Science of the Total Environment 841 (2022) 156539 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Assessment of the health status of the European anchovy (Engraulis encrasicolus) in the NW Mediterranean Sea from an interdisciplinary approach and implications for food safety ⁎ Oriol Rodríguez-Romeu, Anna Soler-Membrives , Francesc Padrós, Sara Dallarés, Ester Carreras-Colom, Maite Carrassón, Maria Constenla Departament de Biologia Animal, de Biologia Vegetal i d'Ecologia, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain H I G H L I G H T S G R A P H I C A L A B S T R A C T • Sampled European anchovies did not show any relevant pathology. • No zoonotic parasites were found in the studied fish. • Half of fish contain anthropogenic items but with no impact on health descriptors. • Higher pollution levels were found in 2019 with respect to 12 years earlier. A R T I C L E I N F O A B S T R A C T Editor: Damià Barceló The European anchovy (Engraulis encrasicolus) is a small pelagic fish with an outstanding commercial value supporting important fisheries and is a key component of pelagic ecosystems in the Mediterranean Sea. Progressive reductions in Keywords: the population size of this species has been observed in the Mediterranean Sea during recent decades, accompanied by Histopathology a decline in the body condition, as well as the size/age of maturation. Nonetheless, the health status has not been yet Plastic pollution assessed using a holistic approach. Herein, we analyse the health status of the European anchovy, integrating distinct Enzyme biomarker Zoonotic parasites indicators from fish condition, enzymatic biomarkers, presence of tissue alterations, and parasite descriptors. In addi- tion, we analyse the presence of anthropogenic items (AIs) in the digestive tract of fish and their potential impact on health status. Additionally, we assess the differences between current AIs values and those recorded over 12 years ago. None of the health indicators studied provided evidence of relevant pathologic conditions affecting this fish species in the studied area. However, changes in the pattern of liver parenchyma were found. Compared with anchovy popula- tions from other distribution areas, no zoonotic parasites were recorded in this study, demonstrating a reduced risks associated with foodborne transmission to humans. AIs, such as fibres and plastic particles, were found in the digestive tract of half of the fish analysed. A significant increase was detected in AIs prevalence between 2007 (40 %) and 2019 (70 %), alongside differences in the abundance and typology of the AIs, though this does not seem to have impacted fish health yet. Therefore, our work underscores the importance of implementing a regular program to monitor the health status of this key species to better understand population dynamics and their drivers. ⁎ Corresponding author. E-mail address: anna.soler@uab.cat (A. Soler-Membrives). http://dx.doi.org/10.1016/j.scitotenv.2022.156539 Received 3 March 2022; Received in revised form 2 June 2022; Accepted 3 June 2022 Available online 7 June 2022 0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/). O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 1. Introduction involved in biochemical and metabolic pathways that are influenced by the presence of xenobiotics — and the effects of this, e.g. alterations in The European anchovy (Engraulis encrasicolus) is a well-known small levels of neurotoxicity, detoxification, and oxidative stress, are highly rec- pelagic fish species widely distributed in the NW Atlantic Ocean, mainly ommended (Mejdoub et al., 2017; Solé and Sanchez-Hernandez, 2018). in near-European and African coastal areas, though also present in the Med- Such markers respond to both natural and anthropogenic stressors, provid- iterranean and Black Sea (Whitehead et al., 1988). This species is highly ing a broader perspective and a better understanding of the observed dy- valued in Mediterranean markets as it supports important local pelagic namics (Cajaraville et al., 2000; Galloway et al., 2002; Matozzo et al., fisheries. Its popularity as a commercial fish species is partly due to the par- 2018). Important knowledge gaps remain; for instance we know of only ticularly rich nutritional profile it boasts, including a high content of essen- one study that has addressed the response of P450 monooxygenase 1A in tial fatty acids, including polyunsaturated fatty acids (PUFAs) — among anchovies from impacted areas (Basilone et al., 2018). which are the omega 3 fatty acids, such as docosahexaenoic acid (DHA) In many cases, pathologies associated with biological agents (parasites, and ecosapentaeonic (EPA) — and omega-6 fatty acids, like arachidonic virus, bacteria, fungi) or non-infectious diseases (neoplasia, pathological acid (ARA) (Zlatanos and Laskaridis, 2007). Due to its significant biomass behavioural changes, genetic diseases) present in wild fish populations at mid-trophic levels, this species is the main prey for numerous predators, are also modulated by environmental impacts. These diseases or alterations thus playing a major role in energy transfer, connecting lower to higher tro- in wild fish populations can be detected using different diagnostic method- phic levels in marine ecosystems (Cury et al., 2000). For these reasons, the ologies and in particular, pathological and histopathological techniques are anchovy is considered an important resource not only for its great value for considered some of the most reliable and suitable tools for a general health human nutrition, but also for its role in the food chains of pelagic ecosys- assessment (Au, 2004; Costa, 2018; Feist et al., 2004; Stentiford et al., tems. Small pelagic fish are widely known for their rapid and significant 2003). They allow for the identification of not only the early warning population fluctuations, rendering their management especially difficult signs of disease and injury in cells, tissues, or organs, but also chronic expo- (Bakun, 1997). Progressive declines of commercially important small pe- sure and the subsequent effects at the population or community level. The lagic fish populations — including anchovies — have been observed in most common target organs used in histopathology are the liver (due to the Mediterranean Sea alongside changes in the population structure its role in transformation, storage, and detoxification) and gills — which (Van Beveren et al., 2014). While the abundance of anchovy remains rela- are in direct contact to the environment. Additionally, the digestive tract, tively high, both biomass and the mean size of individuals have diminished kidney, and gonads may be highly relevant for histopathological diagnos- dramatically, accompanied by a decline in the body condition and size/age tics (Costa, 2018) due to their role in fish metabolism and because of of maturation (Albo-Puigserver et al., 2021; Brosset et al., 2015; Saraux their sensitivity and direct exposure to environmental factors or pathogens. et al., 2019; Van Beveren et al., 2014). The prevailing explanatory hypoth- Apart from a few specific studies describing the histology of the visual esis for this implicates various stressors, stemming from changes in the organ sense (Heß, 2009; Kondrashev et al., 2012) in anchovies, neither environment and food availability. Changes in the plankton community the normal histology of adults nor the specific histopathological alterations affect the diet of this species, resulting in a shift towards feeding on smaller have yet been described. zooplankton species (Queiros et al., 2019; Thoral et al., 2021; Van Beveren Fish parasite communities are widely used as health indicators of both et al., 2014). However, other drivers, such as fishing pressure or increased organisms and ecosystems, since parasitic infestations affect not only fish levels of pollutants including microplastics might also be influencing the in- health, but can also respond to environmental changes depending on the dividual health of fish and therefore the population dynamics (Lefebvre parasites' lifecycle and the nature of pollutants (Mackenzie et al., 1995; et al., 2019; Saraux et al., 2019). Long-term survival, and maintenance of Marcogliese, 2005; Sures, 2001). Moreover, as a species for human con- anchovy stocks is of vital importance for both commercial and ecological sumption, the parasite fauna of European anchovy has been described reasons; this can only be ensured by supporting the good health status of and studied, especially with regard to zoonotic nematodes (Ferrer-Maza populations. et al., 2016; Rello et al., 2009). These zoonotic parasites play a relevant Due to these complex and multifactorial influences, evaluations of the role in human health and may trigger a food safety issue (Cipriani et al., condition and health status of wild fish populations necessitate the integra- 2018). Nematode larvae reach the fish when feeding, penetrate the intesti- tion of multiple distinct indicators, in a holistic approach. This analysis nal wall and then encyst on the surface of the internal organs and/or should include general indices such as overall fish condition, to more spe- migrate towards the musculature (Cipriani et al., 2018; Mattiucci and cific indicators such as the evaluation of potential pathological features Nascetti, 2008). They can be transmitted to humans — who act as paratenic by histopathology, the presence of alterations in enzymatic biomarker hosts — by ingesting infected raw or undercooked fish. Taking into account patterns, and parasite descriptors, among others. the wide range of culinary preparations in many countries of the Mediterra- Fish body/condition indices are based on the relationship that exists nean basin, as well as the traditional method of processing and preserving between the mass of certain organs with respect to the general mass or (brined in salt and preserved in oil and salt mixture, or pickled in vinegar), fish size; they allow comparisons between individuals of the same popula- the presence of these zoonotic parasites in anchovies is considered a risk for tion or between distinct populations. They are considered a simple way to human health (Cipriani et al., 2018). infer overall health of fish and alterations can indicate the occurrence of The Mediterranean Sea is a semi-enclosed, highly populated basin with diseases or other physiological events that may be compromising the a very ancient intensive use of marine and terrestrial resources that results species' fitness. For instance, the Le Cren relative index (Kn) is the main in heavy anthropogenic pressure from land to the sea. During the last few method used to estimate the body condition of an individual or population decades, marine litter has increased exponentially (Ryan et al., 2009), (Brosset et al., 2015), and fasting or feeding intensity can