Investigating the formation and diagnostic value of ω-(o-alkylphenyl)alkanoic acids in ancient pottery

Long-chain omega-(o-alkylphenyl)alkanoic acids (APAAs) derived from the heating of unsaturated fatty acids have been widely used for the identification of aquatic products in archaeological ceramic vessels. To date, little attention has been paid to the diagnostic potential of shorter chain (< C-20) APAAs, despite their frequent occurrence. Here, a range of laboratory and field experiments and analyses of archaeological samples were undertaken to investigate whether APAAs could be used to further differentiate different commodities. The results provide new insights about the conditions for the formation of APAAs and enable the proposition of novel criteria to distinguish different natural products.

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Bondetti, M.; Scott, E.; Courel, B.; Lucquin, A.; Shoda, S.; Lundy, J.; Labra-Odde, C.; Drieu, L.; Craig, O. E.

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pre-publication peer review (FINAL ROUND)
Decision Letter
2020/10/19
19-Oct-2020
Dear Miss Bondetti:
I am writing to inform you that we are happy to accept your manuscript entitled "Investigating the formation and diagnostic value of ω-(o-alkylphenyl)alkanoic acids in ancient pottery" in its current form for publication in Archaeometry.
Thank you for your contribution. On behalf of the Editors of Archaeometry, we look forward to your continued contributions to the Journal.
Yours sincerely,
Prof. Mark Pollard
Managing Editor, Archaeometry
mark.pollard@rlaha.ox.ac.uk
Decision letter by
Mark Pollard
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Author Response
2020/10/16
Comments to the Author
This paper presents the results obtained following a series of laboratory and cooking experiments targeted at better understanding the formation of ω-(oalkylphenyl)alkanoic acids (APAAs) in ancient pottery. The results show that APAAs can form at lower temperatures and require less heating time than previously suggested (Evershed et al. 2008), and will form under aerobic conditions, very significant when applied to archaeological scenarios. Based on the results obtained, the authors suggest that APAA-C20/APAA-C18 ratio can be used to distinguish between aquatic and terrestrial foodstuffs. These results are highly significant within ORA, and will contribute to a better characterisation of the lipid profiles. The manuscript is well structured and written, however there are still some points to consider. Here are my comments:
The following sentence on p. 3 needs rephrasing to emphasise the relevance of studying the shorter-chain APAA homologues: While the use of APAAs to identify aquatic products in pottery represents a significant advance in organic residue analysis, APAAs with a shorter chain-length homologues (i.e.
We do not understand this comment because the literal paragraph is as follows and already explain the relevance of studying the shorter-chain APAAs:
“While the use of APAAs to identify aquatic products in pottery represents a significant advance in organic residue analysis, APAAs with a shorter chain-length homologues (i.e.
Furthermore, the subsequent paragraph (in the manuscript, p.12) explains why we decided to study the isomeric distribution of APAA-C18:
“Controlled heating experiments undertaken by Evershed and co-workers (Evershed et al. 2008), have shown that the distribution of APAA isomers with 18 carbon atoms (APAA-C18) differed according to the number and the position of unsaturations in the fatty acid from which it was derived. Similarly, the difference in the APAA-C18 isomeric distribution in thermally degraded rapeseed oil, cod liver oil and horse adipose fat was interpreted as a direct consequence of the relative amounts of precursor C18:1, C18:2, C18:3 fatty acids present in these products. Furthermore, Shoda et al. (2018) noted the dominance of two APAA-C18 isomers in pottery where starchy plants, such as nuts and cereals, were processed. Based on these observations, it appears that the isomeric distribution of APAA-C18 may provide an additional diagnostic tool for the identification of foodstuffs cooked in pottery. Considering that this has not yet been properly investigated, this research set out to explore the value of APAA-C18 isomeric distribution as a diagnostic tool to identify commodities processed in ancient pottery.”
On p. 9, there is a reference to the heating of rapeseed oil for one hour at 270°C. This is listed in the table, however not in the text under Materials and Methods. It would be good to add it for completeness.
This is mentioned in the Material and Method section, paragraph 2:
“The first series of laboratory experiments was designed to examine the duration of heating on APAA formation. Ca. 65 mg of rapeseed oil (Commercial Organic, cold-pressed, extra virgin rapeseed oil, UK) was sealed under nitrogen in borosilicate glass tubes (Fisherbrand, UK; 12 mL) either with or without the addition of ceramic powder (100 mg). Each tube was heated at 270°C for a duration of 1, 5, 10 or 17 h (Table S1, A). The second series of experiments was designed to examine the effect of temperature on APAA formation. Ca. 65 mg of rapeseed oil was placed in open glass tubes and heated at 100, 150, 200, 250 or 270°C for 5 h with or without the addition of ceramic powder (Table S1, A).”
P. 9, the following sentence needs justification: Interestingly, the proportion of APAAs to other compounds increases following burial, especially for cooked salmon where APAAs were only identifiable after burial. How can APAAs be present only after burial if heating is required for their formation? How are the results obtained justified/interpreted when the experimental data presented before showed that at least 200°C are required for the formation of the APAAs?
The absolute amount of APAAs does not increase during burial, rather their relative abundance compared to other compounds increases. For this reason, APAAs are difficult to detect in samples analysed before burial, as the other lipids (e.g. fatty acids, etc.) partially or totally mask the APAAs signal. However, during burial, these other compounds are subject to preferential degradation than APAAs, due to their different chemical properties, and thus their relative proportion decreases. This implies that APAAs are no longer masked by these other compounds and are therefore detectable.
We believe that this is clear in the paragraph below but we have modified slightly to make it clearer.
“Interestingly, the proportion of APAAs compared to other compounds was observed to increase following burial, especially for the salmon experiment where APAAs were only identifiable after burial. This is due to the relative loss of other more soluble and labile compounds during exposure to the burial environment, enriching the relative abundance of APAAs in the extracts.”
P. 12: why were the APAAs formed in the carbonised residues used for the PCA and not those formed within the ceramic fabric? The latter is the far more common material available for ORA, while carbonised residues less so. Why not do both to see if there is a difference, also keeping in mind the variable introduced by the presence of the metal ions in the ceramic?
For the PCA we used only the samples from the laboratory experiments as these were formed under similar conditions and parameters. This makes it possible to more accurately assess the influence of each food compared to the others. However, these were not ‘carbonised remains’ rather they were the residues formed by food products heated together with ceramic powder in tubes. .
We agree that the use of the terms "carbonised remains" (in the section "Lipid analysis", p. 7) and "carbonised residues" (section "Distinguish different foodstuffs based on APAA-C18 distribution", p. 12) to refer to these samples can lead to confusion. We have therefore replaced or deleted these terms as follows:
P. 7: “ For the experiments undertaken in the laboratory, the residue, formed by heating the food products with the ceramic powder, were used.”
P. 12: “Variability in the distribution of APAAs isomers resulting from the laboratory experiments were investigated using principal component analysis (PCA)”.
The caption to Fig 3 needs to specify that these are carbonised residues. It is misleading as is.
We have specified in the caption of figure 3 that this PCA was done using the results of laboratory experiments.
“Figure 3. Principal component analysis (PCA) scatter plot of the first two principal components (PCs) based on the APAA-C18 isomeric distribution derived from different foodstuffs subjected to heating in the laboratory with the ceramic powder at 270°C for 5 hours.”
P. 13: sample number for the leafy vegetables (n=5) is very small compared to the cereals/non-leafy vegetables (n=20) and animal fats (n=15). Would this affect the categories established?
Our results show a strong correlation between the isomeric distribution of leafy vegetables and heated α-C18:3. According to Pereira et al. (2001) green vegetables are known to contain a relatively high proportion of α-linolenic acid (18:3n-3). Therefore, the isomeric distribution for other leafy vegetables should be very similar to those obtained in this study. Thus, even if the sampling for leafy vegetables is more restricted than for other categories this should not affect the established categories but of course should be tested using the experimental approach we have outlined here.
With regard to the APAA C20/C18 ratio, the data supports the suggestion to use this value as an interim threshold, and I agree with the authors that future investigations will show whether this will hold. I agree with how this has been rephrased.
Table S2: In the caption, it is stated that: The layer of fats formed from salmon and red deer during the cooking was skimmed and gathered in another pot to then be heated on the open fire. These samples are noted with *. This is not very clear. Are the results presented therefore not of cooked salmon and red deer, but specifically of the layer of fat? Where these therefore cooked twice? This needs to be clarified.
We have rephrase the caption in Table S2 to clarify this point:
“Table S2. Table summarizing the results of the simulated cooking in replicate pots of various foodstuffs on an open fire and the field experimental parameters. The layer of fats and oils formed from salmon and red deer during the cooking was skimmed and gathered in another pot and then placed back on the fire to continue their cooking and concentrate lipids. These samples are noted with *.”
Author response by
Bondetti, M.
