Int J Legal Med (2018) 132:487–497

https://doi.org/10.1007/s00414-017-1708-1

ORIGINAL ARTICLE

A valid method to determine the site of drowning

Rafael Carballeira 1,2 & Duarte N. Vieira 3 & Manuel Febrero-Bande 4 &
José I. Muñoz Barús 1,5

Received: 10 May 2017 / Accepted: 11 October 2017 / Published online: 8 November 2017
# Springer-Verlag GmbH Germany 2017

Abstract The diatom test is considered a useful aid in deter-
mining the site of death from drowning. Nevertheless, there is
disagreement within the scientific community concerning its
reliability, and its findings have been challenged and some-
times overturned in courts of law. Using a model based on
animal experimentation, we have developed a diatom test to
discriminate between the locations of drowning sites from
different aquatic systems. We carried out a complementary
combination of quantitative and qualitative analyses together
with a statistical analysis based on the Kullback-Leibler dis-
tance of the samples. A restrictive selection of exclusive dia-
tom species from each reservoir was also made. This approach
allowed us to validate the usefulness of the diatom test in
determining the location of the site of drowning.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00414-017-1708-1) contains supplementary
material, which is available to authorized users.
* José I. Muñoz Barús
joseignacio.munoz.barus@usc.es
1
Institute of Forensic Sciences, University of Santiago de Compostela,
Santiago de Compostela, Spain
2
Department of Botany, Faculty of Biology, University of Santiago de
Compostela, Santiago de Compostela, Spain
3
Department of Forensic Medicine, Ethics and Medical Law, Faculty
of Medicine, University of Coimbra, Coimbra, Portugal
4
Area of Statistics and Operations Research, Faculty of Mathematics,
University of Santiago de Compostela, Santiago de
Compostela, Spain
5
Department of Forensic Sciences, Pathology, Gynecology and
Obstetrics, Pediatrics, University of Santiago de Compostela,
Santiago de Compostela, Spain
Keywords Drowning . Diatom test . Site of drowning .
Forensic pathology
Introduction
The diatom test has long been regarded as a tool with great
potential for its application in cases of death by drowning
[1–3] and particularly for its potential usefulness as an indica-
tor of the site of drowning [4–6].
Diatoms are unicellular photoautotroph microorganisms with
an estimated species diversity as high as 200,000 [7, 8]. The role
as an exogenous indicator is due to the fact that they cannot
survive and proliferate within the human body and are incorpo-
rated with water from the site of drowning. Diatoms are widely
represented in all aquatic systems constituting characteristic pop-
ulations (particular spectra of species) associated with the envi-
ronmental conditions inherent in a particular aquatic system
[8–11]. The ecological affinities of diatoms also allow their use
as ecological indicators in the environmental monitoring of
aquatic systems [9] and can also transfer information about the
characteristics of the aquatic environment to the drowned body.
Several authors [4, 12, 13] have explored the possibility of
determining the correspondence between diatoms founded in
the body and the environment. These studies are based on a
comparison of diatoms in water at the site of the drowning and
the diatoms founded in the drowned corpses [4, 5, 12–15].
However, the ability to determine the site of drowning with
certainty by comparative analysis of diatoms in different aquat-
ic media has not yet been demonstrated. These methodological
proposals to determine the site of drowning are based on diatom
ecology, limnology, or paleoecology with no statistical method
which to guarantee its claims in the courts of justice.
However, the bases of the diatom test have always been
controversial [1–3] and the development of a standardized tool
488
based on a diatom test for forensic cases is difficult, with regard
to reproducibility, repetitions, and the sample size, which is
mainly due to the lack of standardization and validation of
the methodology published so far. Therefore, an experimental
animal model in rat has been developed to overcome these
deficiencies and determine with sufficient certainty the possi-
bility of locating the site of drowning. Experimental animal
models satisfy the statistical requirements necessary to evalu-
ate and validate the applicability of the diatom test in determin-
ing the site of the drowning. The transference of diatoms to the
body occurs is a physical phenomenon, at least in open organs
to the environment, and the differences between rat experimen-
tation models and forensic cases could be minimal.
Material and methods
Study sites and sampling
The water samples came from four freshwater reservoirs in
Galicia (NW Spain): Eume (EU), Portodemouros (PO), Barrié
de la Maza (BM), and As Conchas (AC), (Fig. 1). In each
reservoir, five samples at different sites (1 to 5) were collected.
From each site, 25 l of surface water was collected with a Van
Dorn bottle at a depth of 1 m to obtain a representative sample
without causing sediment re-suspension. Five grams of fresh
superficial sediment from the same site was collected with an
Eckmann dredge. These samples were maintained in a dark
