Journal of Cultural Heritage 15 (2014) 128135

Available online at
www.sciencedirect.com

Original article

Re-treatment of whale bones How to extract degraded fats from weakened

bones?
Elodie Guilminot a, , Gwenal Lemoine a , Charlne Pel a , Laurent Poisson b , Michel Surbled c ,
Isabelle Louvet d , Jean-Yves Mevellec e , Luc Rmy f
a

ArcAntique, 26, rue de la Haute-Fort, 44300 Nantes, France

MMS EA 2160, Universit du Maine, IUT de Laval, 52, rue des Docteurs-Calmette et Gurin, BP 2045, 53020 Laval cedex 09, France
c
Archimex, Parc dinnovation de Bretagne Sud, C.P. no 31, 56038 Vannes cedex, France
d
Laboratoire CEISAM, Universit de Nantes, UMR CNRS 6230, UFR des Sciences et des Techniques, 2, rue de la Houssiniere, BP 92208, 44322 Nantes cedex 3, France
e
Institut des Matriaux Jean-Rouxel (IMN), UMR 6502 CNRS, 2, rue de la Houssinire, BP 32229, 44322 Nantes cedex 3, France
f
Musum dHistoire Naturelle de Nantes (MHNN), 12, rue Voltaire, 44000 Nantes, France
b

a r t i c l e

i n f o

Article history:
Received 21 December 2012
Accepted 19 March 2013
Available online 24 April 2013
Keywords:
Fatty bones
Whale
Skeleton
Degreasing treatments
Conservation

a b s t r a c t
Many whale (baleen whale or toothed whale) skeletons still contain residual lipids even after an initial
osteological preparation. This paper examines the different possibilities of re-treatment. Before a conservation intervention, it was necessary to determine the materials of which bones are made up. The
samples were analyzed by Raman spectroscopy. Different compounds were identied: a mineral part
(apatite), an organic part (collagen) and lipids. Chromatography analysis yielded a detailed composition
of the lipids. It was in fact degraded fat with saturated and unsaturated fatty acids. To remove these
lipids, several techniques were identied and tested: enzymatic treatments, supercritical CO2 , and green
or organic solvents. Esterication catalyzed by lipases could be suitable for a degreasing treatment since
the solubility of esters is higher than that of the corresponding fatty acids. The enzymatic treatment acted
only on the surface and did not appear to be very efcient. The use of supercritical CO2 was even less
effective. Some green solvents can partially extract lipids but prove difcult to eliminate after treatment.
The best results for degreasing were achieved using organic solvents. Different solutions were evaluated
at hot or ambient temperature and in simple immersion or with agitation (Soxhlet or pulsed pressure):
hexane, heptane, a mixture of hexane/isopropanol, or an azeotropic mixture of methanol/chloroform.
Only the mixture of methanol/chloroform succeeded in extracting the overall fat content, but this treatment degraded the organic part of the bones. The other organic solvents extracted mainly colored fat,
which generally corresponded to a weight loss of 20 to 50%. The majority of fat was extracted during
the rst bath. Thus the treatment selected is that of immersion in heptane at ambient temperature. The
degreasing of whole bones is less effective because of the lm of sticky degraded fat on the bones surface.
A pre-cleaning is necessary to eliminate this lm.
2013 Elsevier Masson SAS. All rights reserved.

1. Research aims
The conservation of whale skeletons presents a great challenge due to their uncommon size and their high lipid content.
Fin whale skeletons in particular are very large and fatty. Standard
osteological preparation includes a variety of techniques to clean
and degrease whale bones: dermestid cleaning, burial and maceration. However, all these techniques are empirical. Treatment
often fails to remove all the lipids and residual fats ooze from
the bones during the display of whale skeletons, especially when

Corresponding author.
E-mail address: elodie.guilminot@arcantique.org (E. Guilminot).
1296-2074/$ see front matter 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.culher.2013.03.008

the temperature increases. The extraction of these residual lipids

is problematic. For this reason, a research program was initiated
involving different specialists: curators, conservators and scientists. Several treatments were considered: enzymatic treatment,
the use of supercritical CO2 , and green or organic solvents. Experimental tests were monitored by visual observations and analysis.
This research program evaluated the effectiveness of each of these
methods.
2. Introduction
Bone studies in conservation generally focus on ancient bones
or fossils [1]. The chemical and physical properties of archaeological bones depend on taphonomic processes and the burial

