Method for Spiking Soil Samples with Organic Compounds

Abstract

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.

Effect of pyrene on denitrification activity and abundance and composition of denitrifying community in an agricultural soil

“…Crystalline pyrene (L08162, Alfa Aesar, Lancaster, USA) was dissolved in acetone and uniformly sprayed onto soil to produce sub-samples with pyrene levels of 0 (pyrene-free, with acetone only), 20,50,100,200,400,700,1000,2000 and 5000 mg kg À1 dry weight soil (Brinch et al, 2002;Peng et al, 2010). These subsamples were homogenized by continuous hand shaking in 1000 mL glass bottles and the bottles were closed for 5 min to let the solvent disperse, followed by storage at 25 C in the dark for 16 h. When acetone was evaporated off, sub-samples were mixed with non-spiked soil at the ratio of 1:9 and shaken thoroughly, generating final pyrene levels of 0 (non-spiked control), 2,5,10,20,40,70,100,200 and 500 mg kg À1 dry weight soil (denoted as Pr2, Pr5, Pr10, Pr20, Pr40, Pr70, Pr100, Pr200 and Pr500, respectively).…”

Metabolic and cometabolic degradation of herbicides in the fine material of railway ballast

“…of 'high' (A), 'intermediate' (B) and 'low' (C) microbial biomass were obtained for each track. A fraction of each soil (100 g) was contaminated with diuron (12 mg) dissolved in acetone (5 ml) (procedure adopted from Brinch et al 2002). When the acetone had evaporated the contaminated fraction was mixed thoroughly with the rest of the soil to obtain an approximate diuron concentration of 15 µg g -1 dry wt.…”

Dynamic changes in functional gene copy numbers and microbial communities during degradation of pyrene in soils

“…, following the recommendations of Brinch et al (2002) with some adaptations. Briefly, a subsample of 10% was spiked with the pyrene solution and was mixed by hand shaking in a 1000 ml glass beaker.…”