# Study About the Influence of Diatoms on the Durability of Monumental Limestone
## 2 Materials and Methods
### Methodology  
The main objective of this work is to verify whether or not a positive influence appears on the surface properties and behavior of the limestone rock samples after submerging them in the pond. In addition, the impact on the surface properties that the experiment itself may have caused to obtain the diatoms to act on the samples was also evaluated.  
To achieve these goals, several test methods were selected to identify and quantify possible differences in behavior between the pond samples and the control samples. Given the limited number of specimens available, the priority was to maximize their use and obtain as much information as possible.  
For this reason, the noninvasive tests were performed first, as these do not alter either the samples or the biodeposition of silica on their surface and leave the more invasive and destructive tests for the final stages, as these do cause significant surface wear. Thus, the use of the samples was carefully planned, alternating their use to minimize their deterioration and avoid interference with the biofilm of the diatom biodeposition.  
These details are presented in Table 2, which offers a detailed classification of the noninvasive and destructive tests used.  
To achieve this natural treatment, nine limestone rock samples were introduced in a pond with the presence of diatoms. All the samples introduced were cube shaped, with a side of 50 mm, and originated from Bonar (Spain).  
Diatom colonies are known to grow on submerged substrates in as little time as 4 weeks [29]. For the present work, samples were immersed in an experimental pond, as illustrated in Figure 2a, and exposed to natural environmental conditions for a period of 1 year and 2 months in Leon, Spain. This time period was stipulated because, during this time, it was considered that a sufficiently dense overlay would potentially be generated  
\begin{table}
\begin{tabular}{c c} \hline
**Noninvasive Tests** & **Destructive Tests** \\ \hline Porosity test & Spray water absorption test \\ \hline Water absorption test at atmospheric pressure & Water absorption test by pipette method \\ \hline Water absorption test by capillarity & Frost resistance rest \\ \hline \end{tabular}
\end{table}
Table 2: Classification of the noninvasive and destructive tests used.  
Figure 1: Treated surface of samples.  
due to the biodeposition of the silica frustules of the diatoms on the samples. This process would lead to a surface sealing that would possibly improve the surface properties, as illustrated in Figure 2b. In addition, it should be noted that the key environmental factors influencing diatom growth are the temperature and hours of light received [30], which coincide with positive photoperiods in which hours of light exceed hours without light. For this reason, data on temperature and solar radiation were collected throughout the entire period during which the samples were submerged in the pond (Figure 3), using records obtained from the Spanish State Meteorological Agency (AEMET) [31], specifically from the meteorological station at Virgen del Camino, located at approximately 1 km from the pond.  
As a result of all the aforementioned factors, in particular due to the higher exposure to solar radiation, the upper side, which receives more direct light, shows a higher concentration of diatoms. Conversely, the side faces, with less exposure, show significantly lower growth due to the limited availability of light for photosynthesis and colony development. Therefore, the upper side, which presents the highest silica biodeposition, is studied.  
Figure 3: Temperature and solar radiation data during the diatom biodeposition period.  
Figure 2: (**a**) Samples submerged in the pond and (**b**) SEM image of the surface of the samples showing the diatom frustules.