Screening for potential effects of endocrine-disrupting chemicals in peri-urban creeks and rivers in Melbourne, Australia using mosquitofish and recombinant receptor–reporter gene assays

Sexually mature male mosquitofish (Gambusia holbrooki) were collected from various sites around Melbourne in 2009 to evaluate the performance of gonopodial indices as a biomarker for endocrine disruption in Melbourne's waterways. The mosquitofish indices assessed were body length (BL), gonopodial length (GL)/BL ratio, ray 4:6 ratio and the absence or presence of hooks and serrae, and these varied between sites. The study was complemented by measurements of estrogenic, retinoid, thyroid and aryl hydrocarbon (AhR) receptor activities of the water. Male mosquitofish were 16.3–21.5 mm in length, and although there was a statistically significant positive relationship showing that bigger fish had longer gonopodia than small fish (r2 = 0.52, p < 0.001), there were few significant differences in GL/BL ratio of fish between sites. Measured estrogenic activity was mostly in the range 0.1–1.7 ng/L EEQ, with one site having much higher levels (~12 ng/L EEQ). Aryl hydrocarbon (AhR) receptor activity was observed in all water samples (7–180 ng/L βNF EQ), although there was no consistent pattern in the level of AhR activity observed, i.e., ‘clean’ sites were as likely to return a high AhR activity response as urban or wastewater treatment plant (WWTP)-impacted sites. There was no correlation between measurements of receptor actvity and gonopodial length (GL):BL ratio and BL. We conclude that the mosquitofish gonopodia only fulfills part of the criteria for biomarker selection for screening. The mosquitofish indices assessed were cheap and easy-to-perform procedures; however, there is no baseline data from the selected sites to evaluate whether differences in the morpholical indices observed at a site were a result of natural selection in the population or due to estrogenic exposure.     

Identification and estrogenic characterization of impurities in commercial bisphenol A diglycidyl ether (BADGE)

A sample of commercial BADGE was fractioned by HPLC and eight impurities including novel propyl derivatives (2), (5) and (6) were identified by NMR spectrometry, FAB-MS and GC-MS. The estrogenicity, both agonist and antagonist, of fractions containing these impurities was measured with a yeast two-hybrid assay incorporating the human (hER alpha) and a competitive binding assay for hER alpha (ELISA). In the yeast two-hybrid assay, estrogenic antagonist activity was found in two fractions, while estrogenic agonist activity was not found in any. In the ELISA method, the binding affinity to hER alpha was found in three fractions. It is probable that a comprehensive assessment of the estrogenic properties of commercial BADGE, and their implications for human health, will require examination of all its components as described here.     

Docosahexaenoic acid accumulation in thraustochytrids: search for the rationale

Thraustochytrid protists characteristically accumulate high amounts of lipids, much of which is comprised of the polyunsaturated fatty acid, docosahexaenoic acid (DHA). Since DHA is important in human health, thraustochytrids have attracted much attention from the point of view of biotechnology. However, the biological rationale of DHA production in the storage lipids of these organisms is not clear. We carried out three experiments to study this. (1) The fate of lipids in thraustochytrids during development was studied by staining lipids of vegetative cells with the fluorescent vital stain for lipids, namely Nile blue, following the growth of the cells. The fluorescent lipid bodies decreased in abundance in freshly formed motile limaciform amoeboid cells and subsequently disappeared when they moved away. When vegetative cells produced vast extensions of plasma membrane, the ectoplasmic net elements (EN), the fluorescent lipids appeared to be transported to them. (2) Changes in lipids and DHA during starvation were examined in cells with enhanced lipid and DHA contents and those in which they were not enhanced. Cells in which lipids and DHA were enhanced by refrigerating them for 48 h survived starvation for a longer period. Compared to cells that had not been refrigerated, total fatty acids, as well as DHA were marginally higher in pre-refrigerated cells, while palmitic acid levels were lower. Starvation of cells resulted in a gradual decrease of absolute concentrations and percentage levels of DHA after 8 days, while percentage of palmitic acid levels increased. (3) The relationship between DHA and specific gravity of cells was studied by comparing cells pre-refrigerated as above with those which were not pre-refrigerated. Refrigerated cells with increased lipids and DHA showed higher specific gravities than non-refrigerated cells. We suggest the following roles for storage lipids and DHA in thraustochytrids: (1) lipids serve as energy sources during movement of cells and for production of EN; (2) DHA in storage lipids become distributed in the plasma membrane when EN are formed; (3) DHA is preferably utilized as a fatty acid energy reserve during starvation and (4) an unknown mechanism results in negative buoyancy of cells when total lipids and DHA are marginally enhanced. DHA in storage lipids might be crucial to the survival of thraustochytrid cells.     

