Postmortem Analysis in Drowning-Related Death Determination: Application of the “Diatom Test” in the Forensic Field in Bosnia and Herzegovina

Abstract

Microscopic signs are not specific only to drowning, but to any other type of suffocation, the background of which is mechanical obstruction. What about cases where there are no soft tissues when he has a skeletal body? This pilot research aims to examine for the first time the attendance of diatoms in teeth and bones, with regard to the peripheral vascularization of bones and teeth, in the Bosnia River. The thirty rats were divided into groups: Group A (n = 6; autopsy—1 hour after death; cause of death-hanging); Group B (n = 6; autopsy—72 hours after death; cause of death-hanging); Group C (n = 6; autopsy—immediately after drowning; cause of death-drowning); Group D (n = 6; autopsy—24 hours after drowning; cause of death-drowning) and Group E (n = 6; autopsy—72 hours after drowning; cause of death-drowning). In Group D and Group E, diatoms were found in one rat per group. By optimizing the “Diatom Test” in experimental settings, it could potentially become a routine method in the future. This is just the initial research that leads us toward optimizing testing and taking samples in cases of uncleared etiology, when there are no preserved soft tissue structures for autopsy.

1. Introduction

The primary function of the Agency, under prescribed jurisdiction and scope, is conducted through forensic investigations and examinations in dedicated laboratories staffed with personnel trained in specialized disciplines and equipped with advanced technology, following prescribed standards for each field. According to the type of examination, the Agency has established the following laboratories:

• Laboratory for examining computer systems

• Laboratory for graphological/graphoscopic examinations

• Laboratory for examining photo and video materials

• Laboratory for examining voice and audio recordings

• Laboratory for chemical–physical examinations

• Laboratory for chemical-toxicological examinations

• Laboratory for biological examinations and DNA analysis

• Laboratory for dactyloscopy examinations

Forensic medicine in Bosnia and Herzegovina has been utilizing conventional and available laboratories for DNA and toxicological sample analysis. In the case of unexplained deaths that are sudden, abrupt, or violent, it is within the competence of the prosecutor’s office to issue orders for autopsy and expert examination by an expert either singly or through designated institutes. Considering that diatom analyses are not usually used during the autopsy of drowning victims and are instead reliant on macroscopic assessments by forensic pathologists and microscopic observations, these two methods are mostly insufficient to be established as a definitive. Diatom test method, primarily, requires the development of technical and human resources and a profound knowledge of the classification of silicate algae (diatoms). Pathophysiological processes that occur during drowning have been known for a long time [1]. A fluid medium with sediment and microscopic organisms come through the initial parts of the respiratory and digestive tract and passes through the windpipe into the lungs and then through the alveoli into the circulation. The fluid with all its contents is transported to the organs by large blood flow and settles in their capillaries [2]. Diatoms as unicellular organisms remain present longer in the tissue material. Diatoms are present in freshwater habitat types. These unicellular algae can be easily isolated from organs by acid digestive extractions [3]. From then, until today, there are many conflicting opinions regarding these algae [4]. However, it is also a fact that there is no specific method for diagnosis that would help with the cause of death and the diagnosis of drowning. All types of asphyxia that arose in the background due to mechanical obstruction have nonspecific microscopic findings. Macroscopic signs of drowning on autopsy can be described as pulmonary edema and the presence of a deposit of small microscopic particles that reach the deepest parts of the respiratory system through the bronchial tree. What about situations where there are no soft tissues when he has a skeletal body?

Then, we have a bone and a tooth to identify the DNA and to determine the diatoms from them. Meanwhile, some researchers believe that the diatoms that can be found in other tissues got there from the lungs and that they could not have come through the digestive system [5]. Despite this, the diatom detection test is one of the most commonly used tests to diagnose drowning. Diatoms are a large group of algae that are present in stagnant waters as phytoplankton’s. There are more than 100,000 extant species, either floating freely, plankton forms, or attached to the substrate. Diatoms are a unicellular group of organisms that are formed in almost all water bodies. They occur in the water as plankton or on the bottom where they may be attached to plants or rocks or sand particles. Diatoms are being used excessively as forensic tools for the confirmation of death due to drowning or homicide. The valve-faced diatom frustules are ornamented with pores, spines, hyline areas, and other morphological features. Diatoms are divided into two orders: (1) the central (now called the areola) which tends to appear radially symmetrical and (2) the pennales which have valve striae arranged about a line that tend to appear on both sides symetrically [6].

