Ingestion of microplastics by freshwater Tubifex worms
Rachel R. Hurley*, Jamie C. Woodward, James J. Rothwell
Department of Geography, The University of Manchester, Manchester, M13 9PL, United
Kingdom
ABSTRACT: Microplastic contamination of the aquatic environment is a global issue.
Microplastics can be ingested by organisms leading to negative physiological impacts. The
ingestion of microplastics by freshwater invertebrates has not been reported outside the
laboratory. Here we demonstrate the ingestion of microplastic particles by Tubifex tubifex from
bottom sediments in a major urban waterbody fed by the River Irwell, Manchester, UK. The host
sediments had microplastic concentrations ranging from 56 to 2543 particles kg-1. 87% of the
Tubifex-ingested microplastic particles were microfibres (55 - 4100 µm in length), whilst the
remaining 13% were microplastic fragments (50 - 4500 µm in length). FT-IR analysis revealed
ingestion of a range of polymer types, including polyethylene terephthalate (polyester) and
acrylic fibres. Whilst microbeads were present in the host sediment matrix, they were not
detected in Tubifex worm tissue. The mean concentration of ingested microplastics was 129 ±
65.4 particles g-1 tissue. We also show that Tubifex worms retain microplastics longer than other
components of the ingested sediment matrix. Microplastic ingestion by Tubifex worms poses a
significant risk for trophic transfer and biomagnification of microplastics up the aquatic food
chain.
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Introduction
Microplastics represent a global environmental problem1. Defined as small (<5 mm) plastic
particles, they may be specifically engineered (e.g. microbeads) or can result from the
degradation of larger plastic items 2. These particles can enter the environment via several
pathways including sewage or storm water runoff, and the breakdown of larger plastic litter3.
Multiple implications associated with microplastic ingestion by organisms have been reported,
including: 1) the retention of microplastics in the gut, causing blockages and reducing nutrient
absorption4–6; 2) transferring sorbed contaminants or plastic additives7,8; 3) translocation to other
tissues9,10; and 4) transfer up the food chain11, including to human populations12–14. Whilst early
work focused on microplastic ingestion by marine organisms, researchers have begun to
investigate freshwater ecosystems. Thus far, all studies of river15–18 and lake19 environments have
concentrated on species of fish. Field studies of microplastic ingestion by freshwater
invertebrates are absent, despite them being a key entry point into the food chain.
Several studies have examined the ingestion of microplastic particles by marine7,20–22 and
freshwater23–25 worm species in laboratory settings. This previous work has reported a number of
implications of microplastic ingestion, such as decreased energy reserves and fitness21,22, transfer
of plasticisers and sorbed contaminants7,21, increased oxidative stress7, reduced growth23, and
increased mortality 23. These impacts are linked to the retention of microplastics within the
organism. Van Cauwenberghe et al.20 provide the only study to date of in-field ingestion by a
worm species, the lugworm Arenicola marina, along the French-Belgian-Dutch coastline. They
identified microplastic concentrations of 1.2 ± 2.8 particles g-1 tissue, in addition to 0.3 ± 0.6
particles g-1 in faecal casts, demonstrating the cycling of microplastic by a marine invertebrate
species.
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Tubifex worms are one of the most abundant invertebrates in freshwater systems26. They
inhabit the uppermost layers of freshwater sediment acting as ‘conveyer-belt’ deposit feeders27.
They live partially submerged with their posterior undulating freely in the overlying waters26,28,29.
They ingest sediment particles, typically in the <63 µm fraction, and excrete them as sand-sized
faecal pellets 30,31. Some selectivity in this ingestion has been noted, with a preference for
bacteria-rich substrates 32,33. They typically burrow to depths of 6-10 cm 31; however, this is
reduced to a depth of 2 cm in highly contaminated sediments 26. The entire life cycle of Tubifex
worms take place within sediments 30,34. Tubifex worms are highly tolerant of grossly polluted
settings35 and are one of the last species present under deteriorating environmental conditions36.
Colloquially referred to as the sewage worm37, Tubifex also process raw sewage inputs and other
organic material. They represent primary consumers in the freshwater ecosystem food chain.
Tubifex worms have been shown to accumulate contaminants such as uranium, cadmium, and
copper26–28,38. In fact, Tubifex tubifex have been widely used in bioassays for the assessment of
toxicity and bioaccumulation 39 and have been promoted as an ideal species for sediment toxicity
tests 40. The ingestion of microplastic particles by Tubificidae has not yet been reported. An
improved understanding of the uptake of microplastics by freshwater macroinvertebrates is
essential to better understand microplastic trophic transfer.
