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Analysis of fatty acids extracted from a whale skeleton: Analytical approach

to evaluate the efficacy of degreasing treatment

Article  in  Journal of the American Institute for Conservation · August 2015

DOI: 10.1179/1945233015Y.0000000011

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 ANALYSIS OF FATTY ACIDS EXTRACTED FROM A WHALE
SKELETON: ANALYTICAL APPROACH TO EVALUATE THE
EFFICACY OF DEGREASING TREATMENT
CHARLÈNE PELÉ, BRUNO BUJOLI, ÉLODIE GUILMINOT, GWENAËL LEMOINE,
ISABELLE LOUVET, AND LAURENT POISSON

EPCC Arc’Antique, laboratoire de conservation, de restauration et de recherche

CEISAM, UMR , laboratoire de Chimie Et Interdisciplinarité: Synthèse, Analyse, Modélisation,
UFR sciences et techniques, départements de Nantes, université de Nantes

Laboratoire Mer, Molécules, Santé (EA ), Université du Maine, IUT de Laval

The Natural History Museum of Nantes, France, was encountering difficulties in the display and preservation of an
insufficiently degreased fin whale skeleton. A degreasing method to solve this issue was thus investigated, using bone
samples having problems similar to this skeleton. First, gas chromatography was used to show that the grease
seeping out the bones was constituted of free fatty acids, and the profile of these free fatty acids was then established.
Experimental conditions for the treatment were then optimized to ensure a good efficacy of free fatty acid extraction
while respecting bone integrity. A succession of brief heptane baths at room temperature was found to be a good
compromise. The appropriate number of treatment applications was determined on the basis of the appearance
of the extracts, and gas chromatography analysis was used to rationalize the evolution in their visual appearance.
During the initial baths, extraction of unsaturated fatty acids (brown oil causing brown spots to appear on the bone
surface) occurred. In contrast, from the third bath onwards, mainly saturated fatty acids were removed. However,
extraction of all the saturated fatty acids is not recommended because of their role in the mechanical support of bone
structure. The treatments tested were shown to have no adverse effects on the organic components of bone, as evi-
denced by high-resolution nuclear magnetic resonance spectroscopy, since fragments of collagen were not detected
in the extracts. However, calcium, but only in trace amounts, was detected by atomic absorption spectrometry. This
suggests that the inorganic part of bone, which is composed of hydroxyapatite, had undergone very limited
degradation.
KEYWORDS: Gas chromatography, Atomic absorption spectroscopy, NMR spectroscopy, Unsaturated fatty acids,
Saturated fatty acids, Degreasing treatment, Whale bones

. INTRODUCTION The lipid composition of whale fat has been

Arc’Antique laboratory was approached in  by the
Natural History Museum of Nantes, France, to under-
take the degreasing treatment of a fin whale skeleton
(Balaenoptera physalus) on display since  (fig. ).
Despite an initial degreasing treatment using trichlor-
oethane and chloroform in  (Lemoine and Guilmi-
not ), large brown spots of fat seeping out from the
bone core could be observed on the skeleton. More-
over, a rancid odor caused by aldehydes was detectable
during summer months. In addition to a loss of aes-
thetic quality, the biochemical degradation of lipids
(hydrolysis, oxidation, etc.) along with the development
of micro-organisms might result in the degradation of
the collagen fibers and be detrimental to the long-term
conservation of the specimen.
described in several papers, but the nature of lipids
present in the bones of deceased whales has been
seldom examined (Ackman et al. a, b, ;
Barclay ; Krahn et al. ; Regert et al. ;
Ruchonnet et al. ). In the case of deceased Mediter-
ranean whales (Ruchonnet et al. ), triglycerides
represent – wt% of total lipids, and  types of
free fatty acids (FFAs) were observed. Oils (liquid or
half-solid at room temperature) contain a high percen-
tage of unsaturated FFAs. By contrast, greases (solid at
room temperature) predominantly contain saturated
FFAs (Horie ; Huot and Roy ). Some
authors (Bottino ; Morgan et al. ; Sergeant
and Moyle ; Evershed et al. ; Ruchonnet
et al. ) have studied the grease extraction of

© American Institute for Conservation

of Historic and Artistic Works 
DOI: ./Y. Journal of the American Institute for Conservation , Vol.  No. , –
 ANALYSIS OF FATTY ACIDS EXTRACTED FROM A WHALE SKELETON
FIG. . Image of the whale skeleton exhibited at the Natural
History Museum of Nantes.
living cetaceans or recently deceased whales by gas
chromatography (GC). The chain length of FFAs is gen-
erally between  and  carbons. The molecular
formula of the fatty acids corresponds to the number
of both carbon atoms and insaturations (i.e. 
carbon atoms and  insaturations (C:) for eicosa-
pentaenoic acid). Common whale fat mainly consists
of palmitic acid (C:), stearic acid (C:), and
oleic acid (C:). Highly unsaturated FFAs such as
eicosapentaenoic acid (C:), docosapentaenoic acid
(C:), or docosahexaenoic acid (C:) are also
detected (Morgan et al. ; Ruchonnet et al. ).
If triglycerides are relatively stable (Evershed et al.
), alteration of these molecules can be initiated
by environmental factors (bacteria activity, high temp-
erature, light, oxygen, or fluctuation of relative humid-
ity), resulting in the degradation of grease (Institut de
Conservation Canadien ; Mills and White ;
Williams ). Triglycerides are degraded in FFAs,
as well as in aldehydes which are both very reactive
towards oxygen (Horie ; Reiche et al. ).
This reaction increases in direct proportion to the
degree of unsaturated fats, but remains slow at room
Journal of the American Institute for Conservation , Vol.  No. , –

temperature. Production of free radicals, aldehydes,
and FFAs results in a modification of the properties of
the grease, which becomes more mobile and migrates
through bones, leading to a brown coloration and
rancid odor. Finally, due to their acid character, FFAs
can partially dissolve the mineral (hydroxyapatite)
and organic (collagen) components of bone (Reiche
et al. ; Turner-Walker ; Turner-Walker and
Gau ).
For the fin whale skeleton exhibited at the Natural
History Museum of Nantes, a first series of tests were
carried out to identify the fats found on or within the
bones. Thin-layer chromatography (TLC) was per-
formed on surface and bulk samples taken from an
unknown cetacean vertebra and from the specimen.
The TLC profile of these samples did not substantially
differ (Guilminot et al. ), and no phospholipid
was found to be present nor other polar lipid. While tri-
glycerides were absent, large spots of FFAs could be
observed. Finally, undefined components were also
detected mainly on the surface sample of the fin
whale, likely associated with a non-lipidic component
or a product resulting from lipid degradation. GC ana-
lyses of the samples led to similar FFA profiles, with
only variation in their relative proportion: stearic acid
(C:) was evaluated at  wt% in a fin whale verte-
bra, contrary to other bones ( wt%). Among the
major FFAs present, saturated FFAs were detected,
such as myristic acid (C:), palmitic acid (C:),
stearic acid (C:), in addition to unsaturated FFAs,
such as palmitoleic acid (C:), oleic acid (C:),
gadoleic acid (C:), and erucic acid (C:) (Guilmi-
not et al. ). Within the French Program for the
Knowledge and Conservation of Cultural Heritage
Materials (PNRCC), alternative and effective degreas-
ing of the Nantes fin whale has been investigated by
Arc’Antique laboratory and its partners. The process
was to be applied on greasy areas of bones, while pre-
serving bone integrity and being environmentally
acceptable. Preliminary tests on expendable whale
bones were carried out using environmentally friendly
solvents (limonene), enzymatic degreasing, and
surface treatment of bones with ammonia. However,
none was more efficient than hexane, heptane, and
the azeotropic mixture of chloroform and methanol
that were thus selected to optimize the degreasing treat-
ment (Guilminot et al. ).
The current paper aims to present an analytical
approach to evaluate degreasing treatments on bone
using techniques based on GC, atomic absorption spec-
troscopy (AAS), and high-resolution nuclear magnetic
resonance (NMR) spectroscopy. These techniques can
serve to evaluate the influence of the different par-
ameters and to determine monitoring criteria during
the degreasing stages. To assess treatment efficacy, the
monitoring of FFAs extraction and their quantification
 CHARLÈNE PELÉ ET AL.
FIG. . Samples used for degreasing treatment tests, from right to left: (a) vertebra samples of CRMM; (b) cut samples of
vertebra into a block shape; (c) metacarpus given by Pierre-Henry Fontaine (Québec).
were performed by GC. The impact of the treatment on
the mineral and organic components of bones was
investigated by quantification of the calcium content
in fat extracts by AAS, and by probing the possible
presence of peptides in extracts with NMR
spectroscopy.
. MATERIALS AND METHODS
. SAMPLE COLLECTION
Treatment tests were conducted using spongy blocks
of bones cut from a whale vertebra (fig. a). These
samples were representative of the fin whale skeleton
at the Natural History Museum of Nantes, because
they presented the same problems of bone conservation
and aesthetic appearance due to the presence of grease
on their surface. They were porous and mainly com-
posed of spongiosa, had an approximate weight of
 g, and measured . cm × . cm × . cm (fig. b).
Whole bones (i.e. metacarpus) showing similar degra-
dation were also treated (fig. c). Unlike the other
samples, whole bones were characterized by the pres-
ence of periosteum on the surface.
. EXPERIMENTAL TREATMENTS
Each piece of bone was immersed in successive baths.
Three solvents were chosen according to their efficacy
determined in a previous study (Guilminot et al.
). Hexane and heptane are two hydrocarbons
that limit dehydration which might lead to bone crack-
ing, as observed in the case of acetone or other alcohols.
The azeotropic mixture of chloroform/methanol
(CHCl/MeOH) (/ v/v) gave the best extraction
rate. Nevertheless, it was found that this solution can
degrade the bones because a large amount of saturated
fatty acids necessary for mechanical support of the bone
structure is also extracted. For this reason, this mixture
was only used as a final step in these experiments, to
measure the total amount of fats that can be extracted.
As summarized in table , the different parameters
studied related therefore to the type of solvent used
for extraction (heptane, hexane, and finishing step
Journal of the American Institute for Conservation , Vol.  No. , –
with a mixture of CHCl/MeOH (/ v/v)), tempera-
ture, number, and duration of baths. For example,
sample S was a spongy block immersed in hexane at
room temperature in three successive baths of  days
each. Sample F was also a spongy block immersed in
hexane at room temperature in three successive baths,
each lasting  day. An additional immersion in a sol-
ution of CHCl/MeOH (/ v/v) heated to –°C
for . day (. hours) finished off this treatment.
. ANALYTICAL METHODS
After each treatment, analyses of FFAs were carried
out on the grease extracts removed from the degreasing
baths by evaporating the solution using a rotary evap-
orator. The extracted fats were weighed and analyzed
when sufficient quantities had been collected.
.. GAS CHROMATOGRAPHY
To make FFAs compatible with GC analysis (i.e.
vaporization in a gas mobile phase), their boiling
point has to be reduced. For that purpose, the car-
boxylic acid function of each fatty acid is transformed
in the corresponding methyl ester. This pre-treatment
consists of a saponification step followed by a methyl-
ation of the FFAs, leading to fatty acid methyl esters
(FAMEs) that were subsequently analyzed by GC
(Slover and Lanza ). Saponification was thus
carried out to convert triglycerides and other fatty
acid esters in their methyl ester form. Extracts were
treated with  mL of methanolic sodium hydroxide
(. M). Saponification of fats was conducted at °C
for  minutes. The samples were then allowed to
cool at room temperature and  mL of a  wt%
boron trifluoride–methanol solution was added. FFAs
were methylated at °C for  minutes. The samples
were again allowed to cool at room temperature and
FAMEs were then extracted twice with  mL of isooc-
tane. The upper isooctane layer containing FAMEs
was recovered and rinsed with water until pH . The
lipid mixture was then dried on sodium sulfate before
GC analysis.
 ANALYSIS OF FATTY ACIDS EXTRACTED FROM A WHALE SKELETON 

