Evaluation of Parabens and Bisphenol A Concentration Levels in Wild Bat Guano Samples

Citation: Gonkowski, S.; Martín, J.; Aparicio, I.; Santos, J.L.; Alonso, E.; Rytel, L. Evaluation of Parabens and Bisphenol A Concentration Levels in Wild Bat Guano Samples. Int. J. Environ. Res. Public Health 2023,20, 1928. https://doi.org/10.3390/ ijerph20031928 Academic Editor: Paul B. Tchounwou Received: 3 December 2022 Revised: 17 January 2023 Accepted: 18 January 2023 Published: 20 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Environmental Research and Public Health Article Evaluation of Parabens and Bisphenol A Concentration Levels in Wild Bat Guano Samples Slawomir Gonkowski 1, Julia Martín2, Irene Aparicio 2, Juan Luis Santos 2, Esteban Alonso 2 and Liliana Rytel 3,* 1Department of Clinical Physiology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Street Oczapowskiego 14, 10-719 Olsztyn, Poland 2Departamento de Química Analítica, Universidad de Sevilla, C/Virgen de África, 7, E-41011 Sevilla, Spain 3Department of Internal Diseases with Clinic, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, ul. Oczapowskiego 14, 10-719 Olsztyn, Poland *Correspondence: [email protected] Abstract: Parabens and bisphenol A are synthetic compounds found in many everyday objects, including bottles, food containers, personal care products, cosmetics and medicines. These substances may penetrate the environment and living organisms, on which they have a negative impact. Till now, numerous studies have described parabens and BPA in humans, but knowledge about terrestrial wild mammals’ exposure to these compounds is very limited. Therefore, during this study, the most common concentration levels of BPA and parabens were selected (such as methyl paraben—MeP, ethyl paraben—EtP, propyl paraben—PrP and butyl paraben—BuP) and analyzed in guano samples collected in summer (nursery) colonies of greater mouse-eared bats (Myotis myotis) using liquid chromatography with the tandem mass spectrometry (LC-MS-MS) method. MeP has been found in all guano samples and its median concentration levels amounted to 39.6 ng/g. Other parabens were present in smaller number of samples (from 5% for BuP to 62.5% for EtP) and in lower concentrations. Median concentration levels of these substances achieved 0.95 ng/g, 1.45 ng/g and 15.56 ng/g for EtP, PrP and BuP, respectively. BPA concentration levels did not exceed the method quantification limit (5 ng/g dw) in any sample. The present study has shown that wild bats are exposed to parabens and BPA, and guano samples are a suitable matrix for studies on wild animal exposure to these substances. Keywords: wild animals; environmental pollution; endocrine disruptors; toxicology 1. Introduction Currently, environmental pollution with organic compounds remains on the increase. Among these substances, bisphenol A (BPA) and parabens are of great importance [ 1 , 2 ]. BPA—chemical name 2,2-Bis(4-hydroxyphenyl)propane—is a substance used in production of plastics including polycarbonates and epoxy resins, which in turn is utilized in a wide range of branches of industry [ 2 , 3 ]. The popularity of BPA is because items containing this substance are light, resistant to damage and resilient [ 4 ]. It is not without significance that the synthesis of BPA is relatively easy and cheap [ 2 , 4 ]. Therefore, BPA is present in various everyday issues, such as bottles, electronic elements, furniture, food containers and many others [2–4]. In turn, parabens are a group of the esters of p-hydroxybenzoic acid and differ from each other by the type of substituent, which may be an alkyl chain or an aromatic ring [ 1 ]. Properties of parabens depend on the type of substituent in a molecule, but generally this group of components is characterized by fungistatic, anti-yest and anti-mold activities, as well as antibacterial properties [ 5 , 6 ]. These characteristics of parabens cause their widespread usage as preservatives in cosmetics, personal care products, drugs and food [ 1 ]. The presence of parabens has been described in shampoo, lipsticks, food packaging, baby wipes and many