https://orcid.org/0009-0003-0210-1040
Figures
Abstract
Birch tar is well-documented for its use as an adhesive in the Middle Palaeolithic of Europe, but other uses remain poorly explored. Drawing from recent arguments suggesting multimodal uses of products such as ochre and birch tar, this study tests the antibiotic properties of birch tar produced experimentally with methods reconstructed from Middle Palaeolithic birch tar finds from Europe. Made from the bark of Betula pendula and Betula pubescens, widely documented for the European Late Pleistocene, we produced birch tar samples using an underground pit method, a condensation method, and a modern tin can method. The birch tar samples were tested for antibiotic properties using the modified Kirby-Bauer disc diffusion antibiotic assay. The resulting inhibition zones, ranging from no effect to 10.5 ± 0.7 mm with a mean of 7.5 ± 0.17 mm, indicate a moderate effect against the Gram-positive Staphylococcus aureus, a bacterium widely known for its role in wound infections. We further establish that the efficacy of antibiotic properties is not related to the production method, with all methods showing a degree of variation. This supports a coevolutionary relationship between medicinal and technological use and production of birch tar during the Pleistocene. The antibiotic properties documented in this study are consistent with the use of birch tar as a wound dressing and skin ointment in Mi’kmaq communities in Eastern Canada, and the use of birch tar in Saami communities of Lapland. Arguing from an underexplored angle between experimental archaeology and ethnopharmacology, we suggest that similar to the ethnographic examples, a use of birch tar beyond exclusively technological contexts must be considered for the Middle Palaeolithic.
Citation: Siemssen T, Oludare A, Schemmel M, Puschmann J, Bierenstiel M (2026) Antibacterial properties of experimentally produced birch tar and its medicinal affordances in the Pleistocene. PLoS One 21(3): e0343618. https://doi.org/10.1371/journal.pone.0343618
Editor: Branislav T. Šiler, Institute for Biological Research, University of Belgrade, SERBIA
Received: December 1, 2025; Accepted: February 9, 2026; Published: March 18, 2026
Copyright: © 2026 Siemssen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: Tjaark Siemssen is funded by the Boise Trust of the University of Oxford and the German Academic Scholarship Foundation (Studienstiftung). Aderonke Oludare was funded by Canadian Institutes of Health Research (CIHR) Project Grant 420598 awarded to Matthias Bierenstiel. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1 Introduction
Birch tar is a well-known component of Neanderthal life, and its documented use in the Late Pleistocene primarily includes its use as a hafting agent [1]. As such, traces of it have been found on archaeological sites in Germany, along the Dutch coast, and in Italy [2–7]. Produced under thermolytic and anaerobic conditions in fired clay pits, the complex production process was likely preceded by generations of experimentation and material engagement leading up to production methods with a higher yield [3]. Following pyrolysis, the extract first includes both oily and aqueous fractions. Subsequent open heating vaporises remaining volatile components, resulting in progressively increased viscosity. Depending on intended use, consistency of the extract can thus range from being an oily fluid to a tar that is hard and brittle at room temperature.
In other regions of the world, conifer resin, beeswax, bitumen, Podocarpus tar, and ochre have been found to have been used as hafting agents in Middle Palaeolithic or Middle Stone Age contexts [8–15]. Ochre has not only been discussed as a component of hafting agents but is also thought to have been used in various other contexts, such as body paint, but also regarding its UV protective and insect repellent properties [16,17].
Birch tar, conversely, has received little attention beyond its use as a hafting agent within the Pleistocene [18,19]. This may partly be due to terminological inconsistencies: Whilst being referred to as birch tar in contexts relating to Pleistocene adhesives, it is also discussed as birch extract elsewhere and carries a number of Indigenous names [20]. Its use as an adhesive and wound dressing is known, for example, from Yakut and Saami communities [21,22]. In Mi’kmaq communities Indigenous to Eastern Canada, it is known as maskwio’mi – translated as birch bark oil – and used for its medicinal properties. Here, the production of maskwio’mi is a practice known through oral history passed down from Elders, and the production method involving tins has recently been reconstructed [23]. The practice likely would have involved ceramic production similar to that known from the European Neolithic until a few generations ago [24,25]. Previous studies have indicated broad-spectrum antibiotic properties of maskwio’mi [26,27].
Recent years have seen a surge of interest in Neanderthal life beyond stone tools, and today, the use of medicinal plants by Neanderthals is known from numerous contexts [28–32]. At El Sidrón, Spain, isotope signatures corresponding to chamomile (Matricaria chamomilla) and yarrow (Achillea millefolium) were detected in the dental calculus of a Neanderthal individual and have been suggested to have been used for its medicinal properties [33]. Yarrow was also detected at Shanidar Cave, Iraqi-Kurdistan [32]. Shanidar also shows that communities of care date far beyond the Holocene; remains from a Neanderthal individual with a severed but healed tibia show dependence upon care from the wider group [30,34].
