Message From The Supervisor

In recent years, "sick building syndrome" has been receiving much attention. People affected by this are workers in modern oft`ice buildings who complain of physiological distress. Although this syndrome has chiefly been studied in Europe and America up to now, social awareness of this problem has been growing in Japan. Of all the symptoms that make up this syndrome, Legionella infections have received the most attention. In 1970, before any other country in the world, "Law for maintenance of sanitation in buildings" (hereinafter, Building sanitstion law) was established in Japan to ensure the hygiene of certain buildings used many and various people. As a result, the number of reports of Legionella infections has been low in Japan, but a future increase in the number of the infections is widely predicted and feared. Therefore, the Ministry of Health and Welfare has long been working to develop preventive measures to stop the spread of Legionella infections. After an outbreak of Legionnaires' disease (pneumonia type of Legionella infections) in Philadelphia in 1976, surveys and studies were conducted to prevent further outbreaks of this disease. Then, based on Notification-194 of the Ministry of Health and Welfare in 1982, and subsequently, the notice published by the Environmental Health Bureau on March 18, 1983, the criteria for maintaining cooling towers were established. The Ministry of Health and Welfare has since followed these guidelines to prevent the spread of this disease. Moreover, in order to further tighten the control measures, after a 1988 outbreak of Legionnaires' disease in London, a study was conducted on the risks of indoor air environments, "Disgnostic standard for Legionella pneumonia and guidelines for diagnosis, examination and medical treatment" was published, and much efforts were made for its awareness. Guidance for the relating industries could develop "Self-imposed standard for the safety and effectiveness of anti-Legionella cooling water treatment chemicals". Next, in order to firmly establish preventing measures against Legionella infections, a "Research for the guidelines for prevention and control of Legionnaires' disease (chief researcher: Kazuo Watanabe)" was performed in 1992 as s part of the administrative activity of the Ministry of Health and welfare. Based on the report published by Watanabe's group, the committee for "Guideline of prevention and control of Legionella infections" (chairman: Susumu Yoshizawa, professor at Tokyo Science University) was established last year st the Building Management Education Center to promote the implementation of practical measures for the prevention of Legionella infections.
This book is being published after scrutinizing by specialists in a variety of fields.
I hope that this book will be used widely among building owners, building management agencies, people in charge of air-conditioning facilities, contractors, medical professionals, and government agencies, contributing to the prevention of Legionella infections. And last, but not least, I would like to thank the members of the Building Management Education Center and Anti-Legionella Air-Conditioning water Treatment Chemicals Gonference for their help in the publishing of this book.

February, 1994

Yoshimi Takao, Director
Planning Division, Environmental Health Bureau Ministry of Health and Welfare.

Memorandum of translator

Japanese guideline for Legionella control has three distinct features:

1. Criteria for the evaluation of legionellae measurements is specified.
2. Frequencies of legionellae inspection at cooling towers are indicated.
3. Methods for cleaning of cooling towers and cooling water treatment using chlorine and other chemical agents are described.

A plan for English version of Japanese guideline was started by request of
Mr. L. Staples, Vice President ASHRAE and Mr. D. F. Geary chairman ASHRAE Legionella committee, at the ASHRAE San Diego Meeting in June 1995.
The work was performed with the permission of the officials of the Ministry of Health and Welfare and the Building Management Education Center, and under the sponsorship of following seven companies:
Kajima Corporation,
Nippon Life Insurance C;o.,
Kurita Water Industry Corp.,
AQUAS Corp.,
Showa Corp.,
Dia Water Treatment Service C;o.,
Tokyo Gas Co..
Prof. Yoshizawa, chairman of Guideline Committee gave me special encouragement.

Japanese Guideline is composed of 52 pages including two official messsges (pp.2), contents (4), text (31), appendix (13), and 72 items of references (7).

The English version is abstracted and rearranged to one message, about 70% of text and about 50% of appendix, and eliminated references by financial reason.

November 3, 1995

Kazuo Watanabe, member ASHRAE, secretary Legionella Conference Japan.

Air-Conditioning Engineering Section, Tokyo Gas Co.,
27F Shinjuku Park Tower,
Nishi-Shinjuku Tokyo 163-10,
JAPAN
Tel: 03-5322-7554,
Fax: 03-5322-7561 .

Committee on the Guideline for Prevention and Control of Legionella Infections

Chairman

S. Yoshizawa, Tokyo Science University

Committee

M. Arakawa, Gifu University
J. Kakagawa, Tokyo Women's Medical l;ollege
M. Naemura, Ministry of Health and Welfare
Y. Nakaji, Mitsui Shipbuilding System Research Co.
F. Munakata, Building Mgnagement Education Center
E. Yabuuchi, Osaka City University
K. Watanabe, Tokyo Gas Co.

Secretary

K. Agata, AQUAS Corporation
E. Fiimura, Building Management Education Center

Research Group on the Guideline for Prevention and Control of Legionnaires' Disease

Chief Researcher

K. Watanabe, Air-Conditioning Section, Tokyo Gas Co.

Coworkers

E. Yabuuchi, Osaka City University Medical School
M. Arakawa, Gifu University Medical School
M. Ichihashi, Nippon Life Insurance C;o.
H. NHkgjima, Kgjimg Corporation
E. Kimura, Building Management Education Center
K. Agata, AQUAS Corporation

Secretary

T. Ohida, Environmental Health Bureau, Ministry of Health and Welfare

Center and Anti-Legionella Air-Conditioning Water Treatment Chemicals Conference for their help in the publishing of this book.

February,1994

Yoshimi Takao, Director
Planning Division, Environmental Health Bureau
Ministry of Health and Welfare

