Interdisciplinary Collaborations and Advancements: Amplifying the Potential of Diatom Analysis

International Research Journal of Engineering and Technology (IRJET) e-ISSN:2395-0056

Volume: 11 Issue: 01 | Jan 2024 www.irjet.net p-ISSN:2395-0072

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 ***

Abstract

Forensic Limnology, a specialized subdivision within forensic science, utilizes diatom analysis as an indispensable tool for investigatingcasesnearwaterbodies.Diatoms,minutealgaewithintricatelypatternedsiliceousshells,serveasvitalecological 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 influencesdiatomretrievaldynamics,optimizingtraceevidencecollection.Despiteinherentlimitations,meticulousadherence to standardized protocols ensures reliable and robust results. The cutting-edge Microwave Digestion - Vacuum FiltrationAutomated 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 claritytointricateforensicinvestigations. Diatomanalysiswithinthe fieldofforensic limnologyemergesasanindispensable 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 bestowingclosureuponvictimsandtheirbereavedfamilies.

Key Words: Forensic Limnology, Diatom analysis, Drowning, Postmortem interval, Geographical tracers, DNA barcoding, interdisciplinarycollaborationsetc.

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 objectiveofassistingcriminalinvestigations.Thisdistinctivedomainadroitlyemployslimnologicalprinciplesandtechniques tometiculouslygatherevidentiarymaterial,meticulouslyreconstructsequencesofevents,andprovideprofoundinsightsinto 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]. Theprecisetallyofdiatomspeciesremainselusive,butitisfrequentlyapproximatedtobeapproximately104 [5]. Nevertheless,scholarlyinsightsfrom [6]alludethatadoptingmodernspeciesconceptsmight escalatethis enumeration to no less than 105 [7] These photosynthetic algae are primarily sessile, demonstrating limited motility along a substrate through theexcretionofmucilaginousmaterialalonga well-definedgrooveorchannel knownasaraphe.Diatoms,formally classified withinthe DivisionChrysophyta and ClassBacillariophyceae,are furthertaxonomicallydelineatedintotwo Orders. Thefirst, presently identified as the Biddulphiales but previously denoted as the Centrales, showcases valve striae predominantly

Volume: 11 Issue: 01 | Jan 2024 www.irjet.net p-ISSN:2395-0072

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-basedskeletonsdisplayremarkableresistancetodecay,facilitatingtheirdetectioneveninheavilydecomposed 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,theyfunction asgeographical tracers,greatly assisting investigatorsinhoning in on potential crime scenesorvictimorigins.Furthermore,diatomsplayacrucialroleinextrapolatingthepostmorteminterval,apivotalfactorin forensic investigations, particularly when a cadaver is retrieved from aquatic environments. By meticulously examining the colonizationanddecayprogressionofdiatomsinthebody,forensicscientistscancraftwell-informedassessmentsconcerning thedurationofsubmersion,provingtobeindispensableincriminalinvestigations [8],[9],[10],[11].Theprincipalaimofthis exhaustivereviewistometiculouslyinvestigatetheimportanceofdiatomanalysisinthe fieldofforensicscience,specifically emphasizingitsrelevanceincasespertainingtocadaversdiscoveredinorproximatetoaquaticenvironments. Thearticleaims to illuminate the diverse applications of diatom analysis in forensic investigations, especially in drowning, suspicious deaths nearwaterbodies,andbodiesdisposedofinwater,providinginvaluableinsightsforjustice-seekingefforts.Itwillalsoaddress the challenges and potential advancements in diatom analysis, including emerging research areas and cutting-edge technologiesthatcanenhanceinvestigationreliability.Byculminatingwithaconcisesummaryofdiatomanalysis'importance in forensic limnology and offering insights into interdisciplinary collaboration, the article aims to contribute to the advancementofforensicscienceandinformeddecision-makingforthebenefitofvictimsandtheirfamilies.

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).

Inthestudyof[12] anaciddigestionapproachwasutilizedtoextractdiatomsbyeffectivelyremovingorganicmaterials.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 assiduouslypurifiedusingdiatom-freewaterandsubsequentlytransferredtoacetonetofabricateenduringmicroscopeslides. Theabsenceofdiatomsontheslideconclusivelyrenderedthetestnegative.

