Laboratory Observations of Biocide Efficacy in Model Cooling Tower System.pdf

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Laboratory Observations of Biocide Efficacy in Model Cooling Tower Systems

Ian Smith

Jason Eccles

Elizabeth J. Fricker, Ph.D.

Rosalind Searle, Ph.D.

ABSTRACT

This paper provides an overview of ASHRAE Research Project 954findings of the eflcacy of spec@ oxidizing and nonoxidizing biocides examined using a model cooling tower system inoculated with a microcosm containing an environmental isolate oflegionella pneumophila. The activi9 of three biocides was tested against both planktonic and sessile Legionella against dirty systems, with pre-established bacterial microcosms, and also against clean systems subsequently drip f e d with a Legionella seed. The findings of theproject can be used to better understand the likely reaction of Legionella bacteria to biocide dosage programs and assist in the development of future biocide products and strategies.

INTRODUCTION

Legionellae are a significant cause of respiratory infections. Wet-type heat rejection devices have been shown to transmit aerosolized Legionellae and cause outbreaks of Legionellosis. Publicized outbreaks of Legionellosis and diagnosis of these infections have made this an issue of concern to cooling tower operators and the general public.

Various investigators have examined the efficacy of several biocides against Legionellae in flask or tube cultures of cooling water or buffer (Domingue et al. 1988; McCoy et al. 1986; McCoy and Wireman 1989; Skaily et al. 1980). Others (Fliermans and Harvey 1984; Negron-Alvira et al. 1988; Pope and Dziewulski 1992) have looked at the effect of one or more biocides in the changing environment of an open cooling system. A laboratory model of a domestic hot water system was the topic of one paper (Muraca et al. 1987).

This research project was designed to provide a series of model cooling towers that will more closely simulate true

operating conditions (including makeup, system bleed-off, cycling of water chemistry, biocide holding time, and biocide washout), while minimizing some of the unscheduled events that occur in an operating cooling system. A first phase of trial works was undertaken evaluating biocide efficacy for various biocide products for control of both sessile (within biofilms) and planktonic Legionella (Thomas et al. 1999). This present study follows, investigating the effect of dosage of biocides in combination with dispersants, application to assess biocidal activity against dirty systems with preestablished bacterial microcosms and also against clean systems drip-fed with a Legionella seed.

The biocidal activity of three biocides-tetrakis (hydroxymethyl) phosphonium sulphate (THPS), isothiazolin (ISO), and sodium hypochlorite-were tested in various permutations together with a biodispersant, ethylene oxide/ propylene oxide. The trials were carried out in a pilot-scale wet evaporative cooling system, hereafter referred to as the rig. The efficacy of the biocides was assessed by determining their effect on the survival within the system of Legionella pneumophila.

MATERIALS AND METHODS

Legion ella Test I n oc u I u m

The Legionella test organism was obtained from a sample taken from a domestic hot and cold water service. This source was chosen rather than a cooling tower isolate because of concerns regarding the potential conditioning of the microorganism to biocides used in a cooling tower system. The Legionella test organism was identified as L. pneumophila Serogroup 1 Pontiac, monoclonal antibody pattern 1, 2, 5, 6

lan Smith is director ofthe Built Environment Group, First Environment Ltd., Birmingham, U.K. Elizabeth Fricker i s principal research scientist for innovation and development and Jason Eccles and Rosalind Searle are research assistants at Thames Water Utilities Ltd., Reading, U.K.

Thames Water Utilities Limited is not responsible for the opinions expressed in-Laboratory Observations of Biocide Efficacy in Model Cooling Tower Systems and also accepts no responsiblity for either the accuracy of or use of any data or information contained therein.

31 4 02004 ASHRAE.

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and L. pneumophila Serogroup 10 (as identified by the CDC, Atlanta, Georgia, USA).

Cell A B

Model Cooling System

The test rig is designed to simulate a wet evaporative cooling system. The model cooling system test rig consisted of nine small cross-flow cooling tower cells. The test rig arrangement is described in Appendix I.

C D (Control)

MAIN TRIAL

Legionella

Oxidizing biocide

Nonoxidizing biocide

Biodispersant

Prior to starting each trial, the test cells were sterilized using sodium hypochlorite at 100 parts per million (ppm) neutralized with sodium thiosulphate and thoroughly washed through with water.