GUIDELINE FOR PREVENTION AND CONTROL OF LEGIONELLA INFECTIONS

Introduction

Legionella infections (legionellosis) are a group of infectious diseases caused by bacteria of Legionella species. There are two types of Legionalla infections: The first type is known as Legionella pneumonia (pneumonic type, usually referred to as Legionnaires' disease), in which patients suffer from interstitial pneumonia combined with multiple organ injuries; and the second type is referred to as Pontiac fever, or non-pneumonic type, in which patients suffer from fever similar that of influenza.
After an outbreak that caused the deaths of the American Legion members who were attending a convention at the Bellevue Stratford Hotel in Philadelphia in 1976, Legionella pneumonia was recognized as an independent disease, and the disease-causing bacteria were named Legionella pneumonia in 1977. However, subsequent studies have confirmed that there had been cases of this infections reported since 1943. Ever since the isolation of the disease-causing bacteria for Legionella pneumonia sporadic cases and outbreaks of Legionlla pneumonia have been reported not only in Americs, but all over the world. Many countries issued official announcements regarding the criteria for prevention of this disease, the World Health Organization (WHO) held the World Legionella Conference in Geneva in November 1989 to discuss the epidemiology and prevention and control of Izgionella infections, and issued an official memorandum. In Japan, as a preventive measure, the self-imposed standard for the safety and effectiveness of anti-Legionella cooling water treatment chemicals was established, and the Japan Refrigeration and AirConditioning Industry Association revised the guideline of water quality for refrigeration and air conditioning equipment.
The first outbreak of Pontiac fever was reported at a Health Service in Pontiac, Michigan in 1968, and as the name suggests, patients suffered from fever. Then, in 1978, the disease was clarified as a non-pneumonic type of Legionella infections.
Legionella live naturally in soil, and one outbreak of Legionella pneumonia resulted when the front yard of a hospital was dug up and dust containing Legionella was carried by strong winds and inhaled by patients in the hospital. The disease can also occur when Legionella get inside a cooling tower, multiply in the cooling water, and people inhale the aerosol. Furthermore, it has been reported that when a cooling tower is the source of infection, not only workers who work in the contgminated building, but airborne I.egionella also causes the disease in residents who live in the vicinity of the building. Cooling towers are not the only source of infections; evaporative condensers, whirlpools, humidifiers that are used in hospitals and vegetable sections in supermarkets, or anything that generates a large quantity of aerosol can be the source of infection when water in the equipment is contaminated with Legionella. It has been reported that in one case where the source of infection could not be identified, the spread of infections were stopped when all the cooling towers in the area were cleaned and disinfected. Furthermore, in Japan, a person contracted Legionellg pneumonia after stumbling and accidentally swollowing water in a hot spring; when the water was tested, Legionella pneumophila was isolated. Both pneumonic and non-pneumonic types of Legionella infections spread from a common infection source to a large number of people, but it does not spread from person to person. In order to prevent this disease, it is important to understand how people are infected, and to reduce the number of Legionella in water systems in buildings and the amount of aerosol production.
Problems with indoor air environments have been under discussion in recent years. The energy conservation movement that started in the '70s sparked efforts to reduce the air intake to buildings. Consequently, the quality of indoor air lowered and people in buildings were forced to breathe polluted air for long period of time. All pathological symptoms that are related to buildings are grouped together and referred to as sick building syndrome, and it is believed that chemical substances used in construction, radon, combustion exhaust, fungi, fiberglass, mites and their dead bodies and microorganisms are the causes of sick building syndrome. Sometimes, symptoms that are caused by bacteria are referred to as building-related illness (BRl) to distinguish them frnm the other symptoms. Of these building-related illness, the mortality rate of Legionella pneumonia is particularly high. Therefore, it is important to educate people about Legionella and its epidemiology, and to encourage early checkup and early treatment. It is also necessary to design, install and operate air-conditioning facilities in ways that reduce the risk of Legionella infections.

A. Objectives and Applicability

In order to prevent Legionella infections and maintain the public health, the following guidelines were established to be utilized by medical and health professionals, health service personnel, building envirnnmental sanitation personnel, building owners, and who are in chgrge of designing, constructing, checking, operating, maintaining and managing water systems snd surrnunding equipment in bufldings. In the future, the following guidelines should be revised as advances in technology are made and more data become available.

B. Current State of Legionella Infections

1 . Clinical study of Legionella infections
1-1. Current state of Legionella infections in Japan
The first case of Legionella pneumonia confirmed by a positive culture of an autoptical lung was reported by Saito and coworkers in 1981. According to a report published by the Legionella research group at the Ministry of Health and Welfare, the number of patients diagnosed with Legionella pneumonia between 1979 and 1992 by positive culture was 38, by a significant increase in the level of serum antibody titer another 38, by positive urinary antigen four and by polymerase chain reaction (PCR) six, for a total of 86 patients.
Figure 1 shows the geographic distribution of 80 cases with Legionella pneumonia (the six cases diagnosed by PCR were excluded due to the lack of necessary patient information). Of the 80 cases, 19 cases (24%) were hospital-acquired and 61 (76%) were community-acquired; the number of cases infected at community was more than three times that of at hospitals. The mortality rate of the patients infected at community was 16/61, or 26%, and that of at hospitals was 10/19, or 53%. Both numbers were higher thsn those of other countries. From these findings, it was inferred that high mortality rate corrlated to the lack of early checkup and early treatment. Of the patients infected at community, 18 cases (30%) took a trip that could have been related to the onset of the disease. There were two small scale outbreaks: one when three patients were infected at the same hospital and diagnosed by a significant increase in the level of serum antibody titer, and one when two patients were infected at the same nursing home and diagnosed by positive culture. The number of diagnosed and reported cases of Legionella infections is low in Japan; however, this does not necessarily mean thst the actual number of cases is low. Of the 196 cases, that died of bacterial pneumonia and confirmed clinically and pathohistologically, 42 (20%) [sic) were diagnosed with Legionella pneumonia after direct fluorescent antibody (DFA) tests were performed on their autoptical lung tissues. Therefore, it is thought that Figure 1 does not show the geographical distribution of cases with Legionella pneumonia, but rather the distribution of doctors attuned to this disease.
According to the U.S. Center for Disease Contrnl (CDC), the current average number of reported and confirmed cases of Legionella pneumonia is between 400 and 500 per year. On the other hand, according to a recent survey on the causes of bacterial pneumonia, of all adult bacterial pneumonia patients in America, excluding older patients in nursing homes, it has been estimated that about 11 ,000 patients suffer frnm Legionella pneumonia. This number is about 25 times the reported cases of Legionella pneumonia patients, and it shows that most patients with Legionella pneumonia are never diagnosed.

---

Figure 1 . Distribution of 80 patients with Legionella pneumonia between 1979 and 1992 in Japan Each dot represents one reported patient

The number of patients with Legionella pneumonia is high between May and October, and especially, the number of communal and nosocomial infections in August is about 2 to 3 times as high when compared to that in other months (Figure 2).

Figure 2. Number of reported patients of Legionella pneumonia in each month between 1979 and 1992 in Japan

1-2. Diagnosing Legionella infections The clinical progress for Legionella pneumonia varies greatly. Some patients with acute symptoms die 7 to 10 days after contraction, and other patients are cured by the effective dosage of antibacterial drugs. The risk factors of this disease include, age (people over 60 are likely to contract), sex (male patients are three times as female), smoking, excessive drinking, chrnnic disease (diabetes, chronic obliterative pneumonia), immunosuppressive state, and after organ transplant surgery. If people who fall into these categories have fever and difficulty in breathing, or disturbance of consciousness and wallung after travel or overworking, they should immediately consult a physician at a hospital that can examination and diagnose Legionella pneumonia. The outbreak of Pontiff fever that occurred in Michigan in 1968 was caused by serogroup 1, L. pneumophila. The bacteria got into and multiplied in an air-conditioning system. The disease rate of this outbreak was 95%. In August 1981, L. feeleii multiplied in cutting lubricant at an automobile factory in Ontario, Canada, and 317 workers (46%) were infected by the bacteria. In April 1988, L. anisa multiplied in a decorative fountain at s hotel in Santa Clara, California, and 71% to 94% of people who were dining in the vicinity of the fountain were infected. It was reported that this fountain had never been sterilized. Furthermore, in January 1988, 170 out of 200 children who were in a whirlpool at a resort in Lochgoilhead, Scotland, contracted L. micdadei-induced Pontiac fever. It has also been reported thst people cleaning steam turbines have contracted the fever.

