The Cattedown Hominin fossils of Worth's excavations in 1886-87 were assumed by him and his contemporaries to have been Palaeolithic in origin. Considering the intimate association of the Hominin bones with those
of animals such as the hyaena, rhinoceros and cave lion  -long extinct in this country-  and sealed within and beneath great thicknesses of stalagmite within the lower levels of the cave excavation, this is a tolerable
assumption. The anatomical evaluation of these fossils undertaken in 1903 by Dr. John Beddoe, indicate that the Hominins were shorter in stature than modern man but were otherwise generally the same, except for a
few minor and distinctive cranial characteristics. Dr Beddoe's Report can be viewed in full in Section 7. of the Cattedown Bone Caves Webpages.
Dr Beddoe's interpretations, though very interesting, are insufficient and are unlikely to entirely satisfy our curiosity. We need to know in absolute terms exactly how old "Cattedown Man" actually is.
Eighty eight years later in 1974, the remains of an Arctic Reindeer (Rangifer tarandus) in a cave near Worth's Cattedown Bone Cave, were subjected to a Radiocarbon Dating with an acceptable, though not entirely
satisfactory result. Only one sample was submitted. With the benefit of hindsight, we would have liked to have submitted four samples of bones from the same animal but obtained from different parts of the matrix in
which it was buried.
Since that time in 1974, there have been at least a further two recorded attempts at Radiocarbon Dating the surviving remnants of Worth's Hominin Collection, with (predictably) no satisfactory result. It is necessary,
therefore, to have to consider other methods of dating.

13.2.1....Radiocarbon Dating.
Carbon-14 (expressed hereinafter as ¹⁴C), is produced naturally in the upper atmosphere through the following reaction : ¹⁴N + n => ¹⁴C + p (where nis a neutron and p is a proton). The ¹⁴C formed is rapidly oxidised to
¹⁴CO₂ and enters the earth's plant and animal lifeways through photosynthesis and the food chain as a proportion of the total carbon within the atmospheric carbon dioxide entering plants through the process of
photosynthesis. All living organisms contain the current ratio of the unstable or radioactive ¹⁴C isotope to its other naturally ocurring stable isotopes of ¹²C and ¹³C. These isotopes are present in the following amounts
¹²C = 98.89%, ¹³C = 1.11% and ¹⁴C = 0.00000000010%. Thus, one ¹⁴C atom exists in nature for every 1,000,000,000,000 ¹²C atoms in living material.
The rapidity of the dispersal of ¹⁴C into the atmosphere has been demonstrated by measurements of radioactive carbon produced from thermonuclear bomb testing. ¹⁴C also enters the Earth's oceans in an atmospheric
exchange and as dissolved carbonate (the entire ¹⁴C inventory is termed the "carbon exchange reservoir"). Plants and animals which utilise carbon in biological food-chains take up ¹⁴C during their lifetimes. They exist in
equilibrium with the ¹⁴C concentration of the atmosphere, that is, the numbers of ¹⁴C atoms and non-radioactive carbon atoms stays approximately the same over time. As soon as a plant or animal dies, they cease the
metabolic function of carbon uptake; there is no replenishment of radioactive carbon, only the decay of what was absorbed during its lifetime.

The radiocarbon method is based on the rate of decay of the radioactive or unstable Carbon isotope-14 (¹⁴C), which is formed in the upper atmosphere through the effect of cosmic ray neutrons upon Nitrogen-14 (expressed hereinafter as ¹⁴N). When an organism dies, it stops taking up the ¹⁴C isotope from the environment and that which it has already absorbed begins to decay at a constant value, known as its "half-life".

Libby, Anderson and Arnold (1949) were the first to measure the rate of this decay. They found that after 5568 years, half the ¹⁴C in the original sample will have decayed and after another 5568 years, half of that remaining material will have decayed, and so on. The half-life (t 1/2) is the name given to this value which Libby measured at 5568 ±30 years. This became known as the "Libby" or "conventional" half-life. The application of the Radiocarbon Method for dating has been proven to have an acceptable level of accuracy only as far back in time as 7 half-lives, after which there is a very small amount of radioactive carbon present in a sample. So, at about 40 000 years, the limit of the technique is reached. Beyond this time, other radiometric techniques must be used for dating. By measuring the ¹⁴C concentration or residual radioactivity of a sample whose age is not known, it is possible to obtain the count-rate or number of decay events per gram of Carbon. By comparing this with modern levels of activity (1890 wood corrected for decay to 1950 AD) and using the measured half-life it becomes possible to calculate a date for the death of the sample.
As ¹⁴C decays it emits a weak beta particle (ß), or electron, which possesses an average energy of 160keV.

