Biomonitoring of benzophenones in guano samples of wild bats in Poland.

Benzophenones (BPs) are a group of substances consisting of two phenyl groups linked to a carbonyl group [ 1 ]. Some BPs are of natural origin and are mainly produced by fungi and plants [ 2 ]. Some of them are synthesized for various branches of industry with an estimated annual production of 10,000 tons [ 3 ]. BPs absorb ultraviolet (UV) radiation, and therefore, they are foremost used to produce sunscreens with UV filters, cosmetics, flavorings, perfumes, hair dyes, shampoos, and personal care products [ 4 , 5 ]. Moreover, BPs are part of food containers, bottles, paints, inks, plastics, car polishes, fabrics, and many other items, where they act as UV stabilizers [ 1 , 6 , 7 ]. There are 12 main benzophenone derivatives however, benzophenone 1 (BP-1, 4-dihydroxybenzophenone), benzophenone 2 (BP-2, 2,2’,4,4’-tetrahydroxybenzophenone), benzophenone 3 (BP-3, 2-hydroxy-4-methoxybenzophenone, oxybenzone) and benzophenone 8 (BP-8, dioxybenzone) are the most common in industrial use ( Fig 1 ) [ 3 ]. Structure of studied benzophenones (BPs): A) BP-1; B) BP-2; C) BP-3; D) BP-8. Additionally, the BPs levels in sunscreens and cosmetics, as well as in the environment, can increase significantly under favorable temperature and time conditions, probably due to the degradation of octocrylene—an organic compound commonly used in the beauty industry [ 5 , 8 ]. Due to their widespread industrial use, BPs are increasingly reaching the natural environment. Such pollution by BPs is favored by their chemical properties, namely high stability, resistance to UV radiation, and ability to accumulate in water [ 9 ]. Previous studies have reported the presence of BPs, primarily in the inland surface and oceanic waters [ 10 – 12 ]. BPs also pollute tap water, air and soil, especially agricultural treated with compost from sewage sludge [ 13 – 15 ]. These substances may also penetrate edible for humans and animals plants [ 14 , 16 , 17 ]. BPs levels clearly depend on the urbanization and industrialization of the region, where the studies have been conducted, as well as on human activity in a given area [ 14 , 18 , 19 ]. The presence of BPs in various elements of the environment results in the exposure of humans and animals to these substances, which may get inside the living organisms through the digestive tract, skin, and respiratory system, as well as through the placenta during prenatal period [ 3 , 20 – 23 ]. Exposure to BPs can cause disturbances in the functions of many internal organs since BPs may bind to receptors of various hormones, including estrogen, androgen, and thyroid hormone, and therefore show endocrine disrupting properties, which depend on the type of BP, dose, and animal species or type of cells [ 18 ]. Furthermore, BPs show genotoxic and mutagenic properties [ 24 ]. It is known that BPs affect male, and female reproductive systems, which manifests in perturbation in sperm development, decreased number of oocytes, prolonged estrus cycle, and disorders in the early germ cell development [ 24 – 26 ]. Some studies have also described the connections between BPs and infertility and the risk of endometriosis [ 27 , 28 ]. In turn, BPs cause several changes within the nervous system, including i.e. alterations in the epigenetic state of neuronal cells, mitochondrial membrane potentials, and various levels of neuronal factors, which in turn can lead to abnormal brain development and an increased risk of neurodegenerative diseases [ 24 , 29 ]. Some studies have found that BPs have obesogenic [ 30 ] and carcinogenic properties manifested by increased tumor cell proliferation and vascularity with simultaneous decrease in tumor cell apoptosis [ 31 ]. Due to abovementioned adverse effects of BPs, it is important to monitor the exposure to these substances in humans and animals. Many analytical methods have been developed to determine the concentration of these compounds in various matrices e.g. gas chromatography and high-performance liquid chromatography in combination with tandem mass spectrometry (GC-MS/MS, HPLC-MS/MS) being the most common [ 32 , 33 ]. Other methods include high-performance liquid chromatography with ultraviolet spectroscopy (HPLC-UV), micellar electrokinetic chromatography with ultraviolet spectroscopy (MEKC-UV), ultra-high performance supercritical fluid chromatography- photo diode array detector (UHPSFC-PDA), low temperature plasma ionization mass spectrometry (LTP-MS), thin-layer chromatography with UV spectroscopy (TLC-UV), supercritical fluid chromatography with UV spectroscopy (SFC-UV), and gas chromatography-electron capture detector (GC-ECD) analysis [ 32 , 34 ]. The vast majority of previous studies focused on