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ISSN : 1226-9999(Print)
ISSN : 2287-7851(Online)
Korean J. Environ. Biol. Vol.42 No.1 pp.1-14
DOI : https://doi.org/10.11626/KJEB.2024.42.1.001

Feeding, excretion, survival, and histological alterations in zebrafish Danio rerio from single and combined exposure to microplastics and copper

Hyeon Jin Kim, So Ryung Shin, Jung Jun Park1*, Jung Sick Lee*
Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Republic of Korea
1Aquaculture Industry Research Division, East Sea Fisheries Research Institute, National Institute of Fisheries Science, Gangneung 25435, Republic of Korea
*C0-corresponding author Jung Sick Lee Tel. 061-659-7172 E-mail. ljs@jnu.ac.kr
Jung Jun Park Tel. 033-660-8543 E-mail. pjj515@korea.kr

Contribution to Environmental Biology



▪ This study evaluated biological responses in zebrafish exposed to microplastics and copper alone and in combination is an important study for understanding the biological risk mechanisms of microplastics.


29/11/2023 14/12/2023 08/02/2024

Abstract


This study evaluated the risk of single and combined exposure to microplastics in zebrafish (Danio rerio) through biomarkers, such as survival rate, excretion rate, and histological alterations of organ systems. The experimental groups were the control (Cont.), single microplastics exposure group (MPs, 1.83%/fish total weight (g)), the copper group (Cu, 21.6 μg L-1), and a group with combined exposure to MPs and copper (MPs*Cu). The experiment was conducted with individual exposure (7 days) for MP excretion rate analysis and group exposure (14 days) for biomarker analysis. The daily excretion rate of MPs tended to decrease in a time-dependent manner. The copper concentration in the body was not significantly different between single and combined copper exposure. The degeneration of mucous cells in the skin, capillary dilation of the gill lamella, increased intestinal mucous, hepatocyte hypertrophy, and the degeneration of glomeruli and renal tubules were observed in all exposure groups. These histological alterations showed the highest tendency in the MPs*Cu group. In this study, the changes in biomarkers were attributed to the single effect of copper or the combined effect of copper and MPs rather than being solely influenced by MPs.


polyethylene microplastics , heavy metals , histological biomarker , fish

초록

    1. INTRODUCTION

    Plastic is inexpensive, light, and durable, so it is used in industry and various fields. The production of plastics has increased rapidly since World War II. Production is continuously increasing, with 1.5 million tons in 1950, 322 million tons in 2015, and 390 million tons in 2021 (Geyer et al. 2017;Plastic Europe 2022). Plastics can undergo decomposition into microplastics through processes such as UV radiation, biological degradation, and mechanical degradation (Avio et al. 2017). Microplastics are extensively distributed in freshwater and marine environments, encompassing oceans, seas, bays, coasts, and even deep seas. They can be found not only in surface water but also in sediments, owing to the density of plastics (Digka et al. 2018;Zhu et al. 2018;Kim et al. 2019). The abundance of marine environment varies from 0.00085 to 4137.3 particles m-3 depending on the various ocean environments (Avio et al. 2017). Plastics are delivered to various organisms through the food web in the form of microplastics through decomposition processes (Boerger et al. 2010;Sun et al. 2019).

    Microplastics have physical effects on biological reactions in fish. In a previous study, exposure of PE-MPs (500 mg kg-1) to Clarias gariepinus for 15 days resulted in renal lesions such as glomerular hypertrophy and expansion of Bowman’s space and histological changes in liver such as hemorrhage, cytoplasmic vacuolation, and apoptosis (Sayed et al. 2023). In addition to intestinal distension, intestinal inflammation, reduced swimming ability and growth rate, liver toxicity, and altered nutrient composition and energy storage (Lu et al. 2016;Choi et al. 2018;Jin et al. 2018;Yin et al. 2018;Sun et al. 2019). In addition, microplastics are not only transmitted to organisms through the food web, environmental exposure is also a major influence.

    In the aquatic ecosystem, copper is one of the chemical pollutants and has strong bioaccumulation. This induces changes in metallothionein metabolism (0.1 mg L-1, McCarter and Roch 1983), degeneration of mitochondria in the nervous system (5 mg L-1, Totaro et al. 1986), degeneration of mucous cells and chloride cells in the gill, histological degeneration of livers and kidneys (0.08-0.18 mg L-1, Lee et al. 2001), endocrine abnormalities, and hematological changes in teleosts (0.05 mg L-1, Ciji and Nandan 2014).

