Combined Effects of Diatom-Derived Oxylipins on the Sea Urchin Paracentrotus lividus.

Oxylipins are diatom-derived secondary metabolites, deriving from the oxidation of polyunsatured fatty acids that are released from cell membranes after cell damage or senescence of these single-celled algae. Previous results revealed harmful toxic effects of polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) on sea urchin Paracentrotus lividus embryonic development by testing individual compounds and mixtures of the same chemical group. Here, we investigated the combined effects of these compounds on sea urchin development at the morphological and molecular level for the first time. Our results demonstrated that oxylipin mixtures had stronger effects on sea urchin embryos compared with individual compounds, confirming that PUAs induce malformations and HEPEs cause developmental delay. This harmful effect was also confirmed by molecular analysis. Twelve new genes, involved in stress response and embryonic developmental processes, were isolated from the sea urchin P. lividus; these genes were found to be functionally interconnected with 11 genes already identified as a stress response of P. lividus embryos to single oxylipins. The expression levels of most of the analyzed genes targeted by oxylipin mixtures were involved in stress, skeletogenesis, development/differentiation, and detoxification processes. This work has important ecological implications, considering that PUAs and HEPEs represent the most abundant oxylipins in bloom-forming diatoms, opening new perspectives in understanding the molecular pathways activated by sea urchins exposed to diatom oxylipins.

Molecular and Morphological Toxicity of Diatom-Derived Hydroxyacid Mixtures to Sea Urchin Paracentrotus lividus Embryos.

Oxylipins such as polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) are signaling molecules derived from the oxidation of polyunsaturated fatty acids. They are common in diatoms that constitute a major group of microalgae in freshwater and oceanic ecosystems. Although HEPEs represent the most common oxylipins produced by diatoms, little information is available on their effects on marine invertebrates, and most of the information has been obtained by testing individual HEPEs. Our previous studies reported that four hydroxyacids, i.e., 5-, 9-, 11-, and 15-HEPE, were able to induce malformations and a marked developmental delay in sea urchin Paracentrotus lividus embryos, which had not been reported for other oxylipins. Here, we tested a mixture of 5-, 9-, 11-, and 15-HEPE at different concentrations for the first time. The results showed that mixtures of HEPEs have synergistic effects that are much more severe compared to those of individual HEPEs: The HEPE mixtures induced malformations in sea urchin embryos at lower concentrations. Increasing HEPE mixture concentrations induced a marked increase in the number of delayed embryos, until all embryos were delayed at the highest concentration tested. At the molecular level, the HEPE mixtures induced variations in the expression of 50 genes involved in different functional processes, mainly down-regulating these genes at the earliest stages of embryonic development. These findings are ecologically significant, considering that during diatom blooms, sea urchins could accumulate HEPEs in concentrations comparable to those tested in the present study.

Dispersant approval procedures in France and Italy: A comparative ecotoxicity study.

A research project has been performed to the request of the RAMOGE Executive Secretariat to identify differences between dispersant approval procedures in France and Italy and propose ways to harmonize them. A collaborative study has been conducted by CEDRE (Centre of Documentation, Research and Experimentation on Accidental Water Pollution) and ISPRA (Italian Institute for Environmental Protection and Research) to: a) compare current approval procedures in Italy and France with identification of differences and commonalities; b) carry out toxicity tests using both procedures on two selected dispersants; c) propose a common approach between Italy and France. The results showed that, because of the differences in ecotoxicological tests and in the evaluation criteria used, the outcomes on the same products could be different in Italy and in France. Both tested dispersants met the French requirements for approval (LC50 ≥ 10 times reference toxicant), while only one dispersant met the Italian approval criterion (EC50 > 10mg/L). A possible way of harmonizing the approval procedures could be to increase the number of test organisms in the French procedure, which currently only uses one crustacean species. Furthermore, a common criterion for toxicity assessment should be discussed and agreed.

Lethal and sublethal endpoints observed for Artemia exposed to two reference toxicants and an ecotoxicological concern organic compound.

