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.     

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.     

Modeling recovery of Swedish ecosystems from acidification

Dynamic models complement existing time series of observations and static critical load calculations by simulating past and future development of chemistry in forest and lake ecosystems. They are used for dynamic assessment of the acidification and to produce target load functions, that describe what combinations of nitrogen and sulfur emission reductions are needed to achieve a chemical or biological criterion in a given target year. The Swedish approach has been to apply the dynamic acidification models MAGIC, to 133 lakes unaffected by agriculture and SAFE, to 645 productive forest sites. While the long-term goal is to protect 95% of the area, implementation of the Gothenburg protocol will protect approximately 75% of forest soils in the long term. After 2030, recovery will be very slow and involve only a limited geographical area. If there had been no emission reductions after 1980, 87% of the forest area would have unwanted soil status in the long term. In 1990, approximately 17% of all Swedish lakes unaffected by agriculture received an acidifying deposition above critical load. This fraction will decrease to 10% in 2010 after implementation of the Gothenburg protocol. The acidified lakes of Sweden will recover faster than the soils. According to the MAGIC model the median pre-industrial ANC of 107 microeq L(-1) in acid sensitive lakes decreased to about 60 microeq L(-1) at the peak of the acidification (1975-1990) and increases to 80 microeq L(-1) by 2010. Further increases were small, only 2 microeq L(-1) between 2010 and 2040. Protecting 95% of the lakes will require further emission reductions below the Gothenburg protocol levels. More than 7000 lakes are limed regularly in Sweden and it is unlikely that this practice can be discontinued in the near future without adverse effects on lake chemistry and biology.     

Effects of atmospheric ammonia on vegetation--a review

Atmospheric ammonia does not only cause acute injuries at vegetation close to the source, but significantly contributes to large scale nitrogen eutrophication and acidification of ecosystems because the amount of sources is high and after conversion to ammonium it can reach remote areas by long-range atmospheric transport. Besides having acute toxic potential, NH(3) and NH(4)(+) (= NH(y)) may disturb vegetation by secondary metabolic changes due to increased NH(y) uptake and assimilation leading to higher susceptibility to abiotic (drought, frost) and biotic (pests) stress. Prevention of damage to natural and semi-natural ecosystems will only be achieved if NH(3) emissions are drastically reduced. In this paper, the current knowledge on NH(y) emission, deposition, and its effects on vegetation and ecosystems are reviewed. Critical levels and critical loads for nitrogen deposition are discussed.     

Oil residues in Baltic sediment,mussel and fish. II. Study of the Finnish Archipelago 1979–1981

Aliphatic hydrocarbons and non-polar aromatic compounds have been analyzed after a spill of grude oil at Baltic Sea 1979 from drifting weathered oil glumps, sediment and sediment trap samples and bivalves at five areas of the Finnish Archipelago Sea. $\text{Mytilus}$ and flounder muscles and flounder livers were analyzed 1980 and 1981 from three of the previous five areas. The glumps were shown to be weathered grude oil. Sediment trap samples on the visibly contaminated area contained higher amounts of oil residues and in different ratios than the bottom sediments at the same areas. Extra local aliphatic hydrocarbon pollution was detected from the sediment traps at the reference area. Bivalves contained high amount of oil residues at visibly polluted but in some visibly non-polluted areas also 1979. Any significant decontamination was not detected at summer 1979 but next year the $\text{Mytilus}$ samples contained only traces if any and 1981 no detectable levels of aromatic oil residues. Aliphatic hydrocarbon residues in flounders showed no regional differences and no significant change of levels between 1980 and 1981. In contrary, the aromatic oil residues decreased to the non-detectable level in flounder muscles and to a significantly lower level in flouder livers 1981 related to 1980. Linear correlations of the analysis results are discussed.     

Primary biodegradation of veterinary antibiotics in aerobic and anaerobic surface water simulation systems

The primary aerobic and anaerobic biodegradability at intermediate concentrations (50-5000 microg/l) of the antibiotics olaquindox (OLA), metronidazole (MET), tylosin (TYL) and oxytetracycline (OTC) was studied in a simple shake flask system simulating the conditions in surface waters. The purpose of the study was to provide rate data for primary biodegradation in the scenario where antibiotics pollute surface waters as a result of run-off from arable land. The source of antibiotics may be application of manure as fertilizer or excreta of grazing animals. Assuming first-order degradation kinetics, ranges of half-lives for aerobic degradation of the four antibiotics studied were 4-8 days (OLA), 9.5-40 days (TYL), 14-104 days (MET) and 42-46 days (OTC). OLA and OTC were degraded with no initial lag phase whereas lag phases from 2 to 34 days (MET) and 31 to 40 days (TYL) were observed for other substances. The biodegradation behaviour was influenced by neither the concentrations of antibiotics nor the time of the year and location for sampling of surface water. Addition of 1 g/l of sediment or 3 mg/l of activated sludge from wastewater treatment increased the biodegradation potential which is believed to be the result of increased bacterial concentration in the test solution. Biodegradation was significantly slower in tests conducted in absence of oxygen. Assessments of the toxic properties of antibiotics by studying the influence on the biodegradation rates of 14C-aniline at different concentrations of antibiotics showed that no tests were conducted at toxic concentrations.     

