Forensic studies of phytoplankton ecology of two water bodies of Kurukshetra area of Haryana, State in India

Forensic studies of phytoplankton ecology of two water bodies of Kurukshetra area of Haryana,... Background: In forensic investigations of death cases in which the body has been recovered from a water body, there is an urgent need to prove whether the death is due to drowning or not. Moreover, another important issue arises, whether a person was alive at the time of drowning (anti-mortem drowning) or he was thrown dead into the water body (post-mortem drowning) To answer these questions, phytoplankton species of forensic importance from two water bodies (Braham Sarovar and Prithus Pools) were analyzed to develop the Phytoplankton maps (P- maps). The P-maps are basically a detailed list of categories of species which are common, site-specific, rare and seasonally occurring phytoplanktons along with their photomicrographs. Methods: In the present study, water samples have been collected from Braham Sarovar and Prithus Pools situated at Kurukshetra area of Haryana state, India. The phytoplankton species present in both ponds were extracted by mild acid digestion (5% HCl). The physico-chemical properties of these water bodies such as pH, water temperature, total dissolved solids, electrical conductivity, hardness, alkalinity, calcium, sodium, potassium, and chloride ions have been recorded to establish a correlation with the distribution of phytoplankton using principal component analysis. Results: Total 138 species of 59 genera of phytoplanktons belonging to Cyanophyceae, Chlorophyceae, Bacillariophyceae, Euglenophyceae and Dinophyceae have been observed. In Braham Sarovar, Cylindrospermum sp., Epithemia turgida, Eunotia rhomboidea, and Westella botryoides were the site-specific phytoplanktons whereas in Prithus pool, the site specific phytoplanktons were Leptolynbya granulifera, Arthospira jenerii, Cymbella cymbiformis, Frustulia vulgaris. Conclusion: It is hoped that results/data obtained from the present study (P-maps) may go a long way in solving the mysteries related to the deaths due to drowning and can be usefully used as a reference data for determining the exact site of drowning. Keywords: Phytoplankton, Forensic science, Diatoms, Aquatic bodies, Braham Sarovar, Prithus pool and P-maps, Principal component analysis, Diversity index, Drowning Background The place where actual drowning took place? The recovery of a dead body from any water body does To answer these questions, a diatom test has been not always mean that the death is due to drowning. considered to be the best marker of drowning but it con- There can be other reasons also. In forensic cases, it is fronted with many difficulties and hardships in terms of urgently required to investigate the real cause of death, its reliability. It was shown by some of the workers that whether it is due to drowning or not. In addition, few diatoms were also present in non-drowned victims due important questions arising in relation to it are: to postmortem invasion, autopsy contamination (Lunetta Whether the drowning is anti-mortem or post-mortem? et al. 2013), inhalation of diatoms from the air (Otto 1961; Spitz et al. 1965), water and foodstuff (Yen and Jayaprakash 2007). However, the reliability of diatom test * Correspondence: mukeshk38@gmail.com has been established by the majority of researchers by Department of Forensic Science, Punjabi University, Patiala, India Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 2 of 15 considering qualitative and quantitative criteria (Auer Phytoplanktons are the photoautotrophic organisms and Möttönen 1988; Ludes et al. 1999; Pollanen 1997; which prevail in all types of water bodies such as Singh et al. 2005, 2006a, 2006b; Thakar et al. 2011; Tha- streams, lakes, rivers, and ponds and are an important kar and Singh 2009; Thakar and Singh 2010). In 1988, component of the food chain in the aquatic environ- Auer and Mottonen suggested that minimum 20 num- ment. Phytoplankton groups may be distinguished from ber of diatom per microscope slide from lung samples is each other based on a combination of characteristics, in- required to define the death due to drowning. Similarly cluding photosynthetic pigments, shape, size and cell Ludes et al. (1999) also suggested the limit of 20 diatoms covering. They are microscopic in nature and “invisible” per slide per 100 μL pellet for lungs and 5 diatoms per to perpetrators of crimes, thus it helps in linking a sus- slide per 100 μL of a pellet sediment extracted from 2 g pect to the crime scene (Siver et al. 1994). of tissue samples such as brain, kidney, liver and bone Keeping in mind the needs of environmental and fo- marrow. But under certain environmental conditions, di- rensic experts, the present study has been undertaken to atoms may be scanty or absent in a particular water study the phytoplankton diversity along with seasonal body, leading to a false negative diagnosis of drowning. variations in two forensically important ponds (Braham Many studies on qualitative and quantitative assessment Sarovar and Prithus Pools) of Kurukshetra region of Ha- of diatoms during different seasons and at different time ryana, India. These two ponds are forensically important periods have reported lesser number or absent of diatom because of two reasons: Firstly, sufficiently large amount species in one season and their bloom in the other of water remains in these unguarded ponds throughout (Canini et al. 2013; Cumming and Smol 1993; Singh et the year, and secondly, a large crowd gathers around al. 2010; Sushanth and Rajashekhar 2012). Pollanen them to take a dip during the year on many occasions. (1997) also reported highly positive diatom test outcome The data generated in the present study has been used in the body of drowned victims in the months in which to generate Phytoplankton-maps (P-maps). The charac- diatom population in the water body was at a peak. This teristics Phytoplankton-maps of these two water bodies showed variations in the number of diatoms over a will hopefully help in unfolding the mysteries of deaths period of time in water bodies. due to drowning and the data generated can be utilized The forensic usefulness of phytoplankton came to light as a reference data for determining the exact site of when Chardez and Lambert (1985) (cited in Lunette and drowning. Modell 2005) detected the presence of phytoplankton other than diatoms in the drowned bodies. They also Material and methods found aquatic organisms belonging to division Chloro- Selection of sampling sites phyta, Dinoflagellates, invertebrates, protozoan ciliates In the present study the following two sites which are in the blood of drowned victims. Later, Yoshimura et al. situated in Kurukshetra city and Pehowa city of Haryana, (1995) developed a method to recover phytoplankton India have been selected to collect samples for the ana- from the tissue of drowned victims by using lysis of phytoplanktons: soluene-350. Various species of green algae (Staurastrum and members of Zygnemataceae) as well as diatoms Brahma Sarovar (Navicula, Cymbella and Melosira) were detected in It is the largest man-made water body situated in histor- both putative water samples and tissue samples of ically important Kurukshetra city, Haryana state, India drowned victims. In an another case a serious attempt (29°57′37″ N 76°50′42.2″ E). The water body is rect- has been made to link the suspects and victims to the angular in shape, measuring 3600 ft long, 1500 ft wide crime scene by analyzing the sediment encrusted and 15 ft deep. It has religious significance, every year sneakers from both assailants and victims along with nu- on ‘Somavati Amavasya’(a special day in Hindu religion) merous species of diatoms and scaled Chrysophytes and on the occasion of solar eclipses; millions of people (planktonic algae) from the sneakers as well as reference from different parts of India visit this water body to take samples of pond sediment (Siver et al. 1994). a holy bath (Fig. 1). Further in Díaz-Palma et al. 2009 suggested to extend the diatom test to detect drowning by taking into con- Prithus pool sideration other groups of phytoplanktons. They have It is located in another religiously important city, Pehowa standardized the methods for recovery of microalgae city, Haryana, India (29°58′51.6″N76°34′46.4″E) about from drowned victims by using different extraction pro- 25 Kms from Braham Sarovar. It was constructed by king cedures and concluded that dinoflagellates and some Prithu after the death of his father. This place is known chlorophytes have cell walls and other resistant struc- for funeral ceremonies (pind daan in Hindu religion), tures similar to diatoms. As a result, these organisms therefore, thousands of pilgrims from different places visit can be found in tissues of drowned victims. this pool throughout the year (Fig. 2). Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 3 of 15 behind only a residual material at the bottom of the tube. An excess of water was again mixed with residual material and centrifuged again as above. This process was repeated twice. After discarding the supernatant, the residual material (pellet) containing phytoplankton was poured over two sets of five serially marked (I-V) microscopic slides and smear was prepared. Then, these slides of two sets were permanently mounted using a high refractive index mounting material DPX to study diatoms and Glycerin jelly for phytoplankton analysis under the low and high power of the microscope. Fig. 1 Photograph of Braham Sarovar Analysis of slides prepared All the slides prepared for both the water bodies season Collection of samples wise have been examined with the help of an optical For phytoplankton analysis, the water samples have been compound microscope (Nikon Eclipse microscope) fitted collected during four seasons such as summer (May– with light source under low (10× X 10×), high power June), autumn (August–September), winter (November– (10× X 40×) and oil immersion (10× X 10×) magnifica- December) and spring (February–March) from two se- tions. Nikon E200 series digital camera which has been lected sites from the year 2014 to 2016. The water sam- attached to the microscope was used to prepare Photo- ples were collected in a sterilized plastic bottle of 1 l micrographs for each and every phytoplankton species. capacity and preserved in 4% formaldehyde solution. These Photomicrographs were used for taxonomic iden- tifications of phytoplankton with the help of publications given by Prescott (1962); Komárek et al. (1983); Desi- The procedure adopted to prepare slides for the analysis kachary (1959); Philipose (1967) and Wehr et al. (2015). of phytoplankton Data so obtained have been used to construct a phytoplankton map (p-map) for the both water bodies. Approximately, 50 ml of water sample was taken in Enumeration of phytoplankton was done by using a beaker and digested with 10 ml of 5% HCl for Haemocytometer. The colonial and filamentous microal- 24 h. gae such as Microcystis sp., Merismopedia sp. and Oscil- Next day, after thorough mixing of the water sample latoria sp. have been counted by using plating method and HCl, 10 ml of water sample was taken in a (Stanier et al. 1971). Average of two years (2014–2016) centrifuge tube and centrifuged at 3000 rpm for data has been given in Tables 1, 2 and 3. 10 min. The supernatant was collected out, leaving Physico-chemical properties In the both selected water bodies, the parameters such as total alkalinity, chloride and calcium levels, and total hardness have been determined according to APHA (American Public Health Association) manual (APHA 2005). Sodium and potassium contents in the water samples have also been determined with the help of flame photometer. Electrical conductivity, pH, and temperature have also been determined by using the pH meter cum conductivity meter (Aquapro digital water tester model AP-2). TDS has been determined by using TDS meter (Aquapro digital water tester model AP-1). The correlation between physico-chemical parameters and phytoplankton species was carried out by Principal Component Analysis (PCA) using the Microsoft Excel Fig. 2 Photograph of Prithus Pool Add-Ins software XLSTAT. Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 4 of 15 Table 1 List of phytoplankton species observed in Braham Sarovar and Prithus Pools Family Braham Sarovar Prithus pool Chlorophyceae Ankistrodesmus spiralis, Ankistrodesmus fusiformis, Ankistrodesmus Ankistrodesmus acicularis, Chlorocuccum scabellum, Coelastrum flactum, Chlorococcum sp., Coelastrum microporum, Crucigenia cambricum, Coelastrum microporum, Coelastrum astroideum, lauterbornii, Crucigenia tetrapedia, Dicloster acuatus, Krichneriella Coelastrum morus, Coelastrum pulchrum, Coelastrum sphaericum, sp., Monoraphidium arcuatum, Monoraphidium contortum, Crucigeniella neglecta, Crucigenia lauterbornii, Crucigenia crucifera, Monoraphidium griffithi, Oocystis borgei, Oocytis marssonii, Crucigeniella quadrata, Crucigenia tetrapedia, Dictyochloropsis Scenedesmus quadricauda, Scenedesmus dimorphus, Scenedesmus reticulata, Kirchneriella irregularis, Monoraphidium contortum, ecornis, Scenedesmus bijugatus, Scenedesmus armatus Monoraphidium fontinale, Monoraphidium arcuatum, var.boglariensis, Scenedesmus abundans, Scenedesmus armatus Monoraphidium griffithii, Oocystis sp., Oocystis solitaria, Pediastrum var.bicaudatus, Scenedesmus obliquus, Schroederia indica, duplex, Pediatrum simplex, Scenedesmus bijugatus, Scenedesmus Staurastrum iotanum, Staurastrum sp. Lemmermannia punctata, dimorphus, Scenedesmus abundans, Scenedesmus verrucosus, Tetraedrone lobulatum, Tetraedrone trigonium, Tetrachlorella Scenedesmus accuminatus, Scenedesmus caudato-aculeolatus, alternans, Tetrastrum minimum, Trebouxia sp., Westella botryoides. Scenedesmus obliquus var. dimorphus, Scenedesmus quadricauda, Schroederia setigera, Selenastrum sp., Tetracystis sp., Tetraedrone minimum, Tetraedrone puchtatum, Tetraedron trigonium. Cyanophyceae Aphanocapsa sp., Calothrix sp., Chroococcus cohaerens, Planktosphaeria gelatinosa, Aphanocapsa annulata, Aphanocapsa Chroococcus dispersus, Cylindrospermum sp., Gloeocapsa delicatissima, Planktothrix maougetti, Merismopedia tenuissima, minimum, Merismopedia glauca, Merismopedia minima, Scytonema hofmanni, Spirulina sp., Synechococcus sp., Oscillatoria Microcystis panniformis, Microcystis aerognosa, Oscillatoria agardhii, Oscillatoria rufringe, Oscillatoria limosa, Arthrospira agardhii, Geitlerinema pseuriaccutissimum, pseudoanabena jenneri, Leptolyngbya granuliferat, Geitlerinema amphibium sp.,Synechococcus sp., Synechocystis aqualis Bacillariophyceae Aulacoseira granulata, Cymbella tumida, Epithemia turgida, Frustulia vulgaris, Cymbella cistula, Melosira varians, Nitzschia Eunotia rhomboidea, Navicula capitoradiata, Nitzchia palecea, palea, Nitzschia acicularis, Cymbella cymbiformis, Navicula Nitzchia accicularis, Cymbella laevis, Stephanocyclus gregaria meneghiniana, Pinnularia abaujensis, Navicula cryptocephala, Gomphonema truncatum Euglenophyceae Euglena sp., Lepcinclis fusiformis, Phacus longicauda, Phacus Lepocinclis ovum, Phacus onxy, Phacus pyrum, Euglena acus, tortus,Trachelomonas horrida. Trachelomonas langerdiana, Euglena caudata,Euglena virdis, Lepocinclis elongata, Lepocinclis sternii, Euglena splendens. Dinophyceae Alexanderium catenella, Alexanderium insuetum, Gonyaulax lingulodinium, Gyrodinium fusus, Peridinium cinctum, Peridinium umbonatum. Diversity indices: In the present study, the diversity in- pi = ni/N, ni being the number of individual of ith spe- dices of phytoplankton species have been calculated in cies and. terms of N = total number of individuals in the sample. Species Richness: Species richness is a count of a num- Species diversity method given by Shannon (2001), ber of species in a water body. It is calculated using the Species richness by Menhinick (1964) and following formula: Evenness by Pielou (1975) as follows: D ¼ n=√N Shannon-Wiener diversity index: Shannon diversity index is a quantitative measure of species diversity in a Where D = Species Richness, n = total number of spe- community and can be calculated as follows: cies in a sample. N = Total number of an individual organism in a H ¼ − ðÞ pi log2pi sample. Species Evenness: Species evenness is the measures of Where, H = Shannon and Weaver diversity index. how equals the relative abundance of different species 3 −1 Table 2 Phytoplankton density (individuals× 10 mL ) in Braham Sarovar during different seasons Class Summer Autumn Winter Spring Chlorophyceae 1.180 ± 0.235 0.716 ± 0.206 0.357 × 0.225 0.653 ± 0.234. Cyanophyceae 0.372 ± 0.090 0.322 ± .092 0.714 ± 0.091 0.344 ± 0.293 Bacillariophyceae 0.188 ± 0.10 0.077 ± 0.03 0.256 ± 0.068. 0.190 ± 0.08 Euglenophyceae 0 0.05 ± 0.032 0.024 ± 0.02 0 Dinophyceae 0 0 0.012 ± 0.01 0.098 ± 0.05 Total 1.740 ± 0.52.6 1.162 ± 0.3 1.363 ± 0.28 1.285 ± 0.24 Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 5 of 15 3 −1 Table 3 Phytoplankton density (individuals ×10 mL ) in Prithus Pool during different seasons Class Summer Autumn Winter Spring Chlorophyceae 1.387 ± 0.27 1.078 ± 0.22 0.909 ± 0.11 1.372 ± 0.37 Cyanophyceae 0.346 ± 0.14 0.243 ± 0.006 0.178 ± 0.08 0.231 × ±0.06 Bacillariophyceae 0.192 ± 0.024 0.051 ± 0.034 0.205 ± 0.03 0.128 ± 0.058 Euglenophyceae 0.063 ± 0.057 0.026 ± 0.019 0.101 ± 0.07 0.138 ± 0.04 Total 1.988 ± 0.525 1.398 ± 0.42 1.393 ± 0.32 1.869 ± 0.52 are in a community. It is calculated using formula as These season specific species of a particular water follows: body may help in determining the season of occurrence of crime. J ¼ H= log2ðÞ S ❖ Rarely occurring species: These are those species which are found occasionally or by chance in a Where, J = Evenness H = Shannon and Weaver diver- particular water body. These species are not sity index. important from the forensic point of view. S = Total number of species in a sample. In part-B of P-map for both the water bodies, the pho- tomicrographs of Site-specific species have been shown Phytoplankton-maps (P-maps) (Tables 4 and 5), whereas part – C shows Distribution The data generated in the present study were used to of Phytoplankton species during the different season in prepare the P-maps (Tables 4 and 5) of two selected both water bodies (Tables 4 and 5). water bodies (Braham Sarovar and Prithus Pools). The P-maps depicts the detailed list of phytoplankton genera Results and discussion and species present in the two selected water bodies. Phytoplanktons are the diverse group of photosynthetic P-maps have been divided into three parts- A, B, and organisms present in all the aquatic habitats such as C as shown in Tables 4 and 5. In part – A, phytoplank- lakes, rivers, ponds, canals, sea etc. Moreover, they have tons species have been shown, which have been divided different shapes, sizes, colors and forms, which provide into the following four categories: them individuality. So their presence in water bodies and potential to identify the site of drowning may help in the ▪ Commonly found phytoplanktons forensic investigation. Because of their microscopic na- ▪ Site-specific phytoplanktons, ture and invisibility phytoplankton may adhere to the ▪ Seasonally occurring species of phytoplanktons suspect, victim or their clothes or weapon of offense. In ▪ Rarely occurring phytoplankton addition, different species of phytoplankton present dur- ing different seasons which aids in linking the suspect to The detailed information about these categories has a particular crime scene during the specific season (Hall been given below and is helpful in generating P-map for 1997). Therefore, seasonal variation of phytoplankton a specific water body: has been studied in two selected water bodies of Kuruk- shetra region, continuously for two years to generate a ❖ Commonly found species: Commonly found species baseline database of phytoplankton (Phytoplankton are those which are common in more than one maps) for each water body. water body. They have significance in detecting the In the present study, remarkable changes in diversity death due to drowning and are of little significance and abundance of phytoplankton have been observed in diagnosing drowning site. during different seasons. In Braham Sarovar and Prithus ❖ Site-specific species: Site-specific species are those Pools, a total of 138 species belonging to 59 phytoplank- which are restricted to a particular water body, ton members have been found (Tables 1 and 6). Indi- hence they have great forensic significance in locat- vidually in Braham Sarovar alone 47 genera belonging to ing the exact site of drowning, particularly when a 5 classes i.e. Cyanophyceae (12), Chlorophyceae (18), body is found away from the actual site of drowning Bacillariophyceae (9), Euglenophyceae (4) and Dinophy- and also they help in linking the suspect and victim ceae (4) representing 70 species have been found. Simi- to the crime scene. larly, in Prithus pool, 36 genera belonging to 4 classes’ ❖ Seasonally occurring species: This category includes i.e. Chlorophyceae (15), Cyanophyceae (12), Euglenophy- those species which are dominant in one season and ceae (4), and Bacillariophyceae (5) representing 68 spe- may become extinct or dominated by other species cies found. Similar studies have been conducted by Kim during another season in a particular water body. 2011. He observed 16 species of diatoms, 20 species of Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 6 of 15 Table 4 Phytoplankton -map (P-maps) of Braham Sarovar Commonly occurring Site- specific Seasonally occuring Rarely occurring Part A: List of phytoplanktons genera and species observed in Braham Sarovar Coelastrum microporum,Crucigenia Epithemia turgida, Summer Gloeocapsa sp., Ankistrodesmus fusiformis, Chlorococcum sp., Crucigenia lauterbornii, Crucigenia Eunotia rhomboidea, Monoraphidium arcuatum, Scenedesmus lauterborni, Dicloster acuatus Gloeocystis tetrapedia,Monoraphidium arcuatum, Westella botryoides, obliquus, Tetraedron minimum, sp.,Monoraphidium contortum,Oocystis Monoraphidium contortum, Cylindrospermum sp. Staurastrum iotanum, Choroococcus borgei, Schroederia indica, Staurastrum Monoraphidium griffithi, Nitzschia coherens, Chroococcus dispersus, Nitzschia sp., Tetrachlorella alternans, Tetraedron acicularis,Oscillatoria agardhii, palecea, Merismopedia minima, Gleocapsa lobulatum, Trebauxia sp., Geitlerinema Scenedesmus abundans, Scenedesmus minimum, Pinnularia abaujensis. pseuriaaccutissimum, Microcystis dimorphus, Scenedesmus panniformis, Navicula capitoradiata, Autumn Ankistrodesmus fusiformis, Crucigenia obliquus,Tetraedron minimum, Lepocinclis fusiformis, Lemmermannia Tetrapedia, Monoraphidium griffthii, Tetraedron trigonium punctata,Phacus longicauda, Phacus Lemmermannia punctata. tortus, Ttrachelomonas horrida, Alexanderium acatenella, Alexanderium Winter Merismopedia glauca, Microcsystis insuetum, Gonyaulax lingulodinium, aerognosa, Synechococcus sp., Gyrodinium fusus, Peridinium cinctum, Kritchneriella irregularis, Gomphonema Peridinium umbonatum. truncata, Navicual cryptocephala, Epithemia turgida, Eunotia rhomboidea, Cylindrospermumsp Spring Aphanocapsa sp., Oosystis marsonnii, Scenedesmus bijugatus, Scenedesmus armatus, Pseudoanabaena constrictum Part B: Photomicrographs of some of site-specific species. Part C: Distribution of Phytoplankton during different seasons Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 7 of 15 Table 5 Phytoplankton -map (P-map) for Prithus Pool Commonly occurring Site- specific Seasonally occuring Rarely occurring Part A: List of phytoplanktons genera and species observed in Prithus Pool Coelastrum microporum, Crucigenia Leptolynbya Summer Coelastrum microporum, Crucigenia Ankistrodesmus acicularis, lauterbornii, Crucigenia granulifer, Arthospira quadrata, Tetraedrone minimum, Ankistrodesmus flactum, Coelastrum tetrapedia,Monoraphidium arcuatum, jenerii,Cymbella Chlorococcum sp., Coelastridium astroideum, Coelastrum morus, Monoraphidium contortum, cymbiformis asterodeum, Crucigeniella quadrata, Coelastrum pulchrum, Coelastrum Monoraphidium griffithi, Nitzschia Frustulia vulgaris. Selenastrum sp., Aphanocapsa morus, Crucigeniella neglecta, acicularis,Oscillatoria agardhii, delicattissima, Geitlerinema amphibium, Pediastrum