Forensic Limnology: Current Relevance and Future Outlook

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Literature Thesis

Forensic Limnology: Current Relevance and Future Outlook

Agustina Florencia Brunetti Rolón

Student ID: 11403748

MSc Forensic Science, University of Amsterdam

Supervisor: Dr. J.M.H. (Jolanda) Verspagen Co-assessor: Dr. J.A.J. (Hans) Breeuwer Coordinator: Dr. A.C. (Arian) van Aasten Date: September 2018

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Abstract

Forensic Limnology can be defined as the study of diatoms in a legal setting. Diatoms are microscopic, photosynthesizing algae enclosed by a silica cell wall that makes it possible for them to be detected in drowning victims even in advanced states of decomposition. Such is the purpose of the diatom test, a fairly traditional diagnostic tool used in forensic pathology to determine whether a victim found in water drowned or was submerged post-mortem. This method is regarded as the "golden standard" for the diagnosis of drowning but, as forensics become more DNA- and computer-based, the question remains whether techniques which are simpler in principle will remain relevant. Therefore, the purpose of this thesis was answering the following question: What is the current relevance of Forensic Limnology, and what are its outlooks for the future? After reviewing the latest literature on the topic, it was determined that the relevance of Forensic Limnology lies on the fact that the diatom test is still being used routinely to diagnose drowning when the drowning signs of the victim are destroyed, and recent improvements in methodology and new openings in the field indicate that it is possible for this traditional procedure to adapt to the current times and continue being perfected in the future.

Introduction and Research Questions

What is Forensic Limnology?

To define the discipline of Forensic Limnology, it is best to start by explaining what Limnology entails. To put it simply, Limnology is the study of inland waters as interacting ecosystems, as well as the relationships between the organisms living in them, and their relationship with their medium. [1]

Therefore, Forensic Limnology should be easily defined as the application of Limnology in a legal setting. However, Forensic Limnology refers only to one group of organisms from inland waters in such settings, which are diatoms. [2] The name "Forensic Phycology" would arguably be more suitable for this discipline, while still not being completely accurate, since Phycology does not only study diatoms, but all algae.

Despite the mismatch between the definitions of Limnology and Forensic Limnology, this thesis will focus mainly on diatoms. Their traditional use in forensics, as well as some novel and promising applications of this discipline, will be explored in later sections, as a means of displaying a complete picture of its current relevance and potential outlooks for the future. The

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3 recent use of other aquatic microorganisms will also be discussed, as the field of Forensic Limnology is starting to open up to their study with modern techniques.

What are diatoms?

Diatoms are microscopic, photosynthesizing algae from the Class Bacillariophyceae (Phylum Ochrophyta) which occur in almost any kind of aquatic environment, where they have an important role as primary producers.

Diatom size usually varies from 20 to 200 μm in length, but certain species can reach up to 2 mm. Their most characteristic feature is the frustule, the cell wall they are enclosed by, which is made of silica. The morphology of the frustule is the main basis of classification for these microorganisms. It is divided into two subunits, the valves, which fit together by a connective zone in the middle called "girdle". Some species of diatoms can secrete a mucilaginous substance from their girdles, which makes them capable of reduced movement. However, most diatoms are non-motile, and therefore they live in suspension in their water medium, sediment or soil. The basic structure of a diatom can be seen in Figure 1, while a variety of frustule morphologies is pictured in Figure 2.

The frustule is an extremely robust structure, which makes the diatoms resistant to putrefaction. This trait is particularly interesting for forensic science, since it makes it possible to detect diatoms in heavily decomposed bodies of drowning victims. Such is the purpose of the diatom test, a diagnostic tool traditionally used in forensic pathology that will be presented in later sections of this thesis. [3-5]

Figure 1. Basic diatom structure. The aforementioned frustule, valves and girdle are labeled. Taken and modified from Round et al. [6]

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4 Figure 2. Several genera of diatoms observed under light microscope, as a means of illustrating how

diverse their morphology can be. Taken and modified from Kakizaki et al. [7]

Research question

As it will be discussed throughout the thesis, the common applications of the study of diatoms in forensic science are fairly traditional, and do not make use of the newest developments and technologies. As forensics become more DNA- and computer-based, the question remains whether techniques which are simpler in principle will still have a place in the future. Therefore, the research question that was formulated for the elaboration of this thesis was the following:  What is the current relevance of Forensic Limnology, and what are its outlooks for the

future?

Overview of Studied Literature

1. Use of diatoms in forensic pathology

As was already mentioned, the most well-known application of diatoms in forensic science is in the field of forensic pathology, where they can be used as a means of diagnosis of drowning, both in humans and in animals.

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5 Drowning can be defined as a type of death by asphyxia, where breathing is inhibited by submersion in a fluid. A death by drowning may be either accidental, suicidal, or homicidal. The diagnosis of death by drowning is usually based on drowning signs, such as the presence of frothing at the mouth and/or nostrils, the presence of petechia (red/purple spots caused by fractured vessels), the impression of ribs on the lungs, et cetera. These drowning signs are, of course, absent when the body is decomposed or skeletonized; in those cases, only the diatom test can be used to diagnose death by drowning.[8] The most cited paper on the topic of the diagnosis of drowning, written by Piette and De Letter [9], reviews several diagnosis methods and affirms that the diatom test is still the "golden standard" even though it is not free of controversy. Said paper is now twelve years old, but the diatom test is still in use and being improved, as will be discussed later.

