Micronucleus induction in gill cells of green-lipped mussels (Perna viridis) exposed to mixtures of polycyclic aromatic hydrocarbons and chlorinated pesticides

Different groups of green-lipped mussels (Perna viridis) were exposed to the same net amount of a genotoxicant mixture of four polycyclic aromatic hydrocarbons ([PAHs]; anthracene, fluoranthene, pyrene, and benzo[a]pyrene) and four organochlorine pesticides ([OCs]; alpha-hexachlorocyclohexane (HCH), aldrin, dieldrin, and p,p'-DDT) for four weeks under different regimes that simulated various scenarios of fluctuating toxicant levels in the marine environment. Micronucleus (MN) formation in gill cells was studied at the end of each week. Micronucleus frequencies increased with continual addition of genotoxicants, and did not diminish significantly under conditions of either gradually decreasing concentrations or cessation of exposure for one to two weeks, suggesting that the MN response may persist over relatively long exposure periods. An almost two-fold higher mean MN frequency was recorded in a chronic exposure group than in an acute group that had received the same net nominal dose of genotoxicants, indicating that chronic exposure may lead to a greater genotoxic impact than acute exposure. The results suggested that in field studies, MN response should be monitored at multiple time points in order to elucidate the effects of potentially fluctuating toxicant levels. Finally, MN formation was positively correlated with both nominal contaminant levels and tissue levels of the genotoxicants. These findings suggest that MN responses can be a sensitive indicator of exposure to relatively low levels of genotoxicants and that MN response in mussel gill cells can be a stable biomarker of genotoxicity.     

Antioxidant responses and bioaccumulation in Ictalurus melas under mercury exposure

Laboratory experiment was carried out to determine mercury accumulation in tissues (gills, kidneys, liver, and muscle) and biochemical responses in the liver of freshwater teleost Ictalurus melas. Catfish were subjected to different concentrations of Hg(2+) (35, 70, and 140 microg/L) for 10 days. The chemical analyses showed higher mercury concentrations for all treatments in gills and kidneys followed by liver and muscle. At the lowest mercury concentration a decrease in glutathione (GSH) content and an increase of GSH peroxidase Se-dependent and glyoxalase II enzymes were observed. An increasing trend was observed also for GSH-S-transferase and glyoxalase I, while GSH peroxidase Se-independent enzyme and GSH reductase showed no significant variation in activities. The increase in the enzymes activities of catfish, involved in the inactivation of reactive molecules formed during oxidative stress, could provide an additional protection against the oxidative damage induced by mercury.     

New Zealand green-lipped mussels (Perna canaliculus) enhance non-haem iron absorption in vitro

Fe bioavailability can be manipulated by the nutritional composition of a meal. Ascorbic acid and unidentified components of meat, fish and poultry, but particularly beef, all appear to enhance the absorption of non-haem Fe. The aim of the present study is to identify whether extracts of green-lipped mussels (GLM; Perna canaliculus) enhance non-haem Fe absorption in Caco-2 cells and to compare the effect with that of beef. Raw GLM and raw beef homogenates were digested in vitro with pepsin at pH 2, and pancreatin and bile salts at pH 7. Tracer 55Fe was used to measure cellular Fe uptake. Ascorbic acid was used as a positive control and egg albumin, exposed to the same in vitro digestion process, was used as a negative control. Caco-2 cell monolayers were incubated with treatments for 60?min. All values were standardised per μg of GLM, egg albumin, beef or ascorbic acid. The results showed that ascorbic acid enhanced non-haem Fe absorption to the highest degree. Beef and GLM digestates both significantly enhanced Fe absorption compared with egg albumin. In conclusion, GLM digestate significantly enhances non-haem Fe uptake in Caco-2 cells with a similar magnitude to that of beef.     