E. Guilminot et al. / Journal of Cultural Heritage 15 (2014) 128135

129

Fig. 1. The n whale skeleton exhibited at Natural History Museum in Nantes.

environment [2]. Bone is a complex material: a hierarchically structured composite with the brous protein collagen stiffened by an
extremely dense lling and surrounding of calcium apatite crystals. Collagen accounts for 90% of the organic phase. It is largely
responsible for the exibility of the bone, and its high resistance
to tension and torsion. The hardness of the bone and its resistance
to compression are due to the mineral component hydroxylapatite,
[Ca5 (PO4 CO3 )3 (OH)]. Archaeological bones are degraded: they generally lose part of their organic component, and show an increase
in the crystallinity of the mineral part. Fresh bones preserve all
their components: collagen, apatite and even lipids. Before being
able to exhibit a skeleton, natural history museums must prepare
modern bones. These preparation treatments remove the esh and
lipids. Current methods are burial, dermestids, enzymes, maceration in water or chemical maceration, and boiling [35]. In the case
of fatty skeletons (such as marine mammals), the cleaning of bones
may be followed by degreasing in organic solvents. The preparation
and curation procedures are empirical processes and no approach is
ideal. Some techniques have limited effectiveness and can be dangerous (toxic and often ammable vapours). Modications may also
occur during preparation, affecting the integrity of bones [3,6]. The
damage during composting or burial depends on the environment:
acidic conditions weaken the mineral part whereas an alkaline
environment causes loss of the organic part. The surface of the
bones is damaged and becomes grey. The use of dermestid beetles
leads to an even greater deterioration of the bones: their mandibles
produce grooves and chewing marks. The most frequently used
method is that of aqueous maceration, which results in a slight
degradation of the bones. A prolonged treatment can hydrolyze
proteins and dissolve inorganic components [6]. Aqueous maceration is effective in eliminating the esh but it can take a long time.
The use of additives (enzymes (protease/neutrase), detergent or
soda) may reduce the time but it can be disastrous for the bones.
The use of enzymes (protease/neutrase) leads to an action close to
digestion: the treatment causes the destruction of the bone surface
and is even more aggressive on the mineral part [3]. The detergents
cause decalcication and softening of the bones [4]. The effectiveness of aqueous maceration is limited to extracting the lipids from
the bone. The use of warm water is better but may affect the bones.
Boiling increases bone porosity and may cause irreversible damage
to the collagen [3,6]. When the cleaning is insufcient, the bones are

degreased with organic solvents (alcohol, acetone or chlorinated

solvents) [5,7]. Alcohol and acetone can produce dehydratation and
brittleness causing cracking of the organic component [3]. Chlorinated solvents are toxic and are problematic after treatment. They
also produce hydrochloric acid causing degradation of the mineral
part [6]. All the preparation treatments modify the bones, affecting
thus skeleton assembly and complicating future scientic studies
on the bones.
At the Natural History Museum in Nantes, a n whale skeleton
of more than 18 m long has been exhibited since 1995 (Fig. 1). It
was prepared by the anatomy unit of the Nantes veterinary school.
The water maceration lasted 18 months. The treatment was completed by cleaning with warm water, and by degreasing in a mixture
of organic solvents (chloroform/trichloroethane/two unidentied
solvents) for 6 months. Despite the treatment, some bones still
contain lipids. In hot summers, fats ooze onto the bone surfaces
(Fig. 2) and rancid smells emerge. The Museum would like to retreat the fatty bones. But how should these bones be degreased?
As previously discussed, the current preparation procedures are
imperfect and often incompatible with degreasing. Moreover, the
treated bones are already weakened. A research program was

Fig. 2. A fatty shoulder bone (left scapula) of the n whale skeleton at Natural
History Museum in Nantes.

