INTRODUCTION

Forensic science is the application of scientific meth- odology, knowledge and principles to the resolution of legal questions, whether criminal or civil. Death by drowning is defined as a death due to submersion in a

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*Corresponding Author Received 28th April, 2014

Accepted after revision 21th June, 2014 BBRC Print ISSN: 0974-6455 Online ISSN: 2321-4007

©A Society of Science and Nature Publication, 2014. All rights reserved.

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liquid and the mechanism in acute drowning is hypox- emia and irreversible cerebral anoxia1. 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 bodies. The diagnosis of drowning is one of the most

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Ashiq Hussain Magrey and Mool Raj

difficult diagnoses in forensic pathology and therefore a great number of tests have been proposed to allow a confirmation of death by drowning of a victim. Dia- tom test is one of these tests and works as an important tool in diagnosis of death due to drowning, it count can be used to discriminate between drowning and non- drowning cases. Detection of diatoms in tissues may contribute to diagnosis of drowning, therefore an effi- cient method of extraction and microscopic examina- tion of diatoms from tissues is fundamental, 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. (Peabody, 1978; Hürlimann et al., 2000; James and Nordby, 2003; Thornton and Peterson, 2007; Houck and Seigal, 2010; Saferstein, 2010; Chisum and Turvey, 2011; Verma, 2013).

MATERIAL AND METHODS

The case studies were conducted in Biology Division of Forensic Science Laboratory Jammu (Jammu and Kash- mir), India and all the cases reported here, are fresh water drowning cases. After post-mortem of dead bod- ies, internal organs (sternum, clavicle, femur and lungs) along with the water samples (from where the dead bod- ies were recovered) were sent as crime exhibits to the laboratory. Cases were opened and processed following standard methodology, (Peabody, 1978).

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 stud- ied 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, (Hürlimann et al., 2000)

trated nitric acid (HNO3) was added to The bottles were shaken thoroughly before analyzing. The samples were analyzed with the help of ‘Acid Digestion Method’. These samples were kept as such for two hours. Then the sam- ple was centrifuged at 5000 rpm for 10 minutes in prop- erly labeled plastic centrifuge tubes. The supernatant was pipette off leaving behind only a pellet containing diatom frustules at the bottom of tube. Pellet material was suspended in distilled water and centrifuged twice to remove all traces of acid (Peabody, 1978 and Hürli- mann et al., 2000)

PREPARATION OF SLIDES

After the final centrifugation except for 1ml, the whole supernatant was discarded by using a sterilized drop- per. The pellet along with 1ml of supernatant was stirred well and entire volume of the pellet suspension (1 ml) was aspirated using a Pasteur pipette and droplets of the aspirate were poured. microscopic slides for each sam- ple. Permanent slides were prepared in order to avoid the skipping of any diatom species. This material was completely dried and mounted with help of DPX. Dia- toms were examined with an optical compound micro- scope fitted with light source at magnification of 1500X oil immersion. Slides were fully scanned with help of microscope and representative images of the diatom were captured using a computerized photo-capturing device/camera (Q-Win Leica) fitted on microscope objec- tive lens.

RESULTS AND DISCUSSION

Out of 31 reported cases in the data, only selected few are being reported in the section fo results, (Cases 1-11).

CASE 1

The body of a 19 year old boy was recovered from Dal lake three kms from Lalchowk, Srinagar. No signs of injury were found during the 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.

EXTRACTION OF DIATOMS FROM WATER SAMPLES USING ACID DIGESTION METHOD

About 50 ml of water sample was transferred into a sterilized 100 ml glass beaker and 10-20 ml of concen-

CASE 2

The body of a 26 year old lady was recovered near the bank of Wular lake, the largest lake in Asia. Signs of head and neck injury were traced during autopsy. Nitric acid

Ashiq Hussain Magrey and Mool Raj

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 mur- dered her wife because of having extra-marital affair, thus it was proved that it was not a case of drowning.

CASE 3

The body of a 23 year old lady was recovered from a vil- lage well. No signs of injury were 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, clav- icle and femur showed presence of three diatom spe- cies (Nitzschia gracilis, Cymbella and Navicula radiosa). Same three types of diatom species were detected from the water sample of the 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 Jehlum. No specific reason of death was found at autopsy. Microscopic studies of slides prepared from nitric acid extracts of sternum, clavicle, femur proved the presence of three diatom species (Cymbella cymbiformis, Gomphonema spheroporum and Nitzschia frustulum) which were the same species as traced from the water samples 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 putrefied body of a 30 year old male was recov- ered from a Mansbal lake. No information about causes of death could be 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 dis- tant from the site from where the body was recovered. The cause of death was drowning which was also proved by further police investigation.

CASE 6

The body of a 28 year old lady was recovered from a river Tawi in Jammu. No specific reason of death was

found at autopsy. Microscopic studies of slides prepared from nitric acid extracts of sternum, clavicle, femur proves the presence of three diatom species : Amphora; Apipleura and Aulacoseria, which were the same species as traced from the water samples of river from which the body was recovered. Death was attributed to drowning..

CASE 7

The body of a 16 year old boy was found submerged in a water tank. No sign of injury were found at autopsy. Nitric acid extract of internal organ (sternum) revealed the presence of two types of diatom species Melosira and Hantzschna. The same two 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.

ASE 8

The body of a 34 year old lady was recovered from a canal. Signs of head and neck injury were traced dur- ing autopsy. Nitric acid extract of sternum and clavicle showed the absence of any diatom species. However, a diatom species (Surirella) 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 mother in law that she murdered her.

CASE 9

The body of a 35 year old lady was recovered from a village well. No signs of injury were 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 ster- num, clavicle and femur showed the presence of two diatom species, Eunotia and Epithemia. Same two types of diatoms were detected from the water sample of well in which the body was found. Drowning was the cause of death.

CASE 10

The body of a 45 year old lady was recovered from a river Tawi in Jammu. No specific reason of death was found at autopsy. Microscopic studies of slides prepared from nitric acid extracts of sternum, proved the presence of diatom species Anphora, which was the same species as traced from the water sample of river (from which the body was recovered). Death was attrib- uted to drowning. The family members also accepted that the lady was mentally disturbed and left the home a couple of days.

CASE 11

A highly putrefied body of 25 year old male was recov- ered from a canal. No information about causes of death was obtained at autopsy. Studies from extracts of nitric acid of sternum, clavicle and femur bones proved the presence of Aulacoseria cocconies. Water samples from

Ashiq Hussain Magrey and Mool Raj

multiple sites were sent to the laboratory to trace the original site of drowning. Same diatom species was 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.

Role of diatoms has always remained significant in solving the drowning cases. In cases, where the cause of death cannot be ascertained by conventional post-mor- tem examination in those cases presence of diatoms in sternum and other body tissues plays an important role in determining whether the death is due to drowning or not. Diatoms are unicellular plankton having a cell wall made of silicon dioxide. The glass frustule is composed of two valves, which fit together with the help of a cin- gulum, or set of girdle bands. There are about 10,000 species and 174 genera of diatoms reported which are having different shapes and sizes varying from 1 to 500μm (Rohn and Frade, 2006).

Diatoms are traditionally divided into two orders: cen- tric diatoms (Centrales), which are radially symmetric, and pennate diatoms, which are bilaterally symmetric (Pennales) but Pollanen et al. (1997), classified diatoms into three classes: centric diatoms (Coscinodiscophyceae), pennate diatoms without a raphe (Fragilariophyceae), and pennate diatoms with a raphe (Bacillariophyceae). Most diatoms exist singly, although some join to form colonies. They are usually yellowish or brownish, and are found in fresh and salt water, in moist soil, and also on the moist surface of plants.

Analysis of diatoms present in the body tissues like lungs, liver, spleen, blood and bone marrow has been undertaken as supportive evidence in drowning cases. The theory behind the ‘diatom test’ is that when any per- son drowns, the diatoms present in that water will reach to the lungs and some of them because of their size pen- etrate into the alveoli. If the heart is still beating, the diatoms that have entered into the blood stream travel around the body and may lodge in distant organs such as the kidneys, brain and bone marrow before death (Auer, 1991; Pollanen, 1997).

The presence of diatoms in the internal organs most likely confirms the ante-mortem drowning. If there is any doubt about the drowning site, then water sample from the putative site of drowning can be collected and analysed to determine the similarity of different species of diatoms in the water and the body.

For last 100 years, many scientists have employed various extraction methods individually and in combi- nations in order to isolate diatoms from water and tissues samples successfully. Among these approaches some of them were found to be superior to others. Nitric acid digestion is a worldwide known method for the extrac- tion of diatoms. 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. Again, if specific types of diatoms recovered from the marrow can be matched with those found at the suspected drown- ing site, the results might implicate a particular locale

Ashiq Hussain Magrey and Mool Raj

of submersion and help identify the death scene, which in turn could lead to additional forensic evidence. The presence of diatoms can also corroborate investigative evidence in making accident determinations, (Pollanen, 1997; Rohn and Frade 2006).

CONCLUSION

During the present study it is concluded that among the eximaned cases, 31 human cases were suspected for drowning, 09 cases were positive (death due to drown- ing) while 22 cases were negative. The reason for the positive results was the presence of diatoms in their ster- num which was detected by nitric acid method.

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