others, and the most used parabens are compounds with short-chain substituents, such as methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP) [1,5,6]. Int. J. Environ. Res. Public Health 2023,20, 1928. https://doi.org/10.3390/ijerph20031928 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2023,20, 1928 2 of 17 BPA and parabens, penetrating the body, bind to estrogen receptors and first of all cause disturbances in endocrine system [ 1 , 2 ]. Therefore, these substances are included in the group of compounds called endocrine disruptors. Both BPA and parabens may penetrate to the natural environment [ 6 , 7 ]. The presence of these substances has been described in the surface and tap water, soil, air, house dust and plants around the world [ 1 , 2 , 8 – 10 ]. Moreover, BPA and parabens penetrate the human and animal organisms, mainly through the gastrointestinal tract, but also through respiratory system, transdermal absorption and in utero through placenta [ 1 , 2 , 4 ]. Till now, BPA and parabens have been described mainly in the blood serum and urine [ 1 , 2 , 11 – 13 ]. Apart from these “classic” matrices, their presence has also been found in hair, nail, breast milk, semen and various tissues [1,2,4,14–17]. Endocrine disruptors can impair the functions of many internal organs. Previous studies have reported that BPA adversely affects the nervous system, endocrine glands, immune cells, male and female reproductive organs, heart and many other organs and internal systems [ 2 , 4 , 18 ]. It is also known that high exposure to BPA results in higher risk of diabetes, obesity, hypertension, cancers and neurodegenerative processes [ 2 , 19 – 21 ]. In turn, for many years, parabens were considered substances that had no negative impact on living organisms [ 1 ]. However, latest studies have shown that they have cytotoxic and genotoxic effects and negatively impact on the nervous, reproductive and endocrine systems, as well as immune reactions [ 1 , 5 , 22 , 23 ]. Moreover, some studies have described correlations between parabens and obesity, developmental disorders and neoplasms [22,24]. Contrary to humans, animal exposure to BPA and parabens polluting the environment is much less known. Regarding domestic animals, parabens have been found in the urine of dogs, cats and cows [ 25 , 26 ], raw cow’s milk [ 27 ] and canine fur [ 28 ]. More is known about the exposure of domestic animals to bisphenol A, the presence of which has been reported in the blood serum and urine of dogs and cats [ 29 – 31 ], canine fur [ 32 ], fresh pork meat [33] and cow’s milk [27]. Even less is known about the exposure of wild animals to BPA and parabens. Previous studies have reported the presence of BPA in wild water animals including mussels, fish, prawns and mollusks, as well as sea birds and seals, but knowledge about wild mammal exposure to BPA is rather scanty and fragmentary [ 34 – 37 ]. Parabens have also been described mainly in water animals—fish, birds and mammals [ 38 – 42 ]. Only single previous reports describe parabens in terrestrial birds and mammals [42]. Such a limited number of studies on wild mammals may be related to the fact that it is virtually impossible to collect samples of blood, urine or fur without capturing or killing the animal. The only matrix that can be taken from a wild animal for toxicology studies without significantly interfering with its life are feces/guano samples. The utility of this matrix is also supported by the fact that previous studies have confirmed that both BPA and parabens are excreted from organisms through the digestive tract [43,44]. On the other hand, among terrestrial mammals, bats play an important role in toxicological studies. Bats are an extremely heterogenous group of mammals consisting of above 1400 species living on all continents except Antarctica [ 45 ]. Bats are particularly vulnerable to the impact of factors changing their natural habitats, among which one of the most important is anthropogenic pollutions of the environment [ 46 ]. For this reason, bats are considered to be one of the best bioindicators of quality