Multiple Xenosteroid Pollutants Biomarker Changes in Xultured Nile Tilapia Using Wastewater Effluents as Their Primary Water Source

Abstract

This study was undertaken to screen levels of xenosteroids (estrogenic endocrine disrupting chemicals/E-EDCs) in Nile tilapia (Oreochromis niloticus) fish farms subjected to water fill from the drain at three sites S1 (highly polluted), S2 (moderately polluted), and a putative reference site (RS). Biometric, hormonal, gene expression, and histopathological analysis were investigated. Testosterone, progesterone, and zeranol residues were detected at (0.12-3.44 µg/L) in water samples of different sites. Bisphenol-A (BPA) exhibited a very high concentration (6.5 µg/mL) in water samples from S1. Testosterone, 17β-estradiol residues were detected in fish tissues from all sites at (0.16-3.8 µg/Kg) and (1.05-5.01 µg/Kg), respectively. BPA residues were detected at a very high concentration in the liver and muscle of fish collected from S1 at higher levels of 25.9 and 48.07 µg/Kg, respectively. The detected E-EDCs, at different sites, particularly BPA, reduced the somatic and testicular growth among sites and oversampling time points. Meanwhile, hepatosomatic index (HSI) was significantly increased in S1 compared to S2. All analyzed genes estrogen receptor-type I (er-I, er-ɑ) and II (er-II, er-ß1), polypeptide 1a (cyp19a1), SRY-box containing gene 9 (sox9), and vitellogenin (vtg) and gonadotropin hormones (luteinizing hormone (LH), follicle-stimulating hormone (FSH)), testosterone, 17β-estradiol, and anti-Mullerian hormone (AMH) were significantly expressed at S1 compared to other sites. Histopathology was more evident in S1 than other sites. These findings warrant immediate strategies development to control aquatic pollution and maintain fish welfare and aquaculture sustainability.

Keywords: EDCs; Lake Manzala; fish; histopathology; hormonal disturbances; reproductive gene dysfunction.

Figure 1

The study area of Manzala lagoon in the northeastern Nile delta of Egypt (Bahr El-Baqar drain, sampling sites in red marks). S1: Site 1; S2: Site 2.

Figure 2

(a) Weight (Wt) and Length (L), (b) testicular somatic index (TSI), (c) hepatosomatic index (his), and K-factor of Nile tilapia collected from different sampling sites. Data are presented as the mean of 20 fish ± standard error of mean (SEM). Values with a different uppercase letter superscript indicate a significant difference between sites at the same sampling time. Values with different lowercase letter superscript are significantly different between sampling time points within the same site (p < 0.05, two-way ANOVA).

Figure 3

Vitellogenin (vtg) (a), estrogen receptor-type I (er-I) (b) mRNA levels in the liver; and er-I (c), er–II (d), polypeptide 1a (cyp19a1) (e), SRY-box containing gene 9 (sox9) (f) mRNA levels in testes relative to β-actin mRNA levels analyzed by qRT-PCR in Nile tilapia collected from different sampling sites. Data are presented as the mean of 10 fish ± standard error of mean (SEM). Values with a different letter superscript indicate a significant difference between sites (p < 0.05, one-way ANOVA). RS = control.

Figure 4

Testosterone (a), 17β-estradiol (E2) (b), anti-Mullerian hormone (AMH) (c), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (d) serum hormonal analysis of Nile tilapia collected from different sampling sites. Data are presented as the mean of 10 fish ± standard error of mean (SEM). Values with a different letter superscript indicate a significant difference between sites (p < 0.05, one-way ANOVA). Values with a different dagger superscript indicate a significant difference between FSH and LH levels in the same site (p < 0.05, independent t-test). RS = control.

Figure 5

Microscopic pictures of testes of Nile tilapia from the reference site (RS): (a,c–e)—show accumulation of mature sperms in the lumen of the testicular lobule (black arrows) with vacuolated seminiferous epithelium (arrowheads), (e)—congestion (red arrow) and (b,f)—slightly increased amount of interstitial of connective tissue (orange arrows). hematoxylin and eosin (H&E), ×100: (a,c,e,f) and ×400: (b,d).

Figure 6

Microscopic pictures of testes from site 1 (S1): (a,b)—show vacuolization of seminiferous epithelium (arrowheads) with the absence of spermatozoa from the lumen of the testicular lobule (asterisks), (c)—Developmental stages of oocytes are seen inside testicular lobule (thick arrow), (d,f,g)—with an increased amount of interstitial of connective tissue (orange arrows); (d,e)—congestion (red arrows), (f)—melanomacrophage center (black arrow), (g)—inflammation (green arrow). Testes from site 2 (S2): (h,i)—shows a reduced number of mature sperms (black arrows) with more vacuolization of seminiferous epithelium (arrowheads) and fewer numbers of spermatogonia and spermatocytes (blue arrows). hematoxylin and eosin (H&E), ×100: (a,d,h) and ×400: (b,c,e,f,g,i).

Figure 7

Testicular index of Nile tilapia captured from different sites. Values with a different letter superscript indicate a significant difference between sites (p < 0.05, Kruskal–Wallis). RS = control, S1 = site 1, S2 = site 2.