Interactive effects between multiple stressors, namely climate drivers (e.g., temperature) and chemical pollution (e.g., endocrine disruptors) are poorly studied. Here, it was for the first time evaluated the combinatory effects of temperature and a synthetic progestin, levonorgestrel (LNG), on the fitness and reproductive-related endpoints of zebrafish (Danio rerio). A multi-factorial design was implemented by manipulating both temperature [setting as baseline an ambient temperature of 27 °C, against warming (+3 °C)] and LNG levels (10 ngL−1 and 1000 ngL−1). Groups of males and females were exposed sub-acutely, for 21-days. Increased temperature caused an overall decrease in the females’ gonadosomatic index (GSI), during the pre-reproduction phase, LNG did not affect GSI. In addition, fecundity (number of ovulated eggs) was negatively affected by both temperature and LNG, being the effect of the latter more intense. Fish exposed to the highest LNG concentration (at both temperatures) did not reproduce, but also in those exposed to the lowest dose of progestin at a higher temperature, a complete reproductive failure occurred. These results reflect what was observed in the stereological analysis of the ovary maturation stages prior to reproduction. Accordingly, the higher the LNG concentration, the lower the degree of maturation of the ovary. This was exacerbated by the higher temperature. As to embryonated eggs, they hatched significantly faster at higher temperatures, but exposure to 10 ngL−1 of LNG (at 27 °C) reduced significantly the hatching rate, comparing to control. Further, the recrudescence of the ovary 48 h after spawning seems to be not affected by both stressors. Our data suggest that in a future scenario of global warming and synthetic hormones exposure, the reproduction of fish species, such as the zebrafish, can be endangered, which can put at risk their success, and consequently affect the structure and functioning of associated aquatic ecosystems.

In the past few decades, extensive application of azoxystrobin has led to great concern regarding its adverse effects on aquatic organisms. The objective of the present study was to evaluate the reproductive toxicity of azoxystrobin to zebrafish. After adult zebrafish of both sexes were exposed to 2, 20 and 200 μg/L azoxystrobin for 21 days, egg production, the fertilization rate, the gonadosomatic index (GSI) and hepatosomatic index (HSI), 17β-estradiol (E2), testosterone (T) and vitellogenin (Vtg) concentrations, and histological alterations in the gonads and livers were measured. Meanwhile, expression alterations of genes encoding gonadotropins and gonadotropin receptors (fshb, lhb, fshr and lhr), steroid hormone receptors (era, er2b and ar), steroidogenic enzymes (cyp11a, cyp11b, cyp17, cyp19a, cyp19b, hsd3b and hsd17b) in the hypothalamic-pituitary-gonad (HPG) axis and vitellogenin (vtg1 and vtg2) in the livers were also investigated. The results showed that reduced egg production and fertilization rates were observed at 200 μg/L azoxystrobin. In female zebrafish, reduced E2 and Vtg concentrations, decreased GSI, increased T concentrations, and histological alterations in the ovaries and livers were observed at 200 μg/L azoxystrobin, along with significant down-regulation of lhb, cyp19b, lhr, cyp19a, vtg1 and vtg2, and up-regulation of cyp17, hsd3b and hsd17b. In male zebrafish, increased E2 and Vtg concentrations, reduced T concentration and GSI, and histological alterations in the testes and livers were observed after exposure to 20 and 200 μg/L azoxystrobin, along with significant up-regulations of cyp19b, cyp11a, cyp17, cyp19a, hsd3b and hsd17b, vtg1 and vtg2. Moreover, cyp11a, hsd3b, cyp19a, vtg1 and vtg2 in male zebrafish were significantly up-regulated after treatment with 2 μg/L azoxystrobin. The results of the present study indicate that azoxystrobin led to reproductive toxicity in zebrafish and male zebrafish were more sensitive to azoxystrobin than female zebrafish.

