Heidi Sjursen Konestabo

Recovery of cholinesterase activity in the earthworm Eisenia fetida Savigny following exposure to chlorpyrifos

Organophosphorus (OP) insecticides inhibit cholinesterase activity, an essential process in the nervous system of most animals. Re-establishment of active enzymes is slow and depends on elimination of the insecticide from the body followed by two lengthy processes: Reactivation and/or biosynthesis of new enzymes. Earthworms (Eisenia fetida) were exposed to either clean or chlorpyrifos-containing (240 mg/kg) soil for 48 h. After transfer to clean soil, we monitored two cholinesterases (E1 and E2) and chlorpyrifos content of the earthworms for 12 weeks. After 14 to 21 d of recovery, the exposed and control worms were indistinguishable in terms of appearance and behavior. Chemical analysis showed a rapid elimination of chlorpyrifos from the earthworms, with only minor levels detected after one week. The activities of E1 and E2 were measured spectrophotometrically in whole specimen homogenates using acetylthiocholine as the substrate. Carbaryl, which selectively inhibits E1, was used to discriminate the enzyme activities. Mean +/- standard error of mean of E1 and E2 activity in the controls immediately after exposure were 1.57 +/- 0.18 nanokatal (nkat)/mg protein (n = 3) and 0.95 +/- 0.07 nkat/mg protein, respectively, and 0.48 +/- 0.07 nkat/mg and 0.45 +/- 0.06 nkat/mg, respectively, in exposed worms. After three weeks, E1 had regained an activity comparable to the controls, whereas E2 remained depressed throughout the 12-week monitoring period. The non- or late recovery of E2 makes this enzyme a potential biomarker candidate for previous OP insecticide exposure in Eisenia fetida, provided the protocol for measurements is improved and standardized.

Organic matter flow in the food web at a temperate heath under multifactorial climate change

The rising atmospheric CO(2) concentration, increasing temperature and changed patterns of precipitation currently expose terrestrial ecosystems to altered environmental conditions. This may affect belowground nutrient cycling through its intimate relationship with the belowground decomposers. Three climate change factors (elevated CO(2), increased temperature and drought) were investigated in a full factorial field experiment at a temperate heathland location. The combined effect of biotic and abiotic factors on nitrogen and carbon flows was traced in plant root → litter → microbe → detritivore/omnivore → predator food-web for one year after amendment with (15)N(13)C(2)-glycine. Isotope ratio mass spectrometry (IRMS) measurement of (15)N/(14)N and (13)C/(12)C in soil extracts and functional ecosystem compartments revealed that the recovery of (15)N sometimes decreased through the chain of consumption, with the largest amount of bioactive (15)N label pool accumulated in the microbial biomass. The elevated CO(2) concentration at the site for 2 years increased the biomass, the (15)N enrichment and the (15)N recovery in detritivores. This suggests that detritivore consumption was controlled by both the availability of the microbial biomass, a likely major food source, and the climatic factors. Furthermore, the natural abundance δ(13)C of enchytraeids was significantly altered in CO(2)-fumigated plots, showing that even small changes in δ(13)C-CO(2) can be used to detect transfer of carbon from primary producers to detritivores. We conclude that, in the short term, the climate change treatments affected soil organism activity, possibly with labile carbohydrate production controlling the microbial and detritivore biomass, with potential consequences for the decomposition of detritus and nutrient cycling. Hence, there appears to be a strong coupling of responses in carbon and nitrogen cycling at this temperate heath.

Selection of Nontarget Testing Organisms for ERA of GM Potato with Increased Resistance to Late Blight

The deliberate release of any genetically modified (GM) organism in the European Union requires an environmental risk assessment (ERA) prior to commercialisation, including impact assessment on nontarget organisms. We report from two expert workshops where a newly developed selection procedure for identification of ecologically relevant testing organisms was applied to the case of a GM potato with increased resistance to late blight, planned for cultivation in southern Scandinavia. Species known to contribute to important ecological functions in the receiving environment were selected in a stepwise procedure, to arrive at a practical number of ecologically relevant species that are likely to be exposed to the transgene and suitable for experimental testing. Four ecological functional categories were identified: herbivory and disease transmission, natural enemies, ecological soil processes and pollination. Among these, relevant nontarget species were identified for herbivores and soil living pathogens, natural enemies and decomposers/beneficial soil organisms. Out of a total of 16 herbivores, 17 soil-living pathogens, 49 natural enemies and 14 decomposers/beneficial soil organisms in the initial lists, 8 herbivores, 10 soil-living pathogens, 15 natural enemies and 11 decomposers/beneficial soil organisms were identified as possible testing organisms, based on ecological criteria. These findings are highly relevant for determining the scope and structure of an ERA of this type of GM potato. The selection procedure could not be completed because of insufficient information about tissue- and developmental stage-specific expression levels of the transgenic products for this particular GM potato. Thus, the case study illustrates some of the difficulties and knowledge gaps that limit the relevance and quality of ERA of GM plants.