DOI: 10.5061/dryad.gtht76hpx
title: Evaluating the use of hair as a non-invasive indicator of trace mineral status in woodland caribou (Rangifer tarandus caribou)

description: Trace mineral imbalances can have significant effects on animal health,
 reproductive success, and survival. Monitoring their status in wildlife
 populations is, therefore, important for management and conservation.
 Typically, livers and kidneys are sampled to measure mineral status, but
 biopsies and lethal-sampling are not always possible, particularly for
 Species at Risk. We aimed to: 1) determine baseline mineral levels in
 Northern Mountain caribou (Rangifer tarandus caribou; Gmelin, 1788) in
 northwestern British Columbia, Canada, and 2) determine if hair can be
 used as an effective indicator of caribou mineral status by evaluating
 associations between hair and organ mineral concentrations. Hair, liver,
 and kidney samples from adult male caribou (n­­Hair= 31; nLiver, nKidney=
 43) were collected by guide-outfitters in 2016-2018 hunting seasons. Trace
 minerals and heavy metals were quantified using inductively-coupled plasma
 mass spectrometry, and organ and hair concentrations of same individuals
 were compared. Some organ mineral concentrations differed from other
 caribou populations, though no clinical deficiency or toxicity symptoms
 were reported in our population. Significant correlations were found
 between liver and hair selenium (rho=0.66, p&lt;0.05), kidney and hair
 cobalt (rho=0.51, p&lt;0.05), and liver and hair molybdenum (rho=0.37,
 p&lt;0.10). These findings suggest that hair trace mineral assessment
 may be used as a non-invasive and easily-accessible way to monitor caribou
 selenium, cobalt, and molybdenum status, and may be a valuable tool to
 help assess overall caribou health.
description: <strong>Study Population and Sample
 Collection</strong> We studied 7 herds of the
 Northern Mountain ecotype of woodland caribou (<em>R. t.
 caribou</em>) occurring in the traditional territory of the Tahltan
 Nation (northwestern British Columbia, Canada) (Fig 1). No animals were
 killed for the purposes of this study. Caribou were legally hunted in
 their natural habitat by authorized non-resident hunters during the fall
 hunting season (15 August – 15 October), accompanied by guide-outfitter
 members of the Tahltan Guide and Outfitters Association (TGOA), per
 regulations outlined in the B.C. Hunting and Trapping Synopsis under the
 Wildlife Act (Government of British Columbia). Samples were contributed by
 guide-outfitters participating in a harvest-based wildlife health sampling
 program initiated in 2016 and use for this study was approved per the BC
 Wildlife Permit MRSM 18-285261 (Government of British Columbia Ministry of
 Forests, Lands, Natural Resource Operations, and Rural Development
 (FLNRORD)) and the Animal Use Protocol AC-18-0093 (University of Calgary
 Animal Care Committee).  <em>Fig
 1.</em> Study Area: the Tahltan Nation Traditional Territory with
 herd boundaries of 7 Northern Mountain woodland caribou herds included in
 this study. The study population included adult male
 Northern Mountain Caribou hunted by guided hunters in the fall of 2016,
 2017, and 2018. Sixty-three sample kits were collected from animals hunted
 between 25th August and 11th October from 2016 to 2018. Participating
 guide-outfitters collected a standard set of samples and data from
 harvested animals [18]. This included hair from the dorsal shoulder region
 of the harvested animal, a 10 cm<sup>2</sup> section of skin
 from the dorsal rump area, samples of liver tissue (~ 5
 cm<sup>3</sup> section), and whole left kidneys. In some
 cases, participants submitted partial left kidneys rather than the entire
 organ. Samples were stored in Whirl Pak™ sterile sample bags and frozen at
 -20°C until processing and analysis. Central incisors were submitted to
 Matson’s Laboratory, Manhattan, Montana for aging by cementum ring
 analysis [22]. <strong>Sample
 Analysis</strong> <strong><em>Hair
 Analysis</em></strong> Hair from the
 shoulder was preferentially used. In cases where insufficient sample was
 collected, hair from rump skin sections of the same individual was
 collected in the lab by shaving as close to the skin surface as possible.
 Visible debris such as soil and vegetation were removed from hair samples
 using plastic forceps. Samples were then washed twice in 96% ethanol and
 ultrapure Type 1 reagent-grade water to remove further external
 contamination then placed in clean paper envelopes and oven-dried at 50°C
 for at least 24 hours. 30-50 mg of dried hair was weighed and added to 2
 mL of 70% HNO3 in a plastic vial (TMF Vessel, 100mL; Milestone, Shelton,
 CT, USA). The vials were closed with air-tight caps and digested using a
 high-pressure microwave reactor (ETHOS EZ Microwave Digestion System;
 Milestone, Shelton, CT, USA). The temperature in the reactor was gradually
 increased from room temperature to 220°C over one hour, and then gradually
 cooled to room temperature over one hour. 2 mL of each digested sample was
 transferred to a falcon tube and diluted with ultrapure Type 1 water to a
 total volume of 4 mL and stored at 5C until analysis. Each sample was
 further diluted with Type 1 water to a final dilution of 1:10 and hair
 mineral concentrations were determined using high-resolution inductively
 coupled plasma mass spectrometry (ICP-MS, 8800 Triple Quadrupole ICP-MS,
 Agilent) at the Alberta Center for Toxicology, University of Calgary.
