DIATOMS Guardians of Aquatic Ecosystems and Climate Stabilizers

DIATOMS Guardians of Aquatic Ecosystems and Climate Stabilizers

Registration ID: IJNRD_224313

Published ID: IJNRD2406424

DOI: http://doi.one/10.1729/Journal.40244

Kritika Koundal

Diatom test, Forensic laboratories, Drowning deaths, Ante-mortem, Post-mortem, Penetration, Diatom analysis, Fresh water, Sea water, Lake water, Microbial fingerprint, Dna sequencing, Genetic markers, Extraction, Digestion, Centrifugation, Photosynthesize, Food source, Invertebrates, Small fish

There are so many researches are done on Diatoms and also the case studies: Masahiro Mukaida et al. 1998, they have worked on a case study ‘Detection of bath salts in the lungs of a baby drowned in a bathtub’. This case is one in which a baby was found dead, apparently drowned in a bathtub. To confirm the inhalation of bathwater, an extract taken from the baby’s lungs was analyzed with high performance liquid chromatography. Bath salts contained in the bathwater were detected in the extract. Bath salts are usually used in home bathtubs and, as a dye material, they contain fluorescein which is highly sensitive to detection. The presence of fluorescein in the lung tissue and in the other tissues greatly helps to confirm the bathwater drowning. Death by drowning in which accident, suicide or homicide are involved may occur under various circumstances. Diagnosis of drowning is usually arrived at through the autopsy findings and the detection of diatom in tissues. In the case of a body apparently drowned in a bathtub, inhaled water in the lungs may not necessarily be that of bathwater. Therefore, the precise detection and identification of inhaled substances is of vital importance in determining the actual cause of death. In this case, to prove that the death was caused by drowning in bathwater, the existence or non-existence of bathwater in the lung extracts was ascertained. Analysis proved the existence of bath salts in the lung extract which were contained in the bathwater in which the baby was found dead. The baby was 71 cm long and 7.5 kg in weight. There was a small amount of yellowish exudative liquid found in the pleural cavity. A large amount of bloody fluid was found in the respiratory tract. The vessels of the tract’s mucous membrane were dilated. Both lungs were bulging (left, 60.3 g; right, 69.8 g) and the emphysematous and atelectatic regions were mixed on the surface of the lungs. Microscopic findings revealed a massive alveolar edema, lobular bleeding and emphysema. Red blood cells in the capillaries of the alveolar septa revealed globular form under microscopic inspection. To detect foreign bodies contained in the bathwater, 50 ml of water was filtrated and the residue on the filter was observed under a light microscope. No diatoms were detected in the residue. The diatom test was performed on the lung and kidney tissues with fuming nitric acid. No diatoms were detected in these tissues. In our case, pulmonary edema which may have been caused by the inhalation of water was observed, but the emergency treatment at the hospital such as artificial respiration, cardiac massage and fluid therapy, unfortunately made it impossible to diagnose the exact cause of death. No diatoms were detected in the residue from the bathwater, nor were they detected in the lung or kidney tissues. It cannot be denied that the fluorescein in the liquid inhaled into the lungs was forced into the blood and spread throughout the organs by emergency treatment. But the fluorescent intensity of the renal tissue by UV radiation was almost uniform throughout the cortex. This fact suggests that the fluorescein inflow into the blood and the free fluorescein filtered through the glomerulus was reabsorbed into the epithelial cells of the renal tubule while the baby was alive. To prove that the cause of death was an accidental deglutition of the bathwater, detection of the fluorescein contained in the bathwater is essential. The analysis of lung extract and the identification of the bathwater components, the fluorescein, will greatly help to confirm the baby inhaled bathwater and the drowning occurred in a bathtub. When none were present in the lungs, none were found in other organs. About false-positive results, no diatoms were found in tissue samples from the control subjects. Mei Ming et al. - 2006, they all have A Research related to “Evaluation of Four Digestive Methods for Extracting Diatoms. The cause of death for the decomposed corpses retrieved from water remains a difficult issue in current forensic practice and therefore, a great number of tests have been proposed to support a confirmation or exclusion of drowning. For bodies freshly recovered from water, the indicators of true drowning are the presence of fine froth at the mouth or nostrils, emphysema aquosum and impress of ribs on lungs. In case the putrefaction destroys the above-mentioned ‘‘drowning signs’’, the diagnosis of drowning is rather difficult. Although diagnostic value of diatom test for drowning is controversial among many medicolegal experts, most forensic pathologists still consider it a relatively reliable and useful tool, especially when the qualitative and quantitative diatom analysis is introduced with the prerequisite of avoiding contamination. Experimental animal, diatom and reagent Forty rabbits of either sex weighed 2.5–3.5 kg, were averagely distributed into groups (A, B, C, D and E) and sacrificed by exsanguination. Cyclotella (fresh water, centric diatom), Cybella (fresh water, pinnate diatom) and Navicula (sea water, pinnate diatom) diatoms were obtained from Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. Disorganization Can was presented by Li et al., the inventor. Proteinase K and Soluene - 350 were bought from Merck (Germany) and Packard (Netherlands), respectively. Nitric acid was of analytical grade from China. All the reagents and instruments used were tested periodically to exclude the possibility of contamination with exogenous diatoms. Calculation of the reclaiming ratio of diatoms – The cultured diatoms from Wuhan Institute of Hydrobiology were diluted to be 105 ml determined with counting chamber. Before digestion, a drop of diatoms, the exact number of which had been counted ahead, was added into the minced tissue. After digestion, the precipitate was dissolved to 1 ml and the concentration of diatom was determined under counting chamber. Therefore, the reclaiming ratio of diatom could be calculated with the reclaiming number compared by the added number. Statistical analysis – All values in the text, figures and tables were expressed as the mean S.D. and statistical analyses were performed by one- way analysis of variance (ANOVA) using SPSS11.0 for windows. P < 0.05 was regarded as statistically significant. Time consumption for complete digestion – For digesting 2 g of different tissues, longest time was demanded by Soluene-350, the second by proteinase K, nitric acid plus hydrogen peroxide and nitric acid in Disorganization Can method required the shortest time. Digestive capability – The degree of digestion for different tissues in the limited time 2h was compared by the weight of the precipitate after digestion. Nitric acid in Disorganization Can had the strongest digestive capability, nitric acid plus hydrogen peroxide method took the second place, proteinase K method the third, and Soluene-350 method the weakest. Reclaiming ratio of Diatoms – Cyclotella and Cybella, proteinase K method had the highest reclaiming ratio, nitric acid plus hydrogen peroxide method had the lower reclaiming ratio than proteinase K, Disorganization Can method and Soluene-350 method had very low reclaiming ratios. For Navicula, a majority of diatoms could be extracted by proteinase K method, but only a few of them could be detected by the other three methods. Destruction to Diatoms – Under SEM, the structures of the three different diatoms remained almost intact with clear background and little impurity after digestion by proteinase K. However, they were destroyed to different extent by the other three digestive methods: intact Cyclotella, Cybella and their fragments could be found in the residue, but no intact Navicula could be discovered. The diatom test is based on the inhalation of the diatoms suspended in the fluid medium in the process of drowning. Diatoms can enter the ruptured pulmonary alveolar, capillaries, and reach the organs in the greater circulation. The postmortem extraction and detection of these special algae are possible due to their silica- based extra-cellular coat or frustules which has resistance to various digestive reagents. The reliability of diatom test depends on the exclusion of contamination and the correct interpretation of the result, which involves a complete taxonomic analysis of the diatoms recovered from water surrounding the corpse and from the organs of the deceased. The precondition for qualitative and quantitative diatom analysis demands that the extracted diatoms should be distinguishable and the reclaiming ratio should be high. To some extent, the restricted use of the diatom test for drowning is due to its high false negative ratio. Therefore, Proper digestive method should be chosen to increase the sensitivity of the diatom test to its maximum value, exceeding the current detection rate of 28%. Mukesh Kumar Thakar & Rajvinder Singh – Department of Forensic Science, Punjabi University Patiala, Punjab, India – 2009, they both have research about ‘Diatomological mapping of water bodies for the diagnosis of drowning cases’. Diatoms are group of unicellular algae that have very useful applications in Forensic investigations of drowning cases especially in establishing cause and mode of death i.e. ante-mortem and postmortem drowning. The presence of tiny aquatic diatoms in a dead body has long been held by some to be a clear indicator of death by drowning. If analyzed both quantitatively and qualitatively through a diatom test, can lead not only to a more direct determination of the cause of death, but also pinpoint the site of a suspected drowning. Punjab is a land of canals, rivers, ponds and lakes, which offers an invite for drowning. Every year a large number of drowning cases are reported in Punjab. Sometimes the drowned bodies are found suspiciously floating in these water bodies. In those cases, the Forensic Scientist has to give opinion whether the particular case is of ante-mortem or postmortem drowning. Along with this, another equally important question to be answered is that if the death really took place at that site from where the body was found. It imparts further necessity for the precise localization of site of drowning particularly: (i) when the body is found on land and no reference water body is available, and (ii) when the body is found away from the actual site of drowning may be due to flow of water or any other reason. Therefore search for the putative site of drowning becomes a thrust area in the Forensic investigations. Keeping in mind the sensitivity of the places, the following 10 different water bodies spread over different locations of Punjab state (India) were selected for the Diatomological mapping: 1. Sukhna Lake Chandigarh (SL, CHD-1), 2. Sutlej Water Body, Ropar (SWB-RPR 2), 3. Kanjli Wetland, Kapurthala (KW, KPT-3), 4. National Wildlife Sanctuary & Wetland, Harike (NWS&W, HRK-4), 5. Guru Nanak Dev Thermal Plant, Bathinda (GNDTP, BTH-5), 6. Main Bakhra Branch, Patiala (MBB, PTA-6), 7. Kali Devi Talab, Patiala (KDT, PTA-7), 8. Indira Gandhi Canal, Lambi-Mukatsar (IGC, MKT-8), 9. Bakhra Dam, Nangal (BD, NGL-9), 10. Pong Dam, Nangal (PD, TLR-10). The water samples were collected from 10 different selected water bodies (which include 1 – Lake, 2 – ponds, 3 – canals and 4 – rivers) continuously for two years from June 2005 to March 2007 in two phases. All the samples were collected with a gap of three months covering four seasons i.e. winter, spring, autumn and summer. Eight collections were made during a span of two years. The dates of sample collection in both the phases (years) were kept almost same. Water samples were collected in properly sterilized and serially marked one liter capacity plastic bottles from the all the sides of all water bodies. Lakes have basically four sides, while river and canals had only two sides. The pH of the samples was recorded and preserved as such for the further analysis. Approximately 200 ml of water sample was transferred into an acid washed 250 ml glass beaker. Samples were added with 40 – 45 ml of concentrated nitric acid (HNO3) and a pinch of Potassium dichromate K2Cr2O7. Then samples were allowed to stand undisturbed for 2 h. These samples were transferred to properly label plastic centrifuge tubes and centrifuged at 3000 rpm for 10 min. The supernatant was pipetted out leaving behind only a residual material at the bottom of tube. This residual material was suspended in distilled water and again centrifuged in the same way to ensure that even the traces of acid were removed. Distribution of diatoms based on both season and site specific has been studied in detail. The results of the present study showed that water conditions and seasonal changes are generally important factors affecting changes in the distribution of diatoms across the broad geographical region. Morphological Analysis of Diatoms revealed 51 genera and 126 diatom species, and most of them belonged to order ‘‘Pennales” with few exceptions of ‘‘Centrale” diatoms. The site specific/indicator diatoms exist consistently during all seasons but their percentage keeps on changing depending upon the season in any particular water body. These diatom species were mostly seen in the Lakes with stagnant water conditions, but were mostly absent in running water channels like canals, etc. Stefan Uitdehaag et al. - 2010, they all have A Research about “Extraction of diatoms from (cotton) clothing for forensic comparisons. Diatoms in clothing can be used to determine contact with surface water and contact with a specific water source, which can help link suspects to crime scenes. Linking suspects to a crime scene is a common forensic goal. In several cases at the Netherlands Forensic Institute (NFI) diatoms were extracted from clothing to determine possible contact with surface water and which body of water was the most likely source of the diatoms. Comparisons between diatoms in a piece of clothing and those in a water source depend on an extraction method that does not preferentially lose or damage specific diatom species. In addition, larger numbers of extracted and identified diatoms increase the reliability of the species composition analysis. In this study we investigated the qualitative and quantitative characteristics of three methods for extracting diatoms from cotton clothing. To test if the methods would work under a variety of circumstances, as is usual in forensic work, both old and new clothing and two very different water sources were used. The protocols were tested on cotton clothing, as cotton clothing such as T – shirts and jeans is frequently encountered in casework in the Netherlands. Sample collection – Sampling was performed at two locations by immersing white 100% cotton T-shirts in the middle of the water body and dragging them back to the edge. An old (used) and washed T – shirt (T1) was immersed at location one and a new (unused) T – shirt (T2) at location two. Additionally, used cotton T – shirts with no known surface water contact were subjected to the extraction methods. Location 1 was a canal, roughly 10 meter broad, with regular shipping traffic, few water plants and with slightly brackish water. Reference water samples were taken from the center and the edge of the canal. Location 2 was a fresh water ditch, roughly 2 meters broad and with a large number of water plants on the edges. Water samples were taken from the center and the edge of the ditch. Reference water sample preparation – Formaldehyde was added and after settling for 48 h the supernatant was discarded. The pellet was first treated with 30% H2O2 (Merck, Darmstadt, Germany) and then with 10% HCl (Merck). This was followed by three washing steps. After Rinsing clothing in water (RW) or in 70% ethanol (RE) and Dissolving Clothing, Samples were mounted with a high refractive index medium (NaphraxTM) and were analyzed with a phase contrast microscope at 1000 magnification. The species composition was determined for each method using a sample with an average number of diatoms. Where possible, at least 200 diatoms were identified. The species composition data was compared using the Bray–Curtis similarity index (BCsi). The DI method had the highest diatom yield of the three tested methods for extracting diatoms from cotton clothing, but the values showed a high standard deviation between replicates from the same T – shirt. The high yield indicates that with this method diatoms can be detected where the other methods would fail. This can be important in cases where possible contact of clothing with water is under investigation by reducing the possibility