Marine Environmental Research 186 (2023) 105921 Contents lists available at ScienceDirect Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev Revealing the capability of the European hake to cope with micro-litter environmental exposure and its inferred potential health impact in the NW Mediterranean Sea Laura Muns-Pujadas a, Sara Dallarés a, *, Maria Constenla a, Francesc Padrós a, Ester Carreras-Colom a, Michaël Grelaud b, Maite Carrassón a, Anna Soler-Membrives a a Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193, Barcelona, Spain b Institut de Ciència i Tecnologia Ambientals (ICTA-UAB), Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Prevalence, abundance, concentration, size and composition of anthropogenic items (AIs) (synthetic and non- Merluccius merluccius synthetic) ingested by Merluccius merluccius juvenile specimens and from near-bottom water samples from Microplastics different localities off the Catalan coast (NW Mediterranean), were characterized. The potential effect of AIs on Anthropogenic items fish condition was assessed through different health indicators. Virtually all AIs found in fish and near-bottom Fibre ingestion water samples were fibres. A mean of 0.85 fibres/m3 from the surrounding water was observed. Fish ingested Cellulose Fish health a mean of 1.39 (SD = 1.39) items/individual. Cellulosic fibres were predominant (77.8% of samples), except for Quality assurance Barcelona. No differences in ingested AIs abundance and composition off Barcelona between 2007 and 2019 Bioindicator were found. Small AIs from the environment matched ingested AIs composition. Hakes did not ingest large fibres despite being present in the environment, probably due to their feeding behaviour. No adverse health effects or parasites aggregations were detected to be potentially related to AIs ingestion. 1. Introduction concentrated in ocean gyres (van Sebille et al., 2020). Originated by manufacturing or fragmentation of bigger plastic For the last few decades, one of the topics that have generated materials, plastic particles of less than 5 mm in diameter (i.e. micro­ considerable interest has been plastic litter and its potential impact on plastics (MPs), Arthur et al., 2009) are ubiquitous in the environment marine ecosystems. Presence of plastic pollution in surface waters was (Koelmans et al., 2022). Moreover, the presence of fibres with anthro­ first reported in the 1970s (Carpenter and Smith, 1972). Since then, its pogenic origin in the marine environment has also recently grown evi­ occurrence has been detected throughout the marine ecosystem, dence (Suaria et al., 2020). In fact, it has been reported that as much as resulting in research on the subject (Cole et al., 2011; Eriksen et al., 80–90% of MPs in the marine environment correspond to fibres (Sal­ 2014; Thompson, 2015; Gola et al., 2021). Many of these scientific vador Cesa et al., 2017). Some studies have tended to include the studies deal with marine litter’s impact on the environment and human assessment of synthetic fibres (i.e. those composed of acrylic, polyester, activities all over the world (UNEP/MAP, 2015). This great interest polyamide, polypropylene, etc.) as a particular type of MPs (Salvador stems from concerns about the global massive production of plastics. Cesa et al., 2017; Sanchez-Vidal et al., 2018). However, the great ma­ Even with ongoing efforts to reduce plastic use, almost 370 million jority of fibres found in the oceans are non-synthetic fibres, composed of tonnes of global plastics were still produced in 2019 (PlasticsEurope, processed polymers from natural materials, for instance, regenerated 2021), and it is estimated that almost 60% of all plastics produced could cellulose and dyed cotton (Suaria et al., 2020). Indeed, several studies reach the natural environment (Geyer et al., 2017). Due to their long have evidenced the apparent dominance of both synthetic and environmental residence time and density, they can be vertically non-synthetic textile fibres within the digestive tract of organisms transferred down to the seafloor (Napper and Thompson, 2020) or (Taylor et al., 2016; Compa et al., 2018; Rodríguez-Romeu et al., 2020; horizontally transported by wind-driven circulation until being Carreras-Colom et al., 2020). This hints that studies associated to * Corresponding author. E-mail address: sara.dallares@uab.cat (S. Dallarés). https://doi.org/10.1016/j.marenvres.2023.105921 Received 11 November 2022; Received in revised form 10 January 2023; Accepted 15 February 2023 Available online 18 February 2023 0141-1136/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 anthropogenic items (AIs) ingestion processes should include MPs and Fish parasite communities are widely used as indicators of fish health also pay special attention to the proportions of both synthetic and pro­ and environmental impact (Vidal-Martínez et al., 2010). Yet, few studies cessed cellulosic fibres (Ryan et al., 2019). have examined parasites as bioindicators associated with the effect of In relation to marine litter accumulation, the Mediterranean Sea, AIs in fish (Rodríguez-Romeu et al., 2020, 2022; Pennino et al., 2020). which is a semi-enclosed basin, has become a hotspot for AIs (UNEP/­ The potential parasites-MPs relationships have been studied from MAP, 2015) due to its particular hydrodynamics (Cózar et al., 2015), its different perspectives and particularly Hernandez-Milian et al. (2019) densely populated coastal areas, high industrial and touristic activities, suggested that parasites aggregations in the intestine could favour the intense maritime traffic and the input of important rivers that are also retention and accumulation of MPs, but other mechanisms could not be strongly affected by human activities (e.g. Nile River, Po River). As a disregarded. At a much more detailed level, the use of histopathological result of all these factors, abundance and distribution of micro-litter techniques can help accurately detect alterations and pathologies in show high spatial heterogeneity along the Mediterranean (Simon-Sán­ target organs. These alterations are robust health indicators and, in some chez et al., 2022). In the north-western Mediterranean Sea, AIs presence cases, alterations can be associated to exposure to environmental con­ has been reported in different environments including coastlines (Llorca taminants (Au, 2004). et al., 2020), surface waters (de Haan et al., 2019; Ryan et al., 2019), The present study aims to assess and characterize the presence of AIs shallow seafloor sediments (Simon-Sánchez et al., 2022), deep-sea areas ingested by M. merluccius in relation to the AIs present in the sur­ (Sanchez-Vidal et al., 2018) and also within the digestive tract of mul­ rounding environment in the Balearic Sea, and to assess their potential tiple organisms (Fossi et al., 2018), both benthic (Bellas et al., 2016; impact on the health condition of these animals. To achieve this goal, Carreras-Colom et al., 2018, 2020, 2022a, 2022b; Rodríguez-Romeu prevalence, abundance, size and composition of AIs within the gastro­ et al., 2020) and pelagic (Compa et al., 2018; Rodríguez-Romeu et al., intestinal tract of a representative sample of hake specimens captured in 2022). 