Biomolecular analyses enable new insights into ancient Egyptian embalming

The ability of the ancient Egyptians to preserve the human body through embalming has not only fascinated people since antiquity, but also has always raised the question of how this outstanding chemical and ritual process was practically achieved. Here we integrate archaeological, philological and organic residue analyses, shedding new light on the practice and economy of embalming in ancient Egypt. We analysed the organic contents of 31 ceramic vessels recovered from a 26th Dynasty embalming workshop at Saqqara1,2. These vessels were labelled according to their content and/or use, enabling us to correlate organic substances with their Egyptian names and specific embalming practices. We identified specific mixtures of fragrant or antiseptic oils, tars and resins that were used to embalm the head and treat the wrappings using gas chromatography–mass spectrometry analyses. Our study of the Saqqara workshop extends interpretations from a micro-level analysis highlighting the socio-economic status of a tomb owner3–7 to macro-level interpretations of the society. The identification of non-local organic substances enables the reconstruction of trade networks that provided ancient Egyptian embalmers with the substances required for mummification. This extensive demand for foreign products promoted trade both within the Mediterranean8–10 (for example, Pistacia and conifer by-products) and with tropical forest regions (for example, dammar and elemi). Additionally, we show that at Saqqara, antiu and sefet—well known from ancient texts and usually translated as ‘myrrh’ or ‘incense’11–13 and ‘a sacred oil’13,14—refer to a coniferous oils-or-tars-based mixture and an unguent with plant additives, respectively.

Embalming substances and embalming workshops are documented from three main sources: 1) archaeological deposits, conveniently known as "embalming caches"; 2) written and pictorial sources, e.g.embalming papyri and 3) organic residue analysis of Egyptian mummies.The embalming caches are associated with individual tombs and contain refuse of embalming materials including linen bandages, natron, pottery vessels, sawdust, and other detritus of the embalming procedure [1][2][3][4][5][6][7][8] .They are valuable material for interdisciplinary examinations, which cross the borders of the natural sciences and humanistic disciplines, as in, for example, biomolecular archaeology.The embalming papyri, described as embalming manuals, provide detailed accounts of the practical and ritual treatment of the dead body inside different facilities within the embalming workshop complex (e.g. the per-nefer, interpreted as "house of rejuvenation") 7,[9][10][11][12][13][14][15][16][17][18][19] .The archaeological and textual sources reveal the great economic role of embalming [20][21][22] .Of significance are the wabet-facility (meaning "place/room of purification") and the ibu-purification tent/hall, where the main procedures of evisceration, body preservation, and wrapping were performed.During the mid-third millennium BCE, and based on their representations on tomb walls, both facilities were spatially separated and were possibly for individual use only.However, they were later, in the second millennium BCE, collapsed into the per-nefer workshop complexes, which were administered by entrepreneur priestembalmers 3,12 .
Our knowledge of the per-nefer (pr-nfr) embalming workshops has remained confined to pictorial and textual sources until the archaeologists of the University of Tübingen, Germany, working at Saqqara, uncovered the first embalming workshop complex of its kind.This unprecedented discovery, dating back to the 7 th century BCE.(ca.664-525 BCE), reshapes our knowledge as to the location and design of ancient Egyptian embalming facilities in the necropolis.Conjecture held that they were built aboveground, but the Saqqara workshop offers concrete evidence for embalming procedures having been carried out inside facilities both aboveground and underground.The embalming workshop is located a few meters to the south of the Pyramid of King Unas and includes a subterranean evisceration facility (Egyptian wabet/wʿb.t),a multifunctional aboveground structure (Egyptian ibu/jb.w),and communal burial spaces (i.e.Shaft K24, 30m deep, and catacomb-like galleries, Extended Data Fig. 6).The wabet room (8m × 5m × 3m) is located at the bottom of a 12.5m deep shaft and has an architectural design that could help reduce health risks for the ancient embalmers from microorganisms by means of: i) a ventilation system composed of an air tunnel running through the room's ceiling and connected to a massive network of other subterranean tunnels; ii) a simple two-channel drainage system for bodily fluids, cut on a ledge for evisceration (Extended Data Fig. 7); and iii) an industrial capacity fumigation source (i.e., a large pottery vessel, Extended Data Fig. 8) for repelling insects and deodorizing the room 23,24 .
Since the 35 vessels examined were discovered in two separate loci, they constitute two different groups.Nevertheless, each group stem from a single homogenous closed context.This indicates that the vessels were subject to the same taphonomic/natural alteration.Organic residues were identified in all vessels except for one, and lipid concentrations varied from 5 to 1653 μg per gram of sherd (Extended Data Table 1, Extended Data Fig. 1).

