Effects of phthalates on marine organisms: cytotoxicity and genotoxicity of mono-(2-ethylhexyl)-phthalate (MEHP) on European sea bass (Dicentrarchus labrax) embryonic cell line.

INTRODUCTION: Mono-(2-ethylhexyl) phthalate (MEHP) represents a toxicological risk for marine organisms due to its widespread presence in aquatic environments. METHODS: MEHP effects on cell viability, cell death and genotoxicity were investigated on the DLEC cell line, derived from early embryos of the European sea bass Dicentrarchus labrax L. RESULTS: A dose-dependent cytotoxic effect, with no induction of necrotic process, except at its highest concentration, was observed. Moreover, chromosomal instability was detected, both in binucleated and mononucleated cells, coupled with a minor inhibition of cell proliferation, whereas genomic instability was not revealed. To our knowledge, the overall results suggest the first evidence of a possible aneugenic effect of this compound in the DLEC cell line, that is the induction of chromosomal loss events without the induction of primary DNA damage. CONCLUSIONS: MEHP should be considered more harmful than its parent compound DEHP, because it induces genomic instability in the DLEC cell line without triggering cell death.

Effect of marine antifouling paint particles waste on survival of natural Bermuda copepod communities.

Marine antifouling paints (MAPs) are widely used to prevent organisms from fouling vessel hulls. When scraped from vessels as part of regular maintenance, MAP particles discharged into the seawater become a source of toxic substances, like copper (Cu), to the environment, and biocides leaching from them are known to cause toxic effects on non-target organisms. We investigated the toxicity of MAP particles collected from a Bermuda boatyard on local copepod communities using two experiments. Copepod survival, Chlorophyll a and total dissolved Cu concentrations were measured before and after MAP particles addition. In an acute toxicity test, the addition of 0.3 g/L of MAP particles resulted in 0% copepods survival within 88 h and increased dissolved Cu by 1.8 µM. A significant inverse relationship was observed between copepod survival and MAP particles quantity, highlighting the toxic effects of MAP particles from boat maintenance on copepod communities in the surrounding seawater.

In vitro evaluation of cytotoxic and genotoxic effects of Di(2-ethylhexyl)-phthalate (DEHP) on European sea bass (Dicentrarchus labrax) embryonic cell line.

Marine litter is extensively distributed in the marine environment, and plastic debris, of which litter is mostly composed, can be a major source of pollutants. Among them, Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used plastic additive, and it has been reported to affect biochemical processes both in humans and wildlife; however, studies on its toxicological effects on marine organisms are still scarce. In this survey, we studied the cytotoxic, genotoxic, and mutagenic effects of DEHP in European sea bass embryonic cell line (DLEC) by applying specific in vitro tests. Results showed a significant decrease in cell viability starting at 0.01 mM of DEHP after 24 h together with a significant increase in apoptosis and necrosis, morphological changes and cell detachment. Consistently, we detected a moderate increase in DNA strand breaks from 0.02 mM, and a dose-dependent increase in of micronucleus frequency from 0.01 mM, accompanied by a significant inhibition of cell proliferation, which suggested a possible aneugenic effect of this phthalate. Our results demonstrate that in vitro exposure to DEHP had a dose-dependent cytotoxic and genotoxic effects in DLEC cell line, encouraging further investigation into its effects in in vivo and/or ex vivo cell systems of marine organisms.

Genetic effects in Helix aspersa near a coal plant revealed by the micronucleus test.

