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All issues Volume 57 (2021) Ann. Limnol. - Int. J. Lim., 57 (2021) 6 Full HTML
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Ann. Limnol. - Int. J. Lim.
Volume 57, 2021
Article Number 6
Number of page(s) 9
DOI https://doi.org/10.1051/limn/2021004
Published online 08 February 2021

© EDP Sciences, 2021

1 Introduction

Triclosan (TCS) is a synthetic antimicrobial agent found in a wide range of personal care, household, veterinary, medical, and industrial products (Sewlikar et al., 2015; Zhu et al., 2016). It is a nonionic and phenolic compound with the molecular formula of C12H7Cl3O2 and it is also known as 5-chloro-2-(2,4 dichlorophenoxy) phenol. The chemical structure of TCS is remarkably similar to some other chemicals with two aromatic rings such as bisphenol-A, diethylstilbestrol, and dioxin (Dann and Hontela, 2011). TCS is commonly used due to its high effectiveness against many gram-negative and gram-positive bacteria and some fungi in consumer products such as cosmetics, deodorants, shampoo, shower gels, liquid hand soaps, hand lotions, toothpaste, and mouthrinses (DeSalva et al., 1989; Bhargava and Leonard, 1996; Jones et al., 2000; Dann and Hontela, 2011; Zhu et al., 2016). TCS shows its biocidal activity by destabilizing the cell membranes and blocking fatty acid and lipid biosynthesis of the bacteria (Villalaín et al., 2001; Russell, 2003, 2004; Zhou et al., 2017). Several reasons explain the common containment of TCS in personal care products include: (i) broad-spectrum; (ii) immediate (iii) persistent effectiveness and (iv) the acceptance as a safe, gentle, and tolerated chemical for topical applications to the skin (DeSalva et al., 1989; Bhargava and Leonard, 1996; Jones et al., 2000). This chemical profile makes TCS favored in the skincare products industry for almost 50 yrs (Petersen, 2016). Extensive production and usage of TCS-containing products have resulted in TCS occurrence in household downstream and the surface waters of various freshwater ecosystems such as lakes and rivers (Dhillon et al., 2015; Bera et al., 2020). The high octanol/water partitioning coefficient of TCS (logKow = 4.76) (Bera et al., 2020) indicates the strong hydrophobicity and bioaccumulation potential of this chemical. It is persistent in the environment, and it has been detected in biosolids, soil, and sediments as well as surface water (Heidler and Halden, 2007; Bedoux et al., 2012; Capkin et al., 2017). Detected TCS concentrations range from 1.4 to 40 000 ng/L in surface waters; <0.001–100 ng/L in the sea; <100–53 000 μg/kg in freshwater sediment and 20–133 000 μg/kg in biosolids from wastewater treatment plants (WWTPs) (Dhillon et al., 2015).

Breakdown products of TCS may be generated via both abiotic and biotic processes. Methyltriclosan is the major biodegradation product known to occur in the environment under aerobic circumstances and it shows a relatively higher lipophilic character (log Kow = 5.2) than its parent molecule (Dann and Hontela, 2011). Abiotic transformation of TCS generates dioxins such as 2,8-dichlorodibenzo-p-dioxin (DCDD) via photodegradation (Weatherly and Gosse, 2017). TCS may also be transformed into toxic chlorophenols when it is exposed to chlorine (Dann and Hontela, 2011; Weatherly and Gosse, 2017).

The United States Food and Drug Administration (FDA) has banned TCS usage in soaps due to the risks on human health and the environment in September 2016 (FDA, 2016). However, some other hygiene products such as toothpaste and mouthrinse are not in the content of the ban (Weatherly and Gosse, 2017). Moreover, many of the developed and developing countries have not restricted this antimicrobial agent yet (Mohan and Balakrishnan, 2019), thus TCS still contaminates aquatic ecosystems. Considering all of these, researchers have investigated the potential toxic effects of TCS on aquatic organisms. Previous reports showed acute TCS toxicity in fish. Median lethal concentration (LC50) of TCS for various fish species ranges from 1767 to 260 μg/L. 96-h LC50 value of TCS is 1767 μg/L for Anabas testudineus (Priyatha and Chitra, 2018); 1700 μg/L for Oryzias latipes (Nassef et al., 2009); 1470 μg/L for Xiphophorus helleri (Liang et al., 2013); 1177 μg/L for Pangasianodon hypophthalmus (Bera et al., 2020); 399 μg/L for Oryzias latipes (Ishibashi et al. 2004); 390 μg/L for Labeo rohita (Hemalatha et al., 2019); 370 μg/L for Lepomis macrochirus (Orvos et al., 2002); 340 μg/L for Danio rerio (Oliveira et al., 2009) and 260 μg/L for Pimephales promelas (Orvos et al., 2002).

