By adding both EtOH and H2O2 to the solution, when no electrodeposition was used, a coating similar to that obtained using EtOH alone was found (Figure 5). However, when potential was applied, again isolated crystals with 1–2 μm size were formed. Although smaller than in the case of H2O2 addition alone, these crystals were unable to completely cover the marble surface (in Figure 6, bare calcite grains are clearly visible as darker zones below the lighter CaP crystals). Therefore, combined EtOH + H2O2 addition was discarded in the prosecution of the study.

With regard to the effect of increasing the DAP and, proportionally, the CaCl2 concentrations in the solution (always maintaining a DAP:CaCl2 ratio of 1000:1 and adding the maximum amount of EtOH not leading to immediate CaP precipitation in the solution), the variation in the amount and morphology of the new CaP phases is illustrated in Figure 7.

With no electrodeposition, after 30 min isolated clusters (small lighter particles over the darker marble surface) were found in the case of the solution with the lower concentration and almost no clusters in the case of the more-concentrated solution. This can be explained considering that, in the less-concentrated solution, a much higher amount of EtOH is present (30 vol.%, instead of 10 vol.%), which significantly increases the nucleation of the new CaP phases even at low DAP concentration [7]. Consistently, after 3 h the more-concentrated solution (with lower EtOH addition) caused formation of isolated crystals, relatively big in size (0.5–1 μm), whereas the marble surface was apparently completely covered by a new coating with flower-like morphology in the case of the less-concentrated solution (containing higher EtOH amounts). When potential was applied, formation of new phases was much more abundant for both solutions at both times (Figure 7). After 3 h, in the case of the less-concentrated solution, a very densely packed coating was formed, exhibiting the typical flower-like morphology in a recess in the surface. Also in the case of the more-concentrated solution, almost

complete coverage was obtained after 3 h, but the resulting coating appeared as significantly coarser. In terms of composition of the new phases formed after 3 h at *E* = −2 V, FT-IR spectra exhibit bands attributable to HAP, which were more defined (especially the band at 1028–1031 cm−1) when more EtOH was present in the solution.

**Figure 7.** Effect of the different diammonium hydrogen phosphate (DAP) and CaCl2 concentrations on the new calcium phosphate (CaP) phases formed at different times, with and without electrodeposition (in the FT-IR spectra, bands owing to the substrate are marked by a star, while the position of the new bands owing to CaP phases is reported).

For both DAP/CaCl2 concentrations, formation of an almost continuous coating was obtained. Based on the position of the FT-IR bands (Figure 7), it seems most likely that the coatings are composed of HAP, even though formation of OCP cannot be completely excluded. In either case, both formulations were regarded as promising and hence taken into consideration in the prosecution of the study.

#### *3.3. Influence of Electrokinetic Parameters: Pulsing, Voltage, and Time*

The effect of applying a pulsed potential is illustrated in Figure 8.

**Figure 8.** Effect of pulsed potential on the morphology of the new CaP formed at different times.

In the case of the less-concentrated solution, a limited increase in the density of the new CaP clusters when pulsed potential was applied can be noticed after treatment for 30 min, while the pulsing effect became less evident after 3 h. In fact, after 3 h the flower-like coating was basically complete even with constant potential, so that no increase in coverage thanks to the pulsed potential was visible. In the case of the more-concentrated solution (containing less EtOH), the effect of pulsing was not investigated at 30 min, because almost no coating had been found to form at that time at constant potential (Figure 7). After 3 h, the coating deposited at constant potential appeared to be composed of relatively big, separated crystals, which leave the marble surface partly bare. With pulsed potential, the surface was somewhat improved, although better results were obtained using the less-concentrated solution (containing more EtOH).

Because, in all cases, the effect of pulsing was lower than hoped based on the studies reported in the literature [24,27,29], constant potential was adopted in the prosecution of the study.

The change in the amount and surface coverage of the new phases as a function of the applied voltage and time is illustrated in Figure 9 for the less-concentrated solution and in Figure 10 for the more-concentrated one.

In both cases, it is possible to observe that, at short time (30 min), the number of new phases increased visibly for increasing voltage. In fact, at 30 min, only a few CaP clusters were formed when no voltage was applied (lighter particles over the darker marble surface in Figures 9 and 10). This is consistent with previous results reported in the literature, where CaP formation was found to start to be significant between 3 and 6 h, depending on the formulation of the precursor phosphate solution [12]. When voltage was applied and progressively increased, a progressively more continuous coating was formed. In the case of the more-concentrated solution (Figure 10), the effect was still visible up to 3 h, because the lower amount of EtOH present in this solution (10 vol.%, compared to 30% in the less-concentrated one) implies a longer time for the CaP coating to develop. After 6 h, no significant difference was evident among samples treated with different voltage, including the samples treated without electrodeposition. Apparently, at least in the investigated conditions, after prolonged treatment the effect of time becomes predominant over that of electrodeposition.

**Figure 9.** Effect of voltage and time on the morphology of the new CaP phases formed from the less-concentrated solution (0.1 M DAP + 0.1 mM CaCl2 in 30 vol.% EtOH).

**Figure 10.** Effect of voltage and time on the morphology of the new CaP phases formed from the less-concentrated solution (2 M DAP + 2 mM CaCl2 in 10 vol.% EtOH).

In the case of the less-concentrated solution (Figure 9), a significant change in the amount and morphology of the new phases was clearly visible passing from 0 to −1 and −2 V. On the contrary, the difference between coatings formed at −2 and −3 V was less evident. A reason for this might be that, during electrodeposition, the cathode itself is progressively coated with new CaP phases, as already suggested by the change in current measured during potentiostatic deposition (Figure 5a) and confirmed by SEM observation of the steel grids. As a consequence, after a certain time, the amount of current circulating in the system diminishes, so that electrodeposition of the new phases over the marble sample diminishes as well. Consistently, a reduction was found in the current measured during electrodeposition at −2 and −3 V (Figure 5a), whereas no significant change in current was registered during electrodeposition at −1 V (graph not shown). This is consistent with results previously reported in the literature, indicating that HAP was present only in minor amounts when electrodeposition was carried out at −1.25 V and it became the major phase when electrodeposited at −1.55 V or lower [18].

In the case of the more-concentrated solution, micro-cracks with ~5 μm length and ~1 μm width are visible in the flowery coating deposited at −2 V for 6 h (Figure 10, bottom-right image). This suggests that the film formed in these conditions is likely porous and thick, which leads to cracking during drying. In fact, drying cracks do not form when the film is dense and its thickness is below the critical threshold, below which cracking is thermodynamically impeded [44]. Consequently, electrodeposition at −3 V was not considered for the acid resistance tests in the case of the more-concentrated solution.

Based on all the results reported above, the choice of the formulations for the acid attack tests was made. In the case of the less-concentrated solution, treatment at −2 V for 3 and 6 h was selected, because in both cases a basically complete coverage of the marble surface was achieved (Figure 9). In the case of the more-concentrated solution, the treatment at −1 V for 6 h was taken into account, while the treatment at higher voltage (*E* = −2 V) was discarded, because the resulting coating was heavily cracked (Figure 10). For all these conditions, samples treated for the same time but without electrodeposition were also considered, for comparison's sake.

*3.4. Acid Resistance*

The amounts of Ca and P released in the acidic solutions, measured by ICP at the end of the acid resistance test, are reported in Table 1.

**Table 1.** Weight amounts of Ca and P per unit area of surface exposed to the acid attack, dissolved from untreated and treated marble samples, measured by inductively coupled plasma optical emission spectrometry (ICP) in the solutions collected at the end of the acid resistance tests (marble specimens with bare edges and coated edges were tested).

The surface appearance of untreated and treated marble samples, before and after the acid attack test, is illustrated in Figure 11.

**Figure 11.** Surface morphology of untreated and treated marble samples, before and after the acid resistance test (samples with either bare or coated edges during the acid test are reported).

As expected, after the acid test, the untreated reference exhibited visible surface etching, in the form of grooves and exposed grain boundaries. As a consequence, Ca ions were detected in the solution (Table 1).

In the case of treated samples, tested with bare edges, no formulation was able to completely prevent dissolution of the marble substrate. In fact, the measured Ca losses had the same order of magnitude as those of the untreated reference (Table 1) and cracking of the protective coatings was clearly visible after the acid attack test (Figure 11). Some limited benefits seem to result from treatments applied by electrodeposition for 6 h, with either concentration of DAP and CaCl2 (Table 1). In these conditions, the lowest Ca losses were registered, and limited damage of the coating was observed after the acid attack test. In the case of the less-concentrated formulation, no P was detected in the solutions analyzed after the acid test, which suggests that the coatings were not dissolved during exposure to acid and that the measured Ca losses mostly originate from dissolution of the marble substrate in uncovered zones or cracks in the coating. Differently, in the case of the more-concentrated solution, some small amounts of P were also detected in the solutions, suggesting that some coating dissolution likely took place.

The reason why cracks were visible after the acid resistance test (Figure 11), while no crack was noticed for the same formulations before the acid attack test (Figures 9 and 10), is thought to be the presence of pores inside the coatings, combined with the specific experimental conditions adopted for these specimens. Indeed, before exposure to acid, the bonding at the interface between the coating and the substrate was strong enough to prevent cracking during drying at the end of the treatment application. However, during the acid test, pores likely allowed acid to reach the substrate/coating interface and to weaken the bonding between the two. As a result, the stress arising in the pores during drying at the end of the acid test now led to cracking. To verify the presence of pores in the coatings, further tests would be needed (e.g., observation of cross sections by a focused ion beam microscope, FIB-SEM). A further reason for the weakening of the coating/substrate interface is thought to be the specific experimental conditions adopted for the first set of specimens, i.e., subjecting them to the acid resistance test without coating their edges. In these conditions, the acid attack can start from the edges (where the coating is often damaged during treatment and handling) and then propagate to the rest of the sample. For this reason, the second set of specimens was treated after coating their edges with an impermeable coat, so that only the central, uniform part of the samples was exposed to acid. In these latter conditions, quite different results were obtained.

In fact, when samples were tested after coating the edges with an impermeable coat, the untreated reference exhibited Ca release much higher than the treated samples (Table 1), which indicates that the coatings were able to provide a better acid protection than in the previous case. Accordingly, SEM images show no sign of cracking in any sample (Figure 11). This confirms that the presence of damaged areas near the sample edges allowed acid to reach the substrate/coating interface, weaken the bonding between the two, and trigger cracking. Nonetheless, the effect of pores in the coatings cannot be excluded and further analyses by FIB-SEM are opportune. In some cases, especially in samples treated without electrodeposition, the calcite substrate is sometimes visible in the SEM images (darker areas beneath the lighter flower-like crystals in Figure 11). This indicates that, also in the case of samples tested after coating the edges, the surface coverage and the resulting protection were not complete. Anyway, all the specimens subjected to the acid attack test clearly exhibited bands attributable to HAP after the test (Figure 12), which confirms that the coatings are still present after exposure to acid.

**Figure 12.** FT-IR spectra of untreated and treated samples (tested with coated edges), after the acid test (in the FT-IR spectra, bands owing to the substrate are marked by a star, while the position of the new bands owing to CaP phases is reported).

#### **4. Conclusions**

The results of the present study, aimed at assessing the feasibility of electrodepositing HAP coatings over marble and evaluating the protective ability of the resulting coatings, allow to derive the following conclusions:

• Electrodeposition of HAP over the marble surface can be obtained by placing the marble sample close to the cathode. In these conditions, OH− groups formed near the cathode during electrodeposition favor HAP formation, compared to the situation when no electric potential is applied. Compared to simple increase in pH of the phosphate solution used as HAP precursor, the local increase in OH− near the cathode has the advantage that precipitation of CaP in the bulk solution and excessive film growth are prevented.

All things considered, electrodeposition seems like a technique able to accelerate and improve formation of HAP coatings over the marble surface, even though the resulting protective efficacy is not complete yet. Electrodeposited coatings appear to be mainly composed of HAP, while presence of other CaP was not evidenced. To ascertain the presence of pores in the coatings, further research is needed, for instance observation of cross sections by FIB-SEM.

**Author Contributions:** Conceptualization, E.S.; Methodology, E.S., G.M, M.C.B. and E.F.; Investigation, E.S. and G.M.; Resources, M.C.B. and E.F.; Data Curation, E.S. and G.M.; Writing-Original Draft Preparation, E.S.; Writing-Review & Editing, E.S., G.M., M.C.B. and E.F.; Supervision, M.C.B. and E.F.; Funding Acquisition, E.S.

**Funding:** This work has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement N. 655239 (HAP4MARBLE project, "Multi-functionalization of hydroxyapatite for restoration and preventive conservation of marble artworks").

**Acknowledgments:** George W. Scherer is gratefully acknowledged for fruitful and inspiring discussion. Adelia Albertazzi (Centro Ceramico, Bologna) is gratefully acknowledged for ICP analyses.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Ion-Substituted Carbonated Hydroxyapatite Coatings for Model Stone Samples**

**Rodica-Mariana Ion 1,2,†,\*, Lorena Iancu 1,2,†, Gabriel Vasilievici 1,†, Madalina Elena Grigore 1,†, Ramona Elena Andrei 1,†, George-Ionut Radu 1,†, Ramona Marina Grigorescu 1,†, Sofia Teodorescu 3,†, Ioan Alin Bucurica 3,†, Mihaela-Lucia Ion 4,†, Anca Irina Gheboianu 3,†, Cristiana Radulescu 3,† and Ioana Daniela Dulama 3,†**

Received: 28 February 2019; Accepted: 29 March 2019; Published: 2 April 2019

**Abstract:** Carbonated hydroxyapatite derivatives (CHAp) and its metallic derivatives (Ag, Sr, Ba, K, Zn) have been prepared and characterized in this paper and their coating capacity on some model stone samples have been evaluated and discussed. These compounds were characterized by using several analytical tools, including X-ray diffraction analysis (XRD), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), to determine the purity of the CHAp sample. The XRD and FTIR results confirmed the presence of AB-carbonated type CHAp. The thermal analysis (TGA) established two stages of weight loss that occured during the heating process: The first weight loss between 30–225 ◦C corresponding to the partial carbonate release from OH-channel and the second one between 226–700 ◦C, corresponding to some thermal reactions, possibly to the generation of calcium phosphate. The efficiency and suitability of these products on model stone samples were evaluated by monitoring the resistance to artificial weather (freeze–thaw), and pore structure changes (surface area, pore volume, pore diameter). Meanwhile, optical microscopy (OM) and Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM–EDS) techniques showed the particles size and surface morphology of the samples, as well as information on its chemical composition. Also, the compressive strength of these new compounds as coatings revealed a homogeneity and strengthen of these model stone samples.

