Manganese accumulates in the brain of northern quolls (Dasyurus hallucatus) living near an active mine

Environmental Pollution 233 (2018) 377e386

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Manganese accumulates in the brain of northern quolls (Dasyurus hallucatus) living near an active mine* Ami Fadhillah Amir Abdul Nasir a, Skye F. Cameron a, Frank A. von Hippel b, John Postlethwait c, Amanda C. Niehaus a, Simon Blomberg a, Robbie S. Wilson a, * a b c

School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia Department of Biological Sciences and Centre for Bioengineering Innovation, Northern Arizona University, Flagstaff, AZ 86011, USA Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 February 2017 Received in revised form 20 October 2017 Accepted 23 October 2017

Mining is fundamental to the Australian economy, yet little is known about how potential contaminants bioaccumulate and affect wildlife living near active mining sites. Here, we show using air sampling that fine manganese dust within the respirable size range is found at levels exceeding international recommendations even 20 km from manganese extraction, processing, and storage facilities on Groote Eylandt, Northern Territory. Endangered northern quolls (Dasyurus hallucatus) living near mining sites were found to have elevated manganese concentrations within their hair, testes, and in two brain regionsdthe neocortex and cerebellum, which are responsible for sensory perception and motor function, respectively. Accumulation in these organs has been associated with adverse reproductive and neurological effects in other species and could affect the long-term population viability of northern quolls. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Bioaccumulation Cerebellum Ecotoxicology Hair Neocortex Neurotoxic metal Testes Tissue tropism

1. Introduction Manganese (Mn) is critical for steel and alloy manufacturing, ranking fourth in world metal demand after iron, aluminium and copper (Levy and Nassetta, 2003; ATSDR, 2012). Though it is an essential elementdvital for bone formation and cellular meta€llin and bolism (Oberdoerster and Cherian, 1988; Wedler, 1994; Ro Nogueira, 2011), Mn toxicity is more common than deficiency and causes progressive neurological damage (Mergler and Baldwin, 1997; Andersen et al., 1999; Normandin et al., 2002; Zoni et al., 2007; ATSDR, 2012) that impairs cognition and motor function and alters behaviour in humans (Levy and Nassetta, 2003; Josephs et al., 2005; Klos et al., 2006; Laohaudomchok et al., 2011). Mn is typically ingested via food or drinking water (Kondakis et al., 1989; He et al., 1994; Bouchard et al., 2007, 2011) but is generally more bioavailable and associated with adverse effects when it is inhaled

*

This paper has been recommended for acceptance by Prof. W. Wen-Xiong. * Corresponding author. E-mail address: [email protected] (R.S. Wilson).

https://doi.org/10.1016/j.envpol.2017.10.088 0269-7491/© 2017 Elsevier Ltd. All rights reserved.

(Oberdoerster and Cherian, 1988; Tjalve and Henriksson, 1999; Aschner, 2000; Fechter et al., 2002), making Mn dust a particular hazard near open mine sites where fine particles (<2.5 mm) can be carried large distances through the air to contaminate residential areas and natural ecosystems (WHO, 2006). Unlike dietary Mn, which is poorly absorbed in high doses and eliminated via feces (Andersen et al., 1999; Finley, 1999; Fechter et al., 2002), inhaled fine Mn particles may deposit with high efficiency throughout the entire respiratory tract, accumulate in lung alveoli and pass into the bloodstream (Antonini et al., 2006). Upon entering blood, oxidised Mn was originally found to bind to ironbinding proteins (transferrins) and then to be transported into the brain and other organs via transferrin receptors (Aschner and Gannon, 1994; Andersen et al., 1999). More recently, several reports have also demonstrated the important roles of other binding proteins such as the ZIP8 and ZIP14 of the solute-carrier-39 (SLC39) metal-transporter family, in transporting Mn2þ into the brain (see review by Roth et al., 2013). Via inhalation, Mn particles may also bypass the respiratory system, passing directly into the brain through the olfactory epithelium that lines the nasal cavity, moving up the olfactory nerve into the olfactory bulb (Aschner, 2000;

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Fechter et al., 2002; Dorman et al., 2004; Lucchini et al., 2012). Though Mn is actively transported into the brain, it is removed only by diffusion, which is a much slower efflux process; this can lead to significant Mn accumulation in the brain (Oberdoerster and € llin and Nogueira, 2011). Elevated Cherian, 1988; Aschner, 2000; Ro exposure to Mn in humans increases its concentration in all parts of the brain, but primarily in the caudate putamen, globus pallidus, substantia nigra, and subthalamic nuclei, which are collectively referred to as the basal ganglia (Newland et al., 1989; Mergler, 1999; Aschner, 2000). Accumulation in non-brain tissues, including liver, lung, blood, testes, bone, and kidney has been found in inhalation experiments with laboratory rodents, but the brain is the site of greatest Mn accumulation (Drown et al., 1986; St-Pierre et al., 2001; Salehi et al., 2003). Though Mn has been well studied in the context of human health, little is known about how Mn bioaccumulates and affects organisms within natural environments, where contamination has the potential to affect the longevity or reproductive success of wild species. Here, we evaluate the geographic variation and elemental composition of dust associated with active Mn mining facilities on Groote Eylandt, Northern Territory, Australia; Mn dust visibly blankets the landscape around extraction, transport, and storage facilities. We then quantify accumulation of these elementsdparticularly Mndwithin the hair, brain, liver, kidney, lung, and testes of an endangered carnivorous marsupial, the northern quoll (Dasyurus hallucatus), which is prevalent on the island. Finally, we discuss the potential implications of our findings to the ecology of wildlife living on Groote Eylandt or near active mine sites elsewhere. 2. Materials and methods 2.1. Study site and species More than 900 plant and 330 vertebrate species have been recorded within the Groote Eylandt archipelago, an Indigenous Protected Area and a site of International Conservation Significance (ALC, 2014; DOE, 2014). The area protects 12 threatened and endangered species including northern quolls and is crucially important for Australian mammal conservation because the widespread decline of small and medium-sized mammals occurring across tropical Australia has not yet affected populations on Groote Eylandt. Northern quolls inhabit localised home ranges around denning sites and are the most common predatory species on Groote Eylandt, with a diet consisting primarily of invertebrates (Schmitt et al., 1989; Oakwood, 2002). This species has a rare, suicidal breeding system: all males die after a short annual breeding frenzy, whereas females survive to breed in a second or third year (Oakwood, 2000; Nelson and Gemmell, 2003). Groote Eylandt is also the site of one of the largest Mn ore producers in the world, Groote Eylandt Mining Company (GEMCO, a BHP Billiton subsidiary), which leases mining sites on the island and produces at least 3 million tonnes of Mn ore annually (South32, 2004; USGS, 2016). Mining operations have been active since 1964. Mn is extracted from open pits and then crushed onsite before it is transported in open trailers to the port for open-air storage and shipping. 2.2. Air sampling and analyses We collected air samples from three sites on Groote Eylandt: (1) at the midpoint of Angurugu-Umbakumba Highway (Central 4) in the centre of the island, located >20 km from both the Mn mine sites, where Mn is extracted and crushed, and the port, where Mn is stored in open air prior to shipping (both sites are hereafter

