University of Nova Gorica Graduate School

UNIVERSITY OF NOVA GORICA GRADUATE SCHOOL

THE STUDY OF OPTIMAL TECHNOLOGICAL PROCEDURES OF INTERNAL PLUMBING SYSTEM DISINFECTION FACILITIES IN USE BY THE SENSITIVE HUMAN POPULATIONS

MASTER´S THESIS

Janez Škarja

Mentor: Assis. Prof. Darko Drev

Nova Gorica, 2016

UNIVERZA V NOVI GORICI FAKULTETA ZA PODIPLOMSKI ŠTUDIJ

RAZISKAVA OPTIMALNIH TEHNOLOŠKIH POSTOPKOV DEZINFEKCIJE INTERNIH VODOVODNIH OMREŽIJ OBJEKTOV, KI JIH UPORABLJA OBČUTLJIVEJŠA POPULACIJA LJUDI

MAGISTRSKO DELO

Janez Škarja

Mentor: doc.dr. Darko Drev

Nova Gorica, 2016

SUMMARY

In a developed world, water is used in a variety of installations and devices for the improvement of life standard. It is important for these elements to be suitably managed and maintained, otherwise they can present a risk to people's health. Although potable water from a public plumbing system coming via the water supply into an internal plumbing system normally is compliant with the regulations, the quality of water in an internal plumbing system often changes – water gets contaminated. There are several types of microorganisms that can grow in water. While most of them present no threat, there are some that can induce health issues in people. Internal plumbing systems with heated water and the possibility of aerosol releasing present a special concern in this matter. They carry a great potential for the growth and proliferation of bacteria from the genus Legionella that cause Legionnaires' disease and Pontiac Fever. Even the fact that Legionella infection has for several years been mentioned in the accident insurance conditions of insurance companies in relation to receiving the insurance fee as compensation for bed day, shows the extent and foremost the seriousness of the disease (5–15% mortality rate).

The research part of the master thesis focused mainly on determining the presence of bacteria from the genus Legionella in the water of internal plumbing systems of healthcare facilities. During the entire research period of 8 years, 2,676 samples of cold and hot water were acquired from 23 healthcare facilities.

The main purpose of the research was to determine the efficiency or adequacy of applied approaches, i.e. physical disinfection with heat and chemical disinfection with chlorine, in eliminating or reducing the amount of Legionella present in the water of selected healthcare facilities. Another aim was to evaluate the impact of softening potable water with polyphosphates on the proliferation of certain microorganisms in water, and to determine the by-products of chemical disinfection.

Upon sampling, an organoleptic examination of water (appearance, odour), electrometric measurements of water temperature, and colorimetric measurements of free chlorine concentrations in water were performed. The data for the analysis of

V Legionella presence were acquired by water sampling and the isolation of bacteria found in the water. In order to acquire laboratory results of specific physical and chemical parameters, ion chromatography, gas chromatography, spectrophotometry, titration, and inductively coupled plasma – mass detector were used.

Although water samples from only two healthcare facilities showed no presence of Legionella, the number of water samples with the presence of Legionella decreased for at least 25% from the beginning to the end of period set. Also, after certain general sanitary and technical measures a noticeable improvement within healthcare facilities after 2008 could be observed. Regarding the initial part of the set period, a more favourable relationship between positive and negative samples, a lower number of samples with highest concentrations of present Legionella, and a higher number of samples with lower concentrations of present Legionella were determined. The highest shares of positive samples were observed in the classes with concentrations from 11 to 100 and 101 to 1,000 CFU/mL. Furthermore, the share of most often determined species Legionella pneumophila sg.1 decreased as well. The research has shown that the existing system of ensuring health-compliant water is fairly efficient; however, the results could be improved additionally by investing more into the preparation and renovation of systems. Due to a limited number of samples, a direct impact of adding polyphosphates to potable water could not be linked to the occurrence of Legionella. None of the samples showed an increased concentration of by-products of potable water disinfection.

In its conclusion, the research has shown that the procedures after overheating and after chlorine disinfection result in a similar success. While filtration proved to be most efficient, from the perspective of Legionella infections the use of medical bathtubs can present a high risk to people's health, therefore such devices should be subject to more frequent maintenance and supervision. Not only did medical bathtubs produce a high share of positive samples, the majority of samples with highest concentrations of Legionella were also found in them.

The comparison of results could indeed show the actual effects more thoroughly, had there been a chance to monitor the execution of individual preventive measures at the

VI same collection spots. Therefore, a certain degree of attention needs to be taken into account when interpreting the results.

Keywords: potable water, internal plumbing system, biofilm, health facilities, disinfection, Legionella

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POVZETEK

V razvitem svetu se voda uporablja v raznolikem spektru napeljav in naprav za izboljšanje življenjskega standarda. Pomembno je, da se te elemente ustrezno upravlja in vzdržuje, saj v nasprotnem primeru lahko predstavljajo tudi tveganja za zdravje ljudi. Kljub dejstvu, da je pitna voda iz javnega vodovodnega omrežja, ki prihaja preko vodovodnega priključka v interno vodovodno omrežje običajno skladna s predpisi, se v slednjem kakovost pogosto spremeni – voda se onesnaži. Obstajajo številni mikroorganizmi, ki lahko rastejo v vodi. Medtem ko je večina neškodljivih, nekateri povzročajo obolenja pri ljudeh. Interni vodovodni sistemi z ogrevano vodo in možnostjo sproščanja aerosolov so v tem pogledu še posebej zaskrbljujoči. Omenjeni sistemi imajo velik potencial za rast in širjenje bakterij iz rodu Legionella, ki povzročajo legionarsko bolezen in pontiaško vročico. Tudi to, da najdemo okužbo z Legionella že nekaj let omenjeno tudi v pogojih zavarovalnic za nezgodno zavarovanje, v povezavi z izplačilom zavarovalnine kot nadomestila za bolnišnični dan, kaže na razširjenost, predvsem pa na težo obolenja (5-15% smrtnost).

V raziskovalnem delu smo se osredotočili predvsem na ugotavljanje prisotnosti bakterij iz rodu Legionella v vodi iz internih vodovodnih omrežij zdravstvenih objektov. V celotni raziskavi je bilo v osem letnem obdobju, v 23 zdravstvenih objektih skupaj odvzeto 2676 vzorcev hladne in tople vode.

Glavni namen je bil raziskati učinkovitost oziroma zadovoljivost uporabljenih pristopov, kot so fizikalna dezinfekcija s toploto in kemična dezinfekcija s klorom, za odstranjevanje oziroma zmanjšanje števila Legionella v vodi izbranih zdravstvenih objektov. Namen je bil tudi oceniti vpliv mehčanja pitne vode s polifosfati na razrast nekaterih mikroorganizmov v vodi in določitev stranskih produktov kemične dezinfekcije.

Ob vzorčenju pitne vode iz pip, prh, iztokov hranilnikov, filtrov na mestih uporabe je bil opravljen organoleptični pregled vode (izgled, vonj), elektrometrične meritve temperature vode, kolorimetrične meritve koncentracije prostega klora v vodi.

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Podatke za analizo prisotnosti Legionella smo pridobili z vzorčenjem vode in osamitvijo bakterij iz vode. Za pridobitev laboratorijskih rezultatov določenih fizikalno kemijskih parametrov smo uporabili ionsko kromatografijo, plinsko kromatografijo, spektrofotometrijo, titracijo, induktivno sklopljeno plazmo – masni detektor.

Kljub temu, da v vodi le v dveh zdravstvenih objektov nismo ugotovili Legionella, pa se je od začetka do konca obravnavanega obdobja vsaj za 25% zmanjšalo število vzorcev vode s prisotnostjo Legionella. V letu 2006 je delež pozitivnih vzorcev tako znašal 71,7 %, leta 2013 pa 44,0 %. Tudi z nekaterimi splošnimi sanitarno tehničnimi ukrepi se je po letu 2008 vidno začelo izboljševati stanje v zdravstvenih objektih. Ugotovili smo ugodnejše razmerje med pozitivnimi in negativnimi vzorci, manjše število vzorcev z najvišjimi koncentracijami prisotnih Legionella in večje število vzorcev z nižjimi koncentracijami prisotnih Legionella, glede na začetni del obravnavanega obdobja. Največji delež pozitivnih vzorcev sta predstavljala velikostna razreda s koncentracijami od 11 do 100 in od 101 do 1,000 CFU/mL. Zmanjšal se je tudi delež, sicer najpogosteje ugotovljene vrste Legionella pneumophila sg.1. Raziskava je pokazala, da je obstoječi sistem zagotavljanja zdravstveno ustrezne vode dokaj učinkovit, a menimo, da je možno z večjim vlaganjem v pripravo in obnovo sistemov rezultate, glede prisotnosti Legionella v vodi internih vodovodnih sistemov, še izboljšati. Zaradi omejenega števila vzorcev (N=12), kjer smo izmeril tudi koncentracijo ortofosfatov, nismo mogli povezati neposredni vpliv dodajanja polifosfatov v pitno vodo na razvoj Legionella. Raziskovali smo tudi prisotnost klorida, klorita, klorata, THM – vsota (Kloroform, Bromoform, Bromodiklorometan, Dibromoklorometan) v pitni vodi. V nobenem vzorcu nismo ugotovili povečane koncentracije stranskih produktov dezinfekcije pitne vode.

V zaključku raziskava izkazuje, da sta bila postopka po pregrevanju in dezinfekcija s klorom podobno uspešna glede ugotovljenega števila negativnih vzorcev (57 %) vode na Legionella. Medtem ko je bila filtracija glede ugotovljenega števila negativnih vzorcev (94 %) najbolj učinkovita, ugotavljamo, da uporaba specialnih kadi za nego in terapijo lahko predstavlja visoko tveganje za zdravje ljudi z vidika okužbe z Legionella, zato morajo biti tovrstne naprave še posebej podvržene

IX pogostejšemu vzdrževanju in nadzoru. Kajti pri specialnih kadeh ni bil ugotovljen le visok delež pozitivnih vzorcev (86 %), ampak je bila ugotovljena tudi večina vzorcev (N=8) z najvišjimi koncentracijami (>100,000 CFU/L) Legionella.

Primerjava dobljenih rezultatov glede pozitivnih in negativnih vzorcev vode na prisotnost Legionella, v osem letnem obdobju, bi sicer lahko bolj natančno prikazala dejanske učinke izvajanja preventivnih ukrepov, v kolikor bi lahko spremljali izvajanje posameznih preventivnih ukrepov na istih odvzemnih mestih pred in po izvedbi ukrepov. Učinki zmanjševanja števila Legionella oziroma odstranjevanja le- teh s pregrevanjem, kloriranjem in filtracijo so bili v naši raziskavi izmerjeni tako na stalnih, kot tudi naključnih odvzemnih mestih, saj vedno ni bilo mogoče vzorčiti na predvidenih mestih. Razlike, ki jih z našo raziskavo nismo mogli izmeriti, so bile tudi v času vzorčenja (zgodaj zjutraj in popoldan), ki v neki meri tudi lahko vpliva na končni laboratorijski rezultat.

Ključne besede: pitna voda, hišno vodovodno omrežje, biofilm, zdravstveni objekti, razkuževanje, Legionella

TABLE OF CONTENTS

SUMMARY ...... V POVZETEK ...... VIII 1 INTRODUCTION ...... 1 1.1 Internal plumbing system ...... 4 1.2 Contamination of potable water in the internal plumbing system ...... 5 1.2.1 Physical contamination of potable water within the internal plumbing system ...... 7 1.2.2 Chemical contamination of the internal plumbing system ...... 10 1.2.3 Microbiological contamination of the internal plumbing system ...... 11 1.2.3.1 Microbiological contamination – water phase ...... 15 1.2.3.2 Microbiological contamination – biofilm ...... 16 1.3 Presence of Legionella ...... 21 1.3.1 Legionella in a natural environment ...... 21 1.3.2 Legionella in an artificial environment ...... 23 1.3.3 Biological and ecological characteristics of Legionella ...... 25 1.3.4 Health hazards ...... 28 1.3.4.1 Incidence of legionellosis ...... 33 1.4 Disinfection of internal plumbing systems for the mitigation of legionellosis ...... 40 1.4.1 Physical disinfection ...... 44 1.4.1.1 Thermal disinfection ...... 44 1.4.1.2 Ultraviolet light (UV) ...... 48 1.4.1.3 Filtration with membrane filters ...... 50 1.4.2 Chemical disinfection of potable water in the internal plumbing system ... 54 1.4.2.1 Disinfection of potable water with chlorine ...... 56 1.4.3 Comparison of controlling methods for Legionella in the internal plumbing system ...... 61 1.5 Chemical softening of potable water ...... 66 1.6 Materials in direct contact with potable water within the internal plumbing system ...... 70 1.7 Regulations overseeing the field of Legionella control in plumbing systems ...... 75

1.8 Aim of research, research objectives and working hypotheses ...... 78 1.8.1 Aim of research ...... 80 1.8.2 Research objectives ...... 81 1.8.3 Working hypotheses ...... 81 2 THE EXPERIMENTAL PART ...... 82 2.1 Time schedule ...... 83 2.2 Methods employed ...... 83 2.2.1 Field measurements of potable water ...... 84 2.2.2 Potable water sampling ...... 84 2.2.3 Microbiological analysis methods ...... 86 2.2.4 Physical and chemical analysis methods ...... 87 2.3 Facilities included in research ...... 88 3 RESULTS AND DISCUSSION ...... 90 3.1 Presence of Legionella in the potable water of selected healthcare facilities in the Ljubljana healthcare region (2006–2013) ...... 90 3.1.1 The impact of thermal disinfection on the presence of Legionella in the water of internal plumbing systems ...... 97 3.1.2 The impact of chlorine on the presence of Legionella in the water of internal plumbing systems ...... 99 3.1.3 The impact of point-of-use water filters on the presence of Legionella in the collected water samples ...... 101 3.1.4 The impact of phosphate addition on the presence of Legionella in the water of internal plumbing systems ...... 103 3.1.5 The occurrence of Legionella in the water of internal plumbing systems of bathtubs ...... 106 3.1.6 The comparison of different procedures and types of treatment ...... 107 3.2 Research 2 (2010) ...... 109 3.3 Comparison of research results with hypotheses ...... 119 4 CONCLUSIONS ...... 122 5 REFERENCES ...... 126

ACKNOWLEDGEMENT

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LIST OF FIGURES

Figure 1: Types of information to consider in the risk assessment of potable water pipelines (WHO, 2011a) ...... 7 Figure 2: Positive and negative zeta potential (Drev, 2014) ...... 19 Figure 3: Galvanic cell can occur with zinc-coated sheet metal (Drev, 2006) ...... 19 Figure 4: Water temperature and the increasing risk of Legionella proliferation (ADWG, 2008) ...... 31 Figure 5: Number of reported cases and deaths due to Legionnaires' disease in Slovenia from 1991 to 2014 (IVZ RS, 1997; IVZ RS, 2000; IVZ RS, 2009; IVZ RS, 2010; IVZ RS, 2013; NIJZ, 2014; NIJZ, 2015; ECDC, 2010; ECDC, 2011; ECDC, 2012; ECDC, 2013) ...... 37 Figure 6: Reported cases and notifications of Legionnaires’ disease per million, by reporting country, EU/EEA, 2013 (ECDC, 2015) ...... 38 Figure 7: Reported cases of Legionnaires’ disease by month of onset, EU/EEA, 2013 (ECDC, 2015) ...... 39 Figure 8: Decimal reduction time for Legionella pneumophila sg. 1 at different temperatures (WHO, 2007) ...... 46 Figure 9: Full thickness burns – contact times with water (Henriques and Moritz, 1947) ...... 47 Figure 10: Membrane process classification (EPA, 2001) ...... 50 Figure 11: Bactericidal effect on Legionella pneumophila at different concentrations of chlorine in potable water at a temperature of 21 °C and a pH of 7.6 (Kuchta et al., 1983) ...... 58 Figure 12: Bactericidal effect on Legionella pneumophila at different temperatures of potable water and chlorine concentration 0.1 mg/L and pH 7.6 (Kuchta et al., 1983) ...... 60 Figure 13: Bactericidal effect on Legionella pneumophila at different pH values and at a temperature of 21 °C of potable water and chlorine concentration 0.1 mg/L (Kuchta et al., 1983) ...... 61 Figure 14: The Ljubljana healthcare region on the map of the Republic of Slovenia (http://zora.onko-i.si/) ...... 90 Figure 15: Samples tested positive for Legionella from 2006 to 2013 (in %) from internal plumbing systems of healthcare facilities ...... 93 Figure 16: Classification of collected samples from internal plumbing systems of healthcare facilities which tested positive for Legionella according to size classes in percentages from 2006 to 2013 ...... 94 Figure 17: Classification according to the identified serological group of Legionella pneumophila and the strain Legionella spp. in percentages and by year (from 2006 to 2013) from internal plumbing systems of healthcare facilities ...... 96

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Figure 18: Classification according to the identified serological group of Legionella pneumophila and the strain Legionella spp. in percentages for the entire observed period (from internal plumbing systems of health facilities in 2006- 2013) ...... 97 Figure 19: A comparison of positive samples from internal plumbing systems of healthcare facilities collected before and after overheating from 2006 to 2013 ...... 98 Figure 20: A comparison of the percentages of positive samples without and with chlorine (from internal plumbing systems of health facilities in 2006-2013) 99 Figure 21: Results on the presence of Legionella in water samples from internal plumbing systems of healthcare facilities collected at sites, where outlets are usually equipped with antibacterial filters, from 2006 to 2013 ...... 101 Figure 22: Share of positive samples for Legionella from 2006 to 2013, where there were phosphates added to drinking water from internal plumbing systems of healthcare facilities ...... 104 Figure 23: Share of positive samples for Legionella from 2006 to 2013, where there were no phosphates added to drinking water from internal plumbing systems of healthcare facilities ...... 105 Figure 24: Share of positive and negative samples concerning presence of Legionella from medical bathtub showers in health facilities and share of all samples from 2006 to 2013 ...... 107 Figure 25: Comparison of shares of positive and negative samples concerning presence of Legionella taken after filtration from medical bathtub showers in health facilities, prior to overheating, after overheating, with chlorine, and with phosphates from 2006 to 2013 ...... 108 Figure 26: Measured concentrations of oxidising in water at selected sampling sites of six health facilities in 2010 ...... 111 Figure 27: Measured concentrations of orthophosphates, number of colonies at 22 °C, number of colonies at 36 °C, and Legionellae in water at selected sampling sites of six health facilities in 2010...... 112 Figure 28: Comparison of measured concentrations of nitrates in water with limit value at selected sampling sites of six health facilities in 2010 ...... 114 Figure 29: Measured values of water turbidity at selected sampling sites of six health facilities in 2010 ...... 115 Figure 30: Measured values of free chlorine and chlorate at selected sampling sites of three health facilities in 2010 ...... 116 Figure 31: Measured concentrations of number of colonies at 22 °C, number of colonies at 36 °C, and Legionellae in water, depending on water temperature and free chlorine present in water at selected sampling sites of six health facilities in 2010 ...... 117

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LIST OF TABLES

Table 1: Pathogen bacteria transmitted through potable water (WHO, 2011b) ...... 14 Table 2: Protocol for sampling environment sites for Legionella (Barbaree et al., 1987) ...... 24 Table 3: Legionella species and serogroups (Fünfgeld, 2002) ...... 26 Table 4: Number of cases of Legionnaires' disease in Europe between 1993 and 2013 (EWGLI, 1998; EWGLI, 1999a; EWGLI, 1999b; EWGLI, 2003; EWGLI, 2007; EWGLI, 2005; EWGLI, 2010; ECDC, 2011; ECDC, 2012; ECDC, 2013; ECDC, 2014; ECDC, 2015) ...... 35 Table 5: Main processes in water treatment (Drev, 2011) ...... 42 Table 6: Filter media for microfiltration (Drev, 2011) ...... 52 Table 7: Chemicals most frequently used for the disinfection of potable water and the by-products of disinfection (Drev, 2011) ...... 54 Table 8: Advantages and disadvantages of alternative methods for controlling Legionella in an internal plumbing system (WHO, 2007:50; Tehnična navodila Sanosil) ...... 62 Table 9: Target values for Legionella in internal plumbing systems of selected countries (WHO, 2007:60; EPA, 2009) ...... 77 Table 10: Overview of number of water samples concerning type of water supply system, method of disinfection and chemical softener in health facilities from 2006–2013 ...... 86 Table 11: Selected methods for microbiological testing of potable water samples .. 87 Table 12: Selected work methods for testing water samples in regard to physical and chemical parameters ...... 88 Table 13: Negative and positive samples of cold water concerning presence of Legionella (from internal plumbing systems of health facilities in 2006- 2013) ...... 91

LIST OF ABBREVIATIONS AND SYMBOLS

Abbreviations:

ADWG Australian Drinking Water Guidelines CDC Centers for Disease Control and Prevention CFU Colony-forming unit DBP Disinfectant by-products DHW Domestic hot water DNA Deoxyribonucleic acid DPD N,N-dietil-1,4-fenilendiamin EC European Commission ECDC European Centre for Disease Prevention and Control ELDSNet European Legionnaires' disease Surveillance Network EPA Environmental Protection Agency EPS Exopolysaccharides EU European Union EEA European Economic Area EWGLI European Working Group for Legionella Infections HACCP Hazard Analysis Critical Control Point HTST High temperature short time IVZ RS National Institute of Public Health of the RepubIic of Slovenia LTLT Low temperature long time MF Microfiltration NF Nanofiltration NIJZ National Institute of Public Health NTU Nephelometric turbidimetric units PA Polyamide PCR Polymerase Chain Reaction PE Polyethylene PES Polyethersulfone

XVI

PET Polyetilentereftalat PETF Polyethylene Terephthalate Film PEX Cross-linked polyethylene PEX-Al-PEX A layer of aluminum sandwiched between two layers of PEX POE Point of Entry POU Point of Use PP Polypropylene PPE Personal protective equipment PTFE Polytetrafluorethylene PUR Polyurethane PVC Polyvinyl chloride RO Reverse osmosis THMs Trihalomethanes UF Ultrafiltration UNICEF United Nations International Children's Emergency Fund USA United States of America UV Ultraviolet VBNC Viable but non-culturable VHTST Very high temperature short time WHO World Health Organization

Symbols:

T Temperature °C Celsius pH scale for the concentration of hydrogen ions in solution

XVII

1 INTRODUCTION

Leonardo da Vinci wrote, “Water is the driver of nature” (Smith R.L. and Smith T.M., 2001).

Since prehistoric times, man has been colonising areas where access to potable water is fairly easy. The first settlements and towns were established near watercourses, which were first used only as a source of potable water and later to tend to other needs. People did not know about water contamination at that time or have not yet developed adequate methods to determine whether water was contaminated or not. The (un)suitability of each water source was determined on the basis of experience, passed down from one generation to the next. This is also why various hydric epidemics – water-borne diseases (cholera, typhoid fever, paratyphoid, shigellosis, etc.) – were some of the main causes of early mortality.

Only with the invention of the microscope and the introduction of procedures of acquiring potable water at the end of the 17th century did the safety of the potable water supply start to improve (EPA, 1999). It significantly improved on the brink of the 20th century, when in the process of water treatment man started to use ozone, calcium hypochlorite, and sodium hypochlorite to disinfect water (EPA, 1999). This resulted in a rapid decline of people suffering from typhoid dysentery and cholera. Today, hydric epidemics are practically rooted out in some developed countries (EPA, 1999), which is a result of efficient potable water treatment (with chemical disinfection, filtration) and the managed drainage of wastewater, preventing different types of contamination from intertwining.

Regardless of technological advances and great progress in knowledge, the start of the 21st century in developed countries marked on a world scale still a lot of fatalities (their number is similar to the population number of Slovenia) connected to contaminated potable water, bathroom facilities, and hygiene (WHO, 2010). In 2012, the number of fatalities more than halved (WHO, 2015).

Through different periods in history, man had strong aspirations towards constructing the best possible plumbing system. The internal plumbing system ending with water outlets (faucets) inside people’s homes was known already in early Roman times. Water came from water reservoirs via aqueducts into people’s homes, public wells, and fountains. Running potable water provided by the internal (house) plumbing system, as we know it today, became part of people’s lives in the developed world in the second half of the 19th century (EPA, 1999). Potable water was protected from any external influences and potential secondary contaminants right from the point of pumping water in the well (or elsewhere) to the users' faucets in their homes. Water pipelines were first made out of wood, stone, and later out of lead, concrete, a mixture of cement and asbestos, or plastic. The resourcefulness of man to bring potable water as a life good as close to home as possible was no exception in our part of the world. The old plumbing system, made out of wooden pipes, used on the brink of the 20th century, can still be observed between the towns of Dolenja Trebuša and Gorenja Trebuša and the same can be said for numerous village plumbing systems in the broader area of Trebuša (Podgornik, Mrak; 2008). Already in 1842, the village of Rajndol in the Kočevje region saw the construction of a plumbing system made out of wooden pipes. A unique example of constructing a plumbing system made out of glass pipes was noted in Mačkovec in the Kočevje region (Prelesnik, 2007).

