Environmental Pollution 284 (2021) 117318

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Saline mine-water alters the structure and function of prokaryote

communities in shallow groundwater below a tropical stream☆
Lisa Chandler a, b, *, Andrew J. Harford b, Grant C. Hose c, Chris L. Humphrey b,
Anthony Chariton c, Paul Greenfield d, Jenny Davis a
a
Research Institute for the Environment and Livelihoods, College of Engineering, IT & Environment, Charles Darwin University, Darwin, Northern Territory, Australia
b
Supervising Scientist Branch, Department of Agriculture, Water and the Environment, Darwin, Northern Territory, Australia
c
Department of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia
d
Commonwealth Scientific and Industrial Research Organisation (CSIRO), Canberra, Australian Capital Territory, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Bacteria and archaea (prokaryotes) are vital components for maintaining healthy function of groundwater
Freshwaters ecosystems. The prokaryotic community composition and associated putative functional processes were exam­
Low ionic strength ined in a shallow sandy aquifer in a wet-dry tropical environment. The aquifer had a contaminated gradient of
Hyporheic
saline mine-water, which primarily consisted of elevated magnesium (Mg2+) and sulfate (SO2− 4 ), although other
Metagenomics/community genomics
Microbial ecology
major ions and trace metals were also present. Groundwaters were sampled from piezometers, approximately 2
m in depth, located in the creek channel upstream and downstream of the mine-water influence. Sampling
occurred during the dry-season when only subsurface water flow was present. Next generation sequencing was
used to analyse the prokaryote assemblages using 16S rDNA and metabolic functions were predicted with
FAPROTAX. Significant changes in community composition and functional processes were observed with
exposure to mine-waters. Communities in the exposed sites had significantly lower relative abundance of
methanotrophs such as Methylococcaceae and methanogens (Methanobacteriaceae), but higher abundance in
Nitrososphaeraceae, associated with nitrification, indicating potentially important changes in the biogeochem­
istry of the exposed sites. The changes were most strongly correlated with concentrations of SO2−
4 , Mg
2+
and Na+.
This knowledge allows an assessment of the risk of mine-water contamination to groundwater ecosystem
function and aids mine-water management.

1. Introduction biodiversity, thus altering various biogeochemical cycling processes

Groundwater is a valuable global resource that is under increasing
pressure from disturbances such as extraction and contamination
(Griebler et al., 2019). Groundwaters in uncontaminated aquifers are
often oligotrophic, with little or no dissolved oxygen, limited carbon,
nutrients and energy resources, and the prokaryote communities in
these systems are generally dependent on heterotrophy and lithoauto­
trophy (Goldscheider et al., 2006; Taubert et al., 2019). Prokaryotes are
involved in various biogeochemical cycling processes in groundwater
ecosystems, particularly playing an important role in carbon and
nutrient cycling in groundwaters (Griebler and Leuders, 2009).
Groundwater contamination not only affects the quality of groundwater
directly but causes changes in prokaryote communities, including loss of
(Hemme et al., 2015). The response of groundwater prokaryote com­
munities to contamination has been the focus of a number of studies (e.g.
hydrocarbons, (Grösbacher et al., 2016; Mouser et al., 2005; Stephenson
et al., 2013; Wright et al., 2017); uranium and metals, (He et al., 2018;
Hemme et al., 2010, 2015); general agricultural/industrial pollutants
(Korbel et al., 2013; Smith et al., 2012, 2018); and salinity (Sang et al.,
2018)). Changes in physico-chemical conditions result in changes in
community composition, e.g. Stephenson et al. (2013) and richness, e.g.
Hemme et al. (2015), as well as decreases in functional diversity, e.g. He
et al. (2018). Understanding the ecology of groundwater prokaryote
communities and the impacts of groundwater contamination on these
communities is critical for management and protection of all ground­
water values.

☆
This paper has been recommended for acceptance by Charles Wong.
* Corresponding author. Supervising Scientist Branch, Department of Agriculture, Water and the Environment, Darwin, Northern Territory, Australia.
E-mail addresses: lisa.chandler@environment.gov.au, lisa.chandler@awe.gov.au (L. Chandler).

