Acetylcholinesterase activities in marine snail (Cronia contracta) as a biomarker of neurotoxic contaminants along the Goa coast, West coast of India

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1 Ecotoxicology (2006) 15: DOI /s Acetylcholinesterase activities in marine snail (Cronia contracta) as a biomarker of neurotoxic contaminants along the Goa coast, West coast of India D. Gaitonde Æ A. Sarkar Æ S. Kaisary Æ C. D. Silva Æ C. Dias Æ D. P. Rao Æ D. Ray Æ R. Nagarajan Æ S. N. De Sousa Æ Subhodeep Sarker Æ D. Patill Accepted: 1 February 2006 / Published online: 5 May 2006 Ó Springer Science+Business Media, LLC 2006 Abstract The measurement of acetylcholinesterase (AChE) activity is used worldwide as a biomarker of environmental contamination due to neurotoxic substances. In the present study the AChE activities was measured in marine snails (Cronia contracta) collected seasonally from six sampling sites (viz. Arambol, Anjuna, Dona Paula, Vasco, Velsao and Palolem) along the Goa coast during the pre-monsoon (April, 2004), monsoon (September, 2004) and post-monsoon (November, 2004) periods. The AChE activities in C. contracta showed wide variation along the Goa coast. It was found to be quite high at the reference site, Palolem (23.97, and 24.85) throughout the sampling period (April November, 2004). The AChE activities in C. contracta decreased significantly at Vasco ( % reduction) followed by Dona Paula ( % reduction), Velasao ( % reduction), Arambol ( % reduction) and Anjuna (0 12.7% reduction). Such a significant variation of AChE activities in the marine snail along the Goa coast can be attributed to neurotoxic substances prevalent in those regions. The high concentration of different neurotoxic metals (lead, cadmium, copper, manganese and iron) and petroleum D. Gaitonde Æ A. Sarkar (&) Æ S. Kaisary Æ C. D. Silva Æ C. Dias Æ D. P. Rao Æ D. Ray Æ R. Nagarajan Æ S. N. De Sousa Marine Pollution Assessment and Ecotoxicology Laboratory, National Institute of Oceanography, Dona Paula, Goa , India anupam1_in@yahoo.com S. Sarker Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi Jharkhand, India D. Patill Department of Environmental Science, University of Pune, Pune , India hydrocarbons in the tissues of the marine snails at Dona Paula, Vasco and Velsao clearly substantiate reduction of AChE activities in C. contracta. The in vitro studies on the inhibition of AChE by different metals and PHC indicated that lead, cadmium and copper are the most predominant inhibitor. Based on the AChE activities in C. contracta the sampling sites along the Goa coast can be classified into three major clusters such as highly contaminated sites (Dona Paula, Vasco and Velsao), moderately contaminated sites (Arambol, Anjuna) and least contaminated site (Palolem). Keywords Acetylcholinesterase activity Æ Cronia contracta Æ Biomarker of pollution Æ Marine snails Æ Lead Æ Cadmium Æ Copper Æ PHC Æ Neurotoxic substances Introduction The use of biomarkers to evaluate the biological effects of chemical pollutants in marine organisms is of great significance as diagnostic tool for biomonitoring of pollution (Escartin and Porte 1997 and Sturm et al. 1999). The measurement of acetylcholinesterase (AChE) activity in marine organisms is widely used for biomonitoring of marine pollution due to neurotoxic substances (Pfeifer et al. 2005; Magni et al. 2005; Cajaraville et al. 2000; Escartin and Porte 1997; Lionetto et al. 2003; Galgani et al and Sen Gupta et al. 1991). The inhibition of AChE by neurotoxic substances such as cadmium, copper, lead, organophosphorous, carbamate pesticides, polyaromatic hydrocarbons have been well established (Cajaraville et al. 2000; Sarkar 1992; Mmartinez Tabche et al. 1997; Matozzo et al. 2005; Sturm et al and Wells et al. 2001). The AChE plays a significant role in nerve conduction processes

2 354 Gaitonde et al. at myoneutral junction of the nerve ending of muscle tissue (Cajaraville et al. 2000). AChE is the enzyme that is usually located in membranes of vertebrates and non-vertebrates animals (Bocquené et al, 1997). Cholinesterase enzymes (ChE) are often highly polymorphic enzymes in invertebrates. According to the mechanism of action, the AChE is released at the myoneutral junction in organisms if an action potential is developed at the nerve ending and diffuses through the gap between the nerve and the muscle (the gap is about 100 Å wide). Anticholinetesterases such as organophosphate, carbamate pesticides, toxic elements (Cd, Pb, Cu etc) bind to the catalytic site of the AChE enzyme thus preventing the physiological inactivation of acetylcholine leading to an anomalous protraction of neurotransmission. The AChE activity was used as a biomarker of neurotoxic contaminants in copepods (Tigriopus brevicornis) from the Vilaine River estuary of France (Forget et al. 2003). Marine bivalves such as snails, oysters and mussels were widely used as bioindicators of contamination in the monitoring of pollutant