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Cite this article
* Herrando-Pérez Salvador
2021Bone need not remain an elephant in the room for radiocarbon datingR. Soc.
open sci.8201351201351http://doi.org/10.1098/rsos.201351
SECTION
* Abstract
* 1. Introduction
* 2. Methods
* 3. Results
* 4. Discussion
* 5. Concluding thoughts
* Ethics
* Data accessibility
* Competing interests
* Funding
* Acknowledgements
* Footnotes
* Comments
Supplemental Material
Open AccessResearch articles
BONE NEED NOT REMAIN AN ELEPHANT IN THE ROOM FOR RADIOCARBON DATING
Salvador Herrando-Pérez
Salvador Herrando-Pérez
http://orcid.org/0000-0001-6052-6854
School of Biological Sciences, The University of Adelaide, Adelaide, South
Australia 5005, Australia
salvador.herrando-perez@adelaide.edu.au
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Salvador Herrando-Pérez
Salvador Herrando-Pérez
http://orcid.org/0000-0001-6052-6854
School of Biological Sciences, The University of Adelaide, Adelaide, South
Australia 5005, Australia
salvador.herrando-perez@adelaide.edu.au
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Published:13 January 2021https://doi.org/10.1098/rsos.201351
* Review history
* Bone need not remain an elephant in the room for radiocarbon dating
ABSTRACT
Radiocarbon (14C) analysis of skeletal remains by accelerator mass spectrometry
is an essential tool in multiple branches of science. However, bone 14C dating
results can be inconsistent and not comparable due to disparate laboratory
pretreatment protocols that remove contamination. And, pretreatments are rarely
discussed or reported by end-users, making it an ‘elephant in the room’ for
Quaternary scientists. Through a questionnaire survey, I quantified consensus on
the reliability of collagen pretreatments for 14C dating across 132 experts (25
countries). I discovered that while more than 95% of the audience was wary of
contamination and would avoid gelatinization alone (minimum pretreatment used by
most 14C facilities), 52% asked laboratories to choose the pretreatment method
for them, and 58% could not rank the reliability of at least one pretreatment.
Ultrafiltration was highly popular, and purification by XAD resins seemed
restricted to American researchers. Isolating and dating the amino acid
hydroxyproline was perceived as the most reliable pretreatment, but is
expensive, time-consuming and not widely available. Solid evidence supports that
only molecular-level dating accommodates all known bone contaminants and
guarantees complete removal of humic and fulvic acids and conservation
substances, with three key areas of progress: (i) innovation and more funded
research is required to develop affordable analytical chemistry that can handle
low-mass samples of collagen amino acids, (ii) a certification agency overseeing
dating-quality control is needed to enhance methodological reproducibility and
dating accuracy among laboratories, and (iii) more cross-disciplinary work with
better 14C reporting etiquette will promote the integration of 14C dating across
disciplines. Those developments could conclude long-standing debates based on
low-accuracy data used to build chronologies for animal domestications,
human/megafauna extirpations and migrations, archaeology, palaeoecology,
palaeontology and palaeoclimate models.
1. INTRODUCTION
Radiocarbon (14C) analyses of bone, teeth, antler and ivory—hereafter
‘bone’—answer important research questions in Quaternary sciences [1–3] but also
contribute to a panoply of scientific disciplines including conservation biology
[4], climatology [5], ecology [6], genetics [7,8], and human and wildlife
forensics [9,10] (figure 1). 14C dating determines the geological age of a given
fossil based on the Libby half-life of 14C (5568 years) as it decays into
nitrogen 14N [11,12]. The quantification of a sample's 14C content is done by
either measuring radioactive decay (β-decay counting) or direct counting of 14C
atoms remaining in the sample through accelerator mass spectrometry (AMS) [13].
Higher precision measurements, and ability to date 1/1000th the mass required by
β-decay counting, have made AMS 14C dating the dominant technology for 14C
chronologies. Thus, AMS 14C instrumentation is currently used by more than 150
14C laboratories worldwide [14]. While modern particle accelerators can
technically determine ages to 10 14C half-lives [15], or approximately 55 000–57
000 years, the practical dating limit is eight half-lives (approx. 48 000 years)
due to sample type (inorganic versus organic carbon), pretreatment chemistry and
efficiency to remove contaminants [16]. The development of calibration curves
(IntCal20, SHCal20, Marine20) allows the calibration of 14C dates up to 55 000
calendar years before present (BP, where present is 1950 AD [Anno Domini]) [17].
Figure 1. Publication trends using radiocarbon data and concepts. Annual number
of publications (1950–2019) using the term ‘radiocarbon’ in combination with
generic expressions of skeletal remains (antler, bone, tooth, teeth, ivory,
skelet*) in the title, abstract or key words as recorded in the bibliographic
database Scopus (a). Barplot shows those publications classified according to
Scopus' ‘Subject Areas’ (b). Search done on 8 November 2020 and retrieved 2512
publications.
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Fossil bone has been historically regarded as one of the most difficult and
unreliable materials for 14C dating due to contamination, degradation and
carbon-exchange issues [18–22]. For example, 70% of the oldest 14C dates (mostly
from bone) from Europe's Middle and Upper Palaeolithic sites published before
2010 are now considered to be underestimates of their actual age due to
contamination with more recent 14C [23]. Suffice it to say that a 55
000-year-old sample contaminated with only 1% modern 14C will result in a 40 000
year 14C measurement [2]. If we were characterizing the environment experienced
by the animal or human individual being dated, this 15 000 year error would
place the fossil in any of three different transitions from cold (stadial) to
warm (interstadial) palaeoclimates over the Last Glacial Period [24]. Bone
contamination occurs because both the protein (predominately collagen) and the
mineral phase (carbonate hydroxyapatite or bioapatite) are chemically reactive
with enclosing or overlying soils and sediments, and rain and groundwaters.
During centuries to millennia of burial [25–27], bone protein and its mineral
carbonate can incorporate exogenous organic and inorganic carbon, while collagen
can degrade to levels too low for 14C dating [16,28,29]. Not surprisingly, bone
yields the highest rates of 14C dating failure among datable materials due to
poor preservation and contamination [30]. Overall, the application of different
physico-chemical treatments to remove those contaminants prior to 14C dating
(collectively known as ‘pretreatment’) has been long recognized as a challenging
enterprise [30–33]. Ever since the Nobel-Prize winning conception of 14C dating
by Willard Libby [34], Libby himself foresaw that bone ‘… is a poor prospect
[for 14C dating] for two reasons: the carbon content of bone is extremely low;
and it is extremely likely to have suffered alteration’ [35, p. 45], [36].
