DOI: 10.5061/dryad.tb2rbnzww
title: Feather mercury increases with feeding at higher trophic levels in two species of migrant raptors, Merlin (Falco columbarius) and Sharp-shinned Hawk (Accipiter striatus)

description: Mercury (Hg) is a toxic heavy metal that, when methylated to form
 methylmercury (MeHg), bioaccumulates in exposed animals and biomagnifies
 through food webs. The purpose of this study was to assess Hg
 concentrations in raptors migrating through the upper Midwestern USA. From
 2009-2012, 966 raptors of 11 species were captured at Hawk Ridge, Duluth,
 MN. Breast feathers were sampled to determine the concentration of total
 Hg. Mean Hg concentrations ranged from 0.11 – 3.46 μg g-1 fresh weight
 across species and were generally higher in raptors that feed on birds in
 comparison with those that feed on mammals. To evaluate the effect of
 dietary sources on Hg biomagnification, carbon and nitrogen stable isotope
 ratios were measured in feathers of the two species with the highest Hg
 concentrations, Merlin (Falco columbarius) and Sharp-shinned Hawk
 (Accipiter striatus). Measured δ13C values were similar in both species
 and indicated a primarily terrestrial-derived diet, whereas δ15N values
 suggested that individual Merlin and Sharp-shinned Hawk feeding at higher
 trophic levels accumulated higher concentrations of Hg. The risk to birds
 associated with measured levels of feather Hg was evaluated by calculating
 blood-equivalent values using an established algorithm. Predicted blood
 values were then compared to heuristic risk categories synthesized across
 avian orders. This analysis suggested that while some Merlin and
 Sharp-shinned Hawk were at moderate risk to adverse effects of MeHg, most
 of the sampled birds were at negligible or low risk.
description: Study Area
 All samples were collected
 between 2009-2012 during fall migration (September through November) from
 the Hawk Ridge Bird Observatory banding station at Duluth, MN, USA
 (46.85<sup>o</sup>N, -92.03<sup>o</sup>W) located
 near the westernmost point of Lake Superior. Situated at ~300 m above sea
 level, Hawk Ridge is positioned on basalt rock that extends along the
 north shore of the lake. This unique location concentrates large numbers
 of birds of prey, and on average, the banding station bands over 2,600
 raptors each fall (Evans et al. 2012). Limited band recovery data suggest
 that diurnal raptors migrating past Duluth likely originate from breeding
 grounds in Western Canada and northern Minnesota (Evans et al.
 2012). Raptor
 Sampling  Birds were captured using mist, bow, and dho gaza nets
 (Evans et al. 2012, Table 1). Captured birds were identified, aged, and
 sexed using methods described by Pyle (2008). Two to four breast feathers
 were plucked from each sampled bird, inspected for external debris, and
 placed into envelopes. Feathers were chosen because they represent a
 reliable index of Hg exposure during the time of feather growth (Bearhop
 et al. 2000, Kenow et al. 2007, Condon and Cristol 2009) and because they
 can be sampled non-invasively and easily preserved at room temperature for
 long periods. Hg Analysis One feather per
 individual was analyzed for total Hg concentration by thermal
 decomposition spectrophotometry (EPA method 7473) using a DMA-80 Direct
 Mercury Analyzer (Milestone) at the BRI Toxicology Lab at Biodiversity
 Research Institute in Portland, ME, following methods described by Evers
 et al. (2005). Feathers were prepared for analysis using a protocol
 employed by U.S. Fish and Wildlife Service contract laboratories (e.g.,
 Texas A & M University; R. Taylor, pers. comm.). Only feathers
 with no visual evidence of external debris were analyzed. Feathers were
 not washed, as this protocol does not recommend washing unless external
 contamination is of concern. Quality control methods including the use of
 analytical blanks, sample replicates, and certified reference materials
 DOLT-4, DORM-3 and DORM-4, were employed to evaluate analytical precision
 and accuracy. Total Hg concentrations in all feathers were above the
 method detection limit (0.001 µg g<sup>-1</sup>). Measured Hg
 concentrations in Certified Reference Materials (CRMs, National Research
 Council, Canada and Joint Research Centre, European Union) incorporated
 into each sample run averaged 100% (DOLT-4), 103.7% (DORM-3) and 97.9%
 (DORM-4) of published values. Feather Hg concentrations are presented in
 μg g<sup>-1</sup> on a fresh weight (fw) basis. Previous work
 has shown that Hg concentrations can vary among feathers in different
 parts of the plumage of individual birds (Furness et al. 1986, Braune and
 Gaskin 1987, Peterson et al. 2019). Although we did not sample multiple
 feather tracts, we controlled for variation among feathers within birds
 and standardized comparisons across birds by limiting our sampling to only
 breast feathers. We also analyzed limited duplicates
 (e.g., a separate breast feather from the same individual) to verify
 consistency in Hg concentrations among breast feathers within the same
 individuals. For duplicates we calculated percent relative difference and
 Pearson’s correlation coefficient. Because HY birds were known to have
 grown all feathers within a recent and well-defined timeframe (i.e.,
 nestling development) we also analyzed duplicates and calculated the same
 statistics within HY and AHY age class groups. The fw of a feather is
 nearly equivalent to dry weight (dw; R. Taylor, TERL, Texas A & M
 University, mean % feather moisture &lt;1%, n = 490, reported in
 DeSorbo et al. 2018). We therefore considered fw = dw for the purposes of
 literature comparisons. Stable Isotope Analysis  Ratios of carbon
 (δ<sup>13</sup>C) and nitrogen (δ<sup>15</sup>N)
 stable isotopes in feathers were measured for the two species of raptors
 with the highest mean Hg concentrations: Merlin and Sharp-shinned Hawk.
