Paul Henning Krogh

Effects of the antibiotics oxytetracycline and tylosin on soil fauna.

Antibiotics may enter the terrestrial environment when amending soils with manure. A Note of Guidance on ecological risk assessment of veterinary medicines was issued in January 1998. Hardly any information about ecotoxicological effects of already existing substances are available. This study has tested the effects of two widely used antibiotics, tylosin and oxytetracycline, on three species of soil fauna: Earthworms, springtails and enchytraeids. Neither of the substances had any effect at environmentally relevant concentrations. The lowest observed effect concentration was 3000 mg kg-1 and in many cases no effect was seen even at the highest test concentration of 5000 mg kg-1.

A Comparison of Soil Microbial Community Structure, Protozoa and Nematodes in Field Plots of Conventional and Genetically Modified Maize Expressing the Bacillus thuringiens is CryIAb Toxin

Field trials were established at three European sites (Denmark, Eastern France, South-West France) of genetically modified maize (Zea mays L.) expressing the CryIAb Bacillus thuringiensis toxin (Bt), the near-isogenic non-Bt cultivar, another conventional maize cultivar and grass. Soil from Denmark was sampled at sowing (May) and harvest (October) over two years (2002, 2003); from E France at harvest 2002, sowing and harvest 2003; and from SW France at sowing and harvest 2003. Samples were analysed for microbial community structure (2003 samples only) by community-level physiological-profiling (CLPP) and phospholipid fatty acid analysis (PLFA), and protozoa and nematodes in all samples. Individual differences within a site resulted from: greater nematode numbers under grass than maize on three occasions; different nematode populations under the conventional maize cultivars once; and two occasions when there was a reduced protozoan population under Bt maize compared to non-Bt maize. Microbial community structure within the sites only varied with grass compared to maize, with one occurrence of CLPP varying between maize cultivars (Bt versus a conventional cultivar). An overall comparison of Bt versus non-Bt maize across all three sites only revealed differences for nematodes, with a smaller population under the Bt maize. Nematode community structure was different at each site and the Bt effect was not confined to specific nematode taxa. The effect of the Bt maize was small and within the normal variation expected in these agricultural systems.

The toxicity testing of double-walled nanotubes-contaminated food to Eisenia veneta earthworms.

Nanotechnology is playing an increasing role in the society, but very little is known about the environmental toxicity of nanotechnological products. Few previous studies have been published with regard to the effect of nanoparticles on soil organisms. The present paper deals with the toxicity of carbon double-walled nanotubes (DWNT) and C60 fullerenes to the soil-dwelling earthworm Eisenia veneta, with the aim of evaluating the lethal and sub-lethal toxicity. Reproduction of E. veneta was affected by DWNT administered through food at concentrations above 37 mg DWNT/kg food. The most sensitive toxicological parameter was reproduction (cocoon production), with no effect on hatchability, survival or mortality at up to 495 mg DWNT/kg and 1000 mg C60/kg.

Effects of acclimation temperature on thermal tolerance and membrane phospholipid composition in the fruit fly Drosophila melanogaster

Adaptative responses of ectothermic organisms to thermal variation typically involve the reorganization of membrane glycerophospholipids (GPLs) to maintain membrane function. We investigated how acclimation at 15, 20 and 25 degrees C during preimaginal development influences the thermal tolerance and the composition of membrane GPLs in adult Drosophila melanogaster. Long-term cold survival was significantly improved by low acclimation temperature. After 60 h at 0 degrees C, more than 80% of the 15 degrees C-acclimated flies survived while none of the 25 degrees C-acclimated flies survived. Cold shock tolerance (1h at subzero temperatures) was also slightly better in the cold acclimated flies. LT50 shifted down by ca 1.5 degrees C in 15 degrees C-acclimated flies in comparison to those acclimated at 25 degrees C. In contrast, heat tolerance was not influenced by acclimation temperature. Low temperature acclimation was associated with the increase in proportion of ethanolamine (from 52.7% to 58.5% in 25 degrees C-acclimated versus 15 degrees C-acclimated flies, respectively) at the expense of choline in GPLs. Relatively small, but statistically significant changes in lipid molecular composition were observed with decreasing acclimation temperature. In particular, the proportions of glycerophosphoethanolamines with linoleic acid (18:2) at the sn-2 position increased. No overall change in the degree of fatty acid unsaturation was observed. Thus, cold tolerance but not heat tolerance was influenced by preimaginal acclimation temperature and correlated with the changes in GPL composition in membranes of adult D. melanogaster.