TABLE  CONDITIONS OF TREATMENT FOR EACH SAMPLE

Type of samples Sample Solvent used Temperature Length/ Bath Extract recovered after
bath number each bath (if the quantity
is sufficient)
Spongy block T Hexane –°C . days  Extract : brown and
semi-solid
. days  Extract : white and solid
T Room . days  Extract : brown oil
temperature  days  Extract : white and solid
S Hexane Room  days  Extract : brown oil
temperature  days  Extract : yellow and
semi-solid
 days  Extract : none
S Heptane  days  Extract : brown oil
 days  Extract : yellow and
semi-solid
 days  Extract : none
F Hexane (baths , ,  days  Extract : brown oil
)  days  Extract : brown and
semi-solid
Chloroform/  days  Extract : yellow and
methanol (/ v/v) semi-solid
(bath ) . days  Extract : white and solid
D Heptane Room  days  Extract : brown oil
temperature  days  Extract : yellow and
semi-solid
D  days  Extract : brown oil
 days  Extract : yellow and
semi-solid
D  days  Extract : brown oil
 days  Extract : yellow and
semi-solid
 days  Extract : yellow and solid
 days  Extract : white and solid
D  days  Extract : brown oil
 days  Extract : yellow and
semi-solid
 days  Extract : yellow and solid
 days  Extract : none
D  days  Extract : brown oil
 days  Extract : yellow and
semi-solid
 days  Extract : yellow and solid
D  days  Extract : brown oil
 days  Extract : yellow and
semi-solid
 days  Extract : none
Metacarpus M Room  days  Extract : brown oil
(whole bone) temperature  days  Extract : yellow and
semi-solid
M  days  Extract : brown oil
 days  Extract : yellow and
semi-solid