Diatoms possess two characteristics that make the diatom test such a necessary tool for the forensic pathologist. First of all, they are resistant to putrefaction because of their frustule made of silica. And second, they do not occur naturally in the human body. It is these two traits of diatoms which make them the perfect candidates to be used as diagnostic tool, as will be described in the next section.

1.1. The diatom test – Identifying the cause of death

The diatom test can answer two very important questions that need to be asked when a body is recovered from water:

1. Was the victim still breathing when entering the water?

2. If yes, is the water medium where the victim was found the same medium they drowned in?

Was the victim still breathing when entering the water?

Regarding the first question, it is important to know that when diatoms are present in a drowning medium, they penetrate into the bloodstream of the victim as they breathe in the water. This leads to the distribution of the diatoms into other organs, such as the bone marrow, brain, kidneys and lungs. Therefore, the occurrence of diatoms in these organs (especially in the bone marrow) is considered proof that the victim was breathing and their circulation was still functioning when entering the drowning medium. This is considered to be enough evidence to determine the cause of death as drowning. A comparison of diatom distribution between a still breathing victim and a body that was dumped post-mortem can be found in Figure 3.

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6 Figure 3. Comparison between a drowning victim (left) and post-mortem dumping of remains (right). Diatoms are disseminated into organs only if the victim was alive, and therefore still breathing, when

entering the water medium. Taken from Sasidharan and Resmi. [10]

Is the water medium where the victim was found the same medium they drowned in?

Regarding the second question, to determine the drowning site a correlation must be established between the diatoms extracted from the victim and those of the drowning medium. If said correlation is not observed, it is possible that the body was moved, either by natural currents or by human activity (seasonality might also be the reason, which will be discussed in section 1.2. of this thesis). In those cases, the species of diatoms from the victim may be compared to regional samples to localize the original drowning site. The importance of these regional samples will be discussed further in later sections of the thesis.

1.1.1. How is the diatom test performed?

In order to examine the diatoms from the tissues of a drowning victim, they are usually extracted by acid digestion. This process also leaves the diatoms ready for microscopic examination (that is, clean of any materials that could interfere with their visualization, such as sand or mud). The standard process of acid digestion consists of four steps:

1. Collection of samples from the victim.

2. Removal of femur at autopsy. The bone is washed with distilled water and sectioned longitudinally with a clean band saw. 50g of bone marrow are removed with a spatula and put into a boiling flask.

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7 3. 50 mL of nitric acid (or sulfuric acid, less often) are added to the boiling flask, and the suspension is boiled for 48 hours. Hydrochloric acid is a newer option that is being used quite frequently nowadays.

4. The suspension is cooled and centrifuged. The supernatant is discarded, and the leftover sediment is put on a slide and examined under the microscope.

Nowadays, dark phase or electron microscopy are the preferred methods for microscopic analysis. In fact, the use of electron microscopes are necessary for taxonomic classifications, since the distinctions between species usually depend on punctual morphological characteristics. The Transmission Electron Microscope (TEM) is used to visualize these details of the frustule, while Scanning Electron Microscope (SEM) is used to visualize the entire frustule, both internally and externally. [3]

The microscopic examination determines whether the diatom test is positive or negative. It is positive if unequivocal diatoms are present on the slide, above a minimum of 20 diatoms per 100 microliters of pellet if obtained from lung tissue, or 50 diatoms per 100 microliters of pellet if obtained from other organs. A representation of how diatoms disseminate into organs can be seen in Figure 4.

The test is considered negative if there are no diatoms present on the slide. However, it is worth noting that a lack of diatoms in a sample does not immediately rule out drowning as a possible cause of death.

Figure 4. Schematic representation of diatom dissemination into the organs and use of the diatom test. Taken from Saukko. [11]

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1.1.2. Pitfalls of the diatom test

It is worth noting that the vast majority of drowning cases do not benefit from the performance of the diatom test. There are three main reasons why this might be the case:

1. Not all diatoms present in a sample are extracted with the current methods, and they are completely missed if they are not present in high concentrations. This is especially problematic in cases where the drowning medium is tap water; to pose an example, 71.4% of subjects who had drowned in the bathtub in Muccino et al.’s 2015 research

[12] showed negative results to the diatom test.

2. Drowning victims with circulatory or pulmonary conditions inhale a decreased volume of water, in a shorter timeframe, which leads to a shorter chance of incorporation of diatoms in the tissues. Victims who suffer quick drowning deaths also inhale less water, and in turn present less diatoms in their samples.

3. The volume of blood that circulates through the bone marrow, which is the main analyzed tissue when it comes to the diatom test, is relatively low.

Despite all this, the diatom test still has an enormous value, since there are not many other available tests for drowning diagnoses.

1.1.3. Recent improvements to the diatom test

Over the last few years, some novel developments have surfaced to overcome some of the discussed pitfalls of the diatom test.