1-2-1. Diagnostic criteria for Legionella pneumonia Early general examinations for patients with fever are recommended. People who traveled or worked with soil and dust are at greater risk. (a) Tentative disgnosis (1) Acute bacterial pneumonia is suspected. (2) Pulmonary inf`iltration rapidly advances on chest X-ray, and dyspny is more noticeable. When compared to the degree of infiltration, hypoxemia is more apparent. (3) Bacteria that causes pneumonia cannot be detected in usual bacterial culture. (4) The administration of B-lactam and aminoglycoside has no positive effect.

(b) Confirmed diagnosis Patients who fit the above descriptions and test positive to one ofthe following tests are diagnosed with Legionella pneumonia. (1) Sputum, aspirates from the bronchus, pleural effusion, or bioptic/autoptic lung sgmple is incubated in a BCYE a culture or in a BCYE a culture containing antibacterial and anti-fungal agents for four days. Then, colonies that resemble Legionella colony are selected and after confirming that they are Gram-negative L-Sistine requisite bacilli, identified as Legionella bacteria using diagnostic Legionella anti-serum. (2) The level of the serum antibody titer against SG1 Legionefla pneumophila is increased four-fold or more (>128) in paired serum, or >256 in single serum. There is no established world standard for other serogroups of Legionella pneumophila or other species of Legionella. Therefore, at this point of time, it is necessary to refer to this standard and collect more clinical data. (3) The base sequence that is specif`ic to Legionella is detected from a respiratory tract bioptic sample using PCR (polymerase chain reaction). (4)Legionella-specific antigen is detected in urine.

1-2-2. Diagnosing Pontiac fever Although Pontiac fever is a disease from which patients recover spontaneously and its prognosis is good, the disease rate in an outbreak is greater than 80 - 90%. Patients who show the clinical symptoms of influenza snd a signit"icant increase in the level of serum antibody titer to Legionella are diagnosed with Pontiac fever. Nonetheless, it is difticult to diagnose Pontiac fever without the onset of an outbreak.

1-3. Infection source Soil and dust inhabited by Legionella, and aerosol frnm artificial water contaminated with Legionella are the source of infection. If the diameter of aernsol particles harboring Legionella is between 1 and 5 u m, they can resch the pulmonary alveoli. Larger water droplets will stick to the tracheae, but if evaporation reduces the size of these water droplets, they will reach the pulmonary alveoli.

2. Bacteriology of Legionells 2-1. Genus Legionella Legionella bacteria are found naturally in soil and fresh water (rivers and lakes). Legionella can enter a water system in buildings thrnugh a contaminated water supply or a cloud of dust created by digging up the soil during construction. Also, Legionella csn get into a vater system via dust when the water system is first installed. Legionella bacteria are pathogenic in humans, guinea pigs, embryonated hen eggs and bactenophagic protozoa (e.g. amoebae). As of April 1993, there are 38 species of bacteria in the genus Legionella. Of these, L. pneumophils is most frequently detected in patients and in environmental water.

2-2. Morphology, physiology and biochemical characteristics

Legionella bacterium is an aerobic Gram's negative bacillus, 0.3 - 0.9 x 2 - 20 u m in size. The I..egionella bacterium usually possesses 1 - 2 flagella and numerous pili. Although a freshly separated strain appears long and spindle shaped, it has a definite morphology. However, with each successive cultivation, the number of f`ilament-like cells increases. Also, Legionella bacteria do not grow in regular bacterial culture mediums. They do grnw on a special medium, but the growth rate is low and it takes at least four to f`ive days to form independent colonies. These colonies are grayish-white in color and wet. The size of colonies varies and Legionella colonies have a distinct light acidic smell. The optimal grnwth temperature is 36C, and they can grow in temperatures between 25C and 43C. The optimal pH is 6.9 + 0.05, and C02 does not prnmote grnwth. They can survive for 20 minutes at pH 2.2 or at 50C, and can survive for years when placed in a cooling tower water at 4C. The bacteria do not acidify, nor do they ferrnent glucose or any kind of carbohydrates. Their source of energy and carbon is amino acids. Legionella bacteria lack biochemical characteristics that can distinguish them from other bacteria.

2-3. Distribution and ecology Legionella can be found naturally in soil and fresh water all over the world. They utilize metabolic products of algae and other bacteria. They multiply and spread by parasitic multiplication to amoebae or other bacteriophagic protozoa and killing the hosts. Legionella can multiply in water in cooling towers when they are carried by a cloud of dust or transported via water supply. There have been many studies on the distribution of Legionella in artificial water environments in Japan.

2-4. Pathogenicity The mode of transmission of both types of Legionella infections, pneumonic snd non-pneumonic, is not person-to-person, but when there is an outbreak, many people are infected from a common source of infection. When more than two people are infected at the same site, it is considered an outbreak. Of the 38 species of bacteria in the genus Legionella, only the type strain of nine species are of human origin. However, even though the type strain of some species are of water or soil origin, they sre known to be pgthogenic in humans. Therefore, until the disease-causing factors of Legionella are identif`ied, it is best to assume that all species of the genus Legionella are pathogenic in humans.

C. Role of Interested Parties in Prevention and Control 1 . Role of building constructors and maintenance agencies The Building Standard Law defines the minimum stsndards for the structure, site, facility and use of buildings. When the "Law for Maintenance of Sanitation in Buildings" (hereinafter the Building Sanitation Law) was revised in 1970, parts ofthe Building Stgndards Law were also revised (article 93, item 4 and 5) to permit the manager of the district health services to examine and make recommendations during the planning stage on the sanitation of specific buildings prescribed in the law.

The objectiveo farticle 1 of the Building Sanitation Law is to establish necessary regulations to ensure a sanitary environment in buildings that are used by many and various people and to improve and promote awareness of public sanitation in buildings. This law def`ines the specif`ic buildings of the objective of law and obligate the responsibility on owners and people in charge of maintaining buildings to follow the "building environmental sanitation maintenance standard" (hereinsfter the "maintenance standard"). Article 4, item 3 of the Building Sanitation Law encourages owners and maintenance agencies of buildings that do not f`it the category of "specific buildings" also to follow the maintengnce standard to ensure and maintain a sanitary environment. In this book, we will discuss mainly on the Building Sanitation Law, furthermore, to emphasize the importance ofArticle 4, item 3 of the Building Sanitation Law, all buildings, regardless of use or size, should ensure and maintain a sanitary environment by following these guidelines:

1-1. Construction "Guidelines on environmental sanitation in buildings" is a technical manual on the environmental sanitation used to design buildings, and is used by managers of district health service to assess the design of buildings. When a building verification form is submitted, and an off`icial construction manager determines that the building fits the category of specific building, the necessary paper work will be sent to the concerned distriet health service to be examined by a district health service manager. The manager will then judge whether or not the construction plsn of the building follows the maintenance standard, and make recommendations to rectify deficiencies. The following points Hre therefore important.

