Journal of Forensic and Legal Medicine 41 (2016) 58e64

Contents lists available at ScienceDirect

Journal of Forensic and Legal Medicine

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j fl m

Original communication

Contribution to the determination of the place of death by drowning e

A study of diatoms' biodiversity in Douro river estuary

udia Ribeiro c, Joana Marques d,
Sara Coelho a, Patrícia Ramos b, Cla
Agostinho Santos a, c, e, f, *
a ^ni Monteiro, 4200-319, Porto, Portugal
Faculty of Medicine, Porto University, Al. Prof. Herna
b
LAQV/REQUIMTE, Department of Chemical Sciences, Laboratory of Applied Chemistry, Faculty of Pharmacy, Porto University, Rua de Jorge Viterbo Ferreira
228, 4050-313, Porto, Portugal
c
National Institute of Legal Medicine and Forensic Sciences, North Branch, Jardim Carrilho Videira, 4050-167, Porto, Portugal
d
CIBIO, Center for Investigation of Biodiversity and Genetic Resources, Campus Agra
rio de Vaira
~o, 4485-661, Vaira
~o, Portugal
e
CENCIFOR e Forensic Science Center, Largo da S
e Nova, s/n, 3000-213, Coimbra, Portugal
f
School of Health Sciences, Minho University, Campus Gualtar, 4710-057, Braga, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: The role of the investigation of diatoms' presence in organs and body fluids of an individual found dead
Received 3 December 2015 in a liquid medium and the relevant contribution to the forensic diagnosis of drowning remain
Received in revised form controversial. Furthermore, the absence of an exact and well-defined method for diatoms' analysis makes
5 March 2016
its study a challenging task.
Accepted 1 April 2016
Available online 9 April 2016
Considering this medico-legal problem and the absence of forensic studies on this subject in Portugal,
this work aimed to determine the drowning place of dead individuals based on the analysis of diatom
species found in different tissues (lung, liver, kidney, bone marrow) and stomach content. Diatom species
Keywords:
Diatoms test
found in biological samples were compared with those present in the liquid medium where the corpses
Drowning were found. A total of 37 cases of death by drowning in Oporto metropolitan area were studied. A
Place of death determination seasonal database of the diatom species found in Douro river estuary was built based on water samples
Forensic investigation collected at nine selected places. Diatoms' extractions were performed by a chemical method using 37%
Douro river estuary (w/w) hydrochloridric acid for the biological samples and 96% (w/w) sulfuric acid for water samples.
Diatoms were found in 63% of total cases but only in lung and gastric content samples. The absence of
diatoms in other organs is probably related with a quick death, which may have stopped blood circu-
lation almost immediately, preventing diatom contamination of the other organs.
A strong relationship between the diatom species found in the biological samples and those found in
water samples of the respective drowning place was observed. Due to the high anthropogenic influence
on the Douro estuary no significant differences were observed between the five sampling places, making
it extremely difficult to determine the exact estuary location of the drowning.
The importance of the creation of a diatom database of the potential drowning places (e.g., rivers, seas,
lakes) becomes clear in this study. It also shows that, in cases of drowning, the collection of a water
sample from the drowning place is crucial. This is the only way to allow a rigorous comparison of the
diatom species in water and biological samples.
© 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

1. Introduction
From a forensic point of view, the diagnosis of drowning is a
complex task and requires several complementary diagnostic tests.
Determining whether a corpse found in a liquid medium died as a
* Corresponding author. National Institute of Legal Medicine and Forensic Sci-
ences, North Branch, Jardim Carrilho Videira, 4050-167, Porto, Portugal. Tel.: þ351
222 073 850; fax: þ351 223 325 931.
E-mail address: asantosinml@yahoo.com (A. Santos).
result of drowning or not is a fairly simple task but it is not always
that easy to fully explain what actually happened.
Drowning can be defined as a total submersion of the body or a
partial submersion of the respiratory orifices in a liquid medium.1,2
The drowning often occurs in water, which may be fresh, brackish
or salt water. The pathophysiology of drowning is specific and de-
pends on unique characteristics of the fluid in which the individual
is submerged.

