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.     

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.     

Alteration of monoamine concentrations in the brain of medaka, Oryzias latipes, exposed to tributyltin

We measured the concentrations of monoamines in the brain of Japanese medaka, Oryzias latipes, exposed to tributyltin (TBT). Fish were exposed to 0, 1, 5, 25, or 125 microg g(-1) of TBT via the diet for 21 days. After the administration period, six males and six females in each treatment group were dissected and their brains were collected. The following monoamines were analyzed: dopamine (DA), norepinephrine (NE), epinephrine (E), and 5-hydroxytryptamine (5-HT). The metabolites of DA, 3,4-dihydroxyphenylacetic acid and homovanilic acid, and the metabolite of 5-HT, 5-hydroxyindolacetic acid were also analyzed. The concentration of DA in the brain of male medaka and the concentrations of 5-HT and NE in the female brains were significantly decreased by exposure to 125 microg TBT g(-1). The concentrations of 5-HT and NE in males and of DA in females were slightly decreased by 125 micrg g(-1) of TBT, although the differences were not statistically significant. The present study demonstrates that TBT alters monoamine concentrations in the brain of medaka.     

The utility of the guppy (Poecilia reticulata) and medaka (Oryzias latipes) in evaluation of chemicals for carcinogenicity.

There has been considerable interest in the use of small fish models for detecting potential environmental carcinogens. In this study, both guppies (Poecilia reticulata) and medaka (Oryzias latipes) were exposed in the aquaria water to three known rodent carcinogens for up to 16 months. Nitromethane, which caused mammary gland tumors by inhalation exposure in female rats, harderian gland and lung tumors in male and female mice, and liver tumors in female mice by inhalation, failed to increase tumors in either guppies or medaka. Propanediol, which when given in the feed was a multisite carcinogen in both sexes of rats and mice, caused increased liver tumors in male guppies and male medaka. There was reduced survival in female guppies and no increased tumors in female medaka. 1,2,3-Trichloropropane, which when administered by oral gavage was a multisite carcinogen in both sexes of rats and mice, caused an increased incidence of tumors in the liver of both male and female guppies and medaka and in the gallbladder of male and female medaka. The results of this study demonstrate that for these three chemicals, under these specific exposure conditions, the fish appear less sensitive and have a narrower spectrum of tissues affected than rodents. These results suggest that fish models are of limited utility in screening unknown chemicals for potential carcinogenicity.     

Effects of phytoplankton cell size and chloride concentration on the bioaccumulation of methylmercury in marine phytoplankton

In the current study, the effects of phytoplankton cell size and methylmercury (MeHg) speciation on the bioaccumulation of MeHg by marine phytoplankton were investigated. Volume concentration factors (VCFs) of MeHg were determined in relation to the surface area to volume ratio of the cells for four species of diatom and a cyanobacteria species cultured in unenriched seawater. The VCFs of MeHg, ranging from 7.3 × 10⁴ to 1.6 × 10⁶, increased linearly as the cell surface area‐to‐volume ratio increased. It suggests that pico‐ and nano‐dominated phytoplankton communities may lead to larger MeHg accumulation than the one dominated by microphytoplankton. MeHg VCFs increased with increasing chloride concentration from 0.47 to 470 mM, indicating that MeHg bioaccumulation is enhanced under conditions that facilitate membrane permeability by the formation of neutral MeHgCl species. Overall results suggest that the size distributions of the planktonic community as well as the seawater chemistry affect MeHg bioaccumulation by marine phytoplankton. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 936–941, 2014.     

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.     

Effects of bis(2-ethylhexyl) phthalate and benzo[a]pyrene on the embryos of Japanese medaka (Oryzias latipes)

Effects of two widely found chemical pollutants, bis(2-ethylhexyl) phthalate (DEHP) and benzo[a]pyrene (BaP), on the embryos of Japanese medaka were investigated. The embryos were exposed to different concentrations (0.01, 0.1, 1, and 10 μg/l) of DEHP and BaP. The following were investigated: (1) hatching time and hatching rate in embryos, (2) mortality, sex ratio, body weight and gonadosomatic index (GSI) in adulthood. These two chemicals delayed the hatching time without dose-dependence, but these chemicals had no effect on hatching rate. Mortality was raised and body weight was reduced by DEHP and BaP-treatment; distortion of sex ratio appeared at the lowest concentration of DEHP tested. GSI was decreased because of the BaP-treatment. DEHP and BaP negatively affected Japanease medaka embryos, and the influences of the effects continued into adult stage. Moreover, the effects did not appear to be necessarily dose-dependent.     

