Environmental Science and Pollution Research https://doi.org/10.1007/s11356-018-1477-5
SHORT RESEARCH AND DISCUSSION ARTICLE
Selection of tawny owl (Strix aluco) flight feather shaft for biomonitoring As, Cd and Pb pollution Rita García Seoane 1 & Zulema Varela Río 1,2 & Alejo Carballeira Ocaña 1 & José Ángel Fernández Escribano 1 & Jesús Ramón Aboal Viñas 1 Received: 20 September 2017 / Accepted: 4 February 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract In this study, we determined the concentrations of As, Cd and Pb in the shaft of all primary flight feathers from ten tawny owl (Strix aluco) specimens, with the aim of selecting which shaft of the corresponding primary feather should be used in biomonitoring surveys to enable inter-individual comparisons of the levels of these metals. The birds had died between 2006 and 2013 and their bodies were stored in the various Wildlife Recovery Centres in Galicia (NW Spain). The analyses revealed a high degree of inter-shaft variability, mainly in the concentrations of As and Cd. However, it was possible to identify the most representative samples in each case: for As, the shaft of primary flight feather number 5 (S5) (which represented 11% of the total As excreted in all of the primary flight feathers); for Cd, the shaft of primary flight feather number 2 (S2) (11% of the total excreted); and for Pb, the shaft of primary flight feather number 8 (S8) (14% of the total excreted). However, the difficulties associated with the analytical determination of these pollutants in the shaft should be taken into account when this technique is applied in biomonitoring studies. Keywords Air pollution . Bioaccumulation . Biomonitoring . Feather . Raptor . Terrestrial food chain
Introduction Birds have been used in many biomonitoring studies in the last few decades because of increasing evidence that their populations are strongly affected by environmental pollution of anthropogenic origin, particularly as a result of exposure to heavy metals (Altmeyer et al. 1991; Dauwe et al. 2003; GarcíaSeoane et al. 2017). Birds of prey (raptors) are commonly used as bioindicators of the levels of metals in the environment, for Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-018-1477-5) contains supplementary material, which is available to authorized users. * Rita García Seoane
[email protected] 1
Ecology Unit, Department Functional Biology, Universidade de Santiago de Compostela, Fac. Biología, Lope Gómez de Marzoa s/n, 15702 Santiago de Compostela, La Coruña, Spain
2
Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
the following reasons: (i) they are extremely sensitive to variations in the levels of these pollutants (Furness 1993; Castro et al. 2011), (ii) they occupy the highest trophic levels in terrestrial ecosystems (Martínez et al. 2012), (iii) they are highly territorial and can spatially integrate pollutant levels from across their extensive home ranges (Altmeyer et al. 1991) and (iv) many of the species used in biomonitoring studies are widely distributed throughout the world (Cramp 1978; Varela et al. 2016). Different types of tissue have been extracted from raptors for biomonitoring purposes (e.g. liver and kidney); however, the methods used to obtain the samples are invasive, which may have negative effects on species abundance, population survival and on the corresponding habitats (Cardiel et al. 2011; Castro et al. 2011). Hence, feathers are now often used in a non-invasive approach to monitoring pollutant exposure in terrestrial and aquatic ecosystems. This method has been widely used to detect episodes of environmental pollution, as well as to evaluate spatio-temporal variations in the levels of some elements at both small and large scales (e.g. Dietz et al. 2006; Bustnes et al. 2013; García-Seoane et al. 2017). Different types of feathers have been analyzed in biomonitoring surveys: contour and primary and secondary flight feathers (e.g. Dauwe et al. 2003; Varela et al. 2016); tail
