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.     

Genetic variability evaluation in a Moroccan collection of barley, Hordeum vulgare L., by means of storage proteins and RAPDs

The genetic variation existing in a set of barley (Hordeum vulgare L.) landrace samples recently collected in Morocco was estimated. Two kinds of genetic markers, seed storage proteins (hordeins) and random amplified polymorphic DNA (RAPD), were used. Only six out of 31 landraces were subjected to RAPD analysis. Both kinds of markers, RAPD and storage proteins, yielded similar results, showing that the level of variation observed in Moroccan barley was high: all landraces showed variability; 808 different storage protein patterns (multilocus associations) were observed among 1897 individuals (2.32 seeds per association, on average) with an average of 43 multilocus associations per accession. In general, genetic variation within accessions was higher than between accessions. The 100 polymorphic RAPD bands generated by 21 effective primers were able to generate enough patterns to differentiate between uniform cultivars and even between individuals in variable accessions. One of the aims of this work was to compare the effectiveness of RAPD versus storage protein techniques in assessing the variability of genetic resource collections. On average hordeins were more polymorphic than RAPDs: they showed more alternatives per band on gels and a higher percentage of polymorphic bands, although RAPDs supply a higher number of bands. Although RAPD is an easy and standard technique, storage protein analysis is technically easier, cheaper and needs less sophisticated equipment. Thus, when resources are a limiting factor and considering the cost of consumables and work time, seed storage proteins must be the technique of choice for a first estimation of genetic variation in plant genetic resource collections.     

水肥(氮)对小麦生理生态的影响 (Ⅰ)水肥 (氮 )条件对小麦光合蒸腾与水分利用的影响

光合速率在日出后随着光强的增强而急速升高,4月20日是小麦光合最强。净光合速率日变化随施氮量的增加而下降,水胁迫处理显著低于高水处理;不同时段变化净光合速率随施氮量和土壤含水量的增加而增加,但总的趋势是降低的。小麦蒸腾速率日变化总体趋势是在日出后随着光强的增强而急速升高、随光强的减弱而降低。小麦蒸腾速率日变化曲线为单峰型,中午最高。叶片蒸腾速率日变化随土壤水分供应增加而增加,随氮肥供应增加而下降;不同供水供氮条件下,叶片蒸腾速率的分异在日中尤其是上午比较明显。蒸腾速率从4月10日一直呈现下降趋势。小麦叶片水分利用率日变化曲线为"L"型,即早上较高,中午和下午都非常低。4月20日叶片水分利用率最高,5月10日最低。叶片对不同土壤水分和氮控制的反映为高氮低水高水分利用率,反之,则水分利用率较低。不同水、氮在上午对叶片水分利用率影响差异比较明显,下午则趋于一致。     

Increased emergence of spring wheat after inoculation with Pseudomonas chlororaphis isolate 2E3 under field and laboratory conditions

Inoculation at the time of planting with Pseudomonas chlororaphis strain 2E3 increased the emergence of spring wheat by 8% and 6% at two different sites in northern Utah. Isolate 2E3 strongly inhibited the growth of the wheat pathogen Fusarium culmorum on artificial media. A second isolate of P. chlororaphis (strain O6) also inhibited fungal growth on artifical media but did not increase emergence of the spring wheat at the same field sites. Inoculation of winter wheat by 2E3 did not promote emergence when planted into field soil sterilized by fumigation with methyl bromide. Under laboratory conditions, emergence of spring wheat in sterilized soils from both sites was at least 90%. In the soils that were not sterilized, emergence was below 25% in soil from one site and below 50% in soil from the other. Treating seeds with 2E3 significantly improved emergence in a sterile soil-containing matrix that had been inoculated with the wheat pathogen Fusarium culmorum. Consequently, we propose that increases in wheat emergence can be attributed to the suppression by 2E3 of pathogenic organisms present in the native field soils. A strain of Rhizoctonia solani, shown to the pathogenic on winter wheat, was isolated from one of these soils.     

