Reproductive Toxicity In Fish Research Articles

Combined effects of copper and cadmium exposure on ovarian function and structure in Nile Tilapia (Oreochromis niloticus).

With the rapid development of industrialization and urbanization, the issue of copper (Cu) and cadmium (Cd) pollution in aquatic ecosystems has become increasingly severe, posing threats to the ovarian tissue and reproductive capacity of aquatic organisms. However, the combined effects of Cu and Cd on the ovarian development of fish and other aquatic species remain unclear. In this study, female Nile tilapia (Oreochromis niloticus) were individually or co-exposed to Cu and/or Cd in water. Ovarian and serum samples were collected at 15, 30, 60, 90, and 120 days, and the bioaccumulation, ovarian development, and hormone secretion were analyzed. Results showed that both single and combined exposure significantly reduced the gonadosomatic index and serum hormone levels, upregulated estrogen receptor (er) and progesterone receptor (pr) gene transcription levels, and markedly affected ovarian metabolite levels. Combined exposure led to more adverse effects than single exposure. The data demonstrate that the Cu and Cd exposure can impair ovarian function and structure, with more pronounced adverse effects under Cu and Cd co-exposure. The Cu and Cd affect the metabolic pathways of nucleotides and amino acids, leading to ovarian damage. This study highlights the importance of considering combined toxicant exposure in aquatic toxicology research and provides insights into the potential mechanisms underlying heavy metal-induced reproductive toxicity in fish.

Reproductive toxicity in marine medaka (Oryzias melastigma) due to embryonic exposure to PCB 28 or 4’-OH-PCB 65

Previous studies have shown that juvenile or adult exposure to polychlorinated biphenyls (PCBs) induces alterations in reproductive functions (e.g., reduced fertilization rate) and behavior (e.g., reduced nest maintenance) in fish. Embryonic exposures to other endocrine disrupting chemicals have been reported to induce long-term reproductive toxicity in fish. However, the effects of embryonic exposure to PCBs or their metabolites, OH-PCBs, on long-term reproductive function in fish are unknown. In the present study, we used the marine medaka fish (Oryzias melastigma) as a model to assess the reproductive endpoints in response to embryonic exposure to either PCB 28 or 4’-OH-PCB 65. Our results showed that the sex ratio of marine medaka was feminized by exposure to 4’-OH-PCB 65. Fecundity was decreased in the medaka treated with either PCB 28 or 4’-OH-PCB 65, whereas the medaka from embryonic exposure to 4’-OH-PCB 65 additionally exhibited reduced fertilization and a reduction in the hatching success rate of offspring, as well as decreased sperm motility. Serum 11-KT concentrations were reduced in the PCB 28-treated medaka, and serum estradiol (E2)/testosterone (T) and E2/11-ketotestosterone (11-KT) ratios were decreased in the 4’-OH-PCB 65-treated medaka. To explain these observations at the molecular level, transcriptomic analysis of the gonads was performed. Bioinformatic analysis using Gene Ontology and Ingenuity Pathway Analysis revealed that genes involved in various pathways potentially involved in reproductive functions (e.g., steroid metabolism and cholesterol homeostasis) were differentially expressed in the testes and ovaries of either PCB- or OH-PCB-treated medaka. Thus, the long-term reproductive toxicity in fish due to embryonic exposure to PCB or OH-PCB should be considered for environmental risk assessment.

