DNA strand breakage in mussels (Mytilus edulis L.) deployed in intertidal and subtidal zone in Reykjavík harbour

DNA single-strand breaks were analysed in the blue mussel (Mytilus edulis L.) deployed in intertidal and subtidal zones in the PAH contaminated Reykjavík harbour and at a reference site, Hvalfj?rethur, Iceland. DNA strand breaks were analysed by Comet assay in isolated gill and haemocyte cells from six mussels from each site and depth. Increased DNA damage in both gill cells and haemocytes were observed in mussels deployed in Reykjavík harbour compared to the reference site. Intertidal mussels from Reykjavík harbour had higher DNA damage in haemocytes compared to subtidal mussels. The Comet assay seems to be useful for measuring genotoxic exposure in mussels from the field, and that DNA damage might be higher in the intertidal zone either due to higher exposure to contaminants or because of physiological and biochemical responses to variations in oxygen availability.     

醚菊酯对日本囊对虾肝胰腺细胞DNA损伤效应的研究

为检测中国近海海洋环境中菊酯类农药等污染物对日本囊对虾(Marsupenaeus japonicas)的基因毒性及其危害程度,作者首次使用醚菊酯对日本囊对虾进行毒理实验,将日本囊对虾分为6组进行不同剂量的处理,采用彗星实验分析技术对处理培育25 d的日本囊对虾DNA损伤情况进行检测,并通过Comet Score 1.5分析软件对实验图谱进行拖尾率、尾长、彗尾DNA相对含量、尾距、Olive距等DNA损伤指标的统计分析,在对各指标分别建立一元回归方程的基础上,又通过SPSS软件进行判定分析,筛选影响作用更大的检测指标,建立了多参数的多元回归方程。实验结果发现,与空白对照组相比,日本囊对虾各实验处理组肝胰腺细胞DNA均有不同程度的损伤且各检测指标均有显著性差异(P0.01),各检测指标与处理浓度表现出良好的剂量效应关系,随着处理浓度递增,各检测指标都呈规律性的增长趋势。回归分析和相关系数表明:各检测指标与处理浓度具有高度的相关性,多项式回归方程(P0.05)和多元回归方程(P0.005)均有显著性的统计学意义,通过多元回归方程可以有效地推断醚菊酯处理浓度和处理时间。     

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.     

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.     

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.     

Measurement of DNA single-strand breaks in gill and hemolymph cells of mussels, Mytilus sp., collected on the French Atlantic Coast

DNA single-strand breaks were measured by the comet assay in both gill and hemolymph cells of mussels collected in 3 sampling areas of the French coast (Pointe du Castelli, Pen Bron and Saint-Nazaire Harbour). Whole mussel tissue samples were also collected for the chemical determination of PAH, PCB and heavy metal concentrations. In mussel, a higher level of DNA strand breaks was measured in gill than in hemolymph cells (p < 0.01). Despite a factor of contamination from 2 to 3 between sites, no difference in the extent of mussel DNA strand breaks was shown between sampling locations (p > 0.05), questioning the sensitivity of the assays used in biomonitoring studies.     

Implications of seasonal priming and reproductive activity on the interpretation of Comet assay data derived from the clam, Tapes semidecussatus Reeves 1864, exposed to contaminated sediments

We explore the use of the clam Tapes semidecussatus Reeves 1864 as an indicator for the presence of potentially genotoxic substances in estuarine sediments. The limitations associated with the interpretation of Comet assay data (expressed as % DNA in tail) in terms of clam reproductive state, size (age) and thermal exposure history following laboratory acclimation are discussed. Hatchery-reared clams, subjected to ambient temperature fluctuations during growth, were exposed in vivo under laboratory conditions for three weeks to sediment samples collected from a polluted site and a "clean" reference site. The DNA damage observed in haemocytes, gill and digestive gland cells was significantly higher in animals exposed to contaminated sediment compared to those exposed to sediment from the reference site. The extent of DNA damage recorded was not correlated with size (age). Spawning was not observed during the experiment. Nevertheless, clams with well-developed gonads showed a statistically higher degree of DNA damage in gill and digestive gland cells- but not haemocytes, demonstrating an increased sensitivity to potential genotoxic compounds, possibly caused by impaired DNA repair capacity due to reproductive activity. Furthermore, the degree of DNA damage in clams exposed to contaminated sediments was higher in autumn and winter compared to spring and summer, suggesting an effect of seasonal priming.     