the BPs exposure in humans, and the presence of these compounds has been confirmed in various sample types [ 35 ]. BPs have been described not only in typical matrices such as urine, [ 36 , 37 ] whole blood, serum, and plasma [ 3 , 38 , 39 ], but also i.e. amniotic fluid [ 3 ], seminal plasma [ 39 ], placenta, [ 40 ] and breast milk [ 41 ]. Up to date research on humans have clearly shown that BPs levels depend on the part of the world, where investigations have been performed. Differences in BPs levels are not only linked to urbanization and industrialization, but also the type of work, habits and lifestyle, such as the frequency of cosmetic use [ 35 , 42 , 43 ]. Moreover BPs levels clearly depend on matrix used in analysis [ 37 , 39 , 41 ]. Contrary to humans, knowledge about wildlife exposure to BPs is relatively limited. Studies mainly describe the BPs concentrations in aquatic organisms because BPs, (especially BP-3), are particularly harmful to aquatic ecosystems due to their high water solubility and bioaccumulation potential [ 44 – 46 ]. Previous studies have shown that exposure to BP-3 results i.e. in various disturbances of the nervous system of zebrafish, which manifests in behavioral deficits learning and memory impairment, decreased neurogenesis, increased apoptosis within the brain and alteration in the enzymatic activity [ 47 , 48 ]. Thus far the exposure to BPs have been described in wild marine mammals [ 49 ], turtles [ 50 ], fish [ 18 , 51 ], mussels [ 52 , 53 ], free-living flatworms [ 54 ], prawns, and urchins [ 55 ]. In addition, BPs have also been found in the eggs and internal organs of wild marine and terrestrial birds [ 56 – 58 ]. To the best knowledge of the authors, there are no studies on the exposure of terrestrial wild mammals to BPs with the exception of our earlier publication describing the methodology of examining bat guano for the presence of BPs on a very limited number of animals (n = 5) [ 59 ]. On the other hand, it is known that BPs have a strong harmful effects on terrestrial mammals. The majority of previous studies concerning this issue have been conducted on rodents and have shown that BPs affect blood hormone levels and hematological parameters, have adverse effect on the nervous system, and disrupt the functioning of the female and male reproductive systems [ 60 – 62 ]. However, it is very likely that terrestrial animals are exposed to BPs present in different elements of the natural environment. One group of animals, which play an important role in the ecotoxicology are bats. It is a large group of animals of over 1400 species inhabiting different climate zones [ 63 ]. Bats are highly sensitive to environmental pollution and environmental changes [ 64 , 65 ], which is the reason for the continuous reduction of their population and the protection of many bat species [ 66 – 68 ]. Due to their sensitivity to environmental factors, bats are often used as a bioindicator of environmental quality during toxicological studies [ 69 , 70 ]. Moreover, previous studies have shown that bats are highly exposed to various environmental pollutants, including e.g. heavy metals, organochlorine contaminants, parabens and pesticides, and bat guano is an appropriate matrix for such studies [ 71 – 73 ]. Matrix selection is a substantial problem in studies on wild animal exposure to environmental pollutants, when the levels of the investigated substances are determined. Obtaining vast majority of matrices used in toxicology e.g. urine, blood, or hair is practically impossible without restraining the animals, which is a strong stressor and a significant interference with their life and environment. This is particularly important in studies on protected species. Guano samples are practically the only matrix, which may be collected without significant interference [ 74 , 75 ]. Moreover, our previous study has shown that guano samples can be used to determine BPs levels [ 59 ]. The above facts speak in favor that bats may be exposed and particularly sensitive to the harmful effects of BPs polluting the environment. Therefore this study aimed to answer the question whether and to what extent wild bats are exposed to BPs polluting the environment, as well as to determine whether the degree of this exposure depends on local factors. For this purpose the concentration levels of selected BPs commonly used in the industry (BP-1, BP-2, BP-3, BP-8) were evaluated in guano samples collected from the greater mouse-eared bats ( Myotis myotis ) living in various parts of Poland. This study, to the best of our knowledge, is the first investigation on the exposure of terrestrial mammals to BPs and first to use oguano samples and will contribute to expanding knowledge on BPs in wild animals.