http://dx.doi.org/10.1016/j.jflm.2016.04.007
1752-928X/© 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
 S. Coelho et al. / Journal of Forensic and Legal Medicine 41 (2016) 58e64
A drowning in fresh water, which is a hypotonic medium rela-
tive to plasma, causes hypervolemia, hemodilution with reduced
serum electrolytes, such as sodium (Na) and chlorine (Cl). Hemo-
lysis occurs, leading to release of potassium (K). The increase in the
plasma K concentration promotes fatal arrhythmias, namely ven-
tricular fibrillation. In seawater, which is hypertonic relative to
plasma, it causes an increase in osmotic gradient, thus increasing
the entry of fluid into the alveoli, hypovolemia and hemoconcen-
tration occur with increasing concentration of ions such as Na, Cl
and magnesium (Mg). Since the oxygen regulation did not occurs,
there is hypoxemia, hypercapnia and respiratory and metabolic
acidosis. Pulmonary edema appears within minutes. Cardiac
dysfunction occurs subsequently to changes in arterial pressure
and acid-base balance.3e5
The purpose of a forensic investigation in cases of drowning
focuses on answering the following questions: the identification of
the victim, the estimation of the postmortem submersion period,
determination of the cause and mechanism of death and, finally,
understand if the drowning was vital or not, i.e., if the person was
dead or alive when entering into the water. Diatoms detection
could be interpreted as an unequivocal sign of drowning due to
inhalation of these microorganisms present in almost all aquatic
environments.4,6
Diatoms are unicellular photosynthetic eukaryotic algae. These
organisms are established in a location with regard to the levels of
nutrients, light intensity, mineral composition, temperature and
depth. Changes in seasonality are an important factor leading to
changes in the diatoms' distribution. Winter presents a number of
factors (e.g., short days, low temperature, low light intensity to
penetrate the water column) that cause a decrease in the diversity
of diatoms.6e8 Diatoms can be benthonic (substrates) or planktonic
(water column).7 Diatoms have a characteristic exterior siliceous
skeleton, called frustules formed by two overlapping valves. The
exterior valve is called epitheca, this in turn engages an inner valve
called a hypotheca.9,10 Due to the reduced dimensions of these
organisms, they can pass through pulmonary-alveoli capillaries
through circulation and reach to the different organs of the body.4
Several authors found diatoms in lungs, liver, kidney, bone marrow
and stomach.11e14
Besides taking special care to avoid contamination of material,
reagents, workplace and samples, several other conditions should
be fulfilled in order to consider the diatoms test as reliable and
accepted by the scientific community, namely a significant number
of diatom counts, which according to Sidari et al.15 and Ludes et al.16
should reach at least 20 diatoms (or 5 complete frustules8,15,16) per
100 mL of pellet obtained from 10 g of sample. The chosen method
for sample digestion should guarantee a good quantitative (diatoms
density) and qualitative (number of species) analysis.17 For a full
taxonomic analysis, it is important to develop a comparative study
of diatoms recovered from the tissues/organs of the drowned body
and the diatoms present in the water where the body was
found.5,13,16
The main goal of this study is to contribute to the identification
of the site of drowning in the Douro river estuary through the
comparative study of diatoms in real cases of drowning and those
found in liquid medium.
2. Materials and methods
2.1. Area of study and water samples collection
Water samples (n ¼ 36) were collected based on the most
frequent locations of accidental or suicidal deaths by drowning
followed by forensic autopsy in National Institute of Legal Medicine
and Forensic Sciences, North Branch, Portugal (INMLCF-DN) since
59
October 2012 to June 2013. The specific sampling locations are
signaled in the map (Fig. 1). Samples were collected in a 250 mL
sterile plastic container (DELTALAB, Spain.) and protected with
aluminum foil until further treatment, as described by Taylor et al.9
2.2. Laboratory treatment of water samples
Diatoms were extracted from water samples according to a
protocol developed elsewhere.19 Four replicas for each location
were performed. Samples were centrifuged in a Heraeus-Labofuge
III centrifuge at 3000 rpm during 15 min (Thermo Scientific, USA).
The supernatant was discarded and 20 mL of sulfuric acid at 96%
(Pronalab, Portugal) were added and left at room temperature for
24 h. Successive washes with diatom-free distilled water were
performed with centrifugation at 3000 rpm for 15 min until the
supernatant become completely translucent.
2.3. Biological samples
A total of 37 cases of death by drowning followed by forensic
autopsy in INMLCF-DN between November of 2012 and March of
2014 were studied. Sample collection procedures were identical of
those described by Hurlimann et al.12 All the autopsy tools were
previously washed with a 2 N NaOH solution and special care was
taken to prevent sample contamination.11 Samples from the right
lower and left upper lobes of lung, left side of liver and kidney
(approximately 200 g each), bone marrow (10 mL) and stomach
content (25 mL) were collected in a 250 mL sterile plastic container.
Samples were stored at 10  C until further treatment.
2.4. Laboratory treatment of biological samples
Samples were chopped after defrosting at room temperature
and digested with 20 mL of 37% hydrochloric acid (HCl) in a hot
water bath for 15e20 min accordingly to procedures described by
other authors.17,20 Upon complete tissue digestion, samples were
transferred to centrifuge tubes (DELTALAB, Spain) and centrifuged
at 3000 rpm for 15 min. The supernatant was discarded and the
pellet was washed successively with diatom-free distilled water
until the supernatant become completely translucent.
2.5. Microscopic observation
Naphrax™ (Brunel Microscopes, UK) was the mounting medium
used for permanent slide preparation. Slides were observed on an
optic microscope (Leica DM2500, Portugal), equipped with an im-
mersion lens allowing a magnification of 1000. Observation was
made in a zig zag way to avoid counting the same diatom twice. For
water samples, the number of slides observed ranged up to 300
complete diatoms per sample location.19 Regarding body samples,
the number of slides observed depended on the number of diatoms
founded. An observation of, at least, twenty in the lungs and five
complete diatoms in the other organs was required.16
Note: All reagents and instruments were checked for
contamination.
2.6. Taxonomic determination
Specialized bibliography was used to classify diatom species:
literature21e26 and online databases27e29 regarding diatoms found
in fresh, brackish and salt water environments were consulted.
60 S. Coelho et al. / Journal of Forensic and Legal Medicine 41 (2016) 58e64
Fig. 1. Map of Douro river estuary with target places of water sample collection.18 1: Luz beach; 2: Delta of the estuary; 3: Arra
Arnelas beach; 7: Above Crestuma dam; 8: Po
voa de Varzim beach; 9: Titan beach in Matosinhos.
2.7. Statistical analysis
Multivariate data was analyzed by means of Detrended Corre-
spondence Analysis (DCA) in Canoco for Windows version 4.5
(Microcomputer Power, USA).
3. Results
Forty four genera and 223 species of diatoms were found in
water samples collected along Douro river estuary. Results of DCA
regarding the relationship between the sampling locations and the
diatom species are showed in Fig. 2. Diatom species found in
samples from the estuary locations were very similar. The locations
2, 3, 4 and 7 have high concentration of Nitzschia palea and Aula-
coseira granulata species. Gomphonema parvulum is the most
abundant species in the Ferreira river. Water samples from location
S5 and S6 were rich in Cyclostephanos dubius and Achnanthes
minutissima, and Cyclotella meneghiniana, respectively. High con-
centrations of Cocconeis placentula, Cocconeis neothumensis and
Achnanthes subatomoides species were found in the marine loca-
tions (S1, S8, S9).
Information regarding the 37 cases of death by drowning fol-
lowed by forensic autopsy in INMLCF-DN between November of
2012 and March of 2014 are summarized in Table 1. Diatoms were
found in 63% of these cases. Traumatic injuries were the cause of
death of 9 cases (24.3%). Although samples of lung, liver, kidney,
bone marrow and stomach content were analyzed, diatoms were
found only in lungs and stomach samples.
Corpses regarding cases C1, C15, C17, C18 and C21 were found in
estuarine locations. The drowning of the individual regarding case
C1 occurred in location 3 (Arr
abida bridge). In Fig. 3(d) it is shown
that Fragilaria fasciculata is a diatom specie characteristic of this
location in the autumn. The drowning of the individual regarding
case C15 occurred in location 4 (Luís I bridge) and C17 and C18 in
location 5 (Areinho beach). C. dubius, Fragilaria crotonensis, Cyclo-
tella species and Achnanthes lanceolata characterize these three

