Ecological Indicators 147 (2023) 109966 Contents lists available at ScienceDirect Ecological Indicators journal homepage: www.elsevier.com/locate/ecolind Evaluation of a chronic exposure to nanoplastics in goldfish (Carassius auratus): Analytical validation of automated assays for the measurement of biochemical markers M. Blonç a, 1, I. Brandts a, b, 1, M. Cánovas b, L. Franco-Martínez c, d, C.P. Rubio e, L. Tort a, A. Tvarijonaviciute c, C. Gravato f, M. Teles a, b, * a Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain b Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain c Interdisciplinary Laboratory of Clinical Analysis Interlab-UMU, Regional Campus of International Excellence Mare Nostrum, University of Murcia, Espinardo, Murcia 30100, Spain d Moorepark Animal and Grassland Research Center, Teagasc, Irish Agriculture and Food Development Authority, P61 C996 Cork, Ireland e Department of Animal and Food Science, School of Veterinary Medicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain f Faculty of Sciences of University of Lisbon, Campo Grande 1749-016 Lisboa, Portugal A R T I C L E I N F O A B S T R A C T Keywords: The bulk of plastic pollution is mainly composed of small fragments, micro- and nanoplastics (NPs). Although Automated assay validation many studies are currently published on NPs, research on the effects of NPs in fish after a chronic exposure is still Biomarkers scarce. The present study aimed to validate a series of automated assays to be used in the monitoring of fish Nanoparticles challenged with a chronic exposure to NPs, using Carassius auratus (goldfish) as model species. For this purpose, Fish adult C. auratus were exposed to 100 µg/L polystyrene (PS)-NPs for a 30-day period. Total oxidative status (TOS), total antioxidant capacity (TAC), esterase activity (EA) and adenosine deaminase activity (ADA) were measured in the gills, brain and muscle of fish. In addition, acetylcholinesterase activity (AChE) and creatinine kinase (CK) were measured in the muscle. All biomarkers were successfully validated in goldfish tissues and consequently used to assess the effects of NPs following a chronic exposure. Results showed that EA and TAC significantly increased in gills, while EA decreased significantly in the brain, and no effects were observed in any of these parameters in muscle. These results indicate that both immune and antioxidant responses were triggered by NPs in gills, but not in the brain nor muscle. This suggests that gills may be a primary target for NPs, potentially leading to a cascading effect on gas exchange, or osmo- and ionic regulation that should be further investigated. 1. Introduction and Terova, 2020). NPs appear as emerging contaminants in both nat­ ural and urbanised environments (Cai et al., 2021; Materić et al., 2022), Plastic pollution in aquatic environments is a worldwide problem and conventional wastewater treatments have proven unable to effec­ and has raised increasing concern amongst the scientific community tively remove them from influents (Zhou et al., 2018). One of the most over the last decades (da Costa et al., 2016). As a result of both biotic (e. reported plastic polymers in aquatic environments is polystyrene (PS; g., digestion; Dawson et al., 2018) and abiotic factors (e.g., UV radiation, Cai et al., 2021; Llorca et al., 2021). An increasing number of scientific Lambert and Wagner, 2016), discarded plastics are fragmented into studies have focused on the effects of PS-NPs on the health and perfor­ microplastics (MPs, <5 mm, Browne et al., 2007), and eventually broken mance of aquatic organisms, such as fishes (Barría et al., 2020; Brandts down into nanoplastics (NPs < 1000 nm, Hartmann et al., 2019). In et al., 2021a; Pitt et al., 2018). Moreover, the increasing usage of addition, the presence of NPs in both industrial cleaning and personal Recirculation Aquaculture Systems (RAS) for aquaculture production care products represents an additional pathway for this emerging raise interest on the assessment of NPs in these systems and the impact contaminant to enter aquatic systems (Hernandez et al., 2017; Saroglia on fish reared in such systems. * Corresponding author at: Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain. E-mail address: mariana.teles@uab.cat (M. Teles). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ecolind.2023.109966 Received 20 October 2022; Received in revised form 26 January 2023; Accepted 29 January 2023 Available online 3 February 2023 1470-160X/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). M. Blonç et al. Ecological Indicators 147 (2023) 109966 Most of the published research regarding PS-NPs has explored their these must be previously validated in the appropriate species and ma­ effects on the model species zebrafish (Danio rerio), leaving only a small trix, to ensure their reliability, accuracy, and precision as indicators for a fraction of the literature investigating the response in other fish species specific factor (Franco-Martínez et al., 2021). To this end, the specific (reviewed by Barría et al., 2020). However, when considering relevant objectives of the present study are twofold: 1) to achieve the analytical factors such as rearing and maintenance costs, commercial availability, validation of biochemical markers in goldfish tissues; and 2) to assess the genetic distance with farmed species, and facility to handle, the goldfish effects of a chronic exposure to PS-NPs in the gills, muscle, and brain of (Carassius auratus) emerges as an ideal model organism to investigate C. auratus, using the validated biomarkers. We studied a panel of bio­ the effects of NPs (Blanco et al., 2018; Filice et al., 2021). On the other markers, namely, total oxidative status (TOS), total antioxidant capacity hand, when reviewing NPs’ ecotoxicology, most of the available litera­ (TAC), oxidative status index (OSI), esterase activity (EA), adenosine ture deals with the response of fish to short-term exposures of no longer deaminase (ADA), acetylcholinesterase (AChE) levels, and creatine ki­ than 7 days (Barria et al., 2020). In real-life scenarios, it is more likely nase (CK), with the final aim of providing a set of adequate tools for that organisms are subjected to long-term exposures, with low but giving a first assessment of general fish health status after a chronic persistent concentrations of NPs present in the ecosystems (de Ruijter exposure to NPs. et al., 2020; Weis and Palmquist, 2021). Nevertheless, the effects of chronic exposures to NPs in fish are still poorly understood, although 2. Materials and methods ontogenetic, intestinal, metabolic, or behavioural anomalies, amongst others, have already been reported in some species (Gu et al., 2020; 2.1. Fish husbandry, bioassay and sampling Guimarães et al., 2021; Marana et al., 2022; Mattsson et al., 2015). To the best of the authors’ knowledge only one previous study has explored A total of 32 adult C. auratus (11.16 ± 3.23 cm total length and 7.07 the long-term effects of PS-NPs in goldfish, carried out by this research ± 0.64 g weight) were selected for the present study. Individuals were group (Brandts et al. 2022). In this recent study, the presence of NPs in randomly allocated to experimental aquaria and subjected to either of goldfish liver and muscle after a chronic exposure was confirmed, and two experimental conditions: “Control” (0 µg/L PS-NPs) and “Exposed” genotoxic damage in red blood cells was found. Therefore, further (100 µg/L PS-NPs). To this date, no substantial environmental quanti­ investigating the response to a chronic exposure to PS-NPs in C. auratus fication of PS-NPs has been established, therefore the exposure con­ as a model species is of great interest, as it can provide tools key answers centration selected for this study took into consideration estimated that could be relevant to other fish species. concentrations in the environment (Lenz et al., 2016), in conjunction NPs have been observed to enter fish organs mainly through the gills with those utilised in previous studies (Brandts et al., 2018; Brandts and the gastrointestinal track, under laboratory conditions, (Clark et al., et al., 2020; Brandts et al., 2021b). Regarding the chosen polymer, PS 2022; van Pomeren et al., 2017), being eventually translocated through has been found in real NPs environmental samples (Llorca et al., 2021; blood transport into other organs, where they can accumulate (Ma et al., Ter Halle et al., 2017) and it has also been documented that this polymer 2022). In fish, the exposure to PS-NPs has been found to elicit immune contributed to a significant fraction of plastic waste in monitoring responses, and to have deleterious effects on the digestive, reproductive, studies (de Haan et al., 2019). For the experiment, we used commercial and endocrine systems, as well as to negatively impact behaviour and PS-NPs (44 nm size) obtained from Bangs Laboratories. Dynamic light significantly alter gut microbiota (Brandts et al., 2021a; Marana et al., scattering (DLS) was employed to characterize the NPs’ hydrodynamic 2022; Pitt et al., 2018; Yin et al., 2021). Therefore, investigating bio­ size and zeta potential (Zetasizer Nano ZS, Malvern) in ultrapure water, logical endpoints related to oxidative status, and immune and metabolic and results were as shown in Brandts et al. (2022). Four experimental activities may provide interesting insights on the toxicity of NPs aquaria were prepared for each treatment, each containing 20 L of water (Franco-Martinez et al., 2016; Yin et al., 2021). In addition, nanoplastics and 4 individuals (n = 16; N = 32). Fish were exposed to the experi­ have been demonstrated to enter organisms through trophic transfer mental conditions for a 30-day period, as per guideline 215 of the Or­ (Chae et al., 2018), indicating an additional exposure source for humans ganization