Embryonic exposure to fenbuconazole inhibits gametogenesis in adult zebrafish by targeting gonads not brain

Fenbuconazole (FBZ) is widely used in agriculture. The current study was conducted to evaluate the influence of embryonic exposure to FBZ on reproduction in adult zebrafish. Embryos were exposed to 5, 50 and 500 ng/L FBZ for 72 h and then raised in clean water until adulthood. The result showed that the percentage of mature gametes was significantly reduced in adult zebrafish. The fertilization rate and survival rate of F1 embryos were decreased when the exposed fish were mated with untreated fish. The transcription of brain gnrh3, fshβ and lhγ in adult fish was upregulated, while the levels of 17β-estradiol and testosterone were not significantly changed in all treated groups, indicating that the reproduction-related genes in brain was not responsible for the reduced reproductive ability. The downregulated transcription of fshr, lhr, ar and esr2 in the gonads indicated the dysfunction of Sertoli and Leydig cells. Notably, downregulated transcription and upregulated methylation levels of genes related to germ cells were observed in treated F0 larvae and adult gonads. The elevated methylation levels of piwil1 and dnmt6 in the testes and vasa and dazl in the ovary were matched with the alterations in the expression of these genes, suggesting that germ cells are the main targets of FBZ. These results provide new mechanism underlying reproductive toxicity in fish caused by chemicals, and give potential retroactive biomarkers for monitoring reproductive toxic pollutants.

2-Ethylhexyl Diphenyl Phosphate and Its Hydroxylated Metabolites are Anti-androgenic and Cause Adverse Reproductive Outcomes in Male Japanese Medaka (Oryzias latipes).

Although high concentrations of 2-ethylhexyl diphenyl phosphate (EHDPP) have been detected in wild fish, its reproductive toxicity in fish remains unclear. In this study, we for the first time observed that EHDPP elicited androgen receptor (AR) antagonistic activity with a 50% inhibitory concentration of 37.5 μM. 2-Ethyl-5-hydroxyhexyl diphenyl phosphate was proved to be the dominant metabolite of EHDPP in Japanese medaka and elicited 3.1-fold stronger AR antagonistic activity than that of EHDPP. Medaka larvae (0-day post hatching) were exposed to EHDPP for 100 days, and intersex was observed in males from all exposure groups with significantly increased incidence (13.5-48.6%). 17β-E2 was promoted at 104 ng/L, and androgens were suppressed at 434 ng/L, which account for the intersex incidence in the high-exposure groups but do not explain the significant incidence of intersex in the 29.9 ng/L exposure group. The AR antagonistic activity of EHDPP and its metabolites must therefore play a key role in intersex incidence. EHDPP also significantly (p < 0.05) repressed reproductive behaviors of males in the 434 ng/L group and decreased fertility in high-exposure groups compared with the control. All the adverse outcomes were observed under environmentally relevant concentrations, implying that EHDPP poses an ecological risk for wild fish populations.

Possible role of C60 fullerene in the induction of reproductive toxicity in the freshwater fish, Anabas testudineus (Bloch, 1792).

In recent years, the impact of ultrafine nanomaterials on the aquatic organisms and their ecosystems contributed much concern due to their abundance in environment. Several toxicity studies have reported that nanoparticles induced reproductive stress and resulted in reproductive impairment of fishes. The present study was aimed to investigate the stress-induced toxicity of C60 fullerene nanomaterial on various reproductive parameters of the freshwater fish, Anabas testudineus. Fish were exposed to two sublethal concentrations of fullerene C60, one-tenth (5mg/L) and one-fifth (10mg/L) of LC50-96h, for 4, 7, 15, 30, and 60-day durations. At the end of exposure period, the activities of steroidogenic enzymes, 3β-, and 17β-hydroxysteroid dehydrogenase decreased in the testis and ovary thereby indicated that the nanomaterial affected gonadal steroidogenesis. The level of serum testosterone decreased significantly (p < 0.05) in male whereas the level of estradiol showed significant (p < 0.05) reduction in female fish with significant (p< 0.05) increase in the level of serum cortisol in both sexes in concentration- and time-dependent manner. The analysis of the levels of alkali-labile phosphates, plasma calcium, and total protein showed significant (p < 0.05) reduction in female fish without significant changes in male fish, and this could be due to the antiestrogenic action of fullerene C60 nanomaterial. The activity of aromatase enzyme decreased significantly (p < 0.05) in the ovary and brain of female fish, and the decline in the enzyme activity was prominent only in the brain tissue of male fish. The present results suggested that the stress-induced by fullerene C60 exposure provoked reproductive toxicity in the fish, Anabas testudineus.