    Microplastics have the ability to adsorb endocrinedisrupting chemicals (EDCs) such as dioxin and polychlorinated biphenyl (PCBs), persistent organic pollutants (POPs), and heavy metals from the environment. Due to these physical adsorption characteristics, microplastics affect the absorption and absorption pathway of chemical pollutants, and cause complex physical and chemical hazards to organisms (Hirai et al. 2011;Bakir et al. 2012;Lee et al. 2014;Khan et al. 2015;Avio et al. 2017). Studies of combined exposure to microplastics and chemical pollutants have been performed on fish including the Pomatoschistus microps (Oliveira et al. 2013), zebrafish, Danio rerio (Rainieri et al. 2018), Dicentrarchus labrax (Granby et al. 2018), and Symphysodon aequifasciatus (Wen et al. 2018). According to Santos et al. (2020, 2021, 2022), changes in swimming ability, high inhibition of AChE, and higher degree of Integrated biomarker response index were reported in D. rerio exposed to copper and microplastics. As a result, combined exposure was reported to have a higher risk than single exposure to microplasitcs.

    Zebrafish is an organism used in risk assessment and is being employed to study microplastic exposure (Khan et al. 2015;Lu et al. 2016;Chen et al. 2017;Jin et al. 2018;Lei et al. 2018;Rainieri et al. 2018). This species is physiologically similar to humans (Chen et al. 2009) and is particularly suitable for the risk assessment of microplastics due to its exhibition of sensitive physiological responses, such as food intake and reproductive performance. However, in aquatic organisms, the probability of intake of virgin microplastics is low, and the probability of intake of biofilm-formation microplastics, chemical pollutants-adsorbed microplastics or biofilm-formation and chemical contaminant-adsorbed microplastics is high. Therefore, the risk assessment of microplastics should be performed together with single and combined exposure to microplastics.

    Thus, this study was designed to test the hypothesis that the single and combined exposure to microplastics and copper sulfate can influence the excretory rate of zebrafish and induce changes in their organs.

    2. MATERIALS AND METHODS

    2.1. Exposure materials

    Virgin polyethylene microplastics (PE-MPs; 500-600 μm diameter) were purchased dry from Cospheric LLC (Santa Barbara, CA, USA). The copper (CuSO4·5H2O, 99.9% purity) was purchased from Sigma-Aldrich Corp. (Saint Louis, MO, USA). A 1000 mg L-1 of Cu stock solution in ultra-pure water was prepared.

    2.2. Ethics statement

    All experiments were carried out in accordance with recommendations of the Institutional Animal Care and Use Committee of Chonnam National University (CNU IACUC-YS-2023-1). The samples were anesthetized with ethyl 3-aminobenzoated methane sulfonate (MS- 222, 0.3 mg L-1).

    2.3. Experimental fish

    Adult healthy zebrafish, Danio rerio (Total length 3.50±0.13 cm, total weight 0.37±0.07 g, n=280) were purchased as conventional animals from an ornamental fish aquarium in Korea. The animals cultured in a 30 L glass tank (28×50×30 cm), the water condition were as follows: water temperature 26.0±1.0°C, pH 7.2, dissolved oxygen >7.0 mg L-1, and light/dark (L/D) photoperiod was 12 : 12 h. The fish were fed an pellet feed (Tetra bit complete, Tetra, Blacksburg, VA, USA) at a daily of 2% of initial total weight.

    2.4. Experimental design

    In this experiment, we confirmed the feeding rate by coating type (virgin MPs 6.7%, biofilm-coating MPs 33.3%, feed-coating MPs 100%) during oral administration of microplastics in preliminary experiments. Among them, the feed-coating microplastics with the highest feeding rate was used for the experiment.

    This study was conducted in the following exposure method: 1) The oral administration method was used for MPs exposure, and feed-coating microplastics were administered at every day at 1.83% of the initial total weight, 2) Copper exposure was conducted by the immerged method according to the OECD test guidelines (OECD 1992), the copper concentration was set at the LC5-96 h (21.630 μg L-1) by considering the acute toxicity test data, 3) combined exposure was carried out according to the method of 1 and 2).

    Four experimental groups were set up: 1) control (the absence of copper and MPs, Cont.), 2) and 3) single exposure groups of microplastics (MPs) and copper (Cu), and 4) a combined exposure group of MPs and copper (MPs*Cu). The experiment was performed with duplication.

    2.4.1. Feed-coating microplastics preparation

    To increase the feeding rate of microplastics, feedcoating microplastics were prepared as follows. Commercial pellet feed (Tetra bit complete, Tetra, Blacksburg, VA, USA; Sharma et al. 2021) was ground up and mixed with distilled water. Microplastics were mixed with the feed and then dried a dry oven (50°C, 24 h), feed-coating miroplastics contained one microplastics, fish were dosed with a concentration of 1.83% (g (feed+ MPs)/g fish total weight) (Fig. 1). The feed-coating microplastics were prepared in a similar size (2.21± 0.45 mm) as the pellet feed (2.57±0.31 mm).

