Combined impact of pesticides and other environmental stressors on animal diversity in irrigation ponds.

Rice paddy irrigation ponds can sustain surprisingly high taxonomic richness and make significant contributions to regional biodiversity. We evaluated the impacts of pesticides and other environmental stressors (including eutrophication, decreased macrophyte coverage, physical habitat destruction, and invasive alien species) on the taxonomic richness of freshwater animals in 21 irrigation ponds in Japan. We sampled a wide range of freshwater animals (reptiles, amphibians, fishes, mollusks, crustaceans, insects, annelids, bryozoans, and sponges) and surveyed environmental variables related to pesticide contamination and other stressors listed above. Statistical analyses comprised contraction of highly correlated environmental variables, best-subset model selection, stepwise model selection, and permutation tests. Results showed that: (i) probenazole (fungicide) was a significant stressor on fish (i.e., contamination with this compound had a significantly negative correlation with fish taxonomic richness), (ii) the interaction of BPMC (insecticide; also known as fenobucarb) and bluegill (invasive alien fish) was a significant stressor on a "large insect" category (Coleoptera, Ephemeroptera, Hemiptera, Lepidoptera, Odonata, and Trichoptera), (iii) the interaction of BPMC and concrete bank protection was a significant stressor on an "invertebrate" category, (iv) the combined impacts of BPMC and the other stressors on the invertebrate and large insect categories resulted in an estimated mean loss of taxonomic richness by 15% and 77%, respectively, in comparison with a hypothetical pond with preferable conditions.

Changes in feeding habits of the starspotted smooth-hound, Mustelus manazo, in Tokyo Bay between periods with different stock size levels.

We investigated differences in the feeding habits of the starspotted smooth-hound, Mustelus manazo, in Tokyo Bay between the mid-1990s (low stock size) and the late 2000s (high stock size). The frequency of M. manazo with empty stomachs increased from 5.9% in the mid-1990s to 16.1% in the late 2000s. A decrease in the relative weight of the stomach contents was evident from the mid-1990s to the late 2000s, especially in the small size classes, along with changes in the species composition in the stomach contents. Although crustaceans were the main constituents of the stomach contents, the proportion of crabs increased while those of shrimps and hermit crabs decreased. Changes in the feeding habits of M. manazo may be associated with shifts in the benthic community structure in Tokyo Bay.

Characterization and comparison of transcriptional activities of the retinoid X receptors by various organotin compounds in three prosobranch gastropods; Thais clavigera, Nucella lapillus and Babylonia japonica.

Two cDNAs of RXR were isolated, for the first time, from the ivory shell, Babylonia japonica, and the transcriptional activities were tested in vitro to compare with other gastropod (Thais clavigera and Nucella lapillus) RXR isoforms. The transcriptional activities of all of these RXR isoforms were significantly induced by mammalian RXR agonist, 9-cis retinoic acid (9cRA). The transcriptional activity of T. clavigera RXR-1 was also examined by using 9cRA and 16 organotin compounds, and significant ligand-dependent transactivations were observed by 9cRA and 5 organotins (tributyltin (TBT), tetrabutyltin (TeBT), tripropyltin (TPrT), tricyclohexyltin (TcHT) and triphenyltin (TPhT)). These 5 organotins also induced significant transcriptional activities in N. lapillus and B. japonica RXR isoforms. These 4 organotins, except for TeBT, have been reported to promote the development of imposex after a month of a single injection each, using female T. clavigera. To investigate the function of gastropod RXR isoforms, the effects of mammalian specific pan-agonist, PA024, and pan-antagonist, HX531, were examined, and significant induction of transcriptional activity by PA024 was demonstrated in these gastropod RXR isoforms. The additions of HX531 significantly suppressed the transcriptional activities of these gastropod RXR isoforms by 9cRA and 5 organotins. Using the mammalian two retinoic acid response elements, the transcriptional activities by 2 agonists, 9cRA and PA024, were different among the RXR isoforms of each gastropod species. With retinoid X response element (RXRE), transcriptional activities of TcRXR-1, BjRXR-1, and NlRXRa were significantly higher than those of TcRXR-2, BjRXR-2, and NlRXRb. Transcriptional activities of TcRXR-2, BjRXR-2, and NlRXRb, however, were significantly higher than those of TcRXR-1, BjRXR-1, and NlRXRa with thyroid hormone response element, TREpal. Thus, induction of imposex in prosobranch gastropods is strongly suggested to be triggered by 9cRA and certain organotins, such as TBT and TPhT through the activation of RXRs. These gastropod RXRs might control the different gene transcription by forming homo- or heterodimer complex with their own isoforms. These findings will contribute to our understanding of the fundamentals of the endocrine system in molluscs, particularly on RXR signaling pathway.

