Ecotoxicology and Environmental Safety 106 (2014) 253–261

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Evaluation of impairment of DNA in marine gastropod, Morula granulata as a biomarker of marine pollution A. Sarkar a,b,n, Jacky Bhagat a, Subhodeep Sarker c a

Chemical Oceanography Oceanography Division, CSIR - National Institute of Oceanography Dona Paula, Goa 403004, India Global Enviro-Care, Kevnem, Caranzalem, Goa 403002, India c Clinical Division of Fish Medicine, University of Veterinary Medicine, Veterinarplatz 1, 1210 Vienna, Austria b

art ic l e i nf o

a b s t r a c t

Article history: Received 8 November 2013 Received in revised form 12 April 2014 Accepted 20 April 2014 Available online 24 May 2014

The impairment of DNA in marine gastropod Morula granulata was evaluated in terms of the loss of DNA integrity in the species as a measure of the impact of genotoxic contaminants prevalent in the marine environment along the coast of Goa, India. The extent of DNA damage occurred in the marine gastropods collected from different sampling sites such as Arambol, Anjuna, Sinquerim, Dona Paula, Bogmalo, Hollant, Velsao, Betul and Palolem along the coast of Goa was measured following the technique of partial alkaline unwinding as well as comet assays. The highest DNA integrity was observed at Arambol (F, 0.75), identified as the reference site, whereas the lowest DNA integrity at Hollant (F, 0.33) situated between the two most contaminated sites at Bogmalo and Velsao. The impact of genotoxic contaminants on marine gastropods was pronounced by their low DNA integrity at Sinquerim (F, 0.40) followed by Betul (F, 0.47), Velsao (F, 0.51), Anjuna (F, 0.54), Bogmalo (F, 0.55), Dona Paula (F, 0.67) and Palolem (F, 0.70). The extent of DNA damage occurred in M. granulata due to ecotoxicological impact of the prevailing marine pollutants along the coast of Goa was further substantiated by comet assay and expressed in terms of %head-DNA, %tail DNA, tail length and Olive tail moment. The single cell gel electrophoresis of M. granulata clearly showed relatively higher olive tail moment in the marine gastropod from the contaminated sites, Anjuna, Hollant, Velsao and Betul. The variation in the mean % head DNA at different sampling sites clearly indicated that the extent of DNA damage in marine gastropod increases with the increase in the levels of contamination at different sampling sites along the coast. The stepwise multiple regression analysis of the water quality parameters showed significant correlation between the variation in DNA integrity and PAH in combination with NO3, salinity and PO4 2 (R , 0.90). The measurement of DNA integrity in M. granulata thus provides an early warning signal of contamination of the coastal ecosystem of Goa by genotoxic contaminants. & 2014 Elsevier Inc. All rights reserved.

Keywords: DNA impairment Biomarker Morula granulate Comet assay Alkaline unwinding assay Genotoxic contaminants

1. Introduction The rapid increase in anthropogenic activity and industrial development along the coastal region has resulted in elevated concentration of genotoxic agents in the environment, affecting the biological integrity of the marine ecosystem (Sarkar, 2005; Sarkar and Everaarts, 1998, Sarkar, 1994; Everaarts and Sarkar, 1996). Of all the ecosystems, estuarine and coastal regions are considered as the primary sink for the pollutants and greatly affected by the high degree of contaminations by highly persistent organochlorine compounds (Sarkar and Sen Gupta, 1991; Bowen and Depledge, 2006).

n

Corresponding author at: Global Enviro-Care, Caranzalem, Goa, India. E-mail address: [email protected] (A. Sarkar).

http://dx.doi.org/10.1016/j.ecoenv.2014.04.023 0147-6513/& 2014 Elsevier Inc. All rights reserved.

Marine organisms are exposed to a variety of genotoxic agents like polycyclic aromatic hydrocarbons (PAHs) (Sarkar and Everaarts, 1998), polychlorinated biphenyls (PCBs) (Sarkar et al., 1994; Sarkar et al., 1997), organochlorine pesticides (OCPs), heavy metals, etc. (Fernàndez and Grimalt, 2003; Sen Gupta et al., 1996; Sarkar, 1994). The marine pollutants prevalent in the marine ecosystem may likely to cause severe damage to the genetic material directly or indirectly. Among the direct genotoxicants, alkylating agents, like hydrogen peroxides and pesticides, etc., are significant whereas indirect toxicity depends on the mechanisms of metabolic activation and the formation of reactive oxygen species (ROS) or they consist of substances capable of inhibition of DNA synthesis/repair mechanisms (Everaarts and Sarkar, 1996). Metabolic activation processes generally lead to the formation of electrophilic metabolites which can bind to nucleophilic DNA molecules producing a variety of DNA lesions (Shugart et al., 1989; Shugart, 1999, 1988a, b; Ostling and

254

A. Sarkar et al. / Ecotoxicology and Environmental Safety 106 (2014) 253–261

Johanson, 1984; Lee and Steinert, 2003). The occurrence of DNA strand breaks in various species of marine organisms exposed to enhanced concentration of genotoxic pollutants is a matter of great concern. Thus the measurement of DNA integrity in those species of marine organisms exposed to such pollutants is indeed of great importance for bio-monitoring of pollution of the marine environment (Jha et al., 2005; Sarkar et al., 2006, 2008, 2011, 2013; Deasi et al., 2010; Sarkar, 2011 and Shugart et al., 1989). As regards the study region, Goa is one of the most famous tourism destinations in the world situated on the west coast of India, with a flourishing hotel industry supported by the rich catches of prawns, fishes, crabs, clams, snails, etc. Along with increased numbers of industrial units, shipping, mining and tourism activities keep the living organisms in the coastal region under constant stress. Several investigations have reported that the trace metals in the marine environment can cause serious damage to the physiological status of various species of marine organisms at the molecular level with long-term effects on entire communities (Depledge et al., 1995; Deasi et al., 2010; Trannum et al., 2004; Rhee et al., 2007, 2009; Rygg, 1985; Sarkar, 2011; Won et al., 2011). In view of the environmental stress due to various factors such as ultraviolet rays, nuclear radiations and genotoxic agents, the assessment of the impairment of DNA in marine organisms is of great significance. Among the genotoxic agents, polycyclic aromatic hydrocarbons (PAHs), persistent organic pollutants (POPs) and heavy metals (KrishnaKumari et al., 2006) are of immense importance (Walker, 2009; Sarkar et al., 1997; Sarkar et al., 1994). In order to assess the health of the ‘marine environment’ the quality and the status of marine life especially in coastal areas, it is of urgent necessity for reliable tools to express the effects of anthropogenic impact on biological systems. The condition of environmental health of a marine ecosystem cannot always be diagnosed by only chemical analysis of the water, as it does not provide any information about the physiological status of the organisms exposed to marine pollutants. It only indicates that there might have occurred some undesirable biological effects which may be of great concern, and therefore, studies of biological effects will give us a better understanding of the potential impact on ecosystems (Jha, 2004; Jha et al., 2005; Mustafa et al., 2011, Mosesso et al., 2012; Bauer et al.,1997; Minchin et al.,1997; Widdows et al., 2002; Strand and Asmund, 2003a, b; Rank, 1999). The solution to the above problem is the use of molecular biomarker techniques like measurement of DNA integrity by timedependent partial alkaline unwinding assay (Shugart, 1988a, b, 1999, Sarkar and Everaarts, 1998, Sarkar et al., 2005, 2006, 2008, 2011, 2013) and substantiated by the single-cell gel electrophoresis (SCGE) or comet assay (Sarkar et al., 2013). Since its inception, comet assay has been used in several organisms and plants such as bacteria, (E. coli, Singh et al., 1999), fungi (Saccharomyces cerevisiae, Banerjee et al., 2008), algae (Cryptophyta, Sastre et al., 2001), higher plants (Vicia faba, Menke et al., 2000), bivalves (Limnoperna fortunei, Villela et al., 2007; Nereis virens, (Boeck and Volders, 1997) Drosophila melanogaster, (Siddique et al., 2005), Dreissena polymorpha, (Riva et al., 2007), amphibians (Rana tigrina; Ralph and Petras, 1997), and birds (Ciconia ciconia, Milvus migrans, Baos et al., 2006). The assay has been widely used in assessing DNA damage and repair in healthy individuals, in clinical studies, as well as in dietary intervention studies and in monitoring the risk of DNA damage resulting from occupational, environmental, oxidative DNA damage, exposures or lifestyle. Most of the invertebrate species that are commonly used in environmental monitoring studies have a more limited ability to metabolize aromatic compounds (Livingstone, 1998) and despite intensive research work the estimation of PAH exposure and their

effect in aquatic invertebrate species remain problematic. The choice of marine gastropods as a model sentinel species for environmental bio-monitoring of genotoxic pollutants took into account the fact that the invertebrates represent more than 90 percent of the aquatic species. The phylum Mollusca has the highest number of animal species after arthropods and 80 percent of mollusks species are represented by gastropods. Moreover, gastropods are easy to breed, need little space, can reproduce throughout the year under controlled conditions and have a short life-span. A few data have been observed on the occurrence of DNA damage in marine gastropods (Hagger et al., 2006; Regoli et al., 2006; Benton et al., 2002; Sarkar et al., 2008, 2011, 2013). Gastropods are known to be efficient accumulators of metals, organic pollutants and respond to pollution in a sensitive and measurable manner (Bhagat et al., 2012; Gagnaire et al., 2008; Sarkar et al., 2008). Moreover, because of their characteristics with little mobility, they are very useful as the sentinel species for biomonitoring of pollution and ecotoxicological studies (Angeletti et al., 2013; Noventa et al., 2011). In view of the continuing problem of environmental contamination by various types of genotoxic pollutants it has become an urgent need of the hour to assess the state of pollution of the coastal environment and the ecotoxicological impact on the health of the marine ecosystem. The present study is aimed to evaluate the impact of genotoxic agents on the integrity of DNA as a biomarker of marine pollution.

