Gonadal Soma-Derived Factor Expression is a Potential Biomarker for Predicting the Effects of Endocrine-Disrupting Chemicals on Gonadal Differentiation in Japanese Medaka (Oryzias Latipes).

Chemicals with androgenic or estrogenic activity induce the sex reversal and/or intersex condition in various teleost fish species. Previously, we reported that exposure to 17α-methyltestosterone, bisphenol A, or 4-nonylphenol induces changes in expression of the gonadal soma-derived factor (gsdf) gene accompanied by disruption of gonadal differentiation in Japanese medaka (Oryzias latipes). These findings suggest that gsdf expression might be a useful biomarker for predicting the potential effect of chemicals on gonadal differentiation. We examined the gsdf expression in Japanese medaka exposed to chemicals with estrogenic or androgenic activity. Exposure to the androgenic steroid 17ß-trenbolone at 0.5-22.1 µg/L induced the development of ovotestis (presence of ovarian tissue with testicular tissue) and female-to-male sex reversal in XX embryos, and exposure at 6.32 and 22.1 µg/L significantly increased gsdf expression in XX embryos compared with controls at developmental stage 38 (1 day before hatching). In the present study, no statistically significant difference in gsdf mRNA expression was observed after exposure to 17ß-estradiol, 17α-ethinylestradiol, and 4-t-octylphenol, which have estrogenic activity. In addition, antiandrogenic chemicals or chemicals without endocrine-disrupting activity did not induce changes in gsdf expression in XX or XY embryos. Thus, an increase in gsdf expression after androgen exposure was observed in XX embryos. Together, these findings indicate that gsdf expression might be useful for predicting the adverse effect of chemicals on gonadal differentiation. Environ Toxicol Chem 2022;41:1875-1884. © 2022 SETAC.

Laterally biased diffusion of males of the water flea Daphnia magna.

The water flea, Daphnia magna, is a representative zooplankton that lives in freshwater environments. It primarily propagates via asexual reproduction in normal and healthy environmental conditions. Unsuitable environmental conditions induce D. magna to change its mode of reproduction from asexual to sexual reproduction. During sexual reproduction, D. magna produces special tough eggs (resting eggs) that can survive severe environmental conditions. Despite an increase in our understanding of their mating behavior, the sex-specific characteristics of swimming behavior among daphnid species are poorly understood. In this study, we analysed the swimming patterns and dynamics of female and male adult D. magna using computer modeling. Males displayed laterally biased diffusion in contrast to the homogeneous, nondirectional diffusion of females. Computer modeling analysis using a discrete-time Markov chain simulation, in which the frequencies of turning behavior were evaluated as probability distributions, explained the greater diffusion of males in the horizontal direction. We presumed that high diffusion in the horizontal direction would increase the probability of encountering a distant mate. Our findings suggest that male D. magna increases genotypic heterogeneity by effectively selecting certain motion parameters.

Juvenile hormone synthesis and signaling disruption triggering male offspring induction and population decline in cladocerans (water flea): Review and adverse outcome pathway development.

Juvenile hormone (JH) are a family of multifunctional hormones regulating larval development, molting, metamorphosis, reproduction, and phenotypic plasticity in arthropods. Based on its importance in arthropod life histories, many insect growth regulators (IGRs) mimicking JH have been designed to control harmful insects in agriculture and aquaculture. These JH analogs (JHAs) may also pose hazards to nontarget species by causing unexpected endocrine-disrupting (ED) effects such as molting and metamorphosis defects, larval lethality, and disruption of the sexual identity. This critical review summarizes the current knowledge of the JH-mediated effects in the freshwater cladoceran crustaceans such as Daphnia species on JHA-triggered endocrine disruptive outputs to establish a systematic understanding of JHA effects. Based on the current knowledge, adverse outcome pathways (AOPs) addressing the JHA-mediated ED effects in cladoceran leading to male offspring production and subsequent population decline were developed. The weight of evidence (WoE) of AOPs was assessed according to established guidelines. The review and AOP development aim to present the current scientific understanding of the JH pathway and provide a robust reference for the development of tiered testing strategies and new risk assessment approaches for JHAs in future ecotoxicological research and regulatory processes.

Summary of 17 chemicals evaluated by OECD TG229 using Japanese Medaka, Oryzias latipes in EXTEND 2016.

