Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay

The single-cell microgel electrophoresis assay or the comet assay was used to evaluate DNA damage of dispersed crude oil on sea urchins (Strongylocentrotus droebachiensis) and mussels (Mytilus edulis L.). Sea urchins were exposed to 0.06 and 0.25 mg/L dispersed crude oil in a continuous flow system, while the mussels were exposed to 0.015, 0.06 and 0.25 mg/L dispersed crude oil. Sea urchin coelomocytes and mussel haemocytes were sampled after 4 and 5 weeks exposure, respectively. In the sea urchin coelomocytes, there was a significant concentration-related increase in the percentage of DNA in comet tail. In mussel haemocytes, there was a significantly higher percentage of DNA in comet tail for all treatments compared to the control. The responses were concentration-related up to 0.06 mg/L oil. The two highest exposure concentrations of mussels were not significantly different from each other. These results indicate that the comet assay can be used for biomonitoring of DNA damage in marine invertebrates following oil contamination.     

DNA strand breaks (comet assay) and embryo development effects in grass shrimp (Palaemonetes pugio) embryos after exposure to genotoxicants.

Grass shrimp embryos develop in egg sacs (stages 1-10) attached to the female for 14-20 days after which they 'hatch' from the egg sacs into a swimming zoea stage (stage 11). Until they emerge from the egg sacs, embryos depend on lipids and lipovitellin stored within the egg. The percent of embryos which hatch after exposure to toxicants relative to controls was the basis of an embryo development assay. Exposure of embryos to chromium(III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) resulted in a reduced hatching rate. In addition to effects on embryo development, DNA strand damage tests were carried out on contaminant-exposed embryos, using the single-cell electrophoresis method often referred to as comet assay. Development of stage 4 embryos was more affected by MNQ exposure than stage 7 embryos. The hatching rates of stages 4 and 7 embryos exposed to MNQ (172 micrograms/l) were 0 and 90%, respectively. DNA strand damage, measured as DNA tail moments, were 3.4 and 4.4, respectively. Thus, exposure of an early embryo stage to MNQ prevented full embryo development while development of later embryo stages was not affected. It may be that the DNA repair systems are more efficient in later embryo stages than in early stages and thus DNA damaged in the early stages affects development.     

Contribution of apoptosis to observed dna damage in mussel cells

The comet assay employs the expression of DNA strand breaks by alkaline treatment to measure DNA damage. In contrast to other similar alkaline treatment assays the comet assay incorporates the microscopic examination of damage to individual cell nuclei. Because individual nuclei are examined, features unique to apoptotic cells can be identified. Apoptosis, programmed cell death, is characteristically an orderly sequence of events that ultimately leads to the complete disassembly of a cell. One consequence of apoptosis is the induction of endogenous nucleases which results in the fragmentation of DNA. Apoptosis can be triggered by a variety of stimuli, giving the misleading impression of genotoxic damage if this cytotoxic mechanism of DNA fragmentation is not taken into consideration. In this study mussel cells were exposed to a variety of toxicants and the cytotoxic (apoptotic) contribution to observed DNA damage was determined. A number of stressors induced apoptosis quite rapidly, within hours of exposure. The comet assay detected apoptosis and under certain circumstances apoptosis was found to be the major contributor to observed DNA damage.     

Comet assay in phytoplankton as biomarker of genotoxic effects of environmental pollution

The alkaline comet assay was tested on different microalgae: the dinoflagellates, Karenia mikimotoi and Alexandrium minutum, and the diatom, Chaetoceros gracilis. The microalgae were exposed during their exponential growth to the model direct genotoxicant, hydrogen peroxide (1h, 5 and 100muM H2O2). Following H2O2 exposure, the comet assay was validated only for K. mikimotoi for which genotoxicity was observed from the lowest tested concentration of 5 microM with a concentration-dependent effect. C. gracilis was too small in size (4 microm) to be correctly analysed. For A. minutum, our lysis buffer was not strong enough to digest the cellulosic thecal plates. For K. mikimotoi, the comet assay was thus applied for the study of the genotoxic effects of different pesticides: epoxiconazole (as Opus formulation), chlorpyriphos-ethyl (as Dursban formulation) and endosulfan at 1, 10 and 100 microg of active substance/L for 24h. Exposure to epoxiconazole in formulation resulted in an increase in the extent of DNA strand breaks at the highest tested concentration icro/L. Endosulfan exposure resulted in DNA damage for K. mikimotoi nuclei. Genotoxicity was observed from 1 microg/L of endosulfan and was not concentration dependent.     