1.1 Forensic importance

Diatoms find varied applications in all the fields of science and technology as well as in the field of forensic science. Diatomology is the study of diatoms, and forensic diatomology is the application of diatomology in forensic science. Forensic diamotology has a crucial role in resolving unclear cases of drowning. It is crucial both to solve the source of fatal outcome and to find the place where the drowning occurred. Whether the body was brought, moved, or drowned in the same water and found. Finding diatoms in tissues can play a key role in determining drowning. The species found must correlate and match the species analyzed in the water. The diatom extraction method was improved by acid digestion of the tissue. Way back in 1949, researchers discovered the presence of diatoms in the marrow of bones. When drowning, water is inhaled, and in this way, it reaches the deeper parts of the respiratory system, leading to the expansion of the lung alveoli. These algae pass the barrier between the alveolar walls and small blood vessels and then reach the pulmonary venous blood stream. This pulmonary venous blood, full of diatoms, reaches the left atrium, and then the left heart chamber, where it spreads throughout the circulation, leading to embolization of small blood vessels in numerous organs and tissues such as bone marrow. This supports the fact that fatal drowning was a factor contributing to death and was antemortem. The main purpose of the diatom test is to differentiate death caused by drowning, from submerging an already dead body in water to conceal the actual causes of death. Diatoms are specific indicators in forensics that help medical personnel establish the actual cause of death, especially in cases where personnel are unsure of the cause of death. These algae can be found in numerous organs and tissues, such as lungs, stomach, bone marrow, and other large organs [6].

The morphological characteristics of diatoms remain well preserved even after chemical digestion, which enables forensic scientists to recognize and classify them into groups. Certain types of diatoms are a specific finding in certain types of stagnant water, which enables forensic experts to establish the place of death because suspects cannot easily hide them or remove them from the body. Although a large number of species and subspecies of diatoms are found in different aquatic diversities, this is not encountered in most cases of drowning. Given the high sensitivity to changes in the environment and living conditions, the number of diatoms can vary between different types of water media [7]. If, due to mosquito bites, diatom species from tissue and water match, this will be strong evidence and indicator that the person drowned in that particular place. Limiting factors should be taken into account when analyzing the results of diatom tests, and these test results should be analyzed together with other evidence and indicators in order to determine the cause of death as accurately as possible [8].

1.2 Diatom test

Test with the use of diatoms is a forensic technique used to determine whether a person has died by drowning [9]. In the diatom test, samples of water and tissue from the lungs and other organs of the deceased are collected and examined under a microscope to determine if diatoms are present.

However, it is important to keep in mind that the lack of diatoms in the tissue samples does not necessarily rule out drowning [9, 10]. Overall, the diatom test is just one tool in a forensic investigation and must be used in combination with other findings and data to make a conclusive resolution of the cause of death.

One of the special significances of the diatom test is its possibility to provide strong evidence of drowning when diatoms are found in the lungs but not in the surrounding water.

Despite its usefulness, the diatom test has been criticized for its limitations. For example, the test requires the collection of tissue samples, which may be difficult or impossible in certain circumstances, such as when the body has been severely decomposed or lost at sea. Additionally, diatoms can be present in water sources that are not related to drowning, leading to potential false positives [11].

In conclusion, the diatom test is a valuable forensic tool in deciding about the cause of fatal outcomes in cases of drowning, but it should be used in combination with other data and findings to make a definitive determination about the cause of fatal outcomes.

1.2.1 The positive side of the diatom test

An advantage of the test with diatoms is that it can provide strong evidence that a person has drowned by identifying the presence of diatoms in the lung tissue of the deceased. Diatoms are specific to water and have unique shapes and sizes, which means that they can be used to identify the source of water in which a person may have drowned [12].

In cases where diatoms are found in the lung tissue but not in the sample of the water medium where the body was found, there is strong evidence that the person was alive at the time of submersion and inhaled water into their lungs. This can help forensic investigators to determine the cause of death and provide important evidence for legal proceedings.

The diatom test has also been used successfully in cases where other evidence is lacking, such as when a body has been in water for an extended period or when there are no witnesses to drowning [12].

Overall, the positive side of the diatom test is that it provides a useful and scientifically valid method for determining if a person has drowned and can provide important evidence in forensic investigations.

1.2.2 The negative side of the diatom test

The negative side of the diatom test is that there are certain limitations and the possibility of error that may affect the accuracy of the results of this test.

One of the main limitations of the diatom test is that it can only provide evidence of drowning if diatoms are present in the lung tissue. If the victim does not inhale water into their lungs, the test will not provide any evidence of drowning. Additionally, the absence of diatoms in the tissue does not necessarily rule out drowning, as diatoms may not always be present or may not be preserved in the tissue due to various factors such as the length of time the body was submerged or the temperature of the water [13].

Another limitation of the diatom test is that it relies on the collection of tissue samples from the body of the deceased, which may not always be possible due to various factors such as the condition of the body or the location where it was found.

The diatom test can also be affected by potential sources of error, such as contamination of the tissue samples or the water samples used for comparison. Diatoms may also be present in bodies of water that are not related to drowning, leading to potential false positives [14, 15].

2. Methods

An experimental randomized study was conducted using the Principles for Care of Experimental Animals at the Faculty of Medicine, Veterinary Medicine and Natural Sciences of the University of Sarajevo. After the approval of the Ethics Committee of the Faculty of Medicine of the University of Sarajevo, Bosnia and Herzegovina (number: 02–3-4-AK-6589/23), a total number of 30 adult Albino rats were included in the study (body weight: 250–300 g). The drowning model developed according to knowledge and practice was used. All conditions for the care and feeding of animals ad libitum were met.

Using an experimental model of drowning, we tried for the first time in our country to investigate the presence of diatoms in teeth and bones, immediately after drowning and after 24 and 72 hours.

2.1 Experimental design

Thirty rats were divided into groups: Group A (n = 6; autopsy: 1 hour after death; cause of death: hanging); Group B (n = 6; autopsy: 72 hours after death; cause of death: hanging); Group C (n = 6; autopsy: immediately after drowning; cause of death: drowning); Group D (n = 6; autopsy: 24 hours after drowning; cause of death: drowning), and Group E (n = 6; autopsy: 72 hours after drowning; cause of death: drowning) (Figure 1).

Figure 1.

Postmortem procedure for diatom isolation from tooth and bone samples (a: Rat after drowning; B: Tooth sample; C: Lat. os femur (bone); D: Suspension diatoms; E: Microscopy of diatom suspension; F: Determination of diatoms).

In groups A and B, the cause of death was hanging; then, they were drowned in a container containing river water and taken out 1 h and 72 h after immersion. A complete examination (macroscopic, microscopic, and diatom test) was performed on each animal. Guidelines for diatom analysis were used throughout all phases of the protocol, and they have been tested from the Bosna river. Teeth and bone femur were examined samples.

2.2 Collection of phytobenthos: Study site

The “experimental crime scene” took place at the confluence of the Željeznica river with the Bosna river from the coastal part (coordinates: 43°50′28”N and 18°17′10″E at an altitude of 490 meters). During water sampling for physicochemical analysis and benthic algae, it was observed that the sky was completely cloudy and there was a light breeze. No precipitation was recorded. The water color at the time of sampling was green. Basic physicochemical parameters of the water were measured using a portable multimeter. The conductivity was 337.8 μS/cm, and water turbidity was 2.42 NTU. The pH was 7.35.

In the coastal area where the sampling was conducted, the dominant vegetation consisted of floodplain forests of the Alnetea glutinosae class, where alder (Alnus glutinosa) and willow (Salix alba) were dominant species (Figure 2).

Figure 2.

Location of water and algae phytobenthos sampling.

After collection, the living algal material was transported to the Laboratory of the Biology Department of the University of Sarajevo. Phytobenthos samples were fixed with 4% formalin. For the laboratory approach, the method according to Hustedt [16] was used. Potassium permanganate (KMnO4), sulfuric acid (H2SO4), and oxalic acid (C2H2O4) were used for digestion. After the procedure, the cleaned diatom valves were embedded in Canadian balsam. Microscopy was performed under a magnification of 1000x (Best Scope 2020). The identification of diatoms is supported by the following references (17, 18). The methodology of the study was focused on the sampling and determination of benthic algae and monitoring the physicochemical factors of the water. Light microscope observation was conducted using the Best Scope 2020 microscope.

2.2.1 Determination of physicochemical water factors

Physicochemical factors of the water were measured immediately before the collection of benthic algae samples. Using a portable Orion Star A329 multimeter, the electrical conductivity and pH value were measured, while water turbidity was measured using a portable turbidimeter AQ3010.

3. Results

A total of 12 taxa were identified. Algae from the phyla Chlorophyta [2] and Heterokontophyta (10 taxa) dominated. The species diversity was composed of the following taxa: Nitzschia recta, Mougeotia sp., Meridion circulare, Pediastrum sp., Cymbella sp., Encyonema sp., Fragilaria vaucheriae, Synedra ul., Navicula sp., Surirella sp., Achnanthidium minutissimum, and Gomphonema minutum.

