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.     

Results and evaluation of the first study of organochlorine contaminants (PCDDs, PCDFs, PCBs and DDTs), heavy metals and metalloids in birds from Baja California, México

Organochlorine compounds (OCs) including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p-p'-DDE), heavy metals (Pb, Cd, Zn, Cu), and arsenic were measured in house sparrows (Passer domesticus) and common ground doves (Columbina passerina) from Baja California Sur, México. Concentrations of PCDD/Fs were low, with 21 pg/g for house sparrows, and 7.7 pg/g for common ground doves. Non-ortho-PCB concentrations in house sparrow and common ground doves were 58 and 254 pg/g, respectively, and are within the highest concentrations reported in species that are in the low levels of food webs. The major differences in organochlorine levels between species were found for ortho-PCBs and DDTs. ortho-PCB levels were higher in the seedeater species, whereas DDT levels were higher in the omnivorous species. Heavy metal levels were far below those associated with negative effects.     

Temporal trends of organochlorine contamination in Black Guillemots in Iceland from 1976 to 1996

The levels of several different persistent organochlorines (OCs) in Black Guillemots Cepphus grylle, collected during the summers of 1976-1996 at Breioafj?rour in W-Iceland, were investigated. The levels of about 40 different organochlorines (PCBs, DDTs, chlordanes, toxaphenes, HCH, HCB) were compared with respect to age, sex, fat content, and year of collection. The levels of PCBs correlated very closely with those of DDE, indicating long-range transport as the major source of these contaminants in Iceland, with the ratio PCBs/DDE mostly in the range of 2-5. Unlike the Gyrfalcon Falco rusticolus, the organochlorine levels did not seem to accumulate substantially with age, neither in males nor females. The variation in the levels of OCs at the age of 2 years was even greater than the variation in OC levels over an age range of 12 years. In immature birds the levels of PCBs, DDE, HCB and beta-HCH declined very slowly (T(1/2) from 12 to 20 years) over the years 1976-1996, whereas the levels of alpha-HCH and p,p'-DDT declined much faster. The levels of trans-nonachlor, alpha-chlordane, gamma-chlordane, oxychlordane, and toxaphene did not correlate with the year of collection. As the Black Guillemot is mostly a resident seabird, feeding mainly on small fish and invertebrates, this investigation should give a good indication of the temporal trends of organochlorine pollution at Breioafj?rour, Iceland, during this 20 year period and is likely to reflect baseline trends in the marine environment of the North-Atlantic Ocean.     

Practical considerations for addressing uncertainties in monitoring bulk deposition

The assessment of the deposition of both wet (rain and cloud) and dry sedimenting particles is a prerequisite for estimating element fluxes in ecosystem research. Many nations and institutions operate deposition networks using different types of sampler. However, these samplers have rarely been characterized with respect to their sink properties. Major errors in assessing bulk deposition can result from poor sampling properties and defective sampling strategies. Relevant properties are: sampler geometry and material, in particular the shape of the rim; sink properties for gases and aerosols; and microbial transformations of the collected samples. An adequate number of replicates allows the identification of samples which are contaminated, in particular by bird droppings. The paper discusses physical and chemical properties of the samplers themselves. The dependence of measurement accuracy on the number of replicates and the sampling area exposed is discussed. Recommendations are given for sampling strategies, and for making corrections and substitution of missing data.     

Photo-Fenton-assisted ozonation of p-Coumaric acid in aqueous solution

The degradation of p-Coumaric acid present in olive oil mill wastewater was investigated as a pretreatment stage to obtain more easily biodegradable molecules, with lower toxicity that facilitates subsequent anaerobic digestion. Thus, photo-Fenton-assisted ozonation has been studied and compared with ozonation at alkaline pH and conventional single ultraviolet (UV) and acid ozonation treatments. In the combined process, the overall kinetic rate constant was split into various components: direct oxidation by UV light, direct oxidation by ozone and oxidation by hydroxyl radicals. Molecular and/or radical ozone reaction was studied by conducting the reaction in the presence and absence of tert-butylalcohol at pHs 2, 7 and 9. Ozone oxidation rate increases with pH or by the addition of Fenton reagent and/or UV radiation due to generation of hydroxyl radicals, *OH. Hydrogen peroxide and ferrous ion play a double role during oxidation since at low concentrations they act as initiators of hydroxyl radicals but at high concentrations they act as radical scavengers. Finally, the additional levels of degradation by formation of hydroxyl radicals have been quantified in comparison to the conventional single processes and an equation is proposed for the reaction rate as a function of studied operating variables.     

Modeling the Entrepe?as Reservoir.