bida bridge; 4: Luís I bridge; 5: Areinho beach; 6:
cases and seem to be characteristic of estuaries samples. The
drowning of the individual regarding case C21 occurred near the
location 4 but diatom species found in tissue samples were not
correlated either with this or any other location.
The cases C9 and C28 refer to deaths by drowning in the river.
According to Fig. 3(a), diatoms' profile found in organ samples of
the individual of case C9 is very similar to those found in water from
marine samples and high levels of A. minutissima were found.
Although it is shown in Fig. 4 that diatoms' profile found in this case
is very similar to case C21 (a drowning occurred in the estuary), the
most abundant species in case C9 is A. lanceolata. Both species are
typical from fresh water. The main diatom species found in organ
samples of case C28 were Cysclotephanos dubius and Achnanthes
subatomoide (Fig. 4).
Death by drowning of individuals of cases C33 and C11
occurred at seawaters. In Fig. 3(b) it can be observed that case C11
is characterized by the presence of C. dubius. In Fig. 4 this case is
characterized by presence of C. meneghiniana, Aulacoseira distans
and C. placentula. In Fig. 3(a) it can be observed that the diatom
species found in tissue samples of the individual are the same to
those present in marine locations (A. minutissima and C.
placentula).
The drowning of the individual regarding case C13 occurred in a
lake from a private propriety. The comparison of diatom's profile
found in tissue and water samples allowed establishing, without a
doubt, that this lake was indeed the drowning location. In Fig. 3(b)
it shows that Fragilaria elliptica was found in tissue samples of the
drowned individual and water samples from this lake.
Tissue samples from drowned individual regarding case C12 are
characterized by high concentration of C. dubius (Fig. 3(b)) but it is
impossible to specify undoubtedly the drowning location. Besides,
in Fig. 4 it is clear that tissue samples from this individual have the
same diatom species as other drowning cases occurred in brackish
waters and in water samples taken from the estuaries. The body of
the individual regarding case C19 was recovered from Ferreira
River. In Fig. 4 it can be observed that diatom species found in this
 S. Coelho et al. / Journal of Forensic and Legal Medicine 41 (2016) 58e64 61

