Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Ltd  

Metals are essential elements for human life but may cause disorders when exposure is excessive. Previously, we reported on the acute toxicity of 50 metals
however, the chronic toxicity data of some metals are not available. Therefore, we conducted chronic toxicity tests to determine the effects of 50 metals on the water flea, Ceriodaphnia dubia. The IC20 of 20 metals (Be, Sc, Cr, Co, Ni, Cu, Zn, Y, Ru, Ag, Cd, In, Te, W, Os, Pt, Au, Hg, Tl and Pb) were &lt
100 μg/L
nine metals (Al, V, As, Se, Zr, Nb, Rh, Sb and Bi) were 100 ≤ IC20 &lt
1000 μg/L
16 metals (Li, Mg, K, Ti, Mn, Fe, Ga, Ge, Rb, Sr, Mo, Sn, Cs, Ba, Re and Ir) were 1000 ≤ IC20 ≤ 100 000 μg/L
and two metals (Na and Ca) were &gt
100 000 μg/L. Three metals (Pd, Hf and Ta) did not show IC20 at the upper limit of respective aqueous solubility, and IC20s were not obtained. The maximum test concentrations (almost aqueous solubility) of Pd, Hf and Ta were 83, 2400 and 5.3 μg/L, respectively. These data show the high correlation between our IC50s for C. dubia and those for Dahpnia magna published previously. The IC50s of 47 metals were not correlated with electronegativity, first ionization energy, atomic weight, atomic number, covalent radius, atomic radius or ionic radius.

DOI： 10.1002/jat.4049

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Publisher：Cold Spring Harbor Laboratory  

<title>Abstract</title>The water flea <italic>Daphnia magna</italic> is a representative example of zooplankton living in freshwater environments. They primarily propagate via asexual reproduction under normal and healthy environmental conditions. Environmental stimuli that signal a shift to disadvantageous conditions induce <italic>D. magna</italic> to change their mode of reproduction from asexual to sexual reproduction. During the sexual reproduction phase, they produce special tough eggs (resting eggs), which can survive severe environmental conditions. Despite our increased understanding of their mating behaviours, the sex-specific characteristics of swimming behaviours among daphnid species are poorly understood. In this study, we analysed the swimming patterns and dynamics of female and male adult <italic>D. magna</italic>. First, we found laterally biased diffusion of males in contrast to the homogeneous, nondirectional diffusion of females. Second, computer modelling analysis using a discrete-time Markov chain simulation, in which the frequencies of turning behaviour were evaluated as probability distributions, explained the greater diffusion of males in the horizontal direction. Under the presumption that high diffusion in the horizontal direction increases the probability of encountering a distant mate, these findings led us to hypothesise that male <italic>D. magna</italic> increase genotype heterogeneity by effectively selecting the probability distributions of certain motion parameters.

<sec><title>Summary statements</title>We analysed the swimming behaviours of adult water flea <italic>Daphnia magna</italic>, and found apparent sexual differences: laterally biased diffusion of males in contrast to the nondirectional diffusion of females.

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DOI： 10.1101/2021.01.21.427564

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In the present study, we assessed the negative effects of triphenyltin (TPT) on zebrafish (Danio rerio) by exposing embryos and early-stage larvae to various concentrations of TPT from 2 h after fertilization (haf) until 30 days after hatching (dah). Whether test groups were fed or fasted during ecotoxicity studies using fish models has varied historically, and whether this experimental condition influences test results is unknown. Here, we confirmed that the lethal concentration of TPT to embryo and early-stage larvae (i.e., 3 dah or younger) showed in fed (lowest observed effect concentration (LOEC); 6.34 μg/L) and fasted (LOEC; 6.84 μg/L) groups. In addition, 84% and 100% of the larvae in the 2.95 and 6.64 μg/L exposure groups, respectively, had uninflated swim bladders; all affected larvae died within 9 dah. This finding suggests that morphologic abnormalities in early larval zebrafish are useful as endpoints for predicting the lethality of chemical substances after hatching. We then assessed the expression of several genes in the thyroid hormone pathway, which regulates swim bladder development in many fish species, including zebrafish. Larvae exposed to 6.64 μg/L TPT showed significant increases in the mRNA expression levels of thyroid hormone receptor α (trα) and trβ but not of thyroid stimulating hormone β subunit. These findings suggest that TPT disrupts the thyroid system in zebrafish.

DOI： 10.1016/j.cbpc.2020.108948

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Aquatic heavy metal pollution is a growing concern. To facilitate heavy metal monitoring in water, we developed transgenic <italic>Daphnia</italic> that are highly sensitive to heavy metals and respond to them rapidly. Metallothionein A, which was a metal response gene, and its promoter region was obtained from <italic>Daphnia magna</italic>. A chimeric gene fusing the promoter region with a green fluorescent protein (GFP) gene was integrated into <italic>D. magna</italic> using the TALEN technique and transgenic <italic>Daphnia</italic> named <italic>D. magna</italic> MetalloG were produced. When <italic>D. magna</italic> MetalloG was exposed to heavy metal solutions for 1 h, GFP expression was induced only in their midgut and hepatopancreas. The lowest concentrations of heavy metals that activated GFP expression were 1.2 µM Zn²⁺, 130 nM Cu²⁺, and 70 nM Cd²⁺. Heavy metal exposure for 24 h could lower the thresholds even further. <italic>D. magna</italic> MetalloG facilitates aqueous heavy metal detection and might enhance water quality monitoring.

DOI： 10.1038/s41598-020-78572-z

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Other Link： http://www.nature.com/articles/s41598-020-78572-z

Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.chemrestox.0c00179

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The branched isomer mixture 4-nonylphenol (4-NP) has been used worldwide as a surfactant, and can have endocrine-disrupting effects on aquatic organisms. For instance, 4-NP induces the formation of testis-ova (i.e., testicular and ovarian tissue in the same gonad) or male to female sex reversal of various teleost fishes. Recently, our group revealed that altered gsdf gene expression is associated with disruption of gonadal differentiation in Japanese medaka (Oryzias latipes) embryos exposed to methyltestosterone or bisphenol A, suggesting that gsdf might be useful as a biomarker for predicting the impact of endocrine-disrupting chemicals (EDCs) on gonadal differentiation. Here, we used 4-NP to examine further whether gsdf expression at the embryo stage is useful for predicting EDC impact on gonadal sex differentiation. When fertilized medaka eggs were exposed to 32 or 100 μg/L 4-NP, testis-ova in genetic males and sex reversal from genetic male to phenotypic female were observed. At stage 38 (just before hatching), 4-NP exposure at 1-100 μg/L did not affect gsdf expression in XX embryos compared with the nontreated control; however, in XY embryos, the gsdf expression in the 100 μg/L-exposed group was significantly lower than that in the controls. The 4-NP concentration at which gsdf expression was suppressed was equal to that at which testis-ova and sex reversal were induced. These results indicate that expression of the gsdf gene at the embryonic stage in medaka is a useful biomarker for predicting the impact of EDCs on sexual differentiation.

DOI： 10.1002/jat.4051

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Bisphenol A (BPA), a very important raw material in the plastics industry, is an endocrine-disrupting chemical in teleost fish. Although BPA induces testis-ova and sex reversal in teleost fish species, the molecular mechanism remains unclear. We evaluated the effects of BPA (measured concentrations: 45, 92, 326, 1030 and 3406 μg/L) on Japanese medaka (Oryzias latipes) using OECD TG234 (2011, Fish Sexual Development Test, OECD Guidelines for the Testing of Chemicals, Section 2). BPA at 1030 and 3406 μg/L induced testis-ova and sex reversal with female-type secondary sexual characteristics in XY males at 30 and 60 days posthatching (dph). Then we examined the BPA effect on the expression of sex differentiation genes related to the testis-ova and sex reversal in XY medaka. BPA exposure (1030 and 3406 μg/L) suppressed gsdf mRNA expression and increased cyp19a1a mRNA expression in XY individuals at stage 38 and 30 dph, although foxl2 mRNA expression showed no change. Interestingly, the concentration of BPA that suppressed gsdf mRNA expression at the larval stage was consistent with that needed to induce testis-ova and sex reversal. These results suggest that the gsdf gene at the embryonic stage can be used as a useful biomarker for predicting the impact of estrogenic endocrine-disrupting chemicals on sexual differentiation in Japanese medaka.

DOI： 10.1002/jat.3945

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Diclofenac sodium, a non-steroidal anti-inflammatory drug widely used in both human and veterinary medicine, has been detected in aquatic environments; therefore, its ecotoxicological effects on aquatic organisms need to be clarified. Recently, toxicity testing using zebrafish (Danio rerio) embryos has been recommended from the point of view of animal welfare; therefore, we investigated the suitability of using sub-lethal endpoints observed during embryogenesis for predicting lethal effects in early life stage zebrafish exposed to diclofenac sodium. After exposure to diclofenac sodium (0.4-7.0 mg/L) from 2 hours post-fertilization to 30 days post-hatching, abnormal embryogenesis, characterized by the presence of edema and body curvature, was observed in the 7.0 mg/L exposure group but not in any other groups including controls. The body curvature was found to be the result of abnormal development of the spine. All abnormal embryos hatched without delay, but died within 1 week after hatching, suggesting that the combination of the sub-lethal endpoints of edema and abnormal development of the spine during embryogenesis may predict lethal effects in early life stage zebrafish exposed to diclofenac sodium. Further investigations to verify these findings are needed. The value of the no observed effect concentrations for the embryogenesis, survival and growth endpoints were 3.5, 1.8 and >3.5 mg/L, respectively.

DOI： 10.1002/jat.3752

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The commercial use of metals such as copper, lead, and zinc has markedly increased in recent years, resulting in increased interest in deep-sea mining of seafloor hydrothermal sulfide deposits. However, the full extent of the impact of deep-sea mining at hydrothermal field deposits on the environment remains unclear. In addition to impacting the deep sea, the leaching of heavy metals from extracted sulfide mineral may also affect the upper ocean zones as the sulfide rock is retrieved from the seafloor. Here, we used a delayed fluorescence-based bioassay using the marine cyanobacterium Cyanobium sp. NIES-981 to evaluate the toxicity of three sulfide core samples obtained from three drill holes at the Izena Hole, middle Okinawa Trough, East China Sea. Leaches from two of the cores contained high concentrations of zinc and lead, and they markedly inhibited delayed fluorescence in Cyanobium sp. NIES-981 compared with control. By examining the toxicity of artificial mixed-metal solutions with metal compositions similar to those of the leaches, we confirmed that this inhibition was a result of high zinc and lead concentrations into the leaches. In addition, we conclude that this delayed fluorescence-based bioassay is a viable method for use by deep-sea mining operations because it is quicker and requires less laboratory space and equipment than the standard assay.

DOI： 10.1007/s10646-018-1989-2

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Sex steroid hormones including estrogens and androgens play fundamental roles in regulating reproductive activities and they act through estrogen and androgen receptors (ESR and AR). These steroid receptors have evolved from a common ancestor in association with several gene duplications. In most vertebrates, this has resulted in two ESR subtypes (ESR1 and ESR2) and one AR, whereas in teleost fish there are at least three ESRs (ESR1, ESR2a and ESR2b) and two ARs (ARα and ARβ) due to a lineage-specific whole genome duplication. Functional distinctions have been suggested among these receptors, but to date their roles have only been characterized in a limited number of species. Sexual differentiation and the development of reproductive organs are indispensable for all animal species and in vertebrates these events depend on the action of sex steroid hormones. Here we review the recent progress in understanding of the functions of the ESRs and ARs in the development and expression of sexually dimorphic characteristics associated with steroid hormone signaling in vertebrates, with representative fish, amphibians, reptiles, birds and mammals.

DOI： 10.1016/j.jsbmb.2018.06.002

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The lethal effects of chemicals is a serious concern to the protection of ecosystems, and the OECD TG 212 was established to estimate the lethal and sublethal effects on embryo and sac-fry stages of fish. It is still unclear, however, whether this test can effectively estimate the impacts of chemicals using marine fish. Therefore, this study aimed to use a recognized testing method on the marine fish Oryzias javanicus, and to assess differences in response to organotin compounds between a freshwater congener (Oryzias latipes) and O. javanicus. The lowest observed effect concentration (LOEC) of triphenyltin for lethal effect was the same in both species. The LOEC of tributyltin for lethal and sublethal effects were the same in both species. Our results provide the first evidence that O. javanicus and O. latipes are similarly affected by organotin compounds, suggesting that O. javanicus is a good model marine fish for the ecotoxicological assessment of chemicals.

DOI： 10.1007/s00128-018-2398-1

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Here we developed an analytical means of estimating population-level effects of endocrine disruptors on Daphnia magna. Our approach was based on the fact that the endocrine-disrupting juvenile hormone analogs induce the production of male neonates if they are exposed to the analogs during a particular period in their prenatal development; the method also assumed that the abnormal production of male neonates in the sake of production of female neonates reduces population growth. We constructed a linear toxicodynamics model to elucidate the period in which D. magna neonates are sensitive to exposure to the analog and also the probability of an individual neonate changing sex under specific exposure concentrations. The proposed model was applied to D. magna reproduction test data obtained under time-varying exposure to pyriproxyfen to derive the maximum-likelihood estimates and the posterior distributions of the model parameters. To quantitatively assess the ecological risk at the population level, we conducted a population dynamics simulation under two time-varying exposure scenarios (i.e., constant or pulsed exposure) by using an age-structured population model. When the change in sex ratio was based on the time-weighted average concentration during the period of sensitivity, change in sex ratio caused approximately equivalent population-level effects as did reproductive inhibition (i.e., reduction in the total number of neonates per female parent) regardless of the exposure scenario. In contrast, when change in sex ratio was based on maximum concentration during the sensitive period, change in sex ratio caused only half the population-level effects as did reproductive inhibition under constant exposure, whereas it caused a much larger population-level effect than did reproductive inhibition under pulsed exposure.

DOI： 10.1016/j.ecoenv.2018.03.044

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Tributyltin, an organotin compound, was used worldwide as an antifouling agent in aquatic environments and there has been much concern about the toxicological and ecotoxicological properties of organotin compounds. Even though it has been prohibited worldwide, tributyltin is still detected at low concentrations in aquatic environments. Here we investigated the effects of tributyltin on the early life-stage, reproduction, and gonadal sex differentiation in Japanese medaka (Oryzias latipes). In adults, exposure to tributyltin at 3.82 μg/L suppressed fecundity and fertility and increased mortality. At 10.48 μg/L all medaka died by the sixth day of exposure. Exposure to tributyltin during early life-stages induced no significant differences in mortality or embryonic development, but growth was suppressed in groups exposed to 0.13 and 0.68 μg/L. Furthermore, there was no abnormal gonadal development in Japanese medaka exposed to tributyltin. These results provide evidence of the negative effects of tributyltin on reproduction in a teleost fish. Tributyltin did not affect gonadal sex differentiation in Japanese medaka, but fecundity and fertility were suppressed, although it is not clear whether this suppression resulted from the endocrine-disrupting action of tributyltin or its toxicity.

DOI： 10.1016/j.chemosphere.2018.03.135

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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.