This paper has three principal aims: 1) to investigate the ingestion of microplastics by Tubifex
worms in an urban freshwater environment; 2) to examine the processes related to uptake,
including preferential microplastic particle selection and 3) to explore the relationship between
the uptake of microplastic particles by freshwater invertebrates and the microplastic
concentrations in host sediments.
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Methods
Study site
The Salford Quays basin is located on the edge of Manchester city centre, in the northwest of
England (Figure 1). It was the inland docks of the former Port of Manchester. The basin receives
all of the waters from the River Irwell (793 km2). The Quays are regulated at the distal end by
locks, which mark the start of the Manchester Ship Canal (MSC). The MSC is a large artificial
waterway that opened in 1892 and superseded the former channel of the lower River Irwell. It
permitted the navigation of seagoing vessels 58 km inland from the Irish Sea to the Port of
Manchester. Salford Quays includes a large turning basin with several docks (Figure 1). At
almost 2 km in length, this is a low energy freshwater setting with many similarities to a semi-
open lacustrine environment.
Sampling
Sediment coring
Bottom sediments were sampled via short (< 50 cm) cores from 12 sites across the Salford
Quays basin in spring 2017 using a UWITEC gravity corer (Figure 1c). This permitted the
collection of undisturbed sediment cores (60 mm internal diameter), retaining the sediment water
interface by enclosing a sample of the overlying water. The cores were retained in an upright
position and immediately transported to the laboratory for processing.
Sediment and worm extraction
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The cores were mounted on a UWITEC extrusion rig and the uppermost sediment layer was
brought to the surface. Tubifex worms were abundant in the surface layer of 5 cores (sites 7 to
11). The worms were identified using a number of available taxonomic keys 41–44. In total, 302
worms were extracted. Tubifex tubifex was the only worm species present, – a function of the
very high levels of contamination observed in the Salford Quays sediments 45. A small number of
the worms represent immature individuals that could not be reliably identified as T. tubifex.
However, they were included on the basis that they are tubificid and present in a single species
community. The worms were carefully extracted from the surface sediments and placed into petri
dishes using stainless steel tweezers.
Following the extraction of the Tubifex worms, the surface sediments at all 12 sites were
extruded and isolated for analysis. The uppermost 10 mm was sampled in this study as this
corresponds to the submersion depth of Tubifex worms in highly contaminated sediments, as well
as the key feeding site for T. tubifex – namely the sediment-water interface. Sediments were
freeze-dried and weighed prior to analysis in order to facilitate the quantification of microplastic
concentrations reported by mass and volume.
Microplastic extraction
Sediment samples were placed into pre-washed 50 ml polyethylene tubes and subjected to a
density-based sequential extraction procedure. Three extracts were used: 1.025 g cm-2 NaCl, 1.2
g cm-2 NaCl, and 1.8 g cm-2 NaI. The first density solution (1.025 g cm-2 NaCl) was poured into
the tubes and the contents were agitated continuously for 3 minutes. The sediments were then left
to settle overnight. Following settling, the supernatant was decanted and vacuum-filtered through
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Whatman GF-C filter papers and placed into separate petri dishes. The same density extract was
applied a second time to ensure complete extraction of microplastic particles. This procedure was
repeated sequentially for the denser extracts. Filter papers were oven dried at 40°C.
Microplastics were extracted from both bulk and individual worm samples. To examine the
relationship between worm characteristics and microplastic ingestion, 75 worms from across 4 of
the sites were randomly selected and isolated into separate containers (Table S1). All remaining
worms were combined as bulk samples for each site. All worm samples, bulk and individual,
were carefully cleaned of external debris and placed in deionised water. The worms were then
left for 24 hours to depurate. This has been established as the optimum period for Tubifex gut
clearance 46. The water was changed after 12 hours to prevent repeat ingestion of excreted
material.
Following depuration, the 75 individual worms were measured, weighed (wet weight), and
placed into separate prewashed 15 ml polyethylene tubes. The bulk worm samples for each site
were counted and weighed into falcon tubes. Microplastics were extracted by digestion of the
worm tissue. This was performed using 10% KOH at 60°C 47. This procedure has been
recommended as an effective means of microplastic extraction that does not degrade polymers
during tissue digestion48,49. Complete digestion was achieved in <10 minutes. For all samples, the
resulting slurry was vacuum filtered through Whatman GF-C filter papers and oven dried in petri
dishes at 40°C.
