An Exploratory Study of Airborne Fungal Contamination and Its Association with Microclimate Conditions as Regards Sustainable Zoo Development

Abstract

Air quality management in zoological gardens plays a crucial role in their sustainable development. However, air quality in these settings remains understudied. In addition, previous research has largely focused on airborne microbial contamination merely in animal enclosures. This exploratory study provides preliminary insights into airborne fungal contamination alongside microclimate conditions in the visitor and worker areas of animal premises in the Zagreb Zoo. The study was performed in the Monkey House, Tropical House, Rainy Africa, and Bird House, as well as outdoors in fall. Fungi were identified based on macroscopic and microscopic examinations. Total culturable fungal concentration in indoor air ranged between 50 and 4.25 × 10³ CFU/m³, and in outdoor air between 1.00 × 10² and 1.50 × 10³ CFU/m³. Molds of eight genera and yeasts were isolated from the air. Both indoors and outdoors, the predominant genera were Cladosporium and Penicillium, and also genus Aspergillus indoors. Cladosporium spp. and Penicillium spp. concentrations, as well as total fungal concentration in the air, were on average, highest in Rainy Africa and Bird House, while the highest average Aspergillus spp. concentration was found in the Tropical House. Levels of Cladosporium spp., Penicillium spp., and Aspergillus spp. concentrations were associated with microclimate conditions. Study results suggest that the airborne fungal contamination may depend on the animals housed in the premises, and the design and management of the premises. Although total fungal concentration determined may not necessarily pose a health risk for exposed people, the qualitative composition of fungi signifies the importance of implementing good practices in zoo premises, including optimal microclimate conditions and effective ventilation. The results obtained also indicate the need for air quality monitoring, which concurs with zoo sustainability goals.

1. Introduction

Bioaerosols are suspensions of solid or liquid particles of biological origin in the air, including microorganisms and their components and byproducts, such as endotoxins and mycotoxins [1,2]. It is well established that the industrial animal production facilities are a major source of bioaerosols, which pose a substantial health hazard for animals and workers, as well as the environment [3,4,5]. Moreover, it is estimated that about 60% of common human infectious diseases and 75% of emerging ones originate from domestic or wild animals [6,7]. However, as compared to the animal production facilities, the microbial air quality in zoological gardens has been poorly investigated, even though these places may exhibit thousands of animals and hundreds of species, predisposing health risks from bioaerosols. In addition, zoos are not only animal and working environments but also public spaces characterized by high occupancy density. Thus, good air quality is essential for ensuring healthy and sustainable zoo environments [8], underscoring the critical role of the One Health strategy in managing health risks [9,10].

Considering the fungal air quality in zoos, the study by Grzyb and Lenart-Boroń [11], which was conducted in eight animal enclosures of the Kraków Zoo in all seasons except for summer, showed that the total fungal concentration ranged from 8.40 × 10² to 2.84 × 10⁴ CFU/m³. The concentration significantly differed among particular enclosures, with the highest value recorded in fall. Total fungal concentration in the enclosures did not exceed the recommended permissible value for occupational exposure in Poland, i.e., 5.00 × 10⁴ CFU/m³ [12]; yet, the level of respirable fungal aerosol fraction in the enclosures was quite high, ranging from 62.6 to 89.2%. The study indicated that animals and their enclosures should be thoroughly cleaned, along with the sustaining of favorable microclimate conditions in the enclosures.

In the study performed in the Wroclaw Zoo in 20 animal enclosures over all seasons by Plewa-Tutaj et al. [13], the total fungal concentration in the air varied between 50 and 3.65 × 10⁴ CFU/m³. Consistent with the findings of Grzyb and Lenart-Boroń [11], the total fungal concentration in the enclosures was below the recommended permissible limit for occupational exposure [12], reaching the highest level in fall and lowest in winter. These data also align with the results reported by Rivas et al. [14], who assessed airborne fungal contamination of a penguin exhibit in the Maryland Zoo throughout the year. In their study, Álvarez-Pérez et al. [15] analyzed airborne fungal load in 23 indoor and outdoor locations of the Zoo–Aquarium of Madrid in fall and spring. A significantly lower total fungal concentration was found in fall, with no significant difference in the concentration between indoor and outdoor locations.

Plewa-Tutaj et al. [13] also investigated the qualitative composition of fungi in animal enclosures of a zoo. Air sampling yielded 112 fungal strains representing 50 species within 10 genera. The genus Penicillium prevailed in the air, comprising 58.9% of all strains, followed by the genus Aspergillus (25.9%). Since many detected fungi may endanger animal and human health, the authors concluded with the need for air quality monitoring in zoo premises. A similar conclusion was drawn by Omar et al. [16]. In their study conducted in the National Zoo, Malaysia, Penicillium spp. were also the most abundant airborne fungi in animal environments, with a prevalence of 83.7%.

