Environmental Pollution 314 (2022) 120230 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol Anthropogenic pollutants in Nephrops norvegicus (Linnaeus, 1758) from the NW Mediterranean Sea: Uptake assessment and potential impact on health☆ Ester Carreras-Colom a, Joan E. Cartes b, Oriol Rodríguez-Romeu a, Francesc Padrós a, Montserrat Solé b, Michaël Grelaud c, Patrizia Ziveri c, d, Cristina Palet e, Anna Soler-Membrives a, Maite Carrassón a, * a Departament de Biologia Animal, de Biologia Vegetal i d’Ecologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain b Departament de Recursos Marins Renovables, Institut de Ciències del Mar (ICM-CSIC), 08003, Barcelona, Spain c Institute of Environmental Science and Techonology (ICTA-UAB), Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain d Catalan Institution for Research and Advanced Studies (ICREA), Pg. Lluís Companys 23, 08010, Barcelona, Spain e Departament de Química, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Anthropogenic pollution is considered one of the main threats to the marine environment, and there is an Nephrops norvegicus imperious need to assess its potential impact on ecologically and economically relevant species. This study Plastics characterises plastic ingestion and tissue levels of potentially toxic metallic elements in Nephrops norvegicus and Metals their simultaneous levels in abiotic compartments from three locations of the Catalan coast (NW Mediterranean Health Biomarkers Sea). A multidisciplinary assessment of the health condition of N. norvegicus through condition indices, enzymatic biomarkers and histological techniques is provided, and its relationship with anthropogenic pollutant levels explored. Plastic fibres were commonly found in stomachs of N. norvegicus (85% of the individuals), with higher abundances (13 ± 21 fibres ⋅ ind− 1) in specimens captured close to Barcelona. The presence of long synthetic fibres in near-bottom waters, as well as the mirroring trends in abundance among locations for water and ingested plastics, suggest that uptake from water may be occurring potentially through suspension feeding. The spatial variability in the levels of metallic elements in N. norvegicus was poorly correlated to the variability in sediments. In any case, present levels in abdominal muscle are considered safe for human consumption. Levels of ingested plastics only showed significant, yet weak, correlations with glutathione S-transferase and catalase activities. However, no other health parameter analysed showed any trend potentially associated to anthropo­ genic pollutant levels. Neither the condition indices nor the histopathological assessment evidenced any signs of pathologic conditions affecting N. norvegicus. Thus, it was concluded that presently there is no evidence of a negative impact of the studied pollutants on the health condition of N. norvegicus in the studied grounds. 1. Introduction organisms of plastics and, in particular, microplastics (particles <1–5 mm; Frias and Nash, 2019; Hartmann et al., 2019). In the Mediterranean The impact of human activities on marine ecosystems is undeniable. Sea, a high cumulative human pressure on marine ecosystems has been It encompasses from historical impacts such as overfishing (Jackson highlighted due to its semi-enclosed nature, densely populated coast­ et al., 2001), to the occurrence and bioaccumulation in marine organ­ lines and intensive use of land and marine resources (Micheli et al., isms of toxic chemicals such as persistent organic pollutants and metallic 2013). High levels of pollution, including toxic metallic elements and elements (Habte et al., 2015; Johnson et al., 2013). Most recently, plastics, have been recorded in the northern-western area, extending plastic pollution has raised a great concern within the scientific com­ from the continental shelf to the deep-sea (de Haan et al., 2019; Pal­ munity (Avio et al., 2017; Canals et al., 2021, and references therein) anques et al., 2020; Pinedo et al., 2014; Sanchez-Vidal et al., 2018), because of the persistence, ubiquity and ease of ingestion by marine overlapping in areas of high marine biodiversity (Coll et al., 2012). ☆ This paper has been recommended for acceptance by Maria Cristina Fossi. * Corresponding author. E-mail address: maite.carrasson@uab.cat (M. Carrassón). https://doi.org/10.1016/j.envpol.2022.120230 Received 20 April 2022; Received in revised form 11 September 2022; Accepted 17 September 2022 Available online 22 September 2022 0269-7491/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by- nc/4.0/). E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 Given the ecological and economic importance of marine ecosystems, and even nutritional status (Sreeram and Menon, 2005; Stentiford and there is a critical need for the continued assessment of the health con­ Neil, 2011; Vogt et al., 1986), dition of their key components in order to ensure their conservation and The aim of this study was to characterise simultaneously the biotic sustainability (Österblom et al., 2017). and environmental levels of anthropogenic pollutants of concern and Decapod crustaceans are important members of the megafaunal as­ relate them to the biological and health condition of N. norvegicus from semblages of the deep-sea environments of the Mediterranean Sea three locations in the Balearic Sea. For this purpose, (1) levels of plastics (Cartes et al., 1994; Fanelli et al., 2013). Among them, the Norway in digestive contents, near-bottom water and sediment samples, and lobster Nephrops norvegicus (Linnaeus, 1758), that in Mediterranean levels of potentially toxic metallic elements in abdominal muscle and waters mostly inhabits at depths between 245 and 485 m (Cartes et al., sediments were characterised, (2) a holistic assessment of health con­ 1994), is an excellent candidate for studying the potential impact of dition through condition indices (relative condition factor, hep­ anthropogenic pollutants in this environment. There exists a compre­ atosomatic and gonadosomatic indices), enzymatic biomarkers in hensive knowledge of its biology and ecology (Johnson and Johnson, hepatopancreas (carboxylesterases, catalase, glutathione S-transferase, 2013), it is one of the most abundant decapod crustaceans at traditional pentoxyresorufin-O-deethylase) and muscle (acetylcholinesterase, fishing depths (Abelló et al., 1988), and it has a high economic value in lactate dehydrogenase, citrate synthase) and histological assessment of Mediterranean and Atlantic fisheries (Sbrana et al., 2020). target organs (gills, hepatopancreas, gonad and abdominal muscle) was Past studies targeting the populations of N. norvegicus from different performed, and (3) the correlation between health condition and levels areas in Europe (i.e. the Clyde Sea and the Mediterranean Sea) have of anthropogenic pollutants was explored. The sampling locations were already described remarkable concentrations of plastics in their stom­ chosen to potentially represent areas under different levels and char­ achs (Carreras-Colom et al., 2022; Murray and Cowie, 2011; Welden and acteristics of anthropogenic impact in the context of pollution as dis­ Cowie, 2016a). These levels were comparatively higher than those cussed in previous studies based on the different coverages of land use, commonly reported in fish species from the same areas (Bellas et al., fishing intensity, population density, presence of waste-water treatment 2016; Hermsen et al., 2017; McGoran et al., 2018) and could be related plants and the influence of river discharge (Carreras-Colom et al., 2018; to a higher uptake from the environment due to its ecological features de Haan et al., 2019). The working hypotheses were: i) that levels of (benthic feeder with a close contact with the sediment), in addition to anthropogenic pollutants in biotic and abiotic compartments would be the presence of anatomic traits (the gastric mill) that enhance the correlated and show similar spatial trends, with stronger correlations retention of ingested items (Murray and Cowie, 2011). Long-term ex­ expected between sediment and biotic levels due to the ecological traits posures to plastic ingestion have been related to deleterious effects on of the species and, ii) that if the pollutant levels could cause a negative food consumption and, eventually, the nutritional and energetic status health impact, a variability in the health parameters analysed would of N. norvegicus and other crustacean species (Watts et al., 2015; Welden also reflect this spatial variability. and Cowie, 2016b). Furthermore, plastics exposure has also been related to oxidative stress and inflammatory responses (Suman et al., 2021; 2. Materials and methods Watts et al., 2015), although the mechanisms of impact are still poorly understood. 2.1. Study area and sample collection In addition to plastic ingestion, N. norvegicus may be exposed to other anthropogenic pollutants found in the marine environment. Of these, Samples were collected in summer between June 27th and July 18th, potentially toxic metallic elements have been long recognised as a 2019 from commercial fishing vessels. Three locations along the Catalan critical issue for marine environments (GESAMP, 2001). Moreover, coast in the NW Mediterranean were selected (Costa Brava: 41◦ 29.72′ N, levels of inorganic arsenic (iAs), cadmium (Cd) and lead (Pb), among 3◦ 04.85′ E; 404 m; Barcelona: 41◦ 15.64′ N, 2◦ 26.72′ E, 438 m; Ebro others, in food are frequently surveyed, and maximum admissible levels Delta: 40◦ 09.94′ N, 1◦ 13.80′ E, 339 m; Supplementary Material are regulated in the European Union (Commission Regulation (EC) No Fig. S1). At each location, 30 individuals on a limited size range, with 1881/2006) for safety reasons. Strong evidence exists on the deleterious both chelipeds and at the intermoult stage were selected and processed effects of toxic metallic elements in crustaceans (Rodrigues et al., 2022). onboard for different purposes. Moult stage was determined through For instance, their accumulation has been related to endocrine disrup­ carapace hardness as described by Milligan et al. (2009), with in­ tion, immune suppression, oxidative stress occurrence and histopatho­ dividuals with hard exoskeletons when squeezed behind the eyes logical alterations (Frías-Espericueta et al., 2022; Hernroth et al., 2004; considered to be at the mid-intermoult stage. The choice for individuals Rodríguez et al., 2007; Zhu et al., 2018). Moreover, combined and of these characteristics was based on previous results of Murray and synergistic effects of microplastics and metallic elements, including Cd Cowie (2011) and Welden and Cowie (2016a) on the potential influence or Pb, have been reported in the freshwater crustacean Daphnia magna of these factors on plastic loadings, which were observed from in­ under controlled conditions (Lee et al., 2021; Martins et al., 2022; Yuan dividuals on a wide size range (20–60 mm cephalothorax length), as well et al., 2020). as to reduce their confounding effect on health assessment. A similar Assessing the health condition of wild organisms can be challenging number of females and males per location was also targeted. in natural settings where organisms are subject to multifactorial impacts For ten individuals, portions of the hepatopancreas and the abdom­ and no previous health standards have been developed. For this reason, inal muscle were immediately frozen and stored at 80 ◦ C for biochemical health assessment studies should integrate parameters that range from analysis. These individuals, together with other ten whole individuals, general indicators such as body condition indices, mostly related to the were fixed in Davidson’s fluid for digestive content screening and his­ nutritional status and fitness of organisms, to more specific indicators of tological assessment of target organs (portion of hepatopancreas, gills, impact related to specific physiological pathways such as enzymatic gonad and abdominal muscle). The remaining ten individuals (out of 30) biomarkers (Crespo and Solé, 2016; Wilder et al., 2016). For example, were frozen and stored at − 20 ◦ C for elemental analysis. carboxylesterases are enzymes involved in the metabolization of Environmental parameters (temperature in ◦ C, salinity in ppt, dis­ ester-containing chemicals (Satoh and Hosokawa, 2006) while