**Keywords:** carbonated hydroxyapatite; nanomaterials; coatings

#### **1. Introduction**

Nowadays, one of the main difficulties in preserving objects and heritage monuments is to select the most adequate treatment methods for stone protection, able to preserve their original appearance, without any detrimental effect on their aesthetic properties [1]. The protective products must preserve the stones, to bring it to a state like the original one and prevent further damage. In general, to apply a coating to the surface of the stone.

The requirements for the consolidation of products consist of: Good stability, compatibility with the substrate [2] and adequate penetration depth. A good consolidant should first reduce the rate of degradation of the stone surface and then improve its mechanical properties.

The main challenges in stone protection are related to the creation of a barrier against water penetration and protection of the stone surface against pollutants and the deposition of organic/inorganic particles, while ensuring aesthetic compatibility with the substrate (i.e., color changes at acceptable intervals), a minimal change in water vapor permeability and reversibility of the treatment [3]. In this perspective, hydrophobic coatings [4], antifouling treatments [5] and nanoparticle self-cleaning bands [6] are currently at the center of research on stone conservation [7,8].

However, at present, there are no standard procedures and/or unique assessment methods supporting the effectiveness of enhancers and protection products [9].

A good consolidant should have a good penetration depth and adhesion to the substrate. The bonding capacity depends to a large extent, both the chemical composition of the product (including solvent and concentration) and the stone surface. In particular, the products with a composition-like stone are preferred. Among the new products synthesized for preservation of culture heritage materials, some hybrid formulations have been recently proposed for inorganic [10] and organic materials [11,12].

Another aspect concerning the consolidant is the method of application [11,13], which plays a key role, significantly influencing the transfer of laboratory procedures to on-site practice. There are some application methods: Immersion, which can be easily controlled in the laboratory, but is hardly reproducible on site where it maximizes the penetration depth very hard; poulticing, to control even in laboratory conditions [13]; spraying, which delivers high amounts of consolidant at one time, causing a large consolidant runoff and consumption (besides a higher solvent evaporation due to nebulisation) [11], but a limited penetration depth [10]; and brushing, which often lacks an adequate control of all the parameters that finally determine the treatment efficacy [3,12]. Usually, the brushing method is preferred because it assures an efficient application and a relatively controlled balance between penetration and solvent evaporation [13,14]. Nanomaterials exhibit good chemical–physical features for application in the conservation science of artwork, being able to slow down the degradation processes of artifacts, by comparison with traditional methods, widely described in literature [15]. Up to now, the most used consolidants are nano hydroxyapatite, Ca10(PO4)6(OH)2, already used for travertine, marble and limestone chalk stone consolidation, and used as models for architectural monuments [16].

The aim of this paper is to synthesize and to test some consolidants based on carbonated hydroxyapatite (CHAp), which is quite similar with hydroxyapatite [17–19]. By comparison with Hap, which has a crystal structure very similar to that of calcite, enabling epitaxial growth over calcareous matrices [20], in carbonated hydroxyapatite (CHAp) the CO3 <sup>2</sup><sup>−</sup> ions can replace both OH<sup>−</sup> and PO4 <sup>3</sup><sup>−</sup> ions and thus the last form has a high special surface area than the previous one [21]. In the case of carbonated hydroxyapatite, a decrease in the a-axis and increase in the c-axis in the apatite lattice is registered, due to the incorporation of B-type CO3 <sup>2</sup><sup>−</sup> substitution into the HAp structure [22], a decrease of the crystalinity which suggests a decrease in the crystallite size and crystallinity of the apatite phase [23,24], given by the highest amount of CO3 <sup>2</sup><sup>−</sup> in the apatite structure [25].

Carbonated hydroxyapatite derivatives (CHAp) and its metallic derivatives (Silver (Ag); Strontium (Sr); Barium (Ba); Potassium (K); Zinc (Zn)) have been prepared and characterized by X-ray diffraction analysis (XRD), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR). The efficiency and suitability of these products on model stone samples, applied by brushing (B) and spraying (S), were evaluated by monitoring the resistance to artificial weather (freeze–thaw) and pore structure changes (surface area, pore volume, pore diameter). Also, the mechanical strengths of these new compounds as coatings for model stone samples revealed good adherence, homogeneity and strength of these layers.

#### **2. Materials and Methods**

#### *2.1. Specimens Samples Preparation*

Different samples were prepared from sand and a mixture of gypsum with lime (95%–5%) (1:4) (40 mm × 40 mm × 40 mm) in order to distinguish the difference between non-treated/treated samples and to observe the influence of consolidant used during the treatment. Cubic specimens (diameter: 4 cm, height: 4 cm) have been prepared in a silicon resin matrice in order to avoid the dispersion of the solid matter or evaporation of the solvent from the lateral sides. All specimens were stored under controlled conditions (*T* = 20 ◦C, 65% RH) for 1 month. After the drying process, the substrates were left to cool for two hours in a desiccator and adequately treated with the new prepared compounds, and their appearance was examined.

#### *2.2. Application of the Carbonated Hydroxyapatite Derivatives*

The nanoparticles were suspended in distilled water with conductivity <2 s/cm, due to its higher boiling point and higher surface tension, responsible for a low kinetic stability. They were applied by spraying, an application method commonly adopted in the practice of conservation, for a good penetration and deposition in depth. The procedure was repeated with up to 3 consecutive applications, in order to improve the consolidation effect. The interval between consecutive applications was defined at 6 h, for achieving a complete evaporation of the solvent. The applications were performed under controlled conditions (50% RH, *T* = 20 ◦C, air speed <0.1 m/s). The treated specimens were then stored at 65% RH, *T* = 20 ◦C, air speed <0.1 m/s for over 1 months, in order to enhance the nanoparticles deposition.

#### *2.3. CHAp Synthesis*

Carbonated hydroxyapatite (CHAp), as very fine and uniform sized powder, was obtained by a nanoemulsion technique adapted from Zhou et al. [26]. CHAp nanoparticles were synthesized by mixing aqueous solutions of (NH4)2HPO4 and NH4HCO3, under magnetic stirring. The pH of the aqueous solution was adjusted to 11 using sodium hydroxide 1 M. Then an acetone solution of Ca(NO3)2·4H2O at a molar ratio of Ca2+:PO4 <sup>3</sup>−:CO3 <sup>2</sup><sup>−</sup> = 1.67:1:0.5 was introduced into the flask. The reaction was carried out at room temperature. The precipitate was vacuum filtered using a Buchner funnel and washed with distilled water. The reaction product was freeze-dried overnight and calcined the next day at 900 ◦C for 4 h. Doped CHAp with different metal ions (Ag+, Sr2+, Ba2+, Zn2+, K+) was synthetized by a similar microemulsion method, the metal nitrate solution (AgNO3, Sr(NO3)2, Ba(NO3)2, Zn(NO3)2, KNO3) being added in the Ca(NO3)2·4H2O acetone solution, in concentration of 5 mole% reported to Ca2+. These compounds have been applied by brushing and spraying using an aqueous solution of 0.25g/L on the non-treated samples.

#### *2.4. Chemical Characterization Methods*

X-ray diffraction measurements of powder samples were carried out with a Rigaku Ultima IV diffractometer (Rigaku, Tokyo, Japan) using a Cu Kα radiation (λ = 1.54 Å). In this experiment the accelerating voltage of the generator radiation was set at 40 kV and the emission current at 200 mA. The diffractograms were recorded in parallel beam geometry over 2θ = 5◦ to 100◦ continuously at a scan rate of 4◦/min.

Fourier transformed infrared spectroscopy (ATR–FTIR) was recorded with a Vertex 80 spectrometer (Bruker Optik GMBH, Billerica, MA, USA) equipped with DRIFT accessory, in the range of 2000–400 cm<sup>−</sup>1, because the range 4000–2000 cm−<sup>1</sup> did not show major changes for these samples.

Thermogravimetric analyses of CHAp were performed using a Pyris 1 TGA analyzer (Perkin–Elmer TGA-7, Waltham, MA, USA) with a scan range from 50–700 ◦C and a constant heating rate of 10 ◦C/min under continuous nitrogen.

For porosity determination, the nitrogen adsorption/desorption isotherms were recorded at 77 K in the relative pressure range *p*/*p*<sup>o</sup> = 0.005–1.0 using NOVA2200e Gas Sorption Analyzer (Quantachrome, Boynton Beach, FL, USA). Data processing was performed using NovaWin version 11.03 software. Prior to adsorption measurements, the samples were degassed for 4 h at 180 ◦C under vacuum. The specific surface area was determined by the standard Brunauer–Emmett–Teller (BET) equation. The total pore volume was estimated from the volume adsorbed at a relative pressure *p*/*p*o close to unity. Pore size distribution and mesopores volume were obtained from desorption branch of the isotherm by applying the Barrett–Joyner–Halenda (BJH) model. The t-plot method was used to estimate the micropore surface area and external surface area.

Also, the OM was recorded by a Primo Star ZEISS optical microscope (Carl Zeiss, Oberkochen, Germany) that offers the possibility of investigating the samples in transmitted light at a magnification between 4–100×. The equipment had a digital video camera attached (Axiocam 105, Carl Zeiss, Oberkochen, Germany) which, by the microscope software (Zen Pro), allowed real-time data acquisition. The obtained images could easily be converted from 2D in 3D format through its software for a better view.

The Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM–EDS) was obtained with a SU-70 (Hitachi, Japan) microscope, with a magnification range of 30×–800,000×.

Freeze–thaw aging test: For the freeze-thaw test (20 cycles) STAS 6200/15 83, the sample shall be dried in oven at 105 ± 5 ◦C for 1 h up to the constant mass (*m*1). The samples were immersed in distilled water for 15 min at room temperature. The samples were kept in the freezer for 3 h at −18 ± 5 ◦C and then thawed in water. The freeze–thaw operation is repeated until 20 cycles are performed, after which the mass losses during the freeze–thaw process is expressed as gelivity coefficient, Equation (1).

$$
\mu\_{\text{\%}} = (m\_2 - m\_3/m\_1) \times 100\% \tag{1}
$$

where:

*m*1–the initial mass of the sample, determined after drying at 105 ◦C to the constant mass and before saturation with distilled water, in grams;

*m*2–mass of sample saturated with water, determined before the first freeze–thaw cycle, in grams; *m*3–defrost mass of the sample, determined after drying at 105 ◦C to the constant mass and before saturation with distilled water, in grams.

#### *2.5. Mechanical Testing Method*

The mechanical strength test was performed with a Silver Schmidt Hammer Proceq test hammer, type L-0.735 Nm impact energy, according to ASTM C805. The strength range of the Silver Schmidt test hammer is from 10–100 N/mm2. Ten replicates within the test location with a minimum spacing of 25 mm between each two testing points and a minimum edge distance of 25 mm, have been recorded. The hammer was positioned at 90◦ downward, and the rebound number value is calculated as the average of the readings within this test location, in order to find a relationship between surface hardness and compressive strength with an acceptable error. The compressive strength was calculated using the Equation (2) (for 10th percentile curve range), after apparatus manual indications.

$$\text{Compressive strength} = 2.77 \times \text{e}^{\circ} (0.048 \times \text{Q}) \tag{2}$$

The results have been obtained for the untreated and treated specimens with CHAp, Ag-CHAp, Sr-CHAp, Ba-CHAp, Zn-CHAp, K-CHAp solutions of 0.25 g/L solution applied by brushing and spraying on the stone surface.

#### **3. Results**

#### *3.1. Crystal Structure*

Table 1 summarizes the results of the phase purity and the average crystallite sizes calculated for the most intense peak of CHAp (0 0 2) for all the investigated samples by XRD and Scherrer Equation (3):

$$L = (\mathbb{K} \times \lambda) / (\beta \times \cos \theta) \tag{3}$$

where:

**Table 1.** Summary of the XRD characterization of pure and substituted CHAp.

*L* = crystallite size;

K = Scherrer constant (0.9);

λ = X-ray wavelength (1.54 Å);

β = peak width at half maximum intensity (in radians), from the apparatus programme;

θ = Bragg angle, from the apparatus programme;

By analyzing this table, it could be observed that all the samples matched with the standard JCPDS reference (09-432) file for synthetic carbonated hydroxyapatite. XRD confirmed the phase purity of the synthesized carbonate hydroxyapatite [27] and no additional phases (brushite) were identified, this being an indication for these compounds' purity.

Figure 1 shows the characteristic XRD spectra for all samples. When all the prepared samples were analyzed by X-ray diffraction, their patterns were characteristic of the hexagonal apatite phase [28,29]. Few XRD peaks are characteristic of carbonated hydroxyapatite (with 2θ from 26–40) (Figure 1).