1.1.3.1. New digestive methods

In Fucci et al.’s 2015 paper [13], they compared two different digestive methods for the extraction of diatoms: the currently most used extraction method in the forensic setting (37% HCl) to a method used in Italy for analyzing diatoms in ecological studies (40% H2O2 plus 1M HCl

digestion). The two methods were tested in the brain, lung, liver, kidney and bone marrow of ten subjects recovered from different water mediums. Hydrogen peroxide digestion turned out to be less chemically abrasive, since the diatoms that were extracted from the samples were preserved better than the ones extracted using hydrochloric acid. The lower degree of destruction of the diatoms allowed more genera to be identified in the samples; nine genera were identified in the hydrogen peroxide digestion samples, versus three genera from hydrochloride digestion. The methodology for both techniques was identical to that of the diatom test, except the samples were treated with 37% HCl before sedimentation for one group, and for the other group they were treated with 40% H2O2 and two drops of 1M HCl were added

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9 only downside that the authors mention is that the hydrogen peroxide method left some organic residues in the samples, which resulted in some interference during microscopic observation. However, they do not report this setback as a major issue, and determine that overall the hydrogen peroxide method yielded better results than the one currently used in forensics. While Fucci et al.’s aim was an extraction method that was less abrasive and therefore could preserve a higher quantity of diatoms, Kakizaki et al. [14] opted to develop an acid digestion protocol that was faster and less expensive than the conventional diatom test. The method that they propose was tested only in lung tissue, and it used Proteinase K solution and Buffer ATL in addition to the usual hydrochloric acid for the digestion of the sample. 1mL of Proteinase K solution and 9 mL of Buffer ATL digested the sample first, before heating it in hydrochloric acid and then washing it with ultrapure water and ethanol. The overall procedure only took about 3 hours, and it yielded between 60 and 23000 diatoms in the 20 lung samples that were tested. The authors report no interference during microscopic observation. The greatest advantage of this protocol over the usual diatom test is the quick obtaining of results; while the usual diatom test takes more than 48 hours to process the samples, this new protocol yields results in only 3 hours, which makes it possible to report them in the same day of the autopsy of the victim. However, the authors suggest using this method only on 1g lung samples, since testing other organs such as the liver kidneys requires 10-20g of tissue, and the amount of reagents would make the test too costly. Since detecting diatoms only in the lungs does not provide enough evidence as to confidently diagnose drowning, Kakizaki et al. suggest that this protocol could be used to assist on the diagnostic in a fast an inexpensive way, but not to categorically determine drowning as the cause of death of the victim.

1.1.3.2. Improving sensitivity

The low sensitivity of the diatom test is also an issue that needs to be solved for it to be considered reliable. Zhao et al. [15] developed a method to recover a higher number of diatoms from drowning victims, called the Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) method. As the name says, the method incorporates microwave digestion, membrane filtration and scanning electron microscopy, with the objective to lose less diatoms during the preparation of the samples. The authors tested their method on 128 victims, 115 of which were drowning victims and 13 were postmortem immersions. Lung, liver and kidney tissues were collected from the subjects; the samples were digested in a microwave digestion system with 8 mL of nitric acid and 2 mL of 30% hydrogen peroxide. After digestion, the samples were filtered on a membrane and the membrane was automatically scanned by SEM. Finally, the authors reviewed the pictures taken by the microscope to identify

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10 and quantify the diatoms. Overall, the process took from 1 to 3 h for each sample, and they obtained 112 positive results (97%). It is worth mentioning that they did not detect any diatoms in the liver and kidney of the 13 postmortem immersion cases, while they could detect diatoms in the lungs of all 128 subjects, postmortem immersion cases included. Overall, the use of the MD-VF-Auto SEM method resulted in less diatom loss, as compared to results obtained by the traditional high centrifugation speeds and observation with light microscope, proving to be more sensitive and also more specific than the conventional diatom test. However, the authors mention that further testing is needed, especially on more postmortem immersion cases.

1.1.4. Other uses of the diatom test

Another noteworthy use of the diatom test is for identifying the type of habitat where certain objects, such as clothing or footwear, have been submerged or put into contact with diatoms in any other way. These applications will be discussed further in the following chapter of the literature review.

1.2. Diatom fingerprinting – Identifying the place of death

As it was already pointed out, the diatom test is able to differentiate between death by drowning and post-mortem dumping of the body in water, by comparing the diatoms found in the victim to those in a water sample from the alleged drowning medium. In some cases, however, the drowning location is unclear. That is when diatom fingerprinting comes into play as an extremely useful tool.

Diatom fingerprinting consists of studying a body of water through time in order to characterize its diatom communities. Said communities have to be monitored continuously, since diatom flora fluctuates due to seasonality and other factors. It is known that diatoms are widely distributed in aquatic environments at the generic level, and also that at species level they have narrow tolerances to certain factors such as salinity, temperature and pH. It is these differences in diatom species composition which make it possible to infer characteristics of the water body that a sample was obtained from. [16] However, more in-depth studies are needed to back-up this widely accepted information. Four of such studies will be summarized next; the first one about site specific and seasonal diatoms [8], the second one about stratification of a water body based on its diatom communities [17], the third one about how the size, available nutrients and vegetation of different water bodies of an area can affect concentration and variability of diatoms [4], and the fourth one about the existence of diatom endemism and how changes in taxonomy can alter the perception of species distribution [5].

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1.2.1. Study A: Site specific and seasonal diatoms

In Vinayak et al.’s 2013 article [8], the authors monitored ten different water bodies in Haryana, India, over the span of two years. In order to perform the research, 2 L of water were collected monthly from the different water bodies to be monitored, measuring parameters such as temperature, pH and conductivity each time. The sample was preserved by adding 1 mL of formalin to restrict further diatom growth. Slides were prepared and observed under microscope to study the diatoms’ morphology.