of a false negative result. In this study we tested three methods for extracting diatoms from cotton clothing. The dissolving method produced the highest average yield. The rinsing with ethanol method gave consistent concentration values and enough diatoms were extracted to calculate reliable Bray–Curtis similarity values (BCsi). In the two T – shirts tested the rinsing with ethanol method has the highest BCsis with the reference samples. Therefore, when investigating possible contact between a piece of clothing and surface water either the dissolving method or the rinsing with ethanol method can be used. For comparison studies aimed at determining a specific water source we propose rinsing with ethanol. Article by Kapil Verma about ‘Role of Diatoms in the World of Forensic Science’ – 2013 – Diatom analysis should be considered positive when number of diatoms is above a minimal established limit; 20 diatoms/ 100 μl of pellet (obtained from 10 gm of lung samples) and 50 diatoms from other organs and further matching of diatoms from bone marrow and drowning site can strengthen this supportive evidence and a positive conclusion can be drawn whether person was living or not when drowned. The diatom test, while extremely specific is of immense value considering the limited objective tests available for drowning diagnoses. A fresh outlook is necessary to rekindle use of this important application to medico legal investigations. It has been suggested that marrow of the sternum may be as good of a source of diatoms as femoral tissue. Death of a victim found in water should not always be related to drowning. If the person is still alive when entering the water, diatoms will enter the lungs if the person inhales water and drowns. The diatoms are then carried to distant parts of the body such as the brain, kidneys, and bone marrow by circulation. If the person is dead when entering the water, then there is no circulation and diatoms cannot enter the body. Diatoms do not occur naturally in the body. If laboratory tests show diatoms in the corpse that are of the same species found in the water where the body was recovered, then it may be good evidence of drowning as the cause of death. Bone marrow is described as a sanctuary organ and if diatoms reach this tissue, the diagnostic of drowning could be assessed. Sunlin Hua et al. - Guangzhou Forensic Science Institute, Guangzhou, PR China – 2013, they all have A Research related to “Detection of diatoms in water and tissues by combination of microwave digestion, vacuum filtration and scanning electron microscopy”. At present, the diatom test is considered as the most reliable scientific method or ‘‘the golden standard’’ in solving drowning cases. When the qualitative and quantitative diatom analysis is introduced with the prerequisite of avoiding contamination, the test provides supportive evidence of drowning especially in putrefied bodies where no other tests are possible, and also gives evidence of the probable site of drowning. The conventional methods used in forensic practice for the diatom test often consist of the following three steps: extraction of diatom from water and tissues by digestion using strong acids, enzymes or solubilizers; centrifugation or membrane filtration for collecting diatoms, and detection and identification of diatoms by light microscopy (LM) or scanning electron microscopy (SEM). However, the diatom test with the current methods is restricted to its high false negative ratio for drowning cases and the disadvantage of being rather labor- intensive. Therefore, the study of the detection of diatoms in water and tissues under automated LM or automated SEM with new sample processing techniques will facilitate automatic diatom test as well as improvement of diatom detection sensitivity. In this paper, we describe a new method towards such purposes. Samples of water from the sites where the victims had been found or where the immersion probably had occurred were collected in diatom-free containers. Blocks of 2 g of the organs including lung, liver, kidney and bone marrow of the victims to be studied were cut, avoiding contamination, as well. 2 ml of water samples and 2 g of the organs were transferred into digestion vessels of an MW3000 microwave digestion system (Anton Parr, Austria) respectively. The treatment by microwave digestion, being safer, more rapid and more convenient, provides more clean samples for detection of diatoms in water and tissues. Vacuum filtration instead of centrifugation of the fluid obtained by digestion is capable to achieve the maximum diatom reclaiming ratio, thus improves the sensitivity of the detection of diatoms in water and tissues. Detection and identification of diatoms using the function of automatic field scanning and the features of high resolution and excellent depth of field of automated SEM, allows the diatom test to be more accurate as well as to save labor and time required for analysis. Currently, the diatom test is very tough work and insensitive in forensic practice, and we see this method as the first step towards automation of the diatom test and improvement of diatom detection sensitivity. Provided that the emerging progress in the field of automatic diatom identification, the diatom test with improved methods of automatic identification will play much more important role in the forensic diagnosis of drowning than ever. N. G. CAMERON – 2013, have A Research about “The Use of Diatom Analysis in Forensic Geoscience’. Over 60% of the dry weight of the diatom cell may be silica. However, silica uptake and deposition involve less energy expenditure than formation of the equivalent organic walls found in other classes of algae and higher plants (Round et al. 1990). Each living cell is contained within two valves, one slightly larger than the other and both fitting together like a box and lid. The diatoms are yellow-brown pigmented and photosynthetic, although exceptionally a few species are heterotrophic. The classification of the huge number, perhaps as many as 105 (Mann & Droop 1996), of extant and fossil diatom species is predominantly based on the structure of the valve, its shape, intricate patterning and ornamentation (Round et al. 1990). Living diatoms are distributed in almost all aquatic and damp terrestrial habitats and in many of these the diatoms represent the most abundant and diverse algal class. Different diatom species are highly sensitive to water quality and many species are habitat-specific. Further, diatoms grow rapidly and under favorable conditions produce many cells within a short period. They colonize submerged surfaces quickly and many taxa show seasonality of growth. Diatom cells are mainly solitary but some colonial taxa produce special separation valves during vegetative growth. The formation of resting stages and auxospores can also produce different valve morphologies. These characteristics lead to a number of actual and potential applications of diatoms in forensic science. The 'diatom test' for drowning is probably the most often applied and studied application of diatom analysis in forensic investigations (e.g. Pollanen 1998a) and has become an established forensic technique. Drowning occurs when water enters the lungs and then the bloodstream via ruptures in the alveoli. Any small particles suspended in the water may also be carried into the bloodstream and potentially throughout the body. Such particles are likely to be deposited in the capillaries of the major organs and muscles. In most natural fresh and saltwater bodies this particulate material will include diatoms. However, not all water sources contain diatoms in suspension. A number of characteristics of diatoms, including their widespread presence in water, sensitivity to environmental water quality, high species diversity, habitat specificity, and good potential for preservation, suggest that this group of algae has further potential for use in the field of forensic geoscience. For criminal investigations in particular, the invisibility of valves to the naked eye and the fact that diatoms are not well know to the layman are useful attributes. Standardization of diatom taxonomy and numerical techniques developed in the field of Holocene paleoecology (e.g. Battarbee et al. 2000) will improve the comparison and numerical analysis of diatom assemblages in the forensic context and the presentation of diatom data to a lay audience. Malik MK, Kadian et al. Forensic Science Laboratory, Madhuban, Karnal, Haryana, India - 2013, they all have Case Studies related to “Role of Diatoms in Forensic Investigation. The study was conducted in Biology Division of Forensic Science Laboratory, Madhuban, Karnal (Haryana), India and all the cases reported here, are fresh water drowning cases. After postmortem of dead bodies, internal organs (sternum, clavicle, femur and lungs) along with the water samples (From where the dead bodies were recovered) were sent as crime exhibits to the laboratory. In all cases exhibits were put into different jars. 50 ml of nitric acid was added in each jar containing the sternum, clavicle, femur and lungs. Samples were left undisturbed overnight and boiled for half an hour on next day. A clear yellow solution was obtained with a fat layer at the top. The fat layer was discarded and the remaining samples were centrifuged at 4000 rpm for 10 minutes. The process of centrifugation was repeated three times in the same way. Supernatant was discarded and the pellets were washed with distilled water and re-centrifuged. Microscopic slides were prepared from the pellets after washing, dried on hot plate and studied under the microscope after applying immersion oil. Slides were also prepared from the water sample sent to the laboratory in which possible drowning took place. Comparison of diatom species found in organ sample and water sample was done and correlations were drawn out accordingly. Case 1 – The body of a 19 – year – old boy was found submerged in a water tank. No sign of injury was found at autopsy. Nitric acid extract of internal organs (sternum, clavicle, femur and lungs) revealed the presence of three types of diatom species (Navicula lanceolata, Navicula oblonga and Gomphonema gracile). The same three types of diatom species were found in the water sample from which the body was recovered. So the cause of death was assigned due to drowning. Case 2 – The body of a 26 – year – old lady was recovered from a canal. Signs of head and neck injury were traced during autopsy. Nitric acid extract of sternum and clavicle showed the absence of any diatom species. However, two diatom species (Nitzschia subtilis and Navicula radiosa) were detected in the water sample from where the body was found. The cause of death was attributed to the reasons other than drowning which was later accepted by her husband that he murdered her wife because of having extra-marital affair. Case 3 – The body of a 23 – year – old lady was recovered from a village well. No sign of injury was found during autopsy. Father of the lady claimed that her daughter was being murdered and then thrown into the well to prove the case as suicide. Nitric acid extracts of sternum, clavicle and femur showed presence of three diatom species (Nitzschia gracilis, Cymbella and Navicula radiosa). Same three types of diatom species were detected from the water sample of well in which the body was found. Drowning was the cause of death. Case 4 – The body of a 48 – year – old lady was recovered from a river. No specific reason of death was found at autopsy. Microscopic studies of slides prepared from nitric acid extracts of sternum, clavicle, femur and lungs proved the presence of three diatom species (Cymbella cymbiformis, Gomphonema spheroporum and Nitzschia frustulum) which were the same species as traced from the water sample of river (From which the body was recovered). Death was attributed to drowning. The family members also accepted that the lady was mentally disturbed and left the home a couple of days before the death. Case 5 – A highly putreied body of 30 – year – old male was recovered from a canal. No information about causes of death was obtained at autopsy. Studies from extracts of nitric acid of sternum, clavicle and femur proved the presence of two species of diatom (Cymbella ventricosa and Cocconeis placentula). Water samples from multiple sites were sent to the laboratory to trace the original site of drowning. Same two types of diatom species were found in the water sample from a site distant from the site from where the body was recovered. The cause of death was drowning which was also proved by further police investigation. Pooja Jakhar et al. – 2015, they all have A Case Study “Diatoms as Cardinal Key to Drowning case” Drowning can be defined as death due to full or partial submersion in a fluid If the victim was dead before the body was submerged, the transport of diatom cells of various organs is prevented because of a lack of circulation. The basic principle of the “diatom test” in drowning is based on the diatoms are present in the medium where the possible drowning took place and that the inhalation of water causes penetration of diatoms into the alveolar system and bloodstream and thus, their deposition in the brain, kidneys, and other organs. Diatoms are a widespread group and can be found in the oceans, in fresh water, in soils and on damp surfaces. Most live-in open water, although some live as surface films at the water-sediment interface, or even under damp atmospheric conditions. They are especially important in oceans, where they are estimated to contribute up to 45% of the total oceanic primary production. The presence of a sufficient number of diatoms in vital distant body organs can establish ante-mortem drowning up to a certain extent. In forensic science, diatoms can be extracted from criminal evidence (e.g., From soil adhered to objects, washed from victims and suspects, etc.) and analyzed. This is of particular importance in establishing relationships of both parties and evidence with crime-scenes, and establishing whether multiple locations may have been involved. Following basic experimental protocol was followed for extraction and analysis of diatoms from each of the reported cases: -1. Acid digestion: Concentrated nitric acid was used to extract the diatoms from the exhibits. For this, internal organs in the form of exhibits were put into separate jars and 50 ml of nitric acid was powered in each of the undisturbed and covered. Next day, the digested or semi-digested exhibits were boiled for half an hour or more till the whole of the content changed into yellow liquid and were allowed to cool. 2. Centrifugation: Fat layer was removed and the yellow liquid was subjected to centrifugation at 4000 RPM for about 15 minutes. Centrifugation was repeated three times and the supernatant was discarded every time. Pellets were washed with distilled water and again centrifuged. 3. Microscopic Examination: After discarding the supernatant, pellets were used to prepare the microscopic slides and were examined for the presence of diatoms under the microscope. Slides were also prepared from the water samples from which the dead bodies were recovered. Diatom types found in the organ samples and water samples were compared and analysis was done. Out of 38 case studies, 35 cases were found to be positive for diatom test. The site of drowning, body organs used as exhibits for diatom test and the results of the diatom test in each case. The type of diatom species presents in the body organs (sternum, clavicle, liver, kidney, spleen, intestine, heart) exhibits were the same as detected in the water samples collected from the respective drowning sites. The cause of death was attributed to drowning as diatoms can be carried to the distant parts of the body if the person was alive when entered into the water. These results were also supported by many studies done from time to time. Mahipal Singh Sankhla et al. Vivekananda Global University – 2016, they all have a study about ‘Forensic identifications of Drowning Death by the use of Diatoms Analysis – 2016. Quantitative and qualitative diatom analysis in victims found in the water can give strong evidence of death due to aspiration of water. Diatom’s detection in both drowning and non-drowning cases do not show a definitive relation with time since death. The diatom test, while extremely specific, is of immense value considering the limited objective tests available for drowning diagnoses. If the person is still alive when entering the water, diatoms will enter the lungs if the person inhales water and drowns. The diatoms are then carried to distant parts of the body such as the brain, kidneys, and bone marrow by circulation. If the person is dead when entering the water, then there is no circulation and diatoms cannot enter the body. Diatoms do not occur naturally in the body. If the diatom profiles of the tissues match those of the water, diatom analyses will be very useful for the forensic pathologist to state about the cause of death, such as drowning. The determination