# Study About the Influence of Diatoms on the Durability of Monumental Limestone
## 2 Materials and Methods
### Methodology
#### 2.2.6 Frost Resistance Test  
The standard UNE-EN 12371:2011 [35] describes a test procedure for determining the freeze-thaw resistance of natural stone. This procedure consists in subjecting stone samples to repeated freezing cycles in air, followed by thawing in water. Since the number of samples was limited, only six cubic samples with 50 mm edges were used in the present study: three with diatoms (D1, D2, and D3) and three as control samples (C1, C2, and C3), with the sides opposite to the test surface waterproofed.  
First, the samples were dried until they reached a constant mass; they were then placed in a ventilated oven for 4 days at 68 \({}^{\circ}\)C. After drying, the samples were allowed to cool until reaching room temperature, and each imperfection was marked with a marker to monitor potential visible changes during the cycles.  
Before starting the first freeze-thaw cycle, the samples remained in contact with water for 48 h inside a closed container to ensure they did not begin the first cycle in a dry state. During this time, measurements were taken to record the increase in mass at two 24 h intervals. This enabled the determination of the amount of water each sample absorbed and the comparison with values obtained under the same immersion conditions after the freeze-thaw cycles.  
The freeze-thaw cycle began after the 48 h water immersion. Each cycle consisted in a freezing stage in air at \(-20\)\({}^{\circ}\)C for 6 h, followed by a thawing stage in a closed container in contact with a 3 mm-thick layer of water for another 6 h.  
After each complete cycle, a visual inspection of each sample was performed to check for signs of degradation. When any damage was observed, the cycle number in which this appeared was recorded. After inspection, the samples were subjected again to the freezing stage, starting a new cycle, as shown in Figure 10. This process can be repeated as many times as necessary; here, a maximum of 45 cycles was chosen. Once the maximum number of cycles had been completed, the samples were fully submerged in water for 24 h to allow any loose material particles to detach. Finally, the samples were dried again in a ventilated oven for 4 days at 68 \({}^{\circ}\)C to determine the dry mass of the samples after being subjected to 45 freeze-thaw cycles.  
Figure 9: Samples during the Karsten tube water absorption test.  
With the samples dry and after undergoing the 45 freeze-thaw cycles, they were once again partially submerged in a 3 mm-thick layer of water for 48 h, in the same manner as at the beginning of the test, before starting the freeze-thaw cycles. Measurements of the increase in mass were taken at two 24 h intervals to enable comparison of their behavior before and after the freezing cycles. After the samples had been subjected to the freeze-thaw cycles, a useful comparison could be made to evaluate the behavior changes between the control samples and those with diatoms, independently, to determine the extent at and manner in which these changes occurred.  
The pipette method was used to perform another water absorption test, following the methodology described in Section 2.2.5. Measurements were recorded to determine how long the samples took to absorb each increment of 0.1 mL of water, as well as the total water absorption after 24 h of testing. Figure 11 shows the pipette tubes placed over the samples from the freeze-thaw cycle.  
Figure 11. Samples during the Karsten tube water absorption test.  
Figure 10. Samples ready for freezing.  
From the results obtained in this complementary part of the frost resistance test, the behavioral differences between the upper face of the samples with diatoms and that of the control samples after exposure to low temperatures can be better understood.