At sublethal treatment concentrations, TCS brought about disruption in several tissues of fish. Capkin et al. (2017) noted TCS-induced histopathology in gill, liver, kidney, and spleen tissues of Oncorhyncus mykiss at the concentration of 0.48 ± 0.2 μg/L for 40 days. A. testudineus exposed to 1.6 and 1.8 mg/L of TCS for 96 h showed distinct histopathological alterations in gill and liver (Priyatha and Chitra, 2018). Chronic TCS exposure gave rise to liver injury, hepatocyte atrophy and necrosis, increased hepatic plate gap, and hepatocyte apoptosis in D. rerio at the concentrations of 0.08, 0.16, and 0.25 mg/L (Liu et al., 2019).

Zebrafish (D. rerio) is a well-known cyprinid fish used as a model organism in various research areas including ecotoxicology. Its small size (adults are approximately 3 cm long) and easy husbandry in laboratory conditions serve cost-effective advantages to researchers. Besides, zebrafish is a vertebrate organism that has been shown to have similar toxicokinetic and toxicodynamic processes of xenobiotic metabolism undergoing in the higher mammalians including humans (De Souza Anselmo et al., 2017; Horzmann and Freeman, 2018; Wagmann et al., 2020).

The aim of the present study was to investigate the triclosan-induced acute effects on gill and liver tissues of zebrafish by evaluating the histopathological aspects and apoptosis at sublethal experimental concentrations.

2 Materials and methods

2.1 Experimental design

TCS (CAS No: 3380-34-5) was purchased from Sigma-Aldrich. Adult zebrafish were acclimated to laboratory conditions at 26 ± 2 °C and 14 h:10 h light:dark photoperiod cycle for a week before the experiment. Samples were exposed to three different experimental concentrations of TCS for 120 h in separate tanks. The sublethal concentrations were determined based on the data of 96-h LC50 value of TCS for adult zebrafish (340 μg/L) in the report of Oliveira et al. (2009). Accordingly, zebrafish were exposed to 34 μg/L (10% of LC50), 85 μg/L (25% of LC50), and 170 μg/L (50% of LC50) of TCS. The stock solution was prepared by dissolving TCS in acetone (Sigma-Aldrich) and the test concentrations were diluted from the stock solution. During the experiment, solvent control samples were exposed to 250 μl/L acetone that was the maximum concentration in the stock solution as indicated before (Oliveira et al., 2009). Ten fish (n = 10) were used each in the solvent control and the experimental tanks. Static test method was performed and the test solutions were not renewed.

2.2 Histopathology

At the end of the test duration, fish were euthanized in 250 mg/L of tricaine methanesulfonate (MS-222) solution (Wang et al., 2020). Gill and liver tissues were removed and fixed in Bouin's fluid or 10% neutral buffered formalin for 24 h at room temperature. A standard histological procedure was followed for the paraffin embedding technique. Specimens were dehydrated with increasing concentrations of ethanol (100%, 95%, and 70%, respectively), cleared in xylol, and embedded in paraffin blocks. 5-μm-thick sections were prepared with Leica microtome (RM2125RT). Sections were stained with Harris' hematoxylin and eosin and investigated by light microscopy (Leica DM500). Images were acquired with Leica MC170 HD camera.

2.3 Semiquantitative scoring

Semiquantitative analysis was conducted according to the previous report of Korkmaz et al. (2009). The histopathological lesions in the randomly selected 10 sections of the randomly picked three zebrafish from each control and the test groups were evaluated. The frequency of each alteration was categorized as none (−), mild (±: 0–25% of sections), moderate (+: 25–50% of sections), severe (++: 50–75% of sections), and very severe (+++: 75–100% of sections).