Scott, E.
Courel, B.
Lucquin, A.
Shoda, S.
Lundy, J.
Labra-Odde, C.
Drieu, L.
Craig, O. E.

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pre-publication peer review (ROUND 2)
Decision Letter
2020/09/18
18-Sep-2020
Dear Miss Bondetti:
Manuscript ID ARCH-11-0154.R1 entitled "Investigating the formation and diagnostic value of ω-(o-alkylphenyl)alkanoic acids in ancient pottery" which you submitted to Archaeometry, has been reviewed. The comments of the referee(s) are included at the bottom of this letter.
The referee(s) have recommended some minor revisions to your manuscript. Therefore, I invite you to respond to the referee(s)' comments and revise your manuscript.
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Because we are trying to facilitate timely publication of manuscripts submitted to Archaeometry, your revised manuscript should be uploaded as soon as possible. If it is not possible for you to submit your revision in a reasonable amount of time, we may have to consider your paper as a new submission.
Once again, thank you for submitting your manuscript to Archaeometry and I look forward to receiving your revision.
Yours sincerely,
Prof. Mark Pollard
Managing Editor, Archaeometry
mark.pollard@rlaha.ox.ac.uk
Comments to the Author
This paper presents the results obtained following a series of laboratory and cooking experiments targeted at better understanding the formation of ω-(oalkylphenyl)alkanoic acids (APAAs) in ancient pottery. The results show that APAAs can form at lower temperatures and require less heating time than previously suggested (Evershed et al. 2008), and will form under aerobic conditions, very significant when applied to archaeological scenarios. Based on the results obtained, the authors suggest that APAA-C20/APAA-C18 ratio can be used to distinguish between aquatic and terrestrial foodstuffs. These results are highly significant within ORA, and will contribute to a better characterisation of the lipid profiles. The manuscript is well structured and written, however there are still some points to consider. Here are my comments:
1. The following sentence on p. 3 needs rephrasing to emphasise the relevance of studying the shorter-chain APAA homologues: While the use of APAAs to identify aquatic products in pottery represents a significant advance in organic residue analysis, APAAs with a shorter chain-length homologues (i.e. 2. On p. 9, there is a reference to the heating of rapeseed oil for one hour at 270°C. This is listed in the table, however not in the text under Materials and Methods. It would be good to add it for completeness.
3. P. 9, the following sentence needs justification: Interestingly, the proportion of APAAs to other compounds increases following burial, especially for cooked salmon where APAAs were only identifiable after burial. How can APAAs be present only after burial if heating is required for their formation? How are the results obtained justified/interpreted when the experimental data presented before showed that at least 200°C are required for the formation of the APAAs?
4. P. 12: why were the APAAs formed in the carbonised residues used for the PCA and not those formed within the ceramic fabric? The latter is the far more common material available for ORA, while carbonised residues less so. Why not do both to see if there is a difference, also keeping in mind the variable introduced by the presence of the metal ions in the ceramic?
5. The caption to Fig 3 needs to specify that these are carbonised residues. It is misleading as is.
6. P. 13: sample number for the leafy vegetables (n=5) is very small compared to the cereals/non-leafy vegetables (n=20) and animal fats (n=15). Would this affect the categories established?
7. With regard to the APAA C20/C18 ratio, the data supports the suggestion to use this value as an interim threshold, and I agree with the authors that future investigations will show whether this will hold. I agree with how this has been rephrased.
8. Table S2: In the caption, it is stated that: The layer of fats formed from salmon and red deer during the cooking was skimmed and gathered in another pot to then be heated on the open fire. These samples are noted with *. This is not very clear. Are the results presented therefore not of cooked salmon and red deer, but specifically of the layer of fat? Where these therefore cooked twice? This needs to be clarified.
Decision letter by
Mark Pollard
Cite this decision letter
Reviewer report
2020/09/18
This paper presents the results obtained following a series of laboratory and cooking experiments targeted at better understanding the formation of ω-(oalkylphenyl)alkanoic acids (APAAs) in ancient pottery. The results show that APAAs can form at lower temperatures and require less heating time than previously suggested (Evershed et al. 2008), and will form under aerobic conditions, very significant when applied to archaeological scenarios. Based on the results obtained, the authors suggest that APAA-C20/APAA-C18 ratio can be used to distinguish between aquatic and terrestrial foodstuffs. These results are highly significant within ORA, and will contribute to a better characterisation of the lipid profiles. The manuscript is well structured and written, however there are still some points to consider. Here are my comments:
1. The following sentence on p. 3 needs rephrasing to emphasise the relevance of studying the shorter-chain APAA homologues: While the use of APAAs to identify aquatic products in pottery represents a significant advance in organic residue analysis, APAAs with a shorter chain-length homologues (i.e. 2. On p. 9, there is a reference to the heating of rapeseed oil for one hour at 270°C. This is listed in the table, however not in the text under Materials and Methods. It would be good to add it for completeness.
3. P. 9, the following sentence needs justification: Interestingly, the proportion of APAAs to other compounds increases following burial, especially for cooked salmon where APAAs were only identifiable after burial. How can APAAs be present only after burial if heating is required for their formation? How are the results obtained justified/interpreted when the experimental data presented before showed that at least 200°C are required for the formation of the APAAs?
4. P. 12: why were the APAAs formed in the carbonised residues used for the PCA and not those formed within the ceramic fabric? The latter is the far more common material available for ORA, while carbonised residues less so. Why not do both to see if there is a difference, also keeping in mind the variable introduced by the presence of the metal ions in the ceramic?
5. The caption to Fig 3 needs to specify that these are carbonised residues. It is misleading as is.
6. P. 13: sample number for the leafy vegetables (n=5) is very small compared to the cereals/non-leafy vegetables (n=20) and animal fats (n=15). Would this affect the categories established?
7. With regard to the APAA C20/C18 ratio, the data supports the suggestion to use this value as an interim threshold, and I agree with the authors that future investigations will show whether this will hold. I agree with how this has been rephrased.
8. Table S2: In the caption, it is stated that: The layer of fats formed from salmon and red deer during the cooking was skimmed and gathered in another pot to then be heated on the open fire. These samples are noted with *. This is not very clear. Are the results presented therefore not of cooked salmon and red deer, but specifically of the layer of fat? Where these therefore cooked twice? This needs to be clarified.
Reviewed by
Anonymous
Cite this review
Author Response
2020/07/06
Referee: 1
Comments to the Author
In this paper, Bondetti et al. report the results obtained following a series of laboratory and cooking experiments targeted at better understanding the formation of ω-(o-alkylphenyl)alkanoic acids (APAAs) in ancient pottery. The results show that APAAs can form at lower temperatures and require less heating time than previously suggested (Evershed et al. 2008), and will form under aerobic conditions. Based on the results obtained, the authors suggest the E+F isomer index and APAA-C20/APAA-C18 ratio can be used to distinguish between aquatic and terrestrial foodstuffs.
The research is well executed and the paper is well structured and written. The figures and tables allow further clarification and visual representation of the experimental conditions and results. The results produced provide a novel and significant input to the field of organic residue analysis. I have the following comments:
In the Material and Methods section (p.5), in my view the following sentence: These were left open or sealed under nitrogen and heated for 1, 5, 10 or 17 hours at temperature of 100, 150, 200, 250, or 270°C (Table 1), does not correctly portray the laboratory parameters followed, suggesting that rapeseed oil was heated at different temperatures and for different lengths of time, when in fact, these are two separate parameters, one testing the time it takes for the APAAs to form at 270°C, while the other tested the minimum temperature required to produce APAAs, with the duration of the heating being set at 5 hours.
We agree that the phrasing was ambiguous. This section has been re-written to improve clarity on what experiments were undertaken.
In addition, we have also modified and completed tables in supplementary. There are now:
Table S1. Table summarizing the results of the thermal degradation of (A) rapeseed oil and mono- and unsaturated fatty acids C18 and (B) various foodstuffs carried out in laboratory and the experimental parameters.
Table S2. Table summarizing the results of the simulated cooking in replicate pots of various foodstuffs on an open fire and the field experimental parameters. The layer of fats formed from salmon and red deer during the cooking was skimmed and gathered in another pot to then be heated on the open fire. These samples are noted with *.
Table 1 (in the manuscript) has been moved in the results section to present the experimental parameters carried out in laboratory and thermal conditions required to form APAAs from rapeseed oil and different pure unsaturated fatty acids C18.
It is well known that plants leave a much lower chemical signature than animal fats when cooked in ceramics. For the cooking experiments, what was the reason for cooking chestnut flour for 5 hours, as opposed to 15 hours for the animal fats?
Due to limited possibility of access to the experimental center we were unable to cook the chestnuts for 15 hours. As we found that APAAs were formed after 5 hours, we deemed it unnecessary to repeat the chestnut experiment for 15 hours.
