Long-term exposure to virgin and seawater exposed microplastic enriched-diet causes liver oxidative stress and inflammation in gilthead seabream Sparus aurata, Linnaeus 1758

1 Long-term exposure to virgin and seawater exposed microplastic enriched-diet causes liver 1 oxidative stress and inflammation in gilthead seabream Sparus aurata, Linnaeus 1758. 2 Capo, X1; Company, JJ1; Alomar, C1; Compa, M1; Sureda, A2,3; Grau, A4; Hansjosten, B4; López- 3 Vázquez, J5; Quintana, JB5; Rodil, R5; Deudero, S1. 4 1- Instituto Español de Oceanografía, Centro Oceanografico de Baleares, Muelle de 5 Poniente s/n, 07015, Palma de Mallorca, Balearic Islands. 6 2- Research Group in Community Nutrition and Oxidative Stress, and Health Research 7 Institute of Balearic Islands (IdISBa), University of Balearic Islands, E-07122, Palma de 8 Mallorca, Balearic Islands, Spain. 9 3- CIBEROBN (Physiopathology of Obesity and Nutrition), University of the Balearic 10 Islands, E-07122, Palma de Mallorca, Balearic Islands, Spain. 11 4- Laboratorio de Investigaciones Marinas y Acuicultura, LIMIA-Govern de les Illes 12 Balears, Port d’Andratx, Balearic Islands, Spain. 13 5- Department of Analytical Chemistry, Nutrition and Food Sciences, Institute of Research 14 on Chemical and Biological Analysis (IAQBUS), Universidad de Santiago de 15 Compostela, Constantino Candeira S/N, 15782 Santiago de Compostela, Spain 16 17 18 19 20 21 22 Correspondence 23 Xavier Capó Fiol 24 Instituto Español de Oceanografía, Centro Oceanografico de Baleares, Muelle de Poniente s/n, 25 07015, Palma de Mallorca, Balearic Islands. 26 Email: xavier.cap[email protected] 27 28 29 30 31 32 Accepted manuscript with doi: https://doi.org/10.1016/j.scitotenv.2021.144976 © 2021 Copyright Published by Elsevier B.V. with All Rights Reserved 2 Abstract 33 Plastics accumulation in marine ecosystems has notable ecological implications due to their long 34 persistence, potential ecotoxicity, and ability to adsorb other pollutants or act as vectors of pathogens. The 35 present work aimed to evaluate the physiological response of the gilthead seabream (Sparus aurata) fed for 36 90 days with a diet enriched with virgin and seawater exposed low-density polyethylene microplastics 37 (LDPE-MPs) (size between 100-500 µM), followed by 30 days of depuration, applying oxidative stress and 38 inflammatory markers in liver homogenates. No effects of LDPE-MPs treatments on fish growth were 39 observed throughout this study. A progressive increase in antioxidant enzyme activities was observed 40 throughout the study in both treatments, although this increase was higher in the group treated with seawater 41 exposed MPs. This increase was significantly higher in catalase (CAT), glutathione reductase (GRd), and 42 glutathione-s-transferase (GST) in the seawater exposed MPs group, with respect to the virgin group. In 43 contrast, no significant differences were recorded in superoxide dismutase (SOD) and glutathione 44 peroxidase (GPx) between both groups. Exposure to MPs also caused an increase in the oxidative damage 45 markers (malondialdehyde and carbonyls groups). Myeloperoxidase activity significantly increased 46 because of MPs treatments. After 30 days of depuration, antioxidant, inflammatory enzyme activities and 47 oxidative damage markers returned to values similar to those observed in the control group. In conclusion, 48 MPs exposure induced an increase of antioxidant defences in the liver of S. aurata. However, these elevated 49 antioxidant capabilities were not enough to prevent oxidative damage in the liver since, an increased 50 oxidative damage marker was associated with MPs ingestion. The treatment with seawater exposed MPs 51 caused a more significant antioxidant response (CAT, GRs, and GST). Although after a depuration period 52 of 30 days a tendency to recover the initial values of the biomarkers was observed this does not seem to be 53 time enough for a complete normalization. 54 Keywords 55 Microplastics, Pollution, Oxidative stress, Inflammation, Sparus aurata 56 57 58 3 1. Introduction 59 Nowadays, marine litter contamination is one of the most relevant pressing issues affecting numerous 60 aquatic and marine ecosystems worldwide(UNEP., 2009). Marine litter encompasses elements of different 61 compositions: metals, glass, paper, cloth or plastic, but among these, plastic is considered the most 62 persistent and problematic, representing up to 80-85% of the marine litter present in the oceans (Andrady, 63 2011). In this sense, the amount of plastic released into marine environments is increasing considerably 64 each year (Andrady, 2011; Boteler, 2017; UNEP., 2009). Microplastics (MPs) are plastic particles with a 65 diameter smaller than 5 mm (Andrady, 2011; Cózar et al., 2014). MPs can be classified as primary MPs 66 (that are expressly manufactured with a size less than 5 mm in diameter, commonly used to produce pellets, 67 cosmetics personal care and household products, synthetic fibres from washing clothes, etc.), and 68 secondary MPs (plastics derived from the fragmentation of larger plastics into smaller particles in the 69 marine environment due to the action of erosive agents, including the combination of ultraviolet radiation, 70 the abrasive force of waves and temperature, etc.)(Andrady, 2011; Cózar et al., 2014). 71 MP can be made up of a variety of compounds such as nylon, polypropylene (PP), polyethylene 72 terephthalate (PET), polyethylene (PE), bisphenols, or polyvinyl chloride (PVC) and can be toxic for 73 marine organisms due to contaminants like phthalates and bisphenols added during their manufacturing 74 processes (Hidalgo-Ruz et al., 2012; Rochman et al., 2013; Vert et al., 2012). Also, MPs consist of long 75 hydrophobic chains, which in aquatic and marine environments, can accumulate several pollutants present, 76 at water in higher concentrations than in the sediments (Andrady, 2011). Furthermore, due to its 77 physicochemical properties, MPs can accumulate several environmental pollutants in their surface, which 78 can potentially increase their toxicity across the food web (Rochman et al., 2013; Yuan et al., 2020). 