Comet assays to assess DNA damage and repair in grass shrimp embryos exposed to phototoxicants

Exposure of grass shrimp (Palaemonetes pugio) embryos to four compounds (anthracene, pyrene, alpha-terthienyl, methylene blue) along with solar exposure resulted in extensive DNA strand damage using the comet assay. DNA tail moments of embryos exposed to these chemicals in the dark ranged from 1.8 to 4.3, while exposure to chemicals and solar resulted in tail moments of 14.3-15.3. Reduction of DNA tail moments when solar exposed embryos were transferred to the dark, suggested DNA repair systems were active. The comet assay can be used to follow both DNA damage and repair following exposure to phototoxic chemicals.     

Assessment of putative endocrine disrupters in an in vivo crustacean assay and an in vitro insect assay

Concern over endocrine disrupters in coastal ecosystems has stimulated global efforts to understand their potential impacts on fish and invertebrate communities. Given that marine crustaceans are sensitive to the effects of alkylphenols and other xenobiotics, we are currently investigating whether these responses may be caused via an endocrine mechanism. Tisbe battagliai (Copepoda: Harpacticoida) is representative of an ecologically important group of meiofauna, is sensitive to toxicants and is used as an international test species in marine ecotoxicology. A 21-day life-cycle test that incorporates assessment of survival, development, reproduction and sex ratios, has shown that populations of T. battagliai are not significantly affected by environmentally relevant levels of steroidal oestrogen agonists, or by related synthetic receptor agonists. The absence of in vivo effects due to these steroid agonists and antagonists prompted in vitro studies of ecdysteroid receptor activity of a range of reference compounds and environmental contaminants with an ecdysteroid receptor (EcR/USP)-based screening assay derived from the BII haemocyte line of Drosophila melanogaster. The implications for environmental monitoring of endocrine disrupters are discussed.     

DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis during anoxic stress and aerobic recovery

Anoxia-induced DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis was assessed with the alkaline comet assay (single-cell gel electrophoresis). The alkaline comet assay method for detecting DNA strand breaks and alkali labile sites in individual cells. DNA damage was determened in the scallops (M. yessoensis) gill cells. The scallops were exposed to air for 8 h showing a clear increase in the levels of DNA damage. After the air exposure, M. yessoensis were re-submersed for a period of 12 h, leading values to return to a pre-aerial exposure level. Control animals were kept immersed during the whole period. The resulting data demonstrate that natural influences, such as oxygen depletion (anoxia) in seawater, can be responsible for the induction of DNA damage. If the scallops were re-immersed in oxic conditions, the anoxically induced breaks were repaired. The main mechanisms influencing the integrity of the DNA structure are discussed in this paper.     

A comparison between the physiological effects in fish exposed to lethal and sublethal concentrations of a dispersant and dispersed oil

The acute and sublethal effects of a dispersant and crude oil on anisosmotic and isosmotic regulation in flounder have been tested. Flounder were exposed to different concentrations of Corexit 9527 and crude oil by use of a biotest system. Fourteen days' exposure to 20 ppm of Corexit 9527, alone or in a 1:1 mixture with crude oil, had no effect on the blood parameters. On the other hand, 96 hours' exposure to 80 ppm of the same compounds led to 50 % mortality and significant effects on the blood parameters in surviving fish. The comparability between effects obtained in fish exposed to lethal and sublethal concentrations of toxicants, respectively, is discussed.     