To address those issues, gelatin isolated by the chemical method adapted for 14C
dating by Robert Longin in 1971 [37] (‘gelatinization’ hereafter)—denaturing
collagen in slightly acidic, hot water [38]—has become the primary bone
pretreatment method, and is the minimum if not final pretreatment used by the
vast majority of AMS 14C laboratories (figure 2). However, many authors
acknowledge that gelatinization alone fails to remove mild to severe carbon
contamination from Pleistocene-age bone [39–45]. Consequently, gelatinization is
combined with any of three additional steps: ultrafiltration [46], XAD-2
purification [47] or isolation of individual amino acids (molecular-level
dating) [48,49]. These additional steps are also part of the menu of services
offered by some AMS facilities (figure 2), although they add time and cost to
the sample preparation. Concisely, ultrafiltration assumes that molecules larger
than 30 000 Daltons (30 kDa)—approximately one-third the mass of the
non-cross-linked chains of the heterotrimer collagen type I α1 (2 per molecule)
and α2 (1 per molecule) in bone [50]—are from bone collagen, while smaller
molecules (less than 30 kDa) are presumed to include non-collagenous
contaminants unsuitable for dating [46]. XAD-2 purification uses a non-polar,
hydrophobic resin through which hydrolysed gelatin or hydrolysed collagen
solution is passed, and the eluate is collected and dated. Contaminants,
predominately humic compounds, remain on the resin and are either discarded or
afterwards eluted to determine the fraction modern or ‘apparent’ 14C age of the
contaminant [44]. Lastly, molecular-level dating uses mostly the imino acid
hydroxyproline [32] or, less frequently, amino acids (e.g. glycine, alanine,
aspartic acid; [28,48]) for their AMS 14C dating. The 18 amino acids (including
the imino acids proline and hydroxyproline) comprising collagen range from 75 to
181 Da and are isolated from gelatin hydrolysates by using high-performance
liquid chromatography (HPLC) [51,52]. The focus on 131 Da hydroxyproline occurs
because it is virtually unique to collagen and constitutes 9 molar per cent of
total amino acid content [28,32]. In §4.2, I address 14C research over the last
decades to refine methods dealing with contamination issues.
Figure 2. Canonical pretreatments of collagen gelatin from skeletal remains at
radiocarbon dating facilities using accelerator mass spectrometry (AMS). Bone
demineralization and collagen denaturation (green text) by the so-called Longin
method (after Longin [37]) can be combined with alkali rinses and solvents, and
the resulting gelatin is then optionally subject to one of three additional
pretreatments. The final product is fast-frozen and combusted into graphite or
CO2, which are the ultimate substrates AMS particle accelerators count the
(radio)carbon atoms from. Throughout the chemical pretreatment of bone, solvents
can remove conservation substances from museum specimens, while alkalis can
remove humic contamination non-covalently bound to the collagen fibrils.
Pretreatments include: (i) ultrafiltration separates the molecular fraction
larger than 30 kDa retained through an ultrafilter membrane and only that
fraction is dated; (ii) XAD-2 resins resemble a pile of microscopic beads with a
porous surface that binds to contaminants, only the eluted pool of collagen
amino/imino acids is dated; and (iii) high-performance liquid chromatography
(HPLC) isolates single amino/imino acids into bands by their structure,
hydroxyproline being the most frequently dated given its bone abundance and
specificity. XAD-2 purification and hydroxyproline isolation require the
previous hydrolysis of the collagen gelatin into free amino acids.
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14C age discrepancies obtained from gelatin of the same fossil bone with and
without these additional pretreatment steps range from hundreds to thousands of
years [53–63]. These discrepancies can lead to starkly contrasting conclusions
about the demographic and genetic history of species including domestication,
invasion and extinction events, and placement within discrete climate events,
and, therefore, deserve careful consideration [2,64]. However, researchers in
many disciplines currently ignore whether there is consensus in the research
community about which pretreatment protocols provide the most accurate 14C ages
of fossil bones. Herein, I quantify such a consensus through a questionnaire
survey across 132 researchers (25 countries) at the forefront of the generation,
use and/or publication of 14C dates and associated extraction of collagen from
late-Quaternary human and megafauna bones. Their research demands accurate 14C
dating to examine broad demographic, climatic, cultural and ecological issues. I
initiated this survey because, from my own experience curating 14C data from
multiple literature sources, the issues posed by choice of pretreatment are
rarely discussed by end-users, and pretreatment methods are often not even
reported, making the topic a real ‘elephant in the room’ for Quaternary
sciences. I argue that specialist experience should propel and guide a range of
urgent developments to enhance the accuracy and affordability of bone 14C dating
and its application to the many research disciplines using geochronological data
to unravel the past of human societies and the Earth's biodiversity.
2. METHODS
I invited 267 researchers (potential audience) to participate in a questionnaire
survey by electronic mail, including personnel of AMS 14C facilities (n = 60),
all the editors of the journal Radiocarbon (n = 28), five of the specialists
leading the International Laboratory Intercomparisons (described in §4.3), 13
additional researchers at the front line of research into collagen extraction
for 14C dating and scientists who were top-ranked in Scopus for their
publication record using 14C dates of fossil bone from humans (n = 78, plus 22
combining 14C data and ancient DNA) and megafauna (n = 93) in the primary
literature (research articles in peer-reviewed journals). The former categories
are non-exclusive so a Radiocarbon editor, for example, could also work at an
AMS facility and/or lead publications of megafauna 14C dates. Of the potential
audience, 148 researchers agreed to participate (55%), 19 referred to a
colleague to do the survey in their place (7%), and 28 (10%) and 71 (27%)
declined or did not respond, respectively. A total of 132 submitted their
responses (49% representing 25 countries) and constitute my ‘target audience’.
All respondents completed the survey online via Google Forms. The survey
consisted of four sections (in four successive webpages) totalling 12 questions
that could be completed in 5–10 min. I describe the four sections in the
following, while a copy of the questionnaire layout is provided in the
electronic supplementary material (appendix SA).
In §1—‘Expertise’ (three questions), respondents confirmed their (1.1) area of
expertise and (1.2) focal study taxa (animals and/or humans), and (1.3) whether
they had had any experience working at a 14C laboratory.
In §2—‘Pretreatment’ (four questions), researchers (2.1) ranked the reliability
of four pretreatments (namely, gelatinization alone, and gelatinization with
further steps of ultrafiltration, XAD-2 purification or hydroxyproline
isolation; figure 2) from 1 (low reliability) to 5 (high reliability) in order
to remove contamination of exogenous carbon from a bone sample before AMS 14C
dating (including an ‘I don't know/I am unsure’ option for each pretreatment),
(2.2) chose one of the former four pretreatments should they a priori know that
a bone sample was severely contaminated with exogenous carbon (including an ‘I
don't know/I am unsure’ option) and confirmed whether they customarily (2.3)
request a specific pretreatment when submitting bone samples to a 14C laboratory
(including an ‘I have never submitted bone, tooth or ivory samples to an AMS 14C
dating laboratory’ option) and (2.4) use pretreatment information as a criterion
to rank the reliability of 14C dates collated from the literature (including an
‘I have never collected/used 14C dates from the literature’).
