The efficacy of biocides and other chemical ... - Wiley Online Library

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

The efficacy of biocides and other chemical additives in cooling water systems in the control of amoebae M. Critchley1 and R. Bentham2 1 CSIRO Materials Science and Engineering, Clayton VIC 3168, Australia 2 Department of Environmental Health, Flinders University, Adelaide SA 5001, Australia

Keywords amoebae, biocides, cooling water systems, Legionella. Correspondence Richard Bentham, Department of Environmental Health, Flinders University, PO Box 2100, Adelaide SA 5001, Australia. E-mail: [email protected]

2008 ⁄ 0873: received 22 May 2008, revised 14 July 2008 and accepted 16 July 2008 doi:10.1111/j.1365-2672.2008.04044.x

Abstract Aims: In vitro experiments were undertaken to evaluate biocide formulations commonly used in cooling water systems against protozoa previously isolated from cooling towers. The investigations evaluated the efficacy of these formulations against amoebic cysts and trophozoites. Methods and Results: Laboratory challenges against protozoa isolated from cooling towers using chlorine, bromine and isothiazolinone biocides showed that all were effective after 4 h. The presence of molybdate and organic phosphates resulted in longer kill times for bromine and isothiazolinones. All treatments resulted in no detectable viable protozoa after 4 h of exposure. Conclusions: The chemical disinfection of planktonic protozoa in cooling water systems is strongly influenced by the residence time of the formulation and less so by its active constituent. Bromine and isothiazolinone formulations may require higher dosage of concentrations than currently practiced if used in conjunction with molybdate- and phosphate-based scale ⁄ corrosion inhibitors. Significance and Impact of the Study: Cooling water systems are complex microbial ecosystems in which predator–prey relationships play a key role in the dissemination of Legionella. This study demonstrated that at recommended dosing concentrations, biocides had species-specific effects on environmental isolates of amoebae that may act as reservoirs for Legionella multiplication in cooling water systems.

Introduction Cooling towers provide ideal environments for the proliferation of micro-organisms including bacteria, algae, fungi, protozoa and viruses (Bentham 2000; Thomas et al. 2006). The majority of micro-organisms present are heterotrophic, requiring organic carbon from the environment as a nutrient and energy source. Legionella are commonly isolated from cooling towers and present significant implications for public health through their potential to cause disease (Fields et al. 2002). Free-living protozoa feed predominantly on bacteria, fungi and algae through phagocytosis. However, some micro-organisms have evolved that are able to evade protozoan predation (Matz and Kjelleberg 2005). These organisms are either not able to be ingested by protozoa 784

or are able to survive, multiply and exist within the protozoa after internalization. Legionella demonstrate this capability and can survive and multiply in the cytoplasm of free-living protozoa (Matz and Kjelleberg 2005). In response to environmental variables, this endocytic relationship may range from commensalism to parasitism. Protozoa are important reservoirs for Legionella in cooling waters. There are at least 13 species of amoebae and 2 species of ciliated protozoa that support the intracellular replication of Legionella (Newsome et al. 1998; Little 2003). In many outbreaks of Legionnaires’ disease, protozoa capable of harbouring Legionella have been isolated from the same reservoir of infection. Barbaree et al. (1986) isolated two ciliates, Tetrahymena sp. and Cyclidium sp., from cooling towers associated with outbreaks of Legionnaires’ disease. Laboratory studies showed that

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 784–789

M. Critchley and R. Bentham

both protozoa were capable of supporting the intracellular replication of Legionella pneumophila. Free-living amoebae were also associated with an outbreak of Pontiac fever affecting 24 people in Chicago (Fields et al. 1990). There is insufficient information available to assess the requirements for the chemical control of protozoa in cooling towers, particularly for cyst-forming amoebae and other environmental isolates. There is also little known about biocide performance in the presence of scale and corrosion inhibitors that may have antagonistic or synergistic effects on their activity. Chemical formulations for specific purposes, such as microbial control, have usually been assessed in isolation from other agents used in the same environment (ASTM E645-05a 2005). The combined influences of organic, inorganic, antimicrobial and inhibitory formulations on the microbial community in cooling towers have not been investigated. In situ, the chemical and physical characteristics of the water and the engineered environment, including water temperature and pH, will additionally influence the effectiveness of the biocide (Fields et al. 2002). Chemical treatments for the control of micro-organisms in cooling towers have focused primarily on effects against bacteria and in some instances, specifically Legionella. There is strong evidence to suggest that the presence of protozoa may contribute significantly to the survival of Legionella (Fields et al. 2002). By understanding the chemical treatments required for the effective control of protozoa, informed public health strategies for the risk management of cooling towers can be further developed. The objective of this research was to assess the efficacy of current cooling tower chemical treatment regimes in controlling protozoa. The specific aims were: to determine if biocides commonly used in cooling towers were inhibitory to environmental isolates of amoebae; to assess whether cooling tower chemical additives enhance or adversely affect biocide activity towards protozoa; to determine the role temperature plays in biocide efficacy and amoebal proliferation. Materials and methods Cooling tower sampling and analysis A total of 62 cooling towers within Victoria, Australia were sampled for analysis. The towers were located at universities, small businesses and large industrial sites. Water samples were taken from the basin of operating cooling towers where possible. Biofilm was sampled by aseptically swabbing basin surfaces. Sediment was sampled from the basin when present. Water samples were analysed on site for pH, dissolved oxygen, temperature and chlorine residual. The biocides

