Is blue mussel caging an efficient method for monitoring

environmental microplastics pollution?

Maria Kazour, Rachid Amara

To cite this version:

Maria Kazour, Rachid Amara. Is blue mussel caging an efficient method for monitoring en-
vironmental microplastics pollution?. Science of the Total Environment, 2020, 710, pp.135649.
�10.1016/j.scitotenv.2019.135649�. �hal-03489614�

HAL Id: hal-03489614

https://hal.science/hal-03489614
Submitted on 7 Mar 2022

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est

archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents
entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non,
lished or not. The documents may come from émanant des établissements d’enseignement et de
teaching and research institutions in France or recherche français ou étrangers, des laboratoires
abroad, or from public or private research centers. publics ou privés.

Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International

License
Version of Record: https://www.sciencedirect.com/science/article/pii/S004896971935644X
Manuscript_fdd29f3067f8e5d193d06f6492bdda97

1 Is blue mussel caging an efficient method for monitoring environmental

2 microplastics pollution?

3 Maria KAZOUR1,2*and Rachid AMARA1

1
4 Univ. Littoral Côte d’Opale, CNRS, Univ. Lille, UMR 8187, LOG, Laboratoire d’Océanologie et de

5 Géosciences, 32 Avenue Foch, 62930 Wimereux, France

2
6 CNRS-L, National Center for Marine Sciences, PO Box 534, Batroun, Lebanon

7 *Corresponding author: Maria Kazour

8 E-mail: [email protected]
9

10 Abstract

11 The effectiveness of mussel caging for active microplastics (MPs) biomonitoring was
12 investigated for the first time by comparing abundance and characteristics (shape, size, color
13 and type of polymers) of MPs ingested by caged depurated blue mussels with those ingested
14 by native mussels collected at the same sites and with those found in their surrounding
15 environment (surface water and sediments). Mussels were exposed along a pollution gradient
16 originating from a wastewater treatment plant discharge and near an abandoned coastal
17 landfill. After 6 weeks of deployment, the majority (93%) of clean transplanted mussels had
18 ingested MPs with a mean number of items ranging from 0.61 to 1.67 items/g. The
19 occurrence, abundance and properties of MPs ingested by caged mussels were similar to those
20 found in native mussels. Among the debris items detected in caged and native mussels,
21 fragments were the most predominant type, consistent with the MPs found in their
22 surrounding environment. MPs sizes were very similar whether in the water, sediments and
23 both caged and native mussels, with a dominance of items < 150 µm. Although some
24 polymers were under-represented or totally absent in the caged mussels compared to
25 overlying seawater or surrounding sediment, there was a good overlap in polymer types
26 proportion being found between caged mussels and sediments (Morisita’s index of similarity
27 = 0.93) or seawater (0.86). Polystyrene dominated all samples in all the different matrices.
28 Our study suggests that blue mussels caging may be a promising tool for MPs biomonitoring
29 making monitoring more reliable with an accurate assessment of the biological effects of MPs
30 over a predetermined exposure period. However, further methodological improvements
31 should be considered to define a uniform protocol for blue mussels caging to allow spatial and
32 temporal microplastics active biomonitoring.
33
34
35
36
37 Keywords: Microplastics; Biomonitoring; Caging; Blue mussels; micro-Raman

38
39

1
© 2019 published by Elsevier. This manuscript is made available under the CC BY NC user license
https://creativecommons.org/licenses/by-nc/4.0/
40

41 Introduction
42
43 Filter-feeding bivalves such as mussels of the genus Mytilus have been widely used as
44 sentinel organisms to monitor coastal water pollution (Beyer et al., 2017; Marigómez et al.,
45 2013; Turja et al., 2014). The popularity of mussels as bioindicator stems from their
46 biological and ecological characteristics which make them virtually ideal for pollution
47 monitoring. These sedentary organisms filter large amounts of water efficiently and use
48 waterborne particles as food and, thus, are able to bioaccumulate high amounts of different
49 contaminants (Beyer et al., 2017; Farrington et al., 2016). Their broad geographical
50 distribution, abundance and ability to tolerate a wide range of environmental parameters make
51 them species of choice in large-scale and long-term marine monitoring programs (Andral et
52 al., 2004; Scott et al., 2019; Turja et al., 2014). Most often mussel biomonitoring studies
53 involve collection of samples from natural populations, but the adoption of an active
54 biomonitoring alternative by using transplanted mussel (caging) has gained considerable
55 popularity in ecotoxicology research and monitoring (Andral et al., 2004; Brooks et al., 2018;
56 Galgani et al., 2011; Lacroix et al., 2015; Marigómez et al., 2013; Tsangaris et al., 2011).
57 Mussel caging is particularly useful when indigenous mussels are scarce or absent at the
58 planned study sites (Li et al., 2019). In addition, using caged mussels from a single population
59 minimizes confounding factors such as the genetic variability and the physiological, age or
60 reproductive status of the organisms that influence both contaminant bioaccumulation
61 (Schøyen et al., 2017; Viarengo et al., 2007). However, several studies have previously
62 reported differences in response between caged and native organisms exposed to
63 environmental pollution. Observed differences were attributed to several factors: absence of
64 cumulative long-term effects of pollutants in caged organisms (Nigro et al., 2006), the
65 existence of adaptive traits or compensatory mechanisms in native mussels chronically
66 exposed to pollution, leading for example to lower bioaccumulation levels than in short-term
67 caged organisms (Greenfield et al., 2014; Lacroix et al., 2015; Marigómez et al., 2013;
68 Schøyen et al., 2017). Others studies found that native mussels accumulated higher
69 concentrations of chemicals (Bolognesi et al., 2004; Piccardo et al., 2001). In some studies,
70 the two monitoring approaches showed similar temporal and spatial changes in chemical
71 contaminant concentrations (Hunt and Slone, 2010; Piccardo et al., 2001). The caging
72 influence on mussels contaminants accumulation is still poorly understood (Schøyen et al.,

2
 73 2017). Many studies have suggested that an integrated use of monitoring data from both
74 native and transplanted mussels may provide a more accurate assessment of environmental
75 pollution (Bebianno et al., 2007; Brooks et al., 2012; Hunt and Slone, 2010; Marigómez et al.,
76 2013; Serafim et al., 2011).
77
78 Microscopic particles and fragments of plastics smaller than 5 mm (commonly called
79 microplastics, MPs) represent a matter of growing concern for the marine environment. Their
80 ingestion has been documented in several marine species worldwide (Cole et al., 2013; Watts
81 et al., 2015) particularly in coastal marine environments acting as recipients for plastics and
82 MPs input sources (Leslie et al., 2017; Kazour et al., 2019). The occurrence and sources of
83 MPs in the marine environment and their potential biological impact are subjects of major
84 scientific and public concern. To monitor spatial and temporal trends of MPs pollution and to
85 understand the risk they pose to the health of marine species, it is important to obtain accurate
86 measurements of MPs abundance in environmental compartments (such as water, sediments
87 and biota). However, in highly variable environment as marine coastal areas, the assessment
88 of MPs abundance in water and sediments is often imprecise and tend to be affected by a
89 variety of environmental factors such as winds, tides, currents and bioturbation (Scott et al.,
90 2019). A key challenge for marine MPs biomonitoring is to develop an appropriate and
91 standardized method with a sentinel organism that can be applied over large spatial and
92 temporal scales to allow cross calibration between studies worldwide. Recently, many studies
93 have focused on the uptake of MPs by mussels, both in laboratory experiments (Browne et al.,
94 2008; Van Cauwenberghe et al., 2015) and in their natural habitats (De Witte et al., 2014; Li
95 et al., 2018; Phuong et al., 2018; Qu et al., 2018; Santana et al., 2016; Zhao et al., 2018). The
96 species faces a strong exposure to microplastics because of its high filtering activity (Riisgård
97 et al., 2014). Microplastic abundance in field collected mussels is closely related to human
98 activity (Kazour et al., 2019; Li et al., 2016), and there is evidence for a positive and
99 quantitative correlation between MPs in mussels and surrounding waters (Qu et al., 2018).
100 Their capacity to integrate and amplify the MPs contamination signal (Karlsson et al., 2017)
101 led to their potential consideration as bioindicator for coastal MPs pollution (Beyer et al.,
102 2017; Li et al., 2019; Wesch et al., 2016). The International Council for the Exploration of the
103 Sea (ICES) has suggested blue mussels as suitable sentinels for monitoring of MPs
104 contamination (OSPAR, 2015; Vandermeersch et al., 2015). Although Mytilus spp. controlled
105 deployment has been largely used to study diffuse chemical contaminants and biomarkers,
106 this approach was only recently used to study MPs pollution. To our knowledge, only three