referred to as Mn mining facilities), (2) within the town of Angurugu, located <1 km from Mn mine sites, and (3) at Portside, located <0.5 km from uncovered Mn stockpiles at the port (Fig. 1). Samples were collected during the dry season, between 16 and 21 October 2014, and 14 May-28 August 2015, using a Mini-Vol TAS air sampler (Airmetrics Co. Inc., Eugene, Oregon, USA). No rain occurred during these sampling periods. At each site, we installed a pump at least 2 m off the ground and 50 m from the edge of the road and programmed it to run at a constant flow of 5 L per minutedcollecting data on one filter setting at a time: (1) Total Suspended Particles (TSP; i.e. all airborne particles, regardless of size), (2) Particulate Matter 10 (PM10; i.e. inhalable particulates of 10 mm and smaller, which can enter the upper respiratory tract), or (3) Particulate Matter 2.5 (PM2.5; i.e. respirable particulates of 2.5 mm and smaller, which may enter the lower respiratory tract). Collection of smaller particle sizes was achieved using an inlet impactor designed with a cut-off at 10 mm or 2.5 mm. Each filter run was 24 h in duration, and collected either TSP, PM10, or PM2.5 in a randomised order. After 24 h, we replaced the high-purity quartz microfibre filters (Whatman QM-A, 47 mm diameter, 2.2 mm pore size) with fresh filters and sampled the next particle size, and the next, in a continuous cycle over the course of the sampling period. Used filters were stored individually in sealed containers until analysis. Based on TSP, PM10 and PM2.5 readings, filters were analysed to estimate total, inhalable, and respirable concentrations respectively, of air-borne Mn, and those of 14 other elements: aluminium (Al), arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), magnesium (Mg), molybdenum (Mo), nickel (Ni), phosphorus (P), lead (Pb), selenium (Se) and zinc (Zn). Each filter was closed-vessel digested using an Ethos 1 microwave digester (Milestone, Sorisole, Italy) with aqua regia (3 nitric acid: 1 hydrochloric acid) and hydrofluoric acid. Digested filters were diluted in triple deionized water and analysed using an inductively coupled plasma optical emission spectrometry (ICPOES) instrument (Varian, Santa Clara, California, USA) to determine the element concentrations in each size setting. Another vial with only water and no filter was analysed as a control. The detailed methodology related to sample preparation and chemical analysis was based on USEPA method 3051. Air concentrations of Al, Cr, Cu, Fe, Mg and Mn were log10transformed because data were not normally distributed. Comparisons between all sites for each element at each filter setting were made using analysis of variance (ANOVA) in the statistical software R Studio (R Core Team, 2016). One out of 14 filters for PM10 (Site: Central 4) was excluded from the analysis as it was thought to be mislabeled. 2.3. Hair sampling and analyses We trapped northern quolls in MayeOctober in 2013, 2014, and 2015 during three separate dry-season periods: pre-breeding (MayeJune), breeding (July) and post-breeding (AugusteOctober). Breeding season was determined to have started when female quolls began showing breeding scars. We trapped at seven sites proximate to Mn mining facilities (Alyangula, Angurugu, NEMS, Pole 13, Portside, Rowell and SEMS) and 5 distant sites that served as baseline controls (four centrally located on the island (Central 1, Central 2, Central 3 and Central 4) and one in the most northern tip of the island (Jagged)) (Fig. 1). The order in which we trapped sites was block-randomised within each season and year. Angurugu, Central 4, and Portside were the same sites used for air sampling. Trapping occurred over three consecutive nights at each site, during which we set at least 30 Tomahawk original series cage traps (20  20  60 cm; Tomahawk ID-103, Hazelhurst, Winconsin, USA) at 60 m intervals, baited them with canned dog food, and left them

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Fig. 1. Study sites on Groote Eylandt (inset), located in the Gulf of Carpentaria, Northern Territory, Australia. The study sites where northern quolls were captured (yellow stars) and air sampling conducted (green stars) are located at various distances from the Mn extraction and processing facilities (shaded orange). The communities of Angurugu and Alyangula are located adjacent to the existing Mn mining facilities. Areas of Near-Mn-mining facilities and Far-Mn-mining facilities are shown (shaded purple), highlighting sites where northern quolls were captured for sampling of organs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

