Japanese Medaka Embryos Research Articles

The role of chorion integrity on the bioaccumulation and toxicity of selenium nanoparticles in Japanese medaka (Oryzias latipes)

Selenium nanoparticles (nano-Se) have a wide range of biomedical and agricultural applications. However, there is little information on the potential toxicity of nano-Se once it enters the environment, particularly in fish. The first line of defense from contaminants that embryonic fish have is the chorion, but the degree to which the chorion protects the developing embryo is unknown. Japanese medaka (Oryzias latipes) embryos were exposed to nano-Se in a wide range of concentrations (0.1-400 µM). The importance of chorion integrity was evaluated by exposing embryos to 16 nm nano-Se under four degrees of dechorionation: intact, roughened, partially-dechorionated, fully-dechorionated. Then, effects of particle size on embryos and larvae were determined using four sizes of nano-Se particles (16, 25-50, 50, 100 nm). The results showed that nano-Se exposure reduced survival, development, and hatching. Nano-Se was observed to adsorb on the chorion, with the amount decreasing with increased degree of dechorionation. Toxicity increased with increasing degree of dechorionation, and smaller-sized nano-Se crossed intact chorion more readily and resulted in higher toxicity than larger ones. In larvae, nano-Se accumulated on the skin and was more toxic compared to embryos. This study demonstrated the importance of the chorion in protecting developing embryos and effects of nanoparticle size on its bioavailability and subsequent toxicity.

Injection of Ortho-Functionalized Tetrafluorinated Azobenzene-Containing siRNAs into Japanese Medaka Embryos for Photocontrolled Gene Silencing.

This article describes the detailed methodology of how to inject photoswitchable ortho-functionalized tetrafluorinated short interfering RNAs (F-siRNAs) into a single cell of stage-two Japanese medaka (Oryzias latipes) embryos and how to control gene silencing with different wavelengths of light. Many of the prior papers describing Japanese medaka embryo injections omit key information. As such, this article aims to give an in-depth explanation as to how the NanoJect III microinjector can be used for this purpose. To obtain the embryos for microinjection, adult medaka are housed under a 14-hr light, 10-hr dark cycle to mimic their natural breeding period. This induces mating at approximately the same time each day, when the lights turn on, so recently fertilized eggs can be obtained. Synthetic F-siRNAs are injected into transgenic stage-two single-cell Japanese medaka embryos expressing enhanced green fluorescent protein (eGFP). Our data demonstrate that our F-siRNAs can silence gene activity in Japanese medaka embryos expressing eGFP. Moreover, gene expression can be activated by exposing F-siRNA-injected embryos to blue light and deactivated a few days after exposure to green light. To the best of our knowledge, this marks the first reversible control of a gene-silencing oligonucleotide within an in vivo system. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Medaka maintenance and embryo collection Basic Protocol 2: Injection of stage-two one-cell medaka embryos Basic Protocol 3: Evaluation of the F-siRNA gene-silencing ability through light activation and inactivation using blue and green light by measuring enhanced green fluorescent protein fluorescence.

In Vivo Injection of Reversible Optically Controlled Short Interfering RNA into Japanese Medaka Embryos (Oryzias latipes) to Regulate Gene Silencing.

Photoswitchable ortho-functionalized tetrafluorinated azobenzene-modified siRNAs (F-azo-siRNAs) were synthesized using solid-phase phosphoramidite chemistry. The activity of an F-azo-siRNA targeting enhanced green fluorescence protein (eGFP) in transgenic (Tg) Japanese Medaka (Oryzias latipes) was reversibly photocontrolled with blue (470 nm) and green (530 nm) light, to activate and inactivate the siRNA, respectively. This study highlights the first reversible in vivo study with photoswitchable siRNA. Controlling siRNA function reversibly in vivo could open new opportunities for biotech research to better understand gene function and cellular mechanisms.

Comparative effects of different metals on the Japanese medaka embryos and larvae.

