Environmental Research 212 (2022) 113433 Contents lists available at ScienceDirect Environmental Research journal homepage: www.elsevier.com/locate/envres Nanoplastics are bioaccumulated in fish liver and muscle and cause DNA damage after a chronic exposure I. Brandts a, b, M. Cánovas a, A. Tvarijonaviciute c, M. Llorca d, A. Vega d, M. Farré d, J. Pastor e, N. Roher a, b, M. Teles a, b, * a Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain b Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain c Interdisciplinary Laboratory of Clinical Analysis Interlab-UMU, Regional Campus of International Excellence Mare Nostrum, University of Murcia, Espinardo, Murcia, 30100, Spain d Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Barcelona, Spain e Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain A R T I C L E I N F O A B S T R A C T Keywords: The extent of the widespread, planetary contamination by plastic waste is difficult to fully capture. Nanoplastics Nanoplastics (NPs) are currently in the center of research concerning plastic litter, both for the analytical challenges they pose Quantification and for their potential to provoke hazardous effects in organisms. However, there are still many unanswered Chronic questions in this multidisciplinary field, with a crucial missing piece being the quantification of NPs in fish tissues Genotoxicity Liver after in vivo exposures. Another relevant question that is still greatly unexplored is how a chronic exposure to NPs Muscle will affect fish health. This study aims to provide answers to both of these relevant knowledge gaps. To this end, goldfish (Carassius auratus) were exposed to 44 nm polystyrene (PS)-NPs via water for 30 days. Following the exposure, gastrointestinal tract, liver and muscle were sampled for PS-NPs analysis by means of size exclusion chromatography coupled to high resolution mass spectrometry. PS-NPs were detected in all liver and muscle samples of exposed fish, with higher concentrations in liver than in muscle, whereas no PS-NPs were detected in the gastrointestinal tract. Nevertheless, exposure to PS-NPs did not induce changes in hematology parameters nor in cortisol and glucose levels in plasma. On the other hand, even a relatively low concentration of PS-NPs was able to cause DNA damage, measured by an increase in erythrocyte nuclear abnormalities, suggesting that PS- NPs can reach the cell nucleus and cause genotoxicity. These results show for the first time that PS-NPs find their way to fish muscle after chronic exposure, where they bioaccumulate, but do not alter fish survival nor hematological or physiological stress indicators. The accumulation of PS-NPs in fish muscle can represent a threat to human health as a possible route of exposure to small-sized plastics. The present results in a model fish species open windows for future studies in edible fish species. 1. Introduction addition, micro/nanometer-size particles are also industrially produced for multiple applications, constituting a second source of plastic parti­ Plastic pollution is a major ecological concern that is receiving cles entering the aquatic environment (Mitrano and Wohlleben, 2020). increasing attention from the general public, the scientific community Paradoxically, with this decrease in size that leads to virtual invisibility and governments. Plastic waste and fragments of all sizes reach aquatic in the environment, the concerns on the potential hazardous effects that ecosystems through multiple channels, such as wastewater discharges, these particles may have on living organisms increase (Koelmans et al., littering, illegal dumping, use of plastics for equipment/packaging in 2015). marine activities and coastal human activity. All these sources Nanoplastics (NPs), the particle size-fraction under 1000 nm (Hart­ contribute to the accumulation of plastic litter in the aquatic environ­ mann et al., 2019), are potentially one of the most hazardous marine ment, where it can persist for decades and potentially centuries, litter, as their physicochemical nanoscale properties allow them to cross breaking down to form micro- and nanometric size fragments. In biological barriers, including the intestinal wall and the mammalian * Corresponding author. Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain. E-mail address: mariana.teles@uab.cat (M. Teles). https://doi.org/10.1016/j.envres.2022.113433 Received 25 January 2022; Received in revised form 22 April 2022; Accepted 3 May 2022 Available online 14 May 2022 0013-9351/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). I. Brandts et al. Environmental Research 212 (2022) 113433 placental barrier (Stapleton, 2019). The detection and quantification of chromatography coupled to high-resolution mass spectrometry was set the environmental plastic nanofraction is an ongoing challenge, as the up. In order to evaluate fish general health status, blood and plasma, current analytical techniques to detect NPs in complex biological both non-lethal biological matrices, were collected and hematological matrices are not fully mature (Cai et al., 2021). Nevertheless, the pres­ parameters were determined using an automatic blood cell analyzer; ence of NPs in marine waters has already been confirmed, both in erythrocyte nuclear abnormalities (ENAs) were counted to assess DNA oceanic (Ter Halle et al., 2017) and in Mediterranean waters (Llorca damage; and key stress parameters, such as cortisol and glucose, were et al., 2021). Accurately measuring the concentration of NPs is a major quantified in plasma. Hematological parameters reflect the overall challenge, as precise analytical methods are required, accounting for physiological status of the animal and have been widely used to di­ various types of NPs polymers, as well as for a diverse set of complex agnose health status in several situations, including those of contami­ matrices (soil, sediments, turbid waters and tissues) (Ter Halle et al., nant exposure (Witeska et al., 2022). Moreover, there is evidence that 2017). exposure to NPs can exert DNA damage after a short-term exposure in Studies on the effects of NPs in fish have described alterations in the blood cells in aquatic species (Brandts et al., 2018a, 2021b), which oxidative