S S symmetry Article Higher Number of Yeast-like Fungi in the Air in 2018 after an Emergency Discharge of Raw Sewage to the Gulf of Gda ´nsk—Useof Contingency Tables Małgorzata Michalska 1,* , Piotr W ˛az˙ 2 , Monika Kurpas 1 , Roman Marks 3 and Katarzyna Zorena 1,* 1 Department of Immunobiology and Environment Microbiology, Faculty of Health Sciences with Institute of Maritime and Tropical Medicine, Medical University of Gda´nsk, 80-210 Gda´nsk,Poland; [email protected] 2 Department of Nuclear Medicine, Faculty of Health Sciences with Institute of Maritime and Tropical Medicine, Medical University of Gda´nsk,80-210 Gda´nsk,Poland; [email protected] 3 Institute of Marine and Environmental Sciences, University of Szczecin, 70-383 Szczecin, Poland; [email protected] * Correspondence: [email protected] (M.M.); [email protected] (K.Z.) Abstract: 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´nskin 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 Citation: Michalska, M.; W ˛az,˙ P.; higher than in 2014–2017. The result was significantly positive, p ≤ 2.22 × 10−16. The analysis of the Kurpas, M.; Marks, R.; Zorena, K. General Asymptotic Symmetry Test showed that in Puck and Gda´nsk-Brze´zno,the average number Higher Number of Yeast-like Fungi in of Aspergillus sp. mold fungi was higher in 2018 after an emergency discharge of sewage into the the Air in 2018 after an Emergency Discharge of Raw Sewage to the Gulf Gulf of Gda´nskcompared to the period 2014–2017. The result was not statistically significant. In of Gda´nsk—Useof Contingency addition, the average number of Penicillium sp. molds in 2018 in Gda´nsk-Brze´znowas higher than in Tables. Symmetry 2021, 13, 1522. 2014–2017, but statistically insignificant (p = 0.9593). In 2018, the average number of Cladosporium sp. https://doi.org/10.3390/ molds in Sopot was higher, but also statistically insignificant (p = 0.2114) compared to 2014–2017. sym13081522 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 Academic Editor: discharge of raw sewage. George Papageorgiou Keywords: Gulf of Gdansk; asymptotic symmetry; five-year monitoring of coastal air; contingency tables Received: 24 May 2021 Accepted: 3 August 2021 Published: 19 August 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in In light of research, it has been shown that chemical and biological factors, including published maps and institutional affil- bacteria, mold spores, and viruses, have a significant impact on the quality of atmospheric iations. air (including human health) [1–5]. 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) [6–11]. 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, Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. dry, and sunny weather. The increase in the concentration of spores in the air is observed This article is an open access article with increasing ambient temperature [5–8]. There are hygrophilous species of the class distributed under the terms and Ascomycetes sp. that produce spores in wet weather, often in rainfall at night [10]. For conditions of the Creative Commons many years, our team has been researching the spread of bacteria, mold and yeast-like Attribution (CC BY) license (https:// fungi in the air, with particular emphasis on the air over the Gulf of Gda´nsk[5,13–17]. creativecommons.org/licenses/by/ Previous studies have shown that the spores of mold fungi are transported with air masses 4.0/). from above land areas to the Gulf of Gda´nsk[15]. Moreover, it has also been shown that Symmetry 2021, 13, 1522. https://doi.org/10.3390/sym13081522 https://www.mdpi.com/journal/symmetry Symmetry 2021, 13, 1522 2 of 11 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 Gda´nsk[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 [3,8,18–21]. 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 [15,25–28]. 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 [5,29–32]. 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 [29–31]. 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 Gda´nskat 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 Gda´nsk[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 [34–39]. 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 Gda´nsk[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. 2. Materials and Methods 2.1. Air Sampling Methods Atmospheric air sampling performed in five coastal towns of the Gulf of Gda´nsk(Hel, Puck, Gdynia, Sopot, and Gda´nsk-Brze´zno)(Figure1) 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 Gda´nsk(Hel, Puck, Gdynia, Sopot, and Gda´nsk-Brze´zno).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 Symmetry 2021, 13, 1522 3 of 11 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 Gda´nsk-Brze´zno,while in 2018, 26 duplicate samples were taken [33]. Figure 1. The map shows the sampling points in 5 coastal towns, Hel, Puck, Gdynia, Sopot, Gda´nsk- Brze´zno,on the Gulf of Gda´nsk.