cold room (4 °C) after sampling. Moreover, two replicas at sites
AC1 and PO1 were established to check variability at the same
site, as well as a replica a week later at sites EU1, BM1, PO1 y
AC1, to check short time variability. Site selection was random-
ly generated using geographic information system software
ArcGIS 10.0, to eliminate spatial dependence.
Water samples, acid digestion, and diatom microscopy
For each site, a sample of 1 l of homogenized water was
filtered (0.22 μm) and then re-suspended in 50 ml to concen-
trate and establish a representative quantitative sample of
plankton. An inverted microscope was used to determine the
quantitative concentration of diatoms. This was done using an
aliquot of 10 ml of the concentrated sample of plankton in a
quantitative chamber of sedimentation [16].
A further aliquot of 10 ml was treated for diatom digestion
with 9:1 H2O2 (30%): HCl (35%), in bath water at 60 °C to
remove organic matter, followed by successive centrifugations
at 900×g for 10 min and washed with distilled water to dilute
the remaining acid. All the material and reagents used in the
extraction were evaluated to exclude contamination (inclusive
gloves or filters). Bidistilled water and reagents were 0.22 μm
filtered. Other materials (containers, tubes, surgical instruments,
etc.) were washed with filtered bidistilled water following
Int J Legal Med (2018) 132:487–497
Timpermann [1] and Ludes et al. [5]. In addition, a sample of
0.5 g of fresh superficial sediment was digested following the
same digestion method used for the water sample.
Aliquots of 500 μl of the water and sediment digested
samples were spread on a clean cover glass and allowed to
air-dry for the identification and recount by light microscopy
(LM). Permanent slides were mounted on glass slides using
the Naphrax® resin (refractive index = 1.74; Brunel
Microscopes: http://www.brunelmicroscopes.co.uk/).
Diatoms were examined under LM with Nikon Eclipse E600
equipped with Plan-Apochromat 100× objective (N.A. 1.32)
and a differential interference contrast (Nomarski) optics. LM
photographs were taken with an AxioCam ICc5 Zeiss digital
camera. To characterize the communities and quantify abun-
dances, the species preparations for LM were recounted up to
a 1000 diatom valves (two valves per diatom frustule) for
sample. The identification of diatom species was made ac-
cording to Krammer and Lange-Bertalot [17], Lange-
Bertalot [18], and Hoffman et al. [19].
Animal experimental model
The rats (Rattus norvegicus, Sprague Dawley) were obtained
from the Animalario Central of the University of Santiago de
Compostela 15003AE, from the same origin and raised under
the same conditions. We used a total of 54 rats, weighing 400–
500 g. Thirty-four rats were anesthesized with intraperitoneal
sodic pentobarbital (48 mg/kg) before drowning, and 20 rats
were sacrificed using carbon dioxide euthanasia method. The
procedure with animals was consistent with the care animal
ethics and previously authorized favorable report from the
bioethics committee.
Four groups of treatment were established (I–IV): group I
(control), group II (macerated), group III (drowning), group
IV (spatio and temporal replicates), (Table 1):
Group I, 10 rats control; group II, 10 rats were sacrificed
and then completely submerged in water from As Conchas
(AC) and Portodemouros (PO) for 15 days; group III, 20 rats
were anesthetized and drowning distributed in four different
site sample water in each of the four reservoirs (water samples
were previously kept at room temperature to prevent death of
rats from hypothermia would affect); group IV (spatio and
temporal replicates), in 14 rats, three rats were drowning only
in replicate water samples of AC1 (n = 3) and PO1(n = 3)
points to evaluate the variability at the same point, and other
rats in water sampling of EU1 (n = 2), PO1 (n = 2), BM1
(n = 2), and AC1 (n = 2) sites 1 week after the previous one.
Tissue samples and diatom extraction
Samples were complete organs: lung (right), stomach, heart,
kidney (right), spleen, and bone marrow of femur (right) and
were extracted using uncontaminated material. In groups III
Int J Legal Med (2018) 132:487–497
Fig. 1 Map of the four reservoirs (Eume, Barrié de la Maza,
Portodemouros, and As Conchas) and sampling points. a Location
map of the reservoirs in the Iberian Peninsula (Galicia, NW of
Spain). b Location map of the reservoirs in Galicia and their
and IV, different instruments were used for opening and
manipulating.
The tissue sample was frozen to enhance tissue disorgani-
zation. Acid digestion, as a quantitative method, was modified
and adapted from Ludes et al. [5], Hürliman et al. [12], Ranner
489
watershed; c–f Detailed maps of the reservoirs Eume (EU), Barrié
de la Maza (BM), Portodemouros (PO), and As Conchas (AC) with
location of the five sampling points for each one
et al. [20], Kater [21], and Di Giancamillo et al. [22]. The
samples were treated with HCl (35%) in bath water at 60 °C
to remove organic matter and then with H2O2 (30%), until the
sample stopped reacting at room temperature. Ultrasonic was
then applied for 1 min to boost tissue disorganization. Finally,
490 Int J Legal Med (2018) 132:487–497