DOI： 10.1002/jat.3561

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Short-term pulsed exposure tests have been increasingly used to evaluate the ecotoxicity of pollutants of which concentrations vary over time in the field. In pulsed exposure, time-weighted average (TWA) concentration is often used as an index of exposure. However, there have been few studies to demonstrate whether TWA concentration can be used to evaluate the effect of endocrine-disrupting chemicals on the daphnids. Pyriproxyfen is one of the juvenile hormone analogs that induces daphnids to produce male offspring. To evaluate whether peak or TWA concentration can explain the effects of pyriproxyfen on daphnid reproduction, we measured the number of offspring and the proportion of male offspring produced by Daphnia magna during 21-day under different exposure treatments, constant, single-pulse, and multi-pulse exposure, at an equivalent TWA concentration. Constant exposure of 50 ng/L pyriproxyfen did not affect either the fecundity or the proportion of male offspring, while a single-pulse exposure of 525 ng/L pyriproxyfen over 2 day at four different age did not reduce fecundity, but the proportion of male offspring increased age dependently. Multi-pulses exposure of 131 ng/L pyriproxyfen over two days four times (total eight days) resulted in a decrease in fecundity and the highest proportion of male offspring. Daily observation demonstrated that male offspring was only produced several days after the exposure to a certain concentration of pyriproxyfen. Therefore, neither TWA nor peak concentration accurately evaluated the effects of pulsed exposure of pyriproxyfen on the reproduction of D. magna, particularly its effect on the proportion of male offspring.

DOI： 10.1016/j.chemosphere.2017.12.124

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© 2016 Elsevier B.V. Algal growth inhibition tests are generally used to determine the toxic effects of chemical substances on algae growth. In this report, we describe a rapid and simple test procedure using delayed fluorescence (DF) to determine chemical toxicities more rapidly than the conventional 72 h or 96 h growth inhibition tests. We assess the suitability of DF to serve as an alternative endpoint for biomass production and determine the variability by an interlaboratory ring study using a typical reference toxicant 3,5-dichlorophenol (DCP). The results suggest that DF has the potential to be used as a surrogate measure of photosynthetically-active biomass in the algal growth inhibition tests. The half maximal effective concentration (EC50) values of DCP determined from the DF inhibition test in 6 h and 24 h (1.2 ± 0.3 mg/L and 2.7 ± 0.5 mg/L respectively) are in reasonable agreement with the EC50 value of DCP determined by the 72 h conventional method (1.8 mg/L). In the interlaboratory ring study, the intralaboratory and interlaboratory variabilities of the EC50 of the DF inhibition test for a 24 h exposure period are 12% and 28% respectively. DF intensity can be considered as a surrogate of living biomass with active photosynthesis, and we conclude that a 24 h exposure duration better estimates the toxic effects measured using conventional surrogate measures for dry weight such as cell counts, volume, optical density or fluorescence.

DOI： 10.1016/j.scitotenv.2017.06.228

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© 2017 SETAC The medaka extended one-generation test (MEOGRT) was developed as a multigenerational toxicity test for chemicals, particularly endocrine-disrupting chemicals. Briefly, 3 generations of Japanese medaka (Oryzias latipes) are exposed to a chemical over a 20-wk period: 3 wk in the parental generation (F0), 15 wk in the first generation (F1), and 2 wk in the second generation (F2). The present study reports the first MEOGRT results concerning branched isomer mixtures of 4-nonylphenol (NP). Adult F0 medaka exposed to NP at 5 actual concentrations (1.27, 2.95, 9.81, 27.8, 89.4 µg/L) were unaffected in terms of reproduction, although vitellogenin in the male liver was increased dose-dependently at concentration of 2.95 µg/L and higher. In F1, in contrast, total egg (fecundity), fertile egg, and fertility decreased as NP increased; lowest-observed-effect concentrations (LOECs) for total egg, fertile egg, and fertility were 1.27, 1.27, 27.8 µg/L, respectively. In F1, but not in F0, secondary sex characteristics (i.e., anal fin papillae in males) were suppressed at 27.8 µg/L NP. Vitellogenin induction in adult male fish was slightly weaker in F1 than it was in F0, however. Gonadal sex abnormality and sex reversal occurred at 27.8 and 89.4 µg/L NP in F1 subadults. At 89.4 µg/L NP, all genotypic F1 males in breeding pairs had female phenotype, and some even demonstrated spawning. Concentrations of NP lower than 89.4 µg/L did not affect F2 survival or hatching. The highest detected NP level in environmental freshwater in Japan was approximately a half of the LOEC (1.27 µg/L for F1 fecundity); in other countries, however, environmental concentrations above the LOEC are reported, suggesting that NP may be affecting fish populations. Environ Toxicol Chem 2017;36:3254–3266. © 2017 SETAC.

DOI： 10.1002/etc.3895

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Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America In response to various legislative mandates, the US Environmental Protection Agency (USEPA) formed its Endocrine Disruptor Screening Program (EDSP), which in turn, formed the basis of a tiered testing strategy to determine the potential of pesticides, commercial chemicals, and environmental contaminants to disrupt the endocrine system. The first tier of tests is intended to detect the potential for endocrine disruption mediated through estrogen, androgen, or thyroid pathways, whereas the second tier is intended to further characterize the effects on these pathways and to establish a dose–response relationship for adverse effects. One of these tier 2 tests, the Medaka Extended One Generation Reproduction Test (MEOGRT), was developed by the USEPA for the EDSP and, in collaboration with the Japanese Ministry of the Environment, for the Guidelines for the Testing of Chemicals of the Organisation for Economic Co-operation and Development (OECD). The MEOGRT protocol was iteratively modified based on knowledge gained after the successful completion of 9 tests with variations in test protocols. The present study describes both the final MEOGRT protocol that has been published by the USEPA and the OECD, and the iterations that provided valuable insights into nuances of the protocol. The various tests include exposure to 17β-estradiol, 4-t-octylphenol, o,p′- dichlorodiphenyltrichloroethane, 4-chloro-3-methylphenol, tamoxifen, 17β-trenbolone, vinclozolin, and prochloraz. Environ Toxicol Chem 2017;36:3387–3403. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

DOI： 10.1002/etc.3923

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Triphenyltin (TPT) is an organotin compound used in marine anti-fouling coatings to prevent the attachment and growth of marine organisms, and it has negative effects on aquatic organisms. TPT is still detected at low concentrations, although its use has been prohibited at least in the European Community and is restricted in Japan as well. Studies using Japanese medaka (Oryzias latipes) indicate that TPT has the potential to inhibit reproduction. Although TPT is detected in many aquatic ecosystems, the multi-generational impact of TPT remains unknown. We investigated the two-generational effects of TPT on Japanese medaka and examined the relationships of several such effects between the F0 and F1 generations. Suppression of fecundity was observed in both generations, and fertility and growth were inhibited in the F1 generation. Moreover, delayed hatching and lower hatchability were observed in F1 embryos. Importantly, the value of the lowest observed effect concentration (LOEC) for these influences in F1 was lower than that in F0: that is, the LOEC values of fecundity and mortality were 3.2μg/L in the F0 generation and 1.0μg/L in the F1 generation. Fertility was not affected by TPT in F0, whereas it was significantly suppressed in the 1.0μg/L-exposure group of the F1 generation. Our results provide the first evidence of the effects of TPT on reproduction in a teleost fish across two generations, highlighting the concern that TPT could affect reproduction and mortality at decreasing concentrations in temporally overlapping generations.

DOI： 10.1016/j.aquatox.2017.08.016

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Copyright © 2017 John Wiley & Sons, Ltd. Fish embryo toxicity tests are used to assess the lethal and sublethal effects of environmental chemicals in aquatic organisms. Previously, we used a short-term toxicity test published by the Organization for Economic Co-operation and Development (test no. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages [OECD TG 212]) to assess the lethal and sublethal effects of aniline and several chlorinated anilines in zebrafish embryos and larvae. To expand upon this previous study, we used OECD TG 212 in zebrafish embryos and larvae to assess the lethal and sublethal effects of 20 additional environmental chemicals that included active pharmaceutical ingredients, pesticides, metals, aromatic compounds or chlorinated anilines. Zebrafish embryos (Danio rerio) were exposed to the test chemicals until 8 days post-fertilization. A delayed lethal effect was induced by 16 of the 20 test chemicals, and a positive correlation was found between heart rate turbulence and mortality. We also found that exposure to the test chemicals at concentrations lower than the lethal concentration induced the sublethal effects of edema, body curvature and absence of swim-bladder inflation. In conclusion, the environmental chemicals assessed in the present study induced both lethal and sublethal effects in zebrafish embryos and larvae, as assessed by using OECD TG 212. Copyright © 2017 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.3487

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© 2017 Elsevier Inc. Water samples were collected from effluent-dominated urban streams in Tokushima, Kyoto, and Saitama in Japan to roughly determine the contribution of pharmaceuticals and personal care products (PPCPs) and surfactants to whole toxicity of the water. Approximately 100 PPCPs including anionic surfactants such as linear alkylbenzene sulfonate (LAS), were chemically analyzed. Using 14 water samples, chronic or sub-chronic toxicity tests were conducted on three aquatic species, the green alga Raphidocelis subcapitata, the cladoceran Ceriodaphnia dubia, and the zebrafish Danio rerio. Bioassays for the selected individual PPCPs were conducted using the three species. Assuming the concentration addition (CA) model, the contribution of each PPCP to the whole toxicity of the riverwater was estimated based on toxicity unit (TU). The contribution of PPCPs, which primarily consists of a few antibiotic agents such as triclosan and clarithromycin, ranged from 0.9% to 69% of the whole toxicity of the water samples for algae, whereas the selected LAS congeners accounted for at most 5.3%. In contrast, the contribution of LAS ranged from 0.067% to 86% and from 0.021% to 27% of the whole toxicity for cladoceran and zebrafish, respectively, whereas that of PPCPs for these species was at most 2.1% at all sampling points. Our results suggest a limited contribution of PPCPs except for antimicrobial agents and the possible substantial contribution of LAS to toxicity in cladocerans and zebrafish.

DOI： 10.1016/j.ecoenv.2017.06.032

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© 2017 Informa UK Limited, trading as Taylor & Francis Group. We evaluated the predictivity and applicability of previously proposed models for the reproductive toxicity of chemicals to Daphnia magna [SAR QSAR Environ. Res. 27:10, 833–850] by using external data from the United States Environmental Protection Agency database ECOTOX. These models were based on quantitative structure–activity–activity relationships (QSAARs) and a quantitative activity–activity relationship (QAAR): the models can be categorized as acute-to-chronic models with (QSAAR) and without (QAAR) structural and physicochemical (e.g. distribution coefficients, log D) descriptors. We found that the QSAAR models were suitable for chemicals with an ‘–NH2 attached to aromatic carbon’ sub-structure, whereas the QAAR model was better for multicomponent compounds, coordination complexes, tin compounds and straight-chain primary amines. For chemicals with a known specific mode of action (e.g. pesticides and antibacterial agents and their derivatives), toxicity estimation within the acute-to-chronic framework requires special attention. We evaluated the applicability of the models on the basis of the descriptors in the models. We recommend that chemicals be pre-screened before their toxicities are estimated with these models: pre-screening enabled the estimation of the toxicities of some chemicals within the applicability domains of the models.

DOI： 10.1080/1062936X.2017.1381989

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

© 2017 Zoological Society of Japan. Photoperiodism is a biological seasonal timing system utilized to regulate development and reproduction in organisms. The freshwater micro-crustacean Daphnia pulex displays environmental sex determination, the precise physiological mechanisms of which are largely unknown due to the lack of an experimental system to induce female or male offspring production by alterations of the rearing environment. We recently found that D. pulex, WTN6 strain, produces female or male offspring in response to long-day or short-day conditions, respectively. Taking advantage of this system, here we report the photoperiodic response curve for male offspring production, showing 12 hours as natural critical daylength (50% incidence of male-producing mothers), and that male offspring inducibility is highly sensitive to photoperiodic alterations. By using monochromatic light emitting diode (LED) devices, we found that the effective wavelength is red-light (627 nm), which stably induces male offspring production. This suggests that the red-light photoreceptor may be decisive in the primary step of sex determination process in this strain. Our findings provide the first insights into photoperiodism and red-light as key factors in triggering male offspring production in daphnids.

DOI： 10.2108/zs170005

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© 2017 Japanese Society of Developmental Biologists Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptor (ESR) in all vertebrates. Most vertebrates have two ESR subtypes (ESR1 and ESR2), whereas teleost fish have at least three (Esr1, Esr2a and Esr2b). Intricate functionalization has been suggested among the Esr subtypes, but to date, distinct roles of Esr have been characterized in only a limited number of species. Study of loss-of-function in animal models is a powerful tool for application to understanding vertebrate reproductive biology. In the current study, we established esr1 knockout (KO) medaka using a TALEN approach and examined the effects of Esr1 ablation. Unexpectedly, esr1 KO medaka did not show any significant defects in their gonadal development or in their sexual characteristics. Neither male or female esr1 KO medaka exhibited any significant changes in sexual differentiation or reproductive activity compared with wild type controls. Interestingly, however, estrogen-induced vitellogenin gene expression, an estrogen-responsive biomarker in fish, was limited in the liver of esr1 KO males. Our findings, in contrast to mammals, indicate that Esr1 is dispensable for normal development and reproduction in medaka. We thus provide an evidence for estrogen receptor functionalization between mammals and fish. Our findings will also benefit interpretation of studies into the toxicological effects of estrogenic chemicals in fish.

DOI： 10.1111/dgd.12386

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Copyright © 2017 John Wiley & Sons, Ltd. Environmental risk assessments show increased attention to the sublethal effects of chemicals on aquatic organisms. The Organization for Economic Cooperation and Development (OECD) established the “Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages” (OECD test 212) to predict lethal effects. It is still unclear, however, whether this test can predict sublethal effects. Although their sublethal effects are still unknown, chlorinated anilines are widely used in various fields. The purpose of this study, therefore, is to investigate sublethal effects of chlorinated anilines using OECD test 212 with zebrafish, and to examine the correlation of several sublethal effects between embryo and larval stages. Embryos were exposed to aniline and nine chlorinated anilines until 8 days post-fertilization. A delayed lethal effect was observed from three of the 10 anilines tested. In the control group, the swim bladder inflated after hatching, but there was no swim-bladder inflation after exposure to the chlorinated anilines. Fertilized eggs exposed to lower concentrations of test chemicals showed effects during embryogenesis that did not affect mortality rates, such as changes in body curvature and edema. Our results show that chlorinated anilines induce not only lethal effects but also a variety of sublethal effects. Moreover, a detailed estimate of these effects requires study during both embryonic and larval stages. OECD test 212 may therefore prove useful as a method for screening chemicals for lethal and sublethal effects. Copyright © 2017 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.3431

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Macrolide antibiotics and azole fungicides are detected widely in the aquatic environment as a result of their increased use in humans and animal livestock disease and their incomplete removal by wastewater treatment plants. In most cases, ecotoxicological tests are performed by using individual chemical substances, but because of the coexistence of a number of chemicals in the environment, organisms are exposed to many chemicals simultaneously. Therefore, it is important to evaluate effects of chemical interactions, adding to potential hazards of individual chemical. Here, we investigated the synergetic effects of combined chemicals (the azole fungicide ketoconazole and either of two macrolide antibiotics, erythromycin and clarithromycin) in growth inhibition testing using Pseudokirchneriella subcapitata according to OECD Test guideline 201. Combination index plots, isobolograms, and curve-shift analyses revealed that the combination of macrolide antibiotic and ketoconazole at various ratios resulted in strong synergism that enhanced growth inhibition of P. subcapitata, suggesting the necessity of investigating potential hazard of combined chemicals for regulatory purposes.