Fig. 2. DCA analysis of the relationship between the sampling sites and diatom species. Key: Red line circles: marine samples; black circles: estuarine samples; green rhombs: river
samples; empty circle: diatom species. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1
Summary of the 37 drowning cases followed by forensic autopsy in INMLCF-DN between November 2012 and March 2014.

Case study Diatoms total counting/N species/N genus Discovery site Season Differential diagnosis of death Putrefaction Cause of death

1 36/9/9 3 A S H AS
2 224/17/10 Uk A Uk N AS
3 0/0/0 8 W S H AS
4 0/0/0 9 W Uk H AS
5 0/0/0 4 W S H TI
6 0/0/0 3 W S H TI
7 0/0/0 8 W S H AS
8 49/23/16 9 W Uk N Uk
9 15/9/6 10 W S N AS
10 0/0/0 3 W S H TI
11 91/23/13 1 Sp A N AS
12 52/11/7 5 Sp S N TI
13 99/10/4 11 Sp A N AS
14 0/0/0 1 Sp A N AS
15 38/7/6 4 Su A N AS
16 0/0/0 3 Su S N TI
17 183/28/14 5 Su Uk N AS
18 74/15/9 5 Su A N AS
19 26/7/6 10 Su S N TI
20 108/16/10 5 Su S H Uk
21 61/18/14 4 Su A N AS
22 0/0/0 8 Su Uk H AS
23 0/0/0 3 A S N TI
24 70/22/15 Uk A S N AS
25 0/0/0 10 A A N TI
26 10/6/5 10 A Uk H Uk
27 0/0/0 4 A Uk H AS
28 102/13/6 10 A S H AS
29 5/2/2 Uk W S N AS
30 13/7/7 Uk W S H AS
31 5/3/2 Uk W Uk N AS
32 30/13/12 Uk W A N AS
33 40/17/14 8 W Uk N AS
34 0/0/0 4 W S N TI
35 11/7/6 Uk W A N AS
36 0/0/0 10 W A N AS
37 3/3/3 10 W Uk N AS

