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Article

Advanced Isotopic Techniques to Investigate Cultural Heritage: The Research Activities at the iCONa Laboratory

by
Noemi Mantile
*,
Simona Altieri
,
Maria Rosa di Cicco
,
Valentina Giacometti
and
Carmine Lubritto
*
Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Mediterranean bioArchaeological Research Advances (MAReA) Centre, Università degli Studi della Campania “Luigi Vanvitelli”, Via Vivaldi 43, 81100 Caserta, Italy
*
Authors to whom correspondence should be addressed.
Heritage 2025, 8(8), 296; https://doi.org/10.3390/heritage8080296
Submission received: 13 June 2025 / Revised: 18 July 2025 / Accepted: 23 July 2025 / Published: 24 July 2025
(This article belongs to the Special Issue It's All a Matter of Time: Exploring the Past, Present and Future of Artworks through Dating, Authentication, Material Characterization, Monitoring and Restoration)
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Abstract

Isotopic analyses are useful tools with a wide range of applications, including environmental studies, archaeology and biomedicine. Founded in 2019 at the University of Campania “Luigi Vanvitelli”, the iCONa laboratory specialises in stable isotope mass spectrometry, with a particular focus on cultural heritage. The laboratory performs carbon, nitrogen and oxygen isotopic analyses, including the most recent advances in compound-specific stable isotope analysis of amino acids (CSIA-AAs). In addition to these analytical services, iCONa provides chemical and physical sample preparation for a variety of sample types. This paper focuses on our applications in the field of cultural heritage, exploring how the analysis of stable isotopes performed on archaeological remains can be used to reconstruct past subsistence strategies and human behaviours. We also discuss the challenges inherent in isotopic analysis and recent methodological advances in the field.

1. Introduction

Isotopic techniques have become powerful and versatile tools across a wide range of disciplines, including environmental and ecological sciences [1], archaeology [2], food traceability [3,4], biomedicine [5] and forensic investigations [6,7]. At the core of these applications lies the concept of isotopes, i.e., atoms of the same element which differ in the number of neutrons inside their nucleus [8]. In stable isotope analysis, the isotopic composition of an element is typically expressed as the ratio of its heavier to its lighter isotope [9]. The “delta notation” (δ) expresses the relative difference in this ratio in a sample compared to a defined standard, typically reported in parts per thousand (‰) [10].
A key advantage of isotopic analysis is its ability to differentiate between substances that are chemically identical but originate from different sources, thereby enabling the tracing of specific elements or molecules. The technique is also micro-destructive, requiring only milligram or sub-milligram quantities of material, thus minimising sample loss. This is especially relevant when analysing irreplaceable materials, such as archaeological specimens [11,12,13].
In the framework of these relevant methodologies, the iCONa lab (“isotopic Carbon, Oxygen and Nitrogen analysis laboratory”) was founded in 2019 within the Department of Environmental, Biological and Pharmaceutical Sciences and Technologies at the University of Campania “Luigi Vanvitelli”, with the primary function of performing stable isotope mass spectrometry analysis, with applications principally in archaeometry, environmental, food and biomedical fields. Moreover, the iCONa lab contributes to cultural heritage research within the MAReA (Mediterranean bioArchaeological Research Advances) centre, an inter-university laboratory that is jointly operated by Sapienza Università di Roma and University of Campania “Luigi Vanvitelli”.
The first facilities installation took place in March 2019, with subsequent years witnessing the incorporation of additional instrumentation. This expansion aimed to enable the isotopic analysis of carbon (δ13C), nitrogen (δ15N) and oxygen (δ18O), as well as the determination of carbon and nitrogen concentrations (%C and %N) across various sample types, including solid, liquid and gaseous samples. A recent development has been the expansion of a specific section dedicated to compound-specific analysis, with the result that amino acids and lipids can now be measured.
In addition to the measurement of isotopic ratios, the laboratory provides a section of physical-chemical sample preparation, with specific protocols developed depending on the type of sample to be analysed. This allows organic and inorganic sample preparation also for radiocarbon (14C) dating and strontium isotope analysis (87Sr/86Sr), being performed in collaboration with external research centres.
In this paper, we explore our applications in the cultural heritage field, with specific emphasis on stable isotope analysis conducted on archaeological remains, including human and faunal skeletal samples, and charred botanical materials. This technique is widely employed to investigate research questions concerning ancient subsistence strategies, environmental conditions, mobility, animal management and agricultural practices of past populations, allowing to shed light on sociocultural developments, trade networks and adaptations to environmental changes [9,14].
However, isotopic analyses present inherent challenges and limitations that can compromise the reliability of the data. Challenges associated with sampling—including representativeness, sample size, and ethical and scientific concerns—must be considered [12,15,16]. Standardising pretreatment protocols is also essential to minimise variability and contamination, supported by detailed documentation of all chemical steps to ensure methodological transparency and allowing for data reproducibility [17]. Moreover, data interpretation can vary significantly depending on the scale of analysis, which should be appropriately tailored according to the research question. Consequently, it is imperative to address these issues to ensure the validity of the analytical results and data interpretation.
Here we provide an overview of our applications in the cultural heritage field, including a description of the facilities utilised, the type of samples pretreatment and our progression towards more advanced isotopic techniques. By highlighting these elements, we aim to address the evolving methodologies that can enhance our understanding of past human behaviours through stable isotope analysis, with a specific focus on the investigation of past subsistence strategies.