2. Materials and methods 2.1. Sampling locations In order to assess the impact of environmental contaminants on the health of the marine ecosystem a large number of marine gastropods, Morula granulata, were collected from selected stations such as Arambol, Anjuna, Sinquerim, Dona Paula, Bogmalo, Hollant, Velsao, Betul and Palolem along the Goa coast (Fig. 1). The typical characteristics of the sample of marine gastropods M. granulata are shown in Fig. S1 (supplementary materials). Among the sampling sites, Arambol is one of the most wonderful beaches in the northern tip of Goa along the West coast of India. It is a rocky as well as sandy beach situated in the Pernem Taluka of Goa. Beyond idyllic, rocky-bottom cove, the trail emerges to a broad strip of soft, golden sand hemmed on both sides by steep cliffs. It was chosen as the reference site because of its serene environment. The main cause of pollution of the sampling sites was mainly due to indiscriminate dumping of waste materials as well as discharge of contaminated water from the surrounding restaurants and shacks directly into the coastal water.

Fig. 1. Sampling sites along the coast of Goa, India.

A. Sarkar et al. / Ecotoxicology and Environmental Safety 106 (2014) 253–261 Moreover, some of the beaches are exposed to oil spills released from various types of shipping activities such as fishing trawlers, tourist boats, water scooters, etc. around these regions. It may be noted that a cargo ship, River Princess, has been grounded near the beach for more than a decade, which might also contribute substantially towards coastal pollution of the ecosystem with the oil spills released from the grounded ship (Ingole et al., 2006). Dona Paula is one of the most popular and most crowded tourist spots in Goa, situated just opposite to Mormugao harbor, an open seaport extensively used for various shipping activities such as cargo ships, passenger ships from different parts of the world, fishing trawlers, barges carrying iron ores from the mines, tourist boats, etc. Thus the extent of pollution around Dona Paula is basically contributed by the oil spills from the surrounding region. Some of the sampling sites such as Velsao and Hollant beaches are very close to the discharge points of industrial effluents and waste materials from the surrounding agrochemical industries like Zauri Agrochemicals. Thus the marine organisms inhabited around these regions are greatly affected due to their exposure to various types of genotoxic contaminants released through the industrial effluents into the coastal seawater, leading to serious damage to the physiological status of the marine organisms. 2.2. Collection of marine gastropods The marine gastropods M. granulata were collected from the intertidal rocks scattered around the different sampling sites along the coast of Goa, India, during the period Jan–Feb 2012. The marine gastropods collected from the coast of Goa were identified using the reference sample certified by the Zoological Survey of India (ZSI), Kolkata, India (Subba Rao et al., 1992). Immediately after collection of the marine gastropods they were brought to the laboratory in a plastic container containing seawater from the same location. They were thoroughly washed and preserved with aeration in the seawater from the same sampling site at room temperature to acclimatize them for 48 h under laboratory condition. The average size of the marine gastropods collected was 24.5 mm. 2.3. Lifestyle and feeding behavior of M. granulata M. granulata was found to exist mostly on the oyster (Saccostrea cuccullata) belt spread over the rocky shores along the Goan beaches. It is a predator and feeds on worms, oysters, barnacle and small molluscs (Chim and Ong, 2012). It is reported that Vermetids, oysters and dead molluscs were prey to M. granulata in Hawaii (Wu, 1965; Kay, 1979). M. granulata preyed exclusively on vermetid gastropods at Addu Atoll in the Maldives (Taylor, 1978), while in Guam, they drilled and fed principally upon the mytilid bivalve Modiolus auriculatus and, to a lesser extent, on vermetids and cerithiids (Taylor, 1984). Koh-Siang (2003) studied the feeding behavior of M. granulata and observed that the predominant method of attack used by M. granulata to gain access to oysters was by drilling a hole through their shells. Taylor (1990) reported that there is a strong tendency for Thais clavigera and Morula musiva to drill at or near the margin of the upper (right) valve of Saccostrea. 2.4. Isolation and purification of DNA After acclimatization of the marine gastropods for 48 h they were used for isolation of DNA from their tissues. The marine gastropods collected for the studies were mostly of the same size. The DNA integrity was determined with respect to the composite samples of five individual species of almost the same size and growth. Five individual species of the gastropods from each of the sampling sites were used for isolation of DNA from their tissues. The shells of the gastropods were gently broken to remove the tissues from the shells and were mixed together homogenously using a spatula in pre-cooled Petri dishes placed over ice flakes inside the icebox during the period of sample processing for preservation of the activity of DNA. The composite mixture of tissues was then divided into three parts in order to make triplicate samples of tissues for isolation of DNA. About 300– 400 mg of tissues from the composite mixture of tissues were used for isolation of DNA by treatment with 1 ml of 1 N NH4OH/0.2% TritonX-100 per 200–400 mg of tissue using a dounce homogenizer. After homogenization, 1 ml of triple distilled water was added per 200 mg of tissue. The isolation and purification of DNA were accomplished by extraction with CIP [chloroform/isoamyl alcohol/phenol in the ratio of 24:1:25 (v/v)].The samples were shaken vigorously to fully denature all the proteins and centrifuged at 19,000G at 4 1C for 60 min to separate the different phases. The aqueous phase was pipette out and passed through a molecular sieve column (Sephadex G-50) to isolate DNA from RNA. The sample was stored in a capped eppendorf tube at 4 1C until further processing. 2.5. Measurement of DNA integrity DNA integrity in M. granulata was determined following the technique of time dependent partial alkaline unwinding assay (Shugart, 1988a, b, Everaarts, 1995; Everaarts and Sarkar, 1996; Rao et al., 1996). The three parameters measured in this assay were the amount of double-stranded (dsDNA), single stranded (ss-DNA) and

255

the fraction of double-stranded remaining after alkaline unwinding (au-DNA) for a specified time under defined condition of pH and temperature. After isolation of DNA all the measurements were performed using a spectrofluorometer (Modulus Multimode Microplate Reader, Turner Biosystems Inc.). The DNA integrity is expressed in terms of F-values which are, in fact, the ratio of the expression F¼