In §3—‘Samples’ (three questions), respondents stated whether, before (3.1)
submitting a bone sample to a dating facility (including an ‘I have never
submitted a bone, tooth or ivory sample to a 14C dating facility’ option) or
(3.2) extracting the gelatin (including an ‘I have never extracted collagen from
a bone, tooth or ivory sample’ option), they suspected the bone could be
contaminated with exogenous carbon, then (3.3) ranked from 1 (lowest importance)
to 5 (highest importance) a total of 10 criteria to choose pretreatment before
submitting bone samples to a 14C laboratory—respondents were asked to give rank
= 1 to all 10 criteria if they had never submitted bone samples to a 14C dating
facility (mostly personnel from AMS facilities).
Lastly, in §4—‘Feedback’ (two questions), researchers were given the option of
(4.1) singling out one research paper they would cite to support their choice of
the most reliable bone pretreatment and (4.2) giving constructive criticism
about the survey and their own bone-dating experience.
Overall, my focus is on capturing specialist opinion and experience about bone
14C dating from different perspectives. Thus, §§1, 4, 2.1, 2.2 and 2.4 could be
equally answered by all respondents irrespective of their expertise, §§2.3, 3.1
and 3.3 are directed to users of 14C dates who submit samples to 14C
laboratories, and §3.2 is directed to researchers with experience in extracting
collagen from bone samples whether they do it as part of ongoing investigations
leading to publications and/or as personnel of a 14C laboratory dating samples
for customers.
A draft of the questionnaire was piloted for clarity, completion time and design
with the members of the Australian Centre for Ancient DNA (The University of
Adelaide, Australia) in November 2019, and the final version was distributed to
the target audience in December 2019 to March 2020 according to the periods of
availability communicated by individual respondents. Each respondent submitted
one questionnaire (predefined option in Google Forms), while each submitted
questionnaire was automatically stored online in Google Forms' default
spreadsheet. The raw responses from all respondents are provided in the
electronic supplementary material (appendix SB). The frequency of choices across
respondents per question was plotted in bar plots and pie charts using the
package base from the Comprehensive R Archive Network [65].
The survey fulfills the University of Adelaide's ethical standards (Human
Research Ethics Committee Approval Number H-2019-240 to S.H.-P.) and informed
consent to participate in the survey was obtained from all respondents. To abide
by those standards, the survey was strictly anonymous. Thus, no information
could be retrieved from the Google Forms' default spreadsheet that could be
linked to, or reveal, the affiliation, identity or cultural background of
respondents. The names of the authors in the potential audience were only known
by me.
3. RESULTS
3.1. AUDIENCE PROFILE
Of the 132 researchers who submitted their responses to the survey, 17 (13%), 46
(35%) and 69 (52%) work with human or animal bones or both, respectively
(electronic supplementary material, appendix SC, figure S1a). A total of 6 of
every 10 respondents work or have had previous experience working at a 14C
laboratory (electronic supplementary material, appendix SC, figure S1b), while
8–9 in every 10 respondents have submitted to a 14C laboratory samples of raw
bone (electronic supplementary material, appendix SC, figure S2a) or gelatin
extracted from bone samples (electronic supplementary material, appendix SC,
figure S2b). The predominant areas of expertise across respondents were
archaeology (33%), geochronology (17%) and palaeontology (11%), though the
latter disciplines permeate most of the respondents’ specializations such as
anthropology, (bio)chemistry, genetics, palaeoecology or evolutionary biology
(electronic supplementary material, appendix SC, figure S1c). The profile of the
target audience guaranteed the survey's exposure to a wide range of Quaternary
research and multidisciplinary contexts.
3.2. CONTAMINATION AWARENESS
Whether respondents submit samples of raw bone to 14C laboratories (n = 116) or
do the gelatin preparation themselves as 14C users or personnel of AMS
facilities (n = 105), greater than 95% of them often, sometimes or always
suspect bone contamination prior to 14C dating (figure 3). Of 113 respondents
who have collated ages of fossil bones from the literature for their own
research, 86% regard pretreatment for removing such contamination as a quality
criterion to select or discard individual records (electronic supplementary
material, appendix SC, figure S3). Therefore, researchers are strongly aware of
contamination issues that might impact the results of 14C dating of Quaternary
bone.
Figure 3. Bone contamination awareness in the questionnaire survey. Shown as the
number of respondents (n = 132) who suspect, with four levels of increasing
confidence (never, sometimes, often, always), that bone samples are contaminated
with exogenous carbon prior to radiocarbon (14C) dating. Left and right stacked
bars represent respondents who submit raw samples of bone (n = 116 after
excluding 16 respondents who stated not to have submitted raw bone to a 14C
laboratory; survey question 3.1) or extract the collagen gelatin (n = 105 after
excluding 27 respondents who stated not to have extracted collagen gelatin;
survey question 3.2) for 14C dating, respectively.
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Respondents who have experience submitting samples of raw bone to a 14C
laboratory were asked to rank from low (rank = 1) to high (rank = 5) the
importance of 10 criteria regarding sample pretreatment (figure 4).
Contamination (mean rank = 4.09 ± 0.03 s.e.) and the international prestige of
14C laboratories offering a given pretreatment (4.03 ± 0.04) were the top-ranked
criteria. By contrast, the relatively low ranking of the dating price per sample
(2.46 ± 0.02) and turnaround time for dating results (2.14 ± 0.02) (figure 4)
seem to suggest (somewhat surprisingly) that many researchers are willing to pay
more, and to wait longer, for their 14C results, if contamination can be
appropriately controlled. Remarkably, 52% of the respondents surveyed would not
select pretreatment themselves but ask the 14C laboratory to make the choice for
them (electronic supplementary material, appendix SC, figure S4).
Figure 4. Bone pretreatment reliability in the questionnaire survey. Ranking of
importance (mean ± s.e.) from 1 (lowest) to 5 (highest) assigned to 10 criteria
for choosing pretreatment by respondents (n = 118 after excluding 14 of 132
respondents who stated not to have submitted bone samples) submitting bone
samples for dating to a radiocarbon (14C) laboratory. Criteria include (top to
bottom) a priori knowledge of contamination, pretreatment offered only by
prestigious laboratories, research question under investigation, bone amount
(mass) per sample, pretreatment chosen by the laboratory, geographical locality
of bone find, dating price per sample, study species, return time of dating
results and journal publishing the 14C date (survey question 3.3).
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3.3. PRETREATMENT RELIABILITY
When respondents were asked to pick one single bone pretreatment for its
reliability to remove severe carbonaceous contamination prior to AMS 14C dating
(assuming no limitations of funding or sample size), hydroxyproline isolation
from collagen gelatin was the preferred option (39% of the audience) followed by
ultrafiltration (23%) and XAD-2 purification (9%) (figure 5a). Only fewer than
5% of the respondents chose gelatinization alone, and 23% did not know or were
unsure of what pretreatment to choose (figure 5a). In accord with the previous
results, when researchers were asked to rank each of the four pretreatments from
low (rank = 1) to high (rank = 5) reliability, hydroxyproline isolation (4.05 ±
0.12 s.e.) and ultrafiltration (4.24 ± 0.13 s.e.) were ranked higher than XAD-2
purification (3.39 ± 0.16 s.e.) and, particularly, gelatinization alone (2.55 ±
0.17 s.e.) (figure 5b). Relative to the full set of respondents, best choice,
relative pretreatment rankings and main conclusions prevailed for 74 respondents
with prior or ongoing experience working at an AMS 14C facility, except that
hydroxyproline isolation led mean rankings (4.16 ± 0.23 s.e.) above
ultrafiltration (3.77 ± 0.50 s.e.), XAD-2 purification (3.52 ± 0.22 s.e.) and
gelatinization alone (2.82 ± 0.56 s.e.).
Figure 5. Bone pretreatment preference in the questionnaire survey. Respondents
were asked to choose and rank a given pretreatment if a bone was known to be
severely contaminated with exogenous carbon prior to radiocarbon (14C) dating.
Top panel (a) shows the percentage of respondents selecting one of four
pretreatments (n = 132, survey question 2.2), middle panel (b) shows the mean
reliability [± s.e.] from 1 (lowest) to 5 (highest) ranked by respondents (n =
132, survey question 2.1) and bottom panel (c) shows the percentage of
respondents unable to rank the reliability of a given pretreatment (n = 76,
survey question 2.1). Pretreatment abbreviations: gel, gelatinization alone;
hyp, hydroxyproline isolation; uf, ultrafiltration; xad, XAD-2 purification (see
figure 2).
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A total of 58% of the respondents (n = 76) did not know or were unsure of how to
rank the reliability of at least one of the four pretreatments, and such a
proportion was two to four times larger for the two most complex pretreatments,
XAD-2 purification and hydroxyproline isolation, than for the procedurally
simpler ultrafiltration and gelatinization (figure 5c). Lastly, when researchers
were requested to voluntarily provide a key publication to back their choice of
laboratory pretreatment, 30 of 46 respondents gave any of 19 references (out of
a total of 38 suggested) led or co-authored by past or current personnel of the
School of Archaeology hosting the University of Oxford's Radiocarbon Accelerator
Unit (ORAU) (electronic supplementary material, appendix SC, table S1).
4. DISCUSSION
Using an online questionnaire survey, I show that most respondents suspect that
their studied fossil skeletal materials could be contaminated with exogenous
carbon (figure 3), and acknowledge that the gelatin extracted from the bone
requires additional pretreatments to remove this contamination prior to AMS 14C
dating (figure 5). Of those, hydroxyproline isolation is the preferred option
(39%), followed by ultrafiltration (23%) and XAD-2 purification (9%). Before
submitting bone samples for dating, both contamination and the international
prestige of AMS 14C facilities are the top-ranked criteria respondents ponder to
choose pretreatment (figure 4). One in every two respondents who submit bone
samples for dating to a 14C laboratory do not choose the type of pretreatment
themselves but ask the laboratory to select pretreatment for them (electronic
supplementary material, appendix SC, figure S4), and a similar proportion of the
audience was unable to rank the reliability of at least one pretreatment
(figure 5).
4.1. INTERPRETING EXPERT CHOICES
Researchers perceive that the reliability of dating hydroxyproline is superior
to that of the canonical collagen pretreatments built as modified extensions of
Longin's [37] procedure of demineralization of bone followed by gelatinization
of the collagen (figure 2). Hydroxyproline originates from the
post-translational modification of the other imino acid, proline, and
contributes to the stabilization of the collagen triple helix in animal tissues
[66] where it contributes approximately 13% of the total amino acid carbon [51].
Hydroxyproline occurs in plant cell walls (less than 1% dry weight) [67] and is
enriched in soil organic matter during mineralization [68], but any plant
material attached to a sample of bone will contain negligible amounts of
hydroxyproline and, most importantly, be insoluble during the gelatinization
step and, therefore, unable to contribute any hydroxyproline during AMS 14C
dating [69]. Ultrafiltration is two to three times cheaper per sample than
hydroxyproline isolation by HPLC. Costs can partly explain why ultrafiltration
is the preferred choice, after hydroxyproline isolation, in the survey. Even
though respondents state that they are willing to pay higher prices for
hydroxyproline isolation (figure 4), the fact that I have targeted world-class
research sites might not represent the financial affluence of the average
individual researcher and research team. For the latter, the budget that can be
allocated to AMS 14C dating (relative to other components of a research project)
could be strongly constrained and ultrafiltration or gelatinization alone might
be the most cost-effective options.
Researchers favour pretreatments offered by prestigious 14C laboratories with a
long historical record of developments or refinements of dating procedures, e.g.
‘… the wise decision is to contact a reputable 14C dating laboratory before
sending any samples to discuss with them the best samples to select’ (respondent
#101: electronic supplementary material, appendix SC, table S2). In that
respect, many respondents cite papers produced by ORAU to back their choice of
pretreatment (electronic supplementary material, appendix SC, table S1), which
might reflect ORAU's efficient communication strategy, that 14C chemist Richard
Gillespie at this laboratory was the first to publish hydroxyproline 14C dates
[28] and that ORAU was the first European 14C laboratory to adopt
ultrafiltration as default bone pretreatment [70,71]. This creates a clear
advantage of ultrafiltration over XAD-2 purification. By contrast, I posit that
geographical affiliation is overriding pretreatment reliability against other
criteria determining the use of XAD-2 purification among researchers. Thus,
because XAD-2 purification was introduced to the field of 14C dating by American
geochronologist Thomas W. Stafford [47], it is not part of the default menu of
bone pretreatments offered by non-American 14C laboratories, and has been mostly
used to date skeletal materials found in the Americas (both animal and human).
So, a scientist in the USA (27% of the target audience) is more likely to know
the qualities of, and consequently select, XAD-2 purification of bone for AMS
14C dating than a scientist from other parts of the world. It goes without
saying that a scientist's geographical affiliation should be uncorrelated with
the reliability of a given bone pretreatment. Having said that, sample-shipping
costs, including onerous customs regulations (e.g. sending biological samples
from Europe or America to Australia), might play a role in researchers choosing
pretreatment protocols from AMS facilities located closest to their working
place.
4.2. CONTAMINATION CAVEATS IN CANONICAL PRETREATMENTS
Hydroxyproline isolation, ultrafiltration and XAD-2 purification can fail to
remove some forms of contamination, and can potentially contaminate bone samples
themselves from laboratory equipment and procedures. These pretreatments all
begin with gelatin preparation (figure 2), which might be followed by simple
(0.45–60 µm) through-syringe filters or ultrafiltration requiring
centrifugation. Ultrafiltration has been shown to suffer from carbonaceous
contamination contained in the humectant [72,73] and the constitutive fibrils
[74] of the ultrafilters, and from collagen-humic cross-link complexes [26].