DOI： 10.1016/j.chemosphere.2017.01.071

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Copyright © 2016 John Wiley & Sons, Ltd. The freshwater zooplankton Daphnia magna has been extensively employed in chemical toxicity tests such as OECD Test Guidelines 202 and 211. Previously, it has been demonstrated that the treatment of juvenile hormones (JHs) or their analogues to female daphnids can induce male offspring production. Based on this finding, a rapid screening method for detection of chemicals with JH-activity was recently developed using adult D. magna. This screening system determines whether a chemical has JH-activity by investigating the male offspring inducibility. Although this is an efficient high-throughput short-term screening system, much remains to be discovered about JH-responsive pathways in the ovary, and whether different JH-activators act via the same mechanism. JH-responsive genes in the ovary including developing oocytes are still largely undescribed. Here, we conducted comparative microarray analyses using ovaries from Daphnia magna treated with fenoxycarb (Fx; artificial JH agonist) or methyl farnesoate (MF; a putative innate JH in daphnids) to elucidate responses to JH agonists in the ovary, including developing oocytes, at a JH-sensitive period for male sex determination. We demonstrate that induction of hemoglobin genes is a well-conserved response to JH even in the ovary, and a potential adverse effect of JH agonist is suppression of vitellogenin gene expression, that might cause reduction of offspring number. This is the first report demonstrating different transcriptomics profiles from MF and an artificial JH agonist in D. magna ovary, improving understanding the tissue-specific mode-of-action of JH. Copyright © 2016 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.3368

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Copyright © 2016 John Wiley & Sons, Ltd. 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.

DOI： 10.1002/jat.3365

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Sex determination of Daphnia pulex is decided by environmental conditions. We established a suitable experimental system for this study using D. pulex WTN6 strain, in which the sex of the offspring can be controlled by photoperiod. Long-day conditions induced females and short-day conditions induced males. Using this system, we previously found that methy farnesoate (MF), which is a putative innate juvenile hormone molecule in daphnids, is necessary for male sex determination and that protein kinase C (PKC) is a candidate factor of male sex determiner. In this study, we demonstrated that a PKC inhibitor [bisindolylmaleimide IV (BIM)] application strongly suppressed male offspring induction in the short-day condition. Moreover, co-treatment of BIM with MF revealed that PKC signaling acts upstream of MF signaling for male sex determination. This is the first experimental evidence that PKC is involved in the male sex determination process associated with methyl farnesoate signaling in daphnid species.

DOI： 10.1242/bio.021857

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Effects observed within one generation disregard potential detrimental effects that may appear across generations. Previously we have developed a two generation Daphnia magna reproduction test using the OECD TG 211 protocol with a few amendments, including initiating the second generation with third brood neonates produced from first generation individuals. Here we showed the results of an inter-laboratory calibration exercise among 12 partners that aimed to test the robustness and consistency of a two generation Daphnia magna reproduction test. Pyperonyl butoxide (PBO) was used as a test compound. Following experiments, PBO residues were determined by TQD-LC/MS/MS. Chemical analysis denoted minor deviations of measured PBO concentrations in freshly prepared and old test solutions and between real and nominal concentrations in all labs. Other test conditions (water, food, D. magna clone, type of test vessel) varied across partners as allowed in the OECD test guidelines. Cumulative fecundity and intrinsic population growth rates (r) were used to estimate "No observed effect concentrations "NOEC using the solvent control as the control treatment. EC10 and EC-50 values were obtained regression analyses. Eleven of the twelve labs succeeded in meeting the OECD criteria of producing >60 offspring per female in control treatments during 21days in each of the two consecutive generations. Analysis of variance partitioning of cumulative fecundity indicated a relatively good performance of most labs with most of the variance accounted for by PBO (56.4%) and PBO by interlaboratory interactions (20.2%), with multigenerational effects within and across PBO concentrations explaining about 6% of the variance. EC50 values for reproduction and population growth rates were on average 16.6 and 20.8% lower among second generation individuals, respectively. In summary these results suggest that the proposed assay is reproducible but cumulative toxicity in the second generation cannot reliably be detected with this assay.

DOI： 10.1016/j.scitotenv.2016.11.066

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DOI： 10.1080/1062936X.2017.1365432

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Pseudokirchneriella subcapitata is a sickle-shaped freshwater green microalga that is normally found in unicellular form. Currently, it is the best known and most frequently used species of ecotoxicological bioindicator because of its high growth rate and sensitivity to toxicants. However, despite this organism's, our knowledge of its cell biology-for example, the patterns of nuclear and cytoplasmic division in the mitotic stage-is limited. Although it has been reported that P. subcapitata proliferates by popularity forming four daughter cells (autospores) through multiple fission after two nuclear divisions, here, we report two additional reproductive patterns by which two autospores are formed by binary fission ("two-autospore type") and eight autospores are formed by multiple fission ("eight-autospore type"). Moreover, we found that cell reproductive patterns differed markedly with the culture conditions or with exposure to either of two typical toxicants, potassium dichromate (K2Cr2O7) and 3,5-dichlorophenol (3,5-DCP). The eight-autospore type occurred at the highest frequency in the early phase of culture, but it disappeared under 3,5-DCP at 2.0 mg/L. Under 0.3 mg/L K2CrO7 (Cr(VI)) the eight-autospore type took substantially longer to appear than in control culture. The two-autospore type occurred only in the late phase of culture. To our knowledge, this is the first detailed evaluation of the reproductive patterns of P. subcapitata, which changed dramatically in the presence of toxicants. These findings suggest that observation of the reproductive patterns of P. subcapitata will help to elucidate different cell reactions to toxicants.

DOI： 10.1371/journal.pone.0171259

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The use of delayed fluorescence intensity as an endpoint for rapid estimation of the effective concentration (ECx) has been reported as an alternative to standard growth inhibition (at 72 h after exposure) in some algal species including Pseudokirchneriella subcapitata. In marine algae, although an approach of bioassaying using delayed fluorescence measurements has not been performed yet, its development would provide many benefits for marine environmental risk assessment. In this study, we selected marine cyanobacterium Cyanobium sp. (NIES-981) as our test algal species and demonstrated that this species is valid for the standard growth inhibition test based on criteria provide by Organization for Economic Co-operation and Development guidelines. Furthermore, standard inhibition tests and shorter period test using DF were performed in NIES-981 using five chemicals (3,5-DCP, simazine, diflufenican, K2Cr2O7, and CuSO4), and their EC50 and low-toxic-effect values (EC10, EC5, and NOEC) were determined from two dose-response curves. Based on comparisons of the two dose-response curves and the EC50 values, we conclude that DF intensity is useful as an endpoint for rapid estimation of EC50 in NIES-981.

DOI： 10.1007/s10646-016-1718-7

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Cyanobium sp. NIES-981 is a marine cyanobacterium isolated from tidal flat sands in Okinawa, Japan. Here, we report the complete 3.0-Mbp genome sequence of NIES-981, which is composed of a single chromosome, and its annotation. This sequence information may provide a basis for developing an ecotoxicological bioassay using this strain.

DOI： 10.1128/genomeA.00736-16

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In vertebrates, estrogens play fundamental roles in regulating reproductive activities through estrogen receptors (ESRs), and disruption of estrogen signaling is now of global concern for both wildlife and human health. To date, ESRs of only a limited number of species have been characterized. We investigated the functional diversity and molecular basis or ligand sensitivity of ESRs among ray-finned fish species (Actinopterygii), the most variable group within vertebrates. We cloned and characterized ESRs from several key species in the evolution of ray-finned fish including bichir (Polypteriformes, ESR1 and ESR2) at the basal lineage of ray-finned fish, and arowana (Osteoglossiformes, ESR1 and ESR2b) and eel (Anguilliformes, ESR1, ESR2a and ESR2b) both belonging to ancient early-branching lineages of teleosts, and suggest that ESR2a and ESR2b emerged through teleost-specific whole genome duplication, but an ESR1 paralogue has been lost in the early lineage of euteleost fish species. All cloned ESR isoforms showed similar responses to endogenous and synthetic steroidal estrogens, but they responded differently to non-steroidal estrogenic endocrine disrupting chemicals (EDCs) (e.g., ESR2a exhibits a weaker reporter activity compared with ESR2b). We show that variation in ligand sensitivity of ESRs can be attributed to phylogeny among species of different taxonomic groups in ray-finned fish. The molecular information provided contributes both to understanding of the comparative role of ESRs in the reproductive biology of fish and their comparative responses to EDCs.

DOI： 10.1016/j.jsbmb.2015.12.009

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Principles of concentration addition and independent action have been used as effective tools to predict mixture toxicity based on individual component toxicity. The authors investigated the toxicity of a pharmaceutical mixture composed of the top 10 detected active pharmaceutical ingredients (APIs) in the Tama River (Tokyo, Japan) in a relevant concentration ratio. Both individual and mixture toxicities of the 10 APIs were evaluated by 3 short-term chronic toxicity tests using the alga Pseudokirchneriella subcapitata, the daphnid Ceriodaphnia dubia, and the zebrafish Danio rerio. With the exception of clarithromycin toxicity to alga, the no-observed-effect concentration of individual APIs for each test species was dramatically higher than the highest concentration of APIs found in the environment. The mixture of 10 APIs resulted in toxicity to alga, daphnid, and fish at 6.25 times, 100 times, and 15,000 times higher concentrations, respectively, than the environmental concentrations of individual APIs. Predictions by concentration addition and independent action were nearly identical for alga, as clarithromycin was the predominant toxicant in the mixture. Both predictions described the observed mixture toxicity to alga fairly well, whereas they slightly underestimated the observed mixture toxicity in the daphnid test. In the fish embryo test, the observed toxicity fell between the predicted toxicity by concentration addition and independent action. These results suggested that the toxicity of environmentally relevant pharmaceutical mixtures could be predicted by individual toxicity using either concentration addition or independent action.

DOI： 10.1002/etc.3285

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The freshwater crustacean genus Daphnia has been used extensively in ecological, developmental and ecotoxicological studies. Daphnids produce only female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable conditions and external stimuli, they produce male offspring. Although we reported that exogenous exposure to juvenile hormones and their analogs can induce male offspring even under female-producing conditions, we recently established a male induction system in the Daphnia pulex WTN6 strain simply by changing day-length. This male and female induction system is suitable for understanding the innate mechanisms of sexual dimorphic development in daphnids. Embryogenesis has been described as a normal plate (developmental staging) in various daphnid species; however, all studies have mainly focused on female development. Here, we describe the developmental staging of both sexes during embryogenesis in two representative daphnids, D. pulex and D. magna, based on microscopic time-course observations. Our findings provide the first detailed insights into male embryogenesis in both species, and contribute to the elucidation of the mechanisms underlying sexual differentiation in daphnids.

DOI： 10.2108/zs150116

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Application of herbicides to paddy fields in Japan has strong seasonality, and their environmental concentrations exhibit clear spatiotemporal variation. The authors developed an approach that combines a multimedia environmental exposure model (Grid-Catchment Integrated Modeling System) and density dynamics models for algae. This approach enabled assessment of ecological risk when the exposure concentration shows spatiotemporal variation. First, risk maps of 5 herbicides (pretilachlor, butachlor, simetryn, mefenacet, and esprocarb) were created from the spatial predictions of environmental concentrations and 50% inhibitory concentrations of the herbicides. Simulations of algal density dynamics at high-risk sites were then conducted by incorporating the predicted temporal dynamics of the environmental concentration of each herbicide at the sites. The results suggested that the risk of pretilachlor was clearly the highest of the 5 herbicides, in terms of both the spatial distributions and the temporal durations. The present study highlights the importance of integrating exposure models and effect models to clarify spatial and temporal risk and to develop management plans for chemical exposure that shows high spatiotemporal variation.

DOI： 10.1002/etc.3162

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We propose a three-step strategy that uses structural and physicochemical properties of chemicals to predict their 72 h algal growth inhibition toxicities against Pseudokirchneriella subcapitata. In Step 1, using a log D-based criterion and structural alerts, we produced an interspecies QSAR between algal and acute daphnid toxicities for initial screening of chemicals. In Step 2, we categorized chemicals according to the Verhaar scheme for aquatic toxicity, and we developed QSARs for toxicities of Class 1 (non-polar narcotic) and Class 2 (polar narcotic) chemicals by means of simple regression with a hydrophobicity descriptor and multiple regression with a hydrophobicity descriptor and a quantum chemical descriptor. Using the algal toxicities of the Class 1 chemicals, we proposed a baseline QSAR for calculating their excess toxicities. In Step 3, we used structural profiles to predict toxicity either quantitatively or qualitatively and to assign chemicals to the following categories: Pesticide, Reactive, Toxic, Toxic low and Uncategorized. Although this three-step strategy cannot be used to estimate the algal toxicities of all chemicals, it is useful for chemicals within its domain. The strategy is also applicable as a component of Integrated Approaches to Testing and Assessment.

DOI： 10.1080/1062936X.2016.1174151

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

【目的】残留性有機汚染物質(POPs)である有機フッ素化合物(PFC)は、広く環境中から検出され、野生動物等に蓄積して人や動物への毒性が危惧されている。本研究では、PFCsの汚染実態を把握するため、わが国の魚類、環境水、底質中のPFCs分析を行い、環境中における濃度と魚類への蓄積状況について検討した。&lt;br&gt;【方法】サンプルは、2013～2014年にかけて岩手、茨城、新潟、石川、静岡、兵庫、山口、愛媛、福岡、長崎から採取した。魚類としてはメダカを選定し、福岡ではカダヤシも併せて採取した。環境水及び底質は各地点1サンプル、メダカは各地点20～40匹、カダヤシは12匹採取した。分析対象PFCsは、Perfluorosulfonates(CXS)のC4S、C6S、C7S、C8S、C10S、Perfluorocarboxylates(CXA)のC5A～C14Aとした。&lt;br&gt;【結果と考察】環境水や底質からはPFOA(C8A)～C12Aが、メダカ・カダヤシからはC9A～C13A、PFOS(C8S)が高率に検出された。C8SとC9Aでは、環境水と底質の濃度間で相関が認められた。メダカとカダヤシでは、濃縮係数に差はあるものの蓄積傾向は同様であった。CXAの魚体への蓄積は炭素数に応じて増加し、C8Sの蓄積量はそれより大きかったが、オクタノール/水分配係数を考慮すると、同一の傾向が認められた。C8Sはストックホルム条約等により2009年から使用等が規制され、C8AはUSEPA主導の業界自主規制により2010年までに環境への排出量が95%削減されているにもかかわらず、未だにC8S、C8Aが環境中に残留しており、それ以外のPFCsも検出される実態が明らかとなった。このことから、今後もこの研究を継続していくことが重要であると思われた。

DOI： 10.14869/toxpt.43.1.0_O-16

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We constructed models for acute to chronic estimation of the Daphnia magna reproductive toxicities of chemical substances from their Daphnia magna acute immobilization toxicities. The models combined the acute toxicities with structural and physicochemical descriptors. We used multiregression analysis and selected the descriptors for the models by means of a genetic algorithm. Of the best 100 models (as indicated by the lack of fit score), 90% included the following descriptors: acute toxicity (i.e. an activity parameter), distribution coefficient (log D) and structural indicator variables that indicate the presence of -NH2 attached to aromatic carbon and the presence of a chlorine atom. We compared the predictive abilities of five of these quantitative structure-activity-activity relationship (QSAAR) acute to chronic estimation models with the predictive ability of a simple linear regression model. The comparison revealed that inclusion of structural and physicochemical descriptors such as those in QSAAR models can improve models for extrapolation from acute to chronic toxicity. Our results also provide a QSAAR framework that is expected to be useful for the further development of chronic toxicity estimation models.

