Toxicological Effects Induced by Cadmium in Gills of Manila Clam Ruditapes philippinarum Using NMR‐Based Metabolomics

Cadmium (Cd) has become an important heavy metal contaminant in the sediment and seawater along the Bohai Sea and been of great ecological risk due to its toxic effects to marine organisms. In this work, the toxicological effects caused by environmentally relevant concentrations (10 and 40 µg L^?1) of Cd were studied in the gill tissues of Manila clam Ruditapes philippinarum after exposure for 24, 48, and 96 h. Both low (10 µg L^?1) and high (40 µg L^?1) doses of Cd caused the disturbances in energy metabolism and osmotic regulation and neurotoxicity based on the metabolic biomarkers such as succinate, alanine, branched chain amino acids, betaine, hypotaurine, and glutamate in clam gills after 24 h of exposure. However, the recovery of toxicological effects of Cd after exposure for 96 h was obviously observed in clam to Cd exposures. Overall, these results indicated that NMR‐based metabolomics was applicable to elucidate the toxicological effects of heavy metal contaminants in the marine bioindicator.     

Distribution and bioaccumulation of polychlorinated biphenyls and organochlorine pesticides residues in sediments and Manila clams (Ruditapes philippinarum) from along the Mid-Western coast of Korea

The distribution and bioaccumulation features of PCBs, DDTs, and HCHs were investigated in the sediments and Manila clams collected from along the Mid-Western coast of Korea. The measured concentrations of ΣPCBs, ΣDDTs and ΣHCHs were 1.08–3.5, 0.12–0.35 and 0.090–0.30 ng g⁻¹ dw in sediment, and 33–390, 7.4–46 and 6.3–27 ng g⁻¹ lipid in Manila clam, respectively. Their levels were found to be relatively lower than those of other contaminated areas and the consumption of Manila clam from these areas seems to be safe for human health according to calculated lifetime cancer risk and hazard indices. The ΣPCBs and ΣDDTs concentrations in sediments showed a significant relationship to those in clams. The significant correlation was observed between BSAF in clams and K_ow for each PCB congeners and DDT metabolites. These findings support that the PCBs and DDTs levels, which are highly hydrophobic chemicals, in clam reflect the sediment pollution through bioaccumulation.     

Assessing benthic ecological impacts of bottom aquaculture using macrofaunal assemblages

Bottom aquaculture of bivalves is a high-yield culture method, which is increasingly adopted by shellfish farmers worldwide. However, the effects of bottom aquaculture on benthic ecosystems are not well-known. Manila clam (Ruditapes philippinarum), is a widely distributed bottom aquaculture mollusk species. To assess the ecological impacts of Manila clam bottom aquaculture, clams and other macrofaunal assemblages were investigated during four cruises (July and November 2011, February and May 2012) at six sampling sites in Jiaozhou Bay, China. Correlation analysis showed that macrofaunal assemblages had significant negative correlations with the abundance of Manila clams. However, according to the results of several biotic indices, a low disturbance was detected by Manila clam bottom aquaculture. In conclusion, AMBI (AZTI'S Marine Biotic Index) and M-AMBI (Multivariate AZTI Marine Biotic Index) indices are more suitable for assessing ecological quality than polychaete/amphipod ratios when the disturbance is slight, such as at a bivalve bottom aquaculture.     

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.     

Clam burrowing behaviour and mortality related to sediment copper

Gravel sediment freshly enriched with over 4.4 μg Cu g^?1 significantly increased the burrowing and reburrowing times of Littleneck marine clams (Protothaca staminea). Abnormal burrowing behaviour was observed with time-lapse video. Aged (1 day) copper-enriched sediment did not affect clam burrowing time. Copper in sediment porewater declined three days after sediment enrichment to background levels without change in total sediment copper. In a long-term experiment, clams with increased burrowing time into copper-enriched sediment had up to 25% mortality after 12–29 days burial in the sediment. The majority of clam mortality was found in clams burrowing within the first 48 h. Over 7 weeks there was blackening of sediment and clam shells proportional to sediment copper enrichment.     

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.     

Physiological disturbance of the Manila clam,Ruditapes philippinarum,by altered environmental conditions in a tidal flat on the west coast of Korea

To examine the influence of habitat alteration by tideflat reclamation on the physiological ecology of Ruditapes philippinarum, seasonal variations in its condition, gross biochemical composition, and reproductive cycle were compared for a yearly cycle between a reclaimed flat (Gomsohang, GS) and a natural flat (Hajun, HJ) in Gomso Bay, Korea. Concentrations of chlorophyll a and seston (as well as its energy value as food available to the clam) were consistently higher at HJ than at GS. Condition, dry tissue weight, and energy reserves (proteins and carbohydrates) of the clams were much higher at HJ than at GS during spring–summer, when fast growth and gametogenic development occur. Furthermore, their spring gametogenic development and spawning were advanced at HJ compared with GS. Our results suggest that the Manila clam encounters nutritionally stressful environmental conditions in altered habitats after tideflat reclamation that lead to lowered nutrient accumulation and a changing reproductive cycle.     