of the dominant taxa may also indicate the site of drowning. It has been suggested that marrow of the sternum may be as good of a source of diatoms as femoral tissue. Death of a victim found in water should not always be related to drowning. Drowning is a prevented from entering the lung by submersion of the body in diagnosis of drowning for bodies freshly retrieved from Water is mainly based on some ‘‘drowning signs’’, such as the hemorrhages, impression of ribs on lungs, oedematous lungs however, the diagnosis of drowning is rather difficult because those ‘‘drowning signs’’ were destroyed. Dr. TTK Reddy et al. 2017, they all carried out the study in the Dept. of Forensic Medicine, Guntur medical College, Mortuary, Government General Hospital, Guntur during the year January 2017 to December 2017 on ‘Significance of diatoms test in drowning vs non-drowning cases. After selection of the cases during the post-mortem examination the tissues or organs are taken and diatoms test is done as per the procedures in drowning and non - drowning cases. The sample of drowned water was also examined for presence of diatoms. A comparison is made for similar types of diatoms to get the significance of diatom test. The relevant water samples in the particular rural locality also are examined and compared in non-drowning cases. Diatom test is very valuable in drowning deaths especially in decomposed states as other findings of drowning may be lost or obscured due to decomposition. Although there are some typical signs of drowning known, it is still hard to determine a death by drowning when the post-mortem signs are impossible to find in case of deceased and therefore a great number of tests have been proposed to allow confirmation of death by drowning of a victim. Diatom test is one of these tests and works as an important tool in diagnosis of death due to drowning, it can be used to discriminate between drowning and non-drowning cases. Detection of diatoms in tissues may contribute to diagnosis of drowning, therefore an efficient method of extraction and microscopic examination of diatoms from tissues is necessary, however, it is important to remember that the absence of diatoms does not immediately rule out drowning; the test does not prove the negative, and a thorough investigation is always required. Five cases were found to be negative for the above-mentioned test as no diatom species were detected from the bones and visceral exhibits of the suspected drowning victims. However, diatoms were detected from the water samples of all the three sites. In the present study, the major diatoms found in the drowning cases were centric in shape which followed by small pennate type of diatoms were observed in most of the drowning cases. Diatom test in drowning is significant even though occasional diatoms may be recovered in lungs and stomach mucosa of non-drowning cases. Out of 60 cases, 40 drowning cases and 20 non drowning cases were studied. In cases, where the cause of death cannot be ascertained by conventional postmortem examination in those cases presence of diatoms in lungs and stomach mucosa plays an important role in determining whether the death is due to drowning or not. In the present study, Stomach mucosa and lungs studied in non-drowning cases showed presence of diatoms. Out of 20 non drowning cases, only 10 cases showed diatoms in lungs. During the present study, it is concluded that among 60 sample cases, 40 cases were positive while 20 cases were non drowning cases. The present results revealed that 35 cases show positive for diatoms. And in 20 non drowning cases, only 2 cases show diatoms which is insignificant. Pal S K Sharma & Sehgal Rana Biology and Serology Division, Regional Forensic Science Laboratory Northern Range, Dharamshala, Himachal Pradesh – 2017, both have carried out the study about “Diagnosing Death with Diatoms: A Retrospective Study of Forensic Cases in Himachal Pradesh, India. Diatoms found inside the body of a drowned victim may serve as corroborative evidence in the diagnosis of cause of death. Diatom has proved to be the only golden standard for diagnosis and confirmation of drowning deaths whether the drowning was ante-mortem or post-mortem. The study was based on the cases of death due to drowning received from three districts of Northern Range of Himachal Pradesh, India during the period of five years from 1st January, 2010 to 31st December, 2015 for diatom test. A total of 66 human cases were examined for detection of diatoms. The detailed information regarding cause of death, socio-demographic factors and other associated information was gathered. The acid digestion method accepted worldwide for diatom extraction was used. Accidental drowning was the most common cause of death (37.87%). Highest (51.51%) percentage of drowning cases was noticed during the months of monsoon/rainy season. Blood on mouth and nostrils was present in 14 (21.21%), froth from mouth, nose, larynx and trachea in 35 (53.03%) cases. Diatom-test was found positive in 62 (93.93%) cases. The results of the study revealed the occurrence of various varieties of diatoms in water bodies of northern region of Himachal Pradesh. The most common diatom genera detected were Navicula (86.36%). The study concluded that diatoms are amongst the important biological forensic evidences in diagnosing the cause and place of death due to drowning. Gender based examination revealed higher percentage of males involved in drowning fatalities and the accidental submersion was the commonest manner of death. E.A. Levin, R.M. Morgan & V.J. Jones, 2017, they all have researched about ‘The transfer of diatoms from freshwater to footwear materials: An experimental study assessing transfer, persistence, and extraction methods for forensic reconstruction’. Previous studies have considered the transfer of diatom valves to clothing fabrics, and methods appropriate for their extraction. As of yet, however, no published research has considered their transfer to footwear materials or their persistence. Accordingly, this research seeks to address this lacuna, undertaking a series of exploratory experiments on the transfer and persistence of diatoms upon common footwear materials, a recipient surface that, while valuable, has historically been under-represented in the literature on transfer & persistence. Specifically, this research was designed to assess: Whether diatoms transfer to common footwear materials after short periods of immersion in freshwater, and to what extent the initial levels of transfer are influenced by the recipient material type and duration of immersion; Whether diatoms can be recovered from common footwear materials 168 hours (7 days) after the transfer event, and to what extent the recipient material type appears to influence the timescales over which diatoms persist; Whether diatom valves of different sizes appear to persist at different rates (i.e. whether retention is size-selective). In order to address these questions, it is also necessary to consider how to extract diatoms safely and reliably from common footwear materials. There is, at present, no consensus on how best to extract diatoms for forensic purposes, neither for samples of fabric, or organic tissue. No published research has specifically considered protocols for the extraction of diatoms from footwear. Donated Particles and Recipient Surfaces Where experiments deal with the first principles of transfer and persistence, it is common to opt for highly-controlled simulated contact in order to create a uniform and reproducible distribution of the particles or fibers under investigation. Accordingly, in this experiment, contact between footwear and a water body was simulated by immersing swatches of fabric in a ‘tank’ of diatom suspension. A suite of five fabrics was chosen: canvas, leather, and faux suede (100% polyester), representing uppers, and rubber and polyurethane representing soles. These materials were selected as demonstrative of both synthetic and natural uppers and soles, with variation in surface textures. Pristine (new) 4 cm2 swatches of the five materials were immersed in the ‘tank’ of the collected canal water, and the tank was agitated to keep the algae and sediment in suspension. The modified Wellington boots, which had been immersed in the tank of diatom solution for three minutes, were worn during waking hours over seven days while normal day-to-day activity was conducted (with approximately 15 minutes of activity per hour). Swatches of the five upper and sole materials that had been