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 articulatedtoperpetuallyconductsurveillanceoffreshwaterlocationsandcreatethoroughspecies-levelinventoriesofdiatom flora at these sites, thereby amplifying the effectiveness of medicolegal investigations into drowning fatalities. Until recently, thequalitativeparametersofthediatomtestfordrowningmainlyfocusedonthepresenceandcorrespondenceofdiatomsin 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 oforganic materials.The instrumentcomprised 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 reactionandelevated temperature,organictissuesunderwentliquefactionfordiatomextractionutilizingthisapparatus.The procedure entailed combining 3 grams of tissue sample with 4 milliliters of concentrated nitric acid within the "can,"

01 | Jan 2024 www.irjet.net

subsequentlysubjectingittoheatingat102°Cforadurationof100minuteswithinadrybox.Aftercooling,thedigestedliquid 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 waterbath(60°C-180°C)oronasandtray(80°C-300°C).Subsequenttotheoxidationprocess,arequisitequantityofH2SO4 was introduced to finalize the oxidation of organic materials, resulting in a violet solution upon saturation with potassium permanganateandsubsequentdiscolorationwithoxalicacid.Following48hoursofsedimentation,thesolutionwasadjusted toanapproximatevolumeof100millilitersandsubjectedtocentrifugationat3000revolutionsperminuteforadurationof20 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 analysisinforensicinvestigations.[14]

Inthisstudytheauthorsshowcasedthattheilluminativecharacteristicsoffluorimetrycanbeutilizedtodiscernandseparate 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.Thisinnovativeapproachprovidesavaluabletoolforforensicinvestigationsinvolvingwater-relateddeathsand facilitatestheaccurateidentificationofdiatomsassociatedwithdrowningincidents.[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 controlledconditions.Threediatomextractionmethodologieswereevaluatedongarmentsincontactwithsoilandwatersites: 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 richnessstatisticsfromeachexperimentalsample,alongsidecorrespondenceanalysistoinvestigatesimilarities.Theoutcomes disclose that H2O2 submersion proficiently extracts significant diatom counts with representative species from garments exposedtobothaquaticandterrestrialenvironments,validatedbyscanningelectronmicroscopy(S.E.M.)analysis. Thiswork demonstratesthatdiatomsdotransfertogarmentsinvariousforensicsettingsandhighlightstheH2O2extractionasthemost efficientmethodforcollectingcomparisonsamples.Collectingandanalyzingdiatomcomponentsofgeo-forensicsampleshold potentialvalueinaidingforensicinvestigations.[16]

Acid digestion procedures in diatom analysis have inherent limitations, as they may lead to the destruction of diatom structuresduringthetreatmentprocess.However, thisstudyintroducedagroundbreakingtechniquecalledLefortaquaregia (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 emergesasanoteworthyadvancementinthefield,mitigatingthesedrawbacks.[17]

Accordingtothisstudy,thequantityofdiatomsrecoveredandtheirretentiondynamicsovertimeweresignificantlyinfluenced 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,andviscosefabrics wereidentifiedasthemostdependabletemporalrepositoriesofdiatomtraceevidence,offering a moreextensiveforensicassemblageaccessibleduringthespringforforensiccomparisons.[18]

Accordingtothisstudy,Sternalaspirationhasproventobeahighlyeffectivesampleapproachfordiatomdetection.Oneofits key advantages is its simplicity, as it does not require any specialized expertise or equipment. Performing sternal aspiration beforeautopsycanminimizetheriskofcontamination. Moreover,itisproposedthatsternalaspirateispreferabletofemoral bonemarrowforconductingdiatomtests.[19]

International Research Journal of Engineering and Technology (IRJET) e-ISSN:2395-0056

Volume: 11 Issue: 01 | Jan 2024 www.irjet.net

Study Methodology

Peabody, 1977 AciddigestionusingHClandH2SO4toextractdiatoms.

Ludesetal., 1994 Enzymatic digestion was employed in the diagnostic assessment of decomposed bodies associated with drowning incidents. The studyunderscoredthepotentialofwatermonitoringsystemsand comprehensive diatom databases in elucidating cases involving drowning.

Yangeetal., 1999

Hürlimann etal.,2000

Introduction of the "can" instrument using acid digestion for diatom extraction. Also, the "Disorganization method" for efficientdiatomidentification.