Biocides and amoebae in cooling water

and chemical treatments used in the cooling towers were documented. The samples were analysed in the laboratory for pH, conductivity, total organic carbon and metal content (Fe, Zn, Ni, Cr, Mo) using standard methods. Legionella were analysed according to AS ⁄ NZS 3896 1998 using heat pretreatment at 50C. Nonculturable Legionella were detected by fluorescent in situ hybridization (FISH) using the methods of Declerck and Ollevier (2006). Heterotrophic bacteria were analysed according to AS ⁄ NZS 4276.3.2 2003 with incubation at 25C. All samples were analysed within 24 h of sampling. Protozoan enumeration and isolation Protozoa were enumerated using plaque assays. The samples were vigorously shaken for 60 s and plated in duplicate onto Escherichia coli lawned non-nutrient agar plates (NNE; ATCC 25922). The plates were incubated at both 25 and 30C for 2 days. For isolation of protozoa, water samples reporting no plaques were enriched with E. coli for 2 days at 25C before re-plating on NNE. Biofilm and sediment samples were directly inoculated onto NNE. Protozoan isolates were purified and identified according to morphological characteristics by reference to Page (1988). Identifications were also confirmed to species level using in situ hybridization (Grimm et al. 2001). The cultures were maintained in peptone-yeast extract-glucose broth (PYG) in tissue culture flasks at 25C. Chemical selection Cooling tower management companies operating within Victoria, Australia were surveyed regarding chemical treatment strategies. The most commonly used chemicals were identified for use in antimicrobial experiments. Biocides identified included chlorine, bromine and isothiazolinones. The isothiazolinones were a blend of 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4isothiazolin-3-one. Typical dose concentrations were 1 mg l)1 for chlorine and bromine residuals and 150 mg l)1 for isothiazolinones. Scale and corrosion chemical control additives included phosphate-, molybdate- and zinc-based corrosion inhibitors. The chemicals for use in the antimicrobial experiments were sourced both commercially and from cooling tower treatment companies. Determination of minimum inhibitory concentration (MIC) The MIC of the biocides against trophozoites and cysts was determined using methods of Srikanth and Berk (1994). Tissue culture flasks of 48-h amoebal cultures grown in PYG were washed thrice with Tris-buffered


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saline (TBS; 0Æ05 mol l)1, pH 7Æ5) and the cells harvested. The trophozoites were allowed to stand for 30 min to allow adaptation to the media and enumerated by microscopy. Cysts were obtained from 14-day cultures grown in PYG in tissue culture flasks. Cysts were washed thrice in TBS and incubated for 24 h in 3% HCl to kill residual trophozoites and immature cysts. The washing step was repeated, the cysts harvested and quantified by microscopy. The trophozoites and cysts were tested against a range of biocide concentrations in multi-well plate assays in a total reaction volume of 2 ml at 25C. The starting concentration was in the order of 104 cells or cysts per millilitre. The MIC was reported as the minimum concentration at which growth was inhibited after 8 h incubation with biocide. Viability was determined after neutralization of the biocides in Dey-Engley medium by microscopy (trophozoites) and incubation in PYG for 14 days (cysts). Preliminary tests were performed to ensure the neutralization solution did not influence viability. Viability by microscopy was based on motility and morphological appearance. All experiments were performed in triplicate. Antimicrobial testing experiments Both trophozoites and cysts were assessed for antimicrobial activity based on ASTM E645-05a – Standard Test Method for the Efficiency of Microbicides used in Cooling Towers. The assay used was adapted from this test method as the ASTM standard was not specifically developed for protozoa. For trophozoites, tissue culture flasks of 48-h amoebal cultures were rinsed thrice with TBS and the cells harvested. The cells were allowed to stand for 30 min to allow adaptation to the buffer. The cells were enumerated by microscopy. The cysts were obtained from 14-day cultures in tissue culture flasks. Cysts were washed thrice in TBS and incubated for 24 h in 3% HCl to kill residual trophozoites and immature cysts. The washing step was repeated, the cysts harvested and quantified by microscopy. Biocides were added to 0Æ2-lm filter sterilized cooling tower water (pH 8Æ0) in multi-well plates at various concentrations: chlorine 1 mg l)1 (residual); bromine 1 mg l)1 (residual); and isothiozolone 150 mg l)1. These concentrations were chosen as they are typical concentrations used in cooling tower management. Concentrations of bromine and chlorine were confirmed using HACH test kits. Biocide activity was also assessed in the presence of the corrosion inhibitors at concentrations of 20 mg l)1. Controls containing amoebae in cooling water with no biocides were run simultaneously. The total reaction volume was 5 ml. The experiments were performed at temperatures of 25, 30 and 35C. Viable cell concentrations of trophozoites were determined at 2, 4 and 8 h by 786