environment, including anthropogenic environmental pollution [ 46 – 48 ]. The high sensitivity of bats to environmental pollution is related to their biology, namely, their relatively long lifespan (even more than 30 years) and high metabolic rates resulting in high food intake, as well as frequent establishment of colonies near human habitation [ 49 , 50 ]. Unfortunately, despite the legal protection of most species, the number of bat populations is constantly decreasing [ 45 ], and one of the reasons for this is environmental pollution. Taking these facts into account, the purpose of this study is to assess the exposure of the greater mouse-eared bat (Myotis myotis)—one of the most popular bat species of Poland [ 51 ] to bisphenol A and the most common parabens, such as MeP, EtP, PrP, BuP through guano samples analysis. According to the best knowledge of authors, it is the first study in Int. J. Environ. Res. Public Health 2023,20, 1928 3 of 17 which guano samples are used to evaluate the wild terrestrial mammal exposure to BPA and parabens, as well as the first ever study biomonitoring parabens in wild terrestrial mammals. 2. Materials and Methods 2.1. Reagents The analytical reagents used in this study such as formic acid and ammonium acetate were purchased from Panreac (Barcelona, Spain). C18 disperser sorbent was provided by Scharlab (Barcelona, Spain). HPLC-grade methanol, and water were purchased from Romil (Barcelona, Spain). Paraben and BPA standards ( ≥ 99.0%) were purchased from Sigma-Aldrich (Steinheim, Germany). The internal standards (ISs) BPA-d14 and PrP- 13 C 6 were supplied by Cambridge Isotope Laboratories (Tewksbury, MA, USA). Individual stock standard solutions of 1000 mg/L and working solutions (by dilution of the former) were prepared in methanol. 2.2. Sample Collection Four summer (nursery) colonies of greater mouse-eared bats (Myotis myotis) located in various parts of Poland were included into the study. The localization and characteristics of colonies are presented on Figure 1. The choice of bat colonies was not accidental. The colonies included into the study are some of the largest colonies of a greater mouse-eared bat in Poland. They are located in various environments and in various parts of Poland: colony no. 1 is located in a small village, which is placed relatively close to the most industrialized region in Poland (Upper Silesia); colony no 2 is located in a small village in agricultural land without industry; colony no 3 is located in a medium-sized town with a chemical industry and colony no 4 is located in a small town without industry. Such different localization of bat colonies allowed to better understand, what factors may influence of bat exposure to parabens and BPA. Int. J. Environ. Res. Public Health 2023, 20, x 4 of 17 Figure 1. Location and characteristics of bat colonies included into the study. 2.3. Sample Treatment Samples were lyophilized and homogenized. An aliquot of 1.0 g of sample was fortified with 100 µL of a methanol solution of the ISs (250 ng/mL) in 12 mL glass tubes. The sample procedure involves the extraction of the analytes by ultrasonic solvent extraction, using 7 mL of methanol (0.5% v/v, formic acid) as extraction solvent for 5 min and centrifuged for 5 min (4050× g). The extraction procedure was repeated three times and the supernatants were combined. Given the complexity of the selected biological sample and of the extract obtained in the first stage of ultrasonic solvent extraction (USE), the cleaning process was intended to remove any interference from the extract. For this approach, a cleaning process using dispersive adsorbents (d-SPE) based on the QuEChERs technique (Quick, Easy, Cheap, Effective, Rugged and Safe) was used. This procedure consisted in shaking vigorously for 2 min the combined extracts from USE with 0.3 g of C18 sorbent and then centrifuged at 4050× g for 5 min. Finally, the supernatant was evaporated to dryness, reconstituted in 0.25 mL of a mixture methanol–water (50:50 v/v) and filtered through a 0.22 µm nylon filter prior to injection into the liquid chromatography tandem mass spectrometry (LCMS/MS) instrument (Agilent, Santa Clara, CA, USA). Chromatographic conditions were those previously reported by Martín et al. [52]. Instrument settings and analytical determination parameters are summarized in the Supplementary Material (Table S1). 