Thus, the ¹⁴C decays back to ¹⁴N. There is a quantitative relationship between the decay of ¹⁴C and the production of a beta particle. The decay is constant but spontaneous. That is, the probability of decay for an atom
of ¹⁴C in a discrete sample is constant, thereby requiring the application of statistical methods for the analysis of counting data.
It follows from this that any material which is composed of carbon may be dated. Herein lies the true advantage of the radiocarbon method, it is able to be uniformly applied throughout the world.
The method has been successfully used on materials containing organic (carbon-based) structures, such as bone and charcoal. Greater details on the method and limitations of Radiocarbon Dating can be found in
Burleigh (1972) and is also briefly outlined below

13.2.2....Accelerator Mass Spectrometry Dating.
This is a measuring technique which directly counts the number of carbon atoms in relatively small amounts (5 to 10 milligrams) of a sample to produce a date based on the Radiocarbon Method. Samples from cave paintings and fossil bones can be submitted without noticeable damage to the original object.

13.2.3....Uranium-series Dating.
This technique covers an age range from 1 000 to 800 000 years ago and has been used on dating stalagmite and other cave sinter deposits, coral and teeth. This method relies on the constant accumulation of
electrons, the by-products of radioactive decay of certain elements within the structure of the object. The technique in this case counts the number of electrons trapped over time in the mineral crystals of the object and
assumes a constant radiation dose from the surrounding environment. This method has been successfully used on the dating of cave stalagmite / sinter deposits, wherein the initial crystallization of calcium carbonate
crystals is used as the "zero point" and their subsequent growth can be determined over time and dated.

13.2.4....Thermo-luminescence Dating.
This technique covers an age range from the historic to 250 000 years ago and has been used on dating pottery, burnt rock and sediments. This method also relies on the constant accumulation of electrons, the
by-products of radioactive decay of certain elements within the structure of the object. Here, the technique relies for its "zero point" on an event such as burning or exposure to sunlight.

13.2.5....Electron Spin Resonance Dating.
This technique covers an age range from 1 000's to 100 000's of years ago and has been used on dating tooth enamel and cave sinter deposits such as stalagmite. Once again, this method also relies on the constant
accumulation of electrons, the by-products of radioactive decay of certain elements within the structure of the object. This method has been successfully used on the dating of cave stalagmite / sinter deposits and even
tooth enamel, wherein the initial crystallization of the crystals in the object is used as the "zero point" and their subsequent growth can be determined over time and dated.

13.2.6....Potassium-Argon Dating.
This technique covers an age range from 35 000 to 1 000 000's years ago and has been used on dating volcanic rocks. K-Ar has a long half-life of 1 300 million years and has been best used to date the decay of gases
trapped in volcanic deposits older than 750 000 years. This technique has been crucial in determining the evolution of early Homo in the volcanic regions of Africa and Asia, where archaeological deposits are often
situated between layers of volcanic ash.

13.2.7....Conclusion.
In obtaining the "absolute" age of an object using any of the above methods, there are varying margins of error inherent with the technique used, together with practical limitations on when they can be suitable for use.
All radioactive methods involve a certain amount of statistical uncertainty when calculating a date. Although Radiocarbon Dating is one of the most commonly used techniques, it has its own additional inherent problems.
With reference to para 13.2.1. above, we know that the atmospheric ratio of carbon isotopes has not always been the same as they are now. So, the direct results from the use of this technique have always been further
subjected to calibration graphs which correct the data accordingly (and to the best of our current knowledge) before being issued by the testing laboratory.
It is always recommended, wherever possible, to obtain more than one result using different methods. Even where this is not possible and a result can only be obtained using a single method, several samples should be
submitted.
The Cambridge Radiocarbon Conference in 1962 agreed that : (i) all dates should be calculated using the Libby Half-life value of 5568 years;  (ii) Ages are then quoted as "years BP" (Before the Present) with a modern 
zero point of AD 1950.
A detailed technical overview on the composition of bone and what is required for bone to be dated, is given at the bottom of this page.