In June 2016, the Ministry of the Environment of Japan announced a program "EXTEND2016" on the implementation of testing and assessment for endocrine active chemicals, consisting of a two-tiered strategy. The aim of the Tier 1 screening and the Tier 2 testing is to identify the impacts on the endocrine system and to characterize the adverse effects to aquatic animals by endocrine disrupting chemicals detected in the aquatic environment in Japan. For the consistent assessment of the effects on reproduction associated with estrogenic, anti-estrogenic, androgenic, and/or anti-androgenic activities of chemicals throughout Tier 1 screening to Tier 2 testing, a unified test species, Japanese medaka (Oryzias latipes), has been used. For Tier 1 screening, the in vivo Fish Short-Term Reproduction Assay (OECD test guideline No. 229) was conducted for 17 chemicals that were nominated based on the results of environmental monitoring, existing knowledge obtained from a literature survey, and positive results in reporter gene assays using the estrogen receptor of Japanese medaka. In the 17 assays using Japanese medaka, adverse effects on reproduction (i.e., reduction in fecundity and/or fertility) were suggested for 10 chemicals, and a significant increase of hepatic vitellogenin in males, indicating estrogenic (estrogen receptor agonistic) potency, was found for eight chemicals at the concentrations in which no overt toxicity was observed. Based on these results, and the frequency and the concentrations detected in the Japanese environment, estrone, 4-nonylphenol (branched isomers), 4-tert-octylphenol, triphenyl phosphate, and bisphenol A were considered as high priority candidate substances for the Tier 2 testing.

Toxicity assessment of typical polycyclic aromatic hydrocarbons to Daphnia magna and Hyalella azteca in water-only and sediment-water exposure systems.

While the equilibrium partitioning (EqP) method has been demonstrated to effectively predict the adverse effects of nonionic organic chemicals in sediment on benthic organisms by sediment toxicity tests, only a limited number of studies have been performed both in water-only and whole-sediment toxicity tests using the same species and verified the validity of EqP-based toxicity assessment. To further examine the validity of the EqP method for application in a wide range of hydrophobicity, we conducted sorption/desorption experiments and both water-only and sediment toxicity tests using a popular aquatic crustacean species, Daphnia magna (48 h), and benthic species Hyalella azteca (96 h) for six typical polycyclic aromatic hydrocarbons (PAHs) with three to five rings and an amine derivative: anthracene, phenanthrene, fluoranthene, pyrene, benzo[a]pyrene, dibenzo[a,h]anthracene, and 1-aminopyrene. The linear sorption coefficient was determined and ranged from 2.7 × 102 (phenanthrene) and 1.2 × 104 L/kg (benzo[a]pyrene) highly depending on the hydrophobicity while the aqueous concentrations were stable after 24 h in the desorption test. As result of acute toxicity tests in the water-only exposure system, anthracene and dibenz[a,h]anthracene were found to be nontoxic to both species, while median effect/lethal concentrations (EC50/LC50) were determined as ranging from 0.66 (benzo[a]pyrene) to 330 µg/L (phenanthrene), and from 11 (1-aminopyrene) to 180 µg/L (phenanthrene) for D. magna and H. azteca, respectively. Among these compounds, three PAHs with three, four, and five rings each, and 1-aminopyrene were subjected to sediment-water toxicity tests. In the sediment-water tests, the LC50 of phenanthrene and pyrene was three to six times higher than that of the water-only tests for H. azteca while the EC50 was 1.1 to 2.0 times higher for D. magna. In contrast, the EC50/LC50 of benzo[a]pyrene (BaP) in the sediment-water toxicity test was more than 5 times higher than that in the water-only test for both H. azteca and D. magna. The EC50/LC50 values of 1-aminopyrene were similar in both the sediment-water and the water-only toxicity tests, ranging narrowly from 21 to 28 µg/L and 8.8 to 11 µg/L for D. magna and H. azteca, respectively. The EC50/LC50 based on the body residue (ER50/LR50) was investigated for two of the representative PAHs, pyrene, and BaP. The ER50/LR50 of pyrene in both species was 2.3 and 11 times higher in the water-only toxicity test for D. magna and H. azteca, respectively, while those of BaP in the sediment-water toxicity test were not calculated for the sediment-water toxicity tests, and the highest body concentration in the sediment-water tests was lower than the ER50/LR50 in the water-only toxicity test. Although the experimental results were comparable with the predicted sediment toxicity values based on the EqP method for the selected PAHs in this study, there is a risk of phenanthrene and pyrene being slightly underestimated (1.4-1.9 fold for phenanthrene and 3.7-6.1 fold for pyrene) by the EqP method for H. azteca. These results reaffirm that the bioavailability of poorly water-soluble chemicals is important for sediment toxicity and that the exposure pathway should be further investigated to avoid under- and overestimation via the EqP method.

