Environmental Pollution 253 (2019) 199e206

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Environmental Pollution

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Wing membrane and fur samples as reliable biological matrices to measure bioaccumulation of metals and metalloids in *

* Rúben Mina a, 1, Joana Alves a, , 1, Antonio Alves da Silva a, Tiago Natal-da-Luz a, Joao~ A. Cabral b, Paulo Barros b, Christopher J. Topping a, c, Jose Paulo Sousa a a Centre for Functional Ecology (CFE), Department of Life Sciences, University of Coimbra, Portugal b CITAB - Centre for Research and Technology of Agro-Environment and Biological Sciences, Laboratory of Applied Ecology, University of Tras-os-Montes e Alto Douro, Vila Real, Portugal c Department of Bioscience, Aarhus University, Rønde, Denmark article info abstract

Article history: There is a growing conservation concern about the possible consequences of environmental contami- Received 11 April 2019 nation in the health of communities. Most studies on the effects of contaminants in bats have been Received in revised form focused on organic contaminants, and the consequences of bat exposure to metals and metalloids remain 26 June 2019 largely unknown. The aim of this study was to evaluate the suitability of external biological matrices (fur Accepted 29 June 2019 and wing membrane) for the assessment of exposure and bioaccumulation of metals in bats. The con- Available online 1 July 2019 centration of arsenic, cadmium, cobalt, chromium, copper, manganese, nickel, lead, selenium and zinc was measured in internal organs (liver, heart, brain), internal (bone) and external tissues (wing mem- Keywords: Chiroptera brane, fur) collected from bat carcasses of four species (Hypsugo savii, Nyctalus leisleri, Pipistrellus Metal bioaccumulation pipistrellus, Pipistrellus pygmaeus) obtained in windfarm mortality searches. With the exception of zinc External biological matrices (P ¼ 0.223), the results showed significant differences between the concentrations of metals in the Fur analyzed tissues for all metals (P < 0.05). Significant differences were also found between organs/tissues Wing membrane (P < 0.001), metals (P < 0.001) and a significant interaction between organs/tissues and metals was found (P < 0.001). Despite these results, the patterns in terms of metal accumulation were similar for all samples. Depending on the metal, the organ/tissue that showed the highest concentrations varied, but fur and wing had the highest concentrations for most metals. The variability obtained in terms of metal concentrations in different tissues highlights the need to define standardized methods capable of being applied in monitoring bat populations worldwide. The results indicate that wing membrane and fur, biological matrices that may be collected from living bats, yield reliable results and may be useful for studies on bats ecotoxicology, coupled to a standardized protocol for large-scale investigation of metal accumulation. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction environmental changes due to anthropogenic causes, namely habitat loss through intensive agriculture, forestry, urbanization The populations of bat species are declining across Europe in and industrialization, and/or changes in water quality and trophic response to several environmental stressors. Worldwide, more contamination by and metals, are considered major than 15% of bat species are threatened according to the Interna- drivers of loss of bat species richness worldwide (Mickleburgh tional Union for Conservation of Nature (IUCN, 2017). The ongoing et al., 2002; Walker et al., 2007; Blehert et al., 2009; Jones et al., 2009; Pikula et al., 2010; Hernout et al., 2013; Hernout et al., 2015; Zukal et al., 2015; Hernout et al., 2016a; O'Shea et al., 2016; * This paper has been recommended for acceptance by Prof. Wen-Xiong Wang. Chetelat et al., 2018). There is a growing conservation concern * Corresponding author. Centre for Functional Ecology (CFE), Department of Life about the possible consequences of environmental contamination Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, by metals in the composition and functioning of bat communities Portugal. (Zukal et al., 2015; Flache et al., 2015; Hernout et al., 2016a; Hernout E-mail addresses: [email protected], [email protected] (J. Alves). et al., 2016b; Chetelat et al., 2018). Given their relatively long life 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.envpol.2019.06.123 0269-7491/© 2019 Elsevier Ltd. All rights reserved. 