immersed for three minutes were subjected to three different extraction treatments. After extraction, all sample solutions were treated with the same slide-making process. Slides were examined under phase contrast microscopy. Continuous transects were performed over 50% of the surface area of the slide. Information about the morphology of the valve, and whether it was fragmented or whole was recorded. For the four materials of ‘suede’, leather, rubber, and polyurethane, the three treatments appeared to extract similar numbers of valves. The aim of this study was to identify the transfer of diatom valves to common footwear materials, their persistence on these fabrics, and methods of extracting the valves from these substrates for analysis. It was found that diatoms could be extracted from swatches of canvas, ‘suede’, leather, rubber, and polyurethane after even short periods of immersion, and that valves could be recovered from all three upper materials after 168 hours of wear. (However, some samples produced slides containing no diatoms, and the earliest time after which this was observed was four hours.) A novel extraction technique, involving the heating and agitation of the sample with distilled water, was utilized and led to the extraction of a similar number of valves in comparison to the hydrogen peroxide method adapted from the environmental sciences, without causing a higher level of fragmentation (which might preclude species identification). The implications of the transfer and persistence experiments are fourfold; Footwear is a recipient surface that can be sampled for diatoms in forensic scenarios; Sampling may be viable even when contact with a water body may have been brief; Where shoes are composed of a mixture of fabrics, it may be more fruitful to sample coarser, woven materials; The analysis of diatoms recovered from footwear may be viable even if 168 hours have elapsed between the contact and the evidence being recovered. It appears to be likely that the <200 μm2 fraction will be retained after such time periods. Together, these findings confirm that footwear can represent a useful repository of diatoms in casework scenarios for both forensic reconstruction and the generation of forensic intelligence and evidence. Rafael Carballeira et al. 2017, they all have a researched about ‘ A valid method to determine the site of drowning’. The diatom populations in the water system reservoirs are those that provide the differential elements that help determine the location of the site, hence the need to characterize adequately the diatom populations of the different aquatic systems. While there is a great diversity of species, any aquatic environment of the region is capable of establishing a discriminant analysis. In the water column of the reservoirs, there is a clear dominance of planktonic species (e.g., Fragilaria crotonensis, Asterionella formosa, Cyclotella meneghiniana, Aulacoseira granulata, Aulacoseira dastans). These species are common and abundant in the plankton of reservoirs and other freshwater aquatic systems, coinciding with those in other geographic areas and therefore will have little predictive value, and the least frequent species will be the most useful in determining the drowning site. This has influenced the negative diatom test results obtained by Coelho et al., since the diatom analysis did not allow for differences between the different sites of the same river section at the Douro River estuary. This is because the same aquatic mass is influenced by the same environmental factors and, in general, tends to homogenize the populations of diatoms, as happened in certain stretches of river in which the biological populations settle down in a Bcontinuum. The transfer of diatoms to the body occurs during submersion upon contact with the suspended elements at each site. This process is a physical phenomenon that implies that with the contact with the water, there is a simultaneous contact with all those elements in suspension which transfer to the organism a diatom profile similar to that existing at the site. This profile will be reflected in the different organs according to the selective barriers found within the organism, as reported for rat and humans. The suspension profiles of the organs, either in direct contact or open to the external environment, show a higher fidelity to those in the sites. Also, for this reason, there is very little difference between the animal model in rat and forensic cases. Tissue samples in contact with the immersion medium, such as fur, stomach, and lung, lacking physiological- anatomical barriers, allow a large number of diatoms to be incorporated into the organism and show a better transfer of the spectra and profiles of diatom species. The differential representation of the dominant/indicator species in plankton is probably mediated by the type of sample (fur, stomach, lung) and the active or passive entry in the organism. For example, the lung in group III (drowned) where the active aspiration effects may concentrate the plankton in the sample, thus over-representing the dominant species. Our results indicate that it is possible to determine the site by applying mathematical methods to the quantitative diatom data, such as with EU samples, but this is not always possible. In samples of BM, PO, and some of AC, the similar abundance profiles, the existence of low abundances of exclusive species is a limitation for KL and other mathematical analyses to determine with certainty the provenance of the samples. The use of the indicator species we have selected is especially useful for discerning the origin of the BM samples, and also the samples from PO and AC, but these latter with a lower resolving capacity. On the other hand, although in the literature, the lung is often used as the reference organ to locate the site given that it is the main route of entry into the body, nevertheless, fur has the advantage of being in direct contact with the water medium and has a larger catchment surface without the disadvantages of concentration. A single type of organ sample alone should not be used as a sole reference. The development of a standardized tool based on a diatom test for forensic cases is mainly due to the lack of standardization and validation of the methodology published so far. However, it is possible to determine the precise location (site) where a death by submersion occurred by means of the diatom test as long as the basic premises are fulfilled: namely that there are differential species spectra and abundance profiles in the medium of submersion, and that they are transferred to the body during submersion at that time. Combining the quantitative and qualitative analyses of the diatoms present in the samples and the medium allows us to affirm that the diatom test is a valid tool to determine the location of the submersion site. Species spectra and abundance profiles tend to be homogeneous in relation to the same reservoir and within a period of 1 week in each reservoir. The dominant species in the water column are reduced to a small number of species by the restrictions of living in suspension. The transfer of the diatom abundance patterns from the immersion medium to the organism occurs mostly to those organs in contact with the exterior (fur) or with open routes to the outside (stomach, lung), which are more likely to determine the location of the submersion medium with respect to the internal organs (heart, kidney, spleen, bone marrow). Quantitative analysis of samples of plankton and fur, stomach, and lung allowed us, by means of a statistical model based on distance KL, to verify the probability of provenance of the samples with sufficient success to determine the site of a submersion between different water systems. The fur and stomach show considerable discrimination compare to the lung. The use of many samples of the same organ is not sufficiently precise, for which reason several organs need to be studied to provide sufficient certainty to determine the site of a submersion. Furthermore, the quantitative analysis should be complemented by a selection of indicator species: in the reservoirs studied, the location of the submersion medium could be identified by the indicative species of each reservoir, Achnanthes linearioides (EU), Brachysira brebissonii (EU), Navicula anglica (EU), Achnanthes subhudsonis (BM), Cymbella excisa (PO), Encyonopsis cesati (AC), and Aulacoseira sp1 (AC). Deepali Luthra, Mukesh Kumar Thakar, and Jasvirinder Singh Khattar – 2019, both have case study “Forensic study of the Distribution of Diatoms in Bhakra Canal near Narwana, Haryana, India. Bhakra Canal: Bhakra Canal is the largest multi-purpose irrigation project of Punjab, Haryana and Rajasthan. It harvests the water of Sutlej River and irrigates the 27.41hectare land of Punjab. After irrigating the areas of Punjab, Bhakra Canal enters Haryana near Tohana and irrigates large parts of Hissar, Jind, Fatehabad and Sirsa districts. One liter of water sample was collected from either side of the water body in a sterilized plastic bottle. The water samples for diatom analysis were collected seasonally i.e. during summer, autumn, winter and spring seasons from the year 2014-2016. The collected samples were analyzed by digesting approximately 100 ml of water sample with 10-25 ml of conc. nitric acid (HNO3) and a pinch of Potassium dichromate. The samples were kept overnight for incubation. Next day, 10 ml of sample was centrifuged at 3000 rpm for 10 min. The supernatant was removed off and the residual material left behind was then added with more water and again centrifuged again in the same way. This process was repeated three times and the diatoms at the bottom of tubes were re-suspended with additional washing with distilled water. The shape of the most of diatoms was elongated, fusiform and lanceolate, while some diatoms were cylindrical in shape. The qualitative and quantitative analysis of diatoms of Bhakra Canal showed significant variation in diatoms. The maximum numbers of diatoms were observed during winter while minimum during the summer season. The diversity and abundance of diatoms vary from one season to another due to variation in various environmental parameters (pH, Salinity, alkalinity, total dissolved solids, temperature, etc.) Therefore, different water bodies have different diatom profile. In line with this, there is a great thrust for the baseline database of diatoms of different water bodies. The database generated in this way would be useful to the forensic scientist, forensic medio-legal expert for solving the questions related to drowning cases. Singh AR & Verma, Research Scholar, University Institute of Applied and Health Sciences, Chandigarh University, India - 2019, both have A Research related to “A Systematic Review on Various Diatoms Species Associated with Drowning ”. Drowning is third leading cause of unintentional death worldwide which accounts for 7% of all injury related deaths. Low- and middle-income countries accounts for about 90% of unintentional drowning deaths. Children, males and individuals with more access to water are most at the risk of drowning. About half of the drowning deaths occur in WHO Western Pacific Region and WHO South East Asia Region. There is uncertainty in the estimation of global drowning deaths. Official data categorization methods for drowning deaths exclude intentional drowning deaths (homicide or suicide) and drowning deaths caused by flood disasters and water transport incidents. Diatoms have a siliceous cell wall with distinctive shapes and ornamentations. The ornamentation found on the silica frustules of diatoms is species specific and thus provide important information about their shape and sizes which can be used for their identification and classification. On the basis of shapes diatoms are classified into two specific groups one is centric with radial symmetry and other are pinnate which exhibits bilateral symmetry. Recent years various methods have been developed for diagnosis of diatoms from tissue samples by enzymatic digestion methods by using proteinase kinase K, microwave digestion-vacuum filtration-automated scanning electron microscopy (MD-VF-Auto SEM), Molecular biological techniques and loop mediated isothermal amplification (LAMP). From all these methods acid digestion method is still widely used for the detection of drowning deaths. Diatom test can not only determine the mode of death as drowning but it can also determine the site where the drowning has taken place. Various researches conducted throughout the world showed that some of the species of diatoms are site specific and thus they can be considered as endemic. Diatoms also show variations with temperature, pH and season. Hence Diatomological mapping is of keen interest by the forensic pathologist in the near future. Diatomological mapping helps in generation of baseline data which aids in forensic investigation for diagnosis of drowning deaths, in location of diatoms diversity in particular location and in environmental science and Botany in assessment of water quality in particular region. Continuous monitoring of water bodies for the presence of diatoms is very essential for the detection of diatoms. Sometimes body of a drowned victim get drifted to other sites due to water currents and thus if diatom database is available then we can locate the exact site where actual drowning has taken place. Various biological and thanoto-chemical tests like Hemodilution tests, sinus fluid, microorganisms like algae, paranasal sinuses, Chemical constituents like strontium, diatom test and various markers like magnesium, sodium, chloride, calcium are utilized for the diagnosis of drowning deaths. From all the methods utilized for drowning deaths, diatom test is still considered as “golden standard”. Study showed that detection of diatoms in tissue samples of non-drowned bodies might be due to abundance of diatoms frustules in non-vegetarian cooked food stuff, inhalation of air in a atmosphere containing material used for manufacturing the building like paper, cement and paint etc. and long term exposure of body in a water containing diatoms. Diatom test has emerged as an important test for forensic laboratories to diagnose the cause of death in drowning cases. Presence of diatoms in the tissue samples can act as supportive evidence that death has occurred due to drowning. If a person is already dead before entering into the water the diatoms could not be detected into the internal organs of the drowned victims. Detection and comparison of diatoms with tissue and control sample is required to rule out the positivity or negativity of diatom test. Validity of the diatom test is also based on the number of diatoms valves recovered from the tissue samples of drowned victims. Various methods have been developed so far for the diagnosis of drowning deaths but still diatom test is considered as a golden standard. Molecular biological techniques, ADIAC and phytoplankton mapping may aid up the diatom test in near future. Ekta Saini et al. Department of Genetics, Maharishi Dayanand University, Rohtak – 2020, they all have carried out a study related to ‘Forensic Diatomological Mapping: A Data Base of Diatom Profiling to Solve Drowning Cases – 2020’. Drowning responsible as one of the most common unnatural causes of death in India. Drowning is the occurrence of respiratory impairment when air is prevented from entering into the lungs due to submersion/immersion of mouth and nostrils into water or other types of the fluid medium. Drowning generally occurs in accidental or suicidal cases and sometimes also in homicidal post-mortem drowning. It becomes very important for law enforcement agencies to confirm whether drowning was the actual cause of death or not. The death occurring due to drowning is called antemortem drowning although the death occurred before entering into the water or body is dumped after homicide by any mean termed as post-mortem drowning. Breathing underwater during antemortem drowning draws a lot of water in the lung cavities that exerts high pressure on walls of lungs. Heavy impact ruptures these alveoli networking in lungs and pushes the water deep into the blood circulation. As long as heartbeats, the water keeps on mixing with the blood and also pulls diatoms present in the water medium towards various vital organs such as spleen, brain, liver, kidneys and bone marrow. In case of post-mortem drowning where breath has already ceased before entering the water, the diatoms can be detected up to the lungs due to the passive absorption of water but they remain unreachable to distant body organs due to lack of respiratory thrust. A data revealing 536 cases of death due to drowning happened during 2005-06 was reported from Punjab. The survey made by the National Crime Record Bureau (NCRB) during 2013 in India, explored around 8.0% deaths due to drowning out of all accidental and suicidal deaths. It was observed that on average 82 persons die due to drowning every day in India. It was observed that 73.9% of the cases