Detecting genotoxicity using the Comet assay following chronic exposure of Manila clam Tapes semidecussatus to polluted estuarine sediments

Sediments frequently cause damage to biota due to the accumulation of toxic compounds and the bioavailability of sediment-bound contaminants. Damage can be assessed using biomarkers, such as the degree of genotoxic impact following in vivo exposure to pollutants. Genotoxic damage, expressed as single-strand DNA breaks, was measured in cells isolated from haemolymph, gill and digestive gland from the clam Tapes semidecussatus, using the single cell gel electrophoresis (Comet assay). Clams were exposed for three weeks to sediment samples collected from a polluted site and a ‘clean’ reference site.The level of DNA damage was assessed using an image analysis package and expressed as Tail Moment. Throughout the study, significant differences in DNA damage were recorded for each tissue type between clams exposed to the two sediment samples. We conclude that the Comet assay is a useful tool for the detection of DNA damage in clams chronically exposed to polluted sediments.     

Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons

To investigate the genotoxic effect of marine sediments on aquatic organism, sediment samples were collected from 13 sites along the coast of Gwangyang Bay (Korea). Concentrations of polycyclic aromatic hydrocarbons (PAHs) in sediments were determined and the relationship between exposure of flounder blood cells to sediment extracts and DNA single-strand breakage in the blood cells was examined using the comet assay. Levels of DNA damage were proportionally increased by exposure concentration and the highest sediment-associated DNA damage was observed at the station showing the highest PAHs contamination. DNA damage in blood cells exposed to five types of PAHs (benzo[a]pyrene, fluoranthene, anthracene, pyrene and phenanthrene) and in flounder (Paralichthys olivaceus) exposed to benzo[a]pyrene (BaP) for 0, 2 and 4 days were assessed by measuring comet tail length. The tail lengths of five PAHs-exposed groups at 50 and 100 ppb were significantly different from the non-exposed group, and the genotoxic effect of BaP correlated with both concentration and duration of exposure. Throughout the study, significant differences in DNA breakage were recorded between cells exposed to sediment extracts or PAHs and non-exposed control. This study demonstrated the comet assay as a successful tool in monitoring contamination of marine sediments and assessing genotoxicity of PAHs in marine organisms, either in vitro or in vivo.     

Clam Transplantation and Stress-Related Biomarkers as Useful Tools for Assessing Water Quality in Coastal Environments

In this study, the Active BioMonitoring (ABM) approach has been investigated using species of indigenous (from a pristine site) and transplanted (to a polluted area) hard clam Mercenaria mercenaria as an indicator organism of water/sediment pollution in Tampa Bay, Florida, a highly urbanized and industrialized subtropical estuary. A range of possible induced stress indices was selected and used on the basis of potential pollutant toxicity mechanisms including specific enzymatic responses (MFO as BPH and NADPH cytochrome c reductase, antioxidant enzymes as SOD and caltalase, PDH) and general cellular damage (lysosomal latency, histopathological lesions). The organic sediment contamination compared to the animal body burden revealed bioaccumulation activity in transplanted clams. The marked increases of BPH and catalase activities, together with the significant reduction of lysosomal membrane stability in transplanted animals, suggested their potential for application as biomarkers in subtropical coastal pollution monitoring.     

Use of the clam Asaphis deflorata as a potential indicator of organochlorine bioaccumulation in Hong Kong coastal sediments

