Microplastic particles reduce reproduction in the terrestrial worm Enchytraeus crypticus in a soil exposure

Journal Pre-proof Microplastic particles reduce reproduction in the terrestrial worm Enchytraeus crypticus in a soil exposure Elma Lahive, Alexander Walton, Alice A. Horton, David J. Spurgeon, Claus Svendsen PII:

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DOI:

https://doi.org/10.1016/j.envpol.2019.113174

Reference:

ENPO 113174

To appear in:

Environmental Pollution

Received Date: 21 March 2019 Revised Date:

3 September 2019

Accepted Date: 3 September 2019

Please cite this article as: Lahive, E., Walton, A., Horton, A.A., Spurgeon, D.J., Svendsen, C., Microplastic particles reduce reproduction in the terrestrial worm Enchytraeus crypticus in a soil exposure, Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113174. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical abstract

Microplastic particles reduce reproduction in the terrestrial worm Enchytraeus crypticus in a soil exposure

Elma Lahive*, Alexander Walton, Alice A Horton, David J Spurgeon, Claus Svendsen

Centre for Ecology and Hydrology, Maclean Building, Benson Lane, Crowmarsh Gifford, Wallingford, OX10 8BB

*corresponding author

1

Abstract

2

Terrestrial environments are subject to extensive pollution by plastics and, based on the slow

3

degradation of plastics, are likely to act as long term sinks for microplastic debris. Currently the

4

hazards of microplastics in soil and the potential impacts on soil organisms is poorly understood.

5

Particularly the role of particle characteristics, such a size or polymer type, in dose-response

6

relationships for microplastics is not known. The aim of this study was to assess the ingestion and

7

toxicity of nylon (polyamide) particles, in three different size ranges, to Enchytraeus crypticus in a

8

soil exposure. Effects were also compared with those of polyvinyl chloride (PVC) particles, in a single

9

size range. Nylon particle ingestion was confirmed using fluorescence microscopy, with greatest

10

ingestion for particles in the smallest size range (13-18 µm). To investigate how particle size affected

11

survival and reproduction, E. crypticus were exposed to nylon particles in two well-defined size

12

ranges (13-18 and 90-150 µm) and concentrations of 20, 50, 90 and 120 g/kg (2-12 % w/w). An

13

intermediate nylon size range (63-90 µm) and a larger sized PVC particle (106-150 µm), both at 90

14

g/kg, were also tested. Survival was not affected by either of the polymer types or sizes.

15

Reproduction was significantly reduced, in a dose-dependent manner, by the nylon particles at high

16

exposure concentrations (>90 g/kg). Smaller size ranges (13-18 µm) had a greater effect compared

17

to larger size ranges (>63 µm), with a calculated EC50 for the 13-18 µm size range of 108 ± 8.5 g/kg.

18

This greater hazard could be qualitatively linked with the ingestion of a greater number of smaller

19

particles. This study highlights the potential for toxic effects of plastics in small size ranges to soil

20

organisms at high exposure concentrations, providing understanding of the hazards microplastics

21

may pose in the terrestrial environment.

22 23

Capsule: High concentrations (~10% w/w) of microplastics did not affect survival but did reduce

24

reproduction in Enchytraeus crypticus, particularly in the smaller size range (13-18 µm).

25

Keywords: Nylon, polyamide, earthworms, PVC, plastic, pollution

26

1. Introduction

27

Soil plays a vital role in Earth systems; critical to supporting life on earth, providing food and habitat

28

as well as being important for water filtration and carbon storage. Soils also underpin urban and

29

industrial development and inevitably become the terminus for the disposal of solid wastes. As such,

30

terrestrial environments are subject to extensive pollution by plastics of all sizes. This includes

31

anthropogenic plastic inputs from planned amendments (e.g. sewage sludge application, plastic

32

mulches in agriculture) and diffuse sources (e.g. general littering, atmospheric transport, runoff from

33

roads) (Horton et al. 2017; Nizzetto, Futter, and Langaas 2016b; Rillig, Ingraffia, and Machado 2017;

34

Machado et al. 2018; Corradini et al. 2019; Bläsing and Amelung 2018; Dris et al. 2016; Weithmann

35

et al. 2018). Based on the slow degradation rates of plastics, soils are, therefore, likely to act as long-

36

term sinks for macroplastic and microplastic debris (He et al. 2018; Rillig 2012; Zubris and Richards

37

2005; Hurley and Nizzetto 2018). With mass flow to soils predicted to be 40 times larger than those

38

to aquatic systems, it has been suggested that soils may represent a larger reservoir for plastics in

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the environment than the oceans (Horton et al. 2017; Hurley and Nizzetto 2018; Kawecki and

40

Nowack 2019). As such there is a growing recognition of the need to understand the impacts plastics

41

may have on soil ecosystem functioning.