Swimming speed alteration and mortality assays with the marine crustacean Artemia franciscana were carried out. EC50 and LC50 values after 24-48h exposures were calculated for two reference toxicants, copper sulphate pentahydrate (CuSO4·5H2O) and Sodium Dodecyl Sulphate (SDS), and an ecotoxicological concern organic compound, Diethylene Glycol (DEG). Different end-points have been evaluated, in order to point out their sensitivity levels. The swimming speed alteration (SSA) was compared to mortality values and also to the hatching rate inhibition (literature data). SSA resulted to be more sensitive than the mortality and with a sensitivity comparable to (or even higher than) the hatching rate endpoint.

A comparative toxicity study between an autochthonous Artemia and a non native invasive species.

Acute heavy metal toxicity was compared in nauplii of an autochthonous Artemia parthenogenetica (Branchiopoda, Anostraca) from Saline di Cervia (Ravenna, Italy) and of a non native commercially available A. franciscana. No significant difference in sensitivity was detected between two species.

The under-investigated plastic threat on seagrasses worldwide: a comprehensive review.

Marine plastic pollution is a well-recognised and debated issue affecting most marine ecosystems. Despite this, the threat of plastic pollution on seagrasses has not received significant scientific attention compared to other marine species and habitats. The present review aims to summarise the scientific data published in the last decade (January 2012-2023), concerning the evaluation of plastic pollution, of all sizes and types, including bio-based polymers, on several seagrass species worldwide. To achieve this goal, a comprehensive and critical review of 26 scientific papers has been carried out, taking into consideration the investigated areas, the seagrass species and the plant parts considered, the experimental design and the type of polymers analysed, both in field monitoring and in laboratory-controlled experiments. The outcomes of the present review clearly showed that the dynamics and effects of plastic pollution in seagrass are still under-explored. Most data emerged from Europe, with little or no data on plastic pollution in North and South America, Australia, Africa and Antarctica. Most of the studies were devoted to microplastics, with limited studies dedicated to macroplastics and only one to nanoplastics. The methodological approach (in terms of experimental design and polymer physico-chemical characterisation) should be carefully standardised, beside the use of a model species, such as Zostera marina, and further laboratory experiments. All these knowledge gaps must be urgently fulfilled, since valuable and reliable scientific knowledge is necessary to improve seagrass habitat protection measures against the current plastic pollution crisis.

Effect of biodegradable polymers upon grazing activity of the sea urchin Paracentrotus lividus (Lmk) revealed by morphological, histological and molecular analyses.

In the last years biodegradable polymers (BPs) were largely used as real opportunity to solve plastic pollution. Otherwise, their wide use in commercial products, such as packaging sector, is causing a new pollution alarm, mainly because few data reported about their behaviour in the environment and toxicity on marine organisms. Our previous results showed that embryos of the sea urchin Paracentrotus lividus (Lmk) exposed to poly(ε-caprolactone) (PCL), poly(3-hydroxybutyrate) (PHB) and poly(lactic acid) (PLA) showed delay of their development and morphological malformations, also affecting at the molecular levels the expression of several genes involved in different functional responses. In the present work for the first time, we tested the effects of five microplastics (MPs) obtained from BPs such as PBS, poly(butylene succinate), PBSA, poly(butylene succinate-co-butylene adipate), PCL, PHB and PLA, upon grazing activity of the sea urchin revealed by: i. histological analysis seeing at the gonadic tissues; ii. morphological analysis of the deriving embryos; iii. molecular analyses on these embryos to detect variations of the gene expression of eighty-seven genes involved in stress response, detoxification, skeletogenesis, differentiation and development. All these results will help in understanding how MP accumulated inside various organs in the adult sea urchins, and more in general in marine invertebrates, could represent risks for the marine environment.

Current knowledge of approval procedures of dispersant use at sea: looking for potential harmonization from global to Mediterranean scale.