2. Materials and Methods

2.1. Facilities

The laboratory is equipped with high-precision analytical instrumentation, all branded Thermo Scientific (Bremen, Germany). An Isotope Ratio Mass Spectrometer (IRMS Delta V Advantage, Thermo Fisher Scientific, Waltham, MA, USA) is used to measure isotopic ratios, based on the principle of separations of ions with different masses, permitting the measurement of their relative abundance [18]. The instrument is provided with a dual inlet system for direct measurement of gaseous samples, while is connected through a Helium flow to peripherals for solid and liquid samples, each peripheral depending on the specific isotopic ratio of interest.
For δ13C and δ15N analysis, an Elemental Analyser (Flash EA 1112 series, Thermo Fisher Scientific, Waltham, MA, USA) allows the combustion of solid and liquid samples at 1020 °C by means of O2 gas. Specifically, the samples are weighed in tin capsules, with masses from 0.8 to 2 mg, according to the archaeological material to be processed, and uploaded into an autosampler. After combustion, the gases produced from the bulk samples undergo a red-ox reaction in a quartz tube, manually packed with specific reagents. In particular, we use chromium oxide, silvered cobalt oxide and reduced copper wires, separated by quartz wool. In this way, CO2 and N2 gases are obtained and subsequently separated by a pre-packed chromatographic column, which operates at 60 °C. Before the GC column, a filter is placed to remove water from the sample, filled with magnesium perchlorate. The EA, standing alone, also enables the measurement of carbon and nitrogen concentrations, by means of a Thermal Conductivity Detector (TCD, Thermo Fisher Scientific, Waltham, MA, USA).
For δ18O analysis, the peripheral used is a Thermo Chemical Elemental Analyzer (TC-EA, Thermo Fisher Scientific, Waltham, MA, USA), where pyrolysis occurs at 1350–1450 °C, depending on the organic or inorganic origin of the sample, respectively. In this case, samples are weighed in silver capsules, with weights in the range of a few mg, but still uploaded in an autosampler, as for the EA. The reaction happens in a glassy carbon tube filled with granulate glassy carbon and positioned in a ceramic tube. The catalytic breaking of molecules at high temperature generates CO (and N2, chromatographically separated) gas on which the oxygen isotope ratio is measured.
For compound-specific isotope analysis (CSIA), the laboratory is equipped with a Gas Chromatograph (Trace 1310- GC Isolink II, Thermo Fisher Scientific, Waltham, MA, USA), which allows the selection of specific molecules whose isotope ratios can be measured. The instrument can measure both δ13C and δ15N, as it is equipped with a liquid nitrogen trap that retains the CO2 produced by the combustion of the sample, allowing only N2 to pass through. Depending on the molecule to be measured (amino acids or fatty acids), a specific chromatography column is assembled. In this case, the sample undergoes a specific chemical pretreatment (derivatisation), to enhance the volatility of the molecule to be detected [19]. The system is equipped with a 150-position autosampler.
All the peripherals are connected to the IRMS in continuous flow, by means of a CONFLO IV, which allows the passage of both the sample and reference gases, alternated according to the selected measurement method. The reference gases used are CO2 for δ13C measurements, N2 for δ15N measurements and CO for δ18O one.

