Diatomological Mapping of Aurangabad Region for Unrevealing Drowning Mystery

Purpose: Drowning is the third unnatural cause of death in India. Drowning death is still considered as one of the most dicult to investigate, it requires the expertise in the pathophysiology and limnology. Long procedural protocol in laboratory analysis of vital organs and the site to obtain the victim and site-relation that creates a burden on the investigating agency. The presence of diatoms in the vital organs of the body helps to establish the pre- or post-mortem drowning death and the appearance of site-specic/unique diatoms for a region aids into locating the exact drowning site. The onsite identication can be solved by studying the diversity and preparing the diatoms map (D-map). Availability of D-map can help to reduce the investigation time and helps in conrmation of the drowning site. Methods: In the present study, samples were collected from the 12 water bodies in Aurangabad region M.S. India, and were acid digested, using Aqua regia solution containing HNO 3 : H 2 SO 4 in 1:3 proportion in 20ml of water sample. Slides were prepared, mounted and microscopic analysis was performed using light and inverted phase microscope.


Introduction
Diatoms are unicellular eukaryotic microalgae inhabited in the silica glasshouse. They are cosmopolitan, profoundly seen in fresh and marine habitats. Being phytoplankton, they are the major source of atmospheric oxygen and are a key player in organic carbon xation on earth. Diatoms are very sensitive to the nutrient content, temperature, seasonal variation [1], pH (Potential of hydrogen), Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD) and the availability of silica in water. These factors have an impact on the growth of diatom biomass and show speci city towards all these parameters. Though some species are commonly found across the world, some are unique to location and season. Thus, they are used as one of the "biotic indices" i.e., water quality, aquatic ecosystem health ora and fauna etc. [2]. The global studies have estimated that the number of diatom taxa is around 200,000 [3]. India harbors one of the seventh largest biodiversity of the world, 14,700 taxa from freshwater and marine aquatic [4]. Diatoms are classi ed according to the structure of their siliceous cell wall, based on their valve shapes into centric and pennate diatoms. The centric diatoms have valves with radial symmetry and ribs, central stellate ornamentation, spins, mantle, and stria on the frustules are the features for its identi cation. The Pinnate have narrow elliptical valve shapes such as diverse raphe-, apex-shapes, areolae pattern, longitudinal lines, lineolate, puncta features of their frustules. Further based on the symmetry they are classi ed into Asymmetric Biraphid, Symmetric Biraphid, Monariphid, Epithelioid and Nitzsoid.
The above said species are mainly encountered in the drowning Investigation, the presence or absence of diatoms in the drowned body indicates the suicidal or homicidal death but many times it needs further investigation.
Long procedural protocol in laboratory analysis of vital organs and the site to obtain the victim and site-relation that creates a burden on the investigating agency.
Drowning death possesses a signi cant challenge to forensic investigators, as it requires expertise in the pathophysiology, autopsy report [5]. Three major factors that impede the investigations include putrefaction, pre-and post-mortem drowning and drowning site. Since the diatoms were rst discovered in the lungs by Hofmann in 1896 and were rst used by Revenstor n in 1904 for diagnostic purposes. Since then, diatoms role has been extensively explored and evolved with time. Diatoms plays a signi cant role in the forensic investigation of drowning cases. In such investigations, questions arise about the pre and post mortem drowning, along with suspicion on the drowning site [6]. Diatom test is the "gold standard" method [7]. Many other tests are available for drowning investigation, but they are not widely accepted and appreciated, rarely used as compared to the diatom test [8]- [10].
Diatom test is theoretically based on the presence of diatom in the vital organs of the body such as lungs, bone marrow, kidney and brain [11], [12]. When a person tries to breathe inside water bodies such as lake, ocean and river, the diatoms along with water penetrates in the alveolar system of the human body and gets deposited in vital organs by blood circulation. However, when the dead body is thrown in the water, diatoms enters in the body but cannot be deposited in the vital organs [13], [14]. The occurrence or recovery of diatoms mainly from the organs such as bone marrow and lungs in the body is caused due to aspiration of water and is considered as premortem drowning and the absence indicates nondrowned death. Thus, the accuracy in the diatom identi cation is very much important in drowning death investigation [5].
In the diatom test, a drowning site is located by comparing the species of diatoms extracted from tissue samples and the water samples of suspected water bodies as they are site-speci c [15]. In numerous instances, there is a possibility that the body which has been drowned and moved away from the actual site of drowning, where no other references are available to locate [6], [13] After extraction from the tissue, the forensic scientist will have to compare the species present in the tissue over the putative sample.
As per the National Crime Record Bureau (NCRB) drowning is 3 rd leading cause of unnatural death in India. Drowning death recorded 7.3% (30,187) and 7.8% (32,671) in India while 4,165 and 4,516 in the State of Maharashtra of total cases of unnatural death in 2018 and 2019 respectively [16] [17]. Since the numbers of drowning cases are growing, it is necessary to have a regional diatomic distribution map to validate, identify the drowning site and increase the rate of investigation.
However, as per literature search the current diatomological data is not available for Aurangabad (India) region. Aurangabad region is on the Godavari river basin and the district is located between 19° and 20° north longitudes, and 74°a nd 76° east latitudes ( Figure 3). The region has several large and small water bodies where numerous drowning cases are reported. The present study was intended to study diatom distribution, prevalence and diversity. D-Map is the diatomological map that de ned the diatom diversity of de ned region.

