Higher Number of Yeast-like Fungi in the Air in 2018 after an Emergency Discharge of Raw Sewage to the Gulf of Gda ´nsk—Use of Contingency Tables

: This study aimed to investigate the differences between the number of yeast-like fungi and molds in the coastal air of ﬁve coastal towns of the Gulf of Gda´nsk in 2014–2017 vs. 2018, which saw an emergency discharge of sewage. In 2014–2017, a total of 62 duplicate samples were collected in the coastal towns of Hel, Puck, Gdynia, Sopot, and Gda´nsk-Brze´zno. In 2018, after the emergency disposal of raw sewage, 26 air samples were collected. A Pearson chi-squared test of independence showed that during 2018 in Hel and Sopot, the mean number of molds and yeast-like fungi was higher than in 2014–2017. The result was signiﬁcantly positive, p ≤ 2.22 × 10 − 16 . The analysis of the General Asymptotic Symmetry Test showed that in Puck and Gda´nsk-Brze´zno, the average number of Aspergillus sp. mold fungi was higher in 2018 after an emergency discharge of sewage into the Gulf of Gda´nsk compared to the period 2014–2017. The result was not statistically signiﬁcant. In addition, the average number of Penicillium sp. molds in 2018 in Gda´nsk-Brze´zno was higher than in 2014–2017, but statistically insigniﬁcant ( p = 0.9593). In 2018, the average number of Cladosporium sp. molds in Sopot was higher, but also statistically insigniﬁcant ( p = 0.2114) compared to 2014–2017. Our results indicate that the study of the number of yeast-like fungi in the air may indicate coastal areas that may be particularly at risk of bacterial or mycological pathogens, e.g., after an emergency discharge of raw sewage.


Introduction
In light of research, it has been shown that chemical and biological factors, including bacteria, mold spores, and viruses, have a significant impact on the quality of atmospheric air (including human health) [1][2][3][4][5]. The presence of yeast-like fungi and mold spores can be the etiological cause of many diseases, including allergies, pneumonia, bronchitis, and neoplastic diseases, as well as type 1 diabetes (T1D) [6][7][8][9][10][11].
The intensity of the spread of mold spores depends on the species of fungus and weather conditions [9,10,12]. Typically, higher spore concentrations are found in windy, dry, and sunny weather. The increase in the concentration of spores in the air is observed with increasing ambient temperature [5][6][7][8]. There are hygrophilous species of the class Ascomycetes sp. that produce spores in wet weather, often in rainfall at night [10]. For many years, our team has been researching the spread of bacteria, mold and yeast-like fungi in the air, with particular emphasis on the air over the Gulf of Gdańsk [5,[13][14][15][16][17]. Previous studies have shown that the spores of mold fungi are transported with air masses from above land areas to the Gulf of Gdańsk [15]. Moreover, it has also been shown that processes of bioaerosol emission into atmospheric air have the greatest intensity in the area of the mouth of the Vistula River to the Gulf of Gdańsk [5,15]. A greater number of potentially pathogenic mesophilic bacteria were found in marine bioaerosol compared to the number of psychrophilic bacteria [5]. Exposure to bacterial or fungal bioaerosol may lead to asthma, allergic rhinitis, allergic pneumonia, and many other diseases [3,8,[18][19][20][21].
Mycotoxins, by-products of fungal metabolism, which may cause pain, dizziness, or immunosuppression, are an additional threat related to the presence of mold fungi in the air [22]. Previous studies have shown that fungal spores, bacteria, their endotoxins, and even viruses can be transported over long distances, where meteorological factors play a large role [3,23,24]. It is also known that in the coastal region, the strength and direction of the wind are the greatest factors influencing the microbiological quality of air [15,[25][26][27][28]. Wind speed has a large impact on the process of breaking water waves, causing the formation of bubbles on the water surface and thus contributing to the transport of water pollutants to the atmosphere [5,[29][30][31][32]. A special feature of bubbles is their ability to selectively accumulate hydrophobic matter and microorganisms, which are transported to the water surface and partially emitted into the air [29][30][31]. The research carried out by Michaud et al. showed a clear tendency to enrich marine aerosols in Actinobacteria, certain Gamma-proteobacteria, and lipid-enveloped viruses [32]. Experimental studies showed a high emission of aerosol enrichment with viruses (250×) and Prokaryotes (45×) [30]. On the other hand, Marks et al., in his research carried out in the Gulf of Gdańsk at the mouth of the Vistula River, showed a 12-fold higher coefficient of enrichment of sea aerosol in mesophilic bacteria, potentially pathogenic compared to psychrophilic bacteria [5]. Recent studies have also shown that airborne microorganisms can influence physical and chemical processes in the atmosphere. They affect the Earth's radiation balance, the scattering and absorption of solar radiation directly or through condensation in clouds, and the formation of nuclei (CCN) and ice-nucleating particles (INPs) [30,32]. In our recent research, we searched for hidden relationships and regularities between meteorological factors and the number of mold and yeast-like fungi in the air of five coastal towns of the Gulf of Gdańsk [33]. For this purpose, the Principal Component Analysis (PCA) model was applied, which is one of the numerous methods of factor analysis that has been used for many years in the analysis of environmental samples [34][35][36][37][38][39]. The PCA analysis showed a significant correlation between meteorological factors and the number of molds and yeast-like fungi in the air of five coastal towns in 2014-2017 and in 2018, which saw an emergency discharge of sewage into the Gulf of Gdańsk [33]. However, the PCA model is a relatively simple graphical interpretation of results. The analysis of multivariate PCA data assumes their initial exploration and tries to explain relationships in a large dataset using a smaller set of orthogonal variables, called principal components (PCs), with the minimal loss of original information [40,41]. Therefore, the aim of this study was to assess whether there are differences in the number of mold fungi in different sampling periods, i.e., 2014-2017 vs. 2018 in five coastal towns on the Gulf of Gdansk. The use of contingency tables provided a new interpretation of the results.

