Microplastic Presence in the Digestive Tract of Pearly Razorfish Xyrichtys novacula Causes Oxidative Stress in Liver Tissue

Plastic pollution in the oceans is a growing problem, with negative effects on exposed species and ecosystems. Xyrichtys novacula L. is a very important fish species both culturally and economically in the Balearic Islands. The aim of the present study was to detect and categorise the presence of microplastics (MPs) in the digestive tract of X. novacula, as well as the existence of oxidative stress in the liver. For this purpose, the fish were categorised into two groups based on the number of MPs observed in the digestive tracts: a group with no or low presence of MPs (0–3 items) and a group with a higher presence of MPs (4–28 items). MPs were found in 89% of the specimens analysed, with a dominance of fibre type and blue colour. Regarding the type of polymer, polycarbonate was the most abundant, followed by polypropylene and polyethylene. For the group with a greater presence of MPs, the activities of the antioxidant enzymes glutathione peroxidase and glutathione reductase, as well as the phase II detoxification enzyme glutathione s-transferase, were higher than the activities observed in fish with little to no presence of MPs. The activities of catalase and superoxide dismutase and the levels of malondialdehyde did not show significant differences between both groups. In conclusion, these results demonstrate the presence of MPs in the digestive tract of X. novacula and the existence of an antioxidant and detoxification response, mainly based on the glutathione-based enzymes.


Introduction
One of the most serious problems worldwide is the progressive accumulation of potentially toxic waste in all ecosystems. Among them, plastics account for an important part of these pollutants that end up accumulating as litter in the marine environment [1,2]. Plastic is made up of synthetic or semi-synthetic organic polymers formed by repeating chemical structural units that give them great versatility, durability, and resistance to corrosion. These properties have caused the production of plastics to have increased exponentially in recent years [3]. Due to this extended plastic production and generalized poor waste management, a large proportion of these plastic items end up in the marine environment. Plastic may enter the marine environment in all shapes and sizes, from microplastics (MPs) from cosmetics or clothing to macroplastics [4]. Due to their high resistance, plastics do Formentera (Balearic Islands, Spain), as well as the capability of MPs to induce oxidative stress on these fish through the study of biomarkers in their liver tissues.

Experimental Procedure
During October 2020, 36 razorfish were fished in the southeast region of the island of Ibiza with a hook using worms as bait (Figure 1). The sampling area is far from the main population centres in order to reduce the influence of other possible contaminants. The fish were anesthetised with tricaine methanesulfonate (MS-222) (1 g/10 L water) to minimise stress. The individuals were measured (total length: ±0.1 cm and total weight: ±0.1 g), sexed, and the gastrointestinal tract and the liver were dissected. The gastrointestinal tract was frozen at −20 • C until further analysis in the laboratory, whereas the liver was immediately introduced into liquid nitrogen and kept at −80 • C in the laboratory until biochemical analyses. The experimental procedure followed the EU Directive 2010/63/EU for animal experiments and was revised and approved by the Ethics Committee for Animal Experimentation of the University of the Balearic Islands (Ref. 020/06/AEXP). conditions and not in the natural environment. In this sense, the aim of the present study was to evaluate the presence of MPs in wild X. novacula sampled in the islands of Ibiza and Formentera (Balearic Islands, Spain), as well as the capability of MPs to induce oxidative stress on these fish through the study of biomarkers in their liver tissues.

Experimental Procedure
During October 2020, 36 razorfish were fished in the southeast region of the island of Ibiza with a hook using worms as bait ( Figure 1). The sampling area is far from the main population centres in order to reduce the influence of other possible contaminants. The fish were anesthetised with tricaine methanesulfonate (MS-222) (1 g/10 L water) to minimise stress. The individuals were measured (total length: ±0.1 cm and total weight: ±0.1 g), sexed, and the gastrointestinal tract and the liver were dissected. The gastrointestinal tract was frozen at −20 °C until further analysis in the laboratory, whereas the liver was immediately introduced into liquid nitrogen and kept at −80 °C in the laboratory until biochemical analyses. The experimental procedure followed the EU Directive 2010/63/EU for animal experiments and was revised and approved by the Ethics Committee for Animal Experimentation of the University of the Balearic Islands (Ref. 020/06/AEXP).