catalase solved oxygen in mg/l and turbidity in FTU) were collected with a CTD is recognised as an antioxidant enzyme preventing the action of reactive profiler (ASTD152-ALC) at 5 m above the seafloor. Sediment samples oxygen species (Winston and Di Giulio, 1991). In addition, histopatho­ (two per location) were collected using a stainless steel cylinder logical techniques are considered some of the most reliable tools for attached to the fishing gear in the same fishing trawls where general health assessment (Au, 2004; Costa, 2018). Not only they allow N. norvegicus individuals were collected and in additional trawls per­ for the identification of signs of early disease but also evidence physi­ formed in close proximity (see Supplementary Material, Fig. S1). This ological disturbances, such as effects of chronic exposures to pollutants methodology has been demonstrated to collect superficial sediment 2 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 down to 500 m (Del Rio et al., 2012). Samples were kept frozen at processing were also discarded. No further corrections were applied. − 20 ◦ C for plastic and elemental analysis. The percentages of sand, silt and clay were estimated using a Beckman Coulter LS Particle Size 2.3. Determination of metallic elements Analyser, carbonate content was determined using the Bernard calcim­ eter method (UNE 7–367) and the organic matter content was estimated Abdominal muscle portions were oven-dried and homogenised, and through the Loss-on-Ignition method (LOI; 560 ◦ C, 4 h) at the “Labo­ subsamples of 0.2 g were acid-digested (5 mL HNO3,0.5 mL HF) in an ratori de Sedimentologia” (University of Barcelona). automated microwave system using closed Teflon vessels at 240 ◦ C for Net tow samples for plastic analysis were collected using a WP2-net 15 min. Similarly, sediments were oven-dried, and subsamples of 0.2 g (200 μm mesh) equipped with a flowmeter towed horizontally at 2–3 were acid-digested with 5 mL HNO3 and 2 mL HCl at 200 ◦ C for 15 min knots and 5–10 m above the seafloor for 30 min. Two net tows were (EPA3051a method). Concentrations of Cd, Zn, Li and Ti (only muscle), collected at Barcelona and Ebro Delta and one in the Costa Brava. and Pb, Cu, Co, Cr, Ni, Mn, Al and As (both in muscle and sediment) were analysed by inductively coupled plasma-mass spectrometry (ICP-MS; 2.2. Analysis of plastics and other anthropogenic items Agilent 7500CE). Certified reference materials (ERM-BB422 Fish Mus­ cle, Joint Research Centre; BCR-701 Lake Sediment, European Com­ Stomach and intestine contents were visually screened for the pres­ mission) and laboratory blanks were included in each batch. Percentage ence of any items differing from their regular diet (Cristo, 1998; Cristo recovery from certified reference materials was acceptable (ranging and Cartes, 1998). between 85 and 115%), and no corrections were applied. Extraction of plastics from water and sediment samples was per­ formed by combining already published protocols (Cole et al., 2014; Liu 2.4. Health condition assessment of N. norvegicus et al., 2019; Masura et al., 2015; Simon et al., 2018; Thompson et al., 2004). Details can be found in the Supplementary Material. Briefly, For all individuals, cephalothorax length (CL, in mm), total weight water samples were sieved (1 mm) and the <1 mm fraction was digested with chelipeds (TW, in g) and sex were recorded. Sex was determined at room temperature with 1 M NaOH and the >1 mm fraction underwent based on the morphology of the first pair of pleopods (Farmer, 1974). direct visual inspection. Sediment samples went through a Fenton re­ The hepatopancreas (HW), gonads (GW) and stomach content (SCW) action (50% H2O2, 0.1 M FeSO4, 0.1 M NaOH) and then two successive were also weighted (0.001 g). Condition indices were calculated as density separations using a hypersaline solution. All supernatants and follows: relative condition factor (Kn = TW/EW, where EW is the ex­ fractions resulting from digestions were vacuum-filtered onto poly­ pected weight from the TW-CL regression adjusted for each sex with all carbonate membranes (Millipore, Ø47 mm, 0.45 μm) that were visually sampled individuals), hepatosomatic index (HSI = HW/TW x 100), inspected after drying overnight at 40 ◦ C. gonadosomatic index (only in females, GSI = GW/TW x 100) and All particles of potential anthropogenic origin were separated, clas­ stomach fullness (F = SCW/TW x 100). sified according to their shape (fibres and other-shaped items, including Activities of carboxylesterases (CbE), catalases (CAT), glutathione S- fragments and films), counted and measured. Polymer composition was transferases (GST) and pentoxyresorufin-O-deethylases (PROD) in identified as described in Carreras-Colom et al. (2020) and Rodrí­ hepatopancreas, and activities of acetylcholinesterases (AChE), lactate guez-Romeu et al. (2020). Items were inspected with optical microscopy dehydrogenases (LDH) and citrate synthases (CS) in abdominal muscle (x400) and categorised according to their visual characteristics (diam­ were analysed following the procedures described elsewhere (Antó eter uniformity and cross-section, finishing, striations and signs of wear, et al., 2009; Koenig et al., 2013; Solé and Sanchez-Hernandez, 2018). and surface and backbone textures). A subsample of each category (>4% Details on the preparation of samples and protocols followed for this per category, 32.8% of all items found) was analysed with FTIR (Servei purpose can be found in the Supplementary Material. All enzymatic d’Anàlisi Química, Universitat Autònoma de Barcelona, and CCitUB, activities are expressed in relation to the total protein content of the University of Barcelona) and each visual category was assigned a poly­ sample, which was determined by the Bradford (1976) assay adapted to meric composition based on the results (for each category >80% of a microplate. items were identified as the same polymer). Cellulosic fibres, which are Hepatopancreas, gonads, gills and abdominal musculature from recognised as a form of anthropogenic pollution similar to plastics and Davidson’s fixed individuals were processed in paraffin through routine thus may have a similar impact (Kanhai et al., 2017; Salvador Cesa et al., histological techniques. Qualitative histological examination through 2017), were not excluded from the results, although values are reported light microscopy was conducted on haematoxylin-eosin-stained sections separately. Anthropogenic items include both plastic and cellulosic by comparing the tissue organisation and integrity to that reported as items. normal in decapod crustacean species (Bell and Lightner, 1988; Shields Abundance (number of items) and load (as the sum of lengths of fi­ and Boyd, 2014). The gonadal development stage for females was also bres, in mm, and the sum of areas of fragments and films, in mm2) per determined according to Becker et al. (2018). individual or unit of water and sediment sampled, i.e., m3 and mL, respectively, were calculated. 2.5. Data analysis Preventive measures were taken to reduce contamination. Briefly, dissections were performed in a Class II cabinet (Telstar ® BIO-II-A), Differences among locations in the prevalence, abundance and load water and sediment samples were processed in a clean laboratory con­ of ingested fibres were tested using Generalized Linear Models (GLM; sisting in an 8 m2 hardwall laminar flow chamber ventilated through 4 Poisson distribution for prevalence and Poisson distribution for abun­ laminar flow HEPA filters (H14), all material used was rinsed with dance and load). The overall significance of the fitted models was tested filtered water, cotton or orange lab coats were worn at all times, using an analysis of deviance by comparing the fitted model to a null screening of plastics was performed under covered stereomicroscopes model. Similarly, differences among locations in the levels of metallic (Torre et al., 2016) and procedural blanks and air controls were per­ elements in abdominal muscle and health parameters (condition indices formed to monitor airborne contamination rates and to ensure that po­ and enzymatic biomarkers) were tested using analysis of variance (one- tential contamination was kept at a minimal level (average value 0.78 way ANOVA) considering the sampling location as the independent fibres per sample screened). During the screening of digestive contents, variable and being Costa Brava, Barcelona and Delta three independent fibres identified as floating (deposited on the surface were discarded. For and fixed levels. When ANOVA’s assumptions of normality and homo­ sediments and near-bottom water samples fibres found on the limits of scedasticity were not met (tested through Shapiro-Wilk test for the filters (outside the coloured water mark created during the filtration normality and Levene’s tests for homogeneity of variances, respec­ process) or fibres resembling the orange lab coats used during the tively), Kruskal-Wallis tests were used instead. Significant effects found 3 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 through ANOVA or Kruskal-Wallis tests were examined using the post from Barcelona. Overall, 85% of the individuals had ingested at least one hoc Tukey Honest Significant Difference method and Dunn’s test, plastic fibre, and 12% had cellulosic fibres (Table 1). Tangled fibres in a respectively. Moreover, the potential interaction and main effects of CL ball were observed in 20% of the individuals from Costa Brava and and sex were tested using Multiple Linear Regressions (MLR, for CL) and Barcelona. two-way ANOVA (for sex, using Type II sum of squares) for parametric Differences among sampling locations were only observed for the data, or GLM (both CL and sex) for non-parametric data. abundance and load of ingested plastic fibres (Table 1, Supplementary Further characterisation of plastic pollution was performed by ana­ Material Tables S1 and S2) but not for the prevalence (% of individuals; lysing the differences among locations and matrices (ingested vs water binomial GLM: p > 0.05), with individuals from Barcelona showing and sediment) in the polymer composition and size distribution of higher values compared to the other locations (GLM; abundance: z = plastics through permutational analysis of variance (PERMANOVA; − 6.42 and − 7.70, p < 0.001; load: z = − 5.78 and − 7.71, p < 0.001). “vegan” package) and an adaptation of the Kolmogorov-Smirnoff test, Moreover, fibres found entangled into balls were also more abundant respectively. Differences in the mean fibre length were analysed with and made up for a higher load in individuals from Barcelona compared nonparametric tests (Kruskal-Wallis test) and Dunn’s test was used to to those from the Costa Brava (GLM; abundance: z = − 7.13, p < 0.001; explore significant effects. load: z = − 7.70, p < 0.001). The non-parametric Spearman’s correlation between the abundance Significant differences were identified among locations in terms of of anthropogenic items ingested and health parameters (condition fibre size distribution of ingested plastic fibres (K–S: D = 0.21, p < indices and enzymatic biomarkers) was calculated and significant re­ 0.001; Supplementary Material Table S3), with individuals from Bar­ sults were further explored through MLR to assess the influence of CL, celona showing the highest relative proportion of <1 mm fibres (34% of sex and location. Moreover, potential patterns among locations were the total) compared to individuals from other locations (~13%; Fig. 1). visualized through Principal Component Analysis (PCA), which also In all three locations fibres of 1–5 mm represented more than half of the included physicochemical parameters from near-bottom water (tem­ fibres analysed and fibres >5 mm contributed by 12–19%. Mean fibre perature, salinity, dissolved oxygen and turbidity) and sediment (car­ length was also significantly smaller in Barcelona (2.3 ± 2.1 mm) bonate, organic matter content and granulometry), and potential compared to the Costa Brava and the Ebro Delta (3.1 ± 2.8 and 3.2 ± associations among them were further explored with Redundancy 2.8 mm, respectively, Supplementary Material Tables S4 and S5) (K–W: Analysis (RDA). A backwards stepwise selection procedure based on a χ2 = 28.93, p < 0.001). Differences in the mean length were also permutational test selected the most