Accumulation in trace metals in freshwater macroinvertebrates across metal contamination gradients

metal contamination gradients 2
Amanda Arnolda, John F. Murphya, James L. Prettya, Charles P. Duerdotha, Brian D. Smithb, 3
Philip S. Rainbowb, Kate L. Spencerc, Adrian L. Collinsd and J. Iwan Jonesa* 4
Affiliations and email: 5
a School of Biological & Chemical Sciences, Queen Mary University of London, London, E1 6
4NS, UK. [email protected], [email protected], [email protected] 7
b Department of Zoology, Natural History Museum, Cromwell Road, London, SW7 5BD, UK. 8
[email protected], [email protected] 9
c School of Geography, Queen Mary University of London, London, E1 4NS, UK. 10
[email protected] 11
Devon, EX20 2SB, UK. [email protected] 13
*Corresponding author: [email protected] (J. Iwan Jones) 14
15
freshwater ecosystems. However, measuring trace metal bioavailability has proven difficult, 19
because it depends on many factors, not least concentrations in water, sediment and 20
habitat. Simple tools are needed to assess bioavailabilities. The use of biomonitors has been 21
widely advocated to provide a realistic measure. To date there have been few attempts to 22
identify ubiquitous patterns of trace metal accumulation within and between freshwater 23
biomonitors at geographical scales relevant to trace metal contamination. Here we address 24
this through a nationwide collection of freshwater biomonitors (species of Gammarus, 25
Leuctra, Baetis, Rhyacophila, Hydropsyche) from 99 English and Welsh stream sites 26
spanning a gradient of high to low trace metal loading. The study tested for inter-biomonitor 27
variation in trace metal body burden, and for congruence amongst accumulations of trace 28
metals within taxa and between taxa across the gradient. In general, significant differences 29
in trace metal body burden occurred between taxa: Gammarus sp. was the most different 30
compared with insect biomonitors. Bivariate relationships between trace metals within 31
biomonitors reflected trace metal profiles in the environment. Strong correlations between 32
some trace metals suggested accumulation was also influenced by physiological pathways. 33
Bivariate relationships between insect biomonitors for body burdens of As, Cu, Mn and Pb 34
were highly consistent. Our data show that irrespective of taxonomic or ecological 35
differences, there is a commonality of response amongst insect taxa, indicating one or more 36
could provide consistent measures of trace metal bioavailability. 37
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contamination gradient. 42
1. Introduction 43
Metals are naturally present in freshwater ecosystems and many are essential to the health 44
and survival of living organisms in trace quantities. However, increased use of metals by 45
humans has significantly altered the distribution and availability of trace metals, often 46
resulting in severe impacts on environmental quality. In the UK, long-term legacy effects 47
from past mining activities have resulted in elevated release of trace metals into aquatic and 48
terrestrial environments (Alloway, 2003; Rainbow, 2018). Consequently, 8% of streams and 49
rivers in England and Wales suffer from chronic trace metal contamination, thus failing to 50
reach water quality targets (Environment Agency, 2012). Similar impacts are of concern in 51
metal mining areas across the globe (Kiffney and Clements, 1993; Luoma and Rainbow, 52
2008; Iwasaki and Ormerod, 2012). Importantly, trace metal impacts on local biota are 53
attributable to only certain chemical forms of total trace metal present in the environment, 54
termed the bioavailable fraction. Bioavailability can be defined as a relative measure of that 55
fraction of the total ambient metal an organism actually takes up when encountering or 56
processing environmental media, summated across all possible sources of contaminant, 57
including solution and diet as appropriate (Rainbow, 2018). 58
At any given location, the bioavailable fraction of one or more trace metals is difficult to 59
predict but is initially determined by geological input, and then modified by physical and 60
chemical attributes of the local environment. Typically, dissolved bioavailable concentrations 61
of trace metals are predicted using dissolved metal speciation models such as WHAM 62
(Tipping, 1994; Tipping et al., 1998), WHAM-FTOX (Stockdale et al., 2010) and the BLM 63
(Paquin et al., 2002). However, and crucially, these models are based on the chemical 64
speciation of trace metals in solution, and do not capture the significant role of trace metal 65
uptake from fine-grained sediments by biota particularly obtained through diet (Adams et al., 66
2011; Luoma and Rainbow, 2008; Rainbow, 2018). The development and testing of tools 67
that can capture dietary trace metal uptake which provide a realistic measure of 68
bioavailability is still needed (Jones et al., 2020). Use of aquatic invertebrates as biomonitors 69
is frequently advocated because they accumulate trace metals in their tissues. Accumulated 70
concentration provides a relative measure of the total amount of trace metal taken up by all 71
routes over a preceding time period (Luoma and Rainbow, 2008) which corresponds to local 72