    2.4.2. Individual exposure

    To analyze the MPs excretion rate, in each experimental group, 10 individuals were individually accommodated for 7 days in a 500 mL glass tank.

    2.4.3. Group exposure

    For the analysis of survival rate, copper concentrations in the body tissue, and histological indicators, experiments were conducted on 30 individuals per experimental group for 14 days in a 30 L glass tank. The experiment used a closed circulatory system with aeration, excretions were removed using a siphon once a day, and 30% of the experimental water was exchanged.

    2.5. Analysis

    2.5.1. Survival rate

    For survival rate calculation, individuals that did not exhibit swimming behavior or opening and closing of gill covers when stimulated were deemed dead. Death in each experimental group was confirmed daily at 10:00 and 22:00, and the cumulative mortality rate was converted.

    2.5.2. Excretion rate of microplastics

    The excretion rate was converted by counting the MPs discharged by observing excretion under a stereoscopic microscope at 24 h intervals in an individual exposure tank (n=10) (Eq. 1).

    Excretion rate of MPs (%) =(Number of excreted MPs/Number of supplied MPs)   × 100
    (Eq. 1)

    2.5.3. Analysis of copper concentrations in the body tissue

    The concentration of copper in the body tissue (n=3 for each of two replicates for each group) was determined according to the methodology of Kim et al. (2021). The samples were freeze-dried at -80°C and reduced to a fine powder with an agate mortar. A 0.25 g portion of each sample was measured into a Teflon decomposition container, into which 10 mL of HNO3 (65%), 1 mL of HClO4 (65%), and 1 mL of H2O2 (30%) were added. The mixture was processed with the decomposition sequence of a microwave digestion system (Milestone Srl., Sorisole, BG, Italy). After cooling, the mixture was diluted to 50 mL with 0.1% nitric acid. This sample was used with an inductively coupled plasma mass spectrometer (PerkinElmer Inc., Waltham, MA, USA) to determine the Cu concentration.

    2.5.4. Imaging and histological indicators

    The presence of microplastics in the body was confirmed through computer tomography and histological analysis. The micro-computer tomography (micro-CT) and histological methods were performed after 14 days, the end-point of the exposure experiment.

    After euthanizing the zebrafish for micro-CT imaging, the whole body was fixed in Bouin’s solution for 24 h, rinsed in running tap water, and dehydrated through a graded ethanol series (70-100%). The preparations were then embedded in paraplast (Leica Biosystems, Wetzlar, Germany). Embedded samples were observed using micro-computer tomography (Skycan 1272; Bruker, Belgium) at 8 μm thickness.

    For specimen preparation of histological analysis, the zebrafish were euthanized and dissected and the skin, gills, intestines, liver, and kidneys. After that, the excised organs were fixed, washed, dehydrated, and embedded in the same methods as micro-CT imaging. Embedded tissues were sectioned at a thickness of 4 μm using a microtome (RM2235; Leica Biosystems, Wetzlar, Germany). Sections were executed with a Mayer’s hematoxylin-0.5% eosin (H-E) stain, alcian blue-periodic acid and Schiff’s solution (AB-PAS, pH 2.5) reaction.

    2.5.5. Statistical analysis

    Shapiro-Wilk’s test and Levene’s test were used for assessment of normality of data and homogeneity of variances, respectively. Comparison among groups was performed by analysis of variance (one- or two-way ANOVA), followed by the Tukey’s test. All the data were analysed using SPSS statistics 25.0 (SPSS Inc., Microsoft Co., WA, U.S.A.). The p<0.05 was considered as statistically.

    3. RESULTS

    3.1. Excretion rate of MPs

    As a result of confirming the individuals who ingested microplastics through micro-CT, microplastics were confirmed in the intestine lumen (Fig. 2).

    The cumulative excretion rate of microplastics with exposure conditions increased in a time-dependent manner. The cumulative excretion rate of microplastics was higher than in the MPs*Cu group, with 23.8% in the MPs*Cu group and 17.5% in the MPs group, respectively, but there was no significant difference. In both experimental groups, a rapid increase in daily excretion rate (MPs 16.8%, MPs*Cu 12.8%) was observed until 4 days later, but the daily excretion rate (MPs 5.0%, MPs*Cu 7.1%) showed a tendency to decrease after five days (Fig. 3).

