# Varnishes with Biocidal Activity: A New Approach to Protecting Artworks
## 3 Results and Discussion
### Manila Copal  
Table 4 shows the analytical results for Manila copal. As with the other varnishes studied, the biocide treatments did not lead to any significant change in the composition of the original varnish, so the results for the type 2 samples are not included in the table. The study via microscope indicates that the treatments that were tested inhibited the growth of the fungi _C. sitophila_ and _P. herbarum_ in all such cases (Figure 6). As with the other two varnishes, the data corresponding to these latter samples are not included to simplify the study, since they are very similar to the data from blank samples. As for the GC-MS analysis, in the samples inoculated with microorganisms but not treated with biocide, changes of little relevance occurred compared to the simple blank, most notably a decrease in the relative amount of agathalic acid, which oxidizes to agathic acid. Other trends seen were the moderate but generalized increase in the small peak for 19-norlabda-8 (20), 13-dien-15-oic acid and a decrease in acetoxy agatholic acid in _C. sitophila_, which agrees with the results obtained in previous studies [33]. The MDS statistical study (Figure 7) shows the results from all the samples somewhat grouped together to scale, indicating that in general the chemical changes occurring in the samples inoculated with microorganisms were not as clear as in the other varnishes studied. The results in the samples treated with biocides and inoculated with _A. oxydans_ also appear in an area of the graph close to the uninoculated blank samples, indicating that the composition of the Manila copal was not substantially affected by the microorganisms tested and that the biocides therefore play a minor role in terms of the varnish's chemical composition. However, the treatments tested have shown to be efficient in controlling growth by inhibiting the development of mycelium in the samples inoculated with fungi. Figure 5: MDS analysis of the sandarac samples: blank sample (type 1, red circle); samples inoculated with microorganisms without biocide (type 3, blue diamonds); and samples inoculated with Pc, Cs and Sc, and treated with the studied biocides (type 4 (mix), and 5 (sp), green squares). BC = benzalkonium chloride, TBTN = tributyltin naphenate, and OPP = ortophenyl phenol. Cs = _C. sitophila_; Pc = _P. chrysogenum_; Sc = _S. cellulol flavus_. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & & **19-Norlabda-8** & & & \\
**Test** & **Sample Type** & \begin{tabular}{c} **(20),** \\ **13-Dien-15-oic** \\ **Acid** \\ \end{tabular} & \begin{tabular}{c} **Agathalic** \\ **Acid** \\ \end{tabular} & \
\begin{tabular}{c} **Agathic** \\ **Acid** \\ \end{tabular} &
\begin{tabular}{c} **Acetoxy** \\ **Agatholic Acid** \\ \end{tabular} \\ \hline Manila copal & 1 & **3.24** & **7.35** & **41.66** & **23.28** \\ Copal-Cs & 3 & 3.92 & 3.19 & 46.07 & 17.03 \\ Copal-Ph & 3 & 3.92 & 1.93 & 39.07 & 19.81 \\ Copal-Ao & 3 & 2.88 & 3.58 & 48.14 & 22.27 \\ Copal Benzalkonium & 4 & 3.31 & 1.05 & 46.18 & 28.06 \\ Chloride Ao & 5 & 2.16 & 0.33 & 45.82 & 34.13 \\ Copal Benzalkonium & 4 & 1.30 & 0.49 & 40.59 & 33.85 \\ napthenate Ao & 5 & 2.40 & 0.00 & 50.28 & 26.00 \\ \hline \hline \end{tabular}  
* Sample type: 1: varnish only (blank samples); type 3: varnish inoculated with microorganisms; types 4 and 5: varnish treated with benzalkonium chloride and tributyltin naphthalene mixed (4) or spread (5) and inoculated with microorganisms. Cs = _C. sitophila_; Ph = _P. herbarum_; Ao = _A. oxydans_.  
\end{table} 
Table 4: GC-MS analysis (\(p<0.05\) in all cases) of the diterpenes free of Manila copal and the samples inoculated with microorganisms (values expressed in \(N\)i). Figure 6: Microphotography (400\(\times\)) showing fungal growth of **(a)**_P. chrysogenum_ (sandarac) and **(c)**_C.