Residential water heater cleaning and occurrence of Legionella in Flint, MI

Journal Pre-proof Residential water heater cleaning and occurrence of Legionella in Flint, MI William J. Rhoads, Taylor N. Bradley, Anurag Mantha, Lauren Buttling, Tim Keane, Amy Pruden, Marc A. Edwards PII:

S0043-1354(19)31216-3

DOI:

https://doi.org/10.1016/j.watres.2019.115439

Reference:

WR 115439

To appear in:

Water Research

Received Date: 26 September 2019 Revised Date:

13 December 2019

Accepted Date: 22 December 2019

Please cite this article as: Rhoads, W.J., Bradley, T.N., Mantha, A., Buttling, L., Keane, T., Pruden, A., Edwards, M.A., Residential water heater cleaning and occurrence of Legionella in Flint, MI, Water Research (2020), doi: https://doi.org/10.1016/j.watres.2019.115439. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

City Water Quality

Sediment Mobilization to Outlet? Occurrence of and Impact on Legionella?

Sediments Nutrients Cu Surface Area Fe Zn Cl2 Consumption

Effective Removal?

1

Residential Water Heater Cleaning and Occurrence of Legionella in Flint, MI

2

William J. Rhoads1*, Taylor N. Bradley1,2, Anurag Mantha1, Lauren Buttling1,3, Tim Keane4,

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Amy Pruden1, Marc A. Edwards1*

4

1

Virginia Tech, Civil and Environmental Engineering, Blacksburg, VA

5

2

Current affiliation: The American Cleaning Institute, Washington, DC

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3

Current affiliation: Virginia Tech, Department of Population Health Sciences, Blacksburg, VA

7

4

Legionella Risk Management, Chalfont, PA

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*Corresponding author: Marc A. Edwards, e: [email protected], ph: 540-231-7236; William J.

9

Rhoads, e: [email protected], ph: 417-437-2550

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Abstract

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After the Federal emergency in Flint, MI was declared in early 2016 in response to elevated lead-

12

in-water and incidence of Legionnaires’ disease, concerns arose that contaminants in residential

13

water heaters could continue to contribute to poor quality tap water. Here, a comprehensive field

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survey of residential water heaters (n=30) and associated water quality was conducted and the

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subsequent effects of an aggressive manual water heater clean-out was determined, including

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draining the tank and removing sediments via brushing and flushing. Before cleaning, inorganics

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accumulated in the tank sediments did not serve as a source of metals measured at hot water

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outlets. After cleaning, hardness- (calcium, magnesium, silica) and corrosion-associated

19

inorganics (lead, iron, copper, aluminum, zinc) decreased by 64% in samples from sediment

20

cleanout drain valves. Culturable L. pneumophila was only detected in 1 home (3.3%) prior to

21

cleaning and 2 homes (6.7%) after cleaning, thus quantitative polymerase chain reaction was

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 22

used to quantify potential effects on unculturable strains despite the limitation of detecting live

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and dead cells. After the cleaning protocol, Legionella spp. and L. pneumophila gene numbers

24

decreased or remained non-detectable in 83% and 98% of samples, respectively. Homes with

25

less than 0.4 mg/L influent free chlorine tended to have quantifiable Legionella spp. gene

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numbers in water entering the home and had elevated L. pneumophila and Legionella spp. gene

27

numbers throughout the home plumbing. Also, Legionella spp. and L. pneumophila gene

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numbers were highest for water heaters set at or below ~42 °C and significantly decreased >51

29

°C, consistent with the organism’s preferred temperature range. Examination of the only home

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that had culturable L. pneumophila both before and after the cleaning protocol revealed that the

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organism was culturable from several sample locations throughout the home, including in water

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representative of the water main. Notably, the home was located in close proximity to McLaren

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Hospital, where an outbreak of Legionnaires disease was reported, and the water heater had a

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setpoint within the Legionella growth range of 44.2 °C. Considering that other factors were

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more strongly associated with Legionella occurrence and water heater sediment was not

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detectably mobilizing to tap water, it was concluded that water heater cleaning had some

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benefits, but was not an overarching factor contributing to possible human health risks.

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Keywords: Flint, MI; Legionella, water heater, sediment, flushing, risk factors

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1. Introduction

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After switching from Lake Huron to the Flint River as the drinking water source in April 2014,

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widespread distribution system water quality problems occurred in Flint, MI, ultimately leading

42

to a Federal Emergency declaration (Edwards et al., 2015; Rose, 2015; Masten et al., 2016;

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FEMA, 2016). Citywide lead-in-water contamination and elevated lead in children’s blood

44

(Pieper et al., 2017; Pieper et al., 2018; Hanna-Attisha et al., 2016) prompted a switch back to 2

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 45

Lake Huron source water purchased from Detroit in October 2015. In January 2016, authorities

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announced that outbreaks of Legionnaires’ disease had occurred in summers of 2014 and 2015.

47

Although the switch back to the Lake Huron water source in October 2015 reinstated corrosion

48

control and improved disinfectant residuals, there was lingering concern about persistent public

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health threats.

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All available data collected in early 2016 demonstrated relatively low levels of L. pneumophila

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in small single-story buildings (Schwake et al., 2016; Rhoads et al., 2017a; FACHEP, 2016 as

52

cited in Edwards, 2018), suggesting that L. pneumophila occurrence and Legionnaires’ disease

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incidence were primarily associated with large buildings. However, the State of Michigan and

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the Federal Emergency Management Agency were concerned about possible high levels of

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Legionella bacteria in home water systems, especially due to the high rates of water main breaks

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and red water from 2014-2016. Nationally, it is believed that 96% of reported Legionnaires’

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disease is not associated with outbreaks, with the origin of 64% of reported cases never reliably

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being identified (Hicks et al., 2011; Shah et al., 2018). Residential plumbing systems are a

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potential source of unidentified Legionnaires’ disease (Stout et al., 1987; Stout et al., 1992;

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Pedro-Botet et al., 2002; Straus et al., 1996). Byrne et al. (2018) detected L. pneumophila

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serogroup 6 strains in 12% of Flint homes (n=130) in late 2016, which differed from serogroup 1

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clinical strains detected by the urine antigen test. All available clinical isolates were also found to

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belong to serogroup 1, while isolates recovered from tap water in early 2016 were found to

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belong to serogroup 1 or serogroup 6 (Garner et al., 2019), but of a different sequence type of

65

serogroup 6 than that reported by Byrne et al. (2018).

