Evaluation of the Irradiation Treatment Effects on Ancient Parchment Samples

Round 1

Reviewer 1 Report

General comments:

The article is based on experimental research applied to the disinfection of parchment-archival assets. Specifically, the research aims to evaluate the effectiveness of ionising treatments by means of X-rays on naturally aged and contaminated ancient parchment samples. At the same time, the research intends to assess the possible negative interference of the treatment on the physical and chemical properties of the parchment substrate using various analytical diagnostic techniques.

Strengths: The article is certainly interesting and innovative in that the effects of ionising radiation are studied on naturally aged antique parchment samples. It is rarely possible to conduct such studies on such samples. Furthermore, the diagnostic methods used, with the exception of biological investigation methods, are certainly technologically advanced and informative.

Weaknesses. The article suffers from numerous weaknesses and gaps, which the authors should justify or fill up in order to make this paper publishable.

In particular, the biological investigation does not seem very adequate for the claims made in the conclusions (see specific comments), and its presentation is also unclear.

For some of the diagnostic methods presented, there is no indication of sampling points and number of replications.

It is unclear how the data were statistically processed. The article should contain a specific paragraph under 'Materials and methods' indicating the statistical methods used for the individual analyses.

As rightly noted by the authors, the inevitable inhomogeneity of the parchment material analysed makes it complicated to compare the absolute values obtained from specimens irradiated at different dosages. Indeed, for some non-destructive investigations the comparison between treated and untreated is correctly made on the same sample before and after ionising treatment at the different dosages, thus obtaining for each dose a relative difference to the control. For other analyses, as well non-destructive, however, observations are instead made by comparing absolute values. The authors should justify this choice.

Finally, the authors should indicate in more detail the strengths and weaknesses of the use of X-ray radiation for the sanitisation of archival-library assets, what the possible practical uses of the application might be, the industrial spin-offs and the possible problems associated with its massive application.

 

Specific comments

Line 35: What do you mean for bio-repairing. It doesn’t seem an appropriate term

Line 61. Could you briefly inform the readers which are the most probable biocidal mechanisms induced by X-rays against biodeteriogens and related bibliographic references?

Line 62: Here and below the term “bioremediation” appears inappropriate. Bioremediation is a biotechnology technique employed for restoring or reclaming an environmental matrix using living microbial cells.

Line 75: Among others, one could mention the study of the effect of the treatment on cellulosic material (which makes up the larger part of the archival-library assets) and above all the industrial scale-up to make it truly a system suitable for mass treatment: in particular, studies should investigate the conditions of the material to be treated (water content, possible preliminary dedusting), the way the material is prepared, the operating conditions during treatment (i.e. thermo-hygrometric conditions) and logistical aspects.   

Line 80-82. Not clear. Check English

Line 92: Not "all samples" since the single specimens have been irradiated with a single dose and 0 is not a dose. I'd rather say: "Samples have been irradiated with increasing X-rays doses (350 - 4000 Gy)

Line 133: please refer to your previous publication “The REX irradiation facility and its applications M. Vadrucci, F. Borgognoni, L. Campani, P. Ferrari”, for a better comprehension of the procedures.

Line 133: The reader should better understand how the samples were irradiated, at what distance, under what environmental conditions, at what water content. If they underwent any pre-treatment (dusting, drying...). If, as I think, the samples were treated individually on a single plane, to justify its application at a mass level (as you state several times in the paper), you should also explain (here or in the conclusions) how a mass treatment with this technology could be conducted on archival-library items, often characterised by huge volumes: at what depth does the radiation act? How should a pallet of material be prepared and treated?

Line 142: figures with bad definition

Line 146: Didn't you homogenize in some way the specimens?

Line 148: isolated, not purified

Line 160: use the complete term: phenol-chlorophorm-Iso amyl alcohol

Line 172: not clear at all. What are you exactly treating? Parchment contaminated samples, unknown microbial colonies growing on agar plates or single microbial isolates? If the latter, which species are you treating? How many 1x1 cm portions are you treating at once? Do you have replicates?

Line 176: Despite the lack of clarity on the method used, it appears that ionising treatment was conducted on unspecified microbial colonies growing on agar. Does this methodological choice have bibliographical references? The different biocide resistance of micro-organisms in vivo and in vitro is well known in the literature. Micro-organisms are often more sensitive to treatments when grown on culture media than when treated directly in their natural environment. The authors must justify why biocidal efficacy was not assessed directly on parchment samples. If for contingent reasons in vivo evaluation was not possible (lack of material?), the possible discrepancy between the results obtained in vitro versus in vivo must be emphasised in the conclusions and therefore reporting the efficacy data as preliminary results for future research to be conducted in vivo.

Line 186: how many spots did you measure for each specimen? Are the spectra an average of more readings or just a single spot observation? If so, could you really be confident with the obtained results considering the high degree of variation of the sample substrate?

Line 187: I guess you mean natural aging?

Line 195: As done correctly later for LTA and FORS (but not for ATM) the high degree of variability among the samples suggest to compare the effect of the different dose of irradiation on the same sample, before and after irradiation (relative changes). Could you explain why for FTIR and AFM you didn’t observe the data in the same way?

Line 197: Use in the title the entire term: UV resonant Raman spectroscopy

Line 220: How did you obtain this fibre suspension from your samples?

Line 231: How many measures did you perform? Is the spectra obtained an average of more measurements?

Line 247: Report the number of replicates per sample

Line 261: see what said for FTIR (line 195)

Line 266-268: Sorry, this sentence is not clear to me. Could you explain better the relation among cellulolytic microorganisms and parchment degradation?

Line 298: Ok, variations are not linear but apart for the lower dosage, the ratio-intensities values decrease after irradiation appear statistically significant. You should take it in account.

Line 430-437: As told before is better to refer to these data as preliminary results, since the evaluation should be confirmed directly on parchment on in vivo test

Line 442: LTA deals with relative changes on the same sample, thus in this case inhomogeneity shouldn't be a major concern.

Line 453-455: I get lost! Aren’t the data obtained on the same sample before and after irradiation? So, why inhomogeneity should be a problem?

Line 475: Please illustrate better the weak points of the other radiation techniques respect to X ray.

By the way, could you also inform the readers about any limitations on the use of X-ray, as for example regarding costs, duration of treatment, preparation of material before irradiation (i.e. necessity to treat at a defined water content, necessity to dedust before treatement). Could you also inform if in Europe or others Country do already exist X-ray irradiation facilities intended for a mass disinfection of paper materials at an industrial level? If not, which could be the reasons and limitations?

Author Response

Dear Editor,

we are grateful for the reviewers comments concerning the article “Evaluation of the irradiation treatment effects on ancient parchment samples” by Vadrucci et al., which will help make clearer the contents of the paper.