Method for assessing the chronic toxicity of marine and estuarine sediment-associated contaminants using the amphipod Corophium volutator

Acute sediment toxicity tests do not test key life stage events such as moulting and reproduction and therefore do not reveal the longer-term effects of contaminant exposure. A laboratory method is described for determining the chronic toxicity of contaminants associated with whole sediments. The test is conducted using neonates of the estuarine amphipod Corophium volutator at 15 degrees C, salinity 25 psu and a 12 h light:12 h dark photoperiod. The endpoints are survival and growth after 28 days and survival, growth and reproduction of amphipods upon termination of test i.e. reproduction within all control vessels (ca 75 days). The sediment chronic toxicity test was used to investigate the effects of sediments spiked with environmentally relevant preparations of slightly weathered Alaskan North Slope crude oil, including a water-accommodated-fraction (WAF) and a chemically-dispersed (Corexit 9527) WAF. Sediment oil concentrations were quantified using ultra-violet fluorescence. The amphipods exposed to chemically dispersed oil had higher mortality and lower growth rates than control-, Corexit 9527- and WAF-exposed organisms, resulting in reduced reproduction. The described method supplements the standard acute sediment test and would be particularly useful when long-term ecological effects are suspected but acute tests reveal no significant mortality. The sediment chronic test reported herein has shown that sediment that was not evidently toxic during 10-day acute tests could have population-level effects on sediment-dwelling amphipods.     

Measuring lysosomal stability as an effective tool for marine coastal environmental monitoring

The use of lysosomal stability in the mussel, Mytilus galloprovincialis, as a potential biomarker of environmental contamination has been evaluated along the Portuguese coast. To this end, the neutral red retention (NRR) time was measured in mussel haemocytes gathered from nine different locations reflecting different degrees of anthropogenic contamination. Mussels collected in the vicinity of industrial and urban areas showed the lowest lysosomal stability. Additionally, no significant seasonal variability (winter-spring/summer) for NRR time was observed. In order to further support the usefulness of this method as an integrated tool for monitoring marine coastal environments, we compared the levels of xenobiotics in mussel tissues with the obtained NRR values. The results highlighted a consistent pattern, with the lowest lysosomal stability intimately correlated with the higher contaminant concentrations. In summary, this integrated approach further demonstrated that the NRR assay can provide useful and objective indications of the real health status of organisms subjected to different stress agents, being a valid option for environmental monitoring.     

Metabolic effects and hydrocarbon fate in arctic bivalves exposed to dispersed petroleum

A number of experiments were carried out in the Canadian Arctic on Baffin Island with the purpose of defining the short- and long-term effects of exposure to dispersed crude oil on marine benthic invertebrates. The study reported here assessed metabolic responses by physiological and biochemical indices, and evaluated these in relation to exposure concentration. The overall objective of the study was to evaluate the potential for long-term survival of benthic communities in the Arctic following an oil spill. This objective was consistent with the goals of the larger Baffin Island Oil Spill (BIOS) program, which was implemented to evaluate the relative mitigating effectiveness of chemical dispersants, as compared to conventional oil spill counter-measures.     

Effects of mercury on the feeding behavior of the mummichog, Fundulus heteroclitus from a polluted habitat

Fish living in contaminated environments may exhibit reduced growth which may be due, in part, to the impact of toxicants on prey capture ability. We studied the effects of methylmercury (meHg) and mercuric chloride (Hg) on feeding of Fundulus heteroclitus from Piles Creek, a polluted habitat in Linden, New Jersey. We found that exposure to 0·01 mg/liter of meHg for one week had less impact on the prey capture rate than exposure to a similar concentration of Hg. Exposure to 0·01 mg/liter for two weeks or to 0·02 mg/liter for one week, of either meHg or Hg, produced significant reductions in feeding rate, compared to controls. In general, the fish were affected to a greater degree by exposure to Hg than to meHg. This is the reverse of the usual order of toxicity, but is in keeping with previous observations on this population, in which we have noted that gametes and embryos had developed tolerance to meHg, but were more susceptible to Hg.     