Discovery site: 1: Luz beach; 2: Delta of the of estuary; 3: Downstream Arra
bida bridge; 4: Downstream Luis I bridge; 5: Areinho beach; 6: Arnelas beach; 7: upstream
Crestuma dam; 8: Po
voa de Varzim beach; 9: Titan beach in Matosinhos; 10: Ferreira River; 11: Private lake in Arcozelo; Uk: unknown; Season: W: Winter; Sp: Spring; Su:
Summer; A: autumn; Differential diagnosis of death: S: suicide; H: homicide, A: accident; Uk; Unknown; Putrefaction: H: high; N: normal; L: low; Cause of death: TI:
traumatic injuries; AS: asphyxia by drowning; Uk: unknown.
62 S. Coelho et al. / Journal of Forensic and Legal Medicine 41 (2016) 58e64

Fig. 3. Results of DCA regarding of human samples, water samples and diatom species in (a) winter (b) spring, (c) summer and (d) autumn.

Fig. 4. Results of DCA analysis of cases, water samples and diatom species correlation that occurred during a year. Red line circles are marine cases, black circles are estuaries cases,
green rhombs are river cases, the yellow rectangles are unknown cases and the red circle is the Arcozelo site. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

case are the same to those present in other cases of drowning in
estuary location, with high content of A. minutissima.
Although a few diatoms were found in cases C8, C20 and C26,
the autopsy could not establish the cause of death. Diatom species
found in organs of the individual of case C20, namely F. crotonensis,
were the same to those found in water samples from location 4
(Fig. 3(c)). According to Fig. 3(d), the species Eunotia minor was the
most abundant found in organ samples from the individual
regarding case C26. The body of this individual was recovered at
Ferreira river but only in Fig. 4 the diatom's profile seems identical.
The specific location of death of six cases (C24, C29, C30, C31,
C32 and C35) was unknown but, by comparing the diatom species
found in water samples, an estimation of the drowning place was
performed. The body of individual of case C24 was recovered near
the mouth of the estuary. Fig. 3(d) shows similarity between
diatom species found in this case and location 1 (Luz beach). The
 S. Coelho et al. / Journal of Forensic and Legal Medicine 41 (2016) 58e64
main species found this case were A. distans and C. placentula
(Fig. 4). The body of case C29 was recovered in the Cabedelo beach
and it is possible to observe in Fig. 3(a) that A. minutissima is the
characteristic species of this case. In Fig. 4 it is possible to observe
that the diatom species found in this case was also found in cases
related to fresh and brackish waters. The body of case C30 was
recovered in Sindicato beach, in Valadares, and the main diatoms
found in lung and stomach content samples were C. dubius and C.
meneghiniana (Fig. 3(a)). The corpses of the individuals regarding
case C31 and C32 were both recovered from the beach. Diatoms
profile found in these cases and marine water samples were similar,
with predominance of C. neothumensis (Fig. 4).
4. Discussion
The Douro river estuary suffers from high anthropogenic pres-
sures, namely high maritime traffic and pollution. Mucha et al.30e32
have developed several studies in the estuary focusing on the dy-
namics of phytoplankton and bacterioplankton, aiming to under-
stand the impact of human activity, as well as to evaluate heavy
metal contamination of intertidal banks in the estuary and the
hydrodynamics and salinity intrusion. Given the similarity between
sampling locations (as shown in Fig. 2), possibly due to anthropo-
genic actions, the diatoms' profile in Douro river estuary does not
allow to establish a stratification based on diatoms' composition
that could be useful for determining the drowning place. A more
extensive study could give us a better support regarding the
composition and variation over the years.
Some diatoms species characteristics of fresh waters (A. distans
and C. placentula) were found in some organ samples of individuals
drowned in seawater (case C3 and C11). Their presence can be
explained by the proximity between the drowning site and the
mouth of estuaries, where there is a mixture of fresh and salt water.
Although Fig. 3(b) shows that F. elliptica, a fresh water species,
was found in both samples of organs of the drowned individual
regarding case C13 and water from the lake, in results of DCA
analysis in Fig. 4 are not concordant. This inconsistence can be
explained due to the collection of only one water sample from the
lake and when the DCA was performed, results could be influenced
by other samples. This issue could be overcome with a seasonal
collection of water sample of this location.