2.2. Measurement Methods

The entire instrumentation is managed by Isodat 3.0 Thermo Scientific software, which allows the setting up of specific measurement methods according to sample type and the isotope ratio to be measured. For example, at the iCONa lab, a method was created for measuring the combined δ15N and δ13C of collagen samples for paleodiet investigation. This method exploits the carbon versus nitrogen dilution method and allows also for the measurement of C and N concentrations in a single 480-s run using around 1 mg of each sample. Given the often limited and valuable nature of collagen samples, this method offers a micro-destructive approach that maximises data recovery from approximately 1 mg of each sample, alongside significant time saving.
In our laboratory measurement protocols, internationally certified standards (IAEA standards) are used to accurately calibrate raw measurements. They are included in each run list, every 24 samples, also to check the quality of the measurement and so to calculate analytical precision on precious samples that do not allow for replicate analysis. Typical analytical precision is 0.1‰ for δ13C and 0.2‰ for δ15N.
Figure 1 shows the standardised variable Z calculated for the δ13C measurements of the standard IAEA-CH-6 (sucrose, certified δ13C = −10.449 ± 0.033 ‰) over three years (2020–2022), and of the standard IAEA-CH-3 (cellulose, certified δ13C = −24.724 ± 0.041 ‰) for approximately one and a half years (2023- half 2024). Both the standards are used for the sample’s calibration in the combined measurements of carbon and nitrogen isotopes, whereas the CH-6 standard was replaced with the CH-3 one, due to its lack of availability until recently. The Z value quantifies the distance between a data point and the mean of a dataset; it is therefore obtained by the difference between each observed value and the average, related to its own standard deviation [20].
Figure 1 indicates that all the measurements are in the range of two standard deviations (Z values less than 2) from the mean and most in that of one standard deviation (Z values less than 1).

2.3. Overview of Samples Processed

Figure 2a shows the quantitative distribution of the samples processed at the iCONa lab, from the installation year to date, divided into disciplinary research fields. In addition, the quantity of the standards used for measurement calibrations and quality check is presented.
A first observation clearly shows that the environmental section prevails, specifically because it includes many different applications in this field [21,22,23] and thus there is diversity in the matrices analysed (wood, roots, leaves, soils, microalgal biomass, etc.). The marine biology category specifically regards analysis of different fish species, for studies of changes in trophic levels influenced by environmental and anthropogenic factors.
In addition, it can be observed that biomedical applications represent the smallest slice of the graph, as this field has recently been dealt within our study, but it is clearly expanding due to the numerous collaborations with biological and medical research teams [24].
The typology of archaeological samples processed at the iCONa lab, as presented in Figure 2b, reveals a marked emphasis on osteoarchaeological materials: 57% of the samples consist of bones, while teeth account for 33%. Conversely, archaeobotanical samples, specifically charcoal and seeds, represent a considerably smaller fraction of the laboratory’s workload, at 6% and 4%, respectively. The observed variation in sample frequency directly corresponds to the broad spectrum of research questions addressed by the iCONa lab and its collaborative partners.
Each type of sample necessitates tailored pretreatment protocols to ensure reliable isotopic analyses. The following paragraph will briefly explore the specific preparation techniques needed for each archaeological material.

2.4. Sampling and Pretreating Archaeological Remains for Isotopic Analyses: Challenges and Advantages of Micro-Destructive Techniques

Stable isotope analysis is an inherently micro-destructive technique. However, recent advancements in isotopic methods and sample pretreatment protocols have significantly reduced the amount of archaeological material needed. Despite these improvements, careful sample selection remains essential to balance scientific goals with the preservation of valuable archaeological specimens. Adopting a research-question-driven approach is essential, ensuring that materials are only sampled when non-invasive methods cannot yield the required information. Consequently, an increasing body of research is focusing on establishing best practices for sampling archaeological organic materials for stable isotope analysis, addressing both ethical and scientific concerns associated with the irreversible nature of these procedures [11,12,15,16,25].
Different sampling strategies are required depending on the material type (e.g., archaeological bone or tooth), the specific research question (e.g., early life vs. later life dietary habits) and the type of isotopic analysis being performed (e.g., carbon and nitrogen isotope analysis for diet, or strontium isotope analysis for mobility). Sample preservation also needs to be considered, as diagenesis—chemical, physical and biological alterations occurring in the archaeological record—can impact the integrity of the material and the reliability of isotopic data [26]. Furthermore, effective pretreatment protocols are needed to avoid sample contamination, to promote methodological transparency and to ensure reliable data interpretation [17,27,28,29].
Table 1 provides a summary of the pretreatment protocols, required sample sizes and types of isotopic analysis performed at the iCONa lab, along with the distinct information that each methodology provides. The specified sample sizes are intended for samples in a good state of preservation; samples with poor conservation status usually require increased quantities.
Our laboratory employs a comprehensive analytical approach, integrating established traditional methodologies with advanced isotopic techniques. Notably, our expertise in incremental dentine analysis represents a key differentiator in this field; to our knowledge, our facility is currently the sole laboratory in Italy conducting this specialised methodology in-house. Additionally, our commitment to advancing analytical capabilities is demonstrated by the current development of a dedicated setup for compound-specific isotope analysis of amino acids (CSIA-AAs), a service not currently available in other Italian research facilities.