XðauDNAÞ  XðssDNAÞ XðdsDNAÞ  XðssDNAÞ

where X stands for observed fluorescence. The same procedure was followed with a standard DNA sample (calf thymus) and the DNA integrity (F-value) was determined for quality control of the methodology. 2.6. Single cell gel electrophoresis 2.6.1. Preparation of single cell suspension For preparation of single cell suspension, the shells of the marine gastropods collected from different sampling locations were gently broken and then the gills tissues were carefully transferred into an eppendorf tube containing 1 ml of cold extrusion buffer (71.2 mM NaCl, 5 mM EGTA, 50.4 mM guaiacol glycerol ether, pH 7.5). The tissues were slightly chopped and the suspension was then left for 2–3 min on ice. The supernatants were then centrifuged at 5000 rpm for 3 min using a mini centrifuge. The pellets thus produced were washed thrice with Phosphate Buffer Saline (PBS, 1.2 M NaCl, 0.027 M KCl, 11.5 mM K2HPO4, 0.08 M Na2HPO4, pH 7.3) and suspended in 100 ml of PBS. The cellular suspension was kept on ice to minimize endogenous damage occurring during slide preparation. The trypan blue exclusion test was used for viability assessment. 2.7. Comet assay The comet assay was performed according to the method described by Singh et al. (1988) with minor modifications. Slides were first dipped in methanol and burnt over a blue flame to remove the machine oil and dust. It was then dipped up to one-third in hot one percent Normal Melting Agarose (10 mg/ml in Milli Q water) at the frosted area. Slides were then carefully removed and the underside of it was gently wiped out and laid in a tray on a flat surface to dry. 100 ml of 0.55 percent Low Melting Agarose (LMA, 5.5 mg/ml in PBS) mixed with 20 ml of diluted cells was poured onto the coated slide kept in ice packs. Once the agarose layer hardens, 100 ml of 0.5 percent LMA (50 mg per 10 ml Tris Buffer) was poured on it and allowed to solidify. It was then kept on freshly prepared lysis buffer (2.5 M NaCl, 0.1 M di- sodium EDTA, 0.01 M Tris Buffer, 0.2 M NaOH, pH 10.0) at  4 1C in dark for 1 h. The slides are then removed and placed on the horizontal gel box. It was left for unwinding in the electrophoretic buffer (300 mM NaOH, 1 mM EDTA, pH 13.0) for 15 min. The electrophoresis was carried out in the same buffer at 20 V (cc. 1 V/cm), 300 mA. The current and voltage was made constant throughout the electrophoresis by changing the buffer level. The slides were then neutralized drop-wise using neutralizing buffer (0.4 M Tris, pH 7.5) four times at an interval of 5 min each. It was then kept in cold 100 percent methanol overnight for dehydration. Slides were drained off the excess methanol and then 100 ml of ethidium bromide (20 mg/ml) was added. Excess stain was then gently washed off and overlaid with a cover slip. It was placed in a humid, dark box at 4 1C until analysis (within 24 h). The whole procedure was carried out in yellow light to reduce DNA damage while preparing the slides. 2.7.1. Optimization of the comet assay for M. granulata The provenience of the marine gastropods used for optimization of the comet assay was from the reference site at Arambol. Comet assay was optimized for measurement of the extent of DNA damage in marine gastropod (M. granulata) in terms of lysis time, electrophoresis and unwinding condition. Slides were incubated for a period of 0.5 h, 1 h and 1.5 h in lysis buffer. It was observed that 1 hr of lysis at  41C in dark and electrophoresis at 20 V (1 V/cm) at 300 mA was appropriate for DNA to form an appropriate comet. Time for unwinding was optimized at 15 min in the same electrophoretic buffer. Factors such as lysis, electrophoresis conditions and initial preparation of the single-cell solutions can greatly affect the sensitivity of the method. In order to minimize the variations between experiments during our study we kept the lysis and electrophoresis conditions constant. 2.7.2. Comet capture and image analysis The presence of comets was examined in cells using a DM100 Leica fluorescent microscope (40  magnification). Cells were analyzed and scored using an image analysis package Komet 6.0 (Kinetic Imaging, Liverpool, UK). Human blood was used for measurement of comet control for evaluation of the variation of the comets of the cells from M. granulata. All slides were coded and the whole slide was randomly scanned. Fifty cells per slide were analyzed with two slides per incubation (total 100 cells). All experiments were carried out in triplicate to take

256

A. Sarkar et al. / Ecotoxicology and Environmental Safety 106 (2014) 253–261

Fig. 2. Variation in integrity of DNA in Morula granulata as a function of the concentration of PAH in sediments along the Goa coast. ns ¼non-significant, np o0.01, nn p o 0.001, nnnp o 0.0001 significantly different from the Arambol (Ref) (Dunnett's test).

3. Statistical analysis

Fig. 3. Variation in mean %comet head DNA in the cells of Morula granulata along the coast of Goa. ns ¼ non-significant, np o0.05, nnp o 0.01, nnnp o0.001, significantly different from the Arambol (Ref).

into account possible variations between different cell preparations. All the measurements are expressed in terms of the Mean% comet Head DNA (Fig. 3), percentage of DNA migrated from the comet head to the tail region (mean %tail DNA), mean tail length (TL) and mean olive tail moment (OTM) (Fig. S2 (Multimedia component 4, supplementary materials)) (Anderson et al., 1994). The comet pictures for each of the sampling sites were selected randomly from the 100 cells used for comet assay from the marine gastropods collected from different sites along the coast of Goa.

The statistical analysis was carried out using XLSTAT (Version 2012.5.02, Halseeon Solutions Private Limited, Bangalore, India). The Kolmogorov–Smirnov test was performed to verify whether the results follow a normal distribution. The results of the DNA integrity and comet assay as evaluated with respect to the control and that of the marine gastropods, collected from the reference site (Arambol), were statistically analyzed using analysis of variance (ANOVA) and Dunnett's multiple comparisons of means. Three levels were considered significant: p o0.05 (n), p o0.01 (nn) and po 0.001 (nnn). In order to assess the significance of the role of ambient physico-chemical parameters of the seawater influencing the integrity of DNA of the marine gastropod, a stepwise multiple regression analysis was carried out with DNA integrity as the dependent variable and the temperature, DO, NO2, NO3, turbidity, pH, salinity, PO4, BOD, PAH, etc. as the independent variables (Sarkar, 1991). The stepwise multiple regression analysis was conducted with a statistical software, ‘EASE0 (Essential Applied Statistics for Environmentalists), (Adhikari, 1989). The multiple correlation co-efficient (R2) thus obtained for each of the parameters by stepwise addition showed an increase in the value of the correlation coefficient. However, the correlation coefficient was further adjusted to determine the corrected multiple correla2 tion co-efficient (R )considering the total degrees of freedom (totaldf) and the error of degrees of freedom (errordf) with respect to the number of observations and the number of coefficients according to the following expression: 2

R ¼ 1  ð1  R2 Þ 2.7.3. Measurement of water quality parameter The water quality parameters such as temperature, pH, salinity, dissolved oxygen (DO) biochemical oxygen demand (BOD) and nutrients (nitrate, nitrite and phosphate) were determined following the standard procedure (Grasshoff et al., 1983). PAHs were extracted from the sediments from the sampling sites using bidistilled hexane by homogenization with an Ultra Turrax(R) homogenizer. The moisture content in the solvent extracts was removed by anhydrous sodium sulfate. The solvent extracts were concentrated to 1 ml using a Kuderna Danish evaporator. The concentrated extracts were then passed through deactivated (Ten percent) alumina in order to remove the interfering substances, followed by elution with 15 ml bi-distilled hexane. The purified extracts were then concentrated to 10 ml by evaporation using Snyder column evaporator. The concentration of PAHs in the aliquots was measured by spectrofluorometry (Shimadzu RF-1501 spectrofluorometer with excitation at 310 nm and emission at 360 nm) using the Kuwait oil as the standard (Burns and Kathryn, 1993; Grasshoff et al., 1983; Sarkar et al., 2008).

totaldf errordf

The best fit correlation for DNA integrity was thus obtained collectively with PAH, NO3, salinity and PO4 by evaluating the adjusted correlation co-efficient and the F-statistics due to the newly entered variable (Table 3).

4. Results The impairment of DNA in marine gastropods was measured in terms of the loss of DNA in integrity in M. granulata exposed to various types of genotoxicants prevalent in the marine ecosystem along the coast of Goa. Because of the serene environment and being distantly located from the industrial belt, Arambol was

A. Sarkar et al. / Ecotoxicology and Environmental Safety 106 (2014) 253–261

considered as a reference site for the study. Interestingly, DNA integrity in M. granulata was found to be relatively quite high (F, 0.75) at this site as compared to the other sites. Fig. 2 shows the variation in DNA integrity in M. granulata along the Goa coast. There is a decreasing trend in the DNA integrity in M. granulata from Arambol (F, 0.75) to Sinquerim (F, 0.40), followed by a slight increase at Dona Paula (F, 0.67) and then decreased further up to Hollant (F, 0.33) and finally increased considerably at Palolem (F, 0.70). The extent of DNA damage in marine gastropods was also evaluated by comet assay to substantiate the observation made by alkaline unwinding assay. Thus the impairment of DNA in marine gastropods is clearly indicated by the decrease in integrity of DNA in M. granulata exposed to various types of genotoxic contaminants prevalent at different sites along the coast of Goa. In fact, it has been observed that the DNA integrity in the marine gastropod decreased with the increase in concentration all along the coast of Goa as shown in Fig. 2. Fig. 3 shows the variation in the mean %comet-head DNA in the cells from M. granulata along the coast of Goa as a measure of the integrity of DNA. Multimedia component 2 (supplementary materials) depicts the comet control using human blood for evaluation of the variation of the comets of the cells from M. granulata. Multimedia component 3 (supplementary materials) exhibits the typical characteristics of the comets being produced from the cells of M. granulata from different sampling sites along the coast of Goa. Fig. S2 (Multimedia component 4, supplementary materials) shows the variation in the mean %comet-tail DNA, tail length, and olive tail moment of the comets of the cells from the marine gastropods all along the coast of Goa. Table 2 clearly shows the extent of DNA damage in marine gastropods in terms of variation in different parameters such as mean %head-DNA, mean %tail DNA, mean tail moment, and mean tail length of the comets of the cells from M. granulata along the coast of Goa. Table 1 shows the concentrations of water-quality parameters as well as the distribution of polycyclic aromatic hydrocarbons in sediments at different sampling sites along the coast of Goa during the period from January to February 2012. The pH of the water samples was in the range of 7.45 to 8.33, with Anjuna and Arambol showing the highest pH at 28 1C. The overall temperature was found to be between 26 1C and 29 1C. The highest turbidity (17.5 NTU) was found at Arambol, whereas Palolem water showed the least turbidity (1.65 NTU). The salinity was in the range from 21.39 to 42.97‰. The highest salinity was found at Sinquerim (42.97‰) and the least at Hollant (21.39‰). Velsao seawater was found to be predominantly rich in nutrients (Nitrite 1.718 mmol/L, Nitrate 43.717 mmol/L and Total Phosphate 3.388 mmol/L) compared to all other sampling locations. Dissolved oxygen (DO) was found to be the highest at Sinquerim (4.01 mg/ml) and at Dona Paula (4.01 mg/ ml) and the lowest at Palolem (1.46 mg/ml), whereas the highest BOD was measured at Anjuna (3.782 mg/ml) and the least at

