Metal(loid)s and Rare Earth Elements in Posidonia oceanica (L.) Delile (1813) banquettes.

The Posidonia oceanica (L.) Delile 1813 banquette provides precious ecosystem services for Mediterranean beach nourishment and protection, representing an important way of energy transfer through marine-coastal habitats. It is surprising to note how it is poorly investigated, especially concerning its double role as potential sink and source of chemicals. In particular, few studies exist about the metal (loid)s occurrence and no data are available on emerging contaminants, such as Rare Earth Elements (REEs). The present research investigated for the first time the concentrations of twenty-eight metal(loid)s and fifteen REEs in a well-structured banquette along the Italian coast (Central Tyrrhenian Sea) showing that (i) metal(loid)s and REEs occur in banquettes, with higher relative concentrations of some metal(loid)s (B, Sr, Mn, Fe, Al, Zn) and REEs (Ce, Y, La, Nd) with no statistically significant seasonal variations; (ii) Posidonia banquettes may represent an interesting biological model for chemicals monitoring.

Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax.

The OECD TG 215 method (2000) (C.14 method of EC Regulation 440/2008) was developed on the rainbow trout (Oncorynchus mykiss) to assess chronic toxicity (28d) of chemicals on fish juveniles. It contemplates to use other well documented species identifying suitable conditions to evaluate their growth. OECD proposes the European sea bass (Dicentrarchus labrax, L. 1758) as Mediterranean species among vertebrates recommended in the OECD guidelines for the toxicity testing of chemicals. In this context, our study is aimed to proposing the adaptation of the growth test (OECD TG 215, 2000) to D. labrax. For this purpose toxicity tests were performed with sodium dodecyl sulfate, a reference toxicant commonly used in fish toxicity assays. The main aspects of the testing procedure were reviewed: fish size (weight), environmental conditions, dilution water type, experimental design, loading rate and stocking density, feeding (food type and ration), test validity criteria. The experience gained from growth tests with the sea bass allows to promote its inclusion among the species to be used for the C.14 method.

Insights into the CuO nanoparticle ecotoxicity with suitable marine model species.

Metal oxide nanoparticles, among them copper oxide nanoparticles (CuO NPs), are widely used in different applications (e.g. batteries, gas sensors, superconductors, plastics and metallic coatings), increasing their potential release in the environment. In aquatic matrix, the behavior of CuO NPs may strongly change, depending on their surface charge and some physical-chemical characteristics of the medium (e.g. ionic strength, salinity, pH and natural organic matter content). Ecotoxicity of CuO NPs to aquatic organisms was mainly studied on freshwater species, few tests being performed on marine biota. The aim of this study was to assess the toxicity of CuO NPs on suitable indicator species, belonging to the ecologically relevant level of consumers. The selected bioassays use reference protocols to identify Effect/Lethal Concentrations (E(L)C), by assessing lethal and sub-lethal endpoints. Mortality tests were performed on rotifer (Brachionus plicatilis), shrimp (Artemia franciscana) and copepod (Tigriopus fulvus). While moult release failure and fertilization rate were studied, as sub-lethal endpoints, on T. fulvus and sea urchin (Paracentrotus lividus), respectively. The size distribution and sedimentation rates of CuO NPs, together with the copper dissolution, were also analyzed in the exposure media. The CuO NP ecotoxicity assessment showed a concentration-dependent response for all species, indicating similar mortality for B. plicatilis (48hLC50 = 16.94 ± 2.68mg/l) and T. fulvus (96hLC50 = 12.35 ± 0.48mg/l), followed by A. franciscana (48hLC50 = 64.55 ± 3.54mg/l). Comparable EC50 values were also obtained for the sub-lethal endpoints in P. lividus (EC50 = 2.28 ± 0.06mg/l) and T. fulvus (EC50 = 2.38 ± 0.20mg/l). Copper salts showed higher toxicity than CuO NPs for all species, with common sensitivity trend as follows: P. lividus ≥ T. fulvus (sublethal endpoint) ≥ B. plicatilis >T. fulvus (lethal endpoint) >A. franciscana. CuO NP micrometric aggregates and high sedimentation rates were observed in the exposure media, with different particle size distributions depending on the medium. The copper dissolution was about 0.16% of the initial concentration, comparable to literature values. The integrated ecotoxicological-physicochemical approach was used to better describe CuO NP toxicity and behavior. In particular, the successful application of ecotoxicological reference protocols allowed to produce reliable L(E)C data useful to identify thresholds and assess potential environmental hazard due to NPs.

