Tamoxifen effects on the early life stages and reproduction of Japanese medaka (Oryzias latipes)

Tamoxifen is widely used in the treatment of breast cancer and can enter the aquatic environment in municipal wastewater. To evaluate potential effects on embryonic development of Japanese medaka, fertilized eggs were exposed to tamoxifen at 1-625μg/l for 14 days. Adverse effects on hatchability and time to hatching only occurred at 125 and 625μg/l. Reproductive effects were assessed by exposing adults for 21 days to the same dose range. At all concentrations tested, tamoxifen significantly increased plasma vitellogenin levels in males in a dose-dependent manner. Fecundity and fertility were detrimentally affected at 625μg/l. Additionally, F1 eggs were removed from tamoxifen-contaminated water to evaluate transgenerational effects. Hatchability was affected at 625μg/l but no morphological deformities were observed. A significant dose-dependent increase in the proportion of genotypic males occurred at all concentrations greater than 5μg/l.     

Unexpected effects of zinc pyrithione and imidacloprid on Japanese medaka fish (Oryzias latipes)

Biological effects of the biocide zinc pyrithione (Zpt), used in anti-dandruff shampoos and antifouling paints and the agricultural insecticide imidacloprid on Japanese medaka fish (Oryzias latipes) were assessed in experimental rice fields. Both chemicals are toxic to medaka, in particular Zpt, which also causes teratogenic effects such as spinal cord deformities in embryos at very low, sublethal concentrations. Rates of malformation in medaka fry from paddies treated twice a week with anti-dandruff shampoo (0.18-0.37 microL/L each time) over a period of 4 months were within the natural background, perhaps due to the quick dissipation rate of this chemical in the environment. Both Zpt and imidacloprid caused stress syndrome in juvenile medaka, with fish from Zpt-shampoo fields having a significantly lower weight to body length ratio than those from control fields. As it often happens with stressed fish, a massive infestation by a Trichodina ectoparasite was observed in medaka from imidacloprid fields. However, despite their high stress levels, fish from the Zpt fields did not suffer such infestation, supposedly because the disinfectant action of this biocide.     

Pathological modifications following sub-chronic exposure of medaka fish (Oryzias latipes) to microcystin-LR

Microcystins (MCs) are toxic monocyclic heptapeptides produced by many cyanobacteria. MCs, especially MC-LR, cause toxic effects in animals and are a recognized potent cause of environmental stress and health hazard in aquatic ecosystems when heavy blooms of cyanobacteria appear. Consequently, one of the major problems is the chronic exposure of fish to cyanotoxins in their natural environment. The present experiment involving chronic exposure confirmed initial findings on acute exposure to MC contamination: exacerbated physiological stress and tissue damage in several tissues of exposed medaka fish. The gonads were affected specifically. In female gonads the modifications included reduction of the vitellus storage, lysis of the gonadosomatic tissue and disruption of the relationships between the follicular cells and the oocytes. In the males, spermatogenesis appeared to be disrupted. This is the first report showing that a cyanotoxin can affect reproductive function, and so can impact on fish reproduction and thus fish stocks.     

Effect of trifloxystrobin on hatching,survival, and gene expression of endocrine biomarkers in early life stages of medaka (Oryzias latipes)

Trifloxystrobin is a systemic broad‐spectrum foliar strobilurin fungicides that enters the aquatic environment during agricultural application. It is highly toxic and poses a potential risk to aquatic organisms, whereas the effect on the development of early life stages of fish are unclear. In this study, hatchability, time to hatching, and larval mortality were measured. Additionally, the expression of biomarker genes, including those involved in sex hormone pathways (er, vtg, cyp17, and cyp19a), thyroid hormone pathways (trα and dio2), and aryl hydrocarbon receptor pathways (ahr and cyp1a), was determined after embryos of medaka (Oryzias latipes) were exposed to different levels of trifloxystrobin (0, 0.1, 1, 10, and 100 μg/L) for 28 days. The results showed that there were significant differences between controls and the 100 μg/L treatment group in both hatchability and time to hatching of fertilized eggs (p < 0.05). Larval mortality was significantly increased in the 0.1, 1, and 10 μg/L treatment groups (p < 0.05). These results indicate that embryonic and larval development may be affected by trifloxystrobin exposure. Moreover, the mRNA levels of the er gene were significantly up‐regulated at levels of trifloxystrobin above 1 μg/L treatment groups. Up‐regulation of vtg, cyp17, and cyp19a mRNA levels was observed in the larvae at the lower concentration treatment groups. The mRNA levels of cyp1a genes were significantly up‐regulated at all of the treatment groups. These results suggest that trifloxystrobin is a potential endocrine disruptor through effects on the sex hormone pathway and xenobiotic metabolism. The changes in cyp1a expression can be used as a highly sensitive biomarker to assess trifloxystrobin contamination in the early life stages of fish. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 648–655, 2015.     