42

In recent years the number of studies investigating the effects of microplastics on terrestrial

43

organisms (earthworms, isopods, plants and snails) has started to increase. The majority to-date

44

show limited or no effects on life history traits such as survival, growth or reproduction (Table S1)

45

(Prendergast-Miller et al. 2019; Song et al 2019; Kokalj et al. 2018; Hodson et al. 2017; Rodriguez-

46

Seijo et al. 2017; Wang et al. 2019). Some have gone further to investigate sub-lethal effects, finding

47

altered gene expression, signs of oxidative stress and changes in energy metabolism, as well as

48

evidence of histopathological gut damage as a result of exposure to microplastics (Table S1)

49

(Prendergast-Miller et al. 2019; Song et al. 2019; Rodriguez-Seijo et al. 2017; Wang et al. 2019). One

50

longer term experiment (60 day) found growth of the earthworm L. terrestris was impeded when

51

exposed to plastic concentrations 28-60 % w/w in leaf litter (Huerta Lwanga et al. 2016). Similarly Qi

52

et al. (2018) showed negative impacts on wheat growth in the presence of two plastics, polyethylene

53

(PE) and a biodegradable plastic over four months (Qi et al. 2018). Together these studies suggest

54

hazard associated with exposure to microplastics may be relatively low over a short-term exposure

55

although a systematic assessment of risk would be very difficult given the small number of studies

56

and uncertainty around the concentrations of plastic and diversity of plastic types that occur in the

57

terrestrial environments.

58

Table S1

59

Microplastic ecotoxicology research should adhere to the standard common practice in chemical risk

60

assessment (Koelmans et al. 2017). Although many of the reported studies do set out to assess the

61

effects of microplastics, concentration-response effects are less common. This is likely due to too

62

few concentrations being tested (<3), the highest concentration tested not being sufficient to cause

63

effect or no concentration-response relationships observed (Table S1). Concentration–response

64

analysis is a major part of the hazard characterization within risk assessment and as such tests that

65

establish a concentration-response relationship are needed to provide more appropriate

66

information to assess the present and future risk of microplastics (Everaert et al. 2018; WHO 2009).

67

Further, currently it is difficult to link the observed effects with species traits or particle

68

characteristics (e.g. size, polymer composition). Hence, in order to assess hazard, there is a clear

69

motivation for work to establish exposure-effect relationships that also help elucidate the link

70

between plastic particle characteristics and observed effects.

71

To address some critical gaps in knowledge surrounding the effects of of microplastics on soil

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species, the aim of this study was to assess the ingestion of nylon (nylon 6, polyamide) by the

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enchytraeid worm Enchytraeus crypticus and to assess toxicity across a dose-range, using particles in

74

well-defined size ranges, in a soil exposure. Two dense polymer types were considered, nylon (1.15

75

g/cm3) and polyvinyl chloride (PVC) (1.38 g/cm3). Dense polymers are more likely to associate with

76

sludge in wastewater treatment processes and as such can ultimately reach soils through land

77

application of sludge (Lares et al. 2018). Nylon is a generic name for a family of polymers

78

polyamides. It has a broad range of commercial applications as fabric, fibres and films. PVC is the

79

third most widely produced synthetic plastic polymer, with numerous applications. E. crypticus, is a

80

small worm (~ 1 cm in length), which is a suitable model species for such studies as their small

81

mouthparts allow for likely selective handling and internalisation potential for plastic particles in the

82

micron size range.

83 84

2. Materials and Methods

85 86

2.1 Test organisms

87

Enchytraeus crypticus (Enchytraeidae; Oligochaeta; Annelida) were originally sourced from the

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laboratory of the Department of Ecological Science, Vrije Universiteit, Amsterdam, The Netherlands

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and were maintained in culture at UK Centre for Ecology and Hydrology, Wallingford, UK for six

90

months prior to experiments. The cultures were kept on agar media prepared with Lufa 2.2 soil

91

extract, and maintained in a climate room at constant temperature (18 ± 1 °C) in constant dark. The

92

culture was fed with a mixture of oatmeal, dried baker's yeast, egg yolk powder, and fish oil. Adult E.