Diatoms were divided into commonly occurring/dominating diatoms, least occurring/site specific diatoms, and rarely occurring/seasonal diatoms. Dominating diatoms occurred in a larger percentage and were mostly present all year round. Site specific diatoms were also present in all seasons, but in a lesser percentage than dominating diatoms. Seasonal diatoms were only observed in particular seasons.

A total of 111 taxa were registered over the two years. Even though certain species were very common and were found in most sites (such as Asterionella or Cyclotella), each of the sites presented its own site specific diatoms, some of which were also seasonal. Those are the species that hold the most interest to forensic science, since they could potentially be used to link drowned remains to the drowning medium, in case it was unknown. Therefore, performing diatom fingerprinting in the water bodies of the different regions would be extremely useful, albeit costly, especially in cases when diatom screening is the only available tool and knowing the drowning medium of the remains is key to the case.

1.2.2. Study B: Stratification based on diatom communities

Coelho et al.’s 2016 research [17] attempted to perform diatom fingerprinting by stratifying an estuary based on diatom composition in Oporto, Portugal, instead of focusing on seasonal differences as Vinayak et al. Their study compared the diatom species found in the lungs, liver, kidneys, bone marrow and stomach content of the victims to those in the water body where they were found, which was the Douro river estuary for all 37 cases. The method used for extracting the diatoms was digestion with 37% HCl, centrifugation and observation with an optic microscope.

While they did find a strong correlation between the diatom content of the samples and the drowning medium, they noted that the Douro river estuary has too much anthropogenic influence as to observe significant differences between the diatom content of the five places of the estuary that were sampled during the research. Since determining a stratification of the estuary based on these communities was not possible, diatom fingerprinting would not be useful

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12 to determine the exact location in the estuary where the victim drowned. However, they still built a diatom database for the estuary during their experiment, and confirmed the importance of both collecting water samples along with the body of the victim, and having available databases for water bodies where drowning cases are usual, in order to facilitate the task of confirming whether a body found in water drowned in said medium or anywhere else.

1.2.3. Study C: Effects of different variables over spatial distribution

In Bouchard et al.’s 2004 paper [4], the spatial distribution of diatoms in different water bodies of the Canadian Artic was researched, as well as the relationship between certain taxa of diatoms and different environmental variables.

In that study, variable concentrations of diatoms within regions were reported. These concentrations ranged as many as 5 orders of magnitude. Concerning the variety, as many as 85 taxa were recorded from the largest studied lake. Overall, bigger water bodies yielded a higher variability of species, probably because of the larger range of different habitats available within (so less competitive species of diatoms could thrive as well). On the other hand, the concentration did not seem to be correlated with any of the studied variables. It is worth noting, however, that concentrations where low when there was scarce vegetation and nutrients, as it is to be expected.

Diatoms have an important role in aquatic environments as primary producers. In said environments, they also have certain preferences that affect their concentration and their distribution. For example, their population numbers vary depending on stress and other characteristics of their habitat; these characteristics also have an influence on how diatoms are distributed in different bodies of water.

Diatoms have the ability to disperse [5], using dispersing agents such as the wind or animal vectors. This ability to disperse leads diatoms to be considered as cosmopolitan (i.e. the species is distributed around all or most of the world in their suitable habitats). However, some diatom species have been reported to be endemic (i.e. the species’ distribution is limited to a certain geographic location).

In order to be able to study the distribution of diatoms, it is important to understand their taxonomic classification. The reason is that a wrong assessment of taxa could lead to either an over- or an underestimation of the distribution of diatoms, which would in turn affect the credibility of Forensic Limnology as a discipline.

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1.2.4. Study D: Diatom endemism and the importance of taxonomy

In Vanormelingen et al.’s 2008 paper [5], the existence of diatom endemism was discussed, as opposed to the ubiquity hypothesis. The ubiquity hypothesis states that microorganisms are never endemic because of the size of their population, which equals an ubiquitous dispersal and therefore leaves no room for endemism. The authors proved that the distribution of diatoms can be narrow endemic for certain taxa. Just like Bouchard et al. [4], they also stated that the structure and the diversity of diatom communities depend on geographical factors and the size of the habitat, and that they are regulated, at a minor degree, by the same mechanisms that regulate communities of macroorganisms. One of the cited examples was the distribution of certain taxa of cyanobacteria and archaea which grow exclusively in hot springs.

The management of species taxonomy of diatoms has been a matter of debate for many years. The tendency to lump together many species, based on their morphology alone, has led to an underestimation of the true diversity of these microorganisms, as well as to the belief that they have mostly cosmopolitan distributions. In turn, this poor exploitation of diatom diversity diminishes the potential of diatom fingerprinting, since a more specific classification of diatom species would strengthen the differences between the communities of different water bodies.

2. Use of diatoms in other disciplines

Even though the main application of diatoms in forensic science, as it has been explained thus far, is as a method of diagnosis of drowning as cause of death in humans, it has been proven they have the potential to be used in other disciplines. Such is the case of forensic veterinary, where the diatom test is also "golden standard" for the diagnostic of drowning in animals. On the other hand, diatoms are also applied in forensic geoscience, a discipline that consists mainly of the analysis of trace evidence of soils and sediments, which can provide useful information about the profile of the environment, or can be used to exclude a suspect from being present at a crime scene.