https://doi.org/10.1016/j.envpol.2021.117318
Received 24 November 2020; Received in revised form 30 April 2021; Accepted 3 May 2021
Available online 12 May 2021
0269-7491/Crown Copyright © 2021 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
L. Chandler et al.
The discharge of saline mine-waters has significantly impacted the
quality of many surface- and groundwater ecosystems around the globe
(Lottermoser, 2010), resulting in the modification of hydrogeochemistry
that can impact on biological communities (Cañedo-Argüelles et al.,
2016). Saline mine-waters arise through the dissolution and mobi­
lisation of metals, non-metals and metalloids, and can, depending on the
solutes, range from acidic to alkaline (Nordstrom et al., 2015). Knowl­
edge of salinity effects on prokaryote communities in groundwater
(often measured as total dissolved solids (TDS) or electrical conductivity
(EC)), has mostly been derived from studies of communities along gra­
dients in coastal or estuarine systems, e.g. Héry et al. (2014) and Sang
et al. (2018). These studies observed changes in bacterial community
composition associated with changes in salinity due to seawater intru­
sion, for example Héry et al. (2014) noted increases in relative abun­
dance of Gammaproteobacteria associated with increased salinity.
However, sodium chloride (NaCl), the key constituent of seawater, is
rarely a major component of mine-waters (Nordstrom et al., 2015), and
input of higher salinity waters with different ionic composition can have
different effects on freshwater biota (Cañedo-Argüelles et al., 2016).
To date, little is known of the groundwater prokaryote community in
seasonally-flowing sandy creek channels that are common in the wet-dry
tropics. Moreover, there is little understanding of the community
response to salinisation and/or other associated contaminants of po­
tential concern (COPCs) from anthropogenic sources such as mine-
waters. The impacts of a uranium mine (Ranger) located in the Austra­
lian wet-dry tropics, on downstream surface water communities of
Magela Creek, have been well documented (Humphrey and Chandler,
2018; McCullough, 2006; Mooney et al., 2020; van Dam et al., 2010),
but the impacts on groundwater communities have not been studied.
Although the mine has ceased production (in January 2021), and the site
is undergoing rehabilitation (to 2026), the site will continue to be a
source of salts and other contaminants of potential concern (COPCs)
through surface water runoff and exfiltrating groundwater. The major
contaminants are magnesium (Mg2+) and sulfate (SO2− 4 ), derived from
the waste-rock landform and pit capping. The broad aim of this study
was to examine changes in the structure and function of prokaryote
communities in the shallow groundwater of Magela Creek associated
with inputs of contaminated mine-waters, with two specific objectives:
(1) to determine whether mine-water contaminants (specifically Mg2+
and SO2−4 ) altered the composition of the prokaryote community; and
(2) to assess the implications of any community composition changes on
ecosystem function. Prokaryote communities in the groundwater were
characterised using next generation sequencing technology. Commu­
nities were viewed in terms of taxonomic and functional composition,
and changes to assemblages were related to water quality associated
with mine-water discharge.
2. Methods
2.1. Site description and sampling
The study was conducted in a temporary sand-bed stream arising in
Arnhem Land, with the mid-lower reaches flowing through Kakadu
National Park (KNP), Australia (Supplementary Figure 1). The study
sites were located along a 7 km reach of Magela Creek in the Ranger
Project Area (RPA i.e. in a mine lease excluded from KNP). The tradi­
tional owners of land on which the study was conducted are the Mirrar
people and they rely on the downstream water resources for sustenance.
The region is characterised by a wet-dry tropical climate with clearly
defined summer wet and winter dry seasons, and a mean annual rainfall
of 1550 mm. The wet season prior to the 2018 sampling period had a
higher average mean rainfall (1919 mm) than the wet season preceding
the 2017 sampling period (1701 mm). During the dry season, surface-
flow in the stream ceases for several months (July–December) but per­
manent groundwater flows through sand channel throughout the year.
The sand channel has been filled since the Holocene with medium-
2
Environmental Pollution 284 (2021) 117318
coarse sands (Roberts, 1991). The average thickness of the sands (i.e.
depth to original stream bed) has been reported as 5–12 m in sections
(Ahmad et al., 1982; Nanson et al., 1993; Roberts, 1991). The sand
channel has been described as a strip, unconfined aquifer and has high
transmissivities, ranging between 400 and 800 m2 day− 1 (Ahmad and
Green, 1986). To access the groundwater during the dry season, PVC
piezometers (Enviroequip 50 mm, 1.5 m PVC Bore casings and screens
(Thermofisher Scientific)) were installed to a depth of approximately 2
m within the sand channel (Supplementary Figure 2). A series of pie­
zometers were installed both laterally and longitudinally along the creek
from approximately 3 km upstream of Ranger uranium mine to
approximately 4 km downstream (Supplementary Figure 1). A 300 m
longitudinal section of the shallow groundwaters in Magela Creek is
currently contaminated with mine-waters, most likely arising from
groundwater moving through contaminated sediments from the adja­
cent Coonjimba Billabong. This mine-water source typically contains
high concentrations of Mg2+, SO42− (the dominant contamination
signature in the mine-waters) along with elevated levels of Mn, U and
ammonia (Supplementary Table 1) (Harford et al., 2015; Hart et al.,
1992; Noller, 1991; Trenfield et al., 2019).
2.2. Sample collection
Samples of groundwater for water chemistry and molecular analysis
were collected monthly from 17 sites, during the dry season (August to
November), on 3 occasions in the first sampling year (2017) and 4 oc­
casions in the second year (2018) (Supplementary Table 2). A peristaltic
pump (Series II, Geotech Environmental Equipment, Inc), was used to
purge piezometers prior to sample collection by pumping at least three
casing volumes (Sundaram et al., 2009). In-situ measurements of
physico-chemical variables (Supplementary Table 3) were taken using a
multiparameter sonde and flow-through cell (YSI EXO1, Ohio USA).
Once purged, and the water quality parameter readings had stabilised,
samples for water chemistry and molecular analysis were collected.
Samples for water chemistry were collected in 1 L HDPE bottles,
ensuring that there was no head space in the bottle. Water samples, for
molecular analysis, were collected in sterile 2 L plastic bottles and both
were stored on ice until they were processed back in the laboratory.
2.3. Chemical analysis of groundwater samples
A portion of the 1 L water sample was filtered within 4 h of collection
using a 150 mL Nalgene™ Rapid-Flow™ Sterile Single Use Vacuum
Filter unit (0.45 μm), for analysis of filtered metals and major ions. The
remaining sample was decanted into separate bottles for analysis of