be assessed by the becoming one of the greatest threats that marine ecosystems and their asso- stomach fullness index (Hyslop, 1980). Hepatosomatic and gonadosomatic ciated biota have ever faced, this is an ever-growing cause of concern indices — both widely used in ecological studies and in stock evaluations in worldwide. Between 4 and 12 million tonnes of plastic enters the world's fisheries (Basilone et al., 2020; Brosset et al., 2017, Brosset et al., 2015; oceans annually (Jambeck et al., 2015). Once in the environment, they Stevenson and Woods, 2006) — give information about the physiological are transported long distances thanks to their buoyant and persistent prop- status of the individuals, in regard to the accumulation of short-term re- erties and current evidence suggests that plastics — and the products of serves and reproductive capacity, respectively (Wootton, 1989). their fragmentation — are now ubiquitous in oceans worldwide (Cózar Moreover, environmental stressors can be reflected in alterations at et al., 2014). Microplastics (plastic fragments smaller than 5 mm; Frias different levels, from biochemical pathways, to cells, tissues, organs, sys- and Nash, 2019) have been extensively described in both vertebrate and in- tems, fish and/or fish stocks. Biochemical markers in fish are known to be vertebrate organisms. The ingestion of particles of anthropogenic origin by particularly sensitive to environmental changes (Van der Oost et al., small pelagic fish — including the anchovy — has been widely described in 2003). Thus, studies that combine different sets of biomarker enzymes — the NW Mediterranean (Compa et al., 2018; Lefebvre et al., 2019), 2 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 confirming the ingestion of microplastic fragments, plastic fibres, or cellu- northern and southern locations nearby Blanes and Tarragona, respectively) lose fibres (Capone et al., 2020; Compa et al., 2018; Lefebvre et al., (Fig. 1, Table 1). Fish captured in 2007 were immediately fixed in 10 % buff- 2019). Although the possible hazardous effects have been addressed in lab- ered formalin and stored. In fish captured in 2019, before fixation, a portion oratory experiments, studies inferring the effects in wild populations are of dorsal muscle (0.5 g, w/w) and a portion of liver (0.1 g, w/w) were scarce, though increasing (Rodríguez-Romeu et al., 2020). dissected, weighed and stored in dry ice for subsequent enzymatic biomarker Therefore, the assessment of the effects of active or accidental ingestion analysis. Thereafter, fish were immediately fixed in 10 % buffered formalin of anthropogenic items (AIs) — including microplastics but also other par- and stored in the laboratory. Thirty additional fish per location were frozen ticles from anthropogenic origins such as cellulose fibres — on the health (−20 °C) on board to complement parasitological studies. status of wild marine biota should be properly addressed, in order to better understand their potential impact at the population and ecosystem levels, 2.2. Laboratory procedures as well as the potential risks regarding food safety and human health. For these reasons, the main aim of the present study is to assess the health Prior to dissection in the laboratory, each specimen was measured to the status of the European anchovy sampled from three different geographical nearest mm (total length = TL and standard length = SL) and weighed to areas of the NW Mediterranean Sea using a holistic approach, combining dif- the nearest g (total weight = TW). To minimize airborne contamination, all ferent evaluation methodologies: biological indices, enzymatic biomarkers, procedures were performed in a laminar flow cabinet, which was previ- histological tissue alterations, and parasite descriptors — specifically focus- ously cleaned, and the laboratory equipment and tools rinsed twice with de- ing on zoonotic parasites. In addition, we analyse the presence of AIs in ionized filtered (50 μm) water. Nitrile gloves and exclusive cotton lab coats the digestive tract of fish and their potential impact on the fish condition were used at all times. The gastrointestinal tract was removed by dissection and health status, as well as the differences between current AIs levels and following procedures based on previous work (Lusher et al., 2013), from the those found in fish over 10 years previous. top of the oesophagus to the anus. Stomach (SW), liver (LW), and gonads (GW) were weighed inside the laminar flow hood using a precision scale 2. Materials and methods to the nearest mg. The spleen was also removed, and eviscerated weight (EW) was recorded to the nearest g. All organs including the dissected 2.1. Study area and sample collection gastrointestinal tract (stomach, caeca, and intestine) were stored separately in filtered (50 μm) 70 % ethanol in individual glass vials previously rinsed A total of 150 European anchovies were captured from three distinct lo- with deionized filtered (50 μm) water for subsequent observations. cations (Fig. 1, Table 1) off the Catalan coast (NW Mediterranean) within the framework of the BIOMARE (Spanish Ministry of Science and Innovation), 2.3. Condition and health assessment SOMPESCA (Department of Agriculture, Livestock, Fisheries and Food, Catalonia, Spain) and PLASMAR (Spanish Ministry of Science, Innova- Fish condition was assessed by the gonadosomatic index (GSI = (GW/ tion and Universities project) multidisciplinary projects. Fish were col- EW) x 100), the hepatosomatic index (HSI = (LW/EW) × 100) and Le lected aboard commercial trawling vessels during 2007 (30 specimens) off Cren's relative body condition index (Kn = EW / (α × TLβ)), where Kn is Barcelona and with commercial purse seiner fishing vessels during 2019 at the relative body condition, α and β are the slope and the intercept of the three different sites (30 specimens each; same location off Barcelona, and a weight-length relationship representing the entire dataset of sampled fish Fig. 1. Map of sampling area where sampling for European anchovy occurred. The diamond shape (♦) indicates sampling stations. From north to south, Blanes (BLA), Barcelona (in 2007, BCN1, and in 2019, BCN2), and Tarragona (TAR). Pie charts represent the percentage of each polymer type from the anthropogenic items identified by micro-FTIR in each location. Base map retrieved from the web map services of the EMODnet Bathymetry Consortium (2020). 3 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 Table 1 Cruise data (station, location, year, date, latitude, and longitude) for each sampling site. Mean and standard deviation (SD) of standard length (SL, cm), total weight (TW, g), gonadosomatic index (GSI), hepatosomatic index (HSI), Le Cren relative condition index (Kn), stomach fullness (FULL) and body perimeter in the anterior (P1), middle (P2) and posterior (P3) parts of the body. Mean and standard deviation (SD) of enzymatic activities of Acetylcholinesterase (AChE), Lactate dehydrogenase (LDH), Cytrate synthase (CS), Carboxyl esterase (CbE), Catalase (CAT), Glutatione-S-transferase (GST) and Ethoxyresorufin-O-deethylase (EROD). Significant differences between years (Barcelona 2007–2019) are represented by superscript letters (a and b), while the absence of superscript letter means no significant differences. Differences among localities (Barcelona–Blanes–Tarragona in 2019), are expressed by superscript numbers (1, 2 and 3), while the same superscript number or their absence means no significant differences. Station BCN1 BCN2 BLA TAR Location Barcelona Barcelona Blanes Tarragona Year 2007 2019 2019 2019 Date 20/07/2007 18/07/2019 22/07/2019 25/07/2019 Lat. 41° 8′18.96”N 41°14′44.88” N 41°45′48.12”N 40°55′21.60”N Long. 1°45′41.54″E 2° 9′20.10″E 3° 3′47.40″E 1°16′43.20″E Mean (SD) Mean (SD) Mean (SD) Mean (SD) a b1 2 3 SL 12.04 (0.37) 12.46 (0.71) 11.39 (0.52) 11.09 (0.46) TW 16.97 a (1.58) 18.53 b 1 (3.24) 13.40 2 (1.67) 14.05 2 (1.80) GSI 4.53 (1.60) 4.56 1 (1.22) 4.57 1 (1.24) 3.72 2 (1.11) HSI 1.75 a (0.81) 2.25 b 1 (0.85) 1.53 2 (0.48) 2.25 1 (0.62) Kn 1.04 a (0.06) 0.97 b 1 (0.05) 0.97 1 (0.05) 1.01 2 (0.05) FULL 1.48 a (1.03) 0.48 b (0.32) 0.40 (0.22) 0.58 (0.50) P1 – – 52.55 1 (4.62) 45.4 2 (5.08) 43.97 2 (6.44) P2 – – 47.07 1 (3.59) 41.75 2 (2.39) 41.35 2 (4.02) P3 – – 33.83 1 (2.69) 31.16 2 (1.76) 31.58 2 (2.29) AChE i – – 21.3 1 (4.77) 33.18 2 (10.67) 28.37 2 (8.36) LDH i – – 5915.18 1 (504.87) 5917.09 1 (440.40) 6840.94 2 (409.59) CS i – – 77.09 1 2 (5.63) 81.43 2 (6.99) 75.74 1 (8.53) CbE i – – 46.68 1 (13.81) 50.11 1 (13.75) 76.37 2 (35.40) CAT j – – 311.5 1 (124.54) 456.82 2 (134.69) 451.7 2 (131.13) GST i – – 163.81 1 (59.35) 236.95 2 (65.9) 229.8 2 (66.20) EROD k – – 0.79 1 (0.33) 0.86 1 (0.18) 1.24 2 (0.38) i nmol·min−1·mg protein−1. j μmol·min−1·mg protein−1. k pmol·min−1·mg protein−1. (Le Cren, 1951). Feeding intensity was measured by the stomach fullness adipocytes from the rest of the samples were classified for subsequent index (FULL = (CW/EW) × 100), which was calculated using the total analyses (Fig. 2). stomach content weight (CW) recorded after screening for potential AIs. For the enzymatic biomarker analyses, a muscle portion of around 0.3 g To complement fish condition assessment, height, width, and perimeter wet weight was used for acetylcholinesterase (AChE), lactate dehydrogenase were measured; this was performed in three different parts of the body — (LDH), and citrate synthase (CS) determinations. A portion of liver of around beginning of the dorsal fin (P1), beginning of the anal fin (P2), and 0.07 g wet weight was used to analyse Glutathione-S-transferase (GST), midpoint between the end of the anal fin and the caudal peduncle (P3). catalase (CAT), carboxylesterase (CbE), and ethoxyresorufin-O-deethylase In addition, a semi-quantitative analysis of the perivisceral fat was con- (EROD). Assays were performed following the procedures described in ducted from histological sections. For this purpose, a random subsample past studies in the area (Antó et al., 2009; Koenig et al., 2013; Solé and of 10 fish per location (2019 specimens) were used to determine a semi- Sanchez-Hernandez, 2018). quantitative indicator of size of adipocytes by screening the perivisceral A portion of gonad, liver, spleen, kidney, stomach, caeca, intestine, and fat tissue. The diameter of a minimum of 100 adipocytes was measured gills were embedded in paraffin, sectioned at 5 μm and stained with Haema- using images obtained at 20× magnification by a camera attached to the toxylin and Eosin for histopathological assessment. All histological sections microscope (Leica microscope model: DM 5000 dB) and image-processing were completely screened under the microscope. When alterations were software (ProgRes® C3). According to adipocyte's size, four categories detected, a morphological evaluation of each alteration was performed. were