3. Stable Isotope Analysis to Reconstruct Past Subsistence Strategies with a High Level of Resolution: Recent Methodological Advances

3.1. Stable Carbon and Nitrogen Isotope Analysis: Basic Principles and Limitations

Stable isotope analysis of human bones and teeth has become an essential tool for addressing a wide range of archaeological research questions related to past subsistence strategies and human–environment interactions. The main isotopic proxies utilised to investigate past dietary practices are stable carbon (δ13C) and nitrogen (δ15N) isotopes measured on bone and dentine collagen. Bulk collagen stable carbon values allow us to estimate the proportions of C3 plants and cereals (e.g., wheat, barley, rye, legumes, fruits, vegetables) versus C4 cereals (e.g., millet, sorghum) in the diet, and to distinguish between terrestrial and marine dietary sources, as each has distinct carbon isotopic signatures [2,9,14]. In contrast, nitrogen isotopes offer insights into the trophic levels within the food chain and allow us to infer the protein contribution to the consumer’s diet [9,14,35]. Therefore, the combination of stable carbon and nitrogen analysis is essential for comprehensive dietary reconstruction. To accurately interpret human isotopic measurements, it is also necessary to establish an isotopic baseline of both faunal and plant remains. Local baselines are essential for reconstructing agricultural practices and animal management strategies and for gaining more detailed insight into human dietary choices [36,37,38].
However, stable isotope analysis of human skeletal remains presents limitations that can restrain the reliability of dietary reconstructions. One primary drawback is its averaging nature, which often obscures individual variations in diet and fails to capture short-term or seasonal changes, instead reflecting only longer-term dietary patterns [39]. Additionally, the complexity of isotopic signatures—shaped by factors such as metabolic processes, baseline variability and different access to local resources—complicates result interpretation, particularly when trying to estimate the dietary contribution of different food sources that may exhibit overlapping isotopic signatures [40,41].
To address these limitations and improve the resolution of data interpretation, researchers have developed advanced methodologies over the past few decades, including incremental dentine analysis, compound-specific stable isotope analysis of amino acids (CSIA-AAs) and dietary mixing models. Incremental dentine analysis allows us to explore early life dietary changes, linking isotopic signatures to specific life stages and enabling palaeodietary reconstruction with a high temporal resolution [31,32,42,43]. CSIA-AAs enhance the precision of dietary source identification by analysing the isotopic ratios of individual amino acids, allowing for differentiation of various food sources more effectively compared to bulk collagen stable isotope analysis [44,45]. Furthermore, dietary mixing models, which can incorporate non-isotopic prior information, can quantify the relative contributions of the available food sources for past populations [41,46,47].
In the next paragraph, an overview of these methodological and interpretative advances in the isotopic field, which are also employed at the iCONa lab, will be provided. This discussion will highlight how these innovative techniques enhance insights into dietary patterns and refine isotopic-based historical narratives.