Hayland conversion to wheat production in semiarid eastern Montana: tillage, yield and hay production comparisons

When converting grass- and haylands to cultivated crop production, care must be taken to conserve and maintain soil resources while considering economic issues. Methods of breaking sod can have a bearing on erosivity, physical and chemical properties of soils, and cost of production. Our objective was to compare three methods of converting crested wheatgrass [Agropyron desertorum (Fisch. ex Link) Schult.] hayland to wheat (Triticum aestivum L.) production vs. leaving the land for hay production. We initiated a study in 1990 on Dooley sandy loam (fine-loamy, mixed Typic Argiboroll) near Froid in semiarid eastern Montana, USA. Plots, replicated three times, were 12- by 30-m oriented east to west on a north-facing slope. We converted sod to cultivated crop production by: (1) moldboard plow, (2) toolbar with sweeps, (3) herbicides (no-till). Plots were fallowed until spring 1991 and then seeded to spring wheat each of the next four years. All wheat plots were fertilized with 224 kg ha⁻¹ of 18-46-0 in 1991 and 1992, and 34 kg ha⁻¹ nitrogen as 34-0-0 in 1993 and 1994. Grass was either fertilized same as wheat or not fertilized. Wheat yields averaged 2540 kg ha⁻¹ on tilled treatments and 2674 kg ha⁻¹ on no-till. Fertilized grass consistently out-yielded unfertilized, and averaged 3.2 Mg ha⁻¹ vs. 1.8 Mg ha⁻¹. Toolbar with sweeps had highest economic return of US$169.48 ha⁻¹ to pay for land, labor, and management. Moldboard plow had US$162.05 ha⁻¹. Because of herbicide costs, no-till only returned US$148.64 ha⁻¹. Unfertilized grass hay returned US$67.68 ha⁻¹ and fertilized grass hay, US$97.95 ha⁻¹. Results may be tempered because our wheat yields were high: a 2016 kg ha⁻¹ wheat yield would have returned the same as fertilized grass. Before converting grass- and hay-lands to small grains production, consideration must be given to such variables as sod conversion methods, management practices, labor requirements, market conditions, total precipitation and its temporal distribution, soil conditions, growth environment, soil conservation, and economics.     

参与碳氮磷转化的水解酶对不同施肥响应的差异

本文旨在研究土壤水解酶对不同施肥的响应差异以及影响因素。通过在红壤中添加牛粪有机肥、化肥进行90d的室内土壤培养试验,采用微孔板荧光法动态分析5、30和90d参与碳氮磷转化的土壤水解酶(α-1,4-葡萄糖苷酶、β-1,4-葡萄糖苷酶、纤维素酶、木聚糖酶、亮氨酸氨基肽酶、β-1,4-N-乙酰氨基葡萄糖苷酶、磷酸酶)活性。与不施肥(对照)相比,在30 d后,化肥处理的总酶活性显著下降,对应的参与碳氮磷转化酶活性均有不同程度下降;而有机肥处理的总酶活性在培养期内均未发生显著变化,但是其α-1,4-葡萄糖苷酶显著增加,而磷酸酶活性显著降低。参与碳转化的4种水解酶中,只有α-1,4-葡萄糖苷酶活性对施肥的响应较强,且施加有机肥增加其活性而无机肥则降低其活性;对于参与氮转化的水解酶而言,化肥明显抑制了亮氨酸氨基肽酶活性,而有机肥增加了β-1,4-N-乙酰氨基葡萄糖苷酶活性;磷酸酶活性明显受到有机肥的抑制作用,而对化肥的响应总体不明显。不同水解酶对不同施肥的响应有明显差异,NMDS分析表明,α-1,4-葡萄糖苷酶和亮氨酸氨基肽酶响应最明显,其次为磷酸酶与木聚糖酶;相关和冗余分析显示,土壤p H、可溶性有机碳对酶活性的影响最大,一定程度说明了不同肥料通过影响土壤理化性质进而影响水解酶活性。     

Control of Rainfall-Induced Soil Erosion with Various Types of Polyacrylamide (8 pp)

