La diffusione nell’ambiente di nanoparticelle di molti tipi (metalliche, plastiche, ecc.) e il loro destino ambientale impongono un’accurata valutazione dei loro effetti sugli organismi viventi e sulle dinamiche naturali, in particolare negli ecosistemi marini e salmastri, destinatari ultimi di tutte le contaminazioni ambientali. Saggi ecotossicologici dedicati alla valutazione della tossicità dei nanomateriali che includono i nuovi approcci necessari a valutare le problematiche tipiche della “nanotossicologia”, come i metodi fisici di valutazione dello stato di aggregazione delle nanoparticelle, hanno dimostrato la tossicità di nanoparticelle metalliche nonostante la variazione del loro stato di aggregazione a seconda della salinità del mezzo in cui si trovano. Il saggio su Vibrio anguillarum (vedi Nuovo saggio di ecotossicità per nanomateriali), ha dimostrato chiaramente la relazione dose-effetto in risposta a concentrazioni crescenti di nanoparticelle di ossido di rame (nCuO), ma anche la variazione della tossicità con la salinità. Inoltre, in accordo con le lineeguida dell’OECD, sono state identificate la distribuzione in classi di dimensione e la stabilità delle nanoparticelle tramite due diverse tecniche, Dynamic Light Scattering e Dispersion Analysis by LUMiSizer.

Gli studi sui nanomateriali metallici hanno avuto come obiettivo la costruzione di una batteria di saggi dedicati; a questo scopo non solo sono stati revisionati alcuni saggi già in uso, ma sono anche stati progettati e provati dei nuovi modelli ecotossicologici. I saggi selezionati sono stati utilizzati per identificare le Concentrazioni Efficaci, valutando endpoint letali e sub-letali. Test acuti di mortalità sono stati eseguiti su rotiferi (Brachionus plicatilis) e crostacei (Artemia franciscana, Cymodoce truncata, Gammarus aequicauda, Tigriopus fulvus), mentre con saggi sub-letali sono state studiate le alterazioni della capacità di accrescimento in Tigriopus fulvus (come numero e frequenza di mute) e la capacità riproduttiva del riccio di mare, Paracentrotus lividus (come tasso di fecondazione).

 Oltre ai nanomateriali metallici anche altri materiali a nanoscala possono determinare problemi ambientali, per questo sono stati effettuati test per rilevare i possibili effetti ecotossicologici di microplastiche e nanomateriali a base di Carbonio. Per le microplastiche, sono state studiate risposte letali e sub-letali su organismi marini quali Vibrio anguillarum, Dunaliella tertiolecta, Brachionus plicatilis e Paracentrotus lividus. Invece, i nanomateriali a base di carbonio sono stati saggiati su una pianta acquatica, Lemna gibba.

 Questi studi sono stati possibili grazie alle collaborazioni con: Loredana Manfra, Laboratorio di Ecotossicologia dell’ISPRA; con Linda Prato, del CNR IAMC, Sede di Taranto dell’IRSA e Federica Valentini del Dipartimento di scienze e Tecnologie Chimiche di Tor Vergata.

 

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.