Two-generation exposure to 2-ethylhexyl 4-methoxycinnamate (EHMC) in Japanese medaka (Oryzias latipes) and its reproduction and endocrine related effects

2-Ethylhexyl 4-methoxycinnamate (EHMC) is one of the most widely used UV-filters, and hence has been frequently detected in water environment. EHMC has been reported to induce short-term reproductive toxicity in fish, and thyroid disrupting effects in other animal studies. However, limited information is available for its long-term effects on fecundity, and thyroid disrupting effects in fish. In the present study, effects of EHMC on fecundity, measured as number of eggs, were evaluated in Japanese medaka (Oryzias latipes), and its underlying mechanisms on sex and thyroid hormone disruption were explored. For this purpose, a five-month long (154 d) exposure to F0 generation was conducted on fertilized eggs (<24 h post-fertilization (hpf)), with nominal concentration of 0, 0.05, 0.158, 0.5, 1.58, or 5 mg/L EHMC, followed by a 3-8-d exposure of F1 generation. After >3 months exposure, significant decreases in reproductive performances were observed at all test concentrations as low as 0.05 mg/L. Reproduction effects were not accompanied with sex hormone changes, but up-regulation of vitellogenin gene was observed. Thyroid hormones were decreased by EHMC exposure in F1 fish at -38 day post-fertilization (dpf). In addition, down-regulation of type II iodothyronine deiodinase (dio2) and up-regulation of thyrotropin releasing hormone (trh) were observed in both F0 and F1 juvenile fish, suggesting thyroid disruption potential of EHMC. Our observation suggests that EHMC at the levels one to two orders of magnitude higher than those detected in ambient water may affect reproduction and thyroid hormonal balance of fish.

Testicular transcriptome alterations in zebrafish (Danio rerio) exposure to 17β-estradiol

The hormone 17β-estradiol (E2) can be found in rivers, effluents, and even drinking water. Researches have demonstrated that E2 affects various metabolic pathways through gene activation and may cause reproductive toxicity in fish. Therefore, the aim of this study was to evaluate E2-induced toxicity via testicular transcriptome of zebrafish (Danio rerio) exposed to different concentrations (10 ng L−1, and 100 ng L−1) of E2. A total of >600 significant differentially expressed genes (DEGs) were enriched among the three treatments. Short time-series expression miner analysis revealed five KEGG pathways including drug metabolism, other enzymes, calcium signaling pathway, ECM-receptor interaction, gap junction, and cell adhesion molecules. Twenty genes were selected to verify the accuracy of RNA-Seq. Other reported genes related to sex differentiation, development, energy metabolism, and other processes were found. One set of genes significantly increased/decreased/fluctuated over time, especially 12 h after E2 exposure. Genes associated with ovaries (zp3c), and development (bmp15, gdf9, and sycp2l) were significantly upregulated with increasing E2 concentration. E2 and testosterone was significantly decreased by 10 (except for T) and 100 ng L−1 E2 exposure at 12 h. The current study demonstrated that sex differentiation, development, energy metabolism, immunity, and ribosome biogenesis in male zebrafish were all significantly affected by 17β-estradiol exposure through transcriptional alterations.

Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish

Microcystis aeruginosa, a primary species in cyanobacterial blooms, is ubiquitously distributed in water. Microcystins (MCs) purified from M. aeruginosa can exert reproductive toxicity in fish. However, the effects of M. aeruginosa at environmentally relevant levels on the reproductive and endocrine systems of zebrafish are still unknown. The present study investigated the reproductive and endocrine-disrupting toxicity of M. aeruginosa on female zebrafish (Danio rerio) by short-term exposure (96 h). After exposure, marked histological lesions in the liver or gonads, such as nuclear pyknosis and deformation, were observed, and the fertilization rate and hatchability of eggs spawned from treated females were both significantly lower than they were in females in the control group, suggesting the possibility of transgenerational effects of M. aeruginosa exposure. Moreover, M. aeruginosa exposure decreased the concentration of 17β-estradiol (E2) and testosterone (T) in female zebrafish. Interestingly, the vtg1 transcriptional level significantly decreased in the liver, whereas plasma vitellogenin (VTG) protein levels increased. The present findings indicate that M. aeruginosa could modulate endocrine function by disrupting transcription of hypothalamic-pituitary-gonadal-liver (HPGL) axis-related genes, and impair the reproductive capacity of female zebrafish, suggesting that M. aeruginosa causes potential adverse effects on fish reproduction in Microcystis bloom-contaminated aquatic environments.

Cross‐species sensitivity to a novel androgen receptor agonist of potential environmental concern, spironolactone

Spironolactone is a pharmaceutical that in humans is used to treat conditions like hirsutism, various dermatologic afflictions, and female-pattern hair loss through antagonism of the androgen receptor. Although not routinely monitored in the environment, spironolactone has been detected downstream of a pharmaceutical manufacturer, indicating a potential for exposure of aquatic species. Furthermore, spironolactone has been reported to cause masculinization of female western mosquitofish, a response indicative of androgen receptor activation. Predictive methods to identify homologous proteins to the human and western mosquitofish androgen receptor suggest that vertebrates would be more susceptible to adverse effects mediated by chemicals like spironolactone that target the androgen receptor compared with invertebrate species that lack a relevant homolog. In addition, an adverse outcome pathway previously developed for activation of the androgen receptor suggests that androgen mimics can lead to reproductive toxicity in fish. To assess this, 21-d reproduction studies were conducted with 2 fish species, fathead minnow and Japanese medaka, and the invertebrate Daphnia magna. Spironolactone significantly reduced the fecundity of medaka and fathead minnows at 50 μg/L, whereas daphnia reproduction was not affected by concentrations as large as 500 μg/L. Phenotypic masculinization of females of both fish species was observed at 5 μg/L as evidenced by formation of tubercles in fathead minnows and papillary processes in Japanese medaka. Effects in fish occurred at concentrations below those reported in the environment. These results demonstrate how a priori knowledge of an adverse outcome pathway and the conservation of a key molecular target across vertebrates can be utilized to identify potential chemicals of concern in terms of monitoring and highlight potentially sensitive species and endpoints for testing.

Influence of elevated alkalinity and natural organic matter (NOM) on tissue-specific metal accumulation and reproductive performance in fathead minnows during chronic, multi-trophic exposures to a metal mine effluent

Metal bioavailability in aquatic organisms is known to be influenced by various water chemistry parameters. The present study examined the influence of alkalinity and natural organic matter (NOM) on tissue-specific metal accumulation and reproductive performance of fathead minnows (Pimephales promelas) during environmentally relevant chronic exposures to a metal mine effluent (MME). Sodium bicarbonate (NaHCO3) or NOM (as commercial humic acid) were added to a Canadian MME [45 percent process water effluent (PWE)] in order to evaluate whether increases in alkalinity (3–4 fold) or NOM (~1.5–3mg/L dissolved organic carbon) would reduce metal accumulation and mitigate reproductive toxicity in fathead minnows during a 21-day multi-trophic exposure. Eleven metals (barium, boron, cobalt, copper, lithium, manganese, molybdenum, nickel, rubidium, selenium, and strontium) were elevated in the 45 percent PWE relative to the reference water. Exposure to the unmodified 45 percent PWE resulted in a decrease of fathead minnow egg production (~300 fewer eggs/pair) relative to the unmodified reference water, over the 21-day exposure period. Water chemistry modifications produced a modest decrease in free ion activity of some metals (as shown by MINTEQ, Version 3) in the 45 percent PWE exposure water, but did not alter the metal burden in the treatment-matched larval Chironomus dilutus (the food source of fish during exposure). The tissue-specific metal accumulation increased in fish exposed to the 45 percent PWE relative to the reference water, irrespective of water chemistry modifications, and the tissue metal concentrations were found to be similar between fish in the unmodified and modified 45 percent PWE (higher alkalinity or NOM) treatments. Interestingly however, increased alkalinity and NOM markedly improved fish egg production both in the reference water (~500 and ~590 additional eggs/pair, respectively) and 45 percent PWE treatments (~570 and ~260 additional eggs/pair, respectively), although fecundity over 21 day exposure consistently remained lower in the 45 percent PWE treatment groups relative to the treatment-matched reference groups. Collectively, these findings suggest that metal accumulation caused by chronic 45 percent PWE exposure cannot solely explain the reproductive toxicity in fish, and decrease in food availability (decrease in C. dilutus abundance in 45 percent PWE exposures) might have played a role. In addition, it appears that NaHCO3 or humic acid mitigated reproductive toxicity in fish exposed to 45 percent PWE by their direct beneficial effects on the physiological status of fish.

