Forensic Science International 127 (2002) 198203

Diatoms in forensic expertise of

drowninga Macedonian experience
Svetislav Krstica,*, Aleksej Dumab, Biljana Janevskab,
Zlatko Levkova, Ksenija Nikolovab, Marina Noveskaa
a

Faculty of Natural Sciences, Institute of Biology, P.O. Box 162, 1000 Skopje, Macedonia
b
Faculty of Medicine, Institute of Forensic Medicine, 1000 Skopje, Macedonia
Received 14 March 2002; accepted 15 April 2002

Abstract
Results obtained by examination of 22 human cases suspected for drowning, one human case of death other than drowning and
several tests on laboratory rats were used as a basis for evaluation of diatom method as supportive in forensic expertise of
drowning. The recovery of diatoms from various examined organs, their qualitative and quantitative composition, if properly
treated without the possibility of contamination, can be a reliable proof of the time and place of drowning. The priority of organ
examination (external microflora determination, lungs, brain, heart (and/or blood), stomach, liver and kidney, and finally bone
marrow) is discussed and established as well as the basic future research on cases suspected of drowning, but also on nondrowned victims and laboratory animals.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Diatoms; Drowning; Forensic expertise; Methods

1. Introduction
Drowning can be defined as death due to full or partial
submersion in a fluid. Apart of the fact that precise mechanism of drowning is complex event modified by the medium
involved and other factors, in most cases, inhalation of fluid
resulting in respiratory tract obstruction is the essential cause
of death [1]. Forensic expertise of drowning generally
investigates all the relevant circumstances since there are
several possibilitiesaccident, homicide or suicide. Drowning may occur in deep waters or in as little as 56 cm of fluid,
in which case, the suspicion of decreased consciousness in
the victim should be the predisposing cause as a result of
head injuries, epileptic fit, ingestion of narcotic substance or
most commonly alcoholic intoxication.
Although many signs of drowning, both external and
internal have been described so far, none can be considered
as fully affirmative of the cause of death. Rapid onset of
putrefaction represent additional problem (especially in
warmer waters) that obscures not only those signs regarded
*
Corresponding author.
E-mail address: skrstic@iunona.pmf.ukim.edu.mk (S. Krstic).

as characteristic for drowning, but also renders the assessment of any injuries present extremely difficult. Identification of the victim is often impossible and usually based on
supportive evidences such as clothing, jewelry or physical
deformities or in the worst case only on dental records if such
a database exists.
1.1. Diatom test of drowning
All these controversies regarding the reliable method in
determination of drowning, but also the time and place of
possible drowning, as well as the fact that drowning represents the 4th cause of accidental death in USA [2], as early as
the work of Guy [3], scientific debate on validity of the
diatom test begins. Brouardel and Vibert [4] were conspicuous enough to conduct a serial of drowning on dogs with
findings that a quantity of water as much as one-third or onefourth of subjects blood volume enters the circulation, when
submersion was 20 min or more. Swann and Spafford [5]
later found that it takes only 3 min for the original blood
volume to be diluted by an equal volume of water. The idea
for examining the content of inhaled water in internal organs
is attributed to Revenstorf [6], who examined the penetration

0379-0738/02/$ see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
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S. Krstic et al. / Forensic Science International 127 (2002) 198203

of diatoms into the lungs of drowned subjects, [7]. Since

then, numerous authors have contributed to this dubious
subject reflecting the extreme doubts ranging from: The
presence of single diatom in femoral bone marrow is consistent with drowning as a cause of death [8] to the diatom
method is worthless [9,10]. All the contradictories in using
diatoms as effective method are almost in all references due
to two major points: (a) the presence of diatoms in victims
who were not drowned (an excellent review on subject was
made by Peabody [7]) and (b) the difference in used methods
and implementation of medical approach to the problem that
is entirely a part of applied algology [11].
Amid the doubts, the school that was formed after work
by Incze [12] and his Hungarian co-workers [13,14] has lead
to use of the diatom method routinely in drowning cases by
European pathologists. In addition, several workers have
tested the penetration of diatom frustules into the internal
organs and tissues of animal and human organisms thus,
enlightening the pathways of inert siliceous frustules into the
body ([1518], etc.). Nevertheless, the serious attempt by
Foged [19] for determination of drowning of several cases of
mummies of Greenlanders who were dated about 1460 A.D.,
also supported by analysis of four drowned and four notdrowned persons who died in 19811982, has not lead to
satisfactory results due to a very contradictory facts, forcing
the author to state that diatom analyses give no solid basis
for a reliable diagnosis: drowned or not-drowned.
1.2. Basic principles of diatom method
Fundamental principle of the diatom test in investigation
of drowning is based on postulation that diatoms are present
in the medium where the possible drowning took place and
that inhalation of water causes penetration of diatoms into
alveolar system and blood stream, and consequently their
deposition into brain, kidneys and other organs, like the bone
marrow of large bones. After the autopsy, samples of organs
are digested with strong acids and diatom cells revealed
under the microscope. There is also a test of blood for diatom
content proposed by Yoshioka and Takahashi [20]. In case,
when the victim was already dead before the body was
submerged, the transport of diatom cells to various organs is
prevented due to lack of circulation.
On the other hand, determination of qualitative diatom
composition in victims organs and the surface of the body
(or in the environment where the body was found), enables
valuable conclusions regarding the possible place of drowning and sometimes it is the only valid approach in determining the time of death.
As a basic principles that researchers state against relevant applicability of diatom method in forensic expertise of
drowning we would like to point:
 The presence of diatom cells in post mortem analysis of
victims whose death other than drowning has been confirmed: Tiny diatom cells in human tissues and organs can