2019 were determined from three different sampling areas that corre­ In this context, a common framework in European seas (Marine spond to locations along the Catalan coast with different anthropogenic Strategy Framework Directive (MSFD, 2008/56/EC)) and in the Medi­ pressure levels. Environmental AIs (near-bottom water) were charac­ terranean Sea (IMAP, 2016) was developed, selecting specific marine terized in terms of abundance, concentration, size and composition and species for monitoring spatial and temporal trends regarding marine compared with those ingested in order to assess uptake by hakes. In litter pollution (Bellas et al., 2016; Fossi et al., 2018) for environmental order to assess the potential impact of AIs on M. merluccius health status, risk assessment (Hermsen et al., 2018). This monitoring includes the condition indices, histological alterations and parasite indicators were presence of all kind and size of marine litter in geographical areas but evaluated. Finally, potential changes in ingested AIs within a 12 years- also in ecosystems and organisms. In this sense, the European hake gap were assessed, by comparing samples from the most polluted area (Merluccius merluccius Linnaeus, 1758) has been recommended as a collected in 2007 and in 2019. possible small-scale bioindicator species in FAO-GSAs due to its com­ mercial and ecological importance (Fossi et al., 2018; Giani et al., 2019) 2. Materials and methods and its wide distribution throughout the Mediterranean Sea (Oliver and Massutí, 1995). Moreover, owing to its benthopelagic feeding behaviour 2.1. Study area and data collection (Cartes et al., 2004) and since many of its preys move vertically with nictemeral rhythms, feeding takes place at different levels in the water A total of 82 M. merluccius specimens were captured on board of column (Bozzano et al., 1997), so the hake may be exposed to AIs from commercial fishing trawlers at depths between 62 and 112 m on the the near-bottom environment as well as from the water column. continental shelf of the Catalan coast (NW Mediterranean) in the Researchers have reported a high heterogeneity in the prevalence of framework of the PLASMAR project (Spanish Ministry of Science, AIs ingestion by the European hake in the Mediterranean Sea (Giani Innovation and Universities) and BIOMARE project (Spanish Ministry of et al., 2019; Mancuso et al., 2019; Bianchi et al., 2020; Avio et al., 2020). Science and Innovation). In summer 2007, 20 specimens were collected This high variability could be attributed to the presence of different off Barcelona while, in summer 2019, 62 hakes were collected from concentrations of AIs in the different sampling areas and also to the bias three different sites (Blanes, Barcelona and Ebro Delta coast) (Fig. 1; induced by the lack of standardized methods and protocols for the Table 1). Barcelona coast was selected as the most impacted area due to assessment of the ingested AIs, thus hindering comparisons among its important industrial activity, its densely populated coastline and its studies (Lusher et al., 2017; Hermsen et al., 2018; Cowger et al., 2020). intense maritime transport (Tubau et al., 2015). Blanes coast was Surprisingly, none of these studies have related fish ingestion of AIs to selected as considered a less impacted area owing to its lower industrial the density and profile of AIs in the surrounding environment and, activity and sparse population compared to Barcelona. Ebro Delta coast furthermore, none of them has assessed the potential effect of AIs was selected due to its particular characteristics, clearly different from ingestion on hake’s health condition. Barcelona and Blanes coast. Ebro Delta is influenced by the Ebro River Although some studies carried out under laboratory conditions have (910 km length, 14,000 hm3/y), which receives the inputs of both shown that high levels of MPs ingestion can cause adverse effects due to agricultural and industrial activities (Galimany et al., 2019). the release of their associated toxic additives (Salvador Cesa et al., Immediately upon capture, all individuals were fixed in toto in 10% 2017), and that can interfere with feeding by causing gut blockages or buffered formalin, until further micro-debris analysis was developed. lesions in the digestive system (Lusher et al., 2017; Kögel et al., 2020), Environmental samples of near-bottom water were collected during there is no clear evidence of negative health effects derived from plastic fishing trawls in 2019 in order to ensure that they were taken in exactly ingestion in wild fish populations from areas with specific and real the same surrounding environment as fish. The filtered material from values of AIs in the environment (Rodríguez-Romeu et al., 2020, 2022). water samples was collected using a WP2-net (200 μm mesh size), Assessing an individual’s health condition can be difficult due to the equipped with a mechanical flowmeter that was towed horizontally at a great variety of factors and elements of stress that may influence the velocity of 2–3 knots and at 5–10 m above the seafloor along each trawl physiological responses of the fish. For this reason, it is necessary to (ca. 30 min). Filtered water samples were immediately fixed in 10% integrate different indicators into a multidisciplinary approach. In this buffered formalin until further processing (see below). ASTD152 CTD context, condition indices can provide information on the overall health casts were performed at the same locations and 5 m above the seafloor status which may be related to physiological or disease conditions. As prior to fishing trawls to record environmental variables (temperature, general health indicators, these indices respond to an array of intrinsic salinity, oxygen concentration and turbidity). and/or extrinsic factors including food availability, environmental variables, parasitism and anthropogenic factors (Lloret et al., 2014). 2 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 Fig. 1. Map of the study area. Red circles show the location of the three 2019 sampling points off Blanes at 111.5 m depth, Barcelona at 87 m depth and Ebro Delta at 65 m depth. Red triangle shows the location of the 2007 sampling point off Barcelona at 62.5 m depth. Table 1 Biometric data, indices of fish health condition for each sampling point and the total of Merluccius merluccius examined (n) are shown. Mean and (standard deviation) were calculated for standard length (SL), total weight (TW), Le Cren relative condition index (Kn), hepatosomatic index (HSI) and stomach fullness (FULL). Significant differences among localities are indicated by different superscript letters. Location Year Depth (m) n SL (cm) TW (g) Kn HSI FULL Blanes 2019 111.5 22 18.60 (0.02) 70.20 (25.0) 1.01 (0.12)a 2.68 (0.88)a 1.91 (3.74) Barcelona 2007 62.5 20 16.25 (1.13) 43.10 (9.90) 0.97 (0.05) 2.22 (0.51) 3.44 (3.87) Barcelona 2019 87 21 17.31 (1.35) 54.34 (14.96) 0.97 (0.06)a 2.88 (0.74)a 4.80 (5.35) Ebro Delta 2019 65 19 17.73 (2.55) 65.82 (21.62) 1.09 (0.11)b 3.60 (1.12)b 4.87 (3.80) 2.2. Dissection procedure Barcelona and 0.8 L from samples off Ebro Delta) was filtered through a 1 mm mesh size stainless sieve into two fractions (>1 mm and <1 mm). At the laboratory, standard length (SL) and total weight (TW) were Due to the huge amount of organic material, the <1 mm fraction was recorded for each specimen from 2007 and 2019. The visceral organs split into ten aliquots with a McLane rotary splitter (splitting error were removed and the liver (LW), gonads (GW) and stomach (SW) were <4%). In order to prevent the degradation of cellulosic fibres (as weighed individually to the nearest mg. Then, the eviscerated weight explained by Carreras-Colom et al., 2022b), three of the ten aliquots (EW) of each specimen was recorded to the nearest g. Stomach, intestine were put together and exposed to a soft alkaline digestion (Cole et al., and the rest of the organs (liver, gonads, spleen, heart, kidney and gills) 2014): 20 mL of NaOH (1M) were added to the subsamples and left to were preserved in filtered 70% ethanol and stored separately for further react for 24h at room temperature. Subsamples were split again and two analyses. sub-aliquots were vacuum-filtered onto polycarbonate membranes (Millipore, Ø47mm, 0.45 μm mesh size). The filters were dried overnight 2.3. Visual inspection and collection of anthropogenic items (AIs) at 40 ◦ C and visually inspected for the presence of AIs. For the >1 mm fraction, AIs were directly picked from the stainless sieve under a ste­ 2.3.1. AIs ingested by M. merluccius in 2007 and 2019 reomicroscope. After AIs extraction, all the concentrations obtained for In order to recover AIs from the digestive tract, the stomach and the <1 mm fraction as for the >1 mm fraction were corrected in order to intestine of each specimen were emptied, and their content was placed take into account all the splitting steps. AIs from both fractions were in separate glass Petri dishes. The screening of the gastrointestinal collected, counted and mounted individually between glass slides in content was performed by visual inspection. Each AI found was cleaned, filtered distilled water for their characterization. counted, individually mounted between glass slides in filtered distilled water and observed under the microscope for their characterization. A 2.4. AIs classification and analysis selection criterion adapted from Hidalgo-Ruz et al. (2012) for fibres was followed to prevent misidentification of AIs with organic material (e.g. After AIs isolation from all samples (hake’s digestive contents from seagrass or seaweed). 