Interdisciplinary Collaborations and Advancements: Amplifying the Potential of Diatom Analysis

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Interdisciplinary Collaborations and Advancements: Amplifying the
Potential of Diatom Analysis
Debhjit Mukherjee1*, Bhavika Moza1, Shantanu Pathak2, Anveshion Chaudhury2, Annesha Saha3,
Priyanka Dhondiyal4
1,2,4Post Graduate, Department of Forensic Science, Chandigarh University, Ludhiana, Punjab
3Post graduate, Department of Forensic Medicine and Toxicology, Kasturba Medical College, Manipal Academy of
Higher Education, Manipal, Karnataka
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Abstract
Forensic Limnology, a specialized subdivision within forensic science, utilizes diatom analysis as an indispensable tool for
investigating cases near water bodies. Diatoms, minute algae with intricately patterned siliceous shells, serve as vital ecological
indicators, providing critical evidence in diverse forensic scenarios. This comprehensive review underscores the paramount
importance of diatom analysis in cases involving drowning, suspicious deaths near water bodies, and bodies disposed of in
aquatic environments. Innovative techniques, such as Lefort aqua regia and fluorimetry, significantly enhance diatom
extraction and detection efficiency. Optimal clothing material selection, favoring cotton, acrylic, and viscose, profoundly
influences diatom retrieval dynamics, optimizing trace evidence collection. Despite inherent limitations, meticulous adherence
to standardized protocols ensures reliable and robust results. The cutting-edge Microwave Digestion - Vacuum Filtration -
Automated Scanning Electron Microscopy approach showcases unparalleled precision in the estimation of postmortem
intervals. Diatoms also function as invaluable geographical tracers, expertly aiding crime scene localization and facilitating
meticulous tracking of individuals implicated in criminal investigations. While DNA barcoding holds immense potential in
diatom identification, further database development remains imperative for widespread application. Inclusive case study
demonstrates the diatom test's effectiveness in conclusively establishing drowning as the primary cause of death, bringing
clarity to intricate forensic investigations. Diatom analysis within the field of forensic limnology emerges as an indispensable
reservoir of invaluable evidence, notwithstanding its inherent limitations. Persistent advancements and seamless
interdisciplinary collaborations hold the key to amplifying its potential, thereby fortifying justice-seeking endeavors and
bestowing closure upon victims and their bereaved families.
Key Words: Forensic Limnology, Diatom analysis, Drowning, Postmortem interval, Geographical tracers, DNA barcoding,
interdisciplinary collaborations etc.
1. INTRODUCTION
Forensic science assumes a paramount role in contemporary criminal investigations, furnishing invaluable evidence and
profound insights that facilitate the quest for justice. Within this diverse and interdisciplinary field, diatoms, minute algae
embellished with intricately patterned siliceous shells, have emerged as captivating and irreplaceable tools for forensic
analysis [1][2], [3]. Forensic Limnology, a highly specialized subdivision within forensic science, directs its focus towards the
scrupulous examination of freshwater ecosystems encompassing lakes, rivers, ponds, and wetlands, all with the primary
objective of assisting criminal investigations. This distinctive domain adroitly employs limnological principles and techniques
to meticulously gather evidentiary material, meticulously reconstruct sequences of events, and provide profound insights into
diverse forensic cases involving bodies discovered in or near aqueous environments. An indispensable facet of forensic
limnology resides in the thorough scrutiny of diatoms, singular-celled algae embellished with siliceous shells known as
frustules [4]. The precise tally of diatom species remains elusive, but it is frequently approximated to be approximately 104 [5].
Nevertheless, scholarly insights from [6]allude that adopting modern species concepts might escalate this enumeration to no
less than 105 [7]. These photosynthetic algae are primarily sessile, demonstrating limited motility along a substrate through
the excretion of mucilaginous material along a well-defined groove or channel known as a raphe. Diatoms, formally classified
within the Division Chrysophyta and Class Bacillariophyceae, are further taxonomically delineated into two Orders. The first,
presently identified as the Biddulphiales but previously denoted as the Centrales, showcases valve striae predominantly
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organized around a point, annulus, or central areola, resulting in a distinctive radial symmetry. Conversely, the Pennales
(Bacillariales) exhibit valve striae arranged in relation to a line, contributing to their characteristic bilateral symmetry. The
diatoms' silica-based skeletons display remarkable resistance to decay, facilitating their detection even in heavily decomposed
bodies [1]. The presence and dispersion of diatoms in aquatic environments bestow invaluable insights in forensic
investigations, particularly in cases involving drowning, suspicious deaths near water bodies, or bodies disposed of in water.