alkalinity, nutrients and dissolved organic carbon (Supplementary
Table 4). Samples were analysed were analysed for a suite of metals and
major ions (Supplementary Table 3) by inductively coupled plasma mass
spectrometry, inductively coupled plasma atomic emission spectrometry
and FIA methods, by a NATA-registered laboratory (EnviroLab, Sydney,
Australia). Total and dissolved organic carbon (TOC/DOC) samples
were analysed in-house using a high-temperature combustion method
5310B (TOC-VCSH, Shimadzu Scientific Instruments, Oceania Pty. Ltd,
SD < 0.1 mg L− 1, maximum CV of 2%).
2.4. DNA extraction and amplification
Water samples were filtered within 4 h of collection, through sterile
0.22 μm mixed cellulose membranes using vacuum filtration, 100 mL
increments were added until a total of 1000 mL had been filtered. The
filter membranes were placed in sterile petri dishes and stored at − 80 ◦ C
until DNA extraction. For DNA extraction, membranes were sliced into
thin strips, using a maximum of 75% of each membrane. The weight of
sediment and filter membrane was recorded, as the recommended 0.25
mg of sediment was generally not collected on the filters. Extractions
were performed using the Qiagen DNeasy Powersoil kit, following the
L. Chandler et al.
manufacturers protocol with the following modifications; increase in
time and intensity for bead beating. 30 min incubation period, 4 min
centrifuge period, and only 50 μL of the final C6 solution was used to
concentrate the final volume of DNA extracted.
Polymerase chain reaction (PCR) was conducted on all samples using
the primer set 515F (50–3’: GTGYCAGCMGCCGCGGTAA) (Parada et al.,
2016) and 806R (50–3’: GGACTACNVGGGTWTCTAAT) (Apprill et al.,
2015) for the V4 region of the 16S rRNA gene. Amplification used a
modified Earth Microbiome Project protocol v4.13 (Caporasa et al.,
2012). The sample (2 μL) was added to each reaction, with 15 μL of
AmpliTaq Gold 360 Master Mix (Applied Biosystems), 10 μL of ultrapure
water (UltraPure™ DNase/RNase-Free Distilled Water, Invitrogen) and
3 μL of a unique combination of forward and reverse tag primers (2 μM)
for total PCR reaction volume of 30 μL. A sample specific Golay barcode
was included in the 806R reverse primer. Amplification included both a
positive control (Supplementary Figure 3) and negative controls
(DNA-free water). Thermal cycling included a simplified hot start at
95 ◦ C and an initial denature cycle at 95 ◦ C for 10 min followed by
annealing for 35 cycles of 95 ◦ C for 30 s, 50 ◦ C for 30 s and 72 ◦ C for 1
min, followed by elongation at 72 ◦ C for 7 min and a final hold at 4 ◦ C.
Amplicon quality and quantity was determined by Quant-it Picogreen
assay (Thermofisher Scientific) and agarose gel electrophoresis. PCR
products of all samples were pooled in equimolar amounts before being
purified, using AMPure XP kit (Beckman Coulter, cat#A63880). The
final purified pool was sent to Ramaciotti Centre for Gene Function
Analysis (Sydney, Australia) for paired-end sequencing on the Illumina
MiSeq using the 250 bp v2.2 process.
2.5. Bioinformatics
A custom pipeline was used to process the amplicon sequence data
(Greenfield Hybrid Amplicon Pipeline, GHAP, v2.4) as detailed in Gissi
et al. (2019). Usearch v11.0.667 and RDP Classifier (v2.12) were used to
classify OTU sequences supplemented with a curated reference set
(RefSeq 16S, downloaded September 2019) for the purposes of
species-level classification. Complete with taxonomic classifications,
species assignments and counts for each sample were generated in OTU
tables.
After processing through the GHAP pipeline, and prior to statistical
analysis, data were further processed using the R library phyloseq
(McMurdie and Holmes, 2013). Rare OTUs that occurred in <2 samples
were excluded (10.2% of 37647), as were OTUs that were not classified
to a domain (0.19%), and OTUs that matched Eukaryote organelles (i.e.
chloroplasts). OTUs that had reads less than 0.01% of total number of
sequences were also removed (95.2%) to obtain a total of 1606 different
OTUs. Sequences were rarefied to the lowest common number of se­
quences (7766 sequences/sample) to minimise batch effect due to dif­
ferences in the numbers of total sequences present in the two sampling
years. All subsequent analyses, except the DESeq2 analysis, were based
on these rarefied sequences.
2.6. Functional analysis
Functional group prediction was performed using FAPROTAX (Louca
et al., 2016). Limitations to this approach include: 1) the FAPROTAX
database was constructed primarily to analyse functional profiles of
marine and lentic prokaryote communities; and (2) the database is
limited with the result that only a small percentage of OTUs in this study
could be assigned to at least one functional group. A heatmap illus­
trating the FAPROTAX was generated using the heatmap() function in R.
2.7. Statistical analyses
Samples were classified a priori according to position in the study
reach around a contamination plume (Supplementary Figure 1). Four
exposure groups were defined: all sites upstream of contamination
3
Environmental Pollution 284 (2021) 117318
plume (Upstream Reference), sites laterally adjacent to contamination
plume, in the central channel of the creek (Central Reference), sites
downstream of contamination plume (Downstream Reference), and sites
within the plume (Exposed).
Patterns amongst the environmental characteristics of the shallow
groundwater samples were examined with Principal Components
Analysis (PCA), based on Euclidean distances using Primer v7 (Clarke
and Gorley, 2015). Data were visualised using draftsman plots, and a
log-transformation applied if the data were strongly skewed, before
being normalised to account for different measurement scales.
Differences in prokaryote taxonomic and functional communities
and environmental variables were compared amongst exposure groups
using the Kruskal-Wallis test.
Multivariate statistical analyses were conducted on Hellinger trans­
formed OTU data, and square root transformed functional group data
using Primer v7 (Clarke and Gorley, 2015). Non-metric multidimen­
sional scaling (nMDS) was used to visualise the community assemblages
of Bray-Curtis resemblance matrices for both the OTU and functional
group datasets. PERMANOVA was used to test whether community
composition and function differed between year and a priori “exposure
groups”. Sample sites were included as a random factor nested in
exposure groups. Differences in data dispersion between exposure
groups, sites and years were tested with PermDISP and the association
between environmental variables and prokaryote community composi­
tion and function was determined using DISTLM and dbRDA. Highly
correlated environmental variables (r > 0.90) were removed from the
dataset prior to running DISTLM. DISTLM was used with stepwise se­
lection and Akaike information criterion (AIC). The significance level (α)
for all inferential tests was 0.05.
A negative binomial model applied to the non-rarefied OTU data
with the DESeq2 implementation in phyloseq (McMurdie and Holmes,
2014) was used to test for differences between exposure groups. P-values
were adjusted for multiple tests with the False Discovery Rate
Benjamini-Hochberg method.
3. Results
3.1. Overview of chemistry
The data set included exposed sites (n = 5) within a contamination
plume on the mine-lease area, as well as reference sites that were up­
stream (n = 6), downstream (n = 3) and in the central creek channel (n
= 3) adjacent to the exposed sites (Supplementary Figure 1). The natural
shallow groundwater, observed at the reference sites during the dry
season, was generally acidic (pH 4.8–6.4), with low electrical conduc­
tivity (EC < 50 μS cm− 1), alkalinity (as HCO−3 <10 mg L− 1) and con­
centrations of major ions and metals (Supplementary Figure 4). Mine-
water exposed sites had significantly higher concentrations (Kruskal-
Wallis, P < 0.001) of known mine-water contaminants, SO2− 2+
4 , Mg , and
Mn, significantly higher concentrations of Na+, Cl− and Fe (Supple­
mentary Figure 4) and lower concentrations of Ca2+. The exposed sites
also had generally negative redox values (cf. generally positive redox
measures for reference sites), and a slightly lower range of pH
(4.54–5.93) compared to reference sites. All sites, reference and
exposed, had low concentrations of dissolved oxygen (<1 mg L− 1),
indicating suboxic or anoxic conditions, and were high in temperature
(27–32 ◦ C). The sites were typically low in nitrogen with NO−2 and NO−3
concentrations generally below the detection limit (<0.005 mg L− 1).
Dissolved organic carbon (DOC) was highly variable (0.84–998 mg L− 1),
with a median value of 15 mg L− 1.
Principal Components Analysis (PCA) showed distinct separation of
the environmental characteristics of exposed sites and reference sites
(Fig. 1). The first PCA axis explained 26.4% of the variation in site
environmental variables, and the second axis explained 16%. Separation
of samples along the first axis was positively correlated with Mg2+ and
EC, along with Mn (Pearson correlation coefficient, r > 0.80). The
L. Chandler et al.
second axis separated samples, based on Ca2+ (r > 0.7). Mg concentra­
tion and EC have a strong linear relationship.
A number of chemical variables had lower concentrations in 2018
compared to 2017 (Supplementary Figure 4) presumably due to the
slightly higher antecedent rainfall in 2018 and hence dilution available
from higher recessional flows. However, these differences were not
marked and the PCA is notable for the interspersion of 2017 and 2018
data points within each of the exposure groups indicating relatively low
interannual variability in water quality.
3.2. Taxonomic composition
In the final dataset, 1606 unique OTUs were identified across 17
sites, with Bacteria representing 83% (1332 OTUs) and Archaea 17%
(274 OTUs). OTUs were further assigned to lower taxonomic ranks and
relative abundance for each rank was estimated across different groups.
Across all the sites a total of 31 prokaryote phyla were assigned, con­
sisting of 66 classes, 107 orders, 165 families and 255 genera. Of the 255
genera groups 43% were not classified into a known genus. A large
portion of the reads (~27%) were unclassified at phylum level (24.9%
for Bacteria and 2.5% for Archaea). Excluding the unclassified Bacteria,
the dominant phyla groups across the study were Proteobacteria
(35.6%), Acidobacteria (5.4%), Thaumarchaeota (5%), Firmicutes
(4.9%), Euryarchaeota (4.5%) and Crenarchaeota (4.2%) accounting for
approximately 60% of the total reads.
The Bacteria were represented by 25 known phyla groups, and the
predominant groups included Proteobacteria (43.9%), unclassified
Bacteria phyla (30.7%), Acidobacteria (6.6%) and Firmicutes (6.1%).
Proteobacteria had the highest diversity at genus level (117 assigned
genera in total), with Methylomonas having highest relative abundance
(11%). Acidobacteria were represented by 13 classes with Geothrix the
most abundant genus (27%). The phylum Firmicutes had OTUs divided
between 4 classes, with Clostridium sensu stricto the most abundant genus
(25%).
Five archaeal phyla were represented, with most OTUs within
Archaea assigned to the Thaumarchaeota (9%), Euryarchaeota (29%)
and Crenarchaeota (16%). Nitrososphaera was the dominant genus
(99%) within Thaumarchaeota. Euryarchaeota was the most diverse
4
Environmental Pollution 284 (2021) 117318
Fig. 1. Principal components analysis for samples
based on selected environmental data. To aid visual
interpretation, only vectors with correlations >0.5 are
displayed. Metals and major ions were log trans­
formed. DPW = distance to permanent surface water;
SWL = standing water level. Sites were classified a
priori based on contamination concentrations and the
location of the site within the study reach around the
contaminated (exposed) area (i.e. upstream of
contamination, downstream of contamination, and
adjacent to in the central channel where no contami­
nation was observed).
archaeal phyla with 12 genera across 4 classes. Most OTUs with Eur­
yarchaeota were assigned to the genus Methanobacterium (37%).
The mine-water exposed sites had a significantly higher proportion
of unclassified Bacteria (Fig. 2), compared to reference sites. Methyl­
ococcaceae, as well as other methylotrophic families were absent (or
had < 1% relative abundance) at the exposed sites, as was the Meth­
anobacteriaceae and Clostridiaceae 1 families. Helicobacteraceae were
absent or had <1% relative abundance at the upstream reference sites
but were present at both the downstream and central reference sites and
the exposed sites.
3.3. Community composition
Ordination plots of the prokaryote communities showed a distinct
separation of communities between the exposed and the reference site
groups (Fig. 3 A), with PERMANOVA showing significant differences (p
< 0.01) amongst the exposure groups, sampling years and sites (Sup­
plementary Table 5). Exposure groups, site and the residual were the
largest contributors to variation, and year, although statistically signif­
icant (p = 0.01) was less important than the other factors (Supplemen­
tary Table 5). Pairwise comparisons for exposure groups, indicated that
the exposed sites were significantly different from each reference group,
and reference groups did not significantly differ from each other. Data
dispersion (PermDISP p > 0.05) did not significantly differ between
exposure groups or years (Supplementary Table 5), suggesting that the
results were not influenced by differences in heterogeneity between
these groups. However, data dispersion differed significantly amongst
sites (PermDISP p = 0.016), indicating the PERMANOVA results were
also influenced by differences in dispersion (and Supplementary
Table 5), indicating spatial heterogeneity, which is also shown in a
cluster analysis where samples from each site tended to cluster together
(Supplementary Figure 6).
Examination of the association between community structure and
environmental variables showed the same strong separation between
the exposed and reference groups (Fig. 3 B). Samples from the exposed
group with higher contaminant concentrations were separated along the
first axis (to the right). Na+, SO2−
4 and Mg
2+
concentrations accounted
for the largest portion of variation in the community structure, (12.4,
L. Chandler et al. Environmental Pollution 284 (2021) 117318