Induction of apoptosis in testis of the marine teleost mummichog Fundulus heteroclitus after in vivo exposure to the antifouling biocide 4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (Sea-Nine 211)

4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (Sea-Nine 211) has been widely used as an effective antifouling biocide. However, little is known about its reproductive toxicity in fish. Here we investigated testicular toxicity in a marine teleost, the mummichog Fundulus heteroclitus, after exposure to Sea-Nine 211 for 28d. Although Sea-Nine 211 exposure did not affect germ cell proliferation in testis, terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling revealed that the number of apoptotic spermatocytes was increased in the 1.0- and 3.0-μgL−1 groups, and significant differences emerged between the 1.0-μgL−1 group and control groups. Immunohistochemistry showed that the numbers of cysts expressing caspases 2, 3, 6, and 8 (apoptosis-associated proteins) were significantly increased in the 1.0-μgL−1 group, whereas the signal intensity of an anti-apoptotic protein Bcl-xL was reduced in a dose-dependent manner. Moreover, the number of cysts positive for neuronal nitric oxide synthase was twofold higher in the 1.0-μgL−1 group than in the control groups. These results suggest that long-term exposure to Sea-Nine 211 induces apoptosis in the testicular germ cells of mummichogs via a caspase-dependent pathway and that oxidative stress via nitric oxide synthesized by neuronal nitric oxide synthase is involved in this induction.

Applications of genomic technologies to the study of organochlorine pesticide-induced reproductive toxicity in fish

Microarrays and real time PCR are two molecular methods that are gaining acceptance for ecotoxicology. They will help sort out the complex biochemical pathways that are targeted by exposure of organisms to organochlorine pesticides (OCPs) and other toxicants and may point to the ways that these compounds affect development, reproduction and susceptibility to disease. OCPs are a large group of structurally diverse compounds that are present around the globe and are known to be highly persistent pollutants, for which mechanistic toxicity information is lacking. Genomic technologies such as these will help identify and monitor toxicity and help develop subsequent environmental policies.

Ovarian damage to Channa punctatus after chronic exposure to low concentrations of elsan, mercury, and ammonia

Histopathological changes were induced in the ovary by chronic nonlethal levels of Elsan (211 ppb), mercury (16.7 ppb), and ammonia (15.64 ppm). In the breeding season, treated Channa punctatus revealed a significant decrease in ovarian weight throughout the duration of the experiment. Acute changes were noted in the diameter and percentage occurrence of the different stages of oocytes. In all cases, stage I oocytes revealed no difference in diameter with respect to control although they demonstrated an increase in the percentage occurrence. For stages II and III, a remarkable decrease occurred in both the number and the diameter of these mature oocytes. The preponderance of stage I and destruction of stage II and stage III oocytes in all three treatments indicate that chemically diverse xenobiotics have equal reproductive toxicity in fish, interfering with their breeding.