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Following the Federal Emergency declaration in Flint, there was concern that Legionella could

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have colonized water heater sediments during the water crisis, creating a long-term reservoir for 3

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 68

Legionella. Sediments are known to react with disinfectants and reduce their efficacy, while also

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providing nutrients, shelter, and surface area for attached microbial growth, including for

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Legionella (Lu et al., 2015; Qin et al., 2017). Sediments also decrease heat transfer and energy

71

efficiency (Weingarten, 1992). After the Flint Water Crisis, there were also general concerns that

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accumulation of iron and lead from water main breaks and corrosion, might increase disinfectant

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loss, microbial growth, and lead exposure, although hot water is not considered by EPA to be

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potable water used for consumption (drinking or cooking).

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Current recommendations for water heater cleanout, including flushing, vacuuming, and/or

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dissolving sediments, are designed to remove hardness scale (Weingarten, 1992; Widder and

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Baechler 2013). Water heater flushing protocols were developed following the Elk River 4-

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Methylcyclohexanemethanol (MCHM) spill. However, the protocols were not rigorously

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designed, validated, or verified in the field and there are concerns that flushing might have even

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increased consumer exposure to MCHM (Omur-Ozbek et al., 2016). Eventually, science-based

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recommendations were developed (Casteloes et al., 2015; Hawes et al., 2017; Ragain et al.,

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2019) for the water soluble chemicals of concern, but they were not designed for removal of

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contaminants that settled or deposited in plumbing.

84

In surveying the occurrence of Legionella, it is also important to evaluate all relevant factors that

85

could contribute to its proliferation. In particular, water heater set point has been identified as a

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critical parameter for Legionella colonization of residential plumbing systems, with settings less

87

than 49 °C (120 °F) facilitating Legionella growth in the storage tanks (Lee et al., 2010). While

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Michigan code allows residential water heater temperature settings up to 60 °C (140 °F), default

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or resident-preferred temperatures are typically lower due to a desire to decrease energy demand,

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reduce scaling, and prevent scalding. Gas heaters tend to have lower prevalence of Legionella 4

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 91

than electric heaters, likely due to heating from the tank bottom as opposed to side mounted

92

electrical elements (resistors) that create stratification (Alary and Joly, 1991; Dufresne et al.,

93

2011). In addition, while free chlorine levels in the distribution system had improved markedly

94

by summer 2016 relative to during the period the city was using the Flint River water (Rhoads et

95

al., 2017), residents were using little water due to high water bills, readily available bottled

96

water, and fears about the safety of bath water (Roy and Edwards, 2019). There was also concern

97

that the improved disinfectant residuals were not being consistently delivered to the residential

98

plumbing due to low water use.

99

Here we evaluated the hypothesis that a thorough water heater sediment cleaning event would

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improve water quality at hot water outlets by reducing inorganic contamination, disinfectant

101

demand, and Legionella proliferation and release. Herein we conducted a comprehensive field

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sampling campaign during the Flint, MI Federal Emergency response, deploying a rigorous

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water heater sediment cleaning protocol at 30 single-family Flint residences located near the

104

epicenter of the reported Legionnaires’ disease outbreaks, in a portion of the distribution system

105

with high water age near McLaren Hospital (MDHHS, 2018). The goal was to provide insight

106

into the potential for water heater flushing to reduce risk of exposure to Legionella and other

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water contaminants following major corrosion or other massive contamination events.

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2. Methods

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2.1 Sample Collection

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Two sampling campaigns were conducted in July 2016, including 30 homes near the two

111

hospitals in Flint, MI where the majority of the Legionnaires’ disease cases and highest water

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ages wer reported. Six samples were collected from each home before and 5-6 days after

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Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 113

performing the water heater cleaning protocol. Three of the samples were stagnant first draws

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from the (1) cold kitchen outlet, (2) hot kitchen outlet, and (3) shower head or bathtub spout

115

sample of mixed hot and cold water. Further, samples were collected from the (4) kitchen outlet

116

flushed hot water after a stable hot water temperature was reached, (5) water heater drain valve,

117

and (6) an outside hose bib or outlet nearest to the point-of-entry to the home after 5-minutes of

118

maximum flow rate flushing. The last sample was intended to be representative of influent water

119

to the house from the main distribution system.

120

The four stagnant samples were collected in two sequential 1 L bottles for biological and

121

inorganic chemical analysis, respectively. The first 1 L sterile polypropylene sample bottle was

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pre-dosed with 24 mg of sodium thiosulfate to remove disinfectant residual and, after mixing,

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250 mL was aliquoted for culture into a sterile polypropylene bottle, while the remainder was

124

used for DNA extraction (Appendix A). The second 1 L sample from the outlet was collected in

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an acid-washed high-density polyethylene bottle and temperature, free chlorine, total chlorine,

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and pH were measured in the field. A portion of the sample was filtered in the field with a 0.45

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µm pore size syringe filter (Whatman), where inorganics in the filtrate were operationally

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defined as “soluble”. The remainder of the sample was acid digested to determine total inorganic

129

content. For the hot flushed samples, two consecutive 1 L samples were collected and processed

130

for biological and chemical analysis, as described above, except that an additional 250 mL

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sample was also collected directly from the outlet into a polypropylene bottle dosed with 6 mg

132

sodium thiosulfate for culture. The sampling procedure for cold flushed water was identical to

133

that of the hot flushed samples, except 2 L bottles dosed with 48 mg sodium thiosulfate were

134

collected for molecular analysis to increase the mass of DNA in filter-concentrated extracts from

135

cold water mains, which tend to have less biomass. Samples for culture were shipped overnight

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Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 136

at ambient temperature while molecular samples were shipped overnight on ice. All culture and

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molecular samples were processed within 30 hours of collection.