Acid digestion method using nitric acid for diatom extraction frombonemarrow.

Kumaretal., 2012 Use of fluorimetry to detect and isolate diatoms in bone marrow ortissuesamples.

Scottetal., 2014 Evaluation of diatom extraction procedures (RW, RE, H) on garmentsincontactwithsoilandwater.

Wangetal., 2015

Introduction of Lefort aqua regia technique for diatom recovery fromtissuesamples.

Scottetal., 2021

Influence of clothing material on diatom recovery and retention dynamicsovertime.

Szűcsetal., 2022 Sternal aspiration as an effective sample approach for diatom detection.

3. DIATOMS IN POSTMORTEM SUBMERSION CASES

Key Findings

Effective removal of organic materials, aidingmicroscopicstudyofdiatoms.

Continuous monitoring of freshwater locations and comprehensive diatom species inventories recommended for medico-legalinvestigations.

Improved and efficient means for diatom extraction, overcoming challenges in organic material destruction.

Effective isolation and identification of diatomsfrombonemarrowsamples.

Fluorescent markers facilitate differentiation of diatoms from drowning sites and those naturally occurringintheenvironment.

H2O2 submersion identified as the most efficient method for collecting representativediatomsamples.

Improved method compared to traditional acid digestion, addressing time-consuming and hazardous aspects.

Cotton,acrylic,andviscosefabricswere discerned as dependable temporal reservoirsofdiatomtraceevidence.

Simplicity, lack of specialized expertise or equipment required, and preference overfemur.

The scrutiny of diatoms in pulmonary tissues, internal organs, and bone marrow assumes a pivotal role in corroborating drowningastheprimarycauseofdeath, offeringvaluableinsights intotheparticularaquaticmilieuinvolved. 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 drowningasthecauseofdeathandprovideinsightintotheaquaticsurroundings,solelyrelyingonthepresenceofdiatomsin lungtissuesmaynotalwaysyielddefinitiveresults.Thepost-mortemimmersionofbodiesinwatermayresultintheingressof diatomsintothelungs,potentiallyintroducingdeceptiveevidence.Totacklethischallenge,forensicexpertshaveadvocated for aquantificationmethodology,proposingathresholdconcentrationof20diatomsper10gramsoflungtissueforinvestigative purposes. However, it is essential to acknowledge that the concentration of diatoms in the drowning medium can vary

Table-1:DiatomExtractionTechniquesandProcesses

International Research Journal of Engineering and Technology (IRJET) e-ISSN:2395-0056

Volume: 11 Issue: 01 | Jan 2024 www.irjet.net p-ISSN:2395-0072

significantly,makingafixedcriterionapplicabletoalldrowningcasesimpractical.Diversediatomspeciesdemonstrateevident predilections for either freshwater or saltwater environments. By rigorously scrutinizing the diatom assemblage in postmortemsamples,forensicinvestigatorscandiscriminatebetweencasesinvolvingsubmersioninfreshwaterorsaltwater,a pivotalfactorinascertainingtheprobablelocationoftheincident. Theconventionaldiatomtestingmethodishinderedbyits limitedsensitivity,whichposeschallengesinutilizingquantitativeanalysisto diagnosedrowningcaseseffectively.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 diatomsduringthetestingprocess.Consequently,thisinnovativemethodsignificantlyelevatesthedependability andaccuracy ofdiatomtestsinascertainingdrowningasthecauseofdeath[20],[21],[22],[23],[24],[25].