Table 1 Scheme of the

methodology followed in the Groups Total Treatment Water samples
animal experimental model
Group I (control) (n = 10) 10 rats Sacrified None
Group II (macerated) (n = 10) 5 rats per Sacrified and 2 reservoirs (sites 1–5)
reservoir submerged [AC, PO]
Group III (drowning) (n = 20) 5 rats per Drowning 4 reservoirs (sites 1–5)
reservoir
Group IV (n = 14) 3 rats per Drowning 2 reservoirs (sites 1, 3, 5)
(spatio-temporal reservoir [AC, PO] sampling at the same time
replicates) (n = 6) of Group III water samples
[spatial replicates]
2 rats per Drowning 4 reservoirs (only site 1) sampling
reservoir at the same time of Group III water
(n = 8) samples [temporal replicates]

after acid digestion, successive centrifugations with bidistilled
water were applied (900×g for 10 min).
The final pellet was transferred to 5 ml vials through vortex
washes and previous ultrasonic (max. 1 min). An aliquot of
500 μl of lung and all bone marrow samples were used for the
identification and recount by light microscopy (LM).
Permanent slides were made as cited above. Diatom abun-
dances were estimated using a quantitative approach [12] and
the recount was for 300 diatoms in lung samples and totally of
diatoms present in bone marrow slide samples at a magnifica-
tion of ×1000. General revisions of the slides were made at
×400 to confirm the results with better coverage of the slide.
Statistical methods
We used data relative abundances of diatom counts and R
software for all the statistical analysis [23]. The data are char-
acterized by the sum of relative abundances for every sample,
which is a fixed quantity (in this case 100%). This is known as
compositional data and in mathematical terms, the data are
similar to probability mass function (pmf). Therefore, the spe-
cial nature of the data must be taken into account when ana-
lyzing the differences between observations.
One of the most appropriate distances between densities is
the, so called, symmetrized distance of Kullback-Leibler (KL).
The distance of KL between two pmf (f, g) is given by the
relationship:
distð f ; gÞ ¼ ∑ f i logð f i =gi Þ
i
which is not symmetrical, (dist (f, g) is not equal to the dist (g, f)
but can be easily symmetrized by performing the following
transformation: 0.5dist (f, g) + 0.5dist (g, f). In our application,
we have obviated components lower than 0.02% (components
below this value are not used in the summation) to prevent a
very small component from dominating the whole distance.
This distance is used to graphically separate the different
groups/sites using dendrograms and can be employed as the
distance for a classification method based on distances such
as the k-nearest neighbors (kNN=method). Also, using the or-
igin of the sample as a response variable, we have trained high-
dimensional generalized linear models (GLM) [24] with sam-
ples of plankton to predict the remaining dataset to check the
discriminatory ability of this kind of data. Data field samples
(plankton) have been used as predictors to compare with sam-
ples from the rats [group II (macerated), group III (drowned)],
the values of water samples as training GLMs of high dimen-
sion and predict the remaining dataset (samples from the rats).
Results
Diatom qualitative analysis of waters
The following terms have been defined to clarify the results:
species spectra refer to the list of diatom species; exclusive
species are those that have only been located in a particular
reservoir; species indicators are those species exclusive to
each reservoir as indicators of the location of the site.
The results of the analysis of the spectrum of diatom species
in the reservoirs show a total of 219 different species, of which
181 species are in the water column (Online Resource 1)
representing 82.6% of the total species present in the reservoirs.
The number of species in the sediment was 207 and represented
almost all species present in the water column. As for the spatial
distribution of the diatom species in the water column, no dif-
ferences (< 2%) were observed in the species spectra, nor the
different species present, nor for the different points in each
reservoir, including the replicates on the same points AC1
and PO1, as well as those collected 1 week later at points
EU1, BM1, PO1, and AC1. In the sediment, the number of
species ascended to 207, with almost all species being present
in the water column.
No reservoir houses all species; thus, species spectra are
different for each reservoir. Species spectra yield a different
total number according to the reservoir and with respect to the
Int J Legal Med (2018) 132:487–497
total species in all reservoirs (181): EU 61 has different species
(33.7%) > BM 49 (27.1%) > 44 AC (44.3%) > PO 31 (17.3%).
The reservoirs among them share a maximum of 30% of spe-
cies and present a different number of exclusive species with
respect to the total species in each reservoir: EU 44
(72.1%) > BM 32 (65.3%) > AC 28 (63.6%) > PO 10 (32.3%).
Diatom quantitative analysis of diatom in water samples
The concentration of diatoms in water (valves/ml) has a similar
range in all reservoirs, EU (2446–5072), BM (2648–5072), PO
