Introduction

The growing human activities around the aquatic environments have led to various deleterious effects on the aquatic organisms [1]. Despite that Lake Qaroun was nominated as a protected area back in 1989, it is still suffering from severe pollution problems that were caused by the uncontrolled agricultural, domestic, and industrial waste dumping practices. The increased chemical discharges in addition to the extensive water evaporation from such closed aquatic ecosystem led to obvious elevation in the pollutants content of the lake. Consequently, these concentrated pollutants could change the water quality and badly affected the aquatic biota [2]. The pronounced ecological changes and the heavy pollution load resulted in sharp decrease in fish stock of the lake. Moreover, all suggested solutions that carried out to enrich the fish stock in the lake as transplanting new fish fries into the lake and preventing fishing activities after the transplanting process have been failed [3]. Heavy metals are considered as one of the most abundant pollutants that previously recorded in the lake [4,5,6]. Large numbers of those recorded metals are well classified as endocrine-disrupting chemicals (EDCs) and their adverse effects on the reproductive functions have been reported in many toxicological studies [7, 8]. Nevertheless, there is no study showed their impacts on the reproductive biology and endocrine disruption in the fish inhabiting Lake Qaroun. The most available researches have focused on the measurement of the environmental pollution levels with regard to physicochemical parameters. Evaluation of the harmful effects of the endocrine disruptors on the reproductive functions is very complicated because the endocrine system operates several processes at different levels of biological organization. This complexity is often simplified by assessing the reproductive and developmental abnormalities, leading to conclude the realistic cause of adverse effects [9]. The negative impacts of EDCs on the aquatic biota are well investigated and the feminization of male fish became one of the best pronounced signs [10, 11]. Frequent appearance of intersex symptoms and other gonadal disorders has been recorded in many fish species that chronically exposed to domestic and industrial wastes [12]. Presence of ovotestis (existence of oocytes within the testicular tissues) became a good indicator for the existence of EDCs and could reflect an obvious sign of intersex condition [9]. Therefore, when evaluating the potential effects of EDCs, histological investigation of male gonads has to be considered to observe the ovotestis appearance as these altered gonads often seem normal after the external examination. The histopathological signs of the intersex conditions can be ranged from mild to severe according to many factors as the number, maturity, and distribution of oocytes within the normal testicular tissues [13,14,15]. In severe and prolonged exposure to EDCs, ovarian tissues may clearly occupy large areas of testicular tissues leading to decrease the reproduction capability and threat the population sustainability [16]. It is well reported that reactive oxygen species (ROS) play an important role in the induction of many reproductive disorders and contribute in pathological mechanism of male infertility [17]. Moreover, oxidative stress may cause spermatotoxicity via gonadal and/or extragonadal alterations [18]. The high lipid content in the plasma membrane of the sperms makes them more susceptible to peroxidative damage. Consequently, this peroxidation process will defect the membrane integrity and decrease the motility of the affected sperms [19, 20]. Moreover, the oxidative damages that result from the excessive ROS production play a major role in the dysfunction of the reproductive organs [21]. Thus, the monitoring of antioxidant status of the gonads and its role in ROS inactivation take much attention during the evaluation of the reproductive health. In view of the above, this article was designed to address the impacts of chronic exposure to LQW on the reproductive health of male O. niloticus fish through the assessment of biometal accumulation in testes, histological examination of male gonads (with ovotestis observation), and antioxidant status evaluation of the testicular tissues.

Materials and Methods

Experimental Design

A total number of 60 Nile tilapia fish were purchased from a private fish farm that fed on unpolluted water source from El-Ismailia governorate, Egypt. The transportation process of the fish has been done in well-aerated plastic bags. The fish were maintained in the laboratory conditions for 15 days in glass aquaria with 50 L aerated, dechlorinated tap water. The initial body weight and length were ranged from 43.5–49.8 g and 12.1–13.2 cm, respectively. The water temperature was kept nearby 25 °C; dissolved oxygen was in between 6.5–7.4 mg/L, while the pH ranged from 7.1–7.3. The feeding process was once a day with commercial pellets (20% crude protein, 4% crude fat, 5% crude fiber, 12% crude ash, and 10% crude moisture) during the acclimatization period. The water was continually changed, and any fish debris or remaining pellets were removed. Lake Qaroun water (close to the discharge point of El-Batts drain) was diluted with dechlorinated water at concentrations of 10, 20, and 30%. After the acclimatization period, the fish were distributed in glass aquaria (40 × 70 × 26 cm) in four experimental groups with three replicates for 45 days (n = 5 in each glass aquarium): (a) 10% of LQW, (b) 20% of LQW, and (c) 30% of LQW. The fourth group (0% of LQW) was exposed to pure dechlorinated tap water and served as a control group.

