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All issues Volume 56 (2020) Ann. Limnol. - Int. J. Lim., 56 (2020) 10 Full HTML
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Ann. Limnol. - Int. J. Lim.
Volume 56, 2020
Article Number 10
Number of page(s) 5
DOI https://doi.org/10.1051/limn/2020009
Published online 27 May 2020

© EDP Sciences, 2020

1 Introduction

Pesticides are an essential part of modern agricultural practices and play a vital role in increasing food production. Pesticide usage is one of the most preferred methods because of its short duration of application and ease of use. However, these chemicals can cause various side effects that they may disrupt the endocrine system and/or induced gonadal abnormalities on non-target organisms (Lone et al., 2014).

Dithiocarbamates, the subclass of carbamate pesticides, are mostly preferred in the agricultural applications and form part of a large group of organic pesticides developed in recent years. Ethylene-bis-dithiocarbamates (EBDCs) are widely used as fungicides in agriculture world-wide due to their low cost, relatively lower acute toxicity and shorter persistence in the environment (Ripley and Cox, 1978; Corsini et al., 2005). Mancozeb is a metal EBDC fungicide and it is a mixture of manganese and zinc EBDC (Mn:Zn, 9:1). It is frequently applied to protect fruits, vegetables, vineyards and field crops against a wide range of fungal diseases (Cycoń et al., 2010; Paro et al., 2012).

Mancozeb-induced toxic effects on some species are available in the literature data. It was reported that mancozeb toxicity on aquatic organisms was moderate to high and 48-h EC50 value for Daphnia magna was 1000 μg/L (US EPA, 2000; Černohlávková et al., 2009). Chronic exposure to mancozeb caused alterations in gonadal structure and function in female rats (Baligar and Kaliwal, 2001; Santos et al., 2009). By assessing mouse oocytes, it was noted that mancozeb decreased female reproduction performance at the concentrations of ≤1 μg/ml (Cecconi et al., 2007; Paro et al., 2012). It also induced histopathological changes in the liver and adrenal gland of mice (O'Hara and Didonto, 1985; Sakr and Saber, 2007).

Fish may encounter with various pesticides in their natural habitats (Joseph and Raj, 2011). Non-lethal concentrations of these chemicals in the aquatic environments may cause striking structural and functional changes (Sancho et al., 2003; Ullah and Zorriehzahra, 2015). Reproduction is a sensitive process that can be affected by the pesticides contaminating the water sources. Several studies revealed that pesticides reduced the reproductive activity of fish via various ways such as gonadal histopathology, decreased gonadosomatic indices, endocrine distruption and reduced fertilization capacity (Moore and Waring, 2001; Hanson et al., 2007; Lal, 2007).

Zebrafish (Danio rerio) is an important fish for scientific researches and is frequently used as a vertebrate model organism in genetics, developmental, toxicological and other experimental researches (Sprague et al., 2006; Segner, 2009; Meunier, 2012; Dai et al., 2014). Besides, its small size, easy maintenance in lab conditions and lower cost make this fish beneficial for researchers.

In this study, zebrafish was used as an animal model to reveal acute toxic effects of mancozeb on testicular tissue by assessing the histopathological alterations by light microscopy.

2 Materials and methods

2.1 The test chemical and the organism

Mancozeb (Dikozin M-45) was obtained from an agricultural pharmaceutical company (Agrofarm, Istanbul/Turkey). Sexually mature zebrafish (4–5 cm in length, 1–2 g in weight) were used in the experiments. They were maintained in 20 L capacity glass aquaria with dechlorinated tap water (at 26–28 °C, 7.7 pH, 7.9 mg/L dissolved oxygen). They were kept at 14 h light/10 h dark photoperiod and fed with commercial fish feed (Tetramin Pro Energy®) for one week before the treatment.

2.2 Chemical exposure

Experimental samples were divided into three groups (n = 10) comprising one control and two experimental groups, each were in triplicates. Mancozeb was dissolved in water. The control group was chemical-free and the experimental groups were exposed to 5 ppm and 7.5 ppm mancozeb for five days. Static test system was conducted. Samples were anaesthetised in iced water and testis tissues were removed for histopathological investigations.

2.3 Histological preparation

Specimens were fixed in 10% neutral buffered formalin and processed for routine histology. The ascending series of ethanol concentrations (70%, 80%, 90% and 100%) was used for dehydration. The tissues were cleared in xylene, embedded in paraffin wax and 5 µm sections were prepared by using Leica RM2125RT microtome (Leica, Germany). The sections were mounted on glass slides, stained with hematoxylin and eosin (H&E) and examined under Leica DM500 compound microscope. Microphotographs were taken with Leica MC170 HD camera.