Effects of triclosan on Japanese medaka (Oryzias latipes) during embryo development,early life stage and reproduction

Triclosan has been shown to have endocrine‐disrupting effects in aquatic organisms. In 2016, the US Food and Drug Administration banned the use of triclosan in consumer soaps. Before the ban, triclosan was reported at low concentrations in the aquatic environment, although the effect of triclosan on reproduction in teleost fish species is yet to be clarified. Here we investigated the effects of triclosan on embryo development and reproduction, and during the early life stage, in Japanese medaka (Oryzias latipes) by using Organisation for Economic Co‐operation and Development tests 229, 212 and 210, with minor modifications. In adult medaka, exposure to 345.7 μg l^–1 suppressed fecundity and increased mortality but had no effect on fertility. Exposure to 174.1 or 345.7 μg l^–1 increased liver vitellogenin concentration in females but decreased liver vitellogenin concentration in males. With triclosan exposure, mortality was increased dose dependently during the embryonic and early larval stages, and a particularly steep increase in mortality was observed soon after hatching. The lowest observed effect concentrations of triclosan in Japanese medaka obtained in the present study (mortality [embryonic and larval stages, 276.3 μg l^–1; early life stage, 134.4 μg l^–1; adult stage, 174.1 μg l^–1], growth [134.4 μg l^–1], vitellogenin [174.1 μg l^–1], fecundity [345.7 μg l^–1] and fertility [>345.7 μg l^–1]) were at least 55 times (compared with the USA) and up to 13 400 times (compared with Germany) greater than the detected triclosan levels in the aquatic environment. These results suggest that triclosan may not be affecting fish populations in the aquatic environment.     

Increasing uptake and bioactivation with development positively modulate diazinon toxicity in early life stage medaka (Oryzias latipes).

Diazinon, an organophosphate pesticide, becomes biotransformed to a more potent oxon metabolite that inhibits acetylcholinesterase (AChE). Early life stages (els) of medaka, Oryzias latipes, were used to determine how development of this teleost affects sensitivity to diazinon. With developmental progression, from day of fertilization to 7-day-old larvae, we found that the 96-h LC50 and AChE IC50 values decreased, indicating greater host sensitivity to diazinon upon continued development. We then examined changes in AChE activity, its inhibition by the active metabolite diazoxon, and uptake and bioactivation of the compound. AChE activity remained low during much of development but increased rapidly just prior to hatch. In addition, in vitro incubation of tissue homogenates from embryos or larvae showed no differences in the sensitivity of AChE to diazoxon. Uptake studies with 14C-diazinon revealed greater body burdens of 14C as medaka developed. In addition, AChE IC50 values determined by in vivo exposure to diazoxon were greater in larvae than in embryos. Because diazinon is bioactivated by the P450 enzyme system, two P450 inhibitors were used in vivo to explore the role of metabolism in sensitivity. When exposure to diazinon occurred in the presence of increasing amounts of piperonyl butoxide (PBO), AChE inhibition decreased in a dose-response fashion and 2.0 x 10(-5) M PBO alleviated any difference in inhibition between larvae and embryos. However, PBO did not alter total 14C uptake when exposed simultaneously with 14C-diazinon, nor did it affect AChE inhibition using diazoxon. Controls ruled out differential effects of PBO on uptake and inhibition. In addition, a second general P450 inhibitor, 1-aminobenzotriazole, also decreased AChE inhibition. Finally, using exogenous acetylcholinesterase as a trap for the oxon metabolite, larval microsomes displayed greater bioactivation of diazinon than did a microsomal preparation from embryos. Taken together, results suggest that uptake and bioactivation are working to enhance diazinon sensitivity in this developmental model of a teleost fish.     

