Endocrine disruption of water and sediment extracts in a non-radioactive dot blot/RNAse protection-assay using isolated hepatocytes of rainbow trout

Goal, Scope and Background In order to evaluate the estrogenic activity of sediments and XAD water extracts of selected sites of the catchment area of the River Neckar, a river system in Southern Germany, an integrative assessment approach was used to assess the ecological hazard potential of endocrine-disrupting compounds in sediment and water. Methods The approach is based on estrogen receptor-mediated vitellogenin synthesis induced in isolated hepatocytes of rainbow trout and quantified in a non-radioactive dot blot/RNAse protection-assay in parallel to comprehensive chemical analyses of estrogenic substances. Results and Discussion Numerous investigated extracts revealed an estrogen activity comparable to that of the positive control (1 nM 17?-estradiol corresponding to 270 ng/L in the test medium). Based on a concentration factor of 30 in the extracts and a recovery of XAD resins of approximately 80 %, 17?-estradiol equivalent concentrations between 20 and 26.7 ng/L could be calculated downstream of a sewage treatment plant (< 0.1 ng/L for a reference site). A comparison of the bioassay-derived Bio-TEQs (toxicity equivalents) and the Chem-TEQs revealed a high correlation with a Pearson coefficient of 0.85, indicating that the same ranking of the samples could be obtained with respect to the endocrine disrupting potential with both chemical and bioanalytical analysis. However, the TEQ concentrations computed from chemical analyses were significantly lower than the bioassay-derived TEQ concentrations. In fact, in none of the samples, more than 14 % of the vitellogenin-inducing potency could be attributed to the substances (steroids, alkylphenols, bisphenol A, diethylstilbestrol) analyzed. A comparison of the endocrine disrupting potential of sediments extracted by the solvents acetone and methanol revealed lower biological effects for acetone-extracted samples. Possible reasons may be a masking of endocrine effects in acetone extracts by cytotoxicity, a low extraction efficiency of the solvent acetone, or anti-estrogen potencies of some extracted sediment compounds. Using a mass balance approach, the contribution of the compounds analyzed chemically (Chem-TEQs) to the total endocrine activity (Bio-TEQs) was calculated. Based on the very low detection limits, particularly of the steroids with their high TEF factors, results revealed that a calculation of the Chem-TEQs is associated with considerable scale inaccuracy: Whereas only 7-15 % of the biological effectiveness (Bio-TEQs) could be explained by endocrine substances identified above the detection limits, the assumption of concentrations slightly below the given detection limits would result in a significant over estimation (137-197 %) of the Bio-TEQs. Even the interassay variation of the dot blot assay with different fish donors for primary hepatocyte (factor 2 - 2.5) is relatively low, when compared to the large range of the Chem-TEQ concentrations (factor 20) obtained when applying different modes of calculation. Conclusions and Outlook Overall, only a minor portion of the endocrine activity detected by bioassays could be linked to compounds identified by chemical analysis. In vitro assays for assessment of endocrine activities are useful as sensitive integrating methods that provide quantitative estimates of the total activity of particular receptor-mediated responses. Although discrepancies may also result from different bioanalytical approaches, it is overall likely that bioanalytical and not chemical analytical approaches give the correct estimate of endocrine disrupting potencies in environmental samples. As a conclusion, assessment of endocrine disruption based on chemical analysis alone does not appear sufficient and further research into the spectrum of substances with potential endocrine activity as well as into additive or even synergistic effects in complex environmental samples is urgently needed.     

On the function of warning coloration: a black and yellow pattern inhibits prey-attack by naive domestic chicks

Summary Young chicks were offered a choice of warningly coloured black and yellow and non-warningly coloured green (or olive) prey. Unfed chicks were given palatable painted mealworms on their first day. They directed their first peck at both colour types at the same probability; however, they ate the warningly coloured ones at a much lower rate. This is due to an inhibition of attack which becomes effective after pecking. Chicks which were a few days old showed the same behaviour. Since the control prey was in this case painted with an olive mixture containing the same colours as the warningly coloured mealworms, it can be concluded that the inhibition is caused by the black and yellow coloration. With accumulating positive experience of the chicks, the inhibition decreased. For permanent avoidance it must therefore be supplemented by unpleasant experience. Accordingly, chicks handled the unpalatable black and yellow ringed caterpillars of Tyria jacobaeae only a few times and always for a short period when offered repeatedly. The inhibition caused by the black and yellow pattern is attributed to a genetically fixed predisposition to avoid warningly coloured black and yellow prey which is the result of evolutionary adaptation.     

Under-ice amphipods in the Greenland Sea and Fram Strait (Arctic): environmental controls and seasonal patterns below the pack ice

During three "Polarstern" cruises to the ice-covered Greenland Sea (spring 1997, summer 1994, autumn 1995) studies on the under-ice habitat (morphology, hydrography, ice-algal biomass) and on the macrofaunal, autochthonous under-ice amphipods (species diversity, abundance) were carried out in order to describe environmental controls and seasonal patterns in this community. In spring, the ice underside was rather smooth and whitish, while in summer melting structures and sloughed-off ice-algal threads were observed, in autumn detritus clumps accumulated in depressions at the ice underside. Only in summer, a thin layer of warm (up to -0.6°C) and less saline (as low as S=6.3) water was found at the ice-water interface above Polar Water. Integrated ice-algal biomass was highest during autumn (2.6 mg chl a m^-2) and lowest during summer (1.2 mg chl a m^-2). Four species of under-ice amphipods occurred in spring and summer (Apherusa glacialis, Onisimus glacialis, O. nanseni, Gammarus wilkitzkii), but only the last species was observed at the ice underside in autumn. A. glacialis and G. wilkitzkii were equally abundant in spring; A. glacialis dominated in summer. The highest total abundance of amphipods occurred during summer (31.9 ind. m^-2), compared to lower abundances in spring and autumn (5.3 and 1.1 ind. m^-2, respectively). A factor analysis revealed seasonal patterns in the data set, which mainly influenced A. glacialis, and species-specific relations between several environmental factors and the distribution of under-ice amphipods. Abundance of A. glacialis was closely related to the under-ice hydrography and ice-algal biomass, whereas the other amphipod species were more influenced by the under-ice morphology. It is therefore stated that the observed thinning of the Arctic sea ice and the resulting increased meltwater input and change in morphology of floes will have a profoundly adverse effect on the under-ice amphipods.     

Signs that encourage internalized recycling: Clinical validation, weak messages and “creative elaboration”

Two experiments examined clinical validation's ability to increase examination of a persuasive message and increase long-term recycling. In Experiment 1, validating (acknowledging) recycling's inconvenience decreased criticism of the persuasive message, supporting validation's ability to reduce reactance and open the reader to new ideas. Validation did not improve attitudes towards the sign's author, removing liking for the communicator as an alternate explanation for attitude change. In Experiment 2, different recycling signs were created from a 2(no validation/validation) by 2(weak/strong arguments) factorial design, and placed in university buildings. The validation weak sign increased recycling more than the validation strong sign, especially after the signs were removed. We suggest that validation induced people to scrutinize the weak message and use their existing pro-recycling attitudes to “creatively elaborate” it. Discussion emphasizes clinical validation and the Elaboration Likelihood Model as theoretical tools, as well as the potential for thought provoking signs to have long-term effects.