The curious case of the Dana platypus and what it can teach us about how lead shotgun pellets behave in fluid preserved museum specimens and may limit their scientific value

Abstract

Fluid preserved animal specimens in the collections of natural history museums constitute an invaluable archive of past and present animal diversity. Well-preserved specimens have a shelf-life spanning centuries and are widely used for e.g. anatomical, taxonomical and genetic studies. The way specimens were collected depended on the type of animal and the historical setting. As many small mammals and birds were historically collected by shooting, large quantities of heavy metal residues, primarily lead, may have been introduced into the sample in the form of lead shot pellets. Over time, these pellets may react with tissue fluids and/or the fixation and preservation agents and corrode into lead salts. As these chemicals are toxic, they could constitute a health issue to collection staff. Additionally, heavy element chemicals interfere with several imaging technologies increasingly used for non-invasive studies, and may confound anatomical and pathological investigations on affected specimens. Here we present a case-study based on platypus (Ornithorhynchus anatinus) and other small mammals containing lead pellets from the collection of The Natural History Museum of Denmark.

Full text

RESEARCH ARTICLE
The curious case of the Dana platypus and
what it can teach us about how lead shotgun
pellets behave in fluid preserved museum
specimens and may limit their scientific value
Henrik LauridsenID
1
*, Daniel Klingberg Johansson
2
, Christina Carøe Ejlskov Pedersen
3
,
Kasper Hansen
3
, Michiel Krols
4
, Kristian Murphy GregersenID
5
, Julie Nogel Jæger
5
,
Catherine Jane Alexandra WilliamsID
6
, Ditte-Mari Sandgreen
7
,
Aage Kristian Olsen Alstrup
1,8
, Mads Frost Bertelsen
9,10
, Peter Rask Møller
2
1Department of Clinical Medicine, Aarhus University, Aarhus, Denmark, 2Natural History Museum of
Denmark, University of Copenhagen, Copenhagen, Denmark, 3Department of Forensic Medicine, Aarhus
University, Aarhus, Denmark, 4TESCAN XRE, Ghent, Belgium, 5Institute of Conservation, Royal Danish
Academy, Copenhagen, Denmark, 6Department of Animal and Veterinary Sciences, Aarhus University,
Aarhus, Denmark, 7Givskud Zoo-Zootopia, Give, Denmark, 8Department of Nuclear Medicine & PET,
Aarhus University Hospital, Aarhus, Denmark, 9Center for Zoo and Wild Animal Health, Copenhagen Zoo,
Frederiksberg, Denmark, 10 Department of Veterinary Clinical Sciences, University of Copenhagen,
Copenhagen, Denmark
*henrik@clin.au.dk
Abstract
Fluid preserved animal specimens in the collections of natural history museums constitute
an invaluable archive of past and present animal diversity. Well-preserved specimens have
a shelf-life spanning centuries and are widely used for e.g. anatomical, taxonomical and
genetic studies. The way specimens were collected depended on the type of animal and the
historical setting. As many small mammals and birds were historically collected by shooting,
large quantities of heavy metal residues, primarily lead, may have been introduced into the
sample in the form of lead shot pellets. Over time, these pellets may react with tissue fluids
and/or the fixation and preservation agents and corrode into lead salts. As these chemicals
are toxic, they could constitute a health issue to collection staff. Additionally, heavy element
chemicals interfere with several imaging technologies increasingly used for non-invasive
studies, and may confound anatomical and pathological investigations on affected speci-
mens. Here we present a case-study based on platypus (Ornithorhynchus anatinus) and
other small mammals containing lead pellets from the collection of The Natural History
Museum of Denmark.
Introduction
The first recorded mention of the use of high concentrations of ethanol to preserve whole ani-
mal specimens was on June 4, 1662, when the physician William Croone appeared before the
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OPEN ACCESS
Citation: Lauridsen H, Johansson DK, Pedersen
CCE, Hansen K, Krols M, Gregersen KM, et al.
(2024) The curious case of the Dana platypus and
what it can teach us about how lead shotgun
pellets behave in fluid preserved museum
specimens and may limit their scientific value.
PLoS ONE 19(10): e0309845. https://doi.org/
10.1371/journal.pone.0309845
Editor: Gianniantonio Domina, University of
Palermo, ITALY
Received: May 30, 2024
Accepted: August 20, 2024
Published: October 18, 2024
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0309845
Copyright: ©2024 Lauridsen et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: Raw CT, micro-CT
and MRI data of the Dana platypus NHMD-M01-28

Royal Society of London and “produced two embryos of puppy-dogs, which he had kept eight
days, and were put in spirit in a glass-vial sealed hermetically” [1–3]. Since then, ethanol pres-
ervation has remained a common practice for the long-term storage of animal specimens in
the collections of natural history museums around the globe. When the fixative powers of
formaldehyde were described in the late 19
th
century by another physician, Ferdinand Blum
[4,5], preservation practices were expanded to often include an initial fixation step by injecting
or immersing animal specimens in 4% v/v formaldehyde followed by transferring to gradually
higher concentrations of alcohols e.g. ethanol (EtOH) until usually reaching 70% v/v [3]. In
this state, preserved animal specimens can be stored for centuries and constitute important
and often irreplaceable archives of past and present biodiversity [6].
A useful aspect of museum collections of fluid preserved animals is the possibility to con-
duct both patho-anatomical and comparative anatomy studies across a wide range of species.
With the advent of modern medical imaging techniques such as x-ray computed tomography
(CT) and magnetic resonance imaging (MRI) and their introduction in the zoological field,
non-invasive/destructive studies are now possible on both soft, hard and fossilized tissues [7–
14]. This adds considerable scientific value to collections of fluid preserved animals and drives
present digitization efforts of such collections e.g. the Danish Distributed System of Scientific
Collections (DaSSCo), the European Distributed System of Scientific Collections (DiSSCo),
and the North American Open Exploration of Vertebrate Diversity in 3D (oVert) [6,15].
Collection methods of museum specimens vary by the type of animal and the historical set-
ting. However, to gain maximal scientific value of preserved specimens, the collection method
can be of great importance. In the following, we describe a case study on a specimen of platy-
pus Ornithorhynchus anatinus (Shaw, 1799), in which the discovery with x-rays of widely dis-
tributed dense nodules hugely impacted pathological and anatomical interpretation of the
specimen. We expand this case with further fluid preserved specimens, and demonstrate the
importance of knowing if these specimens were collected by shooting or in other ways contain
shotgun pellets e.g. introduced by previous unsuccessful hunting or by ingestion. Both from a
scientific point of view and potentially also from a health point of view for collection staff and
visiting scientists handling these specimens.
In the winter of 2022, a platypus specimen with the catalogue# NHMD-M01-28 was trans-
ported from the fluid preserved mammal collection at The Natural History Museum of Den-
mark, Copenhagen, to Aarhus University Hospital, Skejby, Denmark, with the intention of
imaging the heart with MRI for the purpose of an ongoing study on cardiac muscle fibre archi-
tecture across mammalian hearts. This was performed outside clinical hours using a sequence
previously optimized for cardiac tissue in ethanol preserved specimens with a field of view
spanning most of the chest- and abdominal cavity. No abnormalities were noted by the opera-
tor of the scanner, who is not a trained veterinarian (author HL). Later, before the specimen
was to be returned to the museum’s collection, it was decided to subject it to another non-inva-
sive imaging technique, CT, in order to digitize the entire specimen for future use and in par-
ticular generate a virtual dataset of bone morphology and bone density of the specimen. On
this occasion, widely distributed centimetre sized hyperintense nodules were observed in the
specimen by the scanner operators, none of whom are trained veterinarians (authors CCEP,
KH and HL). After discussion with the collection manager and the mammal curator at The
Natural History Museum of Denmark (authors DJK and PRM, respectively), it was decided to
ask for advice from highly trained research and zoo veterinarians (authors CJAW, DS, AKOA
and MFB) to form an opinion on what these hyperintense nodules could originate from e.g., a
disease process in the preserved platypus.
Platypus NHMD-M01-28 was formally included in the mammal collection of The Natural
History Museum of Denmark (at that time “Zoological Museum, University of Copenhagen”)
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is available for download on MorphoSource
(https://www.morphosource.org/) project#
000620016 using the link: https://www.
morphosource.org/projects/000620016?locale=en.
Raw digital x-ray images of remaining specimens
can be downloaded from Figshare (https://figshare.
com/) using the link: https://doi.org/10.6084/m9.
figshare.25569099.v1.
