Indoor and outdoor airborne bacterial and fungal air quality in kindergartens: seasonal distribution, genera, levels, and factors influencing their concentration

Journal Pre-proof Indoor and outdoor airborne bacterial and fungal air quality in kindergartens: seasonal distribution, genera, levels, and factors influencing their concentration Farhad Mirkhond Chegini, Abbas Norouzian Baghani, Mohammad Sadegh Hassanvand, Armin Sorooshian, Somayeh Golbaz, Rounak Bakhtiari, Asieh Ashouri, Mohammad Naimi Joubani, Mahmood Alimohammadi PII:

S0360-1323(20)30048-2

DOI:

https://doi.org/10.1016/j.buildenv.2020.106690

Reference:

BAE 106690

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Building and Environment

Received Date: 26 October 2019 Revised Date:

19 December 2019

Accepted Date: 18 January 2020

Please cite this article as: Chegini FM, Baghani AN, Hassanvand MS, Sorooshian A, Golbaz S, Bakhtiari R, Ashouri A, Joubani MN, Alimohammadi M, Indoor and outdoor airborne bacterial and fungal air quality in kindergartens: seasonal distribution, genera, levels, and factors influencing their concentration, Building and Environment, https://doi.org/10.1016/j.buildenv.2020.106690. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Indoor and outdoor airborne bacterial and fungal air quality in kindergartens: seasonal distribution, genera, levels, and factors influencing their concentration Farhad Mirkhond Chegini1, Abbas Norouzian Baghani1, Mohammad Sadegh Hassanvand1,10, Armin Sorooshian2,3, Somayeh Golbaz1, Rounak Bakhtiari4, Asieh Ashouri5,6, Mohammad Naimi Joubani7, Mahmood Alimohammadi1,8,9* 12345678910-

Department of Environmental Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Department of Chemical and Environmental Engineering, University of Arizona, Tucson, Arizona, USA Department of Hydrology and Atmospheric Sciences, University of Arizona, Tucson, Arizona, USA Department of Microbiology, School of Public Health and Institute Health Research, Tehran University of Medical Sciences, Tehran, Iran, Cardiovascular Diseases Research Center, Department of Cardiology, Heshmat Hospital, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran Research center of health and environment, department of health education and promotion, school of health, Guilan University of Medical Sciences, Rasht, Iran Research center of health and environment, School of Health, Guilan University of Medical Sciences, Rasht, Iran Center for Water Quality Research (CWQI), Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran Health Equity Research Center (HERC), Tehran University of Medical Sciences, Tehran, Iran Center for Air Pollution Research (CAPR), Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran

*Corresponding author: Mahmood Alimohammadi; [email protected]; Tel.: +989123573694; fax: +98 2188951583 Address: Department of Environmental Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Address: Center for Air Pollution Research (CAPR), Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran

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Abstract Kindergartens in developing countries are sensitive places for children owing to exposure to bioaerosols that promote a range of infections. This work aimed to determine the concentration of culturable bacteria and fungi aerosols in indoor and outdoor air of twelve kindergartens in Rasht, Iran. The mean concentrations of fungi versus bacteria in indoor air of kindergartens were 7 ± 6 CFU/plate/hr versus 42 ± 29 CFU/plate/hr, respectively, while the mean concentrations of fungi versus bacteria in outdoor air of the same kindergartens were 12 ± 8 CFU/plate/hr versus 24 ± 18 CFU/plate/hr, respectively. The findings revealed that 33% of the concentration of indoor bioaerosols (bacteria and fungi) and 8% of the concentration of outdoor bioaerosols in kindergartens were higher than the recommended value (40 to 84 CFU/plate/hr), indicating medium risk. The main bacteria species detected in indoor and outdoor air in kindergartens were Bacillus spp., Staphylococcus aureus, Micrococcus spp., Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus spp., and Streptococcus spp. The predominant genera of the airborne fungi isolated from indoor and outdoor air in kindergartens were Aspergillus terreus, Aspergillus flavus, Cladosporium spp., Penicillium spp., Rhodotorula spp., Ulocladium spp., and Alternaria spp. Indoor air quality of kindergartens qualified as posing a medium risk level, and strategies should be considered to remove bioaerosol emissions in these susceptible places. Hence, to reduce negative health effects of bioaerosols on children, it is important to have proper ventilation, air conditioning systems, minimalfurniture and textile materials, and application of disinfectants. Keywords: Indoor air quality; Outdoor air quality; Bioaerosols; Kindergarten; Health risk; Iran

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1. Introduction Indoor and outdoor concentrations of bioaerosols (bacteria and fungi), especially in areas with high densities of children such as kindergartens, are a major global public health problem in developing countries [1-4]. Children in the kindergarten are more vulnerable to bioaerosols than adults due to breathing more air than adults [3, 5]. Furthermore, children attend kindergartens for approximately 40 h per week [1, 3, 6-8]. Bacteria and fungal bioaerosols can promote allergic and bacterial diseases, lower respiratory illness, recurrent wheezing, atopic dermatitis, infections, and rhinitis in indoor air of kindergartens [3, 4, 9-12]. For example, previous studies stated that kindergarten attendance can cause an increased risk for illness like acute otitis media, upper respiratory diseases and lower respiratory diseases in children [13-15]. Sick children in kindergarten are also a source of bacteria and fungal bioaerosols that can influence other classmates, teachers, and associated families [15, 16]. Previous work showed that kindergartens infections are elevated by bacteria bioaerosols such as Bacillus, Enterobacter, Micrococcus, Corynebacterium, and Staphylococcus [1]. The same study showed that the predominant airborne bacteria genera measured in indoor air of kindergartens were similar as those in outdoor air of kindergartens [1]. That study also showed that kindergarten infections can be promoted by fungi such as Aspergillus, Zygomycetes, Penicillium, Rhizopus, and Fusarium [17]. The presence of fungi and bacteria aerosols in indoor and outdoor air of kindergartens, particularly in indoor air of kindergartens, is linked to several factors such as the (i) presence, condition, and activities of children in kindergartens and in elementary schools [18, 19], (ii) environmental air parameters such as temperature and relative humidity [5, 20], (iii) air-conditioning systems (operational status (i.e., on/off)) [5], (iv) seasonal factors [2123], (v) sunny, rainy and cloudy days [24, 25], (vi) inadequate ventilation [16, 26], (vii) personal 3