status, changes in hormone levels, dysregulation of energy highlights the importance of evaluating the potential genotoxic effects metabolism, interferences with the immune system, gut dysbiosis and under a chronic scenario. It is relevant to focus on organisms belonging behavioral alterations (reviewed by Barría et al., 2020; Jacob et al., to higher trophic levels, such as are many fish species, as they are more 2020). However, knowledge regarding the occurrence and effects of NPs likely to bioaccumulate xenobiotics and may present a potential risk to in fish is still in its infancy, as research is mainly focused on humans through their ingestion. acute/short-term exposures with high plastic particle concentrations, whereas chronic toxicity has barely been assessed. To the best of the 2. Materials and methods authors’ knowledge, only six studies up to date have evaluated the ef­ fects of NPs in fish for exposure periods of more than 28 days, all pre­ 2.1. 1. Fish maintenance, bioassay and sampling sented in Table 1. NPs have also been observed to be distributed across food webs (Mattsson et al., 2015), but studies assessing the bio­ Adult C. auratus specimens (11.16 ± 3.23 cm length and 7.07 ± 0.64 distribution, bioaccumulation and the effects of chronic exposures to g weight) were randomly distributed in the experimental aquariums, NPs are still scarce. Considering that in nature organisms will most and two experimental conditions were considered: 1) Control group (0 probably be exposed to NPs during long periods of time, chronic studies μg/L PS-NPs) and 2) the group exposed to PS-NPs (100 μg/L PS-NPs). should be prioritized. The mechanisms behind the effects could be For each experimental condition, fish were randomly distributed in 4 different between short- and long-term exposures, and to extrapolate different aquariums (20 L) with 4 fish per tank, n = 16/condition; N = from one to the other might lead the world to wrong conclusions. 32. The number of animals used for each parameter was variable due to In the present study, the model fish Carassius auratus (goldfish) was the small size of the fish and to the variety of parameters determined. exposed for 30 days to polystyrene (PS)-NPs (PS-NPs), attempting to Information concerning the number of animals used for each parameter mimic an environmentally realistic scenario. The main hypothesis of this can be seen in the legends of the figures and in Table 2. Fish were study was that fish can take up PS-NPs from the water after a chronic exposed to PS-NPs for 30 days, following the Organization for Economic exposure and accumulate them in internal organs, causing alterations in Co-operation and Development’s guideline 215 (OECD, 2000). Fish fish health. PS-NPs were chosen, as PS contributes to a significant were fed daily ad libitum with a commercial diet, the bottom of the fraction of marine plastic waste (de Haan et al., 2019). Moreover, PS has aquariums was cleaned, and fish were observed for abnormal behavior. been identified as one of the polymers present in the nano-fraction of the Water physicochemical parameters were monitored daily to ensure few real NPs environmental samples (Llorca et al., 2021; Ter Halle et al., water quality, average values were: temperature (16 C◦ ), carbonate 2017). The exposure concentration chosen for this study takes into ac­ hardness (10 dkH), general hardness (6–7 dGH), pH (8.5), dissolved count predicted environmental concentrations of NPs (Lenz et al., 2016), oxygen (4 mg/L), nitrates (<10 mg/L), nitrite (<0.5 mg/L), ammonia together with concentrations used in other relevant studies with NPs (<0.15 mg/L), (<0.25 mg/L). Every 5 days, 75% of the medium was (Table 1), as well as on the previous results of the authors (Brandts et al., renewed and PS-NPs were added to each aquarium. The concentration of 2018b, 2020; 2021b), as no solid environmental quantifications of NPs available PS-NPs will vary during the 5 days, due to ingestion by the fish have been performed at present. Goldfish was chosen as a model since it and aggregation with food particles or feces, among others, resulting in a is a sturdy and easily manageable species, with demonstrated utility as a lower number of available particles in the medium over time and model organism in several areas of research (Blanco et al., 2018; Filice affecting the accumulation. After 30 days of exposure, all fish were et al., 2022). To test the hypotheses of uptake a protocol for the analysis euthanized by over-anesthetizing them in a tricaine methanesulfonate and quantification of NPs polymers by means of size exclusion (MS-222) bath (1 g/L). Fish were then weighed and measured, and Table 1 Summary of the main effects observed in fish after a chronic exposure to polystyrene nanoplastics (PS-NPs). Species Size Concentration Exposure Exposure time Main effects Reference (nm) route (days) Carassius 250 50, 500 and 5000 Water 28 Accumulation and histological lesions in gill, liver and intestine, activation Abarghouei et al. auratus μg/L of the antioxidant system. Changes in the expression of antioxidant related (2021) genes. C. auratus 500 260 and 690 μg/L Water 28 Inhibition the of olfactory G protein-coupled receptors and ATPases. Shi et al. (2021) Olfactory bulb injury and neurotoxicity. Danio rerio 105 10 μg/L Water 28 Bioaccumulation of EHS and its transfer from parents to offspring. Changes Zhou et al. in thyroid hormone levels. Generation of oxidative stress and damage. (2021) Carassius 53, 29,000 and Diet 67 Accumulation in brain and blood. Behavioral changes, reduced locomotor Mattsson et al. 180 100,000 μg/L activity and weight of loss. (2017) Morphological alteration in the brain (53 nm). C. carassius 24, 27 130 mg particles/ Diet 61 Effects on behavior and metabolism. Weight of loss. Mattsson et al. day (2015) C. carassius 24 0,01% (w/v) Diet 39 Decreased locomotor activity and feeding rate. Cedervall et al. Disturbance of the lipid metabolism and decreased survival rate (2012) 2 I. Brandts et al. Environmental Research 212 (2022) 113433 Table 2 mB. Samples were then homogenized with a Tissulyer II (Quiagen) using Number of fish used for the different analysis performed (n) and p value obtained 5 mm stainless steel spheres and the