2.4 Detection of DNA fragmentation in apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Apoptosis induced by TCS in gill and liver tissues of zebrafish was investigated by the TUNEL assay. The assay was performed according to the manufacturer's guidelines (Apoptag Peroxidase In Situ Apoptosis Detection Kit S7101, Millipore). Briefly, 10% neutral buffered formalin fixed and paraffin embedded tissue sections (5 μm-thick) of randomly picked three individuals were placed on positively charged slides. Following the deparaffinization and rehydration, slides were rinsed in phosphate buffered saline (PBS) (Sigma-Aldrich). Sections were treated with proteinase K (20 µg/mL) (Sigma-Aldrich) and then quenched in 3% hydrogen peroxide in PBS. Samples were incubated with TdT enzyme and antidigoxigenin–peroxidase complex was applied. The negative control slide was without TdT enzyme. Specimens were visualized with 3,3′-diaminobenzidine (DAB) substrate and the nuclei were counterstained with methyl green (Sigma-Aldrich). The apoptotic cells were observed under the light microscope (Leica DM500).

3 Results

During the experiment, no death was observed in the control and the test groups. Control specimens of the gill and the liver of zebrafish showed normal histological structure. No histopathological lesions were noted ( Fig. 1). Histopathological examinations of each tissue and concentrations are given in Table 1.

In the control gills, primary lamellae, central cartilage tissue, and proper secondary lamellae were observed. The lamellae were surrounded by the simple epithelium layer and capillaries were noted (Fig. 1a). In the liver tissues of control zebrafish, hepatocytes and their nuclei were noted. Narrow sinusoids and central veins were observed (Fig. 1b).

TCS exposure caused noticeable alterations in the gills (Tab. 1). The typical gill lesions are presented in Figure 2. 34 μg/L of TCS gave rise to epithelial lifting, desquamation, hyperplasia, aneurysm (Fig. 2a), disorganization of secondary lamellae (Fig. 2a and 2b), and capillary dilation (Fig. 2b). 85 μg/L of TCS exposed samples showed aneurysm, disorganization of secondary lamellae (Fig. 2c), hyperplasia (Fig. 2c and 2d), and capillary dilation (Fig. 2d). In the gills of 170 μg/L of TCS exposed fish, desquamation, aneurysm (Fig. 2e), epithelial lifting, hyperplasia (Fig. 2e and 2f), disorganization of secondary lamellae, and capillary dilation (Fig. 2f) was observed.

Liver histology was also distinctly affected by TCS (Tab. 1). The typical liver lesions are given in Figure 3. 34 μg/L of TCS exposed group samples exhibited sinusoidal dilation, congestion (Fig 3a), vacuolization (Fig. 3a and 3b), and necrosis (Fig. 3b). 85 μg/L of TCS exposure caused congestion, vacuolization (Fig. 3c), and degenerated hepatocytes (Fig. 3d). 170 μg/L of TCS treatment brought about congestion, degenerated hepatocytes (Fig. 3e), vacuole formation, and necrosis (Fig. 3f).

The fragmented DNA of apoptotic cells were labeled by the TUNEL assay. Accordingly, two different colored cells were identified by light microscopy. The nuclei of the non-apoptotic cells were stained with the counterstain (methyl green) and were green-blue color. The nuclei of the apoptotic cells were recognized with brown deposits by DAB staining. The control samples of gills showed no TUNEL-positive cells ( Fig. 4a). No apoptotic cells were detected in the specimens of 34 μg/L of TCS exposed gills (Fig. 4b), while the slides of 85 and 170 μg/L of TCS treated gills showed a high number of TUNEL-positive cells in the secondary lamellae and cartilage tissue (Fig 4c and 4d). No apoptotic cells were identified in the control and 34 μg/L of TCS treated liver samples of zebrafish ( Fig. 5a and 5b). In the groups of 85 and 170 μg/L of TCS, few hepatic parenchymal cells undergoing apoptosis were detected (Fig. 5c and 5d).