Funding: HL is supported by the Carlsberg
Foundation (grant# CF21-0605) and PRM is
supported by the Carlsberg Foundation (grant#
CF21-0435). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: I have read the journal’s
policy and the author Michiel Krols of this
manuscript has the following competing interests:
Employed at TESCAN XRE, a manufacture of one of
the micro-CT systems used in the study (UniTOM
XL Spectral). The remaining authors have declared
that no competing interests exist. This does not
alter our adherence to PLOS ONE policies on
sharing data and materials

on the 1
st
of July 1931. The specimen was obtained during “The Carlsberg Foundation’s
Oceanographical Expedition round the World 1928–30 under the Leadership of Professor
Johannes Schmidt”, also known as the fourth Dana expedition [16]. In Australia, the expedi-
tion was both collecting specimens and receiving donations from local scientists of floral and
faunal samples of natural-historical value [17]. Although platypus were already at that time
prohibited from being traded and/or exported from Australia, the then director of the Queens-
land Museum, Dr. Albert Heber Longman [18], was able to issue a license of export for a single
preserved platypus. In this way platypus NHMD-M01-28, henceforth referred to as “the Dana
platypus”, came into possession of the expedition as a donation when visiting Brisbane
between the 3
rd
and the 10
th
of March 1929 [17]. There is no record of the method of collection
of the Dana platypus, but it was originally collected nearby Eidsvold, South East Queensland
(25˚22’ S, 151˚07’ E) by Thomas Lane Bancroft and donated to the Queensland Museum (orig-
inal catalogue# J2390) on the 21
st
of June 1915 (Janetzki, H., Queensland Museum, personal
communication). There is no fixation or preservation record of the specimen, but it is pre-
sumed that since its inclusion in the collection of the Natural History Museum of Denmark it
has been stored individually and according to custom in 70% v/v EtOH in a glass container for
the last ~92 years until the described imaging event. There is no record of any re-fixation treat-
ments (e.g. short 4% formaldehyde baths) of the specimen during this time.
After being offered several plausible pathological explanations for the dense nodules in the
Dana platypus by veterinarians who based their evaluation on qualitative CT and MRI data, it
was noted that the image intensity of nodules was potentially surpassing biological values. This
could indicate that the nodules may in fact originate from some foreign objects introduced
into the specimen before or after it was collected i.e. perimortem or postmortem. At this point
it was decided to conduct a quantitative analysis to investigate the most likely origin of these
nodules and if similar objects were present in other preserved platypus or other small fluid pre-
served mammals in the museum’s collection. The presence of similar nodules in other platypus
but not in other species would offer support to a pathological origin. This explanation, how-
ever, would be less likely if similar nodules were observed in other unrelated species, which
instead would suggest an external origin e.g. shotgun pellets used for collecting the specimens,
which later corroded to unrecognisable shapes and sizes after long-term storage in preserva-
tion fluids. Since the latter would be of relevance to researchers using fluid preserved museum
specimens for comparative anatomy studies and curators and collection staff handling these
specimens, we decided to dig into this unexpected mystery and investigate the origin of the
dense nodules in the Dana platypus, and the potential effect that corroded shotgun lead pellets
can have on quantitative imaging studies and the safety of handling affected specimens.
Materials and methods
Specimen information
Twenty six ethanol preserved and four pelts of small mammals in the collection of The Natural
History Museum of Denmark collected or registered at the museum between 1849 and 1962
(Table 1) were included in the study. Since the study had its origin in an observation on the
Dana platypus (Ornithorhynchus anatinus catalogue# NHMD-M01-28) all available platypus
specimens in the collection were included except for stuffed specimens and dry bones. The
inclusion of remaining species was determined by the collection era and the perceived likeli-
hood of the specimen having been collected by the aid of a shotgun. Additionally, cadavers of
six freshly culled laboratory rats (Sprague Dawley, 9 weeks of age, body mass = 238.3 ±9.5 g,
not used in other studies), all female, Forum breeding permit, (Aarhus University, breed
under permission from the Danish Animal Experiments Inspectorate) were included to test
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the effect of lead pellets (Danish shot size 5 = 3 mm in diameter, Gyttorp Cartridge Company
AB, Nora, Sweden) on measurements of bone mineral content using quantitative CT imaging.
This study involved only museum specimens and cadavers of previously culled rats not used in
any experiments and did not require an animal experimental permit.
Digital x-ray imaging
All specimens but the Dana platypus were imaged using projection based digital x-ray imaging
using a XPERT 80-L system (KUBTEC Scientific, Stratford, Connecticut, USA) with x-ray
tube voltage = 70–125 kV and x-ray tube current = 42–110 μA.
X-ray computed tomography imaging
The Dana platypus and the six laboratory rat cadavers were imaged with x-ray computed
tomography imaging using a Canon Aquilion Prime SP system with the following parameters:
Table 1. Fluid preserved mammal specimens in the collection of The Natural History Museum of Denmark (NHMD) having undergone x-ray examination for for-
eign objects.
Cat # Binomial name Common name Type Year Nodules Size (mm) #
M07-CN958 Aplodontia rufa Mountain beaver Fluid 1893 No
M03-CN195 Atopogale cubana Cuban solenodon Fluid 1886 No
M07-CN398 Spermophilus citellus European ground squirrel Fluid 1885 No
M02-CN462 Dactylopsila trivirgata Striped possum Fluid 1893 No
M05-CN1074 Eidolon dupreanum Madagascan fruit bat Fluid 1894 No
M07-CN1217 Gymnoromys roberti Voalavoanala Fluid 1895 No
M10-CN1331 Mustela erminea Stoat Fluid 1909 No
M01-3 Ornithorhynchus anatinus Platypus Pelt ukn. No
M01-4 Ornithorhynchus anatinus Platypus Pelt 1879 No
M01-11 Ornithorhynchus anatinus Platypus Pelt 1869 No
M01-29 Ornithorhynchus anatinus Platypus Pelt 1936 No
M01-25 Ornithorhynchus anatinus Platypus Fluid 1915 Yes 13.2 6
M01-10 Ornithorhynchus anatinus Platypus Fluid 1865 Yes 11.7 18
M01-24 Ornithorhynchus anatinus Platypus Fluid 1917 Yes 8.4 5
M01-28 Ornithorhynchus anatinus Platypus Fluid 1915 Yes 10.4 21
M01-34 Ornithorhynchus anatinus Platypus Fluid 1922 Yes 5.0 12
M01-35 Ornithorhynchus anatinus Platypus Fluid 1922 No
M01-9 Ornithorhynchus anatinus Platypus Fluid 1865 Yes 6.4 17
Ucat Ornithorhynchus anatinus Platypus Fluid 1950 No
M02-CN299 Perameles gunnii Eastern barred bandicoot Fluid 1914 No
M02-CN439 Petaurus breviceps Sugar glider Fluid 1931 No
M02-CN297 Phascogale tapoatafa Brush-tailed phascogale Fluid 1902 No
M02-CN265 Philander opossum Gray four-eyed opossum Fluid 1893 No
M02-CN440 Pseudocheirus peregrinus Common ringtail possum Fluid 1931 Yes 7.5 8
M05-CN2901 Pteropus rayneri Solomons flying fox Fluid 1962 Yes 5.2 9
M07-CN13 Ratufa macroura Grizzled giant squirrel Fluid 1865 No
M07-CN1164 Callosciurus finlaysonii Finlayson’s squirrel Fluid 1900 Yes 6.6 5
M07-CN1163 Callosciurus finlaysonii Finlayson’s squirrel Fluid 1900 Yes 6.8 4
M07-CN17 Sciurus colliaei Collie’s squirrel Fluid 1849 No
M06-CN256 Tupaia belangeri Northern treeshrew Fluid 1893 No
Nodule size in mm, number (#) refer to number of large distinct nodules in the specimen.
https://doi.org/10.1371/journal.pone.0309845.t001
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x-ray tube voltage = 80, 100, 120, 140 kVp, x-ray tube current = 260 mA, integration
time = 1000 ms, field-of-view = 256 ×256 ×756 mm
3
, spatial resolution = 0.5 mm isotropic,
convolution kernel = FC18, acquisition time = 60 s pr. scan. Underneath the specimens, a
Mindways QCT Pro bone mineral calibration phantom was positioned to calibrate x-ray atten-
uation values to bone mineral density (mg/mm
3
equivalent aqueous K
2
HPO
4
).
Additionally, the anterior portion (head and shoulder girdle) of the Dana platypus and
excised nodules from the back and the right hind limb were imaged at a higher resolution
using a Medical XtremeCT system (Scanco, Bru¨ttisellen, Switzerland) with the following
parameters: x-ray tube voltage = 59.4 kVp, x-ray tube current = 119 μA, integration time = 132
ms, field-of-view = 70 ×70 ×150 mm
3
, spatial resolution = 0.082 mm isotropic, acquisition
time = 1.5 h pr. scan.
Finally, the neurocranium and part of the bill of the Dana platypus was imaged using a Uni-
TOM XL Spectral system (TESCAN GROUP, Brno, Czech Republic) equipped with a hyper-
spectral detector (channel size of 1 keV) with the following parameters: x-ray tube
voltage = 160 kVp, x-ray tube current = 94 μA, x-ray tube power = 20 W, integration time = 83
ms, field-of-view = 76.4 ×76.4 ×27.1 mm
3
, spatial resolution = 0.108197 mm, acquisition time
= ~10 h. K-edge subtraction to highlight the Pb signal (K-edge at 88.0045 keV) was performed
using 1 channels width and 3 channels separation (91 keV– 85 keV).
Beef samples used to test the corrosion of lead pellets in tissue (see description below) were
imaged using the same UniTOM XL Spectral system as described above, but using its standard
integrating detector and using the following parameters: x-ray tube voltage = 80 kVp, x-ray
tube current = 625 μA, x-ray tube power = 50 W, integration time = 25 ms, field-of-
view = 48 ×48 ×38.35 mm
3
, spatial resolution = 0.05 mm isotropic, acquisition time = 12 min
per sample.