2.4. Method Validation, Quality Assurance and Quality Control The analytical features (linearity, sensitivity and accuracy (trueness and precision)) of the method are presented in Table 1. Table 1. Linearity, method quantification limit, precision and recovery of parabens and BPA in guano matrix . Compound Linearity MDL MQL RSD Rec R 2 (ng/g dw) (ng/g dw) (%) (%) MeP 0.998 0.02 0.05 12.1 98.6 EtP 0.998 0.02 0.05 11.2 108.1 PrP 0.999 0.02 0.05 14.2 107.2 BuP 0.998 0.02 0.05 9.5 118.8 BPA 0.997 2.00 5.00 35.9 139.3 MDL: Method detection limit MQL: Method quantification limit; RSD: Relative Standard Deviation; Rec: Recovery. Figure 1. Location and characteristics of bat colonies included into the study. Guano samples were collected in August–September 2021. To collect samples, flat glass containers were put for 48 h on the floor of the places with bat colonies. After these time containers were removed and samples were transferred to glass jars and frozen at − 20 ◦ C until further analysis. Forty guano samples (ten samples from each colony) collected from different parts of rooms, where bat colonies live, were included into the present study. During collection of samples, special emphasis was placed on not stressing and scaring the bats. Because the sampling procedure was completely non-invasive, according to Act for the Protection of Animals for Scientific or Educational Purposes of 15 January 2015 (Official Gazette 2015, No. 266), applicable in the Republic of Poland, consent of bioethical committee to conduct the present study was not required. Int. J. Environ. Res. Public Health 2023,20, 1928 4 of 17 2.3. Sample Treatment Samples were lyophilized and homogenized. An aliquot of 1.0 g of sample was fortified with 100 µ L of a methanol solution of the ISs (250 ng/mL) in 12 mL glass tubes. The sample procedure involves the extraction of the analytes by ultrasonic solvent extraction, using 7 mL of methanol (0.5% v/v, formic acid) as extraction solvent for 5 min and centrifuged for 5 min (4050 × g). The extraction procedure was repeated three times and the supernatants were combined. Given the complexity of the selected biological sample and of the extract obtained in the first stage of ultrasonic solvent extraction (USE), the cleaning process was intended to remove any interference from the extract. For this approach, a cleaning process using dispersive adsorbents (d-SPE) based on the QuEChERs technique (Quick, Easy, Cheap, Effective, Rugged and Safe) was used. This procedure consisted in shaking vigorously for 2 min the combined extracts from USE with 0.3 g of C18 sorbent and then centrifuged at 4050×g for 5 min. Finally, the supernatant was evaporated to dryness, reconstituted in 0.25 mL of a mixture methanol–water (50:50 v/v) and filtered through a 0.22 µ m nylon filter prior to injection into the liquid chromatography tandem mass spectrometry (LC-MS/MS) instrument (Agilent, Santa Clara, CA, USA). Chromatographic conditions were those previously reported by Martín et al. [ 52 ]. Instrument settings and analytical determination parameters are summarized in the Supplementary Material (Table S1). 2.4. Method Validation, Quality Assurance and Quality Control The analytical features (linearity, sensitivity and accuracy (trueness and precision)) of the method are presented in Table 1. Table 1. Linearity,methodquantificationlimit, precision andrecoveryofparabensandBPAinguanomatrix. Compound Linearity MDL MQL RSD Rec R2(ng/g dw) (ng/g dw) (%) (%) MeP 0.998 0.02 0.05 12.1 98.6 EtP 0.998 0.02 0.05 11.2 108.1 PrP 0.999 0.02 0.05 14.2 107.2 BuP 0.998 0.02 0.05 9.5 118.8 BPA 0.997 2.00 5.00 35.9 139.3 MDL: Method detection limit MQL: Method quantification limit; RSD: Relative Standard Deviation; Rec: Recovery. A matrix-matched calibration curve method was used to overcome the matrix effect. For that, fortified commercial