sources such as clothing [27, 28], (viii) building materials [27-30], (ix) dust (particulate matter) and even soil [1, 31, 32], (x) inadequate disinfection [33, 34], and (xi) geographic location [33, 35-37]. The ratio values of indoor fungi to outdoor fungi (I/O fungi) and indoor bacteria to outdoor bacteria (I/O bacteria) are generally used as an identifier for emission sources of bioaerosols [38-41]. For instance, Kim et al. (2007) and Li et al. (1993) reported that if the I/O ratio of airborne bacteria and fungi concentrations was below one, that outdoor air is the main source of bioaerosols indoor air [38, 42]; if the I/O ratio of airborne bacteria and fungi concentrations was more than one, indoor sources are responsible [38, 42]. Owing to children spending a long time (about 40 h per week) in kindergarten environments and being highly vulnerable to bioaerosols, identifying the types and concentrations of bioaerosols in these places are important for improving the public health of children. To address this knowledge gap, this study aimed to (i) determine the concentration of culturable bacteria and fungi bioaerosols in indoor and outdoor air of 12 kindergartens in Rasht (Iran), (ii) identify the genera and percentage of bioaerosols in indoor and outdoor air of these places, and (iii) study the effects of seasons, density of children (population), and weather conditions on the amount of bioaerosols. 2. Materials and Methods 2.1. Descriptions of Study Area This study was conducted in Rasht, situated in the northwest of Iran on the coast of the Caspian sea (37° 19' N, 49° 37' E). Rasht is the capital of the Gilan province with around 956,971 inhabitants according to a census report in 2016 [43]. Rasht has a temperate climate, is humid, and experiences heavy rainfall with monthly mean minimum and maximum temperatures of 7.8 °C and 28.6 °C, respectively [44, 45]. The monthly average minimum and maximum wind

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speeds in Rasht from 21 March 2018 to 19 March 2019 were 1.1 and 2.6 m s-1, respectively [44]. Furthermore, the monthly average minimum and maximum rainfall/precipitation in Rasht from 21 March 2018 to 19 March 2019 were 25 and 199.8 mm, respectively. Fig. S1 displays a map of Rasht showing the 12 sampling locations coinciding with kindergartens in different areas of the city. More detailed information about the main characteristics of the studied kindergartens in Rasht city, Iran is provided in the supporting information (Section S1 and Table S1). 2.2. Sampling Methods Based on the EPA sampling guideline [46], sampling was performed every twelve days in summer in Iran (52 days) from 1 August to 19 September and in autumn in Iran (52 days) from 1 October to 17 November (Table S2). In total, twelve kindergartens were selected for sampling in Rasht, Iran (Fig. S1). We collected 96 samples in indoor air of twelve kindergartens in summer (48 bacterial and 48 fungal samples) and 96 samples in indoor air of twelve kindergartens in autumn (48 bacterial and 48 fungal samples). Similarity, autumn sampling was performed similar to summer sampling (Table S2). Samples of indoor and outdoor bioaerosols (bacterial and fungal) in 12 kindergartens were collected based on the standard index of microbial air contamination (IMAC for environments at risk) [26, 47] using a passive method (settle plate) in the morning (for one hour in the summer and autumn: 10:00 - 11:00 local time). For sampling indoor and outdoor bioaerosols, petri dishes (9 cm) containing a solid nutrient medium were put at a height of about 0.5 - 0.8 m (at the children breathing zone) from the floor for indoor and 1.5 m from the floor for outdoor and at 1 m from all four sides of the wall or physical barriers [20, 29, 32, 47-49]. Environmental air parameters such as temperature (°C) and relative humidity were also simultaneously recorded using a portable instrument (Preservation Equipment Ltd, UK) to find the relationship between

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bioaerosol concentration and environmental air parameters. After sampling, the plates will be wrapped with masking tape (as a control to minimize unexpected contamination and also to avoid secondary contamination), stored at 4°C (using a portable plastic cooler box) and moved to a laboratory. The fungal samples were incubated in an inverted position at 25–28 °C for 3–7 days, while the bacterial samples were incubated at 37 °C for 24 to 48 h [16, 20, 30, 32, 49, 50]. 2.3. Quantification and Characterization of Bioaerosols Tryptic soy agar (TSA) culture media (Merck Co, Germany) with cycloheximide and sabouraud dextrose agar (SDA) culture media (Merck Co, Germany) with chloramphenicol were used to identify and speciate bacterial and fungal bioaerosols, respectively [16, 48, 49, 51, 52]. The concentration of bioaerosols (bacterial and fungal) in air samples were counted as CFU/plate/hr [29, 48]. Bergey’s Manual and biochemical tests were applied for identification of bacteria species, while the slide culture method in the electronic microscope (Olympus BX60M BF/DF) with a magnification of 100 × and 400 × was utilized for recognition of fungal species [29, 48, 53-55]. 2.4. Quality Control 2.4.1. Quality Control of Culture Media According to previous studies, quality control of culture media is a very crucial parameter for the determination of the quality of media [56, 57]. In this study, there was no growth on the two plates at 37°C for 24 hours and on the plates at least for three days at room temperature. More detailed information about quality control of culture media and samples is provided in the supporting information (Section S2). 2.4.2. Quality Control of Samples

