Echinoderm larvae as bioindicators for the assessment of marine pollution: Sea urchin and sea cucumber responsiveness and future perspectives.

Echinoderms play a crucial role in the functioning of marine ecosystems and due to their extensive distribution, rapid response, and the high sensitivity of their planktonic larvae to a large range of stressors, some species are widely used as biological indicators. In addition to sea urchins, sea cucumbers have recently been implemented in embryotoxicity bioassays showing high potential in ecotoxicological studies. However, the use of this species is still hindered by a lack of knowledge regarding their comparative responsiveness. The present study aimed to investigate the responsiveness of different echinoderm species to environmental pollution in order to develop their integration in batteries of ecotoxicological bioassays. To this end, the embryos of two sea urchins (Paracentrotus lividus and Arbacia lixula) and two sea cucumbers (Holothuria polii and Holothuria tubulosa) were incubated with inorganic and organic toxicants (cadmium, copper, mercury, lead, sodium dodecyl sulphate and 4-n-Nonhyphenol) and elutriates from contaminated marine sediments, chosen as a case study model. The results obtained, expressed through the percentage of abnormal embryos and Integrative Toxicity Indices (ITI), indicated species-specific sensitivities to pollutants, with comparable and correlated responsiveness between sea urchins and sea cucumbers. More specifically, sea cucumber larvae exposed to elutriates appear to be more sensitive than sea urchins, especially when incubated with samples containing trace metals, PCB and TBT. These results indicate that toxic responses in embryos exposed to environmental matrices are probably modulated by interactions between different variables, including additive, synergistic and antagonistic effects. These findings suggest that performing a larval test using different echinoderm classes can integrate the interactive effects of bioavailable fraction of contaminants on various levels, providing sensitive, representative and all year-round batteries of bioassays to apply in ecotoxicological studies.

Comparison of different ecotoxicological batteries with WOE approach for the environmental quality evaluation of harbour sediments.

This study was conducted under the Italian Ministerial Decree D.M. 173/2016 which regulates the assessment of the Sediment Class Quality in Italy using ecotoxicological bioassay and chemical analysis (Weight-Of-Evidence model). The aim of this work was to evaluate the real classification obtained by the theoretically equivalent responses of nine different combinations of batteries based on six different species: Aliivibrio fischeri (inhibition of bioluminescence), Phaeodactylum tricornutum, Skeletonema costatum, Dunaliella tertiolecta (inhibition of algal growth), Paracentrotus lividus and Crassostrea gigas (embryotoxicity). Bioassays, in many cases, showed a non-bioavailability effect of the pollutants; these one highly revealed by the chemical analyses. Algal species showed responses very similar from each other. Otherwise, species used for embryotoxicity produced wide responses, consequently modifying the quality class of sediments and the handling management (i.e. landfill confinement or beach nourishment) allowed by the Law.

Development and validation of new analytical methods using sea urchin embryo bioassay to evaluate dredged marine sediments.

Management of dredged materials disposal is regulated by several environmental normative requirements, and it is often supported by the integration of chemical data with ecotoxicological characterization. The reliability of a bioassay to assess the potential toxicity of dredged sediments requires the selection of quality criteria that should be based on simple analytical methods and easily understandable hazard for politicians and environmental managers. The sea urchin embryo-toxicity bioassay is considered an essential component for evaluating the quality of sediments in harbour areas but its use, when based exclusively on the observation of normal vs. abnormal embryos, may alter the interpretation of the results, overestimating the risk assessment. To improve the reliability of this assay in establishing a causative relationship between quality of sediments and sea urchin embryonic development, here we developed and validated three Integrative Toxicity Indexes (ITI 2.0, ITI 3.0, ITI 4.0), modifying the already-known ITI (here ITI 1.0). Based on this aim, we used Taranto harbour as a model pilot-study to compare results to those obtained from standard criteria. Among the tested indexes, the ITI 4.0, discriminating strictly developmental delay and morphological defects from fertilized egg to gastrula stage, resulted in the most promising.

Integrated characterization and risk management of marine sediments: The case study of the industrialized Bagnoli area (Naples, Italy).