(1804–3088), and AC (2132–3146). The differences in the di-
atom concentration in the water column were similar between
the sampling sites and the replicates, as well as the samples
collected 1 week later (group IV, spatial and temporal repli-
cates). These differences are not greater than the spatial differ-
ence between the different sampling sites in each reservoir.
The analysis of water column samples from reservoirs, as in
their regional comparison, shows a great diversity of species.
The four reservoirs studied represent a high local diversity with
45% of Galicia flora and 10% of flora of the Iberian Peninsula
[25–31]. In fact, species previously uncited in Galicia, such as
Caloneis schumanniana (BM, PO) and Stauroneis
acidoclintata (EU, or even in the Iberian Peninsula, such as
Pinnularia subanglica (BC), Pinnularia anglica (AC), and
Cymbellopsis santasona (EU)) were now discovered.
The most abundant species (greater than 10%) in the water
column are summarized in Online Resource 2. The exclusive
species of each reservoir (even when considered indicator
species) have abundances always less than 10%. These indi-
cator species are predominantly benthic in character, reaching
higher abundances in the sediment samples. However, some
of these indicator species, despite their low abundance, appear
in all spatial samples in the same water column
(Online Resource 2).
Diatom quantitative analysis in rats
The diatom concentration of the samples in groups II
(macerated) and III (drowned) with respect to group I
(control) shows clear differences: in the latter, the presence
of diatoms in all samples analyzed is infrequent and the total
number of valves is less than 10 (Fig. 2).
The internal organs (heart, kidney, spleen, bone marrow),
although in agreement with respect to the species spectrum,
have a maximum number of only 35 valves and show biased
abundance profiles with respect to plankton samples (Fig. 2).
There are clear differences between groups II (macerated)
and III (drowned) in the samples of internal organs. Group III
(drowned) has higher concentrations of valves than the con-
trol, in contrast to group II (macerated).
For group IV drowned rats, spatio and temporal repli-
cates obtained 1 week later are consistent with the
491
abundances and spectra of diatom species in the sample
group III organs (drowned).
The rats of group II (macerated) show abundance profiles
very similar to those of the aquatic environment, with a greater
representation of the dominant species. The rats of group III
(drowned) present a better representation of the low abun-
dance species in relation to the dominant ones in the abun-
dance profiles (Fig. 2).
Statistical determination of site location
There are no significant differences between replicates at the
same point at the same time, nor are there differences with
temporal replicates. As for the presence of species, there are
neither temporal nor spatial differences between the water
samples and therefore neither between water samples of group
II (macerated), group III (drowned), and groups IV (spatio-
temporal replicates).
The types of samples analyzed (fur, stomach, lung) of
group III (drowned) have a higher representation of species
of lower abundances than those of group II (macerated) (Fig.
2). If the types of samples are analyzed independently (wheth-
er fur, stomach, or lung) based on dendrograms, a lower de-
gree of certainty is obtained in the determination of the loca-
tion of the sites (Fig. 3). Also, a single sample type is unable to
determine with certainty the location of BM and PO, as well as
with lung samples in group II (macerated) and in group III
(drowned) from AC (Fig. 3). The greater presence of exclu-
sive species in the samples, although less abundant in the
plankton (Fig. 2), is directly related to the determination of
the location of the submersion medium according to the type
of sample analyzed: fur>stomach>lung (Fig. 3). As a whole,
the statistical model KL determined the location of the site for
the majority of the samples based on the different types of
samples analyzed (fur, stomach, lung): EU (14/15), BM
(7/15), AC (25/30), PO (26 / 30) (Online Resource 3, Table 2).
Although all types of samples can indicate the correct
site (principally in EU), the fur and stomach samples pro-
duce better discrimination than the lung samples (particu-
larly in EU and macerated from AC). However, on the
basis of the independent analysis of each organ (whether
fur, stomach, or lung), it is more difficult to discriminate
between samples from BM and PO, due to the similarities
of the spectra and abundances of diatom species in the
plankton of both reservoirs (Fig. 3).
This is mainly due to the low diversity of species and the
similarity of the dominant species in the BM plankton with
respect to the other reservoirs, which implies an overlap of the
BM samples mainly between the samples of PO and partly
AC. This happens when predicting the origin of samples from
the BM reservoir in which the site location is determined
correctly in 7 of 15 samples in total (Table 2).
492 Int J Legal Med (2018) 132:487–497