Thus, a 29 kDa fragment of collagen bound to a 2 kDa humic-acid molecule would
have an ‘acceptable’ mass of 31 kDa and ostensibly pass the requirement that
only greater than 30 kDa material is dated. Equipment-related contamination has
been attenuated at ORAU with the sonication and rinsing with ultrapure water of
the ultrafilters [70], though the greater than 30 kDa fraction stills retains
less than 30 kDa material along with non-collagenous proteins and
non-proteinaceous organic compounds [75], indicating that the chemical
composition of the ultrafiltered fraction is not yet properly understood. The
ratio of contaminant versus collagen concentrations increased for ORAU's samples
where the collagen yield was low, resulting in offsets of 100–300 years for
bones younger than two 14C half-lives (approx. 12 000 years) [41] because the
apparent age of the humectant (glycerol) was greater than 35 000 years BP. In
fact, later work has shown that the humectant's age has changed between batches
of samples from fossil (approx. 12 000 to greater than 35 000 years BP) in 2006
to modern (post-1950 AD) by 2011 [76,77]. The age of the humectant in
ultrafilters used at the ORAU continues to be post-1950 AD in age and the
cleaning regime removes any trace of humectant below a few micrograms (T. F. G.
Higham, 28 October 2020, personal communication). Other AMS 14C facilities have
not published quantified assessments of this potential source of contamination.
Nevertheless, to diminish or eliminate this problem, washing the ultrafilters
with weak acid (0.01 N HCl) can increase the yield of collagen, hence decreasing
the contaminant-to-protein ratio [78]. It has also been suggested that
ultrafiltration of the gelatin will fail to remove high-molecular-weight
contaminants, which could be eliminated by means of ceramic nanofiltration
(tests applied to nanopores of 368 and 450 Da) of gelatin previously hydrolysed
to amino acids [79,80].
Both the XAD-2 (which is inert in HCl) and hydroxyproline methods mandate HCl
hydrolysis of decalcified collagen or gelatin in 6 M HCl at 110°C for 22–24 h, a
process that yields a solution of individual, free amino acid cations [28,47].
For the XAD-2 procedure, the 5–10 ml of 6 M HCl hydrolysed protein solution are
passed through 1–2 ml of XAD-2 resin in a 2–3 cc, plastic solid phase extraction
(SPE) column, with the eluate being collected in a glass tube. The resin is next
washed with additional, pure 6 M HCl that is added to the initial eluate. Highly
purified XAD-2 is cleaned with solvents by the manufacturer and results in a
resin with no gas-chromatography detectable residues. XAD-1, -2 and -4 are
styrene-divinyl benzene copolymers (SDVB). Other resins in the XAD series (XAD-7
and higher numbers) are acrylic esters that degrade in HCl and are impossible to
use for AMS 14C dating. XAD-2 is sold under different commercial names such as
‘proprietary’ or hydrophobic resin, acronyms like SBD, SDVB or PS-2
(polysterene-2), and in bulk or pre-loaded into SPE cartridges for numerous
biochemical applications [81], but they all use the same copolymer and
principles of purification. At present, no commercial chemical supplier sells
XAD-2 SPE columns engineered exclusively for 14C dating. For the hydroxyproline
isolation procedure, the gelatin or ultrafiltered-gelatin fractions are
hydrolysed as for the XAD-2 process and the total amino acid mixture applied to
a semi-preparative HPLC, mixed-mode hydrophobic/cation-exchange column and
eluted with a binary gradient of water and dilute phosphoric acid. ORAU has
addressed contamination from resin bleed that contributes a finite amount of
degraded resin to the eluted amino acids [51], but further work is required in a
14C dating context.
A major (and largely ignored) chemical reaction relative to successful bone
pretreatment for AMS 14C dating is the Maillard reaction, which was discovered
in the early twentieth century [82,83], and consists of a covalent bonding
between amino acids and reducing carbohydrates. These sugars are present in soil
humic and fulvic acids [84]—major global components of soil organic carbon [85]
and probably the greatest source of fossil-bone contamination [26,86], with
molecular weights varying from a few hundreds to ten thousands kDa and higher
[87–90]. Humic-acid contamination in bone is generally detectable because these
compounds discolour the bone and its collagen brown to black, while the
low-molecular-weight fulvic acids range from colourless to pale yellow and
yellow and can easily go undetected visually. The cleavage of humic and fulvic
acids cross-linked to collagen cannot be accomplished by ultrafiltration
[26,72,91]. Additionally, the widespread use of alkaline rinses (figure 2)
between the demineralization and gelatinization steps [26], customarily
undertaken by many 14C laboratories, increases the solubility of humates but is
ineffective in removing some humic/fulvic acids [92], and comes at the cost of
lowering gelatin yield [93–95]. Humic/fulvic contamination of collagen can be
removed by state-of-the-art proteomic approaches [92,96], or by hydrolysing the
gelatin prior to dating of total or individual amino acids [52,97].
The human and animal late-Quaternary fossil record is inherently rare and mostly
consists of one or, much less frequently, a few bones per individual.
Consequently, fossil specimens from museum collections pose unique (despite
destructive) opportunities for 14C dating and ancient DNA sequencing [98]. Bone
curation in those collections, including embalmed ancient mummies [99,100],
entails the application of a range of conservation substances (adhesives,
coatings, consolidants) that stabilize skeletal materials and prevent
microbiological decomposition [101]. Because those substances contain carbon, it
is critical prior to AMS 14C dating that museum materials be treated with
routine acid–alkali–acid rinses in combination with organic solvents specific to
every conservation substance [102,103] (figure 2). The apparent 14C ages of the
most commonly used solvents group into two classes: ones with modern 14C content
(post-1950 AD) and those with age ranges of 15 000 to more than 40 000 years
[104]. Depending on the age of the fossil, solvents can, therefore, cause bones
to be dated older or younger than their actual age. Tests on synthetic, porous
material indicate that solvents might achieve complete removal of some but not
all types of conservation substances [105] due to a suite of complex
interactions between solvents, conservation substances and the study material
(e.g. cross-links, oxidation, ageing degradation). Consequently, where
contamination is suspected or confirmed from these sources, reliable AMS 14C
dating of museum bones could be guaranteed by dating individual amino acids
and/or by selecting the regions of the bone least impregnated by conservation
substances [103]. This will of course fail if animal hide or bone collagen glues
were used as a preservative [106] because it would be impossible to distinguish
a fossil's collagen amino acids from those in a collagen glue. In addition,
other substances can also be applied in the field to consolidate or preserve
bone and these materials might not have been recorded. These situations
emphasize the importance of maintaining museum records that detail all
treatments a fossil receives, from sampling to storage.
It is always good practice to (pre)screen samples for collagen preservation
[107]. Methods and metrics include percentage yield of collagen after each
pretreatment step, atomic C : N (carbon : nitrogen) ratios, stable C and N
isotope values [29], whole-bone %N [108] and, less frequently, relatively
expensive but extremely quantitative HPLC [47,48,109] and near-infrared
spectroscopic methods [110]. Several respondents (#11, 16, 50, 67, 121 and 127:
electronic supplementary material, appendix SC, table S2) remarked the
importance of those quality indicators in routine research work using 14C data.
And, in the latter context, many stated that bone samples should be a priori
assessed for contamination sources and, subsequently, either chemically treated
on a case-by-case basis or dated using different pretreatments as appropriate
and/or sent to separate 14C laboratories, e.g. ‘… ideally replicate [14C dating
of a given sample] using a different method. Otherwise you are sunk in the “it
is older, it is better” argument (which, I agree with Kuzmin [111], stinks!)’
(respondent #11: electronic supplementary material, appendix SC, table S2).
Dating individual samples several times (see §4.3) will, however, be often
beyond a researcher's budget, and unless done using different pretreatment
methods, could yield a similar but still inaccurate 14C measurement. Clearly,
the preoccupation that a given pretreatment and/or 14C laboratory might fail to
address contamination adequately (often conditional on the type of material
being dated and funding) seems to be pervasive among 14C users.
One can expect that all 14C protocols of collagen purification dealt with in
this study should be reliable under minimal to near-zero humate contamination
for bones free of conservation substances. This best-case scenario will apply to
samples from subarctic to arctic regions or from habitats (e.g. caves) having
limited soil growth and associated humate production. On those grounds,
ultrafiltration might be a valid 14C bone pretreatment for the enormous amount
of past and ongoing palaeochronological research undertaken in Beringia, Canada,
Northern Eurasia and Patagonia, but their reliability remains to be compared to
mid- and equatorial-latitude sites. If bone destruction is to be minimized and
contamination is expected, XAD-2 purification might arguably be the best
compromise because it purifies all collagen amino acids, rather than only
hydroxyproline or the ultrafiltered (high-molecular-weight) fraction.
4.3. QUALITY CONTROL REQUIRED
A major limitation faced by the growing community of scientists using 14C data
is that laboratory protocols vary among AMS 14C facilities, even for the same
bone pretreatment [112]. Such a procedural variance can make 14C dates of
skeletal materials non-comparable from one laboratory to another and from one
research paper to another. The lack of comparability could question the validity
of the increasing number of studies collating 14C dates from multiple sources
(see §4.4) to deal with hotly debated topics such as the causes of extinction of
late-Quaternary megafauna [113,114] or the timing of the global dispersal of
anatomically modern humans [115]. We might have highly sophisticated analytical
and modelling tools to unravel the mechanisms behind those extraordinary
demographic phenomena, but they will be useless if we are unable to time exactly
when those individuals, populations and species (dis)appeared. This rationale
has been put forward by archaeologists whereby the prowess of Bayesian
chronological models [116] can be truncated by the low quality of 14C data,
sample pretreatment and/or reporting etiquette [117,118].
The main attempt to evaluate dating consistency in the 14C field has been the
International Radiocarbon Intercomparison led by the University of Glasgow (UK)
and endorsed by the journal Radiocarbon [64]. This scheme aims to identify
reference materials that can be dated and compared over time as 14C techniques
evolve. In each of the six assessments undertaken to date [119–124], a range of
14C laboratories has been invited to voluntarily participate and date the same
set of samples, then the Glasgow team has quantified dating consensus across
laboratories. The major limitation of this initiative is that these reference
materials either contain no contaminants, or do not contain the levels and types
of contamination found in fossil bones. Bone has been included only in the last
two assessments (and will be part of the next one [125]), not surprisingly
concluding that there is a need for ‘… an investigation of pretreatment effects,
especially for the bone samples' [119, p. 8]. English Heritage has accumulated
385 bone samples with replicate 14C measurements, showing age inconsistencies at
p = 0.05 (probability of the data given the null hypothesis that several
measurements are equal) for (i) 10 out of 60 samples (17%) subjected to
gelatinization (mean offset = 43 ± 22 years), (ii) 34 out of 208 samples (16%)
subjected to ultrafiltration (mean offset = 10 ± 5 years), and (iii) 26 out of
117 samples (22%) subjected to ultrafiltration versus gelatinization (mean
offset = −7 ± 9 years) [126]. None of these offsets have statistical support,
although the data are slightly more dispersed than expected on the basis of
their quoted errors (A. Bayliss, 24 December 2020, personal communication).
Bayliss and Marshall [126, p. 1156] further note that ‘… this dataset consists
of measurements on generally well-preserved bone from a temperate climate, which
is predominantly less than one half-life in age. This reproducibility may not be
obtained on older or poorly preserved material’.
At the heart of this conundrum lies the fact that no international agency
oversees quality control, training and certification in the field of 14C dating.
Currently, should the necessary funding exist, 14C facilities can be
discretionally created with freedom to adopt specific pretreatment protocols to
compete for customers in a competitive market among more than 150 AMS facilities
currently operating globally [14]. We are indeed far from an arguably ideal
scenario whereby 14C pretreatment procedures are universal across laboratories.
Countering that scenario, one respondent (aligning with many 14C laboratory
personnel and palaeo-researchers I have communicated with) stated that ‘… for
the effort a 14C measurement is requiring, every sample deserves the best
individual pretreatment’ (respondent #40: electronic supplementary material,
appendix SC, table S2). The pitfall is that with different 14C laboratories
favouring different bone pretreatments [127], what ‘best’ means for every sample
can have multiple answers. To my knowledge, no comprehensive guidelines have
been published in the primary literature defining what set of consistent
properties make a given (bone) sample suitable for a given chemical protocol
prior to AMS 14C dating.
This is not to say that pretreatment protocols can be expected to reach
infallibility, nor that AMS facilities should not lead or partake in innovation
along with their business activity. The overarching goals of 14C innovation
should be to attain methodological reproducibility and dating consistency across
laboratories and high accuracy (i.e. 14C ages capturing the true age of a
fossil). However, it is unlikely that pretreatment developments led by one AMS
facility are to be promptly adopted by others. It can take time for information
to be disseminated at conferences or through research papers and for AMS
facilities to test promising procedures rather than adopting them directly.
These tests often leave no trace in the literature (P. J. Reimer, 3 November
2020, personal communication). For instance, collagen ultrafiltration for 14C
dating was an initiative of the Simon Fraser University (Canada) published in
1988 [46]. Via flow of personnel and researchers among 14C facilities, the
method reached the Center for Accelerator Mass Spectrometry at the Lawrence
Livermore National Laboratory (which has used ultrafiltration from the early
1990s to date; J.R. Southon, 9 December 2020, personal communication), other
North American laboratories and ORAU in Europe (M.P. Richards, 27 November 2020,
personal communication). ORAU adopted ultrafiltration in 2000, with some
European sites following 6 or 7 years later in some cases when they first
acquired an AMS (e.g. Aarhus, Belfast, Poznań, Zurich), and others never
including it in their default protocols (e.g. Groningen, Kiel, Vienna). By
contrast, the fact that no European AMS facility provides XAD-2 purification
seems surprising by sheer criteria of dating reliability. A different model
could be explored whereby: (i) those AMS facilities interested in innovation
were coordinated within several nodes of research sites (including
universities), each node pushing chemical developments for specialized aspects
of AMS 14C dating (e.g. types of samples versus types of pretreatment); and (ii)
an international certification agency regulated the transition from development
to customer service according to available personnel's expertise and the
equipment hosted by AMS facilities. In such a model, AMS facilities would have
an incentive to participate in innovation, as all would directly contribute to,
and benefit from, developments.