explanatory variables to display in observed regarding polymer composition (K–W: χ2 = 73.63, p < 0.001), RDAs. with PP and PET fibres being the longest and acrylic and cellulosic ones All statistical analyses were performed in R version 4.1.0, and the the shortest (Supplementary Material Tables S4 and S5). No significant significance level was set at p < 0.05. differences were observed in the relative composition of polymers among locations (PERMANOVA; pseudo-F = 1.99, p = 0.07; Supple­ 3. Results mentary Material Table S6). In environmental samples, the most commonly found items were also 3.1. Biological and environmental levels of anthropogenic items plastic (70.3%) and cellulosic fibres (23.3%) (Supplementary Material Fig. S2). Fragments (5.4%) and films (1%) were identified in near- All items identified from digestive contents were fibres, mostly bottom water samples from all locations but only in sediments from synthetic (98%), except for a black film-like particle made of poly­ the Ebro Delta. These items of irregular shape were small (<1 mm) and ethylene (PE) (Supplementary Material Fig. S2). They were found in the were mostly made of PE and polyethylene terephthalate (PET) (Sup­ stomach except for a cellulosic fibre with traces of black pigment found plementary Material Table S6, Fig. S2), and none of them surpassed the among intestine contents. Both exceptions were recorded in individuals 5 mm size in diameter. The highest abundance of plastic fibres was Table 1 Values of prevalence (in %) and abundance (number of items) and load (sum of lengths for fibres, and sum of areas for fragments and films) of anthropogenic items in stomach contents of Nephrops norvegicus and near-bottom water and sediment samples according to the sampling location. n = number of individuals (females)/ samples analysed. Mean values (standard deviation) are presented. Different superscript letters among columns indicate significant differences among locations. Costa Brava Barcelona Ebro Delta Stomach contents n 20 (9) 20 (9) 20 (12) Synthetic fibres % 75 a 85 a 95 a items ⋅ ind− 1 6.2 (6.8) a 13 (21) b 5.2 (4.4) a mm ⋅ ind− 1 20 (22) a 29 (42) b 17 (15) a Cellulosic fibres % 15 a 5a 15 a items ⋅ ind− 1 0.15 (0.37) a 0.050 (0.22) a 0.15 (0.37) a mm ⋅ ind− 1 0.51 (1.3) a 0.020 (0.11) a 0.15 (0.38) a Tangled fibres % 20 a 20 a 0 items ⋅ ind− 1 1.6 (4.1) a 6.5 (18) b 0 mm ⋅ ind− 1 6.2 (14) a 14 (37) b 0 Fragments and films % 0 5 0 items ⋅ ind− 1 0 0.050 (0.22) 0 mm2 ⋅ ind− 1 0 0.070 (0.33) 0 Near-bottom water n 1 2 2 − 3 Synthetic fibres items ⋅ m 0.12 1.1 (0.67) 0.058 (0.037) 3 Cellulosic fibres items ⋅ m− 0.14 0.68 (0.59) 0.10 (0.030) 3 Fragments and films items ⋅ m− 0.13 0.10 (0.13) 0.014 (0.010) Sediments n 1 1 2 − 1 Synthetic fibres items ⋅ ml 0.040 0.080 0.020 (0.030) − 1 Cellulosic fibres items ⋅ ml 0.14 0.020 0.060 (0.010) − 1 Fragments and films items ⋅ ml 0 0 0.060 (0.080) 4 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 observed in Barcelona for both near-bottom water and sediment sam­ ples, while cellulosic fibres were more abundant in Barcelona’s near- bottom waters and Costa Brava’s sediments (no statistical tests could be performed, Table 1 and Supplementary Material S1). The polymer composition of fibres in Costa Brava and Barcelona locations was dominated by polyamide (>70%) in near-bottom water samples and cellulose (>50%) in sediments, while in the Ebro Delta cellulose dominated in water (>50%) and polypropylene and cellulose were almost equally present in sediments (50 and 44.5%, respectively) (Supplementary Material Table S6). Acrylic, PA, PE and PET fibres were also identified in sediments but accounted for <20% of the total. Considered altogether, different patterns on the size distribution of synthetic fibres among locations were observed in near-bottom waters (K–S, D = 0.32–0.52, p < 0.05; Supplementary Material S3), with samples from Barcelona showing the highest relative proportion of >5 mm fibres (63%), the Costa Brava samples being dominated by either short (<1 mm, 52%) or long fibres (>5 mm, 49%) whereas the Ebro Delta samples being equally represented by 1–5 and >5 mm fibres (50% each). In sediments, no significant differences in the size distribution were found among locations (K–S, p > 0.05), although fibres >5 mm were absent in two locations (Costa Brava and Barcelona) (Fig. 1). 3.2. Biological and environmental levels of metallic elements Significant differences among locations were observed in the levels of metallic elements in abdominal muscle (ANOVA; F2,26 = 3.77–11.93, p < 0.05; Supplementary Material Table S7) with the exceptions of Ni, Cu and Pb. The spatial trends were not consistent across all metallic elements (Table 2). Individuals from the Ebro Delta showed the highest levels of Co and As, whereas their levels of Ti, Mn and Cd, were only comparatively higher to those observed in Costa Brava. Both the Ebro Delta and Barcelona individuals also showed higher levels of Al and Zn compared to those from Costa Brava. On the contrary, Li levels were higher in Costa Brava specimens compared to the Barcelona ones. No statistical tests were performed for Cr since its levels in Costa Brava and Barcelona were below the detection limit (LOD 0.31 ppm). No signifi­ cant relationships with size or sex were found for the concentration of any metallic element analysed with the exception of As, for which higher values were recorded in males (ANOVA; F1,26 = 1573.3; p = 0.027; Supplementary Material Table S7). In sediment samples, Barcelona showed the highest concentrations for all elements, with equally high Al, Cr, Ni and Cu levels in the Costa Brava or As in the Ebro Delta samples (no statistical tests were per­ formed; Table 2). The redundancy analysis (RDA) of the content of the metallic ele­ ments in N. norvegicus with stepwise selection of physicochemical pa­ rameters of water and sediment (see Supplementary Material Table S3) suggested that the organic matter content and the percentage of silt and clay were the most explanatory variables for the trends observed among locations (Fig. 2). Sediments from Barcelona showed the highest organic matter content (15.9% compared to 13.9 in the Costa Brava and 13.3% for Ebro Delta), whilst those from the Ebro Delta showed the highest content of silt and clay (50.6% compared to 34.2 in the Costa Brava and 40.9% in Barcelona) (no statistical tests performed; Supplementary Material Table S8). Fig. 1. Size distribution of synthetic (SF) and cellulosic fibres (CF) found in stomach contents and near-bottom water and sediment samples according to 3.3. Health condition assessment and correlation with pollutants the sampling location. Kernel density distribution was used to avoid the con­ founding effect of different concentration values. To improve visualization, SF 3.3.1. Condition indices and CF in sediments were grouped and SF > 20 mm from stomach contents Significant differences among locations were observed in the HSI and from Barcelona and all water samples were not included. GSI mean values (Table 3, Supplementary Material Table S9), with in­ dividuals from the Ebro Delta showing higher HSI values (ANOVA; F2,56 = 9.38, p < 0.001) and Barcelona females showing higher GSI ones (ANOVA; F2,27 = 4.54, p = 0.02). All females, regardless of the sampling location, were considered to be in a late ovary maturation stage (stage 4, Becker et al., 2018). Stomach fullness was also significantly higher in the 5 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 Table 2 Metallic elements in abdominal muscle of Nephrops norvegicus and sediments from the three locations sampled. Mean values (standard deviation) are presented. The sample size is ten individuals per location for N. norvegicus abdominal muscle (although one individual from Barcelona and one from the Ebro Delta were discarded due to analytical inaccuracy for the analysis of Li, Mn, Co, Ni, Cd and Pb content) and two samples per location for sediment. Values are given in mg ⋅ kg− 1 dry weight except for Al, given in mg ⋅ g− 1 dry weight. Different superscript letters indicate differences among locations. BDL = below detection limit. Guideline values in mg ⋅ kg− 1: ERL = effects range-low; ERM = effects range-medium; TET = toxic effect threshold. Li Ti Cr Mn Co Al N. norvegicus a a Costa Brava 0.61 (0.15) 3.5 (2.9) BDL 3.8 (0.9) a 0.06 (0.01) a 47 (53) a b ab Barcelona 0.41 (0.19) 3.7 (1.5) BDL 5.2 (1.8) ab 0.06 (0.01) a 64 (46) b ab b Ebro Delta 0.51 (0.08) 5.1 (1.1) 0.4 (0.2) 5.4 (1.4) b 0.08 (0.02) b 78 (16) b Sediment Costa Brava – – 40 (3.0) 510 (23.0) 10 (1.0) 26 (2.6) Barcelona – – 42 (1.0) 920 (316) 12 (1.0) 25 (10) Ebro Delta – – 23 (9.0) 492 (236) 9.1 (1.3) 11 (6.6) N. norvegicus Ni Cu Zn As Cd Pb a Costa Brava 0.22 (0.10) 16 (5.0) a 53 (3.0) a 110 (22.0) a 0.023 (0.010) a 0.28 (0.23) a a Barcelona 0.17 (0.10) 19 (7.0) a 57 (3.0) b 113 (14.0) a 0.020 (0.010) ab 0.32 (0.17) a a Ebro Delta 0.20 (0.10) 23 (8.0) a 58 (2.0) b 77.0 (18.0) b 0.015 (0.010) b 0.28 (0.13) a Sediment Costa Brava 37 (1.0) 14 (1.0) – 15 (2.0) – 26 (1.0) Barcelona 39 (1.0) 18 (1.0) – 25 (1.0) – 42 (1.0) Ebro Delta 25 (4.0) 9.0 (6.0) – 27 (1.0) – 29 (9.0) ERLi 20.9 34 150 8.2 1.2 46.7 ERM i 51.6 270 410 70 9.6 218 TET j 61 86 540 17 3 170 i Long et al. (1995). j MacDonald et al. (2000). Costa Brava compared to individuals sampled from Barcelona (ANOVA; analysis on the correlation among condition indices and levels of F2,57 = 5.90, p = 0.005). No other spatial, size or sex-related differences metallic elements could be performed at the individual level due to were observed for condition indices, including Kn (MLR; p > 0.05). dataset limitations, and multivariate scaling techniques did not show No significant correlations between condition indices and the any relevant associations (RDA not included). abundance and load of ingested fibres (total and for each polymer type) were identified (rs < 0.20, p > 0.05), nor when then sampling location, 3.3.2. Biomarker responses sex and size were accounted (ANOVA and MLR, p > 0.05). No further No site-related differences were observed for most enzymatic bio­ markers (ANOVA, p > 0.05; Table 3 and Supplementary Material Table S9), except for LDH (ANOVA, F2,27 = 7.35, p = 0.003) and CbE (ANOVA, F2,21 = 6.91, p = 0.005). Mean LDH activities were higher in the Barcelona and Ebro Delta individuals compared to those from the Costa Brava whereas CbE activities were higher in the Costa Brava compared to the Ebro Delta. LDH was the only biomarker showing a positive correlation with CL (rs = 0.60, p = 0.01) and with HSI (rs = 0.43, p = 0.001), whilst CAT and CbE showed a negative correlation with GSI (rs = − 0.78, p = 0.01, and rs = − 0.50, p = 0.043, respectively) (Supplementary Material Fig. S3). Only GST and CAT activities showed significant associations to the values of fibres ingested, although correlations were small (rs = 0.41 and − 0.45, respectively, p < 0.05). Individuals with a higher synthetic fibre load showed higher mean values of GST (MLR, t = 2.84, p = 0.01) and smaller of CAT (MLR, t = − 2.70, p = 0.01), although the proportion of variance explained in both cases was small (R2 = 0.27 and 0.25, respectively). Similar results (levels of significance and coefficient esti­ mates) were observed for the abundance of fibres. A significant inter­ action was observed for LDH between the fibre load and individuals’ size (MLR, t = 2.27, p = 0.032). When only bigger individuals (CL > 31.6 mm) were considered, higher LDH activities were observed in in­ Fig. 2. Redundancy analysis (RDA) on the tissue levels of metallic elements in dividuals with a higher fibre load (MLR; t = 2.19, p = 0.04, R2 = 0.23). N. norvegicus from three locations constrained by the physicochemical param­ When considering the plastic load for each polymer composition, sig­ eters of near-bottom waters and sediments, from which the content of organic nificant, although small, correlations were observed between GST ac­ matter (OM) and the percentage of silt and clay (SC) were selected as the most significant explanatory variables for the ordination. For interpretation of the tivity and PET content (rs = 0.41, p = 0.015), GST and PP (rs = 0.41, p = references to colour in the legend, the reader is referred to the web version of 0.008), CAT and PP (rs = − 0.19, p = 0.046), and LDH and PA (rs = 0.13, the article. (For interpretation of the references to