257

Hollant (0.17 mg/ml). The concentration of PAH in sediments along the coast of Goa ranged from 1.65 to 5.17 μg/g. It was found to be the highest at Hollant (5.17 μg/g), followed by Sinquerim (4.98 μg/g), Betul (3.50 μg/g), Velsao (2.60 μg/g), Bogmalo (2.53 μg/g), Anjuna (2.24 μg/g), Dona Paula (1.89 μg/g), and Palolem (1.78 μg/g) and the lowest at Arambol (1.65 μg/g). The stepwise multiple regression analysis with different physico-chemical parameters showed that among the various water quality parameters, the highest correlation was found 2 between DNA integrity and PAH (R , 0.864) (Table 3). Interestingly, the correlations with DNA integrity increased gradually from 0.864 to 0.901 with the addition of different parameters sequentially. However, the evaluation of the corrected correlation coefficient and the F-statistics due to newly entered variable showed that the best fit correlation of DNA integrity was observed collectively with 2 PAH, NO3, salinity and PO4 (R , 0.90). Such an improved correlation with different physico-chemical parameters clearly indicate that they played a significant role on the impairment of DNA in M. granulata.

5. Discussion The DNA integrity in M. granulata was found to be drastically reduced by 28 percent at Anjuna with respect to that at the reference site (Arambol) which can be attributed to the impact of genotoxic pollutants prevalent in the site due to extensive tourist activity as well as oil spills released from the shipping activity around the site. It has been reported in earlier studies that a considerable amount of genotoxic pollutants such as Polycyclic aromatic hydrocarbons (PAHs) were accumulated in the tissues of

Table 2 Variation in Comet's parameters of the cells from Morula granulata along the coast of Goa. Sl. Sampling No. sites 1 2 3 4 5 6 7 8 9 10

Control Arambol (RS) Anjuna Sinquerim Dona Paula Bogmalo Hollant Velsao Betul Palolem

Mean % head DNA

Mean %tail DNA

Mean olive tail moment (OTM)

Mean tail length (lm)

93.42 7 0.30 6.58 7 0.30 73.80 7 0.11 26.20 7 0.11

0.57 70.07 4.25 70.18

12.46 7 0.36 28.647 0.62

67.767 0.19 32.247 0.19 68.327 0.61 31.687 0.61 74.747 0.45 25.26 7 0.45

9.12 70.16 7.54 70.60 6.52 70.82

45.247 4.22 47.237 1.35 41.90 7 3.77

28.69 7 2.61 4.93 70.55 34.54 7 1.74 7.79 70.51 37.067 1.46 9.51 70.60 47.877 1.06 10.79 70.50 41.36 7 0.62 8.34 70.13

31.86 7 0.30 40.977 1.63 37.677 1.27 36.747 1.58 35.737 1.53

71.317 2.61 65.46 7 1.74 62.94 7 1.46 52.137 1.06 58.647 0.62

Table 1 Variation in water quality parameters (pH, temperature, turbidity, salinity, DO, BOD, nutrients and PAH) along the coast of Goa. Sl. No.

Sites

pH

Temp. (1C)

1 2 3 4

Arambol Anjuna Sinquerim Dona Paula Bogmalo Hollant Velsao Betul Palolem

8.3370.00 8.3370.01 8.30 70.01 7.46 70.04 7.94 70.01 7.81 70.00 7.84 70.05 7.88 70.01 7.86 70.00

5 6 7 8 9

Turbidity (NTU)

NO2 (umol/L)

Total PO4 (umol/L)

PAH in Sediment (μg/g)

1.357 0.06 0.79 7 0.03 0.737 0.05 2.85 7 0.0

1.047 0.05 0.687 0.01 0.93 7 0.02 0.617 0.0

1.65 7 0.014 2.247 0.18 4.98 7 0.476 1.89 7 0.089

1.05 70.07 0.517 0.08 0.59 70.01 5.02 7 0.05 1.72 70.0 43.727 0.03 0.46 70.0 0.09 7 0.0 0.39 70.0 0.117 0.0

1.62 7 0.1 3.29 7 0.56 3.39 7 0.26 0.717 0.12 0.60 7 0.03

2.53 7 0.191 5.177 0.036 2.6 7 0.039 3.5 7 0.037 1.78 7 0.301

Salinity‰

DO (mg/ml)

BOD (mg/ml)

28.25 7 0.25 9.707 0.10 28.007 0.00 11.50 7 0.5 28.50 7 0.00 14.50 7 0.5 26.25 7 0.25 2.50 7 0.10

39.067 0.02 33.62 7 0.0 42.977 0.0 37.317 0.01

3.84 7 0.0 2.94 7 0.0 4.017 0.06 4.017 0.06

1.477 0.0 1.10 70.04 3.78 7 0.0 0.59 70.0 2.88 7 0.0 0.88 70.02 1.52 7 0.06 0.42 70.0

28.75 7 0.25 2.90 7 0.10 29.50 7 0.00 4.90 7 0.10 28.75 7 0.25 17.50 7 0.5 29.007 0.00 3.65 7 0.05 28.007 0.00 1.65 7 0.05

26.34 7 0.75 21.39 7 0.0 28.047 0.96 39.007 0.0 31.917 0.0

2.65 7 0.0 3.90 7 0.06 3.677 0.0 3.22 7 0.06 1.477 0.0

2.03 7 0.06 0.177 0.06 1.92 7 0.06 2.65 7 0.0 3.737 0.0

NO3 (umol/L)

258

A. Sarkar et al. / Ecotoxicology and Environmental Safety 106 (2014) 253–261

Table 3 Stepwise multiple regression analysis of the data on various physico-chemical parameters and PAH influencing the integrity of DNA in marine gastropod, Morula granulata. No. Dependent variable

1 2 3 4 5 6 7 8 9 10

DNA integrity DNA integrity DNA integrity DNA integrity DNA integrity DNA integrity DNA integrity DNA integrity DNA integrity DNA integrity

F-statistics due to newly entered variable

Multiple corr. coefficient R2

Adjusted multiple corr. co-efficient

PAH

0.87

0.86

81.98

NO3

0.89

0.88

3.24

Salinity

0.89

0.88

1.36

PO4

0.92

0.90

5.70

Turbidity

0.92

0.90

1.47

DO

0.93

0.89

0.76

BOD

0.93

0.89

0.54

pH

0.93

0.87

0.48

Temperature

0.93

0.85

0.005

NO2

0.93

0.82

0.0008

Independent variable added

R

2

the marine gastropods Cronia contracta from the ambient marine environment all along the coast of Goa during the pre-monsoon, monsoon and post-monsoon seasons. The concentration of PAHs accumulated into the tissues of the marine gastropods was in the range of 14.31–26.6 μg/g (Arambol), 16.37–19.61 μg/g (Anjuna), 30.45–53.39 μg/g (Dona Paula), 37.83–50.35 μg/g (Vasco), 22.5– 37.77 μg/g (Velsao) and 10.59–18.18 μg/g (Palolem) (Sarkar et al., 2008; Deasi et al., 2010). This clearly indicates that the coasts of Goa were highly polluted by genotoxic compounds. Apart from PAHs, there were many other genotoxic pollutants such as lead (Pb), cadmium (Cd), copper (Cu), manganese (Mn) and iron (Fe) which were accumulated in the tissues of the marine gastropods from the ambient environment. The concentration of the metals in the tissues of marine gastropods (Cronia contracta) was in the range of 0.1–0.6 μg/g (Pb), 0.1–0.8 μg/g (Cd), 0.1–3.2 μg/g (Cu), 0.4–6.2 μg/g (Fe) and 0.4–4.5 μg/g (Mn) (Sarkar et al., 2008). In fact, Anjuna is one of the most popular tourist destinations in Goa and is highly crowded with tourists from all around the world. The large amounts of waste materials containing various toxic substances from the surrounding hotels and restaurants were being regularly discharged into the site through the drainage system. Moreover, the oil spills from the offshore regions might also contribute substantially towards pollution of the site. As regards the pollution of the Sinquerim beach, it is interesting to observe that the DNA integrity in M. granulata was drastically reduced by 46.67 percent at this site. Such a huge reduction of DNA integrity in the gastropod at Sinquerim can be attributed to genotoxic pollutants like polycyclic aromatic hydrocarbons being discharged extensively from various types of shipping activities such as cargo ships, research vessel, tourist vessel, motor boats, fishing trawler, water scooters, barges sailing through this site as well as accidental oil spills, etc.(Deasi et al., 2010). Moreover, it may be noted that one of such cargo ships, MV River Princess, has been grounded near Sinquerim-Candolim beach (100 m away) since February 2001, releasing huge amounts of oil spills all along the coast, leading to serious damage to the marine ecosystem as is evident by the substantial increase in the concentration of

