Template for Electronic Submission to ACS Journals · Web viewThe development of biofilms upon microplastic particles has been observed in a range of aquatic environments 64–66.

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.

1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

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.

2

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

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.

3

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

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

4

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

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

5

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

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

6

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

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

7

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

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:

8

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

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

9

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

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

10

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

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-

11

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

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

12

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

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.

13

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

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

14

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

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.

15

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

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.

16

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

FIGURES:

Abstract art

17

351

352

353

354

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.

18

355

356

357

358

359

360

361

362

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).

19

363

364

365

366

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.

20

367

368

369

370

371

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

21

372

373

374

375

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

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.

REFERENCES

(1) Rochman, C. M.; Browne, M. A.; Underwood, A. J.; van Franeker, J. A.; Thompson, R.

C.; Amaral-Zettler, L. A. The ecological impacts of marine debris: unraveling the

demonstrated evidence from what is perceived. Ecology 2016, 97 (2), 302–312.

(2) Rocha-Santos, T.; Duarte, A. C. A critical overview of the analytical approaches to the

occurrence, the fate and the behavior of microplastics in the environment. TrAC Trends

Anal. Chem. 2015, 65, 47–53.

(3) Browne, M. A. Sources and Pathways of Microplastics to Habitats. In Marine

Anthropogenic Litter; Bergmann, M., Gutow, L., Klages, M., Eds.; Springer International

Publishing: Cham, 2015; pp 229–244.

(4) Cole, M.; Lindeque, P.; Fileman, E.; Halsband, C.; Galloway, T. S. The Impact of

Polystyrene Microplastics on Feeding, Function and Fecundity in the Marine Copepod

Calanus helgolandicus. Environ. Sci. Technol. 2015, 49 (2), 1130–1137.

(5) Lu, Y.; Zhang, Y.; Deng, Y.; Jiang, W.; Zhao, Y.; Geng, J.; Ding, L.; Ren, H. Uptake and

Accumulation of Polystyrene Microplastics in Zebrafish (Danio rerio) and Toxic Effects

in Liver. Environ. Sci. Technol. 2016, 50 (7), 4054–4060.

(6) Welden, N. A. C.; Cowie, P. R. Long-term microplastic retention causes reduced body

condition in the langoustine, Nephrops norvegicus. Environ. Pollut. 2016, 218, 895–900.

22

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

(7) Browne, M. A.; Niven, S. J.; Galloway, T. S.; Rowland, S. J.; Thompson, R. C.

Microplastic Moves Pollutants and Additives to Worms, Reducing Functions Linked to

Health and Biodiversity. Curr. Biol. 2013, 23 (23), 2388–2392.

(8) Bakir, A.; Rowland, S. J.; Thompson, R. C. Enhanced desorption of persistent organic

pollutants from microplastics under simulated physiological conditions. Environ. Pollut.

2014, 185, 16–23.

(9) Browne, M. A.; Dissanayake, A.; Galloway, T. S.; Lowe, D. M.; Thompson, R. C.

Ingested Microscopic Plastic Translocates to the Circulatory System of the Mussel,

Mytilus edulis (L.). Environ. Sci. Technol. 2008, 42 (13), 5026–5031.

(10) Brennecke, D.; Ferreira, E. C.; Costa, T. M. M.; Appel, D.; da Gama, B. A. P.; Lenz, M.

Ingested microplastics (>100 μm) are translocated to organs of the tropical fiddler crab

Uca rapax. Mar. Pollut. Bull. 2015, 96 (1–2), 491–495.

(11) Farrell, P.; Nelson, K. Trophic level transfer of microplastic: Mytilus edulis (L.) to

Carcinus maenas (L.). Environ. Pollut. 2013, 177, 1–3.

(12) Van Cauwenberghe, L.; Janssen, C. R. Microplastics in bivalves cultured for human

consumption. Environ. Pollut. 2014, 193, 65–70.

(13) Li, J.; Yang, D.; Li, L.; Jabeen, K.; Shi, H. Microplastics in commercial bivalves from

China. Environ. Pollut. 2015, 207, 190–195.