obtained (0 = no fat tissue; 1 = small size adipocyte; 2 = medium The prevalence of the lesions (percentage of fish affected by a specific size adipocyte and 3 = large size adipocyte). Using these criteria, all alteration) was calculated. When required, some of these sections were Fig. 2. Optical microscope images giving examples of fat tissue in European anchovy in which adipocytes (indicated by red arrows) of different size classes: a) small size, b) medium size, and c) large size, can be seen. Scale bars at the bottom left of each picture represent 0.1 mm. 4 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 additionally stained with Periodic Acid Schiff (PAS) and Sudan stains. rinsed with filtered deionized water twice before use. Uncovered Petri The spleen was also chosen for a quantitative evaluation of the density dishes containing filtered deionized water were placed inside and outside of melanomacrophage centres (MMCs), due to the ease of dissection of the isolation device to assess airborne contamination. Only fibre-shaped the whole organ and the possibility to obtain complete radial sections items were found in both controls, and the number and morphological char- (Fournie et al., 2001; Manera et al., 2000). For this purpose, three fields acteristics of the airborne fibres were recorded before and after each sample of view (0.23 mm2/screen) were randomly selected from each section of examination. Contamination found in the inside controls (average values of the spleen at 200× and examined microscopically. Area and number of 1.16 fibres per digestive sample screened) was 15.6 times lower than MMCs (mean area = MA.MMC and number = nMMC, respectively) of contamination in outside controls, thus indicating the efficiency of the iso- each field were measured using a MicroComp Integrated Image Analysis lation device in reducing potential contamination. Fibres found in the System, and a size discriminator was used to eliminate objects smaller inside controls were clean and always appeared on the surface of the than 100 μm2. water (indicating that they were deposited from the air). Therefore, fibres External surfaces and gills were checked macroscopically for ectopara- from digestive tracts were only counted if they were clearly embedded sites and the rest of the organs, including stomach, caeca, intestine, gonads, in the digestive content and/or with detritus attached; these were clearly spleen, brain, body cavity, and muscle were carefully inspected for endopar- differentiated from those floating on the surface, which were excluded asites under a stereomicroscope. The location of parasites within the fish was thereafter. Therefore, no correction factor was applied to the final values annotated. Digeneans and cestodes were stained with iron acetocarmine of the fibres reported. All manipulations were carried out under the stereo- and permanently mounted in Canada balsam. Nematodes were temporally microscope. Finally, the content of stomach, caeca and intestine was care- cleared and mounted in glycerine before identification. Parasites were iden- fully separated and stored for a subsequent analysis. tified under an optical microscope to the lowest possible taxonomic level. In The prevalence of fish with anthropogenic items (% AIs; percentage of addition, six digeneans, two nematodes and two monogeneans of thawed fish with AIs within their digestive tract with respect to the total number European anchovy were subjected to molecular analyses. DNA from all sam- of fish analysed) was calculated. AIs detected were collected and mounted ples was extracted with Qiagen TM (Valencia, California) DNeasys Blood and between glass slides in filtrated deionized water and observed under the Tissue Kit. Partial nuclear large subunit ribosomal DNA (28S rDNA) and par- microscope. For the anthropogenic fibres, length, mean cross section tial fragments of mitochondrial cytochrome c oxidase 1 (cox1) gene were (based on three random measures), and area were obtained. For anthropo- amplified (50 μl total volume) using ExcelTaqTM SMOBIO_ PCR Master genic particles, the maximum length and area were recorded. Images were Mix (Taiwan) containing: 5× concentrated master mix, that is, a mixture obtained using a camera attached to the microscope and were subsequently of recombinant Taq DNA polymerase, reaction buffer, MgCl2 (2 mM), measured using image-processing software (ProgRes® C3). dNTPs (0.2 mM), and enzyme stabilizer, 0.25 μM of each PCR primer and Total number of AIs were counted for each individual (nAI) and their 2 μl of extracted gDNA. Primer pairs and amplification conditions were locations within the digestive tract (stomach, caeca, and intestines) were used as follows: partial fragments of the cox1 gene were amplified using annotated. Mean abundance (nAI = number of AIs/total number of indi- the primers LCO1490 (forward, 5′-GGTCAACAAATCATAAAGATATTGG- viduals), and mean intensity (mean intensity = number of AIs/individuals 3′) and HCO2198 (reverse, 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) with ingested AIs) were also calculated. AIs were divided into categories de- (Folmer et al., 1994) under the following thermocycling conditions: initial pending on their form (anthropogenic particles or anthropogenic fibres). denaturation at 95 °C for 15 min followed by 35 cycles (denaturation Total length of AIs (TLAI) was calculated by adding the length of each AIs for 5 min at 80 °C, followed by 1 min 30 s at 92 °C, annealing for 1 min at observed inside the digestive tract of each individual. The percentage of 42 °C, and extension for 2 min at 72 °C), and a final extension step at 72 °C stomach occupation by AIs was calculated (proportion of stomach occupa- for 10 min. Partial fragments of the 28S rDNA gene were amplified using tion = (sum of area of AIs in stomach/stomach area) x (100)). The fibres the primers LSU5F (forward, 5′-TAG GTC GAC CCG CTG AAY TTA AGC A- found in the digestive tract of fish were carefully observed under the micro- 3′) and ECD2R (reverse, 5′-CTT GGT CCG TGT TTC AAG ACG GG-3′) scope, characterized and classified visually into distinct typologies (cellu- (Littlewood et al., 2000) under the following thermocycling conditions: ini- losic and plastic), according to their morphological features, following the tial denaturation at 95 °C for 2 min followed by 30 cycles (denaturation for criteria of Rodríguez-Romeu et al. (2020). Fourier-Transformed Infrared 50 s at 95 °C, annealing for 50 s at 52 °C, and extension for 50 s at 72 °C), Spectrometry (FTIR) was performed on a randomly selected subsample of and a final extension step at 72 °C for 7 min. In every PCR run, a negative 72 anthropogenic items (35 anthropogenic fibres and 37 anthropogenic and a positive control were used to detect any potential contamination, particles) corresponding to a total of 56 % of AIs found. and to have a reliable sample to compare with, respectively. Spectra of fragments and films were recorded using a Tensor 27 FTIR PCR products were visualised on RedGel-stained 1 % agarose gels, puri- spectrometer (Bruker Optik GmbH, Germany) equipped with a diamond at- fied, and sequenced by Macrogen (Amsterdam, Netherlands). Sequencing tenuated total reflectance (ATR) unit (16 scans cm−1, 800–3600 cm−1). primers were the same as for the PCR. Valid sequences were aligned using Fibre spectra were recorded at Scientific and Technological Centres BioEdit 7.0.1 (Hall, 1999), variable sites were checked visually for accuracy, (CCitUB, University of Barcelona) using a micro-FTIR Thermo Scientific and final sequences were submitted to GenBank (Hysterothylacium aduncum Nicolet iN10 MX, equipped with an Imaging Detector (4 scans cm−1, accession numbers ON514619 and ON514624; Lecithaster accession num- 800–4000 cm−1). Resulting spectra were treated (baseline corrections, ber ON524172). Parasite prevalence (P%) and mean abundance (MA) peak normalization, and selection of characteristic band applied) with were calculated following previous studies (Bush et al., 1997), and richness Spectragryph 1.2.11 and compared with reference spectra. Spectra from and Brillouin diversity indices for each individual were also calculated. 11 common reference polymers were included in a custom library (cellu- lose, acrylic, nylon/polyamide, high-density and low-density polyethylene, 2.4. Anthropogenic item (AI) extraction and characterisation polyethylene terephthalate, polypropylene, polyurethane, and polysty- rene). Similarity correlation indices between sample and reference spec- Stomachs were opened longitudinally — with a sagittal cut — and each trum were calculated for characteristic bands (from 1800 to 670 cm−1 half was carefully rinsed, before being stored separately. The area of the wavelengths) and values >70 % similarity were selected. Results were fur- half-sectioned stomach of each fish was calculated. The content of the stom- ther checked by visual correlation of peaks and by using the KnowItAll® ach, caeca, and intestine was carefully screened under a stereoscopic binoc- (Bio-Rad, USA) software, to compare spectra with a broader database. ular at 5× to 40× magnification. To prevent airborne contamination, the stereomicroscope and working area was isolated from the surroundings 2.5. Data analysis using an isolation device adapted from the one proposed by Torre et al. (2016), and the interior was carefully washed before use to minimize the In order to characterize the adipocyte diameter, measured adipocytes of presence of airborne fibres. The laboratory dissection material was also the fat tissue were classified into four clusters by partitioning around 5 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 medoids (PAM). The k-medoids algorithm was applied in order to classify significantly lower values were found in Blanes for the HSI (Barcelona– the dataset into different groups or clusters from a matrix of dissimilarity. Blanes, t = −8.817, p < 0.01; Tarragona-Blanes, t = 8.221, p < 0.01) and Each cluster is represented by one object, which is located in the centre of in Tarragona for GSI index (Barcelona-Tarragona, t = −2.823, p < 0.01; the cluster. The k clusters are established by assigning each object of the Blanes–Tarragona, t = −2.458, p < 0.05). No significant differences in stom- dataset to the nearest representative object — thus objects that show a ach fullness among localities were found. high level of similarity are grouped together, while objects that are dissim- Histologically, differences in the size of the adipocytes in the perivisceral ilar to each other belong to different clusters (Kaufman and Rousseeuw, fat tissue were observed (Fig. 2). Cluster partitioning around medoids of 1990) — using the PAMK function implemented in the package fpc 2.2–3 adipocyte diameter gave three clusters. Small size: Ø <1.52 μm; medium in R Studio (version: 4.0.3). All variables were tested for normality and size Ø >12.52 to <23.18 μm; and large size Ø >23.18 μm. Comparison of equality of variance using the Shapiro-Wilk and Levenne's test respectively. adipocyte clusters showed significant differences among localities, and To assess differences in explanatory and response variables among lo- fish from Barcelona had more small size adipocytes (cluster 1) compared calities (Barcelona, Blanes, and Tarragona) and between years (2007 and to Blanes and Tarragona (z = −2.772, p < 0.05). 