The climate in Burgos province is temperate and hot with rainfall all year long; which would be typical of Cfb in the Köppen-Geiger classification [8][8]. Meteorological data for the period 1981–2010, provided by the Spanish national weather agency, Agencia Española de Meteorología (AEMET), show that Burgos city (865 m above sea level) has an average annual temperature of 10.5 °C and rainfall of 575 l/m². There are no weather data for La Pedraja, but at the weather observatory at Atapuerca (Fig. 1Fig. 1B), which is 966 m above sea level and less than 10 km north-west of the mass graves, the annual average temperature drops to 10 °C and the rainfall rises to 616 mm. The hottest month of the year, averaging 18.5 °C, is August, but by January the average temperature falls to a low of 2.6 °C, with the potential of below-freezing temperatures on several days in January and February.

The AEMET data for the second half of the year 1936 show that the weather was exceptionally cold and wet across the region. For example, at Atapuerca in October of that year, the minimum temperature was −8 °C. Fig. 3Fig. 3 compares the total rainfall figures in Burgos and Atapuerca from June to December 1936 with the 1981–2010 average rainfall figures for Burgos City. This demonstrates that, apart from the month of August, summer and autumn of 1936 were exceptionally wet. In July, Atapuerca received seven times more rain than the current average rainfall for that month in Burgos City. At over 130 m higher than Atapuerca, the average monthly temperatures at the site of La Pedraja 1 would be lower still and this would have been reflected in the soil temperature of the grave.

Fig. 3

The total rainfall figures in Burgos and Atapuerca from June to December 1936 compared with the 1981–2010 average rainfall figures for Burgos City. Meteorological data obtained from AEMET.

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Today, the location of the mass grave is in an open mountain area with a few pine trees. The soil is beige-brown and clayey and pebble inclusions. During the initial excavation of La Pedraja 1 in the summer of 2010, the weather was hot and sunny with a maximum temperature of 38 °C. The soil of the grave was moist, but within several hours of exposure the ground became dry. Surprisingly, during the excavation, a moderate odour of putrefaction could be detected despite 75 years of burial.

The wet conditions at the level of the burials had led to the preservation of some organic material, including fragments of textiles and footwear. These findings confirmed that the deceased were clothed when they were buried but there was no evidence of coffins. Skeletal preservation was very poor with high fragmentation and extensive weathering of the bones, and little survival of bone epiphyses. In some instances, the fragmentation of the skulls was not entirely due to the burial environment but was enhanced by peri-mortem gunshot trauma on some of them. Given the poor skeletal preservation, it was all the more surprising to recover the remains of 45 well-preserved brains from these skulls.

Some of the best-preserved brains were found in the skeletons of individuals 11, 25 and 104 (Fig. 4Fig. 4A ). The colour, texture and smell of these masses were similar to the surrounding moist soil. They were distinguished from soil by the fact that they had retained the neural folds and other gross morphology of the brain, although greatly shrunken to about 20% to 30% of their normal size. The majority of these brains were greasy to the touch but some appeared dry rather than fatty. Most of these were fragmented and bullet debris was recovered in some cases. About 30% of cases retained evidence of both cerebral hemispheres, and in a few of them, portions of the cerebellum could be identified. However, none had retained the brainstem. In the instances where parts of both hemispheres were preserved, one hemisphere appeared laterally flattened. Upon recovery, the brains were transferred to plastic containers that were hermetically sealed and stored under refrigeration at 4 °C, while their study was discussed.

Fig. 4

Gross morphology of the brains. A: Neural folds on the surface of brain 25, (1) central Sulcus, (2) lateral Sulcus and (3) calcarine fissure. B: Plastic 3D printed model of brain 25. C: Magnetic resonance image of brain 104. D: Computed tomography image of brain 25. E: Intracranial haemorrhage on parietal lobule of brain 11 seen as a dark brown discoloration on the surface (arrowed).

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The individuals with brains were distributed across the entire trench and only those from phases with three or fewer corpses contained no brains at all (Fig. 2Fig. 2 and Table 1Table 1). From the study of the skeletal remains, no relationship was found between brain preservation and an individual's sex, age-at-death, height or peri-mortem trauma. No other soft tissues were preserved, except for individual 14 (grave 1B), which had both the brain and heart still surviving [9][9].

Table 1Distribution of brains and ratio of brains/corpses in the different graves (see Fig. 2Fig. 2).