colour in this figure legend, p = 0.02) (Supplementary Material Fig. S3). MLR results showed that the reader is referred to the Web version of this article.) these associations were either poorly fitted (R2 < 0.27, Supplementary 6 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 Table 3 Size range, condition indices and enzymatic activities from hepatopancreas and abdominal muscle of N. norvegicus according to the sampling location. Mean values (standard deviation) are given. Abbreviations for condition indices and enzymatic markers can be found in the corresponding sections throughout the text. Different superscript letters indicate significant differences (p < 0.05) among sampling locations. n = sample size (females), CL = cephalothorax length in mm. Costa Brava Barcelona Ebro Delta Size range nd 30 (13) 30 (10) 30 (16) CL 25.0–39.2 32.5–40.7 28.0–39.1 Condition indices ne 20 (9) 20 (9) 20 (12) Kn 0.97 (0.13) a 1.06 (0.13) a 1.00 (0.13) a HSI 4.0 (0.72) a 4.3 (0.78) a 4.9 (0.84) b GSI 4.4 (1.7) a 6.5 (1.9) b 4.5 (1.5) a F 2.0 (0.75) a 1.1 (0.89) b 1.5 (1.0) ab Abdominal muscle nf 10 10 10 AChE i 9.1 (2.3) a 11 (4.6) a 11 (3.1) a Fig. 3. Principal Component Analysis on the enzymatic response of LDH j 2.1 (0.20) a 2.7 (0.40) b 2.5 (0.40) b N. norvegicus with the total load of synthetic fibres (in mm) used as the re- CS i 20 (4.3) a 18 (5.8) a 16 (5.3) a scaling variable for points size. Letters indicate the sampling location of each observation (C = Costa Brava, B = Barcelona; E = Ebro Delta). Abbreviations Hepatopancreas for enzymatic markers can be found in the materials and methods section. nf 10 10 4 CbE i 369 (76.0) a 294 (55.0) ab 226 (83.0) b CAT j 168 (61.0) a 110 (61.0) a 139 (35.0) a 4. Discussion GST j 2.0 (0.40) a 2.3 (0.50) a 1.6 (0.23) a PROD k 0.25 (0.10) a 0.25 (0.10) a 0.21 (0.13) a 4.1. Ingested and environmental levels of plastics and other anthropogenic i nmol min− 1⋅ mg protein− 1. items j μmol min− 1⋅ mg protein− 1. k pmol min− 1⋅ mg protein− 1. The high prevalence (>80%) and abundance (between 5.2 and 13 d All individuals included in the study (anthropogenic pollutants analysis and fibres ⋅ ind− 1) of ingested plastics observed fall into the range of values health assessment). reported for this species elsewhere (Carreras-Colom et al., 2022; Cau e Subsample of nd with individuals used for both health assessment trough et al., 2019; Hara et al., 2020; Welden and Cowie, 2016a). The pre­ condition indices and anthropogenic ingestion analysis. dominance of fibres (>90%) was highlighted in some of these former f Subsample of ne with individuals used for biochemical analysis. studies, particularly in those carried out in Irish waters and the Clyde Sea (Hara et al., 2020; Joyce et al., 2022; Murray and Cowie, 2011; Material Table S10) or not significant when sampling location was Welden and Cowie, 2016a). On the contrary, in studies conducted in the considered. Mediterranean Sea (i.e., the Sardinian coast and the Adriatic Sea), a The PCA analysis provided an ordination of individuals according to larger proportion of fragments (86%) and films (57%) was identified their sampling location, with individuals with a higher plastic load (Cau et al., 2019; Martinelli et al., 2021). It seems unlikely that these positioned mostly on the left of the axis and, therefore, negatively differences are related to environmental differences as the predomi­ associated with CAT and positively with LDH activities (Fig. 3). RDA nance of fibres has also been reported in sediment and surface waters of analysis did not reveal any significant patterns with physicochemical the Mediterranean Sea (Sanchez-Vidal et al., 2018; Suaria et al., 2016) as parameters (ordination p > 0.05, RDA not included). well as in plastics ingested by organisms (Bellas et al., 2016; Mancuso et al., 2019; Nadal et al., 2016; Tsangaris et al., 2020; Valente et al., 3.3.3. Histological analysis 2019). More likely, the discrepancy could be due to the methodological Histopathological assessment of the targeted organs (gills, hepato­ approach. In both (Sardinian coast and the Adriatic Sea) studies, samples pancreas and abdominal muscle) did not reveal any relevant alterations were digested, which may allow identifying particles of a smaller size except for a granuloma-like structure in the gills of one individual from but may have destroyed fibres (Athey and Erdle, 2021), while Cau et al. Barcelona and a haemocytic nodule within an extensive haemocyte (2019) purposely excluded over 2000 textile fibres from their results infiltration in the abdominal muscle of one specimen from the Ebro because of a potential airborne uncontrolled origin. The overestimation Delta (Supplementary Material Fig. S4). These alterations could not be of fibres due to contamination during processing has been a subject of associated with any biological factor. debate in plastic ingestion studies (Foekema et al., 2013; Pedà et al., Overall, the gill epithelium showed a regular appearance, and no 2020). Although the contribution of airborne fibres cannot be fully ruled alterations of cell morphology were identified in hepatopancreatic or out in our study, the strict quality control procedures implemented muscular cells. The low prevalence of histological findings prevented together with the high contribution of tangled balls of fibres, which are further analysis of any potential relationship with size, health parame­ certainly not a result of sample contamination, support the dominance of ters (condition indices and enzymatic activities) or anthropogenic pol­ fibres in N. norvegicus. lutants (plastics and metallic elements). The site-related differences reported along the Catalan coast, with 7 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 higher values of plastic ingestion in samples collected near Barcelona, do periods, but isolated fibres may be more easily excreted through the not seem related to the slight differences in the size range of the in­ intestine, especially cellulosic ones that may break down thanks to the dividuals. Firstly, because bigger individuals (i.e. the ones from Barce­ stomach grinding action (Cau et al., 2020; Mateos-Cárdenas et al., lona) would be expected to retain less plastic than smaller ones, but most 2020). importantly because the relationship between CL and plastic loadings Until now, it had been hypothesized that plastic ingestion in benthic was described from individuals within a wider size range (20–60 mm CL; crustaceans might occur passively as plastics deposited on the seafloor Welden and Cowie, 2016a). On the contrary, the spatial differences might be accidentally ingested while preying on epibenthic and endo­ observed, that match the patterns described in anthropogenic fibre benthic fauna (Carreras-Colom et al., 2018; Murray and Cowie, 2011). ingestion at shallow areas (~100 m) in red mullet Mullus barbatus However, a former study trying to relate plastic ingestion and diet in (Rodríguez-Romeu et al., 2020), and at greater depth (~800 m) in the N. norvegicus failed to point out strong significant associations with deep-sea shrimp Aristeus antennatus (Carreras-Colom et al., 2018, 2020), benthos (Carreras-Colom et al., 2022). Most recently, Joyce et al. (2022) might be the result of different levels of exposure. The dominance of PET observed that the most common polymers found in sediments mirrored and acrylic fibres, two commonly used polymers in the textile industry, those found in the digestive contents of N. norvegicus from the North-East in addition to the presence in sediments of blue-dyed cellulosic fibres Atlantic Sea, although other abiotic compartments were not sampled. In similar to those pointed out as potentially coming from Jeanswear by the present study, although the absolute concentration of synthetic fi­ other studies (De Wael et al., 2011; Rodríguez-Romeu et al., 2020), bres in sediments highly surpassed that of near-bottom waters, no fibres support a land-based origin. Release from textiles in households is longer than 5 mm were identified in sediments from the Costa Brava and considered one of the main sources of marine plastic pollution (Napper Barcelona locations, and the patterns of abundance among locations did and Thompson, 2016; Stothra Bhashyam et al., 2021), being untreated not mirror those of ingested fibres. Altogether, this suggests that there stormwater and river discharge the most likely major channels for could also be a notable fibre uptake from water, particularly since longer reaching the sea (Athey and Erdle, 2021; Galafassi et al., 2019). fibres (>5 mm) contributed by nearly 30% to all items ingested. Sus­ Therefore, the high population density of the Barcelona area could pension feeding in adults of N. norvegicus is known to occur, with sus­ explain the elevated plastics contribution into the sea (Alomar et al., pended particulate matter representing to up to half of its diet in the 2016; Ruiz-Orejón et al., 2018) and consequently the higher levels of Clew Bay (Santana et al., 2020). In the absence of further evidence on ingested plastics observed. whether Mediterranean populations might share this same feeding Our results further suggest that plastics, mostly synthetic fibres, are strategy and to what extent, it seems plausible that plastics in the common in both near-bottom waters and sediments from all sampled near-bottom water might be ingested during suspension-feeding. In locations, with particularly high concentrations in sediments although addition, although N. norvegicus is an epibenthic species that lives in comparison of absolute values would be inadvisable. Indeed, the sea­ burrows, its diet on the Catalan slope has a higher contribution of floor has been suggested as the ultimate sink of plastics and marine litter pelagic prey (gelatinous plankton, euphausiids) than expected (Cristo (Fischer et al., 2015; Mordecai et al., 2011; Sanchez-Vidal et al., 2018). and Cartes, 1998) which suggests that accidental ingestion of sedi­ To the best of our knowledge, only two studies have characterised the mented plastics while preying on benthos may be less likely to occur. In presence of plastics in deep waters so far (Choy et al., 2019; Courte­ any case, considering the discreet sample size of the present study and ne-Jones et al., 2017). In both cases, the concentrations reported (2.9 the spatial and temporal variability commonly reported for plastic and 70.8 particles ⋅ m− 3, respectively), were much higher than in our pollution (Carreras-Colom et al., 2020), additional studies targeting study (mainly <1 particles ⋅ m− 3), a discrepancy that may be again simultaneously these deep-sea organisms together with their surround­ related, at least to some extent, to methodological (bulk vs filtration) ing environment (near-bottom water and seafloor sediments) at a wider differences (Barrows et al., 2017; Simon-Sánchez et al., 2022). Similarly, geographical frame would be needed to further evaluate the relevance of the comparison with available data in the NW Mediterranean area is each route of uptake. limited as Lefebvre et al. (2019), who reported 0.23 fibres ⋅ m− 3, sampled the whole water column while de Haan et al. (2019), who re­ 4.2. Accumulated and sediment levels of metallic elements ported between 0.039 and 2.057 items ⋅ m− 3 along the Catalan coast, used a bigger mesh size (335 μm) and excluded fibres from the analysis. The levels of most metallic elements (As, Al, Cd, Co, Cr, Cu, Mn, Ni, Regarding sediments, the only comparable study conducted in the area Pb and Zn) analysed in abdominal muscle fell in the same range of values reported slightly higher levels in the Costa Brava, between 0.1 and 0.7 reported in past studies in the Mediterranean Sea and Atlantic Ocean plastic fibres ⋅ mL− 1, (0.1–1.1 fibres ⋅ mL− 1 in the present one) with a (Canli and Furness, 1993a, 1993b; Eriksson, 2000; Iamiceli et al., 2015; similar dominance of cellulosic over synthetic fibres (Sanchez-Vidal Lourenço et al., 2009; Mille et al., 2018; Schuhmacher et al., 1992; et al., 2018). Nonetheless, different instruments were used to collect Storelli et al., 2012). Similarly, metallic elements in sediments were sediment samples hence, given the limited knowledge on the influence either in the same range or below those previously reported at the same of different instrumentation on the analysis of sediments for plastic locations (Jordana et al., 2015; Palanques et al., 2020; Pinedo et al., pollution, caution should be exerted when discussing these values. 