microscopy after neutralization with Dey-Engley broth. Viable cyst concentrations were determined after 8 h by neutralization and plaque assays. Preliminary tests were performed to ensure that the neutralization solution did not influence the viability of cells or cysts. All experiments were performed in triplicate. Statistical analysis Samples were statistically analysed using GraphPad Prism Software. Statistical significance was accepted at P < 0Æ05. Results Cooling tower analysis The majority of cooling towers sampled did not record high microbial populations. Culture results detected L. pneumophila serogroup 1 in 3% of the cooling towers and Legionella spp. in 10% of the towers. In contrast, fluorescent in situ hybridization detected the presence of Legionella spp. in 34% of the cooling towers. There was no relationship between cooling tower chemical composition and Legionella concentration. There was no correlation between heterotrophic plate counts and Legionella. The majority of biocides used in the towers sampled were chlorine, bromine and isothiazolinones. Protozoan analysis Protozoa were detected in 30% of the cooling towers sampled, primarily present as free-living amoebae (Hartmanellidae and Acanthamoebidae). The highest concentration of amoebae reported in basin water was 16 PFU ml)1. Amoebae were more commonly detected in biofilm compared with the basin samples. This was expected as most amoebae are almost exclusively associated with surfaces. There was no relationship between tower chemical composition and protozoan concentration, including the biocide in use (data not shown). There was also no relationship between concentrations of Legionella and protozoa. The amoebal isolates selected for use in the antimicrobial experiments included Acanthamoeba sp., Hartmanella vermiformis and Vahlkampfia sp. These were representatives of the majority of species isolated from the towers. Minimum inhibitory concentrations The Acanthamoeba sp. generally reported the highest resistance to all biocides (Table 1). Trophozoites of Acanthamoeba sp. were inhibited by chlorine at 1 mg l)1 and bromine at 5 mg l)1 over the exposure time of 8 h.




Table 1 Minimum inhibitory concentration of biocides against amoebal trophozoites and cysts after 8 h exposure at 25C (concentrations at mg l)1) Acanthamoeba sp. Trophozoites Chlorine (residual) Bromine (residual) Isothiazolinones Cysts Chlorine (residual) Bromine (residual) Isothiazolinones

H. vermiformis

Vahlkampfia sp.

1 5 150

1 1 50

1 1 25

5 10 200

5 5 150

2 2 100

ence of the phosphate-based inhibitor was significantly less (P < 0Æ05) with only a 2-log reduction reported compared with complete die-off in the other treatments. At 35C, bromine was less effective demonstrating a 2-log decrease after 2 h, with total die-off reported at 4 h. Isothiazolinone was less effective against H. vermiformis at 35C compared with the lower temperatures. There was complete die-off observed at 25 and 30C after 2 h. At 35C, there was a 2-log decrease after 2 h exposure, with complete die-off after 4 h. The biocides were highly effective against Vahlkampfia sp. showing complete die-off with all treatments after 2 h. Antimicrobial testing of amoebal cysts

Chlorine and bromine were equally effective in inhibiting H. vermiformis and Vahlkampfia sp. at concentrations of 1 mg l)1. Inhibition by the isothiazolones was variable. Acanthamoeba sp. trophozoites were the most resistant to isothiozolinones, with an MIC of 150 mg l)1. Vahlkampfia was the most sensitive to isothiazolinones with an inhibitory concentration of 25 mg l)1. Encysted amoebae reported significantly higher inhibitory concentrations compared with trophozoites (P < 0Æ001). Cysts of Acanthamoeba sp. were resistant to chlorine at 5 mg l)1, bromine at 10 mg l)1 and isothiazolinones at 200 mg l)1, well exceeding the recommended doses for tower maintenance. Chlorine and bromine were equally effective against the H. vermiformis cysts at concentrations of 5 mg l)1 for both biocides and Vahlkampfia sp. cysts at 1 mg l)1. Isothiazolinones were inhibitory against cysts of H. vermiformis and Vahlkampfia sp. at 150 and 100 mg l)1, respectively.