PHOTO 13.3.1.  A view of specimen PM 1297, a 7cm long proximal fragment of Human Radius sitting on its
Plymouth City Museum Accession Card, recorded on 20 May 2003.
Donated to the Museum by Robert Burnard on 06 February 1899, this hominin fossil fragment is from Worth's
Cattedown Bone Cave.
The dark-brown "staining" is a thin covering of calcareous sinter or stalagmite.
(The scale is in centimetres.)

13.3.1.    The Problem with Radiocarbon Dating Worth's Hominin Collection :
With the bombing of Plymouth during the Second World War, the Museum of the old Plymouth Institution received a direct hit in 1941. The building became a burning inferno and it was only through the heroic attempts
of one of its Members, who entered the smoking remains of the ruined building afterwards, that the bulk of the Cattedown Hominin Skulls survive today in the care of the Plymouth City Museum. The rest of this fantastic
fossil collection is lost to science forever. So, for the most part, the entire bulk of Worth's extensive donation of the Cattedown Cave Hominin fossils to the Plymouth Athenaeum will never be seen again. In retrospect, if
he had donated them to the Plymouth City Museum, they would have survived the ravages of the bombing.
Owing to their exposure to the heat of the burning Athenaeum building, the surviving Cattedown Hominin fossils would not be suitable for radiocarbon dating, even if they were within the scope of the acceptable limitations
of the Radiocarbon method, ie. < 38 000 years BP in age.

13.3.2.    The 1994 Attempt at ¹⁴C Dating Worth's Hominin Collection.
It has been confirmed that at least one of the Cattedown fossil bones from Robert Burnard's donation to the Plymouth City Museum was of human origin. We have yet to determine if there are any more. Remember that
Robert Burnard's Collection and subsequent donation of Cattedown fossil material did not follow the "ownership route" of Worth's material. Burnard's fossils went from the cave excavation into his own private
Collection, where it remained for about 12 years, before being donated directly to the Plymouth City Museum in 1899. The provenance is quite different.

In one of the attempts at dating the Hominin material in 1994, a proximal fragment of Human Radius from the Burnard Collection (Accession No. PM 1297) and originally derived from Worth's Cattedown Bone Cave,
was removed from the premises of the Plymouth City Museum by M. Bishop and given to K. Ray on 02 March 1994. It was sent to Andrew Chamberlain at Sheffield University. However, the sample bone was found to
be "unsuitable" for dating.
In a personal electronic communication dated 31 January 2005, we have received the following helpful information from Mr Andrew Chamberlain, who sheds some additional light upon this attempt at dating one of the
Cattedown Hominins. We are grateful for his following contribution :-

"Just to correct the record on the Human radius for which I attempted to gain a radiocarbon date (PM 1297). The problem encountered by the Oxford University Radiocarbon Accelerator Unit in 1994 was
that the specimen did not contain enough collagen to allow dating to proceed. While it is possible that the lack of collagen is due to great antiquity, it is more likely that it is a result of the circulation of
alkaline groundwater that has destroyed the collagen, rendering the bones chalky and brittle. (I expect that you may have noticed this on other unburnt bones in the Burnard Collection). Thus the antiquity
of the Human skeletal remains is unresolved. It is possible that in the intervening years the Oxford lab has become more proficient in extracting collagen from bone, so a repeat dating exercise may be
warranted on this material."

13.3.3.    The Problem with Radiocarbon Dating Worth's Faunal Collection :
[To be continued.]

13.3.4.    Exciting New Opportunities of Dating Fossil Material from Worth's Cattedown Bone Cave :
[To be continued.]

RADIOCARBON DATING No. 1....[BM-729. Cattedown Cave, Devonshire] :
A complete tibia from among the new (Rangifer tarandus) Reindeer finds was sent to Richard Burleigh at the Radiocarbon Laboratory at the British Museum (Bloomsbury) who in due course announced a date
of 15 125 ± 390 years B.P. (δ¹³C = -24.7‰).