3
107 studies caged blue mussels in specific areas to investigate the MPs pollution related to
108 specific anthropogenic activity such as wastewater discharge (Railo et al., 2018), wreck
109 removal (Avio et al., 2017) or to assess seasonal changes in plastic pollution (Catarino et al.,
110 2018). This latter study suggested that the use of cage deployed mussels can be an effective
111 method to quantify and assess MPs pollution in the field whereas the study of Avio et al.
112 (2017) highlighted the limited capability of transplanted mussels to discriminate MPs
113 pollution around the wreck area.
114 Mussels are hardy creatures tolerant to handling that are easy to keep in culture, making them
115 suitable for translocation and caging exposure. In addition, the mussels can be depurated in
116 the laboratory before caging and, thus, allow the use of reference individuals to be deployed
117 in different areas. The objective of this research was to investigate the MPs ingestion in caged
118 blue mussels exposed along a pollution gradient originating from a WWTP discharge and near
119 an abandoned coastal landfill (Kazour et al., 2019). The effectiveness of the mussel caging
120 approach to quantify and assess MPs pollution in the field was assessed for the first time by
121 comparing abundance and characteristics (shape, size, color and type of polymers) of MPs
122 ingested by caged mussels with those ingested by native mussels collected at the same site
123 and MPs found in their surrounding environment (surface water and sediments).
124

125 Material and methods

126 2.1 Mussels field deployment and sampling

127 a. Mussels depuration

128 One week prior to field deployment, blue mussels (Mytilus edulis) were collected from
129 farmed Bouchot mussels from Camiers near Boulogne-sur-mer, (50°35'43.1"N, 1°34'41.3"E;
130 French Eastern English Channel coast). Mussels were sorted in the laboratory to get 157
131 individuals of the same size range (5.77 ± 0.5 cm shell length, mean ± standard deviation).
132 Seven of these mussels were immediately frozen at -20°C (reference samples collected prior
133 to the start of the depuration experiment) and the remaining 150 individuals were pre-cleaned
134 to remove any adhering organisms and placed in a 100 L glass aquarium (1 m x 0.45 m x 0.3
135 m) meticulously pre-rinsed with Milli-Q water. The aquarium was maintained in a
136 thermoregulated room with a constant temperature of 8 ± 1°C and a 12 h light-dark
137 illumination regime. The aquarium was equipped with an air pump and was supplied by
138 filtered sea water (filtered on Polycarbonate filters; 0.1 µm) and the water was changed every

4
139 24 hours for 7 consecutive days. During the experiment, the mussels were daily fed (except on
140 Saturday and Sunday) with live microalgae, Rhodomonas sp. and Isochrysis sp. in order to
141 enhance the mussels’ filtration capacity. After 7 days of depuration, seven mussels were
142 frozen at -20°C in order to compare the MPs concentration in both non-depurated and
143 depurated mussels. The rest of the mussels were used for the caging experiment.
144
145 b. Transplantation and caging experiment

146 After the depuration period, mussels were transported to caging sites in a small aquarium (60
147 L, 0.6 m x 0.35 m x 0.3 m) supplied by oxygenated filtered sea water. The translocation
148 experiment was carried out between the 3rd and the 4th of February 2018 and lasted 42 days
149 (until the 4th and 5th of April 2018). Five marine coastal sites were chosen for the caging
150 experiment: four sites in Le Havre harbor along a pollution gradient originating from a
151 WWTP discharge and one site near an abandoned coastal landfill (Figure 1; see Kazour et al.,
152 2019 for more detailed sampling locations). At each site, a stainless-steel cage (50 cm x 50
153 cm x 50 cm, length x width x height) with no plastic materials to avoid contamination was
154 deployed. The cage’s mesh size was 15 mm allowing water circulation and stopping mussels
155 from falling out of the cages. A stainless-steel grid was placed in the mid-height of the cages
156 as a suitable substrate allowing mussels’ attachment and avoiding direct contact with
157 sediments. 17 to 23 mussels were put in each cage. The cages were deployed during low tide
158 in the lower intertidal shore zone and attached to the bottom using four reinforced iron rods.
159 The study area is characterized by semi-diurnal tide with an average tidal range of about 1 m
160 at neap tides and 6 m at spring tides.
161 After retrieval, each mussel was put separately inside an aluminum foil and frozen at -20°C
162 until analysis. Water temperature, salinity and dissolved oxygen were measured using a
163 multiparameter probe (Mutiparameter HI 982, HANNA instruments) during cages
164 transplantation and retrieval.
165
166 2.2 Water, sediment and native mussels sampling

167 Water, sediments and native mussels were taken on the 3rd and 4th of April as described in
168 Kazour et al. (2019). In short, at each site, 1 to 2 m3 of the first 10 cm of the water column
169 was pumped and filtered on stainless steel sieves of different mesh sizes (500 µm, 200 µm, 80
170 µm and 20 µm). Infralittoral sediments were also collected at each site except for site 4 (due

5
171 to its rocky substrate). Sea water and sediments analyses were preformed according to Kazour
172 et al. (2019).
173 Mussels from the French Eastern English Channel coast are dominated by Mytilus edulis, but
174 some areas are also populated by hybrids between M. galloprovincialis and M. edulis. This is
175 the case of Le Havre Harbor where 70% of the mussels are hybrids (J. Couteau, personal
176 communication). Therefore, for native collected mussels the term Mytilus spp. is used in this
177 study. Native mussels (Mytilus spp.) were hand collected from the lower intertidal shore zone
178 from the same sites near the deployed cages. At each site, 10 mussels of similar size were
179 collected and immediately conserved inside an aluminum foil and frozen at -20°C until
180 analysis. In the Harbor (sites 1 to 4), mussels were bigger 6.44 ± 0.73 cm (mean shell length ±
181 standard deviation) than at site 5 (3.2 ± 0.4 cm).
182
183 2.3 Microplastics analyses
184

185 a. Contamination control

186 Microplastics contamination control is an important step during microplastics analysis.

187 Several precautions were followed. Cotton lab coats were worn throughout the experiments,
188 and all steps (filtration, measurements and digestion) were realized under a laminar flow
189 hood. Only laboratory materials (bottles, petri dishes, Erlenmeyers, filtration system) made of
190 glass were used. All used solutions were filtered three times on glass fibers filters (GF/A)
191 Whatman, (France) in order to remove any unwanted particles and fibers. All surfaces and
192 equipment were cleaned using filtered ethanol 70% and MilliQ water. With every
193 manipulation, a control was made following the same conditions as the samples in order to
194 track the contamination.
195
196 b. Samples preparation

197 Water and sediment samples were prepared as described by Kazour et al. (2019). In short,
198 water samples were rinsed inside a separation funnel and underwent a density separation step
199 using zinc chloride (ZnCl2; 1.8 g/cm3) and the supernatant was filtered on GF/A filters.
200 Sediment samples were digested using hydrogen peroxide 30% (H2O2 30%) then they
201 underwent density separation using ZnCl2.

