Alternative Methods for Safety Assessment of Nanomaterials in the Aquatic Environment
Miren P. Cajaraville, University of the Basque Country; Ilaria Corsi, University of Siena; Marta Sendra, Spanish National Research Council (CSIC); and Magalie Baudrimont, University of Bordeaux
The session “Alternative Methods for Safety Assessment of Nanomaterials in the Aquatic Environment” was chaired by Miren P. Cajaraville, University of the Basque Country; Ilaria Corsi, University of Siena; Marta Sendra, Spanish National Research Council (CSIC); and Magalie Baudrimont, University of Bordeaux; and brought together scientists from academia, business and government to discuss novel and established alternative methods for safety assessment of nanomaterials (NMs), including engineered NMs (ENMs) and nanoplastics, in the aquatic environment. The session included 18 on-demand presentations, which covered in vitro approaches using immune cells from marine mussels and sea urchins; in vivo approaches using algae, ascidian and sea urchin embryos, embryos and adults of bivalve mussels, microcrustaceans and amphipods; and in vitro approaches using fish cell lines. Screening and grouping strategies were also proposed. A wide range of ENMs was covered from metal and metal oxide nanoparticles (NPs) to carbon-based graphene family NMs and C60 fullerenes. Finally, several presentations were devoted to in vitro and in vivo toxicity studies of polystyrene (PS) nanoplastics as a proxy of environmental plastics, reflecting the great concern associated to their occurrence from degradation of larger plastic items in the aquatic environment, including polar areas.
The presentation by Julián Blasco and Marta Sendra, CSIC, proposed a new approach to assess the immunotoxicity of PS nano and microplastics to mussel (Mytilus galloprovincialis) hemocytes by using flow cytometry, looking at effects on three different subpopulations of hemocytes. Similarly, Maria Elisabetta Michelangeli and Tania Gomes, Norwegian Institute for Water Research, assessed the effects of polymethyl-methacrylate (PMMA) nano and microplastics in two mussel hemocyte subpopulations by flow cytometry. In both studies it was concluded that different hemocyte subpopulations responded differentially to nano- and microplastic exposures. Manon Auguste and colleagues, University of Genoa and University of Urbino, presented an overview of their recent publications on the effects of surface modified PS nanoplastics on mussel hemocytes in vitro and on mussel embryos and adults in vivo. This integrative approach allowed to conclude that effects of nanoplastics varied, depending on size and surface characteristics as well as on the selected exposure medium and formation of protein corona. The presentation by Alberto Katsumiti and colleagues, University of the Basque Country, focused on the intracellular fate of PS nano- and microplastics alone or in combination with the PAH benzo(a)pyrene (BaP) on mussel hemocytes. The authors concluded that PS nano- and microplastics are internalized in mussel hemocytes by different mechanisms depending on their size and that they can act as vehicles of BaP into mussel hemocytes, causing a differential modulation of the immune function. The presentation by Carola Murano and colleagues, University of Siena and Stazione Zoologica Anton Dohrn, focused on immune cells of sea urchins (coelomocytes of Paracentrotus lividus). Their main finding was that positively charged amino-modified PS nanoplastics (PS-NH2) caused a significantly higher toxicity than carboxyl-modified ones (PS-COOH). They also reported the rapid and time-dependent uptake of PS-COOH by sea urchin coelomocytes. Overall, these works demonstrated the suitability of mussel and sea urchin’s immune cells to study interactions and mode of action of PS nanoplastics and to screen their immunotoxicity potential. Therefore, they represent a fast, accurate, reproducible and cost-effective tool for safety assessment of nanoscale materials, including nanoplastics in the aquatic environment.