Grave sector	Number of corpses	Number of brains	Ratio brains/corpses	Individuals with brain preserved
1A	8	1	12,5	70
1B	10	5(*)	60	8,10,11,12,13,14⁎
1C	14	7	50	17,18.19.23,24,25,28
1D	14	10	64,2	29,30,31,32,33,34,35,36,98,99
2A	6	3	50	39,41,96
2B	3	0	0
2C	4	1	25	47
2D	3	0	0
2E	9	1	11,1	58
2F	32	17	53	67,75,74,80, 82, 83,85, 86, 87, 89,91, 74,75, 84, 104, 88, 95
2G	1	0	0	–
Total	104	45	43,2%	–

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Individual 14 in grave 1B had both the brain and heart surviving.

Historical data indicated that most of the deceased came from regions near to where they were buried and had been illegally detained by supporters of the rebel army at different places and times. They remained kidnapped for a few hours or days and later executed, mostly by handguns. The historical data also indicated that the identities of the dead were not certain, although we know that the majority of the missing were young adult men. It is not surprising to see this at La Pedraja, since this pattern of criminal behaviour was repeated throughout Spain during the first months after the military uprising [[17], [18]]. The details of how these murders occurred is not known, but from the investigation of the mass grave and the analyses of the remains some facts can be deduced.

Guided by the plan of the mass grave in Fig. 2Fig. 2, it is possible to verify that the burials took place at different times, perhaps as many as the number of individual grave sectors identified. Each phase of interment was undertaken within one single trench (no piling of the bodies), where the bodies side by side were buried at a depth of 1.5 m and cut into an almost impermeable clay. The research carried out so far on this grave has suggested that lime was probably not used in all of the graves as there is only evidence of its use in sectors 2F and 2G. The moderate levels of calcium detected in the soil are indicative of the presence of sufficient levels of the cation Ca²⁺. The high levels of CEC (Cationic Exchange Capacity) suggest that the soil surrounding the bodies was susceptible to interaction with the biological organic components, not only with Ca²⁺ but also with other cations, such as Na¹⁺ or Mg²⁺[19][19]. The small number of shell cases found (three in sectors 2C and 2E, see Fig. 2Fig. 2) suggests that only three shots were fired near the mass grave (either to directly kill or to ensure death). In other words, it is possible that the majority of victims were not killed at the foot of the grave. The large quantity of projectiles found in the bodies (37) is further evidence of violent death by execution. The presence of fragmented lead remains in various of the preserved brains, confirms that there was probably an elevated number of deaths that took place as a consequence of shots to the head.

The archaeological data with regard to the number and position of the bodies, the characteristics of the grave, the dating evidence from historical accounts and artefacts, the evidence for bullets confirms this was the scene of a crime. This mass grave was used to clandestinely bury 104 individuals, who had been killed in a place where there had not been a military front. This is important, because knowing the type of mass grave being researched requires the application of forensic methodology including the chain of custody to record, recover, and preserve evidence. The result of this mass grave investigation indicates that the principles of International Human Right Law and International Humanitarian Law (Rights of War) [[1], [5]] should be applied.

Furthermore, historical meteorological data (Fig. 3Fig. 3) shows clearly that the summer and autumn of 1936 were extremely rainy and colder than normal. In these circumstances, the excavated trenches in the impermeable clay would have converted the graves in pools of water, soil and bodies in the initial stages of decomposition. This suggests that the water within the mass grave would have been a key to the preservation of the brains.

There is no apparent correlation between the presence of the preserved brains and the specific location within the grave, the presence of bullets, the pH of the soil, the presence of any skull injury or the preservation of the skeleton. The appearance of these preserved brains is apparently random. Circumstances that may also have contributed to brain preservation may relate to when and how the bodies had been buried. For instance, the burials in sector 2F had been covered in lime and this sector could relate to the second body deposition in July 1936. This sector 2F had 53% of the individuals with preserved brains; compared to sectors or phases 2D and 2B that did not contain lime or preserved brains and could have been made at the end of the summer of 1936 under different meteorological conditions. Possibly, rainfall and the presence of water in the grave during burial could have influenced the preservation or otherwise decay of brains. Therefore, the meteorological data (cold and/or moisture) at the time the grave was produced could be an essential factor in the preservation of these brains. Experimental work shows that water is a key factor in adipocere formation and that cold conditions delay the putrefaction of bodies [[22], [23], [24]].