Magnetic resonance imaging
The Dana platypus was imaged with magnetic resonance imaging using a Siemens Magnetom
Skyra 3 T system equipped with a 15 channel transmit/receive knee coil using a T
2
-weighted
3D spin echo sequence with the following parameters: repetition time = 1000 ms, echo
time = 132 ms, refocusing flip angle = 120˚, field-of-view = 196 ×196 ×108.8 mm
3
, spatial res-
olution 0.34 mm isotropic, number of averages = 4, acquisition time = 2 h.
Image analysis
Image J (version 1.50e) and OsiriX DICOM Viewer were used for image viewing and reslicing.
Bone mineral content analysis was conducted as previously described [11,19].
Chemical analysis of foreign objects
The nodules in the Dana platypus were determined to be foreign objects (FO) based on the
hyperintense appearance on CT images and all 21 major objects were numbered in the order
they appeared from the cranial end of the specimen. Foreign object 11 (dorso-lateral mid tho-
rax), 12 (in the abdomen), 14 (caudal abdomen), 16 (right hind limb, lateral to the tibia) and
21 (lateral tail) were excised and used for chemical analysis (FO 11, 12, 14, 21) and high-resolu-
tion imaging (FO 11, 16). Excision cuts in the specimen were carefully sutured to keep the
integrity of the specimen.
A flame test was conducted on the entire FO 11 as well as on a slurry of the core and crust,
respectively, of FO 12 in double distilled water. The flame test was conducted accordingly to
the Royal Society of Chemistry (United Kingdom) guidelines using wooden splints and double
distilled water as solvent [20]. As a reference of flame colours of common biometals, calcium
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(Ca), potassium (K), sodium (Na) and iron (Fe), salt solutions of CaCl
2
, KCl, NaCl at a concen-
tration of 1 M as well as a saturated slurry of Fe
2
O
3
were used.
To test for traces of common metals in shotgun lead pellet alloys, lead (Pb), antimony (Sb),
tin (Sn) and arsenic (As), a range of test papers and kits were used: Pb, test paper 1, Water-
strips, Joygain, Hong Kong, SAR China; Pb, test paper 2, Plumbtesmo, Macherey-Nagel,
Du¨ren, Germany; Sb, Antimon-Testpapier, Macherey-Nagel, Du¨ren, Germany; Sn, Quantofix
Tin, Macherey-Nagel, Du¨ren, Germany; As, Arsenic Test, MQuant, Supelco/Merck, Darm-
stadt, Germany. The sensitivity of the two used Pb test papers was validated using a serial dilu-
tion of a saturated solution of PbCl
2
in double distilled water. Test paper 2 proved most
sensitive with a sensitivity of 9.7 mg/l PbCl
2
, i.e. 34.9 μM Pb
2+
. Excised foreign objects were
homogenized using a Benchmark BeadBug 6 microtube homogeniser operating at 3500 rpm
for 10 cycles (30 s shaking / 30 s rest) and equipped with 2 ml microtubes containing five 2.8
mm stainless steel beads, 1 ml ultrapure water and ~300 mg sample. Additionally, shotgun
lead pellets (Danish shot size 5 = 3 mm in diameter, Gyttorp Cartridge Company AB, Nora,
Sweden) were used for reference measurements of metal alloys. These shot pellets were treated
in the same way as the samples of excised foreign objects.
Solubility of the whitish core of FO 14 at different temperatures was measured by first incu-
bating the homogenate in double distilled water at 90˚C on a Benchmark Multi-Therm ther-
moshaker at 500 rpm for 2 h. Then the solution was filtered for particles using a strainer with a
100 μm mesh size. The solution was then cooled in steps to 80, 60, 40 and 20˚C without shak-
ing to let the excess of the dissolved salt precipitate as the temperature was lowered and left to
stabilize for 2 h for each temperature. At each temperature step, a dilution series of 1, 2, 4, 8,
16, 32, 64, 128 and 256x was prepared for the salt solution, and the concentration of Pb was
measured according to the calibration dilution series of PbCl
2
on both Pb test paper 1 and 2.
Chemical analysis of preservation fluids
Preservation fluids of specimens abbreviated Dtri CN462, Edup CN1074, Merm CN1331, Oana
M25, Oana M9, Oana M10, Oana M24, Oana M28, Oana M34-35 (stored in same container),
Oana Ucat, Pper CN440, Pray CN2901, Cfin CN1163-1164 (stored in same container), Scol
CN17 and Acub CN195 (see Table 1 for full specimen information) were tested for residues of
the toxic metals Pb and As. First, 40 ml samples were distilled at 79˚C to evaporate the ethanol
content of the samples. Distillation was continued until each sample was concentrated by 30x.
Many concentrated samples consisted of a lipid top layer and a watery bottom lay, thus the pres-
ence of Pb in either the lipid or the water phase was tested by pipetting 10 μl of each phase onto
Pb test paper 2. Subsequently, concentrated samples were diluted to 3.5x initial concentration
by adding double distilled water to allow for a sufficient volume to conduct As-tests.
Corrosion test of lead pellets
A corrosion test of lead shot pellets (Danish shot size 5 = 3 mm diameter, Gyttorp Cartridge
Company AB, Nora, Sweden) stored in either double distilled water (pH = 5.01), 70% v/v
EtOH with remainder being double distilled water (pH = 6.03, but with the uncertainty of
measuring acidity in EtOH/water-solutions [21], 70% v/v EtOH with remainder being phos-
phate buffered saline (pH = 8.61), 100% EtOH, 4% v/v phosphate buffered formaldehyde
(pH = 6.84), 4% v/v unbuffered formaldehyde (pH = 3.97) or 40% v/v unbuffered formalde-
hyde (pH = 3.18) was conducted as previously described [22]. In short, lead pellets were first
freed of any corrosion products or grease by shaking at 2500 rpm for three cycles (30 s shaking
/ 30 s rest) on a Benchmark BeadBug 6 microtube homogenizor equipped with 2 ml micro-
tubes containing 0.5 mm silica glass beads. This was performed first in dilute hydrochloric
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acid (3 M) and then in 100% acetone. Each pellet was weighed with 0.1 mg precision, and for
each of the seven test solutions six pellets were placed in individual 1.5 ml Eppendorf tubes
containing 1 ml of the test solution. Tubes were left in a horizontal position on a laboratory
rocker for 22 days. After that time, corrosion products were removed again as described above
and pellets were reweighed. Corrosion rate was calculated as the mass in μg of lost material per
mm
2
surface area of the spherical pellets per day. Following the corrosion test, test solutions
were centrifuged at 2500 g for 5 min, and supernatants were tested for the presence of dis-
solved Pb, Sb, Sn and As using test papers and kits as described above.
To investigate the corrosion of lead shot pellets within tissue samples stored in various pres-
ervation fluids, six 2 ×2×2 cm
3
pieces of beef sirloin were prepared and a single lead shot pel-
let (Danish shot size 5 = 3 mm diameter, Gyttorp Cartridge Company AB, Nora, Sweden) was
inserted into the centre of the beef cube using a forceps. The beef samples were preserved for a
duration of 256 days (~8.5 months) with gentle rocking in 35 ml of either 70% v/v EtOH (with
remainder being double distilled water), 4% v/v phosphate buffered formaldehyde, 4% v/v
unbuffered formaldehyde, 40% v/v unbuffered formaldehyde, or in either 4% v/v phosphate
buffered formaldehyde or 4% v/v unbuffered formaldehyde for 7 days and then in 70% v/v
EtOH to mimic a typical fixation/preservation procedure. After the preservation period, lead
pellets were carefully removed using forceps with minimum disruption of tissue, and the sam-
ples were imaged using the UniTOM XL Spectral system as described above. The volume of
corrosion products remaining in the beef samples was quantified by counting the number of
voxels with signal intensities surpassing those of unaffected beef tissue (>12800).
Statistical analysis
Data is shown throughout as mean values ±standard deviations. Analyses of significant differ-
ences between two groups were performed using two-tailed Student’s t-test (paired or
unpaired as relevant), and for more than two groups using ANOVA. For repeated measures,
one-way ANOVA with repeated measures was performed with Tukey’s honest significance
test for post hoc test of significant differences between groups. We considered p-values less
than 0.05 to signify statistical significance.
Results
First observations on the Dana platypus and suggested pathologies
Superficially, the Dana platypus presented with no obvious injuries, except from a cavity in the
soft upper bill at the level of the right premaxilla (Fig 1A). Magnetic resonance imaging
focused at the cardiac region did not immediately reveal any abnormalities, however, subse-
quent CT imaging showed widely distributed centimetre sized hyperintense nodules (Fig 1B).