overnight. Early the following morning, we checked the traps and transferred captured quolls into individual cloth bags for processing. The site of each captured quoll was recorded via GPS (Garmin e-Trex 20, Garmin Ltd., Olathe, Kansas, USA). Due to Indigenous fire regimes and cultural practices, regulated by the Traditional Owners of the island, we were not able to access all sites for hair sampling in every season of every year. Quolls were measured and sampled at the Anindilyakwa Threatened Species Centre. Each quoll was individually microchipped by inserting a PIT tag between its shoulder blades (Trovan nano-transponder ID-100, Keysborough, Australia) to ensure correct identification during recaptures. We recorded sex, breeding status, and body mass (±1 g; A & D Company Limited HL200i, Brisbane, Australia), and we estimated age based on incisor wear and reproductive condition (i.e. deepened pouch and swollen teats indicated 2nd or 3rd year females) (Oakwood, 2000). Hair samples of 1.5e2.0 cm in length (10e80 mg) were plucked from between the shoulder blades of each quoll and used to estimate accumulated concentrations of Mn and that of 14 other elements. Quolls were released in the evening of the capture day at their site of collection. All research methods were approved by the University of Queensland animal ethics committee (permit number SBS/541/12/ANINDILYAKWA/MYBRAINSC) and the Northern Territory Parks and Wildlife Commission (permit number 47603). Before analysis of hair samples, external dust was removed by

washing the samples once with deionized water, mixing thoroughly with 1% Triton® X100 non-ionic detergent solution, and rinsing in deionized water at least 8x until detergent residues were removed. Each sample was then dried in an oven at 110  C for an hour and weighed in order to estimate per-kilo element concentrations. This detergent-based hair washing procedure was adapted from that described in Filistowicz et al. (2011) and Kempson and Skinner (2012). To assess the effectiveness of the cleaning methods we adopted, both unwashed and washed hair samples from 20 individual quolls captured in 2014 were sent to the same processing lab in UQ. 10 of these samples were of quolls captured near the mines, and another 10 from sites far from the mines. On average, we found that Mn levels in washed samples to be approximately 75% lower than that of unwashed samples. Hair samples were then mixed with nitric acid and hydrochloric acid solution (3:1 ratio) and closed-vessel digested using an Ethos 1 microwave digester (Milestone, Sorisole, Italy), diluted in triple deionized water, and analysed using an ICPOES instrument (Varian, Santa Clara, California, USA) to determine element concentrations. With each digest batch, two National Institute of Standards and Technology (NIST) QC plant material samples (peach leaf 1547 and tomato leaf 1573; Gaithersburg, Maryland, USA) with known mean concentrations of elements were also micro-digested and analysed alongside the quoll samples to verify the accuracy of the digestion method. Three blanks were also digested with each batch of

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samples. For precision, samples were analysed twice through the instrument, with five replicate readings taken on each ICP run. The mean of readings was used in analyses. The coefficients of variation for control samples were 13.85% (peach leaf 1547) and 12.01% (tomato leaf 1573) across all 15 elements measured. Samples were weighed to five decimal points on a calibrated scale (Sartorius Secura225D-1S semi-microbalance, Goettingen, Germany). All element concentrations are presented in mg/kg units. Hair concentrations of all measured elements were log10transformed because data were not normally distributed. Based on a principal components analysis (PCA) of all transformed elements, two components were derived as an index of element loading in quoll hair (Table 1). Principal component 1 (PC1Load) accounted for 45.84% of the variance, with all element concentrations loaded positively and concentrations of Mn, Cr and Mo loaded most positively. The higher the PC1Load, the higher the concentrations of all elements, particularly Mn, Cr and Mo. Principal component 2 (PC2Load) accounted for 23.05% of the variance and had Mn loaded most positively when compared to the other 14 elements. Thus, a higher PC2Load indicates a higher Mn concentration in quoll hair. Comparisons between all sites were first made for both PCs using analysis of covariance (ANCOVA) to understand the overall distribution of element loadings in quolls across the island. This approach was then repeated for every element separately to identify which of the 15 elements differed most among sites. Body mass (covariate) and sex (fixed factor) were included in all ANCOVAs. 2.4. Tissue sampling and analyses In 2015, we sampled the hair and tissues of 18 post-breeding male quolls from sites near (n ¼ 8) and far (n ¼ 10) from the Mn mining facilities (Fig. 1). Only post-breeding males were used for tissue sampling because their genetic contribution to the population was already complete and they would soon die due to their suicidal breeding strategy. Quolls were humanely euthanized and concentrations of Mn and that of 14 other elements were measured within several target tissues, including the brain (neocortex, cerebellum and olfactory bulb), kidney (left), liver (median lobe), lung (left lobe) and testis (distal). Once processed for body mass and hair samples, an individual male quoll was gently restrained in a canvas bag and killed via decapitation by skilled handlers using a commercially available small-animal guillotine (Kent Scientific, Connecticut, USA). Done quickly and firmly, this process caused Table 1 Principal component loadings of 15 log10-transformed element concentrations measured in hair samples of northern quolls (n ¼ 230; 107 females, 123 males). Percentage of variance explained by each PC is included at the bottom of the table. Element

PC1

PC2

Al As Cd Co Cr Cu Fe Mg Mn Mo Ni P Pb Se Zn Percentage of variance

0.225 0.203 0.028 0.264 0.500 0.053 0.292 0.003 0.484 0.471 0.147 0.019 0.101 0.052 0.093 45.84

0.245 0.143 0.015 0.188 0.401 0.001 0.222 0.104 0.710 0.369 0.110 0.004 0.002 0.049 0.088 23.05

