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.     

Bedeutung der arbuskulären Mykorrhiza (AM) für die Schwermetalltoleranz von Luzerne (Medicago Sativa L.) und Hafer (Avena sativa L.) auf einem klärschlammgedüngten Boden

Effect of arbuscular mycorrhizal fungi (AMF) on heavy metal tolerance of alfalfa (Medicago sativa L.) and oat (Avena sativa L.) on a sewage-sludge treated soil In pot experiments with a sewage sludge treated soil, the influence of two arbuscular mycorrhizal fungi (AMF) isolates of Glomus sp. (T6 and D13) on plant growth and on the uptake of heavy metals by alfalfa (Medicago sativa L.) and oat (Avena sativa L.) was investigated. Alfalfa showed an increase of biomass with mycorrhizal infection only to a small extent. In oat AMF inoculation increased the growth of both root and shoot by up to 70% and 55% respectively. Mycorrhization raised the P-content and -uptake in alfalfa, but not in oat, in both roots and shoots. Mycorrhizal alfalfa showed lower Zn-, Cd- and Ni-contents and uptake in roots and shoots. The root length was significantly decreased in mycorrhizal alfalfa plants (up to 38%). The translocation of heavy metals into the shoot of mycorrhizal alfalfa was slightly increased. Mycorrhizal infection of oat led to higher concentrations of Zn, Cd and Ni in the root but to less Zn in the shoot. The translocation of heavy metals to the oat shoot was clearely decreased by mycorrhizal colonisation. This may be based on the ability of fungal tissues to complex heavy metals at the cell walls, thus excluding metals from the shoot. This conclusion is supported by the enhanced root length (up to 78%) of mycorrhizal oat plants in this experiment. The mycorrhizal infection seemed to protect plants against heavy metal pollution in soils. It was obvious that different host plants reacted in different ways.     

Zur Genese der Marschböden. I. Der Einfluß von Sediment- und Bodengefüge

On the genesis of tidal marsh soils I. The influence of sediment- and soil-structure The significance of sediment structure for soil structure has often been disregarded when discussing the properties and genesis of tidal marsh soils. After defening several terms that are often misinterpreted, three types of tidal-marsh-soils with different structure are described: 1 . the typical clayey tidal-marsh-soils with very various aggregate structures: The well aired and permeable “SEEMARSCH” (marine-tidal-marsh-soil) with polyhedral and prismatic structure, which is formed from the flaky “card-house” structure of marine sediments rich on salt; the densely paked “BRACKMARSCH” (brackish-tidal-marsh-soil), whose moisture is due to perched water, which coarse prismatic to columnar structure, which is formed from the mainly horizontally orientet structure of brackish sediments due to dispersion; the moderately permeable “FLUSSMARSCH” (tidal-river-marsh-soil), whose wetness is often due to groundwater, with medium to coarse prismatic structure, which is formed from the finely aggregated structure of perimarine tidal-river sediments; 2 . the tidal marsh soils poor in clay, rich in silt and very fine sand with coherent structure and low air porosity, due to a strong tendency towards puddling of the top soil by raindrops, causes the formation of “HAFTNÄSSEMARSCH” (marsh soil whose moisture is due to capillar water); 3 . the tidal-marsh-soils rich in plant remains, clay and with a high level of groundwater often extremly acid, with coherent structure, which formed the generally highly permeable “ORGANOMARSCH”. The conditions of formation of these soils and the soil parameters are discussed also.     

Effects of aerobic conditions in the rhizosphere of rice on the dynamics and availability of phosphorus in a flooded soil – A model experiment

The secretion of O₂ by rice roots results in aerobic conditions in the rhizoshere compared to the bulk flooded soil. The effect of this phenomenon on the adsorption/desorption behavior and on the availability of phosphorus (P) in a flooded soil was investigated in a model experiment. An experimental set‐up was developed that imitates both O₂ release and P uptake by the rice root. The results showed that O₂ secretion significantly reduced P adsorption/retention and increased P desorption/release in the “rhizosphere” soil, compared to the anaerobic bulk soil. The P uptake by an anion exchange resin from both unfertilized and P‐amended soil was significantly increased. The results confirm that the O₂ secretion is an important mechanism to enhance P availability and P uptake of rice under flooded conditions, where the “physico‐chemical” availability of P in the anaerobic bulk soil is strongly reduced. The decrease of P availability in the P‐amended flooded bulk soil was mainly associated with the almost complete transformation of the precedingly enriched Al‐P fraction into Fe‐bound P with extremely low desorption/release characteristics during the subsequent flooding.     

Steryl phenolic acid esters in cereals and their milling fractions

The steryl ferulate contents of rye and wheat grains and their milling fractions were analyzed using a reversed-phase high-performance liquid chromatographic (HPLC) method. HPLC-mass spectrometry was used for identification. In addition, steryl ferulates of some selected milling byproducts were determined. The total steryl ferulate contents of rye and wheat grains were 6.0 and 6.3 mg/100 g, respectively. Uneven distribution of steryl ferulates in the grains led to considerable differences in the milling products; their steryl ferulate contents ranged from trace amounts in flours with low ash content to 20 and 34 mg/100 g in rye and wheat brans, respectively. Campestanyl ferulate and sitostanyl ferulate were the main components, followed by campesteryl ferulate and sitosteryl ferulate, whereas sitosterol was the main component in total sterols. Among the other samples, a byproduct of rice milling (pearling dust) was the best source of steryl ferulates, its total steryl ferulate content being 119 mg/100 g, whereas no measurable amounts of steryl ferulates were measured in oat bran or pearling dust of barley. The results indicated that rye and wheat and especially their bran fractions are comparable to corn as steryl ferulate sources.     

Evidence that the reactivity of the martian soil is due to superoxide ions

The Viking Landers were unable to detect evidence of life on Mars but, instead, found a chemically reactive soil capable of decomposing organic molecules. This reactivity was attributed to the presence of one or more as-yet-unidentified inorganic superoxides or peroxides in the martian soil. Using electron paramagnetic resonance spectroscopy, we show that superoxide radical ions (O2-) form directly on Mars-analog mineral surfaces exposed to ultraviolet radiation under a simulated martian atmosphere. These oxygen radicals can explain the reactive nature of the soil and the apparent absence of organic material at the martian surface.     

Cytokinin from soluble RNA of Escherichia coli: 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine

We have isolated a compound responsible for the cytokinin activity of soluble RNA from Escherichia coli. The structure, indicated as 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine, C(16)H(23)N(5)0(4)S, on the basis of low-and high-reso!ution mass spectrometry, was established by unequivocal synthesis. The mass spectra, chromatographic behavior, and ultraviolet spectra of the compounds from natural and synthetic sources were identical.     

Heritable fragile site on chromosome 16: probable localization of haptoglobin locus in man

We have found recurrent chromosome breaks at a site (the "fragile site") on the long arm of chromosome 16. This site segregates in simple Mendelian dominant fashion in a large family. The distal portion of the chromosome sometimes shows selective endoreduplication. Preliminary linkage results reveal only 3 recombinants in 33 opportunities for recombination between the fragile site and the alpha locus of haptoglobin, an indication that the alpha-Hp gene is located near this region on chromosome 16.