In this study, we analyse the antibacterial properties of birch tar produced experimentally using European birch variants, relying on methods reconstructed for use in Middle Palaeolithic contexts against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) using a modified Kirby-Bauer disc diffusion assay and discuss the implications of our results against current perspectives on structures of care in Neanderthal communities.
2 Materials and methods
This section describes several extraction methods for producing birch tar through experimental processes with European birch varieties, namely Betula pendula and Betula pubescens, employing techniques reconstructed for Middle Palaeolithic settings. Using a modified Kirby-Bauer disc diffusion method, the antibacterial activity of the birch tar was evaluated against S. aureus and E. coli, Gram-positive and Gram-negative bacteria, respectively.
2.1 Birch tar production and extraction
To produce the birch tar, we adhered to the reconstructions summarised by Schmidt et al. [3,35] and Bierenstiel et al. [23]. Birch bark was obtained from both B. pubescens and B. pendula, the two species that would have been common during the European Pleistocene. The bark was collected in different areas of Germany, including Lkrs. Lüchow-Dannenberg, Lkrs. Cuxhaven, and Rhein-Sieg-Kreis (Table 1). The raw bark was obtained from dead trees on public land, and no permits were required for the described study, which complied with all relevant regulations. Samples of both B. pubescens and B. pendula have been formally identified and deposited in the herbarium of the University of Bonn (herbarium code BONN), and can be accessed via the accession numbers 4155 (B. pendula) and 4156 (B. pubescens). The bark was transformed into tar using three methods: distillation in a tin, distillation in a raised clay structure, and the condensation method (Fig 1).
1: Birch bark. 2: Birch tar. a: Birch tar piece found at the Middle Palaeolithic site of Königsaue, Germany. b: Cobble grove condensation method. c: Buried bark roll method. d: Condensation method. e: Pit roll method. f: Raised structure method. Methods d and f have been used in this study. Modified after: Schmidt et al. [3].
Destructive distillation of birch tar in a tin involved adding densely packed birch bark to a tin with a lid, and piercing holes into the bottom of the tin. A second, smaller container was placed underneath the larger tin to collect the tar formed during the distillation process. A fire was lit on top of the tin, heating the tin for approximately 120 minutes (Fig 2). A tinfoil spoon was used to extract the birch tar from the bottom container. This method is well-documented in historical contexts and produces reliable and consistent results [26,27].
This method is known from oral accounts of Mi’kmaq Elders and was used to produce maskwio’mi, an ointment used to treat wounds and skin conditions. Photograph by Nicolaas Honig.
Distillation of birch tar in a raised structure involved placing a small container in the bottom of a small hole, then adding densely packed birch tar (c. 150 g dried bark has been used for the setups of this study) on top of it and encapsulating it with a thin layer of clay. A fire was then lit on the structure, burning for approximately 120 minutes. The structure was then taken off, and the birch tar was extracted from the bottom container using a tinfoil spoon to minimise contamination. During the production of birch tar using the raised structure method, any contamination by secondary plant products was avoided by using tin foil instead of foliage for the construction of pits to obtain the cleanest samples possible. A commercially available clay (Claytec® Clay topcoat fine 06) was chosen for the construction of the pits to ensure homogenous fine fibre content across all experiments. For a detailed description of the setup of the raised structure method, see Kozowyk et al. [36].
The condensation method involved burning smaller amounts of birch bark under a fireproof surface, such as stone. The condensed birch tar was subsequently collected on the surface and scraped off using a folded tinfoil spoon to minimize contamination of the sample. This method yields smaller amounts of birch tar compared to the two other methods but has been theorised to have been used in the initial stages of birch tar production in Neanderthal contexts [3,35].