3.2. Incremental Dentine Analysis: Investigating Early Life Dietary Habits with a High Temporal Resolution

Due to different turnover rates, bones and teeth provides distinct types of information tailored to the required research question. Bones undergo continuous remodelling throughout life, influenced by factors such as biological sex, health, nutrition, age and the type of skeletal element, incorporating the isotopic signatures of the diet consumed during that period. Consequently, the turnover rate is an essential factor to consider when selecting bone samples for stable isotope analysis, depending on the research focus. For example, ribs, which remodel quickly, reflect dietary intake from the last few years of life, while femurs, which remodel more slowly, capture a longer-term dietary signal over approximately a decade [48,49].
In contrast, teeth do not remodel after they form, which allows them to preserve an in vivo isotopic record of diet from infancy through to adolescence [32,50]. Each tooth forms during a specific life stage, allowing for the study of dietary patterns at different points in an individual’s early life. For instance, the first molar records dietary information from early childhood, while the third molar provides insight into diet during late adolescence [51]. By sampling multiple teeth from the same individual, it is possible to reconstruct a more detailed dietary history over time [52]. However, bulk collagen analysis of tooth dentine provides an averaged isotopic signal across the entire period of tooth formation, limiting the ability to study short-term dietary changes. Refined methods, such as sectioning dentine increments, allow for more precise insights into dietary shifts over shorter periods [31,32,42,43,53,54].
Dentine is a calcified tissue that grows sequentially from the crown of the tooth towards the root apex, forming a series of incremental layers. In humans, the daily secretion rate of dentine ranges from approximately 2 to 6 micrometres (μm) [55]. By sequentially sampling and performing stable carbon and nitrogen isotope analysis on each dentine increment, high temporal resolution insights into past individuals’ dietary habits can be achieved [54]. Over the past 20 years, multiple sectioning techniques have been developed [31,32,42,43,53,54,55]. At the iCONa lab, we employ both the micro-punch method developed by Czermak et al. [32] and the micro-slice technique introduced by Beaumont et al. [31] for sequential sampling of human dentine. Incremental dentine analysis is particularly useful for reconstructing breastfeeding and weaning practices in past populations using measurements of stable carbon (δ13C) and nitrogen (δ15N) isotope ratios on dentine increments from the first permanent molar, which records isotopic signals from approximately three months postpartum to around nine and half years of age [52,56]. During exclusive breastfeeding, infants consume 15N-enriched milk, placing them at a higher trophic level in the food chain compared to their mothers [57]. A decrease in δ15N identifies the transition from exclusive breastfeeding to the introduction of complementary foods (e.g., cereals, fruits), which are lower in protein compared to human milk [58]. The cessation of the decrease in δ15N values, followed by an isotopic plateau, is employed to estimate the end of breastfeeding [57,59].
Consequently, incremental dentine analysis not only enhances our understanding of past dietary habits at a high temporal resolution but also offers detailed insights into the sociocultural dynamics of infant feeding strategies in ancient populations [57].

3.3. Compound-Specific Stable Isotope Analysis of Amino Acids: Overcoming the Limitations of Bulk Stable Isotope Analysis

The application of compound-specific stable isotope analysis of amino acids (CSIA-AAs) has emerged as a transformative approach for reconstructing ancient diets, offering enhanced resolution compared to bulk isotope methods.
By analysing the isotopic signatures of individual amino acids, CSIA enables the disentanglement of complex dietary inputs and metabolic pathways, providing critical insights into past ecological interactions. For nitrogen isotopes, the ability to distinguish between trophic and source amino acids allows for precise determination of trophic positions, revealing the balance between plant and animal protein consumption in human and animal diets [60]. Similarly, carbon isotope analysis of essential amino acids facilitates the tracing of primary producers within food webs, enabling the differentiation of terrestrial and aquatic dietary sources or the identification of C3 versus C4 plant contributions [61]. This specificity is particularly valuable in archaeological contexts where mixed diets, isotopic mixing zones or environmental variability can obscure signals in bulk isotope data [62].
Furthermore, CSIA’s capability to target individual biochemical compounds reduces the impact of diagenetic alterations, which can significantly affect the reliability of bulk analyses [19]. Advances in preparation techniques and analytical sensitivity have also minimised sample size requirements, making CSIA suitable for rare or fragmented archaeological specimens [45,63].
This methodology not only reconstructs dietary practices with a requirement for smaller sample quantities and reduced susceptibility to contamination but also sheds light on broader ecological and environmental conditions, contributing to a holistic understanding of ancient lifeways and their adaptation to changing environments [60,64].

