International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 49 Brachyuran Crabs as a Biomonitoring tool: A Conceptual Framework for Chemical Pollution AssessmentArya S., Trivedi J.N. and Vachhrajani K.D. Marine Biodiversity and Ecology Lab, Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara- 390002, Gujarat, INDIA Available online at: www.isca.in, www.isca.me Received 22nd November 2013, revised 18th December 2013, accepted 5th January 2014 AbstractMarine ecosystem is very complex in terms of biological and ecological structure and marine organisms are integral part of this structure. In the recent era marine ecosystem has become largest dumping site for effluents of industries and it is creating serious problems at ecosystem level. So the understanding of different direct and indirect sources of pollution and its effect on marine organism population and ecosystem have got lot of ecological importance for conservation. Brachyuran crabs are highly diverse organisms comprising 700 genera and 5000 species. Crabs play important role in the maintenance of ecosystem and also in the marine food web demonstrating prey- predator relationship. The present study provides a protocol or framework to find out the effect of pollution on marine ecosystem using brachyuran crab as a bio indicator species. The present framework was divided into two different aspects viz ecological assessment and toxicological assessment and different methods have been suggested to assess the effect of pollution on brachyuran crabs for both aspects. To check the effectiveness of the framework, pilot study has been done and the results revealed that pollutants do affect different levels of ecosystem and the effect can be traced using brachyuran crab as a bioindicator species. Key words: Brachyuran crabs, pollution, ecotoxicology conceptual framework, bio indicator, Gujarat coast. IntroductionThe marine pollution is mainly contributed by industrial, domestic and agricultural wastes. Effluents, particularly from the industries containing hazardous heavy metals and organic toxicants have posed threat to the marine life. Several heavy metals accumulate within the tissues of the crustaceans at a concentration higher than that is present in the water column and sediment, and such biomagnification results into environmental toxicity. Thus for conserving the inhabiting marine species and to prevent the marine environment from further devastation, certain authentic and sensitive monitoring methodologies must be constructed. There are certain monitoring systems that include the chemical analysis of the abiotic factors such as sediment and water which prove to be accurate enough; but these may not provide the actual ecological state for many reasons. One, the number and range of the pollutant may exceed the capability of the available chemical test. Second, if the pollutants act synergistically, it may become difficult to measure them directly. And finally, the natural availability of the suspected factors that also contribute to the environmental damage cannot be confirmed by chemical analysis. These problems are overcome by bioindicator systems where the physical and physiological responses of organisms that we select are used for monitoring the environment. Bioindicators are the organisms or group of organisms whose interactions are observed to predict the situation of particular system. The bioindicator species for their growth and survival require specific kind of set of environmental variables and changes in such kind of variables, directly affect the number, reproduction and behavior of the species, which implies that changes in the variables are not suitable for the species. The organism to be used as bioindicator has to fulfill many criteria that include ease in sampling, temporal and spatial abundance of the organism and the range in which the biological responses can be detected . There are many species of bivalves and gastropod molluscs that are used as bioindicators6,7. There are many crustacean species also that are used as bioindicators to test the toxicological impacts. The parameters taken into account include percent survival, growth rate of populations, number of adults reaching reproductive age, number of larvae released by females and numbers of progeny surviving. Amongst different crustacean invertebrates, crabs are highly diverse group comprising 5000 species and 700 genera. The species distributed on diverse habitat play important role in the maintenance of the ecosystem. So any problematic variable which affects the crabs can have major effect on the habitat and ecosystem8-11. Studies have been done to assess the health of the particular ecosystem affected by different kinds of pollutions using brachyuran crabs as an indicator species12-17. In Gujarat, various mega industries have been set up in the Gulfs of Kutch and Khambhat as well as along the Saurashtra International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 50 coast18 and there is an immense anthropogenic pressure that affects the marine life. The Common Effluent Treatment Plant (CETP) discharges the treated polluted effluent directly into the sea waters. The CETPs deal about 2000 SSI units in Maharashtra and 2500 SSI units in Gujarat states respectively. The effluent released contains high amount of heavy metals and organics. Heavy metal from the effluent eventually biomagnifies in the estuarine system and gets deposited in sediments and benthic animal tissues. Recently attempts have been made to investigate and develop pollution bioindicator systems19. We carried out some basic ecotoxicological studies in gulf of Khambhat and part of coastal Saurashtra, particularly analyzing the abiotic components. During these studies we realized that macro faunal bioindicators are yet to be explored and studied in Gujarat. In present paper we discuss the basic frame work essential for such faunal indicator studies. Since we have carried out pilot studies on brachyuran crabs, the flagship species of estuarine and inter tidal areas that manipulate and modify the abiotic and biotic status of these communities, in present paper we project brachyuran crabs as prospective bioindicators of coastal pollution. The Concept: The benthic invertebrates show peculiar habitat and substratum preference and thus are indicator of sediment quality. Similarly, water quality also regulates the diversity, abundance and life processes of invertebrates. Macro invertebrates are keystone species in the benthic environment because they play critical role in different functions of the community. Therefore, the diversity, distribution, density, behavior, reproduction, larval settlement and population dynamics of macro invertebrates of the benthic zone can denote whether or not the community is stressed20. Why use crabs?Crabs are the vital component of the marine food chain. Most of the crabs feed on either detritus or the organic matter thus controlling the ecological functioning. In inter tidal area, these organisms work as a super creatures adapting to the harshness of the environment. On the coastal intertidal areas, they are acted upon by variety of environmental factors like desiccation stress, temperature, predation and change in salinity etc. Moreover, the daily tidal fluctuations imply flux in water quality and sediment depositions. These are probable causes of broad scale community pattern variations spatially and temporally. The sediment deposition, grain composition and variations on a diurnal, seasonal and annual basis define the micro habitat conditions21. On inter tidal area, these variations are noted vertically, horizontally and laterally. Tidal fluctuation and changes in water quality influence diversity and distribution of pelagic larval stages of benthic forms22. Tidal variations induce site selection and abundance of benthic forms along inter tidal area23. Crabs play important role in the maintenance, modification and regulation of the benthic environment by influencing both the abiotic and biotic components. They are abundant and serve both as the predator and the prey and hence are located at different trophic levels in the ecosystem. The density of the crabs in mangrove ecosystems can go up to a level of 80-90 crabs per sq. m. Many species of crabs are burrowing in nature and with their burrowing activity they frequently alter the surface characteristics and drive the nutrient cycling25. In mangrove ecosystem, burrowing activity of crabs increase the porosity of the soil which on the other hand increase the regeneration of mangrove seedlings24. The feeding activity and pellet formation by different species of feedler crabs also changes the substratum characteristics and content of organic matter. The bioturbatory processes by crabs on the intertidal area results into variety of biogenic structures and markings. These structures and processes control variety of processes which are important for health of an ecosystem. Even a routine activity of burrowing facilitates series of chemical and physical alterations in the original sediment quality. The facilitation of oxygen to the subsurface depth allows microbial diversity and sediment oxic-anoxic zoning. The feeding activity by scraping the upper organic rich layer of the sediments regulates the organic content and the algal covering and on the contrary byproducts from gut enriches the sediments with few minerals. The multifaceted role of crab in the ecosystem is presented in figure 1. How do crabs fit as bio indicator?Wide range of studies is available on macro invertebrates as an indicator species of aquatic habitat but amongst them specifically, brachyuran crabs are an effective indicator of different changes in both abiotic and biotic factors. Studies have been done with references to changes in different parameters and its effect on crab. Shirley et al.26studied that species of oyster reef crabs live in strict range of salinity and any change in salinity due to presence of any kind of pollutant directly affect the population of the crabs. Pandya25 has also studied the population distribution of brachyuran crab Uca lactea annulipesin Mahi estuary, the results of the study revealed that population density of the particular species was very less at polluted site as compare to the non polluted site. The morphology and physiology of the species also shows the direct effect of pollution. Bergey and Weis14 studied the morphology of fiddler crab Uca pugnax and stated that body size of male and female were significantly greater in sites which were polluted as compared to the sites that were non polluted and on the other hand the population of crab in polluted area was low when compared with the non polluted. The burrowing crabs are mostly filter feeder and they feed on the substratum available. Beltrame et al.17 have used the brachyuran crab Neohelice granulate as an indicator species to study the presence of any heavy metals in estuaries of Atlantic coast of Arghentina. Al-Mohanna and Subrahmanyam27 used Portunus pelagicus as pollution indicator from the Gulf of Kuwait wherein an associated problem of heavy metals along with the oil spill of Gulf War was found. Turoczy et al.28 have given reports of the International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 51 crab Pseudocarcinus gigas showing higher cadmium concentrations from Australia. Results of all these studies suggest that brachyuran crabs have potential to be used as a bioindicator species. Brachyuran crabs have number of advantages as compared to other macro benthic species to be used as a bioindicators. i. Crabs prefer specific kind of substratum to live and if the substratum is polluted by any kind of pollution then it will have effect on crab population so by studying the crab population one can make out the health of the substratum, e.g. rock crabs29. ii. The burrowing crabs are mostly filter feeder or detrivore while crabs of rocky shores are mostly herbivore or carnivore. If the pollutant is present in the substratum, plant and animal body which is food item then it may have effect on feeding behavior of crab. iii. Crabs not only show evidence of environmental features such as salinity and sediment types and content, but they also respond to anthropogenic factors, making them key bioindicators of human impact on benthic communities15. iv. Crab also shows the effect of heavy metal toxicity on the reproductive cycle and different kinds of behavior17. Proposed Studies for Pollution Assessment in Bioindicator SpeciesThe marine organisms when exposed to the toxicants for a longer duration of time accumulate the toxicants in their tissues. The process of metal accumulation in the tissues and organs is species-dependent and hence we find different organisms in the same environment having different concentrations of metals in them30,31,32. Thus there is a need to determine the toxicant concentration in the animal tissues, in this case, crabs. There are different methodologies that are carried out experimentally for the determination of such toxicants (figure 2). Figure-1 Role of brachyuran crabs in the ecosystem International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 52    Toxicological study Ecological study Animal tissue Water analysis Crabs Crab collection Morphometry Laboratory analysis Chemical analysis Elemental analysis Quantitative analysis Emission transmission method Heavy metal analysis Standard procedure APHA Chemical aspect Sediment analysis Distribution Density Intertidal distribution Zonewise distribution Belt-transect method Quadrate method Species specific study Burrow Morphometry External Density (Quadrate method) Opening diameter Distribution Internal Burrow casting X-ray and CT scan Habitat preference Field observation, Photography Feeding area e xploration Photo quadrate Method and measurement Bio turbation Burrow and feeding pellets soil Weighing (on field) Figure-2 Framework of the proposed ecotoxicological studies using crab as pollution indicator Collection of the specimen and taxonomic identification: The crab specimen be collected from the study area by hand, rinsed to remove sediment particles, placed in specimen jars, labeled