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South Africa is one of the most diverse countries in the world but the increase in agricultural, industrial and technological development to meet the needs of the growing human population has led to increased amounts of potentially toxic elements (PTEs) and other chemicals in the environment. As regional and global environmental processes influence local conditions to differing degrees, all organisms within a specific environment are exposed to highly complex, ill-defined PTE and chemical mixtures. Differences in feeding strategies within and between vertebrate trophic levels are likely to influence the degree to which individuals may be exposed to and affected by PTE presence. Using vertebrate faeces as a biological matrix, we investigate and compare quantitative differences in PTE concentrations in herbivorous, omnivorous and carnivorous terrestrial vertebrates from two protected areas in South African savannah. Of the eleven PTEs assessed [aluminium (Al), arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb), antimony (Sb), tin (Sn), strontium (Sr), and vanadium (V)], the highest concentrations of Al, As, Cr, Pb, Sn, and V were found in carnivores. General patterns were evident between groups at each site for specific elements, but absolute values for the same elements were site-specific. This is the first study to non-invasively examine and compare PTE concentrations in a variety of free-ranging mammalian wildlife occupying different trophic levels within South African protected areas. Our results confirm that all individuals across trophic levels within these sites are exposed to multiple and varied PTE mixtures on a continuous basis. Whether PTEs at these concentrations cause synergistic or antagonistic disruption of physiological and biological systems alone or in combination in free-ranging African wildlife species is still unclear and requires further investigation.
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Non-Lethal Assessment of Potentially
Toxic Elements Across Mammalian
Trophic Levels in African Savannahs
Andrea B. Webster
1
*, Javier F. Callealta
2
, Nigel C. Bennett
1
and Andre Ganswindt
1
1
Mammal Research Institute, Department of Zoology and Entomology, University of Pretoria, Pretoria, South Africa,
2
Department
of Economics, Universidad de Alcalá, Alcalá de Henares, Spain
South Africa is one of the most diverse countries in the world but the increase in
agricultural, industrial and technological development to meet the needs of the growing
human population has led to increased amounts of potentially toxic elements (PTEs) and
other chemicals in the environment. As regional and global environmental processes
inuence local conditions to differing degrees, all organisms within a specic environment
are exposed to highly complex, ill-dened PTE and chemical mixtures. Differences in
feeding strategies within and between vertebrate trophic levels are likely to inuence the
degree to which individuals may be exposed to and affected by PTE presence. Using
vertebrate faeces as a biological matrix, we investigate and compare quantitative
differences in PTE concentrations in herbivorous, omnivorous and carnivorous
terrestrial vertebrates from two protected areas in South African savannah. Of the
eleven PTEs assessed [aluminium (Al), arsenic (As), barium (Ba), cadmium (Cd),
chromium (Cr), mercury (Hg), lead (Pb), antimony (Sb), tin (Sn), strontium (Sr), and
vanadium (V)], the highest concentrations of Al, As, Cr, Pb, Sn, and V were found in
carnivores. General patterns were evident between groups at each site for specic
elements, but absolute values for the same elements were site-specic. This is the rst
study to non-invasively examine and compare PTE concentrations in a variety of free-
ranging mammalian wildlife occupying different trophic levels within South African
protected areas. Our results conrm that all individuals across trophic levels within
these sites are exposed to multiple and varied PTE mixtures on a continuous basis.
Whether PTEs at these concentrations cause synergistic or antagonistic disruption of
physiological and biological systems alone or in combination in free-ranging African wildlife
species is still unclear and requires further investigation.
Keywords: environmental pollution, potentially toxic elements (PTEs), non-invasive risk assessment, animal faeces,
non-lethal risk assessment, protected areas (PA), African savannah
INTRODUCTION
Potentially toxic elements (PTEs) have no established biological or physiological function (Järup,
2003). Even when concentrations are too low to be individually effective, the combined action of
multiple PTEs at low concentrations alone and in combination with other organic and synthetic
chemicals can result in adverse effects (Rhind, 2009;Martin et al., 2021). Repeated exposures,
particularly when the interval between exposures is brief, may result in cumulative storage of a
Edited by:
Denina Bobbie Dawn Simmons,
Ontario Tech University, Canada
Reviewed by:
Jean Remy Davee Guimaraes,
Federal University of Rio de Janeiro,
Brazil
Andrew Hursthouse,
University of the West of Scotland,
United Kingdom
*Correspondence:
Andrea B. Webster
andrea.webster@tuks.co.za
Specialty section:
This article was submitted to
Toxicology, Pollution and the
Environment,
a section of the journal
Frontiers in Environmental Science
Received: 13 October 2021
Accepted: 27 December 2021
Published: 27 January 2022
Citation:
Webster AB, Callealta JF, Bennett NC
and Ganswindt A (2022) Non-Lethal
Assessment of Potentially Toxic
Elements Across Mammalian Trophic
Levels in African Savannahs.
Front. Environ. Sci. 9:794487.
doi: 10.3389/fenvs.2021.794487
Frontiers in Environmental Science | www.frontiersin.org January 2022 | Volume 9 | Article 7944871
ORIGINAL RESEARCH
published: 27 January 2022
doi: 10.3389/fenvs.2021.794487
substance and its subsequent increase in body burden until a
permanent state of storage has been reached (Baynes et al., 2012).
The sequential and simultaneous exposure of wildlife to mixtures
of synthetic compounds and PTEs from direct, indirect, past or
contemporary exposures that interact in additive or synergistic
ways, can disrupt various physiological systems resulting in
subsequent negative cascading effects (Kortenkamp and Faust,
2018;Martin et al., 2021).
The Global Assessment of Soil Pollution Report (FAO and
UNEP, 2021) highlights that contamination of soils degrades soil
structure and organic carbon stores, reduces terrestrial ecosystem
resilience and affects the ability to protect and secure water
quality. Trace elements originating from geogenic processes
are naturally occurring constituents within the environment
(Jaishankar et al., 2014). However, increasing anthropogenic
activities have distinctly altered the geochemical cycles and the
biochemical balance of PTEs within the environment (Rzymski
et al., 2015). The differences in geochemical heterogeneity,
coupled with gradients in precipitation, vegetation and
climatic conditions result in the availability of trace elements
at different spatial intensities at a local, regional and global scale
(McNaughton and Georgiadis, 1986;Shorrocks and Bates, 2015).
In addition, the chemical form and abundance at which an
element is present in the environment inuences its
availability for uptake by an organism (Kabata-Pendias, 2011;
Olaniran et al., 2013).
Protected areas are considered the cornerstone of biodiversity
and conservation. As regional and global environmental
processes inuence local conditions to differing degrees,
specic environments contain unique combinations of PTEs
and other chemicals resulting from geogenic processes and
surrounding activities (Webster et al., 2021a). It is reasonable
to assume that all organisms within a specic environment are
exposed to highly complex, ill-dened mixtures of PTEs and
other synthetic compounds (Thrupp et al., 2018). Individual
animal behaviour and species-specic physiology contribute
substantially to potential exposure or bioaccumulation,
particularly when lipophilic substances are involved (Clotfelter
et al., 2004;Gall et al., 2015). Food web magnications occur with
each predator-prey interaction and can result in PTE trophic
increases of 10,000 to 100,000 fold (Gobas, 2008). PTEs that
accumulate in the bones (Pb) or adipose tissue (As, Cd, Hg, and
Pb) can be remobilized during periods when the bodys demand
for calcium increases (e.g., pregnancy and lactation). Early
exposure to PTEs and other chemical contaminants during
critical periods of development can manifest in adverse effects
later in life (World Health Organisation/International
Programme on Chemical Safety, 2002;World Health
Organisation/United Nations Environment Programme, 2012;
Chen et al., 2014). In terrestrial carnivores, successful hunts
are often interspersed with periods of nutritional stress
(Gulson et al., 2003;Hayward and Kerley, 2008). When lipid
reserves are remobilised for energy, circulating PTEs can result in
damage to organs and systems (World Health Organisation/
International Programme on Chemical Safety, 2002).
