Analysis of the effects diclofenac has on Japanese medaka (Oryzias latipes) using real-time PCR

The expression levels of cytochrome P450 1A, p53 and vitellogenin were investigated in three different tissues of male medaka fish after exposure to diclofenac that is one of the main concerns among pharmaceuticals frequently found in sewage treatment plant (STP) effluents. The results showed that cytochrome P450 1A, p53 and vitellogenin were highly expressed in tissue-specific gene expression patterns after exposure to 8 mg/l and 1 μg/l of diclofenac. These elevated expression levels of three biomarkers suggested that diclofenac has potential to cause cellular toxicity, p53-related genotoxicity and estrogenic effects. It is also noteworthy that diclofenac has the potential to cause these effects even at an environmentally relevant concentration of diclofenac, 1 μg/l.     

Effect of 17beta-estradiol on the reproduction of Japanese medaka (Oryzias latipes)

Estrogenic compounds such as 17beta-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis-ova. Males treated with E2 concentrations = 55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis-ova in male medaka exposed to environmentally relevant concentrations of E2.     

Uptake and depuration of the anti-depressant fluoxetine by the Japanese medaka (Oryzias latipes)

The selective serotonin reuptake inhibitor (SSRI) class of anti-depressants is among the most widely prescribed groups of pharmaceuticals. Consequently, aquatic ecosystems impacted by municipal wastewater discharges are predicted to receive substantial annual loadings of these compounds. Although SSRIs have been detected in fish tissues, little is known of their uptake and depuration in freshwater fish species. In this study, Japanese medaka (Oryzias latipes) were exposed to fluoxetine at a nominal concentration of 0.64 microg L(-1) for 7d and subsequently allowed to depurate in clean water over a 21d period. Fluoxetine uptake by medaka was observed within the first 5h of exposure and the biologically active metabolite, norfluoxetine, was also detected in medaka tissues during this timeframe. A maximum fluoxetine concentration was measured in medaka by the third day of the uptake phase, yielding an uptake rate constant (k(1)) of 5.9+/-0.5 (d(-1)). During the depuration phase of the experiment, a half life of 9.4+/-1.1d was determined for fluoxetine. Using these data, bioconcentration factor (BCF) values of 74 and 80 were estimated for fluoxetine and a pseudo-BCF (the ratio of the concentration of norfluoxetine in medaka and the aqueous fluoxetine concentration) of 117 was calculated for norfluoxetine. These results indicate longer persistence and greater potential for the bioaccumulation of fluoxetine and norfluoxetine in fish tissues than would be predicted from prior half life estimates derived using mammalian species.     

Suppression of sexual behavior in male Japanese medaka (Oryzias latipes) exposed to 17beta-estradiol

Mating pairs of Japanese medaka (Oryzias latipes) received 17beta-estradiol (E2; dose, 3 and 30 microg/g body weight daily) in their diet and E2-free diet (control) for two weeks. The effect on reproductive success was assessed in the mating pairs, and sexual and nonsexual behaviors in the male were quantified. The fecundities of the E2-treated groups were reduced significantly (p < 0.05) compared with those of control, and the sexual behaviors (following, dancing, floating, and crossing) of male fish in response to prostaglandin F2alpha (PG)-injected E2-unexposed females were suppressed dramatically. While the fertility of spawned eggs and the frequencies of the nonsexual behaviors of resting and swimming in a straight line performed by male were unchanged in E2-exposed groups. Therefore, sexual behavior in male may be linked with reproductive success in mating pair of madaka exposed to E2. This assay for sexual behavior using PG may provide a useful tool for assessing the effect of endocrine-disrupting chemicals.     

Tributyltin causes abnormal development in embryos of medaka, Oryzias latipes

We examined the effects of tributyltin (TBT) on embryonic development, hatching success and sexual differentiation in Japanese medaka (Oryzias latipes). Embryos (within 8h after fertilization) were exposed to TBT in ovo via nanoinjection at concentrations of 0 (control), 0.16, 0.80, 3.96, 19.2 and 82.1 ng/egg. Embryonic survival, development and hatching were observed. Hatched fry were reared until 60 days when they sexually matured, and sexual differentiation was also examined by accordance of genetic and phenotypic sex, based on existence of DMY (a male determining gene in medaka) and secondary sex characteristics. As results, TBT caused a concentration-dependent mortality and impaired the embryonic development. However, no masculinization was detected at 60 dph medaka adults. Lowest observed effective concentration for inducing abnormal embryonic development was estimated to 0.16 ng/egg (ca. 160 ng/g egg).     