Air Sampling Methods
Atmospheric air sampling performed in five coastal towns of the Gulf of Gdańsk (Hel, Puck, Gdynia, Sopot, and Gdańsk-Brzeźno) ( Figure 1) as well as mycological analysis have been described in detail in the study conducted by Michalska et al. [33]. In short, they can be presented in the following manner: in 2014-2018, air monitoring was carried out in 5 towns of the Gulf of Gdańsk (Hel, Puck, Gdynia, Sopot, and Gdańsk-Brzeźno). All samples were collected for 10 min approximately 100 cm from the waterline at a height of 50 cm. The air samples were collected by impingement with a SAS Super ISO 100 probe (Milan, Italy). The extracted air was then transported through small holes to a head with a Petri dish containing a Sabouraud dextrose agar medium. The maximum efficiency of the collection was for particulate matter of d50 = 2-4 µm. The flow rate was 90 Lpm. All removable parts of the air sampler were sterilized by autoclaving before sampling, and the sterilized sampler head was cleaned between samples with 70% ethanol. In 2014-2017, a total of 62 duplicate samples were collected in the coastal towns of Hel, Puck, Gdynia, Sopot, and Gdańsk-Brzeźno, while in 2018, 26 duplicate samples were taken [33].

Meteorological Conditions
In each year, the samples were always collected from May to July. The air temperature in 2014-2017 ranged from 26 • C to 3 • C in the spring and from 20 • C to 16 • C in the summer. Relative humidity was between 30% and 88% (spring), and from 59% to 82% (summer). Wind speed was between 0 and 31 km/h in the spring and between 7 and 25 km/h in the summer. Air temperature in 2018 fluctuated between 27 • C and 10 • C (spring) and between 27 • C and 15 • C (summer). Relative humidity was between 39% and 93% (spring), and from 44% to 70% (summer). Wind speed varied between 0 and 15 km/h (spring) and between 2.6 and 32 km/h (summer). They were not collected during rain and heavy rainfall [33].

Analysis and Identification of the Mold and Yeast-Like Fungi
Mold and yeast-like fungi were counted after a 120-h incubation at 28 • C on Sabouraud dextrose agar medium by Merck (Germany). Yeast-like fungi were identified by CHROMagar Candida, Graso Biotech (Poland). Mold and yeast-like fungi colonies were identified on the basis of color, texture, topography of the culture surface, smell of the colony, color of the reverse of the colony and the presence of the diffuse pigment. Microscopic features of the fungal colonies were identified based on their microscopic features using the Nikon Eclipse E2000 microscope at magnification 400, 600, 1000× and a fungus identification key [33,42,43].
The number of colonies of fungi was expressed as a colony-forming unit (CFU) per 1 m 3 of the air (CFU/m 3 ). When applying the impact method, we used the Feller table attached to the manual of the air sampler [33,44].
The colonies collected should be revised by the equation: where Pr is the revised colony in stage, N is the number of sieve pores, and r is the number of viable particles counted on the agar plate. The number of colonies of fungi (CFU/m 3 ) was calculated using the following equation: where C-airborne fungi concentration; CFU-colony-forming unit; T-total colonies after application of the Pr statistical correction; t-sampling time; and F-airflow rate.

