Reproductive toxicity assessment of cellulose nanofibers, citric acid, and branched polyethylenimine in sea urchins: Eco-design of nanostructured cellulose sponge framework (Part B).

In the framework of a safe-by-design approach, we previously assessed the eco-safety of nanostructured cellulose sponge (CNS) leachate on sea urchin reproduction. It impaired gamete quality, gamete fertilization competence, and embryo development possibly due to the leaching of chemical additives used during the CNS synthesis process. To extend this observation and identify the component(s) that contribute to CNS ecotoxicity, in the present study, we individually screened the cytotoxic effects on sea urchin Arbacia lixula and Paracentrotus lividus gametes and embryos of the three main constituents of CNS, namely cellulose nanofibers, citric acid, and branched polyethylenimine. The study aimed to minimize any potential safety risk of these components and to obtain an eco-safe CNS. Among the three CNS constituents, branched polyethylenimine resulted in the most toxic agent. Indeed, it affected the physiology and fertilization competence of male and female gametes as well as embryo development in both sea urchin species. These results are consistent with those previously reported for CNS leachate. Moreover, the characterisation of CNS leachate confirmed the presence of detectable branched polyethylenimine in the conditioned seawater even though in a very limited amount. Altogether, these data indicate that the presence of branched polyethylenimine is a cause-effect associated with a significant risk in CNS formulations due to its leaching upon contact with seawater. Nevertheless, the suggested safety protocol consisting of consecutive leaching treatments and conditioning of CNS in seawater can successfully ameliorate the CNS ecotoxicity while maintaining the efficacy of its sorbent properties supporting potential environmental applications.

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.

Nanostructured cellulose sponge engineered for marine environmental remediation: Eco-safety assessment of its leachate on sea urchin reproduction (Part A).

Nanostructured cellulose sponges (CNS) have been developed as eco-friendly and sustainable engineered materials for marine environmental remediation. Despite their functionality, sensitivity, efficiency and specificity have been proved, CNS application is still limited since their environmental safety (eco-safety) has not been completely assessed. In this study, CNS were allowed to leach in natural seawater simulating the remediation process condition and the eco-safety of CNS leachate on sea urchin reproduction has been assessed by carrying out a multi-response integrated approach, combining standardized ecotoxicity tests, innovative bioassays and gamete quality assessment. Overall, the ecotoxicity data indicate that CNS leachate affects gamete quality, gamete fertilisation competence, and embryo development probably associated with the release of chemical additives used during the synthesis process. However, in the framework of the eco-design approach, consecutive leaching treatments and conditioning of CNS in seawater open the route for a new safety protocol successfully solving the ecotoxicity while maintaining CNS sorbent properties. A safe environmental application of the resulting conditioned CNS for seawater pollution remediation is envisaged.

Eco-toxicity assessment of industrial by-product-based alkali-activated binders using the sea urchin embryogenesis bioassay.

New cement-based materials such as alkali-activated binders (AABs) or geopolymers allow the incorporation of waste or industrial by-products in their formulation, resulting an interesting valorization technique. Therefore, it is essential to inquire about the potential environmental and health impacts throughout their life cycle. In the European context, a minimum aquatic toxicity tests battery has been recommended for construction products, but their potential biological effects on marine ecosystems have not been considered. In this study, three industrial by-products, PAVAL® (PV) aluminum oxide, weathered bottom ash (WBA) resulting from incinerator bottom ash and glass cullet recycling waste (CSP), were evaluated as precursors in the AAB formulation from an environmental point of view. To determine the potential effects on marine environment caused by the leaching of contaminants from these materials into seawater, the leaching test EN-12457-2 and an ecotoxicity test using the model organism sea urchin Paracentrotus lividus were conducted. The percentage of abnormal larval development was selected as endpoint of the toxicity test. Based on the results obtained from the toxicity tests, AABs have less damaging impact (EC50 values: 49.2%-51.9%) on the marine environment in general than raw materials. The results highlight the need to stablish a specific battery of toxicity tests for the environmental assessment of construction products on marine ecosystem.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy.