duplex, Pediatrum simplex, Scenedesmus abundans, Syctonema hofmanii, Melosira varians, Schroederia setigera,Geitlerinema Scenedesmus Trachelomonas langerdiana, amphibium,Oscillatoria limosa, dimorphus, Scenedesmus Scenedesmus quadricauda Navicula gregaria, Euglena caudata, obliquus,Tetraedron Euglena splendens, Euglena virdis, Autumn Oocystis solitare, Scenedesmus minimum,Tetraedron trigonium. Lepocinclis elongata, Lepocinclis sternii, versucois, Scenedesmus acumnatus, Phacus onxy. Oscillatoria rufringe, Planktothrix mougetti, Lepocinclis ovum. Winter Tetracystis sp., Kirchneriella sp., Coelastrum asterodeum, Tetracystis sp., Cymbella cistulla, Phacus pyrum Spring Chlorococcum scabellum, Coelastrum cambricum, Dictyochoropsis reticulata, Monoraphidium frontinale, Crucigenia crucifera, Scenedesmus bijugatus, Scenedesmus caudato-aculeolatus, Tet raedrone punchtatum, Oscillatoria agardhii Part B: Photomicrographs of some of site-specific species Part C: Distribution of Phytoplankton during different seasons Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 8 of 15 Table 6 List of genera observed in Braham Sarovar and Prithus Table 6 List of genera observed in Braham Sarovar and Prithus Pools Pools (Continued) S. No Genus Braham Sarovar Prithus Pool S. No Genus Braham Sarovar Prithus Pool Genus related to class Chlorophyceae Genus related to class Euglenophyceae 1. Ankistrodesmus ++ 41. Euglena ++ 2. Chlorococcum ++ 42. Lepocinclis ++ 3. Coelastrum ++ 43. Phacus ++ 4. Crucigeniella ++ 44. Trachelomonas ++ 5. Crucigenia ++ Genus related to class Bacillariophyceae 6. Dicloster +_ 45. Aulacoseira +_ 7. Dictyochloropsis _+ 46. Cymbella ++ 8. Gloeocystis +_ 47. Epithemia +_ 9. Kirchneriella ++ 48 Eunotia +_ 10. Monoraphidium ++ 49. Frustulia _+ 11. Lemmermannia +_ 50. Gomphonema +_ 12. Oocystis ++ 51. Melosira _+ 13. Pediastrum _+ 52. Navicula ++ 14. Scenedesmus ++ 53. Nitzschia ++ 15. Schroederia ++ 54. Pinnularia +_ 16. Selenastrum sp _+ 55. Stephanocyclus +_ 17. Staurastrum +_ Genus related to class Dinophyceae 18. Tetrachlorella +_ 56. Alexanderium +_ 19. Tetracystis _+ 57. Gonyaulax + 20. Tetraedrone ++ 58. Gyrodinium + _ 21. Trebouxia +_ 59. Peridinium +_ 22. Westella +_ 23. Planktosphaeria ++ green algae, 6 species of Cyanobacteria and 6 species of other algae in three aquatic locations of Gwang-ju area Genus related to class Cyanophyceae (South Korea) where drowned victims were found. 24. Aphanocapsa The detailed list of species recovered in the present study 25. Arthrospira _+ has been given in Table 1.Figures 3 and 4 show photomi- 26. Calothrix +_ crographs of different species of phytoplankton observed in 27. Chroococcus +_ Braham Sarovar and Prithus pools water bodies. 28. Cylindrospermum +_ Phytoplankton community 29. Geitlerinema ++ Class - Chlorophyceae 30. Gloeocapsa +_ The members of Chlorophyceae formed the most dom- 31. Leptolyngbya _+ inant group among others in both water bodies which 32. Merismopedia ++ accounted for 46% of total phytoplankton diversity with 33. Microcystis ++ 32 species in the Braham Sarovar (Table 4c) and 54.4% 34. Oscillatoria ++ of total diversity was represented by 37 species of Chlor- ophyceae in the Prithus Pool (Table 5c). Panigrahi and 35. Planktothrix _+ Patra (2013) also reported dominance of Chlorophyceae 36. Pseudanabaena +_ over other phytoplankton groups in terms of numerical 37. Scytonema _+ value and percentage composition in a water body of 38. Spirulina _+ Cuttack city, West Bengal, India. The dominant species 39. Synechococcus ++ of Braham Sarovar were Ankistrodesmus fusiformis, 40. Synechocystis +_ Ankistrodesmus flactum, Crucigenia tetrapedia, Oocystis marsonii, Scenedesmus bijugatus, Scenedesmus abun- dance, Tetrastrum minimum, Coelastrum Microporum. Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 9 of 15 Fig. 3 Photomicrograph of phytoplankton observed during the present study. a. Crucigeniella quadrata, b. Scenedesmus opaliensis, c. Pediastrum duplex, d. Crucigeniella crucifera, e. Tetraedron lobulatum, f. Scenedesmus dimorphus g. Staurastrum iotanum, h. Crucigenia fenestrata, i. Tetraedron minimum, j. Coelastrum microporum, k. Coelastrum cambricum, l. Scenedesmus bijugatus. Scale bar, 10μm Fig. 4 Photomicrograph of phytoplankton observed during the present study a. Phacus pyrum, b. Phacus longicauda, c. Lepocinclis fusiformis, d. Euglena splendens, e. Synechococcus sp., f. Stephanocyclus meneghiniana g. Merismopedia minima, h. Synechocystis aquatilis, i. Nitzschia acicularis, j. Oscillatoria limosa, k. Microcystis aeruginosa.Scale bar,10μm Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 10 of 15 In Prithus pool the most dominant species were Chloro- Seasonal variation in phytoplankton population density coccum scabellum, Coelastrum microporum, Coelastrum Mean density of 1.662 × 10 ± 227 phytoplankton indi- − 1 asteriodeum, Crucigenia crucifera, Crucigenia tetrapedia, viduals mL of water was observed at Prithus pool Crucigeniella quadrata, Crucigeniella reticulata, Dictyo- while in Braham Sarovar mean density has been calcu- 3 − 1 chloropsis reticulata Kritchnella sp., Scenedesmus dimor- lated to be 1.388 × 10 ± 248 individuals mL . In both phus, Scenedesmus obliquus, Scenedesmus quadricauda, water bodies, Chlorophyceae was found to be the most and Tetradron puchtatum. abundant group in summer which declined in autumn and winter. A similar observation has been made by Shinde et al. (2012) in Harsool-savang dam, Aurangabad; Class-Cyanophyceae where maximum density of Chlorophyceae was observed Cyanophyceae was the second most abundant group in in summer season. Nowrouzi and Valavi (2011) while the Braham Sarovar with 21.4% diversity of the total studying phytoplankton of Lake Kaftar in Iran also re- phytoplankton population. Among the 15 species, ported highest density of Chlorophyceae in summer and Aphanocapsa sp., Chroococcus dispersus, Merismopedia spring and the lowest in winter. Cyanophyceae in glauca, Merismopedia minima, Microcystis aeruginosa Prithus pool exhibited maximum growth in summer and and Synechococcus sp. were dominant ones. minimum in winter while, in Braham Sarovar, Cyano- Similarly, in Prithus pool, Cyanophycaeae also ranked phyceae exhibited maximum growth in winter and mini- as the second most abundant group accounting for 21% mum in the autumn. The maximum density of of the total population with 14 species (Tables 4c and Cyanophyceae in Braham Sarovar during winter was due 5c). The dominating species were found to be Merismo- to the overgrowth of Microcystis aeruginosa. Khuantrair- pedia tenuiasima, Spirulina sp., Oscillatoria agardhii ong and Traichaiyaporn (2008) also observed Cyano- and, Plantothrix mougeotti. phyta as the most abundant group in winter and summer in Doi Tao Lake, Thailand. On the contrary, Venkateswarlu (1969) reported the dominance of Cyano- Class- Bacillariophyceae phyceae in summer season in Mossi River, Hyderabad. The class Bacillariophyceae has been ranked third in Singh and Swarup (1980) also reported maximum Braham Sarovar and accounted for 17.1% but the same growth of cyanophyceae during summer in Lake Suraha, class ranked fourth in Prithus Pool and accounted for Uttar Pradesh. The maximum abundance of Bacillario- 10.2% of total phytoplankton diversity found in this phyceae was recorded in winter and minimum in au- water body (Tables 4c and 5c). Total 12 species of dia- tumn. Singh et al. (2010) observed highest density of toms have been found from the Braham Sarovar with Bacillariophyceae in winter in Mawatha lake Rajasthan. Aulacoseira granulata, Nitzschia acicularis, Nitzschia A similar trend has also been observed by Baba and Pan- palecea and Navicula cryptocephala being the dominant dit (2014) while studying the diversity of phytoplankton ones, whereas seven species of diatoms reported from in Wular Lake Kashmir. Members of Euglenophyceae Prithus pool Frustulia vulgaris, Melosira varians Frustu- were observed both in autumn and winter in Braham lia vulgaris and Nitzschia palea were dominant. Sarovar, while in Prithus pool, members of Euglenophy- ceae were maximum during winter and spring and mini- mum in summer. The members of Dinophyceae were Class-Euglenophyceae detected only in Braham Sarovar, that too during win- Euglenophyceae class constituted around 15% (10 spe- ter and spring season. Mean density distribution of cies) of the total phytoplankton population and formed different classes of phytoplankton in Braham Sarovar the third most dominant group in Prithus pool, whereas and Prithus Pools is Part-C of P-map and has been it ranked fifth in Braham Sarovar accounting for 6% (5 showninTables 4 and 5. species). Among them, Phacus longicauda, Phacus tortus Various environmental factors such as light, temperature and Lepocinclis fusiformis were the most abundant and rainfall and physico-chemical factors (pH, temperature, species in the Braham Sarovar, while Lepcinclis ovum, salinity, Nitrogen, Phosphate, Calcium, Sodium, Phosphate, Euglena acus and Euglena virdis were dominant in Chloride, etc) of a water body get changed with seasons Prithus pool (Tables 4c, 5c). which in turn bring about change in composition and abundance of phytoplankton (Figueredo and Giani 2009). Class-Dinophyceae In the present study, the maximum density of Chlorophy- Members of Dinophyceae have been only observed in ceae and Cynaophyceae has been recorded during the sum- the Braham Sarovar water body and constituted 10% (6 mer season and minimum during autumn and winter species) of species diversity with Peridinium cinctum be- season. The increase in the density of phytoplankton may ing the dominant one. be attributed to the increase in temperature during Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 11 of 15 summer, which not only increases the rate of photosyn- physico-chemical parameters was probably due to the thesis, but also increases the concentration of available nu- anthropogenic activity at this pond. The physic-chemical trients due to increase in water evaporation. Whereas a parameters of both aquatic bodies during the different decrease in the density during autumn and winter may be seasons are shown in Tables 8 and 9. due to the increase in the water level of the water body by rainwater which decreases the photosynthetic efficiency of phytoplanktons (Shinde et al. 2012). The density of Bacillar- Correlation between physico-chemical parameters and iophyceae was observed maximum during winter which phytoplankton community may be due to a higher rate of multiplication of Bacillario- Principal component analysis (PCA) phyceae at low temperature (Raibole and Singh 2011). Principal Component Analysis (PCA) is a multivariate data The members of Euglenophyceae were observed during analysis technique which reduces the multi-dimensional autumn and winter season only, may be because of two data into two principal components. In present study Prin- reasons i.e. either they cannot tolerate higher temperature cipal Component Analysis has been used to establish the or increased accumulation of organic matter in the water degree of correlation between physico-chemical parameters bodies due to heavy rainfall, favors the growth of Eugleno- (as mentioned above) and the phytoplankton species pre- phyceae (Tiwari and Chauhan 2006). vailing in the water body. It helps to understand whether phytoplankton has a positive or negative relationship with Diversity index different physico-chemical parameters and what are the key High values of Shannon’s index (H′) were recorded dur- factors responsible for their growth and inhibition? ing summer and lower during winter at both water bod- As in forensic investigations, it is common to find a ies. Shannon’s index has been calculated to be high dead body away from the actual site of drowning due to (3.16 ± 0.013) in Braham Sarovar as compared to Prithus water current, tides, etc. Thus, determination of the actual pool (3.03 ± 0.26) (Table 7). The value of Species rich- site of drowning becomes important for the medico-legal ness of Braham Sarovar was comparatively high during expert. The abundance and diversity of phytoplankton summer (4.55) and minimum during winters (3.87) and vary