Review Reports - Pilot Study on Fingerprinting the Isotopic Signatures of Fossiliferous Limestones as a Tool for Forensic Investigation of Fossil Trafficking (Cretaceous Crato Formation, Brazil)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper addresses the problem of illegal fossil trafficking and suggests a forensic approach based on the δ¹³C and δ¹⁸O isotope composition of limestones containing fossils. The application to the Lower Cretaceous Crato Formation is valuable, particularly in supporting law enforcement efforts to trace the provenance of illegally extracted material. The work establishes an isotopic range for the fossiliferous “sete cortes” bed and demonstrates proof-of-concept by comparing the data obtained with those from the Codó Formation (Brazil) and the Green River Formation (USA).

While the application is promising, its present implementation is limited by a constrained dataset and small geographic scope. Carbonate δ¹³C and δ¹⁸O values are known to vary widely both regionally and within a deposit itself, and may be further changed by processes such as recycling of degraded organic matter from the original fossil material or carcass itself. Thus, the present dataset covering only two Brazilian formations and one North American formation is not sufficent to claim robust global applicability.

I strongly recommend that the study be described as a preliminary investigation establishing methodological groundwork for a much-needed global database of stable isotope signatures in fossiliferous carbonates. To enhance discriminatory power, the authors should also consider integrating inorganic geochemical parameters (e.g., trace and rare earth elements measured by ICP-MS) alongside isotope data. Similar integrated provenance studies in other fields such as Pilgrim et al. (2010) on tea—have achieved high classification accuracy (97.6%) by combining stable isotope and trace element profiles.

In summary, this paper gives a valuable framwork for developing a global forensic framework to combat fossil trafficking. I can recommend publication provided the manuscript is revised to:

Recast the work as a proof-of-concept rather than a fully developed tool.

acknowledge the limitations imposed by the small dataset.

Suggest future expansion to include isotopic and trace element data from fossiliferous carbonates worldwide.

Accept with major revisions.

Author Response

REVIEWER 1

While the application is promising, its present implementation is limited by a constrained dataset and small geographic scope.

REPLY: We agree with Reviewer 1 with the necessity to tune down the implementation of the method because it is based on a limited (geographically, stratigraphically and in terms of depositional environment) dataset. We have modified the text in the Title, Abstract, Introduction, Methods and Discussion highlighting that this first pilot study refers to a constrained dataset. However, the proposed method is applied exclusively to fine laminated micritic limestone containing fossil vertebrates and not to a wide range of marine or lacustrine rocks.

Carbonate δ¹³C and δ¹⁸O values are known to vary widely both regionally and within a deposit itself, and may be further changed by processes such as recycling of degraded organic matter from the original fossil material or carcass itself. Thus, the present dataset covering only two Brazilian formations and one North American formation is not sufficient to claim robust global applicability.

REPLY: As above, we agree with Reviewer 1 and realised that we need to highlight that the implementation of this method is a preliminary scouting of a further wider parametrization of fossil-bearing laminated limestone beds from lacustrine successions because it is based on a limited (geographically, stratigraphically and in terms of depositional environment) dataset. Being aware of the possible diagenetic modification and resetting of stable isotopic data of limestone from deposition to long term burial diagenesis, we have limited our analyses to a specific and distinctive lithology (fine laminated limestone with submillimetre thick laminae) from non bioturbated bottom of a lacustrine basin and fossil-bearing. These lithological and lithofacies criteria, that can be achieved by visual inspection, already restrict the variables for the identification and the field of possible isotopic parameters. The presented data refer only to the laminated limestone embedding the fossil, not to the fossil itself, at a few cm to decimetre from the preserved fossil to try to capture the limestone composition not affected by the carcass itself. The Crato Fm. stands out worldwide as one of the best sites in terms of preservation of fossils, along with some studies pointing a very low deposition rate, thus implying special conditions on the lake bottom and shallow burial to avoid organic matter degradation.

I strongly recommend that the study be described as a preliminary investigation establishing methodological groundwork for a much-needed global database of stable isotope signatures in fossiliferous carbonates.

REPLY: We agree with the Reviewer and we have reported this in the Title, Abstract and Discussion but to our knowledge no other studies applied this methodology to identify smuggled fossils and that we focus on a distinctive lithofacies and depositional environment of lacustrine laminated limestone.