Development and validation of a simulation method, PeCHREM, for evaluating spatio-temporal concentration changes of paddy herbicides in rivers.

In pesticide risk management in Japan, predicted environmental concentrations are estimated by a tiered approach, and the Ministry of the Environment also performs field surveys to confirm the maximum concentrations of pesticides with risk concerns. To contribute to more efficient and effective field surveys, we developed the Pesticide Chemicals High Resolution Estimation Method (PeCHREM) for estimating spatially and temporally variable emissions of various paddy herbicides from paddy fields to the environment. We used PeCHREM and the G-CIEMS multimedia environmental fate model to predict day-to-day environmental concentration changes of 25 herbicides throughout Japan. To validate the PeCHREM/G-CIEMS model, we also conducted a field survey, in which river waters were sampled at least once every two weeks at seven sites in six prefectures from April to July 2009. In 20 of 139 sampling site-herbicide combinations in which herbicides were detected in at least three samples, all observed concentrations differed from the corresponding prediction by less than one order of magnitude. We also compared peak concentrations and the dates on which the concentrations reached peak values (peak dates) between predictions and observations. The peak concentration differences between predictions and observations were less than one order of magnitude in 66% of the 166 sampling site-herbicide combinations in which herbicide was detected in river water. The observed and predicted peak dates differed by less than two weeks in 79% of these 166 combinations. These results confirm that the PeCHREM/G-CIEMS model can improve the efficiency and effectiveness of surveys by predicting the peak concentrations and peak dates of various herbicides.

Inter- and intra-individual variation in urinary concentrations of parabens in male and female Japanese subjects.

We evaluated the representativeness of concentrations of parabens in a spot urine sample for the assessment of long-term exposure levels. Urine sample was taken monthly from 10 male Japanese subjects (35.9 ± 6.8 years) and 12 female Japanese university students (21.1 ± 0.4 years) for 5 months and measured for methyl (MP), ethyl (EP), propyl (PP) and butyl (BP) parabens by liquid chromatography-tandem mass spectrometry. Median (min-max) specific-gravity-adjusted urinary concentrations of the male group (n = 10) were 39.7 (2.99-268), 1.69 (< 0.045-75.2), 0.569 (< 0.11-123) and 0.0264 (< 0.020-24.4) ng mL-1 for MP, EP, PP and BP, respectively. Those of the female group (n = 12) were 283 (5.49-1687), 9.30 (0.290-487), 22.9 (< 0.11-307) and 3.76 (< 0.020-135) ng mL-1, respectively, which were significantly higher than those of the male group. Intra-class correlation coefficients (ICCs) were calculated for the four parabens to find 0.56, 0.58, 0.39 and 0.28 for MP, EP, PP and BP, respectively, in the male group, and 0.40, 0.43, 0.41 and 0.37 for MP, EP, PP and BP, respectively, in the female group. The results suggested that four paraben concentrations in a spot urine sample moderately reflected long-term paraben exposure of Japanese subjects. Source of exposure to parabens is also discussed.

Paraben exposure and semen quality of Japanese male partners of subfertile couples.

OBJECTIVES: Possible relationship between semen quality (semen volume, sperm concentration and sperm motility) and parabens exposure was investigated in male partners of couples who visited a gynecology clinic in Tokyo for infertility consultation (n = 42, 36.8 ± 5.4 years). METHODS: Semen parameters were measured according to WHO guideline at the clinic, and urinary methyl- (MP), ethyl- (EP), propyl- (PP) and butyl (BP) paraben concentrations were measured with liquid chromatography-tandem mass spectrometry. RESULTS: Geometric mean urinary concentrations (geometric standard deviation) of the subjects were 48.2 (4.52), 1.88 (4.72), 1.13 (6.75) and 0.184 (11.1) ng/mL for MP, EP, PP and BP, respectively. No significant association was found between semen parameters and urinary paraben concentrations in multiple regression analyses and logistic regression analyses. CONCLUSIONS: Two reasons of the absence of adverse effects on semen quality might be suggested: lower paraben exposure level of the subjects and small sample size. Further investigation of effect of paraben exposure among general male population at environmental levels is warranted.

Respiratory Uptake and Depuration Kinetics of Perfluorooctanesulfonate (PFOS) in a Marine Sandworm Species.

We determined the respiratory uptake and depuration kinetics of perfluorooctanesulfonate (PFOS) in Perinereis wilsoni, a polychaete sandworm used as a model species to investigate the fate of chemical pollutants in coastal environments. The sandworms were kept in gravel-packed containers, and the water levels were varied cyclically to mimic the tides. We used seawater kept at 17.1°C. A 7-day exposure period was followed by a 9-day depuration period. The dissolved PFOS concentration averaged 28 ng/L during the exposure period. Sandworm samples were collected regularly for analysis of PFOS concentrations, and a first-order-kinetics model was applied to the concentrations. The respiratory absorption efficiency of PFOS was estimated to be 11% that of oxygen, which is higher than the corresponding estimates reported for several fish species. The estimated depuration half-life of 15 days was comparable to previously reported estimates for fish and oligochaete species. The bioconcentration factor was 470.

Maternal Exposure to Pyrethroid Insecticides during Pregnancy and Infant Development at 18 Months of Age.

The possible association between maternal exposure to pyrethroid insecticides (PYRs) during pregnancy and infant development was explored. Levels of exposure to PYRs was assessed by metabolite (3-phenoybenzoic acid, 3-PBA) concentration in maternal spot urine sampled in the first trimester of index pregnancy, and infant development was assessed at 18 months of age using the Kinder Infants Development Scale (KIDS), which is based on a questionnaire to the caretaker. The relationship between KIDS score and maternal urinary 3-PBA levels was examined by a stepwise multiple regression analysis using biological attributes of the mother and infant, breast feeding, and nursing environment as covariates. The analysis extracted 3-PBA and the nursing environment as significant to explain the KIDS score at 18 months of age with positive partial regression coefficients. Inclusion of fish consumption frequency of the mother during pregnancy as an independent variable resulted in the selection of fish consumption as significant, while the two variables were marginally insignificant but still with a positive coefficient with the KIDS score. The result suggested a positive effect of maternal PYR exposure on infant development, the reason for which is not clear, but an unknown confounding factor is suspected.

Effect of water temperature on survival of early-life stages of marbled flounder Pseudopleuronectes yokohamae in Tokyo Bay, Japan.

We investigated factors that might have disturbed the stock recovery of marbled flounder in Tokyo Bay by focusing on the early life stages. Field surveys in Tokyo Bay from 2006 to 2011 revealed that mature adult biomass increased from 2006 to 2008 and decreased thereafter. Meanwhile, larval and juvenile densities were high in 2006 and 2008 but low in other years. Discrepancies in the yearly trends of these parameters suggest that mortality during life stages between spawning and early larval phases might have affected the abundance of the subsequent life stages. Monthly mean water temperature between January and February, in which hatching and pelagic larvae occur in the bay, was lower in 2006 (8.6 °C) and 2008 (9.6 °C) than was observed in other years (10.4-11.4 °C). Significant negative correlation between water temperature and larval density implies that mortality during pre- and post-larval stages would be higher in warmer winter years (>10 °C). To test this hypothesis, we examined the effects of water temperature on mortality and development in egg and larval stages under controlled laboratory conditions. Hatching rate was high in a water temperature range of 9.2-12.7 °C (66.6-82.5%), whereas it decreased in cooler (3.7% at 5.9 °C) or warmer (33.9% at 14.8 °C) conditions. Meanwhile, days from fertilization to hatching, size of larvae at hatching and survival rate of larvae after 18 d from hatching were monotonically and significantly decreased as water temperature was elevated. Combined evidence of the field and laboratory studies suggests that a warmer reproductive season (>10 °C) might induce mortalities of marbled flounder larvae in Tokyo Bay.

The sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate induces ER stress in human astrocytoma cells.

Organophosphorus (OP) compounds such as sarin are toxic agents that irreversibly inhibit the enzyme acetylcholinesterase. A recent study showed that OP compounds also have multiple toxicity mechanisms, and another suggested that endoplasmic reticulum (ER) dysfunction contributes to OP toxicity. However, the signaling pathway and mechanisms involved are poorly understood. We examined whether the sarin-like OP agent bis(isopropyl methyl)phosphonate (BIMP), which exhibits toxicity similar to that of sarin, induced ER stress in human astrocytoma CCF-STTG1 cells. Our results demonstrate that BIMP exposure reduced cell viability. Moreover, it induced changes in mitochondrial membrane potential and increased cleavage of caspase 3. Treatment with BIMP increased the mRNA levels of the ER stress marker genes binding immunoglobulin protein (BiP) and the transcription factor C/EBP homologous protein (CHOP). Furthermore, BIMP increased the protein expressions and phosphorylation of BiP, CHOP, and protein kinase RNA-like ER kinase and the phosphorylation of eukaryotic translation initiation factor 2. Compared to BIMP treatment alone, pretreatment with the CHOP siRNA, siCHOP, decreased BIMP-dependent CHOP expression and improved CCF-STTG1 cell viability. Our findings suggest that BIMP induced mitochondrial dysfunction and apoptotic cell death event mediated by ER stress in CCF-STTG1 cells and that treatment targeted at managing ER stress has the potential to attenuate the toxicity of OP nerve agents.

Association between paraben exposure and menstrual cycle in female university students in Japan.

Parabens have been known to have estrogenic activity in many in vivo and in vitro studies and biomonitoring data indicated ubiquitous exposure in general populations. However, there are few human studies on reproductive effects of parabens. In this study, menstrual cycle length and its intra-individual variation were investigated by bleeding record over the period of 5 months and urinary concentrations of parabens were measured for 128 female Japanese university students. We found significantly negative relationships between menstrual cycle length and urinary estrogen-equivalent total paraben (odds=0.73, 95% CI 0.56-0.96) and butyl paraben concentrations (odds=0.83, 0.70-0.99), which indicated shortened cycle length in women with high urinary paraben concentrations. This study indicated that paraben exposure at not excessive levels is associated with menstrual cycle length or its variability among general female subjects. These results suggest a possibility that parabens exposure is one of the environmental causes of human reproductive problem.

Decline in intertidal biota after the 2011 Great East Japan Earthquake and Tsunami and the Fukushima nuclear disaster: field observations.

In 2011, 2012, and 2013, in the intertidal zones of eastern Japan, we investigated the ecological effects of the severe accident at the Fukushima Daiichi Nuclear Power Plant that accompanied the 2011 Great East Japan Earthquake and Tsunami. The number of intertidal species decreased significantly with decreasing distance from the power plant, and no rock shell (Thais clavigera) specimens were collected near the plant, from Hirono to Futaba Beach (a distance of approximately 30 km) in 2012. The collection of rock shell specimens at many other sites hit by the tsunami suggests that the absence of rock shells around the plant in 2012 might have been caused by the nuclear accident in 2011. Quantitative surveys in 2013 showed that the number of species and population densities in the intertidal zones were much lower at sites near, or within several kilometers south of, the plant than at other sites and lower than in 1995, especially in the case of Arthropoda. There is no clear explanation for these findings, but it is evident that the intertidal biota around the power plant has been affected since the nuclear accident.

Temporal trends for inflow of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) to Tokyo Bay, Japan, estimated by a receptor-oriented approach.