The varied ecosystems, species richness and endemism of the
biomes make South Africa one of the most diverse countries in
the world (Driver et al., 2011). Pollution has however become a
key threat to protected areas and resident vertebrates may be at
risk of associated effects through the consumption of
contaminated vegetation, prey items and drinking water
(Reglero et al., 2008). Traditional methods used for
toxicological risk assessment in wildlife are typically invasive,
involving the sacrice of live animals in laboratory studies
(Egorova and Ananikov, 2017;Kenston et al., 2018), the
chemical immobilization and handling of live animals during
routine management, translocation or assessment procedures
(Facimire et al., 1995;Naidoo et al., 2017) or the opportunistic
collection of various matrices from carcasses after mass mortality
or debilitation events (Richards et al., 2014;Ogada et al., 2016).
Soft tissue is generally considered optimal for accurate
identication of specic toxin(s)/pollutant(s), however,
environmental factors such as temperature and humidity may
cause desiccation of soft tissue, while insect or scavenger activity
may hasten the decomposition of pollutant residues and the
samples themselves. In this regard, the window for recovery,
detection and identication of toxins can be extremely narrow
(Richards et al., 2014). Historical assessments have also primarily
focused on the assessment of single elements, aspects related to
that element (e.g., lipophilicity) or the impact on a specic species
or trophic level within the food web (Streit, 1992). More recent
approaches have, however demonstrated that low dose exposures
to contaminant mixtures are synergistic (Cobbina et al., 2015;
Thrupp et al., 2018;Martin et al., 2021). Interactions between
numerous PTEs and/or steroidal pharmaceuticals can therefore
result in higher toxicities and greater effects than single element
exposures at either high or low concentrations (Rhind, 2009;
Kortenkamp et al., 2019).
Complex intrinsic and extrinsic dynamics including seasonal
variations in PTE contamination within available resources, sex,
age, life stage, and exposure to environmental stressors, can
inuence an individuals ability to acquire and assimilate food
resources and associated PTEs (Warne, 2014). Dietary intake is
closely related to faecal concentrations, which in turn reects
actual utilization of resources (Böswald et al., 2018) making faeces
a practical, non-invasive matrix for assessment. Faecal analysis
has previously been used to assess stress-related and reproductive
hormone concentrations (Kersey and Dehnhard, 2014;Webster
et al., 2018;Palme, 2019), measure nutrient stress (Grant et al.,
2000) and to quantify exposure to toxic substances (Gupta and
Bakre, 2013;Richards et al., 2014;Celis et al., 2015;Sach et al.,
2020) in various species of captive and free-ranging wildlife. As a
consequence of distinct morphological and physiological
differences between species, the partitioning of food sources
within herbivore, omnivore and carnivore groups that facilitate
the sharing of community resources within a specic protected
area (Du Toit and Cumming, 1999) and the degrees to which PTE
mixtures may adversely affect different trophic groups are still
largely unknown.
Given the typically invasive measures associated with sample
collection, management plans of South African wildlife reserves
do not include consistent monitoring and assessment of the
environment or biota for the presence or persistence of
synthetic or trace element contaminants (Mpumalanga
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Webster et al. Non-Invasive Assessment of African Mammals
Tourism and Parks Agency, 2015;Spies, 2018). As a result,
information related to PTE presence within specic protected
areas and the various wildlife species that occur there has been
restricted to pollution events and mass die offs (Ferreira and
Pienaar, 2011;Woodborne et al., 2012). A broad assessment of
environmental matrices within the two protected areas of interest
in this study (Webster et al., 2021a) identify unique geological
signatures within each reserve, but also highlight some PTE
concentrations above acceptable limits outside of geological
inuence. Given these previously established concerns in
environmental matrices, the need for further assessment in
biological components of these systems became evident
(Webster et al., 2021b). The current study aimed to investigate
and compare quantitative differences of eleven PTEs (Al, As, Ba,
Cd, Cr, Hg, Pb, Sb, Sn, Sr, and V) across trophic levels of
terrestrial African vertebrates using faeces as a non-invasive
analytical matrix.
MATERIALS AND METHODS
Study Sites
Tswalu Kalahari Reserve (TKR), consisting of the Korannaberg
(±101,000 ha) and Lekgaba (±20,000 ha) sections, is situated at S
27°2961and E 22°3943in the arid Northern Cape Province of
South Africa (Figure 1A). Prior to the establishment of the
reserve in 1998, historical land-use practices on these owner-
managed properties were restricted to domestic livestock farming
and hunting. Anthropogenic activity in surrounding areas
includes manganese mining, thermal and solar power
generation and livestock farming which may inuence
atmospheric deposition of certain elements. Seasonal pans
(depressions that ll with rainwater in the wet season) are
present on the property but the majority of water is supplied
from articial boreholes. The fenced perimeter prevents
migration to higher resource areas and unpredictable rainfall
patterns inuence vegetation biomass. Supplementary salt and
mineral blocks are however provided at water sources to
somewhat mitigate nutrient stress. Mean temperatures range
from 5 to 24°C in the dry winter months and 2237°C in the
wet summer months with a mean annual rainfall of 325 mm
(World Weather Online, 2020b). Mountain streams provide
seasonal access to water but major rivers do not drain the
reserve (van Rooyen and van Rooyen, 2017).
The ±22,497 ha Manyeleti Nature Reserve (MNR), established
in 1963 from land previously dedicated to pastoral activities by
local indigenous populations, is situated at S 24°6480and E
31°5263in the Mpumalanga Lowveld of South Africa
(Figure 1B). The MNR shares unfenced boundaries with the
Kruger National Park to the east, the Sabi Sand Game Reserve to
the south and the Associated Private Nature Reserves to the
northwest (Mpumalanga Tourism and Parks Agency, 2015).
Given animals can migrate freely to the north, east and south,
provision of articial water and nutrient supplements are not
used as management tools on this property (Personal
communication A WebsterM Bourne). Mean temperatures
range from 7 to 22°C in the dry winter months and 1840°C
in the wet summer months with annual rainfall between 500 and
700 mm (World Weather Online, 2020a). The Nwaswitsontso
and Mthlowa Rivers drain the northern and central regions,
lling the main catchment dams in the centre of the reserve.
The Phungwe, Thorndale, Mhluwati and Tswayini Rivers drain
the southern regions (Cronje et al., 2005). The reserve forms part
of the Kruger National Park western boundary, which abuts a
number of growing rural communities that rely mostly on pit
latrine ablutions and government-controlled spraying of
chemicals for vector control. Anthropogenic activities in
surrounding areas include subsistence agricultural and
livestock activities and small businesses associated with motor
repair, leather tanning and other industries.
Sample Collection, Preparation and
Digestion for ICP-MS Analysis
Fresh faecal matter from 12 herbivorous (n194), 2 omnivorous
(n50), and 7 carnivorous (n137) mammalian species was
collected from Tswalu Kalahari Reserve (TKR), Northern Cape
Province (April-June 2019), and Manyeleti Nature Reserve
(MNR), Mpumalanga Province, (July to September 2019)
(Supplementary Table S1). It is unclear whether specic
analytes are excreted at the same rate within a faecal deposit.