When an individual drowns, they may inhale or ingest water containing diatoms, and these minute organisms can permeate
the body and be conserved in diverse tissues or organs. Scrutinizing diatoms in postmortem samples yields precious clues,
such as the presence of specific diatom species and their abundance, facilitating the determination of the likely location of
drowning or the body's movement within water systems. Given the characteristic ecological predilections and regional
distributions of diatoms, they function as geographical tracers, greatly assisting investigators in honing in on potential crime
scenes or victim origins. Furthermore, diatoms play a crucial role in extrapolating the postmortem interval, a pivotal factor in
forensic investigations, particularly when a cadaver is retrieved from aquatic environments. By meticulously examining the
colonization and decay progression of diatoms in the body, forensic scientists can craft well-informed assessments concerning
the duration of submersion, proving to be indispensable in criminal investigations [8], [9], [10], [11]. The principal aim of this
exhaustive review is to meticulously investigate the importance of diatom analysis in the field of forensic science, specifically
emphasizing its relevance in cases pertaining to cadavers discovered in or proximate to aquatic environments. The article aims
to illuminate the diverse applications of diatom analysis in forensic investigations, especially in drowning, suspicious deaths
near water bodies, and bodies disposed of in water, providing invaluable insights for justice-seeking efforts. It will also address
the challenges and potential advancements in diatom analysis, including emerging research areas and cutting-edge
technologies that can enhance investigation reliability. By culminating with a concise summary of diatom analysis' importance
in forensic limnology and offering insights into interdisciplinary collaboration, the article aims to contribute to the
advancement of forensic science and informed decision-making for the benefit of victims and their families.
2. METHODS FOR EXTRACTION AND DIATOM DETECTION
Acid digestion is commonly used to recover diatoms from the tissues of a drowning victim for examination. This procedure
also prepares the diatoms for microscopic study (by removing any contaminants that would obstruct vision, such as sand or
dirt).
In the study of [12] an acid digestion approach was utilized to extract diatoms by effectively removing organic materials. The
process involved the addition of concentrated hydrochloric acid (HCl) to eliminate residues of calcium carbonate (CaCO3).
Subsequently, the suspension was subjected to boiling with concentrated sulfuric acid (H2SO4) until it turned black. Upon
cooling, solid sodium nitrate (NaNO3) was introduced into the suspension. Subsequent reheating led to the suspension
acquiring a brown hue, eventually transitioning to clarity. This transformation ensued as the nitric acid (HNO3) generated
during the process oxidized the carbon present, converting it into carbon dioxide (CO2). The resultant solution was
assiduously purified using diatom-free water and subsequently transferred to acetone to fabricate enduring microscope slides.
The absence of diatoms on the slide conclusively rendered the test negative.
An enzymatic digestion approach was employed for diagnosing putrefied drowned corpses. The study additionally
underscored the prospective efficacy of implementing a water surveillance system coupled with a diatom species database
compiled from diverse aquatic environments to facilitate the diagnosis of suspected drowning incidents. A proposal was
articulated to perpetually conduct surveillance of freshwater locations and create thorough species-level inventories of diatom
flora at these sites, thereby amplifying the effectiveness of medicolegal investigations into drowning fatalities. Until recently,
the qualitative parameters of the diatom test for drowning mainly focused on the presence and correspondence of diatoms in
tissues. [12]
In the study of [13], authors introduced a novel instrument called the "can," which adopted the acid digestion method for
diatom extraction. The "can" addressed the limitations of previous procedures by simplifying the identification process
through the efficient removal of organic materials. The instrument comprised three parts: the body, the inside cover, and the
outside cover, with Teflon filling the organic material during the destruction process. This design conferred corrosion
resistance, heat resistance, pressure resistance, and leak-proof characteristics to the "can." In the presence of a potent acid
reaction and elevated temperature, organic tissues underwent liquefaction for diatom extraction utilizing this apparatus. The
procedure entailed combining 3 grams of tissue sample with 4 milliliters of concentrated nitric acid within the "can,"

subsequently subjecting it to heating at 102°C for a duration of 100 minutes within a dry box. After cooling, the digested liquid
was centrifuged, and the residue recovered after centrifugation was placed on a slide for further examination. For another
technique termed the "Disorganization method," samples were digested with fuming nitric acid and sulfuric acid either in a
water bath (60°C - 180°C) or on a sand tray (80°C - 300°C). Subsequent to the oxidation process, a requisite quantity of H2SO4
was introduced to finalize the oxidation of organic materials, resulting in a violet solution upon saturation with potassium
permanganate and subsequent discoloration with oxalic acid. Following 48 hours of sedimentation, the solution was adjusted
to an approximate volume of 100 milliliters and subjected to centrifugation at 3000 revolutions per minute for a duration of 20
minutes until achieving a neutral pH. The leftover material was then utilized to create permanent diatom slides. The
introduction of the "can" and the "Disorganization method" provided improved and efficient means for diatom extraction,
overcoming previous challenges in organic material destruction, and facilitating diatom identification in forensic
investigations. [13]
In this research, the acid digestion method was employed using nitric acid for the extraction of diatoms from bone marrow.
