Microplastics induced endocrine disruption, alteration in testicular tissue in tilapia (Oreochromis niloticus) pre-fed on Amphora coffeaeformis

This study examines the potential defending effects of the diatom, Amphora coffeaeformis, as a feed additive against the deleterious effects (mainly on gonads) caused by microplastics (MPs) in the Nile tilapia, Oreochromes niloticus. Male tilapia groups were pre-fed diets with four different supplementation levels of A. coffeaeformis (0%, 2.5%, 5% and 7.5%) for 70 days, then were exposed to 10 mg/L MPs for 15 days. The results showed signicantly (p < 0.05) higher numbers of erythrocytes, but signicant decreases (p < 0.05) in the number of RBCs, Hb, Ht, platelets, and eosinophil percentages. Testicular histological degenerative changes and testis-ova were found in the MPs-exposed sh. Thus, A. coffeaeformis supplementation displayed ameliorative properties that detoxied the negative effects of MPs. This study provides a better understanding of the reproductive injuries caused by MPs and provides evidence for the use of A. coffeaeformis as a natural remedy in freshwater tilapia. 15 days induced hematological and biochemical changes in O. niloticus. Furthermore, MPs prompted oxidative stress, hormonal disruption and testicular damage, and induced testis-ova in male tilapia. The highest tested supplementation level of A. coffeaeformis (A3MP; 7.5%) prevented the oxidative stress, testicular damage, and negative hematological alterations. The present results suggest that MPs indirectly affect the HPG axis and subsequently the gonads through oxidative stress. Additional research work is needed to determine the mechanisms responsible for the endocrine disruption, histomorphological alterations, and possible reproductive impairments induced by MP exposure.


Introduction
In recent years, concerns have been raised about the incorporation of microplastics (MPs) in food webs. MPs are synthetic polymers with a diameter of less than 5 mm . Increased industrial production of plastic has led to excessive quantities of plastic along coasts, and in rivers, seas, and oceans around the world, where they accumulate and fragment into smaller particles of microplastics and nanoplastics (Barnes et al., 2009). MPs have a negative effect on marine organisms, and can combine with other pollutants, such as plastic additives or organic pollutants, to create more damage (Browne et al., 2013;Hammer et al., 2012). At the present time, MPs characterize the greatest category of pollutants in the world (Derraik, 2002;Galgani et al., 2013).
MPs are also a possible cause of sh reproductive stress, which causes reproductive disruption, but the stress response in sh gonads caused by MPs exposure is still poorly understood. It has been reported that MPs cause endocrine impairment and disrupt the hypothalamus-pituitary-gonadal (HPG) axis and steroidogenesis pathway (Karami et al., 2016;Rochman et al., 2014;Wan et al., 2019). MPs have also been found to delay female gonadal maturation, decrease fecundity (Lönnstedt and Eklöv, 2016;Wan et al., 2019), increase the amount of reactive oxygen species (ROS) in the gonads of both male and female zebra sh, cause reproductive stress by increasing apoptosis levels and histological alterations in testes (Qiang and Cheng, 2021), and decrease the plasma 17b-estradiol (E2) and testosterone (T) levels in female marine medaka (Oryzias melastigma) (Wan et al., 2019).
Recent research has focused on nding effective natural antioxidants to replace synthetic antioxidants currently in use (Glodde et al., 2018;Kumosani et al., 2017;Taghvaei and Jafari, 2015). Benthic diatoms are microalgae that are recognized as natural antioxidants (El-Sayed et al., 2018;Lee et al., 2008). Amphora coffeaeformis is a single-celled microalgae present in most aquatic environments that can tolerate various salinities (Sala et al., 1998). Its cell wall is made of opaline silica (Buhmann et al., 2014) and it is an important food source for many invertebrates, sh species, and zooplankton (Kaparapu, 2018). Diatoms such as A. coffeaeformis, have a high capability for metal absorption and a high proliferation rate (Anantharaj et al., 2011), which enables them to resist the toxic effects of environmental contaminants (Gaur and Rai, 2001). These features support the use of diatoms as antioxidant mediators, and detoxifying agents in stress-induced shes (Sheikhzadeh et al., 2012).
This study investigated the effect of A. coffeaeformis supplementation as pre-feeding on the Oreochromis niloticus intoxicated with MPs. This objective accomplished via the assessment of hematological and biochemical parameters, antioxidant enzymes, hormonal levels, and the reproductive status of male sh. This study will help to elucidate the effects of MPs exposure on sh health, with special concern to gonadal health, and attempts to ameliorate the negative impacts of MPs by supplementing A. coffeaeformis in sh diets.