6
202 Mussels were defrosted 4 hours prior to digestion and their total length, width (cm), total and
203 net weight (g) were measured. The condition index (CI) was calculated as follow (AFNOR,
204 1985):
ℎ
205 = ℎ × 100

206 This index is a good indicator for the physiological condition (tissue growth, energy reserves)
207 of mussels (Orban et al., 2002).

208 Mussels were digested using a solution of potassium hydroxide (KOH) 10% (m/v,
209 ChimiePlus, France) (Dehaut et al., 2016). Caged mussels were thawed, dissected and put
210 inside graduated Erlenmeyers equipped with glass stopper containing a volume of 150 mL of
211 KOH 10% (the volume 150 mL was used because it was the necessary volume that led to an
212 optimal digestion efficiency percentage). These Erlenmeyers were maintained on a heating
213 magnetic stirrer at 60 °C for 24 h. After digestion, the digestat of each individual was filtered
214 on a GF/A filter (90 mm diameter). The same procedure was done with native mussels but a
215 volume of 250 mL was added instead (see Kazour et al. 2019).
216
217

218 c. Visual and Raman spectroscopy analysis

219 All filters were observed under 120x magnification using Leica M165 C Stereomicroscope
220 and images of suspected MPs particles were taken with a Leica M170 HD camera and LAS
221 (Leica application suite) software. All suspected MPs particles were counted, categorized by
222 type (fragments, fibers, microbeads or films) and color. The following criteria were taken into
223 consideration while observing the items: (1) absence of cellular or organic structures; (2) a
224 homogenous thickness across the particles; and, (3) homogenous colors (Hidalgo-Ruz et al.,
225 2012). Measurements were done on the suspected items at their longest dimension and they
226 were divided into different size classes of 50 µm.

227 Suspected microplastics were identified using Micro-Raman Xplora Plus (HORIBA
228 Scientific®, France). The machine is protected by a door so that the filter placed inside would
229 not be affected by any airborne contamination. Due to the time consumption that takes when
230 analyzing filters under micro-Raman, five filters of caged and native mussels containing the
231 highest number of potential MPs from each sampling site were chosen and analyzed. All
232 sediments items, and subsamples of water items were also taken for Raman analysis. For
233 identification, two lasers with a wavelength of 532 nm and 785 nm and a range of 200-3400
7
234 cm-1 were used with x10 and x100 objectives (Olympus). Each particle spectrum is compared
235 to a polymer database identification software (KnowItAll, BioRad®) and a personal library
236 made with standard polymers obtained from Goodfellow (France). KnowItAll software gives
237 a resemblance (correlation) between the obtained spectra and the one existing in the database.
238 If the obtained spectrum showed a high fluorescence, several acquisition parameters are
239 changed from one sample to another: whether in the acquisition time (0.2s, 1s, 2s, or 5s), the
240 number of accumulations (2, 5, 10, or 15), the laser wavelength and intensity (0.1%, 1%,
241 10%, 50% or 100%), the slit and the hole, to obtain a better identifiable spectrum. The
242 identification is considered correct when the HQI (Hit Quality Index) was above 70 (ranging
243 from 0 to 100). Baseline correction was realized on spectra in order to have a cleaner
244 identifiable spectrum. Pigments spectra (Copper phthalocyanine, Hostasol Green, etc…)
245 obtained were identified using an option called “Mixture Analysis” existing in “KnowItAll
246 software” that is capable to decompose a mixed spectrum into two spectra consisting of the
247 dye and its associated polymer (see Supplementary Figure 1 for an example).

248 2.4 Statistical and data analyses

249 Microplastics found in water, sediments and mussels were reported in unit volume (per L) and
250 per gram of sample, respectively. Statistics were performed with SPSS software (IBM SPSS
251 STATISTICS 20). The Wilcoxon signed-rank test was used to compare the ingested MPs
252 between depurated and non-depurated mussels and a Student t-test was used to compare the
253 condition index. For inter-site comparisons, if data did not comply with the parametric
254 assumption of normality (Shapiro–Wilk tests) and homogeneity of variance (Levene tests),
255 the non-parametric Kruskall–Wallis test and Mann–Whitney U test for post hoc pairwise
256 comparisons were used. All results are expressed as mean ± SD, and p-value < 0.05 was
257 considered as statistically significant. Morisita’s index of similarity (or Morisita overlap
258 index) is a measure of how similar or different two sets of data are. This index was calculated
259 to compare size and polymer types similarity found in caged mussels and those found in
260 native mussels, water and sediments samples.
261
!
2 ̂ ̂
262 = . .
(!%& )*+ ! )*
" #$%& ' -/" #$%0 1 %0 2
(,& )*+ ,0 )*
% %

263 : Morisita's overlap index between j and k

8
264 j: Caged mussels

265 k: Native mussels or water or sediments

266 ̂ : Proportion of a size class or a polymer i used by j

267 ̂ : Proportion of a size class or a polymer i l used by k

268 3 : Number of size class or polymer j that use i

269 3 : Number of size class or polymer k that use i

270 ; : Total number of size class or polymer in sample

271 This index ranges from 0 (when no common size or polymer types are shared between the two
272 sample types) and 1 (when all sizes or polymer types are common between the two sample
273 types).
274

275 Results
276 3.1 Mussels caging experiment

277 Before translocation, mussels were depurated. There was no significant difference in mussel's
278 condition index after seven days of depuration (p = 0.264 > 0.05). However, a significant
279 difference in the amount of MPs was observed between non-depurated and depurated mussels
280 (p = 0.018 < 0.05). Non-depurated mussels had an average of 2.22 ± 1.35 items/g with
281 fragments constituting 82.3% of the observed items (Figure 2). The number of items
282 significantly decreased by 97.6% after 7 days of depuration reaching 0.05 ± 0.15 items/g
283 consisting only of fibers. The size of observed fibers before depuration ranged from 41.54 µm
284 to 622.04 µm with a mean size of 284 ± 156.8 µm; these fibers were of different colors: black,
285 green, blue and red. Only two blue fibers were observed after depuration with a respective
286 length of 304.06 and 415.73 µm.

287 Even though the cages were transplanted during the winter period when the temperature had
288 been at its lowest (around 6-7°C), the mean mussels survival rate was 73%. After 42 days of
289 caging experiment, mussels shell length (5.7 ± 0.47 cm) and condition index (54.7 ± 3.8%)
290 were not significantly different from the beginning of the experiment (p = 0.621> 0.05). Also,
291 there was no significant inter-sites difference in the caged mussels condition index (p =
292 0.521).

9
293

294 3.2 Microplastics abundance, type and color

295
296 The absence of fragments, microbeads and films in our procedural background blanks
297 indicates that contamination was only limited to airborne fibers. The average fiber
298 contamination during caged mussels dissection and digestion had an average of 0.72 ± 0.78
299 fibers (representing the average found for a batch of 9 mussels representing a total one hour
300 constant laboratory work under the laminar flow hood). Similar values were observed for the
301 other three matrices (see Kazour et al. (2019)). The importance of following procedural
302 blanks is that any potential contamination is then eliminated from the analyzed samples.
303 Microplastic contamination of seawater, sediments, and mussels was evident at all of our
304 sampling sites. The number of suspected MPs in the water decreased with the increasing
305 distance from the effluent of the WWTP and the highest amount was found near the landfill
306 (site 5). Such pattern was not observed for MPs found in the sediments (Figure 3 - A).
307 Suspected MPs were found in mussels of all sites; 93% of native mussels and 94.7% of caged
308 mussels had ingested suspected MPs with a mean number of items ranging from 0.41 to 2.76
309 items/g. The spatial pattern of MP abundances in mussels was different from that observed for
310 water or sediment samples (Figure 3). Caged mussels ingested the same or higher amount of
311 MPs than native mussels at all sites except for site 5; however for the same site, the amount of
312 ingested MPs by caged and native mussels were not significantly different (p > 0.05) (Figure
313 3).
314
315 Both fragments and fibers were found in the water, sediment and in both caged and native
316 mussels. In all the matrices analyzed, fragments dominated the type of MPs except at sites 1,
317 3 and 4 for water samples (Figure 3). Primary microplastics (polystyrene raw microbeads)
318 were found only at site 5 in the water but were not detected in mussels nor in sediment
319 samples. Films were observed only in native mussels. Blue fragments dominated the water
320 (45%) and sediment (69%) samples whereas red and blue fragments dominated in both caged
321 (76%) and native mussels (42% each). For the fibers, black (51%) and blue and green color
322 (71.4%) dominated the water and sediment samples respectively whereas blue and black
323 fibers dominated in caged (85%) and native (80%) mussels. At each site, the proportion of
324 MPs items type or color was quite similar among caged and native mussels. For fragments,
325 the order of dominant colors was blue, red, green, white and transparent; whereas for fibers, it
326 was blue, black, green and red.