In comparison with bacterial and viral diseases, fungal diseases are rarely observed in healthy and immunocompetent animals and humans, but when these diseases do occur, they can be devastating for the host [17,18]. In this regard, fungal contamination and infections have been implicated in cases of morbidity and mortality in zoo animals [19,20]. Accordingly, knowledge of airborne fungal load in zoo premises and understanding the contributing factors are necessary to reduce these pollutants, and to ultimately support the implementation of air quality standards in these places. Thus, except for zoo managers, this may also concern policy decision-makers.

Previous studies conducted in zoos have mostly assessed the fungal air quality in animal enclosures themselves, highlighting the potential health risks from exposure to bioaerosols for animals and zoo workers, who are in direct contact with animals or their enclosures. Therefore, there is limited information on fungal contamination levels in other zoo sections, including visitor areas. Such areas are accessed by the general public, especially vulnerable groups such as children, the elderly, and immunocompromised individuals, usually without protective measures. As such, focusing on visitor areas helps assess the real-world public health risks, and not just the conditions in controlled animal enclosures. Health risks in visitor areas can translate into community-level spread beyond the enclosures.

Following our earlier research on airborne bacterial contamination in the visitor and worker areas of animal premises in the Zagreb Zoo [8], this exploratory study presents preliminary findings on fungal contamination in these areas. The study also investigated fungal contamination of the zoo’s outdoor air, and the association of fungal concentrations with microclimate conditions. The study tested the following hypotheses: (i) quantitative and qualitative composition of airborne fungi will differ among animal premises, including outdoor air, and (ii) microclimate conditions will influence fungal concentrations.

2. Materials and Methods

2.1. Research Framework and Location

The Zoological Garden of Zagreb was used as the setting for the study. Established in 1925, this zoo is situated in Maksimir Park and spans 7 ha. The zoo exhibits around 7000 animals and 400 species from nearly all continents [21]. Annual attendance at the zoo is roughly half a million visitors [22].

The study took six weeks in the first half of fall in 2024. Indoor investigations were carried out in the area frequented by visitors and workers across four animal premises, i.e., Monkey House (MH), Tropical House (TH), Rainy Africa (RA) and Bird House (BH) as part of the Vivarium. These premises were chosen for their convenient accessibility. Location and description of the premises are shown in Figure 1 and

Table 1. Description of the studied animal premises in Zagreb Zoo (reprinted from Ref. [8]).

Parameter	Monkey House	Tropical House	Rainy Africa	Bird House
Year of Construction	1996 renovated	1974	1999	1978
Indoor Area for Animals (m2)	102	350	162	42
Indoor Area for Visitors and Employees (m2)	32	230	20	44
Animals (n)	14	~358 (crocodiles: 3; caimans: 2; other reptiles: 55; amphibians: 10; fish: ~270; birds: 4; mammals: 14)	45 (pygmy hippopotamuses: 3; birds: 2; reptiles: 10; fish: 30)	20
Type of Ventilation	Air conditioning chambers (always on)	Air conditioning chambers (always on)	Air conditioning chambers (always on)	Natural
Type of Bedding in Animal Enclosures	No bedding	Shredded bark + sand	Shredded bark; no bedding (pygmy hippopotamuses)	No bedding
Type of Flooring/Bedding in Area for Visitors and Employees	Ceramic tiles	Concrete + stone	Shredded bark	Concrete

, respectively. Cleaning of the premises always took place between 8 a.m. and 11 a.m., while feeding of the animals, varying by species, occurred five to seven times daily between 8 a.m. and 7 p.m. Outdoor enclosures (MH, BH, and for pygmy hippos in the RA) were occupied by the animals for almost the entire research period. As illustrated in Figure 1, outdoor investigations were carried out approximately 10 m away from one of the studied premises close to the zoo entrance. This site was used as a control, as recommended elsewhere [11].

2.2. Data Collection

Weekly measurements of microclimate parameters and air samplings, both indoors and outdoors, were conducted in five replicates, at the same sites and under identical conditions. Each time, environmental assessments were undertaken between 9.00 a.m. and 12 p.m., following the zoo’s opening and the completion of cleaning and animal feeding activities. The assessments were done 1.5 m from the ground, within the typical inhalation zone for people. In total, 150 air samples were collected and analyzed. During data collection, the premises were free of visitors or workers. All investigations were entirely carried out by the authors holding university degrees, as specified in the Author Contributions section.