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils

Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed.     

Airborne pollutants along a roadside: assessment using snow analyses and moss bags

Vertical snow sampling and moss bag transplants were used to estimate the local inorganic and organic pollutant load deposited from traffic along a major highway in Finland. The pH and concentrations of Cl(-), NO(3)(-), SO(4)(2-), Ca(2+), Na(+) and polyaromatic hydrocarbons (PAHs) were determined from snow samples collected in winter at different sites along the highway. In summer, moss bags containing 20 g of fresh red-stemmed feather moss (Pleurozium schreberi) were transplanted at the same sites. The moss bag transplants remained exposed to roadside traffic for a period of one month following which the samples were collected and the PAH profiles and concentrations were analysed. The deposition of inorganic and organic pollutants from road traffic was observed up to 60 m from the road. The prevailing winds had a significant effect on the dispersion of pollutants. Snow appears to be a good collector of inorganic pollutants from the atmosphere and can be used to monitor local airborne pollution from road traffic. Snow packs can also be used as passive collectors of organic pollutant loads from road traffic on a local scale. To monitor organic PAH deposition from the road traffic, moss bags appeared to be better indicators compared to snow sampling. The efficiency of moss bags in accumulating PAH compounds indicate that vegetation may be an important sink for traffic pollution.     

Modelling stomatal responses of spring wheat (Triticum aestivum L. cv. Turbo) to ozone and different levels of water supply

Spring wheat (Triticum aestivum L. cv. Turbo) was exposed to different levels of ozone and water supply in open-top chambers in 1991. Air was charcoal filtered (CF), non-filtered (NF) and CF plus proportional addition of ambient or twice ambient ozone (CF1, CF2). Seasonal means of O(3), taken over 24 h, were 2.3, 20.6, 17.3, and 34.5 nl litre(-1) for CF, NF, CF1 and CF2 treatments, respectively. A split-plot design was used to obtain two levels of water supply: one-half of the pots was irrigated sufficiently not to show any symptoms of drought stress; the others were exposed to low water supply and received 50% of these amounts. Using a steady-state porometer approximately 800 measurements of stomatal conductance (g(s)) were made on flag leaves from 68 to 106 days after sowing. The measurements yielded only small differences of maximum conductance between the two levels of water supply. Therefore, low water supply did not protect wheat plants against ozone injury via reduced stomatal uptake in this experiment. To describe the effects of environmental variables on the stomatal behaviour, boundary-line analysis and non-linear regression analysis were used. Besides microclimatic parameters, the ozone dose of flag leaves was introduced as an independent variable affecting stomatal aperture. A well-defined boundary line for ozone dose was found, suggesting that increasing ozone dose caused stomatal closure in wheat flag leaves. But at high ozone doses, co-acting senescence seems also responsible for the decrease in stomatal conductance. A multiplicative boundary-line model was used to predict stomatal conductance from combinations of environmental variables. In the test carried out with the measurements of stomatal conductance, the model accounted only for 40% of the variation of g(s). Generalized stomatal response patterns of the herbaceous growth form, the dependence of the variables' age and ozone dose and the lack of an important factor influencing stomatal response (water status of the plant) in the model, are suggested as explanations of the poor results of the test.     

The use of a sequential leaching procedure for heavy metal fractionation in green liquor dregs from a causticizing process at a pulp mill

A five-stage sequential leaching procedure was used to fractionate heavy metals (Cd, Cu, Pb, Cr, Zn, Fe, Mn, Ni, Co, As, V, Ba and Ti) in green liquor dregs into the following fractions: (1) water-soluble fraction (H2O), (2) exchangeable fraction (CH3COOH), (3) easily reduced fraction (HONH3Cl), (4) oxidizable fraction (H2O2 + CH3COONH4), and (5) residual fraction (HF + HNO3 + HCl). The green liquor dregs were derived from a causticizing process at a pulp mill at Kemi, Northern Finland. According to the leaching studies, the leachability of heavy metals in the water-soluble fraction varied between 0.5 and 2 mg kg(-1) expressed on a dry weight (d.w.) basis, indicating relatively low bioavailability of the metals. However, the concentration of Mn (2065 mg kg(-1); d.w.) showed a strong and of Zn (17.6 mg kg(-1); d.w.), Ni (39.7 mg kg(-1); d.w.) and Ba (32.0 mg kg(-1); d.w.) slightly tendency to be extracted in the exchangeable fraction. In addition, Zn, Mn, Ni, Co, V and Ba showed clear leachability in the easily reduced fraction, as well as Cd, Cu, Cr, Zn, Mn, As and Ba in the oxidizable fraction. For Cd, Cu, Cr, Zn, Mn, Ni, Co, Ba and Ti, the sum of leachable heavy metal concentrations in fractions 1-5 agreed relatively well with the "total" heavy metal concentrations. Recoveries of the sum of fractions 1-5 were 84-56% of those obtained by the US EPA method 3052 (i.e. concentrations obtained after microwave oven digestion with a mixture of HF and HNO3).