Coal plants can be a major source of mutagenic pollutants. In this study we used the common land snail Helix aspersa, to detect the mutagenic effect of pollution from a coal plant in central Italy applying the micronucleus test (MN) on snail's haemocytes and evaluating trace elements concentration (As Cd, Pb, Hg, and Zn) in soil and snails. Snails from a biological farm were exposed for 13 days in five locations at different distances from the plant. Wild snails collected in the same locations were also analysed. MN frequency in exposed snails was significantly higher in four locations within 10 km from to the plant, with respect to the control and the farthest location. Comparing the MN frequency between farmed and wild snails, a significantly higher frequency emerged for the exposed snails in all locations except the farthest, likely indicating adaptation or selection of the wild organisms due to chronic exposure to pollutants. In natural snails significantly higher MN frequencies with near the plant emerged as well. Trace elements analysis showed significant correlations between MN frequencies and both Zn and As concentrations in soil, for both exposed and wild snails, and Zn and Pb concentrations in exposed snails. Our results were consistent with those previously obtained when evaluating primary DNA damage in natural snails from the same area and show that the snails near the plant were affected by a permanent cytogenetic damage. Moreover, they confirm the suitability of snails for biomonitoring the presence of pollutants with mutagenic effect.

Amount, composition, and spatial distribution of floating macro litter along fixed trans-border transects in the Mediterranean basin.

Marine litter is a major source of pollution in the Mediterranean basin, but despite legislative requirements, scant information is available for the ongoing assessment of this threat. Using higher size classes as proxy for litter distribution, this study gave a synoptic estimation of the amount, composition, and distribution of floating macro-litter in the Mediterranean. The average amount of macro-litter was in a range of 2-5items/km2, with the highest in the Adriatic basin. Seasonal patterns were present in almost all study areas and were significant in the Ligurian Sea, Sardinian-Balearic basin, and Central Tyrrhenian Sea. Plastic accounted for >80% of litter in all areas and seasons, with the highest proportion in the Adriatic Sea, Ligurian Sea, and Sicilian-Sardinian Channels; in the Bonifacio Strait, Tyrrhenian Sea, and Sardinian-Balearic basin, litter composition was instead more diverse. Spatial analysis suggested an almost homogeneous distribution of litter without evident regular aggregation zones.

Dietary antioxidants, food deprivation and growth affect differently oxidative status of blood and brain in juvenile European seabass (Dicentrarchus labrax).

Compensatory growth may increase molecular oxidative damage, which may be mitigated through the intake of dietary antioxidants. However, dietary antioxidants may also reduce concentration of antioxidant enzymes, which have a key role in regulating the oxidative status. Here we investigated whether feeding on a diet rich in antioxidants (vitamin E) enables juvenile European seabass (Dicentrarchus labrax) to catch up after a period of food deprivation with negligible effects on the oxidative stress to blood and brain as compared to fish feeding on a normal diet (i.e., not enriched in antioxidants). The results show that a higher intake in antioxidants favoured compensatory growth, but this came at a cost in terms of increased oxidative damage. Increased intake of antioxidants also resulted in changes in the activity of enzymatic antioxidant defences and increased protein oxidative damage in both brain and blood. In addition, food deprivation caused increased protein oxidative damage in brain. Our findings show that the beneficial effects of dietary antioxidants on growth may be offset by hidden detrimental effects and that different early life events affect different components of oxidative status of a given tissue.

The role of habitat choice in micro-evolutionary dynamics: An experimental study on the Mediterranean killifish Aphanius fasciatus (Cyprinodontidae).

Habitat choice is defined as a nonrandom distribution of genotypes in different microhabitats. Therefore, it could exert a great impact on the genetic variance of natural populations by promoting genetic divergence, local adaptation, and may even lead to sympatric speciation. Despite this potential role in micro- and macro-evolutionary processes, there is little empirical evidence that the various genotypes within a population may differ in habitat choice-related behaviors. Here, we tested whether habitat choice may have contributed to genetic divergence within a local population of the Mediterranean killifish Aphanius fasciatus, which emerged between groups inhabiting microhabitats with different oxygen concentrations during previous field studies. In a first experiment, we studied the distribution of individuals in conditions of hypoxia and normoxia to test whether they had a different ability to shy away from a hypoxic environment; in a second experiment, we analyzed the individual behavior of fish separately in the two conditions, to verify whether they showed peculiar behavioral responses linked to a possible differential distribution. We then analyzed the six allozyme loci, whose allelic and genotypic frequencies were significantly divergent in the previous studies. In the first test, we found that the distribution of the two homozygote genotypes of the glucose-6-phosphate isomerase-1 locus (GPI-1) was significantly different between the hypoxic and the normoxic conditions. During the second test, all individuals were more active in hypoxic conditions, but the two GPI-1 homozygotes showed a significant difference in time spent performing surface breathing, which was consistent with their distribution observed in the first experiment. These results provide evidence that individual behavioral traits, related to genetic features, may lead to a nonrandom distribution of genotypes in heterogeneous although contiguous microhabitats and, consequently, that habitat choice can play a significant role in driving the micro-evolutionary dynamics of this species.

