An overview of the internalization and effects of microplastics and nanoplastics as pollutants of emerging concern in bivalves.

Microplastic and nanoplastic pollution in aquatic environments is a topic of emerging concern due to the internalization, retention time and effects of these particles in aquatic biota. Bivalves are considered bioindicators due to their wide distribution, sessile behaviour, occupation of ecological niches and ability to filter a large water volume. The study of microplastics and nanoplastics in bivalves has revealed the uptake mechanisms, internalization, distribution and depuration of these particles as well as their effects on physiological parameters, morphological alterations, immunotoxicity and changes in gene expression and proteomic profiles. In this review, we examine the primary characteristics of microplastics and nanoplastics (type of material, size, coating, density, additives and shapes) involved in their possible toxicity in bivalves. Furthermore, secondary characteristics such as the suspension media, aggregation stage and adsorption of persistent pollutants were also recorded to assess the impact of these materials on bivalves. Here, we have highlighted the efforts exerted thus far and the remaining gaps in understanding the extent of microplastic and nanoplastic impacts on bivalves on the basis of laboratory experiments and mesocosm bioassays and in the field. Furthermore, further microplastic and nanoplastic toxicological studies are proposed to facilitate the realistic assessment of environmental risk.

Size matters: Zebrafish (Danio rerio) as a model to study toxicity of nanoplastics from cells to the whole organism.

The contamination of the aquatic environment by plastic nanoparticles is becoming a major concern due to their potential adverse effects in aquatic biota. Therefore, in-depth knowledge of their uptake, trafficking and effects at cellular and systemic levels is essential to understand their potential impacts for aquatic species. In this work, zebrafish (Danio rerio) was used as a model and our aims were: i) to determine the distribution, uptake, trafficking, degradation and genotoxicity of polystyrene (PS) NPs of different sizes in a zebrafish cell line; ii) to study PS NPs accumulation, migration of immune cells and genotoxicity in larvae exposed to PS NPs; and iii) to assess how PS NPs condition the survival of zebrafish larvae exposed to a pathogen and/or how they impact the resistance of an immunodeficient zebrafish. Our results revealed that the cellular distribution differed depending on the particle size: the 50 nm PS NPs were more homogeneously distributed in the cytoplasm and the 1 µM PS NPs more agglomerated. The main endocytic mechanisms for the uptake of NPs were dynamin-dependent internalization for the 50 nm NPs and phagocytosis for the 1 µm nanoparticles. In both cases, degradation in lysosomes was the main fate of the PS NPs, which generated alkalinisation and modified cathepsin genes expression. These effects at cellular level agree with the results in vivo, since lysosomal alkalization increases oxidative stress and vice versa. Nanoparticles mainly accumulated in the gut, where they triggered reactive oxygen species, decreased expression of the antioxidant gene catalase and induced migration of immune cells. Finally, although PS NPs did not induce mortality in wild-type larvae, immunodeficient and infected larvae had decreased survival upon exposure to PS NPs. This fact could be explained by the mechanical disruption and/or the oxidative damage caused by these NPs that increase their susceptibility to pathogens.

Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response.

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.

Nanoplastics: From tissue accumulation to cell translocation into Mytilus galloprovincialis hemocytes. resilience of immune cells exposed to nanoplastics and nanoplastics plus Vibrio splendidus combination.

Plastic litter is an issue of global concern. In this work Mytilus galloprovincialis was used to study the distribution and effects of polystyrene nanoplastics (PS NPs) of different sizes (50 nm, 100 nm and 1 µm) on immune cells. Internalization and translocation of NPs to hemolymph were carried out by in vivo experiments, while endocytic routes and effects of PS NPs on hemocytes were studied in vitro. The smallest PS NPs tested were detected in the digestive gland and muscle. A fast and size-dependent translocation of PS NPs to the hemolymph was recorded after 3 h of exposure. The internalization rate of 50 nm PS NPs was lower when caveolae and clathrin endocytosis pathways were inhibited. On the other hand, the internalization of larger particles decreased when phagocytosis was inhibited. The hemocytes exposed to NPs had changes in motility, apoptosis, ROS and phagocytic capacity. However, they showed resilience when were infected with bacteria after PS NP exposure being able to recover their phagocytic capacity although the expression of the antimicrobial peptide Myticin C was reduced. Our findings show for the first time the translocation of PS NPs into hemocytes and how their effects trigger the loss of its functional parameters.