2007 and 2019, and near-bottom water samples), images were taken at 50x to 400x of magnification and measures of mean diameter (calcu­ 2.3.2. AIs from near-bottom water samples lated from three random measurements) and total length for fibres and In order to analyse the AIs from near-bottom water samples collected mean cross-section (also from three random measurements) for frag­ at the different sampling points, an aliquot of 100 mL of the homoge­ ments were recorded. Images and measurements were taken with a Leica nised mixture of fixed samples (0.5 L from samples off Blanes and camera model CTR5000 connected to a Leica DM500B microscope and 3 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 an image-processing software (ProgRes® C3). For each fish, number and their presumed relationship to MPs exposure was suggested by Pedà (nAI) and total length (TL) of AIs were calculated. Also, to infer the total et al. (2016). Three fields of view were randomly selected and counts of length of AIs occupying the digestive system of fish, the sum of the the number of rodlet cells were performed on each section at 400x of lengths of all AIs (TLAI) found there was calculated for each specimen. magnification, and scored according to cluster partitioning around Mean intensity of AIs was calculated as the mean number of AIs ingested medoids (PAM) as: low (1) (<11 RC per observation field), medium (2) per individual, excluding those without AIs. AIs were firstly classified (12–16 RC per observation field) and high (3) (>17 RC per observation into five different typologies (A to E, Table S1) according to the visual field). Mean intensity was calculated from the average intensity values aspects of their morphology such as colour, length, cross-section, shape of each individual. of the ends and characteristics and texture of the backbone (Zhu et al., Fish external surfaces and right-side gills were inspected macro­ 2019; Carreras-Colom et al., 2020; Rodríguez-Romeu et al., 2020). scopically for ectoparasites, and the rest of organs were checked for Polymer composition of AIs >1 cm was identified by FTIR (Fourier- endoparasites under a stereomicroscope. All parasites found were Transformed Infrared Spectrometry) using a Tensor 27 FTIR spectrom­ counted, identified to the main taxonomic group and stored in 70% eter (Bruker Optik GmbH, Germany) operating in Attenuated Total ethanol. For identification, platyhelminths and acanthocephalans were Reflectance (ATR) mode. Spectra was recorded as 16 scans in the stained with iron acetocarmine, dehydrated through a graded ethanol spectral range of 600–4000 cm− 1 (Servei d’Anàlisi Química, Autono­ series, cleared in clove oil and examined as permanent mounts in Can­ mous University of Barcelona). AIs <1 cm were chemically analysed by ada balsam. Nematodes were cleared and examined as semi-permanent micro-FTIR using a Thermo Scientific™ Nicolet™ iN10 MX Infrared mounts in glycerine. Imaging Microscope, and spectra was recorded as four scans in the spectral range of 800–4000 cm− 1 (CCitUB, University of Barcelona). 2.6. Quality assurance and quality control A randomly selected subsample of the AIs recovered from the digestive tract of hakes and from the environmental samples at each In order to prevent airborne contamination, the dissection of all fish locality (at least 30% of the total AIs) were identified by micro-FTIR and was performed in a laminar flow cabinet. Moreover, all dissection ma­ FTIR spectroscopy. Likewise, all synthetic AIs (belonging to categories B, terial and vials were rinsed three times with filtered distilled water (50 C, D and E), which by their morphological features were doubtful, were μm metal sieve) before use, and nitrile gloves and cotton lab coat were also analysed as well as a representative subsample of cellulosic-like AIs used at all times. from category A in order to represent at least 10% of all cellulosic-like Water samples processing was performed in a clean laboratory, AIs found at each sampling point. consisting in an 8 m2 hardwall laminar flow chamber ventilated through The resulting spectra were treated with Spectragryph 1.2.11 4 laminar flow HEPA filters (H14). All material was previously rinsed (Menges, 2021) in order to apply baseline corrections and select the and distinctive orange lab coats were used at all times. Procedural spectral range of 800–3600 cm− 1. Corrected spectra were compared blanks were performed to check for potential contamination, and a mean with reference spectra (Primpke et al., 2018) and similarity correlation of 0.63 fibres/blank were found. No fibres matching with the coloured values > 70% were accepted for reliable identification. Correlation coat were detected attributable to airborne contamination. values ranging between 60 and 70% were also accepted only when their AIs screening of the gastrointestinal content and environmental spectra matched visually with the reference and the visual appearance of samples was carried out inside an isolation device adapted from the one the AI fitted with the description of the typology (Table S1). More than proposed by Torre et al. (2016), which consisted of a plastic cover 80% of the spectra of all AIs analysed by micro-FTIR and FTIR corre­ isolating the binocular stereomicroscope and the work area from the sponded to the assigned visual category. surrounding environment. Moreover, dissection tools and Petri dishes used were rinsed with filtered distilled water before the examination of 2.5. Analysis of fish health condition in 2019 each sample. The gastrointestinal content of each specimen was diluted in more than one Petri dish when necessary and checked several times in The general health condition of each fish was assessed through two the stereomicroscope to perform a complete inspection of the content physiological indices: Le Cren’s relative body condition index (Kn = and isolate all the AIs present in the samples. Procedural controls (Petri EW/(α × SLβ) (Le Cren, 1951), where Kn is the relative body condition, α dishes containing a few ml of filtered distilled water) were placed inside and β are the slope and the intercept of the weight-length relationship and outside of the isolation device to assess the intensity of background representing the entire dataset of sampled fish, and the hepatosomatic airborne contamination during screenings (Bellas et al., 2016). As an index (HSI = (LW/EW) x 100). The gonadosomatic index was not outcome, contamination found in the outside controls was on average considered as all individuals were immature. Feeding intensity was 5.1 times higher than contamination in the inside controls (average calculated by the stomach-fullness index (FULL = (CW/EW) x 100), values of 0.19 fibres per digestive sample screened), thus demonstrating using the stomach content weight (CW) recorded after AIs inspection. the effectiveness of the isolation device. Contamination in both controls A portion of liver and digestive tract (stomach and intestine after AIs consisted of fibres that always appeared on the surface of the water, inspection) were embedded in paraffin and processed by routine his­ indicating that they were deposited from the air. As a result of this tology techniques. The digestive tract was selected due to its direct observation, fibres found in the examined samples were only counted if contact with AIs and the liver was selected as a target organ for the they were clearly embedded in the digestive content. Thus, no correction assessment of potential changes associated to the presence of toxic ad­ factor was applied to the final values of the AIs obtained. ditives within the AIs. All samples were sectioned at 5 μm and stained with Haematoxylin and Eosin. Each sample was completely screened 2.7. Data analysis and