Fig. 2 Profiles of diatom species abundance (percentage) in water (plankton), fur, lung, stomach (groups II, III), and average of internal organs of group
III (heart, kidney, spleen, bone marrow) of As Conchas (AC) reservoir. Each bar represents a rat (n = 5)

The statistical model based on the distance KL has allowed
us to predict the locations with good results by calculating
probabilities belonging to each site of the reservoirs EU,
AC, and PO. Even so, it is less precise in discriminating BM
samples because they have a lower diversity of exclusive spe-
cies and a similarity in the abundance profiles of the dominant
species in relation to AC and PO (Online Resource 3, Table 2).
In cases where the statistical model shows a lower resolution
in determining the sites, mainly between BM and PO, a com-
plementary analysis based on the use of indicators or exclusive
species for each reservoir can strengthen the determination of
the provenance of the samples indicated in Online Resource 2.
Discussion
The diatom populations in the water system reservoirs are
those that provide the differential elements that help determine
the location of the site, hence the need to characterize ade-
quately the diatom populations of the different aquatic systems.
Int J Legal Med (2018) 132:487–497 493

Fig. 3 Dendrogram of affinities

of the samples of fur (a), lung (b),
stomach (c) relative to plankton
samples from Eume (EU), Barrié
de la Maza (BM), Portodemouros
(PO), and As Conchas (AC) res-
ervoirs based on distance KL
494 Int J Legal Med (2018) 132:487–497