Based on the results from trophozoite testing, the cysts were tested solely with biocides. The highest die-off occurred within 2 h of exposure (Table 2). Cysts of the Vahlkampfia sp. were the most sensitive, demonstrating a 3-log reduction after 2 h. Cysts of the Acanthamoeba sp. were the most resistant, reporting a 2-log reduction after 8 h exposure. Discussion The results obtained were comparable with previous reports of biocide inactivation studies. Variability in the susceptibility of amoebae to cooling tower biocides has been previously documented. Cursons et al. (1980) reported that Naegleria spp. were more sensitive to cooling tower biocides than species of Acanthamoeba. High resistance by Acanthamoeba trophozoites and cysts to nonoxidizing biocides was also reported by Sutherland and Berk (1996). Chlorine and bromine at concentrations

Antimicrobial testing of amoebal trophozoites Increasing water temperature decreased the effectiveness of biocides against Acanthamoeba sp. trophozoites. This may partly be a result of the higher temperature favouring the growth of this species. At 25C, chlorine produced complete die-off of Acanthamoeba sp. after 2 h exposure. Die-off from bromine in the presence of phosphate and molybdate inhibitors was slower, but significant die-off (3-log decrease) was still reported after 2 h. Similarly with isothiazolinones, die-off in the presence of all inhibitors was slower after 2 h exposure compared with the biocide alone (3-log decrease). The results at 30C were comparable with those at 25C. At 35C, a 3-log die-off was reported by all biocides plus additives after 2 h. Total die-off at 35C was reported at 4 h. Bromine and chlorine were highly effective against H. vermiformis demonstrating complete die-off after 2 h exposure at 25 and 30C. At 30C, die-off in the pres-

Table 2 Results of antimicrobial testing against amoebal cysts after 8 h exposure to concentrations of 1 mg l)1 chlorine, 1 mg l)1 bromine and 150 mg l)1 isothiazolinone Viable cyst counts after time (h)

Acanthamoeba sp. Chlorine Bromine Isothiazolinone H. vermiformis Chlorine Bromine Isothiazolinone Vahlkampfia sp. Chlorine Bromine Isothiazolinone