Background   Several types of organic polymers with various molecular weights and surface charges are produced for various industrial and agricultural purposes, and recent studies have shown that high molecular weight anionic polymers are the most effective in reducing runoff and soil loss. However, anionic polyacrylamide (PAM) is marketed under various commercial names with variable molecular weight and length according to its chemistry (Barvenik 1994). Consequently, its interactions with soil and efficiency in reducing soil erosion are expected to vary. Moreover, types of PAM other than those recommended for erosion control may also be effective in reducing soil erosion. Objectives   The objective of this research is to test the influence of 9 types of polyacrylamide (PAM) on water infiltration and soil loss of an arid silt loam soil under simulated rainfall conditions. Method  logy. Nine types of organic polymers varied in surface charge, ranging from +20 to –75, and molecular weight ranging from very low to very high were used in this study. Soil plots with dimensions of 60 cm by 120 cm having moderate slope of3 to 5% were constructed in the field and equipped with runoff collection installations. A specific type of PAM was spread on soil plots at a rate of 10 kg/ha. Rainfall was supplied from a drip type rainfall simulator at constant intensity and duration of 80 mm/h and 50 min, respectively. Runoff, infiltration and soil loss were measured for each plot subjected to a specific type of PAM and compared to control plots. Results and Conclusions   Land application of PAMs significantly increased infiltration prior to runoff but had insignificant effect on total infiltration volume and final infiltration rate under the present experimental conditions. Experimental results confirmed that anionic polymers were more effective in reducing soil loss than that of non-ionic or cationic polymers. In fact the cationic, low molecular weight and low surface charge non-ionic PAMs had insignificant effect on soil loss. High molecular weight anionic PAM with 30% surface charge (A-130) was more effective(46% reduction) than those having a high molecular weight and a lower surface charge of 20% (A-110 and A-120) with an aver-age soil loss reduction of 24%. The PAM A-836, described assoil erosion polymer with 20% surface charge and high molecular weight, reduced soil erosion by 41%. Unexpectedly, the anionic PAM with low molecular weight but very high surface charge was also effective in reducing soil loss (47% reduction). Recommendations and Outlook   Land application of PAM improves water infiltration and highly reduces soil erosion thus improving agricultural production. Application of PAM could be the only viable way to improve arid land farming. It seemed that various types of PAMs, other than those specifically produced for erosion protection can be used to combat soil erosion. This means that farmers in poor countries can use any type of PAM that is available to them to improve agricultural production.     

固定态铵的含量及中国某些耕地土壤NH4+的固定能力

Fixed ammonium contents and NH4^ fixation capacities of some representative cultivated solis collected from 16 provinces of China were studied.Results showed that the contents of fixed ammonium in soils ranged from 35 to 573 mg N kg^-1,with an average of 198mg Nkg^-1.The content of fixed ammonium correlated very significantly with mica content for tropical and subtropical soils,whereas this was not the case for soils in the temperate zone.At the end of K-exhausting experiment the fixed ammonium content increased in most soils studied.However,it decreased in smom temperate soils.Generally,fixation of added NH4^ could not be found either before or after K-exhausting experiment for highly weathered soils,including tropical soils and soils derived from granite-gneiss or Quaternary red clays in the subtropic zone,while for most soils in the Yangtze River dalta the NH4^ fixation capacity was rather high and increased significantly in the K-exhausted soils.     

小麦水分利用效率研究进展

介绍了小麦叶片冠层的水分利用效率估算模型,并阐述了小麦光合与水分利用效率关系的研究进展,为当前节水农业提供了大量信息。     

Gram-negative bacterial flora on the root surface of wheat (Triticum aestivum) grown under different soil conditions

We identified 161 Gram-negative bacterial strains isolated from the root surface of wheat grown under different soil conditions. The strains were divided into seven groups based on major morphological and physiological properties. Taxonomic allocation of the groups was verified by guanine+cytosine contents of DNA. Except for one group, which may be assumed to include bacteria belonging to the genera Flavobacterium and Cytophaga, the various groups were taxonomically united. The distribution of the groups changed with soil improvement. Pseudomonads predominated in unimproved soil, but Flavobacterium and Cytophaga spp. were predominant in the most improved soil. As all the strains were non-fermentative by Hugh and Leifson's test, API 20NE identification was applied. However, many strains were misidentified by this system, especially in the Flavobacterium and Cytophaga spp. group. For ecological studies, the strains were classified to species level by the API 20 NE system and by the results of a combination of guanine+cytosine (mol%) and isoprenoid quinone data. The pattern of distribution of the bacteria on the root surface of wheat varied at species level within one genus depending on soil conditions.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday