DOI: 10.5061/dryad.1g1jwsv20
title: Data from: Mercury concentrations decline with age in the fur of females of an insectivorous terrestrial mammal (Myotis lucifugus)

description: Methylmercury (MeHg) is a toxic form of mercury that bioaccumulates in
 organisms and biomagnifies through food webs. MeHg concentrations can be
 high in aquatic environments, and this puts high trophic-level predators
 who derive energy originating from aquatic environments at risk of toxic
 effects. Due to the potential for bioaccumulation of MeHg over an
 individual’s life, the risk of MeHg toxicity may increase as animals age,
 and this risk may be especially high in species with relatively high
 metabolic rates. Total mercury (THg) concentrations were measured from the
 fur of adult female little brown bats (Myotis lucifugus) collected between
 2012–2017 in Salmonier Nature Park, Newfoundland and Labrador. Using
 linear mixed-effects models, the effects of age, year, and day of capture
 on THg concentrations were evaluated and interpreted with AICc and
 multi-model inference. We expected that THg concentrations would increase
 with age, and that due to annual summer moulting, individuals captured
 earlier in the season would have lower THg concentrations than individuals
 captured later in the season. Contrary to expectations, THg concentrations
 decreased with age, and date of capture did not explain any variation in
 concentration. Among individuals, there was a negative relationship
 between the initial THg concentration of an individual and the rate of
 change in THg concentrations with age. Using a regression analysis, we
 found evidence of a population-level decline in THg concentrations in fur
 over the 6-year study period. Overall, the results indicate that adult
 female bats eliminate enough MeHg from their tissues to affect a decrease
 in THg concentrations in their fur over time and that young adults are
 potentially at the greatest risk of experiencing toxic effects from high
 MeHg concentrations; this could result in reduced reproductive output, and
 warrants further research.
description: Study
 area The fur specimens used
 in this study were collected between 2012 and 2017 in and adjacent to SNP,
 NL, Canada (Lat: 47.3º, Long: -53.3º). Salmonier
 Nature Park is a 1455-ha nature reserve that is part of the Avalon Forest
 Ecoregion which is characterized by its ribbed moraines covered in forests
 that are interspersed with lakes and bogs. The atmospheric deposition rate
 of Hg in Newfoundland is among the highest in Canada (Chételat et al.
 2020) and surface waters in SNP exhibit low pH due to the low acid
 neutralization ability of the soil and rock (Clair et al. 2007). Since low
 pH is correlated with increased methylation of Hg (Zhu et al. 2018), high
 mercury deposition and low surface water pH make wildlife in SNP
 susceptible to accumulating harmful concentrations of MeHg (Little et al.
 2015a). Sample
 collection <em>Myotis
 lucifugus </em>were captured in SNP using
 mist nets (Avinet, Dryden, New York, USA) and harp traps (Austbat Research
 Equipment, Lower Plenty, Victoria, Australia) set near maternity roosts.
 Adult <em>M. lucifugus</em> are sexually segregated during
 their summer active season, and targeted efforts near maternity roosts
 thus resulted in captures comprised almost exclusively of adult females
 and juveniles. Bats were caught throughout their active period in SNP,
 with the earliest capture on May 15 and the latest capture on August 13.
 Age class (adult or juvenile) was determined based on the degree of
 ossification of the third metacarpal-phalangeal joint (Kunz &
 Anthony 1982). Captured bats had a passive integrated transponder (PIT)
 tag (0.09 g; EID-ID100 implantable transponders, EIDAPInc, Sherwood Park,
 Alberta, Canada and Trovan Electronic Identification Systems, UK)
 subdermally injected between the scapulae. Recaptured bats already
 carrying a PIT tag were identified using a handheld PIT tag reader,
 providing data on time between captures. Individuals first captured as
 adults were assigned a ‘minimum age' of 1. In the subsequent captures
 of these individuals, they were assigned a ‘minimum age’ which
 corresponded to the number of years since their first capture. Individuals
 initially captured as juveniles were assigned ‘exact’ ages for each
 capture. Fur samples were taken from between the scapulae using cuticle
 scissors. To prevent cross-contamination of fur, scissors were cleaned
 between individuals by washing in bleach, then water, followed by ethanol.
 Fur was placed in polypropylene vials and frozen at -20 or
 -80<sup>o</sup>C. Differences in storage temperature were a
 result of space limitations; however, given the stability of Hg in fur,
 these differences are not expected to change the THg content (Phelps et
 al. 1980). Forearm length and mass were measured, and sex was determined.