qualitatively examined under the microscope for histopathological assessment and identification of specific disorders (e.g. inflammation, All variables were tested for normality and homoscedasticity using necrosis; see Kögel et al., 2020) which could be potentially related to the the Shapiro-Wilk test and the Levene’s test, respectively. Data distri­ ingestion of AIs or their associated toxic substances, but not to detect the bution was also plotted for visual assessment. AIs themselves. Prevalence of histological alterations found in the Fibres from the digestive tract and from the environment were samples was recorded for each fish. A random subsample of 10 fish per classified into four size clusters by PAM applying the k-medoids algo­ locality was used to determine the intensity of rodlet cells (RC) through rithm on a matrix of dissimilarity. Likewise, RC intensity was also a semi-quantitative analysis from histological sections of the stomach classified into three clusters by PAM. Each cluster was represented by mucosa. RC were considered as their increase in number is related to one object which is located at its centre. The k clusters are established by exposure to physical and chemical injuries (Manera and Dezfuli, 2004), assigning each object of the dataset to the nearest of the central objects, 4 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 thus objects that show a high level of similarity are grouped together, individual (TLAI) was 3.43 (SD = 2.67) mm. while objects which are most dissimilar are assigned to different clusters No differences of nAI, mean intensity or TLAI were observed between (Kaufman and Rousseeuw, 1990). In order to assess differences among stomach and intestine (p > 0.05). Blanes was the location with the sampled localities and between years (in the case off Barcelona) and highest prevalence of non-synthetic AIs (GZM, χ2 = 15.314, p = 0.003) organs as well as between fish and environmental samples in fibres size and mean abundance of both synthetic (GZM, χ2 = 6.388, p = 0.03) and clusters proportions and AIs typologies, Chi-Squared or Fisher-exact non-synthetic (GZM, χ2 = 8.121, p = 0.03), and global AIs (GZM, χ2 = tests (when sample size was too small) were applied. Also, 7.247, p = 0.03) (Table 2). No significant differences among localities Fisher-exact tests were used to detect differences in RC categories among were found for the values of mean intensity, TLAI and TL of either localities. When significant differences were detected, pairwise tests synthetic or non-synthetic AIs (GZM, p > 0.05) (Table 2). were performed to determine which pairs of categories contributed to A total of 7 of the 20 hakes off Barcelona from 2007 analysed con­ the overall significance. Differences in TL of fibres from water samples tained AIs in their digestive tracts. A total of 13 AIs were found within among localities were tested using Kruskal-Wallis test. their digestive tracts. No significant differences in terms of prevalence, Differences among localities and between years (for samples from off TLAI, mean abundance, intensity and length of ingested AIs were found Barcelona) on biological data (SL and FULL) and condition indices (Kn between 2007 and 2019 in Barcelona. and HSI) were tested using Kruskal-Wallis and ANOVA tests, respec­ tively. Post-hoc pairwise comparisons were carried out using Dunn’s 3.2. Environmental water samples multiple comparison test for non-parametric data and TukeyHSD post- hoc tests for parametric data. A total of 180 AIs were found in near-bottom water samples. This is To assess differences in prevalence, mean abundance, mean in­ equivalent to an overall average concentration of 0.85 AIs/m3 (0.52 AIs/ tensity, TLAI and TL of synthetic and non-synthetic AIs among localities m3 off Blanes, 1.53 AIs/m3 off Barcelona and 0.50 AIs/m3 off Ebro Delta) and organs (stomach/intestine), Generalized Linear Models (GZMs) (Table 2). All AIs detected in environmental samples were fibres. The (logistic model for prevalence and negative binomial model for mean average length of these fibres (TL) was 14.65 (SD = 14.29) mm and the abundance, mean intensity, TL and TLAI) were used, with SL and FULL mean diameter was 0.02 (SD = 0.004) mm. Significant higher values of set as covariates. Parasite prevalence (P) and mean abundance (MA) for the mean length of synthetic fibres from off Barcelona were detected each locality were calculated according to Bush et al. (1997) for the total compared to the other localities (K–W, χ2 = 26.15, p < 0.01) (Table 2). of parasites recovered, for each major taxonomic group and only Values of abiotic conditions (temperature, turbidity, salinity and considering parasites within the digestive tract. GZMs were performed oxygen concentration) of the near-bottom water layer from each locality to test differences in P and MA of digestive parasites among localities are given in Table S2. (logistic model for prevalence and negative binomial model for mean abundance). Possible correlations between AIs (nAI and TLAI) and 3.3. AIs size classification prevalence of histological alterations (i.e. granulomas, inflammation areas) were tested using GZMs (binary logistic model). Fibres were classified into four different classes based on the PAM A correlation matrix was built in order to test possible associations of algorithm: small (≤1.15 mm), medium (1.16–2.62 mm), large AIs descriptors (nAI, TLAI and TL), RC and parasite abundance with fish (2.63–6.39 mm) and extra-large (≥6.40 mm). The most abundant size health condition indices (Kn and HSI) and FULL by means of Pearson’s category within the digestive tracts of hakes was small (46.2%), fol­ and Spearman’s correlation tests. lowed by medium (39.8%) and the less abundant size category was large Finally, a Multiple Factor Analysis (MFA) was used to simultaneously (14%). No extra-large fibres were found in fish samples. No significant assess the relationship among AIs parameters, abundance of main differences in the size of the fibres ingested by hakes were found among parasite groups found in the digestive tract, environmental variables and localities nor between organs and years (Chi-squared, p > 0.05). In near- fish health condition indices, and to visualize and differentiate groups of bottom water samples, small was the most abundant fibre size class off samples according to these variables. This multivariate analysis enables Blanes (55.6%) and Ebro Delta (48.5%), contrary to Barcelona, where analysing and visualizing individuals that are characterized by different the most abundant size class was extra-large (79.1%). In this case, sig­ sets of variables (both quantitative and qualitative) which are structured nificant differences in the size of the fibres from water samples were into groups. Thus, the differences within groups are minimized whilst found among localities (Chi-squared, χ2 = 73.86, p < 0.001). Fibre size differences among groups are maximized. distribution from water samples was significantly different to that of Data analysis was performed using RStudio software, version 4.1.2, fibres ingested by hakes in Barcelona (Chi-squared, χ2 = 11.79 p = and using the PAMK function implemented in the package “fpc”, the 0.008), but not in the case of the other localities (Fig. 2, Table S3). packages “dunn.test”, “Hmisc”, “FactoMineR” and “factoextra”. Statis­ tical significance was set at 0.05. 