between different water bodies due to the difference autumn (3.96) while in Prithus pool, species richness in various parameters such as pH, temperature, salinity, was maximum during spring (3.86) and minimum dur- electrical conductivity, hardness, alkalinity etc. of that ing autumn (3.42). Species evenness in Prithus pool water body (Nowrouzi and Valavi 2011). Some of the showed little variation during different seasons, indicat- physico-chemical parameters are favorable for the growth ing that all the species were equally distributed through- of one species while these may not support growth of out the year. In Braham Sarovar, the highest value of others. The different species of phytoplankton get estab- species evenness was 0.93 during summer and lower lished in a particular water body based on their prefer- during winter (0.86). ences for particular physico-chemical components of water. Therefore, a positive test for drowning should not Physico-chemical properties only be based upon the presence or absence of phyto- Physico-chemical parameters of Braham Sarovar and plankton in the body of a drowned victim, rather it should Prithus pools have been assessed to determine the tropical also exhibit the same type of phytoplankton in similar status of the water body and their co-relation with the concentrations of the drowning medium (Sasidharan and phytoplankton species diversity. The physico-chemical Resmi 2014). A detailed study of phytoplanktons and their parameters of Prithus pool were numerically high as relationship with characteristics of the water body can play compared to Braham Sarovar. The high value of a significant role in connecting a drowned dead body to a Table 7 Seasonal variation in phytoplankton diversity indices in Braham Sarovar and Prithus pools Water Body Season Shannon diversity index Evenesss Species richness Braham sarovar Summer 3.32676 0.93 4.55 Autumn 3.31251 0.89 3.96 Winter 2.90562 0.86 3.87 Spring 3.11771 0.89 4.60 Prithus pool Summer 3.139102 0.93 3.68 Autumn 2.951855 0.90 3.42 Winter 2.9244777 0.90 3.44 Spring 3.1210465 0.90 3.86 Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 12 of 15 Table 8 Physico-chemical Parameters of Prithus Pool Prithus pool Season of pH EC TDS HARDNE ALKALINITY SODIUM POTASIUM CHLORIDE CALCIUM WATER TEMPERTU sampling (0 (μs/cm) (mg/l) SS (mg/L) (mg/L) ppm ppm (mg/L) (mg/L) RE c) Summer 7.7 254 500 340 180 20 3.4 122 120 35.2 Autumn 7.1 227 474 250 177 18 4.2 91 104 32.4 Winter 6.9 130 434 230 145 21.5 4.8 57 96 16.5 Spring 7.2 198 587 310 189 28.6 2.8 94 76 22.1 Average 7.2 ± 0.34 198 ± 60 498 ± 64.7 280.7 ± 55.3 172.7 ± 19.1 22 ± 4.6 3.8 ± 0.8 91.0 ± 26 99.0 ± 18.2 26.5 ± 8.7 specific water body source particularly when the body is positively correlated with calcium and Ankistrodesmus found outside or lying adjacent to the water body. fusiformis is positively correlated with chloride. Phacus In the present the study, PCA method has been longicauda, Phacus tortus, Lepocinclis fusiformis, and employed to 10 physico-chemical parameters for 20 Synechococcus sp. are positively co-related with pH, potas- dominant species of phytoplankton for Prithus pool. The sium, hardness and electrical conductivity whereas they all PCA axis 1 and 2 explained 42.81 and 36.70% variation are negatively correlated with TDS and calcium. Similarly, in phytoplankton and physico-chemical parameter. Cru- Synechococcus sp. and Microcystis aeruginosa are posi- cigeniella quadrata, Crucigenia tetrapedia, Scenedesmus tively co-related with pH and alkalinity (Fig. 6). dimorphus, Scenedesmus quadricauda, Planktothrix mougeotti, and Melosira varians are positively co-related Phytoplankton maps (P- maps) of Braham Sarovar and to pH, hardness, alkalinity, chloride, electrical conductiv- Prithus pools ity and temperature of water sample while Crucigenia Among the total 138 species, 13 species were common crucifera is negatively co-related with all these factors. in both the water bodies, therefore, these are categorized Dictyochloropsis reticulata, Tetrastrum punctatum, and as common species and are significant in diagnosing the Frustulia vulgaris are positively co-related to TDS (total drowning death, but not significant in determining the dissolved solids) and sodium while Nitzschia palea is exact site of drowning. Similarly, rarely occurring species negatively co-related to TDS. Lepocinclis ovum and in Braham Sarovar (25 species) and Prithus pools (20 Nitzschia palea are positively correlated to potassium species) are not forensically significant. The site-specific while Coelastrum microporum and Coelastrum astroi- species in Braham Sarovar (4 species) and Prithus pools deum are positively correlated to calcium (Fig. 5). (4 species) are forensically significant in determining the PCA for Braham Sarovar has been drawn by taking in to exact/putative site of drowning. The P-maps with a list account 10 physico-chemical parameters and 14 dominant of common and site-specific phytoplanktons have been species of phytoplankton. The PCA axis 1 explained shown in Tables 4 and 5. The P-map generated of phyto- 50.14% and axis 2 explained 29.03% variations in phyto- plankton species of two water bodies can help in deter- plankton and physico-chemical parameters. Aulacoseira mining not only cause of death, but also helps in linking granulata, Tetraedron minimum, Gloeocapsa minimum, the suspects to a crime scene. Nitzschia palecea, and Coelastrum microporum, is posi- tively correlated to temperature whereas Microcystis aeru- Conclusions ginosa and Synechococcus sp. are negatively correlated. In the present study, phytoplankton data have been generated Chroococcus dispersus and Aphanothceae sp. have been from the season-wise analysis of two water bodies: Braham Table 9 Physico-chemical Parameters of Braham Sarovar Braham Sarovar Lake Season of pH EC TDS HARDNE ALKALINITY SODIUM POTASIUM CHLORIDE CALCIUM WATER TEMPERTU sampling 0 (μs/cm) (mg/l) SS (mg/L) (mg/L) ppm ppm (mg/L) (mg/L) RE ( C) Summer 6.2 85 179 90 20 4.6 0.8 32 54.54 35.3 Autumn 7.6 96 170 110 70 4.2 1.2 25 48.9 29.8 Winter 7.2 98 155 89 76 4.8 0.8 28 44.2 17.5 Spring 6.9 82 184 86 48 3.2 0.6 39 27.8 33.7 Average 6.9 ± 0.59 90.25 ± 7.9 172 ± 12.7 93.7 ± 10.09 53.5 ± 25.3 4.2 ± 0.7 0.8 ± 0.2 31.0 ± 6.0 43.8 ± 29 29.0 ± 8.0 Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 13 of 15 Fig. 5 Principal Component Analysis of physico-chemical parameters and dominant species of Prithus Pool Fig. 6 Principal Component Analysis of physico-chemical parameters and dominant species of Braham Sarovar Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 14 of 15 Sarovar and Prithus pools of the historically important cities Canini ND, Metillo EB, Azanza RV (2013) Monsoon-influenced phytoplankton community structure in a Philippine mangrove estuary. Trop Ecol 54:331–343 (Kurukshetra and Pehowa) of Haryana State, for two years Chardez D, Lambert J (1985) Protozoaires ciliés et thanatologie. Forensic Sci Int continuously. The data analysis differentiated phytoplankton 28:83–101 into common, rare, and site-specific species in order to con- Cumming BF, Smol JP (1993) Development of diatom-based salinity models for paleoclimatic research from lakes in British Coloumbia (Canada). Twelfth struct phytoplankton-map. The P-maps generated for the International Diatom Symposius. Hydrobiologia. Springer, Dordrecht, pp 179- two water bodies of Haryana state, India, are expected to be 96, vol. 269 useful to forensic experts and forensic medicine experts in Desikachary TV (1959) Cyanophyta. Indian Council of Agricultural Research, New Delhi solving drowning cases and unfolding mysteries related to Díaz-Palma P, Alucema A, Hayashida G, Maidana N (2009) Development and the putativesiteofdrowning, especially when diatoms are standardization of a microalgae test for determining deaths by drowning. present in very less number during a particular season. In Forensic Sci Int 184:37–41 Figueredo CC, Giani A (2009) Phytoplankton community in the tropical lake of addition, the data has been successfully used to find the de- Lagoa Santa (Brazil): conditions favoring a persistent bloom of gree of correlation between different environmental parame- Cylindersospermosis raciborskii. Limnologica 39:264–272 ters and phytoplanktons prevailing in a water body with the Hall DW (1997) Forensic botany. Forensic Taphonomy: the postmortem fate of human remains. CRC Press, Inc., Boca Raton help of Principal Component Analysis method. Khuantrairong T, Traichaiyaporn S (2008) Diversity and seasonal succession of the phytoplankton community in Doi Tao lake, Chiang Mai Province, northern Abbreviations Thailand. Tropical. Nat Hist 8:143–156 APHA: America Public Health Association; D: Species richness; EC: Electrical Kim YS (2011) Research for seasonal plankton distribution of in-land water in conductivity; H: Shannon and weaver diversity index; J: Evenness; N: Total Gwang-ju Area.Korean. J Legal Med 35:16–21 number of individuals in the sample; n: Total number of species in the Komárek J, Fott B, Huber-Pestalozzi G (1983) Das Phytoplankton des Süßwassers. sample; PCA: Principal component analysis; P-map: Phytoplankton map; Systematik und Biologie-Teil 7, 1. Hälfte. S: Total number of species in the sample; TDS: Total dissolved solids Ludes B, Coste M, North N, Doray S, Tracqui A, Kintz P (1999) Diatom analysis in victim’s tissues as an indicator of the site of drowning. Int J Legal Med 112: Acknowledgements 163–166 Authors are thankful to U.G.C, New Delhi for providing U.G.C NET/JRF Lunetta P, Miettinen A, Spilling K, Sajantila A (2013) False-positive diatom test: a fellowship to one of authors, Deepali luthra to carry out this research. real challenge? A post-mortem study using standardized protocols. Legal Med 15:229–234 Funding Lunette P, Modell JH (2005) Macroscopical microscopical and laboratory findings U.G.C NET/JRF fellowship. in drowning victims. In: Tsokos M. (eds) Forensic Pathology Reviews, vol. 3. Humana Press, Totowa NJ Availability of data and materials Menhinick EF (1964) A comparison of some species-individuals diversity indices The datasets generated during and/or analyzed during the current study are applied to samples of field insects. Ecology 45:859–861 available from the corresponding authors on reasonable request. Nowrouzi S, Valavi H (2011) Effects of environmental factors on phytoplankton abundance and diversity in Kaftar Lake. J Fish Aquat Sci 6:130–140 Authors’ contributions Otto H (1961) On the demonstration of diatoms in human lung dust. Frankfurter Study conception and design: MKT, JSK, DL. Acquisition of data: DL. Analysis Zeitschrift für Pathologie 71:176–181 and interpretation of data: DL, MKT, JSK. Drafting of manuscript: DL. Critical Panigrahi S, Patra A (2013) Studies on seasonal variations in phytoplankton diversity revision: MKT, JSK, DL. All authors read and approved the final manuscript. of river Mahanadi, Cuttack city, Odisha, India. Indian J Sci Res 4:211–217 Philipose M T (1967) Chlorococcales. Indian Council of Agricultural Research, New Ethics approval and consent to participate Delhi Ethical approval and consent to participate were not required. Pielou E (1975) Indices of diversity and evenness. Ecological Diversity. Wiley, New York Competing interests Pollanen M (1997) The diagnostic value of the diatom test for drowning, II. The authors declare that they have no competing interest. Validity: analysis of diatoms in bone marrow and drowning medium. J Forensic Sci 42:286–290 Prescott GW (1962) Algae of the western Great Lakes area.WC Brown Company Publisher’sNote Dubuque. Springer Nature remains neutral with regard to jurisdictional claims in Raibole M, Singh YP (2011) Impact of physico-chemical parameters on microbial published maps and institutional affiliations. diversity:seasonal study. Curr World Environ 6:71–76 Author details Sasidharan A, Resmi S (2014) Forensic diatomology. Health Sciences 3:1–16 Department of Forensic Science, Punjabi University, Patiala, India. Shannon CE (2001) A mathematical theory of communication. ACM SIGMOBILE Department of Botany, Punjabi University, Patiala, India. Mobile Comput Commun Rev 5:3–55 Shinde S, Pathan T, Sonawane D (2012) Seasonal variations and biodiversity of Received: 18 November 2017 Accepted: 15 May 2018 phytoplankton in Harsool-Savangi dam, Aurangabad, India. J Environ Biol 33: 643–647 Singh M, Lodha P, Singh GP (2010) Seasonal diatom variations with reference to physico-chemical properties of water of Mansagar Lake of Jaipur, Rajasthan. References Res J Agric Sci 1:451–457 APHA (American Public Health Association), American Water Works Associations Singh R, Singh R, Kumar S, Thakar M (2006a) Forensic analysis of diatoms-a (AWWA) and Water Environment Federation (WEF) (2005) Standard methods review. Anil Aggrawal’s Internet J Forensic Med Toxicol 7 for the examination of water and wastewater. APHA (American Public Health Association), American Water Works Associations (AWWA) and Water Singh R, Singh R, Singh R, Thakar M (2006) Diatomological studies from three Environment Federation (WEF), Washington DC water bodies of Jaipur. Indian Internet Journal of Forensic Medicine & Auer A, Möttönen M (1988) Diatoms and drowning. Zeitschrift für Rechtsmedizin Toxicology 4:1-11 101:87–98 Singh R, Singh R, Thakar MK (2005) Drowning associated diatoms. Indian Intern J Baba AI, Pandit AK (2014) Species composition, diversity and population Forens Med Toxicol 3 Dyanamics of phytoplankton at Saderkot in Wular Lake, Kashmir. J Eco Ecogr Singh SR, Swarup K (1980) Studies on the primary production of phytoplankton 4:142–150 in Suraha lake (Ballia, India). Int Rev Hydrobiol 65:709–717 Thakar et al. 