79 Consequently, recent studies have identified that MPs can accumulate on their surface several substances 80 such as heavy metals, polycyclic aromatic hydrocarbons, antibiotics, aromatic and aliphatic compounds, 81 etc (Zhang et al., 2020). Due to their small size, MPs particles can be ingested or filtrated by a wide range 82 of marine organisms, and for this reason, MPs can enter the food web of marine ecosystems (Au et al., 83 2017; Gallo et al., 2018; Li et al., 2019). Moreover, considering their size, colour and buoyancy, MPs can 84 be ingested by many fish species due to their similarity to preys (Güven et al., 2017; Jovanović, 2017). In 85 addition, MPs can be partially digested and their degradation products can be absorbed and distributed 86 throughout the organism and incorporated into different tissues or cells (Barboza et al., 2018). MPs or their 87 derivates can cause adverse health effects in marine organisms, including metabolic disorders, 88 4 neurotoxicity, hepatic stress, oxidative stress, inflammation, decreased growth, blockage of feeding 89 appendages, behaviour alterations, obstruction of the gastrointestinal tract or cause pseudo-satiation 90 resulting in reduced food intake (Barboza et al., 2018). 91 Most pollutants, including MPs, together with additives or other adsorbed elements, accumulated in marine 92 organisms can induce the overproduction of reactive oxygen species (ROS) (Alomar et al., 2017; Barboza 93 et al., 2018; Sillero-Rios et al., 2018; Sureda et al., 2018). If the excess of ROS is maintained along the 94 time, an oxidative stress situation is established that can induce oxidative damage to cellular components, 95 as DNA, proteins and lipids (Livingstone, 2001). Oxidation of biomolecules generates several end products, 96 as malondialdehyde (MDA), carbonyl derivates, and 8-hydroxy-2-deoxyguanosine, that can be used as 97 biomarkers of oxidative stress (Bartoskova et al., 2013; Box et al., 2007). ROS overproduction can disrupt 98 vital functions, altering mitochondrial function and inducing apoptosis (Halliwell, 2007; Regoli et al., 99 2004). However, all organisms, including fishes, have developed a complex network of antioxidant and 100 detoxifying mechanisms to scavenge or avoid ROS generation, detoxify pollutants and repair or remove 101 damaged molecules (Barboza et al., 2018; Livingstone, 2001). This antioxidant system includes enzymatic 102 and non-enzymatic antioxidants. Enzymatic antioxidant mechanisms include enzymes such as superoxide 103 dismutase (SOD), which eliminates superoxide anion; catalase (CAT) and glutathione peroxidases (GPx) 104 that remove hydrogen peroxide; and glutathione reductase (GRd) that regenerates glutathione (GSH), an 105 essential non-enzymatic antioxidant. Glutathione-s-transferase (GST) is a phase II detoxification enzyme 106 that contributes to eliminate xenobiotic compounds and oxidation products conjugating toxic compounds 107 with glutathione, and producing more hydrophilic and less toxic compounds (Cunha et al., 2005; Sureda et 108 al., 2006). 109 The gilthead seabream (Sparus aurata Linnaeus, 1758) is a common species in the Mediterranean Sea, 110 although it is also present along the eastern Atlantic coasts from Great Britain to Senegal. It is a highly 111 appreciated and consumed fish in Southern Europe, and, in this sense, gilthead seabream has experienced 112 an increased growth in the aquaculture industry (FAO, 2001-2020). In addition, this species is one of the 113 most used model fish species in aquaculture studies and in the natural environment. S. aurata is also often 114 used as a model in bioaccumulation studies (Grigorakis, 2007),as a bioindicator for toxicity(DelValls et al., 115 1998; Espinosa et al., 2017),and endocrine studies (Forner-Piquer et al., 2018). Moreover, S. aurata is a 116 5 very voracious predator in the wildlife, which suggests that it could bioaccumulate MPs or other pollutants 117 (Balart et al., 2009). 118 Nowadays, fishing resources are limited and are being overexploited. For this reason, aquaculture is a 119 growing industry supplying the world's increasing demand for seafood (FAO, 2020). According to data 120 from the Food and Agriculture Organization (FAO) of The United Nations (UN), about 82.1 million tons 121 of aquatic animals were produced by aquaculture (FAO, 2020). This data gives an idea of the significant 122 impact aquaculture has in the world. Considering this, fish growing in aquaculture facilities are exposed to 123 the pollutants present in the sea but also from the infrastructure itself. Plastic materials have surrounded 124 these fishes throughout their life cycle as most of the materials used in aquaculture facilities are plastic 125 derived: from the fattening farms, juvenile tanks, nets, ropes, pipes, buoys and the structures that form the 126 cages (Wu et al., 2020; Zhu et al., 2019). Moreover, the continuous maintenance and cleaning to avoid 127 fouling organisms on aquaculture gear (nets, ropes, cages, etc..) also contributes to the release of fibres and 128 particles in the form of microplastics into the environment (FAO, 2020). 129 Consequently, the first objective of this study was to determine long term physiological effects in reared 130 Sparus aurata caused by the ingestion of MPs and assessed throughout oxidative stress and inflammatory 131 markers determined in fish liver. The secondary objective was to assess differential biochemical response 132 to pure polyethylene, compared to seawater exposed polyethylene enriched diets. Additionally, the study 133 also aimed to evaluate the effects of 30 days depuration period on the biochemical response of fish liver. 