Due to their large size and nodular appearance, these objects were initially disregarded as
potential projectiles and their apparently random distribution also suggested that they had not
been inserted for the purpose of mounting the specimen in a certain position. After a qualita-
tive evaluation (i.e. not focusing on x-ray attenuation levels in nodules), it was decided that the
nodules could be of neoplastic or infectious nature. Presenting this specific mind-set (i.e.
potentially inducing preconceived bias), advice was requested of experienced research and zoo
veterinarians. Based on limited and only qualitative imaging material (S1 File) the notion that
nodules could be neoplastic was supported and several additional explanations were suggested
e.g. mucormycosis with mineralisation or tuberculosis (Table 2). However, the scans were not
considered pathognomonic for any of these conditions. Subsequently, subdermal and abdomi-
nal nodules were excised from the Dana platypus for further analysis. High resolution micro-
CT imaging and the physical sectioning of two nodules revealed their consistence of a
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Fig 1. The Dana platypus (NHMD-M01-28) collected during The Carlsberg Foundation’s Oceanographical
Expedition round the World 1928–30. (a) Apart from a single puncture at the right side of the bill (black arrowhead)
the specimen shows no superficial evidence of collection method; (b) Virtual coronal section from x-ray computed
tomography (CT) showing hyperdense foreign objects (FO) embedded in the specimen; (c) Foreign object 16 dissected
free from the thigh musculature appears as a centimetre sized nodule with great visual resemblance to dried chewing
gum, but more brittle in nature; (d) Slice through FO 16 showing a whitish core and a brownish crust; (e) Virtual
section of FO 16 from micro x-ray computed tomography (μCT) showing the crust is more radiodense than the core;
(f) Virtual coronal section (similar to (b) from magnetic resonance imaging (MRI) of FO 11. In contrast to bony
material that appears hypodense in the specific T2-weighted spin echo sequence, the foreign object appears
hyperdense, but with a low level of image artifacts. This indicates that the foreign object does not consist of either bone
mineral or paramagnetic substances; (g) Volume rending from CT of the Dana platypus allowing for the appreciation
of the large number of foreign objects. At least 21 centimetre sized objects can be observed. The volume rending is
calibrated to a 0–5000 Hounsfield unit (HU) calibration bar to demonstrate radiodensity of the skeleton and foreign
objects. Skeletal components rarely exceed 1900 HU, but the average radiodensity of the foreign objects is 4320 HU
indicating a non-biological origin.
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radiodense and physically dense outer crust and a softer core (Fig 1C–1E). A reinspection of
MR images revealed that nodules were in fact observable (Fig 1F), and, in opposition to bone
material presenting hypo intense using the specific T
2
-weighted spin echo sequence, the nod-
ule core presented hyperintense compared to surrounding tissue. Also, nodules did not cause
pronounced image artifacts, suggesting the absence of paramagnetic materials such as iron
(Fig 1F). At this point, a quantitative analysis of x-ray attenuation in nodules was conducted
revealing CT values of 4320 ±531 Hounsfield units (HU) (Fig 1G). This is markedly higher
than most dense bony structures found in humans of approximately 1900 HU [23,24], and
thus a careful investigation for a non-biological origin of the nodules, or more precisely the
foreign objects, was warranted.
Chemical analysis of nodules
The apparently random distribution of nodules suggested that their origin were more likely
that of corroded projectiles e.g. shotgun lead pellets than that of other types of foreign objects
introduced to preserve or mount the specimen. Thus, chemical analysis was conducted to test
the hypothesis that nodules were corroded lead pellets. A flame test of both entire excised nod-
ules as well as core and crust material prepared separately as a slurry in double distilled water,
revealed flame colours in pink, light blue or whitish nuances which is not characteristic for
some of the most abundant biometals: Ca, K, Na and Fe, all with distinct flame colours (Fig
2A). Two different brands of test papers specifically prepared to detect Pb was validated and
calibrated against a serial dilution of PbCl
2
in double distilled water (Fig 2B and 2C), and addi-
tional test papers and kits to detect Sb, Sn and As, revealed the presence of all four metals in
the excised nodules (Fig 2B–2D). Since neither of these metals are usually found in high quan-
tities in healthy biological tissue, this strongly suggested that the nodules were foreign objects
and very likely corroded shotgun pellets. To inform whether lead salts of the corroded lead pel-
lets were reaction products of metallic lead and fixation or preservation chemicals, e.g. formal-
dehyde or EtOH, the solubility of core and crust salts of foreign object14 was tested at 20, 40,
60 and 80˚C. This showed that remaining lead salts were highly insoluble compared to other
relevant lead salts such as lead(II) acetate, lead(II) formate and lead(II) chloride (reference val-
ues from [25]) even at high temperatures (Fig 2E and 2F).
X-ray spectral and hyperspectral analysis of nodules
Different elements have different x-ray attenuation signatures in function of the x-ray photon
energy. Thus, to expand on the chemical analysis suggesting a non-biological origin of the
nodules, an x-ray spectral analysis was conducted on six of the nodules of the Dana platypus
Table 2. Suggested pathologies in Dana platypus based on limited imaging data.
Pathology Observations speaking for Observations speaking against
Osteosarcoma with
secondary metastases
Proliferative mineralised lesions, ‘cannonball’ appearance No obvious destructive process within bony tissue. No obvious primary
lesion, and secondaries often pulmonary in clinical cases
Other bone forming
neoplastic lesions
Multiple mineralised lesions Round and concentrated rather than diffuse appearance
Mucormycosis Well known disease in this species, causing widespread
and potentially extensive nodular lesions
Uncharacteristic for lesions to be mineralised.
Tuberculosis Commonly found in many different species. Lesions can
become calcified.
Typically processes are concentrated in single organs, such as the lungs.
Tumoral calcinosis Multiple calcified nodules. Only described in humans
Tophaceous gout Multiple radiodense nodules. Observed in several species. Typically smaller nodules.
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Fig 2. Chemical tests to reveal elemental components of foreign objects. (a) Flame test of metal salts of most
common biometals and pulverised core and crust of foreign object (FO) 12 and the whole of FO 11; (b) Calibration bar
for lead (Pb) test paper 1, Tin (Sn) test paper, and arsenic (As) test kit. Additionally, test for interactions between As
test kit and common physiological salts and Pb
2+
. No interactions were observed; (c) Serial dilution of PbCl
2
on Pb test
paper 1 and 2 to reveal lower detection limit of Pb on these test papers. Test paper 2 was most sensitivewith a detection
limit of 9.7 mg/l of PbCl
2
equalling 34.9 μM Pb
2+
;(d) Analysis for the presence of Pb, antimony (Sb), Sn and As in
double distilled water, 70% ethanol (EtOH), homogenized lead shot pellet, and homogenized core and crust of FO 14.
No interaction was observed for any metal for water and ethanol whereas all four metals were detected in the lead shot
pellet and in both core and crust of FO 14; (e) Salt solubility of FO 14 core material as a function of temperaturein
comparison with different lead salts (literature values); (f) Magnification of the graph above with a focus on FO 14 core
solubility. The solubility of the FO 14 core material is much lower than would be expected for reaction products
between ethanol and formaldehyde.
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and on lead shotgun pellets (Fig 3A). Conveniently within the energy range of most conven-
tional clinical CT scanners, 80–140 keVp, Pb has a K-edge at 88 kV, resulting in a steep
increase in x-ray absorption at that energy level which is evident in the idealised spectrum of
monochromatic x-rays (Fig 3B) (reference values from [26]). On the other hand, naturally
occurring minerals such as Ca (e.g. in bones), does not have any K-edges in this range, and
thus for bony materials, the x-ray attenuation would be expected to only decrease at increasing
x-ray energies as displayed in Fig 3B. A comparable bone signature was found at regions of
interest positioned at skeletal sites of the left tibia, skull and a thoracic vertebra of the Dana
platypus (Fig 3A and 3C) analysed with the polychromatic x-ray source of the clinical CT scan-
ner when setup to scan the specimen at individual energies. On the other hand, regions of
interest placed within the foreign objects showed a markedly different signature with an initial
decrease in x-ray attenuation from 80 to 100 keVp, followed by an increase in attenuation at
higher energies (Fig 3A and 3D). The attenuation signature of lead shot pellets was most simi-
lar to the idealised monochromatic spectrum of Pb with a steep increase in attenuation
between 80 and 100 keVp, although the subsequent decrease in attenuation at higher energies
was not observed for lead shot pellets with the polychromatic x-ray source and conventional
integrating detector of the used clinical CT scanner (Fig 3A and 3E). To elaborate, we per-
formed hyperspectral CT imaging with a dedicated micro-CT system on the neurocranium of
the Dana platypus (Fig 3F), since foreign objects were also contained within this region. In
contrast to the insensitivity of the clinical CT system to detect K-edges within the foreign
objects, the hyperspectral detector of the micro-CT system clearly detected a K-edge at 88 keV
of regions of interest placed within the foreign objects, which was not observed within bone
voxels (Fig 3G and 3H). This allowed for K-edge subtraction (integrating and subtracting
channels on either side of a K-edge to produce element specific images) and the precise map-
ping of Pb in the neurocranium (Fig 3F middle and right panels). In addition to confirming
the presence of large concentrations of Pb in the foreign objects within the Dana platypus,
hyperspectral imaging also showed that the widely distributed small dense particles within the
fur of the specimen were not corroded lead residues (no Pb K-edge in the particle cloud sur-
rounding the sample in Fig 3F lower right panel).