Methods
Sample collection was performed using the following standard collection protocol [18] 2.1 Selection of Water Bodies and Sample Collection: Twelve major suicidal prone water bodies (see Table-14) from Aurangabad region M.S. (India) were selected and the samples were collected (Benthic, Phytoplankton, Epilithon, Epiphyton and surface) from different sites of water bodies by standard modi ed collection protocol [18] in the autumn season. All the information was documented including sample collection time, the geographical location of the water body i.e., longitude and latitude. Approximately 200-300ml surface water samples were collected and directly transferred into sterile sealed plastic bags. To extract diatoms from substrata stones were collected in bags & were vigorously shaken. The samples were brought to the laboratory for further analysis.

Aqua Regia Acid Digestion Method for Diatom Extraction:
A modi ed standard acid digestion protocol was used for diatom extraction [18]. The increased acid concentration reduced the time required to digest all organic material in the sample Aqua regia is a mixture of two strong acids i.e. nitric acid and sulphuric acid in a 1:3 ratio to digest all the organic material in a sample (HNO 3 : H 2 SO 4 ) (1:3) [18]. About 20ml (milliliter) stored sample was vigorously mixed, then transferred to 100ml clean beaker, to which equal volume of saturated Potassium Permagnet (KMnO 4 ) solution was added, and incubated at room temperature for 3hours. Later, 20ml of aqua regia was added and kept for 24 hours undisturbed for sediment digestion. Next day the solution was placed at 90℃ on a hot plate for 2hours until the solution turns colorless and subsequently transferred into the 15ml centrifuge tube (Tarson Products, Kolkata, India). Later these samples were centrifuged in an Eppendorf tube (Eppendorf Hamburg, Germany) at 3000 (Revolutions per minute) RPM for 10 minutes and the pellets were resuspended and collected with 2ml aqua regia. This step was repeated twice for concentrating the diatoms for slide preparation and analysis.