This approach proved to be effective in isolating diatoms from bone marrow samples, facilitating their identification and
analysis in forensic investigations. [14]
In this study the authors showcased that the illuminative characteristics of fluorimetry can be utilized to discern and separate
diatoms in bone marrow or tissue specimens. Through the integration of distinctive fluorescent markers, this technique
enables the differentiation of diatoms recovered from suspected drowning sites from those naturally occurring in the
environment. This innovative approach provides a valuable tool for forensic investigations involving water-related deaths and
facilitates the accurate identification of diatoms associated with drowning incidents. [15]
Diatom analysis, an underexplored technique in forensic science, provides an autonomous ecological scrutiny of trace
evidence. This study furnishes empirical data to establish a foundational evidentiary framework for comprehending the
transference of diatoms onto clothing and the collection of diatomaceous trace evidence from diverse locations under
controlled conditions. Three diatom extraction methodologies were evaluated on garments in contact with soil and water sites:
rinsing in water (RW), rinsing in ethanol (RE), and submersion in a hydrogen peroxide (H2O2) solution (H). The efficacy of
each method in amassing representative samples for analysis was gauged by scrutinizing the total diatom yield and species
richness statistics from each experimental sample, alongside correspondence analysis to investigate similarities. The outcomes
disclose that H2O2 submersion proficiently extracts significant diatom counts with representative species from garments
exposed to both aquatic and terrestrial environments, validated by scanning electron microscopy (S.E.M.) analysis. This work
demonstrates that diatoms do transfer to garments in various forensic settings and highlights the H2O2 extraction as the most
efficient method for collecting comparison samples. Collecting and analyzing diatom components of geo-forensic samples hold
potential value in aiding forensic investigations. [16]
Acid digestion procedures in diatom analysis have inherent limitations, as they may lead to the destruction of diatom
structures during the treatment process. However, this study introduced a groundbreaking technique called Lefort aqua regia
(composed of a 3:1 ratio of nitric acid to hydrochloric acid) for recovering diatoms from tissue samples. This innovative
method outperforms traditional acid digestion approaches. The traditional acid digestion process for diatom analysis is
recognized for its time-consuming nature, labor-intensive demands, and potential hazards. The Lefort aqua regia technique
emerges as a noteworthy advancement in the field, mitigating these drawbacks. [17]
According to this study, the quantity of diatoms recovered and their retention dynamics over time were significantly influenced
by the type of clothing material used. Complex associations were observed with seasonal variations. Despite fewer diatoms
being collected during winter, the overall retention patterns remained consistent across seasons. The study suggests that
diatoms can be retrieved from clothing even weeks or months after their initial deposition, establishing them as valuable
environmental trace indicators for forensic reconstructions spanning diverse investigation periods. Significantly, cotton,
acrylic, and viscose fabrics were identified as the most dependable temporal repositories of diatom trace evidence, offering a
more extensive forensic assemblage accessible during the spring for forensic comparisons. [18]
According to this study, Sternal aspiration has proven to be a highly effective sample approach for diatom detection. One of its
key advantages is its simplicity, as it does not require any specialized expertise or equipment. Performing sternal aspiration
before autopsy can minimize the risk of contamination. Moreover, it is proposed that sternal aspirate is preferable to femoral
bone marrow for conducting diatom tests. [19]

Table-1: Diatom Extraction Techniques and Processes
Study Methodology Key Findings
Peabody,
1977
Acid digestion using HCl and H2SO4 to extract diatoms. Effective removal of organic materials,
aiding microscopic study of diatoms.
Ludes et al.,
1994
Enzymatic digestion was employed in the diagnostic assessment
of decomposed bodies associated with drowning incidents. The
study underscored the potential of water monitoring systems and
comprehensive diatom databases in elucidating cases involving
drowning.
Continuous monitoring of freshwater
locations and comprehensive diatom
species inventories recommended for
medico-legal investigations.
Yange et al.,
1999
Introduction of the "can" instrument using acid digestion for
diatom extraction. Also, the "Disorganization method" for
efficient diatom identification.
Improved and efficient means for
diatom extraction, overcoming
challenges in organic material
destruction.
Hürlimann
et al., 2000
Acid digestion method using nitric acid for diatom extraction
from bone marrow.
Effective isolation and identification of
diatoms from bone marrow samples.
Kumar et al.,
2012
Use of fluorimetry to detect and isolate diatoms in bone marrow
or tissue samples.
Fluorescent markers facilitate
differentiation of diatoms from
drowning sites and those naturally
occurring in the environment.
Scott et al.,
2014
Evaluation of diatom extraction procedures (RW, RE, H) on
garments in contact with soil and water.
H2O2 submersion identified as the
most efficient method for collecting
representative diatom samples.
Wang et al.,
2015
Introduction of Lefort aqua regia technique for diatom recovery
from tissue samples.
Improved method compared to
traditional acid digestion, addressing
time-consuming and hazardous
aspects.