Journal of the American Institute for Conservation , Vol.  No. , –
 CHARLÈNE PELÉ ET AL.
GC analysis of FAMEs was carried out using an
Agilent HP GC equipped with a split/splitless
injector (°C) and a flame ionization detector
(°C). Samples were separated on a BPX capillary
column (SGE,  m × . mm, . µm film thick-
ness). Nitrogen was used as the carrier gas (. mL
min−), and the gradient of the oven temperature was
set at °C for  minutes, then increased to °C
at a rate of °C min−. Identification of each FAME
was made by comparison of its retention time with
that of the corresponding authentic standards
(Sigma-Aldrich, France). Decanoic methyl ester was
chosen as internal standard to quantify each FAME
because it was never present in the bone samples. In
order to avoid errors due to the injection, the internal
standard (decanoic methyl ester) was added at the last
step of the sample methylation. The amount of each
FAME was calculated on the basis of its relative pro-
portion in each chromatogram by comparison with
the internal standard added to each sample.
In order to monitor the degreasing of whale bones by
GC analysis, it was necessary to validate the protocol.
After checking the retention times and the correct separ-
ation of the peaks of interest (resolution > .), appro-
priate parameters of the method were validated. By
the implementation of four weighted samples between
. and . mg from three different fat samples, the
minimum weight to use for the analysis was deter-
mined. The smaller test sample (. mg) led to an
error of %, while the larger (. mg) led to a relative
error of %. Therefore, a minimum weight of . mg ±
% to be used for the GC analyses was selected.
In order to follow the degreasing treatments, six
samples of similar weight (. mg ± %) were collected
FIG. . Chromatogram of the FAMEs selected to monitor the degreasing treatment: palmitic acid (C:), stearic acid (C:),
oleic acid (C:), erucic acid (C:), and decanoic acid (C:) as the internal standard.
Journal of the American Institute for Conservation , Vol.  No. , –
and analyzed for each fat extract. The reproducibility of
the analysis was found to be satisfactory with a
maximum error of % on each peak of interest. Four
FAMEs (two saturated (palmitic acid (C:) and
stearic acid (C:)) and two unsaturated (oleic acid
(C:) and erucic acid (C:)) (fig. )) were particu-
larly quantified to estimate the efficacy of the degreasing
treatment because of their presence in all the extracts.
.. ATOMIC ABSORPTION SPECTROSCOPY
AAS was specifically used to quantify the possible
presence of calcium in the different extracts after the
degreasing treatments. Quantification of calcium
enabled the impact of the degreasing treatment to be
assessed on the mineral part of bone, mainly composed
of hydroxyapatite (Ca(PO)(OH)).
An atomic absorption spectrometer (Perkin Elmer,
model ) was used with a conventional slit burner
head for an air–acetylene flame (flow:  L min− for
air and  L min− for acetylene) and a hollow cathode
lamp for calcium using  mA of intensity. A cali-
bration curve was plotted, using a commercial standard
solution containing  ppm of calcium (Merck
Chemicals, Germany) that was diluted in the –
 mg L− range, resulting in a linear function. The
relative error for this analysis was estimated at %.
Extract analyses by AAS were carried out after dissol-
ution of samples in acid solution (nitric acid %).
When too concentrated, samples were diluted to
match the calibration curve.
.. NMR SPECTROSCOPY
NMR spectroscopy was used for the structural analy-
sis of organic molecules present in extracts after the
 ANALYSIS OF FATTY ACIDS EXTRACTED FROM A WHALE SKELETON 

degreasing treatment. The objective was to determine .. GENERAL OBSERVATIONS

whether the treatment affects the organic components
of bone (essentially collagen).
Samples were dissolved in dimethyl sulfoxide
(DMSO) or CDCl (deuterated chloroform) according
to their solubility.
NMR experiments were performed on a Bruker
Avance  spectrometer (Bruker Instruments Inc.,
Karlsruhe, Germany) operating at a frequency of 
MHz. Assignment of the signals corresponding to the
different kinds of protons of fatty acids was made
using linoleic acid chosen as a reference for fatty acid,
and on the basis of previously published chemical
shift data (Miyake et al. ).
. RESULTS AND DISCUSSION
. ANALYSIS OF THE EXTRACTION TREATMENTS
The analytical methods presented above aimed to
investigate the effects of the different treatment par-
ameters on FFA extraction. First the extracts were
weighed and compared with bone samples’ weight
(fig. ), to assess which parameters enabled to extract
the maximum quantity of FFAs. The color and
texture of these extracts were also observed after each
degreasing bath. Finally, all extracted FFAs were ana-
lyzed by GC to determine their composition.
The experiments showed that, irrespective of whether
the degreasing treatment was in heptane or hexane, the
first bath successfully extracted the majority of FFAs
(i.e. ca.  wt%), corresponding to around % of
the bone sample weight (fig. ). Indeed, the amount of
extracted FFA sharply decreased with successive ones.
On average, the first bath extracted  times more
FFAs than the second bath. For example, the first
extract of sample S represented  wt% of the bone
sample weight compared to  wt% for the second
extract. Adding a finishing step using a mixture of
CHCl/MeOH resulted in an increase in the amount
of extracted fat (sample F), reaching  wt% of the
bone sample (i.e.  wt% of fat extracted in the first
bath and  wt% in the final bath).
In all degreasing treatments the appearance of the
extracts changed when baths were renewed. Figure 
shows the degreasing treatment of sample T. The
first two extracts (extracted using hexane at room
temperature) presented a liquid texture and a brown
coloration. The last two extracts (obtained after immer-
sion in chloroform and then in a mixture of CHCl/
MeOH) were completely solid and white. Generally,
in heptane or hexane, the first and second extracts
have mainly an oily texture and a brown color
whereas the next extracts are increasingly viscous and

FIG. . Weight ratio (total extract weight/bone sample weight) determined for each degreasing treatment according to the par-
ameters (solvents, temperature, addition of a finishing step, length and number of baths, and type of samples).

Journal of the American Institute for Conservation , Vol.  No. , –
 CHARLÈNE PELÉ ET AL.
FIG. . Successive extracts of the spongy block T (from left
to right: first, second, third, and fourth samples), two first
extractions in hexane at room temperature, followed by two
finishing steps in chloroform and in chloroform/methanol
(/ v/v) at –°C.
white colored. Only the finishing step (CHCl/MeOH)
yielded a white, solid extract (as for sample F).
To rationalize these observations, all extracts were
analyzed by GC. Figure  shows the proportion of satu-
rated (C:, C:) and unsaturated FFAs (C:,
C:) in the extracts. The unsaturated FFAs were
mainly extracted in the first baths and became minor
compared to the saturated FFAs in the last extracts.
The degreasing treatments of sample D can be given
as an example: the first extract was composed of %
of unsaturated FFAs, while the second, third, and
fourth extracts contained , , and  wt% of unsa-
turated FFAs, respectively. Therefore, the last extract
was mainly composed of saturated FFAs. Adding a
finishing step (CHCl/MeOH) confirmed this evol-
ution. The last extract of sample F was composed of
 wt% saturated FFAs.
The visual appearance of extracts was then rational-
ized using GC analyses since their appearance and
texture depend on their composition. The extracts
mainly composed of unsaturated FFAs were brown
and liquid. By contrast, when the proportion of unsatu-
rated vs. saturated FFAs decreased, the appearance of
the extracts became increasingly white and solid.
Given that unsaturated FFAs are chemically unstable,
cause the bone surface to turn brown, and give off a
rancid odor, it was important that the degreasing treat-
ment focused on the extraction of unsaturated FFAs. As
a consequence, removing unsaturated FFAs was con-
sidered a priority. Saturated FFAs, on the other hand,
contribute to the mechanical properties of the bone
structure by pore-filling, and it was therefore detrimen-
tal to extract them. However, no degreasing treatment
can extract unsaturated FFAs selectively. For this
reason, a compromise had to be found between the pro-
portion of extracted unsaturated and saturated FFAs,
Journal of the American Institute for Conservation , Vol.  No. , –
bearing in mind that most of them are extracted
during the first bath.
.. PARAMETER INFLUENCE
The temperature of the degreasing baths influenced
the amount and nature of the extracted FFAs: fats cor-
responding to ca.  wt% of the sample weight were
extracted in the first bath at –°C (fig. , sample
T) vs.  wt% at room temperature (fig. , sample
T). The two first extracts of sample T (–°C,
fig. ) were only composed of  and  wt% of unsatu-
rated FFAs, respectively, vs.  and  wt%, respect-
ively, for sample T (°C, fig. ). The visual
appearance of the extracts confirmed this result. The
first two extracts of sample T were indeed still liquid
and brown, while those of sample T were solid and
white. Although heating the baths led to an increase
of the amount of extracted FFAs, a degreasing treat-
ment at room temperature remains preferable, since
previous studies (Guilminot et al. ) have shown a
higher degradation of bones when they were treated
in a heated extraction solvent.
At room temperature, the best performances were
obtained for the samples treated in heptane: fats corre-
sponding to ca.  wt% of the sample weight were
extracted from samples S, D, and D vs.  wt% in
hexane from samples S, T, and F, without taking
into account the finishing step. However, the compo-
sition of the extracts did not differ between these two
treatments: the first extract was composed of around
 wt% of unsaturated FFAs compared to  wt%
for the second. For both solvents, the appearance of
the first extract was liquid and brown. Then, the
texture became more solid and the color lighter
brown. The amount of fats collected in the third
extract was negligible.
The number of degreasing baths was not found to
influence the extraction in any of the treatments.
Approximately  wt% of the total extraction took
place during the first bath. This rate was observed irre-
spective of the duration of the baths. For example, the
extraction of samples D–D yielded the same results
although the length of the first bath varied between 
and  days. The same observations were made for
samples S, T, and F. Moreover, the composition
of the extracts did not differ. The average composition
of the first and second extracts was, respectively,
around – and  wt% unsaturated FFAs. The
first extract was liquid and brown but its texture
became semi-solid and slightly whitened after the
second bath. Although the first bath presented
the best extraction rate, it seemed necessary to renew
the baths to ensure that the extract became solid and
white, so that the extraction of unsaturated FFAs was
maximized while co-extraction of saturated FFAs
remained low. Simple immersion for short periods
 ANALYSIS OF FATTY ACIDS EXTRACTED FROM A WHALE SKELETON 