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2.2 Water heater flushing protocol

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The water heater cleaning procedure had three progressive “levels” of flushing. First, water was

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repeatedly flushed from the bottom drain valve of the tank until it consistently ran clear. Second,

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the water heater was drained and sediments were repeatedly scoured using gravity and

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pressurized flow sprayed into the drained heater via the cold-water influent dip tube. Third, the

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drain valves were completely removed and the interior of the tank was scrubbed with a coarse

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brush to loosen sediments followed by more pressurized scouring with cold water until water

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from the tank ran clear. The heaters were then re-filled with water and outlets flushed to re-

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pressurize the system.

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Meta data about the system and the home (size, age, primary plumbing material) were collected

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onsite.

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2.3 Sample analysis

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Temperature and pH were measured using an Orion 100 Series pH meter calibrated with a three-

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point calibration curve with automatic temperature correction. Free and total chlorine were

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quantified using a hand-held HACH (Loveland, CO) pocket colorimeter using Standard Method

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4500-Cl. The remaining sample was transported to Virginia Tech and acidified with 2% nitric

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acid (or hydroxylamine, for samples with a large amount of visible iron sediment) for

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quantification of inorganics by inductively coupled plasma mass spectrometry (ICP-MS) using

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Standard Method 3125B (APHA, AWWA, WEF, 1998). A total of 28 inorganics are included in

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the ICP-MS suite (limits of quantification are indicated in Appendix B). 7

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 158

2.4 Culture methods

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Water samples were filter concentrated ~50X using a 0.22 µm pore size mixed cellulose ester

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membrane filter, resuspended in the original unconcentrated sample, and plated onto buffered

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charcoal yeast extract (BCYE) media with 0.4 g/L L-cysteine, 3 g/L ammonium-free glycine,

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80,000 units/L Polymyxin B sulfate, 0.001 g/L vancomycin, and 0.08 g/L cycloheximide. A

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matrix of sample treatments, including heat incubation at 50 °C (122 °F) for 30 minutes as well

164

as 1:10 and 1:100 dilutions, were used to increase Legionella culturability. Potential Legionella

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isolates were culture confirmed on BCYE media with and without 0.4 g/L of L-cysteine, and

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then t-streaked, suspended in 50 µL of molecular grade water, frozen at -20 °C (-4 °F) overnight,

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heated to 90 °C (194 °F) on a heat block to lyse cells, pelleted using a microcentrifuge, and the

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supernatant used directly in polymerase chain reaction (PCR) for isolate confirmation.

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2.5 Molecular methods

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Isolates were confirmed using previously published Legionella spp. (23S rRNA), L. pneumophila

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(mip), L. pneumophila serogroup 1 (wzm), and monoclonal antibody group two (MAb2) positive

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L. pneumophila serogroup 1 specific primers (lag-1; Merault et al., 2011; Wullings et al., 2011;

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Kozak et al., 2009; Appendix C). Positive controls included L. pneumophila strain 130b,

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acquired from the Centers for Disease Control. Negative controls included a non-template

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control of molecular grade purity water and an unknown environmental bacterial isolate that

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readily grew on L-cysteine negative BCYE agar plates. Each PCR reaction was analyzed in

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triplicate and product size was confirmed using gel electrophoresis with low-mass ladder

178

quantification on a 1.2% agarose gel.

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Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 179

Environmental DNA was extracted from filter concentrated (as before) samples using a

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commercially-available DNA extraction kit (FastDNA Spin Kit, MP Biomedicals). Legionella

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spp. (23S rRNA) and L. pneumophila (mip) were enumerated by quantitative PCR (qPCR) using

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previously published methods (Nazarian et al., 2008; Wang et al., 2012). Though qPCR detects

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and quantifies both live and dead cells, causing potential false positive results, relative

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comparisons between levels occurring at the point of entry to a home and at individual outlets

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can be used as an indicator for potential regrowth (e.g., Rhoads et al., 2016). Thus, qPCR was

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applied to make relative rather than absolute comparisons. Briefly, a dilution curve performed on

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a subset of sample types revealed that a 1:10 dilution was adequate to minimize qPCR reaction

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inhibition. For each qPCR run, serially-diluted standards prepared from M13 PCR primer-

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extracted clone plasmids, a non-template control, and a spike and recovery on environmental

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samples to confirm absence of inhibition on a subset of samples were run in triplicate wells. The

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quantification limit was set at 50-100 gene copies/reaction based on the lowest standard DNA

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template that consistently appeared with a standard curve efficiency of greater than 80% and R2

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greater than 0.98. Any sample that had two out of three wells appear above the quantification

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limit was scored as quantifiable, while any sample that appeared to be inhibited was re-analyzed.

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Otherwise, results were scored as non-detectable and entered as 0 gc/mL or below the

196

quantification limit (BQL) and entered as half of the quantification limit (25 gc/mL) for

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statistical analyses, as appropriate. The log(enumeration +1) is reported. Samples for 3 homes

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were inadvertently discarded before analysis and thus only samples from 27 homes were

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included in the DNA analysis portion of this study.

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2.6 Data analysis and statistics

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Plots and statistical tests were constructed in RStudio (R version 3.2.0) and detailed as

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appropriate in the text.

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3. Results and Discussion

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3.1 Qualitative notes on cleaning protocol

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For new heaters (<1 year old) or a few heaters that had been flushed regularly by the homeowner

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(n =3), only 30-45 minutes was required to perform the entire cleaning protocol and achieve

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clear flushed water. However, in the typical case (n=21), the procedure required between 90-120

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minutes before the water from the tank ran clear of sediment. For worst-case heaters (n = 6),

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clear water was never achieved and flushing was discontinued at 2 hours.