Fig. 2. The relative abundances (%) of dominant prokaryote families in the different exposure groups. Taxa that had <1% of total community abundance were
grouped into “Others”.

11.8 and 11.2% of the variation, respectively, Supplementary Table 6). response of individual sites between years (Site(Exp) x Year p = 0.001).
Differential abundance analysis indicated OTUs differed significantly Pairwise tests examining the exposure groups showed that each refer­
(adjusted p < 0.01) in relative abundance between exposed and refer­ ence group differed significantly from the exposed group. Examination
ence sites (Fig. 4). The reference sites had significantly more OTUs (and of associations between functional groups and environmental factors
in greater relative abundance) assigned to an unclassified Betaproteo­ (Fig. 6 B) indicated that Na+ and SO2−4 explained most (15 and 13%,
bacteria family group, Methanobacteriaceae, Methylococcaceae and respectively) of the variation observed in the functional composition,
Methylocystaceae compared to the exposed sites. Whereas the exposed followed by Mg2+ (10%) and Cl− (9%) (Supplementary Table 9).
sites had significantly more OTUs, and in greater relative abundance,
associated with Nitrososphaeraceae, an unclassified Thermoproteales 4. Discussion
family group, Rhodocyclaceae and an unclassified Campylobacterales
family group (Fig. 4). We found changes in groundwater prokaryote community assem­
blages and functional groups strongly correlated with contamination
arising from saline expressions of mine waste waters.
3.4. Prokaryote functional annotation
Samples from the exposed sites had significantly elevated concen­
trations of Mg2+, SO2− 4 , Na , Cl , Fe and Mn, compared to concentra­
+ −
FAPROTAX assigned 479 out 1606 bacterial OTUs (29.8%) to at least
tions in reference sites, adjacent to and upstream and downstream of the
one predicted functional group. A total of sixty-nine functional groups
contaminated area. Elevated concentrations of Mg2+ and SO2− 4 are
were identified and most groups were represented by more than one
indicative of mine-derived water contaminants. However, while Mn is
OTU (Supplementary Table 7). Chemoheterotrophy was the most
elevated in some mine-waters, it also occurs naturally in groundwater
dominant function across all samples (17.7% of total OTUs). OTUs
and samples from one reference site (MCP03) had consistently elevated
assigned to chemoheterotrophy were also associated with other pro­
concentrations of Mn (340–410 μg L− 1). In the reference sites, suboxic
cesses, such as methylotrophy, hydrocarbon degradation, and aromatic
redox conditions and low pH may have favoured elevated concentra­
compound degradation. Exposed sites had significantly (Kruskal-Wallis,
tions of free Mn2+, but there may have also been microbially mediated
p < 0.001) lower relative abundance of OTUs associated with chemo­
dissolution from the sands. The exposed sites also had high concentra­
heterotrophy, hydrocarbon degradation, methylotrophy and methano­
tions of Fe (predominantly as Fe2+, unpublished data), a contaminant
trophy, compared to reference sites (Fig. 5), and significantly (Kruskal-
not associated with mine-waters, and we observed precipitation of iron
Wallis, p < 0.001) higher representation of groups involved with nitri­
oxyhydroxides within the sand channel around these exposed sites,
fication, ureolysis (Fig. 5) and reduction of sulfur compounds along with
indicating activity of iron oxidising bacteria. Therefore, a combination
groups involved in the sulfur cycle (i.e. reduction of sulfur compounds).
of input from mine-waters and the influence of local microbial activity
Multivariate analysis of the functional data separated the exposed
and redox conditions may have created the elevated concentrations of
sites from reference sites (Fig. 6 A), although not as strongly as observed
Mn and Fe at the exposed sites.
for the community composition data. PERMANOVA showed significant
Increases in Mg2+ concentration greater than 3 mg L− 1 have been
difference among the exposure groups, sites and between sampling years
associated with toxic effects on local tropical surface water species
(Supplementary Table 8). Exposure groups, site and the residual
(Hogan et al., 2013; Prouse et al., 2015; van Dam et al., 2010). While
contributed the largest components of variation in the analysis (Sup­
Mg2+ is strongly associated with SO2− 2−
4 in mine-waters, the SO4 ion has
plementary Table 8). Patterns among exposure groups did not vary be­
been shown to have low toxicity to surface water organisms (van Dam
tween years (Exp x Year, p = 0.49) but there was variability in the