Toxicity of eight polycyclic aromatic compounds to red clover (Trifolium pratense), ryegrass (Lolium perenne), and mustard (Sinapsis alba).

The effect of eight polycyclic aromatic compounds (PACs) on the seed emergence and early life-stage growth of three terrestrial plants (Sinapsis alba, Trifolium pratense and Lolium perenne) were studied in a greenhouse, using a Danish agricultural soil with an organic carbon content of 1.6%. After three weeks of exposure, seed emergence and seedling weight (fresh weight and dry weight) were determined. Exposure concentrations were verified with chemical analysis. The substances tested were four polycyclic aromatic hydrocarbons (fluoranthene, pyrene, phenanthrene and fluorene), the N-, S-, and O-substituted analogues of fluorene (carbazole, dibenzothiophene and dibenzofuran, respectively), and the quinoline representative acridine. Seedling growth was a far more sensitive endpoint than seed emergence for all substances. Concentrations estimated to give a 20% reduction of seedling fresh weight (EC20-values) ranged from 36 to 290 mgkg(-1) for carbazole, 43 to 93 mgkg(-1) for dibenzofuran, 37 to 110 mgkg(-1) for dibenzothiophene, 140 to 650 mgkg(-1) for fluoranthene, 55 to 380 mgkg(-1) for fluorene, 37 to 300 mgkg(-1) for phenanthrene, and 49 to 1300 mgkg(-1) for pyrene. For acridine, no toxicity was observed within the concentration range tested (1-1000 mgkg(-1)). As illustrated by the EC20-values, there was a rather large difference in sensitivity between the species, and T. pratense was the most sensitive of the species tested.

NORMA-Gene: a simple and robust method for qPCR normalization based on target gene data.

BackgroundNormalization of target gene expression, measured by real-time quantitative PCR (qPCR), is a requirement for reducing experimental bias and thereby improving data quality. The currently used normalization approach is based on using one or more reference genes. Yet, this approach extends the experimental work load and suffers from assumptions that may be difficult to meet and to validate.ResultsWe developed a data driven normalization algorithm (NORMA-Gene). An analysis of the performance of NORMA-Gene compared to reference gene normalization on artificially generated data-sets showed that the NORMA-Gene normalization yielded more precise results under a large range of parameters tested. Furthermore, when tested on three very different real qPCR data-sets NORMA-Gene was shown to be best at reducing variance due to experimental bias in all three data-sets compared to normalization based on the use of reference gene(s).ConclusionsHere we present the NORMA-Gene algorithm that is applicable to all biological and biomedical qPCR studies, especially those that are based on a limited number of assayed genes. The method is based on a data-driven normalization and is useful for as little as five target genes comprising the data-set. NORMA-Gene does not require the identification and validation of reference genes allowing researchers to focus their efforts on studying target genes of biological relevance.

Effects of the antibacterial agents tiamulin, olanquindox and metronidazole and the anthelmintic ivermectin on the soil invertebrate species Folsomia fimetaria (Collembola) and Enchytraeus crypticus (Enchytraeidae)