    3.2. Copper concentrations in the body tissue

    From the beginning to the 14 day of the experiment, the concentration of copper in the bodies of the MPs group was similar to that of the control group. However, on the 7 and 14 days of exposure, it was higher in the Cu group and MPs*Cu group than in the control and MPs groups. After 14 days of exposure, the Cu group and MPs*Cu group showed with 9.37 mg kg-1 and 12.34 mg kg-1, respectively, there was no significant difference between MPs*Cu group and Cu group (Fig. 4).

    3.3. Survival rate

    After 14 days of the experiment, the survival rate was 100% in the control, MPs group, and Cu group, and 96.7% in the MPs*Cu group, showing no significant difference when compared to the control group.

    3.4. Histological changes

    3.4.1. Skin

    The skin of zebrafish consists of an epithelial layer and a dermal layer, and the epithelial layer was a stratified structure of 3-4 layers. Mucous cells and club cells were round, both were vacuoles in H-E stain, and mucous cells reacted with pink in an AB-PAS (pH 2.5) reaction (Fig. 5a, b). After 14 days of exposure, there were no obvious histological changes except for an increase in chromatophores in the dermal layer in the MPs group (Fig. 5c). However, atrophy of mucous cells and club cells appeared in the Cu group and MPs*Cu group, and the mucous cells showed a change of stainability to light blue and purple upon AB-PAS (pH 2.5) reaction (Fig. 5d, e). In all exposure groups, the thickness of the epithelial layer, and the distribution of mucous cells and club cells showed no significant changes compared to the control group.

    3.4.2. Gill

    The epithelial layer of gill lamellae is a simple squamous epithelium, and the capillaries are separated by pillar cells. Mucous cells and chloride cells are mainly located in the interlamellar epidermis. Mucous cells and chloride cells are oval in shape and are all vacuoles in H-E stain. The mucous cells showed blue in an AB-PAS (pH 2.5) reaction (Fig. 6a, b). After 14 days of exposure, lamella blood sinus dilates, increased blood cells, and hypertrophy of some epithelial cells in gill lamella were observed in the MPs group (Fig. 6c). In the Cu group (Fig. 6d) and MPs*Cu group (Fig. 6e, f), an increase in blood cells, hypertrophy and hyperplasia of epithelial cells, and terminal clubbing of lamella were observed. In all exposure groups, the epithelial layer thickness of gill lamella, and distribution of mucous cells and chloride cells showed increased histological characteristics compared to the control group.

    3.4.3. Digestive tract

    The zebrafish was a stomachless fish without a morphological and functional stomach. The mucosal epidermis of the intestine was a simple layer composed of ciliated columnar epithelium and goblet cells, and the free surface of the mucosal epidermis had a well-developed striated border. Goblet cells was vacuoles in H-E stain, but reacted positively to alcian blue in AB-PAS (pH 2.5) reaction, showing blue (Fig. 7a, b). In the MPs group and the MPs*Cu group, microplastics were confirmed in the lumen of the digestive tract after one day of exposure. After 14 days of exposure, an increase in goblet cells, elimination of striated border, depression of mucosal folds, and disruption of the mucosal layer were observed in the mucosal layer of the intestine in all exposure groups (Fig. 7c-g).

    3.4.4. Liver

    The hepatic cell was roundish polygonal, and the nucleus was round, occupying more than 50% of the cytoplasm, and contained a strong basophilic nucleolus (Fig. 8a). After 14 days of exposure, hypertrophy of hepatic cells, granular degeneration of cytoplasm, congestion, atrophy and necrosis of nucleus, and degeneration of bile ducts were observed in all exposure groups including the MPs group (Fig. 8b-f). The histological change in the liver showed the highest tendency in the MPs*Cu group.

    3.4.5. Kidney

    In the kidney, nephrons composed of glomerulus, renal tubule, and collecting tubule were widely distributed, and other interstitial lymphoid tissues were present. The glomerulus is a numerous loop structure of glomerular capillaries, and the surface of the capillaries is intermittently covered with podocytes. The renal tubule is composed of a columnar epithelial layer, and the free surface of the epithelial layer has a well-developed striated border (Fig. 9a, b). After 14 days of exposure, the MPs group showed dilatation of glomerular capillaries, atrophy of podocytes, and degeneration of renal tubular epithelial cells (Fig. 9c, d). In the Cu group (Fig. 9e, f) and MPs*Cu group (Fig. 9g, h), dilatation and necrosis of glomerular capillaries, closing of renal tubule lumen, and necrosis of some renal tubules and interstitial lymphoid tissue were observed.