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E. Guilminot et al. / Journal of Cultural Heritage 15 (2014) 128135

Fig. 3. Bone samples: cetacean vertebraes, provided by Research Center of Marine Mammals at La Rochelle (France).

conducted to resolve this problem and the results are presented in

this paper. At rst, the degradation of the bones was evaluated and
the lipids to be extracted from the bones were analyzed. Then new
treatments to extract lipids were selected. Attention was paid to
environmentally friendly treatments: enzymatic solutions, supercritical CO2 , green solvents or organic solvents (but ones that are
less toxic than trichloroethane).
3. Materials and methods

(Museum of Skeleton at le-Verte [Canada]) (Fig. 4A). Most experiments were carried out on cut bones from cetacean vertebrae
(blocks about 3 cm 4.5 cm 1 cm) (Fig. 5A). The nal tests were
made with whole bones (n whale metacarpals).
3.1.2. Fat samples
Lipid samples were collected by scratching greasy areas of the
bones with a scalpel. Fats extracted during treatments were also
analyzed. The fats were recovered by evaporating the solutions in
a rotary evaporator.

3.1. Materials
3.2. Analysis techniques
3.1.1. Bone samples
For analysis, bone samples were taken from the n whale skeleton at Nantes Museum. However, the experimental degreasing tests
were conducted on expendable bones. These bones were taken
from cetacean skeletons, prepared by maceration but still showing greasy areas. These were cetacean vertebrae, provided by the
Research Center of Marine Mammals at La Rochelle (France) (Fig. 3),
and n whale metacarpus, provided by Pierre-Henri Fontaine

3.2.1. Lipid analysis by thin layer chromatography (TLC)

Lipid analysis was carried out through TLC on 20 20 cm
silica plates. Fat samples were dissolved in chloroform (concentration approximately 10 g/L). Separation of components was
performed by a double development in one dimension [8]. The
rst development was performed in methyl acetate/propan2-ol/chloroform/methanol/0.25% aqueous KCl (25:25:25:10:9;

Fig. 4. Whale metacarpus. A. Before treatment. B. After treatment of an immersion in heptane at ambient temperature for 4 and 8 days.

E. Guilminot et al. / Journal of Cultural Heritage 15 (2014) 128135

131

Fig. 5. Bone blocks from cetacean vertebraes (3 cm 4.5 cm 1 cm). A. Before treatment. B. After treatment of an immersion in heptane at ambient temperature during 3
and 8 days (sample S07). C. After Soxhlet extraction in hexane with 200 cycles at 6065 C, then in hexane with 400 cycles at 6065 C, and in the azeotropic CHCl3 /MeOH
with 90 cycles at 4550 C (sample S01).

v/v/v/v/v) to half nal distance. The plate was dried at room temperature for about 10 min and the second development was performed
in hexane/diethyl ether/glacial acetic acid (80:20:2; v/v/v) on the
full plate length. After drying at room temperature, components
were visualized by spraying an -naphtol solution (0.25 g naphtol/50 mL ethanol/50 mL sulphuric acid 20%) and incubating
for 10 min at 100 C. Stains were identied by comparison of their
reference values with those of standards analyzed under the same
conditions. The standards were a fatty acid (C22:6), a phospholipid
(phosphatidylcholine), a triglyceride (triolein), a cholesterol ester,
and a wax (oleyl oleate).
3.2.2. Fatty acid analysis by gas chromatography (GC)
Fatty acid composition of lipids was analyzed through saponication followed by methylation of the fatty acids and gas
chromatography (GC) analysis of the esters [9]. The lipid extract was
rst dried under a stream of nitrogen and the residue was collected
in 1 mL of methanolic sodium hydroxide (0.5 M). Saponication of
fatty acids was conducted at 80 C for 15 min.
The sample was then cooled down to room temperature and
2 mL of 12% methanolic boron triuoride (BF3 ) was added. Fatty
acids were methylated at 80 C for 20 min. The sample was cooled
down again to room temperature, and fatty acid methyl esters