134 135 136 137 138 139 140 141 6 2. Material and Methods 142 2.1 Diet preparation 143 For the experiment, three different types of diets were formulated: 1) a control diet (90% food and 10% 144 filler), 2) a diet with virgin MPs (90% food and 10% LDPE-MP) and 3) a diet with seawater exposed MPs 145 (90% food and 10% LDPE-MP previously exposed to seawater for 2 months in an area of high anthropic 146 impact (39º32'38.04" N 2º22'50,97''E). LDPE-MPs 022 pellets f were purchased from a Spanish distributor, 147 Repsol. This plastic was additive free. 148 Seawater exposed MPs were stored in nylon nets, hung from the aquaculture cages located in the Port of 149 Andratx and submerged at a depth of 0.5-1m from the sea surface. Weekly, the nets were manually shaken 150 to remove any large biofouled organisms. After two months, the pellets were removed from the nets, 151 cleaned with distilled water and oven-dried at 60ºC for 48 h. The ingredients for the control treatment 152 consisted of 37 g of fishmeal LT94, 9.2 g wheat gluten, 21.5 g soybean meal, 8.3 g fish oil, 0.9 g soy 153 lecithin, 9.1 g wheat flour, 0.5 g choline chloride, 0.1 g vitamin C, 0.9 g vitamin and mineral mixes, 1.9 g 154 binders (½ guar gum + ½ carboxymethyl), 0.5 g amino acid mixture, mixture (methionine-lysine, 1: 1) and 155 10 g filler (sepiolite-cellulose-guar gum, 1:1:1) in 100 g dw. LDPE pellets for the virgin and seawater 156 exposed MPs enriched diet treatments were dipped in liquid nitrogen and ground. After grinding, plastics 157 were sieved in pre-cleaned stainless-steel metal sieves to collect fragments smaller than 0.5 mm. 158 Additionally, LDPE pellets (thereafter MPs) were ground into 500 - 100 µm fragments, using conical 159 separation grinders for conventional use. MPs obtained were mixed with the same ingredients as the control 160 diet, but for 100g of food, 10 g of it were substituted with 10 g of virgin and seawater exposed MPs. 161 2.2 Pollutants detection 162 A subsample of seawater exposed MPs was collected from the harbour after two months submerged in the 163 water. Pollutants present in MPs (both virgin and seawater exposed) were determined by gas 164 chromatography coupled to quadrupole-time-of-flight mass spectrometry (GC-QTOF) following 165 standardized procedures. In order to decrease background contamination of phthalates, all glass material 166 were baked at 300ºC for 12 hours, 3% (w/w) alumina was added to ethyl acetate (Worsfold, 2019). Next, 167 0.2 g of MPs seawater exposed pellets were sonicated with 2 mL of ethyl acetate for 30 min at room 168 temperature in vials covered with aluminium foil. The ethyl acetate was transferred to a new vial and the 169 7 extraction was repeated with a fresh 2 mL of ethyl acetate aliquot. The 4 mL of solvent was mixed and 170 evaporated under a nitrogen stream and the dry residue was redissolved in 200 µL of ethyl acetate. An 171 aliquot of ethyl acetate extracts was derivatized with N-Methyl-N-(trimethylsilyl) trifluoroacetamide 172 (MSTFA, 60:40) at 60ºC for 1 hour. One microliter of each extract was injected in split less mode using an 173 Agilent 7693B series (Agilent Technologies, Santa Clara, CA, USA) into the gas chromatograph 7890A 174 interfaced with a 7200 QTOF mass spectrometer (Agilent Technologies). The separation was performed in 175 aHP-5MS column (Agilent Technologies J&W, 30 m x 0.25 mm x 0.25 µm) with helium (99.9999%, 176 Nippon gases, Madrid, Spain) as carrier gas at a flow rate1 mL/min. The temperature programme startedat 177 50ºC for 1 minute, then ramped until 290ºC at 10ºC/min and held for 15 minutes. The acquisition was 178 performed in electron impact ionization (EI) mode at 70 eV with an emission current filament set at 5 µA. 179 QTOF MS was operated at 5 spectra/s in the mass range 40 to 1000 Da and the resolution was about 3714 180 FWHM (full width half maximum) at 68.9947 m/z and 7875 FWHM at 501.9708 m/z. Mass calibration 181 was performed each three samples, in accordance with the recommendation of the manufacturer, using 182 perfluoro tributyl amino (PF-43). 183 A suspect screening was performed by comparison of MS spectra for each feature with those of spectral 184 libraries. The libraries used for data processing were (1) an in-house empirical high-resolution pollutants 185 library (356compounds in total, 236 underivatized compounds and 120 derivatized compounds),(2) a 186 commercial high-resolution library of pesticides containing 844 compounds supplied by Agilent, (3) a low- 187 resolution plastic additives composed of 626 compounds created as of the information obtained from the 188 European Chemicals Agency (ECHA, 2020)and AccuStandard pages (Bolgar, 2015) and (4) library 189 NIST.17(306,622 compounds). 190 2.3 Experimental procedure 191 The experimental procedure was carried out at the LIMIA (Laboratorio de Investigaciones Marinas y 192 Acuicultura) in Andratx (Mallorca, Balearic Islands) laboratory facilities between June 2018 and February 193 2019. A total of 2000 specimens of cultured alevine ofS. aurata of about 3 g were allocated into a large 194 2000 L seawater tank and left to acclimate for 4 months prior to commencing the experimental procedure. 195 After the acclimation period, 600 alevins of S. aurata (7 months old, total length 11.8 ± 0.3 cm and weight 196 44.9 ± 3.1 g) were randomly assigned to 12 tanks of 1000 L with an equal stocking rate of 100 fish per 197 tank. Tanks were located inside the laboratory and were protected from climate and weather, ensuring the 198 8 same physical and chemical conditions in each tank. The photoperiod was of 16 h light / 8 h dark cycle. 