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During the second week of sampling, after the heaters had already been cleaned once, we

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selected two heaters that required typical effort to clean during the first week of sampling (one

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tank >6 and one <6 years old) and two heaters that had been identified as worst case for further

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testing. For these heatrs, we repeated a flushing protocol that included draining the tank and

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scouring the sediment with pressurized water twice. The two heaters that had been relatively

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easy to clean remained clear during the follow-up flushing (Figure 1A and B). However, both

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heaters that had been difficult to clean still had noticeable particulates running from the tank

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(Figure 1C – red iron corrosion byproduct; Figure 1D – white particulates). The sediment

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removed during the follow-up flushing in Figure 1C did not settle in a graduated cylinder over a

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5-minute period, unlike the sediment collected from the initial procedure at this home (Figure

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1E), suggesting that sediments removed during the initial cleaning were larger particulates.

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3.2 Water tank sediment quantity and composition

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Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 222

The cleaning protocol decreased the amount of accumulated sediments released from the water

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heater drain valve. The sum of all inorganic constituents measured by ICP-MS were used as a

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measure of total inorganic mass in samples. Cold distribution system water (collected at the hose

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bib after 5 minutes of flushing) is representative of soluble inorganics levels of 42.8-49.9±2

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mg/L both before and after the cleaning protocol was performed. Particulate inorganics coming

227

from water heater drain valves were quantified by subtracting total concentrations of each

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inorganic measured in flushed cold water from the water heater drain valve samples at each

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home to account for inorganics in the cold influent water to each home. Before the first cleaning,

230

the water heater drain valve samples contributed 23.8 mg/L excess inorganics on average (~54%

231

higher than the level dissolved in distribution system water; with high standard deviation 79.0

232

mg/L). In the follow-up sample after the water heater cleaning protocol, the total excess mass

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released from the water heater tank sediment was 8.6 mg/L on average (~19% higher than

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distribution system water; with standard deviation 32.9 mg/L), reflecting lowered concentrations

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of scoured particulates after cleaning (Table D1; Figure 2). Thus, the total mass of particulate

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inorganics released decreased by 64%, or an average of 15.2 mg/L, after the cleaning was

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performed (paired Wilcox Test p-value=0.00086, n=30; Table D1; Figure D1).

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The water heater sediment was found to consist of a combination of hardness-associated

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components (magnesium, silica, calcium) and metal corrosion-related solids (magnesium,

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aluminum, phosphorus, iron, copper, zinc, lead) (Figure 3). These constituents are associated

241

with precipitates due to heating of the water (calcium), corrosion of water heater components

242

(sacrificial magnesium or aluminum anode rod), and precipitated or mobilized corrosion scale

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from iron water mains, service line, or premise plumbing (phosphorus, iron, lead). Before the

244

cleaning, calcium, aluminum, phosphorus, and iron accounted for 70.0% of the particulate

11

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 245

inorganics on average (range: 3.6-97.7%; median: 79.7%). The mass of all particulates decreased

246

significantly after the cleaning protocol (paired Wilcox Test, p-value = 1.32×10-5-0.049, n=30).

247

There was no correlation between water heater age (which ranged from <1 to >10 years old) and

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any particulate inorganics released before or after the cleaning (Spearman Rank Correlation, p-

249

value=0.066-0.18, n =30), with the exception of calcium (rho=0.57, p-value=0.0049, n=30;

250

Appendix E). Primary home plumbing material did not impact the total particulate inorganics,

251

iron, or copper released from the drain valves before or after the cleaning (Kruskal-Wallis Test,

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p-value=0.076-0.86, Table F1-F3). However, there was more zinc particulate released in homes

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with galvanized plumbing before the water heater cleaning was performed (Dunn Test with

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Bonferroni posthoc correction, p-value=0.020), but not after the cleaning protocol (p-

255

value=0.94). Total zinc and iron concentrations were significantly correlated in stagnant cold and

256

all hot water samples (Spearman Rank correlation, p-values=5×10-8-0.008; Table G1), but not in

257

flushed cold water representative of the distribution system. This suggests that a significant

258

source of the accumulated sediments in these homes was due to corrosion of the galvanized

259

plumbing.

260

3.3 Impact of water tank sediment on inorganics at hot outlets

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The only constituents higher in hot stagnant or flushed water samples versus cold water samples

262

before the cleaning were magnesium and aluminum (Mg, Kruskal Wallis, p-values=0.0083-

263

0.0133; Al p-value=0.0007; Table G3), which is expected due to their presence in water heater

264

anodes. The absolute elevation in aluminum (52% increase, or 56 ppb higher relative to 108 ppb

265

in cold distributed water) and magnesium (<3% increase, or 200 ppb higher relative 7,890 ppb in

266

cold distributed water on average) in the hot water lines was relatively low. Only 4% of hot

267

stagnant and 9% of hot flushed samples exceeded the 15 ppb action level for lead, which is in the 12

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 268

range of the 3-9% of cold water samples (Table G2). Although 69% of drain valve samples

269

exceeded 15 ppb, this is not unexpected given that the action level does not apply to hot water,

270

especially drain valves, because it is not meant for human consumption.

271

3.4 Water quality parameters

272

Temperature and chlorine from water main samples were not significantly different before and

273

after the cleaning protocol. Median free chlorine residuals delivered to homes were consistent at

274

0.59-0.60 mg/L during the study (Table 1). Once in the home plumbing, the chlorine residual

275

decayed to varying degrees and levels were slightly higher (0.01-0.07 mg/L, comparing medians

276

before/after cleaning) after the cleaning. This was likely due to flushing (increased water use and

277

high velocity) associated with the cleaning protocol and variable stagnation times, which were

278

not assessed in this study. Chlorine was routinely detected in cold stagnant samples (median 0.41

279

and 0.50 mg/L, before and after the cleaning, respectively), but was lower in hot water samples,

280

as expected, due to the water residence time in the water heater storage tank and faster chlorine

281

decay at higher temperature (median 0.01-0.20 mg/L across all hot water samples). Water heater

282

temperature set point, quantified by the hot flushed water temperature, ranged from 33.4 – 67.4

283

°C (92.1-153.3 °F; median 53.3 °C (127.9 °F); Table 2).

