed rapid initial loss of culturable L. pneumophila subsequent to feeding the SDS waters. However, low colony counts persisted in most SPPR conditions containing copper at the higher pH, representative of the summer 2014 outbreak, for nearly six months. The initial reduction of Legionella CFUs in copper SPPRs may have occurred through limited antimicrobial properties of aged copper at the relatively high pH employed in this study, as observed by others [12,24,42,43].
As Legionella are facultative intracellular bacteria, they are capable of residing in biofilms and replicating in more than 20 species of amoebae [44,45]. In harsh environments, such as the surface of copper pipe material or variable disinfection levels of chlorine, existing biofilms may serve as a protective environment for Legionella to shelter from disinfectants [26,43,45]. However, this study suggests that such protective mechanisms can be overcome by higher chlorine residual and contact time. This is demonstrated by the DET-Enhanced copper condition, which included increased chlorine and corrosion control agent and contained no culturable L. pneumophila from Day 50 forward (with the exception of a single colony obtained from a 50 mL sample of water on Day 82) ( Figure 5D).
Interactions between copper and influent chlorine residuals may provide an environment that allows for the persistence of Legionella in premise plumbing, especially if corrosion control maintains Cu +2 below thresholds controlling Legionella [14,19,46]. A previous study determined that disinfectant potential of free chlorine can be affected by the age of copper pipe material [28]. In both treated Flint River water and Detroit tap water, the presence of aged copper reduced the capacity of the SPPRs to maintain measurable free chlorine residuals ( Figure 5B), but the presence of orthophosphate corrosion control may have allowed Legionella to persist at higher levels in DET or DET-Cold water, whereas the corresponding condition with higher copper in FR helped reduce Legionella.

Experimental Conditions: Hypothetical Effects of Stagnation, Chlorine, and Elevated pH
Comparing results of the current study to those obtained to those from a companion study using the same SPPRs, but with continuous mixing, slightly lower pH, and no disinfectants ( Figure 6) [12] can provide insight into overarching effects of experimental conditions selected for this study. Notably, L. pneumophila in the treated Flint River water with PEX was 2.5-log higher in the previous study. We hypothesize that this is attributable to continuous mixing versus stagnation, since the small pH change is not expected to be influential for PEX, and chlorine levels delivered to this reactor from the SDSs in this work were consistently undetectable. A much smaller increase of 0.4-1.0 log was observed for treated Flint River water copper conditions in the prior study versus this work, mostly likely due to the lower pH significantly enhancing bacteriostatic impacts of cupric ion. In any case, the higher L. pneumophila with more mixing is consistent with prior results in recirculating versus non-recirculating systems [17,19,47,48] in which there were warm temperatures and low disinfectant residuals. In Detroit tap water conditions, comparable conditions between the two studies consistently led to non-detectable L. pneumophila, except for copper pipe with both mixing and corrosion control, where relatively low levels of L. pneumophila persisted.

Conclusion
The findings of this study are consistent with the understanding that L. pneumophila is not uncommon in municipal water flowing into buildings [49], but a range of water chemistry and premise plumbing conditions and disinfectant residuals can prevent their proliferation. Persistent disinfectant, including chlorine, is known to be a critical factor in reducing Legionella risk [35,37,39], and a recent study confirmed predicted associations between low levels of chlorine in Flint's distribution system and observed incidence of LD [5]. Consistent with our prior in-field observations [1,3], we further demonstrate under controlled laboratory conditions the importance of considering interactive effects with flow and pipe materials, particularly with respect to relative water corrosivity and influence on residual chlorine levels, in keeping Legionella levels low. Indeed, many individual factors can act as "two-edged swords" in terms of their net effect of controlling versus enhancing Legionella growth, depending on the status of other factors. For example, copper pipe achieves its best antimicrobial efficacy without corrosion control, but absence of corrosion control also leads to elevated iron and depleted chlorine residual, which in turn enhance Legionella growth. Such interactive effects can help explain why prior studies reported relatively low levels of Legionella in single family homes, which tend to have greater stagnation and more copper in water from copper service lines and plumbing, compared to large multi-story buildings during the Flint Water Crisis.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: PCR confirmation of unique morphologies that were identified morphologically as Legionella.

Conclusions
The findings of this study are consistent with the understanding that L. pneumophila is not uncommon in municipal water flowing into buildings [49], but a range of water chemistry and premise plumbing conditions and disinfectant residuals can prevent their proliferation. Persistent disinfectant, including chlorine, is known to be a critical factor in reducing Legionella risk [35,37,39], and a recent study confirmed predicted associations between low levels of chlorine in Flint's distribution system and observed incidence of LD [5]. Consistent with our prior in-field observations [1,3], we further demonstrate under controlled laboratory conditions the importance of considering interactive effects with flow and pipe materials, particularly with respect to relative water corrosivity and influence on residual chlorine levels, in keeping Legionella levels low. Indeed, many individual factors can act as "two-edged swords" in terms of their net effect of controlling versus enhancing Legionella growth, depending on the status of other factors. For example, copper pipe achieves its best antimicrobial efficacy without corrosion control, but absence of corrosion control also leads to elevated iron and depleted chlorine residual, which in turn enhance Legionella growth. Such interactive effects can help explain why prior studies reported relatively low levels of Legionella in single family homes, which tend to have greater stagnation and more copper in water from copper service lines and plumbing, compared to large multi-story buildings during the Flint Water Crisis.