Impact of oil-related environmental pollutants on the ovary structure in the freshwater leech Erpobdella johanssoni (Johansson, 1927) (Clitellata: Hirudinea)

Abstract Toxicity of organic chemical compounds, including benzene, toluene, ethyl benzene and xylene (BTEX), is a major concern because of their induction of adverse effects in organisms, including reproductive abnormalities. In the present study, we investigated impacts of chronic exposure to BTEX at 25 µg L−1 on the ovaries of the freshwater leech Erpobdella johanssoni at the cytological and molecular level. Based on light and transmission electron microscopy, we found that somatic cells and vitellogenic oocytes of the treated animals underwent degenerative changes, such as cytoplasmic vacuolization, mitochondrial alterations, and nuclear DNA condensation, as compared with normal. The comet test supported histological and ultrastructural results and showed that BTEX exposure induced significantly more DNA fragmentation in the ovary cells of treated leeches than in controls (p < 0.0001). Overall, we concluded that BTEX-induced deterioration in ovarian cells suggests the genotoxicity of BTEX on oogenesis in leech, which could impair their reproduction.

They are environmental toxicants that exert their toxicity and disrupt cellular processes via induction of apoptosis and genotoxicity (Ross 2000;Nakai et al. 2003;Fan et al. 2009;Zhang et al. 2010;Ayan et al. 2013;Li et al. 2013;Neuparth et al. 2014). The reproductive toxicity of these compounds has recently received much attention, and many studies have been performed to address the ability of BTEX to impair reproductive functions (Chen et al. 2000;Hannigan & Bowen 2010;Sirotkin et al. 2012;Webb et al. 2014;Djemil et al. 2015). In particular, numerous studies have shown that female organisms accumulate 3.7-6.8 times more xylene than males, and ovaries are the important accumulation organ of this hydrocarbon (Suter-Eichenberger et al. 1998;Wan et al. 2007;Lin et al. 2013).
Although there is concern regarding the influence of BTEX on reproductive processes in aquatic invertebrates, to our knowledge, only one study has reported the effects of toxic insult by BTEX on the gonads of freshwater leeches (Khaled et al. 2016); most studies have addressed effects on mammals (Zhang et al. 2010;Singh et al. 2011;Saxena & Ghosh 2012;Kumar et al. 2014), whereas those on invertebrates are lacking. In a previous study (Khaled et al. 2016), we demonstrated that considerably highconcentration exposure of BTEX (1.4 and 2.8 mg L −1 ) for a short term (1 h) had a significant effect on male and female reproduction of the leech Limnatis nilotica, primarily on the previtellogenic and vitellogenic oocytes. In the present study, our goal was to investigate the effects of chronic low-concentration exposure of BTEX (26 days) on the histopathology and the ultrastructure of the ovaries of the freshwater leech Erpobdella johanssoni. Indeed, many studies have demonstrated that pollutants could have adverse effects when used at high enough concentrations but have no effects at considerably lower concentrations Zheng et al. 2012;Neuparth et al. 2014). The acute toxicity of BTEX is between 1 and 10 mgL −1 in terms of LC 50 or EC 50 to aquatic organisms including fish, invertebrates and aquatic plants (OECD 2002;Zheng et al. 2012;Avramov et al. 2013). We used the species E. johanssoni as a model because it has been regarded as a suitable organism for generating valuable information on various behavioral and physiological parameters (Petrauskienė 2004). This macrophagous predator of aquatic invertebrates has been much investigated and is considered an excellent model for the ecological assessment of invertebrate species interactions and as a bioindicator species for aquatic toxicology (Siddall 2002). Hence, investigation of the effect of this group of pollutants on the reproduction of freshwater invertebrates, in particular leeches, will be informative.

Sample collection
Adult leeches were carefully collected from the Tamerza waterfall (a small village and mountain oasis south of Tunisia) during the 2014 breeding season (May-June; 34°12′ 31.302″N, 7°54′56.555″E). For adaptation to the laboratory conditions, worms were kept for 1 month in an aerated glass aquarium at room temperature (20°C) and fed crushed chicken livers weekly.
Chronic exposure of E. johanssoni to BTEX Benzene, toluene, ethyl benzene and xylene with greater than 99% purity were obtained from Labo chimie PVT. Ltd. 107. To investigate the chronic toxicity of a mixture of BTEX with equal amounts of each component, a concentration of 25 µg L −1 was applied in triplicate to 15 leeches per replicate for 26 days. A control group containing 15 leeches was kept in uncontaminated water. The test conditions were consistent with the acclimation conditions, but aeration was removed to avoid volatilization of these pollutants. During chronic exposure, treated leeches were fed weekly and the entire exposure solution was changed daily to maintain the desired concentration of BTEX. At the end of the exposure period, all of the specimens were transferred into a pre-aerated tank and BTEX-free freshwater. The choice of this concentration was based primarily on our preliminary test results and values derived from the literature regarding chronic exposure on other aquatic invertebrates and fish (Fan et al. 2009;Li et al. 2013).