The presentations of Maria Concetta Eliso and colleagues, University of Siena and Stazione Zoologica Anton Dohrn, and Leticia Palmeira Pinto and colleagues, University of Sao Pauloand University of Siena, reported effects of either positively charged PS nanoplastics (PS-NH2) and titanium dioxide (TiO2) NPs respectively on the embryogenesis of the marine ascidian Ciona robusta and the tropical sea urchin Lytechinus variegatus. Transcriptomic and adverse outcome pathway approaches applied in C. robustaembryos revealed several gene functions and pathways adversely affected by PS-NH2 nanoplastics. TiO2NPs were revealed to affect fertilization rate at environmentally realistic concentrations while at highest exposure concentration malformations and delays in the development of sea urchin embryos and larvae were found. Nagore González-Soto and colleagues, University of the Basque Country, presented the effects of graphene oxide (GO) nanoplatelets alone or combined with BaP on adult mussels M. galloprovincialis. They demonstrated that GO can adsorb BaP and act as a carrier of this compound to mussels. While GO alone and GO with BaP caused genotoxicity and inflammatory reactions in digestive gland and gonad, GO with BaP provoked additional effects, such as neurotoxicity, inhibition of biotransformation, changes in antioxidant enzymes, and oocyte atresia. Andrea Rivero Arze and colleagues, (INERIS and Université Catholique de l’Ouest, evaluated the kinetics of bioaccumulation and depuration of 12 different ENMs (6 TiO2, 3 SiO2, 2 CeO2 and 1 ZnO) on Daphnia magna. The results showed that differences in kinetics relied on the physico-chemical properties of the ENM and, notably, the zeta potential, which was correlated to the elimination rate constants. Similar observations were made with microalgae, offering the possibility of grouping ENMs based on their physico–chemical properties, driving nano-biointeractions. Arianna Bellingeri and colleagues, University of Siena and University of Rome, tested the acute and chronic effects of Ag NPs coated with citrate and L-cysteine for Hg remediation on microalgae and microcrustaceans from fresh and marine waters. Low or no acute toxicity to both microalgae and microcrustaceans was observed, while chronic exposure revealed severe effects in both Ceriodaphnia dubia and brine shrimp Artemia franciscana, attributed to a nano-related effect. The results highlighted that the surface coating plays a predominant role in reducing toxicity of Ag NPs in both fresh and marine waters, and the need to apply chronic tests for proper risk assessment of Ag NPs. An overview of testing strategies for bioaccumulation assessment of metal ENMs was presented by Christian Schlechtriem and Sebastian Kuehr, Fraunhofer IME, RWTH Aachen University and University of Siegen. They proposed the use of the freshwater benthic amphipod Hyalella azteca bioconcentration and biomagnification tests to integrate the process of sedimentation occurring after agglomeration of metal ENMs in the aquatic systems. A literature overview was also presented by Amalie Thit Jensen and colleagues, Roskilde University and Université Catholique de l’Ouest, on bioaccumulation and adverse effects of NPs in aquatic sediments. The challenge posed to extrapolate the effects on sediment from water studies was underlined in terms of NP behavior, bioaccumulation and internal distribution, but also the integration of non-conventional toxicity endpoints and chronic and population-level effects.
As far as studies on fish cell lines and mixtures toxicity, Nicolas Martin and colleagues, University of Gothenburg, investigated the effect of UV filters, including three different coatings of TiO2 NPs alone and in binary combination with traditional organic molecules on rainbow trout gill cell line (RTgill-W1). While no cytotoxicity for all TiO2 NPs and dose-dependent effects for organic UV filters were observed, mixtures resulted either less or more toxic based on NP-type and concentration. Furthermore, Francis Okoroigbo and colleagues, University of Gothenburg, reported the effects of three silica NPs (SiO2 NPs, anionic spherical, anionic rod-shaped and cationic spherical) and three pesticides (hexadecylpyridinium, pentachlorophenol and diflufenican) on RTgill-W1 cell line. Binary exposure combinations revealed that surface area and charge of SiO2 NPs affect pesticides interaction causing lower or higher cell toxicity compared to single exposures. In addition, Marta Sendra and colleagues, CSIC, reported different mechanisms of uptake, trafficking and depuration/degradation of 50 nm and 1 µm PS particles in ZF4 cell line. 50 nm PS nanoplastics showed higher internalization, with several active endocytic mechanisms and passive mechanisms involved, whereas 1 µm particles were internalized by phagocytic mechanisms. 50 nm PS NPs were also degraded and depurated faster at cellular level than 1 µm particles.