200 R. Mina et al. / Environmental Pollution 253 (2019) 199e206 span and high metabolic rates, requiring high daily food intake 1967; Vahter, 1981; Lansdown, 1995; Magelsir, 2016; Rose, 2016), rates, bats can be particularly prone to chemical exposure, espe- and is supplied with a high volume of blood flow which can transfer cially to contaminants such as metals that accumulate through the metals to the wing; third, as wing membrane has a high regener- food chain (Hernout et al., 2016a). The coexistence of bats with ative ability (Faure et al., 2009; Weaver et al., 2009), healing humans in urban, industrial and/or intensive agricultural land- completely in a few weeks, its use as biological matrix does not scapes (Zukal et al., 2015), combined with the fact that some bat exercise a lasting negative effect on the bats, similar to fur. species feed on emerging insects that spend their larval stages in The aim of this study was to evaluate the metal accumulation in sediment and water where contaminants may have accumulated carcasses of four insectivorous bat species, and to investigate the (Hickey et al., 2001; Flache et al., 2016), are other factors which suitability of fur and wing membrane samples, to monitor metal make bats particularly susceptible to bioaccumulate metals. exposure in bats by comparing metal concentrations from these Furthermore, bats are usually at relatively high trophic levels, matrices with those from several internal organs (liver, heat, brain) which can contribute to the high accumulation of metals through and tissue (bone). We hypothesize that metal concentrations on biomagnification (Yates et al., 2014). Bats may be exposed to metals wing membrane provide a suitable indication of the overall expo- through different pathways, such as inhalation, contact with sure of bats to metals, being correlated with the metal concentra- contaminated soil, ingestion of contaminated water, and con- tions found in internal organs and fur. Furthermore, we expected to sumption of contaminated prey (Clark and Shore, 2001; Zocche provide valuable practical information to help design future sam- et al., 2010; Hernout et al., 2013). pling protocols for assessing metal exposure on bats. Until now, several studies on the effects of contaminants in bats have focused on organic contaminants (Hickey et al., 2001; Walker 2. Materials and methods et al., 2007; Zukal et al., 2015), with the consequences of exposure to other substances, particularly metals (Hernout et al., 2013), 2.1. Study area and sample collection remaining largely unknown. Even so, some studies have reported effects of metal accumulation on bats, such as hepatopathy, DNA In this study, we used bat carcasses collected in north and damage, hemochromatosis, renal inclusion bodies, ascending pa- central Portugal between August and October of 2006e2014, dur- ralysis, tremors, spasms, general slowness, lack of control in body ing ecological monitoring programs to estimate the impact of wind movement and mortality (Sutton and Wilson, 1983; Hariono et al., farms on bat communities. We collected a total of 56 individuals of 1993; Skerratt et al., 1998; Hoenerhoff and Williams, 2004; Farina four species (Hypsugo savii, Nyctalus leisleri, Pipistrellus pipistrellus, et al., 2005; O'Shea and Johnston, 2009; Zocche et al., 2010; Nam Pipistrellus pygmaeus) and froze then at 20 C for subsequent et al., 2012). More recently, Lovett and McBee (2015) reported analyses. The four species studied are insectivorous and belong to behavioral effects on bats caused by metal contamination. These the family , and occur in a wide range of habitats authors found a possible alteration on circadian rhythms of bats, and with synurbic habits. The exception is N. leisleri, which is a tree- wherein bats from a site contaminated with lead (Pb), cadmium dwelling bat (further details on Table S1). (Cd) and zinc (Zn) exhibited a different pattern of emergence when We divided the biological samples collected from bat carcasses compared with bats from uncontaminated locations. into internal (liver, heart, bone and brain) and external (wing