The following extract of data is taken from the publication "Radiocarbon", Volume 21. (3). pp. 339 and 341., from information provided by Richard Burleigh and Andrew Hewson of the Research Laboratory, the British
Museum, London;

-..Methodology :
The date was obtained by liquid scintillation counting of benzene using a Model 3315 Hewlett Packard 2100A computer system for on-line processing of counting data. Sample materials were pre-treated
with dilute acid and alkali as appropriate; only the Collagen fraction of bone was used for dating.
The Date is expressed in radiocarbon years relative to AD 1950 based on the Libby half-life for ¹⁴C of 5570 years and is corrected for isotope fractionation ( δ¹³C values are relative to PDB). No corrections
have been made for natural ¹⁴C  variations. The modern reference standard is NBS oxalic acid. The error quoted with the date is based on counting statistics alone and is equivalent to ± 1 standard deviation (± 1σ).
-..Sample Description :
BM-729. CATTEDOWN CAVE, Devonshire :
Collagen from tibia of reindeer [Rangifer tarandus L.] from Cattedown Cave, Plymouth, Devonshire, England. Tibia (ref. CBR 15.6.74/3) was from an associated group of bones representing the hind part of a single skeleton from late Pleistocene fill. Collected 1974 by B. Lewarne; submitted by A.J. Sutcliffe, Dept. Palaeontology, British Museum (Natural History) as part of programme for dating late-glacial and
post-glacial mammals in British Isles.
-..Comment..(A.J. Sutcliffe) :
"date corresponds with time of glacial advance and low sea level inferred from field evidence (Sutcliffe & Lewarne, 1977); human skeletal remains of possible Pleistocene age were found in 1886 in cave
filling ca 60 metres away in same Devonian Limestone formation (Worth, 1887; 1888)."

.
RADIOCARBON DATING No. 2....[OxA-17160. Cattedown Reindeer Rift, UK.] :
In April 2005., the Society was approached by Dr. R. Jacobi, a Member of the AHOB Project, with a request for the Society to submit a sample of the Cattedown Reindeer skeleton for a more accurate dating procedure,
which would involve the removal of only 500 mg. bone material by drilling into the heart of a bone. As a consequence, on Friday 22 April 2005., the Society submitted the fossil Reindeer's Right Calcaneum for testing.
It was to be sent to the Oxford University Accelerator Unit of their Research Laboratory for Archaeology and the History of Art. On 11 July 2007., the Society received a reply. 
The Oxford Accelerator Unit announced a date of 14 550 ± 55 years B.P. The data-set calibration information is reproduced in the Image to the left.
The full data is published in "Archaeometry" journal, about which we shall give more information when we have it.
In the accompanying communication, Dr. Jacobi offered the following information :-

"The new radiocarbon determination fits not too badly with the original age obtained by the British Museum Research Laboratory. Both dates indicate that your Reindeer lived during the time of extreme cold
near the end of the DIMLINGTON STADIAL (26,000 - 13,000 radiocarbon years ago). Interestingly, we have just obtained a date of OxA-14826 = 14,395.± 60 years B.P. for an Astragulus of a reindeer
from Kent's Cavern, which is almost identical to the Cattedown result and overlaps with it at 20. The Kent's Cavern date came as something of a surprise as we had expected the bone to be of younger
age."

Photo 13.5.4...The MicroScribe G2X 3-D Morphometric digitizer in use during the
osteo-morphometric analysis of the Cattedown Hominin Bones.
(Photo :  B. Lewarne, 19 Nov. 2004.)

13.5.1.    OSTEO-MORPHOMETRIC ANALYSIS :
Since the time of their donation to the Plymouth City Museum after their rescue from the burning ruins of the Plymouth Athenaeum, some pieces of the Hominin fossils have undergone a very primitive "reconstruction", which in some cases, has served only to join mis-matched pieces of bone together and additionally, in every case, to further damage the already fragile bone fragments.
The glue that was used seems to have been a type of "Evostick" which uses a very powerful solvent. These very damaging and futile attempts at reconstruction will shortly be "de-constructed" by the Museum.