 This was done to evaluate the potential relationship between feather Hg
 and trophic status (as measured by δ<sup>15</sup>N), and
 aquatic versus terrestrial carbon source in the raptor diet (as measured
 by δ<sup>13</sup>C). Samples from 20 individuals within each
 age and sex class were selected for stable isotope analysis, with 10
 feathers each from the lowest and highest Hg results for each class. Each
 feather sample was cleaned with a 2:1 chloroform:methanol solution (Hobson
 1999), placed in a pre-combusted scintillation vial, and dried at
 50<sup>o</sup>C for at least 24 hours. After drying, the
 sample was minced and 0.7 μg analyzed with a Costec 4010 EA and Thermo
 Delta Plus XP isotope ratio mass spectrometer. Stable isotope ratios are
 reported in standard δ notation, wherein Vienna Pee Dee Belemnite and air
 are standards for δ<sup>13</sup>C and
 δ<sup>15</sup>N, respectively. Analytical error, calculated as
 the mean standard deviation of replicate reference material, was &lt;
 0.1‰ for δ<sup>13</sup>C and
 δ<sup>15</sup>N.  Statistical Analyses Measured feather
 Hg concentrations were natural log-transformed, and geometric mean
 concentrations (± SE) were calculated for all 11 species. For a subset of
 six species with greater sample sizes (n ≥20), analysis of variance was
 used to compare ln-transformed Hg concentrations, ln(feather Hg), by sex
 within species, age within species, and their interaction (cohort = all
 combinations of age and sex) within species. Additional
 statistical analyses were performed for Merlin and Sharp-shinned Hawk, our
 two largest samples. A set of 18 general linear models was developed to
 compare ln(feather Hg) to the year in which birds were captured, ordinal
 day on which birds were captured, bird age class, sex, and all bivariate
 interaction terms (Table 2). Year was modeled as a quantitative covariate
 to test whether feather Hg concentrations were changing over the course of
 the four years of study. Date was also modeled as a quantitative covariate
 to explore seasonal variation in feather Hg. Age and sex were modeled as
 factors, each with 2 levels. Variable importance was calculated based on
 Akaike’s Information Criterion, corrected for small sample size
 (AIC<sub>c</sub>), as the sum of AIC<sub>c</sub>
 weights for each model in which the variable occurred (Burnham and
 Anderson 2002). A list of all models considered for Merlin and
 Sharp-shinned Hawk is provided in Table 2. Correlations
 between ln(feather Hg) and δ<sup>13</sup>C, and between
 ln(feather Hg) and δ<sup>15</sup>N by sex, age, and cohort
 within each species were evaluated using Pearson’s correlation
 coefficient. All analyses were conducted in R (version 2.14.0, R
 Development Core Team 2013). 
description: There are three worksheets within the Excel file: Hg, Hg_Dups,
 and Isotope. Hg contains the methylmercury data and should be
 self-explanatory except perhaps for the 4-letter alphanumeric species
 codes, which can be easily looked up, and age codes (HY = Hatch Year, AHY
 = After Hatch Year, SY = Second Year, ASY = After Second Year). Hg_Dups
 contains the Hg lab duplicate data. Isotope contains the C and N stable
 isotope data, which should also be self-explanatory, although there are
 only two species (SS = Sharp-shinned Hawk and ML = Merlin).