5
L. Chandler et al.
Fig. 3. nMDS ordination (A) of the prokaryote communities. Dashed line circles represent significant clusters identified by a SIMPROF test for 45% similarity. dbRDA
ordination (B) showing relationship between prokaryote communities and environmental variables. Vector overlay of environmental variables identified as
significantly correlated using a stepwise DISTLM with AIC.
et al., 2010). The sensitivity of Mg2+ has been attributed to the
extremely soft waters of Magela Creek (van Dam et al., 2010). The high
toxicity and high mobility of Mg2+ places this major ion as the toxicant
of most concern in mine-waters released, or dispersed, to the Magela
Creek system. However, elevated concentrations of SO2− 4 were more
likely to be influencing the groundwater prokaryote communities in this
system than Mg2+ concentrations, although the two major ions were
highly correlated.
Increasing solutes such as Mg2+ and SO2−4 can have diverse mecha­
nisms of action on prokaryote organisms, which will be dependent on
the concentration. Increased levels of Mg2+ have been reported to
decrease survival of Staphylococcus aureus cells by disrupting membrane
function (Xie and Yang, 2016), as well as decreasing adherence of bac­
teria to surfaces, impairing biofilm assembly (Demishtein et al., 2019),
which can decrease resistance to external stressors. However, the
6
Environmental Pollution 284 (2021) 117318
concentrations at which these effects occurred were markedly higher
than observed in our samples, indicating direct toxicity from Mg2+ isn’t
likely to be occurring in these waters. SO2−4 is used by a number of
prokaryotes as an energy source, through electron donation, and
increasing SO2−
4 has been linked to changes in prokaryote community
structure and function (discussed further below). While specific mech­
anisms of influence of increased concentrations of Mg2+ and SO2−4 were
not examined in this study, we hypothesise that the most likely mech­
anism for community changes in the shallow groundwater of Magela
Creek is through preferential growth of species responding to sulfate,
which probably leads to complex biogeochemical processes with other
species in synergistic relationships.
Elevated SO2−
4 concentrations have been correlated with changes in
prokaryote community structure in field (Flynn et al., 2012, 2013) and
laboratory (Bethke et al., 2008; Raskin et al., 1996) studies, and our
L. Chandler et al.
Fig. 4. The 26 most abundant prokaryote families showing a significant differential abundance between the reference and exposed sites, based on the mean
abundance change in log2fold (p < 0.01). The log2fold change has been adjusted for multiple tests and it is based on a negative binomial model using DESeq2. Circle
size reflects the mean base abundance of OTUs aggregated to family-level and the grey scale indicates the number of different OTUS within the corresponding family.
results concur with these findings. OTUs associated with sulfate
-reducing bacteria (SRB) (i.e. Desulfovibrionaceae, Desulfobulbaceae
and Desulfobacteraceae) were more prevalent in the exposed sites
(Supplementary Figure 7), however they represented only a small per­
centage (<0.5%) of the overall relative read abundance in the samples.
Flynn et al. (2013) observed that SRB were 4x more abundant in as­
semblages attached to sediments within an aquifer, compared to as­
semblages suspended in the groundwater fraction. In this study, sulfate
reduction was obviously occurring based on observations of strong H2S
odour associated with the exposed sites, but organisms associated with
the process were not in high abundances and thus were not highlighted
as major discriminators in analyses between the exposed and reference
sites. However, this study only examined filtered groundwater samples
and other attached biota may have been involved in the reduction of
sulfur compounds. Future studies aimed at establishing the contribution
of attached microbes in these processes would be valuable.
Samples from our exposed sites had reduced relative abundances of
OTUs assigned to taxa associated with methanogenesis (e.g. Meth­
anobacteriaceae). Flynn et al. (2013) suggested that the relatively low
abundance of methanogens they observed in wells with greater than
0.03 mM of SO2− − 1
4 (~3 mg L ) could be explained by methanogens being
unable to respire quickly enough to maintain a viable population in the
presence of active SO2−
4 reduction. Raskin et al. (1996) indicated addi­
tion of 30 mg L− 1 of SO2− 4 resulted in decreased concentrations of
methanogens (from 25% initial concentration to 8%) and these low
concentrations persisted for at least a year. This could explain why, even
with measured SO2−4 concentrations decreasing in the second year of our
study, these groups were still absent, or in very low abundances at sites
within the exposed group.
OTUs assigned to taxa involved with oxidation of methane (i.e.
methanotrophs) were also poorly represented in samples from the
exposed sites, particularly taxa in the Methylococcaceae family. These
taxa were associated with several putative functional groups including
7
Environmental Pollution 284 (2021) 117318
chemoheterotrophy and those involved with methane oxidation (i.e.
methanotrophy) and oxidation of other carbon compounds (i.e. meth­
ylotrophy), and these functions were also lower at the exposed site.
Redox potential is one of the most important controls of methane
oxidation (Chowdhury and Dick, 2013), therefore the exposed sites
having generally negative Eh (ORP) values) would not favour growth of
methanotrophs.
In addition to Mg2+ and SO2−4 , we identified Na as a possible driver,
+
influencing the differences observed between exposed and reference
prokaryote communities and functions. Na+ is highly correlated with
Mg2+ and SO2− 4 (r > 0.80), and elevated concentrations of Na in the
+
shallow groundwater of the exposed sites in Magela Creek appeared to
be mine-water related, as concentrations were higher than reference
sites. However, Na+ is not elevated in surface-water discharges from the
mine and has not been identified as a Contaminant of Potential Concern
for the Ranger mine surface water monitoring program. Hence, there has
been limited site-specific toxicity testing and it is unknown at what
concentration Na+ may result in adverse effects to local surface water
organisms. If elevated concentrations of Na+ were discharged to the
surface-waters of the Magela Creek, it is possible the ecosystem may be
sensitive to the additions due the extremely soft waters of the stream (i.
e. <5 mg L− 1 CaCO3). There is also limited information on the influence
of Na+ on prokaryote communities, more broadly. Two studies that have
focused on the effect of Na+ (as NaCl) (i.e. (Baldwin et al., 2006;
Beyer-Robson, 2014) on sediment prokaryote communities in fresh­
water ecosystems applied NaCl experimentally at up to 100 times higher
concentrations than those observed in the Magela Creek shallow
groundwaters. Baldwin et al. (2006) observed shifts in community
structure in wetland sediment bacterial communities only at the highest
concentration of NaCl (equivalent to approximately 2352 mg L− 1 Na+).
However, they observed significant changes with increasing NaCl con­
centrations in the archaeal communities and noted decreased produc­
tion of methane even at low Na+ levels (57 mg L− 1). The decreased
L. Chandler et al. Environmental Pollution 284 (2021) 117318