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QC samples consist of field blanks and shipping blanks. The precision of the measurement is assessed by duplicate sampling [58]. Hence, one of the main methods for QC of samples was field blanks [57, 58]. The possible contamination resulting from handling of the sample media is assessed by analyzing a field blank [57-59]. Blank values for bacteria and fungi were lower than 10% of the post sampling values for all samplers. In addition, the sterility of the plates is checked by returning one unexposed shipping blank of each medium (TSA and SDA). The shipping blank is prepared by taking an unused plate (without opening the Petri dish) and submitting it to the laboratory with the other samples [58]. The sterility of the plates was checked and contamination was not observed on the shipping blank. The repeatability (precision) of sampling and analysis is assessed by sample duplicates. One set of duplicate samples is collected at a specified indoor location and another set of duplicate samples is collected at the outdoor location [58]. Sampling in this study was performed according to duplicate samples. In fact, the reported concentrations of each sampling site was the mean of duplicate samples. 2.5. Statistical Analysis Statistical analysis was conducted using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY) and R Statistical Software version 5.3.1 (Free and open source). For tests of between-subject effects (i.e., differences between indoor airborne bacteria and fungi levels with outdoor airborne bacteria and fungi levels as well as differences between indoor airborne bacteria and fungi levels in twelve kindergartens) and also for tests of within-subject effects (i.e., differences between airborne bacteria and fungi levels at four months (August, September, October and November)) was performed based on repeated measures ANOVA. One of the assumptions of using analysis of variance for repeated measurements is the equality of covariance between dependent variables as assessed using Mauchly's Test of Sphericity in this 7

study. If this assumption were true, the Sphericity test should be used, otherwise the HuynhFeldt test should be applied. In addition, the relationship between bioaerosol concentration and environmental air parameters such as temperature (°C) and relative humidity (%) were represented with Spearman's correlation coefficient. Figures were developed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, San Jose, CA, USA) and R Statistical Software version 5.3.1 (Free and open source).

3. Results and Discussion 3.1. Bioaerosol Concentrations 3.1.1. Indoor and Outdoor Bacteria The mean (± SD), minimum, and maximum concentration of indoor and outdoor bacteria according to CFU/plate/hr in different sampling sites are summarized in Table S3. The total mean (± SD) concentration of bacteria for indoor and outdoor were 42 ± 29 and 24 ± 18 CFU/plate/hr, respectively, for the 12 kindergartens in Rasht, Iran. The minimum and maximum bacteria concentrations were 2 and 143 CFU/plate/hr for indoor air, respectively, and 4 and 85 CFU/plate/hr for outdoor air. For comparison, the range of culturable bacteria in indoor and outdoor air of child daycare centers in Edirne (Turkey) was 3 to 74 CFU/plate/10-15 min and 2 to 141 CFU/plate/10-15 min, respectively [32]. The ratio values of indoor fungi to outdoor fungi (I/O fungi) and indoor bacteria to outdoor bacteria (I/O bacteria) are generally used as an identifier for emission sources of bioaerosols [3840]. Hence, in order to correctly determine the ratio values of I/O fungi and I/O bacteria, bioaerosols samples were measured in similar conditions for all kindergartens under study. In

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other words, kindergartens were chosen that used natural ventilation (Table S1). For this reason, children and staff in kindergartens had to open their windows to have proper ventilation or regulate air flow. In this work during four months (August, September, October and November) for 12 kindergartens, the ratio of I/O bacteria ranged from 0.2 to 13.6, which implies that outdoor air is the major source of bacterial bioaerosol (due to 88% of 12 kindergartens in different months had I/O ratio of airborne bacteria more than one (I/O bacteria > 1)), which is consistent with the findings of Canha et al. (2015) in urban and rural primary schools in Lisbon (Portugal) (2.1), by Faridi et al. (2015) in the retirement home in Tehran (Iran) (1.77), and by Faridi et al. (2015) in the school dormitory in Tehran (Iran) (1.44) [18, 48]. Hence, in this work, the indoor air could be contaminated with outdoor airborne bacteria by natural ventilation, which is consistent with the findings of former works [17, 38, 42, 60]. In addition, the lack of proper ventilation and high density of children in kindergartens (human sources) (due to 12% of 12 kindergartens in different months had I/O ratio of airborne bacteria lower than one (I/O bacteria < 1)) were other reasons for the enhanced number of bacterial aerosols in indoor air, which is consistent with the findings of Dehghani et al. (2018), Canha et al. (2016), Tesfaye et al. (2015) , Önoğlu et al. (2011), Park et al. (2004), Tolabi et al. (2019), and Aydogdu et al. (2010) [16, 19, 25, 26, 32, 35, 61]. In addition, Brągoszewska et al. (2016) and Lee et al. (2012) demonstrated that the main source of bacterial aerosols in kindergarten in Gliwice (Poland) and bacterial in public restrooms in Korea were humans [62, 63]. Harbizadeh et al. (2019) reported that the highest and lowest I/O bacteria were measured in the spring at residential areas with values of 2.51 and 1.89, respectively, which is consistent with our results. In addition, the same study based on I/O bacteria (1.89 - 2.51) declared that environmental and human sources (children) are mainly responsible for distribution of airborne bacteria in the kindergartens [64]. As mentioned in Table S1, floor covering in the

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six of twelve kindergarten was covered by carpets; hence, other sources of bacterial aerosol in kindergarten were bacteria associated with dust on indoor surfaces such as carpets and furnishings that can be resuspended into the indoor air through activities such as walking or cleaning (due to 12% of 12 kindergartens in different months had I/O ratio of airborne bacteria lower than one (I/O

bacteria

< 1)). Deng et al. (2016) reported that when the dust concentration at

three kindergartens in Hong Kong (China) increased, the concentration of bacterial aerosols went up too [1]. In addition, a positive correlation was observed between the concentrations of urban outdoor bacterial aerosol and the concentration of fine particulate matter (PM2.5) in Xi’an (Northwest China) (p < 0.05 and r = 0.402) [24]. A method to decrease bacterial aerosol levels in indoor air of kindergartens is use of artificial ventilation and air conditioning systems and application of a disinfectant such as ClO2 spray, which is common for schools, hospitals, and office buildings [65].