Microclimate parameters were measured with handheld digital instruments Testo 625 thermohygrometer (air temperature, °C, and relative humidity, %) and Testo 425 thermal anemometer (airflow rate, m/s). The thermohygrometer had an accuracy of ±0.5 °C for temperature and ±2.5% (5 to 95%) for humidity, whereas the anemometer’s accuracy was ±(0.03 m/s + 5% of measuring value) (Testo SE & Co. KGaA, Lenzkirch, Germany). The values were recorded as instantaneous readings.

To determine the quantitative and qualitative composition of culturable fungi, air sampling was performed using a handheld SAS Super 100^TM instrument (PBI International, Milan, Italy) and Petri plates containing Sabouraud dextrose agar (Biolife, Milan, Italy). A 20 L air volume was obtained per each sample, in line with previous zoo-based studies [23,24]. The instrument operated at an airflow rate of 100 L/min. Prior to each day of sampling, the head of the instrument was sterilized, and between every two measurements, it was disinfected by applying 70% ethanol (T.T.T. d.o.o., Sveta Nedelja, Croatia) with cotton balls. Once sampling was completed, the plates were bagged in sterile containers (Bürkle GmbH, Bad Bellingen, Germany) and delivered to the laboratory in an insulated cooler within an hour. Incubation of the plates and identification of fungi were conducted in the laboratory of the Department of Poultry Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, adhering to good laboratory practice principles. The plates underwent aerobic incubation at 25 °C for 5–7 days. Media sterility was validated by a 3-day incubation of Sabouraud agar plates at 25 °C. For each incubation series, blank plates were used as controls and subjected to the same incubation conditions as sample plates. A digital colony counter (J.P. Selecta, Barcelona, Spain) was employed for quantification of the developed fungal colonies. The counts were subsequently corrected according to the formula below: X = (Pr × 1000)/V, where X represents colony-forming units per m³ of air (CFU/m³), Pr the probable number of fungal colonies determined using positive hole correction (taken from the instrument manufacturer’s table based on the counted colonies), and V the volume of sampled air (L), noting that 1000 L equals 1 m³. Identification of fungi included macroscopic and microscopic examinations. Macroscopic examination was done by observing colony morphology, i.e., size, color, shape, and texture, on the front and back sides of the plate. Microscopic examination was done by a tease mount method using lactophenol cotton blue stain (Sigma-Aldrich, St. Louis, MO, USA) [25]. Fungal isolates were presumptively identified by comparing the phenotypic features with those described in the reference text [26].

All instruments used in the study undergo regular calibration by an authorized service technician.

2.3. Statistical Analysis

Statistica v. 14.1.0.8 reference software (Cloud Software Group, Inc., Palo Alto, CA, USA, 2023, Data Science Workbench, http://tibco.com, accessed on 17 December 2025) was utilized for data analyses. The normality of data distribution was evaluated using Kolmogorov–Smirnov test, followed by application of suitable parametric and non-parametric tests. One-way ANOVA was employed to test for significant differences in the total fungal concentration and microclimate parameter values among particular premises, including outdoor air as a control. Kruskal–Wallis test was employed to test for significant differences in the concentrations of particular fungi among the premises. Statistical significance was assumed at p < 0.05. Multivariate association between microclimate parameters and fungal concentrations was assessed by principal component analysis (PCA). The analysis considered air temperature, relative humidity and airflow rate, and Cladosporium spp., Penicillium spp., and Aspergillus spp. concentrations. After standardizing the variables, PCA was applied using a correlation matrix. Principal components were retained based on eigenvalues greater than 1 and inspection of the scree plot. The first two principal components (PC1 and PC2) were selected for interpretation, as they explained 53.1% of the total variance. Additional components (PC3 and higher) were not interpreted, as they explained a smaller proportion of the variance and did not provide ecologically meaningful patterns.

3. Results

Total fungal concentration in indoor air of the premises and outdoor air is illustrated in Figure 2. On an average, total fungal concentration in indoor air varied between 7.07 × 10² and 1.36 × 10³ CFU/m³ (range 50–4.25 × 10³ CFU/m³), while accounting 7.02 × 10² CFU/m³ in outdoor air (range 1.00 × 10²–1.50 × 10³ CFU/m³). The RA and BH exhibited a significantly higher total fungal concentration relative to the MH and outdoor air (p < 0.001 all), with no significant differences determined among the TH and other premises, including outdoor air. The ratio between indoor and outdoor values (I/O) for total fungal concentration was as follows: 1.0 (MH), 1.5 (TH), and 1.9 (RA and BH).

