1、4723 (RP-954) 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 biocide
2、s examined using a model cooling tower system inoculated with a microcosm containing an environ- mental 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 a
3、lso against “clean” systems subsequently drip fed 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 Legionella
4、e are a significant cause of respiratory infec- tions. 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
5、 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; N
6、egron-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 provid
7、e 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
8、 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, appl
9、ication 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 hypochlor
10、ite-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 surviv
11、al 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
12、 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,
13、U.K. Elizabeth Fricker is 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
14、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. - and L. pneumophila Serogroup 10 (as identified by the CDC, Atlanta, Georgia, USA). Cell A B Model Cooling System The
15、 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
16、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 complement
17、ary trials based on the manner of dosing the Legionella into the cells (Tables 1 and 2), one trial to evaluate biocidal activity against prees- tablished Legionella and the other against drip-fed Legionella. The biocide and biodispersant dosing regimes for each trial are summarized in Tables 1 and 2
18、. 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
19、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
20、 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
21、 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 c
22、ells 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- colon
23、ized 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 wher
24、e 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
25、 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
26、l ASHRAE Transactions: Research 31 5 P la n ko n IC Leg io n e IIa in ._ Cell A Il I I I 11 I I II II II II 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
27、 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) w
28、ere 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 on
29、to 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
30、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 w
31、ere 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
32、 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
33、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
34、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 supplemen- tary trial to repeat Cells B and C. At the start of the supple- mentary trial, the number of Legionella in the control cell
35、 was 1.9 log. Over the next two weeks, the population was main- tained 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) th
36、e 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 IS0 +
37、 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 le
38、ss 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 31 6 ASHRAE Transactions: Research Planktonic Legion
39、ella in Cell B -Cell B -Dispersant -THPS Day from start of Trial Figure 2 Planktonic levels of Legionella in Cell B after the addition of biocides. Planktonic Legionella in Cell C *n Figure 3 Planktonic levels of Legionella in Cell C after the addition of biocides. with Cell C and the control cell,
40、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 t
41、he 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
42、 Legionella per mL of 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
43、 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 lo
44、g 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 appli
45、cation 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 pr
46、ior 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 ASHRAE Transactions: Research 31 7 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 Hete
47、rotrophs 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 g
48、rowth 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 Cel
49、l 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