Microplastic Analysis
The gastrointestinal tracts were digested in Erlenmeyer flasks with 20 mL of 10% KOH solution per gram of digestive tissue following a previously described procedure [47]. Briefly, after complete digestion of the tissue, the samples were filtered with a Büchner funnel, using filtering paper of 47 mm in diameter and a 10 µm pore size (Albet Lab-Science, Barcelona, Spain), and the filters were then dried at 60 °C. Control blanks were also carried out simultaneously under the same conditions as the samples to account for possible environmental contamination. MPs captured in the filters were visually identified and photographed through a Leica EZ4 stereomicroscope with an HD camera (optical magnification up to 11.5x). During the handling process, measures to avoid microplastic contamination were adopted, such as working under a laminar flow hood, rinsing and cleaning material and surfaces using filtered ethanol 70% and MilliQ water, and wearing

Microplastic Analysis
The gastrointestinal tracts were digested in Erlenmeyer flasks with 20 mL of 10% KOH solution per gram of digestive tissue following a previously described procedure [47]. Briefly, after complete digestion of the tissue, the samples were filtered with a Büchner funnel, using filtering paper of 47 mm in diameter and a 10 µm pore size (Albet Lab-Science, Barcelona, Spain), and the filters were then dried at 60 • C. Control blanks were also carried out simultaneously under the same conditions as the samples to account for possible environmental contamination. MPs captured in the filters were visually identified and photographed through a Leica EZ4 stereomicroscope with an HD camera (optical magnification up to 11.5×). During the handling process, measures to avoid microplastic contamination were adopted, such as working under a laminar flow hood, rinsing and cleaning material and surfaces using filtered ethanol 70% and MilliQ water, and wearing lab coats made of 100% cotton. The MP particles were categorised by colour, size, and type (fibre or fragment).
The polymer composition of the MPs (N = 46 items) was analysed using microattenuated total reflection micro-Fourier-transform infrared spectroscopy (µ-ATR-FTIR) (Bruker, OPTICS, Germany). Due to the small size of the MPs, analysis was carried out with the Hyperion ATR microscope. FTIR absorption spectra were recorded as an average of 250 scans in the mid-infrared range of 400-4000 cm −1 . The obtained spectrum was compared with commercial and in-house spectral databases, and a minimum of 700 hit quality index was necessary to accept a confirmed polymer [48].

Biochemical Analysis
Liver sections of X. novacula were homogenised in ten volumes (w/v) of Tris-HCl buffer 100 mM, pH 7.5 using a dispersing system (Ultra-Turrax ® Disperser, IKA, Staufen, Germany). The samples were then centrifuged at 9000× g for 10 min at 4 • C, and supernatants were recovered and used for biochemical analyses.
The activities of the antioxidant enzymes CAT, SOD, GPx, and GRd, and the detoxification enzyme, GST, were monitored using a spectrophotometer (Shimadzu UV-2100, Kyoto, Japan) at 25 • C. CAT activity (mK/mg prot) was determined following the method of Aebi based on the decomposition of H 2 O 2 at 240 nm [49]. SOD activity (pKat/mg prot) was determined according to previously described method at 550 nm, using cytochrome as an indicator [50]. GPx activity was determined at 340 nm using H 2 O 2 and reduced glutathione (GSH) as substrates [51]. GRd activity was monitored at 340 nm with oxidised glutathione (GSSG) as a substrate [52] method. GST activity (nKat/mg prot) was determined at 340 nm using GSH and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates [53]. MDA concentration (nM/mg protein) was quantified using a colorimetric assay kit (Merck, Madrid, Spain), following the manufacturer's instructions.
All results were normalised by the protein content of the samples (Bio-Rad ® Protein Assay, Alcobendas, Spain) using bovine serum albumin as a standard.

Statistical Analysis
The effects of MP ingestion on oxidative stress biomarkers were evaluated using a statistical analysis package (SPSS 27.0 for Windows ® ) (IBM ® SPSS Inc., Chicago, IL, USA). For the statistical analysis, the samples were categorised into two groups according to the number of MPs observed in the gastrointestinal tract: low MPs (N = 18) and high MPs (N = 18). The normality of the data was confirmed by the Shapiro-Wilk test and homogeneity of variance by the Levene's test. Statistical differences between the groups were carried out with a Student's t-test for unpaired data. The correlations between the presence of MPs and stress biomarkers were calculated using the bivariate correlation Pearson test. The results are presented as mean ± standard error of the mean (SEM) and p < 0.05 was considered statistically significant.