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feathers (e.g. Zolfaghari et al. 2007; Behrooz et al. 2014); and primary and secondary cover feathers (e.g. Honda et al. 1985; Debén et al. 2012). A high degree of inter-feather variability in the concentrations of pollutants has been observed. Some authors have even found that different sections of the same feather accumulate different amounts of pollutants, depending on the elements considered (e.g. Dauwe et al. 2003; Cardiel et al. 2011; Rodriguez-Ramos et al. 2011). These variations may be due to the origin of the pollutants themselves, as elements such as As, Cd and Pb may be derived largely from atmospheric deposition and their uptake will depend on the degree of exposure of the feather to the pollutant (Hahn et al. 1993; Dauwe et al. 2003), whereas elements such as Hg are taken up almost exclusively via the diet (Denneman and Douben 1993; Hahn et al. 1993). In the case of Hg, which reaches the feathers mainly via endogenous routes, the vanes (barbs) can be used in biomonitoring studies to determine the amount of pollutant that is incorporated during the growth of feathers (Varela et al. 2016). However, the use of barbs is not recommended for biomonitoring Pb, as external contamination on the surface of feathers can lead to overestimation of the amount of the metal incorporated via the diet (Dauwe et al. 2003; Cardiel et al. 2011). For biomonitoring Pb and other metals and metalloids (e.g. As and Cd), shafts have been found to be more representative than barbs of the uptake and tissue deposition within bird tissues during growth of feathers (e.g. Dauwe et al. 2003; Cardiel et al. 2011). This may be due to the smaller specific surface area of the shaft (and the consequent lower level of exposure to external pollution) and to the fact that the shaft can be cleaned more easily and efficiently than the barbs (Jaspers et al. 2004; Cardiel et al. 2011). Goede and de Bruin (1984) observed a slight increase in As concentrations in barbs, but not in shafts, and a substantial increase in the Pb concentration in barbs relative to the small increase in shafts over time in waders, suggesting that the shaft is rather insensitive to external contamination by As and Pb. Likewise, Altmeyer et al. (1991) reported much higher concentrations of Cd and Pb in the barbs than in the shafts of feathers of the white-tailed eagle (Haliaeetus albicilla). Dauwe et al. (2003) also reported significantly higher concentration of As, Cd and Pb in barbs than in the shaft of the outermost tail feathers of Strix aluco and the raptor Accipiter nisus after feather formation. More recently, Cardiel et al. (2011) also found that the outermost primary feather barbs contained significantly higher amounts of Pb than the shafts in four scavenging raptors. Dauwe et al. (2003) and Cardiel et al. (2011) concluded that as the shaft is not affected by atmospheric deposition, it is more representative of internal deposition via the bloodstream than the barb and is therefore more appropriate for studying these elements. Although the feather shaft has been demonstrated to be ideal for biomonitoring pollutants that are mainly incorporated
via endogenous routes (e.g. As, Cd and Pb), no attempt has been made to determine which primary flight feather shaft should be used for this purpose, as done for the barbs of raptor feathers used for biomonitoring Hg pollution (Martínez et al. 2012; Varela et al. 2016). Harmonization of this aspect of the process would enable inter-individual comparison of the levels of pollutants in different feathers obtained from specimens of S. aluco. Therefore, taking into account all of the abovementioned considerations, the aim of the present study was to harmonize the primary flight feathers of the tawny owl (Strix aluco) used to obtain the shaft analyzed for biomonitoring environmental levels of As, Cd and Pb.
Material and methods Sampling Bird specimens were obtained from the Wildlife Recovery Centres in Galicia (NW Spain). A total of ten tawny owls (Strix aluco L., 1758) were collected. The shafts (consisting of the rachis and the calamus, Bachmann et al. 2012) of all ten primary flight feathers of one wing from each bird were analyzed with the aim of establishing which yields the metal concentration that is most representative of the body burden. The primary flight feathers were chosen because they are the first to moult (Cramp 1978) and are probably the most representative of the body burden. In addition, tawny owls, which are some of the most common forest-dwelling nocturnal raptors in the temperate and southern boreal zone of Europe (Karell et al. 2011), are also common in forests from the north of Spain and are used extensively to biomonitor metal pollution in Galicia and throughout Europe (Castro et al. 2011; Lourenço et al. 2011; Debén et al. 2012; Bustnes et al. 2013; Varela et al. 2016; García-Seoane et al. 2017). Most of the birds had died between 2006 and 2013 as a result of physical injuries, and all had been held in the centres for no longer than 1 month before dying. The bodies were stored frozen at − 30 °C. Only adult specimens were used in order to minimize any possible variation in the results due to the age of the birds (Gochfeld et al. 1996).
Sample processing All primary flight feathers obtained from each individual were washed for 15 min with 5 L of bidistilled water and 5 mL of Triton X-100 detergent in an ultrasound bath (Branson 5200) to remove superficial contamination. The feathers were then rinsed in bidistilled water and blotted on filter paper before being dried in a forced air oven (45 °C) until constant weight. The shaft was separated from each feather and the barbs were discarded. The shafts were cut into small pieces with stainless steel scissors and oven-dried at 45 °C.
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Sample analysis To determine the concentrations of As, Cd and Pb, a sample of 100 mg of each shaft was weighed on a precision balance (Mettler Toledo XP26) and digested with 2 mL of HNO3 (65%) in temperature-controlled Teflon bombs in a sand bath (at 90 °C for 48 h). The bombs were cooled to room temperature and the solutions were then made up to 10 mL with milliQ water in Falcon tubes and stored at 4 °C. All laboratory materials used for sample mineralization and storage were washed overnight with 20% HNO3 and with abundant tap water and distilled water, to prevent contamination of the samples. After the acid digestion process was completed, the concentrations of As, Cd and Pb in the samples were determined. The concentrations of As, Cd and Pb in the shafts were expressed relative to the weight of each individual shaft, following the recommendations of Bortolotti (2010). Inductively coupled plasma mass spectrometry (ICP-MS Agilent 7700×) was conducted at the Research Support Services Unit (University of Santiago de Compostela), and the more sensitive high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC Flexar PerkinElmer, ICP-MS NexION 300× Perkin-Elmer) was conducted by the Research Group in Trace Elements, Spectroscopy and Speciation (University of Santiago de Compostela). To ensure the analytical quality of data, a sample of certified reference material (human hair, NCS DC73347a, supplied by the Chinese National Analysis Centre for Iron and Steel (Beijing, China), was analyzed, once every ten samples, following the same procedure used for the shaft samples. The concentrations of As, Cd and Pb were within the range of the certified values for each element. Likewise, an analytical blank (65% HNO3) was also analyzed, once every ten samples, to check for contamination during the analytical procedures. In the case of As and Cd, only those specimens analyzed by HPLC-ICP-MS (n = 5) were considered further as the concentrations determined by ICP-MS were below the limit of quantification (LOQ), 0.46 and 0.37 ng g−1, respectively. Thus, the LOQs for As and Cd determined by HPLCICP-MS were 0.01 and 0.001 ng g−1, respectively. For Pb, the LOQs were 0.03 (n = 5, HPLC-ICP-MS) and 0.59 ng g−1 (n = 5, ICP-MS). For other feathers, those concentrations below the LOQ (more than 22% of the total) were assigned a value corresponding to half of the LOQ. The percentage recoveries of the reference material were 81, 108 and 96% for respectively As, Cd and Pb.
Data treatment Box plots were constructed to enable descriptive analysis of the data. Major axis regression (Zar 2009) was used to study the relationship between the metal content of each shaft (S1– S10) and the total amount of each metal in the shaft of all
primary flight feathers. As the data were not normally distributed, and thus could not be normalized by log transformations, the relationships between the metal contents in the shaft of five of the bird specimens were determined by nonparametric Spearman’s rank coefficient correlation procedure. Relationships were considered significant at p < 0.05. All statistical analyses were carried out with R software.