Scott et al.,
2021
Influence of clothing material on diatom recovery and retention
dynamics over time.
Cotton, acrylic, and viscose fabrics were
discerned as dependable temporal
reservoirs of diatom trace evidence.
Szűcs et al.,
2022
Sternal aspiration as an effective sample approach for diatom
detection.
Simplicity, lack of specialized expertise
or equipment required, and preference
over femur.
3. DIATOMS IN POSTMORTEM SUBMERSION CASES
The scrutiny of diatoms in pulmonary tissues, internal organs, and bone marrow assumes a pivotal role in corroborating
drowning as the primary cause of death, offering valuable insights into the particular aquatic milieu involved. Diatoms, being
microscopic algae, accurately reflect the unique water quality of the environment where they flourish. Their presence and
specific types can provide valuable information about the location where the drowning incident occurred. Variables such as
salinity, pH spectrum, water depth, substrate characteristics, and aquatic vegetation exert influence over the composition of
diatomaceous species present in the medium. While qualitative and quantitative analysis of diatoms can help establish
drowning as the cause of death and provide insight into the aquatic surroundings, solely relying on the presence of diatoms in
lung tissues may not always yield definitive results. The post-mortem immersion of bodies in water may result in the ingress of
diatoms into the lungs, potentially introducing deceptive evidence. To tackle this challenge, forensic experts have advocated for
a quantification methodology, proposing a threshold concentration of 20 diatoms per 10 grams of lung tissue for investigative
purposes. However, it is essential to acknowledge that the concentration of diatoms in the drowning medium can vary

significantly, making a fixed criterion applicable to all drowning cases impractical. Diverse diatom species demonstrate evident
predilections for either freshwater or saltwater environments. By rigorously scrutinizing the diatom assemblage in
postmortem samples, forensic investigators can discriminate between cases involving submersion in freshwater or saltwater, a
pivotal factor in ascertaining the probable location of the incident. The conventional diatom testing method is hindered by its
limited sensitivity, which poses challenges in utilizing quantitative analysis to diagnose drowning cases effectively. However, a
groundbreaking and revolutionary methodology, recognized as the Microwave Digestion - Vacuum Filtration - Automated
Scanning Electron Microscopy method (MD-VF-Auto SEM method), has been innovatively devised. This state-of-the-art
technique substantially enhances sensitivity, thereby enabling more meticulous and precise extraction and detection of
diatoms during the testing process. Consequently, this innovative method significantly elevates the dependability and accuracy
of diatom tests in ascertaining drowning as the cause of death [20], [21], [22], [23], [24], [25].
4. DIATOMS AS INDICATORS OF GEOGRAPHICAL ORIGIN
Numerous meticulous studies have dedicated efforts to reconstruct historical sea level variations through detailed analysis of
diatom data sourced from diverse sedimentary deposits dating back to the late Quaternary period. The profound significance
of diatoms as sea-level indicators emanates from their nuanced ecological classifications, particularly regarding salinity.
Diatoms are adeptly classified into polyhalobian and mesohalobian classes, distinctly indicating marine and brackish
conditions, respectively. Oligohalobian forms unequivocally denote freshwater environments. In the context of salt marshes,
salinity fluctuations give rise to vertically zoned diatom assemblages, intricately linked to the tidal frame. These assemblages
encompass a diverse community transitioning from a predominance of freshwater taxa to salt-tolerant forms like Amphora
salina, notably under highmarsh conditions. Canonical correspondence analysis (CCA) is employed to delve deeper into the
intricate relationship between diatom species and salt-marsh environments. This sophisticated multivariate statistical
technique establishes a direct correlation between community composition and known environmental variations. CCA, with
meticulous precision, extracts synthetic environmental gradients from comprehensive ecological datasets, offering a concise
yet illustrative representation of diverse habitat water diatom taxa, especially aerophilous forms like Pinnularia microstauron.
This analytical approach significantly contributes to a comprehensive comprehension of diatoms' responses to ever-changing
salt-marsh conditions, solidifying their potential as exceptional indicators for sea-level variations throughout historical epochs.