199 There was no difference in the average daily temperature between the tanks, and it was maintained at 19 ± 200 2 °C, with a water flow rate of ~1L/minute to ensure 1.5 water-cycles per day, and the oxygen concentration 201 was of 5.9-6.1 ppm. Three different diet treatments were applied: control diet (Control) without MPs, virgin 202 MPs diet enriched with 10% MPs, and seawater exposed MPs diet (enriched with 10% of seawater exposed 203 MPs). Three replicate tanks were randomly assigned to each treatment. The experimental procedure lasted 204 120 days, including a 90 days exposure period in which the MPs treatment group was exposed to a diet 205 enriched in MPs and followed a 30 days depuration period. During the depuration period, all tanks were 206 fed with the same original control diet. During the exposure, feeding of fish was done twice daily at a 2% 207 body weight rate ·day−1 using an automatic feeder (FIAP Belt Feeder with clock (12 hours). 208 2.4 Fish sampling 209 For each sampling period (T0, T30, T60, T90 and T120) and treatment, 9 randomly selected fish (3 from each 210 tank) were sampled after a period of 24 h of feed deprivation from each of three treatments. Individuals 211 from each treatment were randomly caught using a hand net, placed into a smaller tank and anesthetized 212 with tricaine methanesulfonate/MS-222 (1g in 10 L of water) to minimize stress. Individuals were sacrificed 213 by decapitation and immediately dissected. From each fish, the liver was placed into a microtube and stored 214 individually at −80 °C until further processing. One liver slice of each fish was stored at room temperature 215 in 10% phosphate-buffered formalin until histological analysis. The total weight (g) and the total length 216 (cm) of each individual were recorded at 0, 30, 60 90 and 120 days of the beginning of the experiment. To 217 investigate the possible effect of MPs ingestion on fish's fitness or physical condition, Fulton's condition 218 factor (CF) was calculated (Nash et al., 2006). CF was subsequently calculated individually (total weight 219 in g / (total length in cm)3) x 100. 220 2.5 Histological analysis 221 Portions of liver which had been fixed in 10%(w/v) phosphate-buffered formalin, were dehydrated using a 222 graded series of ethanol solutions and cleared with X-Free® (Bio-Optica), embedded in paraffin wax at 223 60ºC and sectioned at 3-4 μm thickness, using a rotation microtome (MICROM HM330). One section of 224 each sample was taken and stained with Mayer's Hematoxylin and Eosin (H&E). All sections were then 225 9 mounted using Eukitt ® (Sigma-Aldrich), and observed under a light microscope (Olympus BX51) with an 226 attached photographic camera (Olympus DP20). 227 2.6 Gastrointestinal tract analysis for microplastic ingestion 228 Gastrointestinal tract samples were used to determine MPs ingestion. All instruments were cleaned with 229 acetone previously before the start of each analysis. To isolated MPs in fish's gastrointestinal tract, tissues 230 were dissolved in 10% of potassium hydroxide(KOH) (20 mL of KOH / g tissue) as described by Dehaut 231 and collaborators (Dehaut et al., 2016). The chemical digestion took place during 48-96 h at room 232 temperature inside a fume hood. The digested solution was filtered through polycarbonate membrane filters 233 (20.0 μm, diameter 47 mm) and left to dry at room temperature for 24 h. Filters were observed under the 234 stereomicroscope for microplastic identification. Given the high number of plastic pellets observed in the 235 filters, the ingestion of MPs by S. aurata was assessed by applying a Covering Index following the Braun- 236 Blanquet system (Boudouresque, 1971). For this, the filter was divided into quadrants and the percentage 237 coverage of the quadrants by MPs was estimated (microplastic ingestion index). 238 2.7 Sample preparation for biochemical determinations 239 Around 0.15 g of liver sample was homogenized in ten volumes (w/v) of 100 mM Tris-HCl buffer pH 7.5 240 using a small sample dispersing system (ULTRA-TURRAX® Disperser,IKA). Each homogenate was 241 briefly sonicated (2–3 s) using an ultrasonic processor. Homogenates were centrifuged at 9000 ×g, 10 min, 242 4 °C and supernatants were recovered and used for biochemical assays. All biochemical analyses were 243 normalised per mg of total protein, measured with a commercial kit (Biorad®), using bovine serum albumin 244 as a standard. 245 2.8 Enzymatic activities 246 Antioxidant (CAT, SOD, GRd and GPx), inflammatory (Myeloperoxidase (MPO)) and detoxification 247 (GST) enzymatic activities were measured with a Shimadzu UV-2401 PC spectrophotometer at 25°C. CAT 248 activity was measured following the method described by Aebi (Aebi, 1984). GRd activity was measured 249 by a modification of the Goldberg and Spooner's spectrophotometric method (Goldberg DM Spooner R. , 250 1984). GPx activity was determined using the method described by Flohe (Flohe and Gunzler, 1984). 251 16 per mL for 14 days (Ding et al., 2018), zebrafish (Danio renio, Hamilton, 1822) (Lu et al., 2016) and the 408 sea bass (Dicentrarchus labrax) fed with a diet enriched with between 0,25-0,69 mg of MPs per L for 96 409 hours (Barboza et al., 2018). An increase in SOD activity was also found in O. niloticus liver samples after 410 an exposure to 0.1 µm polystyrene MPs for 14 days (Ding et al., 2018). Similarly, an increase in liver CAT 411 and SOD activities has been reported in D. renio after 7-day exposure to5 μm polystyrene-MPs (Lu et al., 412 2016). In the same line, a study performed with sea bass observed an increase in muscle and brain oxidative 413 damage after ingestion of MPs at 0.25-0.69 mg/L, during 96 hours (Barboza et al., 2018). On the other 414 hand, studies performed with D. labrax after 3 weeks of PVC-MPs supplementation did not show an 415 increase in antioxidant enzymes (Espinosa et al., 2019). The absence of differences in antioxidant enzymes 416 was also observed in striped red mullet (Mullus surmuletus; Linnaeus, 1758) when comparing fish with 417 MPs present in their digestive tract to fish without MPs (Alomar et al., 2017). However, all these studies 418 were carried out within short periods of exposure to MPs, with a wide range of MPs concentrations, or with 419 samples collected from the wild, while our study consisted of 90-day MPs exposure. Our results show that 420 antioxidant (CAT, GRd and GPx) and detoxification (GST) activities are slightly higher in seawater 421 exposed MPs, treatment in comparison to virgin MPs. In this sense, a previous study has reported an 422 increase in the toxicity response of fish species as a consequence of chemicals adsorbed on the surface of 423 plastics (Rochman et al., 2013). In this work, several compounds were found adhered to the MPs after two 424 months exposed to seawater in an area of high anthropogenic impact, and between these compounds a wide 425 variety of compounds as plastic, food and paint additives, aromatic hydrocarbons and industrial 426 antioxidants were detected. The presence of these compounds in seawater exposed MPs is a consequence 427 and indication of anthropic pressure in marine environments, which is particularly high in port areas 428 (Buruaem et al., 2012; Solis-Weiss et al., 2004). These results are in accordance with several studies that 429 have detected several compounds as metals, organic pollutants, or polycyclic aromatic hydrocarbons 430 sorbing and accumulating on the surface of marine MPs, typically in concentrations higher than those 431 present in the marine environment (Rios et al., 2007; Rochman et al., 2013; Rochman et al., 2014). 432 MDA is a biomarker widely used to determine lipid oxidative damage (Capo et al., 2015b; Sureda et al., 433 2011). A significant increase in liver MDA levels was found after the MPs exposure, and it is interesting 434 to highlight that this increase was more evident in the seawater exposed MPs group. Similarly, carbonyl 435 protein derivates are used to indicate oxidative protein damage (Capó et al., 2020). We have evidenced a 436 considerable increase in liver protein damage as a consequence of MPs exposure; however, this increase 437 17 was only significant from T60, both in virgin and seawater exposed treatments. These results are in 438 accordance with previous studies (Alomar et al., 2017; Barboza et al., 2018; Ding et al., 2018) suggesting 439 that the increase in antioxidant defences observed after MPs ingestion was not enough to prevent oxidative 440 damage in S. aurata liver. 441 Several studies have demonstrated a direct relationship between pollutants and inflammation (Auffret, 442 1988; Bayne and Lowe, 1985), and it has also been observed that MPs can induce inflammation and immune 443 system alterations (Brown et al., 2001). MPO is a pro-inflammatory enzyme located in neutrophils, and its 444 primary function is to produce hypochlorous acid, with antimicrobial activity, which is released into 445 extracellular space during degranulation (van der Veen et al., 2009). It is evidenced that an excessive MPO 446 activity is related to an increase in oxidative damage and inflammation (Capo et al., 2015a; van der Veen 447 et al., 2009). An increase in MPO activity in tissues is directly related to immune cell infiltration and 448 inflammation (Solomando et al., 2020). Our results showed an increase in MPO activity as a consequence 449 of MPs exposure, suggesting that MPs induces an inflammatory response in S. aurata liver. Our results are 450 in accordance with previous studies evidencing that microplastics exposure induces an inflammatory 451 response in S. aurata and D. renio gut (Qiao et al., 2019; Solomando et al., 2020), in copepods (Jeong et 452 al., 2017) and bivalves gills (von Moos et al., 2012). 453 MPs ingestion not only causes oxidative stress but also can causes toxicity (Barboza et al., 2018; Rochman 454 et al., 2013; Rochman et al., 2014). Toxic molecules should be rapidly metabolized and/or excreted by 455 organisms in order to avoid its accumulation in their body (Sureda et al., 2006). The metabolization of toxic 456 compounds is carried out in two phases. In the first phase, compounds are metabolized by cytochrome P450 457 system, which catalyses the addition of an oxygen atom to the molecule; in the second phase, the compound 458 generated by the cytochrome P450 system is conjugated with endogenous substances as glutathione, 459 glucuronic acid or sulphate, to convert them in more hydrophilic compounds (Falfushynska et al., 2019; 460 Uno et al., 2012). GST is an enzyme involved in phase II detoxification process that produces a more 461 hydrophilic glutathione conjugate of the toxic compound, therefore favouring their elimination (Sureda et 462 al., 2006). An increase in GST activity has been reported as a consequence of MPs ingestion in wild M. 463 surmuletus showing MPs in their gastrointestinal tract (Alomar et al., 2017) and in D. labrax juveniles, 464 exposed during 96 h to MPs (Barboza et al., 2018). Our results showed an increase in GST activity as a 465 consequence of MPs diet supplementation. In this sense, the higher GST activity was observed after 90 466 18 days of MPs exposure. However, no significant differences were found between virgin or seawater exposed 467 MPs, even though GST activity was slightly higher in the seawater exposed treatment in all sampling times. 