following cycle: 1 min at 25 Khz, 1 for each parameter (control group versus polystyrene nanoplastics, PS-NPs, min in ice, followed by another cycle of 1 min at 25 Khz. subsequently, exposed group. Statistically significant results are marked in bold and consid­ samples were immediately stored at − 80 ◦ C until further analysis. Next, ered when p < 0.05. NPs were extracted from fish tissues and purified according to the Parameter n p value methodology described by Schirinzi et al. (2020). Briefly, the samples PS-NPs concentration in the liver 9 0.0057 were digested for 6 h with KOH (10%) in a proportion 1:3 (w/v) in a PS-NPs concentration in the muscle 9 < 0.0001 stove at 60 ◦ C and left to rest at room temperature overnight. After­ Hematocrit 7 0.5423 wards, the samples were filtered through fiberglass filters and the Hemoglobin 7 0.7568 clogged filters were digested with HNO3 at 20% for 1 h at room tem­ Red blood cell count 7 0.5303 Mean corpuscular cell volume 7 0.8329 perature, and the acid was eliminated through filtration. These filters White blood cells count 7 0.5697 were then cleaned with water and dried overnight at 60 ◦ C. Finally, the Thrombocyte count 7 0.2571 extraction of NPs from the filters was carried out with 10 mL of toluene Lymphocytes count 7 0.6014 by means of ultrasonic assisted extraction (USAE) for 30 min and af­ Monocytes count 7 0.5934 terwards collected and transferred to amber glass vials. This procedure Neutrophils count 7 0.5594 Eosinophils count 7 0.2231 was repeated twice, and the final extracts were combined and evapo­ Fish weight 9 0.5624 rated under nitrogen stream to concentrate the samples to a volume Fish length 9 0.3220 close to 1.5 mL. The extracts were then vortexed, centrifuged (5376 g, Fulton’s condition factor 9 0.3711 10 min), and the supernatant transferred to LC-vials and evaporated Hepatosomatic index 9 0.5917 Micronucleus 13 0.0002 again until 1.0 mL remained. Reniform nucleus 13 0.0003 Segmented nucleus 13 0.8571 2.4. Analysis and quantification of polystyrene nanoplastics using size Lobed nucleus 13 0.3834 exclusion chromatography coupled to high-resolution mass spectrometry Total erythrocytic nuclear anomalies 13 0.0002 Cortisol 7 0.7207 Glucose 7 0.2655 The analysis of NPs was performed by size exclusion chromatog­ raphy (SEC) coupled to high-resolution mass spectrometry (HRMS), equipped with an atmospheric pressure photoionization (APPI) working heparinized syringes were used to immediately draw the blood from the under negative conditions. The chromatographic system Acquity LC caudal vein. Blood smears were made immediately after collection, and (Waters, Milford) was equipped with an advanced polymer chromato­ the remaining blood was kept in Eppendorf tubes (in ice) with heparin graphic column (Acquity APC XT45 1.7 μm) working with isocratic for posterior hematological analysis. Liver, muscle and the gastrointes­ conditions with toluene according to (Schirinzi et al., 2019). The chro­ tinal tract were excised, weighted, snap-frozen in liquid nitrogen and matographic system was coupled to a Q-Exactive (Thermo Fisher Sci­ kept at − 80 ◦ C until further analysis. All experimental procedures entific) hybrid quadrupole-Orbitrap mass spectrometer. Data involving fish were carried out according to the 3 R’s of Animal acquisition was performed in full scan mode (m/z 500–3000) with a Experimentation (Replacement, Reduction, and Refinement), following resolution of 17,500 FWHM (Llorca et al., 2021). The whole system is Spanish legislation (law 32/2007 and RD53/2013) that agrees with the controlled by Xcalibur 3.0 software. The identification was performed International Guiding Principles for Biomedical Research Involving by matching with a calibration curve of PS’ standard (MW~1200Da, Animals (EU, 2010/63). supplied by Polymer Standard Service GmbH (PSS, Mainz, Germany) in toluene. An example of chromatogram and spectra can be seen in Fig. 2a. 2.2. Polystyrene nanoplastics characterization Finally, the quantification was done by means of equivalent concen­ tration according to the procedure described by Llorca et al. (2021). Polystyrene nanoplastics (PS-NPs, 44 nm size) were purchased from Calculations were done with respect to the wet weight of the organs. Bangs Laboratories (ref. PS02002). The particles were characterized in ultrapure water (MiliQ) and in the water used in the experiment (AQ 2.5. Fitness indicators water), by dynamic light scattering (DLS) (Zetasizer Pro, Malvern). For this purpose, five dilutions were prepared (1:102, 1:103, 1:104, 1:105 and Fish survival was 100% in both experimental groups during all the 1:106) from the original stock of PS-NPs (100 mg/mL) in both media and exposure trial. At the end of the experiment, fish length was determined hydrodynamic size, polydispersity index (PDI) and the zeta potential (cm) by measuring from the anterior end to the tip of the caudal fin. Fish were measured. The hydrodynamic size measures the diameter of a were also weighted (g) and length and weight were used to calculate particle by measuring the scattered light from a dispersion containing Fulton’s condition factor (K factor). Afterwards, liver was excised and diffusing particles, while de PDI estimates the width of this particle size weighted (g) and the values used to calculate the hepatosomatic index distribution (Ragheb and Nobbmann, 2020). The zeta potential mea­ (HSI). K factor and HSI were calculated as follows (Rizzo and Bazzoli, sures the electrostatic repulsion/attraction between particles and is an 2020): indicator of stability or dispersion in colloidal suspensions (Parupudi ( / ) et al., 2022). In order to observe changes in PS-NPs through time, K = weight(g) length(cm)3 × 100 nanoparticle hydrodynamic size and the zeta potential were assessed at 0, 24, 48, 72, 96 and 120 h. To further characterize particle size and HSI = (liver weight / total weight) × 100 morphology in MiliQ and AQ water, transmission electron microscopy (TEM, Jeol 1400 120 KV) was used. Following acquisition, images were 2.6. Hematological profile processed using ImageJ, where a minimum of 150 particles for each condition were measured. Blood samples were analysed using an automated laser flow blood cell analyzer (Sysmex XN-1000 