All nine cells of the rig were set up to run concurrently. The experiment was split into two complementary trials based on the manner of dosing the Legionella into the cells (Tables 1 and 2) , one trial to evaluate biocidal activity against preestablished Legionella and the other against drip-fed Legionella. The biocide and biodispersant dosing regimes for each trial are summarized in Tables 1 and 2. Cells A to D (Table 1) contained a population of Legionella pneumophila established prior to the commencement of biocide dosing (preestablished Legionella trial). Cells E to I (Table 2 ) had no established populations of Legionella prior to dosing; instead, Legionella pneumophila was drip-fed into each cell on a daily basis for the duration of the experiment (drip-fed Legionella trial).

Established Established Established Established

1 -3ppm x 2 daily

THPS (50 ppm) THPS (50 ppm) THPS (50 ppm) IS0 (613 ppm) IS0 (6/3 ppm)

5 PPm 5 PPm

Biocidal activity was assessed against planktonic bacteria by analyzing samples of water taken from each cell at regular intervals throughout the trial. Activity against sessile bacteria was assessed using galvanized steel coupons. Six galvanized steel coupons (surface area of 4.9cm2), attached to titanium wire, were suspended within the circulating waters of each cell. These easily removable coupons were set up to study the buildup of a bacterial biofilm and to measure the effect of the biocides on sessile bacteria, both Legionella and heterotrophs.

For Cells A to D, a biofilm was allowed to develop on the coupons prior to the first biocide dose. For cells E to I, the coupons were placed in the cells immediately prior to the first biocide dose.

Cell

Dosing Regimes Each test cell was subjected to a specific dosing regime as

summarized in Table 1 and Table 2 . The two nonoxidizing biocides were tested under a variety ofpermutations in the pre- colonized and drip-fed cells, i.e., withiwithout an oxidizing agent, chlorine, and withiwithout biodispersant, ethylene oxidelpropylene oxide. In cells where the oxidizing agent was dosed, this was done twice daily to maintain a consistent level, of approximately 2.5 ppm, as total chlorine. In cells where the biodispersant was dosed, this was at the start of the trial at 5 ppm and topped up on a weekly basis to allow for removal of any dispersant through sampling. Details of the dosing regime and concentrations achieved are shown in Figures 1 to 7. Cells D and I were positive controls and no dosing was carried out on these two cells.

E F G H I (Control)

Legionella Drip-fed Drip-fed Drip-fed Drip-fed Drip-fed

~

Nonoxidizing biocide

Biodispersant

Oxidizing biocide 1-3ppm x 2 dailv I

THPS (70/50 pprn) THPS (70150 ppm) THPS (70/50ppm) IS0 (6/3 ppm) IS0 (6/3 ppm)

5 PPm 5 PPm

l 1 -3ppm x 2 dailv 1 I l

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P la n k o n I C L e g i o n e I I a in ._

C e l l A

..Il I I I 1\1 I I I I I I I I I I

Figure I Planktonic levels of Legionella in Cell A after the addition of biocides.

Water Samples

Three 200 mL samples of water were taken from each cell twice a week; 100 mL from these samples was filtered and resuspended in 5 mL of sterile distilled water.

From each 5 mL sample a 100 pL aliquot was removed and a 1 O-fold dilution series was set up ranging from 10" to 10- 6; 100 L of each dilution was plated onto R2A agar and incu- batedat 30C for three days. Colony-forming units (cfu) were then enumerated at the appropriate dilution level.

A 250 yL aliquot was removed from the 5 mL samples and heated to 50C to destroy all non-Legionella bacteria. A 100 uL aliquot was taken from each sample and a dilution series set up ranging from lo-' to lo-*; 100 pl of each dilution was plated onto Legionella-GVPC Selective Medium (Oxoid) plates and incubated at 37C for at least 10 days. Colony- forming units (cfu) were then enumerated.

Biofilm Samples

The coupons were very gently rinsed prior to being placed in a test-tube with 5 mL of sterile distilled water. The test tube was mixed on a vortex for 1 minute. All subsequent analyses were carried out as described above for heterotrophic counts and Legionella counts. For Cells A to D, three coupons were analyzed just before the first biocide dose and the remaining three were analyzed at the end of the trial. For cells E to I, coupons were analyzed at the start of dosing from three of the cells and from all cells part way through the trial and at the cessation of the trial.

Environmental Parameters

monitored on a daily basis by direct reading instruments.

RESULTS

The Legionella results from the water samples taken during the trial are presented graphically with brief text descriptions. For each biocide dosing regime, the

Total dissolved solids (TDS), temperature, and pH were

microbiological data from the cell receiving the biocide can be compared on the same figure with the data from the control cell. Results for planktonic heterotrophs are described for each cell. Data for the biofilm monitoring are presented in Tables 3 and 4.

