Effect of freezing on the microstructure of a highly 1 decomposed peat material close to water saturation when used 2 prior to X-ray micro computed tomography 3 4

The modelling of peatland functioning, in particular the impact of anthropogenic warming and direct 13 human disturbance on CO2, CH4 and N2O, requires detailed knowledge of the peat structure and of both water 14 and gas flow with respect to the groundwater table level. To this end, freezing is nowadays increasingly used to 15 obtain small size peat samples for X-ray micro computed tomography (X-ray μ-CT) as required by the need to 16 increase the resolution of the 3D X-ray CT images of the peat structure recorded. The aim of this study was to 17 analyze the structure of a peat material before and after freezing using X-ray μ-CT and to look for possible 18 alterations in the structure by investigating looking at the air-filled porosity. A highly decomposed peat material 19 close to water saturation was selected for study and collected between 25 and 40 cm depth. Two samples 20 4 ́4 ́7 cm in volume were analyzed before and after freezing using an X-ray μ-CT Nanotom 180NF (GE 21 Phoenix X-ray, Wunstorf, Germany) with a 180 kV nanofocus X-ray tube and a digital detector array 22 (2304 ́1152 pixels Hamamatsu detector). Results showed that the continuity and cross section of the air-filled 23 tubular pores several hundreds to about one thousand micrometers in diameter were altered after freezing. Many 24 much smaller air-filled pores not detected before freezing were also recorded after freezing with 470 and 474 25 pores higher than one voxel in volume (60 ́60 ́60 μm in volume each) before freezing, and 4792 and 4371 air26 filled pores higher than one voxel in volume after freezing for the two samples studied. Detailed analysis showed 27 that this increase resulted from a difference in the whole range of pore size studied and particularly from a 28 dramatic increase in the number of air-filled pores ranging between 1 voxel (216 10 μm) and 50 voxels 29 (10.8 10 μm) in volume. Theoretical calculation of the consequences of the increase in the specific volume of 30 water by 8.7% when it turns from liquid to solid because of freezing led to the creation of a pore volume in the 31 organic matrix which remains saturated by water when returning to room temperature and consequently to the 32 desaturation of the largest pores of the organic matrix as well as the finest tubular pores which were water-filled 33 before freezing. These new air-filled pores are those measured after freezing using X-ray μ-CT and their volume 34 is consistent with the one calculated theoretically. They correspond to small air-filled ovoid pores several voxels 35 https://doi.org/10.5194/soil-2021-86 Preprint. Discussion started: 24 August 2021 c © Author(s) 2021. CC BY 4.0 License.

volume and the kerozene method developed by Monnier et al. (1973). The total porosity was obtained by 112 dividing the volume of water contained in a saturated sample by the known volume of the sample as described 113 by Boelter (1976) and Nimmo (2013). The water content of the collected samples was determined after oven 114 drying at 105°C for 24h. The degree of peat decomposition was characterized with the pyrophosphate index 115 (Kaila 1956) which was determined following Gobat et al. (1986). The C and N contents were determined by 116 combustion of dried and crushed samples at 1100°C, using a CNS-2000 LECO apparatus.

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In order to have samples of the appropriate size for X-ray µ-CT, sub-samples of peat materials 4´4´7 cm 3 in 119 volume corresponding to the depth of 30-37 cm were prepared by cutting with a scalpel blade to limit 120 disturbance of the peat structure as far as possible. Then, each sample was placed in a transparent plastic tube 5 121 cm in diameter which was then hermetically sealed to avoid water loss. They were first submitted to X-ray µ-CT 122 and then, on the basis of the methodology developed and used by Rezanezhad et al. (2010), Ramirez et al. (2016) 123 and Moore et al. (2017), they were frozen at -10°C for 48 h, defrosted for 48 h at 20°C and submitted again to X-124 ray CT. Each sealed plastic tube with its peat material was weighed at the different steps of the process to check 125 the absence of water loss. Measurements showed that the weight variation between two successive steps and 126 between the first and last step was <0.1 g for the two samples studied.

X-ray Computed Tomography imaging (2D and 3D images)
128 X-ray µ-CT was performed for the sub-samples 4´4´7 cm 3 in volume cut between 30 and 37 cm depth using a 129 micro X-ray µ-CT device Nanotom 180NF (GE Phoenix|x-ray, Wunstorf, Germany). This equipment has a 180-130 kV nanofocus X-ray tube and a digital detector array (2304×1152 pixels, Hamamatsu detector). Samples were 131 placed in the chamber and rotated by 360 degrees during acquisition. The resulting projections were converted 132 into a 3D image stack using a microcluster of four personal computers (PCs) with the Phoenix 3D reconstruction 133 software (filtered back projection Feldkamp algorithm (Feldkamp et al., 1984)). The reconstruction software 134 contains several different modules for artifact reduction (beam hardening, ring artifacts) to optimize the results.

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Finally, the 16-bit 3D image was converted into an 8-bit image (256 grey levels) before preprocessing. The 136 samples were mounted and waxed on a glass rod. An operating voltage of 110 kV and a filament current of 137 59 µA were applied. The distance between the X-ray source and the sample and between the X-ray source and 138 the detector was 300 and 350 mm, respectively, giving a voxel size of 60 µm. The 2000 projection images 139 (angular increment of 0.18°) were acquired during stone rotation (with an acquisition time of 4 hours). As the 140 cone beam geometry created artifacts, the first and the last cross-sectional images were removed (Le Trong et al., 141 2008;Rozenbaum and Rolland du Roscoat, 2014).