199

be introduced in several ways. (i) Up-to-day described

more than 20 000 recent and fossil diatom taxa [21],
generally abundant in water ecosystems where their
number and composition vary in time and space, but also
as a result of antropogenic impacts during eutrophication
and pollution [22]. Certain species (sometimes in very
high numbers) are lifted up in the air [23] and transported
at great distance [2426], thus, representing an continual
source of diatom cells that might be inhaled (Dayan et al.
[18] determine that monkeys inhabiting dry and dusty
regions in Africa show high content of diatom cells in
lungs comparing to those from tropical rain forests). A
special source of diatom cells in respiratory organs is also
the cigarette paper [27] of bad quality. (ii) Second major
source of diatom cells in human tissues are food and water
[28]. (iii) The third and most difficult to cope with source
of diatom cells reported by [29] are laboratory glass and
reagents used during analysis.
 Contamination of submerged bodies with diatoms that
enter into tissues due to their degradation (passive contamination): During prolonged submersion of the body in
water, apart of severe decomposition of tissues that
enables close contact of organs and water, Ludes and
his coworkers [30] state the importance of the difference
of the hydrostatic pressure, relaxation of muscles and
subsequent transport of diatoms into closed organs.
 Lack of diatom cells in analyzed organs: Regarding this
problem, there basically two reasons stated in literature.
(a) A rapid death that prevents the penetration of diatoms
into circulation and organs [31], and (b) the cause of death
is not drowning [32] or the drowning occurred in water
deprived of diatoms [33].
Aiming scientifically, to determine the applicability of the
diatom method in forensic expertise of possible drownings in
Macedonia, algological team at the Institute of Biology,
Faculty of Natural Sciences in Skopje in cooperation with
the Institute of Forensic Medicine in Skopje, starts a serial of
experiments in the frame of two projects (diatoms in forensic
expertise of drowning I and II, 19942000) on a total of
23 victims of drowning and tests on laboratory rats. This
paper represents the obtained results and possible future
directions of research in this extremely controversial field of
applied algology.
2. Materials and methods
In investigated period, 22 human cases of possible drowning, one control case of death with other etiology and two
series of test on rats (Irats that were given water ad libitum
saturated with diatom suspension isolated from river Vardar
during 1 week (two individuals), and IIrats (two individuals) drowned in suspension of diatoms from river Vardar
and have been performed left in the water for a week; control
group (two individuals) sacrificed and then submerged into
diatom suspension for a week.

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S. Krstic et al. / Forensic Science International 127 (2002) 198203

Analyzed tissues and organs both of human (which tissues

and organs depended on available autopsy material) or
animal origin were subjected to same procedure:
I. Approximately 2 g of tissue or part of specific organ
(in vicinity of the major blood vessel that enters the
organ) were placed in H2O2 for 24 h.
II. After finishing the oxidation process, H2SO4 was
added in amount necessary to complete the oxidation
of organic matter.
III. Then, saturated solution of permanganate was added
till the color turned violet.
IV. The sample is discolored with saturated solution of
oxalic acid.
V. After 48 h of sedimentation, the probe is leveled to
approximately 100 ml that are centrifuged at
3000 rpm for 20 min, until the pH is neutral.
VI. At the end, the supernatant is aspirated to approximately 100 ml that are used for preparing the serial of
permanent slides in kanada balsam medium with
refraction index 1.53.
VII. Microscope Nikon Eclipse E-800 was used for
analyzing the slides, usually at 1500 magnification.

