Yoshifumi Horie

Assessment of the lethal and sublethal effects of 20 environmental chemicals in zebrafish embryos and larvae by using OECD TG 212

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

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

The effects of endocrine disruptors, including anti‐androgenic chemicals, on aquatic environments have received increased attention in recent years. Currently, the method used to screen chemicals for anti‐androgenic activity is called the androgenized female stickleback screen, and it was established by the Organization of Economic Cooperation and Development in 2011 using the three‐spined stickleback. However, screening chemicals for anti‐androgenic activity has yet to be established using Japanese medaka. Thus, the purpose of this study was to establish a screening method for anti‐androgenic activity utilizing the number of papillary processes in Japanese medaka (Oryzias latipes) as an indicator of the chemicals 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

Lethal and sublethal effects of aniline and chlorinated anilines on zebrafish embryos and larvae

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

Cell reproductive patterns in the green alga Pseudokirchneriella subcapitata (=Selenastrum capricornutum) and their variations under exposure to the typical toxicants potassium dichromate and 3,5-DCP

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.

Effects of triphenyltin on reproduction in Japanese medaka (Oryzias latipes) across two generations

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.

Rapid ecotoxicological bioassay using delayed fluorescence in the marine cyanobacterium Cyanobium sp. (NIES-981)

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.

Effects of triclosan on Japanese medaka (Oryzias latipes) during embryo development, early life stage and reproduction: The negative effects of triclosan on reproduction in a teleost fish.

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.

Effects of tributyltin on early life-stage, reproduction, and gonadal sex differentiation in Japanese medaka (Oryzias latipes)

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.

Medaka extended one‐generation reproduction test evaluating 4‐nonylphenol

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.

Evaluation of the toxicity of leaches from hydrothermal sulfide deposits by means of a delayed fluorescence-based bioassay with the marine cyanobacterium Cyanobium sp. NIES-981

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.