Light microscopy
After exposure to the pollutants at room temperature, five specimens of E. johanssoni were dissected, and ovaries were removed and fixed in neutral buffered formalin (NBF) 4%. Then, they were embedded in paraffin wax, sectioned to 6-µm thickness, and stained with eosin and hematoxylin. Photos were taken with a camera (SONY DSC-S3000) and digitized with Adobe Photoshop 9.

Transmission electron microscopy
After 26 days of exposure under constant conditions, five specimens of E. johanssoni were dissected and ovaries were removed and fixed in 3% glutaraldehyde at 4°C for 3 days. Thereafter, ovaries were rinsed with 0.1 M sodium phosphate buffer, followed by fixation with 1% osmium tetraoxide (O S O4) for 2 h at 4°C. Then, they were dehydrated in a graded series of ethanol and finally embedded in Epon-Araldite. Semi-thin sections (0.8 µm thick) were taken and stained with toluidine blue. Ultrathin sections were prepared using a Reicheirt-Jung Ultracut E ultramicrotome, stained with uranyl acetate and lead citrate, and examined using a Joel 100SX transmission electron microscope.

Comet assay
Five animals from each treatment were used for the comet assay. Gonads were extracted and immersed in 100 µL of phosphate buffered saline (PBS). They were minced with very fine scissors to obtain a cell suspension; 60 µL of the cell suspension was mixed with 60 µL of 1.0% low-melting point agar (LMA) at 37°C and spread on slides previously coated with a layer of 1% (w/v) normal-melting-point agarose prepared in PBS. After solidification at 4°C for 5-10 min, the slides were immersed in a lysis solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris, and NaOH to pH 10.0) with freshly added 1% Triton X-100 and 10% dimethyl sulfoxide (DMSO) at 4°C overnight. Slides were then randomly placed in an electrophoresis tank containing 0.3 M NaOH and 1 mM EDTA for 20 min. Electrophoresis was conducted for 20 min at 25 V and 300 mA at ambient temperature. The slides were then neutralized using 0.4 M Tris, at pH 7.5, and immersed in absolute ethanol for 10 min. During the assay, the slides were stained with 40 µL ethidium bromide and viewed under a fluorescent microscope. The experiment was repeated 3 times for each sample. A total of 100 comets on each slide were visually scored according to the relative intensity of fluorescence in the tail, and placed in one of five classes. Comet classes were given values (0, 1, 2, 3 or 4; from undamaged, 0, to maximally damaged, 4) as previously described by Collins et al. (1996). The total score was calculated using the following equation: (percentage of cells in class 0 × 0) + (percentage of cells in class 1 × 1) + (percentage of cells in class 2 × 2) + (percentage of cells in class 3 × 3) + (percentage of cells in class 4 × 4). Consequently, the total score ranged from 0 to 400.

Statistical analysis
Statistical analyses were performed using GraphPad prism version 5. Data were represented as mean ± standard deviation (SD). The Mann-Whitney U test was used to compare differences between groups. Differences were considered statistically significant at P < 0.01.

Light microscopy
Light microscopy identified several morphological changes that occurred in the ovary of E. johanssoni during BTEX exposure. In fact, chronic exposure resulted in significant effects on both nurse cells and vitellogenic ovarian cells compared to control cells, thereby increasing the number of degenerating oocytes in the ovarian cord, and decreasing the number of normal oocytes (Figure 1(a) and (b)). The morphological appearance of affected vitellogenic oocytes was characterized by the clumping, shrinkage, extensive alteration and vacuolization of the cytoplasm, especially in the cortical zone (Figure 1(b)).