    4. DISCUSSION

    Among the exposure methods of MPs, the oral administration method is divided into administering only virgin MPs and administering a mixture of MPs in a feed. The latter has the advantage that the test organism’s rejection of the exposure substance is small, and the dose and exposure time can be standardized. Therefore, recently, a method of exposure to MPs by mixing them with feed has been widely used (Pedà et al. 2016;Granby et al. 2018;Jabeen et al. 2018;Rainieri et al. 2018). As a result of exposure of Pomatoschistus microps to PE-MPs, alone and in combination with Artemia nauplii, the feeding rate was higher in the combination exposure than in the single exposure (de Sá et al. 2015). In this study, the feeding rates of biofilm-coated MPs and feed-coated MPs were higher than those of virgin MPs, showing similar results to previous studies. Therefore, in order to increase the MPs selectivity of test organisms in the oral administration method, it is worth considering adding a feed-coating or feed attractant to the MPs.

    It was difficult to find a study on the excretion rate and residual time in the body of MPs. Okamoto et al. (2022) reported that over 50% experimental species (Japanes medaka, zebrafish, Indian medaka, and clown anemonefish) exposed to PE-MPs excreted all MPs contained in gastrointestinal tracts within 24 h. Some MPs persist in the gastrointestinal tract even after 24 hours in some individuals. Excretion time patterns can be broadly divided into three categories: (1) immediate excretion, in which most MP is excreted within the first 4 h; (2) gradual excretion, in which MP is excreted gradually over 24 h; and (3) delayed excretion, in which most MP is excreted after 16 h. In this study, the daily excretion rate of MPs tended to decrease in a time-dependent manner. The daily excretion rate of both the MPs group and the MPs*Cu group showed a rapid increase until the 4th day, but the increase tended to tail off after the 5th day of exposure. In relation to the histological structure on the 14th day, the increase in excretion rate is thought to be due to the swelling of the digestive tract and the increasing secretion of mucus in the digestive tract. Conversely, the subsequent decrease in excretion is thought to occur due to the structural degeneration of the mucosal folds in the digestive tract. In the future, it will be necessary to analyze three excretion patterns when single and combined exposed to MPs.

    In an aquatic ecosystem, MPs have the ability to adsorb pathogens as well as various chemical pollutants including heavy metals. Due to these characteristics, aquatic animals are subject to complex physical, chemical, and biological effects when exposed to MPs (Lu et al. 2018;Rainieri et al. 2018;Gao et al. 2021;Liu et al. 2021;Iqbal et al. 2022). As a result of single and combined exposure of zebrafish to cadmium and PS-MPs for three weeks, cadmium concentrations in the body increased as the concentration of PS-MPs increased (Lu et al. 2018). Also, as a result of single and combined exposure of zebrafish to PE-MPs, and penthiopyrad for three weeks, the penthiopyrad concentration in the body increased after combined exposure compared to single exposure (Zhao et al., 2024). But, Santos et al. (2022), the concentration of copper accumulated in the body was similar when zebrafish were single or combined exposed to MPs and Cu. In polluted water, trace metals accumulate in organisms consumed by fish, or directly in fish through the skin and gills (Sinha et al. 2002;Sures 2003). In this study, the Cu concentration in the body also increased in both the Cu group and the MPs*Cu group compared to the control group, and there was no significant difference between MPs*Cu group and Cu group. This is thought to be due to the direct absorption of copper through the skin and gills of the fish and the increased accumulation of copper in the body due to the ability of MPs to adsorb.

    In a risk assessment of an aquatic ecosystem, the survival rate is an important indicator that can evaluate acute or chronic risk. As a result of previous studies, the survival rate did not show a significant difference regardless of the type, size, and exposure time of MPs (Khan et al. 2015;Lu et al. 2016;Choi et al. 2018;Jabeen et al. 2018;Jin et al. 2018). As a result of the exposure of zebrafish to 0.001-10.0 mg L-1 PA-MPs, PEMPs, PS-MPs, and PVC-MPs, there was no significant difference in survival rate (Lei et al. 2018). No mortality was observed in all experimental groups in which Cyprinodon variegatus was exposed to 150-180 μm spherical and 300-355 μm irregularly shaped PE-MPs for 96 h (Choi et al. 2018). In addition, when Carassius auratus was exposed to ethylene vinyl acetate (EVA) fibers, PS-MPs fragments, and PA-MPs pellets for six weeks, no mortality was observed in all experimental groups (Jabeen et al. 2018). Mortality was not confirmed when zebrafish were exposed to 0.5 μm and 50 μm PS-MPs for 14 days (Jin et al. 2018). In this study, the survival rate of zebrafish was reduced to 96.7% in the MPs*Cu group compared to the control group, but there was no significant difference, with similar results to previous studies.