3.1.2. Indoor and Outdoor Fungi Table S3 summarizes the mean (± SD), minimum, and maximum concentration of indoor and outdoor fungi at the different sampling sites. The total mean (± SD) concentration of fungal for indoor and outdoor ranged from 7 ± 6 and 12 ± 8 CFU/plate/hr, respectively. The lowest and highest fungal concentration across the 12 kindergartens were 1 and 31 CFU/plate/hr for indoor air, respectively, and 2 and 39 CFU/plate/hr for outdoor air. In this work during four months (August, September, October and November) for 12 kindergartens, the ratio of I/O fungi ranged from 0.1 to 4.5, suggestive of conditions conducive for indoor fungal growth (due to 86% of 12 kindergartens in different months had I/O ratio of airborne fungi lower than one (I/O bacteria < 1)), consistent with the findings by Canha et al. (2015) for urban and rural primary schools in Lisbon

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(Portugal) (0.7) [18]. Similarity, Kim et al. (2007) reported the ratio of I/O fungi for hospital (0.56), childcare center (0.72), elderly welfare facility (0.33), and maternity recuperation center (0.66) in Kyunggi-do province (Korea) was below one, further suggestive of favorable conditions for indoor fungal growth [38]. Additionally , humidity, insufficient disinfection, dust (particulate matter), cracks in the ceiling and surfaces, wallboard, wallpaper, and natural ventilation are generally the main sources of fungal bioaerosols in indoor air in our study region, which is consistent with the findings of former works [16, 22, 24, 25, 66-71]. As mentioned in Table S1, floor covering in the six of twelve kindergarten was covered by carpets; hence, other sources of fungal aerosols in kindergartens were fungi associated with dust on indoor surfaces such as carpets and furnishings and from indoor plants that can be resuspended into the indoor air through such activities as walking or cleaning. According to the finding of this work, I/O ratio of airborne bacteria and fungi were higher than one (I/O bacteria >1) and lower than one (I/O fungi <1), respectively. Thus, it can be concluded that the indoor airborne bacteria and fungi could be contaminated with outdoor airborne bacteria by natural ventilation and inside generative sources, respectively. It should be noted that these findings were related to summer and autumn and if other researchers investigate bioaerosols (bacterial and fungal) in this study areas at other seasons such as spring and winter, different results may be observed. For example, Reponen et al. (1989) reported that the outdoor levels of airborne bacteria and fungi in Finnish homes were the highest in spring and autumn rather than in the extreme weather conditions like summer and winter [72]. 3.2. Comparison of Bioaerosol Concentrations with Proposed Guidelines With regards to the health risks of the exposure to bioaerosols (bacteria and fungal ), standard values of bioaerosol concentrations in kindergartens have not yet been established in Iran [16, 11

20, 49, 51]. For reasons noted earlier (e.g., children breathing more than adults, long duration at kindergarten), measuring the concentrations of bioaerosols in the kindergartens and comparison of bioaerosol concentrations with recommended guidelines are essential for enhancing the public health of children. Pasquarella et al. (2000) has recommended guidelines (maximum levels of the index of microbial air contamination (MAL of IMA)) for the concentration of bioaerosols in environments at risk such as kindergartens [47]. If the concentration of bioaerosols is either less than 9 CFU/(plate)/hr, between 10 to 39 CFU/(plate)/hr, between 40 to 84 CFU/(plate)/hr, between 85 and 124 CFU/(plate)/hr, or exceeds 125 CFU/(plate)/hr, it qualifies as very low, low, medium, high, and very high risk, respectively [47]. Hence, the concentration of indoor bioaerosols was higher than the recommended value (40 to 84 CFU/plate/hr) in 33% of kindergartens, indicative of medium risk. The concentration of outdoor bioaerosols exceeded the recommended value (40 to 84 CFU/plate/hr) in 8% outdoor air of kindergartens, indicative of medium risk. Generally, indoor air quality of kindergartens is at a medium risk level and strategies should be considered to reduce bioaerosol emissions in these susceptible places. Useful strategies include designing proper ventilation, having air conditioning systems, minimal furniture and textile materials, and applying disinfectants. 3.3. Interrelationships between Bioaerosol Concentrations and Environmental Factors 3.3.1. Bacterial Interrelationships Table S4 shows Spearman's correlations between indoor and outdoor bacteria concentrations based on average concentrations (CFU/plate/hr). Relationships between bacteria concentrations, population (N) and environmental air parameters (relative humidity (%) and temperature (°C)) 12

are shown too. The results of Spearman's correlation analysis demonstrate that the number of children in kindergartens (population) had a significant positive correlation with the concentration of indoor bacterial bioaerosols (p < 0.05 and r = 0 .741), which is consistent with the findings of previous works in public buildings of Kyunggi-do (Korea) and in some educational settings in Warsaw (Poland), and in Danish classrooms [38, 73, 74]. The relationships between bacterial concentrations and population (N) can be linked to childrens’ behavior such as high activity in kindergartens. In addition, Bartlett et al. (2004), Madureira et al. (2015), and Nygaard et al. (2018 a and b) stated that the number of children, occupancy patterns, and activity levels in 39 elementary schools in British Columbia (Canada) had the highest effect on the indoor air concentrations of bacterial bioaerosols [11, 75-77], which is consistent with our results. Relative humidity (p = 0.66 and r = 0.05) and temperature (p = 0.23 and r = 0.12) did not exhibit a significant relationship with indoor bacterial bioaerosols. Similarly, relative humidity (p = 0.68 and r = 0.04) and temperature (p = 0.79 and r = 0.027) had no significant relationship with outdoor bacterial bioaerosols. For comparison, Qi et al. (2007) reported that relative humidity (p < 0.01 and r = 0.986) had a significant relationship with bacterial bioaerosols [22]. In addition, a negative correlation was observed between the concentrations of urban outdoor bacterial aerosol and temperature in Xi’an (Northwest China) (p < 0.01 and r = -0.403) [24]. The mean concentrations of bacterial aerosols indoors were higher than those outdoors, but no significant difference was found (P = 0.17 and r = 0.139), which is consistent with the findings of the previous work by Madureira et al. (2015) and Paciência et al. (2016) [76, 78]. 3.3.2. Fungi Interrelationships