To enhance discriminatory power, the authors should also consider integrating inorganic geochemical parameters (e.g., trace and rare earth elements measured by ICP-MS) alongside isotope data. Similar integrated provenance studies in other fields such as Pilgrim et al. (2010) on tea—have achieved high classification accuracy (97.6%) by combining stable isotope and trace element profiles.

REPLY: We thank the Reviewer for this suggestion that we have implemented in the text quoting the Pilgrim et al. paper.

In summary, this paper gives a valuable framework for developing a global forensic framework to combat fossil trafficking. I can recommend publication provided the manuscript is revised to:

Recast the work as a proof-of-concept rather than a fully developed tool.

REPLY: this was implemented in Introduction at lines 146-150 and Discussion from lines 539 to 579.

acknowledge the limitations imposed by the small dataset.

REPLY: this was implemented in Discussion from lines 539 to 579.

Suggest future expansion to include isotopic and trace element data from fossiliferous carbonates worldwide.

REPLY: this was implemented in Discussion from lines 539 to 579.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Review Report

Manuscript Title: Fingerprinting the Isotopic Signatures of Fossiliferous Limestones as a Tool for Forensic Investigation of Fossil Trafficking (Cretaceous Crato Formation, Brazil)

General Assessment

This well-structured manuscript presents a novel study that applies stable carbon and oxygen isotope analysis of fossiliferous limestones as a forensic tool to combat the illegal trafficking of fossils. The study focuses on the Cretaceous Crato Formation in Brazil. This is a timely and relevant topic, both scientifically and socially, as it combines sedimentary geochemistry, palaeontology and forensic geoscience. The study is original in its targeting of the 'sete cortes' ethnostratum, a lithostratigraphic unit closely associated with valuable vertebrate fossils, and in its proposal to develop a global isotopic database for provenance verification.

The paper is well-written, sound in its methodology, and logically structured. The integration of published data with new isotopic analyses is handled appropriately, and the statistical treatment of the datasets is thorough. The graphical outputs (e.g. scatterplots and boxplots) are clear and relevant. However, some figure captions could be expanded to improve their interpretability when viewed in isolation.

In my opinion, the main strengths of the manuscript are:

In terms of originality and significance, the forensic application of isotopic fingerprinting to fossil-bearing limestones is a novel and significant concept. The authors successfully demonstrate the potential for use in practical law enforcement cases involving the repatriation of fossils.

In terms of methodological rigour, the selection of data from the literature and the new analyses is systematic, and the stratigraphic and ethnostratigraphic criteria applied to group the data are well justified.

In terms of interdisciplinary relevance, the study effectively integrates sedimentary geology, stable isotope geochemistry and forensic science, making it accessible to a broad Geosciences readership.

The inclusion of IQR, standard deviation and correlation coefficients in the statistical analysis strengthens the robustness of the interpretations.

Before publication, the authors should make the following improvements:

The abstract is informative but could be more concise. This could be achieved by reducing the background detail and emphasising the key findings (e.g. specific isotopic ranges and forensic applicability).

Some figures (e.g. Figures 5–9) could be improved by making the forensic implications of the observed trends clearer.

Captions should summarise the main message of each plot and be fully self-contained.

Comparative Framework – While the Codó and Green River formations are valid analogues, the manuscript could briefly justify why these were chosen over other fossiliferous lacustrine formations worldwide.

The discussion acknowledges that some isotopic datasets were derived from non-fossiliferous samples. This limitation should be stated earlier, for example, in the 'Materials and Methods' section, to manage reader expectations.

Although meteoric alteration is discussed in the context of previous studies, the manuscript could expand on the implications of diagenetic overprinting for the reliability of forensic discrimination.

The conclusion is strong but could provide more specific guidance on the practical next steps for law enforcement agencies and policymakers, such as recommended database structures, sample storage and analytical protocols.

Specific Minor Comments

Line 393–397: When discussing potential overlaps with other formations, briefly state whether these overlaps pose a practical limitation for the method.

Table 2: It may be useful to highlight the “sete cortes” statistical parameters in bold to make them easier to locate.

Author Response

REVIEWER 2

General Assessment

The paper is well-written, sound in its methodology, and logically structured. The integration of published data with new isotopic analyses is handled appropriately, and the statistical treatment of the datasets is thorough. The graphical outputs (e.g. scatterplots and boxplots) are clear and relevant. However, some figure captions could be expanded to improve their interpretability when viewed in isolation.

REPLY: Figure captions of Figures from 5 to 9 have been expanded as well as their reference in the text.

