The diffusion in the environment of several types of nanoparticles (metallic, plastic, etc.) and their environmental fate require a careful evaluation of the effects on living organisms or natural ecosystem dynamics, particularly the marine and brackish ones, final sink of all contaminants. Dedicated tests for the ecotoxicological evaluation of nanomaterials demonstrated the toxicity of metal nanoparticles and included the physical methods to evaluate the aggregation state of nanoparticles, according to salinity of the medium.
The assay on Vibrio anguillarum (see New ecotoxicity tests for nanomaterials) has shown clearly the dose effect relationship in response to increasing concentrations of copper oxide nanoparticles (nCuO), but also the variation of toxicity with salinity. In agreement with the OECD guidelines, the distribution in size classes and the stability of nanoparticles have been identified by two different techniques, Dynamic Light Scattering and Dispersion Analysis by LUMiSizer.

The studies on metallic nanomaterials are aimed to build a battery of dedicated assays. To this end, some already used assays have been revised to cope with the specific needs of nanomaterials, and new ecotoxicological models have also been tested. The selected assays were used to identify the Effective Concentrations, by evaluating lethal and sub-lethal endpoints. Acute mortality tests were performed on rotifers (Brachionus plicatilis) and crustaceans (Artemia franciscana, Cymodoce truncata, Gammarus aequicauda, ​​Tigriopus fulvus), while the alteration of growth capacity in Tigriopus fulvus (as number and frequency of moults) and the reproductive capacity of the sea urchin, Paracentrotus lividus (as fertilization rate) were analysed by sub-lethal assays.

 In addition to metallic nanomaterials, other nanosized materials can pose environmental concerns. For this reason, the possible ecotoxicological effects of carbon-based microplastics and nanomaterials were studied. As regard microplastics, lethal and sub-lethal responses of marine organisms, as Vibrio anguillarum, Dunaliella tertiolecta, Brachionus plicatilis and Paracentrotus lividus were studied. Conversely, carbon-based nanomaterials were tested on an the aquatic plant, Lemna gibba.

 These studies were made possible thanks to the collaborations with: Loredana Manfra, ISPRA Ecotoxicology Laboratory; Linda Prato, CNR IAMC (Taranto headquarter of the IRSA), and Federica Valentini, Department of Chemical Sciences and Technologies, Tor Vergata University.

 

Valentini F., Di Giacobbe M., Bertuccini L., Iosi F., Krasojevic K., Calcaterra A., Migliore L., Botta M., Talamo M. (2021) - Chemical interactions and ecotoxicity effects between graphene oxide and Lemna gibba. Fullerenes, Nanotubes & Carbon Nanostructures 29(9): 746-753.  DOI: 10.1080/1536383X.2021.1891046

 The features of Graphene Oxide (GO) could produce unexpected interactions with ecosystem, triggering negative effects on biota. In this work we evaluated the effects of two different GO samples (chemically synthesized GO_C and electrochemically synthesized GO_E) on Lemna gibba L., after incubation with different concentrations over a period of 21 days. The stress conditions were evidenced by the measurements of frond number, fresh weight, dry weight, dry/fresh weight ratio, and inhibition of growth rate. Furthermore, a morphological characterization was also carried out to verify the mechanical effects on L. gibba plants of these two GO. Results demonstrated that the smallest GO_E nano sheets were very well dispersed on L. gibba fronds than GO_C. This effect could be explained considering the lower GO_E functionalization degree and, as a result, a less electrostatic adhesion among nanosheets. The presence of electrically charged functionalities mainly provides electrostatic interactions with plants causing growth inhibition as shown by GO_C.

 

Prato L., Fabbrocini A., Libralato G., Migliore L., Parlapiano I., D’Adamo R., Rotini A., Manfra L., Lofrano G., Carraturo F., Trifuoggi M., Biandolino F. (2021) - SHORT-TERM EXPOSURE AND EFFECTS OF ZnO NANOPARTICLES AND ZnSO₄ TO CYMODOCE TRUNCATA, GAMMARUS AEQUICAUDA, AND PARACENTROTUS LIVIDUS. Environmental Science and Pollution Research, 28(31): 42891-42900. DOI: 10.1007/s11356-021-13712-0

Due to the continuous development and production of manufactured nanomaterials or nanoparticles (NPs), their effects in marine coastal environment represent a major concern. The aim of this study was to evaluate the toxicity of ZnO nanoparticles (ZnO NPs) and compare it to the toxicity of ZnSO₄ on three macroinvertebrates: the isopod Cymodoce truncata and the amphipod Gammarus aequicauda, and in gametes and embryos of the sea urchins Paracentrotus lividus. These organisms are sensitive species to environmental contamination and, for this reason, routinely used as bioindicators to monitor aquatic systems in standard toxicity tests. The standard protocols were applied and results showed comparable results in the three species, with a dose- and time-dependendent relationship for both ZnO NPs and ZnSO₄, suggesting that the zinc ions may play a role in generating toxicity. Both Zn forms elicit high toxicity to G. aequicauda and C. truncate, but ZnO NPs induce comparable toxicity to both species (LC₅₀ = 0.30 and 0.37 mg/L, respectively; p <  0.05), whilst differences were found after 96 h exposure to ZnSO₄ (LC₅₀ = 0.28 and 0.63 mg/L for G. aequicauda and C. truncata; p < 0.05). Moreover, ZnO NPs elicit sub-lethal effects on P. lividus embryos (EC₅₀ = 0.04 (0.03, 0.05) mg/L after 72 h), not significantly different from ZnSO4 ones (EC50 = 0.06 (0.05, 0.07) mg/L after 72 h). The importance in assessing the possible environmental risks due to Zn forms contamination and the intrinsic toxicity of ZnO NPs, probably related to ions dissolution is discussed.

