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Experiment 2: Biodegradation of TPH and PAM The soil was taken from Department of Agronomy, Kansas State University. The soil contained 24% sand, 50% silt, 26% clay, 0.086% N, 0.95% C, and 1.4% organic matter (Soil Testing Lab, Dept. of Agronomy, Kansas State). superfloc (solid crystal PAM) and Cydril (a mixture of PAM and TPH) were obtained from Cytec industries for the experimental work. Soil moisture was calculated by drying 10 g of soil and reweighing it after the soil was dried. The difference in initial and final weights gave the amount of soil moisture. The value obtained for the soil moisture was about 1% for the soil as received. The whole soil was grounded, sieved and dried for 24 hours. About 200 grams of soil was sterilized in an autoclave with steam at 1 atm gauge pressure and a temperature of 120° C for 30 minutes. Various formulations of Anti Traction Material (ATM) are being investigated, and some of the ingredients used in the formulations are used in this experimental work. Ten grams of soil was transferred to each of eighty-seven 120 ml bottles. The following type of treatments made were: A) sterilized soil with 8 g water, 0.27 g cydril, and 0.13 g superfloc, B) 8 g water, 0.27 g cydril, 0.13 g superfloc in soil, C) 4 g water, 0.135 g cydril, 0.065 g superfloc in soil, D) 0.27 g cydril and 8 g water in soil, E) 0.13 g superfloc and 8 g water in soil and F) control with no contaminant in the soil. Ten grams of dry soil was used in each sample. Eighteen replicates were made of the type B and D formulations in 120 ml bottles. Fifteen replicates were made of the type A, C, E and F in 120 ml bottles. All the 120 ml bottles with treatments inside were transferred to 1L masonjars. Approximately 100 ml water is added to each jar to create moisture inside thejars. All the jazs had a rubber septum in the lid to allow the gas phase to be sampled. Extraction of TPH in the soil was performed at regular time intervals using the entire mass of soil from a jar to get an estimate of amount of TPH present at any instant. At time [=0, six replicates of treatments - B, C, D, E and 3 replicates of treatment F were taken for extraction. Tetracosene was used as an extraction standard for the TPH and 500 µl of it was added to each treatment being extracted. After adding tetracosene, the treatments were left for 10-15 minutes for the sorption of tetracosene to soil. The procedure formulated for extraction employs mixing and shaking with acetone three times to extract the TPH sorbed from soil to acetone phase. Twenty ml of acetone was added in each extraction. Afer twenty ml of acetone was added for each extraction, the TPH was allowed to separate by shaking for approximately 2 hours so that the hydrocarbon phase from soil moves into the acetone. After that, centrifuging was done at 500 rpm for 10 minutes. Empty 60 ml bottles were taken and weighed with caps. Extract from a soil sample was poured into a 60 ml bottle and then the bottle was closed with a cap. After three extractions, approximately 60 ml of extractant was accumulated in each 60 ml bottle. All the bottles with extractant were weighed with caps. Total weight of extractant was calculated for each bottle after subtracting the final weight from the initial empty bottle weight with cap. Sub sampling of the extract was done with a plastic syringe capped with a filter. Next, filtered extract was placed in GC vial and was capped tightly using a capper. Hydrocarbon peaks were determined using the GC and temperature program as given in Experiment 1. This experimental method resulted in more than 95% recovery of the tetracosene (23). The left over soil was left open to the environment for 1 week at room temperature to evaporate any trace of acetone. The acetone-dried soil was sent to soil testing laboratory for total organic carbon (TOC) analysis. At subsequent time intervals t= 3, 6, 13, and 23 weeks, the same procedure of extraction and GC analysis was repeated for three replicates of each treatment. The