Transmission electron microscopy
The ultrastructure study showed obvious effects of chronic exposure of the freshwater leech E. johanssoni to a 25 µg L −1 concentration of BTEX. Indeed, and in comparison to control (Figure 2(a)), many ultrastructure alterations in somatic cells and vitellogenic oocytes were indicated (Figure 2(b) and (c)). The somatic cells showed a degenerating ooplasm characterized by damaged mitochondria and developing autophagosomes, which were observed on occasion amalgamating into a larger autophagic vacuole (Figure 2(b)). The vitellogenic oocytes were the most damaged cells because no glycogen granules were found in their ooplasm (Figure 2(c)). Moreover, the vitelline envelope was damaged in some zones, whereas the microvilli were damaged in other zones (Figure 2(c)).

BTEX-induced DNA fragmentation in ovaries of E. johanssoni
Results of the visual scoring of total DNA damage in ovaries of the leech E. johanssoni after 26 days of treatment with BTEX are illustrated in Figure 3. We observed an increase in DNA fragmentation caused by BTEX treatment for 26 days in ovary cells (p < 0.0001). In fact, DNA damage was higher by approximately 7-fold than in the control group.

Discussion
BTEX has been identified as potential groundwater pollutants, which cause deleterious effects on organisms, including humans (Chen et al. 2008;Li et al. 2013;Lim et al. 2014;Liu et al. 2014). Our histological findings demonstrated obvious alterations in the progression of oogenesis in E. johanssoni with BTEX treatment of 25 µg L −1 for 26 days. Our findings agreed with previous studies that also investigated the effect of BTEX on the sensitivity of freshwater animals (Dórea et al. 2007;Avramov et al. 2013). However, the level of sensitivity of organisms to pollutants seems to vary among species, during exposure, and by concentration and life stage. Reproductive toxicity in leeches has seldom been addressed. In fact, it was shown that cadmium  exposure induced a detrimental effect on the structure and development of reproductive tissue in the freshwater predatory leech Nephelepsis obscura (Westcott 1997). Davies and Gates (1991) also found a decrease in reproductive potential in Nephelepsis obscura exposed to cadmium with reduced production of ova and spermatozoa as well as reduced masses of the ovisacs, epididyms and testisacs. Rao et al. (1983) observed that exposure to reserpine and chlorpromazine induces a decrease in ovary and testis indices in the leech Poecilobdella viridis. It has also been noted that pesticides such as mercuric chloride and copper sulfate decreased testis and ovarian indices and inhibited testis and ovarian maturation in the leech Poecilobdella viridis (Kulkarni & Deshpande 2007). Kiceniuk and Khan (1983), and Khan and Kiceniuk (1989), reported that water-soluble fractions (WSF) of a crude oil affected the reproduction process, egg production and hatching of egg, and survival in the marine leech Johanssonia arctica.
Our results show that the majority of somatic cells and vitellogenic oocytes were affected by BTEX treatment. This result is especially important because the toxic effect of BTEX may disrupt the later phases of vitellogenesis because of the accumulation of reserve material (mainly proteins, lipids and saccharides) for use during the later stage of embryonic development (Świątek 2005;Ben Ahmed et al. 2010. It is known that BTEX are highly lipid-soluble toxicants, and they can be easily accumulated in high levels in the ovaries. It has been shown that exposure to benzene, toluene and xylene induced pathological alterations in the earthworm Eudrilus eugeniae (Eseigbe et al. 2013), whereas exposure to benzene only induced severe histopathological injuries in rat ovaries (Singh & Rana 2010). Volatile organic compounds (VOCs), especially BTEX, lead to potential damage to the reproductive and developmental health of women, resulting in preterm birth, infertility, birth defects and spontaneous abortion in women exposed to toluene and benzene in their work environment (Lin et al. 2013;Webb et al. 2014). Exposure to toluene and formaldehyde led to several injuries in the ovaries of adult female mice, causing disruption in the follicular growth process and alteration of their histological structure (Kareem et al. 2014). Chen et al. (2000) reported that many solvents including toluene and benzene damage ovaries by inducing disruption in the follicular growth process, whereas Sirotkin et al. (2012) demonstrated that exposure to BTEX induced apoptosis of porcine and bovine ovarian cells.