Rapid evaluation of the toxicity of metals using fish embryo acute toxicity is facilitative to ecological risk assessment of aquatic organisms. However, this approach has seldom been utilized for the comparative study on the effects of different metals to fish. In this study, acute and sub-chronic tests were used to compare the toxicity of Se(IV) and Cd in the embryos and larvae of Japanese medaka (Oryzias latipes). The embryos with different levels of dechorionation and/or pre-exposure were also exposed to Se(IV) and Cd at various concentrations. The results showed that the LC50-144 h of Cd was 1.3-5.2 folds higher than that of Se(IV) for the embryos. In contrast, LC50-96 h of Se(IV) were 200-400 folds higher than that of Cd for the larvae. Meanwhile, dechorionated embryos were more sensitive to both Se and Cd than the intact embryos. At elevated concentrations, both Se and Cd caused mortality and deformity in the embryos and larvae. In addition, pre-exposure to Cd at the embryonic stages enhanced the resistance to Cd in the larvae. However, pre-exposure to Se(IV) at the embryonic stages did not affect the toxicity of Se(IV) to the larvae. This study has distinguished the nuance differences in effects between Se(IV) and Cd after acute and sub-chronic exposures with/without chorion. The approach might have a potential in the comparative toxicology of metals (or other pollutants) and in the assessment of their risks to aquatic ecosystems.

Detrimental impacts and QSAR baseline toxicity assessment of Japanese medaka embryos exposed to methylparaben and its halogenated byproducts

Despite the theoretical risk of forming halogenated methylparabens (halo-MePs) during water chlorination in the absence or presence of bromide ions, there remains a lack of in vivo toxicological assessments on vertebrate organisms for halo-MePs. This research addresses these gaps by investigating the lethal (assessed by embryo coagulation) or sub-lethal (assessed by hatching success/heartbeat rate) toxicity and teratogenicity (assessed by deformity rate) of MeP and its mono- and di-halogen derivatives (Cl− or Br−) using Japanese medaka embryos. In assessing selected apical endpoints to discern patterns in physiological or biochemical alterations, heightened toxic impacts were observed for halo-MePs compared to MeP. These include a higher incidence of embryo coagulation (4–36 fold), heartbeat rate decrement (11–36 fold), deformity rate increment (32–223 fold), hatching success decrement (11–59 fold), and an increase in Reactive Oxygen Species (ROS) level (1.2–7.4 fold)/Catalase (CAT) activity (1.7–2.8 fold). Experimentally determined LC50 values are correlated and predicted using a Quantitative Structure Activity Relationship (QSAR) based on the speciation-corrected liposome-water distribution ratio (Dlipw, pH 7.5). The QSAR baseline toxicity aligns well with (sub)lethal toxicity and teratogenicity, as evidenced by toxic ratio (TR) analysis showing TR < 10 for MeP exposure in all cases, while significant specific or reactive toxicity was found for halo-MeP exposure, with TR > 10 observed (excepting three values). Our extensive findings contribute novel insights into the intricate interplay of embryonic toxicity during the early-life-stage of Japanese medaka, with a specific focus on highlighting the potential hazards associated with halo-MePs compared to the parent compound MeP.

Nano-graphene oxide particles induce inheritable anomalies through altered gene expressions involved in oocyte maturation

The inheritable impact of exposure to graphene oxide nanoparticles (GO NPs) on vertebrate germline during critical windows of gamete development remain undetermined to date. Here, we analyzed the transgenerational effects of exposure to nano-graphene oxide particles (nGO) synthesized in house with lateral dimensions 300–600 nm and surface charge of −36.8 mV on different developmental stages of germ cells (GCs): (1) during GCs undergoing early development and differentiation, and (2) during GCs undergoing gametogenesis and maturation in adulthood. Biocompatibility analyses in Japanese medaka embryos showed lethality above 1 µg/ml and also an aberrant increase in germ cell count of both males and females at doses below the lethal dose. However, no lethality or anomalies were evident in adults up to 45 µg/ml. Long term exposure of embryos and adults for 21 days resulted in reduced fecundity. This effect was transmitted to subsequent generations, F1 and F2. Importantly, the inheritable effects of nGO in adults were pronounced at a high dose of 10 µg/ml, while 1 µg/ml showed no impact on the germline indicating lower doses used in this study to be safe. Further, expressions of selected genes that adversely affected oocyte maturation were enhanced in F1 and F2 individuals. Interestingly, the inheritance patterns differed corresponding to the stage at which the fish received the exposure.