We have accordingly made all the required changes to the manuscript to address the raised issues. In the following we reply to the reviewers comments reporting, where necessary, the changes made in the manuscript.

We hope we have been exhaustive and detailed in our replies to the valuable comments of the reviewers.

General remarks:

-the text has been revised by an english native speaker;

-the number of references judged necessary has been increased and the number of self-citations reduced.

Following are the replies to each of the reviewers’ comments.

Reviewer 1

General comments:

The article is based on experimental research applied to the disinfection of parchment-archival assets. Specifically, the research aims to evaluate the effectiveness of ionising treatments by means of X-rays on naturally aged and contaminated ancient parchment samples. At the same time, the research intends to assess the possible negative interference of the treatment on the physical and chemical properties of the parchment substrate using various analytical diagnostic techniques.

Strengths: The article is certainly interesting and innovative in that the effects of ionising radiation are studied on naturally aged antique parchment samples. It is rarely possible to conduct such studies  on such samples. Furthermore, the diagnostic methods used, with the exception of biological investigation methods, are certainly technologically advanced and informative.

Weaknesses. The article suffers from numerous weaknesses and gaps, which the authors should justify or fill up in order to make this paper publishable.

In particular, the biological investigation does not seem very adequate for the claims made in the conclusions (see specific comments), and its presentation is also unclear.

For some of the diagnostic methods presented, there is no indication of sampling points and number of replications.

More information has been added in the paragraph dedicated to each technique.

It is unclear how the data were statistically processed. The article should contain a specific paragraph under 'Materials and methods' indicating the statistical methods used for the individual analyses.

More information has been added in the paragraph dedicated to each technique. 

As rightly noted by the authors, the inevitable inhomogeneity of the parchment material analysed makes it complicated to compare the absolute values obtained from specimens irradiated at different dosages. Indeed, for some non-destructive investigations the comparison between treated and untreated is correctly made on the same sample before and after ionising treatment at the different dosages, thus obtaining for each dose a relative difference to the control. For other analyses, as well non-destructive, however, observations are instead made by comparing absolute values. The authors should justify this choice.

It is now indicated in lines 105-124 the introduction that ”Based on the results of the previous investigations carried out in samples of modern parchment which had been irradiated and artificially aged it turned out that only Light Transmission Analysis (LTA)  proved sensitive enough to detect not only the changes induced in the parchment collagen stability by the artificial ageing treatments but also those associated with the different degree of irradiation damage, while the other employed techniques (Attenuated Total Reflectance-Fourier Transformed Infrared (ATR-FTIR) spectroscopy and Reflectance Spectroscopy) were only able to account for the ageing effects. So in this work the effects of the various irradiation doses on the naturally aged parchment is analyzed primarily by LTA which also was enabled to detect the effects of aging. The other techniques, listed in the following, were used to investigate mainly the deterioration effects associated with the ageing. Fiber Optic Reflectance Spectroscopy (FORS) has been used to evaluate macroscopic alteration in the reflectance properties of the sample surface induced by the deterioration processes ATR-FTIR spectroscopy has been used to characterize the changes induced on the collagen protein. The UV Resonant Raman (UVRR) spectroscopy at an excitation wavelength of 213 nm has been used in order to evaluate the effect of the natural aging on the collagen proteins. Finally, the Atomic Force Microscopy (AFM) mechanical analysis was applied to evaluate  the changes induced in the elastic properties of the parchment, in particular in its stiffness”

Also in the results section, lines 380-386 it is stated that “As stated earlier on, the ATR-FTIR analysis was carried out to characterize mainly the effects of natural aging of the samples, and, since no additional specific deterioration effects associated with the X-ray irradiation alone could be detected by the previously reported analysis, the characterization  was carried out only in the samples after the irradiation treatment to highlight possible phenomena of hydrolysis and/or denaturation associated with the aging. As shown in Table 1, the variation of values of the intensity ratio of the Amide I and Amide II bands do not follow the irradiation dose trend.”

Finally, the authors should indicate in more detail the strengths and weaknesses of the use of X-ray radiation for the sanitisation of archival-library assets, what the possible practical uses of the application might be, the industrial spin-offs and the possible problems associated with its massive application.

The discussion paragraph has been improved as suggested (lines 523-552) and, moreover, a paragraph with conclusions and perspective (lines 554-580) has been added in order to describe the advantages that this kind of disinfection treatment offers and the possible future application and developments of the proposed method.

Specific comments

Line 35: What do you mean for bio-repairing. It doesn’t seem an appropriate term

We thank the Reviewer for the suggestion. The term has been change with “disinfection”.

Line 61. Could you briefly inform the readers which are the most probable biocidal mechanisms induced by X-rays against biodeteriogens and related bibliographic references?

We thank the Reviewer for the suggestion. The information has been added in the Introduction section, lines 61-66.

Line 62: Here and below the term “bioremediation” appears inappropriate. Bioremediation is a biotechnology technique employed for restoring or reclaiming an environmental matrix using living microbial cells.

We thank the Reviewer for the suggestion. The term has been change with “disinfection”.

Line 75: Among others, one could mention the study of the effect of the treatment on cellulosic material (which makes up the larger part of the archival-library assets) and above all the industrial scale-up to make it truly a system suitable for mass treatment: in particular, studies should investigate the conditions of the material to be treated (water content, possible preliminary dedusting), the way the material is prepared, the operating conditions during treatment (i.e. thermo-hygrometric conditions) and logistical aspects.   

Regarding the collagen-based materials, the object of our research, the required information has been added in the introduction section that has been improved and revised accordingly.

Line 80-82. Not clear. Check English

The paragraph, and the entire text, has been revised by a native english speaker

Line 92: Not "all samples" since the single specimens have been irradiated with a single dose and 0 is not a dose. I'd rather say: "Samples have been irradiated with increasing X-rays doses (350 - 4000 Gy)

We thank the Reviewer for the suggestion. The text has been corrected following the suggestion.

Line 133: please refer to your previous publication “The REX irradiation facility and its applications M. Vadrucci, F. Borgognoni, L. Campani, P. Ferrari”, for a better comprehension of the procedures.

The suggested reference has been added.

Line 133: The reader should better understand how the samples were irradiated, at what distance, under what environmental conditions, at what water content. If they underwent any pre-treatment (dusting, drying...). If, as I think, the samples were treated individually on a single plane, to justify its application at a mass level (as you state several times in the paper), you should also explain (here or in the conclusions) how a mass treatment with this technology could be conducted on archival-library items, often characterised by huge volumes: at what depth does the radiation act? How should a pallet of material be prepared and treated?

According to the suggestion of the reviewer, the requested information about the experimental irradiation procedure has been added in the paragraph (2. Materials and methods - 2.2. The disinfection by irradiation), lines 195-204.

Line 142: figures with bad definition

The figure has been replaced with a higher resolution one and its legend has been modified accordingly.