We have embarked on a programme of joint collaboration with the Plymouth City Museum and a Bio-anthropologist from Oxford Brookes University to undertake a detailed morphological examination of Worth's
Cattedown Cave Hominin skulls. Certain absolute measurements between standard "reference points" on various parts of the human skull, when compared with measurements between those same "reference points"
obtained from a wide variety of fossil Hominin skull-types, can lead to a useful "morphological typing" of the skull under investigation. A database of such measurements is necessary for such a comparative study.
The larger the database, the more useful and valid the interpretation. Such work needs to be undertaken before any future "reconstructive" work is carried out on the Cattedown Hominin Fossils.

We hope to be able to report on the outcome of this attempt in due course in 2005, however, for the interim, we can present some images recorded during the first stage of data-collection, undertaken on 19 November
2004 and confined to the mandibles of the Hominin skulls excavated from Worth's Cattedown Bone Cave. The next stage of data collection will involve the maxillae of the same Hominin skulls.

13.5.2.    DNA TESTING :
[Further information to follow.]

13.6.1.    A Direct Dating Technique using Extant Ossiferous Material from Worth's 1886-87 Excavation now in the Plymouth City Museum Collections :
We believe that there may be sufficient Hominin fossil material in existence within the Plymouth City Museum Collections and which originates from Worth's excavation. Even if we discount the use of material which has
already been sent for dating, it would appear that there may still exist other components of the same Collection, which came into the Museum's ownership without going through the damaging route of the Athenaeum
Museum. An example of such material which has recently come to light is that of a mass of highly-ossiferous stalagmitic breccia. This is illustrated in Section 14a. of the Cattedown Bone Caves Webpages as Image PCM-HC/191104-1. and through an accompanying short video sequence PCM-HC/191104-Mpeg 5. These images show the possibility of using a previously un-used direct source of raw material containing both
exposed (contaminated) samples and totally embedded (uncontaminated) samples of Hominin fossil material.

13.6.2.    A Direct Dating Technique using Extant Excavated Ossiferous Material in Worth's Cattedown Bone Cave :
Considering the quantity of ossiferous stalagmitic material that was originally uncovered and removed by Worth during his excavation, there is a good chance that some of it was thrown back into the excavated cave when it was backfilled and levelled to the new post-1886-87 quarry floor after the excavation was completed. We believe that there may exist quantities of contaminated and uncontaminated ossiferous stalagmitic breccia amongst this back-fill material, which may be of some use in a series of Dating applications.

13.6.3.    A Direct Dating Technique using Extant Superficial in situ Unexcavated Ossiferous Material in Worth's Cattedown Bone Cave :
Considering the survival of stalagmitic breccia on the exposed East Wall of the North Chamber, we are of the opinion that there is a good probability of the existence of small quantities of unexcavated ossiferous stalagmitic breccia in situ on the lower levels of the East and West Walls of the North Chamber. The possibilities of what may lie below in the totally unexcavated parts of Worth's Catttedown Bone Cave will not be considered here.

13.6.4.    An Indirect Dating Technique using Extant Unexcavated in situ Stalagmite in Worth's Cattedown Bone Cave :
Again, in consideration of the survival of stalagmite on the exposed East Wall of the North Chamber, we are of the opinion that there is a good probability of the existence of small quantities of unexcavated stalagmite floor in situ on the lower levels of the East and West Walls of the North Chamber. Worth records that because some of the lower stalagmite floors joining the East and West Walls of the North Chamber were so thick, they had to be "dynamited" to remove them. If they were of such great thicknesses, then they should be in evidence when the Cave is exhumed. Samples can then be removed for the application of the Uranium-series Dating Technique. These massive stalagmite floors sealed in a large part of the Hominin Collection. If an age could be determined for the stalagmite at this level in the cave, this would certainly provide a minimum age
for the fossil bones contained within it and sealed below it.

Similarly, this analytical process has been applied more directly to "track the movement" (demography) of Hominins by analysing the chemical / mineral composition of bones and teeth. On the basis (though less so in
modern times) that "you are what you eat", it has been possible to determine through the chemical analysis of the mineral content of bones and teeth, that the remains of individuals discovered by archaeologists in a
given location may have ended their lives far from their geographical origins.