Shot trajectory
In the Dana platypus, one large foreign object was positioned in the posterior portion of the
right cerebral hemisphere (Figs 1G and 3A (“FO 3”), 3f and 4a, 4b). Since the neurocranium is
a nearly closed bony structure, an encapsulated foreign object would most likely leave a trace
of entry and thus carry information about the shot trajectory. A micro-CT scan of the head
region revealed bone fragments and hyperintense fragments or smears in the skull (Figs 3F
and 4A and 4B). The position of fragments and the large encapsulated foreign object suggested
a shot trajectory with a vertical component close to parallel or just a few degrees lower than the
bill and a horizontal component approximately 30˚ to the right of facing the animal directly
(Fig 4B). Although entry wounds were not immediately visible in the wet specimen and had
been completely overlooked during the initial handling of the specimen, after 10 minutes of
drying when most EtOH at the surface was evaporated, an entry wound was clearly visible on
the leftmost portion of the leathery upper bill flap (Fig 4C, right) at the position where it would
be expected from the micro-CT determined shot trajectory. Likewise, a small nearly sealed
entry wound just left of the midline was observed at the lower bill after the preservation fluid
had evaporated from the surface (Fig 4C, lower left). The position of this entry wound was
consistent with the interpreted shot trajectory and the obvious cavity on the right side of the
upper bill being an exit wound (Fig 4C).
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Fig 3. X-ray spectral analysis of foreign objects in the Dana platypus and in lead pellets. (a) Overview of the Dana
platypus and magnified lead pellets showing regions (circles) or points (crosshair) of interest; (b) Idealised x-ray
attenuation spectra for lead (Pb) (primary second axis), cortical bone and calcium (Ca) (both on secondary second
axis) at monochromatic x-ray photon energies from 60–200 keV. Lead has a sharp K-edge at 88 keV allowing for
material identification in this range; (c) Polychromatic x-ray computed tomography (CT) attenuation spectra for peak
energies between 80 and 135 keVp for the Dana platypus bone at regions of interest placed at the tibia, skull and a
vertebra, respectively. All three spectra follow the expected decrease in attenuation at higher energies as predicted by
the idealised spectrum for bone (b); (d) Polychromatic CT attenuation spectra for six foreign objects (FO) in the Dana
platypus. The spectra are distinct from the bone spectra (c) showing an increase in attenuation at higher peak energies,
but they are also not similar to the idealised lead spectrum (b) probably due to the polychromatic nature of
conventional CT imaging; (e) Polychromatic CT attenuation spectra for the peripheral partial volume effected region
of three lead pellets. In spite of the partial volume effect (necessary to avoid the Hounsfield unit threshold) and the
polychromatic nature of conventional CT imaging, the lead pellet spectra are comparable to the idealised spectrum of
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Foreign objects in other fluid preserved mammals
Although chemical analysis and x-ray spectral analysis of nodules in the Dana platypus and the
discovery of two entry wounds and bone fragments with a consistent shot trajectory all
strongly suggested the nodules to be foreign objects, very likely corroded lead shot pellets
where tissue infiltration by corrosion products made them much larger than the original pel-
lets, more support for this hypothesis would be gained if similar nodules were found in other
unrelated mammals collected in other locations. Therefore, another 29 mammal specimens
(25 fluid preserved and four dry pelts) were imaged with two-dimensional digital x-ray imag-
ing (Table 1). This included the entire collection of platypus at the Natural History Museum of
Denmark totalling eight fluid preserved platypus (including the Dana platypus), six of which
contained hyperintense nodules (Fig 5A), and four pelts without any nodules. In addition,
hyperintense nodules were found in an Australian opossum (Pseudocheirus peregrinus catalog#
NHMD-M02-CN440), two Southeast Asian squirrels (Callosciurus finlaysonii
NHMD-M07-CN1163 and M07-CN1164) and a Solomon Islands flying fox (Pteropus rayneri
M05-NHMD-CN2901) (Fig 5A and Table 1). The close resemblance of nodules in these mar-
supial and placental mammals to the monotreme platypus, suggested a shared origin as foreign
objects rather than a pathological nature, and very likely that of shot pellets. The Pteropus ray-
neri NHMD-CN2901 specimen was collected during the Danish Noona Dan Expedition to the
Pacific in 1961–62, and the shotgun and its custom-made adapter sleeve which were most
likely used for the collection of the flying fox was until 2023 in the weapons collection at the
Natural History Museum of Denmark but was recently transferred to the Danish National
Museum (Fig 5B).
Chemical analysis of Pb and As in preservation fluids
Both Pb and As are highly toxic metals, and since both metals are common components of
lead shot alloys and both were detected in the chemically analysed nodules of the Dana platy-
pus, it was of interest to test the presence of these metals in the preservation fluid of the Dana
platypus and the like of the several other fluid preserved specimens found to contain corroded
shot pellets (n = 9) and compare with preservations fluids of specimens that were seemingly
lead pellet free (n = 6). Fluid samples were heated to 79˚C to ensure evaporation of ethanol
and concentrated to 30x to measure residual Pb. Several concentrated samples consisted of a
lipid upper phase and watery bottom phase, and both phases were tested on the most sensitive
brand of Pb test paper. Lead was not detected in either phase of the concentrated samples
meaning that original preservation fluids contained <1.2 μM Pb
2+
(Fig 6A). To produce a suf-
ficient volume to run the applied As test kit, concentrated fluid samples were diluted with dou-
ble distilled water to reach a 3.5x concentration of the original preservation fluid. In contrast
to the negative Pb test results, all but two samples tested positive for the presence of As with a
peak concentration of 114 μg/l (1.5 μM As) (Fig 6B and 6C). Although both of the two preser-
vation fluid samples without traces of As were found within the specimens seemingly without
shot pellets, many other preservation fluids of specimens without pellets still contained traces
of As (Fig 6B). There was no significant difference between As concentration in preservation
lead (b) showing a pronounced increase in attenuation between 80 and 100 kVp; (f) Hyperspectral CT imaging of the
neurocranium of the Dana platypus presented as coronal slices (top row) and maximum intensity projections (MIP,
bottom row) of an averaged image of the 20–120 keV energy channels. K-edge subtraction at the K-edge of lead (88
keV) allows for material specific mapping of lead within the CT volume; (g) X-ray attenuation spectra (linear
attenuation coefficient, μ, over x-ray energy) for five regions placed within FO 2 and FO 3 and five regions with skull
bone. The sharp K-edge at 88 keV in the foreign objects confirms the presence of lead in foreign objects; (h) Derived
curves of attenuation spectra in (g) showing maximum increase in attenuation at 88 keV.
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Fig 4. Shot trajectory. (a) Coronal (left) and oblique sagittal (right) virtual micro-CT sections in the head of the Dana
platypus reveal a large foreign object in the right cerebral hemisphere and lead fragments and/or smears show the most
likely shot trajectory; (b) Three-dimensional reconstructions of the head in a dorsal view (left) and viewed along the
probable shot trajectory (right). Red and blue structures are hyperintense foreign objects; (c) Photos of the Dana
platypus from the same viewpoints as in (b) and from a ventral viewpoint in addition (lower left). White arrowhead
point to the exit wound on the dorsal portion of the bill. Magnifications show entry wounds. In the 70% ethanol
soaked specimen (upper right) it is easy to overlook the small entry wound, whereas it is obvious after superficial
preservation fluid has evaporated off (lower left and lower right).
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fluids containing specimens with or without visible foreign objects (unpaired t-test, n = 6 and
n = 9, p = 0.29) (Fig 6C).
Corrosion rate of lead shot pellets in different fluids
To test the corrosion rate of lead shot pellets in different fluids with relevance to fluid preserva-
tion, lead pellets were stored for 22 days in either double distilled water, 70% EtOH (diluted
from 100% with double distilled water), 70% EtOH (diluted from 100% with phosphate buff-
ered physiological saline solution), 100% EtOH, 4% phosphate buffered formaldehyde, 4%
unbuffered formaldehyde and saturated 40% unbuffered formaldehyde. After the storage
period, the different fluids were tested for the presence of Pb, Sb, Sn and As. All but the 70%
EtOH (PBS), 100% EtOH and the 4% buffered formaldehyde solution tested positive for the
presence of Pb, although only barely for the 70% EtOH sample (Fig 7A). All solutions apart
from the 100% EtOH solution tested positive for Sb (Fig 7A). Tin was only detected with cer-
tainty in the 40% unbuffered formaldehyde solution (Fig 7A). Arsenic was detected at low con-
centrations in the double distilled water and the 4% buffered formaldehyde solutions and at a
high concentration in the 70% EtOH (PBS) solution (Fig 7A).
Corrosion rate, measured as the mass of lost pellet material per surface area per day, was
highest when storing shot pellets in 4% unbuffered formaldehyde, followed by double distilled
water and 40% unbuffered formaldehyde with significantly lower corrosion rates in 70%
EtOH, 70% EtOH (PBS), 100% EtOH and 4% buffered formaldehyde (Fig 7B).
Based on corrosion rate, the extrapolated time it would take to completely corrode shot pel-
lets of the given size (3 mm in diameter) and mass (165.4 ±8.4 mg) was calculated. This ranged
from 5.1 years in 4% unbuffered formaldehyde to 64.6 years in 70% EtOH (Fig 7C).