Comparative ecotoxicity of polystyrene nanoparticles in natural seawater and reconstituted seawater using the rotifer Brachionus plicatilis.

The impact of nanoplastics using model polystyrene nanoparticles (PS NPs), anionic (PS-COOH) and cationic (PS-NH2), has been investigated on the marine rotifer Brachionus plicatilis, a major component of marine zooplanktonic species. The role of different surface charges in affecting PS NP behaviour and toxicity has been considered in high ionic strength media. To this aim, the selected media were standardized reconstituted seawater (RSW) and natural sea water (NSW), the latter resembling more natural exposure scenarios. Hatched rotifer larvae were exposed for 24h and 48h to both PS NPs in the range of 0.5-50µg/ml using PS NP suspensions made in RSW and NSW. No effects on lethality upon exposure to anionic NPs were observed despite a clear gut retention was evident in all exposed rotifers. On the contrary, cationic NPs caused lethality to rotifer larvae but LC50 values resulted lower in rotifers exposed in RSW (LC50=2.75±0.67µg/ml) compared to those exposed in NSW (LC50=6.62±0.87µg/ml). PS NPs showed similar pattern of aggregation in both high ionic strength media (RSW and NSW) but while anionic NPs resulted in large microscale aggregates (Z-average 1109 ± 128nm and 998±67nm respectively), cationic NP aggregates were still in nano-size forms (93.99 ± 11.22nm and 108.3 ± 12.79nm). Both PDI and Z-potential of PS NPs slightly differed in the two media suggesting a role of their different surface charges in affecting their behaviour and stability. Our findings confirm the role of surface charges in nanoplastic behaviour in salt water media and provide a first evidence of a different toxicity in rotifers using artificial media (RSW) compared to natural one (NSW). Such evidence poses the question on how to select the best medium in standardized ecotoxicity assays in order to properly assess their hazard to marine life in natural environmental scenarios.

Long-term toxicity of surface-charged polystyrene nanoplastics to marine planktonic species Dunaliella tertiolecta and Artemia franciscana.

Plastic pollution has been globally recognized as a critical issue for marine ecosystems and nanoplastics constitute one of the last unexplored areas to understand the magnitude of this threat. However, current difficulties in sampling and identifying nano-sized debris make hard to assess their occurrence in marine environment. Polystyrene nanoparticles (PS NPs) are largely used as nanoplastics in ecotoxicological studies and although acute exposures have been already investigated, long-term toxicity on marine organisms is unknown. Our study aims at evaluating the effects of 40nm PS anionic carboxylated (PS-COOH) and 50nm cationic amino-modified (PS-NH2) NPs in two planktonic species, the green microalga Dunaliella tertiolecta and the brine shrimp Artemia franciscana, respectively prey and predator. PS NP behaviour in exposure media was determined through DLS, while their toxicity to microalgae and brine shrimps evaluated through 72h growth inhibition test and 14 d long-term toxicity test respectively. Moreover, the expression of target genes (i.e. clap and cstb), having a role in brine shrimp larval growth and molting, was measured in 48h brine shrimp larvae. A different behaviour of the two PS NPs in exposure media as well as diverse toxicity to the two planktonic species was observed. PS-COOH formed micro-scale aggregates (Z-Average>1µm) and did not affect the growth of microalgae up to 50µg/ml or that of brine shrimps up to 10µg/ml. However, these negatively charged NPs were adsorbed on microalgae and accumulated (and excreted) in brine shrimps, suggesting a potential trophic transfer from prey to predator. On the opposite, PS-NH2-formed nano-scale aggregates (Z-Average<200nm), caused inhibition of algal growth (EC50=12.97µg/ml) and mortality in brine shrimps at 14 d (LC50=0.83µg/ml). Moreover, 1µg/ml PS-NH2 significantly induced clap and cstb genes, explaining the physiological alterations (e.g. increase in molting) previously observed in 48h larvae, but also suggesting an apoptotic pathway triggered by cathepsin L-like protease in brine shrimps upon PS-NH2 exposure. These findings provide a first insight into long-term toxicity of nanoplastics to marine plankton, underlining the role of the surface chemistry in determining the behaviour and effects of PS NPs, in terms of adsorption, growth inhibition, accumulation, gene modulation and mortality. The use of long-term end-point has been identified as valuable tool for assessing the impact of nanoplastics on marine planktonic species, being more predictable of real exposure scenarios for risk assessment purposes.