Extreme care was taken to limit contamination from the laboratory environment during all
stages of extraction. The worm samples were examined for external debris prior to, and
following, depuration and the KOH solution was vacuum-filtered (1.2 µm) prior to use. All of
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the samples were kept covered during all stages of digestion and a foil lid was used to prevent
atmospheric contamination of filter papers during filtration. Several procedural blanks were
included. These were prepared simultaneously and followed an identical procedure to the worm
digestion and microplastic extraction. No particles were identified as plastic in the blanks.
Microplastic identification, quantification and characterisation
Microplastic particles, from individual worms, bulk worm samples, and sediment samples,
were visually identified using a Zeiss Axio Zoom.V16 at 20-50x magnification. Particles were
then tested for plastic composition using the hot needle test50. Only particles that responded
unambiguously to the application of a hot needle were extracted and quantified. Counts were
produced for each density extract for the sediment samples. Plastic particles were measured
along their longest axis using the Zeiss Zen imaging software and characterised by shape
(fragment, fibre, bead, other) and colour. Particles were removed from the filter papers and
placed into pre-weighed pots for each extract or worm digest at each site. Due to the very low
weight of individual microplastic particles extracted from both the worms and surface sediments,
it was not possible to obtain a weight for some extracts and so the microplastic particles were
aggregated into bulk samples for each substrate (sediment or worm) at each site for the purposes
of recording mass.
Microplastic particles were then characterised by polymer type using FT-IR spectroscopy. All
of the microplastics extracted from the surface sediments and the individual worms were
analysed using FT-IR. 50% of the microplastic particles from the bulk worm samples were also
analysed in order to verify the identification technique. FT-IR analysis was performed with a
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Perkin Elmer Spotlight 400 imaging system using a diamond ATR crystal. The spectrum range
was set at 4000 to 650 cm-1, with a resolution of 4 cm-1. 16 co-scans were obtained for each
particle and background scans were run between each sample. Spectra were compared to the
Perkin Elmer ATR Polymers library for identification of polymer type (sample spectra shown in
Figure S1).
Statistical analysis
Variability around the mean is reported as ± standard deviation. Relationships between
microplastic concentrations in sediments and tissues were examined using a bivariate Pearson
correlation coefficient. Analysis of the differences between worm characteristics (length, mass,
number of ingested particles), sediment associated microplastics (length, shape, polymer) and
ingested microplastics (length, shape, polymer) were assessed using one-way ANOVAs. All
statistical tests were performed using SPSS Statistics 20 software.
Results and discussion
Microplastics in the sediment matrix
The concentrations of microplastics in the host sediments at each site are shown in Figure 2a.
All 12 sites were contaminated by microplastics. The mean (914 ± 844 particles kg-1; 1793 ±
1275 particles m-2) and maximum (2543 particles kg-1; 3891 particles m-2) microplastic
concentrations are comparable with shore and bottom sediments from lacustrine environments in
Europe, such as the Swiss lakes (mean: 1300 particles m-2; 51) and Lake Bolsena, Italy (mean:
1922 particles m-2; 52). These are higher than those observed in Lake Ontario sediments (mean:
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980 particles kg-1; 53), or sediments from lakes in China (max: 235 particles kg-1 and 563 particles
m-2; 54,55). We have estimated that the mean concentration of microplastics (m-2) indicates a load
of 421 million particles in the surface sediments (top 10 mm) in the Salford Quays basin (0.24
km2).
The spatial distribution of microplastics across the Salford Quays basin varies with respect to
microplastic density (Figure 2b) and shape (Figure 2c). Across the Salford Quays basin, 43% of
microplastics were characterised as fragments (76 to 3910 µm in length), 29% as microbeads
(124 to 1050 µm), 24% as fibres (91 to 4330 µm) and 3% as ‘other’ (1026 to 2500 µm) (Table
S2). The spatial distribution of microplastic types across the basin is highly variable (Figure 2c)
and this corresponds with variability in polymer composition (Figure 2d). No clear spatial
patterns could be observed. The density of the extracted plastic particles shows greater variability
close to the River Irwell inflow. This includes a higher proportion of particles extracted at
seawater density. Towards the locks and the outflow to the MSC, the density of microplastic
particles increases where the majority of particles sampled close to the locks was extracted at 1.8
g cm-2. The accumulation of large floating plastic debris in the Salford Quays basin is a
significant management problem. There is more variability in the density composition of
microplastic contamination at sites 6 and 12, which may partly reflect the breakdown of this
larger plastic debris.