2019) when possible, general linear models (GLM, gaussian models) or gen- When comparing fish from 2007 and 2019 (Table 1), significantly higher eralized linear models (GZM, gamma models) were applied for biological values of standard length (t = −2.907, p < 0.05), TW (t = −2.719, p < indices (SL, TW, Kn, GSI, HSI and FULL), for enzymatic biomarkers, body 0.05) and HSI (t = −2.021, p < 0.05) were observed in anchovies sampled perimeters, diversity parasite descriptors (richness and Brillouin diversity in 2019 when compared with fish obtained in 2007. Meanwhile significantly index), and percentage of AIs occupation. Differences among localities higher values of Kn condition index (t = −9.069, p < 0.01) and stomach full- and years for the prevalence of AIs, and between localities for the adipocyte ness (t = 5.450, p < 0.01) were detected in 2007 when compared to 2019. diameter and prevalence of histological alterations and parasites were No significant differences in GSI were found between years for Barcelona. tested using a GZM (binomial model, link logit). For mean parasite abun- dance (total parasites, endoparasites, and only parasites from digestive 3.1.2. Enzymatic biomarkers tract) a GZM based on negative binomial models (link log) was applied. Fi- When compared to other locations, fish obtained in Barcelona showed nally, mean intensity and abundances of AIs were tested for differences significantly lower values for AChE (Barcelona–Blanes, t = −3.568, p < among localities and year using GZMs (poisson models, link log). When 0.005; Barcelona–Tarragona, t = −2.181, p < 0.05); CAT (Barcelona–Blanes, necessary, SL was considered as covariate, including stomach fullness for t = −2.842, p < 0.05; Barcelona–Tarragona, t = −2.534, p < 0.05) and GST AI approximations. (Barcelona–Blanes, t = −2.92, p < 0.05; Barcelona–Tarragona, t = −2.468, Correlations between condition indices, biomarkers, parasites, and AIs p < 0.05) (Table 1). Instead, Tarragona showed significantly higher values for variables were tested in order to detect any possible relationships among LDH (Barcelona–Tarragona t = −4.430 p < 0.001; Blanes–Tarragona t = them. This was done using Pearson's correlation tests and non-parametric −5.231, p < 0.001); CbE (Barcelona–Tarragona t = −3.160, p < 0.05; Spearman's correlation tests (when normality was not satisfied), and plot- Blanes–Tarragona t = 2.070, p < 0.05) and EROD (Barcelona–Tarragona ted with the corrplot R package (Wei and Simko, 2017). Parasites have t = −3.081, p < 0.005; Blanes–Tarragona t = −3.081, p < 0.005). Blanes been suggested as a possible factor affecting the retention of microplastics showed significantly higher values for CS when compared to Tarragona (Hernandez-Milian et al., 2019; Pennino et al., 2020). Thus, the total num- (Blanes–Tarragona, t = 2.070, p < 0.05). None of these differences were ber of parasites, and those found in the digestive tract only, were consid- related to SL. ered as explanatory variables in subsequent analyses. These variables were included in models to test the association between parasite load and 3.1.3. Histological observation and alterations levels of ingestion of AIs. Melanomacrophage centers (MMCs), when present, were mostly lo- Multiple factor analysis (MFA) was used to assess the possible effects of cated in the spleen, but also in a smaller number and size in the kidneys AIs on fish health condition indices (including biomarkers). This multivar- and liver. Total prevalence of fish with splenic MMCs was 68 %; this iate data analysis enables the evaluation and identification of individuals as was significantly higher in Barcelona when compared to Tarragona (z = characterized by sets of variables (both quantitative and qualitative) which −2.061, p < 0.05). The number of MMCs (nMMC) observed per fish ranged are structured into groups on this basis. Thus, the differences within groups from 0 to 7, with a mean value of 1.23 MMC/ind. (SD = 1.58). The area of are minimized, whilst differences between groups are maximized. Distinct MMCs ranged from 246.40 μm2 to 6046 μm2, and mean area (MA.MMC) MFAs were conducted using the following as explanatory variables: nAI, was 2253.92 μm2/ind. (SD: 1442.74). Comparing nMMC among localities, TLAI, abundance of fibres (plastic/non-plastic), abundance of particles, fish sampled in Barcelona showed significantly higher values than Tarra- and abundance of plastic and non-plastic anthropogenic items. Data gona (t = 2.417, p < 0.05; Table 2). No significant differences in MA. analysis was performed using R Studio software (version: 4.0.3). For each MMC were found among localities. statistical hypothesis test, significance was set at 0.05. No relevant histopathological alterations were found in the analysed samples, although some minor histological alterations were found in the 3. Results livers of some fish. Vacuole-like structures were observed in the cytoplasm of hepatocytes in 65 % of the fish (Fig. 3a). Fish from Barcelona showed a 3.1. Health status higher prevalence of these structures when compared to Tarragona (t = −2.212, p < 0.05 (Table 2). These structures were not reactive to PAS, 3.1.1. Condition indices nor to Sudan staining. Other liver alterations consisted of the occasional Body condition indices of fish are shown in Table 1. For 2019, signifi- presence of small patches of inflammatory foci, mainly composed of cantly higher values were found in Barcelona with respect to other locations lymphocytes and sometimes a few macrophages (Fig. 3b). These foci in standard length (Barcelona–Blanes t = −7.165 p < 0.01; Barcelona– were found in 15 % of individuals, and their prevalence was higher in Tarragona t = −10.150, p < 0.01) and total weight (Barcelona–Blanes t = Barcelona than in Blanes, though these differences were not significant −8.817, p < 0.01; Barcelona–Tarragona t = 8.221, p < 0.01); therefore (Table 2). No inflammatory foci were found in Tarragona (Table 2). In ad- these results call for further analyses. In addition, the Barcelona fish demon- dition, gill epitheliocystis were observed in the gills of 15 % of fish sampled strate significantly higher values in body circumferences P1 (Barcelona– from Tarragona, though always in a very low intensity. No alterations or Blanes, t = 6.905, p < 0.01; Barcelona–Tarragona, t = 7.140, p < 0.01); damage (epithelial erosion, haemorrhages, or inflammation) potentially as- P2 (Barcelona–Blanes, t = 7.090 p < 0.01; Barcelona–Tarragona, t = sociated to mechanical abrasion by AIs were detected in the intestinal or 7.918, p < 0.01); P3 (Barcelona–Blanes, t = 4.673, p < 0.01; Barcelona– gastric structures. Other histological observations were explained by the Tarragona, t = 4.338, p < 0.01). Fish sampled in Tarragona showed signifi- parasite infestation — typically by nematodes and digenean larvae — de- cantly higher values of the Kn condition index (t = 2.667, p < 0.01). Whereas tected in the parasitological study. These lesions were mainly observed as 6 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 Table 2 Parasite descriptors, and histopathological alterations found in European anchovy (Engraulis encrasicolus). Prevalence (P%) of parasites, epitheliocystis, inflammatory foci and vacuoles-like structures; mean abundance (MA) and standard deviation (SD) of parasites, and melanomacrophage centres (MMCs). Mean and standard deviation (SD) of the area (μm2) of melanomacrophage centers (MA. MMC in) and number of melanomacrophage centers (nMMC). Different superscript numbers (1 and 2) show significant differences (p < 0.05) among localities, while the same superscript number or their absence means no significant differences. Locality Barcelona Blanes Tarragona Year 2019 2019 2019 PARASITES P% MA (SD) P% MA (SD) P% MA (SD) METAZOA CNIDARIA Myxozoa 25 – – 30 – – 25 – – NEMATODA 70 1.701 (1.62) 63 1.072 (1.22) 63 0.972 (0.96) PLATYHELMINTHES Trematoda 100 32.031 (23.94) 100 27.571 (21.67) 100 15.302 (12.89) Digenea 97 30.831 (24.14) 100 27.2312 (21.77) 100 14.572 (12.71) Monogenea 501 0.871 (1.04) 132 0.132 (0.35) 401 0.701 (1.06) Cestoda 271 0.331 (0.61) 101 0.2012 (0.66) 32 0.032 (0.18) PROTISTA APICOMPLEXA 40 – – 15 – 20 – – CILIOPHORA 10 – – 5 – – – – Parasites Species Richness 4.201 (1.16) 3.472 (1.2) 3.302 (0.92) MSR/MD (SD) MSR/MD (SD) MSR/MD (SD) Brillouin Diversity Index (H′) 0.781 (0.27) 0.711 (0.3) 0.612 (0.19) HISTOPATHOLOGY P% P% P% Epitheliocystis – – 15 Inflammatory foci 25 20 – Vacuoles-like structures 801 7012 452 Melanomacrophage centers (MMCs) 801 7612 472 nMMC (SD) MA.MMC (SD) nMMC (SD) MA.MMC (SD) nMMC (SD) MA.MMC (SD) 1.571 (1.71) 1865.04 (1663.53) 0.921 (0.80) 1496.04 (1284.66) 0.392 (0.60) 1206.07 (1761.04) small granulomas or cysts in stomach and intestinal walls, pancreatic tissue, and Blanes when compared to values from Tarragona (t = 2.897, p < and the liver, and contained whole or degraded forms of the parasites. 