3. Results and discussion

We tried to analyze the possible pathways and accumulation of diatom cells in tissues and organs by allowing
laboratory rats to drink water saturated with diatom cells
for 7 days, but we also drowned a control group in the same
saturated water (Table 1). Total number of determined
diatom cells in analyzed organs of rats enabled the basic
information for the accumulation pathways, pointing out the
bone marrow (34 cells), but quite interestingly the brain as
well (32 cells) as dominant deposition sites. Diatom cells
abundance is also proven important in kidney (22), stomach
(19) and lungs (11), pointing out these organs as important in
regular forensic expertise of drowning. Low number of
determined diatom cells in heart (2) is also indicative.
On the other hand, the analysis of organs of the control
group of rats drowned in water saturated with diatoms shows
clear confirmation of drowning: huge number (533) of

diatom cells in lungs and stomach (60) as direct result of

inhalation and swallowing of water; similar number of
revealed diatom cells in kidney (23) and brain (23); appearance of diatoms in liver (11) and strong confirmation of
diatoms in blood circulation through relatively high number
of cells in heart (18).
Analysis conducted on groups III and IV was orientated to
resolving the basic problem cited in literature against reliability of diatom method, regarding the possibility that diatom cells can be found in victims that are not drowned while
existing in medium of submersion. In investigated period of
7 days, in rats that were killed outside and submerged in
diatom suspension, diatoms were revealed from stomach
(38) and less in lungs (7) what confirms the postulation for
passive introduction of diatom cells after releasing the
muscles. Analysis of other organs showed much less diatoms
(2.3 in heart) as direct proof of the real cause of death. On the
other hand, results on concentration of diatom cells in organs
of drowned rats were as much as expected, maximal in lungs
(352.2), heart (213) and stomach (168), and minimal in brain
(0.1), kidney (3.3) and liver (6.1), supporting the evidence of
a rapid death and almost no deposition in terminal organs
due to lack of blood circulation.
Table 2 summarizes hitherto analyzed human cases in
19972000 period. Some of the stated problems with the
application of the diatom method are immediately evident
and should be overcome in future:
 all the organs were not analyzed in some cases, especially
from the surface and stomach;
 lack of diatom cells on the surface of the cadaver, but their
presence in organs (case 20);
 relatively high abundance of diatoms on the surface, but
extremely low abundance in analyzed organs (cases 12,
14, 18 and 19).
According to determined abundance of diatom cells in
analysed human organs, it is evident that primary organ to be
checked for diatom presence are by all means lungs in which
(except case 8) diatoms have always been recorded. As
exceptionally important are findings of diatom cells in the
brain which in certain cases (4, 5, 20) were more abundant
then those detected on the surface of the body. These results
point for the first time (at least in literature) this specific

Table 1
Total number of determined diatom cells in analyzed tissues and organs of rats

Group
Group
Group
Group

I
II
III (killed)
IV (drowned)

Lungs

Heart

Brain

Liver

Kidney

Stomach esophagus

Bone marrow

11
533
7
352.2

2
18
2.3
213

32
23
0.2
0.1

11
0.5
6.1

22
23
0.2
3.3

19
60
38
168

34
6

Group I: control group that drunk water saturated with diatoms; group II: test group drowned in water saturated with diatoms; group III:
control group of rats killed outside and then submerged in water saturated with diatoms for 7 days; group IV: rats drowned in water saturated
with diatoms and left for 7 days.

S. Krstic et al. / Forensic Science International 127 (2002) 198203

201

Table 2
Total number of determined diatom cells in analyzed human organs in 19972000 period
Case
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.

(26.05.1997)
10712/89 (5.06.1997)
10647/24 (5.02.1997)
10914/72 (6.05.1998)
10719/97 (6.05.1998)
10908/66 (6.05.1998)
11032/190 (9.02.1999)
11033/? 99.02.1999)
11132/33(18.02.1999)
11136/37 (27.02.1999)
11153/54 (30.03.1999)
11164/65 (9.04.1999)
11169/70 (12.04.1999)
11180/81 (23.04.1999)
11401/302 (29.12.1999)
11477/75 (10.05.2000)
11478/76 (11.05.2000)
11524/122 (16.07.2000)
11573/171 (31.08.2000)
11562/160 (18.09.2000)
11607/205 (15.10.2000)
11660/258 (15.12.2000)
11664/262 (17.12.2000)

