J Forensic Sci, November 2015, Vol. 60, No.

6
doi: 10.1111/1556-4029.12850
PAPER Available online at: onlinelibrary.wiley.com

PATHOLOGY/BIOLOGY; ANTHROPOLOGY

Elizabeth N. Celata1, M.Sc.

Postmortem Intervals in Mice Submerged in

Aqueous Environments at 20°C*

ABSTRACT: Aqueous submersion can impede decay and produce decomposition stages not seen in terrestrial burials. To further understand
the variances, fifty-four mice were submerged in freshwater, marine water, and a control environment at 20°C. The mice displayed sequential
stages at differing rates over 6 weeks. Regression plots and comparative t-tests demonstrated that internal putrefaction, weight difference, and
abdominal circumference of the aqueous environments varied significantly from the control group. The aqueous subjects did not vary signifi-
cantly from each other quantitatively. The postmortem intervals were not consistent regardless of temperature or environment although a clear
variance was noted between the control and the submerged groups.

KEYWORDS: forensic science, aqueous decomposition, liquefaction, skin slippage, bloating, mice

Aqueous environments offer a unique challenge in determin-
ing postmortem intervals (PMI). Submerged bodies are more
likely to have accelerated skin slippage and preservation of par-
ticular fatty tissues (1–5). However, freshwater and marine water
affect external and internal decomposition in different manners.
Freshwater often denatures body fluids, whereas marine water’s
salinity dilutes them (6). There are specimen-specific variables
which also influence decomposition in addition to environmental
factors. Bloating and liquefaction will be of interest due to fluid
absorption, skin slippage, and floating (7,8).
This study determined the extent and significance of weight
change and abdominal circumference in freshwater and marine
water submersion. To establish the variation in core temperature
between the three environments, each specimen had its tempera-
ture recorded prior to dissection. To determine the extent and
variation of liquefaction between the two aqueous environments
and the control group, each mouse was dissected in order to
observe the capacity to identify the organs over the 6 weeks. To
ascertain the differences in skin slippage rates between the three
groups, the skin was tested to determine the malleability or
observe whether sections had completely separated from the
body. To establish the rates at which each environment contrib-
uted to weight loss or gain during the decomposition process,
the weights of the mice were taken prior to and following sub-
mersion in their assigned environment. The abdominal circum-
ferences were taken pre- and postsubmersion to ascertain the
affects of each environment.
1
Monroe County Office of the Medical Examiner, 740 East Henrietta Rd,
Rochester, NY 14623.
*Funding provided by Bournemouth University. Presented at the 15th
Annual Conference of the British Association for Biological Anthropology
and Osteoarchaeology BABAO, September 13–15, 2013, in York, U.K
Received 14 April 2014; and in revised form 6 Sept. 2014; accepted 6
Nov. 2014.
The control group should reach skeletonization within fewer
weeks than either aqueous environment if any of the three reach
that point during the 6-week experiment due to the shortened
bloating phase. The comparison of the three environments will
provide an improved understanding of submersion’s effect on
decomposition rates.
Methods
Following an 8-day pilot study in January 2013, a 6-week
decomposition experiment was undertaken utilizing an incubator to
maintain a controlled environment with 12 day hours and 12 night
hours at 20°C. The fifty-four mice acquired for the experiment
were divided into three groups of eighteen. The mice were labeled
E1-E54 with E1-E18 in the control group, E19-E36 in freshwater,
and E37-E54 in marine. One group was placed into airtight plastic
containers on their own to function as a control group. The second
group was placed in airtight plastic containers lined with Osnaburg
linen over which 400 mL of freshwater was poured. The final
group of eighteen mice were placed in airtight plastic containers
lined with Osnaburg linen over which 400 mL of marine water was
added. The abdominal circumference and weight of the mice were
recorded prior to insertion into the assigned environment. The con-
tainers were stacked in columns of three in lines of three and a row
of six on a middle shelf of the incubator.
Mice were used rather than pigs to increase environmental
control and sample size. Additionally, the use of pig and human
specimen are regulated by the Department for Environment,
Food and Rural Affairs and the Horticultural Trades Association.
Mice were a suitable alternative to humans with similar skin per-
meability, biochemical, cellular, and physiological pathways
(9,10). The availability and size permitted a sample size of fifty-
four within a relatively small space without causing risk to other
experiments or researchers utilizing the same facilities. The mice
supplied were killed prior to the experiment and frozen (11). As
all specimens had been frozen including the control group, the