environmental bioavailability integrated over space and time (Rainbow et al., 2011). In this 73
respect, quantifying the trace metal content of aquatic invertebrates offers promise as a 74
robust and simple means of determining trace metal bioavailability (Cain et al., 1998; Jones 75
et al., 2020; Luoma and Rainbow, 2008). 76
Thus far, exploration of biomonitor trace metal accumulation patterns have been limited to 77
relatively few, localised geographical areas and river catchments (Costas et al., 2018; De 78
Jonge et al., 2013; Kiffney and Clements, 1993; Luoma et al., 2010; Fialkowski et al., 2012; 79
Rodriguez et al., 2018). However, scale of observation can have a profound impact on the 80
description and interpretation of measured patterns (Levin, 1992). Covering a wide range of 81
trace metals and bioavailabilities, from a broad geographical area overcomes these 82
limitations of scale, and enables the exploration and identification of general patterns of trace 83
metal bioaccumulation in biota. 84
Potential exposure routes, although including dissolved trace metals, occur primarily through 85
contact and/or ingestion of inorganic and organic matter, associated biofilm, and prey 86
exposed to such material (Cain et al., 2011; Croteau and Luoma, 2009; Croteau et al., 2011; 87
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Custer et al., 2016; Hare, 1992; Smock, 1983b; Sola and Prat, 2006). The strength of 88
association of organic and inorganic substrates with trace metals will influence subsequent 89
supply to biota, and to subsequent consumers. Consequently, trace metal exposure will vary 90
depending on an organism’s ecological interaction with its environment. Interaction extent 91
will be determined by habitat preferences (interstitial vs surface dwelling), feeding style 92
(shredder, collector-gatherer, filter feeder, sediment-ingestor, predator) and dietary 93
preferences (seston, detritus, biofilm, sediment and prey). Hence, trace metal accumulation 94
patterns will tend to differ depending on local environment, trace metal and invertebrate 95
species, even amongst closely related taxa (Luoma and Rainbow, 2008). 96
Aquatic invertebrates are typically net accumulators of metals, with uptake occurring at the 97
rate of environmental bioavailability (Jones et al., 2020). Strong metal accumulators show a 98
wider range of accumulated trace metal concentrations than weak accumulators over the 99
same trace metal bioavailability range (Luoma and Rainbow, 2008). Irrespective of 100
accumulated variation, when biomonitors are exposed to locally high or low trace metal 101
bioavailabilities, concurrent increases or decreases in relative patterns of trace metal 102
accumulation are expected (Rainbow, 2018). 103
Invertebrate species are not uniformly distributed and may be absent from a site because 104
conditions other than metals limit establishment. Therefore, reliance on a single biomonitor 105
to assess bioavailability limits the number of sites that can be compared. Indeed, relying on 106
one biomonitor may also result in the under- or over-representation of relative measures of 107
metal bioavailability, as different species may be weak or strong trace metal accumulators 108
(Rainbow, 2018). 109
A suite of biomonitors accumulating trace metals from a variety of sources can be utilised to 110
increase geographical coverage to include a wider range of trace metals and concentrations. 111
This approach facilitates the comparison of biomonitor taxa with overlapping distributions, 112
uptake routes for trace metals and patterns of trace metal accumulation. To establish how 113
comparable different biomonitors are in terms of accumulated trace metal concentration, we 114
considered differences in body burdens among taxa or trace metals could be due to: 115
a) differences associated with physiology and/or ecology 116
b) differences in the bioavailability of metals (affected by geology (metal ore), mining 117 activity and local conditions (hydrology, pH, FPOM, carbonate content)) 118
To establish if differences in body burdens among taxa or trace metals occur due to 119
physiology and/or ecology, it is essential to initially consider differences in the environmental 120
bioavailabilities of trace metals. The co-occurrence of trace metals in ores and gangue 121
material is well documented. For example, Cu ores are associated with As, whilst Pb ores 122
are associated with Ag, Zn and/or Cd (Alloway, 2003). If trace metal uptake occurs at the 123
level of environmental bioavailability, internal associations of trace metals in biomonitors 124
should correspond with geological associations as determined by mined material. Trace 125
metal uptake may, however, be moderated by physiology/ecology and consequently patterns 126
of trace metal accumulation may correspond with species-specific responses to trace metal 127
bioavailability. 128
The aim of this field study was to compare accumulated trace metal concentrations in a suite 129
of biomonitor taxa that varied in their exposure to trace metal contamination. All taxa 130
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collected were widespread and cosmopolitan genera (species of Gammarus, Leuctra, 131
Baetis, Rhyacophila, Hydropsyche) and although utilised in other studies as biomonitors, 132
have never been compared concomitantly. We measured the body burden of 11 trace 133
metals (Ag, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V, Zn) and 1 metalloid (As) - for brevity all are 134
subsequently referred to as trace metals. This enabled the following objectives to be met. 135
Firstly, body burdens of trace metals were assessed in each of the five biomonitor taxa 136
across the contamination gradient. Secondly, relationships among trace metals within a 137
species were compared to identify if biomonitor metal body burdens reflect geological 138
associations commonly co-occurring in ores and gangue material. Finally, relationships 139
among species for a given trace metal were tested to compare the consistency of change in 140
metal body burdens amongst biomonitors. Through these objectives we were able to 141
address our hypothesis: patterns of trace metal accumulation are consistent for different 142
biomonitors across the metal contamination gradient. 143
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Twenty spatially independent, replicate river catchments were identified across England and 146
Wales in areas affected by abandoned metal mining facilities such that mining is the 147
dominant stressor on the aquatic system (Figure 1). Details of the mining facilities 148
associated with each catchment are given in Table S1. Within each catchment, five 149
monitoring sites were selected; one upstream of the mining facility, one on an adjacent but 150
un-impacted watercourse of comparable geology, one immediately downstream of the 151
mining facility and two other sites successively further downstream on the same impacted 152
watercourse. This design ensured a gradient of trace metal pollution, from natural 153
background levels to extreme contamination, was evenly captured. At one catchment in 154
south west England it was not possible to sample upstream of the mining facility: in total, 99 155
sites were sampled across 20 catchments. 156
At each site a sample of biomonitor specimens was collected for trace metal analysis. Each 157
site was visited once, either in spring (March-May) or autumn (September-November), 158
between autumn 2013 and spring 2015. 159
2.2 Geochemical Gradient 160
Sediment samples were also collected at each site and analysed for metals to corroborate 161
metal gradients within each catchment (see Table S2 for methods). Environmental quality 162
standards for trace metals in sediment were included to identify significant gradients. 163
2.3 Selection of biomonitors 164
Biomonitor species were selected based on high levels of co-occurrence and abundance 165
across all sites to obtain sufficient individuals for trace metal analysis. Species fitting these 166
criteria were: two caddisflies (Hydropsyche spp. and Rhyacophila spp.), a mayfly (Baetis 167
spp.), a stonefly (Leuctra spp.) and an amphipod crustacean (Gammarus pulex group). The 168
taxa varied in their ecological niches and, thus, potentially their exposure to trace metal 169
contamination (see Table S3). 170
2.4 Biomonitor sampling 171
A separate sample of >20 individuals from each of the five biomonitor genera were collected 172
from each site (where present). Kick samples were collected until sufficient individuals were 173
found. Specimens were rinsed in stream water to remove any attached material, individually 174
placed in small re-sealable polythene bags and frozen in the field in a portable -18 °C 175
freezer. Upon return to the laboratory, specimens were transferred to a -20 °C freezer. 176
Larger individuals were collected in preference to smaller individuals to ensure enough 177
material for trace metal analysis. 178
Collection of individuals did not allow the specific identification of each specimen (see Table 179
S3). Whilst it is ideal to identify biomonitors to the species level (Rainbow, 2018), it is usually 180
pragmatic in the field to identify biomonitors to genus level. The assumption is that any 181
difference in accumulated trace metal concentrations between closely related species at the 182
same site is less than between individuals of the same species between sites. 183
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In the laboratory, biomonitor specimens were rinsed in double-distilled water and dried to 185
constant mass in acid-washed Pyrex tubes. They were then digested in Aristar grade 186
concentrated nitric acid (BDH Ltd., Poole, UK) at 100 °C, made up to volume with double-187
distilled water and analysed for trace metals on a Thermo iCAP 6500 ICP-OES . Analyses 188
involved routine use of blanks and certified reference materials (TORT-2 Lobster 189
Hepatopancreas (NRC Canada), Mussel Tissue 2976 (NIST, USA) and Prawn NCS ZC 190
80006 (China)) to assess accuracy (within 5%). Eleven trace metals (of silver (Ag), cadmium 191
(Cd), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead 192
(Pb), vanadium (V), zinc (Zn)) and one metalloid (arsenic (As)) were measured. 193
Bioaccumulated trace metal concentrations in aquatic invertebrates, including insect larvae 194
(Darlington et al., 1987) and amphipod crustaceans (Rainbow and Moore, 1986), typically 195
decrease with size. The concentration [C] to dry weight [W] relationship can be described by 196