Development of an in vivo anti‐androgenic activity detection assay using fenitrothion in Japanese medaka (Oryzias latipes)

The effects of endocrine disruptors, including anti‐androgenic chemicals, on aquatic environments have received increased attention in recent years. Currently, the method used to screen chemicals for anti‐androgenic activity is called the androgenized female stickleback screen, and it was established by the Organization of Economic Cooperation and Development in 2011 using the three‐spined stickleback. However, screening chemicals for anti‐androgenic activity has yet to be established using Japanese medaka. Thus, the purpose of this study was to establish a screening method for anti‐androgenic activity utilizing the number of papillary processes in Japanese medaka (Oryzias latipes ) as an indicator of the chemical's anti‐androgenic activity. Thus, at 35 days post‐fertilization, medaka were exposed to fenitrothion, an anti‐androgenic compound, for 28 days. In the control group, the formation of papillary processes was observed in XY medaka, but not in XX medaka. However, after fenitrothion exposure, the number of papillary processes was significantly decreased in a dose‐dependent manner in XY medaka; in the 300 μg l⁻¹ concentration group, four of 11 XY medaka showed no papillary processes even if there were no significant effects on total length and wet body weight compared with the control group. Our results indicate that the number of papillary processes in Japanese medaka can be used as an indicator of anti‐androgenic activity and that this model may prove useful as a chemical screening method. Copyright © 2016 John Wiley & Sons, Ltd.     

Behavioral and sex ratio modification of Japanese medaka (Oryzias latipes) in response to environmentally relevant mixtures of three pesticides

We exposed Japanese medaka (Oryzias latipes) to environmentally relevant concentrations of azinphos-methyl, chlorothalonil, endosulfan, and mixtures of all three to determine if combinations of these pesticides result in additive, less-than-additive, or more-than-additive effects. Medaka were exposed from fertilization until 7 days posthatching, and end points included survival, time to hatch, size at 7 days posthatching, activity level (as measured by distance swam) and foraging ability at 3 weeks posthatching, and adult size, liver size, and sex ratio at 5 months posthatching. Although exposure to individual pesticides or pesticide mixtures did not affect survival, hatching time, or foraging ability, fry exposed to azinphos-methyl were significantly smaller at 1 week of age, and those exposed to chlorothalonil and a combination of the chemicals showed reduced activity. Adult sex ratios were biased toward females in all groups exposed to pesticides, with those exposed to azinphos-methyl, chlorothalonil, and the pesticide mixture departing significantly from an even sex ratio. There was no evidence of additive or synergistic effects of pesticide mixtures.     

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.     

Fish multigeneration test with preliminary short‐term reproduction assay for estrone using Japanese medaka (Oryzias latipes)

The most potent chemicals potentially causing adverse effects on fish species are estrogens in human waste. Sewage is a source of these estrogens and it is difficult to reduce. In particular, although the bioactivity of estrone is estimated to be about half of that of estradiol, multiple studies report that more than 100 ng l^–1 of estrone can be detected in urban rivers, including discharges from sewage treatment works; approximately two times as high as estradiol. Few studies have been conducted to investigate the long‐term effects of estrone on wildlife; therefore, we conducted fish multigeneration test using Japanese medaka (Oryzias latipes). Medaka were exposed to estrone for 27 weeks across three generations in environmentally relevant concentrations, being 5.74, 11.4, 24.0, 47.1 and 91.4 ng l^–1. No effects on reproduction were observed in the first generation; however, a decline in egg production and fertility was observed in the second generation exposed to 91.4 ng l^–1 estrone, which is lower than some known environmental concentrations in urban environments. Furthermore, histopathological abnormalities were observed in the third generation exposed to both 47.1 and 91.4 ng l^–1, suggesting that estrone possibly exerts severe effects on the third or later generations. However, appearances of testis–ova were observed in the second and third generation they were not consistent with actual effects on reproduction, notwithstanding the testis‐ova is regarded as the key evidence for endocrine disruption. Accordingly, we consider that qualitative measurement of abnormalities using histopathological observations is required for appropriate evaluation of endocrine disruption. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of 17beta-estradiol and testosterone on the expression of flavin-containing monooxygenase and the toxicity of aldicarb to Japanese medaka, Oryzias latipes.

