M. Sendra

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.

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.

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.

Cytotoxicity of CeO 2 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.

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.

Erythromycin sensitivity across different taxa of marine phytoplankton. A novel approach to sensitivity of microalgae and the evolutionary history of the 23S gene

Erythromycin has been recorded in coastal waters and could pose a severe threat to marine microbial life. Macrolides such as erythromycin may affect microalgae by inhibiting the pathways involved in protein synthesis. Toxicological testing of microalgae has proven to be a useful tool for the risk assessment of a substance affecting phytoplankton. Due to the controversial results concerning the sensitivity of microalgal species to erythromycin found in the literature, the goals of this work were, initially, to assess the erythromycin sensitivity of different species of marine microalgae from different and representative taxonomic groups; and, secondly, to examine whether the sensitivity to erythromycin could be explained by the differences in the phylogenetic evolution. We chose eight species: two green algae, four heterokonts, one haptophyte and one dinoflagellate, which were then exposed to erythromycin (0.1 to 10 mg L-1). Our results showed a wide range of sensitivities indicating that the biology of each species was primarily responsible for the variation observed. To test the second objective, we contrasted different ecotoxicological endpoints (growth, cellular properties and status of the photosynthetic apparatus) with the phylogenetic distribution [eukaryotic host (concatenated nuclear tree), evolutionary history of the chloroplast (16S tree), efficiency and repair of photosystem II (psbA tree), and the binding site of erythromycin (23S tree)] of the species. We found that the growth inhibition of microalgae as a toxicological endpoint was the endpoint best explained by the topology of the 23S rRNA gene tree when it was modelled following a non-stationary evolutionary process.