93

crypticus with white spots in the clitellum region were selected for the ingestion and toxicity

94

experiments.

95 96

2.2 Experimental particles

97

Three different size ranges of nylon 6 (polyamide) were used for Enchytraeid tests: 13-18 µm

98

(particles <50 µm, mean size = 13-18 µm, density = 1.15 g cm-3) (Goodfellow Scientific, United

99

Kingdom) and two larger sizes of 63-90 µm and 90-150 µm. These were virgin microplastics and were

100

not reported to contain additives. As it was not possible to find a commercial supplier of nylon

101

particles in the two larger sizes, these particles were generated in-house by grinding nylon-6 pellets

102

(Sigma Aldrich, United Kingdom) under liquid nitrogen. An amount of 5 g of the nylon pellets were

103

placed in grinding jars with stainless steel ball bearings also containing liquid nitrogen and pulverised

104

in a Tissue lyser (Qiagen) for two minutes. The powder which resulted from the grinding was then

105

suspended in water and filtered to produce the two size fractions used for testing. High molecular

106

weight PVC (polyvinyl chloride) particles (106-150 µm) were obtained from Sigma Aldrich, UK.

107

Nylon particles with a nominal size of 63-90 µm and 90-150 µm, and PVC particles with a nominal

108

size of 106-150 µm were mapped using an FTIR (Perkin Elmer Spotlight 400) at UK Centre for Ecology

109

& Hydrology to confirm the size distribution of these particles. Dry powder was finely distributed by

110

hand onto a 5 µm silver filter disc and an area of 10 mm x 10 mm was mapped using four

111

accumulations (i.e. four scans per pixel) at a resolution of 25 µm per pixel. This produced an infrared

112

heat map in which distinct polymer particles were visible (Fig S1). These mapped images could then

113

be analysed using image analysis software (Image J) based on known scale and pixel size. This

114

spectral data was also used to confirm our known polymer types of nylon and PVC. The smallest

115

nylon particle (13-18 µm) were measured Coulter Counter (Multisizer 3, Beckman, USA) as the FTIR

116

resolution was not sensitive enough for individual particle analysis.

117 118

2.3 Nylon particle ingestion

119

To qualitatively assess the ingestion of nylon particles, E. crypticus were exposed to the 13-18, 63-90

120

and 90-150 µm sized particles at a concentration of 20 g/kg (2 % w/w) in Lufa 2.2 soil and a control

121

soil, also Lufa 2.2, without added microplastics. To detect and track ingestion, the particles were

122

initially dyed with RADGLO fluorescent dye (Radiant color, Belgium). Dyed particles were initially

123

mixed with a 50 g aliquot of dry soil that was then made up to 150 g dry weight by further test soil

124

addition. The soil was then transferred to a plastic container (96 x 45 cm, polypropylene, Graham

125

Tyson, UK) and wetted to 50 % water holding capacity (WHC). After one day soil equilibration, adult

126

E. crypticus were added to each particle size treatment and exposed for 20 hours, approximately ten

127

times an estimated E. crypticus gut transition time (van Vliet, van der Zee, and Ma (2005). Following

128

the exposure, the worms were removed from soil by gentle washing over a fine sieve. Individuals

129

were rinsed, mounted onto a slide and examined for fluorescence using a Horiba HR800-UV

130

fluorescence microscope (Olympus, UK) using a BX41 object U-RFL-T Halogen light unit (Olympus,

131

UK). Images were captured along the length of each organism and composite images created for

132

individuals.

133 134

2.4 Toxicity tests with nylon and PVC particles

135

Nylon particles from two size ranges, 13-18 µm and 90-150 µm, were spiked in the Lufa 2.2 soil as

136

for the ingestion study, to establish concentration ranges for each of the two microplastic sizes of:

137

20, 50, 90 and 120 g nylon/kg (equivalent to 2-12 % w/w of soil dry weight). For the intermediate-

138

sized particles (63-90 µm) there was insufficient material available for a full concentration range, and

139

so only the 90 g/kg (9% w/w) concentration could be run. In addition to testing the effects of two

140

differently sized nylon microplastics, a further toxicity test was also run with PVC at the 90 g/kg

141

concentration (Table S2). This allowed a robust comparison of nylon toxicity across the particle sizes,

142

and between the two polymer types at the exposure concentration 90 g/kg. The concentration

143

ranges were based on pilot experiments which saw effects at 90 g/kg or greater (data not shown).