Infaunal macrobenthic community dynamics in a manipulated hyperhaline ecosystem: a long-term study.

BACKGROUND: Understanding the responses of ecological communities to human-induced perturbations is crucial for establishing conservation goals. Ecological communities are dynamic entities undergoing fluctuations due to their intrinsic characteristics as well as anthropogenic pressures varying over time. In this respect, long-term studies, based on large spatial and temporal datasets, may provide useful information in understanding patterns and processes influencing the communities' structure. Theoretical evidence suggests that a role of biodiversity is acting as a compensatory buffer against environmental variability by decreasing the temporal variance in ecosystem functioning and by raising the level of community response to perturbations through the selection of better performing species. Therefore, the spatial and temporal changes in the specialization of the community components may be used as an effective tool to monitor the effects of natural and anthropogenic alterations of the environment in dynamic systems. We examined the temporal dynamics of macroinvertebrate community structure in the hyperhaline habitat of Tarquinia Saltworks (central Italy). We aimed at: (i) investigating the relationships between the level of community specialization and the alterations of the environment across fourteen years; (ii) comparing the ability of aggregate community parameters such as the average abundance vs. species specialization in describing patterns of community composition. RESULTS: We arranged the data in three sub-sets according to three periods, each characterized by different environmental conditions. The mean abundance of sampled macroinvertebrates showed a significant change (p < 0.01) only in the community inhabiting the saltwork basin closely connected to the sea, characterized by the highest environmental variation (i.e. the coefficient of variation, CV, of the aggregate environmental variability over the study period, CVrange = 0.010 - 0.2). Here we found marine species like Modiolus adriaticus (Lamarck, 1819), Neanthes irrorata (Malmgren, 1867), and Amphiglena mediterranea (Leydig, 1851), which inhabited the saltworks during the halt period but disappeared during the subsequent eutrophication phase. Conversely, species specialization showed a significant decrease for each sampled community in the presence of habitat degradation and a recovery after ecological restoration. The widest fluctuations of specialization were recorded for the community inhabiting the saltwork basin with the highest long-term environmental variability. CONCLUSIONS: Recent advances have shown how the increased temporal and spatial variability of species' abundance within the communities may be a signal of habitat disturbance, even in the absence of an apparent decline. Such approach could also be used as a sensitive monitoring tool, able to detect whether or not communities are subjected to increasing biotic homogenization. Also, the increased functional similarity triggered by habitat degradation may impact on species at higher trophic levels, such as the waterbirds wintering in the area or using it as a stopover during migration.

Invertebrate welfare: an overlooked issue.

While invertebrates make up the majority of animal species, their welfare is overlooked compared to the concern shown to vertebrates. This fact is highlighted by the near absence of regulations in animal research, with the exception of cephalopods in the European Union. This is often justified by assumptions that invertebrates do not experience pain and stress while lacking the capacity for higher order cognitive functions. Recent research suggests that invertebrates may be just as capable as vertebrates in experiencing pain and stress, and some species display comparable cognitive capacities. Another obstacle is the negative view of invertebrates by the public, which often regards them as pests with no individual personalities, gastronomic entities, or individuals for scientific experimentation without rules. Increasingly, studies have revealed that invertebrates possess individual profiles comparable to the personalities found in vertebrates. Given the large economic impact of invertebrates, developing certain attitude changes in invertebrate welfare may be beneficial for producers while providing higher welfare conditions for the animals. While the immense number and type of species makes it difficult to suggest that all invertebrates will benefit from increased welfare, in this review we provide evidence that the topic of invertebrate welfare should be revisited, more thoroughly investigated, and in cases where appropriate, formally instituted.