It would have been beneficial to know the temperature of the internal surface of the pottery during the cooking experiments, as this would have provided the exact temperature at which APAAs were forming in real time. I am assuming that there would be a temperature discrepancy between the outer and inner ceramic surfaces, but I do not know how significant this difference would have been. I am also aware of the difficulties in fixing the thermocouples to record the temperature on the interior surface, so this is more of a general comment.
Indeed it would have been interesting to measure the temperature of the internal surface of the pots but there are practical difficulties in doing this. We can assume the temperature was between the boiling temperature of water and the temperatures recorded on the outside of the vessel. We conducted laboratory experiments to assess the temperature at which APAAs are formed, these experiments were rather to assess formation during simulated cooking.
Further experiments exploring specifically this point according to different aspects (cooking type, pottery wall thickness, ceramic type) would be very interesting to better understand the heat transfers between outer and inner ceramic surfaces during its use.
I cannot fully follow the logic of the following sentence (p. 8): Interestingly, the relative abundance of APAAs is higher following burial, especially for cooked salmon where APAAs were only clearly visible after burial. This is probably due to the relative loss of other more soluble compounds. If the APAA recovery percentages reported here are relative to the total lipid quantified, then this is to be expected since one would expect less lipid after burial, so the percentages of the APAAs will of course be higher. It would be interesting to know the actual quantification, and how and if the quantities of APAAs formed are significantly affected by burial.
We have revised this sentence to clarify, especially to point out that we are referring to proportion rather than absolute abundance (Section “Time and temperature”, 1st paragraph):
“Interestingly, the proportion of APAAs to other compounds increases following burial, especially for cooked salmon where APAAs were only identifiable after burial. This is due to the relative loss of other more soluble and labile compounds during exposure to the burial environment, enriching the relative abundance of APAAs in the extracts.”
However, we agree that it would be interesting to quantify the absolute abundance of APAAs before and after the burial. Unfortunately, it was difficult to accurately quantify the APAAs for all samples prior to burial, other than noting their presence, due to their low relative abundance compared to other compounds. Ideally, we would need to purify the APAAs in the extracts for this task but this would also risk loss and present other issues for accurate quantification.
On p. 9, it is reported that heating or the duration of the heating does not affect the formation process of the APAAs, however what about burial? Do we know for a fact that after their formation during heating, the isomer distributions and the APAA-C20/APAA-C18 ratio are not affected by decay during burial and differential preservation?
We address this point page in section “Evacuated vs aerobic conditions”, however we have amended the following sentences to clarify.
“Interestingly, the distribution of the APAA-C18 isomers obtained by heating salmon, chestnut flour and red deer undertaken in the field experiments are not significantly different to those carried out in the laboratory in open tubes (Kruskal-Wallis test: chestnut flour, Chi2 = 1.22; p = 1; salmone; Chi2 = 0.93; p = 0.99; red deer; Chi2 = 0.19; p = 0.91); either before or after burial. These findings suggest that the formation of APAAs during cooking is more likely to occur under aerobic conditions, and the isomeric distribution remains stable over time even when subjected to natural degradation processes.”
However, we do not really know how much the APAAs C18 to C20 ratio is altered during burial since the C20 APPAs were not detectable in the pre-burial samples due to the very high amounts of other lipids. However, as we mentioned in the manuscript, the ratio for pots used to cook salmon and buried remains significantly higher than other non-aquatic products and therefore it appears to be still reliable for distinguishing aquatic and terrestrial resources.
We have modified the sentence in the manuscript to clarify the section “Distinguishing aquatic from terrestrial resources (APAA-C20 vs. APAA-C18)”, paragraph 3
“However, in the burial experiments conducted here on pots used to cook salmon, the APAA C20/C18 ratio was still greater than 0.06 (n = 3; x̄ = 0.10 ± 0.00) following 6 months burial (Fig. 5). Nevertheless, differential preservation of APAAs C18 and C20 in different burial contexts should be a focus of future investigations.”
We also address this point in the conclusions to highlight the fact that further analyses are required to better grasp APAAs behaviour subjected to different environment contexts:
“While APAAs are frequently identified in archaeological pottery, they are also notably absent in many archaeological contexts despite large systematic investigation (Whelton et al. 2018; Cubas et al. 2020). This is surprising given that APAAs are so easily formed from a wide range of products and, even more so, considering that other fatty acid thermal degradation products are frequently encountered in vessels from these contexts (e.g. long-chain ketones C33 and C35; Raven et al. 1997; Cubas et al. 2020). Further investigations are therefore needed to examine the formation of APAAs in relation to the physical and chemical properties of the ceramic matrices and to examine whether all burial conditions are conducive to their preservation.”
In the results section, the mean is used when comparing E+F isomer indices, while in the boxplots (Figs 3 & 4), the median is reported. The values reported appear to be the same. Please clarify.
This section has been extensively re-written.
After further investigation it has been found that the ratio of isomers E over H (E/H ratio) allowed a better distinction of 3 product groups: leafy vegetables, cereals/fruits/non-leafy vegetables and animal fats. Furthermore, additional archaeological case studies to compare with experimental data have been added. This section was therefore revised according to this and figures adjusted.
In box plot figures (Figure 4 and 5) we have added mean values (represented by dashed line) and the captions have been amended accordingly.
“Figure 4. Boxplots of E/H ratio of modern references thermally degraded in the laboratory and archaeological samples. Samples with aquatic biomarkers are indicated by an asterisk, samples with plant and beeswax biomarkers are in orange and grey respectively. Plots represent median (solid line), mean (dashed line), ranges and quartiles. The arrow (thermal impact) shows the effect of increasing temperature on the E/H ratio.”
“Figure 5.Boxplots of APAA C20/C18 ratio of modern references, heated either in the laboratory or during field experiments after 6 months burial (#), and archaeological samples containing aquatic sources. Plots represent median (solid line), mean (dashed line), ranges and quartiles.”
With regard to the E+F isomer index, how do mixtures of foodstuffs influence this ratio? What should the contribution of mixtures of aquatic, plant and animal fats/oils be to have an effect on the ratio between E+F and C+I isomers recovered?
We have not conducted experiments to evaluate mixtures between different commodities here. There are two factors to take into account in case of mixing, the APAAs profile and the amount of unsaturated fatty acids in the original sources (especially unsaturated fatty acid). It is most likely that both these factors would influence the ratio in the event of mixing.
We expect that if non-leafy vegetables were mixed with either leafy vegetables or animal fats then the E/H ratio would be lower. Likewise, leafy plants mixed with other products should lead to produce higher E/H ratio values. Nevertheless extreme values (very high or very low) can still be useful for distinguishing these products.
We have made this point clearer in the manuscript and also suggested that additional experiments increasing the reference set with different mixtures is needed for further clarification:
Section “Distinguish different foodstuffs based on APAA-C18 distribution”, paragraph 3:
“Further experiments are needed to investigate how mixing of different foodstuffs may affect the E/H ratio or else theoretical values could be crudely predicted based on the proportion of unsaturated fatty acids in the original foodstuffs. A seemingly obvious limitation is that mixing for cereals/fruits/non-leafy vegetables and leafy vegetables is likely to produce intermediate E/H ratios matching animal fats.”
Moreover, on p. 13 it is stated that: it may not be possible to predict the APAA-C18 distribution based on a product’s original UFAs content, because contrary to work on UFA standards, the isomeric distribution of the foodstuffs after heating showed no clear correlation with their fatty acid content. How does this support the E+F isomer index? Can we therefore still apply this index to general categories, i.e. cereals/nuts, vegetables/fruits, terrestrial animals and aquatic animals, or will this change depending on the species and therefore requires more experimental work before we can confidently use the E+F isomer indices?
We point out that we can’t accurately predict the distribution of APAAs when complex mixtures are heated just based on the initial fatty acid content of products. However, there is a tendency that leafy plants (specifically spinach and cabbage), which are particularly high in α-C18:3 form a greater relative amount of the F, G and H isomers (in-line with the experiments on purified compounds). Therefore, it appears that both the original proportion of UFA and the position of their unsaturations are involved in APAA isomeric distribution. Other than that we agree with the referee that the range of food commodities experimentally tested for their C18 APAA distribution should be enlarged.
To better clarify these points we have done correlation tests, the results of which have been added into the supplementary material (Table S4). We have also made a table summarizing the proportion of unsaturated fatty acid C18:X detected in products prior to being heated (Supplementary Table S3), and completed figure S3 (in supplementary) with the APAAs-C18 isomeric distribution of spinach and cabbage.
The criteria for establishing an APAA-C20/APAA-C18 ratio of >0.006 for distinguishing aquatic resources from terrestrial ones have not been clearly defined in the text, but need to be inferred by the reader.
We have amended the paragraph 3 in this section to clarify this point as well as adjusted figure 5.