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Academic Press, New York Yen LY, Jayaprakash P (2007) Prevalence of diatom frustules in non-vegetarian foodstuffs and its implications in interpreting identification of diatom frustules in drowning cases. Forensic Sci Int 170:1–7 Yoshimura S, Yoshida M, Okii Y, Tokiyasu T, Watabiki T, Akane A (1995) Detection of green algae (Chlorophyceae) for the diagnosis of drowning. Int J Legal Med 108:39–42 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Egyptian Journal of Forensic Sciences Springer Journals

Forensic studies of phytoplankton ecology of two water bodies of Kurukshetra area of Haryana, State in India

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Abstract

Background: In forensic investigations of death cases in which the body has been recovered from a water body, there is an urgent need to prove whether the death is due to drowning or not. Moreover, another important issue arises, whether a person was alive at the time of drowning (anti-mortem drowning) or he was thrown dead into the water body (post-mortem drowning) To answer these questions, phytoplankton species of forensic importance from two water bodies (Braham Sarovar and Prithus Pools) were analyzed to develop the Phytoplankton maps (P- maps). The P-maps are basically a detailed list of categories of species which are common, site-specific, rare and seasonally occurring phytoplanktons along with their photomicrographs. Methods: In the present study, water samples have been collected from Braham Sarovar and Prithus Pools situated at Kurukshetra area of Haryana state, India. The phytoplankton species present in both ponds were extracted by mild acid digestion (5% HCl). The physico-chemical properties of these water bodies such as pH, water temperature, total dissolved solids, electrical conductivity, hardness, alkalinity, calcium, sodium, potassium, and chloride ions have been recorded to establish a correlation with the distribution of phytoplankton using principal component analysis. Results: Total 138 species of 59 genera of phytoplanktons belonging to Cyanophyceae, Chlorophyceae, Bacillariophyceae, Euglenophyceae and Dinophyceae have been observed. In Braham Sarovar, Cylindrospermum sp., Epithemia turgida, Eunotia rhomboidea, and Westella botryoides were the site-specific phytoplanktons whereas in Prithus pool, the site specific phytoplanktons were Leptolynbya granulifera, Arthospira jenerii, Cymbella cymbiformis, Frustulia vulgaris. Conclusion: It is hoped that results/data obtained from the present study (P-maps) may go a long way in solving the mysteries related to the deaths due to drowning and can be usefully used as a reference data for determining the exact site of drowning. Keywords: Phytoplankton, Forensic science, Diatoms, Aquatic bodies, Braham Sarovar, Prithus pool and P-maps, Principal component analysis, Diversity index, Drowning Background The place where actual drowning took place? The recovery of a dead body from any water body does To answer these questions, a diatom test has been not always mean that the death is due to drowning. considered to be the best marker of drowning but it con- There can be other reasons also. In forensic cases, it is fronted with many difficulties and hardships in terms of urgently required to investigate the real cause of death, its reliability. It was shown by some of the workers that whether it is due to drowning or not. In addition, few diatoms were also present in non-drowned victims due important questions arising in relation to it are: to postmortem invasion, autopsy contamination (Lunetta Whether the drowning is anti-mortem or post-mortem? et al. 2013), inhalation of diatoms from the air (Otto 1961; Spitz et al. 1965), water and foodstuff (Yen and Jayaprakash 2007). However, the reliability of diatom test * Correspondence: mukeshk38@gmail.com has been established by the majority of researchers by Department of Forensic Science, Punjabi University, Patiala, India Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 2 of 15 considering qualitative and quantitative criteria (Auer Phytoplanktons are the photoautotrophic organisms and Möttönen 1988; Ludes et al. 1999; Pollanen 1997; which prevail in all types of water bodies such as Singh et al. 2005, 2006a, 2006b; Thakar et al. 2011; Tha- streams, lakes, rivers, and ponds and are an important kar and Singh 2009; Thakar and Singh 2010). In 1988, component of the food chain in the aquatic environ- Auer and Mottonen suggested that minimum 20 num- ment. Phytoplankton groups may be distinguished from ber of diatom per microscope slide from lung samples is each other based on a combination of characteristics, in- required to define the death due to drowning. Similarly cluding photosynthetic pigments, shape, size and cell Ludes et al. (1999) also suggested the limit of 20 diatoms covering. They are microscopic in nature and “invisible” per slide per 100 μL pellet for lungs and 5 diatoms per to perpetrators of crimes, thus it helps in linking a sus- slide per 100 μL of a pellet sediment extracted from 2 g pect to the crime scene (Siver et al. 1994). of tissue samples such as brain, kidney, liver and bone Keeping in mind the needs of environmental and fo- marrow. But under certain environmental conditions, di- rensic experts, the present study has been undertaken to atoms may be scanty or absent in a particular water study the phytoplankton diversity along with seasonal body, leading to a false negative diagnosis of drowning. variations in two forensically important ponds (Braham Many studies on qualitative and quantitative assessment Sarovar and Prithus Pools) of Kurukshetra region of Ha- of diatoms during different seasons and at different time ryana, India. These two ponds are forensically important periods have reported lesser number or absent of diatom because of two reasons: Firstly, sufficiently large amount species in one season and their bloom in the other of water remains in these unguarded ponds throughout (Canini et al. 2013; Cumming and Smol 1993; Singh et the year, and secondly, a large crowd gathers around al. 2010; Sushanth and Rajashekhar 2012). Pollanen them to take a dip during the year on many occasions. (1997) also reported highly positive diatom test outcome The data generated in the present study has been used in the body of drowned victims in the months in which to generate Phytoplankton-maps (P-maps). The charac- diatom population in the water body was at a peak. This teristics Phytoplankton-maps of these two water bodies showed variations in the number of diatoms over a will hopefully help in unfolding the mysteries of deaths period of time in water bodies. due to drowning and the data generated can be utilized The forensic usefulness of phytoplankton came to light as a reference data for determining the exact site of when Chardez and Lambert (1985) (cited in Lunette and drowning. Modell 2005) detected the presence of phytoplankton other than diatoms in the drowned bodies. They also Material and methods found aquatic organisms belonging to division Chloro- Selection of sampling sites phyta, Dinoflagellates, invertebrates, protozoan ciliates In the present study the following two sites which are in the blood of drowned victims. Later, Yoshimura et al. situated in Kurukshetra city and Pehowa city of Haryana, (1995) developed a method to recover phytoplankton India have been selected to collect samples for the ana- from the tissue of drowned victims by using lysis of phytoplanktons: soluene-350. Various species of green algae (Staurastrum and members of Zygnemataceae) as well as diatoms Brahma Sarovar (Navicula, Cymbella and Melosira) were detected in It is the largest man-made water body situated in histor- both putative water samples and tissue samples of ically important Kurukshetra city, Haryana state, India drowned victims. In an another case a serious attempt (29°57′37″ N 76°50′42.2″ E). The water body is rect- has been made to link the suspects and victims to the angular in shape, measuring 3600 ft long, 1500 ft wide crime scene by analyzing the sediment encrusted and 15 ft deep. It has religious significance, every year sneakers from both assailants and victims along with nu- on ‘Somavati Amavasya’(a special day in Hindu religion) merous species of diatoms and scaled Chrysophytes and on the occasion of solar eclipses; millions of people (planktonic algae) from the sneakers as well as reference from different parts of India visit this water body to take samples of pond sediment (Siver et al. 1994). a holy bath (Fig. 1). Further in Díaz-Palma et al. 2009 suggested to extend the diatom test to detect drowning by taking into con- Prithus pool sideration other groups of phytoplanktons. They have It is located in another religiously important city, Pehowa standardized the methods for recovery of microalgae city, Haryana, India (29°58′51.6″N76°34′46.4″E) about from drowned victims by using different extraction pro- 25 Kms from Braham Sarovar. It was constructed by king cedures and concluded that dinoflagellates and some Prithu after the death of his father. This place is known chlorophytes have cell walls and other resistant struc- for funeral ceremonies (pind daan in Hindu religion), tures similar to diatoms. As a result, these organisms therefore, thousands of pilgrims from different places visit can be found in tissues of drowned victims. this pool throughout the year (Fig. 2). Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 3 of 15 behind only a residual material at the bottom of the tube. An excess of water was again mixed with residual material and centrifuged again as above. This process was repeated twice. After discarding the supernatant, the residual material (pellet) containing phytoplankton was poured over two sets of five serially marked (I-V) microscopic slides and smear was prepared. Then, these slides of two sets were permanently mounted using a high refractive index mounting material DPX to study diatoms and Glycerin jelly for phytoplankton analysis under the low and high power of the microscope. Fig. 1 Photograph of Braham Sarovar Analysis of slides prepared All the slides prepared for both the water bodies season Collection of samples wise have been examined with the help of an optical For phytoplankton analysis, the water samples have been compound microscope (Nikon Eclipse microscope) fitted collected during four seasons such as summer (May– with light source under low (10× X 10×), high power June), autumn (August–September), winter (November– (10× X 40×) and oil immersion (10× X 10×) magnifica- December) and spring (February–March) from two se- tions. Nikon E200 series digital camera which has been lected sites from the year 2014 to 2016. The water sam- attached to the microscope was used to prepare Photo- ples were collected in a sterilized plastic bottle of 1 l micrographs for each and every phytoplankton species. capacity and preserved in 4% formaldehyde solution. These Photomicrographs were used for taxonomic iden- tifications of phytoplankton with the help of publications given by Prescott (1962); Komárek et al. (1983); Desi- The procedure adopted to prepare slides for the analysis kachary (1959); Philipose (1967) and Wehr et al. (2015). of phytoplankton Data so obtained have been used to construct a phytoplankton map (p-map) for the both water bodies. Approximately, 50 ml of water sample was taken in Enumeration of phytoplankton was done by using a beaker and digested with 10 ml of 5% HCl for Haemocytometer. The colonial and filamentous microal- 24 h. gae such as Microcystis sp., Merismopedia sp. and Oscil- Next day, after thorough mixing of the water sample latoria sp. have been counted by using plating method and HCl, 10 ml of water sample was taken in a (Stanier et al. 1971). Average of two years (2014–2016) centrifuge tube and centrifuged at 3000 rpm for data has been given in Tables 1, 2 and 3. 10 min. The supernatant was collected out, leaving Physico-chemical properties In the both selected water bodies, the parameters such as total alkalinity, chloride and calcium levels, and total hardness have been determined according to APHA (American Public Health Association) manual (APHA 2005). Sodium and potassium contents in the water samples have also been determined with the help of flame photometer. Electrical conductivity, pH, and temperature have also been determined by using the pH meter cum conductivity meter (Aquapro digital water tester model AP-2). TDS has been determined by using TDS meter (Aquapro digital water tester model AP-1). The correlation between physico-chemical parameters and phytoplankton species was carried out by Principal Component Analysis (PCA) using the Microsoft Excel Fig. 2 Photograph of Prithus Pool Add-Ins software XLSTAT. Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 4 of 15 Table 1 List of phytoplankton species observed in Braham Sarovar and Prithus Pools Family Braham Sarovar Prithus pool Chlorophyceae Ankistrodesmus spiralis, Ankistrodesmus fusiformis, Ankistrodesmus Ankistrodesmus acicularis, Chlorocuccum scabellum, Coelastrum flactum, Chlorococcum sp., Coelastrum microporum, Crucigenia cambricum, Coelastrum microporum, Coelastrum astroideum, lauterbornii, Crucigenia tetrapedia, Dicloster acuatus, Krichneriella Coelastrum morus, Coelastrum pulchrum, Coelastrum sphaericum, sp., Monoraphidium arcuatum, Monoraphidium contortum, Crucigeniella neglecta, Crucigenia lauterbornii, Crucigenia crucifera, Monoraphidium griffithi, Oocystis borgei, Oocytis marssonii, Crucigeniella quadrata, Crucigenia tetrapedia, Dictyochloropsis Scenedesmus quadricauda, Scenedesmus dimorphus, Scenedesmus reticulata, Kirchneriella irregularis, Monoraphidium contortum, ecornis, Scenedesmus bijugatus, Scenedesmus armatus Monoraphidium fontinale, Monoraphidium arcuatum, var.boglariensis, Scenedesmus abundans, Scenedesmus armatus Monoraphidium griffithii, Oocystis sp., Oocystis solitaria, Pediastrum var.bicaudatus, Scenedesmus obliquus, Schroederia indica, duplex, Pediatrum simplex, Scenedesmus bijugatus, Scenedesmus Staurastrum iotanum, Staurastrum sp. Lemmermannia punctata, dimorphus, Scenedesmus abundans, Scenedesmus verrucosus, Tetraedrone lobulatum, Tetraedrone trigonium, Tetrachlorella Scenedesmus accuminatus, Scenedesmus caudato-aculeolatus, alternans, Tetrastrum minimum, Trebouxia sp., Westella botryoides. Scenedesmus obliquus var. dimorphus, Scenedesmus quadricauda, Schroederia setigera, Selenastrum sp., Tetracystis sp., Tetraedrone minimum, Tetraedrone puchtatum, Tetraedron trigonium. Cyanophyceae Aphanocapsa sp., Calothrix sp., Chroococcus cohaerens, Planktosphaeria gelatinosa, Aphanocapsa annulata, Aphanocapsa Chroococcus dispersus, Cylindrospermum sp., Gloeocapsa delicatissima, Planktothrix maougetti, Merismopedia tenuissima, minimum, Merismopedia glauca, Merismopedia minima, Scytonema hofmanni, Spirulina sp., Synechococcus sp., Oscillatoria Microcystis panniformis, Microcystis aerognosa, Oscillatoria agardhii, Oscillatoria rufringe, Oscillatoria limosa, Arthrospira agardhii, Geitlerinema pseuriaccutissimum, pseudoanabena jenneri, Leptolyngbya granuliferat, Geitlerinema amphibium sp.,Synechococcus sp., Synechocystis aqualis Bacillariophyceae Aulacoseira granulata, Cymbella tumida, Epithemia turgida, Frustulia vulgaris, Cymbella cistula, Melosira varians, Nitzschia Eunotia rhomboidea, Navicula capitoradiata, Nitzchia palecea, palea, Nitzschia acicularis, Cymbella cymbiformis, Navicula Nitzchia accicularis, Cymbella laevis, Stephanocyclus gregaria meneghiniana, Pinnularia abaujensis, Navicula cryptocephala, Gomphonema truncatum Euglenophyceae Euglena sp., Lepcinclis fusiformis, Phacus longicauda, Phacus Lepocinclis ovum, Phacus onxy, Phacus pyrum, Euglena acus, tortus,Trachelomonas horrida. Trachelomonas langerdiana, Euglena caudata,Euglena virdis, Lepocinclis elongata, Lepocinclis sternii, Euglena splendens. Dinophyceae Alexanderium catenella, Alexanderium insuetum, Gonyaulax lingulodinium, Gyrodinium fusus, Peridinium cinctum, Peridinium umbonatum. Diversity indices: In the present study, the diversity in- pi = ni/N, ni being the number of individual of ith spe- dices of phytoplankton species have been calculated in cies and. terms of N = total number of individuals in the sample. Species Richness: Species richness is a count of a num- Species diversity method given by Shannon (2001), ber of species in a water body. It is calculated using the Species richness by Menhinick (1964) and following formula: Evenness by Pielou (1975) as follows: D ¼ n=√N Shannon-Wiener diversity index: Shannon diversity index is a quantitative measure of species diversity in a Where D = Species Richness, n = total number of spe- community and can be calculated as follows: cies in a sample. N = Total number of an individual organism in a H ¼ − ðÞ pi log2pi sample. Species Evenness: Species evenness is the measures of Where, H = Shannon and Weaver diversity index. how equals the relative abundance of different species 3 −1 Table 2 Phytoplankton density (individuals× 10 mL ) in Braham Sarovar during different seasons Class Summer Autumn Winter Spring Chlorophyceae 1.180 ± 0.235 0.716 ± 0.206 0.357 × 0.225 0.653 ± 0.234. Cyanophyceae 0.372 ± 0.090 0.322 ± .092 0.714 ± 0.091 0.344 ± 0.293 Bacillariophyceae 0.188 ± 0.10 0.077 ± 0.03 0.256 ± 0.068. 0.190 ± 0.08 Euglenophyceae 0 0.05 ± 0.032 0.024 ± 0.02 0 Dinophyceae 0 0 0.012 ± 0.01 0.098 ± 0.05 Total 1.740 ± 0.52.6 1.162 ± 0.3 1.363 ± 0.28 1.285 ± 0.24 Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 5 of 15 3 −1 Table 3 Phytoplankton density (individuals ×10 mL ) in Prithus Pool during different seasons Class Summer Autumn Winter Spring Chlorophyceae 1.387 ± 0.27 1.078 ± 0.22 0.909 ± 0.11 1.372 ± 0.37 Cyanophyceae 0.346 ± 0.14 0.243 ± 0.006 0.178 ± 0.08 0.231 × ±0.06 Bacillariophyceae 0.192 ± 0.024 0.051 ± 0.034 0.205 ± 0.03 0.128 ± 0.058 Euglenophyceae 0.063 ± 0.057 0.026 ± 0.019 0.101 ± 0.07 0.138 ± 0.04 Total 1.988 ± 0.525 1.398 ± 0.42 1.393 ± 0.32 1.869 ± 0.52 are in a community. It is calculated using formula as These season specific species of a particular water follows: body may help in determining the season of occurrence of crime. J ¼ H= log2ðÞ S ❖ Rarely occurring species: These are those species which are found occasionally or by chance in a Where, J = Evenness H = Shannon and Weaver diver- particular water body. These species are not sity index. important from the forensic point of view. S = Total number of species in a sample. In part-B of P-map for both the water bodies, the pho- tomicrographs of Site-specific species have been shown Phytoplankton-maps (P-maps) (Tables 4 and 5), whereas part – C shows Distribution The data generated in the present study were used to of Phytoplankton species during the different season in prepare the P-maps (Tables 4 and 5) of two selected both water bodies (Tables 4 and 5). water bodies (Braham Sarovar and Prithus Pools). The P-maps depicts the detailed list of phytoplankton genera Results and discussion and species present in the two selected water bodies. Phytoplanktons are the diverse group of photosynthetic P-maps have been divided into three parts- A, B, and organisms present in all the aquatic habitats such as C as shown in Tables 4 and 5. In part – A, phytoplank- lakes, rivers, ponds, canals, sea etc. Moreover, they have tons species have been shown, which have been divided different shapes, sizes, colors and forms, which provide into the following four categories: them individuality. So their presence in water bodies and potential to identify the site of drowning may help in the ▪ Commonly found phytoplanktons forensic investigation. Because of their microscopic na- ▪ Site-specific phytoplanktons, ture and invisibility phytoplankton may adhere to the ▪ Seasonally occurring species of phytoplanktons suspect, victim or their clothes or weapon of offense. In ▪ Rarely occurring phytoplankton addition, different species of phytoplankton present dur- ing different seasons which aids in linking the suspect to The detailed information about these categories has a particular crime scene during the specific season (Hall been given below and is helpful in generating P-map for 1997). Therefore, seasonal variation of phytoplankton a specific water body: has been studied in two selected water bodies of Kuruk- shetra region, continuously for two years to generate a ❖ Commonly found species: Commonly found species baseline database of phytoplankton (Phytoplankton are those which are common in more than one maps) for each water body. water body. They have significance in detecting the In the present study, remarkable changes in diversity death due to drowning and are of little significance and abundance of phytoplankton have been observed in diagnosing drowning site. during different seasons. In Braham Sarovar and Prithus ❖ Site-specific species: Site-specific species are those Pools, a total of 138 species belonging to 59 phytoplank- which are restricted to a particular water body, ton members have been found (Tables 1 and 6). Indi- hence they have great forensic significance in locat- vidually in Braham Sarovar alone 47 genera belonging to ing the exact site of drowning, particularly when a 5 classes i.e. Cyanophyceae (12), Chlorophyceae (18), body is found away from the actual site of drowning Bacillariophyceae (9), Euglenophyceae (4) and Dinophy- and also they help in linking the suspect and victim ceae (4) representing 70 species have been found. Simi- to the crime scene. larly, in Prithus pool, 36 genera belonging to 4 classes’ ❖ Seasonally occurring species: This category includes i.e. Chlorophyceae (15), Cyanophyceae (12), Euglenophy- those species which are dominant in one season and ceae (4), and Bacillariophyceae (5) representing 68 spe- may become extinct or dominated by other species cies found. Similar studies have been conducted by Kim during another season in a particular water body. 2011. He observed 16 species of diatoms, 20 species of Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 6 of 15 Table 4 Phytoplankton -map (P-maps) of Braham Sarovar Commonly occurring Site- specific Seasonally occuring Rarely occurring Part A: List of phytoplanktons genera and species observed in Braham Sarovar Coelastrum microporum,Crucigenia Epithemia turgida, Summer Gloeocapsa sp., Ankistrodesmus fusiformis, Chlorococcum sp., Crucigenia lauterbornii, Crucigenia Eunotia rhomboidea, Monoraphidium arcuatum, Scenedesmus lauterborni, Dicloster acuatus Gloeocystis tetrapedia,Monoraphidium arcuatum, Westella botryoides, obliquus, Tetraedron minimum, sp.,Monoraphidium contortum,Oocystis Monoraphidium contortum, Cylindrospermum sp. Staurastrum iotanum, Choroococcus borgei, Schroederia indica, Staurastrum Monoraphidium griffithi, Nitzschia coherens, Chroococcus dispersus, Nitzschia sp., Tetrachlorella