**Figure 1.** XRD diagrams for CHAp and Me-CHAp (Me = Sr, Ag, Ba, Zn, K).

#### *3.2. Molecular Structure*

The major functional groups in this case are the carbonate, hydroxyl, phosphate groups, identified by FTIR. The observed vibrational peaks, summarized in Figure 2, are characteristic of carbonated hydroxyapatite compounds, similar to other literature data [27,30]. For a proper identification of the specific bands of these compounds, the IR region was chosen between 2000–400 cm<sup>−</sup>1.

**Figure 2.** FTIR spectra for CHAp and Me-CHAp (Me = Sr, Ag, Ba, Zn, K).

#### *3.3. Thermal Properties*

The thermogravimetric analysis of the samples carried out from 30–1000 ◦C in air atmosphere using a heating rate of 10 ◦C/min is shown in Figure 3. The TGA result shows that there are two stages of weight loss that occurs during the heating process: The first weight loss between 30–225 ◦C and the second one between 226–700 ◦C, corresponding to some thermal reactions [31].

**Figure 3.** The TGA diagram for CHAp and Me-CHAp (Me = Sr, Ag, Ba, Zn, K).

#### *3.4. The Efficiency and Suitability of These Products on Model Stone Samples*

The efficiency and suitability of these products on model stone samples were evaluated by compressive strength, to artificial weather (freeze–thaw), water absorption test in relationship with porosimetry measurements. Except the pore structure changes (surface area, pore volume, pore diameter), the variation of the surface area and pore diameter (by brushing and spraying), is represented in Figure 4.

**Figure 4.** Surface area and pore diameter parameters for CHAp and Me-CHAp (Me = Sr, Ag, Ba, Zn, K).

With the Silver Schmidt test hammer measures the speed of the impact as well as of the rebound immediately before and after the impact, calculating the amount of energy that can be recovered. It is one of the traditionally used methods for evaluating the mechanical properties of the rocks (compressive strength). The rebound number measurements for the untreated and treated with carbonated hydroxyapatite (by brushing and spraying the specimens) are shown in Figure 5. The measurements of rebound number have been taken and then the compressive strength determined as per ASTM C805.

**Figure 5.** Mechanical strength for control, CHAp and Me-CHAp (Me = Sr, Ag, Ba, Zn, K).

*Coatings* **2019**, *9*, 231

For the water absorption test, the samples were dried in the oven at 40 ◦C for 8 h, cooled at room temperature and then weighed (*W*1). After that, the samples were immersed in distilled water for 24 h. At the end, they were removed from distilled water and weighed (*W*2). The water absorption content was calculated with the Equation (4), and graphical represented in Figure 6.

$$\text{water absorption} = \left[ (\mathcal{W}\_2 - \mathcal{W}\_1) / \mathcal{W}\_1 \right] \times 100\% \tag{4}$$

**Figure 6.** Water absorption for control, CHAp and Me-CHAp (Me = Sr, Ag, Ba, Zn, K).

The freeze–thaw operation is repeated until 20 cycles are performed, after which the mass losses during the freeze–thaw process is expressed as gelivity coefficient (Figure 7).

**Figure 7.** Gelivity coefficient for control, CHAp and Me-CHAp (Me = Sr, Ag, Ba, Zn, K).

#### **4. Discussion**

The XRD patterns shown in Figure 1, indicated that Me-CHAp samples are very similar to that of pure CHAPp and were in accordance with ASTM data (Card 9-432). The diffraction peaks (2θ = 25.879◦ for (002), 31.775◦ for (211), 31.196◦ for (112), 32.902◦ for (300), 33.684◦ for (202), 35.597◦ for (301) and 39.853◦ for (130) respectively), are sharp and well resolved, indicating the obtained phase pure, well crystallized hydroxyapatites [32]. The unit cell parameters of all samples are presented in Table 2.

**Table 2.** The lattice parameters a and c for synthesized carbonated hydroxyapatites.

The lattice parameters a and c increased with increasing ionic radium of metallic ions. The substitution of Ca (0.99 Å) with Zn (0.74 Å), Sr (1.12 Å), Ag (1.26 Å), Ba (1.35 Å), and K (1.38 Å) in CHAp lattice could be a cause of increasing of a and c lattice parameters. Changes in cell parameters may imply that all the metals substituted for calcium in the CHAp lattice corresponded with an increase in lattice parameters (except for Zn, which has a smaller ionic radius than Ca). It is also possible that lattice parameter changes occurred due to silver ion and other site substitutions in the crystal lattice. These results are in accordance with previous reports by [30]. Moreover, the inclusion of the metals with a larger ionic radius compared to Ca2+, caused the expansion of the lattice parameters (mainly along the *c* axis) and the increase of cell volume [33–35]. According to results given in Table 2, all Me-CHAp samples have lower crystallinity than pure hydroxyapatite.

On the other hand, the substitution of strontium and silver can cause phase shifting to lower 2θ indicating an increase in the lattice parameters, which can be attributed to the higher ionic radius of Sr (1.12 Å) and Ag (1.26 Å), as compared to Ca2+ [35].

Broadening of the peaks due to the reduction in the crystallite size and increase in the lattice disorder, are attributed to the divalent metal+ substitution in the HAp lattice [36]. Also, the average crystallite sizes, calculated after Scherrer equation suggests that the hydroxyapatite samples could be considered as nano-CHAp (Table 1). The results show that insertion of the metals into CHAp lattice led to a higher SBET that is correlated with smaller crystallite sizes of these samples [37].

The bands from 1455 and 1419 cm−<sup>1</sup> have been ascribed to carbonate and phosphate IR bands. When the carbonate IR bands are at 1455 and 1419 cm−<sup>1</sup> (ascribed to CO3 <sup>2</sup><sup>−</sup> and PO3 <sup>4</sup>−) a B type substitution is identified, whereas the bands are at 1550 and 1530 cm−<sup>1</sup> (ascribed to OH and PO3 <sup>4</sup>−) a A type substitution is present (Figure 2).

The bands from 879, 873 and 866 cm−<sup>1</sup> are similar and characteristic of all the samples. This means that the carbonate ions are present in the CHAp structure either carbonate ions substituting OH− groups (type A–879 cm−1) or carbonate ions substituting the phosphate ions (type B-873 cm−1). Phosphate bands could be identified at 605, 575 and 563 cm<sup>−</sup>1. The FTIR bands obtained confirm that the synthesized samples could be AB type: CO3 <sup>2</sup><sup>−</sup> is substituting PO4 <sup>3</sup>−. Similar results have been reported by Padila [30].

The TGA result shows that there are two stages of weight loss that occur during the heating process. The first weight loss of 8.3 wt.% is observed from 30 to 225 ◦C due to the removal of trapped water in the sample. The second weight loss of approximately 31.6 wt.% occurs from 226 to 700 ◦C and was attributed to the decomposition of the other components (CO3 <sup>2</sup><sup>−</sup> according to the weight loss observed in TGA curves between 550–950 ◦C [31,38].

The results obtained for the non-treated and treated specimens with carbonated hydroxyapatite solutions 0.25 g/L concentration, revealed that by brushing, acicular crystals of approximately 500 nm in length and 10 nm in width were obtained (Figure 8); and the second one, when the solution was sprayed on the stone surface, agglomerates of spheroidal crystals of approximately 10 ± 20 nm in diameter were obtained (marked by arrows).

**Figure 8.** SEM images for BaCHAp applied by brushing and spraying: (**a**) Ba-CHAp applied by brushing, (**b**) Ba-CHAp applied by spraying.

By optical microscopy (Figure 9) it was possible to observe that the untreated specimen presents a whitish, highly porous surface (Figure 9a–c), while the treated specimen shows a more homogeneous surface with no chromatic alteration macroscopically visible. Looking more in detail to the microstructure, it can be observed that the nanoparticles partially filled the superficial pores of the original matrix. The thickness of the deposited layer depends on the consolidant type, higher for Ag-CHAp and mostly similar for Ba-CHAp, Sr-CHAp and CHAp. The layer thickness of the other consolidants is very thin, between 8–10 μm, this being a possible reason for the low efficacy of them.

**Figure 9.** Optical microscopy of the untreated and treated by spraying the specimens: (**a**) Ag-CHAp, (**b**) Ba-CHAp, (**c**) SrCHAp, (**d**) CHAp.

*Coatings* **2019**, *9*, 231

The layer varies between 30–90 μm, which is in good agreement with other literature report [39]. By brushing, the layer is thickened, as SEM images shown (Figure 10a–d).

**Figure 10.** SEM images for brushed specimens: (**a**) CHAp, (**b**) Ba-CHAp, (**c**) Sr-CHAp, (**d**) Ag-CHAp.

As humidity plays a key role in stone deterioration, a detailed understanding of the processes involved and imposed is required. The amount of water presented in a building stone, its actual material moisture, is a function of several parameters. The specific inner surface represents the interface for any process between the atmosphere and the solid material. There is a close interrelation between material moisture and mechanical properties [40].

From the above images it can be observed that the layer obtained by brushing the carbonated hydroxyapatite derivatives on model samples has different thicknesses: 53–59 μm for CHAp (Figure 10a) and thicker from 245 to 524 μm for its metallic derivatives (Figure 10b–d). For CHAp derivatives with divalent metals (Sr, Ba), the layer obtained by application is between 262 and 378 μm, while for the monovalent derivative of CHAp (Ag) the thickest layer of 540 μm is obtained. The reason lies in the tendency to agglomerate of these metallic derivatives, due to the large surface-to-volume ratio (Figure 4), more accentuated at Ag-CHAp than Sr-CHAp and Ba-CHAp.

If the pore volume, determined by porosity, is quite similar (0.008–0.01 cc/g), the surface area and pore diameter are different and depend on the consolidant type: It is highest for Ag-CHAp applied by brushing and for CHAp and Ba-CHAp applied by spraying. Meanwhile, the pore diameter is the smallest for Sr-CHAp and Ba-CHAp applied by spraying. From these data, it could be concluded that the application type is decisive and contribute to the layer homogenity applied on the stone surface. For the control specimens, these values are not different at all. They are similar both for brushing and for spraying (Figure 4).

Measurement of mechanical properties of the samples in all stages (i.e., by brushing and by spraying) was achieved by the test of compression strength. It could be observed that by comparison

with the control samples, all the treated samples presented higher compressive strength values for the samples treated by brushing than those treated by spraying (Figure 11a–d) correlated with an increased rebound number (Figure 5). The addition of the nanoparticles on the specimen surface enhanced the durability of stone compared to the samples not treated, due to the role of nanoparticles in reinforcing the stone, improving their interaction with the stone grains.

It was observed that the results of Zn-CHAp nanoparticles gave the highest values of compressive strength. This can be attributed to the effect of the high reactivity, crystallites size, nanoparticles size and high compatibility with the substrates, similar with other cases reported in the literature [41]. The higher the crystallites size, the higher the compressive strength. The chemical composition of nanosized consolidant can exhibit improvements in thermal, physical, and mechanical properties, because of the strong synergistic effects between the stone surface and CHAp on both the molecular and nanometric scales [42].

(**b**)

**Figure 11.** *Cont*.

**Figure 11.** The compressive strength vs. rebound index relationship for model stones coated with carbonated hydroxyapatite derivatives. (**a**) samples treated by brushing, exponential graphical representation; (**b**) samples treated by brushing, linear graphical representation; (**c**) samples treated by spraying, exponential graphical representation; (**d**) samples treated by spraying, linear graphical representation.

This paper presents the compressive strength vs. rebound index relationship for model stones. Linear and exponential relationship between average rebound number and compressive strength was established using the least square method. The linear model was found to be the better one with a regression coefficient of 1.0 than the exponential model with a regression coefficient of 0.9996, which indicates the acceptability of the linear model for predicting compressive strength of model stones, Figure 11a–d.

As humidity plays a key role in stone deterioration, a detailed understanding of the processes involved and imposed is required. The amount of water presented in a building stone, its actual material moisture, is a function of several parameters. The specific inner surface represents the interface for any process between the atmosphere and the solid material. There is a close interrelation between material moisture and mechanical properties [40].

Transport and distribution of H2O, fluid water and vapor in porous materials are governed by different processes. In the assumed case of a dry material, as a first step, its surface starts to adsorb single H2O molecules from the atmosphere to form a layer. The only transport mechanism which occurs is vapor transport. With rising air humidity, the number of layers of water molecules on the surface increases. Within those multimolecular layers of the water, surface flow occurs as a secondary and more efficient transport mechanism. Additionally, at a certain content, the water layers merge into the small pores due to capillary condensation, which is effective up to some 10−<sup>7</sup> m pore radius. With an increase of water content in the system, capillary forces, which are active in the pore radius range between some 10<sup>−</sup>7–10−<sup>3</sup> m, start to govern the internal flow. Finally, when the material is completely filled with water, saturation flow occurs. Similar, but inverse are the processes when a water saturated material is subjected to drying. The removal of H2O stops when desorption out of the material and adsorption of air humidity reach equilibrium.

When water from the stone surface evaporates faster, a larger active evaporation surface cools occur. Partial saturation enhances this effect. This shows that the surface roughness expressed in the specific inner surface is a determining parameter. The amount of water sorption uptake correlates mainly with the inner surface area, determined by the N2-BET method.

Samples with medium specific surface areas have a lower uptake and the lowest values can be observed in samples with the lowest specific surface areas. So, the samples with high specific inner surfaces, exhibit the highest moisture uptake values.

The specimens that supported the freeze–thaw cycles were first dried, weighed, and later immersed in H2O for saturation throughout 48 h, consistent with the standardized procedure represented in EN12371. The freeze–thaw aging was performed on 15 specimens and lasted 20 cycles. At the end of freeze–thaw aging procedure the dry mass of the specimens was assessed (Figure 12).