Experimental design
Nile tilapia ngerlings (360) were obtained from the Baltim experimental farm (NIOF), Egypt and were transferred to sh nutrition laboratory (NIOF) and fed on a commercial diet (25% crude protein, 4.5 L, Aller Aqua, Egypt) for one week to adjust to the experimental conditions. The sh (average body weight ca. 4 g) were distributed in 12 tanks with 200 L capacity on triplicate basis. Water temperature was 27.4 ± 1.2°C. Oxygen was supplied and kept above 7.0 mg/L. The natural photoperiod was 11-h light: 13-h dark throughout the experiment period. The experimental diets contained ascending levels of A. coffeaeformis (0%, 2.5%, 5 % and 7.5%) to their diets, as described by Saleh et al. (Saleh et al., 2020) (Table 1). The test diets were given three times a day to satiation, and the sh were fed the experimental diets for 70 days. After this period 10 male sh were selected from each tank and placed into separate aquaria for extra 15 days under MPs treatment on triplicate basis. Microplastic was added to the each aquarium at a concentration of 10 mg/ L according to Hamed et al. (2019). The experimental sh groups included the control group (A0MP0) where sh were fed a control diet and reared in normal conditions, a group (A0MP) where the sh were fed the control diet, but exposed to MP-polluted water, and an additional three test groups (A1MP, A2MP and A3MP), where sh were reared in MP-polluted water while receiving increasing concentrations of A. coffeaeformis (2.5, 5 & 7.5%, respectively) in their diets.
At the end of the day 15 of MPs exposure, six sh from each group were randomly chosen and sampled. Blood was collected from the caudal vein to measure antioxidant enzymes, hormone levels, and lipid peroxidation. After sh were anesthetized and weighed separately, they were directly killed by decapitation and then dissected. Gonads were excised from each sh, weighed and GSI calculations were completed using the following formula: GSI = gonads weight/gutted weight × 100. Afterwards, the gonads were xed in neutral buffered formalin (10%).
Fish maintenance and experimental procedures were approved by the Research Committee of the NIOF, Egypt, and were in accordance with the Guide for Use and Care of Laboratory Animals (European Communities Council Directive 2010/63/EU).

Diatom preparation
A. coffeaeformis powder was obtained from the Microalgae Culture Center at the National Research Center, NRC, Egypt. The production procedure was carried out according to El-Sayed et al. (2018). Analysis of the A. coffeaeformis powder samples was conducted to determine their biochemical composition (AOAC, 1995), which included 26.6% crude protein, 9.9% crude lipid, 45.4% ash, 3.3% ber and 14.8% total carbohydrate content. The diets were isonitrogenous (ca. 30% CP), isolipidic (8% L) and isoenergetic (19 MJ/kg).

Microplastics
The MPs consisted of raw powder with irregular-shaped particles. More than 90% of the MPs were > 100 nm in size. The MP powder was purchased from Toxemerge Pty Ltd. (Melbourne, Australia). A stock solution was prepared from the powder using puri ed water (Milli-Q) according to the manufacturer's instructions and stored at 4 ºC in the dark. The stock solution (2.5 g MP/L) was sonicated before each use. Additional dilutions were prepared from this stock immediately every time the rearing water was changed. The characterization of the MP particles was performed using light and transmission electron microscopy at TEMU, Assiut University (JEOL JEM-1200 EX II) (Hamed et al., 2019).

Hematological parameters
The blood samples were collected from the caudal vein of the sh using heparinized syringes, and immediately smeared onto clean glass slides. Blood smears (six slides from each sh) were dried, then xed in absolute methanol for 1 min and stained with hematoxylin and eosin stain (H&E) per Mekkawy et al. (2011). In each slide group, 10,000 cells (minimum of 1,000 cells per slide) were examined under a 40× objective lens with a 10× eyepiece using an OMAX microscope with a 14 MP USB digital camera (CS M837ZFLRC140U) to identify any micronucleated, morphological, or nuclear abnormalities in the red blood cells (RBCs) per Al-Sabti and Metcalfe (1995). The RBCs and white blood cell (WBCs) counts, hematocrit (HCT), and hemoglobin (HB) were estimated using an automated hematology analyzer (BCC-3000B; Changchun Dirui Industrial Co., Ltd). Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated using the formulae mentioned by Dacie and Lewis (1991).