4. DIATOMS AS INDICATORS OF GEOGRAPHICAL ORIGIN

Numerous meticulousstudieshavededicatedeffortstoreconstructhistoricalsealevelvariationsthroughdetailedanalysisof diatomdatasourcedfromdiversesedimentarydepositsdatingbacktothelateQuaternaryperiod.Theprofoundsignificance 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, salinityfluctuationsgiveriseto verticallyzoned diatom assemblages,intricatelylinkedtothetidal frame. Theseassemblages 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 yetillustrativerepresentationofdiversehabitatwaterdiatomtaxa,especiallyaerophilousformslikePinnulariamicrostauron. Thisanalyticalapproachsignificantlycontributestoacomprehensivecomprehensionofdiatoms'responsesto ever-changing salt-marshconditions,solidifyingtheirpotentialasexceptionalindicatorsforsea-levelvariationsthroughouthistoricalepochs. Notably,thesubstantialvariationsobservedindiatomassemblagesacrossdiversewaterbodies,coupledwiththeirexceptional 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 andassociationsof individualsimplicatedincriminal investigations. Theidentificationof diatomvalvesfromSachPassatan 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 environmentalconditions.Theresearchpresentsinsightsintodiatomdiversityintheregionbasedonfivesamples.Taxonomic examinationsofdiatomassemblagesarenotablyscarceinthewesternHimalayas,withpredominantresearchfocusedonthe eastern Himalayas, central, and southern regions of India. The establishment of diatom databases in remote areas holds the potentialtocontributetoconservationeffortsandunveilpreviouslyunknowndiversityintheregion.Future investigationsare 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 diatomspeciesdiminishes,offeringcrucialinsightsforPMIassessment.Accordingto[31],relyingsolelyonaquaticinsectsfor 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 researchuncoveredintriguingtrends,illustratingthattheconcentrationsof elemental componentsinherent to teeth,suchas Calcium(Ca)andPhosphorus(P),exhibitadeclineovertime.Conversely,levelsofSilicon(Si),Magnesium(Mg),andPotassium

01 | Jan 2024 www.irjet.net p-ISSN:2395-0072

(K) experience an increase. Noteworthy findings from the study indicate that roughly 30 days post-mortem, a substantial portionoftheenamelbecameadornedwithphytoplankton,predominantlycharacterizedbydiatoms.Furthermore,aroundthe 60-day mark after death, the entirety of the enamel surface exhibited coverage by these microscopic organisms. These remarkablefindingsofferinvaluableinsightsandholdsubstantialpotentialforprecisepostmorteminterval(PMI)estimation inaquaticenvironments.Moreover,thetemporalsuccessionofdiatombiotasonacadaveremergesasarobustanddependable 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 fromthe soilsurroundingthedecedent'sbodyholdsignificant evidentiary valueforPMI estimation. Their presence and diverse characteristics furnish further critical insights into the precise timing of death, bolstering the accuracy andreliabilityoftheinvestigativeprocess. [33] The meticulous examinationof diatombiodiversityand 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

Overthepastdecade,theconceptofDNAbarcodinghasemergedasapromisingapproachforidentifyingdiatomtaxa. ADNA barcode functions as a tool to align an unidentified individual with a taxon possessing a congruent genetic sequence in a referencedatabase. Several generegions,including cox1,rbcL,ITS,and others,have been proposedaspotential barcodes for diatoms.Theidentificationofasuitablebarcodenecessitatesacomprehensivereferencedatabasewithasubstantialvolumeof 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,hasbeen widely exploredasa potential barcoding markerforvariousorganismgroups.Notably, recentlyDNA 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, hinderingthetechnology'swidespreadapplication.Consequently,manyoperationaltaxonomicunits(OTUs)arenotaccurately identified, resulting in molecular identification success rates lower than those achieved through microscopic identification. EstablishingareliablediatomDNAbarcodereferencesequencedatabaseisapressingtaskinthecontextofdrowningforensics [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 consistentmethods.Inanoteworthypositivecase,a23-year-oldwoman,foundfromawellwithoutapparentinjuries,became subjecttoherfather'sallegationsofhomicideand thendisposalinthewell.Despitetheabsenceof externalsigns,thediatom test unveiled the presence of Nitzchia, Cymbella, and Navicula species both in her bone marrow and the well water. This findingsubstantiatedthedeterminationofdrowningasthecausativefactorinherdemiseandnothomicide.

In contrast, an instance involving a 26-year-old female rescued from a canal, exhibiting evidence of head and neck injuries, yieldednegativeresultsfordrowninguponapplicationofthediatomtest. AlthoughNitzschiaandNaviculaspecieswerefound intheaquaticenvironment,nodiatomsweredetectedinthevictim'sremains,rulingoutdrowningasthecauseofdeath.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 provedvitalindeterminingtheactualcauseofdeathandprovidingclarityinforensicinvestigations.[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 explanationsforthislimitation.Firstly,thecurrenttechniquesusedfordiatomanalysismaynotcompletelyremovealldiatoms present in a sample, leading to potential underestimation. Additionally, in cases where the drowning medium is tap water,