To limit this possible inuence on trace element concentrations
being measured, faecal deposits were homogenised in situ before
collection of sub-samples from the centre of multiple faecal boli
or pellets from monogastric and ruminant herbivores respectively
(Ganswindt et al., 2002). All faecal samples (20 g) were collected
from observed defecation events to ensure samples were not
contaminated with urine. The majority of fresh carnivore faecal
samples were collected opportunistically from observed
defecation events around kills, near dens or latrines. However,
tracks and other sign around overnight faecal deposits and at
latrines left by nocturnal species were used to locate and identify
faecal samples belonging to specic species and collected before
07h00 each morning to minimize UV degradation of samples
(Liebenberg, 1990a;Liebenberg, 1990b). In the event faecal
deposits could not be assigned to a specic species (through
observation or track interpretation), samples were not collected.
Steenbok (Raphicerus campestris) faecal samples were uncovered
after burial and lightly brushed with a paintbrush to remove
excess soil. Additionally, to avoid contamination from metal-
containing implements, all faecal samples were collected using
gloves, subsequently placed into individual metal-free screw-top
plastic sampling containers, labelled and stored at 20°Cto
prevent microbial or chemical degradation of samples post-
defaecation.
Faecal samples were lyophilized at 50°C for 58 days. Dried
faecal samples from all species were subsequently pulverized
using a ceramic grater to separate any undigested material and
sieved through a plastic-mesh (37 µm) strainer to remove large
particles of bone, sinew and vegetation. The use of metal
equipment was avoided to prevent contamination of samples
during all phases of preparation. Individual herbivore and
carnivore 0.3 g dry mass (DM) samples were microwave
digested (MARS
®
5) in 6.5 ml (65%) HNO
3
: 0.5 ml (30%)
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Webster et al. Non-Invasive Assessment of African Mammals
FIGURE 1 | Tswalu Kalahari Reserve (1A) within the arid Northern Cape Province South Africa, showing fenced boundaries and the Korannaberg (white:
±101,000 ha) and Lekgaba sections (grey: ±20,000 ha). Manyeleti Nature Reserve (1B) within the mesic Mpumalanga Lowveld of South Africa with unfenced boundaries
to the northwest (Associated Private Nature Reserves, east (Kruger National Park) and South (Sabi Sand Game Reserve).
Frontiers in Environmental Science | www.frontiersin.org January 2022 | Volume 9 | Article 7944874
Webster et al. Non-Invasive Assessment of African Mammals
HCl. Post-digestion, de-ionized water was added to make 50 ml
nal volume of each sample (United States Environmental
Protection Agency, 2007). All faecal concentrations are given
as microgram per kilogram (µg/kg) faecal dry mass (DM).
Trace Element Analysis and Quality Control
Trace element analyses was performed on an Agilent 7900
quadrupole Inductively Coupled Plasma-Mass Spectrometer
(ICP-MS) equipped with a High Matrix Introduction system
and Agilent Mass Hunter software (version 4.4) for instrument
control and data processing (Agilent Technologies, 2009).
Detailed parameters for method quantication, adjustments
and validation are outlined in Webster et al. (2021c),in
Webster et al. (2021a) for analysis of environmental samples
and in Webster et al. (2021b) for analysis of essential elements
11
B,
55
Mn,
56
Fe,
59
Co,
60
Ni,
63
Cu,
66
Zn,
95
Mo, Se. In brief, before
sample introduction into the plasmas, argon as the dilution gas
was added. Helium collision cell mode using the 4
th
generation
Octopole Reaction System was used for analysis of element
isotopes
11
B,
27
Al,
51
V,
52
Cr,
55
Mn,
56
Fe,
59
Co,
60
Ni,
63
Cu,
66
Zn,
75
As,
88
Sr,
95
Mo,
111
Cd,
118
Sn,
121
Sb,
137
Ba,
202
Hg, and
208
Pb except Se. Inter-element corrections were made for possible
isobaric interferences of high
115
Sn on
115
In used as an internal
standard. Optimization for sensitivity and low oxide ratios (CeO/
Ce <0.3%) were performed daily.
Respective sets of digestions included dual quality controls of a
blank acid mixture and a matrix-suitable Certied Reference Material
obtained from the National Institute for Science and Technology,
Gaithersburg, United States. NIST 1573a tomato leaf 0.3 g (dm) and
NIST 1577c bovine liver 0.2 g (dm) were used as controls for
herbivore and carnivore faecal sample digestions respectively. In
addition, every third set of carnivore digestions contained 0.3 g
(dm) of NIST 1573a tomato leaf CRM control to ensure consistency
throughout the digestion sequence of carnivore faecal samples.
Accuracy (% recovery) and precision (% RSD) of replicate
measurements for certied reference material controls fell within
20% of expected value at lower concentrations and 15% at higher
concentrations for all elements except Al (United Nations Ofce on
Drugs and Crime, 2009).
Data Analysis
All statistical analyses were conducted using the R software: v
3.6.1 (R Core Team, 2019). In total, 381 faecal samples were
collected from 21 different terrestrial mammal species belonging
to three trophic groups (herbivore, omnivore and carnivore) at
two geographically different sites (MNR and TKR)
(Supplementary Table S1). To balance the comparison
between sitesand groups, species present at both study
sites were selected for evaluation. In the event a given species
was present at only one site (e.g., Meerkat/Brown hyaena) a
similar surrogate species (e.g., Dwarf mongoose/Spotted hyaena)
representing the same trophic group was selected at the other site.
Faecal concentrations are given as microgram per kilogram
(µg/kg) faecal dry mass (DM). Descriptive statistics for all
measured potentially toxic elements (PTEs) were determined
using all samples from each species and evaluated at each site.
To exclusively facilitate a visual comparison, the scales of measure
for each PTE were homogenized (mean 1), dividing each
elements concentration by its mean across sites and species.
Subsequently, plots (Figures 2,3) were created to compare
relative concentrations of elements within animal groups and
at each site (ggplot2 R package). Consequently, 24 outliers that
produced extensive compression of scale for a specic element,
thus obscuring specic differences between speciesboxplots
within animal groups were identied and removed, one by
one, before further analyses.
Before testing for differences between trophic groups and/or
study sites, individual effects (with the potential to affect element
means) were considered in the estimation model. To therefore
account for the variability of measured element concentrations
FIGURE 2 | Overall prole of potentially toxic element concentration measured in herbivorous (white), omnivorous (light grey), and carnivorous (dark grey) mammals
relative to the mean for both sites (dashed line). Dark lines within boxes represent median values; the box represents the upper (3rd) and lower (1st) quartiles. Whiskers
represent minimum and maximum values excluding outliers while points represent outliers. Concentrations are relativized and given as microgram per kilogram (µg/kg)
faecal dry mass (DM).