alternans, Tetraedron acicularis,Oscillatoria agardhii, palecea, Merismopedia minima, Gleocapsa lobulatum, Trebauxia sp., Geitlerinema Scenedesmus abundans, Scenedesmus minimum, Pinnularia abaujensis. pseuriaaccutissimum, Microcystis dimorphus, Scenedesmus panniformis, Navicula capitoradiata, Autumn Ankistrodesmus fusiformis, Crucigenia obliquus,Tetraedron minimum, Lepocinclis fusiformis, Lemmermannia Tetrapedia, Monoraphidium griffthii, Tetraedron trigonium punctata,Phacus longicauda, Phacus Lemmermannia punctata. tortus, Ttrachelomonas horrida, Alexanderium acatenella, Alexanderium Winter Merismopedia glauca, Microcsystis insuetum, Gonyaulax lingulodinium, aerognosa, Synechococcus sp., Gyrodinium fusus, Peridinium cinctum, Kritchneriella irregularis, Gomphonema Peridinium umbonatum. truncata, Navicual cryptocephala, Epithemia turgida, Eunotia rhomboidea, Cylindrospermumsp Spring Aphanocapsa sp., Oosystis marsonnii, Scenedesmus bijugatus, Scenedesmus armatus, Pseudoanabaena constrictum Part B: Photomicrographs of some of site-specific species. Part C: Distribution of Phytoplankton during different seasons Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 7 of 15 Table 5 Phytoplankton -map (P-map) for Prithus Pool Commonly occurring Site- specific Seasonally occuring Rarely occurring Part A: List of phytoplanktons genera and species observed in Prithus Pool Coelastrum microporum, Crucigenia Leptolynbya Summer Coelastrum microporum, Crucigenia Ankistrodesmus acicularis, lauterbornii, Crucigenia granulifer, Arthospira quadrata, Tetraedrone minimum, Ankistrodesmus flactum, Coelastrum tetrapedia,Monoraphidium arcuatum, jenerii,Cymbella Chlorococcum sp., Coelastridium astroideum, Coelastrum morus, Monoraphidium contortum, cymbiformis asterodeum, Crucigeniella quadrata, Coelastrum pulchrum, Coelastrum Monoraphidium griffithi, Nitzschia Frustulia vulgaris. Selenastrum sp., Aphanocapsa morus, Crucigeniella neglecta, acicularis,Oscillatoria agardhii, delicattissima, Geitlerinema amphibium, Pediastrum duplex, Pediatrum simplex, Scenedesmus abundans, Syctonema hofmanii, Melosira varians, Schroederia setigera,Geitlerinema Scenedesmus Trachelomonas langerdiana, amphibium,Oscillatoria limosa, dimorphus, Scenedesmus Scenedesmus quadricauda Navicula gregaria, Euglena caudata, obliquus,Tetraedron Euglena splendens, Euglena virdis, Autumn Oocystis solitare, Scenedesmus minimum,Tetraedron trigonium. Lepocinclis elongata, Lepocinclis sternii, versucois, Scenedesmus acumnatus, Phacus onxy. Oscillatoria rufringe, Planktothrix mougetti, Lepocinclis ovum. Winter Tetracystis sp., Kirchneriella sp., Coelastrum asterodeum, Tetracystis sp., Cymbella cistulla, Phacus pyrum Spring Chlorococcum scabellum, Coelastrum cambricum, Dictyochoropsis reticulata, Monoraphidium frontinale, Crucigenia crucifera, Scenedesmus bijugatus, Scenedesmus caudato-aculeolatus, Tet raedrone punchtatum, Oscillatoria agardhii Part B: Photomicrographs of some of site-specific species Part C: Distribution of Phytoplankton during different seasons Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 8 of 15 Table 6 List of genera observed in Braham Sarovar and Prithus Table 6 List of genera observed in Braham Sarovar and Prithus Pools Pools (Continued) S. No Genus Braham Sarovar Prithus Pool S. No Genus Braham Sarovar Prithus Pool Genus related to class Chlorophyceae Genus related to class Euglenophyceae 1. Ankistrodesmus ++ 41. Euglena ++ 2. Chlorococcum ++ 42. Lepocinclis ++ 3. Coelastrum ++ 43. Phacus ++ 4. Crucigeniella ++ 44. Trachelomonas ++ 5. Crucigenia ++ Genus related to class Bacillariophyceae 6. Dicloster +_ 45. Aulacoseira +_ 7. Dictyochloropsis _+ 46. Cymbella ++ 8. Gloeocystis +_ 47. Epithemia +_ 9. Kirchneriella ++ 48 Eunotia +_ 10. Monoraphidium ++ 49. Frustulia _+ 11. Lemmermannia +_ 50. Gomphonema +_ 12. Oocystis ++ 51. Melosira _+ 13. Pediastrum _+ 52. Navicula ++ 14. Scenedesmus ++ 53. Nitzschia ++ 15. Schroederia ++ 54. Pinnularia +_ 16. Selenastrum sp _+ 55. Stephanocyclus +_ 17. Staurastrum +_ Genus related to class Dinophyceae 18. Tetrachlorella +_ 56. Alexanderium +_ 19. Tetracystis _+ 57. Gonyaulax + 20. Tetraedrone ++ 58. Gyrodinium + _ 21. Trebouxia +_ 59. Peridinium +_ 22. Westella +_ 23. Planktosphaeria ++ green algae, 6 species of Cyanobacteria and 6 species of other algae in three aquatic locations of Gwang-ju area Genus related to class Cyanophyceae (South Korea) where drowned victims were found. 24. Aphanocapsa The detailed list of species recovered in the present study 25. Arthrospira _+ has been given in Table 1.Figures 3 and 4 show photomi- 26. Calothrix +_ crographs of different species of phytoplankton observed in 27. Chroococcus +_ Braham Sarovar and Prithus pools water bodies. 28. Cylindrospermum +_ Phytoplankton community 29. Geitlerinema ++ Class - Chlorophyceae 30. Gloeocapsa +_ The members of Chlorophyceae formed the most dom- 31. Leptolyngbya _+ inant group among others in both water bodies which 32. Merismopedia ++ accounted for 46% of total phytoplankton diversity with 33. Microcystis ++ 32 species in the Braham Sarovar (Table 4c) and 54.4% 34. Oscillatoria ++ of total diversity was represented by 37 species of Chlor- ophyceae in the Prithus Pool (Table 5c). Panigrahi and 35. Planktothrix _+ Patra (2013) also reported dominance of Chlorophyceae 36. Pseudanabaena +_ over other phytoplankton groups in terms of numerical 37. Scytonema _+ value and percentage composition in a water body of 38. Spirulina _+ Cuttack city, West Bengal, India. The dominant species 39. Synechococcus ++ of Braham Sarovar were Ankistrodesmus fusiformis, 40. Synechocystis +_ Ankistrodesmus flactum, Crucigenia tetrapedia, Oocystis marsonii, Scenedesmus bijugatus, Scenedesmus abun- dance, Tetrastrum minimum, Coelastrum Microporum. Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 9 of 15 Fig. 3 Photomicrograph of phytoplankton observed during the present study. a. Crucigeniella quadrata, b. Scenedesmus opaliensis, c. Pediastrum duplex, d. Crucigeniella crucifera, e. Tetraedron lobulatum, f. Scenedesmus dimorphus g. Staurastrum iotanum, h. Crucigenia fenestrata, i. Tetraedron minimum, j. Coelastrum microporum, k. Coelastrum cambricum, l. Scenedesmus bijugatus. Scale bar, 10μm Fig. 4 Photomicrograph of phytoplankton observed during the present study a. Phacus pyrum, b. Phacus longicauda, c. Lepocinclis fusiformis, d. Euglena splendens, e. Synechococcus sp., f. Stephanocyclus meneghiniana g. Merismopedia minima, h. Synechocystis aquatilis, i. Nitzschia acicularis, j. Oscillatoria limosa, k. Microcystis aeruginosa.Scale bar,10μm Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 10 of 15 In Prithus pool the most dominant species were Chloro- Seasonal variation in phytoplankton population density coccum scabellum, Coelastrum microporum, Coelastrum Mean density of 1.662 × 10 ± 227 phytoplankton indi- − 1 asteriodeum, Crucigenia crucifera, Crucigenia tetrapedia, viduals mL of water was observed at Prithus pool Crucigeniella quadrata, Crucigeniella reticulata, Dictyo- while in Braham Sarovar mean density has been calcu- 3 − 1 chloropsis reticulata Kritchnella sp., Scenedesmus dimor- lated to be 1.388 × 10 ± 248 individuals mL . In both phus, Scenedesmus obliquus, Scenedesmus quadricauda, water bodies, Chlorophyceae was found to be the most and Tetradron puchtatum. abundant group in summer which declined in autumn and winter. A similar observation has been made by Shinde et al. (2012) in Harsool-savang dam, Aurangabad; Class-Cyanophyceae where maximum density of Chlorophyceae was observed Cyanophyceae was the second most abundant group in in summer season. Nowrouzi and Valavi (2011) while the Braham Sarovar with 21.4% diversity of the total studying phytoplankton of Lake Kaftar in Iran also re- phytoplankton population. Among the 15 species, ported highest density of Chlorophyceae in summer and Aphanocapsa sp., Chroococcus dispersus, Merismopedia spring and the lowest in winter. Cyanophyceae in glauca, Merismopedia minima, Microcystis aeruginosa Prithus pool exhibited maximum growth in summer and and Synechococcus sp. were dominant ones. minimum in winter while, in Braham Sarovar, Cyano- Similarly, in Prithus pool, Cyanophycaeae also ranked phyceae exhibited maximum growth in winter and mini- as the second most abundant group accounting for 21% mum in the autumn. The maximum density of of the total population with 14 species (Tables 4c and Cyanophyceae in Braham Sarovar during winter was due 5c). The dominating species were found to be Merismo- to the overgrowth of Microcystis aeruginosa. Khuantrair- pedia tenuiasima, Spirulina sp., Oscillatoria agardhii ong and Traichaiyaporn (2008) also observed Cyano- and, Plantothrix mougeotti. phyta as the most abundant group in winter and summer in Doi Tao Lake, Thailand. On the contrary, Venkateswarlu (1969) reported the dominance of Cyano- Class- Bacillariophyceae phyceae in summer season in Mossi River, Hyderabad. The class Bacillariophyceae has been ranked third in Singh and Swarup (1980) also reported maximum Braham Sarovar and accounted for 17.1% but the same growth of cyanophyceae during summer in Lake Suraha, class ranked fourth in Prithus Pool and accounted for Uttar Pradesh. The maximum abundance of Bacillario- 10.2% of total phytoplankton diversity found in this phyceae was recorded in winter and minimum in au- water body (Tables 4c and 5c). Total 12 species of dia- tumn. Singh et al. (2010) observed highest density of toms have been found from the Braham Sarovar with Bacillariophyceae in winter in Mawatha lake Rajasthan. Aulacoseira granulata, Nitzschia acicularis, Nitzschia A similar trend has also been observed by Baba and Pan- palecea and Navicula cryptocephala being the dominant dit (2014) while studying the diversity of phytoplankton ones, whereas seven species of diatoms reported from in Wular Lake Kashmir. Members of Euglenophyceae Prithus pool Frustulia vulgaris, Melosira varians Frustu- were observed both in autumn and winter in Braham lia vulgaris and Nitzschia palea were dominant. Sarovar, while in Prithus pool, members of Euglenophy- ceae were maximum during winter and spring and mini- mum in summer. The members of Dinophyceae were Class-Euglenophyceae detected only in Braham Sarovar, that too during win- Euglenophyceae class constituted around 15% (10 spe- ter and spring season. Mean density distribution of cies) of the total phytoplankton population and formed different classes of phytoplankton in Braham Sarovar the third most dominant group in Prithus pool, whereas and Prithus Pools is Part-C of P-map and has been it ranked fifth in Braham Sarovar accounting for 6% (5 showninTables 4 and 5. species). Among them, Phacus longicauda, Phacus tortus Various environmental factors such as light, temperature and Lepocinclis fusiformis were the most abundant and rainfall and physico-chemical factors (pH, temperature, species in the Braham Sarovar, while Lepcinclis ovum, salinity, Nitrogen, Phosphate, Calcium, Sodium, Phosphate, Euglena acus and Euglena virdis were dominant in Chloride, etc) of a water body get changed with seasons Prithus pool (Tables 4c, 5c). which in turn bring about change in composition and abundance of phytoplankton (Figueredo and Giani 2009). Class-Dinophyceae In the present study, the maximum density of Chlorophy- Members of Dinophyceae have been only observed in ceae and Cynaophyceae has been recorded during the sum- the Braham Sarovar water body and constituted 10% (6 mer season and minimum during autumn and winter species) of species diversity with Peridinium cinctum be- season. The increase in the density of phytoplankton may ing the dominant one. be attributed to the increase in temperature during Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 11 of 15 summer, which not only increases the rate of photosyn- physico-chemical parameters was probably due to the thesis, but also increases the concentration of available nu- anthropogenic activity at this pond. The physic-chemical trients due to increase in water evaporation. Whereas a parameters of both aquatic bodies during the different decrease in the density during autumn and winter may be seasons are shown in Tables 