We estimated inflow rates of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) to Tokyo Bay, Japan, between February 2004 and February 2011 by a receptor-oriented approach based on quarterly samplings of the bay water. Temporal trends in these inflow rates are an important basis for evaluating changes in PFOS and PFOA emissions in the Tokyo Bay catchment basin. A mixing model estimated the average concentrations of these compounds in the freshwater inflow to the bay, which were then multiplied by estimated freshwater inflow rates to obtain the inflow rates of these compounds. The receptor-oriented approach enabled us to comprehensively cover inflow to the bay, including inflow via direct discharge to the bay. On a logarithmic basis, the rate of inflow for PFOS decreased gradually, particularly after 2006, whereas that for PFOA exhibited a marked stepwise decrease from 2006 to 2007. The rate of inflow for PFOS decreased from 730kg/y during 2004-2006 to 160kg/y in 2010, whereas that for PFOA decreased from 2000kg/y during 2004-2006 to 290kg/y in 2010. These reductions probably reflected reductions in the use and emission of these compounds and their precursors in the Tokyo Bay catchment basin. Our estimated per-person inflow rates (i.e., inflow rates divided by the estimated population in the basin) for PFOS were generally comparable to previously reported per-person waterborne emission rates in Japan and other countries, whereas those for PFOA were generally higher than previously reported per-person waterborne emission rates. A comparison with previous estimates of household emission rates of these compounds suggested that our inflow estimates included a considerable contribution from point industrial sources.

Comparison of different urine pretreatments for biological monitoring of pyrethroid insecticides.

During a cross-validation practice of urinary analysis of pyrethroid insecticide metabolite (3-phenoxybenzoic acid, 3-PBA) by two laboratories (Lab A and Lab B), difference in 3-PBA concentration was noticed. The analytical methods used in the exercise were enzymatic deconjugation (glucuronidase/sulfatase) followed by solid phase extraction and isotope dilution LC-MS-MS determination (Lab A) and acidic deconjugation followed by liquid-liquid extraction and gas chromatography-mass spectrometry determination (Lab B). A significant difference was found for one of the five samples: lower value was obtained in Lab A. Use of acidic deconjugation in Lab A resulted in comparable value with the analytical result for this sample originally obtained in Lab B. The comparison was extended to 42 urine samples taken from Japanese males in Lab A by using the two different deconjugation methods and LC-MS-MS determination. Significantly lower value was obtained for enzymatic deconjugation in some of the 42 urine samples. The results suggested the presence of other conjugated species of 3-PBA than glucuronide and sulfate in human urine. Although the overall agreement between the values obtained by the deconjugation methods was fair, it appears that urine samples should be pretreated by acidic deconjugation for the analysis in biological monitoring of pyrethroid exposure.

Prenatal pyrethroid insecticide exposure and thyroid hormone levels and birth sizes of neonates.

Pyrethroid insecticides have been shown to possess thyroid hormone disrupting properties in previous animal studies. In this study, the relationship between maternal exposure to pyrethroid insecticides during pregnancy and neonatal thyroid hormone status (free thyroxine (fT4) and thyroid stimulating hormone (TSH) in whole blood) and birth sizes were explored in 147 mother-neonate pairs in Tokyo. The concentration of 3-phenoxybenzoic acid (3-PBA) in maternal urine, sampled in the first trimester of gestation, was used for pyrethroid exposure assessment. Neonatal fT4 and TSH were within the normal range except for one elevated TSH (but normal fT4) in a neonate. Multiple regression analyses with stepwise variable selection did not extract maternal 3-PBA as significant for neonatal fT4 and TSH, indicating that maternal pyrethroid exposure had no apparent effect on the neonatal thyroid hormone status of the neonate subjects. For birth weight and head circumference, maternal 3-PBA was selected as significant with a positive partial regression coefficient along with other factors known to increase birth sizes of neonates (gestational weeks or maternal BMI). It was not clear if this was causal because no biological mechanism was apparent.

Disturbance of recruitment success of mantis shrimp in Tokyo Bay associated with effects of hypoxia on the early life history.