Identifying patterns of exposure and analyzing the potential membrane and fur) samples. Although we used only carcasses from bioaccumulation of contaminants in bats is difficult due to the lack freshly dead bats (1e2 days in the landscape; unaltered by scav- of non-lethal or less intrusive sampling methods. Given the con- enging animals and without signs of fly larva infestation; Grodsky servation status of bats worldwide, the use of experimental in-vivo et al., 2012), we could not collect all the organs from all the car- bat models to obtain standard toxicological data may not be ethi- casses due to the internal damages caused by the impact of the cally feasible. Thus, we believe that the use of external tissue wind turbines. These constraints resulted in differing numbers of samples, such as fur and wing membranes, may be a good strategy samples for different organs/tissues. to understand contaminant loads in bat populations. Several ad- Stainless steel dissection tools were used for dissection of bats. vantages have been listed specifically for the use of fur in moni- All tools were cleaned in acetone after use on each sample to avoid toring studies. Firstly, fur sampling is easy and involves minimal cross-contamination. Internal samples were always composed of stress to individuals (Schramm, 1997). Secondly, concentrations of the entire organ. The bone used for this study was the forearm (i.e. metals in fur are usually higher than those in biological fluids like ulna and radius), which was measured during the dissection of the blood and urine, and sometimes even higher than organ concen- individuals. Small samples of fur were clipped from the mid-dorsal trations (e.g., Hariono et al., 1993; Halbrook et al., 1994; Liu, 2003). region of each bat, about 1e2 mm above the skin, corresponding to Finally, some studies confirmed that fur may be an indicator of an area of approximately 1 cm2. The sampling of the wing mem- internal organ concentrations for several metals found in other brane consisted of the collection of four punches with a diameter of mammals (Hariono et al., 1993; Halbrook et al., 1994; Nolet et al., 4 mm from each wing, between the 4th and the 5th digits (dacty- 1994). As such, fur has been increasingly used in ecotoxicological lopatagium major). studies in bats (e.g. Hickey et al., 2001; Flache et al., 2015; Hernout Fur and wing membrane, being external samples, were washed et al., 2016a; Åkerblom and de Jong, 2017; Moreno-Brush et al., once with detergent (Triton.X-100), twice with acetone and three 2018). times with distilled water. This process was carried out to eliminate However, another potentially useful external biological matrix is external contamination and thus, to ensure that the concentration the punch of the wing membrane of bats to collect tissue, usually of metals obtained came only from bioaccumulation. After dissec- used for molecular analyses or to mark animals in the field. To our tion, the samples were oven dried at 45 C for 72 h and then knowledge, the wing membrane has not been previously used to weighed (±0.0001 g). quantify the concentration of metals in bats. Wing membrane samples may be a good non-lethal method for three main reasons: 2.2. Metal extraction and quantification first, wing membrane is highly exposed to the external environ- ment, potentially leading to direct accumulation of contaminants Once all the biological samples were dried, we mixed them with through skin absorption; second, the skin of bats has the capacity to 1 ml of 65% nitric acid (except for the wing membrane that was accumulate metals, as it occurs in other mammals (Schroeder et al., mixed with 0.5 ml) and left under pressure in PDS-6 pressure R. Mina et al. / Environmental Pollution 253 (2019) 199e206 201 digestion systems (Loftfields analytical solutions, Neu Eichenberg, using Bonferroni correction. Model validation was performed by Germany) at 150 C for 10 h. The resulting solutions were diluted inspecting the residuals for normality, homogeneity, and with ultrapure water to a final volume of 6.5 ml (for internal sam- independence. ples and fur samples) or 3.25 ml for wing membrane samples, to Correlations between the metal concentrations in the different obtain a final extract within the calibration range and with an acid sample types were measured using