The chemical analysis of the three preserved brains confirmed the existence of a specific qualitative profile of fatty acids, described by many authors as characteristic of saponification [[20], [21], [22], [23]]. Some authors (Fielder) [24][24] argue that of all the fatty acids present in adipocere, palmitic acid and stearic acid are the most important, since their point of fusion is 63 °C (Palmitic) and 68 °C (Stearic). Therefore, in the thermal ranges of La Pedraja (annual average temperature of 10 °C), these acids crystallise, solidifying and hardening the tissues that comprise them. Nevertheless, this argument proposed years ago by Takatori and Yamaoka [26][26], also extends to other fatty acids of high melting points such as Myristic (54.4 °C), Lauric (43 °C), Heptadecanoic (61.3 °C), Pentadecanoic (52 °C), Eicosanoic (75.5 °C) or Lignoceric (84 °C) [25][25]. The La Pedraja brains contained all these fatty acids, therefore it is reasonable to think that this may have significantly contributed to saponification.

The histological study, as expected, showed that the encephalic microscopic structure is not preserved. However, the immunohistochemical detection of some vessels and especially the existence of myelinated structures observed under the electron microscope can be highlighted, which confirms that these masses contain nerve structures and are therefore human brains, as shown by other authors [[14], [27], [28], [29]]. Every rehydration of desiccated tissues is a unique experiment on a case-by-case basis. Good results were obtained with the preserved brains using two hydration protocols, Ruffer's and Eklektos's, however Ruffer's solution provided a 3.6% increase in hydration (brain weight) compared to Eklektos's solution, in a shorter time.

Furthermore, the identification of the extensive hemosiderin deposits on the left hemisphere in brain 11, following staining with Prussian blue, are consistent with an ante-mortem injury. These deposits indicate an intracerebral (subarachnoid) haemorrhage but as it takes at least 36 h for the haemoglobin to breakdown to hemosiderin [30][30], the individual may have suffered a traumatic brain injury several days prior to death. From a forensic perspective, it is particularly significant that an ante-mortem lesion can be identified after a post-mortem interval of 77 years. Historical data reveal that the victims were arrested some days before death. In this context, this finding of hemosiderin deposits could be considered as an indication of abuse or torture. The bones of individual 11 were in very poor condition and thus the presence of other pathological and/or traumatic findings cannot be confirmed. Nevertheless, the intracerebral (subarachnoid) haemorrhage could be the result of spontaneous, accidental but also the result of a criminal (traumatic) act, therefore the violation of Human Rights and Torture cannot be excluded, which could be a case to be considered by the Spanish or International Courts.

By revising the forensic and archaeological literature about the preservation of brains, some authors find that there are more than 200 published and unpublished cases described since 1960 [31][31]. In addition, at the end of the 18th century, cases of preserved brains from the Cemetery of the Innocents of Paris are described, but without photographs or sufficient descriptions [32][32]. The most recent literature provides more detail and greater depth of study. However, it is very difficult to compare all cases thoroughly to obtain a reliable interpretation of the factors influencing brain tissue preservation, although the comparison itself helps the understanding and discussion of the main factors that determine the preservation of these brains. The most important cases in the literature are listed in Table 2Table 2.

Table 2Published cases of preserved brains and some additional information regarding their probable mechanism(s) of preservation.