In vivo immunomodulatory and antioxidant properties of nanoceria (nCeO2) in the marine mussel Mytilus galloprovincialis.

Cerium nanoparticles (nCeO2) are increasingly utilized in a wide variety of industrial, environmental and biomedical applications, and are therefore expected to be released in the aquatic environment. Due to its peculiar redox properties, nCeO2 may present unique hazards to environmental and human health. Previous data showed that in the hemocytes of the marine bivalve Mytilus galloprovincialis, in vitro exposure to a particular type of nCeO2 (9 nm, characterized by negative ζ-potential, high H2O2 scavenging capacity and Ce3+/Ce4+ surface ratio) reduced basal ROS production, lysosomal membrane stability and phagocytic activity in the presence of hemolymph serum; the effects observed were partly ascribed to the formation of a SOD-protein corona in the hemolymph. In this work, the in vivo effects of this type of nCeO2 were investigated in mussels exposed to 100 µg/L nCeO2 for 96 h; several lysosomal, immune, inflammatory and antioxidant biomarkers were measured at cellular (hemocytes) and tissue (gills, digestive gland) level. Molecular responses were evaluated in hemocytes and digestive gland by determining expression of 11 selected genes related to known biological functions. The results show specific immunomodulatory and antioxidant effects of nCeO2 at different levels of biological organization in the absence of Cerium tissue accumulation. These data further support the redox mechanisms at the basis of the physiological effects of nCeO2. Finally, in order to evaluate the possible impact at the whole organism level, the effects of nCeO2 were evaluated in the 48 h embryotoxicity assay in a wide concentration range. However, nCeO2 exposure resulted in a small reduction in normal embryo development. Overall, the results demonstrate that in mussels nCeO2 can selectively modulate different physiological processes at different levels of biological organization.

Comparative effects of seawater acidification on microalgae: Single and multispecies toxicity tests.

In order to gain knowledge about the potential effects of acidification in aquatic ecosystems, global change research based on microalgae as sentinel species has been often developed. However, these studies are limited to single species tests and there is still a research gap about the behaviour of microalgal communities under this environmental stressor. Thus, the aim of this study was to assess the negative effects of CO2 under an ecologically realistic scenario. To achieve this objective, two types of toxicity tests were developed; i) single toxicity tests and ii) multispecies toxicity tests, in order to evaluate the effects on each species as well as the interspecific competition. For this purpose, three microalgae species (Tetraselmis chuii, Phaeodactylum tricornutum and Nannochloropsis gaditana) were exposed to two selected pH levels (7.4, 6.0) and a control (pH 8.0). The pH values were choosen for testing different scenarios of CO2 enrichment including the exchange atmosphere-ocean (pH 7.4) and natural or anthropogenic sources of CO2 (pH 6.0). The effects on growth, cell viability, oxidative stress, plus inherent cell properties (size, complexity and autofluorescence) were studied using flow cytometry (FCM). Results showed that T. chuii was the most resistant species to CO2 enrichment with less abrupt changes in terms of cell density, inherent cell properties, oxidative stress and cell viability. Although P. tricornutum was the dominant species in both single and multispecies tests, this species showed the highest decrease in cell density under pH 6.0. Effects of competence were recorded in the multispecies control (pH 8) but this competence was eclipsed by the effects of low pH. The knowledge of biological interactions made by different microalgae species is a useful tool to extrapolate research data from laboratory to the field.

Cytotoxicity of CeO2 nanoparticles using in vitro assay with Mytilus galloprovincialis hemocytes: Relevance of zeta potential, shape and biocorona formation.