International Research Journal of Engineering and Technology (IRJET) e-ISSN:2395-0056

Volume: 11 Issue: 01 | Jan 2024 www.irjet.net

p-ISSN:2395-0072

diatoms maybe presentinlower concentrations,andtheir significance mightbeoverlooked. Thisisparticularlyproblematic forvictimswithcirculatoryorpulmonaryissueswhoinhalelesseramountsofwateroverashorterduration,resultinginfewer diatomsintegratedintotheirtissues.Anotherchallengearisesfromthefactthatthevolumeofbloodpassingthroughthebone marrow,a majortissuestudiedinthediatomtest,isrelativelylow.Asaresult,the diatomcountmaybecomparativelylower 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 analysisisessential to minimize potential sourcesof contamination, suchasinstruments, gloves, papers, water supplies, and reagents,astapwatermaycontaindiatoms.[39],[40],[41]

This study underscored the potential for false-positive outcomes in diatom testing utilizing strong acid when employing Kjeldahlflasksthathavebeenreused.Thoroughwashingdidnoteliminatethepresenceof290falsepositivediatomsinreused flasks. False positives occurred in 11 to 20 cases when diatom concentrations exceeded 10,000. The reused flasks mainly contained common diatomswith a sizelessthan 30 µm,suchasDiatoma,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 suggestedthatclosedorgansmaycontainfewerdiatomsindrowningcasesthanpreviouslyreported.Asapreventivemeasure, freshflasksshouldexclusivelybeusedwhenexaminingbothdrowningandnon-drowningvictims.[41]

9. CONCLUSION

ForensicLimnology,withaspecializedfocusondiatomanalysis,emergesasan invaluabletoolininvestigatingcasesoccurring 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 undeniableutility, the diatom test exhibits limitations encompassingcontamination risks andchallenges 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 FiltrationAutomatedScanningElectronMicroscopymethodshowsgreatpromiseinaugmentingsensitivityandprecision,specificallyin estimating postmortem intervals [20], [21], [22], [23], [24],[25].Moreover,diatoms serve asinvaluablegeographical tracers, facilitating the exact localization of crime scenes and providing invaluable insights into the movements of individuals implicatedincriminalinvestigations[26],[27],[28].Despiteitsoccasionalconstraints,thediatomtestretainsitspivotalstatus inforensiclimnology,furnishingcriticalinformationtoascertainthetruecauseofdeath.Furthermore,DNAbarcodingexhibits vastpotentialindiatomidentification,althoughfurtherdatabasedevelopmentisimperativetoachievewidespreadapplication [42],[43] ImpeccablecasestudiesfromHaryanaexemplifythediatomtest'sefficacyindiscerningtheveritablecauseof 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 therelentlesspursuitofjustice.

10. REFERENCES

[1] K.Verma,“Roleofdiatomsintheworldofforensicscience,” J. Forensic Res,vol.4,no.2,pp.118–181,2013.

[2] A.S.RanaandP.Verma,“DigestionofConcentratedWaterSamplesofDiatomsforEnvironmentalPurposebya Newly DesignedMethod,”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,andZ.Cakar,“Theuseofdiatomsinforensicscience:advantages andlimitationsofthediatomtestincasesofdrowning,” The Archaeological and Forensic Applications of Microfossils: A Deeper Understanding of Human History. McLean (Va), GeoScienceWorld,pp.261–277,2017.

International Research Journal of Engineering and Technology (IRJET) e-ISSN:2395-0056

Volume: 11 Issue: 01 | Jan 2024 www.irjet.net p-ISSN:2395-0072

[5] R.R.L.&K.P.Guillard,“Theecologyofmarineplanktonicdiatoms.InTheBiologyofDiatoms.BotanicalMonographs,” D. Werner, Oxford: Blackwell Scientific Publications, vol.13,pp.372–469,1977.

[6] D.G.MannandS.J.M.Droop,“Biodiversity,biogeographyandconservationofdiatoms,”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.,“Researchadvancesinforensicdiatomtesting,” Forensic Sci Res,vol.5,no.2,pp.98–105,2020.

[9] C.R.HardyandJ.R.Wallace,“Algaeinforensicinvestigations,” Forensic Botany: A practical guide, Wiley, Oxford,pp.145–174,2012.