We investigated effects of severe hypoxia (dissolved oxygen <1 ml l(-1)) on recruitment of mantis shrimp Oratosquilla oratoria in Tokyo Bay. Ten-year field surveys were conducted to examine quantitative relationships in annual mean densities of larvae and juveniles, and spatial distribution of juveniles and severe hypoxia. There was no significant correlation between annual mean densities of larvae and juveniles, suggesting that mortality during larval or juvenile stages varies among years, which might have regulated abundance of young-of-the-year juveniles. Juvenile density was low in the severely hypoxic area, implying that hypoxia could affect survivals and spatial distribution of juveniles. Meanwhile, there are yearly fluctuations in juvenile density in normoxic areas of both northern and southern part of the bay. This evidence suggests that abundance of post-settled juveniles might have been determined by not only effects of hypoxia, but also other factors influencing mortality during the early life stages.

[Cross-reactivity of Instant-View M-1 for detection of benzodiazepine-related drugs and their metabolites in urine].

Immunoassays are useful methods for the determination of regulated drugs in clinical and forensic laboratories. Although the Instant-View M-1 (IV M-1) immunoassay kit is frequently used to screen drugs in laboratories in Japan, basic information about the IV M-1 such as its specificity and reactivity is not available. In this study, we determined the specificity and cross-reactivity of IV M-1 for the detection of benzodiazepine-related drugs and their metabolites in urine. The IV M-1 could detect triazolobenzodiazepines such as triazolam in urine at concentrations > or = 300 ng/mL. However, thienodiazepines such as etizolam could not be detected because of lack of cross reactivity. A correlation was observed between the structure of the metabolites and the reactivity of the kit; 4-hydroxy metabolites of alprazolam and triazolam were detectable, whereas a-hydroxy metabolites were not. Furthermore, 7-amino metabolites such as nitrazepam could not be detected at any concentration, including high concentrations. The specificity and reactivity of various kits used for detection of drugs in urine are different. Therefore, it is necessary to consider the basic features of the kit used while assessing the results obtained.

Changes in growth and reproductive traits of dragonet Callionymus valenciennei in Tokyo Bay, concurrent with decrease in stock size.

We examined changes in the growth and reproductive traits of dragonet Callionymus valenciennei concurrent with decrease in stock size in Tokyo Bay. Stock size in mid 2000s decreased to 14% of that recorded in early 1990s. The minimum standard length at which dragonet attain gonadal maturation was smaller in 2000s (4.8 cm) compared to that in 1990s (6.0 cm). In addition, timing of the onset of the first spawning became earlier in 2000s (starting from spring) than that in 1990s (from summer). We also found significant changes in growth for both sexes from 1990s to 2000s; growth of dragonet after they attained sexual maturation showed a significant decrease in 2000s against that in 1990s. Changes in the life history traits may reflect trade-off for allocating available energy resource to reproduction rather than to somatic growth under limited prey abundance for enhancing a chance for stock recovery. However, these changes in the life history traits might have not led to the resilience of the population due to negative effects of hypoxia on settlement of larvae from the spring spawning season.

Development of reproductive organs in the ivory shell Babylonia japonica: observations from wild populations and laboratory-reared juveniles.

We histologically examined normal differentiation and development of genital tract and gonad in the ivory shell Babylonia japonica (Buccinidae) to determine whether the formation of male-type genitalia in imposex-exhibiting females mimics the normal development of male genitalia in prosobranch gastropods. We used a wild-caught 2-year-old specimen and laboratory-reared juveniles aged 0-24 months. Gonad differentiation was unclear before age 14 months, but progressed after 16 months. Both sexes had complete genital tract and mature gonad at 20 months. However, differentiation and development occurred earlier in females than in males. Development of genital tract preceded gonad differentiation. Vas deferens morphogenesis in males resembled that in imposex-exhibiting females. These findings help to understand the morphogenesis of genital tract and gonad in prosobranch gastropods and will contribute to more in-depth studies of the mode of action of organotin compounds such as TBT and TPhT in imposex development in female prosobranch gastropods.