Pearson's correlations (r). This concentration of about 10%. analysis was performed individually for each metal and for all the The concentration of arsenic (As), cadmium (Cd), cobalt (Co), possible combinations of tissues (i.e. bone vs brain; bone vs heart; chromium (Cr), copper (Cu), manganese (Mn), nickel (Ni), lead (Pb), bone vs liver; bone vs wing; bone vs fur; brain vs heart; brain vs selenium (Se) and zinc (Zn) was measured in an ICP-MS spectro- liver; brain vs wing; brain vs fur; heart vs liver; heart vs wing; heart photometer (Model iCAP Q, Thermo Fisher Scientific, Bremen, vs fur; liver vs wing; liver vs fur; wing vs fur). Germany). Accuracy and precision of the extraction and analytical All the statistical tests were considered significant when ® methods were evaluated by analyzing a certified reference material P < 0.05. The statistical analysis was performed using IBM.SPSS , (DOLT-3 - Dogfish Liver Certified Reference Material for Trace version 23, and R 3.3.2 (R Core Team 2017). Metals certified by National Research Council ) and blanks. Standard solutions were prepared by appropriate dilutions of a 3. Results multielement standard (92091, Periodic table mix 1 for ICP, Sigma- Aldrich). The calibration of ICP-MS measurements was ensured by There were no significant differences between metal concen- using a 5-point calibration curve per element. The detection limits trations obtained in the different sampling locations (F(9,45) ¼ 1.241; obtained were 0.015, 0.002, 0.003, 0.007, 0.242, 0.045, 0.086, 0.077, P ¼ 0.295). Significant differences were found between the con- m 0.207 and 0.534 g/g for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Se, and Zn, centrations of metals in each sample type (organ/tissue) for all respectively. The average recoveries reached were 91.6, 87.8, 94.4, species and for all metals (P < 0.05), except for Zn (F(3, 64) ¼ 1.501; 86.7, 98.7, 153, 103 and 111% for As, Cd, Cr, Cu, Ni, Pb, Se, and Zn, P ¼ 0.223). Despite these differences, the organs/tissues with the respectively. Average recoveries of Co and Mn could not be calcu- highest and the lowest metal concentrations in each species were lated since we did not have the reference values for these elements. similar (Fig. 1). In general, Nyctalus leisleri had lower concentrations For statistical analyses, metal concentrations below the detection in all the organs/tissues than the other species (Fig. 1). limit were replaced by the value of the detection limit. All metal Significant differences were also found between sample types concentrations are expressed in mg of metal/g of tissue dry weight. (F(5,2371) ¼ 536.125; P < 0.001), metals (F(9,2346) ¼ 1521.095; P < 0.001) and the interaction between sample type and metals 2.3. Data analysis (F(45,2346) ¼ 27.519; P < 0.001). Depending on the metal, the type of sample presenting the highest concentration varied, but generally Where error distributions were not normal, we transformed the fur and wing membrane had the highest concentrations for most concentration of metals in each sample type (organ/tissue) using a metals (Fig. 2, Table 1). log transformation to achieve an approximation of a normal dis- Depending on the metal analyzed, the accumulation level in the tribution and to reduce heteroscedasticity. We tested the effect of different organs/tissues varied, and consequently, the correlations sample type, species and sampling sites (independent variables) on between them also changed. Significant positive correlations were the different metal concentrations (dependent variables) using found between the concentrations of some metals in external general linear mixed models (GLMM), where the individuals were (wing membrane and fur) and internal organs/tissue samples added as a random effect. Pairwise comparisons were performed (Table 2). Regarding correlations between the two external

Fig. 1. Boxplots of the log-transformed metal concentrations (ng/g dw) in the different organs/tissues for the four bat species studied (Hypsugo savii (N ¼ 10), Nyctalus leisleri (N ¼ 13), Pipistrellus pipistrellus (N ¼ 21), Pipistrellus pygmaeus (N ¼ 12)). 202 R. Mina et al. / Environmental Pollution 253 (2019) 199e206

Fig. 2. Boxplots of the log-transformed metal concentrations (ng/g dw) in the different sample types (organs/tissues) from bat species (N ¼ 56).