Nowadays, we are all well aware that the supply of potable water is a constituent element of the quality of life and the human right to being healthy. Water is used for drinking, personal hygiene, preparation of food and a whole array of other activities. We usually become aware of how important it is and how much we actually need it only when we are deprived of it. Potable water is one the most basic elements of health. With regard to health, organised society requests two demands to be met simultaneously: the supply of water of such quality that meets the standards for potable water and the supply of water in the needed quantity at any given moment in any given part of a settlement or to any inhabitant. This is also why these issues cannot be solved by individuals, but rather by society as a whole. Natural resources are not enough to provide sufficient quantities of quality potable water, which means that an adequately constructed and managed plumbing system is of great importance. The most significant aspect in the construction of an adequate plumbing system is the usage of suitable materials (Drev et al., 2008). 2

Due to its composition, water can be directly or indirectly connected to acute and chronic diseases. Water needs to have adequate physical characteristics and must not contain any chemical, biological, and radioactive substances above the permitted limit or in values which would represent danger to people's health. With regard to water constituents, not only their ingestion, but also the possibility of them acting through skin and the respiratory system should be taken into account. In the past, mainly the microbiological contamination of potable water was given greater importance due to acute consequences. In the past few decades, the focus has been shifted towards contamination with various chemicals (pesticides, heavy metals, nitrates, mineral oils, etc.) which are present in water due to the excessively contaminated environment and consequently overly exploited water resources, as well as in many cases due to artificially inflicted secondary contamination. Such an example can be seen in the construction of plumbing pipelines or in the uptake of different substances from materials in direct contact with potable water (Drev et al., 2008). Modern analytics enables the discovery of these substances, which often have toxic or even carcinogenic effects and influence the genetic material.

Slovenian potable water regulations are overseen by several sectors of different ministries (Ministry of Health, Ministry of the Environment and Spatial Planning, Ministry of Agriculture, Forestry and Food, and Ministry of Defence). Managing the quality of potable water in Slovenia are Rules on Drinking Water (2004) – a statutory instrument of the Act Regulating the Sanitary Suitability of Foodstuff, Products and Materials Coming into Contact with Foodstuffs (ZZUZIS, 2000). This regulation is in line with the corresponding Council Directive 98/83/EC on the quality of water intended for human consumption (Council Directive 98/83/EC, 1998). Water quality must be controled (Rules on Drinking Water, 2004). The Slovenian legislation demands external and internal monitoring of potable water supply. The external monitoring is handled by the state – the so-called monitoring or control of the quality of potable water – which conducts it in accordance with an annual programme passed in advance. The other aspect of external monitoring is carried out by the inspection services in charge. The internal monitoring is in the hands of the manager of the plumbing system and must be in accordance with the HACCP system which demands that the quality of water must be controlled all the way from the capture to 3 the consumption of water – to the water gauge or to the outlet (faucet) of a consumer. From there onwards, the care for the preservation of the adequate quality of potable water, as part of the household or internal plumbing system, is in the hands of the owner or manager of the building. Rules on Drinking Water determine that the internal plumbing system covers pipelines, equipment, and all the devices included in the network between the potable water supply station and the system for the supply of potable water and water distribution outlets. If the plumbing system manager provides the building with pristine water, the building owner or manager is obliged to preserve the quality of water, which needs to meet the prescribed regulations, from the water gauge to each outlet in the building (Rules on Drinking Water, 2004). The latter especially binds managers of public buildings, such as medical institutions, educational institutions and nurseries, social assistance institutions, residential buildings, sports and leisure buildings, health resorts, etc. In some industrial plants (food processing industry, pharmaceutical industry, etc.), additional care for the preservation of the quality of potable water is often needed, which demands additional technological processes of water treatment (WHO, 2012).

1.1 Internal plumbing system

The building’s plumbing system, often regarded as an internal plumbing system, includes pipelines, equipment, and all the devices which are part of the network between the potable water supply system and water distribution outlets (Rules on Drinking Water, 2004). The potable water supply system, also known as a plumbing system, is a system of plumbing elements, such as pipelines, water pumps, forebays, wastewater treatment plants, and equipment, including installations and hydrants; the potable water supply system is for the most part of its regular activity acting as an independent plumbing system, hydraulically separated from other plumbing systems (Action Plan for Water Supply, 2006).

According to data provided by WHO and UNICEF from 2013, at the end of 2011 89% (6.74 billion) of the world population had access to potable water through an outlet on the internal plumbing system or to improved and managed water sources supplied by other means, such as public faucets, water reservoirs, water springs, and

4 protected wells. 55% of the entire world population had access to potable water only via an outlet within the internal plumbing system. 11% (884 million) of the world population did not have access to potable water, thus they consumed water from unprotected wells and water springs, lakes, rivers, and canals (WHO and UNICEF, 2013).

The majority of people in Slovenia has access to potable water via an outlet within the internal plumbing system. In 2013, 93% of all the inhabitants of Slovenia had access to potable water via the public plumbing system, where quality measurements (monitoring) were made on the spot of consumption – the water outlet of an end- consumer. The quality of potable water was not determined for around 7% of Slovenia’s inhabitants, pertaining to systems which provide water to less than 50 people (i.e. own sources of potable water, rainwater) or those not included in the monitoring for some other reason (Environmental Indicators in Slovenia, 2015).

1.2 Contamination of potable water in the internal plumbing system

The term water contamination entails a reduction in water quality, an adulteration of its physical, chemical, biological, and radiological characteristics due to added substances, which may result in changes for the worse regarding all or only one of the present parameters. The quality and health compliance of potable water are controlled through different laboratory parameters. We furthermore define the contamination of potable water as the consumption of potable water endangering people’s health (Žerovnik, 2009). Water may contain dissolved and suspended substances which represent a health risk (Roš et al., 2005). People must be aware that potable water as a medium within the internal plumbing system also brings and spreads dangerous substances, such as microorganisms and their secretions, chemicals, etc., which end-consumers come in contact with.

The contamination of potable water within the internal plumbing system is a result of water getting contaminated after entering the internal plumbing system either due to the impacts of the structure of the materials forming the internal plumbing system and coming into direct contact with potable water or due to the existence of 5 favourable conditions for the growth and proliferation of microorganisms in the internal plumbing system. The internal plumbing system with an outlet at the end always represents the last section of the plumbing system in its entirety. The rate of water discharge in the building’s plumbing system depends on the frequency of use of a specific water outlet. Once water started running through the plumbing system to public water outlets, wells, and troughs on town squares and village centres, man's wish to bring water into his own house grew even stronger. Within internal plumbing systems water often stagnated, in comparison with public outlets, where water was constantly flowing. Rusty and old public as well as internal plumbing systems are some of the most frequent causes of different types of potable water contamination (WHO, 2011a). As such, they do not only represent a problem regarding the quality but also the quantity of water as a consequence of water loss from the plumbing system. According to data by the Statistical Office of the Republic of Slovenia (SURS) from 2013, in 2012 Slovenia's losses in the water distribution system amounted to 30% or 50 million m3 of water. It is especially discouraging to see the losses equalling those of 2002.

The thesis focuses primarily on the issue of physical, chemical, and microbiological contamination of potable water within the internal plumbing system, the latter being especially important from the perspective of endangering people’s health. Figure 1 shows an array of information which needs to be taken into account in the risk assessment of water safety in buildings.

Figure 1: Types of information to consider in the risk assessment of potable water pipelines (WHO, 2011a)

1.2.1 Physical contamination of potable water within the internal plumbing system

Concerning the physical contamination of potable water, mainly the following parameters are determined: turbidity, colour, odour, taste, visible impurities, pH values, etc. Organoleptic parameters, taste, odour, and colour of the water are determined with sense organs, and are as such detected by water consumers as one of the first changes in the water. The cause of changes occurring or limit values of mentioned parameters being exceeded in the internal plumbing system is in most cases connected to the wear-and-tear of built-in materials and the non-maintenance of the internal plumbing system. Generally speaking, potable water (taste, odour, colour) must be acceptable for the majority of end-consumers. The change in the taste or odour of water can be attributed to the plumbing system (water can dissolve materials and substances in contact with potable water, as for example metals, 7 plastic, and lubricants) and to the changes in the source of water supply and water treatment (i.e. use of disinfection substances) as well as to the presence and activity of microorganisms in potable water. Changes in the colour of potable water, detected on an outlet, point to inadequate water treatment, damage to the plumbing system, rise in water sediments or to the detachment of biofilm in the system. The colouring of water is a consequence of the rise in water sediments due to a change of directions or water flow rate, pipes breaking, or the opening and closing of valves in different parts of the potable water supply system. Water sediments can be a result of corrosion or impurities invading the system via the water supply. End-consumers often perceive the white colour of potable water coming from their faucets as an irregularity. In most cases, the white colouring of water is merely transient as a consequence of micro air-bubbles in potable water. The brown colouring of potable water in the internal plumbing system is mostly caused by iron as a consequence of corrosion of built-in materials. Rules on Drinking Water (2004) classify colour, taste, and odour among indicator parameters in Appendix 1, part C. The limit values of said parameters are set as being "acceptable for end-consumers and without uncharacteristic changes”. These indicators are grouped together because their limit values are not based on data on the immediate dangers to people’s health (NIJZ, 2015).

The cause of any changes in the taste, odour, and colour of potable water must be determined. Since changes in taste, odour, and colour provide only little information, they are evaluated in correlation with the values of other parameters. The evaluation of parameters demands knowledge of the internal plumbing system and the quality of inbound potable water. Until the cause and the effect on people’s health are not determined and appropriate measures are not taken, such water is not suitable for drinking (limitation of water usage for drinking) (NIJZ, 2015).

In the majority of natural waters, pH values are tied to the balance of carbon dioxide, hydrogen carbonate, and carbonate, as well as water hardness (soft waters have a lower and hard waters a higher pH value). A deviation of pH value can either represent an indirect cause of water contamination with a specific substance or it is the water itself that naturally possesses such values. The regulation defines the limit pH value to be between ≥6.5 and ≤9.5 and states that water must not be too 8 aggressive. The group of indicator parameters includes pH because its limit value is not based on the data about the dangers for the health of people, but on the impact on the materials in contact with water and the efficiency of disinfecting water (NIJZ, 2015).

Extreme values in potable water may be a consequence of accidents, errors in water treatment or the release of substances from materials in contact with water (e.g., cement pipes). The impact of a fluctuating concentration of hydrogen ions (pH value) on human health can be direct or indirect. Direct exposure to extremely high or low pH values can cause irritations of eyes, mucosa, skin, and tissue damage. Extreme values, which could cause such damage (e.g., pH values under 4 or above 11), are not common in potable water supply systems. Included among indirect impacts is the increase in corrosion of materials in contact with water with a low pH value; the consequence of corrosion can be contamination of water, change in its taste and appearance, and damage to the material. An adequate pH value is especially important in the process of water treatment to ensure efficient disinfection and coagulation. For efficient disinfection of water with chlorine, a pH value of less than 8 is recommended. Due to its impact on corrosion and the processes of water treatment, it is one of the most important operative (technological) parameters (NIJZ, 2015).

The turbidity of water is an indicator of the presence of particles sized between 1 nm and 1 mm. Particles are formed by inorganic and organic substances and microorganisms (clay particles, silt, colloidal particles, humic substances, algae, plankton, bacteria). Individual components can bind among each other, and form for example a clay-organic part. Particles can result from inadequate treatment, a rise of sediment or a detachment of biofilm in the water distribution system. The turbidity of water is expressed in NTU (nephelometric turbidity units). Particles can represent direct or indirect danger to people’s health. They protect pathogenic microorganisms from the effects of disinfection and increase the use of disinfectants. Particles also stimulate the growth of bacteria in the water distribution system as nutrients adsorb onto them. The absorbable ability of some particles can contribute to the presence of harmful inorganic and organic components in potable water. The consumption of

9 turbid water therefore presents or points to the possibility of a higher health risk (NIJZ, 2015).

Rules on Drinking Water (2004) classify turbidity among indicator parameters, which means that its limit value is not determined on the basis of data about the dangers for health. The limit value demands the “turbidity to be acceptable for end- consumers and for the water to have no uncharacteristic changes.” In the case of potable water treatment from surface water, water turbidity must not surpass 1.0 NTU after it has been treated. According to data by WHO (WHO, 1996), the appearance of water with a turbidity level of up to 5 NTU is usually still acceptable for end-consumers, although lower values of turbidity are recommended due to the microbiological safety of water. Turbidity is one of the parameters which on its own gives little information, thus changes in turbidity are evaluated in connection with values of other parameters. Turbidity contributes to the global evaluation of the quality of water and is an important parameter in the process of monitoring, treatment, and distribution of water. An increase in the turbidity of water from an outlet can indicate contact with surface water, errors or inadequate water treatment, damages in the pipelines, contamination, rise of sediments or detachment of biofilm in the distribution system. The process of water quality evaluation demands adequate knowledge of the system of water supply and the internal plumbing system (NIJZ, 2015). States et al. (1987) stated that Legionellae grew mainly and more visibly in the corners and bottoms of tanks, sedimentation basins, and reservoirs than anywhere else because of excessive sediments and scale found in the mentioned areas. Deducing from this, it is clear that turbidity and organic carbon also contribute to Legionella growth, as these impacts can be found in water areas with excessive sediments (EPA, 1999).

1.2.2 Chemical contamination of the internal plumbing system

Factors that represent a health risk can be biological or chemical. In most cases, chemical contamination contribute to people suffering from chronic diseases, which can manifest themselves in a span of a couple of years or decades.

Chemical toxicity occurs due to chemical substances, which include inorganic substances, such as lead, mercury, hydrofluoric acid, and chlorine gas, and organic substances, such as methanol and most medicines and toxins produced or secreted by biotic organisms (Szabo and Minamyer, 2014).

1.2.3 Microbiological contamination of the internal plumbing system

The microbiological contamination of the internal plumbing system manifests itself in the presence of various microorganisms and their secretions (products- metabolites) in potable water provided by an internal plumbing system and on the inner surfaces of the internal plumbing system elements that the potable water comes in direct contact with. The causes of the microbiological contamination of the internal plumbing system can be divided onto external and internal causes. In most cases, potable water burdened by microorganisms already upon its entrance into the internal plumbing system (via the water supply), consequently contaminates the supply (external cause). In most cases, there is a one-way contamination route, even though contamination can theoretically also occur via an outlet due to the water flow in the internal plumbing system (contaminated pipe surfaces, aerosols). Potable water in public plumbing systems is not sterile, thus, if there are no sanitary obstacles, water running from the water supply also brings various microorganisms into the internal plumbing system. They usually occur in very low (also immeasurable) concentrations and are in most cases non-pathogenic. Even though the number of present microorganisms upon entrance is low, favourable conditions (areas where water stagnates and has a temperature between 20° C and 45° C) can result in a quick increase in the number of microorganisms. In the mentioned areas, microorganisms attach themselves to the inner surfaces of the internal plumbing system elements and form a biofilm (Wingender and Flemming, 2011). Regarding the general and health compliance of potable water with the Rules on Drinking Water (2004), two parameters need to be determined (Escherichia coli and enterococci), which upon confirmation display a certain faecal contamination of potable water. Most definitely, the biggest and most frequent microbiological health risk involved in the

11 consumption of potable water is its contamination with faecal microorganisms. Nevertheless, other sources and manner of exposure should not be neglected. Some microorganisms (e.g., Legionella) proliferate and grow also in the internal plumbing system, once they find their way into the system (WHO, 2011a).

Classical water-borne infections are caused by different microorganisms, deriving from the intestine of a human being or an animal. Potable water may also contain numerous other microorganisms which in certain circumstances may lead to diseases. Such are, for example, protozoa Naegleria fowleri, numerous bacteria, including Pseudomonas, Klebsiella, and some species of environmental Mycobacteria and Legionella spp.. Apart from that, contaminated water may also contain parasites, fungi, and viruses (ADWG, 2011).

Hydric infections are the main but not the only issue brought about by microorganisms in potable water. Microorganisms also excrete toxins, which represent another health risk. Certain microorganisms cause a deterioration of the organoleptic characteristics of water and encourage sediment deposition and corrosion (ADWG, 2011).

Obligate water-related pathogens, which derive from faeces, may cause disease in people regardless of their health status. Other pathogens (the so-called opportunistic pathogens) may cause disease in those population groups which are more disease- prone or exposed to diseases, e.g., the elderly, children, people with a compromised immune system, and patients with other diseases or other predisposing conditions which facilitate infection by these organisms. The most significant opportunistic bacterial pathogens connected to diseases deriving from water are currently Legionella pneumophila and some other Legionella species, Pseudomonas aeruginosa and non-tuberculous mycobacteria (Wingender and Flemming, 2011).

Potable water in compliance with the Rules on Drinking Water (2004) may occasionally contain different microorganisms that may be present below the level of detection of laboratory testing or do not reach the highest value allowed – the latter might not even be set, since not all microorganisms in potable water are subject to routine detection practices. The Rules on Drinking Water (2004) mention 6 12 microbiological parameters (Escherichia coli, Enterococci, coliform bacteria, colony count 22 °C, colony count 36 °C, Clostridium perfringens including spores and their limit values. Among the infectious disease agents in Table 1, the Rules on Drinking Water (2004) directly name only bacterium Escherichia coli.

Table 1: Pathogen bacteria transmitted through potable water (WHO, 2011b)

Pathogen Health Persistence Resistance Relative Important significance in water to chlorined infectivity animal b suppliesc e source Burkholderia High May Low Low No pseudomallei multiply Campylobacter High Moderate Low Moderate Yes jejuni, C.coli Escherichia coli – High Moderate Low Low Yes Pathogenicf E.coli – High Moderate Low High Yes Enterohaemorrhagic Francisella High Long Moderate High Yes tularensis Legionella spp. High May Low Moderate No multiply Leptospira Long Low High Yes Mycobacteria (non- Low May High Low No tuberculous) multiply Salmonella Typhi High Moderate Low Low No Other salmonellae High May Low Low Yes multiply Shigella spp. High Short Low High No Vibrio cholerae High Short to Low Low No longg

(Source: WHO, 2011b) a This table contains pathogens for which there is some evidence of health significance related to their occurrence in potable water supplies. b Health significance relates to the incidence and severity of disease, including association with outbreaks. c Detection period for infective stage in water at 20 °C: short, up to 1 week; moderate, 1 week to 1 month; long, over 1 month. d When the infective stage is freely suspended in water treated at conventional doses and contact times and pH between 7 and 8. Low means 99% inactivation at 20 °C generally in <1 min, moderate 1–30 min and high >30 min. It should be noted that organisms that survive and grow in biofilms, such as Legionella and mycobacteria, will be protected from chlorination. e From experiments with human volunteers, from epidemiological evidence and from experimental animal studies. High means infective doses can be 1–102 organisms or particles, moderate 102–104 and low >104. f Includes enteropathogenic, enterotoxigenic, enteroinvasive, diffusely adherent and enteroaggregative. g Vibrio cholerae may persist for long periods in association with copepods and other aquatic organisms.

1.2.3.1 Microbiological contamination – water phase

Besides contaminated water, hands, accessories and clothing, potable water as a medium is one of the possible ways of transferring disease agents which are transmitted via the faecal-oral route. Breakdown in water supply safety (source, treatment and distribution) may result in a contamination of a larger magnitude, possibly leading to detectable disease outbreaks. On some occasions, potentially repeated low level contaminations may lead to sporadic diseases, although the public health monitoring services are likely not to identify the contaminated potable water as the source of disease (WHO, 2011b).

The mentioned manner of contaminating potable water with microorganisms in the internal plumbing system is often discovered in the process of monitoring the quality of potable water by laboratory testing of taken samples of potable water. This means that in a specific volume of sampled potable water from an internal plumbing system (water phase) the presence of microorganisms is detected. The latter are not evenly distributed according to the volume of water in the internal plumbing system and represent only a part of all the microorganisms present. The cause behind it may be the invasion of microorganisms into water already in the process of water acquisition at the water supply source (primary contamination) or their invasion into water during distribution due to damages on the pipes (secondary contamination). Part of such classification is also the contamination of potable water with microorganisms which proliferate and grow mainly in the biofilm in the plumbing system.

Usually, cultural methods are used to detect pathogenic bacteria in water. Nevertheless, they may make a transition into a viable but non-culturable (VBNC) state. In the VBNC state, bacteria do not grow on conventional microbiological media, where they would usually grow and form colonies; however, they are still alive and marked by low levels of metabolic activity (Oliver, 2010). Their reply to unfavourable environmental conditions, e.g., nutrient deficiency, unfavourable water temperature, presence of disinfectants or toxic metal ions, e.g., copper (Dwidjosiswojo et al., 2011), is converting to the VBNC state. Favourable conditions can upon resuscitation lead to VBNC bacteria becoming culturable again. Oliver

(2010) named all the pathogens which are known to enter the VBNC state and which include all significant water-associated bacterial pathogens, such as Pseudomonas aeruginosa, Legionella pneumophila, Salmonella typhi, or Vibrio vulnificus. Planktonic cells are used to investigate VBNC states, thus this is largely unknown. It can be hypothesised that the biofilm environment can also induce the VBNC state. From the perspective of health, the importance of pathogens in the VBNC state may be underestimated, as they are able to regain their virulence and as such are able to contribute to the start of infection when they revert to the culturable state in favourable conditions. As such, VBNC cells carry a potential to infect when they are present in biofilms of potable water systems (Wingender and Flemming, 2011).

1.2.3.2 Microbiological contamination – biofilm

In many medical, industrial and environmental settings, the forming of waterline microbial biofilms – microorganism communities which attached to a solid surface within an aquatic environment – is an issue. They can be formed where there are enough organic nutrients and sufficient moisture. Biofilm may be found on endotracheal tubing, dental unit waterlines, venous, arterial, and urinary catheters and on in-dwelling devices in a medical environment; even dental plaque can be considered a biofilm (Rowland, 2003).

Biofilms are formed when planktonic microorganisms adhere to a solid surface. Firstly, those microorganisms that have attachment structures such as fimbriae adhere to the surface. Next in line are those microorganisms that need longer contact with the surface for adhesion to occur. Biofilm is formed when the adherent bacteria replicate and the colonies start to merge. When bacteria secrete exopolysaccharides (EPS), adhesion cannot be reversed anymore (Rowland, 2003). EPS can represent anywhere between 50–90% of biofilm’s total organic carbon; EPS is also highly hydrated since it can, by hydrogen bonding, incorporate large amounts of water into its structure. The biofilm is protected from desiccation (Walker, 2007), harsh chemicals, and predation by the hydrated EPS (Rowland, 2003).

The detachment of biofilm can appear constantly, but it should be taken into account that it does not occur in regular intervals. Apart from that, parts of biofilm may detach, leading to a locally increased density of cells in the water phase (the so-called clouds). It needs to be emphasised that in the water phase a great number of bacteria do not display either the quantity of biofilm or its location (Wingender & Flemming, 2011).

Data shows that 95% of bacteria in a potable water system can be found on the surface, while only 5% can be found in the water phase and can be, as such, detected by sampling, which is usually used for water quality control (Flemming et al., 2002).

The biofilm structure is complex as it includes channels that allow the flow of nutrients through the community. Biofilms may include protozoa, algae, bacteria, fungi, nematodes, and diatoms. Means of various signalling compounds enable a symbiotic relationship and even some interspecies communication among biofilm members (Rowland, 2003).

Porteous et al. (2004) note that after their establishment the removal of biofilms is difficult due to their complex structure. Upon establishment, the Legionella colonisation can exist in waterlines for years (Pankhurst, 2007).

Natural potable water also contains organic and inorganic substances, representing nutrients for the microorganisms present. A stronger biofilm may be formed at higher or elevated nutrient levels. Biodegradable compounds from synthetic polymers, e.g., plasticisers, antioxidants, etc., can also serve as nutrients, when they are employed in potable water systems (Keevil, 2002; Rogers et al., 1994).

In favourable conditions in the plumbing system (mainly waterlines where the flow of water is worse or where water stagnates), microorganisms can contribute to the formation of a covering, the so-called biofilm. The biofilm is formed on all surfaces which come in contact with non-sterile water (Flemming, 2011). The formation and growth of biofilm are influenced by water temperature, nutrients availability, hydraulic conditions, and the type and concentration of disinfectant residues (Norton and LeChevallier, 2000). 17

The formation of biofilm is a survival strategy of certain microorganisms in tough conditions. Apart from that, older plumbing waterlines often include various sediments of impurities (smaller pieces of sand, corrosion particles, etc.) and scale, which represent a habitat and food to microorganisms in the water. Potable water often contains calcium carbonate and other minerals, which deposit themselves in the plumbing system (Cotruvo et al., 2009). After the formation of the biofilm, the latter can become a permanent or occasional potential source of microbiological water contamination. The exposure to microbiologically contaminated potable water represents a health risk especially for a more sensitive population of people. Infection may occur by consuming contaminated water, inhaling aerosols which contain disease agents or by contact through skin or the mucous membranes of the eyes and ears (WHO, 2006).