for Economic Co-operation and Development (OECD; Fish, through the ingestion of contaminated food. Furthermore, previous 2000). Throughout the experimental period, fish were fed ad-libitum at studies have shown the ability of these nanoparticles to cross the the same hour every day with a commercial diet (TROPICAL Goldfish blood–brain barrier, not only in fish (Ma et al., 2022), but also in Colour Pellet; crude protein 45.0 %, crude fat 7.0 %, crude fibre 3.0 %, mammals (Shan et al., 2022). Thus, investigating the effects of PS-NPs in moisture 10 %; fish and fish derivatives, derivatives of vegetable origin, fish, particularly their brains may allow for a better understanding of the oils, fats, and minerals), and the bottom of the aquaria was cleared of potential risks that this contaminant may pose to human health and impurities which could negatively affect water quality. Fish mortality, as welfare. well as behavioural parameters (e.g., aggressiveness, activity rate: Biomarkers, defined as biochemical, physiological, or histological hyperactivity/lethargy/erratic swimming, feeding behaviour/appe­ indicators of either exposure to, or effects of, anthropogenic stress tite) were recorded daily. The physicochemical parameters of the water (Huggett, 2018), have been applied in ecotoxicology and ecological risk were kept at optimal levels for the studied species (Table 1). assessment for>20 years (van der Oost et al., 2003). One of their pri­ Approximately 75 % of the medium was replaced once every fifth mary strengths is that they can provide early signs of response to day and PS-NPs were added, aiming to maintain the contaminant’s level exposure, allowing us to determine damage and defence processes at a as close to the starting concentration as possible. At the end of the 30- sub-cellular level (Roméo et al., 2013), measurable before effects on day experimental period, all individuals were over-anesthetized in a individual performance and population/community dynamics occur tricaine methane-sulfonate bath (MS-222; 1 g/L). Each fish was subse­ (Forbes et al., 2006). Thus, developing biomarkers to monitor NPs’ quently measured and weighted, and blood was successively extracted pollution can be extremely valuable to indicate that organisms have through caudal puncture using heparinized syringes. Brain, gills, and been or are being exposed to these pollutants, and could suffer future muscle samples were collected from 7 randomly selected individuals impairments of ecological relevance. Generally, biomarkers are deter­ from each group (n = 7; N = 14), snap-frozen with liquid nitrogen, and mined using manual techniques and often without being validated for stored at − 80 ◦ C until analysis. The remaining fish were either used to the species under study (Franco-Martinez et al., 2016; Oliveira et al., test the reliability of the employed techniques or stored at − 80 ◦ C until 2018). Determining biomarkers via automated analysers and assays further analysis. The entirety of the experiment was carried out presents significant advantages compared to manual techniques, being following the 3 Rs of Animal Experimentation (Replacement, Reduction, more time- and cost-effective, eliminating operator-tied errors and, and Refinement), under Spanish legislation (law 32/2007 and RD53/ consequently, increasing the repeatability and reliability of the obtained 2013), and in agreement with the International Guiding Principles for results (Franco-Martinez et al., 2018; Oliveira et al., 2018). However, Biomedical Research Involving Animals (EU 2010/63). 2 M. Blonç et al. Ecological Indicators 147 (2023) 109966 Table 1 Parameters of water quality maintained throughout the acclimatisation and experimental period. Temperature (T◦ ) Alkalinity Dissolved Oxygen pH Nitrate (NO–3) Nitrite (NO–2) Ammonia (NH3) Ammonium (NH4+) Hardness 16 C ◦ 6.7 dkH 4 mg/L 8.5 <10 mg/L <0.5 mg/L <0.15 mg/L <0.25 mg/L 6–7 dGH 2.2. Sample processing prior to analysis did not follow a normal distribution. The results are presented as bar graphs, displaying the mean ± standard deviation (sd; n = 7), consid­ Samples of gill, brain and muscle were homogenized in potassium ering the threshold for significance at p < 0.05. Ordinary linear phosphate buffer 0.1 M (pH 7.2), at a weight:volume ratio of 1:4. regression analysis, comparing measured and expected concentrations Following a 30-min incubation at room temperature, the samples were of analytes in different samples, was used to evaluate the linearity under centrifuged at 15,000 × g for 90 min, at 4 ◦ C, and the obtained super­ dilution, and Runs-test was performed to determine whether data natant translocated to a clean tube, and placed at − 80 ◦ C until further deviated significantly from the linearity. use. 3. Results 2.3. Validation of the biochemical parameters in goldfish tissues 3.1. Parameter validation Precision and accuracy were evaluated for all biomarkers in tissue homogenates. Precision was assessed by the determination of intra-assay The precision and accuracy data of the methods are presented in coefficients of variation. For this, biomarkers were measured in one Table 2. All methods showed a coefficient of variation below 15 %. sample with high and one sample with low values three times in the Serial dilutions of samples resulted in linear regression equations in same run. Coefficient of variation (CV) was calculated using the which correlation coefficients did not differ from 1, the slope and following equation (1): intercept were close to 1 and 0, respectively, and the Runs test revealed σ no deviation from linearity (P > 0.05). Recovery between observed and CV(%) = *100 (1) expected concentrations ranged from 81 to 114 %, in all cases. x where: σ = standard deviation; x = average. 3.2. Biochemical parameters in gills, brain and muscle Accuracy was evaluated indirectly by performing linearity and re­ covery tests. To assess linearity under dilution, samples with high con­ The analysed biochemical parameters for all three studied tissues are centrations were serially diluted with a phosphate buffer. To evaluate shown in Fig. 1. No significant differences were found in TOS nor OSI the ability of the assays to recover the amount of added analyte, spiking recovery was performed by mixing two samples (one with high and one Table 2 with low concentrations) at different rates. Test recovery (percentage) Precision and accuracy of employed techniques. Intra-assay coefficient of vari­ was calculated for each dilution for comparison of expected versus ation (CV), linearity under dilution and spiking recovery rates of total oxidant measured concentrations of each analyte in each tissue homogenate. status (TOS), total antioxidant capacity (TAC), esterase activity (EA), adenosine deaminase (ADA), acetylcholinesterase activity (AChE), and creatinine kinase 2.4. Measurement of biochemical biomarkers (CK) in homogenates from the gills, brain and muscle of the teleost fish Carassius auratus. TOS was measured as previously described (Erel, 2005). TAC was Tissue Analyte CV, % Linearity under dilution* Recovery, % assessed by evaluating ferric reducing ability of the samples as described (Range) Linear Regression R2 (Range) by Benzie and Strain (1996). EA was measured using p-nitrophenyl ac­ Equations etate as substrate (Haagen and Brock, 1992) adapted to automatic Gills TOS 3.6–7.3 y = 0.566x + 0.999 90.3–108.7 analyser (Tvarijonaviciute et al., 2012a). AChE was measured following 0.0177 the methodology described by Tecles et al. (2000). CK and ADA activity TAC 2.2–7.2 y = 1.1713x − 0.979 100.0–103.5 were determined using commercially available kits (Creatine Kinase, 0.0109 EA 1.9–5.4 y = 0.6892x − 0.995 81.5–100.0 Olympus Systems Reagents; Olympus life and Material Science Europe 0.0289 GmbH, Hamburg, Germany; Adenosine Deaminase assay kit, Diazyme ADA 0.6–2.5 y = 0.6684x + 0.999 98.4–100.0 Laboratories, Poway, CA, USA) following manufacturer’s indications. 3.3182 All parameters were determined with an automatic analyser (Olympus Brain TOS 7.1–10.3 y = 0.8569x − 0.999 90.8–100.0 Diagnostica, GmbH). 0.4469 TAC 0.5–2.8 y = 1.0883x + 0.990 96.2–108.9 0.0066 2.5. Oxidative stress index EA 8.0–14.1 y = 0.9623x − 0.983 89.8–100.0 0.0102 The oxidative stress index (OSI) was determined through the ADA 0.9–1.9 y = 1.0136x − 0.998 95.9–100.0 0.9661 following equation: Muscle TOS 9.1–13.0 y = 1.9064x − 0.989 100.0 – 106.5 7.4016 OSI = TOS/TAC TAC 6.5–9.4 y = 0.9191x + 0.968 94.9–100.0 0.0206 2.6. Data processing and analysis EA 7.7–12.4 y = 0.8232x + 0.998 96.3–101.1 0.0105 ADA 6.2–11.8 y = 0.9993x − 0.996 95.7–106.4 Data manipulation and analysis were carried out through GraphPad 0.0085 Prism 8.0.1 (GraphPad Software, Inc.). Homogeneity of variance was AChE 4.4–9.1 y = 0.9567x + 0.994 87.4–113.3 tested with an F-test, and the normality of the data was tested with the 1.4078 Shapiro-Wilk test. Data complying with normal distribution and ho­ CK 1.5–5.3 y = 0.7845x + 0.998 96.8–101.9 12.435 moscedasticity were analysed with a student’s t-test. The non- parametric Mann-Whitney U test was employed to analyse data that *, Runs-tests were not significant (P > 0.05) in all cases. 3 M. Blonç et al. Ecological Indicators 147 (2023) 109966 Fig. 1. Effects of PS-NPs on the gills, brain, and muscle of Carassius auratus (goldfish). Control conditions (ctrl = 0 µg/L PS NPs; n = 7) are compared to the treatment group (PS NPs = 100 µg/L PS NPs; n = 7). Variations in Total Oxidative Status (TOS), Total Antioxidant Capacity (TAC), Oxidative Status Index (OSI), Esterase Activity (EA), Adenosine Deaminase (ADA), Acetylcholinesterase activity (AChE), and Creatine Kinase (CK) are depicted in the corresponding organs. Significant differences, as well as degree of significance between control and treatment groups are indicated (* = P-value < 0.05, P-value < 0.01 ***). 