Histological analysis
The xed testicular samples were dehydrated in ascending grades of ethanol, then were cleared in methyl benzoate, and embedded in para n wax. Para n sections of 5 µm thickness were cut and stained with H&E stain, then examined microscopically.
2.9. Statistical analysis Data are expressed as mean ± standard deviation. Signi cant differences between the treated and control groups were tested using one-way analysis of variance (ANOVA) followed by the Tukey-HSD test for multiple comparisons. Probability of a signi cant difference was set at p < 0.05. The analysis was carried out using the SPSS® version 23.0 package (SPSS, 1998).

Hematological parameters
The blood smears from all treated groups had altered erythrocyte shapes, including acanthocytes, crenated cells, teardrop-like cells, and sickle cells. Altered nucleus shapes were also found, including blebbed nuclei, notched nuclei, vacuolated nuclei, and bilobed nuclei (Fig. 1b-f).
The sh group that was only exposed to MPs (A0MP) had a signi cantly high number of altered erythrocytes (p < 0.05) compared to the other groups (Fig. 2). The group fed the highest level of A. coffeaeformis (A3MP) had a signi cantly (p < 0.05) smaller number of altered erythrocytes compared to the other two A. coffeaeformis fed groups (A1MP and A2MP), but still displayed a signi cantly higher (p < 0.05) amount than the control group (Fig. 2).
The sh blood analysis results displayed a signi cant decrease (p < 0.05) in RBCs, HB, Ht and platelets in the control group (A0MP0), A1MP and A2MP sh groups. In contrast, these blood biomarkers were not signi cantly different in the A3MP group compared to the control (p < 0.05; Table 2). WBCs were signi cantly (p < 0.05) increased in A0MP and A1MP compared to A2MP; however, MCV, MCH, and MCHC percentages were not signi cantly different between the different treatment groups. WBCs differentiation demonstrated a remarkable decrease in the percentage of eosinophils in A0MP and A1MP, which coincided with a signi cant increase in neutrophils (p < 0.05). No change was found in the percentage of monocytes and lymphocytes. Data are presented as mean ± SD, n = 6; means with different superscript letter within the same row for each parameter are signi cantly different (p < 0.05).

Biochemical parameters
No signi cant differences (p < 0.05) were found in liver enzyme levels (AST and ALT) among the treatment groups (Table 3). ALP levels were the highest in A0MP but remained at control levels in sh fed A. coffeaeformis. The serum glucose levels signi cantly increased (p < 0.05) in A0MP, A1MP, and A2MP compared to the control, but A3MP had serum glucose at control levels. The cholesterol and serum total protein levels were unchanged between the different treatment groups (p < 0.05). An obvious elevation in serum uric acid values were recorded in A0MP, A1MP, and A2MP, but A3MP had uric acid levels at control levels. The uric acid levels displayed a negative correlation with increasing amounts of A. coffeaeformis (Table 3). Serum albumin levels and albumin-to-globulin ratios (A/G) were signi cantly elevated (p < 0.05) in A0MP and A1MP. A2MP and A3MP had the same serum albumin and A/G values as the control. Data are presented as mean ± SD, n = 6, means with different superscript letter within the same row for each parameter are signi cantly different (p < 0.05). Abbreviations: A/G, albumin-globulin ratio; ALT, alanine aminotransferase; AST, aspartate aminotransferase 3.3. Antioxidant enzyme activities TAC levels were unchanged between (A1MP, A2MP, A3MP) and control, while TAC level was only signi cant higher in A0MP than A1MP group (Fig. 3a). A signi cant elevation (p < 0.05) in the serum levels of SOD and CAT enzymes in A0MP were observed compared to the control, while the other three sh groups fed A. coffeaeformis had levels that were comparable to the control group (Fig. 3b, c).

Hormonal activity and GSI
The serum LH and T pro les demonstrated pronounced suppression in A0MP. The LH and T serum levels in A3MP were close to those of the control group. The FSH serum levels showed insigni cant (p < 0.05) changes between the different groups (Fig. 4a, b and c). Moreover, the GSI displayed signi cant increases (p < 0.05) in A3MP (Fig. 4d).