V) with the veterinary software version 2.3. Extraction of polystyrene nanoplastics from biological samples 3.04 with a betta version for bird blood analysis, adapted for fish. He­ matocrit, red blood cell count, leucocyte count, hemoglobin concen­ Liver, gastrointestinal tract and muscle samples (n = 9; N = 18) were tration, mean corpuscular cell volume, and thrombocyte count were lyophilized using a CRYODOS-80 (Telstar) for 72 h at − 75 ◦ C and <0.15 determined. Blood smears for differential leucocyte count were stained 3 I. Brandts et al. Environmental Research 212 (2022) 113433 with panoptic fast staining. Manual count of lymphocytes, monocytes 2.7. Erythrocytic nuclear abnormalities neutrophils and eosinophils were determined in blood smears counting 100 leukocytes per smear. Erythrocytic nuclear abnormalities (ENAs) were classified into four categories (micronuclei, reniform-shaped, segmented and lobed nuclei) Fig. 1. a) Characterization of polystyrene nanoplastics (PS-NPs) by dynamic light scattering at 0, 24, 48, 72, 96 and 120 h. Hydrodynamic diameters, polydispersity index (PDI) and zeta potential (mean ± standard deviation) of PS-NPs in ultrapure water (MQ) and aquarium water (AQ) over time, at 104 μg/L b) Transmission Electron Microscopy images of the PS-NPs in MQ and c) AQ, with the corresponding size distribution histograms. 4 I. Brandts et al. Environmental Research 212 (2022) 113433 according to Pacheco and Santos (1996). Blood smears were prepared on glass slides and fixed with methanol for 10 min. Slides were stained with 2.8. Cortisol and glucose 5% Giemsa for 30 min. Subsequently, blood smears were stained with panoptic fast staining. A total of 1000 erythrocytes per sample were The levels of cortisol were measured using a commercially available examined using an optical microscope (100X magnification). Only solid-phase, competitive chemiluminescence enzyme immunoassay erythrocytes with intact nuclear and cytoplasmic membranes were (COR Cortisol, REF LKC01, Siemens Health Diagnostics, Deerfield, IL) in considered. The results were expressed as the frequency of ENAs the automatic analyzer (Immulite® 1000 analyser; Immulite System; observed (‰) in a total of 1000 cells. Differential anomalies were also Siemens Health Diagnostics, Deerfield, USA) (Franco-Martinez et al., presented in the figures. The following formula was used to determine 2019). Plasma glucose levels were determined using the hexokinase the total number of ENAs: G-6-PDH based commercially available method (OSR6121, Beckman ENAs(%0 ) = Number of cells with ENAs/Total number of cells counted Coulter Ireland Inc, Ireland) and following the manufacturer’s in­ dications in an automatic analyzer (Olympus Diagnostica, GmbH, Fig. 2. a) Polystyrene nanoplastics (PS-NPs) chromatogram and mass spectra of the common loss for PS of 104 Da. b) PS-NPs concentration in the liver and muscle of goldfish (Carassius auratus) after 30 days of exposure to PS-NPs. Values are presented as mean ± standard deviation. Asterisk (*) denotes significant differences versus control (p < 0.05). 5 I. Brandts et al. Environmental Research 212 (2022) 113433 Freiburg, Germany). test. Significant differences between the control group and the group exposed to NPs are marked in figures with an asterisk (*) and considered when p < 0.05. 2.9. Data analysis 3. Results Results are expressed as mean ± standard deviation (for n informa­ tion see Table 2). The statistical data analysis was done using GraphPad 3.1. Polystyrene nanoplastics characterization Prism version 8.0.1 for Windows (GraphPad Software, Inc, www.gra phpad.com). The data was first checked for normality using the The DLS measurements show that the hydrodynamic diameter of PS- Shapiro-Wilk test. Data that followed a normal distribution was further NPs and PDI increased as the concentration of PS-NPs decreased in both analysed with a t-test and the data that did not follow a normal distri­ tested media. However, in MiliQ, the hydrodynamic diameter of PS-NPs bution was further analysed using the Mann-Whitney nonparametric Fig. 3. a) Hematological indicators determined in control group and in goldfish (Carassius auratus) exposed to polystyrene nanoplastics (PS-NPs) for 30 days. Values are presented as mean ± standard deviation. b) Blood cell identification in goldfish blood using an optical microscope (100 x). A) Erythrocytes; B and C) Lym­ phocytes; D and E) Neutrophils; F and G) Monocytes; H and I) Thrombocytes; J) Eosinophils. 6 I. Brandts et al. Environmental Research 212 (2022) 113433 remained more stable than in AQ water (Fig. 1). As shown in Fig. 1, the 3.2. Polystyrene nanoplastics concentration levels in muscle, liver and hydrodynamic diameter of PS-NPs and PDI also increased over time in gastrointestinal tract both media. In ultrapure water, the hydrodynamic diameter of PS-NPs ranged from 46 to 51 nm. However, in AQ the values ranged from 73 PS-NPs were not detected in the gastrointestinal tract of exposed fish. to 349 nm. Zeta potential analysis showed that, in MiliQ water, PS-NPs In contrast, PS-NPs were detected in all liver and muscle samples of the were negatively charged with values between − 14 and − 25 mV, but exposed fish (Fig. 2b). In liver, 7 out 9 samples had concentrations levels these decreased to − 6 to − 9 mV after 48 h. At the AQ medium, zeta ranging from 2.23 to 265.89 ng/g liver wet weight; in the other two liver potential ranged between − 12 and 13 mV. This points to a lower sta­ samples PS-NPs were below the limit of detection. In 6 out of 9 muscle bility of dispersion in MiliQ water after 48 h and in AQ water at all samples, PS-NPs were detected at concentrations ranging from 9.58 to measured times; however, PDI values indicate higher polydispersity in 18.64 ng/g of muscle wet weight. The concentrations of PS-NPs were AQ water. significantly higher in the liver and muscle of fish exposed to PS-NPs for 30 days when compared to the control group. Fig. 4. Biological indicators determined in control group and in goldfish (Carassius auratus) exposed to polystyrene nanoplastics (PS-NPs) for 30 days. Values are presented as mean ± standard deviation. Asterisk (*) denotes significant differences versus control, p < 0.05. a) Biometric indicators. b) Genotoxicity indicator. Different erythrocytic nuclear anomalies (ENAs) found and total ENAs (per 1000) scored in the blood of the goldfish. c) Stress indicators. Cortisol and glucose levels determined in plasma of the goldfish. 