4 M. Blonç et al. Ecological Indicators 147 (2023) 109966 between the control and the fish exposed to NPs in any of the studied increase of TAC in gills of Sparus aurata exposed to poly­ organs. However, gill samples from the individuals exposed to 100 µg/L methylmethacrylate (PMMA) NPs concentrations ranging from 0.001 to of PS-NPs displayed significantly higher TAC when compared to the 0.1 mg/L. This suggests a similar effect of PMMA-NPs and PS-NPs, both control group (p-value = 0.016). Moreover, EA was also significantly causing oxidative stress in gills. Nonetheless, in yellow croaker (Lar­ increased in the gills of fish exposed to PS-NPs (p-value < 0.001). In imichthys crocea), dietary exposure to PS-NPs did not cause any signifi­ contrast, the results indicated a significant decrease of EA in the brain cant variations in TAC in liver, but did on muscle (Lai et al., 2021), and (p-value = 0.01) of fish exposed to NPs compared to the control group. waterborne exposure led to oxidative stress in the liver, with evident In muscle, no significant variations were observed in any of the bio­ rises in levels of CAT, SOD, and glutathione peroxidase (GPx; Gu et al., markers assessed. 2021). This points towards variations in the effects of NPs linked to species, organ, and exposure type. Furthermore, a significant increase 4. Discussion was found in TOS in the muscle and liver of S. aurata exposed to similar concentrations of PMMA-NPs (Balasch et al., 2021; Brandts et al., Analytical validation of given biomarkers is essential to ensure that 2021b). Nevertheless, no significant differences in TOS were found in test results reflect the condition of an animal, rather than variations gills nor skin under the same experimental conditions (Brandts et al., caused by the laboratory itself due to method imprecision and inaccu­ 2021c). In addition, Brandts et al. (2021a) reported no significant dif­ racy, among others (Jensen and Kjelgaard-Hansen, 2010; Tvar­ ferences in TAC or OSI when examining skin mucus and plasma of ijonaviciute et al., 2012b). Therefore, validation studies must ensure Dicentrarchus labrax exposed to PS-NPs. This further indicates inter- that analytical methods can detect the corresponding analyte in a pre­ organ variations in the response to NPs, although these differences cise and accurate way in a target sample. This practice is of particular could also be explained by the specificities of each polymer, such as size, importance in novel fields like the effect of exposure to NPs in living composition, and shape, and the way these factors affect the organ-NPs organisms, in order to avoid under- or over-estimating the extent of a interaction (Guerrera et al., 2021). These results suggest an additional problematic, and to enable for standardisation of techniques, allowing inter-specific component that could explain the observed differences, by for the correct comparison between results of different studies (Dor which different organs in different species display varying responses to et al., 1999). Furthermore, validating biomarkers for a particular con­ similar NPs polymers and concentrations. The present results also sug­ dition is of utmost importance to ensure the accuracy of ecotoxicological gest that gills are one of the main pathways for NPs to be internalized in studies monitoring nanoplastics pollution and its effect on both wild and freshwater fish, as shown previously for other xenobiotics (Bhagat et al., cultured populations. In this study, automated methods for oxidative 2020). It should be considered that the ingestion of food during chronic stress markers (TAC, TOS, EA), biomarkers of immune response (EA, bioassays might modify the nutritional status of fish and help to coun­ ADA), neurotoxicity (AChE), and a biomarker of muscle damage (CK), teract the effects induced by NPs, notably oxidative stress (Sinha et al., were validated in gills, brains and muscle homogenates of C. auratus. All 2015), on internal organs; but not in gills, which would serve as a portal the evaluated methods presented adequate precision in tissue homoge­ of entry and internalization. In addition, the differences in findings nates with intra-assay CVs lower than 20 %, the limit for an objective regarding the potential of oxidative stress or oxidative damage caused analytic performance standard for precision (US Department of Health by NPs exposure between the current study and the available literature and Human Services, FDA, CDER, CVM, 2018). In the same line, the could be attributed to differences in targeted biomarkers (Han et al., accuracy of the methods has fulfilled the established criteria consisting 2021). of (1) obtaining a regression equation approximating R2 to 1.0 in the ADA has been widely investigated as an indicator for immune regression analysis of the relationship of the measured and expected response and oxidative stress management (Baldissera et al., 2018; analyte values in the linearity under