Testicular histology
Light microscopic examination showed that transverse sectioned testes from the control group consisted of lobules with different spermatogenic stages that were separated from each other by interstitial tissue. An accumulation of mature spermatozoa was observed in the lumen of the testicular lobules (Fig. 5a).
The A0MP group had a small number of spermatozoa or no spermatozoa in the lumen of the testicular lobule. Vacuolated seminiferous epithelium and a slight proliferation of the interstitial connective tissue were also detected in the testes, but the testes still displayed considerable amounts of spermatogonia and spermatocytes in the tubule cysts. Other sections of the testes had perinucleolar oocytes inside the testicular lobules (testis-ova) (Fig. 5b, c & d).
A1MP had proliferated interstitial connective tissue, a marked decrease of the spermatozoa in the lumen of the testicular lobules, and vacuolization of the seminiferous epithelium. The development of a considerable amount of early perinucleolar oocytes were found inside the testicular lobules (testis-ova), and degenerative changes with necrotic areas were also observed (Fig. 5e &f).
A2MP and A3MP had no testicular alterations. Sections showed a large number of spermatozoa occupying most of the testicular lobules (Fig. 5g&h).

Discussion
Hematological parameters are the primary indicators of sh health (Thummabancha et al., 2016). In this study, MPs induced erythrocyte alterations in all tested groups compared to the control (A0MP0). Fish groups fed A. coffeaeformis-supplemented diets expressed less signi cant erythrocyte alterations than the A0MP0 group. A remarkable reduction in RBCs, Hb, Ht, platelets, and eosinophil percentages after exposure to MPs were detected. Furthermore, the neutrophil percentage signi cantly increased after MPs exposure. In general, the hematological results indicated that the highest amphora supplementation level (7.5%) ameliorates the effects of MPs, since they maintained the hematological parameters at control levels. These results concur with those of Hamed et al. (2019), who found that MPs exposure induced hematological deterioration in juvenile Nile tilapia. Moreover, immune system e ciency is affected by MPs uptake, which disturbs the organism's defense and health (Espinosa et al., 2017).
Serum biochemical parameters are good indicators for the general health status of animals and can provide early signs of serious changes in stressed organisms (Folmar, 1993;Garima and Himanshu, 2015;Jacobson-Kram and Keller, 2001). It has been reported that MPs exposure can induce changes in the biochemical parameters of various sh species, including the common goby (Pomatoschistus microps) (Oliveira et al., 2013), the common carp (Cyprinus carpio) (Haghi and Banaee, 2016), and Nile tilapia (Hamed et al., 2019). In general, the different serum biomarkers act as indicators for organ dysfunction and the impairment of biological processes. In this study, some serum biomarkers (ALP, glucose, uric acid, albumin, and A/G ratio) were signi cantly increased due to MPs exposure. These biomarkers were not signi cantly changed and maintained as control levels when A. coffeaeformis supplementation used with 5% and 7.5% diets.
The results of this study agree with those of Mekkawy et al. (2020), who recorded that A. coffeaeformis dietary supplementation improved arsenic-induced hematological parameters in African cat sh (Clarias gariepinus), and those of El-Sayed et al. (2018), who proved that dietary supplementation of A. coffeaeformis removed elements causing liver tissue damage induced by paracetamol induction in rats. The ameliorative effect of A. coffeaeformis may be attributed to its bioactive components, which have many health bene ts Saleh et al., 2020) that protect organisms against stressors. Ayoub et al. (Ayoub et al., 2019a) found that A. coffeaeformis supplementation in sh diets can increase disease resistant and improve hematological and biochemical parameters in Nile tilapia.
Antioxidant biomarkers are used to identify the effects of multiple environmental stressors in aquatic organisms (Hook et al., 2014). SOD and CAT levels in this study were signi cantly elevated attributable to MPs exposure, while TAC had close to control levels in all tested groups, but were signi cantly different between A2MP and A0MP. The disruption of antioxidant enzyme activities triggered by MPs exposure has been recorded in some studied aquatic organisms (Prinz and Korez, 2020) and sh, including the common goby (Luís et al., 2015), zebra sh (Lu et al., 2016), sheepshead minnows (Choi et al., 2018), and red tilapia (Ding et al., 2018). Opposing the present results, Karami et al. (2016) found that MPs had no effects on the antioxidant enzyme activities in zebra sh. Changes in CAT and SOD enzymes were limited by A. coffeaeformis supplementation. These results agree with El-Sayed et al. (2018) and Moneeb et al. (2020), who suggested that A. coffeaeformis extract can restore antioxidant enzymes to control levels. A. coffeaeformis has antioxidative properties and is considered a natural antioxidant (Lee et al., 2008;Lee et al., 2009) that is able to stimulate a speci c set of biochemical and physiological actions to repel the toxic effects of environmental pollutants (Gaur and Rai, 2001).
