Forensic limnology

Forensic limnology is a sub-field of forensic botany, which examines the presence of diatoms in crime scene samples and victims. Different methods are used to collect this data but all identify the ratios of different diatom colonies present in samples and match those samples with locations at the crime scene.^[1][2][3]

Use of diatoms

Diatoms are diverse microscopic algae with silica cell walls that have different characteristics such as color, shape, and size. There are 8,000 known species of diatoms (without taking into account the variety of subspecies that have specialized within their specific environments). Diatoms do not have specialized nutrient and water conducting tissues, which affects their dispersion throughout ecosystems.^[4] These microscopic organisms mainly inhabit freshwater environments because of their inability to survive with the cleaning agents present in domestic water sources.^[1]

Benefits of Diatoms

Diatoms are identifiable based on each species unique silica cell walls and vary depending on their environment. Because they have determinant properties diatoms create flora profiles for scientists. When these microscopic algae die their frustules (silica cell walls) becomes a part of the water sediment. The presence of the frustules enables scientists to compare the cell walls of the deceased organisms with the living diatoms to determine characteristics of their environment. In early spring and the fall the ratio of living diatoms to dead diatoms is high, whereas, in summer and winter the amount of dead diatoms out populates the living.^[1] Based on this known information diatoms can verify the time of year samples were taken. Different types of diatoms can also determine the properties of a samples ecosystem. For example, the higher ratio of periphytic (type of platonic diatom), the higher the vegetation concentration and shallower the water, but the higher the ratio of scaled chrysophytes a sample means the environment of that organism contains more open water.^[4] Forensic scientists can create differentiated markers on environments in the area they are collecting data based on the diatoms population growth rate, which is affected by pollutants in their water sources because the simple tissues of diatoms make them sensitive environmental changes.^[5][6] The reason diatoms are a common tool to match water environments is because of the variability of their populations are predictable and constant, the organisms can be identified by using the light microscope, and their silica cell walls allow for preservation.^[2]

Disadvantages of Diatoms

Out of the 771 cases in 2001, only 28% were fresh water drowning. Living diatoms do not inhabit domestic water sources, which limits the situations that diatoms can be used to create flora profiles or time of death estimations.^[1] Diatoms can only tell when or where evidence was found in some situations and not the time of death if there is no body fluid sample available to be collected. If a body is placed in freshwater post mortem then diatoms cannot be used to evaluate time of death. Without the inhalation of water and some circulation present in the victim, the diatoms will not be able to enter the alveolar system and blood stream making it difficult to extract a reliable sample. Another issue with the use of diatoms in order to provide evidential support is that diatoms can also be found on clothes, in food and drink, or air. In a study conducted by Spitz and Schneider in 1964, 500 cubic meters of air was filtered for three days in April and there was between 662 and 1564 individual diatoms present on the filtrate.^[7] Because the body can preserve these microscopic algae, the presence of diatoms may not only be on a victim or suspect through their relation to a crime scene, which affects the reliability of the results collected from a scene. Diatoms can also be destroyed based on the biological make up of the body it encounters, this could affect the results in a criminal investigation.^[3]

Diatom testing

In order to use diatom testing there are some guidelines that scientists must follow. To get a more accurate result there has to be at least 20 diatoms in a 100 microliter sample. When dealing with testing on a human body having 5 complete diatoms from more than 2 different organs will also give a positive diagnosis.^[2] Samples are taken from bone marrow, lung, spleen, liver, kidney, brain tissue, or from the area where the crime was discovered or occurred.^[5]

Body Test

When a body has been drowned diatoms go into the lungs and then are circulated to other internal organs through the individuals circulation.^[1] A forensic scientist will extract 100 g of either a tissue sample or bone marrow from a femur still attached to the victim. There are many different methods of extracting diatoms but the most commonly used method is acid digestion. This method is acknowledged worldwide, less expensive, and takes less time to get results.^[7] Scientists boil the sample and then add 50 ml of nitric acid solution. The color of the mixture will turn from yellow to transparent. After 48 hours when the sample has reached room temperature it is centrifuged, which allows the organic matter to completely oxidize, and the pellet is extracted, mixed with distilled water, and examined on a slide.^[1] The ratios of the different specimen of diatoms are counted, which allows scientists to match the area where the victim entered the water.^[4]

Testing a Sample

Materials taken from victims, suspects, or the crime scene can also be tested to match locations of where the samples derived. Environmental products, such as mud, are placed in a test tube with a sulfuric acid and potassium dichromate solution for 24 hours. After centrifuging the tube, it is then placed in a boiling water bath for an hour. After the pellet is extracted it is mixed with distilled water and mounted on a slide with Hyrax. When diatom testing on an organic sample scientists use phase contrast microscopy. While observing the diatoms are tallied and organized based on their different species. The ratio of specific specimen of diatoms in the water will have a similar ratio to the sample that is taken from the site where the diatoms were transferred. Scientists use this to match materials and people to specific locations at a crime scene. An example of when this test was used was in Connecticut, July 1991. Two boys were fishing along a river when they were bound, beaten with a baseball bat, and attempted drowning by other three teenage boys. The boys escaped to go report the attack. Forensic scientists took the clothes of the victims and the clothes of the suspects that the boys had identified. At the College of Connecticut the samples taken from the clothing and crime scene underwent diatom testing and then analyzed. The observation of the samples showed that the ratios of algae populations from each sample were about 80% similar.^[4] The transfer function approach also was applied to this case and three specific species of Eunotia were considered unique to the crime scene. All three species were found in both the victim and suspect’s clothes. The results were then used to get confessions from the suspects.^[3]

Time of death estimation

When using diatom testing, scientists observe the amount of diatoms present in the organism and determine a generalized time of death. If there are less than 20 different types of colonizing diatoms, then the organism's death was within 7 to 12 days, but, if there are more than 50 different colonies of diatoms then it is determined that the time of death occurred several weeks ago. Certain taxa of diatoms narrow the time frame to more exact dates. For example, Ankistrodesmus algae is a type of diatom that does not start to colonize on an organism till 30 days after its death. This taxon is also referred to as "late colonizers".^[2]

Extraction methods

When using the ‘’’acid digestion method’’’ scientists add sulfuric acid to the sample, let sit for 48 hours, centrifuge, mix with distilled water, and mount on slide for examination. Because this method has the least amount of steps, it gives scientists faster and less expensive results. The ‘’’Soluene-350 method’’’ is used only for freshwater samples. The sample is mixed with formalin, rinsed and centrifuged 3 times with distilled water. Then the pelletis removed from the test tube and added with 8ml of Soluene-350, incubated at 50 degrees Celsius for 2 hours, centrifuged again, and finally mounted on a slide.^[7] The ‘’’Enzymatic method’’’ is used for tissue samples and because of the use of enzyme chemicals it is considered an environmentally safe method and has a smaller percentage of damaging the diatoms collected than that of the acid digestion method.^[2] Concentrated nitric acid and Proteinase-K, or peroxide, are added to the sample for 12 hours, centrifuged twice, rinsed with distilled water, and then mounted on a slide with Naphrax. Using this form of extraction, observers can use a light microscope when examining the results. The ‘’’Membrane Filter method’’’ is used when there is a presence of either damaged diatoms in the sample (result of pollutants) or inorganic materials. The blood of the victim is taken and filtered through Sodium Dodecyl Sulfate and nitrocellulose membranes with decreasing pore sizes. The membranes are then dried and filtered with distilled water. After being dried the pieces of membrane that filtered the blood are examined for diatoms and pollen grains. ‘’’Colloidal Silica Gradient Centrifugation method’’’ can be used with liver and lung samples. The tissue collected is added to saline solution, centrifuged at a temperature of 12 degrees Celsius, and then added to distilled water to then be mounted on a slide and observed. ‘’’Dry Ash method’’’ can only be applied to a bone marrow sample. Five grams of marrow is taken from a victim to be placed in a nitric acid solution and then bunt in a furnace allowing the release of diatoms to then be analyzed.^[7]

Transfer function approach

In order to determine the reliability of a conclusion, scientists use the transfer function approach. The sample taken from a crime scene is matched with a location at a crime scene based on the ratio of diatom species present. Because diatoms can also be in the air and transferred to people through contact, breathing, eating, and drinking forensic scientists want to establish a base line or percent of error. Samples are taken off of clothing and objects of suspects and around the area of the crime scene and then tested to see the ratio of diatom colonies present before the alleged incident occurred. Specific species of diatoms will be only common to the sample taken from the crime scene. If the person or object was at the crime scene then the presence of that specific taxa should be on the sample taken from the suspects or material. After calculating the ratio scientists then cross-examine the similarities. If there are diatom colonies present at both the scene and in the sample that are different from base line samples then it concludes the information viable.^[3]

Diatom database

In 2006, scientists began creating a diatom database. It is the 'fingerprint system' for diatoms. The computer recognizes different diatom species based on shape and color characteristics. The tallying of colonies present in a sample, as well as the preferences of flora for diatom colonies are in the system so that scientists can access the information. But, as of 2012 the database is not complete and not used in courts.^[5]

Legal application

As of 2012, a small percentage of forensic limnology is used as evidence in courts. The presence of diatoms in air, food, drink, and close contact is not variable enough to be supportive evidence in determining locations of events. Even so, an investigation requires the use of forensic limnology in order to estimate time of death, location of drowning, and the determining of suspect. If the results of forensic limnology are not used in prosecution, the results are used to understand the crime.^[5]

References

1. Coyle, Heather Miller; Carll Ladd; Timothy Palmbach; Henry C. Lee (2001). "The Green Revolution: Botanical Contributions to Forensics and Drug Enforcement". Croatian Medical Journal (2001): 340–345.
2. Dommelen, Jennifer Van (17 June 2005). "Limnology". Forensic Botany. Dalhousie University. Retrieved 29 September 2012.
3. Horton, Benjamin P. (27 October 2007). "Diatoms and Forensic Science". Paleontological Society Papers: 14–22.
4. Siver, P. A.; Lord W. D.; McCarthy, D. J. (May 1994). "Forensic Limnology: The Use of Freshwater Algal Community Ecology to Link Suspects to an Aquatic Crime Scene in Southern New England". Journal of Forensic Sciences. 39 (3): 847–853.
5. Rohn, Edward J.; Peter D. Frade (22 December 2006). "The role of Diatoms in medico-legal investigations II: a case for the development and testing of new modalities applicable to the diatom test for drowning". Forensic Examiner.
6. Bijaya, Kumar Padhi; Jnanendra Rath; Pratap Kumar Padhy (2010). "Diatoms for assessing the ecological condition of inland freshwater bodies". World Review of Science. 7 (4): 352. doi:10.1504/WRSTSD.2010.032743.
7. Singh, Rajvinder (2006). "M.K. Thakar". EXTRACTION METHODS OF DIATOMS. Department of Forensic science, Punjabi University.