10
327
328

329 3.3 Microplastics size and polymer types

330
331 Suspected MPs sizes were very similar whether in the water, sediments and both caged and
332 native mussels, with a dominance of items < 150 µm (Figure 4). This was confirmed by the
333 very high overlaps between caged mussels vs native mussels (0.98), vs sediments (0.9) or vs
334 water samples (0.84). Smaller size classes (< 200 µm) were more abundant in native mussels
335 compared to caged mussels (Figure 4 - A). Caged mussels showed greater number of items
336 with a size class > 200 µm compared to native mussels. Sediments and water samples showed
337 higher proportion of items of size class < 150 µm compared to caged mussels (Figure 4 - B-
338 C).
339 Of the 503 items analyzed with the micro Raman, 422 (83.9%) were positively identified as a
340 known polymer (examples of various parameters used to identify samples are indicated in
341 Supplementary Table 2). Among the non-polymers items, only 2% were identified as
342 cellulose-based fibers. The rest of the spectra were highly fluorescent and non-identifiable
343 (for example if a sample is too thin, Raman tends to detect the underlying substrate instead of
344 the sample (Käppler et al., 2016)).
345 Five polymers were identified in caged mussels, by order of dominance: polystyrene (PS),
346 acrylonitrile butadiene styrene (ABS), polyurethane (PUR) and polyethylene (PE),. A higher
347 number of polymers were identified in native mussels (9), water (11) and sediments (8)
348 (Figure 5). PS dominated all samples in all the different matrices with ABS and PA the
349 second most abundant polymers. The highest overlap in polymer types was found between
350 caged mussels and sediments (0.93) followed with native mussels (0.88) and water samples
351 (0.86).

352 Discussion

353 The key advantage of using blue mussels for microplastics biomonitoring, is that they can be
354 depurated in the laboratory, reducing initial contamination bias, and thus, clean reference
355 individuals will reflect the MPs contamination of their specific deployment sites. In mussels,
356 MPs may be retained for extended periods of time, for example, complete clearance of MPs
357 was not achieved after a seven-days depuration period under laboratory conditions with
358 microbeads (2,6 µm) being retained within the digestive tracts (Paul-Pont et al., 2016). Our
359 results showed that after 7 days of depuration, 97.6% of the MPs were eliminated by the

11
360 mussels. Similar depuration rate (85%) was observed in the Mediterranean mussel, M.
361 galloprovincialis, after seven days depuration period (Fernández and Albentosa, 2019). Lower
362 depuration period seems however to be insufficient for total depuration; in a three days
363 depuration experiment of farmed M. edulis, Van Cauwenberghe and Janssen (2014) found a
364 reduction of ca. 33% in the MPs quantity. Since depuration significantly decreased the
365 quantity of MP in blue mussels, the eliminated particles were probably located in the digestive
366 system as suggested for the brown mussels (Perna perna) by Birnstiel et al. (2019).
367
368 The main objective of the study was to test effectiveness of the mussel caging for active
369 microplastics biomonitoring, and this was investigated by comparing MPs ingested by caged
370 blue mussels with those ingested by native mussels collected at the same site and MPs found
371 in their surrounding environment (surface water and sediments). Two aspects can be
372 compared: the quantitative aspect (MPs concentrations in the different matrices) and the
373 qualitative aspect (shape, size, color and type of polymers) of MPs.
374
375 4.1 Quantitative MPs comparison
376
377 A key question for microplastics caging studies is how long the blue mussels should stay
378 deployed to be equalized with native mussels and to reflect MPs concentrations of their
379 surrounding environment. For chemical pollution, the reported caging periods lasted generally
380 between 3 to 6 weeks but may last 18 - 24 weeks or up to 2 years (see review in Beyer et al.,
381 2017). Short term mussel deployments, such as one or two months, has been shown to be
382 suitable for chemical pollutant monitoring in coastal waters, e.g. metals (Greenfield et al.,
383 2014; Lacroix et al., 2015; Piccardo et al., 2001). To date, only three studies used caged blue
384 mussels to investigate the MPs pollution (Avio et al., 2017; Catarino et al., 2018; Railo et al.,
385 2018). These studies showed that mussels transplanted for 4 weeks period have accumulated
386 MPs in their tissues. However, they have not compared the ingested MPs with those found in
387 native mussels or in their surrounding environment. In the present study, even though mussels
388 were not transplanted during their optimal condition (the seawater temperature has been at its
389 lowest: around 6-9°C), mussels’ condition and growth were maintained after 6 weeks, and
390 their survival rate was high (73%). More importantly, the majority (93%) of transplanted
391 mussels had ingested MPs. This percentage was similar to that of native mussels (94,7%)
392 collected in the same areas. In addition, the spatial pattern in the amount of ingested MPs by
393 native and caged was quite the same except in site 5 where the native mussels ingested higher
12
394 number of MPs. This difference in ingested MPs can be due to the significantly lower body
395 weight of native mussels from the site 5 compared to that of transplanted mussels (1.07 ± 0.35
396 g and 5.15 ± 1.11 g, respectively). The increase on the retention time of plastics particles with
397 decreasing size in mussel has been reported (Fernández and Albentosa, 2019; Van
398 Cauwenberghe and Janssen, 2014). Using caged mussels from a single population avoids bias
399 related to the age, the size or the reproductive status of the organisms that can influence
400 ingestion, retention or clearance rates.
401
402 Among the detected debris items in native and caged mussels, fragments were the most
403 predominant type observed, consistent with the MPs found in mussels and cockles
404 (Hermabessiere et al., 2019) and juvenile fish (Kazour et al., 2018) collected along the
405 Eastern English Channel coast. This high occurrence and abundance of fragments in mussels
406 reflect those found in their surrounding environment (seawater and sediments). A recent study
407 showed a positive and quantitative correlation of MPs in mussels (M. edulis and Perna
408 viridis) and their surrounding waters (Qu et al., 2018). The authors suggested that the amount
409 of MPs in mussels can reflect the real abundance of MPs in the coastal water but that mussels
410 were more likely to ingest smaller MPs. In our study, the spatial pattern of MPs
411 concentrations in mussels (both caged and native) was different from that observed for water
412 or sediment samples. Similar results were observed along the coastal waters of the U.K., with
413 no quantitative correlation between MPs in mussels and their ambient seawaters (Li et al.,
414 2018; Scott et al., 2019). Such absence of correlation may be due to the limited sampling sites
415 (only five in our study and seven in Li et al. (2018) study) which are insufficient for robust
416 correlation analyses. In addition, it is likely that a range of factors influence mussel encounter
417 rates with particles within their immediate environment, including hydrodynamics processes
418 in the study area, particle behavior in the water column and the mussels or seawater sampling
419 location in the water column (Avio et al., 2017; Scott et al., 2019). In the study of Avio et al.
420 (2017) mussels were caged for 4 weeks at 2 different depths, approximately at 1.5 m from the
421 surface and from the bottom (30-45 m). They found some differences in the occurrence of
422 ingested MPs as a function of caging depth, with higher ingested particles (32%) in surface
423 deployed mussels compared to those caged in proximity of the bottom (12%). In addition,
424 some variations were observed in terms of typology and size of particles between surface and
425 bottom-caged mussels highlighting the influence of a different distribution of MPs along the
426 water column. The density of the plastic particles will determine their position in the water
427 column, and, therefore, the likelihood of encounter by an organism (Desforges et al., 2014). In