Estimation of sources and inflow of dioxins and polycyclic aromatic hydrocarbons from the sediment core of Lake Suwa, Japan

To elucidate the historical changes in polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF), coplanar polychlorinated biphenyl (co-PCB), and polycyclic aromatic hydrocarbon (PAH) inflows in Lake Suwa, their concentrations in the sediment core were analyzed in 5 cm interval. The maximum concentrations (depth cm) of PCDDs/DFs, co-PCBs, and PAHs were 25.2 ng/g dry (30-35 cm), 19.0 ng/g dry (30-35 cm), and 738, 795 ng/g dry (50-55 cm, 30-35 cm), respectively. Age and sedimentation rate of the sediment were estimated from the vertical changes in apparent density. Deposition rate of dioxins and PAHs were calculated from the concentration and sedimentation rate of the sediment. The results indicate that large amounts of dioxins and PAHs flowed into the lake in flood stage compared to normal stage.     

Persistent organochlorines in human breast milk collected from primiparae in Dalian and Shenyang, China

The present study determined the concentrations of organochlorines (OCs) such as polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), chlordane compounds (CHLs), hexachlorobenzene (HCB) and tris(4-chlorophenyl)methane (TCPMe) in human breast milk collected from primiparae in Dalian and Shenyang, northeastern China during 2002. In addition, dioxins and related compounds in pooled samples of human breast milk from Dalian and Shenyang were also analyzed. OCs were detected in all the human breast milk samples analyzed in this study. The predominant contaminants in human breast milk were HCHs, DDTs and HCB, and the levels were relatively higher than those in other countries. On the other hand, concentrations of dioxins and related compounds, PCBs, and CHLs were relatively low. Concentrations of OCs in human breast milk from Dalian, which is located along the coast of Bo Hai Strait, were significantly higher than those from Shenyang, implying that the residents in Dalian might be mainly exposed to these contaminants from seafood. When the relationship between concentrations of OCs in human breast milk and age of primiparae was examined, no significant correlation was observed. This might be caused by the limited sample numbers and narrow range of mother's age and/or recent ban of DDT and HCH production and use. Significant correlation between concentrations of TCPMe and DDTs in human breast milk suggested that technical DDT might be a source of TCPMe in the Chinese population. When daily intakes of DDTs and HCHs to infants through human breast milk were estimated, human breast milk from Dalian showed significantly higher contribution than Shenyang, implying that infants in Dalian might be at higher risk by these contaminants.     

Genetic variation in marine teleosts: High variability in habitat specialists and low variability in habitat generalists

Genetic variation was reviewed in 106 species of marine teleosts. Two heterozygosity estimates were used, one including all protein and enzyme loci and a second excluding the non-enzymatic protein loci. Mean heterozygosities are 0.055±0.036 based on all loci in 106 species and 0.060+0.038 based on enzymatic loci in 89 species. A significant negative correlation was noted between heterozygosity and the proportion of general protein loci included in the estimate. A comparison was made of heterozygosities among taxonomic orders and families, life zones, reproductive mode, geographical range and size. High levels of genetic variation are found in Clupeiformes, Atheriniformes, Pleuronectiformes, temperate pelagic, tropical, intertidal-sublittoral and wide-range species. Low levels of genetic variation are found in Gadiformes, Scorpaeniformes, temperate demersal, polar, and narrowrange species. The most striking differences in heterozygosities are between temperate demersal flatfishes and temperate demersal round fishes. It is suggested that much of the data can be explained by a habitat specialist-generalist model, with high heterozygosities in specialists and low heterozygosities in generalists, but that this is only one of a mosaic of factors which influence genetic variation.     

Assessment of thyroid hormone activity of halogenated bisphenol A using a yeast two-hybrid assay

The thyroid hormone agonist/antagonist activities of halogenated derivatives of bisphenol A (BPA) were assessed using a yeast two-hybrid assay incorporating the human thyroid hormone α (TRα), both with and without possible metabolic activation by rat liver S9 preparation. In the absence of the rat liver S9 preparation, 3,3′,5,5′-tetrabromobisphenol A (TBBPA), 3,3′,5,5′-tetrachlorobisphenol A (TCBPA), and 3,3′,5-trichlorobisphenol A (3,3′,5-triClBPA) exhibited agonist activity, whereas 3-chlorobisphenol A (3-ClBPA), 3,5-dichlorobisphenol A (3,5-diClBPA), 3,3′-dichlorobisphenol A (3,3′-diClBPA), and BPA did not. The activities of TBBPA and TCBPA increased markedly (7.6-fold and 3.1-fold, respectively) after their metabolic activation with the rat liver S9 preparation. TBBPA, TCBPA, and 3,3′,5-triClBPA inhibited the binding of triiodothyronine (T3) to TRα at 2 × 10⁻⁵ M without rat liver S9 treatment and 4 × 10⁻⁶ M with rat liver S9 treatment, demonstrating their T3 antagonist activity. These results revealed that metabolic activation by rat liver S9 significantly increased the agonist/antagonist potential of some halogenated BPAs.     