144

Given it is difficult to know what the environmental concentrations for plastics in different size

145

ranges will be these concentrations were chosen to reflect where effects on organisms might be

146

expected and hazard is expected. Four replicates were used per treatment except for the 90 g/kg

147

treatments where six replicates were used. Eight soil control replicates without added microplastics

148

were used.

149

The test protocol followed Enchytraeid reproduction test OECD 220 (OECD 2004). Spiked Lufa 2.2

150

soils were wetted to 50 % of WHC and 30 g wet weight transferred to each experimental container

151

(50 ml centrifuge tubes) (115 x 30 mm, polypropylene, Fisher Scientific, UK). As food, 0.1 g of finely

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ground oats was added to the soil surface. To initiate the test, ten adult E. crypticus were added to

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the soil surface and the weight of each tube recorded. Fine cotton mesh was added between tube

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rim and lid to prevent excessive moisture loss. Lids had small holes inserted to allow for ventilation.

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The tubes were kept at 18 ± 1 °C, in constant dark for 21 days. Water content (mass loss from water

156

evaporation) and food availability (visually assess amount of oats left on the surface) were checked

157

weekly and additional oats or water added as necessary.

158

After 21 days , the test was terminated by adding 20 ml of 97% ethanol (Sigma Aldrich, UK) and 300

159

µl of Rose Bengal lipid stain (Sigma Aldrich, UK) (1% in ethanol). Tubes were left overnight to allow

160

staining, after which samples were passed through a 160 µm mesh and juveniles counted.

161 162

2.5 Data analysis

163

Survival and reproduction data were checked for normality using Shapiro-Wilks test. Survival and

164

reproduction data were fitted to a three parameter log-logistic model (Equation 1):

165

y = ymax/(1+cc/EC50)exp(b))

166

where ymax is the upper asymptote; cc the exposure concentration; EC50 the concentration resulting

167

in 50% effect on measured endpoint and b the slope parameter. Dose-response curves for juvenile

168

production were plotted as a change from the mean of the controls. Responses at the 90 g/kg (9%

169

w/w) concentration used for all particle types were compared using ANOVA, with Tukey’s post-hoc

170

test used to compare responses between treatments.

171 172

3. Results

Equation 1

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3.1 Particle characterisation

174

The nominal size ranges for the nylon particles were confirmed by FTIR and Coulter counter analysis

175

(Figure S1). The three particles were sized 19.95 ± 3.6, 77.6 ± 21.4 and 150.9 ± 36.4 µm, respectively

176

(Figure 1). The PVC particle was sized as 140.4 ± 27.9 µm (Figure 1). The particles were non-spherical

177

and polymorphic in shape (Figure S1).

178 179

Figure 1, Figure S1

180 181

3.2 Ingestion of nylon particles by E. crypticus

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Nylon particles of all size fractions were ingested by E. crypticus (Figure 2). The number of particles

183

ingested was qualitatively greater in the worms exposed to 13-18 µm compared to the other two

184

size fractions, most noticeably the largest (90-150 µm) materials which showed low ingestion.

185 186

Figure 2

187 188

3.3 E. crypticus survival and reproduction

189

Enchytraeid survival was not significantly reduced by either sized nylon particle after 21 days at any

190

of the exposure concentrations tested (one-way ANOVA: 13-18 µm, F4, 21 = 0.48, P = 0.75; 90-150 µm,

191

F4, 21 = 1.7, P = 0.18) (Figure S2).

192 193 194

Figure S2

195

Reproduction, measured as the number of juveniles produced per worm over 21 days, was

196

significantly reduced by exposure concentrations used for both the 13-18 µm and 90-150 µm nylon

197

particles (Figure 3). Increasing concentrations of 13-18 µm nylon concentration in the soil

198

significantly reduced the production of juveniles (one-way ANOVA, F4, 21 = 16.09, P < 0.0001). For this

199

particle size, juvenile production was significantly reduced compared to the controls in the 90 g/kg

200

(9% w/w) and 120 g/kg (12% w/w) treatments (post-hoc Tukey: P < 0.001 for both concentrations).