3 Results

3.1 Control group

In the control group, testis consisted of numerous seminiferous tubules and each tubule was covered by a thin basement membrane. Dark, small and round-shaped Leydig cells were noted in the intersititial tissue among the seminiferous tubules. The germ cells were observed as spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and sperm cells. Spermatogonia were the biggest cells of the spermatogenic cells and they were noticed with their oval-shaped faded nuclei. Primary spermatocytes were smaller than the spermatogonia and they had smaller, dense nuclei covering the majority of the cytoplasm. Secondary spermatocytes had large nuclei and they were smaller than the primary spermatocytes. Spermatids were the smallest cells among the all spermatogenic cells and had dense nuclei. Sperms were observed in the luminal area of the seminiferous tubules. These cells were dark, round-shape nucleated and their cytoplasm could not be noticed (Fig. 1).

thumbnail Fig. 1

Testicular histology of the control zebrafish. A seminiferous tubule (ST) (surrounded) covered by a basement membrane (BM) and containing spermatogenic cells peripherally as spermatogonia (1), primary spermatocytes (2), secondary spermatocytes (3), spermatids (4) and sperms (5) in the luminal area (Lu). Intertubular area was full with intersitial tissue (IT) and Leydig cells (Le) were also noticed. (H&E staining).

3.2 Experimental groups

3.2.1 5 ppm mancozeb exposed group

The testis histology of 5 ppm mancozeb treated samples showed thinning of the basement membrane that surrounded some seminiferous tubules, fibrosis (Fig. 2A), fusion of some tubules (Fig. 2B), degenerative appereance of developing spermatogenic cell clusters, separations between the sperms and spermatogenic cells (Fig. 2A,C,D), hemorrhage in the intertubular space (Fig. 2B,C) and vacuole formations (Fig. 2D).

thumbnail Fig. 2

Testicular histopathology of 5 ppm mancozeb exposed samples. (A) Degenerated spermatogenic cell clusters (encircled), thinned basement mambrane (arrows), separations between the sperms and other developing spermatogenic cell clusters (asterisks) and fibrosis (F). (B) Fusion of the some seminiferous tubules (ellipse) and hemorrhage in the intertubular area (arrows). C) Degenerative appereance of spermatogenic cells (encircled), hemorrhage in the intertubular area (arrows) and separations (asterisks) between the sperms and other developing spermatogenic cell clusters. (D) Degenerated spermatogenic cell clusters (encircled), separation (asterisk) and vacuolization (arrowheads). (H&E staining).

3.2.2 7.5 ppm mancozeb exposed group

The testis of 7.5 ppm mancozeb exposed zebrafish exhibited severe histopathological alterations. Seminiferous tubules were disorganized and their boundaries were not clear (Fig. 3A–B–C). Prominent vacuolization (Fig. 3B,D) and edema (Fig. 3C) were observed. Basement membranes of some tubules were very thin and hypertrophy of spermatocytes were noticed (Fig. 3C). Separations of intertubular area strikingly increased (Fig. 3A–C). Degenerative spermatogenic cell clusters were clearly noticed (Fig. 3A–C). Among these cells, some of them had pyknotic or karyolytic nuclei (Fig. 3D). Even some tubules showed dramatically decreased developing spermatogenic cells and in these tubules only sperm cells were noted in the luminal area (Fig. 3D). Sperms were also severely decreased in some of the seminiferous tubules (Fig. 3B).

thumbnail Fig. 3

Testicular histopathology of 7.5 ppm mancozeb exposed samples. (A) Degenerative appereance of spermatogenic cells (encircled) in the disorganized tubules and increased intertubular spaces (asterisks). (B) Vacuole formations (arrowheads), degenerated spermatogenic cells (encirled), decreased sperm cells (squares) and increased separations (asterisks). (C) Edema (E), degenerated spermatogenic cells (encircled), hypertrophy of primary spermatocytes (arrowhead), thinned basement membrane (arrows) and separations (asterisks) (H&E staining). (D) Tubules without developing spermatogenic cells (ST), vacuolization (arrowheads), pyknotic nuclei (arrows) and karyolytic nuclei (white arrows). (H&E staining).

4 Discussion

Pesticides are discharged by human activity and contaminate water sources. Fish are exposed to these chemicals directly or indirectly. Pesticides have adverse effects on fish reproduction. Hormonal processes, behavior, gametes and/or gonads can be impacted by pesticide exposure. Several studies have pointed that gonads showed pathological conditions following pesticide treatment (Ewing, 1999; Khan and Law, 2005). Histology is an useful and reliable technique to investigate and evaluate the toxic effects of pesticides on gonads at tissue and cellular level (McHugh et al., 2011).

There are limited data on mancozeb toxicity on testis, and researchers have focused on the effects of this chemical particularly in rodents. Joshi et al. (2005) reported that mancozeb treated Wistar albino rats exhibited weight loss of testis, epididymis, seminal vesicle, prostate and significantly decreased sperm cells. In albino mice, mancozeb inhibited the spermatogenesis histologically and spermatogenic cells and sperm counts decreased (Ksheerasagar and Kaliwal, 2010). Another study with albino mice revealed that mancozeb exposure gave rise to decreased sperm cell number and motility; while it increased lipid peroxidation, it reduced antioxidant enzyme activities. The authors also noted that testis was histopathologically altered (Mohammadi-Sardoo et al., 2018). The testis of zebrafish also exhibited altered histology and decreased sperm cell numbers; however in this work enzymatic processes were not investigated.