Results and discussion The concentrations of As, Cd and Pb (ng g−1 dry weight (d.w.)) in the shaft of each primary flight feather from the S. aluco specimens under study are shown in Fig. 1 and Table S1 (Supplementary material). The concentrations of all three elements were extremely low, especially those of As and Cd. The concentration of As was highest in the shafts closest to the body (S1–S4). A similar relation was observed for Cd but not for Pb, the maximum concentrations of which remained constant among all of the shafts analyzed. The highest median values of both As and Pb were found in S10, and for Cd, the highest median value corresponded to S2. As the interest in determining the concentrations of As, Cd and Pb was related to the levels of excretion via the feathers, the concentrations can also be represented as the amount of each metal present in each shaft, taking into account the weight of each feather. If the quantity of metal in the shaft is expressed relative to the total quantity for each wing (Fig. 1), the maximum concentrations corresponded to S7 and S8 for As, in S2 and S5 for Cd and in S7 and S8 for Pb. These values are consistent with the median values. Studies of the levels of these heavy metals in raptor flight feather shafts are rather scarce and therefore few comparisons with already published data can be made. The median concentrations of Cd and Pb (ng g−1 d.w.) in the S. aluco flight feather shafts under study are respectively 5 and 4 orders of magnitude lower than those reported for flight feather barbs of adult specimens in the same study region (Castro et al. 2011). Likewise, the concentrations of Pb determined in different whole-body feathers of S. aluco (Debén et al. 2012) are much higher (one order of magnitude) than in the shafts analyzed in this study. On the other hand, the concentrations of As, Cd and Pb determined in the present study are lower (up to 4 orders of magnitude) than those reported for primary flight feather shafts from different raptor species (e.g. tawny owl, whitetailed eagle, griffon and cinereous vultures and black kite), i.e. for As in Belgium (Dauwe et al. 2003), Cd and Pb in Poland (Altmeyer et al. 1991) and Pb in Spain (Cardiel et al. 2011; Rodriguez-Ramos et al. 2011). Regarding the selection of the flight feather, the coefficients of determination were calculated for the As, Cd and Pb concentrations in each flight feather shaft (i.e. S1 to S10; Fig. 1). The feather shaft which shows maximum
y2=67.204y1-0.2227
0.8
0.6
0.4
0.2
100 Percentage respect to the maximum amount of As in each wing
As
3.2 1.9 1.3
As concentration (ng g-1)
n.s.
n.s.
n.s.
n.s.
n.s.
0.868
0.994
0.992
1.0
0.990
r2
0.0 n.s.
0.08
0.06
0.04
0.02
0.00 0.509
0.521
0.528
0.505
0.782
0.613
0.468
n.s.
n.s.
Pb
y2=6.313y1-0.0061
4
3
2
1
0
25
Cd
75
50
25
1
2 3 4 5 6 7 8 9 Number of primary feather
determination coefficient with respect to the amounts of metal immobilized in the whole primary feather shafts of a wing can be determined, which would better represent the total variability present in the wing. The total amount of As, Cd and Pb present in all shafts from the ten flight feathers revealed significant relationships. For As, the highest coefficient was obtained for S4, which therefore best represented the As content in the wing. The highest coefficient for Cd corresponded to
100 Percentage respect to the maximum amount of Pb in each wing
5
50
0 n.s.
r2
75
100 Percentage respect to the maximum amount of Cd in each wing
n.s.
n.s.
0.830
n.s.
n.s.
0.857
0.960
0.955
Cd
y2=3.842y1-0.0023
Cd concentration (ng g-1)
0.10
As
0 0.909
r2
Pb concentration (ng g-1)
Fig. 1 Box plots of the As, Cd and Pb concentrations (ng g−1 d.w.) in the shaft of the ten primary flight feathers and of the median (M) value for all the shafts from the Strix aluco specimens analyzed (n = 5 (As, Cd); n = 10 (Pb)). Box plots of the percentage amount of As, Cd and Pb for each primary flight feather shaft relative to the total amount in each primary flight feather from all S. aluco specimens under study (n = 5 (As, Cd); n = 10 (Pb)). Shaft number 1 is the closest to the body and shaft number 10 is the farthest from the body. The boxes represent the interquartile range, the line within the boxes represents the median value and the whiskers represent the minimum and maximum values. The upper boxes show the coefficients of determination (r2) for the concentrations of As, Cd and Pb (ng g−1 d.w.) in the shaft of each primary flight feather (S1– S10) and the summed concentrations of each metal in all primary flight feather shafts in the S. aluco specimens under study. For each of the shafts finally recommended for biomonitoring As, Cd and Pb purposes, the corresponding SMA regression is shown in the lower boxes, where y1 = As, Cd, Pb (ng) in a single shaft and y2 = sum of As, Cd, Pb (ng) in all primary flight feather shafts from the same wing. Bold italics p < 0.005, bold p < 0.01, italics p < 0.025, regular typeface p < 0.05, n.s. not significant (p > 0.05)
0.989
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10 M
Pb
75
50
25
0
1
2 3 4 5 6 7 8 9 10 Number of primary feather/median
S3. Finally, the highest coefficient in the case of Pb corresponded to S6. For As and Cd, the highest coefficients of determination corresponded to the shafts closest to the body (S1–S5). The opposite was found for Pb, for which the highest coefficients of determination corresponded to the shafts furthest from the body (S5–S10). If the concentrations of As, Cd and Pb accumulated in the shaft represent the total amount of the elements accumulated
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by endogenous routes, the present results should accurately describe the moulting pattern in S. aluco. According to Cramp (1978), moulting of flight feathers begins in early May to late June in this raptor and ends between the beginning of September and the end of November. Although the moult is complete, not all flight feathers moult at the same time: in the primary moult, feathers 2 to 10 are lost sequentially, but 1 and 3 are often lost almost simultaneously. Hence, the metal concentrations should be highest in the shaft of the feathers that are replaced earliest (Altmeyer et al. 1991); however, this was not observed for any of the three elements considered (Fig. 1). This further confirms the suggestion made by Dauwe et al. (2003) that the final concentrations of elements in the shafts may be influenced by external pollution (e.g. atmospheric deposition). Nonetheless, the shafts are still more representative than the barbs as regards the endogenous levels of As, Cd and Pb in raptors. For the identification of which shaft should be used to enable comparison of the levels of As, Cd and Pb among different flight feathers, the shaft in which the maximum amounts were accumulated and the shaft with the highest determination coefficient should be taken into consideration. Thus, S2 was selected as the most representative shaft for biomonitoring Cd because after S5, it contained the highest amounts of this metal and the determination coefficient was the second highest (Fig. 1). In the case of Pb, S8 would be the most appropriate for use in biomonitoring studies because after S7, it contained the highest amounts of this metal and had the second highest determination coefficient (Fig. 1). The best shaft for biomonitoring As was selected by balancing the determination coefficient and the amount of As excreted, as it was difficult to choose the most representative from among the ten primary flight feathers in the wing. Thus, in this case, S5 was selected as the most appropriate as the corresponding determination coefficient was high and the amounts of As excreted were fairly large. Finally, significant correlations (**p < 0.01) were obtained for all three possible metal-pair comparisons (r = 0.609** for As-Cd, r = 0.497** for As-Pb, r = 0.665** for Cd-Pb). Significant correlations between Al and Pb concentrations in the barbs of feathers from four different raptor species in Spain (r = 0.782**) have also been reported by Cardiel et al. (2011). This suggests that the accumulation of As, Cd and Pb may be influenced by the same factors, such as a common route of uptake of these metals (ingestion) and atmospheric deposition, or by synergic interaction between these factors (Cardiel et al. 2011).
Conclusions The study findings show that the concentrations of As, Cd and Pb in flight feather shafts are much lower than those found in
barbs. However, analysis of shaft remains the best option for determining these elements to prevent the results from being skewed by the influence of atmospheric deposition. As already done for other elements such as Hg, the protocols for use of feathers for biomonitoring pollutants (either with the shaft or other parts of the feather) should be harmonized in order to refine the technique and obtain more accurate and comparable results. Despite the high level of intraspecific variability in the data and the differences in interpretation of the results arising from the determination coefficients and the total amount of each metal found in each shaft relative to the total amount in the whole wing, a shaft was selected in each case. Thus, the shaft of primary flight feather number 5 (S5) is recommended for use in biomonitoring levels of As in S. aluco, S2 for biomonitoring Cd levels and S8 for biomonitoring Pb levels. Finally, the difficulties associated with the analytical determination of these pollutants in the shaft may represent a disadvantage to the use of this technique in biomonitoring studies which should be taken into account. Acknowledgments We thank all the personnel at the Wildlife Recovery Centres of the Xunta de Galicia for helping in obtaining the samples. Funding information The authors are members of the Galician Competitive Research Group GRC/GPC2016-002 and of the CRETUS Strategic Partnership (AGRUP2015/02), which are co-funded by FEDER (EU).
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