From an ultrastructural perspective, oogenesis in the normal ovary of E. johanssoni starts with the oogonia that undergoes a marked increase in volume because of the accumulation of reserve material to form previtellogenic oocytes and huge vitellogenic oocytes (Ben Ahmed et al. 2013). The ooplasm formed during vitellogenesis is filled with cytoplasmic constituents primarily composed of cell organelles, lipid droplets and yolk. At a later stage, the vitellogenic oocyte has an ooplasm filled with glycogen granules and lipid droplets, and is limited by a dense layer of microvilli and the vitelline envelope (Ben Ahmed et al. 2013). However, in the ovary of treated leeches, the majority of somatic cells and vitellogenic oocytes underwent degenerative changes, such as cytoplasmic vacuolization and nuclear condensation, resulting in these oocytes not completing oogenesis and degenerating instead. Using histological outcomes combined with ultrastructure results, we can conclude that BTEX induced deterioration of somatic cells and vitellogenic oocytes of treated E. johanssoni. To determine whether BTEX induced DNA fragmentation in ovarian cells of treated E. johanssoni, we used the alkaline comet assay, which is a sensitive, rapid and simple method that has been widely used for detection of DNA damage (Chen et al. 2008;Azqueta et al. 2016). The comet assay outcomes were clearly associated with the histopathological and ultrastructural results because they demonstrated the ability of BTEX to induce DNA damage in leech ovaries. The latter result is in agreement with that found during the investigation of aluminum pollution and the level of DNA damage in hemocytes of the medicinal leech (Mihaljević et al. 2009). It has also been shown that the gasoline water-soluble fraction (GWSF) induced genotoxic and mutagenic toxicity in the bivalve mollusk Corbicula fluminea (Fedato et al. 2010) and in the freshwater amphipod Quadrivisio afflutzi (Lacaze et al. 2011;Weber et al. 2013). Siu et al. (2004) observed that benzo [a] pyrene leads to genotoxic damage in hemocytes of the green mussel Perna viridis.
Several previous studies focused on the ability of a wide variety of environmental toxicants to trigger cell death, including cadmium, chromium, methyl mercury, organotin compounds and volatile organic compounds (Robertson & Orrenius 2000;Mathur et al. 2011;Fouad & Jresat 2015;Zhuang et al. 2016). Moreover, most of the mitochondria in the ovary of treated animals lost their integrity as compared to those in control animals. The affected mitochondrial integrity increased the permeability of the outer mitochondrial membrane, allowing the release of cytochrome, thereby inducing the formation of apoptosomes and the subsequent activation of the caspase-dependent mitochondrial pathway (Chang & Yang 2000;Servais et al. 2006;Zhang et al. 2010). This suggestion is in agreement with several studies that demonstrated the pivotal role of mitochondria in BTEX-induced apoptosis. Indeed, benzene induced apoptosis in epithelial cells of respiratory tracts, as evidenced by nicking of DNA, endonucleolytic degradation of genomic DNA and increased caspase activity (Weaver et al. 2007;Weaver & Liu 2008). Zhang et al. (2010) showed that ethylbenzene induced apoptosis of renal tubular epithelial cells via the mitochondria-mediated apoptotic pathway, whereas Singh et al. (2011) demonstrated that benzene, toluene and xylene caused genotoxicity and apoptosis in Drosophila larvae through the mitochondria-mediated caspase-dependent pathway of cell death. Our findings are also consistent with those observed for several kinds of chemicals that induce apoptosis. Indeed, Ateeq et al. (2006) indicated that treatment with 2,4-D butachlor induced apoptosis and disrupted the mitochondrial integrity of the ovaries and testis of the catfish Clarias batrachus, whereas Wu et al. (2011) found that the T-2 toxin induced apoptosis in ovarian granulosa cells of rats via a reactive oxygen species-mediated mitochondrial pathway.
In summary, and due to environmental contamination, it has been demonstrated that the biodiversity of freshwater ecosystems is reducing very quickly at a global level (Sala et al. 2000;Knakievicz 2014). On the other hand, the acute toxicity of benzene, toluene, ethylbenzene and xylene is between 1 and 10 mgL −1 in terms of LC 50 or EC 50 to aquatic organism (Zheng et al. 2012;Avramov et al. 2013). Many studies have demonstrated that pollutants could have adverse effects when used at high enough concentrations but that they have no effects in considerably lower concentrations Zheng et al. 2012;Neuparth et al. 2014). To our knowledge, no reports are available concerning the effect of low doses of BTEX on the reproduction of aquatic organisms. Using histological, ultrastructural and molecular analysis, the present study elucidated that chronic exposure to a low dose of BTEX might also cause a serious risk to female reproduction of the freshwater leech E. johanssoni. The current study may increase awareness about the tangible risk of chronic exposure to BTEX, even at low concentrations, on the reproduction of aquatic organisms. Therefore, actions are needed in order to restore altered ecosystems in response to environmental conditions.