Previous studies in our laboratory indicated gender differences in salinity-enhanced acute toxicity of aldicarb in Japanese medaka with females being more susceptible. In the current study, the effects of the sex steroids, 17beta estradiol (E2) and testosterone (T) on aldicarb toxicity was examined. Adult Japanese medaka were separated by sex and exposed to 100 microg/l E2 or T for 6 days followed by exposure to the 96-h LC50 (0.5 mg/l) of aldicarb. The toxicity of aldicarb to adult males was significantly lowered by E2 and T whereby the mortality percentage was reduced to 23.3 +/- 5.8% and 3.3 +/- 5.8%, respectively, compared to the fish not receiving steroids (46.7 +/- 5.8% mortality). In females, T caused significant reduction in aldicarb toxicity to 16.7 +/- 5.8%, while E2 significantly enhanced the toxicity to 96.7 +/- 5.8% mortality. Since the flavin-containing monooxygenase (FMO) enzyme system had been shown to play a critical role in aldicarb toxicity, the effect of E2 and T on FMO expression was examined. Gill FMO activity showed a direct correlation with the overall toxicity of aldicarb in both male and female medaka. Expression of FMO1-like protein was significantly reduced by T in male livers and gills, and T did not affect the expression of FMOs in female tissues. In contrast, E2 significantly reduced FMO1-like protein expression in male gills and female livers, as well as FMO3 expression in both male and female livers, but significantly increased gill FMO1 expression in females. Since aldicarb acts by inhibiting the enzyme cholinesterase (ChE), the effect of sex hormones on the activity of this enzyme was also examined. In both male and female medaka, T counteracted the inhibitory effect of aldicarb on muscle ChE. In male fish, E2 had similar effects but did not seem to counteract the ChE inhibition in females. In conclusion, E2 and T modulation of aldicarb toxicity in Japanese medaka seems to be mediated via alteration of gill FMO and ChE actitivies.     

Stage sensitivity of medaka (Oryzias latipes) eggs and embryos to permethrin

The effects of exposure to permethrin on gametes, fertilization and embryonic development were examined in medaka (Oryzias latipes). Following range finding (25, 50, 100, 200 or 300 microg/l) and duration of exposure (0, 120, 144, 168, 192, 216, or 240-h) assays, the relative sensitivity was studied when initiation of exposure to permethrin (100 microg/l, for 192-h) occurred at one of four different stages, i.e., unfertilized egg (0-h), late morula (5-h), early neurula (24-h), and early organogenesis (40-h). The later exposure interval proved the most sensitive. Also, differences were observed in rates of recovery in larvae initially affected following the earliest exposure treatment (0-h, gametes prior to fertilization). Permethrin (100 microg/l) did not affect fertilization success and no lethal effects were observed in embryos. Sublethal effects were primarily observed at hatch. Toxicity endpoints in larvae included: delayed swim bladder inflation; inability of hatchling to respond to stimulus; uncoordinated movements, myoskeletal defects and transient enlargement of gall bladder. These changes were characteristic for all hatchlings exposed to nominal concentrations of 50 microg/l. While certain of the above alterations were reversed within 72-h after hatching, lack of swim bladder inflation and inability to respond to stimuli were two features that persisted with significant incidences. Based on persistence of sublethal effects, results from this work indicate the importance of exposures to gametes and to embryos prior to water hardening. The approach taken herein may better reflect environmental risk conditions than assays limited to exposure of embryonated eggs.     