thumbnail Fig. 1

Control (250 μl/L acetone) tissues. (a) Normal gill histology of the control zebrafish. PL: Primary lamella, SL: Secondary lamella, C: Cartilage, Ca: Capillary lumen, E: Epithelial cells. (b) Normal liver histology of the control zebrafish. H: Hepatocytes, arrowheads: nuclei of hepatocytes, CV: Central vein, S: Sinusoids.
Table 1

Semiquantitative scoring of gill and liver tissue lesions of Danio rerio exposed to different concentrations (34, 85, and 170 μg/L) of triclosan.

thumbnail Fig. 2

Representative images of zebrafish gill histopathology. (a and b) 34 μg/L of TCS-induced lesions: Aneurysm (A), epithelial lifting (asterisks), desquamation (black arrow), hyperplasia (H), disorganization of the secondary lamellae (circles), and capillary dilation (white arrow). (c and d) 85 μg/L of TCS-induced lesions: Aneurysm (A), hyperplasia (H), disorganization of the secondary lamellae (circles), and capillary dilation (white arrow). (e and f) 170 μg/L of TCS-induced lesions: Aneurysm (A), desquamation (black arrows), epithelial lifting (asterisks), hyperplasia (H), disorganization of the secondary lamellae (circles), and capillary dilation (white arrow).
thumbnail Fig. 3

Representative images of zebrafish liver histopathology. (a and b) 34 μg/L of TCS-induced lesions: Sinusoidal dilation (arrow), congestion (C), vacuolization (arrowheads), and necrosis (N). (c and d) 85 μg/L of TCS-induced lesions: Congestion (C), vacuolization (arrowheads), and degenerated hepatocytes (circle). (e and f) 170 μg/L of TCS-induced lesions: Congestion (C), degenerated hepatocytes (circles), vacuolization (arrowheads), and necrosis (N).
thumbnail Fig. 4

Detection of apoptosis in gills of zebrafish using TUNEL assay. (a) Control. (b) 34 μg/L of TCS exposed group. (c) 85 μg/L of TCS exposed group. (d) 170 μg/L of TCS exposed group.
thumbnail Fig. 5

Detection of apoptosis in livers of zebrafish using TUNEL assay. (a) Control. (b) 34 μg/L of TCS exposed group. (c) 85 μg/L of TCS exposed group. (d) 170 μg/L of TCS exposed group.

4 Discussion

In the modern world, aquatic ecosystems suffer from pollution originating from various types of chemicals which are anthropogenically released into the environment. Water pollution threatens the aquatic organisms and severe adverse effects may occur in fish (Corcoran et al., 2010; Murthy et al., 2013). In the present study, TCS-induced acute toxicity in gill and liver tissues of zebrafish were evaluated at the concentrations of 34, 85, and 170 μg/L. Although the environmental concentrations of TCS in freshwater ecosystems ranging from ng/L to low μg/L, environmentally measured concentrations could not always represent accurate exposure. Because vertical translocation of the organisms between the water column and the sediment may result in temporal and/or nonequilibrium exposure to the chemical (Chalew and Halden, 2009). The bioaccumulation tendency of TCS is another issue that has to be considered that TCS exposure may also occur by transferring the chemical via the food web in the aquatic ecosystems. From this viewpoint, to avoid any complexity, the sublethal test concentrations of the current work were determined based on the previously calculated exact LC50 value of TCS for adult zebrafish as mentioned above. This method of experimental concentration determination was also applied in recently published studies (Bera et al., 2020; Gyimah et al., 2020; Wang et al., 2020).

Fish gills take part in multiple important metabolic functions such as gas exchange, osmoregulation, ion regulation, and nitrogenous waste excretion (Camargo and Martinez, 2007; Tabassum et al., 2016). Gill surface directly in contact with the pollutants. Due to the vital multifunctions and being the primary contact area of the xenobiotics, gills are considered critical indicators for assessing the water-borne exposure to environmental contaminants (Velmurugan et al., 2009). On the other hand, liver is mainly responsible for the detoxification of toxicants and some other important metabolic processes (Dutta et al., 1993; Velmurugan et al., 2009). Chemical exposure may affect the normal histological structure of the liver, thus structural alterations give critical clues about the water-borne harmful effects of the chemicals (Tabassum et al., 2016). Gill and liver histopathology have been frequently evaluated together to reveal the toxicity of various types of chemicals contaminating the aquatic ecosystems (Velmurugan et al., 2009; Ostaszewska et al., 2016; Macêdo et al., 2020).

