Current Status of Antifouling Biocides Contamination in the Seto Inland Sea, Japan.

A monitoring survey of antifouling biocides was conducted in the Harima Nada Sea and Osaka Bay of the Seto Inland Sea, Japan to assess contamination by organotin (OT) compounds and alternative biocides. The concentrations of tributyltin (TBT) compounds in surface water ranged from 1.0 to 2.8 ng/L, and the detected TBT concentrations in the bottom water layer were higher than those in the surface water. The concentrations of TBT compounds in sediment samples ranged from 2.0 to 28 ng/g dry weight (dw), respectively. The concentrations of alternative biocides in the water and sediment were lower than those before the banning of TBT by the International Maritime Organization (IMO). Although triphenyltin (TPT) compounds were not detected in water samples, TPT compounds were detected in the range of < 0.1-2700 ng/g dw in sediment samples. Their concentrations in the water samples were as follows: diuron, < 1-53 ng/L; Sea-Nine 211, < 1-1.8 ng/L; Irgarol 1051, < 1-4.0 ng/L; dichlofluanid, < 1-343 ng/L; and chlorothalonil, < 1-1 ng/L, and the ranges of these alternative compounds in sediment samples were diuron, 32-488 ng/g dw; Sea-Nine 211, 47-591 ng/g dw; Irgarol, 33-128 ng/g dw; dichlofluanid, 67-8038 ng/g dw; and chlorothalonil, 31-2975 ng/g dw. Thus, the OTs and alternative biocides have still been detected in water and sediment samples from closed sea areas.

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.

Toxicity and Environmental Risk Assessment of Polycarbamate and Its Main Components to Marine Algae and Crustaceans.

Polycarbamate is commonly used as an antifoulant coating on fishing nets in Japan. Although its toxicity to freshwater organisms has been reported, its toxicity to marine organisms is currently unknown. We conducted algal growth inhibition and crustacean immobilization tests to assess the effects of polycarbamate on marine organisms. We also evaluated the acute toxicity of the main components of polycarbamate, namely, dimethyldithiocarbamate and ethylenebisdithiocarbamate, to algae, which are the most sensitive tested organisms to polycarbamate. The toxicities of dimethyldithiocarbamate and ethylenebisdithiocarbamate partially explain that of polycarbamate. To assess the primary risk, we derived the predicted no-effect concentration (PNEC) for polycarbamate in a probabilistic manner using species sensitivity distributions. The 72 h no observed effect concentration (NOEC) of polycarbamate to the alga Skeletonema marinoi-dohrnii complex was 0.45 µg/L. The toxicity of dimethyldithiocarbamate may have contributed up to 72% of the toxicity observed for polycarbamate. The fifth percentile of hazardous concentration (HC5) derived from the acute toxicity values was 0.48 µg/L. Comparison of previously reported environmental polycarbamate concentrations in Hiroshima Bay, Japan, to the PNEC estimated using the minimum NOEC and HC5 suggest that polycarbamate currently poses a high ecological risk. Therefore, reducing the risk by restricting polycarbamate use is necessary.

Integrated transcriptomic and metabolomic analyses reveal mechanism underlying higher resistance of the marine oligochaete Thalassodrilides cf. briani (Clitellata: Naididae) to heavy contamination of sediments with polycyclic aromatic hydrocarbons.

In some coastal areas, sediments are contaminated with various chemical compounds, causing significant threats to marine organisms. Therefore, the development of remediation techniques is important. Here, we focused on bioremediation using marine benthic animals such as aquatic oligochaetes. The oligochaete Thalassodrilides cf. briani is highly resistant to contamination of sediments with toxic chemicals. We examined whether T. cf. briani could decompose high-concentration polycyclic aromatic hydrocarbons (PAHs) in sediments. Furthermore, relevant genes expressed in T. cf. briani exposed to contaminated sediment were comprehensively examined using next-generation sequencing, and its metabolites were identified by metabolomic analysis using gas chromatography-mass spectrometry. T. cf. briani reduced the concentration of 16 PAHs in the sediment from 55,900 to 45,200 ng/g dry weight in 50 days, thereby reducing total PAH concentrations by approximately 20%. The results of transcriptomic analysis suggest that activation of a drug-metabolizing enzyme system may promote the metabolism of harmful chemical substances during excretion of chemicals from the body. According to the results of principal component analysis based on the values of 43 types of metabolomes identified by metabolomic analysis, groups were divided according to the difference in the number of exposure days. In addition, levels of glutamine, which is important for maintaining digestive tract functions, increased. This suggests that the digestive tract function promotes the metabolism and detoxification of foreign substances. Furthermore, transcriptome analysis revealed that glutamate dehydrogenase increased 1.3-fold and glutamine synthetase increased 1.7-fold, confirming the increase in glutamine. Thus, we conclude that T. cf. briani adapted to the polluted sediment by regulating its metabolism.