(14) Galloway, T. S. Micro- and Nano-plastics and Human Health. In Marine Anthropogenic

Litter; Bergmann, M., Gutow, L., Klages, M., Eds.; Springer International Publishing,

2015; pp 343–366.

23

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

(15) Sanchez, W.; Bender, C.; Porcher, J.-M. Wild gudgeons (Gobio gobio) from French rivers

are contaminated by microplastics: Preliminary study and first evidence. Environ. Res.

2014, 128, 98–100.

(16) Peters, C. A.; Bratton, S. P. Urbanization is a major influence on microplastic ingestion by

sunfish in the Brazos River Basin, Central Texas, USA. Environ. Pollut. 2016, 210, 380–

387.

(17) Phillips, M. B.; Bonner, T. H. Occurrence and amount of microplastic ingested by fishes

in watersheds of the Gulf of Mexico. Mar. Pollut. Bull. 2015, 100 (1), 264–269.

(18) Jabeen, K.; Su, L.; Li, J.; Yang, D.; Tong, C.; Mu, J.; Shi, H. Microplastics and

mesoplastics in fish from coastal and fresh waters of China. Environ. Pollut. 2017, 221,

141–149.

(19) Biginagwa, F. J.; Mayoma, B. S.; Shashoua, Y.; Syberg, K.; Khan, F. R. First evidence of

microplastics in the African Great Lakes: Recovery from Lake Victoria Nile perch and

Nile tilapia. J. Gt. Lakes Res. 2016, 42 (1), 146–149.

(20) Van Cauwenberghe, L.; Claessens, M.; Vandegehuchte, M. B.; Janssen, C. R.

Microplastics are taken up by mussels (Mytilus edulis) and lugworms (Arenicola marina)

living in natural habitats. Environ. Pollut. 2015, 199, 10–17.

(21) Besseling, E.; Wegner, A.; Foekema, E. M.; van den Heuvel-Greve, M. J.; Koelmans, A.

A. Effects of Microplastic on Fitness and PCB Bioaccumulation by the Lugworm

Arenicola marina (L.). Environ. Sci. Technol. 2013, 47 (1), 593–600.

(22) Wright, S. L.; Rowe, D.; Thompson, R. C.; Galloway, T. S. Microplastic ingestion

decreases energy reserves in marine worms. Curr. Biol. 2013, 23 (23), R1031–R1033.

24

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

(23) Huerta Lwanga, E.; Gertsen, H.; Gooren, H.; Peters, P.; Salánki, T.; van der Ploeg, M.;

Besseling, E.; Koelmans, A. A.; Geissen, V. Microplastics in the Terrestrial Ecosystem:

Implications for Lumbricus terrestris (Oligochaeta, Lumbricidae). Environ. Sci. Technol.

2016, 50 (5), 2685–2691.

(24) Rodriguez-Seijo, A.; Lourenço, J.; Rocha-Santos, T. A. P.; da Costa, J.; Duarte, A. C.;

Vala, H.; Pereira, R. Histopathological and molecular effects of microplastics in Eisenia

andrei Bouché. Environ. Pollut. 2017, 220, Part A, 495–503.

(25) Hodson, M. E.; Duffus-Hodson, C. A.; Clark, A.; Prendergast-Miller, M. T.; Thorpe, K. L.

Plastic Bag Derived-Microplastics as a Vector for Metal Exposure in Terrestrial

Invertebrates. Environ. Sci. Technol. 2017, 51 (8), 4714–4721.

(26) Lagauzère; Terrail, R.; Bonzom, J.-M. Ecotoxicity of uranium to Tubifex tubifex worms

(Annelida, Clitellata, Tubificidae) exposed to contaminated sediment. Ecotoxicol.

Environ. Saf. 2009, 72 (2), 527–537.

(27) Lagauzère; Boyer, P.; Stora, G.; Bonzom, J.-M. Effects of uranium-contaminated

sediments on the bioturbation activity of Chironomus riparius larvae (Insecta, Diptera) and

Tubifex tubifex worms (Annelida, Tubificidae). Chemosphere 2009, 76 (3), 324–334.