little discomfort and distress for these animals. Immediately after decapitation, target tissues were dissected and stored at 80  C. Carcasses were disposed of using standard biological waste protocols. All research methodologies were approved by the University of Queensland animal ethics committee (SBS/541/12/ANINDILYAKWA/MYBRAINSC) and the Northern Territory Parks and Wildlife Commission (permit number 47603). Tissue samples were digested and analysed using a method similar to that used for hair samples, except with a nitric acid and perchloric acid solution (5:1 ratio). Tissues ranged in mass from 20-300 mg and were too small to separately test for moisture content and convert to dry-weights. For this reason, we report tissue metal concentrations on a wet weight basis. Because sample sizes were limited, capture sites were analysed as categorical variables, relative to distance from Mn mining facilities (far vs. near). We calculated three measures for contaminated hair in euthanised quolls (as above) in order to relate metal concentrations in hair to metal concentrations in tissues: the PC1Load, PC2Load and log10-transformed Mn concentration (hereafter referred to as hair[Mn]). We used an information-theoretic approach to develop the most parsimonious model for each mea , 2016; R package MuMIn, R sure and tissue combination (Barton Core Team, 2016). For each element assessed, we first fitted a global model with the following variables: Body mass þ Measure, and a second order interaction term: Body mass:Measure. We then ranked each sub-model (fitted under the global model) according to its Akaike Information Criterion (AIC; with small-sample adjustment), and calculated Akaike weights (wi) to estimate the relative support/likelihood for each sub-model. We then extracted the most parsimonious model (with the highest wi) and examined the significance of effects using F-tests. When the most parsimonious model retained a low wi, we averaged all sub-models produced under the same global model to obtain conditional and unconditional parameter estimates and relative importance values for each variable. Lastly, we used Pearson product-moment correlation coefficients to assess the relationship between PC1Load, PC2Load and hair[Mn] with log10-transformed Mn concentration in each tissue separately. We also conducted Wilcoxon rank-sum tests to compare the log10-transformed Mn concentrations in the hair of male quolls that were euthanised to those concentrations of male quolls measured during catch and release within the same post-breeding period, both near and far from the mine. 3. Results 3.1. Air samples We analysed 131 filters for the total (TSP; n ¼ 44), inhalable (PM10; n ¼ 43), and respirable (PM2.5; n ¼ 44) concentrations of Mn and that of 14 other elements. Only five elements differed significantly across sites in at least one filter setting, and Mn was the only element to differ significantly between sites in all three filter settings (particle size groups: total, inhalable and respirable): TSP (F(2,41) ¼ 46.687, p < 0.0001), PM10 (F(2,40) ¼ 25.967, p < 0.0001), and PM2.5 (F(2,41) ¼ 9.382, p < 0.001) (Fig. 2 A, D, G). Al and Fe concentrations differed between sites in the two largest particle size groups: TSP (F(2,41) ¼ 28.305, p < 0.0001 and F(2,41) ¼ 54.806, p < 0.0001, respectively) (Fig. 2 B, C) and PM10 (F(2,40) ¼ 12.923, p < 0.0001 and F(2,40) ¼ 15.227, p < 0.0001, respectively) (Fig. 2 E, F). Se differed between sites in PM10 (F(2,40) ¼ 4.528, p ¼ 0.016) and Zn differed between sites in TSP (F(2,41) ¼ 8.810, p < 0.001). Descriptive statistics and post hoc significance between sites for these five elements are presented in Table 2. The concentration of total, inhalable and respirable Mn particles

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Fig. 2. Atmospheric concentrations (mg/m3) of total, inhalable, and respirable (TSP, PM10 and PM2.5 respectively) Mn (A, D, G), Al (B, E, H) and Fe (C, F, I) were recorded at three sites on Groote Eylandt between 16 and 21 October 2014 and 14 May to 28 August 2015. Horizontal lines represent the median with hinges representing the 1st and 3rd quartiles; two outliers were removed from the boxplot that represented unusually high PM10 values during Mn loading at the port (values of 4.234 and 4.537 mg/m3), but were not excluded from analyses. The differences between sites were analysed using post hoc Tukey test and the significance values are displayed in asterisks (**p < 0.01, ***p < 0.001).

were significantly higher near the mine at Angurugu and the Mn stockpile at Portside than at central island (Central 4), but did not differ between Angurugu and Portside (Fig. 2 A, D, G). Total Mn concentration (TSP) was 13x higher at Angurugu (1.200 mg/m3) and 8x higher at Portside (0.738 mg/m3) than at central island (0.095 mg/ m3) (Table 2, Fig. 2 A). Inhalable Mn concentration (PM10) in Portside (1.223 mg/m3) was 3x and 15x higher than that in Angurugu (0.437 mg/m3) and central island (0.075 mg/m3) respectively. Respirable Mn concentration (PM2.5) was 3x higher at Angurugu (0.181 mg/m3) and 4x higher at Portside (0.261 mg/m3) than at central island (0.075 mg/m3). No other element showed differences in concentrations at this smallest, respirable size. Total and inhalable Al and Fe concentrations were all significantly higher at

Angurugu than at central island (Fig. 2 B, C, E, F), and for total Al and Fe, the concentrations in Angurugu were also significantly higher than those in Portside. 3.2. Hair samples Hair samples from 230 individual quolls (107 females, 123 males) were analysed to quantify concentrations of Mn and that of 14 other elements. We found that animals living near Mn mining facilities had higher overall concentrations of elements (especially of Mn, Cr and Mo) in their hair, as indicated by the first component of the PCA conducted on all 15 elements, PC1Load. PC1Load was greater in the hair of quolls captured at Portside and Alyangula,

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Table 2 Atmospheric concentrations of total (TSP, all suspended particles), inhalable (PM10, <10 mm), and respirable (PM2.5, <2.5 mm) Mn and four other elements found to be significantly different across three sites (in at least one filter setting) using air filters (n ¼ 131) on Groote Eylandt between 16 and 21 October 2014 and 14 May to 28 August 2015. SD ¼ standard deviation. TSP (total suspended particles) Element(mg/m3)

Angurugu (n¼16) Mean

Mn Al Fe Zn Se

1.200 2.064 1.827 0.119 0.025

h** p**, h** p**, h** p**

Portside (n¼14) Min

Max

SD

Mean

0.297 0.309 0.531 0.060 0.007

2.791 4.146 3.916 0.199 0.041

0.683 1.212 0.917 0.036 0.008

0.738 0.482 0.480 0.064 0.025

h** a**, h* a**, h** a**, h*

Central 4 (n¼14) Min

Max

SD

Mean

0.174 0.056 0.097 0.002 0.001

2.502 1.043 1.281 0.113 0.048

0.645 0.309 0.317 0.032 0.014

0.095 0.249 0.226 0.094 0.018

a**, p** a**, p* a**, p** p*

Min

Max

SD

0.011 0.080 0.109 0.029 0.007

0.170 0.803 0.327 0.147 0.030

0.047 0.209 0.079 0.038 0.007

Min

Max

SD

0.004 0.075 0.061 0.006 0.008

0.148 0.279 0.550 0.235 0.041

0.039 0.066 0.119 0.058 0.010

Min

Max

SD

0.003 0.083 0.126 0.013 0.007

0.118 0.614 0.614 0.207 0.041

0.033 0.140 0.134 0.049 0.011

PM10 (inhalable suspended particles) Element(mg/m3)