DOI： 10.1080/1062936X.2016.1243151

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Sex-determination systems can be divided into two groups: genotypic sex determination (GSD) and environmental sex determination (ESD). ESD is an adaptive life-history strategy that allows control of sex in response to environmental cues in order to optimize fitness. However, the molecular basis of ESD remains largely unknown. The micro crustacean Daphnia pulex exhibits ESD in response to various external stimuli. Although methyl farnesoate (MF: putative juvenile hormone, JH, in daphnids) has been reported to induce male production in daphnids, the role of MF as a sex-determining factor remains elusive due to the lack of a suitable model system for its study. Here, we establish such a system for ESD studies in D. pulex. The WTN6 strain switches from producing females to producing males in response to the shortened day condition, while the MFP strain only produces females, irrespective of day-length. To clarify whether MF has a novel physiological role as a sex-determining factor in D. pulex, we demonstrate that a MF/JH biosynthesis inhibitor suppressed male production in WTN6 strain reared under the male-inducible condition, shortened day-length. Moreover, we show that juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme of MF/JH biosynthesis, displays MF-generating activity by catalyzing farnesoic acid. Expression of the JHAMT gene increased significantly just before the MF-sensitive period for male production in the WTN6 strain, but not in the MFP strain, when maintained under male-inducible conditions. These results suggest that MF synthesis regulated by JHAMT is necessary for male offspring production in D. pulex. Our findings provide novel insights into the genetic underpinnings of ESD and they begin to shed light on the physiological function of MF as a male-fate determiner in D. pulex.

DOI： 10.1016/j.jinsphys.2015.02.002

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Metals are essential for human life and physiological functions but may sometimes cause disorders. Therefore, we conducted acute toxicity testing of 50 metals in Daphnia magna: EC50s of seven elements (Be, Cu, Ag, Cd, Os, Au and Hg) were < 100 µg l(-1) ; EC50s of 13 elements (Al, Sc, Cr, Co, Ni, Zn, Se, Rb, Y, Rh, Pt, Tl and Pb) were between 100 and 1000 µg l(-1) ; EC50s of 14 elements (Li, V, Mn, Fe, Ge, As, In, Sn, Sb, Te, Cs, Ba, W and Ir) were between 1,001 and 100,000 µg l(-1) ; EC50s of six elements (Na, Mg, K, Ca, Sr and Mo) were > 100,000 µg l(-1) ; and. 7 elements (Ti, Zr, Bi, Nb, Hf, Re and Ta) did not show EC50 at the upper limit of respective aqueous solubility, and EC50s were not obtained. Ga, Ru and Pd adhered to the body of D. magna and physically retarded the movement of D. magna. These metals formed hydroxides after adjusting the pH. Therefore, here, we distinguished this physical effect from the physiological toxic effect. The acute toxicity results of 40 elements obtained in this study were not correlated with electronegativity. Similarly, the acute toxicity results of metals including the rare metals were also not correlated with first ionization energy, atomic weight, atomic number, covalent radius, atomic radius or ionic radius.

DOI： 10.1002/jat.3078

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Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.

DOI： 10.1021/acs.est.5b00704

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The chronic toxicity of 12 compounds of parabens and their chlorinated by-products was investigated using 7-day Ceriodaphnia dubia test under static renewal condition in order to generate information on how to disinfect by-products of preservatives that are discharged in aquatic systems. The mortality and inhibition of reproduction tended to increase with increasing hydrophobicity and decreased with the degree of chlorination of parabens. The EC50 values for mortality, offspring number, and first brood production ranged between 0.30-3.1, 0.047-12, and 1.3-6.3 mg L-1, respectively. For the number of neonates, the most sensitive endpoint, the no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) values ranged from 0.63 to 10 mg L-1 and from 1.2 to 19 mg L-1, respectively. Methylparaben (MP), benzylparaben (BnP), and dichlorinated BnP (Cl2BnP) elicited a significant decrease in offspring numbers even at their lowest concentration tested; the NOEC for these compounds was determined to be less than the lowest test concentration (1.3, 0.04, and 0.63 mg L-1 for MP, BnP, and Cl2BnP, respectively). Propylparaben (PP), chlorinated PP, isopropylparaben (iPP), and chlorinated iPP exhibited nonmonotonic concentration-dependent response; their NOEC and LOEC values could not be determined. The multivariate approach involving principal component analysis and hierarchical cluster analysis revealed four groups that corresponded to the toxicological profiles of parabens. Our results suggested that disinfection of parabens by chlorination could reduce aquatic toxicity of original compounds. The findings obtained in our study together with the data available on paraben concentrations in aquatic systems can be used to perform preliminary risk assessment by comparing the predicted environmental concentration (PEC) with the predicted no-effect concentration (PNEC) for the marine aquatic environment. The calculated PEC/PNEC ratios ranged from 0.0012 to 0.2, with the highest value observed in MP. This suggested that there are negligible environmental risks for aquatic organisms at current use levels. (c) 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 664-673, 2015.

DOI： 10.1002/tox.21944

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We conducted a battery of toxicity tests using photo bacterium, algae, crustacean and fish to evaluate acute toxicity profile of coking wastewater, and to evaluate the performance of a novel wastewater treatment process, vertical tubular biological reactor (VTBR), in the removal of toxicity and certain chemical pollutants. A laboratory scale VTBR system was set up to treat industrial coking wastewater, and investigated both chemicals removal efficiency and acute bio-toxicity to aquatic organisms. The results showed that chemical oxygen demand (COD) and phenol reductions by VTBR were approximately 93% and 100%, respectively. VTBR also reduced the acute toxicity of coking wastewater significantly: Toxicity Unit (TU) decreased from 21.2 to 0.4 for Photobacterium phosphoreum, from 9.5 to 0.6 for Isochrysis galbana, from 31.9 to 1.3 for Daphnia magna, and from 30.0 to nearly 0 for Danio rerio. VTBR is an efficient treatment method for the removal of chemical pollutants and acute bio-toxicity from coking wastewater.

DOI： 10.1016/j.ecoenv.2015.02.017

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BACKGROUND: The cladoceran crustacean Daphnia pulex produces female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable external stimuli, it produces male offspring (environmental sex determination: ESD). We recently established an innovative system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be controlled simply by changes in the photoperiod: the long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we demonstrated that de novo methyl farnesoate (MF) synthesis is necessary for male offspring production. These results indicate the key role of innate MF signaling as a conductor between external environmental stimuli and the endogenous male developmental pathway. Despite these findings, the molecular mechanisms underlying up- and downstream signaling of MF have not yet been well elucidated in D. pulex. RESULTS: To elucidate up- and downstream events of MF signaling during sex determination processes, we compared the transcriptomes of daphnids reared under the long-day (female) condition with short-day (male) and MF-treated (male) conditions. We found that genes involved in ionotropic glutamate receptors, known to mediate the vast majority of excitatory neurotransmitting processes in various organisms, were significantly activated in daphnids by the short-day condition but not by MF treatment. Administration of specific agonists and antagonists, especially for the N-methyl-D-aspartic acid (NMDA) receptor, strongly increased or decreased, respectively, the proportion of male-producing mothers. Moreover, we also identified genes responsible for male production (e.g., protein kinase C pathway-related genes). Such genes were generally shared between the short-day reared and MF-treated daphnids. CONCLUSIONS: We identified several candidate genes regulating ESD which strongly suggests that these genes may be essential factors for male offspring production as an upstream regulator of MF signaling in D. pulex. This study provides new insight into the fundamental mechanisms underlying how living organisms alter their phenotypes in response to various external environments.

DOI： 10.1186/s12864-015-1392-9

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Various receptor bioassays, including estrogens, androgens and thyroid hormones, have been developed and applied successfully for assessing hormone function in a wide range of animal species, including fish. In fish, corticosteroids play a pivotal role in physiology as they do in mammals, but far less is known about the corticosteroid receptor system in fish compared with in mammals. Here we established a transient transactivation assay using the Japanese medaka, Oryzias latipes, glucocorticoid receptors (olGRs) and mineralocorticoid receptor to analyse their functional properties in a fish. We found that olGR2 was highly responsive to glucocorticoids, similar to the human GR, whereas the olGR1 subtype was minimally responsive. Thus, olGR2 most likely mediates glucocorticoid signaling in medaka. We further tested crosstalk between GRs and other steroid hormones, and found that progestins could activate or inactivate olGR2-mediating transcription, depending on the presence or absence of cortisol. The transactivation assays developed for medaka GRs provide tools to gain useful insights into corticosteroid signaling in fish and for in vitro screening of environmental substances activating GRs.

DOI： 10.1002/jat.3020

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Juvenile hormone (JH) and JH agonists have been reported to induce male offspring production in various daphnid species including Daphnia magna. We recently established a short-term in vivo screening assay to detect chemicals having male offspring induction activity in adult D. magna. Diofenolan has been developed as a JH agonist for insect pest control, but its male offspring induction activity in daphnids has not been investigated yet. In this study, we found that the insect growth regulator (IGR) diofenolan exhibited a potent male offspring induction activity at low ng/L to μg/L concentrations, as demonstrated by the short-term in vivo screening assay and the recently developed TG211 ANNEX 7 test protocol. A two-hybrid assay performed using the D. magna JH receptor confirmed that diofenolan had a strong JH activity. Global whole body transcriptome analysis of D. magna exposed to 10 ng/L diofenolan showed an up-regulation of JH-responsive genes and modulation of several genes involved in the ecdysone receptor signaling pathway. These results clearly demonstrate that diofenolan has strong JH activity and male offspring induction activity, and that a combination of modified standardized regulatory testing protocols and rapid in vitro and in vivo screening assays are able to identify potential endocrine disruptors in D. magna. The observation that diofenolan modulates multiple endocrine signaling pathways in D. magna suggests that further investigation of potential interference with growth, development and reproduction is warranted.

DOI： 10.1016/j.aquatox.2014.11.015

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Juvenile hormone (JH) and JH agonists have been shown to induce male offspring production in various daphnids, including Daphnia magna using OECD TG211. The critical period (about 1h) for JH action on ova in the parent's ovary to induce male offspring is existing at 7-8h later from ovulation. Therefore, we considered that adult D. magna could be used to produce a short-term screening method for detecting JH analogs. Using this method, we successfully demonstrated male offspring induction in the second broods after exposure to JH or JH agonists. After investigating the exposure time, the number of repetitions and the exposure concentration, we established a short-term, in vivo screening method for detecting JH analogs using adult D. magna. We examined positive and negative control chemicals using a previously developed method and verified the validity of our new testing method.

DOI： 10.1002/jat.2989

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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-ovais 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.

DOI： 10.1002/jat.2981

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Language：Japanese   Publisher：Japan Society for Environmental Chemistry  

The current worldwide regulatory guideline requires evaluation of the chronic effects of chemicals on water flea &lt;i&gt;Daphnia magna&lt;/i&gt;. Based on the life cycle of &lt;i&gt;D.magna&lt;/i&gt;, chronic tests with survival and reproductive endpoints requires a 21 days exposure period. &lt;i&gt;Ceriodaphnia dubia&lt;/i&gt;, a common freshwater invertebrate similar to the &lt;i&gt;D.magna&lt;/i&gt;, has a shorter life cycle and therefore reproductive (and survival) endpoints have been established following about 8 days of exposure. No observed effect concentrations for reproduction were obtained for &lt;i&gt;D.magna&lt;/i&gt; and &lt;i&gt;C.dubia&lt;/i&gt; following exposure to general 10 organic compounds (ie. aromatic amines as pharmaceutical, chlorinated aromatic compound, polyaromatic compound and alkyl ester) according to the OECD TG 211 and the biological test method of Environmental Canada. These data were evaluated to determine whether one organism might be considered more sensitive. Reproduction data obtained from &lt;i&gt;C.dubia&lt;/i&gt; chronic test provide similar sensitivity to &lt;i&gt;D.magna&lt;/i&gt; in this study. Based on these data, &lt;i&gt;C.dubia&lt;/i&gt; may be used as a cost-effective alternative and representative invertebrate species when assessing the potential risk on the organic compounds.

DOI： 10.5985/jec.25.55

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Language：Japanese   Publisher：Japan Society for Environmental Chemistry  

The current worldwide regulatory guideline requires evaluation of the chronic effects of chemicals on water flea <i>Daphnia magna</i>. Based on the life cycle of <i>D.magna</i>, chronic tests with survival and reproductive endpoints requires a 21 days exposure period. <i>Ceriodaphnia dubia</i>, a common freshwater invertebrate similar to the <i>D.magna</i>, has a shorter life cycle and therefore reproductive (and survival) endpoints have been established following about 8 days of exposure. No observed effect concentrations for reproduction were obtained for <i>D.magna</i> and <i>C.dubia</i> following exposure to general 10 organic compounds (ie. aromatic amines as pharmaceutical, chlorinated aromatic compound, polyaromatic compound and alkyl ester) according to the OECD TG 211 and the biological test method of Environmental Canada. These data were evaluated to determine whether one organism might be considered more sensitive. Reproduction data obtained from <i>C.dubia</i> chronic test provide similar sensitivity to <i>D.magna</i> in this study. Based on these data, <i>C.dubia</i> may be used as a cost-effective alternative and representative invertebrate species when assessing the potential risk on the organic compounds.

DOI： 10.5985/jec.25.55

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Language：Japanese   Publisher：Japan Society for Environmental Chemistry  

In view of developing a bioassay for use into effluent management system in Japan, short-term toxicity tests using fish, daphnids, and algae were selected as a battery of bioassays, which evaluate the integrated effect of effluent, and "a draft protocol of bioassays for effluent testing" has been prepared. To examine the robustness, sensitivity and reproducibility of the draft protocol, we conducted an inter-laboratory trial where the same effluent was tested by 9 laboratories in Japan. No observed effect concentrations (NOEC) of the test effluent in all the laboratories were within the median +/-1 dilutions, suggesting that the variability between laboratories was at acceptable level. Furthermore, testing of reference chemicals by 10 testing laboratories showed that the reproducibility of the bioassays was relatively higher than that exhibited in the inter-laboratory validation of similar bioassays conducted by US EPA. However, the percentage of minimum significant difference (PMSD) and control coefficient of variation (CV) in the almost all the Japanese laboratories were at acceptable levels (as appears in the US EPA report), indicating that the intra-laboratory precision and the sensitivity of each test was sufficient. We investigated the value of using IC₂₀ for fish, IC₂₅ for daphnids, and IC₅ for algae as an alternative to NOEC. With this approach, toxicity survey of 91 effluent samples conducted from 2008 to 2013 found that 36% of effluents were toxic to at least one of the three test species even after 10-fold dilution. The test species that demonstrated toxic effects were different in each sample; thus, we suggest that effluent toxicity evaluation should be based on more than one species due to different sensitivities.