FIG. . Percentage of unsaturated and saturated fatty acids extracted from each bath as a function of treatment tests.

seemed sufficient, and for the spongy block samples,
three baths were enough to obtain this result.
However, the degreasing treatment in heptane on the
whole bones (M and M) seemed slower than on the
spongy bone samples. The treatments led to an inter-
mediate amount of the extracted fats, ranging
between  and  wt% vs.  wt% for the spongy
blocks, and the proportion of unsaturated FFAs in the
first and second extracts was, respectively,  and
 wt%, slightly lower than those of spongy samples.
The presence of periosteum probably slowed the
exchange between the solvent and the fats and seemed
to limit the extent of the extraction: the amount col-
lected during the second extraction was too small to

Journal of the American Institute for Conservation , Vol.  No. , –
 CHARLÈNE PELÉ ET AL.

FIG. . Percentage of calcium extracted during treatment with respect to the sample weight.

be of any further use. However, for all the samples mainly composed of hydroxyapatite of approximate
tested in this study, the successive immersions in composition Ca(PO)(OH) (Turner-Walker ).
heptane were found to restore a clean appearance of In this compound, the calcium content represents
the bones (white color and non-sticky surface) and around  wt%. In bone, calcium may represent
remove the unpleasant odor. Since the majority of unsa- between  and  wt% of the total weight. The
turated FFAs have been extracted using this protocol, amount of calcium found in the extracts can thus be
we believe that this recommended treatment should considered as low, and can be assumed to result from
confer long stability of the treated bones. a slow degradation of the bone by the fatty acids
released over time (i.e. dissolution of apatite). This is
. COMPLEMENTARY ANALYSIS: IMPACT OF DEGREASING supported by the absence of calcium in the second
TREATMENTS extraction bath that confirmed that no damage to the
bone microstructure arose from the degreasing
AAS analyses were performed to quantify the process itself. In addition, the amount of calcium
amount of calcium extracted during the degreasing released was higher for spongy block samples cut
process of either partial or whole bones, in order to
provide information on the degradation of the
mineral component of the bone structure. Analyses 
were carried out on the first extracts of samples D TABLE  PEAK ASSIGNMENT OF THE H NMR SPECTRUM
OF THE FIRST EXTRACTION BATH OF A WHOLE BONE (SAMPLE
and F and the two first extracts of the whole bone
M) USING HEPTANE AT ROOM TEMPERATURE, RECORDED IN
M (metacarpus). The results obtained are shown in
figure . Percentages refer to the mass of calcium CDCl. THE RESONANCE AT . ppm CORRESPONDS TO
present in the corresponding extracts with respect to CDCl IN THE DEUTERATED SOLVENT.
the mass of bone samples. The amount of calcium in Chemical shift (δ in ppm) Peak assignment
the first extracts of the spongy blocks (D and F)
is higher than in the case of the whole bone (M): . –HC=CH–
. wt% for D and . wt% for F vs. . wt% . –CH–COO–
for M. For the second extract, the amount of . –CH–HC=CH–CH–
calcium was under the detection limit of the technique . –CH–CH–COO–
( µg L−). . –(CH)n–
The mineral part of a whale bone represents %– . CH–(CH)n–
% of its total weight (Williams ). This part is