guano samples were prepared containing the analytes at eight different concentration levels in the range from method quantitation limit (MQL) to 100 ng/g dw. Note that the commercial guano samples used for matrix-matched calibration were simultaneously analyzed and their signals were subtracted to spiked sample extract signals. BPA and parabens (except MeP) were not detected in the commercial guano samples. Method detection limits (MDLs) and method quantification limits (MQLs) were calculated as the concentrations of each compound corresponding to a signal-to-noise ratio of 3:1 and 10:1, respectively, using guano spiked samples at low concentration levels. Accuracy (trueness and precision) of the method was assessed using commercial guano samples spiked at 25 ng/g dw. Accuracy was evaluated by a recovery control over the whole procedure, which included extraction from the matrix, d-SPE and concentration step. The precision is expressed through the relative standard deviation (%, RSD) of measurements on different days. To guarantee the quality assurance of results, a protocol involving the use of control spiked samples (fortified commercial guano samples at 50 ng/g dw), solvent (methanol:water 50:50 v/v) injections, standards containing a mixture of the target compounds (12.5 ng/mL) and procedural blanks (processed in the same way as the samples) were included into each analytical batch (5 samples). Int. J. Environ. Res. Public Health 2023,20, 1928 5 of 17 2.5. Statistical Analysis The statistical analysis of the differences in concentration levels of substances studied between particular bat colonies was made up with GraphPad Prism version 9.2.0 (GraphPad Software, San Diego, CA USA) and a Kruskal–Wallis test was used. The differences were considered statistically significant at p< 0.05. 3. Results During the present investigation, parabens were present in guano samples collected in all bat colonies (Table 2). Among parabens, MeP was a substance that was quantified in all samples analyzed in concentration levels. Its levels fluctuated from 14.00 ng/g dry weight (dw.) to 142.00 ng/g dw. The second most common paraben was EtP, which had concentration levels higher than MQL, was observed in 62.5% of guano samples, and its concentration levels fluctuated from <0.05 ng/g dw to 239 ng/g dw. In turn, PrP in concentration levels above MQL was noted in 42.4% of samples with a range from <0.05 ng/g dw. to 229 ng/g dw. The least common paraben in the samples included into the study was BuP. The concentration levels of this paraben higher than the method detection limit (MDL) were noted only in two guano samples (5% of all samples). In one of them BuP concentration levels achieved 3.61 ng/g dw, and in the second 27.5 ng/g dw. Contrary to parabens, BPA concentration levels in the vast majority of samples did not exceed MDL. Only in one sample the value was slightly higher but did not exceed MQL (Tables 2and 3). During the present study, clear differences in parabens concentration levels between particular animals in one colony were noted. The most visible such differences were noted in bat colony no. 1 (Table 2). Moreover, statistically significant differences in MeP concentration levels were noted between colonies. The highest mean concentration of this paraben was found in colony no. 3, where this value was statistically significantly higher than values noted in colonies no. 1 and no. 4 (Table 4). Differences in concentration levels of other parabens between colonies were not statistically significant (Table 4). Moreover, differences in the frequency of occurrence of EtP, PrP and BuP in concentrations higher than MQL were noted between colonies. The highest frequency was noted in colony no. 1, and the lowest in colony no. 4 (Table 4). Table 2. Concentration levels (ng/g dw) of parabens and bisphenol A in bat guano samples. Bat Colony No. Sample No. Concentration (ng/g) MeP EtP PrP BuP BPA 1 1 31.1 0.68 0.41 <MDL <MDL 2 25.5 0.65 0.30 <MDL <MDL 3 27.8 0.75 0.48 <MDL <MDL 4 20.7 0.23 0.08 <MDL <MDL 5 52.7 35.3 18.6 <MDL <MDL 6 25.1 1.48 0.81 <MDL <MDL 7 25.6 1.89 2.09 <MDL <MDL 8 