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Applied and Environmental Microbiology, April 2002, p. 1808-1816, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1808-1816.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Method for Spiking Soil Samples with Organic Compounds

Ulla C. Brinch,1 Flemming Ekelund,2 and Carsten S. Jacobsen1*

Department of Geochemistry, Geological Survey of Denmark and Greenland,1 Department of Terrestrial Ecology, Zoological Institute, Copenhagen University, Copenhagen, Denmark2

Received 10 July 2001/ Accepted 21 December 2001

We examined the harmful side effects on indigenous soil microorganisms of two organic solvents, acetone and dichloromethane, that are normally used for spiking of soil with polycyclic aromatic hydrocarbons for experimental purposes. The solvents were applied in two contamination protocols to either the whole soil sample or 25% of the soil volume, which was subsequently mixed with 75% untreated soil. For dichloromethane, we included a third protocol, which involved application to 80% of the soil volume with or without phenanthrene and introduction of Pseudomonas fluorescens VKI171 SJ132 genetically tagged with luxAB::Tn5. For both solvents, application to the whole sample resulted in severe side effects on both indigenous protozoa and bacteria. Application of dichloromethane to the whole soil volume immediately reduced the number of protozoa to below the detection limit. In one of the soils, the protozoan population was able to recover to the initial level within 2 weeks, in terms of numbers of protozoa; protozoan diversity, however, remained low. In soil spiked with dichloromethane with or without phenanthrene, the introduced P. fluorescens VKI171 SJ132 was able to grow to a density 1,000-fold higher than in control soil, probably due mainly to release of predation from indigenous protozoa. In order to minimize solvent effects on indigenous soil microorganisms when spiking native soil samples with compounds having a low water solubility, we propose a common protocol in which the contaminant dissolved in acetone is added to 25% of the soil sample, followed by evaporation of the solvent and mixing with the remaining 75% of the soil sample.

* Corresponding author. Mailing address: Department of Geochemistry, Geological Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV, Denmark. Phone: 45 3814 2314. Fax: 45 3814 2050. E-mail: csj{at}geus.dk.

Applied and Environmental Microbiology, April 2002, p. 1808-1816, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1808-1816.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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