Ludes et al.16 previously reported that, if the location where the
drowning occurred is known, the comparison between diatoms
found in water samples from the site and lungs sample shows high
concordance; if the drowning location is unknown, the matching is
not so easy to perform. This could be explained by environmental
changes occurring in the liquid medium (e.g., seasonal precipita-
tion). The difficulty in the comparison could be overcome only with
a database of diatoms.16 In the present study the majority of
drowning locations was known. It was possible to confirm the
drowning location using the data from the database built in this
study. However, regarding the cases where the local of drowning
was unknown, the matching was not as easy to be established.
If a person is still alive when the body enters the water, the
diatoms will enter through the inhalation of water and by blood
circulation they will set to several organs as the brain, kidneys, liver
and bone marrow. Several authors argue that if no diatoms are
found in bone marrow, the diatom test can be questioned. However,
it is possible that the death occurred very quickly and, as the blood
circulation stopped, it did not allow the diatoms to reach to the
bone marrow. Krstic et al.33 explain that a quick death could avoid
the penetration of diatoms in the bloodstream, and, consequently,
to the rest of the organs. We believe that this can explain the
absence of diatoms in liver and kidney samples but more studies
should be performed to explain the process.
63
The absence of diatoms in cases where they were expected to be
found could be explained by several factors, including low con-
centration of diatoms in the liquid medium, low liquid volume
inhaled, and diatom loss during sample preparation, as described
by Lunetta et al.34 Penetration of the diatoms in alveoli and blood
circulation during the drowning process depends partly on its
shape and size, as well as on the possibility of their capacity to form
colonies. Therefore, the absence of diatoms in body samples should
not be sufficient by itself to exclude the possibility of drowning. On
the other hand, the presence of these organisms in the human body
should not automatically point drowning as the cause of death.
Several authors demonstrated that a dead body placed into water
could present diatoms in lungs and stomach because a reduced
number of diatoms can enter by passive transport.33 In the present
study, diatoms were found in two out of nine cases which death
occurred by traumatic injuries and not by drowning (C12 and C19).
It is possible that diatoms entered the human body by passive
transport or, although the cause of death was traumatic injuries, the
individual could have swallowed some water while still alive.
The existence of a specific protocol to avoid contamination
during sample preparation and the exclusion of false positive and
negative results is also important.4,34 Díaz-Palma et al.20 demon-
strated that a digestion with strong acids was less aggressive if the
acid concentration were not too high and selected 20% (w/w) HCl.
However, 37% (w/w) HCl was chosen in the present study since that
gave us better results than 20, 25 and 30% (w/w) HCl
concentrations.
The number of intact diatoms found in water samples from
marine locations was very low. This can be explained by the in-
tensity of waves, which can rupture a great number of diatoms.
Furthermore, silica is less available in marine waters than in fresh
waters and, consequently, marine diatoms are more sensitive to
strong acids used in extraction procedures. Therefore, two protocol
modifications should be considered in future studies: the selection
of another method of extraction (e.g., enzymatic method with
proteinase K, as suggested by Ming et al.13) and the analysis of the
chemical substances, present in seawater drowning medium, such
as Na, Cl, K and bromide as proposed by Takahashi et al.35 and Inoue
et al.36
As other authors have previously reported,4,34 our results also
confirm that the validity and reliability of the test of diatoms de-
pends on a quantitative and qualitative taxonomic concordance of
diatoms found in the corpse and in the liquid medium.
This study demonstrated the importance of collecting water
samples from the site where the drowning occurred in the moment
of death, but it is also very important to have a database of the
pelagic diatoms in the area around Porto, to compare it with cases
where the precise site of drowning is unknown (not only in the
estuaries but also in the small rivers around it and the beaches
nearby).
5. Conclusion
In most of the cases of death by drowning analyzed in this study