The diversity of fungi in the indoor air of the premises and outdoor air is presented in

Table 2. Qualitative composition of culturable airborne fungi in indoor visitor and worker areas of the studied animal premises (I) and outdoor area (O) of Zagreb Zoo.

Fungi	Monkey House (I)	Tropical House (I)	Rainy Africa (I)	Bird House (I)	Control (O)
	CFU/m3 Median (Min–Max)
Alternaria spp.	0 (0–1.50 × 102)	0 (0–1.00 × 102)	0 (0–2.00 × 102)	0 (0–1.00 × 102)	0 (0–2.00 × 102)
Aspergillus spp.	50 a (0–3.50 × 102)	1.75 × 102 b (0–1.70 × 103)	50 a (0–2.50 × 102)	50 a (0–3.00 × 102)	0 a (0–1.50 × 102)
A. section Flavi	0 (0–1.00 × 102)	25 (0–2.00 × 102)	0 (0–2.50 × 102)	0 (0–1.00 × 102)	0 (0–1.00 × 102)
A. section Fumigati	0 (0–2.50 × 102)	0 (0–1.50 × 102)	0 (0–2.50 × 102)	0 (0–1.50 × 102)	0 (0–1.50 × 102)
A. section Nigri	0 (0–1.00 × 102)	0 (0–2.00 × 102)	0 (0–1.00 × 102)	0 (0–1.00 × 102)	0 (0–50)
A. section Circumdati	0 (0–2.00 × 102)	50 a (0–1.70 × 103)	0 (0–2.00 × 102)	0 b (0–50)	0 b (0–0)
A. section Terrei	0 (0–50)	0 (0–1.00 × 102)	0 (0–1.50 × 102)	0 (0–50)	0 (0–0)
Cladosporium spp.	3.25 × 102 a (0–1.10 × 103)	4.25 × 102 a (50–1.20 × 103)	5.25 × 102 b (2.50 × 102–2.25 × 103)	7.00 × 102 b (1.50 × 102–2.85 × 103)	4.50 × 102 (0–1.25 × 103)
Diplosporium spp.	0 (0–0)	0 (0–0)	0 (0–0)	0 (0–1.00 × 102)	0 (0–0)
Fusarium spp.	0 (0–0)	0 (0–0)	0 (0–0)	0 (0–0)	0 (0–50)
Mucor spp.	0 (0–50)	0 (0–50)	0 (0–1.50 × 102)	0 (0–1.00 × 102)	0 (0–50)
Penicillium spp.	1.50 × 102 (0–5.00 × 102)	1.75 × 102 (0–1.10 × 103)	2.75 × 102 a (0–8.50 × 102)	2.75 × 102 a (0–7.50 × 102)	1.00 × 102 b (0–2.50 × 103)
Rhizopus spp.	0 (0–50)	0 (0–1.00 × 102)	0 (0–50)	0 (0–50)	0 (0–0)
Yeasts	0 (0–2.50 × 102)	0 (0–1.00 × 102)	0 (0–5.50 × 102)	0 (0–1.50 × 102)	0 (0–2.50 × 102)
Unidentified	0 (0–0)	0 (0–0)	0 (0–1.00 × 102)	0 (0–50)	0 (0–0)

n = 30 air samples per each of the premises. a, b: values within a row carrying different letter labels are significantly different at p < 0.05 according to Kruskal–Wallis test.

. Molds of eight genera along with yeasts were identified in the air. Fusarium spp. were not found indoors, and Rhizopus spp. and Diplosporium spp. outdoors. The most frequently detected molds indoors were Cladosporium spp., Penicillium spp., and Aspergillus spp. An average Cladosporium spp. concentration indoors varied between 3.25 × 10² and 7.00 × 10² CFU/m³ (range 0–2.85 × 10³ CFU/m³), Penicillium spp. concentration between 1.50 × 10² and 2.75 × 10² CFU/m³ (range 0–1.10 × 10³ CFU/m³), and Aspergillus spp. concentration between 50 and 1.75 × 10² CFU/m³ (range 0–1.70 × 10³ CFU/m³). Cladosporium spp. (average concentration 4.50 × 10² CFU/m³, range 0–1.25 × 10³ CFU/m³) and Penicillium spp. (average concentration 1.00 × 10² CFU/m³, range 0–2.50 × 10³ CFU/m³) were the most common molds found outdoors as well. The RA and BH exhibited a significantly higher Cladosporium spp. concentration relative to the MH and TH, and a significantly higher Penicillium spp. concentration than outdoor air (p < 0.05 all). The TH exhibited a significantly higher Aspergillus spp. concentration relative to all other premises, including outdoor air (p < 0.05 all). The genus Aspergillus encompassed five sections, i.e., Flavi, Fumigati, Nigri, Circumdati, and Terrei, with the Aspergilli from the latter two sections not being detected outdoors. The concentration of the Aspergilli from the section Circumdati only differed among the premises, with a significantly higher value found in the TH relative to the BH, as well as outdoor air (p < 0.05 both).