Pathological effects in freshwater fish Poecilia reticulata (guppy) and Oryzias latipes (medaka) following methyl bromide and sodium bromide exposure

To study the effects of the fumigant methyl bromide (CH₃Br) and its anorganic residue, bromide (Br⁻), young guppies and medakas were exposed to these chemicals for 1 and 3 months. The concentration ranges were 10–32 000 mg/l (guppy) and 180–5 600 mg/l (medaka) for NaBr and 0.032–3.2 mg/l for CH₃Br. Besides monitoring of general toxicological parameters, histopathology was performed on total body sections. In addition, a short-term study with the two highest concentrations CH₃Br was conducted for the evaluation of (lethal) effects, in which scanning electron microscopy of the gills was included.In the guppy, a NaBr concentration of 100 mg/l and higher caused paresis and paralysis. Histopathology of these animals, exposed to concentrations of 3 200 mg/l, showed muscle changes indicative of a functional neuromuscular disturbance. In the thyroid, the epithelium appeared to be stimulated (at 32 and 100 mg/l and higher after 1 and 3 months' exposure, respectively), and the thyroid stimulating hormone producing cells in the pituitary were also activated. The development of gonads in both sexes was more advanced than in the controls after the 3-month exposure period, and there was inflammation in and around the larger blood vessels in the abdomen at concentrations of 3 200 mg/l and higher after 3 months. Medakas showed only activation of the thyroid epithelium even at the lowest concentration tested (180 mg/l).In the CH₃Br study, no specific effects were observed in the long-term study of either species. In the short-term study, however, major degenerative and regenerative changes were found in the superficial epithelia, especially of the gills and oral mucosa. These were apparently caused by the local irritating action of this compound. Moreover, at these lethal concentrations, necrotic alterations were seen in the thymic cortex and the testis which were considered stress-related. In this context, the importance of gill lesions is discussed.It is concluded that Br⁻ is goitrogenic in both species at low concentrations, and in guppies, at higher concentrations, it also affects skeletal muscle, blood vessels and gonads. Methyl bromide has no specific action but is irritating for superficial epithelia at relatively high concentrations.     

Frequency and size of micronuclei induced in gill cells of medaka fish (Oryzias latipes) after whole-body exposure to clastogenic chemicals

Medaka fish (Oryzias latipes) were whole-bodily treated with various doses of mitomycin C (MMC), ethylmethanesulfonate (EMS), cyclophosphamide (CP), diethylnitrosamine (DEN), or colchicine (COL) for 24?h, and the frequency of micronucleated cells (MNCs) was measured in the gills at 24 and 48?h after treatment. In the present experiments, MMC, CP, and DEN were recorded as efficient inducers of micronuclei at both sampling times, and none of the MNC frequencies recorded with these agents at 24?h significantly exceeded the corresponding frequency at 48?h. For EMS and COL, positive responses were recorded only 48?h after treatment. By comparison with the time-course data reported for radiation-induced MNCs in the same MN assay system, the clear responses observed at the 48-h time point for all the chemicals used were regarded as evidence of their delayed effects on micronucleus (MN) formation. The mean sizes of micronuclei induced after exposure to COL was significantly larger by a factor 2 as compared with that induced by X-irradiation, whereas those determined for the other four chemicals were almost equal to that induced by X-irradiation. These results demonstrate that the medaka gill-cell MN assay can detect chemically-induced chromosome damage, either directly or after metabolic activation, and spindle malfunction, and provide a basis for further development of the present assay system for testing cytogenetic activities of chemical agents.     

