MEASURED FUNGAL INDEX DETERMINED USING FUNGAL GROWTH AND COMPUTED FUNGAL INDEX BASED ON TEMPERATURE AND RELATIVE HUMIDITY IN HOUSES

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1 MEASURED FUNGAL INDEX DETERMINED USING FUNGAL GROWTH AND COMPUTED FUNGAL INDEX BASED ON TEMPERATURE AND RELATIVE HUMIDITY IN HOUSES Keiko Abe 1, Yuji Kawakami 2, Takuya Hamano 3 Yuichi Imai 4 and Kenichi Iso 3 1 Institute of Environmental Biology, JDC Corporation, Aikou-gun Kanagawa, Japan 2 FCG Research Institute, Inc., Tokyo, Japan 3 JDC Co., Ltd., Aikou-gun Kanagawa, Japan 4 Nippon Living, Co., Ltd., Tokyo, Japan ABSTRACT The fungal index is a biological climate-parameter, which represents the environmental capacity to allow fungal growth. The author developed software that determines the computed fungal index, which was estimated using the Excel software "INDEX" from the measured temperature and relative humidity. The computed fungal index and the measured fungal index, determined using a fungal detector encapsulating fungal spores, were determined in 10 rooms in six dwelling houses. The computed fungal index values were generally close to those of the measured fungal index in rooms. The computed and measured fungal index values showed a positive correlation, and the correlation coefficient between these fungal index values was 0.91 (p=0.01). KEYWORDS Computed fungal index, Fungal index, Fungi, Climate, Relative humidity INTRODUCTION Fungi induce allergies, and fungal contamination in buildings has adverse effects on the health of residents. Fungi usually spread in buildings by repeating the following process: (1) Attachment of fungal spores to a region, (2) germination of the spores, (3) growth of hyphae from the spores, (4) formation of new spores on the hyphae, and (5) dispersal of the newly formed spores. Indoor dampness is the most common cause of fungal contamination in buildings. However, fungi are living organisms, so time is required for the growth of fungi that adhere to building materials. Fungal contamination does not immediately occur in buildings, even when humidity is high. Residents do not observe fungal growth everyday in their building, and notice fungal contamination at some stage. If it is recognized that the building has an indoor environment suitable for fungal growth, they can take measures before the occurrence of fungal contamination. Thermohygrometers are widely used for the measurement of temperature and relative humidity in the general indoor environment. However, fungal growth cannot be directly estimated using these physical climatic parameters. The author considered that environments in which fungi easily grow can be accurately predicted by evaluating the environment using fungal growth itself, and proposed a fungal index (Abe 1993, Abe et al 1996, Abe 2001). The index is a biological climatic parameter showing the capacity of the climate to facilitate fungal growth at the test site. The index is measured by placing a fungal detector, in which dried fungal spores and their nutrients are encapsulated, at a test site for a certain period. If the test site is suitable for fungal growth, the spores encapsulated in the detector grow in response to the Corresponding Author: Tel: , Fax: address: abekeiko@kamakuranet.ne.jp

2 environment. If the test site is unsuitable for fungal growth, the spores in the detector do not grow. The test environment is quantitatively evaluated by the index using the growth response of fungal spores encapsulated in the detector and the period of exposure. Fungi grow more easily in regions with a higher fungal index, and fungal contamination advances more rapidly in such regions, so that environmental susceptibility to fungal contamination can be estimated by measuring the index. It has been clarified that the fungal index is determined by the temperature and relative humidity, and the relationship between the fungal index and physical climatic parameters (temperature and relative humidity) was reported in a previous paper (Abe 1993). In this study, the author developed software to compute the index from temperature and relative humidity using the Excel software function INDEX and a database concerning the relationship between the fungal index and the climatic parameters of temperature and relative humidity. The usefulness of the developed software was evaluated by comparing the computed fungal index and the measured fungal index, the index determined biologically using the growth response of the fungal spores enclosed in the detector. MATERIALS AND METHODS Test regions and periods Examinations were performed at one or two sites at six dwellings in Tokyo, Japan: one at a low region of a north-side wall in a living room, and one at a low region of a north wall in a bedroom. These measurement sites on the walls were 10 cm above the floors in the rooms. The examination was performed from June to November Fungal detector A fungal detector, in which the conidia of Eurotium herbariorum, strain J-183 (Abe 1993) were encapsulated, was produced to examine the environment. The maximal length of hyphae measured in the detector was about 3,000-m in the present study. Figure 1 shows the fungal detector we prepared. The detector was produced as follows. One surface of a double-stick sheet frame (outer size, mm; width, 3 mm; thickness, 0.3 mm) was applied to a transparent plastic plate (13 50 mm; thickness, 0.4 mm), and the spore suspension was spotted on the plastic plate within the frame and air-dried (spot diameter, about 3 mm). The plastic plate was covered with gas-permeable film (13 23 mm), and the film was stuck on the double-stick sheet frame. Since the inoculated spots of fungal spores were encapsulated, newly generated spores did not disperse to the test environment, even though fungi grew markedly within the detector, i.e., a large number of spores were newly Figure 1. Fungal detector. formed on the extended hyphae that emerged after germination of the spores in the spots. Measured fungal index Each fungal detector was fixed with a clip and hung on a nail on each wall. Each fungal index was measured after exposing the detector to the test environment for one month. The index measured using the fungal detector was defined as the measured fungal index. The measured fungal index was obtained using the following procedures: (1) The fungal detector was