3.4. Bayesian Dietary Mixing Models: Estimating Quantitative Dietary Reconstructions

Bayesian dietary mixing models represent a cutting-edge approach for quantitatively reconstructing individual diets by analysing the relative contributions of various food sources through isotopic data from consumers and their dietary sources [41,46,47,65]. In the field of archaeology, stable carbon and nitrogen isotopic ratios measured on bone collagen serve as the primary isotopic proxies for reconstructing past diets; however, other isotopic ratios and tissue types can also be utilised for this purpose [41,66].
Accurate dietary reconstruction through dietary mixing models requires careful consideration of several key factors. One significant challenge is the isotopic fractionation that occurs during metabolic processes, resulting in diet-to-tissue isotopic offsets [67,68]. These offsets, which accounts for the isotopic differences between ingested foods and the consumer tissues, are typically estimated through controlled feeding experiments [46,47,69].
Another consideration in quantitative dietary reconstructions is the estimation of the isotopic composition of macronutrients—specifically, proteins compared to lipids and carbohydrates—in food remains. Potential variations between the isotopic values obtained from food remains (e.g., faunal bone collagen) and those of the edible parts of the food (e.g., meat protein) require the use of offset corrections [47,69]. Additionally, when dealing with plant and cereal remains, it is essential to account for charring effects that may have altered their in vivo isotopic signatures [46,47].
It is also necessary to consider the macronutrient composition of the food groups available for the studied population [69]. Animal-based foods generally contain higher protein levels, while plant-based foods are predominantly carbohydrate-rich [70]. Moreover, macronutrient composition can vary significantly across different animal species [69]. By considering the average macronutrient profiles of each food group, and their relative uncertainties, dietary mixing models can more accurately reflect the dietary contributions of food sources [47].
Lastly, it is essential to acknowledge dietary routing mechanisms by which carbon and nitrogen from dietary macronutrients (proteins, carbohydrates and lipids) are incorporated into bone collagen [71]. Controlled feeding studies have shown that nitrogen isotopes are sourced from dietary protein, while carbon isotopes predominantly originate from protein, with lesser contributions from carbohydrates and lipids [46,47,71,72].
Advanced models, such as ReSources (previously FRUITS; [46]), effectively address these parameters and their associated uncertainties. Notably, ReSources allows for the integration of non-isotopic prior constraints, which can be derived from historical records, zooarchaeological findings and archaeobotanical data [73,74]. This incorporation significantly enhances the precision of dietary estimates, especially when it comes to distinguishing between food sources that exhibit overlapping isotopic signatures [46,47].
Bayesian dietary mixing models therefore represent a significant advancement in stable isotope analysis, offering a quantitative approach to reconstructing past diets. By incorporating multiple parameters, including diet-to-tissue offsets, macronutrient and isotopic composition of food groups, dietary routing and the possibility to integrate non-isotopic prior information, these models provide more detailed insights into past human subsistence strategies [40,46,69,75,76].
However, it is essential to also acknowledge the limitations of dietary mixing models. The reliability of dietary reconstructions heavily relies on the quality of the input data, including isotopic values for potential food sources to reconstruct local baselines [39,41,70]. Furthermore, while quantitatively reconstructing paleodiets, assumptions of the linearity of isotopic fractionation and the homogeneity of food sources within the selected population may not always accurately reflect the complexities of the human diet [70].