0.005) (Table 2). Parasite Richness showed significantly higher values in Barcelona than other locations (Barcelona–Tarragona t = 3.345, p < 3.1.4. Parasitological load 0.005; Barcelona–Blanes t = 2.960, p < 0.005) (Table 2). Nematodes Every fish individual assessed (n = 90) showed at least one parasite. were significantly more abundant in Barcelona (z = −3.007, p < 0.005) Globally, a total of 1360 parasites belonging to at least ten different as compared to the other two locations, whereas digeneans and cestodes taxa were identified: four digeneans (Aphanurus virgula, Lecithaster spp., where higher only when compared to Tarragona (z = −2.109, p < 0.05 unidentified metacercariae and an unidentified juvenile digenea), three and z = −2.062, p < 0.05, respectively). Monogeneans were significantly nematodes (Hysterothylacium aduncum, H. fabri and unidentified encysted less abundant in fish from Blanes (Blanes–Barcelona, z = 2.380, p < 0.05; nematodes), two monogeneans (one unidentified Monopystochotylea Blanes–Tarragona z-value: 2.973, p < 0.01). Regarding the prevalence and the Poliopystochotylea Pseudacanthocotyloides heterocotyle) and of parasite taxa, cestodes showed lower values in Tarragona compared to Tetraphyllidea fam. Gen. sp. larvae (Table 2). None of the unidentified nem- Barcelona, (Barcelona–Tarragona z = −2.146, p < 0.05), while Blanes atodes matched the morphological characteristics of an Anisakidae species. showed significantly lower values for monogeneans compared to other Regarding differences across locations, Brillouin Diversity index locations ((Barcelona–Blanes, z = −2.882, p < 0.005; Tarragona–Blanes, showed significantly higher values of parasites in fish sampled in Barcelona z = 2.243 p < 0.05) (Table 2). Fig. 3. Optical microscope images of the main histological alterations found in livers of European anchovy. Showing a) vacuole-like structures (indicated by red arrows) in the cytoplasm of hepatocytes, and b) inflammatory foci formed by patches of immune cells (surrounded by dashed white circles). Scale bars at the bottom left of each picture represent 0.1 mm. 7 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 Table 3 Prevalence (%) of fish ingesting anthropogenic items. Mean and standard deviation (SD) of mean intensity (number of items per fish ingesting anthropogenic items), mean abundance of anthropogenic Items (nAI), total plastic items, number of particles (n Particles), number of fibres (n Fibres), number of plastic fibres (n Plastic Fibres), number of cellulosic fibres (n Cellulose fibres, total length of anthropogenic items (TLAI) and percentage of stomach occupation by anthropogenic items (proportion of stomach occupation). Significant differences between years (Barcelona 2007–2019) are represented by superscript letters (a and b), while the absence of superscript letter means no significant differences. Differences among localities (Barcelona–Blanes–Tarragona in 2019), are expressed by superscript numbers (1 and 2), while the same superscript number or their absence means no significant differences. Locality Barcelona Blanes Tarragona Year 2007 2019 2019 2019 ANTHROPOGENIC ITEMS Prevalence (%) 40a 70b1 5312 332 Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean Intensity 1.58 (0.67) 2.71 (1.76) 2.00 (1.41) 2.00 (1.33) nAI 0.63a (0.89) 1.9b1 (1.94) 1.0712 (1.44) 0.672 (1.21) Total plastic 0.43a (0.63) 1.23b1 (1.68) 0.372 (0.72) 0.402 (0.81) n Particles 0.23a (0.50) 1.17b1 (1.68) 0.332 (0.66) 0.232 (0.68) n Fibres 0.40 (0.72) 0.73 (1.17) 0.73 (1.28) 0.43 (0.73) n Plastic fibres 0.20 (0.48) 0.07 (0.25) 0.03 (0.18) 0.17 (0.46) n Cellulose fibres 0.20a (0.48) 0.67b (1.09) 0.70 (1.24) 0.27 (0.52) TLAI 2.16 (5.73) 4.201 (3.07) 1.762 (2.53) 2.372 (3.89) Proportion (%) of stomach occupation 0.31a (0.88) 1.05b1 (1.66) 0.182 (0.53) 0.432 (1.18) 3.2. Ingestion of anthropogenic items by European anchovy and polymer found in the intestine. Only fibres were found in the pyloric caeca. The pro- characterisation portion of the stomach occupied by AIs ranged from 0.004 to 4.95 % (Table 3). Half of the anchovies analysed (59 of 120; total prevalence 49.17 %) The size of 95 % of the items found ranged between 0.25 mm to 4.82 mm, contained AIs in their digestive tract (Table 3), with a total mean intensity with a mean size of 1.99 mm (SD = 1.21); this fits within the definition of of ingested AIs of 2.17 (SD = 1.46). A total of 128 AIs were found in the di- microplastic (Frias and Nash, 2019). Moreover, six items larger than 5 mm gestive tract of analysed fish, 69 were fibres (53.4 %), including cellulosic were also found, ranging from 5.23 mm to 6.88 mm, with a mean size of and plastic fibres, and 59 (46.6 %) were particles, englobing films, and 6.31 mm (SD = 0.63) and one exceptional item of 13.82 mm. Due to their fragments (Fig. 4a). Most of the items (102) were found in the stomach, plastic basis, the items bigger than 5 mm were also considered in subsequent while nine were found in the pyloric caeca, and 17 in the intestine. Particles analyses. The TLAI was 2.37 mm/ind. (SD = 3.89). From the 59 particles were located mainly in the stomach (55) and only four small particles were (46.1 % of total AIs), 40 items (31.3 %) were fragments and 19 (14.8 %) Fig. 4. Values of ingestion of anthropogenic items (AIs) by European anchovy showing A) Prevalence as a percentage of the AIs ingested by typology (fibres and particles), composition of fibres (plastic or cellulosic) in the sampling stations. B) mean intensity (number of AIs/number of fish ingesting AIs). Data are shown for Barcelona in 2007 (BCN1) and 2019 (BCN2), and in Blanes and Tarragona in 2019 (BLA and TAR, respectively). Different letters and numbers show significant differences between years (Barcelona 2007–2019) and localities (Barcelona–Blanes–Tarragona in 2019), respectively (p < 0.05). 8 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 were films. Moreover, 69 items (53.9 %) were fibres, 14 of these (10.9 %) polyethylene was the most abundant polymer in both years. However, with were plastic-like fibres, while 55 (42.97 %) were cellulose-like fibres. fibres, polyethylene terephthalate was the most abundant polymer in 2007, All fibres visually classified as plastic were correctly identified as syn- whereas cellulose fibres were more abundant in 2019 (z = 2.587, p < 0.005). thetic polymers by micro-FTIR, while all those classified as cellulosic were identified as cellulose — which corresponded to 36.6 % of all polymers 3.4. Relationship between health status and potential stressors identified. Synthetic polymer identification showed polyethylene (38 %) as the most abundant polymer, followed by polyethylene terephthalate MFA for the different locations explained 30.32 % of variability with (11.3 %), polypropylene (8.5 %), polyamide (2.8 %), and acrylic (1.4 %). the first two axes (Fig. 5). The quantitative variables that explained the Polyethylene, polypropylene, and polyamide were identified only in parti- variability in the first dimension concerned mainly anthropogenic items cles, while polyethylene terephthalate and acrylic were in fibres. (AIs abundance, TLAI, and percentage of stomach occupation by AIs). When comparing the three sampled stations in 2019, Barcelona showed Regarding the second dimension, abundance of parasites of the diges- the higher prevalence (z = −2.77, p < 0.005) of fish ingesting AIs, and AIs tive tract — which were slightly associated with fish size — explained were also more abundant (z = −2.798, p < 0.05); this was followed by most of the variability, followed by enzymes (Fig. 5a). Blanes and Tarragona, although significant differences were only found be- A slight separation of data according to the sampling location was ob- tween Barcelona and Tarragona (Fig. 1, Table 3). Barcelona was also the lo- served (Fig. 5b). In general, individuals of each locality were similar to cation with a significantly higher abundance of particles (Blanes–Barcelona, each other, but some individuals were more differentiated in Barcelona. z = −2.657, p < 0.05; Tarragona–Barcelona, z = −2.832, p = 0.005). Dispersion along the first dimension means fish from Barcelona had higher Total length of AIs was higher in Barcelona (TLAI, Barcelona–Blanes, t = values of descriptors of anthropogenic items (AIs abundance, AIs length 2.087, p < 0.05; Barcelona–Tarragona, t = 2.515, p < 0.05), as was the and proportion of stomach occupation by AIs); dispersion along the proportion of stomach occupation by AIs (Barcelona–Blanes t = 2.818, second-dimension indicates bigger fish and higher parasite abundances. p < 0.005; Tarragona–Barcelona, t = −2.551, p < 0.05). None of these dif- The correlation matrix showed that fish size was not correlated with para- ferences were related to fish size or to stomach fullness. Mean intensity was site descriptors: parasite Richness (rho = 0.44, p < 0.01) and Brillouin also higher in Barcelona compared to the other two locations, but with no Diversity Index (rho = 0.30, p < 0.01), nor with the abundance of the significant trend (p > 0.05) (Fig. 4b, Table 3). different taxonomic groups of parasites identified in the digestive tract, such as digeneans (rho = 0.32, p < 0.01); cestodes (rho = 0.27, p < 3.3. Differences in anthropogenic items ingestion over a 12-year gap 0.05); nematodes (rho = 0.34, p < 0.01) or with the total abundance of par- asites in the digestive tract (rho = 0.35, p < 0.01). When comparing the AIs ingestion in Barcelona between two distinct No significant relationships were found among parasite descriptors and sampling occasions separated by 12 years, a significant increase was de- enzymatic biomarkers or histological alterations (Fig. S1). Likewise, no sig- tected in AIs prevalence (2007: 40 %; 2019: 70 %) (z = 2.296, p < 0.05) nificant relationships were found between AIs and response variables, in- (Fig. 1). Mean intensity and TLAI also showed higher values in 2019 than cluding health status descriptors, condition indices, enzymatic biomarkers, in 2007, but this difference was not significant (Fig. 4b, Table 3). adipocyte diameters, total parasite abundance, abundance of parasites in The abundance of AIs, and the proportion of stomach occupation by AIs the digestive tract, or histological alterations. Moreover, when parasites were significantly higher in 2019 as compared to 2007 (nAI, z = 2.473, were considered as variables in models testing for their potential impact p ≤0.05; z = 2.337, p < 0.05; respectively) (Table 3). Fibre abundances on fish health, no significant relationship with the response variables was were not significantly different between years, while particles were more found. abundant in 2019 (z = 2.515, p ≤0.05). Likewise, the percentage of parti- cles from the total AIs was higher in 2019 (61.5 % respect to 37 % in 3.5. Temporal comparison 2007). If only particles were considered, fragments were the most abundant shape in both years. None of these differences were related to SL or stomach When considering data obtained of fish sampled in Barcelona in 2007 fullness. Regarding polymer identification, considering only particles, and 2019, the MFA performed explained 60.92 % of the variability (Fig. 6a Fig. 5. Multiple factor analysis (MFA) among fish from locations in 2019. (A) MFA among body condition indices (standard length (SL); total weight (TW); Le Cren relative condition index (Kn); stomach fullness (Fullness); hepatosomatic index (HSI); and gonadosomatic index (GSI)), abundance of anthropogenic items (AI abundance, (total anthropogenic items (AI tot)), length of anthropogenic items (AI length), percentage of stomach occupation by anthropogenic items (% AI stm), enzymes (catalase (CAT), glutathione-S-transferase (GST), lactate dehydrogenase (LDH)) and parasites (cestodes in the digestive tract (Ces. dig), digeneans in the digestive tract (Dig. dig), nematodes in the digestive tract (Nem. dig) and total parasites in the digestive tract (Tot. dig)). (B) Factor map of the MFA, individuals are represented by dots and locations by colours. 