The aim of the present work is to demonstrate the practical importance of a multidisciplinary approach and weighted criteria to synthesize and integrate different typologies of data (or lines of evidence, LOEs), including chemical levels in marine sediments, their bioavailability to specific indicator species, ecotoxicological effects measured through subcellular biomarkers and batteries of bioassays, and potential impacts of pollution on local benthic communities. The area of Bagnoli (Gulf of Naples, Southern Italy) was selected as a model case-study, as it is a coastal area chronically impacted by massive industrial contamination (trace metals and hydrocarbons), and dismissed decades ago without any subsequent remediation or habitat restoration. The results of each LOE were elaborated to provide specific hazard indices before their overall integration in a weight of evidence (WOE) evaluation. Levels of some trace metals and PAHs revealed a severe contamination in the entire study area. Bioavailability of hydrocarbons was evident particularly for high molecular weight PAHs, which also caused significant variations of cellular biomarkers, such as cytochrome P450 metabolization in fish, lysosomal membrane destabilization in mussels, genotoxic effects both in fish and molluscs. The results of a battery of bioassays indicated less marked responses compared to those obtained from chemical and biomarkers analyses, with acute toxicity still present in sediments close to the source of contamination. The analysis of benthic assemblages showed limited evidence of impact in the whole area, indicating a good functioning of local ecosystems at chronic contamination. Overall, the results of this study confirm the need of combining chemical and biological data, the quantitative characterization of various typologies of hazard and the importance of assessing an integrated environmental WOE risk, to orientate specific and scientifically-supported management options in industrialized areas.

Sea cucumber Holothuria polii (Delle Chiaje, 1823) as new model for embryo bioassays in ecotoxicological studies.

The sea cucumbers are common members of marine benthic communities, widespread distributed, easily available and handled. Nevertheless, no data are available on embryo toxicity assays using sea cucumbers, despite some of these species could fully meet the requirements for model test organisms. Holothuria polii is a key species in soft sediments and seagrass meadows; the aim of the present study was the standardization of a new embryo bioassay with this species, as an ecologically relevant test to evaluate the effects of environmental stressors. Sequential experiments were carried out, allowing to define the test acceptability, and a minimum sample size of 240 embryos. Temperature of 26 °C, salinity at 36‰ and a density of 60 eggs/ml were identified as optimum experimental conditions for performing the bioassay. The EC50 calculated for Cd2+ and Cu2+ in dose-response experiments indicated a good sensitivity of H. polii, with comparable values with those obtained in embryo toxicity bioassays of other marine invertebrates. An Integrative Toxicity Index (ITI) was calculated by integrating the frequency of abnormal embryos with the severity of observed abnormalities. The index allows to better discriminate different levels of toxicity, appearing particularly relevant for validating the usefulness of H. polii in embryo assays and ecotoxicological studies on environmental quality.

First molecular evidence of the toxicogenetic effects of copper on sea urchin Paracentrotus lividus embryo development.

Bioassays with sea urchin embryos are widely used to define the environmental quality of marine waters. Anomalies during embryogenesis are generally considered as end-points, whereas a toxigenomic approach, despite it is wide use in other species, is yet in its infancy. In the present study we evaluated toxigenic effects induced by copper on the sea urchin Paracentrotus lividus embryo, combining morphological observations with gene expression analysis. Many anthropogenic activities release copper in the marine environment, with harmful effects on aquatic organisms. In the present study P. lidivus embryos were exposed to different concentrations of copper (24, 36, 48 µg/L) and the activation of fifty specific marker genes, involved in different biological processes (stress, skeletogenesis, development/differentiation, detoxification) was investigated at early blastula, late gastrula and pluteus stage. At blastula stage no morphological anomalies were found, with early down-regulation of genes involved in development/differentiation and a moderate up-regulation of some detoxification genes. At gastrula stage a slight increase in developmental anomalies (up to 19% of malformed embryos) was followed by an increased number of targeted genes belonging to the same two classes, relative to the blastula stage. At pluteus stage morphological anomalies increased in a dose dependent manner. All the analyzed genes were strongly up-regulated, stress and skeletogenic genes showing a "late response" and almost all genes were targeted by copper at all the concentrations tested. The present study represents the first molecular report on the potential negative effect of copper on P. lividus embryos in the environment. Gene expression analysis should be considered as a promising tool for future environmental biomonitoring programs.

In situ embryo toxicity test with sea urchin: Development of exposure chamber for test execution.

In situ toxicity tests represent a technique rarely performed owing to the lack of standard methodologies or to technical or economic problems. Nevertheless, its application would allow for a more realistic interpretation of pollution effects than those obtained by standard laboratory tests. The goal of this study is to develop and validate a specific exposure chamber for in situ exposition of Paracentrotus lividus embryos to obtain a defined methodology to perform the sea urchin embryo toxicity test in field conditions. After a first part of the study to verify the feasibility of the test chamber and the methodology, this approach was used as a tool to investigate whether the cruise ship "Costa Concordia" shipwrecked on Giglio Island (Tuscany, Italy), could have acted as a source of pollution. The results obtained for in situ tests showed, on average, percentages of normal embryos lower than those obtained in laboratory conditions and a greater sensitivity than for those obtained in the laboratory owing to the time-integration of results. Thus the exposure chamber and the in situ methodology so far developed would appear to be suitable tools for future application in the environmental quality evaluation of marine waters.