Preserving beef samples containing a single lead pellet in various preservation fluids also
demonstrated differences in the formation of corrosion products over the course of 8.5 months
(Fig 7D and 7E). Whereas preservation directly in 70% EtOH or initial fixation in buffered 4%
formaldehyde followed by storage in 70% EtOH, which can be considered normal practice for
Fig 5. Museum specimens collected with the shotgun. (a) X-ray overview of small mammals in the fluid collection of The Natural History Museum of Denmark
containing shotgun pellets. Oana = Ornithorhynchus anatinus, Pray = Pteropus rayneri, Pper = Pseudocheirus peregrinus, Cfin = Callosciurus finlaysonii. Six 3 mm lead
pellets are shown (middle to the left) for comparison of size with foreign objects in the specimens; (b) Historical shotgun previously housed in the weapons collection of
the The Natural History Museum of Denmark. This weapon and the custom made adapter sleeve (red arrow) was used at the Danish Noona Dan Expedition to the Pacific
in 1961–62 and the Solomon Islands flying fox (Pray M05-CN2901) was most likely collected using this weapon.
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modern long-term preservation of biological specimens, and to a lesser degree continued stor-
age in 4% buffered formaldehyde only yielded a small volume of corrosion products within the
sample, contact with any unbuffered formaldehyde solution, even followed by transfer to 70%
EtOH resulted in much larger volumes of corrosion products (Fig 7D and 7E).
Radiodensity of lead shot pellets and the effect on measurements of bone
mineral content
The Hounsfield scale is a linear quantitative scale for describing radiodensity of CT scanned
objects and it is formally defined with distilled water having a CT number of 0 HU and atmo-
spheric air a CT number of -1000 HU and in the 16-bit version it ranges from -32768 to
Fig 6. Chemical analysis of preservation fluids for lead (Pb) and arsenic (As). (a) Analysis for the presence of Pb in 30x concentrated samples of
preservation fluids of mammal specimens (see Table 1 for full names and catalogue#) with (+) or without (-) hyperintense nodules on x-ray images. Some
specimens were stored in the same jars as indicated with multiple + or -. Both the lipid (L) and the water (W) phase was analysed but the presence of Pb
was not detected in any sample; (b) Analysis for the presence of As in 3.5x concentrated samples of preservation fluids (mixed lipid and water phase). In
all but two samples As was detected; (c) Arsenic concentration in different samples of preservation fluids. There was no significant difference between
[As] is samples with and without hyperintense nodules.
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Fig 7. Corrosion test of lead pellets in different solutions. (a) Analysis for the presence of lead (Pb), antimony (Sb),
tin (Sn) and arsenic (As) after storing lead pellets for 22 days in 1 ml of either double distilled water, 70% ethanol
(EtOH) with and without phosphate buffed saline (PBS), 100% EtOH, phosphate buffered 4% formaldehyde and
unbuffered 4% and 40% formaldehyde. All but the 70% EtOH (PBS), 100% EtOH and the buffered 4% formaldehyde
solutions tested positive for Pb. All solutions except the 100% EtOH tested positive for Sb. Tin was only detected in the
unbuffered 40% formaldehyde solution. The water, the buffered 4% formaldehyde and in particular the 70% EtOH
solution with PBS contained a detectable amount of As; (b) corrosion rate of lead pellets in different solutions. There
was significant difference between the corrosion rate in different solutions (one way ANOVA with post hoc Tukey
honest significance test). Significantly different groups are marked with different capital letters (A, B, C and D) i.e. a
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+32767 HU [27]. It follows that lipid rich tissues are slightly less radiodense than water (-120
to -90 HU), lean tissues are slightly more radiodense than water (0 to +100 HU) and mineral-
ised tissues like bone are even more radiodense (+300 to +1900 HU). However, the radioden-
sity of lead shot pellets surpasses the conventional Hounsfield scale (>32767 HU) (Fig 8A and
8B). Thus, to estimate the actual Hounsfield value of lead shot pellets, we conducted a profile
analysis of free lead pellets, taking advantage of the partial volume effect i.e. the fact that voxels
containing both pellet material and surrounding air have averaged Hounsfield values in the
measurable range, and thus the actual Hounsfield value of voxels only containing pellet mate-
rial can be estimated by extrapolating the profile (Fig 8B). The estimated Hounsfield value of
lead shot pellets at 120 keVp was 43584 ±4670 HU. By comparison to the size of the
4320 ±531 HU foreign objects found in the Dana platypus, this corresponds to the Dana platy-
pus having been shot with lead pellets (disregarding the small density effect of the trace metals
Sb, Sn and As within the alloy) with an average diameter of 2.6 ±0.7 mm equalling a shot size
of an English bird shot size #6 (2.59 mm in diameter).
The presence of very radiodense lead shot pellets is of importance when translating
acquired CT numbers to measurements of bone mineral density and bone mineral content
(BMC) using a calibration phantom with known concentrations of calcium hydroxyapatite.
Due to the much higher radiodensity of Pb than Ca (see Fig 3B), measurements of total BMC
in a specimen is strongly affected by the presence of metallic Pb and even more so of Pb salts.
To demonstrate this, we conducted an experiment on six fresh cadavers of laboratory rats.
These were first CT-scanned without any lead shot pellets, and then they were each injected
with six lead pellets (Fig 8C). Total BMC was calculated based on a calibration procedure to
translate CT numbers to bone mineral density for both the lead shot pellet free state, the state
with pellets, and a state in which pellets were digitally removed by enforcing an image thresh-
old of 1900 HU, resulting in most voxels with pellets being removed. Absolute BMC per ani-
mal was measured to 5.5 ±0.4 g in pellet free rats, 8.9 ±0.4 g in the same rats with pellets, and
5.8 ±0.4 g when pellets were digitally removed. This was significantly different between all
groups (One way ANOVA with repeated measures, F(2,10) = 3917.5, p <0.001 and post hoc
Tukey HSD for tests between each group all with p <0.001). Likewise, the body mass adjusted
BMC (BMC / body mass fraction) was also significantly different between each group (F(2,10)
= 1165.0, p <0.001 and post hoc Tukey HSD for tests between each group all with p <0.05)
(Fig 8D). Thus the presence of lead pellets caused a significant overestimation of BMC, and
although a digital removal of most voxels within lead shot pellets resulted in a reduction of
BMC overestimation (Fig 8D), both absolute BMC and body mass adjusted BMC were still sig-
nificantly overestimated due to partial volume effects of voxels in the border zone of lead shot
pellets that could not be digitally removed by thresholding without also potentially removing
actual bone containing voxels (Fig 8C, see magnification of digitally removed pellet in the
lower right image with remaining partial volume border zone).
Naturally, the effect of BMC overestimation due to encapsulated lead shot pellets is more
dominant in smaller species with a generally low BMC, and it becomes more apparent the
higher the number of pellets found in the individual. To demonstrate this, percentage error of
the total BMC of animals ranging in BMC from 1–100 g (representing species roughly the size
group labelled with A is significantly different from a groups labelled with B but not from one labelled with AC; (c)
Lead pellet mass over time at the corrosion rate of different solutions. The point at which all pellet material has been
corroded is marked with a circle in the respective line colours (or by an arrow in cases where circles are close); (d)
Virtual cross sections of beef cubes previously hosting a lead pellet (top rows) or maximum intensity projections (MIP,
bottom row). Varying degrees of corrosion products remain within the sample after 8.5 month of storage; (e) Volume
(V) of corrosion products within beef samples preserved in different preservation fluids.
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Fig 8. Determination of lead Hounsfield value and the effect on measurements of bone mineral content in animal
specimens. (a) Virtual section of 3 mm lead shot pellet from x-ray computed tomography (CT). Due to the high
radiodensity of the lead alloy, the pellet core exceeds the maximum Hounsfield value on conventional 16-bit CT
systems (from -32768 to +32767 HU) and actual radiodensity of lead pellets cannot be measured directly. However,
peripheral voxels suffer from partial volume effects from the averaging of signal contribution from lead and
surrounding air (-1000 HU); (b) By extrapolating the maximum slope of 12 longitudinal profile plots originating in air
and terminating at the core of six lead pellets, the partial volume effect can be exploited to indirectly reveal the average
radiodensity (43584 ±4670 HU) of lead pellets allowing for original pellet size calculations of foreign objects in the
Dana platypus; (c) Laboratory rat before (top) and after (middle) injection of six lead pellets, and after pellets have
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of laboratory mice to domestic cats) was calculated for 1, 5 and 10 pellets (Fig 8E). Since the
radiodensity of the metallic lead alloy found in shotgun pellets surpasses the maximum value
of the conventional Hounsfield scale (Fig 8B), the full effect of lead pellets on BMC measure-
ments cannot be observed when pellets are still in the metallic state, since most voxels values
within the shot pellet are capped by the maximum Hounsfield value. As lead pellets corrode,
however, radiodensity of the corrosion salts falls below the Hounsfield scale maximum, and
the full effect of encapsulated lead will act on the BMC measurement. To illustrate this point,
we calculated both the full effect (lead pellet Hounsfield value = 43584 HU) and the limited
effect (lead pellet Hounsfield value = 32767 HU) of 1, 5 and 10 lead shot pellets imbedded in
specimens with BMC in the range of 1–100 g (Fig 8E).
Discussion
Recently it was estimated that the wet biomass of terrestrial wild mammals (~22 Mt) currently
constitute about 2.1% of the biomass of all terrestrial mammals when including humans (~390
Mt) and livestock (~630 Mt) (Greenspoon et al. 2023). Of this, the majority of wild land mam-
mal biomass is found in large-bodied species (>10 kg) such as even-hoofed mammals with a
dominance of a few species of deer [28]. In this light, the fluid preserved small mammals in the
collections of natural history museums are as important as ever to document past small mam-
mal diversity [6]. However, for anatomical and pathological studies, preserved animals are only
as valuable as their collection and preservation record. In this study, we use the Dana platypus
to demonstrate a case in which limited information on how a sample was collected could easily
have led to erroneous conclusions. At first, the widely distributed large nodules in this specimen
bore no resemblance to projectiles (e.g. compare Figs 1B and 3A to Figs 5and 6of [29], and Fig
6in [30]). This indicated a pathological origin. Several pathologies could potentially explain
large and dense nodules (Table 2). Although the most obvious aetiology in general would be
tuberculosis, since our initial observation of nodules was in a platypus, mucormycosis (also
referred to as ulcerative dermatitis, mycotic granulomatous dermatitis and ulcerative mycotic
dermatitis was of special interest [31,32]. This disease caused by the fungus Mucor amphi-
biorum can cause severe granulomatous and ulcerative dermatitis in affected individuals and is
a leading cause of morbidity and mortality in wild platypus of Tasmania [30–33]. Although this
disease does not usually present with mineralised nodules, the appearance of subdermal and
muscular dense nodules in the Dana platypus seemed mostly in agreement with the clinical
description of the disease (Table 2). If this had been the case, it would be of significant interest,
since the Dana platypus was collected at least 53 years before the first reported observation of
mucormycosis in wild platypus in the austral autumn of 1982 [34].
However, after careful analysis we conclude that the nodules in the Dana platypus are for-
eign objects and not caused by a pathology. When inspected with CT and micro-CT the
been digitally removed by subtracting all voxels with CT values >1900 HU (bottom). To the left are maximum
intensity projections, and to the right are virtual sagittal sections. Red boxes on the right are 2.5x magnifications of a
pellet in the chest region; (d) Body mass adjusted bone mineral content (BMC) measured using quantitative CT on
laboratory rats before and after injection of lead pellets and after digital removal of these. The presence of lead pellets
increased body mass adjusted BMC significantly, and even after digital removal of lead pellets, the measurement was
significantly affected due to the partial volume voxels with CT-values of a little less than 1900 HU that were still present
in the images (see magnification in (c) lower right); (e) Percentage error (difference in estimated and actual value) of
total BMC as a function of total BMC and the presence of either 1, 5 or 10 lead pellets. Due to the CT-value threshold
of 32767 HU on most clinical CT systems, metallic lead alloys of lead shot pellets with CT values of 43584 ±4670 HU
do not express the maximum effect on BMC measurements. However, as lead pellets corrode and radiodensity of lead
salts fall below the CT-value threshold, the effect of lead pellets is increased as these corrode. This is demonstrated
using solid graph lines for the full effect of imbedded lead pellets and dashed lines for the limited effect when pellets are
still in the metallic state.
https://doi.org/10.1371/journal.pone.0309845.g008
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nodules appear more radiodense than most mineralised tissues (Fig 1), and the x-ray attenua-
tion spectrum is unlike that of calcified bone (Fig 3). Further, hyperspectral CT revealed a K-
edge at 88 keV matching the spectrum of lead (Fig 3G and 3H). A simple flame test revealed
that neither an entire nodule nor the core or crust material burned with similar flame colours
as the most abundant biometals, Ca, K, Na and Fe (Fig 2A), ruling out some form of naturally
occurring precipitation of biometal salts caused by a pathology (e.g., metastatic calcification)
or as a result of the preservation process. Instead, both core and crust material contained Pb,
Sb, Sn and As (Fig 2B–2D). These metals are uncommon in healthy biological tissues but have
for centuries been used in lead alloys for the manufacturing of lead shot pellets: Sb (0.5–6.5%
of alloy mass) increases the hardness of lead shot pellets, Sn (~0.1%) increases the malleability
of the pellet and reduces fragmentation and As (0.1–0.2%) facilitates the spherical formation of
pellets [35–37]. The finding of these four metals strongly suggests the nodules in the Dana
platypus to be foreign objects and specifically those of lead shot pellets. This was further sup-
ported when inspecting the head of the Dana platypus more closely. A shot pellet trapped in
the right cerebral hemisphere had left a trace of fragmented bone and pellet material along the
proposed shot trajectory (Fig 4A and 4B), and entry wounds were located at the furless bill
when the specimen was left to dry i.e. reducing reflections at the surface (Fig 4C). Finally, simi-
lar nodules as in the Dana platypus were observed in six out of the eight fluid preserved platy-
pus in the collection of the Natural History of Denmark collected between 1865 and 1950. In
itself, this does not rule out a previous pathology causing mineralised nodules, but it is none-
theless consistent with platypus being harvested by shooting up until 1941–1960 [38]. Also,
similar nodules were found in unrelated marsupial and placental mammals in the fluid pre-
served collection (Fig 5). One of these, the common ringtail possum Pseudocheirus peregrinus
cat# NHMD-M440, was also collected or donated in Australia during the fourth Dana expedi-
tion, and another one, the Solomons flying fox Pteropus rayneri cat# NHMD-CN2901, was
most certainly collected using a shotgun with a custom-made adapter sleeve (Fig 5B), during
the later Noona Dan Expedition. In conclusion, the overwhelming amount of evidence points
to the large and irregularly shaped nodules in the Dana platypus and the other inspected mam-
mal specimens being corroded shot pellets. In the Dana platypus, these are with certainty lead
shot pellets (Fig 3F–3H). Even though the potentially toxic effects of lead pellets on wildlife
were recognised as early as in the late 19
th
century [39,40], lead alloys were the most popular
materials for shot pellet manufacturing up until a few decades ago [41]. Accordingly, the pel-
lets found in the remainder of the studied specimens are most likely also lead shot pellets.
Although collection practises using firearms are well known, and often associated with high
collection outputs e.g. in the collection of birds, the use of shotguns can increase outputs by up
to 50% compared with the use of traps and nets (Hosner, P.A., Natural History Museum of
Denmark, personal communication), the potential effects of preserving specimens containing
shot pellets remain underreported. Apart from a brief mention in an ornithological protocol
paper of the preference of collecting bird specimens for fluid collections with other methods
than shooting since shot pellets can interfere with modern imaging techniques [42], and the
interesting discovery of very small lead pellets in the skull of the best preserved dodo specimen
in existence, the Oxford dodo [43], we have not been able to locate any other relevant literature
on this subject apart from a mention of the corrosion of metal tags in fluid preserved collec-
tions [3,44]. Veterinary forensic pathology, while an increasing speciality, deals usually with
recent projectile impacts with different characteristic appearance to the corroded tissue
embedded features described here [29], or lead shot or bullets as a toxicology hazard when
ingested, fragmented and absorbed in the gastrointestinal tract of wildlife (e.g. [22,45]. Apart
from the risk of misinterpreting corroded shots as pathologies there are two potential concerns
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when handling affected specimens and including these in comparative studies: The potential
risk of unknowingly handling toxic metals and the effect on quantitative measurements.
Both Pb and As are toxic metals, and both are recognised as elements of concern in
museum collections introduced via collection items such as lead objects and pigments or pre-
viously used preservation substances e.g. arsenic soaps [46–49]. The lead shot pellets found in
the Dana platypus and additional specimens were clearly no longer in the metallic state (Fig
1C and 1D), but consisted of corrosion products much larger than the original shot pellets
(Table 1 and Fig 5). The calculated average size of original shot pellets of 2.6 ±0.7 mm (English
bird shot size #6) in the Dana platypus seems to match a reasonable shot size for dispatching
an animal of that size, in particular since the large number of incorporated pellets indicates a
fairly short shot distance with a tight grouping. Thus, only a limited amount of pellet material
would be expected to have dissolved in the preservation fluid. In regards of Pb, this was also
the case, since no trace of Pb was detected in upconcentrated preservation fluids of any of the
included samples, demonstrating that the concentrations of Pb in the original samples
were <1.2 μM (Fig 6A). Even at high temperatures, water solubility of the lead salt which
made up the nodules was low (Fig 2E and 2F) indicating that the corrosion product did not
consist of lead(II) acetate or lead(II) formate, which have been suggested as common corrosion
products of lead in museum collection [50], nor a precipitation of lead(II) chloride after reac-
tion with widely present chloride ions in the tissue. Most likely the corroded lead pellets con-
sisted of another highly insoluble corrosion product such as e.g. lead(II) oxide, lead(IV) oxide,
lead(II) carbonate, lead (II) phosphate or a mixture. Small particles were encapsulated in the
fur of the entire specimen which could be observed with CT (S1 File) and micro-CT imaging
(Fig 3F, arrow). At first there was a concern that these particles could be precipitated Pb salts,
however hyperspectral CT with K-edge subtraction revealed no traces of Pb in these particles
(Fig 3F, arrow), which are more likely to consist of sand grains. In the case of As, traces of this
metal were found in preservation fluids of both specimens with and without shot pellets with
no significant differences (Fig 6C). This may be a result of As leakage from other sources e.g.
previously used arsenic soaps, or due to repeated use and mixing of preservation fluids
between samples over time. Although As exposure should be minimised using personal pro-
tection equipment such as nitrile gloves [45], even the highest concentration of 114.0 μg/l
found in the preservation fluid of Pteropus rayneri cat# NHMD- M05-CN2901 (Fig 6B and
6C), is well below the 500 μg/l threshold considered safe for bathing/showering in household
water in many places [51,52]. In summary, our analysis showed that handling biological speci-
mens containing corroded lead pellets does not directly expose collection staff to large concen-
trations of Pb and As, but care is warranted if performing dissection which could directly
expose encapsulated lead nodules.