The polymer composition of the sediment microplastics is presented in Figure 2d and Table S3.
Polyethylene and polypropylene were the dominant polymer types observed for the fragments.
Microbeads were almost entirely composed of polystyrene (87%). Fibres had more variable
polymer composition which included polyethylene terephthalate (polyester), acrylic, and vinyl
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polymers. Finally, the ‘other’ fraction generally referred to pieces of glitter, which were found to
be coated in polyethylene terephthalate.
Tubifex worms in Salford Quays
Tubifex worms were identified in the surface sediments at 5 sites (7 to 11). Only those sites
with water depths >5 m exhibited Tubifex populations. The density of worms in the surface
sediment ranged from 1,061 to 32,543 individuals m-2 (Figure 3a). Site 9, located at the base of a
former dock, showed the lowest worm abundance, whereas all other sites in the main part of the
basin exhibited densities >20,000 individuals m-2. The density of Tubifex communities has been
shown to vary considerably between environments and in response to seasonal changes56.
Densities up to 600,000 individuals m-2 have been observed57. The individually analysed worms
were 3 – 56 mm in length (mean: 16.5 ± 9.77 mm) and weighed between 0.1 and 23.9 mg (mean:
4.0 ± 3.99 mg) (Table S1). This exceeds the mean weight recorded for Tubifex worms in the
River Thames (2.5 mg; 57) or in laboratory experiments at comparable water temperatures (2.3 –
3.15 mg; 58).
Microplastic ingestion
A total of 131 microplastic particles were extracted from 302 Tubifex worms across 5 cores.
This includes the 75 worms individually analysed. The mean concentration of ingested
microplastics was 129 ± 65.4 particles g-1 tissue. The density of microplastic particles g-1 tissue is
higher than those reported for other freshwater or marine species12,13,20,59,60. This partly relates to
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the very low mass of Tubifex worms compared to other animal species so far analysed for
microplastic contamination.
The vast majority of microplastics ingested by Tubifex worms were characterised as fibres
(87%). This is in agreement with other field studies of microplastic ingestion in freshwater and
marine environments22,60. The remaining proportion was made up of microplastic fragments
(Figure 3c). Interestingly, no microbeads were identified in the digested worm tissue, despite
their presence in the host sediment matrix (Figures 2 and 3). Extracted fibres ranged from 55 to
4100 µm in length (mean: 847 ± 673 µm) and were mostly blue (50%), black (22%) or red (9%).
Fragments were between 50 and 4500 µm along their longest axis (mean: 676 ± 1260 µm) and
were all blue, with the exception of two pieces of clear film.
Individual worm analysis
Seventy five Tubifex worms from sites 8-11 were analysed individually for microplastic
ingestion (Table S1). On average, ingestion of microplastics was 0.8 ± 1.01 particles per worm.
48% of the worms had ingested microplastic. The individual worm analysis demonstrated that
some worms had ingested multiple microplastic particles; 14 worms had ingested 2 microplastic
particles, 2 worms had ingested 3 microplastic particles and 2 worms had ingested 4 microplastic
particles.
All of the microplastics extracted from the individually sampled worms were analysed using
FT-IR (60 particles from 36 worms). This was performed to test the efficacy of the identification
protocol and to provide an assessment of the relative proportions of different polymer types
ingested across the Salford Quays basin. All of the analysed particles were confirmed to be
plastic, with the exception of a small number of fibres that could not be characterised using FT-
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IR as they were too small or were transparent. All of these particles did, however, respond
unambiguously to the hot needle test and it is therefore highly likely that these particles also
represent microplastics. The ingested fragments were composed of polystyrene (44%),
polyethylene (44%), or polypropylene (11%). Ingested plastic fibres consisted of
polyester/polyethylene terephthalate (30%), acrylic/polyacrylonitrile (28%), polypropylene
(26%), polyethylene (9%), and poly(vinyl) alcohol (9%) (Table S3).
Selectivity of microplastic ingestion
The majority of microplastics extracted from the individual worms were found to be fibres,
with some fragments (15%) (Figure 3c). There was no significant difference in the type of plastic
(length, fragment/fibre, or polymer composition) consumed by worms of different length or mass
(ANOVA p > 0.34). This was the case within and across sites. There was no significant
correlation between the size (length or mass) of worms and the number or size of microplastics
ingested (Pearson’s: p > 0.20). This indicates that there is limited selectivity in the ingestion of
microplastic particles within fragments or fibres.