    MPs and copper induce histological changes in the integumental system of teleosts. Various histological changes were observed in the skin of Ictalurus nebulosus exposed to copper for one month. These changes included mucous cell proliferation and degeneration, a reduction in club cell number in epithelial layer (Benedetti et al. 1989). In this study, mucous cell stainability changes, mucous cells and club cells increase, and hypertrophy of some superficial epidermal cells were observed in the Cu group and MPs*Cu group. These results are judged to be influenced by copper rather than microplastics. In addition, chromatophores were increased in the dermal layer of zebrafish in all exposure groups. An increase in chromatophores is representative of nonspecific reactions showing physiological changes. When fish are exposed to chemical stress in the external environment, the secretion of melanophore- stimulating hormone (MSH) is increased in the adenohypophysis of the pituitary gland by the absorbed chemicals, and MSH induces color change by increasing melanophores in the dermal layer (Fujii 1969).

    Accumulation of MPs was observed in zebrafish gills exposed to 5 μm and 20 μm of PS-MPs (Lu et al. 2016). PE and PS-MPs induced lamellae fusion and mucous hypersecretion in zebrafish gills (Limonta et al. 2019), and fusion and terminal clubbing in gill lamellae were also observed when Oncorhynchus mykiss was immersed in PS-MPs (Karbalaei et al. 2021). However, Carassius auratus was immersed in 0.1-1,000 μm PVCMPs, and no histological changes were observed in the gills (Romano et al. 2020). In copper-exposed Puntius parrah, degeneration such as, epithelium proliferation, swelling of the tip of gill lamellae, and hyperplasia were reported (Ciji and Nandan 2014). As a result of co-exposure of the Nile tilapia, Oreochromis niloticus, to copper and PS-MPs, hyperplasia of gill filaments and lamellae and clubbed tips of lamellae appeared (Zhang et al. 2022). After exposure of zebrafish to cadmium and PS-MPs, gill lamella fusion by epithelial hyperplasia was observed (Lu et al. 2018). As a result of this study, histological alterations such as epithelial hypertrophy, terminal clubbing of gill lamellae, and degeneration of chloride cells in the gills were highest in the MPs*Cu group, similar to the results of previous studies. Epithelial hypertrophy and hyperplasia are mechanisms that protect the gills by increasing the barrier distance from external chemical factors (Mallatt 1985). However, this mechanism is thought to reduce the gas exchange of the epithelial cells and the flow of water in the interlamellar space, thereby reducing the respiration and osmoregulation of the gills.

    When zebrafish were exposed to 5 μm and 20 μm of PS-MPs, accumulation of MPs was observed in the intestine (Lu et al. 2016). When Carassius auratus was exposed to EVA fibers, intestinal mucosal epithelial cells were shed (Jabeen et al. 2018). In addition, when Cyprinodon variegatus was exposed to PE-MPs, intestinal distension due to the accumulation of MPs was observed (Choi et al. 2018). Abdominal distension, deformation of mucosal fold, decay of mucosal epithelia, and increase of mucous in intestine were observed when zebrafish were exposed to PA-MPs, PE-MPs, PP-MPs, PVC-MPs, and PS-MPs (Lei et al. 2018). In the digestive tract of Oncorhynchus mykiss exposed to 20 μg L-1 CuSO4, atrophy of goblet cells, mucosal layer necrosis and vacuolation, necrosis of mucosal epithelium, and elimination of striated borders were observed (Al-Bairuty et al. 2013). Similarly, in 15 mg L-1 CuSO4-exposed Oreochromis niloticus, intestinal examination revealed erosion of the villi and infiltration of mononuclear inflammatory cells (Soliman et al. 2021). Disruption of intestinal mucosal folds was observed when zebrafish was exposed to cadmium and PS-MPs (Lu et al. 2018), and lifting of edema, necrosis, and intestinal epithelium of the digestive tract was observed when Oncorhynchus mykiss was exposed to PS-MPs and chlorpyrifos combined (Karbalaei et al. 2021). When the combined effects of cadmium and PS-MPs were confirmed in the digestive tract of Ctenopharyngodon idellus, the lifting of the mucosal epithelium, degeneration and fusion of villi, leukocyte infiltration, and fibrosis were observed (Zuo et al. 2022). In this study, in the MPs group and MPs*Cu group, microplastics were found in the lumen of the digestive tract from day 1 of exposure, and an increase in goblet cells and degeneration of some mucosal folds were observed in all exposure groups after 14 days.