Line 146: Didn't you homogenize in some way the specimens?

The specimens were shaken by a vortex for 2 min. The information has been added in the Material and Methods section (2. Materials and Methods - 2.1. Bacterial and fungal strains identification), lines 139-140.

Line 148: isolated, not purified

The text has been modified according to the Reviewer’s suggestion. (Line 141)

Line 160: use the complete term: phenol-chlorophorm-Iso amyl alcohol

The text has been modified according to the Reviewer’s suggestion. (Line 153)

Line 172: not clear at all. What are you exactly treating? Parchment contaminated samples, unknown microbial colonies growing on agar plates or single microbial isolates? If the latter, which species are you treating? How many 1x1 cm portions are you treating at once? Do you have replicates?

We apologise for the lack of information.  For irradiation treatments, 1 × 1 cm² portions of NB agar plates covered with bacterial and fungal colonies after biodeteriogens isolation, were cut and placed in 3.5 cm Petri plates for subsequent irradiation tests. The text has been modified accordingly. (Lines 168-170)

Line 176: Despite the lack of clarity on the method used, it appears that ionising treatment was conducted on unspecified microbial colonies growing on agar. Does this methodological choice have bibliographical references? The different biocide resistance of micro-organisms in vivo and in vitro is well known in the literature. Micro-organisms are often more sensitive to treatments when grown on culture media than when treated directly in their natural environment. The authors must justify why biocidal efficacy was not assessed directly on parchment samples. If for contingent reasons in vivo evaluation was not possible (lack of material?), the possible discrepancy between the results obtained in vitro versus in vivo must be emphasised in the conclusions and therefore reporting the efficacy data as preliminary results for future research to be conducted in vivo.

We agree with the Reviewer that biodeteriogens can be more sensitive to treatments in lab conditions than when treated directly in their natural environment. In this study, X-ray efficacy was not assessed directly on parchment samples, first of all to verify that the radiation could not further damage the document dating back to the 16th century. The microorganisms’ isolation allows identifying five bacterial and three fungal species, which have colonised agar plates used during isolation in lab conditions. The same plates have been used for irradiation treatment, so in  vitro assays, X-ray results were efficient against the microbial community identified. As suggested by the Reviewer, in the Conclusion section the possible discrepancy between the results obtained in vitro versus in vivo have been emphasised. However, more information about methods used have been provided in the Materials and Methods section (lines 205-212).

Line 186: how many spots did you measure for each specimen? Are the spectra an average of more readings or just a single spot observation? If so, could you really be confident with the obtained results considering the high degree of variation of the sample substrate?

We apologise for the lack of clarity. We have added more information as requested. (Lines 233-234)

Line 187: I guess you mean natural aging?

We apologise for the lack of clarity. The sentence has been changed as requested. (Line 264)

Line 195: As done correctly later for LTA and FORS (but not for ATM) the high degree of variability among the samples suggest to compare the effect of the different dose of irradiation on the same sample, before and after irradiation (relative changes). Could you explain why for FTIR and AFM you didn’t observe the data in the same way?

See the reply to the general comment of reviewer 1.

Line 197: Use in the title the entire term: UV resonant Raman spectroscopy

 

The title has been corrected as suggested.

 

Line 220: How did you obtain this fibre suspension from your samples?

 

We apologise for the lack of information.  The description of the procedure to obtain the suspension of fibres has been added in the text at paragraph 2.5. LTA (Lines 218-219)

 

Line 231: How many measures did you perform? Is the spectra obtained an average of more measurements?

 

We apologise for the lack of information.  In this work, the reported T_d values are obtained as an average of three different measurements. The sentence has been added to the text as suggested. (2.5. LTA, lines 233-234)

 

Line 247: Report the number of replicates per sample

 

We apologise for the lack of information. In this work, the reflectance spectra have been obtained by averaging the data from 5 measurements. The sentence has been added to the text as suggested. (2.6. FORS, lines 241-242).

 

Line 261: see what said for FTIR (line 195)

See the reply to the general comment of reviewer 1.

Line 266-268: Sorry, this sentence is not clear to me. Could you explain better the relation among cellulolytic microorganisms and parchment degradation?

 

We apologise for the lack of clarity. Cellulolytic enzymes refer to the metabolism of microorganisms involved in paper and book biodeterioration, while parchment damage is associated with collagen degradation by bioteteriogens’ collagenases. The concept has been explained more clearly in the Introduction section (Lines 86-92).

 

Line 298: Ok, variations are not linear but apart for the lower dosage, the ratio-intensities values decrease after irradiation appear statistically significant. You should take it into account.

 

Authors thank the reviewer for the clarification. The variation of the ratio between the intensities of the bands relating to Amides I and II are not significant since the experimental error associated with each measurement must be considered, which is better highlighted in the histogram in the attached file.

Figure 1. FTIR data of the ratio between the intensities of the Amide I and II bands as a function of X-ray dose. Data are normalized with respect to non irradiated one.

Line 430-437: As told before is better to refer to these data as preliminary results, since the evaluation should be confirmed directly on parchment on in vivo test.

We thank the Reviewer for the suggestion. In the conclusion section, the efficacy data have been reported as preliminary results, useful for future research to be conducted in vivo (lines 560-569).

Line 442: LTA deals with relative changes on the same sample, thus in this case inhomogeneity shouldn't be a major concern.

 

That is correct. Actually, the aim of the sentence was to highlight that, despite of their uneven initial state of preservation, in all the samples “... the natural deterioration associated with hygrothermal and bioagents agents effects overshadowed the damage related to the X-ray irradiation process”. This is now introduced in the text (line 480-490).

 

Line 453-455: I get lost! Aren’t the data obtained on the same sample before and after irradiation? So, why inhomogeneity should be a problem?

 

Inhomogeneity could be a problem when one wants to compare results from different samples. We didn’t do it and this has been now clarified in the text (lines 486-490): “In fact, the different samples showed intrinsic deterioration inhomogeneity despite having been retrieved from neighbourong areas, no correlation being found between the absolute Td values and the applied X-ray dose, and no change could be found in the Td values obtained  on each sample before and after the respective  irradiation treatment, even at the largest employed dose.”

 

Line 475: Please illustrate better the weak points of the other radiation techniques respect to X ray.

By the way, could you also inform the readers about any limitations on the use of X-ray, as for example regarding costs, duration of treatment, preparation of material before irradiation (i.e. necessity to treat at a defined water content, necessity to dedust before treatment). Could you also inform if in Europe or others Country do already exist X-ray irradiation facilities intended for a mass disinfection of paper materials at an industrial level? If not, which could be the reasons and limitations?

The discussion paragraph has been improved as suggested and, moreover, a paragraph with conclusions and perspective has been added in order to describe the advantages that this kind of disinfection treatment offers and the possible future application and developments of the proposed method. (Lines 523-552 and lines 570-580)  

Author Response File: Author Response.docx

Reviewer 2 Report

The authors executed a straightforward study on evaluating irradiation effects on aged parchment. This is a good reference paper based on the detail paid to sample prep and the different methods attempted to evaluate different features. However, the paper is rather cursory in explaining background and experimental details, often relying on references (even though ¼ of the references are the authors’ papers, that is not acceptable). Useful background or details have also been found in sections or in an order that doesn’t sit well with the flow of the paper. The authors might consider the comments in the detailed bulleted list for this.

 

Additionally, this is a study on one historical parchment. Not finding radiation effects does not strongly suggest irradiation does not have an impact. As an outsider to this topic, the reviewer would not be convinced on these results. There are many variables (the authors even indicated the inhomogeneity of this one object) and it does not seem appropriate that the next step is to begin irradiation on archival samples, but perhaps instead to test different types of parchment at different states of conservation, and to test the limits of modern prepped samples. The authors should reconsider the impact of their study and new tests in this line of research in their discussion/conclusion. Perhaps it might be useful to talk about developing reliable protocols in evaluating parchment/paper condition and testing and how their work follows it.

Comments for author File: Comments.pdf

Author Response

Dear Editor,

we are grateful for the reviewers comments concerning the article “Evaluation of the irradiation treatment effects on ancient parchment samples” by Vadrucci et al., which will help make clearer the contents of the paper.

We have accordingly made all the required changes to the manuscript to address the raised issues. In the following we reply to the reviewers comments reporting, where necessary, the changes made in the manuscript.

We hope we have been exhaustive and detailed in our replies to the valuable comments of the reviewers.

General remarks:

-the text has been revised by an english native speaker;

-the number of references judged necessary has been increased and the number of self-citations reduced.

Following are the replies to each of the reviewers’ comments.

Reviewer 2

The authors executed a straightforward study on evaluating irradiation effects on aged parchment. This is a good reference paper based on the detail paid to sample prep and the different methods attempted to evaluate different features. However, the paper is rather cursory in explaining background and experimental details, often relying on references (even though ¼ of the references are the authors’ papers, that is not acceptable). Useful background or details have also been found in sections or in an order that doesn’t sit well with the flow of the paper. The authors might consider the comments in the detailed bulleted list for this.

 

Additionally, this is a study on one historical parchment. Not finding radiation effects does not strongly suggest irradiation does not have an impact. As an outsider to this topic, the reviewer would not be convinced on these results. There are many variables (the authors even indicated the inhomogeneity of this one object) and it does not seem appropriate that the next step is to begin irradiation on archival samples, but perhaps instead to test different types of parchment at different states of conservation, and to test the limits of modern prepped samples. The authors should reconsider the impact of their study and new tests in this line of research in their discussion/conclusion. Perhaps it might be useful to talk about developing reliable protocols in evaluating parchment/paper condition and testing and how their work follows it.

 

• 33 – the damage is due to conservation treatment or natural phenomena? Not all parchment damage is anthropogenic. This should be discussed more clearly.

 

Thanks for the suggestion. The paragraph has been revised accordingly (lines 29-31).

 

• 45 – incomplete sentence: “as all the collagen-based materials eventually exhibit (?)…”

 

Thanks for the suggestion. The paragraph has been revised (lines 43-44).

 

• It would be useful to clearly lay out the molecules and features that are looked at in the parchment constituents to determine types of degradation, which degradation effects are acceptable, if any, and which specific structures are being targeted in the evaluation techniques. I think the intro is a bit vague in this regard.

 

The introduction section has been revised and improved according to the reviewers suggestions (in particular lines 104-124).

 

• Place UVRR before spectroscopy

 

The text has been corrected according to the suggestion

 

• 110 – lowercase b in bioremediation

 

The term has been replaced according to the suggestion of the reviewer 1

 

• 112 – collagen-based

 

The suggestion has been accepted

 

• 116 – really long sentence. Best to break up. Line 121 may be best to preface it.

The paragraph, and the entire text, has been revised by a native speaker.

• 173 – petri

 

The text has been modified accordingly.

 

• 185 – Full width at half maximum (FWHM) is the conventional term. It is the reviewer’s opinion that would be better to replace FWHH with that.

 

The text has been modified accordingly (lines 261-262).

 

• 209 – HV?

 

HV is a common way in Raman spectroscopy to indicate a specific geometrical configuration in which the collected diffused radiation is polarised perpendicular to the incident one (see chapter 1 and 2 of https://doi.org/10.1016/B978-0-12-254105-6.X5000-8). To better explain this point we amended the text as follows, with the addition of the new citation: “To remove the elastic contribution an HV (horizontal-vertical) geometrical configuration has been adopted, i.e. it has been selectively collected only the diffused radiation with polarization perpendicular (horizontal with respect to  the optical plane) to the incident one (vertical with respect to the optical plane) [https://doi.org/10.1016/B978-0-12-254105-6.X5000-8]” (lines 292-295).

 

• 213 – 60 min/ sample is not a count rate. It’s a cumulative data acquisition time

 

We modify the text in agreement with the reviewer indication (line 297).

 

• 220 – Td – please change throughout paper

 

Td has been replaced with T_d in the text

 

• 220 – 0,1 to 0.1

 

The text has been corrected (line 219)

 

• 233 – at this point it’s unclear how FORS is actually useful here. Up to 1100 nm, you are just focusing on color measurements? Why not just use a colorimeter? What other features might you need otherwise? FORS up to 2500 nm would actually provide molecular information.

 

In this work FORS analyses were performed to highlight possible effects of irradiation on chromatism and brightness. Given the substantially observed invariability of the spectra obtained on the samples after the irradiation, which induced no significant change in the colorimetric  coordinates  it was decided not to present the colorimetric data.

Spectroscopic analysis in the IR at longer wavelengths have been performed by means of  ATR-FTIR spectroscopy which results to be much more sensitive and accurate.

For clarity, the information has been added to the text (lines 350-253)

 

• 244 - what are these other measurements? Are they relevant to this paper?

 

The sentence has been corrected in order to not be misleading (lines 250-253).

 

• 259 – please remove the parentheses. It’s an entire line on its own.

 

The text has been corrected (lines 309-311).

 

• Section 2.1 should probably come immediately after the sample prep discussion, and the conditions under which the samples were irradiated should be listed in this section.

 

We thank the Reviewer for the suggestion. The paragraphs have been modified accordingly.

 

• 271 – how is mortality determined? We eventually see in Figure 3 that CFU counting analysis (whatever this means) was used.

 

We apologise for the lack of clarity. As specified in the Material and Methods section, colonies obtained were counted by colony forming units (CFU) method, measuring the capacity of a single viable cell to form a colony. The test measured the survival rate of biodeteriogens after irradiation, compared to the untreated control (lines 205-212).