Nitrogen niches revealed through species and functional group removal in a boreal shrub community

Most theories attempting to explain the coexistence of species in local communities make fundamental assumptions regarding whether neighbors exhibit competitive, neutral, or positive resource-use interactions; however, few long-term data from naturally assembled plant communities exist to test these assumptions. We utilized a 13-year experiment consisting of factorial removal of three shrub species (Vaccinium myrtillus, V. vitis-idaea, and Empetrum hermaphroditum) and factorial removal of two functional groups (tree roots and feather mosses) to assess how neighbors affect N acquisition and growth of each of the three shrub species. The removal plots were established on each of 30 lake islands in northern Sweden that form a natural gradient of resource availability. We tested the hypotheses that: (1) the presence of functionally similar neighbors would reduce shrub N acquisition through competition for a shared N resource; (2) the removal of functional groups would affect shrub N acquisition by altering the breadth of their niches; and (3) soil fertility would influence the effects of neighbor removals. We found that the removal of functionally similar neighbors (i.e., other shrub species) usually resulted in higher biomass and biomass N, with the strength of these effects varying strongly with site fertility. Shrub species removals never resulted in altered stable N isotope ratios (delta(15)N), suggesting that the niche breadth of the three shrubs was unaffected by the presence of neighboring shrub species. In the functional group removal experiment, we found positive effects of feather moss removal on V. myrtillus biomass and biomass N, and negative effects on E. hermaphrotium N concentration and V. vitis-idaea biomass and biomass N. Tree root removal also caused a significant shift in foliar delta(15)N of V. myrtillus and altered the delta(15)N, biomass, and biomass N of E. hermaphroditum. Collectively, these results show that the resource acquisition and niche breadth of the three shrub species are often affected by neighbors, and further that both the identity of neighbors and site fertility strongly determine whether these interactions are positive, negative, or neutral. These findings have implications for understanding species coexistence and the reciprocal relationships between productivity and species diversity in this ecosystem.     

Dioxin concentration in human milk in Hebei province in China and Tokyo, Japan: potential dietary risk factors and determination of possible sources

Very limited information is available on body burdens and environmental levels of dioxins and dioxin-like PCBs (dl-PCBs) in mainland China. In the current studies, human milk samples were collected from 30 breastfeeding mothers in Shijiazhuang city (industrialized) and 11 in the Tanshan countryside (agricultural) of Hebei Province in northern China. An additional 20 samples were obtained from mothers in Tokyo, Japan. PCDDs, PCDFs, and dl-PCBs in human milk were analyzed by high-resolution gas chromatography/high-resolution mass spectrometry. Our results show that arithmetic means for body burdens of PCDDs/Fs and dl-PCBs in Hebei were 3.6 and 1.9 pg TEQ g⁻¹ fat, respectively, which were only about one fourth of the levels in Japan. In addition, no difference was found in the chemical levels except dl-PCBs between the urban and rural areas. Based on the results of an in-person interview of the Chinese mothers using a 59-item questionnaire, freshwater fish consumption was found to correlate with the body burden of dioxins. Principal component analysis of dioxin congeners revealed that the patterns of dioxins in the Hebei urban and rural areas are quite similar; however, they are clearly different from those in Japan. Collectively, our results suggest that the lower body burdens of dioxin in Hebei may be due in part to the relatively slow industrialization and a lower consumption of marine foods. Finally, the results indicate that comprehensive monitoring of dioxins and dl-PCBs in humans as well as in the environment and foods is necessary in China.     

Long-term aboveground and belowground consequences of red wood ant exclusion in boreal forest

Despite their ubiquity, the role of ants in driving ecosystem processes both aboveground and belowground has been seldom explored, except within the nest. During 1995 we established 16 ant exclusion plots of approximately 1.1 x 1.1 m, together with paired control plots, in the understory layer of a boreal forest ecosystem in northern Sweden that supports high densities of the mound-forming ant Formica aquilonia, a red wood ant species of the Formica rufa group. Aboveground and belowground measurements were then made on destructively sampled subplots in 2001 and 2008, i.e., 6 and 13 years after set-up. While ant exclusion had no effect on total understory plant biomass, it did greatly increase the relative contribution of herbaceous species, most likely through preventing ants from removing their seeds. This in turn led to higher quality resources entering the belowground subsystem, which in turn stimulated soil microbial biomass and activity and the rates of loss of mass and carbon (C) and nitrogen (N) from litter in litterbags placed in the plots. This was accompanied by losses of approximately 15% of N and C stored in the humus on a per area basis. Ant exclusion also had some effects on foliar stable isotope ratios for both C and N, most probably as a consequence of greater soil fertility. Further, exclusion of ants had multitrophic effects on a microbe-nematode soil food web with three consumer trophic levels and after six years promoted the bacterial-based relative to the fungal-based energy channel in this food web. Our results point to a major role of red wood ants in determining forest floor vegetation and thereby exerting wide-ranging effects on belowground properties and processes. Given that the boreal forest occupies 11% of the Earth's terrestrial surface and stores more C than any other forest biome, our results suggest that this role of ants could potentially be of widespread significance for biogeochemical nutrient cycling, soil nutrient capital, and sequestration of belowground carbon.