Our results highlight that the preservation history of museum specimens may be of impor-
tance for the corrosion of embedded shot pellets. By measuring corrosion rate of lead shot pel-
lets, we found that solutions with a close to neutral or slightly alkaline pH (70% EtOH,
pH = 6.03; buffered 4% formaldehyde, pH = 6.84; 70% EtOH (PBS), pH = 8.61) or a water free
solution (100% EtOH) displayed a significantly lower rate of corrosion on lead pellets than
more acidic and unbuffered solutions (double distilled water, pH = 5.01; unbuffered 4% form-
aldehyde, pH = 3.97; unbuffered 40% formaldehyde, pH = 3.18) (Fig 7B). This translates to a
much faster corrosion of internalised lead pellets if specimens are stored in e.g. unbuffered 4%
formaldehyde than 70% EtOH (Fig 7C–7E). However, when approaching and surpassing cen-
turies of storage, lead pellets may eventually corrode fully (Fig 5A). It is of interest to note,
however, that no clear pattern of age and the degree of corrosion is evident in the studied spec-
imens e.g. compare Ornithorhynchus anatinus NHMD-M01-10 collected in 1865 and
Ornithorhynchus anatinus NHMD-M01-25 collected in 1915, both displaying large and highly
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corroded pellets, to Ornithorhynchus anatinus NHMD-M01-9 collected in 1865 with much
smaller pellets (Fig 5A). Likewise, the introduction of formaldehyde fixation following the dis-
covery in 1893 (the first reported use at the Natural History Museum of Denmark was in 1902
[53]), also does not seem to influence the corrosion state of pellets (Fig 5A). In opposition to
Pb, the corrosion test revealed that storing lead pellets in slightly alkaline 70% EtOH (PBS)
containing sodium phosphate salts resulted in a much higher concentration of As in the pres-
ervation fluid compared to other solutions (Fig 7A). It has been suggested to replace the pure
water content of widely used 70% EtOH in museum collections with more physiological rele-
vant salt solutions for long-term storage of fluid preserved specimens [21], however in cases of
specimens containing lead shot pellets our results demonstrate that caution is warranted, since
this could lead to increased solubility of As.
The other major concern of lead shot pellets in fluid preserved specimens comes into play
when using these specimens for quantitative imaging analyses of tissue components in com-
parative studies. In our experience, mineralised bony material is well-preserved in well-man-
aged fluid collections although some demineralisation can take place in unbuffered
preservation fluids over time. Thus, fluid preserved specimens are useful in studies of compar-
ative osteology. Important parameters when comparing skeletons are bone mineral density
and BMC. These parameters are directly linked to bone strength and for semi-aquatic and
fully aquatic species mineral content plays an important role in buoyancy regulation [11]. Tra-
ditionally, BMC is measured by ashing i.e. heating bone samples to >500˚C to burn off all
organic matter, but quantitative CT allows for a much faster and non-destructive method of
estimating both BMC in addition to density and volume of bones of interest [11]. However,
this method is sensitive to the presence of foreign objects with a much higher radiodensity
than calcified bones. In this study, we estimated radiodensity of lead shot pellets to
43584 ±4670 HU. This is ~23 times denser than the densest bones in the human organism,
and most likely even more so in smaller mammals considering allometric scaling of skeletal
strength in terrestrial mammals [54]. This means that, if overlooked, a single or just a few lead
shot pellets can have significant effect on the estimation of BMC in small mammals (Fig 8C–
8E). At increasing body sizes and bone mineral contents, this effect is decreased (Fig 8E), but
lead pellets are also easier to overlook in large specimens. If pellets are recognised and they
remain in the very radiodense metallic state, their effect on BMC can be alleviated to some
degree by applying a low pass image filter e.g. removing all pixels with Hounsfield
values >1900 HU. But due to the partial volume effect at the interface between pellets and sur-
rounding tissue, the BMC estimate will still be affected (Fig 8C and 8D). If pellets are corroded,
this task becomes even more difficult since the radiodensity of corrosion products of Pb is con-
siderably closer to the radiodensity of calcified bone (Fig 1G), making image filtering proce-
dures much more challenging or even unsuited. In these cases, affected specimens may be
excluded for analysis.
In summary, a coincidental observation of dense nodules in an old fluid preserved speci-
men of platypus of the mammal collection at the Natural History Museum of Denmark
inspired us to thoroughly examine the origin of these structures. This work led to an increased
understanding of the behaviour of corroding lead shot pellets during long-term preservation
of museum specimens, and potential caveats when using affected specimens to draw patho-
anatomical veterinary and osteology information. Most of the applied analysis methods are
simple and inexpensive, e.g. flame test and metal test papers and kits, and are applicable to test
samples for the presence of lead shot pellets at museums and research institutions throughout
the world. These simple methods can be complemented with more sophisticated methods
such as quantitative CT and hyperspectral CT based on availability. Of importance, we found
that leakage of toxic Pb and As from corroding lead shot pellets may not constitute a direct
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hazard when handling intact specimens and their preservation fluids. However, caution is war-
ranted especially if dissecting previously shot specimens, thus exposing toxic metal salts. In
such cases, and before undertaking large comparative analysis sensitive to the presence of lead
pellets, high throughput screening for residual foreign objects using e.g. digital x-ray imaging
should be considered. Further studies should investigate the fate of the steel, bismuth, and
tungsten pellets that have in some places replaced lead in shotgun pellets.
Supporting information
S1 File. Collection of photos, images, slice videos and virtual renders produced from CT
and MRI. This material was originally presented to highly trained research and zoo veterinari-
ans (authors CJAW, DS, AKOA and MFB) to form an opinion on what disease the hyperin-
tense nodules in the Dana platypus could originate from.
(ZIP)
Acknowledgments
We wish to thank the Department of Forensic Medicine, Aarhus University, specifically Pro-
fessor Lene Warner Thorup Boel for kindly providing access to the x-ray computed tomogra-
phy system. Likewise, we wish to thank the MR-research Center, Aarhus University,
specifically Professor Christoffer Laustsen for providing affordable access to magnetic reso-
nance imaging. We also wish to thank the Clinic for Osteoporosis, Hormone and Bone Dis-
eases, Aarhus University Hospital, specifically Lars Rejnmark and Tove Stenum for providing
access to the extremity CT system used to image nodules. Finally, we wish to thank Daniel
Kjær Andersen for help with the rat cadaver experiment.
Author Contributions
Conceptualization: Henrik Lauridsen, Daniel Klingberg Johansson,
Christina Carøe Ejlskov Pedersen, Kasper Hansen, Peter Rask Møller.
Data curation: Henrik Lauridsen.
Formal analysis: Henrik Lauridsen, Catherine Jane Alexandra Williams,
Ditte-Mari Sandgreen, Aage Kristian Olsen Alstrup, Mads Frost Bertelsen,
Peter Rask Møller.
Funding acquisition: Henrik Lauridsen, Peter Rask Møller.
Investigation: Henrik Lauridsen, Daniel Klingberg Johansson, Christina Carøe
Ejlskov Pedersen, Kasper Hansen, Michiel Krols, Kristian Murphy Gregersen,
Julie Nogel Jæger, Catherine Jane Alexandra Williams, Ditte-Mari Sandgreen,
Aage Kristian Olsen Alstrup, Mads Frost Bertelsen, Peter Rask Møller.
Methodology: Henrik Lauridsen, Daniel Klingberg Johansson, Michiel Krols,
Kristian Murphy Gregersen, Julie Nogel Jæger.
Project administration: Henrik Lauridsen.
Resources: Henrik Lauridsen, Peter Rask Møller.
Software: Henrik Lauridsen.
Supervision: Peter Rask Møller.
Validation: Henrik Lauridsen.
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Visualization: Henrik Lauridsen, Peter Rask Møller.
Writing – original draft: Henrik Lauridsen.
Writing – review & editing: Daniel Klingberg Johansson, Christina Carøe Ejlskov Pedersen,
Kasper Hansen, Michiel Krols, Kristian Murphy Gregersen, Julie Nogel Jæger,
Catherine Jane Alexandra Williams, Ditte-Mari Sandgreen, Aage Kristian Olsen Alstrup,
Mads Frost Bertelsen, Peter Rask Møller.
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PLOS ONE
The Dana platypus and corroding shotgun pellets in museum specimens
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