Veterinary pharmaceutical products such as antibacterial agents and antiparasitics are widely used to control diseases and promote production in the agricultural sector. Exposure of non-target organisms are a likely result of using manure from treated live stocks or from dung dropped on the field by grazing animals. The aim of this study was to determine the toxic threshold levels of three antibacterial agents (tiamulin, olanquindox and metronidazole) and one anthelmintic (ivermectin) to two species of soil dwelling organisms (springtails and enchytraeids), that are often found in bio-solids such as manure or dung. The antibacterial agents were not toxic to adults and effects on reproduction occurred generally above concentrations normally found in soil or dung. The threshold values for toxicity (10% reduced reproduction or EC10 values) were in the range of 61-111 mg kg(-1) dry soil for springtails and 83-722 mg kg(-1) dry soil for enchytraeids. Ivermectin was significantly more toxic with EC10 values of 0.26 mg kg(-1) dry soil for the springtails and 14 mg kg(-1) dry soil for the enchytraeids. A comparison of these results with rough estimates of likely and worse case environmental concentrations indicates a potential risk of ivermectin to non-target species such as springtails and enchytraeids, whereas direct toxic effect of antibacterial agents is very unlikely to occur at environmental realistic concentrations. However, indirect effects of antibacterial agents driven through changes in the food web cannot be abolished at this stage.

Indicators for Monitoring Soil Biodiversity

A Bispo,3 D Cluzeau,4 R Creamer,1 M Dombos,I U Graefe,# PH Krogh,33 JP Sousa,44 G Peres,4 M Rutgers,11 A Winding,33 and J Rombke*II 7French Agency for Environment and Energy Management, France 8University of Rennes, France 6Teagasc, Ireland IResearch Institute for Soil Science and Agricultural Chemistry, Hungary #Institut fur Angewandte Bodenbiologie, Germany 77Aarhus University, Denmark 88University of Coimbra, Portugal 66National Institute for Public Health and the Environment, Netherlands IIECT Oekotoxikologie GmbH, Germany * J-Roembke@ect.de

Automatic counting of collembolans for laboratory experiments

A new procedure involving digital image processing and image analysis for determination of the number of individuals and size distribution of a collembolan population is presented. It is applicable to experiments conducted with a single species either on plaster or in soil. Collembolans are transferred to an even, black surface and immobilised by anaesthetisation with carbon dioxide. By scanning the surface with the collembolans, the number and size of individuals are quantified. In subsequent data treatment steps, adults and clutches of juveniles can be discriminated using an ordination or clustering technique. The method is illustrated by the weekly population development of Folsomia candida Willem in the laboratory.

Interactions between saprotrophic fungi, bacteria and protozoa on decomposing wheat roots in soil influenced by the fungicide fenpropimorph (Corbel®): a field study.

Abstract Litterbags containing freshly harvested wheat roots with adhering rhizosphere soil were placed in their native soil. Four levels of the fungicide fenpropimorph were applied: 0, 1, 10 and 100 mg kg −1 soil. The experimental set-up was a randomised block design with five replicates. Litterbags were sampled six times over a 4-month period and microbial numbers determined. Bacteria (colony forming units, CFUs) and protozoa (most probable number) followed a similar pattern: a sudden decrease at the onset of the study period was followed by a fast recovery and increase. This ended at maximum mean values of ≈7.5 × 10 7 bacteria g −1 root litter by day 7 and 3 × 10 6 protozoa g −1 root litter by day 20, respectively. Fenpropimorph had no significant effect on either of these two groups, for which mean population sizes seemed to interact in a prey–predator manner. The fungi (stained by calcofluor white and fluorescein diacetate) were unaffected by the pesticide until day 20. Hereafter, fluorescein diacetate-active (FDA-active) hyphae were significantly inhibited in all fungicide treated plots, compared to the untreated plots 50 and 114 days after the application of fenpropimorph. Fungal colony forming units (fungal CFUs) on fenpropimorph-containing agar demonstrated a selection towards more fenpropimorph tolerant fungi in the 100 mg kg −1 fenpropimorph treated plots. The delayed effect of fenpropimorph on FDA-activity could be explained as a combined effect of the pesticide and the degradation product itself, and the fluctuating water stressing field conditions of this year. Our results, thus, demonstrated long term effects of fenpropimorph on the activity of saprotrophic fungi at recommended field dose, and that high dose fenpropimorph can select for fenpropimorph tolerant fungal populations.