Da Vinci's famous self portrait was drawn in chalk on paper in 1510 and survived well until the 1950's but has long shown signs of deterioration that curators are keen to stop! Given the delicacy of the object (and its huge value, it is insured for $50 million) investigations into what was causing the progressive growth of multiple rust coloured spots across the image has been very careful and causes of the 'foxing' have been the subject of much speculation. Since 1975 it has been suggested that the cause is microbial growth and in 2005 chemical damage was postulated but a recent study has gone much further into the matter.
Earlier attempts to culture microbes from the painting in order to identify them were unsuccessful so the new attempt used non- and very minimally invasive techniques to try to identify. Electron microscope images clearly show structures consistent with Aspergillus halophilicum
The authors state
Regarding the role of fungi in the formation of foxing stains, it can be assumed that the efflorescences developed preferentially on cellulose fibres already undermined by a chemical attack (i.e. iron impurities and dust deposition) and were, therefore, the result of chemical foxing preceding the fungal attack. This hypothesis is consistent with the detection of higher concentrations of iron in the foxing spots than in the background (Bicchieri, 2014). However, it can be also hypothesized that fungi have developed following the onset of storage conditions without aeration and characterized by high relative humidity and high temperatures (Mosca Conte et al ., 2014). These authors assert that the type of chromophores present in Leonardo’s self-portrait are similar to those found in ancient and modern paper samples aged in extremely humid conditions or within a closed environment
DNA was also extracted from microbial samples taken from the image and attempts made to identify species via PCR amplification were carried out. Several fungi were detected including Phialosimplex, Penicillium and Acremonium species
The authors conclude
The role of E. halophilicum, and possibly of other fungi such as Phialosimplex, Penicillium and Acremonium species, in the browning of the paper was indirect and possibly took place (and thus, it is still taking place) according to the phenomena described by Arai (2000, and references therein), which do not depend on the vitality of the organisms, but on the remaining compounds that fungi leave on cellulose fibres after death.
The direct role of fungi in cellulose oxidation and the formation of foxing stains could be attributed to the production of oxalic acid, taking into account the presence of crystals of calcium oxalate and surface erosion connected to some foxing spots. The presence of fungal material, oxalates and other biological compounds undoubtedly represents a very real threat to the conservation of the drawing because the browning phenomenon could be caused by slow ongoing chemical reactions that are independent of the viability of the detected microorganisms, and the current conditions of preservation.