Journal of the American Institute for Conservation , Vol.  No. , –
 ANALYSIS OF FATTY ACIDS EXTRACTED FROM A WHALE SKELETON
FIG. . H NMR spectrum of the first extraction bath of a whole bone (sample M) using heptane at room temperature,
recorded in CDCl.
from a vertebra with a saw. The sample preparation
and their manipulation could also be responsible for a
slight degradation of the mineral components.
Complementary analyses by NMR spectroscopy
were carried out to identify a potential alteration of
the organic part (collagen) of the bone. It is important
to note that attempts to solubilize different commercial
samples of collagen in organic solvents (CDCl, MeOD,
or even deuterated DMSO) were unsuccessful (no H
NMR signal detected), suggesting that the dissolution
of collagen as a result of degreasing treatment is unli-
kely. Moreover, if very small collagen fragments were
however found, H resonances corresponding to
glycine and proline should be present (i.e. at ca.
 ppm for the NH amide proton (–NH–CH(R)–CO–)
and ca. –. ppm for the methylene proton α to the
carbonyl and nitrogen groups (–NH–CH(R)–CO–)),
since collagen is rich in these two amino acids. The
absence of these signals in all spectra clearly demon-
strates that degreasing treatments do not affect the
organic component of bones. Indeed, whatever the
nature of the extracts (first extraction bath of a
spongy block (sample S) or a metacarpus bone
(sample M) using heptane, or the finishing bath of a
spongy block (sample F) using a CHCl/MeOH
mixture), the H NMR were similar, showing the
characteristic resonances of FFAs (table ) by compari-
son with published data (Miyake et al. ). One
example is given in figure , corresponding to the first
extraction bath of a metacarpus bone, using heptane
at room temperature (sample M). As expected, a
Journal of the American Institute for Conservation , Vol.  No. , –

lower intensity of the resonances at . and . ppm
was observed in sample F compared to samples S
and M, due to a lower relative amount of unsaturated
FFAs in the extract.
. CONCLUSIONS
On the basis of GC, AAS, and NMR analyses per-
formed on extracts obtained from degreasing bones,
the best conditions for the degreasing treatment were
selected with respect to whale bone integrity. The
degreasing treatment recommended consists of succes-
sive and brief immersions in heptane (heavier and less
volatile than hexane) at room temperature, which was
found to be the solvent of choice, since its degreasing
efficacy is higher than the one observed for hexane.
Nevertheless, adequate ventilation and appropriate dis-
posal of the solvent after use are recommended, since
heptane is highly flammable and toxic for aquatic life.
The number and length of each bath then depend on
the nature and condition of the specimen. However, the
experiments showed that a long immersion did not lead
to an improvement in degreasing. To monitor the
degreasing treatments, conservators can simply
observe the extracts. Indeed, the GC analyses enabled
the correlation of the texture and coloration of the
extracts resulting from each degreasing bath with
their composition. When extracts were liquid and
brown, they were mainly composed of unsaturated
FFAs and baths needed to be renewed until the extracts
become solid and light brown. This protocol enabled
 CHARLÈNE PELÉ ET AL.
the specific extraction of the majority of unsaturated
FFAs (responsible for the brown color, rancid odor,
and chemical instability of bones), with limited
co-extraction of saturated FFAs which are considered
to contribute positively to the mechanical properties
of the bone structure by pore-filling. Although no
fixed amount of unsaturated FFAs to be extracted or
saturated FFAs to be retained was established, a
degreasing treatment in heptane until extracts were
composed of  wt% saturated FFAs (i.e. typically
the case after the second extraction) seemed to be satis-
factory. Indeed, the mass of extracted fats after the
second extraction only represented . wt% of the
total weight of samples. Only a finishing step with a
mixture of chloroform/methanol enabled the complete
extraction of fats, but for the reasons mentioned
above it is not recommended: the use of this mixture
at –°C is toxic, degrades the bone (Guilminot
et al. ), and requires specific safety equipment. In
the next future, the fin whale skeleton exhibited at the
Natural History Museum of Nantes will be disas-
sembled to treat all bone pieces individually, following
the recommendations resulting from the present study.
ACKNOWLEDGMENTS
We would like to thank the Natural History Museum of
Nantes, where this project initiated, together with the
French Ministry of Culture (through the French PNRCC
research program “Programme national de recherche sur la
connaissance et la conservation des matériaux du patrimoine
culturel”) for their financial support.
We are grateful for the contribution of many partners:
Michel Surbled, Olivier Crosnier (Polytech’Nantes) and
Willy Dabin (Research Center on Marine Mammalians, La
Rochelle) and Pierre-Henry Fontaine (Museum of skeletons
at Ile-Verte, Canada) for providing fin whale bone samples.
We would also like to thank Delphine Morvan, Laurence
Dubreuil, Thibaut Foucher, Sabine Le Blond, and Claire
Musso, students who participated in this project.
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AUTHOR BIOGRAPHIES
CHARLÈNE PELÉ has worked at Arc’Antique: a conservation laboratory for archaeological materials since . An engineer since
, her study interests include the optimization of conservation treatments for different materials (wood, bone, metal, and
ceramic). Address: EPCC Arc’Antique, laboratoire de conservation, de restauration et de recherche, , rue de la Haute
Forêt,  Nantes, France. E-mail: arcantique.recherche@wanadoo.fr.