(28) Bouché, M.-L.; Habets, F.; Biagianti-Risbourg, S.; Vernet, G. Toxic Effects and

Bioaccumulation of Cadmium in the Aquatic Oligochaete Tubifex tubifex. Ecotoxicol.

Environ. Saf. 2000, 46 (3), 246–251.

(29) Hare, L.; Tessier, A.; Warren, L. Cadmium accumulation by invertebrates living at the

sediment–water interface. Environ. Toxicol. Chem. 2001, 20 (4), 880–889.

25

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

476

477

(30) Méndez-Fernández, L.; Martínez-Madrid, M.; Rodriguez, P. Toxicity and critical body

residues of Cd, Cu and Cr in the aquatic oligochaete Tubifextubifex (Müller) based on

lethal and sublethal effects. Ecotoxicology 2013, 22 (10), 1445–1460.

(31) Karickhoff, S. W.; Morris, K. R. Impact of tubificid oligochaetes on pollutant transport in

bottom sediments. Environ. Sci. Technol. 1985, 19 (1), 51–56.

(32) Coler, R. A.; Gunner, H. B.; Zuckerman, B. M. Selective feeding of tubificids on bacteria.

Nature 1967, 216 (5120), 1143–1144.

(33) Rodriguez, P.; Martinez-Madrid, M.; Arrate, J. A.; Navarro, E. Selective feeding by the

aquatic oligochaete Tubifex tubifex (Tubificidae, Clitellata). Hydrobiologia 2001, 463 (1–

3), 133–140.

(34) Reynoldson, T. B.; Thompson, S. P.; Bamsey, J. L. A sediment bioassay using the

tubificid oligochaete worm Tubifex tubifex. Environ. Toxicol. Chem. 1991, 10 (8), 1061–

1072.

(35) Wiederholm, T.; Dave, G. Toxicity of metal polluted sediments to Daphnia magna and

Tubifex tubifex. Hydrobiologia 1989, 176–177 (1), 411–417.

(36) Milbrink, G. Biological characterization of sediments by standardized tubificid bioassays.

Hydrobiologia 1987, 155 (1), 267–275.

(37) Kaonga, C. C.; Kumwenda, J.; Mapoma, H. T. Accumulation of lead, cadmium,

manganese, copper and zinc by sludge worms; Tubifex tubifex in sewage sludge. Int. J.

Environ. Sci. Technol. 2010, 7 (1), 119–126.

(38) Mosleh, Y. Y.; Paris-Palacios, S.; Biagianti-Risbourg, S. Metallothioneins induction and

antioxidative response in aquatic worms Tubifex tubifex (Oligochaeta, Tubificidae)

exposed to copper. Chemosphere 2006, 64 (1), 121–128.

26

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

(39) Chapman, P. M. Utility and relevance of aquatic oligochaetes in ecological risk

assessment. In Aquatic Oligochaete Biology VIII; Springer, 2001; pp 149–169.

(40) Chapman, K. K.; Benton, M. J.; Brinkhurst, R. O.; Scheuerman, P. R. Use of the aquatic

oligochaetes Lumbriculus variegatus and Tubifex tubifex for assessing the toxicity of

copper and cadmium in a spiked-artificial-sediment toxicity test. Environ. Toxicol. 1999,

14 (2), 271–278.

(41) Brinkhurst, R. O. A guide for the identification of British aquatic Oligochaeta; Freshwater

Biological Association Kendal, Wilson, 1971.

(42) Brinkhurst, R. O. Guide to the freshwater aquatic microdrile oligochaetes of North

America. Canada. Spec Publ Fish Aquat Sci 1986, 84.

(43) Brinkhurst, R. O.; Jamieson, B. G. M. Aquatic Oligochaeta of the world. 1971.

(44) Klemm, D. J. A guide to the freshwater Annelida (Polychaeta, Naidid and tubificid

Oligochaeta, and Hirudinea) of North America; Kendall Hunt Publishing Company, 1985.