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Webster et al. Non-Invasive Assessment of African Mammals
between species, numbered blocks (blockvariable in
Supplementary Table S1) were assigned to each species (or
comparable species) unique to each study site. Following this a
3-way ANOVA randomized block design was used that
considered sitesand groupsas respective xed effect
factors and blocksas a random effect factor. Subsequently, a
linear mixed-effects model assuming heterogeneous variances
and including an interaction effect between sitesand
groupswas built for each PTE and estimated by maximum
likelihood (nmle and car R-packages). Using these estimated
models (summary of tted models Supplementary Table S2),
marginal and conditional means as well as standard errors (SE)
were determined for groupsand sitesusing the predicted
marginal means method (emmeans R-package). Mean differences
between groupsor siteswere investigated by applying Tukeys
pairwise post-hoc test (multcomp R-package). Basic results are
reported as mean ±SE and statistical signicance was determined
at the p<0.05 αlevel (Table 1). Associations between PTEs were
investigated using their corresponding Pearsons r correlation
coefcients and p-values (Tables 2,3).
RESULTS
Overall Comparison of PTEs Between
Trophic Groups
Overall PTE concentrations varied between individuals and
between animal groups (Figure 2). Measured mean
concentrations for respective PTEs were four to six times
higher than their respective means in some individuals. When
measured concentrations from animal groups from both sites
were compared, Al, As, Cd, Pb, Sn, and V were signicantly
higher in carnivores compared to herbivores. Measured mean
element concentrations in omnivores did not differ signicantly
from those in either herbivores or carnivores. Measured mean
concentrations of Ba, Cd, Hg, Sb and Sr did not differ signicantly
overall between trophic groups (Table 1: left Gr column).
Comparison of PTEs in Trophic Groups
Within Sites
Differences between mean measured PTE concentrations in
trophic groups within and between sites are shown in
Figure 3. Within the TKR site, concentrations were
signicantly higher for Al, As, Cr, Pb, Sn, and V in carnivores
compared to herbivores (Table 1: right Gr column).
Concentrations in omnivores did not differ signicantly to
those measured in either herbivore or carnivore groups. There
was no signicant difference in concentration between groups for
Ba, Cd, Hg, Sb, or Sr at the TKR site. Within the MNR site,
concentrations were signicantly higher for Al, Cr, Pb, Sn and V
in carnivores compared to herbivores, but concentrations in
omnivores did not differ signicantly from either herbivores
or carnivores (Table 1: right Gr column). In omnivores, Ba
concentrations were signicantly higher than those in
herbivores, but concentrations in carnivores were not
signicantly different to those measured in other groups. In
herbivores, Sb concentrations were signicantly higher to
those measured in carnivores but did not differ signicantly in
omnivores compared to other groups. There was no signicant
difference in concentration between groups for As, Cd, Hg, or Sr
at the MNR site.
Comparison of PTEs in Trophic Groups
Between Sites
When mean measured PTE concentrations in trophic groups
were compared between sites (Table 1:rightScolumn),animal
FIGURE 3 | Differences in measured potentially toxic element concentrations between sites (MNR: solid borders, TKR: dashed borders) in herbivorous (white),
omnivorous (light grey), and carnivorous (dark grey) mammals relative to the mean for both sites (dashed line). Dark lines within boxes represent median values; the box
represents the upper (3rd) and lower (1st) quartiles. Whiskers above and below the box represent minimum and maximum values excluding outliers. Points represent
outliers. Concentrations are relativized and given as microgram per kilogram (µg/kg) faecal dry mass (DM).
Frontiers in Environmental Science | www.frontiersin.org January 2022 | Volume 9 | Article 7944876
Webster et al. Non-Invasive Assessment of African Mammals
groups at the TKR site showed signicantly higher
concentrations of As, Cd, and Hg while those in the MNR
site had signicantly higher concentrations of Al, Cr, and Sr.
High As, Cd, and Hg concentrations were mirrored in
carnivores and omnivores from the TKR site. Sb was also
signicantly high in carnivores. Herbivores at the TKR site had
signicantly higher concentrations of As, Ba, and Cd than
those measured in their MNR counterparts. At the MNR site,
Al, Cr, and Sr in carnivores, Ba, Cr, and Sr in omnivores and
Al,Cr,Hg,Sb,andSrinherbivoresweresignicantly higher
than those measured in the same animal groups in the TKR.
There was no signicant difference in concentrations for Pb,
Sn, and V in any group between sites. Clear patterns emerge
between groups for certain elements within sites but absolute
values for the same elements differ between sites. Detailed
results and summary of tted models for each PTE are
presented in Table 1 and Supplementary Table S2 of
supplementary material.
Comparison of PTE Associations in Trophic
Groups Between Sites
Mutually associated (considerable correlation for any PTE pair)
and correlated (between elements) PTEs for each trophic group at
the TKR site can be seen in Table 2. In herbivores at the TKR site
a mutual association between Al + As + V + Cr + Pb + Sn
(minimum correlation: r+0.563) was evident. The highest
correlation within mutually associated PTEs was between Al + As
TABLE 1 | Estimates for potentially toxic element concentrations between animal groups overall (left) and between sites for any group (right). Note: Overall differences
between groups are shown on the left of the table. Comparisons between sites (overall and in any group) and between groups (within each site) are shown on the right.
Within row letter subscripts a, b indicate a signicant difference between sites (p<0.05) and within column letter superscripts a, b, c indicates signicant differences between
groups (p<0.05). Faecal concentrations are given as microgram per kilogram (µg/kg) faecal dry mass (DM).
Element Group n Overall Gr Group n Tswalu KR mean ±SE S Gr hn Manyeleti NR mean ±SE S Gr h
Al Overall n mean ±SE hOverall 193 3748 ±398.1
a
186 4762.1 ±436.6
b
Herbivore 194 2366 ±384.1
a
Herbivore 100 2143.5 ±382.5
aa
94 2588.5 ±400.6
ba
Omnivore 50 4578.5 ±919.5
ab
Omnivore 25 4387.1 ±976.7
aab
25 4769.8 ±1000.9
aab
Carnivore 135 5820.7 ±601.9
b
Carnivore 68 4713.4 ±570.8
ab
67 6928 ±743.9
bb
As Overall n mean ±SE hOverall 192 419.1 ±35.5
b