DOI： 10.5985/jec.25.43

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DOI： 10.1016/j.envsoft.2015.06.013

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Whole Effluent Toxicity (WET) testing evaluates the toxic effect of unknown or multiple substances in wastewater using aquatic organisms. It has already been implement in the US. When the result of WET testing indicates the effect exceeding a certain standard, Toxicity Reduction Evaluation (TRE) and Toxicity Identification Evaluation (TIE) are required for identifying the effluent characteristics causing toxicity and causing the toxicity.<br>In this study, we evaluated effluents from the metal manufacturing plant using three types of aquatic organisms (<i>Danio rerio, Ceriodaphnia dubia,</i> and <i>Pseudokirchneriella subcapitata</i>) with reference to the draft testing methods for effluent using bioassays. The result for one of effluents indicated toxic effect on <i>C. dubia</i>.<br>We conducted chelate resin absorption test and flocculation test as TRE/TIE. As a result, we identified Zn and Ni in the effluent as possible major toxicants and studied improvement of wastewater treatment for reducing toxicity.

DOI： 10.5985/jec.25.35

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Language：Japanese   Publisher：Japan Society for Environmental Chemistry  

In 2009, Ministry of the Environment, Japan, started the discussion about the implementation of bioassay to evaluate and control toxic chemical compounds in effluent and ambient water to complement the conventional chemical analysis of individual compounds, and is aimed to start the new system under Water Pollution Control Act as early as 2016. In this manuscript, the summary of the draft test manual, publicized by National Institute for Environmental Studies (NIES) in 2013, is presented, and then the current status of effluent management in other countries is summarized. There are still several difficulties in implementing the new system using bioassay such as the increase of the cost, the shortage of testing laboratories, the establishment of the benchmark, and toxicity reduction evaluation. Some of the solution measures are presented in addition to the authors' expectation for the implementation of this approach and the contribution of Reference Laboratory in NIES.

DOI： 10.5985/jec.25.3

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Language：Japanese   Publisher：Japan Society for Environmental Chemistry  

Compliance with chemical-based effluent standards does not always ensure ecological risk management of industrial effluent which contains a variety of chemicals used in a chemical manufacturer. To evaluate the biological effects of effluent discharged from our factories, we have applied Whole Effluent Toxicity (WET) test on effluent management system since 2007. Effluent samples were collected at least once a year from four factories and subjected to a battery of bioassays using fish, daphnids, algae, and luminescence bacteria. Our results showed that the most sensitive test species demonstrating toxic effects including annual changes were different for the four effluents. Considering the dilution factor of effluent after discharge, the ecological risk of all samples, which includes the highest toxic effluent from factory D, was assumed negligible in the receiving waters. To reduce effluent toxicity in factory D, we investigated the weekly and daily fluctuation of the effluent toxicity, suggesting that the effluent toxicity was different depending on products. Toxicity assessment of effluent samples collected from each drainage system inside the factory D identified the in-plant source of toxicity, which could be suppressed by reducing the amount of sodium hypochlorite. Moreover, C18 resin treatment in lab-scale reduced the toxicity of the effluent to the daphnids, indicating that at least some of the toxic chemicals were organic compounds.

DOI： 10.5985/jec.25.27

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Language：Japanese   Publisher：Japan Society for Environmental Chemistry  

Short-term chronic toxicity tests were conducted using a battery of three aquatic organisms (fish, daphnia, and alga) for effluents of five selected factories in Tokushima, Japan, and relatively strong toxicity was found for two factories (X and Y) for <i>Ceriodaphnia dubia</i>. The source water, the major processes to discharge toxicants, and their water treatment processes were examined by the interview with effluent managers. For Factory X, source water and the influent of the water treatment facility did not exert strong toxicity for <i>C. dubia</i> while the treated effluent was strongly toxic. As results of bio-assay for two major water additives, the inorganic additive was found to be toxic. The additional bio-assay for the major components aluminum and sulfate, and chemical analysis of these ions identified aluminum ion as a major causative agent. For Factory Y, source water was non-toxic while the influent of the water treatment facility was found to be strongly toxic. As results of interview with the effluent manager, bio-assay and chemical analysis, sulfate was found as a major causative agent. Toxicity reduction measure was under process for both factories using improvement of water treatment processes.

DOI： 10.5985/jec.25.11

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Exposure to estrogenic endocrine disrupting chemicals (EDCs) induces a range of adverse effects, notably on reproduction and reproductive development. These responses are mediated via estrogen receptors (ERs). Different species of fish may show differences in their responsiveness to environmental estrogens but there is very limited understanding on the underlying mechanisms accounting for these differences. We used custom developed in vitro ERα reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17β (E2) were similar to only a 3-fold difference in ERα sensitivity between species. Diethylstilbestrol was the most potent estrogen (∼ 10-fold that of E2) in transactivating the fish ERαs, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ERαs to weaker environmental estrogens varied, and for some considerably. Medaka, stickleback, bluegill and guppy showed higher sensitivities to nonylphenol, octylphenol, bisphenol A and the DDT-metabolites compared with cyprinid ERαs. Triclosan had little or no transactivation of the fish ERαs. By constructing ERα chimeras in which the AF-containing domains were swapped between various fish species with contrasting responsiveness and subsequent exposure to different environmental estrogens. Our in vitro data indicate that the LBD plays a significant role in accounting for ligand sensitivity of ERα in different species. The differences seen in responsiveness to different estrogenic chemicals between species indicate environmental risk assessment for estrogens cannot necessarily be predicted for all fish by simply examining receptor activation for a few model fish species.

DOI： 10.1021/es5002659

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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 mu g l(-1) vinclozolin and 1000 mu g 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 (c) 2013 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.2934

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Daphnia magna has been used extensively to evaluate organism- and population-level responses to pollutants in acute toxicity and reproductive toxicity tests. We have previously reported that exposure to juvenile hormone (JH) agonists results in a reduction of reproductive function and production of male offspring in a cyclic parthenogenesis, D. magna. Recent advances in molecular techniques have provided tools to understand better the responses to pollutants in aquatic organisms, including D. magna. DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to JH agonists: methoprene (125, 250 and 500 ppb), fenoxycarb (0.5, 1 and 2 ppb) and epofenonane (50, 100 and 200 ppb). Exposure to these JH analogs resulted in chemical-specific patterns of gene expression. The heat map analyses based on hierarchical clustering revealed a similar pattern between treatments with a high dose of methoprene and with epofenonane. In contrast, treatment with low to middle doses of methoprene resulted in similar profiles to fenoxycarb treatments. Hemoglobin and JH epoxide hydrolase genes were clustered as JH-responsive genes. These data suggest that fenoxycarb has high activity as a JH agonist, methoprene shows high toxicity and epofenonane works through a different mechanism compared with other JH analogs, agreeing with data of previously reported toxicity tests. In conclusion, D. magna DNA microarray is useful for the classification of JH analogs and identification of JH-responsive genes. Copyright (c) 2013 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.2922

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Androgens play key roles in the morphological specification of male type sex attractive and reproductive organs, whereas little is known about the developmental mechanisms of such secondary sex characters. Medaka offers a clue about sexual differentiation. They show a prominent masculine sexual character for appendage development, the formation of papillary processes in the anal fin, which has been induced in females by exogenous androgen exposure. This current study shows that the development of papillary processes is promoted by androgen-dependent augmentation of bone morphogenic protein 7 (Bmp7) and lymphoid enhancer-binding factor-1 (Lef1). Androgen receptor (AR) subtypes, AR alpha and AR beta, are expressed in the distal region of outgrowing bone nodules of developing papillary processes. Development of papillary processes concomitant with the induction of Bmp7 and Lef1 in the distal bone nodules by exposure to methyltestosterone was significantly suppressed by an antiandrogen, flutamide, in female medaka. When Bmp signaling was inhibited in methyltestosterone-exposed females by its inhibitor, dorsomorphin, Lef1 expression was suppressed accompanied by reduced proliferation in the distal bone nodules and retarded bone deposition. These observations indicate that androgen-dependent expressions of Bmp7 and Lef1 are required for the bone nodule outgrowth leading to the formation of these secondary sex characteristics in medaka. The formation of androgen-induced papillary processes may provide insights into the mechanisms regulating the specification of sexual features in vertebrates.

DOI： 10.1210/en.2013-1507

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A novel measurement system to determine oxygen consumption rates via respiration in migrating Zebrafish (Danio rerio) has been developed. A signal equalization system was adapted to detect oxygen in a chamber with one fish, because typical electrochemical techniques cannot measure respiration activities for migrating organisms. A closed chamber was fabricated using a pipet tip attached to a Pt electrode, and a columnar Vycor glass tip was used as the salt bridge. Pt electrode, which was attached to the chamber with one zebrafish, and Ag electrode were immersed in 10 mM potassium iodide (KI), and both the electrodes were connected externally to form a galvanic cell. Pt and Ag electrodes act as the cathode and anode to reduce oxygen and oxidize silver, respectively, allowing the deposition of insoluble silver iodide (AgI). The AgI acts as the signal source accumulated on the Ag electrode by conversion of oxygen. The amount of AgI deposited on the Ag electrode was determined by cathodic stripping voltammetry. The presence of zebrafish or its embryo led to a decrease in the stripping currents generated by a 10 min conversion of oxygen to AgI. The conversion of oxygen to AgI is disturbed by the migration of the zebrafish and allows the detection of different equalized signals corresponding to respiration activity. The oxygen consumption rates of the zebrafish and its embryo were estimated and determined to be similar to 4.1 and 2.4 pmol.s(-1), respectively. The deposited AgI almost completely disappeared with a single stripping process. The signal equalization system provides a method to determine the respiration activities for migrating zebrafish and could be used to estimate environmental risk and for effective drug screening.

DOI： 10.1021/ac402962f

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna, these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments.
Results: We found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds) RNA specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene.
Conclusions: We developed a microinjection system for RNAi studies in D. pulex. These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.

DOI： 10.1186/1472-6750-13-96

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Acute and chronic (or sub-chronic) toxicity of five selected antimicrobial agents, including triclosan (TCS), triclocarban (TCC), resorcinol, phenoxyethanol and p-thymol, was investigated using the conventional three-aquatic-organism battery. These compounds are widely used in cosmetics and other personal care products and their ecological risk has recently become a significant concern. As results of toxicity tests, TCS was found to be most strongly toxic for green algae [e.g. 72h no observed effect concentration (NOEC) of 0.50 mu gl(-1)] among the selected compounds, followed by TCC, while TCC was more toxic or similar to TCS for Daphnia and fish (e.g. Daphnia 8day NOEC of 1.9 mu gl(-1)). Having compared the predicted no effect concentration (PNEC) determined from the toxicity data with measured environmental concentrations (MEC), the preliminary ecological risk assessment of these five antimicrobials was conducted. The MEC/PNEC ratios of TCS and TCC were over 1 for some monitoring data, especially in urban streams with watershed areas without sewage service coverage, and their potential risk for green algae and Daphnia might be at a level of concern, although the contribution of TCS/TCC on the total toxicity of the those sites needs to be further investigated. For the three other antimicrobials, the maximum MEC/PNEC ratio for resorcinol was 0.1-1, but those for phenoxyethanol and p-thymol were &lt;0.1 and their risk to aquatic organisms is limited, although the additive effects with TCS, TCC and other antimicrobial agents, such as parabens, need to be further examined in future studies. Copyright (c) 2012 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.2771

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Ltd  

Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRβ) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3: 10-11 M in TRα and 10-10 M in TRβ) and thyroxine (T4: 10-9 M in TRα and 10-8 M in TRβ). Analogs of thyroid hormone (3,5,3′,-triiodothyroacetic acid and 3,3′,5,5′-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4. These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3-stimulated transcriptional activity of the O. latipes TRα was inhibited by 10-5 M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10-5 M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation. © 2012 John Wiley &amp
Sons, Ltd.

DOI： 10.1002/jat.2825

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Juvenile hormone is an essential regulator of major developmental and life history events in arthropods. Most of the insects use juvenile hormone III as the innate juvenile hormone ligand. By contrast, crustaceans use methyl farnesoate. Despite this difference that is tied to their deep evolutionary divergence, the process of this ligand transition is unknown. Here we show that a single amino-acid substitution in the receptor Methoprene-tolerant has an important role during evolution of the arthropod juvenile hormone pathway. Microcrustacea Daphnia pulex and D. magna share a juvenile hormone signal transduction pathway with insects, involving Methoprene-tolerant and steroid receptor coactivator proteins that form a heterodimer in response to various juvenoids. Juvenile hormone-binding pockets of the orthologous genes differ by only two amino acids, yet a single substitution within Daphnia Met enhances the receptor's responsiveness to juvenile hormone III. These results indicate that this mutation within an ancestral insect lineage contributed to the evolution of a juvenile hormone III receptor system.

DOI： 10.1038/ncomms2868

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Background: The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication
therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species.Results: We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5' upstream regulatory elements are preserved in D. magna and D. pulex.Conclusions: The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans. © 2013 Toyota et al.
licensee BioMed Central Ltd.

DOI： 10.1186/1471-2164-14-239

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ERa, ER beta 1 and ER beta 2) and applied an estrogen-responsive (ERE)-luciferase reporter assay system to characterize the interactions of these receptors with steroidal and synthetic estrogens. DNA fragments encoding all three ERs in carp, ERa, ER beta 1 and ER beta 2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% (a vs beta 1), 49% (a vs beta 2) and 53% (beta 1 vs beta 2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cER beta 2 showed a higher sensitivity to the natural steroid oestrogen, 17 beta-estradiol, than cERa. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptorenvironmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution. Copyright (C) 2011 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.1707

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV HAWAII PRESS  

Life history and growth of the surf clam, Mactra veneriformis, were examined in a tidal flat area of Tokyo Bay from November 2002 to November 2004. Gonads of both sexes began to develop in March, and animals with mature gonads (ripe phase) were observed from spring to summer (April August). Most of the animals showed signs of having finally spawned by September, indicating that spawning occurs from spring to summer. Moreover new cohorts were established several times in both years, with the largest settlement occurring in July. These findings suggest that surf clams in this area spawn several times a year. By monitoring increase in shell length of these cohorts, we found that shell growth was rapid in the first year, reaching approximately 35-40 mm 1 yr after settlement. In the second year, shell lengths exceeded 40 mm. Most of the animals died after the spawning season in the second year. The observation of several settlement events per year and a short life span differed considerably from characteristics of M. veneriformis along the Korean coast.