2.1. Forensic veterinary

The diagnosis of drowning in animals poses the same challenges as it does for humans. Questions such as "was the animal still alive when entering the water?", "was the body of the animal submerged in water post-mortem?" or "was the animal drowned in the same water body where it was found?" are just as relevant to investigations as they are for human victims, and therefore the diagnosis of drowning presents all of the same difficulties. [18]

In McEwen and Gerdin’s 2016 paper [19], they refer to the already mentioned downfalls of the diatom test, such as the relatively low sensitivity of the test and the high possibility of

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14 contamination of the samples during their preparation. However, they mention that the diatom test has been used in animals before, and how it is necessary to validate the use of this technique to be reliably applied to animals.

One of such cases is that of a 7-year-old German Wirehaired Pointer dog, explained in detail in Benson et al.’s 2013 paper. [20] While the dog did not drown (it was found having a seizure in a drainage ditch and it was euthanized a few days later due to its worsening condition), the finding of a low number diatoms in the dog’s tracheal wash was interpreted as a consequence of the previous aspiration of water from the ditch. The conclusion was further supported by the fact that the diatom species that were found (Planothidium lanceolatum, Craticula cuspidata, Nitzschia sigmoidea, Surirella minuta, etc.) are not species which are found in the water column but species that live attached to plants or on sediments, which were to be expected to be found in water from the ditch. Overall, the relevance of the findings comes from the fact that the diatoms extracted from the lungs were not reported as a possible contamination of the sample, but as a consequence of a previous inhalation of water. While a diagnosis of drowning was not necessary in this case, it was an indicator of the potential of the diatom test to function as evidence of water aspiration, and therefore of drowning in certain cases in animals.

The applicability of the diatom test to animals is further supported by the findings of Fucci et al. reported in their 2017 paper. [21] In this case, the authors performed the diatom test in ten carcasses: two magpies, a deer, two roes, and five otters. Four of the carcasses were recovered from water (one magpie, one otter, one deer and one roe); three of those were actual drowning cases (the magpie, the deer and the roe), and the other seven had different causes of death, such as gunshot injuries or vehicle collisions. The method they chose was the traditional hydrogen peroxide digestion, and the tissues that were chosen were the standard lung, liver and kidney. Diatoms were detected in eight carcasses: the five otters, and the three actual drowning cases. This lead the authors to conclude that the diatom test could be a valid tool for diagnosing drowning in animals, as long as ante-mortem contamination by diet during life is taken into account (as it was the case for the five otters), and rigorous validation of the method is undertaken, just as McEwen and Gerdin suggested in their 2016 article.

2.2. Forensic geoscience

2.2.1. Study on clothing

A possible application for diatoms was deemed to be that of pollen evidence in forensic palynology: being recovered from items which are frequently found at crime scenes, such as clothing, as a means of placing (or not) a suspect at the scene of the crime. In 2014, Scott et al.

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[22] tested three different methods on cotton t-shirts that had been in contact with water and soil, to test which method was the most efficient when it came to recovering diatoms from fabric. The methods were rinsing with water, rinsing with ethanol, and hydrogen peroxide (H2O2)

extraction. Different submersion times were also tested, as to reproduce different forensic scenarios. The times were 3 min, 30 min, 3 h and 24 h submersion. The samples were then examined using electron microscopy, and the efficacy of the methods was assessed by considering diatom count, species richness, and similarity of composition to the submersion medium. The tests were run on samples of only 1 cm2_{of fabric.}

Overall, a greater number of diatoms was recovered when using hydrogen peroxide extraction, no matter the submersion time, regarding which the highest number of diatoms were always recovered past 3 h of submersion. The reason why this was the most useful method could be that, in water mediums, the diatoms are weaved through the fabric instead of just lying on the surface, which means they need a more "vigorous" method than just rinsing with water or ethanol to be recovered. After 24 h the diatom counts decreased marginally, but they were still high; however, it must be pointed out that the use of this method would only be useful for instances when short contact (~3 h) between the piece of clothing and the water medium has taken place.

It is also worth noting that hydrogen peroxide extraction was the quickest method, and also that all three methods were non-destructive, which would allow the clothing to be tested for different traces after being tested for diatoms.

Regarding species richness, it was the highest in samples that were submerged in streams as compared to ponds. Composition data of the clothing samples showed the same trends as the submersion medium regarding relative abundance of the different species of diatoms.

Soil samples also yielded high counts of recovered diatoms, the highest also being when using hydrogen peroxide extraction despite most diatoms likely remaining on the surface of the fabric, as opposed to weaved through it.

Scott et al.’s paper received a reply from Dragutinovic et al., from the Netherlands Forensic Institute (NFI), in 2015 [23]. In their reply, they point out that while the H2O2 method seems very

promising, the way they performed the rinsing with ethanol method (published by them in 2010

[24]) was different in methodology, and therefore the results were not comparable. Scott et al. published a reply to Dragutinovic et al. in the same year [25], confirming that indeed there were some differences in methodology when replicating their rinsing with ethanol method, especially in the use of a weaker flask shaker, which in turn could account for the lower number of diatoms

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16 that they could extract with their method. However, they point out that the H2O2 method could

be an alternative when the kind of flask shaker that they used is not available, and also that they worked with smaller samples than Dragutinovic et al. did (1 cm2_{samples vs. 1 dm}2_{samples). In}

their case, the H2O2 method proved to be sensitive enough to extract enough valves to perform

a proper investigation even from such small samples, and therefore Scott et al. maintain that their method remains effective and practical for forensic casework involving recovery of diatoms from clothing.