3 exposed to the test environment for a certain period (one month in this study); (2) after exposure, the detector was placed in a container with silica gel and the development of hyphae was immediately terminated by desiccation with silica gel; (3) photographs of fungal growth in the detectors were taken using microscopy; (4) the length of hyphae was measured on the photographs (up to 3000 m in this study); (5) the fungal response unit (ru, which was proposed as a measure of the fungal growth response) during the exposure period was determined by the length of hyphae using the standard curve (Figure 2); and (6) the fungal index was obtained by dividing the fungal response unit (ru) by the exposure period (week). Figure 2 shows the standard curve indicating the relationship between the length of hyphae and the fungal response unit (ru). The length of hyphae shown in Figure 2A was the distance between the spore and the hyphal tip, which was used when the hyphae were short, and this was less developed outward from the edge of spore inoculated spot. The length of hyphae shown in Figure 2B was the distance between the edge of the inoculated spot and the hyphal tip, which was used when the hyphae developed from the spot s edge outward into the region without nutrients by more than 100 m. The standard curve was obtained using the following method: The standard fungus (Eurotium herbariorum Figure 2. Standard curve. The hyphal length is the distance from the spores to the tips of hyphae in A, and the distance from the edge of the spore-containing spot to the tips of hyphae in B Figure 3. Typical fungal-responses to environment. The hyphal length from the spores to the tips of hyphae was ca. 60µm in A, and the hyphal length from the edge to the tips of hyphae was ca. 2000µm in B.

4 J-183) was incubated under a standard climate (temperature, 25C; relative humidity, 93.6%; within an airtight container in which humidity was controlled using KNO 3 and its saturated solution), and the growth curve (hyphal extension curve) under the standard climate was obtained. The standard curve was obtained by substituting the fungal response unit (ru) for the incubation time (h) on the growth curve at a ratio of 1 : 1. Fungal growth response was thus provided in fungal response units (ru), which are proportional to the incubation time under the standard climate. Figure 3 shows the typical fungal response to test environment of the standard fungus Eurotium herbariorum. In Figure 3A, the length of hyphae from spores to the tips was about 60µm. The response was 10ru by applying the standard curve in Figure 2A. In Figure 3B, the length of hyphae from the edge to the tips of hyphae was about 2000µm. The response was 58ru by applying the standard curve in Figure 2B. The fungal index was determined by dividing the fungal response unit (ru) by the exposure period (week), showing the fungal response unit per week. For example, when the distance between the inoculated spot edge and the hyphal tip was 3000 m after exposing the fungal detector to the environment, a fungal response unit of 80 ru was obtained from the standard curve B, which is the same growth response as the standard fungus incubated for 80 hours under the standard climate. When the exposure period was 7 days (one week), the fungal index in the test environment was 80, 80 divided by 1 is 80. When the same response of 80 ru was obtained after an exposure period of 31 days (4.4 weeks), the fungal index in the test environment was 18, 80 divided by 4.4 is about 18. Measurement of temperature and relative humidity The thermohygrosensor (RSH-1010, ESPEC MIC Inc.) of a thermohygrorecorder (thermorecorder RS11, ESPEC MIC Inc.) was hung immediately next to the fungal detector at each test region, and temperature and relative humidity were automatically measured every hour and recorded during the examination period. Estimation of computed fungal index from measured temperature and relative humidity A database to extract fungal index values from specified temperature and relative humidity values was prepared using fungal index measurements obtained under various constant climatic conditions in the temperature range of 0-35C and relative humidity range of 0-100%. Software using the database with the Excel function INDEX was developed to estimate the index from a specified temperature and relative humidity. The index transformed from the temperature and relative humidity value was defined as the computed fungal index. The temperature and relative humidity values obtained every hour by thermohygrorecorders exposed at each test region were transformed into the index using the developed software. Then, the mean value of these transformed values during the exposure period of each fungal detector (one month) was calculated. RESULTS AND DUSCUSSION Figure 4 shows the computed and measured fungal index values in all exposure sites at six houses. The computed and measured fungal index values at each survey site a positive correlation. Higher computed index values Figure 4. Comparison of the computed and measured fungal indices.