BP-1 was found in all samples included in the study (S3 Table in S1 File ). Furthermore, its concentrations were the highest among the tested substances ( Table 2 ). BP-1: benzophenone 1; BP-2: benzophenone 2; BP-3: benzophenone 3; BP-8: benzophenone 8; MQL—Method Quantification Limit ((0.10 ng/g dw); MDL—Method Detection Limit (0.04 ng/g dw) BP-1 concentration fluctuated from <0.1 ng/g to 259 ng/g (mean 41.50 ng/g) and its concentration above MQL was observed in 97.5% of studied samples (S3 Table in S1 File ). The second most common BP was BP-3, with concentration higher than MQL observed in 40% of the samples ( Table 2 ). Its concentration levels fluctuated from <0.1 ng/g to 19 ng/g with a mean value of 6.67 ng/g. Noticeable differences in the BP-1 and BP-3 levels were observed even between samples collected from the same colony. Colony no.2 had the most prominent differences in BP-1 levels, which fluctuated from 30.5 ng/g to 259 ng/g (S3 Table in S1 File ). Contrary to BP-1 and BP-3, the concentration of BP-2 and BP-8 did not exceed the method detection limit (MDL) (0.1 ng/g) in any studied sample (S3 Table in S1 File ). Furthermore, clear differences in BP-1 levels were found between studied bat colonies ( Fig 3 ). Higher concentrations of this substance were found in bat colony no. 2 and no. 3, with the mean values (±SD)76.71±65.51 ng/g and 56.97±14.42 ng/g, respectively. In colonies no. 1 and no. 3 BP-1levels were statistically significantly lower than in colonies no. 2 and no. 3 and achieved 18.08±6.81 ng/g (colony no.1) and 11.23±13.13 ng/g (colony no. 4) ( Fig 3A ). Mean concentration levels of BP-3 amounted to 5.03±6.03 ng/g in colony no. 1, 8.02±9.31 ng/g in colony no. 2, 5.76 ng/g in colony no. 3 and 9.18±7.65 ng/g in colony no. 4 ( Fig 3B ). There were no statistically significant differences in BP-3 between colonies. Mean concentration (±SD) of benzophenone 1 (BP-1) (A) and benzophenone 3 (BP-3) (B) in studied bat colonies. Statistically significant differences (p < 0.05) were marked with different letters, values not showing such differences—with the same letters. Bat colony in Brenna (1), Sliwice (2), Pulawy (3) and Opole Lubelskie (4). The figure was created using GraphPad Prism version 9.2.0 (GraphPad Software, San Diego, California USA).

The present research is the first one to focus on the exposure of wild terrestrial mammals to BPs through the analysis of guano samples. Obtained results demonstrated that wild bats are exposed to BP-1 and BP-3, which confirms environmental contamination with these substances. These observations also strongly suggest that BPs polluting the environment may affect the health of wild terrestrial mammals and bats seem to be a good bioindicator for monitoring BPs levels in the environment. Furthermore, our study has shown that despite some limitations guano samples are a good alternative to urine or blood samples in the assessment of the wild animal exposure to BPs. Such samples are of particular importance in research on protected wild animals, because collection of such samples is simple and does not require (unlike other matrices) capturing specific animals. However, due to intraspecies differences between BPs metabolism and toxicokinetics, and the lack of knowledge about the correlation between the presence of BPs in the guano and their distribution in internal organs, it is difficult to accurately assess the BPs levels in guano that are a sign of exposure to these substances harmful to the animal health. Therefore, further studies regarding this matter is crucial.