Case	Country	Brains	Grave	Adipocere	Chemical study	Soil	Moisture	Causes
Thouret [33][33] 1791	France	Many	Mass grave Holy Innocents	Yes	No	–	High	Brain's fat and moisture
Oackley [34][34] 1960	UK	1	Buried at depth of 10 ft and wooden coffin	Yes	No	Clay	High	Moisture exclusion O2
Pilleri [35][35] 1970	Switzerland	18	Mass grave	Unknown	No	Acid	High	Unknown
Tkocz [36][36] 1979	Denmark	57	Burials in earth cut graves (no coffins)	Yes	No	Clay alkaline	High water salted	Moisture reduced bacterial growth exclusion O2
Doran [37][37] 1986	USA	91⁎	Individual graves	Yes	No	Bog and peat	High. Mixture of fresh and salted water	Not published
Radanov [38][38] 1992	Bulgary	3	Mass grave 30–40 cm underground	?	No	Loose and stony	Low	Dehydration
Gerszten [39][39] 1995	Chile	15	Desert of Arica	–	No	–	Low	–
Eklektos [14][14] 2006	South-Africa	1	On the ground	Yes	No	Veld	High/low⁎⁎	Hot and dry at time of death⁎⁎⁎
Karlic [40][40] 2007	Egypt	1	Mummy	Yes	Yes	Desert	Very low	Drying
Papageorgo-poulou [29][29] 2010	France	1	Underground wooden coffin and leather wrapped around the body	Yes	Yes	Clay acid	High. Salted and fresh water	Water immersion
O′Connor [31][31] 2011	UK	1	Waterlogged pit	No	Yes⁎⁎⁎⁎	Soft sandy clay	High	Rapid burial into wet sediment in study
Altinoz [41][41] 2014	Turkey	4	Burned	Yes	Yes	Carbonates clay	Very low	Drying
Serrulla 2016	Spain	45	Mass grave	Yes	Yes	Clay acid	High. Rain water	Moisture exclusion O2

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Personal communication (not published). Glen Doran found 91 brains in Windover (Florida). Adovasio et al. (2001) [42][42] add the reference of more than 90 brains also found in Windover.

Eklektos explained that the specimen appeared to have survived to several veld fires and a high annual rainfall and moisture.

Eklektos et al. suggested that the brains became preserved because of the hot and dry conditions at the time of death.

O'Connor reported that there was no evidence for the formation of adipocere. After the chemical analysis of the brains, she found lower proportions of extractable lipids, almost complete absence of phospholipids and only a trace of cholesterol.

The comparative study of similar cases found worldwide is quite difficult but reveals that there may be two kinds of preserved brains: those that have been preserved in an environment with high humidity (and perhaps therefore relative exclusion of oxygen) and whose bodies do not retain other tissues; and those that have been preserved as a result of dehydration [[43], [44]]. The cases of La Pedraja and many others belong to this former group [[29], [33], [34], [35], [36], [37]]. The case of Tkocz (Denmark) was found close to the sea (salt water). Cases such as Papageorgopoulou (France) and Doran (Miami, United States of America) were found in places near the sea, but with salt and fresh water. The case described by Pillery (Switzerland), stated that all brains were found in a river under a bridge (only fresh water). The presence at Oackley (United Kingdom) was associated with a place with high concentration of salt. In general, the degree of preservation of brains is better in La Pedraja compared to others. The majority of brains preserve at least one hemisphere in all cases and many authors describe the presence of adipocere formation. Only recent studies (five of 13) have carried out chemical investigation and this is another difficulty when comparing cases. Those brains that seem to have been preserved by a process of intense dehydration were found in places of high temperatures and low humidity, as in the case of Egypt, South Africa and the Arica desert in Chile [[14], [38], [39]] (these examples showed preservation of the brain and other tissues by processes of desiccation). In the case of O'Connor [31][31], the preservation mechanism is still being researched, although it may be that the brain was preserved in wet conditions, which would then lead to the formation of adipocere. The Altinoz case in Turkey [41][41] is surprising in that it could clearly be a case of dehydration, where the presence of a lipid profile of saponification is demonstrated. In this case, it seems that an earthquake and subsequent fire were the cause of the death for four people. Perhaps, this case can be explained by the ‘oven’ effect of the skull, which would allow slow desiccation of the tissue and would produce both the hydrolysis of brain lipids and of the cerebral lipids, resulting in the characteristic profile of saponification, aided no doubt, by the high levels of aluminium and magnesium carbonates in the soil where they were found.

The study of the brains preserved in the mass grave of La Pedraja 1, and the comparative study with similar published cases, allows the development of a hypothesis to explain the preservation of the brains. However, and in agreement with Ubelaker [45][45], there needs to be more research on the taphonomic processes of saponification, to better clarify the physical, chemical and biological factors that cause the transformation of the fatty tissues into adipocere.