In order to test its ability as a bioaccumulator, the deposit feeding clam Asaphis deflorata, collected from a clean coastal area in Hong Kong (Ding Chau, Ma On Shan), was exposed to field-collected sediments representing a gradient of contamination. Sediments were collected from four sites, namely Shek O in the southeast of Hong Kong Island, Ap Lei Chau in Aberdeen district, Tai Kok Tsui in the Mong Kok waterfront, and To Kwa Wan near Kowloon City pier. Shek O was the cleanest and To Kwa Wan was the most polluted site. Replicate 30 L fiberglass tanks containing equal numbers of organisms were maintained for 28 days, with equal quantities of sediment and filtered seawater. Prior to this the animals were depurated for 10 days in clean sediment. During the 28-day holding period, sampling of both sediment and clams was performed on days 0, 10 and 28, along with condition index measurements. Concentrations of organochlorine pesticides and PCBs in sediments showed that Shek O was the cleanest of the sites, followed by Ap Lei Chau, Tai Kok Tsui and To Kwa Wan. There were marked differences among the sites, especially in PAH concentrations, with To Kwa Wan showing extremely high sediment concentrations.Following exposure, the biota concentrations of various organochlorine pesticides and PCBs suggested that there was sediment concentration-dependent accumulation by A. deflorata on day 10 of the experiment. However, there was no significant increase on day 28 for any of the compounds tested. Contaminant concentrations in clams feeding on Shek O sediments were minimal and almost the same on all sampling days, indicating the presence of only baseline contaminant concentrations. The condition indices (CI) of the clams on various sampling days showed that the animals were relatively more stable in sediment from Shek O. There were some deaths in all the test tanks (including those containing Shek O sediments). In addition, clams appeared to bury faster in the cleaner Shek O sediment and remained buried, unlike those in the sediment from the other locations, where some remained predominantly on the surface. Nevertheless, despite not completely burying, the clams in other test sediments still exhibited sediment concentration-dependent tissue accumulation. The results of the investigation indicate the possibility of developing a standard test protocol for bioaccumulation by sediment dwelling invertebrates, for practical use in Hong Kong, using A. deflorata.     

Use of the clam Asaphis deflorata as a potential indicator of organochlorine bioaccumulation in Hong Kong coastal sediments

In order to test its ability as a bioaccumulator, the deposit feeding clam Asaphis deflorata, collected from a clean coastal area in Hong Kong (Ding Chau, Ma On Shan), was exposed to field-collected sediments representing a gradient of contamination. Sediments were collected from four sites, namely Shek O in the southeast of Hong Kong Island, Ap Lei Chau in Aberdeen district, Tai Kok Tsui in the Mong Kok waterfront, and To Kwa Wan near Kowloon City pier. Shek O was the cleanest and To Kwa Wan was the most polluted site. Replicate 30 L fiberglass tanks containing equal numbers of organisms were maintained for 28 days, with equal quantities of sediment and filtered seawater. Prior to this the animals were depurated for 10 days in clean sediment. During the 28-day holding period, sampling of both sediment and clams was performed on days 0, 10 and 28, along with condition index measurements. Concentrations of organochlorine pesticides and PCBs in sediments showed that Shek O was the cleanest of the sites, followed by Ap Lei Chau, Tai Kok Tsui and To Kwa Wan. There were marked differences among the sites, especially in PAH concentrations, with To Kwa Wan showing extremely high sediment concentrations.Following exposure, the biota concentrations of various organochlorine pesticides and PCBs suggested that there was sediment concentration-dependent accumulation by A. deflorata on day 10 of the experiment. However, there was no significant increase on day 28 for any of the compounds tested. Contaminant concentrations in clams feeding on Shek O sediments were minimal and almost the same on all sampling days, indicating the presence of only baseline contaminant concentrations. The condition indices (CI) of the clams on various sampling days showed that the animals were relatively more stable in sediment from Shek O. There were some deaths in all the test tanks (including those containing Shek O sediments). In addition, clams appeared to bury faster in the cleaner Shek O sediment and remained buried, unlike those in the sediment from the other locations, where some remained predominantly on the surface. Nevertheless, despite not completely burying, the clams in other test sediments still exhibited sediment concentration-dependent tissue accumulation. The results of the investigation indicate the possibility of developing a standard test protocol for bioaccumulation by sediment dwelling invertebrates, for practical use in Hong Kong, using A. deflorata.     

Polycyclic aromatic hydrocarbons (PAHs) in transplanted Manila clams (Tapes philippinarum) from the Lagoon of Venice as assessed by PAHs/shell weight index: a preliminary study

Variation of polycyclic aromatic hydrocarbons (PAHs) levels was assessed in Tapes philippinarum from the Lagoon of Venice. Clams were transplanted from a polluted area next to Porto Marghera to two rearing areas of the Southern Lagoon. Analyses of PAHs were made in sediments and clams by GC/MS at first sampling and after 30, 60 and 180 days. Principal component analysis was performed to elucidate bioaccumulation and depuration pattern and input sources. Biota-Sediment-Accumulation-Factor (BSAF) was applied to evaluate the PAHs input sources from sediment. Condition index was calculated to compare the seasonal variation of clam tissue to PAHs levels. To propose results not affected by seasonal changes in flesh weight of clams, the approach based on the calculation of PAHs/SW index was applied. From the results, it was concluded that PAHs/SW index is more recommendable to asses temporal variation of PAHs levels in Manila clams.     