In the current paper, gill and liver tissues of zebrafish exhibited prominent histopathological changes following 120 h TCS exposure at the concentrations of 34, 85, and 170 μg/L. In the gills, TCS caused aneurysm, capillary dilation, lamellar disorganization, hyperplasia, epithelial lifting, and desquamation. These types of alterations are not chemical-specific and have been reported in various studies with certain fish species in response to other chemicals that have a similar structure to TCS. One or more of these lesions were also noted in the fish exposed to bisphenol A (Elshaer et al., 2013), polychlorinated biphenyls (Kim et al., 2003), 2,2′,4,4′-tetrabromodiphenyl ether (Barja-Fernández et al., 2013), and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (Arellano et al., 2001). Priyatha and Chitra (2018) reported acute TCS toxicity in the gills of A. testudineus at the concentrations of 1.6 and 1.8 mg/L as epithelial lifting and epithelial hyperplasia, aneurysm, disorganization of secondary lamellae, and hyperplasia in gill arches. The results of the present work were substantially in accordance with the report; however while additionally desquamation and capillary dilation was observed in zebrafish, gill arch hyperplasia was not diagnosed. Histopathological changes observed in the lamellar epithelium like epithelial lifting, desquamation, and hyperplasia have been thought of as barrier-like defense mechanisms to extend the route of the chemical to the blood circulation (Poleksić and Mitrović-Tutundžić, 1994; Hassaninezhad et al., 2014). Besides the direct response of gill epithelium, chemical stressors may also induce more severe injuries in blood vessels such as dilation and aneursym (Camargo and Martinez, 2007; Hassaninezhad et al., 2014). Previous reports associated the lesion of aneurysm with the collapse of pillar cells leading to vascular integrity breakdown (Martinez et al., 2004). It was noted that pollutant-induce epithelial disruption of the gills might be reversible while vascular damage was probably irreversible (Poleksić and Mitrović-Tutundžić, 1994; Hassaninezhad et al., 2014).

In the liver, TCS exposure gave rise to sinusoidal dilation, congestion, vacuolization, hepatocellular degeneration, and necrosis. TCS also caused irregular or anucleated hepatocytes, vacuolization, cytoplasmic degeneration, and melanomacrophage centers in A. testudineus (Priyatha and Chitra, 2018). These hepatic lesions are very common that they have been reported frequently in chemical exposed fish livers (Camargo and Martinez, 2007). Sinusoidal dilation and congestion might be associated with venous outflow impairment (Kakar et al., 2004), heart failure, and extrahepatic inflammatory conditions (Brancatelli et al., 2018). Vacuolization has been thought as a result of several conditions such as unbalance in the synthesized and released material ratios of the hepatocytes (Jiraungkoorskul et al., 2003), lipid dystrophy (Abdel-Moneim et al., 2012), protein synthesis inhibition, microtubule disaggregation, and energy depletion (Hinton and Lauren, 1990; Younis et al., 2013). According to Agamy (2012), chemicals directly affect the organs and lead to degenerative lesions that are not reversible. Cellular degeneration and necrosis are closely related in the context of oxidative stress. Necrosis might be related to oxidative stress and oxidative stress is associated with cellular disruption via the free radicals react with the cell membrane lipids and damage the membrane structure irreversibly (Avci et al., 2005; Abdel-Moneim et al., 2012; Mela et al., 2013). TCS-induced oxidative stress was reported previously in mice (Yueh et al., 2014), rats (Tamura et al., 2012), and zebrafish (Liu et al., 2019; Gyimah et al., 2020). Gyimah et al. (2020) noted that TCS gave rise to oxidative stress in the liver and brain of adult zebrafish. The authors histologically observed atrophy and necrosis of hepatocytes after TCS treatment.