The toxicity of titanium dioxide nanopowder to early life stages of the Japanese medaka (Oryzias latipes)

In this study, fertilized Japanese medaka (Oryzias latipes) embryos were exposed from fertilization to 5 d post-hatch using static non-renewal assays to aqueous suspensions of titanium dioxide nanoparticles (nTiO₂) ranging in nominal concentrations between 0 and 14 μg mL⁻¹. The average size of the nTiO₂ in the stock solution before addition to the test treatments was 87 nm (±14 nm). TiO₂ materials accumulated in a concentration dependent manner on the chorionic filaments of developing medaka embryos with evidence of pericardial edema occurring during embryo development. However, no significant (p > 0.05) increases in mortality relative to control treatments were observed for the nTiO₂ exposed embryos. A concentration dependent increase in cumulative percent hatch was observed at 11 d, indicating that exposure to increasing concentrations of nTiO₂ resulted in the premature hatch of medaka embryos. Post-hatch, a significant proportion of sac fry from the nTiO₂ exposure groups exhibited moribund swimming behavior and these individuals also experienced greater mortality at 15 d post-hatch. Combined, these results demonstrate that exposure to nTiO₂ can impact the development of early life stages of fish.     

Developmental effects in Japanese medaka (Oryzias latipes) exposed to nonylphenol ethoxylates and their degradation products

The endocrine modulating potency of five alkylphenol compounds to fish, including nonylphenol (NP), three nonylphenol ethoxylate mixtures (NP1EO, NP4EO, NP9EO) and one nonylphenol ethoxycarboxylate (NP1EC) was assessed using in vivo tests conducted with Japanese medaka (Oryzias latipes). Medaka exposed to test materials from 1 day to 100 days post-hatch were monitored for alterations to sex ratios and secondary sex characteristics and development of gonadal intersex (i.e., testis-ova). The treatment with 100 microg l-1 NP (measured 29 microg l-1) induced gonadal intersex in over 80% of exposed males, mixed secondary sex characteristics in over 40% of exposed fish and suppression of the development of papillae on the anal fin of 100% of males. The 30 microg l-1 NP (measured 8.7 microg l-1) treatment induced gonadal intersex in only one of the 22 exposed males and mixed secondary sex characteristics in approximately 20% of the exposed fish. An elevated incidence of fish with mixed secondary sex characteristics and suppression of papillae development was also observed in the treatment with NP1EO at the highest test concentration of 300 microg l-1 (measured 105 microg l-1). There was no evidence of mixed secondary sex characteristics or gonadal intersex in treatments with the remaining test mixtures. This study confirms that NP is an estrogenic compound that could affect gonadal development in fish chronically exposed to concentrations in the range of 10 microg l-1. NP1EO is very weakly estrogenic at concentrations that are an order of magnitude higher than the lowest observed effect concentration for nonylphenol.     

Influence of sediment composition on PAH toxicity using zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) embryo-larval assays

Due to hydrophobic and persistent properties, polycyclic aromatic hydrocarbons (PAHs) have a high capacity to accumulate in sediment. Sediment quality criteria, for the assessment of habitat quality and risk for aquatic life, include understanding the fate and effects of PAHs. In the context of European regulation (REACH and Water Framework Directive), the first objective was to assess the influence of sediment composition on the toxicity of two model PAHs, benzo[a]pyrene and fluoranthene using 10-day zebrafish embryo-larval assay. This procedure was undertaken with an artificial sediment in order to limit natural sediment variability. A suitable sediment composition might be then validated for zebrafish and proposed in a new OECD guideline for chemicals testing. Second, a comparative study of toxicity responses from this exposure protocol was then performed using another OECD species, the Japanese medaka. The potential toxicity of both PAHs was assessed through lethal (e.g., survival, hatching success) and sublethal endpoints (e.g., abnormalities, PMR, and EROD) measured at different developmental stages, adapted to the embryonic development time of both species. Regarding effects observed for both species, a suitable artificial sediment composition for PAH toxicity testing was set at 92.5 % dry weight (dw) silica of 0.2–0.5-mm grain size, 5 % dw kaolin clay without organic matter for zebrafish, and 2.5 % dw blond peat in more only for Japanese medaka. PAH bioavailability and toxicity were highly dependent on the fraction of organic matter in sediment and of the K _ow coefficients of the tested compounds. The biological responses observed were also dependent of the species under consideration. Japanese medaka embryos appeared more robust than zebrafish embryos for understanding the toxicity of PAHs following a sediment contact test, due to the longer exposure duration and lower sensitivity of sediment physical properties.     