Biometric Parameters
A total of 36 fish were included in the study, with an average size of 15.6 ± 0.5 cm (15.7 ± 0.8 low MPs, 15.5 ± 0.7 high MPs) and weight of 55.5 ± 5.7 g (55.8 ± 8.9 low MPs, 55.1 ± 7.4 high MPs). Moreover, 60% of the caught fish were females. The fish were caught at a depth of approximately 15-20 m with water at 19 • C and 36 psu.

Microplastics in the Gastrointestinal Tract
MPs were reported in 32 of the 36 (89%) the gastrointestinal tract of fish analysed with a total of 171 plastic items, while in the remaining 4, there was no evidence of MPs (11%), and in 7 of the fish, only one plastic element was found (19%). Some representative images of the MPs found in the gastrointestinal tracts of X. novacula are presented in Figure 2. Collectively, the fish presented an average of 4.75 ± 0.90 items/individual in their gastrointestinal tract.  To assess whether the increased presence of MPs is related to variations in oxidative stress markers, the fish were divided into two groups according to the number of MPs. Thus, the group with low MP content presented 1.28 ± 0.23 items/individual (ranging between 0 and 3 items per individual) and the group with high MP content exhibited 8.22 ± 1.36 items/individual (ranging between 4 and 28 items per individual).
When analysing the characteristics of the MPs found, 81.7% corresponded to a fibre type, while the remaining 18.7% were fragments. Regarding the colours, the most common MP colour in the samples was blue (50.9%, 87 items), followed by black (25.1%, 43 items), red (7.6%, 14 items), and grey (5.3%, 9 items).

Biomarker Analysis
The activity of the antioxidant enzymes is shown in Figure 4. All of the activities tended to be higher in the group with the highest presence of MPs, although the differences were only statistically significant for GPx and GRd (p < 0.05) and not for catalase and SOD. To assess whether the increased presence of MPs is related to variations in oxidative stress markers, the fish were divided into two groups according to the number of MPs. Thus, the group with low MP content presented 1.28 ± 0.23 items/individual (ranging between 0 and 3 items per individual) and the group with high MP content exhibited 8.22 ± 1.36 items/individual (ranging between 4 and 28 items per individual).
When analysing the characteristics of the MPs found, 81.7% corresponded to a fibre type, while the remaining 18.7% were fragments. Regarding the colours, the most common MP colour in the samples was blue (50.9%, 87 items), followed by black (25.1%, 43 items), red (7.6%, 14 items), and grey (5.3%, 9 items).
Chemical polymer identification was carried out in 46 items using µ-ATR-FTIR ( Figure 3). The most common polymers were polycarbonate (21.7%), followed by polypropylene (19.6%) and polyethylene (19.6%). The remaining polymers were polyester (15.2%), polystyrol (13.0%), polystyrene (8.7%), and cellophane (2.2%).  To assess whether the increased presence of MPs is related to variations in oxidative stress markers, the fish were divided into two groups according to the number of MPs. Thus, the group with low MP content presented 1.28 ± 0.23 items/individual (ranging between 0 and 3 items per individual) and the group with high MP content exhibited 8.22 ± 1.36 items/individual (ranging between 4 and 28 items per individual).
When analysing the characteristics of the MPs found, 81.7% corresponded to a fibre type, while the remaining 18.7% were fragments. Regarding the colours, the most common MP colour in the samples was blue (50.9%, 87 items), followed by black (25.1%, 43 items), red (7.6%, 14 items), and grey (5.3%, 9 items).

Biomarker Analysis
The activity of the antioxidant enzymes is shown in Figure 4. All of the activities tended to be higher in the group with the highest presence of MPs, although the differences were only statistically significant for GPx and GRd (p < 0.05) and not for catalase and SOD.