© 2015 American Academy of Forensic Sciences 1495

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experiment was still useful in determining the difference in

decomposition rates between the three environments.
Every 7 days, three specimens of each treatment were
removed for analysis over the 6 weeks (11,12). Buoyancy was
observed visually prior to the removal of the specimen. Weight
was taken to the hundredth of a gram. The temperature of the
specimens was taken orally (13–15). The abdominal circumfer-
ence of each specimen was measured to determine the extent of
bloat in centimeters (8). Skin slippage was rated as “none,”
“mild,” and “extreme.” “None” was observed when skin slippage
was not visible on a specimen. “Mild” described slight skin slip-
page, wherein the majority of the skin remained attached to the
specimen. “Extreme” occurred when large sheets of skin were
no longer attached to the specimen. Liquefaction was taken as a
measurement of internal decomposition (8). The scale focused
on the general visibility and identifiability of the organs. The liq-
uefaction rates were measured as organs discernible, intestines
visible, and indistinguishable.
Regression formulae were used to determine whether a line of
best fit could be estimated to predict the progression of decom- FIG. 1––Scatter plot of control group core temperature.
position (1). R-squared values of 0.95–1.00 were considered sig-
nificant. Normal distributions were confirmed via one-sample
Kolmogorov–Smirnov tests, and with a confirmed distribution,
parametric tests, specifically t-tests, were determined appropriate
to perform on the available data. Correlating equations were
tested to examine the possibility of a predicting progression line.
The extent and time of liquefaction, buoyancy, and skin slippage
were noted and compared between the two aqueous environ-
ments. All data were compared to a control environment to
examine the variance and the significance of that variance.

Results
Skin Slippage
All but four aqueous samples had extreme skin slippage. The
three freshwater that were not extreme presented in the third and
fourth week without fluid retention. The fourth was a marine sam-
ple that was the single mild skin slippage within the fourth week
of testing. None of the control specimens had skin slippage.

Core Temperature
Core temperature was charted through the oral cavity. The
control and freshwater samples fell within the same range 18–
22°C although the control had a spike in the fifth week (Fig. 1).
While control and freshwater appeared to have a total gradual
increase, the samples submerged in marine had an overall
decrease in temperature (Fig. 2). None of the three environments
had significant regression results.
Liquefaction
Internal decomposition was faster in the control group with
the majority of specimens having unidentifiable organs by the
second week, whereas the freshwater specimens took 3 weeks
and the marine took until the fifth and sixth week (Fig. 3). The
process of liquefaction was followed through the graying then
blackening of the organs in the submerged specimens. The con-
trol group’s abdominal cavity flattened and became indistin-
guishable around the fourth week. Marine water had the slowest
rate of liquefaction overall with a third of the specimens still
having visible intestines in the final 2 weeks.
FIG. 2––Scatter plot with regression lines for marine core temperatures.
Abdominal Circumference
Abdominal circumference measurements were compared to
those taken prior to submersion to calculate the absolute differ-
ence of abdominal circumference. Increases and decreases were
taken into account for comparisons between absolute differences
and difference including negative when the postsubmersion
circumference was greater than the presubmersion circumfer-
ence.
The abdominal circumference of the control group steadily
and significantly decreased over time (Fig. 4). The freshwater
and marine water specimens also had a significant variance
between pre- and postsubmersion; however, the aqueous speci-
mens’ circumferences increased. The spread of the marine
circumference differences began wide before narrowing toward
1.00 (Fig. 5). Neither control nor freshwater narrowed around
a particular point. The two aqueous environments differed
significantly from the control; however, the two were not signifi-
cantly different from each other.