8 and 9. due to the increase in the water level of the water body by rainwater which decreases the photosynthetic efficiency of phytoplanktons (Shinde et al. 2012). The density of Bacillar- Correlation between physico-chemical parameters and iophyceae was observed maximum during winter which phytoplankton community may be due to a higher rate of multiplication of Bacillario- Principal component analysis (PCA) phyceae at low temperature (Raibole and Singh 2011). Principal Component Analysis (PCA) is a multivariate data The members of Euglenophyceae were observed during analysis technique which reduces the multi-dimensional autumn and winter season only, may be because of two data into two principal components. In present study Prin- reasons i.e. either they cannot tolerate higher temperature cipal Component Analysis has been used to establish the or increased accumulation of organic matter in the water degree of correlation between physico-chemical parameters bodies due to heavy rainfall, favors the growth of Eugleno- (as mentioned above) and the phytoplankton species pre- phyceae (Tiwari and Chauhan 2006). vailing in the water body. It helps to understand whether phytoplankton has a positive or negative relationship with Diversity index different physico-chemical parameters and what are the key High values of Shannon’s index (H′) were recorded dur- factors responsible for their growth and inhibition? ing summer and lower during winter at both water bod- As in forensic investigations, it is common to find a ies. Shannon’s index has been calculated to be high dead body away from the actual site of drowning due to (3.16 ± 0.013) in Braham Sarovar as compared to Prithus water current, tides, etc. Thus, determination of the actual pool (3.03 ± 0.26) (Table 7). The value of Species rich- site of drowning becomes important for the medico-legal ness of Braham Sarovar was comparatively high during expert. The abundance and diversity of phytoplankton summer (4.55) and minimum during winters (3.87) and vary between different water bodies due to the difference autumn (3.96) while in Prithus pool, species richness in various parameters such as pH, temperature, salinity, was maximum during spring (3.86) and minimum dur- electrical conductivity, hardness, alkalinity etc. of that ing autumn (3.42). Species evenness in Prithus pool water body (Nowrouzi and Valavi 2011). Some of the showed little variation during different seasons, indicat- physico-chemical parameters are favorable for the growth ing that all the species were equally distributed through- of one species while these may not support growth of out the year. In Braham Sarovar, the highest value of others. The different species of phytoplankton get estab- species evenness was 0.93 during summer and lower lished in a particular water body based on their prefer- during winter (0.86). ences for particular physico-chemical components of water. Therefore, a positive test for drowning should not Physico-chemical properties only be based upon the presence or absence of phyto- Physico-chemical parameters of Braham Sarovar and plankton in the body of a drowned victim, rather it should Prithus pools have been assessed to determine the tropical also exhibit the same type of phytoplankton in similar status of the water body and their co-relation with the concentrations of the drowning medium (Sasidharan and phytoplankton species diversity. The physico-chemical Resmi 2014). A detailed study of phytoplanktons and their parameters of Prithus pool were numerically high as relationship with characteristics of the water body can play compared to Braham Sarovar. The high value of a significant role in connecting a drowned dead body to a Table 7 Seasonal variation in phytoplankton diversity indices in Braham Sarovar and Prithus pools Water Body Season Shannon diversity index Evenesss Species richness Braham sarovar Summer 3.32676 0.93 4.55 Autumn 3.31251 0.89 3.96 Winter 2.90562 0.86 3.87 Spring 3.11771 0.89 4.60 Prithus pool Summer 3.139102 0.93 3.68 Autumn 2.951855 0.90 3.42 Winter 2.9244777 0.90 3.44 Spring 3.1210465 0.90 3.86 Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 12 of 15 Table 8 Physico-chemical Parameters of Prithus Pool Prithus pool Season of pH EC TDS HARDNE ALKALINITY SODIUM POTASIUM CHLORIDE CALCIUM WATER TEMPERTU sampling (0 (μs/cm) (mg/l) SS (mg/L) (mg/L) ppm ppm (mg/L) (mg/L) RE c) Summer 7.7 254 500 340 180 20 3.4 122 120 35.2 Autumn 7.1 227 474 250 177 18 4.2 91 104 32.4 Winter 6.9 130 434 230 145 21.5 4.8 57 96 16.5 Spring 7.2 198 587 310 189 28.6 2.8 94 76 22.1 Average 7.2 ± 0.34 198 ± 60 498 ± 64.7 280.7 ± 55.3 172.7 ± 19.1 22 ± 4.6 3.8 ± 0.8 91.0 ± 26 99.0 ± 18.2 26.5 ± 8.7 specific water body source particularly when the body is positively correlated with calcium and Ankistrodesmus found outside or lying adjacent to the water body. fusiformis is positively correlated with chloride. Phacus In the present the study, PCA method has been longicauda, Phacus tortus, Lepocinclis fusiformis, and employed to 10 physico-chemical parameters for 20 Synechococcus sp. are positively co-related with pH, potas- dominant species of phytoplankton for Prithus pool. The sium, hardness and electrical conductivity whereas they all PCA axis 1 and 2 explained 42.81 and 36.70% variation are negatively correlated with TDS and calcium. Similarly, in phytoplankton and physico-chemical parameter. Cru- Synechococcus sp. and Microcystis aeruginosa are posi- cigeniella quadrata, Crucigenia tetrapedia, Scenedesmus tively co-related with pH and alkalinity (Fig. 6). dimorphus, Scenedesmus quadricauda, Planktothrix mougeotti, and Melosira varians are positively co-related Phytoplankton maps (P- maps) of Braham Sarovar and to pH, hardness, alkalinity, chloride, electrical conductiv- Prithus pools ity and temperature of water sample while Crucigenia Among the total 138 species, 13 species were common crucifera is negatively co-related with all these factors. in both the water bodies, therefore, these are categorized Dictyochloropsis reticulata, Tetrastrum punctatum, and as common species and are significant in diagnosing the Frustulia vulgaris are positively co-related to TDS (total drowning death, but not significant in determining the dissolved solids) and sodium while Nitzschia palea is exact site of drowning. Similarly, rarely occurring species negatively co-related to TDS. Lepocinclis ovum and in Braham Sarovar (25 species) and Prithus pools (20 Nitzschia palea are positively correlated to potassium species) are not forensically significant. The site-specific while Coelastrum microporum and Coelastrum astroi- species in Braham Sarovar (4 species) and Prithus pools deum are positively correlated to calcium (Fig. 5). (4 species) are forensically significant in determining the PCA for Braham Sarovar has been drawn by taking in to exact/putative site of drowning. The P-maps with a list account 10 physico-chemical parameters and 14 dominant of common and site-specific phytoplanktons have been species of phytoplankton. The PCA axis 1 explained shown in Tables 4 and 5. The P-map generated of phyto- 50.14% and axis 2 explained 29.03% variations in phyto- plankton species of two water bodies can help in deter- plankton and physico-chemical parameters. Aulacoseira mining not only cause of death, but also helps in linking granulata, Tetraedron minimum, Gloeocapsa minimum, the suspects to a crime scene. Nitzschia palecea, and Coelastrum microporum, is posi- tively correlated to temperature whereas Microcystis aeru- Conclusions ginosa and Synechococcus sp. are negatively correlated. In the present study, phytoplankton data have been generated Chroococcus dispersus and Aphanothceae sp. have been from the season-wise analysis of two water bodies: Braham Table 9 Physico-chemical Parameters of Braham Sarovar Braham Sarovar Lake Season of pH EC TDS HARDNE ALKALINITY SODIUM POTASIUM CHLORIDE CALCIUM WATER TEMPERTU sampling 0 (μs/cm) (mg/l) SS (mg/L) (mg/L) ppm ppm (mg/L) (mg/L) RE ( C) Summer 6.2 85 179 90 20 4.6 0.8 32 54.54 35.3 Autumn 7.6 96 170 110 70 4.2 1.2 25 48.9 29.8 Winter 7.2 98 155 89 76 4.8 0.8 28 44.2 17.5 Spring 6.9 82 184 86 48 3.2 0.6 39 27.8 33.7 Average 6.9 ± 0.59 90.25 ± 7.9 172 ± 12.7 93.7 ± 10.09 53.5 ± 25.3 4.2 ± 0.7 0.8 ± 0.2 31.0 ± 6.0 43.8 ± 29 29.0 ± 8.0 Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 13 of 15 Fig. 5 Principal Component Analysis of physico-chemical parameters and dominant species of Prithus Pool Fig. 6 Principal Component Analysis of physico-chemical parameters and dominant species of Braham Sarovar Thakar et al. Egyptian Journal of Forensic Sciences (2018) 8:38 Page 14 of 15 Sarovar and Prithus pools of the historically important cities Canini ND, Metillo EB, Azanza RV (2013) Monsoon-influenced phytoplankton community structure in a Philippine mangrove estuary. Trop Ecol 54:331–343 (Kurukshetra and Pehowa) of Haryana State, for two years Chardez D, Lambert J (1985) Protozoaires ciliés et thanatologie. Forensic Sci Int continuously. The data analysis differentiated phytoplankton 28:83–101 into common, rare, and site-specific species in order to con- Cumming BF, Smol JP (1993) Development of diatom-based salinity models for paleoclimatic research from lakes in British Coloumbia (Canada). Twelfth struct phytoplankton-map. The P-maps generated for the International Diatom Symposius. Hydrobiologia. Springer, Dordrecht, pp 179- two water bodies of Haryana state, India, are expected to be 96, vol. 269 useful to forensic experts and forensic medicine experts in Desikachary TV (1959) Cyanophyta. Indian Council of Agricultural Research, New Delhi solving drowning cases and unfolding mysteries related to Díaz-Palma P, Alucema A, Hayashida G, Maidana N (2009) Development and the putativesiteofdrowning, especially when diatoms are standardization of a microalgae test for determining deaths by drowning. present in very less number during a particular season. In Forensic Sci Int 184:37–41 Figueredo CC, Giani A (2009) Phytoplankton community in the tropical lake of addition, the data has been successfully used to find the de- Lagoa Santa (Brazil): conditions favoring a persistent bloom of gree of correlation between different environmental parame- Cylindersospermosis raciborskii. Limnologica 39:264–272 ters and phytoplanktons prevailing in a water body with the Hall DW (1997) Forensic botany. Forensic Taphonomy: the postmortem fate of human remains. CRC Press, Inc., Boca Raton help of Principal Component Analysis method. Khuantrairong T, Traichaiyaporn S (2008) Diversity and seasonal succession of the phytoplankton community in Doi Tao lake, Chiang Mai Province, northern Abbreviations Thailand. Tropical. Nat Hist 8:143–156 APHA: America Public Health Association; D: Species richness; EC: Electrical Kim YS (2011) Research for seasonal plankton distribution of in-land water in conductivity; H: Shannon and weaver diversity index; J: Evenness; N: Total Gwang-ju Area.Korean. J Legal Med 35:16–21 number of individuals in the sample; n: Total number of species in the Komárek J, Fott B, Huber-Pestalozzi G (1983) Das Phytoplankton des Süßwassers. sample; PCA: Principal component analysis; P-map: Phytoplankton map; Systematik und Biologie-Teil 7, 1. Hälfte. S: Total number of species in the sample; TDS: Total dissolved solids Ludes B, Coste M, North N, Doray S, Tracqui A, Kintz P (1999) Diatom analysis in victim’s tissues as an indicator of the site of drowning. Int J Legal Med 112: Acknowledgements 163–166 Authors are thankful to U.G.C, New Delhi for providing U.G.C NET/JRF Lunetta P, Miettinen A, Spilling K, Sajantila A (2013) False-positive diatom test: a fellowship to one of authors, Deepali luthra to carry out this research. real challenge? A post-mortem study using standardized protocols. Legal Med 15:229–234 Funding Lunette P, Modell JH (2005) Macroscopical microscopical and laboratory findings U.G.C NET/JRF fellowship. in drowning victims. In: Tsokos M. (eds) Forensic Pathology Reviews, vol. 3. 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Egyptian Journal of Forensic SciencesSpringer Journals

Published: May 29, 2018

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