Skin, hair,
surface

Lungs

Heart

Brain

Liver

Kidney

Stomach,
esophagus

Bone
marrow

144.3

171.5
107.8

298.2
261.3

15.8
118.5
95.3
145
181.2
99.3
55.7
49.6
92.8

6.7

4.5

37.2
266.4
81.4
110.6

82.5

0.2a
49.5
102.1
36
16.7
2.4
2.4
4.4
2.3
32.5
0.2
2.2


15.2

29.6
21.6
20.1


2.3
0.1
10.2


2.5

5.6
5.1
0.2
7.5
3.4
2.5
0.4


6.1

196.4
121.2
40.9
76.5
82.5
0.5
25.8

0.4


3.4
8.7
8.8
6.6

39.2


0.3

7.7
17.5

5.3
37
12.9
0.1



0.6

0.3
0.3
4.1
6
14.2

0.1
32.6

0.5
0.1

25.5
29.6

8.6
61.9
28.9
2.2
2.2
0.2
0.1
2.1


4.1

2.8
0.3
0.1

18
0.3

0.3

11.4

0.5


6.1b
0.5
0.1

19.1

0.1

5.5
9.5
0.1

6.6

57.8
0.5

22.1



0.1


0.3

0.4
8.5
4.9
0.2
0.2
2.5
2.2

Organ not analyzed (); no diatoms present (); commas present single finding.
a
4.4 trachea.
b
Pancreas.

organ as most effective filter for diatom cells in the blood and
support the results obtained in tests with rats (Table 1).
Analysis furthermore showed that heart, kidney and liver
are somehow isolated group of organs which express more or
less similar diatom cells concentration (except in cases 5 and
20, where high diatom concentrations in both kidney and
liver have been found). As was also determined in rats, these
organs should always be analyzed for determination time
when death occurred, but also for determination of overall
diatom penetration in organs.
Especially, doubtful for their diatom composition according to obtained results certainly are stomach and esophagus,
as well as the bone marrow in cases of suspected drowning.
Highly irregular results in analyzing of these organs are
partly due to incorrectly sampled material during autopsy,
but also due to lack of experience in analyzing these organs
and the possibility the victim does not swallow sufficient
amount of water, or diatoms, during drowning. The bone
marrow on the other hand represents a problem which,
according to literature data (Peabody, 1980; Foged, 1982,
1983), still remains a mystery, mainly due to accumulation
of the inert silica diatom cells that originate from air, food or
water consumed by the possible victims. Future analysis of
this organ, both on drowned and non-drowned persons and
laboratory animals, would have to determine its real value

for diatom test, especially in cases of advanced putrefaction

of the cadaver.
Finally, obtained results of the only confirmed case of
non-drowned person (no. 6), who has died of poisoning and
had numerous findings of diatom cells in all available
organs, once more, points out the need of basic approach
to diatom method control in future which has to be conducted through principal data base in relation to:
 concentration of diatom cells in air;
 abundance of diatom cells in food (vegetables especially)
and drinking water;
 the possibility of contamination with diatom cells during
laboratory analysis of tissues and organs.

4. Conclusions
Conducted analyses on laboratory rats, available cases of
possible drowned persons and the control, on abidance and
composition of diatom cells in tissues and organs during
forensic expertise of drowning, enable the postulation of
following conclusions and future research trends:
 Affirmative diagnosis of drowning is one of the most
difficult in forensic pathology what has led to proposition

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S. Krstic et al. / Forensic Science International 127 (2002) 198203

of numerous test for confirmation of the real cause of

death.
The diatom method represents one of the most valid for
detecting the cause, place and time of drowning, if conducted properly on the field and in the laboratory with
maximal exclusion of possible mistakes.
In case, when analyzed tissues and organs are deprived of
diatom cells, possible death by water aspiration must not
be excluded. Final judgement must be issued by experts in
forensic medicine and diatomology.
Search for possible diatom presence should be conducted
in following order: field material from the exact place of
possible drowning (depending of the water ecosystem
samples from plankton, bents . . .), cadaver surface, lungs,
brain, heart (blood), stomach, liver, kidney, bone marrow.
Analyzes of possible victims of drowning should be
imperatively followed by the same in cases other than
drowning, as well as tests on laboratory animals, aiming to
formation of data base that will include information on
qualitative and quantitative composition of diatoms in air,
water and food in the region.
Overall analyzes of diatom cells abundance in organs
should have quantitative figures, like the total number of
diatom cells per gram tissue or organ, which in some cases
is the only reliable differential analysis for final decisiondrowning or no.
Qualitative analysis of diatom microflora from the place
of possible drowning, surface of the cadaver and internal
organs, actually enables valuable data on place, time and
real cause of death.
Reliability and validity improvement of diatom method in
future forensic expertise of drowning should inevitably
include testing of different methods for extraction of
diatom cells from tissues and organs (enzymatic method)
and certainly implementation of recent technologies like
electron microscopy and image processing.

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