(1) In order to ensure a ssnitary environment in the building, a building owner must permit a building maintenance manager to be involved with the planning of the building, so that the building will be constructed with a well-defined sanitation management plan.

(2) With the particular goal of preventing Legionella pneumonia, the district health senrice manager must indicate that the structure of the water system that generates aerosol must be designed to minimize the amount of aerosol, that such water system is installed in a place ivhere it will be difficult for aerosol to come into open-air intake, snd that cleaning and maintenance of such water system will be performed safely and easily. (3) After completion, the building owner must inspect and record whether or not all facilities were installed as planned.

1-2. Maintenance As far as air environment is concerned, the maintenance standard has been established to regulste the following six categories: dust concentration, carbon dioxide, temperature, humidity and air current. However, there is no maintenance standard to prevent bacterial pollution. Nonetheless, Notification 194, 1-1 "Maintenance of central air-conditioning system" of the Ministry of Health and Welfare published on November 16, 1982, sets the technical standard for maintaining air cleaners, cooling- heating equipment, humidifying-dehumidifying equipment, air duct and cooling towers. These standards established by the Ministry are the basis of I.egionella infections prevention and the following guidelines complement the exzsting standards.

1-2-1 . Role of building owners

(1) When s building owner entrusts maintenance duties that are specified in a contract to a maintenance agency, the owner must clearly state in the contrsct the details snd frequency of work that will be performed to prevent Legionella infections. (2) The building owner must ensure that the building environmental sanitation engineer is aware and capable of taking actions to prevent Legionellg infections. (3) The owner must request periodic reports from the appointed maintensnce engineer.

1-2-2. Role ofmaintenance engineers (1) The maintenance engineer must prepare s yearly plan based on the preventive measure specified by the owner, and perform daily and periodic msintenance duties. (2) The details of maintenance duties must be clearly written in a work log and kept for five years. (3) The maintensnce engineer must ensure that water system cleaning and sanitation workers wear prntective masks for the safeguard of Legionella infections.

1-2-3. Outbreak When an outbreak is confirmed, contact medical organizations and district health services, and take necessary measures such as emergency cleaning and other requirements.

2. Role of medical facilities gnd prnfessionals 2-1 . Msintaining a sanitary envirnnment in hospitals Since hospitals house many people who are susceptible to infections, it is necessary to manage hospitals to prevent Legionella infections.

2-2. Role of medical professionals Medical professionals must be familiar with Legionella infections, and they must correctly assess the spread of the disease, and be ready to diagnose and treat patients early.

2-3. Outbreak

Although the law does not require that an outbreak of I.egionella infections to be reported, when more than two people are infected at the same facility or area , the situation is considered an outbreak, and the medical supervisor should report to the head of the district health service and request assistance.

3. Role of district health services, regional health laboratories and local autonomous governments District health services must perform guidance with consideration of the following:

(1) Promote the "Guideline for prevention and control of Legionella infections". (2) Promote proper methods for maintaining cooling towers. (3) In case of an outbreak, identify the source of infection and perform preventive measures to stop further spresd of the disease. It is desirable that when building owners and people in charge of maintenance request a Legionella test, the district health service or the regional health laboratory is in charge of organizing the test to be conducted in the regional health laboratory. Also, the local government environmental health agency must promote and enforce measures to prevent Legionella infections, and in case of an outbreak (when more than two people are infected at the same site), the agency must guide the district health service to perform studies to identify the source of infection and measures to prevent the further spread of the disease, and report to the Planning Division of the Environmental Health Bureau at the Ministry of Health and Welfare.

D. Assessment and Quantitative Analysis of Legionella

1 . Significance of the detected cell number of Legionella The cause-and-effect relationship between pathogenesis in humans and the concentration of Legionella in cooling water and other artificial water system is not clearly established. Experimental research on the pathogenesis of Legionella infections cannot be performed directly for humans, and the number of Legionella at the time when an outbresk is conf`irmed does not necessarily reflect the number of cells at the actual time of the outbreak. However, judging from the results of epidemiological studies in the past, it is believed that following the direction in Table 1 will help in the prevention of Legionella infections. This table also incorporates information from a 1987 outbreak in Australia, where the cell count of Legionella in s cooling tower was 2 x 105 CFU/100 mL.

2. Detection, identification and cell count of Legionella

2-1. Fundamentals for Legionella analysis (Safety standard) A Legionella sample collected from a water system in a building must be tested by an experienced laboratory technician familiar with pathogenic microbiology at a well-equipped laboratory. Test results are meaningful only when samples are collected and analyzed properly.
According to the pathogen classification system listed in the Psthogen Safety Contrnl Regulations (revised on September 3, 1992) by the National Institute of Health, all species of the genus Legionalla and Legionella-like organisms are classified as level 2 pathogens.
Therefore, people who handle Legionella bacteria must be familiar with the Legionella's characteristics and its pathogenicity in humans, range of possible biohazard, prnper handling methods, emergency procedures in case of accident, the use and organizHtion of designsted laboratories, and they must have technical experience. Furthermore, any jobs that may generate aerosol must be performed in biological safety cabinet.

2-2. Practices st Legionella test

2-2-1 . Necessary information and record in Legionella test The cooling tower's belonging, location, manufacturer, and model type; the system's water holding capacity; sampling position, day and time; water temperature; properties and amount of water treatment chemicals used; and date of last injection must be clearly recorded in writing and accompany a water sample bottle. It is also recommended to measure the pH and electric conductivity of cooling water.

2-2-2. Requirements for a proper Legionella test

(1) For the quantitative analysis of Legionella the method listed in Appendix A in this book or other methods that give equivalent results should be applied.
(2) Although the frequency of testing varies with each cooling tower, as a general rule, cooling towers in hospitals should be tested once every other month while cooling towers are in operation, and once every six months in other buildings.

E. Legionella Control Measures for Water Systems in Buildings

1. Possible problem in cooling tower and cooling wster system If Legionella multiplies in the cooling water of an air-conditioning system consisting of equipment shown in Figure 3, the cooling tower can become an important source of infection, depending on the location of the cooling tower, the distance of the open.air intake of the air-conditioning system and wind-direction. When windows of residences and heavy foot traffic are in the vicinity of the cooling tower and to which water droplets could be carried by wind, special attention must be paid to the design of construction arrangement in these buildings.

Figure 3: Air-conditioning system

1-1. Proliferation of Legionells

1-1-1. Temperature
Legionella grows well in artificial cultures at temperatures between 35 and 37C, and the temperature of cooling water nears this range between May and September. In cooling water systems that do not regulate water temperature, the temperature of cooling water drops significantly in winter. On the other hand, when the motion and rntation speed of air-inducing fan and flow rate of cooling water are regulated to maintain water temperature, Legionella can multiply in the water systems because the temperature of the cooling water in the system does not decrease even when the outside temperature is low .