13
428 our study, we sampled surface water whereas mussels were near the bottom. When comparing
429 different matrices or compartments, a more adapted sampling strategy should take into
430 account numerous parameters such as the sampling time according to the tide, the time lag
431 necessary for MPs to sink in the water column, to be ingested by mussels and to be
432 sedimented at the bottom (Schmidt et al., 2018). As these conditions have not been
433 implemented in the present study, it is therefore difficult to compare the MP concentrations
434 found in mussels, water and sediments. Thus, the present data should be considered as semi-
435 quantitative until the influence of environmental factors on the measured MP concentrations
436 found in the seawater and sediments and those ingested by blue mussels have been further
437 investigated. In addition, an understanding of MPs bioaccumulation is a prerequisite for the
438 use of mussel as biomonitoring organisms in the aquatic environment.
439
440
441 4.2 Qualitative (size, color and polymers) MPs comparison
442
443 Comparison between native and caged mussels showed that, at each sampling site, the
444 proportion of MPs items type, size, color and to some extent their composition (polymer
445 types) were quite the same with a high overlap index. Both native and caged mussels from all
446 sites have mainly ingested small sized MPs (< 200 μm). This was similar to other studies that
447 demonstrated that MPs below 300 μm were the most found in mussels (Leslie et al., 2017;
448 Naji et al., 2018; Phuong et al., 2018). Blue fragments were the dominant items found in all
449 the analyzed compartments. Although small sized MPs dominated the water and sediments
450 samples, it is currently unknown whether their abundance in mussels are due to mussels size
451 preferences in regards to prey, or because the majority of MPs in the environment were
452 typically in this size range (Bråte et al., 2018; Scott et al., 2019).
453
454 Concerning polymer types, a good overlap was found between caged mussel and the others
455 compartment analyzed. The highest overlap in polymer types being found between caged
456 mussels and sediments (0.93). Polystyrene (PS) dominated all samples in all the different
457 matrices. This polymer was among the most common plastics used worldwide whether in
458 packaging or other household materials. We found that some polymers were under-
459 represented or totally absent in the caged mussels compared to overlying seawater or
460 surrounding sediment. More polymer types (N=13) were identified in the surface water
461 compared to sediments (N=9) or caged mussels (N=6). In the South West of England, Scott et
14
462 al. (2019) found significant differences in the relative abundance of polymer types and
463 particle sizes between seawater, sediment, and mussels. However, they found that particles
464 within mussels reflected more closely those found in the intertidal surface sediment compared
465 to those found in the surface seawater. This is also the case in our study where particles found
466 within caged mussels presented the highest similarity with those found in the sediments. The
467 bioavailability of MPs to aquatic organism depends on their characteristics, such as polymer
468 type, chemical composition, size, shape, density, etc. (Carbery et al., 2018). The shape of MPs
469 may also influences their accumulation (persistence inside mussels) or elimination (Browne et
470 al., 2011; Thompson et al., 2004). As suggested by Scott et al. (2019), it is likely that both
471 environmental and biological partitioning of microplastic particles and the selective feeding
472 ecology of this species is responsible for the under-representation of certain polymer types
473 and particle sizes within the mussels. This should be taken into consideration when using
474 mussels for MPs pollution biomonitoring.
475
476 4.3 Mussels biomonitoring as a standardized tool for microplastics assessment
477
478 In this work, we were able to demonstrate the potential use of mussels as a MPs
479 biomonitoring tool. But a lot of factors should be considered to optimize and standardize this
480 tool. The first issue is that mussels represent different filtration rates depending on the region
481 and genotype, for example (Lüskow and Riisgård, 2018; Riisgård et al., 2013). The difference
482 in the uptake rate for analogous species needs to be further studied to appropriately adjust
483 interspecific difference. Algal abundance, temperature and salinity of water, as well as the
484 depth of the transplanted cages could alter mussel's filtration and clearance rates (Comeau et
485 al., 2008; Riisgård et al., 2014, 2013) and, therefore, their MPs uptake. For example,
486 differences in MPs ingestion have been observed between caged mussel's transplanted at 1.5m
487 and 30-45m depth (Avio et al., 2017). In addition, seasonal variation for MPs intake has been
488 observed in caged mussels but it has been more linked to an important water flow from a river
489 rather than the different seasons (Catarino et al., 2018). More progression in this area is highly
490 needed to obtain a standardized protocol. Such tool would constitute plenty of advantages for
491 MPs evaluation such as allowing a microplastics assessment while using similar population
492 (genetic and physiological) in areas lacking mussel beds, controlling exposition period and to
493 potentially study the toxicological impact of microplastics on mussels exposed in the natural
494 environment.
495

15
496 Conclusion
497
498 In summary, the present study demonstrated for the first time the effectiveness of the mussel
499 caging approach. The abundance and properties of MPs found in caged and native mussels
500 were quite similar. If we rely on the assumption that MPs concentrations in native mussels
501 have reached steady-state (balance between intake and defecation/egestion), the results of this
502 study indicate that short term mussel deployments (i.e. 6 weeks) appear to be suitable to
503 reflect native mussels MPs ingestion. Although microplastics monitoring is a complex task
504 that it is in its initial phases (Carbery et al., 2018), caging presents many advantages including
505 the selection of well-characterized homogenous organisms (number, age, size, weight, sex)
506 and the control of exposure (location, time, season). To the best of our knowledge, mussels
507 caging as tool to study microplastics pollution has not been investigated before. Though the
508 abundance and properties of microplastics in mussels does not exactly match those in their
509 environment (seawater and sediments), our study suggests that blue mussels caging may be a
510 promising tool for MPs biomonitoring in marine coastal environments. The caging approach
511 makes monitoring more reliable and may allow accurate assessment of the biological effects
512 of MPs over a predetermined exposure period. However, further methodological
513 improvements should be considered, such as the choice of the deployment time and the caging
514 location (with respect to the shore and the depth of their implantation in the water column), to
515 define a uniform protocol for blue mussels caging allowing an accurate spatial and temporal
516 microplastics biomonitoring.
517
518 Funding

519 Maria Kazour is financially supported by a PhD fellowship from the National Council for
520 Scientific Research (Lebanon) and Université du Littoral Côte d’Opale (France). This work
521 has been partially financially supported by the European Union (ERDF), the French
522 Government, the Région Hauts-de-France and IFREMER, in the framework of the project
523 CPER MARCO 2015- 2020.
524
525 Authors Contributions
526

16
527 R.A. conceived of the presented idea. R.A. and M.K. planned and carried out the experiment.
528 M.K. performed the samples analyses. All authors discussed the results and contributed to the
529 final manuscript.
530
531 Acknowledgments
532
533 The authors would like to thank the administration of Le Havre harbor (especially Miss.
534 Magali HAUTOT who helped obtaining permissions to enter the harbor and was our
535 companion during sampling), Khalef Rabhi and Sarah Terki for field assistance. The author
536 would like also to thank Sami Souissi and Capucine Bialais for providing use live microalgae
537 cultures.
538