201

The 21 day EC50 calculated for the 13-18 µm nylon particles was 108 ± 8.5 g/kg (Figure 3). E. cryticus

202

reproduction was also significantly reduced by nylon particles in the size range 90-150 µm (one-way

203

ANOVA, F4, 21 = 8.42, P = 0.0003). However, production of juveniles was reduced by less than 50% at

204

the maximum exposure concentration (120 g/kg) and consequently an EC50 value could not be

205

reliably estimated.

206 207

Figure 3

208 209

The two nylon particles exposed over a full concentration series were compared with the

210

intermediate-sized nylon (63-90 µm) and the PVC particles (106-150 µm) in exposures with the four

211

materials conducted at a concentration 90 g/kg (Figure 4). There was no significant difference in

212

adult survival when comparing the three sizes of nylon (13-18 µm, 63-90 µm and 90-150 µm), the

213

PVC particle type at 90 g/kg and the control group (one-way ANOVA, F4,

214

Production of juveniles was significantly lower in all nylon particle size treatments at 90 g/kg than in

215

the control (one-way ANOVA F4,

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reduction (25-35%) when compared to the controls. No significant effect of PVC exposure on E.

217

crypticus reproduction was found (P >0.05).

218

27

19

= 1.174, P =0.34).

= 17.182, P < 0.0001); each treatment resulting in a similar

219

Figure 4

220 221

4. Discussion

222

4.1 Ingestion of microplastics in relation to particle size

223

Earthworms living in soils are in constant contact with particles of various kinds, be it soil particles

224

such as sand, silt and clay, organic material such leaf litter that becomes fragmented in soils or

225

anthropogenic material such as plastics. Given their regular exposure to particles, a major question

226

surrounding microplastics is whether they will be ingested by organisms along with, or instead of,

227

soil or food particles and, thereafter, whether they will exert negative effects. This study showed

228

clearly that enchytraeids exposed in soil will ingest microplastic particles in a size-dependant

229

manner. Previous research has shown E. crypticus can also ingest nanoplastics (0.05–0.1 μm) (Zhu et

230

al. 2018), and although they did not observe negative effects on reproduction, they found worm

231

biomass reduced. The pharyngeal bulb, the mouth part which draws in food particles to the fore gut,

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in E. crypticus is roughly 72 µm across and 90 µm in length (Westheide and Graefe 1992). Based on

233

these physiological constraints, it would be expected that particles at, or below, these dimensions

234

could potentially be ingested by feeding enchytraeids. Qualitative assessment of internalised

235

fluorescence confirms such potential size limitation. A higher number of 13-18 and 63-90 µm sized

236

particles were ingested compared to particles in the larger 90-150 µm size range. However, despite

237

these potential physiological limitations, some particles were ingested in this larger size range. The

238

fact that the lower end of the larger size range was close to the size dimensions of E. crypticus

239

mouthparts and that particles were not spherical, and so could have been smaller in other

240

dimensions, may explain the observed ingestion of some larger size range particles. Although given

241

the reduced amounts, size exclusion based on enchytraeid mouthpart size remains a likely limiting

242

mechanism.

243

Size has previously been shown to affect microplastic ingestion and egestion (Hurley, Woodward,

244

and Rothwell 2017; Redondo-Hasselerharm et al. 2018), with ingestion most affected by particle size

245

and feeding traits (Windsor et al. 2019; Scherer et al. 2017). Our observation of potential

246

physiological limitations on ingestion by enchytraeids are consistent with previous evidence. For

247

example in sediment dwelling Tubifex worms which normally ingest natural particles <63 µm; plastic

248

particles above this size range were also not found to be ingested (Hurley, Woodward, and Rothwell

249

2017). L. terrestris showed preference for particles <50 µm when multiple sizes were available,

250

based on analysis of the casts, suggesting some selectivity in the sizes of particles ingested, or indeed

251

egested (Huerta Lwanga et al. 2016; Huerta Lwanga et al. 2017). Following ingestion, egestion is

252

likely to also be governed by the shape or form of the plastic particles (Frydkjaer, Iversen, and Roslev