2014). The polymer composition of ingested fibres (PET > PA and Acrylic > The most relevant differences observed for tissue levels with avail­ PP and PE) did not match the proportions reported for the abiotic able data for the species were the lower concentration of Cd in our study compartments in this study, with an almost exclusive dominance of PA (0.02 mg ⋅ kg− 1 d. w.) compared to the values reported in the Gulf of in waters and unclear trends in sediments. However, some similarities Lion in 2012 (0.33 mg ⋅ kg− 1 d. w.; Mille et al., 2018) or in areas of could be found with the composition of fibres described in the literature significant industrial activity (e.g., the Clyde Sea, >1 mg ⋅ kg− 1 d. w.; for deep-sea sediments (Cellulose > PET > Acrylic > PA > PE > PP; Canli and Furness, 1993a). For some metallic elements, comparison with Sanchez-Vidal et al., 2018). The almost absence of PET in our environ­ literature data was either limited for comparison to other species (Li; mental samples while being the most abundant ingested items, and the Leblanc et al., 2005) or not possible at all (Ti). Titanium, which was absence of cellulosic fibres in the digestive contents while being pre­ detected in our muscle samples at variable concentrations (0.9–7.0 mg ⋅ dominant in sediments is noteworthy. This discrepancy may be related kg− 1 d. w.), is receiving increasing attention in plastic studies as it is to differences in their retention times once ingested. PET fibres not only widely used in the form of TiO2 as a plastic additive (pigment) and were the longest but also the most predominant in tangled balls of fibres because some studies have suggested that it could enhance the adsorp­ (~80%) whilst other synthetic polymers were equally present as isolated tion of metals to plastic surfaces (Godoy et al., 2019). Moreover, the or entangled. Tangled balls of fibres may only be removed through European Food Safety (EFSA) no longer considers it safe as a food ad­ ecdysis (Welden and Cowie, 2016a, 2016b), thus being retained for long ditive (EFSA, 2021). However, the lack of information on its current 8 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 environmental levels prevents further discussion. suitable for the construction and maintenance of extensive burrow Spatial trends in the tissue levels of metallic elements in N. norvegicus complexes, and a significant correlation between areas with a finer were varied, with individuals from Barcelona, exposed to overall high composition, higher population densities and also larger individuals was environmental concentrations of metallic elements in sediments, not observed in the Clyde Sea (Bailey et al., 1986; Campbell et al., 2009). In mirroring a clear higher accumulation pattern. Body levels of metallic our case, the highest percentage of silt and clay was indeed found in elements in aquatic invertebrates depend on numerous factors (e.g., the sediments from the Ebro Delta, where the higher HSI values were also relative bioavailability of these elements in solution and prey or envi­ reported. ronmental conditions such as the organic matter content or sedimenta­ Other significant correlations between pollutants and health were tion rates) that determine the balance between uptake, excretion, those observed for antioxidant enzymes, with individuals with the detoxification, and accumulation (Rainbow, 2002; Rainbow and Luoma, highest levels of plastics ingested showing lower activities of CAT and 2011). Analysing other tissues that may have a higher tissue burden (i.e. higher activities of GST. Similar trends in these biomarkers have been hepatopancreas) may shed some light. Nevertheless, a certain correla­ correlated with plastic exposure in experimental studies with fish tion among the levels in different tissues would be anticipated, as (Alomar et al., 2017), small crustaceans (Jeong et al., 2017, 2016), bi­ indicated in experimental and observational studies in the Clyde Sea valves (Avio et al., 2015; Paul-Pont et al., 2016; Ribeiro et al., 2017), (Canli and Furness, 1993a, 1993b). and nematodes (Lei et al., 2018). These findings suggest that plastics Regarding human safety, reported levels of Cd and Pb in abdominal might induce the formation of reactive oxygen species, thus triggering muscle (0.004 and 0.006 mg ⋅ kg− 1 in w. w., respectively) could be an oxidative stress response, and that assessing antioxidant enzymes considered safe according to the EFSA limit (0.50 mg kg− 1 in w. w. for might be suitable for environmental monitoring of plastic pollution crustaceans). Values of As (mean 21 mg ⋅ kg− 1 in w. w.), for which there (Suman et al., 2021). Nevertheless, it should be considered that, in our is no regulation on safety levels, were in the range of those previously study, the levels of correlation between enzymatic activities and plastic reported in studies assessing dietary exposures in the Mediterranean Sea ingestion levels were low and with a large proportion of variability not (Fontcuberta et al., 2011; Ramon et al., 2021; Uneyama et al., 2007), explained by the statistical models and, most importantly, restricted to where the consumption of crustaceans was not considered a high only these biomarkers. Therefore, as with condition indices, the asso­ exposure route thanks to the low ratio (<10%) of inorganic arsenic ciations observed might be coincidences resulting from the natural (toxic form) in respect to the total arsenic content (Storelli and Marco­ variability of the species (Antó et al., 2009). Indeed, individuals from the trigiano, 2001). Costa Brava, with intermediate levels of plastic ingestion, showed higher Likewise, current levels of metallic elements in the environment CbE and lower LDH levels, which indicate the good detoxification ca­ could be considered non-toxic based on reference levels from ecotoxi­ pacity of the species in this area and that is not using the anaerobic cological studies (Long et al., 1995; MacDonald et al., 2000), with the pathway for energy production. Although individuals from Costa Brava exceptions of Ni, above the effects range-low (ERL) concentration in all showed the highest tissue levels of Cd and As, the absence of other locations, and total As, surpassing the toxic effect threshold (TET) in enzymatic responses, including antioxidant enzymes, much related to Barcelona and the Ebro Delta sediments. This could suggest that sedi­ metallic exposure (Frías-Espericueta et al., 2022), does not allow for ments in the area could be responsible of adverse effects on conclusions on the impact of current levels of metallic elements either. sediment-dwelling organisms (MacDonald et al., 2000). However, cur­ No relevant histological alterations were identified in line with the rent research is being undertaken to re-evaluate the ecological thresh­ small biochemical responses observed. The occasional findings of al­ olds in European Atlantic and Mediterranean waters since natural terations, including the granuloma-like structure and the remains of a sources of As in the Mediterranean areas seem to be more relevant than haemocytic nodule, could be regarded as common host responses in in North America, where the ecotoxicological threshold studies had been crustaceans, probably involving haemocytes surrounding and encapsu­ developed (V. Besada, pers. comm.). Moreover, since tissue levels of As in lating an unidentified element that could go from fungal spores to inert individuals from the Ebro Delta were the lowest despite the high sur­ material (Battistella et al., 1996). It should also be noted that Hema­ rounding sediment levels, it might be argued that sequestration and todinium spp., parasitic dinoflagellates commonly found in northern detoxification mechanisms might be playing a key role in preventing populations of this decapod species (Stentiford and Neil, 2011), were not higher values of accumulation (Ahearn et al., 2004). identified in individuals from the Mediterranean Sea. However, no specific methods such as ELISA or PCR assays, recommended as the most 4.3. Overview of Norway lobster’s health condition and relationship with sensitive diagnostic methods, particularly in summer when subpatent anthropogenic pollutant levels infections may dominate (Beevers et al., 2012), were performed in our survey. Although site differences in biological condition indices (HSI and GSI) were observed in N. norvegicus, no correlations with any of the 5. Conclusions pollutants analysed could be confidently established. The high GSI values observed in females from Barcelona might be likely related to This study is the first to simultaneously characterise the levels of slight size differences among locations, as the onset of maturity is esti­ plastics and metallic elements in the Norway lobster, N. norvegicus, and mated to occur at 30–32 mm CL in the Catalan Coast (Orsi-Relini et al., its surrounding environment (near-bottom water and sediments) and the 1998). Similarly, the higher HSI values reported in individuals from the potential relationship with its health condition. Our results suggest that Ebro Delta may likely respond to the area’s more favourable environ­ both plastic fibre ingestion and bioaccumulation of metallic elements mental conditions. Although enlarged hepatopancreas (higher HSI) has are common phenomena in the population of N. norvegicus from the NW been related to chronic toxic responses due to the increased contribution Mediterranean Sea, with the highest levels of plastic ingestion in the of biotransformation enzymes (Crespo and Solé, 2016), no further his­ area close to the most populated Barcelona but with varied spatial topathological alterations or signs of an enhanced detoxification effort patterns for metallic elements. Contrary to what was hypothesized, the (elevated PROD, CbE or GST activities) were here observed to support correlation between the biotic and abiotic levels of pollutants is limited this hypothesis. Instead, it seems plausible that thanks to the nutrients for anthropogenic items and unclear for metallic elements. The simul­ fluxes from the Ebro river’s discharge, there is an increased biological taneous quantification and characterisation of plastics from biotic and productivity in the area that could result in a better nutritional status. abiotic compartments has provided valuable information on the likely Moreover, Bailey et al. (1986) described how the sediment’s particle size routes for plastics uptake and questions the relative importance of sed­ distribution and organic carbon content seemed to largely influence the iments and water. High levels of plastics encountered in sediments biological characteristics of this species. Muddy sediments are more support an uptake from this matrix while the presence of long fibres and 9 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 matching spatial trends of abundance in near-bottom waters suggest 2019-000928-S) and CSIC Interdisciplinary Thematic Platform (PTI+) that suspension-feeding may also be involved in plastics uptake from for Sustainable Plastics towards a Circular Economy+ (PTI-SusPlast+). water. Moreover, concurrent trends in the abundance of plastic fibres in We also thank the management of sample elemental analysis of the the environment and in the stomach contents of N. norvegicus provides Servei d’Anàlisi Química (SAQ, Chemistry Analysis Service) from UAB. further support on the use of this species as an indicator for plastic fibre The manuscript benefited from the suggestions made by three anony­ pollution in the deep-sea environment. In terms of the potential impact mous reviewers, to whom the authors are especially grateful. of anthropogenic pollutants, only higher activities of GST and lower activities of CAT have been identified in individuals with high plastic Appendix A. Supplementary data loads while higher activities of CbE and lower of LDH have been re­ ported in the location where individuals showed the highest tissue Supplementary data to this article can be found online at https://doi. content of Cd and As. However, a clear spatial variability of the health org/10.1016/j.envpol.2022.120230. parameters assessed in relation to the general trends on environmental levels of pollutants has not been established. Given the absence of any References relationship of pollutant levels with other biomarkers, condition indices or significant pathological alterations, the individuals of N. norvegicus Abelló, P., Valladares, F.J., Castellón, A., 1988. Analysis of the structure of decapod crustacean assemblages off the Catalan coast (North-West Mediterranean). Mar. Biol. analysed are considered to be in overall good health status. Nonetheless, 98, 39–49. caution should be kept in extrapolating these results given the natural Ahearn, G.A., Mandal, P.K., Mandal, A., 2004. Mechanisms of heavy-metal sequestration variability of wild organisms and the potential future changes in the and detoxification in crustaceans: a review. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 174, 439–452. https://doi.org/10.1007/s00360-004-0438-0. levels and characteristics of pollutants. Taking advantage of its potential Alomar, C., Estarellas, F., Deudero, S., 2016. Microplastics in the Mediterranean Sea: as indicator species, further studies encompassing larger spatial ranges deposition in coastal shallow sediments, spatial variation and preferential grain size. and temporal variations are fully encouraged to provide a more exten­ Mar. Environ. Res. 115, 1–10. https://doi.org/10.1016/j.marenvres.2016.01.005. Alomar, C., Sureda, A., Capó, X., Guijarro, B., Tejada, S., Deudero, S., 2017. Microplastic sive comprehension of the potential impact of anthropogenic pollutants ingestion by Mullus surmuletus Linnaeus, 1758 fish and its potential for causing on other health parameters. Regarding food safety, limits established by oxidative stress. Environ. Res. 159, 135–142. https://doi.org/10.1016/j. the EFSA on metallic elements were not exceeded, and thus, Norway envres.2017.07.043. Antó, M., Arnau, S., Buti, E., Cortijo, V., Gutiérrez, E., Solé, M., 2009. Characterisation of lobsters sampled at these locations can be considered safe for con­ integrated stress biomarkers in two deep-sea crustaceans, Aristeus antennatus and sumption. The nearly absence of plastics in the intestine also indicates a Nephrops norvegicus, from the NW fishing grounds of the Mediterranean sea. low risk of exposure for humans through the consumption of this species. Ecotoxicol. Environ. Saf. 72, 1455–1462. https://doi.org/10.1016/j. ecoenv.2009.02.007. Athey, S.N., Erdle, L.M., 2021. Are we underestimating anthropogenic microfiber CRediT author statement pollution? A critical review of occurrence, methods, and reporting. Environ. Toxicol. Chem. 1–16. https://doi.org/10.1002/etc.5173, 00. E. Carreras-Colom: Investigation, Formal analysis, Methodology, Au, D.W.T., 2004. The application of histo-cytopathological biomarkers in marine pollution monitoring: a review. Mar. Pollut. Bull. 48, 817–834. https://doi.org/ Writing – Original DraftJ. E. Cartes: Conceptualization, Writing – Re­ 10.1016/j.marpolbul.2004.02.032. view & Editing.O. Rodríguez-Romeu: Investigation, Methodology, Avio, C.G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., D’Errico, G., Pauletto, M., Writing – Review & Editing. F. Padrós: Investigation, Validation, Writing Bargelloni, L., Regoli, F., 2015. Pollutants bioavailability and toxicological risk from microplastics to marine mussels. Environ. Pollut. 198, 211–222. https://doi.org/ – Review & Editing. M. Solé Methodology, Resources, Validation, 10.1016/j.envpol.2014.12.021. Writing – Review & Editing. M. Grelaud: Methodology, Investigation, Avio, C.G., Gorbi, S., Regoli, F., 2017. Plastics and microplastics in the oceans: from Writing – Review & Editing. P. Ziveri: Validation, Resources, Writing – emerging pollutants to emerged threat. Mar. Environ. Res. 128, 2–11. https://doi. org/10.1016/j.marenvres.2016.05.012. Review & Editing. C. Palet: Methodology, Resources, Validation. Bailey, N., Howard, F.G., Chapman, C.J., 1986. Clyde Nephrops: biology and fisheries. Writing – Review & Editing. A. Soler-Membrives: Conceptualization, Proc. R. Soc. Edinburgh. Sect. B. Biol. Sci. 90, 501–518. https://doi.org/10.1017/ Investigation, Writing – Review & Editing. M. Carrassón: Conceptuali­ s0269727000005194. Barrows, A.P.W., Neumann, C.A., Berger, M.L., Shaw, S.D., 2017. Grab: vs. neuston tow zation, Supervision, Project administration and Funding acquisition, net: a microplastic sampling performance comparison and possible advances in the Writing – Review & Editing. field. Anal. Methods 9, 1446–1453. https://doi.org/10.1039/c6ay02387h. Battistella, S., Bonivento, P., Amirante, G.A., 1996. Hemocytes and immunological reactions in crustaceans. Ital. J. Zool. 63, 337–343. https://doi.org/10.1080/ Declaration of competing interest 11250009609356156. Becker, C., Cunningham, E.M., Dick, J.T.A., Eagling, L.E., Sigwart, J.D., 2018. A unified The authors declare that they have no known competing financial scale for female reproductive stages in the Norway lobster (Nephrops norvegicus): interests or personal relationships that could have appeared to influence evidence from macroscopic and microscopic characterization. J. Morphol. 279, 1700–1715. https://doi.org/10.1002/jmor.20852. the work reported in this paper. Beevers, N.D., Kilbride, E., Atkinson, R.J.A., Neil, D.M., 2012. Hematodinium infection seasonality in the Firth of Clyde (Scotland) Nephrops norvegicus population: a re- Data availability evaluation. Dis. Aquat. Org. 100, 95–104. https://doi.org/10.3354/dao02473. Bell, T.A., Lightner, D.V., 1988. A Handbook of Normal Penaeid Shrimp Histology. World Aquaculture Society, Baton Rouge, Louisiana. Data will be made available on request. Bellas, J., Martínez-Armental, J., Martínez-Cámara, A., Besada, V., Martínez-Gómez, C., 2016. Ingestion of microplastics by demersal fish from the Spanish Atlantic and Mediterranean coasts. Mar. Pollut. Bull. 109, 55–60. https://doi.org/10.1016/j. Acknowledgements marpolbul.2016.06.026. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram This study was supported by the Spanish Ministry of Science, Inno­ quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. https://doi.org/10.1006/abio.1976.9999. vation and Universities project ‘PLASMAR’ (RTI 2018-094806-B-100) Campbell, N., Allan, L., Weetman, A., Dobby, H., 2009. Investigating the link between and by the Catalan Department of Agriculture, Livestock, Fisheries and Nephrops norvegicus burrow density and sediment composition in Scottish waters. Food (European Maritime and Fisheries Fund (EMFF)) project ‘SOM­ ICES J. Mar. Sci. 66, 2052–2059. https://doi.org/10.1093/icesjms/fsp176. Canals, M., Pham, C.K., Bergmann, M., Gutow, L., Hanke, G., van Sebille, E., PESCA’ (ARP059/19/00003). We thank all fishermen from commercial Angiolillo, M., Buhl-Mortensen, L., Cau, A., Ioakeimidis, C., Kammann, U., fishing vessels involved in the ‘PLASMAR’ and ‘SOMPESCA’ projects. Lundsten, L., Papatheodorou, G., Purser, A., Sanchez-Vidal, A., Schulz, M., Vinci, M., ECC benefited from an FPU PhD student grant from the Spanish Ministry Chiba, S., Galgani, F., Langenkämper, D., Möller, T., Nattkemper, T.W., Ruiz, M., of Science, Innovation and Universities (FPU16/03430). MG and PZ Suikkanen, S., Woodall, L., Fakiris, E., Molina Jack, M.E., Giorgetti, A., 2021. The quest for seafloor macrolitter: a critical review of background knowledge, current acknowledge financial support from the i-plastic project (JPI-Ocean­ methods and future prospects. Environ. Res. Lett. 16 https://doi.org/10.1088/1748- s—Grant PCI 2020-112059). JC and MS acknowledge their institutional 9326/abc6d4. support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 10 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 Canli, M., Furness, R.W., 1993a. Heavy metals in tissues of the Norway lobster Nephrops comparison by stable isotope analysis. Mar. Ecol. Prog. Ser. 490, 199–221. https:// norvegicus: effects of sex, size and season. Chem. Ecol. 8, 19–32. https://doi.org/ doi.org/10.3354/meps10430. 10.1080/02757549308035297. Farmer, A.S., 1974. The development of the external sexual characters of Nephrops Canli, M., Furness, R.W., 1993b. Toxicity of heavy metals dissolved in sea water and norvegicus (L.) (Decapoda: nephropidae). J. Nat. Hist. 8, 241–255. https://doi.org/ influences of sex and size on metal accumulation and tissue distribution in the 10.1080/00222937400770231. Norway lobster Nephrops norvegicus. Mar. Environ. Res. 36, 217–236. https://doi. Fischer, V., Elsner, N.O., Brenke, N., Schwabe, E., Brandt, A., 2015. Plastic pollution of org/10.1016/0141-1136(93)90090-M. the kuril–kamchatka trench area (NW pacific). Deep Sea Res. Part II Top. Stud. Carreras-Colom, E., Cartes, J.E., Constenla, M., Welden, N.A., Soler-Membrives, A., Oceanogr. 111, 399–405. https://doi.org/10.1016/j.dsr2.2014.08.012. Carrassón, M., 2022. An affordable method for monitoring plastic fibre ingestion in Foekema, E.M., De Gruijter, C., Mergia, M.T., Van Franeker, J.A., Murk, A.J., Nephrops norvegicus (Linnaeus, 1758) and implementation on wide temporal and Koelmans, A.A., 2013. Plastic in North Sea Fish. Environ. Sci. Technol. 47, geographical scale comparisons. Sci. Total Environ. 810 https://doi.org/10.1016/j. 8818–8824. https://doi.org/10.1021/es400931b. scitotenv.2021.152264. Fontcuberta, M., Calderon, J., Villalbí, J.R., Centrich, F., Portaña, S., Espelt, A., Duran, J., Carreras-Colom, E., Constenla, M., Soler-Membrives, A., Cartes, J.E., Baeza, M., Nebot, M., 2011. Total and inorganic arsenic in marketed food and associated health Carrassón, M., 2020. A closer look at anthropogenic fiber ingestion in Aristeus risks for the Catalan (Spain) population. J. Agric. Food Chem. 59, 10013–10022. antennatus in the NW Mediterranean Sea: differences among years and locations and https://doi.org/10.1021/jf2013502. impact on health condition. Environ. Pollut. 263 https://doi.org/10.1016/j. Frías-Espericueta, M.G., Bautista-Covarrubias, J.C., Osuna-Martínez, C.C., Delgado- envpol.2020.114567. Alvarez, C., Bojórquez, C., Aguilar-Juárez, M., Roos-Muñoz, S., Osuna-López, I., Carreras-Colom, E., Constenla, M., Soler-Membrives, A., Cartes, J.E., Baeza, M., Páez-Osuna, F., 2022. Metals and oxidative stress in aquatic decapod crustaceans: a Padrós, F., Carrassón, M., 2018. Spatial occurrence and effects of microplastic review with special reference to shrimp and crabs. Aquat. Toxicol. https://doi.org/ ingestion on the deep-water shrimp Aristeus antennatus. Mar. Pollut. Bull. 133, 10.1016/j.aquatox.2021.106024. 44–52. https://doi.org/10.1016/j.marpolbul.2018.05.012. Frias, J.P.G.L., Nash, R., 2019. Microplastics: finding a consensus on the definition. Mar. Cartes, J.E., Company, J.B., Maynou, F., 1994. Deep-water decapod crustacean Pollut. Bull. 138, 145–147. https://doi.org/10.1016/j.marpolbul.2018.11.022. communities in the Northwestern Mediterranean: influence of submarine canyons Galafassi, S., Nizzetto, L., Volta, P., 2019. Plastic sources: a survey across scientific and and season. Mar. Biol. 120, 221–229. https://doi.org/10.1007/BF00349682. grey literature for their inventory and relative contribution to microplastics Cau, A., Avio, C.G., Dessì, C., Follesa, M.C., Moccia, D., Regoli, F., Pusceddu, A., 2019. pollution in natural environments, with an emphasis on surface water. Sci. Total Microplastics in the crustaceans Nephrops norvegicus and Aristeus antennatus: flagship Environ. 693, 133499 https://doi.org/10.1016/j.scitotenv.2019.07.305. species for deep-sea environments? Environ. Pollut. 255, 113107 https://doi.org/ Godoy, V., Blázquez, G., Calero, M., Quesada, L., Martín-Lara, M.A., 2019. The potential 10.1016/j.envpol.2019.113107. of microplastics as carriers of metals. Environ. Pollut. 255 https://doi.org/10.1016/ Cau, A., Avio, C.G., Dessì, C., Moccia, D., Pusceddu, A., Regoli, F., Cannas, R., Follesa, M. j.envpol.2019.113363. C., 2020. Benthic Crustacean digestion can modulate the environmental fate of Habte, G., Choi, J.Y., Nho, E.Y., Oh, S.Y., Khan, N., Choi, H., Park, K.S., Kim, K.S., 2015. microplastics in the deep sea. Environ. Sci. Technol. 54, 4886–4892. https://doi.org/ Determination of toxic heavy metal levels in commonly consumed species of shrimp 10.1021/acs.est.9b07705. and shellfish using ICP-MS/OES. Food Sci. Biotechnol. 24, 373–378. https://doi.org/ Choy, C.A., Robison, B.H., Gagne, T.O., Erwin, B., Firl, E., Halden, R.U., Hamilton, J.A., 10.1007/s10068-015-0049-4. Katija, K., Lisin, S.E., Rolsky, C., , S., Van Houtan, K., 2019. The vertical distribution Hara, J., Frias, J., Nash, R., 2020. Quantification of microplastic ingestion by the and biological transport of marine microplastics across the epipelagic and decapod crustacean Nephrops norvegicus from Irish waters. Mar. Pollut. Bull. 152, mesopelagic water column. Sci. Rep. 9, 7843. https://doi.org/10.1038/s41598-019- 110905 https://doi.org/10.1016/j.marpolbul.2020.110905. 44117-2. Hartmann, N.B., Hüffer, T., Thompson, R.C., Hassellöv, M., Verschoor, A., Daugaard, A. Cole, M., Webb, H., Lindeque, P.K., Fileman, E.S., Halsband, C., Galloway, T.S., 2014. E., Rist, S., Karlsson, T., Brennholt, N., Cole, M., Herrling, M.P., Hess, M.C., Ivleva, N. Isolation of microplastics in biota-rich seawater samples and marine organisms. Sci. P., Lusher, A.L., Wagner, M., 2019. Are we speaking the same language? Rep. 4, 1–8. https://doi.org/10.1038/srep04528. Recommendations for a definition and categorization framework for plastic debris. Coll, M., Piroddi, C., Albouy, C., Ben Rais Lasram, F., Cheung, W.W.L., Christensen, V., Environ. Sci. Technol. 53, 1039–1047. https://doi.org/10.1021/acs.est.8b05297. Karpouzi, V.S., Guilhaumon, F., Mouillot, D., Paleczny, M., Palomares, M.L., Hermsen, E., Pompe, R., Besseling, E., Koelmans, A.A., 2017. Detection of low numbers Steenbeek, J., Trujillo, P., Watson, R., Pauly, D., 2012. The Mediterranean Sea under of microplastics in North Sea fish using strict quality assurance criteria. Mar. Pollut. siege: spatial overlap between marine biodiversity, cumulative threats and marine Bull. 122, 253–258. https://doi.org/10.1016/j.marpolbul.2017.06.051. reserves. Global Ecol. Biogeogr. 21, 465–480. https://doi.org/10.1111/j.1466- Hernroth, B., Baden, S.P., Holm, K., André, T., Söderhäll, I., 2004. Manganese induced 8238.2011.00697.x. immune suppression of the lobster, Nephrops norvegicus. Aquat. Toxicol. 70, Costa, P.M., 2018. The Handbook of Histopathological Practices in Aquatic 223–231. https://doi.org/10.1016/j.aquatox.2004.09.004. Environments. Elsevier, London, UK. https://doi.org/10.1016/C2016-0-01962-9. Iamiceli, A.L., Ubaldi, A., Lucchetti, D., Brambilla, G., Abate, V., De Felip, E., De Courtene-Jones, W., Quinn, B., Gary, S.F., Mogg, A.O.M., Narayanaswamy, B.E., 2017. Filippis, S.P., Dellatte, E., De Luca, S., Ferri, F., Fochi, I., Fulgenzi, A.R., Iacovella, N., Microplastic pollution identified in deep-sea water and ingested by benthic Moret, I., Piazza, R., Roncarati, A., Melotti, P., Fanelli, R., Fattore, E., di invertebrates in the Rockall Trough, North Atlantic Ocean. Environ. Pollut. 231, Domenico, A., Miniero, R., 2015. Metals in Mediterranean aquatic species. Mar. 271–280. https://doi.org/10.1016/j.envpol.2017.08.026. Pollut. Bull. 94, 278–283. https://doi.org/10.1016/j.marpolbul.2015.02.034. Crespo, M., Solé, M., 2016. The use of juvenile Solea solea as sentinel in the marine IMO/FAO/UNESCO/WMO/WHO/IAEA/UN/UNEP Joint Group of Experts on the platform of the Ebre Delta: in vitro interaction of emerging contaminants with the Scientific Aspects of Marine Pollution, 2001. Protecting the Oceans from Land-Based liver detoxification system. Environ. Sci. Pollut. Res. 23, 19229–19236. https://doi. Activities: Land-Based Sources and Activities Affecting the Quality and Uses of the org/10.1007/s11356-016-7146-7. Marine. Coastal and Associated Freshwater Environment. No. 71). Cristo, M., 1998. Feeding ecology of Nephrops norvegicus (Decapoda: nephropidae). Jackson, J.B.C., Kirby, M.X., Berger, W.H., Bjorndal, K.A., Botsford, L.W., Bourque, B.J., J. Nat. Hist. 32, 1493–1498. Bradbury, R.H., Cooke, R., Erlandson, J., Estes, J.A., Hughes, T.P., Kidwell, S., Cristo, M., Cartes, J.E., 1998. A comparative study of the feeding ecology of Nephrops Lange, C.B., Lenihan, H.S., Pandolfi, J.M., Peterson, C.H., Steneck, R.S., Tegner, M.J., norvegicus (L.), (Decapoda: nephropidae) in the bathyal Mediterranean and the Warner, R.R., 2001. Historical overfishing and the recent collapse of coastal adjacent Atlantic. Sci. Mar. 62, 81–90. https://doi.org/10.3989/ ecosystems. Science 84 293, 629–637. https://doi.org/10.1126/science.1059199. scimar.1998.62s181. Jeong, C.B., Kang, H.M., Lee, M.C., Kim, D.H., Han, J., Hwang, D.S., Souissi, S., Lee, S.J., de Haan, W.P., Sanchez-Vidal, A., Canals, M., 2019. Floating microplastics and aggregate Shin, K.H., Park, H.G., Lee, J.S., 2017. Adverse effects of microplastics and oxidative formation in the western Mediterranean Sea. Mar. Pollut. Bull. 140, 523–535. stress-induced MAPK/Nrf2 pathway-mediated defense mechanisms in the marine https://doi.org/10.1016/j.marpolbul.2019.01.053. copepod Paracyclopina nana. Sci. Rep. 7, 1–11. https://doi.org/10.1038/srep41323. De Wael, K., Baes, C., Lepot, L., Gason, F., 2011. On the frequency of occurrence of a Jeong, C.B., Won, E.J., Kang, H.M., Lee, M.C., Hwang, D.S., Hwang, U.K., Zhou, B., peculiar polyester fibre type found in blue denim textiles. Sci. Justice 51, 154–162. Souissi, S., Lee, S.J., Lee, J.S., 2016. Microplastic size-dependent toxicity, oxidative https://doi.org/10.1016/j.scijus.2011.02.001. stress induction, and p-JNK and p-p38 activation in the monogonont rotifer Del Rio, J.L., Acosta-Yepes, J., Cristobo, J., Martinez, J., Parra-Descalzo, S., Tel, E., (Brachionus koreanus). Environ. Sci. Technol. 50, 8849–8857. https://doi.org/ Viñas, L., Muñoz, A., Vilela, R., Jiménez, E.E., Patrocinio, T., Ríos, P., Almón- 10.1021/acs.est.6b01441. Pazos, B., Blanco, R., Murillo, J., Polonio-Povedano, V., Fernández, J., Cabanas, J.M., Johnson, L.L., Anulacion, B.F., Arkoosh, M.R., Burrows, D.G., da Silva, D.A.M., Gago, J., Gonzalez-Nuevo, G., Cabrero-Rodríguez, Á.H., Besada, V., Schultze, F., Dietrich, J.P., Myers, M.S., Spromberg, J., Ylitalo, G.M., 2013. Effects of legacy Franco-Hernández, M.Á., Bargiela, J., García-Blanco, X., 2012. Estudio de los persistent organic pollutants (POPs) in fish-current and future challenges. In: Fish Ecosistemas Marinos Vulnerables en aguas internacionales del Atlántico Physiology, first ed. ed. Elsevier Inc. https://doi.org/10.1016/B978-0-12-398254- Sudoccidental. Boletin Oceanografico. 4.00002-9. EC, 2006. Commission Regulation (EC) No 1881/2006 of 19 December 2006 Setting Johnson, M.L., Johnson, M., 2013. The Ecology and Biology of Nephrops norvegicus. Maximum Levels for Certain Contaminants in Foodstuffs (Text with EEA Relevance) Advances in Marine Biology, London, UK. 5–24. Jordana, E., Pinedo, S., Ballesteros, E., 2015. Macrobenthic assemblages, sediment EFSA, P. on F.A. and F., 2021. Safety assessment of titanium dioxide (E171) as a food characteristics and heavy metal concentrations in soft-bottom Ebre Delta bays (NW additive. EFSA J. 19, e06585 https://doi.org/10.2903/j.efsa.2021.658. Mediterranean). Environ. Monit. Assess. 187 https://doi.org/10.1007/s10661-015- Eriksson, S.P., 2000. Temporal variations of manganese in the haemolymph and tissues 4315-y. of the Norway lobster, Nephrops norvegicus (L.). Aquat. Toxicol. 48, 297–307. Joyce, H., Frias, J., Kavanagh, F., Lynch, R., Pagter, E., White, J., Nash, R., 2022. Plastics, https://doi.org/10.1016/S0166-445X(99)00035-1. prawns, and patterns: microplastic loadings in Nephrops norvegicus and surrounding Fanelli, E., Papiol, V., Cartes, J.E., Rumolo, P., López-Pérez, C., 2013. Trophic webs of habitat in the North East Atlantic. Sci. Total Environ. 826, 154036 https://doi.org/ deep-sea megafauna on mainland and insular slopes of the NW Mediterranean: a 10.1016/j.scitotenv.2022.154036. 11 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 Kanhai, L.D.K., Officer, R., Lyashevska, O., Thompson, R.C., O’Connor, I., 2017. Österblom, H., Jouffray, J.B., Folke, C., Rockström, J., 2017. Emergence of a global Microplastic abundance, distribution and composition along a latitudinal gradient in science–business initiative for ocean stewardship. Proc. Natl. Acad. Sci. U.S.A. 114, the Atlantic Ocean. Mar. Pollut. Bull. 115, 307–314. https://doi.org/10.1016/j. 9038–9043. https://doi.org/10.1073/pnas.1704453114. marpolbul.2016.12.025. Palanques, A., López, L., Guillén, J., Puig, P., 2020. Trace metal variability controlled by Koenig, S., Fernández, P., Company, J.B., Huertas, D., Solé, M., 2013. Are deep-sea hydrodynamic processes in a polluted inner shelf environment (Besòs prodelta, NW organisms dwelling within a submarine canyon more at risk from anthropogenic Mediterranean). Sci. Total Environ. 735, 139482 https://doi.org/10.1016/j. contamination than those from the adjacent open slope? A case study of Blanes scitotenv.2020.139482. canyon (NW Mediterranean). Prog. Oceanogr. 118, 249–259. https://doi.org/ Paul-Pont, I., Lacroix, C., González Fernández, C., Hégaret, H., Lambert, C., Le Goïc, N., 10.1016/j.pocean.2013.07.016. Frère, L., Cassone, A.L., Sussarellu, R., Fabioux, C., Guyomarch, J., Albentosa, M., Leblanc, J.C., Guérin, T., Noël, L., Calamassi-Tran, G., Volatier, J.L., Verger, P., 2005. Huvet, A., Soudant, P., 2016. Exposure of marine mussels Mytilus spp. to polystyrene Dietary exposure estimates of 18 elements from the 1st French total diet study. Food microplastics: toxicity and influence on fluoranthene bioaccumulation. Environ. Addit. Contam. 22, 624–641. https://doi.org/10.1080/02652030500135367. Pollut. 216, 724–737. https://doi.org/10.1016/j.envpol.2016.06.039. Lee, Y., Yoon, D.S., Lee, Y.H., Kwak, J. Il, An, Y.J., Lee, J.S., Park, J.C., 2021. Combined Pedà, C., Battaglia, P., D’Alessandro, M., Laface, F., Malara, D., Consoli, P., Vicchio, T.M., exposure to microplastics and zinc produces sex-specific responses in the water flea Longo, F., Andaloro, F., Baini, M., Galli, M., Bottari, T., Fossi, M.C., Greco, S., Daphnia magna. J. Hazard Mater. 420, 126652 https://doi.org/10.1016/j. Romeo, T., 2020. Coupling gastro-intestinal tract analysis with an airborne jhazmat.2021.126652. contamination control method to estimate litter ingestion in demersal Lefebvre, C., Saraux, C., Heitz, O., Nowaczyk, A., Bonnet, D., 2019. Microplastics FTIR elasmobranchs. Front. Environ. Sci. 8, 1–13. https://doi.org/10.3389/ characterisation and distribution in the water column and digestive tracts of small fenvs.2020.00119. pelagic fish in the Gulf of Lions. Mar. Pollut. Bull. 142, 510–519. https://doi.org/ Pinedo, S., Jordana, E., Flagella, M.M., Ballesteros, E., 2014. Relationships between 10.1016/j.marpolbul.2019.03.025. heavy metals contamination in shallow marine sediments with industrial and urban Lei, L., Wu, S., Lu, S., Liu, M., Song, Y., Fu, Z., Shi, H., Raley-Susman, K.M., He, D., 2018. development in Catalonia (Northwestern Mediterranean Sea). Water. Air. Soil Pollut. Microplastic particles cause intestinal damage and other adverse effects in zebrafish 225 https://doi.org/10.1007/s11270-014-2084-9. Danio rerio and nematode Caenorhabditis elegans. Sci. Total Environ. 619–620. Rainbow, P.S., 2002. Trace metal concentrations in aquatic invertebrates: why and so https://doi.org/10.1016/j.scitotenv.2017.11.103, 1–8. what? Environ. Pollut. 120, 497–507. https://doi.org/10.1016/S0269-7491(02) Liu, F., Vianello, A., Vollertsen, J., 2019. Retention of microplastics in sediments of urban 00238-5. and highway stormwater retention ponds. Environ. Pollut. 255, 113335 https://doi. Rainbow, P.S., Luoma, S.N., 2011. Metal toxicity, uptake and bioaccumulation in aquatic org/10.1016/j.envpol.2019.113335. invertebrates-Modelling zinc in crustaceans. Aquat. Toxicol. 105, 455–465. https:// Long, E.R., Macdonald, D.D., Smith, S.L., Calder, F.D., 1995. Incidence of adverse doi.org/10.1016/j.aquatox.2011.08.001. biological effects within ranges of chemical concentrations in marine and estuarine Ramon, D., Morick, D., Croot, P., Berzak, R., Scheinin, A., Tchernov, D., Davidovich, N., sediments. Environ. Manag. 19, 81–97. https://doi.org/10.1007/BF02472006. Britzi, M., 2021. A survey of arsenic, mercury, cadmium, and lead residues in Lourenço, H.M., Anacleto, P., Afonso, C., Martins, M.F., Carvalho, M.L., Lino, A.R., seafood (fish, crustaceans, and cephalopods) from the south-eastern Mediterranean Nunes, M.L., 2009. Chemical characterisation of Nephrops norvegicus from Sea. J. Food Sci. 86, 1153–1161. https://doi.org/10.1111/1750-3841.15627. Portuguese coast. J. Sci. Food Agric. 