2.2.2. Study on footwear

Footwear is an often overlooked but valuable source of trace evidence in forensic contexts. As a surge of interest in environmental trace evidence is currently taking place, Levin et al.’s 2017 article [16] reports a series of experiments that investigated the recovery, transfer and persistence of diatoms from footwear materials. Three different methods were tested: one of them was the hydrogen peroxide digestion method, and the other two were novel techniques which were safe for organic materials, jet rinsing and heating and agitation with distilled water. As there is no consensus on what the best method is for diatom extraction, performing experiments with novel methods has shed some new light onto the issue. Three materials were used to represent the upper materials of footwear (canvas, leather and suede), and two were used as sole materials (rubber and polyurethane). The water was collected from the Oxford canal, in the Upper Thames Valley; the water from the canal was well characterized species-wise, and all the genera that were present are usually found in British freshwater environments. The experiments yielded some very encouraging results. To begin with, the novel extraction methods did not yield lower counts of recovered diatoms than the established hydrogen peroxide digestion method, and it did not cause an increase in the fragmentation of valves. This suggests that these methods could be viable as alternatives when the use of hydrogen peroxide might be hazardous, due to the nature of the footwear material. It is especially useful for the cases when the examiner does not know what material the sample is made of.

It is also worth noting that diatoms could be recovered from all the materials after short immersion times (30 seconds being the shortest period), and that more diatoms were recovered from the rougher materials (canvas yielding the most) than from the smoother ones (polyurethane yielding the least). Therefore, in cases where the questioned footwear is made of different materials, it is most likely safer to sample the woven fabrics from the upper part of the footwear than the soles, which are made of smoother materials. Diatoms persisted in detectable quantities a week after immersion, in all materials too. However, after 168h, diatoms of bigger

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17 size (>200 μm2_{) could not be found in any of the materials. This size-selective retention is also}

commonly observed in other trace materials, such as fibers or glass.

It must be noted that the experiments were performed by removing the samples continuously (the pieces of fabric were attached to a boot and removed in the determined intervals), which made it impossible to monitor a single sample through time.

Lastly, longer immersion intervals yielded higher diatom counts, even though periods as short as 30s yielded an useful number of diatoms. This suggests that footwear may be sampled for diatoms even if the time of contact with a water body has been short. In turn, this flexibility of options means that there is potential for diatoms to be used in footwear trace analysis as a useful and reliable tool.

3. Recent use of other aquatic microorganisms

Even though diatoms are still the staple microorganism when it comes to aiding the diagnosis of drowning, the field is starting to open up to new possibilities, in the search for ways to palliate the shortcomings of this mostly traditional method.

3.1. Heterotrophic bacteria

Bacteria are beginning to be studied in the context of drowning diagnosis, especially using PCR techniques to detect their DNA in the organs of the victims. One of them is Aeromonas hydrophila, a Gram-negative, facultatively anaerobic bacteria that is ubiquitous to aquatic environments and even drinking water, and is also associated with gastroenteritis and other infections. [26] In Xu et al.’s 2017 paper [27], the hemolytic genes of this bacteria were amplified by PCR capillary electrophoresis in 36 drowning cases, in the lungs, liver and kidneys of the subjects. The performance of this method was compared to the detection of diatoms in the same subjects, which were detected with MD-VF-Auto SEM, the method developed by Zhao et al. [15] Overall, diatoms were detected in 100%, 97.2% and 91.7% of cases in the lungs, liver and kidneys respectively, while A. hydrophila was detected in 97.2%, 63.9% and 55.5% of cases. The authors considered these results to be indicative of a high potential for A. hydrophila to be used as a drowning diagnostic tool.

In Lee et al.’s 2017 paper [28], the microbiota of the lungs as a whole was analyzed, using a rat model and forming two groups: post-mortem submersion and drowning. The detection was performed by amplifying the 16S rRNA genes of the samples, and then analyzing it by high-throughput sequencing, as it is common in environmental microbiology. It was found that

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18 several aquatic microorganisms were present in the drowning group, but not in the post-mortem submersion group. These microorganisms belonged to the genera Morganella, Escherichia and Enterococcus. Therefore, it was determined that those microorganisms could be good indicators to help diagnose drowning. However, the authors do not consider the possibility of these microorganisms belonging to the microbiota of the victims instead of being exclusive to the drowning medium (these three genera belong to the family Enterobacteriaceae, some of whose members are normal inhabitants of the human microbiota [29]). No comments are made regarding the translatability of this study to humans either. The authors also pointed out that, since bacteria are smaller in size than diatoms, they move more easily into the circulation of the victims and are therefore easier to recover in bigger quantities than diatoms.

3.2. Phototrophic bacteria

In Rácz et al.’s 2016 paper [30], a PCR-based method was also used, this time to extract DNA from spleen tissues of four drowning victims and test it with cyanobacteria-specific primers. Cyanobacteria are an important group of phytoplankton (just like diatoms); they are the only prokaryotes which are photosynthetic, and they are widely distributed in aquatic environments.