468 In addition, the effects of 30-day depuration period on oxidative stress and detoxification markers were also 469 investigated. A decrease in oxidative stress and detoxification markers was identified in the liver of S. 470 aurata after the depuration period. CAT activity also decreased after the depuration period, but this decrease 471 was only significant in the seawater exposed MPs treatment. In the same way, GRd activity also decreased 472 after the 30-day depuration period. However, this decrease was only significant in virgin MPs treatment, 473 whereas SOD, GPx and MPO activities did not decrease as a consequence of the depuration period. 474 Carbonyl protein derivates also decreased after a 30 days depuration period, reaching levels like those 475 detected on the 30th day of the study. On the other hand, no effects of the depuration period were found in 476 MDA levels. 477 Our results did not show evident histological damage due to the MPs exposure and intake at T90. However, 478 the increase in the infiltration of immune cells observed in T90 could explain the increase in MPO activity 479 observed in T90. In this sense, histological analysis in samples from T120 shows a reduction in the infiltration 480 of immune cells in the liver that would be related to the decrease in enzymatic activities observed at the 481 same time. 482 Moreover, a significant decrease in GST activity was also found after the depuration period, although this 483 decrease was only significant in the virgin MPs treatment. The GST activity in the virgin MPs treatment 484 after 30 days of depuration period is similar to the GST activity measured at day 30 of the study. The results 485 suggest that 30-days of depuration can slightly reduce the oxidative stress and hepatotoxicity status in S. 486 aurata, but it is not enough to observe a complete recovery from MPs exposure in the liver. In this sense, 487 there is a lack of literature considering the effects of depuration periods in the antioxidant and detoxification 488 mechanism in fish. A study performed in liver from juveniles of catfish (Clarias gariepinus,Burchell, 1822) 489 did not find any differences in the antioxidant defences after a depuration period (Iheanacho and Odo, 490 2020). Other studies performed on bivalves exposed to MPs for 14 days, followed by 7 days period 491 depuration, did not find effects in SOD and CAT activities, while a decrease in GPx and GST activities 492 after the depuration period was reported (Ribeiro et al., 2017). 493 19 In conclusion, long-term MPs exposure induced an increase in the antioxidant defences in the liver of S. 494 aurata. However, this increase in antioxidant capabilities was not enough to prevent oxidative damage since 495 there was an increase in MDA levels due to MPs ingestion. MPs exposure also caused an increase in GST 496 activity; these results suggest an activation of detoxification systems as a consequence of MPs ingestion. 497 Seawater exposed MPs cause a higher antioxidant response (CAT, GRd and GST). A depuration period of 498 30 days induces a tendency to recover initial values of biomarkers determinations in fish liver, although it 499 does not seem to be enough for its complete normalization. 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 20 5. Acknowledgements 515 José Maria Valencia, Elvira Álvarez, Beatriz Ríos, Alberto Aparicio, Karlos Garcia for all of the laboratory 516 support. Aurelio Ortega from the Centro Oceanográfico de Murcia for supplying fish. 517 Acknowledgments to the research project Plásticos derivados de la acuicultura: impactos y efectos en las 518 redes tróficas marinas” (ACUIPLASTIC), with reference number CTM2017-88332-R and funded by the 519 Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad, en el 520 marco del Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016, from the Spanish 521 Government. X Capó was supported by a postdoctoral contract with Juan de la Cierva-Formación (2018- 522 037395-I). A. Sureda was supported by the Instituto de Salud Carlos III (CIBEROBN - CB12/03/30038). 523 MC is the recipient of a pre-doctoral FPI Fellowship from the Conselleria d’Innovació, Recerca i Turisme, 524 of the regional government of the Balearic Islands co-financed by the European Social Fund as part of the 525 FSE 2014-2020 operational programme. We extend the acknowledgements to Xunta de Galicia 526 (ED431C2017/36), the Spanish Agencia Estatal de Investigación (ref. CTM2017-84763-C3-R-2 and 527 CTM2017-90980-REDT) and FEDER/ERDF funds. Acknowledgements to Net4sea: Red científica de 528 análisis de riesgo y exposición a contaminantes emergentes en el medio marino de la Agencia Nacional de 529 Investigación, Ministerio de Ciencia, Innovación y Universidades (CTM2017-90890-REDT). 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 21 6. References 547 Aebi H. Catalase in vitro. Methods Enzymol 1984; 105: 121-6. 548 Alomar C, Sureda A, Capó X, Guijarro B, Tejada S, Deudero S. Microplastic ingestion by Mullus 549 surmuletus Linnaeus, 1758 fish and its potential for causing oxidative stress. Environ Res 2017; 159: 135- 550 142. 551 Andrady AL. Microplastics in the marine environment. Mar Pollut Bull 2011; 62: 1596-605. 552 Au SY, Lee CM, Weinstein JE, van den Hurk P, Klaine SJ. Trophic transfer of microplastics in aquatic 553 ecosystems: Identifying critical research needs. Integr Environ Assess Manag 2017; 13: 505-509. 554 Auffret MM. Histopathological changes related to chemical contamination in Mytilus edulis from field 555 and experimental conditions MARINE ECOLOGYPROGRESS SERIES. Mar. Ecol. Prog. Ser 1988; 46: 556 101-107. 557 Balart E, Pérez-Urbiola J, Campos-Dávila L, Monteforte-Sanchez M, Ortega-Rubio A. On the first record 558 of a potentially harmful fish, Sparus aurata in the Gulf of California. Biological Invasions 2009; 11: 547- 559 550. 