DOI： 10.2984/66.3.7

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The occurrence of oocytes in the testis (testis-ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis-ova remain to be elucidated. To develop marker genes for detecting testis-ova in the testis, adult male medaka were exposed to nominal concentration of 100 ng L-1 of 17 alpha-ethinylestradiol (EE2) for 3-5 weeks, and 800 ng estradiol benzoate (EB) for 3 weeks (experiment I), and a measured concentration of 20 ng L-1 EE2 for 1-6 weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis-ova were induced in the testis after 4 weeks of exposure to 100 ng L-1 EE2, 3 weeks of exposure to 800 ng EB, and 6 weeks of exposure to 20 ng L-1 EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis-ova in male medaka. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2011.12.047

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Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ER alpha activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17 alpha-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ER alpha was found to be the most responsive and carp and stickleback ER alpha the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ER alpha-mediated response sensitivity order of zebrafish &gt; medaka &gt; roach &gt; fathead minnow &gt; carp &gt; stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ER alpha activation assays)&gt; zebrafish &gt; fathead minnow &gt; medaka &gt; roach &gt; stickleback &gt; carp. Overall, the responses to steroid estrogens in vitro via ER alpha compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ER alpha-mediated responses indicative of estrogenic responses (VTG induction) in vivo. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquatox.2011.09.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

In the present study, aquatic concentrations of seven parabens were determined in urban streams highly affected by treated or untreated domestic sewage in Tokushima and Osaka, Japan. The detected highest concentrations were 670, 207, and 163 ng l(-1) for methylparaben, n-propylparaben, and n-butylparaben, respectively in sampling sites with watershed area of no sewer system in Tokushima. Conventional acute/chronic toxicity tests were conducted using medaka (Oryzias latipes), Daphnia magna, and Psuedokirchneriella subcapitata for four parabens, which was consistent with our previous study on three parabens, n-butylparaben, butylparaben, and benzylparaben. The aquatic toxicity on fish, daphnia, and algae was weaker for the parabens with a shorter alkyl chain than those with a longer alkyl chain as predicted by their hydrophobicity. Medaka vitellogenin assays and DNA microarray analysis were carried out for methylparaben and found induction of significant vitellogenin in male medaka at 630 mu g l(-1) of methylparaben, while the expression levels of genes encoding proteins such as choriogenin and vitellogenin increased for concentrations at 10 mu g l(-1) of methylparaben. Measured environmental concentrations (MECs) of seven parabens in Tokushima and Osaka were divided by predicted no effect concentrations (PNECs) and hazard quotient (MEC/PNEC) was determined for individual parabens. The MEC/PNEC was highest for n-propylparaben and was 0.010 followed by n-butylparaben (max. of 0.0086) and methylparaben (max. of 0.0042). The sum of the MEC/PNEC for the seven parabens was 0.0049. Equivalence factors were assigned for each paraben on the basis of the toxicity of n-butylparaben for each species, and n-butylparaben equivalence was calculated for the measured environmental concentrations. The MEC/PNEC approach was also conducted for the n-butyl-paraben-based equivalence values. The maximum MEC/PNEC was 0.018, which is lower than the trigger level for further detailed study such as large-scale monitoring for chronic toxicity tests including full-life cycle tests for fish. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scitotenv.2011.09.040

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Nanoparticles are particles with diameters of 100 nm or less. As the applications of these particles have increased in recent years, their potential impact on the physiology of humans and animals has also increased. However, little is known regarding the effect of nanoparticles on the physiology of aquatic organisms. In this study, we investigated the effect of nano-sized, fluorescent, latex particles on the freshwater fish, medaka (Oryzias latipes). Medakas were exposed to four different types of fluorescent latex particles and the uptake, excretion, and the effect of nanoparticle accumulation on survival rate in medaka larvae were examined. These are fluorescent latex particles, which are non-functionalized 50 and 500 nm in diameter and carboxyl-group functionalized 50 and 500 nm in diameter. Fluorescence intensity in fish embryos exposed to non-functionalized and carboxyl-group functionalized particles measuring 50 nm in diameter (Particle 50 nm and Particle c50 nm) was markedly higher compared to when embryos were exposed to particles measuring 500 nm in diameter (Particle 500 nm and Particle c500 nm). Moreover, the excretion of nano-sized particles (Particle 50 and Particle c50 nm) from embryos was considerably slow, compared to larger particles (Particle 500 and Particle c500 nm). In addition, the survival rate of larvae exposed to nano-sized particles in small cups was significantly lower than the survival rates of fish maintained in larger containers. The findings suggested that although the nano-sized fluorescent latex particles were not intrinsically toxic, a synergistic toxic effect arose in combination with other factors, which is not favorable for fish larvae. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquatox.2011.08.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Terpenoid hormones in insects (i e juvenile hormones) have various effects on physiology morphology and behavior producing a wide range of phenotypic variation Recent studies have shown that sex determination in cladoceran crustaceans is under the strong control of a major terpenoid hormone of crustaceans methyl farnesoatote (MF) It can be easily conceived that MF is also a major determinant of other traits in cladocerans In the present study morphological changes known as antipredatory responses in a cladoceran Daphnia galeata in response to exposure to MF and a juvenile hormone mimicking pesticide fenoxycarb were investigated Morphological change was studied using neonates less than 24 h old exposed either to MF at the concentrations from 1 9 to 30 mu g/L or fenoxycarb at the concentrations from 13 to 200 ng/L for 6 d Animals developed a longer helmet at I 9 mu g/L of MF and 25 ng/L of fenoxycarb and showed a concentration dependent elongation However the tail spine was reduced in size in a concentration dependent manner Results of the present study not only give new insight into the mechanisms of inducible defenses in cladocerans but also provide invaluable information to understand ecological and evolutionary consequences of endocrine disruption through the shift in biological interaction between predator and prey Environ Toxicol Chem 2011 30 232-238 (C) 2010 SETAC

DOI： 10.1002/etc.378

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPEKTRUM AKAD VERLAG  

In October 2010, a group of experts met as part of the transatlantic think tank for toxicology (t(4)) to exchange ideas about the current status and future of safety testing of nanomaterials. At present, there is no widely accepted path forward to assure appropriate and effective hazard identification for engineered nanomaterials. The group discussed needs for characterization of nanomaterials and identified testing protocols that incorporate the use of innovative alternative whole models such as zebrafish or C. elegans, as well as in vitro or alternative methods to examine specific functional pathways and modes of action. The group proposed elements of a potential testing scheme for nanomaterials that works towards an integrated testing strategy, incorporating the goals of the NRC report Toxicity Testing in the 21(st) Century: A Vision and a Strategy by focusing on pathways of toxic response, and utilizing an evidence-based strategy for developing the knowledge base for safety assessment. Finally, the group recommended that a reliable, open, curated database be developed that interfaces with existing databases to enable sharing of information.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Environmental decline triggers a switch in reproductive strategy of Daphnia magna from asexual to sexual reproduction; however, the molecular basis of such environmental sex determination remains largely unknown. In insects most closely related to branchiopod crustaceans, orthologs of the Drosophila transformer gene such as dipteran transformer (tra), honeybee feminizer (fern) and complementary sex determiner (csd) function as sex determining factors. Therefore, we cloned a D. magna transformer gene (dmagtra) and analyzed its expression. The predicted amino acid sequence has 380 amino acids including an arginine serine-rich region, which is characteristic of insect orthologs of Tra. Residues 180 to 205 are highly conserved with known Tra orthologs. The domain organization of DmagTra is distinct from known Tra orthologs; moreover, dmagtra does not display any detectable sexual dimorphic differences in expression or splicing patterns. We infer from these results that dmagtra may not be responsible for sex determination in D. magna. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygeno.2009.12.005

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Language：English   Publisher：Japan Society for Environmental Chemistry  

We examined the influence of combination chemical exposure on estrogenic activity, <I>in vivo</I> using Japanese medaka (<I>Oryzias latipes</I>), by a continuous-flow exposure system for six dayexposure period. We co-exposed the fish to various combination of a CYP1A inducer &beta;-naphthoflavone (&beta;NF) and <I>trans</I>-stilbene (t-S) which is known as a proestrogen that acquires estrogenic activity through biotransformation. We measured blood VTG concentration and EROD activity as indices of the exposure effects. We found that t-S induced both blood VTG and EROD activity itself, but inhibited &beta;NF induced EROD activity when co-exposed with &beta;NF. In contrast, &beta;NF induced EROD activity with no alteration to blood VTG induction itself, and inhibited t-S induced blood VTG when co-exposed with t-S. The results of present study indicate that the combined exposure to a CYP1A inducer and a proestrogen that is known to be estrogenically active by CYP1A does not necessarily increase the estrogenic activity in vivo. Moreover, the results of present study also suggest that blood VTG inhibition can be mediated by factors other than aryl hydrocarbon receptor.

DOI： 10.5985/jec.19.371

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LTD  

The acute toxicity of 21 parabens and their chlorinated derivatives was investigated by means of two toxicity bioassays: Daphnia magna immobilization test and the inhibition of bioluminescence of Vibrio fischeri. The median effective concentration (EC(50)) values of the tested parabens ranged from 2.2 to 62 mg I(-1) in the D. magna test and from 0.0038 to 5.9 mg I(-1) in the V. fischeri test at 15 min after exposure. The toxicity of dichlorinated methyl- and n-propylparaben, the most commonly used preservatives in cosmetics, toward D. magna was 3.9- and 2.8-fold that of their corresponding parent compounds. Toxicity toward D. magna showed a linear relationship with log P, indicating that toxicity increases with increasing hydrophobicity. On the other hand, the correlations of toxicity toward V. fischeri with hydrophobicity and with the degree of chlorination were poor. In addition, the results of the present study indicated that the V. fischeri test was more sensitive than the D. magna test for the determination of the acute toxicity of parabens. A complete assessment of the ecological and toxicological risks of parabens may require the examination of chlorinated parabens as well as the parent pollutants, as described in the present study. Copyright (C) 2008 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.1402

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We evaluated the effect of combination chemical exposure on estrogenic activity, in vivo using Japanese medaka (Oryzias latipes) during co-exposure to estrone (N) and beta-naphthoflavone (beta NF) or alpha-naphthoflavone (alpha NF). We used E1 because which is one of the major estrogenic substances in the environment, and beta NF or alpha NF are both AhR-ligands and usually used as a model substance of common environmental pollutants such as PAHs from physiological response standpoint. The results Of present study demonstrate that both beta NF and alpha NF inhibit the E1-induced production of plasma VTG in a concentration-dependent manner. These results indicate that estrogenic activity is Suppressed by AhR-ligands in aquatic environment. At the same time, significant increase of EROD was observed in groups of beta NF co-exposed with E1, conversely no significant alteration of EROD was observed in groups of alpha NF co-exposed with E1. Therefore, we conclude that the inhibition of VTG synthesis effect was not directly related to EROD. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.etap.2008.09.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Chemicals released into the environment have the potential to affect various species and it is important to evaluate such chemical effect on ecosystems, including aquatic organisms. Among aquatic organisms, Daphnia magna has been used extensively for acute toxicity or reproductive toxicity tests. Although these types of tests can provide information on hazardous concentrations of chemicals, they provide no information on their mode of action. Recent advances in toxicogenomics, the integration of genomics with toxicology, have the potential to afford a better understanding of the responses of aquatic organisms to pollutants. In a previous study, we developed an oligonucleotide-based DNA microarray with high reproducibility using a Daphnia expressed sequence tag (EST) database. In this study, we increased the number of genes on the array and used it for a careful ecotoxicogenomic assessment of Daphnia magna. The DNA microarray was used to evaluate gene expression profiles of neonate daphnids exposed to beta-naphthoflavone (bNF). Exposure to this chemical resulted in a characteristic gene expression pattern. As the number of the genes on an array was increased, the number of genes that were found to respond to the chemicals was also increased, which made the classification of the toxic chemicals easier and more accurate. This newly developed DNA microarray can be useful for a obtaining a better mechanistic understanding of chemical toxicity effects on a common freshwater organism.

DOI： 10.1007/s10565-008-9108-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Daphnia magna is known to switch between sexual and asexual reproduction depending on the environment. It reproduces asexually when in an optimal environment for food, photoperiod, and population density. Once the environment declines, it changes reproductive strategy from asexual to sexual reproduction. However, the molecular bases of environmental sex determination are largely unknown. To understand the molecular mechanisms of environmental sex determination in Daphnia, it is essential to isolate the genes related to sex determination. As DM-domain genes are well known as sex-related genes, we aimed to identify DM-domain genes from Daphnia. Based on degenerate PCR of conserved DM domains using Daphnia cDNA, we identified three DM-domain genes that corresponded to DMRT11E, DMRT93B, and DMRT99B of Drosophila melanogaster Quantitative gene expression analysis in gonads revealed that DMRT93B was expressed only in the testis. This finding contributes to an improved understanding of the switching mechanism from an asexual to a sexual life cycle depending on the environment. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygeno.2007.09.002

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Recent detection of fluoxetine in the aquatic environment and fish suggests a possibly high accumulation of fluoxetine; however, no report is available on the bioaccumulation of fluoxetine in aquatic organisms. Since bioaccumulation of fluoxetine was probably dependent on pH near the pK(a) value of 10.1, experiments were conducted approximately at pH 7, 8, and 9. Distribution coefficients between I octanol and water (D-ow), and those between synthetic membrane vesicles (liposomes) and water (Dlip-wat)were determined at pH 7, 8, and 9. The D-ow and Dlip-wat values increased significantly with increasing pH. Acute toxicity tests were performed using Japanese medaka (Oryzias latipes) prior to the bioaccumulation test, and 96-h LC50 values were 5.5, 1.3, and 0.20 mg l(-1) at pH 7, 8, and 9, respectively. In the bioaccumulation test, concentrations of fluoxetine and its major metabolite, norfluoxetine, in the fish body and liver were measured. The bioconcentration factors (BCF) of fluoxetine for Japanese medaka were 8.8, 3.0 x 10, and 2.6 x 10(2) in the body and 3.3 x 10(2), 5.8 x 10(2), and 3.1 X 10(3) in the liver at pH 7, 8, and 9, respectively. The BCF values were lower at pH 7 and higher at pH 9 mainly because of the increase in nonionized species with significantly higher hydrophobicity than the ionized species at pH values closer to pKa. A similar trend was obtained for the concentration of norfluoxetine in the fish but the pseudo-BCF values (the ratio of the norfluoxetine concentration in the fish and the fluoxetine concentration in test water) were higher than the BCF value of fluoxetine at all pH conditions. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2007.06.089

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Language：Japanese   Publisher：Japan Society for Environmental Chemistry  

The luminescent <i>umu</i> test was examined as a technique for monitoring genotoxic agents in river water. The specimen was condensed using a C18 filter and the extract was fractionated into three using a florisil column. When this method was applied to a specimen from the Ayase River, detection at a sensitivity level higher than that of the micro-suspension method was accomplished. This method was applied to specimens from 20 rivers in Japan. The majority of activity was obtained from 10 % acetone/dichloromethane elution fraction, and specific activity could be calculated on 18 specimens. The maximum value of specific activity was 1709 count/m<i>l</i> in a metabolic activating enzyme non-added system and 825 count/m<i>l</i> in a metabolic activating enzyme added system. No correlation was observed among the genotoxicity, total organic carbon, total nitrogen and total phosphorous of river water. This method can also be applied to multi-sample specimens, and is considered to be a method appropriate for monitoring.