petroleum hydrocarbon in the sediment from Sinquerim beach (total petroleum hydrocarbon, 89 mg/g) compared to the previously reported values (Ingole et al., 2006). The DNA integrity in the marine gastropods from Dona Paula was found to decrease by 10.67 percent with respect to that in the reference site. Among the sampling sites, Velsao was considered as one of the most contaminated sites along the Goa coast as the DNA integrity in M. granulata reduced significantly by 32 percent at this particular site whereas it was reduced by 26.67 percent at Bogmalo with respect to those at the reference site. Such a drastic reduction in DNA integrity at Velsao and Bogmalo could be due to industrial discharge into the coastal water from the surrounding industries. In fact, one of the leading agrochemical industries of India, the Zuari Agrochemical Industries, situated on top of the plateau behind the Velsao beach could be regarded as the main source of contamination of the site at Velsao. Interestingly, it has been observed on several occasions that the stringent smell of industrial effluents emanated from the discharge point at Velsao straight down the plateau while sampling at this site. This clearly indicates the indiscriminate discharge of industrial effluents from the surrounding industries during sampling at Velsao. The drastic reduction of DNA integrity in M. granulata at Velsao clearly indicated the prevailing state of coastal pollution at the site. The most significant reduction of DNA integrity was observed at Hollant (56 percent). Such a drastic reduction of DNA integrity (F, 0.33) at this site could possibly be due to oil pollution caused by extensive shipping activities as well as industrial discharges. Hollant is located in between the Mormugua harbor and Zuari Agrochemical Industry and is greatly influenced by the discharge of waste materials at these sites. It has been observed in an earlier in-vivo experiment that the DNA integrity of marine gastropod Nerita chameleon was greatly affected due to exposure to series of concentrations of genotoxic pollutant, cadmium chloride (10; 25; 50; 100 mg/L), for a period of five days (Sarkar et al., 2013). Moreover, an onsite field experiment showed that the DNA integrity in marine gastropod Cronia contracta was significantly affected due to their exposure to ambient genotoxic pollutants along the Goa coast (Sarkar et al., 2008). DNA integrity was found to be quite high at Palolem (F, 0.70) and Dona Paula (F, 0.67). There is an insignificant reduction of DNA integrity (6.67 percent) at Palolem. The high DNA integrity at Palolem could due to less pollution of the site as it is far away from shipping and mining activities. The extent of DNA damage in marine gastropods as measured by the comet assay is expressed in terms of the proportion of DNA present in the comet-head, comet-tail, comet tail-length, and olive tail moment (OTM). Fig. 3 shows the proportion of DNA in comet-head along the coast of Goa. It follows the same trend as observed by alkaline unwinding assay. The proportion of DNA in the comet-head was significantly reduced in M. granulata collected from the different sampling sites along the coast of Goa as compared to the control (human blood) DNA. The majority of control cells consisted of a nucleoid core with zero or minimal DNA migrating toward the anode (Fig. 3). A portion of the control cells (average twenty percent) exhibited a small but measurable comet, which is indicative of some strand breaks produced endogenously or during cell isolation and processing (Multimedia component 2 (supplementary materials)). The comet assay allows the quantitation of single cells so that a population profile for an experiment may be visualized, as depicted in Multimedia components 2 and 3 (supplementary materials). Multimedia component 2 (supplementary materials) shows the comets for the control samples while Multimedia component 3 (supplementary materials) shows the variation in the sizes of the comets produced due to DNA damage as appeared after staining with ethidium bromide under the fluorescent

A. Sarkar et al. / Ecotoxicology and Environmental Safety 106 (2014) 253–261

microscope for M. granulata from different sampling sites along the coast of Goa. The comparison of the mean percentage of DNA in comet tail, mean tail length and mean olive tail moment (OTM) is shown in Multimedia component 4 (supplementary materials). The most pronounced DNA damage as estimated by comet assay in terms of %tail DNA for M. granulata was 47.8 percent tail-DNA at Betul as compared to 26.2 percent tail-DNA at the reference site (Arambol). The same trend was observed in terms of OTM, with Betul showing the highest OTM at 10.79 while the lowest OTM was found at Arambol (4.25). M. granulata from Sinquerim showed the longest tail length of 47.2 mm compared to that at Arambol with the shortest tail length of 28.6 mm. Enhanced DNA damage was observed in M. granulata with respect to those from the reference site (Arambol) for most of the study regions as believed to be contaminated by genotoxic compounds with the exception at Dona Paula. The decrease in the proportion of DNA in comet head illustrates the increase in the amount of genotoxic pollutants along these sites. Polycyclic aromatic hydrocarbons (PAHs) being the most predominant components of oil contaminants may be likely to cause severe DNA damage in marine invertebrates either by direct DNA strand breakage or are metabolized into reactive intermediates that can form unstable DNA adducts (Nacci et al., 1992, Nacci et al., 1996). Moreover, heavy metals have a tendency to bind to phosphates and a wide variety of organic molecules, including base residues of DNA, which can lead to mutations by altering primary and secondary structures of the DNA (Wong, 1988). Exposure to such anthropogenic chemicals can have mutagenic and carcinogenic effects, often inducing cancerous diseases (Mix, 1986). There have been few studies concerning the assessment of genotoxic risks on marine snails due to the difficulty in quantifying exposure levels, bioavailability and effects (Hamers et al., 2004; Regoli et al., 2006). Previous studies reporting the changes in DNA integrity caused by contaminants have referred to DNA damage (comet assay) as a biomarker of contaminant stress (Nigro et al., 2002; Rahman et al., 2002; Regoli et al., 2003; Clément et al., 2004; Reinecke and Reinecke, 2004). It has been observed that the impact of genotoxic contaminants like PAH and metals was greatly pronounced by the extent of DNA damage in marine gastropods Cronia contracta (Sarkar et al., 2008) and Planaxis sulcatus (Bhagat et al., 2012) along the coast of Goa. Comet assay previously has been used to investigate the levels of DNA damage in marine and freshwater bivalves exposed to water-borne pollutants (Nacci et al., 1992, 1996; Pavlica et al., 2001). Various studies have applied the comet assay to assess the DNA damage in cells from a single type of tissue, following the exposure of bivalves to a variety of contaminants, (Nacci et al., 1996; Wilson et al., 1998; Steinert et al., 1998; Steinert, 1999; Mitchelmore and Chipman, 1998). Others have used the comet assay to measure DNA damage in hemolymph and gill cells in mussels (Rank, 1999). Recently a few studies have been carried out in snails using the comet assay (Itziou and Dimitriadis, 2011; Osterauer et al., 2011 and Mohamed, 2011). A severe DNA damage was reported after the treatment of snails with organophosphate insecticides (Itziou and Dimitriadis, 2011). Therefore, DNA damage resulting from contaminant exposure is a key factor when assessing the general health of an organism, as is the need to recognize the cause, seriousness of genotoxicity and the consequences on populations and communities (Depledge et al., 1995). PAHs are suspected of having a genotoxic effect and some of the PAHs such as Benzo(a)pyrene, Indeno[1,2,3-c,d]pyrene, Benzo[g,h,i]perylene, etc. are well known for their genotoxic effects (Chen and White, 2004; Perez-Cadahia et al., 2004; Lemiere et al., 2005; Woo et al., 2006a, b). There are strong evidences that some of them are carcinogenic (Diguilio et al., 1995) with the capacity to cause various types of DNA damage. Benzo (a) pyrene, a representative

259

PAH, is reported to be converted at cellular level to chemically reactive oxygen species, diol-epoxide (BaPDE), which can form stable adduct with DNA resulting into DNA strand breaks (Pisoni et al., 2004; Sarkar et al., 1997).

6. Conclusion The impairment of DNA in marine gastropods, M. granulata, was found to be quite prominent along the coast of Goa due to their exposure to various types of genotoxic pollutants such as PAHs, metals, etc. The impact of genotoxic contaminants on the integrity of DNA in M. granulata was studied by determining the extent of DNA strand breaks in the marine gastropods exposed to various types of genotoxicants all along the coast of Goa. The extent of DNA damage was evaluated in terms of the loss of DNA integrity in the marine gastropods at different sites along the coast of Goa with respect to those from a reference site. For this purpose marine gastropods were collected from selected stations such as Arambol, Anjuna, Sinquerim, Dona Paula, Bogmalo, Hollant, Velsao, Betul and Palolem along the coast of Goa. The extent of DNA damage occurred in marine gastropods was measured by the partial alkaline unwinding assay and further ascertained by the single cell gel electrophoresis or comet assay by determining the %head-DNA, %tail-DNA, Tail length and Olive tail moment. The highest DNA integrity was found at Arambol (F, 0.75), whereas the lowest DNA integrity was at Hollant (F, 0.33) followed by Sinquerim (F, 0.40), Betul (F, 0.47), Velsao (F, 0.51), Anjuna (F, 0.54), Bogmalo (F, 0.55), etc. The decreasing trend in the integrity of DNA in marine gastropods was corroborated by the increase in concentration of PAHs in the sediments along the coast of Goa. Moreover, the various water quality parameters also a played significant role in the variation of integrity of DNA in marine gastropods as observed by the stepwise multiple regression analysis. Based on the evaluation of impairment of DNA integrity in marine gastropods, M. granulata, along the coast of Goa, it can be considered as useful sentinel species for biomonitoring of pollution by genotoxic contaminants and ecotoxicological studies.

Acknowledgments The authors are thankful to the Director, NIO, for the wholehearted cooperation and keen interest in the work. They are also thankful to the Department of Biotechnology, Govt. of India, New Delhi, for financial support in the form of fellowship to Jacky Bhagat. They are grateful to the Council of Scientific and Industrial Research (C.S.I.R.) for providing the infrastructural facilities.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.04.023. References Adhikari, A.K., 1989. Essential applied statistics for environmentalist (EASE) computer package. Indian Statistical Insitute, Calcutta, India. Angeletti, D., Sebbio, C., Carere, C., Cimmaruta, R., Nascetti, G., Pepe, G., Mosesso, P., 2013. Terrestrial gastropods (Helix spp) as sentinels of primary DNA damage for biomonitoring purposes: a validation study. Environ. Mol. Mutagen. 54 (3), 204–212. Anderson, D., Yu, T.W., Phillips, B.J., Schmezer, P., 1994. The effect of various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in the Comet assay. Mutat. Res. 307 (1), 261–271.

260

A. Sarkar et al. / Ecotoxicology and Environmental Safety 106 (2014) 253–261

Banerjee, P., Talapatra, S.N., Mandal, N., Sundaram, G., 2008. Genotoxicity study with special reference to DNA damage by Comet assay in fission yeast, Schizosaccharomyces pombe exposed to drinking water. Food Chem. Toxicol. 46, 402–407. Baos, R., Jovani, R., Pastor, N., Tella, J.L., Jimenez, B., Gomez, G., González, María J., Hiraldo, F., 2006. Evaluation of genotoxic effects of heavy metals and arsenic in wild nestling white storks (Ciconia ciconia) and black kites (Milvus migrans) from southwestern Spain after a mining accident. Environ. Toxicol. Chem. 25, 2794–2803. Bauer, B., Fioroni, P., Schulte-Oehlmann, U., Oehlmann, J., Kalbfus., W., 1997. The use of Littorina littorea for tributyltin (TBT) effect monitoring results from the German TBT survey 1994/1995 and laboratory experiments. Environ. Pollut. 96, 299–309. Benton, M.J., Malott, M.L., Trybula, J., Dean, D.M., Guttman, S.I., 2002. Genetic effects of mercury contamination on aquatic snail populations: allozyme genotypes and DNA strand breakage. Environ. Toxicol. Chem. 21, 584–589. Bhagat, J., Ingole, B., Sarkar, A., Gunjikar, M., 2012. Measurement of DNA damage in Planaxis sulcatus as a biomarker of genotoxicity. Ecoscan 1 (01–04,2012), 219–223. Boeck, M.D., Volders, K.M., 1997. Nereis virens (Annelida: Polychaeta) is not an adequate sentinel species to assess the genotoxic risk (Comet assay) of PAH exposure to the environment. Environ. Mol. Mutagen. 30, 82–90. Bowen, R.E., Depledge, M.H., 2006. Rapid Assessment of Marine Pollution (RAMP). Mar. Pollut. Bull. 53, 631–639. Burns, Kathryn, A., 1993. Evidence for the importance of including hydrocarbon oxidation products in environmental assessment studies. Mar. Pollut. Bull. 26 (2), 77–85. Chen, G., White, P.A., 2004. The mutagenic hazards of aquatic sediments: a review. Mutat. Res.-/Rev. Mutat. 567, 151–225. Chim, C.K., Ong, Y.Y.B., 2012. Diet of an intertidal predator, Morula fusca (neogastropoda: muricidae) on St. John's Island, Singapore. Contrib. Mar. Sci., 153–158. Clément, B., Devaux, A., Perrodin, Y., Danjean, M., Ghidini-Fatus, M., 2004. Assessment of sediment ecotoxicity and genotoxicity in freshwater laboratory microcosms. Ecotoxicology 13 (4), 323–333. Depledge, M.H., Aagaard, A., Gyö rkö s, P., 1995. Assessment of trace metal toxicity using molecular, physiological and behavioural biomarkers. Mar. Pollut. Bull. 31 (1–3), 19–27. Deasi, S.R., Verlecar, X.N., Ansari, Z.A., Jagtap, T.G., Sarkar, A., Vashistha Deepti, Dalal, S.G. 2010. Evaluation of genotoxic responses of Chaetoceros tenuissimus and Skeletonema costatum to water accommodated fraction of petroleum hydrocarbons as biomarker of exposure. Water Res., 44: 2235–2244. Published online doi:10.1016/J.Watres.2009.12.048. ISSN: 0043-1354. Diguilio, R.T., Benson, W.H., Sanders, B.M., van Veld, P.A., 1995. Biochemical mechanisms: metabolism, adaptation and toxicity. In: Rand, G.M. (Ed.), Fundamentals of Aquatic Toxicology, Effects, Environmental Fate and Risk Assessment. Taylor and Francis, Washington, USA, pp. 523–561. Everaarts, J.M., 1995. DNA integrity as a biomarker of marine pollution: strand breaks in seastar (Asterias rubens) and dab (Limanda limanda). Mar. Pollut. Bull. 31, 431–438. Everaarts, J.M., Sarkar, A., 1996. DNA damage as a biomarker of marine pollution: strand breaks in seastars (Asterias rubens) from the North Sea. Water Sci. Technol. 34, 157–162. Fernàndez, P., Grimalt, J.O., 2003. On the global distribution of persistent organic pollutants. Chimia 57 (9), 514–521. Gagnaire, B., Geffard, O., Xuereb, B., Margoum, C., Garric, J., 2008. Cholinesterase activities as potential biomarkers: characterization in two freshwater snails, Potamopyrgus antipodarum (Mollusca, Hydrobiidae, Smith 1889) and Valvata piscinalis (Mollusca, Valvatidae, Müller 1774). Chemosphere 71 (3), 553–560. Grasshoff, K., Ehrhardt, M., Kremling, K. (Eds.), 1983. Methods of Sea Water Analysis. Verlag Chemie, Weinheim. Hagger, J.A., Depledge, M.H., Oehlmann, J., Jobling, S., Galloway, T.S., 2006. Is there a causal association between genotoxicity and the imposex effect? Environ. Health Persp. 114, 20–26. Hamers, T., Kalis, E.J., van den Berg, J.H., Maas, L.M., van Schooten, F.J., Murk, A.J., 2004. Applicability of the black slug Arion ater for monitoring exposure to polycyclic aromatic hydrocarbons and their subsequent bioactivation into DNA binding metabolites. Mutat. Res. 552 (1–2), 219–233 (August 18). Itziou, A., Dimitriadis, V.K., 2011. Introduction of the land snail Eobania vermiculata as a bioindicator organism of terrestrial pollution using a battery of biomarkers. Sci. Total Environ. 409, 1181–1192. Livingstone, D.R., 1998. The fate of organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 120 (1), 43–49. Ingole, B., Sivadas, S., Goltekar, R., Clemente, S., Nanajkar, M., Sawant, R., D’Silva, C., Sarkar, A., Ansari, Z., 2006. Ecotoxicological effect of grounded MV River Princess on the intertidal benthic organisms off Goa. Environ. Int. 32 (2006), 284–291. Jha, A.N., 2004. Genotoxicological studies in aquatic organisms: an overview. Mutat. Res. 552 (1-2), 1–17. Jha, A.N., Dogra, Y., Turner, A., Millward, Geoffrey, E., 2005. Impact of low doses of tritium on the marine mussel, Mytilus edulis: Genotoxic effects and tissuespecific bioconcentration. Mutat. Res./Genet. Toxicol. Environ. Mutagen. 586 (1), 47–57. Kay, E.A., 1979. Hawaiian Marine Shells. Reef and Shore Fauna of Hawaii: Section 4. Mollusca. Bernice P. Bishop. Museum Special Publication, 64. Bishop Museum Press, Hawaii, pp. 1–653.

KrishnaKumari, L., Kaisary, S., Rodrigues, V., 2006. Bio-accumulation of some trace metals in the short-neck clam Paphia malabarica from Mandovi estuary, Goa. Environ. Int. 32, 229–234. Koh-Siang, T.a.n., 2003. Feeding ecology of common intertidal Muricidae (Mollusca: Neogastropoda) from the Burrup Peninsula, Western Australia. In: Perth., E., Wells, D.I., Walker, Jones, D.S. (Eds.), The Marine Flora and Fauna of Dampier, Western Australia. Western Australian Museum, pp. 173–192. Lee, Richard F., Steinert, Scott, 2003. Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat. Res./Rev. Mutat. Res. 544 (1), 43–64. Lemiere, S., Cossu-Leguille, C., Bispo, A., Jourdain, M.J., Lanhers, M.C., Burnel, D., Vasseur, P., 2005. DNA damage measured by the single-cell gel electrophoresis (Comet) assay in mammals fed with mussels contaminated by the ‘Erika’ oilspill. Mutat. Res. 581 (1-2), 11–21. Menke, M., Meister, A., Schubert, I., 2000. N-Methyl-N-nitrosourea-induced DNA damage detected by the Comet assay in Vicia faba nuclei during all interphase stages is not restricted to chromatid aberration hot spots. Mutagenesis 15, 503–506. Minchin, D., Bauer, B., Oehlmann, J.U., 1997. Schulte-Oehlmann, C.B., Duggan. Biological indicators used to map organotin contamination from a fishing port, Killybegs, Ireland. Mar. Pollut. Bull. 34, 235–243. Mitchelmore, C.L., Chipman, J.K., 1998. DNA strand breakage in aquatic organisms and the potential value of the Comet assay in environmental monitoring. Mutat. Res.––Fundam. Mol. Mech. Mutagen. 399, 135–147. Mix, M.C., 1986. Cancerous diseases in aquatic animals and their association with environmental pollutants: a critical literature review. Mar. Environ. Res. 20, 1–141. Mohamed, A.H., 2011. Sublethal toxicity roundup to immunological and molecular aspects of Biomphalaria Alexandria to Schistosoma mansoni infection. Ecotoxicol. Environ. Saf. 74, 754–760. Mosesso, P., Angeletti, D., Pepe, G., Pretti, C., Nascetti, G., Bellacima, R., Cimmaruta, R., Jha, A.N., 2012. The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem. Mutat. Res: Genet. Toxicol. Environ. Mutagen. 742 (1–2), 31–36. Mustafa, S.A., Al-Subiai, S.N., Davies, S.J., Jha, A.N., 2011. Hypoxia-induced oxidative DNA damage links with higher level biological effects including specific growth rate in common carp Cyprinus carpio L.,. Ecotoxicology 20, 1455–1466. Nacci, D.E., Cayula, S., Jackim, E., 1996. Detection of DNA damage in individual cells from marine organisms using single cell gel assay. Aquat. Toxicol. 35, 197–210. Nacci, D., Nelson, S., Nelson, W., Jackirn, E., 1992. Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bilvalves. Mar. Environ. Res. 33, 83–100. Nigro, M., Frenzilli, G., Scarcelli, V., Gorbi, S., Regoli, F., 2002. Induction of DNA strand breakage and apoptosis in the eel Anguilla anguilla. Mar. Environ. Res. 54 (3–5), 517–520. Noventa, S., Pavoni, B., Galloway, T.S., 2011. Periwinkle (Littorina littorea) as a sentinel species: a field study integrating chemical and biological analyses. Environ. Sci. Technol. 45 (7), 2634–2640. Osterauer, R., Faßbender, C., Braunbeck, T., Kö hler, H.R., 2011. Genotoxicity of platinum in embryos of zebrafish (Danio rerio) and ramshorn snail (Marisa cornuarietis). Sci. Total Environ. 409 (11), 2114–2119. Ostling, O., Johanson, K.J., 1984. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123, 291–298. Pavlica, M., Klobuèar, G.I.V., Moja, N., Erben, R., Pape, D., 2001. Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutat. Res. 490, 209–214. Perez-Cadahia, B., Laffon, B., Pasaro, E., Mendez, E., 2004. Evaluation of PAH bioaccumulation and DNA damage in mussels (Mytilus galloprovincialis) exposed to spilled Prestige crude oil. Comp. Biochem. Physiol. C 138, 453–460. Pisoni, M., Cogotzi, L., Frigeri, A., Corsi, I., Bonacci, S., Iacocca, A., Lancini, L., Mastrototaro, F., Focardi, S., Svelto, M., 2004. DNA adducts, benzo(a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy). Environ. Res. 96 (2), 63–175. Rahman, M.F., Mahboob, M., Danadevi, K., Saleha Banu, B., Grover, P., 2002. Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes. Mutat. Res. 516 (1–2), 139–147. Ralph, M., Petras, S., 1997. Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (Comet) assay. Environ. Mol. Mutagen. 29, 418–430. Rank, J., 1999. Use of Comet assay on the blue mussel, Mytilus edulis, from coastal waters in Denmark. Neoplasma 46, 9–10. Rao, S.S., Neheli, T.A., Carey, J.H., 1996. DNA alkaline unwinding assay for monitoring the impact of environmental genotoxins. Environ. Toxicol. Water Qual. 11, 351–354. Regoli, F., Gorbi, S., Fattorini, D., Tedesco, S., Notti, A., Machella, N., Bocchetti, R., Benedetti, M., Piva, F., 2006. Use of the land snail Helix aspersa as sentinel organism for monitoring ecotoxicological effects of urban pollution: an integrated approach. Environ. Health Persp. 114, 63–69. Regoli, F., Winston, G.W., Gorbi, S., Frenzilli, G., Nigro, M., Corsi, I., Focardi, S., 2003. Integrating enzymatic responses to organic chemical exposure with total oxyradical absorbing capacity and DNA damage in the European eel Anguilla anguilla. Environ. Toxicol. Chem. 22 (9), 2120–2129.