“We suggest that a value of 0.06 for the APAA C20/C18 ratio could be used as an interim threshold to distinguish aquatic sources from terrestrial products, since this is the lowest value observed for aquatic products and remains higher than any other type of resources (e.g. terrestrial animals and plants; Fig. 5). Preferential degradation processes differentially acting on the two homologous potentially could compromise the utility of this approach, for example due to differences in solubility. However, in the burial experiments conducted here on pots used to cook salmon, the APAA C20/C18 ratio was still greater than 0.06 (n = 3; x̄ = 0.10 ± 0.00) following 6 months burial (Fig. 5). Nevertheless, differential preservation of APAAs C18 and C20 in different burial contexts should be a focus of future investigations.”
The authors rightly question the stability of this ratio on p. 14, as differential degradation could lead to this number changing depending on climatic and environmental conditions influencing decay during burial. While recognising that this is an important finding and should be published, in my view, it should be made more evident that a value of >0.06 is obtained under specific burial conditions. This experiment needs to be expanded to encompass different burial conditions, as generalising such a ballpark figure now might risk erroneous interpretations in future research.
We agree with the referee that this an empirical observation based on the experiments presented in the article. We agree that further burial experiments are needed and that this proxy is evaluated against others in archaeological materials, such as the presence of other biomarkers or isotopic measurements made on fatty acids. We have amended or added the following sentences to that effect.
Section “Distinguishing aquatic from terrestrial resources (APAA-C20 vs. APAA-C18)”, paragraph 3:
“However, in the burial experiments conducted here on pots used to cook salmon, the APAA C20/C18 ratio was still greater than 0.06 (n = 3; x̄ = 0.10 ± 0.00) following 6 months burial (Fig. 5). Nevertheless, differential preservation of APAAs C18 and C20 in different burial contexts should be a focus of future investigations.”
Conclusion:
“The stability of APAA C20/C18 ratio should be assessed under a range of different environmental scenarios. Investigations should also examine the correspondence of this ratio with other molecular and isotopic data in archaeological samples.”
There is a significant sample bias between the archaeological samples included in this study: Zamostje 2 having 46 samples and Joto only 2 samples, as reported in Figure 3.
While this is clearly the case, we sought to include samples with APAAs where there is clear complementary corroborative evidence of cereal processing. The Joto site where SEM evidence also points to rice embedded in the pots clearly stands out and for that reason we argue it warrants inclusion.
We have expanded on the description of Joto and referenced the work where the rice was identified by SEM.
“Two samples were obtained from the early agricultural site of Joto where SEM has previously identified the charred remnants of a layer of rice pericarp tissue in two surface deposits. Bulk isotope analysis from these samples exhibit values consistent with starchy plants (δ15Nmean = 0.6 ± 1.8 ‰; C:Nmean = 17.9 ± 4.6) (Shoda et al. 2011; Yoshida et al. 2013).”
In addition, we have supplemented the archaeological comparison with another case study (Tianluoshan site) which shows evidence of pottery use for both the processing of starchy plants and animal products. This case study confirms what has been observed for the Joto site.
In Table 1 there are two entries for Rapeseed oil being heated for 5 hours at 200°C, both unsealed and with ceramic powder present, yet in one APAAs are reported and in the other not.
Corrected 200 °C replaced by 100°C and the table 1 has been completely revised.
In the Table S1, only three experiments are reported for this category, as opposed to five in Table 1. Moreover in the text, four tests are reported on p. 4 and five on p.5. Please check.
We have completely revised the description of the method and Table S1.
Tables S1 (A and B) have been revised and the text explaining the laboratory experiment extensively re-written to improve clarity.
In Figure 4, it is recommended that the plot representations are added, even if they are the same as Figure 3, i.e. median, range and quartiles. (Also, please see point 5.)
We think the referee is referring to figures 4 and 5 (the boxplots). We have amended the captions to both these plots (see response to point 5).
In Table S2, APAAs-C18 are reported in samples for which the isomer distribution is not reported. Why is this?
The isomer distribution is not given when APAAs-C18 were found only in trace amounts and could not be accurately integrated. Therefore we report their presence but not the isomer distribution.
To clarify this we have added a note at the end of the table:
“NB. In some samples APAAs, although present, were at very low abundance. In this case the isomeric distribution of APAAs-C18 could not be obtained.”
For the purposes of furthering this research, in my view, a fully labelled chromatogram should be presented in Figure 1, with the suite of APAAs highlighted as an inset or vice versa.
For the purpose of the study and to better detect the APAAs, samples were analysed in SIM (Selected Ion Monitoring) mode. Therefore, we cannot provide a complete chromatogram labelling all molecules present in the samples.
What about the results from the unheated fats? Where APAAs found here?
They were absent. We have now added this result to the supplementary Material Table S1, B and to the text, section “Distinguish different foodstuffs based on APAA-C18 distribution”; paragraph 1:
“Analysis of the foodstuffs prior to heating found no evidence of APAAs.”
Referee: 2
Comments to the Author
Review of Investigating the formation and diagnostic value of ω-(o-alkylphenyl) alkanoic acids in ancient pottery
Summary of paper:
This paper explores important questions that underpin the diagnostic value of APAAs found in lipid extracts in archaeological pottery, often used to identify the processing of aquatic products. It conducts several relevant experiments: firstly, a series that assess the conditions under which APAAs form, such as time; temperature; in the presence/absence of pottery; and oxic versus anoxic conditions. The paper demonstrates that the relationship between heating of unsaturated fatty acids and formation of APAAs is complex and is not only contingent on heating up to temperatures of 270°C as suggested by Evershed and colleages (Evershed et al. 2008). The question about whether APAAs are formed due to contact with pottery matrix is also of relevance to archaeological contexts. Secondly, the authors also test the degree of resolution offered by APAAs, particularly APAA-C18, by heating different types of foodstuff. They confirm previous investigations that suggest there is no correlation between a product’s original unsaturated fatty acid content and the distribution of APAA-C18 isomers after heating, recommending further investigation. Finally, the authors discuss how the presence of APAA-C20 is not unique to aquatic products in ancient pottery. They suggest that the ratio of APAA C20/C18 could provide an additional tool for identifying aquatic products in ancient pottery and provide a comparison with archaeological pottery to demonstrate its application.
Significance of paper:
Overall, the paper is an important contribution as it provides new knowledge about the formation of APAAs, highlights the need for caution for their use as a diagnostic marker and proposes novel criteria to distinguish between aquatic and terrestrial products. These results have implications for how scholars in the field of organic residue analysis characterise the processing of aquatic products in ancient pottery around the world.
General comments:
This paper is a good fit for Archaeometry, however, the statistical analyses are problematic, and several conclusions in the paper hinge on inappropriate statistical tests (detailed in the comments below).
Comments on structure and clarity:
A clear explanation of the rationale and research questions explored by the experiments before Methods and Materials would bring clarity to the reader.
We have modified the introduction to state our aims more clearly and the methods section to clarify the experiments that were undertaken.
To better clarify our research questions we have added in the introduction:
Paragraph 5: “Furthermore, Shoda et al. (2018) noted the dominance of two APAA-C18 isomers in pottery where starchy plants, such as nuts and cereals, were processed. Based on these observations, it appears that the isomeric distribution of APAA-C18 may provide an additional diagnostic tool for the identification of foodstuffs cooked in pottery. Considering that this has not yet been properly investigated, this research set out to explore the value of APAA-C18 isomeric distribution as a diagnostic tool to identify commodities processed in ancient pottery. This was done through a series of experiments involving the heating of different fats and oils, and through the comparison with the distribution of APAAs observed in archaeological samples.”
Paragraph 6: “To that extent, our experiments gave us an additional opportunity to reassess the conditions for APAAs formation in order to better interpret results, particularly with respect to ancient culinary practices.”
A table summarising the different types of experiments conducted would also improve understanding as there are several of them.
Tables (S1 A and B, and S2) in Supplementary Material, summarizing the results and the experimental parameters of the experiments, have been reworked to better show the experiments conducted in this research.
Furthermore, the section “Material and Method”, “Cooking experiments”, paragraph 2 has been completely re-written to improve clarity:
“The first series of laboratory experiments was designed to examine the duration of heating on APAA formation. Ca. 65 mg of rapeseed oil (Commercial Organic, cold-pressed, extra virgin rapeseed oil, UK) was sealed under nitrogen in borosilicate glass tubes (Fisherbrand, UK; 12 mL) either with or without the addition of ceramic powder (100 mg). Each tube was heated at 270°C for a duration of 1, 5, 10 or 17 h (Table S1, A). The second series of experiments was designed to examine the effect of temperature on APAA formation. Ca. 65 mg of rapeseed oil was placed in open glass tubes and heated at 100, 150, 200, 250 or 270°C for 5 h with or without the addition of ceramic powder (Table S1, A). The third series examined the relationship between APAA formation and precursor fatty acids. Ca. 20 mg of pure fatty acids C18:0, C18:1 (cis-9-Octadecenoic acid), C18:2 (cis-9,cis-12-Octadecadienoic acid) and α-C18:3 (α-Lnn, cis,cis,cis-9,12,15-Octadecatrienoic acid) were heated in duplicate in open glass tubes with or without powdered ceramic (100 mg) for 5 hours at 270 °C (Table S1, A). Finally, in the last series of laboratory experiments, a selection of foodstuffs, including meat, fish and edible plants (leafy vegetables, fruits, nuts and cereals) were heated for 5 hours at 270°C with the presence of ceramic powder (Table S1, B). Prior to heating, a sub-sample of each raw foodstuffs was retained for analysis.”