1-1-2. Coexsting microbes Legionella is parasitic in a variety of protozos, and utilizes metabolic prnducts of algae and other bacteria. Since algae , prntozos and other bacteria live in cooling water, Legionella can multiply easily in cooling towers. These microbes, including Legionella, enter cooling water system via dust in the atmosphere or water supply. A positive correlation between the number of general bacteria and that of Legionella in cooling water system has not been drawn.

1-2. Dispersement ofcooling water It has been reported that 0.85"/o of the circulating cooling water in an air-conditioning system is evaporated, and even if an eliminator is installed, 0.45% of the circulating water is entrained outside with air-flow as a splash. This mechanism disperses Legionella in the droplet of splash into the atmosphere.

1-3. Actuals ofLegionella detection The sensitivity of the testing method described in this book is 10 CFU/l00mL. Therefore, a negative test result only mesns that the number of cells per 100 mL of water is less than 10; it does not necessary mean that the water is germ-free. This explains why Legionella in a cooling tower return to pre-sterilization levels within about 10 days, even though the test result has once come out negative after bacteriocidal washing. For six years, between 1987 and 1992, a total of 1407 non-treated cooling towers in Japan were tested for Legionella, and 856 cooling towers (60.8%) tested culture positive. Of these 856 cooling towers, about 26% contained 101 - <102 Legionella (CFU/100 mL), about 74% >10² - <10⁵, and 0.5% >10⁵

(Table 2).

As the need for air-conditioning increases and more cooling towers are in operation, the number of I,egionella in cooling water also increases, and detection rate soars. The average annual detection rate of Legionellg in this survey is about 60%: For five month, February through May and October, detection rate is about 50%, and that between June and September is about 67%. These findings suggest that outside temperature, load of air-conditioning chiller, an increase in the temperature of cooling water and accumulation of slimy substances are correlated with the growth of Legionella.

2. Preventive measures in cooling towers and cooling water system
2-1. Cooling towers
2-I-1. Selecting cooling tower types There are two kind of open type cooling tower: round type towers utilize the counter flow of water and air, and water splash is inavoidably entrained with air in the upward direction. On the other hand, the cross flow of water and air in square type towers means that aerosol is less likely to be generated in the upward direction. Due to this structural difference, there is about a 10-fold difference in the amount of splashed wster between the two types of cooling towers. Therefore, when installing a large capacity cooling tower, it is better to select a square type cooling tower, which is also preferable for a purposes of water saving.
It is also important that a cooling water system be designed that the inside of the cooling tower can be easily cleaned, and that packing materials are removable for cleaning and exchange. Furthermore, install an appropriately sized drainage valve at the lowest point of the cooling water system, for easy and complete drainage of the cooling water.

2-I-2. Choosing proper material Woods, certain types of rubber, adhesives and packing materials promote the growth of bacteria and fungi. Therefore, it is important to choose proper materials.

2-1-3. Improving eliminator When selecting a round type cooling tower, due to cooling capacity limitations or financial reasons, and to further reduce the amount of splashed water from square type cooling towers, use a different eliminator. Eliminators can reduee the amount of splashed water if their thickness, thereby increasing the deflection stage, or by increasing the deflection angle. It is possible to choose an eliminator that reduces the amount of splashed water by 50% or more from any number of manufacturers. However, one must be careful, as this process can cause a change in pressure loss and increase the power requirement of fan-motor.

2-1-4. Installation site of cooling tower

2-1-4-1 . Open-air intake
In order to avoid the influences of exhaust gas from automobiles , as a general rule, install a open-air intake at least 10 m off the ground on a roof. This guideline may not always be useful, such as when highways are elevated, or depending on the structure of buildings or the location of an air exit on neighboring buildings; it is important to install a open-air intake where there is the lesst amount of pollution, regardless of height. Position the open-air intake of an underground market at a high place where there is the least amount of auto-traffic. Furthermore, it is important to place an open-air intake on a wall different from the wall where an air exit of a parking area or ventilation holes of various drainage tanks are located. When they must be placed on the same wall, maximize the distance between the open-air intake and the other air outlets, and when an open-air intake is installed on a roof, place the intake at least 10 m away from the other outlets. These numbers may be insufficient, in one case in Australia, even though the distance between the cooling tower identified as the source of infection and an open-air intake was about 50 - 70 m, an outbreak claimed the lives of nine out of 44 people infected by Legionella pneumonia. Therefore it is important to consider wind factors when preventing the spread of Legionells infections.

2-1-4-2. Location of cooling tower
Exhaust from cooling towers contains a large quantity of water droplets. The concentration of these water droplets are three to five times that of makeup water and contain water treatment chemicals, so they can cause staining to windows and walls of buildings and paint on cars, as well as carry Legionella inside. In sum, it is recommended that a cooling tower be installed at least 10 metres away from open-air intake, windows and areas where there are people.

2-1-5. Maintenance work When installing a cooling tower, place the tower so as to facilitste easy inspection, cleaning and sterilization, and set up a hydrant and drainage system in the vicinity ofthe tower for cleaning purposes. It is necessary to keep the interior of cooling tower clean to prevent pollution, proliferation of algae, contamination and corrnsion of cooling water system. Therefore, perform chemical washing before and after the operation of the year, and during air-conditioning season, inspect snd physically clean the tower periodically, e.g. once a month. Furthermore, in order to maintain washing effect, it is thought that some other chemicals gnd methods thst are known to be effective in preventing the growth of Legionella should be used in between the procedures mentioned above.

2-2. Maintenance of general water quality in cooling water system The maintenance of the cooling water in an air-conditioning system is generally performed as follows. These steps are the basis of Legionella prevention, and it is important to understand how Legionella grows under these circumstances, and to take the measures necessary for prevention of I.egionella infections.

2-2-1. Concentration control In order to avoid scaling , do not allow excessive concentration of circulating water. When a large quantity of corrosive ion is in cooling wster, it will cause corrosion in the tower. Usually, Langelier index is used to assess concentrstion limit, but for chemical treatment, use an spprnpriate water quality standard of concentration value according to the indication of proprietgry agent manufacturer. In concentration control, use an arrangement, such as an automatic discharge, for enforced blow down of over-concentrated cooling water.

2-2-2. Chemical treatment In order to prevent usual problems in cooling water systems, generally, the following chemicals are utilized:

(1) Scale control:
To prevent calcium carbonate scaling, phosphonic acid, synthetic organic polymer compounds and phosphate polymers are used.
(2) Corrosion prevention:
Different types of chemicals are used depending on the types of metals used. Phosphates and bivslent metal chemicals are used to protect iron, and azole chemicals are used to protect copper.
(3) Slime control:
Depending on what kind of slime (bacteria or algae) is the problem, different types of chemicals, e.g. chlorine; hydrogen perniude, quaternsry ammonium and organic bmmides are used. These chemicals must be maintained in appropriate concentrations. Therefore, operate a chemical injection pump by connecting it to an automatic discharge of cooling wster, or proportional to makeup water. Sometimes, chemicals with different functions are injected separately, but it is possible to mix three of the chemicals mentioned above and apply them as one-injector type agent. In small cooling towers, a simple battery-operated chemical injector can be applied.

2-3. Control measures for Legionella in cooling wster system In order to suppress the growth of Legionella in s cooling water system, periodically clean the system physically or use a combination of chemical washing and anti-bacterial agent. When chemical washing by anti-bacterial agent is used, the whole cooling water system, including the pipes, is sterilized, and the number of Legionella will fall below that which can be detected by a Legionella test. After that, however, the number of Legionella gradually increases, and depending on the conditions of cooling water system, the number of bacteria cells will return to pre-sterilization levels in one to two weeks. Therefore, in order to keep the number of Legionella in the desirable range (under 102 CFU/100 mL) by utilizing chemical washing by anti-bacterial agent alone, it is necessary to repeat the chemical washing at least once a month. As s result, it is recommended that, as mentioned in section E-2-3-4 below, chemical washing be applied before and after operating period of cooling tower in the air-conditioning season, and that anti-bacterial agent be successively added during operation.

2-3-1. Physical washing (cleaning) It is important periodically clean cooling towers physically and change and renew the cooling water for the maintenance of the facility. However, the effects of physical cleaning do not last long, and the number of bacterial cells in cooling water will usually start to increase right after operation is resumed. Generally, cooling towers are washed as follows:

(1) After stopping the circulation of cooling water, drain the cooling water in the basin at the bottom of the cooling tower.
(2) Clean the interior ofthe tower using a yard boom.
(3) Clean the packing material using a high-pressure wster nozzle.
(4) Drain the dirty water collected in the basin through the drainage at the bottom of the cooling tower, ensuring that it will not contaminate the rest of the cooling water line.
(5) After thoroughly rinsing the interior of cooling tower, f`ill with fresh water, and resume operation.
(6) During this time, ensure that workers are wearing protective masks to avoid aerosol inhalation.

2-3-2. Chemical disinfection washing Hydrogen peroxide, anti-bacterial agent, hydrochloric acid or organic acids are circulated to chemically disinfect cooling water systems. When a cooling water system is disinfected by chemical washing, the whole cooling water system is substantislly disinfected, and the number of Legionella drops to less thsn detection levels. However, the effect of chemical disinfection washing does not lsst long, and one must not forget that the number of Legionella often returns to pre-washing levels in about two weeks. Depending on the type ofchemicals used, scale snd slime are eliminsted at the same time, but when using chemicals that are highly corrnsive, it is necessary to take sppropriate measures to prevent corrosion of metal parts in the system. Types of chemicals for washing are shown below.

(l) Hydrogen peroxide:
For washing the slime and disinfection, several percent of hydrogen pernade solution is circulated for several hours. This will decompose organic substances, and make disinfection by oxidation and washes by foaming.
(2) Glutaraldehyde:
Since glutgraldehyde is not corrnsive to metals, it is widely used. Legionella can be effectively eliminated using a several hundred mg/L solution.
(3) acids:
Hydrochloric acid and sulfamic acid are mainly used to eliminate scale. A several percent of acidic solution is circulated to disinfect by low pH. Since acids are highly corrosive against iron and copper, anti-corrosive agents must be used simultaneously. In particular, be aware that hydrochloric acid is highly corrosive to stainless steel. When hydrogen peroxide is used, the cleaning method of a cooling water system is as follows:

(1) Lower the water level in the cooling tower according to the amount of hydrogen peroxide to be added.
(2) While circulating the cooling water, add hydrogen peroxide gradually.
(3) While hydrogen peroxide is circulating, turn off the fan on the cooling tower.
(4) To avoid excessive foam, be careful not to add hydrogen peroride at once, and relieve air pressure in the pipe as necessary.
(5) Measure the concentration of hydrogen peroxide in the cooling water system to assess the status of chemical washing as necessary.
(6) After circulating hydrogen peroxide for two to three hours, add sult`ite or other chemicals to neutralize hydrogen peroxide, drain the washing water, and rinse with water.
(7) If the rinse water is markedly dirty, repeat these rinsing procedures.
(8) After rinsing, fill the system with fresh water and resume normal operation.

2-3-3. Chemical water treatment in cooling water systems In order to control the number of Legionella in a cooling water system at all times, it is desirable to chemically wash the cooling tower before and after airconditioning season, and to successively add anti-bacterial agents during operation.

2-3-3-1 . Kind of chemical treatment agents

Many different kinds of chemicals are used to treat Legionella in cooling water, and the effective concentration, functional mechanism and persistence of each chemical are all different.

(1) Hydrochloric acid: When hydrochloric acid is added to cooling water at a concentration of 2 - 5 mg/L, Legionella can be effectively eliminated. However, since hydrochloric acid is highly corrosive against metals, such as iron and copper, it is necessary to utilize anti-corrosive agent to prevent corrosion.
(2) Various organic compounds: The majority of anti-bacterial agents used in cooling water are synthetic organic compounds, and their compositions, functions and effective concentrations vary (Table 3).
(3) Copper:
Copper possesses anti-bacterial abilities, and it is believed to be toxic to many species of bacteria. However, it has been reported that L. pneumophila forms a round-shaped colony on copper. Therefore, it will not be expected that copper act as an anti-bacterial agent in cooling towers.
(4) Silver:
Silver ion is a potent disinfectant, and a small quantity of silver ion can quickly sterilize Legionella suspended in distilled water. However, in
environments, e.g. cooling water, where a variety of materials coexist
silver ions combine with chloride and precipitate, thereby failing to disinfect for Legionella. Therefore, it is difficult to utilize silver ion to disinfect for Legionella in cooling towers.

Values show the concentration that can eliminate Legionella within 24 hours in a test tube.

The disinfection ability of these anti-bacterial agents in a test tube are not necessarily equal to those in an actual cooling Water. Therefore, when utilizing an anti-bacterial agent, it is necessary to examine the anti- bacterial effect of the agent not only in test tube, but in actual cooling towers as well. Furthermore, it is important to consider the effect of corrosion on metals in the cooling water system, when utilizing anti- bacterial agents. If it is necessary, use any anti-corrosion agent.

2-3-3-2. Use of chemieals As for the dosage and injecting method of chemicals, refer to the specifications established by pharmaceutical manufacturers and water treatment companies. Users must be informed of the safety and effect of chemicals from the supplier before use. In particular, make cextain that chemicals are not corrosive against metals in the cooling water system and do not obstruct scale control activities.

(1) Intermittent injection: In intermittent injection, that sometimes referred to as "shock injection", anti-bacterial agents are added every five to seven days. A period of time, that takes for the CFU of Legionella to return to pre- treatment levels, is called the "duration of disinfection effect".

(2) Continuous injection: Continuous injection suppresses the growth of Legionella by maintaining the concentration of anti-bacterial agent in cooling water at s certain effective level. In this method, the previously mentioned multiple agent solution is continuously added frnm one injector to cooling water. By maintaining the concentration of anti-bacterial agents at an appropriate level, the cooling water is constantly disinfected, obtaining reliable results. However, the risk of facilitating the growth of chemical-resistant bscteria is increased. When the number of Legionella starts to increase, even when anti- bacterial agents are being added continuously, this means that the effectiveness of the anti-bacterial agents is diminished. In these case, it is important to change the type of anti-bacterial agents used, apply the shock injection of high concentration agent, and carry out the chemical washing, in adding to consulting a specialist at the district health service.