BRUNO BUJOLI obtained his chemistry engineer degree in  from the National Institute of Industrial Chemistry of Rouen
(France). He received his PhD in  from the University of Nantes. He then joined Laboratoire de Synthèse Organique (Uni-
versity of Nantes) as CNRS Charge de Recherche in . He was awarded the Brass Medal of CNRS in , and in , he
was promoted to CNRS Director of Research. He is known for pioneering work on metal phosphonates at the early stage of this
emerging field in the early s, and most of his activity is focused on the use of phosphonic acid for the surface modification of
inorganic surfaces, as a route for the preparation of functional materials. In particular, he has an international expertise in the
field of organic–inorganic materials applied to biotechnologies (DNA and protein microarrays) and biomaterials, and has
designed a series of original concepts related to drug–device combinations, which have been patented in the area of “biomaterials
for orthopedics.” He is also interested in the chemistry of lipids, in particular for the conversion of biomass residues into biobitu-
men for road paving. Address: Laboratoire CEISAM, UMR , laboratoire de Chimie Et Interdisciplinarité : Synthèse,
Analyse, Modélisation, UFR Sciences et Techniques, départements de Nantes, université de Nantes,  rue de la Houssinière,
BP ,  Nantes, France. E-mail: Bruno.Bujoli@univ-nantes.fr.

ÉLODIE GUILMINOT graduated as an engineer from the school Polytech’Nantes (France) in , and earned a PhD in electrochem-
istry from the Institut National Polytechnique de Grenoble (France) in . Her doctoral research focused on the conservation
of waterlogged wood/metal composites in collaborative French conservation–restoration laboratories (Arc’Antique and ARC
Nucleart). She worked at different research institutes (French Institute of the sea (IFREMER) and the University of Grenoble)

Journal of the American Institute for Conservation , Vol.  No. , –
  CHARLÈNE PELÉ ET AL.

on the corrosion and the characterization of materials. She joined the Arc’Antique laboratory (Nantes, France) as a research
engineer in . Her research interests include the corrosion of metals and the development of restoration treatments.
Address: As for Pelé. E-mail: elodie.guilminot@arcantique.org.

GWENAËL LEMOINE has worked as a conservator at Arc’Antique since . She specializes in the treatment of organic archaeo-
logical materials. Address: As for Pelé. E-mail: arcantique.organique@orange.fr.

ISABELLE LOUVET has been Assistant Engineer at CNRS for  years and she runs the chromatography service at the laboratory
CEISAM, UMR . As an expert in separation by GC, HPLC, and coupled methods, she has carried out molecule character-
ization from organic synthesis and develops enantioselective separation by liquid chromatography. She also develops quantitat-
ive methods using GC, GC–MS, especially of lipid compounds or polycyclic aromatic hydrocarbons, and assay methods for
molecules at therapeutic use (bisphosphonates, etc.). Address: As for Bujoli. E-mail: isabelle.louvet@univ-nantes.fr.

LAURENT POISSON is teacher–researcher at the Université du Maine. He splits his time between teaching biochemistry at the tech-
nical institute and researching in the multi-site Laboratory Mer, Molécules, Santé. His research focuses on the valorization of
marine lipids using enzymes. Laurent also spent  months in Quebec City at the Université Laval to take part in a research
program on lipase-catalyzed production of waxes from anhydrous milk fat. Address: Laboratoire Mer, Molécules, Santé (EA
), Université du Maine, IUT de Laval,  rue des Docteurs Calmette et Guérin, BP ,  Laval Cedex , France.
Email: laurent.poisson@univ-lemans.fr.

Journal of the American Institute for Conservation , Vol.  No. , –

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