560 Barboza LGA, Vieira LR, Branco V, Figueiredo N, Carvalho F, Carvalho C, et al. Microplastics cause 561 neurotoxicity, oxidative damage and energy-related changes and interact with the bioaccumulation of 562 mercury in the European seabass, Dicentrarchus labrax (Linnaeus, 1758). Aquat Toxicol 2018; 195: 49- 563 57. 564 Bartoskova M, Dobsikova R, Stancova V, Zivna D, Blahova J, Marsalek P, et al. Evaluation of ibuprofen 565 toxicity for zebrafish (Danio rerio) targeting on selected biomarkers of oxidative stress. Neuro 566 Endocrinol Lett 2013; 2: 102-8. 567 Bayne BL, Brown, D.A., Burns, K., Dixon, D.R., Ivanovici, A., Livingstone, D.A.,, Lowe DM, Moore, 568 M.N., Atebbing, A.R.D., Widdings, J. The Effects of Stress and Pollution on Marine Animals. Praeger, 569 New York. 1985. 570 Berglund E, Fogelberg V, Nilsson PA, Hollander J. Microplastics in a freshwater mussel (Anodonta 571 anatina) in Northern Europe. Sci Total Environ 2019; 697: 30. 572 Bolgar MH, J; Groeger J; Meronek, S. Handbook for the chemical analysis of plastic and polymer 573 additives: CRC Press, 2015. 574 Boteler BS, LO; Bodle, R; Donat, L. The UN Ocean Conference - June 2017, Guidance to the ENVI 575 Committee of the European Parliament. European Parliament: Brussels 2017. 576 22 Boudouresque C. Méthodes d'étude qualitative et quantitative du benthos (en particulier du 577 phytobenthos). Tethys 1971; 3: 79-104. 578 Box A, Sureda A, Galgani F, Pons A, Deudero S. Assessment of environmental pollution at Balearic 579 Islands applying oxidative stress biomarkers in the mussel Mytilus galloprovincialis. Comp Biochem 580 Physiol C Toxicol Pharmacol 2007; 146: 531-9. 581 Brown DM, Wilson MR, MacNee W, Stone V, Donaldson K. Size-dependent proinflammatory effects of 582 ultrafine polystyrene particles: a role for surface area and oxidative stress in the enhanced activity of 583 ultrafines. Toxicol Appl Pharmacol 2001; 175: 191-9. 584 Buruaem LM, Hortellani MA, Sarkis JE, Costa-Lotufo LV, Abessa DM. Contamination of port zone 585 sediments by metals from Large Marine Ecosystems of Brazil. Mar Pollut Bull 2012; 64: 479-88. 586 Capeillère-Blandin C. Oxidation of guaiacol by myeloperoxidase: a two-electron-oxidized guaiacol 587 transient species as a mediator of NADPH oxidation. Biochem J 1998; 336: 395-404. 588 Capo X, Martorell M, Sureda A, Tur JA, Pons A. Effects of docosahexaenoic supplementation and in 589 vitro vitamin C on the oxidative and inflammatory neutrophil response to activation. Oxid Med Cell 590 Longev 2015a; 187849: 19. 591 Capo X, Tejada S, Box A, Deudero S, Sureda A. Oxidative status assessment of the endemic bivalve 592 Pinna nobilis affected by the oil spill from the sinking of the Don Pedro. Mar Environ Res 2015b; 110: 593 19-24. 594 Capó X, Tejada S, Ferriol P, Pinya S, Mateu-Vicens G, Montero-González I, et al. Hypersaline water 595 from desalinization plants causes oxidative damage in Posidonia oceanica meadows. Sci Total Environ 596 2020; 736: 22. 597 Cole M, Lindeque P, Fileman E, Halsband C, Goodhead R, Moger J, et al. Microplastic ingestion by 598 zooplankton. Environ Sci Technol 2013; 47: 6646-55. 599 Cózar A, Echevarría F, González-Gordillo JI, Irigoien X, Ubeda B, Hernández-León S, et al. Plastic 600 debris in the open ocean. Proc Natl Acad Sci U S A 2014; 111: 10239-44. 601 Cunha I, Garcia LM, Guilhermino L. Sea-urchin (Paracentrotus lividus) glutathione S-transferases and 602 cholinesterase activities as biomarkers of environmental contamination. J Environ Monit 2005; 7: 288-94. 603 Dehaut A, Cassone AL, Frère L, Hermabessiere L, Himber C, Rinnert E, et al. Microplastics in seafood: 604 Benchmark protocol for their extraction and characterization. Environ Pollut 2016; 215: 223-233. 605 23 DelValls TA, Blasco J, Sarasquete MC, Forja JM, Gomez-Parra A. Evaluation of heavy metal sediment 606 toxicity in littoral ecosystems using juveniles of the fish Sparus aurata. Ecotoxicol Environ Saf 1998; 41: 607 157-67. 608 Ding J, Zhang S, Razanajatovo RM, Zou H, Zhu W. Accumulation, tissue distribution, and biochemical 609 effects of polystyrene microplastics in the freshwater fish red tilapia (Oreochromis niloticus). Environ 610 Pollut 2018; 238: 1-9. 611 Duncan EM, Broderick AC, Fuller WJ, Galloway TS, Godfrey MH, Hamann M, et al. Microplastic 612 ingestion ubiquitous in marine turtles. Glob Chang Biol 2019; 25: 744-752. 613 ECHA. Describing uses of additives in plastic material for articles and estimating related exposure - 614 Practical Guide for Industry 2020: 1-19. 615 Espinosa C, Cuesta A, Esteban M. Effects of dietary polyvinylchloride microparticles on general health, 616 immune status and expression of several genes related to stress in gilthead seabream (Sparus aurata L.). 617 Fish Shellfish Immunol 2017; 68: 251-259. 618 Espinosa C, Esteban M, Cuesta A. Dietary administration of PVC and PE microplastics produces 619 histological damage, oxidative stress and immunoregulation in European sea bass (Dicentrarchus labrax 620 L.). Fish Shellfish Immunol 2019; 95: 574-583. 621 Falfushynska H, Sokolov EP, Haider F, Oppermann C, Kragl U, Ruth W, et al. Effects of a common 622 pharmaceutical, atorvastatin, on energy metabolism and detoxification mechanisms of a marine bivalve 623 Mytilus edulis. Aquat Toxicol 2019; 208: 47-61. 624 FAO. Aquaculture topics and activities. Acuicultura. In: Departamento de Pesca y Acuicultura de la FAO 625 2001-2020. 626 FAO. The State of World Fisheries and Aquaculture 2020. . 2020. 627 Flohe L, Gunzler WA. Assays of glutathione peroxidase. Methods Enzymol 1984; 105: 114-21. 628 Flohe L, Otting F. Superoxide dismutase assays. Methods Enzymol 1984; 105: 93-104. 