Biometric Measurement

Body weight (g), total length (cm), and gonadal weight (g) were determined using digital weighing balance and caliper after that the gonadosomatic index (GSI) was calculated as gonad weight/body weight × 100 [22].

Metal Bioaccumulation Levels in Gonads

Metal bioaccumulation levels in testes of the studied fish were assessed using flam atomic absorption spectrophotometry (Model, PerkinElmer-2280). The measurement of the recorded metals (Cu, Mn, Fe, Pb, and Cd) was done according to APHA [23]. The isolated tissues were oven-dried at 80 °C for 8 h, acid-digested by concentrated HCl using the dry ashing method, then the mixture was gently shacked and diluted with deionized water to known volume (25 mL). The detection limits of the instrument in ppm were < 0.002 for Cu, < 0.005 for Pb, < 0.01 for Fe & Mn, and < 0.0005 for Cd. In order to correct the background absorption, blank samples were prepared accurately in the same way as the samples. To obtain a straight line standard curve, working standard solutions (for each metal) were aspirated into the flame in different concentrations before the aspiration of the tissue samples. Moreover, a standard reference material (Lake Superior fish 1946 NIST, National Institute of Standards and Technology, USA) was used to check the accurateness of the measurement process and the metal recovery ranges were 97–110%. The recorded metal concentrations were expressed as mg/kg dry weight of gonads.

Histological Examination of the Testicular Tissues

The testes that collected from the different studied groups were washed in a saline solution then preserved in Bouin’s fixative. Tissues were processed, sectioned at 4 μm, and then stained using hematoxylin and eosin as described by Bernet et al. [24], then examined by light microscopy. Specimens of 15 fish per group (six slides for each fish) were examined and the common alterations were recorded to calculate the percentage of each histopathological alteration in the studied fish groups.

Ovotestis Observation and Ovotestis Severity Index (OSI) Calculation

To avoid the inaccurate ovotestis observation, six transversal sections (at least) per fish were sectioned at 0.2-mm intervals throughout the testicular tissues. Each section was accurately examined and any sign of ovotestis was recorded. The ovotestis severity index (OSI) was used as a severity ranking system as described in detail by Bateman et al. [25]. Under a total magnification of × 100 (× 10 eyepiece & × 10 objective lens), six sections for three intersex cases in each group (18 sections/group) were examined. Each histological section was divided into different number of fields depending on the size of the testicular tissue. Each field was scored for the occurrence, distribution pattern, and the developmental stages of oocytes. Then, the following equation was applied to calculate the OSI for an individual section of gonad:

$$ \mathrm{OSI}=\left(\frac{\sum \left[D1\bullet D2\right]}{X}\right) $$

where, D1 is the most advanced developmental stage of oocytes within a field of view (score 1–5; Table 1), D2 is the distribution pattern of oocytes within a field of view (score 1–4; Table 1), and X is the total number of fields of view that examined.

Table 1 The scoring system that described by Bateman et al. [25] to calculate OSI
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As noticed in Table 1, the OSI was depended on both the oocytes developmental stages and the distribution pattern of these oocytes within the testicular tissues. But in this study, the all-observed oocytes were immature previtellogenic oocytes; therefore, the calculated OSI was based on the distribution pattern of oocytes only. The means of the calculated OSI were statically analyzed and compared among the different studied groups at a significant level of p < 0.05.