No microbeads were identified in the Tubifex worm tissue (302 worms in total). This strongly
suggests that microbeads are not ingested by Tubifex worms in the Salford Quays. Selective
feeding by T. tubifex has been reported by Rodriguez et al.33. A size selectivity of sediments <63
µm was recognised, suggesting that microbeads, which range between 124 and 1050 µm in the
Salford Quays sediments (Table S2), are too large for ingestion by Tubifex worms. Whilst the
majority of the fibres and fragments exceed 63 µm along their major axis, they are typically
significantly smaller in diameter, allowing them to be ingested along with similarly fine-grained
sediments and organic matter. This particle size selectivity has also been demonstrated in
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earthworms (Lumbricus terrestris), whereby a preference for particles <50 µm within a matrix of
mixed microplastic sizes was observed by Huerta Lwanga et al.23.
Rodriguez et al.33 also identified the preferential ingestion of particles associated with organic
material. Tubifex worms ingest benthic sediment and pass it through their gut, absorbing
nutrients from associated organic material. Higher concentrations of organic matter in the faeces
of T. tubifex than in surrounding sediments have been reported in a number of studies 61–63. It is
entirely possible that microplastic particles that have been biofouled or exhibit biofilms may be
preferentially ingested by Tubifex worms. Microplastics have been shown to host unique and
abundant bacterial assemblages 64 and may therefore represent a potential nutrient source. The
selective feeding of Tubifex on bacteria has been established 32. The development of biofilms
upon microplastic particles has been observed in a range of aquatic environments 64–66. During
the initial visual identification step, organic coatings were observed on some microplastic
particles and many particles were incorporated into organic-rich aggregates. This may lead to a
preferential ingestion of these microplastics by Tubifex worms. Further investigation (e.g.
through laboratory exposure studies) is required to confirm this form of microplastic particle
selectivity.
The spatial distribution of polymer types ingested by the worms across the 4 sites is fairly
uniform, with the exception of site 9 where only a single polystyrene fragment was found in the
3 worms sampled (Figure 3cd). This suggests limited selectivity related to polymer composition.
However, the polymer composition of ingested particles (Figure 3d) differs slightly from the
polymer types extracted from the host sediments (Figure 2d). For example, worms at sites 8, 10,
and 11 contained several polyester, acrylic, and polypropylene fibres which were not well
represented within host sediments and this may indicate a preference for these polymers.
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Sediment microplastic concentration and microplastic ingestion
There were no significant differences between the size characteristics of the worms across the
5 sites (ANOVA, p > 0.45) despite variability in microplastic concentrations in the host
sediment. Moreover, there is no correlation between sediment microplastic concentration and
worm population density (Pearson’s: p = 0.39) or the concentration of ingested particles g-1 tissue
(Pearson’s: p = 0.28). Our data suggest that sediment microplastic concentrations do not
influence worm abundance or growth in this setting.
The density of microplastics within host sediments at the sites exhibiting Tubifex populations
is higher than observed across the rest of the Salford Quays basin (Figure 2b). This may be
linked to bioturbation processes. If microplastics pass through the gut, they will be ejected as
mucus-bound faecal particles into the overlying waters. This may lead to the mixing of
microplastics in the water column and the resuspension of less dense particles. Moreover, during
digestion any associated biofilm or biofouling may be fully or partially absorbed and so the
density of egested particles may be reduced. This could explain the enrichment of higher density
microplastics at these sites. The difference in fibre polymer composition ingested by the worms
compared to fibres in the sediment may be influenced by this process, whereby the irregular
shape of microfibres involves slower settling velocities. This may deplete sediments of lower
density fibres composed of polyethylene, polypropylene, or acrylic, for example. Further work is
required to elucidate more fully the potential for a bioturbation influence on the microplastic
particle assemblage within freshwater sediments.