there was a positive correspondence between the diatom species
found in victim's tissues and the diatoms present in water samples
collected at the drowning location. Although the results demon-
strated that it is not possible to identify the precise location of
drowning in Douro river estuary, this study shows that, considering
diatom composition over the seasons and at different points of the
Douro river estuary and the sea coast it is possible to clearly
distinguish whether the drowning occurred in fresh water or salt
water. Our results suggest that it is important to collect water
samples in the same day and at the same place where the drowning
occurred. Only combining these two procedures, the identification
64 S. Coelho et al. / Journal of Forensic and Legal Medicine 41 (2016) 58e64
of the site of drowning can be successfully achieved. Our results
demonstrate the interest and the necessity to have a local diatoms
database (diatoms characteristic from fresh water and salt water)
since it can be an useful tool to establish the site of drowning in
cases of advanced state of putrefaction and when the body is found
far from where the drowning occurred.
Conflict of interest
The authors have no conflict of interest to declare.
Funding
None declared.
Ethical approval
The samples were collected from individuals submitted to
forensic autopsy exam at INMLCF-DN after verifying all current
legal regulations for human tissue collection for scientific research
purposes.
References
1. Calabuig G. Fisiopatología general de las asfixias. In: Can ~ adas EV, editor.
Medicina Legal Y Toxicología. 6th ed. Barcelona: Masson S.A; 2004.
2. CRdS Lopes. Guia de Perícias M
edico-Legais. 7th ed. 1982. Porto.
3. Piette MH, De Letter EA. Drowning: still a difficult autopsy diagnosis. Forensic
Sci Int 2006;163(1e2):1e9.
4. Lunetta P, Modell JH. Macroscopical, microscopical, and laboratory findings in
drowning victims: a comprehensive review. In: Tsokos M, editor. Forensic Pa-
thology Reviews, 3. Totowa, New Jersey: Humana Press; 2005.
5. Dolinak D, Matshes E, Lew E. Forensic pathology: principles and practice. 1st ed.
Amsterdam: Elsevier Academic Press; 2005.
6. Thakar MK, Singh R. Diatomological mapping of water bodies for the diagnosis
of drowning cases. J Forensic Leg Med 2010;17(1):18e25.
7. Falciatore A, Bowler C. Revealing the molecular secrets of marine diatoms. Plant
Biol 2002;53:109e30.
8. Horton BP, Boreham S, Hillier C. The development and application of a diatom-
based quantitative reconstruction technique in forensic science. J Forensic Sci
2006;51(3):643e50.
9. Taylor J, Harding W, Archibald C. A methods manual for the collection, preparation
and analysis of diatom samples - version 1.0. Water Research Commission; 2007.
10. Rovira L, Trobajo R, Ib
anez C. Periphytic diatom community in Mediterranean
salt wedge estuary: the Ebro Estuary (NE Iberian Peninsula). Acta Bot Croat
2009;68(2):285e300.
11. Ludes B, Quantin S, Coste M, Mangin P. Application of a simple enzymatic
digestion method for diatom detection in the diagnosis of drowning in putri-
fied corpses by diatom analysis. Int J Leg Med 1994;107(1):37e41.
12. Hurlimann J, Feer P, Elber F, Niederberger K, Dirnhofer R, Wyler D. Diatom detection
in the diagnosis of death by drowning. Int J Leg Med 2000;114(1e2):6e14.
13. Ming M, Meng X, Wang E. Evaluation of four digestive methods for extracting
diatoms. Forensic Sci Int 2007;170(1):29e34.
14. Takeichi T, Kitamura O. Detection of diatom in formalin-fixed tissue by pro-
teinase K digestion. Forensic Sci Int 2009;190(1e3):19e23.
15. Sidari L, Di Nunno N, Costantinides F, Melato M. Diatom test with Soluene-350
to diagnose drowning in sea water. Forensic Sci Int 1999;103(1):61e5.
16. Ludes B, Coste M, North N, Doray S, Tracqui A, Kintz P. Diatom analysis in
victim's tissues as an indicator of the site of drowning. Int J Leg Med
1999;112(3):163e6.
17. DiGiancamillo A, Domeneghini C, Gibelli D, Cattaneo C. Diatom extraction with
HCl from animal tissues: a technical note. Leg Med (Tokyo) 2011;13(5):268e71.
18. Vieira MEC, Bordalo AA. The Douro estuary (Portugal) - a mesotidal salt wedge.
Oceanol Acta 2000;23(5):585e94.
19. Karthick B, Taylor JC, Mahesh MK, Ramachandra TV. Protocols for collection,
presevation and enumaration of diatoms from aquatic habitats for water
quality monitoring in India. Icfai Univ J Soil Water Sci 2010;III(1).
20. Diaz-Palma PA, Alucema A, Hayashida G, Maidana NI. Development and stan-
dardization of a microalgae test for determining deaths by drowning. Forensic
Sci Int 2009;184(1e3):37e41.
21. Germain H. Flore Des Diatom
ees, eaux douces et saum^
te
etres. Paris: Socie
Nou-