Interactions of silver, cadmium, and copper accumulation in green mussels (Perna viridis)

Metal interaction is vital for assessing the use of aquatic organisms in monitoring metal contamination. The present study examined the interactions between Ag and Cd accumulation and between Ag and Cu accumulation in the green mussel (Perna viridis). Accumulation of Ag and Cd in the whole tissue of green mussels exposed to 5 microg/L of Ag and 20 microg/L of Cd for two weeks was independent; however, interaction was observed at the subcellular level. Approximately 25% of Ag shifted from the insoluble fraction (IF) to the metallothionein-like protein in the presence of Cd, which probably resulted from the competition of Cd on IF in the Ag-Cd coexposure. On the other hand, coexposure of the mussels to Ag (5 microg/L) and Cu (30 microg/L) for two weeks increased the Ag and Cu concentrations in the tissue synergistically (two- to fivefold), but Ag and Cu subcellular distributions were similar in the coexposed and the singly exposed mussels. Exposure to Ag alone increased the dietary uptake of Ag by 30%, but the effect was reduced in the presence of Cd. No interaction, however, was observed between uptake rates of metals from the dissolved phase. To conclude, a significant interaction was observed for total Ag and Cu accumulation, but not for total Ag and Cd accumulation, in the mussels. Metal interaction is more likely to be observed at the subcellular level than at the whole-tissue level.     

Tissue-specific toxicological effects of cadmium in green mussels (Perna viridis): nuclear magnetic resonance-based metabolomics study

Toxicity tests for metals have traditionally focused on selected biomarkers to characterize the biological stress induced by metals in marine organisms. Here nuclear magnetic resonance (NMR)-based metabolomics, a system biology tool, was applied to the marine green mussel, Perna viridis, to investigate the toxicological effects of Cd in both digestive gland and adductor muscle tissues. After Cd exposure for either two or four weeks, there was no significant metabolic change in the mussels exposed to Cd at 2?μg/L. At 20?μg/L, there were major metabolite changes related to amino acids, osmolytes, and energy metabolites. Digestive gland tissue was more sensitive to Cd than adductor muscle tissue. The adductor muscle tissue showed elevated levels of glutamine, glutamate, and lactate, and reduced levels of branched chain amino acids, aspartate, phenylalanine, and tyrosine. Overall, four weeks of Cd exposure produced neurotoxicity and metabolic disturbances and disturbed osmoregulation. These results suggest that the adductor muscle tissue of mussels may be a suitable supplemental biomarker for exposure to toxicants. In addition, the results demonstrate that (1) H-NMR-based metabolomic analysis can provide a systematic view of the toxicological effects of metals on mussels, suggesting that it might be employed to investigate the toxicological effects of other marine pollutants.     

Modeling of cadmium bioaccumulation in two populations of the green mussel Perna viridis

This study attempted to quantify differences in Cd biokinetics from two populations of green mussels (Perna viridis) from two sites (eastern and western) in Hong Kong with contrasting hydrological conditions. Body Cd concentrations were modeled using a simple biokinetic model coupled with measurements of dissolved Cd concentrations at each site. Mussels collected from the western site had three to six times higher Cd tissue concentration than the eastern population collected during two seasons (summer wet and winter dry), but the salinity was only lower in the western site during the summer. More Cd was distributed in the metallothionein-like and heat-sensitive proteins in the western population than the eastern population, and Cd predominantly was distributed in the insoluble fraction during summer. The Cd uptake rate constant from the dissolved phase was higher in the western population during summer due to a much lower salinity, but was comparable during winter. Dietary uptake of Cd was similar in both populations, and assimilation was lower from ingested radiolabeled seston than from diatoms. Efflux of Cd remained comparable between the two populations from two seasons (0.02-0.03 /d). Kinetic modeling demonstrates that the faster influx of Cd from aqueous phase caused the higher body Cd concentrations in the western population. The predicted Cd concentrations in mussels were comparable to those observed in the field. Our study highlights differences in Cd accumulation kinetics in different populations of mussels likely caused by the different physical environments.     

Occurrence of polychlorinated biphenyls and polybrominated diphenyl ethers in green mussels (Perna viridis) from Singapore, Southeast Asia

The green mussel, Perna viridis, was used in this study to measure levels of polychlorinated biphenyls (PCBs) and, for the first time, polybrominated diphenyl ethers (PBDE) in the marine environment. Samples were collected from eight different locations in the coastal waters of Singapore between April and May 2002. Forty-one PCB and 21 PBDE congeners were quantified by gas chromatography-mass spectrometry and were all positively detected in the mussel tissues. Total concentrations in green mussel tissues ranged from 6.1 to 82 ng/g and 2.0 to 38 ng/g on a dry-weight basis for PCBs and PBDEs, respectively. Such levels reflect the ubiquity of these persistent organic pollutants in a tropical marine environment. Principal component analysis was applied to the PCB data and revealed similarities in the congener composition of mussel tissues to that of the commercial PCB mixture, Aroclor 1254. The PBDE levels, to date, were approximately one order of magnitude greater than the upper concentrations reported for blue mussel (Mytilus edulis) tissues in Europe. At some sampling sites, the congener composition of PBDEs in P. viridis tissues indicated recent exposure to a commercial pentabrominated flame retardant.     