Feminization of Japanese medaka (Oryzias latipes) exposed to 17beta-estradiol: formation of testis-ova and sex-transformation during early-ontogeny

Gonad histological changes were examined in Japanese medaka exposed to 17beta-estradiol (E2) during early-life stages. Two experiments were conducted at different concentrations of E2 (33.5 and 140.6 ng/L, mean value of measurement) and larvae and juveniles were observed for histological changes in the gonad. Differentiation of ovary and testis in control fish was apparent 12 days post-hatch (dph). At 12 dph, normal testes were observed in male fish that had been exposed to 33.5 ng/L E2, but at 14 and 20 dph, testis-ova was recognized in male fish. Male fish exposed to 140.6 ng/L E2 had testis-ova at 12 dph and gradual transformation to ovary was observed in male fish until 20 dph. In both experiments, the ovarian tissue in testis of male fish exposed to E2 was frequently distributed along the central transverse axis of the gonad, expanding into the transverse axis. The results indicated that 17beta-estradiol can induce testis-ova in male medaka during the larval period and sex-transformation is more frequent at higher (140.6 ng/L) than lower concentrations (33.5 ng/L) of estradiol. The results also demonstrated that testis-ova first appear in the central area of the transverse axis of testis.     

Methylmercury accumulation, histopathology effects, and cholinesterase activity alterations in medaka (Oryzias latipes) following sublethal exposure to methylmercury chloride

A series of specific toxicological effects including bioaccumulation of the pollutant, histological changes and influences on cholinesterase (ChE) activities were examined in the adult Japanese medaka after the exposure to graded sublethal concentrations (40, 20, 10, 5, 2.5ngHg/mL) of methylmercury chloride (MMC). Methylmercury (MeHg) contents in the exposed medaka tissues ranged from 0.03 to 64.4μgHg/g (wet weight, w.w.). High concentrations of MeHg were accumulated in the liver and brain, while the concentrations in muscle and fat were relatively low. A dose-dependent and exposure time-dependent increase of MeHg contents in tissues was observed. Histopathological changes, such as oedema, vacuolization, pyknotic nucleus, telangiectasis, and degenerative sperm, can clearly be observed in the slices from the liver, gill, and male gonad of the exposed medaka. Inhibition of ChE activity was common in the exposed fish's brain, liver, gill, and muscle. The serious intoxication of MMC to medaka was definitely demonstrated herein.     

Global quantitative analysis of protein expression and phosphorylation status in the liver of the medaka fish (Oryzias latipes) exposed to microcystin-LR I. Balneation study

Microcystins (MCs) are hepatotoxins with potent inhibitor activity of protein phosphatases PP1 and PP2A. These non-ribosomal peptides are getting more and more attention due to their acute toxicity and potent tumor-promoting activity. These toxins are produced by freshwater cyanobacteria. The most toxic and most commonly encountered variant in aquatic environment is MC-LR (MC Leucine-Arginine). It has been used for toxicological investigations on the liver of intoxicated medaka. Differential proteome as well as differential phosphoproteome analyses have been performed for providing new information on early responses to the toxin. The experiments are also aiming at selecting biomarkers of MC-LR exposure. In the 2D electrophoresis gel protein maps from cytosol of liver cells of animals exposed or non-exposed to the cyanotoxin, 15 spots showed a significant increase or decrease of their stain signal either in specific phosphoprotein stain or total protein stain. Thirteen of these proteins have been identified by mass spectrometry. Among them, phenylalanine hydroxylase (PAH) and keratin 18 type I showed variations in phosphorylation stain in possible agreement with inhibition of PP2A activity. The other identified proteins exhibited variations in their expression level. The identified proteins appear to be involved in cytoskeleton assembly, cell signalling, oxidative stress and apoptosis. Such results confirm that proteomics and phosphoproteomics approaches may become valuable tools to identify signalling pathways implied in MC-LR effects. From accumulated data, specific pools of biomarkers could possibly be selected as specific for toxin exposure.     

Feminization of Japanese medaka (Oryzias latipes) exposed to 17beta-estradiol: effect of exposure period on spawning performance in sex-transformed females

Two groups of Japanese medaka (Oryzias latipes) were exposed to 17beta-estradiol (E2: 150ng/L, nominal concentration) for either a short-term exposure (STE: 0-31 days after fertilization (daf); egg-larval period) or a long-term exposure period (LTE: 0-81 daf; egg-adult period) and their subsequent spawning performance was compared in terms of fecundity, spawning time, and fertility. Most genetic males were transformed to phenotypic females by E2 following both short-term and long-term exposure, but spawning performance and gonad somatic index (GSI) of sex-transformed females (XY females) following long-term exposure were lower than those of sex-transformed females following short-term exposure and those of normal females (XX) in the control group. Sex-transformed females in the STE group and normal females possessed mature ovary, whereas most of the sex-transformed females in the LTE group possessed immature ovary, with most oocytes being in the pre-vitellogenic phase. Moreover, the chromosome types of first filial generation delivered from sex-transformed female in STE group composed with 51.9% as XY, 18.5% as YY, and 29.6% as XX. From these results, it seems that exposure to E2 until the end of the larval period produces sex-transformed medaka with high reproductive ability, similar to normal females, but longer exposure to E2 may inhibit sexual maturation in the sex-transformed female.     