dilution test; and (2) obtaining Capiotti et al., 2016; Zhang et al., 2022). This enzyme, involved in pu­ percentages between 80 % and 120 % in the spiking recovery test rine metabolism (Cristalli et al., 2001), has been reported to play a major (Jensen and Kjelgaard-Hansen, 2010). These findings confirm all the role in neutrophil regulation, and in controlling the immune response to methods to be precise and accurate when quantifying their respective bacterial stressors (Kälvegren et al., 2010). The lack of response of this analytes in C. auratus tissue homogenates. parameter to long-term exposure to PS-NPs in any of the studied organs This study showed that, in C. auratus, immune and antioxidant re­ suggests that the specific mechanisms regulated by this enzyme were not sponses occur in the gills following a chronic waterborne exposure to PS- significantly affected. Similarly, Brandts et al. (2021b) and Balasch et al. NPs, but not in brain nor muscle. To the best of the authors’ knowledge, (2021) did not report significant alterations in ADA in gilthead seabream no study has investigated the effects of PS-NPs on the considered bio­ subjected to short-term exposure to PMMA-NPs. Moreover, Brandts et al. markers in the gills, muscle, and brain of C. auratus, after a chronic (2021a) did not detect significant differences in ADA in skin mucus nor exposure. For these reasons, the results hereby presented are mostly plasma of D. labrax exposed to PS-NPs, further corroborating the present compared with previous findings with different species or polymers, findings. Moreover, the esterase activity (EA) was significantly with some exceptions. increased in the gills of fish exposed to PS-NPs (p-value < 0.001), which The increase in TAC detected in gills of C. auratus in the present suggests that the mechanism to counteract the oxidative stress caused by experiment indicates a boosted production of antioxidant compounds an anti-inflammatory response due to PS-NPs was successfully triggered. aiming to inhibit the effects of reactive oxygen species (ROS), which EA is also often employed as a proxy for immune activity (Marcos-López have been documented in fish exposed to different types of NPs (Jacob et al., 2017), and the observed response could indicate that the gills were et al., 2020). Similarly, a recent study indicates that waterborne, chronic developing an anti-inflammatory response to the contact and internali­ (28-day) exposure to PS-NPs (250 nm, 0.05–5 mg/L) induces oxidative zation of NPs. The present results are in contradiction with the findings stress in C. auratus, although different biomarkers as those hereby published by Brandts et al. (2021c), describing a significant decrease of considered were investigated (Abarghouei et al., 2021). Indeed, the EA in both gills and liver of S. aurata exposed to PMMA-NPs, but, once authors reported significant increases in the levels of superoxide dis­ again, this could point not only to inter-specific variations, but also mutase (SOD) and catalase (CAT) in serum. In addition, PS-NPs differences linked to the nature of the polymer, the exposure period, and appeared to have size-, and concentration-dependent effects on the the absence of food throughout the acute tests. The significant decrease health of C. auratus, with smaller particles at higher concentrations in EA observed in brain, could indicate an alteration in the immune causing severe histopathological changes in gills, liver, and gut. More­ capacity of this organ (Oliveira et al., 2018), and could be attributed to over, a number of previous studies have reported evidence that exposure the direct effect of the PS-NPs on this enzyme, or to its relocation as a to PS-NPs, and other NPs, cause oxidative stress in fish (Reviewed by response to oxidative stress in gills (Brandts et al., 2021c). Interestingly, Han et al., 2021). Similarly, Brandts et al. (2021c) detected a significant besides the immune and antioxidant responses observed in gills, 5 M. Blonç et al. Ecological Indicators 147 (2023) 109966 previous studies reported a general genotoxic response in blood cells considered and further investigated in these chronic scenarios, as both (Brandts et al., 2021b), which might mean that NPs are entering blood oxidative-stress and general stress related responses have been shown to circulation through the gills, inducing oxidative stress in blood cells, differ between sexes and could condition the response to NPs (Balasch reaching preferentially the liver due to a fenestrated endothelium, and Tort, 2019; Niksirat et al., 2021). compared to muscle or brain. In accordance with previous experiments Although estimates on the environmental concentrations of NPs carried out in a different species (S. aurata) and NPs polymer (PMMA) don’t have consensus among researchers on the field, it has been sug­ (Balasch et al., 2021), EA did not result in significant differences in gested that the