629 Forner-Piquer I, Mylonas CC, Calduch-Giner J, Maradonna F, Gioacchini G, Allarà M, et al. Endocrine 630 disruptors in the diet of male Sparus aurata: Modulation of the endocannabinoid system at the hepatic 631 and central level by Di-isononyl phthalate and Bisphenol A. Environ Int 2018; 119: 54-65. 632 Gallo F, Fossi C, Weber R, Santillo D, Sousa J, Ingram I, et al. Marine litter plastics and microplastics 633 and their toxic chemicals components: the need for urgent preventive measures. Environ Sci Eur 2018; 634 30: 018-0139. 635 24 Goldberg DM Spooner R. p. Glutathione reductase. Methods of enzymatic analysis. Enzymes 1: 636 oxidoreductases, transferases, 1984; 3: 258-265. 637 Grigorakis K. Compositional and organoleptic quality of farmed and wild gilthead sea bream (Sparus 638 aurata) and sea bass (Dicentrarchus labrax) and factors affecting it: A review. Aquaculture 2007; 272: 639 55-75. 640 Güven O, Gökdağ K, Jovanović B, Kıdeyş AE. Microplastic litter composition of the Turkish territorial 641 waters of the Mediterranean Sea, and its occurrence in the gastrointestinal tract of fish. Environ Pollut 642 2017; 223: 286-294. 643 Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic 644 acid formation. J Biol Chem 1974; 249: 7130-9. 645 Halliwell B. Biochemistry of oxidative stress. Biochem Soc Trans 2007; 35: 1147-50. 646 Hara J, Frias J, Nash R. Quantification of microplastic ingestion by the decapod crustacean Nephrops 647 norvegicus from Irish waters. Mar Pollut Bull 2020; 152: 15. 648 Hidalgo-Ruz V, Gutow L, Thompson RC, Thiel M. Microplastics in the Marine Environment: A Review 649 of the Methods Used for Identification and Quantification. Environmental Science & Technology 2012; 650 46: 3060-3075. 651 Iheanacho SC, Odo GE. Dietary exposure to polyvinyl chloride microparticles induced oxidative stress 652 and hepatic damage in Clarias gariepinus (Burchell, 1822). Environ Sci Pollut Res Int 2020; 27: 21159- 653 21173. 654 Jeong CB, Kang HM, Lee MC, Kim DH, Han J, Hwang DS, et al. Adverse effects of microplastics and 655 oxidative stress-induced MAPK/Nrf2 pathway-mediated defense mechanisms in the marine copepod 656 Paracyclopina nana. Sci Rep 2017; 7. 657 Jovanović B. Ingestion of microplastics by fish and its potential consequences from a physical 658 perspective. Integr Environ Assess Manag 2017; 13: 510-515. 659 Levine RL, Williams JA, Stadtman ER, Shacter E. Carbonyl assays for determination of oxidatively 660 modified proteins. Methods Enzymol 1994; 233: 346-57. 661 Li J, Lusher AL, Rotchell JM, Deudero S, Turra A, Bråte ILN, et al. Using mussel as a global 662 bioindicator of coastal microplastic pollution. Environ Pollut 2019; 244: 522-533. 663 Livingstone DR. Contaminant-stimulated reactive oxygen species production and oxidative damage in 664 aquatic organisms. Mar Pollut Bull 2001; 42: 656-66. 665 25 Lu Y, Zhang Y, Deng Y, Jiang W, Zhao Y, Geng J, et al. Response to Comment on "Uptake and 666 Accumulation of Polystyrene Microplastics in Zebrafish (Danio rerio) and Toxic Effects in Liver". 667 Environ Sci Technol 2016; 50: 12523-12524. 668 Lusher AL, Hernandez-Milian G, O'Brien J, Berrow S, O'Connor I, Officer R. Microplastic and 669 macroplastic ingestion by a deep diving, oceanic cetacean: the True's beaked whale Mesoplodon mirus. 670 Environ Pollut 2015; 199: 185-91. 671 Mancia A, Chenet T, Bono G, Geraci ML, Vaccaro C, Munari C, et al. Adverse effects of plastic 672 ingestion on the Mediterranean small-spotted catshark (Scyliorhinus canicula). Mar Environ Res 2020; 673 155: 14. 674 Nash R, Valencia AH, Geffen A. The origin of Fulton's condition factor - Setting the record straight. 675 Fisheries 2006; 31: 236-238. 676 Qiao R, Sheng C, Lu Y, Zhang Y, Ren H, Lemos B. Microplastics induce intestinal inflammation, 677 oxidative stress, and disorders of metabolome and microbiome in zebrafish. Sci Total Environ 2019; 662: 678 246-253. 679 Regoli F, Frenzilli G, Bocchetti R, Annarumma F, Scarcelli V, Fattorini D, et al. Time-course variations 680 of oxyradical metabolism, DNA integrity and lysosomal stability in mussels, Mytilus galloprovincialis, 681 during a field translocation experiment. Aquat Toxicol 2004; 68: 167-78. 682 Ribeiro F, Garcia AR, Pereira BP, Fonseca M, Mestre NC, Fonseca TG, et al. Microplastics effects in 683 Scrobicularia plana. Mar Pollut Bull 2017; 122: 379-391. 684 Rios LM, Moore C, Jones PR. Persistent organic pollutants carried by synthetic polymers in the ocean 685 environment. Mar Pollut Bull 2007; 54: 1230-7. 686 Rochman CM, Hoh E, Kurobe T, Teh SJ. Ingested plastic transfers hazardous chemicals to fish and 687 induces hepatic stress. Sci Rep 2013; 3. 688 Rochman CM, Kurobe T, Flores I, Teh SJ. Early warning signs of endocrine disruption in adult fish from 689 the ingestion of polyethylene with and without sorbed chemical pollutants from the marine environment. 690 Sci Total Environ 2014; 493: 656-61. 691 Sillero-Rios J, Sureda A, Capo X, Oliver-Codorniu M, Arechavala-Lopez P. Biomarkers of physiological 692 responses of Octopus vulgaris to different coastal environments in the western Mediterranean Sea. Mar 693 Pollut Bull 2018; 128: 240-247. 694 Figure 2 Click here to access/download;Figure;Figure 2.jpg Figure 3 Click here to access/download;Figure;Figure 3.jpg Figure 4 Click here to access/download;Figure;Figure 4.jpg Authors contribution XC, MC, CA AS, and SD conception and design of research; XC, JJC, MC, AG, BH, JLV, JBQ, RR and SD, performed experiments; XC, JJC, MC, CA, AS, AG, BH, JLV, JBQ, RR and SD analysed data; XC, JJC, MC, CA, AG, BH and SD interpreted results of experiments; XC, MC, CA, AS and SD drafted manuscript; XC, CA, MC, AS and SD edited and revised manuscript; XC, JJC, CA, MC, AS, AG, BH, JLV, JBQ, RR and SD approved final version of manuscript. All authors read and approved the final manuscript Credit Author Statement Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Interest Statement