56.1 55.3 46.4 3.61 <MDL 9 142 239 229 27.5 <MQL 10 26.9 3.48 4.38 <MDL <MDL 2 1 33.6 2.27 2.59 <MDL <MDL 2 50.3 1.73 1.51 <MDL <MDL 3 60.4 2.38 1.59 <MDL <MDL 4 43.8 0.95 0.99 <MDL <MDL 5 35.9 0.60 0.42 <MDL <MDL 6 40.6 0.41 <MQL <MDL <MDL 7 38.6 0.55 <MQL <MDL <MDL 8 35.9 0.46 <MQL <MDL <MDL 9 37.2 0.67 <MQL <MDL <MDL 10 43.7 <MQL <MQL <MDL <MDL Int. J. Environ. Res. Public Health 2023,20, 1928 6 of 17 Table 2. Cont. Bat Colony No. Sample No. Concentration (ng/g) MeP EtP PrP BuP BPA 3 1 70.2 1.60 1.33 <MDL <MDL 2 72.7 0.92 <MQL <MDL <MDL 3 46.6 <MQL <MQL <MDL <MDL 4 80.3 0.84 <MQL <MDL <MDL 5 73.5 1.07 <MQL <MDL <MDL 6 96.4 <MQL <MQL <MDL <MDL 7 86.4 <MQL <MQL <MDL <MDL 8 100 1.28 1.34 <MDL <MDL 9 71.2 <MQL <MQL <MDL <MDL 10 119 0.70 <MQL <MDL <MDL 4 1 50.8 <MQL <MQL <MDL <MDL 2 24.4 <MQL <MQL <MDL <MDL 3 14.0 <MQL <MQL <MDL <MDL 4 22.6 <MQL <MQL <MDL <MDL 5 22.9 <MQL <MQL <MDL <MDL 6 24.2 <MQL <MQL <MDL <MDL 7 24.4 <MQL <MQL <MDL <MDL 8 25.4 <MQL <MQL <MDL <MDL 9 45.3 <MQL <MQL <MDL <MDL 10 24.3 <MQL <MQL <MDL <MDL Compound acronyms: MeP: methylparaben; EtP: ethylparaben; PrP: Propylparaben; BuP: butylparaben; BPA: bisphenol A; <MQL: Below Method Quantification Limit EtP = 0.05 ng/g dw; PrP = 0.05 ng/g dw.; BPA = 5 ng/g dw.); <MDL: Below Method Detection Limit (BuP = 0.02 ng/g dw.; BPA = 2.00 ng/g dw.). Table 3. (ng/g dw.) and frequency of detection of parabens and bisphenol A in the analyzed guano samples (n = 40)—cumulative data. Compound Range (ng/g) Arithmetic Mean Geometric Mean Median Frequency of Detection above MQL MeP 14.00–142.00 48.70 41.81 39.6 100 EtP <0.05–239 14.21 1.61 0.95 62.5 PrP <0.05–229 18.37 1.78 1.45 42.5 BuP <0.02–27.5 15.56 9.96 15.56 5 BPA <2.00–<5.00 - - - 0 Compound acronyms: MeP: methylparaben; EtP: ethylparaben; PrP: Propylparaben; BuP: butylparaben; BPA: bisphenol A; MQL—Method Quantification Limit. Table 4. Concentration values (ng/g dw.) and frequency of detection of parabens and bisphenol A in the analyzed guano samples taking into account the place of collection. Bat Colony Number 1 2 3 4 MeP Number of samples, in which levels were higher than MQL 10 10 10 10 Minimum 20.70 33.60 46.60 14.00 Maximum 142 60.40 119 50.80 Mean ±SD 43.35 ±36.7 A42.00 ±8.13 81.63 ±19.97 AB 27.83 ±11.21 B EtP Number of samples in which levels were higher than MQL 10 9 6 0 Minimum 0.23 0.41 0.7 - Maximum 239 1.97 1.6 - Mean ±SD 33.88 ±74.51 1.11 ±0.79 1.07 ±0.33 - PrP Number of samples in which levels were higher than MQL 10 5 2 0 Minimum 0.08 0.42 1.33 - Maximum 229 2.17 1.34 - Mean ±SD 30.26 ±71.34 1.42 ±0.8 1335 ±0.01 - BuP Number of samples in which levels were higher than MDL 2 0 0 0 Minimum 3.61 - - - Int. J. Environ. Res. Public Health 2023,20, 1928 7 of 17 Table 4. Cont. Bat Colony Number 1 2 3 4 Maximum 27.5 - - - Mean ±SD 15.56 ±16.89 - - - BPA Number of samples in which levels were higher than MQL 0 0 0 0 Minimum - - - - Maximum - - - - Mean ±SD - - - - Statistically significant differences A —between bat colony 1 and 3 ( p= 0.0084 ), B —between bat colony 3 and 4 ( p< 0.0001 ). Compound acronyms: MeP: methylparaben; EtP: ethylparaben; PrP: Propylparaben; BuP: butylparaben; BPA: bisphenol A; MQL—Method Quantification Limit; MDL—Method Detection Limit. 4. Discussion The results obtained during the present investigation have shown that parabens are present in guano samples, which indicates the exposure of wild bats to these substances. MeP, a paraben with the shortest functional group, has been noted in the highest concentration levels in all samples studied. In turn, other parabens studied in this investigation have been found less frequently and in lower concentration levels. Generally, it is in agreement with the previous investigations, which have reported that MeP is the paraben that is most commonly and abundantly found in the natural environment, including water, soil and air [8,53,54], as well as in humans [55,56] and wild marine animals [39–42]. Moreover, in the present study differences in parabens concentration levels in guano samples between particular bat colonies were clearly visible. It is in agreement with previous observations