[31] Their size, just like previously mentioned bacteria, also makes it easier for them to enter blood circulation than diatoms. In all of the four reported drowning cases, the diatom test was negative both in the spleen and in the drowning medium sample; however, when applying PCR method cyanobacteria were detected in all cases, and diatoms were detected in three cases. The water body types were groundwater, firewater storage, an adventure pool, and a ditch. The idea behind this article was tackling the issue of false-negative diatom tests, which are an issue due to the usually low number of diatoms which are recovered from the victims. The authors also mention how PCR-based methods are sensitive to contamination; in the case of cyanobacteria, contamination can be due to the victim’s skin or to tap water in the autopsy room; however, they tested the tap water and it turned out negative, and contamination because of contact with the skin can be avoided if the preparation of the samples is done carefully. Four other cases where victims were found in water but had not drowned in it were also assessed, and no cyanobacteria were found in their spleen tissue. They affirm that this PCR method could be used to supplement the diatom test in the diagnosis of drowning.

In Uchigasaki et al.’s 2016 paper [32], the widespread genus of cyanobacteria Synechococcus was targeted for detection in the lung, liver, kidney and blood of both drowned rabbits and rabbits that were dumped in water post-mortem. Again, the method that was used consisted on the amplification of the 16S rRNA genes of the cyanobacteria by PCR. In short, the authors found cyanobacteria DNA in the lungs of all six drowned rabbits, but also in two of the six non-drowned

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19 rabbits (in very small quantities). However, cyanobacteria DNA was only detected in the liver, kidney and blood only of the drowned rabbits. They speculate that cyanobacteria enter the lungs during the process of drowning, since drowned rabbits were noticed to aspirate a large amount of water before dying. The stopping of the circulation, however, makes it impossible for cyanobacteria to reach other organs after having drowned, and that is why they suggest that detecting phytoplankton DNA in organs such as the liver and kidney is more efficient to diagnose drowning than doing so in the lungs. They also considered the seasonal differences in phytoplankton composition in water (which were discussed in section 1.2. of this thesis), but they indicated than even so, they could detect phytoplankton in winter when they performed the study, the season when the phytoplankton count is the lowest.

3.3. Refinement of PCR-based methods

In Xu et al.’s 2016 paper [33], the authors went a step further and used a more refined PCR-based method for the detection of algal DNA in drowning victims. Magnetic nanoparticles (MNPs) were used first to extract DNA from the tissues of 20 drowned subjects, and then were run through a multiplex PCR-Capillary electrophoresis (MPCE) system specific for the detection of two microalgae as a possible means of diagnosis method. Overall, the objective of the authors was improving the detection rate of previous studies by incorporating the MNPs to the process. And they did, both compared to other PCR-based methods and to the MD-VF-Auto SEM method.

4. Real cases

After exploring how the diatom test works and how useful this traditional tool of forensic pathology can be in certain scenarios, all that remains is presenting some real cases where its use was essential to solve the case at hand.

In Malik et al.’s 2013 article [34], five fresh water drowning cases in Haryana (India) are presented where the diatom test was used during the examination. In the five cases, the samples (from the alleged drowning place, and the sternum, clavicle, femur and lungs of the victims) were processed for diatom testing using the same methodology.

One of the positive cases for drowning was that of a 23 year old female, recovered from a well, presenting no signs of injury. Even though her father claimed she had been murdered and then disposed of in the well, the diatom test showed presence of Nitzchia, Cymbella and Navicula species in her remains, as well as in the well water. Her death was confirmed as a drowning case.

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20 On the other hand, a negative case for drowning was that of a 26 year old female, recovered from a canal, with signs of head and neck injury. The diatom test was performed, and no diatom species were recovered from the victim’s remains, even though species of Nitzschia and Navicula were found in the water medium. Therefore, her death was ruled out as not drowning. Later on, her husband confessed that he had murdered her.

These two cases are perfect examples on how the diatom test is a powerful tool to clarify the circumstances of certain cases, where death by drowning is a possibility but not always the real cause of death of the victim.

Critical Discussion

After going through an overview of some of the recent literature regarding Forensic Limnology, what remains is taking a step back and addressing certain aspects of the discipline which are essential for any field that is applied to legal contexts. The critique will therefore be focused on answering three basic questions:

1. What are the strengths and pitfalls of Forensic Limnology as a discipline? 2. What is the reliability of Forensic Limnology as a discipline?

3. Is Forensic Limnology a discipline that is still relevant?

What are the strengths and pitfalls of Forensic Limnology as a discipline?

Since the current application of Forensic Limnology is based mostly on the use of the diatom test, the strengths and pitfalls of the discipline are firmly correlated with the strengths and pitfalls of the technique itself.

As we have reviewed, one of the main strong points of the diatom test is that it is not difficult to perform. It is not necessary to own expensive equipment to perform acid digestion and microscope examination (unless electron microscopy is chosen), and the laboratory technicians only need to be trained extensively if identification to species level by microscopy is necessary, instead of just detection.

However, the diatom test also has a number of drawbacks. To begin with, it has been reported that the test has yielded a considerable number of negative results in cases where drowning is the confirmed cause of death of the victim. Therefore, the sensitivity of the test is the main concern; nevertheless, efforts are being made to improve this traditional method and to incorporate more modern technology to it. Simple changes to the protocol, such as replacing

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21 the nitric acid from the extraction process to hydrogen peroxide, have proven to yield a higher number of diatoms from both organic and inorganic samples, such as clothing. The recently developed MD-VF-Auto SEM technique also appears to have strengthened the diatom test. On the other hand, it has been proposed that PCR technologies could be used to enhance the amount of diatom DNA recovered from a sample when it is too scarce, but the current taxonomy of diatoms is based on the morphology of the frustule, which would make it impossible to classify the microorganisms based on 16S rDNA sequencing alone. [35] Previous treatment of samples with acid or hydrogen peroxide would be detrimental to DNA, too. Therefore, incorporating PCR technologies does not seem to be the right way to go, while introducing changes to the standard diatom test protocol regarding acid digestion and microscopy appears to be more effective. PCR-based methods are, however, yielding positive results in studies on microorganisms other than diatoms, which should not be disregarded.