Multi-level assessment of chronic toxicity of estuarine sediments with the amphipod Gammarus locusta: I. Biochemical endpoints

We report on biomarker responses conducted as part of a multi-level assessment of the chronic toxicity of estuarine sediments to the amphipod Gammarus locusta. A companion article accounts for organism and population-level effects. Five moderately contaminated sediments from two Portuguese estuaries, Sado and Tagus, were assessed. Three of them were muddy and two were sandy sediments. The objective was to assess sediments that were not acutely toxic. Three of the sediments met this criterion, the other two were diluted (50% and 75%) with clean sediment until acute toxicity was absent. Following 28-d exposures, the amphipods were analysed for whole-body metal bioaccumulation, metallothionein induction (MT), DNA strand breakage (SB) and lipid peroxidation (LP). Two of the muddy sediments did not cause chronic toxicity. These findings were consistent with responses at organism and population levels that showed higher growth rates and improvement of reproductive traits for amphipods exposed to these two sediments. Two other sediments, one muddy and one sandy, exhibited pronounced chronic toxicity, affecting SB, MT induction (in muddy sediment), survival and reproduction. Potential toxicants involved in these effects were identified. The last sandy sediment exhibited some loss of DNA integrity, however growth was also enhanced. Present results, together with the organism/population-level data, and also benthic communities information, were analysed under a weight-of-evidence approach. By providing evidence of exposure (or lack of it) to contaminants in sediments, the biomarkers here applied assisted in distinguishing toxicants' impacts in test organisms from the confounding influence of other geochemical features of the sediments.     

Exposure of grass shrimp to sediments receiving highway runoff: Effects on reproduction and DNA

A grass shrimp bioassay was carried out on sediments from three estuarine stations which were different distances from a highway storm drain. Total polycyclic aromatic hydrocarbon (PAH) concentrations were 29, 1.5 and 0.1 μg/g sediment at stations A (next to drain), B (100 m from drain) and C (500 m from drain), respectively. Lower embryo production and embryo hatching rates and a higher level of DNA strand breaks (comet assay) were observed in grass shrimp exposed to stations A and B sediments. There appeared to be an association between reproduction abnormalities and increased DNA strand breaks as a result of grass shrimp exposure to estuarine sediments receiving highway runoff.     

Genotoxicity of copper oxide and silver nanoparticles in the mussel Mytilus galloprovincialis

Though there is some information on cytotoxicity of copper nanoparticles and silver nanoparticles on human cell lines, there is no information on their genotoxic and cytotoxic behaviour in bivalve molluscs. The aim of this study was to investigate the genotoxic impact of copper oxide and silver nanoparticles using mussels Mytilus galloprovincialis. Mussels were exposed to 10 μg L⁻¹ of CuO nanoparticles and Cu²⁺ and Ag nanoparticles and Ag⁺ for 15 days to assess genotoxic effects in hemocytes using the comet assay. The results obtained indicated that copper and silver forms (nanoparticles and ionic) induced DNA damage in hemolymph cells and a time-response effect was evident when compared to unexposed mussels. Ionic forms presented higher genotoxicity than nanoparticles, suggesting different mechanisms of action that may be mediated through oxidative stress. DNA strand breaks proved to be a useful biomarker of exposure to genotoxic effects of CuO and Ag nanoparticles in marine molluscs.     

Impact of the Sea Empress oil spill on lysosomal stability in mussel blood cells.

Coastal zones are among the most productive and vulnerable areas on the planet. An example of impact on these fragile environments was shown in the case of the "Sea Empress" oil tanker, which ran aground in the Bristol Channel in 1996, spilling 72,000 tons of "Forties" crude oil. The objective was to investigate the sub-lethal cellular pathology and tissue hydrocarbon contamination in marine mussel populations, 4 months after the initial spill, using the neutral red retention (NRR) assay for lysosomal stability in blood cells. NRR was reduced in mussels, and indicative of cell injury, from the two sites closest to the spill in comparison with more distant and reference sites. Lysosomal stability was inversely correlated with polycyclic aromatic hydrocarbon concentrations in mussel tissues. Reduced lysosomal stability has previously been shown to contribute to impaired immunocompetence and to autophagic loss of body tissues. The use of this type of technique is discussed in the context of cost-effective, ecotoxicological tools for Integrated Coastal Zone Management.     