Slide Preparation and Microscopic Analysis:
A drop of diatom sample was evenly spread over a clean grease-free glass slide, covered with a coverslip and air dried.
Subsequently, the microscopic slide was permanently mounted with Dibutylphthalate Polystyrene Xylene (DPX), (Thermo Fisher Scienti c, Massachusetts, United States) and observed under the inverted phase contrast compound microscope (Magnus OPTO systems Microscope, New Delhi, India) 100x, oil immersion objective for species identi cation. The slides were scanned and analyzed by the inverted phase contrast compound microscope. Photographic analysis for the identi cation of taxa of the diatom was carried out using available literatures of commonly found diatoms of India and the available guides from web portals such as diatoms.org, diatombase.org and An illustrated guide to a Common Daitoms of Peninssular India [19]- [21] 3. Results In current study generated data shows the distribution pattern of diatom species in 12 different sites (Table No. 1 to 12) that were taken for sampling. Distribution of diversity helps to identify the key indicator species of the drowning site. The unique and key indicator species such as Craticula, Gomphonema, Tibetiella, Melosira, Pleurosira, and Mastogloia were identi ed in (Water body) WB1, WB4, WB5, WB8 and WB12 respectively (Figure No. 4,Table No. 13). This study has generated data that will assist in the investigation and to locate a site of drowning in the Aurangabad region (Table  No.14) [22].
While the characterization of all sites, we found that WB1 has the highest diversity with 17 identi ed species of 8 genus, while in WB3, 11 species were identi ed. However, in both sites, we noticed Nitzschia, Navicula, and Cymbella were the most common genus. In WB7 and WB9, 6 species were detected at both the sites. In WB8 the 5 species were identi ed. In WB4, WB10, WB11 and WB12, 4 diatom species were reported. 3 Species were identi ed in WB6 with 2 species in WB2 and WB5 ( Figure No. 1) In the genus distribution Nitzschia was the most common species found in 10 different sites followed by Navicula and Cymbella, observed in 7 and 8 different sites. Ulnaria and Cyclotella were found in the 6 different sites whereas Pinnularia, Diploneis and Encyonema, were comparatively rare as were found in only in 2 sites. Craticula, Gomphonema, Tibetiella, Melosira, Pleurosira, and Mastogloia were unique diatoms and their presence is restricted to only one location (

Discussion
The clear image of diatom thus obtained helped in accurate identi cation of the species even without the highly sophisticated instruments. A lucid and conspicuous image are required to identify the taxa and demarcated based on cell wall (valves) shape as well as the number stria present.
D-map is a concept of making representative information of all known and unknown identi ed species in the area of study, all data regarding the particular species Microscopic and invisible diatoms accidentally adhere to the victims' clothing in addition to their footwear that can also lead to identi cation or to co-relate the drowning site and also can discover the victim/suspect relation [23], [24]. The availability of the D-map results can assist in quick correlation to the victim/suspect as well as to the drowning site. Diatoms are only evidence to be considered in the putre ed body to conclude the drowning death [25]. The autopsy contamination and non-vegetarian foodstuff can give false positive diatom tests in case of non-drowned death [26].

Conclusion
Diatoms are the key indicators of drowning death and can be used to locate the site of the drowning. The present study demonstrates that the acid concentration is inversely related to the time required for organic matter digestion and helps to improve image clarity. The current study intends to identify predominant diatoms in suicide prone water bodies of the Aurangabad region and prepare a database.
The diatoms restricted to only single water body are unique diatoms and can be used as site-speci c indicators and marker in drowning investigation for the Aurangabad region. This study generated diatomic map for experimental water body present in Aurangabad region. These D-maps will be helpful in dispense a signi cant information about water bodies and their common as well as unique ora with a qualitative and quantitative distribution of diatoms. D-Map generated in this study can be harnessed to update knowledge of diatomology or diatoms database and generated data will aid in drowning investigation. Further studies are required to generate D-map data at global level to speed up the investigation and reduce the burden of investigators. Further the method of extraction and identi cation of diatoms in both putative and the visceral sample is arduous and time consuming, requires experience and knowledge in the eld of limnology to minimize the errors in the Diatomic test. This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.

Con icts of interest/Competing interests:
The authors declare that they have no competing interests Availability of data and material: All data generated or analyzed during this study are included in this published article.
Code availability: