Radiocarbon dating bone collagen

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Archaeological bone collagen is highly useful for radiocarbon 14 C dating and palaeodietary reconstruction. Strong progress has been achieved in the past two decades by 14 C and stable isotopic laboratories in removing contamination from archaeological bones, but different pretreatment protocols have been proven to produce varying. Here we compare three collagen extraction protocols used for palaeodietary studies and 14 C dating, considering collagen yield, elemental and stable isotopic data, FTIR analysis, and 14 C dates.

We focus on the impact of ultrafiltration on the yield and quality of the extracted material.

Radiocarbon dating bone collagen

The again underline the importance of rigorous decontamination methods to gain accurate 14 C dates and demonstrate that different protocols have ificant effects on the quality and yield of extracted collagen. Collagen extracted from archaeological bones and teeth is one of the most important biomolecules for radiocarbon 14 C dating and palaeodietary studies. A key concern of laboratories specialising in 14 C dating or palaeodietary analysis of archaeological bone is the refinement of methods to extract and purify collagen for analysis.

This is hampered by three key issues: 1 The degradation of collagen through the rapid or gradual breakup of the peptide chains. This is strongly influenced by environmental conditions, with tropical or arid environments particularly detrimental to the preservation of proteins.

The attack of fungi and bacteria can further alter the triple-helix sequence of mammalian collagen Collins et al. Contaminants may derive from the burial environment such as humic acids or bacteria from the soilduring post-excavation handling and storage including the application of conservatives or the laboratory pretreatment and measurement Nielsen-Marsh and Hedges ; Higham To correctly determine the age of any archaeological bone sample, these issues need to be carefully considered.

The age of any exogenous carbon contaminants matters. Fossil contamination is, therefore, less problematic for Palaeolithic samples where 80 years is usually less than the standard error associated with the measurement. However, for Neolithic or younger samples, the offset exceeds the typical radiocarbon error for this time range of 25—40 years. In contrast, the addition of modern carbon will make 14 C ages younger, with the effects becoming increasingly catastrophic with rising age due to the exponential decay of 14 C.

For these reasons, when considering 14 C dates from Palaeolithic bones, older ages are generally viewed as more likely to be accurate Higham Due to the high risk of producing erroneous 14 Cmodern carbon contamination should therefore be kept below 0. The increasing effect of modern carbon contamination with age. Figure 1. Most 14 C labs employ a collagen extraction method based on the method developed by Longin Although the resulting gelatin solution is often called collagen, in fact, it is a mixture of soluble collagen and non-collagenous proteins DeNiro ; Ambrose ; van Klinken ; Wworth and Buckley The addition of an ultrafiltration step UF following gelatinisation, first suggested by Brown et al.

Ultrafiltration will therefore also retain, and even concentrate, large molecular weight contaminants, including humic acids Szpak, Krippner, and Richards a and lipid contaminants Guiry, Szpak, and Richards which may alter stable isotopic values and 14 C dates. For this reason, it is important to incorporate the NaOH step to remove humic acids contaminants prior to ultrafiltration. It has been demonstrated that the ultrafilter membrane needs to be thoroughly washed prior to use to remove the humectant coating on the filter itself to avoid contamination Bronk Ramsey et al.

Particularly for Pleistocene samples, in many cases, the addition of this step has resulted in older ages usually deemed to be more accurate compared to non-ultrafiltered extracts Hajdas et al. Prior to attempting a costly radiocarbon date, the quality of the collagen extract is a crucial consideration. If the collagen is well preserved, the C:N ratio should fall between 2. Collagen samples with C:N ratios falling outside of this range are considered unsuitable for dating.

Although these ranges are useful quality indicators, they are not infallible and low levels of contamination may be present in an extract without causing the values to fall outside of accepted ranges so a range of quality indicators should be considered Schoeninger et al. Nitrogen isotopes are tracers of trophic level, while carbon isotopes can distinguish diets from marine or terrestrial environments, and if the subsistence relies on C 4 or C 3 plants Schoeninger, DeNiro, and Tauber The use of these tracers in archaeological contexts reveals differences in subsistence strategies, such as highlighting the more variable diets of Homo sapiens compared to Neanderthals Richards and Trinkausthe onset of agriculture in the Americas Tykot, Burger, and Van der merwe ; Kennett et al.

Collagen extracts with C:N ratios falling outside the biogenic range are also discarded for dietary interpretation, due to 1 isotopic fractionation due to the loss of amino acids and protein hydrolysis Bada, Schoeninger, and Schimmelmann ; Ambrose ; Grupe, Balzer, and Turban-just and 2 the potential for contaminants altering the stable isotopic values Sealy et al.

As it has been extensively documented that different collagen extraction techniques yield variable in terms of collagen yield and quality Chisholm et al. Details are given below and shown in Figure 2. Two tests Experiment A and Experiment B were conducted to compare the different collagen extraction methods. Comparison of the collagen extraction protocols used in this study. The red cross indicates exclusion of a step. Figure 2. The bone sample is decalcified in HCl 0. Prior to use, the ultrafilters are cleaned by centrifuging once with NaOH 0.

Bone samples are decalcified in 0. The outer surface of the bone sample was cleaned by a sandblaster and then ca. The first Method 1 has been in use since for collagen extraction for stable isotopic studies of palaeodiet e. Richards and Schmitz ; Mannino et al. Initially, the protocol followed Richards and Hedges but was updated to include an additional step to purify the collagen with Ezee-filters and ultrafiltration Sealy et al.

Radiocarbon dating bone collagen

The second protocol Method 2 has been in use since Talamo and Richards ; Fewlass et al. Hublin et al. Methods 1 and 2 are both modified versions of the Longin protocol with the addition of ultrafiltration, but vary in the strength, temperature and duration of the different steps. For each bone, the outer surface was first cleaned by a shot blaster to eliminate impurities from the surface.

Generally, Method 1 is performed on powdered bone, whereas Method 2 uses whole bone pieces, although this can vary depending on what is available. Therefore, for each bone, three aliquots of ca. In order to assess the effects of ultrafiltration on 14 C dates, Experiment B compares Method 2 with an identical protocol that excludes the ultrafiltration step e. All collagen extracts were assessed based on their collagen yield, elemental and stable isotopic values and were analysed with FTIR.

For Experiment B, we included 13 archaeological bone samples spanning different time-periods and environments Table 1 and Supplementary Table S2. All 13 samples we pretreated once with our standard 14 C dating collagen extraction protocol Method 2 with ultrafiltration and once without ultrafiltration Method 3. To assess the quality of each extract, collagen ca. Repeated analysis of both internal and international standards indicates an analytical error of 0.

For the collagen extracts from Experiment B, ca. The spectra were analysed and compared to library spectra of well-preserved collagen and bone. This confirms a trend reported in Colleter et al. Squares are whole pieces of bone and circles are bone powder samples. Figure 3.

Radiocarbon dating bone collagen

It has been noted in several studies that the NaOH step can lead to collagen loss Chisholm et al. In this test, the addition of the NaOH step in Method 2 did not cause a decrease in the yield of collagen compared to Method 1, although in general, we consider these bones to be well-preserved for their age. It has also been noted ly that the pretreatment of powdered or ground bone in lower collagen yields compared to whole bone fragments Schoeninger et al.

The of this study again demonstrate this difference, with the extraction of collagen from whole pieces of bone median 7. The stable isotopic values obtained were in keeping with species dietary expectations. Squares are whole bone samples, and circles are bone powder samples. The error bars show an analytical error of 0. Figure 4. More recently, Guiry and Szpak reported a C:N value of 3.

Based on these criteria, the mammoth and woolly rhino bones from Methods 1 and 2 would both be considered perfectly suitable for 14 C dating and for dietary isotope studies. Figure 5. The only exception was the Mammoth sample 2 as already noted above. It is likely that the longer duration and higher temperature of the gelatinisation step used in Method 1 lead to increased hydrolysation of collagen Semal and Orban ; Beaumont et al.

Figure 6. The 14 C dates obtained from the mammoth and woolly rhino collagen extracted using Method 2 dating protocol are in keeping with dates obtained from these bones, from powdered and whole bone pieces Fewlass et al.

In contrast, the 14 C dates obtained from collagen extracted using Method 1 Palaeodiet protocol are younger for both bones Figure 7 and Supplementary Table S1indicating that the Method 1 extracts still contained a modern C contribution following pretreatment. As the Method 1 and 2 extracts were graphitised and measured in the AMS at the same time, it is unlikely that this contamination occurred only for Method 1 extracts during graphitisation or measurement.