Effect of certain chemicals on the reproduction of medaka (Oryzias latipes)

In order to understand the effects of estrogenic chemicals on fish reproduction, we exposed male medaka (Oryzias latipes) to a natural estrogen [17 beta-estradiol (17 beta-E2)] and three estrogenic chemicals [bisphenol-A, nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP)]. After two weeks' exposure, one male medaka was kept together with two female medaka for spawning, and the number of eggs and hatchings were compared to those of a negative control group. The results indicated that exposure to 17 beta-E2 caused a significant decrease in the number of eggs and hatchings as compared to the negative control group at and above 3 nmol/l. Also, the highest concentrations of bisphenol-A and NP caused a decrease in the number of hatchings, but no decrease in hatchings was observed in DEHP treatments. In the treatment using these chemicals the decrease in egg numbers was not so much as in hatching numbers. When compared to other in vitro studies, concentrations observed to have adverse effects on reproduction in this study are generally lower. In addition, it was suggested that physical alterations, such as an induction of plasma vitellogenin, were caused at much lower concentrations than those at which a decline in reproductivity was actually induced.     

Effects of formulations of the fungicide, pentachloronitrobenzene on early life stage development of the Japanese medaka (Oryzias latipes)

Quintozene is a fungicide containing the active ingredient, pentachloronitrobenzene (PCNB) that is used to control "snow mold" on golf courses in temperate regions of North America. In this study, quintozene and a formulation of quintozene widely used on golf courses, FFII were tested for toxicity to early life stages of the Japanese medaka, Oryzias latipes. For medaka exposed in static non-renewal assays to quintozene for 17d from the fertilized egg stage to yolk resorption at the fry stage, the LC(50) for mortality was a nominal concentration of 707 microgl(-1) and the effective concentration for 50% hatch (i.e. EC(50)) was a nominal concentration of 71 microgl(-1). Eggs and fry showed developmental abnormalities, including ocular malformations and retarded development of the brain, notochord, organs and body segmentation, which were interpreted as teratogenic responses to exposure to PCNB. For medaka exposed to quintozene, the LOECs for abnormalities of the eye and all other developmental abnormalities were 750 and 100 microgl(-1), respectively. In medaka exposed to the FFII formulation, similar patterns of mortalities, reduced hatching success and developmental abnormalities were observed, but at higher test concentrations that were consistent with the proportion of quintozene in the formulation. For medaka exposed to the formulation, the LOECs for abnormalities of the eye and all other developmental abnormalities were 10,000 and 1,000microgl(-1), respectively. Overall, these data indicate that studies should be conducted to assess the risk of exposure of early life stages of fish to quintozene in watersheds impacted by golf courses.     

Reproductive effects and bioconcentration of 4-nonylphenol in medaka fish (Oryzias latipes)

The objective of this study was to investigate the reproductive success (fertility and fecundity) of medaka (Oryzias latipes) exposed to 4-nonylphenol (4-NP) in water and the viability of their F(1) offspring. In addition, we measured the bioconcentration of 4-NP in eggs. After a 21-d exposure to 100microg/l 4-NP, medaka showed reduced egg production and fertility. Hepatic vitellogenin levels were increased significantly in males treated with 10, 50 and 100microg/l of 4-NP. In the F(1) generations, the hatchability and time to hatching of embryos in the 100microg/l treatment group were adversely affected, and 2-7microg 4-NP/g egg was found in spawned eggs (the bioconcentration factor: 30-100). These results indicate that (1) 4-NP adversely affected the reproduction (fecundity and fertility) of adult medaka, (2) 4-NP accumulated in eggs through maternal transfer and (3) these levels of 4-NP were associated with adverse effects in the F(1) offspring.     