Does Manila clam cultivation affect habitats of the engineer species Lanice conchilega (Pallas, 1766)?

The major French site of Manila clam Ruditapes philippinarum (Adams and Reeves, 1850) cultivation is located in the Chausey Archipelago where the associated practices are highly mechanized: every steps of production are made with tractor-driven machinery. The Manila clam concessions are concentrated on Lanice conchilega (Pallas, 1766) bioherms, which are known to increase alpha-diversity and to locally modify sediment dynamics. This study focus on the impacts of Manila clam cultivation on (i) the natural populations of L. conchilega and on (ii) the structure of the associated benthic assemblages during the different steps of the farming production cycle. We found that the L. conchilega populations are significantly affected within the concessions where their total abundances drastically decrease, their spatial patterns are modified and the associated benthic assemblages are significantly altered. Our results are discussed in a context of a sustainable management of the Manila clam cultivation in coastal areas.     

The effects of salinity on the Manila clam (Ruditapes philippinarum) using the neutral red retention assay with adapted physiological saline solutions

This study investigated the internal osmotic regulatory capabilities of the Manila clam (Ruditapes philippinarum) following in vivo exposure to a range of salinities. A second objective was to measure the health status of the Manila clam following exposure to different salinities using the neutral red retention (NRR) assay, and to compare results using a range of physiological saline solutions (PSS). On exposure to seawater of differing salinities, the Manila clam followed a pattern of an osmoconformer, although they seemed to partially regulate their circulatory haemolytic fluids to be hyperosmotic to the surrounding aqueous environment. Significant differences were found when different PSS were used, emphasizing the importance of using a suitable PSS to reduce additional osmotic stress. Using PSS in the NRR assay that do not exert additional damage to lysosomal membrane integrity will help to more accurately quantify the effects of exposure to pollutants on the organism(s) under investigation.     

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.     

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.     

Exposure of the clam Tapes philippinarum to 4-nonylphenol: changes in anti-oxidant enzyme activities and re-burrowing capability

The effects of 4-nonylphenol on anti-oxidant enzyme (superoxide dismutase and catalase) activity in both gills and digestive gland and re-burrowing capability of the clam Tapes philippinarum were investigated after 7 days' exposure to various sublethal concentrations of nonylphenol (NP). NP caused a significant decrease in superoxide dismutase activity in both tissues from the lowest concentration tested, whereas no significant alteration with respect to controls was observed in catalase activity. NP effects on re-burrowing of clams were evaluated in two experiments. In the first, clams exposed to the highest NP concentrations showed a marked delay in re-burrowing 48 h after the end of exposure. Modifications in normal burrowing behaviour were also observed, suggesting that NP acts via narcosis and/or neurotoxic mechanisms. In the second experiment, the elapsed time for 50% of the exposed clams to re-burrow into the sediment (RT(50)) was also calculated in a 24-h re-burrowing response test. At the highest NP concentrations, it was not possible to estimate RT(50) values, as the percentage of clams showing complete burial was less than 50%. The present study demonstrates that NP causes oxidative stress by inhibiting superoxide dismutase activity and greatly affects the re-burrowing capability of T. philippinarum, even at environmentally realistic concentrations.     

Effects of hypoxia in the gills of the Manila clam Ruditapes philippinarum using NMR-based metabolomics

Coastal hypoxia affects the survival, behavior, and reproduction of individual local marine organisms, and the abundance, biomass, and biodiversity of coastal ecosystems. In this study, we investigated the chronic effects of hypoxia on the metabolomics in the gills of Ruditapes (R.) philippinarum. The results indicated significant alterations in the metabolite profiles in the gills of the hypoxia-treated clams, in comparison with those maintained under normoxia. The levels of betaine, taurine, glycine, isoleucine, and alanine were significantly reduced, suggesting a disturbance of osmotic balance associated with hypoxia. Meanwhile, metabolites involved in energy metabolism, such as alanine and succinate, were also affected. Dramatic histopathological changes were observed in the gills and hepatopancreases of R. philippinarum grown in hypoxic waters, demonstrating tissue damages apparently caused by long-term exposure to hypoxia. Our findings suggest that hypoxia significantly affects the physiology of R. philippinarum, even at a sub-lethal level, and impedes health of the clams.     

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.     