284

3.5 Culturable Legionella pneumophila

285

Culturable L. pneumophila was detected in only two homes sampled in this study (6.6%). Before

286

the cleaning protocol, one home tested positive for culturable L. pneumophila in the flushed hot

287

water, the water heater drain valve, and even the cold flushed water sample thought to be

288

representative of the distribution system. After the cleaning, this same home still yielded

289

culturable L. pneumophila in all the stagnant samples collected from distal locations (cold and

13

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 290

hot water at the kitchen outlet and at the showerhead). Another home was found to have

291

culturable L. pneumophila in the hot stagnant sample only; interestingly, this was after the

292

cleaning. All isolates identified in this study were MAb2-positive serogroup 1 strains (i.e.,

293

positive for lag-1 and wzm genes by PCR) and classified as Sequence Type 192 via whole-

294

genome sequencing described elsewhere (Garner et al., 2019).

295

The home that was positive both before and after the cleaning was re-sampled in August 2016

296

(Garner et al., 2019). In this follow up sampling, only the stagnant shower sample was positive

297

and the isolate was again identified as a Mab2-positive L. pneumophila serogroup 1 strain by

298

PCR, but was classified as Sequence Type 1 instead of 192 (Garner et al., 2019). There is

299

precedence for multiple sequence types of L. pneumophila being identified at the same location

300

(Bedard et al., 2019). Notably, the August 2016 Sequence Type 1 isolate is the same sequence

301

type identified in 3 (of 10) Legionnaires’ disease clinical isolates collected from 2015 examined

302

by Garner et al. (2019) and differed from one clinical isolate by only 38 SNPs via single

303

nucleotide polymorphism analysis. This was the highest similarity of any environmental isolate

304

compared to clinical isolates examined by Garner et al. (2019).

305

The one home with culturable L. pneumophila both before and after the cleaning, including in

306

the sample representative of water entering the home, was located in close geographical

307

proximity to McLaren Hospital (~0.3 miles direct sight; 1.6-1.9 miles by road), which was

308

associated with 51 of the 90 Legionnaires’ disease cases that occurred during 2014-2015

309

(MDHHS, 2018). It also had several risk factors that deviated somewhat from other homes in

310

this study. The free chlorine levels entering the home (0.36 ppm as Cl2) was on the lower end of

311

that entering other homes (median = 0.59-0.60 mg/L). This home was also located towards the

312

end of an hydraulically isolated neighborhood within a high water age region of the Flint 14

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 313

distribution system (estimated to be 9 or more days; Arcadis, 2018), which likely contributed to

314

lower residuals and potentially the isolate detected in water entering the home. The hot water set

315

point (44.2 °C) was more than 9 °C below the average water heater temperatures in this study

316

and in the high risk range for Legionella growth (Yee & Wadowsky et al., 1982). Several

317

samples had elevated iron levels (>250 µg/L) compared to other homes (median=41.3-50.9 µg/L

318

before/after cleaning). In addition, our team found that this water heater was one of the three that

319

were very difficult to clean. While all of these factors, individually or in combination, could

320

increase the likelihood of Legionella detection, there were at least some other homes that had

321

greater individual risk factors, but which did not yield culturable Legionella or elevated levels of

322

Legionella spp. or L. pneumophila gene numbers, which are presented in section 3.6.

323

The other home that had culturable Legionella, and only in the hot stagnant sample after

324

cleaning, had no significant risk factors given the high water temperature set point (>52 °C) and

325

influent free chlorine concentrations (0.6-0.71 ppm as Cl2 before/after cleaning). This home also

326

had relatively low iron levels (only one sample slightly elevated at 88 µg/L and spikes of iron did

327

not occur as in the other home with culturable L. pneumophila). However, the water heater was

328

again one of three heaters judged very difficult to clean. Even after six attempts to clean the

329

heater, the water was still turbid. In sum, 2 of 6 of homes with difficult to clean heaters also

330

happened to yield culturable L. pneumophila.

331

The reported positivity rates for culturable L. pneumophila serogroup 1 of 1.8% of samples (n=7

332

of 372) and 6.7% of homes (n=2 of 30) are comparable to some prior reports. Byrne et al. (2018)

333

detected L. pneumophila serogroup 1 in only one of 130 homes (0.7%) in Flint from September-

334

October 2016. However, Byrne et al. identified L. pneumophila serogroup 6 in 12% of residences

335

(n=130) sampled. In general, L. pneumophila serogroup 1 is thought to be the dominant agent of 15

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 336

Legionnaires’ disease in the US, as observed in a survey of sputum cultures collected from 508

337

patients (L. pneumophila serogroup 1 was identified in 84.2% sputum cultures; Yu et al., 2002).

338

Further, MAb2 is considered to be a strong indicator of virulence, e.g., in on study it was

339

detected in 75% of 100 clinical isolates and only 8% of 50 environmental isolates (Kozak et al.,

340

2009). Still, it must be acknowledged that clinical diagnostics of Legionnaires’ disease is

341

inherently biased towards serogroup 1 strains, both because of the urine antigen test and non-

342

pneumophila species tend to be more difficult to isolate (Mercante and Winchell, 2015; Lee et

343

al., 1993; Lucas et al., 2011).

344

Given that Flint had major Legionnaire’s disease outbreaks in 2014 and 2015 that were largely

345

linked to a hospital building (MDHHS, 2018), the fact that this study and Byrne et al. (2018)

346

independently report very low rates of L. pneumophila serogroup 1 in homes sampled in 2016,

347

and a third study reported non-detection in household water samples in late 2015 (Rhoads et al.,

348

2017), is somewhat of a mystery because the residential water systems in Flint had multiple risk

349

factors for colonization by Legionella (Table 3). In particular, 60% of homes in this study had

350

water heater temperature settings lower than the recommended levels to inhibit Legionella

351

growth and one quarter of home water heater temperature settings were at very high-risk

352

temperatures of 33.4-46.3° C (92-115° F). We speculate that one reason incidence of Legionella

353

was so low is because the majority of water heaters (86%) sampled in this study were gas, even

354

though efforts were made to identify and sample the higher risk electric water heaters (Alary and

355

Joly, 1991).

356

3.6 Molecular markers for Legionella

357

3.6.1 Impact of the cleaning protocol

16

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 358

The proportion of samples with detectable Legionella gene copies (i.e., positivity) decreased

359

after the water heater tank cleaning. Legionella spp. positivity decreased from 73% (before) to

360

40% (after) and L. pneumophila positivity decreased from 23% to 4% (Table 4). Legionella spp.

361

positivity decreased across all sample types after the cleaning protocol except cold stagnant

362

samples (Table 4; McNemar Test p-value = <0.0001-0.023). The change in L. pneumophila

363

positivity was less frequently significant among individual sample types, likely due to the low

364

number of positive samples both before and after the cleaning protocol. Samples that were

365

negative before the cleaning tended to remain negative after the cleaning. Overall, only 5

366

samples that were negative for Legionella spp. before the cleaning were positive after the

367

cleaning, and only 1 sample negative before cleaning was positive afterwards for L. pneumophila

368

(Table H1).

369

3.6.2 Trends in Legionella gene marker occurrence

370

Though there were low levels of Legionella spp. and L. pneumophila gene copy numbers in

371

samples overall (Table H2-H4; Figure H1 and H2), influent conditions appeared to be an

372

important factor for detecting sporadically high levels in individual homes. For instance, there

373

were 20 homes (nbefore=12; nafter=8) with quantifiable levels of Legionella spp. in the cold flushed

374

water sample representative of water entering the home (i.e., 5 minutes flushing at hose bib or

375

nearest outlet to the point of entry). These homes had significantly more Legionella spp. and L.

376

pneumophila DNA within the home plumbing compared to homes without quantifiable

377

Legionella spp. DNA in water entering the home (Kruskal Test, p-value=1.68×10-16 and

378

3.98×10-8, respectively; Figure 4 A & B). However, Legionella spp. and L. pneumophila gene

379

copies did not increase within these homes relative to the cold water entering the home (Figure 4

380

A & B). 17

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 381

In addition, while there was no significant correlation between free chlorine and overall

382

Legionella spp. or L. pneumophila gene copy numbers (Spearman rank p-value=0.65-0.76;

383

Figure H3), or even amongst the different types of samples (p-value=0.15-0.85), homes with

384

influent free chlorine <0.4 mg/L (n=11) contained significantly higher Legionella spp. (but not L.

385

pneumophila) copy numbers than homes that had influent free chlorine >0.4 mg/L (Kruskal Test,

386

p-value 1.21×10-6 and 0.68, respectively; Figure 4 C & D). Of the eight homes with median

387

Legionella spp. gene copy numbers from all samples above the quantification limit, six of them

388

had free chlorine levels less than 0.4 mg/L. The home with culturable L. pneumophila both

389

before and after the cleaning had low influent free chlorine (0.24-0.36 mg/L). This home also

390

had the highest levels of Legionella gene markers, on average, an order of magnitude more

391

Legionella spp. and L. pneumophila gene copies than the 90th percentile of all samples (104.1 vs

392

103.2 gc/mL and 102.6 vs below the quantification limit of ~101.4 gc/mL, respectively).

393

It is widely recognized that lower chlorine residuals at the point of entry to buildings can be a

394

risk factor for Legionella colonization (e.g., Strauss et al., 1996), but a target disinfectant residual

395

for controlling Legionella in homes has not been specified. There is no work in residential water

396

heaters specifically, or residential hot water systems in general, that specifies a residual to be

397

achieved and maintained to inhibit Legionella colonization. The American Society of Heating,

398

Refrigeration, and Air-conditioning Engineers (ASHRAE) once proposed a minimum cold water

399

influent chlorine residual of 0.5 mg/L is desirable in a public draft version of ASHRAE 188

400

(ASHRAE, 2011), but this was not included in the final version likely because it was based on

401

anecdotal evidence and was unclear how this would be enforced (Rhoads et al., 2012). Though

402

the proposed ASHRAE target influent chlorine concentration appears consistent with this work,

403

it still is not clear how this would translate to large building plumbing systems with more

18

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 404

complicated plumbing and higher water retention time. However, a recent survey of main water

405

distribution systems reported that 69.2% of L. pneumophila culture-positive samples (only 13 of

406

317 total samples were positive) also had free chlorine residuals <0.5 mg/L (LeChevallier, 2019),

407

which is consistent with the findings of this study.

408

Elevated stored hot water temperatures were associated with decreased positivity of Legionella

409

spp. and L. pneumophila gene copy numbers in hot water samples. Samples were binned into

410

four broad categories based impacts of temperature on Legionella growth predicted based on a

411

literature survey: <32 °C (survival or moderate growth up to 32 °C; <89.6 °F), ≥32 °C and <42

412

°C (optimal growth range; ≥89.6-107.6 °F), ≥42 °C and <51 °C (moderate growth or survival;

413

≥107.6-123.8 °F), and ≥51 °C (death; ≥123.8 °F). Though not statistically significant, Legionella

414

spp. and L. pneumophila levels trended higher with increasing temperatures up to approximately

415

42 °C (107.6 °F; Figure 5). Above 42 °C (107.6 °F), quantifiable Legionella spp. (Spearman

416

Rank, p42-51=0.017; p>51=0.045) and L. pneumophila (p>51=0.039) significantly decreased as

417

temperature increased. Samples with temperatures ≥51 °C (≥123.8 °F) also had significantly less

418

Legionella spp. gene copies than either <32 °C or 32-42 °C (<89.6 °F or 89.6-107.6 °F; Dunn

419

Test with Bonferroni correction, p-value = 0.0002 and 0.0031, respectively). An identical result

420

showing lower levels of Legionella spp. gene copy numbers >42 °C (>107.6 °F) was reported in

421

an Australian survey of 68 homes (Hayes-Phillips et al., 2019).