Dispersants are approved for use in many countries (UK, South Korea, Australia, Egypt, France, Greece, Indonesia, Italy, Japan, Malaysia, Norway, Singapore, Spain, Thailand, and several coastal African, South American, and Middle Eastern countries). Here, the protocols of the most advanced (France, Norway, UK, Spain, Greece, Italy, USA, and Australia) are compared for identifying possible harmonization of approval procedures. Pre-toxicity testing, recognized oil datasets, common thresholds, standardized protocols, zoning, and monitoring are some of the aspects that can be discussed between countries.

Biodegradation of Plastics Induced by Marine Organisms: Future Perspectives for Bioremediation Approaches.

Plastic pollution is a distinctive element of the globalized world. In fact, since the 1970s the expansion and use of plastics, particularly in the consumer and commercial sectors, has given this material a permanent place in our lives. The increasing use of plastic products and the wrong management of end-of-life plastic products have contributed to increasing environmental pollution, with negative impacts on our ecosystems and the ecological functions of natural habitats. Nowadays, plastic pollution is pervasive in all environmental compartments. As aquatic environments are the dumping points for poorly managed plastics, biofouling and biodegradation have been proposed as promising approaches for plastic bioremediation. Known for the high stability of plastics in the marine environment, this represents a very important issue to preserve marine biodiversity. In this review, we have summarized the main cases reported in the literature on the degradation of plastics by bacteria, fungi, and microalgae and the degradation mechanisms involved, to highlight the potential of bioremediation approaches to reduce macro and microplastic pollution.

Effects of biodegradable-based microplastics in Paracentrotus lividus Lmk embryos: Morphological and gene expression analysis.

Plastic pollution is a remarkable environmental issue. In fact, plastic is widespread in the lifetime and serious environmental problems are caused by the improper management of plastic end of life, being plastic litter detected in any environment. Efforts are put to implement the development of sustainable and circular materials. In this scenario, biodegradable polymers, BPs, are promising materials if correctly applied and managed at the end of life to minimize environmental problems. However, a lack of data on BPs fate and toxicity on marine organisms, limits their applicability. In this research, the impact of microplastics obtained from BPs, BMPs, were analyzed on Paracentrotus lividus. Microplastics were produced from five biodegradable polyesters at laboratory scale by milling the pristine polymers, under cryogenic conditions. Morphological analysis of P. lividus embryos exposed to polycaprolactone (PCL), polyhydroxy butyrate (PHB) and polylactic acid (PLA) showed their delay and malformations, which at molecular level are due to variation in expression levels of eighty-seven genes involved in various cellular processes, such as skeletogenesis, differentiation and development, stress, and detoxification response. Exposure to poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) microplastics showed no detectable effects on P. lividus embryos. These findings contribute with important data on the effect of BPs on the physiology of marine invertebrates.

Biodegradable polymers: A real opportunity to solve marine plastic pollution?

Plastic is a ubiquitous material in our life, and its durability represents a great problem for the environment. Several studies reported the occurrence of plastic litter in different environmental compartments and, consequently, numerous efforts are currently focused on how improving its recycling process and produce environmentally friendly solutions. In recent years, biodegradable polymers/plastics (BPs) have been proposed to reduce environmental impacts in specific applications (e.g., when conventional plastics are difficult or expensive to remove from the environment). Their wide use in commercial products, especially in the packaging sector, is causing new pollution alarm. Research studies are ongoing to improve BPs manufacturing and characteristics, but few data are reported about their behavior and toxicity into the marine environment. This paper reviewed the current state of the art highlighting that, even though the degradation of BPs in simulated or real marine environments is quite investigated, only eleven papers reported their effects on marine organisms (e.g., behavioral and oxidative stress and potential cascading effects on marine ecosystems). Presently, the main benefits of BPs are linked to waste management (including collection and recycling of organic waste). Due to the existing knowledge gaps, BPs cannot be deemed yet as a solution to marine plastic pollution.

Comparative toxicity of ionic and nanoparticulate zinc in the species Cymodoce truncata, Gammarus aequicauda and Paracentrotus lividus.