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.     

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.     

Physiological response of the sea urchin Paracentrotus lividus fed with the seagrass Posidonia oceanica and the alien algae Caulerpa racemosa and Lophocladia lallemandii

The aim was to determine the effects of alien algae feeding on biomarkers of oxidative stress in the sea urchin Paracentrotus lividus. Sea urchins were fed during three months with the native seagrass Posidonia oceanica, and the alien macroalgae Caulerpa racemosa and Lophocladia lallemandii and biochemical analysis were performed in the gonads. A control group was immediately processed after sampling from the sea. Antioxidant enzyme and glutathione S-transferase activities and GSH concentration were significantly higher in sea urchins fed with alien algae when compared with the control group and the one fed with P. oceanica group. This response was more intense in the group fed with L. lallemandii respect to the C. racemosa group. The concentration of MDA, protein carbonyl derivates and 8-OHdG reported no significant differences between treatments. In conclusion, the invasive algae C. racemosa and L. lallemandii induced an antioxidant response in P. lividus without evident oxidative damage.     

Relationships among predatory fish, sea urchins and barrens in Mediterranean rocky reefs across a latitudinal gradient

Previous studies conducted on a local scale emphasised the potential of trophic cascades in Mediterranean rocky reefs (involving predatory fish, sea urchins and macroalgae) in affecting the transition between benthic communities dominated by erected macroalgae and barrens (i.e., bare rock with partial cover of encrusting algae). Distribution patterns of fish predators of sea urchins (Diplodus sargus sargus, Diplodus vulgaris, Coris julis and Thalassoma pavo), sea urchins (Paracentrotus lividus and Arbacia lixula) and barrens, and fish predation rates upon sea urchins, were assessed in shallow (3-6m depth) sublittoral rocky reefs in the northern, central and southern sectors of the eastern Adriatic Sea, i.e., on a large spatial scale of hundreds of kilometres. No dramatic differences were observed in predatory fish density across latitude, except for a lower density of small D. sargus sargus in the northern Adriatic and an increasing density of T. pavo from north to south. P. lividus did not show any significant difference across latitude, whereas A. lixula was more abundant in the southern than in the central Adriatic. Barrens were more extended in the southern than in the central and northern sectors, and were related with sea urchin density. Fish predation upon adult sea urchins did not change on a large scale, whereas it was slightly higher in the southern sector for juveniles when predation rates of both urchins were pooled. Results show that: (1) assemblages of predatory fish and sea urchins, and barren extent change across latitude in the eastern Adriatic Sea, (2) the weak relations between predatory fish density and predation rates on urchins reveal that factors other than top-down control can be important over large scale (with the caveat that the study was conducted in fished areas) and (3) patterns of interaction among strongly interacting taxa could change on large spatial scales and the number of species involved.     

pH variability off Goa (eastern Arabian Sea) and the response of sea urchin to ocean acidification scenarios

The increasing atmospheric CO₂ concentration in the last few decades has resulted in a decrease in oceanic pH. In this study, we assessed the natural variability of pH in coastal waters off Goa, eastern Arabian Sea. pH_T showed large variability (7.6–8.1) with low pH conditions during south-west monsoon (SWM), and the variability is found to be associated with upwelling rather than freshwater runoff. Considering that marine biota inhabiting dynamic coastal waters off Goa are exposed to such wide range of natural fluctuations of pH, an acidification experiment was carried out. We studied the impact of low pH on the local population of sea urchin Stomopneustes variolaris (Lamarck, 1816). Sea urchins were exposed for 210 days to three treatments of pH_T: 7.96, 7.76 and 7.46. Our results showed that S. variolaris at pH_T 7.96 and 7.76 were not affected, whereas the ones at pH_T 7.46 showed adverse effects after 120 days and 50% mortality by 210 days. However, even after exposure to low pH for 210 days, 50% organisms survived. Under low pH conditions (pH_T 7.46), the elemental composition of sea urchin spines exhibited deposition of excess Sr²⁺ as compared to Mg²⁺ ions. We conclude that although the sea urchins would be affected in future high CO₂ waters, at present they are not at risk even during the south-west monsoon when low pH waters reside on the shelf.     

Movement patterns of the sea urchin Paracentrotus lividus in a marine reserve and an unprotected area in the NW Mediterranean

Sea urchins can alter the composition and dynamics of algal communities by grazing. Changes in their displacement capability can influence their grazing and thus their effect on algal communities. The daily and monthly movement of Paracentrotus lividus inside and outside a marine reserve in the NW Mediterranean Sea was studied in order to determine the role of predation in sea urchin movements and its potential grazing impacts. During the 3‐month study, the total distance travelled by sea urchins ranged from 71.8 to 673.6 cm (mean = 379.2 cm, SD = 221.3). Home ranges (distances from the initial position) were between 50 and 302 cm (mean = 150.7 cm, SD = 99.4). Movements were not directional but random, with a mean of 150 cm around the initial position. The activity of Paracentrotus lividus was significantly higher at night than during daylight. Total displacement at the end of the experiment was significantly higher in the unprotected area than in the reserve, as was the home range. The linear distance travelled in a 24‐h period ranged between 6 and 220 cm with a mean home range of 51 cm. These results agree with those of other authors, and allow us to rule out major migratory processes of sea urchin populations in this area. Nevertheless, these results underline the role of predatory fishes in reducing herbivory pressure by trophic cascades, reinforcing the evidence that sea urchin escape behaviour may be an important factor in structuring algal communities.     

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.     

Effect of oil and dispersants on the growth of Mussels

Mussels (Mytilus edulis L.) were exposed to North Sea crude oil, microencapsulated oil and dispersants, singly and in combination, and growth rates measured at 24–48 h intervals.Exposure to microencapsulated pure oil (2·0–2·1 mg litre⁻¹) and to microencapsulated mixtures of oil (2·2−2·5 mg litre^-1+5 % of the different dispersants (FINASOL OSR 5, COREXIT 9527, DISPOLENE 36 S) gave approximately the same reduction in growth rate (80–90%) within 170h.Oil chemically dispersed with DISPOLENE 36 S and a pure oil mechanically dispersed in water were significantly less toxic. In high concentrations (2 mg litre⁻¹) all disperants are toxic, DISPOLENE 36 S ssignificantly more than the others.Mussels exposed for 170 h to microencapsulated oil and to microencapsulated oil dispersant mixtures recovered to control growth within 300 h in clean seawater, while in those given pure oil-in-water suspension, the recovery was slower.It is concluded that the toxicity of oil is mainly related to size and concentration of oil particles, while the effect of 5% dispersants added is negligible.     

Genotoxicity in two bivalve species from a coastal lagoon in the south of Portugal

DNA damage was evaluated by comet assay in the haemolymph of two bivalve species Ruditapes decussatus and Mytilus galloprovincialis from the Ria Formosa lagoon (south Coast of Portugal). Clams and mussels were sampled from sites close to each other to determine interspecific responses to similar environmental conditions, considering also seasonal and gender differences. Coupled with genotoxic effect, another damage biomarker (lipid peroxidation) was analysed to verify if the conditions that instigate DNA damage can be related with injury to cell membranes. For both species, DNA damage was low, reflecting the low levels of genotoxic contaminants in the lagoon, and no interspecific differences were found. However, seasonal differences were established for both bivalve species, reflecting higher environmental stress in summer. Regarding gender susceptibility, only clams showed differences in percentage of Tail DNA, with females more sensitive to DNA damage than males. Additionally, results for clams point out that factors responsible for LPO may not be the same as those causing genotoxicity. This study demonstrated that DNA damage is a sensitive biomarker to discriminate spatial, temporal and gender differences, being an appropriate biomarker for genotoxicity evaluation even in places of low contamination, such as the Ria Formosa lagoon.     

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.     

Differential recruitment of introduced Pacific oysters and native mussels at the North Sea coast: coexistence possible?