More favourable conditions for the development of microorganisms can be found in systems constructed of zinc-coated steel pipes than in those constructed of plastic pipes. Once the system has already started to corrode, even more favourable conditions for the development of microorganisms are guaranteed. The corroded metal surface has a positive, while microorganisms have a negative zeta potential; thus, the biological coating starts to stick to the metal surface. The zinc coating, which starts to dissolve, already has a fairly high positive zeta potential. This results in the surface being covered by zinc compounds (Zn2+). When the zinc coating vanishes, the corrosion of iron begins and leads to the formation of iron compounds (Fe3+). In the process of clarifying potable water and wastewater, compounds with 3+ Fe are known as flocculants (FeCl3, Fe2(SO4)3). Their main role is connecting fine particles of impurities with negative zeta potential into big flocculants, which can be removed by sedimentation or filtration. In water, all organic impurities (including mineral oils), humic acids, bacteria, fungi, viruses, parasites, etc. have a negative zeta potential (Figure 2) (Drev, 2014).

negatively charged surace

layer of positive particles

sliding layer

surface potential

layer potential

potential

distance between particles

Figure 2: Positive and negative zeta potential (Drev, 2014)

With an aggressive electrolytic solution, a galvanic cell (shown in Figure 3) appears on the zinc-coated steel pipe. A disinfectant is a very aggressive electrolytic solution.

electrolyte solution

zinc coating steel pipe

anode cathode anode

Figure 3: Galvanic cell can occur with zinc-coated sheet metal (Drev, 2006)

Galvanic cells and metal corrosion can also occur with lead, copper, brass, etc. The following contacts are very common: iron-copper (boiler pipelines), iron-brass (fixture pipelines), etc (Drev, 2006).

The impact of biofilm on the quality of water is directly visible also due to the formed metabolic products, such as hydrogen sulphide, nitrite or endotoxins. Their presence may affect the organoleptic characteristics of water (colour, turbidity, and odour). In some cases, biofilms even influence the trophic food chain, leading to the occurrence and growth of protozoa and eventually non-vertebrates (Wingender and Flemming, 2011).

Environmental microorganisms which bear no relevance to man’s health predominantly form the autochthonous microflora of biofilms in potable water systems. This natural population is usually non-pathogenic and contributes to the development and establishment of biofilms (Wingender and Flemming, 2011). An especially serious issue are hospitals’ and other healthcare facilities’ water systems, where water-associated nosomical infections can be greatly influenced by biofilm- borne pathogens (Exner et al., 2005). Biofilms may be a source of pathogens during a continual exposure of patients, care-givers and all surfaces which may come into contact with contaminated water (Ortolano et al., 2005).

It is evident that the biofilm mode of existence of pathogens is of grave importance and as such needs to be part of the risk assessment within water-related pathogens.

To ensure microbiologically safe potable and other types of water, knowledge pays a key role in proper operating and maintaining of water systems. The goal is to minimise the disease potential, deriving from man-made water systems for the human population (Wingender and Flemming, 2011).

One of the first steps in reducing the health hazards for patients and medical staff is to employ the general knowledge about nature as well as the knowledge on the formation and elimination of biofilm (Szymanska, 2003).

1.3 Presence of Legionella

The research part of the thesis focuses mainly on the assessment of the presence of Legionella bacteria in potable and sanitary hot water in the internal plumbing systems of buildings occupied primarily by a more sensitive population.

1.3.1 Legionella in a natural environment

Legionella can be found in moist environments. It can be found mainly in surface waters, such as rivers, lakes, ponds, as well as in mud. From there, it enters a shallow aquifer, where it is present in very low concentrations – a few CFU/1000 mL or even in concentrations below the limit of detection. Legionellae are sensitive to salt, thus they only rarely appear in salt and sea water; nevertheless, some studies note Legionella being fairly common also in marine waters (Ortiz-Roque and Hazen, 1987; Palmer et al., 1993). The temperature span in which Legionellae can be found is pretty wide. They were found in water under the ice as well as in the hot water springs in Yellowstone National Park at a temperature of 63 °C. A nutrient-rich environment enables their proliferation (multiplication) even at 5 °C, while they can also survive in an environment with only a few nutrients (Fünfgeld, 2002). Certain Legionella species can also be found in the ground but their values are fairly low. Legionella manages to proliferate extremely quickly in an environment with favourable conditions. The basic condition which needs to be met for the development of Legionella is an increased water temperature. Legionella can survive in varied water conditions, in temperatures of 0-63 °C, a pH range of 5.0-8.5, and a dissolved oxygen concentration in water of 0.2-15 ppm (Nguyen et al. 1991).

Legionella growth is temperature dependent (ADWG, 2008). The impact of temperature characteristics are: <20 °C dormant 20–25 °C virtually dormant although very slow growth is possible 25–30 °C slow growth if other criteria are met 30–37 °C increase in growth rate

optimum temperature range for multiplication of 37–43 °C Legionella 45 °C maximum temperature for growth 46 °C stationary phase (dies in about 1 week) 50 °C dies slowly (about 10 hours) 55 °C dies in about 1 hour 63 °C dies in a few minutes 70 °C dies in seconds

On some occasions Legionella was also found in groundwater (Lieberman et al., 1995; Lye et al., 1997; Riffard et al., 2001).

Some studies make evident that the number of Legionella spp. found in groundwater was lower than the number of that generally found in man-made waters (Atlas, 1999; Miller and Kenepp, 1993; Wadowsky et al., 1982). This may be due to smaller amounts of biofilm found in groundwater in comparison to the occasionally massive development of biofilms in surface waters and man-made water environments (Marrão, 1993; Colbourne and Dennis, 1988).

A Portuguese research study noted that Legionellae were isolated from any groundwater samples from borehole over a period of seven years. The number of Legionellae varied between 3×102 and 2.4×104 CFU liter−1. Legionella oakridgensis and Legionella sainthelensi were the most often recovered isolates, whereas Legionella pneumophila were recovered only rarely (Costa et al., 2005).

The increase in the use of groundwater as a source of potable water calls for more comprehensive data regarding the occurrence and distribution of Legionellae in such an environment.

212 samples were taken from a public plumbing system – a bigger plumbing system supplied by groundwater – in the period between 2012 and 2015. A review of the laboratory results of the collected potable water samples did not show any proof of the presence of Legionella spp. Conversely, the concurrent sampling of water from 22 the internal plumbing systems of end-consumers included in the aforementioned plumbing system resulted in 16 discovered cases of Legionella spp. (Žitnik, 2015).

1.3.2 Legionella in an artificial environment

Legionellae transferred via water from a natural to an artificial water environment, the so-called man-made waters, created by man to meet his needs. Legionella may be found in a wide array of man-made water sources, including internal plumbing system components. It may enter the water distribution system as a sporadic contaminant within the municipal water supply and find small microscopic pits, which are formed as pipes get older (Singh, 2005).

It could be said that proportionally to the increase in man’s needs related to the use of water, the conditions for the growth of Legionella grew more favourable. One of the most significant factors involved in the increased growth rate of Legionella is water temperature. For example, dental unit waterlines supply water which is frequently still heated to the temperature of 30 °C to 37 °C. Such dental practice is now advised against, since warm water increases the possibility of microbial contamination. Warm water is used for bathing, washing, swimming, cleaning, heating, and the preparation of steam. Domestic hot water (DHW) in domestic installations is drinking water which is heated for uses other than heating of rooms.

Table 2 shows in which artificial environments Legionella may be found. In addition, Legionella may also be found in other artificial water environments, such as air conditioning systems, cooling towers, dental unit waterlines, respiratory-therapy equipment, birthing pool water, humidifiers, whirlpools, vehicle-wash, high-pressure water devices for cleaning, different irrigation systems, misting devices, decorative fountains, home birthing pools, chilled water dispensers, ice machines, etc (EPA, 1999; WHO, 2007).

Table 2: Protocol for sampling environment sites for Legionella (Barbaree et al., 1987)

Site and description Potable water outside or on the boundaries of hospital property 1. Treatment plant (raw and refined water) 2. Guard house or outlying facility if water is not fed there from the hospital 3. Fire hydrant(s) General potable water system for hospitals 4. Incoming water pipe(s) 5. Water softener (pre and post) 6. Pre-heater (discharge side) 7. Primary heater (discharge side) 8. Circulating pump(s) 9. Reservoir tanks (cold water, discharge side) 10. Expansion tank for hot water (if possible)

11. Back drain on sprinkler system(s) (a trap to prevent back-flushing may be present and should be sampled) 12. Fireline where it branches off from the main system (may be multiple) Pharmacy 13. Water used for respiratory therapy equipment Air compressor system 14. Vacuum water source Positive pressure equipment side 15. Condensate from tank(s) 16. Water separator(s) (directly off compressors) 17. Water source(s) near air intake(s) 18. Air samples where patients were ill with legionellosis Potable water final distribution outlets Haemodialysis water source 19. Before demineraliser 20. After demineraliser -Table continues-

-Continuation of the Table 2- Site and description Intensive care units 21. Respiratory therapy (patient rooms) 22. Cardiac 23. Services with different geographical locations 24. Ice-maker (entry water) Air-conditioning system 25. Air handling unit servicing the point of disease occurrence (drain pan) Cooling towers 26. Blowdown 27. Water supply Whirlpools 28. Whirlpool (one nearest air intake system) 29. Whirlpool drain Other 30. Decorative fountain(s) 31. Creeks, ponds, and sites of stagnant water

1.3.3 Biological and ecological characteristics of Legionella

Legionellae are a group of bacteria, which differ from each other partially in their shape and pathogenicity, as shown in Table 3 (Fünfgeld, 2002). They belong to the Legionellaceae family, genus Legionella. There are 49 species of Legionella and 70 serogroups, which differ in the polysaccharide antigens on the cell surface. 70% to 90% of infections in people are caused by Legionella pneumophila of serogroup 1 (WHO, 2007).

Table 3: Legionella species and serogroups (Fünfgeld, 2002)

Legionella species Legionella species

serotypes serotypes

Number of of Number of Number

Pathogenicity Pathogenicity

Legionella pneumophila 15 + »LLAP« 1 + Legionella adelaidensis 1 - Legionella londiniensis 1 - Legionella anisa 1 + Legionella longbeachea 2 + Legionella 1 + Legionella maceachernii 1 + birminghamensis Legionella bozemanii 2 + Legionella micdadei 1 + Legionella brunensis 1 - Legionella moravica 1 - Legionella 1 + Legionella nautarum 1 - cincinnatiensis Legionella cherri 1 - Legionella oakridgensis 1 + Legionella dumoffii 1 + Legionella parisiensis 1 - Legionella erythra 2 - Legionella quaterensis 1 - Legionella fairfieldensis 1 - Legionella quinlivanii 2 - Legionella feeleii 2 + Legionella rubrilucens 1 - Legionella geestiana 1 - Legionella sainthelensis 2 - Legionella gormanii 2 + Legionella santicrucis 1 - Legionella gratina 1 - Legionella shakespearei 1 - Legionella hackeliae 2 + Legionella spiritensis 1 - Legionella israelensis 1 - Legionella steigerwaltii 1 - Legionella 1 - Legionella tucsonensis 1 + jamestowniensis Legionella jordanis 1 + Legionella wadsworthii 1 + Legionella lansingensis 1 + Legionella worsleiensis 1 -

Legionella can be found in water, as a gram of negative, not sporogenic rod-shaped bacterium (0.3–0.9 µm x 2–20 µm or longer), with one or more flagella on opposite ends. Literature prefers the use of the term Legionella instead of the descriptive form – a group of bacteria. Potable water which contains Legionella bacteria generally does not lead to infection, unless it comes to aspiration upon the consumption of such water. Legionella causes disease mainly in people and domestic and farm animals (horses, pigs, small cattle, and dogs) if it enters the lungs with inhalation of contaminated aerosols. Among the infection risk factors is also wound infection, which is regarded as a hospital infection (WHO, 2007). Legionella proliferates intracellularly, meaning that it needs a host – usually amoebas, as it is protected from 26 bactericides and antibiotics by the amoeba’s structure. Thus, it is very important to be aware of the dangers presented by the formation of biofilms in water supply systems.

A significant ecological factor which can importantly increase the risk of legionellosis is the interaction of Legionella with amoebae. Therefore, the redefinition of risk assessment models calls for further consideration (Singh, 2005).

Mycobacterium and Legionella species may be harboured as endosymbionts by Acanthamoeba, Hartmanella, and Naegleria species (Rowland, 2003). Freshwater amoebae are ubiquitous. Some amoeba species may cause infections in people while others can ingest and protect opportunistic bacteria. Hartmanella and Acanthamoebae spp. can ingest Legionella spp. as well as stimulate their proliferation and increase their virulence (Barbeau, 2001).

An important habitat of Legionella and other similar pathogens are free-living amoebae, which can protect the microorganism living within them from chlorine, increased temperature, and biocides. The transmission potential of amoebae is increased due to the production of vesicles filled with pathogens. How and why Legionella manages to survive may be explained by endosymbiosis resulting in the protective intracellular inclusion of Legionellae in the amoebae (Singh, 2005).

The presence, development of, and exposure to Legionella in an internal plumbing system may be caused by several factors, such as (WHO, 2007): - poor water quality and treatment failures (presence of other bacteria, algae, sediments), - presence of biofilm, - aerosol production, - inefficient or ineffective disinfection, - water temperature of 25–50 °C, - distribution system problems, such as stagnation and low flow rate, - pH value of water, - oxygen, - calcium coatings (fur), 27

- construction materials that contribute to microbial growth and biofilm formation (corrosion, organic compounds), - the mere composition of water.

1.3.4 Health hazards

Almost 40 years ago, the Legionnaires’ disease was first described upon an outbreak of pneumonia among members of the American Legion attending a convention in Philadelphia, which received widespread media attention. The unexplained outbreak of pneumonia gave way to research studies, which soon showed that the disease was caused by a bacterium called Legionella pneumophila. It was discovered in January 1977 by Joseph McDade of the U.S. Centers for Disease Control (CDC) (Brenner, 1987). Consequently, the condition was named Legionnaires’ disease. The source of the infection was supposedly located in the air conditioning system. It is of some interest that the same hotel also saw an outbreak of pneumonia two years prior to the outbreak in question (EPA, 1999). Legionella pneumophila received the name Legionella to honour the stricken American legionnaires, while pneumophila is Greek for “lung-loving” (Fang et al., 1989). Soon after its discovery, Legionella pneumophila began to be not only carefully studied but also employed in the development of biological weapons (Miller et al., 2002).

In retrospect, however, it must be noted that the first successful case of isolating a Legionella strain occurred in 1947, from the blood of a soldier stationed in Fort Bragg (USA), who suffered from a febrile illness, the so-called “Fort Bragg fever” (Fünfgeld, 2002). Aside from outbreaks, sporadic cases of legionellosis were furthermore detected in 1943, 1947, and 1959 (Brenner, 1987).

The first reported outbreak of hospital-acquired Legionnaires’ disease occurred in a psychiatric hospital in Washington DC in 1965. 81 patients contracted pneumonia and 15 of them died. Retrospective studies on stored serum samples showed antibody seroconversion for Legionella pneumophila in 85% of the patients (Thacker et al., 1975).

At the time, Legionella pneumophila was a newly discovered bacterium, the search for which was spurred on by its fatal effect on people. The immunity of particular individuals also varies considerably, whether the disease be acquired by contact with a pathogen or influenced by such factors as age, sex, state of health, and living conditions.

Most at risk are: - men rather than women (*the male-to-female ratio was 2.4:1), - people under constant stress (at home and in the work environment), - smokers, - patients with diabetes, - alcohol and drug addicts, - athletes having undergone considerable physical strain, - people with a compromised immune system, - older people (*people older than 50 years of age accounted for 81% of the cases reported), - immobile people (especially in hospitals), - people who have undergone surgery, - people with bronchitis and/or asthma, - medicated people, - patients with AIDS, - patients with rheumatic diseases, - travellers and tourists with a compromised immune system (*travel-associated cases accounted accounted for 19% of the total). *(ECDC, 2015)

Although the consumption of contaminated potable water represents the greatest risk, other routes of transmission can also lead to disease, with some pathogens being transmitted by multiple routes (e.g., the adenovirus). Certain serious illnesses may result from the inhalation of water droplets (or aerosols – i.e. air-borne water particles less than 10 μm in diameter) in which causative organisms have multiplied due to warm waters and the presence of nutrients. These include illnesses caused by the amoebae Naegleria fowleri (primary amoebic meningoencephalitis) and

Acanthamoeba spp. (amoebic meningitis, pulmonary infections), and by Legionella spp., which causes legionellosis (WHO, 2011b).

Aerosol-generating systems, such as faucets, showerheads, cooling towers, and nebulisers aid in the transmission of Legionella from water to air. The inhalation of contaminated aerosols leads to infections with Legionella and outbreaks of disease. Historically, many of the reported outbreaks were nosocomial (i.e. hospital- acquired), resulting from the adulteration of hospital potable water supplies, air conditioning systems, or cooling towers (EPA, 1999).

In terms of risk assessment, it is important to realise that the most prevalent source of Legionella transmission is potable water from large buildings, particularly hospitals. Thus, although Legionella is widely distributed in both natural and artificial water systems, the transmission from a water source to human consumers results mainly from inhalation or the aspiration of aerosolised contaminated potable water (EPA, 1999).

Legionella is an important pathogen in hospital-acquired (nosocomial) pneumonia, particularly in immunocompromised patients. Legionella spp. can also cause community-acquired pneumonia, which has a high rate of hospital admission. Legionnaires’ disease is recognised as a major form of travel-associated pneumonia, and about 20% of the cases of legionellosis detected in Europe are considered to be related to travel; these cases present a particular set of problems due to the difficulties in identifying the source of infection (ECDC, 2015).

The main types of disease caused by Legionella are:  Legionnaires’ disease,  Pontiac fever, and  extrapulmonary syndrome (caused when L. pneumophila spreads from the respiratory system to the body) (WHO, 2007).

Legionellosis can occur as sporadic cases or as outbreaks. The majority of cases of Legionnaires’ disease are sporadic rather than outbreak-related (Stout et al., 1992).

Due to the increased awareness of the disease, numerous community-acquired and travel-acquired outbreaks are now reported each year as well.

Outbreaks of legionellosis are typically categorised as (WHO, 2007):  nosocomial (i.e. hospital-acquired),  travel-acquired,  or community-acquired.

Depending on the use of water, the load of Legionellae can be either tolerated or incompatible. The risk is high in intensive care units, neonatology, and transplant wards. Hence a correlation between the use of water, the tolerated presence of Legionellae, the environment, and the host’s sensitivity is essential, especially with regard to immunocompetence (WHO, 2007).

Figure 4 shows the relationship between the increasing risk of Legionella proliferation and the temperatures of various water systems. The bars represent the average range of operating temperatures of various manufactured water systems.

Figure 4: Water temperature and the increasing risk of Legionella proliferation (ADWG, 2008)

In the case of Legionella, disease prevention is based on averting the proliferation of a large number of microorganisms in warm water and aerosol systems, as well as diminishing contact with aerosols. This is frequently not entirely feasible.

When carrying out a decontamination procedure, appropriate personal protective clothing and equipment (PPE) must be worn. When using the pasteurisation method to decontaminate a warm water system, procedures must be implemented to prevent scalding (ADWG, 2008). In general, we must minimise the generation and spreading of any aerosols, which will reduce the risk of disease in the community.

When assessing the risk and pinpointing the possible sources of Legionella infection in healthcare buildings, other systems, such as cooling towers, vaporising condensers, respirators, medicinal humidifiers (filled with potable water), birth pools, water drinkers, and dental chairs must be taken into account besides water from the internal plumbing system (WHO, 2007).

Concerning the causes of Legionnaires’ disease, we may conclude that the causal chain consists of six parts (Likar, 2002): - the natural container of the infection (water), - multiplying factors (nourishment, temperature, water stagnation), - spread of the bacteria (aerosol origin), - virulent strain of Legionella, - entry into the human being by suitable route (inhalation, aspiration), and - host sensitivity (immune system).

There is evidence that virulence is an important factor in the survival of Legionella in aerosols, with the most virulent strains surviving longer than their less virulent counterparts (Dennis & Lee, 1988). Legionella infections have frequently been associated with sources at distances of up to 3.2 kilometres (Addiss et al., 1989).

1.3.4.1 Incidence of legionellosis

Legionellosis has been reported to occur in North and South America, Asia, Australia, New Zealand, Europe, and Africa (Edelstein, 1988).

Although Legionella is a well-recognised problem in developed countries, data from developing countries are scarce. Since risk environments and susceptible populations are found worldwide, it is likely that the problem of Legionella is under-appreciated in developing countries (WHO, 2007).

The exact incidence of legionellosis worldwide is unknown, since there are great differences in the methods countries use to ascertain whether the disease has been contracted as well as in reporting those cases. The reported incidence of Legionnaires’ disease varies widely according to the intensity of investigation and the diagnostic methodology applied (WHO, 2007).

According to the data of the National Institute of Public Health of the Republic of Slovenia (NIJZ, 2015), the number of reported cases of legionellosis in Slovenia is increasing. The same increase in number can also be detected elsewhere in Europe and in the USA. In 2013, Slovenia was ranked first in the number of reported cases of legionellosis in the EU. Slovenia also leads the EU in the six-year average (2008– 2013), with 33.1 reported cases per one million inhabitants. The high reported incidence rate suggests very good cooperation among diagnostic laboratories and does not imply a high infection rate, as it may seem at first glance (NIJZ, 2015). In 2013, most cases of Legionnaires’ disease in the EU were community-acquired (73%), 19% were travel-associated, and 8% were linked to healthcare facilities. In the same year in Slovenia, 94% cases were community-acquired, 6% were travel- associated, and none were linked to healthcare facilities. In 2013, travel-associated Legionnaires’ disease was 5% lower than in 2012 and seems to continue on a slightly decreasing trend, which has been observed since 2007 (ECDC, 2015). One of the possible reasons for this development is also the onset of a global crisis, which has altered many peoples’ travelling habits.

The disease is diagnosed primarily based on a urine test, with a positive Legionella antigen. Other methods used include: Culture, Fourfold titre rise, PCR, Direct immunofluorescence, Single high titre. If necessary, additional testing is conducted on the appropriate harvested cultures (Maiwald et al. 1998; ECDC, 2015).

The incidence of legionellosis in the EU, Iceland, and Norway is monitored by the European Legionnaires’ Disease Surveillance Network (ELDSNet). The beginnings of data gathering go back to 1986. It includes the monitoring of risk factors in public healthcare and guidelines regarding measures for the prevention of the emergence and proliferation of legionellosis. Slovenia started to contribute its reports in 1998. Table 4 shows the number of cases of Legionaires’ disease in Europe between 1993 and 2013. Number of cases also makes evident the increase in the incidence rate and the prevalence rate. The incidence rate in the past 20 years almost tripled (EWGLI, 1998; EWGLI, 1999a; EWGLI, 1999b; EWGLI, 2003; EWGLI, 2007; EWGLI, 2005; EWGLI, 2010; ECDC, 2011; ECDC, 2012; ECDC, 2013; ECDC, 2014; ECDC, 2015).