Biomarkers of exposure and effect in the Brazilian flounder Paralichthys orbignyanus (Teleostei: Paralichthyidae) from the Patos Lagoon estuary (Southern Brazil)

Biomarkers of exposure (liver metallothionein-like proteins content and catalase and glutathione S-transferase activities) and effect (liver lipoperoxidation and blood cell DNA damage) of contaminants were analyzed in the Brazilian flounder Paralichthys orbignyanus from the Patos Lagoon estuary (Southern Brazil). Flounders were collected for a year in two sites: "Coroa do Boi" (polluted site) and "Saco do Justino" (non-polluted site). Results indicated that micronucleated cells frequency was the best biomarker to distinguish flounders from the two sites. Taken together, data from DNA damage analyses (micronucleus test and comet assay) indicated that flounders from the non-polluted site efficiently repaired the DNA breaks, contrary to those from the polluted site, which probably had their DNA repair system inhibited or exhausted. Furthermore, data from enzyme activities (catalase and GST) and lipid peroxidation indicated that flounders from the polluted site were under oxidative stress in summer and autumn.     

Polycyclic aromatic hydrocarbons (PAHs) in transplanted Manila clams (Tapes philippinarum) from the Lagoon of Venice as assessed by PAHs/shell weight index: A preliminary study

Variation of polycyclic aromatic hydrocarbons (PAHs) levels was assessed in Tapes philippinarum from the Lagoon of Venice. Clams were transplanted from a polluted area next to Porto Marghera to two rearing areas of the Southern Lagoon. Analyses of PAHs were made in sediments and clams by GC/MS at first sampling and after 30, 60 and 180 days. Principal component analysis was performed to elucidate bioaccumulation and depuration pattern and input sources. Biota-Sediment-Accumulation-Factor (BSAF) was applied to evaluate the PAHs input sources from sediment. Condition index was calculated to compare the seasonal variation of clam tissue to PAHs levels. To propose results not affected by seasonal changes in flesh weight of clams, the approach based on the calculation of PAHs/SW index was applied. From the results, it was concluded that PAHs/SW index is more recommendable to asses temporal variation of PAHs levels in Manila clams.     

Polycyclic aromatic hydrocarbons (PAHs) in transplanted Manila clams (Tapes philippinarum) from the Lagoon of Venice as assessed by PAHs/shell weight index: A preliminary study

Variation of polycyclic aromatic hydrocarbons (PAHs) levels was assessed in Tapes philippinarum from the Lagoon of Venice. Clams were transplanted from a polluted area next to Porto Marghera to two rearing areas of the Southern Lagoon. Analyses of PAHs were made in sediments and clams by GC/MS at first sampling and after 30, 60 and 180 days. Principal component analysis was performed to elucidate bioaccumulation and depuration pattern and input sources. Biota-Sediment-Accumulation-Factor (BSAF) was applied to evaluate the PAHs input sources from sediment. Condition index was calculated to compare the seasonal variation of clam tissue to PAHs levels. To propose results not affected by seasonal changes in flesh weight of clams, the approach based on the calculation of PAHs/SW index was applied. From the results, it was concluded that PAHs/SW index is more recommendable to asses temporal variation of PAHs levels in Manila clams.     

Induction of DNA strand breaks in the mussel (Mytilus trossulus) and clam (Protothaca staminea) following chronic field exposure to polycyclic aromatic hydrocarbons from the Exxon Valdez spill

In 2002, 13 years after the Exxon Valdez spill, mussels and clams were examined for lingering oil exposure and damage. Known oil patches were sampled at four locations, and compared to nearby reference areas (same bay), and were also compared to "hot reference" sites to verify the methods used (Cordova harbor and fresh diesel spill at Port Chalmers). Passive samplers deployed for a month at the sites, along with tissue samples, confirmed that the oiled sites were oiled (fingerprinting back to Exxon Valdez oil) and that reference sites were clean. The highest PAH loads were detected in sub-surface interstitial waters at oiled sites. Exposure at the surface was generally low level, and probably intermittent. DNA damage was assessed in blood cells using sensitive comet analyses. DNA strand breakage was detected in both mussels and clams, with the highest level of damage detected at "hot reference" sites of Cordova harbor and Port Chalmers. Bioavailability and DNA damage at the oiled sties was low, indicating there has been substantial progress in recovery from the spill 13 years before, yet low level bioavailability and damage were still detectable.     