In my opinion, the main strengths of the manuscript are:

In terms of interdisciplinary relevance, the study effectively integrates sedimentary geology, stable isotope geochemistry and forensic science, making it accessible to a broad Geosciences readership.

The inclusion of IQR, standard deviation and correlation coefficients in the statistical analysis strengthens the robustness of the interpretations.

Before publication, the authors should make the following improvements:

REPLY: Abstract has been reduced as requested from 234 to 192 words integrating also the comments by the other reviewers.

Some figures (e.g. Figures 5–9) could be improved by making the forensic implications of the observed trends clearer.

REPLY: we explain these implications in the Dicussion text eferring to the Figures.

Captions should summarise the main message of each plot and be fully self-contained.

REPLY: Figure captions of Figures from 5 to 9 have been expanded as well as their reference in the text.

REPLY: These lacustrine units were selected for the similarities of depositional environment and lithofacies of laminated limestones and for their broad range of geographic and stratigraphic features. This is explained in the Methods at lines 333-351.

REPLY: This is reported in the Methods, Table 2 and figures there are both fossiliferous and non fossiliferous limestones.

Although meteoric alteration is discussed in the context of previous studies, the manuscript could expand on the implications of diagenetic overprinting for the reliability of forensic discrimination.

REPLY: Being aware of the importance of diagenetic modifications, we have included in the Discussion the necessity of petrographic analysis of thin sections. However, the proposed approach aims at constraining the oxygen and carbon isotope data of the fossil-bearing laminated limestone independently from the pristine lacustrine or diagenetic character of this signature. Independently from the origin, the goal is to restrict the range of the distinctive isotopic values of the fossil-bearing laminated limestone.

REPLY: We thank the reviewer for this suggestion and we have expanded the Discussion with some suggestions from line 554 to line 554.

Specific Minor Comments

Line 393–397: When discussing potential overlaps with other formations, briefly state whether these overlaps pose a practical limitation for the method.

REPLY: Limitations of the methods are reported now in the Methods and Discussion.

Table 2: It may be useful to highlight the “sete cortes” statistical parameters in bold to make them easier to locate.

REPLY: Thank you for the suggestion, Table 2 has been modified accordingly.

Reviewer 3 Report

Comments and Suggestions for Authors

The article discusses how to identify fossil material from a natural deposit in a South American location using stable carbon and oxygen isotopes. This identification would allow the competent authorities to reclaim it if it were looted for illegal sale. In essence, the aim of this work is to verify whether the isotopic composition of the continental carbonate containing the local fossil heritage allows for its identification. The authors also claim to have found in the isotopic technique an innovative solution that can be generally used for the identification of looted fossil material (Lines 18-21 and 54-57).

Whatever the authors say, the purpose of the work fails because the isotopic composition of the deposit in question is very common among continental carbonates and specifically among carbonates deposited in lacustrine environments. The fact that the isotopic variability of the deposit is low has no impact on the attribution of an unknown sample that has an isotopic composition similar to that of the deposit. In this case, isotopic analysis cannot provide a crucial contribution to the identification of a fossil of unknown provenance, but rather a further confirmation of an ancillary nature to a previous general study. However, isotopic correspondence alone would not be sufficient, if not misleading.

Furthermore, the C and O isotope technique might be useful only in their specific case and should be verified every time.

That said, the article could be significantly shortened, sticking closely to its purpose, rather than delving into various descriptions, which might be interesting to those unfamiliar with the context of the fossil deposit, but which are not scientifically relevant.

Minor flaws are the following:

- it is not at all clear what criteria were used to select the two deposits to be compared, which also have a much smaller amount of data than the deposit of interest;

- the statistics are not well presented;

- since the data are entirely from literature, there is no specific sampling strategy for the purpose of the work.

Perhaps a different approach, based on comparing various indicators (including isotopes) among the major fossil deposits with which the deposit studied in this work could be confused, might be more interesting. However, I am not aware of the existence of a context involving fossil material deposits of international importance.

While I believe that combating the plundering of a country's historical and natural heritage is of primary importance, I believe that isotopic technique for the specific purpose presented in this study should be framed differently.

Author Response

REVIEWER 3

Whatever the authors say, the purpose of the work fails because the isotopic composition of the deposit in question is very common among continental carbonates and specifically among carbonates deposited in lacustrine environments. The fact that the isotopic variability of the deposit is low has no impact on the attribution of an unknown sample that has an isotopic composition similar to that of the deposit. In this case, isotopic analysis cannot provide a crucial contribution to the identification of a fossil of unknown provenance, but rather a further confirmation of an ancillary nature to a previous general study. However, isotopic correspondence alone would not be sufficient, if not misleading.

REPLY: In line with the comments of the other reviewers, we have modified the presentation of the proposed approach as a proof-of-concept tool highlighting the preliminary character of the method, the limitation of the dataset in terms of geographic and stratigraphic parameters, and the necessity of integrating fundamental distinctive character as the palaeontology (taxonomy of the fossils), depositional environment and lithofacies (lacustrine laminated limestone), diagenesis (petrographic analysis of thin sections) and other geochemical parameters.

We are aware of the range of isotopic values of lacustrine (and in general non marine) carbonates (see Figure below from Della Porta 2015), but the identified range of the fossiliferous laminated limestone matches with few known examples and specifically with the Eocene Green River Formation from Wyoming.

We do not claim that the isotopic signature of fossiliferous limestone should be used alone as an identification parameter, we propose that this must be included together with sedimentologic, palaeontologic and depositional environment features as a discriminating tool.

We also do not claim that our study is unique but, so far and to our knowledge, we did not find similar published studies on lacustrine fossiliferous limestones.

Compilation of oxygen and carbon isotope data from various types of non marine carbonates redrafted and modified from Della Porta (2015). Della Porta, G. Carbonate build-ups in lacustrine, hydrothermal, and fluvial settings: comparing depositional geometry, fabric types, and geochemical signature. In Microbial Carbonates in Space and Time: Implications for Global Exploration and Production Bosence DW, Gibbons KA, Le Heron DP, Morgan WA, Pritchard T, Vining BA, Eds.. London: Geological Society of London, Special Publications, 418, 2015. p. 17-68.

Furthermore, the C and O isotope technique might be useful only in their specific case and should be verified every time.

REPLY: The purpose is in fact that the technique should be used when other observations, lithological and paleontological, are insufficient for the non expert, non palaeontologist observer.

REPLY: We appreciate this advice and we tried to reduce the text where redundant but we think that a contextualization of the illegal trade of fossils is instrumental to comprehend the significance of the proposed approach.

Minor flaws are the following:

- it is not at all clear what criteria were used to select the two deposits to be compared, which also have a much smaller amount of data than the deposit of interest;

REPLY: We have explained this in the Methods (lines 321-342): the selection is based on the similarity of laminated limestone and isotopic signature combined with a wide geographical and stratigraphic spectrum.

- the statistics are not well presented;

REPLY: We would appreciate more explanatory details on this comment and guidelines on how we can improve the current presentation of the statistical data.

- since the data are entirely from literature, there is no specific sampling strategy for the purpose of the work.

REPLY: The use of literature data is aimed to have a broad and unselected dataset to identify the correct groupings to constrain the fossil-bearing laminated limestone.

REPLY: We have highlighted that the isotopic data are proposed as a supportive tool after lithological, paleontological and facies inspection and might help the police officers, who are untrained in palaeontology and vertebrate taxonomy, to identify the geographical origin of the smuggled fossils. In fact, in the Introduction we wrote “These signatures, which can be obtained with a non-destructive and easily accessible technique [24], will be used as an additional identifier of the geographical origin of specimens seized by the Brazilian Federal Police.”

REPLY: We hope that the changes made to the reviewed paper, remarking the necessity of lithological, lithofacies, depositional environment, petrographic, diagenetic and varied geochemical parameters, make the proposed approach more adequate for the reviewer evaluation.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The comments on the first version of this article are largely the same. The authors did not address any of the points raised, but presented a revised version very similar to the first version.
What are those fossil deposits that the deposit studied in this work might be confused with? Essentially, how should the database be structured to compare an unknown sample? There is no answer to these questions in the article.
The authors argue that one of these deposits could be the Green River Formation (but only for an inexperienced person! It is unclear why an inexperienced person would judge whether or not a fossil belongs to a particular provenance). That said, the authors do not even dig into this comparison - it appears that the Green River Formation data overlap to some extent with those of the Sete Cortes.
The other crucial aspect that cannot be resolved is that the isotopic composition of the deposit in question is very common among continental carbonates.
I had suggested drastically reducing the length of the article, and although the authors agree, it seems even longer than the first version.
The data presentation is cluttered, and many of the data groupings are useless to me. What is the point of comparing regression lines across data groups? the purpose of data analysis should be to demonstrate the discriminatory ability of the method(s) used in identifying groups, not trends.
For these reasons I still believe that the article cannot be accepted for publication.