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.     

A lysimeter experiment to investigate the leaching of veterinary antibiotics through a clay soil and comparison with field data

Pharmaceuticals used in livestock production may be present in manure and slurry as the parent compound and/or metabolites. The environment may therefore be exposed to these substances due to the application of organic fertilisers to agricultural land or deposition by grazing livestock. For other groups of substances that are applied to land (e.g. pesticides), preferential flow in clay soils has been identified as an extremely important mechanism by which surface water pollution can occur. This lysimeter study was therefore performed to investigate the fate of three antibiotics from the sulphonamide, tetracycline and macrolide groups in a clay soil. Only sulphachloropyridazine was detected in leachate and soil analysis at the end of the experiment showed that almost no antibiotic residues remained. These data were analysed alongside field data for the same compounds to show that soil tillage which breaks the connectivity of macropores formed over the summer months, prior to slurry application, significantly reduces chemical mobility.     

Bioregeneration of powdered activated carbon in the treatment of alkyl-substituted phenolic compounds in simultaneous adsorption and biodegradation processes

The role of bioregeneration process in renewing the adsorbent surface for further adsorption of organics during simultaneous adsorption and biodegradation processes has been well recognized. The extent of bioregeneration of powdered activated carbon (PAC) as an adsorbent loaded with phenol, p-methylphenol, p-ethylphenol and p-isopropylphenol, respectively, in the simultaneous adsorption and biodegradation processes were quantitatively determined using oxygen uptake as a measure of substrate consumption. Bioregeneration phenomenon was also evaluated in the simultaneous adsorption and biodegradation processes under sequencing batch reactor (SBR) operation to treat synthetic wastewater containing 1200 mg l(-1) phenol and p-methylphenol, respectively. The SBR systems were operated with FILL, REACT, SETTLE, DRAW and IDLE periods in the ratio of 4:6:1:0.75:0.25 for a cycle time of 12 h. The results show that the percentage of desorption from loaded PAC decreased in the order phenol>p-methylphenol>p-ethylphenol>p-isopropylphenol. For the treatment of phenol and p-methylphenol in the SBR reactors, respectively, the simultaneous adsorption and biodegradation processes were able to produce a consistent effluent quality of COD < or = 100 mg l(-1) when the applied PAC dosage was 0.115 and 0.143 g PAC per cycle, respectively. When no further PAC was added, the treatment performance deteriorated to that of the case without PAC addition after 68 and 48 cycles of SBR operation, respectively, for phenol and p-methylphenol. This observation is consistent with the greater extent of bioregeneration for phenol-loaded PAC as compared to p-methylphenol-loaded PAC.     

Interpretation of heavy metal data from mussel by use of multivariate classification techniques

Different strategies of multivariate analysis of metals concentrations (Mn, Fe, Ni, V, Co, Cu, Cd, Hg, Pb, Na, K) in mussel samples from different spanish markets are used to interpret a data base and identify differences between species and origin of the samples. Principal Component Analysis and Potential curves are applied to properly classify unknown samples from representative mussels samples (Mytilus edulis and Perna canaliculus). Also, Principal Components Analysis is used as display method to visualize the relation between the variables and objects of interest.     

Laboratory simulation of biodegradation of chemicals in surface waters: closed bottle and respirometric test

Microbial degradation is the most dominant elimination mechanism of organics from the environment. For evaluation of biodegradability of pure chemicals many standardized tests are available, but no standardized procedure for assessment of biodegradability of chemicals in surface water is agreed upon. Rates of in-situ biodegradation are usually estimated in laboratory simulation where environmental factors are reproduced to some extent. The aim of our study was to compare standardised ready biodegradability assessment, test (Closed bottle test) and its modifications employing the basic agreements on test conditions to simulate biodegradation in surface water. Standard test was modified using various natural river waters to simulate the natural environment in a simplified way. The impact of different types and amounts of nutrients and microorganisms on biodegradation was confirmed. The conditions in the recipient should be examined to extrapolate the results from ready biodegradability tests to real surface water.     

Methane emission from lakes

A physico-chemical model to describe methane and carbon dioxide emission from lakes was developed. The model describes the emission by diffusion, bubbles and plants. The intensity of the fluxes can be calculated either for total or particular emission in dependence on gas bubbles composition. It was found, that diffusional (Q) and bubble (J) fluxes depend on the methane molar ratio (X) in bubbles as follows: [formula: see text]. The model allows to estimate the role of the methane oxidation by atmospheric oxygen in the total methane flux. It was shown, that the methane oxidation does not influence much methane fluxes to the atmosphere for most of the experimentally observed situations.     

Levels of dioxin-like compounds in sewage sludge determined with a bioassay based on EROD induction in chicken embryo liver cultures

A bioassay for the detection of dioxin-like compounds was used to estimate levels in sewage sludge from Swedish sewage treatment plants (STPs). The sludge extracts were HPLC-separated into three fractions containing a) monoaromatic/aliphatic, b) diaromatic (e.g. polychlorinated biphenyls [PCBs], polychlorinated dibenzodioxins and polychlorinated dibenzofurans [PCDDs/Fs]), and c) polyaromatic compounds (e.g. polycyclic aromatic hydrocarbons [PAHs]). The bioassay, which is based on EROD (7-ethoxyresorufin O-deethylase) induction in cultured chicken embryo livers detected dioxin-like activity in all unfractionated extracts and in the di- and polyaromatic fractions of all sludge extracts, but not in the monoaromatic/aliphatic fractions. The levels ranged between 6 and 109 pg bio-TEQ/g sludge (d.w.). In sediment samples from rural lakes in Sweden, levels of about 5 pg bio-TEQ/g (d.w.) have been found. The polyaromatic fractions of the sludge samples were potent in the bioassay, probably due to various PAHs and other polyaromatics in the sludge. The levels of six PAHs that are screened for in the sludge at Swedish STPs accounted for only 3-10% of the observed EROD-induction by the polyaromatic fractions. Consequently, many other polyaromatic EROD-inducing compounds were present in the sludge. Inclusion of a biological test like the chicken embryo liver bioassay in the screening of sludge would improve the ability to detect the presence of bioactive dioxin-like compounds. A theoretical estimation of bio-TEQ concentrations in farm-soil following long-term application of sludge with bio-TEQ concentrations similar to those observed in this investigation indicated that the bio-TEQ levels in soil would increase very slowly over time. The chicken embryo liver bioassay proved useful in assessing levels of dioxin-like compounds in sewage sludge and it gives valuable complementary information to chemical analysis data.     

Tissue distribution of planar and non-planar chlorobiphenyls, 4,4'-DDE and hexachlorobenzene in sheep and lambs

The bioconcentration and distribution pattern of individual PCB congeners (IUPAC Nos: -54, -80, -155 and -169) and organochlorine pesticides (HCB and 4,4'-DDE) in blood, adipose tissue, liver and brain were examined in sheep two months after administration and in their offspring continuously exposed during the two months lactation period. Analyses were performed by high resolution gas chromatography. The levels of individual organochlorines varied significantly between tissues; the tissue/blood ratio (on a fat basis) varied by two orders of magnitude for individual congeners. The bioconcentration of the toxic planar PCB-169 congener was the highest in the liver and the lowest in the brain of the sheep.     

Degradation of three phenylurea herbicides (chlortoluron, isoproturon and diuron) by micromycetes isolated from soil

As part of a study conducted on the fate of xenobiotics in the environment, a selection of 100 strains of micromycetes (Ascomycetes, Basidiomycetes and Yeasts) have been cultivated in liquid synthetic medium with 3 phenylurea herbicides: chlortoluron and isoproturon (100mg L-1) and diuron (20mg L-1). While 17 strains depleted isoproturon over 50% only 4 depleted diuron and 2 chlortoluron at the same level. The best results were obtained with Bjerkandera adusta and Oxysporus sp which were the most efficient towards the 3 substrates. After 2 weeks Bjerkandera adusta depleted chlortoluron 98%, diuron 92% and isoproturon 88%.     

Clearance of PCDD/Fs via the gastrointestinal tract in occupationally exposed persons.

A digestive tract mass balance was performed on six men with high body burdens of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). Intake via food was measured by analyzing duplicate portions of the food consumed by the volunteers and excretion via feces was determined by quantitative collection and analysis of the feces. Blood samples were taken to determine the current body burden. The results showed that the quantity of non-metabolized chemical excreted in the feces clearly exceeded the uptake via food for all of the 2,3,7,8-substituted PCDDs and some of the PCDFs, indicating a significant clearance across the gastrointestinal tract. The concentrations of these PCDD/F congeners in blood and feces were highly correlated (r > 0.8), demonstrating that the fecal PCDD/F content was determined by the body burden. The half lives in the test persons due to fecal clearance of non-metabolized chemical were estimated from the excretion rate and the current body burden and ranged between 10 years (Cl8DD) and 33 years (2,3,4,7,8-Cl5DF). These were compared with the overall contaminant half-lives due to all clearance processes which were calculated from the body burden and the decrease in blood concentrations measured over several years. The fecal clearance of non-metabolized PCDD/F contributed on average between 37% (2,3,7,8-Cl4DD) and 90% (Cl8DD) to the total elimination. This indicates that the gastrointestinal pathway plays a decisive role in the clearance of most 2,3,7,8-subsituted PCDD/F congeners.