Environmental estrogens are capable of interfering with the spermatogenesis and fertility of fish. However in natural waters, these chemicals are more likely to occur as a combination rather than a single stressor. Whether and how the mixture of xenoestrogens with environmental relevant concentrations may affect fish spermatogenesis remains largely unknown. In this study, male zebrafish adults were administered to 17alpha-ethinylestradiol (EE2) and a mixture of xenoestrogens (Mix (E2, EE2, DES, 4-t-OP, 4-NP and BPA)), with the estrogenic potency equivalent to EE2. After a 60-day exposures, elevated mRNA levels of vitellogenin 1 (vtg1) and estrogen receptor 1 (esr1) in the liver of fish in both treated groups were observed. Moreover, the plasma level of E2 declined significantly in the Mix group and the ratio of 11-KT/E2 was significantly elevated in both treated groups. Consistently, the mRNA level of P450 side-chain cleavage (scc) in the EE2 group and ovarian type aromatase (cyp19a1a) in the Mix group was significantly suppressed. In addition, decreased gonadosomatic index and sperm count in the fish of Mix group were present. Furthermore, increased number of the proliferating germ cells (such as spermatogonia and spermatocytes) was observed in the fish of both groups, suggesting a stimulated germ cell proliferation and meiosis. Accordingly, both exposures significantly up-regulated the mRNA levels of genes in mitosis (cyclinb1) and meiosis (cyp26a1 in EE2 group, aldh1a2, cyp26a1, sycp3 and spo11 in Mix). In addition, decreased number of spermatozoa and increased number of TUNEL-positive signals were present in the testis of fish in the Mix group, indicating an enhanced apoptosis. Further analyses demonstrated the significant elevated expressions of tnfrsf1a and the ratio of tnfrsf1a/tnfrsf1b in the Mix group, suggesting an elevated apoptosis in the testis of fish in the Mix group via extrinsic pathway. The present study greatly extends our understanding of the underlying mechanisms of the reproductive toxicity of xenoestrogens on fish.

The interaction of xenobiotics is common in aquatic ecosystems; therefore, we wanted to evaluate if trenbolone (TB) modulates the effects of 17α-ethinylestradiol (EE2). Male eelpout (Zoarces viviparus) were exposed to 5 ng L−1 EE2 continuously for 19 d (EE2-C) or discontinuously (11 d, EE2-D) alone or in combination with low (50 ng L−1, TBL) or high (500 ng L−1, TBH) concentrations of TB (19 d). Exposure to EE2 caused reduced gonadosomatic index, increased plasma vitellogenin concentrations, up-regulated vtg and era mRNA expression and severe alterations in gonadal histology. TBL and TBH did not affect plasma vitellogenin, era or vtg mRNA expression. TBL and TBH did not counteract the EE2-induced increase in plasma vitellogenin and reduction in 11-ketotestosterone whereas TBH counteracted the EE2 induced increase in vtg and era mRNA expression. Exposure to TBH and EE2-C + TBH lead to severe gonadal histology alterations. TBL and EE2-D + TBH exposed fish showed less histopathological alterations.

Bisphenol A (BPA), a monomer used for polycarbonate manufacture, has been widely reported as an endocrine-disrupting chemical (EDC). Among other alterations, BPA induces reproductive dysfunctionalities. Changes in the endocannabinoid system (ECS) have been recently shown to be associated with reproductive disorders. The ECS is a lipid-based signaling system (cannabinoid receptors, endocannabinoids and enzymatic machinery) involved in several physiological functions. The main goal of the present study was to assess the effects of two environmental concentrations of BPA (10 and 20 μg/L) on the ECS in 1-year old zebrafish gonads. In males, BPA increased the gonadosomatic index (GSI) and altered testicular levels of endocannabinoids as well as reduced the testicular area occupied by spermatogonia. In male liver, exposure to 20 μg/L BPA significantly increased vitellogenin (vtg) transcript levels. In female zebrafish, BPA altered ovarian endocannabinoid levels, elevated hepatic vtg mRNA levels as well as increased the percentage of vitellogenic oocytes in the ovaries. In conclusion, exposure to two environmentally relevant concentrations of BPA altered the ECS and consequently, gonadal function in both male and female zebrafish.

This work aimed at predicting the toxic effects of phenolic compounds in Ba River on the health of female sharpbelly (Hemiculter lucidus) by the de novo transcriptomic analysis of the liver. Sharpbelly, a native fish living in freshwater ecosystem of East Asia, were sampled upstream, near, and downstream of a wastewater discharge to the Ba river. Based on the occurrence of bisphenol A (BPA), nonylphenol (NP), and 4-tert-octylphenol (4-t-OP) in the water and fish sampled from each site, up-, mid-, and down-stream were interpreted as control, high, and low treatment groups, respectively. In the mid-stream group the Fulton’s condition factor (CF) and body weight were remarkably increased by approximate 20%; the gonado-somatic index (GSI) and hepatosomatic index (HSI) in mid-stream fish showed a similar increasing trend but lacking of statistical difference. Exposure to wastewater effluent caused 160 and 162 differentially expressed genes (DEGs) in up-mid and down-mid stream groups, respectively. Two sets of DEGs were primarily enriched in the signaling pathways of drug metabolism, endocrine system, cellular process, and lipid metabolism in the mid-stream sharpbelly, which may alter the fish behavior, disrupt the reproductive function, and lead to hypothyroidism, hepatic steatosis, etc. Taken together, our results linked the disrupted signaling pathways with activities of phenolic compounds to predict the potential effects of wastewater effluent on the health of wild fish.