 Instrument calibration verification for Quality Assurance (QA) were
 performed before, during, and after sample analyses using certified
 reference materials (Trace Elements in Natural Water (NIST1640a);
 Multi-Element Standard (SCP Science); and Environmental Calibration
 Standard (Agilent)). For each digestion (15 vials per run), 1 sample was a
 blank sample containing only acid to check for any contamination in
 laboratory procedure; 1 sample consisted of reference material for QA; and
 13 vials contained samples. Of these samples, 1 sample was randomly
 selected to be run in duplicate for QA for each run. A maximum deviation
 limit of 20% between duplicates was set for the results in the run to be
 accepted, and all samples run in duplicate met these criteria when amount
 of mineral detected was greater than the method Limit of Quantitation
 (LOQ). For samples run in duplicate, the average of the two mineral
 concentration values was used for analysis. The LOQ (wet weight, digested
 sample) for Co, Pb, and Mo was 0.005 mg/L, for Mn and Se was 0.001 mg/L,
 for Cd, Cu, Zn was 0.005 mg/L, and for Fe was 0.05 mg/L. Mineral
 concentrations detected but falling below LOQ were included in the
 analysis. In cases where concentrations fell below detection limits,
 values of half the detection limit were assigned to assess correlations
 [23], and omitted for reporting baseline hair concentrations. Quality
 assurance was further confirmed in each batch, using certified reference
 materials (NIST2976 freeze-dried mussel tissue, National Institute of
 Standards and Technology; and NRC DORM-4 “Fish Protein Certified Reference
 Material for Trace Metals”, National Research Council Canada) as positive
 controls, and blank samples as negative controls. Blanks were negligible
 for all samples, and the laboratory positive controls were measured within
 acceptable ranges of certified reference values for all elements studied.
 Results are reported in mg/kg dry weight.
 <em><strong>Kidney and Liver Tissue
 Analysis</strong></em> The outermost
 portions of liver samples and partial kidney samples were removed to
 minimize external trace mineral contamination from handling. Sterile
 stainless-steel scalpel blades were used, with efforts made to minimize
 instrument use where possible. Extra tissues and the renal capsule were
 removed from kidneys. When only partial kidney samples were submitted,
 those samples with unequal cortex:medulla ratios were discarded. Samples
 (minimum 5 g tissue, wet weight) were submitted to a commercial laboratory
 (ALS Environmental, Vancouver, BC). Metals analysis was conducted as
 described in by Horvath [24], where tissues were homogenized and then
 subsampled prior to being hot block digested with nitric and hydrochloric
 acids combined with hydrogen peroxide. Analysis for tissue concentrations
 of various trace minerals was done by collision cell - inductively coupled
 plasma - mass spectrometry (CC-ICP-MS), modified from the standard US-EPA
 Method 6020A [25]. Moisture content of tissues (% Moisture) was determined
 gravimetrically by drying each sample at 105°C for a minimum of 6
 hours.  Samples were run in duplicate. A maximum
 deviation of 20% between duplicates was applied to qualify samples to be
 included in data analysis, and all samples met these criteria. Quality
 assurance was confirmed in each batch, using certified reference materials
 (NRC DORM-4 “Fish Protein Certified Reference Material for Trace Metals”,
 National Research Council Canada) and laboratory control samples as
 positive controls and method blank samples as negative controls. Blanks
 were negligible for all samples, and the concentrations measured were
 within acceptable ranges of certified reference values for all elements,
 meeting data quality objectives set by the commercial laboratory. In cases
 where concentrations fell below detection limits, values of half detection
 limit were assigned [23].  Results are reported in mg/kg dry weight
 (DW). <strong>Statistical
 analysis</strong> Liver and kidney trace mineral
 concentrations were compared to those published for other Canadian caribou
 herds/ecotypes using t-tests with Bonferroni multiple comparisons
 corrections. Shapiro-Wilk tests and visualization of residual plots were
 used to assess normality and homoscedasticity in the dataset. Where
 necessary and possible, data were logarithmically transformed to meet
 normality assumptions. Spearman rank correlations between hair, liver, and
 kidney mineral concentrations were assessed (Tables 2 and 3). The Cook’s
 distance test was used to identify influential multivariate outliers based
 on a standard cut-off (4 x mean), and univariate outliers were identified
 as values deviating from the mean by greater than 3 standard deviations.
 Outliers were maintained in the dataset and impacts of these outliers on
 relationships found between tissue mineral concentrations were assessed by
 removal for a sensitivity analysis. ANOVAs with Tukey’s post-hoc tests
 were used to evaluate differences in hair trace mineral concentrations
 based on body location of hair collection (rump versus shoulder). When
 bivariate normality was not possible, non-parametric tests were used
 instead (Kruskal-Wallis rank tests and Pairwise-Wilcoxon rank sum tests in
 place of 1-way ANOVA and Tukey post-hoc tests). In cases where significant
 differences were noted between the means of element concentrations in
 samples from different body locations, Spearman rank correlations were
 reassessed separately for different sample types as a further sensitivity
 analysis of the initial relationships found based on pooled data.
 Statistical tests were applied using the following packages: ‘tidyverse’
 Version 1.2.1 (2019); ‘dplyr’ Version 0.8.3 (2019); and ‘ggplot2’ Version
 3.2.1 (2019). All statistical analyses were done in R Version 3.6.0
 [26].
description: Microsoft Excel