Notably, the substantial variations observed in diatom assemblages across diverse water bodies, coupled with their exceptional
capacity to serve as distinctive geographical markers, underscore the pivotal role of diatoms as indicators of geographical
origin within forensic science. These empirical findings emphasize the remarkable potential of diatom analysis in furnishing
indispensable insights to investigators, facilitating precise geolocation of crime scenes and shedding light on the movements
and associations of individuals implicated in criminal investigations. The identification of diatom valves from Sach Pass at an
altitude exceeding 4000 meters opens up novel avenues for diatom research in the region. This study delves into diatoms
within a poorly documented geographic area, underscoring the necessity for extensive exploration across diverse
environmental conditions. The research presents insights into diatom diversity in the region based on five samples. Taxonomic
examinations of diatom assemblages are notably scarce in the western Himalayas, with predominant research focused on the
eastern Himalayas, central, and southern regions of India. The establishment of diatom databases in remote areas holds the
potential to contribute to conservation efforts and unveil previously unknown diversity in the region. Future investigations are
warranted to delve into the relationship between diatoms and glaciations, identifying species capable of thriving in extreme
conditions. [3], [26], [27], [28], [29], [30]
5. DIATOMS AS FORENSIC TIME MARKERS
The diversity of diatoms colonizing a corpse immersed in water undergoes a discernible sequence of changes, underscoring
their potential as a valuable tool for postmortem interval (PMI) estimation. As decomposition advances, the abundance of
diatom species diminishes, offering crucial insights for PMI assessment. According to [31], relying solely on aquatic insects for
postmortem interval (PMI) estimation may not consistently provide accurate results. To enhance the precision of estimates,
they proposed the use of semi-qualitative and qualitative approaches using aquatic plant models. Incorporating these
alternative methods can significantly improve the accuracy of PMI estimation. In a pioneering study conducted by [32] ,a
groundbreaking method for estimating the time since death at sea was introduced. This innovative technique involves a
comprehensive analysis of water elemental content and a meticulous examination of phytoplankton present on teeth. The
research uncovered intriguing trends, illustrating that the concentrations of elemental components inherent to teeth, such as
Calcium (Ca) and Phosphorus (P), exhibit a decline over time. Conversely, levels of Silicon (Si), Magnesium (Mg), and Potassium

(K) experience an increase. Noteworthy findings from the study indicate that roughly 30 days post-mortem, a substantial
portion of the enamel became adorned with phytoplankton, predominantly characterized by diatoms. Furthermore, around the
60-day mark after death, the entirety of the enamel surface exhibited coverage by these microscopic organisms. These
remarkable findings offer invaluable insights and hold substantial potential for precise postmortem interval (PMI) estimation
in aquatic environments. Moreover, the temporal succession of diatom biotas on a cadaver emerges as a robust and dependable
indicator for estimating the postmortem interval (PMI). By closely monitoring the dynamic changes in diatom composition
over time, investigators gain valuable supplementary clues to determine the duration since death accurately. Additionally,
diatoms extracted from the soil surrounding the decedent's body hold significant evidentiary value for PMI estimation. Their
presence and diverse characteristics furnish further critical insights into the precise timing of death, bolstering the accuracy
and reliability of the investigative process. [33]. The meticulous examination of diatom biodiversity and temporal succession,
complemented by state-of-the-art methodologies like water elemental analysis and phytoplankton examination on teeth,
constitutes a substantial enhancement to the precision and dependability of postmortem interval (PMI) estimation. These
scientific breakthroughs equip forensic experts with invaluable tools for ascertaining the timing of death in aquatic
environments, thereby strengthening the foundations of justice and facilitating informed decision-making in criminal
investigations. [34]
6. DIATOM DNA BARCODING IN FORENSIC APPLICATIONS
Over the past decade, the concept of DNA barcoding has emerged as a promising approach for identifying diatom taxa. A DNA
barcode functions as a tool to align an unidentified individual with a taxon possessing a congruent genetic sequence in a
reference database. Several gene regions, including cox1, rbcL, ITS, and others, have been proposed as potential barcodes for
diatoms. The identification of a suitable barcode necessitates a comprehensive reference database with a substantial volume of
sequences. Among the available sequence databases, the Silva ribosomal RNA database stands out for containing the most
extensive collection of nuclear small ribosomal subunit (16S/18S, SSU) sequences for diatoms[35]. The 18S rRNA gene, in
particular, has been widely explored as a potential barcoding marker for various organism groups. Notably, recently DNA has
been utilized barcodes from the 18S rDNA v7 region to distinguish nine independent diatom species in this study [36].
However, the current DNA barcode reference sequence database for diatoms is still significantly lacking and contains errors,
hindering the technology's widespread application. Consequently, many operational taxonomic units (OTUs) are not accurately
identified, resulting in molecular identification success rates lower than those achieved through microscopic identification.
Establishing a reliable diatom DNA barcode reference sequence database is a pressing task in the context of drowning forensics
[37].
7. CASE STUDY
In Haryana (India), the diatom test was applied in five freshwater drowning cases for assessment. In each instance, samples
were taken from the alleged drowning site, as well as the victims' sternum, clavicle, femur, and lungs, and processed using
consistent methods. In a noteworthy positive case, a 23-year-old woman, found from a well without apparent injuries, became
subject to her father's allegations of homicide and then disposal in the well. Despite the absence of external signs, the diatom
test unveiled the presence of Nitzchia, Cymbella, and Navicula species both in her bone marrow and the well water. This
finding substantiated the determination of drowning as the causative factor in her demise and not homicide.