and kept in ice for transportation33,34. Identification and further analysis be done in the laboratory. Specimen having similar size belonging to each species be categorized for analyzing metals within the tissues35. Morphometric analysis including weighing, measurements of carapace size etc. and length of the chelipede (using vernier calipers) should be done34. The crab samples should be dried at 105-110°C in an oven33-36. The percentage dry matter of the samples can be calculated by using the formula33: Percent dry matter = (W3-W1) / (W2-W1) X 100 Assessment of the Water Quality Water quality is an important factor that is responsible for the toxicant accumulation in the marine organisms. Water samples should be collected using Nikson bottle from the surface. A two liter capacity bottle can be used for sampling and for physical and chemical analysis. The heavy metals in the samples be analyzed by the methods described below for toxicant analysis in crabs. Assessment of the Sediment QualitySediment samples should be collected by using a polyethylene corer, by following standardized methodologies. Samples can be digested in the microwave vessels, acid cleaned; with a standardized mixture of hydrofluoric acid and nitric acid37. The heavy metals in the samples can be analyzed by the methods described below for toxicant analysis in crabs. Assessment of Toxicants in Crabs Microwave acid digestion of dried samples should be done by using a standardized chemical combination of concentrated nitric acid and hydrogen peroxide33,36,38,39. There are various analytical methods that can be applied for determination of concentration: i. The heavy metal content of the crabs collected can be determined by using Energy Dispersive X-Ray Fluorescence (EDXRT) Technique34. ii. For most heavy metals; analysis can be done by using Inductively Coupled Plasma – Mass Spectrometer (ICP-MS) instrument33. iii. Particularly for Fe, techniques including Inductively Coupled Plasma – Emission Spectrometer (ICP-ES) can be used33. iv. An Open tube block digestion method for determining Arsenic can be used. This includes digestion of the dried specimen sample in a concentrated mixture of nitric acid and perchloric acid. Analysis International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 53 can be done by using VG/ICP-MS; where VG is Vapour Generation33. v. In wet digestion method, to determine the heavy metals concentrations: Cd, Cu, Zn, Pb; analysis can be done by using an atomic absorption spectrophotometer GBC-AVANTA36,40. vi. For Hg metal, analysis can be done by Atomic absorption spectrophotometer (Perkin Elmer)39. Quantitative analysis: Quantitative analysis of the pollutants in the samples should be done by using the Emission Transmission (E-T) method, which is already having a number of quantification methods, previously applied41-46. Studies of Physiological Indicators in Crabs: The physiological indicators include metabolites and enzymes associated with important functions like energy production. The metals and other pollutants are reported to induce oxidative stress as one of the mechanism of action. We propose to study Succinate dehydrogenase47, acid phosphatase48 cholesterol49, superoxide dismutase50, glutathione peroxidase51, glutathione- s-transferase52, oxidative degradation of lipid53, reduce glutathione54, ascorbic acid55 and steroidogenic parameters in various tissues of crabs. Studies of Behavioral Indicators in Crabs: The activities of crabs exhibit distinct behavioral processes. The burrowing, bioturbatory, feeding, territoriality, inter and intra specific interactions, mating displays and reproduction8,9, larval settlement and juvenile establishment etc activities and processes are integral life processes of crabs and also significant behavioral responses to the pollution. These activities will be observed in polluted and reference non polluted regions and compared for selected crab species which will serve as indicators. The burrowing and bioturbatory activities should be studied through C T Scan X-ray studies of cores and resin burrow casts. All other activities will be studied in situ by visual observations and video recordings. Studies carried out so far: Studies were carried out in the polluted region of Gulf of Khambhat, as well as part of Saurashtra coast along Junagadh district, Gujarat where the crab species were considered as bioindicators. Study area: Gujarat, the leading industrialized state, has longest coast line among Indian states and extensive industrialization is further proposed. The Gulf of Khambhat is very specific in studying sedimentology as well as water quality because of its geomorphology and hydrodynamics. During low tide vast area remain open for