Acetylcholinesterase as a biomarker of exposure to neurotoxic compounds in the clam Tapes philippinarum from the Lagoon of Venice

Measurement of acetylcholinesterase (AChE) is widely used as a biomarker of exposure to neurotoxic compounds in aquatic environments. In the present study, AChE activity was monitored in the clam Tapes philippinarum in the Lagoon of Venice, with the aim of evaluating the possible exposure of animals to neurotoxic compounds. Two sampling strategies were followed. In the first case, temporal and spatial variations of AChE activity were measured in clams collected seasonally (from October 2003 to June 2004) in 3 sites of the Lagoon: Campalto (site 1), near a sewage treatment plant, Marghera (site 2), a highly polluted area, and Poveglia (site 3), a reference site. The condition index (CI) of clams was also calculated. In the second case, AChE activity was measured in clams collected at the end of seasonal samplings in 12 sites located throughout the Lagoon: areas licensed for clam farming (sites 5 to 11), nearby canals characterised by waste waters from agricultural areas (sites 4, 12, 13, 14), and a reference site outside the Lagoon (site 15). Results revealed a significant reduction in AChE activity in clams from Marghera in October, January and April, indicating their probable exposure to neurotoxic compounds originating in the industrial zone of Porto Marghera. A seasonal trend in AChE activity was also observed, enzyme activity being higher in January and lower in June. Clams from Campalto generally had higher CI values than those from Marghera and Poveglia. However, no particular correlation between AChE activity and CI was found. Lastly, marked differences in AChE activity were recorded when comparing enzyme activity of clams from various sites in the Lagoon of Venice with those of clams collected at site 15. Significant reductions in AChE activity were observed in animals collected in both nearby canals and licensed areas, indicating the homogeneous spatial distribution of potentially neurotoxic compounds throughout the Lagoon. It is probable that sediment mobilisation caused by clam harvesting and the passage of shipping plays an important role in resuspension of persistent contaminants having neurotoxic activity.     

Clam burrowing behaviour: Inhibition by copper-enriched sediment

Burrowing behaviour is adaptive and allows clams to escape predation; yet the effects of potentially toxic metals on such behaviour have not been adequately investigated. In natural marine sediment contaminated with copper the time for littleneck clams (Protothaca staminea) to achieve complete burial was recorded. Above a threshold of 5.8 μg g⁻¹ Cu added to dry sediment, the time for 50% of the clams to burrow (ET₅₀) increased logarithmically with increasing sediment copper concentration according to:

logET₅₀ = 0.15 (CU) - 1.37 (n = 4, r² = 0.98)

where ET₅₀=time in hours for 50% of clams to burrow and Cu=μg g⁻¹ Cu in dry sediment. Previously exposed clams had both a lower threshold to Cu and a longer reburrowing time (ET₅₀). Clams exposed to sediment mixed with Chelex-100®-sorbed copper showed no significant change in burrowing time. Bioassays based on claim burrowing behaviour can measure both bioeffectiveness of sediment-sorbed metals and a sublethal effect with ecological meaning.     

Use of the clam Asaphis deflorata as a potential indicator of organochlorine bioaccumulation in Hong Kong coastal sediments

