Relationship between nitrate, heavy metal, and sterols contents in Japanese agricultural soils with risk of groundwater pollution.

In Japanese agricultural lands, nitrate-nitrogen contamination of soil and groundwater often occurs due to the application of livestock excrements and compost. Therefore, rural soils in Japan were sampled and analyzed for nitrate-nitrogen leaching, heavy metal content, and sterols associated with livestock excrement and compost to calculate contamination risk indicators. The results were analyzed using self-organizing maps and cluster analysis. Nitrate-nitrogen content using water extraction was detected in most of the sampled soils. In addition, many samples from areas that were already severely contaminated with nitrate-nitrogen showed particularly high concentrations. Coprostanol, an indicator of fecal contamination, was detected in more than half of the samples. The main source of nitrate-nitrogen contamination in these areas is livestock excrement and compost. Self-organization maps showed that areas with high nitrate-nitrogen contamination also corresponded to areas with high copper and zinc soil contents. The self-organization maps and cluster analysis resulted in five clusters: a nitrate-contaminated group mainly originating from livestock excrement and compost, a heavy metal-contaminated group, a general group, a nitrate-contaminated group mainly originating from chemical fertilizers, and a contaminated group with potentially hazardous substances requiring attention. Authorities and decision-makers can use the results to prioritize areas requiring remediation.

First evaluation of genotoxicity of strong bases and zwitterions in treated household effluents.

Various genotoxic substances in household effluents have not been sufficiently studied. The purpose of this study is to evaluate them using the umu test after dividing them based on the acid-base properties of their functional groups by solid-phase extraction cartridges. The results of the samples concentrated with reverse-phase cartridges showed that the substances with acid functional groups had stronger genotoxicity as 4.1-12.1 ng-4-NQO/mL without S9 enzyme and 17.4-51.8 ng-2-AA/mL with S9 enzyme, while the basic substances also showed a certain degree of toxicity. The results of dividing the effluents by acid-base properties using ion-exchange cartridges showed that chemical substances with strong acid functional groups did not demonstrate genotoxicity. It was found that the genotoxicity of chemicals with functional groups of weak acids was half of that of the total amount. The genotoxicity of the neutral substance was not strong, and the genotoxicity of the weak basic substances was negligible. The zwitterions and substances with strong basic functional groups showed about half the total genotoxicity. This is the first report that has investigated the genotoxicity of zwitterions in effluents.

Seasonal Fluctuation of Polycyclic Aromatic Hydrocarbons and Aerosol Genotoxicity in Long-Range Transported Air Mass Observed at the Western End of Japan.

In order to clarify the level transboundary air pollution caused by polycyclic aromatic hydrocarbons (PAHs) and genotoxic substances, aerosols were collected from forest and suburban sites in Nagasaki, west Japan, for 6 years. The PAH concentration was measured, and the genotoxicity of the substances were evaluated using the umu test. The results showed no notable trends in the concentration or toxicity of either sites throughout the study period. The suburban and forest sites shared similar seasonal fluctuation patterns and quantitative values, suggesting that the western end of Japan might be affected by long-range transported pollutants, especially in winter. PAH concentration and genotoxicity showed the same seasonal patterns of increased levels in winter and lower levels in summer. This suggests that PAHs and genotoxic substances were correlated and share common sources. Back trajectory and source analyses were conducted using the diagnostic ratios of PAHs. It was predicted that air pollution by PAHs at the forest site arise predominantly as a result of biomass or coal combustion in continental regions, such as northern parts of China and the Korean Peninsula. This is particularly expected in winter. Therefore, genotoxic substances would also be strongly influenced by transboundary pollution from the continental region. In addition, it was estimated that the contribution of transboundary PAH pollution could reach 70% at the suburban site in winter.

Evaluation of genotoxicity potential of household effluents from onsite wastewater treatment systems using umu test.