Biomarker Analysis
The activity of the antioxidant enzymes is shown in Figure 4. All of the activities tended to be higher in the group with the highest presence of MPs, although the differences were only statistically significant for GPx and GRd (p < 0.05) and not for catalase and SOD.
GST activity also showed significantly higher values in the fish with higher MP content (p < 0.05) ( Figure 5). For MDA, as an indicator of lipid peroxidation, although the group with more MPs had higher values, these did not reach significance. No significant differences were found for either MP quantity or biomarker variations between male and female fish. GST activity also showed significantly higher values in the fish with higher MP tent (p < 0.05) ( Figure 5). For MDA, as an indicator of lipid peroxidation, althoug group with more MPs had higher values, these did not reach significance. No signi differences were found for either MP quantity or biomarker variations between ma female fish.   GST activity also showed significantly higher values in the fish with higher MP content (p < 0.05) ( Figure 5). For MDA, as an indicator of lipid peroxidation, although the group with more MPs had higher values, these did not reach significance. No significant differences were found for either MP quantity or biomarker variations between male and female fish. The correlation analysis revealed the existence of a positive association between high levels of MPs and the activities of the GST (r = 0.738; p < 0.001), GPx (r = 0.385; p < 0.025), and GRd (r = 0.400; p < 0.017). The remaining biomarkers did not present correlations in relation to the MPs. The correlation analysis revealed the existence of a positive association between high levels of MPs and the activities of the GST (r = 0.738; p < 0.001), GPx (r = 0.385; p < 0.025), and GRd (r = 0.400; p < 0.017). The remaining biomarkers did not present correlations in relation to the MPs.