Angurugu (n¼16) Mean

Mn Al Fe Zn Se

0.437 0.939 0.739 0.149 0.028

h** h** h**

p*, h**

Portside (n¼14) Min

Max

SD

Mean

0.115 0.137 0.156 0.070 0.013

1.000 2.484 2.021 0.873 0.045

0.264 0.795 0.482 0.196 0.008

1.223 0.412 0.548 0.089 0.020

Min

Max

SD

Mean

h** h** h**

a*

Central 4 (n¼13)^ Min

Max

SD

Mean

0.162 0.077 0.153 0.004 0.004

4.537 0.964 1.386 0.146 0.035

1.425 0.266 0.403 0.037 0.010

0.075 0.177 0.188 0.086 0.018

Min

Max

SD

Mean

a**, p** a**, p** a**, p**

a**

PM2.5 (respirable suspended particles) Element(mg/m3)

Angurugu (n¼16) Mean

Mn Al Fe Zn Se

0.181 0.311 0.278 0.084 0.021

h**

Portside (n¼14)

0.089 0.085 0.136 0.004 0.002

0.562 1.545 0.822 0.129 0.043

0.113 0.353 0.161 0.033 0.011

0.261 0.251 0.246 0.084 0.021

h**

Central 4 (n¼14)

0.073 0.074 0.115 0.037 0.007

0.726 0.742 0.432 0.151 0.042

0.211 0.194 0.086 0.032 0.011

0.075 0.188 0.211 0.081 0.021

a**, p**

Mean concentrations are significantly different from a(Angurugu), p(Portside) and/or h(Central 4) at *(p  0.05) or **(p  0.01). ^One filter was excluded from the analysis because it was thought to be mislabeled (value of 0.793 mg/m3).

located near the open-air Mn storage facility at the port, than at eight other sites further from the Mn mining facilities (F(11,205) ¼ 7.615, p < 0.0001). PC2Load, which represents Mn loading in hair, was also significantly higher in quolls living closer to Mn mining facilities (F(11,205) ¼ 7.415, p < 0.0001), and higher in females than males (F(1,205) ¼ 9.928, p ¼ 0.002). When each element was assessed separately in hair, we found that Mn was the most different among sites (Table 3). Mean Mn concentration was significantly higher in the hair of quolls at Portside than at 8 other sites (post hoc Tukey test; Fig. 3 A), and significantly lower at Highway 4 than at 5 other sites. Fedthe second most different element among sitesdwas also significantly higher in the hair of quolls living near Mn mining facilities versus

samples from the more distant sites, with some exceptions (post hoc Tukey test; Fig. 3 B). Females had significantly higher concentrations of Cu, Fe, Mg and Mn than males, and heavier quolls had lower concentrations of Cu and Mg for both sexes (Table 3). 3.3. Tissue samples Tissues for 18 male quolls were analysed for concentrations of 15 elements (Table S1). Euthanised male quolls near the mine (n ¼ 8) did not differ in hair[Mn] from male quolls captured and released near the mine (n ¼ 6) within the same post-breeding period (Wilcoxon rank-sum tests; W ¼ 23, p ¼ 0.95), and similarly euthanized male quolls far from the mine (n ¼ 10) did not

Table 3 The F and p-values derived from analysis of co-variance (ANCOVA) which compared the differences in concentrations of all log10-transformed elements in hair of northern quolls (n ¼ 230; 107 females, 123 males) between all sites and both sexes, and across varying body mass. Element concentrations that are significantly related to site, sex and/or body mass are bolded. Element

Site

Sex

Body mass

Al As Cd Co Cr Cu Fe Mg Mn Mo Ni P Pb Se Zn

F(11,205) ¼ 3.664, p ¼ 9.173 x 10¡5 F(11,205) ¼ 4.753, p ¼ 1.704 x 10¡6 F(11,205) ¼ 4.476, p ¼ 4.708 x 10¡6 F(11,205) ¼ 2.932, p ¼ 1.275 x 10-3 F(11,205) ¼ 5.254, p ¼ 2.724 x 10¡7 F(11,205) ¼ 1.109, p¼0.356 F(11,205) ¼ 6.870, p ¼ 7.957 x 10¡10 F(11,205) ¼ 2.491, p ¼ 5.914 x 10-3 F(11,205) ¼ 15.202, p < 2.200 x 10¡16 F(11,205) ¼ 4.200, p ¼ 1.294 x 10¡5 F(11,205) ¼ 0.771, p ¼ 0.669 F(11,205) ¼ 2.443, p ¼ 6.963 £ 103 F(11,205) ¼ 1.061, p ¼ 0.395 F(11,205) ¼ 2.401, p ¼ 8.028 £ 103 F(11,205) ¼ 2.770, p ¼ 2.251 £ 103

F(1,205) ¼ 2.102, p¼0.149 F(1,205) ¼ 0.015, p¼0.902 F(1,205) ¼ 0.036, p¼0.851 F(1,205) ¼ 0.220, p¼0.639 F(1, 205) ¼ 2.284, p¼0.132 F(1,205) ¼ 7.598, p ¼ 6.37 x 10-3 F(1,205) ¼ 4.526, p ¼ 0.035 F(1,205) ¼ 12.473, p ¼ 5.100 £ 104 F(1,205) ¼ 10.126, p ¼ 1.689 £ 103 F(1,205) ¼ 0.084, p ¼ 0.773 F(1,205) ¼ 0.001, p ¼ 0.981 F(1,205) ¼ 0.163, p ¼ 0.688 F(1,205) ¼ 1.879, p ¼ 0.172 F(1,205) ¼ 0.543, p ¼ 0.462 F(1,205) ¼ 2.205, p ¼ 0.139