**Figure 12.** *Cont*.

**Figure 12.** Stone samples with metallic hydroxyapatite applied by brushing and spraying.

The deterioration of the stones involves mechanical ruptures along the edges, fractures due to the weathering, grooves in depth, shrinkage cracks and brown patina. Salt incrustation may cover the stone surface and fill the fissures of blocks. The deterioration/weathering condition could be poorly weathered stone surfaces and extremely weathered stone surfaces.

By correlation between the visual appearances of samples treated with gelivity values for the samples treated with various carbonated hydroxyapatite a similar degradation tendency could be observed. The control samples undergo similar degradations after the 20 freeze–thaw cycles, as shown in Figure 7, and more pronounced in the samples treated by spraying with two exceptions of the samples treated with Zn-CHAp and K-CHAp.

The most pronounced degradations are observed in the case of Ba-CHAp, applied by spraying, most probably due to Ba ionic radium 1.35 Å, too high to cover the homogeneous samples.

The higher the gelivity value, the higher the degradation rate and smaller the protection capacity of the carbonated hydroxyapatite derivatives coatings.

The spraying procedure is a fast application, requires one application, leads to a smooth finish free of brush/roller marks, can get into hard to reach areas, but has some disadvantages: Long prep and clean up time, uneven coverage (sometimes too thick), uses 2–3 times as much paint as brushing, poor adhesion, cannot paint on windy days. By comparison, brushing has an excellent control, very good adhesion, even uniform coverage, gets paint into nooks and crannies better than spraying. But it is a slow and laborious application, can require 2 or more coats, can leave brush marks and obstructions which make for a difficult application. It is slow, but an even, uniform coat of paint could be obtained and much better adhesion than with spraying alone [20,43,44].

After analyzing these results, it could be concluded that the brushing procedure could offer a more efficient coating of the model stone specimens, while the spraying procedure, evenly induces good compressive strength, produces mechanical ruptures along the edges (Zn-CHAp, Ba-CHAp, Ag-CHAp), fractures (Zn-CHAp, Ba-CHAp, Ag-CHAp), grooves in depth (Zn-CHAp, K-CHAp, Ba-CHAp, Sr-CHAp), shrinkage cracks (CHAp), brown patina (Zn-CHAp).

#### **5. Conclusions**

Carbonated hydroxyapatite derivatives (CHAp) and its metallic derivatives (Ag, Sr, Ba, K, Zn) have been prepared and characterized in this paper and their coating capacity has been evaluated and discussed. These compounds were characterized using several analytical tools, XRD, TGA, FTIR, OM and SEM–EDS techniques.

The efficiency and suitability of these products on model stone samples were evaluated by monitoring the resistance to artificial weather (freeze–thaw), and pore structure changes (surface area, pore volume, pore diameter). Also, the mechanical strengths of these new compounds as coatings for model stone samples revealed homogeneous and strong layers deposited on the stone surface. By correlation, between the visual appearance of samples treated with gelivity values and the samples treated with various carbonated hydroxyapatite, a similar degradation tendency could be observed that is more pronounced in the samples treated by spraying with two exceptions of the samples treated with Zn-CHAp and K-CHAp. The higher the gelivity value, the higher the degradation rate and smaller protection capacity of the carbonated hydroxyapatite derivatives coatings.

The brushing procedure could offer a more efficient coating of the model stone specimens, while the spraying procedure, evenly induces good compressive strengths, produces mechanical ruptures along the edges (Zn-CHAp, Ba-CHAp, Ag-CHAp), fractures (Zn-CHAp, Ba-CHAp, Ag-CHAp), grooves in depth (Zn-CHAp, K-CHAp, Ba-CHAp, Sr-CHAp), shrinkage cracks (CHAp) and brown patina (Zn-CHAp).

**Author Contributions:** Conceptualization, R.-M.I. and M.-L.I.; Formal analysis, R.-M.I. and M.-L.I.; Funding acquisition, R.-M.I.; Investigation, R.-M.I., L.I., G.V., M.E.G., R.E.A., G.-I.R., R.M.G., S.T., I.A.B., M.-L.I., I.D.D., A.I.G. and C.R.; Project administration, R.-M.I.; Software, G.-I.R.; Supervision, R.-M.I. and M.-L.I.; Visualization, R.-M.I. and L.I.; Writing—original draft, R.-M.I.; Writing—review & editing, R.-M.I. and L.I.

**Funding:** This paper received the financial support of the projects: PN-III-P1-1.2-PCCDI-2017-0476 from UEFISCDI-MECI, Romania.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Diammonium Hydrogenphosphate Treatment on Dolostone: The Role of Mg in the Crystallization Process**

#### **Elena Possenti 1,\*, Claudia Conti 1, G. Diego Gatta 2, Marco Realini <sup>1</sup> and Chiara Colombo <sup>1</sup>**

Received: 28 January 2019; Accepted: 26 February 2019; Published: 4 March 2019

**Abstract:** The diammonium hydrogenphosphate (DAP, (NH4)2HPO4) reaction with calcite has been extensively investigated. The availability of free calcium ions in the reaction environment has been acknowledged as a crucial factor in the crystallization of calcium phosphates with a high (hydroxyapatite, Ca/P 1.67) or low Ca/P molar ratio (dicalcium phosphate dihydrate, Ca/P 1.00; octacalcium phosphate, Ca/P 1.33). On the contrary, no data are available on the DAP interaction at room temperature with dolomite in terms of reaction mechanism and composition of the reaction products. Here, a multi-analytical approach based on scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectrometry (EDS) and X-ray powder diffraction before and after heating treatments is proposed to explore how the formation of calcium phosphates occur on Mg-enriched substrates and if the presence of magnesium ions during the reaction influences the crystallization process of calcium phosphates. The DAP reaction with polycrystalline dolomite gives rise to the formation of struvite and of poorly crystalline hydroxyapatite. Calcium and magnesium ions mutually interfered in the crystallization of magnesium and calcium phosphates, respectively, whose effects influenced the properties (size, micro-morphology, composition and crystallinity) of the newly-formed phases.

**Keywords:** calcium phosphate; hydroxyapatite; magnesium phosphate; struvite; dolomite; consolidating treatment; cultural heritage; ammonium phosphate

#### **1. Introduction**

Diammonium hydrogenphosphate (DAP, (NH4)2HPO4) is a promising inorganic consolidating treatment for decayed carbonatic stones [1–4]. The DAP consolidating treatment aims at restoring the stone matrix through a partial transformation of the original material in newly-formed phosphate phases. Ideally, crystalline hydroxyapatite (HAP, Ca5(PO4)3OH) is formed by combining calcium ions of the substrate with phosphate groups of the reagent [5,6].

The DAP reaction with calcite (CaCO3) of carbonatic stones has been extensively investigated over the last years, [7,8] and recent findings showed that the DAP reaction with polycrystalline calcite of carbonatic stones is non-stoichiometric [9], which implies that many crystalline phases may form in complex mixtures with a nanocrystalline and/or partially-substituted HAP [1,5,6,9–12]. Furthermore, it has been demonstrated that the composition of the new phases depends on several variables, as e.g., the reaction condition (pH, ion strength), the stone substrate (lithotype, microstructure) and the treatment protocol (duration, application method, DAP molarity) [10,13–16].

The DAP reaction in the presence of low amount of calcium ions (e.g., when DAP is used with a low molarity [9] or on calcite substrates with Mg-containing veins [17]) follows a different crystallization process. In particular, the availability of free calcium ions is a crucial factor in the crystallization; phases with a low Ca/P molar ratio (e.g., dicalcium phosphate dihydrate DCPD CaHPO4·2H2O, Ca/P molar ratio 1.00, and octacalcium phosphate OCP Ca8(HPO4)2·5H2O, Ca/P molar ratio 1.33 [13,14]) are formed instead of HAP [9,18] (Ca/P molar ratio 1.67) in reaction environments with a low concentration of calcium ions.

Over the centuries, several carbonatic stones with magnesiac nature have been used for artefacts and buildings. A crucial issue is the challenging conservation of Angera stone, a sedimentary dolostone widely used as ornamental lithotype in the North of Italy and severely affected by decay [19]. The literature on the investigation of DAP treatments applied to calcite-based materials of cultural heritage is progressively increasing, while, surprisingly, the DAP reaction with magnesium carbonates is still quite unexplored.

Clearly, the DAP reaction with magnesium carbonates is expected to give rise to a set of crystalline phases different to those formed with calcium carbonate. However, even though the transformation of magnesium carbonates into magnesium phosphates with hydrothermal conditions [20] or by calcination [21] is well-known in literature, only a few studies investigated the carbonate-to-phosphate conversion at room temperature [22,23], which normally is the operative condition of conservation treatments.

This study focused, for the first time, on the reaction of polycrystalline dolomite with DAP solutions at room temperature in order to explore how: (i) DAP treatments react with dolostones; (ii) the nucleation of calcium phosphates occurs in presence of Mg-enriched substrates; (iii) the presence of magnesium and calcium ions during the reaction influences the crystallization process of calcium and magnesium phosphates.

The DAP treatments were carried out by using DAP water solutions with two different molarities on quarry specimens. The formation of specific crystalline phases and their diffusion inside the stone matrix are investigated following a multi-analytical approach (scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectrometry (EDS), X-ray diffraction before and after heating treatments) and critically discussed.

#### **2. Materials and Methods**

#### *2.1. Materials*

The Angera stone, a dolostone quarried in the northern Italy (Piedmont) and widely used in the Lombard architecture as ornamental building material since the Roman age, was used for this study [24]. The lithotype is characterized by a very fine grain size and a high porosity (18–26%, depending on the Angera stone variety). The stone is mainly composed of dolomite (CaMg(CO3)2) in association with a low fraction of clay minerals and iron oxides [19,25–28].

The experiments were performed on the white variety of Angera stone, as it is the variety that undergoes the most severe decay processes in the environmental conditions. They were carried out on a set of freshly quarried prismatic specimens (50 mm × 50 mm × 20 mm) in order to explore the crystallization of phosphates from DAP solutions at room temperature on dolostones.

The Angera stone specimens were treated by a 0.76 M or of a 3.00 M aqueous solutions of DAP (CAS Number 7783–28-0, assay ≥99.0%, reagent grade, Merck, Darmstadt Germany). The concentration 0.76 M (corresponding to a 10% w/w) was selected on the basis of previous experiments [10] and on the consolidating practice in conservation worksites [29]; the choice to also include 3.00 M concentration was suggested in previous studies available in the literature [7,30], where this value was used to enhance the crystallization of calcium phosphates. The consolidating DAP solution was applied by poultice (dry cellulose pulp, MH 300 Phase, Italy; ratio ~ 5:1 DAP solution:dry cellulose pulp), as it is one of the most common application methods in the conservation

field. The treatment time was 48 h, during which the specimens were wrapped in a plastic film to avoid the evaporation of the solvent. After 48 h, the plastic film was removed and the specimens were left drying at room temperature for other 24 h with the poultice on top. The DAP poultice was then removed and the specimens were rinsed three times by poultice made with deionized water and dried at room temperature.

#### *2.2. Methods*

The crystalline phases of the Angera stone specimens before and after the consolidating treatments were investigated by X-ray powder diffraction (XRD) (Malvern, UK) in Bragg–Brentano geometry with a Panalytical X'Pert PRO diffractometer, equipped with a PW 3050/60 goniometer, anti-scatter slit and divergence slit (1◦ and 1/2◦ respectively), a PW3040/60 generator and a X'Celerator solid state detector PW3015/20 nickel filtered. The samples were finely pulverized and spread on silicon zero background holders. The diffraction patterns were collected with a Cu Kα radiation source (λ ~ 1.54 Å), accelerating voltage 40 kV and electric current at the Cu anode of 40 mA, in the 2 Theta angular range 4.5–65◦ with a stepsize of 0.17◦and time per step of 130 s.

A set of thermal treatments were carried out in order to fully explore the composition of the newly-formed calcium phosphates, which showed ambiguously interpretable X-ray diffraction patterns. For this reason, untreated and treated specimens were initially analyzed at room temperature (rT); after that, the samples were heated at 400, 600 and 900 ◦C, and re-analyzed at rT after each heating process. The selection of the temperature steps used to investigate the *T*-induced "parent-to-product" phase transformation is driven by literature data [9,16,31–36].

The micro-morphology of the specimens before and after the DAP treatments were investigated by zenithal observations using a JEOL 5910 LV scanning electron microscope (SEM) (Tokyo, Japan) coupled with energy dispersive X-ray spectrometer (EDS) IXRF-2000 (0–20 keV) (Austin, Texas) in high vacuum mode on carbon coated samples.

#### **3. Results**

#### *3.1. Analysis of the Newly-Formed Phases on Angera Stone*

Figure 1 shows the X-ray diffraction patterns of quarry Angera stone before and after the DAP consolidating treatment. The XRD pattern of the untreated substrate shows well-defined peaks of dolomite (main peak at 31.02◦ and secondary peaks at 24.13◦, 33.61◦, 35.33◦, 37.42◦, 41.18◦, 44.96◦, 50.59◦ and 51.11◦ of 2Theta, λ = Cu Kα) and weaker peaks of secondary minerals (quartz at 26.60◦ of 2Theta, phyllosilicates at 9.41◦ and 25.37◦, feldspar at 26.96◦ and plagioclase at 27.48◦).

**Figure 1.** (**a**) X-ray powder diffraction(XRD) patterns of untreated Angera stone (NT) and Angera stone treated by poultice with 0.76 M and 3.00 M DAP solutions. (**b**) Detail in the range 12–30 ◦ showing the phase variations occurred after the consolidation. D = dolomite, S = struvite, HAP? = possible hydroxyapatite, DAP = diammonium hydrogen phosphate, ADP = ammonium dihydrogen phosphate.