Discussion
The ubiquity and small size of MPs make them easily ingestible by many taxa, with potentially negative physical, physiological, and toxicological impacts [9,12,54]. The presence of MPs in the digestive tract can cause intestinal obstruction, inflammation, reduce appetite, and impair nutrient absorption [55]. In addition, smaller MPs, as well as adsorbed substances, can cross endothelial cells, enter the circulatory system, and move to other organs such as the liver, inducing toxic effects on physiological functions [56]. The present results evidenced the presence of MPs in almost 90% of the sampled individuals of X. novacula, suggesting that this species is prone to MP ingestion. Such a high percentage of MP-polluted individuals has been reported in several papers: 100% of the sampled deep-sea fish (N = 35, 11 species) from south China [46] and 77% of Engraulis japonicus (Temminck and Schlegel 1846) (N = 64) in Tokyo Bay [44]. In the Mediterranean Sea, high values, close to 100%, have been observed in Scomber scombrus and Trachurus trachurus in the south Iberian coast [57] and in Seriola dumerili caught around Eivissa and Formentera [29], and approximately 70% in Boops boops (Linnaeus 1758) (N = 337) sampled in the coastal waters of Mallorca [58]. Moreover, the presence of MPs in a benthic fish that lives on sandy and muddy bottoms similar to razorfish, such as Solea solea (Linnaeus, 1758) from the northern and central Adriatic Sea, reached 95% of the specimens analysed [59]. Other studies, however, have reported lower values for the percentage of fish presenting with MPs, such as 19.8% of commercial fish (N = 263, 26 different species) from Portuguese waters [43], 27.3% for Mullus surmuletus (Linnaeus 1758) (N = 417) captured around the coasts of Mallorca [24], and 58% of sampled species in Turkish waters (N = 1337) [60]. X. novacula feeds both from the water column and directly from the sediment, feeding mainly on benthic invertebrates such as molluscs, shrimps, crabs, and polychaetes [36,61], which makes it vulnerable to ingestion from both environments. In addition, it is a species that lives in coastal areas, between 5 and 50 m deep, on sandy bottoms and in Cymodocea nodosa seagrass meadows, generally with high anthropic pressure associated with tourism, which can favour its exposure to MPs, mostly of land-source origin [1].
The colours and types of plastics (fibre or fragment) are consistent with previous scientific literature [24,58,60,62]. Plastics are generally described as blue and black, which might be due to the main colours from the source (packaging, clothing, etc.), or mistaken for prey since they resemble blue copepod species, for example [57,63]. As for fibres, they are found in significant quantities both in the water column and in sediment [64], as well as in other species in various studies [58,62]. The main sources of fibres have been recognized to be washing machines, but also fragments from fishing nets and the textile industry [65,66].
MPs and associated components, such as plasticisers, additives, and other adsorbed pollutants, make them dangerous as they could potentially disrupt the immune and antioxidant systems [67]. This occurs when these alien components affect the cells and induce an excess and accumulation of ROS, creating a pro-oxidative state [68]. ROS are highly reactive and can interact with biomolecules, altering their structure and function and establishing a situation of oxidative stress [69]. To protect themselves, organisms have an elaborate antioxidant and detoxification system to deactivate and eliminate pollutants and to maintain ROS at physiological levels. The present study highlights the potential of MPs to induce a physiological response in X. novacula, since higher values of antioxidant enzymes and GST have been observed for fish with a higher number of MPs in their digestive tracts. Specifically, a greater activity of GPx and GRd, enzymes that participate in the glutathione redox cycle with a key role in the reduction of intracellular hydroperoxides and H 2 O 2 , has been observed [25]. In this sense, the correlation analysis showed a direct relationship between the number of MPs found in the gastrointestinal tract of X. novacula and the activities of GPx and GRd, and especially of GST. GPx catalyses the reduction of H 2 O 2 and other peroxide radicals, preventing lipid peroxidation and avoiding cell membrane damage, whereas GRd converts the oxidised form of glutathione (GSSG) to GSH [70]. In this sense, the presence of MPs can induce oxidative stress with an increase in the production of ROS that translates into an activation of antioxidant mechanisms, especially those related to the GSH cycle. In a study carried out in the liver of juvenile large yellow croaker Larimichthys crocea, GPx was observed to be the first enzyme to significantly increase in the presence of nanoplastics, whereas CAT, SOD, and MDA only increased with the highest MP concentrations [71]. These results match those observed in this study, since despite all of the activities being higher in the group with a greater abundance of MPs, the activities of SOD and CAT were not significantly higher, which suggests that the presence of MPs was not important enough. These results make sense, since in most studies carried out under controlled conditions, the exposure to MPs is much higher than in the natural environment. Similarly, GPx was also reported to increase in Cyprinus carpio liver when exposed to a plasticiser (di-n-butyl phthalate) [72], as well as in Carassius auratus, exposed to similar plasticisers [73]. GRd activity has also been found to increase at low to moderate levels of plastic contamination, as seen in the gut of Holothuria tubulosa, where for three areas with different plastic contamination (pristine, intermediate, and contaminated), is the only biomarker tested with significantly higher values for the intermediate location [74]. Likewise, the results for Sparus aurata showed that both GPx and GRd increase in liver tissues when exposed to plastics [75]. In addition, the MDA levels do not show significant differences between the two groups, which would indicate that the antioxidant mechanisms prevent an increase in the oxidation of biomolecules. Similar to what occurs with antioxidants, markers of the oxidative damage associated with MPs tend to show few changes in organisms obtained from the natural environment [24,29].
GST, a metabolic isozyme involved in phase II detoxification process, favours the elimination of pollutants by conjugating GSH and transforming them into a more hydrophilic product [27]. GST activity has been found to increase when in the presence of plastic pollution, suggesting an induction of the detoxification system [24,76]. Similarly, in the present study, GST activity significantly increased in the group with high MP presence, matching the results from both Alomar et al. (2017) [20] and Solomando et al. (2020) [24], which also reported increased GST activity related to the presence of a larger amount of ingested MPs in M. surmuletus and S. aurata, respectively. Furthermore, increased GST activity was also observed in the livers of M. surmuletus and the brain of B. boops when exposed to microplastics [77], as well as in the brains of S. aurata subjected to an MP-enriched diet [30]. Therefore, plastic presence seems to be closely linked to GST, GRd, and GPx in the case of the studied X. novacula liver samples. Although further studies are needed to confirm the biomarker that is most related with MP presence, these results suggest that enzymes such as GST, GRd, and GPx could be primary indicators of plastic pollution effects on fish for small concentrations of MP ingestion. Due to the small sample size, more studies are necessary to fully understand the long-term effects of microplastic exposure fish and ecosystem health. In addition, the difficulty of finding areas without anthropic influence is a limitation, since it does not allow knowledge of the baseline activity of the measured enzymes in this fish species for comparison. In fact, significant levels of MPs have been found on the coasts of the Marine Protected Area of the Cabrera Archipelago National Park, in the south of Mallorca Island [78]. Ongoing studies with other similar species will be able to better explain the effect of MP presence in the ecosystem.

Conclusions
In conclusion, X. novacula is exposed to MPs, since plastic items have been found in the gastrointestinal tract of practically all of the specimens analysed. A greater presence of MPs is associated with an activation of antioxidant enzymes, especially those related to the GSH cycle, as well as the GST detoxifying enzyme. These results highlight the potentially dangerous effects of ingesting MPs on X. novacula, and also emphasise possible early warning enzymes for oxidative stress. However, more research is required to provide information on the short-and long-term physiological effects of the ingestion of MPs on marine species, which biomarkers are most related to the presence of MPs, and to determine whether they may affect the fish population.