concerning both human [ 5 , 6 , 8 , 54 ] and wild animal exposure (Table 5) to parabens, which unequivocally confirm that paraben levels noted in the living organisms depend to a large extent on the place, where the study is performed. It relates to the human origin pollution of the natural environment, human population density, industrialization, as well as the frequency of using cosmetics, personal care products and medicines by the inhabitants of a given area [ 1 , 5 , 6 ]. Moreover, clear differences in parabens concentration levels in particular guano samples collected in the same colony were also noted in the present study. This situation has been also observed in previous studies on parabens levels in wild animals (Table 5) and in humans living in the same area. For example, the levels of MeP in the human urine samples collected in Queensland (Australia) ranged from 74.4 to 1180 ng/mL [ 55 ], and samples collected in Seville (Spain) achieved from 68.3 to 14,187 ng/g [ 56 ]. The present results strongly confirm that local factors may influence on the parabens levels in particular individuals and also in wild bats. Table 5. Selected previous studies on paraben concentration levels in wild animals. Paraben concentration levels are shown in ng/g (in solid matrices) or ng/mL (in liquid matrices). Species Localization Matrix nConcentration Levels Ref. MeP EtP PrP BuP Dolphin (various species) Gulf of Mexico, Florida (USA) liver 17 <41.1–865 n.d. <2.05–3.47 n.d. [39] blubber 17 n.a.–<8.21 n.d. n.d. n.d. Pygmy sperm whale Atlantic ocean/Anciote river liver 3 <20.5–37.7 n.d. <4.10 n.d. blubber 3 n.a. n.d. n.d. n.d. Sea otter California Coast (USA) liver 25 n.a.–126 <4.1–11.8 <1.03–3.95 n.d. kidney 10 n.a.–360 n.d. n.d. n.d. brain 10 5.99–77.2 <1.03–3.26 n.d. n.d. Washington Coast (USA) liver 18 <20.5–358 n.d. n.d. <4.1–31.8 Northern sea otter Alaskan Coast (USA) liver 14 n.a.–686 <8.21–31.6 n.d. n.d. Polar bear Alaska (USA) liver 10 <4.10–16.9 n.d. n.d. n.d. Int. J. Environ. Res. Public Health 2023,20, 1928 8 of 17 Table 5. Cont. Species Localization Matrix nConcentration Levels Ref. MeP EtP PrP BuP Common dolphin Korean coastal waters blubber 6 1.3–7.9 [40] muscle 6 13–121 melon 6 4.8–12 stomach 6 44–118 liver 6 13–224 testis/ ovary 6 26–76 brain 5 4.2–50 uterus 2 37–74 Finless porpoises Korean coastal waters blubber 6 6.4–21 muscle 6 12–88 melon 6 21–71 stomach 6 75–228 kidney 6 181–359 liver 6 235–569 testis/ovary 6 44.3–256 stomach content 2 59–76 Fish (various species) Greater Pittsburgh Area (USA) brain 58 n.d. n.d. n.d. n.d. [41] Fish (various species Taihu Lake (China) muscle 199 88.1–1200 33.6–450 55.3–543 <LOQ–40.0 [57] Fish and bivalves (various species) Atlantic ocean, pacific Ocean, Mediterranean Sea, Victoria lake (Uganda) body 64 0.8–32 n.d. n.d. n.d. [58] Fish (various species) New York State (USA) muscle 50 n.a.−690 n.d. n.d. n.d. [42] Fish (various species) Florida (USA) liver 6 <2.01–44.3 n.d. n.d. n.d. muscle 6 n.a.–43.9 n.d. n.d. n.d. kidney 1 18.8 n.d. n.d. n.d. gill 1 71 n.d. n.d. n.d. brain 1 735 n.d. n.d. n.d. Black bear Michigan (USA) liver 2 33.5–58.2 n.d. n.d. n.d. kidney 2 24.0–37.6 n.d. n.d. n.d. Sea eagle Baltic Sea area liver 20 <8.01–657 n.d. n.d. n.d. Albatrosses (various species) Sand Island, Midway (USA) liver 15 <8.01–23.4 n.d. n.d. n.d. kidney 16 <8.01–18.2 n.d. n.d. n.d. brain 12 <1.99–6.66 n.d. n.d. n.d. fat 10 n.a. n.d. n.d. n.d. muscle 12 <2.01–15.7 n.d. n.d. n.d. eggs 13 n.a. n.d. n.d. n.d. Bald eagle Michigan (USA) liver 1 796 n.d. n.d. n.d. plasma 15 <0.2–0.37 n.d. n.d. n.d. kidney 1 580 n.d. n.d. n.d. muscle 2 87.4–169 n.d. n.d. n.d. Herring gull Michigan (USA) eggs 9 <1.99–14.0 n.d. n.d. n.d. Common cormorant Michigan USA) eggs 4 n.a. n.d. n.d. n.d. Loon Carteret County (USA) liver 10 <8.01–336 n.d. n.d. n.d. Loon Maine and New Hampshire (USA) eggs 15 <1.99–22.2 n.d. n.d. n.d. Fish (various species) Yangtze river (China) plasma 36 11.6–39.5 0–10.5 0–6.97 0–b1.46 [38] Fish (various species) Yangtze river (China) bile 35 8.17–21.9 0–31.6 2.19–112 0–4.42 [59] Fish (various species) Spain/various rivers muscle 59 n.d.–84.69 n.d.