Conifer by-products: oils/tars of cedar and juniper/cypress
1.1.Cupressaceae by-product: oils/tars of juniper/cypress (n=21) Totarols, and in some cases their derivatives (oxo-totarol), were identified in 21 samples (Fig. 2 and Extended Data Fig. 4).These phenolic diterpenoids are generally associated with the presence of members of the Cupressaceae family, in particular junipers or cypresses 25,26 .The absence of sandaracopimaric acid rules out the Tetraclinis (e.g., sandarac resin).Cuparenerelated sesquiterpenes (especially cuparene and cuparenic acid) were identified in the 21 samples mentioned above (Fig. 2).They confirm the presence of juniper or cypress byproducts [27][28][29] .Oxo-cuparene, an oxidised product of cuparene 28 , was identified in seven vessels.Traces of α-muurolol (δ-cadinol) reported in juniper wood oil 27 and cypress oil 30 were also identified in five samples.In archaeological samples, the presence of low molecular weight sesquiterpenoids in quantities equal to or higher than that of the diterpenoids indicates that the substances were not resins but could have been prepared by steam-distillation / extractive immersion of odoriferous plant material (fragrance/essential oil 28,31,32 ) or by pyrolysis (wood tar 33 ).
In two additional samples (SSTP 33 and 104), cuparene-related sesquiterpenes were identified without totarol derivatives.They could also be associated with juniper/cypress oil as they are present in significant amounts in Cupressaceae.However, the identification of cuparenes alone is not always characteristic as they can be identified in different plants exudates/distillates 31,34,35 and cuparene may even be derived from the degradation of himachalenes 36 and therefore Cedrus sp.

Pinaceae by-product (n=20): oils/tars of cedar (n=19)
Abietane-type diterpenes (e.g., dehydroabietic and 7-oxo-dehydroabietic acids) were identified in 20 vessels.In four of these vessels isopimaric acid was also detected (SSTP 006, 020, 001,  104).This indicates the presence of conifer by-products (e.g. 37), and Pinaceae by-products in archaeological contexts 25,26 .Sesquiterpenoid derivatives of the himachalene series were also identified in 19 of these vessels (Fig. 2 and Extended Data Fig. 4).γ-Dehydro-ar-himachalene, ar-himachalene and α-dehydro-ar-himachalene were detected in 17 vessels and only traces of the latter in two vessels.Traces of himachalol were also identified in nine vessels.The himachalenes are significant in cedar by-products 27,28,33,[38][39][40] and can be considered as cedar oil markers in archaeological contexts 28,41,42 .Oxidation products of ar-himachalenes, especially oxo-γ-dehydro-ar-himachalene and nor-ar-himachalone were also identified in 15 of the pots tested, along with oxo-ar-himachalol in three cases.These components attest to the intense oxidative alteration processes of the cedar by-product 28 .As for the Cupressaceae by-products, the predominance of sesquiterpenoids indicates the presence of an oil or tar.
Although, one or a few himachalenes have been detected in several plant extracts (e.g., Abies sp. 43, Pimpinella sp. 446][47] , Juniperus sp. 27,29,48,49), these sesquiterpenes were to our knowledge 1/ never reported altogether in quantities large enough (always one or a few as traces / very low concentration in modern oil) to be relevant in the context of archaeological samples and 2/ rarely display the complete spectrum of himachalenes derivatives found in Cedrus SP (modern and artificially degraded 28,42 ) and in the Saqqara samples.They can be considered as valid biomarkers because they are both 1/ originally present in Cedrus sp.byproducts and 2/ formed by aromatisation/dehydrogenation of the native major sesquiterpenes of Cedrus sp., namely α-, β-and γ-himachalenes 42 .
We have also identified an additional marker of dammar which is the 20,24-epoxy-25hydroxydammaren-3-ol (product of the oxidation of hydroxydammarenol 73 ).Although derivatives of this compound have been identified in modern extract of Commiphora sp 69,71 , the amount of dammaranes reported by these authors precludes their relevance as markers of Commiphora sp. in an archaeological context.Therefore, 20,24-epoxy-25-hydroxydammaren-3-ol can reasonably be used as another argument for the presence of dammar in our samples.

Animal fats, plant oil and beeswax
Fatty acids, especially palmitic and stearic acids were identified in 30 vessels, which may suggest the presence of animal fat, plant oil and/or beeswax according to their distribution.

