Development of a microplate-based novel toxicity bioassay using Chlorophyta and Phaeophyceae macroalgae.

Macroalgae are one of the main producers in marine environments. However, only a few toxicity test methods have been established that use reference strains of macroalgae to evaluate the effects of chemicals on the growth and reproduction of macroalgae to monitor water quality. We selected reference strains of Chlorophyta, Ulva aragoënsis; Phaeophyceae, Ectocarpus siliculosus; and wakame, Undaria pinnatifida, as test species to establish a microplate-based method to investigate the toxicity of potassium dichromate, 3,5-dichlorophenol, and two common herbicides (diuron and simazine). We determined the growth of the three macroalgae in their early life stages and during the sporangia formation stage in E. siliculosus under laboratory conditions. We observed that the growth and sporangia formation in these algae were impaired in a dose-dependent manner. Additionally, we investigated the sensitivity of these macroalgae by comparing the toxicity values of toxicants used in this study with those obtained from a database. Compared to other microalgae and plant species, macroalgae showed a relatively high sensitivity to organic compounds, including herbicides. Growth tests using U. aragoënsis and E. siliculosus produced reliable results at 0-32 and 25-32 practical salinity units (PSU), respectively. The tests established in this study could test the toxicity of chemical substances in macroalgae and are thus expected to contribute to a better understanding of the environmental risks of chemical substances on aquatic biota. The tests could be applied to all effluent toxicity tests used for the management of seawater and brackish water quality.

Environmental RNA as a Noninvasive Tool for Assessing Toxic Effects in Fish: A Proof-of-concept Study Using Japanese Medaka Exposed to Pyrene.

Although environmental RNA (eRNA) is emerging as a noninvasive tool to assess the health status of aquatic macroorganisms, the potential of eRNA in assessing chemical hazards remain largely untested. In this study, we investigated the ability of eRNA to detect changes in gene expression in Japanese medaka fish (Oryzias latipes) in response to sublethal pyrene exposure, as a model toxic chemical. We performed standardized acute toxicity tests and collected eRNA from tank water and RNA from fish tissue after 96 h of exposure. Our results showed that over 1000 genes were detected in eRNA and the sequenced read counts of these genes correlated with those in fish tissue (r = 0.50). Moreover, eRNA detected 86 differentially expressed genes in response to pyrene, some of which were shared by fish RNA, including the suppression of collagen fiber genes. These results suggest that eRNA has the potential to detect changes in gene expression in fish in response to environmental stressors without the need for sacrificing or causing pain to fish. However, we also found that the majority of sequenced reads of eRNA (>99%) were not mapped to the reference medaka genome and they originated from bacteria and fungi, resulting in low sequencing depth. In addition, eRNA, in particular nuclear genes, was highly degraded with a median transcript integrity number (TIN) of <20. These limitations highlight the need for future studies to improve the analytical methods of eRNA application.

The complete mitochondrial genome of water flea Ceriodaphnia dubia (Crustacea: Cladocera) NIES strain.

Water flea Ceriodaphnia dubia has been widely used for risk assessments of chemicals and environmental contamination. In this study, the complete mitochondrial genome (mitogenome) of this species NIES strain was determined using short-read high throughput and long-read sequencing technologies. The mitogenome of C. dubia was 15,170 bp in length and consisted of 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs), and 22 transfer RNAs (tRNAs). The gene order was identical to the pattern conserved across crustaceans. The complete mitogenome of the NIES strain will serve as genetical reference in ecological risk assessments in Japan, as well as resources for future phylogenetical studies using cladocerans.

Adverse effects of thyroid-hormone-disrupting chemicals 6-propyl-2-thiouracil and tetrabromobisphenol A on Japanese medaka (Oryzias latipes).

Thyroid-hormone-disrupting chemicals are increasingly attracting attention because of their potential harmful effects on animal health, including on fishes. Here, we investigated the effects of exposure to the thyroid-hormone-disrupting chemicals 6-propyl-2-thiouracil (PTU) and tetrabromobisphenol A (TBBPA) on swim bladder inflation, eye development, growth, swimming performance, and the expression of thyroid-related genes in Japanese medaka (Oryzias latipes). PTU exposure resulted in reductions in eye size, growth, and swim bladder inflation, and these effects led to poorer swimming performance. These phenotypic effects were accompanied by increased expression of the thyroid-stimulating hormone subunit beta (tshß) paralog tshß-like, but there were no significant changes in expression for tshß, deiodinase 1 (dio1), deiodinase 2 (dio2), and thyroid hormone receptor alpha (trα) and beta (trß). For PTU exposure, we identified the key event (swim bladder inflation reduction) and an adverse outcome (swimming performance reduction). No significant effects from TBBPA exposure were seen on swim bladder inflation, eye development, growth, or swimming performance. However, expression of tshß-like and tshß (significantly enhanced) and trα and trß (significantly reduced) were affected by TBBPA exposure albeit not in dose-dependent manners. There were no effects of TBBPA on the expression of dio1 and dio2. We thus show that the two thyroid-hormone-disrupting chemicals PTU and TBBPA differ in their effect profiles with comparable effects on the studied phenotypes and thyroid-related gene expression to those reported in zebrafish.

Rapid transcriptomic and physiological changes in the freshwater pennate diatom Mayamaea pseudoterrestris in response to copper exposure.

Diatoms function as major primary producers, accumulating large amounts of biomass in most aquatic environments. Given their rapid responses to changes in environmental conditions, diatoms are used for the biological monitoring of water quality and for performing ecotoxicological tests in aquatic ecosystems. However, the molecular basis for their toxicity to chemical compounds remains largely unknown. Here, we sequenced the genome of a freshwater diatom, Mayamaea pseudoterrestris NIES-4280, which has been proposed as an alternative strain of Navicula pelliculosa UTEX 664 for performing the Organisation for Economic Co-operation and Development ecotoxicological test. This study shows that M. pseudoterrestris has a small genome and carries the lowest number of genes among freshwater diatoms. The gene content of M. pseudoterrestris is similar to that of the model marine diatom, Phaeodactylum tricornutum. Genes related to cell motility, polysaccharide metabolism, oxidative stress alleviation, intracellular calcium signalling, and reactive compound detoxification showed rapid changes in their expression patterns in response to copper exposure. Active gliding motility was observed in response to copper addition, and copper exposure decreased intracellular calcium concentration. These findings enhance our understanding of the environmental adaptation of diatoms, and elucidate the molecular basis of toxicity of chemical compounds in algae.

Severity Classification of Clinical Signs and Defining the Moribund State as an Experimental Endpoint for Acute Toxicity Testing Using Japanese Medaka (Oryzias latipes).

Use of the moribund state as an endpoint for acute toxicity testing in fish is complicated by the lack of consensus about the sublethal signs that define the state or that can predict imminent death. Defining the moribund state in fish requires determination of the association between each observable sublethal sign and transition to imminent death. To establish a criterion for defining the moribund state in Japanese medaka (Oryzias latipes), we determined death/clinical-sign ratios, defined as the proportion of fish with each clinical sign that transitioned to imminent death, for 13 clinical signs observed in individually housed fish exposed to one of two typical toxicants, three pharmaceuticals, two pesticides, and one metal. Also, the time from onset of each clinical sign to transition to imminent death (referred to as the survival time) was determined by continuous monitoring. Three of the observed clinical signs-immobility, lethargy, and immobility at the surface-were found to be indicators of the moribund state, with each of these signs having a death/clinical-sign ratio of 1.0. Evaluation of the survival time after onset of the other 10 signs enabled determination of whether timely euthanasia would be appropriate, thereby providing a means of reducing the suffering of laboratory fish in the period before death. Environ Toxicol Chem 2022;41:1089-1095. © 2022 SETAC.

Potential differences in chitin synthesis ability cause different sensitivities to diflubenzuron among three strains of Daphnia magna.

Ecotoxicity testing of crustaceans using Daphnia magna has been implemented in the chemical management systems of various countries. While the chemical sensitivity of D. magna varies depending on genetically different clonal lineages, the strain used in ecotoxicity tests, including the acute immobilization test (OECD TG202), has not been specified. We hypothesized that comprehensive gene expression profiles could provide useful information on phenotypic differences among strains, including chemical sensitivity. To test this hypothesis, we performed mRNA sequencing on three different strains (NIES, England, and Clone 5) of D. magna under culture conditions. The resulting expression profile of the NIES strain was clearly different compared to the profiles of the other two strains. Gene ontology (GO) enrichment analysis suggested that chitin metabolism was significantly enriched in the NIES strain compared to that in the England strain. Consistent with the GO analysis, evidence of high levels of chitin metabolism in the NIES strain were observed across multiple levels of biological organization, such as expression of chitin synthase genes, chitin content, and chitinase activity, which suggested that the different strains would exhibit different sensitivities to chemicals used to inhibit chitin synthesis. We found that among all strains, the NIES strain was more tolerant to diflubenzuron, a chitin synthesis inhibitor, with a 14-fold difference in the 48 h-EC50 value for the acute immobilization test compared to the England strain. The present study demonstrates that the differences among strains in chitin metabolism may lead to sensitivity difference to diflubenzuron, and serves as a case study of the usefulness of comprehensive gene expression profiles in finding sensitivity differences.

The complete mitochondrial genome of the non-biting midge Chironomus yoshimatsui (Diptera: Chironomidae).

A non-biting midge Chironomus yoshimatsui has been widely used in ecotoxicology and chemical risk assessments. In this study, the complete mitochondrial genome (mitogenome) sequence of C. yoshimatsui was determined using short-read next-generation sequencing technologies. The mitogenome was 15,734 bp in length and consisted of 13 protein coding genes, 2 ribosomal RNAs, and 22 transfer RNAs. The A + T content was 77.8%. The gene order was identical to the pattern conserved across Diptera. The mitocgenome sequence obtained in this study provides a useful resource for further evolutionary and ecological studies.

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.

Arteriolar hyalinosis is related to rapid GFR decline and long-standing GFR changes observed on renal biopsies in normo-microalbuminuric type 2 diabetic patients.

AIMS: Whether or not renal structural changes, especially arteriolar hyalinosis, are related to the rate of renal functional decline and increase in urinary albumin excretion (UAE) at the early stage of diabetic nephropathy in patients with type 2 diabetes is still unknown. Our previous study determined that arteriolar hyalinosis is an independent risk factor for low GFR. We sought to determine whether arteriolar hyalinosis is also a risk factor for rapidly progressive decline in GFR. METHODS: We evaluated 22 type 2 diabetic patients with normo- or microalbuminuria who took part in the previous study, to clarify which renal histological factors were associated with changes in UAE and the glomerular filtration rate (GFR) during 11.0 ±â€¯3.0 years of follow-up. Light and electron microscopy-based morphometric analyses were performed to quantitatively evaluate the glomerular and interstitial structural changes. RESULTS: In all 22 patients, the GFR was significantly decreased from baseline to follow-up, while the UAE did not change markedly between the 2 periods. After 11 ±â€¯3 years of follow-up, the GFR was significantly lower in the rapid decliner group (annual rate of GFR decline ≥3.0%) than in the non-rapid decliner group (p = 0.017). The index of arteriolar hyalinosis (IAH) at baseline in the rapid decliners was significantly larger than in the non-rapid decliners (p = 0.015). The IAH showed a significant negative correlation with the GFR at follow-up (r = 0.50, p = 0.018) and the annual rate in the GFR decline (r = 0.47, p = 0.027) and significant positive correlations with UAE at follow-up (r = 0.46, p = 0.034) and the annual rate in the UAE increase (r = 0.57, p = 0.005). The GFR at follow-up in patients with IAH ≥2.0 was significantly decreased from baseline (p = 0.042) and significantly lower than that of the patients with IAH <2.0 (p = 0.026), which did not decrease significantly from baseline. The frequency of rapid decliners was larger in the IAH ≥2.0 than in the IAH <2.0 patients (p = 0.037). CONCLUSIONS: Aggravated arteriolar hyalinosis was a risk factor for a rapid GFR decline. This finding might reflect initial changes in early diabetic nephropathy.

Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide.

Raphidocelis subcapitata is one of the most frequently used species for algal growth inhibition tests. Accordingly, many microalgal culture collections worldwide maintain R. subcapitata for distribution to users. All R. subcapitata strains maintained in these collections are derived from the same cultured strain, NIVA-CHL1. However, considering that 61 years have passed since this strain was isolated, we suspected that NIVA-CHL1 in culture collections might have acquired various mutations. In this study, we compared the genome sequences among NIVA-CHL1 from 8 microalgal culture collections and one laboratory in Japan to evaluate the presence of mutations. We found single-nucleotide polymorphisms or indels at 19,576 to 28,212 sites per strain in comparison with the genome sequence of R. subcapitata NIES-35, maintained at the National Institute for Environmental Studies, Tsukuba, Japan. These mutations were detected not only in non-coding but also in coding regions; some of the latter mutations may affect protein function. In growth inhibition test with 3,5-dichlorophenol, EC50 values varied 2.6-fold among the 9 strains. In the ATCC 22662-2 and CCAP 278/4 strains, we also detected a mutation in the gene encoding small-conductance mechanosensitive ion channel, which may lead to protein truncation and loss of function. Growth inhibition test with sodium chloride suggested that osmotic regulation has changed in ATCC 22662-2 and CCAP 278/4 in comparison with NIES-35.

Theory of coevolution of cytoplasmic male-sterility, nuclear restorer and selfing.

Gynodioecy is a sexual polymorphism in angiosperms, where hermaphroditic and female individuals coexist. This is often caused by a cytoplasmic genetic element (CGE) that destroys male functions, which is called cytoplasmic male-sterility (CMS). On the other hand, nuclear genes tend to evolve the ability to restore male function. The coevolutionary process of CMS and the restoration has been studied theoretically. Recently, a theoretical study suggested that these coevolutionary dynamics could be influenced by the rate of selfing within populations, although it assumed that the selfing rate of a population was a fixed parameter. Accordingly, we theoretically study the coevolution of three traits in this paper: CMS, nuclear restorer and selfing rate, in which we hypothesize that selfing evolution can suppress CMS evolution under some conditions. The analysis indicates three significant properties of the system; (1) CMS-restorer evolution can result in bistability under a given selfing rate, (2) the coevolution of three traits can realize intermediate levels of selfing, and (3) the evolution of high levels of selfing is conditionally associated with no CMS and/or no restoration, which may support our hypothesis.

Serial renal biopsies in normo- and microalbuminuric patients with type 2 diabetes demonstrate that loss of renal function is associated with a reduction in glomerular filtration surface secondary to mesangial expansion.

AIMS: The aim of the present study was to explore the relationship between changes in renal structure in patients with type 2 diabetes at an early stage of diabetic nephropathy using serial renal biopsies, and change in renal function. METHODS: The study population comprised 10 patients with type 2 diabetes with normo- or microalbuminuria at baseline. Light and electron microscopy-based morphometric analyses were performed to quantitatively evaluate glomerular and interstitial structural changes. Urinary albumin excretion (UAE) and glomerular filtration rate (GFR) were measured annually. A second renal biopsy was performed after a mean of 6.1 ±â€¯2.4 years of follow-up. RESULTS: UAE, GFR, blood pressure and severity of diabetic retinopathy (DR) did not change between the baseline and follow-up. The annual decrease in the surface density of the peripheral glomerular basement membrane (GBM) (Sv[PGBM/glom]) was correlated with the rate of loss of GFR during the six-year follow-up period. The annual change in the Sv(PGBM/glom) was negatively correlated with the change in mesangial volume fraction. CONCLUSIONS: Decreases in the GFR in patients with type 2 diabetes with normo- or microalbuminuria at baseline were associated with a decreased glomerular filtration surface, as a result of mesangial expansion during a mean six years of observation. These findings confirm ongoing pathological progression of glomerulopathy despite no significant change in albuminuria or retinopathy status.

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.

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.

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.

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.

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 © 2017 John Wiley & Sons, Ltd.