The XRD patterns of the Angera stone treated with 0.76 M and with 3.00 M DAP solution exhibit the formation of magnesium phosphates and calcium phosphates. In fact, the XRD peaks of struvite (MgNH4PO4·6H2O, peaks at 14.99◦, 15.81◦, 16.47◦, 20.85◦, 21.45◦, 27.07◦ and 31.94◦) and most likely apatite (peak at ~25.90 ◦) were revealed in mixture with the phases of the substrate.

The XRD peaks of struvite were sharp and well-resolved, indicating the formation of a well crystalline phase, whereas the peak of the possible apatite was extremely weak and broad, most likely due to a poorly-ordered structure. No other peaks ascribable to calcium phosphates were detectable in the XRD patterns, likely due to overlapping with the peaks of other phases or to their severe weak intensity. Several calcium phosphate phases had a peak at ~25.90◦ of 2Theta, as many of them were characterized by a similar crystalline structure (e.g., hydroxyapatite; calcium-deficient hydroxyapatite and its partially-substituted forms; octacalcium phosphate; amorphous calcium phosphates, ACPs, CaxHy(PO4)z·*n*H2O, with *n* = 3–4.5 and 15–20% H2O) [13,37–41]. The peaks of struvite were more intense in the XRD patterns of the specimens treated with the 3.00 M DAP, while the peak of the possible apatite was more visible in the 0.76 M ones.

Moreover, the XRD pattern of the specimens treated with the 3.00 M DAP solution showed evidence of residual traces of the reagent (DAP peaks at 17.55◦, 17.94◦, 23.56◦, 26.43◦, 27.70◦, 28.41◦ and 29.16◦) and of ammonium dihydrogen phosphate (ADP, NH4H2PO4, at 16.63◦), a by-product of the reaction; these two phases were still present within the specimens even though they were rinsed. No peaks of DAP and ADP were detected on the specimens treated with the 0.76 M DAP solution.

In order to further explore the microstructural-crystallographic feature of these poorly-ordered calcium phosphate phases, a sequence of investigations were carried out by electron backscatter diffraction (EBSD), Fourier Transform Infrared spectroscopy (FTIR) and Raman spectroscopy. However, the outcomes of these supplementary techniques did not supply any further information and their results are not reported in this paper.

In particular: (i) for EBSD, it was not possible to obtain the XRD patterns of the investigated area due to the poorly crystalline nature of the phases, their nanometric size and the boundary effects, deriving from the alteration during the sample preparation of the interface between the new phases and the substrate; (ii) for Raman spectroscopy, phosphate phases had a weak Raman scatter, especially when submicrometric and/or poorly ordered, and in mixture with carbonates (phases characterized by a very high Raman cross section); (iii) as for FTIR data, the superimposition of the characteristic stretching and bending PO4 vibrational bands of both the phosphate phases prevented their unambiguous identification.

#### *3.2. Micro-Morphological Investigations of the Stone Surface*

The micro-morphology of untreated Angera stone, showing the well-shaped rhombohedral crystals habit of dolomite is appreciable in Figure 2a, while that of the reacted dolomite is shown in Figure 2b,c. After the DAP treatments, the stone surface showed the presence of a newly-formed coating characterized by a pseudo-amorphous aspect. This coating was prevalently adherent to the stone matrix together with the crystallization of spherical particles agglomerates. Both of them covered the characteristic morphological features of the lithotype.

In the case of the specimens treated with the 0.76 M DAP solution; the newly-formed phases appeared as a pseudo-amorphous thin film (thickness ~ 0.6 μm) that covered the micritic dolomite (Figure 2b, left). On the film profiles, an incipient formation of spherical particles in an elongated arrangement was detected. On dolomite rhombohedral grains (dolomite crystal size ~ 15–20 μm), a shell of spherical crystallites with a bigger size was distinguishable (Figure 2b, right).

In the case of the specimens treated by DAP 3.00 M, the profiles of the dolomite crystals appeared affected by pronounced corrosion marks, with the consequent crystal nucleation of spherical structures combined to form elongated chain individuals (detail of Figure 2c).

Phosphorous is detected by EDS microanalysis in correspondence of the crystals overgrown on the substrate and morphologically different from the dolomite grains of the substrate. By measuring these newly-formed phases, calcium, magnesium and phosphorous are always present, even though the ratio of their elemental abundance varies.

The SEM investigations suggested the presence of two different phosphates phases: a magnesium and a calcium phosphate, which exhibited differences in morphology and particle size (Figure 2b,c). These phases (struvite and a possible apatite, as suggested by the XRD data) did not show their typical orthorhombic and rose-like morphologies [9,42]. However, on the basis of XRD results and on literature data about the mutual influence of Ca2+ and Mg2+ ions on the size and habit of hydroxyapatite and struvite, it is possible to hypothesize the following correlation: (i) the pseudo-amorphous aspect of the coating could be ascribed to nano-sized agglomerates of Ca-phosphate spherical particles; (ii) the spherical particles (consistently in the range of 10–50 nm; dimension slightly affected by the treatment concentration) often characterized by an aggregate prismatic aspect (Figure 2c, right) could be correlated to the formation of magnesium phosphate nuclei.

**Figure 2.** SEM images of Angera stone. (**a**) Untreated substrate, showing micritic dolomite on the left and bigger grains of well-shaped rhombohedral dolomite on the right; (**b**) treated lithotype after the poultice with a 0.76 M DAP solution. On the left, pseudo-amorphous coating formed on micritic dolomite and incipient formation of spherical structures in an elongated arrangement (white rectangle); on the right, big dolomitic crystal with the overgrowth of crystal agglomerates in spherical structures, some of them having prismatic elongations; (**c**) treated Angera stone after the poultice with a 3.00 M DAP solution, showing detail of spherical crystallites and their aggregation in chains - elongated arrangement (arrows).

#### *3.3. Evidence of HAP Formation by Thermal Treatments*

Thermal treatments are an acknowledged tool to investigate the nature of calcium phosphate phases showing at room conditions (rT) hardly distinguishable X-ray diffraction patterns [31], by promoting *T*-induced phase transitions.

More specifically, heating of: (i) poorly-crystalline stoichiometric HAP generated crystalline stoichiometric HAP; (ii) partially-substituted HAP (e.g., carbonated-hydroxyapatite) produced crystalline HAP in mixture with β-tricalcium phosphate (β-TCP, β-Ca3(PO4)2), and with β-TCP predominant versus HAP; (iii) OCP did not generates HAP, but only β-TCP in mixture with β-calcium pyrophosphate (β-CPP, Ca2P2O7) [9,31].

Figure 3 shows the parent-to-product phase transformations occurred in Angera stone treated with the 0.76 M DAP solutions. Identical results are obtained on the specimens treated with the 3.00 M one. Thermal treatments were carried out also on untreated Angera stone for comparison and the principal XRD patterns are provided as well.

**Figure 3.** (**a**) XRD patterns of untreated (grey patterns) and treated (black patterns) Angera stone at room temperature (rT) and after heating at 400, 600 and 900 ◦C. (**b**) zoom of (a) showing the phase transformation occurred with heating. D = dolomite, L = lime, M = MgO, S = struvite, H = hydroxyapatite.

The heating at 400 ◦C induces the first phase variation, namely the total disappearance of the XRD peaks of struvite, due to the collapse of struvite into amorphous magnesium phosphate phases. Previous studies describe that heating induces a chain decomposition, involving the initial formation of amorphous magnesium hydrogen phosphate hydrate (MgHPO4·3H2O [36]), followed by its transformation into amorphous anhydrous magnesium hydrogen phosphate (MgHPO4 [32,35,36]). In this study, it was not possible to discriminate between the two amorphous products; thus, they are referred to a generic amorphous magnesium phosphate phases.

The heating at 600 ◦C acts on poorly-crystalline calcium phosphates, as the weak broad peak at ~25.90◦ of 2Theta becomes more pronounced. The principal phase transformation occurs after heating at 900 ◦C, where dolomite of the substrate is decomposed into magnesium oxide (MgO, peaks at 42.92◦ and 62.30◦) and lime (CaO, peaks at 32.27◦, 37.48◦ and 53.97◦; phase transformation probably occurred between 600–820 ◦C [33,34]) and the peaks of calcium phosphates become more defined. More precisely, the growth of sharp, well-defined peaks at 2Theta of 25.90◦ (*d*002), 31.77◦ (*d*211), 32.96◦(*d*300) and 34.08◦ (*d*202) unambiguously identify crystalline HAP as heating by-product. Only the very weak peak at 28.09◦ could be attributed to β-TCP.

Magnesium oxide and lime, the calcinations by-products of dolomite, are active components that might interact with the calcium phosphates at high temperature, resulting in a further calcium source. However, even if the occurrence of this phenomenon cannot be a priori excluded, there is no evidence

of other high-temperature calcium phosphate phases nor of high-temperature magnesium phosphates formed by the reaction of active oxides with amorphous phosphates.

These results demonstrate that apatite formed after DAP treatments is poorly-crystalline, but mainly stoichiometric. Moreover, the formation of minor fraction of β-TCP indicates that a partially-substituted nonstoichiometric apatite and OCP were formed by the DAP treatment, but with poorly-crystalline stoichiometric HAP predominant versus these phases. No phase variations were observed for silicates and phyllosilicates of the Angera stone matrix.

#### **4. Discussion**

The DAP reaction with the Angera stone involves the partial dissolution of the dolomite grains, with the release of calcium and magnesium ions from the stone substrate. It was followed by the interaction of these bivalent ions with the reagent ions, with the consequent nucleation and growth of newly-formed phosphate phases. This dissolution-recrystallization process is topotactic and forms magnesium phosphates and calcium phosphates: (struvite and hydroxyapatite, respectively) arranged in a coating on dolomite grains. The micro-morphology of this coating, which resembles almost the features of polymeric products used for the conservation, is quite different from the crystalline shell observed on calcite-based lithotypes [9,10], even where the reaction occurred in presence of Mg-containing veins [9,16,17]. The crystallization of these phases is not merely a superficial film, but it is a binding network which connects different dolomite grains.

The crystallization of the newly-formed phosphates is influenced by the microstructure and the composition of the lithotype. In particular, the microstructure of the substrate acts on the micro-morphology of the new phases. In fact, the newly-formed phosphates are well shaped when they grow on dolomite in large crystals, while on micritic dolomite, the phosphates mainly develop tiny crystals with irregular morphologies.

Mg-phosphates are crystalline, as demonstrated by the sharp well-defined XRD peaks of struvite. On the contrary, the apatite is formed as poorly ordered partially-substituted crystals. It is worth underlying that hydroxyapatite formed after DAP treatments by using only calcium ions of the substrate is never highly crystalline, even when the reaction occurs on calcium carbonate stones. However, the DAP reaction on dolomite of Angera stone is particularly non-stoichiometric, and the formed apatite is so poorly ordered that its identification before heating is assumed only by a weak broad XRD peak. It is conceivable that Mg2+ ions destabilizes the crystalline structure and growth sequence of apatite, which shows a morphology characterized by an amorphous-spherical aspect similar to that reported by Ren et al. [43]. In any case, the thermal treatments shed light on the nature of this phase and, for clarity's sake, the poorly ordered calcium phosphate phase formed after the DAP treatment will be labeled as HAP in the following discussion.

The HAP X-ray diffraction peak is more intense in the specimens treated with the DAP 0.76 M, while struvite peaks are more evident in the specimens treated with the DAP 3.00 M, even though it is only a semi-quantitative evaluation. The higher the DAP molarity, the higher the ion dissolution from the substrate; consequently, the more pronounced the formation of struvite and the more poorly crystalline HAP. Regarding to the DAP molarity, the "coating-like" morphology is more evident in the specimens treated with the DAP 0.76 M, whereas the rounded morphology in elongated chains is prevalent on the specimens treated with the DAP 3.00 M. Considering these features, it is conceivable to hypothesize that these morphological and compositional differences depend on the reaction variables, first of all the pH and the ionic strength. Moreover, dolomite is less reactive than calcite to DAP solutions [20]; thus, the reaction kinetic may be different as well.

The minimum solubility of struvite is documented in the pH range 9–11 [42], while HAP crystallizes in aqueous solutions at pH > 8.5–9 [14].

At the beginning of the reaction the pH is 8.8, which is close to the ideal crystallization pH for both the phases. Actually, even though it is not possible to measure the evolution of pH on grain boundaries, it is reasonable that pH decreases as long as the reaction evolves, due to the: (i) dissociation of the reagent into PO4 <sup>3</sup><sup>−</sup> and H<sup>+</sup> ions [9,44], (ii) nucleation of HAP which consumes OH<sup>−</sup> ions [41]; iii) precipitation of struvite which subtracts NH4 <sup>+</sup> ions [36].

Furthermore, close to dolomite boundaries where the dissolution-recrystallization process is ongoing, Ca2+ and Mg2+ ions compete for PO4 <sup>3</sup><sup>−</sup> groups to nucleate calcium phosphates or magnesium phosphates. In the microscale variations of the pH and of the ionic strength at the grain boundaries, carbonate and bicarbonate ions are reasonably involved as well [45].

The prediction of struvite/apatite crystallization is particularly challenging when this reaction occurs on dolomite stone. A combination of conditions governed by thermodynamics of solid-liquid equilibrium, kinetics of reaction, pH of the solution from which struvite and hydroxyapatite may precipitate, super saturation and presence of foreign ions influence their nucleation and crystallization process.

In particular, the influence of foreign ions on struvite/hydroxyapatite nucleation and their crystallization is a crucial issue because of Ca and Mg are at the same time "impurities" in that solution from which the two Mg/Ca phosphate phases may precipitate. This mutual interference affects the growth rate, which in turn inhibits the increase of crystal size [36].