Table 1 Metal concentrations (mg/g dry weight) in each sample type (organ/tissue) of bat carcasses collected from wind farms mortality searches carried out in North and Centre of Portugal.

Bone (N ¼ 54) Brain (N ¼ 42) Heart (N ¼ 36) Liver (N ¼ 14) Fur (N ¼ 51) Wing (N ¼ 50) F (df) P value

Median Range Median Range Median Range Median Range Median Range Median Range

As 0.05 (0.01e0.26) 0.20 (0.05e1.61) 0.17 (0.01e3.05) 0.30 (0.07e6.85) 0.88 (0.20e7.62) 0.44 (0.04e2.10) 116.124 (5, <0.001 175) Cd 0.01 (0.00e0.06) 0.04 (0.01e0.10) 0.16 (0.03e1.04) 0.58 (0.17e1.77) 0.06 (0.02e0.57) 0.08 (0.02e0.24) 200.991 (5, <0.001 185) Co 0.05 (0.01e2.66) 0.09 (0.03e8.62) 0.18 (0.03e7.78) 0.12 (0.05e1.05) 0.34 (0.07e7.82) 0.33 (0.10e3.24) 25.803 (5, <0.001 175) Cr 0.56 (0.12e4.10) 1.04 (0.23e4.05) 0.95 (0.07e5.63) 0.39 (0.12e0.79) 2.52 (0.82e8.67) 6.87 (1.29e43.99) 96.233 (5, <0.001 182) Cu 1.23 (0.07e4.33) 13.42 (7.84e28.75) 21.83 (8.70e50.49) 18.23 (11.37 12.56 (6.86e49.63) 18.03 (2.41e54.76) 281.050 (5, <0.001 e33.17) 179) Mn 1.65 (0.63e5.81) 2.15 (1.00e5.25) 6.49 (2.12e15.66) 8.25 (1.34e22.10) 11.37 (3.70e110.99) 7.84 (2.49e27.09) 139.036 (5, <0.001 177) Ni 0.37 (0.05e38.30) 0.76 (0.11e8.68) 0.58 (0.03 0.51 (0.16e3.45) 2.60 (0.58e44.86) 7.76 (1.44 40.989 (5, <0.001 e179.74) e323.29) 184) Pb 0.46 (0.13e14.87) 0.65 (0.15e2.20) 0.50 (0.07e2.99) 0.27 (0.15e0.98) 2.50 (1.18e57.56) 3.51 (0.82e21.87) 68.164 (5, <0.001 183) Se 0.23 (0.10e0.45) 1.25 (0.77e2.05) 1.45 (1.00e3.38) 2.72 (1.32e4.15) 3.42 (0.95e21.91) 2.85 (0.72e10.95) 289.136 (5, <0.001 183) Zn 67.86 (27.87 56.5 (33.66 59.1 (32.92 77.14 (39.28 239.33 (149.21 92.82 (37.07 125.955 (5, <0.001 e106.86) e131.16) e104.19) e212.45) e827.16) e428.97) 179) samples, significant values were found for As (r ¼ 0.64, p < 0.001), 4. Discussion Mn (r ¼ 0.43; p < 0.01), Se (0.63; p < 0.001) and Zn (r ¼ 0.70; p < 0.001). The need to validate and define standardized methods to eval- When considering only the internal samples, at least one strong uate the exposure of bat populations to contaminants is well significant positive correlation was found between them documented (Zukal et al., 2015). Considering the high conservation (Figs. S1eS6; S8eS10), except for Ni, where the most relevant status of bat species, a standardized protocol should be defined correlation was found between liver and bone (r ¼0.58; Fig. S7). using biological matrices that can be collected from living animals, The correlations varied according to the metal analyzed (see and that can provide reliable results. According to our results, both Fig. S1eS10 for further details). Metal concentrations in bone were wing membrane and fur presented the highest metal concentra- less correlated with the concentrations found in other organs/tis- tions and, depending on the metal analyzed, presented at least one sues (Figs. S1eS10). significant correlation with the metal concentration in internal R. Mina et al. / Environmental Pollution 253 (2019) 199e206 203

Table 2 membrane samples were correlated with the metal concentrations Pearson's correlations (r) between log-transformed metal concentration (ng/g dw) found in internal organs/tissues, particularly in the bone. Fur in external (wing and fur) and internal samples (bone, brain, heart and liver). sampling is highly suited as a non-invasive sampling technique, *p < 0.05, **p < 0.01, ***p < 0.001. since it is easily accessible and transportable, does not require Metal Bone Brain Heart Liver particular storage conditions, and can be sampled from live animals As Fur 0.43** 0.45** 0.17 0.61* (Pereira et al., 2006; Hernout et al., 2016b). Mammalian fur is Wing 0.57*** 0.56*** 0.48** 0.54* predominantly composed of keratin, a protein rich in cysteine Cd Fur 0.24 0.05 0.05 0.07 sulfhydryl (thiol) containing amino acids that avidly bind certain Wing 0.50*** 0.30 0.37* 0.00 Co Fur 0.58*** 0.69*** 0.60*** 0.62* metals (Burger et al., 1994; McLean