(FAME) were then extracted twice with 2 mL of isooctane. The

upper isooctane layer (containing FAME) was recovered and rinsed
with water to wash out the remaining sodium hydroxide and BF3 .
Rinsing was performed until the pH of the water reached 7. The lipid
mixture was then dried on sodium sulphate before GC analysis.
FAME analyses were carried out by GC using an Agilent HP6890
GC equipped with a split/splitless injector (250 C) and a ame ionization detector (280 C). Separation was performed with a BPX70
capillary column (SGE, 30 m 0.32 mm, 0.25 m lm thickness).
Nitrogen was used as a carrier gas (1.3 mL/min), and the gradient of the oven temperature was set rst at 120 C for 4 min, then
increased to 220 C at a rate of 6 C/min. Peaks were identied by
comparison of their retention times to those of standards analyzed
under the same conditions. These fatty acid standards were C16:1,
C18:0, C18:1, C18:2, C18:3, C20:0, C20:4, C20:5, C21:2.
3.2.3. Bone analysis by Raman spectroscopy
Raman spectra were obtained at room temperature under an
excitation wavelength of 1064 nm (Nd-YAG laser) to limit the
uorescence effect, using a FT-Raman Bruker RFS 100 spectrophotometer. To avoid any damage, the nominal power was modulated
between 100 and 340 mW. Spectra were recorded at 4 cm1 resolution over the wavenumber range 1003500 cm1 , with 100 scan

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E. Guilminot et al. / Journal of Cultural Heritage 15 (2014) 128135

Table 1
Description of treatments in organic solvants.
No of samples

Extracta

Treatment

S01

Soxhlet extraction in hexane 200 cycles at 6065 C

Soxhlet extraction in hexane 400 cycles at 6065 C
Soxhlet extraction in the azeotropic CHCl3 /MeOH, 90 cycles at 4550 C

36.0% of colored fat

0.4% of white fat
1.0% of white fat

S02

Immersion in an agitated hexane 20 C during 18 h

Immersion in an agitated hexane 20 C during 170 h
Soxhlet extraction in CHCl3 350 cycles at 5560 C
Soxhlet extraction in the azeotropic CHCl3 /MeOH, 40 cycles at 4550 C

21.8% of colored oil

2.3% of colored fat
4.5% of white fat
6.9% of white fat

S03

Immersion in a stirred mixture of heptane and isopropanol (80/20 v/v) 80 C during 6 h

Soxhlet extraction in the azeotropic CHCl3 /MeOH, 14 cycles at 4550 C

39.7% of colored fat

1.6% of white fat

S04

Immersion in a mixture of hexane and isopropanol (80/20 v/v) at 75 C under pulsed pressure
Soxhlet extraction in the azeotropic CHCl3 /MeOH, 25 cycles at 4550 C

36.2% of colored fat

0.3% of white fat

S05

Immersion in a mixture of hexane and isopropanol (80/20 v/v) at 75 C under pulsed pressure
Immersion in a mixture of hexane and isopropanol (80/20 v/v) at 75 C under pulsed pressure

50.3% of colored fat

10.6% of colored fat

S06

Immersion in hexane at ambient temperature during 24 h

Immersion in hexane at ambient temperature during 24 h
Immersion in hexane at ambient temperature during 24 h

31.0% of fat
2.2% of fat
0.4% of fat

S07

Immersion in heptane at ambient temperature during 3 + 8 days

30.5% of fat

S08

Immersion in heptane at ambient temperature during 2 + 3 + 2 + 2 days

28.7% of fat

S09

Immersion in heptane at ambient temperature during 7 + 7 + 7 days

32.1% of fat

Mass percentage versus the initial total mass of the sample.