Similarly, it is not clear at the start that the experimental research is supplemented with comparisons to archaeological examples. This should be specified in the introduction. I recommend improving language and precision wherever possible
This point has been emphasized in the Introduction at the end of the paragraph 5:
“This was done through a series of experiments involving the heating of different fats and oils, and through the comparison with the distribution of APAAs observed in archaeological samples.”
And in the abstract:
“ Here, a range of laboratory and field experiments and analyses of archaeological samples were undertaken to investigate whether APAAs could be used to further differentiate different commodities.”
Comments on statistical analyses:
The dataset is very interesting, but I recommend the stats are checked carefully by an expert in statistics. For example, it is important to first test if there is an effect (heating/no heating; lab/field; cereals, nuts/animal) and/or an interaction between these main effects using, for example, a linear mixed effects model. Useful reference: Kuznetsova, Alexandra, Per B. Brockhoff, and Rune Haubo Bojesen Christensen. "lmerTest package: tests in linear mixed effects models." Journal of statistical software 82.13 (2017). Only if there is an effect is it possible to look at post-hoc test such as t-tests between groups.
We agree that using a multivariate approach might be appropriate if we had all these data i.e. if we had subjected each foodstuff (n=33) to differential temperatures (n=5), times (n= 4), burial or not (n=2), aerobic/anaerobic conditions (n=2) and with/without ceramics (2). However, this would have required the analysis of over 5,000 samples in replicates not including the field experiments! We argue that such an approach is not warranted, we are reporting an experimental study (rather than an observational study) where we choose to keep one variable constant and change other variables resulting in empirical statements regarding the findings. Our objective was not to define a linear model to explain APAA formations for all parameters but merely to identify some limits of their formation as we have clarified in the introduction. The experiments are also based on prior knowledge that protracted heating is required for APAA formation reactions and we assume that their formation is independent of foodstuff (although we comment on this point in the text with respect to the isomers formed).
Additionally, the interpretation of the PCA requires further explanation, as does the reason behind the summing of E+F isomers.
Following the remark of the reviewer we have reevaluated our PCA and changed the ratio used. We have provided the rationale of the ratio within the text (section “Distinguish different foodstuffs based on APAA-C18 distribution”, paragraph 2)
Specific comments:
Page 1:
Line 6 and Line 12: The ‘biomarker concept’ should be clarified, and the term ‘biomarker’ or ‘lipid marker’ should be used with caution
We have clarified the term “biomarker concept” in the manuscript using a definition closer to Everhsed’s (2008):
“Based on the archaeological biomarkers concept (Evershed 2008), it is possible to trace organic molecules, or suites of molecules, extracted from pots to organisms likely to have been exploited in the past.”
Line 19: remove ‘and’ before ‘beehive products’
We have removed “and”
Line 21: replace ‘range’ with ‘types’
We have replaced “range” with “types”.
Page 3:
Line 8: “long-chain”
We have made the correction.
Line 12: ‘amount’ should be ‘amounts’
The language has been corrected.
Line 16: should be: “the detection of APAAs in Early Woodland hunter-gatherer pottery in North America has shown that…”
We have decided to keep our sentence as it was originally written since it better reflects what we are saying.
Line 32: “chain-length”
The language has been corrected
Page 4:
Line 3-21: Can the rationale for the current study be expanded upon? What is missing from our understanding of APAA-C18? How is this study different from Evershed et al. 2008 and Shoda et al. 2018?
To better emphasize the novelty of our study, we have further expanded the 5th paragraph of the introduction. Please see the above comments (Referee 2, first comment).
Line 28: Remove ‘In addition’
We have removed “In addition”.
Line 28: which experiments? The ones cited or the ones in the paper?
We have revised this paragraph (last paragraph of the introduction):
“Previous studies (Matikainen et al. 2003; Hansel et al. 2004; Evershed et al. 2008) involving different natural commodities (rapeseed oil, horse adipose fat and cod liver oil) have shown that APAAs were formed when UFAs are subjected to protracted heating (≥17 hours at temperatures above 270°C), although a shorter cooking time and lower temperatures have so far not been assessed. Yet, understanding the minimum time and temperature needed to form these compounds is often important for interpretative purposes. Secondly, previous studies have shown that APAAs are only formed in the presence of fired clay, containing the metal ions (Redmount and Morgenstein 1996; Mallory-Greenough et al. 1998) required for the prior alkali isomerization step. And thirdly, anaerobic conditions are regarded as necessary to produce APAAs, promoting the cyclization process. To that extent, our experiments gave us an additional opportunity to reassess the conditions for APAAs formation in order to better interpret results, particularly with respect to ancient culinary practices.”
Line 46: remove ‘the’ before ‘metal ions’
The language has been corrected
Line 52: remove ‘first’ and comma after ‘undertaken’ in “thermal degradation experiments were first undertaken”
This sentence has been completely modified:
“And thirdly, anaerobic conditions are regarded as necessary to produce APAAs, promoting the cyclization process. To that extent, our experiments gave us an additional opportunity to reassess the conditions for APAAs formation in order to better interpret results, particularly with respect to ancient culinary practices.”
Page 9: Line 5: Rationale behind Kruskal-Wallis tests?
We have added an explanation regarding the choice of statistical tests used in this article in the supplementary material as well as a note in the section “Material and method”:
“Statistical tests were conducted using PAST3 software (version 3.25 for Windows). Detailed explanations on the choice of statistical tests applied are given in the Supplementary Material.”
In the Supplementary Material: “To test the normality of the distributions, Shapiro–Wilk statistical tests were conducted, since the populations were less than 50 cases for all tests. To compare two samples, when the results show that either data were normally distributed, parametric tests were then applied (T-test) or not normally distributed, non-parametric tests were used (Mann-Whitney test). For statistical tests involving more than two samples, in all cases the normality tests results show that data were not normally distributed. Therefore, the non-parametric tests were applied (Kruskal-Wallis). A p value of 0.05 (H1 acceptance with p ≤ 0.05) was used as the significance threshold.”
Line 21: Table 1 suggests that APAAs were not formed when rapeseed oil was heated at 150°C or 200°C in evacuated conditions, but instead at 270°C in the absence of pottery powder. Can this be addressed in the discussion?
These are not our results, but we agree that Table 1 and Table S1 (in the supplementary) are confusing. We have amended them for increased clarity. Please see previous comments (Referee 1, point 1).
Line 25: should be written as “described by Matikainen and colleagues (Matikainen et al. 2003).”
We have changed by “described by Matikainen et al. (2003)” to keep consistency throughout the manuscript.
Line 39: Full stop required after (Fig S2) and remove ‘and’ before ‘overall’
We have accepted this modification.
Page 10:
Line 12: comma to be added after “fully aerobic conditions”
We have added the comma.
Line 26: ‘salmon’ not ‘salmone’
We have corrected this mistake.
Lines 26-29: Suitability of Kruskal-Wallis test?
The choice of statistical tests used in this article is explained in more detail in the supplementary material (section “Statistical Analysis”).
Page 11:
Line 23-28: Figure 2: The image is too small; could be bigger and clearer.
We have modified the image format to make it bigger.
Lines 37-39: It is not clear how the samples coloured in orange (n=9) ‘stand out’ from all the others, or how the PCA suggests that the E and F isomers should be summed. PCA seems to indicate that it is mostly E that defines the group that ‘stands out.’ What is the rationale behind summing E + F isomers?
We agree that this section had some ambiguity. Therefore this entire section has been re-written. Please see above comment, referee 1, point 5.
Line 55-60: Suitability of t-test? Is there an effect of group of foodstuffs?
This section has been completely revised and the statistical test has been adjusted to reflect the new variables used. In addition, as stated above the choice of statistical tests used in this article is explained in more detail in the supplementary material (section “Statistical Analysis”).
Page 12:
Lines 8-12: Figure 3: The PCA scatter plot could be larger. Figure caption should explain what the points in orange signify.
We have completely modified Figure 3 and the colour choice has been explicitly indicated in the figure itself.
Lines 19-24: Is it meaningful to compare the mean E+F isomer contribution to archaeological samples when the sample sizes are so different? I suggest removing Joto entirely.
See response to referee 1 (point 9) for our justification for analysing the Joto samples.