Pacific oysters (Crassostrea gigas Thunberg 1793) have been introduced into the Wadden Sea (North Sea), where they settle on native mussel beds (Mytilus edulis L.), which represent the only extensive insular hard substrata in this soft-sediment environment. As abundances of C. gigas rose, some mussel beds became increasingly overgrown with oysters, whereas others did not. Field experiments revealed that recruitment of C. gigas was higher in the lower intertidal than in the upper subtidal zone, that it was higher on conspecifics than on mussels, and that it was not affected by barnacle epigrowth except when settling on mussels. Mussel recruitment is known from inter- and subtidal zones. It occurred equally on oyster and mussel shells but showed a clear preference for barnacle epigrowth over clean shells. Assuming that settlement and recruitment are key processes for species abundances on the North Sea coast, it is predicted that the positive feedback in oyster settlement will lead to rapid reef formation of this invader at the expense of mussel beds. Mussels, however, may escape competitive exclusion by settling between or on the larger oysters especially when barnacles are abundant. Experimental patches with mussels were more often covered by fucoid algae (Fucus vesiculosus forma mytili Nienburg) than patches with oysters, and oyster recruitment was poor underneath such algal canopies. Thus, fucoids may provide the native mussels with a refuge from the invading oysters and the two bivalves may coexist, provided food is not limiting.     

Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis): effect of low temperatures.

Expanding industrial activities in the Arctic require an urgent assessment of the toxicity of chemicals at low temperatures. Organisms acclimatized to low temperature exhibit specific adaptations. For example, the amount of unsaturated lipids is increased to maintain the fluidity of the cell membranes. It has been hypothesized that such temperature-induced alterations in membrane lipid composition may affect the stability of lysosomal and cell membranes in the common mussel, Mytilus edulis, an organism exposed to seasonal temperature extremes. As mussels may be exposed to petroleum compounds along industrialized coastlines, we tested the combined effects of exposure to low temperature and the petroleum compound, phenanthrene, on haemocyte membrane stability. Test animals, acclimated to either 0 or 10 degrees C, were exposed to phenanthrene (0 = control or 500 micrograms l-1) and haemocytes were examined using the neutral red retention assay (lysosomal stability) and a fluorescence assay (cell membrane stability). At 0 degree C, lysosomal and cell membranes from uncontaminated mussels were destabilized compared with 10 degrees C (P = 0.0005). No significant effects (P > 0.05) of phenanthrene were detected at either temperature. Possible mechanisms underlying membrane destabilization include a weaker physical resistance of the membrane due to a higher amount of unsaturated lipids, a potentially higher level of reactive oxygen radicals at low temperature and the higher susceptibility of unsaturated lipids to oxidative stress. More work is required to better understand the consequences of this membrane destabilization at low temperature on the susceptibility of the organism to pollutants.     

Habitat modification in tidepools by bioeroding sea urchins and implications for fine‐scale community structure

By creating novel habitats, habitat‐modifying species can alter patterns of diversity and abundance in marine communities. Many sea urchins are important habitat modifiers in tropical and temperate systems. By eroding rocky substrata, urchins can create a mosaic of urchin‐sized cavities or pits separated by exposed, often flat surfaces. These microhabitats appear to harbor distinct assemblages of species. We investigated how a temperate rocky intertidal community uses three small‐scale (<100 cm²) microhabitats created by or adjacent to populations of the purple sea urchin (Strongylocentrotus purpuratus): pits occupied by urchins, unoccupied pits, and adjacent flat spaces. In tidepools, flat spaces harbored the highest percent cover of algae and sessile fauna, followed by empty pits and then occupied pits. The Shannon diversity and richness of these sessile taxa were significantly higher in flat spaces and empty pits than in occupied pits. The composition of these primary space holders in the microhabitats also varied. Unlike primary space holders, mobile fauna exhibited higher diversity and richness in empty pits than in flat spaces and occupied pits, although results were not significant. The protective empty pit microhabitat harbored the highest densities of most trophic functional groups. Herbivores, however, were densest in flat spaces, concordant with high algal coverage. These results suggest the habitats created by S. purpuratus in addition to its biological activities alter community structure at spatial scales finer than those typically considered for sea urchins.