A bioluminescence enzymatic assay based on bacterial luciferase and NAD(P)H:FMN-oxidoreductase was used by Arina Kicheera and colleagues and Akaterina Kovel and colleagues, Siberian Branch of the Russian Academy of Sciences, Siberian Federal University and National Research University, to screen and compare toxicity and antioxidant properties of hydrated fullerenes (HyFn), magnetite (Fe3O4) NPs and fullerenols. Higher toxicity of HyFn compared to fullerenols was found and moderate antioxidant activity thus raising concern on its use in biomedicine. Suspensions of bare Fe3O4 provided toxic effects at higher concentration probably associated to the release of Fe2+ in exposure media oxidated to Fe3+ in water solutions. The work of Richard Cross and colleagues, Centre for Ecology & Hydrology, demonstrated that extrinsic properties of nanoforms can modulate their fate, exposure and toxicity in case that the coating does not face away under degradation processes. For this, authors developed a colorimetric carbon substrate utilization assay as indicator of microbial respiration.
The live discussion of the session was followed by an average of 85 participants. Cajaraville, briefly introduced the topic and identified several key issues andquestions that were further discussed. Since mussels represent one of the most widely studied model species for the assessment of ENM and nanoplastic toxicity in the marine environment, it was pertinent to discuss whether mussels collected in the field represent a suitable model as they can show different genetic backgrounds, physiological adaptations and variability related to sex, size, tidal level, season and levels of pollutants. Harmonized and well-defined protocols for mussel collection and laboratory acclimatization could successfully contribute towards standardization and robustness of their use as reliable in vitro and in vivo models. It was agreed that production of a guideline could be very useful to allow comparisons between different studies and experiments. In addition to mussel sampling and acclimatization, the guideline should cover protocols for preparation and characterization of ENMs and nanoplastics in exposure media, controlled exposure conditions, cell isolation and culture, and relevant lethal and sub-lethal endpoints, such as immune function in hemocytes.
In fish, a high number of cell lines are available for toxicity testing of ENMs and other nano-scale environmental pollutants. Recently the RTgill-W1 cell line has been validated by the OECD as alternative to the fish acute toxicity test, being the first alternative method validated in the ecotoxicology and aquatic toxicology fields. Whereas the need to replace in vivo fish toxicity tests is widely recognized and under regulation, in vitro methods using aquatic invertebrates should be seen as a promising cost-effective and reliable alternative. Therefore, advancements towards their further development by solving current challenges should be made so to increase their wider application. Not only they significantly reduce the numbers of animals needed for hazard and risk assessment of ENMs and other emerging pollutants in the aquatic environment but they also provide tools to elucidate mechanisms and mode of action, better linking the observed exposure to biological effects.
Overall, there is the need to bridge the gap between in vitro testing results and those in vivo so that the first could be predictive of overall toxicity exerted at whole organism level. Furthermore, links with consequences at populations and communities levels up to ecosystem services and functions should be provided. This could facilitate categorization of risks for aquatic ecosystems based on current in vitro studies. In this context, developing more realistic exposure scenarios using environmentally relevant concentrations and exposure conditions was seen as crucial for both in vitro and in vivo approaches. It was considered fundamental to define precisely exposure media, exposure conditions and to characterize the behaviour of studied NMs in environmental and biological matrices.
Finally, it was discussed whether nanoplastics should be considered as ENMs for environmental risk assessment. Research on nanoplastics is blooming, but we still lack a consensus even regarding a definition for nanoplastics. Most investigations on the fate and effects of nanoplastics both in vitro and in vivo on aquatic animals have tested polystyrene spherical beads as proxy for environmental nanoplastics. However, it has become clear that most prevalent micro- and nanoplastics in the aquatic environment are nanofibers. It was pointed out that there is a need to develop methods to obtain nanofibers from environmental plastics under controlled laboratory conditions in order to use them as standardized nanoplastic material.
Overall, the session served as a forum to share and discuss advances in the field of environmental risk assessment of ENMs and nanoplastics in the aquatic environment based on alternative methods.
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