DOI： 10.5985/jec.17.453

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Acute and reproductive toxicity tests were conducted on seven strains of Daphnia magna from six laboratories in five countries. 3,4-Dichloroaniline (DCA) and fenoxycarb were used as test chemicals. Acute toxicity tests revealed that estimated EC50 (50% effective concentration) values for DCA varied by a factor of 2.1 among strains (310-640 mu g/L), whereas the EC50 values for fenoxycarb varied by a factor of 4 (210-860 mu g/L). EC50 values for reproductive toxicity tests with DCA ranged from 5.9 to 38 mu g/L among strains. Fenoxycarb exposure induced the production of male neonates in all the strains used in the present study. Estimated EC50 values for the induction of male offspring were highly variable among strains: sensitivity to fenoxycarb differed by a factor of approximately 23 overall (0.45-10 mu g/L). The present pre-validation tests suggest that induction of male sex in neonates by a juvenile hormone analog is universal among genetically different strains. Decreased total numbers of neonates at increased concentrations of fenoxycarb as well as other Juvenoids may, however, obscure the incidence of male neonates production in the 21-day reproduction tests due to the low statistical power. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ecoenv.2006.12.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY ENVIRONMENTAL TOXICOLOGY & CHEMISTRY-SETAC  

Toxic chemical contaminants have a variety of detrimental effects on various species, and the impact of pollutants on ecosystems has become an urgent issue. However, the majority of studies regarding the effects of chemical contaminants have focused on vertebrates. Among aquatic organisms, Daphnia magna has been used extensively to evaluate organism- and population-level responses of invertebrates to pollutants in acute toxicity or reproductive toxicity tests. Although these types of tests can provide information concerning hazardous concentrations of chemicals, they provide no information about their mode of action. Recent advances in molecular genetic techniques have provided tools to better understand the responses of aquatic organisms to pollutants. In the present study, we adapted some of the techniques of molecular genetics to develop new tools, which form the basis for an ecotoxicogenomic assessment of D. magna. Based on a Daphnia expressed sequence tag database, we developed an oligonucleotide-based DNA microarray with high reproducibility. The DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to several different chemicals: Copper sulfate, hydrogen peroxide, pentachlorophenol, or P-naphthoflavone. Exposure to these chemicals resulted in characteristic patterns of gene expression that were chemical-specific, indicating that the Daphnia DNA microarray can be used for classification of toxic chemicals and for development of a mechanistic understanding of chemical toxicity on a common freshwater organism.

DOI： 10.1897/06-075R.1

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cDNAs encoding the ecdysone receptor (EcR) and ultraspiracle (USP) protein were cloned from the water flea Daphnia magna (Crustacea: Cladocera). The deduced EcR and USP amino acid sequences showed a high degree of homology to those of other crustaceans as well as insects. We isolated three isoforms of EcR that differ in the A/B domain. Quantitative PCR analysis indicated differing temporal expression patterns of the EcR isoforms during the molting period and demonstrated that the expression of one subtype correlated well with the timing of molt. Using cDNAs encoding EcR and USP we constructed a Daphnia EcR/USP reporter based on a two-hybrid system. The gene fusions encoded the EcR ligand-binding domain (LBD) fused to the Gal4 DNA-binding domain, and the USP-LBD fused to the Vp16 activation domain. These! chimeric genes were transfected with a luciferase reporter gene. Dose-dependent activation of the reporter gene could be observed when transfectants were exposed to Ec and other chemicals known to have Ec-like activities. This two-hybrid system may represent a useful reporter system for further examination of hormonal and chemical effects on Daphnia at the molecular level.

DOI： 10.1677/JOE-06-0228

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The water flea Daphnia magna (Crustacea, Cladocera) is a cyclical parthenogen, which can reproduce both by parthenogenesis and by sexual reproduction. With its ease of handling in the laboratory, several testing methods using D. magna exist for regulatory toxicity testing. Recently, several studies revealed that one of the major hormone groups in insects and crustaceans, juvenile hormones, are involved in the shift of reproductive mode from parthenogenesis to sexual reproduction (production of male neonates). Using offspring sex ratio as a new endpoint has made it possible to identify chemicals with juvenile hormone-like effects on crustaceans. The testing method using D. magna, in which offspring sex ratio is incorporated as a new endpoint, is now being proposed to the OECD as an enhanced version of the existing OECD Test Guideline 211: Daphnia magna reproduction test. No other clear-cut endpoint for identifying juvenile-hormone disrupting effects has ever been found in crustaceans than the induction of male neonates production in cladocerans. In this regard, it is expected that testing methods using D. magna are suitable for screening and risk assessment of chemicals with juvenile-hormone disrupting effects.

DOI： 10.1007/s10646-006-0120-2

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Eight pharmaceuticals were selected on the basis of their domestic consumption in Japan, the excretion ratio of the parent compound and the frequency of detection in the aquatic environment or wastewater treatment plant effluent. Toxicity tests on these pharmaceuticals were conducted using Japanese medaka (Oryzias latipes), daphnia (Daphnia magna), and green algae (Psuedokirchneriella subcapitata). Predicted no effect concentration (PNEC) was calculated using lethal or effect concentration 50 (LC50 or EC50) values and no effect concentration (NOEC) obtained in the toxicity tests for these compounds. Predicted environmental concentration (PEC) was also calculated from annual consumption, the excretion rate of the parent compound, and removal rate in the preliminary batch activated sludge treatment performed in this study. Maximum concentrations found in the aquatic environment or sewage effluent in Japan or foreign countries were also used for another calculation of PEC. Initial risk assessment on the selected pharmaceuticals was performed using the PEC/PNEC ratio. The results of initial risk assessment on the eight selected pharmaceuticals suggest neither urgent nor severe concern for the ecological risk of these compounds, but further study needs to be conducted using chronic toxicity tests, including reproduction inhibition and endocrine disruption assessments.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Two groups of Japanese medaka (Oryzias latipes) were exposed to 17 beta-estradiol (E2: 150 ng/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. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquatox.2006.06.018

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We studied the susceptibility of three genetically different strains of the cyclical parthenogen Daphnia magna (Cladocera, Crustacea) in producing male neonates following exposure to juvenile hormone analogs. In experiment 1, NIES, Clone A, and Belgium A strains were exposed to the insect growth regulators (IGRs) fenoxycarb or epofenonane in a 21-day reproduction experiment. Fenoxycarb exposure decreased the total number of neonates and increased production of male neonates in a concentration-dependent manner in the NIES strain. The decrease in the total number of neonates was so great in Clone A following fenoxycarb exposure that male neonates were not observed, even at the highest concentration, where the total number of neonates was only 2% of the control. In the Belgium A strain, male neonates were observed at a rate of about 20% following exposure to the highest fenoxycarb concentration, but the total number observed was small. Epofenonane did not decrease reproduction in the NIES and Belgium A strains as dramatically as did fenoxycarb, but the neonatal sex ratio changed in a concentration-dependent manner. Although the ratio of males was as low as about 10%, induction of male neonates was also observed in Clone A following epofenonane exposure. In experiment 2, gravid females were exposed to high concentrations (5 or 10 mu g/1) of fenoxycarb or pyriproxyfen for 12 h. These treatments induced the production of male neonates in all strains, with a small decrease in the total number of neonates. Although induction of male neonates by juvenile hormones and their analogs was universal among genetically different strains, care is needed in interpreting the results of the 21-day reproduction tests, because decreased numbers of neonates at higher concentrations could obscure the presence of male neonates. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2005.09.026

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To investigate the environmental safety of waste disposal landfill sites and of land reclaimed from such sites, we evaluated the toxicity of leachate from these sites by a combination of bioassays in the Japanese killifish medaka Oryzias latipes. We tested for lethal toxicity in adult and larval medaka and for hatching inhibition of embryos from eggs. As biochemical evidence of the effects of leachate exposure, CYP1A (EROD activity) and vitellogenin (Vtg) were induced. We also bioassayed water-treated leachate and downstream river water. Leachate solution was lethal to larval and adult medaka. Embryo hatchability was inhibited, and abnormal hatching, spinal deformity and anisophthalmia occurred in embryos exposed to leachate solution. CYP1A was induced by exposure to leachate solution diluted to 1.0%, and EROD activity was significantly higher than in control. Vtg and unknown proteins were induced in the sera of male medaka exposed to the diluted leachate solution. Conventional water treatments worked effectively to remove toxic compounds but did not work well to remove element ions, including heavy metals. Treated leachate produced neither lethal toxicity nor hatching abnormalities during the exposure period. Fish toxicity tests for leachate would be useful for monitoring the environmental safety of landfill sites.

DOI： 10.1007/s10661-006-7670-x

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Gonad histological changes were examined in Japanese medaka exposed to 17 beta-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 mate 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 17 beta-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. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquatox.2005.11.001

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Bioconcentration factors (BCF) for pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP) in Japanese medaka (Oryzias latipes) were determined at five different concentrations of the chemicals, between 0.1 and 10 mu g/l (PCP), 0.3 and 30 mu g/l (2,4-DCP), in the ambient water. Medaka were exposed to each chemicals in a continuous-flow system during the embryonic development period and 60 days after hatching from eggs collected in the laboratory. Both the exposure time and the aqueous concentrations are much more realistic and closer to natural aquatic environments than those used in conventional BCF studies.
The BCF values of PCP were from (4.9 +/- 2.8) x 10(3) at the aqueous concentration of 0.074 +/- 0.028 mu g/l to (2.1 +/- 1.4) x 10(3) at 9.70 +/- 0.56 mu g/l. The BCF value of 2,4-DCP were from (3.4 +/- 3.0) x 10(2) at 0.235 +/- 0.060 mu g/l to 92 +/- 27 at 27.3 +/- 1.6 mu g/l. Generally, BCF values increased as the aqueous concentrations of PCP or 2,4-DCP decreased. This finding suggests that a relatively low and realistic aqueous concentration of these compounds is necessary to more accurately determine their BCF values in natural aquatic environments. Conventional BCF experiments at higher aqueous concentrations may underestimate the BCF values. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2005.03.058

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We exposed the water flea Daphnia magna (Cladocera, Crustacea) to either juvenile hormone I (JH I), juvenile hormone II (JH II), or the juvenile hormone-mimicking insecticides kinoprene, hydroprene, epofenonane, or fenoxycarb. By 21-day reproduction tests, we investigated the effects on the number of neonates born per female and the offspring sex ratio. All six chemicals induced D. magna to produce male neonates; the male sex ratio of the offspring increased as the chemical concentration increased. EC50 values for production of male neonates were estimated as 400 (JH I), 410 (JH II), 190 (kinoprene), 2.9 (hydroprene), 64 (epofenonane), and 0.92 (fenoxycarb) mu g/l. The number of neonates produced was reduced with all chemicals at the concentrations investigated. At the EC50 for male production, five of the six chemicals reduced the reproductive rate to less than 50%; the exception was epofenonane, which caused only a slight reduction in reproductive rate. These results were similar to those obtained for five juvenoids studied previously, one of which was studied here again. There are now 10 chemical substances-all juvenile hormones or their analogs-that are known to induce D. magna to produce male neonates. This suggests that juvenile hormone is involved in initiating male production followed by sexual reproduction in D. magna, and probably in most cladocerans that exhibit cyclic parthenogenesis. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2005.02.075

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL RESEARCH COUNCIL CANADA  

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.

DOI： 10.1139/G05-038

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To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.

DOI： 10.1139/G05-038

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Previous studies have found that exposure of a cyclic parthenogen, the water flea Daphnia magna (Cladocera, Crustacea), to juvenile hormones and their analogs results in the production of neonates of male sex at concentration-dependent rates. We conducted reproduction experiments in four different species (Moina macrocopa, M. micrura, Ceriodaphnia dubia and C reticulata) of cladoceran to test for the first time whether the occurrence of this phenomenon after exposure of the parent to such hormones is a generalized phenomenon. In the presence of a juvenile hormone analog, fenoxycarb, all four species produced male neonates and showed reduced rates of reproduction. The estimated median effective concentration (EC50) for the production of male neonates varied with species, ranging from 0.60 x 10(3) to 9.3 x 10(3) ng/l. Although there was a wide range of sensitivity to fenoxycarb, the production of male neonates in all four species demonstrates that this phenomenon is a common response to juvenile hormone analogs and further suggests that these hormones are capable of initiating sexual reproduction in cladocerans, most of which exhibit cyclic parthenogenesis. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2004.12.080

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Language：English   Publisher：The Pharmaceutical Society of Japan  

An enzyme-linked immunosorbent assay (ELISA) was validated for the measurement of vitellogenin (VTG) in the cyprinid model species, medaka (<I>Oryzias latipes</I>). Both polyclonal antibody- and monoclonal antibody-based kits showed very high correlation between VTG values in unknown samples when we used standardized VTG protein. This was confirmed by 5 laboratories which participated in the ring test. The use of one gold standard VTG is essential to measure VTG values using ELISA kits in future OECD ring tests to screen for estrogenic chemicals in the 3 fish species medaka, zebrafish and fathead minnow. <br>

DOI： 10.1248/jhs.50.301

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The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 mug/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 mug of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 mug of NP/ L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 mug/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 mug/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.

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Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is widely used as an antibacterial agent in various industrial products, such as textile goods, soap, shampoo, liquid toothpaste and cosmetics, and often detected in wastewater effluent. However, there is a paucity of data on the toxicity of triclosan and its effects on aquatic organisms. In this study, the acute toxicity of triclosan to the Microtox bacterium (Vibrio fischeri), a microalga (Selenastrum capricornutum), a crustacean (Ceriodaphnia dubia) and fish (Danio rerio and Oryzias latipes) was examined. As a result, the MicrotoxR bacterium, crustacean and fish had similar sensitivities towards triclosan toxicity (i.e., IC25 from 0.07 to 0.29 mg/L triclosan). In contrast, the microalga was about 30-80-fold (IC25 = 0.0034 mg/L triclosan) more sensitive to triclosan toxicity than the bacterium and fish. Therefore, triclosan is quite highly toxic to aquatic animals, and is particularly highly toxic to the green alga used as a test organism in this study. This result indicates that triclosan exerts a marked influence on algae, which are important organisms being the first-step producers in the ecosystem; therefore, the possible destruction of the balance of the ecosystem is expected if triclosan is discharged into the environment at high levels.

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The water flea Daphnia magna reproduces primarily by cyclic parthenogenesis. Environmental stimuli that signal a change to adverse conditions induce the organisms to switch from parthenogenesis to gamogenetic reproduction. During the gamogenetic period, they produce male daphnids and dormant resting eggs, which can survive prolonged periods of environmental adversity. However, little is known about the mechanisms associated with the switch from parthenogenesis to gamogenetic reproduction. We investigated the effects of several juvenoids on sex determination in Daphnia. Females less than 24 h old were exposed to various concentrations of the test substance and were observed for 21 days. It was found that they can trigger the appearance of male daphnids: the percentage of males in the population increases to a level greater than what occurs under ordinary environmental conditions. We found that methylfarnesoate, juvenile hormone III, methoprene, and the phenoxyphenoxy derivatives pyriproxyfen and fenoxycarb (both insecticides) reduced the production of offspring and produced sex ratios dominated by male daphnids. Pyriproxyfen and fenoxycarb showed striking effects at low concentrations. Exposure to either of these chemicals at a concentration of 330 ng I-1 caused adult females to produce almost all male neonates. Methylfarnesoate, juvenile hormone III, and methoprene showed an effect in inducing male production at higher concentrations (3.7x 10(3), 3.3x10(5), and 1.3x10(5) ng1(-1), respectively). Our findings suggest that juvenile hormone agonists, including some insecticides, affect the chemical signaling responsible for inducing the production of male offspring. (C) 2003 Elsevier Ltd. All rights reserved.