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Table S4 shows Spearman's correlations between indoor and outdoor fungi concentrations based on average concentrations (CFU/plate/hr). Relationships between fungi concentrations, population (N), and environmental air parameters (relative humidity (%) and temperature (°C)) are shown too. Relative humidity did not exhibit a significant correlation with the concentration of outdoor fungi bioaerosols (p = 0.58 and r = 0.06), which is opposite of with the findings of previous works on the roof of a research building at the Chinese Academy of Sciences, during non-haze and haze days in Beijing (North China) and in different indoor environments in Ljubljana (Slovenia) [50, 79, 80]. Temperature had a significant relationship with outdoor fungi bioaerosols (p = 0.047 and r = 0.75), which is consistent with the findings of Zhong et al. (2016) in the outdoor environment of the Qingdao region (China), Du et al. (2018) in bacteria-PM2.5 in Beijing (China), Genitsaris et al. (2017) in an urban Mediterranean area in Thessaloniki (Greece), and in outdoor air of several child daycare centers in Edirne (Turkey) [12, 21, 81, 82]. Relative humidity (p = 0.44 and r = 0.08) exhibited no significant relationship with indoor fungi bioaerosols, whereas temperature (p = 0.049 and r = -0.2) and population (p = 0.047 and r = 0.19) did have a significant relationship with indoor fungi bioaerosols. In addition, temperature (p = 0.047 and r = 0.75) exhibited a significant relationship with outdoor fungi bioaerosols. In contrast to bacterial levels, a significant difference existed between fungi bioaerosol levels indoors versus outdoors (P < 0.05 and r = 0.407). 3.4. Bioaerosol Concentrations on Sunny and Cloudy/ Rainy Days 3.4.1. Bacteria 14

Fig. S2 displays the concentration of indoor and outdoor bacterial aerosols based on CFU/plate/hr on sunny and cloudy/rainy days during summer (from 1 August to 19 September) and autumn (from 1 October to 17 November) at the 12 kindergartens. The mean concentration of indoor and outdoor bacterial bioaerosols (CFU/plate/hr) on sunny days exceeded concentrations on cloudy/rainy days due to less sensitivity to wet scavenging [83, 84], which is in line with the findings of the past work [23]. Qi et al. (2018) showed that outdoor bacteria aerosols on sunny and foggy/cloudy days in Qingdao (China) were higher due to fog/cloud events, which is in line with the findings of the present study [22]. For comparison, outdoor bacterial aerosols on hazy, foggy, and rainy days in Qingdao (China) exceeded levels on sunny days in winter, autumn, and spring [21]. In addition, Aydogdu et al. (2010) described that the rainfall days can decrease concentrations of bacteria aerosols in child daycare centers in Edirne (Turkey), which is in line with the findings of the this study [32]. The main reason for these differences can be originated from variations in meteorological condition in the different seasons of study area [21-23]. In addition, Jones et al. (2004) reported that meteorological conditions can have a variety of effects on the biological aerosol concentrations [85]. Hence, in this work, different meteorological/environmental conditions could affect the concentrations of bacteria aerosols. The results showed that a statistically insignificant difference was observed between the mean concentration of indoor bacterial aerosol on sunny and cloudy/rainy days (p = 0.388), while Aydogdu et al. (2010) reported that positive correlations (p < 0.05) were observed between the monthly average amount of sunlight on sunny days and culturable bacteria indoor air of child daycare centers in Edirne (Turkey) [32]. 3.4.2. Fungi

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Fig. S2 also repeats the same information from section 3.4.1 for fungi aerosols. Accordingly, the mean concentration of indoor and outdoor fungi aerosols (CFU/plate/hr) on cloudy/rainy days exceeded mean concentration of indoor and outdoor fungi aerosols on sunny days. The reasons for a higher fungal concentration in this study area on cloudy/rainy days compared to sunny days could be explained with the higher temperature and precipitation in summer and autumn [12, 86]. Additionally, this study area has abundant dead plant material that facilitate production of fungi aerosols, which is consistent with the results of the previous studies by Aydogdu et al. (2008) in child day-care centers in Edirne (Turkey) and Sakiyan et al. (2003) in outdoor air of Ankara (Turkey) [12, 86]. In addition, the findings of this work showed that the concentrations of indoor and outdoor fungi aerosols were highest during the period of most vegetation growth in summer, which is in line with the findings of the past work [12, 87]. Furthermore, Jones et al. (2004) declared that environmental conditions can have a variety of effects on the biological aerosol concentrations [85]. Hence, in this study, various environmental conditions could affect fungi concentrations. The results showed that a statistically insignificant difference was observed between the mean concentration of indoor fungi aerosol on sunny and cloudy/rainy days (p = 0.388). For comparison, Topbaş et al. (2006) declared that cloudy/rainy days in autumn had significant effects on the concentration of urban outdoor fungi aerosol in Trabzon (Turkey) [88]. Similarly, Li et al., (2017) reported that sunny, rainy, and cloudy days in four seasons had no significant effects on the concentration of urban outdoor fungi aerosols in Xi’an (Northwest China) (p>0.05) [24]. 3.5. Temporal Variations of Bioaerosols 3.5.1. Bacteria 16

The results of Huynh-Feldt test for examining the within-subjects variables (different months (August, September, October and November)) on the concentrations of bacteria showed that different months have a significant effect on the concentrations of bacteria aerosols (p < 0.05). The results also showed that although the concentrations of bacteria in indoor and outdoor air (i.e. between-subjects variables) were significantly different (p value <0.05), differences between air situation (the concentrations of fungi in indoor and outdoor air) and time (different months (August, September, October and November)) were not significant. In other words, the interaction between locations and different months was not significant at the 0.05 level . This may be because in some months indoor air showed more pollution and in other months outdoor air displayed more pollution. For example, in case of Fereshteh Kochooloo, the concentrations of indoor bacteria aerosols in August were higher than in outdoor air, while this trend was reversed in September, for which the concentrations of outdoor bacteria aerosols were higher than indoors. Again in October, the concentrations of indoor bacteria aerosols were higher than in outdoor air. However, the results showed that there was a significant difference between the concentrations of bacteria in indoor and outdoor air (i.e. between-subjects variables) as well as between the concentrations of bacteria in indoor air of different kindergartens (i.e. between subjects factors) (p < 0.05). Profile plots of bacteria in indoor and outdoor air and twelve kindergartens based on CFU/plate/hr in different months (August, September, October and November) is shown Fig. 1. Fig. 1a show the mean concentrations of indoor (blue line) and outdoor bacteria (red line) for twelve kindergartens during four months (August, September, October and November). Based on Fig. 1a, concentrations of bacteria in the indoor air of kindergartens exceeded those in outdoor air and it increased with the proportion of 0.2-13.6 compared to outdoor air (Table S3 and Fig. 17

1). Additionally, the highest concentrations of outdoor bacteria aerosols were observed in August and September, while the lowest concentrations of outdoor bacteria aerosols were observed in October and November. The concentration of outdoor bacteria is likely higher in August and September than October and November due to increased temperatures (Table S3), which promotes growth of bacteria [21, 89]. For comparison, Goudarzi et al. (2018) reported that the lowest and highest bacteria concentrations in kindergartens in Ahvaz (Iran) were detected in November and February, respectively [40]. Furthermore, the maximum concentrations of indoor bacteria aerosols were observed in September and November, while the lowest concentrations of indoor bacteria were measured in August and October. The high density of children in kindergartens (population) in September and November may be responsible for the number of bacteria aerosols in indoor air, which is consistent with the findings of Faridi et al. (2015) in the retirement home and the school dormitory in Tehran (Iran), findings of Kim et al. (2007) in the public buildings in Kyunggi-do province (Korea), and results from Canha et al. (2016) in nursery and elementary schools in France [19, 38, 48]. Another reason why the number of bacteria aerosols in indoor air of kindergartens in September and November was higher than other months may be explained by opening of windows in September due to this month having warm weather in Rasht city (Iran), promoting natural ventilation. This leads to more dust on indoor surfaces such as carpets and furnishings that can be resuspended into the indoor air through such activities as walking or cleaning in November. The results of Fig. 1b showed that the concentrations of bacteria in various kindergartens was different and each kindergarten had its own trend. In addition, the concentrations of bacteria in some of those kindergartens such as Maho Setareh, Atre Zendegi, Orkideh, and Samereh were 18

highest in September, which could be attributed to the different factors such as environmental air parameters, especially relative humidity and natural ventilation.

3.5.2. Fungi The results of Huynh-Feldt test for examining the within-subjects variables (different months (August, September, October and November)) on the concentrations of fungi showed that different months have a significant effect on the concentrations of fungi aerosols (p < 0.05). The results also showed that although the concentrations of fungi in indoor and outdoor air (i.e. between-subjects variables) was significantly different (p value <0.05), their concentrations have changed similarly throughout the different months. The difference between the air situation (the concentrations of fungi in indoor and outdoor air)

and time (different months (August,

September, October and November)) was not significant. In other words, the interaction between locations and different months was not significant at the 0.05 level . This may be due to the fact that in some months indoor air showed more pollution and in other months outdoor air is more polluted and this can be related to variations in environmental air parameters, cold and hot weather, population density, and natural ventilation. For example, in case of Fereshteh Kochooloo, the concentrations of outdoor fungi aerosols in August and September were higher than in indoor air, while this trend was reversed in October, which the concentrations of indoor fungi aerosols being higher than outdoor, which could be attributed to that the weather in Rasht city (Iran) in October was relatively cold and children like to play in indoor air and the lack of proper ventilation. In addition, the results showed that there were a significant difference between the concentrations of fungi among different kindergartens (i.e. between subjects factors) (p < 0.05) and this difference occurred between different times at different kindergartens. In 19

other words, the interaction between locations and different months was significant at the 0.05 level. Profile plots of fungi in indoor and outdoor air and twelve kindergartens based on CFU/plate/hr in different months (August, September, October and November) are shown in Fig. 1. Fig. 1c show the mean concentrations of indoor (blue line) and outdoor fungi (red line) for twelve kindergartens during four months (August, September, October and November). According to Fig. 1c, the minimum concentrations of both indoor and outdoor fungi aerosols were observed in August, after which their concentrations increased and reached a maximum in September and October. Finally, the concentrations of both indoor and outdoor fungi aerosols were decreased in November. The reason for these variations in bioaerosol concentrations can be explained by different weather conditions in the study area. It should be noted that although the concentrations of fungi dropped in November, their concentrations were still higher than in August. Additionally, based on Fig. 1c clearly showed that the concentrations of fungi in the outdoor exceeded that of indoor air of kindergartens and increased with the proportion of 0.1- 4.5 compared to indoor air (Table S3 and Fig. 1). As can be seen in Fig. 1d, the difference between different kindergartens was noticeable. Each kindergarten had own trend, which can be influenced by different factors like environmental air parameters, and population density. Additionally, according to Fig. 1d, the concentrations of fungi at Fereshteh Kochooloo and Negin kindergartens during October were higher than other kindergartens, which could be attributed to the high green space coverage at near those kindergartens and environmental air parameters, especially high relative humidity (Table S3). However, the concentrations of fungi at the rest of kindergartens was higher in September. 3.6. Frequency of Occurrence of Bioaerosols 20