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is predominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Ecological niche models for the assessment of site suitability of sea cucumbers and sea urchins in China.

In the present study, the maximum entropy model (MaxEnt) based on the data of sea surface temperature (SST) and published information was used to assess the site suitability for the aquaculture expansion of the sea cucumber Apostichopus japonicus and the sea urchin Strongylocentrotus intermedius in China. According to the current assessment, the coastal areas of Hebei province and Tianjin have great prospects for A. japonicus aquaculture, while is currently being underutilized. In the south, more than 94% of the coastal areas in Zhejiang, Fujian, and Guangdong provinces are suitable for the growth of A. japonicus for six months, especially the coastal areas of Lianjiang, Changle, Fuqing and Putian in Fujian province. The water temperatures in more than 94% of China's coastal areas are higher than 25 °C in July and August, which probably results in the mortality of S. intermedius in aquaculture. This clearly indicates that high water temperature is the bottleneck of S. intermedius aquaculture and well explains the limited expansion of this commercially important exotic species since the introduction in 1989. We suggest a new aquaculture model of S. intermedius that extends the seed production to November to avoid the mass mortality in summer. In the south, 64% of coastal areas in Zhejiang and Fujian provinces are suitable for the transplantation of S. intermedius to the south. The present study suggests the ecological niche model MaxEnt based on the data of SST and published information as a new tool for the assessment of the site suitability of sea cucumbers and sea urchins in China. This provides new insights into the aquaculture expansion of native and exotic species.

Fitness costs of alarm cues in sea urchins: Fertilization, hatching, and larval size.

Sea urchins sense alarm cues extracted from crushed conspecifics and perform anti-predation behaviors in exposure to alarm cues. This indicates of the fitness benefits of alarm cues in sea urchins. The present study investigated whether fitness costs of alarm cues exist in fertilization, hatchability, deformity, and larval size of the sea urchin Mesocentrotus nudus. In the present study, we found that fertilization and hatching rates were significantly lower in the group with alarm cues than those in the group without alarm cues, indicating that fitness costs of alarm cues exist in sea urchins. However, there was no significant difference in deformity rate, larval length, stomach length, and stomach width of M. nudus with and without alarm cues. The group with alarm cues showed significantly shorter larval width than the group without alarm cues. This indicates that smaller larvae of sea urchins more probably survive in the environment with alarm cues. The present study reveals that fitness costs of alarm cues exist in sea urchins and sheds light on the ecological roles of alarm cues in kelp bed ecosystems.

Interactive effects of ocean acidification, ocean warming, and diurnal temperature cycling on antioxidant responses and energy budgets in two sea urchins Strongylocentrotus intermedius and Tripneustes gratilla from different latitudes.

To accurately predict the fitness of marine ectotherms under the climate change scenarios, interactive effects from multiple environmental stressors should be considered, such as ocean acidification (OA), ocean warming (OW) and diurnal temperature cycling (DTC). In this work, we evaluated and compared the antioxidant capacity and metabolism homeostasis of two sea urchins, viz. the temperate species Strongylocentrotus intermedius and the tropical species Tripneustes gratilla, in response to oceanic conditions under a climate change scenario. The two species were treated separately/jointly by acidic (pH 7.6), thermal (ambient temperature + 3 °C), and temperature fluctuating (5 °C fluctuations daily) seawater for 28 days. The activities of antioxidant enzymes (catalase and superoxide dismutase) and the cellular energy allocation in the urchins' gonads were assessed subsequently. Results showed that exposure to OA, OW, and DTC all induced antioxidant responses associated with metabolism imbalance in both S. intermedius and T. gratilla. The physiological adjustments and energy strategies towards exposure of OA, OW, and DTC are species specific, perhaps owing to the different thermal acclimation of species from two latitudes. Moreover, decrease of cellular energy allocation were detected in both species under combined OA, OW, and DTC conditions, indicating unsustainable bioenergetic states. The decrease of cellular energy allocation is weaker in T. gratilla than in S. intermedius, implying higher acclimation capacity to maintain the energy homeostasis in tropical urchins. These results suggest that climate change might affect the population replenishment of the two sea urchins species, especially for the temperate species.