 

• Information in paragraph starting with line 282 is not relevant to the results. It’s general information that is better suited for the introduction, which it does need.

 

We thank the Reviewer for the suggestion. The paragraph has been removed from the results section and, in a revised form, added in the introduction section (lines 86-92).

 

• 297 – why is the ratio of value? Previously it was only discussed that each band was evaluated to look at different structures – reading further it is described in 304 but a lot of this information should not be mentioned earlier on to justify why you are doing certain analyses.

 

We apologise for the lack of clarity. We have added information about the ratio between the vibrational band intensities (I_AmideI/I_AmideII) also in materials and methods section to facilitate the understanding of the results (L 273-279).

 

• Figure 4 – the bands should be marked and this figure doesn’t clearly show the effects of natural aging as there is no reference for that.

We apologise for the lack of clarity. The figure has been modified accordingly.

• 346/353 – please be consistent when referring to labels such as Amides (Amide I vs Amide-I vs AmI)

 

We apologise to the reviewer. The terms have been corrected in the text so that they now result to be consistent.

 

• Figure 5 – what does it mean that many of the aromatic amino acid bands are still present?

 

We apologise for the lack of clarity. The required information and the relative references have been added in the text (lines 435-437).

 

• Figure 6 – Would a plot of Td before and after irradiation on a modern prepared sample, unaged, be a useful comparison to justify the statement made in L381?

 

The sentence finds its justification in the results of previous measurement campaigns dedicated to the characterization of the damage induced by radiation on samples of modern parchment. The denaturation temperatures obtained on samples of modern parchment treated with the same protocol and at the same doses of radiation with which the present work was performed are available in the cited references

[https://doi.org/10.1016/j.apradiso.2019.04.021; https://doi.org/10.1140/epjp/s13360-021-01766-5].

 

• 386 – FORS is not a macroscopic investigation tool It is one that is used to look at molecular structure, electronic transitions, and vibrational absorptions. All that seems to be said here is that the overall reflectance has not changed, which means there hasn’t been much color change.

 

In this work, the FORS analysis was mainly employed in order to evaluate chromatic effects of alteration due to the irradiation treatment. For the analysis of the molecular alterations FITR-ATR spectroscopy was used, which certainly shows greater sensitivity. As far as the FORS analysis is concerned, we can certainly say that in the VIS-IR we do not observe spectral variations, i.e. molecular alterations. In particular, in the VIS we do not observe spectrum alterations associated with yellowing and loss of brightness phenomena.

 

• 423 – this line sounds confusing – hardly?

The paragraph, and the entire text, has been revised by a native speaker.

• Would it not be useful to find the dosage and exposure time that would cause a change in the Young’s modulus in the modern sample?

 

The objective of this work is to identify the radiation dose threshold that disinfects the artefact without further damaging it. The change in the Young's modulus in the modern sample, thus not deteriorated by time and storage conditions, was obtained with the measures of a dedicated work (Vadrucci, M.; Cicero, C.; Parisse, P.; Casalis, L.; De Bellis, G. Surface evaluation of the effect of X-rays irradiation on parchment artefacts through AFM and SEM. Appl. Surf. Sci. 2020, 513. https://doi.org/10.1016/j.apsusc.2020.145881).

 

• It is the opinion of the reviewer that the discussion should be called a conclusion

According to the reviewers 1 and 2 suggestions, a paragraph with conclusions and perspective has been added in order to describe the advantages that this kind of disinfection treatment offers and the possible future application and developments of the proposed method.

Author Response File: Author Response.docx

Reviewer 3 Report

This is an interesting and important contribution to the literature on the conservation of medieval manuscripts.  The results are also relevant to other types of analysis of manuscripts that utilize ionizing radiation, such as x-ray imaging.

 

-          the manuscript would benefit from a close edit by a native English speaker. 

o   line 43 and 424 – “pelt” should be “skin” (a pelt includes skin and hair)

o   “X-rays radiation” should be “X-ray radiation” throughout

o   the sentence from line 80 to 83 is awkward

-          last paragraph of the first section

o   it would be good to indicate here why these particular analytical methods were chosen, and what damage to the parchment would look like in each case. 

-          line 126 – “other ionizing radiation sources”… such as?

-          line 190 - “commonly used in conformational protein studies”… for example?

-          section 2.4 – could background fluorescence contribute to the spectra?

-          line 219 – should “peculiar” be “particular”?

-          line 222 – “sample goes to” should probably be “sample goes from”

-          section 3.1 – are these species of bacteria and fungi expected?

o   the irradiation doses of hundreds of Gray.. most sources suggest that a dosage of 8+ Grays is fatal to humans.  Is it any surprise that these doses are lethal to the bacteria and fungi? 

-          line 282 – “undesirable and irreversible damage”… it would be helpful to have this in the introduction along with a clear indication of what “damage” is (see the comment above re the analytical methods and expected results)

-          paragraph starting line 294 - is the random trend expected?

-          line 312 – “du”  should be “due”

-          line 321 – considering the fact that the authors only looked at naturally aged samples, how can they make this claim?

-          paragraph starting on line 364 – does the inhomogeneity noted in the sample here have implications for the other analytical methods and the presentation of their results in absolute values?  This might be relevant to the unexpected results in section 3.6?  This is noted with regard to the FTIR results in the paragraph starting in line 438.  Perhaps this should be more explicitly discussed throughout.  And would the results presented in Table 4 and elsewhere be improved by taking more readings?

-          line 393 – samples “before” the radiation – should this not be “after” the radiation?

 

Author Response

Dear Editor,

we are grateful for the reviewers comments concerning the article “Evaluation of the irradiation treatment effects on ancient parchment samples” by Vadrucci et al., which will help make clearer the contents of the paper.

We have accordingly made all the required changes to the manuscript to address the raised issues. In the following we reply to the reviewers comments reporting, where necessary, the changes made in the manuscript.

We hope we have been exhaustive and detailed in our replies to the valuable comments of the reviewers.

General remarks:

-the text has been revised by an english native speaker;

-the number of references judged necessary has been increased and the number of self-citations reduced.

Following are the replies to each of the reviewers’ comments.

Reviewer 3

This is an interesting and important contribution to the literature on the conservation of medieval manuscripts.  The results are also relevant to other types of analysis of manuscripts that utilize ionizing radiation, such as x-ray imaging.

 -          the manuscript would benefit from a close edit by a native English speaker. 

 The manuscript has been revised by a native speaker.

o   line 43 and 424 – “pelt” should be “skin” (a pelt includes skin and hair)

We thank the Reviewer for the suggestion. The term has been replaced as suggested.

o   “X-rays radiation” should be “X-ray radiation” throughout

We apologise to the reviewer. The terms have been corrected

 

o   the sentence from line 80 to 83 is awkward

The paragraph, and the entire text, has been revised by a native speaker

-          last paragraph of the first section

o   it would be good to indicate here why these particular analytical methods were chosen, and what damage to the parchment would look like in each case. 

The introduction section has been revised and improved according to the reviewers suggestions (lines 104-124)

-          line 126 – “other ionizing radiation sources”… such as?

The reference to other ionizing radiation sources (i.e. machines that deliver directly with accelerated particles or the radioactive sources) has been added in the text (lines 188-189).

-          line 190 - “commonly used in conformational protein studies”… for example?

Thanks to the reviewer for the question. Examples of conformational studies are: monitoring protein denaturation and/or aggregation. Some references have been added in the text to answer the question. (Nebojsa, P.; Nils, K.A.; Ragni, O.; Achim, K. Monitoring Protein Structural Changes and Hydration in Bovine Meat Tissue Due to Salt Substitutes by Fourier Transform Infrared (FTIR) Microspectroscopy. J. Agric. Food Chem. 2011, 59, 10052-10061. https://doi.org/10.1021/jf201578b ; Shivu, B.; Seshadri, S.; Li, J.; Oberg, K.A.; Uversky, V.N.; Fink, A.L. Distinct β-Sheet Structure in Protein Aggregates Determined by ATR–FTIR Spectroscopy.  Biochemistry 2013, 52, 5176-5183. https://doi.org/10.1021/bi400625v.) (line 268)

-          section 2.4 – could background fluorescence contribute to the spectra?

The missing of fluorescence contribution on the Raman spectra collected in the deep UV it is actually well known (see for example:        Asher, S.A.; Johnson, C.R. Raman Spectroscopy of a Coal Liquid Shows That Fluorescence Interference Is Minimized with Ultraviolet Excitation. Sci. 1984, 225, 311-313DOI: 10.1126/science.6740313). Employing an excitation wavelength of 213 nm, the UVRR spectra shown in the manuscript falls in the wavelength range of 217-222 nm, therefore far from the typical fluorescence spectral region of biomolecules. For clarity, the reference has been added to the text (line 415).

-          line 219 – should “peculiar” be “particular”?

The text has been corrected.

-          line 222 – “sample goes to” should probably be “sample goes from”

The text has been corrected.

-          section 3.1 – are these species of bacteria and fungi expected?

As highlighted in the text and supported by the literature, isolated bacterial and fungal species are known to be involved in parchment biodeterioration (lines 331-332)

o   the irradiation doses of hundreds of Gray.. most sources suggest that a dosage of 8+ Grays is fatal to humans.  Is it any surprise that these doses are lethal to the bacteria and fungi? 

Thanks to the reviewer for the question. The cellular inactivation of microorganisms is more complex because they have a different cellular duplication process which is instead more rapidly inhibited in the tissue of animal species by radiation. Moreover, they are characterised by the presence of a cell wall, which is not present in animal cells. This is the reason for the higher dose needed.

-          line 282 – “undesirable and irreversible damage”… it would be helpful to have this in the introduction along with a clear indication of what “damage” is (see the comment above re the analytical methods and expected results)

We thank the Reviewer for the suggestion. The paragraph has been moved to the introduction and it has been modified accordingly (lines 86-88).

-          paragraph starting line 294 - is the random trend expected?

We apologise for the lack of clarity. Yes, the random trend of this parameter is expected for very heterogeneous samples, such as naturally aged ones, because of the inner complexity of the deterioration processes.

-          line 312 – “du”  should be “due”

The entire paragraph has been revised according to the suggestions of reviewer 1.

-          line 321 – considering the fact that the authors only looked at naturally aged samples, how can they make this claim?

The authors apologize to the reviewer. We have removed the sentence.

-          paragraph starting on line 364 – does the inhomogeneity noted in the sample here have implications for the other analytical methods and the presentation of their results in absolute values?  This might be relevant to the unexpected results in section 3.6?  This is noted with regard to the FTIR results in the paragraph starting in line 438.  Perhaps this should be more explicitly discussed throughout.  And would the results presented in Table 4 and elsewhere be improved by taking more readings?

The entire paragraph has been revised according to the suggestions of reviewer 1. In the following, the reply to the comment of reviewer 1:

It is now indicated in lines 105-124 the introduction that ”Based on the results of the previous investigations, carried out in samples of modern parchment which had been irradiated and artificially aged, it turned out that only Light Transmission Analysis (LTA)  proved sensitive enough to detect not only the changes induced in the parchment collagen stability by the artificial ageing treatments, but also those associated with the different degree of irradiation damage; the other employed techniques (Attenuated Total Reflectance-Fourier Transformed Infrared (ATR-FTIR) spectroscopy and Reflectance Spectroscopy) were only able to account for the ageing effects. So in this work, the effects of the various irradiation doses on the naturally aged parchment is analyzed primarily by LTA which also was enabled to detect the effects of aging. The other techniques, listed in the following, were used to investigate mainly the deterioration effects associated with the ageing. Fiber Optic Reflectance Spectroscopy (FORS) has been used to evaluate macroscopic alteration in the reflectance properties of the sample surface induced by the deterioration processes ATR-FTIR spectroscopy has been used to characterize the changes induced on the collagen protein. The UV Resonant Raman (UVRR) spectroscopy at an excitation wavelength of 213 nm has been used in order to evaluate the effect of the natural aging on the collagen proteins. Finally, the Atomic Force Microscopy (AFM) mechanical analysis was applied to evaluate  the changes induced in the elastic properties of the parchment, in particular in its stiffness”

Also in the results section, lines 380-386 it is stated that “As stated earlier on, the ATR-FTIR analysis was carried out to characterize mainly the effects of natural aging of the samples, and, since no additional specific deterioration effects associated with the X-ray irradiation alone could be detected by the previously reported analysis, the characterization  was carried out only in the samples after the irradiation treatment to highlight possible phenomena of hydrolysis and/or denaturation associated with the aging. As shown in Table 1, the variation of values of the intensity ratio of the Amide I and Amide II bands do not follow the  irradiation dose trend. “

-          line 393 – samples “before” the radiation – should this not be “after” the radiation?

The text has been corrected.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

General comment:

After the first review, the authors have clarified some obscure aspects and provided some answers. However, there are still some unresolved points, which will be indicated in this second revision point by point. In general, the conclusions still seem too trenchant and sometimes unjustified. In particular, the statement regarding the application of this technology as a mass treatment is disputed: the data provided are in fact still too preliminary and not sufficient to suggest an application in this sense. Furthermore, in general it seems that the authors are not completely informed on the state of the art concerning the specific sector of biodeterioration of cultural heritage.

 

Specific comments

Line 51: in Italy and other European countries disinfection of cultural assets with ethylene oxide is still permitted

Line 66: substitute bioremediation with disinfection

Line 69-75: these sentences are not clear: is there any synergic effect among aging and irradiation? Please be more clear.