a negative power relationship [C] = a [W]b where b is negative, with a considerable rise in 197
total bioaccumulated concentration below a threshold dry weight of about 0.002 g (Rainbow 198
and Moore, 1986). Consequently, concentrations derived from samples ≥ 0.002 g dry weight 199
(dw) were eliminated to mitigate mass effects. 200
2.6 Statistical Analysis 201
Body burdens of trace metals in individual biomonitor samples were log10 transformed to aid 202
comparison and to ensure equal variances. Based on multiple samples taken at each site, 203
the mean log10 body burden was calculated for each trace metal and biomonitor taxon at 204
each site. Boxplots were drawn to summarise the range and distribution of site average 205
biomonitor trace metal body burdens. For each trace metal, differences in the median body 206
burden between biomonitors was assessed, using Kruskal-Wallis followed by Dunn’s Test 207
for multiple comparisons (Zar, 1996). By sampling across a metal contamination gradient, a 208
broad range of trace metal body burdens was expected. As such, comparisons between 209
biomonitors were of median body burden as it is less affected by extreme low and high 210
values than the arithmetic mean. Finally, using Spearman’s rank-abundance correlations (rs) 211
the strength of association was evaluated: (a) among pairs of trace metals within each 212
species, and; (b) among pairs of species for each trace metal. It is acceptable to use rank 213
correlations to make interspecific comparisons between biomonitors collected from the same 214
site but it is not valid to compare absolute accumulated metal concentrations between 215
interspecific taxa because of mechanistic differences in the bioaccumulation of metals by 216
different species (Rainbow, 2018). To reduce the chance of Type I errors, the Holm–217
Bonferroni method (Holm, 1979) to correct for family-wise error rates was applied to all tests. 218
219
3.1 Trace metal body burdens of biomonitor taxa 221
None of the five biomonitor taxa were present at all 99 sampling sites. The most complete 222
data sets were for Hydropsyche (88 sites), Rhyacophila (86 sites) and Baetis (86 sites), 223
whilst Leuctra (69 sites) and Gammarus (30 sites) were found at the fewest sites (Table S4). 224
Body burdens of metals across species varied by up to six orders of magnitude (Figure 2): 225
the minimum body burden of Ag in Baetis was 0.13 µg g-1 dw relative to the maximum value 226
for Zn in Hydropsyche of 23,140 µg g-1 dw (Table S4). 227
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Amongst biomonitors, the range between minimum and maximum values indicated the least 228
variable metal body burdens differed by two orders of magnitude (e.g., Ag, Mn, V) whilst the 229
most variable differed by four to six orders of magnitude (e.g., As, Cu, Pb: Figure 2). The 230
highest median concentration of trace metal body burdens across taxa was Fe (651-2,840 231
µg g-1 dw), followed by Zn (110-480 µg g-1 dw) and Mn (99-174 µg g-1 dw) whilst the lowest 232
was Ag (0.4-1.2 µg g-1 dw: Table S4). The general order of median trace metal body burden 233
concentrations amongst insect taxa was similar: Fe > Zn > Mn > Cu/Ni/Pb > As > 234
Cr/V/Co/Cd > Ag (Table S4). For Gammarus, the order differed although similarities with the 235
insects were apparent: Fe > Mn > Zn > Cu > Ni > As > Pb > Cr > V > Co > Cd > Ag. 236
Comparing across taxa for each trace metal (Figure 2; Table S4), minimum body burdens 237
occurred within the same order of magnitude (one-off exceptions occurred for: Fe, Mn, Pb). 238
The low median, mean and minimum body burdens of Ag, Co, Cr and V indicate low metal 239
bioavailabilities, a pattern consistent across all biomonitor taxa. All taxa also had low 240
minimum body burdens of As, Cd, Ni and Pb, but median and maximum body burdens were 241
relatively higher indicating a wider range of bioavailabilities across the sites sampled than for 242
Ag, Co, Cr and V (Figure 2). Cu, Fe, Mn and Zn had relatively high median and minimum 243
body burdens compared with other trace metals, indicating greater bioavailability across all 244
sites (Figure 2). 245
No significant differences were detected between trace metal body burdens of Leuctra and 246
Hydropsyche (Figure 2). Furthermore, both these taxa (but in particular Hydropsyche) 247
accumulated significantly higher median body burdens for most trace metals when 248
compared with the other taxa (Figure 2). The median trace metal body burden of 249
Rhyacophila was significantly lower than Hydropsyche for all trace metals apart from Ni and 250
Zn. Median trace metal body burden of Gammarus was either significantly lower than (Pb, 251
Fe, Zn) or similar to Baetis and/or Rhyacophila (Ag, Cd, Co, Cr, Ni, V), apart from the body 252
burdens of As, Cu and Mn which were accumulated in quantities equivalent to those found in 253
Leuctra and Hydropsyche (Figure 2). Median trace metal body burdens of Baetis were the 254
most variable, either equivalent to Gammarus and/or Rhyacophila (Ag, Cr, Fe, Mn, Ni, Pb, V) 255
or Leuctra and/or Hydropsyche (As, Cd, Co, Cu, Zn). 256
3.2 Relationships between pairs of trace metals within taxa 257
Within individual taxa, there were significant positive correlations between body burdens of 258
many pairs of trace metals (Figure 3). Of 66 possible…