2-3-3-3. Confirming the effectiveness of treatment
It is important to confirm the result of disinfection after treating cooling water systems. Messure cell count in cooling water before and after treating. This confirmation process takes about 10 days, and even if the result is negative, that is under detection limit, or in the desirable range, strict water quality control management should be maintained in succession.

2-3-3-4. Safety of chemicals

The safety of anti-bacterial agents added to cooling water system is just as important as the effectiveness of the anti-bacterial agents against Legionella. The Drugs, Cosmetics and Medical Instruments Act and other laws and regulations do not apply to water treatment chemicals used in cooling water systems. Therefore, the Anti-Legionella AirConditioning Water Treatment Chemicals Conference has established its own self-imposed standard for the effectiveness and safety of water treatment chemicals utilized to disinfect for Legionella. When disinfecting for Legionella in cooling water, use water treatment chemicals that are in agreement with this standard. Furthermore, users are sdvised to request information on the effectiveness and safety of chemicals under the conditions of their operation in different systems from the suppfier.

2-3-4. Other treatment methods for cooling water systems

(1) Ozone treatment: Experiments have confirmed that adding ozone to the concentrations of 0.2 - 0.3 mg/L disinfects for Legionella.

(2) Ultraviolet treatment: It has been reported that a immersion type ultraviolet lamp can be used to continuously disinfect for Legionella when it is placed in the basin of a cooling tower where cooling water is returned. Since a desirsble disinfection effect cannot be obtained when the permeability of ultraviolet rays in cooling water diminishes, continuous disinfection for Legionella requires that the quality of water in cooling towers be monitored by measuring electric conductivity. Electric conductivity should be close to 100 mS/m.

2-3-5. Emergency washing

If an outbreak of Legionella infections is suspected or confirmed, perform emergency washing. Chlorine (hypochlorite) is usually used in emergency washing, but other effective anti-bacterial agents can also be used.

(1) Close the drainage valve, and stop the air-conditioning chiller and cooling tower fan.
(2) Before injecting new anti-bacterial agents, tske a water sample and immediately measure Legionella cell count.
(3) Keep operating the cooling water pump, and circulate the cooling water.
(4) Continue adding gnti-bacterial agents after initial injection as necessary to maintain an appropriste level of concentration (for chlorine, a residual concentration of a 5 to 10 mg/L) for 12 to 24 hours.

3. Possible problem and preventive measures in other water systems

3-1 . Humidifier
Since the temperature of the water in steam and pan type humidifiers is high, the risk of Legionella contamination is low. On the other hand, since ultrasonic, spray, and ventilation- evaporation type humidifiers utilize water at room temperature, if periodical inspections are not performed, Legionella can grow and multiply in these types of humidifiers and pollute the indoor air environment. It is recommended to periodically clean humidifiers when they are in use, and to drain water when they are not in use.

3-2. Water and hot-water supplies

3-2-I. Water supply facility
Tap water is required by law to be disinfected with chlorine, and since nutrients conditions and the temperature do not promote bacterial growth, the risk for contamination is low. When a water tank is placed high above the ground, the tank must be built with materials that sunlight can not penetrate. Furthermore, even in small buildings (less than 3,000 m2), the Building Sanitation Law should be followed, e.g. periodical cleaning of water tanks, to maintain sanitary conditions.

3-2-2. Hot-water supply facility

In comparison to cold water supply, the risk of Legionalla proliferation in hot-water supply facilities is higher because the water temperature is higher and retention time is longer. In hospitals where cases of nosocomial Legionellg pneumonia infection were reported throughout the year, even in winter season when cooling towers were not being used, the growth and multiplication of Legionella were detected in the hot-water supplies, e.g. hot-water storage tank and shower head. Legionella can not survive in water hotter than 60C.
The temperature of hot water between the hot-water source and terminals should be over 55C. Moreover, it is important to design a hot-water system so that the capacity of the facility does not significgantly exceed the required amount ofhot water.

3-3. Thermal storage tank

Thermal storage systems operate heat source machine during the night hours to store hot or cold water. The capacity ofthese tanks ranges from 100 to >2,000 m3. When these tanks are used to store hot water, they became saturated with oxoygen, and bacteria are more likely to proliferate. It has been reported in Japan that sanitation workers contracted Legionella infections after cleaning the interior of a hot-water storage tank. Therefore, it is necesssry to disinfect these tanks using regular disinfecting chemicals before physical cleaning.
Do not clean chemically untreated thermal storsge tanks with ahigh-pressure water nozzle. Furthermore, sanitation workers must wear necessary protective appliances.

3-4. Whirlpool

In recent years, whirlpools have been installed in many exercise facilities and public baths. Since air is blown into the hot water that is being circulated, bacteria, including I.egionella, can easily multiply in whirlpools. Furthermore, when air bubbles are injected, aerosol is generated on the surface of hot water, and the contaminated aerosol is inhaled by people . Whirlpools have been popular in Europe snd America for a long time, but unfortunately, they are a breeding ground for Legionella infections. Currently, there is no continuous disinfection apparatus on these tubs, so chlorine tablets are widely used instead. Because people are actually in the water in these tubs, it is necessary to pay special attention to their disinfection and cleaning.

3-5. Decorative fountain

There has been some cases of decorative fountains in hotel lobbies and cutting lubricants at automobile factories becoming contaminated and causing outbreaks of Pontiac fever. Since fountains are generally out in the open, when they are contaminated with Legionella, it is highly possible that bacteria are dispersed into the atmosphere via aerosol generated by these fountains. It is advised that these fountains be periodically cleaned, tiltered and disinfected using chlorine or water treatment chemicals.

3-6. Hand-washing hot-water

Even though hand-washing hot-water supplies sre widely used in Japan, there is no standard to regulate these facilities. In the case of hot-water of suitable temperature for hand-washing is directly supplied to a faucet, it is suitable for the growth snd multiplication of Legionella and other bacteria in these facilities. It is advised that combination taps be installed, and that the temperature of hot-water source and terminals be maintained at over 55 degrees C

Appendix A. Analytical Procedures with Relation to Legionella Control

1 . Practice of Legionella analysis

1-1. Collecting a water sample

(1) Collect a water sample in a 500 mL heat-resistant polypropylene bottle with a screw cap. The bottle should have been sterilized in an autoclave.
When sampling, keep about 10% of the air space at the top of the bottle. If additional water quality tests must be conducted, take another water sample in another sterile bottle. It is important not to seal the collection bottle with rubber or cork, as these substances can facilitate the growth of bacteria. To create steam when sterilizing collection bottles in an autoclave, place a small quantity of distilled water and loosen caps slightly. After sterilization, tighten the caps so that these sterilized collection bottles can be stored for a long period of time.
(2) Collect g water sample from the surface of the water layer in the center of the basin in a cooling tower. The showering water in the tower is more homogeneous and contain s less debris, but depending upon cooling tower design, showering water can not be collected in some cooling towers.
(3) When collecting a water sample, measure the on site temperature of the cooling water. It is preferable that pH and electric conductivity also be measured at the same time.
(4) Kind of cooling water, sampling site, date, time and name of person who collected the sample must be clearly written on the sample bottle.
(5) Water sample gre transported in a cooler or insulated box, and should be tested as soon as possible. When water samples must be sent to a testing laboratory, keep the temperature of the cooler between 5 - l00C and send the sHmples immediately.
(6) In order to perform Legionella tests smoothly, it is important to communicate with the testing laboratory regarding the date of collecting and sending water samples.