The determined values of microclimate parameters are presented in

Table 3. Microclimate data in indoor visitor and worker areas of the studied animal premises (I) and outdoor area (O) of Zagreb Zoo.

Parameter	Monkey House (I)	Tropical House (I)	Rainy Africa (I)	Bird House (I)	Control (O)
	Mean ± SD (Min–Max)
Air Temperature (°C)	21.89 a ± 1.51 (19.30–24.50)	21.30 a, b ± 1.98 (16.50–24.40)	19.76 b ± 2.11 (16.00–23.30)	22.24 a ± 2.43 (16.10–25.50)	15.82 c ± 4.31 (6.90–20.20)
Relative Humidity (%)	59.01 a ± 8.02 (43.00–67.20)	68.95 b ± 9.36 (53.30–82.80)	71.45 b ± 8.51 (55.90–83.20)	56.79 a ± 5.67 (46.00–64.30)	71.33 b ± 9.48 (51.80–81.40)
Airflow Rate (m/s)	0.08 a ± 0.04 (0.01–0.17)	0.08 a ± 0.05 (0.01–0.18)	0.11 a ± 0.08 (0.01–0.40)	0.06 a ± 0.05 (0.01–0.25)	0.49 b ± 0.22 (0.23–1.16)

n = 30 measurements per parameter and each of the premises. a, b, c: values within a row carrying different letter labels are significantly different at p < 0.05 (air temperature) and p < 0.001 (relative humidity and airflow rate) according to one-way ANOVA.

. Indoor air temperature averaged between 19.76 and 22.24 °C (range 16.00–25.50 °C), relative humidity between 56.79 and 71.45% (range 43.00–83.20%), and airflow rate between 0.06 and 0.11 m/s (range 0.01–0.40 m/s). Outdoor air temperature averaged 15.82 °C (range 6.90–20.20 °C), relative humidity 71.33% (range 51.80–81.40%), and airflow rate 0.49 m/s (range 0.23–1.16 m/s). Air temperature inside all premises was significantly higher (p < 0.05) than outdoor air temperature. Upon comparing the values among the premises, the MH and BH exhibited a significantly higher air temperature than the RA (p < 0.05 for both). Relative humidity in the TH and RA, as well as in the outdoor air, was significantly higher relative to the MH and BH (p < 0.001 all). The airflow rate inside all premises was significantly lower (p < 0.001) than outdoor airflow rate.

As illustrated in Figure 3, the PCA revealed that the PC1, explaining 32.6% of the variance, was primarily associated with microclimate conditions. It showed strong positive loadings for airflow rate (0.85) and relative humidity (0.57), and strong negative loadings for air temperature (−0.84) and Cladosporium spp. concentration (−0.39), indicating a gradient from warm environments with higher Cladosporium spp. concentration to more humid and ventilated zoo environments. The PC2, explaining 20.5% of the variance, was mainly associated with fungal concentrations. Aspergillus spp. concentration (0.74) and Penicillium spp. concentration (0.47) showed strong positive loadings along with relative humidity (0.53), while airflow rate exhibited a negative loading (−0.30). Therefore, the PC1 reflected a broader microclimate gradient, and PC2 pointed to specific fungal responses to environmental conditions.

4. Discussion

In the present study, an exploratory, initial assessment of airborne fungal contamination was performed in the visitor and worker areas of the Zagreb Zoo. Total fungal concentration in the indoor air of the studied premises concurs with the findings from previous research conducted in zoos, although some studies reported levels in animal enclosures as high as 10⁴ CFU/m³ [11,13,14,15,24]. In accordance with Polish guidelines on permissible total airborne fungal concentration in working spaces (5.00 × 10⁴ CFU/m³) and living areas and public spaces (5.00 × 10³ CFU/m³) [12], total fungal concentration in the indoor air did not exceed the recommended values. However, by observing differences among the premises, a significantly higher total fungal concentration was found in RA and BH as compared to the MH. In our previous study performed in the Zagreb Zoo, the highest total bacterial concentrations in the air were also found in RA and BH, suggesting that premises exhibiting larger animals and birds may represent a significant source of bioaerosols [8]. In addition, that study demonstrated that a glass wall protection in MH and TH might be effective in reducing bacterial emissions from animal enclosures into the visitor areas, which can also explain the results obtained in the present study, although total fungal concentration in the TH as compared to the RA and BH was not significantly different.