The Entrepe?as Reservoir is a monomictic reservoir located in River Tagus (Spain). The aim of this work is to establish a feasible model to predict the depth of the thermocline that is developed in the reservoir during the period of natural thermal stratification. Entrainment, eddy diffusion, inflow of external energy, and other factors are considered to calibrate the parameters of the model. The methodology involves the measure of actual temperature and electrical conductivity profiles, use of meteorological data and reservoir parameters, and selection and application of several models from the literature. The calculations and predictions are integrated to a software packet that is able to predict thermocline depth and water temperature profile during a 1-year period on a day-by-day basis. In the thermocline depth, the prediction error, on the basis of real data, is less than 6% and, in the water temperature, it is 2 degrees C.     

A European survey of atmospheric heavy metal deposition in 2000-2001

The background, administration and current status of the monitoring programme "European Survey of Atmospheric Heavy Metal Deposition" is described. All European states are invited to join the survey 2000-2001. The co-ordination and responsibility for the survey will gradually be transferred from the Nordic Council of Ministers to the International Cooperative Programme on Effects of Air Pollution on Natural Vegetation and Crops (ICP Vegetation).     

Mercury pollution and macrophage centres in pike (Esox lucius) tissues

From June 1993 to October 1994, studies have been carried out on the effects of mercury in the Oder River and pike tissue contamination (muscle, kidney, liver). The mean mercury contents in the sediment range from 0.03 to 1.1 mg/kg dry weight. In the pike muscle, between 0.22 and 0.85 mg/kg, on a wet weight basis, were found. The measured mercury concentrations were analysed in relation to the number of macrophage centres of the liver, spleen and kidney of the pike. Positive correlations between mercury and MC response (0.54 ≤ r ≤ 0.79, p < 0.05) were found in all of these organs. The suitability of the macrophage-centre-response as a possible bioindicator for mercury pollution is discussed in the literature. In our study, the response of MCs was found to be suitable as a biomarker for the impairment of fish health.     

Monoterpene emissions and carbonyl compound air concentrations during the blooming period of rape (Brassica napus)

An increasing percentage of agricultural land in Germany is used for oil seed plants. Hence, rape has become an important agricultural plant (in Saxony 1998: 12% of the farmland) in the recent years. During flowering of rape along with intensive radiation and high temperatures, a higher production and emission of biogenic VOC was observed. The emissions of terpenes were determined and more importantly, high concentrations of organic carbonyl compounds were observed during this field experiment. All measurements of interest have been carried out during two selected days with optimal weather conditions. It is found that the origin or the mechanism of formation of different group of compounds had strong influence on the day to day variation of their concentrations. The emission flux of terpenes from flowering rape plants was determined to be 16–32 μg h⁻¹ m⁻² (30–60 ng h⁻¹ per g dry plant––540–1080 ng h⁻¹ per plant), in total. Limonene, -thujene and sabinene were the most important compounds (about 60% of total terpenes). For limonene and sabinene reference emission rates (M_S) and temperature coefficients were determined: β_limonene=0.108 K⁻¹ and M_S=14.57 μg h⁻¹ m⁻²; β_sabinene=0.095 K⁻¹ and M_S=5.39 μg h⁻¹ m⁻².

The detected carbonyl compound concentrations were unexpectedly high (maximum formaldehyde concentration was 18.1 ppbv and 3.4 ppbv for butyraldehyde) for an open field. Possible reasons for these concentrations are the combination of primary emission from the plants induced by high temperature and high ozone stress, the secondary formation from biogenically and advected anthropogenically emitted VOC at high radiation intensities and furthered by the low wind speeds at this time.     

Application of the Microtox test and pollution indices to the study of water toxicity in the Albufera Natural Park (Valencia, Spain)

The toxic effects of waters collected from irrigation channels in a Mediterranean wetland (Albufera Natural Park, Valencia, Spain) were tested with the Microtox assay and compared with six pollution indices (PIs) defined from analytical parameters. Chemical oxygen demand (COD), biological oxygen demand (BOD), nutrients, heavy metals and pesticides were measured. The bioassay result (concentrations of the water sample (% V/V) that reduced light emission to 10%, 20% and 50%, EC10, EC20 and EC50, respectively (ECs)) was compared with the PIs. This comparison has demonstrated a general agreement between ECs and PIs, except in the case of irrigation channels affected by herbicides used in rice farming (molinate and thiobencarb). No pronounced inhibition was detected in the bioluminescence in relation to the eutrophic parameters in the irrigation waters for EC50 values, indicating that this parameter does not suffice to detect eutrophic waters. Data derived from irrigation water pollution and bioassay were assembled by multivariate statistical techniques (principal component analysis). These components were associated with various contamination sources.