The majority of victims buried in the mass grave of La Pedraja 1, were not killed by the side of the grave, although certainly only a short time (possibly a few hours) would have elapsed between death and burial. Most of the people buried at La Pedraja were shot in the head with handguns, in various groups and their bodies deposited there between the second half of July and November of 1936. The various sectors of the graves were dug in an area where some mountain water flowed. The soil of the mass grave was clay and impermeable. In the months of July to November 1936 in La Pedraja, it rained so much that the graves would have become bathtubs full of decaying corpses, leading to the important process of saponification in many of the bodies. The temperature at which the bodies were exposed was probably low, averaging between 5 °C and 10 °C. Due to the manner and cause of death (gunshot trauma to the head), the brains would have lost a considerable amount of blood; therefore the initial saponification processes would have begun before bacterial putrefaction could liquefy the brains. It is reasonable to assume that the absence of blood in the intracranial vessels would have made the putrefaction of the brain difficult. The wounds perforating the skin and the skull probably allowed water to enter the brain, facilitating the hydrolysis of phospholipids and the formation of free fatty acids. Rainwater charged with dissolved cations from the soil, would have permitted the esterification of those fatty acids resulting in saponification. Therefore, it is possible to suggest that the preservation of these brains occurred in cases that met the following conditions:

• 1)

  Brains with empty blood vessels or some other alteration which delayed cephalic putrefaction. This would imply specific and/or low microbiological charge, very low temperatures or bodies with low body fat and a high degree of dehydration at the time of death, or those who had a violent death resulting in agonising deaths in which there would be minimal cerebral circulation.

• 2)

  Brains where the skull had been perforated due to ballistic trauma, allowing water within the grave to get inside the skull and thus favouring the hydrolysis of phospholipids.

• 3)

  In those burials where there was plenty of water containing cations capable of esterifying the fatty acids, which would then produce adipocere.

If the buried corpse met these three conditions, the phenomenon of saponification would have extended sufficiently to allow the preservation of the external brain morphology a few weeks after death.

The slow and gradual depletion of water from the graves over the years would have dehydrated the bodies and brains, while, at the same time, the ground would have become acidified. It is possible that acidification was too slow and a changeable process, according humidity and evolution of decay. Seventy years after an intense acidification of the soil, the skeletons would have slowly decomposed, whereas the skull would have served as a protective barrier against this high acidity, therefore protecting the brains. Several authors have stated that adipocere could be produced in acid, neutral and alkaline soil [[21], [23], [24]].

In summary, the hypothesis about the unusual preservation of brains maintains that there are three classes of factors, which intervene and act in the proper order and proportion:

• –

  Microbiological factors. It appears that nerve tissues are preserved because initially the processes of cephalic putrefaction are delayed for one of the following reasons: low or inadequate microbiological load, and/or low or very low temperatures and/or high dehydration and/or the absence of the intracranial blood circulatory system. Autolysis begins, however, when the liquefaction of the brain fails to occur. In cases of artificial mummification, obviously putrefaction is stopped by the application of chemicals (e.g. natron, formaldehyde or other biocides). Recent experimental studies on the formation of adipocere, have deepened our understanding of these microbiological factors [46][46], relating the adipocere production in the first phase of decomposition with the reduction of the microbiological load.

• –

  Biochemical factors. In the case of humid conservation of the brain, the hydrolysis of brain lipids is required, especially phospholipids, which are the most abundant lipids in nerve tissue, and which contain in their complex molecule, fatty acids bound to diphosphoglycerol [47][47]. This hydrolysis is carried out through lipid contact with water, which implies that there must be some communication with the outside intracranial space and water has to be present. The biochemical factors of saponification have been studied for many years and are relatively well characterised [[26], [48]]. The phospholipid hydrolysis produces free fatty acids, and if esterified with cations it gives rise to a compact, creamy and moist soap-like substance, which preserves the shape of the tissue, and decays much slower than tissue that is not saponified. This biochemical process receives the name of saponification. The cation exchange capacity of the medium in which the body is located, could be an important element that determines the saponification.

• –

  Physical factors. Temperature and humidity are the principal factors that influence the preservation of these brains. These factors, due to their ability to affect others, have to act at the right time and temperature. Low temperatures reduce the rate of biological decomposition and high or very high temperatures accelerate dehydration, which in turn can reduce the rate and extent of decomposition. The presence of a high proportion of water is a key element in wet preservations while the relative absence of water is key to dry preservations. Finally, we believe the slow dehydration produced for years in the mass grave has helped to reduce the size of these brains.

Hopefully, this hypothesis will be verified when other cases of preserved brains are found and studied using similar methods and with other research designs and experimental investigations. This will lead to a better understanding of the causes, mechanisms and circumstances that are involved in the process of adipocere formation.