应用两种彗星试验方法检测饮用水源水的遗传毒性

应用人外周血淋巴细胞彗星试验和小鼠睾丸细胞彗星试验,对苏南地区代表性饮用水源水的遗传毒性进行了监测。彗星试验的结果表明,各水样中有机浓集物均能对人外周血淋巴细胞和小鼠睾丸细胞产生不同程度的DNA损伤。研究表明,上述两种试验方法可有效地检测水中有机污染物的遗传毒性。     

Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.     

Biomarker responses and reproductive toxicity of the effluent from a Chinese large sewage treatment plant in Japanese medaka (Oryzias latipes)

The present study was conducted to assess the potential toxicity of the effluent from a large sewage treatment plant (GBD-STP) in Beijing. Japanese medakas (Oryzias latipes) at reproduction active period were exposed to a serial of graded concentrations of the effluent or 100 ng l-1 of 17-alpha-ethinylestradiol (EE2, positive control). Growth, gonadosomatic index (GSI), hepatosomatic index (HSI), reproductive success, induction potency of vitellogenin (VTG) in male fish and that of 7-ethoxyresorufin-o-deethylase activity (EROD) in male fish liver were used as test endpoints. The growth suppression of fish was observed in a dose-dependent manner, resulting in significant differences in both body length and body weight of medaka above 5% effluent. This effluent can inhibit the growth of gonad of medakas and are more sensitive to male than to female. At exposure concentration of 40% and higher, there was an unexpected decrease of HSI values, which may be resulted from sub-lethal toxicity of effluent to fish liver. VTG of plasma in males were induced in all exposure concentration levels, but not in a dose-dependent manner. The concentration of 5% effluent would be the lowest observed adverse effect level (LOAEL) affecting reproductive success when examining fertile individuals, fecundity and fertilization rate. The overt CYP1A response and higher reproductive toxicity may be indicative of low process efficiency of this STP.     

Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.     

Influence of water hardening of the chorion on cadmium accumulation in medaka (Oryzias latipes) eggs

This report describes a study in which in vitro fertilization methods were used to expose medaka (Oryzias latipes) eggs to cadmium (Cd(2+)). This approach was applied to address the differential sensitivity and cumulative potential of Cd(2+) when exposure was initiated early (before fertilization and water hardening of the chorion) versus later during embryo development (i.e., well after the chorion has undergone water hardening). Following range finding exposures (2.5, 10, 20, 40 or 80 mg/l) under artificially controlled experimental procedures, results from hatching success and embryo malformations showed the earlier exposure interval more sensitive than the assay involving only the embryonated egg. Subsequent accumulation studies have shown that the exposure initiated before fertilization apparently led to more Cd(2+) deposition in the chorion compared to the exposure during embryonated stages of the eggs. Similarly, values for total Cd(2+) indicated higher concentrations in those eggs exposed prior to--and during--water hardening. Results suggest an alteration of the properties of the zona radiata in the early-stage eggs, making it more permeable to the potential exit or entrance of waterborne agents even after water hardening. Ongoing studies must now address the development of more realistic exposure conditions of the gametes by using incubation media with osmolarities similar to surface waters, and by shortening duration for gamete exposure. Also, sensitive methods to localize Cd(2+) and to delineate the transfer from the chorion to the embryo are needed.     

Avoidance by olfaction in a fish, medaka (Oryzias latipes), to aquatic contaminants

Behavioral tests using a fish, medaka (Oryzias latipes), from which the olfactory organs were resected confirmed that fish avoid aquatic contaminants such as surfactants and pesticides mainly by olfaction. Control medaka (non-resected) exhibited significant avoidance to 10, 20 and 30 microg liter(-1) of LAS (sodium linear laurylbenzene sulfonate), 90 and 100 microg liter(-1) of fenitrothion (dimethyl 4-nitro-m-toryl phosphorothionate), and 500 microg liter(-1) of POE-ether (polyoxyethylene lauryl ether). By contrast, medaka with bilateral nose resections (BNRM) exhibited no avoidance to 10-50 microg liter(-1) of LAS, 90 and 100 microg liter(-1) of fenitrothion, and 500 microg liter(-1) of POE-ether. Although medaka with unilateral nose resection (UNRM) avoided some concentrations of these toxicants, the UNRM exhibited no avoidance to 10 and 40 microg liter(-1) of LAS and 90 microg liter(-1) of fenitrothion. That is, the avoidance response of the UNRM was less distinct than that of the controls. This difference may have been caused by the deterioration of detecting ability and the paralyzation or adaptation of olfaction.