7 I. Brandts et al. Environmental Research 212 (2022) 113433 3.3. Biological effects of polystyrene nanoplastics in goldfish after 20 days of exposure to 34 μg/L PS-NPs (26 nm) (Guimarães et al., 2021a). The concentration values observed in the present study, with an In this study, PS-NPs exposure did not affect fish fitness indicators, as approximate average of 100 ng/g liver wet weight, would match that fish survival was 100%, food consumption and fish behavior were obtained in C. idella and fall lower than the other two estimated quan­ similar in both control and exposed fish. The effects of PS-NPs in the tifications. Nevertheless, the available quantifications for NPs have been hematological parameters (hematocrit, hemoglobin concentration, red estimated by measuring the fluorescence emitted by fluorescently blood cell count, mean corpuscular cell volume, leucocyte count, labeled MPs and NPs, which might not always faithfully reflect the mass thrombocyte count, and the differential count of lymphocytes, mono­ of plastic particles (Catarino et al., 2019). In some cases, very small MPs cytes neutrophils and eosinophils) are shown in Fig. 3a. No statistical (5 μm) have also been found in liver, with Lu et al. (2016), Ding et al. differences were observed in the studied hematological parameters in (2020) and Qiao et al. (2019) documenting the presence of 5 μm plastic PS-NPs exposed fish compared to the control group. Fig. 3b shows the particles in the fish liver after exposures of 7, 14 and 21 days, respec­ morphology of the different types of leucocytes found in the blood of tively. There is contradictory information on the size of plastic particles goldfish. The exposure to PS-NPs had no significant effects in the body that could be translocated to the liver, as some studies report that par­ weight, length, condition factor nor liver weight of the fish (Fig. 4a). A ticles over 20 μm cannot be found in the liver (Lu et al., 2016; Su et al., significant increase in the total number of ENAs, a genotoxicity indica­ 2019), whereas in other studies particles of up to 90 μm have been found tor, was found in the group of fish exposed to PS-NPs, when compared to in liver (Ding et al., 2020). The liver is one of the largest internal organs control (Fig. 4b). The number of erythrocytes presenting reniform nuclei in fish and has a significant role in multiple vital processes, such as and micronuclei was significantly higher in fish exposed to PS-NPs than nutrient processing and storage, protein synthesis, detoxification of in control fish. However, lobed and segmented nuclei values were autogenous metabolites, bile production and secretion, as well as drug similar in both groups of fish. The levels of cortisol and glucose in and xenobiotic metabolism (Olsvik et al., 2007). The accumulation of plasma, were not statistically different between exposed and control fish NPs in fish hepatocytes has been previously documented in vitro, with (Fig. 4c). the intracellular localization mainly located in lysosomes (Brandts et al., 2020). The accumulation in this organelle is probably related to its role 4. Discussion in xenobiotic metabolism (Gu and Manautou, 2012), but could also lead to alteration in liver metabolisms, due to this organelle’s role in lipid 4.1. Polystyrene nanoplastics bioaccumulate in liver and muscle of metabolism (Thelen and Zoncu, 2017). Changes in liver metabolomics goldfish profile have been previously seen in Carassius carasssius and in D. rerio after exposure to NPs, showing a disruption in lipid and energy meta­ In this study, protocols for the extraction and quantification of NPs in bolism (Mattson, 205; Lu et al., 2016). Moreover, alterations in key fish tissues were set up. Following these protocols, the present data transcripts related to lipid metabolism have been found in liver of both demonstrates that 44 nm NPs can be taken up from the water, crossing Sparus aurata and Dicentrarchus labrax (Brandts et al., 2018b, 2021b). contact surfaces (gills, gastrointestinal tract), translocated and accu­ Oxidative stress, one of the main mechanisms of action of NPs (Hu and mulated in inner organs, after a 30-day exposure quantifiable levels of Palić, 2020), has also been found in both Larimichtys crocea and S. aurata PS-NPs were found in liver and muscle of goldfish. However, no PS-NPs livers after exposure to NPs, described as altered gene expression, could be found in gastrointestinal tract samples. Although NPs have not enzyme activity and lipid peroxidation (Brandts et al., 2021b; Lai et al., yet been detected in tissue samples of animals captured from the envi­ 2021; Li et al., 2021). The action of oxidative species generated due to ronment, microplastics (MPs) have been sampled from the gastrointes­ NPs exposure could be related to tissue damage, which has been re­ tinal tracts of multiple fish species (Azevedo-Santos et al., 2019). ported in both goldfish and Epinepehlus coioides, following NPs exposure Furthermore, Dawson et al. (2018) showed that MPs could be turned (namely vacuolization, infiltration and necrosis). into NPs inside the digestive tract of Antarctic krill (Euphausia superba), a Due to its role as part of the digestive system, the liver has an optimal process that could potentially also take place in fish (Dawson et al., position for gathering, transforming, accumulating, and eliminating 2018). Recently, Clark et al. (2022) documented the translocation of substances, as substances absorbed in the gastrointestinal tract have a palladium-doped PS-NPs (ca. 200 nm) through an ex vivo gut sac direct highway to the liver via the portal vein, which transports 70–80% exposure system, with the highest passage occurring in the anterior in­ of the blood that reaches the organ (Akiyoshi and Inoue, 2004). testine, and up to 700 million particles passing through the intestinal Therefore, following this absorption route and considering the role of barrier after 4 h. The results from the present study suggest that PS-NPs the liver in