89, 2572–2580. https://doi.org/10.1002/ Ribeiro, F., Garcia, A.R., Pereira, B.P., Fonseca, M., Mestre, N.C., Fonseca, T.G., jsfa.3754. Ilharco, L.M., Bebianno, M.J., 2017. Microplastics effects in Scrobicularia plana. Mar. MacDonald, D.D., Ingersoll, C.G., Berger, T.A., 2000. Development and evaluation of Pollut. Bull. 122, 379–391. https://doi.org/10.1016/j.marpolbul.2017.06.078. consensus-based sediment quality guidelines for freshwater ecosystems. Arch. Rodrigues, P.D.A., Ferrari, R.G., Conte-junior, C.A., Hauser-davis, R.A., Conte-junior, C. Environ. Contam. Toxicol. 39, 20–31. https://doi.org/10.1007/s002440010075. A., 2022. A Systematic Review on Metal Dynamics and Marine Toxicity Risk Mancuso, M., Savoca, S., Bottari, T., 2019. First record of microplastics ingestion by Assessment Using Crustaceans as Bioindicators 881–903. European hake Merluccius merluccius from the Tyrrhenian Sicilian coast (Central Rodríguez-Romeu, O., Constenla, M., Carrassón, M., Campoy-Quiles, M., Soler- Mediterranean Sea). J. Fish. Biol. 94, 517–519. https://doi.org/10.1111/jfb.13920. Membrives, A., 2020. Are anthropogenic fibres a real problem for red mullets (Mullus Martinelli, M., Gomiero, A., Guicciardi, S., Frapiccini, E., Strafella, P., Angelini, S., barbatus) from the NW Mediterranean? Sci. Total Environ. 733, 139336 https://doi. Domenichetti, F., Belardinelli, A., Colella, S., 2021. Preliminary results on the org/10.1016/j.scitotenv.2020.139336. occurrence and anatomical distribution of microplastics in wild populations of Rodríguez, E.M., Medesani, D.A., Fingerman, M., 2007. Endocrine disruption in Nephrops norvegicus from the Adriatic Sea. Environ. Pollut. 278, 116872 https://doi. crustaceans due to pollutants: a review. Comp. Biochem. Physiol. Mol. Integr. org/10.1016/j.envpol.2021.116872. Physiol. 146, 661–671. https://doi.org/10.1016/j.cbpa.2006.04.030. Martins, A., da Silva, D.D., Silva, R., Carvalho, F., Guilhermino, L., 2022. Long-term Ruiz-Orejón, L.F., Sardá, R., Ramis-Pujol, J., 2018. Now, you see me: high concentrations effects of lithium and lithium-microplastic mixtures on the model species Daphnia of floating plastic debris in the coastal waters of the Balearic Islands (Spain). Mar. magna: toxicological interactions and implications to ‘One Health. Sci. Total Environ. Pollut. Bull. 133, 636–646. https://doi.org/10.1016/j.marpolbul.2018.06.010. 838 https://doi.org/10.1016/j.scitotenv.2022.155934. Salvador Cesa, F., Turra, A., Baruque-Ramos, J., 2017. Synthetic fibers as microplastics in Masura, J., Baker, J., Foster, G., Arthur, C., 2015. Laboratory methods for the analysis of the marine environment: a review from textile perspective with a focus on domestic microplastics in the marine environment. NOAA Mar. Debris Progr. Natl. 1–39. washings. Sci. Total Environ. 598, 1116–1129. https://doi.org/10.1016/j. Mateos-Cárdenas, A., O’Halloran, J., van Pelt, F.N.A.M., Jansen, M.A.K., 2020. Rapid scitotenv.2017.04.172. fragmentation of microplastics by the freshwater amphipod Gammarus duebeni Sanchez-Vidal, A., Thompson, R.C., Canals, M., De Haan, W.P., 2018. The imprint of (Lillj.). Sci. Rep. 10, 1–12. https://doi.org/10.1038/s41598-020-69635-2. microfibres in Southern European deep seas. PLoS One 13, 1–12. https://doi.org/ McGoran, A.R., Cowie, P.R., Clark, P.F., McEvoy, J.P., Morritt, D., 2018. Ingestion of 10.1371/journal.pone.0207033. plastic by fish: a comparison of thames estuary and firth of Clyde populations. Mar. Santana, C.A. da S., Wieczorek, A.M., Browne, P., Graham, C.T., Power, A.M., 2020. Pollut. Bull. 137, 12–23. https://doi.org/10.1016/j.marpolbul.2018.09.054. Importance of suspended particulate organic matter in the diet of Nephrops norvegicus Micheli, F., Halpern, B.S., Walbridge, S., Ciriaco, S., Ferretti, F., Fraschetti, S., (Linnaeus, 1758). Sci. Rep. 10, 3387. https://doi.org/10.1038/s41598-020-60367-x. Lewison, R., Nykjaer, L., Rosenberg, A.A., 2013. Cumulative human impacts on Satoh, T., Hosokawa, M., 2006. Structure, function and regulation of carboxylesterases. Mediterranean and Black Sea marine ecosystems: assessing current pressures and Chem. Biol. Interact. 162, 195–211. https://doi.org/10.1016/j.cbi.2006.07.001. opportunities. PLoS One 8. https://doi.org/10.1371/journal.pone.0079889. Sbrana, M., Zupa, W., Ligas, A., Capezzuto, F., Chatzispyrou, A., Follesa, M.C., Mille, T., Cresson, P., Chouvelon, T., Bustamante, P., Brach-Papa, C., Bruzac, S., Gancitano, V., Guijarro, B., Isajlovic, I., Jadaud, A., Markovic, O., Micallef, R., Rozuel, E., Bouchoucha, M., 2018. Trace metal concentrations in the muscle of seven Peristeraki, P., Piccinetti, C., Thasitis, I., Carbonara, P., 2020. Spatiotemporal marine species: comparison between the Gulf of lions (North-West Mediterranean abundance pattern of deep-water rose shrimp, Parapenaeus longirostris, and Norway Sea) and the Bay of biscay (North-East Atlantic Ocean). Mar. Pollut. Bull. 135, 9–16. lobster, Nephrops norvegicus, in European Mediterranean waters. Sci. Mar. 83, 71. https://doi.org/10.1016/j.marpolbul.2018.05.051. https://doi.org/10.3989/scimar.04858.27A. Milligan, R.J., Albalat, A., Atkinson, R.J.A., Neil, D.M., 2009. The effects of trawling on Schuhmacher, M., Domingo, J.L., Bosque, M.A., Corbella, J., 1992. Heavy metals in the physical condition of the Norway lobster Nephrops norvegicus in relation to marine species from the tarragona coast, Spain. J. Environ. Sci. Heal. Part A Environ. seasonal cycles in the Clyde Sea area. ICES J. Mar. Sci. 66, 488–494. https://doi.org/ Sci. Eng. Toxicol. 27, 1939–1948. https://doi.org/10.1080/10934529209375833. 10.1093/icesjms/fsp018. Shields, J.D., Boyd, R.A., 2014. Atlas of Lobster Anatomy and Histology. Virginia Mordecai, G., Tyler, P.A., Masson, D.G., Huvenne, V.A.I., 2011. Litter in submarine Institute of Marince Science. canyons off the west coast of Portugal. Deep. Res. Part II Top. Stud. Oceanogr. 58, Simon-Sánchez, L., Grelaud, M., Franci, M., Ziveri, P., 2022. Are research methods 2489–2496. https://doi.org/10.1016/j.dsr2.2011.08.009. shaping our understanding of microplastic pollution? A literature review on the Murray, F., Cowie, P.R., 2011. Plastic contamination in the decapod crustacean Nephrops seawater and sediment bodies of the Mediterranean Sea. Environ. Pollut. 292, norvegicus (Linnaeus, 1758). Mar. Pollut. Bull. 62, 1207–1217. https://doi.org/ 118275 https://doi.org/10.1016/j.envpol.2021.118275. 10.1016/j.marpolbul.2011.03.032. Simon, M., van Alst, N., Vollertsen, J., 2018. Quantification of microplastic mass and Nadal, M.A., Alomar, C., Deudero, S., 2016. High levels of microplastic ingestion by the removal rates at wastewater treatment plants applying Focal Plane Array (FPA)- semipelagic fish bogue Boops boops (L.) around the Balearic Islands. Environ. Pollut. based Fourier Transform Infrared (FT-IR) imaging. Water Res. 142, 1–9. https://doi. 214, 517–523. https://doi.org/10.1016/j.envpol.2016.04.054. org/10.1016/j.watres.2018.05.019. Napper, I.E., Thompson, R.C., 2016. Release of synthetic microplastic plastic fibres from Solé, M., Sanchez-Hernandez, J.C., 2018. Elucidating the importance of mussel domestic washing machines: effects of fabric type and washing conditions. Mar. carboxylesterase activity as exposure biomarker of environmental contaminants of Pollut. Bull. 112, 39–45. https://doi.org/10.1016/j.marpolbul.2016.09.025. current concern: an in vitro study. Ecol. Indicat. 85, 432–439. https://doi.org/ Orsi-Relini, L., Zamboni, A., Fiorentino, F., Massi, D., 1998. Reproductive patterns in 10.1016/j.ecolind.2017.10.046. Norway lobster Nephrops norvegicus (L.), (Crustacea Decapoda Nephropidae) of Sreeram, M., Menon, N., 2005. Histopathological changes in the hepatopancreas of the different Mediterranean areas. Sci. Mar. 62, 25–41. penaeid shrimp Metapenaeus dobsoni exposed to petroleum hydrocarbons. J. Mar. Biol. Assoc. India 47, 160–168. 12 E. Carreras-Colom et al. Environmental Pollution 314 (2022) 120230 Stentiford, G.D., Neil, D.M., 2011. Diseases of Nephrops and Metanephrops: a review. Valente, T., Sbrana, A., Scacco, U., Jacomini, C., Bianchi, J., Palazzo, L., de Lucia, G.A., J. Invertebr. Pathol. 106, 92–109. https://doi.org/10.1016/j.jip.2010.09.017. Silvestri, C., Matiddi, M., 2019. Exploring microplastic ingestion by three deep-water Storelli, M.M., Marcotrigiano, G.O., 2001. Total, organic, and inorganic arsenic in some elasmobranch species: a case study from the Tyrrhenian Sea. Environ. Pollut. 253, commercial species of crustaceans from the Mediterranean sea (Italy). J. Food 342–350. https://doi.org/10.1016/j.envpol.2019.07.001. Protect. 64, 1858–1862. https://doi.org/10.4315/0362-028X-64.11.1858. Vogt, G., Quinitio, E.T., Pascual, F.P., 1986. Leucaena leucocephala leaves in formulated Storelli, M.M., Normanno, G., Barone, G., Dambrosio, A., Errico, L., Garofalo, R., feed for Penaeus monodon: a concrete example of the application of histology in Giacominelli-Stuffler, R., 2012. Toxic metals (Hg, Cd, and Pb) in fishery products nutrition research. Aquaculture 59, 209–234. https://doi.org/10.1016/0044-8486 imported into Italy: suitability for human consumption. J. Food Protect. 75, (86)90005-0. 189–194. https://doi.org/10.4315/0362-028X.JFP-11-212. Watts, A.J.R., Urbina, M.A., Corr, S., Lewis, C., Galloway, T.S., 2015. Ingestion of plastic Stothra Bhashyam, S., Nash, R., Deegan, M., Pagter, E., Frias, J., 2021. Microplastics in microfibers by the crab Carcinus maenas and its effect on food consumption and the Marine Environment: Sources, Impacts and Recommendations. energy balance. Environ. Sci. Technol. 49, 14597–14604. https://doi.org/10.1021/ Suaria, G., Avio, C.G., Mineo, A., Lattin, G.L., Magaldi, M.G., Belmonte, G., Moore, C.J., acs.est.5b04026. Regoli, F., Aliani, S., 2016. The mediterranean plastic soup: synthetic polymers in Welden, N.A.C., Cowie, P.R., 2016a. Environment and gut morphology influence mediterranean surface waters. Sci. Rep. 6, 1–10. https://doi.org/10.1038/ microplastic retention in langoustine, Nephrops norvegicus. Environ. Pollut. 214, srep37551. 859–865. https://doi.org/10.1016/j.envpol.2016.03.067. Suman, K.H., Haque, M.N., Uddin, M.J., Begum, M.S., Sikder, M.H., 2021. Toxicity and Welden, N.A.C., Cowie, P.R., 2016b. Long-term microplastic retention causes reduced biomarkers of micro-plastic in aquatic environment: a review. Biomarkers 26, 13–25. body condition in the langoustine, Nephrops norvegicus. Environ. Pollut. 218, https://doi.org/10.1080/1354750X.2020.1863470. 895–900. https://doi.org/10.1016/j.envpol.2016.08.020. Thompson, R.C., Olson, Y., Mitchell, R.P., Davis, A., Rowland, S.J., John, A.W.G., Wilder, S.M., Raubenheimer, D., Simpson, S.J., 2016. Moving beyond body condition McGonigle, D., Russell, A.E., 2004. Lost at sea: where is all the plastic? Science 80–, indices as an estimate of fitness in ecological and evolutionary studies. Funct. Ecol. 304–838. https://doi.org/10.1126/science.1094559. 30, 108–115. https://doi.org/10.1111/1365-2435.12460. Torre, M., Digka, N., Anastasopoulou, A., Tsangaris, C., Mytilineou, C., 2016. Winston, G.W., Di Giulio, R.T., 1991. Prooxidant and antioxidant mechanisms in aquatic Anthropogenic microfibres pollution in marine biota. A new and simple organisms. Aquat. Toxicol. 19, 137–161. https://doi.org/10.1016/0166-445X(91) methodology to minimize airborne contamination. Mar. Pollut. Bull. 113, 55–61. 90033-6. https://doi.org/10.1016/j.marpolbul.2016.07.050. Yuan, W., Zhou, Y., Chen, Y., Liu, X., Wang, J., 2020. Toxicological effects of Tsangaris, C., Digka, N., Valente, T., Aguilar, A., Borrell, A., de Lucia, G.A., microplastics and heavy metals on the Daphnia magna. Sci. Total Environ. 746, Gambaiani, D., Garcia-Garin, O., Kaberi, H., Martin, J., Mauriño, E., Miaud, C., 141254 https://doi.org/10.1016/j.scitotenv.2020.141254. Palazzo, L., del Olmo, A.P., Raga, J.A., Sbrana, A., Silvestri, C., Skylaki, E., Vighi, M., Zhu, Q., Zhou, Z., Tu, D., Zhou, Y., Wang, C., Liu, Z., Gu, W., Chen, Y., Shu, M., 2018. Wongdontree, P., Matiddi, M., 2020. Using Boops boops (osteichthyes) to assess Effect of Cadmium Exposure on Hepatopancreas and Gills of the Estuary Mud Crab microplastic ingestion in the Mediterranean Sea. Mar. Pollut. Bull. 158, 111397 (Scylla Paramamosain): Histopathological Changes and Expression Characterization https://doi.org/10.1016/j.marpolbul.2020.111397. of Stress Response Genes 195, pp. 1–7. https://doi.org/10.1016/j. Uneyama, C., Toda, M., Yamamoto, M., Morikawa, K., 2007. Arsenic in various foods: aquatox.2017.11.020. cumulative data. Food Addit. Contam. 24, 447–534. https://doi.org/10.1080/ 02652030601053121. 13