539

540 Declaration of interests: None

541

542
543 References
544
545 Andral, B., Stanisiere, J.Y., Sauzade, D., Damier, E., Thebault, H., Galgani, F., Boissery, P.,
546 2004. Monitoring chemical contamination levels in the Mediterranean based on the use
547 of mussel caging. Mar. Pollut. Bull. 49, 704–712.
548 https://doi.org/10.1016/j.marpolbul.2004.05.008
549 Avio, C.G., Cardelli, L.R., Gorbi, S., Pellegrini, D., Regoli, F., 2017. Microplastics pollution
550 after the removal of the Costa Concordia wreck: First evidences from a biomonitoring
551 case study. Environ. Pollut. 227, 207–214. https://doi.org/10.1016/j.envpol.2017.04.066
552 Bebianno, M.J., Lopes, B., Guerra, L., Hoarau, P., Ferreira, A.M., 2007. Glutathione S-
553 tranferases and cytochrome P450 activities in Mytilus galloprovincialis from the South
554 coast of Portugal: Effect of abiotic factors. Environ. Int. 33, 550–558.
555 https://doi.org/10.1016/j.envint.2006.11.002
556 Beyer, J., Green, N.W., Brooks, S., Allan, I.J., Ruus, A., Gomes, T., Bråte, I.L.N., Schøyen,
557 M., 2017. Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution
558 monitoring: A review. Mar. Environ. Res. 130, 338–365.
559 https://doi.org/10.1016/j.marenvres.2017.07.024

17
560 Birnstiel, S., Soares-Gomes, A., da Gama, B.A.P., 2019. Depuration reduces microplastic
561 content in wild and farmed mussels. Mar. Pollut. Bull. 140, 241–247.
562 https://doi.org/10.1016/j.marpolbul.2019.01.044
563 Bolognesi, C., Frenzilli, G., Lasagna, C., Perrone, E., Roggieri, P., 2004. Genotoxicity
564 biomarkers in Mytilus galloprovincialis: Wild versus caged mussels. Mutat. Res. -
565 Fundam. Mol. Mech. Mutagen. 552, 153–162.
566 https://doi.org/10.1016/j.mrfmmm.2004.06.012
567 Bråte, I.L.N., Hurley, R., Iversen, K., Beyer, J., Thomas, K. V., Steindal, C.C., Green, N.W.,
568 Olsen, M., Lusher, A., 2018. Mytilus spp. as sentinels for monitoring microplastic
569 pollution in Norwegian coastal waters: A qualitative and quantitative study. Environ.
570 Pollut. 243, 383–393. https://doi.org/10.1016/j.envpol.2018.08.077
571 Brooks, S., Harman, C., Soto, M., Cancio, I., Glette, T., Marigómez, I., 2012. Integrated
572 coastal monitoring of a gas processing plant using native and caged mussels. Sci. Total
573 Environ. 426, 375–386. https://doi.org/10.1016/j.scitotenv.2012.03.059
574 Brooks, S.J., Escudero-Oñate, C., Gomes, T., Ferrando-Climent, L., 2018. An integrative
575 biological effects assessment of a mine discharge into a Norwegian fjord using field
576 transplanted mussels. Sci. Total Environ. 644, 1056–1069.
577 https://doi.org/10.1016/j.scitotenv.2018.07.058
578 Browne, M.A., Crump, P., Niven, S.J., Teuten, E., Tonkin, A., Galloway, T., Thompson, R.,
579 2011. Accumulation of Microplastic on Shorelines Woldwide: Sources and Sinks -
580 Environmental Science & Technology (ACS Publications). Environ. Sci. Technol 9175–
581 9179. https://doi.org/10.1021/es201811s
582 Browne, M.A., Dissanayake, A., Galloway, T.S., Lowe, D.M., Thompson, R.C., 2008.
583 Ingested Microscopic Plastic Translocates to the Circulatory System of the Mussel,
584 Mytilus edulis (L.). Environ. Sci. Technol 42, 5026–2031.
585 https://doi.org/10.1021/es800249a
586 Carbery, M., O’Connor, W., Palanisami, T., 2018. Trophic transfer of microplastics and
587 mixed contaminants in the marine food web and implications for human health. Environ.
588 Int. 115, 400–409. https://doi.org/10.1016/j.envint.2018.03.007
589 Catarino, A.I., Macchia, V., Sanderson, W.G., Thompson, R.C., Henry, T.B., 2018. Low
590 levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is
591 minimal compared to exposure via household fibres fallout during a meal. Environ.
592 Pollut. 237, 675–684. https://doi.org/10.1016/j.envpol.2018.02.069
593 Cole, M., Lindeque, P., Fileman, E., Halsband, C., Goodhead, R., Moger, J., Galloway, T.S.,

18
594 2013. Microplastic ingestion by zooplankton. Environ. Sci. Technol. 47, 6646–6655.
595 https://doi.org/10.1021/es400663f
596 Comeau, L.A., Pernet, F., Tremblay, R., Bates, S.S., Leblanc, A., 2008. Comparison of
597 eastern oyster (Crassostrea virginica) and blue mussel (Mytilus edulis) filtration rates at
598 low temperatures. Can. Tech. Rep. Fish. Aquat. Sci. 2810 1, 11.
599 De Witte, B., Devriese, L., Bekaert, K., Hoffman, S., Vandermeersch, G., Cooreman, K.,
600 Robbens, J., 2014. Quality assessment of the blue mussel (Mytilus edulis): Comparison
601 between commercial and wild types. Mar. Pollut. Bull. 85, 146–155.
602 https://doi.org/10.1016/j.marpolbul.2014.06.006
603 Dehaut, A., Cassone, A.L., Frère, L., Hermabessiere, L., Himber, C., Rinnert, E., Rivière, G.,
604 Lambert, C., Soudant, P., Huvet, A., Duflos, G., Paul-Pont, I., 2016. Microplastics in
605 seafood: Benchmark protocol for their extraction and characterization. Environ. Pollut.
606 215, 223–233. https://doi.org/10.1016/j.envpol.2016.05.018
607 Desforges, J.P.W., Galbraith, M., Dangerfield, N., Ross, P.S., 2014. Widespread distribution
608 of microplastics in subsurface seawater in the NE Pacific Ocean. Mar. Pollut. Bull. 79,
609 94–99. https://doi.org/10.1016/j.marpolbul.2013.12.035
610 Farrington, J.W., Tripp, B.W., Tanabe, S., Subramanian, A., Sericano, J.L., Wade, T.L.,
611 Knap, A.H., 2016. Edward D. Goldberg’s proposal of “the Mussel Watch”: Reflections
612 after 40 years. Mar. Pollut. Bull. 110, 501–510.
613 https://doi.org/10.1016/j.marpolbul.2016.05.074
614 Fernández, B., Albentosa, M., 2019. Insights into the uptake, elimination and accumulation of
615 microplastics in mussel. Environ. Pollut. 249, 321–329.
616 https://doi.org/10.1016/j.envpol.2019.03.037
617 Galgani, F., Martínez-Gómez, C., Giovanardi, F., Romanelli, G., Caixach, J., Cento, A.,
618 Scarpato, A., Benbrahim, S., Messaoudi, S., Deudero, S., Boulahdid, M., Benedicto, J.,
619 Andral, B., 2011. Assessment of polycyclic aromatic hydrocarbon concentrations in
620 mussels (Mytilus galloprovincialis) from the Western basin of the Mediterranean Sea.
621 Environ. Monit. Assess. 172, 301–317. https://doi.org/10.1007/s10661-010-1335-5
622 Greenfield, R., Brink, K., Degger, N., Wepener, V., 2014. The usefulness of transplantation
623 studies in monitoring of metals in the marine environment: South African experience.
624 Mar. Pollut. Bull. 85, 566–573. https://doi.org/10.1016/j.marpolbul.2014.03.032
625 Hermabessiere, L., Paul-Pont, I., Cassone, A.L., Himber, C., Receveur, J., Jezequel, R., El
626 Rakwe, M., Rinnert, E., Rivière, G., Lambert, C., Huvet, A., Dehaut, A., Duflos, G.,
627 Soudant, P., 2019. Microplastic contamination and pollutant levels in mussels and

