Pollution Induced Community Tolerance (PICT) in Soil Microorganisms Subjected to the Oxy-tetracycline

Background and Objectives
Microorganisms make changes to their lives, when they are exposed to contaminants. These changes include physiological and genetically changes. Antibiotics are widely used in the livestock industry, agriculture, as well as in medicine, and enter soils via waste waters and manure applications. Oxy-tetracycline (OTC) is one of the most consumed antibiotics in the livestock industry. Its half-life in soil has been reported up to 79 days depending on soil conditions. Increasing levels of antibiotics in soil environment will results in physiological or genetically changes (tolerance) in microbial communities. Although, physiological changes are usually reversible, but the genetically ones are almost permanent. Several methods have been suggested for assessing the risks of contamination of soil ecosystems, among them the pollution induced community tolerance (PICT) has been accepted as valuable procedure. The principle of the procedure is as follow. The increasing levels of a contaminant are added to the soil and incubated for a defined time period. At several time points, the microbial populations are extracted and subjected to the same levels of contaminant. The amount of microbial activity is determined using appropriate procedure, for example, determination of dehydrogenase activity in soil. The IC₅₀ (contaminant level at which the microbial activity is inhibited by 50%) is calculated for soil samples treated with different levels of contaminant. A contaminant level at which a sharp change occurs in microbial tolerance to the contaminant at defined time point, is called PICT.    
Methodology
In this study, the effects of different levels of oxy-tetracycline (OTC) on microbial activity were investigated in a silty loam soil. Different levels of OTC (10, 20, 30, 40 and 50 mg.kg^-1) were applied to the pots containing 2 kg of soil with three replications and kept at room temperature (25±2℃) for 120 days. The soil moisture level was adjusted to 50-70% of field capacity by daily weighing and adding distilled water. Soil dehydrogenase activity as an indicator of soil microbial activity was measured at 3, 7, 15, 30, 60, 90 and 120 days of incubation. Tri-phenyl tetrazolium chloride (TTC) was use as a substrate for dehydrogenase enzyme. The amount of tri-phenyl formazan produced by the reduction of TTC, was accounted for dehydrogenase activity.  The ΔIC₅₀ diagram (PICT) was plotted against different soil OTC concentrations for each time point and the trend of microbial tolerance changes was evaluated.
 
Findings
The results showed that with increasing incubation time and concentration of OTC, induction of tolerance in the microbial population was gradually enhanced. The amount of PICT was increased by increasing OTC level in soil on day 3, but without showing critical point on the diagram. However, it was declined toward the day 7. By increasing OTC level from 30 to 40 mg.kg^-1, a marked decrease in ΔIC₅₀ was seen on day 15.  Overall, the increase of tolerance in microbial population started after day 15 and reached the highest values on days 30 and 60 at OTC concentration of 50 mg.kg^-1. After that, PICT levels gradually increased toward the 50 mg/kg OTC, but were very low on days 90 and 120. A marked change (critical point) in the slope of ΔIC₅₀ was occurred on day 30 at 30 mg.kg^-1 OTC.
Conclusion
Based on obtained results it can be concluded that the increasing of OTC concentration to 30 mg.kg^-1 in soil would likely led to occurring permanent tolerance to OTC in soil microbial community after 30 days. Therefore, this concentration can be regarded as critical level of OTC in soil at this time point. Continues application of manures containing OTC residue will results in accumulation of OTC in agricultural soils, hence we should regularly assay the concentration of OTC in soil to avoid of occurring permanent tolerance (also regarded as genetically resistance) in microbial community against OTC. Although this critical concentration and time point may differ for other soils and antibiotics