422

There were no significant differences in Legionella spp. or L. pneumophila gene copy numbers

423

based on primary plumbing material (Kruskal-Wallis Test, p-value=0.51-0.91), nor were there

424

differences in categories of water heater ages (≤2, 2 to ≤5, 5 to ≤10, and >10 years old) (Kruskal-

425

Wallis Test, p-value=0.081-0.13). Among the inorganics analyzed, only the copper concentration

426

was a consistent and significant positive predictor of Legionella spp. gene concentration across 19

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 427

all

samples and

ordinary least

squares (OLS)

multiple linear regression

models

428

(ols_step_forward(), ols_step_backward(), ols_step_best_subset, p-value=0.0016). However, all

429

possible models were poor in predicting the variability of in Legionella spp. gene numbers

430

(Adjusted R2<0.05) and copper was not generally correlated with Legionella spp. gene numbers

431

(Spearman Rank Correlation, p-values by sample type ranged 0.06-0.29). This suggests that

432

levels of Legionella spp. gene copy numbers in the water collected may co-vary with elevated

433

copper and low chlorine residuals, which may simply serve as indicators for stagnation within

434

domestic plumbing drinking water systems. During stagnation, free chlorine can react with

435

cupric hydroxide solids on copper pipe walls (Nguyen et al., 2011), rapidly eliminating the

436

disinfectant residual in stagnant building plumbing. Other researchers have sporadically

437

documented that inorganic or metal concentrations have played a role in Legionella building

438

colonization, including zinc and copper (e.g., Borella et al., 2004), but do not typically collect

439

enough information to evaluate if the incidence of these metals are simply co-factors during

440

stagnation.

441

4. Conclusions

442

The one-time cleanout of sediment accumulated in Flint, Michigan water heater tanks:

443

•

Reduced the amount of particulate inorganics released from the drain valve;

444

•

Had no major impact on the level of inorganics sampled at other outlets (kitchen, shower)

445

within homes, indicating that water heater sediments do not serve as a constant source

446

(i.e., sink) of inorganics at hot water point of use outlets; and

447

•

At least temporarily reduced the occurrence and levels of Legionella spp. and L.

448

pneumophila gene copy numbers, possibly due to scouring of particulate (sediment) and

449

biofilms by high velocity flushing of cold water from the distribution system associated 20

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 450

with the heater flushing protocol. However, the flushing protocol also introduced large

451

amounts of distribution system water to the home, which delivered free chlorine residual

452

and may have also contributed to the lower levels of gene copy numbers.

453 454

Quantifiable levels of Legionella spp. and L. pneumophila within homes: •

455

Co-occurred with free chlorine levels that were below 0.4 mg/L in flushed cold influent water to the home from the main distribution system;

456

•

Were negatively correlated with temperature when water temperature was >42 °C, and;

457

•

Co-occurred with quantifiable levels of Legionella spp. in flushed cold influent water.

458

Finally, the detection of culturable L. pneumophila serogroup 1 (sequence type 192) in flushed

459

cold influent water of one home in this study occurred in a part of the system with high water age

460

and relatively low chlorine residual, indicating distributed water can be a source of Legionella to

461

buildings. At-risk buildings, as defined by ASHRAE 188, should consider routine monitoring of

462

water quality at the point of entry as well as within the building plumbing.

463

5. Acknowledgments

464

We would like to thank the Flint residents that participated in this study. We would also like to

465

thank members of the Flint Water Study team for dedicating their time to conduct the sampling,

466

in particular: Pan Ji, Rebekah Martin, Laurel Strom ,Owen Strom, Ni Zhu, Kimberley Hughes,

467

Mariana Martinez, David Otto Schwake, Jeffrey Parks, Dongjuan Dai, Haniyyah Chapman,

468

Philip Smith, Ethan Edwards, Ailene Edwards, Connor Brown, and Kandance Donalson.

469

6. Funding

21

Rhoads et al., Residential Water Heater Cleaning and Legionella Occurrence 470

This work was funded by a grant from the Michigan Department of Environmental Quality

471

(MDEQ). The author's views expressed in this publication do not necessarily reflect the views of

472

the MDEQ.

473

7. Conflicts of Interest

474

Dr. Edwards has been subpoenaed as a fact witness in several criminal cases related to the Flint

475

Water Crisis. Otherwise, we have no conflicts to declare.

476

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Figure 1. Drain valve samples collected 4-6 days after water heater cleaning procedure occurred; Photographs (top view of 1-L graduated cylinder) were taken after draining the water heater and scouring one time with cold pressurized water for A) a gas heater <6 years old that was easy to clean, B) a gas heater >6 years old that was easy to clean, C) an electric heater >6 years old that was difficult to clean, D) an electric heater >6 years old that was difficult to clean, E) original cleaning of water heater sediments collected from same tank as C, fully settled during the first sampling but they remained suspended during the follow up sampling.

100%

Percentile

75%

50%

25%

Dist. Sys. Water Drain Valve Before Cleaning Drain Valve After Cleaning

0% 40

50 60 Total Inorganics (mg/L)

70

Figure 2. Cumulative distribution function (CDF) of the total mass of inorganics measured in the distribution system water and water heater drain valve samples before and after cleaning the water heaters. Total inorganics are defined as sum of all inorganic constituents measured by ICP-MS (Appendix B). The distribution system water samples from both before and after the cleaning are included in the CDF.