PREESTABLISHED LEGIONELLA TRIALS (CELLS A-D)

Planktonic Legionella

Dosing Regime: Control (Cell D). At the start of the trial the numbers of Legionella in the control cell were at 3.5 log. The numbers gradually decreased over the weeks until on day 20 there were 1.4 log, which subsequently increased and leveled off at 2.8 log. Cell D was rerun as part of a supplementary trial to repeat Cells B and C. At the start of the supplementary trial, the number of Legionella in the control cell was 1.9 log. Over the next two weeks, the population was maintained at around 2 log after which it dropped to 1.1 log on day 14 and 0.8 log on day 22 before it increased to 2.0 log by the end of the trial.

Dosing Regime: THPS + Oxidizing Biocide + Biodis- persant (Cell A). In Cell A (Figure 1) the dosing regime worked very well. Prior to dosing the biocide, the Legionella population was 2.9 log. Following dosing, there was a rapid reduction in numbers of the Legionella. From day eight onward, no Legionella were recorded in any of the samples taken.

Dosing Regime: THPS Alternating with I S 0 + Biodispersant (Cell B). Dosing started with 6 ppm of IS0 in week 1 followed by 50 ppm of THPS in the second week. In the third week IS0 was dosed at 3 ppm and in the fourth week THPS was dosed at 50 ppm. However, the Legionella in Cell B never established and by the start of the trial there were less than 1 log of Legionella in Cell B as compared to 3.5 log in the control cell (Cell D). With dosing, the numbers rapidly dropped off so that by day six no bacteria were recorded. The biocide regime and setup for Cell B were rerun in conjunction

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P l a n k t o n i c L e g i o n e l l a in C e l l B

- C e l l B -D ispersant -THPS

D a y from start of T r i a l

Figure 2 Planktonic levels of Legionella in Cell B after the addition of biocides.

P l a n k t o n i c L e g i o n e l l a i n C e l l C * n

Figure 3 Planktonic levels of Legionella in Cell C after the addition of biocides.

with Cell C and the control cell, Cell D, at a later date as a supplementary trial. At the start ofthe supplementary trial, the number of Legionella in Cell B was at 1.4 log (Figure 2). There was no discernible change in numbers until day 14 onward, when the numbers of Legionella dropped below detectable levels. On the last day of the trial , some Legionella were recorded (0.8 log).

Dosing Regime: THPS Alternating with IS0 (Cell C). The dosing regime in Cell C was the same as in Cell B but without the biodispersant. Legionella never established in Cell C and the highest numbers recorded (after dosing with ISO) were less than 3 Legionella per mL o f water. Toward the end of the trial, the cell was emptied, cleaned ,and re-spiked with Legionella, but again, the bacteria did not establish. The biocide regime and setup for this cell were rerun as part of a supplementary test.

At the start of the supplementary trial there were more Legionella in Cell C (2.5 log) than in the control cell (1.9 log),

and this rapidly dropped to 1.9 log on day 1 and to 1.3 log on day 6. There was a slight increase in the number of Legionella on day 8 to 2.0 log, after which there were further reductions in the Legionella population to 0.5 log on day 14 and 0.1 log on day 16. For the last two samples taken during the trial, the numbers of Legionella started to gradually increase. It appeared that the numbers of Legionella dropped dramatically after the first and second dose of THPS. Although the numbers appear to recover after the application of ISO, the Legionella numbers subsequently dropped again after the third and fourth doses of THPS.

Planktonic Heterotrophs

Dosing Regime: Control (Cell D). Heterotrophic bacteria are an integral part of any water system and a natural population of these bacteria was established in the rig prior to the start of the trials. In the control cell (Cell D) the natural population was 5.9 log per mL of water. This population



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Legionella Day from start of trial

O 27

Cell A 3.20 0.00

Cell B (Supplementary Tnal) 3.70 2.00

Cell C (Sumlementaw Trial) 3.40 1.50

Heterotrophs Day from start of trial

O 27

5.01 4.18

5.20 6.00

6.30 5.60

remained stable over the trial period, peaking at 6.1 log on day 1 and dropping to 4.6 log on day 27. For the supplementary trial in the control cell, there were 4.2 log number of bacteria at the start of the trial. There was some growth between day 6 and day 8 (peaking at 5.1 log), but the population dropped down to just below 4.0 log on day 14 after which the population stabilized over 4 log for the remainder of the trial.