In contrast, an instance involving a 26-year-old female rescued from a canal, exhibiting evidence of head and neck injuries,
yielded negative results for drowning upon application of the diatom test. Although Nitzschia and Navicula species were found
in the aquatic environment, no diatoms were detected in the victim's remains, ruling out drowning as the cause of death. The
victim's spouse later confessed to the murder. These two cases exemplify how the diatom test can elucidate the truth in
situations where drowning is a plausible but not definitive cause of death. The utilization of the diatom test for drowning
proved vital in determining the actual cause of death and providing clarity in forensic investigations. [38]
8. DRAWBACK AND LIMITATIONS
It is crucial to acknowledge that the diatom test may not be effective in most drowning cases, and there are three plausible
explanations for this limitation. Firstly, the current techniques used for diatom analysis may not completely remove all diatoms
present in a sample, leading to potential underestimation. Additionally, in cases where the drowning medium is tap water,

diatoms may be present in lower concentrations, and their significance might be overlooked. This is particularly problematic
for victims with circulatory or pulmonary issues who inhale lesser amounts of water over a shorter duration, resulting in fewer
diatoms integrated into their tissues. Another challenge arises from the fact that the volume of blood passing through the bone
marrow, a major tissue studied in the diatom test, is relatively low. As a result, the diatom count may be comparatively lower
than expected in some cases. Despite these limitations, the diatom test remains valuable as there are few alternative tests
available for diagnosing drowning incidents. However, careful adherence to all specifications during sample processing and
analysis is essential to minimize potential sources of contamination, such as instruments, gloves, papers, water supplies, and
reagents, as tap water may contain diatoms. [39], [40], [41]
This study underscored the potential for false-positive outcomes in diatom testing utilizing strong acid when employing
Kjeldahl flasks that have been reused. Thorough washing did not eliminate the presence of 290 false positive diatoms in reused
flasks. False positives occurred in 11 to 20 cases when diatom concentrations exceeded 10,000. The reused flasks mainly
contained common diatoms with a size less than 30 µm, such as Diatoma, Cymbella, Cocconeis, Gomphonema, Nitzschia, and
Navicula. To prevent false positives, it is crucial to use new and unused flasks for diatom analysis. Moreover, the study
suggested that closed organs may contain fewer diatoms in drowning cases than previously reported. As a preventive measure,
fresh flasks should exclusively be used when examining both drowning and non-drowning victims. [41]
9. CONCLUSION
Forensic Limnology, with a specialized focus on diatom analysis, emerges as an invaluable tool in investigating cases occurring
in proximity to water bodies. Diatoms, minute algae adorned with intricate siliceous shells, assume a paramount role as
ecological indicators, furnishing compelling evidence across diverse forensic scenarios[1], [4]. The comprehensive review
accentuates the profound significance of diatom analysis in cases involving drowning, suspicious deaths in aquatic settings,
and bodies submerged in water. Innovative techniques like the groundbreaking Lefort aqua regia and the illuminating
fluorimetry elevate the efficiency and accuracy of diatom extraction and detection[15], [17]. Optimal clothing material
selection, with a predilection for cotton, acrylic, and viscose, profoundly influences diatom retrieval and retention dynamics.
Notwithstanding its undeniable utility, the diatom test exhibits limitations encompassing contamination risks and challenges
in precise quantification. Nonetheless, scrupulous adherence to established protocols and methodologies is paramount to
guarantee the reliability and validity of obtained results. The revolutionary Microwave Digestion - Vacuum Filtration -
Automated Scanning Electron Microscopy method shows great promise in augmenting sensitivity and precision, specifically in
estimating postmortem intervals [20], [21], [22], [23], [24], [25]. Moreover, diatoms serve as invaluable geographical tracers,
facilitating the exact localization of crime scenes and providing invaluable insights into the movements of individuals
implicated in criminal investigations [26], [27], [28]. Despite its occasional constraints, the diatom test retains its pivotal status
in forensic limnology, furnishing critical information to ascertain the true cause of death. Furthermore, DNA barcoding exhibits
vast potential in diatom identification, although further database development is imperative to achieve widespread application
[42], [43]. Impeccable case studies from Haryana exemplify the diatom test's efficacy in discerning the veritable cause of death
in drowning cases, effectively distinguishing them from alternative causes of demise, thereby conferring clarity to forensic
inquiries. Overall, diatom analysis within the purview of forensic limnology yields invaluable evidence despite certain
limitations. Substantive advancements and interdisciplinary collaborations hold the key to further unlocking its potential in
the relentless pursuit of justice.
10. REFERENCES
[1] K. Verma, “Role of diatoms in the world of forensic science,” J. Forensic Res, vol. 4, no. 2, pp. 118–181, 2013.
[2] A. S. Rana and P. Verma, “Digestion of Concentrated Water Samples of Diatoms for Environmental Purpose by a Newly
Designed Method,” Jan. 2022.
[3] A. S. Rana, “Investigation of Diatom Communities in Highlands of Western Himalayas: Geographical and Ecological
Patterns,” pp. 1347–1367, Jan. 2022.