Due to the continuous development, production and consumption of nanoparticles (NPs), their release, fate and effects in marine coastal environment can represent a major concern. The aim of this study was to evaluate the toxicity of ZnO nanoparticles (ZnO NPs) and compare it to bulk ZnSO4 on three macroinvertebrates: the isopod Cymodoce truncata (i.e. used for the first time in ecotoxicology), the amphipod Gammarus aequicauda and the sea urchin Paracentrotus lividus. This study showed concentration- and time-dependent relationships for all biological models for both ZnO NPs and ZnSO4. Both Zn forms elicited high toxicity to G. aequicauda and C. truncata juveniles, but ZnO NPs induced comparable responses to both species (96h-LC50 = 0.30 and 0.37 mg/L for G. aequicauda and C. truncata, respectively; p > 0.05), while differences were found after ZnSO4 exposure (96h-LC50 = 0.28 and 0.63 mg/L, respectively; p < 0.05). ZnO NPs generated sub-lethal effects on P. lividus embryos (72h-EC50 = 0.04 (0.03, 0.05) mg/L), not significantly different from ZnSO4 ones (72h-EC50 = 0.06 (0.05, 0.07) mg/L). Effects of ZnO NPs were similar to existing literature data for other testing species. C. truncata can be considered as a promising new biological model in (nano)ecotoxicology.

Toxicity of nanoplastics during the embryogenesis of the ascidian Ciona robusta (Phylum Chordata).

Nanoplastics are considered contaminants of emerging concern at the global scale. The recent evidence of their occurrence in seawater from the Mediterranean Sea calls for a thorough evaluation of their impact on marine life and in particular on vulnerable life stages such as planktonic embryos. Here, we investigated the impact of increasing nominal concentrations of 50 nm amino-modified (PS-NH2) and 60 nm carboxy-modified (PS-COOH) polystyrene nanoparticles (PS NPs) on the embryonic development of the ascidian Ciona robusta (phylum Chordata), a common benthic invertebrate living in Mediterranean coastal areas with the peculiarity of being an early chordate developmental model. A strong agglomeration of PS-COOH (approx. 1 µm) was observed in natural sea water (NSW) already at time 0, while PS-NH2 resulted still monodispersed (approx. 130 nm) but largely aggregated after 22 h with a microscale dimension similar to those negatively charged. However, their effect on C. robusta embryos development largely differed at 22 h: PS-COOH did not affect larvae phenotypes nor their development, while PS-NH2 caused a dose-dependent effect (EC50 (22 h) of 7.52 µg mL-1) with various degrees of phenotype malformations (from mild to severe) and impairment of larval swimming. Embryos (up to 30%) exposed to 15 µg mL-1 PS-NH2 resulted not developed and the majority was unable to hatch. Calculated PS-NH2 EC50 resulted higher than those available for other marine invertebrate species, suggesting a protective role of the egg envelopes surrounding C. robusta embryos toward nanoplastics exposure.

New approaches on the use of tunicates (Ciona robusta) for toxicity assessments.

After the accidental release of crude oil in marine environment, dispersants are applied on sea surface transferring oil into the water column where it can be broken down by biodegradation, thereby reducing potential pollution to coastal areas. Before they can be used in the wild, the ecotoxicity of such dispersants is usually evaluated with toxicity assays using algae, crustacean and fishes. Nowadays, there is a need to find alternative species to reduce the use of vertebrates both for ethical considerations and for reducing the cost of bioassays. Ciona robusta is a solitary ascidian that inhabits shallow waters and marine coastal areas. This species has been recently adopted as valuable biological model for ecotoxicity studies, thanks to its rapid embryonic and larval development, resemblance to vertebrates, and low risk of ethical issues. On this ground, the lethal and sublethal toxicity of two dispersants has been evaluated on Ciona juveniles. At this stage, the organisms become filter-feeders and the morphological alterations of the organs can be easily observed. The median lethal concentrations at 96 h (96hLC50) for Dispersant 1 (non-ionic surfactant) and for Dispersant 2 (mixture of non-ionic surfactants and anionic surfactants) are 41.6 mg/L (38.6-44.9) and 92.5 mg/L (87.7-97.5), respectively. The Ciona juvenile model was compared to Dicentrarchus labrax fish juveniles test, and it showed increased sensitivity for Ciona to these compounds. These results suggest that 96 h mortality test bioassay could be a good alternative method to 96 h mortality assay with D. labrax, limiting the use of vertebrates for dispersant toxicity.