Antioxidant Biomarkers of the Testicular Tissues

The gonadal tissues were washed in phosphate-buffered saline solution (pH 7.4 containing 0.16 mg/mL heparin) to remove any RBCs and blood clots. Gonads were homogenized in 5 mL cold buffer (50 mM potassium phosphate, pH 7.5, 1 mM EDTA) per gram tissue then centrifuged (100,000×g) for 15 min at 4 °C. The supernatant was preserved at − 80 °C until use. Superoxide dismutase activity was measured as described by Nishikimi et al. [26] who relied on the ability of the SOD enzyme to inhibit the phenazine methosulphate-mediated reduction of nitroblue tetrazolium dye. The change in absorbance at 560 nm over 5 min was associated with the inhibition rate that is directly proportional to SOD activity. As demonstrated by Aebi [27], catalase reacts with a known amount of H2O2, then the reaction is stopped after exactly 1 min with catalase inhibitor. In the presence of peroxidase, remaining H2O2 reacts with 3, 5-Dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone to form a chromophore with intensity of color (at 510 nm) inversely proportional to the amount of catalase. Both SOD and CAT are expressed as U/mg protein. The assay of reduced glutathione levels was measured by the method of Beutler et al. [28] which is based on the reduction of 5,5´dithiobis 2-nitrobenzoic acid with GSH to produce a yellow chromogen whose absorbance is directly proportional to GSH concentration (at 405 nm) and expressed as mg/mg protein. The lipid peroxidation level was assayed according to the method described by Ohkawa et al. [29], in which the malondialdehyde (index of LPO) reacts with thiobarbituric acid forming thiobarbituric acid reactive species. The absorbance at 534 nm of the resultant pink product was directly proportional to LPO level and expressed as nmole/g tissue.

Statistical Analyses

The results were expressed as mean ± SE. Data were statistically analyzed with analyses of variance F test and Duncan’s multiple range to estimate the comparability between means as indicated by different case letters in the descending order a, b, c, and d at p < 0.05 using Statistical Processor Systems Support, SPSS software, version 16.0, IBM, Chicago, IL, USA.

Results

Biometric Measurements

Table 2 showed the total body weight, total length, and GSI after 45 days of exposure to LQW. There were no significant (p < 0.05) differences in body weight and fish total length among the all-studied groups. Compared to the control group, the GSI was significantly decreased in fish that exposed to LQW.

Table 2 Fish biometric measurements of O. niloticus after 45 days of exposure to different concentrations of Lake Qaroun water
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Bioaccumulation of Metals in the Testicular Tissues

Bioaccumulation levels of five metals (Cu, Mn, Fe, Pb, and Cd) in the testicular tissues of the studied fish are shown in Table 3. These results showed significant elevations in metal accumulation levels after exposure to LQW. The studied metals were unequally distributed within the testes and the distribution pattern was Cu > Fe > Mn > Pb > Cd. Moreover, the highest accumulation levels were recorded in the 30% LQW-exposed groups while the lowest levels were in 10% LQW-exposed group which confirm a significant concentration-dependent metals elevation.

Table 3 Bioaccumulation of Cu, Mn, Fe, Pb, and Cd in testes of O. niloticus after 45 days of exposure to different concentrations of Lake Qaroun water
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Testicular Histopathological Study

Figure 1 showed the representative histopathological alterations that observed in the testicular tissues of the studied fish. The testicular histology of the control fish (Fig. 1 a) showed normal testicular structure with normal seminiferous tubules that surrounded by several Leydig cells in the interstitium between the tubules and packed with spermatozoa near the central region. In LQW exposed fish, the testicular histology (Fig. 1 b, c) showed different degrees of degeneration that represented in presence of lacuna, degeneration of Leydig cells, and loss of tubular organization; furthermore, absence of sperms in many seminiferous tubules (azospermia) was also observed. To evaluate the degree of degeneration among the LQW-exposed groups, the percentages of all recorded abnormalities were considered and listed in Table 4 . Consistent with the results of the metal bioaccumulation, the highest percentages of all recorded histopathological lesions were recorded in 30% LQW-exposed group compared to the other groups.

Fig. 1
figure 1

The testicular tissues of O. niloticus after 45 days of exposure to different concentrations of Lake Qaroun water. a showed the normal testicular structure of the control group with normal seminiferous tubule (ST), primary spermatocytes (Sc), spermatogonia (Spg), Leydig cells (L), intertubular tissues (IT), and spermatozoa (Sp) stored in the testicular lumen (Lu). b & c showed the observed alterations in the fish groups that exposed to the lake water as azospermia (asterisks), testicular degeneration (arrows), Leydig cells degeneration (circles), and appearance of ovotestis (arrowheads)

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Table 4 The percentage of the observed histological abnormalities and ovotestis appearance in the testes of O. niloticus after 45 days of exposure to different concentrations of Lake Qaroun water, n = 15 fish for each concentration
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Ovotestis Appearance and OSI Evaluation