By investigating airborne fungal contamination in animal enclosures of a zoo, Grzyb and Lenart-Boroń [11] recorded the highest total concentration in the exotarium (which would correspond to our TH) and the lowest in the enclosures for pygmy hippos (which would correspond to RA), i.e., contrary to the results of this study. Several factors may account for these differences. The lower total fungal concentration in the TH in this study could be, along with a larger surface area per animal excluding fish, attributed to the constant operation of the ventilation system. On the other hand, the number of pygmy hippos, the surface area per animal and the ventilation regime in the enclosures were similar in both studies, but, in the present study, the fungal concentration was measured in the visitor area, which has bedding on the floor (shredded bark), and not merely in the hippo enclosure where there is no bedding. In this respect, previous studies conducted in zoos have shown about a three-fold lower airborne bacterial level in the enclosures for monkeys with no bedding [27,28]. The Zagreb MH enclosures likewise lack bedding, which may further explain the lower total fungal concentration found in the air of the MH in this study, as compared to the concentration determined in the enclosures for monkeys by Grzyb and Lenart-Boroń [11]. In the latter study, a high concentration of airborne fungi was also recorded in the enclosures for ostriches, which is in agreement with the results of this study. Although the rooms for birds in the BH where measurements were taken have no bedding, as they did in that study, it is known that the bird housing and activity generate large amounts of dust, which favors the spread of microorganisms through the air [8,29]. The present findings also corroborate the results reported by Plewa-Tutaj et al. [24], who found the highest total airborne fungal concentration in the Kongo Pavilion with crocodiles, manatees, and many species of birds, and the lowest in the MH.

The control site included in this study was based on earlier zoo research by Grzyb and Lenart-Boroń [11]. Considering the potential impact of nearby zoo premises and related activities on aerosol distribution and fungal presence in the air, this site did not serve as a true environmental control. Preferably, a control site should be established outside the zoo, within a representative background environment. As such, the purpose of the control site was not to establish a pristine environmental baseline, but rather to enable relative comparison of fungal concentrations between indoor environments and an outdoor area of the zoo, which is equally used by visitors and workers. Our study showed that the total fungal concentration measured in the zoo outdoor air is consistent with the results presented by Grzyb and Lenart-Boroń [11]. By comparing the total fungal concentration in the air inside the premises and in outdoor air, significantly higher values were found in the RA and BH, while the concentration in the MH and TH was not significantly different as compared to outdoor air. According to Grzyb and Lenart-Boroń [11], the total fungal concentration in the indoor and outdoor air of a zoo did not differ significantly in the case of all enclosures observed either. Indeed, total fungal concentration in a smaller number of enclosures was significantly higher than in outdoor air. Moreover, Álvarez-Pérez et al. [15] demonstrated that the total airborne fungal load was similar across the indoor and outdoor locations of a zoo.

In the present study, the I/O ranged from 1.0 (MH) to 1.9 (RA and BH), while in the previous study performed in the Zagreb Zoo, the I/O for total airborne bacterial concentration ranged from 4.3 (MH) to 16.6 (RA) [8], thus being manifold higher. Accordingly, comparison of the results of this study and the previous one [8] reveals that the total fungal concentration is two-fold higher than the total bacterial concentration in outdoor air. This tendency is also shown by the results of the studies by Grzyb and Lenart-Boroń [11,27], which were conducted in the Kraków Zoo, the comparison of which discloses a three-fold higher concentration of fungi than bacteria in outdoor air. These findings may result from fungi, relative to bacteria, being often more resistant to stresses, using different strategies such as spore formation to survive and disperse in the environment [30,31].

There are no uniform international standards regarding the acceptable level of mi-crobial air pollutants in indoor environments. For instance, the U.S. Occupational Safety and Health Administration states that microbial air concentration above 1.00 × 10³ CFU/m³ may indicate indoor pollution; yet, the qualitative composition of bioaerosols should be determined to identify health risks [32]. In this study, the most abundant fungi both indoors and outdoors were molds belonging to the genera Cladosporium and Penicillium, and the genus Aspergillus indoors as well. The results obtained correspond with those reported in previous studies, although, in these studies, molds of the genus Penicillium predominated in zoo air [13,14,16,24]. On this matter, differences in findings across studies may stem from variations in methodology, such as the sampling season, the air sampling method, the type of air sampler and volume of sampled air, or the method of fungal identification and the culture medium used.