Lines 48-53: Figure 4: Could you provide data points within the boxplots? The boxplot does not match up with sample numbers provided in PCA.
We agree that it brings confusion. Therefore we have modified figure 3 and defined one colour for each category. Figure 4 has been completely redone and data points added the next to the boxplots. Color codes used in the PCA have been re-used in figure 4 to match up and facilitate comparison.
Page 14:
Lines 10-17: Rationale behind Mann-Whitney test?
Please see earlier comments (referee 2 for comment Page 9: Line 5).
Why are the sample numbers so small for the experimental samples?
For the experiments only salmon have been cooked and as stated in the manuscript p. 8 APAAs for salmon were not visible before burial:
“Interestingly, the proportion of APAAs to other compounds increases following burial, especially for cooked salmon where APAAs were only identifiable after burial. This is due to the relative loss of other more soluble and labile compounds during exposure to the burial environment, enriching the relative abundance of APAAs in the extracts.”
Furthermore, after burial foodcrusts formed during the cooking were gone and for one of the pots APAAs C20 were present only in trace amounts.
So we were able to compute the APAA C20/C18 ratio only for 3 samples (EFB1-cer-bur, EFC1-cer-bur, EFFA1-cer-bur, Table S1).
Lines 28-39: Interesting proposition, however, groups have uneven and very small sample sizes. Defining a 0.06 criterion seems premature. Results appear to only indicate that this ratio should be investigated further.
We agree on this point. We have slightly qualified our remarks:
“We suggest that a value of 0.06 for the APAA C20/C18 ratio could be used as an interim threshold to distinguish aquatic sources from terrestrial products, since this is the lowest value observed for aquatic products and remains higher than any other type of resources (e.g. terrestrial animals and plants; Fig. 5). Preferential degradation processes differentially acting on the two homologous potentially could compromise the utility of this approach, for example due to differences in solubility. However, in the burial experiments conducted here on pots used to cook salmon, the APAA C20/C18 ratio was still greater than 0.06 (n = 3; x̄ = 0.10 ± 0.00) following 6 months burial (Fig. 5). Nevertheless, differential preservation of APAAs C18 and C20 in different burial contexts should be a focus of future investigations.
Lines 46-53: Circular reasoning? It is not clear how pottery at Zamostje 2 was mostly used to process freshwater resources.
We agree that further clarification could be given regarding the independent criteria used to establish freshwater resources. We have further expanded this point in section “Distinguish different foodstuffs based on APAA-C18 distribution” in order to better explain our criteria for samples selection:
“The samples (n=35) of Middle Neolithic pottery (ca. 5000–4000 cal BC) from Zamostje were found in close association with freshwater fish (Bondetti et al. 2020) and all met established molecular criteria for the identification of aquatic products (Hansel et al. 2004; Evershed 2008; Cramp and Evershed 2014; Lucquin et al. 2016a) and are associated with charred surface deposits with high bulk δ15N values (x̄ = 10.7 ± 2.2 ‰), also characteristic of aquatic resources (Dufour et al. 1999; Craig et al. 2013; Choy et al. 2016).”
Page 15:
Lines 3-6: Figure 5: Could you provide data points within the boxplots?
We have added the data points next to the boxplots in figure 5.
Supplementary materials:
Table S1: The tables were cut off after APAAs-C18 isomer E or H.
We think that the table provided with the paper was complete, so this comment may refer to a trouble in the file provided to the reviewer. In any case, we have provided an amended version of the table S1 with this submission.
(page 33) ‘Leak’ should be “leek”; ‘sesam’ should be “sesame”
We have made the corrections
Bondetti et al 2020_Minor Comments - pdf doc
Typing mistakes and rephrasing comments have been considered and corrected
Author response by
Bondetti, M.
Scott, E.
Courel, B.
Lucquin, A.
Shoda, S.
Lundy, J.
Labra-Odde, C.
Drieu, L.
Craig, O. E.

Cite this author response
pre-publication peer review (ROUND 1)
Decision Letter
2020/03/02
02-Mar-2020
Dear Miss Bondetti:
Manuscript ID ARCH-11-0154 entitled "Investigating the formation and diagnostic value of ω-(o-alkylphenyl)alkanoic acids in ancient pottery" which you submitted to Archaeometry, has been reviewed. The comments of the referee(s) are included at the bottom of this letter.
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Referee(s)' Comments to Author:
Referee: 1
Comments to the Author
In this paper, Bondetti et al. report the results obtained following a series of laboratory and cooking experiments targeted at better understanding the formation of ω-(o-alkylphenyl)alkanoic acids (APAAs) in ancient pottery. The results show that APAAs can form at lower temperatures and require less heating time than previously suggested (Evershed et al. 2008), and will form under aerobic conditions. Based on the results obtained, the authors suggest the E+F isomer index and APAA-C20/APAA-C18 ratio can be used to distinguish between aquatic and terrestrial foodstuffs.
The research is well executed and the paper is well structured and written. The figures and tables allow further clarification and visual representation of the experimental conditions and results. The results produced provide a novel and significant input to the field of organic residue analysis. I have the following comments:
In the Material and Methods section (p.5), in my view the following sentence: These were left open or sealed under nitrogen and heated for 1, 5, 10 or 17 hours at temperature of 100, 150, 200, 250, or 270°C (Table 1), does not correctly portray the laboratory parameters followed, suggesting that rapeseed oil was heated at different temperatures and for different lengths of time, when in fact, these are two separate parameters, one testing the time it takes for the APAAs to form at 270°C, while the other tested the minimum temperature required to produce APAAs, with the duration of the heating being set at 5 hours.
It is well known that plants leave a much lower chemical signature than animal fats when cooked in ceramics. For the cooking experiments, what was the reason for cooking chestnut flour for 5 hours, as opposed to 15 hours for the animal fats?
It would have been beneficial to know the temperature of the internal surface of the pottery during the cooking experiments, as this would have provided the exact temperature at which APAAs were forming in real time. I am assuming that there would be a temperature discrepancy between the outer and inner ceramic surfaces, but I do not know how significant this difference would have been. I am also aware of the difficulties in fixing the thermocouples to record the temperature on the interior surface, so this is more of a general comment.
I cannot fully follow the logic of the following sentence (p. 8): Interestingly, the relative abundance of APAAs is higher following burial, especially for cooked salmon where APAAs were only clearly visible after burial. This is probably due to the relative loss of other more soluble compounds. If the APAA recovery percentages reported here are relative to the total lipid quantified, then this is to be expected since one would expect less lipid after burial, so the percentages of the APAAs will of course be higher. It would be interesting to know the actual quantification, and how and if the quantities of APAAs formed are significantly affected by burial.
On p. 9, it is reported that heating or the duration of the heating does not affect the formation process of the APAAs, however what about burial? Do we know for a fact that after their formation during heating, the isomer distributions and the APAA-C20/APAA-C18 ratio are not affected by decay during burial and differential preservation?
In the results section, the mean is used when comparing E+F isomer indices, while in the boxplots (Figs 3 & 4), the median is reported. The values reported appear to be the same. Please clarify.
With regard to the E+F isomer index, how do mixtures of foodstuffs influence this ratio? What should the contribution of mixtures of aquatic, plant and animal fats/oils be to have an effect on the ratio between E+F and C+I isomers recovered?
Moreover, on p. 13 it is stated that: it may not be possible to predict the APAA-C18 distribution based on a product’s original UFAs content, because contrary to work on UFA standards, the isomeric distribution of the foodstuffs after heating showed no clear correlation with their fatty acid content. How does this support the E+F isomer index? Can we therefore still apply this index to general categories, i.e. cereals/nuts, vegetables/fruits, terrestrial animals and aquatic animals, or will this change depending on the species and therefore requires more experimental work before we can confidently use the E+F isomer indices?
The criteria for establishing an APAA-C20/APAA-C18 ratio of >0.006 for distinguishing aquatic resources from terrestrial ones have not been clearly defined in the text, but need to be inferred by the reader. The authors rightly question the stability of this ratio on p. 14, as differential degradation could lead to this number changing depending on climatic and environmental conditions influencing decay during burial. While recognising that this is an important finding and should be published, in my view, it should be made more evident that a value of >0.06 is obtained under specific burial conditions. This experiment needs to be expanded to encompass different burial conditions, as generalising such a ballpark figure now might risk erroneous interpretations in future research.
There is a significant sample bias between the archaeological samples included in this study: Zamostje 2 having 46 samples and Joto only 2 samples, as reported in Figure 3.
In Table 1 there are two entries for Rapeseed oil being heated for 5 hours at 200°C, both unsealed and with ceramic powder present, yet in one APAAs are reported and in the other not. In the Table S1, only three experiments are reported for this category, as opposed to five in Table 1. Moreover in the text, four tests are reported on p. 4 and five on p.5. Please check.