The histopathological signs of intersex condition were studied based on the ovotestis appearance as shown in Fig. 1c. Table 4 showed that the incidence of ovotestis increased in the fish that exposed to high concentrations of LQW. The prevalence of ovotestis ranged from 0% (in control group) to 66.7% (in 30% LQW-exposed group). Moreover, the severity of ovotestis occurrence was studied depending on the OSI scores (OSI = 0 it means absent; OSI > 0–5 it means stage1; OSI > 5–10 it means stage2; OSI > 10–20 it means stage3). Mean ovotestis severity indices were statically compared among the studied groups and a significant elevation was observed in 30% LQW-exposed group (Fig. 2). Based on the severity ranking system is that used in this study [25], the all studied intersex specimens remained within the same severity stage (stage1).

Fig. 2
figure 2

Mean ovotestis severity indices in six gonadal histological sections taken at 0.2-mm intervals for each fish sample. n = 3 intersex fish cases for each group (18 slides for each group). The severity ranked according to OSI values as follows: Absent (OSI = 0), stage 1 (OSI > 0–5), stage 2 (OSI > 5–10), and stage 3 (OSI > 10–20)

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Antioxidant Status of the Testicular Tissues

As a result of chronic exposure to different concentrations of LQW, the activities of SOD and CAT in addition to GSH levels were significantly decreased in all exposed fish groups, while, GPx activity and MDA content were significantly increased in the testicular tissues of the LQW-exposed fish in comparison with the control group (Fig. 3).

Fig. 3
figure 3

Activities and levels of different oxidative stress biomarkers (SOD, CAT, GPx, GSH, & MDA) in testes of O. niloticus after 45 days of exposure to different concentrations of Lake Qaroun water (n = 6). Means with the same letter for each oxidative stress biomarker are not significantly different, otherwise they do (Duncan’s test)

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Discussion

The environmental endocrine disruptors may affect the gonadal growth either directly through the growth inhibition of testes or indirectly through impairing the gonadotrophin homeostasis and spermatogenesis [30]. Therefore, the testicular growth inhibition that indicated by the decreased GSI value is considered as a good biomarker for fish exposure to EDCs [31]. It is not evident if this inhibition is caused by cytotoxic disruption of the gonadal cells and loss of cellular integrity of the testicular tissues or via triggering endocrine malfunction due to the interference with pituitary-hypothalamic system [32]. Moreover, Billard et al. [33] found that the growth inhibition of the testes varied depending on the concentration of EDCs, stage of testicular development, and the time of exposure.

Wild fish are rarely exposed to single metal, but instead, they are subjected to combination of several metals with various concentrations. Thus, this work focused on reproductive consequences of environmentally relevant metal exposure. The aqueous metals are reported to be one of the most hazardous toxicants to the reproductive system of fish through disruption of hypothalamic–pituitary–gonadal axis [34] or disruption of other endocrine organs [35]. Moreover, Matthiessen and Johnson [36] found that endocrine disruptor metals could block hormone receptors, mimic endogenous hormones, and/or alter the hormonal biosynthesis processes. Copper has been confirmed to be an interferer with many crustacean hormones that stimulate reproduction as well as with gonadal secretion of inhibiting hormones [37]. The decreased motility of sperms and altered level of sex hormones were observed in human and animals after high manganese exposure at environmentally relevant concentrations [38, 39] suggesting an endocrine disrupting effect. In addition, Ramamoorthi et al. [40] found that the combination of manganese with cadmium or copper was negatively correlated with total motile sperms and the correlations of the combined metals were stronger than those of the individual ones. Iron metal might act as a mediator of oxidative damage and play an important role in male infertility and spermatogenesis [41]. Exposure to lead reported to be a deactivator for many steroidogenic enzymes in testicular tissue of rats and had the ability to decrease sperm quality parameters via oxidative stress generation [42]. Lafuente et al. [43] suggested that cadmium may interfere directly with sex steroid hormone productions with dose-dependent pattern. Moreover, Cd is described to be reproductive toxicant as it can reduce spermatogenesis, initiate testicular necrosis, and modify the morphology of testes [44, 45]. All above-mentioned reproductive toxicant metals were recorded in the LQW with high concentrations and highly accumulated in the testicular tissues of the studied fish, suggesting their potent roles in the testicular damage and endocrine disruption that observed in LQW exposed fish.