Over the last decades, the growth in nanotechnology has provoked an increase in the number of its applications and consumer products that incorporate nanomaterials in their formulation. Metal nanoparticles are released to the marine environment and they can interact with cells by colloids forces establish a nano-bio interface. This interface can be compatible or generate bioadverse effects to cells. The daily use of CeO2 nanoparticles (CeO2 NPs) in industrial catalysis, sunscreen, fuel cells, fuel additives and biomedicine and their potential release into aquatic environments has turned them into a new emerging pollutant of concern. It is necessary to assess of effects of CeO2 NPs in aquatic organisms and understand the potential mechanisms of action of CeO2 NP toxicity to improve our knowledge about the intrinsic and extrinsic characteristic of CeO2 NPs and the interaction of CeO2 NPs with biomolecules in different environment and biological fluids. The conserved innate immune system of bivalves represents a useful tool for studying immunoregulatory responses when cells are exposed to NPs. In this context, the effects of two different CeO2 NPs with different physico-chemical characteristics (size, shape, zeta potential and Ce+3/Ce+4 ratio) and different behavior with biomolecules in plasma fluid were studied in a series of in vitro assays using primary hemocytes from Mytilus galloprovincialis. Different cellular responses such as lysosome membrane stability, phagocytosis capacity and extracellular reactive oxygen species (ROS) production were evaluated. Our results indicate that the agglomeration state of CeO2 NPs in the exposure media did not appear to have a substantial role in particle effects, while differences in shape, zeta potential and biocorona formation in NPs appear to be important in provoking negative impacts on hemocytes. The negative charge and the rounded shape of CeO2 NPs, which formed Cu, Zn-SOD biocorona in hemolymph serum (HS), triggered higher changes in the biomarker of stress (LMS) and immunological parameters (ROS and phagocytosis capacity). On the other hand, the almost neutral surface charge and well-faceted shape of CeO2 NPs did not show either biocorona formation in HS under tested conditions or significant responses. According to the results, the most relevant conclusion of this work is that not only the physicochemical characterization of CeO2 NPs plays an important role in NPs toxicity but also the study of the interaction of NPs with biological fluids is essential to know it behavior and toxicity at cellular level.

Are the TiO2 NPs a "Trojan horse" for personal care products (PCPs) in the clam Ruditapes philippinarum?

In recent years, increasing quantities of personal care products (PCPs) are being released into the environment. However, data about bioaccumulation and toxicity are scarce; and extraction and analytical approaches are not well developed. In this work, the marine clam Ruditapes philippinarum, selected as model organism, has been employed to investigate bioaccumulation, antioxidant enzyme activities and DNA damage due to exposure to TiO2 nanoparticles and bulk TiO2 (inorganic compounds that are frequent components of PCPs, plastics, paints and coatings, foods and disinfectant water treatments). We have also studied the joint effect of both forms of inorganic TiO2 combined with four organic compounds (mixture exposures) commonly used in PCPs: an antimicrobial (triclosan), a fragrance (OTNE) and two UV filters (benzophenone-3 and octocrylene). Bioaccumulation of the inorganic compound, TiO2, was almost immediate and constant over exposure time. With respect to the organic compounds in mixtures, they were mediated by TiO2 and bioaccumulation is driven by reduced size of the particles. In fact, nanoparticles can be considered as a vector to organic compounds, such as triclosan and benzophenone-3. After a week of depuration, TiO2 NPs and TiO2 bulk in clams showed similar levels of concentration. Some organic compounds with bioactivity (Log Kow >3), like OTNE, showed low depuration after one week. The joint action of the organic compound mixture and either of the two forms of TiO2 provoked changes in enzyme activity responses. However, for the mixtures, DNA damage was found only after the depuration period.

Toxicity of TiO2, in nanoparticle or bulk form to freshwater and marine microalgae under visible light and UV-A radiation.

Use of titanium dioxide nanoparticles (TiO2 NPs) has become a part of our daily life and the high environmental concentrations predicted to accumulate in aquatic ecosystems are cause for concern. Although TiO2 has only limited reactivity, at the nanoscale level its physico-chemical properties and toxicity are different compared with bulk material. Phytoplankton is a key trophic level in fresh and marine ecosystems, and the toxicity provoked by these nanoparticles can affect the structure and functioning of ecosystems. Two microalgae species, one freshwater (Chlamydomonas reinhardtii) and the other marine (Phaeodactylum tricornutum), have been selected for testing the toxicity of TiO2 in NP and conventional bulk form and, given its photo-catalytic properties, the effect of UV-A was also checked. Growth inhibition, quantum yield reduction, increase of intracellular ROS production, membrane cell damage and production of exo-polymeric substances (EPS) were selected as variables to measure. TiO2 NPs and bulk TiO2 show a relationship between the size of agglomerates and time in freshwater and saltwater, but not in ultrapure water. Under two treatments, UV-A (6 h per day) and no UV-A exposure, NPs triggered stronger cytotoxic responses than bulk material. TiO2 NPs were also associated with greater production of reactive oxygen species and damage to membrane. However, microalgae exposed to TiO2 NPs and bulk TiO2 under UV-A were found to be more sensitive than in the visible light condition. The marine species (P. tricornutum) was more sensitive than the freshwater species, and higher Ti internalization was measured. Exopolymeric substances (EPS) were released from microalgae in the culture media, in the presence of TiO2 in both forms. This may be a possible defense mechanism by these cells, which would enhance processes of homoagglomeration and settling, and thus reduce bioavailability.