Radiocarbon dating bone collagen

Comparison of radiocarbon dates from mammoth a and woolly rhino b collagen extracts pretreated with the Method 1 orange Method 2 black extraction protocols. Squares are whole bone samples and circles are bone powder samples. Figure 7. Two factors in the pretreatment protocols are the most likely candidates for causing these age discrepancies. Firstly, the inclusion of the NaOH step in Method 2 to remove humic acid contamination. Talamo and Richards reported similarly young ages from the same bones using extraction protocols that also excluded the NaOH step.

We observed a distinct colour change from clear to yellow during the NaOH wash during the Method 2 pretreatment for both bones. We, therefore, consider this the most likely cause for the under-estimated ages, as ly demonstrated in DeNiro and EpsteinAmbroseSzpak et al. Alternatively, different brands of ultrafilter and filter cleaning protocols were used in the two methods. It could be that the cleaning protocol used did not sufficiently clean the Amicon filter, but this hypothesis requires further testing.

Overall, the from Experiment A indicate that both collagen extraction protocols are suitable for palaeodietary studies. The 14 C protocol Method 2 yields a higher amount of collagen than the palaeodietary protocol Method 1. However, the most important insight was that Method 1 did not produce accurate 14 C dates, likely due to the omission of the NaOH step, indicating that this method is unsuitable for 14 C dating. The indistinguishable isotopic compositions between the methods indicates that any humic acid contaminants remaining in the Method 1 extracts were sufficiently low in quantity not to impact the stable isotopic values.

As a caveat, this test is based on only two bone samples with unknown levels of humic acid contamination. For bones without humic acid contaminants, Method 1 may well yield accurate. As it is not possible to determine the level of humic acid contamination in advance, the Method 2 protocol should be used for all bone samples to be dated.

In those cases, the inclusion of the ultrafiltration step may not have as dramatic an impact on the yield as those observed here. Figure 8. As both methods include the same conditions for the NaOH and gelatinisation stages, it could be that a small amount is also lost during filtering with the Ezee filter not used in Method 3 or is retained on the ultrafilter itself.

This indicates that the inclusion of the ultrafiltration step did not affect the stable isotopic values, in agreement with the findings of Cersoy et al. Figure 9. However, the C:N values of all fractions fall within the accepted range of 2. Figure All should be reported as quality indicators in publications, rather than just the commonly reported C:N value.

This indicates that the ultrafilters removed some contaminants that remained in Method 3 extracts, resulting in a more accurate older age. It is not known why one bone yielded an older age using Method 3. The Milli-Q water from the cleaning steps of the ultrafilters is regularly measured with an elemental analyser on chromosorb to monitor carbon content.

Radiocarbon dating bone collagen

These measurements indicate that any carbon remaining on the filter after the cleaning steps is below the level of detection in the EA. Our demonstrate that this material, having passed through the filter, should not be used for 14 C dating under any circumstances as it will not provide accurate. Further testing with other filters is necessary to determine if this is the case only for the Sartorius Vivaspin Turbo15 ultrafilters or other brands.

Since the ultrafilter membrane is coated with glycine to maintain flexibility, we performed FTIR analyses on the filter membrane before and after washing Figure The uncleaned membrane filter presented peaks in the same regions as collagen, which could interfere with the analysis of collagen extracts. However, after the cleaning procedure, the majority of the bands disappeared. FTIR spectra of the Sartorius Vivaspin Turbo 15 ultrafilter membrane used in the Method 2 prior to cleaning red and after cleaning black.

As FTIR analysis detects the composition of the material regardless of the quantity, in theory even low levels of contamination should be visible in the spectra as additional peaks. Alternatively, it may be the case that peaks from the contaminant overlap with the collagen peaks and are therefore masked. This demonstrates that exogenous carbon was present in the extracts without this being apparent in the quality criteria. This possibility was discussed by van Klinken and demonstrates that multiple quality indicators should be considered for each extract.

Radiocarbon dating bone collagen

In some cases, contamination causing high C:N ratios can be the result of material endogenous to the bone or exogenous material that is contemporaneous to the dated material. In these cases, 14 C ages of contaminated material will not necessarily be inaccurate and the limit imposed by van Klinken should be taken as a simple warning. We note and report here two cases Table 2 where bones with C:N ratios outside of accepted ranges produced 14 C dates in keeping with expectations and historical data. Her birth and death dates are known AD Le Cloirec

Radiocarbon dating bone collagen

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Bone need not remain an elephant in the room for radiocarbon dating