253

2017; Hurley, Woodward, and Rothwell 2017; Redondo-Hasselerharm et al. 2018). Regular shaped

254

spheres have been shown to be more rapidly egested by aquatic organisms than irregular-shaped

255

particles (Frydkjaer, Iversen, and Roslev 2017) or fibres (Hurley, Woodward, and Rothwell 2017; Au

256

et al. 2015). Further plastic particles have been found to be retained longer that sediment materials

257

or natural food items aquatic organisms (Hurley, Woodward, and Rothwell 2017; Watts et al. 2014;

258

Welden and Cowie 2016; Woods et al. 2018). For the particles used in this study which were

259

irregularly shaped, the possibility of increased retention time for particles in the gut might be

260

associated with the observed effects.

261 262

4.2 Effects of microplastics

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Enchytraeus crypticus survival was not affected by microplastic exposure, with >85% survival across

264

all treatments. This is in line with previous results from studies conducted with other soil organisms

265

exposed to microplastics in short term tests (≤4 weeks) (Hodson et al. 2017; Kokalj et al. 2018;

266

Huerta Lwanga et al. 2016; Rodriguez-Seijo et al. 2017) but also from extended bioassays (60 days)

267

with < 0.2% v/v microplastics in the soil (from 7% in leaf litter) (Huerta Lwanga et al. 2016). Survival

268

of Eisenia fetida was not affected by exposure to polyethylene particles (up to 1 g/kg) (Rodriguez-

269

Seijo et al. 2017). Although Huerta Lwanga et al. (2016) found no effect of microplastics up to < 0.2%

270

v/v of polystyrene microplastics in the soil (from 7% in leaf litter) on the survival of Lumbricus

271

terrestris, effects on mortality and growth were found at concentrations above 28% and up to 60%

272

microplastics in added plant litter (equivalent to 0.4-1.2 % v/v in soil). Mortality (~40%) was also

273

increased in E. fetida exposed to 2 g/kg (2% w/w) polystyrene in soil (Cao et al. 2017). As these

274

effects were found at concentrations ten-fold below the maximum concentration used in this study

275

(12% w/w, 120 g/kg), this suggests the potential for greater microplastic effects in lumbricids,

276

especially under longer exposure periods, such as the 60 day duration used by Huerta Lwanga et al.

277

(2016). Such observations highlight the importance of studies conducted across different species and

278

also the need to consider both exposure concentration and time in assessments of microplastic

279

toxicity.

280

Clear dose-response effects of microplastic exposure on earthworm reproduction has not been

281

reported previously. Rodriguez-Seijo et al. (2017) found no effects on juvenile production in Eisenia

282

andrei, but some inflammation of the gut was observed. Similarly, Huerta Lwanga et al. (2016) found

283

no effect on L. terrestris reproduction (cocoon production) at exposure concentrations up to 1.2%

284

v/v soil. In their study, earthworms were exposed by mixing microplastic spiked leaf litter into the

285

upper soil layers and although higher concentrations of microplastics were observed in earthworms

286

with increasing leaf litter concentration, evidence was found that, at the highest exposure

287

concentrations earthworms, were avoiding the surface layers. Hence, the nature of exposure in their

288

system is uncertain. However, avoidance of plastics contaminated soils has also been observed for E.

289

fetida (Rodríguez-Seijo, 2019). Zhu et al. (2018) did observe effects of nanoplastics on E. crypticus

290

reproduction after seven days exposure through addition to food. However, there was not a dose-

291

response relationship for the observed effects. Thus, the highest concentration (10% w/w diet) did

292

not significantly change reproduction, while the lower concentration (0.5%, w/w diet) showed a

293

significant increase in reproduction compared to the control. The above studies assume dietary

294

exposure scenarios, Huerta Lwanga et al. (2016) (leaf litter exposure) and Zhu et al. (2018) (diet

295

exposure via oats), and this

296

concentration dependent effects on reproduction observed in this study where exposure was

297

through the soil itself, possibly more realistic exposure pathway. The reproductive effects seen here

298

for E. crypticus at high soil concentrations (108 g/kg (10.8 %), 13-18 µm nylon) do indicate that there

299

is possibility for reproductive output to be depressed, even in the absence of mortality. Ultimately,

300

exposure to high plastic concentrations in soils, therefore, do have the potential for sub-lethal

301

effects that may to lead to changes in population dynamics.