187 200.9 ±31.1
a
Herbivore 193 223.6 ±31.7
a
Herbivore 99 279.4 ±32.5
ba
94 167.8 ±32.4
aa
Omnivore 50 319.1 ±73.8
ab
Omnivore 25 431.4 ±82.6
bab
25 206.8 ±73.1
aa
Carnivore 136 387.2 ±49.1
b
Carnivore 68 546.4 ±58.9
bb
68 228 ±48.3
aa
Ba Overall n mean ±SE hOverall 193 172227.2 ±17299.9
a
187 192913.6 ±22959.4
a
Herbivore 194 163724.5 ±17847.3
a
Herbivore 100 221517.3 ±19915.4
ba
94 105931.7 ±17443.5
aa
Omnivore 49 234516.7 ±45413.1
a
Omnivore 25 153882.6 ±39874.4
aa
24 315150.8 ±61481.2
bb
Carnivore 137 149469.9 ±25090.6
a
Carnivore 68 141281.6 ±26589.1
aa
69 157658.1 ±25690.5
aab
Cd Overall n mean ±SE hOverall 192 130.9 ±17.7
b
186 53.5 ±16
a
Herbivore 194 92.6 ±16.9
a
Herbivore 100 150.9 ±19
ba
94 34.4 ±16.2
aa
Omnivore 49 91.1 ±38.2
a
Omnivore 24 125 ±42
ba
25 57.2 ±38.2
aa
Carnivore 135 92.9 ±24
a
Carnivore 68 116.8 ±26.2
ba
67 69 ±23.9
aa
Cr Overall n mean ±SE hOverall 192 7379.2 ±761.8
a
182 17063.4 ±1323.4
b
Herbivore 192 8228 ±894.5
a
Herbivore 100 4754.5 ±738.2
aa
92 11701.4 ±1283.3
ba
Omnivore 48 13399.5 ±2123.1
ab
Omnivore 25 8005.7 ±1720.1
aab
23 18793.2 ±3163.9
bab
Carnivore 134 15036.5 ±1403
b
Carnivore 67 9377.5 ±1311.4
ab
67 20695.5 ±2026.2
bb
Hg Overall n mean ±SE hOverall 193 37.7 ±7.1
b
187 29.9 ±7
a
Herbivore 193 34.7 ±7.3
a
Herbivore 100 27.1 ±7.2
aa
93 42.3 ±7.8
ba
Omnivore 50 30.7 ±16.2
a
Omnivore 25 41.9 ±16.9
ba
25 19.5 ±16.4
aa
Carnivore 137 36 ±10.5
a
Carnivore 68 44.3 ±11.2
ba
69 27.8 ±10.6
aa
Pb Overall n mean ±SE hOverall 192 2222.9 ±289.7
a
188 1729.5 ±229.6
a
Herbivore 194 967 ±154
a
Herbivore 100 1001.2 ±156.1
aa
94 932.8 ±157.1
aa
Omnivore 50 2370.2 ±540.6
ab
Omnivore 25 2717.8 ±745.7
aab
25 2022.5 ±615
aab
Carnivore 136 2591.3 ±291.3
b
Carnivore 67 2949.6 ±418.3
ab
69 2233.1 ±267.5
ab
Sb Overall n mean ±SE hOverall 191 18.3 ±2.2
a
187 20.3 ±3
a
Herbivore 194 21.1 ±2.5
a
Herbivore 100 15.1 ±2.5
aa
94 27 ±3.3
bb
Omnivore 49 19.6 ±5.5
a
Omnivore 24 14.2 ±5
aa
25 25.1 ±7.8
aab
Carnivore 135 17.1 ±2.9
a
Carnivore 67 25.5 ±3.4
ba
68 8.7 ±2.8
aa
Sn Overall n mean ±SE hOverall 193 149.3 ±13.5
a
186 156.5 ±14.4
a
Herbivore 194 100.5 ±12.8
a
Herbivore 100 101.5 ±13.2
aa
94 99.5 ±13.4
aa
Omnivore 49 150.2 ±29.6
ab
Omnivore 25 156.5 ±31.9
aab
24 143.8 ±31.3
aab
Carnivore 136 208 ±21.1
b
Carnivore 68 189.8 ±21.5
ab
68 226.2 ±26.6
ab
Sr Overall n mean ±SE hOverall 193 113009.5 ±22604.6
a
188 199519.9 ±23788.1
b
Herbivore 194 153918.4 ±24138.2
a
Herbivore 100 139415.3 ±24277.2
aa
94 168421.5 ±24980.7
ba
Omnivore 50 157369 ±53685.3
a
Omnivore 25 97800.9 ±53406.5
aa
25 216937 ±55555.1
ba
Carnivore 137 157506.8 ±34559.2
a
Carnivore 68 101812.2 ±34016
aa
69 213201.4 ±37181.3
ba
V Overall n mean ±SE hOverall 193 7002.1 ±725.6
a
186 7183.3 ±730.7
a
Herbivore 193 4538.5 ±675.8
a
Herbivore 100 4685.8 ±685.6
aa
93 4391.2 ±696.5
aa
Omnivore 50 7841.6 ±1631.2
ab
Omnivore 25 7906.1 ±1812.5
aab
25 7777.1 ±1723.3
aab
Carnivore 136 8898 ±1010.6
b
Carnivore 68 8414.2 ±991.9
ab
68 9381.8 ±1161.9
ab
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Webster et al. Non-Invasive Assessment of African Mammals
(r+0.918; p<0.001), while the highest correlation for other
PTEs was between Ba + Sr (r+0.0.699; p<0.001). In TKR
omnivores, V + Al + As + Cr + Sn (minimum correlation; r
+0.901) were mutually associated with Al + V (r+0.962; p<
0.0001) being the highest. In addition, Cd + Ba + Sr (minimum
correlation: r+0.850) were also mutually associated in
omnivores, with Ba + Cd (r+0.935; p<0.001) being the
highest. The highest signicant correlations for other PTE in
omnivores was between Cr + Sr (r0.695; p<0.001). In
carnivores at the TKR site, mutual association between PTEs
V + Al + As + Sn (with a minimum correlation; r+0.690) was
evident. Within mutually associated PTE in carnivores, the
correlation between Al + V (r+0.958; p<0.001) was the
highest, while the highest other correlated PTEs for carnivores at
the TKR site were Ba + Sr (r0.757; p<0.001).
Mutually associated and correlated PTEs for each trophic
group at the MNR site can be seen in Table 3. At the MNR
site, mutual association between PTEs V + Al + Cr (minimum
correlation r+0.603) was evident in herbivores with Al + V (r
0.926; p<0.001) being the highest. The highest other PTE
correlations in herbivores were Ba + Sr (r+0.834; p<
0.001). In MNR omnivores mutual associations were evident
between PTE As + Sb + Cd (minimum correlation r+0.556)
with the strongest mutual association being between Sb + As (r
+0.780; p<0.004). In addition, mutual associations were found
between V + Al + Cr + Sn (minimum correlation r+0.537) with
Al + V (r+0.946; p<0.010) being the highest. Ba + Sr (r
+0.872; p<0.001) were other highly correlated PTEs for
omnivores at this site. In MNR carnivores, a mutual
association between PTEs As + Pb + V + Al + Sn (minimum
correlation; r+0.709) was evident with Al + V (r+0.941; p<
0.001) being the highest. Cd + Hg (r+0.648; p<0.001) were
other highly correlated PTEs in MNR carnivores.
DISCUSSION AND CONCLUSION
Heavy metalpollution rated highest of 22 themes related to
emerging chemical management issues in developing countries
that are not covered by international treaties (STAP, 2012).
Compared to marine and aquatic environments, information
related to the presence and effects of PTEs and other
pollutants in terrestrial systems is limited (Rodríguez-Jorquera
et al., 2017). Although valuable information has been gained from
previously used invasive sampling approaches, much of what has
been learnt has occurred as a result of opportunistic sampling of
fresh carcasses at mass die offs and debilitation events (Ferreira
and Pienaar, 2011). Faecal material is a readily produced waste
product and is an effective matrix for trace element assessment in
free-ranging wildlife species. With the exception of Hg, which can
undergo chemical change in the liver, rumen and digestive tract,
most elements may be stored within the body after ingestion but
are not chemically changed through metabolism in the liver or
kidney. In this regard, many trace elements are detectable in their
original form within excreta (Das et al., 2019). This study presents
the rst quantitative assessment and comparison of multiple
TABLE 2 | Mutually associated PTEs and other PTE correlations at the Tswalu Kalahari Reserve (TKR) in different trophic groups. Grey highlighted blocks with bold lettering
show highest mutually associated PTE for respective trophic groups. Bold text represents the highest mutually correla ted elements and other correlated PTEs for specic
trophic groups. Faecal concentrations are given as microgram per kilogram (µg/kg) faecal dry mass (DM).