Direct and indirect effects of silver nanoparticles on freshwater and marine microalgae (Chlamydomonas reinhardtii and Phaeodactylum tricornutum).

The last decade has seen a considerable increase in the use of silver nanoparticles (AgNPs), which are found in many every-day consumer products including textiles, plastics, cosmetics, household sprays and paints. The release of those AgNPs into aquatic environments could be causing ecological damage. In this study we assess the toxicity of AgNPs of different sizes to two species of microalgae, from freshwater and marine environment (Chlamydomonas reinhardtii and Phaeodactylum tricornutum respectively). Dissolution processes affect the form and concentration of AgNPs in both environments. Dissolution of Ag from AgNPs was around 25 times higher in marine water. Nevertheless, dissolution of AgNPs in both culture media seems to be related to the small size and higher surface area of NPs. In marine water, the main chemical species were AgCl2- (53.7%) and AgCl3-2 (45.2%). In contrast, for freshwater, the main chemical species were Ag+ (26.7%) and AgCl- (4.3%). The assessment of toxicological responses, specifically growth, cell size, cell complexity, chlorophyll a, reactive oxygen species, cell membrane damage and effective quantum yield of PSII, corroborated the existence of different toxicity mechanisms for microalgae. Indirect effects, notably dissolved Ag ions, seem to control toxicity to freshwater microalgae, whereas direct effects, notably attachment onto the cell surface and the internalization of AgNPs inside cells, seem to determine toxicity to the marine species studied. This research contributes to knowledge on the role of intrinsic and extrinsic factors in determining the behavior of NPs in different aquatic environments and the interaction with microalgae.

Homoagglomeration and heteroagglomeration of TiO2, in nanoparticle and bulk form, onto freshwater and marine microalgae.

TiO2 nanoparticles (TiO2 NPs) are employed in many products (paints, personal care products, especially sunscreens, plastics, paper, water potabilization and food products) and are then released into the environment from these products. These nanoparticles present potential risk to freshwater and marine microalgae. The primary toxicity mechanism is adsorption between NPs and microalgae (heteroagglomeration); however, studies of interactions of this kind are scarce. We investigated the heteroagglomeration process that occurs between two forms of TiO2 material, nanoparticles and bulk, and three different microalgae species, and under different environmental conditions (freshwater and marine water), in order to assess the influence of pH and ionic strength (IS). The heteroagglomeration process was examined by means of co-settling experiments and the Derjaguin-Landau-Verwey-Overbeek (DLVO) approach. The homoagglomeration process (only NPs to NPs) did not show differences between culture media (freshwater and marine water). However, in the heteroagglomeration process between NPs and cells, IS played an important role. Ions can compress the electro-double layer between NPs and microalgae, allowing a heteroagglomeration process to take place, as shown by settling experiments. TiO2 NPs presented a settling rate higher than bulk TiO2. The DLVO theory could only partially explain heteroagglomeration because, in this model, it is not considered that NP-NP and Cell-Cell homoagglomeration co-occur. In this study neither the role of exopolymeric substances in the interaction between NPs and cells nor detoxification are considered. The authors suggest that the interaction between NPs and microalgae could be considered as the first stage in the process by which nanoparticles affect microalgae.

CeO2 NPs, toxic or protective to phytoplankton? Charge of nanoparticles and cell wall as factors which cause changes in cell complexity.