effects. As filter feeders, these species are known to be good general indicators of chemical contamination. The decrease in AChE activity in the oyster was efficiently used as a biomarker of exposure to neurotoxic compounds (Bocquené et al. 1997). The inhibition of AChE in Zebra mussels from the Italian Great Lakes was used as biomarkers for the persistent organic pollutants (POPs) biomonitoring of aquatic environments (Binelli et al. 2005). Lionetto et al (2003) studied AChE activities in Mytilus galloprovincialis and a benthic teleost fish, Mullus barbatus from a coastal marine area around Salento Peninsula (Italy) for detection of the possible exposure/effect induced by chemical pollutants in native marine organisms. It has been reported that AChE activity in the clam Tapes philippinarum was used to evaluate the impact of exposure of animals to neurotoxic compounds in the Lagoon of Venice (Matozzo et al. 2005). A year-round study was conducted to assess the seasonal variations and potential influence of the riverine discharge from the Pearl River on AChE activities in the green mussel, Perna viridis as a biomarker of exposure to contaminants in Hong Kong waters (Lau et al. 2004) In view of rapid industrial development all around Goa as well as extensive shipping and mining activities the coastal environment is highly stressed by various types of pollutants. In order to evaluate the environmental stress of neurotoxic contaminants along the Goa coast the AChE activities in marine snails (Cronia contracta) was measured. Materials and methods In order to measure the seasonal variation of AChE activities marine snails (C. contracta) were collected from different locations along the coasts of Goa (Fig. 1) during the pre-monsoon, monsoon and post-monsoon period. The rationale for selection of this particular species of marine snails is that they are considered to be very good sentinel organisms and was available in all the sampling sites throughout the year. Description of C. contracta The shells of the snails are solid with angulate whorls sculptured with strong axial folds. About 8 12-folds could be found on body whorl with scaly spiral ribs on the whole surface crossing the axial folds. Sometimes there is a varix 3 4-folds before the aperture. The outer lip is finely crenulated with 6 8 internal denticles in mature shells. Anterior canal short are slightly recurved. Aperture colour is white with exterior fawn and sometimes with brown spiral bands. The sizes of the snail are generally up to 45 mm in length. They are abundantly found mainly in the intertidal and subtidal zones on rocky shores. A typical structure of the snail (C. contracta) on the rocky beach of Goa is shown in Fig. 2. They were found to lay eggs capsules in clusters on rock surfaces or on the shells of other snails, the same or different species from which crawling young were hatched. Interestingly, the egg clusters vary in size, shape, colour, and number of individual capsules. Since the mortality rate of eggs was usually high the eggs were laid in very large numbers. The snails are most probably carnivores and scavengers. The snails are probably nocturnal and feed on a variety of invertebrates by immobilizing the prey with secretions from the proboscis gland. They use a gill to feed, filter pollutants present in the water too and accumulate them inevitably. Description of the sampling locations The sampling sites were selected on the basis of the locations of the estuaries along the Goa coast. The six sampling sites selected for collection of marine snails (C. contracta) along the Goa coast are Arambol, Anjuna, Dona Paula creek, Vasco, Velaso and Palolem. There are four major estuaries situated along the coast of Goa namely, Zuari estuary, Mandovi estuary, Chapora estuary and Tiracol estuary. The rivers and the estuaries are the main vector of pollution along the Goa coast. Arambol beach is situated in the extreme north of Goa, near Tiracol River while Anjuna beach is far from the other sites and the two major rivers, Tiracol and Chapora. The Dona Paula creek is situated between Mandovi and Zuari estuaries opposite to the Mormugao Harbour. Because of its tourist attraction the creek is over crowded with large numbers of hotels, restaurants, motor vehicles, boats, water scooters contributing extensively towards contamination of the site. Vasco beach is very closed to the Murmugao harbour and highly

3 Acetylcholinesterase activities in marine snail 355 Fig. 1 Locations of the sampling sites along the Goa coast, India Fig. 2 A typical marine snail (C. contracta) on the rock, Goa coast, India

4 356 Gaitonde et al. contaminated due to rigorous shipping activities and dumping of waste materials. Velsao beach situated south of the Zuari estuary is contaminated mainly because of the discharge of industrial waste materials from the peripheral region. Palolem beach is far away from the harbour and considered as the reference site for its pristine environment. Isolation of AChE enzyme In order to isolate AChE enzymes the whole tissues were removed from the snails and pooled together to make composite samples of five individual animals. The composite samples were then divided into three replicate samples for acetycholinesterase study. The AChE enzymes were isolated from the tissues by homogenization with 0.01 M phosphate buffer (ph ) spiked with Triton-X-100 (0.2%) and 0.25 M sucrose (1:1) solution using an Ultra Turrax homogeniser (T-25). The homogenates were centrifuged by refrigerated centrifuge (Eltek) at 20,000 g for about 35 min at 4 C. The protein concentration of each of the sample extract was determined according to Lowry s method (1951) using bovine serum albumin as the standard. Determination of AChE activity The AChE activity was measured following modified D-pH metric method (Sarkar 1992) using bromothymol blue and acetylcholine bromide as the indicator and the substrate respectively for the reaction kinetics. The changes in ph (D-pH) was measured at an interval of 10 min over a period of 1 h of incubation corresponding to the amount of acetic acid liberated due to interaction with the AChE enzyme. The unit of activity of AChE was calibrated (using the best fit equation, Y = 57.23X)1.66, R 2 = 0.99) with respect to D-pH (changes in ph) against the micromoles of acetic acid liberated due to hydrolysis of acetylcholine. The AChE activity is expressed in terms of units corresponding to micromoles of acetic acid liberated per minute per mg of protein. The concentrations of the metals in the tissues of marine snails were determined by atomic absorption spectrophotometer (AAS) while the organic contaminants such as organochlorine pesticides by GC-ECD (Varian GC-3380) and petroleum hydrocarbons by spectroflurometer. Results and discussion The variation of AChE activities in marine snails (C. contracta) during the pre-monsoon (April, 2004), monsoon (September-04), and post-monsoon periods (November, 2004) are shown in Fig. 3. It clearly shows that the AChE activities decreased significantly from the normal value at the reference site, Palolem (23.97) during the pre-monsoon period at Arambol (21.5% reduction), Dona Paula (36.2% reduction) and Vasco (44.6% reduction). Because of its pristine environment the Palolem beach is considered as the reference site. Interestingly, the AChE activities at Anjuna was found to be very close to that of Palolem during the pre-monsoon period probably because of its location and away from the river mouths. The low AChE activities at Dona Paula and Vasco can be attributed to the levels of contamination at these locations due to extensive shipping activities, dumping of untreated waste materials, riverine input of contaminants through the Mandovi and Zuari estuaries. Moreover, extensive trawling of barges carrying crude ore materials from the mines to the harbour through the waterways contributed largely towards contamination of the sampling sites. It has been observed that the AChE activities of C. contracta decreased further during the monsoon period at Vasco (52.4% reduction) and Velsao (35.9% reduction) probably due to increased contamination of the sampling sites with toxic metals such as lead, cadmium, copper etc. Figure 4 clearly shows the levels of contamination of the sampling sites by different toxic metals. Interestingly, the concentrations of the metals at Dona Paula, Vasco and Velsao were relatively higher than those in the rest of the sampling sites. The analysis of the samples does not show the presence of any pesticides in the tissues of the marine snails. The concentration of PHC was found to be in the range of ppm. However, the in vitro studies on inhibition of AChE activities clearly indicated the anticholinesterase properties of the metals and PHC in the decreasing order: lead > cadmium > copper > PHC. In fact, PHC did not show any significant effect on inhibition of AChE activities. The same trend of the reduction in AChE activities was observed at Dona Paula, Vasco and Velsao during the post-monsoon period. However, significant decrease in AChE activities could be observed at Arambol (37% reduction) and Anjuna (12.7%) during this period. Such a variation of AChE activities at Arambol and Anjuna during the post-monsoon season could be due to rigorous shipping and trawler activities as well as industrial discharge along the Goa coast. Figure 4 clearly illustrates the levels of contamination of the marine snails with toxic metals at different sampling sites. Among the most potent neurotoxic metals (Pb, Cd and Cu) detected in the marine snail tissues, cadmium was predominant at different sampling sites with highest concentration at Vasco (3.37 ppm) followed by Dona Paula (1.67 ppm), Velsao (1.43 ppm), Arambol (0.8 ppm), Anujuna (0.13 ppm) and Palolem (0.17 ppm). Copper was found to be highest at Dona Paula (2.4 ppm) followed by Arambol (0.90 ppm), Vasco (0.73 ppm), Anjuna (0.70 ppm) and Palolem (0.1 ppm).

5 Acetylcholinesterase activities in marine snail 357 Fig. 3 Seasonal variation of AChE activities in marine snail (C. contracta) along the Goa coast AChE activity (units) Pre-monsoon-04 Monsoon-04 Post-monsoon-04 0 Arambol Anjuna Dona Paula Vasco Velsao Palolem Control Sampling locations Fig. 4 Variation of concentration of metals in the tissues of marine snail (C. contracta) along the Goa coast Concentration (ppm) Pb Cd Cu Fe Mn Arambol Anjuna Dona Paula Vasco Velsao Palolem Sampling locations This clearly substantiated the fact that the prevalence of neurotoxic metals (Pb, Cd and Cu) in snail tissues could play a major role in significant decrease in AChE activities in C. contracta at Vasco, Dona Paula and Velsao. Thus on the basis of the AChE activities in marine snail (C. contracta) the Goa coastal region can be classified into three clusters: the most contaminated sites (Vasco, Dona Paula and Velsao), moderately contaminated sites (Arambol and Anjuna), and least contaminated site (Palolem). Acknowledgment The authors are thankful to Dr. S. R. Shetye, Director, NIO, Dr. M. D. Zingde, Dr. S. W. A. Naqvi and Dr. C. G. Naik for their constant encouragement and interest in this study. They also thank Mr. A. Mahale, Mr. R. Uchil and Mr. Sham Akerkar for their cooperation in computation of the sampling sites. References Binelli A, Ricciardi F, Riva C, Provini A (2005) Screening of POP pollution by AChE and EROD activities in Zebra mussels from the Italian Great Lakes. Chemosphere 61(8): Bocquené G, Roig A, Fournier D (1997) Cholinesterases from the common oyster (Crassostrea gigas): evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors. FEBS Lett 407(3): Cajaraville MP, Bebianno MJ, Blasco J, Porte C, Sarasquete C, Viarengo A (2000) The use of biomarkers to assess the impact of pollution in coastal environment of the Iberian Peninsula: a practical approach. Sci Total Environ 247: Escartin E, Porte C (1997) The use of cholinesterase and carboxylesterase activities from Mytilus galloprovincialis in pollution monitoring. Environ Toxicol Chem 16: Forget J, Beliaeff B, Bocquené G (2003) Acetylcholinesterase activity in copepods (Tigriopus brevicornis) from the Vilaine River estuary, France, as a biomarker of neurotoxic contaminants. Aquatic Toxicol 62(3): Galgani F, Bocquené G, Cadiou Y (1992) Evidence of variation in cholinesterase activity in fish along a pollution gradient in the North Sea. Mar Ecol Prog Ser 91:77 82 Lau PS, Wong HL, Garrigues Ph (2004) Seasonal variation in antioxidative responses and acetylcholinesterase activity in Perna viridis in eastern oceanic and western estuarine waters of Hong Kong. Continent Shelf Res 24(16): Lionetto MG, Caricato R, Giordano ME, Pascariello MF, Marinosci L, Schettino T (2003) Integrated use of biomarkers (acetylcholinesterase and antioxidant enzymes activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian coastal marine area. Mar Pollut Bull 46(3): Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: Magni P, De Falco G, Falugi C, Franzoni M, Monteverde M, Perrone E, Sgro M, Bolognesi C (2005) Genotoxicity biomarkers and acetylcholinesterase activity in natural populations of Mytilus

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