Incubation Methodology

The methodology used for the experiment was based on SNIFFER (2008), which used sediment from green SUDS, in order to enable comparison of results. This involved spiking soil samples with a known concentration of PAH (Sigma Aldrich PAH Mix A, 500μg, 13 compounds dissolved in DCM), and then incubated. Variations in the incubation were introduced, including temperature, concentration of PAH and moisture content of the soil.

Slight changes were made with regards to the spiking procedure, as Brinch et al. (2002) determined a superior method, applying the pollutant to 25 % of the soil, and then adding the remaining 75 % once the solvent had dissolved. This was done in order to reduce the proportion of soil subjected to the solvent (acetone), in order to minimise the effect on the indigenous microorganisms. First, the samples were dried at 30 ˚C for 24 h, and stored at 5 ˚C. Half of the sediment was then sterilised by autoclaving at 121 ˚C at 103.4 kPa for 15 minutes. Sediment was then weighed out into 48 x 25 g samples (24 sterilised and non-sterilised), of which 75 % (18.75 g) was removed and stored. The sample bottles used were amber glass, in order to prevent the effects of light exposure, such as photolysis of the PAH.

Moisture content (by mass) of the samples was then adjusted to 40 %. A mixture of 13 constituent PAH dissolved in acetone (Sigma Aldrich EPA 525 PAH Mix B) was then applied to the sub sample, which was shaken to mix. The bottle lid was then left on for 5 minutes in order for the pollutant to dissipate, and then removed for 16 h to allow evaporation of the acetone. The remaining 75 % of the sediment was then added, the lids put on, and the entire sample was shaken to mix. This meant that each sample received approximately 15 mg/kg of PAH. Once the samples had been spiked with PAH, they were incubated at a constant temperature of 15 ˚C.

In addition to the ‘Control’ condition, detailed above, several treatments were introduced to further batches of samples. The first of these was variation in temperature. Whereas the ‘Control’ samples were kept at 15 ˚C, these were

kept at 4 ˚C, in order to simulate the cool seasonal temperature that the sediment would be subject to in the real world. Secondly, moisture content was doubled, to 80 %, in order to replicate wet and dry periods associated with channel drains. Finally, PAH concentration was doubled, representing a situation such as an influx of PAH after a storm event.

Six bottles (3 sterilised and non –sterilised) were then removed at 0,1,3,7,14,28,42 and 60 days after treatment (DAT), and stored at -60 ˚C in order to prevent any further degradation until all samples had been taken and analysis took place.

6.2.3 Analytical Methods

Samples were removed from frozen storage and left to defrost overnight in a refrigerator, as to minimise further degradation. Once defrosted, ultrasonic sequential extraction took place.

For the PAH analysis of the samples, they were first extracted by ultrasonic sequential extraction using a method based on (Risdon et al., 2008). Firstly, 5.0 g +/- 0.1 g of sample, and also an equal amount of Na2SO4 was weighed out and manually blended with the sediment in order to remove moisture. The sample was then transferred to a centrifuge tube and 1 ml of 100µg/ml surrogate standards were added and allowed to equilibrate for 12hrs at 20oC. 6 ml of DCM was added, and the sample sonicated for 2 minutes at 20oC. 10 ml hexane and 4 ml DCM were then added, and the sample sonicated for 10 minutes, then manually shaken. Samples were then centrifuged for 5 minutes at 750 rpm, then passed through a filter column with the filtrate retained.

The residue was re-suspended in 10 ml Hexane:DCM mix (1:1), sonicated for 15 minutes at 20oC and centrifuged for 5 minutes at 750 rpm. The sample was then filtered through a filter column with the filtrate retained. This was then repeated, and the final extract made up to 40 ml with a Hexane:DCM mix (1:1).

The extract was then cleaned up using Sigma SPE-18 Solid Phase Extraction cartridges. Once done, 900 µl of sample and 100µl of the deuterated PAH mix was transferred to a GC-MS vial.

The sample was then analysed using an Agilent GC-MS, using the ‘alkane.std’ program.

The full analytical method used is detailed in Appendix A.

6.2.4 Statistical Analysis

An unpaired T-test was applied to the results obtained from the experiment. This was carried out using GraphPad Ystat (2015).

6.3 Results

6.3.1 Explanatory Note

Unfortunately, 127 of the 192 samples were contaminated/destroyed. All of the samples were extracted and analysed using the methods described above. However, when the results of the GC-MS analysis were interpreted, a lack of PAH was displayed. The analysis was repeated several times, with blanks and standards for quality control, but no data was obtained, despite the machine functioning correctly.

It is believed that during storage at -18 ˚C, the freezer was left open, the samples defrosted, and the PAH in the samples was lost, due to its volatile nature. Due to the expense of analysis, and time constraints, it was not possible to repeat the experiments.

The results of the successful samples have been used in this chapter, however it is acknowledged that these are unfortunately, and unavoidably lacking in number and depth.

6.3.2 Results Table

Results of the three replicates for each sample were averaged, the results of which are recorded below.

Table 6.1: PAH Conc. Over 60 Day Incubation Period

Table 6.2: % Reduction of PAH Concentration

6.3.3 Graphs

Graphs of the results were plotted in Figure 6.1 in order to show the concentration of PAH over time under the different conditions. Error bars were added to show standard deviation.

DAT Non-Sterilised Sterilised Non-Sterilised Sterilised Non-Sterilised Sterilised Non-Sterilised Sterilised 0 - - 2.1747 1.66 2.59 2.51 3.57 3.91 1 - - 1.65 - 1.78 2.66 3.55 1.7 3 - - 2.1 1.42 1.62 2.53 3.46 2.11 7 - - 2.83 1.59 1.61 2.14 2.7 1.68 14 - - - 2.26 - 2.24 28 - - - - 42 - - - - 60 - - - -

Control Temperature Moisture Content PAH Conc. Condition

Variable Non-Sterilised Sterilised

Temperature 30.13 -4.22

Moisture Content -37.84 -9.96

PAH Conc. -24.37 31.76

Figure 6.1: PAH Conc. Over Incubation Period

6.3.4 Control

Samples from the ‘Control’ condition were destroyed as per 6.3.1.