Dosing Regime: THPS + Oxidizing Biocide + Biodis- persant (Cell A). The heterotrophic population in Cell A was 6.4 log at the start of the trial. It dropped to 5 log on day 1 following dosing and rose to over 7 log on day 6. Subsequently the population appeared to be affected by the biocides and dropped to 2.0 log on day 15, after which a stable population was maintained at this lower level.

Dosing Regime: THPS Alternating with IS0 + Biodis- persant (Cell B). The heterotrophic population in Cell B was 6.5 log at the start of the trial. It dropped to 5.4 log on day 1 following dosing and rose to over 7 log on day 6. The numbers dropped to 5.0 log on day 8 after which they increased and leveled off at around 6 log. The heterotrophic population in Cell B was maintained at a higher level than that in the control cell (Cell D). The biocide regime and setup for Cell B was rerun as a supplementary trial. In the supplementary trial, Cell B had a thriving heterotrophic population throughout. The bacterial population rose from 3.9 log at the start of the trial to 5.5 log on day 8 and 6.0 log on day 16 before gradually dropping to 5.1 log by the end of the trial. Overall a higher population of bacteria was maintained in Cell B than in the control cell, with a mean population of 5.0 log (as compared to a mean population of 4.3 log in the control cell).

Dosing Regime: THPS Alternating with IS0 (Cell C). The heterotrophic population in Cell C was 6.3 log at the start of the trial. It dropped to 5.2 log on day 1 following dosing and rose to over 7 log on day 6. The numbers dropped to 3.0 log on day 8 after which they started to increase prior to the cell being shut down and cleaned. After the cell was restarted, the heterotrophic population in Cell C started to grow rapidly and by the end of the trial (day 27) had reached over 7 log. The biocide regime and setup for Cell C were rerun as a supplementary trial. In the supplementary trial, the heterotrophic population in Cell C started at 4.0 log. It rose to

~ ~~

Cell D (First Trial) 5.04 5.39 5.20 6.00

Cell D Supplementary Trial 2.80 2.50 5.10 5.70

5.7 log on the first day before it dropped back to 4.0 log on day 6. Although the population fluctuated over the first part of the trial, the population numbers stabilized after day 14 at between 4.5 and 4.8 log.

Sessile Legionella and Heterotrophic Bacterial Levels

Results for the sessile Legionella and heterotrophic bacteria for Cells A-D are presented in Table 3. Sampling for sessile bacteria was carried out at the start and at the end of the trial.

In the control cell (Cell D) for the first trial run, Legionella had established well and had reached 5 log by the start of the trial. This population remained stable throughout the duration of the trial; at the end of the trial, the population was at 5.39 log, slightly higher than at the start.

In Cell A the Legionella did not grow as well as in the control cell with just over 3 log of Legionella established. In Cell A the biocide regime totally reduced detectable Legionella by the end of the trial. In Cell B and Cell C for the first trial it was considered that the numbers of Legionella established were insufficient to assess the effectiveness of the biocide, and the biocide regime and setup for Cells B and C were rerun as a supplementary trial in conjunction with the control ceil, Ceil D.

In the control cell (Cell D) for the second trial run, the preestablished Legionella had reached 2.8 log by the start of the trial. The population remained stable throughout the duration of the trial; at the end of the trial, the number of sessile Legionella was 2.5 log, 0.3 log lower than at the start.

In cells B and C, the Legionella had established at higher levels than in the control cell by the start of the trial (3.7 and 3.4 log, respectively). In Cell B the biocide regime had reduced detectable Legionella significantly to 2.0 log by the end of the trial, and in Cell C the Legionella was reduced to 1.5 log by the end of the trial.

The sessile heterotrophic bacteria rapidly established on the coupons in all cells. The population remained stable in the control cell (Cell D) throughout the trial for the initial and supplementary trial runs. There was a small drop in numbers in Cells A and C, indicating a possible effect from the dosing.

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P l a n k t o n i c L e g i o n e l l a i n C e l l E

'i 1

2 3

I E I < b

5 > f s I <

n

w2

D a y s f r o rn 5 t a r t o f T r i a I

Figure 4 Planktonic levels of Legionella in Cell E after the addition of biocides.

t " I

P l a n k t o n i c L e g i o n e l l a in C e l l F r

Figure 5 Planktonic levels of Legionella in Cell F after the addition of biocides.

In Cell B the population increased. At the end of the trial, the heterotrophic populations in Cell B and Cell C were comparable to Cell D for the supplementary trial.

DRIP-FED LEGIONELLA TRIALS (CELLS E-I)

Planktonic Legionella

Dosing Regime: Control (Cell I). Continuously adding Legionella into the control cell resulted in rapid population growth of over 3 log in two weeks with peaks at just over 4 log at three weeks. The control population varied in number between 1.5 to over 4.0 log.

Dosing Regime: THPS + Oxidising Biocide + Biodis- persant (Cell E). In Cell E (Figure 4) the Legionella population did not establish during the trial, indicating

-C e II F

-*- T H P S

effective control of the Legionella using this regime. On day 37 a small number of Legionella were detected (less than 10 bacteria per mL of water), after which no more Legionella were recorded.

Dosing Regime: THPS Alternating with I S 0 + Biodis- persant (Cell F). The first dose, 8 ppm IS0 followed by 70 ppm THPS, was effective in preventing the Legionella from establishing in the cell (Figure 5). Subsequent doses were lower, 3 ppm IS0 followed by 50 ppm THPS, and some Legionella survived (approximately 1 bacterium per mL of sample water).

Dosing Regime: Oxidizing Biocide (Sodium Hypochlorite) (Cell G). Sodium hypochlorite was very effective as a biocide at a concentration level of between 1.5 ppm and 3 ppm (Figure 6 ) . Complete kill (below the level of



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P l a n k t o n i c L e g i o n e l l a in C e l l G *,

, 5 IO 2, $0 D a y s from s ta r t o f Trial ' I - - - -0x id isr 1

Figure 6 Planktonic levels of Legionella in Cell G after the addition of sodium hypochlorite.

P l a n k t o n i c Legionella in C e l l H

D a y f rom s t a r t o f Tr ia l

Figure 7 Planktonic levels of Legionella in Cell H after the addition of biocides.

16 - C e i l H

I - T H P S - c o n t r o l

- I S 0 I detection) was achieved for the majority of the trial; some bacteria (i bacterium per mL of sample water) were occasionally detected.

Dosing Regime: THPS Alternating with IS0 (Cell H). This regime was the same as in Cell F but the biodispersant was omitted (Figure 7). The results are very similar to Cell F with limited Legionella survival (approximately 1 bacterium per mL of sample water).

Planktonic Heterotrophs

Dosing Regime: Control (Cell I). Heterotrophic bacteria are an integral part of any water system, and a natural population of these bacteria was established during the setup of the rig prior to the start of the triais. In the control cell (Cell I) the natural population was 6.3 log per mL of water. This

320

population fluctuated over the trial period, peaking at 7.2 log on day 21 and dropping to 5.0 log on day 35.

Dosing Regime: THPS + Oxidizing Biocide + Biodis- persant (Cell E). The heterotrophic bacteria in Cell E were low at the beginning of the trial (4.1 log) and dropped after the first dose of THPS to 3.2 log. The population then recovered, and although it fluctuated during the trial, a steady population level of 4.6 log was maintained, 1 log lower than that in the control cell.

Dosing Regime: THPS Alternating with IS0 + Biodis- persant (Cell F). The population of heterotrophic bacteria in Cell F was initially lower than that in the control cell, 3.4 log as compared to 6.3 log. The population increased to peak at 6.2 log on day 14, and although numbers fluctuated during the triai, a steady population level was maintained at an average of 5.4 log, the same as in the control cell.

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Table 4. Log,,, cfu per cm2 of Legionella Bacteria and Heterotrophic Bacteria Grown on Coupons Suspended within Cells E to I.

Legionella Day from start of trial

4

23

E F G H I

O O 2.29

O 0.53 O 1.53 5.44

I 43 I 2.29 I 2.28 I O I 1.61 1 4.34 1

4

23

Heterotrophs Day from start of trial

5.3 1 3.31 6.64

4.73 3.95 2.49 4.95 6.44

I 43 I 5.63 I 6.24 I 3.91 I 7.21 I 4.54 I Dosing Regime: Oxidizing Biocide (Sodium

Hypochlorite) (Cell G). At the start of the trial, the heterotrophic bacteria in Cell G were lower than the control (4.1 log as compared to 6.3 log in the latter). The population gradually increased to 6.4 log on day 21. Although the numbers of heterotrophs fluctuated, a steady population was maintained, an average of 4.6 log, 1 log lower than that maintained in the control cell (5.6 log).