    In Dicentrarchus labrax exposed to PVC and PE-MPs using an oral administration method, morphological changes, hypertrophy, vacuolation, and congestion of hepatic cells were observed (Espinosa et al. 2019). In zebrafish with immerged exposure to 5 μm and 70 nm PS-MPs, hepatic cell necrosis and lipid droplet accumulation were observed (Lu et al. 2016). Synechogobius hasta immerged with copper showed congestion, necrosis, hypertrophy, and vacuolation of hepatic cell (Liu et al. 2010). In the liver of Oncorhynchus mykiss exposed to 20 μg L-1 CuSO4, pycnosis and melano-macrophages increased (Al-Bairuty et al. 2013). As a result of combined exposure of medaka, Oryzias latipes, to PE-MPs, PAHs, and PCBs, fatty granules and necrosis of hepatic cells appeared in the liver (Rochman et al. 2013). In the liver of Nile tilapia, co-exposure to copper and PS-MPs resulted in congestion and vacuolar degeneration (Zhang et al. 2022), and combined exposure to cadmium and PS-MPs resulted in infiltration in hepatocytes (Lu et al. 2018). In this study, various histological degenerations, including hypertrophy of hepatic cells and degeneration of bile ducts, were observed in all exposure groups, most severely in the MPs*Cu group. The liver performs important functions such as metabolism and detoxication of toxic substances (Olsvik et al. 2007). As a toxic indicator of chemical substances, liver is useful for studying the effects of exposure to aquatic organisms (Fernandes et al. 2008), and exposure to polluted substances causes histological changes such as atrophy of hepatic cell. This histological degeneration is caused by chronic exposure, and tissue alteration and physiological alteration seem to be related (Olurin et al. 2006).

    No histological alterations were observed in the kidneys of Oryzias latipes (Hu et al. 2020) and zebrafish (Lei et al. 2018) immerged in MPs of various shapes. In the kidneys of Oncorhynchus mykiss exposed to 20 μg L-1 CuSO4, degeneration of epithelial cells in renal tubules and increasing Bowman’s space were confirmed (Al-Bairuty et al. 2013). As a result of co-exposure of Ctenopharyngodon idellus to cadmium and PS-MPs, hemorrhage in the interstitial lymphoid tissue, atrophy of glomerulus, and renal tubular necrosis were observed (Chen et al. 2022). In this study, various histological alterations including dilatation of glomerular capillaries and degeneration of epithelial cells in renal tubule were identified in all groups. This was the most severe in the MPs*Cu group. In teleosts, the kidney has functions such as filteration, osmotic regulation, and excretion of metabolites (Hinton et al. 1992). Kidney undergoes histopathological changes depending on the contamination of the aquatic environment (Silva and Martinez 2007), and this structural degeneration can cause malfunction (Jezierska and Witeska 2006).

    The histological alterations identified in this study are judged to be indirect effects of MPs in the digestive tract and direct or indirect effects of copper rather than direct effects of MPs. MPs of a size that unabsorbed by organisms are anticipated to increase the risk due to the long-term exposure and adsorption of chemical toxic substances. This risk is expected to stem more from the adsorption process rather than exerting a direct impact on aquatic animals. Continued research on the risks and absorption pathways of nanoplastics, which can be absorbed at the cellular level, is essential to comprehensively grasp the biohazards associated with microplastics.

    As a result of this study, it is judged that the daily excretion rate and histological alteration of the digestive tracks reflect the influence of microplastics, and the histological degeneration of skin, gill, liver, and kidney reflect the influence of copper. This effect tended to be high in the combined exposure group.

    ACKNOWLEDGEMENTS

    This work was supported by a grant from the National Institute of Fisheries Science (R2024046) of South Korea.

    CRediT authorship contribution statement

    HJ Kim: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - Original draft, Writing- Review & editing. SR Shin: Data curation, Investigation, Software, Validation, Visualization. JJ Park: Conceptualization, Formal analysis, Funding acquisition, Investigation, Resources, Supervision, Validation, Visualization. JS Lee: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - Original draft, Writing - Review & editing.

    Declaration of Competing Interest

    The authors declare no conflicts of interest.

    Figure

    KJEB-42-1-1_F1.gif

    Formation of feed-coating microplastics (MPs).

    KJEB-42-1-1_F2.gif

    Micro-CT image of zebrafish Danio rerio exposed to MPs. (a) Control and (b) MPs exposure group. In: intestine, *: microplastics.

    KJEB-42-1-1_F3.gif

    Excretion rate in zebrafish Danio rerio according to exposure conditions for 7 days. Vertical bar: SD. MPs: single microplastics exposure; MPs*Cu: combined exposure to microplastics and copper.

    KJEB-42-1-1_F4.gif

    Copper concentrations in zebrafish Danio rerio according to exposure conditions. Vertical bar: SD. Cont.: control; Cu: single copper exposure; MPs: single exposure to microplastics; MPs*Cu: combined exposure to microplastics and copper.