Tswalu kalahari reserve (TKR)
Herbivore Omnivore Carnivore
Mutually associated PTE Mutually associated PTE Mutually associated PTE
Al +As +V+Cr +Pb +Sn (r+0.563; p<0.001) V +Al +As +Cr +Sn (r+0.901; p<0.001) V +Al +As +Sn (r+0.690; p<0.001)
Pearsons correlation and p-values Pearsons correlation and p-values Pearsons correlation and p-values
As Cr Pb V Al Sn As Cr V Al Sn As V Al Sn
As 00000As 0000As 000
Cr 0.829 0 0 0 0 Cr 0.916 0 0 0 V0.773 0 0
Pb 0.793 0.669 0 0 0 V0.923 0.901 0 0 Al 0.793 0.958 0
V0.887 0.826 0.754 0 0 Al 0.950 0.943 0.962 0Sn 0.718 0.690 0.746
Al 0.918 0.824 0.794 0.911 0 Sn 0.926 0.936 0.903 0.955
Sn 0.726 0.563 0.641 0.666 0.727
Cd + Ba + Sr (r+0.850; p<0.001)
Cd Ba Sr
Cd 00
Ba 0.935 0
Sr 0.850 0.891
Tswalu Kalahari Reserve (TKR) other PTE correlations
Herbivore Omnivore Carnivore
Ba +Sr: r=+0.699; p<0.001 Cr +Sr: r=-0.695; p<0.001 Ba +Sr: r=+0.757; p<0.001
Ba + Cd: r+0.521; p<0.001 Pb + Sn: r+0.668; p<0.001 Cd + Hg: r0.605; p<0.001
Sn + Sr: r-0.641; p0.001 Sb + Sn: r0.596; p<0.001
Cr + Cd: r-0.624; p0.001 V + Sr: r-0.556; p<0.001
Al + Ba: r-0.504; p0.010 Al + Sr: r-0.523; p<0.001
Frontiers in Environmental Science | www.frontiersin.org January 2022 | Volume 9 | Article 7944878
Webster et al. Non-Invasive Assessment of African Mammals
PTEs in herbivorous, omnivorous and carnivorous terrestrial
mammals in a mesic and arid protected area of South Africa,
using faeces as a non-invasive matrix.
When measured concentrations from animal groups at both
sites were compared, Al, Cd, Pb, Sn, and V as well as As at the
TKR site were signicantly higher in carnivores than in other
animal groups. Site-specic geology contributes to natural
background levels of metals and metalloids within the
environment (FAO and UNEP, 2021), however anthropogenic
activities can increase bioavailability and bio-accessibility of
PTEs. The physical interaction between water, geologic
substrate and soils (Knoop and Walker, 1985) additionally
inuences vegetation structure and PTE concentrations in
woody and C
4
plant species, making soils a key component of
terrestrial ecosystems (Herselman, 2007;Vaughn et al., 2015).
Relatively high levels of PTEs As, Cd, Cr, Pb, and Sb are
associated with the banded iron ores of the Kalahari region
surrounding the TKR site (Varentsova and Kuleshovb, 2019).
In contrast, Archaean gneiss and prominent intrusions of
Timbavati gabbroic rock underlie much of the MNR site,
particularly in the west (Walraven, 1986). The gabbro
intrusions closest to the MNR are associated with relatively
high levels of Cr and V as well as elements Co, Fe and Zn
(Walraven, 1986), which are considered essential for biological
function (Webster et al., 2021b). In addition to site-specic
geochemical signatures that may in part account for high
concentrations of specic PTE particularly in carnivores,
results indicate dietary-niche peculiarities and possible
biomagnication of environmental concentrations at both sites.
When trophic groups within sites were compared, Al, As, Cr,
Pb, Sn, and V were highest in carnivores at the TKR site. At the
MNR site, Al, Cr, Pb, Sn, and V were highest in carnivores, Ba was
higher in omnivores and Sb was highest in herbivores than in any
other trophic groups at that site. Aluminium is an abundant
element in the Earths crust (Kabata-Pendias, 2011) and its
occurrence in carnivores at both sites may in part be
attributed to its presence in underlying geological signatures,
but may also be attributed to one or a combination of behavioural
factors. These include the ingestion of Al-containing soil during
consumption of kills, the grooming habits of social meso- and
apex predators or consumption of ground-dwelling invertebrates
and arthropods by insectivorous mesocarnivores. High levels of
Al in carnivores may also be indicative of trophic
biomagnication from ingestion of herbivorous/omnivorous
prey species with high body burden (Ali and Khan, 2019). Al
is not generally available for participation in biogeochemical
reactions given its insoluble nature (Driscoll and Schecher,
1990), however environmental sources of exposure to this
element remain speculative. Whether concentrations at the
levels measured here have deleterious effects alone or in
combination with co-occurring PTE in wildlife is uncertain.
The presence of arsenic (As) and many of its water-soluble
compounds (Agency for Toxic Substances and Disease Registry
(ATSDR), 2007a) in the geological signature of the TKR,
TABLE 3 | Mutually associated PTEs and other PTE correlations at the Manyeleti Nature Reserve (MNR) in different trophic groups. Grey highlighted blocks with bold lettering
show highest mutually associated PTE for respective trophic groups. Bold text represents the highest mutually correla ted elements and other correlated PTEs for specic
trophic groups. Faecal concentrations are given as microgram per kilogram (µg/kg) faecal dry mass (DM). Tswalu Kalahari Reserve (TKR) other PTE correlations"
Herbivores - "Ba +Sr: r=+0.699 ; p<0.001.
Manyeleti nature reserve (MNR)
Herbivore Omnivore Carnivore
Mutually associated PTE Mutually associated PTE Mutually associated PTE
V+Al +Cr (r+0.603; p<0.001) V +Al +Cr +Sn (r+0.537; p<0.010) As +Pb +V+Al +Sn (r=>+0.709; p<0.001)
Pearsons correlation and p-values Pearsons correlation and p-values Pearsons correlation and p-values
Cr V Al V Al Cr Sn As Pb V Al Sn
Cr 00V000As 0000
V0.684 0 Al 0.946 00Pb 0.789 0 0 0
Al 0.603 0.926 Cr 0.777 0.732 0.010 V0.714 0.835 0 0
Sn 0.707 0.755 0.537 Al 0.759 0.839 0.941 0
Sn 0.709 0.838 0.848 0.847
As + Sb + Cd (r+0.556; p<0.004)
As Sb Cd
As 00
Sb 0.780 0
Cd 0.556 0.678
Manyeleti Nature Reserve (MNR) other PTE correlations
Herbivore Omnivore Carnivore
Ba +Sr: r=+0.834; p<0.001 Ba +Sr: r=+0.872; p<0.001 Cd +Hg: r=+0.648; p<0.001
Sr + Cd: r+0.603; p<0.001 As + Sn: r+0.689; p<0.001 Ba + Sr: r+0.647; p<0.001
As + Sb: r+0.543; p<0.001 Pb + Al: r+0.573; p0.003 Cr + V: r+0.637; p<0.001
Sb + Sr: r+0.531; p<0.001 Pb + V: r+0.569; p0.003 As + Hg: r+0.631; p<0.001
V + Pb: r+0.506; p<0.001 Sr + V: r-0.544; p0.005 Al + Cd: r+0.586; p<0.001
Pb + Sn: r+0.533; p0.007 Pb + Cd: r+0.579; p<0.001
Sr + Al: r-0.529; p0.007 V + Cd: r+0.515; p<0.001
Frontiers in Environmental Science | www.frontiersin.org January 2022 | Volume 9 | Article 7944879
Webster et al. Non-Invasive Assessment of African Mammals
translated to signicantly high measured concentrations across
animal groups and a clear pattern of trophic accumulation.
Drinking from contaminated water sources is a major route of
exposure for wildlife species. Between 70 and 90% of inorganic As
is absorbed through the gastrointestinal tract and distributed
primarily to the liver, kidneys, bladder and lungs and secondarily
to muscle and nerve tissue (Palma-Lara et al., 2020). Herbivores
must access water regularly to meet the physiological demands of
thermoregulation and rumination (Boyers et al., 2019). Species
such as buffalo (Syncerus caffer) and impala (Aepyceros
melampus) are water dependant and must access water daily
(Redfern et al., 2003). Gemsbok (Oryx gazella), springbok
(Antodorcas marsupialis), and steenbok (Raphicerus
campestris) are adapted to higher temperatures and limited
surface water availability and are able to concentrate
excretions to minimise water loss (Eloff, 1973;Knight, 2013;
Boyers et al., 2019). As the mechanistic adaptations of this trophic
group may translate into As-accumulation in the kidneys, liver
and bladder, herbivores are at high risk of repeated and continual
exposure through consumption of As-contaminated water.
Carnivores, particularly those living in arid environments,
access surface water when available, obtaining the majority of
their moisture through the prey items they consume (Eloff, 1973).
Because of their high nutrient content, blood rich organs such as
the liver and kidneys are often consumed rst (Kohl et al., 2015).
Toxins within these organs then add to the body burden of
carnivorous predators. The assessment of long-term exposure to
inorganic arsenic is complicated by the duration of exposure,
pathway of ingestion, physicochemical properties of the
compound and symptoms that differ between individuals,
species and geographical areas (World Health Organisation,
2019;Palma-Lara et al., 2020). Long-term As exposure in
animals has been linked to pregnancy-related complications in
females including miscarriage, low foetal birth weight and foetal
deformity (Agency for Toxic Substances and Disease Registry
(ATSDR), 2007a) all of which affect survival and reproductive
ability of carnivore populations.
Chromium (Cr) the most abundant element measured in
environmental matrices (Webster et al., 2021a) at the MNR
site was reected across animal groups. In part, concentrations
could be attributed to the geochemical signature of the region
however, in mesic environments, interactions between sulphate
and iron carriers in plants facilitate high rates of Cr uptake in
vegetation (Shankar et al., 2005). Herbivores and omnivores
feeding on this vegetation daily (Reglero et al., 2008) and
social species that exhibit allo-grooming behaviour are likely
to accumulate concentrations of this element to a greater
extent than solitary species. Damage to the reproductive
system in male animals and increased complications during
parturition have been documented in females (World Health
Organisation/International Agency for Research on Cancer,
1990;Agency for Toxic Substances and Disease Registry
(ATSDR), 2012a). Biomagnication in end-consumers would
therefore have negative effects on reproductive success.
Signicantly high levels of lead (Pb) were measured in
carnivores at both sites. In biological systems, Pb and calcium
act in a similar manner, facilitating Pb-storage within bone
(Gulson et al., 2003) and remobilisation of Pb into the blood
stream during periods of physiological or nutritional stress.
Consumption of bones by scavenging and obligate carnivores
may contribute to Pb concentrations within carnivores and other
species such as vultures (Ogada et al., 2012;Naidoo et al., 2017).
Foraging opportunities for suitable prey species are
unpredictable, so carnivores are particularly vulnerable to the
effects of Pb remobilisation during periods of nutrient stress. Lead
is a systemic toxicant and there is growing evidence to suggest
that low-level environmental exposure from various sources
impairs renal function and neurological development in
juveniles, which can result in life-long neurological decits,
endocrine disruption and reproductive failure (Jadhav et al.,
2007;Assi et al., 2016;Agency for Toxic Substances and
Disease Registry (ATSDR), 2019b).
Carnivores within both sites also had signicantly high
measured concentrations of Tin (Sn) and vanadium (V). Sn is
found in fungicides, insecticides and pesticides and given the
increased conversion of land for agricultural purposes, has
become a threat to the environment. Although Sn has
generally low solubility in soil, in response to a decrease in
soil pH, uptake in plants increases (Nakamaru and Uchida
2008). Whether V is an essential element is still being debated
however, it accumulates in plant roots and is a known insulin
mimic (Agency for Toxic Substances and Disease Registry
(ATSDR), 2012b). Although site-specic geochemistry may be
linked to Sn and V concentrations at both sites, Sn presence at the
TKR site may be related to historical legacy of livestock farming
while at the MNR site, increasing subsistence agricultural activity
in communities surrounding the reserve may be inuencing
concentrations. Whether Sn and V alone, in combination with
other elements or combined with exogenous toxins cause
deleterious effects in different free-ranging wildlife species is
unclear.
Signicantly high concentrations of antimony (Sb) were
measured in herbivores at the MNR site and are most likely
related to geological signature and soil to plant transfer (Agency
for Toxic Substances and Disease Registry (ATSDR), 2019a). Ba
concentrations were signicantly higher in omnivores at this site
than in other groups at either site while herbivores at the TKR site
had higher concentrations of Ba than other groups. Although
these differences may be attributed to feeding ecology between
species at the different sites, Ba compounds are mostly insoluble
in water so persist in the environment. Although barium-
carbonate is insoluble in water, it is soluble in the acid
environment of the GI tract resulting in toxic effects. Ba
compounds are often associated with waste sites, which
contaminate surrounding soil, plants and ground water
through leaching (Agency for Toxic Substances and Disease
Registry (ATSDR), 2007b). Historical land use and waste
disposal methods are unknown for the TKR site but previous
disposal of waste in landlls may play a role in Ba concentrations
in herbivores utilizing rehabilitated sites on this property. In
contrast, omnivores at the MNR site frequent the landll site
situated in close proximity to the riverine stretches along the main
catchment area during daily foraging forays. Established
vegetation and sources of fruit, berries and other food items
Frontiers in Environmental Science | www.frontiersin.org January 2022 | Volume 9 | Article 79448710
Webster et al. Non-Invasive Assessment of African Mammals
may be contributing to concentrations in this trophic group.
Further research is however required to determine site-specic
sources of this element and the negative effects they may have (if
any) in the species with high concentrations.
Mercury is an agricultural, industrial, mining and waste-
related pollutant and is classied as one of the top 10
contaminants of health concern (FAO and UNEP, 2021). Hg
was found in concentrations above the TKR mean in
environmental matrices (Webster et al., 2021a) and in
carnivores (this study) and may be related to anthropogenic
activities associated with the landing strip. Although
contamination at the TKR site was localised, highest levels of
Hg at the MNR site were measured in herbivores and may result
from water-transport of mercury-containing fertilizers and anti-
parasitic veterinary drugs used in substance farming by
communities in close proximity to the reserve. Additionally,
direct runoff from contaminated livestock manure into surface
water may be a contributing factor to contamination of grazing
areas in close proximity to water catchment areas (Richards et al.,
2014;Webster et al., 2021a). Cadmium is used in multiple
industries and agricultural fertilizers and when bound to ne
particles, can be transported atmospherically (Burger, 2008). Cd
concentrations were signicant in environmental matrices at the
TKR site, which is reected across animal groups (Webster et al.,
2021a). Plants have the ability to accumulate Cd in roots and
shoots at levels that are toxic to most other organisms. Although
this mechanism possibly developed as a defence against herbivory
(Godinho et al., 2018), species consuming Cd-containing forage
are likely to accumulate Cd in the liver and kidneys. Toxicity is
exacerbated by low rates of excretion, accumulation in the
proximal renal tubules and high rate of reabsorption through
the kidneys (Azeh Engwa et al., 2019).