2.2 Antimicrobial assay
We obtained S. aureus ATCC 25923 and E. coli ATCC 25922 strains from the American Type Culture Collection (ATCC®; USA). Using these two strains allowed the comparison of efficacy against both a Gram-positive and a Gram-negative strain, since earlier analysis of birch tar (maskwio’mi) produced in a controlled laboratory environment showed broad-spectrum antibiotic properties against both (see section 4.1). The strains were inoculated onto TSA (Sigma-Aldrich) plates at 37 °C for 24 h. The obtained pure cultures were suspended in 0.85% sterile normal saline. The bacterial suspensions were adjusted to achieve a 0.5 McFarland standard. All work about the handling of bacterial cultures was performed in a Labconco® Purifier Microbiological Safety Cabinet Class II type A2. The antibacterial activities of the birch tar were determined by using the modified Kirby-Bauer disc diffusion antibiotic assay [37,38]. We saturated a blank, sterile Whatman Filter paper disc (6 mm in diameter) with 20 µL of birch tar with a concentration ranging from 30-400 mg/mL in DMSO overnight. Suitable bacterial suspensions were uniformly distributed on Mueller-Hinton agar (MHA, Sigma-Aldrich) plates using a sterile disposable cotton swab. Discs containing bark extracts were placed on the inoculated agar plates with sterile tweezers. The standard antibiotics – Gentamicin (10 µg/ disc), BBL, 6 mm Sensi-disc – were used as the positive control, and DMSO as the negative control. The entire antibacterial assay was carried out under strict aseptic conditions, and followed Clinical and Laboratory Standards Institute (CLSI) guidelines [39]. Experiments were performed in duplicate. The plates were incubated at 37 °C for 24 hours. A clear zone of inhibition (ZOI) was measured in millimetres with a ruler and reported descriptively as average measurements. Detailed figures of ZOI experiments (Figures S1 to S6 in S1 File) can be found in the supplementary materials section.
3 Results
The birch tar (BT) yield of the six samples, BT1 - BT6, varied significantly across samples, ranging from 0.06 g to 0.69 g (Table 1). We tested the antibacterial potential of birch tar using a modified version of the Kirby-Bauer disc diffusion method. The results are reported as the mean of zones of inhibition (ZOI, mm). The results (Table 2) showed that birch tar was selectively active and worked against S. aureus but had no effect on E. coli. None of the samples produced any measurable inhibition zones for E. coli (6 ± 0.0 mm), indicating no antibacterial activity against this Gram-negative bacterium. In contrast, the birch tar samples showed varying inhibition levels against S. aureus (Fig 3). The effectiveness depended on both the extraction method and the concentration used. Sample BT5 stood out, showing the highest antibacterial activity with a zone of inhibition measuring 10.5 ± 0.7 mm. This sample was made using the raised structure method with B. pendula bark at a concentration of 133 mg/mL. Other samples (BT1, BT2, BT4, and BT6) showed moderate activity, with inhibition zones between 7.0 and 7.5 mm. One sample, BT3, showed no activity, mirroring its lack of effect on E. coli. As expected, the standard antibiotic Gentamicin (10 μg/disc) used as a positive control was more effective, producing clear zones of 22–23 mm against both bacteria, which is within the CLSI-established quality control ranges for both S. aureus ATCC 25923 and E. coli ATCC 25922. The negative control (DMSO) showed no inhibition.
a: Zone of inhibition of BT5 showing antibacterial activity against Gram-positive S. aureus ATCC 25923. b: Zone of inhibition of BT5 with no antibacterial activity against Gram-negative E. coli ATCC 25922.
4 Discussion
Here, we discuss our samples in direct comparison with samples obtained by other studies to establish differences related to the production method and birch species and consider the implications of our results for Middle Palaeolithic healthcare practices.
4.1 Comparative analysis
Our results show that birch tar has a selective antibacterial effect, working specifically against Gram-positive bacteria like S. aureus. The inhibition zones we recorded ranged from 7.0 ± 0.0 mm to 10.5 ± 0.7 mm, indicating measurable antibacterial activity (Table 2).
This pattern aligns with evidence from other plant-based antimicrobials, which generally exhibit more potent effects against Gram-positive bacteria. The primary explanation for this is their structural variation: Gram-positive bacteria show a thicker peptidoglycan layer, whereas Gram-negative bacteria have an additional outer membrane that can prevent antimicrobial entry [40].
Our findings not only reinforce what past scientific studies have reported, but they also echo traditional knowledge. Birch tar has long been recognised for its antiseptic properties, particularly in treating skin infections, and our study adds new evidence to support its potential use in modern and historical healthcare practices. Similarly, the outcomes align with the traditional practices of the L’nu (Mi’kmaq) people, who have used birch bark extract to address skin infections commonly caused by S. aureus [41–45].