Being ubiquitous in the environment, Cladosporium molds are often found in high concentrations in the air. Although these molds are primarily saprophytes that do not cause infections, they are among the most important fungi inducing allergic reactions, such as allergic rhinitis and asthma [33,34,35]. In the present study, there were significant differences in the Cladosporium spp. concentration among the premises, with higher values being recorded in the RA and BH as compared to the MH and TH.

Infections caused by Penicillium molds are also rare in animals and humans [36]. However, these molds are well-established producers of potent mycotoxins, with adverse health effects [37,38,39]. The study by Plewa-Tutaj et al. [24] showed that the concentration of these molds was among the highest in the Kongo Pavilion and lowest in the MH, which aligns with present findings, despite the concentrations in this study being lower as compared to theirs. Penicillium spp. concentration did not differ significantly among the premises, but the concentration was significantly higher in the RA and BH than in outdoor air.

Aspergillus molds may represent a serious risk to animal and human health based on infection, allergy and toxicity [17,18]. In terms of infection risk in working spaces, Aspergillus spp. are categorized as group 2 biological agents [40]. Aflatoxins, which are mainly produced by the species of this genus, are the most potent mycotoxins that can harm animal and human health [41]. Although mycotoxins in the air are generally not present in the form of aerosol, they may attach to dust particles and fungal spores that facilitate their transport to the respiratory system [42,43], posing a significant but often neglected health risk [44]. The study by Plewa-Tutaj et al. [24] revealed that the Aspergillus molds in most of the zoo enclosures reached a concentration of 10² CFU/m³, which is in agreement with the results of this study. In comparison with the Aspergillus spp. concentration determined in the air of an indoor–outdoor penguin exhibit by Rivas et al. [14], in our study, the concentration inside all premises and in outdoor air reached higher values. According to Plewa-Tutaj et al. [24], Aspergillus species from the Nigri, Flavi and Fumigati sections were highly represented in the air of a zoo, reflecting their adaptability to such an environment. Their findings are consistent with the results of this study, showing the Aspergilli belonging to these sections, as well as sections Circumdati and Terrei to be present in the zoo air. The concentrations of the Aspergilli from almost all sections observed did not differ significantly among the premises or between indoor and outdoor air. The exception was the Aspergillus section Circumdati. The concentration of the Aspergilli from this section was significantly higher in the TH than in the BH, as well as outdoor air, where they were not detected. Apparently, the concentration of the Aspergilli from the section Circumdati contributed to the significantly higher Aspergillus spp. concentration in the TH as compared to other premises, including outdoor air, and eventually to the nonsignificant differences in the total fungal concentration among the TH and other premises, including outdoor air.

In the present study, molds of the genera Alternaria, Fusarium, Mucor, Rhizopus, and Diplosporium, and yeasts were also identified in indoor air of the premises and/or outdoor air, with no significant differences in the concentrations of these fungi among the premises or between indoor and outdoor air. Alternaria spp. and Fusarium spp. are known to cause both allergies and mycotoxicoses, as well as mycoses in animals and humans [18,45]. Fusariosis ranks as the second most frequent mold infection in immunocompromised people, following aspergillosis [46]. Mucor spp. and Rhizopus spp. belong to fungi causing opportunistic infection mucormycosis in humans and animals, etiologic agents of which show high resistance to most common antifungals used in the treatment of systemic mycoses [18]. Reports suggest that Diplosporium spp. may act as causative agents of fungal keratitis in humans [47,48]. Finally, yeasts are also known to pose a health risk, at least as allergens [49,50].

Although air temperature and relative humidity differed significantly among particular premises, with relative humidity in the TH and RA reaching values above 80%, the values of microclimate parameters, including airflow rate, were generally in line with recommendations regarding human comfort and health [51,52,53]. Outdoor air temperature was significantly lower and airflow rate significantly higher as compared to the values recorded indoors. Outdoor relative humidity was significantly higher as compared to the values recorded in the MH and BH. Plewa-Tutaj et al. [24] reported on air temperature to be a critical factor affecting the Aspergilli concentration in the air of zoo enclosures, while the impact of humidity was less pronounced. In this study, the PCA showed the strong positive association of Aspergillus spp. concentration, as well as Penicillium spp. concentration with relative humidity, and negative association with airflow rate. These results support the relevance of humidity in promoting fungal proliferation [11,13], whereas increased airflow rate may reduce their concentrations. However, the distinct pattern of Cladosporium spp., the concentration of which was negatively associated with relative humidity and more closely related to air temperature, may indicate ecological requirements different from those of other fungal genera. Nevertheless, the observed data signify the role of microclimate control in managing fungal air contamination in zoo environments, with further research needed to elucidate the impact of microclimate parameters on specific fungal concentrations.