The growing use of octocrylene (OC) in sunscreens has posed a great threat to aquatic organisms. In the present study, to assess its reproductive toxicity and mechanism, paired Japanese medaka (Oryzias latipes) (F0) were exposed to OC at nominal concentrations of 5, 50, and 500 μg/L for 28 d. Significant increases were observed in the gonadosomatic index (GSI) and hepatosomatic index (HSI) of F0 medaka at 500 μg/L OC (p < 0.05) without significant differences in fecundity. The fertility was significantly decreased at all treatments (p < 0.05). Significant increases in the percent of mature oocytes were observed at 5 and 500 μg/L OC, in which contrary to the percent of spermatozoa (p < 0.05). The plasma sex hormones and vitellogenin levels significantly increased in males at all treatments and in females at 50 and 500 μg/L OC (p < 0.05). In addition, the levels of fshβ and lhβ in the brains and the levels of fshr, lhr and cyp17α in the gonads were significantly upregulated in males at all treatments (p < 0.05), in line with those of ar, erα, erβ and cyp19β in the brains of male and female. The upregulation of vtg in male and female livers was observed only at 500 μg/L OC and upregulation of star and hsd3β was observed in testis at all treatments (p < 0.05). Continued exposure to OC significantly induced increases in the time to hatching, morphological abnormality rates, and cumulative death rates of F1 embryos, inconsistent with body length of F1 larvae (p < 0.05). Therefore, the responses of the exposed fish at the biochemical and molecular levels indicated reproductive toxicity and estrogenic activity of OC, providing insights into the mechanism of OC.

A large variety of anthropogenic chemicals present in the aquatic environment have been shown to be able to alter the endocrine system of exposed organisms, potentially impacting their reproductive function. The aim of this study was to assess the effects of environmental pollution on the reproductive system of wild female roach (Rutilus rutilus) from the Seine River (Normandy, France). A suite of biomarkers of endocrine disruption including gonado-somatic index, plasmatic vitellogenin, gonadal aromatase activity and histological parameters (oocyte diameter and gonad maturation) were studied. Female fish from the polluted sites showed a number of reproductive alterations, including inhibited gonad maturation, reduced oocyte growth, reduced levels of plasmatic vitellogenin and 3-fold lower gonadal aromatase activity than females collected in the reference site. Overall, these results highlight the presence of endocrine disruption in female roach from the Seine River.

Endocrine disrupting chemicals (EDCs) found in wastewater treatment plant (WWTP) effluents have been shown to cause adverse effects, but the uptake of EDCs from effluents (measured in fish muscle) are not known. In this study, the biological effects and bioaccumulation of steroidal and phenolic EDCs were assessed in high-back crucian carp (Carassius auratus) exposed to WWTP effluents for 141 days. Compared with fish controls caged in Dianchi Lake, a significant reduction in gonadosomatic index (GSI) and increase in hepatosomatic index (HSI) and plasma vitellogenin (VTG) levels were observed in effluent-exposed fish. The concentrations of steroids and phenols in effluent-exposed fish showed time-dependent increase during the exposure. In addition, bioconcentration factors (BCFs) for steroids and phenols were between 17 and 59 on day 141. The results confirm that steroids and phenols bioconcentrate in fish muscle and this accumulation may account for the biological effects associated with exposures to WWTP effluents.

To evaluate the health status at six different study areas, we used the generalized linear model approach with selected biochemical markers in resident fish from uncontaminated and contaminated sites. We also confirmed the independence between the biochemical indices and the morphometric indices including the hepato-somatic index (HSI), gonado-somatic index (GSI), and condition factor (CF) in fish from the sampling areas. The effect of area on the presence of biotransformation markers (ethoxyresorufin-O-deethylase activity; EROD) was significantly high in Masan Bay. The area with the greatest effect on acetylcholinesterase (AChE) activity was Jindong Bay, while there was no significant effect of GSI, HSI, CF, and sex in the EROD model and HSI, CF and sex in the AChE model. These results clarify that fish from Masan, Gwangyang and Jindong Bay were affected by pollutant stress, and the analysis of sensitive biochemical responses allowed for an improved interpretation of the results.