Assessment of microplastic content in Diadema africanum sea urchin from Tenerife (Canary Islands, Spain).

Sea urchins are highly abundant in the marine ecosystem where they graze limiting algal biomass and also serving as food for other predators. In this work, the presence of microplastics in the digestive tracts and gonads of 33 Diadema africanum sea urchins collected at two sampling points in Tenerife (Canary Islands, Spain) was studied. After separation and digestion of the digestive tracts and the gonads, the visualization of the filtrates under the stereomicroscope revealed the presence of 320 items which were microfibers (97.5%), fragments (1.9%) and films (0.6%), mainly blue (43.3 and 47.0% in the two sampling points, Tajao and El Porís, respectively) and translucent white (32.5 and 39.5%, respectively). Statistical analysis revealed that there were no significative differences in the contents of gonads and digestive tracts between both sampling locations. Regarding microfibers lengths, significative differences were only observed between the two sampling points, not between tissues. µRaman analysis showed that they were mainly cellulosic (46.0%), polypropylene (24.3%) and polyethylene terephthalate (24.3%). This study confirms for the first time the presence of microplastics in sea urchins from the Macaronesian region and also from Spain.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Biomonitoring Assessment of Toxic and Trace Elements in Sterechinus neumayeri Sea Urchins from the Comandante Ferraz Station in Antarctica.

In the present study, sea urchin Sterechinus neumayeri tissues were used for the passive biomonitoring of toxic and trace elements at the Comandante Ferraz Station, Antarctica and compared to a pristine region (Botany). As, Ba, Br, Ca, Co, Cr, Fe, K, Na, Rb, Sc, Se and Zn concentrations were determined by instrumental neutron activation analysis (INAA), while toxic metals (Cd, Hg, Ni and Pb), and Cu were determined by atomic absorption spectrometry (GF-AAS). The findings were compared to other organisms commonly applied for biomonitoring purposes and to the sediment concentrations of each sampling region. Urchins from the Ferraz Station area presented higher Br, Co, Cr, Cs, K, Se and Zn levels than the pristine location. The results obtained herein suggest S. neumayeri can be applied to the biomonitoring of Cr and Zn. The present study also contributes to knowledge of the mineral composition of the sea urchin S. neumayeri.

Blue growth: Sea urchin sustainable aquaculture, innovative approaches / Crecimiento azul: acuicultura sostenible de erizos de mar, enfoques innovadores

Introduction: "Blue Economy" refers to ocean-based economies with a sustainable approach. It focuses in smaller carbon footprints and efficiency, principles that can be applied to aquaculture. However, it has been difficult to develop successful blue economy projects in sea urchin aquaculture. Objective: To compare URCHINOMICS (Norway) and ARBACIA (Argentina), two aquaculture projects with different business models. Methods: We used publicly available information to compare both companies on the basis of their value proposition and tensions (e.g. cultural, social economic and technological). Results: To be successful, sea urchin aquaculture requires development of appropriate technology, open innovation and cooperation of people with different academic, business and organizational backgrounds. Conclusion: The ultimate success of these and similar companies will depend on free interaction of experts from multiple fields and on technological innovation.

Introducción: "Economía azul" se refiere a las economías basadas en el océano con un enfoque sostenible. Se enfoca en menores huellas de carbono y eficiencia, principios que se pueden aplicar a la acuicultura. Sin embargo, ha sido difícil desarrollar proyectos exitosos de economía azul en la acuicultura de erizos de mar. Objetivo: Comparar URCHINOMICS (Noruega) y ARBACIA (Argentina), dos proyectos de acuicultura con diferentes modelos de negocio. Métodos: Utilizamos información disponible públicamente para comparar ambas empresas sobre la base de su propuesta de valor y tensiones (por ejemplo, culturales, socioeconómicas y tecnológicas). Resultados: Para tener éxito, la acuicultura de erizos de mar requiere el desarrollo de tecnología adecuada, innovación abierta y cooperación de personas con diferentes antecedentes académicos, empresariales y organizativos. Conclusión: El éxito final de estas y otras empresas similares dependerá de la libre interacción de expertos de múltiples campos y de la innovación tecnológica.