Line 90: In fact the more frequent colonizers of ancient parchment are actinomycetes (halophilc actinomycets in particular). Native collagen is degradate by collagenases produced by a very low amount of bacterial species, such as Clostridium in anerobiosis. It is not clear if fungi are really able to produce collagenases (see for example https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5220638/). It is more probable that they are present on parchment only degrading some other residual non-structural proteins and fats, or hydrolysed collagen already degraded by other agents.

Line 92: in the ref. 32 purple discoloration is associated with actinobacteria, not fungi.

Line 112: spectroscopy

Line 135: Substitute characterized with composed

Line 234. In this chapter you should better explain your experimental plan: being LTA a destructive analysis the comparison among treated and not treated is made on different samples (absolute differences): you should mention this in the text

Line 279: you should always specify, here and in the other presented analysis, if the comparison among treated and not treated are made on the same sample (in case of non-destructive analysis) or on different samples (that is unavoidable if the analysis is destructive).

Line 321: Cell viability tests after irradiation of agar colonized specimens.

Line 334: why speaking of paper if you are working only with parchment?

Line 350: how could you perform your analysis before and after irradiation on the same sample if the analysis is destructive? How could be a relative change?

Line 380-386: I don’t understand, maybe it’s my fault… If, as you say, FTIR is performed only to characterize the effects of natural ageing, why are you analysing the irradiated samples? Not irradiated samples are not sufficient. Sorry but to me this sentence is a little bit non-sense.

Line 522: … material, at least for heavy contaminated and deteriorated parchment assets

Line 523-25: not clear. Why? You did perform the treatment only on these specific isolates. You can't generalize for all the possible species on parchment assets. Furthermore you treated species growing on the generic NB media. On this media most of the time you underestimate the presence of peculiar but typical xerophylic species growing on parchment. How can you speculate on the effect on these specific species?

 

Line 525: Which are the other disinfection techniques on cultural assets in which the identification is propaedeutic?

 

Line 527: so finally which dosage do you suggest for a complete sterilisation? Could you be confident with its activity against the majority of biodeteriogens potentially present on parchment?

 

Line 547: how do you prepare the assets employing the other techniques, as for example Gamma or uv? Maybe do you already know the effect of x ray on asset at different condition such as level of humidity or dirtiness?

 

Line 563: what is the effect of the radiation on the other possible materials present in archival and book assets, primarily paper, but also pigments and adhesives? If you intend it as a mass treatment, its efficacy and safety must also be guaranteed for the other possible materials present

 

Line 577: The analyses were conducted on already degraded material. What happens to a more recent and less degraded parchment? For a mass treatment usually it is not possible to make huge preventive distinctions between the different ages and conditions of the materials. The treatment must also be safe for less deteriorated and more recent parchments, as well as for the other associated material components, especially paper, as already mentioned.

 

 

 

 

 

 

 

Author Response

Dear Editor,

we are grateful for the further reviewer's comments concerning the article “Evaluation of the irradiation treatment effects on ancient parchment samples” by Vadrucci et al.

In the following we reply to the reviewer’s comments reporting, where necessary, the changes made in the manuscript.

 

Specific comments

Line 51: in Italy and other European countries disinfection of cultural assets with ethylene oxide is still permitted

We thank the reviewer for pointing this out. It is now stated in lines 51-53: “Though the use of ethylene oxide is still permitted in Europe, it is strongly discouraged because of its toxic nature for the environment, the operator and even for some of the library and archival substrates [12].”

Line 66: substitute bioremediation with disinfection

We apologize to the reviewer. The term has been replaced (line 66).

Line 69-75: these sentences are not clear: is there any synergic effect among aging and irradiation? Please be more clear.

We apologize to the reviewer for the lack of clarity. It is now specified in lines 74-76:  “It was found that, when the irradiation was performed in samples of originally undamaged modern parchment, the ageing induced damage added up on top of that caused by the irradiation pretty much independently of the irradiation dose [30].”

Line 90: In fact the more frequent colonizers of ancient parchment are actinomycetes (halophilc actinomycets in particular). Native collagen is degradate by collagenases produced by a very low amount of bacterial species, such as Clostridium in anerobiosis. It is not clear if fungi are really able to produce collagenases (see for example https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5220638/). It is more probable that they are present on parchment only degrading some other residual non-structural proteins and fats, or hydrolysed collagen already degraded by other agents.

In agreement with the reviewer observations the sentence has been modified as follows (line 91): “The major biodeteriogens of parchment produce collagenases, weak acids and pigments, causing localized collagen damage.”

Line 92: in the ref. 32 purple discoloration is associated with actinobacteria, not fungi.

We apologize for the mistake, the sentence has been changed accordingly as follows (line 93): “Specifically, Actinobacteria parchment discoloration and degradation and subsequent purple spots formation [32]”.

Line 112: spectroscopy

The term has been corrected (line 113).

Line 135: Substitute characterized with composed

The term has been replaced (line 136).

Line 234. In this chapter you should better explain your experimental plan: being LTA a destructive analysis the comparison among treated and not treated is made on different samples (absolute differences): you should mention this in the text

We apologize to the reviewer for the lack of clarity. In lines 351- 356 it is now stated that:  “In this respect it is worthwhile pointing out that, even though LTA is a destructive technique, it involves such a small sample area (1 mm²) that, by performing the measurements before and after the irradiation on neighbouring areas of the sample, the results  can  be considered obtained on the same area before and after irradiation.“

Line 279: you should always specify, here and in the other presented analysis, if the comparison among treated and not treated are made on the same sample (in case of non-destructive analysis) or on different samples (that is unavoidable if the analysis is destructive).

According to the reviewer suggestion, specific comments have been added in correspondence of the results sections for each adopted technique (lines: 347-350, 375-377, 387-392, 433-436 and 462-465)

Line 321: Cell viability tests after irradiation of agar colonized specimens.

We thank for the suggestion, the text has been modified accordingly (line 323).

Line 334: why speaking of paper if you are working only with parchment?

In agreement with the reviewer the text has been modified and the term cancelled (line 336).

Line 350: how could you perform your analysis before and after irradiation on the same sample if the analysis is destructive? How could be a relative change?

See response to objection corresponding to content of line 234.

Line 380-386: I don’t understand, maybe it’s my fault… If, as you say, FTIR is performed only to characterize the effects of natural ageing, why are you analysing the irradiated samples? Not irradiated samples are not sufficient. Sorry but to me this sentence is a little bit non-sense.