FIG. 3––Bar charts with the stages of liquefaction per week in the three environments.
FIG. 4––Scatter plot with regression lines for control abdominal circum-
ferences.
Weight
The control specimens decreased in weight. There were no
negative values permitting for singular testing performed on all
CELATA . PMI IN SUBMERGED SPECIMENS 1497
FIG. 5––Scatter plot with regression lines of the marine abdominal cir-
cumference percentages.
positive weight differences. The correlation between duration
within the environment and the weight difference was stronger
than that of circumference difference. The positive relation
between time and weight difference indicated the weight was
decreasing significantly while the time increased (Fig. 6).
1498 JOURNAL OF FORENSIC SCIENCES
FIG. 6––Scatter plot with regression lines of the weight differences in the
control group.
The weight of the freshwater specimens increased during the
first 2 weeks due to water absorption via passive intake into the
stomach and lungs. Specimens removed in later weeks had a
smaller difference than earlier specimens in weight pre- and
postsubmersion with the freshwater specimens having significant
variance when compared as absolute; however, the differences
wherein negative values were included were not significant.
The specimens submerged in marine water varied significantly
before and after submersion in both absolute differences and
those in which negative values were maintained. When all three
environments were compared, the two aqueous environments dif-
fered significantly from the control; however, the two did not
vary significantly from each other.
Discussion
The stages observed in the control, freshwater and marine water
environments were sequential for individual specimens passing
through observed phases (14,16–18). The control group went
through a more internal decomposition process: fresh, minimal
bloat in the first few days, active decay, advanced decay. Due to
their environment and the resulting water absorption, bloating was
predictably greater in the aqueous environments as noted with
weight and circumference differences. There was a distinct differ-
ence in one particular stage that submerged bodies went through
in both waters: gelatinous decay. Although a current likely would
have prevented this particular stage, a still body of water could
produce this form of external and internal viscosity. The freshwa-
ter entered into this stage within the fifth and sixth week prevent-
ing circumference measurements. The skin of these specimens had
completely slipped off and floated elsewhere in the containers.
The marine water sample had a similar state in the sixth week;
however, the marine gelatinous mice were more malleable.
At 20°C, the control group was in a different stage of decay
with dried epidermis leaving external features visible. The con-
trol specimens had liquefied leathery internal organ during the
later weeks although they were externally decaying at a more
gradual pace. The submerged bodies were not more or less
decayed than the control although previous arguments suggest
submersion slows decomposition (6). The higher salinity water
showed greater signs of slowed internal decay with delayed
onset of liquefaction than the freshwater environment. Humph-
reys’ freshwater reservoir experiment was confounded due to an
increase in algae presence as a result of the insertion of carcasses
which was prevented in this experiment with a more carefully
controlled environment (11). Extraneous factors were further
limited by taking three specimens per week per group instead of
removing a single specimen for pathological analysis as in
Humphreys’ study (11).
Submersion in either type of aqueous environment produces
increased skin slippage. The samples submerged in marine water
had a greater amount of slippage than freshwater. Although the
categories of skin slippage were divided into defined sections,
the placement of each sample would be subjective. Evaporation
likely influenced the skin slippage on the five specimens that
were waterless when tested. A single environment experiment
working on a singular shelf to determine the variation between
different placements would be the best method to determine the
extent to which incubator placement influenced the decomposi-
tion process.
Weight change either increased or decreased overall; however,
there was not a strong enough pattern to have a significant line
of best fit. The same was true of abdominal circumference
although there was a significant difference in both cases between
the aqueous environments and control. Experiments observed
over longer periods of time indicate that the somewhat erratic
rise and fall was common in submerged decomposition (19).
Fitzgerald suggested that decomposition stages are not sequential
(1). Contrary to this claim, the weekly tested mice progressed in
such a way that while each specimen did not pass through the
same stage at exactly the same time, the different environments
each had stages that all specimens passed through suggesting a
sequential decomposition progression (14,17,19–21).
Although it might be desirable to simplify decomposition into
few stages to cover a greater number of variables, this would
not satisfy the multitude of varying substages that result from
particular environments. Four stages denoting fresh, bloat, putre-
faction, and putrid dry may cover the overall reality of decompo-
sition; however, putrefaction can ultimately be skipped in an
environment similar to the control which had a short period of
bloat with an extended period of putrid dry. Oppositely, the
gelatinous form which might be considered under putrefaction in
the freshwater and marine water sample lasted longer with little
likelihood of putrid dry.
As a comparison of postmortem intervals, the samples were
frozen and thawed for the same period of time. The experiment,
therefore, focused on frozen bodies thawed prior to insertion into
the particular environment. It was not intended as an attempt to
replicate homicide cases but to improve understanding of decom-
position as affected by submersion including whether water type
affects the process differently. The lack of significant variance
between marine and freshwater suggests that further studies
would have to either have higher temperatures or extend over
the 6-week timeline to show a greater difference between the
two. The collected data demonstrated that submersion alters the
rates and stages of decay in mammals.
Acknowledgments
The author thanks Dr. Karina Gerdau-Radonic for supervising
the MSc thesis from which this article was based.
References
1. Fitzgerald CM, Oxenham M. Modelling time-since-death in Australian
temperate conditions. Aust J Forensic Sci 2009;41(1):27–41.