3.4. AIs polymer characterization 3. Results The 100% of the resulting spectrums from AIs belonging to category A were identified as cellulose. From category B, 86.3% of the spectrums 3.1. AIs ingested by M. merluccius in 2007 and 2019 were identified as polyethylene terephthalate (PET), 9.1% as polyamide and 4.5% as cellulose. In the case of category C, 100% of the resulting A total of 41 of the 62 hakes from 2019 analysed contained AIs in spectra corresponded to acrylic. Regarding category D, 96.2% of the their digestive tracts. A significant major prevalence of AIs was noticed resulting spectra matched to polyamide (PA) and 3.8% to PET. Finally, off Blanes (90.9%), followed by Ebro Delta (63.1%) and Barcelona 75% of the resulting spectra from AIs belonging to typology E were (42.8%) (GZM, χ2 = 10.289, p = 0.006). A total of 95 AIs were found identified as polypropylene (PP) and 25% as PET (Fig. 3). within the digestive tracts of M. merluccius. The vast majority of AIs were Of the 108 AIs found in the hake’s digestive tract in 2007 and 2019, fibres (97.8% of the total). Only one fragment (0.03 mm2) and one film cellulose was the most frequent polymer in all localities (overall prev­ (2 mm2) were detected in the intestine of two different individuals in alence 71.3%). The rest of the AIs were of synthetic composition, mostly Barcelona and Blanes, respectively. The mean number of AIs found per PET (overall prevalence ca. 20%), but also acrylic and polypropylene. individual (nAI) was 1.39 (SD = 1.39) and the mean intensity of AIs per No significant differences among localities nor years were noticed in individual was 2.13 (SD = 1.18). The average length of the fibres (TL) terms of polymer composition (Chi-squared, p > 0.05). found was 1.56 (SD = 1.24) mm, their mean diameter was 0.03 (SD = Regarding the 180 AIs found in near-bottom water samples, cellulose 0.07) mm and the mean sum of the total length of the AIs found per was the most frequent polymer in Blanes and Ebro Delta. However, 5 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 Table 2 Prevalence of AIs ingested by fish and mean (standard deviation, SD) of the abundance, intensity, sum of total lengths (TLAI) and total length (TL) of the AIs (synthetic and non-synthetic) found in digestive contents of hakes in 2007 and 2019, as well as mean (SD) of the abundance, TLAI and TL in water samples, from each sampled locality in 2019. Different superscript letters indicate significant differences among localities. Blanes Barcelona Ebro Delta Total Year 2019 2019 2007 2019 2019 Digestive content n 22 21 20 19 Prevalence (%) Synthetic 40.91 14.28 10.00 21.05 25.80 Non-synthetic 86.36b 38.09a 35.00 57.89a 61.29 Mean abundance (n/ind) Synthetic 0.60 (0.85)b 0.14 (0.36)a 0.15 (0.49) 0.26 (0.56)a 0.34 (0.65) Non-synthetic 1.50 (1.14)b 0.52 (0.75)a 0.50 (0.76) 1.11 (1.41)a 1.05 (1.18) Mean intensity (n/ind) Synthetic 1.44 (0.73) 1.00 (0.00) 1.50 (0.71) 1.25 (0.50) 1.31 (0.60) Non-synthetic 1.74 (1.05) 1.38 (0.52) 1.43 (0.53) 2.22 (1.20) 1.78 (1.02) TLAI (mm/ind) Synthetic 3.78 (2.54) 1.37 (0.66) 2.29 2.99 (2.99) 2.71 (2.52) Non-synthetic 2.41 (1.97) 1.52 (1.07) 1.43 (0.95) 2.99 (2.22) 2.36 (1.91) TL (mm/ind) Synthetic 2.54 (1.29) 1.37 (0.66) 1.15 2.11 (1.58) 2.27 (1.30) Non-synthetic 1.34 (0.84) 1.35 (1.16) 0.87 (0.37) 1.32 (0.69) 1.34 (0.87) Environmental samples n 1 1 1 Mean Abundance (n/m3) Synthetic 0.21 1.33 0.06 0.53 Non-synthetic 0.31 0.19 0.44 0.32 3 TLAI (mm/m ) Synthetic 0.07 0.27 0.11 0.15 Non-synthetic 0.12 0.03 0.05 0.07 TL (mm/AI) Synthetic 1.25 (0.49)a 22.40 (12.92)b 6.58 (5.63)a 20.68 (13.56) Non-synthetic 2.00 (1.76) 1.34 (0.99) 1.60 (1.13) 1.66 (1.30) Fig. 2. Density standardized charts showing the distribution of the total length (TL, mm) of fibres from the digestive tract of Merluccius merluccius in 2019 (red colour), 2007 (yellow colour) and from near-bottom water samples (blue colour) according to the sampled locality. polyamide fibres were predominant in Barcelona. In this case, signifi­ No significant differences among localities were found for parasite cant differences were found in polymer composition among localities total prevalence and mean abundance, either considering the total (Chi-squared, χ2 = 134, p < 0.001). parasite load or only digestive parasites. However, a higher prevalence Composition of the AIs found in near-bottom water samples was of cestodes (GZM, χ2 = 8.424, p = 0.05) and a higher abundance of significantly different from that of the AIs ingested by hakes in Barcelona digeneans (GZM, χ2 = 6.424, p = 0.03) were detected off Barcelona, and (Chi-squared, χ2 = 97.8, p < 0.001) (Fig. 4). a significantly lower prevalence of digeneans was observed off Blanes (GZM, χ2 = 6.404, p = 0.008) (Table 3). Histopathological analyses did not reveal alterations in the mucosa 3.5. Trends on fish biological parameters, histopathology and of the stomach or the intestine potentially associated to AIs abrasion. parasitological indicators However, a particular high number of RC in the stomach mucosa and some granulomas were detected. No significant differences in RC in­ SL of the individuals examined ranged between 14.5 and 24.6 cm tensity and prevalence of granulomas were detected among localities. and no significant differences were found among localities nor between Findings and changes observed in the histopathologic evaluation of the years (p > 0.05). Fish from off Ebro Delta presented significantly higher liver consisted of lipidic metamorphosis, granulomas, macrophages ag­ values of Kn (ANOVA, F(79, 2) = 10.575, p < 0.001) and HSI (ANOVA, gregates and presence of small inflammation areas, as well as contained F(59, 2) = 4.915, p < 0.001). No significant differences among localities degraded forms of nematodes larvae (Table 3). However, none of them nor between years were detected regarding stomach fullness (p > 0.05) could be related to AIs ingestion since no clear statistical relationships (Table 1). were found between the prevalence of these observations and the levels A total of 208 parasites were found in hakes of 2019. High total of AIs ingested by individuals (GZM, p > 0.05). prevalence values were detected in all localities: 100% off Barcelona, The correlation matrix indicated no clear relationships among fish 86.4% off Blanes and 94.7% off Ebro Delta. Among the most frequently condition indices, stomach fullness, AIs parameters, parasite and RC found parasite groups, nematodes were the most abundant taxon, fol­ abundance. General AIs parameters (abundance, sum of lengths and lowed by digeneans and cestodes, and the least abundant groups were mean length) were positively correlated among them (rho >0.6), with monogeneans and copepods (Table 3). A single isopod and one acan­ abundance and sum of length of cellulosic fibres (i.e. non-synthetic) thocephalan were detected in two different individuals from Blanes and showing high correlation values with total AIs abundance (rho = 0.93, Ebro Delta, respectively. 6 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 Fig. 3. Images of representative AIs of the five different typologies of polymers found in the digestive tract of Merluccius merluccius and in the surrounding near- bottom water samples. Each category (A to E) is described in Table S1. 7 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 Fig. 4. Mean size (TL) and polymer composition of the AIs found in the digestive content of Merluccius merluccius and in water samples from each sampling area: off Blanes, off Barcelona and off Ebro Delta. Table 3 Parasite descriptors and histopathological alterations found in Merluccius merluccius from each locality in 2019. Location within host and prevalence (P%) of main parasite taxa and histopathological alterations are shown. Mean abundance (MA) for parasite taxa and mean intensity (MI) for rodlet cells, followed by standard deviation (SD), are also displayed. Keys for locations within hosts: DT, digestive tract; G, gills; H, heart; L, liver; M, mesentery; S, stomach. In the case of Nematoda, Digenea and total parasites, total values and only the values from the DT are shown. Different superscript numbers and letters indicate significant differences in P% and MA, respectively, among localities. Only significant differences are indicated. Blanes Barcelona Ebro Delta Total PARASITE GROUP Location P% MA (SD) P% MA (SD) P% MA (SD) P% MA (SD) NEMATODA DT, L, M 54.55 0.95 (1.11) 61.90 0.95 (0.94) 78.95 1.63 (1.50) 64.52 1.16 (1.22) DT 40.90 0.68 (0.99) 61.90 0.95 (0.97) 73.68 1.47 (1.50) 58.06 1.02 (1.19) PLATYHELMINTHES Digenea DT, H 22.72 0.68 (1.43) 52.38 1.33 (1.71) 57.89 1.21 (1.72) 45.16 1.06 (1.62) DT 9.091 0.32 (1.13)a 52.382 1.33 (1.71)b 42.102 0.79 (1.32)a 33.87 0.81 (1.45) Monogenea G 4.55 0.04 (0.21) 14.29 0.14 (0.36) 5.26 0.05 (0.22) 8.06 0.08 (0.27) Cestoda DT 45.451 0.57 (0.67) 71.432 1.00 (0.77) 26.321 0.26 (0.45) 50.00 0.60 (0.69) ARTHROPODA Copepoda G 9.09 0.18 (1.41) 9.52 0.09 (0.28) – – 6.45 0.09 (1.15) TOTAL DT, G, H, L, M 94.74 3.09 (3.05) 100.00 3.76 (2.60) 86.36 3.21 (2.34) DT 81.81 2.00 (2.39) 95.24 3.29 (2.10) 84.21 2.58 (2.41) HISTOPATHOLOGICAL ALTERATIONS P% MI (SD) P% MI (SD) P% MI (SD) P% MI (SD) Lipidic metamorphosis L 59.09 47.61 68.42 58.06 Granuloma L, S 45.45 38.09 47.37 43.54 Macrophages aggregates L 4.54 4.76 5.26 4.84 Inflammatory focus L 13.63 9.52 15.78 12.90 Rodlet cells (RC) S 90.00 1.70 (0.95) 100.00 1.60 (0.84) 100.00 2.10 (0.88) 98.38 1.80 (0.89) p < 0.001 and rho = 0.92, p < 0.001), respectively. Mean length of bottom water layer. A multidisciplinary approach, as in the present synthetic AIs displayed high correlation values with mean total AIs case, offers valuable opportunities to appreciate the simultaneous length (rho = 0.82, p < 0.001) (Fig. 5). behaviour of a number of factors of different nature, which is usually The MFA explained 36.2% of the variability by the two first axes. A complex and difficult to assess. differentiation was detected among fish from the three localities, as shown in Fig. 6B. The variables that explained most of the variability 4.1. Characterization of environmental and ingested AIs along the first dimension were the abundance of non-synthetic fibres ingested and abiotic parameters. Individuals off Blanes were charac­ Microplastics studies have demonstrated that a wide range of AIs terized by a larger body size and higher AIs abundance (Fig. 6A). shapes can be found in the marine environment (Hidalgo-Ruz et al., Regarding the second axis, specimens off Barcelona were related to 2012; Lusher et al., 2017; Hartmann et al., 2019). Present results higher abundance of cestodes and to temperature, and those off Ebro highlight that fibre-shaped items are the most abundant AIs (i.e. 97.8%) Delta were characterized mainly by the condition factor, abundance of found both in fish digestive tracts and in near-bottom water samples, as nematodes, oxygen concentration and stomach fullness. Overall, abiotic has already been proved in several studies from the Mediterranean Sea conditions were the group of variables that contributed most to explain (Bellas et al., 2016; Compa et al., 2018; Suaria et al., 2020; Rodrí­ the model (Fig. 6A). guez-Romeu et al., 2020; Carreras-Colom et al., 2020, 2022a, 2022b), making clear that they dominate micro-litter composition in this area, 4. Discussion both in fish and near-bottom water. Given that most of the fibres found in the marine environment are This is the first study that reports and quantifies AIs ingestion by the made of cellulose (Avio et al., 2020; Suaria et al., 2020), in the present European hake off the Catalan coast (NW Mediterranean) in relation to study non-synthetic polymers (cellulosic-like fibres) have been charac­ the exposure of the AIs from the surrounding environment, the near- terized and quantified in addition to the synthetic polymers. However, 8 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 Fig. 5. Correlation matrix indicating positive (blue) and negative (red) significant (p < 0.05) correlations among total AIs abundance (nAI), non-synthetic fi­ bres (Non-synthetic) and synthetic fibres (Synthetic) abundance, sum of total AIs lengths (TLAI), non- synthetic fibres (Non-synthetic) and synthetic fibres (Synthetic) sum of lengths, mean of total AIs lengths (TL), non-synthetic fibres (Non-synthetic) and syn­ thetic fibres (Synthetic) mean of lengths, fish stan­ dard length (SL), stomach fullness (FULL), Le Cren relative condition index (Kn) and hepatosomatic index (HSI), and total abundance of parasites (nPAR), and of main parasite taxa (i.e. nematodes, digeneans and cestodes), found within Merluccius merluccius digestive tracts. Circle size and colour intensity are proportional to the value of the correlation coefficient. Fig. 6. (A) Multiple Factor Analysis (MFA) displaying relationships among AIs abundance (synthetic and non-synthetic) in the hake digestive tract, fish health condition indices (Kn, HSI), stomach fullness and standard length (SL), abundance of main parasite taxa (Nematoda, Digenea and Cestoda) and abiotic con­ ditions measured on the near-bottom water layer (temperature, salinity, turbidity and O2 concentra­ tion). (B) Factor map of the MFA results for Merluccius merluccius individuals from each locality. Each indi­ vidual is represented by a dot. given that most of the available studies on the subject have only focused (2016) reported in the digestive tract of hakes from the Gulf of Cadiz on reporting MPs (Hidalgo-Ruz et al., 2012; Hartmann et al., 2019), (fibres size <3.1 mm). This high abundance of cellulosic fibres found in comparison of present results regarding the total amount of AIs with the present study has indeed been reported in other marine environ­ published data can be inaccurate. Thus, these studies are compared to ments such as the deep-sea (Sanchez-Vidal et al., 2018) and inside the only the synthetic fibre data. Furthermore, although some studies have digestive tract of other fish and crustacean species (Carreras-Colom excluded non-synthetic fibres from their results to avoid overestimation et al., 2018, 2020; Rodríguez-Romeu et al., 2020, 2022). Its origin is of AIs due to airborne contamination (Foekema et al., 2013), the present mainly associated to textile industries and households, from which fi­ study demonstrates that working on isolation devices can effectively bres are released through wastewater treatment plants, or to discharges prevent background contamination of the samples and thus provide from domestic washing machines into the seas (Browne et al., 2011). reliable results. As a matter of fact, several studies focusing on The long-term accumulation of cellulose in the marine environment micro-litter and avoiding digestion methods have highlighted a much could be explained by the fact that natural fabrics release more fibres greater presence of cellulosic fibres both in biotic and abiotic compart­ during laundering than synthetics, and although they are considered ments (Sanchez-Vidal et al., 2018; Suaria et al., 2020; Rodríguez-Romeu biodegradable, their degradation rate is poorly understood and may be et al., 2020). For all this, it is of major importance to avoid destructive slowed down if these fibres have been dyed and processed with chemical digestion techniques that can potentially degrade cellulose-based fibres products (Suaria et al., 2020). (Dehaut et al., 2016; Kühn et al., 2017; Treilles et al., 2020) and to Literature on the abundance of MPs in seawater has highlighted a establish a standardized methodology to characterize micro-debris heterogeneous spatial distribution of marine debris in the Mediterra­ (Athey and Erdle, 2022). nean Sea (Suaria and Aliani, 2014; Spedicato et al., 2019), which is Most of the AIs ingested by hakes were cellulosic fibres smaller than corroborated by present results, mostly regarding the higher abundance 5 mm, which corresponds to the traditional microplastic size-based of synthetic AIs in polluted areas (Alomar et al., 2016) such as off Bar­ definition. Similar sizes of AIs (<2.6 mm) were found both in the celona. Available studies have mostly focused on quantifying MP con­ digestive tract of hakes and in the near-bottom water layer off Blanes centrations from surface waters (Cózar et al., 2015; Suaria et al., 2020) and Ebro Delta. This result is in accordance with what Bellas et al. and from the seafloor sediments (Woodall et al., 2014; Sanchez-Vidal 9 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 et al., 2018). Few studies regarding their vertical distribution have heterogeneous distribution of AIs in the water column (Bour et al., demonstrated that, due to their properties, which include shape, density 2018). and chemical composition, most MPs are likely to spread into deeper Furthermore, AIs have been impacting the area off Barcelona for the layers of the water column, where they can interact with marine biota past few years, as our results indicate, since AIs ingested by hakes from (Reisser et al., 2015; Avio et al., 2020). 