 Reproductive status (pregnant, lactating, post-lactating, not obviously
 pregnant, or non-reproductive) was determined by palpitation of the
 abdomen and visual inspection of the nipples for signs of lactation (Racey
 & Swift 1985). Following processing, all animals were released at
 the site of capture. All animal handling and sample transport was done in
 accordance with the protocols approved by St. Mary’s University Animal
 Care Committee and the Government of Newfoundland and Labrador (SMU-MSVU
 protocols<strong> </strong>11-18<strong>,
 </strong>12-17, 13-15, 14-10A, 15-12, 16-12; Government of
 Newfoundland and Labrador protocols IW2011-14, IW2012- 26, IW2013-10,
 IW2014-31, IW2015-52, IW-2015-11, WLR2016-12, WLR2017-16,
 WLR2017-17). Mercury
 analysis Due to a limited sample
 size of male bats, only fur from adult female bats was analysed. Prior to
 Hg analysis, fur samples were visually examined for external sources of
 contamination such as dirt. Fur with visible contamination was cleaned
 with a Kimwipe (Kimberly-Clark, Mississauga, ON). To avoid loss of sample
 mass, fur was not washed to remove other potential sources of exogenous
 contamination. Chételat et al. (2018) and Little et al. (2015b) both found
 that washing then drying bat fur does not alter THg content. Thus, we
 assumed any exogenous contamination was negligible for THg and MeHg
 analyses. Fur samples were weighed
 (± 0.001 mg) and analysed for THg content using a direct mercury analyser
 in accordance with Environmental Protection Agency (EPA) method 7473 in
 the Biotron Centre for Experimental Climate Change Research at Western
 University, London, Ontario (Milestone Tricell DMA 80, Milestone, Inc.,
 Shelton, CT). The mean weight of fur samples analysed for THg was 1.192 mg
 (SD = 0.626). For quality control, IAEA-086 human hair was used as an
 external certified reference material (CRM) to determine total mercury
 recovery (average relative percent recovery = 101 %). Analytical precision
 was determined using sample duplicates (n = 10; average relative percent
 difference = 5%). As determined using the definition and procedure
 outlined in EPA (2016), the method detection limit for THg was 0.07 ng and
 no samples fell below this limit. THg concentrations are reported in µg/g
 fresh weight (fw). Methylmercury was
 analysed using a modified version of EPA method 1630 in an ISO 17025
 accredited laboratory in the Biotron Centre for Experimental Climate
 Change Research at Western University using a
 Tekran<sup>®</sup> 2700 automated methylmercury analyser. The
 mean weight of fur samples analysed for MeHg was 1.696 mg (SD = 0.265).
 Percent MeHg recovery was determined using a human hair CRM (IAEA-086)
 (average relative percent recovery = 110 %). Analytical precision was
 determined using a sample duplicate (n = 1; relative percent difference =
 2%). As determined using the definition and procedure outlined in EPA
 (2016), the method detection limit for MeHg was 0.051 ng/g and no samples
 fell below this limit. MeHg concentrations are reported in µg/g
 fw. Statistical
 analysis We used THg
 concentrations in 159 fur samples (representing 104 individuals), 9 of
 which were subsampled and analysed for MeHg concentration. A regression
 analysis was used to estimate the proportion of THg in the fur comprised
 of MeHg. An alpha value of 0.05 was used, and residuals of analyses were
 assessed to check assumptions. All data analysis was performed in R
 version 4.0.3 (R Core Team 2020). To assess trends in THg
 concentrations among calendar years, a regression analysis was performed
 on THg data collected from the fur of 60 randomly selected (10
 samples/calendar year) adult females. To avoid sample bias towards older
 individuals, only bats that had a ‘minimum age’ of 1, and thus of unknown
 age, were selected. To minimize within-year variability that may result
 from factors such as moulting and/or insect availability, the analysis was
 limited to samples collected from adult females captured between July 29
 and August 7 (2012–2017). These dates were selected based on the
 coincidence of sampling periods in all years. To characterize how THg
 concentrations in fur vary with bat age, we used fur samples from 47 adult
 female bats that were captured at least twice over the study period (mean
 = 2.21). Using the R package <em>lme4</em> (Bates et al.
 2015), we created multiple linear mixed-effects models from three
 <em>a priori </em>selected parameters (Burnham &
 Anderson 2002) including <em>minimum age</em>,
 <em>Julian day</em>, and<em> year</em>.
 <em>Julian day</em> is the day-of-year the fur sample was
 taken (day 1 to day 365) and was included to account for annual moulting,
 which has been suggested to occur during summer months (Fraser et al.
 2013). If moulting occurs in the summer months, individuals captured
 earlier in the year may not have moulted at the time of capture. If THg
 increases with age, we would thus expect earlier captures to have a
 slightly lower THg relative to captures later in the year. <em>Year
 </em>was included to account for variability among calendar years.
 To account for differing intra-annual trends among years, an interaction
 term including <em>year </em>and<em> Julian day
 </em>was included. There was no evidence of multicollinearity among
 the parameters, <em>minimum age</em>,
 <em>year</em>, and <em>Julian day</em> (variance
 inflation factor &lt; 5) as determined using the <em>vif
 </em>function in the R package, <em>car</em> (Fox
 & Weisberg 2019). Results from nine
 <em>a priori</em> linear mixed-effects models and one null
 random effects model (Table 2) were evaluated using Akaike’s information
 criterion corrected for small sample size (AIC<sub>c</sub>)
 (Burnham & Anderson 2002) with the <em>MuMIn</em>
 package in R (Bartoń 2019). Following the procedure in Zuur et al. (2007),
 and using AIC values, the optimal random effect structure was identified
 as a random intercept and slope structure (Table 3). Thus, each
 mixed-effects model included random intercepts and slopes, with individual
 as the random effect. Using multimodel inference, we calculated
 unconditional estimates for parameters included in models that comprised ≥
 95 % of the confidence set.