–0.82 n.d.–7.43 [60] Striped Catfish Colombia/various rivers muscle 20 <20–32.0 [61] Int. J. Environ. Res. Public Health 2023,20, 1928 9 of 17 Table 5. Cont. Species Localization Matrix nConcentration Levels Ref. MeP EtP PrP BuP Clams Antarctica body 7 <2.1–5.8 n.d. n.d.–5.3 n.d. [62] Sea urchin body 1 5.7 n.d. n.d. n.d. Fish (Emerald rockcod) body 7 5.1–26.9 n.d. n.d n.d. liver 1 2.4 n.d. n.d. n.d. MeP: methylparaben; EtP: ethylparaben; PrP: Propylparaben; BuP: butylparaben; n.a.—not available; n.d.—not detected; Ref.—references. The comparison of present results with previous investigations is difficult, because in spite of the fact that paraben levels have been determined in some species of wild animals (Table 5), till now, according to the best knowledge of authors, feces/guano samples were not used at all in this type of research. Moreover, the majority of previous studies concern marine animals (fish, birds or mammals), and only very limited observations have been conducted on terrestrial animals (Table 5). In addition previous studies have been conducted in in completely different parts of the world than the present research. For these reasons, it is difficult to relate the results concerning MeP concentration levels in bat guano to data from previous literature (Table 5). On the other hand, levels of other parabens observed in bat guano samples are higher than those noted in the majority of previous studies on wild animals, in which these parabens were usually not detected (Table 5). Due to such a high correlation between the study site and exposure to parabens, it seems advisable to analyze the levels of parabens in Poland to better understand the obtained results. It should be pointed out that knowledge about this issue is relatively limited in comparison to studies in other parts of the world. It is known that parabens are present in surface water in Poland. In lake water, MeP concentration level amounted from 1.7 to even 1578 ng/L [ 63 ]. The levels of other parabens are clearly lower and achieved values 0.8–27.5 ng/L for EtP, 0.5–93.9 ng/L for PrP and 0.6–22.6 ng/L for BuP [ 63 ]. Much higher levels of parabens have been observed in wastewater, where limits of MeP, EtP, PrP and BuP achieved 2235.0–40,898.6 ng/L, 791.2–8169.4 ng/L, 542.2–7803.3 ng/L and 68.8–710.7 ng/L, respectively [ 64 ]. Moreover, parabens have also been found in the fur of dogs living in Poland. In this case mean concentration levels of MeP, EtP and PrP achieved 176 ng/g, 48.4 ng/g and 79.8 ng/g, respectively [28]. Despite other matrices used in previous studies and the comparative difficulties resulting from this, it can be concluded that the parabens levels in wild bat guano in Poland are generally lower than the levels of these compounds in humans and domestic animals [ 28 , 65 – 67 ]. This is logical due to the fact that humans create these environmental pollutants and are exposed to them through the use of cosmetics, personal care products, medicines and food containers [ 1 , 5 , 6 ]. In turn, companion animals, which live in close proximity to humans, have direct contact with the owners and are exposed to similar factors as humans [ 28 ]. Unlike humans and domestic animals, bats that are exposed to parabens, which can be found in the environment such as in water, air or food, tend to have a lower degree of exposure, even if their summer colonies are located near human populations. In addition to parabens, the presence of BPA in bat guano samples was also found in the present study. However, BPA concentration levels were lower than MQL in all samples investigated. To date, only one study has described bat exposure to BPA. In that study, BPA in the median concentration level of 397 ng/g was described in bat carcasses collected in the northeastern United States [ 68 ]. Therefore, BPA concentration levels noted in the present study are clearly lower. Other previous studies (more numerous than studies on parabens) have described BPA in various aquatic animals including fish, mussels, prawns, mollusks, as well as seabirds [34–37]. 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