Statistical Analysis
Statistical analysis of the results was carried out using R software [45,46], and for the symmetry, the test using the coin [47].
The non-parametric Friedman rank-sum test was used to analyze the difference between the mean number of mold and yeast-like fungi in the air in 2014-2017 and 2018. For the analysis using the χ 2 test, contingency tables were prepared for the qualitative variable "Years of sampling" (two subgroups; "2014-2017" and "2018") and the qualitative variable "Place" (five subgroups: Hel, Puck, Gdynia, Sopot, and Gdańsk-Brzeźno). The average number of mold and yeast-like fungi is responsible for the presence or absence of correlation between the qualitative variables. The expected values for individual cells (from the row i and column j) were calculated based on the following formula: where r i is the sum of the values from row i, c j is the sum of the values from column j, and n is the sum of all values from the contingency table. The formula shows that the appearance of large values in the cells of the contingency tables has an impact on the expected values. It also affects the share of each cell in the total value χ 2 , which is calculated using the following formula: where O ij is the cell value from the contingency table (average number of fungi). The scripts took into account the fact that the χ 2 test may present difficulties with contingency tables if the numbers in individual cells of the tables are too small. In the event of a warning message, the algorithm automatically decides to use an exact probability, such as Fisher's exact test. We have also calculated the measure of dependence between the factors placed in lines and columns (Pearson's χ 2 ): (where w is the number of rows, and k is the number of columns), which-in combination with the results determined from the previous formulas-allowed the figures corresponding to the contingency tables to be created (Figures 2-5). The bars on the plot represent the Pearson residuals, the color code and the height of the bars denote the significance level and magnitude of the residuals, and their widths are proportional to the sample size. In each of the association figures, the blue color indicates that the observed value is higher than the expected value if the data were random. The red color specifies that the observed value is lower than the expected value if the data were random. The grey color represents the data where the concentrations are similar to observations under the null hypothesis where a test of independence is true.  Table 1.     In our study, statistical analysis based on contingency tables was used to evaluate the number of mold fungi Aspergillus sp. (Figure 3), Penicillium sp. (Figure 4), and Cladosporium sp. (Figure 5) in 2014-2017 and in 2018.

The Number of Molds and Yeast-Like Fungi (CFU/m 3 ) in the Seaside Air over the Gulf of Gdańsk
In 2018 in Puck and Gdańsk-Brzeźno, the number of Aspergillus sp. mold fungi was higher compared to 2014-2017. The result was statistically significantly positive, p ≤ 2.22 × 10 −16 (Figure 3).
The analysis of contingency tables demonstrated that in 2018, which saw the emergency disposal, the number of mold fungi Penicillium sp. in the coastal town of Gdańsk-Brzeźno was significantly higher than in 2014-2017 (p = 8.9631 × 10 −14 ) (Figure 4).   Moreover, in Sopot, also in 2018, the number of mold fungi Cladosporium sp. was significantly higher compared to 2014-2017 (p ≤ 2.22 × 10 −16 ) ( Figure 5).
An Asymptotic General Symmetry Test was used and we checked how the average number of mold and yeast-like fungi changed in the two studied groups. These groups were "2014-2017" and "2018", and the binding element has five coastal towns located on the Gulf of Gdańsk. In 2014-2017 in the coastal towns Hel and Sopot, the number of molds and yeast-like fungi decreased, and in 2018 it increased compared to the expected value. In Gdynia, the number of molds and yeast-like fungi in 2018 decreased and was lower than in 2014-2017. The result was not statistically significant, p = 0.05957.
In 2018, the average number of Aspergillus sp. molds in Hel and Gdynia was lower compared to 2014-2017. On the other hand, in Puck and Gdańsk-Brzeźno it was higher compared to 2014-2017. However, the result was not statistically significant, p = 0.1346. The average number of Penicillium sp. in 2018 in Puck and Gdynia was lower than that reported in 2014-2017, and in Gdańsk-Brzeźno the number was higher than in 2014-2017. The result obtained using the Asymptotic General Symmetry Test was also not statistically significant, p = 0.9593. In 2018, a lower average number of Cladosporium sp. was detected in Gdynia and a higher number in Sopot, but not statistically significant, p = 0.2114 (in both towns) compared to the number of Cladosporium sp. in 2014-2017.