et al., 2009). Each fur shaft is Wing 0.11 0.23 0.18 0.31 continuously in contact with the bloodstream at the fur root, and Cr Fur 0.41** 0.19 0.21 0.13 thus may incorporate metals circulating through the blood during Wing 0.20 0.06 0.18 0.08 growth. Mammalian fur has been successfully utilized as indicator Cu Fur 0.21 0.35* 0.37* 0.64* Wing 0.39** 0.21 0.04 0.26 of the exposure to a range of metals and metalloids including As, Al, Mn Fur 0.44** 0.34* 0.21 0.01 Cd, Cu, Fe, Mn, Pb, Se and Zn in species such as racoons Procyon lotor Wing 0.39** 0.31 0.37* 0.59* (Clark et al., 1989); opossum Didelphis virginiana (Burger et al., Ni Fur 0.39** 0.05 0.34* 0.01 1994); ringed seals Phoca hispida ladogensis, ringed seals Phoca Wing 0.06 0.26 0.23 0.03 hispida and bearded seals Erignathus barbatus (Medvedev et al., Pb Fur 0.54*** 0.35* 0.12 0.62 Wing 0.48*** 0.03 0.25 0.23 1997); rodents (wood mice Apodemus sylvaticus, bank voles Cleth- Se Fur 0.09 0.30 0.49** 0.14 rionomys glareolus, black rats Rattus rattus and Algerian mice Mus Wing 0.10 0.76*** 0.61*** 0.11 spretus)(Erry et al., 2005; Pereira et al., 2006; Tete^ et al., 2014); Zn Fur 0.39** 0.21 0.18 0.03 European hedgehog Erinaceus europaeus (D'Have et al., 2006; Wing 0.48*** 0.22 0.12 0.23 Vermeulen et al., 2009); sled dogs Canis lupus familiaris (Dunlap e ¼ ¼ ¼ ¼ Sample sizes wing: N 50; fur: N 51; bone: N 54; brain: N 42; heart: et al., 2007); flying foxes Pteropus sp. (Hariono et al., 1993); and N ¼ 36; liver: N ¼ 14. insectivorous bats (Myotis lucifugus, Myotis leibii, Myotis septen- trionalis, Myotis bechsteinii, Myotis daubentonii, Myotis myotis, samples, proving to be useful matrices for studies on bat Eptesicus fuscus, Pipistrellus pipistrellus and Pipistrellus pygmaeus) ecotoxicology. (Hickey et al., 2001; Flache et al., 2015; Hernout et al., 2016b). More The interspecific differences in the bioaccumulation of metals recently, Becker et al. (2018) and Chetelat et al. (2018) showed that found in this study highlight the importance of using different fur was a good indicator of Hg accumulation in bats, and that it species in ecotoxicological studies and risk assessment when the reflected the dietary connectivity to aquatic food webs. objective is to evaluate the potential impact of metals on a The high concentration of some metals found in the wing mammalian community, like bats. Using a range of species with membrane supports our hypothesis that this external tissue can be different biological features and ecological niches increases the used as a good proxy for metal contents in internal organs/tissues chance that particularly vulnerable species and ecological contexts and as a non-lethal sampling method for monitoring metal expo- can be identified. As shown previously by Hickey et al. (2001) and sure on bats. The histological constitution of the wing membrane of Flache et al. (2015), different bat species have different levels of bats is similar to the skin of other mammals but comparatively metal bioaccumulation. These differences can be explained by the thinner (Gupta 1967; Madej et al., 2013) and highly perfused (Faure diversity of physiological requirements among different species, et al., 2009). After the metal absorption, its distribution through the which implies differential feeding behaviors, different patterns of entire organism is largely dependent on the blood flow (Roberts foraging habitat use and distinct specialization levels of diet et al., 2015). Metals may bind to plasma proteins and other com- composition (Hickey et al., 2001; Pereira et al., 2006; Hernout et al., ponents in the blood (Roberts et al., 2015), thus tissues that receive 2015). For example, bat species that feed on insects with high a high blood flow are prone to have higher metal concentrations exposure to metal contamination are expected to have higher (Roberts et al., 2015; Rose, 2016). The uptake of metals into tissues