1.2.    Some Extracted Fractions of Bone :
- COLLAGEN :
Around 30% of bone is composed of organic compounds, of which 90 to 95% is collagen, the rest being non-collagenous proteins. Collagen is a fibrous protein which provides the bone with strength and flexibility, and is an important component of many other tissues,
including skin and tendon. Individual collagen molecules contain three polypeptides of about 1000 amino acids per chain with a high glycine and hydroxyproline content. Bundles of these collagen molecules are arranged in fibrils with a molecular weight close to 97.1
Daltons. These fibrils are twisted into a right handed coil (fibre) with a total weight of between 95 000 and 102 000 Daltons.
For the collagen fibre to fully mature a number of chemical bonds must form. These include hydrogen bonds involving hydroxyproline, which stabilise the helix, and cross linkages involving hydroxylysine and lysine, which stabilise the fibrillar structure. These processes
occur throughout the growth and maturity of an individual, consequently the density and stability of the bone tends to increase while the solubility decreases. Once these bonds form only a small fraction of collagen can be extracted by neutral salt solutions and organic acids or acid-citrate buffers. The insoluble collagen which remains from such dissolutions, can however, be solubilised by heating above 58'C. At this temperature the triple helix denatures, but will partly reform into a gel when cooled.
Collagen in its unaltered state is also very resistant to proteolytic enzymes, however a group of enzymes exist which degrade native collagen fibrils under physiological conditions of temperature and pH; these are the collagenases. An enzyme secreted by the gas
gangrene bacteria (Clostridium perfringens and Cl. histolyticum) and Bacteroides melaninogenicus, a bacterium common in the gingival crevice of the tooth, will also cleave the triple helix. The peptide's produced in such cleavage are then open to proteolytic attack
from the more conventional enzymes.
- AMINO ACIDS :
Collagen molecules are composed of linear, unbranching sequences of approx 20 naturally occurring amino acids. The structure of the molecule is stabilised by hydrogen bonds; the most common being between the amino group (-NH2) of one residue and the carboxyl
group (-COOH) of a second residue, resulting in both acidic and basic properties. Uncharged side-chains also interact with one another, but by excluding water from their mutual interfaces (i.e. hydrophobic reaction). All amino acids, except glycine, exhibit optical
activity, existing in the natural state as laevo-rotary compounds, a property apparently restricted to amino acids of a biological origin, a property which is exploited in amino acid racemisation dating.
In general the composition of mammalian collagens shows little variability. Of special significance to recent AMS works is the amino acid hydroxyproline. Hydroxyproline is found rarely in other proteins but comprises about 10% of all amino acids in collagen. However,
from a practical point of view the use of hydroxyproline for Carbon-14 analysis is limited as it does not occur in large quantities in fossil bones, has been detected in natural waters, is excreted in the urine, and can be found in some plants.
- APATITE :
Seventy percent of bone is made up of the inorganic mineral hydroxyapatite, which includes calcium phosphate, calcium carbonate, calcium fluoride, calcium hydroxide and citrate. This inorganic component [Ca₃(P)₄)₂]₃.Ca(OH)₂ is predominantly crystalline, though
may be present in amorphous forms. The crystals are platelets or rods, about 8 to 15Å thick, 20 to 40Å wide and 200 to 400Å long. The substitution mechanisms that occur in the hydroxyapatite of bone include intercrystalline exchange and a recrystallisation due to
dissolution and reformation of crystals, with the addition of new ions into the crystal structure replacing Ca²+ or being adsorbed on the crystal surfaces.

1.3.    Teeth
The tooth is constructed of three layers; the pulp cavity, containing blood vessels and nerves; this is covered with the dentine; where the tooth is exposed the dentine is covered by enamel; and the submerged roots are covered with cementum. The cementum closely
resembles cortical bone in composition, except that dentine is hard and dense, being almost 75% mineral but with a higher collagen content than bone (30% compared to 15% in bone). The enamel is denser and harder and is almost 98% mineral with the
hydroxyapatite crystals being much larger than those of dentine, cementum or bone and consequently more resistant. Fully formed enamel contains a small amount of low-molecular weight peptides that are almost devoid of proline and hydroxyproline.