 

 Prato E., Parlapiano I., Biandolino F., Rotini A., Manfra L., Berducci M.T., Maggi C., Libralato G., Paduano L., Carraturo F., Trifuoggi M., Carotenuto M., Migliore L. (2020) - CHRONIC SUBLETHAL EFFECTS OF ZNO NANOPARTICLES ON TIGRIOPUS FULVUS (COPEPODA, HARPACTICOIDA). Environmental Science and Pollution Research, 27(25): 30957-30968 DOI: 10.1007/s11356-019-07006-9

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.

 

Gambardella C., Morgana S., Bramini M., Rotini A., Manfra L., Piazza V., Garaventa F., Migliore L., Faimali M. (2018) - ECOTOXICOLOGICAL EFFECTS OF POLYSTYRENE MICROBEADS IN A BATTERY OF MARINE INVERTEBRATES BELONGING TO DIFFERENT TROPHIC LEVELS. Marine Environmental Research, 141: 313-321. DOI: 10.1016/j.marenvres.2018.09.023

 The aim of this study was to detect ecotoxicological effects of 0.1 μm polystyrene microbeads in marine organisms belonging to different trophic levels. MP build up, lethal and sub-lethal responses were investigated in the bacterium Vibrio anguillarum (culturability), in the green microalga Dunaliella tertiolecta (growth inhibition), in the rotifer Brachionus plicatilis (mortality and swimming speed alteration) and in the sea urchin Paracentrotus lividus (immobility and swimming speed alteration) exposed to a wide range of microplastic (MP) concentrations (from 0.001 to 10 mg L-¹). Survival was not affected in all organisms up to 10 mg L-¹, while algal growth inhibition, rotifer and sea urchin larvae swimming behaviour alterations were observed after exposure to MPs. Ingestion was only observed in rotifers and it was directly correlated with sub-lethal effects. These results account for the ecotoxicological risk associated to the polystyrene microbeads, which are able to affect different endpoints in primary producers and consumers (rotifers and sea urchins) since no effects were observed in decomposers. This study points out the importance of using a battery of marine organisms belonging to different trophic levels by studying acute toxicity of MPs at low and high contamination levels, and investigating sub-lethal responses. Further investigations aimed at studying the transfer of these materials through the web are particularly recommended.

 

Gallo A., Manfra L., Boni R., Rotini A., Migliore L., Tosti E. (2018) - CYTOTOXICITY AND GENOTOXICITY OF CuO NANOPARTICLES IN SEA URCHIN SPERMATOZOA THROUGH OXIDATIVE STRESS. Environmental International, 118: 325-333 DOI: 10.1016/j.envint.2018.05.034

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.

 

Rotini A., Gallo A., Parlapiano I., Berducci M.T., Boni R., Tosti E., Prato E., Maggi C., Cicero A.M., Migliore L., Manfra L. (2018) - INSIGHTS INTO THE CuO NANOPARTICLE ECOTOXICITY WITH SUITABLE MARINE MODEL SPECIES. Ecotoxicology and Environmental Safety, 147:852-860. DOI: 10.1016/j.ecoenv.2017.09.053

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). Ecotoxiticy 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 standard protocols to identify Effective Concentrations, 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 Cu2+ dissolution, were also analysed in the exposure media. The CuO NP ecotoxicity assessment showed a dose-dependent response for all species, indicating similar mortality for B. plicatilis (48hLC50=16.94±2.68 mg/l) and T. fulvus (96hLC50=12.35±0.48 mg/l), followed by A. franciscana (48hLC50=64.55±3.54 mg/l). Comparable EC50 values were also obtained for the sub-lethal endpoints in P. lividus (EC50=2.28±0.06 mg/l) and T. fulvus (EC50=2.38±0.20 mg/l). Cu²⁺ 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 test media, with different particle size distributions depending on the medium. The Cu²⁺ dissolution was about 10 % of the initial concentration, comparable to literature values, but not accounting for the toxicity of NP suspensions. The integrated ecotoxicological-physicochemical approach was used to better describe CuO NP toxicity and behavior. In particular, the successful application of ecotoxicological standard protocols allowed to produce reliable L(E)C₅₀ data useful to identify thresholds and assess potential environmental hazard due to NPs.

 

This work is a starting point in the evaluation of nanoparticles toxicity on marine or brackish organisms. It is supported by the Research Program "NanoBioTech Environment and Health" (funded by Regione Lazio and Consorzio Hypazia) and include project agreement with ISPRA. The work is done in collaboration with the staff of the Consorzio Hypazia, in particular with Flavio Lucibello, President of the Consorzio, with Loredana Manfra (Laboratory of Ecotoxicology, ISPRA) and Linda Prato (CNR IAMC, Taranto).