Could a harmonized tiered approach assess dispersant toxicity in Italy and France?

In recent years, EU countries have recognized national policies to authorize dispersant use to mitigate the petroleum hydrocarbon contamination in case of unintentional oil spills at sea. A harmonization of dispersant approval procedures is needed because the application of different methodologies agrees on dissimilar toxic responses for the same dispersant in different European countries. Actually, different dispersant approval procedures are applied in France and Italy with one French mandatory toxicity test and three Italian bioassays accompanied with different criteria of toxicity classification. In this paper, a harmonized tiered approach is proposed to address the dispersant ecotoxicological assessment in these two nations. Our approach, applicable at the European level, introduces two mandatory tests (algal growth inhibition test and mortality test with crustaceans) and one discretionary test (fish mortality test), by reducing use of vertebrates as much as possible in accordance with humane principles and animal welfare.

Chronic sublethal effects of ZnO nanoparticles on Tigriopus fulvus (Copepoda, Harpacticoida).

This study investigated for the first time the effects of ZnO nanoparticle (NP) chronic exposure (28 days) on Tigriopus fulvus. Acute toxicity (48 h) of three Zn chemical forms was assessed as well including the following: (a) ZnO nanoparticles (NPs), (b) Zn2+ from ZnO NP suspension after centrifugation (supernatant) and (c) ZnSO4 H2O. Physical-chemical and electronic microscopies were used to characterize spiked exposure media. Results showed that the dissolution of ZnO NPs was significant, with a complete dissolution at lowest test concentrations, but nano- and micro-aggregates were always present. Acute test evidenced a significant higher toxicity of Zn2+ and ZnSO4 compared to ZnO NPs. The chronic exposure to ZnO NPs caused negative effects on the reproductive traits, i.e. brood duration, brood size and brood number at much lower concentrations (≥ 100 µg/L). The appearance of ovigerous females was delayed at higher concentrations of ZnO NPs, while the time required for offspring release and the percentage of non-viable eggs per female were significantly increased. ZnO NP subchronic exposure evidenced its ability to reduce T. fulvus individual reproductive fitness, suggesting that ZnO NPs use and release must be carefully monitored. Graphical abstract Graphical Abstract.

Toxicity of diatom-derived polyunsaturated aldehyde mixtures on sea urchin Paracentrotus lividus development.

Diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal, derive from the oxidation of fatty acids and have cytotoxic and anticancer effects. PUAs, tested separately, induce malformations in sea urchin Paracentrotus lividus embryos. Decadienal induces the worst malformations and lowest survival rates. Interestingly, decadienal, heptadienal and octadienal place in motion several genes to counteract their negative effects. To date, no studies are available reporting on the effects of PUA mixtures on marine invertebrates. Here we test binary and ternary mixtures on embryonic development of P. lividus. Our findings demonstrate that mixtures of PUAs act (i) at morphological level in synergistic way, being much more severe compared to individual PUAs; (ii) at molecular level also reveal an additive effect, affecting almost all fifty genes, previously tested using individual PUAs. This study is relevant from an ecological point of view since diatoms are a major food source for both pelagic and benthic organisms. This work opens new perspectives for understanding the molecular mechanisms that marine organisms use in reacting to environmental natural toxin mixtures such as diatom PUAs.

Sediment concentrations of trace metals in the Berre lagoon (France): an assessment of contamination.