Dosing Regime: THPS Alternating with IS0 (Cell H). The population of heterotrophic bacteria in Cell H was initially lower than that in the control cell, 3.4 log as compared to 6.3 log. The population increased to peak at 5.4 log on day 14. The numbers of heterotrophs fluctuated for the rest of the trial with an average of 5.0 log.

Sessile Legionella and Heterotrophic Bacterial Levels

Results for the sessile Legionella and heterotrophic bacteria for Cells E-I are presented in Table 4.

The first samples were taken on day 4 after the coupons had been introduced into the cells. In the control cell, Legionella had reached 2.3 log. By day 23 they had increased to over 5 log and by the end of the trial were above 4 log.

In contrast, the coupons from the cells dosed with biocide had much lower growth. The most effective regime was the oxidizing biocide in Cell G, as no Legionella were recorded. All other cells showed growth of sessile Legionella but in very low numbers compared to the control.

The sessile heterotrophic bacteria became established on the coupons much faster in the control cell than in the dosed cells. However, during the trial the heterotroph numbers gradually increased in all of the dosed cells, so that by the end of the trial the populations in Cells E, F, and H had increased to such an extent that they are much higher than the control. At the end of the trial, the sessile bacteria in the control cell had dropped slightly, indicating that the sessile populations are prone to rapid fluctuations in numbers similar to the way the planktonic populations fluctuate. The only cell in which the

dosing seems to have strongly affected the sessile heterotroph population was in Cell G, where the oxidizing agent was dosed on its own.

Environmental Parameters

Total dissolved solids (TDS) were generally maintained between 650 and 950 ppm. The TDS in Cell A increased over time as water evaporated from the cell but remained more constant in Cells B, C, and the control cell. The temperature fluctuated in response to changes in the ambient temperature, but an average temperature of around 27C was maintained. The pH was consistently within a very limited range between 8.8 and 9.1.

DISCUSSION

Preestablished Legionella Trial (Cells A-D)

Planktonic Legionella. Dosing THPS with sodium hypochlorite in the presence of a biodispersant was successful in reducing the numbers of a preestablished Legionella population and preventing any reestablishment of the organism.

Dosing THPS in alternation with IS0 in the presence of a biodispersant produced a reduction in the numbers of a preestablished Legionella population to below detectable levels; however, Legionella recovered to detectable levels at the end of the trial.

Dosing THPS in alternation with I S 0 without the presence of a biodispersant produced a reduction in the numbers of a preestablished Legionella population, which then fluctuated with a downward trend but remaining at detectable levels throughout. There was consistently a drop in Legionella numbers following the dosing of THPS compared with a general increase following dosage of ISO. It would appear that THPS had a stronger biocidal effect than IS0 against Plank- tonic Legionella.

Planktonic Heterotrophs. In Cells A, B, and C the heterotrophic populations were detected in moderate to high

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numbers, and in Cells B and C they appeared to remain unaffected by the dosing regimes. In Cell A, some effect of the biocide on the heterotrophs was seen as the population was reduced and maintained at a much lower level.

Sessile Legionella and Heterotrophic Bacterial Levels. The sessile Legionella did not establish as well in the test cells as in the control cell. However, the numbers of Legionella were reduced below detection in Cell A and showed just under a 2 log reduction for Cells B and C. Significant numbers of sessile Legionella survived throughout the trial for Cells B and C, indicating that Legionella within the biofilm were buffered against the effects of THPS and ISO. The presence of dispersant did not appear to have any significant effect, as there was no significant difference between Cells B and C.

Drip-Fed Legionella Trials (Cells E-I)

Planktonic Legionella. In the drip-fed cells, all of the biocides significantly reduced the numbers of Legionella pneumophila and prevented increases throughout the trials. The effectiveness of the biocides can be compared:

THPS on its own (Cell E) compared with the alternating regime of THPS one week followed by IS0 the next week (Cells F and H) indicates that THPS performs better than ISO. This is seen by the 0.1 log survival of the Legionella after the dosing of IS0 compared with below detection of the Legionella after dosing of THPS in all three cells. The activity of THPS and IS0 in the presence of biodispersant is no different than the activity of THPS and IS0 without the presence of a biodispersant (Cells F and H). The effect of the biocides THPS and IS0 together with sodium hypochlorite on the planktonic Legionella is similar to dosing with sodium hypochlorite on its own (Cells E and G). The effect of the biocides THPS and IS0 without sodium hypochlorite on the planktonic Legionella is similar to dosing with sodium hypochlorite on its own (Cells F and G).