Table 4: Number of cases of Legionnaires' disease in Europe between 1993 and 2013 (EWGLI, 1998; EWGLI, 1999a; EWGLI, 1999b; EWGLI, 2003; EWGLI, 2007; EWGLI, 2005; EWGLI, 2010; ECDC, 2011; ECDC, 2012; ECDC, 2013; ECDC, 2014; ECDC, 2015)

Year Number of Number of Populations Rate per Death case cases countries (millions) million – fatality- contributing data rate (%) 1993 *1 1242 19 300 4.1 / 1994 *1 1161 20 346 3.4 / 1995 *1 1255 24 339 3.7 / 1996 *7 1563 24 350 4.5 / (4.9) 1997 *7 1360 24 351 3.9 136 (10) 1998 *6 1442 28 333 4.3 188 (13) 1999 *5 2136 28 398 5.4 193 (9) 2000 *4 2156 28 400 5.4 234 (11) 2001 *4 3470 29 455 7.6 284 (8) 2002 *4 4696 32 467 10.1 283 (6) 2003 *3 4578 34 468 9.8 352 (7.7) 2004 *3 4588 35 550 8.3 396 (8.6) 2005 *2 5700 35 551 10.3 377 (6.6) 2006 *2 6280 35 563 11.2 387 (6.2) 2007 *1 5907 33 523 11.3 391 (6.6) 2008 *1 5960 34 506 11.8 388 (6.5) 2009 *8 5518 27 493 11.2 404 (9) 2010 *9 6296 28 506 12.4 438 (10) 2011 *10 4897 28 507 9.7 358 (11) 2012 *11 5852 29 509 11.5 419 (10) 2013 *12 5844 29 512 11.4 461 (10)

Source: *1 Joseph CA, Ricketts KD, on behalf of the European Working Group for Legionella Infections. Legionnaires’ disease in Europe 2007–2008. Euro Surveill. 2010;15(8):pii=19493.Available online: http://www.eurosurveillance.org/images/dynamic/EE/V15N08/art19493.pdf *2 Ricketts KD, Joseph CA. Legionnaires’ disease in Europe: 2005-2006. Euro Surveill. 2007;12(12):pii=753. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=753 *3 Ricketts KD, Joseph CA, on behalf of the European Working Group for Legionella Infections. Legionnaires’ disease in Europe: 2003-2004. Euro Surveill. Available online: http://ecdc.europa.eu/en/healthtopics/legionnaires_disease/ELDSNet/Documents/1001_ELSN_Legionnaires_disease_in_Europe _in_2003-2004.pdf *4 Joseph CA, on behalf of the European Working Group for Legionella Infections. Legionnaires’ disease in Europe: 2000- 2002. Euro Surveill. Available online: http://ecdc.europa.eu/en/healthtopics/legionnaires_disease/ELDSNet/Documents/1001_ELSN_Legionnaires_disease_in_Europe _in_2000-2002.pdf *5 Prepared by the PHLS Communicable Disease Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, United Kingdom; on behalf of the European Working Group for Legionella Infections. See No. 33, 1999, pp. 273-277. Available online:

35 http://ecdc.europa.eu/en/healthtopics/legionnaires_disease/ELDSNet/Documents/1001_ELSN_Legionnaires_disease_in_Europe _in_1999.pdf *6 Weekly Epidemiological Record. WHO, 1999. Legionnaires’ disease, Europe, 1998. Euro Surveill. Available online: http://ecdc.europa.eu/en/healthtopics/legionnaires_disease/ELDSNet/Documents/1001_ELSN_Legionnaires_disease_in_Europe _in_1998.pdf *7 Weekly Epidemiological Record. WHO, 1998. Legionnaires’ disease, Europe, 1997. Euro Surveill. Available online: *8European Centre for Disease Prevention and Control. Legionnaires’ disease in Europe 2009. Stockholm: ECDC; 2011 *9European Centre for Disease Prevention and Control. Legionnaires’ disease in Europe 2010. Stockholm: ECDC; 2012 *10European Centre for Disease Prevention and Control. Legionnaires’ disease in Europe 2011. Stockholm: ECDC; 2013 *11European Centre for Disease Prevention and Control. Legionnaires’ disease in Europe 2012. Stockholm: ECDC; 2014 *12European Centre for Disease Prevention and Control. Legionnaires’ disease in Europe 2013. Stockholm: ECDC; 2015

The rate of reported cases after 2000 has most likely also grown due to improved diagnostics, more precise in identifying the origin of pneumonia. In addition, the awareness of the disease spreads quickly and a greater number of reports is submitted due to its recognisability. The problem with Legionnaires’ disease is that it is quite similar to regular pneumonia, so it often went by undiagnosed in the past. It is assumed that a high number of cases went unnoticed in the past.

Legionnaires’ disease is a severe and sometimes fatal form of infection with Legionella spp. According to ECDC data (2015), 461 people died of legionellosis in 2013 in the EU, which is comparable to data on fatality rates since 2008. The highest percentages of fatalities with regard to the number of infections in 2013 can be traced to Poland (45%), Estonia (40%), Hungary (24%), and Sweden (19%). With a 6% fatality rate, Slovenia comes in at the rear of these rankings. Figure 5 shows the number of reported cases and deaths due to Legionnaires’ disease in Slovenia from 1991 to 2014. Although most cases of Legionnaires’ disease in the EU were community-acquired, the case–fatality ratio was more than two times higher in healthcare-associated cases (nosocomial and other healthcare settings) than in community-acquired cases (ECDC, 2015).

Figure 5: Number of reported cases and deaths due to Legionnaires' disease in Slovenia from 1991 to 2014 (IVZ RS, 1997; IVZ RS, 2000; IVZ RS, 2009; IVZ RS, 2010; IVZ RS, 2013; NIJZ, 2014; NIJZ, 2015; ECDC, 2010; ECDC, 2011; ECDC, 2012; ECDC, 2013)

Reviewing the data of the ten largest reported clusters of Legionnaires’ disease from 2008 until 2013, it is noticeable that, with regard to the setting of the infection, most cases were community-acquired (ECDC, 2015). Out of these, in at least two cases the probable origin of infection was the plumbing system in the home environment. One cluster was located in Spain and the second in Poland. It is of some note that in the aforementioned time period the case–fatality ratio was more than two times higher in healthcare-associated cases (nosocomial and other healthcare) than in community-acquired cases. Travel-associated cases had the lowest case–fatality ratio (ECDC, 2015). Reviewing the reported cases of Legionnaires’ disease by reporting country in the EU/EEA (Figure 6), an uneven pattern is noticeable. The majority of the reported cases of Legionnaires’ disease are located in the western and partly in the northern part of Europe. According to experts in the field, this does not reflect the actual state

37 of events and more attention needs to be paid to certain countries in order to improve the clinical awareness, laboratory diagnosis, and reporting of Legionnaires’ disease (ECDC, 2015).

Figure 6: Reported cases and notifications of Legionnaires’ disease per million, by reporting country, EU/EEA, 2013 (ECDC, 2015)

A survey of the number of reported cases of Legionnaires’ disease in the EU by month of onset reveals that almost 60% of the cases fall into the time period between June and October (Figure 7) (ECDC, 2015). Studies suggest that the incidence of Legionnaires’ disease may be higher under certain environmental conditions, such as warm and wet weather. Considering that, due to external causes, in the warmer part of the year the temperature of potable water in the internal plumbing system of many a building rises, this means, according to Kucht et al. (1983) at most an enhanced

38 bactericidal effect of chlorine on Legionella pneumophila, insofar as the water is chlorinated, of course. From this perspective, a study of the incidence rate of Legionnaires’ disease in the warmer part of the year where the cause is a chlorinated water system might be of some value.

This correlation should be supplemented with the following probable causes: - greater exposure of the population to risk factors, since many spend their holidays in this time period at public pools, in hotels, spas, camps, etc.; - greater exposure to air conditioning systems, fountains, etc. in said time period; - more favourable conditions for the growth and proliferation of microorganisms, since internal plumbing systems see a rise in water temperatures and water stagnation due to the absence of consumers (brought on by holidays, vacations); - due to heating water supplied by the district heating network in this time period, it is often not feasible to perform thermal disinfections, which are probably the most frequently used preventive measure in buildings.

Figure 7: Reported cases of Legionnaires’ disease by month of onset, EU/EEA, 2013 (ECDC, 2015)

1.4 Disinfection of internal plumbing systems for the mitigation of legionellosis

In principle, ensuring quality water is often an issue, even when the supply of sufficient quantities is not in question. According to potable water quality standards (Rules on Drinking Water, 2004), water treatment constitutes water processing by which its compliance with health standards is guaranteed. The main water treatment methods are displayed in Table 5. The state-of-the-art technology enables the production of pristine potable water from even the most contaminated water sources. This is, however, also tied to enormous costs, which is why in water-supply planning technological as well as economic aspects need to be taken into account. The conservation of water sources is economically much preferable to constant water treatment with very demanding technological purifying procedures. Potable water quality standards also prioritise water in no need of any kind of treatment (Rules on Drinking Water, 2004).

Despite the fact that potable water from a public water supply system, arriving into the internal plumbing system by way of a water supply connector, conforms to accepted standards, the quality in the internal plumbing system frequently shifts (Lautenschlager et al., 2010). Since potable water in the internal plumbing system often undergoes microbiological contamination, which makes it a greater health risk to its users, preventive measures need to be taken. This means performing different water treatment processes and maintaining the individual elements of the plumbing system.

Water treatment processes are either physical, chemical or a combination of both (Benjamin and Lawler, 2013). Water from a water supply system is treated until it reaches the quality necessary for drinking or other household purposes. These methods, although used less frequently and only when necessary, are also employed in the treatment of potable water in internal plumbing systems. The selection of a specific process depends primarily on the type, state, and complexity of the internal plumbing system, the type of microbiological contamination, water composition, the criteria that need to be met, etc.

Water is often not so microbiologically sound as to be drunk without prior treatment. Disinfection is the most important process water undergoes in such treatment. While it occurs through natural processes of disinfection in nature, in water supply systems it must be subjected to technological processing, which can either mimic natural processes or be completely artificial. In nature as in technological processing in water plants, microorganisms in water can be eliminated in a number of ways. The most important is filtration. As water flows through different layers of soil, different particles are removed from it, either through physical containment or chemical bonding. Plant species, which retain microorganisms and use them for their growth, play an important role in water purification. Even one thousand years ago, people knew that it is better to drink water from a river or a lake where there are water lilies or other plants, because water there is cleaner. In many respects, the technological treatment of water in reservoirs is not dissimilar to natural processes. Sedimentation basins or sand bed filters are merely somewhat adapted natural processes. Even membrane filters in water treatment are similar to cell membranes in plant and animal species. Furthermore, UV sterilisation bears similarities to sun rays, the difference being that wavelengths in the visible spectrum are prevalent in sunlight. Chemicals used in disinfection can also be compared to natural processes. For example, iron or aluminium ions dissolved in water set off the coagulation of microorganisms. The presence of certain chemicals inhibits the growth of specific microorganisms. Raising the temperature above 60 °C also has disinfectant effects, while temperatures above 80 °C mean that the disinfection is complete. Therefore, heating it to high temperatures is the oldest means of disinfecting water. When it boils, there are certainly no bacteria or viruses left in it, which means that water has been sterilised. Sterilisation completely renders inoperative or eliminates all kinds of microorganisms, including their stages of development, while disinfection only disables the vegetative forms of microorganisms and not necessarily also their developmental stages (spores, for instance, are much tougher to kill) (Drev, 2011).

Table 5: Main processes in water treatment (Drev, 2011)

process process description equipment and accessories sedimentation sedimentation due to gravity and sedimentation basin particle weight pasteurisation thermal treatment of water (62 °C–85 heat exchangers, °C) heaters

sterilisation thermal treatment of water – boiling heat exchangers, (above 100 °C) heaters

hydrocyclone separation of particles through hydrocyclone centrifugal force flocculation aggregation of smaller particles into Fe+3 and Al+3, etc. flocs traditional retention of larger particles on the sand filter, various filtration lattice of the filter medium sieves, textile filters, etc. flotation process suspended impurities adhering to air flotation units bubbles and floating to the surface adsorption physical bonding of impurities to the activated carbon, active surface of the filter medium diatomaceous earth, silica sinter, chemisorption chemical bonding of impurities limestone, ion exchangers, etc. microfiltration retention of particles of up to 0.1 μm microfiltration on the filter medium modules -Table continues-

-Continuation of the Table 5- process process description equipment and accessories ultrafiltration retention of particles with ultrafiltration ultrafiltration membranes (0.1–0.001 membrane modules μm) reverse osmosis retention of particles with reverse reverse osmosis osmosis membranes (0.005–0.0001 membrane modules μm) plant purification plant-based purifying system fixed or floating plants chlorination disinfection liquid, gaseous, or solid form

The flushing of water from the faucets of the internal plumbing system, insofar as the water in it stagnates, could also be considered a potable water treatment technique. Flushing will help eliminate stagnant water and minimise the proliferation of bacteria that may be present. Flushing can to a degree also minimise the concentration of microorganisms present in potable water, although it does not completely eliminate them from the internal plumbing system. Australian guidelines for reducing the risks at home of Legionnaires’ disease suggest that all warm water faucets that are not used on a daily basis should be flushed weekly by turning the tap on at full flow with hot/warm water for at least 15 seconds once they reach the correct operating temperature (ADWG, 2008). Based on prior experience, the same preventive measure should be employed in the case of cold water taps.

The common disinfection processes, also included in this study, are as follows (Benjamin and Lawler, 2013):  heating – thermal disinfection,  exposure to UV light – physical disinfection,  chemical treatment – chemical disinfection,  membrane filter filtration – physical disinfection.

Technological processes of disinfection can be grouped into physical, chemical, and biological processes, which often interact (can be combined) and supplement one another.

1.4.1 Physical disinfection

Drev (2011) includes the following processes as different types of physical disinfection: - thermal disinfection (boiling, autoclave sterilisation process), - mechanical treatment (filtration; MF, UF, NF, RO), - adsorption (activated carbon, diatomaceous earth, asbestos), - light treatment (UV), - ultrasound treatment (ultrasound cavitation), - hydromechanical cavitation, - plant-based clarification systems, - special ferments and bacterial clusters.

Out of the enumerated methods, thermal disinfection, treatment with UV irradiation, and mechanical treatment shall be further covered below.

1.4.1.1 Thermal disinfection

Heating water to boiling point eradicates all the most important germs. In the interest of safety, water is usually boiled for a couple of minutes. This is practical for individual purposes, but not for the long-term public potable water supply. There is a precise set of criteria, which mandates when this measure should be employed and it almost always accompanies states of emergency and catastrophes.

Thermal technologies’ primary mechanism for the destruction of microbes in water is heat produced by burning fuel. These include boiling and heating to pasteurisation temperatures (WHO, 2011a). This water treatment process usually involves only the internal plumbing system of sanitary hot water.

Pasteurisation is a thermal disinfection process which (Drev, 2011):  destroys all pathogen organisms,  destroys as many technologically damaging microbes as possible,  deactivates enzymes.

The types of pasteurisation processes for the treatment of food and drink differ in temperature regimes and the length of treatment. The LTLT (low temperature long time) approach takes 30 minutes at 62–65 °C, the HTST (high temperature short time) approach takes 15–45 seconds at 72–76 °C, and the VHTST (very high temperature short time) approach takes 5–15 seconds at 85 °C (Drev, 2011).

Although the term pasteurisation is more commonly used in food technology and processing, it has the same value and achieves the desired effect in the thermal disinfection of potable water. The only difference lies in the fact that potable water in an internal plumbing system is subsequently not stored or packaged for longer periods of time, since it is usually in constant flow due to steady usage.

For the development of pathogenic bacteria in water, temperatures of up to 55 °C are the most favourable. To destroy all bacteria in thermal disinfection, sanitary water must therefore be heated above 60 °C. Figure 8 shows the necessary contact time and temperature to kill 90% of the population of Legionella. To eliminate them all, a different contact time is required. It should also be taken into consideration that the bacteria often form a slim biofilm on the walls of the pipe and like to keep to areas that are difficult to access (e.g., the threads of pipe couplings, the housings of fittings), which considerably diminishes the effectiveness of this process. Bacteria of the genus Legionella are especially problematic. They do not develop in cold water, but rather at temperatures ranging from 20 to 45 °C (WHO, 2007).

Decimal reduction time (D) = time in minutes to kill 90% of the population of Legionella

Source: data combined from Dennis, Green & Jones (1984); Schulze-Robbecke, Rodder & Exner (1987)

Figure 8: Decimal reduction time for Legionella pneumophila sg. 1 at different temperatures (WHO, 2007)

Concerning the properties of Legionella, it is important to maintain the recommended temperatures of sanitary hot water (at least above 50 °C) in the entire internal plumbing system of a building after a thermal disinfection has been carried out. The recommended boiler temperature is upward of 60 °C. Due to the hydraulic imbalance of the internal plumbing system of a building or the insufficient capacities of the treatment process and the hot water reservoir, divergences in the temperature of hot water may occur, especially in larger buildings.

While maintaining high hot water temperatures in the internal plumbing system kills numerous microbes or inhibits their growth and proliferation, it also represents a greater risk of scalding in users, as well as greater damage to the materials of the

46 plumbing system and its equipment. The risk with regard to the intensity or degree of the burn depends on the water temperature to contact time ratio. The higher the water temperature to which human skin is exposed, the shorter the contact time required for a deep burn. Compared to surface scalding, deep burns damage all skin layers. A study conducted as far back as 1947 (Harvey et al., 2010) determined that deep burns occur at a contact time of no more than 1 second at 70 C, about 5 seconds at 60 C, and about 5 minutes at 50 C. Figure 9 provides details of the relative contact times leading to full thickness burns at varying water temperatures. The burn risk limit at a given contact time with water at temperatures just below 50 C is also determined by the sensitivity of an individual’s epidermis. For this reason, some regulations require that water temperatures be kept below 50 C (ADWG, 2008).

Figure 9: Full thickness burns – contact times with water (Henriques and Moritz, 1947)

The Rules on Norms and Minimal Technical Conditions for Premises and Equipment of Pre-school Institutions (2000) which set the standards and minimal technical criteria that must be met in the building and equipment of nursery schools in Slovenia, for example, state that water temperature on faucets used by children must not exceed 35 C, in order to ensure the children’s safety and prevent scalding. Hot- water distribution systems may be maintained at lower temperatures (warm water) or

47 have thermostatic mixing valves installed before the outlets to reduce the risk of scalding (typically 41–45 °C). However, caution should be applied, since warm- water systems or pipework downstream of mixing valves can provide environments for the growth of environmental pathogens (WHO, 2011b). Therefore, it is necessary that all pipework downstream of thermostatic mixing valves and tempering valves be decontaminated (for example, by using a chlorine solution or by pasteurisation) at least once every 12 months (ADWG, 2008).

A study conducted in the USA by Alden et al. (2007), reports a 22% fatality rate and a considerable number of injuries due to burns of this sort. Hospitalisation of the injured also meant that high costs ensued (Alden et al. 2007).

Due to the danger of hot water burning and scalding, warnings must be communicated to all users when maintaining water temperatures exceeding 55 °C on the faucets of the internal plumbing system (Harvey et al., 2010; Durand et. al 2012).

1.4.1.2 Ultraviolet light (UV)

Sunlight is a natural disinfectant, which was already well-known in ancient India. Ultraviolet radiation is the energetic part of the electromagnetic spectrum located between X-rays and visible light. Ultraviolet irradiation enhances disinfectant effects and is also a technologically useful method. Irradiation with ultraviolet light is an alternative method for the disinfection of potable water (Drev, 2011).

Lamps of different wavelengths are used (Drev, 2011): 1. VACUUM UV with a wavelength between 100 and 200 nm; 2. UV-A or long-wave ultraviolet radiation with a wavelength between 315 and 400 nm; 3. UV-B or medium-wave ultraviolet radiation with a wavelength spanning between 280 and 315 nm – radiation of up to 300 nm has a lesser germicidal effect, so the desired effect can be achieved by combining with a longer contact period. Lamps of this wavelength are used in medicine; 48

4. UV-C or short-wave ultraviolet radiation spanning between 200 and 280 nm – lamps with these properties have a very high germicidal effect.

Disinfection with UV irradiation is a water disinfection method free of oxidation chemicals, so no hazardous oxidised by-products of disinfection are formed in the water, as is, for instance, typical of chemical-based processes. The application of ultraviolet light has no adverse effects on the taste or potability of the water and does not damage the piping (EWGLI, 2011). In disinfection with UV radiation, water flows through a reactor, where microbes are exposed to the energy of photons in the UV-C spectrum (200–280 nm). This damages the genetic code (by producing thymine dimmers in their DNA) of microbe cells, so they can no longer successfully multiply and cause diseases. The degree of inactivation of the microbes with UV irradiation depends on the UV dose (mWs/cm2). The contact time required for UV disinfection is typically less than 1 min (Benjamin and Lawler, 2013). In addition to water (potable water, wastewater, pool water, mineral water, thermal water, sea water, process water), UV irradiation can also be used to disinfect other fluids and solutions, air as well as surfaces (Drev, 2011).

The ultraviolet light produced by a mercury lamp is effective if the water is free from suspended and colloidal substances, which adsorb light and thus obscure the microbes. The water layer must be slim in order to minimise adsorption by water itself. An important aspect in water disinfection is also the placement of the equipment, since its effectiveness can only be guaranteed close to the point of use. Because it does not spread any residual disinfectant throughout the water systems, it has no effect on possible subsequent microbial contamination. The use of UV as a stand-alone biocide does not fulfil the requirements of the Legionella regulations, because Legionella remains in the biofilms, the dead ends and stagnant areas of the system. Therefore, thermal shock and chlorination methods can be used prior to the application of ultraviolet light in order to control the Legionella present in the system (ADWG, 2008).

1.4.1.3 Filtration with membrane filters

Since membrane filtration of potable water was also used in the present study, the following paragraphs provide some information on this topic.

Membrane-based separation processes comprise those types of filtration which retain only very fine particles. The basic classification of the types of membrane filtration is as follows: conventional filtration, microfiltration, ultrafiltration, nanofiltration, and reverse osmosis (Drev, 2011). Figure 10 provides a parallel display of the approximate molecular weight of specific contaminants found in potable water and the types of filtration that dispose of them.

Anti-microbial water filters are designed to stop microbes suspended in the water. They are usually installed at points of use.

Figure 10: Membrane process classification (EPA, 2001)

Filtration membranes can be (Drev, 2011):  symmetrical (porosity is evenly distributed throughout the entire cross section of the membrane),  asymmetrical (at the top there is a very thin micro-porous structure, followed by a thicker, rougher porous layer).

With regard to their chemical composition, filtration membranes can be polymeric, composite (polymer + reinforcement), metallic, ceramic, glassy (Drev, 2011). Polymeric and composite membranes are the most common. With an asymmetric membrane, filtration occurs only on the micro-porous layer, while with the symmetric membrane the entire cross section is utilised. Reverse osmosis membranes are particularly interesting because they enable the removal of dissolved substances in water, making it, for example, possible to procure “freshwater” out of the sea. Water retrieved in this way is not salty, but it also lacks other minerals, which potable water should have (Ca, Mg, etc.) (Drev, 2011).

Various other filters can also be employed in microfiltration, ones that do not fall into the category of membrane filters. Table 6 shows the main types of filter media used in fine filtration. Some of the enumerated filter media cannot be grouped among membrane filter media, although their properties include the retention of fine particles (Drev, 2011).

Membrane filter modules can be used for the disinfection of potable, technological, or waste-water. In the filtration of potable water, membrane filters for different types of filtration are used. Relatively rough membranes suffice for disinfection (microfiltration), since the bacteria that need to be retained are bigger than 0.1 μm. Ultrafiltration membranes, which in addition to bacteria also dispose of viruses and various big organic molecules (remnants of pesticides), are often employed in the treatment of potable water (Drev, 2011).

Table 6: Filter media for microfiltration (Drev, 2011)

Filter medium Primary material Type of filtration

microporous filter paper microfibres, binding microfiltration agent melt-blown textile materials polymeric microfibres microfiltration

textile rolls shaped like fibres microfiltration candles

filter mats cellulose, asbestos fibres, microfiltration surface-active agents polymeric and composite polymers, reinforcement microfiltration, membranes materials as carriers ultrafiltration, reverse osmosis metallic membranes stainless steel castings, microfiltration, aluminium, precious ultrafiltration, reverse metals osmosis

porous ceramic SiC, Al2O3, clay, etc. microfiltration, ultrafiltration, reverse osmosis porous glass glass microfiltration

With microporous filter media, filtration usually hinges only on the mechanical retention of particles. However, this is not the only available particle-retention process. Besides the mechanical retention of particles, additional forces may be present, which accelerate the filtration effect. Included among these additional clarifying effects are (Drev, 2011): - enhanced adsorption capability (physical bonding), - chemical binding of impurities,

- electrical force (electrodialysis), - magnetic force, - other.

A membrane filter installed at a point of use of potable water is another way of preventing infection with Legionella via potable water in public institutions, such as hospitals, retirement homes, hotels, sports and leisure centres, and other public buildings. Anti-microbial water filters can be installed on the existent faucets as well as showerheads. They certainly constitute one of the most simple solutions in ensuring quick and very effective protection from infection with Legionella. WHO guidelines (WHO, 2007) for hot and cold water systems in general hospitals in high- risk areas state that water from outlets should be free of Legionella. If this cannot be achived within the system, point-of-use filters need to be installed at the outlets (WHO, 2007).

A membrane filter with an immediate barrier against water-borne microorganisms can last up to 31 days. The filter has a bacteriostatic additive throughout the housing, which minimises the risk of retrograde contamination and is compatible with thermal and chemical systemic treatments. The latter is especially important for building managers, who also perform other preventive, i.e. counter-Legionella measures. At first, these filters had a water temperature limit (<50 °C) in order to prevent damage to the filters themselves, which meant that they had to be removed before flushing with sanitary hot water >50 °C. Tehnologically more advanced filters can now withstand an operating pressure of 5 bar and a constant water temperature of 60 °C, even 75 °C for shorter periods of time, and have a guaranteed lifetime of 1 or 2 months, after which they must be replaced. They consist of double-layered 0.2 μm sterilising grade filter membranes. These filters retain fungi, protozoa, and bacteria, such as Legionella spp., Pseudomonas spp., non-tuberculous Mycobacteria, and Escherichia coli (WHO, 2007; Technical Sheet QPoint™ Tap Water Filter, 2015).

In the permeate of these membrane filters (disposable showerhead or faucet filters) we can also monitor the presence of Legionella in potable water. Additional information can be found in the chapter titled Results. 53

1.4.2 Chemical disinfection of potable water in the internal plumbing system

This process entails the biocidal treatment of potable water with an oxidative effect in order to ensure the microbiological purity of potable water. The disinfectant is added to destroy present microbes or as preventive residual maintenance.

In chemical disinfection, effectiveness depends on the type of the selected disinfectant and its concentration, water temperature, water pH, contact time, contamination of the materials in direct contact with water, and the organic and inorganic load of the water. When choosing a disinfectant, possible by-products of disinfection need to be taken into account, which is shown in Table 7.

Table 7: Chemicals most frequently used for the disinfection of potable water and the by-products of disinfection (Drev, 2011)

Disinfectant By-products of disinfection Chlorine / hypochlorite trihalomethanes (THMs) chloroacetic acids chloroacetic nitriles chloroketones chlorohydrates (trichloroacetaldehydes) chloropicrin (trichloronitromethane) cyanogen chloride chlorates chloramines -Table continues-

-Continuation of the Table 7-

Ozone bromaes aldehydes ketones ketoacids carboxylic acids bromoform propanone Chlorine dioxide chlorites chlorates Bromine / hypobromite/BCDMH bromoform bromine hydrates bromates bromamines Hydrogen peroxide with an addition oxygen of silver water silver

Since ozone has a high oxidation potential, it oxidises the cell components of the bacterial cell wall. When it enters a cell, all the essential components of the cell are oxidised (enzymes, protein, DNA, and RNA). The aforementioned mechanism differs from that of chlorination. Ozone first damages the cell walls and only then breaks into the cell, while chlorine enters the cell by means of diffusion and reacts with different enzymes within the cell. The contact time of the disinfectant is of paramount importance.

Most microbes found in water are heterotrophs, which means that they need organic compounds for carbon and energy. In their study, Miettinen et al. (1997) mention that when using various disinfectants to control the growth of microorganisms in potable water, organic compounds with a high molecular weight break down into

55 simple organic acids due to oxidation processes, which increases the chances for the growth of heterotrophic microbes in the internal plumbing system.

Furthermore, chemical disinfection is often the only means of disinfecting the entire internal plumbing system of sanitary hot water where temperatures for a thermal disinfection cannot be reached or of disinfecting the internal plumbing system of cold water. Despite ensuring the recommended cold water temperatures, Legionella can still frequently be found in it.

The study Contamination of the Cold Water Distribution System of Health Care Facilities by Legionella Pneumophila has determined that 35% of cold water samples under 20 °C were contaminated at collection. Those data highlight the importance of assessing the cold water supply of healthcare facilities for Legionella (Arvand et al., 2011).

When using biocides in the treatment of potable water instead of maintaining the recommended regime of potable water temperatures in the internal plumbing system (storage of hot water at 60 °C and distribution at 50 °C), careful monitoring should be conducted in order to check if the biocide process is just as effective. Regular monitoring of this process is furthermore required because a lowering of the temperature of hot water with possible errors in the biocide process can lead to serious issues in coping with Legionella.

1.4.2.1 Disinfection of potable water with chlorine

Because of its high effectiveness and ease of use, chlorine is the most widespread disinfectant (Drev, 2011). In our study, its effectiveness in the control of Legionella in potable water was monitored by using sodium hypochlorite for the purposes of disinfection. Chlorine has a high oxidation potential, which causes the degradation of organic substances and inhibits the metabolism in microorganisms, effectively causing their demise. In water, chlorine first binds with fast oxidising agents (e.g., Fe2+, Mn2+) and organic substances. Subsequently, it forms chloramines with

56 ammonia (monochloramines NH2Cl, and dichloramines NHCl2), which can further oxidise into trichloramines, NCl3 (nitrogen trichlorides), or nitrogen and its oxides. The chlorine in chloramines represents bonded residual chlorine. The limit point is reached when all these chemical reactions have occurred and the continuous addition of chlorine causes the formation of free (excess or residual) chlorine – i.e. hypochlorous acid (HOCl) and the hypochlorite ion (OCl-). A generally effective disinfection is possible if there is at least 0.1 mg/L of free residual chlorine in the form of HOCl in the water (Drev, 2011). However, this frequently does not suffice for a complete elimination of Legionella, which is well-protected in amoebas and biofilm. In their study, Kuchta et al. (1983) note that Legionella pneumophila is more resistant than Escherichia coli when exposed to a water temperature of 21 °C at a pH of 7.6 and a free chlorine concentration of 0.1 mg/L. To kill 99% of Legionella pneumophila, a contact time of 40 minutes is required, compared to 1 min of contact time to achieve a similar effect on Escherichia coli.

The HClO molecule is neutral, whereas the bacterium has a negative charge. HClO penetrates into the cell and oxidises it. While ClO- is also an oxidant, it is negatively charged and therefore less effective. At higher concentrations of H+ more HClO is produced, or rather, at a lower pH the concentration of H+ is higher, which in turn means a more effective disinfection. Consequently, slightly alkaline waters demand higher concentrations of chlorine. The usual dose is 0.1–0.3 mg Cl2/L. The precise volume of chlorine to be added must be gauged, since excessive quantities will degrade the organoleptic properties of the water, whereas insufficient quantities will fail to produce the desired disinfective effect (Drev, 2011). Figure 11 shows the effectiveness of chlorination, which can be enhanced by extending the contact time or elevating the concentration of chlorine in the water.

Figure 11: Bactericidal effect on Legionella pneumophila at different concentrations of chlorine in potable water at a temperature of 21 °C and a pH of 7.6 (Kuchta et al., 1983)

With organic compounds, chlorine forms chlorinated carbohydrates – trihalomethanes, which are carcinogenic. This process can be prevented by using stronger doses of Cl2. This entails a shorter contact time, which does not enable the formation of trihalomethanes, while the residual chlorine is removed from the water by adding SO2 (Drev, 2011).

It is a well known fact that the disinfectant effect of Cl2 hinges on the reaction of Cl2 with enzymes that are crucial in the metabolic processes of cells. The cells die if crucial enzymes are deactivated. Because enzymes are produced within the cell, the disinfection process proceeds in two phases (Drev, 2011): 1. penetration into the walls of the microorganism,

2. reaction of Cl2 with enzymes.

The technology of water disinfection with Cl2 depends on (Drev, 2011):  the types of microorganisms,  the type and the distribution of the disinfectant, as well as products resulting from reactions with the disinfectant in the water, 58

 the properties of water being disinfected,  contact time,  the temperature of disinfected water.

The temperature of the water being disinfected with chlorine solutions is a very important parameter influencing the effectiveness of the entire process (Kuchta et al. 1983; Cooper and Hanlon, 2010). It is often the case that different processes of disinfection have contrary effects if performed simultaneously. For example, the disinfection of water with sodium hypochlorite (NaOCl) with concurrent thermal disinfection will not enhance the final effect. The recommended water temperature when disinfecting with sodium hypochlorite is up to 30 C (Kuchta et al. 1983). The frequently used sodium hypochlorite has its boiling point at >40 C, which is also the point of its thermal degradation (Varnostni list Natrijev Hipoklorit). Impacted by high temperatures and sunlight, sodium hypochlorite in contact with air decomposes into NaCl and Na2(CO3) (Varnostni list Natrijev Hipoklorit). Comparing similar water treatment technologies, different kinds of dependence of individual disinfectants on water temperature can also be noted. The effectiveness of chlorine, chlorine dioxide, ozone, and peracetic acid is highly dependent on water temperature. This is also evident in Figure 12, which shows the degree of bactericidal effectiveness of chlorine at different temperatures of water being disinfected.

Figure 12: Bactericidal effect on Legionella pneumophila at different temperatures of potable water and chlorine concentration 0.1 mg/L and pH 7.6 (Kuchta et al., 1983)

To remove Legionella pneumophila, it takes twice the contact time at 4 °C than at 21 °C, meaning that Legionella can survive in low chlorine values for a relatively long period.

The amount of Cl2 necessary for disinfection depends on the amount of Cl2 dissolved and used in water, which again depends on the amount of HOCl in water. The amount of HOCl depends on the pH value. The higher the amount of acid in water, the lower the consumption of chlorine. The consumption of chlorine can also be interpreted as a part of chlorine being used for decreasing the pH value of water, whereas the other part is being used for disinfection itself. If water is already acidulated, a correspondingly lower amount of Cl2 is used (Drev, 2011). Also in this case a high dependence of chlorine on the pH level is estimated, as shown in Figure 13. While the difference between pH levels 6.0 and 7.0 for a specific amount of chlorine regarding bactericidal activity is small, the dependence on pH rapidly becomes significant already at the pH level 7.6.

Figure 13: Bactericidal effect on Legionella pneumophila at different pH values and at a temperature of 21 °C of potable water and chlorine concentration 0.1 mg/L (Kuchta et al., 1983)

1.4.3 Comparison of controlling methods for Legionella in the internal plumbing system

Table 8 shows the most widely used preventive controlling methods for Legionella in an internal plumbing system. The comparison indicates no costs. There is no ideal solution; however, science should strive for the most optimal technique which will prove most efficient in following the preventive measures in a specific internal plumbing system.

Table 8: Advantages and disadvantages of alternative methods for controlling Legionella in an internal plumbing system (WHO, 2007:50; Tehnična navodila Sanosil)

Method Advantages Disadvantages

Keeping  Simple, effective  Only really applicable to temperature and easily monitored potable water systems <20 °C  Less significant growth of Legionella

Keeping  Simple, effective and  Does not eliminate temperature easily monitored Legionella >50 °C  Requires circulation temperature to be near 60 °C  Difficult to maintain temperatures in old systems  Requires protection against scalding

Periodic flushing  Simple, effective and  Not applicable in cold-water with easy to monitor systems hot water at 50–60  Requires protection against °C scalding (usually an  Must be maintained and essential part of inspected to achieve control by high consistent control temperature)  Re-colonisation occurs within days

Dosing with  Proven, effective  Formation of trihalomethanes sodium disinfection technique  Needs protection (e.g., hypochlorite  Simple to use carbon filter) for dialysis  Relatively patients inexpensive  Toxic to fish  Affects taste and odour  Not stable, particularly in hot water  Increases corrosion of copper

-Table continues-

-Continuation of the Table 8- Method Advantages Disadvantages

Dosing with  More persistent than  Needs protection (e.g., monochloramine chlorine carbon filter) for dialysis  Simple to use in patients mains distributions  Toxic to fish  Penetrates into  Affects rubber components biofilms  No commercial kit available for dosing small water systems

Dosing with  Proven disinfection  Formation of chlorite chlorine technique  Needs protection (e.g., dioxide  Simple to use carbon filter) for dialysis  Low dependence on patients pH value  Safety considerations (depending on the method of generation)

Dosing with  Simple to use  Weak disinfectant hydrogen peroxide  Suspected of mutagenicity

Dosing with  Does not form any  Frequent monitoring of silver hydrogen peroxide side compounds needed with silver  Simple to use  Does not alter the taste of treated medium  Efficient at temperatures between 0 °C and 95 °C  Very little effect on natural environment due to high biodegradability  Low dependence on pH value and temperature

-Table continues-

-Continuation of the Table 8-

Method Advantages Disadvantages

Copper and  Effective when  Frequent monitoring of silver ionisation prescribed copper and silver needed concentrations  Pre-treatment needed (pH, are maintained hardness)  Increased concentrations of copper and silver in water

Anodic oxidation  Disinfection  Pre-treatment needed demonstrated (depending on the effect of pH and hardness)  Effect on Legionella in biofilms not known

UV (ultraviolet)  Proven disinfection  Effective only at point of disinfection technique application; no control  Simple to use downstream (no residual)  Not suitable for turbid waters  No effect on biofilm formation

Ultrafiltration at  Physical disinfection  No inactivation of Legionella point of entry to the barrier downstream of filter within building or system  Effective removal of the system biomass and particles  Effect on the formation of biofilms and sediment not known

Point-of-use filters  Physical barrier  Only suitable at point of use  Easy to install (may  Must be replaced regularly require some  Particulates in water may modifications of the reduce flow and operational outlet) life  Suitable for hot and  Expensive cold-water systems  Good for use in systems where high- risk patients are -Table continues-

-Continuation of the Table 8-

Method Advantages Disadvantages

Pasteurisation heat  Disinfection barrier  Transient effect on with flushing  Useful as short-term Legionella remedial measure  No limitation of biofilm  Simple to apply in formation hot water  Scalding risk installations

Non-oxidising  Proven technique for  Not suitable for potable water biocides cooling systems systems  Mostly not applicable to spa pools  Resistant populations may develop  Need to alternate two different biocides  Often concentrations cannot be readily monitored  Difficult to neutralise for sampling purposes

A half of the abovementioned methods have been applied in preventive measures, monitored in the research. It should be noted that experience shows a considerable amount of cases where it is actually harder to apply the method of keeping the temperature <20 C than keeping the temperature >50 C.

1.5 Chemical softening of potable water

Water containing a considerable amount of calcium hydrogen carbonate

(Ca(HCO3)2) and magnesium hydrogen carbonate Mg(HCO3)2) is referred to as hard water. The hardness of water is a natural characteristic of potable water. According to the concentration of calcium carbonate in millimoles or milligrams per litre of water, we use the water hardness scale to determine the water hardness category: soft, medium hard and hard. The level of hardness of potable water is important for end-consumers chiefly from the aesthetic acceptability, economic, and operative points of view (Cotruvo et al., 2009).

Due to the increased content of minerals in water, the increase in temperature results in an increased accumulation of scale, i.e. fur, on heating surfaces, on the walls of reservoirs and pipelines, and on plumbing fixtures because of the low solubility of calcium carbonate and magnesium carbonate (Matičič, 2010). Heating the water promotes the reaction (Equation 1.1)

– 2– 2 HCO3 → CO3 + CO2 (Equation 1.1)

2– 2+ 2+ by driving off the carbon dioxide gas. The CO3 reacts with Ca or Mg ions, to form insoluble calcium and magnesium carbonates (Equation 1.2) which precipitate out (Earland and Raven, 1971).

Ca(HCO3)2 ↔ CaCO3 + H2O + CO2 (Equation 1.2)

Often, the forming of scale results in serious issues, e.g., clogged pipes, rough surfaces of pipes, which makes it easier for biofilm to attach, increased energy consumption when heating the water, and faulty operation of plumbing fixtures (Roš et. al., 2005; Godskesen et al., 2012). At the same time, especially in steel pipes, corrosion can appear due to aggressive carbon dioxide (Cotruvo et al., 2009). In certain cases, it is necessary to soften the water before using it as sanitary hot water. Softening of water occurs by decreasing or completely eliminating its entire hardness. Demineralisation occurs when the entire amount of salt and ions from

66 potable water is eliminated. Two most appropriate methods of demineralisation are reverse osmosis (physical method) and ion exchange (chemical method). When using the ion exchange and reverse osmosis method to soften the water, we need to be aware of the fact that certain microorganisms can colonise the artificial exchange resin and membranes (Herzberg and Elimelech, 2007). Therefore, besides appropriate maintenance, preventive disinfection of water after such a softening process is recommended. In ion exchange, the most frequent exchange occurs when calcium ions are replaced by sodium ions (Cotruvo et al., 2009). An increase in sodium in potable water is especially problematic in child nutrition and with people on strict diets (NIJZ, 2015).

When pressure changes occur (foremost changes in temperature), firstly small crystals of calcite emerge from crystal cores of calcium and later on continue to grow until they reach a certain size and start depositing from water. Polyphosphates (condensed phosphates) in water hinder the growth of calcite crystals (Roš et. al., 2005). By adding polyphosphate to water (chemical method), the deposition of scale is prevented, at the same time creating a protective layer against corrosion on the walls of pipes and fixtures (Roš et. al., 2005). Mineral polyphosphate solutions for micro-softening were often used within the facilities included in the research in order to prevent the deposition of scale and to decrease corrosion in water installations and sanitary hot water units. A similar effect to the one of polyphosphates can be achieved with other chemical means, for example, sodium silicate, chromium salts, calcium hydroxide, etc. When using polyphosphates in order to prevent scale from forming within sanitary hot water systems it is important to know that, especially when performing heat shocks, polyphosphates will dissolve at temperatures above 80 °C (Matičič, 2010).

Adding mineral polyphosphate solution to potable water with the intention of preventing the forming of scale and the corrosion in steel pipes can benefit the growth and proliferation of microorganisms in the potable water of an internal plumbing system. The second part of the research was dedicated to defining and confirming the abovementioned hypothesis. In nature, phosphorus plays an important ecological role, since it presents an essential element for microorganisms and comes in quantities, usually fairly smaller than carbon (Wetzel, 1975). In their research, 67

Miettinen et al. (1997) state that the microbial growth is increased up to a phosphate -1 concentration of 10 µg of PO4-P liter and that besides other factors (other nutrients, disinfectants, etc.) the microbiological growth is also induced by pipeline corrosion. Although LeChevallier et al. (1996) state that high concentrations of phosphate used for preventing pipeline corrosion proved to be successful in reducing the occurrence of total coliform bacteria in a plumbing system, past results show that the use of phosphate for preventing corrosion could induce issues regarding the microbiological quality of potable water. Therefore, it is advised to keep the levels of phosphates in potable water at lowest possible quantities (Miettinen et al., 1997). Another significant source of possible phosphorus in potable water should be taken into account; the amount of phosphorus released from corroded pipes presents an important basis for the formation of biofilm, even though phosphorus is not added to potable water. Iron or steel water pipes usually contain 0.03–0.2% phosphorus by weight (Morton et al., 2005).

Among the physical methods for softening potable water there is also the use of the devices for magnetic water treatment (Roš et. al., 2005). Compared to chemical softening procedures, the magnetic treatment does not remove calcium and magnesium from water; instead, this process makes the minerals around the magnetised cores flow further together with water without attaching to installations, devices, heaters, wash basins and bathtub surfaces, dishes, cutlery, and other (Roš et. al., 2005). Although this procedure is often characterised as magnetic water softening, this term actually should not be used, as water does not get any softer after this type of treatment. The basis for this is the impact of magnetic or electrostatic field on the microstructure of foreign substances in water (Matičič, 2010). The operation of the device for the magnetic water treatment results in the recrystallisation of calcium carbonate (Matičič, 2010). Matičič (2010) states that the efficiency of this process depends on several parameters (temperature and composition of water, characteristics of the heat transmitter and of the device for magnetic water treatment as well as the position of its instalment) and on the fact that not all the impacts of magnetic field on the process of scale formation are known; the devices for magnetic water treatment seem to be randomly or partially efficient. However, ecologically they are far more acceptable, presenting as low operating

68 costs as systems with liquid polyphosphates already after two years of operation, considering the replacement of heat transmitter due to clogging (Matičič, 2008).

Different terminology for potable water in Slovenian and European legislation should be noted when utilising the chemical process of softening potable water. In Slovenia, the Rules on Drinking Water (2004) do not determine any limit value of water hardness as a tested laboratory chemical parameter. Therefore, even eventually low or high values of potable water hardness do not affect the evaluation of water's adequacy and do not determine its wholesomeness. The same regulations (Rules on Drinking Water, 2004) state no other matter should be added to water except for additives that are necessary for the preparation of water. The preparation of water is seen as the treatment of water which ensures its adequacy and wholesomeness. Therefore, according to the Rules on Drinking Water (2004), adding chemical means for softening potable water does not ensure its adequacy and wholesomeness, and the process of chemical softening itself in this case cannot be seen as water treatment.

Given the fact that in Slovenia there are numerous facilities where potable water is chemically softened for the needs of preparing sanitary hot water, these procedures contravene our legislation; at the same time, the Council Directive (Council Directive 98/83/EC, 1998) includes no specific definition on this matter.

Even in the Technical requirements for the construction of hot water plumbing system and heat stations (2012) as well as for connecting facilities to the hot water system it is stated that at discharging outlets (faucets) sanitary hot water should meet all the conditions defined by the Rules on Drinking Water (2004) and by the SIST EN1212:2005 Standard, which require a certain level of quality of additives used for the preparation of potable water. For the preparation of sanitary hot water the technical requirements for softening the water recommend the installation of a dosage delivery device for liquid polyphosphates that should be adequate for use in potable water preparation as stated by the Rules on Drinking Water (2004). Discrepancies that occur could be attributed to the change of Slovenian legislation in the field of potable water as it was translated from the European legislation in 2004. The previous regulation regarding the softening of water by adding phosphates tolerated the value of common phosphorus to be 6.7 mg/L PO4 which was 20-times 69 higher than usual. The new and present regulation no longer mentions them and does not even allow them as means of preparation – a fact that is obviously still not taken into consideration by every stakeholder.

The good-practice guidelines (Guidelines for the Control of Legionella in Manufactured Water Systems in South Australia / Health Protection Programs, 2008) justify the need to soften hard potable water especially for specific purposes. They state that epidemiological studies have shown a higher incidence of cardiovascular diseases in the areas with soft potable water supply, as opposed to the areas with hard potable water supply. There is no known explanation for this fact; however, drinking water that has previously been artificially softened to the concentration level below

150 mg/L (as CaCO3) is not recommended. Furthermore, there is a possibility of softened water causing the metals in pipes to dissolve.

Although known impacts of hard water on end-consumers are mostly negative, hard water can indirectly also be seen as beneficial to health preservation, given the fact that potentially toxic metals, such as copper and lead, are more soluble in soft rather than in hard water (NIJZ, 2015).

1.6 Materials in direct contact with potable water within the internal plumbing system

Depending on specific requirements within an individual facility, there are several types of materials suitable for the construction of an internal plumbing system. The materials used must meet the legal criteria (Act Regulating the Sanitary Suitability of Foodstuff, Products and Materials Coming into Contact with Foodstuffs, 2000), since potable water is regarded as a foodstuff.

Potable water in an internal plumbing system must meet the regulation criteria as well (Rules on Drinking Water, 2004). The adequacy of potable water must also be ensured at faucets or other areas where water is used for drinking. Besides that, potable water complies with the health regulations when it does not contain

70 concentrated substances that alone or in combination with other substances could present a hazard to people's health (Rules on Drinking Water, 2004). The same Regulations state that the materials and substances, coming into contact with potable water, must not affect the adequacy of potable water regarding its physical, chemical or microbiological characteristics.

Legally, this field is regulated by the Decree on the implementation of Regulation of the European Parliament and of the EC Council on materials and articles intended to come into contact with foodstuffs and on the repeal of Directives 80/590/EEC and 89/109/EEC (2004), the purpose of which is to ensure the safety of materials and products intended for contact with foodstuffs.

The legislation requires each material or product coming into direct or indirect contact with potable water to be inert enough so that its constituents are not transferred into potable water in amounts that could present a hazard to people's health or induce unacceptable changes to the composition of potable water or deteriorate its organoleptic characteristics (Decree …, 2004).

When constructing and maintaining public potable water supply systems, it is necessary to follow the Standard provisions (SIST EN 805) and other regulations, concerning this field. When plumbing is concerned, all built-in materials have to be considered, starting from the primary facility (a water reservoir – a tank or a well, etc.) to the end device (outlet) where potable water for human needs is poured out. Materials that were usually used to construct plumbing systems can be classified into the following groups (Drev et al., 2008):  inorganic materials (concrete, brick, ceramics, glass, asbestos, sand, silica sinter, limestone, etc.),  polymeric materials (PE, PP, PVC, PET, PETF, PA, PUR, PES, PEX, PEX- Al-PEX, rubber, silicone, etc.),  natural organic materials (jute, wood, charcoal, etc.),  metals (stainless iron, iron, aluminium, brass, zinc, copper, lead, chromium, nickel, etc.).

Water reservoirs within internal plumbing systems are usually built of reinforced concrete which is often protected by polymeric coatings to increase chemical resistance and to make the surfaces that come in contact with potable water smoother (Drev et al., 2008). Individual reservoirs and parts of technological equipment for the preparation of water are normally made of reinforced polymeric materials, sometimes even of stainless steel (Drev et al., 2008). Rarely, standard steel sheet metal coated with appropriate materials is used. Suitable protective coatings of corrodible steel surfaces are (Drev et al., 2008):  inorganic enamels (often in heaters),  hot-dip galvanised surfaces (pipes, etc.),  galvanic deposits of metal (chromium, nickel, zinc),  polymeric protective layers (epoxy coatings, alkyd coatings, acrylate coatings, etc.),  ceramic tile coverings.