1.4.    Bone Weathering :
Little attention has been given to the environmental conditions of bone preservation. However, the quantities and composition of surviving organic materials in a specimen are dependent on their burial environment. Environmental factors which have been suggested
as influencing the rate at which collagen degrades include the composition, pH and hydrology of the matrix; oxygenation; temperature; and changes brought about by soil flora and fauna.
In a generalised view of bone degradation the protein component undergoes relatively slow hydrolysis to peptides, which then break down into amino acids. At the same time there is spontaneous rearrangement of the inorganic crystalline matrix which weakens the
protein-mineral bond and leaves the bone susceptible to dissolution by the action of internal and external agents. Alterations during diagenesis are believed to include random cross-linking, humification of parts of the molecule, attachment of exogenous humic
materials, and hydrolysis with preferential loss of some amino acids.
Two major groups of contaminants exist; humic and non-humic substances. Humic substances are dark coloured acids moderately high-molecular-weight polymers of indefinite structure. They represent an extremely heterogeneous mixture of molecules, including amino
acids which, in any given soil or sediment may range in molecular weight from 2,000 to over 3 000 000 and have a range of properties depending on size and attached functional group. While humic substances are generally insoluble in acid and soluble in alkali, one
component, the fulvic acids, are soluble in both mediums making them extremely difficult to separate from collagen. Non-humic substances include all classes of organic compounds. The major contaminants are polyphenols, polysaccharides, lignins as well as degraded
collagen and other broken down bone components.

1.5.    Radiocarbon Dating :
The first radiocarbon measurements on bone were on naturally burned bone. Soon after Libby (1952) stressed concern over the low organic carbon content, porous structure and possible effects of putrefaction and chemical alteration on the bone. Only two samples of
whole bone had been measured at this time, and both gave young dates. Consequently, while there had been little work in this area, bone did not appear in Libby's 1952 listing of suitable sample materials, though burned bone was ranked alongside charcoal at the top.
However, the obvious importance of bone to the chronology of many sites saw a continued interest in bone as a dating medium. The major problem was traced to the use of whole bone to generate Carbon Dioxide for Carbon-14 measurements, whereby contamination
from both carbonates and organics could enter the date. Initial efforts to remove the indigenous organics from the bone included techniques such as the artificial pyrolysis of bone by May (1955) whose process was designed to minimise loss of residual organics, acid
digestion and dialysis and the gelatinization of "collagen". Despite these attempts problematic dates still persisted. In a review of the literature up to 1960, Olson noted that bone dates were most often rejected. Proof that humates were the predominant contaminant in
decalcified bone was finally given by De Vries. A variety of techniques were developed to remove this matter: Initially the pre-treatment procedure used on charcoal was adopted whereby decalcified bone is extracted with 0.1 to 0.5 M NaOH; conversion of the sample
to gelatin by Longin (1971); and later Protsch (1975) combined the HCl, NaOH and gelatinization steps. This is the general "collagen" extraction procedure used today in carbon dating and dietary analysis.

By the mid-70's a number of reviews and evaluations of bone dating were being undertaken. One group at the Uppsala laboratory proposed the use of different fractions (acid soluble and acid insoluble) for the majority of bones on the basis that it would be improbable
for contaminants to cause the same error in different fractions. Unfortunately the yields from the different fractions were often insufficient for conventional dating techniques.
Initial descriptions of experiments demonstrating the feasibility of accelerator or cyclotron-radiocarbon-based isotopic measurements appeared in 1977. The advent of AMS enabled dating of small amounts of material, of material with very low organic carbon content,
and multiple Carbon-14 determinations of different organic fractions. While this was a clear advantage in the dating of bone, the use of smaller samples required a clearer separation of the organic and inorganic portions. Therefore, more emphasis had to be placed on
the purity of the sample.
Cation exchange chromatography had initially been introduced for the dating of problem samples. The large sample sizes and excellent preservation of collagen in the bones at the La Brea tar pits, California, made these Carbon-14 measurements possible, but proved
to be too expensive and impractical for the large samples required with conventional dating methods. The advent of AMS changed this. Initial chromatographic techniques involved the hydrolysis of the extracted "collagen", but the incomplete removal of humic acids
by gelatinization, alkali and acid treatments often resulted in cross linkages with residual impurities when hydrolysed. Attempts to remove humates prior to hydrolysis using XAD resins and decolourising charcoal still failed to remove exogenous amino acids associated
with soil contaminants. More recently to aid in the understanding of the series of reactions that can take place during diagenesis and pretreatment, van Klinken (1994) has used sample yields during enzymatic cleavage to screen the degree of cross linking.