Earlier research has shown that birch tar contains a range of phenolic derivatives, such as catechols and guaiacols [27,44,45], which are likely responsible for its antibacterial and antifungal effect [46,47]. Indeed, Richert et al. [48] reported that phenolic compounds in birch tar contribute substantially to its antimicrobial activity, with pronounced effects on Gram-positive bacteria. The selective inhibition of S. aureus found in our research thus aligns well with the known antimicrobial capabilities of phenols [46,49,50]. This relationship has also been investigated by Agarwal et al. [51], who assessed how polyphenolic content relates to antibacterial effects by comparing bark extracts of wild cherry, larch, and sweet chestnut against S. aureus and E. coli. Their bacterial growth curves indicated that the extracts did not suppress E. coli; in fact, bacterial proliferation was enhanced. In contrast, S. aureus was strongly inhibited. Their sweet chestnut extract was most effective which they linked to its high antioxidant potential and highest total phenol content compared to wild cherry and European larch. In turn, none of our samples showed measurable inhibition of E. coli (6 ± 0 mm), implying limited efficacy against Gram-negative species, likely due to lower phenolic levels and the protective barriers of their outer membrane. This selectivity may also be a result of the distinctions in cell wall structures between Gram-positive and Gram-negative bacteria, reflecting variations in cell surface [52] and the outer membrane structure [53].
Hitherto unpublished data by Kaliaperumal et al. [26], who have assessed the antibacterial properties of industrially produced maskwio’mi made from Betula papyrifera, compares well to the results presented here. The antibacterial activity of their extracts TE1 and TE2 revealed higher inhibition zones against S. aureus strains (18–20 mm for TE1 and 11–12 mm for TE2) compared to our BT samples, which ranged from 7.0 to 10.5 mm. The higher inhibition zones of their extracts may suggest that TE1 and TE2, particularly TE1, possess more potent bioactive compounds than our BT samples. Kaliaperumal et al. [26] identified over 60 bioactive compounds, including phenols, monoterpenes, and triterpenes, in the chemical analyses (GC-MS) results of their extracts. Based on the GC-MS analysis, the authors attributed the higher bioactivity of TE1 to its elevated triterpene concentration, including betulin and lupeol derivatives, which are known for their antimicrobial effects [51].
In contrast, their extract TE2 exhibited lower activity, potentially due to its higher naphthalene derivative content, which may be less effective against Gram-positive bacteria. This may explain the highest antibacterial activity against S. aureus recorded for our sample BT5 (10.5 ± 0.7 mm ZOI) in comparison with other samples BT1, BT2, BT4, and BT6 (7.0 ± 0.0 to 7.5 - ± 0.7 mm) in our study, which suggests that BT5 contains higher concentrations of bioactive compounds than others. Phenolic compounds appear to be key contributors to the antimicrobial activity of birch tar, although volatile compounds may also have an effect [54]. Further analysis of GC-MS on the birch tar samples would be ideal to validate the compounds present in our samples. Just like our samples, the samples TE1 and TE2 extracts displayed weaker activity against Gram-negative bacteria, with minimal inhibition against E. coli, and Klebsiella pneumoniae,.
This further supports the hypothesis that the antimicrobial compounds primarily target Gram-positive bacterial species. Several previous studies have examined the antibacterial properties of related extracts. Acquaviva et al. [55] reported that the extract of Betula aetnensis leaves inhibited S. aureus but had a lower impact on Gram‑negative bacteria. Similarly, Emrich et al. [56] reported that water-based extracts of B. pendula were highly effective against Staphylococcus epidermidis and MRSA. Vandal et al. [57], too, showed that ethanol extracts from plant material of B. papyrifera inhibited S. aureus but not E. coli.
Along with the mixture of compounds like phenols, terpenoids and organic acids discussed above, previous in vitro assessment of extracts made from birch bark tar has also evidenced toxic and mutagenic properties [58]. Specifically, birch bark tar extracts have induced apoptosis and DNA damage when tested using Comet, micronucleus, and sister-chromatid-exchange assays [58].
In addition, the combustion of biofuels like birch bark is a known source for polycyclic aromatic hydrocarbons (PAH), exposure to which has been shown to cause a range of respiratory conditions, including carcinogenic effects. [59,60]. Despite this, medicinal use of birch tar is documented across a wide range of communities, and dental ingestion of birch tar has been particularly common in the European Mesolithic, between c. 11 ka and 7 ka before today [20]. In this study, the evaluation of birch tar only included in vitro antibacterial activity using the agar-based diffusion assays. Future studies combining detailed chemical characterization, PAH quantification and standardized toxicological testing of birch tar to analyse its cytotoxicity, mutagenicity, or therapeutic suitability would be needed to fully understand the relationship between the antimicrobial properties and the potential risks of birch tar.
