Diatoms in lung tissue : first investigation in Brazil in proving death by drowning

Diatoms are microalgae widely distributed in inland and marine waters and during drowning they are inhaled together with water, passing through the bloodstream to various internal organs of a victim. Our investigation aimed to evaluate the diatoms extracted from the lung tissues of an individual with drowning diagnosis and compare them to those found in the environment, in order to prove the cause of death and the drowning site. A left lung fragment of 17.9 grams removed from the victim was oxidized and prepared for diatoms identification and quantification using inverted microscope. A total of 133 diatoms valves was found, being 85% belong to pennate forms and 15% to centric forms. Pinnularia, a bentonic genus, had greatest number of valves, followed by Fragilaria, Actinocyclus, Cocconeis and Encyonema. These results revealed that the victim drowned at the bottom of the lake, inhaling both water and sediments with diatoms. This is the first investigation of this nature carried out in Brazil .


INTRODUCTION
The use of diatoms to diagnose drowning began in Europe in the nineties with investigations by coroners [1][2][3][4][5][6][7].They found that human body parts such as the femur, brain, heart and lung used in the autopsy of victims by drowning had diatoms, a microalga that has cell wall (valves) with silica, thus resistant to degradation.These organisms are widely distributed in inland and marine waters, and during the drowning process, are inhaled together with the water passing through the bloodstream to various internal organs of the victim.
Successful evidences of diatoms in the pulmonary cavities, tissues and bone marrow greatly depends on their concentration in the water.The first investigations by Timperman [7] were not successful because in some rivers of Belgium, planktonic diatoms were sparse.
Investigations advanced in Europe encouraging diatomologists to study this theme and several case studies can be found in the book of Ludes & Coste [8].
v. 6, n. 3, 13-16, 20176, n. 3, 13-16, ISSN 2237-9223 -9223 Some researchers in forensic medicine, non-diatom specialists, questioned the value of the diatom test due to detecting these algae in peripheral organs of people who did not drown or their absence in cases of drowning [9].The absence can be explained by lack or shortage of diatoms in certain water bodies and the presence of these algae in the gut of people and animals can originate from raw water intake and not completely cleaned food containing diatoms.For a true diagnosis of drowning, these aspects must be observed, as well as possible external contamination on the autopsy table during the extraction of the body parts or during the storage and material preparation in the laboratory [10,11].
In recent years, drowning diagnostic studies using diatoms have advanced with the use of new methods of preparation and material analysis [12][13][14].A review about the diagnosis of death by drowning using diatoms can be found in Donadel et al. [15].For future applications and automatic use of diatoms in forensic medicine, studies mapping water bodies in India and the inventory of diatoms in major rivers in China are already being undertaken [16,17].
This study aimed to evaluate diatoms extracted from lung tissues of an individual with drowning diagnosis and comparing them to those found in the environment, in order to prove the cause of death and drowning site.This is the first reported case associating diatoms to death by drowning in Brazil.

Fact Description
The victim, a 20 year old male, was found on the shores of Sailors' Island in Guaiba Lake.Upon arriving at the Medical Legal Department (DML) of Porto Alegre, Brazil, he had a wound on left eyebrow, according to witnesses, by a hitting his head on a stone at the time he dove, a fact that probably led to a partial loss of consciousness and subsequent drowning.The diagnosis given by the medical examiner on duty on the date of November 25, 2012, was death by drowning.

Sample preparation
A left lung fragment of the victim located near the windpipe was collected and stored in a flask with formalin 4%.In the laboratory the fragment was weighed in a precision electronic weighing scale and oxidized with concentrated nitric acid in a glass Becker directly on fire.After fifteen minutes of preparation, the degraded material was placed in test tubes for washing and acid removal.We washed the material 12 times with distilled water, using a centrifuge (2,500 rotations/minute) for ten minutes.Later using a Pasteur pipette, the sample was distributed in sedimentation chambers with 2 ml capacity, allowed to settle for two hours for observation under an inverted microscope Zeiss (640 X magnification).

Analysis and Identification of diatoms
We analyzed all material in 12 chambers for identification and valves quantification.The results was expressed in numbers of valves per 17.9 grams.The diatoms were measured and recorded in digital imaging with equipment attached to the microscope (Power Shot G10).
The identification of genera and species was based on the reference collection (iconography) of the diatoms from Rio Grande do Sul at the Herbarium of the Museu de Ciências Naturais, Fundação Zoobotânica do Rio Grande do Sul.

RESULTS AND DISCUSSION
The analysis of 17.9 grams of oxidized lung fragment showed 133 diatoms.The valve concentration can be considered fairly high compared with literature [18][19][20].One can consider the diatom test as positive, since the minimum amount required for diagnostic confirmation is five valves per 10 grams sample of cadaveric tissue [12].Of the total number of taxa present, 85% belong to pennate forms of bilateral symmetry and 15% to centric forms of radial symmetry (Fig. 1).The latter occurs mainly in plankton (suspended in the water column) while the other originates from the benthos (sediment) or periphyton (marginal zone of the lake).Regarding the taxonomic composition (Fig. 2), the genera that had greatest number of valves were Pinnularia (38 specimens), which are benthic (epipelic) organisms [21], followed by Fragilaria (14), Actinocyclus (12), Cocconeis (5) and Encyonema (4).In the lung fragment, we found 32 genera, 6 species and 11 morphotypes, which due to its position in the chamber could not be identified.The taxa, in general, are known to be present in the Guaiba Lake Basin where the diatom community has been extensively studied [22][23][24] as well as in some rivers that flow into this lake [24][25][26][27].This knowledge allowed us to identify some abundant species (Plate 1, Figures 1-4) and some pennate diatoms belonging to several genera (Plate 2, Figures 5-16).Comparative analyzes of diatoms in lungs with those found in the water samples were also used successfully in rivers from France to indicate drowning sites [27].Our investigation revealed that the victim drowned at the bottom of the lake, inhaling both water and sediments with diatoms.The presence of the diatoms from Guaíba Lake in the victim's lung fragment confirms the drowning site.
This research has unprecedented character in Brazil and supports the use of diatom tests in forensic medicine.

Fig. 1 .
Fig. 1.Number of diatoms observed in the victim's lung fragment.

Fig. 2 .
Fig. 2. Valve numbers of the main genera observed in the victim's lung fragment.