Short-Term Exposure to Nanoplastics Does Not Affect Bisphenol A Embryotoxicity to Marine Ascidian Ciona robusta

Plastic pollution is recognized as a global environmental threat and concern is increasing regarding the potential interactions of the smallest fragments, nanoplastics (1 m), with either physical and chemical entities encountered in the natural environment, including toxic pollutants. The smallest size of nanoplastics (<100 nm) rebounds to their safety associated with remarkable biological, chemical and physical reactivity that allow them to interact with cellular machinery by crossing biological barriers and causing damage to living beings. Recent findings on nanoplastic occurrence inmarine coastal waters, including the Mediterranean Sea, leave open the question on their ability to act as a vector of other contaminants of emerging concerns (CECs) concomitantly released by wastewater treatment plants and reaching marine coastal waters. Here, we assess for the first time the role of non-functionalized polystyrene nanoparticles (PS NPs, 20 nm) as a proxy for nanoplastics (1 and 10 g/mL) alone and in combinationwith bisphenolA(BPA) (4.5 and 10 m) on Ciona robusta embryos (22 h post fertilization, hpf) by looking at embryotoxicity through phenotypic alterations. We confirmed the ability of BPA to impact ascidian C. robusta embryo development, by affecting sensory organs pigmentation, either alone and in combination with PS NPs. Our findings suggest that no interactions are taking place between PS NPs and BPA in filtered sea water (FSW) probably due to the high ionic strength of seawater able to trigger the sorption surface properties of PS NPs. Further studies are needed to elucidate such peculiarities and define the risk posed by combined exposure to BPA and PS NPs in marine coastal waters

Eco-interactions of engineered nanomaterials in the marine environment: Towards an eco-design framework

Marine nano-ecotoxicology has emerged with the purpose to assess the environmental risks associated with engineered nanomaterials (ENMs) among contaminants of emerging concerns entering the marine environment. ENMs’ massive production and integration in everyday life applications, associated with their peculiar physical chemical features, including high biological reactivity, have imposed a pressing need to shed light on risk for humans and the environment. Environmental safety assessment, known as ecosafety, has thus become mandatory with the perspective to develop a more holistic exposure scenario and understand biological effects. Here, we review the current knowledge on behavior and impact of ENMs which end up in the marine environment. A focus on titanium dioxide (n-TiO2) and silver nanoparticles (AgNPs), among metal-based ENMs massively used in commercial products, and polymeric NPs as polystyrene (PS), largely adopted as proxy for nanoplastics, is made. ENMs eco-interactions with chemical molecules including (bio)natural ones and anthropogenic pollutants, forming eco- and bio-coronas and link with their uptake and toxicity in marine organisms are discussed. An ecologically based design strategy (eco-design) is proposed to support the development of new ENMs, including those for environmental applications (e.g., nanoremediation), by balancing their effectiveness with no associated risk for marine organisms and humans

“Ciona robusta (formerly Ciona intestinalis type A) as model system for ecotoxicological studies”

The invertebrate urochordate Ciona robusta (formerly Ciona intestinalis type A) is a sessile marine benthic organism distributed worldwide that attaches to the surfaces of both natural and artificial substrates, from shallow water to the deep sea. Based on its phylogenetic position as basal chordate, from more than a century Ciona represented an excellent model system for studying developmental biology, thanks to the rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, low risk of ethical issues and a number of techniques and genomic resources developed in the course of the years. Recently, in light of such interesting features, Ciona has been used to evaluate the embriotoxicity of legacy pollutants (e.g. heavy metals, pesticides, organic compounds). Notably, the genetic, genomic and molecular tools available for Ciona allow a deeper investigation of the molecular mechanisms affected by pollutants and could provide insights on their mode of actions (MoA). Within this thesis, the effects of two classes of contaminants of emerging concerns (CECs) have been tested on the embryogenesis of C. robusta. CECs are a group of natural and synthetic chemicals including nanoscale particles and transformation products, which have been increasingly found at low levels in surface waters. These compounds may pose a risk to aquatic life and, thus, it is fundamental to assess their potential effects on marine organisms. In detail, the effects of two dispersants named as A and B, used for cleaning up the petroleum hydrocarbon contamination in case of accidental oil spills at sea, and polystyrene nanoparticles (PS NPs), as proxy for nanoplastics have been investigated on C. robusta larval development. The four chapters of the thesis report findings on the effects of the two dispersants and PS NPs bearing different surface charges by using two approaches: 1) embryotoxicity, by looking at adverse effects in developing embryos and sub-lethal biological responses on functional proteins and enzymes; 2) mechanisms of action (MoA) at molecular level using different techniques as Real-Time PCR, RNA sequencing and bioinformatics. We demonstrated how embriotoxicity in C. robusta could represent a useful tool to evaluate the impact of dispersants on marine species. The data obtained indicated a different toxicity between dispersants A and B, confirmed also by phenotype alterations. Moreover, the evaluation of the expression of selected genes involved in stress response (SODa, SODb, MnSOD, GPx, HSP60, HSP70), detoxification (Cyp450, GST, GluR) and cell survival (p38, Cas8) indicated dispersant B as teratogen while dispersant A having less impact on C. robusta larvae. Regarding nanoplastic, surface charges seem to play a significant role in the observed embryotoxicity of the amino-modified PS NPs (PS-NH2) in agreement with their behavior in exposure media. No effects were found for carboxyl-modified PS NPs (PS-COOH) on C. robusta embryo development while from mild to severe 2 phenotype alterations were observed upon exposure to PS-NH2, including behavioral traits (e.g. swimming performances). Among those mostly evident, embryos resulted unable to hatch and several abnormal phenotypes were found. In addition, induction of oxidative stress linked to an increase of ROS production and the down-regulation of some representative genes involved in stress response (HSP70, HSP60, MnSOD, cytochrome b, p-38 mapk and caspase 8) were observed. The analysis of transcriptome, through differential RNA-seq, allowed to identify altered pathways affected by PS-NH2. Several genes resulted dysregulated upon the exposure to PS-NH2, while the GO analysis, which classified genes in three different subclasses, revealed that the number of genes affected, belonging to different subclasses, have a dose-response relationship with the concentration tested. Going deeply into the bioinformatic analysis, “glutathione synthesis and recycling pathway”, “neurotransmitter clearance pathway”, “passive transport by aquaporins” and “fructose and mannose metabolism”, “starch and sucrose metabolism” and “glycolysis” pathways resulted affected. The alteration of these pathways could be related to the hypoxic microenvironment due to the dense coating of PS-NH2 around the egg envelopes of Ciona embryos. Similar findings in terms of embryotoxicity and phenotype alterations have been observed in another ascidian species, Phallusia mammillata, exposed in similar conditions to amino-modified PS NPs (PS-NH2). Furthermore, the quantitative analyses of Phallusia phenotype using the software Toxicosis8, revealed the affection of both central and peripheral nervous system. The use of C. robusta embryos as a model to study the effects of dispersants and PS NPs as proxy for nanoplastics proved to be instrumental in shedding light on different aspects of developmental toxicity exerted by those CECs. Moreover, these results will hopefully provide important information useful for higher and more complex chordates

Application of transcriptome profiling to inquire into the mechanism of nanoplastics toxicity during Ciona robusta embryogenesis

: The growing concern on nanoplastics (<1 μm) impact on marine life has stimulated a significant amount of studies aiming to address ecotoxicity and disclose their mechanisms of action. Here, we applied an integrative approach to develop an Adverse Outcome Pathway (AOP) upon acute exposure to amino-modified polystyrene nanoparticles (PS-NH2 NPs, 50 nm), as proxy for nanoplastics, during the embryogenesis of the chordate Ciona robusta. Genes related to glutathione metabolism, immune defense, nervous system, transport by aquaporins and energy metabolism were affected by either concentration tested of 10 or 15 μg mL-1 of PS-NH2. Transcriptomic data and in vivo experiments were assembled into two putative AOPs, identifying as key events the adhesion of PS-NH2 as (molecular) initiating event, followed by oxidative stress, changes in transcription of specific genes, morphological defects, increase in reactive oxygen species level, impaired swimming behavior. As final adverse outcomes, altered larval development, reduced metamorphosis and inhibition of hatching were identified. Our study attempts to define AOPs for PS-NH2 without excluding that chemicals leaching from them might also have a potential role in the observed outcome. Overall data provide new insights into the mechanism of action of PS-NH2 NPs during chordate embryogenesis and offer further keys for a better knowledge of nanoplastics impact on early stages of marine life

Application of transcriptome profiling to inquire into the mechanism of nanoplastics toxicity during Ciona robusta embryogenesis

The growing concern on nanoplastics (<1  μm) impact on marine life has stimulated a significant amount of studies aiming to address ecotoxicity and disclose their mechanisms of action. Here, we applied an integrative approach to develop an Adverse Outcome Pathway (AOP) upon acute exposure to amino-modified polystyrene nanoparticles (PS-NH2 NPs, 50 nm), as proxy for nanoplastics, during the embryogenesis of the chordate Ciona robusta. Genes related to glutathione metabolism, immune defense, nervous system, transport by aquaporins and energy metabolism were affected by either concentration tested of 10 or 15 μg mL-1 of PS-NH2. Transcriptomic data and in vivo experiments were assembled into two putative AOPs, identifying as key events the adhesion of PS-NH2 as (molecular) initiating event, followed by oxidative stress, changes in transcription of specific genes, morphological defects, increase in reactive oxygen species level, impaired swimming behavior. As final adverse outcomes, altered larval development, reduced metamorphosis and inhibition of hatching were identified. Our study attempts to define AOPs for PS-NH2 without excluding that chemicals leaching from them might also have a potential role in the observed outcome. Overall data provide new insights into the mechanism of action of PS-NH2 NPs during chordate embryogenesis and offer further keys for a better knowledge of nanoplastics impact on early stages of marine life

A broad-taxa approach as an important concept in ecotoxicological studies and pollution monitoring

Aquatic invertebrates play a pivotal role in (eco)toxicological assessments because they offer ethical, cost-effective and repeatable testing options. Additionally, their significance in the food chain and their ability to represent diverse aquatic ecosystems make them valuable subjects for (eco)toxicological studies. To ensure consistency and comparability across studies, international (eco)toxicology guidelines have been used to establish standardised methods and protocols for data collection, analysis and interpretation. However, the current standardised protocols primarily focus on a limited number of aquatic invertebrate species, mainly from Arthropoda, Mollusca and Annelida. These protocols are suitable for basic toxicity screening, effectively assessing the immediate and severe effects of toxic substances on organisms. For more comprehensive and ecologically relevant assessments, particularly those addressing long-term effects and ecosystem-wide impacts, we recommended the use of a broader diversity of species, since the present choice of taxa exacerbates the limited scope of basic ecotoxicological studies. This review provides a comprehensive overview of (eco)toxicological studies, focusing on major aquatic invertebrate taxa and how they are used to assess the impact of chemicals in diverse aquatic environments. The present work supports the use of a broad-taxa approach in basic environmental assessments, as it better represents the natural populations inhabiting various ecosystems. Advances in omics and other biochemical and computational techniques make the broad-taxa approach more feasible, enabling mechanistic studies on non-model organisms. By combining these approaches with in vitro techniques together with the broad-taxa approach, researchers can gain insights into less-explored impacts of pollution, such as changes in population diversity, the development of tolerance and transgenerational inheritance of pollution responses, the impact on organism phenotypic plasticity, biological invasion outcomes, social behaviour changes, metabolome changes, regeneration phenomena, disease susceptibility and tissue pathologies. This review also emphasises the need for harmonised data-reporting standards and minimum annotation checklists to ensure that research results are findable, accessible, interoperable and reusable (FAIR), maximising the use and reusability of data. The ultimate goal is to encourage integrated and holistic problem-focused collaboration between diverse scientific disciplines, international standardisation organisations and decision-making bodies, with a focus on transdisciplinary knowledge co-production for the One-Health approach