Animal fats (n=18)
A narrow distribution of saturated triacylglycerols (TAGs) was identified in 13 vessels and shows the presence of animal fats (e.g. 78).TAGs 46:0 or 48:0 to 54:0 are identified in 10 vessels (SSTP 001, 103, 031, 017, 037, 030, 038, 016, 039, 040, Extended Data Fig. 2).TAGs with up to 52:0 were present in one sample (SSTP 014), and only TAGs 48:0 and 50:0 were identified in two others (SSTP 008 and 018).A distribution of saturated DAGs (36:0, 34:0 and 32:0 isomers) resulting from the first step of degradation of the TAGs was also identified in all of these samples, and in 6 additional vessels (SSTP 006, 024, 005, 007, 032).This shows the presence of animal fat in 18 vessels (Extended Data Fig. 1).Furthermore, traces of saturated TAGs with an odd number of carbon atoms (53:0, 51:0 and 49:0) were identified in 7 vessels (SSTP 016, 038, 037, 039, 040; only TAGs 51 for SSTP 001 and TAGs 47 in STPP 030; Extended Data Fig. 2 and Extended Data Fig. 1).These are characteristic of ruminant animal fats 32,79 .The major saturated fatty acids present in animal fat, namely palmitic, stearic and myristic acids (e.g. 80), were identified in all 18 samples and in 8 additional ones for which the animal or plant origin could not be clearly determined (Extended Data Fig. 1).A mixture of positional isomers of octadecenoic acid (C18:1) and minor straight and branched pentadecanoic and heptadecanoic acids was also identified in the 7 samples associated with the odd-numbered TAGs, as well as in 9 additional samples (SSTPP 006, 023, 024, 005, 007, 008, 031, 036 and 103; Extended Data Fig. 1 and 4).Minor straight and branched pentadecanoic and heptadecanoic acids are known to be formed in the rumen by bacterial synthesis, and thus might be assigned to ruminant animal fats (e.g. 78).The formation of several positional C18:1 can result from the biohydrogenation of dietary fats which occurs in the rumen of ruminant animals 81 .

Plant oil (n=5)
Unsaturated triacylglycerols (54:3, 52:2 and 50:1) were detected in 4 samples (SSTP 010, 037,  014, 031).Unsaturated diacylglycerols (DAGs 34:1 and 36:2 isomers) and di-palmitin (DAGs 32:0 isomers) were also identified (Extended Data Fig. 3).They result from the degradation of the unsaturated TAGs.Unsaturated TAGs and DAGs are extremely scarce in archaeological residues, as they are rarely preserved.Only plants that contain high quantities of oils will lead to the preservation of unsaturated TAGs.Despite their very poor preservation in most of archaeological contexts, these markers are almost the only ones that can be used to identify the nature of archaeological plant oils 79,82 .The distribution of TAGs in SSTP 010, 037 and 014 corresponds to modern olive oil.However, other oils containing the same TAGs co-occurring with other even more easily degraded polyunsaturated TAGs (e.g., argan and hazelnut oil) cannot be totally excluded 79,82 .In a fifth pot (SSTP 020), ricinoleic acid was identified (Extended Data Fig. 4).This marker suggests the presence of castor oil in ancient Egyptian contexts 80,83 .However, it could also derive from lipids biosynthesized by the ergot fungi which are common pests of Graminea, and therefore also could be associated with the presence of cereal 84 .In the Saqqara embalming workshop, the presence of castor oil (in mixture with cedar oil, Pistacia resin, elemi and possibly mixed with other oils) is most plausible as the vessels are dedicated to the preparation of balsams for mummification, which required antiseptic/antifungal properties.Palmitic and oleic acids, identified in the five samples, were the major fatty acids in SSTP 010, 014 and 020.Traces of linoleic acid were also identified in SSTP 010 and 014.Azelaic acid and the two isomers of 9,11-dihydroxy-stearic acids were also identified in SSTP 020 as well as in SSTP 031 and SSTP 037.The formation of the hydroxy acids can result from oxidation by dihydroxylation of the double bond in oleic acid 85 .The presence of 9-and 10-hydroxyoctadecenoic acids can result from the dehydration of the two isomers 85 .

Beeswax (n=5)
Long-chain palmitic esters with an even number of carbon atoms from C40 to C48 and saturated long-chain even-numbered fatty acids (C22:0 to C28:0, maximising at C24:0) characteristic of beeswax 86,87 (Fig. 3) were identified in 5 vessels Extended Data Fig. 1).The presence of palmitic acid in each, and that of even-numbered n-alcohols with 22 to 30 carbon atoms in 3 samples (SSTP 39, 43 and 103) can be attributed to a process of preferential hydrolysis of shorter-chain esters 87,88 .The series of odd-number n-alkanes which are indicative of beeswax were not identified in the five vessels.This suggests that the bee-products were subjected to a heat treatment 87 .

Markers of concoctions/mixtures of bio-products
Triterpenic palmitates were identified in 3 vessels.They can result from the intentional mixing of triterpenic tar/resins and fats 96 .11-Oxo-β-amyrin palmitate, 11-oxo-α-amyrin palmitate and α-amyrin palmitate were characterised in 2 white beakers (SSTP 011 and SSTP 037).Their identification in association with the other markers of elemi, animal fat and/or plant oil indicates an intentional preparation involving the mixing and heating of these substances (Fig. 3 and Extended Data Fig. 1).Another recipe based on dammar resin, beeswax and/or animal fat markers can be proposed based on the presence of their specific markers as well as α-amyrin palmitate and oxo-oleanene palmitate in a red bowl (SSTP 001; Fig. 3 and Extended Data Fig. 1).