F(1.205) ¼ 0.824, p¼0.365 F(1,205) ¼ 1.098, p¼0.296 F(1,205) ¼ 0.059, p¼0.809 F(1,205) ¼ 0.325, p¼0.569 F(1,205) ¼ 2.794, p¼0.096 F(1,205) ¼ 4.050, p ¼ 0.045 F(1,205) ¼ 1.180, p ¼ 0.279 F(1,205) ¼ 15.282, p ¼ 1.258 £ 104 F(1,205) ¼ 0.024, p ¼ 0.877 F(1,205) ¼ 0.457, p ¼ 0.499 F (1,205) ¼ 1.118, p ¼ 0.292 F(1,205) ¼ 0.164, p ¼ 0.686 F(1,205) ¼ 0.773, p ¼ 0.380 F(1,205) ¼ 2.742, p ¼ 0.099 F(1,205) ¼ 1.318, p ¼ 0.252

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Fig. 3. The mean concentrations (with standard error) of (A) Mn and (B) Fe in hair of northern quolls from 12 sites that vary in distance from Mn extraction and processing facilities across Groote Eylandt. Values plotted are not log10-transformed. The sample size for each site is noted above the mean bars. Each site is assigned an alphabet for post hoc comparisons and subscript alphabet(s) above each mean bar represents the site(s) in which the means are statistically different from one another based on the post-hoc Tukey test (p < 0.05).

differ in hair[Mn] from male quolls captured and released far from the mine (n ¼ 8; W ¼ 30.5, p ¼ 0.42). We fitted 28 global models for each element, including all possible interaction combinations between tissue type (seven different types) and measure (proximity to Mn mining facilities, hair PC1Load, hair PC2Load and hair[Mn]). Overall, Mn was the predominant element accumulating in quoll tissues, primarily in the brain and testes (Table S2). Zn and Al also accumulated in the brain. Animals living closer to Mn mining facilities had greater concentrations of Mn in their cerebellum and neocortex. Quolls with higher PC2Load (therefore more Mn in hair) had higher Mn concentrations in cerebellum (r ¼ 0.742, p < 0.001) and testes (r ¼ 0.492, p ¼ 0.038), but a lower concentration of Mn in the olfactory bulb (r ¼ 0.552, p ¼ 0.017) (Table 4). Similarly, hair[Mn] was positively correlated with Mn concentration in the cerebellum (r ¼ 0.650, p ¼ 0.003) and negatively correlated with Mn concentration in the olfactory bulb (r ¼ 0.487, p ¼ 0.040). Both Zn and Al accumulated in the cerebellum and neocortex regions of the brain.

Fe did not accumulate in any target tissues assessed. Details on the statistics for the most parsimonious models (wi, F and p-values) for all elements that are significantly related to proximity to Mn mining facilities, hair PC1Load, hair PC2Load and/or hair[Mn] across the seven target tissues are included in Table S2. Outside of the brain, only testes Mn concentration was found to be significantly positively correlated with hair PC2Load; the higher the PC2Load (therefore more Mn in hair), the higher the testes Mn concentration. Because the most parsimonious model only carried 0.42 wi, followed by 0.34 and 0.15 wi in the second and third models, we averaged all models and found that hair PC2Load still had the highest conditional and unconditional parameter estimates of 0.244 and 0.198 respectively, and relative variable importance of 0.81 when compared to body mass and the interaction term body mass:PC2Load. The statistical output from this model averaging analysis is included in Table S3. On its own, body mass was positively associated with concentrations of Cu and Mo in neocortex (F(1,16) ¼ 6.672, p < 0.05 and

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Table 4 The Pearson product-moment correlation coefficient (r) and the significance of the correlation (p) between hair PC1Load, hair PC2Load and log10-transformed hair Mn concentration (hair[Mn]) with log10-transformed Mn concentration in each target tissue (*p < 0.05, **p < 0.01, ***p < 0.001) in male quolls caught at sites that are far from the Mn mining facilities (n ¼ 10) and sites that are near Mn mining facilities (n ¼ 8) (Fig. 1) during 2015 post-breeding on Groote Eylandt. Tissue [Mn]

PC1Load

Brain: Cerebellum Brain: Neocortex Brain: Olfactory Bulb Kidney Liver Lung Testes

r r r r r r r

¼ ¼ ¼ ¼ ¼ ¼ ¼

0.246, p ¼ 0.326 0.482*, p ¼ 0.043 0.020, p ¼ 0.938 0.320, p ¼ 0.196 0.369, p ¼ 0.132 0.182, p ¼ 0.485 0.153, p ¼ 0.543

F(1,16) ¼ 6.980, p < 0.05, respectively) and Cu in liver (F(1,16) ¼ 9. 582, p < 0.01) and was negatively associated with concentrations of Mn in the olfactory bulb (F(1,16) ¼ 7.821, p < 0.05), Cd, Cr and Pb in testes (F(1,15) ¼ 9.323, p < 0.01; F(1,16) ¼ 16.528, p < 0.001; F(1,15) ¼ 7.527, p < 0.05, respectively) and lastly, Co, Cr, Pb and Se in lung (F(1,15) ¼ 7.286, p < 0.05; F(1,15) ¼ 5.097, p < 0.05; F(1,15) ¼ 6.831, p < 0.05; F(1,15) ¼ 4.786, p < 0.05, respectively).