302

The microplastic concentrations used in this study were high (highest = 120 g/kg, 12% w/w),

303

however, it is difficult to compare the levels with environmental concentrations as these still remain

304

relatively unknown. Soils around landfill sites or on agricultural land where plastic mulches are used

305

have the potential to become long-term sinks for plastics and as such have the potential to

306

accumulate high (micro)plastics concentrations (Rillig 2012; Nizzetto, Futter, and Langaas 2016a(Liu

307

et al. 2018; Huerta Lwanga et al. 2017; Zubris and Richards 2005). Indeed, of the few studies

308

available which report mass concentration and not particle concentration, the concentrations used

309

here do correspond to plastic mass in some industrial soils (max. reported = 67.5 g/kg) (Fuller and

310

Gautam 2016), but much higher than those reported for floodplain soils (Scheurer and Bigalke 2018).

311

It should be noted though that environmental studies are limited by the size detection limits of the

312

analytical techniques (for particle quantification procedures this is usually <100 µm, with lower limits

313

around 10-20 µm, even using the most advanced techniques) (Käppler et al. 2016). Consequently

314

microplastic concentrations may be underestimated, particularly in the most biologically relevant

315

size fractions (Mintenig et al. 2017; Simon, van Alst, and Vollertsen 2018). Due to the complex

316

structure and high organic content of soils, particle analysis is more challenging in soils than in

317

water, effluent or even sediment-based analyses, and the limited research to date has tended not to

318

capture particles in the smaller size ranges (Wang et al. 2018; Liu et al. 2018). Although the

319

concentrations tested, and which caused effect, in this study were high compared to most reported

may explain the differences observed compared to the clear

320

environmental concentrations, in an assessment of future risk, where increased plastic production

321

and progressive fragmentation of plastic particles would be accounted for, higher than present

322

concentrations could be considered relevant (Koelmans et al. 2017).

323 324

4.3 Effect of different polymer types

325

In addition to size and shape, the type of polymer could also play a role in the extent to which

326

microplastics affect soil invertebrates (Lei et al. 2018, Wang et al 2019). It has been shown in a

327

number of aquatic studies that different polymer types can have different toxic effects on

328

organisms, based on the chemical composition or the presence of chemical additives (Lithner,

329

Nordensvan, and Dave 2012a; Li et al. 2016; Rochman 2015). From these studies it has been

330

observed that PVC is frequently one of the most toxic polymers, mainly due to plasticiser and the

331

vinyl chloride monomer itself (Li et al. 2016; Lithner, Nordensvan, and Dave 2012b). In this current

332

study, although comparison could only be made at a single exposure concentration, nylon in the size

333

range 90-150 um reduced reproduction by 25% whereas the PVC polymer in a comparable size range

334

(106-150 um) did not. This could be an indication of a chemical effect exerted by the nylon particles

335

not found for PVC, although more in depth studies would be needed to further elucidate this. In soil

336

there may be other processes acting which may limit the extent to which these chemical effects

337

occur, including the potential that the PVC particle size excludes this particle from being ingested or

338

lack of plasticisers in these particular particles. There are few comparable studies using nylon (or

339

polyamides). In the limited number of soil exposure studies, polyethylene is the most commonly

340

tested microplastic polymer type. Polyethylene is a significant polymer, being the most commonly

341

used polymer in the world, with many applications directly associated with agriculture, most notably

342

in plastic mulches. To date this polymer has been associated with toxicity only in a longer-term L.

343

terrestris study and caused subtle effects in E. fetida (Huerta Lwanga et al. 2016; Rodriguez-Seijo et

344

al.2017; Rodriguez-Seijo et al. 2018, Wang et al. 2019). Given the aim of this study was assess

345

toxicity of dense polymers that may be associated with sludge application, nylon and PVC were

346

chosen as our test particles. However applying the concentration-response approach using restricted

347

size ranges as used in this study to polyethylene would be highly relevant.

348 349

5. Conclusions

350

Microplastics entering the terrestrial environment can either be transported to waterways through

351

surface runoff or the soil can act as a sink, retaining particles in the soil ecosystem. The fate of

352

microplastics accumulated in soils is still largely unknown, with smaller size fractions (<100 µm) not

353

commonly reported largely due to the challenge of detecting materials in this size range in this

354

complex media. However, from this study there is evidence that nylon microplastics in the

355

biologically relevant size fractions (i.e. sizes that can be ingested by soil organisms) can result in a

356

dose-response reduction in worm reproduction compared to larger sized nylon particles.