extended period. A channel brings the industrial effluent in this region from the industrial area of Vadodara. The general and ecotoxicological hazards of such industrial effluent and its components have been studied in our laboratory56-60. The habitat characterization and distribution of brachyuran crab have been studied in the Gulf of Khambhat estuaries that demonstrate differences in crab distribution and diversity in the polluted and non polluted regions61-63. Saurashtra region is located in the south west part of the state and has been attractive location for industrial and associated infrastructure development. The coastal zone has a cliffed shoreline and a flat coastal plain dotted with milioite limestone. The Saurashtra intertidal region is primarily rocky in nature having upper margins of the sandy shore. This intertidal area is narrow in width, has lesser slope gradient, high tidal currents and very rich in marine biodiversity64,67. The present study area is selected on the basis of presence of different kinds of industries, pollution level, habitat type and marine fauna. Our recent studies demonstrated occurrence of several brachyuran and anomuran crab species in the widely diverse habitats of Gulf of Kachchh and on Saurashtra coast65-68. Veraval is amongst the largest fish landing sites of India. Veraval is surrounded by different kind of small and large industries like Rayon industry and fish processing units. The place is also known for boat manufacturing and the coastal area receives different kinds of waste from these units. Sutrapada is a small village which is situated 30 km from Veraval. The place harbors a large soda ash manufacture unit and the coastal water receives wide variety of chemical pollutants. Kodinar is situated 45 km south from Vearval and has largest cement manufacture unit in the state while Alang is the world’s biggest ship breaking yard. Till now, around 70% of ships are sent to the Alang-Sosiya ship recycling yard69. During the breaking or dismantling of the ship the coastal water receives different kinds of wastes including all solids, liquids and gaseous wastes. The study areas were surveyed during 2010 to 2012 for habitat categorization and crab sampling. The samples were collected for water and sediment analysis and several specific studies like sediment composition, quantitative faunal studies, burrow casting, radiological studies, behavioural studies etc. Water, sediment, crabs and plant samples were collected from different stations along the Mahi estuary from Gulf of Khambhat. Sediment samples were taken up to 15 inch depth. Samples were analyzed for their heavy metal content using Atomic Absorption Spectrophotometer (tables- 1, 2, 3). Table-1 Pollutant analysis in water samples from Mahi estuary Stations C. O. D. mg/l B. O. D. mg/l Oil & Grease mg/l Cd Co Cu Cr Ni Pb Sarod-1 780 230 38 18 7 6 10 16 21 Sarod-2 436 107 26 5 1 3 1 3 6 Kamboi-1 189 86 24 0 0 1 2 4 3 Kamboi-2 123 45 18 3 0 3 0 2 3 International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 54 Table-2 Pollutant analysis in sediment samples from Mahi estuary Station C. O. D. B. O. D. Oil and Grease Cd Co Cu Cr Ni Pb mg/g mg/g mg/g ΅g/g ΅g/g ΅g/g ΅g/g ΅g/g ΅g/g Sarod-1 0.36 1 0.02 50 50 18 10 12 80 Sarod-2 0.11 0.5 0.06 11 0 20 9 7 50 Kamboi-1 0.02 0.4 0.08 0 0 15 8 20 15 Kamboi-2 0.01 0.5 0.03 0 0 10 7 6 30 Table-3 Heavy metal analysis in crab, plant, effluent and sediment samples from Mahi estuary Sample Zn Fe Co Cr Ni Pb Cd ppm ppm ppm ppm ppm ppm ppm Sample-1 crab tissue 0.459 34.421 0.023 1.088 0 0.017 0 Sample-2 crab tissue 0.39 13.489 0.02 4.324 0 0.032 0.134 Sample-3 vegetation 0.317 123.895 0.044 7.919 0.012 0.01 0.015 sample-4 effluent 0.036 0 0.067 0 0 0.026 0 Sample-5 sediment 1.419 882.597 0.316 0.278 4.726 0 11.92 Note: Values for all heavy metals are in ppm, “0” indicates Values below detection limit Conclusion The results revealed that the heavy metals which are present in the effluent of industries are accumulated in the ecosystem at different levels like water, sediments, vegetation and animal tissue. The results suggest that crabs are prone to heavy metal accumulation. The sources of the heavy metal accumulation in crabs are water and sediment. The heavy metal accumulation and other pollution ingredients the marine environment do affect the ecology, physiology and behavior of marine organisms and crab could be used as an indicator species to reveal the effect of pollution on biota. The reasons for use of crab as an bio indicator species are: i. Crabs are the most important organisms to be used as bio indicators because of their huge diversity and density, ii. Crabs play important role in maintenance of different marine ecosystems so any factor affecting their health will cause damage to whole ecosystem, iii. Crabs as a prey and predators play important role in marine food web so tracing of pollutant in food chain will be very easy using crabs. iv. Crabs also show peculiar preference to micro habitats. So health of different habitats can be predicted using crabs, v. The toxicological assessment at different levels of biological organization with special reference to crabs strongly increases the sensitivity of pollution assessment in the concerned area. References1.Kasmin S., Enforcing Ship-Based Marine Pollution for Cleaner Sea in the Strait of Malacca, Environment Asia, 3, 61-65 (2010) 2.Rainbow P. S., Trace metal bioaccumulation: Models, metabolic availability and toxicity, Environment International,33, 576–582 (2007) 3.Bresler V., Abelson A., Fishelson L., Feldstein T., Rosenfeld M. and Mokady O., Marine molluscs in environmental monitoring, Helgol mar. res.,57, 157–165 (2003) 4.Phillips D. J. H. and Rainbow P. S., Biomonitoring of trace aquatic contaminants, Chapman and Hall, London, (1994) 5.Hellawell J. M., Biological Indicators of Freshwater Pollution and Environmental Management, Applied science publishers, London, (1986) 6.Boening D.W., An evaluation of bivalves as biomonitors of heavy metals pollution in marine waters, Environmental Monitoring and Assessment, 55, 459–470 (1999) 7.Davies-Coleman H. D. and Palmer C. G., The use of a freshwater mollusc, Burnupia stenochorias (Ancylidae) as an ecotoxicological indicator in whole effluent toxicity testing, Proceedings of the water institute of southern Africa (WISA) Biennial Conference, (2004) 8.Strachan P. H., Smith R. C., Hamilton D. A. B., Taylor A. C. and Atkinson R. J. A., Studies on ecology and behaviour of the ghost crab Ocypode cursor (L.) in northern Cyprus, Scientia marina, 63, 51-60 (1999) 9.Siddon C. E. and Witman J. D., Behavioral indirect interactions: Multiple predators effects and prey switching in the rocky subtidal, Ecology, 85, 2938-2945 (2004) 10.Pandya P. J. and Vachharajani K. D., Life under ecological stress: An estuarine case study. In: Animal Diversity, Natural History and Conservation (Gupta VK, Verma AK, eds). Daya Publication House, New Delhi, India, 1, 427-436 (2011) International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 55 11.Trivedi J. N., Gadhavi M. K. and Vachhrajani K. D., Diversity and habitat preference of brachyuran crabs in Gulf of Kutch, Gujarat, India, Arthropods, 1, 13-23 (2012) 12.Cunningham P. A. and Myers L. E., Effects of Diflubenzurin (Dimilin) on Survival, Molting, and Behavior of Juvenile Fiddler Crabs, Uca pugilator, Archives of environmental contamination and toxicology, 16, 745-752 (1987) 13.Culbertson J. B, Vahiela I., Peacock E. E, Reddy C. M, Carter A, and Vanderkruik R., Long-term biological effects of petroleum residues on fiddler crabs in salt marshes, Marine pollution bulletin, 54, 955-962 (2007)14.Bergey L. L. and Weis J. S., Aspects of population ecology in two populations of fiddler crab, Uca pugnax. Marine Biology, 154, 435-442 (2008)15.Bartolini F., Penha-Lopes G., Limbu S., Paula J. and Cannicci S., Behavioral Responses of the mangrove fiddler crabs (Uca annulipes and U. inversa) to urban sewage loadings: Result of a mesocosm approach. Marine pollution bulletin, 58, 1860-1867 (2009)16.Oosterom J., King S.C., Negri A., Humphrey C. and Mondon J., Investigation of the mud crab (Scylla serrata) as a potential bio-monitoring species for tropical coastal marine environments of Australia, Marine pollution bulletin,60, 283-290 (2010)17.Beltrame M.O., Marco S.G.D. and Marcovecchio J.E., The burrowing crab Neohelice granulata as potential bioindicator of heavy metals in estuarine systems of the Atlantic coast of Argentina, Environ monitor assess, 172, 379–389 (2011)18.Bhadja P. and Kundu R., Status of the seawater quality at few industrially important coasts of Gujarat (India) off Arabian Sea, Indian journal of geo-marine sciences, 41, 90-97 (2012)19.Mohandass C., Nair S., Achuthankutty C.T. and Loka Bharathi P.A., Pollution monitoring of coastal and estuarine areas: I. Bacterial indicators along the south Gujarat coast, Indian journal of marine sciences, 29, 43-47 (2000) 20.Pandya P.J. and Vachhrajani K.D., Predicting vulnerability of estuarine brachyuran taxa using animal-sediment relationship as an effective tool, Student Conference on Conservation Science, University of Cambridge, Cambridge, UK, (2009)21.Johannesson L.T., Stevens R.L. and Alexanderson J.H., Sediment character in a micro-tidal, harbor-estuary environment, Goteborg, Sweden, Estuaries, 23(3), 400-410 (2000) 22.Levin L. A., Boesch D. F., Covich A., Dahm C. and Erseus C., The function of marine critical transition zones and the importance of sediment biodiversity, Ecosystems, 4, 430-451 (2001) 23.Talley T. S., Dayton P. K. and Obando S. E., Tidal flat macrofaunal communities and their associated environments in estuaries of Southern California and Northern Baja California, Mexico, Estuaries, 23, 97-114 (2000) 24.Khan A. S., Raffi S. M., Lyla P. S., Brachyuran crab diversity in natural (Pichavaram) and artificially developed mangroves (Vellar estuary), Current Science, 88, 1316-1324 (2005) 25.Pandya P. J., Benthic community structure of Mahi River estuary with special reference to animal-sediment relationship. Ph. D. Thesis, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India, (2011) 26.Shirley M., McGee V., Jones T., Anderson B., and Schmid J., Relative abundance of stenohaline and euryhaline oyster reef crab populations as a tool for managing freshwater inflow to estuaries, Journal of coastal research,45, 195-208 (2004) 27.Al-Mohanna S. Y. and Subrahmanyam M. N. V., Flux of heavy metal accumulation in various organs of the intertidal marine blue crab, Portunus pelagicus (L.) from the Kuwait coast after the Gulf War, Environment International, 27,321–326 (2001)28.Turoczy N. J., Mitchell B. D., Levings A. H., and Rajendram V. S., Cadmium, copper, mercury and zinc concentrations in tissues of the king crab (Pseudocarcinus gigas) from southeast Australian waters, Environment international, 27, 327–334 (2001)29.Wong M. C., Wright L. D. and Barbeau M. A., Sediment selection by juvenile sea scallops (Placopecten magellanicus (Gmelin)), sea stars (Asterias vulgaris verrill) and rock crabs (Cancer irroratus say). Journal of shellfish, 25, 813-821 (2006)30.Ritterhoff J. and Zauke G. P., Trace metals in the field samples of zooplankton from the Fram Strait and the Greenland Sea, Sci. Total environ., 199, 255 (1997)31.Kahle J. and Zauke G. P., Trace metals in Antarctic copepods from the Weddell Sea (Antarctica), Chemosphere, 51, 409 (2003) 32.Prowe F., Kirf M. and Zauke G. P., Heavy metals in crustaceans from the Iberian deep sea plain, Sci. mar.,70,271 (2006) 33.Andersen L. E. and Melzer A., Intertidal crabs as potential biomonitors in Port Curtis, Centre for environmental management, Central Queensland University, Technical report 12 (2002) 34.Abolude D. S., Davies O. A. and Avong D. W., Level of Heavy Metals in Freshwater Crab (Cardisoma guahumi) obtained from Ahmadu Bello University Reservoir, Zaria Nigeria, International journal of animal and veterinary advances,1, 54-58 (2009) International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 56 35.Mitra A., Barua P., Zaman S. and Banerjee K., Analysis of trace metals in commercially important Crustaceans collected from UNESCO protected world heritage site of Indian Sundarbans, Turkish journal of fisheries and aquatic sciences,12, 53-66 (2012)36.Stanciu G., Mititelu M. and Gutaga S., Pesticides and heavy metals determination in marine organisms from Black Sea, Chem. bull. "Politehnica" univ. (Timioara), 50(64), 1-2 (2005) 37.Abida B., HariKrishna S. and Khan I., Analysis of heavy metals in water, sediments and fish samples of Madivala lakes of Bangalore, Karnataka, Int. J. chem. tech. res.,, 245-249 (2009)38.Hsiao-Chien Y., Chen I. M., Chen P. and Wang W. H., Heavy metal concentrations of the soldier crab (Mictyris brevidactylus) along the inshore area of Changhua, Taiwan, Environ monit assess, 153, 103-109 (2009)39.Mukke V. K., Bioaccumulation study of mercury chloride in selected tissue of fresh water crab, Barytelphusa guerinifrom Aurangabad region, J. chem. pharm. res.,, 398-401 (2012) 40.Kumar K. A. and Achyuthan H., Heavy metal accumulation in certain marine animals along the East Coast of Chennai, Tamil Nadu, India, Journal of environmental biology, 28,637-643 (2007) 41.Giauque R.D., Garreth R.B. and Goda L.Y., Determination of trace elements in light element matrices by X- ray fluorescence with incoherent scattered radiation as an internal standard, Anal. Chem., 51, 512-516 (1979) 42.Markowicz A. A., A method for correction for absorption matrix effects in samples of intermediate thickness in EDXRF analysis, X – ray Spectrum, 8, 14-18 (1979) 43.Tang S.M., Kump P., Yap C.T. and Bilal M.G., Calculation of relative fluorescence intensity for annular source geometry by Monte Carlo method, X- ray Spectrum, 15, 289-293 (1986) 44.Markowicz A. A. and Van Grieken R. E., Quantification in XRF Analysis of Intermediate Thickness Samples. In: Handbook of X- ray Spectroscopy, R. E. Van Grieken and A.A. Markowicz. (Eds), Marcel Decker Inc. New York,339 – 358 (1993) 45.Bernasconi G.B., Bamford S.A., Dosan B., Haselberger N., Markowicz A., Mahmoud A. and Valcovic V., Applicability of annular source excited systems in quantitative XRF analysis, X- ray Spectrum, 23, 65–70 (1996) 46.Kump P., Quantitative Analysis of Environmental Samples , Instruction Manual, Ljubljana, (1996) 47.Beatty C.H., Basinger G.M., Dully C.C. and Bocek R.M., Comparison of red and white voluntary skeletal muscles of several species of primates, J. histochem. Cytochem.,14, 590-600 (1966) 48.Bessey O.A., Lowry O.H. and Brock M.J., A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum, J boil. Chem., 164, 321-329 (1946)49.Pearson S., Stern S. and McGavak T.H., Rapid, accurate method for the determination of total cholesterol in serum, Anal Chem., 25, 813-814 (1953)50.Marklund S. and Marklund G., Involvement of the superoxide anion radical in the auto oxidation of pyrogallol and a convenient assay for superoxide dismutase, Eur. j. biochem, 47, 469-474 (1974) 51.Rotruck J. T., Pope A. L., Ganther H. E., Swanson A. B., Hafeman D. G. and Hoekstra W. G., Selenium biochemical role as component of glutathione peroxidase, Science, 179588-590 (1973) 52.Habig W. H., Pabst M. J. and Jakoby W. B., Glutathione -s-transferases: the first enzymatic step in mercapturic acid formation, J biol chem, 249, 7130-7139 (1974)53.Buege J.A. and Aust S.D., Microsomal lipid peroxidation, Meth enzymol, 52, 302–310 (1978)54.Beutler E., Duron O. and Kelley B. M., Improved method for the determination of blood glutathione, J. lab. clin. Med.,61, 882-888 (1963) 55.Roe J.H. and Kuether C.A., The determination of ascorbic acid in whole blood and urine through the 2, 4-dinitrophenyl hydrazine derivatives of dehydro ascorbic acid, J. biol. chem, 147, 399-407 (1943) 56.Vachhrajani K.D., Makhija S., Chinoy N.J. and Roy Chowdhury A., Structural and functional alterations in the testis of rats after mercuric chloride treatment,J Reprod Biol Comp Endocrinol,8, 97-104 (1988) 57.Nanda A., Vachhrajani K. D and Mankodi P.C., Crustacean community structure of Mahi River receiving industrial effluent, Poll Res.,24, 243-246 (2005)58.Vachhrajani K.D. and Verma S., Impacts of Industrial pollution on the status of the lentic ecosystem, Elec J Environ Sci., 3, 15-21 (2010)59.Morya K. and Vachhrajani K.D., Low dose heterogeneous chemical mixture exposure for the duration of one seminiferous epithelial cycle Impairs spermatogenesis and induce oxidative stress in rat testis,J Cell Tissue Res., 11,2899-2908 (2011)60.Patel B.K. and Vachhrajani K.D., Pollution status in mangrove ecosystem of Mahi and Dadhar river estuaries, Proceedings of National Conference on Biodiversity: Status and Challenges in Conservation, 163-172 (2013) 61.Pandya P.J. and Vachhrajani K. D., Spatial distribution and substratum preference of brachyuran crab Macrophthalmus International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(1), 49-57, January (2014) Int. Res. J. Environment Sci. International Science Congress Association 57 depressus (Decapoda: Ocypodidae) along the lower estuarine mudflats of Mahi River, Gujarat, India,Crustaceana 83, 1055-1067 (2010)62.Pandya P.J and Vachhrajani K.D., On the diversity of brachyuran crabs from lower estuarine mud flats of Mahi River with new record of two species (Decapoda: Brachyura) from Gujarat, India, Arthropods, 2(4)242-250(2013) 63.Shukla M. L., Patel B. K., Trivedi J. N. and Vachhrajani K. D. Brachyuran crabs diversity in mangrove habitat of Gulf of Khambhat, Gujarat, India, Research Journal of Marine Sciences,1(2), 8-11 (2013) 64.Trivedi J.N. and Vachhrajani K.D., Distribution and diversity of brachyuran crabs along the coastal region of Junagadh district, Gujarat, Proceedings of the Biodiversity and Conservation of Coastal and Marine Ecosystems of India,8-14 (2012) 65.Trivedi J.N. and Vachhrajani K.D., First record of Cryptopodia angulata (H. Milne Edwards & Lucas, 1841) from Saurashtra coast, Gujarat, India (Decapoda, Brachyura, Parthenopidae), Check List, 9(4), 897-898 (2013) 66.Trivedi J.N. and Vachhrajani K.D., First record of two porcellanid crabs from Gujarat state, India (Crustacea: Decapoda: Porcellanidae), J. Mar Biol Assoc India, 55(1), 55-58 (2013) 67.Trivedi J.N. and Vachhrajani K.D., Study of intertidal distribution of Cerithium scabridum, Philippi, 1848 (Mollusca, Gastropoda) along the coastal Saurashtra Gujarat, India, Proceedings of National Conference on Biodiversity: Status and Challenges in Conservation, 130-134 (2013) 68.Trivedi J.N., Arya Sumedha and Vachhrajani K.D., Gastropod shell utilization preferences of hermit crab Clibanarius zebra (Dana, 1852) (Diogenidae, Anomura), Taprobanica,5(1), 7-13 (2013)69.Reddy M.S., Basha S., Joshi H.V. and Ramachandraiah G., Seasonal distribution and contamination levels of total PHCs, PAHs and heavy metals in coastal waters of the Alang–Sosiya ship scrapping yard, Gulf of Cambay, India,Chemosphere 61, 1587–1593 (2005)