The Thermal Stability of the Collagen Triple Helix Is Tuned According to the Environmental Temperature.

Triple helix formation of procollagen occurs in the endoplasmic reticulum (ER) where the single-stranded α-chains of procollagen undergo extensive post-translational modifications. The modifications include prolyl 4- and 3-hydroxylations, lysyl hydroxylation, and following glycosylations. The modifications, especially prolyl 4-hydroxylation, enhance the thermal stability of the procollagen triple helix. Procollagen molecules are transported to the Golgi and secreted from the cell, after the triple helix is formed in the ER. In this study, we investigated the relationship between the thermal stability of the collagen triple helix and environmental temperature. We analyzed the number of collagen post-translational modifications and thermal melting temperature and α-chain composition of secreted type I collagen in zebrafish embryonic fibroblasts (ZF4) cultured at various temperatures (18, 23, 28, and 33 °C). The results revealed that thermal stability and other properties of collagen were almost constant when ZF4 cells were cultured below 28 °C. By contrast, at a higher temperature (33 °C), an increase in the number of post-translational modifications and a change in α-chain composition of type I collagen were observed; hence, the collagen acquired higher thermal stability. The results indicate that the thermal stability of collagen could be autonomously tuned according to the environmental temperature in poikilotherms.

Structural optimization of cyclic peptides that efficiently detect denatured collagen.

To develop a facile method for detecting denatured collagen, we investigated the structure-activity relationship of cyclic collagen-mimetic peptides (cCMPs). Reported cCMP prototypes tend to self-assemble and they must be disassembled just before use. Introducing charge repulsion and a deformation in the peptide backbone structure enabled cCMPs to detect denatured collagen without a pre-treatment for disassembly. Using the optimized cCMP, types I-V collagen were detected by western blotting and denatured collagen fibrils were visualized in a cell culture system.

Cyclic Peptides for Efficient Detection of Collagen.

We report here a new class of collagen-binding peptides, cyclic collagen-mimetic peptides (cCMPs), that efficiently hybridize with the triple-helix-forming portions of collagen. cCMPs are composed of two parallel collagen-like (Xaa-Yaa-Gly)n strands with both termini tethered by covalent linkages. Enzyme-linked immunosorbent assays and western blotting analysis showed that cCMPs exhibit more potent affinity toward collagen than reported collagen-binding peptides and can specifically detect different collagen polypeptides in a mixture of proteins. Collagen secreted from cultured cells was detected by confocal microscopy with fluorescein-labeled cCMP. The cCMP is also shown to detect sensitively folding intermediates in the endoplasmic reticulum, something that was difficult to visualize with conventional collagen detectors. Molecular-dynamics simulations suggested that a cCMP forms a more stably hybridized product than its single-chain counterpart; this could explain why cCMP has higher affinity toward denatured collagen. These results indicate the usefulness of cCMPs as tools for detecting denatured collagen.

Reproductive toxicity of 1-nitronaphthalene and 1-nitropyrene exposure in the mummichog, Fundulus heteroclitus.

After pregnant mummichog were implanted with 1-nitronaphthalene or 1-nitropyrene via cholesterol pellet, we investigated the effects of the chemicals on embryo normality, hatchability and days to hatch of normal embryos, and growth and survival of hatched larvae from normal embryos of the implanted mummichog. Hatchability was the parameter most sensitive to the effects of both 1-nitronaphthalene and 1-nitropyrene. The 4-week lowest-observed-effect concentration (LOEC) of 1-nitronaphthalene, based on the actual concentrations in the eggs in the test, was 447 ng g(-1) wet wt.; and the LOEC and no-observed-effect concentration (NOEC) of 1-nitropyrene were 958 and 344 ng g(-1) wet wt., respectively. The 4-week LOEC of 1-nitronaphthalene, based on the concentration in the water, was estimated at 4.8 µg L(-1) by using the reported bioconcentration factor; and the LOEC and NOEC of 1-nitropyrene, based on the concentration in the water, were estimated at 3.1 and 8.6 µg L(-1), respectively. The reported environmental concentrations of 1-nitropyrene and 1-nitronaphthalene are over three magnitudes lower than the toxicity values we obtained. Therefore, the effects of environmental levels of 1-nitropyrene and 1-nitronaphthalene on fish reproduction, not including genomic effects on embryos, appear to be almost negligible. However, DNA damage has been detected in marine organisms exposed to 1-nitropyrene. Further studies of the genotoxicity of nitrated polycyclic aromatic hydrocarbons at environmental levels are therefore needed to evaluate their ecotoxicological risks.