This complex ionic equilibrium, with pH fluctuations toward not ideal reaction conditions, generates two effects. The first one is a clear inhibiting effect of Mg2+ ions on the crystallinity, morphology and crystal size of HAP, which nucleates as a poorly-crystalline phase and with a pseudo-amorphous coating aspect or in agglomerated structures (depending on the employed DAP molarity). A partial intra-crystalline Mg2+ versus Ca2+ substitution, as ab initio simulated by [43], is also possible. Referring to previous findings, this phenomenon should induce a slight variation of the unit-cell constants. In the case of HAP formed after the DAP treatment on dolomite, the possible generation of a Mg-HAP is not clearly documented, as it is most likely a very minor phase.

The second aspect is the interference of Ca2+ ions in the crystallization of struvite, which occurred in sub-micrometric nuclei of crystals, more than well-structured micrometric prismatic crystallites. The possible presence of ions substitutions in struvite is also highly likely [36].

Focusing on conservation evaluations, the newly-formed crystalline phases nucleate in a coating on dolomite grains by forming a covering that provides new functional properties to the substrate.

Irrespective of their composition, the newly-formed phases nucleate on the dolomite grain surface and among dolomite grains; hence, their crystallization provide a clear bonding action on the stone microstructure. Further experiments are scheduled in order to explore the quantitative ratio of the two phases and their arrangement within the stone pores.

#### **5. Conclusions**

The DAP reaction with polycrystalline dolomite of the Angera stone determines an interaction among calcium and magnesium ions, which compete each other to form phosphate phases. The variation of the solution pH and of the ionic strength during the DAP reaction with dolomite generate a complex crystallization processes of the phosphate phases, which are formed as Mg-phosphates and Ca-phosphates crystals with morphologies different from those commonly described in literature. The mutual interference of the ions involved in reaction (NH4+, Mg2+, PO4 <sup>3</sup>−, Ca2+, CO3 <sup>2</sup>−, HCO3 −) determines an irregular crystal growth pattern with respect to the primary nucleation process. The consequence is a clear effect in the crystallization of the new phases (struvite and hydroxyapatite) in terms of crystal size, micro-morphology, composition and crystallinity.

In particular, the Mg2+ ions presence destabilizes the hydroxyapatite well-ordered structure, causing a structural variation that inhibits the growth of well-shaped crystals and, on the contrary, promotes the formation of an amorphous coating on the dolomite grains. The higher the DAP molarity, the higher the Mg molar fraction in the solution and the lower the crystallinity of the formed calcium phosphate. In any case, heating treatments demonstrated that the newly-formed apatite is poorly crystalline but mainly stoichiometric; thus, with a Ca/P molar ratio quite close to the ideal one (1.67).

On the other hand, the struvite formation is affected by the presence of free calcium ions, and the descending effects are observed in the crystal morphology and crystal size.

Regarding the effects induced by the DAP treatment on the Angera lithotype, it is important to consider that the new phases formed on the dolomite grains create a crystalline network that likely improves the cohesion of the lithotype.

**Author Contributions:** Conceptualization, data curation, investigation, methodology, writing—original draft writing—review & editing, E.P. and C.C. (Chiara Colombo); writing—review & editing, C.C. (Claudia Conti); supervision, M.R. and G.D.G.

**Funding:** This research received no external funding

**Conflicts of Interest:** The authors declare no conflict of interest

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Comparative Study Between Ammonium Phosphate and Ethyl Silicate Towards Conservation of Prehistoric Paintings in the Magura Cave (Bulgaria)**

**Enrico Sassoni 1,2,\*, Elisa Franzoni 1,2, Milyana Stefanova 3, Zdravko Kamenarov 3, Paolo Scopece <sup>4</sup> and Emanuele Verga Falzacappa <sup>4</sup>**

Received: 16 January 2020; Accepted: 6 March 2020; Published: 9 March 2020

**Abstract:** This study aimed at evaluating the effectiveness and the compatibility of two alternative treatments, in view of their possible use for conservation of prehistoric paintings in the Magura cave (Bulgaria). The paintings are made of bat guano applied over limestone; therefore, two sets of laboratory specimens were considered: stone specimens and stone specimens covered with a layer of sterilized bat guano. The two investigated treatments were a commercial product based on ethyl silicate ("ES") and a solution of diammonium hydrogen phosphate ("DAP"), aimed at forming calcium phosphates. The results of the study indicated that both treatments were able to increase the mechanical properties of stone, the increase being higher for "DAP". Both consolidants caused acceptable color changes, but the "ES" treatment significantly decreased stone wettability, water absorption, and water vapor permeability, while the "DAP" treatment slightly affected those properties. In the stone + guano specimens, the presence of the guano layer affected the penetration of the consolidants, thus partly reducing their effectiveness. Compared to the stone samples, the guano layer experienced a more intense color change, alongside visible cracking. However, the adopted methodology to replicate the cave paintings was not completely successful, as the so-deposited guano layer was very prone to detachment when dry, unlike cave paintings. Future work will be dedicated to assessing the consolidant performance onto samples that resemble even more closely the conditions of the cave paintings, by improving the methodology for the guano layer deposition and by contaminating specimens with soluble salts before consolidant application.

**Keywords:** cave painting; inorganic consolidant; hydroxyapatite; calcium phosphate; ammonium phosphate; ethyl silicate; TEOS; non-thermal plasma; wettability

#### **1. Introduction**

The present paper aimed at investigating the effectiveness and the compatibility of two treatments that have been taken into consideration for the conservation of prehistoric paintings in the Magura cave (northwest Bulgaria). The Magura cave contains impressive prehistoric paintings, displaying both figurative and abstract compositions [1]. Religious ceremonies, hunting scenes, signs, and graphic motifs decorate one of the cave chambers—the Gallery with the paintings (Figure 1).

**Figure 1.** General view of the Gallery with the paintings in the Magura cave (Bulgaria) (**a**,**b**), details of the cave paintings (**c**,**d**,**e**), and details of the deterioration patterns ((**f**,**g**,**h**)—cracks, powdering, blistering, and fragmentation of the limestone; (**i**,**l**)—biological colonization; (**m**)—cracking and loss of the original guano painting due to the drying of the surface and powdering of the stone).

These artworks, dating back to some 5500 years ago, are made of fossilized guano of cave-dwelling bats applied over limestone [2–5]. After opening to the public in 1961, with the consequent presence of visitors and the use of artificial light, the delicate balance of environmental conditions inside the cave has been altered. Despite the cultural relevance of the Magura Cave, unfortunately, the micro-environmental data, such as temperature, relative humidity, CO2 concentration, and atmospheric pressure, have never been professionally measured and controlled. Today, the cave suffers from biological colonization and stone pulverization, originated by crystallization of soluble salts present in rising damp from the ground. Deterioration patterns, such as green biofilm and white and black colorations over the stone/paintings surface, together with limestone powdering, blistering, and fragmentation [6] (Figure 1), led to the closure of the Gallery in May 2019.

The preservation of the cave paintings requires challenging interventions to face the growth of bio-organisms and the deterioration of the stone. As for biological growth, the use of biocides is dangerous because they may completely alter the balance of microbial communities in the cave with negative consequences [7]. For this reason, biodeactivation by non-thermal plasma sterilization has been attempted, and some encouraging results have been obtained [8]. As for stone consolidation, the issue is particularly challenging for the following reasons:

In these conditions, the selection of consolidants to be tested and possibly applied in the Magura cave is very hard, as none of the commercially available treatment seems ideal for application onto the limestone and the paintings in the cave. The two consolidants tested in this study, namely, a commercial product based on ethyl silicate and diammonium hydrogen phosphate, were selected based on the following reasoning.

Organic consolidants, such as acrylic resins, were discarded because, in the past, the use of polymeric treatments often led to enhanced deterioration to such an extent that removal of polymers applied in the past is nowadays one of the greatest issues in materials science for cultural heritage conservation [13]. The failure of organic consolidants often originates from water vapor being impeded to move from the interior of the consolidated substrate towards the exterior [14]. This risk would be particularly high in the case of the Magura cave, where water is continuously supplied from the ground, and systems currently used in building for damp reduction are not applicable.

Among inorganic treatments, nanolimes were first considered because of their good chemical-mineralogical compatibility with limestone and because they have shown a good performance when applied onto lime-based frescoes and wall paintings [9,13–16]. However, as already mentioned, a substantial difference exists between wall paintings and paintings in the Magura cave, made of bat guano. Moreover, in the case of the Magura cave, the purpose was the consolidation of the powdering stone with the bat guano painting on the surface, and not of the painting layer alone; therefore, a consolidant able to penetrate in depth in the substrate was needed. In several cases of application of nanolimes onto stones, a poor penetration depth (<1 mm) has been reported [14]. Nanolime accumulation below the surface is likely not due to nanoparticle coagulation preventing penetration, but rather to nanoparticle re-transport towards the surface during drying [14]. When applied onto the salt-laden stone, conflicting results have been reported about the ability of nanolimes to increase stone durability to salt weathering [17,18]. In fact, in some cases, nanolimes have been reported to increase the crystallization pressure in the treated stone, as a result of the alteration in pore size distribution after consolidation, with a consequent decrease in durability [17]. Considering the limitations exhibited by nanolimes in terms of penetration depth and durability to salt weathering, which are both very important aspects for the conservation of the Magura cave limestone and paintings, nanolimes were discarded, and further options were explored.

Ammonium oxalate was considered as this treatment was specifically developed for the protection and consolidation of carbonate substrates, like wall paintings, marble, and limestone [19]. A positive aspect of the ammonium oxalate treatment is that encouraging results have been reported in the literature about its application onto salt-laden stones [20,21]. Removal of salts before application of ammonium oxalate solutions was found to be not as crucial as in the case of organic treatments because ammonium oxalate does not completely occlude pores (although forming a surface coating) [20]. However, ammonium oxalate was finally discarded because of its reduced availability in the market. The method based on barium hydroxide [19] was discarded as well due to its recommended poultice application (aimed at reducing the risk of surface whitening) and its possible reaction with nitrate salts (giving barium nitrate, a very damaging salt upon crystallization). Given the impossibility to apply the consolidant in the cave by poultice and the presence of several salts in the stone, barium hydroxide was not considered for the present application.

Ammonium phosphate was proposed years ago as an improvement of the ammonium oxalate treatment for the consolidation and protection of carbonate stones [22,23]. The treatment is based on the formation of new calcium phosphates (ideally hydroxyapatite, the least soluble calcium phosphate at pH > 4 [24]) from the reaction between an aqueous solution of ammonium phosphate and the stone [22]. This reaction occurs in 24–48 h during which the stone is kept impregnated with the ammonium phosphate solution; therefore, the environmental conditions of the cave are not expected to negatively affect the treatment success. Thanks to the low viscosity of the phosphate solution and to the absence of nanoparticles, a good penetration depth is usually achieved (>5 cm in highly porous limestone) [25]. So far, the treatment has provided good results on a variety of substrates [25], including many types of natural stones [26–32], mortars [33,34], and stuccoes [35]. Recently, the interaction between ammonium phosphate solutions and pigments used in wall paintings has been investigated, and, in most of the cases (but not in all), the treatment has been found to cause negligible color changes [36]. This is encouraging towards the use of this treatment for the conservation of wall paintings, even though the substantial difference of the Magura cave paintings remains. Another positive aspect of the ammonium phosphate treatment is that the presence of salts in the stone pores has proven to be not detrimental for the treatment success [30,37], but, of course, this depends on the amount and type of salts and on the features of the stone pore system. Moreover, accelerated durability tests have shown that ammonium phosphate treatment is able to increase the durability of treated stone to salt crystallization cycles [38–40]. Furthermore, considering the presence of hydroxyapatite in the Magura cave limestone (although not in the limestone used for the laboratory tests), the chemical-compatibility of the ammonium phosphate treatment can be considered as excellent. For all these reasons, the phosphate treatment was regarded as highly promising for application in the Magura cave and hence was investigated in this study. As described in Section 2.3.2, two alternative formulations of the phosphate treatment were initially considered, and, after some preliminary screening tests, the most promising one was chosen.