(45) Williams, A. E.; Waterfall, R. J.; White, K. N.; Hendry, K.; others. Manchester Ship Canal

and Salford Quays: industrial legacy and ecological restoration. Ecol. Ind. Pollution–

Cambridge Univ. Press Camb. 2010, 276.

(46) Gillis, P. L.; Dixon, D. G.; Borgmann, U.; Reynoldson, T. B. Uptake and depuration of

cadmium, nickel, and lead in laboratory-exposed Tubifex tubifex and corresponding

changes in the concentration of a metallothionein-like protein. Environ. Toxicol. Chem.

2004, 23 (1), 76–85.

(47) Rochman, C. M.; Tahir, A.; Williams, S. L.; Baxa, D. V.; Lam, R.; Miller, J. T.; Teh, F.-

C.; Werorilangi, S.; Teh, S. J. Anthropogenic debris in seafood: Plastic debris and fibers

from textiles in fish and bivalves sold for human consumption. Sci. Rep. 2015, 5.

27

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

(48) Dehaut, A.; Cassone, A.-L.; Frère, L.; Hermabessiere, L.; Himber, C.; Rinnert, E.; Rivière,

G.; Lambert, C.; Soudant, P.; Huvet, A.; et al. Microplastics in seafood: Benchmark

protocol for their extraction and characterization. Environ. Pollut. 2016, 215, 223–233.

(49) Karami, A.; Golieskardi, A.; Choo, C. K.; Romano, N.; Ho, Y. B.; Salamatinia, B. A high-

performance protocol for extraction of microplastics in fish. Sci. Total Environ. 2017, 578,

485–494.

(50) De Witte, B.; Devriese, L.; Bekaert, K.; Hoffman, S.; Vandermeersch, G.; Cooreman, K.;

Robbens, J. Quality assessment of the blue mussel (Mytilus edulis): Comparison between

commercial and wild types. Mar. Pollut. Bull. 2014, 85 (1), 146–155.

(51) Faure, F.; Demars, C.; Wieser, O.; Kunz, M.; Alencastro, L. F. de. Plastic pollution in

Swiss surface waters: nature and concentrations, interaction with pollutants. Environ.

Chem. 2015, 12 (5), 582–591.

(52) Fischer, E. K.; Paglialonga, L.; Czech, E.; Tamminga, M. Microplastic pollution in lakes

and lake shoreline sediments – A case study on Lake Bolsena and Lake Chiusi (central

Italy). Environ. Pollut. 2016, 213, 648–657.

(53) Ballent, A.; Corcoran, P. L.; Madden, O.; Helm, P. A.; Longstaffe, F. J. Sources and sinks

of microplastics in Canadian Lake Ontario nearshore, tributary and beach sediments. Mar.

Pollut. Bull. 2016, 110 (1), 383–395.

(54) Su, L.; Xue, Y.; Li, L.; Yang, D.; Kolandhasamy, P.; Li, D.; Shi, H. Microplastics in Taihu

Lake, China. Environ. Pollut. 2016, 216, 711–719.

(55) Zhang, K.; Su, J.; Xiong, X.; Wu, X.; Wu, C.; Liu, J. Microplastic pollution of lakeshore

sediments from remote lakes in Tibet plateau, China. Environ. Pollut. 2016, 219, 450–455.

28

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

(56) Lazim, M. N.; Learner, M. A. The life-cycle and productivity of Tibifex tubifex

(Oligochaeta; Tibificidae) in the Moat-Feeder Stream, Cardiff, South Wales. Ecography

1986, 9 (3), 185–192.

(57) Palmer, M. F. Aspects of the respiratory physiology of Tubifex tubifex in relation to its

ecology. J. Zool. 1968, 154 (4), 463–473.

(58) Anlauf, A. Some characteristics of genetic variants of Tubifex tubifex (Müller, 1774)

(Oligochaeta: Tubificidae) in laboratory cultures. Hydrobiologia 1994, 278 (1), 1–6.