4. Discussion We were not surprised to find that levels of airborne Mn were higher near Mn mining facilities on Groote Eylandt (Table 2), given that a black dust settles over the landscape near the mine sites, along the Mn transport roads, and near the storage mounds. However, we detected respirable Mn particles at levels exceeding international recommendations for non-occupational exposure (0.05 mg/m3; USEPA, 1984) even 20 km from these areas. In fact, nearly 80% of the Mn dust at our remote central island site was of the smallest particle sizes (
PC2Load r r r r r r r

¼ ¼ ¼ ¼ ¼ ¼ ¼

0.742***, p < 0.001 0.285, p ¼ 0.252 0.552*, p ¼ 0.017 0.065, p ¼ 0.797 0.452, p ¼ 0.059 0.268, p ¼ 0.299 0.492*, p ¼ 0.038

Hair[Mn] r r r r r r r

¼ ¼ ¼ ¼ ¼ ¼ ¼

0.650**, p ¼ 0.003 0.296, p ¼ 0.233 0.487*, p ¼ 0.040 0.051, p ¼ 0.839 0.315, p ¼ 0.203 0.329, p ¼ 0.198 0.412, p ¼ 0.090

were associated with reduced attention (Solis-Vivanco et al., 2009) and declines in motor function in adult humans (RodriguezAgudelo et al., 2006). Even low levels, Mn can be toxic if inhaled over a long period of time. For example, Baldwin et al. (1999) and Mergler et al. (1999) studied four communities exposed to Mncontaining dust emitted from a former Mn alloy plant in southern Quebec. They found that people exposed to this dustdwhich had total Mn of 0.009e0.035 mg/m3 and inhalable Mn (PM10) of 0.007e0.019 mg/m3dhad higher Mn levels in their blood and, in many cases, exhibited brain dysfunction. The highest levels detected in that study were substantially lower than even the mean air Mn concentrations detected at our site in the centre of the island, which is farthest from mining activities (TSP ¼ 0.095 mg/m3, PM10 ¼ 0.075 mg/m3; Table 2). Rats exposed to Mn dust in the laboratory also significantly accumulate it within the cerebellum (St-Pierre et al., 2001; Salehi et al., 2003; Yu et al., 2003; Dorman et al., 2004; Tapin et al., 2006) and show elevated locomotor behaviour and impaired motor function (St-Pierre et al., 2001; Salehi et al., 2003; Tapin et al., 2006; but see Saputra et al., 2016). Even subtle changes to the motor performance or behaviour of wild animals can have serious implications for their survival, because movement underlies food acquisition, mating, and predator escape (Husak and Fox, 2006; Husak et al., 2006; Wilson et al., 2007; Nathan et al., 2008). Therefore, if Mn acts in the brains of quolls as it does in other mammals, its accumulation might be associated with population declines in contaminated areas. Outside the brain, Mn levels in the testes were significantly and positively correlated with Mn in the hair (PC2Load), suggesting a potential impact on reproduction in this species. On its own, the model associating hair and testes Mn concentrations had a low relative likelihood of being the best model (Akaike weight of 0.42; Tables S2 and S3), yet it had high relative value when all models were averaged, suggesting that the relationship will only strengthen with increasing sample size. Northern quolls have a rare reproductive strategy: males breed suicidally, investing so much energy into a frenetic three-week rut that they die soon after (Oakwood, 2000; Nelson and Gemmell, 2003). Females mate with multiple males, and fertilisation of the embryos occurs via sperm competition within the female reproductive tract (Fisher et al., 2013). Laboratory studies demonstrated decreased sperm count and motility and increased sperm deformities in Mn-treated rats (Ponnapakkam et al., 2003; Lee, 2009). Effects of Mn on sperm in northern quolls and other Groote Eylandt animals is therefore of concern and needs investigation due to the potential negative effects on reproductive success and population viability. Most of what is known about Mn toxicity comes from studies of workplace exposure in humans or controlled experiments in laboratory animals; the effects of lower-level, chronic exposure are poorly studied, despite the fact that fine, inhalable Mn particles € llin and may settle outside workplace settings (WHO, 2006; Ro

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Nogueira, 2011). Mn has been actively mined on Groote Eylandt, Northern Territory, for more than half a century, yet almost nothing is known about how its emancipation from the soil affects the island's ecology. These finer Mn particles can travel large distances from the source and are small enough to be inhaled by humans and €llin and Nogueira, 2011), though to our knowledge, only wildlife (Ro one study to-date has assessed Mn contamination in wildlife. In that study, Loranger et al. (1994) focused on Methylcyclopentadienyl Manganese Tricarbonyl (MMT)dan organic derivative of Mn used in unleaded gasoline and which, upon combustion, forms fine Mn oxide particles. They found that feral pigeons (Columba livia) living in urban areas accumulated 30% and 45% more Mn in their liver and feces, respectively, compared with pigeons living in rural areas, though the implications to the pigeon's health or ecology remain unclear. Though we specifically focused on Mn as a potential contaminant, in reality, organisms (including people) living near Mn mining facilities are simultaneously exposed to an array of toxic metals that may have deleterious effects on their own and/or in combination. To account for this, we used PCA to derive cumulative measures of contaminationdPC1Load and PC2Load dwhich together explained almost 70% of the variance in 15 different elements. This approach enables a reduction in data by describing a given multidimensional system by means of a small number of new variables (Loska and Wiechula, 2003; Boruvka et al., 2005). In this way, it is possible for us to analyse the whole data set consisting of 15 element loadings, instead of analysing based on Mn exposure alone. Unsurprisingly, given the extensive Mn mining activities taking place on the island, both analyses demonstrated similar elemental composition trendsdPC1Load and PC2Load showed disproportionally higher Mn concentration compared to most of the other elements in quoll hair. The same approach has been useful in many studies of contaminants (Loska and Wiechula, 2003; Boruvka et al., 2005; Singh et al., 2005; Passos et al., 2010). In addressing effects of environmental Mn exposure, future research should also account for all toxic metals present at environmentally relevant concentrations, and their interactions. We demonstrated that inhalable and respirable Mn particles released by Mn mining activities move great distances through the air and accumulate in the hair, brains and testes of wild quolls on Groote Eylandt, Australia. Because we established that Mn in the hair of quolls was positively correlated with Mn in the cerebellum (Table 4), in future work, hair can be useddas it is in human studiesdas a non-invasive proxy for brain Mn contamination (Kondakis et al., 1989; He et al., 1994). Similarly, PC2Load in hair can be used as a non-invasive proxy for Mn accumulation in the testes. Given the endangered status and unusual breeding ecology of this species, further work should focus on assessing the impacts of Mn contamination on the motor performance, cognition, and reproductive function of wild quolls. Funding sources This research was funded by an Australia Research Council (ARC) Linkage Grant to RSW, FVH and SB, a grant from the Anindilyakwa Land Council (ALC) to RSW, and a PhD scholarship by the Malaysian Ministry of Education to AFAAN. The ARC and Malaysian Ministry of Education had no role in any aspect of this study. Employees of the ALC did participate in data collection, but were not involved in the study design, analysis and interpretation of data, writing, or the decision to submit the article for publication. Acknowledgements We thank members and volunteers of the Wilson Performance