4.2 Implications for Neanderthal healthcare
The differences among the six samples collected for our study indicate varying extraction efficiencies, which could be due to factors including the composition of the raw materials, the exact conditions during pyrolysis, or natural variability in the properties of birch bark. Importantly, we observed no clear relationship between the extraction method and antibacterial efficacy. Samples produced using the condensation method and the raised structure method result in similar ZOI diameters against gram-positive S. aureus widely known for its role in wound infections. Consequently, the application of birch tar to the skin specifically for the treatment of wounds and skin conditions would have been afforded as early as exploration of its hafting properties has occurred, minimally during MIS 7 from c. 191–243 ka [5], and does not rely on underground pit production. Given the low viscosity of birch tar produced in underground pits, and adhesive properties of birch tar, contamination of the skin during handling is nearly inevitable, regardless of production technique.
Indeed, quantities of birch tar sufficient for skin application are low, with 0.2 g of birch tar covering as much as 100 cm2 of skin (19; Fig 4), and are thus easily obtained as a by-product of production for its use in a hafting context, regardless of the production process. In this regard, it might prove useful to consider further uses beyond skin application, as previously discussed for ochre [16,17]. For example, studies have suggested the efficacy of birch tar as an insect-repellent [61]; Faraone et al., unpublished data), and the Late Pleistocene environments of Europe saw considerable ecological and epidemiological pressure with respect to insect populations. This is evidenced by, for example, the extensive flying insect assemblages known from Neanderthal sites such as Lichtenberg, or Salzgitter-Lebenstedt [19,62,63] as well as by genetic material relating to immune response introgressed from Neanderthals [64–67].
Approximately 0.2 g of birch tar are sufficient to cover c. 100 cm2 of skin surface.
This study on birch tar’s affordances for wound care sits in the context of a surge in interest in Neanderthal life beyond stone tools. Structures of care are increasingly recognised as an essential part of Pleistocene life, and numerous scholars have now published on neglected aspects of Neanderthal care. Spikins et al. [29,30] have explored structures of care in the Neanderthal context. Houldcraft and Underdown [68] have documented the plethora of pathogens that Neanderthals were subjected to during the Middle Palaeolithic. Hardy [31] and Hardy et al. [33] have documented the manifold plants used potentially medicinally found in Neanderthal contexts. Weyrich et al. [69] have documented dental care in a Neanderthal individual from El Sidròn. Trinkaus and Villotte [70] and Conde-Valverde et al. [71] have reported on structures of care related to disability in Neanderthal communities. Today, practices of care are understood to be a key component of Neanderthal life. As such, our study contributes more explicit data on the multimodal affordances of pyrotechnological birch tar production, shedding light on the healthcare of deep time.
5 Conclusion
Our experimental findings demonstrate that birch tar possesses selective antibacterial properties, showing consistent inhibitory effects against S. aureus but no detectable activity against E. coli. Among the six birch tar samples tested, only BT5,extracted via the raised structure method from B. pendula, showed a comparatively strong response, producing an inhibition zone of 10.5 ± 0.7 mm. Other samples exhibited mild to moderate activity, while one (BT3) showed no effect. This variability underscores how differences in extraction method, bark species, and possibly compound concentration influence the antibacterial efficacy of birch tar. The complete absence of inhibition against E. coli across all samples aligns with the known structural resistance of Gram-negative bacteria. Further, it supports the tar’s specificity toward Gram-positive strains. Our results reinforce the hypothesis that birch tar’s antibacterial properties may have been exploited intentionally in both deep time and Indigenous contexts for wound care, and bear potential for targeted therapeutic development in the present day. As today’s world is facing an antibiotic crisis, seeing increased antibiotic tolerance of bacterial strains, engagement with traditional remedies becomes ever more important.
Our study permits exploration of the co-evolutionary relationship between technology and medicine as early as MIS 7, since all three tested production methods showed some level of antibacterial properties, and offers more explicit data to support the medicinal use of antibacterial birch tar in deep time. Whilst drawing from arguments previously proposed for the use of ochre in the Pleistocene, such multimodal uses may indeed be considered for other aspects and localities of Pleistocene lifeways. Yet, further research going beyond the exploratory character of this study is necessary, that isolates the parameters that affect the antibiotic activity of birch tar produced using different methods, and by checking results with the cultivation method or using absorbance measurements, the accuracy of the results could be further improved.
Supporting information
S1 File. Supporting Information File.
Document containing photos of sample plate BT_2024_001–01 (BT1), BT_2024_005–01 (BT2), BT_2024_006–01 (BT3), BT_2024_011–01 (BT4), BT_2024_012–01 (BT5) and BT_2024_013–01 (BT6) showing varying levels of antibacterial activity against Staphylococcus aureus.
https://doi.org/10.1371/journal.pone.0343618.s001
(PDF)
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