Planktonic Heterotrophs. All biocides had a limited effect on the overall heterotrophic populations. At most, suppression by 1 log was achieved in Cell E; otherwise, no sustained effect was apparent for any of the other biocides and biocide combinations.

Sessile Legionella and Heterotrophic Bacterial Levels. The biocides had a greater effect on the numbers oflegionella in comparison with the heterotrophic populations. Dosing with sodium hypochlorite on its own resulted in no Legionella (i.e., below detection). Although the nonoxidizing biocide dosing was effective in reducing numbers, some Legionella survived during the trial, and toward the end, there were indications of regrowth. This suggests that the Legionella

within the biofilm were more protected from the effects of the biocides THPS and ISO.

The sessile heterotrophic population remained virtually unaffected by the biocide dosing. The sodium hypochlorite had the greatest effect with some reduction in numbers, but the THPS and IS0 had no effect on the sessile heterotrophic population over the long term.

CONCLUSIONS

This study has shown that both planktonic and sessile Legionella can be established and maintained in a model cooling tower system and be maintained over extended periods. The control cells exhibit characteristics expected of operating cooling tower systems, enabling the effectiveness of biocide applications against Legionella to be effectively measured. The ability of LegoneZla to survive readily in the test cells and its resilience to treatment chemicals to some extent in the planktonic phase and more so in the sessile phase reinforce the importance of applying biocide programs to all operating cooling tower systems. Treatment programs should be established as soon as a system is brought on line after disinfection to prevent Legionella bacteria from colonizing a system at the outset.

The biocides were all effective against planktonic Legionella, with numbers reduced to, and maintained at, low numbers in all of the drip-fed cells and in the preestablished Legionella trials. The heterotrophic planktonic bacteria remained largely unaffected by the biocides, which is not unexpected given current industry knowledge. Against preestablished Legionella populations, only chlorine was effective in reducing and maintaining planktonic Legionella to below detectable levels for the duration of the trial.

There is no apparent difference in the effectiveness of the biocides, both oxidizing and nonoxidizing, against Legionella when dosed in conjunction with a dispersant as in comparison to the effectiveness when dosed without dispersant. Although the study showed no significant difference in this respect, it should not be concluded that dispersants are not required. The model cooling tower system used does not mimic the larger and more complex distribution pipework of many systems or the ingress of airborne contaminant from many industrial operating environments. For these reasons, in many circum- stances the use of dispersants will be a necessary consider- ation.

The numbers of sessile Legionella were significantly reduced by the biocides, but the developing biofilm possibly provided some protection for Legionella. Only dosing with chlorine on its own resulted in total elimination of LegionelZa (below the level of detection). The sessile heterotrophic bacteria remained largely unaffected by the dosing regimes in both the drip-fed and the preestablished trials.

In choosing a biocide program for effectiveness against planktonic and sessile Legionella, the results of this study indi- cate that, where system conditions and water chemistry allow

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it, a continuous or semi-continuous low-level dosing of an oxidizing biocide to achieve a free oxidant residual in the circulating water is likely to be the more effective approach. The levels of total oxidizing biocide necessary to maintain effective free residual levels for a given pH as shown from this study illustrate the conflict between maintaining microbial control and maintaining protection of the system components against corrosion, with levels of total oxidizing biocide in this study maintained in excess of 2.0 ppm. Continuous use of chlorine at 2.0 ppm and above can lead to corrosion of water systems. In areas with elevated pH levels, use of acid dosing in conjunction with the biocide may be necessary to enable effective oxidizing biocide levels to be achieved while balanc- ing the need to protect system components against corrosion.

The test rig has proved a useful tool to enable the quali- tative testing of biocide effectiveness against both Legionella and heterotrophic bacteria under realistic operating conditions. The test rig has been thoroughly cleaned and the packing material replaced prior to each trial to provide a common base against which to compare results. Results are therefore repre- sentative of Clean systems. Further study would be benefi- cial to test the effect of the biocides against systems with a level of deposited material, such as sand or scale, that can be present in many tower systems. It would also be valuable to see if oxidizing biocides other than chlorine are as effective against planktonic and sessile Legionella given the need identified above to balance microbial control against the protection of system waterside components. Finally, improvement in the reliability of the test rig would enable trial runs of extended duration to explore the long-term effects of biofilm development and to assess the level, if any, by which bacteria can become resistant to the biocides applied.