Uptake and depuration of paralytic shellfish toxins in the green-lipped mussel, Perna viridis: a dynamic model

Uptake and depuration of paralytic shellfish toxins in the green-lipped mussel, Perna viridis, were investigated by exposing the mussels to dinoflagellates (Alexandrium tamarense, ACTI01) under laboratory conditions for 8 d, then depurating them in clean seawater for 14 d. First-order linear differential equations were set up for five tissue compartments: Viscera, gill, hepatopancreas, adductor muscle, and foot. The solutions to these equations were used to fit the experimental data. We then estimated the parameters governing the model, which depend on the elimination rate from each compartment and the transfer coefficient between compartments. An assumption of the model is that the gills transport the dinoflagellates directly to the mouth and then to the viscera, where the ingested cells are broken down, releasing the toxins. The toxins absorbed are transferred to other tissues. During the uptake phase, the transfer coefficients from viscera to gill, hepatopancreas, adductor muscle, and foot were 0.03, 0.24, 0.01, and 0.004 per day, respectively. During the depuration phase, the transfer coefficients were 0.01, 0, 0.01, and 0.003 per day, respectively. In terms of the anatomical distribution of N-sulfocarbamoyl-11-hydroxysulfate (C2) toxins in various tissues, viscera and hepatopancreas contained the highest percentages (47-74% and 8-41%, respectively). Together, these two tissue compartments accounted for 71 to 96% of all C2 toxins present. The biokinetic model allows a quantitative prediction of C2 toxins in whole ussel as well as individual tissue compartments based on the density estimates and toxin load of dinoflagellate cells in the surrounding waters over time.     

Nutritional composition of three estuarine bivalve mussels,Perna viridis,Donax cuneatus and Meretrix meretrix

The present study determined the nutrient composition of three bivalve mussels. The biochemical constituents (protein, carbohydrate, lipid and vitamins), minerals (iron, copper, zinc, calcium, magnesium, and manganese) and carbon, nitrogen and calorific values were evaluated. There was a high degree of variation in biochemical constituents and mineral components among the tested mussels. Perna viridis exhibited good nutritional composition when compared with Donax cuneatus and Meretrix meretrix. The results of this study showed that the samples possessed appreciable quantities of all the dietary elements tested, which could make them partial or complete substitutes for conventional seafood.     

Nutritional composition of three estuarine bivalve mussels, Perna viridis, Donax cuneatus and Meretrix meretrix

The present study determined the nutrient composition of three bivalve mussels. The biochemical constituents (protein, carbohydrate, lipid and vitamins), minerals (iron, copper, zinc, calcium, magnesium, and manganese) and carbon, nitrogen and calorific values were evaluated. There was a high degree of variation in biochemical constituents and mineral components among the tested mussels. Perna viridis exhibited good nutritional composition when compared with Donax cuneatus and Meretrix meretrix. The results of this study showed that the samples possessed appreciable quantities of all the dietary elements tested, which could make them partial or complete substitutes for conventional seafood.     

Benzo[a]pyrene absorption and exposure pathways in the green mussel Perna viridis