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Lab for assistance with running the experiments in the field. We also thank the Anindilyakwa Land and Sea Rangers of Groote Eylandt for their generous assistance, logistical support and use of laboratory facilities. We also thank the traditional owners of Groote Eylandt for their generous support and access to their land. This project was supported by the Anindilyakwa Land Council, a University of Queensland collaboration and Industry Engagement Fund (UQ-CIEF) grant awarded to R.S.W., an Australian Research Council (ARC) DECRA (DE130101410) awarded to A.C.N., an ARC Linkage (LP160100736) Grant awarded to R.S.W, FvH and S.B., and an ARC Future Fellowship (FT150100492) awarded to R.S.W. This manuscript was substantially improved by two anonymous reviewers. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.envpol.2017.10.088. References Andersen, M.E., Gearhart, J.M., Clewell, H.J., 1999. Pharmacokinetic data needs to support risk assessments for inhaled and ingested manganese. Neurotoxicology 20, 161e171. Anindilyakwa Land Council (ALC), 2014. Groote Eylandt in the region. Retrieved from http://www.anindilyakwa.com.au. . Antonini, J.M., Santaimaria, A.B., Jenkins, N.T., Albini, E., Lucchini, R., 2006. Fate of manganese associated with the inhalation of welding fumes: potential neurological effects. Neurotoxicology 27, 304e310. Aschner, M., 2000. Manganese: brain transport and emerging research needs. Environ. Health Perspect. 108, 429e432. Aschner, M., Gannon, M., 1994. Manganese (Mn) transport across the rat-blood brain barrier - saturable and transferrin-dependent transport mechanisms. Brain Res. Bull. 33, 345e349. Agency for Toxic Substances and Diseases Registry (ATSDR), 2012. Toxicological Profile for Manganese. U.S. Department of Health and Human Services, Public Health Service, Atlanta, GA. Baldwin, M., Mergler, D., Larribe, F., Belanger, S., Tardif, R., Bilodeau, L., Hudnell, K., 1999. Bioindicator and exposure data for a population based study of manganese. Neurotoxicology 20, 343e353.  , K., 2016. MuMIn: Multi-model Inference. R Package Version 1.15.6. https:// Barton CRAN.R-project.org/package¼MuMIn. Boruvka, L., Vacek, O., Jehlicka, J., 2005. Principal component analysis as a tool to indicate the origin of potentially toxic elements in soils. Geoderma 128, 289e300. Bouchard, M.F., Laforest, F., Vandelac, L., Bellinger, D., Mergler, D., 2007. Hair manganese and hyperactive behaviors: pilot study of school-age children exposed through tap water. Environ. Health Perspect. 115, 122e127. Bouchard, M.F., Sauve, S., Barbeau, B., Legrand, M., Brodeur, M.E., Bouffard, T., Limoges, E., Bellinger, D.C., Mergler, D., 2011. Intellectual impairment in schoolage children exposed to manganese from drinking water. Environ. Health Perspect. 119, 138e143. Department of the Environment (DOE), 2014. Dasyurus hallucatus in Species Profile and Threats Database. Department of the Environment, Canberra. Retrieved from http://www.environment.gov.au/sprat. Dorman, D.C., McManus, B.E., Parkinson, C.U., Manuel, C.A., McElveen, A.M., Everitt, J.I., 2004. Nasal toxicity of manganese sulfate and manganese phosphate in young male rats following subchronic (13-week) inhalation exposure. Inhal. Toxicol. 16, 481e488. Drown, D.B., Oberg, S.G., Sharma, R.P., 1986. Pulmonary clearance of soluble and insoluble forms of manganese. J. Toxicol. Environ. Health 17, 201e212. Fechter, L.D., Johnson, D.L., Lynch, R.A., 2002. The relationship of particle size to olfactory nerve uptake of a non-soluble form of manganese into brain. Neurotoxicology 23, 177e183. Filistowicz, A., Dobrzanski, Z., Przysiecki, P., Nowicki, S., Filistowicz, A., 2011. Concentration of heavy metals in hair and skin of silver and red foxes (Vulpes vulpes). Environ. Monit. Assess. 182, 477e484. Finley, J.W., 1999. Manganese absorption and retention by young women is associated with serum ferritin concentration. Am. J. Clin. Nutr. 70, 37e43. Fisher, D.O., Dickman, C.R., Jones, M.E., Blomberg, S.P., 2013. Sperm competition drives the evolution of suicidal reproduction in mammals. Proc. Natl. Acad. Sci. 110, 17910e17914. He, P., Liu, D.H., Zhang, G.Q., 1994. Effects of high-level manganese sewage irrigation on children's neurobehavior. Chin. J. Prev. Med. 28, 216e218. Huang, C.C., Lu, C.S., Chu, N.S., Hochberg, F., Lilienfeld, D., Olanow, W., Calne, D.B., 1993. Progression after chronic manganese exposure. Neurology 43, 1479e1483. Husak, J.F., Fox, S.F., 2006. Field use of maximal sprint speed by collared lizards (Crotaphytus collaris): compensation and sexual selection. Evolution 60, 1888e1895.

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