accumulation. The laser spot size was 1 mm2 . Several measurements (at least four measurements for each analyzed zone) were
obtained to check reproducibility. Raman spectra were studied
with Opus software. They were background-corrected and decomposed. Spectral ratios were calculated with the intensities of raw
bands. Each component was characterized by one or two peaks:
960 cm1 for the mineral part; 1270 cm1 and 2940 cm1 for the
organic part; 2850 cm1 and 1296 cm1 for the lipids.
3.3. Degreasing methods
3.3.1. Enzymatic treatment
During the preparation, the usual enzymes are proteases or/and
neutrases. They mainly act on the esh, not on lipids. Lipases
are generally used to degrade triglycerides to fatty acids (by lipid
hydrolysis reaction). But these enzymes are ineffective on degraded
fat. So for the fatty bone re-treatments, we thought of using the
reversed reaction: the esterication of fatty acids. The resulting
esters are more soluble in alcohol than fatty acids. Thus it was
expected that it would be easier to extract the esters from bones
than fatty acids. Several lipases obtained from Novozymes (Denmark), Amano (Japan) or Sigma-Aldrich (USA) were tested on the
mixture of fatty acids [C14:0/C18:1 (1:5)] in ethanol. The solutions were mixed at ambient temperature for 15, 30, 45, 60 or
120 min. The resulting esters were quantied by GC. The most effective enzymes selected were: DF Amano 15 and lipozyme TL 100 L
(Novozyme). These two lipases were tested on fatty bones. The bone
block was immersed in 40 mL of ethanol without lipases, or with
40 mg of DF Amano 15, or 4 mL of lipozyme TL 100 L. A positive
blank was made in acetone. Each sample was incubated at 30 C
and shaken at 170 rpm for 72 or 100 h.
3.3.2. Supercritical CO2
Supercritical CO2 is a non-toxic, inert and odorless gas. It
is used in the degreasing of metal components in industry. At
250300 105 Pa and 3540 C it will dissolve approximately 1%
of triglycerides. It produces no contaminative waste solvents
and its only disadvantages are cost and access to the equipment. One bone block was processed in the following way: the
rst cycle at 300 105 Pa and 3739 C with 19.4 kg of CO2 (CO2

density: 0.910.94); then the second cycle at 300 105 Pa and

3638 C and 1.45 g of ethanol with 12 kg of CO2 (CO2 density:
0.920.94). Each cycle lasted 24 h (maceration was static for 16 h
and stirred for 7 h).
3.3.3. Green solvents
Green solvents are derived from plants. For degreasing, terpene
derivatives or vegetable esters are used. Green solvents can be toxic.
In this study, two green solvents were tested: limonene and methyl
oleate from colza. Bone blocks were immersed for several days at
ambient temperature.
3.3.4. Organic solvents
For degreasing, solvents should have a low dielectric
constant, e.g. hydrocarbons (heptane, hexane), chloroform,
dichloromethane, acetone or ethyl acetate. The azeotropic mixture
of chloroform and methanol (CHCl3 /MeOH) assumed the total
extraction of lipids from bones. Due to these harmful effects on
bones, this mixture was reserved for use at the end of the other
treatments to evaluate the efciency of extraction. This enabled us
to evaluate the mass of total lipids present in the sample and then
to dene the mass balance between the lipids extracted during the
treatment and the initial mass of lipids in samples. The extraction
of lipids can also be promoted by heating and/or stirring (by using
Soxhlet extraction or elevated pressure). Different parameters
were tested on the bone blocks and are described in Table 1. Three
samples (S06, S07 and S08) were treated under pulsed pressure of
N2 . The processing was: the increase of temperature (75 C), the
increase of pressure (5 105 Pa), the impregnation of the solvent
by increasing the pressure up to 10 105 Pa, application of pulses
(low value: 5 105 Pa/high value: 10 105 Pa) for a duration of
2 h, then the rapid depressurization.
4. Results and discussion
4.1. Analysis of bones
4.1.1. Bones
Despite the preparation and the degreasing of the n whale
skeleton at Nantes Museum, some bones remain fatty, whereas

E. Guilminot et al. / Journal of Cultural Heritage 15 (2014) 128135

133

Fig. 6. Raman spectra of whale bones: the black spectrum was made on a treated
weakened bone of the whale skeleton at the Nantes Museum; the grey spectrum was
made on a fatty cetacean vertebra, provided by Research Center of Marine Mammals
at La Rochelle (France).