Fig. 5. A heatmap showing distribution of the top 35 functional groups predicted by FAPROTAX. Sites are grouped by exposure, with reference group sites ordered
from the most upstream site (MCP02) to most downstream (MCP15) site. Exposed group sites are ordered by mean concentration of SO42-, with MCP11 having the
lowest concentration and MCP08 the highest. The square root of the relative abundance of each functional group is represented by colour intensity according to the
legend. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

production of methane was attributed to inhibition of acetoclastic
methanogenesis, a process undertaken by archaeal genera Meth­
anosarcina and Methanothrix. In our study we noted these taxa had
generally reduced relative abundance at the exposed sites compared to
reference sites but they were in low abundances (<1%) across all sam­
ples, indicating that acetoclastic methanogenesis was not a dominant
process in the shallow groundwater ecosystem of Magela Creek, and any
responses to Na+ are presumably associated with other taxa and pro­
cesses. Increased salinity (associated with NaCl) has also been impli­
cated in reduction of nitrification activity (i.e. (Nelson et al., 2016) but
at concentrations of Na+ much higher than those measured in our
samples. In laboratory studies, using saturated sandy sediments from
ephemeral creeks in north Queensland, Australia, Beyer-Robson (2014),
noted reduced potential rates of nitrification processes with salinities of
10000 μS cm− 1 (NaCl concentration not reported). In our study, we
observed the opposite, with taxa associated with inferred nitrification (i.
e. Nitrososphaera sp.) having higher relative abundance in the exposed
sites (mean of 150 μS cm− 1) compared to the reference sites. It could also
be that the high correlations amongst the major ions in our samples are
confounding results and reflect a statistical, rather than a true causal
effect. Nevertheless, Na+ and Mg2+ are present in equivalent concen­
trations in our samples and could be exerting effects independently,
working together or could be antagonising any effects observed on the
prokaryote communities. The roles of different ions are complex, and it
would require further studies to tease out the effects of elevated con­
centrations of the different ions on the prokaryote communities in these
shallow groundwater systems.
Sampling location also had a strong influence on community as­
semblages, and to a lesser extent functional groups, indicating there was
considerable spatial heterogeneity in the prokaryote community struc­
ture within the shallow groundwater of the sand channel that was not
necessarily associated with water chemistry. The study sites were spread
over a 7 km reach of the creek channel, with minimum distance of 10 m
between the closest sites (Supplementary Figure 1). Although the sand
channel comprises medium-coarse sands (Roberts, 1991) with similarly
high hydraulic conductivities along the reach (unpublished data), there
is likely to be localised physical differences at each site associated with
factors such as differing porosity, and organic content including buried
leaf litter and riparian plant roots. Brad et al. (2008) observed location
specific grouping of bacteria communities in sediment samples from an
aquifer impacted by landfill leachate, even over sampling depth dis­
tances of 1 m. Lin et al. (2012) noted spatial heterogeneities, both
laterally and vertically in prokaryote communities collected from bores
approximately 30 m apart in an unconfined aquifer, and hypothesised
the variation was associated with habitat heterogeneities such as voids
between gravelly cobble within the aquifer, the study also observed a
strong temporal variation governed by fluctuations in groundwater
elevation which influenced both geophysical and hydrochemical
variables.
While PERMANOVA showed a small but significant contribution of
sampling year to variation in the prokaryote community composition,
these year-to-year differences were not marked in ordination space
(Fig. 3). The ordination depicted a general pattern of interspersion of
2017 and 2018 data points within each of the exposure groups, indi­
cating relatively low interannual variability in community composition.
The interannual variability in communities, may be due to changes in
water chemistry with differences particularly evident in sites within the
contamination plume. These exposed group sites had mean