The following identified limitations may be considered when interpreting the findings of the present study. While aligned with previous research, the study was confined to a single zoo at a unique location, with characteristic management practices and spatial configuration and design of the premises, including animals, which may limit the generalizability of the results. Data were collected in the morning hours. As animal activity patterns can vary significantly across different periods of a day, study results may not reflect behaviors occurring in the afternoon or evening. No visitors or workers were present in the premises at the time of data collection. Fungal concentration may have varied depending on the occupancy density. The results obtained are season-specific, although the study design was based on the fact that the highest airborne fungal concentrations in zoo premises in earlier studies were predominantly recorded in fall. The volume of air sampled for fungal assessments using impactor samplers may range up to 1000 L [54], whereas this study employed a 20 L volume. In environments with potentially high fungal loads, such as animal facilities including zoo enclosures, this approach may offer relevant insights [23,24]. Using larger volumes can lead to overgrowth on culture plates, causing colony merging, compromised quantification, and misidentification. Collecting a smaller volume helps preserve colony morphology, facilitating accurate enumeration and presumptive identification with culture-based methods. We recognize, however, that a 20 L volume represents a small fraction of air and may not fully capture spatial and temporal variability in airborne fungal concentrations. Consequently, this approach may result in an underestimation of fungal load, particularly for less abundant or heterogeneously distributed taxa. Moreover, variability between sampling points may be amplified when using smaller volumes, as localized fluctuations in fungal concentrations can disproportionately influence the results. To address these issues, all samples were collected in replicates using consistent, uniform procedures, allowing for reliable comparative analysis within the study design. We further note that future studies should adopt larger sampling volumes to improve accuracy and representativeness of fungal assessments. Fungal identification was based solely on colonial and microscopic morphology, without the use of molecular methods. We clarify that reliance on morphological identification alone limits taxonomic resolution. This raises the potential for misidentification, particularly among closely related taxa, which could influence interpretation of fungal composition and abundance patterns. In addition, non-culturable fungi remained undetected by culture-based methods, reducing overall comprehensiveness of the data. Accordingly, without molecular confirmation, the results provide an indicative assessment of fungal presence. Incorporating molecular approaches in subsequent studies is essential for reliable and high-resolution fungal identification. Finally, we explicitly acknowledge that the statistical analysis was exploratory and limited by the sample size, due to the six-week monitoring period and the number and design of the chosen premises. Comparable sampling approaches have been used in similar studies conducted in zoos, where practical and logistical constraints, such as number of sites, replicates, and sampling events feasible within the study period often limit the sample size. Regardless, the limited sample size restricts statistical power and representativeness, thereby affecting the robustness and generalizability of the findings and requiring careful interpretation. To account for this, we applied appropriate statistical methods, including PCA, to maximize the extraction of meaningful patterns from the available data and interpreted the results with restraint. Future zoo investigations with larger sample sizes and more intensive sampling designs are encouraged to strengthen statistical reliability and confirm observed patterns. Based on the above, Plewa-Tutaj et al. [23] suggest that the scientists should collaborate on developing standardized experimental methods and conducting large-scale studies in zoos, with these recommendations being in accordance with zoo sustainability goals [55].

5. Conclusions

The findings of this exploratory study, which was carried out in the visitor and worker areas of the animal premises of a zoo, may contribute to the current knowledge on fungal air quality in zoos, given that previous studies have mainly focused on air contamination within animal enclosures themselves. The study showed that the total culturable fungal concentration, as well as the concentrations of particular fungi in the air, differed among certain premises, including outdoor air, suggesting that quantitative and qualitative composition of these microbes may depend on the animals exhibited in the premises, and the design and management of the premises. There was an association between microclimate parameters and fungal concentrations. Considering the total fungal concentration inside the premises, the determined values are consistent with those reported in earlier studies conducted in zoos and may not necessarily indicate air contamination; however, the qualitative composition of fungi warrants attention, as the identified fungi may pose health concerns. Therefore, the implications of the study point to the importance of implementing good practices in zoo premises, including optimal microclimate conditions and effective ventilation, in order to reduce the potential risk from exposure to fungal aerosols. Study results also imply the need for air quality monitoring, which may be performed periodically, such as monthly or quarterly. This may help in assessing the effectiveness of measures to mitigate health risks from bioaerosols and identifying areas for improvement, thus supporting sustainable zoo development.