Representatives of the zoobenthos were also observed in the sample: Gammarus cf. bosniacus and larvae of aquatic insects (genus Baetis) (Figures 3–6).

Figure 3.

Species diversity.

Figure 4.

Valve view of Navicula cryptocephala (SEM microphotographs).

Figure 5.

Valve view of Cymbella cymbiformis (SEM microphotographs).

Figure 6.

Valve view of Pinnularia borealis (SEM microphotograph).

In Group D and Group E, diatoms were found in one rat per group, but in groups A, B, and C, no diatoms were found. The diatom test is positive when considering the tooth analysis (Figures 7–9).

Figure 7.

Results of the diatom analysis from the tooth (positive diatom test for groups D and E). NA: Diatoms are not analyzed. -: Diatoms are not found in samples; +: Diatoms are found in samples.

Figure 8.

Diatoms detected in the tooth (positive diatom test for groups D and E) (species detected: Denticula kützingii, Cocconeis placentula, and Gomphonema minutum).

Figure 9.

Results of the diatom analysis from the bones (negative diatom test for all analyzed groups). NA: Diatoms are not analyzed. -: Diatom is not found in samples.

Within all investigated groups, no diatoms were found. The diatom test is negative when considering the bone analysis.

3.1 Comparative analysis of benthic algae in the sample and isolated parts of rats (teeth and bones)

The water sample used for experimental purposes (rat drowning in laboratory conditions) was analyzed under a light microscope (temporary and permanent slides), and the presence of several algal species was confirmed, with diatoms being among the most dominant. Within the experimental group of rats in the tooth (groups D and E), diatoms were confirmed, including Denticula kützingii, Cocconeis placentula, and Gomphonema minutum.

A negative diatom test was observed within the experimental group in the bone. Diatoms were not found in any of the rats. It is important to note that the number of diatoms found in the teeth within the experimental groups D and E was extremely scarce, and the reason for this should be attributed to the quality and type of the collected water sample. The water sample for experimental purposes was collected during the summer season and was free of mud, fine sand, detritus, and stone pebbles. Unfortunately, the quantity of algae in the phytoplankton was not significant, which is evident in the investigated samples (Figure 10).

Figure 10.

Comparative representation of the presence of algae in the investigated samples.

4. Discussion

Whether drowning is the cause of death or the place where the body was found is just the final stage of a person’s disappearance is still becoming a very interesting and completely unanswered question in forensic medicine practice. However, dead bodies in water sometimes do not have predictable marks of drowning, which makes the diagnosis of drowning tremendously controversial. Since diatoms are photoautotrophic organisms, they can enter the human body through the respiratory tract during drowning and spread through the bloodstream to all organs in the body [8, 9, 10, 11, 12, 13, 14]. Meanwhile, the presence of diatoms in the tissues can be considered a vital reaction that indirectly proves that the deceased went through the drowning process [15]. The ability of diatoms to adapt and reproduce results in large differences in the distribution of diatoms in different biodiversities. Thanks to this knowledge, the potential determination of the place of drowning would have great forensic-medical significance. At the moment, the most common method of morphological examination is based on digestion with strong acids [16].

There are also certain limitations, which as examining a larger sample, in different parts of the year, and applying this methodology to the bodies that were pulled out of the water at the site of the investigation and examining the appearance of diatoms in the teeth. Despite all the applied methods in forensic death investigation, the use of diatoms can provide valuable support, and it is essential to consider the limitations of this method.

In river ecosystems, it is best to compare the composition of benthic algae, while in lake ecosystems, both phytoplankton and benthic algae composition can be considered.

In addition to soft tissues, for rapid screening, teeth or bones can be used. If the body has been in the water for a short time, diatoms may not reach the bones, only soft tissues and possibly teeth. Water and benthic algae sampling should follow the guidelines of the Water Framework Directive (BAS EN 15708:2011). Tissue sampling for analysis should also be optimized to minimize possible sample contamination [17, 18, 19].

Water and benthic algae, as well as phytoplankton, must be sampled by a well-trained person. The same applies to tissue sampling, preferably in laboratory conditions, using sterile equipment and adhering to safety measures for the sampler (biohazard).

5. Conclusion

The mentioned method has proven to be highly successful in assessing the location and time of drowning on a global scale, but it has not yet been widely adopted in local contexts. By optimizing the “diatom test” method in experimental settings, it could potentially become a routine method in the future. This is just the initial research that leads us toward optimizing tests in cases of unresolved etiology, when there are no preserved soft tissue structures for autopsy, and teeth and bones become available materials for diagnosing the cause of death with standardized nonspecific findings when there are no organs for micro- and macroanalysis.