[4] Z. Levkov, D. M. Williams, D. Nikolovska, S. Tofilovska, and Z. Cakar, “The use of diatoms in forensic science: advantages
and limitations of the diatom test in cases of drowning,” The Archaeological and Forensic Applications of Microfossils: A
Deeper Understanding of Human History. McLean (Va), GeoScienceWorld, pp. 261–277, 2017.

[5] R. R. L. & K. P. Guillard, “The ecology of marine planktonic diatoms. In The Biology of Diatoms. Botanical Monographs,”
D. Werner, Oxford: Blackwell Scientific Publications, vol. 13, pp. 372–469, 1977.
[6] D. G. Mann and S. J. M. Droop, “Biodiversity, biogeography and conservation of diatoms,” in Biogeography of Freshwater
Algae: Proceedings of the Workshop on Biogeography of Freshwater Algae, Held During the Fifth International
Phycological Congress, Qingdao, China, June 1994, Springer, 1996, pp. 19–32.
[7] J. P. Smol and E. F. Stoermer, The diatoms: applications for the environmental and earth sciences. Cambridge University
Press, 2010.
[8] Y. Zhou et al., “Research advances in forensic diatom testing,” Forensic Sci Res, vol. 5, no. 2, pp. 98–105, 2020.
[9] C. R. Hardy and J. R. Wallace, “Algae in forensic investigations,” Forensic Botany: A practical guide, Wiley, Oxford, pp. 145–
174, 2012.
[10] E. J. Armstrong and K. L. Erskine, “Investigation of drowning deaths: a practical review,” Acad Forensic Pathol, vol. 8, no.
1, pp. 8–43, 2018.
[11] K. L. Erskine and E. J. Armstrong, Water-related death investigation: practical methods and forensic applications. CRC
press, 2021.
[12] B. Ludes, S. Quantin, M. Coste, and P. Mangin, “Application of a simple enzymatic digestion method for diatom detection
in the diagnosis of drowning in putrified corpses by diatom analysis,” Int J Legal Med, vol. 107, pp. 37–41, 1994.
[13] L. Yange, H. Chuanying, W. Chengxing, and W. Xu, “Development of can for destruction of organic material in use for
forensic diatom examination,” Forensic Sci Int, vol. 101, no. 3, pp. 163–166, 1999.
[14] J. Hurlimann, P. Feer, F. Elber, K. Niederberger, R. Dirnhofer, and D. Wyler, “Diatom detection in the diagnosis of death by
drowning,” Int J Legal Med, vol. 114, pp. 6–14, 2000.
[15] M. Kumar, J. Deshkar, S. K. Naik, and P. K. Yadav, “Diatom test-past, present and future: A brief review,” IJRRMS, vol. 2, no.
3, pp. 28–32, 2012.
[16] K. R. Scott, R. M. Morgan, V. J. Jones, and N. G. Cameron, “The transferability of diatoms to clothing and the methods
appropriate for their collection and analysis in forensic geoscience,” Forensic Sci Int, vol. 241, pp. 127–137, 2014.
[17] H. Wang et al., “A simple digestion method with a Lefort aqua regia solution for diatom extraction,” J Forensic Sci, vol. 60,
pp. S227–S230, 2015.
[18] K. R. Scott, V. J. Jones, N. G. Cameron, J. M. Young, and R. M. Morgan, “Freshwater diatom persistence on clothing I: A
quantitative assessment of trace evidence dynamics over time,” Forensic Sci Int, vol. 325, p. 110898, 2021.
[19] D. Szucs, V. Fejes, Z. Kozma, V. S. Poor, and K. Sipos, “Sternal aspirate sampling of Bacillariophyceae (diatoms) and
Cyanobacteria in suspected drowning cases,” J Forensic Leg Med, vol. 85, p. 102298, 2022.
[20] J. M. Lang, R. Erb, J. L. Pechal, J. R. Wallace, R. W. McEwan, and M. E. Benbow, “Microbial biofilm community variation in
flowing habitats: potential utility as bioindicators of postmortem submersion intervals,” Microorganisms, vol. 4, no. 1, p.
1, 2016.
[21] C. R. Hardy and J. R. Wallace, “Algae in forensic investigations,” Forensic Botany: A practical guide, Wiley, Oxford, pp. 145–
174, 2012.
[22] M. E. Benbow, J. L. Pechal, J. M. Lang, R. Erb, and J. R. Wallace, “The potential of high‐throughput metagenomic
sequencing of aquatic bacterial communities to estimate the postmortem submersion interval,” J Forensic Sci, vol. 60,
no. 6, pp. 1500–1510, 2015.

[23] G. C. Dickson, R. T. M. Poulter, E. W. Maas, P. K. Probert, and J. A. Kieser, “Marine bacterial succession as a potential
indicator of postmortem submersion interval,” Forensic Sci Int, vol. 209, no. 1–3, pp. 1–10, 2011.