Table 2 Results of the prediction by high-dimensional GLM models the contact with the water, there is a simultaneous contact with
on the dataset of fur, lung, and stomach samples from groups II
all those elements in suspension which transfer to the organ-
(macerated) and III (drowned) from reservoirs using plankton samples
as training. (No=means it does not belong to the group; Yes=means it ism a diatom profile similar to that existing at the site. This
belongs to the group) profile will be reflected in the different organs according to the
selective barriers found within the organism, as reported for
Predict EU AC BM EU PO
rat and humans [12, 34, 42, 43].
No 30 15 1 30 The suspension profiles of the organs, either in direct
Yes 0 0 14 0 contact or open to the external environment, show a higher
Predict BM fidelity to those in the sites [2, 3, 5, 12, 34–36]. Also, for
No 30 8 15 30 this reason, there is very little difference between the animal
Yes 0 7 0 0 model in rat and forensic cases. Tissue samples in contact
Predict AC with the immersion medium, such as fur, stomach, and
No 5 15 15 30 lung, lacking physiological-anatomical barriers, allow a
Yes 25 0 0 0 large number of diatoms to be incorporated into the organ-
Predict PO ism and show a better transfer of the spectra and profiles of
No 30 15 15 4 diatom species [2–4, 6, 12–15, 35–41].
Yes 0 0 0 26
The differential representation of the dominant/indicator
species in plankton is probably mediated by the type of sample
(fur, stomach, lung) and the active or passive entry in the
organism. For example, the lung in group III (drowned) where
A comparison between water column and sediment sam- the active aspiration effects may concentrate the plankton in
ples has shown a greater diversity of species in sediment. The the sample, thus over-representing the dominant species.
sediment is adding the main differential species although with Our results indicate that it is possible to determine the site
low (< 10%) or very low (< 5%) abundances in the water by applying mathematical methods to the quantitative diatom
column. This is due to the fact that the water column is a more data, such as with EU samples, but this is not always possible.
homogeneous environment and imposes a series of restric- In samples of BM, PO, and some of AC, the similar abun-
tions (e.g., buoyancy, nutrients) to which only a small number dance profiles, the existence of low abundances of exclusive
of diatom species are adapted and show a great dominance species is a limitation for KL and other mathematical analyses
when the BSurface of the sediment/Volume of water^ relation- to determine with certainty the provenance of the samples
ship is very low [10, 32, 33], as in the case of the very deep (Online Resources 2 and 3). The use of the indicator species
analyzed reservoirs (50–200 m). we have selected (Online Resource 2) is especially useful for
While there is a great diversity of species, any aquatic en- discerning the origin of the BM samples, and also the samples
vironment of the region is capable of establishing a discrimi- from PO and AC, but these latter with a lower resolving ca-
nant analysis. In the water column of the reservoirs, there is a pacity (Online Resource 3, Table 2).
clear dominance of planktonic species (e.g., Fragilaria These results contradict many authors [2–4, 6, 12–14, 35,
crotonensis, Asterionella formosa, Cyclotella meneghiniana, 36, 38, 40, 41]. These authors stated that a direct comparison
Aulacoseira granulata, Aulacoseira distans). These species of the spectrum of diatom species could be sufficient to dis-
are common and abundant in the plankton of reservoirs and criminate the sites in cases where the differences between the
other freshwater aquatic systems [27, 28, 31], coinciding with diatom populations are not obvious. For this reason, the sta-
those in other geographic areas [12, 15], and therefore will tistical approach based on the species spectrum and their
have little predictive value, and the least frequent species will abundance profiles may be a valid criterion but only if there
be the most useful in determining the drowning site. are sufficient differences. The presence is as important as the
This has influenced the negative diatom test results obtain- abundance of the species as seen when we compare EU sam-
ed by Coelho et al. [15], since the diatom analysis did not ples to those other reservoirs.
allow for differences between the different sites of the same On the other hand, although in the literature, the lung is
river section at the Douro River estuary. This is because the often used as the reference organ to locate the site given that it
same aquatic mass is influenced by the same environmental is the main route of entry into the body [4, 13]; nevertheless,
factors and, in general, tends to homogenize the populations fur has the advantage of being in direct contact with the water
of diatoms, as happened in certain stretches of river in which medium and has a larger catchment surface without the disad-
the biological populations settle down in a Bcontinuum^ [44]. vantages of concentration. A single type of organ sample
The transfer of diatoms to the body occurs during submer- alone should not be used as a sole reference.
sion upon contact with the suspended elements at each site. The different approaches made by other authors based on
This process is a physical phenomenon that implies that with simple comparative of pairs of samples through similarity
Int J Legal Med (2018) 132:487–497
indices (Jaccard) [4, 6] or even the combination separately
of the same index of similarity and abundances [12] are not
enough. Combined statistical analyses of the spectra spe-
cies and abundance profiles are necessary to determine the
site [13–15]. Horton [13] used an appropriate dissimilarity
or similarity measure based on modern analog techniques
[45], which is a numerical comparison, but was designed