A review of studies on androgen and estrogen exposure in fish early life stages: effects on gene and hormonal control of sexual differentiation

Teleost fish are unique among vertebrates in that phenotypic sex or onset of sex inversion can be easily manipulated by hormonal treatments. In recent years, researchers have begun reporting concentrations of synthetic and natural hormones in the environment. Although concentrations are very low (in the parts per trillion to low parts per billion), they are still of concern because of the high potency of synthetic hormones and the enhanced susceptibility of teleost fishes, especially early life stages, to hormonal exposures. In this review, we will focus on sex differentiation in teleost fishes and how these processes in fish early life stages may be impacted by environmental hormones which are known to contaminate aquatic environments. We will start by reviewing information on sources and concentrations of hormones in the environment and continue by summarizing the state of knowledge of sex differentiation in teleost gonochoristic fishes, including information on genes involved (e.g. cyp19, dmrt1, sox9 and foxl2). We will end our review with a summary of studies that have examined the effects of androgens and estrogens on fish sex differentiation after exposure of fish embryos and larvae and with ideas for future research.     

Prenatal exposure to polychlorinated biphenyls and dioxins from the maternal diet may be associated with immunosuppressive effects that persist into early childhood

We investigated whether prenatal exposure from the maternal diet to the toxicants polychlorinated biphenyls (PCBs) and dioxins is associated with the development of immune-related diseases in childhood. Children participating in BraMat, a sub-cohort of the Norwegian Mother and Child Cohort Study (MoBa), were followed in the three first years of life using annual questionnaires (0–3 years; n = 162, 2–3 years; n = 180), and blood parameters were examined at three years of age (n = 114). The maternal intake of the toxicants was calculated using a validated food frequency questionnaire from MoBa. Maternal exposure to PCBs and dioxins was found to be associated with an increased risk of wheeze and more frequent upper respiratory tract infections. Furthermore, maternal exposure to PCBs and dioxins was found to be associated with reduced antibody response to a measles vaccine. No associations were found between prenatal exposure and immunophenotype data, allergic sensitization and vaccine-induced antibody responses other than measles. Our results suggest that prenatal dietary exposure to PCBs and dioxins may increase the risk of wheeze and the susceptibility to infectious diseases in early childhood.     

CYP1A induction and blue sac disease in early life stages of white suckers (Catostomus commersoni) exposed to oil sands

The objectives of this study were to evaluate the influence of natural oil sands on the early developmental stages of white sucker (Catostomus commersoni) and to determine whether biochemical responses in this species were similar to native fish caught in the Athabasca Oil Sands area. Early life stage (ELS) sediment toxicity tests were conducted using controls, reference sediments, natural oil sands, and industrially contaminated (wastewater pond) sediments collected from sites along the Athabasca River, Alberta (Canada). Eggs and larvae were observed for mortality, hatching, deformities, growth, and cytochrome P-4501A (CYP1A) activity using immunohistochemistry. E-Nat-, S-Nat-, and wastewater pond sediment-exposed groups showed significant premature hatching, reduced growth, and exposure-dependent increases in ELS mortality and larval malformations relative to controls. The most common larval deformities included edemas (pericardial, yolk sac, and subepidermal), hemorrhages, and spinal defects. Juveniles exposed to oil sands and wastewater pond sediments (96 h) demonstrated significantly increased 7-ethoxyresorufin-O-deethylase (EROD) activity (30- to 50-fold) as compared to controls. Reference sediment-exposed groups and water controls demonstrated reliable embryo and larval survival, minimal malformations, and negligible CYP1A staining. These observed signs of blue sac disease (ELS mortality, malformations, growth reductions, CYP1A activity induction) may produce deleterious reproductive effects in natural fish populations exposed to oil sands mixtures.