other bones are weakened and white (no traces of lipids). This difference is due to a technical problem during the degreasing. Some
bones were not totally immersed in organic solvents. Thus, the rst
thoracic vertebrae, some lumbar vertebrae, the rst thoracic ribs
and the scapulae still contain lipids (Fig. 2). These fatty bones of
the n whale skeleton are like the expendable bones (cetacean
vertebrae and n whale metacarpus) (Fig. 3). The different components were detected by Raman analysis of fat bones (Fig. 6). The
main peak associated with the mineral part is the 1 PO4 3 vibration at 960 cm1 . As regards the organic component, the peak at
2940 cm1 ( CH2 ) is dependent on proteins present in the bone.
However, the CH3 vibration of the lipids also contributes to this
peak. The best peak associated with collagen (the organic part) is
the amide III vibration, displaying a doublet at 12451270 cm1 (
NH) [10]. On the fatty cetacean vertebra spectrum (Fig. 6), the peaks
related to collagen are masked by the lipid peaks. Lipids are associated with a peak at 1296 cm1 , close to the amide III peaks; but the
most intense peak associated with lipids is situated at 2850 cm1
[10]. On the treated bones of the Museum n whale (Fig. 6), the
proportions of different parts change. The peak related to lipids
(2850 cm1 ) has almost disappeared, we just see a shoulder on the
peak at 2880 cm1 . The proportion mineral/organic also varies: the
peak related to the apatite at 960 cm1 becomes the major peak. The
Raman results showed that the degreasing was effective but some
solvents (especially chloroform and trichloroethane) degraded the
organic part of bones. Any subsequent treatment should avoid these
solvents.

Fig. 7. Two-dimensional thin layer chromatography of lipid collected on

cetacean bones. A. Whole chomatographic plate. 1. Free fatty acid22:6. 2.
Phospholipidsphosphatidyl choline. 3. Triglyceridstriolein. 4. Cholesterol ester.
5. Waxoleyl oleate. 6. Surface lipids of cetacean vertebra. 7. Surface lipids collected
on a blade of the whale exhibited at the Nantes Museum. 8. Inner lipids of cetacean
vertebra. 9. Inner lipids collected on a spare bone of the whale exhibited at the
Nantes Museum. B. Detail of upper part of chromatograph.

4.1.2. Lipids
Lipids collected from the Museum skeleton and test bones were
analyzed by two-dimensional TLC (Fig. 7). The rst development
allowed the separation of polar lipids on the bottom of the plate
whereas the second development allowed the separation of nonpolar lipids on the top of the plate. All samples (run 6, 7, 8 and 9)
exhibited a similar prole. No spot seems to correspond to triglycerids (run 3 on Fig. 7) or phospholipids (run 2 on Fig. 7). The main
spot can be assigned to free fatty acids (run 1 on Fig. 7). Ambiguous spots present near the solvent front suggest that waxes are
probably present in all samples (detail shown on Fig. 7B). In the
lipids collected on/in the vertebra of the unknown cetacean, a component corresponding to none of the spotted standards can also
be observed, which may correspond to pigments by analogy with

4.2.1. Enzymatic treatment

After incubation, samples were observed and weighed. The
results of weight loss are shown in Table 3. The efciency of the
enzymatic degreasing is very poor, especially with lipozyme TL
100 L. The lipases act in the initial treatment. In fact, the enzymes
seem efcient only on the surface and are unable to reach the inner
core of the bone. As the results show, increasing the treatment
time is unnecessary. After 72 h of incubation, the treatment with
DF Amano 15 is close to the acetone solution with regard to weight
loss as well as visual aspect. The enzymatic treatment is slightly
more efcient than acetone on the surface but less efcient in the
interior. According to this study, the enzymes cannot penetrate the
bone. Therefore, the enzymatic treatment was not selected for the
degreasing treatment of the Nantes Museum n whale.

previous studies by Henderson and Tocher [8]. The TLC analysis shows that lipids from the bones are totally degraded; no
triglyceride is detected. The gas chromatography (GC) analysis may
indicate the fatty acid composition (Table 2). We cannot identify all
the compounds. The main fatty acids are C18:1, C16:0, C16:1, C14:0,
C20:1 and C18:0. This prole is fairly consistent with data published
in literature [11,12]. The saturated fatty acids (e.g. C16:0, C14:0 and
C18:0) are characteristic of degraded lipids. We nd the same fatty
acids in the different samples but the proportions change. For the
n whale skeleton exhibited at Nantes Museum, the major fatty
acid is C18:1 (about 25%). This major compound was also found
in the sample of the n whale metacarpus. But for the vertebra
sample, the major fatty acid is C16:0 (about 32%). These variations
can be due to the animal (species, age, sex) and the conditions of
bone conservation. Despite these differences, the test bones were
considered as representative of the n whale skeleton exhibited at
Nantes Museum.
4.2. Evaluation of treatments

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E. Guilminot et al. / Journal of Cultural Heritage 15 (2014) 128135

Table 2
GC analysis of fat: fatty acid composition (%, w/w) from cetacean skeletons (n = at least 3 replicates).
Cetacean

Whale

Whale exhibited at Nantes Museum

Fatty acid

Vertebra surface

Metacarpus surface

Blade surface

Vertebra surface

12:0

1.20 0.06

0.16 0.03

0.37 0.01

0.20 0.01

14:0

10.1 0.3

3.71 0.05

7.24 0.09

6.20 0.09

16:0

31.7 0.5

7.33 0.08

11.3 0.1

8.8 0.1
1.23 0.04

11.4 0.1

13.1 0.1

3.4 0.2

16:1

3.8 0.1

17:0

1.55 0.01

0.69 0.01

0.5 0.4

0.3 0.2
0.96 0.02
0.75 0.02

18:0

6.9 0.1

1.47 0.01

2.06 0.02

11.3 0.2

18:1

12.5 0.1

22.31 0.06

28.1 0.6

25.6 0.3

18:2

0.81 0.05

0.44 0.04

1.0 0.4

0.81 0.03

0.18 0.02

18:3
9.4 0.3

20.3 0.3

22:1

6.7 0.3

18.48 0.07

20:5

0.47 0.01

20:1

5.4 0.3
1.64 0.07
6.8 0.2

2.6 0.1

2.34 0.03

0.5 0
1.14 0.02

0.29 0.02

0.16 0.08

4.5 0.4

0.07 0.01
0.12 0.01

0.56 0.05
5.3 0.1

0.13 0.01
5.0 0.3

93 2

88.1 0.8

22:6
Totala

0.1 0

6.3 0.2

79 2

81 2

GC: gas chromatography.

a
Total does not sum to 100% because minor peaks were not assigned.

4.2.2. Treatments by supercritical CO2

After the rst cycle (without co-solvent), the weight loss of the
bone sample is only 6.0%. After the second cycle (with co-solvent,
ethanol), the weight loss is 7.9%. The treatment could only extract
brown oil. The visual aspect of the bone sample does not change.
The treatment by supercritical CO2 is ineffective.

solvents are very toxic and are hazardous to the bones. Indeed, the
Raman analysis shows an alteration of the organic part: collagen
peaks decrease after a treatment in the CHCl3 /MeOH. The other
organic solvents tested (hexane, heptane or alcane/isopropanol)
can extract the majority of the lipids: the weight loss is between 20
and 50% (Table 1). The degreasing is visible (Fig. 5B) but the bone
pores remain clogged with white grease. The variation of treatment parameters modies the efciency (Table 1). The extraction
dynamic improves the efciency: the Soxhlet extraction in hexane
at 60 C (sample S01) is better than the simple immersion in hexane
at ambient temperature (sample S02). Heating improves the solubility and the diffusion of lipids. The best extraction is obtained with
the action of the pulsed pressure (samples S04 and S05; sample
S05 was very fatty before treatment). With the simple immersion
in hexane or heptane, the weight loss is about 30% (samples S06,
S07, S08 and S09). The treatment time does not seem to inuence
the efciency of degreasing. The majority of fat is extracted during the rst bath. The Raman analysis shows that the bones are not
degraded by the treatments with these organic solvents (heptane
or hexane). The organic solvents offer good treatment solutions but
the use of heating or pressure requires expensive equipment to conform to safety and security standards. To treat a whole bone, we
selected the simplest treatment: immersion in alcane at ambient
temperature. Heptane is as effective as hexane but heavier and less
volatile. Heptane was also preferred because of its lower toxicity.

4.2.3. Treatments in green solvents

The efciency of treatments with limonene or the methyl oleate
is limited. We cannot measure the weight loss because these green
solvents are not volatile. After treatment, these green solvents
remain in the bone. To extract them, we must use another volatile
organic solvent. Bones impregnated with solvents cannot be exhibited, mainly because limonene is allergenic. The only solution to
extract degraded lipids from the n whale skeleton would therefore
seem to be the use of organic solvents.
4.2.4. Treatments in organic solvents
All organic solvents tested were effective in extracting colored
oil and grease (Table 1). But only the mixture of chloroform and
methanol (CHCl3 /MeOH) can extract the white grease. The GC
analysis shows a difference in composition between colored oil
and white grease. The colored oil contains unsaturated fatty acids
and the white grease is mainly saturated fatty acids. The mixture
of CHCl3 /MeOH allows complete degreasing (Fig. 5C). But these

Table 3
Weight loss after 72 or 100 h in different solutions with or without lipases at 30 , stirred at 170 rpm.
Treatment

Weight loss (%)

Ethanol without lipase

Acetone

Ethanol with DF Amano 15

Ethanol with lipozyme TL 100 L

72 h

100 h

72 h

100 h

72 h

100 h

72 h

100 h

10.7

9.5

13.4

19.1

14.6

9.6

6.3

5.6

E. Guilminot et al. / Journal of Cultural Heritage 15 (2014) 128135

4.3. Application
Tests on n whale metacarpus bones in heptane were disappointing. Despite two baths (3 and 8 days) in heptane, residual fat
remained visible on the bone surface after treatment (Fig. 4). The
degreasing in heptane on whole bones is much less effective than
on bone samples with open porosity: in the metacarpus, the weight
loss is limited to between 10 and 16%, while it is about 30% in the
bone blocks. The presence of the periosteum (compact part in the
outer whole bone) may limit the extraction of fat. Another difference between the whole bone and the bone block is the nature of
the lipids. On the bone surface, the triglyceride molecules react with
the oxygen in the air and form a polymeric network [13]. The process of autoxidation followed by a polymerization is well known
and studied in art, especially in painting conservation [14]. This
lm on the bone surface may act like a varnish. Thus a thick lm
of sticky oil can accumulate on the bones surface and impede further diffusion of oxygen into the bone. Fat that is deep inside the
porous structure of the bone degrades slowly. This has implications
for the cleaning of the fatty bones [15]. G. Turner-Walker [15] eliminates the polymeric lm of sticky oils by aqueous ammonia via a
saponication reaction.
5. Conclusion
This study shows the complexity and difculty of the retreatment of fatty bones. The problem is quite widespread due to
the difculty in preparing large skeletons like those of n whales.
Current methods are still empirical and cannot guarantee complete degreasing. In our research program, we selected different
solutions of re-treatment. But several methods were found to be
inadequate. Degreasing by lipases, by supercritical CO2 or in green
solvents proved to be ineffective. Only the use of organic solvents
appears to be suitable. Hexane, Heptane or a mixture of hexane/isopropanol can extract degraded fat without deteriorating the
bone. Heating or pressure increases the effectiveness of treatment.
A simple immersion in hexane or heptane at ambient temperature
already allows for the extraction of the majority of colored fat if the
porosity of bones is open. The effectiveness of treatment decreases
on whole bones. Our proposal for treatment can be improved upon
and further assays are essential. In particular, a pre-cleaning of
the bones surface is necessary to remove the lm of sticky fat. In
the case of the n whale skeleton at Nantes Museum, some bones
should not be re-treated. The most weakened bones should be the
object of surface cleaning only. But for the fattiest bones, such as the
shoulder bone, extraction in an organic solvent is also necessary.
Acknowledgments
The authors would like to express their gratitude to Pierre
Watelet, conservator in Nantes Museum who has initiated this

135

study. We also wish to thank Willy Dabin (Research Centre

on Marine Mammalians, La Rochelle) and Pierre-Henri Fontaine
[Museum of Skeleton at le-Verte (Canada)] for providing n whale
bone samples.
We are very grateful to Sabine Le Blond, Thibaut Foucher, Claire
Musso, Hugues Badet, Laurence Dubreuil and Delphine Morvan, the
students who participated to this work.
We sincerely thank reviewers, Lindsay Le Mtais and Angela
Arden for reviewing this text and correcting English.
The study was nancially supported by the French Ministry of
Culture through a national research program on knowledge and
preservation of materials of the cultural heritage.
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