The Influence of Meteorological Parameters on the Number of Mold and Yeast-Like Fungi in the Air of the Towns Located in the Gulf of Gdańsk
The research into the number of mold and yeast-like fungi in the air of seaside towns in the Gulf of Gdańsk (Hel, Puck, Gdynia, Sopot, and Gdańsk-Brzeźno) included relative air humidity, air temperature, wind speed, and direction. The analysis based on Spearman's correlation coefficient showed a statistically significant relationship between the relative air humidity in the Gulf of Gdańsk and the number of mold and yeast-like fungi (p = 0.009). However, no relationship was found between air temperature and the number of mold and yeast-like fungi (p = 0.526).
On the other hand, a statistically significant relationship was found between the wind speed in the seaside towns in the Gulf of Gdańsk (Hel, Puck, Gdynia, Sopot, and Gdańsk-Brzeźno) and the number of mold and yeast-like fungi (p = 0.002). The analysis conducted based on the Kruskal-Wallis test demonstrated a statistically significant relationship between the wind direction and the number of mold and yeast-like fungi (p = 0.007).

Discussion
There are many environmental studies in which multivariate statistical methods were used, for instance, physicochemical analysis of surface waters [34,37,39], waters in retention reservoirs [36], bottom sediments [48,49] and atmospheric air [31,50], as well as the assessment of the microbiological quality of air [3,33,38]. In our current study, the multivariate data analysis with the use of contingency tables showed a statistically significant relationship between the number of molds and yeast-like fungi in the coastal towns in 2014-2017 and 2018. In 2018, a statistically significantly higher total number of mold and yeast-like fungi was detected in the atmospheric air of the seaside town of Hel and Sopot, compared to the period 2014-2017. Moreover, in 2018, there was a higher, statistically significant (p ≤ 2.22 × 10 −16 ) number of mold fungi Aspergillus sp. in Puck and Gdańsk-Brzeźno. Furthermore, in 2018, in the seaside town of Gdańsk-Brzeźno, a statistically significantly higher number of Penicillium sp. mold fungi was detected (p ≤ 8.9631 × 10 −14 ). On the other hand, in the seaside town of Sopot, a higher, statistically significant (p ≤ 2.22 × 10 −16 ) number of mold fungi of Cladosporium sp. was identified. According to research conducted so far, the spores of the mold fungi Aspergillus sp., Penicillium sp., Cladosporium sp. and their mycotoxins contribute to the development of allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, chronic necrotic aspergillosis (CNA), invasive pulmonary aspergillosis (IPA), asthma and anemia [6,10,51]. Therefore, seasonal monitoring of the microbiological quality of atmospheric air should be carried out in seaside towns as an important tool to estimate the exposure to allergens. Current studies on air pollution focused on monitoring the concentrations of plant pollen, PM 2.5 , PM 10 dust, and gases do not reflect the actual situation [50]. The authors of this study are also convinced that these determinations can be used to develop a prognostic model for the concentration of mold spores in the coastal region.
We also know that the spread of mold fungi is influenced by atmospheric conditions, especially relative humidity, wind speed, temperature, and insolation [18,24,52]. Our research also attempted to assess the impact of meteorological conditions on the presence of mold and yeast-like fungi in the seaside air of the towns of Hel, Puck, Gdynia, Sopot, and Gdańsk-Brzeźno. The results of our study indicate a statistically significant relationship between the relative air humidity (p = 0.009) and the number of mold and yeast-like fungi. We have also shown a statistically significant relationship between the wind direction (p = 0.097), wind speed (p = 0.002), and the number of mold and yeast-like fungi. Recent bioaerosol studies confirm that the number of mold fungi depends on air temperature, relative humidity, and wind speed [18,[53][54][55]. In 2018, the maximum number of mold and yeast-like fungi was detected in the atmospheric air of the seaside town of Hel, when the relative humidity was 60% and the wind from the Gulf of Gdańsk was blowing at a speed of 3 ms −1 . On the other hand, in the seaside town of Sopot, a significantly higher number of mold fungi Cladosporium sp. was observed when the relative air humidity was 53% and the wind was blowing along the shore at a speed of 4.5 ms −1 . In addition, in 2018, we detected a significantly higher number of mold fungi Aspergillus sp. and Penicillium sp. in the seaside town of Gdańsk-Brzeźno. The maximum number of Aspergillus sp. was detected in the atmospheric air, at a relative humidity of 55%, and when the wind was blowing along the shore with a speed of 4.2 ms −1 . The maximum number of Penicillium sp. was detected in the atmospheric air when the relative humidity was 65% and the wind from the Gulf of Gdańsk was blowing at the speed of 8.9 ms −1 . Our previous research results [8,33] and the present study outcomes confirm the influence of atmospheric conditions on the number of yeast-like fungi in coastal bioaerosol.

Conclusions
The higher number of mold and yeast fungi in 2018 due to the failure of the sewage treatment plant may pose a risk to people visiting seaside bathing areas. Therefore, we suggest air monitoring in coastal towns to quickly detect higher concentrations of potentially pathogenic microorganisms.

Conflicts of Interest:
The authors declare no conflict of interest.