CURRENT RESEARCH ACTIVITIES

WP 1.1.: Validation of established methods on new matrices (e-mail to WP 1 Leader: )
In WP1, the development of cutting edge technologies are continued to detect and quantify not only the presence, but also the activity of pathogenic microorganisms and organic contaminants in environmental waters using new mass spectrometric methods capable of analyzing steroid estrogens, pharmaceuticals, antibiotics and antiparacetics and their degradation products
simultaneously and introduce modern microbiological techniques to the field.
The methods are used to provide analysis of the compounds formed in the different matrices using the new manure handlings techniques in WP 2 and WP 3 for assessing the fate of compounds and microorganisms. Similarly, established quantitative PCR techniques for Campylobacter spp.developed for chicken dung are adapted and employed for the matrices soil, manure and water(Keramas et al. 2004, Lund et al. 2004, Lund & Madsen 2006, Li et al. in press).


WP 1.2.: Methods to quantify viable and infective pathogens
The potential of using molecular quantification technologies based on quantitative PCR of cDNA generated from soil mRNA are exploited. In our present technology targeting Salmonella spp. a sequence specific purification technology is used (Jacobsen & Holben, 2007,Journal of Microbiological Methods).
This technology confers high specificity, but the capture technology, due to its principles, excludes genes not used as capture probes. We have recently successfully used another purification system to quantify indigenous soil microbial functional genes on mRNA level (Bælum et al unpublished).
Using this new approach we make a cDNA copy of the full transcript from microorganisms present in environmental waters since the reverse transcript is generated using random hexamer primers.The potential for using this technology in automated high throughput systems targeting not onlySalmonella sp. invA genes is pursued.


WP 1.3.: Fate of steroid estrogens or metabolites
The fate of these compounds is very different from that of the parent compounds. In many cases metabolites are more polar than the parent compound, and thus are more mobile. Especiallythe need of expanding the methods for also analyzing the conjugates in existing methods arepursued (Lykkeberg et al 2006. Journal of Pharmaceutical and Biomedical Analysis. vol.  42: pp.223-231). GC-MS-MS are used for the analysis of transformation processes and metaboliteformation in model systems (known bacteria and sorbents), abiotic degradation and liverhomogenates (microsomes of pigs) under different redox conditions (Lykkeberg et al 2006. Journalof Pharmaceutical and Biomedical Analysis. vol.  42: pp. 223-231).
Accelerated Solvents Extraction are used combined with solid phase extraction and other clean-upmethods before analysis (Jacobsen and Halling-Sørensen 2006. Analytical and BioanalyticalChemistry. vol. 384: pp. 1164-1174). Main metabolites are identified by the use of NMR and areincluded in the final analytical method if they possess activity.


WP 2.1.: Information on distribution of contaminants (e-mail to WP 2 Leader: ) 
Information about pathogens and natural estrogenes as well as chemical (nutrients, particle sizedistribution) and biological (biochemical composition, degradability) parameters in liquid and solidfractions from operating farm slurry separation units with a range of different technologies, slurrytypes and animal species (pig, cattle, mink, raw/biogassed slurry) are collected and evaluated.This is based on an existing database and sample archive of about 40 operating slurryseparation facilities (from a concurrent VMP-III project) as well as information from theparticipating industrial partners, previous projects and the literature.


WP 2.2.: Separation technology effects on contaminants
Comparisons of contaminant distribution, fate and chemical and biological proporties of separates between commercially available manure separation technologies from the industry partners of theproject are carried out at a few farm operations treatments with slurry spiked with different contaminants. To address special contaminant problems, laboratory studies are conducted on different contaminated animal slurries, by applying newly emerging separation methodologies (e.g.various enzymes and biological additives, Novozymes A/S). Test pathogens of different size andwith different surface properties are spiked into manure samples for precise determination ofdistribution. 


WP 2.3.: Storage effects on contaminants
Knowledge on the persistence of contaminants in the liquid fractions of separated manure isobtained in storage experiments with different fractions of separated slurry including degradation ofnatural estrogenes and survival of pathogens.


WP 3.1.: Redistribution (e-mail to WP Leader 3: )
The mechanisms controlling the redistribution of slurry or separation products and associatedcontaminants as affected by dry matter content of separation products and soil water potential areidentified. This is accomplished by quantifying effects of and interactions between slurryproperties and soil water potential using dialysis (Petersen et al., 2003a). The mobilization and fate
of estrogens are studied in incubation experiments, and by amendment of slurry to repacked soiladjusted to different soil water potentials.


WP 3.2.: Leaching
The effect of soil type and slurry properties on the potential for leaching of contaminantsthrough intact soil is investigated. Leaching of natural estrogens and pathogens is investigated inintact soil columns in a set-up with realistic lower boundary conditions in either small columns inthe laboratory or large soil monoliths in a semi-field set-up (Leagdsmand et al. 2007. Geoderma.vol. 93: pp. 33-59.).
The intact soil is amended with slurry and then subjected to realistic leaching events.


WP 3.3.: Field experiment 
The transport of manure applied pathogens is assessed through a controlled field experimentmonitoring the leaching of pathogens for 1 year after manure application. The field experimenttakes place at two experimental field sites used for monitoring of pesticide leaching withinthe frames of the Danish Pesticides Leaching Assessment Programme (Lindhardt et al., 2001).
This project benefits from on-going monitoring activities, including state-of-the-art monitoring equipment and a well-proven sampling strategy for analyzing leaching processes of contaminants(Kjær et al., 2005. Geological Survey of Denmark and Greenland, Copenhagen: ISBN87-7871-1665; Kjær et al., 2007. Environmental Science & Technology. vol.  42: pp. 3911-3917).
The field sites are managed by normal agricultural practice and the type of slurry or slurry separates
and application.