2. Baccino E, Cattaneo C, Jouineau C, Poudoulec J, Martrille L. Cooling
rates of the ear and brain in pig heads submerged in water – implications
for post-mortem interval estimation of cadavers found in still water. Am
J Forensic Med Pathol 2007;28(1):80–5.
3. Goff ML. Early post-mortem changes and stages of decomposition in
exposed cadavers. Exp Appl Acarol 2009;49(1/2):21.
4. Cotton GE, Aufderheide AC, Goldschmidt VG. Preservation of human
tissue immersed for five years in fresh water of known temperature. J
Forensic Sci 1987;32(4):1125–30.
5. Notter SJ, Stuart BH, Rowe R, Langlois N. The initial changes of fat
deposits during the decomposition of human and pig remains. J Forensic
Sci 2009;54(1):195–201.
6. Di Maio DJ, Di Maio VJM. Death by drowning. In: Di Maio DJ, Di
Maio VJM, editors. Forensic pathology, 2nd edn. Boca Raton, FL: CRC
Press, 2001;399–408.
7. Pickering RB, Bachman DC. The use of forensic anthropology, 2nd edn.
Boca Raton, FL: CRC Press, 2009.
8. Stejskal SM. Death, decomposition, and detector dogs: from science to
scene. Boca Raton, FL: CRC Press, 2013.
9. Lee JJ, Jacobsen EA, Ochkur SI, Mcgarry MP, Condjella RM, Doyle
AD, et al. Human versus mouse eosinophils: “that which we call an
eosinophil, by any other name would stain as red”. J Allergy Clin Immu-
nol 2012;130(3):572.
10. Takeuchi H, Mano Y, Terasaka S, Sakurai T, Furuya A, Urano H, et al.
Usefulness of rat skin as a substitute for human skin in the In vitro skin
permeation study. Exp Anim 2011;60(4):373–84.
11. Humphreys MK, Panacek E, Green W, Albers E. Comparison of proto-
cols for measuring and calculating post-mortem submersion intervals for
human analogues in freshwater. J Forensic Sci 2013;58(2):513–7.
12. Davis JB. Lee Goff M. Decomposition patterns in terrestrial and inter-
tidal habitats on O’ahu Island and Coconut Island, Hawai’i. J Forensic
Sci 2000;45(4):836–42.
13. Honjyo K, Yonemitsu K, Tsunenari S. Estimation of early post-mortem
intervals by a multiple regression analysis using rectal temperature and
non-temperature based post-mortem changes. J Clin Forensic Med
2005;12(5):249–53.
14. Komar DA, Buikstra JE. Forensic anthropology: contemporary theory
and practice. Oxford: Oxford University Press, 2008.
15. Henssge C, Knight B. The estimation of the time since death in the early
post-mortem period. London, UK: Arnold, 1994.
16. Heaton V, Moffatt C, Simmons T, Lagden A. Predicting the post-mortem
submersion interval for human remains recovered from U.K. waterways.
J Forensic Sci 2010;55(2):302–7.
CELATA . PMI IN SUBMERGED SPECIMENS 1499
17. Gunn A. Essential forensic biology, 2nd edn. Oxford, UK: Wiley-Black-
well, 2009.
18. Klepinger LL. Fundamentals of forensic anthropology. Hoboken, NJ: Wi-
ley, 2006.
19. Statheropoulos M, Agapiou A, Zorba E, Mikedi K, Karma S, Pallis GC,
et al. Combined chemical and optical methods for monitoring the early
decay stages of Surrogate human models. Forensic Sci Int 2011;210(1–
3):154–63.
20. Zhou C, Byard RW. Factors and processes causing accelerated decompo-
sition in human cadavers – an overview. J Forensic Leg Med 2011;18
(1):6–9.
21. Pakosh CM, Rogers TL. Soft tissue decomposition of submerged, dis-
membered pig limbs enclosed in plastic bags. J Forensic Sci 2009;54
(6):1223–8.
Additional information and reprint requests:
Elizabeth N. Celata, M.Sc.
Monroe County Office of the Medical Examiner
740 East Henrietta Rd
Rochester, NY 14623
E-mail: celatae@gmail.com
Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Table S1. Independent sample t-test comparing the control
and freshwater specimens’ abdominal circumference differences.
Table S2. Independent sample t-test comparing the control
and marine specimens’ abdominal circumference differences.
Table S3. Independent sample t-test comparing the marine
and freshwater specimens’ abdominal circumference differences.
Table S4. Independent sample t-test between the control and
freshwater groups’ weight.
Table S5. Independent sample t-test between the control and
marine groups’ weight.
Table S6. Independent sample t-test between the freshwater
and marine groups’ weight.
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