Single and mixture toxicity of cadmium and copper to swim bladder in early life stages of Japanese medaka (Oryzias latipes).

Toxicity observed in aquatic ecosystems often cannot be explained by the action of a single pollutant. Likewise, evaluation standards formulated by a single effect cannot truly reflect the environmental quality requirements. The study of mixtures is needed to provide environmental relevance and knowledge of combined toxicity. In this study, the embryos of Japanese medaka (Oryzias latipes) were treated with individual and binary mixture of copper (Cu) and cadmium (Cd) until 12days post-fertilization (dpf). Hatching, mortality, development, histology and gene expression were assessed. Our results showed that the highest concentration mixture of Cd (10mg/L) and Cu (1mg/L) affected survival, hatching time and hatching success. Occurrence of uninflated swim bladder was the highest (value) with exposure to 10mg/L Cd. Swim bladder was commonly over-inflated in a mixture (0.1mg/L Cd + 1.0mg/L Cu) exposure. Individuals exposed to the mixture (0.1 Cd + 1.0 Cu mg/L) showed up to a 7.69% increase in swim bladder area compared to the control group. The mixtures containing 0.1 or 10mg/L Cd, each with 1.0mg/L Cu resulted in significantly increased of Pbx1b expression, higher than any Cd or Cu alone (p < 0.01). In the co-exposure group (0.1/10 Cd + 1.0 Cu mg/L), Pbx1b expression was found at 12dpf but not 7dpf in controls. Higher concentrations of Cd may progressively reduce Pbx1b expression, potentially explaining why 75% of individuals in the 10mg/L Cd group failed to inflate their swim bladders. Additionally, the swim bladder proved to be a valuable bio-indicator for biological evaluation.

Relationships between Isomeric Metabolism and Regioselective Toxicity of Hydroxychrysenes in Embryos of Japanese Medaka (Oryzias latipes)

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are ubiquitous contaminants that can be formed through oxidation of parent PAHs. Our previous studies found 2-hydroxychrysene (2-OHCHR) to be significantly more toxic to Japanese medaka embryos than 6-hydroxychrysene (6-OHCHR), an example of regioselective toxicity. We have also previously identified a sensitive developmental window to 2-OHCHR toxicity that closely coincided with liver development, leading us to hypothesize that differences in metabolism may play a role in the regioselective toxicity. To test this hypothesis, Japanese medaka embryos were treated with each isomer for 24 h during liver development (52-76 hpf). Although 6-OHCHR was absorbed 97.2 ± 0.18% faster than 2-OHCHR, it was eliminated 57.7 ± 0.36% faster as a glucuronide conjugate. Pretreatment with cytochrome P450 inhibitor, ketoconazole, reduced anemia by 96.8 ± 3.19% and mortality by 95.2 ± 4.76% in 2-OHCHR treatments. Formation of chrysene-1,2-diol (1,2-CAT) was also reduced by 64.4 ± 2.14% by ketoconazole pretreatment. While pretreatment with UDP-glucuronosyltransferase inhibitor, nilotinib, reduced glucuronidation of 2-OHCHR by 52.4 ± 2.55% and of 6-OHCHR by 63.7 ± 3.19%, it did not alter toxicity for either compound. These results indicate that CYP-mediated activation, potentially to 1,2-CAT, may explain the isomeric differences in developmental toxicity of 2-OHCHR.

Effects of adrenergic α-antagonists on the early life stages of Japanese medaka (Oryzias latipes).