5 were obtained at the sites with higher measured index values. The differences between the values of computed and measured fungal indices were within 5. The correlation coefficient between the computed and measured fungal indices during the examination period between June and December was 0.91 (p=0.01). The computed fungal index is considered to be applicable to practical environmental evaluations, as well as the measured fungal index, because the computed fungal index was closely correlated with the measured fungal index. Although the database used in the developed software to determine the computed fungal index was prepared using the measured fungal indices determined under steady climates, constant temperatures and relative humidities, the computed fungal index values were generally close to those of the measured fungal index, despite the fluctuating climate in the room environments of the dwelling houses. In the outdoor air, the differences between the computed and measured fungal indices were larger; sometimes the differences exceeded more than 30, and the computed indices during the exposure periods of fungal detectors were always higher than the measured indices if the measures values were lower than 30 (Abe 2006). However, in room climates where the maximal value of measured fungal indices was 18, similar values for measured and computed indices were obtained. The fluctuations in climate are generally smaller indoors than outdoors. Smaller fluctuations in indoor climates thus resulted in almost equal values for measured and computed indices. Figure 5 shows typical computed and measured fungal index values in two houses. The climate was different in each house. House A showed high fungal indices and house B showed low fungal indices. Measured and computed fungal indices exceeded 18 in June, July, September and October in room 1 (A-1) and in July in room 2 (A-2) in house A, while measured indices were not detected and computed indices were low (below 2) in both room 1 (B-1) and room 2 (B-2) in house B. A-1 A-2 B-1 B-2 Figure 5. Typical Computed and measured fungal indices in two houses from June to November 2006.

6 The climatic conditions affecting fungal growth could be evaluated using the computed fungal index. These conditions could not be detected by conventional measurements of temperature and humidity. Estimation of fungal growth directly from temperature and humidity is considered difficult. Although we could predict the presence of fungal growth from temperature and relative humidity data if the temperature is maintained at around room temperature and relative humidity is always higher than 70%, we could not predict the rate of fungal growth without fungal indices. Also, without fungal indices, prediction was difficult under varying environmental conditions that included both suitable and unsuitable climates for fungal growth. Only by transformation to the computed fungal index from the data of temperature and relative humidity measured continuously could we observe the climate that affected fungal growth. The susceptibility of buildings to fungal contamination must be evaluated by examining the computed fungal index. Using this index, routine measurements of temperature and relative humidity in building management will become an applicable tool to prevent fungal contamination. Furthermore, we must be able to simulate the index using simulated temperature and relative humidity in the design stage before the construction of buildings to create a comfortable indoor environment free from fungal contamination. CONCLUSION 1. The author developed software that computes the fungal index (a biological climatic parameter) from temperature and relative humidity (physical climatic parameters). 2. The computed fungal index values were generally close to those of the measured fungal index in rooms. The computed and measured fungal index values showed a positive correlation. The correlation coefficient between the computed and measured fungal indices was 0.91 (p=0.01). 3. We can develop countermeasures against fungal contamination by estimating the computed fungal index using records of temperature and relative humidity. Also, we can construct buildings in which fungal contamination is unlikely to occur by evaluating the computed fungal index using simulated distribution of temperature and humidity in the building design stage. ACKNOWLEDGMENTS The authors would like to thank R. Sekiguchi, Y. Matsushita, and K. Katsuta for their assistance. REFERENCES 1) K. Abe (1993) A Method for numerical characterization of indoor climates by a biosensor using a xerophilic fungus Indoor Air, Vol. 3, ) K. Abe, Y. Nagao, T. Nakada, and S. Sakuma (1996) Assessment of indoor climate in an apartment by use of a fungal index, Appl. Environ. Microbiol, Vol. 62, ) K. Abe (2001) Fungal index evaluation of indoor environments, Bokin Bobai, Vol. 29, ) K. Abe (2006) Comparison of a measured fungal index determined using fungal growth and a computed fungal index based on temperature and relative humidity J. Soc. Indoor. Environ, Japan. Vol. 9,