Molecular cloning and gene expression of mummichog (Fundulus heteroclitus) Runx2 during embryogenesis.

In our previous study, we clarified the toxicity of 2,2'-dipyridyldisulfide [(PS)2], one of photodegradation products of a metal pyrithione that is used as an alternative antifouling paint biocides to organotin compounds in Japan. In early life stage toxicity tests, we exposed the mummichog, (Fundulus heteroclitus) to (PS)2, and the hatched larvae subsequently displayed notochord undulations and skeletal deformities ( Mochida et al., 2012 ). Runx2, a transcription factor of the runt family, is a key regulator in skeletal development in mammals. It is possible that (PS)2 inhibits Runx2 gene expression, inducing the skeletal deformities in mummichog. In the present study, we cloned two Runx2 cDNAs (type I and type II) from mummichog embryos. The deduced amino acid sequences of type I and type II contain an open reading frame encoding 450 and 464 amino acid residues, respectively. The derived amino acid sequence of Fundulus Runx2 type I showed the highest identity (93.8%) with Takifugu Runx2 type I, and Fundulus Runx2 type II showed 94.6% homology with medaka Runx2. The expression level of Runx2 mRNA in the early stage series was measured using a real-time quantitative PCR assay. Expression levels tended to increase in both the blastula-gastrula and the retinal pigmentation stage. To examine the effect of toxic compounds on skeletal formation, mummichog embryos in the late blastula to retinal pigmentation stage were exposed to (PS)2. After exposure to (PS)2 for one week, the expression level of Runx2 mRNA was unchanged. These results suggest that there is no inhibition of Runx2 gene expression due to (PS)2 exposure.

Toxicity of degradation products of the antifouling biocide pyridine triphenylborane to marine organisms.

We evaluated the acute toxicities of the main degradation products of pyridine triphenylborane (PTPB), namely, diphenylborane hydroxide (DPB), phenylborane dihydroxide (MPB), phenol, and biphenyl, to the alga Skeletonema costatum, the crustacean Tigriopus japonicus, and two teleosts, the red sea bream Pagrus major and the mummichog Fundulus heteroclitus. DPB was the most toxic of the degradation products to all four organisms. The acute toxicity values of DPB for S. costatum, T. japonicus, red sea bream, and mummichog were 55, 70, 100, and 200-310 µg/L, respectively. The degradation products were less toxic than PTPB to S. costatum and T. japonicus; however, the toxicities of DPB and PTPB to the fish species were similar. We also examined changes in the inhibition of growth rate of S. costatum as well as the percentage of immobilization of T. japonicus as end points of toxicity of PTPB after irradiation of PTPB with 432 ± 45 W/m(2) of 290-700 nm wavelength light. After 7 days of irradiation with this light, the concentration of PTPB in the test solutions decreased markedly. A decrease in toxic effects closely coincided with the decrease in the concentration of PTPB caused by the irradiation. PTPB probably accounted for most of the toxicity in the irradiation test solutions. Because the concentrations of PTPB that were acutely toxic to S. costatum and T. japonicus were <10 % of the corresponding concentrations of its degradation products, PTPB probably accounted for most of the toxicity in the irradiation test solutions.

[Contribution of clinical laboratory to the diabetes care team: efforts in physiological laboratory].

A large number of clinical laboratory technologists are qualified as diabetes educators; however, few of them actually participate in teaching patients. This is partially because it is difficult to balance their routine laboratory tasks and the work of a diabetes educator. We have introduced ultrasonic examinations of the ophthalmic artery, inspection of the R-R interval and current perceptual-threshold inspection for the early diagnosis of diabetic complications, and we have been contributing to the good medical care for diabetes. Furthermore, to care for diabetic foot lesions, clinical laboratory technologists have participated in checking diabetic patients' feet since 2007. In concrete terms, we examine the feet of diabetic patients, take digital pictures of the feet, and write a report, while preparing for thermographic examination of the patient. At the same time, we give simple guidance about foot care. Technologists cannot perform medical treatment; however, this has been accepted by medical staff because we only check foot lesions. We make use of existing medical imaging and reporting systems in the physiological laboratory, so doctors and nurses on the diabetic care team can always obtain information about the patients. Such actions have a good reputation not only among medical staff but also among diabetic patients.

Toxicity of metal pyrithione photodegradation products to marine organisms with indirect evidence for their presence in seawater.

We evaluated the acute toxicities of the metal pyrithiones (MePTs)--copper pyrithione (CuPT) and zinc pyrithione (ZnPT)--to four species of marine algae and a marine crustacean (Tigriopus japonicus). We also performed acute toxicity tests using six of the main MePT photodegradation products: pyridine-N-oxide (PO); 2-mercaptopyridine (HPS); pyridine-2-sulfonic-acid (PSA); 2-mercaptopyridine-N-oxide (HPT); 2,2'-dithio-bis-pyridine ([PS](2)); and 2,2'-dithio-bis-pyridine-N-oxide ([PT](2))-and three marine organisms representing three trophic levels: an alga (Skeletonema costatum), a crustacean (T. japonicus), and a fish (Pagrus major). The acute toxicity values (72-h EC(50)) of CuPT, ZnPT, HPT, (PT)(2), (PS)(2), HPS, PO, and PSA for S. costatum, which was the most sensitive of the test organisms to the chemicals tested, were 1.5, 1.6, 1.1, 3.4, 65, 730, >100,000, and >100,000 microg l(-1), respectively. CuPT was detected in the growth media used for S. costatum tests and in seawater containing HPT or (PT)(2); the concentration of CuPT in seawater containing HPT was highly dependent on the Cu(2+) concentration. These results indicate that in the presence of sufficient Cu(2+), the toxicities of HPT and (PT)(2) should be assessed as CuPT because in Japan MePTs are most frequently used as antifouling booster biocides in conjunction with cuprous oxide.

Lowering the culture temperature corrects collagen abnormalities caused by HSP47 gene knockout.

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes triple helical portions of procollagens. The chaperone function of HSP47 is indispensable in mammals, and hsp47-null mice show an embryonic lethal phenotype accompanied by severe abnormalities in collagen-based tissue structures. Two leading hypotheses are currently accepted for the molecular function of HSP47 as a procollagen-specific chaperone. One is facilitation of procollagen folding by stabilizing thermally unstable triple helical folding intermediates, and the other is inhibition of procollagen aggregation or lateral association in the ER. The aim of this study was to elucidate the functional essence of this unique chaperone using fibroblasts established from hsp47-/- mouse embryos. When the cells were cultured at 37 °C, various defects in procollagen biosynthesis were observed, such as accumulation in the ER, over-modifications including prolyl hydroxylation, lysyl hydroxylation, and further glycosylation, and unusual secretion of type I collagen homotrimer. All defects were corrected by culturing the cells at a lower temperature of 33 °C. These results indicated that lowering the culture temperature compensated for the loss of HSP47. This study elucidated that HSP47 stabilizes the elongating triple helix of procollagens, which is otherwise unstable at the body temperature of mammals.

Early life-stage toxicity test for copper pyrithione and induction of skeletal anomaly in a teleost, the mummichog (Fundulus heteroclitus).

We used a teleost fish, the mummichog (Fundulus heteroclitus), to conduct early life-stage toxicity testing for copper pyrithione (CuPT). Fertilized mummichog eggs were exposed to CuPT at various concentrations for 50 d under continuous flow-through conditions. Hatchability, survival, growth, and morphologic abnormalities were measured. Hatchability did not differ significantly between any experimental group and control groups. Survival and growth were significantly reduced at 50 d in the groups exposed to 2 or 4 microg/L CuPT. During the test, morphologic abnormalities, such as vertebral deformity and formation of inflammatory masses in the lateral muscles, occurred in fish exposed to CuPT. Light and electron microscopic studies indicated that muscle dysfunction played a role in the vertebral deformity and revealed that the inflammatory mass was composed mainly of macrophages and necrotic myocytes. We consider that macrophages infiltrated and phagocytized necrotic cells, thus forming the inflammatory mass. In addition, acetylcholinesterase activity was markedly decreased in the 2- and 4-microg/L exposure groups, suggesting the skeletal deformity was due to mechanisms similar to those proposed for organophosphorous pesticide exposure.

Molecular cloning of two estrogen receptors expressed in the testis of the Japanese common goby, Acanthogobius flavimanus.

We isolated cDNA clones of two estrogen receptors (ER1 and ER2) from testis of the Japanese common goby (Acanthogobius flavimanus). The cDNAs of ER1 contained 3,796 nucleotides, including an open reading frame encoding 564 amino acids (Mr: 61.9 kDa); the cDNA for ER2 was 3,274 nucleotides long, with an open reading frame of 567 amino acids (Mr: 63.5 kDa). The deduced aminoacid sequences of ER1 and ER2 each had a characteristic ER structure consisting of five domains (A/B, DNA-binding, D, ligand-binding, and F) and were homologous to ERalpha and ERbeta of other vertebrates. We therefore named ER1 and ER2 as goby ERalpha (gERalpha) and goby ERbeta (gERbeta), respectively. The DNA- and ligand-binding domains in each gER showed high similarity to the corresponding domains of other vertebrates. Analysis of the distribution of gERalpha and gERbeta mRNAs in tissue by reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that gERalpha was expressed at high levels in brain and testis, and gERbeta was expressed most strongly in testis. In situ hybridization showed that the mRNA of each gER was expressed mainly in the Sertoli cells of goby testis.

Molecular and histological evaluation of tributyltin toxicity on spermatogenesis in a marine fish, the mummichog (Fundulus heteroclitus).

There is still concern about the effects of organotin compounds (OTs) on marine organisms, and especially on their reproductive systems. We investigated the toxicity of tributyltin oxide (TBTO) on spermatogenesis in a marine fish, mummichog, Fundulus heteroclitus. TBTO exposure caused serious histological damage to the testis, including reduction in counts of spermatids and spermatozoa and malformation of somatic cells around the seminal duct. Analysis of the incorporation of bromodeoxyuridine into spermatogenic cells revealed inhibition of the proliferation of germ cells. To find a biomarker for evaluation of the effects of TBTO on fish spermatogenesis, we cloned genes downregulated by TBTO exposure in the mummichog testis, and identified mummichog creatine kinase (mCK). The cDNA sequence of mCK contained an open reading frame encoding 387 amino acid residues (M(r)=43,344). The derived amino acid sequence of mCK was very similar to that of the testicular-type CK of the rainbow trout, Oncorhynchus mykiss. Furthermore, Northern blot analysis revealed that mCK was produced specifically in the testis. We therefore identified mCK in the mummichog as a testicular-type CK. Real-time PCR revealed that exposure of the fish to TBTO significantly reduced mCK expression in the testis. To some extent, this reduction was coincident with that of bromodeoxyuridine incorporation into spermatogenic cells. The mCK gene can therefore be used as a biomarker for evaluating the effects of TBTO on fish spermatogenesis. In addition, levels of expression of the mCK gene in control fish were well correlated with increments in the gonad somatic index (GSI) below 4%. Individuals that were thought to have testicular damage caused by TBTO could be discriminated from those considered normal. The results suggest that TBTO is involved in the suppression of fish spermatogenesis and that analysis of both GSI values and mCK gene expression is useful for evaluating the levels of xenobiotic pollution in coastal areas.

Effects of estrone on full life cycle of Java medaka (Oryzias javanicus), a new marine test fish.

Estrone is a natural estrogen detected in sewage treatment works effluents and in estuarine waters. However, there is little information on the effects of estrone on marine fish. This study investigated the effects of estrone on reproduction of the estuarine fish, Java medaka (Oryzias javanicus). Java medaka were exposed to concentrations of 39, 198, 484, 1,188, and 3,701 ng/L of estrone from embryonic stages up to adult stages for 239 d after hatching. The fertility and egg numbers of Java medaka exposed to 1,188 and 3,701 ng/L were significantly lower than that of control. The hepatic vitellogenin concentrations in male Java medaka exposed to estrone greater than 484 ng/L were significantly higher than that of control. Oocytes in testis (testis-ova) were not detected in the males in any of the exposure groups. The lowest-observed-effect concentration and no-observed-effect concentration for Java medaka were 484 and 198 ng/L of estrone. These results suggest that in relatively low estrone concentrations, 39 and 198 ng/L, Java medaka will not be affected by exposure to estrone.

Photo-induced toxicity and oxidative stress responses in Tigriopus japonicus exposed to nitro-polycyclic aromatic hydrocarbons and artificial light.

Photo-induced toxicity is an important phenomenon in ecotoxicology because sunlight reaches many organisms in their natural habitats. To elucidate whether sunlight enhances the toxicity of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs), the acute toxicities of 10 nitro-PAHs and the related compound 1-nitropyrene (1-NP) to Tigriopus japonicus were assessed in darkness or under light conditions. In addition, the relationships among the toxicity of 1-NP to T. japonicus, lighting condition, and the concentration of reactive oxygen species (ROS) formed were investigated in the presence or absence of the ROS scavenger ascorbic acid in the test solutions. Light irradiation increased the toxicity of all tested nitro-PAHs except 1,5-dinitronaphthalene. Among the compounds tested, 1-NP was the most phototoxic: it was more than 1000 times more toxic under the light conditions than in darkness. In contrast, at the same light levels, pyrene was not phototoxic. Light irradiation induced the generation of ROS in the 1-NP exposure groups, and the immobilization rate of T. japonicus increased with the amount of ROS produced. The addition of ascorbic acid to the test solutions suppressed both the generation of ROS and the light-induced immobilization of T. japonicus. To accurately assess the ecotoxicologic risk of nitro-PAHs, their overall photo-induced toxicity must be considered.

Acute toxicity of pyrithione antifouling biocides and joint toxicity with copper to red sea bream (Pagrus major) and toy shrimp (Heptacarpus futilirostris).

We evaluated the median lethal concentrations (LC50s) of the pyrithione (PT) antifoulants copper pyrithione (CuPT) and zinc pyrithione (ZnPT) to a teleost, red sea bream (Pagrus major), and a crustacean, toy shrimp (Heptacarpusfutilirostris). The 96-h LC50 values of CuPT and ZnPT, on the basis of actual concentrations, were 9.3 and 98.2 R.g/L, respectively, for red sea bream and 2.5 and 120 microg/L, respectively, for toy shrimp. Histological observations revealed that the secondary lamellae of the gill filaments of the experimental fish were heavily damaged after exposure to the PTs, suggesting that fatal hypoxemia was one cause of death. Because CuPT and ZnPT are usually used in combination with Cu, we also estimated the joint toxicities of the PTs with Cu using the LC50 values of the PTs and those of Cu (84.4 and 113 microg/L for red sea bream and toy shrimp, respectively). The results suggested that the joint toxicity of the ZnPT and Cu mixture is more than the additive toxicities of CuPT and Cu, especially in toy shrimp. The enhancement of toxicity in the mixture was inferred to be caused by conversion of ZnPT to the more toxic CuPT in the presence of Cu.

Evaluation of bioremediation potential of three benthic annelids in organically polluted marine sediment.

This study aimed to evaluate the possible remedial effects of three marine benthic annelids on organically polluted sediments from the waters of Hatsukaichi Marina, Hiroshima, Japan. Two polychaetes, Perinereis nuntia and Capitella cf. teleta, and an oligochaete, Thalassodrilides sp., were incubated in sediments for 50 days. Their effects on physicochemical properties such as organic matter (loss on ignition), redox potential (Eh), acid volatile sulfides (AVS), and degradation of polycyclic aromatic hydrocarbons (PAHs) were assessed. The polychaetes P. nuntia and C. cf. teleta significantly increased Eh level and decreased AVS level compared with the oligochaete Thalassodrilides sp. and control (without benthic organisms). Total PAH concentration significantly decreased from the initial level with all three groups; Thalassodrilides sp. had a marked ability to reduce PAHs in sediment. These results indicate that benthic organisms have species-specific remediation properties and ecological functions in organically polluted sediments.

Differences in the ability of two marine annelid species, Thalassodrilides sp. and Perinereis nuntia, to detoxify 1-nitronaphthalene.

Bioremediation is a promising method for remediating environmentally polluted water. We investigated the abilities of two benthic annelid species to biotransform 1-nitronaphthalene, a nitrated polycyclic aromatic hydrocarbon. We used an oligochaete, Thalassodrilides sp. (Naididae), collected from the sediment beneath a fish farm and a polychaete, Perinereis nuntia, which was obtained from a commercial source. Populations of both organisms were exposed to 1400 µg L(-1) of 1-nitronaphthalene in seawater for 3 days in the dark at 20 °C. The concentration of the pollutant decreased to 12 µg L(-1) in the seawater containing the Thalassodrilides sp. and to 560 µg L(-1) in the seawater containing P. nuntia. The 1-nitronaphthalene concentration in the bodies of the animals increased from 12 to 94 µg kg(-1) in Thalassodrilides sp. and from 0.90 µg kg(-1) to 38,000 µg kg(-1) in P. nuntia. After 3 days, 99% and 40% of the 1-nitronaphthalene had been biotransformed in the Thalassodrilides sp. and P. nuntia experimental groups, respectively. We then tested the acute toxicity of residual 1-nitronaphthalene from the same water using mummichog (fish) larvae. After the larvae had been exposed for 96 h, the percentage of apparently unaffected larvae remaining was 83.3% in Thalassodrilides sp. group but only 16.7% in the P. nuntia group. Clearly, of the two species we studied, Thalassodrilides sp. had a superior ability to convert 1-nitronaphthalene into substances that were nontoxic to mummichog larvae. Therefore, we recommend the use of this species for bioremediation of chemically polluted sediments.

Effects of 17beta-estradiol on the reproduction of Java-medaka (Oryzias javanicus), a new test fish species.

17beta-estradiol (E2) is a female hormone which is known to be one of the strongest estrogenic chemicals in the environment. The present study investigated the effects of E2 on the reproduction of the estuarine fish, Java-medaka (Oryzias javanicus). Starting from the embryonic stages, Java-medaka was exposed for 6 months to 9.5, 16, 68, 159 and 243 ng/l of E2. The fecundity of Java-medaka exposed to E2 levels >16 ng/l was significantly lower than that of the control. The appearance of secondary sexual characteristics seemed to be inhibited by exposure to 159 and 243 ng/l E2. Vitellogenin concentrations in the liver of male fish exposed to 68, 159 and 243 ng/l were significantly higher than that of the control. Thirty-three and sixty percent of male fishes exposed to 159 and 243 ng/l, respectively, had testis-ova. These results suggest that reproduction in the Java-medaka, especially the male fish, could be affected by exposure to E2 concentrations greater than 16 ng/l.

Spatial analysis of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (Sea-Nine 211) concentrations and probabilistic risk to marine organisms in Hiroshima Bay, Japan.

We analyzed the spatial distribution of an antifouling biocide, 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (Sea-Nine 211) in the surface water and sediments of Hiroshima Bay, Japan to determine the extent of contamination by this biocide. A quantitative estimate of the environmental concentration distribution (ECD) and species sensitivity distributions (SSDs) for marine organisms were derived by using a Bayesian statistical model to carry out a probabilistic ecological risk analysis, such as calculation of the expected potentially affected fraction (EPAF). The spatial distribution analysis supported the notion that Sea-Nine 211 is used mainly for treatment of ship hulls in Japan. The calculated EPAF suggests that approximately up to a maximum of 0.45% of marine species are influenced by the toxicity of Sea-Nine 211 in Hiroshima Bay. In addition, estimation of the ecological risk with a conventional risk quotient method indicated that the risk was a cause for concern in Hiroshima Bay.

Primary risk assessment of dimethyldithiocarbamate, a dithiocarbamate fungicide metabolite, based on their probabilistic concentrations in a coastal environment.

The primary ecological risk of dimethyldithiocarbamate (DMDC), a dithiocarbamate fungicide (DTC) metabolite, was evaluated based on their probabilistic environmental concentration distributions (ECDs) in the coastal environment, Hiroshima Bay, Japan. And their behavior and temporal trends was further considered. This is the first report of the identification of DMDC from environmental seawater and sediment samples. DMDC concentrations in bottom seawater were substantially higher than those in surface seawater, which are associated with the leachability from sediments in bottom seawaters, and with photodegradation in surface seawaters. Furthermore, seasonal risks are dominated by higher concentrations from April to June, indicating temporal variation in the risk to exposed species. Hierarchical Bayesian analysis offered DMDC ECD medians and range (5th to 95th percentiles) of 0.85 ng L(-1) (0.029, 22), 12 ng L(-1) (3.2, 48) and 110 ng kg dry(-1) (9.5, 1200) in surface seawater, bottom seawater and sediment, respectively. Considering that DMDC and DTCs have similar toxicological potential to aquatic organisms, the occurrence of the compound in water is likely to be of biological relevance. In summary, this work provides the first demonstration that the ecological risk of DMDC and its derived DTCs in Hiroshima Bay is relatively high, and that DTCs should be a high priority for future research on marine contamination, especially in bottom seawaters.