Although strontium (Sr) is a substantial component of igneous
rock (Kabata-Pendias, 2011), electronic waste sites containing
broken visual display units and television screens and the
improper disposal of mechanical greases contribute to its
presence in the environment. All stable isotopes behave in a
similar chemical manner to each other, but also behave in a
chemically similar way to Ba and calcium (Pathak and Gupta,
2020). Prolonged Sr exposure in plants causes reduced
phytoestrogen content resulting in decreased antioxidant
capacity and impaired uptake of other micronutrients (Dresler
et al., 2018). When Sr-containing plants are consumed by
herbivorous or omnivorous species, Sr presence can affect
bone growth, the ability of bone marrow to produce blood
cells and compromised clotting (Agency for Toxic Substances
and Disease Registry (ATSDR), 2004).
The highest mutually associated PTEs were Al and V in all
trophic groups at both sites, and are likely indicative of their
abundance in the environment. However, site-specic patterns in
mutually associated PTE were in contrast between sites for other
correlated PTEs (Tables 2,3). At the TKR site mutual
associations between these elements were highest in omnivores
>carnivores >herbivores. In addition, a mutual association
between Ba and Cd in omnivores was also high at this site. Ba
and Sr were other highly correlated PTEs in omnivores and
herbivores at this site, while Ba and Sr followed by Cd and Hg
were other highly correlated elements in TKR carnivores. At the
MNR site, a mutual association between Al and V was highest in
carnivores >herbivores >omnivores. Sb and As were also
mutually correlated in omnivores at this site. Ba and Sr were
other highly correlated PTEs in omnivores and herbivores,
followed by Cr and V. Cd and Hg as well as Ba and Cr were
highly correlated in MNR carnivores. In biological terms, PTE
associations at each site are likely inuenced to some degree by
localized geochemistry but clear patterns also emerge related to
feeding ecology, PTE transfer via trophic interactions and
biomagnication in carnivores and soil-plant transfer in
herbivores and omnivores. Additionally, interactions between
essential and potentially toxic elements are well known for
some elements including (Fe and Cd; Akesson et al., 2002,Ca
and Pb; Gulson et al., 2003, Fe, Cu, and Zn; Hooser, 2018), but
require more specic investigation in free-ranging wildlife
species.
Previous studies have been conducted in heavily polluted
protected areas (Gupta and Bakre, 2013), but this is the rst
assessment of PTE in African savannahs that considers
differences between trophic groups in relatively pristine
protected areas. Our understanding of the mechanisms by
which PTEs affect biological systems has increased with new
technology and the development of environmental assessment
frameworks. Much of what we know is related to established
mechanisms associated with essential and toxic elements but
there is still much to learn about how these elements interact with
one another in wildlife species of different body size, age, sex, and
life stage living in habitats with site-specic contaminant
signatures. Additionally, more detailed analyses related to the
effects different diets and consumption of various food items may
have on PTE accumulation in different trophic groups (and
species) are required. Addressing these gaps in our knowledge
will require a One-Health approach and the sharing of
information between veterinary, medical and ecological
professionals as well as application of invasive methods
simultaneous to the use of a non-invasive approach. Faecal
material is one of numerous matrices to consider for non-
lethal environmental monitoring but it is important to
remember that all methods have limitations. We know faecal
material closely reects actual intake; we also know that
chemically intact elemental compounds can be measured in
faecal material. Although some physiological mechanisms
related to elimination of toxins from the body are known
what we do not yet know without lethal investigation is how
much of the actual intake is being retained as body burden in the
organs of different species occupying different dietary niches.
Additionally, although faecal material is a suitable metric for the
measurement of essential and potentially toxic trace elements in
wildlife, it may not be an effective tool for the monitoring of other
pollutants not excreted in the faeces or for those that are
chemically altered through metabolism in the body.
Given trace element concentrations within protected areas are
inuenced by background geochemical signatures, PTE pollution
is site, species-as well as diet-specic. Although faecal analysis
cannot tell the full story related to the presence and/or persistence
of hazardous pollutants and their effects on ecosystem integrity, it
Frontiers in Environmental Science | www.frontiersin.org January 2022 | Volume 9 | Article 79448711
Webster et al. Non-Invasive Assessment of African Mammals
can help to identify species at high risk and hot spots for more
intensive environmental monitoring within protected areas,
acting as an early warning system for potentially devastating
pollution events. In addition to mammalian species, faecal
samples can practically be collected in a non-invasive manner
from birds, insects, bats and reptiles in conjunction with
environmental matrices such as water, soil/sediment and
vegetation samples to provide a more complete assessment of
ecosystem integrity. Information gathered in this way would
allow us to draw general conclusions related to animal
exposure that are relevant to species biodiversity assessments,
wildlife management and species conservation. Further research
is required at both plant and animal species-specic levels to
provide greater insight into how metabolism changes pollutant
compounds for more accurate determination of different toxins
using faecal material, the possible sources of pollutants within
specic protected areas and the routes of exposure that result in
wildlife exposure to toxins in protected areas.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories.
The names of the repository/repositories and accession number(s) can
be found below: Doi:10.25403/UPresearchdata.14675388.
ETHICS STATEMENT
The animal study was reviewed and approved by the University of
Pretoria Research and Animal Use and Care Committee
(Reference EC043-18 and EC043-18-A1) and the South
African Department of Agriculture, Forestry and Fisheries
(DAFF-18/02/2019). The MNR site lies within the infected
zonefor the control of Foot and Mouth Disease (FMD) in
South Africa. As a result, faecal subsamples from all samples
collected were transported under Red Cross Veterinary Permit to
the Agricultural Research Council, Trans-boundary Animal
Diseases facility at the Onderstepoort Veterinary Research
Campus, South Africa, for testing prior to removal from the
MNR site. Upon receipt of negative FMD results and termination
of eldwork, frozen faecal samples were transported under Red
Cross Veterinary Permit to the University of Pretorias Endocrine
Research Laboratory for preparation and subsequent diagnostic
testing.
AUTHOR CONTRIBUTIONS
ABW: Principal investigator, Conceptualisation, Methodology,
Data collection and analysis, Visualisation, Project
administration, Data curation and WritingOriginal draft. JFC:
Formal analysis, Data curation, WritingReview and editing.
NCB: Conceptualisation, Academic supervision, Funding,
WritingReview and editing. AG: Conceptualisation, Academic
Supervision, WritingReview and editing.
FUNDING
This research was supported by the Department of Science and
Technology and National Research foundation SARChI chair of
Mammalian Behavioural Ecology and Physiology, South Africa
(GUN number 64756), The University of Pretoria Post-graduate
Scholarship Programme and The Tswalu Foundation, South
Africa. The National Institute for Science and Technology,
Gaithersburg, United States of America is acknowledged for
donation of domestic sludge and tomato leaf Certied
Reference Materials.
ACKNOWLEDGMENTS
Tswalu Kalahari Reserve and Mpumalanga Tourism and Parks
Agency, Manyeleti Game Reserve are thanked for facilitating this
research. Central Analytical Facilities, ICP-MS Laboratory,
University of Stellenbosch is thanked for sample analysis. The
University of Pretoria Endocrine Research laboratory and CSIR
Stellenbosch are thanked for assistance with sample preparation.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fenvs.2021.794487/
full#supplementary-material
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