CeO2 nanoparticles (CeO2 NPs) are well-known for their catalytic properties and antioxidant potential. Recent uses in therapy are based on the Ce+3 ions released by CeO2 NPs. Reactions involving redox cycles between Ce+3 and Ce+4 oxidation stage seem to promote scavenging of reactive oxygen species (ROS), thus protecting cells from oxygen damage. However, the internalization of CeO2 NPs and release of Ce+3 could be responsible for a toxic effect on cells. The literature reports controversial results on the toxicity of CeO2 NPs to phytoplankton. Therefore, we have tested the potential toxic effect of two CeO2 NPs (with positive and negative zeta potential) and bulk CeO2 (at 0.1, 1, 10, 100 and 200mg·L-1) on three species of microalgae from different environments: marine diatom (Phaeodactylum tricornutum), marine chlorophyte (Nannochloris atomus) and freshwater chlorophyte (Chlamydomonas reinhardtii) over 72h in batch cultures. Responses measured in the microalgae population are: growth, chlorophyll a, cell size, cell complexity, percentage of ROS, and percentage of cell membrane damage. Positive zeta potential CeO2 NPs provoked greater cell complexity (up to 78, 172 and 23 times more cell complexity than in controls found for C. reinhardtii, P. tricornutum and N. atomus respectively) than negative zeta potential CeO2 NPs. The SSC signal detected by flow cytometry measured increases of particles entering cells, and this is related to cell viability and levels of intracellular ROS (correlation between SSC and percentage of ROS of 0.72 and 0.97 found for C. reinhardtii and P. tricornutum). When increased cellular complexity over controls is between 2 and 6 times greater, CeO2 (in bulk or nanoparticulate form) seems to protect against ROS. When increased cellular complexity is from 7 to 23 times greater, CeO2 does not provoke toxic responses; however, when increased cellular complexity over controls is very high, from 61 to 172 times, increased ROS production and toxic responses are found. Results show that two factors, the charge of CeO2 NPs and cell wall structure, constitute the primary barrier to the possible accumulation of CeO2 NPs within phytoplankton cytosol.

Effects of TiO2 nanoparticles and sunscreens on coastal marine microalgae: Ultraviolet radiation is key variable for toxicity assessment.

Given the large numbers of sunbathers on beaches, sunscreen compounds are being released into the coastal aquatic environment in significant amounts. Until now the effect of these potential pollutants on microbiota has been not well-known. Phytoplankton is a key component of the microbiota community. It forms the basis of the aquatic trophic networks, and any change in the natural population of phytoplankton can affect the structure of aquatic biota. This paper describes an experiment performed outdoors (in natural sunlight conditions including ultraviolet radiation (UVR) and with UVR blocked) on mixed microalgae populations (four species from different key marine taxonomic groups, Nannochloropsis gaditana, Chaetoceros gracilis, Pleurochrysis roscoffensis and Amphidinium carterae), for three days, exposed to a range of concentrations of three commercial sunscreens (with variable TiO2 concentrations: highest concentration for sunscreen C, followed by sunscreen A; and sunscreen B did not contain TiO2 in its composition). With regard to UVR effect, in the absence of sunscreens, the most sensitive species is the centric diatom, Chaetoceros gracilis, and the least is Nannochloropsis gaditana; this last species presented the same behavior in the absence of UVR and with high sunscreen concentrations. The toxicity gradient obtained for sunscreens and nanoparticles under UVR is: TiO2 NPs>Sunscreen C>Sunscreen A>Sunscreen B. The differential sensitivity of microalgae to sunscreens and TiO2 NPs can produce a change in the dynamics of phytoplankton populations and provoke undesirable ecological effects (such as giving dinoflagellates more prominence). The effects of UVR, commonly neglected in bioassays, could alter the results in important ways and should be considered when performing environmentally-relevant bioassays. The toxicity mediated by hydrogen peroxide production associated with the concentration of TiO2 NPs cannot be considered the only factor responsible for the toxicity: the organic compounds in the sunscreens must also be taken into account.

Cadmium effects on development and reproduction of Oncopeltus fasciatus (Heteroptera: Lygaeidae).

Newly hatched nymphs of the insect Oncopeltus fasciatus were exposed to various concentrations of CdCl2 administered in drinking water until the end of adult life. Significant nymphal mortalities were observed at concentrations above 30 mg Cd/l (corresponding to the LC50). The duration of the nymphal stages increased in proportion to the Cd concentration; at the lowest Cd concentration of 10 mg Cd/l, the median duration was significantly prolonged by one day, while at the highest concentration of 100 mg Cd/l it was increased by 10 days over the control group. The weight of newly emerged adults lineally decreased with Cd concentration, being reduced to half the weight of controls at 100 mg Cd/l. In addition, a proportionality between delay in development and weight reduction was found in Cd-treated adults. Survival of adult females was decreased at concentrations higher than 10 mg Cd/l, while males were only affected at 30 mg Cd/l or higher doses. Reproduction was the most affected parameter. Oviposition rate, fecundity and fertility of females exposed to 10 mg Cd/l were significantly lower than controls (73%, 58% and 55% relative to controls, respectively). Hatchability of the eggs laid by treated females was also reduced. These results show that development and reproduction of O. fasciatus are seriously impaired at sublethal Cd concentrations.