0

2

4

8

1Æ2 · 104 1Æ2 · 104 1Æ2 · 104

8Æ6 · 102 6Æ5 · 102 9Æ2 · 102

2Æ3 · 102 3Æ1 · 102 5Æ4 · 102

1Æ9 · 102 1Æ8 · 102 3Æ3 · 102

2Æ3 · 104 2Æ3 · 104 2Æ3 · 104

3Æ2 · 102 5Æ1 · 102 7Æ4 · 102

2Æ1 · 101 2Æ4 · 101 3Æ9 · 101

2Æ0 · 101 2Æ0 · 101 2Æ5 · 101

1Æ8 · 104 1Æ8 · 104 1Æ8 · 104

9Æ6 · 101 8Æ6 · 101 9Æ9 · 101

5Æ0 · 101 4Æ4 · 101 6Æ0 · 101

– – –


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up to 10 mg l)1 have previously demonstrated effective against amoebal trophozoites and cysts over various exposure times (Kilvington and Price 1990). The results demonstrated that at the recommended dose concentrations, biocides had species-specific effects on environmental isolates of amoebae. It was clear from all laboratory determinations that maintenance of an effective residual concentration of biocide for at least 2 h was an essential prerequisite for effective disinfection. In practice, the operation of cooling systems results in continual dilution and neutralization of added chemicals. The residence time of the chemical formulations in the system must be considered when calculating initial dose concentrations so that effective residuals are maintained for appropriate time periods. The current predominantly used biocides (chlorine, bromine and isothiazolinones) are effective as disinfectants against the commonly isolated protozoa in cooling water provided they have sufficient exposure time. The results demonstrated efficacy was also dependant on temperature as well as time and concentration. Generally, systems operating at higher temperatures (above 30C) may require longer biocide residence times for effective disinfection. The operating temperatures at the warmest parts of the system should also be considered. Other chemical additives used in cooling water treatment may also compromise the efficacy of some biocide formulations. This is especially true for phosphate and molybdate additives and the efficacy of bromine and isothiazolinone. This study identified these two additives as the most commonly used scale and corrosion inhibitor formulations in the cooling towers sampled. This effect was also enhanced by elevated temperatures. Systems using these additives may require longer biocide exposure times to ensure effective disinfection. It is also possible that the protozoa may have suffered stress from the exposure that may have also affected the results. Of the protozoa screened, Acanthamoeba species were most commonly isolated and least susceptible to cooling water treatment. Vahlkampfia spp. were most susceptible and least commonly isolated. This suggests that currently employed disinfection processes favour the colonization by Acanthamoeba spp. while limiting colonization by Vahlkampfia spp. It is also possible that the protozoa may have suffered stress from the exposure that may have also affected the results. The efficacy of cooling water biocides in controlling protozoa may be compromised by common chemical additives used to control scale and corrosion. Whether a similar decrease in biocide efficacy applies for other microbial populations in the systems has not been reported. The normal temperatures commonly found at the heat exchange surfaces of systems (35C) are optimal for survival of 788


protozoa exposed to cooling water biocides. These heat exchanger surfaces have been demonstrated to be the major reservoir for Legionella multiplication in cooling systems (Bentham 1993). This suggests that system temperature may favour Legionella multiplication in two ways: by providing optimum growth temperature for Legionella and by reducing the effectiveness of biocides against their protozoan hosts. Cooling water systems are complex microbial ecosystems in which predator–prey relationships play a key role in the dissemination of Legionella. Understanding the relative physical, chemical and biological contributions to these ecosystems is the prerequisite for providing informed management strategies to protect public health. This study has investigated biocide performance against planktonic protozoan populations. Chemical control of protozoa in the presence of biofilm would be more indicative of the efficacy of the treatments in situ. Acknowledgements This work was funded by the Victorian Department of Human Services. References AS ⁄ NZS 3896. (1998) Waters – Examination for Legionellae Including Legionella pneumophila. Sydney, Australia: Standards Australia. AS ⁄ NZS 4276.3.2. (2003) Water Microbiology – Method 3.2: Heterotrophic Colony Count Methods - Plate Count of Water Containing Biocides. Sydney, Australia: Standards Australia. ASTM E645-05a. (2005) Standard Test Method for the Efficiency of Microbicides used in Cooling Towers. American Society for Testing and Materials. Barbaree, J.M., Fields, B.S., Feeley, J.C., Gorman, G.W. and Martin, W.T. (1986) Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intercellular multiplication of Legionella pneumophila. Appl Environ Microbiol 51, 422–424. Bentham, R.H. (1993) Environmental factors affecting the colonization of cooling towers by Legionella spp. in South Australia. Int Biodeter Biodegrad 31, 55–63. Bentham, R.H. (2000) Routine sampling and the control of Legionella spp. in cooling tower water systems. Curr Microbiol 41, 271–275. Cursons, R.T.M., Brown, T.J. and Keys, E.A. (1980) Effect of disinfectants on pathogenic free-living amoebae in axenic conditions. Appl Environ Microbiol 40, 62–66. Declerck, P. and Ollevier, F. (2006) Whole cell fluorescent in situ hybridisation (FISH) of Legionella in various kinds of samples. In Diagnostic Bacteriology Protocols, Vol. 345 ed. O’Connor, L. pp. 175–184. Totowa, NJ: Humana Press.



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Matz, C. and Kjelleberg, S. (2005) Off the hook – how bacteria survive protozoan grazing. Trends Microbiol 13, 302–307. Newsome, A.L., Scott, T.M., Benson, R.F. and Fields, B.S. (1998) Isolation of an amoeba naturally harboring a distinctive Legionella species. Appl Environ Microbiol 64, 1688–1693. Page, F.C. (1988) A New Key to Freshwater and Soil Gymnamoebae. Cambria, UK: Freshwater Biological Asssociation. Srikanth, S. and Berk, S.G. (1994) Adaption of amoeba to cooling tower biocides. Microbial Ecol 27, 293–301. Sutherland, E.E. and Berk, S.G. (1996) Survival of protozoa in cooling tower biocides. J Ind Microbiol 16, 73–78. Thomas, V., Herrera-Rimann, K., Blanc, D.S. and Greub, G. (2006) Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network. Appl Environ Microbiol 72, 2428–2438.


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