This paper reports the concentrations of eight trace metals (Cr, Zn, Cu, Pb, Ni, As, Cd, and Hg) in the surficial sediments of the Berre lagoon. This is a typical Mediterranean lagoon, seriously affected by human activities since the 1930s, when it became one of the most important industrial sites in France. The overall range of concentrations is 38-428 mg kg(-1) for chromium, 50-151 mg kg(-1) for zinc, 11-48 mg kg(-1) for copper, 18-82 mg kg(-1) for lead, 18-56 mg kg(-1) for nickel, 4-10 mg kg(-1) for arsenic, 0.2-1.6 mg kg(-1) for cadmium, and 0.15-0.40 mg kg(-1) for mercury. The spatial distribution of concentrations largely mirrors the localization of major impact sources, with the most affected area restricted to a radius of a few kilometers from point sources, due to the environmental conditions of the outfall zone (fine sediments, low hydrodynamic regime, confinement), which favor the in situ accumulation of pollutants. The extent of contamination from trace metals in the lagoon sediments is evaluated through a three-pronged approach: (i) by comparison with other areas (see Table 3 for an up-to-date review of trace metal concentrations in lagoon systems and coastal regions of the world ocean); (ii) by evaluating the metal enrichment in the sediments through the calculation of concentration factors (CF) for each trace metal and by combining the CFs to estimate a Metal Pollution Index (MPI) taking into account the differences in toxicity of trace metals; and (iii) by defining a potential level of biological risk by the use of quality criteria such as the Threshold Effect Level (TEL) and Effects Range-Low (ER-L) benchmarks. On the basis of the calculated CFs, sediments appear as particularly enriched with Pb and Cd throughout the lagoon and with Cr and Zn at some sites. Although no site can be classified as "unpolluted" when looking at the calculated MPIs, two regions--the Vaïne pond and the southwestern area--have been identified as critical points of contamination. Trace metal inputs to the lagoon need to be kept under strict control in the future, with particular regard to Ni, Cr, Pb, and Cu that, on the basis of TEL and ER-L benchmarks, already appear associated with a potential biological risk.

Numerical-ecotoxicological approach to assess potential risk associated with oilfield production chemicals discharged into the sea.

Several different chemical products are used on oil platforms to aid oil-water separation during the production process. These chemicals may enter into the sea by means of production water (PW), the main discharge derived from oil and gas offshore platforms. Consequently, toxic effects may occur in the marine environment, causing reductions in wildlife numbers, degrading ecosystem functions and threatening human health. For most of these chemicals, environmental toxicity and safety thresholds in marine ecosystems have not been fully investigated as yet. In this work, a numerical-ecotoxicological approach is proposed to assess the potential environmental risk associated with the discharge of five oilfield production chemicals (deoiler, scale inhibitor, corrosion inhibitor, catalyst, dehydrating agent) from a platform in the southern Adriatic Sea (Mediterranean Sea). Their concentrations in the seawater are numerically predicted, under different seasonal conditions, starting from the real concentrations used during the production process. The predicted concentrations are then evaluated in terms of possible toxic effects in order to assess the potential risk of oilfield production chemicals discharged into the sea.

Cytotoxicity and genotoxicity of CuO nanoparticles in sea urchin spermatozoa through oxidative stress.

Copper oxide nanoparticles (CuO NPs) are extensively used in various industrial and commercial applications. Despite their wide application may lead to the contamination of marine ecosystem, their potential environmental effects remain to be determined. Toxicity assessment studies have primarily focused on investigating the effects of CuO NPs on fertilization success and embryo development of different sea urchin species while the impact on sperm quality have never been assessed. In this line, this study aims to assess the effects of CuO NPs on the spermatozoa of the sea urchin Paracentrotus lividus. After sperm exposure to CuO NPs, biomarkers of sperm viability, cytotoxicity, oxidative stress, and genotoxicity as well as morphology were evaluated. Results showed that CuO NPs exposure decreased sperm viability, impaired mitochondrial activity and increased the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, CuO NPs exposure caused DNA damage and morphological alterations. Together with the antioxidant rescue experiments, these results suggest that oxidative stress is the main driver of CuO NP spermiotoxic effects. The mechanism of toxicity is here proposed: the spontaneous generation of ROS induced by CuO NPs and the disruption of the mitochondrial respiratory chain lead to production of ROS that, in turn, induce lipid peroxidation and DNA damage, and result in defective spermatozoa up to induce sperm cytotoxicity. Investigating the effects of CuO NPs on sea urchin spermatozoa, this study provides valuable insights into the mechanism of reproductive toxicity induced by CuO NPs.