(59) Hämer, J.; Gutow, L.; Köhler, A.; Saborowski, R. Fate of Microplastics in the Marine

Isopod Idotea emarginata. Environ. Sci. Technol. 2014, 48 (22), 13451–13458.

(60) Li, J.; Qu, X.; Su, L.; Zhang, W.; Yang, D.; Kolandhasamy, P.; Li, D.; Shi, H.

Microplastics in mussels along the coastal waters of China. Environ. Pollut. 2016, 214,

177–184.

(61) Brinkhurst, R. O.; Chua, K. E.; Kaushik, N. K. Interspecific Interactions and Selective

Feeding by Tubificid Oligochaetes1. Limnol. Oceanogr. 1972, 17 (1), 122–133.

(62) Brinkhurst, R. O.; Austin, M. J. Assimilation by Aquatic Oligochaeta. Int. Rev. Gesamten

Hydrobiol. Hydrogr. 1979, 64 (2), 245–250.

(63) Matisoff, G.; Wang, X.; McCall, P. L. Biological Redistribution of Lake Sediments by

Tubificid Oligochaetes: Branchiura sowerbyi and Limnodrilus hoffmeisteri/Tubifex

tubifex. J. Gt. Lakes Res. 1999, 25 (1), 205–219.

(64) McCormick, A.; Hoellein, T. J.; Mason, S. A.; Schluep, J.; Kelly, J. J. Microplastic is an

Abundant and Distinct Microbial Habitat in an Urban River. Environ. Sci. Technol. 2014,

48 (20), 11863–11871.

29

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

(65) Lagarde, F.; Olivier, O.; Zanella, M.; Daniel, P.; Hiard, S.; Caruso, A. Microplastic

interactions with freshwater microalgae: Hetero-aggregation and changes in plastic density

appear strongly dependent on polymer type. Environ. Pollut. 2016, 215, 331–339.

(66) Oberbeckmann, S.; Löder, M. G. J.; Labrenz, M. Marine microplastic-associated biofilms

– a review. Environ. Chem. 2015, 12 (5), 551–562.

(67) Carpenter, E. J.; Anderson, S. J.; Harvey, G. R.; Miklas, H. P.; Peck, B. B. Polystyrene

spherules in coastal waters. Science 1972, 178 (4062), 749–750.

(68) Shaw, D. G.; Day, R. H. Colour- and form-dependent loss of plastic micro-debris from the

North Pacific Ocean. Mar. Pollut. Bull. 1994, 28 (1), 39–43.

(69) Moore, C. J. Synthetic polymers in the marine environment: A rapidly increasing, long-

term threat. Environ. Res. 2008, 108 (2), 131–139.

(70) Wright, S. L.; Thompson, R. C.; Galloway, T. S. The physical impacts of microplastics on

marine organisms: A review. Environ. Pollut. 2013, 178, 483–492.

(71) Rochman, C. M. The Role of Plastic Debris as Another Source of Hazardous Chemicals in

Lower-Trophic Level Organisms; The Handbook of Environmental Chemistry; Springer

Berlin Heidelberg, 2016; pp 1–15.

(72) Reisser, J.; Proietti, M.; Shaw, J.; Pattiaratchi, C. Ingestion of plastics at sea: does debris

size really matter? Front. Mar. Sci. 2014, 1.

(73) Young, J. O.; Ironmonger, J. W. A laboratory study of the food of three species of leeches

occurring in British lakes. Hydrobiologia 1980, 68 (3), 209–215.

(74) Egeler, P.; Meller, M.; Roembke, J.; Spoerlein, P.; Streit, B.; Nagel, R. Tubifex tubifex as

a link in food chain transfer of hexachlorobenzene from contaminated sediment to fish.

Hydrobiologia 2001, 463 (1–3), 171–184.

30

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

590

(75) Dunbrack, R. L. Feeding of Juvenile Coho Salmon (Oncorhynchus kisutch): Maximum

Appetite, Sustained Feeding Rate, Appetite Return, and Body Size. Can. J. Fish. Aquat.

Sci. 1988, 45 (7), 1191–1196.

31

591

592

593

594

595