2007 have remained at a similar level as in 2019. Previous studies in the Due to the limited number of studies characterizing AIs in the near- same area detected similar levels of AIs when compared to those bottom water layer in the Mediterranean Sea, comparisons with other ingested in 2007 and 2018 in fish and crustaceans, but reported differ­ data from close regions are difficult to establish. However, taking into ences in polymer composition between these two years, highlighting a consideration only synthetic AIs in order to compare present outcomes shift in production and usage trends of textile polymers (Carreras-Colom with those of other studies carried out in the Mediterranean area, similar et al., 2020; Rodríguez-Romeu et al., 2020, 2022). However, considering AIs concentrations to those observed off Blanes have been detected by that local effects on AIs dynamics may exist in this area, no clear evi­ Baini et al. (2018) in the north Tyrrhenian Sea (0.22 (SD = 0.57) dence of temporal trends for the levels of AIs could be drawn from the items/m3), and by Lefebvre et al. (2019) in the Gulf of Lions (0.23 (SD = present results. 0.20) items/m3), whose samplings were performed at different depths of the water column. 4.2. Comparison between environmental and ingested AIs Further research should be done in quantifying the abundance of micro-debris in different layers of the water column to better understand For a complete and contextualized interpretation of the levels of AIs the behaviour and fate of these pollutants. For this purpose, it is ingested by hakes, it is important to take into account the abundance important to consider the factors that can determine the distribution of and distribution of plastic debris present in the associated environment. AIs in the environment such as urbanization, industry, river inputs, It has been demonstrated that higher concentrations of micro-debris in different strategies of wastewater treatment plants, fishing activities, marine ecosystems from impacted areas imply that fish and other local local pollution sources, density properties of micro-debris and resus­ organisms are easily exposed to them, with the consequent active or pension processes likely caused by bottom-trawl fishing vessels (Paradis passive ingestion of these particles while feeding (Fossi et al., 2018), et al., 2018). Furthermore, the observed differences among localities in foraging or drinking (Roch et al., 2020). AIs concentration from water samples could also be explained by the Although some studies have detected a clear relationship between different morphology of the associated continental shelves. Indeed, the the levels of AIs ingested by marine species and their proximity to highly depositional continental shelf off Barcelona, characterized by a smooth anthropized areas (Andrady, 2011; Giani et al., 2019; Rodríguez-Romeu bathymetry, is influenced by the Llobregat and Besós rivers that drain et al., 2020), in the present study, lower levels of ingested AIs were densely urbanized areas, and thus discharge huge amounts of AIs, found off Barcelona, a densely populated coastal area with high con­ compared to the other two localities, that can easily be accumulated centrations of AIs in the environment. However, these inconsistent re­ there (Durán et al., 2014). In the case off Blanes, the nearby submarine sults could be explained by the size distribution of fibres from the canyon could have a role in the AIs that arrive from inland sources and environment and by the particular feeding behaviour of hakes. probably accumulate them in the deep sea (Tubau et al., 2015). Ontogenetic shifts in the feeding behaviour and diet in juvenile hakes Regarding the area off Ebro Delta, the low abundance of AIs/m3 allow the transition from an opportunistic feeding behaviour based on detected in water samples suggests that this sampling area might not euphausiids, mysids and decapods crustaceans (Modica et al., 2011) to a receive the direct influence of Ebro river discharge and consequently a more selective foraging behaviour (D’Iglio et al., 2022) dominated by low land-based AIs input, due to its distance from the coast compared to small and fast-moving fishes and suprabenthic crustaceans of the sub­ the other areas. Besides, in this microtidal and low-energy continental order Gammaridea, the orders Mysidacea, Euphausiacea and natantian shelf, resuspension processes are rare (Palanques et al., 2002), so AIs decapods (Carrassón et al., 2019). This high specialization on certain could sediment more rapidly and thus would not be detected at the prey in hakes from sizes as those in the present study, could be related to near-bottom water. In fact, this is in accordance with the turbidity levels a low probability of ingesting large fibres from their surrounding recovered from the present study, which were lower compared to the environment. other two sampling areas (Table S2). In fact, according to present results, the size and polymer composi­ In the present study, hakes captured off the Catalan coast seem to be tion of ingested fibres were similar to those of fibres from the environ­ easily exposed to the ingestion of AIs due to the frequent presence of ment off Blanes and Ebro Delta. However, in Barcelona the fibre size these elements within their digestive tracts. Similar prevalence values of distribution from water samples was much wider than that of fibres ingested synthetic AIs in Barcelona and Ebro Delta were reported by ingested by hakes due to the predominance of extra-large polyamide Bellas et al. (2016) in hakes from the Gulf of Cadiz (16.7%), but higher fibres in the environment, which were absent in the fish digestive tracts. mean abundances of AIs per individual were detected in this case (1 Considering only the total abundance of fibres within the observed size AIs/ind) compared to what is reported in the present work (see Table 2). range of ingested AIs for hakes, the concentration of AIs in Barcelona Although hakes from off Blanes presented the highest prevalence and would be reduced from 1.53 to 0.26 items/m3, a lower value than the abundance of ingested synthetic AIs in the present study, these values concentration of AIs from the other two localities, but consistent with are, by far, lower than what was reported on hakes from the Tyrrhenian the lower levels of AIs ingested by hakes from this area. Thus, hakes Sea (82.5%) (Bianchi et al., 2020), from the Ionian Sea (48%, 1.75 (SD could overcome the exposure to large fibres, and only ingest accidentally = 2) AIs/ind) and from the Adriatic Sea (30.6%, 1.09 (SD = 0.30) those below a certain size threshold. AIs/ind) (Giani et al., 2019), probably due to the different impacts of To sum up, for a mean of 0.85 items/m3 of AIs from the surrounding human activity on the sampling areas and to the different methodology water from the three localities considered together, examined fish used for AIs quantification. ingested 1.39 (SD = 1.39) items/individual. However, important dif­ Considering only fibres, differences in the levels of ingested fibres ferences, due to the high differences in the size range of environmental have also been detected among distinct fish species captured in the same AIs depending on the sampling station, were observed at a local scale. or close to the sampling areas as those considered in the present study: Overall, levels of micro-debris detected both in the environment and higher levels of ingested fibres were detected in benthic species ingested by hakes are consistent for fibres below 6 mm size, indicating (Rodríguez-Romeu et al., 2020) compared to the present results, but that this species could be useful as a bioindicator species for monitoring similar or even lower levels were recorded from pelagic species (Compa small-sized micro-debris present in the near-bottom water layer. et al., 2018; Pennino et al., 2020; Rodríguez-Romeu et al., 2022). These differences could be explained by different fish feeding modes or by the 10 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 4.3. Association of ingested AIs to hake health status be needed to assess the potential relationship between MPs and parasite aggregations. Present results suggest that ingested AIs do not significantly affect in Differences found in Kn and HSI values among localities could be a negative way the general health condition, nutritional state or feeding related to the variability regarding food availability and environmental activity