Arsenic bioaccessibility and speciation in clams and seaweed from a contaminated marine environment

The bioaccessible concentration and speciation of arsenic (soluble in a gastrointestinal medium and available for absorption into the bloodstream) were determined in softshell clams (Mya arenaria), harvested by local residents until 2005, and in seaweed (Fucus sp.) from an arsenic-contaminated marine site in Seal Harbour, Nova Scotia, Canada. Bioaccessibility extractions to simulate the human gastrointestinal environment (pH 1.5 and glycine for 1h followed by pancreatin, bile extract and pH adjustment to 7 for an additional 4h) and speciation of arsenic in extracts (HPLC-HG-AAS to target inorganic arsenic species) and whole samples (XANES) were carried out. Total arsenic for the clams from the contaminated area ranged from 218 to 228 ppm wet weight, with a bioaccessible fraction of 34-46%, and the major bioaccessible species of arsenic were inorganic. The seaweed from the contaminated area contained 27-43 ppm wet weight total arsenic, with the bioaccessible fraction ranging from 63% to 81%, and inorganic arsenic was also predominant. The predominantly inorganic nature of arsenic in the whole samples was confirmed by XANES. In concurrence with the closure of the area for clam harvesting, the clams and seaweed from Seal Harbour should probably not be used for human consumption.     

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.     

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.     

Potential link between exposure to fungicides chlorothalonil and mancozeb and haemic neoplasia development in the soft-shell clam Mya arenaria: A laboratory experiment

The aetiology of haemic neoplasia (HN) is unknown, so far but many causative factors are suggested such as viral, pollution and genetics. The aim of this study was to determine if, under chronic exposure, two major pesticides (chlorothalonil and mancozeb) which are used in potato production could induce HN in soft-shell clams (Mya arenaria). Short-term experiments with acute exposure were also performed. Clams were collected from an epizootic site (North River, PEI) and from a site free of the disease (Magdalen Islands, Quebec). The tetraploid level of haemocytes was assessed by flow cytometry for each clam to determine the HN status. The bioaccumulation of pesticides in tissues was quantified by gas chromatography/mass spectrometry (GC/MS) for chlorothalonil while mancozeb and manganese were quantified by inductively coupled plasma-mass spectrometer (ICP/MS). Long term exposure to fungicide Bravo 500^® did not induce high tetraploid levels on negative calm from North River and the analysis of the digestive gland and the mantle did not reveal any detectable level of chlorothalonil. In the Manzate 200 DF^®, some clams revealed high level of tetraploid cells but no difference were observed between the treatments and the control. The analysis of the digestive gland and the mantle for manganese did not highlight any significant difference in tissue concentration (p = 0.05). For the acute exposure, chlorothalonil analysis showed that the active ingredient is distributed between four chlorinated compounds: 99.5% for chlorothalonil isomers, 0.4% for pentachlorothalonil and 0.1% for trichlorothalonil isomers. For a 72 h experiment, the accumulation was within 4 h; the higher tissue concentration of chlorothalonil was 59.2 μg g⁻¹ in the mantle after 48 h, following by a decrease to an undetectable level at the end. For the manganese, the accumulation was detected after 4 h; the higher tissue concentration was 48.8 μg g⁻¹ in the mantle after 24 h and, over the following 48 h, the accumulation decreased until the end of the trial. Based on the data, the accumulation of these fungicides seems to be transitory. Chlorothalonil and mancozeb are both oxidative-stress promoters and could have induced cell dysfunction while in the tissue. Study on the effect of these fungicides on the p53 protein system is an example of strategy that would provide information on cellular events promoting neoplasia.     

Abnormal features of Macoma balthica (Bivalvia) in the Baltic Sea: alerting symptoms of environmental adversity?

Recent studies of the Baltic clam Macoma balthica (L.) from the southern Baltic (the Gulf of Gdansk) have revealed striking morphological, histological and cytogenetic features. Strong deformation of the shell, including elongation of the posterior end and the appearance of an easily visible flexure in this part, has been recorded. The population contribution of the deformed blunt shelled ("irregular") clams ranged from 0% to 65% and tended to increase with depth. The morphologically "irregular" clams had higher accumulated tissue concentrations of trace metals (As, Ag, Cd, Pb, Cu and Zn), indicating a different metal handling ability. Adverse conditions in deeper water regions of the Gulf (e.g. hypoxia, hydrogen sulphide, elevated bioavailability of contaminants) have been suggested as inducers of the phenotypical changes (morphological deformation) in part of the population and, in parallel, of the specific physiological adaptations that result in higher metal accumulation in the "irregular" clams. Cytogenetic and histological analyses showed the presence of tumours in gill cells and digestive system of the affected clams, the prevalence of disseminated neoplasia ranging from 0% to 94% depending on the site. The disease was manifested by a modified karyotype (i.e. an abnormal number and morphology of chromosomes), a higher activity of nucleolar organizer regions (AgNORs), and tissue lesions (enlarged cells, actively proliferative with pleomorphic nuclei). Bottom sediments showed acute toxicity and have been proposed as a source of an initialising carcinogenic factor. However, none of the ecotoxicological studies provided was successful in the clear demonstration of a single (or multifactorial) agent that can account for the disseminated neoplasia.