9 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 Fig. 6. Multiple factor analysis among fish from Barcelona between a 12-year interval (2007 vs 2019). (A) Multiple factor analysis between body condition indices (standard length (SL); total weight (TW); Le Cren relative condition index (Kn); stomach fullness (Fullness); hepatosomatic index (HSI); gonadosomatic index (GSI) and stomach fullness (Fullness)), abundance of anthropogenic items (AI abundance, total anthropogenic items (AI tot), anthropogenic particles (AI), anthropogenic fibres (AF), cellulosic anthropogenic fibres (AFC), plastic anthropogenic fibres (AFP), total plastic items (PL. tot)), length of anthropogenic items (AI length) and percentage of stomach occupation by anthropogenic items (proportion (%) AI stm). (B) Factor map of the MFA, individuals are represented by dots and years by colours. and b). AIs length and the proportion of stomach occupation by AIs were the several authors have highlighted how small pelagic populations are able variables that explained most of the variability in the first dimension, while to spawn in a preferred geographical area and within a range of environ- SL and TW were the variables with a higher contribution in the second di- mental conditions (Bakun, 1997; Motos et al., 1996). Among these vari- mension (Fig. 6a). Between years (Fig. 6b), all individuals were similar ables, depth appears to play a key role, together with oceanic features except some individuals in 2019 that were dispersed along the first dimen- and food availability (Giannoulaki et al., 2013; Motos et al., 1996; sion, meaning higher values of anthropogenic items (TLAI and proportion Somarakis et al., 2006). of stomach occupation). Previous studies revealed that sardines (Sardina pilchardus) with more anthropogenic particles ingested had the lowest body condition in the 4. Discussion Mediterranean Sea (Compa et al., 2018). We find no clear evidence of that in our study, in keeping with other work on anchovies in the Balearic 4.1. Current body condition and health status of European anchovy Sea or the Gulf of Lion (Compa et al., 2018; Lefebvre et al., 2019); though in the Ligurian Sea, a positive correlation between body condition and The results of this study indicate that the European anchovy populations number of ingested fragments was found (Capone et al., 2020). Given the from the NW Mediterranean Sea are not affected by relevant pathologies. contradictory nature of these results, the potential negative effects of AIs Moreover, the absence of zoonotic parasites in the analysed fish strongly on body condition of European anchovy in the wild remain unclear. suggests a low prevalence of these kind of parasites in the studied area, Results from the histological analysis of fat tissue indicate differences when compared with fish stocks in other geographical locations. However, in adipocyte size clusters among localities, but these differences were not substantial declines in the stock size, mean body size and/or condition have related to condition indices nor to enzymatic biomarkers. The size of adipo- been observed in the NW Mediterranean Sea (Albo-Puigserver et al., 2019; cytes has been widely used for analysis of fat indices in humans or mam- Brosset et al., 2017, Brosset et al., 2016, Brosset et al., 2015; Ferrer-Maza mals (Salans et al., 1973) and changes in size of adipocytes (hypertrophy) et al., 2016; Saraux et al., 2019; Van Beveren et al., 2014) resulting in pro- of the mesenteric fat tissue have been described in relation to fat con- found changes in the structure of the stocks and a major decline in landings tent and composition of diets in farmed fish (Cruz-Garcia et al., 2011; and fishing activity (Brosset et al., 2017; Coll and Bellido, 2020; Saraux Landgraf et al., 2017; Oka et al., 2010). It is known that adipose tissue un- et al., 2019). Studies using condition indices, lipid content, and the fatty- dergoes dynamic remodelling in short periods of time, in response to acid profile of the muscle of European anchovy have revealed that the changes in the nutritional status in farmed fish (Navarro and Gutiérrez, health status of this species is impaired due to malnourishment (Biton- 1995). Visceral fat in particular may respond to the mobilization of lipid Porsmoguer et al., 2020). This situation seems to be linked to changes in storage during periods of physiologically high-energy demand that necessi- plankton abundance and the community composition over recent decades tate catabolism of body reserves — such as during reproduction — or when (Zarubin et al., 2014). high levels of lipids are included in the diet (Company et al., 1999). In wild Differences in condition indices (HSI, GSI and Kn) among localities fish, it is much more difficult to assess how environment, diet, and fish analysed by Multi-factorial analysis (MFA) and Spearman's correlation physiology can affect lipid storage dynamics in fat tissue. Therefore in test clearly indicate that these are not related to any of the potential this case, only specific differences in food availability among different sam- stressors analysed (parasites or AIs); and the variability observed may be pling areas could be used to explain these differences, although the high due to the sampling design used to ensure sample size uniformity. The ob- variability in fish condition found within a population prevents any further served changes in body condition are probably driven by physiological con- conclusions. ditions related to the reproductive stage, as well as environmental-related Although parasitism has been identified as a further factor affecting the changes such as season, environmental fluctuations, or food availability body condition (Kn) of several fish species in the Mediterranean (e.g. Ferrer- (Brosset et al., 2015). Specimens of the present study were sampled during Maza et al., 2016, Ferrer-Maza et al., 2015), our results do not show a neg- their reproductive period. This species is known to display daily spawning ative relationship between parasite infestation and condition indices, or synchronicity (Basilone et al., 2015) which can affect daily variability in the with other health indicators such as biomarkers, clearly indicating that population, such differences are evident in the somatic and reproductive the effect on these fish populations is negligible. These findings also support states (Basilone et al., 2006; García Lafuente et al., 2002). Moreover, previous observations from the same study area (Biton-Porsmoguer et al., 10 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 2020; Dallarés et al., 2014; Pérez-i-García et al., 2017; Rodríguez-Romeu 4.2. Parasitological load et al., 2020). The different values of enzymatic biomarkers among localities could not Our results indicate that the parasite community of European anchovies be clearly correlated with any potential stressors (AIs, parasites) nor with along the Catalan coast has a very low prevalence of zoonotic parasites, far fish size (SL), which suggests that this variability could be linked to a smaller than in other Mediterranean areas and the Atlantic waters surround- high levels of variation among individuals (Antó et al., 2009). The usage ing the Iberian Peninsula. Moreover, since all endoparasites found were lo- of biomarkers related to oxidative stress (CAT, GST), detoxification of xeno- cated within the digestive tract, mesenteric tissue, or visceral cavity, and no biotics (CbE), neurotoxicity (AChE,) tissue damage (EROD), aerobic- (CS) parasites were found in the flesh of this species, these findings indicate little and anaerobic- (LDH) metabolism have been widely employed as biomark- risk to humans and thus give an added value for the fishing and commercial- ers in aquatic organisms to evaluate the effects of contaminants like ization of anchovies from the Catalan coast. The parasite fauna of the microplastics (Prokić et al., 2019). However, since most of the patterns ob- European anchovy in the Mediterranean and Atlantic waters has been served in biomarkers response are limited to experimental exposures under extensively described, particularly regarding nematode taxa, but also controlled conditions, it is extremely difficult to draw a parallel in the field, digeneans to some extent (Dessier et al., 2016). Aphanurus virgula — in ad- where conditions are highly variable and usually not adequately moni- dition to affecting anchovies — is considered a main parasite of Boops boops, tored. In addition, concentrations used in these experimental studies are but is also described in other sparids such as Pagellus erytrhinus or clupeids usually very high, as the experimental approach is purely toxicological, so such as Sardina pilchardus (Kostadinova et al., 2004). Lecithasater confusus the levels of plastics used are unrealistically outside the range of concentra- has been previously described in anchovies (Dessier et al., 2016), but our tions typically reported in the natural environment (Burns and Boxall, genetic and morphological analyses on unidentified Lecithaster spp. did 2018). In natural conditions, many factors may be influencing response of not confirm the species, and indicates therefore a possible new species for fish concurrently, making it difficult to find strong correlations unless fish the genus. Regardless, both digeneans found in our samples were adults, populations had been exposed to severe stress conditions during a certain which indicates that they were in their definitive host and should not period (e.g. an oil spill; Penela-Arenaz et al., 2009). have a zoonotic potential. Regarding the nematodes species observed — In the present study, the differences in the number of splenic MMCs Hysterothylacium aduncum and H. fabri — these have been cited in a wide identified in fish among the different locations do not show correlations number of fish species. The Hysterothylacium genera has been reported with size or with the presence of potential stressors. The assessment of only once as non-invasive anisakidosis (Yagi et al., 1996) and may have MMCs in liver of European anchovies has been previously proposed as a been involved in some cases of food allergies (Valero et al., 2003). Contrary biomarker of environmental pollutant exposure in the Tyrrhenian Sea to other species such as Anisakis spp., Hysterothylacium species are not recog- (Basilone et al., 2018). The increase in size and number of MMCs has nized as truly pathogenic for humans, possibly because their final host are been related to various histopathological and inflammatory conditions not mammals but fish (Cipriani et al., 2018) — which do not have the ability (Manrique et al., 2014), as well as to pollutant exposure, environmental to thermoregulate and maintain high body temperatures — and therefore degradation (Carrassón et al., 2008; Carreras-Colom et al., 2022b), and have body temperatures that depend on the environment and are usually parasitological infections (Carrassón et al., 2008; Pérez-i-García et al., lower. Furthermore, considering that Hysterothylacium larvae does not mi- 2017). However, it should be noted that in addition to the response to grate into the flesh of the fish host, nematodes