To counteract possible contamination products, techniques based on the molecular weight and size of the collagen molecule have been used. Brown, Nelson, Vogel and Southon (1988) modified the Longin method of "collagen" extraction by adding an ultrafiltration
step (gel electrophoresis) designed to exclude low molecular weight species. Another approach developed to purify collagen for stable isotope analysis involves the use of collagenase, which preferentially isolates tripeptides of known length from the surviving collagen
fragments.
Other attempts have concentrated on identifying relatively uncontaminated parts of bone. The isolation of "aggregates" which were identified as having potentially a better protected environment for collagen survival was undertaken by DeNiro and Weiner, but do not
give reliable results from bone with a low collagen content. Recently several researchers have noted the use of non-collagenous components for dating seriously degraded bone. Long, Wilson, Ernst, Gore and Hare have suggested that phospho-proteins may be
protected from degradation as they bond to the apatite structure. Gillespie noted the existence of osteocalcin, osteonectin and other phosphoproteins, proteoglycans, and glycoproteins as well as blood proteins, which may display differential survival characteristics to
collagen.

The first suite of Carbon-14 measurements of a non-collagenous protein were undertaken on osteocalcin by Ajie, Kaplan, Slota and Taylor (1990). Osteocalcin makes up 1% of total bone protein and appears to bind tightly to hydroxyapatite, suggesting a good
possibility of being protected from contamination. Further, it has not been detected in many species of bacteria, plants or invertebrates. However, osteocalcin values on two skeletons from the Haverty site (Los Angeles) gave disproportionably old values, which if correct
would signify the oldest human remains in the western hemisphere. It may be that for osteocalcin to be a suitable medium, isolation of essential amino acids may need to be performed.

APATITE FRACTION :
Early Carbon-14 studies using the inorganic or carbonate fraction of bone were in most cases clearly false, usually too young. Haynes investigated the reliability of using the bone apatite fraction and concluded that erroneous apatite dates can result from carbon
exchange in the apatite structure during recrystallization, and/or surface exchange reactions. Studies into separation of the in situ primary apatite fraction from diagenetic carbonates were initiated in the 1960's and 1970's. Hass and Banewics (1980) reported more
encouraging results, and the demonstration that careful etching with acetic acid can enable the residual carbonate to maintain a biogenetic d13C signal suggests possibilities. But no-one has so far demonstrated that the indigenous carbonate can be extracted reliably
and separated from diagenetic carbonate.
TEETH :
Good results have generally been obtained from teeth, though Carbon Dioxide exchange with the atmosphere may be more efficient in teeth than initially thought. Recent studies on Carbon Dioxide from teeth do, however, indicate that secondary carbonates may be
identified from stable isotope values, suggesting that reliable Carbon-14 determinations may possibly be obtained on tooth enamel.

1.6.    The AHOB Project Approach to Carbon Dating :
The AHOB (= ANCIENT HUMAN OCCUPATION OF BRITAIN) Project, funded by the Leverhulme Trust, is using a new improved methodology developed by project workers.

All these analytical techniques for collagen assessment have met with only limited success, depending on the preservation state of the bone itself. In an attempt to achieve a better chemical characterisation of the fraction selected for dating Stafford, Brendel and
Duhamel (1988) used a number of these criteria to classify bone preservation. Unfortunately, there is currently no consensus as to bio-geochemical methods which can be routinely used in bones exhibiting very low or trace amounts of collagen (i.e. lost >95% of their
protein). As a consequence Hedges and van Klinken suggest an age limit of 18ka as older dates are more sensitive to modern contamination.