The discharge of EDCs into the aquatic bodies was considered as an environmental inducer of intersex fish (ovotestis) through laboratory and field studies [46]. Therefore, the prevalence rate of ovotestis has been used as a biological endpoint of endocrine disruption in fish [25, 47]. To my knowledge, the ovotestis appearance in fish that inhabits Lake Qaroun has never been studied. In the present study, the ovotestis appearance was observed in all LQW-exposed fish and the percentage of appearance was ranged from 40 up to 66.7% intersex fish. Based on the OSI, the prevalence of ovotestis was directly proportioned with the concentration of LQW and this result was consistent with that of Dietrich and Krieger [48] who stated that intersex gonads appear to vary according to the concentrations of the EDCs. The testes of developing male fish are very sensitive to the environmental EDCs and this was clearly verified by ovotestis recognition after the examination of testicular tissues histologically. The EDCs have the ability to disrupt the normal hormonal mechanisms of the fish. For example, EDCs can bind to the androgen and estrogen receptors that disturb the normal sexual differentiation, sexual development, and reproductive health of fish [9]. Moreover, the occurrence of empty seminiferous tubules (without sperms) appeared to be related to one of EDCs action. Jobling et al. [31] reported the role of EDCs in spermatogenesis inhibition by interfering with gonadotropin-releasing hormone synthesis in the hypothalamus and/or gonadotropin synthesis in the pituitary gland that is responsible for the testicular development. Furthermore, the chronic exposure to LQW induced other pathological alterations as vacuolar degeneration of testicular tissues, loss of tubular organization, and degeneration of Leydig cell. These alterations could be resulted from the injurious effect of the xenobiotic metals in LQW. Pathological alterations in testes of fish have been reported after metal exposure due to the direct injuries and disruption of the blood-testes barrier that caused by the accumulated metals and/or the oxidative damage initiated by ROS generation [49]. The observed histopathological alterations were recorded in small proportions in 10% LQW-exposed groups and became more prominent with the increase in LQW concentrations.

Many authors [50, 51] have reported the functional implications of the oxidative stress in the reproductive organs and its harmful impacts on the male reproductive health. Many environmental pollutants, especially heavy metals, exert their toxicological impact through ROS generation. Therefore, the enzymatic and non-enzymatic components of the antioxidant system play its protective role via ROS scavenging. The enzymatic antioxidants, SOD and CAT, alternately associated with each other against the excess ROS to convert them into less harmful form (H2O2), then into non harmful compounds as H2O and O2. The decreased activities of both enzymes in the testes after chronic exposure to LQW may result from the excessive generation of ROS and consequently excessive H2O2 production. The GSH is one of non-enzymatic antioxidant components on which GPx enzyme is rely during the process of hydroperoxides detoxification [19]. The significant increase in GPx activity that accompanied by sharp decrease in GSH level indicated that the detoxification process facilitated by GPx enzyme was provoked along with an ineffective GSH regeneration. This observation was supported by Jozefczak et al. [52] who found that GSH depletion could possibly be triggered because of metal-induced toxicity. The inefficient defense capability of the antioxidant components in testicular tissues is indicated by an elevation in MDA (a key product of lipid peroxidation) levels. The elevated MDA levels confirmed that the antioxidant defense system of testicular tissues was damaged after the exposure to LQW. Oxidative injury of the testicular tissues that resulted from LQW-induced oxidative stress might clearly confirm the histopathological alterations that observed in the testes of exposed fish.

Conclusion

In the animal model used in this work, the chronic exposure to untreated LQW could be highly detrimental for the reproductive capability of male fish. The exposure to LQW had a potential endocrine disruption effects as indicated by frequent appearance of intersexual fish. The severity of intersexuality among fish, based on the mean values of OSI, was directly proportioned with the concentration of LQW. The increased oxidative stress and the inefficient defense mechanisms of the testicular antioxidant system were accompanied by histopathological lesions in the testes, as showed by the disorganized seminiferous tubules with decreased number of sperm in its lumen in addition to vacuolar degeneration of the testicular and Leydig cells. It may be concluded that testicular damage induced by LQW was due to metal-related oxidative stress. Finally, mitigation measurements and monitoring protocols have to be applied in Lake Qaroun to reduce the harmful consequences of the anthropogenic contaminates.