of M. merluccius, since no association with condition indices or conditions such as temperature or oxygen concentration, which are stomach fullness was detected. Similar outcomes have been reported in regarded as the most important factors impacting fish health (Lloret other fish species like Sardina pilchardus, Engraulis encrasicolus (Compa et al., 2014). The significantly higher values of Kn and HSI observed in et al., 2018; Rodríguez-Romeu et al., 2022) and Mullus barbatus the individuals from off Ebro Delta could be related to a higher oxygen (Rodríguez-Romeu et al., 2020). concentration and to a greater quantity and/or availability of prey in Negative effects related to AIs ingestion on fish health have been this locality linked to a higher food consumption, as demonstrated by described in experimental studies working with acute exposures to high the higher values of the fullness index (yet not significant) in this lo­ levels of MPs (Kögel et al., 2020). At the histopathological level, the cality, which could enhance the general condition of hakes in this area mucosa of the digestive tract is expected to be affected due to its direct (Hidalgo et al., 2008). Moreover, Lloret et al. (2002) reported that in­ contact with the AIs. In fact, intestinal damage and inflammatory dividuals inhabiting shallower habitats, as in the case of Ebro Delta, changes were detected in Sparus aurata exposure study with 100–500 mg were in better condition than those in deeper waters given that shallow of microparticles (with sizes ranging between 40 and 150 μm) per kg of areas are important habitats for food resources. Nevertheless, in­ feed (Espinosa et al., 2017; Kögel et al., 2020). Also, an increase in rodlet dividuals from off Barcelona were in a poorer condition compared to cells abundance in the intestinal mucosa of Dicenthrarchus labrax was individuals from the other two localities. Although individuals in this noticed in exposure experiments with diets containing 0.1% (w/w) of area were slightly more parasitized than in other areas, no significant MPs for 90 days (Pedà et al., 2016). However, these outcomes are hardly correlations were found between condition indices and parasite in­ comparable to patterns observed in the natural environment, where dicators. It could be hypothesized that certain environmental conditions concentrations of AIs are much lower. In the wild, no consistent effects or factors related to anthropogenic pollution affecting this area could on health condition have been found in Aristeus antennatus (Carrer­ cause a detrimental impact on fish condition. as-Colom et al., 2018), M. barbatus (Rodríguez-Romeu et al., 2020) nor E. encrasicolus (Rodríguez-Romeu et al., 2022). Similar to these later 5. Conclusions studies, in the case of M. merluccius, AIs seem to pass through the digestive tract in the same way as digested prey contents since no sig­ The present study demonstrates the widespread presence of AIs in nificant differences were detected between the abundance of AIs from the near-bottom water layer and their ingestion by M. merluccius off the the stomach and intestine. Besides, no alterations in the digestive tract Catalan coast (NW Mediterranean Sea). Barcelona was the sampling potentially associated with AIs were noticed which is in accordance with point with the greatest abundance of AIs present in the environment, other studies (Jovanović, 2017; Jovanović et al., 2018; Batel et al., however, the total abundance of AIs ingested by hakes (determined by 2020). Although rodlet cells have been reported as immune cells and AIs size range) was lower than in the other localities, probably due to the biomarkers of exposure to contaminants, such as MPs (Pedà et al., 2016), feeding behaviour of juvenile hakes, which is paramount to take into their origin and function are still controverted (Manera and Dezfuli, account in marine litter studies. The most abundant type of AIs found 2004), and in the present study their presence is not associated with AIs were cellulosic fibres despite the predominant abundance of large abundance. polyamide fibres in the water samples from off Barcelona. No potential Although some studies have detected MPs in the liver or muscle impact of AIs could be detected by fish condition indices, histological (Collard et al., 2017; Haave et al., 2021), the processes by which MPs assessment or parasitological descriptors. Despite this, it is essential to from the digestive system could be translocated into other organs are not keep developing tools to accurately assess plastic pollution’s impact on yet demonstrated and well understood as methodological limitations ecosystems so that potential harmful effects can be detected and eval­ prevent the precise localization of MPs within the organs (Burns and uated as early as possible. Boxall, 2018). AIs found in this study are unlikely to pass through the intestinal barrier due to their size (Burns and Boxall, 2018), but poten­ CRediT authorship contribution statement tially toxic additives and substances that could be present in the AIs (Amelia et al., 2021), can be released and absorbed in the intestine, and Laura Muns-Pujadas: Methodology, Formal analysis, Investigation, therefore could reach other organs, such as the liver. Some experimental Writing – original draft, Writing – review & editing, Visualization. Sara studies have recorded some alterations detected in the liver which Dallarés: Methodology, Investigation, Writing – original draft, Writing include decreased energy storage of glycogen (Rochman et al., 2013), – review & editing, Supervision. Maria Constenla: Conceptualization, effects on lipid tissues, inflammation (Lu et al., 2016), oxidative damage Methodology, Investigation, Writing – review & editing, Supervision. and necrosis (Chen et al., 2017). However, the findings found in the liver Francesc Padrós: Conceptualization, Methodology, Investigation, sections in the present study are not correlated with the presence or Writing – review & editing, Supervision. Ester Carreras-Colom: levels of AIs, so they are probably associated with natural metabolic Methodology, Investigation, Writing – review & editing. Michaël Gre­ changes related to different feeding regimes and/or by the presence of laud: Methodology, Investigation, Resources, Writing – review & edit­ nematode parasites. This is not surprising considering that studies ing, Funding acquisition. Maite Carrassón: Conceptualization, finding tissue alterations associated with MPs have been conducted Methodology, Investigation, Resources, Writing – original draft, Writing using very high levels of MPs, much higher than the levels detected in – review & editing, Supervision, Project administration, Funding this study. acquisition. Anna Soler-Membrives: Conceptualization, Methodology, No clear trends were found between the abundance of parasites with Formal analysis, Investigation, Writing – original draft, Writing – review fish size or condition indices nor with AIs ingestion. So, parasites seem & editing, Supervision. not to interfere in the accumulation of AIs within the digestive tract, as reported by Rodríguez-Romeu et al. (2020). This contrasts with what Hernandez-Milian et al. (2019) suggested in grey seals. Recently, Pen­ Declaration of competing interest nino et al. (2020) reported a positive relationship between MPs and parasites in anchovies, and explained their results on the basis that in­ The authors declare that they have no known competing financial dividuals from more polluted areas ingested more MPs and thus, were interests or personal relationships that could have appeared to influence more likely to be infected by parasites. However, further research would the work reported in this paper. 11 L. Muns-Pujadas et al. Marine Environmental Research 186 (2023) 105921 Data availability Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on its own terms: margolis et al. Revisited. J. Parasitol. 83, 575. https://doi.org/ 10.2307/3284227. Data will be made available on request. Carpenter, E.J., Smith, K.L., 1972. Plastics on the sargasso sea surface. Science 175, 1240–1241. https://doi.org/10.1126/science.175.4027.1240. 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