Androgen disruption of early development in Qurt strain medaka (Oryzias latipes)

Androgen disrupting chemicals (ADCs) are widespread in the aquatic environment, where they may pose a risk to aquatic organisms during critical periods of development. In this study, static renewal 96-h toxicity tests were followed over a 3-month growth period to investigate the endocrine disrupting effects of the androgen 11-ketotestosterone (11-KT), and the antiandrogen flutamide (FLU) on 1-week-old Qurt strain medaka larvae. The measured endpoints included: survival, growth performance (i.e., body weight, body length, condition index), and histopathology. There was no significant acute mortality, except for males treated with the highest FLU concentration (96 h-LC(50)=1.92 mg/l). Gender-specific effects in growth were identified after 11-KT and FLU treatments. Histopathological alterations including thyroid follicular hypertrophy (TFH), germ cell necrosis (GCN), ovarian atresia (OA), and testis-ova (TO) were observed in medaka at 90-day post-exposure. We observed TFH in all 11-KT treatment levels. The incidence of TFH in males was double that in females 11-KT or FLU treatment. Females showed GCN at lower 11-KT concentrations (0.01, 0.1mg/l) than males (1.0mg/l). Severe OA was observed at low (0.01 mg/l) and high (1.0mg/l) 11-KT concentrations in females. Flutamide induced TO (0.32, 1.0mg/l), ovarian cell necrosis (0.32 mg/l), and disrupted spermatogenesis (3.2mg/l) in males. The lowest observed effective concentration (LOEC) for TO induction in Qurt medaka males was 0.32 mg/l. The present study underscores the importance of fish early life stage tests for detecting the interaction of ADCs with the reproductive and thyroid glands in both genders.     

Verification of responses of Japanese medaka (Oryzias latipes) to anti‐androgens,vinclozolin and flutamide,in short‐term assays

Various testing methods for the detection of the endocrine disruptive activities of chemicals have been developed in freshwater fish species. However, a few relatively easier specific methods for detecting anti‐androgenic activities are available for fish. The aim of this study was to verify the papillary process in Japanese medaka (Oryzias latipes) as an indicator of the anti‐androgenic activity of chemicals. Japanese medaka were exposed to two types of anti‐androgenic compounds, vinclozolin and flutamide, using two short‐term assays; one was conformed to the existing short‐term reproduction assay using adult fish (adult test) and the other was a test based on the same methods but using juvenile fish at the beginning of exposure (juvenile test). Significant decreases in male papillary processes were observed in the juvenile test treated with the highest concentration of both antiandrogens (640 µg l^–1 vinclozolin and 1000 µg l^–1 flutamide); however, no significant effects were observed in the adult test. Consequently, our results indicate that papillary processes in Japanese medaka can be used as the end‐point for screening the anti‐androgenic activity of chemicals using juvenile fish for a specific period based on the existing short‐term reproduction assay. Copyright © 2013 John Wiley & Sons, Ltd.     

The cytotoxicity and genotoxicity of hexavalent chromium in medaka (Oryzias latipes) cells

Chromium is an increasing health concern for aquatic environments, however, the mechanism of chromium toxicity in aquatic species is yet unknown. We used a medaka (Oryzias latipes) fin cell line to investigate the cytotoxicity and genotoxicity of sodium chromate, a soluble form of hexavalent chromium. We used a clonogenic cytotoxicity assay to measure sodium chromate cytotoxicity, gamma-H2A.X immunofluoresence to measure DNA double-strand breaks, and chromosome damage to measure clastogenicity. We found that sodium chromate is cytotoxic to medaka fin cells, with toxicity increasing in a concentration-dependent manner. Treatments of 0.5, 1, 5, 10, 25, 50 and 100 microM sodium chromate caused 100, 103.5, 87.8, 77.5, 40.9, 15 and 2.7% survival, respectively, relative to the control. We visualized DNA double-strand breaks in medaka cells through the formation of gamma-H2A.X foci. Breaks could be detected at concentrations as low as 1 microM. We also found that sodium chromate induces chromosomal aberrations, causing chromatid lesions and exchanges that increase with concentration. Treatments of 0, 1, 5, 10 and 25 microM sodium chromate damaged 10.3, 17, 32.3, 43 and 51.6% of metaphases and induced 13, 23, 44, 69 and 118 total aberrations in 100 metaphases, respectively. These data show that hexavalent chromium is both cytotoxic and genotoxic to fish cells. Our results set the context for future work in the medaka cell culture model and provide important tools for investigating mechanisms of toxicity in aquatic organisms.     

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.