of this genera cannot be con- anthropogenic stressors, MMCs can also respond to natural variability sidered as a concern for food safety (Levsen and Karl, 2014). No nematodes such as species, age (Stentiford et al., 2003), sex (Fournie et al., belonging to the Anisakis genera were found in our study, despite being pre- 2001), spawning phase (Kumar et al., 2016), diseases, or even unspe- viously described in the studied area — although in low numbers (Ferrer- cific environmental factors such as seasonality (Carreras-Colom et al., Maza et al., 2016; Biton-Porsmoguer et al., 2020). The higher presence of 2022b). Therefore, before using it as an indicator in environmental Anisakis spp. in anchovies from the East Atlantic Ocean as compared to monitoring programs, it is necessary to thoroughly characterize and evalu- the West Mediterranean Sea (Rello et al., 2009) is well-known, as happens ate the natural variability of MMCs in the target species. In this sense, the in other pelagic (Molina-Fernández et al., 2015) and demersal fish species present work may be foundational for future studies of anchovy-based (Gómez-Mateos et al., 2016, and references therein). Regarding the Medi- MMC assessment. terranean Sea, higher values of Anisakis spp. have been found in the Ligurian The absence of relevant histological alterations in the fish examined in Sea compared to southern areas (Rello et al., 2009), and Roca-Geronès et al. this study is particularly interesting, as this confirms the evident lack of a (2020) reported a higher prevalence of A. simplex in the Adriatic Sea when clear impact from diseases on the health of the anchovies sampled. Only comparing it with the Western Mediterranean. On the Catalan Coast, the very mild alterations were observed, and all of them affected a very limited absence of Anisakis spp. herein is in agreement with the low prevalence re- area of the tissues or were minor metabolic changes. Although inflamma- ported by Ferrer-Maza et al. (2016) in anchovies sampled off the northern- tory foci have been described as a possible response to a polluted ecosystem most coast of Catalonia, and Biton-Porsmoguer et al. (2020) reported a (Bernet et al., 1999; Feist et al., 2004), in the present study, they are found prevalence of 23 % infection rate of the fish studied. Results by Cipriani with very low prevalence and intensity; this is similar to the observations of et al. (2018) on anchovies parasitized by A. pegreffi are also in concordance other wild fish in the same area, which also found no relationship between with our results, since this parasite was not reported in the Mediterranean these foci and AIs or parasites (Rodríguez-Romeu et al., 2020). These Spanish coast (Alboran and Balearic Seas), while a prevalence of over changes are part of the natural variability of populations, and can be due 70 % was observed in the Adriatic Sea. to factors related to the biology or normal development of the species. The high percentage of fish with vacuole-like structures in the liver is note- 4.3. Anthropogenic items (AIs) worthy, but unfortunately their nature and cause are still unknown. The negative PAS and Sudan stains indicate that they are neither composed of Concerning the detection of AIs in the sampled anchovy populations, lipids nor glycogen. However, as many lipids are usually removed during 50 % of the analysed fish presented AIs — including particles and fibres — histological processing, the vacuole-like structures may correspond in in their digestive tract. Despite the high variability observed between fact, to lipidic droplets. As the liver plays a key role in lipid metabolism in studies, our results are in the average range of values reported by similar many fish species, these changes in the hepatic parenchyma structure analyses of anchovies in the Mediterranean Sea. This variability is notewor- may be related to changes in the nutritional status of fish. For instance, po- thy in the prevalence of ingestion, with values of >90 % in the Adriatic Sea tential transient accumulations of lipids that is associated with lipid absorp- or the Gulf of Lion, while in other areas — such as the Ligurian Sea — the tion, or lipid mobilization in certain physiological conditions such as prevalence is lower than 45 % (Capone et al., 2020; Lefebvre et al., 2019; vitellogenesis. Thus, further studies on this aspect are needed to understand Misic et al., 2022; Renzi et al., 2019). This wide range of reported values the significance of these changes. is found not only between different Mediterranean areas, but also in the 11 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 same geographical area as in the present study, where prevalence ranged relationship between the abundance of parasites and the number of from 6.6 to 60 % (Compa et al., 2018; Pennino et al., 2020). Moreover, microplastics in stomachs of European anchovy was found. These au- our average ingestion values (1.07 AIs/ind.; SD = 1.49) are slightly higher thors suggested that the individuals distributed in more polluted than those obtained in previous studies, in which the average values re- areas — and thus feeding on more microplastics — also had a higher ported are usually <1 item/ind. Those differences could be due to variation probability of being infected by parasites. However, previous studies in environmental concentrations, but also to the different extraction and using a similar approach in red mullet fish (Mullus barbatus) did not identification methodologies used, such as digestion and filtration, visual find a relationship between AIs/microplastics and parasite infestation selection or AIs size selection; this unfortunately hinders the comparison (Rodríguez-Romeu et al., 2020). In large marine mammals, such as of results (Simon-Sánchez et al., 2022). When considering differences grey seals (Halichoerus grypus), the interacting aggregation of both par- among our sampled locations, the prevalence and mean intensity of plastic asites and microplastics (fragments and fibres) along the intestine was ingestion are higher in Barcelona, due to the higher prevalence of particles, not conclusive, since the number of microplastics were not significantly which are also larger, with no differences in the number of ingested fibres related to parasites (Hernandez-Milian et al., 2019). among locations. During the last decade, public discourse flagging fish as a significant Particles found in our anchovies are made of PP and PE — polymers that route of exposure to plastics in humans has grown. However, considering abound on the surface due to their low density — which allows for a natu- the amounts of AIs found in fish (1.07 AIs/fish), and the fact that digestive rally buoyancy and makes them more available for organisms inhabiting tracts (containing the AIs) of fish are generally discarded before cooking, the water column (like this pelagic fish species). The sinking process is de- it should be stressed that fish consumption may be a far less significant termined by the weathering and biofouling, this allows AIs to sink and be route of exposure than previously thought. Moreover, the amounts of AIs available to sub-surface waters and organisms inhabiting deeper habitats ingested from consuming fish would be much lower than the amounts po- (Andrady, 2011). Fibres of cellulose or PET are expected to have a faster tentially ingested from other sources; Catarino et al. (2018) calculated sinking rate due to their higher density, reaching the bottom faster. Once that, within a year, humans ingest 13,700–68,400 microplastics from there, they may be retained in the sediments where they have been seen household dust, while ingesting a mean of 123 microplastics/year via mus- dominating the polymer composition of the fibres present (Woodall et al., sels (equivalent to 190 AIs/year via fish). Furthermore, these values are 2014), and are available to the benthonic organisms inhabiting these envi- also far lower than those found in bottled water (10–1000 micropastics/L). ronments (Carreras-Colom et al., 2022a, Carreras-Colom et al., 2018; Rodríguez-Romeu et al., 2020). 4.3.1. Changes in AIs levels over a 12-year interval We found that the stomach is the organ with the greatest accumulation The percentage of fish ingesting AIs in Barcelona is higher in 2019 than of AIs, particularly of larger fragments that were only observed in this loca- 2007, which could indicate an increasing trend during years. Not only is tion; these findings are similar to those of Capone et al. (2020). Conversely, prevalence higher, but also the number and size of AIs also increased. The fibres were found throughout the entire digestive system, including the presence of AIs in the gastrointestinal tracts of the studied fish captured in pyloric caeca. However, the greatest amount of AIs found in the stomach 2007 demonstrates that ingestion of this type of debris has been occurring still does not represent >1 % occupation of this organ, suggesting that par- for at least twelve years. This trend was also described in previous works ticles do not accumulate and are eventually egested (Grigorakis et al., 2017; in the same area, both in fish and crustacean species (Carreras-Colom Roch et al., 2021). The presence of microplastics in tissues is a controversial et al., 2018; Rodríguez-Romeu et al., 2020). Moreover, changes in the topic (Burns and Boxall, 2018). Microplastics are measured using extraction proportion of fibres and particles, and their polymer composition, could techniques that are based on organ disintegration such as powdering or di- be revealing a change in this type of pollution. This could be due to various gestion (Avio et al., 2015b). However, these techniques are prone to influ- factors, especially since the study area is very close to a highly populated ence from airborne contamination during processing and do not allow the area (Andrady, 2011; Derraik, 2002; Jambeck et al., 2015), hence the char- observation or assessment of the exact location of AIs in the tissues, and acteristics of ingested AIs likely reflects changes in the debris accumulated the possible interaction and effects. The presence of anthropogenic particles in the marine environment over the last decade (Avio et al., 2015a). How- (323 μm) in livers of anchovies captured in the Mediterranean Sea was de- ever, those changes could also be the result of oscillations of climatic and scribed by Collard et al. (2017). In this paper, authors argued that due to the meteorological factors (Misic et al., 2022), among others, occurring in the technique used (cryosections of tissue and observation in a polarized light same area at different times and resulting in a non-predictable increase microscope), it was not possible to precisely locate microplastics within in anthropogenic fibres. For instance, Carreras-Colom et al. (2020) reported the tissue. Recent studies in intestinal cell cultures have demonstrated a thirty-fold increase in the values of fibre ingestion in shrimp over a three- that nano-sized plastic particles (beads of 50 nm of diameter) can be inter- month period, inferring that this could be the result of increased arrival of nalized by intestinal human cells, but without any evident detrimental terrestrial pollutants into the sea due to increased rainfall events. Therefore, effect in the cells (Domenech et al., 2020). This is not an unexpected finding it is not possible to clearly establish an upward trend over the years based on due to cells having mechanisms — such as pinocytosis — by which they can our data alone, and further investigations should be performed to establish a intake substances into their cytoplasm through vesicles of 100 to 200 nm in clear temporal trend. diameter (Guyton and Hall, 2001). However, for larger-sized particles, as is the case for the identified particles from the anchovies' digestive tracts, the 5. Conclusions mechanisms of absorption, transport, and distribution into the blood stream are much more difficult and thus likely happen more rarely. For these Despite clear changes in anchovy populations observed during the last items, entry into the body and subsequent presence within tissues would decade — including a steady decline in their abundance and size — our be dealt with far more aggressively — as with any foreign bodies that gen- work indicates that in general terms, the fish studied do not show relevant erally cause tissue damage — and an associated inflammatory response pathologies that directly affect their health status. Moreover, the negligible would follow. In our study, no such signs were observed. presence of zoonotic parasites in the studied area confirms this species as Some authors have hypothesized that parasite aggregations within suitable and safe for human consumption, thus giving added commercial the digestive tract could retain microplastics and cause their aggrega- value to anchovies caught in this Mediterranean area. tion or accumulation. In the present study, no relationship between As with many other species, anchovies ingest anthropogenic plastic AIs abundance (including plastic or non-plastic items) and total parasite particles, as well as plastic- and cellulosic-fibres. This is not a new phenom- infestation was found. Not even when considering only parasites found enon; ingestion of plastics was also observed in individuals captured in the digestive tract or when considering only parasites found in the in 2007. The ingestion of this kind of debris appears to show geographical stomach. Our results contrast with the results obtained by Pennino trends, with individuals from Barcelona showing higher levels of AIs when et al. (2020) in the same area one year previous, where a positive compared to other locations along the coast. Although several studies have 12 O. Rodríguez-Romeu et al. Science of the Total Environment 841 (2022) 156539 highlighted the potential negative effects of these kind of pollutants, this Nephrops norvegicus, from the NW fishing grounds of the Mediterranean Sea. Ecotoxicol. Environ. Saf. 72, 1455–1462. https://doi.org/10.1016/j.ecoenv.2009.02.007. study demonstrates that no signs of health impacts were associated with Au, D.W.T., 2004. The application of histo-cytopathological biomarkers in marine pollution AIs ingestion in wild fish at the present level. In conclusion, the problematic monitoring: a review. Mar. Pollut. Bull. 48, 817–834. https://doi.org/10.1016/j. population dynamics of this species do not seem to be due to pathological marpolbul.2004.02.032. Avio, C.G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., D’Errico, G., Pauletto, M., reasons, but are instead likely multifactorial and may be linked to wider Bargelloni, L., Regoli, F., 2015a. Pollutants bioavailability and toxicological risk from changes in anchovy community structure. In a broader sense, our results re- microplastics to marine mussels. Environ. Pollut. 198, 211–222. https://doi.org/10. inforce the utility of the European anchovy as an epipelagic species suitable 1016/j.envpol.2014.12.021. Avio, C.G., Gorbi, S., Regoli, F., 2015b. Experimental development of a new protocol for ex- for the monitoring of this type of marine pollutant, both spatially and tem- traction and characterization of microplastics in fish tissues: first observations in commer- porally. This species could therefore be an important addition to future cial species from Adriatic Sea. Mar. Environ. Res. 111, 18–26. https://doi.org/10.1016/j. studies and monitoring programmes that aim to include fish with varied marenvres.2015.06.014. Bakun, A., 1997. Patterns in the ocean: ocean processes and marine population dynamics. feeding behaviours and habitats. Oceanogr. Lit. Rev. 5, 530. Supplementary data to this article can be found online at https://doi. Basilone, G., Ferreri, R., Barra, M., Bonanno, A., Pulizzi, M., Gargano, A., Fontana, I., org/10.1016/j.scitotenv.2022.156539. Giacalone, G., Rumolo, P., Mazzola, S., Genovese, S., McBride, R., Aronica, S., 2020. Spawning ecology of the european anchovy (Engraulis encrasicolus) in the strait of Sicily: linking variations of zooplankton prey, fish density, growth, and reproduction in an up- CRediT authorship contribution statement welling system. Prog. Oceanogr. 184, 102330. https://doi.org/10.1016/j.pocean.2020. 102330. Basilone, G., Ganias, K., Ferreri, R., D’Elia, M., Quinci, E.M., Mazzola, S., Bonanno, A., 2015. Oriol Rodríguez-Romeu: Conceptualization, Methodology, Formal Application of GAMs and multinomial models to assess the spawning pattern of fishes analysis, Investigation, Writing - original draft, Writing - review & editing. with daily spawning synchronicity: a case study in the european anchovy (Engraulis Anna Soler-Membrives: Conceptualization, Methodology, Validation, encrasicolus) in the Central Mediterranean Sea. Fish. Res. 167, 92–100. https://doi. Formal analysis, Resources, Writing - review & editing, Supervision. org/10.1016/J.FISHRES.2015.01.017. Basilone, G., Gargano, A., Corriero, A., Zupa, R., Santamaria, N., Mangano, S., Ferreri, R., Francesc Padrós: Formal analysis, Writing - review & editing. Sara Pulizzi, M., Mazzola, S., Bonanno, A., Passantino, L., 2018. Liver melanomacrophage cen- Dallarés: Methodology, Formal analysis, Writing - review & editing. tres and CYP1A expression as response biomarkers to environmental pollution in euro- Ester Carreras-Colom: Methodology, Formal analysis, Writing - review pean anchovy (Engraulis encrasicolus) from the western Mediterranean Sea. Mar. Pollut. Bull. 131, 197–204. https://doi.org/10.1016/j.marpolbul.2018.04.028. & editing. Maite Carrassón: Writing - review & editing, Funding acquisi- Basilone, G., Guisande, C., Patti, B., Mazzola, S., Cuttitta, A., Bonanno, A., Vergara, A.R., tion. Maria Constenla: Conceptualization, Methodology, Validation, For- Maneiro, I., 2006. Effect of habitat conditions on reproduction of the european anchovy mal analysis, Resources, Writing - review & editing, Supervision, Funding (Engraulis encrasicolus) in the strait of Sicily. Fish. Oceanogr. 15, 271–280. https://doi. org/10.1111/J.1365-2419.2005.00391.X. acquisition. Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P., Wahli, T., 1999. Histopathology in fish: proposal for a protocol to assess aquatic pollution. J. Fish Dis. 22, 25–34. https:// Declaration of competing interest doi.org/10.1046/j.1365-2761.1999.00134.x. Biton-Porsmoguer, S., Bou, R., Lloret, E., Alcaide, M., Lloret, J., 2020. Research article fatty acid composition and parasitism of european sardine (Sardina pilchardus) and anchovy The authors declare the following financial interests/personal relation- (Engraulis encrasicolus) populations in the northern catalan sea in the context of chang- ships which may be considered as potential competing interests: Maite ing environmental conditions. Conserv. Physiol. 8, 1–14. https://doi.org/10.1093/ conphys/coaa121. Carrasson reports financial support was provided by Autonomous University Brosset, P., Fromentin, J.M., Ménard, F., Pernet, F., Bourdeix, J.H., Bigot, J.L., Van Beveren, of Barcelona. Maria Constenla reports financial support was provided by E., Pérez Roda, M.A., Choy, S., Saraux, C., 2015. Measurement and analysis of small pe- Autonomous University of Barcelona. lagic fish condition: a suitable method for rapid evaluation in the field. J. Exp. Mar. Bio. Ecol. 462, 90–97. https://doi.org/10.1016/j.jembe.2014.10.016. Brosset, P., Fromentin, J.M., Van Beveren, E., Lloret, J., Marques, V., Basilone, G., Bonanno, Acknowledgements A., Carpi, P., Donato, F., Čikeš Keč, V., De Felice, A., Ferreri, R., Gašparević, D., Giráldez, A., Gücü, A., Iglesias, M., Leonori, I., Palomera, I., Somarakis, S., Tičina, V., Torres, P., Ventero, A., Zorica, B., Ménard, F., Saraux, C., 2017. Spatio-temporal patterns This study was supported by the Spanish Ministry of Science and Tech- and environmental controls of small pelagic fish body condition from contrasted Mediter- nology “BIOMARE” project (CTM2006-13508-C02-01MAR) and the Com- ranean areas. Prog. Oceanogr. 151, 149–162. https://doi.org/10.1016/J.POCEAN.2016. plementary Action (CTM2006-28145-E/MAR); the project “SOMPESCA” 12.002. (ARP059/19/00003) by the Catalan Department of Agriculture, Livestock, Brosset, P., Le Bourg, B., Costalago, D., BǍnaru, D., Van Beveren, E., Bourdeix, J.H., Fromentin, J.M., Ménard, F., Saraux, C., 2016. Linking small pelagic dietary shifts with Fisheries and Food and European Maritime and Fisheries Fund (EMFF)) ecosystem changes in the Gulf of Lions. Mar. Ecol. Prog. Ser. 554, 157–171. https:// and the Spanish Ministry of Science, Innovation and Universities project doi.org/10.3354/MEPS11796. “PLASMAR” (RTI2018-094806-B-100). Authors would like to thank Andrea Burns, E.E., Boxall, A.B.A., 2018. Microplastics in the aquatic environment: evidence for or against adverse impacts and major knowledge gaps. Environ. Toxicol. Chem. 37, Millán Sánchez for her invaluable research assistance. We also thank the 2776–2796. https://doi.org/10.1002/etc.4268. fleet of commercial fishing vessels of Barcelona, Tarragona, and Blanes, Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on its and the fisherman associations “Confraria de Pescadors de Barcelona”, own terms: Margolis et al. Revisited. J. Parasitol. 83, 575. https://doi.org/10.2307/ 3284227. “Confraria de Pescadors de Tarragona”, and “Confraria de Pescadors de Cajaraville, M.P., Bebianno, M.J., Blasco, J., Porte, C., Sarasquete, C., Viarengo, A., 2000. The Blanes” for their support during sampling cruises. We are very grateful to use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Amy MacLeod (EditingZoo) for the English editing. Finally, authors would Peninsula: a practical approach. Sci. 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