Haruna Watanabe

Chronic toxicity of an environmentally relevant mixture of pharmaceuticals to three aquatic organisms (alga, daphnid, and fish)

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.

Reduction in toxicity of coking wastewater to aquatic organisms by vertical tubular biological reactor

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.

Application of whole sediment toxicity identification evaluation procedures to road dust using a benthic ostracod Heterocypris incongruens.

Road dust is considered to be an important source of sediment contamination in receiving water bodies; however, few studies have evaluated the toxicity of road dust to benthic organisms. This study evaluated the toxicity of road dust to a benthic ostracod, Heterocypris incongruens, using a six-day direct exposure experiment. We applied whole sediment toxicity identification evaluation (TIE) methods to identify the primary group of toxicants in road dust. Three road dust samples from Tokyo caused high ostracod mortality. The addition of hydrophobic adsorbents, Ambersorb and XAD, eliminated toxicity in all samples, suggesting that hydrophobic compounds were the main toxicants in road dust samples. A cation exchange resin, Chelex, also reduced the toxicity of two samples, although the measured concentrations of dissolved heavy metals in the test solution did not exceed the LC(50) values in the literature. In addition, the sum of toxic unit (TU=measured concentration/LC(50)) of each individual metal which predicts the toxicity of the metal mixtures did not exceed 1.0 in all samples, suggesting that heavy metal mixtures did not have additive effects. We hypothesized that the toxicity reduction by XAD and Chelex was due to the removal of hydrophobic compounds, rather than heavy metals. Thus, a toxicity test was conducted on fractions eluted with organic solvents from the XAD and Chelex recovered from one of the road dust samples. Methanol-eluted fractions of XAD and Chelex showed 100 percent ostracod mortality, indicating that the hydrophobic organic compounds removed by these adsorbents were the principal toxicants in the road dust sample.

Establishment of a short-term, in vivo screening method for detecting chemicals with juvenile hormone activity using adult Daphnia magna.

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 parents 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. Copyright

Diofenolan induces male offspring production through binding to the juvenile hormone receptor in Daphnia magna

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.

Towards modelling of the environmental fate of pharmaceuticals using the QSPR-MM scheme

Pharmaceuticals are considered as a new, important group of pollutants. These compounds can enter the environment via several routes and can disturb the natural balance of ecosystems. Factors affecting the environmental fate of medical substances can be determined with computational modelling. The routine application of the modelling methodology in the environmental risk assessment for newly designed pharmaceuticals would enable prediction of their important physical/chemical properties and forecasting their long-range transport and fate. In this contribution, we present the existing state-of-the-art and review the currently available modelling tools of two groups: Quantitative Structure-Property Relationship techniques and Multimedia Mass-balance models. We discuss the current research needs in the context of extending the applicability of the existing tools onto pharmaceuticals, being a more structurally diversified group of contaminants than persistence organic pollutants, for which the majority of the existing models have been originally developed. Multimedia fate models applied for pharmaceuticals are reviewed.QSPR approach to predict physical/chemical properties of pharmaceuticals is reviewed.QSPR-MM combined model is suggested to predict Pov and LRTP of chemical pollutants.

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

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.

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.

Estimation of population-level effect of the endocrine disruptor pyriproxyfen in Daphnia magna by using changes in sex ratio and reproductive output

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.