Significance of ingested particles
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Laboratory exposure studies report various outcomes regarding the biophysical significance of
microplastic ingestion 23–25. At high microplastic concentrations, the growth rate of earthworms
(Lumbricus terrestris) is affected23. However Hodson et al.25 reported that where microplastics
are derived from fragmented HDPE bags, the particles are not retained within the gut and have
limited effect. Rodriguez-Seijo et al. 24 recorded no significant effects in earthworms (Eisenia
andrei) after 28 days of exposure to microplastic contamination. It is important to note that these
species of worm differ from Tubifex in a number of key respects including size, feeding habits,
and environment. However, due to their smaller size, it is likely that Tubifex worms are more
likely to retain microplastics within their gut. Such retention has the potential to cause
inflammation, reduce absorption of nutrients from other ingested particles and increase the
residence time of microplastic particles, and therefore increase the potential for the stripping of
any microplastic-borne contaminants7,69.
The presence of microplastics in Tubifex worm tissue following a depuration period indicates
that microplastics have higher residence times in the gut than non-plastic sediment particles. The
preference for ingesting microplastic fibres is significant given their potential for the transfer of
contaminants associated with their shape 70. Despite this, the ingestion of microplastics has no
statistically significant effect on the population density of Tubifex communities in this
environment. Tubifex worms in Salford Quays are larger than those observed in many other
freshwater settings, so it is unlikely that microplastics are hindering worm growth. Tubifex
worms are known to be extremely tolerant of environmental contamination. Thus, the transfer of
plastic additives or sorbed contaminants is less likely to influence their health or mortality.
Having said that, laboratory exposure studies are needed to fully assess the tolerance of Tubifex
worms to microplastics and associated contaminants.
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The key significance of microplastic ingestion and retention in Tubifex worms is linked to their
trophic level and the concentration of microplastic particles g-1 tissue. Despite their small size,
Tubifex worms ingest microplastic particles of up to 4500 µm in length and present higher
microplastic concentrations g-1 tissue than other freshwater or marine organisms. These particles,
and any associated additives or contaminants, may biomagnify at higher trophic levels71 due to
the higher concentrations, the low mass, and the position of Tubifex worms at the base of the
food chain. The high tolerance of Tubifex may also increase the risk of bioaccumulation as the
worms will survive the transfer of high concentrations of plastic additives and desorbed
contaminants. The faecal pellets egested by Tubifex worms may form another pathway of
microplastic transfer to higher aquatic organisms, since some species consume these within
overlying waters72. T. tubifex is a food source for many macroinvertebrates, such as leeches73 as
well as small benthivorous fish74. They are also consumed by salmon and trout75, and thus
represent a potential direct link to the human food chain.
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FIGURES:
Abstract art
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Figure 1. The distribution of sampling sites across Salford Quays (A) and the location of the
Salford Quays basin in relation to the UK (B) and the River Irwell catchment (C). The basin (A)
receives waters from the River Irwell (right) and drains into the Manchester Ship Canal through
a series of locks (top left). An aerial photograph of the area is also provided (D), showing the
Salford Quays basin in relation to the cities of Manchester (top centre) and Salford (middle left).
Aerial photograph by M J Richardson (2010) licensed under CC BY-SA 2.0.
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Figure 2. Concentrations of microplastics in the bottom sediments of Salford Quays. These are
provided as total concentrations in particles kg-1 (A), in addition to the relative proportions of
each density extract (B), microplastic type (C) and polymer composition (D).
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Figure 3. Density of Tubifex worm populations (A) and concentrations of ingested microplastics
(B) in Salford Quays. Concentrations are also broken down by microplastic type (C) and
polymer composition (D). *Polymer composition refers to the microplastic ingested by the 75
worms analysed individually.
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ASSOCIATED CONTENT
Supporting Information. Details of the worms selected for individual analysis; overview of
particle size data; overview of polymer composition of microplastic; sample FT-IR spectra from
the most commonly identified polymer types.
AUTHOR INFORMATION
Corresponding Author
* [email protected]; Arthur Lewis Building, The University of Manchester,
Manchester, M13 9PL.
Author Contributions
All authors contributed equally to the design of the project and fieldwork. RRH performed all the
analyses. The manuscript was written with contributions from all authors. All authors have given
approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors would like to thank APEM Ltd for fieldwork access and assistance We also wish
to acknowledge Dr. Tom Bishop who provided invaluable fieldwork assistance, Prof. Roy
Wogelius and Dr. Heath Bagshaw for access and training in FT-IR analysis, and John Moore and
Jonathan Yarwood in the Geography Laboratories, and Nick Scarle who provided assistance with
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the figures We would also like to thank three anonymous reviewers for helpful comments on the
manuscript. R.R.H. was in receipt of a University of Manchester President's Doctoral Scholar
Award which helped to fund this research.
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