accumulation levels than species occurring in more natural habitats depends on several physiological factors, such as the rate of organ where prey are less prone to contaminants (Walker et al., 2007; perfusion, membrane permeability and physicochemical factors, Hernout et al., 2015). The lower levels of metals recorded in Nyc- like lipid solubility of metals and presence of any organic sub- talus leisleri seemed to corroborate the hypothesis of a lower stituents (Rose, 2016). It is known that bat wings have a high exposure in species inhabiting natural habitats since it may be number of blood vessels and have a high regenerative capacity related to their use of trees as roosting sites, instead of the mines (Faure et al., 2009; Weaver et al., 2009). These features and the high and urban structures used by the other three species. Nevertheless, concentrations of Cr, Cu, Mn, Ni, Pb, Se, and Zn found in the wing depending on the mass of the species, the same exposure to metals membrane corroborate our hypothesis that wing membranes in may not express necessarily the same concentrations/effects. This bats are particularly likely to accumulate some metals more than prompts a careful analysis of data obtained for N. leisleri, as this fur. species, by having a higher average biomass when compared with Our results clearly demonstrate that fur and wing membrane the other three species, the same exposure levels may lead to lower may be used to assess metal exposure to bats and their accumu- metal concentrations in the organs/tissues (Table S1). Despite the lation in internal organs and tissues. However, when comparing the absence of metal toxicity thresholds for bats, considering the limits performance of both matrices, results are not linear. Significant reported for other mammals, some metal concentrations obtained correlations were found between both fur and wing membrane in this study may indicate a potential risk of metal contamination with bone, but no clear trend was found with the other internal for bats occurring in Portugal. These results highlight the need for organs. However, also no clear trend was found when correlating large-scale studies on the effects of metal bioaccumulation, with between different internal samples, highlighting the existence of larger sample sizes, and for the definition of specific threshold different pathways of accumulation for different metals/metalloids. levels for these potential bioindicators. The wing membrane presented significant correlations in key ele- As hypothesized, some metal concentrations in fur and wing ments like As, Cd and Se, while fur was better correlated with some 204 R. Mina et al. / Environmental Pollution 253 (2019) 199e206 internal organs for other important metals like Ni, Zn and Cu. may be useful for studies on wildlife ecotoxicology, and may help to So, despite the fact that both external matrices are promising define a protocol capable of being applied at large-scale to inves- tools to monitor exposure and internal accumulation of metals and tigate the ecological consequences of the accumulation of metals or metalloids in bats, it is important to be aware of the constraints other contaminants (e.g. pesticides) on bat communities. associated to this type of data when interpreting results from monitoring studies, especially those based on fur samples. 