Household effluents are predominantly treated by wastewater treatment plants (WWTPs). Other treatment methods, which were examined in this study, are also used in the countryside. These treatment modes include (1) onsite toilet wastewater treatment system (OTWTS), (2) onsite wastewater treatment system (OWTS), (3) community wastewater treatment system (CWTS), and (4) onsite vault toilet (OVT). Household effluents consist of excrements and urine released from toilets as well as wastewater released from kitchens and bathrooms. In the present study, household effluents that were discharged from the residential areas having undergone similar treatment methodologies were compared using the umu test, an in vitro bioassay to assess genotoxicity potential. The different treatment methodologies were categorized based upon whether the two kinds of wastewater were mixed or not mixed and treated or not treated. Treated wastewater containing excrements and urine from the OTWTS exhibited the strongest genotoxicity potential compared to other effluents, whereas most of the kitchen and bathroom wastewater from OVT did not display genotoxicity. Data indicated that the genotoxicants in the effluents originated primarily from excrements and urine, and may increase an adverse environmental risk.

Field survey of environmental estrogen pollution in the coastal area of Tokyo Bay and Nagasaki City using the Japanese common goby Acanthogobius flavimanus.

Endocrine disrupting chemicals (EDCs) are common pollutants in coastal waters. To investigate the estrogen risk of EDCs in the coastal areas of Japan, the Japanese common goby, which is a commonly observed species in these waters, was used as the target fish. Plasma 17ß-estradiol (E2) and vitellogenin (VTG) levels were analyzed and the gonads of fish collected from the Taira River (northern Nagasaki, reference site), Nagasaki Port, and two sites in Tokyo Bay were observed. Abnormal levels (>150 ng/mL, p < 0.05) of plasma VTG and high levels of plasma E2 were detected in the fish from Nagasaki Port and Tokyo Bay, whereas the levels of both were low in the fish from the Taira River. The target EDCs, including natural estrogen [estrone (E1), and E2] and alkylphenols [4-t-octylphenol (4-t-OP), 4-nonylphenol (4-NP), and bisphenol-A (BPA)] in water samples were quantified using gas chromatography tandem mass spectrometry (GC/MS/MS), respectively. It was observed that the E2-equivalent (EEQ) in Nagasaki Port and Tokyo Bay, which was calculated from the actual EDC measurement value, were almost 20- and 150-fold higher, respectively, than that at the reference site (Taira River, 0.021 ng/L). The EEQs mostly comprised natural estrogen in the sampling sites, although there was some influence of alkylphenols. There was an association between the EEQ and the E2 in environmental water, suggesting a high estrogen risk in Japan coastal waters. Moreover, the results indicated that abnormal VTG synthesis was induced by environmental estrogen (EE) pollution in Nagasaki Port and Tokyo Bay.

Occurrence and seasonal variation of equine estrogens, equilin and equilenin, in the river water of Japan: Implication with endocrine-disrupting potentials to Japanese medaka (Oryzias latipes).

In this study, we determined the concentration of equine estrogens, such as equilin (Eq) and equilenin (Eqn), in the river water collected from nine research stations in Hokkaido, Japan. The LC-MS/MS analysis revealed that Eq concentrations were 2.7 ±â€¯6.7, 0.22 ±â€¯0.12, and 1.2 ±â€¯0.64 ng/L in Sep 2015, Feb 2016, and Jul 2016, respectively. Eqn had concentration levels similar to those of Eq. Comparison of the concentrations at nine research stations showed that seasonal variation was observed in the detected Eq and Eqn concentration levels. This study was the first to show the occurrences and seasonal variation of Eq and Eqn in the river water of Japan. We further investigated the reproductive and transgenerational effects of Eq in Japanese medaka (Oryzias latipes) exposed to 10, 100, and 1000 ng/L for 21 days and assessed the transcriptional profiles of the estrogen-responsive genes in the livers of both sexes. The reproduction assay demonstrated that 1000 ng/L of Eq adversely affected the reproduction (i.e. fecundity) in the F0 generation and that the hatching of F1 generation fertilized eggs was reduced in the 100 and 1000 ng/L treatment groups. Our qRT-PCR assay revealed that the mRNA expression levels of hepatic vitellogenin 1 and 2, choriogenin L and H, and estrogen receptor α were significantly up-regulated in males exposed to 100 and/or 1000 ng/L of Eq. In contrast, the transcriptional levels of several genes, such as pregnane X receptor and cytochrome P450 3A, were down-regulated in the livers of males after the 21-d exposure. These results suggest that Eq has endocrine-disrupting potential such as reproductive and transgenerational effects by the modulation of hepatic estrogen-responsive genes expression on medaka.

Size distribution and sources of 37 toxic species of particulate polycyclic aromatic hydrocarbons during summer and winter in Baoshan suburban area of Shanghai, China.