[10] E.J.ArmstrongandK.L.Erskine,“Investigationofdrowningdeaths:apracticalreview,” 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,andP.Mangin,“Applicationofasimpleenzymaticdigestionmethodfordiatomdetection inthediagnosisofdrowninginputrifiedcorpsesbydiatomanalysis,” 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 forensicdiatomexamination,” Forensic Sci Int,vol.101,no.3,pp.163–166,1999.

[14] J.Hurlimann,P.Feer,F.Elber,K.Niederberger,R.Dirnhofer,andD.Wyler,“Diatomdetectioninthediagnosisofdeathby drowning,” Int J Legal Med,vol.114,pp.6–14,2000.

[15] M.Kumar,J.Deshkar,S.K.Naik,andP.K.Yadav,“Diatomtest-past,presentandfuture:Abriefreview,” 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 appropriatefortheircollectionandanalysisinforensicgeoscience,” Forensic Sci Int,vol.241,pp.127–137,2014.

[17] H.Wang et al.,“AsimpledigestionmethodwithaLefortaquaregiasolutionfordiatomextraction,” 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 quantitativeassessmentoftraceevidencedynamicsovertime,” 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 Cyanobacteriainsuspecteddrowningcases,” J Forensic Leg Med,vol.85,p.102298,2022.

[20] J.M.Lang,R.Erb,J.L.Pechal,J.R.Wallace,R.W.McEwan,andM.E.Benbow,“Microbialbiofilmcommunityvariationin flowinghabitats:potentialutilityasbioindicatorsofpostmortemsubmersionintervals,” Microorganisms,vol.4,no.1,p. 1,2016.

[21] C.R.HardyandJ.R.Wallace,“Algaeinforensicinvestigations,” 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.

International Research Journal of Engineering and Technology (IRJET) e-ISSN:2395-0056

Volume: 11 Issue: 01 | Jan 2024 www.irjet.net p-ISSN:2395-0072

[23] G. C. Dickson, R. T. M. Poulter, E. W. Maas, P. K. Probert, and J. A. Kieser, “Marine bacterial succession as a potential indicatorofpostmortemsubmersioninterval,” Forensic Sci Int,vol.209,no.1–3,pp.1–10,2011.

[24] B.Ludes,M.Coste,N.North,S.Doray,A.Tracqui,andP.Kintz,“Diatomanalysisinvictim’stissuesasanindicatorofthe siteofdrowning,” 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 indicatorofdrowning,” 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,“Theuseofdiatomanalysisinforensicgeoscience,” Geological Society, London, Special Publications,vol. 232,no.1,pp.277–280,2004.

[27] K.PyeandD.J.Croft,“Forensicgeoscience:principles,techniquesandapplications,”GeologicalSocietyofLondon,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, “DiatomsFlora From Sach Pass Glacier of Himachal Pradesh:SpeciesComposition And Their RelationshipToEnvironmentalVariables,”p.20038,Feb.2023.

[31] K. A. Zimmerman and J. R. Wallace, “The potential to determine a postmortem submersion interval based on algal/diatomdiversityondecomposingmammaliancarcassesinbrackishpondsinDelaware,” 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,“Forensicbotany:who?,how?,where?,when?,” Science & Justice,2023.

[35] Z.Li et al.,“BarcodingfordiatomsintheYangtzeRiverfromthemorphologicalobservationand18SrDNApolymorphic 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,andA.Kadian,“Roleofdiatomsinforensicinvestigation:CasestudiesfromHaryana,” 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 drowningdeathsinbathwater,” 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 suspectstoanaquaticcrimesceneinsouthernNewEngland,” J Forensic Sci,vol.39,p.847,1994.

Volume: 11 Issue: 01 | Jan 2024 www.irjet.net p-ISSN:2395-0072

[41] E. Kakizaki, N. Shinkawa, A. Sonoda, and N. Yukawa, “Conventional diatom testing using strong acid: Notable falsepositive resultscausedbyanunderestimatedcontaminationsource(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.,“BarcodingfordiatomsintheYangtzeRiverfromthemorphologicalobservationand18SrDNApolymorphic analysis,” Forensic Sci Int,vol.297,pp.81–89,2019,doi:https://doi.org/10.1016/j.forsciint.2019.01.028.