Considering that ammonium phosphate has the limitation that experience on its use dates back only to a few years ago and that only a limited number of applications onto real case studies have been reported, a possible alternative consolidant was searched. Products based on ethyl silicate were taken into consideration as they are known to generally exhibit a good penetration depth into porous substrates [41], often sensibly higher than that of nanolimes [14]. One major limitation of ethyl silicate when applied onto carbonate stones, as would be the case in the Magura cave, is that its consolidating effectiveness is lower than in the case of silicate stones. In fact, in the latter case, chemical bonding can take place between the silicate substrate and the silica gel resulting from hydrolysis-condensation of ethyl silicate, while, in the case of carbonate stones, only physical bonding can occur [12]. Nonetheless, significant increases in mechanical properties of carbonate stones treated by ethyl silicate have been reported [42], especially when the stone contains even limited quarzitic fractions that may allow for some chemical bonding between the consolidant and the substrate [43]. To improve the performance of ethyl silicate onto carbonate stones, several routes have been investigated, such as the use of coupling agents [41] or the addition of organic molecules (e.g., polyethylene glycol chains end-capped with carboxylic acid groups [44]) or inorganic nanoparticles (e.g., amorphous calcium carbonate and amorphous calcium oxalate [45]) directly into the ethyl silicate solution. Cases of application of ethyl silicate products onto salt-laden stones have been reported in the literature, with mixed results [18,37,41,46], also due to the variable nature of the ethyl silicate products in the market in terms of oligomer size, solvent nature, catalyst, etc. When salts are present in the pores of the treated stone, the consolidating effectiveness is not dramatically diminished [18,37]. However, the alterations induced by ethyl silicate in the stone pore size distribution may increase the salt crystallization pressure, thus decreasing the resistance to salt crystallization cycles, as assessed on both initially salt-contaminated [18] and uncontaminated [38,39] stone. In comparative studies, the salt resistance of carbonate stones treated by ethyl silicate proved to be lower compared to ammonium phosphate (but still higher than untreated stone) [38,39]. Another import factor when evaluating the suitability of ethyl silicate for the case of the Magura cave is the temporary hydrophobicity induced by ethyl silicate [12]. In fact, until hydrolysis-condensation reactions are completed, the treated stone remains hydrophobic because of the presence of residual ethoxy groups [12]. Completion of hydrolysis-condensation reactions can take as long as 6–7 months [12,47,48], even though the technical data sheet of commercial products usually report that the product reaction is completed in 1 month. The hydrophobic behavior of stone treated by ethyl silicate, which generally preserves permeability to water vapor but prevents the transport of liquid water through the consolidated layer, may be an issue in the case of Magura cave, where rising damp from the ground is present. On the other hand, the high relative humidity (65–90%) inside the cave is expected to speed up the hydrolysis-condensation reactions, which is positive. To accelerate the curing of ethyl silicate, thus re-establishing a hydrophilic behavior in a shorter time, various methods have been proposed, which basically consist of supplying water [47] or water-ethanol mixtures [48] to promote hydrolysis reactions. However, such methods seem unfeasible in the case of the Magura cave, as they involve the application of a poultice onto the treated surface, which may lead to damage and detachment of the cave paintings. All things considered, even though not ideal, ethyl silicate was regarded as worthy of investigation as a possible alternative to ammonium phosphate. Among the numerous products available in the market, a commercial formulation enhanced with antimicrobial ability was used, as described in detail in Section 2.3.1.

For testing the two selected consolidants, the complex situation of the Magura cave had to be necessarily simplified, to isolate variables and better evaluate the consolidant performance. With regard to the three aspects discussed above, the following decisions were made:

(1) To evaluate the effects of the two consolidants not only on limestone but also on the cave paintings, two types of laboratory specimens were considered: (i) specimens made of a limestone similar to that in the cave and (ii) specimens made of the same limestone with a layer of sterilized bat guano deposited on top, to resemble the prehistoric paintings in the cave. The procedure for the deposition of the guano layer, described in detail in Section 2.2, was developed in such a way to likely resemble the process of creation of the paintings, even though, of course, freshly prepared samples could not be identical to cave paintings that have undergone about 5500 years of fossilization.

In the frame of the experimental choices described above, in the present paper, the performance of the two consolidants was characterized in terms of effectiveness (penetration depth, dynamic elastic modulus, and resistance to abrasion) and compatibility (alterations in color, pore size distribution, contact angle, water sorptivity, water absorption, water vapor permeability). The results obtained in the present study will be used in future research to evaluate also the durability of the two treatments, also in salt-contaminated stone.

#### **2. Materials and Methods**

#### *2.1. Stone Specimens*

Two small samples of the Magura cave limestone were available for characterization, namely, a stone piece affected by severe powdering and a hard surface crust. Although not sufficient to fully characterize the stone in the cave, these samples were characterized to select a stone for laboratory testing that could suitably resemble the limestone in the cave. The minerals present in the two samples were determined by powder X-ray diffraction (XRD), using a Philips diffractometer (Milan, Italy) with PW1830 generator and PW1820 goniometer (40 kV and 30 mA, 2θ range 3◦–80◦, step size 0.025 2θ, time per step 1 s). Additional minerals, present in quantities below the sensitivity of XRD, were investigated by Fourier transform infrared spectrometry (FT-IR), using a Perkin Elmer Spectrum Two spectrometer (Milan, Italy) (ATR mode, 2000–400 cm−<sup>1</sup> range, spectral resolution 2 cm−1, 32 scans, data interval 1 cm−1). The amount of calcite in the samples was quantified by Dietrich–Frühling gas volumetric method, i.e., by measuring the CO2 volume released by reacting the powdered sample with HCl. The open porosity of the samples was assessed by mercury intrusion porosimetry (MIP), using a Pascal 140 and 240 instrument. The Magura cave limestone was found to be mainly composed of calcite (the only mineral detected by XRD), quantified as 90.0 ± 1.8 wt.% (by gas volumetric method). Based on FT-IR, the remaining fraction was composed of hydroxyapatite and possibly traces of quartz. In terms of microstructure, the open porosity was found to range significantly between the two samples, from 2% for the hard crust to almost 48% for the powdering sample.

The stone selected for the laboratory tests was Vratsa limestone, which is quarried very close to the Magura cave (Oreshets quarry, Municipality of Vidin, Bulgaria, 20 km from the cave). In terms of mineralogical composition (assessed as above), Vratsa limestone was mainly composed of calcite (80.0 ± 0.1 wt.%), the remaining fraction being quartz (the only other phase detected by XRD and FT-IR). The open porosity of Vratsa limestone (assessed as above) exhibited some variability, ranging from 11% to 18%. Although not identical to the cave limestone, the Vratsa limestone was considered as still suitable, as it exhibited mineralogical composition similar to the Magura cave limestone (although the minor phases were different) and open porosity that was intermediate between the values registered for the samples from the cave (which, however, were very different from each other).

Specimens of Vratsa limestone to be used for the tests (labeled "ST") were sawn in the form of prisms (7 <sup>×</sup> <sup>7</sup> <sup>×</sup> 3 cm3) and cubes (5 cm side).

#### *2.2. Stone*+*Guano Specimens*

To resemble the cave paintings, the original guano from the Gallery with the paintings, which is home of 8 different bat species, was used. The guano has an organic matter content greater than 40%, visible chitinous insect remains and is a favorable environment for many different types of microorganisms [49,50]. For safety reasons, the original bat guano was exposed to autoclave sterilization (Laboratory of Geological Microbiology, Department of Microbiology, Sofia University St. Kliment Ohridski, Bulgaria).

The stone+guano specimens (labeled "ST+G") were prepared, starting from the "ST" specimens, by depositing a layer of sterilized bat guano on top, following the procedure illustrated in Figure 2. First, a small amount of guano was added with deionized water so as to reach a suitable consistency (guano:water ratio of 1:2 by weight). After mixing the guano with water, the obtained mixture was applied onto one face of the specimens by brush. After about 2 min, the second layer of guano was applied in the same way.

Considering that the so-deposited guano layer becomes dusty and starts to detach from the substrate when it dries, the consolidant application was carried out 30 min after the guano application to avoid the stone+guano specimens from becoming too powdery.

**Figure 2.** Procedure for guano application: sterilized dry guano (**a**) is added with water (**b**); after mixing (**c**), stone specimens (**d**) are covered with the first layer of guano (**e**) applied by brush; after about 2 min, the second layer of guano (**f**) is applied.

#### *2.3. Consolidants*

The two consolidants selected for testing were a commercial product based on ethyl silicate (labeled "ES") and diammonium hydrogen phosphate (labeled "DAP"). In both cases, the consolidants were applied by brushing onto one face of the specimens (the face already covered with the guano layer, in the case of the "ST+G" specimens). For each consolidant, 7 prismatic and 2 cubic specimens were treated.

#### 2.3.1. Ethyl Silicate-Based Consolidant

The commercial product "Bio Estel New" by CTS Srl (Florence, Italy) was selected. According to the manufacturer's technical data sheet, "Bio Estel New" is based on ethyl silicate and isopropyl alcohol as a solvent, and it combines consolidating ability with protective ability against microorganisms. The technical data sheet also reports that the product is suitable for application onto silicate and carbonate stones, especially underwater stones and stones in a very humid environment, and that the product reaction is completed in about 4 weeks at T = 20 ◦C and RH = 40–50%.

The ES consolidant was applied by brushing until apparent refusal (7 brush strokes), waiting for the product to be absorbed between two consecutive brush strokes. At the end of the consolidant application, specimens were left to cure in laboratory conditions (T = 21 ± 2 ◦C, RH = 50% ± 5%) for at least 4 weeks before characterization.

#### 2.3.2. Phosphate Consolidant

For the phosphate treatment, diammonium hydrogen phosphate (DAP, (NH4)2HPO4), calcium chloride (CaCl2·2H2O), and ethanol (EtOH), all purchased from Sigma-Aldrich (Milan, Italy, assay > 99%) were used. Based on results obtained in previous studies [22,25,51], two formulations of the phosphate treatment were initially considered for the tests: (i) 1 M DAP + 1 mM CaCl2 and (ii) 0.1 M DAP + 0.1 mM CaCl2 in 30 vol.% EtOH. Calcium chloride has the beneficial effect of accelerating the formation of calcium phosphates [51], while ethanol has been found to promote densification of the new calcium phosphates [52]. To identify the most promising treatment condition in the specific case of the specimens resembling paintings in the Magura cave, preliminary tests were carried on the "ST+G" specimens. The surface alteration following consolidant application, carried out 30 min after guano application, was visually evaluated. As illustrated in Figure 3, the formulation containing 0.1 M DAP + 0.1 mM CaCl2 in 30 vol.% EtOH caused lower alteration (darkening and cracking) of the specimen surface, compared to the alternative formulation. Consequently, that formulation was selected and applied to the "ST" and "ST+G" specimens.

**Figure 3.** Alteration of the specimen surface caused by consolidant application 30 min after guano application: 1 M DAP + 1 mM CaCl2 (**a**) and 0.1 M DAP + 0.1 mM CaCl2 in 30 vol.% (**b**).

The "DAP" treatment was applied by brushing until apparent refusal (10 brush strokes), waiting for the product to be absorbed between two consecutive brush strokes. At the end of the consolidant application, specimens were wrapped in a plastic film to prevent evaporation and left to react for 48 h. Then, specimens were unwrapped, rinsed with deionized water (only in the case of the "ST" specimens), and left to dry at room temperature until constant weight.

#### *2.4. Pre-treatment by Plasma*

To evaluate whether penetration of the two consolidants into the stone might be enhanced, with a consequent improvement in the consolidating efficacy, part of the stone specimens was pre-treated by plasma, before consolidant application. Indeed, an improvement in wettability is a well-known effect of a wide range of plasma sources on different materials. Mainly, thanks to oxygen atoms implantation and the subsequent formation of polar groups on the treated surface, an increase in surface energy is typically observed together with a reduction in water contact angle [53].

To evaluate whether pre-treatment by plasma might be beneficial for the consolidant performance, right before consolidant application, part of the "ST" specimens was subjected to pre-treatment by plasma, carried out with a dual-frequency atmospheric plasma jet device (Stylus Plasma Noble provided by Nadir Srl, Venice, Italy) [54]. The following working parameters were adopted: 5 W power at 16 kHz, 30 W power at 27 MHz, argon gas flow as working gas at 10 L/min, and compressed air as cooling gas at 12 L/min. The 7 <sup>×</sup> 7 cm2 surface of prismatic specimens were manually treated for a total exposure time of 2 min, maintaining a constant working distance of 3 mm between the plasma source and the specimen.

The effects of the plasma pre-treatment were evaluated in terms of static contact angle, amount of product absorbed, and increase in dynamic elastic modulus, assessed as described in the next paragraph.

#### *2.5. Sample Characterization*

The effectiveness and the compatibility of the two consolidants were investigated by comparing the properties of untreated ("UT") and treated specimens ("ES" and "DAP"), as described in the following.

#### 2.5.1. Morphology of the New Phases

The morphology of the new phases formed over the treated surface was evaluated by observing samples (about 10 <sup>×</sup> 10 <sup>×</sup> 2 mm3, obtained by wet sawing from the prismatic specimens), using a field emission gun scanning electron microscope (FEG-SEM, Tescan Mira3, Brno, Czech Republic, WD = 10 mm, Voltage = 10 kV). Before SEM observation, the sample surface was made conductive by sputtering with aluminum.

#### 2.5.2. Composition of the New Phases

The composition of the new phases formed over the treated surface was evaluated by analyzing powdered samples (obtained from the specimen surface using a spatula) by FT-IR, using the same instrument as in Section 2.1.

#### 2.5.3. Penetration Depth

The penetration depth of the two consolidants into the "ST" and "ST+G" prismatic specimens was evaluated by fracturing them with a chisel to expose a cross-section, right at the end of the consolidant application (7 brush strokes in the case of ES, 10 in the case of DAP). The consolidant penetration depth was visually identified as the darker zone. Two replicate specimens were considered for each condition.

#### 2.5.4. Dynamic Elastic Modulus

Dynamic elastic modulus (*Ed*) was measured on prismatic specimens, perpendicular and parallel to the plane of the slab (in the "ST+G" samples, perpendicular and parallel to the face covered with guano). Given the absence of any clear anisotropy in the stone, values measured in the two directions parallel to the plane of the slab were averaged, so that, in the end, each specimen was characterized in terms of *Ed* and *Ed*//. *Ed* was calculated according to the formula *Ed* <sup>=</sup> <sup>ρ</sup> <sup>×</sup> *<sup>v</sup>*2, where <sup>ρ</sup> is the density and *v* is the ultrasonic pulse velocity. *v* was measured with a Matest instrument with 55 kHz transducers, using a rubber sheet to improve the contact between the transducers and the specimen. A rubber sheet instead of grease was used to improve the contact (as would be the standard procedure), in order not to stain the samples that later had to be consolidated. For instrument calibration, a standard bar with fixed traveling time was used. Five replicate specimens were tested.

#### 2.5.5. Resistance to Abrasion

Resistance to abrasion was measured on prismatic specimens, in terms of material loss after an accelerated abrasion test, carried out following a previously developed method [55]. By partially modifying the PEI (Porcelain Enamel Institute) abrasion test, samples were tested by keeping steel spheres and corundum powder in rotatory motion over one of the 7 <sup>×</sup> 7 cm<sup>2</sup> faces of the specimens (the face already covered with the guano layer, in the case of the "ST+G" specimens), for a total of 15,000 rounds. The amount of material abraded during the test was then determined by comparing the sample weight before and after the test. Five replicate specimens were tested.

In the case of the "ST+G" samples, because guano flakes were prone to detach also from the area not directly subjected to the abrasion test, before the test all the guano-covered surface, not directly in contact with the steel spheres, was covered with scotch tape, so that no material loss could take place from that area.

#### 2.5.6. Color Change

The color change after consolidation was measured on prismatic specimens by making use of a NH310 colorimeter. For the untreated and treated conditions, the LabCIE\* color parameters (*L*\* = black–white, *a*\* = green–red, *b*\* = blue–yellow) were determined on duplicate specimens, repeating the measurements in three different spots for each specimen. For instrument calibration, a standard white sample supplied with the instrument was used. The color change (Δ*E*\*) between untreated and treated specimens was then calculated as Δ*E*\* = (Δ*L*\*<sup>2</sup> + Δ*a*\*<sup>2</sup> + Δ*b*\*2) <sup>1</sup>/2, considering the average *L*\*, *a*\*, and *b*\* values obtained for each condition.

#### 2.5.7. Pore Size Distribution

The alterations in open porosity and pore size distribution following the treatments were determined by mercury intrusion porosimetry (MIP), using a Porosimeter 2000 by Carlo Erba (Milan, Italy) with Fisons Macropore Unit 120. MIP samples were obtained by chisel from prismatic specimens, always including the treated surface (where alterations were expected to be most pronounced). For each condition, two replicate samples were tested.

#### 2.5.8. Static Contact Angle

For the untreated and treated conditions, the static contact angle was measured on prismatic specimens by making use of a DSA30 instrument by Krüss (Hamburg, Germany), calibrated according to its standard procedure. A drop of deionized water (4 μL volume) was released on the specimen surface, and then the drop profiles were recorded using a camera and analyzed by SCA20 software 1.0. The static contact angle was determined on duplicate specimens, repeating the measurements in three different spots for each specimen.

#### 2.5.9. Water Sorptivity and Water Absorption

Water sorptivity (*S*) of untreated and treated specimens was measured on cubic specimens, according to the European Standard EN 15801 [56]. Deionized water was let penetrate the specimens

through the treated face, and the weight increase over time was periodically monitored. The amount of water absorbed by capillarity into the samples after 24 h (*WA24h*) and 7 days (*WA7d*) was determined. Two specimens were tested for each condition.

#### 2.5.10. Water Vapor Permeability

The water vapor permeability of untreated and treated specimens was measured on prismatic samples and expressed in terms of water vapor diffusion resistance coefficient (μ), according to the European Standard EN 15803 [57]. A glass container was filled with a saturated aqueous solution of KNO3 up to 3 cm from the top and the specimen was placed on top of the container, with the treated face towards the solution. The specimen was then glued to the container, and the lateral sides of the samples were sealed. The coefficient μ was calculated as μ = δa/δp, where δ<sup>a</sup> is the water vapor permeability of air, and δ<sup>p</sup> is the water vapor permeability of the specimen. Two specimens were tested for each condition.

#### 2.5.11. Field Exposure

Considering the importance of evaluating also the durability of stone consolidants [38] and that small differences in microclimatic conditions may have a strong impact on stone durability [58], untreated and treated stone specimens were exposed in the cave for 9 months (from April to December 2019) to obtain a preliminary evaluation of the effects of the specific environmental conditions inside the Magura cave (T = 12–25 ◦C, RH = 65–90%). The exposed samples were periodically monitored by visual observation.

#### **3. Results and Discussion**

#### *3.1. Morphology of the New Phases*

The morphology of the new phases formed after treatment is illustrated in Figure 4.

**Figure 4.** Morphology of untreated and treated stone ("ST") and stone+guano (ST+G") specimens.

In the case of the "ST" specimens, the untreated stone was characterized by large pores among the calcite crystals. After consolidation with "ES", a coating of amorphous silica was formed, as expected [41]. Because of the porous nature of the substrate, the coating was not continuous, and bare areas were largely visible. After treatment with "DAP", small clusters of the new calcium phosphates were visible on the stone surface, which, also in this case, was not continuously coated with the new phases. Generally, new calcium phosphates formed in carbonate stones from the reaction with ammonium phosphate solutions show a flower-like morphology [25], but, in the case of dilute solutions (such as that adopted in the present case), the formation of small clusters has been reported before [59,60].

In the case of the "ST+G" specimens, observation of the untreated sample at low magnification allowed to distinguish the scales constituting the guano layer, which was cracked and prone to detachment from the substrate. After consolidation with "ES", cracks were still visible in the guano layer. The tendency of the silica gel formed from ethyl silicate to crack upon drying is well known [41]. An increase in the cohesion of the guano layer seemed to be present in the case of the "DAP" specimen, where tiny crystals, ascribable to the formation of the new calcium phosphates, were visible over the treated surface.

#### *3.2. Composition of the New Phases*

The FT-IR spectra of untreated and treated specimens are reported in Figure 5.

**Figure 5.** FT-IR spectra of untreated and treated stone ("ST") and stone+guano (ST+G") specimens. C = Calcite; S = Silica; new bands at 602 and 561 cm<sup>−</sup>1, owing to the formation of new calcium phosphate phases, are highlighted in the inset.

In the "ST" specimens, bands owing calcite and silica were present in the untreated reference. After treatment with "ES", no new band was visible because "ES" led to the formation of amorphous silica, whose bands overlapped with those of the substrate. After treatment with "DAP", small new bands appeared at 602 and 561 cm<sup>−</sup>1, which indicated the formation of new calcium phosphates [61]. The fact that these new bands were very small was due to the low concentration of DAP used for the treatment (0.1 M), which was consistent with a previous study where FT-IR bands of new calcium phosphates were found to be more pronounced for more concentrated DAP solutions [60]. To distinguish between formation of hydroxyapatite [HAP, Ca10(PO4)6(OH)2] and octacalcium phosphate

[OCP, Ca8H2(PO4)6·5H2O], which both could be formed as the reaction product between calcareous stones and DAP solutions [51] and both have FT-IR bands at 560–563 and 600–602 cm−<sup>1</sup> [61], the position of the other strong bands (at 1031 cm−<sup>1</sup> for HAP and 1023 cm−<sup>1</sup> for OCP [61]) should be considered. However, in the present case, no other band was clearly visible because of the overlapping with the strong band at 1080 cm−<sup>1</sup> owing to quartz in the substrate. Consequently, no conclusive identification of the new phase was possible. However, the formation of either hydroxyapatite or octacalcium phosphate was expected to have a durable consolidating ability, as both minerals have lower water solubility than calcite [25,51].

In the case of the "ST+G" specimens, the spectrum of the untreated reference exhibited some new shoulders at 1580, 1000, and 700 cm−<sup>1</sup> (not present in the untreated "ST" sample), which could be attributed to the guano layer. These shoulders were present also in the FT-IR spectra of the treated "ST+G" samples, which exhibited no additional clear band owing to new phase formation. In the case of "ES", this was not surprising, for the reasons discussed above. In the case of "DAP", the lack of new bands (contrary to the case of the "ST" sample) was most likely due to the very low amount of new phases formed after treatment. In fact, very little amounts of new phases are usually formed [22], especially when diluted DAP solutions are used [25]. Moreover, in the present case, the guano layer likely acted as a barrier, limiting the consolidant penetration (cf. Section 3.3) and reducing the number of new phases. Although below the detection limit of FT-IR, new phases were, however, formed after treatment, as indicated by the significant mechanical improvement (cf. Sections 3.4 and 3.5).

#### *3.3. Penetration Depth*

The penetration depth of the "ES" and "DAP" treatments into the "ST" and "ST+G" samples, measured by fracturing the samples right at the end of the consolidant application, is illustrated in Figure 6.

**Figure 6.** Examples of penetration depth of the treatments into the stone and stone+guano samples.

In the case of the stone specimens, the penetration depth was 4.5 ± 1 mm for "ES" and 7.0 ± 1.0 mm for "DAP", the limit of the wet fringe being more easily distinguishable in "ES" specimens. Such penetration depth of both consolidants was in line with previous results if the porosity of the substrate was considered: a penetration >10 mm was reported for both ES [42] and DAP [26,42,62] for highly porous limestones (open porosity 35–50%), while values between 3 and 5 mm were reported for less porous stones (open porosity 5–17%) [30,40]. It is worth noting that previous studies highlighted that, during curing, consolidants are progressively absorbed into finer pores [12], so that the final depth of formation of new binding phases is generally higher than assessed right at the end of the consolidant application [38,63].

In the case of the "ST+G" specimens, the penetration depth was 3.5 ± 1 mm for "ES", hence slightly lower compared to the "ST" specimens. This reduction in penetration depth was likely due to the guano layer acting as a barrier and altering the penetration into the substrate. Moreover, because the consolidants were applied 30 min after application of the guano layer, some water used to apply the guano might have been still present in the pores of the samples, thus reducing the ability of the consolidants to penetrate in depth. In the case of "DAP", no evident sign of a wet fringe could be observed. Nonetheless, this was not an indication that no penetration took place because, otherwise, there would be no mechanical improvement, contrary to what assessed in Sections 3.4 and 3.5.

#### *3.4. Dynamic Elastic Modulus*

For untreated and treated "ST" and "ST+G" specimens, *Ed* values measured in the directions perpendicular and parallel to the plane of the slabs are reported in Figure 7.

**Figure 7.** Dynamic elastic modulus (*Ed*) of untreated ("UT") and treated ("ES" and "DAP") stone ("ST") and stone+guano ("ST+G") specimens in the directions perpendicular and parallel to the plane of the slab.

In the "ST" specimens, both treatments caused an improvement in *Ed*, the increase being higher in the case of the "DAP" treatment. Indeed, the "ES" treatment caused an *Ed* increase of +8% in the perpendicular direction and +8% in the parallel direction, whereas the "DAP" treatment caused an *Ed* increase of +11% in the perpendicular direction and +14% in the parallel direction. The fact that the DAP treatment resulted more effective than ES was in line with several previous studies on limestone consolidation by these two consolidants: the increase in *Ed* was +15% for DAP and +11% for ES in Pietra Serena [28], +9–11% for DAP and +7% for ES in Arenisca Ronda [39], +90–110% for DAP and +42% for ES in Indiana limestone [22,48]. In other comparative studies, ES caused higher *Ed* increases than DAP: +25% for DAP and +39% for ES in Giallo di Siena [28] and +47% for DAP and +62% for ES in Globigerina limestone [42]. In any case, when comparing results obtained in different studies, it should be borne in mind that the properties of the stones tested in each study (mineralogical composition, open porosity, and pore size distribution) and the properties of the tested consolidants (concentration of the active principle, nature and amount of the solvent, application technique, etc.) play a fundamental role, determining the final outcome of the consolidating treatment.

As for the "ST+G" specimens, in the untreated condition, they exhibited a lower *Ed* value in the perpendicular direction compared to the "ST" specimens (41.3 ± 3.9 GPa against 45.8 ± 2.3 GPa, respectively), while, in the parallel direction, the values were much closer (48.3 ± 10.1 GPa against

47.2 ± 3.8 GPa, respectively). This was thought to be a consequence of the guano presence in the "ST+G" specimens, where the guano layer worsened the contact between the specimen and the transducers in the perpendicular direction, while no alteration was present in the parallel direction. The guano presence also slightly reduced the consolidating effectiveness. In fact, the "ES" treatment caused an *Ed* increase of +1% in the perpendicular direction and +7% in the parallel direction, while the "DAP" treatment caused an *Ed* increase of +7% in the perpendicular direction and +4% in the parallel direction. This reduction, compared to the "ST" samples, was most likely due to the guano layer, limiting the consolidant penetration in depth and thus the consolidating ability in the "ST+G" specimens.

#### *3.5. Resistance to Abrasion*

The weight losses of untreated and treated specimens after the abrasion test are reported in Figure 8.

**Figure 8.** Weight loss after the abrasion test of untreated ("UT") and treated ("ES" and "DAP") stone ("ST") and stone+guano ("ST+G") specimens.

In the case of the "ST" specimens, both consolidants improved the stone resistance to abrasion, again the benefit being higher for the "DAP" treatment, consistent with the *Ed* results. Indeed, the "ES" treatment caused a reduction in weight loss by 26%, whereas the "DAP" treatment caused a reduction by 47%. The higher efficacy of DAP to increase stone resistance to abrasion was consistent with previous results obtained on marble (reduction in the weight loss by 86% for DAP and 60% for ES [64]), while substantially similar results were obtained for the two treatments on highly porous limestone (reduction in the weight loss by 37% for both DAP and ES [42]).

In the case of the "ST+G" specimens, the untreated references exhibited a higher weight loss compared to the "ST" specimen because the guano layer could be easily detached from the stone substrate. In the case of the treated specimens, as already noticed in the case of *Ed*, the guano layer interfered with the consolidant penetration and hardening, so that a lower benefit was found, compared to the "ST" specimens. In particular, the "DAP" treatment caused an improvement in the resistance to abrasion (weight loss reduced by 18%), while the "ES" treatment apparently worsened the resistance to abrasion. In fact, compared to the untreated condition, the "ES" treatment apparently caused an increase in weight loss by 49%. This was thought to occur because the "ES" treatment deposits a relatively high amount of new material in the porous guano layer so that the removal of the guano layer during the abrasion test results in a high weight loss.

#### *3.6. Color Change*

The appearance of an illustrative specimen for each condition of the "ST" and "ST+G" specimens is shown in Figure 9. The measured CIELab color parameters are reported in Table 1.