    KJEB-42-1-1_F5.gif

    Histopathological changes in the skin of zebrafish Danio rerio according to MPs and copper exposure for 14 days. (a) and (b) Control, showing the stratified epithelial layer (El) and dermal layer (Dl). Note the alcian blue-positive mucous cell (Mc) and vacuolar club cell (Cc). (c) Single MPs exposure group and (d) single copper exposure group. Note the atrophy of mucous cells and club cells and the increase in melanophores (M) in the dermal layer. (e) Combined exposure to MPs and copper, showing the atrophy of mucous cells and club cells and the acidification of mucous cells. Ec: epithelial cell; S: scale. (a) H -E stain; (b)-(e) AB-PAS (pH 2.5) reaction.

    KJEB-42-1-1_F6.gif

    Histopathological changes in the gill of zebrafish Danio rerio according to MPs and copper exposure for 14 days. (a) and (b) Control, showing simple squamous epidermis of the gill lamellae (Gl), an alcian blue -positive mucous cell (Mc), and a vacuolar chloride cell (Chc). (c) Single MPs exposure group, showing the hypertrophy of epithelial cells (Ec) and increases in hemocytes. (d) Single copper exposure group, showing the hypertrophy (circle) of epithelial cells and terminal fusion of the lamellae. (e) and (f) Combined MPs and copper exposure group, showing hemocyte increases, the hypertrophy of epithelial cells, and terminal clubbing (circle) in the lamellae. Bc: blood capillary; Gf: gill filament; and Sc: secretory cell. (a), (c), and (e) H -E stain; (b), (d), and (f) AB-PAS (pH 2.5) reaction.

    KJEB-42-1-1_F7.gif

    Histopathological changes in the intestine of zebrafish Danio rerio according to MPs and copper exposure for 14 days. (a) and (b) Controls. (a) Ciliated columnar epithelium and vacuolar goblet cell (Gc) in the mucosal layer (Ml) and (b) alcian blue -positive goblet cell and striated border (Sb). (c), (d), and (e) Single MPs exposure group. Note the MPs in the intestinal lumen. (e) Disruption of mucosal folds (Mf) (circle). (f) Single copper exposure group, showing increases in goblet cells and lifting of the striated border (Sb) (circle). (g) Combined MPs and copper exposure group, showing increases in mucous and the disruption of the mucosal layer (circle). Ec: epithelial cell. (a), (c), and (d) H-E stain; (b), (e)-(g) AB-PAS (pH 2.5) reaction.

    KJEB-42-1-1_F8.gif

    Histopathological changes in the liver of zebrafish Danio rerio according to MPs and copper exposure for 14 days. (a) Control, showing the well-developed hepatic cord (Hc). (b) Single MPs exposure group, showing hypertrophy and cloudy swelling of some hepatocytes (H). (c) and (d) Single copper exposure group, showing the hypertrophy of hepatocytes, pycnosis, and necrosis. (e) and (f) Combined MPs and copper exposure group, showing the granular degeneration and vacuolation (*) of hepatocytes, pycnosis, necrosis, and the degeneration of the bile duct (Bd). H -E stain.

    KJEB-42-1-1_F9.gif

    Histopathological changes in the kidney of zebrafish Danio rerio according to MPs and copper exposure for 14 days. (a) and (b) Control, showing Bowman’s capsule (Bc), podocytes (Pc), and hemocytes (Hc) in the glomerulus (G) and renal tubules (Rt). (c) and (d) Single MPs exposure group, showing (c) the dilation of glomerular capillaries (circle), the atrophy of podocytes, and (d) Alcian blue-positive necrosis and striated border (Sb) in the renal tubules. (e) and (f) Single copper exposure group, showing (e) the early necrosis (circle) of glomeruli and (f) renal tubules. (g) and (h) Combined MPs and copper exposure group, showing the necrosis of glomeruli, renal tubules, and interstitial tissue. (a) and (b) H -E stain; (c)-(h) AB-PAS (pH 2.5) reaction.

    Table

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    Vol. 40 No. 4 (2022.12)

    Journal Abbreviation 'Korean J. Environ. Biol.'
    Frequency quarterly
    Doi Prefix 10.11626/KJEB.
    Year of Launching 1983
    Publisher Korean Society of Environmental Biology
    Indexed/Tracked/Covered By

    Contact info

    Any inquiries concerning Journal (all manuscripts, reviews, and notes) should be addressed to the managing editor of the Korean Society of Environmental Biology. Yongeun Kim,
    Korea University, Seoul 02841, Korea.
    E-mail: kyezzz@korea.ac.kr /
    Tel: +82-2-3290-3496 / +82-10-9516-1611