Estimation of the uptake and gut retention of microplastics in juvenile marine fish: Mummichogs (Fundulus heteroclitus) and red seabreams (Pagrus major).

We investigated the impact of microplastics (MPs) on marine fish by estimating the uptake and retention by mummichogs (Fundulus heteroclitus) and red seabreams (Pagrus major) of MPs similar in size (≥0.25 mm) and composition (polyethylene) to MPs detected in fish intestines. Results revealed a correlation between MP concentrations in aquarium water and the content of MPs in the gastrointestinal tracts of exposed fish. More than 95% were excreted from both species within 25 h; this retention time is similar to the processing time of food items. The rate of excretion showed little dependence on MP size, but there was some dependence on fish species and MP shape. These results suggest that MPs similar to those we studied have little direct adverse impact on these two marine fish species.

Ecological risk assessment of an antifouling biocide triphenyl (octadecylamine) boron in the Seto Inland Sea, Japan.

In this study, we derived the predicted no-effect concentrations (PNEC) for triphenyl (octadecylamine) boron (TPB-18) and investigated the occurrence of triphenylboranes (TPBs), including TPB-18, for ecological risk assessment in the Seto Inland Sea, Japan. We tested algal growth inhibition, crustacean immobilization, and reproductive toxicity and performed toxicity tests in fish to assess acute and chronic toxicity and generate the PNEC for TPB-18. The minimum toxicity value was 0.30 µg/L, as determined by the 72-h no-observed-effect concentration (NOEC) for the alga Chaetoceros gracilis. The 5th-percentile of hazardous concentration (HC5), derived from NOECs using the species sensitivity distributions approach, was 0.059 µg/L, which indicated the PNEC of 0.0059 µg/L. In comparison, the highest concentration in seawater sampled from the Seto Inland Sea was 0.00034 µg/L, suggesting that the ecological risks posed by TPB-18 are currently low.

Occurrence of neonicotinoids and fipronil in estuaries and their potential risks to aquatic invertebrates.

This study aimed to evaluate and qualify field-based potential risks of seven neonicotinoid and phenylpyrazole (fipronil) insecticides on aquatic invertebrates, including estuary-resident marine crustaceans. One hundred and ninety-three estuarine water samples, with salinity ranging from 0.5 to 32.7, were collected from four estuarine sites in the Seto Inland Sea of Japan, in 2015-2018 and the insecticide levels were measured. Five neonicotinoid and fipronil insecticides were successfully identified, and their occurrence varied temporally. Marine crustaceans were simultaneously harvested every month from one of the estuarine water sampling sites in 2015-2017. Three predominant crustacean species, kuruma prawn (Penaeus japonicus), sand shrimp (Crangon uritai), and mysid (Neomysis awatschensis), were captured and their seasonal presence was species independent. A 96-h laboratory toxicity study with the insecticides using kuruma prawn, sand shrimp, and a surrogate mysid species (Americamysis bahia) indicated that fipronil exerted the highest toxicity to the three crustaceans. Using both toxicity data and insecticide occurrence in estuarine water (salinity ≥10, n = 169), the potential risks on the three marine crustaceans were quantified by calculating the proportion of mixture toxicity effects (Pmix). The Pmix of seven neonicotinoids on the crustaceans was less than 0.8%, which is likely to be too low to indicate adverse effects caused by the insecticides. However, short temporal detection of fipronil (exclusively in June and July) significantly affected the Pmix, which presented the maximal Pmix values of 21%, 3.4%, and 72% for kuruma prawn, sand shrimp, and mysid, respectively, indicating a significant effect on the organisms. As for estuarine water (salinity <10), some water samples contained imidacloprid and fipronil exceeding the freshwater benchmarks for aquatic invertebrates. The present study provides novel insights into the seasonally varying risks of insecticides to estuarine crustaceans and highlights the importance of considering whether ecological risk periods coincide with crustacean presence.

Physiological responses of eelgrass (Zostera marina) to ambient stresses such as herbicide, insufficient light, and high water temperature.

This study aimed to elucidate the biological responses of eelgrass (Zostera marina) to artificially induced stresses such as herbicide (Irgarol 1051, Irg) exposure, insufficient light, and high water temperature (27 ± 1.0 °C) by evaluating growth inhibition, photosynthetic activity, and metabolomic profiles. After 14 days, all treatments inhibited growth, but photosynthetic activity was only reduced in the Irg-exposed group. In the Irg-exposed and insufficient light groups, the metabolomic profiles were characterized by decreased levels of sugar (sucrose) and increased levels of amino acids such as glutamine, glycine, and leucine. Biochemical and ultrastructural analyses revealed that the loss of sugar-derived metabolic energy was compensated for by energy generated during autophagic protein degradation. Furthermore, the level of myo-inositol, which has various biological roles and participates in several cellular processes such as cell wall synthesis, stress response, and mineral nutrient storage, was markedly increased in the Irg-exposed and insufficient light groups. A combination of metabolomic analysis with other analyses such as measurement of photosynthetic activity might further elucidate the response of eelgrass to ambient stresses in the natural environment.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species.

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ±â€¯36 mg) were about 12 and 630 µg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 µg/L and a no-observed-effect concentration of 2.1 µg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 µg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

Detection and localization of the thyroid hormone receptor beta mRNA in the immature olfactory receptor neurons of chum salmon.

Thyroid hormone (TH) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in various organs and tissues of vertebrates. It is generally accepted that anadromous Pacific salmon (Oncorhynchus spp.) imprint odorants from their natal stream during their seaward migration, and they then use olfaction to discriminate their natal stream during the spawning migration. Both serum TH levels and the specific binding values of TH in the salmon olfactory epithelium were markedly increased during the seaward migration. However, thyroid hormone receptor (TR) expression in the olfactory epithelium has not been confirmed in vertebrates. We investigated gene expression of TR isoforms in chum salmon (O. keta) by both molecular biological and histochemical techniques. Expression of TRß mRNA was detected in the olfactory epithelium by reverse transcriptase polymerase chain reaction (RT-PCR). Nucleotide sequencing demonstrated the existence of a remarkable homology between the RT-PCR product and part of the ligand-binding domain of other teleost TRß isoforms. By in situ hybridization using a digoxygenin-labeled salmon olfactory TRß cRNA probe, signals for salmon olfactory TRß mRNA were observed preferentially in the perinuclear regions of immature olfactory receptor neurons (ORNs), as protein gene product 9.5 (PGP9.5)-immunopositive ORNs. Our results provide the first detection of TRß gene expression in the olfactory epithelium, and suggested the possibility that TRß may be involved in cell maturation and/or cell differentiation of the ORNs in Pacific salmon.

Development of a method to assess the ichthyotoxicity of the harmful marine microalgae Karenia spp. using gill cell cultures from red sea bream (Pagrus major ).

The present study reports the development of a method to investigate ichthyotoxicity of harmful marine microalgae using cultured red sea bream (Pagrus major) gill cells. The cultured gill cells formed adherent 1-2 layers on the bottom of the culture plate and could tolerate seawater exposure for 4 h without significant alteration in cell survival. The microalgae Karenia mikimotoi, Karenia papilionacea, K. papilionacea phylotype-I, and Heterosigma akashiwo were cultured, then directly exposed to gill cells. After K. mikimotoi and K. papilionacea phylotype-I exposure, live cell coverage was significantly lower than in the cells exposed to a seawater-based medium (control cells; P < 0.05). Toxicity of K. mikimotoi cells was weakened when cells were ruptured, and was almost inexistent when the algal cells were removed from the culture by filtration. Significant cytotoxicity was detected in the concentrated ruptured cells, and in the concentrated of ruptured cells after freezing and thawing though cytotoxicity was weakened; whereas, cytotoxicity almost disappeared after heat treatment. In addition, examination of the distribution of toxic substances from the ruptured cells showed that cytotoxicity mainly occurred in the fraction with the resuspended pellet after centrifugation at 3000×g.

Effect of taurine supplementation on hepatic metabolism and alleviation of cadmium toxicity and bioaccumulation in a marine teleost, red sea bream, Pagrus major.

This study was performed to unravel the mechanism of the beneficial action of taurine on marine teleost fish, red sea bream (Pagrus major), by analyzing the hepatic metabolism. Moreover, the ameliorative effects of the nutrient against cadmium toxicity and bioaccumulation were further evaluated. The fish were fed a diet containing 0 % (TAU0 %), 0.5 % (TAU0.5 %), or 5.0 % (TAU5.0 %) taurine for 40-55 days (d) and subjected to cadmium acute toxicity and bioaccumulation tests. Taurine deficiency in feed severely affected growth and the hepatic metabolic profiles of the fish, including a remarkable increase in myo-inositol, aspartate, and ß-alanine in the TAU0 % group, which indicates a complementary physiological response to taurine deficiency. For the acute toxicity test, fish were fed the test diets for 55 d and were then exposed to different dose of cadmium ranging from 0 to 5.6 mg/L for 96 h. Fish fed taurine had a higher tolerance to cadmium than those not fed taurine. For the bioaccumulation test, fish were fed the test diets for 40 d and then were chronically exposed to 0.2 mg/L of cadmium for 28 d followed by depuration for 21 d. Cadmium concentrations in the liver and muscle of fish fed TAU5.0 % were significantly lower than those of fish fed TAU0 % for the first 7 d of exposure and the first 7 d of elimination. Our findings suggest a possible mechanism for the beneficial role played by taurine and that the inclusion of taurine in fish aquaculture feed may reduce cadmium contamination of fish intended for human consumption.

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.

A hepatic metabolomics-based diagnostic approach to assess lethal toxicity of dithiocarbamate fungicide polycarbamate in three marine fish species.

The present study was performed to evaluate the toxic effect of the dithiocarbamate fungicide polycarbamate (PC) on the hepatic metabolic profiles of three marine fish species, red sea bream (Pagrus major), spotted halibut (Verasper variegatus), and marbled flounder (Pleuronectes yokohamae). First, juvenile fish were exposed to graded concentrations of PC for 96h; the 96-h LC50 values obtained were 22-29, 239-553, and 301-364µgL-1 for red sea bream, spotted halibut, and marbled flounder, respectively, indicating that red sea bream possessed higher sensitivity to PC than the two benthic species. Second, the fish were exposed to lethal-equivalent concentration (H group) or sub-lethal (one-tenth of the H group concentrations; L group) for 24 and 96h and gas-chromatography based metabolomics approach was employed to explore the crucial biomarker metabolite associated with lethal toxicity. Of the 53 metabolites identified, only reduced glutathione (GSH) was consistently elevated in the H group for the three fish species at 96h. The calculated cut-off value of GSH (mM) based on receiver operating curve analysis between H group and the other treatment groups (control, solvent control, and L group) was obtained at 0.56mM, which allowed to distinguish between the groups with high confidence for the three fish species. These results are the first to demonstrate the potential of using GSH as a possible biomarker metabolite and its usefulness of threshold cut-off value for diagnosing life-threatening health conditions of fish.

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.

Transcription of a novel P450 gene varies with some factors (pollutant exposure, temperature, time, and body region) in a marine oligochaete (Thalassodrilides sp.).

Cytochrome P450 (CYP) enzymes play important roles in the metabolism of exogenous compounds such as polycyclic aromatic hydrocarbons (PAHs). A novel, full-length CYP gene (CYP4V30) was identified in the oligochaete Thalassodrilides sp. CYP4V30 mRNA expression was studied in worms exposed to PAH-polluted (Σ16PAHs; 37441ng/g dry weight) or unpolluted (Σ16PAHs; 19ng/g dry weight) sediment. CYP4V30 expression was much higher in worms exposed to contaminated sediments than in those exposed to unpolluted sediments at some temperatures (20 and 25°C) and exposure durations (11-fold increase at 20°C, 10-day exposure), but not at 15°C or other exposure durations (P<0.05). CYP4V30 mRNA expression was higher in the middle of the body than in the posterior (P<0.05). The variation in transcriptional response with exposure time, temperature, and body region indicates that these factors should be considered when monitoring marine sediment pollution.

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.

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.

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.