The absorption efficiency (AE) of benzo[a]pyrene (BaP), a representative polycyclic aromatic hydrocarbon compound, in the green mussel Perna viridis was measured under different biological and food conditions (body size, food quantity, and food species). Uptake of BaP from the aqueous phase by different species of phytoplankton and green mussels was also experimentally quantified. A bioenergetic-based kinetic model was then used to separate the exposure pathways of BaP accumulation in the green mussels. Our results demonstrated that the AE of BaP in the green mussels ranged between 17 and 47%. Body size did not significantly affect the AE of BaP in the mussels. The AEs increased with a decrease in food availability, largely as a result of lengthening the gut passage of chemicals. The AEs differed by up to 2.3 times (17-39%) among the different food diets tested (four phytoplankton diets and one sediment diet). The BaP associated with the sediment was assimilated least efficiently. The difference in gut passage also appeared to account partially for the difference in AEs observed among different food types. No correlation between the AE and the distribution of BaP in the cytoplasm of phytoplankton cells was found. The concentration factor of BaP measured in four different marine phytoplankton species was in the range of 10(4)-5 x 10(4) (L/kg). The calculated uptake rate constant of BaP in the green mussels was 6.37 L/g/d, and the efficiency of absorption of BaP from the aqueous phase was 6.6 to 8.8%. The model calculation indicated that under most circumstances, >56% of BaP in mussels can indeed be derived from the uptake from the aqueous phase. However, the relative importance of aqueous and dietary exposure can be affected by the variation of AE, mussel's ingestion rate, and concentration of BaP in ingested food particles.     

Metal and oxygen uptake in the green mussel Perna viridis under different metabolic conditions

Coupled respirometric and radiotracer techniques were applied to simultaneously measure the rates of oxygen and metal uptake in the green mussel Perna viridis. This was performed under different metabolic conditions by varying the ambient oxygen partial pressure (P(O2)), temperature, air exposure, and body size. When the mussels were tested under different hypoxic and anoxic conditions, Cd and Zn uptake decreased with decreasing P(O2), accompanied by a decrease in the ventilation activity of mussels. Significant reduction in metal uptake was observed at a P(O2) level of 3 kPa. Under anoxic conditions, the uptake of Cd and Zn was 1.6 to 2.7 times and 2.8 times, respectively, lower than those measured under normoxia. In contrast, both the absorption efficiencies of Cd and Zn and the oxygen extraction efficiency increased significantly with decreasing P(O2). There were significant correlations between the rates of Cd/Zn and O2 uptake by the mussels when quantified under various P(O2) levels. The uptake of Cd and Zn was temperature dependent and increased with temperature over a range of 15 to 30 degrees C. Significant correlations between the rates of Cd/Zn and O2 uptake were also found in the temperature experiments. With reimmersion of mussels after aerial exposure, the mussels experienced an apparent O2 debt. Metal uptake also increased within the first 15 min followed by gradual recovery to the control levels. Similarly, the quantified uptake rates of Zn were significantly correlated with the O2 uptake in experiments with different sizes of mussels. These results strongly suggest that Cd and Zn uptake are coupled with oxygen uptake in the mussels; thus, physiological processes need to be considered in studying metal accumulation.     

Heavy Metals in Green Mussel (Perna viridis) and Oysters (Crassostrea sp.) from Trinidad and Venezuela

Heavy metal concentrations were monitored in edible soft tissues of shellfish from Trinidad and Venezuela. Oysters (Crassostrea sp.) and the green mussel (Perna viridis), which is a recently transplanted species to the Caribbean from the Far East, were collected at six locations in Venezuela and five in Trinidad, the latter along the coast line of the Gulf of Paria. Simple and low-cost methods of analysis were optimized and validated using standard reference materials. Cadmium, copper, lead, nickel, and zinc were analyzed by flame atomic absorption spectrometry. Mercury was determined by cold-vapor atomic absorption spectrometry. The present study has confirmed that oysters have a much greater capacity for accumulation of copper and zinc than does green mussel. In addition, concentrations of copper and zinc in oysters (Crassostrea sp.) at many of the sites in the Gulf of Paria exceeded local and international standards, whereas green mussel P. viridis contained generally acceptable levels for human consumption. Received: 13 June 2001/Accepted: 10 December 2001     

Antioxidant responses in the nereidid Laeonereis acuta (Annelida, Polychaeta) after cadmium exposure

The objective of this study was to evaluate the effects of the exposure to cadmium on the antioxidant responses in the polychaeta Laeonereis acuta. The worms were submitted to 0, 5, and 100 microg of Cd/L during a period of test of 7 days. Cadmium was significantly (p<0.05) accumulated in L. acuta in both concentrations assayed, but the concentration of reactive oxygen and nitrogen species (RONS) increased (p<0.05) only in the group submitted to the highest concentration of cadmium (100 microg/L). At this concentration, a decrease in the activity of the superoxide dismutase and an increase of glutathione-S-transferase activity (p<0.05) was observed. The levels of both lipid peroxides and the activities of catalase and glutathione peroxidase were not affected (p>0.05) by the exposition to cadmium. Thus, cadmium can augment RONS levels and can interfere with the antioxidant defense system of the polychaete L. acuta, although cadmium does not directly induce oxidative stress unlike copper and iron.     