19
628 cockles collected along the channel coasts. Environ. Pollut. 250, 807–819.
629 https://doi.org/10.1016/j.envpol.2019.04.051
630 Hidalgo-Ruz, V., Gutow, L., Thompson, R.C., Thiel, M., 2012. Microplastics in the marine
631 environment: A review of the methods used for identification and quantification.
632 Environ. Sci. Technol. 46, 3060–3075. https://doi.org/10.1021/es2031505
633 Hunt, C.D., Slone, E., 2010. Long-term monitoring using resident and caged mussels in
634 Boston Harbor yield similar spatial and temporal trends in chemical contamination. Mar.
635 Environ. Res. 70, 343–357. https://doi.org/10.1016/j.marenvres.2010.07.002
636 Käppler, A., Fischer, D., Oberbeckmann, S., Schernewski, G., Labrenz, M., Eichhorn, K.J.,
637 Voit, B., 2016. Analysis of environmental microplastics by vibrational
638 microspectroscopy: FTIR, Raman or both? Anal. Bioanal. Chem. 408, 8377–8391.
639 https://doi.org/10.1007/s00216-016-9956-3
640 Karlsson, T.M., Vethaak, A.D., Almroth, B.C., Ariese, F., van Velzen, M., Hassellöv, M.,
641 Leslie, H.A., 2017. Screening for microplastics in sediment, water, marine invertebrates
642 and fish: Method development and microplastic accumulation. Mar. Pollut. Bull. 122,
643 403–408. https://doi.org/10.1016/j.marpolbul.2017.06.081
644 Kazour, M., Terki, S., Rabhi, K., Jemaa, S., Khalaf, G., Amara, R., 2019. Sources of
645 microplastics pollution in the marine environment: Importance of wastewater treatment
646 plant and coastal landfill. Mar. Pollut. Bull. 146, 608–618.
647 https://doi.org/10.1016/j.marpolbul.2019.06.066
648 Lacroix, C., Richard, G., Seguineau, C., Guyomarch, J., Moraga, D., Auffret, M., 2015.
649 Active and passive biomonitoring suggest metabolic adaptation in blue mussels (Mytilus
650 spp.) chronically exposed to a moderate contamination in Brest harbor (France). Aquat.
651 Toxicol. 162, 126–137. https://doi.org/10.1016/j.aquatox.2015.03.008
652 Leslie, H.A., Brandsma, S.H., van Velzen, M.J.M., Vethaak, A.D., 2017. Microplastics en
653 route: Field measurements in the Dutch river delta and Amsterdam canals, wastewater
654 treatment plants, North Sea sediments and biota. Environ. Int. 101, 133–142.
655 https://doi.org/10.1016/j.envint.2017.01.018
656 Li, J., Green, C., Reynolds, A., Shi, H., Rotchell, J.M., 2018. Microplastics in mussels
657 sampled from coastal waters and supermarkets in the United Kingdom. Environ. Pollut.
658 241, 35–44. https://doi.org/10.1016/j.envpol.2018.05.038
659 Li, J., Lusher, A.L., Rotchell, J.M., Deudero, S., Turra, A., Bråte, I.L.N., Sun, C., Shahadat
660 Hossain, M., Li, Q., Kolandhasamy, P., Shi, H., 2019. Using mussel as a global
661 bioindicator of coastal microplastic pollution. Environ. Pollut. 244, 522–533.

20
662 https://doi.org/10.1016/j.envpol.2018.10.032
663 Li, J., Qu, X., Su, L., Zhang, W., Yang, D., Kolandhasamy, P., Li, D., Shi, H., 2016.
664 Microplastics in mussels along the coastal waters of China. Environ. Pollut. 214, 177–
665 184. https://doi.org/10.1016/j.envpol.2016.04.012
666 Lüskow, F., Riisgård, H.U., 2018. In Situ Filtration Rates of Blue Mussels (Mytilus edulis)
667 Measured by an Open-Top Chamber Method. Open J. Mar. Sci. 08, 395–406.
668 https://doi.org/10.4236/ojms.2018.84022
669 Marigómez, I., Garmendia, L., Soto, M., Orbea, A., Izagirre, U., Cajaraville, M.P., 2013.
670 Marine ecosystem health status assessment through integrative biomarker indices: A
671 comparative study after the Prestige oil spill “mussel Watch.” Ecotoxicology 22, 486–
672 505. https://doi.org/10.1007/s10646-013-1042-4
673 Naji, A., Nuri, M., Vethaak, A.D., 2018. Microplastics contamination in molluscs from the
674 northern part of the Persian Gulf. Environ. Pollut. 235, 113–120.
675 https://doi.org/10.1016/j.envpol.2017.12.046
676 Nigro, M., Falleni, A., Barga, I. Del, Scarcelli, V., Lucchesi, P., Regoli, F., Frenzilli, G.,
677 2006. Cellular biomarkers for monitoring estuarine environments: Transplanted versus
678 native mussels. Aquat. Toxicol. 77, 339–347.
679 https://doi.org/10.1016/j.aquatox.2005.12.013
680 OSPAR, 2015. Request on Development of a Common Monitoring Protocol for Plastic
681 Particles in Fish Stomachs and Selected Shellfish on the Basis of Existing Fish Disease
682 Surveys, in: ICES Special Request Advice. pp. 1–6.
683 Paul-Pont, I., Lacroix, C., González Fernández, C., Hégaret, H., Lambert, C., Le Goïc, N.,
684 Frère, L., Cassone, A.L., Sussarellu, R., Fabioux, C., Guyomarch, J., Albentosa, M.,
685 Huvet, A., Soudant, P., 2016. Exposure of marine mussels Mytilus spp. to polystyrene
686 microplastics: Toxicity and influence on fluoranthene bioaccumulation. Environ. Pollut.
687 216, 724–737. https://doi.org/10.1016/j.envpol.2016.06.039
688 Phuong, N.N., Zalouk-Vergnoux, A., Kamari, A., Mouneyrac, C., Amiard, F., Poirier, L.,
689 Lagarde, F., 2018. Quantification and characterization of microplastics in blue mussels
690 (Mytilus edulis): protocol setup and preliminary data on the contamination of the French
691 Atlantic coast. Environ. Sci. Pollut. Res. 25, 6135–6144. https://doi.org/10.1007/s11356-
692 017-8862-3
693 Piccardo, M.T., Coradeghini, R., Valerio, F., 2001. Polycyclic aromatic hydrocarbon pollution
694 in native and caged mussels. Mar. Pollut. Bull. 42, 951–956.
695 https://doi.org/10.1016/S0025-326X(01)00057-1

21
696 Qu, X., Su, L., Li, H., Liang, M., Shi, H., 2018. Assessing the relationship between the
697 abundance and properties of microplastics in water and in mussels. Sci. Total Environ.
698 621, 679–686. https://doi.org/10.1016/j.scitotenv.2017.11.284
699 Railo, S., Talvitie, J., Setälä, O., Koistinen, A., Lehtiniemi, M., 2018. Application of an
700 enzyme digestion method reveals microlitter in Mytilus trossulus at a wastewater
701 discharge area. Mar. Pollut. Bull. 130, 206–214.
702 https://doi.org/10.1016/j.marpolbul.2018.03.022
703 Riisgård, H.U., Larsen, P.S., Pleissner, D., 2014. Allometric equations for maximum filtration
704 rate in blue mussels Mytilus edulis and importance of condition index. Helgol. Mar. Res.
705 68, 193–198. https://doi.org/10.1007/s10152-013-0377-9
706 Riisgård, H.U., Lüskow, F., Pleissner, D., Lundgreen, K., López, M.Á.P., 2013. Effect of
707 salinity on filtration rates of mussels Mytilus edulis with special emphasis on dwarfed
708 mussels from the low-saline Central Baltic Sea. Helgol. Mar. Res. 67, 591–598.
709 https://doi.org/10.1007/s10152-013-0347-2
710 Santana, M.F.M., Ascer, L.G., Custódio, M.R., Moreira, F.T., Turra, A., 2016. Microplastic
711 contamination in natural mussel beds from a Brazilian urbanized coastal region: Rapid
712 evaluation through bioassessment. Mar. Pollut. Bull. 106, 183–189.
713 https://doi.org/10.1016/j.marpolbul.2016.02.074
714 Schmidt, N., Thibault, D., Galgani, F., Paluselli, A., Sempéré, R., 2018. Occurrence of
715 microplastics in surface waters of the Gulf of Lion (NW Mediterranean Sea). Prog.
716 Oceanogr. 163, 214–220. https://doi.org/10.1016/j.pocean.2017.11.010
717 Schøyen, M., Allan, I.J., Ruus, A., Håvardstun, J., Hjermann, D., Beyer, J., 2017. Comparison
718 of caged and native blue mussels (Mytilus edulis spp.) for environmental monitoring of
719 PAH, PCB and trace metals. Mar. Environ. Res. 130, 221–232.
720 https://doi.org/10.1016/j.marenvres.2017.07.025
721 Scott, N., Porter, A., Santillo, D., Simpson, H., Lloyd-Williams, S., Lewis, C., 2019. Particle
722 characteristics of microplastics contaminating the mussel Mytilus edulis and their
723 surrounding environments. Mar. Pollut. Bull. 146, 125–133.
724 https://doi.org/10.1016/j.marpolbul.2019.05.041
725 Serafim, A., Lopes, B., Company, R., Cravo, A., Gomes, T., Sousa, V., Bebianno, M.J., 2011.
726 A multi-biomarker approach in cross-transplanted mussels Mytilus galloprovincialis.
727 Ecotoxicology 20, 1959–1974. https://doi.org/10.1007/s10646-011-0737-7
728 Thompson, R.C., Olson, Y., Mitchell, R.P., Davis, A., Rowland, S.J., John, A.W.G.,
729 McGonigle, D., Russell, A.E., 2004. Lost at Sea: Where Is All the Plastic? Science (80-.