8
L. Chandler et al. Environmental Pollution 284 (2021) 117318

Fig. 6. nMDS (A) and dbRDA (B) ordinations of the predicted functional groups. Dashed line circles represent significant clusters identified by a SIMPROF test for
80% similarity. Vector overlays on the on the nMDS (A) are predicted functional groups with >0.5 correlation, and similar functional processes have been grouped to
primary function (indicated by bold font) for clarity. dbRDA has a vector overlay of the environmental variables identified as significantly correlated using a stepwise
DISTLM with AIC.

concentrations of contaminants such as Mg2+ and SO2− 4 at least 50%
lower in the second year of the study. This difference was likely due to
the greater rainfall and longer duration of the wet season that preceded
the first sampling, which led to larger and longer surface flow, and
would have contributed to longitudinal dilution of the contaminant
plume. Alternatively, there was a considerable difference in the read
depth of samples between the two years (Supplementary Figure 5),
which, although rarefaction was done to minimise this batch effect, may
have also contributed to the statistical difference between years.
There was considerable taxonomic variability within the putative
functional groups, and a number of OTUs were assigned to four or more
different functional groups indicating a functional redundancy within

9
L. Chandler et al.
these shallow ground water communities. This might explain why the
separation observed between exposure groups was not as strong in the
functional data compared to the community composition data. How­
ever, much of the diversity that was observed in the prokaryote com­
munity ordinations was associated with OTUs that were unclassified
beyond phyla level, and FAPROTAX ignored OTUs that were not iden­
tified at species or genus level because many functions are only
conserved at this level. In studies of prokaryote communities in sediment
samples from floodplains (Nelson et al., 2016) and a billabong (Sutcliffe
et al., 2017) within Kakadu National Park large proportions of unclas­
sified sequences were also reported, and it was suggested that these
sequences may be unique to tropical systems. Sutcliffe et al. (2017)
compared shotgun metagenomics with 16S rRNA amplicon data and
observed that shotgun metagenomics identified a greater number of
genera groupings than the 16S amplicon data. Future studies examining
groundwater communities in these understudied regions would benefit
from undertaking whole metagenome sequencing.
Given this is the first report, to our knowledge, examining ground­
water prokaryote communities in the region, it provides a baseline
characterisation of these communities for sand channel streams, as well
as empirical evidence that mine-waters with elevated levels of Mg2+ and
SO42− changed important biogeochemical processes such as (increased)
nitrification and (decreased) methane oxidation, albeit in a localised
area. Groundwater prokaryotes are not widely used in assessment of
mining impacts, despite having a direct link to changes in biogeo­
chemistry, and this study shows they can provide a useful line of evi­
dence to inform whole of mine-water management.
5. Conclusion
Despite a considerable history of work investigating impacts of
mining in the Alligator Rivers Region, NT, this is the first study of re­
sponses of groundwater prokaryotes in the region to mine-water
contamination. The study demonstrated clear differences in prokary­
ote community structure and function in response to a contamination
plume on the Ranger Project Area. The contamination was associated
primarily with Mg2+ and SO2− 4 , although Na was identified as the
+
variable most strongly correlated with prokaryote structure and func­
tion. This knowledge assists in the assessment of potential mining im­
pacts on groundwater ecosystems and will aid ongoing mine-water
management.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We pay our respects to all Traditional Owners of Kakadu National
Park where we conducted this study and acknowledge Elders past,
present and emerging. This study was funded by the Supervising Sci­
entist Branch (SSB) of the Department of Agriculture, Water and the
Environment and through an Australian Government Research Training
Program Scholarship awarded to Lisa Chandler. We would like to thank
Andrew Jansen, Julie Hanley, Costi Buccella, Ian Douglas, Matt Piamnok
and Sam Walker (SSB) for assistance in the field and collection of sam­
ples. We are grateful to Brodie Sutcliffe for her support and guidance
with the DNA extraction and amplification process, along with other
members of the Macquarie University EGEEL Laboratory. We’d also like
to thank Mirjam Kaestli (Charles Darwin University) for advice with
statistical analysis. The authors declare that they have no conflicts of
interest.
10
Environmental Pollution 284 (2021) 117318
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envpol.2021.117318.
Author contribution
Lisa Chandler: Conceptualization, Methodology, Project adminis­
tration, Formal analysis, Investigation, Visualisation, Writing - Original.
Andrew J Harford: Supervision, Conceptualization, Methodology, Proj­
ect administration, Writing -Reviewing and Editing. Grant C Hose: Su­
pervision, Conceptualization, Methodology, Writing -Reviewing and
Editing. Chris L Humphrey: Supervision, Conceptualization, Methodol­
ogy, Writing -Reviewing and Editing. Anthony Chariton: Resources,
Methodology, Writing -Reviewing and Editing. Paul Greenfield: Soft­
ware, Formal analysis, Writing -Reviewing and Editing. Jenny Davis:
Supervision, Conceptualization, Methodology, Writing -Reviewing and
Editing.
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