Figure 3. Particulate concentrations for the ten most abundant inorganic constituents measured in the water heater drain valve samples (n=30) (i.e., Particulate Mg = total Mg from drain valve – soluble Mg present in flushed water main sample). Boxplots represent the interquartile range (IQR), whiskers represent a deviation of ±1.5*IQR from the median, and outliers to those ranges are plotted as individual points. y-axis truncated for convenience (truncated high levels include: Mg 31.3 mg/L; Al 5.7, 8.1, 12.5, 21.3, 27.5, 27.6,133.8 mg/L; Si 10.9; P 6.9, 13.9, 14.0, 61.4; Ca 5.7, 5.9, 6.0, 8.8, 10.5, 29.0, 288.5; Fe 7.0, 8.5, 10.8, 14.7; Mn 21.2.

Table 1. Summary statistics for free chlorine levels (ppm as Cl2) in each sample (n=30 for each sample before and after cleaning)

Sample Cold Stagnant Hot Stagnant Shower Stagnant Hot Flush Drain Valve Cold Flushed

Avg 0.44 0.06 0.09 0.13 0.22 0.60

Chlorine (ppm as Cl2) Before Cleaning After Cleaning Min Max Median Avg Min Max 0.0 0.93 0.50 0.37 0.0 0.80 0.0 0.59 0.01 0.07 0.0 0.45 0.0 0.53 0.03 0.14 0.0 0.81 0.0 2.0 0.03 0.10 0.0 0.38 0.02 0.96 0.18 0.21 0.01 0.68 0.01 1.16 0.59 0.55 0.14 0.87

Median 0.41 0.04 0.10 0.08 0.20 0.6

Table 2. Summary statistics for temperature (°C) of each collected sample; data from before and after cleaning events is combined because there were no differences between the two sampling events (n = 30 for each sample, before and after cleaning). Sample Cold Stagnant Hot Stagnant Shower Stagnant Hot Flush Drain Valve Cold Flushed

Avg 21.2 35.4 43.1 52.6 43.4 17.9

Min 17.2 17.3 15.5 33.4 20.6 16.2

Max 26.9 55.2 64.0 67.4 58.3 21.2

Median 19.8 35.1 47.0 53.3 44.5 17.5

* Note: Temperature in stagnant samples was measureed from the second 1 L sample (see Appendix A); therefore, temperature in these samples is impacted by amount of time the outlet was stagnant prior to sampling and the size of the building plumbing system (in some systems, 2 L could potentially contain water from the water heater).

Table 3. Colonization factors in residential hot water systems identified by two seminal studies Risk factor Electric (vs oil or gas) heater

Condition in Flint Homes 17% of heaters (n=5 of 29)

Reference Alary and Joly, 1991; Straus et al., 1996; Dufresne et al., 2012

Lower water heater set point <46-48 °C highest risk

20-24% (n=6-7 of 29)

<54 °C moderate risk Heater Age >7.2 years

58% (n=17 of 29) 45% of heaters (n=11 of 24)a

Lee et al., 1988; Stout et al., 1992; Mathys et al., 2012 Alary and Joly Alary and Joly, 1991

Influent Cl2 <0.5 ppm

32% of Cold Flushed (n=19 of 60)b

ASHRAE, 2011c

a

Some water heater age data was not available. If not available, experienced plumbers assisting our team estimate the age based on manufacturing and installation practices (see Appendix E); bIncludes both before and after samples; c 0.5 mg/L as Cl2 was suggested in a draft version of ASHRAE 188, but was not included in the final published version.

Table 4. Number (n+) and percent (%) of samples positive for Legionella spp. and L. pneumophila by sample type. McNemar test pvalue for contingency tables are presented. P-value<0.05 (bold) indicates that the proportion of samples positive for Legionella spp. or L. pneumophila decreased after cleaning the water heaters. Legionella spp. Before Sample

Total n All Samples 162 Cold, Stagnant 27 Hot, Stagnant 27 Hot, Flushed 27 Shower/Bathtub 27 Water Heater 27 Hose Bib 27

n+

(%)

118 18 18 23 15 20 24

(73%) (67%) (67%) (85%) (56%) (74%) (89%)

After n+ 64 13 11 14 6 6 14

L. pneumophila McNemar

(%)

p-value

(40%) (48%) (41%) (52%) (22%) (22%) (52%)

3.5×10-11 0.131 0.023 0.016 0.016 0.002 0.004

Before n+ 38 6 6 7 6 6 7

After

(%)

n+

(23%) (22%) (22%) (26%) (22%) (22%) (26%)

6 1 2 1 1 1 0

McNemar

(%)

p-value

(4%) (4%) (-7%) (4%) (4%) (4%) (0%)

1.1×10-7 0.131 0.134 0.041 0.074 0.074 0.013

Figure 4. Legionella spp. and L. pneumophila gene copy numbers across all sample types (A & B) in homes with < limit of quantification (LOQ) versus >LOQ levels of Legionella spp. in the influent water (n<500=24; n>500=6) and (C & D) in homes with less than or greater than 0.4 mg/L Cl2 in the influent water (n<0.5=17, n>0.5=13). Boxplots represent the interquartile range (IQR), whiskers represent a deviation of ±1.5*IQR from the median, and outliers to those ranges are plotted as individual points.

Figure 5. Scatter plots of (A) Legionella spp. and (B) L. pneumophila gene copy numbers as a function of temperature by category: Survival or Moderate Growth (<32 °C), Optimal Growth (32-42 °C), Moderate Growth or Survival (42-51°C), and Death (>51 °C). Spearman correlation p-values are indicated for each temperature overall (and only considering samples with quantifiable levels of DNA are provided parenthetically). A LOESS local regression (blue line) was fit to quantifiable data with standard error (gray shading) to aid visual data interpretation.

• • • •

L. pneumophila culturability rates were low in Flint, MI homes in 2016 Removing sediment from water heaters decreased Legionella gene markers in some homes Influent chlorine <0.4 ppm was associated with Legionella gene markers in homes Water temperature was a key factor controlling levels of Legionella gene markers

Declaration of interests The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Edwards has been subpoenaed as a fact witness in several criminal cases related to the Flint Water Crisis. Otherwise, we have no conflicts to declare.