for another purpose and only compares samples of the res-
ervoir with those of the body.
Also, the statistical methods applied by other authors [4, 5,
12–15] are comparative methods developed in limnology,
ecology, or paleoecology of diatoms and not designed to de-
termine the location of a site of drowning with the minimum
certainty required in the medico-legal field. Moreover, the
forensic cases analyzed in other studies [4, 5, 12–15] only
demonstrate the transference of diatom spectra and abundance
profiles from the environment to the organism during drown-
ing because they were compared within the same aquatic sys-
tem; therefore, there is a total correspondence between organs
and plankton samples. In these cases, the samples should be
tested in different water bodies to demonstrate their utility.
However, diatom counts are compositional data, and
one of the most appropriate methods to compare distances
between densities is the symmetrized distance of Kullback-
Leibler (KL) which was used in this study as means of
combining species spectra and abundance profiles of the
samples. The KL statistical method applied in this study
shows the same results. However, other authors are using
chi-square test (χ2) based on goodness of fit between the
distribution of pairs of samples [14], or applying descrip-
tive statistical methods such as detrended correspondence
analysis (DCA) [15], although this is inadequate to deter-
mine the location of the drowning site because it is an
exploratory data analysis.
In this study, we have combined a qualitative analysis to
complement the limitations of mathematical methods applied
to quantitative data. Our complementary qualitative analysis
was based on the criterion of concordance, the presence of the
same species among the environmental samples, and those
incorporated into the organism, but used differently from that
applied by other authors [1–4, 35, 36, 46].
Not only have we taken into account the existence of
concordance, but also restricted the criterion of concor-
dance to establish with sufficient certainty the discrimina-
tion of the submersion medium from the different types of
samples (plankton, internal organs, fur, stomach, lung). We
have taken into account the exclusivity regarding each res-
ervoir, ubiquity in the aquatic mass, correspondence with
the samples of samples of rat organs, and regional charac-
ter, such as rare or infrequent species in the reservoir. In
addition, these samples are also compared with the sedi-
ment samples, since the sediment contains an annual aver-
age of the species present in the aquatic mass [9].
495
Conclusions
The development of a standardized tool based on a diatom test
for forensic cases is mainly due to the lack of standardization
and validation of the methodology published so far. However,
it is possible to determine the precise location (site) where a
death by submersion occurred by means of the diatom test as
long as the basic premises are fulfilled: namely that there are
differential species spectra and abundance profiles in the me-
dium of submersion, and that they are transferred to the body
during submersion at that time. Combining the quantitative
and qualitative analyses of the diatoms present in the samples
and the medium allows us to affirm that the diatom test is a
valid tool to determine the location of the submersion site.
Species spectra and abundance profiles tend to be homo-
geneous in relation to the same reservoir and within a period
of 1 week in each reservoir. The dominant species in the water
column are reduced to a small number of species by the re-
strictions of living in suspension.
The transfer of the diatom abundance patterns from the
immersion medium to the organism occurs mostly to those
organs in contact with the exterior (fur) or with open routes
to the outside (stomach, lung), which are more likely to deter-
mine the location of the submersion medium with respect to
the internal organs (heart, kidney, spleen, bone marrow).
Quantitative analysis of samples of plankton and fur, stom-
ach, and lung allowed us, by means of a statistical model based
on distance KL, to verify the probability of provenance of the
samples with sufficient success to determine the site of a sub-
mersion between different water systems. The fur and stomach
show considerable discrimination compare to the lung.
The use of many samples of the same organ is not suffi-
ciently precise, for which reason several organs need to be
studied to provide sufficient certainty to determine the site of
a submersion. Furthermore, the quantitative analysis should
be complemented by a selection of indicator species: in the
reservoirs studied, the location of the submersion medium
could be identified by the indicative species of each reservoir,
Achnanthes linearioides (EU), Brachysira brebissonii (EU),
Navicula anglica (EU), Achnanthes subhudsonis (BM),
Cymbella excisa (PO), Encyonopsis cesati (AC), and
Aulacoseira sp1 (AC).
Acknowledgments We wish to thank Demarcaciones Hidrográficas
del Duero, Cantábrico, Miño-Sil, Galicia-Costa, and Augas de Galicia
of the Autonomous Government of Xunta de Galicia for providing infor-
mation on diatom communities in Galician rivers. Thanks also to the
Instituto José Cornide de Estudios Coruñeses for providing the unpub-
lished documents of López-Seoane (1893) and thanks to V. Reynolds for
his support.
Funding Information Rafael Carballeira is grateful for the financial
support (2012–2013) of the Instituto de Ciencias Forenses BLuis
Concheiro Carro^ (USC) and for the Galician research plan innovation
and development 2011–2015 (Plan I2C) of the autononamous
496
Government Xunta de Galicia and co-financed by European Social Fund.
The research of Manuel Febrero-Bande is partially supported by
MTM2016-76969-P (Spanish State Research Agency, AEI) and co-
funded by the European Regional Development Fund (ERDF).
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