Summary of reference chemicals evaluated by the fish short-term reproduction assay, OECD TG229, using Japanese Medaka, Oryzias latipes.

Under the Organisation for Economic Co-operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARß of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.

Exposure to 4-nonylphenol induces a shift in the gene expression of gsdf and testis-ova formation and sex reversal in Japanese medaka (Oryzias latipes).

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.

Influence of triphenyltin on morphologic abnormalities and the thyroid hormone system in early-stage zebrafish (Danio rerio).

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 trß but not of thyroid stimulating hormone ß subunit. These findings suggest that TPT disrupts the thyroid system in zebrafish.

Chronic toxicity of 50 metals to Ceriodaphnia dubia.

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 <100 µg/L; nine metals (Al, V, As, Se, Zr, Nb, Rh, Sb and Bi) were 100 ≤ IC20 < 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 >100 000 µg/L. Three metals (Pd, Hf and Ta) did not show IC20 at the upper limit of respective aqueous solubility, and IC20 s 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 IC50 s for C. dubia and those for Dahpnia magna published previously. The IC50 s of 47 metals were not correlated with electronegativity, first ionization energy, atomic weight, atomic number, covalent radius, atomic radius or ionic radius.

Production of genome-edited Daphnia for heavy metal detection by fluorescence.

Aquatic heavy metal pollution is a growing concern. To facilitate heavy metal monitoring in water, we developed transgenic Daphnia 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 Daphnia magna. A chimeric gene fusing the promoter region with a green fluorescent protein (GFP) gene was integrated into D. magna using the TALEN technique and transgenic Daphnia named D. magna MetalloG were produced. When D. magna 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. D. magna MetalloG facilitates aqueous heavy metal detection and might enhance water quality monitoring.

Molecular Insights into Structural and Ligand Binding Features of Methoprene-Tolerant in Daphnids.

Juvenile hormone (JH) is an important endocrine factor regulating many biological activities in arthropods. In daphnids, methoprene-tolerant (Met) belongs to a basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family protein which has recently been confirmed as a JH receptor and can bind and be activated by JHs and JH agonists. Although the activation of the JH signaling pathway causes many physiological effects, the molecular basis for the structural feature and ligand binding properties of Daphnia Met are not fully understood. To study the ligand preference in terms of structural features of Daphnia Met, we built in silico homology models of the PAS-B domain of Daphnia Mets from cladoceran crustaceans, Daphnia pulex and D. magna. Structural comparison of two Daphnia Met PAS-B domain models revealed that the volume in the main cavity of D. magna Met was larger than that of D. pulex Met. Compared with insect Met, Daphnia Met had a less hydrophobic cavity due to polar residues in the core-binding site. Molecular docking simulations of JH and its analogs with Daphnia Met indicated that the interaction energies were correlated with each of the experimental values of in vivo JH activities based on male induction and in vitro Met-mediated transactivation potencies. Furthermore, in silico site-directed mutagenesis supported experimental findings that Thr292 in D. pulex Met and Thr296 in D. magna Met substitution to valine contribute to JH selectivity and differential species response. This study demonstrates that in silico simulations of Daphnia Met and its ligands may be a tool for predicting the ligand profile and cross species sensitivity.

Bisphenol A induces a shift in sex differentiation gene expression with testis-ova or sex reversal in Japanese medaka (Oryzias latipes).

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.

The non-steroidal anti-inflammatory drug diclofenac sodium induces abnormal embryogenesis and delayed lethal effects in early life stage zebrafish (Danio rerio).

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.

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.

Ecotoxicological Test Assay Using OECD TG 212 in Marine Java Medaka (Oryzias javanicus) and Freshwater Japanese Medaka (Oryzias latipes).

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.

Functional distinctions associated with the diversity of sex steroid hormone receptors ESR and AR.

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 ARß) 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.

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.

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.

Comparison of the effects of constant and pulsed exposure with equivalent time-weighted average concentrations of the juvenile hormone analog pyriproxyfen on the reproduction of Daphnia magna.

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.

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

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