xenobiotic metabolism, the accumulation of NPs in this ingested by goldfish effectively crossed the intestinal barrier, not organ, as well as the high levels found in some of the samples, could be remaining in the gastrointestinal epithelia but reaching inner tissues, expected. Blood coming from the digestive system is processed by the namely liver and muscle, potentially after being distributed through the liver as it flows through the sinusoids and is finally collected by the blood. The translocation of NPs to inner tissues has been documented in central vein, which will eventually proceed to the systemic circulation. teleost fish (Brun et al., 2018; Chae et al., 2018; Skjolding et al., 2017), Through this route, NPs that have not been intercepted in the liver could although mainly through the observation of fluorescence after the be transported to other organs. administration of fluorescent NPs particles. To the best of the authors’ In this study, results show that PS-NPs can find their way to fish knowledge, the direct quantification of nanometric plastic polymer muscle, potentially through said circulatory deliverance. To the best of particles in fish organs has not been previously reported. the authors’ knowledge, only one laboratory study has quantified NPs in Previous studies have documented the presence of NPs in fish liver fish muscle, and it was done through the indirect quantification of after controlled laboratory exposures, and several of them have esti­ fluorescent NPs. In that study, Chen et al. (2017) found accumulation of mated NPs’ concentrations by indirect measurement of fluorescence. In NPs in the muscle of zebrafish after 3 days of exposure to 1000 μg/L of a study with red tilapia (Oreochromis niloticus), Ding et al. (2020) 50 nm fluorescent PS-NPs, with estimated concentrations ranging from calculated a concentration of 98,200 ng/g liver of 300 nm PS-NPs after 64,000 to 49,000 ng/g of muscle weight. These values are a thousand 14 days of exposure to 100 μg/L. With a similar methodology, Sar­ times higher than those found in the present study, with NPs concen­ asamma et al. (2020) estimated a concentration of 2000 μg/g protein of trations ranging from 9.58 ng/g to 18.64 ng/g of muscle weight. A large PS-NPs (70 nm) in zebrafish liver after 30 days of exposure to 1500 μg/L number of dissimilarities between both studies could explain such dif­ of PS-NPs. In a recent study, Guimarães et al. (2021a) estimated the ference in order of magnitude; including the fish species, the exposure concentration of NPs through indirect fluorescence quantification, concentrations, and the quantification methodology, as Chen et al. obtaining values of 100 ng/g liver wet weight in Ctenopharyngodon idella (2017) estimated the concentration from the emission of fluorescence 8 I. Brandts et al. Environmental Research 212 (2022) 113433 while the values presented here directly quantified polymer concentra­ 4.2. Polystyrene nanoplastics do not alter fish morphological nor tion. The accumulation of NPs in fish muscle can have detrimental ef­ hematological indices but induces DNA damage fects on this tissue, as shown by Yang et al. (2020) in goldfish larvae, where they found damage and mitochondrial vacuolization in muscle In this study, the Fulton’s condition factor (K) values were higher mesenchymal cells, nerve fiber atrophy and decreased acetylcholines­ than 1 in both experimental groups, which indicates that all fish were in terase (AchE) activity, after a short-term exposure to PS-NPs (70 nm). good health condition during the experiment and no changes in the The generation of reactive oxygen species found by the authors could nutritional condition were found between control and exposed fish. This explain this induced cell injury. In a study with carnivorous fish was also confirmed by the behavior that the animals presented during L. crocea, Lai et al. (2021) found alterations in fish muscle flesh quality the experiment, i.e., no changes in feeding behavior, ventilatory activity, after an exposure to NPs (80 nm), which could be a consequence of the swimming behavior and no signs of aggressiveness. Interestingly, described tissue damage. Moreover, a previous study by the authors although fish liver accumulated PS-NPs in all exposed fish, no changes in found that a short-term exposure to NPs (45 nm) induce an antioxidant HSI under the present experimental conditions were found. The HSI is a response, altered AchE activity and inhibited key transcripts related to widely known indicator of contaminant exposure due to the critical role muscle development in muscle of S. aurata (Balasch et al., 2021). Effects of the liver in the detoxification of xenobiotics, which can lead, e.g., to an on fish growth after exposure to NPs have been previously reported in increase in liver size due to hypertrophy or hyperplasia of the hepato­ other fish species (Gu et al., 2020; Lai et al., 2021; Wang et al., 2022). cytes (Ahmed and Sheikh, 2019). HSI, together with K, are also used as The diversion of energy reserves to respond to oxidative stress and indirect indicators of the energy status. Therefore, it seems that under inflammation, both systemically and locally in muscle tissue, could the assessed experimental conditions, the global health condition and partially explain this decrease in growth, together with the alterations in energy status of the animal was not affected by the exposure to PS-NPs. muscle growth regulators and in nutrient metabolism (i.e., lipids and These results agree with previous findings obtained for S. aurata exposed carbohydrates.). to a concentration range (0, 0.001, 0.01, 0.1, 1 and 10 mg/L) of 45 nm The ingestion of NPs has been suggested as the main route of expo­ polymethylmethacrylate (PMMA)-NPs for 96 h (Brandts et al., 2021b), sure for humans, as MPs have already been found in edible products, where neither HSI nor K were altered. Even if changes in K and HSI in the such as seafood, honey, beer, salt and sugar, as well as in both tap and present study could have been expected, due to the longer duration of bottled water (Lehner et al., 2019). Nevertheless, fish have not been the exposure to PS-NPs (acute vs. chronic), this was not the case. strongly considered