4. Research Lines and Collaborations of iCONa Lab in the Archaeological Field

At the iCONa lab we explore a range of research questions related to past human subsistence strategies, mobility, agricultural and animal management practices across different historical periods and geographic areas, in collaboration with academic and cultural institutions.
A key focus of our research is isotope-based past dietary reconstruction, which we explore with different levels of resolution. One significant line of research delves into the exploration of socioeconomic practices of the Basque region (Spain) during the Middle Ages. Through stable carbon and nitrogen isotope analyses of human, animal and plant remains, we reconstructed the dietary patterns as well as agricultural and animal husbandry techniques utilised by medieval rural communities within this region. This work has been conducted in the framework of an ongoing collaboration with the University of the Basque Country [77,78].
Another research pathway aims at investigating palaeodietary practices with a higher level of resolution, by employing advanced isotopic techniques. We utilised incremental dentine stable carbon and nitrogen isotope analysis to gain detailed insights into breastfeeding and weaning practices across the Roman Empire, as part of a collaborative effort with the Max Planck Institute of Geoanthropology, the Archaeological Park of Pompeii and the Archaeological Park of Ostia Antica. This study inferred a possible link between infant feeding strategies and settlement complexity across the Roman Empire. The results suggested shorter breastfeeding durations in highly complex urban centres like Pompeii (79 CE), compared to rural sites like Ostia AVM (1st–2nd centuries CE), potentially influenced by factors such as access to medical knowledge and socioeconomic conditions [57].
Our collaboration with the Archaeological Park of Ostia Antica also included the investigation of subsistence practices of the Late-Antiquity individuals buried in the Antemurale Area of Portus Romae. The employment of a multidisciplinary approach allowed for the reconstruction of the cultural and biological traits of this population. Stable carbon and nitrogen isotope analyses suggested a diet primarily based on C3 terrestrial resources, while strontium isotope analysis implied their local geographic origins [79].
In pursuit of exploring past human behaviours with multi-scale approaches, one of our research efforts focuses on the collection of extensive isotopic data across various chronological and geographical contexts, particularly emphasising Europe and the Mediterranean Basin. Within the framework of the Mediterranean Bioarchaeological Research Advances (MAReA) centre’s efforts, several isotopic databases have been published in Open Access, serving as valuable resources for bioarchaeological research. These include CIMA: Compendium Isotoporum Medii Aevi [74,80], IsoMedIta: A Stable Isotope Database for Medieval Italy [81], Isotòpia: A Stable Isotope Database for Classical Antiquity [82] and MAIA: Mediterranean Archive of Isotopic dAta [83]. These databases serve as comprehensive isotopic archives that help identify research gaps and investigate a variety of archaeological questions using multi-scale approaches. These activities are framed within the Pandora & IsoMemo initiative “https://isomemo.gea.mpg.de/ (accessed on 22 July 2025)”.
Our ongoing collaboration with the University of Foggia and the University of Salento is contributing to a broader understanding of dietary practices, resource management, past mobility and environmental adaptation in medieval communities from the Apulia region (Southern Italy). A collaborative effort, led by the Max Planck Institute of Geoanthropology, highlighted a farming economy based on cereal production complemented by intensive pig and ovicaprid husbandry in Late Medieval Capitanata. Dietary differences reflected a socioeconomic hierarchy, with elites likely to have greater access to animal protein and higher-quality grains [73].
While palaeodietary studies provide valuable insights into subsistence strategies of ancient populations, radiocarbon dating serves as a useful tool for establishing chronological frameworks within archaeological studies. Radiocarbon dating performed on mortars and charcoals was employed to investigate and reassess the chronologies of the encastellation phenomenon (incastellamento) in medieval Italy. A multidisciplinary approach was employed to establish more precise timelines of and provide novel insights into the formation and transformation processes of fortified structures between the 11th and 12th centuries CE, thereby enhancing our understanding of the historical context and significance of encastellation in northern Italy. This line of research has been carried out within the framework of PRIN 2020 Project “The Times of CASTLES: Multidisciplinary Research for a New Chronology of the Building Sites of Incastellamento (XI–XII centuries) (MIUR protocol: 20203YX58R, CUP: B53C2100029000, PI: Prof. Giovanna Bianchi).
By employing a combined approach of stable carbon and nitrogen isotope analyses alongside radiocarbon dating of bone collagen, we also investigated the individuals buried in the Selvicciola Necropolis, a notable Copper Age burial site in Central Italy. This integrative methodology revealed a burial chronology spanning approximately 2000 years and provided valuable insights into the dietary practices and burial patterns of this community [84].
Our laboratory’s publication record showcases the breadth of research questions we address, employing diverse levels of resolution. These investigations collectively encompass multiple archaeological contexts, with multidisciplinary approaches made possible by our engagement with numerous institutions and collaborative partners.

5. Conclusions

This paper has emphasised the contributions, in the context of the iCONa lab, of stable isotope analysis to the field of cultural heritage, particularly focusing on archaeological remains such as bones, teeth and charred botanical materials. These analytical techniques provide valuable insights into ancient subsistence strategies, past mobility and agricultural and husbandry practices, allowing us to explore how past populations adapted to environmental, political and socioeconomic changes.
The research undertaken herein underscores the importance of acknowledging and addressing the inherent challenges associated with isotopic analyses. Key issues such as the representativeness of sampling and the necessity for methodological standardisation must be carefully examined to uphold the validity and integrity of our results. Tackling these challenges is essential for enhancing the reliability of data interpretation. Furthermore, the ongoing advancement of isotopic methodologies has proven essential in refining our understanding of past subsistence strategies with heightened resolution level.
The iCONa lab distinguishes itself from facilities operating primarily as service laboratories through its dual commitment to methodological advancement and active engagement in original research. This approach, enhanced by our collaborative efforts with a variety of academic and institutional partners, ensures that our analytical advances translate into contributions which address multiple research questions in the cultural heritage field.

Author Contributions

Conceptualisation, N.M., S.A. and C.L., methodology, N.M., S.A., M.R.d.C. and V.G.; software, N.M., S.A., M.R.d.C. and V.G.; validation, N.M., S.A., M.R.d.C., V.G. and C.L.; formal analysis, N.M. and S.A.; investigation, N.M. and S.A.; resources, C.L.; data curation, N.M., S.A., M.R.d.C. and V.G.; writing—original draft preparation, N.M., S.A., M.R.d.C. and V.G.; writing—review and editing, N.M., S.A., M.R.d.C., V.G. and C.L.; visualisation, N.M., S.A., M.R.d.C. and V.G.; supervision, C.L.; project administration, C.L.; funding acquisition, C.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Heritage 08 00296 g001
Figure 1. Variable Z values calculated for the standards IAEA-CH-6 (blue circles) and IAEA-CH-3 (orange circles).
Figure 1. Variable Z values calculated for the standards IAEA-CH-6 (blue circles) and IAEA-CH-3 (orange circles).
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Figure 2. Pie charts representing the distribution of samples analysed at iCONa lab between 2019 and 2024 according to the disciplinary research field (a) and the typology of archaeological samples (b).
Figure 2. Pie charts representing the distribution of samples analysed at iCONa lab between 2019 and 2024 according to the disciplinary research field (a) and the typology of archaeological samples (b).
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Table 1. Summary of the sample types, the required mass for each sample, the pretreatment protocols utilised, the isotopic analyses conducted at the iCONa lab and the types of information that can be obtained from these analyses.
Table 1. Summary of the sample types, the required mass for each sample, the pretreatment protocols utilised, the isotopic analyses conducted at the iCONa lab and the types of information that can be obtained from these analyses.
Sample TypeSample WeightPretreatment ProtocolInformation
Bone300–500 mgBone collagen extraction described in [29], omitting the ultrafiltration stepPaleodiet and
animal management practices reconstruction (δ13C, δ15N)
Radiocarbon dating (14C)
ToothVariableDentine collagen extraction [30]Paleodiet and
animal management practices reconstruction (δ13C, δ15N)
Radiocarbon dating (14C)
VariableIncremental dentine sampling [31,32]Infant feeding practices and early life dietary habits (δ13C, δ15N)
5–10 mgEnamel sampling and strontium extraction [33]Spatial mobility (87Sr/86Sr)
Charcoal/SeedsMin. 5–10 mgStandard ABA protocol [34] or its modified versions [27], depending on the material type and preservation stateAgricultural practices, paleoenvironmental and paleoclimate reconstruction (δ13C, δ15N)
Radiocarbon dating (14C)
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MDPI and ACS Style

Mantile, N.; Altieri, S.; di Cicco, M.R.; Giacometti, V.; Lubritto, C. Advanced Isotopic Techniques to Investigate Cultural Heritage: The Research Activities at the iCONa Laboratory. Heritage 2025, 8, 296. https://doi.org/10.3390/heritage8080296

AMA Style

Mantile N, Altieri S, di Cicco MR, Giacometti V, Lubritto C. Advanced Isotopic Techniques to Investigate Cultural Heritage: The Research Activities at the iCONa Laboratory. Heritage. 2025; 8(8):296. https://doi.org/10.3390/heritage8080296

Chicago/Turabian Style

Mantile, Noemi, Simona Altieri, Maria Rosa di Cicco, Valentina Giacometti, and Carmine Lubritto. 2025. "Advanced Isotopic Techniques to Investigate Cultural Heritage: The Research Activities at the iCONa Laboratory" Heritage 8, no. 8: 296. https://doi.org/10.3390/heritage8080296

APA Style

Mantile, N., Altieri, S., di Cicco, M. R., Giacometti, V., & Lubritto, C. (2025). Advanced Isotopic Techniques to Investigate Cultural Heritage: The Research Activities at the iCONa Laboratory. Heritage, 8(8), 296. https://doi.org/10.3390/heritage8080296

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