A. Sarkar et al. / Ecotoxicology and Environmental Safety 106 (2014) 253–261

Reinecke, S.A., Reinecke, A.J., 2004. The comet assay as biomarker of heavy metal genotoxicity in earthworms. Arch. Environ. Contam. Toxicol. 46 (2), 208–215. Rhee, J.S., Lee, Y.-M., Hwang, D.S., Won, E.-J., Raisuddin, S., Shin, K.H., Lee, J.S., 2007. Molecular cloning, expression, biochemical characteristics, and biomarker potential of theta class glutathione S-transferase (GST-T) from the polychaete Neanthes succinea. Aquat. Toxicol. 83, 104–115. Rhee, J.S., Raisuddin, S., Lee, K.W., Seo, J.S., Ki, J.S., Kim, I.C., Park, H.G., Lee, J.S., 2009. Heat shock protein (Hsp) gene responses of the intertidal copepod Tigriopus japonicus to environmental toxicants. Comp. Biochem. Physiol., Part C 149 (1), 104–112 (ISSN 1532-0456). Riva, C., Binelli, A., Cogni, D., Provini, A., 2007. Evaluation of DNA damage induced by decabromodiphenyl ether (BDE-209) in hemocytes of Dreissena polymorpha using the comet and micronucleus assays. Environ. Mol. Mutagen. 48 (9), 735–743. Rygg, B., 1985. Distribution of species along pollution induced diversity gradients in benthic communities in Norwegian Fjords. Mar. Pollut. Bull. 16, 469–474. Sarkar, Anupam, Bhagat, J., Ingole, B., Markad, Vijay, Rao, D.P., 2013. Genotoxicity of cadmium chloride in marine gastropod, Nerita chamaeleon using comet assay and alkaline unwinding assay. In: Tchounwou, P.B. (Ed.), Environmental Toxicology. John Wiley & Sons, http://dx.doi.org/10.1002/tox.21883 (Online ISSN: 1522-7278 online publication). Sarkar, A., 2011. Assessment of the impact of xenobiotic pollutants on the marine organisms: molecular biomarker approach. In: Bhattacharya, B., Ghosh, A., Majumdar, S.K. (Eds.), Environmental Pollution: Ecological Impacts, Health Issues and Management. Institute of Ecotoxicology and Environmental Sciences and Mudrakar, pp. 70–81 (ISBN 978-81-910222-4-7). Sarkar, A., Vashistha, Deepti, Gupta, Nitin, Malik, Kirti, Gaitonde, Dipak, C.S., 2011. Measurement of DNA integrity in marine gastropods as biomarker of genotoxicity environmental pollution: ecological impacts. In: Bhattacharya, B., Ghosh, A., Majumdar, S.K. (Eds.), Health Issues and Management. Institute of Ecotoxicology and Environmental Sciences and Mudrakar, pp. 108–112 (ISBN 978-81-910222-4-7). Strand, Jakob, Asmund, Gert, 2003a. Tributyltin accumulation and effects in marine molluscs from West Greenland. Environ. Pollut. 123 (1), 31–37. Deasi, S.R., Verlecar, X.N., Ansari, Z.A., Jagtap, T.G., Sarkar, A., Vashistha, Deepti, Dalal, S.G., 2010. Evaluation of genotoxic responses of Chaetoceros tenuissimus and Skeletonema costatum to water accommodated fraction of petroleum hydrocarbons as biomarker of exposure. Water Res. 44, 2235–2244. Sarkar, A., Gaitonde, D.C.S., Sarkar, A., Vashistha, D., D’Silva, C., Dalal, S.G., 2008. Evaluation of impairment if DNA integrity in marine gastropods (Cronia contracta) as a biomarker of genotoxic contaminants in coastal water around Goa, West coast of India. Ecotox. Environ. Saf. 71, 473–482. Sarkar, A., Ray, D., Shrivastava, A.N., Sarker, S., 2006. Molecular biomarkers: their significance and application in marine pollution monitoring. Ecotoxicology 15, 333–340. Sarkar, A., 2005. Detection of pollutants in marine organisms and the environment as marker of environmental quality. In: Majumdar, S.K., Huffman, J.E., Brenner, F.J., Panah, A.I. (Eds.), Wildlife Diseases: Landscape Epidemiology, Spatial Distribution and Utilization of Remote Sensing Technology, Chapter27. Pub. Sheridan Printing Company Inc. Alpha, NJ, USA, ISBN: 0945809190, pp. 398–414 (ISBN 0-945809-19-0, A publication of The Pennsylvania Academy of Science). Sarkar, Everaarts, J.M., 1998. Riverine input of chlorinated hydrocarbons in the coastal pollution. In: Majumdar, S.K., Miller, E.W., Brenner, Fred J (Eds.), Ecology of Wetlands and Associated Systems, Chapter 27. Pub: The Pennsylvania Academy of Science, pp. 400–423 (ISBN-10: 094580914X). Sarkar, A., Nagarajan, R., Singbal, S.Y.S., Chhapadkar, S., Pal, S., 1997. Contamination of organochlorine pesticides in sediments from the Arabian Sea along the west coast of India. Water Res. 31 (2), 195–200. Sarkar, A., Singbal, S.Y.S., Fondekar, S.P., 1994. Pesticide residues in the sediments from the lakes of Schirmacher Oasis, Antarctica. Polar Rec. 30 (172), 33–38 (ISSN (printed): 0032-2474). Sarkar, A., 1994. Occurrence and distribution of persistent chlorinated hydrocarbons in the seas around India. In: Majumdar, S.K., Miller, E.W., Forbes, G.S., Schmalz, R.F., Assad, A. Panah (Eds.), The Oceans: Physico-chemical Dynamics and Resources. The Pennsylvania Academy of Science, ISBN: 0945809107, pp. 445–459. Sarkar, A., Sen Gupta, R., 1991. Pesticide residues in sediments from the west coast of India. Mar. Pollut. Bull. 22 (1), 42–45 (Impact factor: 2.503) ISSN: 0025326X).

261

Sen Gupta, R., Sarkar, A., Kureshey, T.W., 1996. PCBs and organochlorine pesticides in krill, birds and water from Antarctica. Deep Sea Res., II 43, 119–126. Sastre, M.P., Vernet, M., Steinert, S., 2001. Single-cell gel/Comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta). Photochem. Photobiol. 2001, 55–60. Shugart, L.R., Gustin, M.K., Laird, D.M., Dean, D.A., 1989. Susceptibility of DNA in aquatic organisms to strand breakage: effect of x-rays and gamma radiation. Mar. Environ. Res. 28 (1-4), 339–343. Shugart, L.R., 1999. Structural damage to DNA in response to toxicant exposure. In: Forbes, V.E. (Ed.), Genetics and Ecotoxicology, pp. 151–168. Shugart, L.R., 1988a. An alkaline unwinding assay for the detection of DNA damage in aquatic organisms. Mar. Environ. Res. 24, 321–325. Shugart, L.R., 1988b. An alkaline unwinding assay for the detection of DNA damage in aquatic organisms. Mar. Environ. Res. 24, 321–325. Siddique, H.R., Chowdhuri, D.K., Saxena, D.K., Dhawan, A., 2005. Validation of Drosophila melanogaster as an in vivo model for genotoxicity assessment using modified alkaline Comet assay. Mutagenesis 20, 285–290. Singh, N.P., Stephens, R.E., Singh, H., Lai, H., 1999. Visual quantification of DNA double-strand breaks in bacteria. Mutat. Res. 429, 159–168. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Steinert, S.A., Streib-Montee, R., Leather, J.M., Chadwick, D.B., 1998. DNA damage in mussels at sites in San Diego Bay. Mutat. Res. Fundam. Mol. Mech. Mutagen. 399, 65. Steinert, S.A., 1999. DNA damage as a bivalve biomarker. Biomarkers 4, 492–496. Strand, J., Asmund, G., 2003b. Tributyltin accumulation and effects in marine molluscs from West Greenland. Environ. Pollut. 123, 31–37. Subba Rao, N.V., Dey, A., Barua, S. (Eds.), 1992. Estuarine and Marine Molluscs Fauna of West Bengal. Pub.: Zoological Survey of India, Calcutta (State fauna Series 3). Taylor, J.D., 1978. Habitats and diet of predatory gastropods at Addu Atoll, Maldives. J. Exp. Mar. Bio. Ecol. 31, 83–103. Taylor, J.D., 1984. A partial food web involving predatory gastropods on a Pacific fringing reef. J. Exp. Mar. Biol. Ecol. 74 (3), 273–290. Taylor, J.D., 1990. Field observations of prey selection by the muricid gastropods Thais clavigera and Morula musiva feeding upon the intertidal oyster Saccostrea cucullata. In: Morton, B. (Ed.), The Marine Flora and Fauna of Hong Kong and Southern China II. University Press, Hong Kong, pp. 836–855 (Hong Kong). Trannum, H.C., Olsgard, F., Skei, J.M., Indrehus, J., Øverås, S., Eriksen, J., 2004. Effects of copper, cadmium and contaminated harbor sediments on recolonisation of softbottom communities J. Exp. Mar. Biol. Ecol. 310, 87–114. Villela, I.V., de Oliveira, I.M., Silveira, J.C., Dias, J.F., Henriques, J.A., Da Silva, J., 2007. Assessment of environmental stress by the micro-nucleus and Comet assays on Limnoperna fortunei exposed to Guai´ba hydrographic region samples (Brazil) under laboratory conditions. Mutat. Res. 628, 76–86. Walker, C.H., 2009. Organic pollutants: an ecotoxicological perspective, 2nd ed. CRC Press, Boca Raton, FL p. 408. Widdows, J., Donkin, P., Staff, F.J., Matthiessen, P., 2002. Measurement of stress effects (scope for growth) and contaminant levels in mussels (Mytilus edulis) collected from the Irish Sea. Mar. Environ. Res. 53, 327–356. Wilson, J.T., Pascoe, P.L., Parry, J.M., Dixon, D.R., 1998. Evaluation of the comet assay as a method for the detection of DNA damage in the cells of a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda). Mutat. Res. 399, 87–95. Woo, S., Kim, S., Yum, S., Yim, U.H., Lee, T.K., 2006a. Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons. Mar., Pollut. Bull. 52, 1768–1775. Won, E.J., Kim, R.O., Rhee, J.S., Park, G.S., Lee, J.H., Shin, K.H., Lee, Y.M., Lee, J.S., 2011. Response of glutathione S-transferase (GST) genes to cadmium exposure in the marine pollution indicator worm, Perinereis nuntia. Comp. Biochem. Physiol. C 154, 82–92. Wong, P.K., 1988. Mutagenicity of heavy metals. Bull. Environ. Contam. Toxicol. 40 (1988), 597–603. Woo, Seonock, Kim, Sojung, Yum, Seungshic, Yim, U.n., Hyuk, Lee, Taek, Kyun, 2006b. Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons, Mar. Pollut. Bull. 52 (12), 1768–1775. Wu, S.K., 1965. Comparative functional studies of the digestive system of the muricid gastropods Drupa ricina and Morula granulata. Malacologia 3, 211–233.