Overall, Forensic Limnology does have a number of setbacks that still need to be addressed. However, these setbacks do not seem to be as severe as those of other disciplines which are often questioned by forensic practitioners (such as bitemark analysis [36]), and it is also worth remembering that even well-established techniques such as DNA or fingerprint analysis have their own disadvantages too [37][38]. The lack of an alternative diagnostic tool for cases where the remains recovered from water are too decomposed to rely on drowning signs, gives the diatom test a guaranteed place in the forensic pathologist’s toolbox for now.

What is the reliability of Forensic Limnology as a discipline?

As it was mentioned previously, the diatom test has been reported to yield false negative results in cases where drowning is confirmed as the cause of death. However, the standard protocol of the test is being modified so that more diatoms can be recovered from samples, by using less abrasive acid digestion methods or automating microscopic observation. This augment in sensitivity will most likely reduce the number of false negatives, and the reliability of the test will increase.

Nowadays, Forensic Limnology has enough credibility as to still be used in forensic settings, despite its drawbacks. The standardization of an optimized protocol would help solidify the foundation of the discipline, and therefore it would be an advisable next step towards clearing out remaining controversies.

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22 Is Forensic Limnology a discipline that is still relevant?

Forensic Limnology is still a useful tool nowadays, since its use is quite common especially for diagnosing drowning in decomposed remains.

As for the investment in research, several recent studies concerning developments in the field have been reviewed in this thesis, which indicates that there is still an interest in improving the methods. In Figure 5 the number of articles about Forensic Limnology published per year are graphed; as it can be seen, the tendency is mostly positive, with remarkable peaks in 2013 and 2015, and an already higher number of publications than last year, at the moment of the writing of this thesis.

Figure 5. Number of publications about Forensic Limnology published per year, from 2010 to September 2018. Searched in PubMed (queue: "forensic" AND "diatoms").

Recommendations and Conclusion

A few general recommendations can be suggested after delving into the world of current Forensic Limnology.

One of the pressing issues concerns the optimization and standardization of the diatom test protocol. Several of the reviewed studies indicate that the use of nitric or hydrochloric acid for acid digestion is not the most suitable option, with less abrasive methods such as the hydrogen peroxide method or the Proteinase K method yielding a higher count of diatoms from samples

0 2 4 6 8 10 12 14 16 2010 2011 2012 2013 2014 2015 2016 2017 2018 (until Sep)

Publications

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23 and preserving more genera. The introduction of automated microscopy (in the MD-VF-Auto SEM method) also seems very promising. Therefore, it would be a great step to standardize the protocol using these more suitable options, to grant more reliability and repeatability to the diatom test. Another interesting observation is that the diatom test focusses mainly on the detection of diatoms in bone marrow, but it has been reported that in quick drowning deaths the amounts of inhaled water and short circulation time sometimes is the reason behind obtained false negatives. It would be best to diagnose drowning from other organs, such as the kidneys or the liver, which have yielded better results in recent studies than bone marrow. As long as the organs are not completely liquified, they could be sampled and diatoms should still be detected, since their silica cell wall would make them persist.

Nowadays, more microorganisms than just diatoms are being researched as possible aids for the diagnosis of drowning. It is advisable to focus on those microorganisms most likely to be waterborne than to belong to the microbiota of the subject, such as phototrophic bacteria, instead of those whose precedence might be more difficult to elucidate. An added recommendation would be to perform these studies preferably in human subjects, since human and animal microbiota are not the same, and this might translate into the obtained results not being applicable to human victims.

Lastly, investing in more research towards advances in diatom fingerprinting would be very valuable. Characterizing the diatom communities of water bodies and creating databases will help tremendously in those cases where the drowning location is unclear, or where the suspect needs to be linked to a water body based on trace evidence. Further studies regarding transferability of diatoms to items such as clothing or footwear are recommended, too. To wrap it up, let us address the initial research question of this thesis one more time. The question was the following: What is the current relevance of Forensic Limnology, and what are its outlooks for the future? The final answer to that question is the following: the relevance of Forensic Limnology lies on the fact that the diatom test is still the golden standard to diagnose drowning when the drowning signs of the victim are destroyed, and recent improvements in methodology and new openings in the field indicate that it is possible for this traditional procedure to adapt to the current times and continue being perfected in the future.

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Appendix: Search Strategy

For the articles, I used the Web of Science and PubMed, and the following combination of keywords: "diatom" and "forensics". "Forensic limnology" was searched as well, but not many authors use this terminology.

The authors with more published works about the topic, such as Zhao J and Fucci N, were researched individually. Some articles were found by looking at the references of other reviewed studies.

For the books, they were searched in Science Direct using the necessary keywords for the concerning topic (such as "diatom morphology" or "waterborne disease").

Forensic Limnology: Current Relevance and Future Outlook

Literature Thesis