In Figure 4, it is recommended that the plot representations are added, even if they are the same as Figure 3, i.e. median, range and quartiles. (Also, please see point 5.)
In Table S2, APAAs-C18 are reported in samples for which the isomer distribution is not reported. Why is this?
For the purposes of furthering this research, in my view, a fully labelled chromatogram should be presented in Figure 1, with the suite of APAAs highlighted as an inset or vice versa.
What about the results from the unheated fats? Where APAAs found here?
Referee: 2
Comments to the Author
Review of Investigating the formation and diagnostic value of ω-(o-alkylphenyl) alkanoic acids in ancient pottery
Summary of paper:
This paper explores important questions that underpin the diagnostic value of APAAs found in lipid extracts in archaeological pottery, often used to identify the processing of aquatic products. It conducts several relevant experiments: firstly, a series that assess the conditions under which APAAs form, such as time; temperature; in the presence/absence of pottery; and oxic versus anoxic conditions. The paper demonstrates that the relationship between heating of unsaturated fatty acids and formation of APAAs is complex and is not only contingent on heating up to temperatures of 270°C as suggested by Evershed and colleages (Evershed et al. 2008). The question about whether APAAs are formed due to contact with pottery matrix is also of relevance to archaeological contexts. Secondly, the authors also test the degree of resolution offered by APAAs, particularly APAA-C18, by heating different types of foodstuff. They confirm previous investigations that suggest there is no correlation between a product’s original unsaturated fatty acid content and the distribution of APAA-C18 isomers after heating, recommending further investigation. Finally, the authors discuss how the presence of APAA-C20 is not unique to aquatic products in ancient pottery. They suggest that the ratio of APAA C20/C18 could provide an additional tool for identifying aquatic products in ancient pottery and provide a comparison with archaeological pottery to demonstrate its application.
Significance of paper:
Overall, the paper is an important contribution as it provides new knowledge about the formation of APAAs, highlights the need for caution for their use as a diagnostic marker and proposes novel criteria to distinguish between aquatic and terrestrial products. These results have implications for how scholars in the field of organic residue analysis characterise the processing of aquatic products in ancient pottery around the world.
General comments:
This paper is a good fit for Archaeometry, however, the statistical analyses are problematic, and several conclusions in the paper hinge on inappropriate statistical tests (detailed in the comments below).
Comments on structure and clarity:
A clear explanation of the rationale and research questions explored by the experiments before Methods and Materials would bring clarity to the reader. A table summarising the different types of experiments conducted would also improve understanding as there are several of them. Similarly, it is not clear at the start that the experimental research is supplemented with comparisons to archaeological examples. This should be specified in the introduction. I recommend improving language and precision wherever possible
Comments on statistical analyses:
The dataset is very interesting, but I recommend the stats are checked carefully by an expert in statistics. For example, it is important to first test if there is an effect (heating/no heating; lab/field; cereals, nuts/animal) and/or an interaction between these main effects using, for example, a linear mixed effects model. Useful reference: Kuznetsova, Alexandra, Per B. Brockhoff, and Rune Haubo Bojesen Christensen. "lmerTest package: tests in linear mixed effects models." Journal of statistical software 82.13 (2017). Only if there is an effect is it possible to look at post-hoc test such as t-tests between groups. Additionally, the interpretation of the PCA requires further explanation, as does the reason behind the summing of E+F isomers.
Specific comments:
Page 1:
Line 6 and Line 12: The ‘biomarker concept’ should be clarified, and the term ‘biomarker’ or ‘lipid marker’ should be used with caution
Line 19: remove ‘and’ before ‘beehive products’
Line 21: replace ‘range’ with ‘types’
Page 3:
Line 8: “long-chain”
Line 12: ‘amount’ should be ‘amounts’
Line 16: should be: “the detection of APAAs in Early Woodland hunter-gatherer pottery in North America has shown that…”
Line 32: “chain-length”
Page 4:
Line 3-21: Can the rationale for the current study be expanded upon? What is missing from our understanding of APAA-C18? How is this study different from Evershed et al. 2008 and Shoda et al. 2018?
Line 28: Remove ‘In addition’
Line 28: which experiments? The ones cited or the ones in the paper?
Line 46: remove ‘the’ before ‘metal ions’
Line 52: remove ‘first’ and comma after ‘undertaken’ in “thermal degradation experiments were first undertaken”
Page 9: Line 5: Rationale behind Kruskal-Wallis tests?
Line 21: Table 1 suggests that APAAs were not formed when rapeseed oil was heated at 150°C or 200°C in evacuated conditions, but instead at 270°C in the absence of pottery powder. Can this be addressed in the discussion?
Line 25: should be written as “described by Matikainen and colleagues (Matikainen et al. 2003).”
Line 39: Full stop required after (Fig S2) and remove ‘and’ before ‘overall’
Page 10:
Line 12: comma to be added after “fully aerobic conditions”
Line 26: ‘salmon’ not ‘salmone’
Lines 26-29: Suitability of Kruskal-Wallis test?
Page 11:
Line 23-28: Figure 2: The image is too small; could be bigger and clearer.
Lines 37-39: It is not clear how the samples coloured in orange (n=9) ‘stand out’ from all the others, or how the PCA suggests that the E and F isomers should be summed. PCA seems to indicate that it is mostly E that defines the group that ‘stands out.’ What is the rationale behind summing E + F isomers?
Line 55-60: Suitability of t-test? Is there an effect of group of foodstuffs?
Page 12:
Lines 8-12: Figure 3: The PCA scatter plot could be larger. Figure caption should explain what the points in orange signify.
Lines 19-24: Is it meaningful to compare the mean E+F isomer contribution to archaeological samples when the sample sizes are so different? I suggest removing Joto entirely.
Lines 48-53: Figure 4: Could you provide data points within the boxplots? The boxplot does not match up with sample numbers provided in PCA.
Page 14:
Lines 10-17: Rationale behind Mann-Whitney test? Why are the sample numbers so small for the experimental samples?
Lines 28-39: Interesting proposition, however, groups have uneven and very small sample sizes. Defining a 0.06 criterion seems premature. Results appear to only indicate that this ratio should be investigated further.
Lines 46-53: Circular reasoning? It is not clear how pottery at Zamostje 2 was mostly used to process freshwater resources.
Page 15:
Lines 3-6: Figure 5: Could you provide data points within the boxplots?
Supplementary materials: Table S1: The tables were cut off after APAAs-C18 isomer E or H.
(page 33) ‘Leak’ should be “leek”; ‘sesam’ should be “sesame”
Decision letter by
Mark Pollard
Cite this decision letter
Reviewer report
2020/02/27
Review of Investigating the formation and diagnostic value of ω-(o-alkylphenyl) alkanoic acids in ancient pottery
Summary of paper:
This paper explores important questions that underpin the diagnostic value of APAAs found in lipid extracts in archaeological pottery, often used to identify the processing of aquatic products. It conducts several relevant experiments: firstly, a series that assess the conditions under which APAAs form, such as time; temperature; in the presence/absence of pottery; and oxic versus anoxic conditions. The paper demonstrates that the relationship between heating of unsaturated fatty acids and formation of APAAs is complex and is not only contingent on heating up to temperatures of 270°C as suggested by Evershed and colleages (Evershed et al. 2008). The question about whether APAAs are formed due to contact with pottery matrix is also of relevance to archaeological contexts. Secondly, the authors also test the degree of resolution offered by APAAs, particularly APAA-C18, by heating different types of foodstuff. They confirm previous investigations that suggest there is no correlation between a product’s original unsaturated fatty acid content and the distribution of APAA-C18 isomers after heating, recommending further investigation. Finally, the authors discuss how the presence of APAA-C20 is not unique to aquatic products in ancient pottery. They suggest that the ratio of APAA C20/C18 could provide an additional tool for identifying aquatic products in ancient pottery and provide a comparison with archaeological pottery to demonstrate its application.
Significance of paper:
Overall, the paper is an important contribution as it provides new knowledge about the formation of APAAs, highlights the need for caution for their use as a diagnostic marker and proposes novel criteria to distinguish between aquatic and terrestrial products. These results have implications for how scholars in the field of organic residue analysis characterise the processing of aquatic products in ancient pottery around the world.
General comments:
This paper is a good fit for Archaeometry, however, the statistical analyses are problematic, and several conclusions in the paper hinge on inappropriate statistical tests (detailed in the comments below).