Ecotoxicological assessment of chemotherapeutic agents using toxicity tests with embryos of Mellita quinquiesperforata.

The consumption of anticancer agents has increased in the recent decades, and these substances may be present in sewage. Consequently, they may reach the environment when sanitation infrastructure is ineffective. This study evaluated the toxicity of three anticancer agents-Tamoxifen (TAM), Cisplatin (CisPt), and Cyclophosphamide (CP)-on the development of embryos of the sand-dollar Mellita quinquiesperforata. Adult individuals were collected in sandy beaches, and gametes were obtained. Freshly-fertilized eggs were exposed to increasing sets of concentrations of each compound, and the effective concentrations needed to cause a 50% effect in the organisms (EC50) were calculated. The three compounds were toxic, and their EC50 values were 16.78 ± 2.42 ng·L-1 (TAM), 27.20 ± 38.26 ng·L-1 (CisPt), and 101.82 ± 70.96 ng·L-1 (CP). There is no information on the environmental levels of these compounds in Brazil, but as they were already detected in ng·L-1 levels worldwide, it can be expected that these substances pose environmental risks to the marine biota.

A Novel In Situ Toxicity Identification Evaluation (iTIE) System for Determining which Chemicals Drive Impairments at Contaminated Sites.

Human-dominated waterways contain thousands of chemicals. Determining which chemical is the most important stressor is important, yet very challenging. The Toxicity Identification Evaluation (TIE) procedure from the US Environmental Protection Agency uses a series of chemical and physical manipulations to fractionate compounds within a matrix and systematically identify potential toxicants through laboratory bioassay testing. Although this may provide useful information, it lacks ecological realism because it is subject to laboratory-related artifacts and is resource intensive. The in situ Toxicity Identification Evaluation (iTIE) technology was developed to improve this approach and has undergone a number of modifications over the past several years. The novel prototype 3 consists of an array of iTIE ambient water fractionation units. Each unit is connected to a peristaltic pumping system with an organism exposure chamber that receives water from a resin chamber to chemically fractionate test site water. Test organisms included freshwater and marine standard toxicity test species. Postfractionation waters are collected for subsequent chemical analyses. Currently, the resins allow for separation of ammonia, metals, and nonpolar organics; the subsequent toxicity responses are compared between treatments and unfractionated, ambient exposures. The iTIE system was deployed to a depth of 3 m and evaluated in streams and marine harbors. Chemical analyses of water and iTIE chemical sorptive resins confirmed chemical groups causing lethal to sublethal responses. The system proved to be as sensitive or more so than the traditional phase 1 TIE test and required almost half of the resources to complete. This iTIE prototype provides a robust technology that improves stressor-causality linkages and thereby supports strong evidence for ecological risk weight-of-evidence assessments. Environ Toxicol Chem 2020;39:1746-1754. © 2020 SETAC.

Ocean warming and acidification pose synergistic limits to the thermal niche of an economically important echinoderm.

To make robust projectios of the impacts of climate change, it is critical to understand how abiotic factors may interact to constrain the distribution and productivity of marine flora and fauna. We evaluated the effects of projected end of the century ocean acidification (OA) and warming (OW) on the thermal tolerance of an important living marine resource, the sea urchin Loxechinus albus, a benthic shallow water coastal herbivore inhabiting part of the Pacific coast of South America. After exposing young juveniles for a 1-month period to contrasting pCO2 (~500 and 1400 µatm) and temperature (~15 °C and 20 °C) levels, critical thermal maximum (CTmax) and minimum (CTmin) as well as thermal tolerance polygons were assessed based on self-righting success as an end point. Transcription of heat shock protein 70 (HSP70), a chaperone protecting cellular proteins from environmental stress, was also measured. Exposure to elevated pCO2 significantly reduced thermal tolerance by increasing CTmin at both experimental temperatures and decreasing CTmax at 20 °C. There was also a strong synergistic effect of OA × OW on HSP70 transcription levels which were 75 times higher than in control conditions. If this species is unable to adapt to elevated pCO2 in the future, the reduction in thermal tolerance and HSP response suggests that near-future warming and OA will disrupt their performance and reduce their distribution with ecological and economic consequences. Given the wider latitudinal range (6 to 56°S) and environmental tolerance of L. albus compared to other members of this region's benthic invertebrate community, OW and OA may cause substantial changes to the coastal fauna along this geographical range.