DOI： 10.1016/S0045-6535(03)00761-6

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We investigated the subacute toxicity of 2 wood preservatives, DDAC (principal component: 37.5% didecyldimethyl ammonium chloride) and BAAC (principal components: 40% DDAC, 13.4% boric acid), in a number of aquatic invertebrates: a green alga (Selenastrum capricornutum), 2 cladocerans (<i>Ceriodaphnia dubia</i> and <i>Daphnia magna</i>), 2 fishes (<i>Danio rerio</i> and <i>Oryzias latipes</i>), and photobacteria (Microtox^&reg;). DDAC and BAAC inhibited biological functions in all of the organisms tested; the order of sensitivity was green alga > cladocerans > fish. Growth of <i>S. capricornutum</i> was inhibited by both preservatives at very similar levels of exposure. DDAC inhibited reproduction in <i>C. dubia</i> at lower levels of exposure than for <i>D. magna</i>, and the reverse was true for BAAC. Both DDAC and BAAC reduced the survival rate of <i>D. rerio</i> at lower exposure levels than for <i>O. latipes</i>. However, phylogenetic class differences in sensitivity to the preservatives were much greater than species differences.<br>

DOI： 10.1248/jhs.48.359

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The endocrine disruptor activity of styrene in humans and other vertebrates appears to be negligible. However, offspring numbers were reduced in Ceriodaphnia dubia bred in polystyrene cups. Styrene dimers and trimers were found to be eluted from the polystyrene cups by hexane and methanol with gas chromatography-mass spectrometry. Styrene dimers and trimers at concentrations of 0.04-1.7 mug/l affected C dubia fertility (25% reduction after seven days), suggesting that styrenes have the potential to impair crustacean populations in the aquatic environment. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0045-6535(02)00119-4

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A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals. Copyright (C) 2000 Elsevier Science B.V.

DOI： 10.1016/S0378-4347(00)00278-4

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The bioaccumulation potential of 3,4,5-trichloroguaiacol (345-TCG), a representative chlorophenol present in bleached kraft mill effluents of Japanese pulp and paper industry, in zooplankton (Daphnia magna) was evaluated by using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) in the presence of tetramethylammonium hydroxide (TMAH). This TMAH/Py-GC/MS was used to determine the amount of 345-TCG accumulated in individual zooplankton that were exposed to a 345-TCG aqueous solution for 3 weeks. TMAH/Py-GC/MS could detect approximately 0.5 ppm of 345-TCG, an amount that is below the detection level of conventional GC or LC methods, which require pre-extraction of organisms. The repeatability was also good among three TMAH/Py-GC/MS runs (RSD: approximately 1.96-2.61%). Static exposure experiments with 345-TCG (0.3 ppm: mg/l) showed a rapid uptake in D. magna reaching a level of 3.95 ppm (μg/g) after 3 weeks, which was ten times higher than the acute toxicity (48 h LC50) of D. magna. Compared with the concentration in the original incubation solution, the concentration in individual D. magna after a 3-week exposure increased about approximately 13-fold. The TMAH/Py-GC/MS approach for assessing the bioaccumulation of chlorophenols in individual zooplankton is simple, requires only a small sample (0.2-0.25 mg/trace), and is rapid, with a total analysis time of less than 1 h/sample. TMAH/Py-GC/MS is therefore useful for assessing the bioaccumulation of toxic components in industrial mill effluents.

DOI： 10.1016/S0165-2370(99)00087-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Science Publishers B.V.  

The bioaccumulation potential of 3,4,5-trichloroguaiacol (345-TCG), a representative chlorophenol present in bleached kraft mill effluents of Japanese pulp and paper industry, in zooplankton (Daphnia magna) was evaluated by using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) in the presence of tetramethylammonium hydroxide (TMAH). This TMAH/Py-GC/MS was used to determine the amount of 345-TCG accumulated in individual zooplankton that were exposed to a 345-TCG aqueous solution for 3 weeks. TMAH/Py-GC/MS could detect approximately 0.5 ppm of 345-TCG, an amount that is below the detection level of conventional GC or LC methods, which require pre-extraction of organisms. The repeatability was also good among three TMAH/Py-GC/MS runs (RSD: approximately 1.96-2.61%). Static exposure experiments with 345-TCG (0.3 ppm: mg/l) showed a rapid uptake in D. magna reaching a level of 3.95 ppm (μg/g) after 3 weeks, which was ten times higher than the acute toxicity (48 h LC50) of D. magna. Compared with the concentration in the original incubation solution, the concentration in individual D. magna after a 3-week exposure increased about approximately 13-fold. The TMAH/Py-GC/MS approach for assessing the bioaccumulation of chlorophenols in individual zooplankton is simple, requires only a small sample (0.2-0.25 mg/trace), and is rapid, with a total analysis time of less than 1 h/sample. TMAH/Py-GC/MS is therefore useful for assessing the bioaccumulation of toxic components in industrial mill effluents.

DOI： 10.1016/S0165-2370(99)00087-X

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<p>Perfluoroalkyl acid (PFAA) is diffused into the environment and detected in wildlife and its environmental persistence and accumulation is becoming a major concern. In this study, we determined the homolog distribution of 15 PFAA in samples of medaka (<i>Olyzias</i> <i>latipes</i>) , surrounding water, and sediment collected from 5 sites in Japan. PFAA concentrations in environmental water, sediment, and medaka differed depending on the sampling site; however, each PFAA composition ratio was almost constant within these sampling sites. The PFAA bioconcentration factor (BCF) in medaka was related with the octanol/water partition coefficient (Log <i>K_ow</i>) of PFAA. PFAA concentrations in the water samples had better correlations with those in the sediment samples not per unit dry weight, but per unit ignition loss (IL) . In addition, between sediment and medaka, good correlations were found when the comparison was done with the PFAA concentrations of sediment per unit IL. The PFAA accumulation in medaka was estimated by multiregression analysis using the PFAA concentrations in sediment per unit IL, gender, and body length. The PFAA concentrations in sediment per unit IL were significantly related to accumulation in medaka.</p>

DOI： 10.2965/jswe.41.61

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Perfluorinated compounds (PFCs) have been used widely, detected worldwide in the environment, and have accumulated highly in animals. As far as we know, there have been no reports which relate the PFC concentration in wild animals to the physicochemical properties. Therefore, we measured the concentrations of 15 currently available PFCs (perfluorocarboxylic acids with x carbons: Cx, perfluorosulfonic acids with x carbons: CxS) in medaka and the environmental water where medaka live. Samples were obtained from 7 points in Japan (Iwate, Ibaraki, Niigata, Hyogo, Yamaguchi, Ehime, and Nagasaki) from July to September in 2013. Twenty to forty medaka were collected from each point, as well as 2 L of water in a clean PET bottle. PFCs were extracted and concentrated using a solid-phase cartridge, and were measured by LC/MS/MS. The medaka samples were treated individually. C5-C9 and C8S were detected mainly in the water, C11-C13 and C8S were detected mainly in medaka. C8S was always detected in high concentrations in the water and medaka. The bioconcentration factors (BCFs) of PFCs were calculated from PFC concentrations of the water and the medaka. The BCFs of C8-C11 were increased exponentially with the length of carbon chain. The BCF of C8S (approx. 5,500) was far greater than C8 (approx. 330) or C9 (approx. 480). However, the BCFs of C8-C11 and C8S tended to increase in proportion with octanol/water partition coefficient (log <i>K</i><i>_ow</i>).

DOI： 10.2131/fts.2.201

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DOI： 10.2208/jscejer.69.III_375

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DOI： 10.2208/jscejer.69.III_401

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DOI： 10.18999/stulc.34.2.3

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Language：Japanese   Publisher：産業用水調査会  

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Language：Japanese   Publisher：日本比較内分泌学会  

DOI： 10.5983/nl2008jsce.39.106

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DOI： 10.5983/nl2008jsce.39.106

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&nbsp;Japan Ministry of Environment started discussion on the management of industrial and municipal effluent using the bio-assays, and the test manual was publicized in March, 2013 as well as continuous discussion on the system installation. In the present study, short-term chronic toxicity tests were conducted for the effluents collected in Tokushima using the publicized method mainly referred to the USEPA's Whole Effluent Toxicity Test Methods, and the toxicants for three aquatic organisms, fish, daphnia, and algae, were characterized using the technique listed as Phase 1 in Toxicity Identification Evaluation (TIE) manual. As results, adverse effects were found for all three organisms for Plant A while relatively strong toxicity was found for algae and daphnia for Plant C effluent. The major toxicant for algae in both effluents was found to be residual chlorine confirmed by the significant toxicity reduction by the pretreatment using sodium thiosulfate and toxicity test for sodium hypochlorite.

DOI： 10.2208/jscejer.69.III_375

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Language：Japanese   Publisher：Japan Society of Civil Engineers  

&nbsp;Various chemical substances are used in our industrial, agricultural, and daily activities, and are continuously discharged into the aquatic environment. Since the mixture effects of these compounds have become growing concerns, direct measurement of adverse effects of effluents or ambient waters on aquatic organisms has been frequently used. We had conducted short term chronic toxicity tests using algae, daphnia and fish refered to USEPA's whole effluent toxicity (WET) test method to evaluate the river water samples in our previous studies. However, no toxicant was identified in these investigations. So, in the present study, we collected seven samples in Tokyo metropolitan area, Osaka/Kyoto and Tokushima, and toxicants were characterized using the procedure of Phase I of Toxicity Identification Evaluation such as pre-treatment using solid phase extraction cartridges. As a result, cations such as metal ions are found to mainly contribute to the growth inhibition of algae. For daphnia, both cations such as metals and organic compounds are found to be major contributors to inhibit the reproduction, while organic compounds are major contributors for the inhibition of hatching and survival of fish.

DOI： 10.2208/jscejer.69.III_401

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Language：Japanese   Publisher：JAPAN TECHNICAL ASSOCIATION OF THE PULP AND PAPER INDUSTRY  

It is related with the Whole Effluent Toxicity Testing (WET) which is the evaluation and the management technique using the living thing response of drainage or environmental water.&lt;BR&gt;WET regulation has already been performed in the U. S. and there are also countries currently performed in Europe.&lt;BR&gt;The WET regulation currently performed in the U. S. is the method of performing effluent regulation using the result of the bioassay of in vivo without specifying the chemical substance contained in place-of-business drainage.&lt;BR&gt;Since it was difficult to manage the toxicity of the contaminant under drainage by an independent chemical substance, the U. S. did the WET examination.&lt;BR&gt;In the U. S., when a WET examination is introduced and the criterion of judgment of drainage is set up, you have to reduce the toxicity in the drainage which exceeds a standard. A series of procedures are defined by EPA as toxic reduction evaluation (TRE : Toxicity Reduction Evaluation). The technique of looking for a toxic causative agent in TRE is called toxic identification evaluation (TIE : Toxicity Identification Evaluation). &lt;BR&gt;From 2009, it refers to U. S. WET and the Ministry of Environment is considering construction of the evaluation managerial system of drainage using a living thing response.

DOI： 10.2524/jtappij.66.1362

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Language：Japanese   Publisher：JAPAN TECHNICAL ASSOCIATION OF THE PULP AND PAPER INDUSTRY  

It is related with the Whole Effluent Toxicity Testing (WET) which is the evaluation and the management technique using the living thing response of drainage or environmental water.<BR>WET regulation has already been performed in the U. S. and there are also countries currently performed in Europe.<BR>The WET regulation currently performed in the U. S. is the method of performing effluent regulation using the result of the bioassay of in vivo without specifying the chemical substance contained in place-of-business drainage.<BR>Since it was difficult to manage the toxicity of the contaminant under drainage by an independent chemical substance, the U. S. did the WET examination.<BR>In the U. S., when a WET examination is introduced and the criterion of judgment of drainage is set up, you have to reduce the toxicity in the drainage which exceeds a standard. A series of procedures are defined by EPA as toxic reduction evaluation (TRE : Toxicity Reduction Evaluation). The technique of looking for a toxic causative agent in TRE is called toxic identification evaluation (TIE : Toxicity Identification Evaluation). <BR>From 2009, it refers to U. S. WET and the Ministry of Environment is considering construction of the evaluation managerial system of drainage using a living thing response.

DOI： 10.2524/jtappij.66.1362

CiNii Books

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DOI： 10.2208/jscejer.68.III_217

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Language：Japanese   Publisher：環境システム計測制御学会  

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&nbsp;Various chemical substances are used in our industrial, agricultural, and daily activities, and are continuenly discharged into the environment. Several toxic chemical substances have been detected from wastewater and surface water, and their additive or synergistic effects have become growing concerns. Thus, there is an apparent limitation in the risk assessment and management based on the individual chemical assays. In the present study, 28 samples were collected at first class rivers in Japan between October 2011 and April 2012. We conducted the short term chronic toxicity test using fish, daphnia and algae of the USEPA's whole effluent toxicity (WET) method to evaluated the toxic effects of the river water samples, and investigated distribution and trend of the toxicity all over Japan. As results, the significant growth inhibition for green algae (Pseudokirchnerella subcapitata) was found from three samples. The significant mortality and reproductive effects were found for Ceriodaphnia dubia from eight samples. The significant inhibition of hatching and survival was found for zebra fish (Danio rerio) from four samples.

DOI： 10.2208/jscejer.68.III_217

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Language：Japanese   Publisher：The Japanese Society of Printing Science and Technology  

In Ministry of the Environment, the way of the future water environmental conservation was examined, and an interim report was performed in December, 2009. The effectiveness of new wastewater management technique (Whole Effluent Toxicity: WET) using bioassay is considered in that. This report introduces the need of the introduction of the WET system in Japan and an examination status to date.

DOI： 10.11413/nig.49.034

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Language：English   Publisher：SPRINGER-VERLAG BERLIN  

The effect of gas composition on the properties of slag generated from high temperature gasification was studied. The proportion of crystalline phase in the slag was measured by X-ray diffraction (XRD) and the microstructure of the slag was quantitatively analyzed by CCSEM. Metal ions in acid -digested slag samples were analyzed by ICP-AES. The results indicate that, in a strongly reducing atmosphere such as 10/90 (volume %) mixtures of CO2/H-2, the Fe content of bulk slag was reduced due to the precipitation of metallic iron in molten slag. The profile of the iron content of the slag was related to the dissolved oxygen transfer in molten slag. In addition, slag generated in mildly gas composition is more stable under than that of strongly atmosphere. Some heavy metals showed the same behavior as iron in the strongly reducing atmosphere were extracted by 1M/HCl, which results in higher extraction rates for Fe, Zn, and As, Cr.

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DOI： 10.11413/nig.49.034

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In October 2010, a group of experts met as part of the transatlantic think tank for toxicology (t 4) to exchange ideas about the current status and future of safety testing of nanomaterials. At present, there is no widely accepted path forward to assure appropriate and effective hazard identification for engineered nanomaterials. The group discussed needs for characterization of nanomaterials and identified testing protocols that incorporate the use of innovative alternative whole models such as zebrafish or C. elegans, as well as in vitro or alternative methods to examine specific functional pathways and modes of action. The group proposed elements of a potential testing scheme for nanomaterials that works towards an integrated testing strategy, incorporating the goals of the NRC report Toxicity Testing in the 21 st Century: A Vision and a Strategy by focusing on pathways of toxic response, and utilizing an evidence-based strategy for developing the knowledge base for safety assessment. Finally, the group recommended that a reliable, open, curated database be developed that interfaces with existing databases to enable sharing of information.

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