Nowadays, plumbing pipes are mostly made of polymeric materials (PE, PEX, PEX- Al-PEX, PP, PVC, etc.) and partly of steel pipes. Many plumbing pipes and other parts of internal plumbing systems in older facilities, however, are still made of steel, usually galvanised steel pipes. All these parts are more or less exposed to corrosion which directly leads to chemical and physical contamination of potable water with possible organoleptic changes (Drev et al., 2008; WHO, 2011a). From the perspective of human health protection most issues occur with lead, but at the same time copper and galvanised pipes also present a threat for water contamination (NIJZ, 2015; Lytle and Liggett, 2016). The side products of corrosion cause water contamination either by directly contaminating it or indirectly by providing a nutrient medium for microorganisms. The connection between iron corrosion and a repeated growth of bacteria has been known for a while (Larson, 1939). One of the studies finds that higher levels of iron corrosion increase the number of bacteria in biofilm (LeChevallier et al., 1996). In form of a nutrient, iron presents one of the more important factors for the growth of Legionella (Earhart, 2009; Bargellini et al., 2011). The fact is that nutrient media containing iron (ferric phosphate) are used for the growth of Legionella in laboratories.

All metal pipes and parts of technological equipment are sensitive to corrosion when galvanic cells appear (Drev, 2006). This often happens when different metals come in contact or there is a temporary increase or decrease in pH value. A temporary but considerable decrease in pH value appears with every chlorine shock in an internal plumbing system (EWGLI, 2011; Drev, 2011). Every galvanised steel pipe – still the most common material installed in internal plumbing systems – is also sensitive to corrosion (Drev, 2006). The zinc coating is there to dissolve in water before iron does. With iron, many different mechanisms of corrosion can appear, depending on the composition of water and the type of galvanic cell that enables corrosion. With galvanised pipes, galvanic cell is created when zinc coating – being a less precious metal and dissolving – acts as anode and iron as cathode which is not used as long as enough zinc is available (Drev et al., 2008). This is the basic purpose of zinc coating – protecting iron from corrosion by dissolving itself first. The consequence of such dissolution are zinc ions that appear in water (Peng et al., 2010; WHO, 2011a).

Inadequate seals or materials for water preparation, etc. can also be seen as significant sources of water contamination (Drev et al., 2008). Often there is no attention paid to the end-parts of an internal plumbing system which come in contact with water before it is used, e.g., outlets, seals or various cleaning elements at the location where water is used. These materials can also deposit toxic substances or substances that accelerate the growth of microorganisms into water (Drev et al., 2008). The latter is noted in the article by Kilb et al. (2003) where water samples from drinking water distribution systems repeatedly showed the presence of coliform bacteria. In several cases, inspecting the internal surfaces of the systems showed visible biofilms on rubber-coated valves where coliform bacteria were found. These valves seem to be the spots responsible for water contamination. Usually, low molecular weight additives of polymers can support microbial growth. The key role of materials regarding drinking water quality is demonstrated in this example (Wingender and Flemming, 2011).

In heavy water flows there is no significant amount of substances that come into water by depositing from the materials the water has been in contact with (Drev et al., 2008). Often, this does not apply to individual points within the internal

73 plumbing system with light and rare flows which can be the cause of an increased concentration of depositions (Drev et al., 2008; WHO, 2011a).

When constructing plumbing systems, the use of different plastic materials (PE, PP, PVC, PES, etc.) has become a constant in the past twenty years. Chemically and mechanically, these materials are extremely resistant and easy to install; however, they come with some degree of possibility for water contamination (Drev et al., 2008). The materials that come in contact with water need to have fundamentally different composition than other types of plastic. For example, PVC that is used in windows and doors contains toxic substances as stabilisers, which must not be present in the PVC that is used in plumbing (Pb, Bi, Cd, Zn, etc.) (Drev et al., 2008). The same goes for other types of plastic as well. For the purpose of easier thermal modification of plastic, plastic materials can contain softeners that are not suitable for use in products that come in contact with water (Drev et al., 2008). In products that are made of regenerates or low-quality polymers, decomposition processes of plastic can occur. All such processes can cause the formation of toxic substances that can dissolve in water (Drev et al., 2008). An even bigger issue than standard plastic materials are materials used for sealing (different types of rubber, soft plastic, and textile fibres). Also PTFE seals which are chemically very resistant can contain substances that could present an issue (Drev et al., 2008). In the production of PTFE foils and seals, oil derivatives are namely used as sliding agents (Drev et al., 2008). If the treatment of PTFE products was not done correctly (i.e. the removal of sliding agents using heat or solvents), such material may not be suitable for use in plumbing systems, since it can contain up to 20% oil-based sliding agents (Drev et al., 2008). It is important that the materials used in plant construction can resist corrosion and withstand extreme conditions when exposed to different levels of water quality or chemicals for water treatment (WHO, 2011a).

1.7 Regulations overseeing the field of Legionella control in plumbing systems

Potable water is regarded as a foodstuff; therefore, it has to meet certain conditions of the Act Regulating the Sanitary Suitability of Foodstuff, Products and Materials Coming into Contact with Foodstuffs (2000). There are no individual microbiological parameters noted in this act; there is only a general demand that a foodstuff should not contain microorganisms, parasites or any of their developmental forms or secretions that may be harmful to people's health. Based on the Act Regulating the Sanitary Suitability of Foodstuff, Products and Materials Coming into Contact with Foodstuffs (2000), the Rules on Drinking Water (2004) were passed, which oversee the adequacy and wholesomeness of potable water in the Republic of Slovenia. The Rules on Drinking Water (2004) are only indirectly concerned with Legionella, since there is no specific mentioning of it. Regarding this aspect, the Regulations consider potable water to be wholesome, as long as it contains no microorganisms, parasites or any of their developmental forms in amounts that may be harmful to people's health (Rules on Drinking Water, 2004), which takes into consideration the broadest area of all mentioned microbiological organisms. On the contrary, a narrower meaning can be attributed to the evaluation of adequacy, meeting the demands of set 6 microbiological parameters for limit values of the Rules on Drinking Water (2004). The list of parameters can eventually be supplemented by additional parameters and their limit values. Although the presence of Legionellae in potable water may present a potential risk for people's health, they have not yet been added as an additional parameter. The case would certainly be different, if Legionella presented a risk for people's health in the sense of ingesting potable water.

The legislation concerned with the field of controlling Legionellae in potable and bathing water systems, and measures arising from this legislation differ in different countries across the world as well as in the European Union. A few years ago, in Austria a special emphasis was put on preventive measures regarding bathing waters (WHO, 2007), while England (WHO, 2007), the Netherlands (WHO, 2007), and

Ireland (WHO, 2007) also saw these measures in the field of safety at work. In Ireland, special attention in their guidelines (WHO, 2007) was given to preventive measures regarding dental chairs and high risks in hospitals. Some European countries include the field of risk management regarding Legionella infections in their Contagious Diseases Act. For reference, among 20 European countries Slovenia places alongside Belgium, England, Germany, Italy, Latvia, the Netherlands, and Sweden (WHO, 2007) as far as preventive measures against legionellosis are concerned. These measures include potable water, thermal water, bathing water, cooling towers, air conditioning, and process water, the only difference being that at this time our legislation does not mention the field of process water. Different countries have differently set target values (Table 9) for Legionella in plumbing systems. In Slovenia, there have been no such recommendations legislated on the state level yet; health facility operators set the target values for the prevention of legionellosis in their plans for the prevention and management of hospital-acquired infections.

Table 9: Target values for Legionella in internal plumbing systems of selected countries (WHO, 2007:60; EPA, 2009)

Country Unit (CFU/L) Commentary Source France <1000 Target value for public Ministère de facilities la Sante et <100 Target value for the des prevention of hospital Solidarités infections (2005) <50 Target value for facilities where immunocompromised patients are hospitalised Germany 1000 DVGW (2004) Netherlands 100 Target guideline value VROM (2002) Great Britain <100 Target guideline value HSE (2004) Italy 1000 Target guideline value Gazzetta Ufficiale 2000 Denmark - No guideline value has been set for Legionella in potable water Ireland - No guideline value has been set for Legionella in potable water Croatia - No guideline value has been set for Legionella in potable water Slovenia - No guideline value has been - set for Legionella in potable water Australia - No guideline value has been ADWG set for Legionella in potable (2015) water Serbia - No guideline value has been set for Legionella in potable water Bosnia & - No guideline value has been Herzegovina set for Legionella in potable water USA 0 Public Health Goal EPA (2009) CFU – Colony forming units

In Slovenia, the field of monitoring, controlling, and preventing infectious diseases is regulated by the Contagious Diseases Act (2006) and by the Rules on Reporting Infectious Diseases and Specific Measures for their Prevention and Control (1999). These regulations define the diseases that must be reported as well as the manner of reporting them (among them also legionellosis). These are all infectious diseases with which there is a high possibility of spreading; all of them also demand fast action in order to reduce the possibility of disease occurrence, infectious diseases, against which people get vaccinated, and infectious diseases which may represent a substantial burden to society (IVZ RS, 2013).

To protect the residents, the Contagious Diseases Act (2006) also includes legionellosis among the infectious diseases which call for the employment of general and special measures for their prevention and management. The measures also include providing medically adequate potable water and the adequate quality of air in closed areas.

If Legionella is found in potable water, the suitability of water is not determined; however, limit values are specified in the Rules on Minimum Hygiene Requirements to be met by Baths and Bathing Water in Swimming Pools (2011) and in the Rules on the Ventilation and Air-conditioning of Buildings (2002).

1.8 Aim of research, research objectives and working hypotheses

Despite the fact that potable water flowing from the public plumbing system via the water supply into the internal plumbing system may be in full compliance with the current regulations, its quality may often change within the internal plumbing system – water becomes contaminated – and may, as such, represent a great health risk to its end-consumers. In order to provide greater safety, preventive measures and corrective actions must often be undertaken. Physical, chemical, and microbiological contamination of water are the most frequent. This goes especially for microbiological contamination, which is the main focal point of this master's thesis. The treatment procedures of potable water may be physical, chemical or a

78 combination of both. They help treat the water in the plumbing system and increase its quality to a higher level, meeting the health standards for drinking or other household purposes. In some rare cases, these procedures are also employed in potable water treatment within internal plumbing systems, though none of the mentioned procedures can be used universally. The physical procedure of water treatment by heating (heat disinfection), which helps maintain the high temperatures of sanitary hot water in the internal plumbing system, is often employed. This manner of water treatment destroys numerous microorganisms in water or slows down their growth and proliferation. At the same time, maintaining high water temperatures (above 50 ºC) consequently leads to a higher risk of burns in end- consumers and damage to the materials of plumbing fixtures and devices. The mentioned treatment procedure can only be employed with sanitary hot water in an internal plumbing system.

The master's thesis mainly focuses on the microbiological aspect, especially on the presence of Legionella bacteria in the internal plumbing system of selected medical facilities in the Ljubljana region. Medical facilities were chosen for the purposes of this research, since their “users” (patients) represent the most sensitive group of the population, due to their often immunocompromised condition (health condition, diseases, illnesses, etc.) and are, as such, more prone to various infections. The study focuses on Legionella bacteria which are often present in the water environment of an internal plumbing system, representing an immediate health risk to water- consumers.

Besides being found in the water of internal plumbing installations, Legionella may also be found in the water of numerous other sources: cooling towers, air conditioning devices, air humidifiers, pools (bathing water), wells, fountains, devices for sprinkling water on fresh vegetables, devices for respiratory therapies, dental chairs, etc.

The presence of Legionella in internal plumbing systems of medical facilities is a known issue around the world, but one which is mainly, as expected, registered in

79 developed countries only. Researched scientific articles from this field expose this as a topical issue and one which is tackled by researchers in different manners. Needless to say, their success in managing and preserving the adequate quality of water differs. As it is often the case, at the expense of safety, the financial input plays an important role also in this field. It should not be forgotten that human life, which can be exposed to great risk in such cases, has no price.

1.8.1 Aim of research

The aims of Master's thesis:  study the efficiency and adequacy of employed approaches such as physical disinfection with heat and chemical disinfection, for the elimination or reduction of the number of Legionella in potable (cold) water and sanitary hot water of selected medical facilities in use mainly by a more sensitive part of the population. One of the aims was also to check some other technological procedures of disinfecting internal plumbing systems, such as filtration and UV radiation;  evaluate the impact of potable water softening with polyphosphates on the growth of some microorganisms in sanitary hot water;  produce an overview of the use of different materials (hot-dip galvanised iron pipes, stainless pipes and PEX-Al-PEX pipes) that come in immediate contact with potable and sanitary hot water as well as check the possibilities of water contamination due to the composition of said materials;  evaluate the efficiency of chemical disinfection using chlorine products regarding the presence of Legionella in the water of selected systems of sanitary hot water (in correlation with water temperature);  critically evaluate the existing regulations in Slovenia regarding the management and prevention of legionellosis occurring in the water of internal plumbing systems.

1.8.2 Research objectives

Research objectives:  determine the presence of Legionella in the water of internal plumbing systems of selected medical facilities after an 8-year monitoring;  determine the advantages and disadvantages of performing different types of water disinfection in internal plumbing systems; specifically, the comparison of physical disinfection – heat disinfection and chemical disinfection;  determine the by-products (chloride, chlorite, chlorate, trihalomethanes-sum, chloroform, bromoform, bromodichloromethane, and dibromochloromethane) of chemical disinfection in selected water samples;  determine orthophosphates, nitrates and nitrites in selected water samples.

In most cases, higher values of nitrates present in water point to water contamination as a consequence of human activity. By determining the presence of nitrates in water, their immediate impact on the growth of some microorganisms in cold and sanitary hot water can be evaluated. Nitrites in water may also be a nutrient for certain water organisms, which can lead to favourable conditions for the growth and proliferation of other organisms such as Legionella. Algae and amoebae, which are often present also in artificial water environments (internal plumbing system), present supporting organisms for the occurrence and proliferation of Legionella.

1.8.3 Working hypotheses

 A preventive measure of physical disinfection by increasing the temperature of sanitary hot water in internal plumbing systems of selected medical facilities is an efficient way of managing the issue of Legionella occurrence.  From a technological perspective, providing suitable conditions for heat disinfection is a demanding process, which directly affects the reduction of Legionella in drinking water.  Softening of potable water with chemical means that contain phosphorus (polyphosphates) may accelerate the growth of microorganisms in water.

2 THE EXPERIMENTAL PART

Controlling the quality of water regarding the presence of Legionella was performed in the scope of the internal monitoring of potable water in internal plumbing systems of selected medical facilities. The monitoring included determining the sanitary technical conditions of elements and devices within internal plumbing systems, field measurements, and taking of water samples for microbiological and physical and chemical tests in compliance with the ISO 5667 standard (2006). Altogether, 2676 samples of cold and sanitary hot water were taken.

The research was focused mainly on determining the suitability of performed preventive measures regarding the management of the Legionella issue in internal plumbing systems of selected medical facilities in use mainly by a more sensitive part of the population.

In the research period, the following preventive measures were undertaken in the majority of internal plumbing systems:  maintaining the temperature of sanitary hot water above 50 ºC;  maintaining the temperature of cold water below 20 ºC;  flushing the water out from rarely used outlets;  cleaning of faucet aerators and showerhead nozzles; if needed, the replacement of an entire faucet or a showerhead;  flushing out water from sanitary hot water reservoirs at least once a month;  the system is thoroughly cleaned whenever an inspection reveals sludge, slime, scale, foam, rust, dirt, dust or other impurities or foreign material present in the system;  physical disinfection (overheating) – heat disinfection of the sanitary hot water system at least once a year by maintaining the water temperature within the reservoir above 70 ºC for more than 24 hours and ending the process by flushing the water out with a temperature above 65 ºC through the end-outlets for at least 5 minutes. The efficiency of the disinfection process was evaluated by comparing the results of laboratory testing of taken water samples before

performing the heat disinfection and the results of samples taken a week or two after the performed heat disinfection.

2.1 Time schedule

The first part of the research included a general overview of internal plumbing systems of selected medical facilities, a selection and processing of data from the existing database of an 8-year period, an overview of acquired results of microbiological laboratory testing of water samples, and an overview of regulations dealing with the control of Legionella presence in plumbing systems.

The second part of the research included practical work – additional field measurements, sampling and laboratory testing of water samples. The impact of softening potable water with polyphosphates on the proliferation of some microorganisms in sanitary hot water was evaluated. Water samples were taken from the internal plumbing systems of four medical facilities in Ljubljana.

The third part of the research included the processing and evaluation of data, and an estimation of microbiological quality of water (presence of Legionella) from internal plumbing systems of selected medical facilities in the Ljubljana region, and a study of possible risks and measures in preventing or reducing them.

2.2 Methods employed

By using Microsoft Word, Microsoft Excel and SPSS for MAC 20.0.0 statistical software, the collected data from the existing database of taken water samples were processed and evaluated, and are presented in tables and figures below. Regarding the second database, additional field measurements, sampling, and laboratory testing of 12 water samples were made on 9 physical and chemical parameters (nitrite, nitrate, ammonium, orthophosphates, oxidising, pH, electrical conductivity (20 C), turbidityiron and 6 samples additionally on chloride, chlorite, chlorate, 83 trihalomethanes-sum, chloroform, bromoform, bromodichloromethane, and dibromochloromethane) and 12 samples on 6 microbiological parameters (Escherichia coli, coliform bacteria, number of colonies at 22 C, number of colonies at 36 C, Pseudomonas aeruginosa, and Legionella).

2.2.1 Field measurements of potable water

Upon sampling the water, individual field measurements were made. Besides the measured water temperature (electrometric measurements) upon sampling, normally the dynamics of water temperature in correlation with the time of letting the water run was observed. An organoleptic review (appearance, odour) of water was made. In time of water sampling at sampling points, concentrations of disinfectant means (free chlorine) in water were measured within the systems, where potable water was disinfected using chlorine means. Measurements were made according to the method pertaining to the SIST EN ISO 7393-2:2000 standard, which determines the colorimetric method for determining free and total chlorine in potable water, bathing water, and wastewater by employing the DPD reagent. Free chlorine is chlorine, present in the form of hypochlorous acid, hypochlorite or dissolved elemental chlorine. Free chlorine was determined with a direct reaction with N,N-Diethyl-1,4 Phenylenediamine Sulfate (DPD) and the formation of red component, followed by direct colorimetric measuring of acquired colour complex with the HACH POCKET

COLORIMETER measuring device. The result, expressed in mg/L of Cl2, represents a direct reading from the HACH colorimeter screen.

2.2.2 Potable water sampling

From 2006 to 2013, altogether 2648 water samples were collected from the internal plumbing systems of diverse healthcare facilities, which were then tested for the presence of bacteria from the genus Legionella. In the majority of cases (N=2626), the samples were collected from the internal plumbing systems of sanitary hot water;

84 only 22 samples were collected from the internal plumbing systems of cold water. As a result of thermostatic settings, samples of a mixture of cold and sanitary hot water were taken in some cases. Said samples are included in the sum of samples of sanitary hot water.

However, it is also worth noting that after 2011 there was a larger share of samples collected after overheating, whereas until 2010 more samples were collected before overheating.

One of the reasons for the pronounced decrease in the number of positive samples in 2008 and 2009 is also the fact that building managers adopted some changes in the implementation of overheating (i. e. doing it for longer periods of time at higher temperatures) and the flushing of hot water. In 2008, 2009 and 2010, samples were generally collected 3 days after overheating the system, whereas over the remaining years the interval between overheating and sample collection was longer. In 2011, 2012 and 2013, sample collection thus took place 14 to 21 days after overheating.

A number of samples was also collected as permeate via antibacterial filters installed on outlets and declared for use in the case of high concentrations of Legionella (i. e. water treatment at a point of use with point-of-use filters). The samples were collected from filters with 0.2 µm filter membranes with a lifespan of one month.

The samples were taken also from the showers of medical bathtubs (mobile, supported or therapeutic) that in the form of autonomous devices possess their own plumbing system, connected to an internal plumbing system within a facility. Medical bathtubs are used in healthcare facilities for the care of patients and functionally disabled and for rehabilitation programmes (therapy).

Most of the samples were collected in compliance with professional guidelines for the collection of potable water samples (IVZ RS). Sampling of potable water was performed in compliance with the ISO 5667 (2006) standard. Water samples were mainly taken after a prior, at least 2-minute flushing of cold or hot sanitary water through different types of outlets (faucets, showerheads, pipe outflows, etc.).

Table 10: Overview of number of water samples concerning type of water supply system, method of disinfection and chemical softener in health facilities from 2006– 2013

Internal plumbing systems Chlorination Filtration Chemical softener YES NO YES NO YES NO Cold water systems 1 21 0 22 0 22 Sanitary hot water systems 211 2415 31 2595 2334 292

For the second database the sampling deliberately from the two mentioned sampling sites were located at the end of the internal plumbing system.

Regarding Legionella presence, water sampling was performed as part of the planned annual control according to the Recommendations on Sampling of Water in regard to Legionella (IVZ RS, 2008).

2.2.3 Microbiological analysis methods

Laboratory testing of taken water samples in regard to specific microbiological parameters was performed in the laboratory of the Institute of Public Health of the Republic of Slovenia (IVZ RS) and in the laboratory of the Institute of Public Health Celje (ZZV CE) in accordance with the methods named in Table 10. Both laboratories were accredited in compliance with the SIST EN ISO/IEC 17025:2005 standard.

Table 11: Selected methods for microbiological testing of potable water samples

PARAMETER METHOD Escherichia coli SIST EN ISO 9308-1:2001 Cor1:2007 Standard test Coliform bacteria SIST EN ISO 9308-1:2001 Cor1:2007 Standard test Number of colonies at 22 C SIST EN ISO 6222:1999 Number of colonies at 36 C SIST EN ISO 6222:1999 Pseudomonas aeruginosa SIST EN ISO 16266:2008 Legionella SIST ISO 11731:1999, SIST EN ISO 11731- 2:2008

The isolated strain of Legionella spp. was confirmed with the Oxoid Legionella Latex test, which means that it belonged to one of the following species of Legionella: L.longbeachae sg. 1 and 2, L. bozemanii sg. 1 and 2, L. dumoffii, L. gormanii, L. jordanis, L. micdadei, or L. anisa.

2.2.4 Physical and chemical analysis methods

The laboratory testing of taken water samples in regard to specific physical and chemical parameters was performed in the laboratory of the Institute of Public Health Celje in accordance with the methods named in Table 11. The mentioned laboratory was accredited in compliance with the SIST EN ISO/IEC 17025:2005 standard. In order to acquire the laboratory results, the following analysis methods were employed after sample preparation: ion chromatography, gas chromatography, spectrophotometry, titration, inductive coupled plasma – mass detector, and electrode measurement.

Table 12: Selected work methods for testing water samples in regard to physical and chemical parameters

PARAMETER METHOD Nitrite /ISO 26777 Nitrate 283/SIST EN ISO 10304-1 Ammonium 263/ Orthophosphates 283/SIST EN ISO 10304-1 Oxidising 262/ISO 8467 pH 238/SIST ISO 10523 Electrical conductivity (20 C) 261/ISO 7888 Turbidity 260/SIST EN ISO 7027 Iron /SIST EN ISO 17294-2:2005 Chloride 283/SIST EN ISO 10304-1 Chlorite 283/SIST EN ISO 10304 Chlorate 283/SIST EN ISO 10304 TRIHALOMETHANES-sum 320/DIN 38407-30:2003 Chloroform 320/DIN 38407 Bromoform 320/DIN 38407 Bromodichloromethane 320/DIN 38407 Dibromochloromethane 320/DIN 38407

2.3 Facilities included in research

The study included 23 buildings and their respective internal plumbing systems of different ages and different approaches to water treatment. The internal plumbing systems of these buildings consisted mainly of zinc-coated pipes. A smaller proportion of the materials used in construction was taken up by PEX-Al-PEX pipes, stainless steel and enamelled metal. In the majority of the buildings, sanitary hot water was stored in tanks with a joint volume of more than 100 m3.

Included medical facilities were mainly larger structures and a complex with a vast internal plumbing system. On average, these facilities are around 50 years old. The age of each individual facility generally agrees with the age of its internal plumbing system, which is mainly made from zinc-coated metals, whereas in some facilities

88 heating stations for the preparation of sanitary hot water were renovated already years ago. To provide greater safety in treating immunocompromised people, some medical facilities additionally treat potable water by filtration or disinfection after it enters the facility. In most cases (in 22 out of 23 medical facilities), potable water is chemically softened before it is heated for the purposes of sanitary hot water. The issue with larger facilities is that individual spaces are not always evenly burdened. Locally, this results in merely sporadic flows and water stagnation in the internal plumbing system.

There are immunocompromised patients with a weakened immune system located in different units within health facilities, such as intensive care unit, oncology department, transplant department, haematology department, nephrology department – dialysis, etc. They are regarded as patients with an increased risk of diseases caused by contaminated water, whether by drinking it, coming in contact with it, or aspirating it.

3 RESULTS AND DISCUSSION

3.1 Presence of Legionella in the potable water of selected healthcare facilities in the Ljubljana healthcare region (2006–2013)

The study was conducted in the Republic of Slovenia, in the Ljubljana healthcare region, from 2006 to 2013. The mentioned region spans more than 23% of the surface area of the Republic of Slovenia and encompasses more than 30% (or 600,000) of the inhabitants of the Republic of Slovenia. Figure 14 shows the size and location of the Ljubljana healthcare region within the Republic of Slovenia.

LJUBLJANA HEALTHCARE REGION

Figure 14: The Ljubljana healthcare region on the map of the Republic of Slovenia (http://zora.onko-i.si/)

With the cold water samples, as much as 77% of positive samples with an average concentration of approximately 300 CFU/L (min. 10 CFU/L, max. 3000 CFU/L) were detected. The temperatures of 12 collected positive cold water samples were measured as ranging from 13.2 °C to 18.3 °C (max. 600 CFU/L), while the other 5 such positive samples had temperatures ranging from 24.5 °C to 27.2 °C (max. 3000 CFU/L) (Table 13).

Table 133: Negative and positive samples of cold water concerning presence of Legionella (from internal plumbing systems of health facilities in 2006-2013)

Number of Water Negative Positive The highest samples of temperature at concentration cold water sampling (°C) - not found - found Legionella spp. (Legionella spp.) (Legionella spp.) (CFU/ 1000 mL)

11,2 - 18,3 5 12 600 22 24,5 - 27,2 0 5 3000

In Germany (Arvand et al., 2011), there are no special protocols in place for a routine testing of cold water for the presence of Legionella in the internal plumbing systems of healthcare facilities, unless cold water temperatures at distal sites exceed 25 °C. In their study, Arvand et al. (2011) find that cold water samples collected at distal sites of the internal plumbing systems of healthcare facilities are contaminated with Legionella more often than sanitary hot water samples (at a ratio of 40% to 23%). They also detect higher concentrations of Legionella in cold water samples. Our results show that there were more than 70% of positive samples in the temperature of cold water within the limit of the recommended temperature for cold water (<20 °C). In our experience, it would certainly be more sensible in this respect to periodically test and verify cold water for the presence of Legionella in the internal water distribution system in medical facilities, irrespective of water temperature.

For the most part, the laboratory results of samples collected after flushing showed lower concentrations of Legionella than samples collected before flushing. For example, a concentration of 130 CFU/L of Legionella (with a collection temperature of 24.5 °C) was detected in a cold water sample collected before flushing, while the concentration of Legionella after flushing was 0 CFU/L (with a collection temperature of 17.0 °C). Similarly, a sanitary hot water sample collected before flushing displayed a concentration of 13,000 CFU/L of Legionella (the temperature upon collection being 47.7 °C), whereas the concentration of Legionella after flushing amounted to 450 CFU/L (the temperature upon collection being 53.3 °C).

Figure 15 shows the presence of Legionella in the samples collected from the internal plumbing systems of selected healthcare facilities in the period from 2006 to 2013. Evidently, the percentage of contaminated (positive) samples underwent a statistically significant drop (Χ2 = 150.7, df = 7, p <0,001), from 71.7 % in 2006 to 44.0 % in 2013. In the time period under consideration, the amount of samples which tested positive for Legionella was thus reduced by at least 25%.

Figure 15: Samples tested positive for Legionella from 2006 to 2013 (in %) from internal plumbing systems of healthcare facilities

The prime reason for the significant drop in positive samples after 2007 is the improved implementation of preventive measures by building managers, who in the majority (all of the 23 healthcare facilities) of cases took up systematic overheating of the internal plumbing systems of sanitary hot water. It is imperative that overheating be conducted in accordance with the relevant guidelines (like EWGLI Technical Guidelines), which also require keeping records of the operations performed and the subsequent findings. Compared to others (for example, chemical disinfection), overheating is a relatively simple procedure, which building managers can perform on their own. In buildings where sanitary hot water systems and the internal plumbing system permitted overheating at higher temperatures maintained for longer periods of time, followed by hot water flushing, the measures were usually successul and led to a lower concentration of Legionella with fewer positive samples.

The number of Legionella (CFU) in 1,000 mL of the potable water sample was grouped into six size classes: up to 10 CFU per sample, from 11 to 100, from 101 to 1,000, from 1,001 to 10,000, from 10,001 to 100,000, and more than 100,000 CFU/L. Figure 16 shows that results shows significant drop (Χ2 = 57.3, df = 21, p <0,001) of positive samples in percentages and according to the aforementioned size classes for the period from 2006 to 2013.

Figure 16: Classification of collected samples from internal plumbing systems of healthcare facilities which tested positive for Legionella according to size classes in percentages from 2006 to 2013

The presence of Legionella in concentrations exceeding 100,000 CFU/1,000 mL is a good showcase of the drop in concentrations by year. Figure 16 shows that in 2006 1.6% of samples contained more than 100,000 CFU/L, in 2007 the percentage of such samples fell to 1.4%, it further dropped to 0.3% in 2008, while between 2009 and 2013 no such samples could be detected. From a total of 1281 positive samples were values exceeding 100,000 CFU/1,000 mL in 8 samples. In the literature (Rota et

94 al., 2004), values exceeding 100,000 CFU/1,000 mL are deemed highly conducive to legionellosis. In the observed 8-year-long period, it is evident from Figure 16 that concentration values fluctuate most in the lower three size classes (up to 10 CFU per sample, from 11 to 100, and from 101 to 1,000 CFU/L). The reason why 2008, when there was a pronounced decrease in the share of overall positive samples (from 71.7% in 2006 to 55.3% in 2008), also saw the highest share of positive samples in the lowest size class (up to 10 CFU) can be identified in the joint effect of all preventive measures (systematic overheating of the internal plumbing systems of sanitary hot water, regular preservation of recommended water temperature, flushing pipes of the internal plumbing systems). In this time period, a decrease of positive samples in all size classes can be detected, with a logical concomitant increase of the positive sample percentage in the lowest size class (up to 10 CFU), which represents the final boundary line delimiting a positive from a negative sample. This means that since the gradual implementation of systematic preventive measures, more samples with Legionella concentrations ranging from 1 to 10 CFU/1,000 mL were detected. By contrast, a 12% decrease of positive samples in the lowest size class (up to 10 CFU) could be observed in 2009. The overall trend shows a decrease in positive samples after 2009 (from 71.7% in 2006 to 37.6% in 2009), which is, as already noted, a consequence of the implementation of systematic preventive measures. The same can be said for the concentrations of Legionella: after 2009, the percentage of samples with high concentrations dropped significantly. Assessing the final three years of the observed period, it is evident that the positive samples from one size class (up to 10,000 CFU) began to migrate into another class (up to 1,000 CFU), which is certainly advantageous for building managers. In general, the majority of positive samples in the observed period fall into size classes spanning from 11 to 1000 CFU/mL (Figure 16).

Figures 17 and 18 show that in the observed period the majority share (67,7%) , calculated by the number of all positive samples (N=1281), is represented by the species Legionella pneumophila sg. 1. This species and serotype are considered to be the most virulent strain of Legionella, which causes from 70% to 90% of all infections in people. Given its health hazard to the human population, the finding that it is precisely the species Legionella pneumophila sg. 1 that was detected in all samples with high concentrations of Legionella (>100,000 CFU/L) is even more 95 alarming. In 2009, compared to other years in the observed period, the proportion of Legionella pneumophila sg. 1 decreased the most (50,4%) (Χ2 = 142.93, df = 7, p <0,001). The proportion of an unidentified strain of Legionella spp., data fluctuation in 2009 is primarily a consequence of detecting a higher number of samples (83.3%) of the strain Legionella spp. in the potable water of a newly opened healthcare facility (Χ2 = 48.26, df = 7, p <0,001). In 2006 the proportion of the species Legionella pneumophila sg. 2–14 was highest, significant diferencess in distribution of samples through the years were observed (Χ2 = 114.52, df = 7, p <0,001).

Figure 17: Classification according to the identified serological group of Legionella pneumophila and the strain Legionella spp. in percentages and by year (from 2006 to 2013) from internal plumbing systems of healthcare facilities

While Legionella pneumophila sg. 1 was predominant among all positive samples for the entire period from 2006 to 2013 with a 67% share, the proportions of Legionella pneumophila sg. 2–14 and Legionella spp. were similar (Figure 18).

3.1.1 The impact of thermal disinfection on the presence of Legionella in the water of internal plumbing systems

Figure 19 shows that for the entire observed period of 8 years, the number of positive samples collected after overheating was smaller (43%) than the number of positive samples (57%) collected before overheating. Within the samples taken before termal overheating we found statisticaly significant diferencess in distribution of positive and negative samples through the years (Χ2 = 73.27, df = 7, p <0,001). The same applied for the samples taken after termal overheating (Χ2 = 78.75, df = 7, p <0,001). Laboratory test results of the water samples were generally better (with a lower number of positive samples, lower values of Legionella, or even its absence) after an overheating of the internal plumbing system of a building had been performed. In 2009, for instance, three days after overheating and flushing the internal plumbing system of a selected building, laboratory tests of the collected water samples showed a negative sample rate of 73%. By contrast, samples collected from the same building a couple of months later without prior overheating and flushing were negative only in 57% cases (i.e. down by 16%).

80%

70%

60%

50%

40%

30%

20% 2006 2007 2008 2009 2010 2011 2012 2013 (N=187) (N=283) (N=396) (N=364) (N=401) (N=388) (N=345) (N=284)

positive samples pre heat positive samples po heat

Figure 19: A comparison of positive samples from internal plumbing systems of healthcare facilities collected before and after overheating from 2006 to 2013

The decrease in the proportion of positive samples collected before overheating in the years 2008 and 2009 furthermore suggests that the drop is also due to other, non- thermal measures (chlorination, filtration, flushing). Additional confirmation of this is provided by differences in the proportions of positive samples, which fluctuated between 0.6% and 12.4%. Only in the last three years is there a visible trend in the increasing difference between positive samples collected before and after overheating.

The result analysis suggests that it would be prudent for future research to survey individual outlets and thereby evaluate the actual effectiveness of the implemented measures at selected sites. Since the results comprised in this study outline a general picture of the condition of the water in selected buildings, it is not at this point possible to analyse individual points of use and, for instance, compare the state of potable water at the very same tap before and after specific measures have been implemented.

3.1.2 The impact of chlorine on the presence of Legionella in the water of internal plumbing systems

The internal plumbing systems of 3 out of 23 buildings were exposed to permanent chemical disinfection of potable water with sodium hypochlorite (14–16% solution).

Figure 20: A comparison of the percentages of positive samples without and with chlorine (from internal plumbing systems of health facilities in 2006-2013)

In the observed 8 year period, the ratio of the number of positive samples with chlorine to positive samples without chlorine was 92/1189. The number of collected samples, however, is also significantly higher for unchlorinated water. Within the facilities that did not use clorine, we found statisticaly significant diferencess in distribution of positive and negative samples through the years (Χ2 = 122.27, df = 7, p <0,001). The same applied for the facilities that used clorine (Χ2 = 43.15, df = 7, p <0,001). The ratio of the overall number of samples with chlorine to the number of samples without chlorine was 212/2436. This data is especially significant in analysing results obtained in the years 2006–2008, where the number of positive 99 samples with chlorine exceeded the number of positive samples without chlorine (Figure 20). In 2007, out of the 15 chlorinated samples 14 tested positive, which equals 93%. The small amount of positive samples with chlorine is therefore not realistically comparable with positive samples without chlorine. This is another case where there is a noticeable drop in the number of positive samples without chlorine in the years 2008 and 2009, which again suggests that the decrease is also due to other preventive measures. One of the causes could be the already mentioned better, systematic approach of building managers to preventive operations.

The highest recorded concentration of free chlorine was 0.29 mg Cl2/L; on average, it did not exceed 0.10 mg Cl2/L. Low concentrations around 0.10 mg Cl2/L have a weaker bactericidal effect on Legionella pneumophila. Kuchta et al. (1983) found that at a concentration of 0.10 mg Cl2/L and an exposure interval of 30 minutes, 4% of Legionella pneumophila survive. The same effect can be achieved at a 15-fold shorter exposure interval if the concentration is 0.50 mg Cl2/L. Judging by the recorded concentrations of free chlorine in buildings where water is chlorinated, building managers should increase these concentrations for a greater bactericidal effect. However, increasing the concentration of free chlorine in potable water also increases DBP concentrations. In the building where the concentration of free chlorine in potable water was measured at 0.18 mg Cl2/L, for example, the water sample also revealed the following concentrations: 11.5 mg/L of Cl, <0.1 mg/L of

ClO2, 0.58 mg/L of ClO3, and 5.4 µg/L of all THMs. Meanwhile, in another building, where the concentration of free chlorine was measured at 0.06 mg Cl2/L, the water sample also revealed these other concentrations: 8.7 mg/L of Cl, <0.1 mg/L of ClO2,

<0.1 mg/L of ClO3, and <0.5 µg/L of all THMs. The formation of DBP does not hinge only on higher concentrations of free chlorine in the water, but also on the types and concentrations of organic substances in raw water, temperature, and pH value. It also depends on the reaction rate, which consequently leads to increasing THM concentrations along the distribution system.

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3.1.3 The impact of point-of-use water filters on the presence of Legionella in the collected water samples

Results on the presence of Legionella in water samples collected at sites, where outlets are usually equipped with antibacterial filters, are presented in Figure 21.

Figure 21: Results on the presence of Legionella in water samples from internal plumbing systems of healthcare facilities collected at sites, where outlets are usually equipped with antibacterial filters, from 2006 to 2013

Out of the 31 collected filtrate samples, 2 tested positive for Legionella. Legionella was also detected in 10 samples which were collected after the installed antibacterial filters were removed. In 3 of the cases, no presence of Legionella was found after the removal of previously installed antibacterial filters. It is of note, however, that in some instances the bacteria Pseudomonas aeruginosa could be isolated from the filtrate, which was not the case for water sampled ahead of the filter. Given the size of the membrane pores of the used filters (0.2 µm), Pseudomonas aeruginosa (1.5– 101

3.0 µm long and 0.5–0.8 µm wide bacillae) was expected to be removed. Probable causes for the failed removal are water retention ahead of the filter and in the filter itself or the contamination of the outer, outflow surfaces of the antibacterial filter. Furthermore, water retention or lower waterflow speeds caused by antibacterial filters often lead to heightened concentrations of Legionella in the internal plumbing system ahead of an installed filter. Antibacterial filters were installed in individual healthcare facilities on those points of use where patients with a compromised immune system come into direct or indirect contact with water. Although these types of filters are used primarily as an immediate barrier against Legionella in the case of outbreaks, building managers use them permanently if they cannot ensure the safety and health of patients with a compromised immune system with regular preventive measures. Within the facilities that use point-of-use water filters, we found statisticaly significant diferencess in distribution of positive and negative samples through the years (Χ2 = 13.07, df = 5, p <0,023). The same applied for the facilities that do not used such filters (Χ2 = 150.70, df = 7, p <0,001).

Antibacterial filters are a welcome solution in exceptional circumstances, but only for shorter periods of time, since they do not eliminate the presence of Legionella in the internal plumbing system of a building – on the contrary, Legionella concentrations could be even higher than before. The installation of antibacterial filters is a short-term solution, similar to boiling water when faced with a public plumbing network contaminated with faecal bacteria. What is needed in these cases are comprehensive long-term solutions. Antibacterial filters also need to be regularly replaced with new filters, depending on the lifespan determined by the manufacturer. The replacement deadline can be seen on the technical sheet of the product; with some filters it can be found on the filter's casing. In rare cases, either filter substitution was found to be a couple of days late or the date of first filter use was not noted on the filter. Due to the diminished waterflow, sedimentation and microorganisms may accumulate ahead of the filter, which means that when performing intensive flushing and overheating (prior to which filters need to be removed), absolute safety of the users should be guaranteed and the formation of aerosols prevented. On the basis of personal experience it can be said that despite its quick and simple application the filtration process requires careful maintenance. In buildings with a high number of points of use the installation of antibacterial filters is 102 also cost-intensive. On the basis of personal experience, the price for a single piece of such an antibacterial filter can cost up to EUR 100.00. Presuming that a healthcare facility includes, for example, 1500 points of use where in theory a filter would be installed to every single one and replaced on a monthly basis, the expenses of such a project would amount to EUR 1,800,000.00 per year. However, no matter the amount, no price should be put on human life.

3.1.4 The impact of phosphate addition on the presence of Legionella in the water of internal plumbing systems

With the aim of preventing scale deposition and decreasing the corrosion in plumbing installations or devices, in the majority (in 22 out of 23) of the selected buildings the procedure for the treatment of sanitary hot water included microsoftening of the water, using mineral polyphosphate solutions. The majority of samples were taken from sanitary hot water systems. Therefore, a slight similarity between the curve presenting the number of positive samples with phosphates (Figure 22) and the curve showing the number of all positive samples (Figure 15) can be observed. Within the facilities that use microsoftening of the hot water systems, using mineral polyphosphate solutions, we found statisticaly significant diferencess in distribution of positive and negative samples through the years (Χ2 = 159.65, df = 7, p <0,001). The same applied for the facilities that do not used such softening procedures (Χ2 = 31.4, df = 7, p <0,001).

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Figure 22: Share of positive samples for Legionella from 2006 to 2013, where there were phosphates added to drinking water from internal plumbing systems of healthcare facilities

Although only 11% of 2,648 taken samples contained no added phosphates, Figure 23 shows that with the exception of the years 2006 and 2010, the number of samples without added phosphates presented a higher share of positive samples than the number of positive samples with phosphates. A lower number of positive samples without the addition of phosphates, compared to the number of negative samples without the addition of phosphates was determined only in 2006 and 2010. Unfortunately, regarding the addition of phosphates, there is only a single piece of data available which shows that there was indeed such water treatment (chemical softening) used in the selected facility, however there are no specific data available regarding the concentrations of dosing polyphosphates in the internal plumbing system. Based on the measurements of Research 2, it can be ascertained that the measured values of phosphates present were very low (maximum 0.74 mg/L PO4). Unfortunately, the research in question was carried out on a small sample (N=12) 104 which only leaves space for speculations that the actual phosphate concentrations in water are low or are not monitored precisely for that matter. The manufacturer's recommendation for an effective use of phosphates in the softening of potable water amounts to concentrations up to 5 mg/L PO4. The connection between the presence of phosphates and the increased presence of Legionella has been researched primarily due to many authors' beliefs (Miettinen et al., 1997; Morton et al., 2005) that phosphorus could be one of the limiting factors for the proliferation of bacteria. If during the potable water treatment process phosphorus is added single-handedly, this limiting factor no longer exists and consequently it is reasonable to expect increased concentrations of Legionella. Given the variable and foremost low concentrations of phosphates in water the mentioned correlation cannot be confirmed. From the research that extended over 8 years, we could not acquire more precise data on the added phosphates in potable water; nevertheless, the research was carried out in 2010, albeit, on a rather small sample.

Figure 23: Share of positive samples for Legionella from 2006 to 2013, where there were no phosphates added to drinking water from internal plumbing systems of healthcare facilities

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3.1.5 The occurrence of Legionella in the water of internal plumbing systems of bathtubs

In 8 (0.3%) out of the total of 2,648 samples, more than 100,000 CFU/1000 mL Legionella (maximum of 820,000 CFU/1000 mL) were present, with another 2 samples coming very close to that value (720,000 and >300,000 CFU/1000 mL). There were additional samples with high concentrations of Legionella, i.e. 240,000 CFU/1000 mL, 140,000 CFU/1000 mL, 120,000 CFU/1000 mL, 110,000 CFU/1000 mL, 97,000 CFU/1000 mL, and 96,000 CFU/1000 mL. The determined values in the mentioned samples generally did not present the state of the internal plumbing systems of healthcare facilities. Figure 24 shows comparative shares of positive and negative samples, retrieved from medical bathtubs, and all the positive and negative samples. Within the medical bathtub showers, we found statisticaly significant diferencess in distribution of positive and negative samples through the years (Χ2 = 20. 76, df = 6, p <0,002). 73 samples were taken from the showers of medical bathtubs, merely 10 of which showed no presence of Legionella, with more than half of them taken after the cleaning and chemical disinfection of medical bathtub plumbing systems. When comparing, the results show that Legionella was present in the water of medical bathtub plumbing systems in concentrations several times higher than in the internal plumbing system to which they were connected. For example, the water sample taken from the external ball valve of the internal plumbing system of the medical bathtub showed the presence of Legionella in the concentration of 150 CFU/1000 mL, whereas the sample taken from the shower of the medical bathtub showed the concentration of 820,000 CFU/1000 mL. Tuttlebbe C.M. et al. (2002) and Walker (2004) state the same in the case of dental chairs and other diagnostic and therapeutic devices that use potable water without previous treatment. The most probable reason for this may be longer stagnation of water in such closed systems at favourable temperatures (for different microorganisms) and consequently the proliferation or a more intensive growth of Legionella. Besides that, the pipes are often made of rubber, which presents additional favourable conditions for the growth of Legionella. Usually, in medical bathtubs there is an option of adjusting the water temperature with a thermostatic mixing valve. These valves need

106 to be checked for sanitary hygiene and maintained regularly, since the inner surfaces of the thermostatic mixing valves present favourable conditions for the colonisation of bacteria (WHO, 2011a).

Figure 24: Share of positive and negative samples concerning presence of Legionella from medical bathtub showers in health facilities and share of all samples from 2006 to 2013

3.1.6 The comparison of different procedures and types of treatment

Figure 25 shows that there was a higher share of negative samples compared to positive samples in the process of filtration, thermal disinfection – after overheating, chemical disinfection with chlorine, and phosphate softening. The ratio between positive and negative samples in the last procedure was equal to the share of all negative and positive samples. As previously mentioned, the reason lies in the fact that the majority of water samples was subject to the softening of water with phosphates.

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Figure 25: Comparison of shares of positive and negative samples concerning presence of Legionella taken after filtration from medical bathtub showers in health facilities, prior to overheating, after overheating, with chlorine, and with phosphates from 2006 to 2013

Disregarding the number of taken samples, the results in Figure 25 show that both procedures, after overheating and chlorine disinfection were similarly successful. While the filtration proved to be the most successful, it can be ascertained that the use of medical bathtubs can present a high risk for people's health from the perspective of Legionella infection. Not only was the share of positive samples high; in this case also the majority of samples with the highest concentrations of Legionella was determined.

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3.2 Research 2 (2010)

Besides standard testing, an additional laboratory testing on 26 potable water samples, taken from six health facilities in 2010, was carried out. Tested were the presence and the values of the following microbiological parameters: Escherichia coli, coliform bacteria, number of colonies at 22 °C, number of colonies at 36 °C, Pseudomonas aeruginosa, Legionella, and physical and chemical parameters: nitrite, nitrate, ammonium, orthophosphate, oxidising, pH, electrical conductivity, turbidity, iron, chloride, chlorite, chlorate, THM – sum (chloroform, bromoform, bromodichloromethane, dibromochloromethane). In Figures 5–8, some results of laboratory testing of specific physical and chemical parameters of taken water samples are presented.

The regrowth of bacteria in an internal plumbing system could present an issue if all the key nutrients are available and there are no disinfectants present (Morton et al., 2005).

Organic Matter + Nitrogen + Phosphate + Trace Nutrients → Regrowth (Morton et al., 2005)

The adjustment of pH, the increase in alkalinity or in hardness or the adding of corrosion inhibitors, such as polyphosphates, silicates and orthophosphates, are methods that are applied most commonly in order to control the corrosion in water distribution networks. Regarding the specifications for such water treatment chemicals (WHO, 2011), the quality and maximum dose should be taken into account. Determining the presence of orthophosphates in water, the aim was to assess the possible impact of using polyphosphates for potable water softening on the proliferation of certain microorganisms in sanitary hot water. Roš and Zupančič (2010) state that only orthophosphates are suitable for biological growth since polyphosphates have to hydrolyse before they can be used.

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Phosphates definitely represent one of the important nutrients, which are in some places added to water in order to prevent the excessive deposition of scale within internal plumbing systems and to ensure a certain level of protection from internal corrosion within the installation. The impacts of a higher amount of phosphorus, as a water pollutant, stem fundamentally from the overuse of phosphorus fertilisers and powders (Smith R. L, Smith T. M., 2001). Namely, when present in excessive amounts in water, phosphorus represents one of the nutrients that can induce excessive growth of algae and bacteria in water (Roš and Zupančič, 2010; Rismal, 1995; Šolar, 2011). Algae and bacteria can use all the oxygen in water for their growth and proliferation (Ećimović et al., 1998). Regarding this correlation, the parameter of oxidising was also researched, showing the organic burdening of water. A quick and simple detection of changes in water characteristics can be detected by following the value of the oxidising parameter in potable water. Organic compounds in potable water, which can represent a direct or indirect health risk, since many among them are toxic, can be used as food for the growth of unwanted microorganisms, and they can react with the present disinfectants into toxic by- products (DBPs), etc. The oxidising parameter (5 mg/L O2) standard (Rules on Drinking Water, 2004) was not exceeded in any case (Figure 26). The highest observed value was more than 15-times lower.

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Figure 26: Measured concentrations of oxidising in water at selected sampling sites of six health facilities in 2010

Figure 27 presents the results of laboratory testing of water samples regarding the

presence of orthophosphates (PO4) in connection to the number of colonies at 22 °C, the number of colonies at 36 °C, and the presence of Legionellae. The presence of orthophosphates has only been confirmed in four samples of sanitary hot water. In the remaining eight samples of cold water, as expected, there has been no presence of orthophosphates confirmed, since they are not added into a cold water system. Two out of four samples containing orthophosphates have also confirmed the presence of Legionellae, while the number of colonies at 22 °C and the number of colonies at 36 °C have not increased. The connection between phosphates and the occurrence of microorganisms or the impact of the presence of orthophosphates in water and an increase in the occurrence of Legionellae could not be confirmed due to a small sample included in the research. The Rules on Drinking Water (2004) determine no standards regarding orthophosphates, just as there are no standards regarding the softening of potable water mentioned in the current legislation.

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Figure 27: Measured concentrations of orthophosphates, number of colonies at 22 °C, number of colonies at 36 °C, and Legionellae in water at selected sampling sites of six health facilities in 2010

From the perspective of installed materials within an internal plumbing system, the retrieved water samples were checked for the presence of iron as well. According to the known natural background of water sources that supply the selected health facilities, increased values of iron in potable water would point to an anthropogenic origin. All measured values were <20 µg/L.

Higher values of nitrates present in water mostly show the contamination of water as a result of man's activity (Smith and Smith, 2001). Normally, increased content of nitrates occurs as a result of the overuse of mineral fertilisers and as a result of the impact of communal wastewaters. By determining the presence of nitrates in water, the aim of the research was to evaluate their indirect impact on the proliferation of certain microorganisms in cold and sanitary hot water. Nitrates in water can also 112 represent a nutrient for certain water organisms, which indirectly could be regarded as a favourable condition for the growth and proliferation of other microorganisms, e.g., Legionellae. Algae and amoebae, often present also in an artificial water environment (internal plumbing systems), are host organisms for Legionellae (Rowland, 2003). Legionellae multiply intracellularly (WHO, 2007). The research results show (Figure 28) that the values of nitrate presence in water are very similar

(from 15.0 to 16.5 mg/L NO3), and are perfectly comparable to the nitrates of the known burdening of water sources. Water sources that supply the inspected health facilities are burdened with nitrates, but their values are 3-times lower than the approved limit. The Rules on Drinking Water (2004) set the limit value for nitrate at

50 mg/L NO3. The research could not link the direct impact of the nitrate to the occurrence of Legionellae and other microorganisms; however, it has shown minor differences in the concentrations of present nitrates in cold and hot water. In sanitary hot water, the values ranged from 0.1 to 1.4 mg/L lower than in cold water (perhaps the heating effect – thermal decomposition or the reaction with added polyphosphates). The Miettinen et al. (1997) research finds that adding the remainder of inorganic nutrients, like nitrogen, compared to adding phosphorus, has an insignificant impact on the growth of microorganisms. At all sampling sites, the content of found nitrates in the research is large enough to meet the needs for microorganism development.

Given the oxidative and reductive state of nitrogen, besides nitrates, the research also tested for ammonium and nitrites. The results have shown that there was no increase in the value of ammonium or nitrite (<0.02 mg/L NH4 and <0.01 mg/L

NO2).

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Figure 28: Comparison of measured concentrations of nitrates in water with limit value at selected sampling sites of six health facilities in 2010

An important criterion for determining the purity of water is turbidity (ADWG, 2011). Usually, turbidity is represented by suspended particles of inorganic or organic origin that are found in water. Particles of organic origin can represent a nutrient for the development of microorganisms (EPA, 1999). Inorganic particles can also have a role of a nutrient (phosphates, etc.) or can merely offer favourable conditions for the attachment and camouflage of microorganisms from different disinfection effects (UV disinfection, chemical disinfection). Sediments in water, seen as increased turbidity and total organic carbon, are a factor that encourages the growth of Legionellae, since sediments contain necessary nutrients and at the same time provide conditions for the growth of other micro flora (States et al., 1987; EPA, 1999; WHO, 2007). As an important nutrient for the development of microorganisms, carbon cannot only be present as an organic contaminant in water, but also as dissolved CO2 from air. Upon heating sanitary hot water, CO2 is released into water also from water-soluble calcium hydrogen carbonate. The reaction sequence is shown with the following chemical equation (Equation 3.1):

Ca(HCO3) → heating → CaCO3 + H2O + CO2 (Equation 3.1) 114

1 0,9 0,8 0,7 0,6 0,5

NTU 0,4 0,3 0,2 0,1 0 1 2 3 4 5 6 7 8 9 10 11 12 Sampling site (N=12)

Figure 29: Measured values of water turbidity at selected sampling sites of six health facilities in 2010

Figure 29 shows the measured values of water turbidity at selected sampling sites. Slightly increased water turbidity was determined only in one sample in the value of 0.4 NTU. Compared to other samples, this one also included the highest value of the number of colonies at 36 °C and confirmed the presence of Legionellae. It can be assumed that the determined slightly increased water turbidity could be the result of water flow-induced sediment rising in pipes. Especially in facilities with an outdated internal plumbing system (zinc-coated steel pipes), there is often turbid and coloured potable water present upon sampling as the aerator is removed, the reason being the outflow of the upstream-accumulated sediments behind the aerator. Aerators, shower heads, point of use filters and other types of outlet nozzles present an obstacle and cause a decrease in the speed of water flow to such outlets. From this perspective, aerators are neither adequate nor desired. Their basic purpose is to block the impurities that flow with water and to save water running through them by mixing it with air. From the epidemiologic perspective, the increase in a resulting formation of aerosol could be seen as an issue.

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High content of chlorine in organically contaminated water can cause the occurrence of halogenated organic compounds, usually determined as trihalomethanes (THM). Some other compounds from the AOX group (adsorbable organic halogens) can be formed as well (Drev, 2011). An overly intense chlorination of organically contaminated water does in fact lower the possibility of the microbiological contamination of water, but also increases the possibility of chemical contamination with the compounds from the AOX group. Regarding this matter, the Rules on Drinking Water (2004) state that it is necessary to ensure that the level of contamination with disinfection by-products is as low as possible. Figure 30 shows that the measured values of chlorate were in most cases proportionate to the measured values of free chlorine. The Rules on Drinking Water (2004) determine no limit values for free chlorine and chlorate, while WHO permits values for chlorate up to 0.7 mg/L.

Figure 30: Measured values of free chlorine and chlorate at selected sampling sites of three health facilities in 2010

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Figure 31 shows the impact of temperature and free chlorine on the development of certain microorganisms in water samples at sampling sites included in the research. The impact of temperature on the development of microorganisms in this research is not as evident as could be expected, based on the existing literature (WHO, 2007; Kramer et al., 2005). The development of microorganisms is also affected by the content of free chlorine and the contamination of water with nutrients (WHO, 2007). The water at sampling sites 1, 2, 3, 5, and 6 was chlorinated by adding a 12–16% solution of sodium hypochlorite.

0,5 70 0,45 60 0,4

0,35 50 ºC

/ L / 0,3

2 l 0,25

30 CFU ; mg mg C 0,2

0,15 20 0,1 10 0,05 0 0 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Sampling site (N=12)

Free chlorine Number of colonies at 22 °C (1 mL) Number of colonies at 36°C (1 mL) Legionella (1 L) Temperature of cold water Temperature of hot water

Figure 31: Measured concentrations of number of colonies at 22 °C, number of colonies at 36 °C, and Legionellae in water, depending on water temperature and free chlorine present in water at selected sampling sites of six health facilities in 2010

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Figure 31 shows that three out of twelve taken samples contained Legionellae. What raises great interest are the highest values of 15 and 49 CFU/L in the sanitary hot water samples with a temperature around 59 °C. At this temperature, Legionellae should be destroyed in approximately 10 minutes. Already in the introduction of this thesis, the importance of the impact of heat disinfection was mentioned, comprising all end parts of the installation with an adequately high temperature and sufficient contact time. The two mentioned sampling sites were located at the end of the internal plumbing system. The survival of Legionellae could in this case be caused by the presence of biofilm. Legionellae were also found in water samples with a temperature below 15 °C, where they can survive up to one year in an idle state (Bentham, 1993). Two sites showed the presence of Legionellae in water samples from an internal plumbing system, where the water is constantly chemically disinfected with chlorine. Figure 31 also shows that at the selected sampling sites in facilities, the recommended temperatures of cold and sanitary hot water were provided as given in Slovenian recommendations. There is no clear relationship between heterotrophic colony count results and the presence of Legionella; however, they can show an indication of how effective the maintenance and treatment are. If the heterotrophic colony counts in warm water systems are in excess of 100 CFU/mL, the maintenance and treatment can be regarded as unsatisfactory (Guidelines for the Control of Legionella in Manufactured Water Systems in South Australia / Health Protection Programs, 2008).

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3.3 Comparison of research results with hypotheses

H1: “The preventive measure of physical disinfection with increased temperature of sanitary hot water in internal plumbing systems of selected medical facilities represents a successful way of managing the issue of Legionella.”

The research determined that the preventive measure of physical disinfection with an increased temperature of sanitary hot water in internal plumbing systems of selected medical facilities is a successful way of managing the issue of Legionella. It was expected for the results to confirm this hypothesis even more clearly. As an individual preventive step, the mentioned measure cannot be completely satisfactory in all facilities, since often the wanted effect is achieved not only by systematic overheating, but also by the implementation of additional measures (filtration, chemical disinfection). It was also determined that for the physical disinfection with an increased temperature of sanitary hot water in internal plumbing systems to be efficient, the manner of its realisation is also of importance. Despite the lack of immediate data within this research, we can on the basis of personal experience distinguish that a successful overheating requires an appropriate water temperature, contact time, the inclusion of the entire internal plumbing system (dead end pipes), the release of thermostatic mixing valves, and sufficient rinsing. This calls for appropriate technology of the treatment of sanitary hot water, providing adequate quantity and temperature of the said water. In this process, the potential negative impacts for water-consumers (risk of burns) should also be taken into consideration. The research results partially confirm the hypothesis, since the mentioned measure results in the complete removal of Legionella only rarely, and when it is achieved, it is usually not long-term. In some cases in the spring, for example, after the carried out thermal disinfection of the system of sanitary hot water, the laboratory results of taken water samples concerning the presence of Legionella were at first negative and after 6 months positive. This became a reoccurring matter. In buildings used by a more sensitive population, where Legionella is present in internal plumbing systems, the health safety calls for additional preventive measures and for a more thorough and frequent overheating measure, while the condition of water quality needs to be

119 monitored long-term, since potential additional measures in bigger complex internal plumbing systems can be planned and realised only in this manner.

H2: “From the technological perspective, the ensuring of adequate conditions for heat disinfection is a demanding process, which directly affects the reduction of Legionella in drinking water.”

The hypothesis claiming that from the technological perspective the ensuring of adequate conditions for heat disinfection is a demanding process is confirmed. It was determined that in buildings, which were connected to the hot water supply (remote heating), an efficient heat disinfection is not possible, due to the delivered water temperature being too low, especially in the summer. In most cases, the mentioned buildings did not possess a secondary heating option. Where the latter was available, it either did not function properly or it did not provide adequate temperature for the expected preventive measure. It was also determined that the technology of sanitary hot water treatment, especially in buildings where there were older boiler rooms (heat stations), does not enable the realisation of heat disinfection as mentioned in different preventive recommendations. In most cases, the issue lies in the quantity of water at the disposal and in the ensuring of fast heating of larger quantities of water. In some cases, the issue is the speed of the adaptation or re-establishment of recommended sanitary hot water temperature in the time of bigger daily consumption of sanitary hot water. Some technical limitations, such as the provider’s limit of highest allowed temperature of stored sanitary hot water at 60 °C, were also determined. According to the established assumption, a more suitable method of thermal disinfection systems of hot water, would also improve the results in terms of the presence of Legionella in drinking water. This process also has negative impacts on the internal plumbing system (increase in the formation and deposition of scale within the pipes, on heat exchangers and storage, expansion of materials, damage to water seals and outlets, while the chemical disintegration also leads to the reduced impacts of added disinfectants as well as added means for chemical softening of the water).

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H3: “Softening of potable water with chemical means that comprise phosphorus (polyphosphates) can accelerate the proliferation of microorganisms in the water.”

Although scientific literature confirms that the softening of potable water with chemical means that comprise phosphorus (polyphosphates) can accelerate the proliferation of microorganisms in water (Miettinen et al., 1997), the research did not reliably confirm this hypothesis. The majority of water samples (N=2334) included in the research comprised added polyphosphates, meaning that the number of samples without phosphates was too low. In order to confirm the hypothesis, measurements of the phosphate concentration in the water would need to be made, since there is a possibility (regarding the phosphate measurement results in Research 2, which included some buildings from the research in the period between 2006 and 2013) that the concentrations of phosphates in the water were very low and probably also very different and as such did not have a significant impact on the increased proliferation of bacteria. Nevertheless, it is possible for the presence of polyphosphates to contribute to favourable conditions for the growth and proliferation of bacteria in the water.

To conclude, potential guidelines for further research are as follows.

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4 CONCLUSIONS

The research has shown that the water in the internal plumbing system of medical facilities was often not in compliance with the health regulations. The research which focused on the presence of bacteria from the genus Legionella has shown that the existing system of ensuring medically pristine water is in most cases not efficient enough. Usual concentrations of free chlorine, as well as procedures of heat disinfection of water and regular preservation of recommended water temperature are not efficient enough for a complete elimination of Legionella. None of the mentioned procedures removes biofilm. A complete and permanent elimination of Legionella was achieved only by using antibacterial filters at points of use, but only in the water phase (permeate-filtrate) and not in the entire internal plumbing system ahead of filters. If disinfection of the internal plumbing system is performed with overheating, no problematic chemicals should be added to the water. The problem lies in the fact that suitable conditions for the realisation of such disinfection are hard to ensure, since the latter often results also in negative impacts (burdening of materials within the internal plumbing system, increased formation of water scale, corrosion, risk of burns). After the performed disinfection, the recolonisation of Legionella may occur again, if nutrients and other favourable conditions are present. Besides that, this procedure of preventive measures encompasses only the system of sanitary hot water, which is in most cases problematic; nevertheless the presence of Legionella in systems of cold water should not be neglected. The additives for the prevention of water scale formation and corrosion of steel pipes (phosphates) are a nutrient for the bacteria, and can, as such, even worsen the contamination with Legionella in the water of internal plumbing systems. It needs to be pointed out that the softening of potable water by adding different chemicals is not in compliance with the Slovenian law and at the same time is not subdued to medical monitoring. For water softening purposes it would be more sensible to resort to other, non-chemical procedures or, in the case of chemical water softening, previously assess the health risks posed by each individual chemical. Comparative results show that Legionella can be present in the plumbing system of medical baths in amounts vastly exceeding the number of Legionella found in the internal plumbing systems of buildings. In some such cases,

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Legionella dwindled only after multiple cleanings of the plumbing system of medical baths.

With the introduction of general sanitary measures, such as regular monitoring and the maintenance of recommended water temperatures, scheduled implementations of perfected overheating, the removal of dead legs, the cleaning and disinfection of individual elements of the internal plumbing system and the renewal of heating stations, the conditions in healthcare buildings observed in this study started to improve after 2008 (a decrease in the number of positive samples and a lowering of Legionella concentrations could be detected). Results show that the trend toward diminishing positive samples stopped at around 44% in the final part of the observed period (447 positive samples found among 1017 collected samples). The percentage of samples with high concentrations of Legionella (>10,000 CFU/mL) also underwent a visible drop. Although the proportion of samples in which the presence of Legionella was detected decreased significantly from 2006 to 2013, it remained relatively large. There should be no Legionella in the internal plumbing systems of healthcare facilities. This can be achieved by preventing the development of conditions conducive to their proliferation or improving the procedures of disinfection. From the point of view of enhanced end-consumer safety, the erection of multiple sanitary barriers is particularly important in the internal plumbing systems of buildings where it is difficult to prevent favourable conditions for the proliferation of Legionella. These barriers are most effective if they are installed and rendered operational right from the beginning, in every newly built or renovated building, since they may thus prevent the entry of microorganisms into the internal plumbing system of a building. Hence, in the case of a newly built building or a building whose mechanical installations have undergone a complete overhaul, the prevention of water system colonisation by Legionella would foremost entail water processing at the very site where potable water enters the building, in order to prevent the entry of Legionella into its internal plumbing system. An adequate approach to water processing in this case is ultrafiltration, with secondary UV disinfection. Should the internal plumbing system already be contaminated with Legionella, measures must involve the identification of dead legs (branches without

123 outlets) and their subsequent removal. If so required, all mechanical installations, including the heating station, if it does not meet the requirements for the implementation of preventive measures involving the maintenance of recommended water temperature, must be renovated. The plumbing system must be brought into a hydraulic equilibrium, which means that at any point in the system the adequate water flow velocity and temperature has been reached. In addition to dead legs, other factors contributing to water retention should also be taken into account, such as fire hydrant access pipes, pre-installed uninsulated pipes (installed prior to regular usage), persistent low water flow velocity segments, and enlarged pipe dimensions due to fire safety and other requirements. Occasional flushing should also be foreseen, in case what was described in a couple of last sentences cannot be avoided. On the return system (recirculation), before the entry of sanitary warm water into the sanitary warm water tank, it is sensible to implement UV disinfection. An individual assessment of the internal plumbing system of a building determines the level of cleaning and chemical disinfection the internal plumbing system should be exposed to. If necessary, a continued chemical disinfection lasting a couple of months is set up for a complete removal of the biofilm. All things considered, if the chemical disinfection of water ensures the microbiological safety of end-consumers, it is not in all cases necessary to also maintain the recommended temperature of sanitary warm water (>50 °C). Furthermore, it is only chemical disinfection that also has a preventive effect on the cold water system, though it brings with it other difficulties, as previously mentioned.

When interpreting the results presented in this study, it should be noted that it was not possible to monitor the implementation of every individual preventive measure at one and the same site of sample collection, which would yield a more accurate representation of the actual effects of these measures and a better understanding of the obtained results. Moreover, the study could not accurately assess the economic differences between the current implementation of preventive measures by building managers, as observed in this study, and the possible measures described in the final part of the previous paragraph.

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In order to improve the interpretation of results of the presence of Legionella in the water of internal plumbing systems of selected healthcare facilities, further research should be conducted so that:  Only the same sampling sites in exactly the same conditions would be monitored (the same water outlet).  The sampling would be carried out at the same time of day or possibly consecutively in the morning and in the afternoon of the same day, providing insight into possible differences from this perspective as well.  The number of cold water samples would be increased and would be compared along with samples of sanitary hot water to determine the difference in acquired laboratory results regarding the presence of Legionella in winter and in summer, and compare the results with the occurrence of Legionellosis during the entire year. The highest number of Legionellosis can be observed in August and September (ECDC, 2015).  All samples collected at sites where polyphosphates are added to the water of an internal plumbing system would be checked for the concentration of polyphosphates at every individual water outlet. At the same time, at least one of the facilities should simultaneously be monitored for the presence of Legionella at sites where polyphosphates are added to the water in a part of internal plumbing system and at sites where they are not added.  All the pre-overheating sampling would be done a week prior to the scheduled overheating and all the post-overheating sampling a week after overheating.  In all instances of water samples, collected at sites with installed filters, samples would also be collected before the filter.

Given suggestions transcend the boundaries and capabilities of our research; however, they might present an additional support to researchers who wish to examine certain phenomena from this perspective.

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ACKNOWLEDGEMENT

Firstly, I would like to thank all the employees at the University of Nova Gorica who put together, offered and realised the post-graduate study programme Environmental studies, for which I immediately knew that it is the right choice for me. The study programme gave me new knowledge and experiences, as well as new connections with people – something that means a lot to me.

For accepting the mentorship and for all his professional help, which on numerous occasions has surpassed this master's thesis, I want to sincerely thank Asst Prof Darko Drev (University of Ljubljana Faculty of Civil and Geodetic Engineering, Institute of Sanitary Engineering).

Senior lecturer Gregor Jereb, M. Sc., (University of Ljubljana, Faculty of Health Sciences, Department of Sanitary Engineering) offered me great support in writing this master's thesis. His help could without a doubt be called co-mentorship, which he, as a great friend, unselfishly offered to me. His advice and especially his moral support were key encouragements for me to finally finish my studies successfully.

For their help in laboratory testing of samples and result interpretation I want to thank Maja Gošnjak, M. Sc., Andrej Planinšek, M. Sc., and Aleš Zagajšek, Chemical Engineer (all from Institute of Public Health of Celje).

For all the technical help in data processing I want to thank Asst Prof Iztok Tomažič (University of Ljubljana, Biotechnical Faculty, Department of Biology, Chair of Biological Education).

For the final additions regarding the format of my master's thesis I want to sincerely thank my friend, Damjan Kovačič, Engineer (Milan Vidmar Electric Power Research Institute, Ljubljana).

For the received critical evaluations that are proof that my thesis was well organised and written, I want to thank all the members of the commission at the defence of my master's thesis.

How could I forget my father, whom I still carry close to my heart, remembering how he was secretly proud and happy upon my diploma graduation? Somewhat secretly, I enrolled in the post-graduate master's programme just a day after we buried him. I am still deeply saddened that I was not able to share my plans with him. Maybe I wanted to surprise him, but in the end, he surprised me. Mom and dad, thank you for helping me also on my educational path.

Since my studies lasted longer than I anticipated, I want to sincerely thank my wife's parents and especially my wife Metka and my daughters Martina, Manca, and Maja for their support and patience. I want to pay my daughters back with my support in their education, so that they can once make me happy in a similar way.

The greatest use of a life is to spend it on something that will outlast it.

William James

ZAHVALA

Najprej bi se rad zahvalil zaposlenim na Univerzi v Novi Gorici, ki so na zelo visokem nivoju pripravili, ponudili in ne navsezadnje izvajali podiplomski študijski program Znanosti o okolju, v katerem sem se po preučitvi le-tega takoj videl. Študij mi je dal nova znanja in izkušnje. Nastale pa so tudi nove povezave z ljudmi, kar mi veliko pomeni.

Za sprejem mentorstva in vso strokovno pomoč, ki je večkrat segla tudi preko te naloge se iskreno zahvaljujem doc.dr. Darko Drevu (Univerza v Ljubljani, Fakulteta za gradbeništvo in geodezijo, Inštitut za zdravstveno hidrotehniko).

Višji predavatelj mag. Gregor Jereb (Univerza v Ljubljani, Zdravstvena fakulteta, Oddelek za sanitarno inženirstvo, Katedra za zdravstveno ekologijo in nadzorstvo) je ob nalogi nosil zelo veliko podporno težo. Njegova pomoč je zagotovo vredna somentorstva, ki mi ga je kot dober prijatelj nesebično nudil. Njegovi nasveti, predvsem pa moralna spodbuda, da se le spravim uspešno zaključiti študij še z nalogo, so bili zame ključni.

Za pomoč pri laboratorijskem preskušanju vzorcev vode in interpretaciji rezultatov se zahvaljujem mag. Maji Gošnjak, mag. Andreju Planinšku in inženirju Alešu Zagajšku (vsi Zavod za zdravstveno varstvo Celje).

Za tehnično pomoč pri obdelavi podatkov za potrebe naloge se zahvaljujem doc.dr. Iztoku Tomažiču (Univerza v Ljubljani, Biotehnična fakulteta, Oddelek za Biologijo).

Da je naloga na koncu dobila ustrezen izgled, se za oblikovno pomoč iskreno zahvaljujem prijatelju, inženirju Damjanu Kovačiču (Elektroinštitut Milan Vidmar).

Za prejete kritične ocene, ki mi dajejo potrditev, da sem nalogo dobro izdelal, se zahvaljujem tudi vsem članom komisije za zagovor magistrskega dela.

Proti koncu bi ob tem rad izpostavil tudi svojega očeta, katerega imam še vedno v spominu, kako je bil na skrit način ponosen in vesel ob slovesni podelitvi moje diplome, ob zaključku predhodnega študija. Tako nekako skrito, sem študij, ki ga s to nalogo zaključujem, uradno vpisal dan po tem, ko smo ga pokopali. Iskreno hudo mi je, da mu tega nisem uspel povedati v oči. Morda sem ga želel presenetiti, a je on prej mene. Oče in mama, hvala, da sta mi pomagala tudi na življenjski poti mojega izobraževanja.

Ker je študij trajal dlje, kot sem si želel, se iz vsega srca za podporo in potrpljenje zahvaljujem ženinim staršem, še posebej pa ženi Metki in hčerkam Martini, Manci in Maji. Še posebej zadnjim trem se želim, s svojo pomočjo pri njihovem izobraževanju, oddolžiti vsaj tako, da bi se mi lahko morda tudi one, kdaj zahvalile na podoben način.

Življenje najbolje uporabimo, če ga porabimo za nekaj, kar traja dlje kot življenje samo.

William James