The study has shown significant differences between the effectiveness of different biocides, particularly different nonoxidizing chemical formulations. The correct choice of the active chemical constituents for treatment regimes and verifi- cation of their effectiveness in use are key factors in achieving and maintaining a successful control program.

REFERENCES

Domingue, E.L., R.L. Tyndall, W.R. Mayberry, and O.C. Pancorbo. 1988. Effects of three biocides on L.pneumophila SG 1. Applied and Environmental Microbiology 54: 741-747.

Fliermans, C.B., and R.S. Harvey. 1984. Effectiveness of 1- Bromo-3-Chloro-5, 5-Dimethylhydantoin against L. pneumophila in a cooling tower. Applied and Environ- mental Microbiology 47: 1307- 13 1 O.

McCoy, W.F., J.W. Wireman, and E.S. Lasher. 1986. Effi- cacy of methylchloro/methyl -isothizolone biocide against L. pneumophila in cooling tower water. Journal of Industrial Microbiology 1:49-56.

McCoy, W.F., and J.W. Wireman. 1989. Efficacy of bromo- chlorodimethyl hydantoin against L. pneumophila in

cooling tower water. Journal of Industrial Microbiology

Muraca, P., J.E. Stout, and V.L. Yu. 1987. Comparative assessment of chlorine, heat, ozone and UV Light for killing Legionella Pneumophila within a model plumb- ing system. Applied and Environmental Microbiology 53:447-453.

Negron-Alvira, A., I. Perez-surez, and T.C. Hazen. 1988. Legionella spp. In Puerto Rico cooling towers. Applied and Environmental Microbiology 54: 233 1-2334.

Pope, D.R., and D.M. Dziewulski. 1992. Efficacy of biocides in controlling microbial populations, including Legionella, in cooling systems (RP-586). ASHRAE Transactions 1998 (1):24-39.

Skaily, P., T.A. Thompson, G.W. Gorman, G.K. Morris, H.V. McEachem, and D.C. Mackel. 1980. Laboratory studies for disinfectants against Legionella pneumophila, Applied and Environmental Microbiology 40: 697700.

Thomas, W.M., J. Eccles, and C.R. Fricker. 1999. Laboratory Observations of Biocide Efficiency against Legionella in Model Cooling Tower Systems. ASHRAE Transactions: Symposia 1999: 607-623.

41403 -408.

APPENDIX A

DETAILS OF THE MODEL COOLING TOWER SYSTEM

The test rig consists of nine test chambers, or cells, situated above a communal heated water bath. It is made from galvanized steel, commonly used for the construction of cooling towers. Twelve liters of water is circulated around each chamber where it is allowed to drip through a block of pack material. Air is drawn into each cell from the surrounding atmosphere and passes through this pack. However, as the air drawn into this test system is at ambient temperature, it is cooler than the air circulating within the systems that it is set up to simulate. To allow for this, and to simulate the temperature increase usually associated with cooling tower water, the water in the test system is heated through a copper coil submerged into the communal hot water bath. To provide the necessary airflow through the test rig, cells were coupled to an air scrubber unit located outside the building. Once air has been through the test chamber ,it is exhausted through a drift eliminator and sterilized in the air scrubber (Figure Al).

Water loss by evaporation is replaced with water from a header tank filled with a blend of town mains water and town mains water that has passed through a reverse osmosis (RO) unit. The use of this blended makeup water is important in helping to prevent the total dissolved solids (TDS) in the test chambers rising to excessive levels. Alkalinity and hardness levels for the town mains water where the rig was located are high. Blending of the makeup water allows the cycles of concentration for the water to be increased beyond the 2.0 cycle limit that can otherwise be achieved. Scale and corrosion

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Deconiaminnted Air Out

Tonl 'O" I Test Cooling Tower ' i) Contaminated Air Flow D Cleanair

Figure A l Diagram of the model cooling tower system.

1 OOppm Chiorinnicd w.ilbi

l i

Not to scale

Air Scobber

inhibitors are added to the header tank to ensure a consistent of operating parameters: a :mperature drop of 3C o 5C across the test cell packing, output and return water tempera- tures of 24C to 27C and 27C to 32"C, an evaporation rate of 15 to 20 liters per cell per day, and an air face velocity for each test cell of 1.5 to 2.0 m s .

dose to each test chamber. Physiochemical parameters (pH, TDS, and temperature) were determined throughout the trial.

Each test rig cell has a sump of 10 liter capacity with its own water makeup ball valve. Initial commissioning and hydraulic testing of the test rig confirmed the following ranges

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