3.6.1. Bacteria The frequency of occurrence of indoor and outdoor bacteria species measured in 12 kindergartens in Rasht (Iran) is presented in Table 1. The predominant genera of airborne bacteria isolated in indoor air of 12 kindergartens in Rasht were Bacillus spp. (32.6%), Staphylococcus aureus (29.9%) and Micrococcus spp. (17.3%). Similarly, Goudarzi et al. (2018) showed that the main measured bacterial species in indoor air of kindergartens in Ahvaz (Iran) were Staphylococcus spp. and Bacillus spp. [40]. In addition to Bacillus spp., Staphylococcus spp. is useful as indoor air pollution intensity index in kindergartens [11, 32, 90]. These species have yellow pigments, which protects the airborne bacteria from the negative effects of ultraviolet radiation [32, 91, 92]. Based on Table 1, the predominant genera of airborne bacteria identified in outdoor air of 12 kindergartens in Rasht were Bacillus spp. (26.7%), Staphylococcus aureus (25.3%) and Micrococcus spp. (23.2%). For comparison, Goudarzi et al. (2014) reported that the predominant genera of the airborne bacteria isolated from the ambient air of Ahvaz, Iran were Bacillus spp. (24.2%), Streptomyces spp. (18%), Kocuria spp. (14.3%), Corynebacterium spp. (9.5%), and Paenibacillus spp. (2.55%) [71]. Moreover, the dominant bacterial species isolated from indoor air of child daycare centers in Edirne (Turkey) were Staphylococcus spp. (39.16%), Bacillus spp. (18.46%), Corynebacterium spp. (16.25%), and Micrococcus spp. (7.21%) [32], while the dominant bacterial species identified for outdoor air in Edirne (Turkey) were Bacillus, Corynebacterium, and Staphylococcus [32]. According to Bartlett et al. (2004), the dominant bacterial species in the outdoor air of school classrooms in Canada were Bacillus spp., Corynebacterium spp., and Micrococcus spp. [75].

21

Finally, our findings showed that the dominant bacterial species in indoor and outdoor air of twelve kindergartens were Bacillus spp., which is consistent with the findings of previous studies [32, 40, 71, 75, 93]. As Bacillus spp. are resistant to unfavorable conditions, these species can be found in water, dust, and even soil [32, 94, 95]. The difference between results in various studies can most likely be explained by differences in the sampling site, meteorological conditions, and geographic location (Edirne (Turkey), Rasht (Iran), and Ahvaz (Iran), and Canada), duration of incubation, and seasonal factors [32, 40, 71, 75, 91-95]. 3.6.2. Fungi The frequency of occurrence of indoor and outdoor fungal species measured in 12 kindergartens in Rasht (Iran) is presented in Table 1. As can be seen in Table 1, the predominant genera of airborne fungi isolated in indoor air of 12 kindergartens in Rasht were Aspergillus (14.3 16.0%), Cladosporium spp. (10.9%) and Penicillium spp. (8.6%). Similar results were also reported by Aydogdu et al. (2008) that the predominant fungal species in child daycare centers in Edirne (Turkey) were Cladosporium spp., Penicillium spp., Alternaria spp., and Aspergillus spp. [12]. In addition, Önoğlu et al. (2011) reported that the most common fungal species in indoor air of

kindergartens in Fatih District of Istanbul (Turkey) were Aspergillus spp.

and Cladosporium spp. [35]. In addition, besed on Table 1, the predominant genera of airborne fungi identified in outdoor air of 12 kindergartens in Rasht were Aspergillus flavus (18.7%), Penicillium spp. (18.6%) and Cladosporium spp. (16.6%). Similarly, Aydogdu et al. (2008) reported that the dominant fungal species isolated from the indoor air of child daycare centers in Edirne (Turkey) were Cladosporium spp., Penicillium spp., Alternaria spp., and Aspergillus spp. [12]. In addition, the 22

predominant genera of fungi isolated in indoor air of five daycare centers and five elementary schools in Seoul (Korea) were Cephalotrichum spp., Alternaria spp., Penicillium spp., and Aspergillus spp. [3]. In addition, Aspergillus spp. (31.3%), Cladosporium spp. (22.1%), Penicillium spp. (13.8%), and Alternaria spp. (12.2%) were measured to be the most common fungi species in the ambient air of Tehran (Iran) [96]. For comparison, with regard to seasons, Topbaş et al. (2006) found that the main fungal species measured from the outdoor air of Trabzon (Turkey) in summer versus autumn were Penicillium spp., Alternaria spp. and Fusarium spp. versus Penicillium spp. and Alternaria spp. [88]. Differences exist between studies in the results of this section for the same reasons provided in Section 3.6.1. 4. Conclusions This work reports on the nature of fungi and bacteria species in 12 kindergartens in different regions of Rasht for various seasons. The findings of this work showed that 33% and 8% of the concentration of indoor and outdoor bioaerosol (bacteria and fungi), respectively, were higher than the recommended value (40 to 84 CFU/plate/hr), indicating medium risk. In addition, the results of Huynh-Feldt test showed that although the concentrations of bioaerosols (fungi and bacteria) in indoor and outdoor air (i.e. between-subjects variables) were significantly different (p value <0.05), while the contrast between air situation (the concentrations of fungi in indoor and outdoor air) and time (different months (August, September, October and November)) was not significant. The main bacteria species detected in indoor and outdoor air in kindergartens were Bacillus spp., Staphylococcus aureus, Micrococcus spp., Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus spp., and Streptococcus spp. The predominant genera of the airborne fungi isolated from indoor and outdoor air in kindergartens were

23

Aspergillus terreus, Aspergillus flavus, Aspergillus niger, Cladosporium spp., Penicillium spp., Rhodotorula spp., Ulocladium spp., and Alternaria spp. Based on the results of this work, indoor air quality of kindergartens qualifies as being at a medium risk level, and strategies should be considered to remove bioaerosol emissions in these susceptible places. Methods include designing proper ventilation, having air conditioning systems, minimal furniture and textile materials, and using disinfectants. Acknowledgments This research was part of Msc degree thesis in environmental health engineering Tehran University of Medical sciences. The authors sincerely acknowledges Tehran University of Medical Sciences for their superb academic support. Conflict of Interest: The authors declare that they have no conflict of interest. References [1] W. Deng, Y. Chai, H. Lin, W.W. So, K. Ho, A. Tsui, R. Wong, Distribution of bacteria in inhalable particles and its implications for health risks in kindergarten children in Hong Kong, Atmospheric Environment 128 (2016) 268-275. [2] E. Hoseinzadeh, P. Taha, A. Sepahvand, S. Sousa, Indoor air fungus bioaerosols and comfort index in day care child centers, Toxin Reviews 36(2) (2017) 125-131. [3] S.-K. Shin, J. Kim, S.-m. Ha, H.-S. Oh, J. Chun, J. Sohn, H. Yi, Metagenomic insights into the bioaerosols in the indoor and outdoor environments of childcare facilities, PLoS One 10(5) (2015) e0126960. [4] S.S. Hamzavi, A. Amanati, P. Badiee, M.R. Kadivar, H. Jafarian, F. Ghasemi, S. Haghpanah, M. Dehghani, A.N. Baghani, Changing face of Candida colonization pattern in pediatric patients with hematological malignancy during repeated hospitalizations, results of a prospective observational study (2016–2017) in shiraz, Iran, BMC Infectious Diseases 19(1) (2019) 1-9. [5] A.J. Prussin II, A. Vikram, K.J. Bibby, L.C. Marr, Seasonal dynamics of the airborne bacterial community and selected viruses in a children’s daycare center, PloS one 11(3) (2016) e0151004. [6] R. Crameri, M. Garbani, C. Rhyner, C. Huitema, Fungi: the neglected allergenic sources, Allergy 69(2) (2014) 176-185. [7] W.A. Suk, K. Murray, M.D. Avakian, Environmental hazards to children’s health in the modern world, Mutation Research/Reviews in Mutation Research 544(2) (2003) 235-242. [8] M. Neri, D. Ugolini, S. Bonassi, A. Fucic, N. Holland, L.E. Knudsen, R.J. Šrám, M. Ceppi, V. Bocchini, D.F. Merlo, Children's exposure to environmental pollutants and biomarkers of genetic damage: II. 24

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29

Table 1. Frequency of occurrence of bacteria and fungal species measured in 12 kindergartens in Rasht (Iran) based on CFU/plate/hr.

Bacteria aerosols Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Micrococcus spp. Streptococcus spp. Enterococcus spp. Bacillus spp. Mean total

Fungi aerosols Aspergillus flavus Aspergillus niger Aspergillus terreus Mucor Alternaria spp. Cladosporium spp. Rhodotorula spp. Penicillium spp. Ulocladium spp. Scopulariopsis spp. Rhizopus spp. Aureobasidium spp. Fusarium spp. Mean total

Frequency of occurrence of bacteria species Indoor Outdoor MaxMean±SD (frequency %) Mean±SD (frequency %) min 14±11 (29.9%) 1-68 7±5 (25.3%)

Max-min 1-30

10±9 (12.4%)

1-43

4±2 (7.1%)

1-19

7±6 (6.2%)

1-34

5±3 (9.8%)

23-1

11±8 (17.3%) 1-35 6±5 (23.2%) 4±3 (0.2%) 1-6 2±1 (0.3%) 5±2 (1.4%) 1-8 5±3 (7.5%) 14±12 (32.6%) 1-63 8±5 (26.7%) 42±27 (100%) 1-143 24±16 (100%) Frequency of occurrence of fungal species Indoor Outdoor MaxMean±SD (frequency %) Mean±SD (frequency %) min 3±2 (15%) 1-10 5±4 (18.7%) 3±2 (14.3%) 1-12 3±2 (9.9%) 5±2 (16.0%) 1-12 3±1 (13.3%) 2±1 (2.3%) 1-4 3±2 (1.8%) 3±2 (4.7%) 1-10 2±1 (3.7%) 3±2 (10.9%) 1-8 5±3 (16.6%) 3±2 (5.4%) 1-10 4±3 (8.2%) 3±1 (8.6%) 1-8 5±4 (18.6%) 2±1 (6.7%) 1-7 2±1 (1.4%) 4±2 (5.7%) 1-16 2±1 (1.0%) 2±1 (2.1%) 1-3 2±1 (2.1%) 2±1 (4.1%) 1-4 3±2 (2.9%) 2±1 (4.2%) 1-8 2±1 (2.0%) 7±5 (100%) 1-31 12±8 (100%)

25-1 1-4 1-28 1-26 4-85

Max-min 1-26 1-8 1-14 1-14 1-8 1-34 1-15 1-17 1-4 1-3 1-4 1-7 1-4 2-39

Fig. 1. Monthly profiles of bacteria in indoor and outdoor air (CFU/plate/hr) (a), bacteria in twelve kindergartens (CFU/plate/hr) (b), fungi in indoor and outdoor air (CFU/plate/hr) (c), and fungi in twelve kindergartens (CFU/plate/hr) (d).

Highlights •

Bacteria and fungi were measured in kindergartens for different regions/seasons.

•

Mean concentration of fungi/bacteria in indoor air was 7 ± 6/42 ± 29 CFU/plate.

•

Mean concentration of fungi/bacteria in outdoor air was 12 ± 8/24 ± 18 CFU/plate.

•

The I/O bacteria and fungi ratios were 0.5 -13.6 and 0.1- 4.5, respectively.

•

The main bacteria and fungi species were Bacillus spp. and Aspergillus spp., respectively.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: