Experimental variation in the spatial deposition of trace metals in feathers revealed using synchrotron X-ray fluorescence

Feathers can be used to investigate exposure to pollution in birds because they are a secondary route for the excretion of trace elements. Evidence based on analytical imaging and spectroscopy suggests that the spatial distribution of the essential trace element zinc within feathers is related to melanin pigmenta-tion. However, our understanding of how trace elements are deposited into growing feathers is poor and has been hampered by a lack of analytical tools to examine the localization of trace elements within a feather. Here, synchrotron micro X-ray fluorescence spectroscopy was used to map zinc directly within the barb and barbules of lesser scaup ( Aythya affinis ) feathers grown after experimental increases in dietary zinc. The results showed distinct spatial variation in zinc within barbs and barbules, with higher levels observed in the latter. Furthermore, increases in dietary zinc were found to increase the relative levels of zinc throughout the barbules from the base to the tip of the feather. Finally, analysis of feather cross sections revealed that regions of the feather barb and barbules with higher melanosome density also contained higher levels of zinc. These results provide a more detailed understanding of zinc and melanosome arrangement within the feather barb and barbules. Moreover, these results provide further support for the use of feathers as a noninvasive tool to study exposure to trace elements and highlight the utility of X-ray spectroscopy in studies investigating impacts of a rapidly changing environment on wild bird health.

Feathers can be used to investigate exposure to pollution in birds because they are a secondary route for the excretion of trace elements. Evidence based on analytical imaging and spectroscopy suggests that the spatial distribution of the essential trace element zinc within feathers is related to melanin pigmentation. However, our understanding of how trace elements are deposited into growing feathers is poor and has been hampered by a lack of analytical tools to examine the localization of trace elements within a feather. Here, synchrotron micro X-ray fluorescence spectroscopy was used to map zinc directly within the barb and barbules of lesser scaup (Aythya affinis) feathers grown after experimental increases in dietary zinc. The results showed distinct spatial variation in zinc within barbs and barbules, with higher levels observed in the latter. Furthermore, increases in dietary zinc were found to increase the relative levels of zinc throughout the barbules from the base to the tip of the feather.
Finally, analysis of feather cross sections revealed that regions of the feather barb and barbules with higher melanosome density also contained higher levels of zinc. These results provide a more detailed understanding of zinc and melanosome arrangement within the feather barb and barbules. Moreover, these results provide further support for the use of feathers as a noninvasive tool to study exposure to trace elements and highlight the utility of X-ray spectroscopy in studies investigating impacts of a rapidly changing environment on wild bird health.

| INTRODUCTION
Pollution is a global environmental problem with farreaching consequences for ecosystems, biodiversity, and human and wildlife health. [1,2] Trace elements are pollutants that are typically present in the environment in minute quantities; however, mining, industry, agriculture, and urbanization can further release these elements into the environment at concentrations that are toxic to wildlife. [3,4] Feathers are increasingly used to measure a variety of biomarkers of exposure to pollution in birds because feathers are a secondary route of excretion of trace elements. [5][6][7] Research often focuses on nonessential trace elements (e.g., mercury and lead) because they are toxic at low concentrations [8] and can have wideranging detrimental effects. [9][10][11] Less is known about the consequences of exposure to essential trace elements (such as zinc, iron, and copper), which are necessary for proper biological functioning, and only become toxic when ingested at high levels. For example, zinc is essential for normal development, immune function, and reproduction [12,13] and is sequestered in feathers during the production of melanin pigments. [14][15][16][17][18] Melanins are responsible for black, brown, and rufous plumage colors, and in some species, the size and quality of melanin plumage ornaments can play important roles in signaling, mate acquisition, and components of fitness. [19,20] Another route for trace elements to be sequestered in feathers is via metallothionein, a protein that binds trace metals in detoxification/excretion. [21][22][23] Previous studies conducted on trace elements in feathers have measured bulk deposition of the elements in homogenized tissues. [14,[24][25][26][27] Although these studies provided useful information on the overall concentrations of trace elements, the distribution of trace elements within feathers is poorly understood. Recently developed synchrotron microprobe X-ray fluorescence (μ-XRF) techniques have the potential to provide additional information on distribution and potential physiological mechanisms associated with the deposition of trace elements within feather structures. Synchrotron μ-XRF techniques can measure the distribution and level of trace elements at minuscule spatial scales (<5 μm), directly within intact feathers, and synchrotron can image multiple elements simultaneously against the sulfur (S)-dominated keratin matrix. [28] This technique has been used effectively to better understand the elemental composition of feathers from both extinct and extant species, providing powerful insights into the evolution of the structure and color of feathers. [29][30][31][32] μ-XRF uses a micro-focused X-ray beam to excite corelevel electrons in a sample, resulting in the emission of fluorescence X-ray. Different elements emit fluorescence X-rays at different characteristic energies that can be used to identify and quantify the elements present in a sample. [33] Using a micro-size X-ray beam enables mapping the distribution of various elements in heterogeneous samples by rastering the sample with a fixed Xray beam.
Previous work has shown that gradients in the darker regions of feathers are related to the density of melanincontaining organelles called melanosomes. [34] μ-XRF mapping has revealed that darker areas of feathers contain more zinc, which is assumed to be related to the presence of feather melanins. [15,31] However, to our knowledge, μ-XRF has not been used to assess fine-scale spatial variation in trace elements in feathers under conditions of experimentally increased levels of essential trace elements (e.g., zinc). Furthermore, the distribution of trace elements within the barbs and barbules of feathers has not been investigated. In this paper, the effects of experimentally increased dietary zinc on the level and spatial distribution of this essential trace element within feathers were investigated using μ-XRF spectroscopy. Both longitudinal and cross-sectional μ-XRF maps of feather barbs were studied to provide detailed spatial information on the distribution of zinc. It was predicted that more zinc would be present in the experimentally increased dietary zinc bird's feather compared with a control, and because melanin is a known ligand of zinc during feather growth, [35] zinc was expected to correlate with areas containing more melanosomes. A common migratory diving duck (lesser scaup, Aythya affinis) was used as a model because zinc pollution of waterways is a concern for birds that use aquatic ecosystems for breeding and foraging, [4,36] and it has been hypothesized that contaminants are responsible for scaup population declines. [37] The study will provide further support for the use of feathers as noninvasive samples to study exposure of trace elements and the usefulness of μ-XRF in detecting elemental deposition within feather in microstructure levels.

| Sampling
Four lesser scaup (two males and two females, mean ± SD mass: 566 ± 28 g) were selected from a captive flock based on degree of molt of the tail feathers. Two randomly selected scaup (one male and one female) were each administered one 50-mg-crushed zinc tablet (Jamieson Laboratories, Canada) mixed with 20 ml of molasses ("zinc-fed"), and the remaining two scaup (one male and one female) were each administered 20 ml of molasses only ("control"). Birds received their treatments once daily for 4 days. Throughout the experiment, ducks were fed a commercial poultry grower ad libitum and had access to water at all times. The location and length of tail pin feathers (i.e., developing feathers with all or a portion encased in a sheath containing blood) were recorded for each duck. Size was measured to the nearest millimeter with calipers. Feathers were marked by notching the vane at the level of the shaft so that the portion of the feather grown during the experimental period could be identified later. Three weeks after administration of treatments, two tail feathers with identifiable notches per bird were collected for analyses. Feathers were placed in labeled paper envelopes until time of analysis. All scaup were treated according to the Canadian Council on Animal Care Guidelines (available online at: http://www.ccac.ca/en_/standards/guidelines), and the University of Saskatchewan Animal Care Committee approved the protocols.

| X-ray fluorescence spectroscopy, electron, and light microscopy
The significant amount of time required to conduct finescale μ-XRF mapping, and the small amount of beamtime allocated to our study, required us to limit our sample to one control and one experimental feather, which were randomly selected for X-ray fluorescence spectroscopy, electron, and light microscopy. Individual feather barbs located approximately halfway along the length of the rachis (shaft) below the notch ensuring that the barbs were grown during administration of the zinc. The barbs were removed from each of these feathers using plastic tweezers, washed in a dilute soap solution to remove any surface contamination, rinsed three times in ultrapure water, and air-dried completely before analysis. Barbs were mounted linearly on Rinzyl plastic slides using strips of Kapton tape at the base and tip. Mounted samples were stored in a small plastic box when not in use to reduce exposure to airborne dust. μ-XRF maps were collected from the proximal (base), middle, and distal (tip) regions of feather barbs using the Very Sensitive Elemental and Structural Probe Employing Radiation from a Synchrotron beamline (VESPERS; 07B2-1) [38] at the Canadian Light Source (Saskatoon, Canada). Barbs mounted on the Rinzyl plastic slides were placed on a motorized sample stage that was rotated 45 with respect to the incident X-ray beam. A four-element silicon drift Vortex detector was used to collect the XRF spectra and placed in the horizontal polarization plane at 90 to the incident X-ray beam with a sample-to-detector distance of 50 mm. All measurements were performed in air using the "Pink Beam" mode, which included all Xray energies from 2 to 30 KeV (with a significant drop in flux occurring near 20 KeV) and an X-ray spot size of 2-5 μm. The flux can be estimated to be significantly greater than 10 [11] photons per second. Elemental maps of zinc and sulfur were collected using a step size of 5 μm and a dwell time of 1 s per point. The resolution of the XRF maps is assumed to be the same as the step size (5 μm). Air attenuation is expected to affect the X-ray fluorescence signals of the low-energy elements (i.e., sulfur and calcium). However, the concentration of these elements in the feather, particularly with respect to sulfur, is large enough that the effects of air attenuation are negligible. Also, multiple μ-XRF maps collected from the same area of the feather resulted in no changes in elemental distribution, indicating that the feather samples do not suffer from X-ray beam damage. All maps were normalized to the intensity of an ion chamber positioned in front of the sample to eliminate the effects of X-ray beam flux variation.
Electron and light microscopy work was conducted at the Western College of Veterinary Medicine Imaging Centre of the University of Saskatchewan. For scanning electron microscopy (SEM), barbs were mounted on an SEM stub with double-sided sticky tape at the ends and coated with gold (Edwards S150B) at 20-mA current (1 kV) for 2 min. Barbs were examined under low vacuum, and surface morphologies were recorded. Internal structures of the barbs were studied using light microscopy of resin-embedded cross sections. An adjacent cross section of barb was also analyzed using μ-XRF spectroscopy to investigate the distribution of zinc within the barb and barbules. In brief, approximately 2-mm sections were excised from the base, mid, and the tip of the barbs and dehydrated in an ascending series of 70%, 100%, and 100% ethanol, with a 2-hr incubation at each step. The sections were left in 100% ethanol for approximately 17 hr. Ethanol was gradually replaced with Spurr's resin by placing the section in a series of solutions with an ascending resin: ethanol ratio (1:1, 3:1), with a 3-hr incubation at each step. The samples were left in 100% resin at room temperature for 36 hr to ensure complete impregnation of the barbs and then embedded in a fresh batch of resin before polymerizing them at 65 C for 24 hr. Two adjacent cross sections were prepared from each of the base, mid, and tip sections using new glass knives. One section (300 nm) was stained with toluidine blue and immediately examined under a light microscope. Another section (1 μm) was mounted onto a Rinzyl plastic slide and analyzed at the VESPERS beamline at the Canadian Light Source. The same beamline settings were used as described above, except that a longer dwell time (5 s) was used to map the cross section, to account for the thinner samples of cross sections compared with the whole barb.

| Data analysis
Synchrotron μ-XRF data were analyzed using the PyMCA software program (version 5.1.2). [39] Spatial maps were constructed by fitting the X-ray fluorescence spectra collected at each data point using a Levenberg-Marquardt algorithm to remove the scattering background and deconvolute the X-ray fluorescence peaks for each element of interest. The Levenberg-Marquardt algorithm is a standard technique used for nonlinear least squares regression and can be considered a combination of steepest descent and Gauss-Newton methods. [40,41] The relative levels of the elements were measured by normalizing the intensity of each element with the intensity of the incoming beam and using sulfur levels in the keratin matrix as an internal standard. [28] The density of melanosomes were calculated using the cell counter plugin of Fiji ImageJ. [42] Melanosome density were computed from three technical replicates (i.e., separate cross sections), and results were presented as means ± standard error. Heat maps and statistical graphs were developed in SigmaPlot, version 12.0.

| RESULTS
Using synchrotron μ-XRF spectroscopy, zinc was detected within the feathers of a control bird, and a bird fed a diet with increased levels of zinc. A typical μ-XRF map showing the spatial distribution of zinc within feather barbs and barbules is shown in Figure 1.
Overall, relative zinc levels were generally higher in the barbules than the barbs in both birds with slightly higher levels observed in the zinc-fed bird ( Figure 2). Also, zinc levels of the barbs were generally constant from the base to the tip of the barb for both birds. In barbules, relative zinc levels decreased from the base to the tip of the feather barb in the control bird, whereas the levels remained constantly high in the zinc-fed bird. SEM micrographs revealed the presence of sporadic particles between barbules (Figure 3). These could have been dust particles (which are a known external source of zinc [15] ) or broken pieces of feather, both of which may inflate the measured zinc levels. However, it was clearly visible from SEM micrographs that the particles were confined to the troughs between the barbules ( Figure 3) and were not detected in the selected area under μ-XRF (Figure 1).
Scans of barb and barbules cross sections from control and zinc-fed birds revealed that zinc was distributed within feather sections (rather than on the surface of feathers) and was concentrated within the barbules and dorsal ridge of the barb, whereas sulfur, as expected, was distributed throughout the barb and barbules (Figure 4).
The distribution of zinc within the feather was similar to the distribution of melanosomes. As illustrated in Figure 5, light microscopy (100×) of cross sections from the zinc-fed bird revealed that the number of melanosomes was higher in the dorsal ridge of the barb ramus compared with the ventral ridge, where they were nearly absent. The density of melanosomes in the barbules was~3.7 times greater than in dorsal ridge of the barb ramus (1.48 ± 0.21/ μm 2 vs. 0.40 ± 0.05/μm 2 ). This arrangement of melanosomes matched the distribution of zinc observed under μ-XRF (Figure 4). The pattern was the same in the control bird with a greater number of melanosomes in the dorsal ridge of the barb ramus compared with the ventral ridge, and the density of melanosomes in the barbules was~3.2 times greater than in dorsal ridge of the barb ramus (2.034 ± 0.22/μm 2 vs. 0.633 ± 0.11/μm 2 ). Furthermore, there were no differences in the density of melanosomes between control and zinc-fed birds in either barb, t(4) = 1.44, p = .22, or barbules, t(4) = 0.74, p = .50.