died due to accidental drowning while suicidal and homicidal cases were 16.1% and 0.1% respectively. Yuanyuan Zhou et al. 2020, they all had Research about ‘Advances in Forensic Diatom Testing. Diatom cell walls are composed of a hydrated silicon dioxide called a frustule, which is not easily destroyed under extreme conditions such as strong acid digestion. Digestion with strong acids (e.g. nitric acid) is the most commonly used method for diatom detection. Although it is effective to eliminate organic components, the remaining waste liquid may cause serious environment pollution. The improvements are beneficial for separating diatoms more efficiently and environmentally from multiple organ tissues with a higher detectable rate. Comparing four chemical digestion methods, Ming et al. found that proteinase K digestion provided a more satisfactory result with fewer samples and less pollution. However, proteinase K is too expensive to use for diatom testing with numerous samples. Zhao et al. developed a new method called Microwave Digestion-Vacuum Filtration- Automated Scanning Electron Microscopy (MD-VF Auto SEM) by integrating and improving methods that had been used for diatom testing over a long period of time. In this method, microwave digestion and vacuum filtration replaced traditional acid digestion and centrifugation, respectively. The highlight of this method is Auto SEM, which could easily and efficiently identify diatoms. In recent years, molecular methods such as DNA sequencing have been used in diatom testing in drowning cases. DNA sequencing analyzes the base sequence of a specific DNA fragment, and has the ability to identify specific species using genetic markers. Diatom species are abundant in water and are extremely sensitive to their living environments. There are large differences in the abundance of. The fragments of diatom under 400 light microscopy. Excessive digestion will cause the structure of most diatoms to be destroyed, resulting in an amount of diatom fragments. Reprinted with permission. 100 Y. ZHOU ET AL. diatom species among nearby water environments. Diatom species abundance patterns in victims’ organs may be similar to those at drowning sites. Currently, traditional chemical digestion methods are widely considered as the “gold standard” for drowning diagnosis. The primary task of forensics in cases involving a dead body in water is to determine whether the individual died due to drowning or whether the body was thrown into the water after death. However, due to adverse factors such as corpse decomposition and gnawing by fish or worms in the water, dead bodies in water often lack the characteristic signs of drowning, making the diagnosis of drowning extremely difficult. The microwave digestion device and electron microscope in the MD-VF-Auto SEM method are not common in conventional forensic laboratories. The introduction of the above methods and systems can reduce and prevent false-positive results and improve the reliability of diatom testing results. Elisa Falasco et al. 2010 - 2020, they all have A research related to “Looking back, looking forward: A Review of the New Literature on Diatom Teratological Forms. Over the last years, issues concerning diatom teratological forms and environmental stress have received growing interest within the scientific community. Diatom teratological forms are one of the best individual-level biomarkers since they provide a rapid response to several environmental stressors, including new emerging pollutants. The mechanisms involved in teratological valve likely involve both cytoskeleton and silicon metabolic pathway impairments. Presently, there is still a wide debate about the most appropriate indicators of response to environmental stress in aquatic environments. Recent studies have highlighted the limitations of some metrics commonly used for detecting the effects of pollutants. In total, it is analysed that 409 papers, 222 already contained in Falasco et al. (2009), 187 in the present paper. Of these 409 articles: 185 cited in their text at least one diatom species showing teratologies (about 45% of the total analysed literature); 154 hypothesized one or more causes leading to the deformations (about 38%). In total, from this literature review we were able to obtain a list of 298 taxa, which showed some kind of deformation and the corresponding hypothesized cause. Most of the research focusing on diatom teratologies suggested that abnormal form (presence/absence) detection is a good and reliable signal for the identification of an environmental stress. Deformity frequency ranging from 0 to 0.5% should be considered as naturally occurring (Morin et al., 2008, 2012; Arini et al., 2012), while higher percentages can be considered warning signals of contamination. But the absence of deformity can help in the interpretation of certain results. This paper intends to give continuity to a project started in 2009 and fruitfully pursued in 2017, when several issues related to diatom teratological forms were examined throughout the collaboration of many experts (Lavoie et al., 2017). Even though an index based on teratological forms is still far from being developed, we hope the present paper can help researchers in the interpretation of the results obtained from their future research. The four-step procedure we proposed in this article is based on theoretical hypotheses, and most of the readers certainly know that natural processes are never so linear in their responses. Indeed, many other important issues necessarily need to be considered in the stress-response analysis. Among them, the interspecific competitions within diatom community and among the other primary producers inhabiting the biofilm, which could drive and shape species composition and induce the production of teratological forms. Junaid Shaikh et al. Government Institute of Forensic Science Nagpur, 2021, they all carried out study in 2021, about ‘Diatomological Mapping of Aurangabad Region for Unrevealing Drowning Mystery’. Drowning is the third unnatural cause of death in India. Drowning death is still considered as one of the most difficult 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- specific/unique diatoms for a region aids into locating the exact drowning site. The onsite identification 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 confirmation of the drowning site. 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 HNO3: H2SO4 in 1:3 proportion in 20ml of water sample. Slides were prepared, mounted and microscopic analysis was performed using light and inverted phase microscope. The D-map was generated and it featured 55 identified species, amongst the members of Nitzschia, Navicula and Cymbella were commonly found in many water bodies, while Craticula, Gomphonema, Tibetiella, Melosira, Pleurosira, and Mastogloia were restricted to only single water body. These unique diatoms can be used as site-specific indicators in drowning investigation of Aurangabad region. This study generated diatomic map for experimental water body present in Aurangabad region. Diatom test is theoretically based on the presence of diatom in the vital organs of the body such as lungs, bone marrow. Thus, the accuracy in the diatom identification is very much important in drowning death investigation. In the diatom test, a drowning site is located by comparing the species of diatoms extracted from tissue samples and the to have a regional diatomic distribution map to validate, identify the drowning site and increase the rate of investigation. In current study generated data shows the distribution pattern of diatom species in 12 different sites that were taken for sampling. Distribution of diversity helps to identify the key indicator species of the drowning site. Diatoms are the key indicators of drowning death and can be used to locate the site of the drowning. The diatoms restricted to only single water body are unique diatoms and can be used as site-specific indicators and marker. These D-maps will be helpful in dispense a significant information about water bodies and their common as well as unique flora 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.

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"DIATOMS Guardians of Aquatic Ecosystems and Climate Stabilizers", IJNRD - INTERNATIONAL JOURNAL OF NOVEL RESEARCH AND DEVELOPMENT (www.IJNRD.org), ISSN:2456-4184, Vol.9, Issue 6, page no.e267-e270, June-2024, Available :https://ijnrd.org/papers/IJNRD2406424.pdf

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