The occurrence of pharmaceuticals in the aquatic environment has stimulated considerable research efforts into their potential ecotoxicological consequences. There are a number of pharmaceuticals targeting adrenergic receptors; however, relatively few studies have explored the effects of adrenergic α-antagonists (or α-blockers) on fish. In this study, moxisylyte was selected as a representative α-blocker, and Japanese medaka embryos were exposed to moxisylyte (1-625 μg/L) for 44 days. Moxisylyte caused no significant or only marginal effects on the mortality, development, and growth; however, most genes involved in detoxification and antioxidant were transcriptionally upregulated, and antioxidant enzymes activities were induced as well. Moxisylyte exposure resulted in transcriptional downregulation of most of the steroidogenesis genes, and accordingly, the mRNA levels of steroid hormone receptors and vitellogenin decreased, particularly in males, indicating that moxisylyte disrupts the hypothalamic-pituitary-gonadal (HPG) axis in a gender-specific manner. Therefore, the risk of α-blockers on fish should not be overlooked and deserves further investigation.

Developmental toxicity and thyroid hormone-disrupting effects of acetyl tributyl citrate in zebrafish and Japanese medaka

Chemical pollution from plasticizers in water environments is a serious environmental problem worldwide. Phthalate plasticizers have potential endocrine-disrupting effects in vertebrates. In this study, the effects of a non-phthalate acetyl tributyl citrate (ATBC) plasticizer on endocrine hormone activity (according to thyroid-related gene expression) and the developmental toxicity of ATBC were determined in zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) embryos. ATBC exposure increased the mortality of zebrafish and Japanese medaka at concentrations of 443.05 and 1,937.41 μg/L, respectively. Body curvature, edema, and growth inhibition were also observed after ATBC exposure in both species. Importantly, both species exhibited suppressed thyroid-related gene mRNA expression after ATBC exposure. ATBC exposure significantly suppressed the expression of thyroid-stimulating hormone beta subunit (tshβ), iodothyronine deiodinase 1 (dio1), dio2, and thyroid hormone receptor alpha (trα) in zebrafish. In Japanese medaka, ATBC exposure suppressed the mRNA expression of dio2, trα, and trβ but not tshβ and dio1. In summary, ATBC exposure inhibited thyroid-related gene expression and led to improper swim bladder inflation in the test species. This is the first report of a non-phthalate ATBC plasticizer inducing abnormal embryo development and disrupting thyroid hormone activity in fish.

Potential contribution of intrinsic developmental stability toward body plan conservation

BackgroundDespite the morphological diversity of animals, their basic anatomical patterns—the body plans in each animal phylum—have remained highly conserved over hundreds of millions of evolutionary years. This is attributed to conservation of the body plan-establishing developmental period (the phylotypic period) in each lineage. However, the evolutionary mechanism behind this phylotypic period conservation remains under debate. A variety of hypotheses based on the concept of modern synthesis have been proposed, such as negative selection in the phylotypic period through its vulnerability to embryonic lethality. Here we tested a new hypothesis that the phylotypic period is developmentally stable; it has less potential to produce phenotypic variations than the other stages, and this has most likely led to the evolutionary conservation of body plans.ResultsBy analyzing the embryos of inbred Japanese medaka embryos raised under the same laboratory conditions and measuring the whole embryonic transcriptome as a phenotype, we found that the phylotypic period has greater developmental stability than other stages. Comparison of phenotypic differences between two wild medaka populations indicated that the phylotypic period and its genes in this period remained less variational, even after environmental and mutational modifications accumulated during intraspecies evolution. Genes with stable expression levels were enriched with those involved in cell-cell signalling and morphological specification such as Wnt and Hox, implying possible involvement in body plan development of these genes.ConclusionsThis study demonstrated the correspondence between the developmental stage with low potential to produce phenotypic variations and that with low diversity in micro- and macroevolution, namely the phylotypic period. Whereas modern synthesis explains evolution as a process of shaping of phenotypic variations caused by mutations, our results highlight the possibility that phenotypic variations are readily limited by the intrinsic nature of organisms, namely developmental stability, thus biasing evolutionary outcomes.

Transient Copper Exposure During Embryogenesis and Temperature Affect Developmental Rate, Survival, and Fin Regeneration in Japanese Medaka (Oryzias latipes).

Combined environmental stressors that an organism experiences can have both immediate and lasting consequences. In the present study, we exposed Japanese medaka (Oryzias latipes) embryos to sublethal copper sulfate (CuSO4 ; 0, 10, and 100 ppb) in combination with different rearing temperatures (27, 30, and 33 °C) to assess acute and latent effects on development, growth, and regenerative capacity. Embryos exposed to CuSO4 and/or higher temperatures hatched significantly earlier. At 4 months post-exposure, fish exposed to low levels of CuSO4 during development had higher survival, whereas fish exposed to both 100 ppb CuSO4 and 33 °C temperatures had significantly lower survival. In addition, a sex-specific effect of embryonic CuSO4 exposure was observed as female mass decreased with increasing Cu dose. We also assessed caudal fin regenerative capabilities in both embryo-exposed fish at 4 months of age and adult medaka that were exposed to 0, 10, and 100 ppb CuSO4 at room temperature during a 14-day trial. Whereas fin regeneration was unaffected by adult exposure to Cu, fish transiently exposed during embryogenesis displayed an initial increase in fin growth rate and an increased incidence of abnormal fin morphology following regrowth. Collectively, these data suggest that developmental Cu exposure has the potential to exert long-lasting impacts to organismal growth, survival, and function. Environ Toxicol Chem 2022;41:748-757. © 2021 SETAC.

1,2,3-benzotriazole adversely affects early-life stage of Oryzias latipes

1,2,3-benzotriazole (BT) is used in large amounts around the world and is one of the substances derived from household chemicals that are of concern for risk when discharged to aquatic environments. Therefore, several studies have been conducted on the aquatic toxicity effects of BT, but the chronic impact assessment studies to evaluate the developmental effects on the early-life stage of fish are insufficient. In this study, the acute toxicity test and subchronic toxicity test (fish, early-life stage toxicity test, ELS test) using embryos of Japanese medaka (Oryzias latipes) were performed to evaluate the acute toxicity, developmental toxicity, growth (indicated by total length and weight at the end of the test), and histopathological effect of BT. In the short-term toxicity test on embryo and sac-fry stage, toxicity value was calculated to be 41 mg/L (NOEC). Based on this value, the exposure concentration of the ELS test was determined as 0.04, 0.4, 4 and 40 mg/L, and total exposure duration was 42 days. At the highest concentration group (40 mg/L), failure of swim bladder inflation and decrease of survival and size (total length and weight) were observed. Moreover, in the histopathological analysis, abnormal findings were detected in swim bladders from the 40 mg/L group such as inflammation and tumor changes. On the other hands, condition index (weight-length relationships, CI) was statistically significantly lower in all exposed groups compared to the control group. NOEC for the survival of BT was calculated to be 4 mg/L. LOEC for CI was 0.04 mg/L, which means BT inhibited weight gain relative to its length on larvae of medaka.

Toxicity evaluations using Japanese medaka embryos in sediments collected from Kahoku-gata, Ishikawa prefecture

Toxicity evaluations using Japanese medaka embryos in sediments collected from Kahoku-gata, Ishikawa prefecture

Inhibition of swim bladder inflation in Japanese medaka (Oryzias latipes) embryos following exposure to select pharmaceuticals alone and in combination

This study leveraged the Japanese medaka fish embryo model for the assessment of effects of select contaminants on early development in fish. Fish embryos were exposed to various pharmaceutical contaminants including synthetic hormones and non-steroidal anti-inflammatory drugs and their effects on development were observed. Initial screening determined that swim bladder inflation failure was the most common endpoint detected. Swim bladder inflation failure was first explored in a study demonstrating that medaka require access to the air-water interphase to inflate their swim bladders in a time-dependent manner, and swim bladder inflation failure was correlated with mortality. Fish embryos were exposed 24-hours post fertilization until hatch to concentration ranges of various pharmaceutical contaminants including: 17β-estradiol, 17α-ethinylestradiol, and levonorgestrel (1 to 1000 µg/L), or diclofenac (0.32 to 100 mg/L). The main effect observed across all four compounds was a significant increase in failure of swim bladder inflation with increasing exposure concentration (24 to 72-hours post-hatch). Following single compound experiments combinatorial exposures using no-observed-effect concentrations were conducted. The main effect observed was a significant decrease in inflation success 24-hours post-hatch following a binary mixture of levonorgestrel and 17α-ethinylestradiol, as well as a significant decrease in swim bladder inflation success at all times following exposure to a quaternary mixture of all four compounds. This study demonstrated that embryonic exposure to pharmaceutical compounds, both alone and in combination, resulted in failure of swim bladder inflation in larval Japanese medaka.

Stage-dependent and regioselective toxicity of 2- and 6-hydroxychrysene during Japanese medaka embryogenesis

Exposure to oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) at critical developmental time-points in fish models impairs red blood cell concentrations in a regioselective manner, with 2-hydroxychrysene being more potent than 6-hydroxychrysene. To better characterize this phenomenon, embryos of Japanese medaka (Oryzias latipes) were exposed to 2- or 6-hydroxychrysene (0.5, 2, or 5 μM) from 4 h-post-fertilization (hpf) to 7 d-post-fertilization. Following exposure, hemoglobin concentrations were quantified by staining fixed embryos with o-dianisidine (a hemoglobin-specific dye) and stained embryos were imaged using brightfield microscopy. Exposure to 2-hydroxychrysene resulted in a concentration-dependent decrease in hemoglobin relative to vehicle-exposed embryos, while only the highest concentration of 6-hydroxychrysene resulted in a significant decrease in hemoglobin. All tested concentrations of 2-hydroxychrysene also caused significant mortality (12.2 % ± 2.94, 38.9 % ± 14.4, 85.6 % ± 11.3), whereas mortality was not observed following exposure to 6-hydroxychrysene. Therefore, treatment of embryos with 2-hydroxychrysene at various developmental stages and durations was subsequently conducted to identify key developmental landmarks that may be targeted by 2-hydroxychrysene. A sensitive window of developmental toxicity to 2-hydroxychrysene was found between 52–100 hpf, with a 24 h exposure to 10 μM 2-hydroxychrysene resulting in significant anemia and mortality. Since exposure to 2-hydroxychrysene from 52 to 100 hpf, a window that includes liver morphogenesis in medaka, resulted in the highest magnitude of toxicity, liver development and function may have a role in 2-hydroxychrysene developmental toxicity.

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.

Effects on Trout Alevins of Chronic Exposures to Chemically Dispersed Access Western Blend and Cold Lake Blend Diluted Bitumens

The present study assessed the chronic toxicity of 2 chemically enhanced water accommodated fractions (CEWAFs) of diluted bitumens (dilbits), Access Western Blend (AWB) and Cold Lake Blend (CLB), to rainbow trout alevins. Chemical dispersion was used to overcome the resistance to dispersion of dilbits and to generate test solutions that contained more and smaller oil droplets for increased partitioning of petroleum hydrocarbons into water. Test solutions were characterized by fluorescence spectroscopy, a rapid and inexpensive analytical tool to compare toxicity endpoints measured by fluorescence (total petroleum hydrocarbons measured by fluorescence [TPH-F]). Cumulative mortality and the prevalence and severity of malformations increased following exposure of alevins to dispersed dilbits. Toxicity curves overlapped for AWB and CLB when expressed as TPH-F and 22- to 24-d median lethal and effect concentrations ranged from 0.36 to 1.5 mg/L. Gene expression in alevins was also altered following exposure to dispersed dilbit, with relative cytochrome P450-1A mRNA levels increasing up to 170-fold for AWB and up to 240-fold for CLB. Access Western Blend and CLB caused similar toxicity to rainbow trout alevins as light to medium conventional crude oils, and rainbow trout alevins were more sensitive than yellow perch, Japanese medaka, and fathead minnow embryos exposed to dispersed AWB and CLB. The present study is the first to assess the embryotoxicity of dilbits to a Canadian freshwater salmonid species. Environ Toxicol Chem 2020;39:1620-1633. © 2020 SETAC.

Transcriptional responses in newly-hatched Japanese medaka (Oryzias latipes) associated with developmental malformations following diluted bitumen exposure

Japanese medaka embryos were exposed to water accommodated fractions (WAF) and chemically-enhanced WAF of two types of diluted bitumen (dilbit) at concentrations bracketing the EC50s for developmental malformations. Within these treatments, fish were grouped based on the presence or absence of developmental malformations (e.g., blue sac disease (BSD)), and analyzed for novel transcriptomic responses. Microarray analyses identified novel biomarkers and gene networks in dilbit-exposed malformed embryos that were not evident in dilbit-exposed fish without BSD or in controls without dilbit. The top differentially expressed genes (DEGs) included cytochrome P450 transcripts (cyp1) in fish from all dilbit treatments (malformed and non-malformed fish), as well as: fibroblast growth factor (fgf7), AHR repressor (ahrr), and squalene monooxygenase (sqle). In dilbit-exposed fish that did not develop BSD, the only reported individual DEG was eukaryotic translation initiation factor 3 subunit D (eif3d). However, a number of other pathways were enriched, including melatonin effects on circadian clock and the antioxidant response, estrogen and androgen metabolism as well as many receptor signaling pathways. Pathways associated with hedgehog, steroid biosynthesis, and Wnt signaling were significantly altered between low and high concentrations of dilbit exposure. An effect of the dispersant control on swim bladder development was observed at concentrations 10-fold higher than those used to disperse dilbit, and a number of gene targets unique to fish in this comparison were affected. This suggests that the toxic effects of dispersant may involve alternative mechanisms to dilbit, but cause similar phenotypic responses. This study identified novel biomarkers in fish exposed to dilbit, with or without visual malformations, that can be used to assess the risks of dilbit to aquatic ecosystem health.

Transient exposure to environmentally realistic concentrations of di-(2-ethylhexyl)-phthalate during sensitive windows of development impaired larval survival and reproduction success in Japanese medaka

Di-(2-ethylhexyl) phthalate (DEHP) is a well-known endocrine disruptor and it is ubiquitously distributed in the environment. However, very few studies have investigated the effects of short-term exposure to environmentally realistic concentrations of DEHP during early developmental stages and its chronic effects. This study monitored the long-term effects of transient exposure to DEHP in early life stages (F0 generation) and its subsequent fertilization success in F1 generation using Japanese medaka, Oryzias latipes, as model organism. Embryos (4 h post-fertilization, 4 hpf) of Japanese medaka were exposed to 0.001 ppb, 0.1 ppb, or 10 ppb DEHP for 21 days and returned to control water (without DEHP) for maturation (4 months old). At day 9 of the exposure study, mortality was significantly increased in medaka embryos (before hatching) treated with 0.001 ppb and 10 ppb DEHP. Continual exposure of young hatchlings for an additional 12 days (a total of 21 days of exposure) resulted in a significant increase in mortality in fish exposed to 0.001 ppb, 0.1 and 10 ppb DEHP. Significant reduction in egg production was observed in adult female medaka (4 months old) with prior exposure to 0.1 ppb and 10 ppb DEHP for 21 days during early development. Fertilization and hatching success were also significantly reduced in breeding pairs with prior exposure to 0.001 ppb, 0.1 ppb and 10 ppb DEHP during early life stage. Histological analysis of adult male gonads revealed a significant decline in mature sperm count accompanied by an increase in interstitial space in fish exposed to 0.1 ppb and 10 ppb DEHP during early development. Likewise, the amount of vitellogenic (mature) oocytes observed in the ovaries of adult female with transient exposure to 0.1 ppb and 10 ppb DEHP was significantly reduced when compared with the solvent control group. Our data suggest that transient exposure to ultra low concentrations of DEHP during sensitive time windows of development results in irreversible reproductive impairment which may impact fish populations negatively.