In order to test its ability as a bioaccumulator, the deposit feeding clam Asaphis deflorata, collected from a clean coastal area in Hong Kong (Ding Chau, Ma On Shan), was exposed to field-collected sediments representing a gradient of contamination. Sediments were collected from four sites, namely Shek O in the southeast of Hong Kong Island, Ap Lei Chau in Aberdeen district, Tai Kok Tsui in the Mong Kok waterfront, and To Kwa Wan near Kowloon City pier. Shek O was the cleanest and To Kwa Wan was the most polluted site. Replicate 30L fiberglass tanks containing equal numbers of organisms were maintained for 28 days, with equal quantities of sediment and filtered seawater. Prior to this the animals were depurated for 10 days in clean sediment. During the 28-day holding period, sampling of both sediment and clams was performed on days 0, 10 and 28, along with condition index measurements. Concentrations of organochlorine pesticides and PCBs in sediments showed that Shek O was the cleanest of the sites, followed by Ap Lei Chau, Tai Kok Tsui and To Kwa Wan. There were marked differences among the sites, especially in PAH concentrations, with To Kwa Wan showing extremely high sediment concentrations. Following exposure, the biota concentrations of various organochlorine pesticides and PCBs suggested that there was sediment concentration-dependent accumulation by A. deflorata on day 10 of the experiment. However, there was no significant increase on day 28 for any of the compounds tested. Contaminant concentrations in clams feeding on Shek O sediments were minimal and almost the same on all sampling days, indicating the presence of only baseline contaminant concentrations. The condition indices (CI) of the clams on various sampling days showed that the animals were relatively more stable in sediment from Shek O. There were some deaths in all the test tanks (including those containing Shek O sediments). In addition, clams appeared to bury faster in the cleaner Shek O sediment and remained buried, unlike those in the sediment from the other locations, where some remained predominantly on the surface. Nevertheless, despite not completely burying, the clams in other test sediments still exhibited sediment concentration-dependent tissue accumulation. The results of the investigation indicate the possibility of developing a standard test protocol for bioaccumulation by sediment dwelling invertebrates, for practical use in Hong Kong, using A. deflorata.     

Biomarkers in croakers Micropogonias furnieri (Teleostei: Sciaenidae) from polluted and non-polluted areas from the Patos Lagoon estuary (Southern Brazil): evidences of genotoxic and immunological effects

Biomarkers of exposure and effect of pollutants were analyzed in croakers Micropogonias furnieri (Teleostei: Sciaenidae) captured in winter and summer in a polluted and in a non-polluted site at the Patos Lagoon estuary (Southern Brazil). Catalase and glutathione S-transferase activities (exposure biomarkers) and lipid peroxidation (effect biomarker) were analyzed in liver samples. Other two effect biomarkers were also studied: blood cells DNA damage (through comet assay and micronucleus test) and respiratory burst measurements. In a broad view, results point to an important seasonal variation of the biochemical biomarkers analyzed. However, data obtained clearly indicate that croakers collected in winter at the polluted site were subjected to a level of clastogenic agents sufficient to generate irreversible genetic damages (mutations) and impair the fish immune system.     

Oxidative stress in the mussel Mytella guyanensis from polluted mangroves on Santa Catarina Island,Brazil

Digestive glands of the mangrove mussel Mytella guyanensis, collected at one non-polluted site (site 1) and two polluted sites (sites 2 and 3), were analysed for different antioxidant defenses, lipid peroxidation and DNA damage. Thiobarbituric acid-reactive substance (TBARS) and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels were enhanced at the polluted sites. With the exception of superoxide dismutase, the activities of catalase and glutathione peroxidase were also higher at the polluted sites. Greater increases were observed in glutathione reductase, glutathione S-transferase and etoxyresorufine-O-deethylase activities at the polluted sites. Conversely, reduced glutathione content was higher at the control site. Trace metal contents in mussels collected at polluted sites were increased compared to the control site, and there were strong positive correlations between TBARS and Cu and Pb contents. M. guyanensis is routinely exposed to an oxidative stress condition at both polluted sites, and considering xenobiotic bioaccumulation in bivalve molluscs, the mangrove mussel represents an excellent bioindicator for environmental monitoring studies.     

Characteristic analysis of phospholipid fatty acids(PLFAs) in typical nutrient polluted lake sediment in Wuhan