Comments on structure and clarity:
A clear explanation of the rationale and research questions explored by the experiments before Methods and Materials would bring clarity to the reader. A table summarising the different types of experiments conducted would also improve understanding as there are several of them. Similarly, it is not clear at the start that the experimental research is supplemented with comparisons to archaeological examples. This should be specified in the introduction. I recommend improving language and precision wherever possible
Comments on statistical analyses:
The dataset is very interesting, but I recommend the stats are checked carefully by an expert in statistics. For example, it is important to first test if there is an effect (heating/no heating; lab/field; cereals, nuts/animal) and/or an interaction between these main effects using, for example, a linear mixed effects model. Useful reference: Kuznetsova, Alexandra, Per B. Brockhoff, and Rune Haubo Bojesen Christensen. "lmerTest package: tests in linear mixed effects models." Journal of statistical software 82.13 (2017). Only if there is an effect is it possible to look at post-hoc test such as t-tests between groups. Additionally, the interpretation of the PCA requires further explanation, as does the reason behind the summing of E+F isomers.
Specific comments:
Page 1:
Line 6 and Line 12: The ‘biomarker concept’ should be clarified, and the term ‘biomarker’ or ‘lipid marker’ should be used with caution
Line 19: remove ‘and’ before ‘beehive products’
Line 21: replace ‘range’ with ‘types’
Page 3:
Line 8: “long-chain”
Line 12: ‘amount’ should be ‘amounts’
Line 16: should be: “the detection of APAAs in Early Woodland hunter-gatherer pottery in North America has shown that…”
Line 32: “chain-length”
Page 4:
Line 3-21: Can the rationale for the current study be expanded upon? What is missing from our understanding of APAA-C18? How is this study different from Evershed et al. 2008 and Shoda et al. 2018?
Line 28: Remove ‘In addition’
Line 28: which experiments? The ones cited or the ones in the paper?
Line 46: remove ‘the’ before ‘metal ions’
Line 52: remove ‘first’ and comma after ‘undertaken’ in “thermal degradation experiments were first undertaken”
Page 9: Line 5: Rationale behind Kruskal-Wallis tests?
Line 21: Table 1 suggests that APAAs were not formed when rapeseed oil was heated at 150°C or 200°C in evacuated conditions, but instead at 270°C in the absence of pottery powder. Can this be addressed in the discussion?
Line 25: should be written as “described by Matikainen and colleagues (Matikainen et al. 2003).”
Line 39: Full stop required after (Fig S2) and remove ‘and’ before ‘overall’
Page 10:
Line 12: comma to be added after “fully aerobic conditions”
Line 26: ‘salmon’ not ‘salmone’
Lines 26-29: Suitability of Kruskal-Wallis test?
Page 11:
Line 23-28: Figure 2: The image is too small; could be bigger and clearer.
Lines 37-39: It is not clear how the samples coloured in orange (n=9) ‘stand out’ from all the others, or how the PCA suggests that the E and F isomers should be summed. PCA seems to indicate that it is mostly E that defines the group that ‘stands out.’ What is the rationale behind summing E + F isomers?
Line 55-60: Suitability of t-test? Is there an effect of group of foodstuffs?
Page 12:
Lines 8-12: Figure 3: The PCA scatter plot could be larger. Figure caption should explain what the points in orange signify.
Lines 19-24: Is it meaningful to compare the mean E+F isomer contribution to archaeological samples when the sample sizes are so different? I suggest removing Joto entirely.
Lines 48-53: Figure 4: Could you provide data points within the boxplots? The boxplot does not match up with sample numbers provided in PCA.
Page 14:
Lines 10-17: Rationale behind Mann-Whitney test? Why are the sample numbers so small for the experimental samples?
Lines 28-39: Interesting proposition, however, groups have uneven and very small sample sizes. Defining a 0.06 criterion seems premature. Results appear to only indicate that this ratio should be investigated further.
Lines 46-53: Circular reasoning? It is not clear how pottery at Zamostje 2 was mostly used to process freshwater resources.
Page 15:
Lines 3-6: Figure 5: Could you provide data points within the boxplots?
Supplementary materials: Table S1: The tables were cut off after APAAs-C18 isomer E or H.
(page 33) ‘Leak’ should be “leek”; ‘sesam’ should be “sesame”
Reviewed by
Anonymous
Cite this review
Reviewer report
2020/01/16
In this paper, Bondetti et al. report the results obtained following a series of laboratory and cooking experiments targeted at better understanding the formation of ω-(o-alkylphenyl)alkanoic acids (APAAs) in ancient pottery. The results show that APAAs can form at lower temperatures and require less heating time than previously suggested (Evershed et al. 2008), and will form under aerobic conditions. Based on the results obtained, the authors suggest the E+F isomer index and APAA-C20/APAA-C18 ratio can be used to distinguish between aquatic and terrestrial foodstuffs.
The research is well executed and the paper is well structured and written. The figures and tables allow further clarification and visual representation of the experimental conditions and results. The results produced provide a novel and significant input to the field of organic residue analysis. I have the following comments:
In the Material and Methods section (p.5), in my view the following sentence: These were left open or sealed under nitrogen and heated for 1, 5, 10 or 17 hours at temperature of 100, 150, 200, 250, or 270°C (Table 1), does not correctly portray the laboratory parameters followed, suggesting that rapeseed oil was heated at different temperatures and for different lengths of time, when in fact, these are two separate parameters, one testing the time it takes for the APAAs to form at 270°C, while the other tested the minimum temperature required to produce APAAs, with the duration of the heating being set at 5 hours.
It is well known that plants leave a much lower chemical signature than animal fats when cooked in ceramics. For the cooking experiments, what was the reason for cooking chestnut flour for 5 hours, as opposed to 15 hours for the animal fats?
It would have been beneficial to know the temperature of the internal surface of the pottery during the cooking experiments, as this would have provided the exact temperature at which APAAs were forming in real time. I am assuming that there would be a temperature discrepancy between the outer and inner ceramic surfaces, but I do not know how significant this difference would have been. I am also aware of the difficulties in fixing the thermocouples to record the temperature on the interior surface, so this is more of a general comment.
I cannot fully follow the logic of the following sentence (p. 8): Interestingly, the relative abundance of APAAs is higher following burial, especially for cooked salmon where APAAs were only clearly visible after burial. This is probably due to the relative loss of other more soluble compounds. If the APAA recovery percentages reported here are relative to the total lipid quantified, then this is to be expected since one would expect less lipid after burial, so the percentages of the APAAs will of course be higher. It would be interesting to know the actual quantification, and how and if the quantities of APAAs formed are significantly affected by burial.
On p. 9, it is reported that heating or the duration of the heating does not affect the formation process of the APAAs, however what about burial? Do we know for a fact that after their formation during heating, the isomer distributions and the APAA-C20/APAA-C18 ratio are not affected by decay during burial and differential preservation?
In the results section, the mean is used when comparing E+F isomer indices, while in the boxplots (Figs 3 & 4), the median is reported. The values reported appear to be the same. Please clarify.
With regard to the E+F isomer index, how do mixtures of foodstuffs influence this ratio? What should the contribution of mixtures of aquatic, plant and animal fats/oils be to have an effect on the ratio between E+F and C+I isomers recovered?
Moreover, on p. 13 it is stated that: it may not be possible to predict the APAA-C18 distribution based on a product’s original UFAs content, because contrary to work on UFA standards, the isomeric distribution of the foodstuffs after heating showed no clear correlation with their fatty acid content. How does this support the E+F isomer index? Can we therefore still apply this index to general categories, i.e. cereals/nuts, vegetables/fruits, terrestrial animals and aquatic animals, or will this change depending on the species and therefore requires more experimental work before we can confidently use the E+F isomer indices?
The criteria for establishing an APAA-C20/APAA-C18 ratio of >0.006 for distinguishing aquatic resources from terrestrial ones have not been clearly defined in the text, but need to be inferred by the reader. The authors rightly question the stability of this ratio on p. 14, as differential degradation could lead to this number changing depending on climatic and environmental conditions influencing decay during burial. While recognising that this is an important finding and should be published, in my view, it should be made more evident that a value of >0.06 is obtained under specific burial conditions. This experiment needs to be expanded to encompass different burial conditions, as generalising such a ballpark figure now might risk erroneous interpretations in future research.
There is a significant sample bias between the archaeological samples included in this study: Zamostje 2 having 46 samples and Joto only 2 samples, as reported in Figure 3.
In Table 1 there are two entries for Rapeseed oil being heated for 5 hours at 200°C, both unsealed and with ceramic powder present, yet in one APAAs are reported and in the other not. In the Table S1, only three experiments are reported for this category, as opposed to five in Table 1. Moreover in the text, four tests are reported on p. 4 and five on p.5. Please check.
In Figure 4, it is recommended that the plot representations are added, even if they are the same as Figure 3, i.e. median, range and quartiles. (Also, please see point 5.)
In Table S2, APAAs-C18 are reported in samples for which the isomer distribution is not reported. Why is this?
For the purposes of furthering this research, in my view, a fully labelled chromatogram should be presented in Figure 1, with the suite of APAAs highlighted as an inset or vice versa.
What about the results from the unheated fats? Where APAAs found here?
Reviewed by
Anonymous
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Investigating the formation and diagnostic value of ω-(o-alkylphenyl)alkanoic acids in ancient pottery

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