Bioassay-directed Fractionation of Organic Extracts of Marine Surface Sediments from the North and Baltic Sea - Part II: Results of the biotest battery and development of a biotest index (8 pp)

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Part I: Determination and identification of organic pollutants Part II: Results of the biotest battery and development of a biotest index

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Preamble. This series of two papers presents the results of an interdisciplinary research project (ISIS) dealing with bioassay-directed fractionation of marine sediment extracts. Part I presents the extraction and fractionation procedure as well as the results of chemical analysis, including non-target analysis of sediments. Part II describes the results of the biotest battery in relation to chemicals possibly causing parts of the observed effects. A biotest index is used to compare the toxicities of the samples.

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AUTHORS / AFFILIATIONS Ninja Reineke (3), Werner Wosniok (4), Dirk Danischewski (1), Heinrich Hühnerfuss (3), Angelika Kinder (5), Arne Sierts-Herrmann (5), Norbert Theobald (2), Hans-Heinrich Vahl (6), Michael Vobach (1), Johannes Westendorf (6) and Hans Steinhart (5).

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(1) Federal Research Centre for Fisheries, Institute for Fishery Ecology, Palmaille 9, 22767 Hamburg, Germany (2) Federal Maritime and Hydrographic Agency, Bernhard-Nochtstr. 78, 20359 Hamburg, Germany (3) University of Hamburg, Institute for Organic Chemistry, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany (4) University of Bremen, Institute of Statistics, Bibliothekstr. 1, 28334 Bremen, Germany (5) University of Hamburg, Institute for Food Chemistry, Grindelallee 117, 20146 Hamburg, Germany (6) University of Hamburg, University Hospital Hamburg-Eppendorf, Department for Toxicology, Vogt-Kölln-Str. 30, 22527 Hamburg, Germany (7) Eurofins Wiertz-Eggert-Jörissen, Stenzelring 14b, 21107 Hamburg, Germany

Goal, Scope and Background

The ecological relevance of contaminants in mixtures is difficult to assess, because of possible interactions and due to lacking toxicity data for many substances present in environmental samples. Marine sediment extracts, which contain a mixture of environmental contaminants in low concentrations, were the object of this study. The extracts were investigated with a set of different biotests in order to identify the compound or the substance class responsible for the toxicity. For this goal, a combination of biotests, biotest-directed fractionation and chemical analysis has been applied. Further on, a strategy for the development of a biotest index to describe the toxicity of the fractions without a prior ranking of the test results is proposed. This article (Part II) focuses on the biological results of the approach.

Methods

The toxicological potential of organic extracts of sediments from the North Sea and the Baltic Sea was analyzed in a bioassay-directed fractionation procedure with a set of biotests: luciferase reporter gene assays on hormone receptor and Ah receptor, arabinose resistance test, fish embryo test (Danio rerio), comet assay, acetylcholinesterase inhibition test, heat-shock protein 70 induction, oxidative stress and luminescence inhibition test (Vibrio fischeri). The test results provided the basis for the calculation of a biotest index by factor analysis to compare the toxicity of the samples and fractions.

Results and Discussion

Results of 11 biotests on different fractionation levels of the samples were described and discussed with regard to the occurrence of contaminants and their toxic potentials. Polychlorinated biphenyls, polycyclic aromatic hydrocarbons, quinones, brominated indoles and brominated phenols were in the focus of interest. A biotest index was constructed to compare the toxic responses in the samples and to group the biotest results.

Conclusion

The procedure presented in this study is well suited for bioassay-directed fractionation of marine sediment extracts. However, in relatively low contaminated samples, high enrichment factors and sufficient fractionation is necessary to allow identification of low concentrations of contaminants which is required to link effects and possible causes. In the present case, the relation between substances and effects was difficult to uncover due to relatively low concentrations of pollutants compared to the biogenic matrix and to the remaining complexity of the fractions. The results, with respect to the brominated phenols and indoles in the samples, highlight the successful use of bioassay directed fractionation in the case of high concentrations and high toxicity.

Recommendation and Outlook

In general, it has been shown that a marine risk assessment requires focusing on the input of diffuse sources and taking into account the fact of mixture toxicity. Effects resulting from biogenic substances will make the assessment of the influence of anthropogenic substances even more difficult.     