Metagenomic assessment of body surface bacterial communities of the sea urchin, Tripneustes gratilla.

Sea urchins, including Tripneustes gratilla, are susceptible to a disease known as bald sea urchin disease, which has the potential to lead to economic losses in this emerging aquaculture industry in South Africa. This disease is characterized by lesions that form on sea urchin exoskeletal surfaces. This study aimed to characterize the body surface bacterial communities associated with T. gratilla, using a 16S rDNA gene metagenomics approach, to provide insight into the bacterial agents associated with this aquaculture species, as well as with this balding disease. Bacterial samples were collected from non-lesioned healthy animals obtained from natural locations along the eastern coast of South Africa, as well as from different cultured cohorts: non-lesioned healthy-, lesioned diseased- and non-lesioned stressed animals. A total of 1,067,515 individual bacterial operational taxonomic units (OTUs) were identified, belonging to 133 family-, 123 genus- and 113 species level OTU groups. Alpha diversity analyses, based on Chao1, Shannon and Simpson indices, showed that there were no statistically significant differences (ANOVA; P > 0.05) between the respective cohorts, as all cohorts displayed a high degree of bacterial diversity. Similarly, beta diversity analyses (Non-metric multidimensional scaling) showed a large degree of overlapping OTUs across the four cohorts. Within each cohort, various OTUs commonly associated with marine environments were found, predominantly belonging to the families Vibrionaceae, Saprospiraceae, Flavobacteriaceae and Sphingomonadaceae. Differential abundance analysis (DESeq2) revealed that OTUs that are differentially abundant across cohorts were likely not responsible for this balding disease, suggesting that complex bacterial agents, rather than a specific pathogenic agent, are likely causing this disease. Furthermore, the putative metabolic functions assigned to the bacterial communities showed that heterotrophic bacteria appear to be responsible for tissue lysis of degrading animal matter. The results from this study, obtained through univariate and multivariate-based approaches, contributes to future management strategies of this emerging aquaculture species by providing insight into the bacterial communities associated with both natural and cultured environments.

Annual assessment of the sea urchin (Paracentrotus lividus) humoral innate immune status: Tales from the north Portuguese coast.

Innate immune status of the sea urchin Paracentrotus lividus population from two different rocky shore beaches in the northern Portuguese coast was evaluated for a period of one year. Although some ecological studies regarding the effect of toxics on the immune parameters of the sea urchin were made in Portuguese waters, there is a current lack of knowledge concerning their immune status all over the year. In perspective of a changing ecosystem in these waters due to global warming and colonization of new species, it is important to assess the status of the major species living in the area. In this way, immune parameters such as total protein content, nitric oxide concentration, haemolytic activity, protease activity, lysozyme concentration and bactericidal activity were evaluated in the perivisceral coelomic fluid, and were correlated with the gonadal index of the population and water parameters. Also, the spawning period can upset some immune status parameters, and others such as haemolytic activity and bactericidal activity against Vibrio anguillarum, showed a clear correlation with the gonad maturation status. The knowledge of the basal immune status of the species could serve as ecological indicator of some stress agent or contaminant into the field; also, coelomic fluid is suggested as good quality marker to assess the immune status of sea urchins.

Seasonal assessment of biological indices, bioaccumulation, and bioavailability of heavy metals in sea urchins Paracentrotus lividus from Algerian west coast, applied to environmental monitoring.