The process of urbanization aggravates the endogenous pollution of urban lake sediment,and polluted sediment may seriously affect the quality of the water in lakes.At present,it is difficult to distinguish the difference between sediment that is heavily polluted by nitrogen(N)and phosphorus(P)when using an analysis based on a physicochemical index classification.The current study applied phospholipid fatty acid(PLFA)technology to further analyze the sediment characteristics from the perspective of microbiology.Surface sediment from five urban lakes that are heavily polluted with N and P in Wuhan were sampled.Statistical approaches were used to analyze the microbial community structure in the sampled sediment,and to determine the correlations between the microorganisms and physicochemical indices.The most severely polluted lake sediment had similar PLFA structures,the highest saturated fatty-acid content,and bacteria as the dominant microorganism.However,there were differences between the microbial biomass of the various sediment samples,which may have been related to the degree of N and P pollution.Analysis of the microbial diversity in the sediment samples indicated that the bacteria were experiencing starvation and nutrient pressure,which may have been due to the dissolved oxygen concentration of the heavily polluted lake sediment.A correlation analysis showed that the endogenous N and P had different effects on the microbes of the polluted sediment.A redundancy analysis(RDA)demonstrated that the N/P ratio had the greatest influence on the PLFA species,accounting for 83%of the cumulative interpretation.To effectively promote the role of sediment microorganisms on circulating elements,it is necessary to regulate the N/P ratio of the sediment to some extent.When the N/P ratio in sediment exceeds 6,N pollution should be prioritized.     

Field validation of antioxidant enzyme biomarkers in mussels (Perna viridis) and clams (Ruditapes philippinarum) transplanted in Hong Kong coastal waters

Green-lipped mussels, Perna viridis, and Manila clams, Ruditapes philippinarum were sourced from “clean” sites in the Hong Kong region, depurated in a laboratory using uncontaminated filtered seawater for 8 days, and transplanted to a suspected gradient of chemically polluted sites in Hong Kong. After 14- and 28-days of field exposure, several antioxidant parameters including glutathione S transferase (GST), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH) were quantified in gill and hepatopancreas tissues. Whole body tissue concentrations of polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons (PHCs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs) were determined in pooled site samples. Chemical analyses indicated that: (a) clams had higher levels of PAHs, PHCs, DDTs and PCBs, whereas mussels had higher hexachlorocyclohexane (HCHs) and there was no difference between species for dieldrin and remaining OCs; (b) Kat O should not be continued as a “clean” reference site for Hong Kong, because of the levels of contaminants measured and (c) PAH concentrations in the current survey were similar to those previously measured. Toxicological conclusions were: (a) antioxidant responses were different between species; (b) CAT and GST have highest utility in clams for field use in Hong Kong, whereas CAT in both gill and hepatopancreas tissue showed most potential in mussels; (c) significant induction of antioxidant responses over day 0 (excluding GPx in both tissues, and GST in mussel hepatic tissue); (d) groups of contaminants do not consistently induce antioxidant responses and (e) organochlorines and PCBs correlated significantly with CAT and GST in clam hepatopancreas and with CAT in mussel gill and hepatic tissue. Multivariate statistical techniques indicated little relationship between the site patterns for antioxidant responses and the contaminant gradients identified in body burden analysis.     

Drug-resistance transfer in Escherichia coli in New York Bight sediment

Sediment from the sewage sludge dump site area in the New York Bight contains bacteria resistant to antibiotics and heavy metals. The purpose of this study was to determine if antibiotic resistance could be transferred from donor to recipient Escherichia coli strains inoculated into glass vessels containing sediment and seawater obtained near the dump site. Temperature was maintained at 10°C, the mean winter temperature of benthic water at the dump site. Transconjugants (recipients which inherit donor genes for tetracycline resistance) were isolated from the seawater and sediment within one hour after inoculation and were found in all subsequent sediment samples for one month. Donor and recipient E. coli remained viable in the sediment for at least one month. Our results indicate that sewage sludge polluted sediment may serve as an environment conducive to conjugal transfer of antibiotic resistance genes.     

Bioindicative potential of shell abnormalities occurring in the clam Macoma balthica (L.) from the Baltic Sea

This study reports the occurrence of shell deformities in the Baltic clam Macoma balthica from the Northern Baltic Sea (Trösa Archipelago, Sweden). The functional significance and the bioindicative potential of observed exoskeleton’ anomalies was assessed using a suite of physiological and morphological biomarkers.Analyzed shell deformations (SD) included damages of the dorsal margin visible as shell excavations, destruction of the beak, umbo and hinge ligament and decreased shell’ transparency. Deformed clams exhibited worse physiological conditions and increased frequency of micronuclei. Skewed sex ratio towards male domination was observed in all studied populations. Spatial differences in the occurrence of deformed clams are reported, with more than 50% of deformed clams inhabiting polluted locations and 8% the reference station. These results are encouraging for the use of described SDs as initial and cost effective indicators of environmental health.