The Reviewer is right; the sentence is not clear. We apologize for this fault. We have changed the sentence to “Previously reported analysis indicates that no deterioration effects (i. e. conformational changes due to gelatinization), due to X-ray exposure, occurred. Therefore, it is reasonable to assume that every change in FTIR spectra are attributable to heterogeneity of parchments, rather than X-ray treatments.  Data reported in table 1, indicate that the variation of values of the intensity ratio of the Amide I and Amide II bands does not follow the irradiation dose trend, thus confirming our hypothesis.” (lines 387-392) 

For further explanation of how much natural aging complicates the understanding of the phenomenon, authors have added a sentence in which a comparison with a modern parchment is made: “The extent of degradation of the parchment, due to aging alone, is better highlighted when compared to that of a modern parchment in good condition. Indeed, as can be seen from table 1, a naturally aged parchment, not irradiated (0 Gy), shows a value of the ratio between the intensities of the Amide I and Amide II bands of 3.0 ± 0.1, while in the case of modern parchment this value is 1.2 ± 0.1. Furthermore the relative distance between the bands of Amides I and II is about 93 cm^-1 and 89 cm^-1, for ancient and modern parchment respectively. These results clearly indicate that natural aging has a marked effect on the material, producing its hydrolysis and denaturation and/or gelatinization.” (lines 409-417)

Line 522: … material, at least for heavy contaminated and deteriorated parchment assets

We thank for the suggestion, the text has been modified accordingly (line 542)

Line 523-25: not clear. Why? You did perform the treatment only on these specific isolates. You can't generalize for all the possible species on parchment assets. Furthermore you treated species growing on the generic NB media. On this media most of the time you underestimate the presence of peculiar but typical xerophylic species growing on parchment. How can you speculate on the effect on these specific species?

 

We agree with the reviewer that NB is a generic medium and we could have missed some species, although it has been also used in other studies [Pinzari, F., Cialei, V., & Piñar, G. (2012) A case study of ancient parchment biodeterioration using variable pressure and high vacuum scanning electron microscopy. In: Historical technology, Materials and conservation: SEM and microanalysis. Meeks N, Cartwright C, Meek A, Mongiatti A (eds). Archetype Publications - International Academic Projects, 1 Birdcage Walk, London, ISBN: 9781904982654; A. M. Omar, A. Salah Taha, and A. AA Mohamed,―Microbial Deterioration of Some Archaeological Artifacts: Manipulation and Treatment, Eur. J. Exp.Biol., vol. 08, no. 04, pp. 1–7, 2018; Akmal Sakr, et.al, Characterization of Microbiota Deteriorating Specific Coptic Manuscripts, Coptic Museum, EgyptInternational Journal of Research Studies in Biosciences. 2018, 6(9) : 1-15.]. However, we generalize the X-rays application as a sanitization procedure independent of microbial colonization since, as stated in the introduction,  the effect of the irradiation is the damage of DNA macromolecules that are present in all microorganisms.

Line 525: Which are the other disinfection techniques on cultural assets in which the identification is propaedeutic?

We apologize to the reviewer for the lack of clarity. We particularly refer to the fact that the prevention of the spread of bacteria and other microorganisms is a delicate task in disinfection treatments employing radiation systems based on radionuclides (such us the Gamma rays). The use of machines that generate X-rays does not make critical the contamination of the source, which can be accessed without the strict security regulations, even for any bonification actions.

Nevertheless, the authors recognize that in order to find the more efficient disinfection treatments for biodeteriorated cultural heritage items, it is surely appreciable the knowledge of the species carrying out the infection. Among all the possible chemical and non-chemical treatments employable in the disinfection of library and archival materials (from the traditional one to the most recent developed such us the use of essential oils, the nanoparticles or the biological methods), the knowledge of the biodeteriogens as well as their possible effect on the specific substrate is the essential starting point for an effective treatment.

For clarity, the sentence has been modified as follows (lines 546-550): “One of the strong points of the use of X-rays for the sanitization of the archival-library assets is the possibility to directly act on the artifacts to be preserved without first identifying the infesting biodeteriogen. In particular, this constitutes an advantage with respect to other treatments for which it is necessary to know the infesting agent both for the specific treatment actions (in order to find the more effective one) and to avoid any contamination of the environment in which the process takes place (such us the radiation systems based on radionuclides).”

Line 527: so finally which dosage do you suggest for a complete sterilisation? Could you be confident with its activity against the majority of biodeteriogens potentially present on parchment?

It is now stated in lines 539-541 : When the parchment is not originally heavily deteriorated, the minimum dose sufficient to sterilize the sample without introducing additional damage (i.e. ≈ 350 Gy) would be advisable. “

Line 547: how do you prepare the assets employing the other techniques, as for example Gamma or uv? Maybe do you already know the effect of x ray on asset at different condition such as level of humidity or dirtiness?

We apologize to the reviewer for the lack of clarity.

Thus, the sentence has been modified as follows (lines 572-574): “Finally, an additional advantage of the applications of this X-ray source is that the objects to be treated can be irradiated as they are, without any specific preparation (i.e. preliminary dry cleaning) or environmental conditions control since the treatments are carried out in the air, under normal temperature and pressure conditions.”

Line 563: what is the effect of the radiation on the other possible materials present in archival and book assets, primarily paper, but also pigments and adhesives? If you intend it as a mass treatment, its efficacy and safety must also be guaranteed for the other possible materials present

We apologize to the reviewer for the lack of clarity. It is now stated in lines 580-588: “It must be finally remarked that additional research is required finalized at promoting X-rays irradiation as a possible standard disinfectant remedy against bio-infections for library and documentary assets. In fact, it remains to be established what are the possible damaging effects of the radiation on the materials, other than parchment, of which books are constituted and also the possible damaging effects on parchment which is initially not as heavily degraded as the one considered in this work. To date, as stated earlier on, only investigations on undamaged modern parchment have been carried out, and experiments on parchment with a progressively increasing initial degree of degradation is under way.”

Line 577: The analyses were conducted on already degraded material. What happens to a more recent and less degraded parchment? For a mass treatment usually it is not possible to make huge preventive distinctions between the different ages and conditions of the materials. The treatment must also be safe for less deteriorated and more recent parchments, as well as for the other associated material components, especially paper, as already mentioned.

We apologize to the reviewer for the lack of clarity. As explained in the reply to the previous comment, it is now stated in lines 580-588: “It must be finally remarked that additional research is required finalized at promoting X-rays irradiation as a possible standard disinfectant remedy against bio-infections for library and documentary assets. In fact, it remains to be established what are the possible damaging effects of the radiation on the materials, other than parchment, of which books are constituted and also the possible damaging effects on parchment which is initially not as heavily degraded as the one considered in this work. To date, as stated earlier on, only investigations on undamaged modern parchment have been carried out, and experiments on parchment with a progressively increasing initial degree of degradation is under way.”

 

Kind Regards,

Cristina Cicero, on behalf of all the authors.

 

Corresponding author details

Cristina Cicero: Researcher

Department of Literary, Philosophical and Art History Studies

University of Rome “Tor Vergata”

Via Columbia 1, 00133 Rome, Italy

Mail: [email protected]

 

Author Response File: Author Response.docx