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The resulting 3D images were cropped for sample A to a size of 430´600´800 voxels corresponding to 143 2.6´3.6´4.8 cm 3 before and after freezing, and for sample B to a size of 430´530´850 voxels before and after freezing corresponding to 2.6´3.2´5.1 cm 3 , each image in a local 3D coordinate system with a voxel size of 145 60´60´60 µm 3 for samples A and B before and after freezing. 146 2.5 X-ray image analysis (segmentation and attenuation) slices containing these features were sought. The other slices were discarded. This sets the height of the 3D 151 images. Each image was then horizontally cropped so as to keep only the interior of the samples. The cropped 152 region was defined again with respect to clearly identifiable features in the images before and after freezing.

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Smoothing the 3D images with a moving average filter over a window of 5´5´5 voxels increased their signal-to-154 noise ratios from the range [7.6-9.2] to [12.5-15.0]. They were then segmented by thresholding. The threshold 155 value used was the absolute minimum between the two peaks of the bimodal distribution of the grey levels of the 156 voxels of each image ( Fig. 1) (Rozenbaum et al., 2012). The grey level corresponding to that threshold value for 157 sample A before and after freezing was 93 and 78, respectively. For sample B before and after freezing, it was 80 158 and 68, respectively. This simple procedure has no adjustable parameter and therefore introduces no bias when 159 comparing the images. In each binary image, each pore (i.e. group of contiguous foreground voxels surrounded 160 by background voxels) was identified by scanning the image, and its volume (in terms of number of voxels) 161 recorded.

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The grey level on the images was determined by the absorption of the incident X-ray radiation by the different 187 phases of the peat material. The absorption of each phase depends on its density and mean atomic number 188 resulting from its chemical composition (Youn et al., 2015). It is described by the Beer-Lambert Law: where I is the transmitted X light, I o the incident X light, µ the absorption coefficient, and x the path length.

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Consequently, the intensity of the transmitted X light which results in a grey level of the pixel in the 2D images 192 and of the voxel in the 3D images depends on the proportion of air, water and organic compounds in the pixel or 193 voxel considered. Because of the weak difference between the mean atomic number assumed for the porous 194 organic matrix of a highly decomposed and water-saturated peat material (Table 1)

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For each pair of 2D X-ray µ-CT images, comparison showed the presence of pores recognizable on the images 204 before freezing which were still present after freezing but exhibiting a different morphology, of pores 205 recognizable on the images before freezing which were not present after freezing, and the presence of pores 206 recognizable after freezing and which were not present before freezing. However, the use of pairs of 2D X-ray µ-

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CT images does not enable an accurate estimation of the possible evolution of the porosity of peat materials 208 during the freezing process since it was not possible to say whether the pairs of 2D images corresponded exactly 209 to the same slice in the sample before and after freezing. Only a 3D analysis is able to establish whether the 210 porosity of the peat materials is different before and after freezing. The 3D X-ray µ-CT binary images of the two samples A and B were first morphologically compared globally by 213 comparing the porosity characterized in X-ray µ-CT before and after freezing (Figs. 3a and d, 4a and d). Results

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showed that the air-filled pores measured corresponded to a very small proportion of the total porosity of the peat material studied, less than 0.02, whereas the total porosity of samples A and B was 0.918 and 0.904 before 216 freezing, respectively (Tables 1 and 2). Most of the porosity corresponded to both water-filled pores associated 217 to the highly decomposed organic compounds and potentially to larger water-filled pores occupied by water and 218 consequently indistinguishable from the porous organic matrix.

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The number of air-filled pores composing the very small proportion of the total porosity described with the X-220 ray µ-CT used was however very different before and after freezing for the two samples studied.

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As freezing leads to an 8.7% increase in the specific volume of the water, the possible consequences of this 244 increase on the changes recorded for the peat material studied were analyzed. The total porosity before freezing 245 (f T, BF ) can be written as follows:

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where V V, BF is the total specific volume of pores of the peat material before freezing in cm 3 g -1 , V S is the specific 248 volume of the organic solid phase dried at 105°C in cm 3 g -1 and equal to 0.591 cm 3 g -1 and 0.562 cm 3 g -1 for 249 samples A and B, respectively (reciprocal of the particle density measured for peat materials A and B) ( Table 1).

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Thus, using equation (2): organic matrix, all saturated with water before freezing, is no longer located in these pores when the water turns 288 from solid to liquid after thawing. The porosity newly occupied by air was measured using 3D X-ray µ-CT 289 ( Table 2)

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Our results show that the freezing technique that can be used prior to peat material sub-sampling as required by 295 3D X-ray CT altered the structure of the highly decomposed and close to water saturation peat material studied.

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The tubular pores from several hundreds to about one thousand micrometers in diameter were indeed altered, 297 with both their continuity and cross section being different before and after freezing. These pores were several 298 hundred to several thousand voxels in volume in the 40 cm 3 in volume highly decomposed peat material studied,      showing the whole pores detected before (a) and after (d) freezing, the pores smaller than 500 voxels in volume 454 before (b) and after (e) freezing, and the pores larger than 500 voxels in volume before (c) and after (f)    showing the whole pores detected before (a) and after (d) freezing, the pores smaller than 500 voxels in volume 478 before (b) and after (e) freezing, and the pores larger than 500 voxels in volume before (c) and after (f) freezing. showing the whole pores detected before (a) and after (d) freezing, the pores smaller than 500 voxels in volume 484 before (b) and after (e) freezing, and the pores larger than 500 voxels in volume before (c) and after (f)