| DISCUSSION
This study highlighted spatial variation in the deposition of the essential trace element zinc within feather barbs and barbules using synchrotron X-ray fluorescence spectroscopy. The advantage of this technique over traditional analytical methods (such as inductively coupled plasma-mass spectroscopy) is that it is nondestructive and involves minimum sample preparation. This ensures the identification of elements in the regions of the feather where they were originally deposited during active feather growth. Because feathers are increasingly used to measure biomarkers for wildlife health and environmental monitoring, such information is crucial to understanding mechanisms of elemental deposition within feathers in response to various dietary and environmental conditions faced by birds. Our findings confirmed the preferential distribution of zinc in feather barb and barbules. Also, constantly high relative zinc levels from the base to the tip of the feather barbules in the zincfed bird provide limited but intriguing evidence that manipulating levels of an essential trace element in the diet may alter levels deposited within a feather. To our knowledge, this is the first study where preferential distribution and relative levels of zinc are reported in feather barbs and barbules of an extant bird in an experimental context.
The similarities between the patterns of distribution of zinc and melanosomes in feathers supports the hypothesis of melanosome sequestration of zinc, as suggested in previous studies. [34,[43][44][45] Keeping in mind that logistical constraints necessitated a small sample size in this study and that further study is needed to confirm the generalizability of our results, it appears that the deposition of zinc within the feather barbs and barbules depends on the presence, distribution, and density of melanosomes. Increased dietary zinc apparently did not change the relative proportions of melanosomes within the feather, as similar melanosome densities and patterns of distributions were detected in both control and zinc- fed birds. This finding is encouraging from a biomarker standpoint because it suggests that the mechanisms of zinc deposition in lesser scaup feathers were not affected by the excess zinc administered to the zinc-fed bird.
Imaging and mapping evidence conducted on the cross sections of feather barbs and barbules supported a link between the distribution of zinc and melanosomes. Melanosomes are lysosome-related organelles of melanin pigment granules that provide tissues with color and photoprotection. [46,47] Melanin consists of black and brown eumelanin and red and yellow pheomelanin [47] that can serve as a reservoir (for storage and release of elements) or a sink (for sequestration of toxic elements) of metals depending on the type and levels of metals present in a biological system. [43] For example, a comparison of bulk analysis of zinc in the feathers of melanic pigeons demonstrated higher levels of zinc compared with their paler counterparts. [14] Furthermore, a recent X-ray absorption spectroscopy study of whole feathers has established that melanin pigments control the distribution of zinc and other elements (e.g., calcium and copper) in the feather and that the arrangement of zinc and sulfur differs depending on whether the portion of feathers is dominated by eumelanin or pheomelanin. [31] The μ-XRF zinc distribution map from the cross section of a feather barb in the present study added to this finding by providing a more detailed understanding of how zinc and melanosomes were distributed within the feather structure.
Previous studies mentioned surface contamination [27,[48][49][50] and variations within and between feathers [27,51] as drawbacks in feather-based environmental monitoring studies. We addressed the former by additionally mapping distribution of zinc in cross sections of feather barb and barbules which confirmed internal deposition of zinc within feather microstructures. Regarding the latter, larger sample sizes are necessary to ensure variation in trace element deposition is properly attributed to experimental manipulation. Applying additional synchrotron technologies, such as micro X-ray absorption near edge structure and X-ray absorption fine structure spectroscopy to the study could also be helpful. These techniques would identify chemical speciation of zinc inside feather barb and barbules and provide a better understanding of the reasons for variations of specific elements within and between feathers as well as the mechanisms of element deposition in feathers in general. Thus, future work should build on the current study by including a large sample size and by investigating the chemical speciation of zinc to improve our understanding of the mechanism of zinc uptake in bird feathers, as well as how melanins control elemental distribution within a feather or vice versa. Indeed, μ-XRF works on the melanosomes of mice suggested that a zinc-and calcium-rich pheomelanin core was surrounded by a copper-rich eumelanin shell, [52] indicating a complex interplay between the melanin pigments and trace element deposition.

| CONCLUSIONS
This study demonstrated the usefulness of synchrotron X-ray fluorescence spectroscopy in examining the spatial deposition of trace elements within feathers developing under experimental dietary conditions. The study also demonstrated the importance of using a combination of imaging techniques to identify whether dietary changes affect the level and spatial variation of trace elements within feathers. Synchrotron μ-XRF spectroscopy has advantages over traditional analytical methods (e.g., inductively coupled plasma-mass spectroscopy) by focusing on the elements of interest, even at low levels, and provides the necessary spatial resolution to image metals within the feather barb and barbules. Some drawbacks of this technology are longer dwell times needed per point during sample analysis as well as the relative quantification of elements instead of full quantification. Although the former depends on the advancement of synchrotron technology, the latter has the potential to be addressed by comparing element-specific signal intensity with known standards. When conducted in conjunction with minimally invasive feather sampling, this technology has great potential for use in environmental monitoring of other trace element contaminants, such as mercury, arsenic, selenium, and lead in areas impacted by increasing urbanization and industrial activity.

DATA ACCESSIBILITY
Data used in the paper have been uploaded to Dryad, at https://doi.org/10.5061/dryad.vq83bk3pb. Additional data are available upon request, from corresponding authors.