357

Understanding the consequences of microplastic accumulation in soil ecosystems means having a

358

good understand of the hazards posed by plastics in the terrestrial environment in order to allow risk

359

assessors and regulators to make decisions regarding policies for plastic usage and disposal. Indeed

360

applying the approach taken in this study to one of the mostly commonly used plastics in agriculture

361

and elsewhere, polyethylene, would be a pertinent step for assessing risk of plastics in terrestrial

362

systems. Further research is necessary in order to discern the potential for long-term effects or

363

trophic transfer by understanding the factors determining ingestion and egestion of different sizes

364

and shapes of particles by soil organisms.

365 366

Acknowledgements

367

The authors wish to acknowledge Andrew Watt for his assistance in preparing the nylon particles for

368

the experiments. This work was supported with funds from the NERC GW4+ Doctoral Training

369

Partnership under grant NE/L002434/1 and NERC National Capability Funding to the Centre for

370

Ecology and Hydrology under the UK-SCAPE project grant NE/R016429/1.

371 372

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373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413

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Figures

598 599

Figure 1. The size distributions of nylon particles in the three different size ranges and a single size

600

range of PVC used for the toxicity tests, imaged using FTIR and Coulter counter (images in Figure S1).

601

The smallest particle (13-18 µm) was imaged by coulter counter due to the size limitations

602

associated with the FTIR imaging.

603 604

Figure 2. Composite images from fluorescence microscope of individual E. crypticus after 20 hours

605

exposure to soil spiked with fluorescently labelled particles (a) 13-18 µm nylon. (b) 63-90 µm nylon.

606

550 (c) 90-150 µm nylon. The images on the left show the red rectangles enlarged from the whole

607

worm images on the right. The red arrows indicate the presence of microplastics in the organism.

608

The anterior and posterior ends of the worms are indicated above the images of the whole worms.

609

The average length of the worms was 7-10 mm.

610 611

Figure 3. The number of juveniles produced per worm in soil spiked with nylon particles in two size

612

ranges, 13-18 and 90-150 µm after 21 days exposure in spiked soil. Points represent data for each

613

replicate; the solid lines represent the logistic model fit; EC50 values and slopes predicted by the

614

model are included for both particle sizes.

615 616

Figure 4. The number of juveniles produced per worm in soil spiked with nylon particles in three size

617

ranges and PVC particles at the same exposure concentration, 90 g/kg, after 21 days exposure. *

618

indicates treatments where reproduction was significantly different from the control.

619 620

Table S1. Summary of toxicological studies carried out for micron and nano-sized plastics for

621

terrestrial organisms, detailing exposure, endpoints measured and concentrations that resulted in an

622

endpoint response.

623 624

Figure S1: The four particles used in the experimental set up imaged using FTIR and Coulter counter.

625

A = Nylon (13-18 µm), B = Nylon (63-90 µm), C = Nylon (90-150 µm), D = PVC (106-150 µm). B-D were

626

characterised via FTIR. The smallest particle (13-18 µm) (A) was imaged by coulter counter due to

627

the size limitations associated with the FTIR imaging.

628 629

Figure S2. The number of surviving adults after 21 days exposure to nylon of two size fractions (13-

630

18 and 90-150 µm) at four different concentrations (20-120 g/kg, 2-12 % w/w) and a joint control

631

(no added plastic) of eight replicates used across both treatments Bars represent the mean survival

632

from four replicates for 20, 50 and 120 g/kg. Error bars represent standard deviations.

633 634 635 636 637 638 639 640

Table S2: The polymer types, nominal sizes and the concentration ranges tested in the toxicity test.

Figure 1

Figure 2

Figure 3

Figure 4

Highlights •

Enchytraeid survival was not affected by exposure to nylon or PVC microplastics.

•

Enchytraeid reproduction was reduced by smaller-sized nylon microplastics.

•

Enchytaeids ingested more particles in the smaller size ranges (13-18 µm).

•

Nylon reduced reproduction significantly more compared to PVC.

There are no conflicts to declare.