Microcosm tributyltin bioaccumulation and multibiomarker assessment in the blue mussel Mytilus edulis

Blue mussels, Mytilus edulis, were exposed to two different concentrations of tributyltin (TBT) in seawater, 1,000 ng Sn/L (C1 experiment) and 10 ng Sn/L (C2 experiment), for 4 d, in order to evaluate the bioaccumulation of TBT by mussels Mytilus edulis in microcosms and to test the ability of a multimarkers analysis to determine the effects of TBT on the biochemical parameters in mussels. Tissue burdens of Mytilus edulis were 204 +/- 7 and 2,120 +/- 4 ng Sn/g TBT after the 4-d tests for the C2 and C1 experiments, respectively. Analyses of dissected organs and/or tissues demonstrated that TBT accumulated to the greatest extent in gills in the C1 experiment and in the digestive gland in the C2 experiment. Bioconcentration factors (BCFs) were 12,100 +/- 300 and 2,000 +/- 10 for mussels exposed in the C2 and C1 experiments, respectively. The four biomarkers used in this work were acetylcholinesterase (AChE), glutathione S-transferase (GST), catalase (CAT) activities, and thiobarbituric acid-reactive substances (TBARS) contents. No significant changes were observed in the measured enzyme activities or in TBARS concentration after the 4-d TBT exposure.     

Dietary mercury exposure and bioaccumulation in southern leopard frog (Rana sphenocephala) larvae

Aufwuchs was collected from three reservoirs, a constructed wetland used for groundwater treatment, and mercury (Hg)-enriched mesocosms to examine the relationship between inorganic Hg and methylmercury concentrations in the diet of tadpoles. Four diets were then formulated with Hg-enriched aufwuchs to concentrations that bracketed those of Hg observed in aufwuchs from the field and reported in the literature from sites contaminated by atmospheric deposition. The diets were fed to southern leopard frog tadpoles in the laboratory for the entire larval period (60-254 d). Metamorphs and tadpoles were analyzed for inorganic Hg and methylmercury contents by gas chromatography-cold-vapor atomic fluorescence spectrophotometry. Methylmercury concentration increased with total Hg concentration in aufwuchs, but the proportion of methylmercury to inorganic Hg decreased with increasing total Hg concentration. In the feeding experiment, there was an inverse relationship between Hg exposure concentration and the bioaccumulation factor for each Hg species. We concluded that neither methylmercury nor inorganic Hg in aufwuchs is highly bioavailable to tadpoles and that bioaccumulation is not well explained by a simple partitioning model. This suggests that bioaccumulation factors as currently used are not the best predictors of dietary Hg bioaccumulation.     

Immunotoxicity in channel catfish,Ictalurus punctatus,following acute exposure to tributyltin

Tributyltin (TBT) is a trialkylated organotin formulated for use primarily as a biocide for aquatic and agricultural industries. Although macrophages isolated from toadfish (Opsanus tau) are sensitive to exposure, very little is known about the effects of TBT on fish humoral immunity and non-specific cytotoxic cell (NCC) functions. To evaluate the effects of TBT on these parameters, channel catfish (Ictalurus punctatus) were given a single intraperitoneal injection of corn oil with 0 (vehicle control) or with 0.01, 0.1, and 1.0 mgTBT/kg as TBTCl. Three and seven days later, NCC activity and phagocyte oxidative burst were evaluated and related to allometric indices and hematology. The humoral immune response to Edwardsiella ictaluri was evaluated fourteen days after treatment. Peripheral blood neutrophilia and specific antibody secreting cell (SASC) numbers were the most sensitive parameters and were affected in all three TBT treatment groups. Allometric indices, peripheral blood lymphocyte and monocyte percentages, NCC activity, and phagocyte oxidative burst were less sensitive and were affected only at the highest dose of TBT.