Fitness Evaluation of Ruditapes philippinarum Exposed to Ni.

In this study, long-term effects of Ni, a widespread heavy metal in the aquatic ecosystems, have been determined on growth and lethality of the clam Ruditapes philippinarum, a known bioindicator of the marine environment. Three/four-month-old bivalves have been exposed to different concentrations of Ni dissolved in synthetic seawater. Growth and lethality as endpoints after 28 days of treatment have been observed. Obtained results are the following: EC25 = 3.97 ± 0.94 and 9.45 ± 1.59 mg/L and NOEC = 1.56 and 6.25 mg/L for growth and mortality, respectively. Moreover, this study can be considered a new tool for the evaluation of fitness of bivalve clam, together with other biological responses following to the biological impacts of metal pollution.

Can Artemia Hatching Assay Be a (Sensitive) Alternative Tool to Acute Toxicity Test?

Artemia sp. is extensively used in ecotoxicity testing, despite criticisms inherent to both acute and long-term tests. Alternative endpoints and procedures should be considered to support the use of this biological model. The hatching process comprises several developmental steps and the cyst hatchability seems acceptable as endpoint criterion. In this study, we assessed the reliability of the hatching assay on A. franciscana by comparing with acute and long-term mortality tests, using two chemicals: Diethylene Glycol (DEG), Sodium Dodecyl Sulphate (SDS). Both DEG and SDS tests demonstrated a dose dependent hatching inhibition. The hatching test resulted more sensitive than acute mortality test and less sensitive than the long-term one. Results demonstrate the reliability and high sensitivity of this hatching assay on a short time lag and support its useful application in first-tier risk assessment procedures.

Ecotoxicity of diethylene glycol and risk assessment for marine environment.

Diethylene glycol (DEG) is a chemical compound used during offshore oil activities to prevent hydrate formation, and it may be released into the sea. A full ecotoxicological characterization is required according to European and Italian regulations for chemical substances. We have evaluated long-term toxic effects of DEG on indicator species of the marine environment as algae (Phaeodactylum tricornutum), crustaceans (Artemia franciscana), molluscs (Tapes philippinarum) and fish (Dicentrarchus labrax). A range of no observed effect concentrations (365-25,000 mg/L) has been identified. Based on the toxicity results and the ratio between predicted environmental concentration and predicted no-effect concentration, we have estimated the maximum allowable value of DEG in the marine environment.

Near-field dispersion of produced formation water (PFW) in the Adriatic Sea: an integrated numerical-chemical approach.

Produced formation waters (PFWs), a by-product of both oil and gas extraction, are separated from hydrocarbons onboard oil platforms and then discharged into the sea through submarine outfalls. The dispersion of PFWs into the environment may have a potential impact on marine ecosystems. We reproduce the initial PFW-seawater mixing process by means of the UM3 model applied to offshore natural gas platforms currently active in the Northern Adriatic Sea (Mediterranean Sea). Chemical analyses lead to the identification of a chemical tracer (diethylene glycol) which enables us to follow the fate of PFWs into receiving waters. The numerical simulations are realized in different seasonal conditions using both measured oceanographic data and tracer concentrations. The numerical results show the spatial and temporal plume development in different stratification and ambient current conditions. The analytical approach measures concentrations of the diethylene glycol at a maximum sampling distance of 25 m. The results show a good agreement between field observations and model predictions in the near-field area. The integration of numerical results with chemical analyses also provides new insight to plan and optimize PFW monitoring and discharge.

Metal content and toxicity of produced formation water (PFW): study of the possible effects of the discharge on marine environment.

A preliminary chemical and ecotoxicological assessment was performed on the produced formation water (PFW) and superficial sediment around a gas platform (Fratello Cluster), located in the Adriatic Sea (Italy), in order to evaluate the effects of PFW discharged from the installation. The ecotoxicological bioassays, with the marine bacterium Vibrio fischeri and the sea urchin Paracentrotus lividus, were associated with chemical data to estimate the possible effects on living organisms. PFW collected on the platform was toxic, but no significant effect was recorded on marine sediment. Only the sediment station nearest to the discharge point showed higher values of some contaminants (zinc and arsenic) in comparison to other sites and only some stations showed low toxicity.