Soil organic matter dynamics after the conversion of arable land to pasture

 Conversion of arable land (maize) to pasture will affect the soil organic matter (SOM) content. Changes in the SOM content were studied using a size- and density-fractionation method and ¹³C analysis. Twenty-six years of maize cropping had resulted in a depletion of carbon stored in the macro-organic fractions (>150 μm) and an increase in the <20 μm fraction. Maize-derived carbon in the upper 20 cm increased from 10% in the finest fraction (<20 μm) to 91% in the coarse (>250 μm), light (b.d. <1.13 g cm^–3) fractions. Pasture installation resulted in a rapid recovery of the total SOM content. Up to 90% of the pasture-derived carbon that was mineralized during maize cropping was replaced within 9 years. Especially the medium and coarse size (>150 μm) and light (b.d. <1.13 g cm^–3) fractions were almost completely regenerated by input of root-derived SOM. The amount of medium-weight and heavy macro-organic fractions (>150 μm; b.d. >1.13 g cm^–3) in the 0- to 20-cm layer was still 40–50% lower than in the continuous pasture plots. Average half-life times calculated from ¹³C analyses ranged from 7 years in the light fractions to 56 years in heavy fractions. Fractionation results and ¹³C data indicated that mechanical disturbance (plowing) during maize cropping had resulted in vertical displacement of dispersed soil carbon from the 0- to 20-cm layer down to 60–80 cm. Conversion of arable land to pasture, therefore, not only causes a regeneration of the soil carbon content, it also reduces the risk of contaminant transport by dispersed soil carbon. Received: 10 March 1998     

Investigating the mechanisms for the opposing pH relationships of fungal and bacterial growth in soil

Soil pH is one of the most influential variables in soil, and is a powerful factor in influencing the size, activity and community structure of the soil microbial community. It was previously shown in a century old artificial pH gradient in an arable soil (pH 4.0-8.3) that bacterial growth is positively related to pH, while fungal growth increases with decreasing pH. In an attempt to elucidate some of the mechanisms for this, plant material that especially promotes fungal growth (straw) or bacterial growth (alfalfa) was added to soil samples of the pH gradient in 5-day laboratory incubation experiments. Also, bacterial growth was specifically inhibited by applying a selective bacterial growth inhibitor (bronopol) along the entire pH gradient to investigate if competitive interaction caused the shift in the decomposer community along the gradient. Straw benefited fungal growth relatively more than bacterial, and vice versa for alfalfa. The general pattern of a shift in fungal:bacterial growth with pH was, however, unaffected by substrate additions, indicating that lack of a suitable substrate was not the cause of the pH effect on the microbial community. In response to the bacterial growth inhibition by bronopol, there was stimulation of fungal growth up to pH 7, but not beyond, both for alfalfa and straw addition. However, the accumulation of ergosterol (an indicator of fungal biomass) during the incubation period after adding alfalfa increased at all pHs, indicating that fungal growth had been high at some time during the 5-day incubation following joint addition of alfalfa and bronopol. This was corroborated in a time-series experiment. In conclusion, the low fungal growth at high pH in an arable soil was caused to a large extent by bacterial competition, and not substrate limitation.     

Permanence of soil surface crusts on abandoned farmland in the Central Ebro Basin/Spain

Surface crusts are frequently found on fallow land in the semi-arid Ebro Basin (Spain) and are an important factor in land degradation. Soil surface sealing leads to a decrease in infiltration rates and a consequent increase in runoff, thereby accelerating sheet wash and rill erosion. Thin sections were used to analyse the development and structures of the different crusts found across the ridge/furrow field pattern. Rainfall simulations experiments and infiltration measurements show the runoff generation and the soil erosion rates on the crusts. The spatial distribution of crusts was documented using large-scale aerial photographs, taken from a remote-controlled hot air blimp.     

Growth measurements of saprotrophic fungi and bacteria reveal differences between canopy and forest floor soils

Canopy-held organic matter develops into a distinct soil system separate from the forest floor in wet temperate coniferous forests, creating a natural microcosm. We distinguished between fungal and bacterial components of the decomposer community in one site with Maple (Acer macrophyllum) and one site with Alder (Alnus rubra) by using direct measurements of growth; acetate incorporation into ergosterol, and leucine incorporation for fungi and bacteria, respectively. The higher organic matter content of the canopy soils correlated with higher fungal growth. The relative importance of fungi, indicated by fungal:bacterial growth ratio, was higher in the canopy soil of the Maple site, while there was no difference in the Alder site. The high C:N ratio of the Maple canopy soil likely contributed to this difference. These results demonstrate a divergence between canopy and forest floor that should be explored to gain insights in decomposer ecology using the natural microcosms that the canopy soils provide.     

Einfluss von konventioneller und biologisch-dynamischer Bewirtschaftungsweise auf die mikrobielle Biomasse und deren Stickstoff-Dynamik in Parabraunerden der Friedberger Wetterau

Effect of conventional and biological farming on microbial biomass and its nitrogen turnover in agriculturally used luvisols of the Friedberg plains The influence of conventional and biological farming on microbial biomass-carbon (Bio-C) and nitrogen (Bio-N) of agriculturally used fields was compared. The fields were located on luvisols of the Friedberg plains and tilled according to each system for many years. Bio-C and Bio-N were measured using the chloroform-fumigation-extraction technique. During early spring and autumn (1990) [Bio-CI% C_org] and Bio-N [%N₁] were slightly lower in sites of conventional farming than on fields tilled by biological farming. During the vegetation period Bio-C and Bio-N increased continuously in both systems. This increase, however was higher on conventional than on biological fields and may be ascribed to a higher C-input (rhizodeposition) and availability of mineral nutrients. In the soils of the biological farming system N-deficiency apparently decreased Bio-N (but not Bio-C) in spring time. Consequently the Bio-C/N-ratio increased. It seems as if plant growth affects the quality of microbes (Bio-C/N). The average turnover-time of Bio-N was about 1 year in all soils. However, the average turnover-time of Bio-C was 1.6 years, being 2 times faster in the conventional than in the biological farming system. It seems as if long-term management practices may affect the amount of labile organic matter by changing the biomass pool and its turnover rate.     

Polyuria and Polydipsia and Disturbed Vasopressin Release in 2 Dogs With Secondary Polycythemia

In dogs, secondary polycythemia (SP) may be associated with polyuria and polydipsia (PU/PD). The pathogenesis of this PU/PD has not yet been explained. We hypothesized that hyperviscosity and increased blood volume in SP might affect vasopressin (VP) release, resulting in PU/PD. This hypothesis was tested in 2 dogs with SP caused by renal neo-plasia and PU/PD. Osmoregulation of VP release was studied by a modified water deprivation test and by investigating the VP response to hypertonic saline infusion.
Water deprivation test results were consistent with an inability to produce concentrated urine despite increasing plasma osmolality. During hypertonic saline infusion, the osmotic threshold of VP release was markedly increased in both dogs, resulting in a delayed VP response to increasing plasma osmolality. The sensitivity of VP release was low normal in both dogs. We conclude that blood hyperviscosity and increased blood volume led to impaired VP release and polyuria.     

Ultrasonographic characterization of the urinary bladder in sows with and without urinary tract infection

The urinary bladders of sows (n=10) without urinary tract infection (UTI) were longitudinally transrectally scanned after emptying and refilling with 200, 400, 600 and 800mL saline, and a volume dependence was found for bladder depth (BD), dorsal (dWT) and ventral wall thicknesses (vWT), wall regularity (WR) and mucosal wall surface (mWS). When another 31 sows without and 15 with UTI (as defined on the basis of high bacterial count and macroscopic/biochemical urine abnormalities) were compared for these parameters using BD as volume equivalent, no differences were found. Sows with UTI more often had moderate to high amounts of sediment than animals without UTI. Ultrasonographic assessment of dWT, vWT, WR and mWS of the urinary bladder of sows requires knowledge of bladder volume, and BD may be used as a volume equivalent. However, the parameters are inappropriate for the diagnosis of UTI as defined in this study, while moderate/high amounts of sediment seem to be indicative. Sediment can be visualized by transrectal scanning, but this is also possible using the transcutaneous route.