4.4. FUTURE RESEARCH
Molecular-level dating seems the way to go to advance the accuracy of bone 14C
dating. The rationale is obvious in that, rather than using the gelatin from a
bone sample, or a purified version of it, the safest way of avoiding
carbonaceous contamination is to date the molecular bricks forming the chemical
architecture of collagen. Only molecular-level dating appears to accommodate all
known bone contaminants and can guarantee complete removal of humic and fulvic
acids, conservation substances and any other contaminant of bone collagen. How
the amino acids are separated from the contaminants following collagen
hydrolysis, and how to maximize the datable mass of amino acids given a fossil's
initial mass and degree of collagen preservation, are the steps requiring
research innovation. If dating of collagen amino acids is to galvanize a future
revolution in the chronological study of skeletal remains from the Quaternary
fossil record, chemistry procedures need to be developed that are
contamination-free, affordable by the majority of 14C users across scientific
disciplines and able to handle low-mass fractions of amino acids and valuable
specimens.
Anyone familiar with the primary literature reporting 14C data will know that
molecular-level dating is in practice far less used than gelatinization alone,
ultrafiltration or XAD-2 purification to date fossil bone. Molecular-level
dating is time-consuming, costly and procedurally challenging, reflecting its
dearth of application [51]. To circumvent those limitations, simpler, faster and
more affordable methods are needed to replace HPLC—which has prevailed as the
standard procedure to separate amino acids over the last three decades [51,52]
despite being expensive in terms of the equipment, experienced staff and
reagents required [128]. One possible route is using N-phenacylthiazolium
bromide to cleave glucose-derived protein cross-links [129]. Using this reagent
has allowed researchers to improve the amplification of ancient DNA from
megafauna dung [130,131], but remains to be applied to bone samples for AMS 14C
dating. Another possible route involves first derivatizing the amino acids in a
gelatin hydrolysate with a reagent that does not react with the imino acids
(proline and hydroxyproline)—such as o-phthaldialdehyde as employed for amino
acid racemization dating [132]—combined with SPE cartridges [133]. Those SPE
cartridges have been successfully employed to extract collagen from bone [134]
and should be simpler and cheaper than HPLC methods. Lastly, the specific
chemical reaction of ninhydrin with the α-carboxyl group of free amino acids
(hence not interacting with humates [26]) produces CO2 that has been used for
isotopic fractionation [135] and bone 14C dating [136–138]. This 14C
pretreatment also uses collagen hydrolysed to amino acids in 1 M HCl and is
simpler and cheaper than HPLC, but has been criticized for requiring abundant
glassware and a minimum bone mass of approximately 1 g per sample and remains
open for improvement [139]. Any new developments should of course gauge the
extent by which novel reagents might add carbonaceous contaminants.
On the other hand, the mass of datable amino acids will always be much lower
than the mass of datable gelatin per sample unit. The use of different bone
amino acids can increase datable mass and, in that direction, the established
practice of separating bone proline from hydroxyproline for AMS 14C dating (so
dwarfing the sample mass per imino acid) remains to be comprehensively examined.
For bones that are severely degraded during burial, and/or consist of one or a
few small fragments (the majority of the late-Quaternary fossil record!), and/or
belong to small body-sized taxa such as shrews or mice, and/or have cultural
value (e.g. ancient humans or unique animal specimens), molecular-level dating
might remain unfeasible unless AMS 14C dating incorporates methods of protein
enrichment that could increase the yield of collagen amino acids while aiding in
the removal of potential contaminants [92,140].
Should authors compile sets of ages of fossil bone from multiple sources and
publication years to test hypotheses and make broad inferences about ancient
populations and species? Without data-quality control or ways to rank 14C bone
chemistry, the enterprise is certainly risky but keeps attracting the attention
of high-profile journals. If a widespread standardization of bone pretreatment
protocols came true, we might have to be ready to face the eventuality that many
14C dates of fossil bone (as well as the inferences made from them) published in
the scientific literature over the last seven decades might be inaccurate or
wrong, hence hardly comparable with new 14C dates. XAD-2 purification protocols
have remained procedurally constant since its conception in the 1980s, so bone
14C ages generated through this method should arguably share a similar degree of
reliability over time. Unfortunately, there does not exist a year or time
interval before and after which 14C ages should be deemed (un)reliable (but see
[23,126]) partly because individual AMS 14C facilities have incorporated new
chemical and physical protocols at their own pace (see §4.3) and might have not
recorded how and when these chemical protocols have changed.
The published evidence for the reliability of bone ages obtained through
different pretreatments is sketchy, definitely not comprehensive and would
benefit from a global experiment using skeletal materials of known age from
multiple geological deposits, latitudes and time periods. Although I partly
concur with the view that ‘… the most important criterion, far more important
than pretreatment, and one that is often not considered (as exemplified in this
survey) is “context” of the specimen. That is, clear and unambiguous control of
association and context of the sample with respect to the cultural activities in
question’ (respondent #3: electronic supplementary material, appendix SC, table
S2), the reality is that the stratigraphic integrity of most archaeological and
palaeontological sites cannot be confirmed with 100% confidence. So the
selection of sites for the global experiment suggested above would have to be
based on a careful selection of reliably dated fossil-containing deposits (e.g.
volcanic tephras) and/or deposits showing high chronological agreement by
several dating methods (e.g. electro-spin resonance, optical techniques,
[thermo]luminescence, uranium-series—reviewed by Walker [13]). The latter would
require to frame fossil dates into a comparable ranking of reliability across
multiple dating methods, which has so far only been applied to megafauna fossils
from Australia and Papua New Guinea [141–144] and awaits developments with
global scope. A complementary approach would be to apply different pretreatments
to a comprehensive set of samples whose age (determined by other chronological
methods and/or stratigraphic evidence) conclusively exceeds the limit of 14C
dating. For such old samples, the presence of 14C would be an unequivocal
signature of contamination given an appropriate selection of background samples
(see [145]).
5. CONCLUDING THOUGHTS
14C dating has meritoriously established itself as one of the most powerful
tools for dating cultural and palaeontological deposits from the late Quaternary
[3,56]. The method is conceptually simple and well understood (see
Introduction). Along with its prominence in the Quaternary sciences, its
importance in modern research has been, and will be even more, heightened by the
growing application of palaeoarchives and fossil materials to understand ongoing
global ecosystem shifts and anthropogenic impacts on biodiversity and the
environment [4,146].
While precision and accuracy of the 14C measurement are controlled by AMS
physics, a sample's absolute age accuracy is controlled by its chemical
purification, geologic provenance and taxonomic identification. Precision (how
well we measure 14C content in AMS facilities) determines the magnitude of the
error bars of 14C dates, while accuracy (how well we remove contamination)
determines how far 14C dates depart from the true age of skeletal materials.