Terrestrial gastropods (Helix spp) as sentinels of primary DNA damage for biomonitoring purposes: a validation study.

We validated the alkaline comet assay in two species of land snail (Helix aspersa and Helix vermiculata) to test their suitability as sentinels for primary DNA damage in polluted environments. The study was conducted under the framework of a biomonitoring program for a power station in Central Italy that had recently been converted from oil to coal-fired plant. After optimizing test conditions, the comet assay was used to measure the % Tail DNA induced by in vitro exposure of hemocytes to different concentrations of a reactive oxygen species (H2 O2 ). The treatment induced significant increases in this parameter with a concentration effect, indicating the effectiveness of the assay in snail hemocytes. After evaluating possible differences between the two species, we sampled them in three field sites at different distances from the power station, and in two reference sites assumed to have low or no levels of pollution. No species differences emerged. Percent Tail DNA values in snails from the sites near the power station were higher than those from control sites. An inverse correlation emerged between % Tail DNA and distance from the power station, suggesting that the primary DNA damage decreased as distance increased away from the pollution source. Detection of a gradient of heavy metal concentration in snail tissues suggests that these pollutants are a potential cause of the observed pattern. The comet assay appears to be a suitable assay and Helix spp. populations suitable sentinels to detect the genotoxic impact of pollutants.

The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem.

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprinodont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability of this fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxic mixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectiveness of the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) induced by (a) in vivo exposure of individual fish to X-rays (dose, 3Gy) and (b) following in vitro challenge of erythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strand breaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circulating fish blood contained actively proliferating cells that could influence the extent of DNA damage in control (untreated) fish, we measured the number of "comets" positive for 5-bromo-2'-deoxyuridine (BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. with X-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) values compared with the relevant untreated controls, indicating the effectiveness of the comet assay in the erythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibody labelling of erythrocytes indicated a very low percentage (5%) of "comets" positive for BrdU. Following optimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetello lagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically significant increases for tail DNA (%) compared with corresponding values observed in erythrocytes of fish caught in the unpolluted reference site "Saline di Tarquinia". The effects of physico-chemical parameters of the water (i.e., salinity, pH and oxygen content) did not significantly influence the induction of DNA damage. These results indicate that the comet assay provides a reliable parameter and that A. fasciatus is a promising "sentinel organism" to detect the genotoxic impact of complex mixtures in coastal lagoon ecosystems.

Genetic diversity of the killifish Aphanius fasciatus paralleling the environmental changes of Tarquinia salterns habitat.

The habitat in the Natural Reserve of the Tarquinia salterns, located on the Tyrrhenian coast of central Italy, has undergone dramatic alterations over the last 10 years. After salt production was terminated in 1997 the site was abandoned until 2002, with consequent degradation of habitat quality and stiffening of the environmental conditions. From 2003 to 2006 ecological rehabilitation of the site was carried out, restoring water circulation to its previous equilibrium. The genetic variation in the killifish Aphanius fasciatus inhabiting the salterns was monitored using allozymes from 1998. The results showed that the genetic variability of the killifish strongly reduced through time: a high number of rare alleles were lost and both heterozygosity and allele richness were significantly decreased. The most recent samples, taken after the ecological restoration, showed that to date the genetic erosion of A. fasciatus gene pool has slowed down, since no significant differences have been detected for any genetic variability parameter. Concerning the mechanisms leading to the impoverishment of the genetic variability, the strong loss of rare alleles suggests a role of genetic drift, which accords with the fluctuation of the effective population size recorded over the period of study and with the low gene flow typical of this species. The low levels of gene flow reported for this species imply that once lost, the genetic variability can rarely be restored through immigration from highly variable populations.