The following reagents were used: formic acid and ammonium acetate from Panreac (Barcelona, Spain); C18 disperse sorbent from Scharlab (Barcelona, Spain); HPLC-grade methanol, and water from Romil (Barcelona, Spain); Standards of benzophenone-1 (BP-1, purity: 99%), benzophene-2 (BP-2, purity: 97%), benzophenone-3 (BP-3, purity: 98%), benzophenone-8 (BP-8, purity: ≥98%) from Sigma-Aldrich (Steinheim, Germany). Isotopically labelled BP used as internal standard (BP-d 10 ) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Individual stock standard solutions were prepared at 1000 mg/L in methanol and kept at -18°C. Working solutions were made up by dilution of the stock standard solution in methanol. Bat guano samples were collected from August to September 2021 from four summer (nursery) colonies of greater mouse-eared bats ( Myotis myotis ) located in various regions of Poland ( Fig 2 ). Localization of studied bat colonies 1) Brenna; 2) Śliwice; 3) Pulawy; 4) Opole Lubelskie. The greater mouse-eared bat was chosen because it is the most common bat species in Poland despite being endangered species. This fact made sample collection easier, and such research can contribute to the protection of its population because the greater mouse-eared bat is especially sensitive to the environmental pollution with substances of human origin due to the fact that it establishes summer colonies in close proximity to human settlements. The characterization of bat colonies included into the study is presented in Table 1 . Data according to statistical yearbooks published by the Central Statistical Office in Poland, https://stat.gov.pl/obszary-tematyczne/roczniki-statystyczne/ Sample collection was performed as follows: flat glass containers were distributed throughout the floor where bat colony was located. After 48 hours, containers were removed and bat guano samples were placed in glass jars, frozen, and stored at -20°C for further analysis. Ten guano samples collected from each bat colony were included in the investigation. Sample collection was carried out in a way that did not scare or stress the animals. Due to the non-invasive character of guano collection this study did not need the approval of the Local Ethics Committee for Animal Experiments based on the Polish law (the Act for the Protection of Animals for Scientific or Educational Purposes of 15 January 2015 Journal of Laws 2015, No. 266), Moreover, building administrators and personnel responsible for the protection of specific bat colonies gave verbal consent to collect samples. The samples were transferred to dark glass using sterile glass vanes, frozen at -22°C and stored at this temperature until further analysis. Sample preparation was carried as previously described by Martin et al. [ 59 ]. Guano samples were lyophilized, homogenized and grounded into powder. Then 1g of each sample was weighed into a 12 mL glass tube and supplemented with 100 μL of a methanol solution of BP-d 10 (250 ng/mL). At that point sample was sonicated with 7 mL of methanol (0.5% v/v, formic acid) as extraction solvent in a bath for 5 mins and centrifuged for 5 mins (4050 × g). The extraction procedure was repeated three times, and the supernatants were combined. To remove interferences from the matrix, a clean-up procedure based on dispersive solid phase extraction (d-SPE) was performed by adding 0.3 g of C18 sorbent. The tubes were shaken vigorously for 2 mins and then centrifugated at 4050× g for 5 mins. The supernatant was evaporated under a nitrogen stream, reconstituted in 0.25 mL of a mixture methanol:water (50:50 v/v) until dry and filtered through a 0.22 μm nylon filter. A 10 μL aliquot was injected into the liquid chromatography tandem mass spectrometer (LC-MS/MS). Chromatographic analyses were performed using an Agilent 1260 Infinity II (Agilent, Santa Clara, CA, USA) as described by Martín et al. [ 59 ]. Separation was performed with a HALO C18 Rapid Resolution (50 x 4.6 mm i.d., 2.7 μm) (Teknokroma, Barcelona, Spain). The mobile phase consisted of methanol (solvent A) and a buffer solution acetic acid/ammonium acetate (pH 4.4) (solvent B). The elution program was as follows: 0–14 min, linear gradient from 28 to 70% of solvent A, increased to 80% of A in 5 min and to 100% of A in 6 min and held for 2 min. Flow rate was 0.6 mL/min. The LC system was coupled to a 6495 triple quadrupole mass spectrometer with electrospray ionization source operated in negative mode. Two multiple reaction monitoring (MRM) transitions, for identification and quantification purposes, were selected for each benzophenone. Instrument settings and analytical determination parameters are summarized in the Supplementary Material (S1 Table in S1 File ). The analytical features (linearity, sensitivity and accuracy (trueness and precision)) of the method are presented in S2 Table in S1 File . A matrix-matched calibration was performed for quantification purposes and to correct matrix effect. To this end, commercial guano samples were fortified with the analytes at eight different concentrations within the range from method quantitation limit (MQL) up to 100 ng/g dry weight—dw (0.1, 0.25, 0.5, 1.00, 5.00, 25.0, 50.0 and 100 ng/g dw). The commercial guano (“SuperGuano” from Top Crop - www.topcropfert.com ) used for matrix-matched calibration was simultaneously analyzed. Commercial guano used in the study is 100% natural, collected in caves, where bats live far from the human influence. Note that the origin of the commercial guano used in matrix-matched comes from insectivores, like those analyzed in this study. BPs were not detected in the commercial guano samples. To guarantee the quality of the obtained results, a protocol involving the use of control spiked samples including fortified commercial guano samples (25 ng/g dw), solvent, standards containing a mixture of the target compounds in pure solvent (100 ng/mL) and procedural blanks (without guano, processed the same way as the studied samples)were included into each analytical batch (5 samples). The statistical analysis was performed using GraphPad Prism version 9.2.0 (GraphPad Software, San Diego, CA USA) and a Kruskal–Wallis test was used to determine the differences in BP-1 and BP-3 levels in selected bat colonies. Differences were considered statistically significant at p < 0.05.

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