1-2. Pretreatment and incubation Figure shows the steps for pretreatment and incubation of water samples.
(1) Apply an already pretreated water sample to a selective medium.
(2) Incubate the medium at 370C for five to seven days (up to 10 days).
(3) Colonies that are found within 24 to 48 hours are not Legionella. Independent colonies of Legionella begin to appear in four to five days, and they can be positively identified in six to seven days. Nonetheless, these culture media must be inspected each day.
(4) Colonies thst grayish white, slightly transpsrent, wettish, and various sized are presumed Legionella, if they have a distinct acidic smell.

(5) When colonies that fit the above descriptions are found, perform the procedures necessary to positively identify Legionella.

2. Measurement for counting the number of general bacteria

The cell count of general bacteria is measured with ordinary quantitative culture method. Choose a culture medium that permits the grnwth of both Gram's positive and negative bacteria, and in order to obtain more meaningful results, incubate the culture medium at temperature between 25 and 30C for two to three days. A dip slide test is easy to perform, but the results obtained are not highly reliable.

3. Examining the effectiveness of anti-Legionella water treatment chemicals In order to confirm the effectiveness of water treatment chemicals on Legionella, it is recommended thst the following tests be performed: Before conducting a field test using an actual cooling tower, the effectiveness of a water treatment chemical must be confirmed in test tubes. First, confirm the effectiveness of the water treatment chemical in a test tube containing Legionella suspended in distilled water. Next, suspend Legionella in actual cooling water to test the effectiveness of the water treatment chemical by series of stepped concentrations. Finally, only water treatment chemicals that can eliminate Legionells suspended in actual cooling tower in 24 to 48 hours are used to conduct a field test using an actual cooling tower.

3-1 . Experiment in test tube

3-1-1. Distilled water experiment

(1) Suspend a reference strain of Legionella pneumophila in sterile distilled water with the concentration of 108 CFU/mL.
(2) Take the test water treatment chemical and prepare a stepped concentration series of diluted solution with sterile distilled water.
(3) Add the suspended bacteria to the diluted test water treatment chemical solutions and incubate them at 300C. Count the number of viable cells after
1 , 3, 6, 24, 48 and 72 hours. For the reference, use distilled water instead of chemical solution and count the number of viable cells.

3-1-2. Cooling water experiment

(1) Make certain to use cooling water free of Legionella.
(2) Suspend a reference strain of Legionella pneumophila in the cooling water. (3) Based on the results of the distilled water experiments, prepare three different concentrations of the test water treatment chemical solution using the cooling water, e.g. 1, 5 and 10 mg/L.
(4) Add the suspended bacteria to the test chemical solutions and incubate them at 30C. Count the number of viable cells by measuring CFU Hfter 6, 24, 48, 72 and 168 hours.

3-2. Field test using cooling towers

(1) Just after the beginning of air-conditioning season, select a cooling tower that is inhabited with Legionella and count the number of viable cells.
(2) Clean the cooling tower using hydrogen peroxide and other appropriate disinfecting chemicals.
(3) The next day, conduct a measurement to make sure that Legionella cannot be detected.
(4) Add the predetermined quantity of the test water treatment chemical continuously or periodically to the cooling water line.
(5) After cleaning and chemical injection, collect a water sample every two weeks to examine the chsnges in the number of Legionella.
(6) Continue counting the number of viable cells until the end of air-conditioning season.
(7) If the undetectable condition of after cleaning is succeeded, or the cell number is maintained at less than 100 CFU/100 mL, then the effectiveness of the test water treatment chemical is verified. However, if the number of Legionella increases in the course of the testing, then the test water treatment chemical is determined to be ineffective.

Appendix B.
Self imposed Standard for Anti-Legionella Air-Conditioning Cooling Water Treatment Chemicals

Since the Drugs, Cosmetics and Medical Instruments Act does not cover chemicals such as disinfectant for cooling water systems, each water treatment company has establ.ished its own criteria and confirmed by itself the effectiveness and safety of the chemicals. In June 1991 , thirteen companies which are related to the industry of water treatment chemicals for air conditioning systems organized a working group named the Anti-Legionella Air-Conditioning Water Trestment Chemicals Conference aiming to supply effective and safe anti-Legionella chemicals and spread correct knowledge of environmental pollution by Legionella bacteria.

The Conference established a Self imposed Standard for Anti-Legionella Air-Conditioning Cooling Water Treatment Chemicals, in July 1992. The standard consists of a preamble and six articles. An outline of the articles is as follows:

Article 1: Objective
This standard is established to ensure the effectiveness and safety of anti- Legionella cooling water treatment chemicals, which are used for the prevention and contrnl of Legionella multiplication in cooling water of Air-conditioning system.

Article 2: Coverage
This standard is applied to water treatment chemicals which are used for cooling water of building air-conditioning systems and have main function of prevention and control of Legionella multiplication.

Article 3: Confirmation of effectiveness
Anti-Legionella chemicals for air-conditioning cooling water must be tested and confirmed for bacteriocidal or growth-suppressing effects against Legionella.

Article 4: Confirmation of safety
Chemicals must satisfy the following criteria.
(1) Components of the chemicals must be the existing and publicated chemical substances proclaimed in the "Law on Examination of Chemical Substances and Production Regulations" and the "lndustrial Safety and Hygiene Act".

(2) Chemicals must not be poisonous substances prescribed in the "Regulations for the Control of Poisonous and Deleterious Substances".

(3) Chemicals must have data accumulstion on the following toxicity tests.
(a) Acute oral and dermal toxicity tests for the msin components (active ingredients against Legionellg). Data for toxicity tests considered necessary from the properties of the main compounds (especially dermal sensitivity tests) must be accumulated as possible.
(b) Dermal primary stimulating tests using chemicals at the same concentrations as in actual utilization and ten times concentrations.

Article 5: Notations
Notations of anti-Legionella cooling water treatment chemicals must be stated on labels and manuals. The chemicals must be registered in the record of the Conference.

Article 6: Prevention of environmental pollution by drainage
The water quality of drainage from air-conditioning systems with ordinary use of water treatment chemicals to public water area and sewerage system must satisfy the respective effluent standards.

Use of water treatment chemicals which meet this self-imposed standard is recommended for disinfecting Legionella in cooling water. Users should request suppliers for data on the effectiveness and safety of chemicals under each utilization condition.