[24] B. Ludes, M. Coste, N. North, S. Doray, A. Tracqui, and P. Kintz, “Diatom analysis in victim’s tissues as an indicator of the
site of drowning,” Int J Legal Med, vol. 112, pp. 163–166, 1999.
[25] J. Zhao et al., “A quantitative comparison analysis of diatoms in the lung tissues and the drowning medium as an
indicator of drowning,” J Forensic Leg Med, vol. 42, pp. 75–78, 2016, doi: https://doi.org/10.1016/j.jflm.2016.05.021.
[26] N. G. Cameron, “The use of diatom analysis in forensic geoscience,” Geological Society, London, Special Publications, vol.
232, no. 1, pp. 277–280, 2004.
[27] K. Pye and D. J. Croft, “Forensic geoscience: principles, techniques and applications,” Geological Society of London, 2004.
[28] B. P. Horton and Y. Sawai, “Diatoms as indicators of former sea levels, earthquakes, tsunamis, and hurricanes,” The
diatoms: applications for the Environmental and Earth Sciences, vol. 2, pp. 357–372, 2010.
[29] A. S. Rana, “Diatomological Profile at Discrete Depths of Powai Lake, Mumbai for Medico-Legal Purposes,” May 2023,
doi: 10.23880/ijfsc-16000301.
[30] A. S. Rana and P. Verma, “Diatoms Flora From Sach Pass Glacier of Himachal Pradesh: Species Composition And Their
Relationship To Environmental Variables,” p. 20038, Feb. 2023.
[31] K. A. Zimmerman and J. R. Wallace, “The potential to determine a postmortem submersion interval based on
algal/diatom diversity on decomposing mammalian carcasses in brackish ponds in Delaware,” J Forensic Sci, vol. 53, no.
4, pp. 935–941, 2008.
[32] N. Ishikawa, Y. Miake, K. Kitamura, and H. Yamamoto, “A new method for estimating time since death by analysis of
substances deposited on the surface of dental enamel in a body immersed in seawater,” Int J Legal Med, vol. 133, pp.
1421–1427, 2019.
[33] M. Wanner, E. Betker, S. Shimano, and R. Krawczynski, “Are soil testate amoebae and diatoms useful for forensics?,”
Forensic Sci Int, vol. 289, pp. 223–231, 2018.
[34] I. Kasprzyk, “Forensic botany: who?, how?, where?, when?,” Science & Justice, 2023.
[35] Z. Li et al., “Barcoding for diatoms in the Yangtze River from the morphological observation and 18S rDNA polymorphic
analysis,” Forensic Sci Int, vol. 297, pp. 81–89, 2019, doi: https://doi.org/10.1016/j.forsciint.2019.01.028.
[36] Y. Zhao et al., “Potential forensic biogeographic application of diatom colony consistency analysis employing
pyrosequencing profiles of the 18S rDNA V7 region,” Int J Legal Med, vol. 132, pp. 1–10, Nov. 2018, doi:
10.1007/s00414-018-1849-x.
[37] M. Liu et al., “Comparative study on diatom morphology and molecular identification in drowning cases,” Forensic Sci
Int, vol. 317, p. 110552, 2020, doi: https://doi.org/10.1016/j.forsciint.2020.110552.
[38] M. K. Malik, P. Jakhar, and A. Kadian, “Role of diatoms in forensic investigation: Case studies from Haryana,” Int J Forensic
Sci Pathol, vol. 1, no. 3, pp. 302–304, 2013.
[39] K. Ago, T. Hayashi, M. Ago, and M. Ogata, “The number of diatoms recovered from the lungs and other organs in
drowning deaths in bathwater,” Leg Med, vol. 13, no. 4, pp. 186–190, 2011.
[40] P. A. Siver, W. D. Lord, and D. J. McCarthy, “Forensic limnology: the use of freshwater algal community ecology to link
suspects to an aquatic crime scene in southern New England,” J Forensic Sci, vol. 39, p. 847, 1994.

[41] E. Kakizaki, N. Shinkawa, A. Sonoda, and N. Yukawa, “Conventional diatom testing using strong acid: Notable false-
positive results caused by an underestimated contamination source (blind spot),” Forensic Sci Int, vol. 330, p. 111131,
2022.
[42] Y. Zhao et al., “Potential forensic biogeographic application of diatom colony consistency analysis employing
pyrosequencing profiles of the 18S rDNA V7 region,” Int J Legal Med, vol. 132, pp. 1–10, Nov. 2018, doi:
10.1007/s00414-018-1849-x.
[43] Z. Li et al., “Barcoding for diatoms in the Yangtze River from the morphological observation and 18S rDNA polymorphic
analysis,” Forensic Sci Int, vol. 297, pp. 81–89, 2019, doi: https://doi.org/10.1016/j.forsciint.2019.01.028.

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