5. Conclusion Furthermore, considering the limited sample size used in this study, more work using larger sample sizes is needed to confirm the Given the need for large-scale studies to establish whether suitability of external tissues for metal evaluation evidence in this environmental contamination is indeed one of the factors associ- study, especially at low exposure levels. When fur is used to mea- ated with the decline of bat populations, species with a high level sure metal exposure, molting effects should be taken into consid- conservation status, external biological matrices are the best option eration. As fur incorporates metals while growing, older hair is to assess exposure and accumulation of different contaminants. As presumed to contain higher levels of metals due to a longer demonstrated, this type of samples can yield reliable results in growing time than newer hair. Consequently, the fur probably environmental contexts where contamination with metals may contains higher amounts of metals just prior to molting than dur- represent a threat to the conservation of these vulnerable species. ing, or just after molt (Beernaert et al., 2007; Hernout et al., 2016b). However, accumulation of metals in these tissues seems to be In general, bat species grow new fur once a year (usually in late higher than in some internal organs, which may give a more con- summer-fall) and males tend to grow new fur before females servative indication of the overall risk of metals to bats. Therefore, (Fraser et al., 2013). The timing and development of the molt cycle our results provide valuable insights for the implementation of vary substantially among bat species, as well as among sex and age further surveys aiming to monitor exposure of bats to metals and classes (Fraser et al., 2013). It is important to note that the bats other contaminants. In this perspective, we highlight the interplay analyzed in this study were collected between August and October, between ecotoxicological research and ecological monitoring, the period of greatest activity of bats in Europe, and some of them which will make the external samples more informative and were collected during molt, which may explain the lower metal credible to decision-makers and environmental managers. concentrations obtained from fur comparatively with those found in the wing membrane. Moreover, different metals have different Declarations of interest accumulation pathways, and consequently, they will not have the same accumulation pattern in the entire organism. This may be None. further complicated for metals considered essential elements or micronutrients. For these metals, no correlations were found be- Acknowledgements tween fur and internal organs, which may possibly be due to different exposure levels, as also reported by D'Have et al. (2006). This research was supported by POPH/FSE funds from the Por- Therefore, to a certain extent, essential elements can be regulated tuguese Foundation for Science and Technology (FCT) through the in living organisms by homeostatic mechanisms (Talmage and fellowship of J. Alves (SFRH/BPD/123087/2016), A. Alves da Silva Walton, 1991; D'Have et al., 2006). Thus, the excretion of essential (SFRH/BD/75018/2010) and T. Natal da Luz (Ref. SFRH/BPD/110943/ elements in hair may remain low and uncorrelated with internal 2015). This study was funded by IATV-UC. The biological material concentrations until a certain threshold of concentration is was collected in field campaigns supported by European Invest- reached, and only thereafter excretion will increase. If this is the ment Funds from FEDER/COMPETE/POCI e Operational Competi- case then this would suggest that bats have an effective regulation tiveness and Internationalization Program, under the project UID/ process of essential metals similar to that described for other AGR/04033/2019 (Centre for the Research and Technology of Agro- mammals (Johnson et al., 1978; McLean et al., 2009). Environment and Biological Sciences), and INTERACTeIntegrative To the best of our knowledge, the wing membrane has not been Research in Environment, Agro Chains and Technology, under the used previously to quantify the accumulation of metals in bats. “Programa Norte 2020, FEDER Aviso Norte 45-2015-02”. However, its use seems to be a promising sampling location and may possibly be a better indicator of metal bioaccumulation than Appendix A. Supplementary data fur. 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