Effects of benzene and dimethylnaphthalene on homeostatic processes in two species of crabs

Long-term exposure of animals to sub-lethal doses of toxicants such as benzene (B) and dimethylnaphthalene (DMN) may result in subtle changes in their physiology and biochemistry. In crustaceans such changes include decreased rate of limb regeneration, extended time to molt and decreased growth increment at molt.^1,2 These processes depend upon an adequate supply of stored nutrients in the tissues and appropriate release of neuroendocrine substances from the central nervous system.^3,4 We are examining the effects of sub-lethal doses of B and DMN on osmotic and ionic regulation and on nutrient storage in the blue crab, Callinectes sapidus, and the green crab, Carcinus maenas. Disturbances in these physiological processes may reflect alterations in neuroendocrine functions brought about by the presence of toxicants. We find that exposure of C. maenas to B or DMN, followed by transfer to a dilute medium, results in impaired osmoregulatory capacity, while addition of these compounds to a dilute medium to which the animal is already acclimated does not affect their regulatory ability. Storage of carbohydrate in the midgut gland (hepatopancreas) is decreased by B or DMN, while the accessory storage tissues (gill, muscle) are not significantly affected.     

Evaluation of neutral red retention assay,micronucleus test,acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring

The neutral red lysosomal retention assay (NRR) of the haemocytes, and the acetylcholinesterase activity (AChE) in the haemolymph, the digestive gland, the gills and the mantle/gonad complex have been evaluated on mussels Mytilus galloprovincialis collected from Thermaikos and Strymonikos gulfs (northern Greece) in June and October 2001. The validity of performing the above core biomarkers is supported, firstly by their ability to respond to different pollution levels and, secondly, by the significant linear correlation among them. The evaluation of the micronuclei frequency (MN) has been performed in gill tissue and haemocytes of the same mussels and, according to the results, it needs more research in order its use as stress indices to be validated. In addition, the first results on cAMP levels in the gills, the mantle/gonad complex and the digestive gland, whose concentrations correlated to both, NRR and AChE introduce this signal transduction molecule as a new, promising biomarker.     

Long-term effects of tributyltin compounds on the baltic amphipod, Gammarus oceanicus

Amphipod larvae, Gammarus oceanicus, were exposed for 8 weeks to bis(tributyltin) oxide (TBTO) or tributyltin fluoride (TBTF), or for 5 weeks to leachates from tributyltin-containing antibiofouling paints. No larvae survived 8 weeks' exposure to 3 μg/litre TBTO or TBTF. Differential larval survival occurred during exposure to 0·3 gmg/litre of these same compounds. Paint leachates were shown to cause similar toxicity responses at comparable aqueous tributyltin concentrations. An examination of sublethal responses showed slight decreases in growth for larvae exposed to TBTO and to paint leachates, although responses at tributyltin levels below 1 μg/litre were not marked. Whole-animal oxygen consumption rates were not affected by low tributyltin exposure levels. While these studies demonstrate low level toxicity of tributyltins, effects on oxygen consumption and growth do not indicate a mode of action for these compounds.     

Quantitative determination of lysozyme in the hemolymph of Mytilus edulis by the enzyme-linked immunosorbent assay (ELISA)

The defensive responses of the bay mussel, Mytilus edulis, to invading organisms and toxicants is primarily cellular. Hemolymph cells as well as the other organs and tissues of the mussel through their normal functioning and interactions with toxic substances influence the chemical composition of the animal's hemolymph. It has been found that the levels of specific molecules in the hemolymph fluctuate as a consequence of trace metal insult. It was proposed that one way in which trace metal toxicity may be expressed is through an influx of free lysosomal hydrolases in the hemolymph.Reported here is the development of an enzyme-linked immunosorbent assay (ELISA) with which micro-samples of mussel hemolymph can be analyzed quantitatively for the lysosomal hydrolytic enzyme, lysozyme. During development comparisons were made of the results acquired by spectrophotometric determinations of lysozyme activity and ELISA-determined lysozyme concentrations in the hemolymph of mussels. The hemolymph from unstressed and copper-stressed mussels was examined. It was found that the overall trends in lysozyme fluctuations were indicated by both techniques. However, on occasion in copper-stressed populations a divergence between the two sets of data was observed. This suggested an effect of copper on lysozyme molecular configuration.