Previous reports represent histopathological effects of various pesticide types on the testis of different fish species. Diazinon brought about damaged cells and connective tissue, disrupted seminiferous tubules, decreased count of spermatogenic and sperm cells in the testis of Lepomis macrochirus (Dutta and Meijer, 2003). Diazinon exposure also caused testicular degeneration in Clarias gariepinus (Ola-Davies et al., 2015). L. macrochirus testis tissues were affected by endosulfan. Connective tissue and tubule damage, decreased count of spermatids and sperms were noted (Dutta et al., 2006). Mancozeb also caused distinct sperm cell decrease in zebrafish at 7.5 ppm while it was not noted in 5 ppm mancozeb exposed group. It gave rise to seminiferous tubule malformations as fusion and disorganization with uncertain tubule boundaries. Cypermethrin treatment induced cytotoxic damage, condensed spermatogenic cells, vacuole formation and distruped interstitial cells in the testis tissues of Heteropneustes fossilis (Singh and Singh, 2008). Vacuolization was also observed in the current study, both experimental specimens showed vacuoles, while it was more prominent in 7.5 ppm treated zebrafish testis and edema was also accompanied. Fenitrothion exposed Oreochromis niloticus testis showed sterile seminiferous tubules (Benli and Özkul, 2010). In this work, seminiferous tubules without spermatogenic cells were noted in the samples of 7.5 ppm mancozeb treatment. Deltamethrin exposure gave rise to histopathological alterations in the testis of Xiphophorus helleri such as not defined seminiferous tubule shape, decreased count of spermatogenic cells and arrested spermatogenic cycle (Koc et al., 2012). Another study revealed that deltametrin treated Oreochromis niloticus testis exhibited decreased numbers of spermatogenic cells and sperms, degenerative and hypertrofied spermatogonia, pyknosis and cellular necrosis (Bayar et al., 2014). Degenerative spermatogenic cells were observed in both of the experimental groups; however hypertrohy of spermatocytes, pyknotic and karyolytic cells were noted only in the highest mancozeb concentration treated samples of the current paper.

In the present study, water-borne effects of mancozeb on the testicular structure of zebrafish were noted as degenerative spermatogenic cells, seminiferous tubule disorganizations, fibrosis, tubular fusion, vacuolization, edema, hemorrhage, decreased spermatogenic cell clusters and sperms, hypertrophy of spermatocytes, pyknotic and karyolytic nuclei. In general terms, comparing to previous reports mentioned above, pesticides induced similar hazardous histological changes in male gonads. It is clear that arrested spermatogenic processes and decreased sperm number interrupt and fall through the fertilization potential.

Acknowledgement

This study is based on the MSc thesis of the first author which was supervised by the last author.

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Cite this article as: Abar Gürol M, Arman S, Yön ND. 2020. Effects of mancozeb on the testicular histology of the zebrafish (Danio rerio). Ann. Limnol. - Int. J. Lim. 56: 10

All Figures

thumbnail Fig. 1

Testicular histology of the control zebrafish. A seminiferous tubule (ST) (surrounded) covered by a basement membrane (BM) and containing spermatogenic cells peripherally as spermatogonia (1), primary spermatocytes (2), secondary spermatocytes (3), spermatids (4) and sperms (5) in the luminal area (Lu). Intertubular area was full with intersitial tissue (IT) and Leydig cells (Le) were also noticed. (H&E staining).
In the text
thumbnail Fig. 2

Testicular histopathology of 5 ppm mancozeb exposed samples. (A) Degenerated spermatogenic cell clusters (encircled), thinned basement mambrane (arrows), separations between the sperms and other developing spermatogenic cell clusters (asterisks) and fibrosis (F). (B) Fusion of the some seminiferous tubules (ellipse) and hemorrhage in the intertubular area (arrows). C) Degenerative appereance of spermatogenic cells (encircled), hemorrhage in the intertubular area (arrows) and separations (asterisks) between the sperms and other developing spermatogenic cell clusters. (D) Degenerated spermatogenic cell clusters (encircled), separation (asterisk) and vacuolization (arrowheads). (H&E staining).
In the text
thumbnail Fig. 3

Testicular histopathology of 7.5 ppm mancozeb exposed samples. (A) Degenerative appereance of spermatogenic cells (encircled) in the disorganized tubules and increased intertubular spaces (asterisks). (B) Vacuole formations (arrowheads), degenerated spermatogenic cells (encirled), decreased sperm cells (squares) and increased separations (asterisks). (C) Edema (E), degenerated spermatogenic cells (encircled), hypertrophy of primary spermatocytes (arrowhead), thinned basement membrane (arrows) and separations (asterisks) (H&E staining). (D) Tubules without developing spermatogenic cells (ST), vacuolization (arrowheads), pyknotic nuclei (arrows) and karyolytic nuclei (white arrows). (H&E staining).
In the text

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