Fish reproductive success has a long-term effect on population constancy and the integrity of entire aquatic ecosystems. The current results demonstrated that exposure to MPs pollution can negatively impact male tilapia fertility, and induce oxidative stress. The potential negative impacts of MPs were reduced in sh fed higher A. coffeaeformis levels.
It is suggested that MPs indirectly act on the hypothalamus-pituitary axis to alter the synthesis and secretion of gonadotropin, which leads to the interruption of sex steroid production and testicular degeneration. The alterations seen in this study have also been recorded in male marine medaka (Oryzias melastigma) and may be explained by the up-regulation of gene expression for genes involved in the HPG axis and the steroidogenesis pathway (Wan et al., 2019). MPs also down-regulated the transcription of gonadotropin-releasing hormone (GnRH) in the hypothalamus of African cat sh (Clarias gariepinus) (Karami et al., 2016), and vitellogenin and choriogenin in the liver of male Japanese medaka (Oryzias latipes) (Rochman et al., 2014). In accordance with the histological alterations described in this study, Rochman et al. (2014) de ned abnormal male germ cell growth and suggested that this abnormal growth may be due to small clusters of oogonia that undergo atrophy when male Japanese medaka were exposed to environmentally relevant MPs concentrations. Wang et al. (2019) explained different histological alterations in testicular tissues after MPs exposure, including an increase in the interstitial tissue, a disorder of the arrangement of seminiferous lobules, the dissolution of the basal membrane, and a loose arrangement of spermatocytes. Moreover, a reduction in the testis basement membrane thickness was also recorded (Qiang and Cheng, 2021). In contrast to the present results, MPs exposure signi cantly increased the level of T in male marine medaka (Wan et al., 2019).
This study indicated that the testis histological alterations found in sh exposed to MPs were the most improved by the higher two doses of A. coffeaeformis supplementation in A2MP (5%) and A3MP (7.5%), which maintained the LH and T levels as in control sh and improved GSI values.
Altogether, the current results suggest that MP exposure reduces reproductive performance in Nile tilapia indirectly through oxidative stress, which agrees with Wang et al. (2019). Qiang and Cheng (2021) proposed that overall stress in detoxi cation and the induction of antioxidant enzyme activities are contributing factors in reproductive impairments. Gonads are most predisposed to oxidative stress, since gonads display a higher frequency of cell division, mitochondrial oxygen intake, and unsaturated fatty acids compared to other tissues (Asadi et al., 2017;Ciani et al., 2015). It has been previously shown that stressors, such as chemical exposure, can induce testicular oxidative stress, cause apoptosis in germ cells, and negatively affect spermatogenesis (Asadi et al., 2017).
In conclusion, 10 mg/L MPs exposure in sh for 15 days induced hematological and biochemical changes in O. niloticus. Furthermore, MPs prompted oxidative stress, hormonal disruption and testicular damage, and induced testis-ova in male tilapia. The highest tested supplementation level of A. coffeaeformis (A3MP; 7.5%) prevented the oxidative stress, testicular damage, and negative hematological alterations. The present results suggest that MPs indirectly affect the HPG axis and subsequently the gonads through oxidative stress. Additional research work is needed to determine the mechanisms responsible for the endocrine disruption, histomorphological alterations, and possible reproductive impairments induced by MP exposure. Funding Wan, Z., Wang, C., Zhou, J., Shen, M., Wang, X., Fu, Z., Jin, Y., 2019. Effects of polystyrene microplastics on the composition of the microbiome and metabolism in larval zebra sh. Chemosphere 217, 646-658.

Figure 3
Antioxidant enzyme activity (TAC, SOD and CAT) in Nile tilapia (Oreochromis niloticus) fed with different A. coffeaeformis concentrations, then exposed to 10 mg/L microplastics. Data are presented as mean ± SD. n = 6. Means with different superscript letters are signi cantly different (p < 0.05).
Page 22/24 Hormonal activity (LH, FSH and T) and GSI% value in Nile tilapia (Oreochromis niloticus) fed different A.
coffeaeformis concentrations, and then exposed to 10 mg/L microplastics. Data are presented as mean ± SD. n = 6. Means with different superscript letters are signi cantly different (p < 0.05).