The aim of the present work was to extend our knowledge on the variability of trace metals in sea urchin tissues, focusing on seasonal fluctuations (2010 February for "winter," May for "spring," August for "summer," November for "autumn") in the three different sampling sites of Algerian west coast (Oran Harbor (S1), Ain Defla (S2), and Hadjaj (belonging to Mostaganem City S3)). For this purpose, the bioavailability (metal indices) and bioaccumulation (metal concentrations in soft tissues) of heavy metals (Zn, Cu, Pb, and Cd), the physiological characteristics (e.g., biological indices such as condition index (CI), repletion index (RI), gonad index (GI)), and the biometric parameters (diameter (D) and the height (H)) of sea urchins Paracentrotus lividus were assessed and related to seasons and sites. To investigate the metal bioavailability to sea urchins more precisely, the metal indices were used as a reliable tool in the present work, instead of the metal concentrations only. The interest to standardize metal concentrations with the weight of the urchin test is to overcome the metal burden variations in the soft tissues of urchin related to the seasonal weight changes of the soft body of animal. We evidence that the most contaminated sites were S1 and S2. Furthermore, it should be noted that the bioavailability of metals, corresponding to the values of metal indices, is also more pronounced in S1 and S2 compared to that measured in S3. Thus, a correlation is observed between seasonal metal content in urchin tissues from the three sites and the corresponding metal indices. The high metal concentrations were obtained during the period when RI and CI were highest. So, it appears that the bioaccumulation of metals in sea urchins of the three sites studied is significantly influenced by the reproductive cycle and diet, feeding activity, and physiological state of these organisms. We noticed that the sea urchins from the sites S1 and S2 were small in size. It is probable that these animals, whose internal tissues contained high concentrations of metals, have been exposed to metal pollution, which might have affected both their growth and altered their physiological capacity. This approach is very original and might be used in the monitoring of the quality of coastal waters, and the present work provided a useful data set for Mediterranean monitoring network.

Assessment of the individual and mixture toxicity of cadmium, copper and oxytetracycline, on the embryo-larval development of the sea urchin Paracentrotus lividus.

Multiple pollutions by trace metals and pharmaceuticals have become one of the most important problems in marine coastal areas because of its excessive toxicity on organisms living in this area. This study aimed to assess the individual and mixture toxicity of Cu, Cd, and oxytetracycline frequently existing in the contaminated marine areas and the embryo-larval development of the sea urchin Paracentrotus lividus. The individual contamination of the spermatozoid for 1 h with the increasing concentrations of Cd, Cu, and OTC decreases the fertility rate and increases larvae anomalies in the order Cu > Cd > OTC. Moreover, the normal larva frequency and the length of spicules were more sensitive than the fertilization rate and normal gastrula frequency endpoints. The mixture toxicity assessed by multiple experimental designs showed clearly that concentrations of Cd, Cu, and OTC superior to 338 µg/L, 0.56 µg/L, and 0.83 mg/L, respectively, cause significant larva malformations.

Response of rocky shore communities to anthropogenic pressures in Albania (Mediterranean Sea): Ecological status assessment through the CARLIT method.

The lower mid-littoral and shallow subtidal communities were studied in the district of Vlora (Albania), three years after the establishment of a Marine Protected Area, with particular attention to the long-lived species. The bioconstructions built in the mid-littoral zone by the calcified rhodobiont Lithophyllum byssoides were in poor condition and sometimes even dead. In contrast, the brown alga Cystoseira amentacea constituted lush stands. For assessing the ecological status of the studied area, the CARLIT method, based upon macroalgal communities, was applied. The observed range of ecological status was wide ('high' through 'bad') and was overall among the lowest assessed to date in the Mediterranean Sea. The occurrence of extensive sea-urchin barren-grounds, though not taken into consideration by the CARLIT index, confirmed the poor condition of large sectors of the study area. Overall, the CARLIT index is well correlated with anthropogenic pressures, as assessed by the LUSI index.