environmental concentration range varying between muscles between treatment groups. This absence in variations of EA in ca.1 pg/L and ca. 20 μg/L (Lenz et al., 2016). Moreover, to the best of the muscle indicates that esterase did not take part in counteracting the author’s knowledge, the only existing studies that report actual envi­ oxidative stress caused by PS-NPs in this organ. ronmental NPs quantifications, report concentration ranging from 7 to AChE activity is widely used as a biomarker for neurotoxicity of 52 μg/L (Llorca et al. 2021; Materić et al. 2022). On the other hand, environmental pollutants (Fulton and Key, 2001), and alterations in this reports giving information on MPs concentrations found in industrial factor are known to lead to motor problems, potentially causing severe effluent discharges account for concentrations of ≤ 30 mg/L (Lechner paralysis and, eventually, death (Modesto and Martinez, 2010). No and Ramler, 2015). Considering that secondary NPs are constantly abnormal behaviour of any kind was reported in goldfish in the present released by the fragmentation and degradation of macro and micro study, and the lack of alterations in AChE activity seems to match these plastic debris, concentrations are bound to be increasing and some au­ observations. Previous studies have reported either an upregulation or thors point to NPs concentrations 1014 times higher than those measured downregulation in AChE activity following acute exposure of S. aurata to for MPs (Besseling et al., 2019). Furthermore, future predictions visu­ PMMA-NPs, and D. rerio and Oreochromis niloticus to PS-NPs (Balasch alize increases of at least 50-fold from present-day concentrations during et al., 2021; Chen et al., 2017a; Chen et al., 2017b; Ding et al., 2018). this 21st century (Everaert et al., 2018). Considering this, the dose used However, the differences in previously reported results might be linked in this study, (100 μg/L) would fall close within the range of estimated to the characteristics of each particular polymer (Guerrera et al., 2021), concentrations of NPs in the environment and only one order of the exposure period (Atli and Canli, 2011; Kögel et al., 2020), or the magnitude greater than real environmental quantifications. Future species of interest and their developmental stage when subjected to the studies should further explore more environmentally realistic chronic- stressor (Barton, 2002; Fowler et al., 2009). exposure scenarios, leaning towards lower doses of NPs and plastic CK is a reliable indicator for muscle damage (Wallimann et al., 2011; nanoparticles resulting from breakdown of larger plastic objects instead Yousaf and Powell, 2012), and high levels of this protein could cause of model spherical particles, to better understand potential ecosystem changes in the swimming behaviour. Therefore, the lack of alterations in effects. More data is essential for risk assessment, risk management, and the activity of this enzyme supports the fact that the exposed fish did not risk communication. display any sort of abnormal behaviour throughout the experimental period. This result is in accordance with what Balasch et al. (2021) 5. Concluding remarks recorded, stating that no significant effect of PMMA-NPs on CK was observed. The main difference between the present study and the liter­ New automated methods for biomarkers of oxidative stress (TAC, ature described above, other than the exact type of contaminant used TOS, EA), immune response (EA, ADA), neurotoxicity (AChE), and and the species of interest, is the exposure period. In a recent study from muscle damage (CK), were successfully validated in gills, brain, and the authors’ research team, it was observed that NPs were preferentially muscle homogenates of C. auratus, showing their adequate analytical accumulated in muscle of C. auratus (Brandts et al., 2022), but the performance. These methods enabled the assessment of the impact of PS- complete absence of response observed in this organ during the present NPs on gills, where it possibly caused oxidative stress and subsequently experiment indicates that the number of particles was below the triggered antioxidant mechanisms. It appears as if PS-NPs had a heavier threshold to trigger an immune or antioxidant response. impact in gills when compared to brain or muscle, where they had little Altogether, it appears as if the gills sustained greater oxidative to no evident effect. The obtained results, compared with those available damage compared with the brain or muscle, which could be justified by in the published literature, confirm strong inter-specific and inter-organ gills, as one of the major entry portals, being subjected to direct exposure variations. Even though some responses seem generalised, specific or­ to xenobiotics (Arellano et al., 2001). Thus, gills are generally more gans react differently to the presence of PS-NPs, although some of these susceptible