the book. The books for ethylene oxide measure-ment were chosen aiming to get a representative average of all these features. This is especially important when it comes to transfer data into application Europe-wide and on an “average archive” at the end of the project. 28 volumes bound in leather, parchment, paper and textiles, dating from the 18th, 19th and 20th century, thus representing also different sorts of paper as book block, ranging from folio to quart were selected by the Parish in Swidnica and brought to the European Research Centre for Book and Paper Conservation-Restoration in Horn. There these books were put into a chamber of inert synthetic material of 1 m3 volume and stored under closed conditions for 60 days at 20°C and 50% R. H.
Two different analytical methods were chosen to detect the off gassing ethylene oxide, a digi-tal handheld device, Dräger X-am 5000, which turned out to be too rough (measuring range 0 - 200 ppm, resolution 0.5 ppm, smallest possible detection 1 ppm; expected concentration below) and a GC/FID. For the later the measurement was passive with ORSA-tubes filled with activated carbon. The measurement lasted for 336 hours.
Then the tubes were taken out, closed and sent to gas chromatography. The analysis was made with GC/FID with retention time catalogues and quantification via external calibration function.
This standard working instruction is a modified NIOSH 1612. Working conditions:
Gaschromato-graph “Doppelsäulengerät Sichromat 1-4”; Injek-tion: split/splitless; 2.5 μL splitted auf 2 wide-bore colums, 250°C, Colums: 30 m OPTIMA-WAX; 0.53 mm * 1.0 μm and 30 m OPTIMA 1; 0.53 mm *1.0 μm, Both detectors FID (250°C), Carrier gas Nitro-gen: 2.5 mL/min, burning gas synth. air: 300 mL/
min, Hydrogen: 25 mL/min, stove temperature 45°C isotherm 10 min, 45°C to 180°C in 5°C/
min; 180°C isotherm 13 min, 180°C to 200°C in 10°C/min, 200°C isotherm 13 min.
Results and conclusions
The books in Swidnica were fumigated with ethylene oxide in the 1990s. Nowadays, after 20 years, no ethylene oxide could be detected with the methods described above. A risk for human heath is thus rather improbable.
The results of the fungal sampling are shown in Table 1. From some samples, especially from the inner part of the books, no fungi could be isolat-ed. Only very few fungi that are directly related to the destruction of the materials where isolat-ed (Sterflinger, 2010; Sterflinger & Pinzari, 2011):
Nigrospora and Alternaria are known to degrade cellulose and Scedosporium is a keratinolytic fungus, able to degrade leather. Species of Sce-dosporium are known to be pathogenic for hu-mans, causing a skin infection called phyaeohy-phomycosis (de Hoog et al., 2000). Most abundant were species of the genus Cladosporium which were isolated from nearly all samples except for some inner parts of books. Cladosporium, how-ever, is not regarded a true contaminant of books and cannot be related to the phenomenology of the sampled areas. Cladosporium rather is an en-vironmental fungus which is extremely common in the air and spores of which settle down in dirt and dust. Thus, these fungi are isolated readily from the materials because of the spore load and not because they are actively growing and established on the books. Typical contaminants of humid paper, like species of Chaetomium or Trichoderma are missing.
As a preliminary conclusion from the project it can be stated that the fungal community isolated 15-20 years after fumigation can only to a small extend be related to the biogenic fungal phenom-ena like white and dark floccose spots and areas visible on and in the books. It can be concluded that this is due to the ethylene oxide treatment that killed especially those fungi that formed
Fig. 2: Sampling of books by cellulose nitrate membranes.
hyaline white mycelia on and in the books. Hy-line, non pigmented fungi are generally more susceptible to biocides than darkly pigmented fungi. Most of the fungi that were isolated now can either be related to air borne spores or be-long to darkly pigmented species that might have had a higher resistance against the ethylene oxide treatment. Although the amount of ethyl-ene oxide degassing from the books was under the detection level, certain prevention against re-colonization by fungi seems likely also after 15-20 years. The analysis of the non-viable fungal community based on DNA extraction could help to support this hypothesis. However, ethylene oxide fumigation might have intercalated into the DNA of the original fungal micro-flora thus hampering this type of analysis.
References
Meier, C., Petersen, K. (2006) Schimmelpil-ze auf Papier, ein Handbuch für Restaura-toren, Der andere Verlag, 198 pp.
Sterflinger, K. (2010): Fungi: Their role in the deterioration of cultural heritage.
Fungal Biology Reviews, 24, 47-55 Sterflinger, K; Pinzari, F (2012): The revenge of time: fungal deterioration of cultural heritage with particular refer-ence to books, paper and parchment.
Environ Microbiol. 14(3):559-566
de Hoog, GS; Guarro GJ, Figueras MJ (eds.) (2000) Atlas of Clinical Fungi, ASM press, 2nd edition, 1160 pp.
Acknowlegement
We kindly acknowledge the financial support of the project by EU-grant 2012 – 0920 / 001-001 (Culture Program 2007-2013, http://menandbooks.icar-us.eu/)
Notes
1 EN 1422 and EN 550, which allow the use of ethylene oxide for sterilization of certain medicine materials. Since 1981 Germany law which forbade to fumigate food with ethylene oxide. For books no regulations exist.
Authors Katja Sterflinger
Department of Biotechnology, Vienna In-stitute of Bio Technology (VIBT), Universi-ty of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria.
Katja.Sterflinger@boku.ac.at Patricia Engel
European Research Centre for Book and Paper Conservation-Restoration, Wiener Straße 2, 3 580 Horn
ercbookpaper@gmail.com
Signature of the book
description fungal isolates according to
morphological identification 00029 1 leather, inner part of back side book cover,
white fungal mycelium
Aureobasidium sp.
Cladosporium spp.
Penicillium sp.
Scedosporium sp.
2 leather, front of book cover, white floccose mycelium
Cladosporium spp.
Eurotium rubrum
00084 3 leather, inner part of book cover no cultivable fungi
4 leather, book cover no cultivable fungi
R0184 5 suede, outside of book cover, white to yellowish, granular colonies
Cladosporium spp.
Penicillium sp.
6 leather, inner side of book cover Alternaria sp.
Cladosporium sp.
Eurotium sp.
Penicillium sp.
Scedosporium sp.
7 textile, glued part inside of book, white fungal colonies
no cultivable fungi
8 paper inside, white fungal colonies Cladosporium sp.
Penicillium sp.
R0103 9 paper inside, fist page behind book cover, white fungal colonies
no cultivable fungi
02426 10 paper inside of book, dark spots Cladosporium sp.
R0176 11 paper inside of book, dark spots no cultivable fungi
12 paper, inner part of book cover Cladosporium sp.
R0232 13 paper inside of book, dark stains and colonies no cultivable fungi 14 textile, book cover, white spots and areas no cultivable fungi
15 textile, book cover, white spots Cladosporium sp.
Nigrospora sp.
Penicillium sp.
R0029 16 parchment, book cover Cladosporium sp.
Eurotium sp.
Penicillium sp.
08122 17 parchment, book spine Alternaria sp.
Cladopsorium sp.
R0103 18 textile, outside Penicillium sp.
dark pigmented fungus with meristematic growth Penicillium sp.
white sterile mycelium
01695 19 paper inside of book Cladosporium sp.
red-brown sterile mycelium reference 20 reference from sampling environment
(table in book center Horn)
Cladosporium spp.
Table 1: List of books and materials sampled for microbiological analysis with morpho-logical identification of cultivable fungi.*
* The molecular identification of all stains by sequencing of the barcode region (ITI-5.8S-ITSII) is in progress.