22
730 ). 304, 838. https://doi.org/10.1126/science.1094559
731 Tsangaris, C., Hatzianestis, I., Catsiki, V.A., Kormas, K.A., Strogyloudi, E., Neofitou, C.,
732 Andral, B., Galgani, F., 2011. Active biomonitoring in Greek coastal waters: Application
733 of the integrated biomarker response index in relation to contaminant levels in caged
734 mussels. Sci. Total Environ. 412–413, 359–365.
735 https://doi.org/10.1016/j.scitotenv.2011.10.028
736 Turja, R., Lehtonen, K.K., Meierjohann, A., Brozinski, J.M., Vahtera, E., Soirinsuo, A.,
737 Sokolov, A., Snoeijs, P., Budzinski, H., Devier, M.H., Peluhet, L., Pääkkönen, J.P.,
738 Viitasalo, M., Kronberg, L., 2014. The mussel caging approach in assessing biological
739 effects of wastewater treatment plant discharges in the Gulf of Finland (Baltic Sea). Mar.
740 Pollut. Bull. 97, 135–149. https://doi.org/10.1016/j.marpolbul.2015.06.024
741 Van Cauwenberghe, L., Claessens, M., Vandegehuchte, M.B., Janssen, C.R., 2015.
742 Microplastics are taken up by mussels (Mytilus edulis) and lugworms (Arenicola marina)
743 living in natural habitats. Environ. Pollut. 199, 10–17.
744 https://doi.org/10.1016/j.envpol.2015.01.008
745 Van Cauwenberghe, L., Janssen, C.R., 2014. Microplastics in bivalves cultured for human
746 consumption. Environ. Pollut. 193, 65–70. https://doi.org/10.1016/j.envpol.2014.06.010
747 Vandermeersch, G., Van Cauwenberghe, L., Janssen, C.R., Marques, A., Granby, K., Fait, G.,
748 Kotterman, M.J.J., Diogène, J., Bekaert, K., Robbens, J., Devriese, L., 2015. A critical
749 view on microplastic quantification in aquatic organisms. Environ. Res. 143, 46–55.
750 https://doi.org/10.1016/j.envres.2015.07.016
751 Viarengo, A., Lowe, D., Bolognesi, C., Fabbri, E., Koehler, A., 2007. The use of biomarkers
752 in biomonitoring: A 2-tier approach assessing the level of pollutant-induced stress
753 syndrome in sentinel organisms. Comp. Biochem. Physiol. - C Toxicol. Pharmacol. 146,
754 281–300. https://doi.org/10.1016/j.cbpc.2007.04.011
755 Watts, A.J.R., Urbina, M.A., Corr, S., Lewis, C., Galloway, T.S., 2015. Ingestion of Plastic
756 Microfibers by the Crab Carcinus maenas and Its Effect on Food Consumption and
757 Energy Balance. Environ. Sci. Technol. 49, 14597–14604.
758 https://doi.org/10.1021/acs.est.5b04026
759 Wesch, C., Bredimus, K., Paulus, M., Klein, R., 2016. Towards the suitable monitoring of
760 ingestion of microplastics by marine biota: A review. Environ. Pollut. 218, 1200–1208.
761 https://doi.org/10.1016/j.envpol.2016.08.076
762 Zhao, S., Ward, J.E., Danley, M., Mincer, T.J., 2018. Field-Based Evidence for Microplastic
763 in Marine Aggregates and Mussels: Implications for Trophic Transfer. Environ. Sci.

23
764 Technol. 52, 11038–11048. https://doi.org/10.1021/acs.est.8b03467
765

24
Figure 1: Mussels caging site location with respect to the wastewater treatment plant (WWTP, Le Havre harbor) and the coastal landfill (Sainte-
Adresse).
 4
Number of suspected microplastics (items/g)

3.5

2.5

1.5

(*)
0.5

0
T0 T7d

Fragment Fibers

Figure 2: Average (+SD) number of suspected microplastics (items/g of mussels' wet weight) by shape
categories found in the non-depurated mussels (T0: farmed mussels) and the 7 days depurated mussels
(T7d).
 A)

6 1.6

Suspected microplastics in sediments

Suspected microplastics in water

1.4
5
1.2
4
1

(items/g)
(items/L)

3 0.8

0.6
2
0.4
1
0.2

0 0
Sediments

Sediments

Sediments

Sediments

Sediments
Water

Water

Water

Water

Water
B)

6
Suspected microplastics abundance

4
(items/g)

0
Caged Native Caged Native Caged Native Caged Native Caged Native
Site 1 Site 2 Site 3 Site 4 Site 5

Fragments Films Fibers Microbeads

Figure 3: Average (+SD) number of suspected microplastics by shape categories found in (A) the surface
water (items/L) and sediments (items/g), and in (B) the caged and native mussels (items/g of mussels' wet
weight).
A) Overlap index: 0.98
0.2

0.1

0.0

0.1

0.2

0.3
< 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 >
µm 1000
µm
B)
0.2
Overlap index: 0.84

0.1

0.0

0.1

0.2

0.3
< 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 >
µm 1000
µm
C) Overlap index: 0.89

0.2

0.1

0.0

0.1

0.2

0.3
< 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 >
µm 1000
µm

Caged Sediments Native Water Difference

Figure 4: Back to back histograms of size classes of suspected microplastic items found between A) caged mussels
(in blue) and native mussels (orange), B) caged mussels and the surface water (in red) and C) caged mussels and
sediments (in green). The difference in size classes frequency between samples is shown with the dotted black lines,
while the overlap index (Morisita’s index) is indicated within the graph.
 A)
0.8 Overlap index: 0.88
0.6
0.4
0.2
0.0
0.2
0.4
0.6

B)
0.8
0.6
Overlap index: 0.86
0.4
0.2
0.0
0.2
0.4
0.6

C)

0.8

0.6
Overlap index: 0.93
0.4

0.2

0.0

0.2

0.4

0.6

Caged Sediments Native Water Difference

Figure 5: Back to back histograms of polymer types found between A) caged mussels (in blue) and native mussels
(orange), B) caged mussels and the surface water (in red), C) caged mussels and sediments (in green). The difference
in polymer frequency between samples is shown with the dotted black lines, while the overlap index (Morisita’s
index) is indicated within the graph.