to external contaminants than brain or muscle, as it could differences might be due to the specificities of the particles used (e.g., have been expected. Furthermore, differences regarding the main organs size, shape). Further research is needed to fully understand the effect of accumulating nanoplastics, and therefore displaying responses to this chronic waterborne exposure of fish to PS-NPs. However, although the contaminant, could be expected between freshwater and marine species. effects observed in brains are relatively mild, the results could have Indeed, marine fishes tend to drink large quantities of seawater to implications for human health, as humans are in constant exposure to compensate for the important osmotic loss through the gills, due to their these contaminants, and might suffer from the NPs ability to cross the euryhaline environment (Takei, 2021), making the intestinal tract a blood–brain barrier. greater portal of entry than gills. In contrast, freshwater fishes are sub­ jected to osmotic gain of water through the gills due to their oligohaline CRediT authorship contribution statement surroundings (Edwards and Marshall, 2012), therefore, gills play a major role as a portal of entry for xenobiotics. This should be taken into M. Blonç: Writing – original draft, Writing – review & editing. I. consideration when selecting organs of interest for investigating both Brandts: Conceptualization, Investigation, Writing – original draft, the accumulation potential, and the effects of NPs in fish. Writing – review & editing. M. Cánovas: Investigation. L. Franco- Research concerning NPs still has multiple knowledge gaps, which Martínez: Methodology. C.P. Rubio: Methodology. L. Tort: Funding limits the assessment of risk of exposure and potential effects of NPs in acquisition, Supervision, Writing – review & editing. A. Tvar­ biota and humans (European Commission and Directorate-General for ijonaviciute: Funding acquisition, Methodology, Writing – review & Research and Innovation, 2019). The accurate quantification of NPs in editing. C. Gravato: Writing – review & editing. M. Teles: Conceptu­ the environment in order both to establish environmental concentra­ alization, Funding acquisition, Supervision, Writing – review & editing. tions and to be able to quantify the bioaccumulation of NPs in organism and studies evaluating the sub-lethal effects of chronic exposures to NPs Declaration of Competing Interest are crucial stepping-stone in the field. Moreover, species-specific effects and potential sex-specific effects within each species should also be The authors declare that they have no known competing financial 6 M. Blonç et al. Ecological Indicators 147 (2023) 109966 interests or personal relationships that could have appeared to influence (Sparus aurata) to polymethylmethacrylate nanoplastics causes effects at cellular and molecular levels. J. Hazard. Mater. 403, 123590. the work reported in this paper. Brandts, I., Solà, R., Martins, M.A., Tvarijonaviciute, A., Barreto, A., Teles, M., Oliveira, M., 2021c. 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This research was supported by the European Union’s Horizon 2020 Capiotti, K.M., Siebel, A.M., Kist, L.W., Bogo, M.R., Bonan, C.D., Da Silva, R.S., 2016. research and innovation programme under the Marie Skłodowska-Curie Hyperglycemia alters E-NTPDases, ecto-5′ -nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (MSCA) grant agreement No 956481 through the RASOPTA received by (Danio rerio). Purinergic Signall. 12 (2), 211–220. MB. MT was supported by a Ramon y Cajal contract (ref. RYC2019- Chae, Y., Kim, D., Kim, S.W., An, Y.J., 2018. Trophic transfer and individual impact of 026841-I), and the Plan Nacional de Investigación with reference nano-sized polystyrene in a four-species freshwater food chain. Sci. Rep. 8 (1), 1–11. Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., Hollert, H., 2017a. PID2020-113221RB-I00. IB was supported by a PhD grant from Gen­ Quantitative investigation of the mechanisms of microplastics and nanoplastics eralitat de Catalunya (2018FI_B_00711). LFM was granted a Margarita toward zebrafish larvae locomotor activity. Sci. Total Environ. 584, 1022–1031. Salas postdoctoral contract by the Ministerio de Universidades of the Chen, Q., Yin, D., Jia, Y., Schiwy, S., Legradi, J., Yang, S., Hollert, H., 2017b. Enhanced uptake of BPA in the presence of nanoplastics can lead to neurotoxic effects in adult Government of Spain, financed by the European Union­ zebrafish. Sci. Total Environ. 609, 1312–1321. —NextGenerationEU. CPR has a post-doctoral fellowship “Juan de la Clark, N.J., Khan, F.R., Mitrano, D.M., Boyle, D., Thompson, R.C., 2022. Demonstrating Cierva Formación” supported by the “Ministerio de Economía y Com­ the translocation of nanoplastics across the fish intestine using palladium-doped petitividad” (FJC2019-042475-I), Spain. 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