The objectives of this study were to assess the size-segregated distribution and sources of 37 different species of particulate polycyclic aromatic hydrocarbons (PAHs) in a suburban area of Shanghai metropolitan City, China. The ambient particulate sampling was carried out on the rooftop of a five-stories building in Baoshan campus of Shanghai University. An Andersen high-volume air sampler was employed to collect ambient size-segregated particulate matter during summer of August to September and winter of November to December 2015. The high toxic PAHs were determined by a gas chromatography mass spectrometry. The concentrations of total PAHs in suspended particulate matter (SPM) and PM1.1 (suspended particulate matter below 1.1µm in diameter) in the suburban area of Shanghai were 4.58-14.5ng/m(3) and 1.82-8.56ng/m(3), respectively in summer, and 43.6-160ng/m(3) and 23.2-121ng/m(3), separately in winter. 1,8-Naphthalic anhydride (1,8-NA) showed the highest concentration among 37 different species of PAHs in the suburban area of Shanghai. The concentrations of high molecular PAHs (e.g. 5-6 ring PAHs) followed a nearly unimodal size distribution with the highest peaks in PM1.1. The diagnostic ration qualitatively indicated that PAHs in SPM of Shanghai were mainly derived from motor-vehicle or petroleum combustion in summer and from coal and biomass combustion in winter. According to the calculated toxicity equivalency factors based on the methods of Nisbet and Lagoy and the potency equivalency factors (PEF) recommended by U.S. EPA, the highest contributors in the total carcinogenicity of the PAHs in SPM and PM1.1 were dibenzo[a,h]pyrene (46.2% and 45.0% in summer), benzo[a]pyrene (44.4% and 43.8% in winter) and benz[j]aceanthrylene (80.2% and 83.1% in summer and 83.1% and 84.0% in winter), respectively. Therefore, benzo[a]pyrene seemed to be a lower contributor than other carcinogenic PAHs.

Accumulation and pharmacokinetics of estrogenic chemicals in the pre- and post-hatch embryos of the frog Rana rugosa.

Amphibian eggs spawned in water are exposed immediately to various chemicals present in their water. The present study aimed to investigate the accumulation and pharmacokinetics of 17α-ethynylestradiol (EE(2)), bisphenol A (BPA), and nonylphenol (NP), as well as 17ß-estradiol (E(2)), in the pre-hatch and post-hatch embryos of the frog Rana rugosa. Fertilized eggs were exposed to chemicals at a final concentration of 500 nM in breeding water for two days, then the embryos with jelly coats were reared in fresh-breeding water without supplementation of the xenoestrogens for six more days. All exogenous chemicals were concentrated in the embryo body at two days after fertilization, whereas their concentrations in the jelly coat were the same as those in the breeding water. The bioconcentration factors for E(2), EE(2), BPA, and NP were 217.9, 170.2, 382.3, and 289.1, respectively, suggesting that the estrogenic chemicals were concentrated in the embryo body through the jelly coat.

Survey of contamination of estrogenic chemicals in Japanese and Korean coastal waters using the wild grey mullet (Mugil cephalus).

We monitored the contamination by environmental estrogens (EEs) of coastal areas in Korea and Japan using the wild grey mullet. The grey mullet were collected from Ansan, Jeju, Yeosu, Tongyeong, and Busan in Korea and Nagasaki, Omuta, and Fukuoka in Japan. Contamination by EEs was determined by measuring vitellogenin (VTG) levels in serum and identifying gonadal abnormalities histologically (i.e., testis-ova). In four sites in Korea (Ansan, Yeosu, Tongyeong, and Busan) and two sites in Japan (Nagasaki and Fukuoka), serum VTG in immature and male grey mullet was detected at levels greater than 1.0 microg/ml, which is considered to be an abnormal level. Although, testis-ova were observed in some individuals collected in Ansan, Tongyeong, and Busan in Korea and Omuta in Japan, there was no correlation between individuals with testis-ova and individuals with abnormal levels of VTG. Furthermore, in Japan, serum VTG levels of fish collected from Nagasaki and Fukuoka were also greater than 1.0 microg/ml. Although individuals with testis-ova were found in Omuta, these fish expressed normal levels of serum VTG. Our results suggest that the grey mullets living in these coastal areas are influenced by EEs in the environment. Furthermore, it appears that the production of VTG and the occurrence of testis-ova are caused by different mechanisms.

Acute toxicity of pharmaceutical and personal care products on freshwater crustacean (Thamnocephalus platyurus) and fish (Oryzias latipes).

Pharmaceutical and personal care products (PPCPs) enter aquatic environments via sewage treatment facilities and their potentially toxic effects on biota, particularly aquatic organisms, are of considerable concern. In this study, we investigated the acute toxicity of selected PPCPs on a freshwater crustacean (Thamnocephalus platyurus) and a fish species (Oryzias latipes). The 24-hr median lethal concentration (LC(50)) values of ibuprofen, mefenamic acid, indometacin, carbamazepine, propranolol, ifenprodil, clarithromycin and triclosan for T. platyurus were estimated to be 19.59, 3.95, 16.14, > 100, 10.31, 4.43, 94.23 and 0.47 mg/l respectively. Conversely, the 96-hr LC(50) values for these PPCPs were estimated at > 100, 8.04, 81.92, 45.87, 11.40, 8.71, > 100 and 0.60 mg/l for O. latipes, respectively. The toxic sensitivity of T. platyurus to these PPCPs, except for carbamazepine, was therefore higher than for O. latipes. No acute toxicity effects were associated with PPCPs, such as atenolol, disopyramide, famotidine, fluconazole, erythromycin and levofloxacin, in the two aquatic organisms at the concentrations tested in this study (> 100 mg/l). These findings may help us to understand the potential biological effects and risks associated with PPCP exposure in aquatic organisms. Further long-term studies are required to fully assess the growth and reproduction of these compounds on aquatic biota.

Initial ecological risk assessment of eight selected human pharmaceuticals in Japan.

Eight pharmaceuticals were selected on the basis of their domestic consumption in Japan, the excretion ratio of the parent compound and the frequency of detection in the aquatic environment or wastewater treatment plant effluent. Toxicity tests on these pharmaceuticals were conducted using Japanese medaka (Oryzias latipes), daphnia (Daphnia magna), and green algae (Psuedokirchneriella subcapitata). Predicted no effect concentration (PNEC) was calculated using lethal or effect concentration 50 (LC50 or EC50) values and no effect concentration (NOEC) obtained in the toxicity tests for these compounds. Predicted environmental concentration (PEC) was also calculated from annual consumption, the excretion rate of the parent compound, and removal rate in the preliminary batch activated sludge treatment performed in this study. Maximum concentrations found in the aquatic environment or sewage effluent in Japan or foreign countries were also used for another calculation of PEC. Initial risk assessment on the selected pharmaceuticals was performed using the PEC/PNEC ratio. The results of initial risk assessment on the eight selected pharmaceuticals suggest neither urgent nor severe concern for the ecological risk of these compounds, but further study needs to be conducted using chronic toxicity tests, including reproduction inhibition and endocrine disruption assessments.

Hormonal activity of polycyclic musks evaluated by reporter gene assay.

Synthetic musk fragrance compounds, such as polycyclic musks (PCMs), are a group of chemicals used extensively as personal care products, and can be found in the environment and the human body. PCMs, such as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (AHTN), are known to have agonistic activities toward human estrogen receptor alpha (hERalpha) and hERbeta, and have antagonistic activity toward the human androgen receptor (hAR), as shown in several reporter gene assays. However, little is known about the interaction of PCMs with the human thyroid hormone receptor (hTR), and the hormonal effects of other PCMs except for HHCB and AHTN. In this study, we focus on the interactions of six PCMs, namely, HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butyl-indan (ADBI), 6-acetyl-1,1,2,3,3,5-hexamethylindan (AHMI), 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI), and 5-acetyl-1,1,2,6-tetramethyl-3-isopropy-lindan (ATII) with hERalpha, hAR, and hTRbeta by in vitro reporter gene assay using Chinese hamster ovary cells. All the samples were found to be agonists toward hERalpha, whereas no agonistic activities of these PCMs for hAR and hTRbeta were observed. No antagonistic activities for hERalpha and hTRbeta were observed at the concentrations tested. However, several PCMs, namely, HHCB, AHTN, ATII, ADBI, and AHMI, showed dose-dependent antagonistic activities for hAR, and the IC50 values of these compounds were estimated to be 1.0 x 10(-7), 1.5 x 10(-7), 1.4 x 10(-7), 9.8 x 10(-6), and 1.4 x 10(-7) M, respectively. The results suggest that these PCMs interact with hERalpha and hAR but have no hormonal effect on hTRbeta. This is the first report on the agonistic and antagonistic activities of ATII, ADBI, AHMI, and DPMI for hERalpha and hAR as determined by in vitro reporter gene assay using stably transfected Chinese hamster ovary cells.

In vitro evaluation of atmospheric particulate matter and sedimentation particles using yeast bioassay system.

Little information on the evaluation of airborne particulate matter (APM) and sedimentation particles from subway stations is available. The thermal metamorphism of train wheels generating toxic particles in subway stations is a possibility. In this study, the toxicity and physiological effects of particles from subway stations were evaluated using a yeast bioassay system. Estrogenic and antiestrogenic activities of APM in APM extracts from subway stations were determined. No estrogenic activity was found in the APM fractions and their S9-activated APM samples. Sedimentation dust samples also showed no estrogen activity. In contrast, extracts from sedimentation dust samples showed antiestrogen activity. Marked yeast toxicity was observed in the samples extracted from sedimentation dust. Potent yeast toxicity was also found in the S9-activated extracts from sedimentation dust. The results suggest that sedimentation dust from a semiclosed area of a subway system has antiestrogen activity, although both the origin and generation system of this activity are uncertain. These pollutants in sedimentation dust may change to a more toxic form in vivo by S9 activation.

Reproductive effects and bioconcentration of 4-nonylphenol in medaka fish (Oryzias latipes).

The objective of this study was to investigate the reproductive success (fertility and fecundity) of medaka (Oryzias latipes) exposed to 4-nonylphenol (4-NP) in water and the viability of their F(1) offspring. In addition, we measured the bioconcentration of 4-NP in eggs. After a 21-d exposure to 100microg/l 4-NP, medaka showed reduced egg production and fertility. Hepatic vitellogenin levels were increased significantly in males treated with 10, 50 and 100microg/l of 4-NP. In the F(1) generations, the hatchability and time to hatching of embryos in the 100microg/l treatment group were adversely affected, and 2-7microg 4-NP/g egg was found in spawned eggs (the bioconcentration factor: 30-100). These results indicate that (1) 4-NP adversely affected the reproduction (fecundity and fertility) of adult medaka, (2) 4-NP accumulated in eggs through maternal transfer and (3) these levels of 4-NP were associated with adverse effects in the F(1) offspring.

Effects of triclosan on the early life stages and reproduction of medaka Oryzias latipes and induction of hepatic vitellogenin.

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is widely used as antibacterial agent in various industrial products, such as textile goods, soap, shampoo, liquid toothpaste and cosmetics, and often detected in wastewater effluent. In this study, the effects of TCS on the early life stages and reproduction of medaka (Oryzias latipes) were investigated. The 96-h median lethal concentration value of TCS for 24-h-old larvae was 602 microg/l. The hatchability and time to hatching in fertilized eggs exposed to 313 microg/l TCS for 14 days were significantly decreased and delayed, respectively. An assessment of the effects of a TCS 21-day exposure period on the reproduction of paired medaka showed no significant differences in the number of eggs produced and fertility among the control and 20, 100 and 200 microg/l TCS treatment groups. However, concentrations of hepatic vitellogenin were increased significantly in males treated with TCS at 20 and 100 microg/l. In the F(1) generations, although the hatching of embryos in the 20 microg/l treatment showed adverse effects, there was no dose-response relationship between hatchability and TCS treatment levels. These results suggest that TCS has high toxicity on the early life stages of medaka, and that the metabolite of TCS may be a weak estrogenic compound with the potential to induce vitellogenin in male medaka but with no adverse effect on reproductive success and offspring.

Styrene dimers and trimers affect reproduction of daphnid (Ceriodaphnia dubia).

The endocrine disruptor activity of styrene in humans and other vertebrates appears to be negligible. However, offspring numbers were reduced in Ceriodaphnia dubia bred in polystyrene cups. Styrene dimers and trimers were found to be eluted from the polystyrene cups by hexane and methanol with gas chromatography-mass spectrometry. Styrene dimers and trimers at concentrations of 0.04-1.7 microg/l affected C. dubia fertility (25% reduction after seven days), suggesting that styrenes have the potential to impair crustacean populations in the aquatic environment.