vell Des Editions Boube
e; 1981.
22. Krammer K, Lange-Bertalot H. Bacillariophyceae. In: Süßwasserflora von Mit-
teleuropa. Stuttgart: Gustav Fischer Verlag; 1986.
23. Krammer K, Lange-Bertalot H. Bacillariophyceae. In: Süßwasserflora von Mit-
teleuropa. Stuttgart: Gustav Fischer Verlag; 1988.
24. Krammer K, Lange-Bertalot H. Bacillariophyceae. In: Süßwasserflora von Mit-
teleuropa 2/3. Stuttgart: Gustav Fischer Verlag; 1991.
25. Krammer K, Lange-Bertalot H. Bacillariophyceae. In: Süßwasserflora von Mit-
teleuropa. Stuttgart: Gustav Fischer Verlag; 1991.
26. Ricard M. Atlas Du Phytoplancton Marin. In: Sournia A, editor. Editions
Du
Centre Nacional De La Recherche Scientifique. Paris; 1987.
27. http://www.algaebase.org/ [cited 20 February 2015].
28. http://www.marinespecies.org/index.php [cited 20 February 2015].
29. http://diatoms.lifedesks.org/ [cited 20 February 2015].
30. Mucha AP, Bordalo AA, Vasconcelos MT. Sediment quality in the Douro river
estuary based on trace metal contents, macrobenthic community and elutriate
sediment toxicity test (ESTT). J Environ Monit 2004;6(7):585e92.
31. Mucha AP, Vasconcelos MT, Bordalo AA. Macrobenthic community in the
Douro estuary: relations with trace metals and natural sediment characteris-
tics. Environ Pollut 2003;121(2):169e80.
32. Mucha AP, Vasconcelos MT, Bordalo AA. Spatial and seasonal variations of the
macrobenthic community and metal contamination in the Douro estuary
(Portugal). Mar Environ Res 2005;60(5):531e50.
33. Krstic S, Duma A, Janevska B, Levkov Z, Nikolova K, Noveska M. Diatoms in
forensic expertise of drowningea Macedonian experience. Forensic Sci Int
2002;127(3):198e203.
34. Lunetta P, Penttila A, Hallfors G. Scanning and transmission electron micro-
scopical evidence of the capacity of diatoms to penetrate the alveolo-capillary
barrier in drowning. Int J Leg Med 1998;111(5):229e37.
35. Takahashi M, Kinoshita H, Nishiguchi M, Nishio H. Bromide detection in blood
using energy dispersive X-ray fluorescence; a chemical marker supportive of
drowning in seawater. Leg Med (Tokyo) 2010;12(3):132e6.
36. Inoue H, Ishida T, Tsuji A, Kudo K, Ikeda N. Electrolyte analysis in pleural
effusion as an indicator of the drowning medium. Leg Med (Tokyo) 2005;7(2):
96e102.