Apoptosis is a well-controlled and critical form of programmed cell death naturally occurring in both normal developmental and adulthood stages to maintain homeostasis in multicellular organisms. This cell death process that works completely for the benefit of the organism can be stimulated by various intrinsic and extrinsic factors. Fish are considered promising tools to investigate apoptosis stimulated by environmental contaminants (AnvariFar et al., 2017, 2018). Several studies have shown chemical-induced apoptosis in gill and liver tissues of fish. Apoptosis by inhibiting glucose-6-phosphate dehydrogenase in gill and liver samples of O. mykiss exposed to chlorpyrifos was noted (Topal et al., 2014). Cadmium exposure caused necrotic and apoptotic chloride cells in the gills of Oreochromis mossambicus (Pratap and Bonga, 1993). Cadmium was also triggered apoptosis in the gills and livers of Atherinops affinis (Rose et al., 2006). In the current paper, TUNEL assay showed that exposure to 85 and 170 μg/L of TCS for 120 h caused apoptosis in zebrafish gills. However, the samples of the lowest TCS concentration group (34 μg/L) did not show TUNEL-positive cells. In the liver of zebrafish from 34 μg/L of TCS group, no signs of DNA fragmentation were noticed. The other experimental groups exhibited a very low proportion of apoptotic cells. However, Liu et al. (2019) reported that chronic exposure to TCS induced significant hepatocyte apoptosis compared to control in zebrafish. This may probably due to the duration of exposure. Taking together with the histopathological examinations of the present study, acute TCS exposure related cell death occurred via necrosis instead of apoptosis. Since molecular pathways were not investigated in the present work, further studies are required to reveal the mechanism of apoptosis in gill and liver tissues of adult zebrafish exposed to TCS.

Acknowledgments

The author would like to thank Dr. Sezen Toksoy Köseoglu for providing triclosan. Also, special thanks to Dr. Cansu Akbulut for technical support during the laboratory work.

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Cite this article as: Arman S. 2021. Effects of acute triclosan exposure on gill and liver tissues of zebrafish (Danio rerio). Ann. Limnol. - Int. J. Lim. 57: 6

All Tables

Table 1

Semiquantitative scoring of gill and liver tissue lesions of Danio rerio exposed to different concentrations (34, 85, and 170 μg/L) of triclosan.

In the text

All Figures

thumbnail Fig. 1

Control (250 μl/L acetone) tissues. (a) Normal gill histology of the control zebrafish. PL: Primary lamella, SL: Secondary lamella, C: Cartilage, Ca: Capillary lumen, E: Epithelial cells. (b) Normal liver histology of the control zebrafish. H: Hepatocytes, arrowheads: nuclei of hepatocytes, CV: Central vein, S: Sinusoids.
In the text
thumbnail Fig. 2

Representative images of zebrafish gill histopathology. (a and b) 34 μg/L of TCS-induced lesions: Aneurysm (A), epithelial lifting (asterisks), desquamation (black arrow), hyperplasia (H), disorganization of the secondary lamellae (circles), and capillary dilation (white arrow). (c and d) 85 μg/L of TCS-induced lesions: Aneurysm (A), hyperplasia (H), disorganization of the secondary lamellae (circles), and capillary dilation (white arrow). (e and f) 170 μg/L of TCS-induced lesions: Aneurysm (A), desquamation (black arrows), epithelial lifting (asterisks), hyperplasia (H), disorganization of the secondary lamellae (circles), and capillary dilation (white arrow).
In the text
thumbnail Fig. 3

Representative images of zebrafish liver histopathology. (a and b) 34 μg/L of TCS-induced lesions: Sinusoidal dilation (arrow), congestion (C), vacuolization (arrowheads), and necrosis (N). (c and d) 85 μg/L of TCS-induced lesions: Congestion (C), vacuolization (arrowheads), and degenerated hepatocytes (circle). (e and f) 170 μg/L of TCS-induced lesions: Congestion (C), degenerated hepatocytes (circles), vacuolization (arrowheads), and necrosis (N).
In the text
thumbnail Fig. 4

Detection of apoptosis in gills of zebrafish using TUNEL assay. (a) Control. (b) 34 μg/L of TCS exposed group. (c) 85 μg/L of TCS exposed group. (d) 170 μg/L of TCS exposed group.
In the text
thumbnail Fig. 5

Detection of apoptosis in livers of zebrafish using TUNEL assay. (a) Control. (b) 34 μg/L of TCS exposed group. (c) 85 μg/L of TCS exposed group. (d) 170 μg/L of TCS exposed group.
In the text

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