Both parameters are different sides of the same coin.
However, progress in the physics of modern AMS 14C dating has driven a
revolutionizing transition from β-decay counting to particle accelerators [147]
(see Introduction) and from there to the prompt incorporation of the latest
accelerators MICADAS [148] already functioning in many 14C laboratories (e.g.
[149–151]). The focus of those developments has been put on minimizing the
required amount of datable mass [152–154]. By contrast, one of the respondents
bluntly expressed that ‘… if AMS labs spent as much money on chemistry and
biology as they do on physics, the inherent inaccuracy in most 14C bone ages
would have been eliminated years ago’ (respondent #4: electronic supplementary
material, appendix SC, table S2). Indeed, the chemistry of modern AMS 14C dating
still rests on refined versions of procedures developed during the 1960s–1980s
(figure 2) and awaits a revolution of its own. We cannot expect this revolution
to be prompted by AMS personnel, geochronologists and Quaternary scientists
alone, given the multidisciplinary applications of 14C data (figure 1).
Additionally, although contamination of fossil samples with modern carbon might
be most problematic for Late-Pleistocene bone, scientists should not be
acquiescent with contamination issues in modern and Holocene-age materials as
science should always strive for reducing uncertainty. More cross-disciplinary
communication and research, particularly with chemists, is a critical endeavour
to better understand the factors that drive the accuracy of AMS 14C dating and
to unite efforts towards integrating chemical protocols and 14C research with
our own fields of specialization. Those efforts should go hand in hand with
funding agencies supporting research projects focusing on the improvement of
less expensive 14C chemistry.
How blasé scientists might be about how bone samples are processed prior to 14C
dating can be inferred from the poor reporting standards of 14C laboratory
protocols in the literature. This deficiency might even curtail the chances
researchers might have to collaborate with world-class geochronologists and
integrate their 14C results with those from other dating methods, e.g. ‘… If
someone asks me: is this [tooth sample] 5000 years or 10 000 years [old]? I
would even date enamel for them if there was no protein preserved, so long as I
know they will either publish the limitations of the enamel method appropriately
or include me as a co-author. If they just want a “number”, or I suspect that
they will publish the date as a number, I will not date enamel for them’
(respondent #46: electronic supplementary material, appendix SC, table S2). Wood
[155, p. 68] painstakingly asserts that ‘No other isotope [14C] measurements can
be so regularly accompanied by such scant description of methods within refereed
journal articles without catching the eye of a reviewer or editor’. This problem
is by no means new. Journal editors [156] and 14C authorities [12] chronicled
early reporting deficiencies from the personnel of 14C facilities who then
routinely published their 14C dates in a range of peer-reviewed journals. The
lack of reporting etiquette is nowadays commonplace among scientists who publish
14C dates, and among the editors handling research manuscripts from specialized
Quaternary to the top multidisciplinary journals, and has prompted authoritative
recommendations [157] that hardly transcend to the array of scientists and
disciplines that consume geochronological data.
Surely, if an author does not report a piece of information, it must be because
it is deemed to be unimportant. One respondent in the survey noted that ‘I am
basically a consumer [of 14C data], but I learn that I need to be more involved
[in how the data are generated]’ (respondent #126: electronic supplementary
material, appendix SC, table S2). And when in my work, I have requested
unpublished pretreatment details of published 14C dates a typical type of
response has been ‘Your request can only be answered by the radiocarbon lab! I
am palaeontologist and morphologist’ (confidential personal communication, 15
August 2019), or ‘I have not the faintest idea what you are asking. I am an
archaeologist and I use dating to contextualize archaeological levels and at
most generate population models' (confidential personal communication, 7
November 2020). These attitudes align with the greater than 50% of the surveyed
experts who ask 14C laboratories to choose bone pretreatment for them. This is
not an inappropriate approach per se as the personnel of AMS 14C facilities
should be the true chemistry, geochronology and physics experts. The problem is
when authors fail to acknowledge the importance of 14C protocols relative to the
importance of the research questions they attempt to answer. No modelling
approach (no matter how sophisticated it is) and no research hypothesis (no
matter how global, trendy or scientifically novel it is) should subjugate the
use of high-quality data, even if less but more reliable data should decrease
the power of a statistical analysis and the scope of the emerging inferences. I
contend that scientists using 14C data should be conceptually more involved in
the chemical processes of data generation—without such involvement, bone
pretreatment might yet remain for many years an elephant in the room of 14C
dating.
ETHICS
The survey fulfills the University of Adelaide's ethical standards (Human
Research Ethics Committee Approval Number H-2019-240 to S.H.-P.) and informed
consent to participate in the survey was obtained from all respondents.
DATA ACCESSIBILITY
Layout of questionnaire survey (electronic supplementary material, appendix SA)
and all responses provided by the expert audience (electronic supplementary
material, appendix SB) uploaded as electronic supplementary material.
COMPETING INTERESTS
I declare I have no competing interests.
FUNDING
The research was funded by an Australian Research Council Discovery Project
(DP170104665). The School of Biological Sciences (The University of Adelaide,
Australia) covered the open-access publication fees.
ACKNOWLEDGEMENTS
I am grateful to all respondents who participated in the questionnaire survey.
Chris S. M. Turney (University of New South Wales, Australia) endorsed the
research ethics application (see ‘Ethics Statement’) and along with members of
the Australian Centre for Ancient DNA (The University of Adelaide, Australia)
piloted the questionnaire survey leading to improvements of survey content and
design. Celia José Herrando kindly made the drawings of antler, bone, ivory and
teeth. Corey J. A. Bradshaw (Flinders University, Australia) revised the first
Abstract. Matthew J. Collins (University of Cambridge, UK) and Richard Gillespie
(Australian National University) gave valuable feedback on drafts of the
original manuscript. Paula J. Reimer (Queen's University Belfast, Northern
Ireland), H. Gregory McDonald (Bureau of Land Management, USA), Kieren J.
Mitchell (University of Adelaide, Australia) and Thomas W. Stafford (Stafford
Research Laboratories Incorporated, USA) revised the final draft following
peer-review. A. Bayliss (Historic England, UK), Thomas F. G. Higham (Oxford
University, UK), Michael P. Richards (Simon Fraser University, Canada) and John
R. Southon (University of California-Irvine, USA) clarified a range of 14C
developments, and Thomas W. Stafford provided feedback on XAD resins and humate
chemistry.
FOOTNOTES
Second e-mail: salherra@gmail.com
Electronic supplementary material is available online at
https://doi.org/10.6084/m9.figshare.c.5249550.
© 2021 The Authors.
Published by the Royal Society under the terms of the Creative Commons
Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited.
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* Manuscript accepted08/12/2020
* Published online13/01/2021
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Keywords
* Quaternary
* hydroxyproline
* ultrafiltration
* chronology
* collagen
* XAD
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Subjects
* biochemistry
* ecology
* palaeontology
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