a risk for plastic particle exposure for humans, as it Blood analysis is a reliable and inexpensive tool for the assessment of has been assumed that MPs ingested by fish will remain in the gastro­ animal health via non-lethal sampling; therefore, reducing animal sac­ intestinal tract and, therefore, be discarded before the meat is ingested. rifice in experiments as recommended by the European Union (Direc­ However, when NPs, and possibly small MPs (for example 5 μm) are tive, 2010/63 EU). In the present study, the hematological indicators considered, we cannot assume that particles will stay in the gastroin­ were unchanged in goldfish blood chronically exposed to PS-NPs. It is testinal tract. This study provides evidence that fish species of which difficult to compare the present data with previous results since, to the only the meat is ingested should be considered as another relevant best of the authors’ knowledge, there are no available studies assessing source of NPs for humans. Fish muscle represents a large proportion of the effects of NPs on fish hematology. A few previous studies where fish the body mass, of up to 60% depending on the fish species (SäNger and were exposed through the water to MPs, showed changes in some he­ Stoiber, 2001) and, therefore, could potentially store a large amount of matological parameters (Hamed et al., 2019; Hodkovicova et al., 2021). the NPs accumulated in fish. Moreover, as trophic transfer of NPs within Hamed et al. (2019) evaluated the effects of a 15-day exposure to MPs aquatic organisms has been demonstrated (Cedervall et al., 2012; through the water on juveniles of the freshwater fish Oreochromis nilo­ Mattsson et al., 2015), eating predator fish from higher trophic level ticus (Nile tilapia). They found changes (increases or decreases, might increase the risk of exposure to relevant concentrations of the depending on the parameter) in hematological indices, such as the red pollutants, as occurs with heavy metals. blood cell count, hemoglobin concentration, hematocrit value and white It is relevant to note that most of the detections or approximate blood cell count. These authors do not specify the nature of the MPs used quantifications of NPs in fish organs up to date are indirect and based on in the study. Hodkovicova et al. (2021) also found changes in total red the use of fluorescently marked MPs and NPs and not on the detection of blood cells count and hematocrit in rainbow trout (Oncorhynchus mykiss) the polymer itself. Fluorescence is very useful to visualize particles, and exposed to 50 μm MP for six weeks. In mammals, mice that received it can be used to localize the plastic particles in tissue. However, some PS-NPs daily during 28 days by oral gavage presented changes in he­ authors have reported fluorophore leaching from fluorescent-labeled matological indices, such as a decrease in white blood cell count (Xu nanoplastics, which PS can lead to the accumulation of fluorophore et al., 2021). Taking together these findings, changes in blood parame­ alone within in internal tissues, potentially overestimating NPs con­ ters of the exposed goldfish could be expected in the present study, centration (Catarino et al., 2019). Moreover, with fluorescence, the especially because the exposure time was longer than the exposure smallest particles (or aggregated) can be challenging to be detected and duration carried out in the other studies. However, this was not the case. the tissue distribution can also influence the result. The advantage of In fact, comparing the effects of NPs with that of MPs could be a mistake, size-exclusion coupled to high-resolution mass spectrometry is that it as NPs differ from MPs with respect, for example, to their transport allows identifying the different types of polymers and quantifying them properties, bioavailability and diffusion times, and effects of both con­ in terms of the weight of a polymer by gr of tissue, simultaneously. This taminants could be different (Gigault et al., 2021). Moreover, other technique solubilizes the plastic particles independently of the size or differences between studies, such as the species, animal age (adults vs. their distribution. Moreover, the method proposed shows high sensi­ juveniles), dynamic of the particles inside the organism, concentration tivity, enough to be used with real samples or low concentrations in or exposure time, are all factors that make this issue extremely complex. exposure experiments, as in the present study, in which realistic con­ In conclusion, based on the available studies it was very difficult to ditions of exposure are considered. This method has been already vali­ predict the effects of PS-NPs on blood parameters of goldfish and the dated and applied for real samples (Llorca et al., 2021). On the other absence of hematological changes could be due to a capacity of the hand, some other studies rely on metal-doped NPs, in order to be able to animal to cope with the stress caused by the absorbed PS-NPs. monitor their accumulation in tissues. While both fluorescent and Present data suggest that a chronic exposure to 44 nm NPs can induce metal-doped particles have proven useful to contribute to understanding DNA damage in goldfish red blood cells. This is evidenced by the re­ the behavior of NPs inside organisms, they do not provide appropriate ported increase in ENAs of exposed fish, with micronuclei being the most tools to detect and monitor NPs in real environment of food samples. frequently recorded abnormality. Micronuclei and other nuclear ab­ normalities can form during the proliferative phase of the cell cycle, 9 I. Brandts et al. Environmental Research 212 (2022) 113433 inducing permanent damage that can only be fixed through DNA repair one referenced above (Brun et al., 2019), where the results differed from or apoptosis. ENAs have been extensively used as indicators of exposure the present data. Considering the few available data on the effects of NPs to mutagenic or genotoxic contaminants in numerous fish species for a on cortisol and glucose concentrations, variability of these responses variety of chemicals such as PCBs, PAHs, heavy metals, and pesticides may depend on the fish species and experimental protocol; more studies (Braham et al., 2017). It can be hypothesized as to whether the increase in this direction could be valuable. in ENAs observed in this study is due to oxidative stress provoked by the PS-NPs or to the interaction of NPs with the chromosomal material, as 5. Concluding remarks both are plausible explanations. Oxidative stress has been widely re­ ported as a consequence of exposure to NPs (Jacob et al., 2020), and this Taken together, present data suggest for the first time that: 1) fish imbalance between radical species and the antioxidant defense systems chronically exposed to 44 nm PS-NPs internalize these emergent con­ can lead to damage to biomolecules, including DNA; therefore poten­ taminants from the water. PS-NPs are then distributed in the organism, tially inducing ENAs (Juan et al., 2021). On the other hand, NPs have potentially through blood, and bioaccumulate in internal organs, such as shown the ability to be internalized by multiple cell types (e.g., Brandts the liver and the muscle. 2) Higher levels of PS-NPs were found in the et al., 2020; Rubio et al., 2020; Sendra et al., 2020; Xu et al., 2019), thus, liver when compared to the muscle. No bioaccumulation was found in their direct interaction with chromosomes is also a possible scenario. the gastrointestinal tract which agrees with the main function of this These results corroborate previous findings showing that short-term organ, i.e., absorption. Considering that fish consumption is increasing exposures to a concentration range (from 0.05 up to 50 mg/L) of 100 steadily worldwide, the fact that PS-NPs are bioaccumulated in fish nm PS-NPs were able to induce DNA strand breaks (comet assay) in the muscle, the edible part of the animal, means that these contaminants hemocytes of the Mediterranean mussel (Mytilus galloprovincialis) and may be ingested by humans, presenting a potential threat to human chromosome damage (scoring the ENAs frequency) in erythrocytes of health. 3) Despite the capability of internalization and bioaccumulation, the S. aurata exposed to 45 nm PMMA-NPs (0.01–10 mg/L) (Brandts PS-NPs did not induce changes in fish growth, nor in hematological and et al., 2018a, 2021b). Therefore, the present data confirm that NPs have stress indicators. 4) PS-NPs induced genotoxicity in fish blood cells, genotoxic potential, able to induce DNA damage, after both short and which can escalate to mutagenicity and is related to more serious con­ long-term exposures. Results obtained by other authors in mussels ditions such as cancer or degenerative conditions. (Gonçalves et al., 2022), fish (Estrela et al., 2021; Guimarães et al., 2021b) and human blood cells (Ballesteros et al., 2020; Gopinath et al., Credit author statement 2019) support previous and current findings, reinforcing the idea that PS-NPs are genotoxic contaminants. This is of concern, since the accu­ I. Brandts: Research conception and design, Data acquisition, mulation of DNA damage in cells play a role in their mutagenic poten­ Formal analysis, Manuscript preparation, Final approval. M. Cánovas: tial, which can lead to cancer, as well as to the occurrence of Data acquisition, Formal analysis, Manuscript preparation, Final degenerative conditions such as accelerated aging and immune approval. A. Tvarijonaviciute: Resources, Data acquisition, Final dysfunction. approval. A. Vega: Data acquisition, Final approval. M. Llorca: Data acquisition, Formal analysis, Final approval. M. Farré; : Resources, Data 4.3. Polystyrene nanoplastics do not affect goldfish response to stress acquisition, Formal analysis, Final approval. J. Pastor: Resources, Data acquisition, Formal analysis, Final approval. N. Roher: Research Generally, fish respond to stress with increases in plasma cortisol, conception and design, Resources, Data acquisition, Formal analysis, their main corticosteroid. In teleost fish, cortisol plays a major role in the Final approval. M. Teles: Research conception and design, Resources, stress response and plasma cortisol levels are considered a marker for Data acquisition, Formal analysis, Manuscript preparation, Final the intensity of hypothalamo-pituitary-interrenal (HPI) axis regulation approval under stressful situations (Balasch and Tort, 2019). This hormone is also known to increase the energy availability during stress. This task is Declaration of competing interest accomplished primarily through gluconeogenesis, resulting in higher levels of plasma glucose. Nevertheless, fish responses to stressors The authors declare that they have no known competing financial demonstrated to be more complex, since different patterns of response interests or personal relationships that could have appeared to influence have been observed either concerning the cortisol variation (Pacheco the work reported in this paper. and Santos, 2001; Teles et al., 2004) or its relationship with the sec­ ondary stress responses (Teles et al., 2017). In the present study, a Acknowledgements chronic exposure to PS-NPs resulted in unchanged levels of plasma cortisol, which agrees with previous results concerning a short-term This research was supported through Plan Nacional de Investigación exposure of D. labrax to 0.20 and 20 mg/L of PS-NPs (Brandts et al., with reference PID 2020-113221RB-I00 and through the project with 2021a). Despite the importance of cortisol, to the best of our knowledge, reference PLAS-MED; CTM 2017-89701-C3-2-R. MT is supported by a there is only one more study, where the effects of PS-NPs in cortisol and Ramon y Cajal contract (ref. RYC 2019-026841-I) and IB is supported by glucose levels in fish are evaluated. In that study, Brun et al. (2019) a PhD grant from Generalitat de Catalunya (2018FI_B_00711). ML ac­ exposed zebrafish larvae to three concentrations of 25 nm PS-NPs for 2 knowledges DEGBIOPLAST (RTI 2018-097860-J-I00) and AV his PhD days and measured cortisol and glucose levels, finding increased grant PRE 2018-083989, both from the Spanish Ministry of Science and whole-body cortisol levels but decreased glucose (Brun et al., 2019). Innovation. However, it is difficult to compare a short-term exposure in larvae with a long-term exposure of adult fish from another species. 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